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In: Food and Beverage Consumption and Health Series
HANDBOOK OF GREEN TEA AND

HEALTH RESEARCH
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FOOD AND BEVERAGE
CONSUMPTION AND HEALTH SERIES
Handbook of Green Tea and Health Research

Helen McKinley and Mark Jamieson
2009. ISBN 978-1-60741-045-4
In: Food and Beverage Consumption and Health Series
HANDBOOK OF GREEN TEA AND

HEALTH RESEARCH
HELEN MCKINLEY
AND
MARK JAMIESON
EDITORS
Nova Science Publishers, Inc.

New York
Copyright © 2009 by Nova Science Publishers, Inc.
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LIBRARY OF CONGRESS CATALOGING-IN-PUBLICATION DATA
Handbook of green tea and health research / [edited by] Helen McKinley and Mark Jamieson.
p. ; cm. -- (Food and beverage consumption and health)
Includes bibliographical references and index.
ISBN 978-1-60876-202-6 (E-Book)
1. Green tea--Health aspects. I. McKinley, Helen. II. Jamieson, Mark. III. Series: Food and beverage consumption and
health.
[DNLM: 1. Tea. 2. Camellia sinensis. 3. Catechin--analogs & derivatives. 4. Phytotherapy--methods. WB 438 H236
2009]
RM251.H36 2009
615'.321--dc22
2009000178
Published by Nova Science Publishers, Inc. Ô New York

CONTENTS
Preface ix
Chapter 1 Central Functions of Green Tea Components 1
M. Furuse,, N. Adachi, S. Tomonaga, H. Yamane and
D. M. Denbow
Chapter 2 Green Tea Catechins: A Class of Molecules with Antimicrobial
Activity 23
P. Buzzini, P. Vignolini, M. Goretti, B. Turchetti, E. Branda,
E. Marchegiani, P. Pinelli and A. Romani
Chapter 3 Lipid-soluble Green Tea Polyphenols: Stabilized for Effective
Formulation 45
Ping Chen, Douglas Dickinson and Stephen Hsu
Chapter 4 Assessment of the Antioxidant Capacity of Green Teas:
A Critical Review 63
Camilo López-Alarcón and Eduardo Lissi
Chapter 5 Design and Assessment of the in Vitro Anti-oxidant Capacity of a
Beverage Composed of Green Tea (Camellia Sinensis L.) and
Lemongrass (Cymbopogon Citratus Stap) 81
D. Fernando Ramos Escudero, Luis Alberto Condezo Hoyos,
Mónica Ramos Escudero and Jaime A. Yáñez
Chapter 6 Teas Are not All the Same: In Vitro and in Vivo Antioxidant
Activity and Appetite Modulation in Rats of Green Teas with High
and Low Levels of Organic Selenium 103
Abdul L. Molan, Zhuojian Liu and Wenhua Wei
Chapter 7 Anti-obesity Effects of (-)-Epigallocathchin-3-gallate and its
Molecular Mechanism 125
Cheol-Heui Yun, Gi Rak Kim, Min Ji Seo, Hyun-Seuk Moon
and Chong-Su Cho
vi Contents
Chapter 8 Green Tea: Protective Action against Pesticides and other

Xenobiotics Present in Human Diet 157
Geetanjali Kaushik, Poonam Kaushik and Shivani Chaturvedi
Chapter 9 New Method to Improve the Function and Industrial Applicability
of Green Tea and Its Byproducts Using Irradiation Technology 177
Cheorun Jo and Myung Woo Byun
Chapter 10 Green Tea Catechin as Angiogenesis Inhibitor 197
Kiminori Matsubara and Yoshiyuki Mizushina
Chapter 11 Neuroprotective Effect of Theanine on Cerebral Ischemia 207
Nobuaki Egashira,, Kenichi Mishima, Katsunori Iwasaki,
Ryozo Oishi and Michihiro Fujiwara
Chapter 12 Characterization of the Neuroprotective Activity of the Polyphenol
(-)-Epigallocatechin-3-gallate in the Brain 219
Orly Weinreb, Tamar Amit, Moussa B. H. Youdim and
Silvia Mandel
Chapter 13 Cardiovascular and Metabolic Effects of Green Tea 243
Kamilla Kelemen
Chapter 14 Molecular Basis for the Anti-cancer Activity of EGCG in Vivo:
Molecular-Targeting Prevention of Cancer by Green Tea Catechin 257
Yoshinori Fujimura and Hirofumi Tachibana
Chapter 15 Utility of Epigallocatechin Gallate in the Treatment and Prevention
of Breast Cancer: Molecular Mechanisms for Tumor Suppression 301
R. J. Rosengren
Chapter 16 Green Tea Catechins in Colorectal Cancer 325
Seung Joon Baek and Mugdha Sukhthankar
Chapter 17 Inhibitory Effect of Catechin Derivatives from Green Tea on DNA
Polymerase Activity, Human Cancer Cell Growth, and TPA
(12-O-tetradecanoylphorbol-13-acetate) -induced Inflammation 347
Yuko Kumamoto-Yonezawa, Hiromi Yoshida and
Yoshiyuki Mizushina
Chapter 18 Telomerase Regulation in Response to Green Tea 363
Huaping Chen and Trygve O. Tollefsbol
Chapter 19 Green Tea and Chronic Obstructive Pulmonary Disease: A Case-
control Study in Japan 383
Fumi Hirayama and Andy H. Lee
Chapter 20 Green Tea and Diabetes 393
Dongfeng Wang, Linge Wang and Li Zhang
Chapter 21 Green Tea and Type 2 Diabetes 411
Jae-Hyung Park, Hye-Young Sung and Dae-Kyu Song
Contents vii
Chapter 22 Biocatalytic Conversion of Green Tea Catechins to Epitheaflagallin,

Epitheaflagallin, 3-O-gallate, and Theaflavins: Production of
Promising Functional Foods 419
Nobuya Itoh and Yuji Katsube
Chapter 23 Preventive Effects of Green Tea Catechins on Dementia 429
Michio Hashimoto, Md Abdul Haque, Kohinoor Begum Himi
and Yukihiko Hara
Short Commentary
Green Tea and Potential Human Health Effects 451
James E. Trosko
Index 463
PREFACE
After water, tea signifies the second most frequently consumed beverage worldwide.
Teas are not all the same; among the many areas of research that are included in this book are
the effects of selenium-containing green tea on food consumption and body weight gain.
Research shows that tea consumption may have its strongest effect among patients with
cardiovascular disease. A specific chapter investigates whether green tea intake can reduce
the risk of chronic obstructive pulmonary disease.
Research is presented to show that green tea and its major constituent epigallocatechin
gallate (EGCG) have a potential chemopreventative and/or treatment for a variety of diseases
including breast cancer. Other research sheds new light on the molecular basis for the cancer-
preventive activity of EGCG in vivo and helps in the design of new strategies to prevent
cancer. A further study presents an analysis assessing the progress of research on the
mechanisms pertaining to how telomerase activity is regulated by green tea in cancer cells.
Further chapters look at the relationship of tea to diabetes and a description of the
beneficial effects of green tea catechins on neuronal functions and neuronal diseases such as
dementia. To improve biological functions and industrial applicability of green tea and its by-
products, research is presented showing irradiation as a useful method.
Chapter 1 - Tea (Camellia sinensis) is widely consumed throughout the world and has a
number of biologically active substances such as caffeine, catechins, and L-theanine (γ-
glutamylethylamide). Tea consumption is generally known to induce a feeling of relaxation
which may be mediated by either catechins, L-theanine, or both, since caffeine stimulates
locomotor activities.
The catechin (-)-epigallocatechin gallate (EGCG) occurs abundantly in tea. Moreover,
frequent consumption of green tea results in high levels of EGCG in the blood and brain.
Catechins, which are flavonoids, affect the central nervous system (CNS). The therapeutic
effects of flavonoids may involve their binding to γ-aminobutyric acid (GABA)A receptors,
which is a major inhibitory neurotransmission system. Recently, EGCG was shown to bind to
GABAA receptors in vitro and to induce a sedative effect through GABAA, but not GABAB,
receptors in the brain.
L-Theanine, a derivative of glutamate, is a unique amino acid occurring only in green tea
and a few other plants. After administration L-theanine concentrations were increased in
serum, liver and brain, suggesting that L-theanine can cross the blood-brain barrier.
Intravenous administration of L-theanine was shown to affect the cortex, hippocampus and
amygdala, and increase the alpha-band component of electroencephalograms in rats. More
x Helen McKinley and Mark Jamieson
recently, it was shown that L-theanine could reduce stress via either inhibiting cortical neuron
excitation in human subjects or influencing the secretion and function of neurotransmitters in
the CNS. We discuss the central functions of green tea components such as EGCG and L-
theanine in the CNS.
Chapter 2 - A significant part of scientific interest of academy or industry is focused on
discovering novel natural antimicrobial drugs. This attention is essentially justified by the
expectation that a few of them could play a role in supporting (or even in substituting) some
antibiotics of current use. It has been estimated that, although about some tens of novel
antimicrobial drugs (either of biological or synthetic origin) are currently launched each year,
due to the increasing development of resistant microbial genotypes, their downturn is
becoming very rapid.
Taking into account these considerations, the enormous scientific and commercial
interest in discovering and developing novel classes of molecules exhibiting more or less
pronounced antimicrobial properties has oriented the work of a growing part of the scientific
community toward large-scale screening programs aimed at discovering novel classes of
bioactive molecules.
The occurrence in some plants of secondary metabolites exhibiting a more or less
pronounced antimicrobial activity is a well-known phenomenon. Among them, green tea
polyphenols represent a reservoir of molecules characterized by antioxidant, antiradical and
antimicrobial activity. In particular, catechins have proven to be effective towards both
prokaryotic and eukaryotic microorganisms. Despite the large number of studies published so
far, their actual potentialities and limitations as antimicrobial (mainly antibacterial and
antimycotic) drugs have not been critically evaluated. The present chapter represents an
overview of the recent literature on the antiviral and antimicrobial properties exhibited by
polyphenols, particularly catechins, occurring in green tea composition.
Chapter 3 - Green tea polyphenols (GTPs), also referred to as green tea catechins, possess
properties that can provide unique health benefits to humans. As indicated in other chapters of
this book, studies using molecular, cellular, and animal models, and in human subjects, have
demonstrated that these phytochemicals from non-oxidized tea leaves have anti-cancer, anti-
oxidant, anti-microbial, and anti-inflammatory properties. Recently, investigations in our and
other laboratories indicated that topical application of GTPs could protect the epidermis
against autoimmune disorders, such as psoriasis, prevent or repair UV-induced damage, and
suppress scar tissue overgrowth. In addition, specific gene regulation by GTPs, especially
epigallocatechin-3-gallate (EGCG), promotes skin cell differentiation, which could lead to
improved homeostasis of the skin.
Based on these facts, the topical use of products containing GTPs has become more
popular, and manufacturers of cosmetic, health care, and household products are adding GTPs
or EGCG to their formulations. However, it is important to note that studies described in this
book always use “freshly prepared” GTPs or green tea, instead of “pre-prepared” materials.
This is because GTPs are potent antioxidants that react rapidly with reactive oxygen species
(ROS). As a result, GTPs in most commercially available products have been oxidized and/or
epimerized; the biological effects of the resulting compounds are largely unknown. In
addition, due to the highly water-soluble nature of these compounds, GTPs in their original
form are not lipid-soluble, and therefore not permeable to the skin, a water-proof barrier.
Another problem with formulation of GTPs for topical application is the coloration change
and precipitation caused by oxidation. Thus, GTPs for topical application (e.g., on skin and
Preface xi
mucous membranes) must be prepared and used immediately prior to oxidation, coloration
and precipitation. These properties of GTPs make it difficult to formulate products containing
them that have a reasonable shelf life and maintain their activity and effectiveness. In other
words, most of the commercially available “green tea”–containing products are without the
full benefits of green tea or GTPs. Therefore, strategies to stabilize and increase the
bioavailability of GTPs are needed to provide the full benefits of GTPs to consumers or
patients.
Recently, it has been shown that lipid esters of GTPs can be formed either enzymatically
or chemically. These green tea polyphenol-lipid esters, also referred to as lipid-soluble tea
polyphenols (LTPs), could significantly improve formulations of consumer or health care
products. We hypothesized that fatty acyl esterification of green tea polyphenol would protect
the hydroxyl groups from oxidation and improve skin permeability. In the current study, we
compared the activities of LTPs to GTPs for their anti-cancer and gene regulation properties.
We examined whether LTPs can be converted into a free GTP (EGCG) in human skin
keratinocyte cultures. In addition, the effects of LTPs in a mouse model for psoriasis were
evaluated. The results indicate that LTPs effectively cause cancer cell death, induce caspase
14 gene expression both in vitro and in vivo, and improve the skin condition in an animal
model for psoriasis. Consistent with these observations, HPLC analysis demonstrated that
EGCG in its original form was released from LTPs in situ by human epidermal keratinocytes.
These results suggest that LTPs, under appropriate conditions, function similarly to GTPs.
More importantly, since the most reactive hydroxyl group(s) is/are protected, and the lipid
solubility is dramatically increased by the fatty acyl groups, the biological activity of these
compounds can be stabilized, and their bioavailability increased significantly.
In conclusion, LTPs are a novel and more effective form of green tea polyphenols for
topical applications and other purposes, especially in formulations that require a reasonable
shelf life. In addition, LTPs can be a natural additive to consumable products such as salad
oil, fish oil, and cooking oil as antioxidants.
Chapter 4 - In the last decades, the beneficial influence of green tea on human health has
been related to the antioxidant capacity (AC) of its phenolic constituents. The latter has
originated systematic studies of the AC of green tea and/or its pure antioxidants. Different
methodologies have been used with this purpose. The methods are based on: (1) Estimation
of the consumption by additives of stable free radicals (DPPH, and ABTS radical cation);
(2) Evaluation of the protection given by antioxidants to a target being oxidized by free
radicals (ORAC, TOSC, LDL oxidation assay); (3) Estimation of the steady state of free
radicals before and after addition of additives (TAR); (4)Estimation of the reducing power
capacity of the additives (FRAP, CUPRAC).
The assays differ in the experimental conditions and their chemistry. Therefore, different
conclusions could be obtained depending on the methodology used. For example, green tea
presents a lower AC than peumus boldus by ORAC (oxygen radical absorbance capacity)
method when fluorescein is used as target molecule. However, if pyrogallol red is used as
probe, green tea appears with an ORAC index six times higher than peumus boldus.
In the present review, we discuss the advantages, and disadvantages of the different
methodologies employed to evaluate the AC of green tea.
Chapter 5 - Tea is one of the most popular and widely consumed beverages in the world
and it is derived from the infusion of tea leaves (Camellia sinensis L.). Different commercial
types of tea are available, including black tea, oolong tea (semi-fermented) and green tea,
xii Helen McKinley and Mark Jamieson
which differ on their processing and chemical composition. All these types of tea have been
reported to prevent multiple diseases such as cancer, heart conditions, among others. On the
other hand, lemongrass (Cymbopogon citratus Stap) is a rich source of essential oils, widely
employed in infusions, soaps, and perfumes, and it has been reported to possess
gastrointestinal and analgesic properties. In the present study, green tea (Camellia sinensis L.)
and lemongrass (Cymbopogon citratus Stap) leaves were collected from Río Azul and
Porvenir de Marona, Perú. The anti-oxidant capacity of green tea and lemongrass extracts was
evaluated using the DPPH method and it was observed that the IC50 values for green tea was
32.4 ± 0.39 μg/mL and 1350 ± 47 μg/mL for lemongrass. These two plants (green tea and
lemongrass) were employed to design multiple infused beverages and it was determined that
an infused beverage containing 10 mg/mL total extract (50% green tea and 50% lemongrass)
reported a total catechin content of 24.4 ± 0.65 mg/100mL, a DPPH inhibition percentage of
88.6%, and exhibited the greatest acceptance for sensory attributes such as flavor, color, and
aroma (values of 6.8, 9.0, and 8.0 respectively) based on Friedman Multiple Comparisons
test. The taste panel results also indicated that the optimized acidity and sweetness were to be
set at pH 3.1 and 11°Brix, while the optimum infusion time based on the total catechin
content was 7 minutes. The pasteurization profile at 90°C for 5 minutes achieved mesophilic
microorganisms counts of <10 cfu/mL. The maximum shelf-life of the beverage was achieved
at 15ºC based on total catechin content and absence of browning (catechin degradation).
Furthermore, the formulated beverage was well accepted by the test panel and presented
similar anti-oxidant capacity than commercial green tea based beverages.
Chapter 6 - Green tea is a good source of various polyphenolic compounds and minerals
which are powerful antioxidants. The effects of selenium-containing green tea (Se-GTE; 1.4
mg selenium/kg) and China green tea (CH-GTE; 0.13 mg selenium/kg) on food consumption
and body weight gain were investigated using a rat model. In addition, the total phenolic
contents (TPC), antioxidant/antiradical activities of these teas were determined in vitro. Both
teas had a satiating influence on experimental rats, as evidenced by their ability to decrease
food intake by 4.9% (CH-GTE) and 13.8% (Se-GTE), although a statistically significant
decrease over the control rats was achieved only for Se-GTE treatment. In addition, the final
body weight of rats gavaged with Se-GTE was significantly lower (P = 0.0063) than that of
the water-gavaged control rats and this corresponds to 8.5% reduction in body weight relative
to the control group. In contrast, rats gavaged with CH-GTE showed only 1.8% reduction in
the final body weight relative to the control group. The reduction in food intake over a short
period compared to a control rats preloaded with the same volume of water suggests that the
decrease in food intake was mainly a consequence of a satiating effect, rather than a stomach
distension effect. The underlying mechanism responsible for this satiating effect was not
identified as part of this study. It is also important to mention that water intake for the groups
given the tested teas was similar to that of rats given water only and no significant differences
were observed.
Se-GTE had significantly higher TPC (P < 0.0001), higher ferric reducing antioxidant
power (FRAP) (P < 0.01), higher diphenyl-picrylhydrazyl (DPPH) free radical scavenging
activity (P < 0.05), higher ferrous-ion chelating activity (P < 0.05-0.01), and higher selenium
contents (P < 0.0001) than CH-GTE. A strong positive correlation was found between the
TPC, and the FRAP, DPPH, and the ferrous-ion chelating activities in both teas, indicating
Preface xiii
that the polyphenolic compounds are the major contributors of the antioxidant/antiradical
activities.
When rats were gavaged with water-soluble tea extract (10 ml/kg/day) of Se-GTE for
four consecutive days, serum FRAP increased significantly (P = 0.0002) as compared to
water-gavaged controls. The level of serum FRAP in rats gavaged with CH-GTE increased
slightly when compared with the control rats but not to a significant extent. These results
indicate that green tea may have the ability to elevate circulating antioxidant potentials in vivo
and this ability is dependent on the type of tea used.
The observed results suggest that the reduction in food intake and decrease in body
weight in experimental animals may be a consequence of antioxidant mechanisms played by
the polyphenolic compounds and the minerals found in green teas. Green tea, especially the
one with a high level of organic selenium may provide a good satiety inducer and weight
management modulator.
Chapter 7 - Development of obesity appears to be influenced by a complex array of
genetic, metabolic, and neural frameworks, together with one’s behavior, eating habit, and
physical activity. The incidence of obesity is significantly increasing in virtually all societies
of the world and causes important pathological consequences such as cardiovascular diseases
and type 2 diabetes mellitus. Furthermore, rates of pediatric obesity have increased
dramatically over the past decade resulting cardiovascular, metabolic, and hepatic
complications. Since ancient times, green tea has been considered as a traditional medicine as
a healthful beverage. Major components of green tea including epigallocathchin gallate
(EGCG), epigallocatechin (EGC), epicatechin gallate (ECG) and epicatechin (EC) have been
received significant scientific attention and public awareness for its beneficial effects on
prevention and therapeutic treatment of cardiovascular diseases and cancer.
Anti-obesity effects of EGCG have been demonstrated in various in vitro and in vivo
models showing that EGCG treatment could reduce food intake, glucose uptake, blood
glucose level, and differentiation and growth activity of fat cells together with modulation of
lipolytic and lipogenic activities. In these actions, large number of molecules including
laminin receptor, fatty acid synthase, glucose-6-phosphate dehydrogenase, cyclin-dependent
kinase 2, CCAAT/enhancer binding proteins, mitogen activated protein kinases, AMP-
activated protein kinase, phosphatidylinositol-3-kinase, peroxisome proliferator activated
receptor, and number of transcription factors including NF-kB were suggested to be involved.
This review will focus on the effects of ECGC at molecular level in terms of (1) receptor
recognition, (2) modulation of signaling pathways, (3) lipid metabolism and altering the
lipogenic and lipolytic activities leading to metabolic changes and apoptosis, and (4) final
outcome in vitro and in vivo.
Chapter 8 - The indiscriminate usage of synthetic chemicals and pesticides has lead to a
widespread contamination of land, water and air with harmful xenobiotics. The exposure to
these toxicants results in severe health effects on organisms. Even some natural foods contain
harmless chemical species (nitrate) which however become toxic upon certain conditions.
Hence it is pertinent to focus attention on commonly consumed plant food materials that
can potentially neutralize the toxicity damage caused by environmental agents. One of the
most important sources of antioxidants is green tea. This review focuses on the mechanisms
of oxidative damage caused by different xenobiotics and the defensive action of green tea in
mitigating the damage. It is concluded that tea polyphenols, catechins and flavonoids
scavenge reactive oxygen species (ROS) and render the hepato-protective effect. However it
xiv Helen McKinley and Mark Jamieson
is important to note that the protective effects of green tea extract are rendered irrespective of
the xenobiotic involved thereby suggesting the involvement of a common biochemical
pathway.
Chapter 9 - Green tea is a well-known biomaterial with various high biological functions.
Irradiation was introduced to develop a new processing method to improve color of the
extract, resulting in a higher applicability without any adverse change to the beneficial
functions such as inhibitory effects of oxidation, melanin hyperpigmentation on the skin, and
others. To investigate the application of irradiated green tea leaf extract for a real cosmetic
composition, the physiological activities of irradiated green tea leaf extract powder dissolved
in butylenes glycol and ethanol were compared to a commercial green tea extract product.
Furthermore, a cream lotion was manufactured using the powder and the physiological
activities were compared. Results showed that the irradiation of the green tea leaf extract and
the freeze-dried powder from the extract had the same physiological activities as the
commercial product in a cosmetic composition. Addition of irradiated green tea leaf in a patty
can retard lipid oxidation and add the biological functions green tea possessed, however, the
intensity of off-odor produced from green tea is a concern. Using irradiated green tea powder,
the off-odor problem can be solved. Some research utilizing green tea byproducts were
investigated. Irradiation of green tea byproducts extract showed higher biological functions
than that of non-irradiated counterparts. Therefore, irradiation technology can be a useful
method to improve biological functions and industrial applicability of green tea and its
byproducts.
Chapter 10 - Many studies suggest that beneficial effect of green tea for human health is
mainly attributed to catechins, polyphenol compounds, having anti-oxidative activity. In the
last decade, new aspect of green tea catechin has emerged as angiogenesis inhibitor.
Angiogenesis is forming new blood vessels from a pre-existing blood vessel and involved in
various diseases including tumor growth and metastasis, diabetic retinopathy and
atherosclerosis. In addition, it has been demonstrated that obesity is prevented by
angiogenesis inhibitor and suggested that angiogenesis is closely associated with Alzheimer’s
disease. Thus, angiogenesis inhibitors in food would be expected to prevent these diseases
and give beneficial effect on our health. Interestingly, inhibitory effect of green tea catechin
on angiogenesis has been demonstrated in various models suggesting green tea catechin could
suppress cancer. Furthermore, it has been revealed that a higher consumption of green tea
catechin reduces human body fat and is associated with a lower prevalence of cognitive
impairment. These evidences suggest that anti-angiogenic activity of green tea catechin might
play important roles in human health.
Chapter 11 - The present article introduces our study related to the neuroprotective effect
of γ-glutamylethylamide (theanine), a component Japanese green tea (Camellia sinensis), on
cerebral ischemia. Theanine (1 mg/kg) significantly decreased the size of the cerebral infarcts
in a 4 h middle cerebral artery (MCA) occlusion model in mice. However, theanine did not
affect the cerebral blood flow, brain temperature and physiological variables (pH, pCO2, pO2
and hematocrit) in this model. Theanine also reduced the alterations of NeuN (neuron), GFAP
(astrocyte) and Iba 1 (microglia) expression levels at 24 h after MCA occlusion. This
neuroprotective effect of theanine was prevented by bicuculline, γ-aminobutyric acidA
(GABAA) receptor antagonist, but not 3-mercaptopropionic acid, glutamate decarboxylase
inhibitor. Furthermore, theanine (0.3 and 1 mg/kg) significantly prevented the impairment of
spatial memory in rats subjected to twice-repeated cerebral ischemia, 7 days after the second
Preface xv
reperfusion. In addition, theanine (1 mg/kg) significantly inhibited the decrease in the number
of surviving cells in the hippocampal CA1 field in the same rats. These results suggest that
theanine directly provides neuroprotection against cerebral ischemia and its neuroprotective
effect is mediated, at least in part, by GABAA receptors, and that it may be clinically useful
for preventing cerebrovascular disease.
Chapter 12 - Standing after water, tea signifies the second most frequently consumed
beverage worldwide, which varies its status from a simple ancient drink and a cultural
tradition to a nutrient endowed with possible neurobiological-pharmacological actions
beneficial to human health. Accumulating evidence suggests that oxidative stress resulting in
reactive oxygen species generation plays a pivotal role in neurodegenerative diseases,
supporting the implementation of radical scavengers and metal chelating agents, such as
natural tea polyphenols for therapy. Vast epidemiology data indicate a correlation between
occurrence of neurodegenerative disorders, such as Parkinson’s and Alzheimer’s diseases and
green tea consumption. In particular, literature on the putative novel neuroprotective
mechanism of the major green tea polyphenol, (-)-epigallocatechin-3-gallate (EGCG)
strengthens the notion that diverse molecular signaling pathways, participating in the
neuroprotective activity of EGCG, thus rendering this natural compound as potential agent to
reduce the risk of various neurodegenerative diseases. In the present article, we review the
studies concerning the mechanisms of action implicated in EGCG-induced neuroprotection in
the brain and discuss the vision to translate these findings into a lifestyle arena.
Chapter 13 - Green tea (Camelia sinensis), widespread in the whole world, possesses
many health protective properties. Its polyphenolic compounds, mostly flavonols, better
known as catechins (e.g. epigallocatechin-3-gallate [EGCG]), are considered to be
responsible for the health protective effects. Tea consumption may have its strongest effect
among patients with cardiovascular disease. Recently, studies suggested that high flavonoid
intake may reduce coronary heart disease by lowering blood lipid levels and inhibiting the
oxidation of low-density lipoproteins. Trials showed that short- and long-term tea
consumption could reverse endothelial dysfunction in subjects with documented coronary
heart disease, providing one possible mechanism for an effect of tea in patients with
cardiovascular disease. Interestingly, EGCG has also been shown to have electrophysiological
effects by blocking HERG potassium channels, the most important repolarizing potassium
channel in the human ventricle that forms the α-subunit of the rapid delayed rectifier current
IKr. Inhibition of HERG channels may have profound effects on cardiac repolarization.
Furthermore, tea flavonoids have been reported to exhibit metabolic effects in terms of anti-
diabetic properties. This review summarizes the latest studies on cardiovascular effects of
green tea and discusses the possible cardiac health benefits of green tea consumption.
Chapter 14 - For the past two decades, many researchers have been investigated the
potential cancer-preventive and therapeutic effects of green tea. (–)-Epigallocatechin-3-O-
gallate (EGCG) has been shown to be the most active and major polyphenolic compound
from green tea. The mechanisms of action of EGCG have been extensively investigated, but
the mechanisms for the cancer-preventive activity of EGCG are not completely characterized
and many features remain to be elucidated. Recently we have identified 67-kDa laminin
receptor (67LR) as a cell-surface EGCG receptor that confers EGCG responsiveness to many
cancer cells at physiological concentrations. This article reviews some of the reported
mechanisms and possible targets for the action of EGCG. Especially, we focus the current
understanding of signaling pathway for physiologically relevant EGCG through the 67LR for
xvi Helen McKinley and Mark Jamieson
cancer prevention. This information shed new light on the molecular basis for the cancer-
preventive activity of EGCG in vivo and helps in the design of new strategies to prevent
cancer.
Chapter 15 - Green tea and its major constituent epigallocatechin gallate (EGCG) have
been extensively studied as a potential chemopreventative and/or treatment for a variety of
diseases including breast cancer. Experimental evidence is supported by epidemiological
studies that have shown an inverse relationship between green tea consumption and the
incidence of breast cancer. Numerous studies have demonstrated that EGCG is cytotoxic
toward both estrogen receptor-positive and estrogen receptor-negative breast cancer cell lines.
These studies have highlighted potential mechanisms for the actions of EGCG, such as the
induction of apoptosis, the alteration of the expression of cell cycle regulatory proteins critical
for cell proliferation, inhibition of the chymotrypsin-like proteasome, inhibition of
angiogenesis, as well as inhibition of cell invasion and metastasis. Importantly, these effects
occur independently of estrogen receptor expression. This chapter will provide evidence for
these events and other molecular mechanisms that significantly contribute to the actions of
EGCG in vitro. The utility of green tea extract or EGCG as a breast cancer treatment and
chemopreventative has also been extensively investigated using various in vivo models of
estrogen receptor-positive and estrogen receptor-negative breast cancer (ie., chemical
carcinogenesis and xenograft models). Evidence for EGCG-mediated tumor suppression and
the major molecular mechanisms for this effect, such as the induction of apoptosis, the
inhibition of angiogenesis and modulation of the expression of cell signaling proteins are also
fully examined. These in vivo studies have led to investigations which have focused on ways
to improve the actions of EGCG, by either using it as part of a combination therapy or by
synthesizing pro-drugs of EGCG. Both of these have been done in order to enhance the
bioavailability, stability and efficacy of EGCG. These new compounds and drug
combinations have significantly improved the tumor suppression potential of EGCG and
provide an exciting future for this multi-faceted phytochemical in the prevention and
treatment of breast cancer.
Chapter 16 - Colorectal cancer is a global problem that accounts for over 50,000 cancer-
related deaths each year in the United States. Americans have about a one in 20 lifetime risk
of developing colorectal cancer. It affects primarily those over 65, but risk starts increasing at
age 40. Colorectal cancer develops following disruptions in key cancer-causing genes
(oncogenes) like K-ras and β-catenin and tumor suppressor genes like gate keeper APC and
p53, and early detection greatly increases the chances of survival. Most cancers are related to
a combination of hereditary and environmental factors, and such factors can either contribute
to the initiation of cancer or the prevention of tumor development. There is persuasive
epidemiological and experimental evidence that a phytochemical-enriched diet may be
involved in the prevention of colon cancer. Therefore, the use of dietary compounds for
prevention and therapy of colorectal cancer would be of major importance with potentially
fewer side effects than therapeutic drugs. Green tea has received much attention as a suitable
dietary agent because of its anti-tumorigenic activity. The most active constituents of green
tea are catechins, including epigallocatechin 3-gallate (EGCG), epigallocatechin (EGC),
epicatechin-3-gallate (ECG) and epicatechin (EC). Many laboratories, including ours, have
reported preventive effects with green tea components in cancers of the gastrointestinal tract,
lung, skin, prostate, and breast. A mechanistic study indicated that green tea decreased the
total levels of early carcinogenesis biomarkers and increased tumor suppressor proteins; in
Preface xvii
addition, reports related to new molecular targets affected by green tea in chemoprevention
study have been increased. Since the preponderance of the data strongly indicates significant
anti-tumorigenic benefits from green tea polyphenols, this chapter will summarize the current
knowledge of molecular targets of green tea research in human colorectal cancer prevention.
Chapter 17 - Green tea obtained from the leaves of the plant, Camellia sinensis, is one of
the most popular beverages in the world. The major polyphenolic compounds in green tea are
catechin derivatives (i.e., flavan-3-ols), and their composition varies depending on the season
of harvest and the manufacturing process. The inhibitory activities against DNA polymerases
(pols) of catechin derivatives such as (+)-catechin (C), (-)-epicatechin (EC), (-)-gallocatechin
(GC), (-)-epigallocatechin (EGC), (+)-catechin gallate (Cg), (-)-epicatechin gallate (ECg), (-)-
gallocatechin gallate (GCg), and (-)-epigallocatechin gallate (EGCg) were investigated.
Among these eight catechins, several inhibited mammalian pols with EGCg being the
strongest inhibitor of pols and with IC50 values of 5.1 and 3.8 M, respectively. EGCg
did not influence the activities of plant pols, such as pols and , or prokaryotic pols, and
had no effect on the activities of DNA metabolic enzymes such as calf primase of pol , T7
RNA polymerase, T4 polynucleotide kinase, or bovine deoxyribonuclease I. Some tea
catechins also suppressed human cancer cell growth and/or TPA (12-O-tetradecanoylphorbol-
13-acetate)-induced inflammation, and the tendency of the pol inhibitory activity for these
compounds was the same as that of their anti-inflammatory activity rather than their anti-
cancer activity. Based on these results, the relationship between the structure of tea catechins
and their bioactivities is discussed.
Chapter 18 - The anti-tumor effect of green tea, especially its major constituent EGCG,
has been demonstrated in several animal experiments and its ability to induce apoptosis of
most of cancer cell lines has been further documented in cell culture models. A number of
mechanisms for how green tea impacts cancer have been proposed. These mechanisms
basically include intervention of cell signal transduction pathways or changes of cell
epigenetic processes. Telomerase has been recognized as a novel target of green tea. This
important enzyme is largely localized to cancer cells, is responsible for the maintenance of
telomeres so that cancer cells can escape the replication problem due to their linear
chromosomes, and it has been shown to be reactivated in almost all tumor tissues. Telomerase
has been found to be inhibited by green tea in telomerase-positive cancer cell lines. This
analysis assesses the progress on research of the mechanisms pertaining to how telomerase
activity is regulated by green tea in cancer cells. A number of mechanisms for how green tea
works through this pathway have been proposed. Since telomerase has been identified as a
potential molecular target for cancer treatment, and green tea has been shown to inhibit
telomerase, clarifying the specific mechanisms for how green tea functions in this pathway
should shed new light on the potential to design effective and novel preventive or anti-cancer
approaches using green tea.
Chapter 19 – Background: Chronic obstructive pulmonary disease (COPD) is a common
cause of morbidity and leading cause of death in the world. Cigarette smoking has been
established as the principal risk factor for COPD. While 95% of COPD patients are, or have
been, cigarette smokers, only 20% of smokers develop COPD. Therefore, other factors may
protect against or contribute to the development of the disease. Objective: To investigate
whether green tea intake can reduce the risk of COPD. Study design: Case-control study
conducted in Aichi, Gifu and Kyoto during 2006. Subjects: A total of 278 eligible patients
(244 men and 34 women; mean age 66.5 (SD 6.7) years), with COPD diagnosed by
xviii Helen McKinley and Mark Jamieson
respiratory physicians as the primary functionally limiting illness within the past four years,
were referred from the outpatient departments of six hospitals in central Japan. During the
same period, 335 age-matched community-dwelling controls (267 men and 68 women; mean
age 65.3 (SD 5.5) years) were recruited from the same catchment areas as the cases. Methods:
Interviews were conducted face-to-face to collect information on demographic characteristics
and habitual green tea consumption using a structured questionnaire. The reference recall
period was set at 5 years before diagnosis for cases or 5 years before interview for controls.
Tea drinkers were defined as persons who drunk both Sencha, Bancha, Hojicha and
Genmaicha types of green tea once a week or more often. The effect of green tea intake on
the COPD risk was assessed by multivariate logistic regression analysis. Results: The
prevalence of regular green tea consumption was significantly higher (p < 0.01) among
controls (n = 64, 19.3%) than cases (n = 27, 9.7%). Among drinkers, life-long exposure (years
of drinking) was similar between the two groups (p = 0.70), and about half of them drank one
to three cups of green tea daily. The risk of COPD appeared to decrease with green tea
drinking. The adjusted odds ratio was 0.45 (95% confidence interval 0.25-0.81) for tea
drinkers relative to non-drinkers after accounting for confounding factors including age, body
mass index, gender, marital status, residential location, education level, retirement status,
smoking status and alcohol consumption. Conclusion: The preliminary evidence suggests that
green tea intake may be protective against COPD for Japanese adults. More research is
required to confirm the observed finding and to understand the biological mechanism
underlying the disease prevention role of green tea.
Chapter 20 - Providing that there has been a dramatic increase in the incidence of
diabetes mellitus associated with long-term complications, it is critical to find a natural
nutritional material aimed at reducing the prevalence of diabetes which threatens human
health over the world .
Currently, green tea, only next to water, is the most widely consumed beverage in the
world and exerts beneficial bioabilities, such as anti-inflammative, anti-oxidative, anti-
mutagenic, etc. With increasing interest in the health promoting properties of tea and a
significant rise in scientific investigation, in vitro and animal studies provide more and more
strong evidence that green tea consumption has a great prophylactic and therapeutic effect on
diabetes and its complications, which is intensively associated with its mainly bioactive
components, such as tea polyphenols and tea polysaccharides.
In this article, we will summarize effect of green tea on diabetes, especially anti-diabetic
ability of tea polyphenols and polysaccharides, discuss possible mechanisms, and make
perspectives and future directions in this area.
Chapter 21 - Green tea, which is consumed world wide as a beverage, is known to have
many beneficial effects on human health. Green tea contains various biologically-active
materials including catechins, flavonols and caffeine. Of them, catechins are the major
constituents consisting of 30% of water-extractable materials. Pharmacological implication
has been mainly made upon (-)-epigallocatechin-3-gallate (EGCG), as it is the most abundant
catechin in green tea extracts. The second biologically important catechin is (-)-epicatechin-3-
gallate (ECG), which is called one of gallated catechins as EGCG. To date, there is no
prominent evidence for green tea consumption to determine whether be beneficial or harmful
in metabolic diseases, such as type 2 diabetes and obesity. It may be due to catechins having a
variety of function in human body. This chapter will focus on action mechanism of EGCG on
ATP-sensitive potassium channels, which manifests as actual phenotypes in cardiac and beta-
Preface xix
cell function. Additionally, changes by green tea or gallated catechins in insulin resistance
will be reviewed. Thereafter, a right method to use EGCG as a supportive regimen for
diabetic care and obesity will be introduced. New era should come with modifying natural
products fit to human well-being.
Chapter 22 - World-wide tea production has reached 2.97 x 106 metric tons/year, and
more than 75% of tea products are black tea. In recent years, green tea which contain catechin
derivatives such as (-)-epicatechin (EC) (1), (-)-epicatechin gallate (ECg) (2), (-)-
epigallocatechin (EGC) (3), and (-)-epigallocatechin gallate (EGCg) (4) has been recognized
as a useful functional food. However, recent development of enzymatic processes now makes
it possible to produce epitheaflagallin (5), epitheaflagallin 3-O-gallate (6) and theaflavin (TF)
(7) derivatives from catechins, which are components of black tea. Epitheaflagallin and
epitheaflagallin 3-O-gallate are preferentially synthesized from EGC (3) and EGCg (4) in
green tea extracts in the presence of laccase and gallic acid. Takemoto and colleagues have
developed a Camellia sinensis cell culture system containing peroxidase and hydrolases of
ECG (3) and EGCg (4) to produce theaflavin (7) from tea catechins. These biocatalytic
processes allow us to preferentially convert catechin derivatives in a crude mixture of green
tea into different compounds, and, thus, to improve the composition of the catechins present
in green tea.
Chapter 23 - Tea is rich in polyphenols which are contained in the leaves and stems of the
tea plant. (-)-Epigallocatechin gallate (EGCG), the major and most active component of green
tea catechins, acts as an antioxidant in the biological system, and is absorbed and distributed
mainly into the mucous membranes of the small intestine and liver; more interestingly it can
cross the blood brain barrier. Oxidative stress, a condition of cellular prooxidant-antioxidant
disturbance which favors the prooxidant state, induces the production of lipid peroxide
(LPO), reactive oxygen species (ROS) and free radicals in membrane lipids. Oxidative stress
causes deterioration of a wide variety of cellular enzymes, subsequently exacerbating
neurodegenerative process.
Aging leads to a decline in memory-related learning ability. Oxidative damage to the
brain is associated with age-related cognitive dysfunction but some antioxidants are effective
in alleviating this dysfunction for Alzheimer’s disease (AD) model animals. Moreover, a
decrease in hippocampal LPO level improves spatial cognition learning in aged rats and an
increase in antioxidative activity in the hippocampus prevents or ameliorates the impairment
of learning ability in AD model rats produced by the infusion of amyloid-β (1-40) (Aβ1-40)
into their cerebral ventricle. Catechins have a protective effect against age-related
neurological diseases caused by oxidative damage. Epidemiological studies report that a
higher consumption of green tea is associated with lower prevalence of cognitive impairment
in elderly people. This chapter describes the beneficial effects of green tea catechins on
neuronal functions and neuronal diseases such as dementia.
Short Commentary - The scientific understanding of many biological functions involved
in maintaining human health (e.g., role of gap junctions in homeostatic control of cell
behaviors; multi-state nature of carcinogenesis; role of gap junctions in the tumor promotion
step of carcinogenesis; toxicant induced oxidative stress in many human diseases; role of
oxidative stress by tumor promoting chemicals; role of green tea components as anti-oxidants;
preventive effects of green tea components on toxicant-inhibition of gap junction function and
tumor promotion) provides some evidence that green tea could prevent some oxidative stress-
linked human diseases. However, as has been demonstrated in experimental examples, it will
xx Helen McKinley and Mark Jamieson
be unlikely that any agent, such as green tea, will be a “silver bullet” to protect against all
toxic agents. On the other hand, it does seem it would cause “no harm” and might, in some
cases, even be beneficial.
In: Handbook of Green Tea and Health Research ISBN 978-1-60741-045-4
Editor: H. McKinley and M. Jamieson © 2009 Nova Science Publishers, Inc.
Chapter 1
CENTRAL FUNCTIONS OF GREEN TEA COMPONENTS
M. Furuse 1,2,*, N. Adachi2, S. Tomonaga2,

H. Yamane2 and D. M. Denbow3
Laboratory of Advanced Animal and Marine Bioresources,
1
Faculty of Agriculture and 2Graduate School of Bioresources and Bioenvironmental
Sciences, Kyushu University, Fukuoka 812-8581, Japan
3
Department of Animal and Poultry Sciences, Virginia Polytechnic Institute and State
University, Blacksburg, VA 24061-0306, USA
ABSTRACT
Tea (Camellia sinensis) is widely consumed throughout the world and has a number
of biologically active substances such as caffeine, catechins, and L-theanine (γ-
glutamylethylamide). Tea consumption is generally known to induce a feeling of
relaxation which may be mediated by either catechins, L-theanine, or both, since caffeine
stimulates locomotor activities.
The catechin (-)-epigallocatechin gallate (EGCG) occurs abundantly in tea.
Moreover, frequent consumption of green tea results in high levels of EGCG in the blood
and brain. Catechins, which are flavonoids, affect the central nervous system (CNS). The
therapeutic effects of flavonoids may involve their binding to γ-aminobutyric acid
(GABA)A receptors, which is a major inhibitory neurotransmission system. Recently,
EGCG was shown to bind to GABAA receptors in vitro and to induce a sedative effect
through GABAA, but not GABAB, receptors in the brain.
L-Theanine, a derivative of glutamate, is a unique amino acid occurring only in
green tea and a few other plants. After administration L-theanine concentrations were
increased in serum, liver and brain, suggesting that L-theanine can cross the blood-brain
barrier. Intravenous administration of L-theanine was shown to affect the cortex,
hippocampus and amygdala, and increase the alpha-band component of
electroencephalograms in rats. More recently, it was shown that L-theanine could reduce
stress via either inhibiting cortical neuron excitation in human subjects or influencing the
*
Correspondence author. Tel: +81-92-642-2953; Fax: +81-92-642-2953; E-mail address: furuse@brs.kyushu-
u.ac.jp (M. Furuse)
2 M. Furuse, N. Adachi, S. Tomonaga et al.
secretion and function of neurotransmitters in the CNS. We discuss the central functions
of green tea components such as EGCG and L-theanine in the CNS.
INTRODUCTION
Green tea, the most consumed beverage in the world except for fresh water, is a drink
made from the steamed and dried leaves of the Camellia sinensis plant, a shrub native to Asia.
Green tea is widely consumed in Japan, China, and other Asian nations, and is becoming
more popular in Western nations. Black tea is also made from the plant, but unlike green tea,
it is made from leaves that have been fermented. Due to differences in the fermentation
process, a portion of the active compounds are destroyed in black tea, but remain active in
green tea.
The active constituents in green tea are a family of polyphenols (catechins) and flavonols
which possess potent antioxidant activity. Tannins, large polyphenol molecules, form the bulk
of the active compounds in green tea, with catechins comprising nearly 90%. Several forms of
catechin are present in the plants (Figure 1). Among them, epigallocatechin-3-gallate (EGCG)
is the most abundant flavonoid in tea (Arts et al., 2000) and particularly in green tea
(Campbell et al., 2004). Approximately 26% of the solid weight of green tea extract is tea
polyphenols, of which 11% are EGCG (Suganuma et al., 1998). Thus, EGCG is primarily
responsible for the pharmacological actions of tea. Moreover, frequent consumption of green
tea results in high levels of EGCG in the blood and brain (Suganuma et al.., 1998; Kim et al.,
2000). Some reports indicate green tea may have the ability to help prevent cancers of the
skin, esophagus, stomach, colon, pancreas, lung, bladder, prostate, and breast. Green tea
contains chemicals known as polyphenols, which have many beneficial effects such as
antioxidant, anticarcinogenic, and antiviral activity (Frei and Higdon, 2003; Tachibana et al.,
2004). Compared with the peripheral actions of catechins, the information for the central
effect is limited to date.
L-Theanine (γ-glutamylethlamide) is a unique amino acid only occurring in green tea and
a few other plants, and is a derivative of glutamate (Figure 2). After administration, L-
theanine concentrations increased in the serum, liver and brain (Yokogoshi et al., 1998a, b),
suggesting that L-theanine can cross the blood-brain barrier. Intravenous administration of L-
theanine was shown to affect the cortex, hippocampus and amygdala and increase the alpha-
band component of electroencephalograms (EEG) in rats (Kakuda et al., 2000a). More
recently, it was shown that L-theanine could reduce stress via either inhibiting cortical neuron
excitation in human subjects (Kimura et al., 2007) or influencing the secretion and function of
neurotransmitters in the central nervous system (CNS) (Terashima et al., 1999).
Green tea also contains other active substances such as caffeine. While caffeine
stimulates locomotor activities in rats (Mukhopadhyay and Poddar, 1995), tea consumption is
generally known to induce a feeling of relaxation (Juneja et al., 1999) which may be mediated
by either catechins, L-theanine, or both. We review here the central effects of EGCG and L-
theanine with reference to pharmacology, pharmacokinetics, and toxicology.
Central Functions of Green Tea Components 3
CENTRAL FUNCTION OF EGCG

The effects of flavonoids on the CNS have been studied (Zanoli et al., 2000), with
particular reference to their role as therapeutics involving γ-aminobutyric acid (GABA)A
receptors (Huen et al., 2003). The characteristic structure of flavonoids, which have binding
capacity for GABAA receptors, is similar to that of EGCG. Recently, EGCG has been
confirmed to bind to GABAA receptors in vitro (Campbell et al., 2004). According to Adachi
et al. (2006), intracerebroventricular (i.c.v.) injection of EGCG clearly suppressed the number
of vocalizations (Figure 3). The cumulative number of vocalizations increased with time, but
those of EGCG-treated groups remained low during the experimental period. Behavioral
patterns are shown in Table 1. Central EGCG increased sleep-like behavior, but the reverse
was true for active wakefulness.
Figure 1. Structures of tea catechins.

NH2
H
HO N
O O
L-Theanine
NH2
HO O
OHH
O O
L-Glutamate
Figure 2. Structures of L-theanine and L-glutamate.
1200 Control
EGCG 50 μg
EGCG 100 μg
EGCG 200 μg
900
Vocalization (count)
600
300
0
1 2 3 4 5 6 7 8 9 10
Time (min)
Figure 3. Effect of i.c.v. injection of EGCG (0, 50, 100 or 200 μg) on cumulative number of
vocalizations for 10 min in response to social separation stress in chicks. Data are expressed as
means±S.E.M. Significant regression equations were obtained between number of vocalizations and
EGCG (μg) as follows: vocalization (count) = 0.001 (SE 2.063 x 10-4) EGCG3 + 0.243 (SE 0.059)
EGCG2 – 26.036 (SE 3.895) EGCG + 875.571 (SE 55.016), R2 = 0.872, P<0.0001. Reproduced from
Adachi, N., Tomonaga, S., Tachibana, T., Denbow, D.M. & Furuse, M. (2006). (-)-Epigallocatechin
gallate attenuates acute stress responses through GABAergic system in the brain. Eur. J. Pharmacol.,
531, 171-175, with permission from Elsevier as the authors’ right.
10
Corticosterone concentration
8
(ng/ml)
6
* *
2
0
EGCG (μg) 0 50 100 200
Figure 4. Effect of i.c.v. injection of EGCG (0, 50, 100 or 200 μg) on the plasma corticosterone
concentration after social separation stress for 10 min in chicks. Data are expressed as means±S.E.M.
*, Significantly different from control at P<0.05. Reproduced from Adachi, N., Tomonaga, S.,
Tachibana, T., Denbow, D.M. & Furuse, M. (2006). (-)-Epigallocatechin gallate attenuates acute stress
responses through GABAergic system in the brain. Eur. J. Pharmacol., 531, 171-175, with permission
from Elsevier as the authors’ right.
Table 1. Effect of i.c.v. injection of EGCG (0, 50, 100 or 200 μg) on various behavioral
categories in response to social separation stress for 10 min in chicks
EGCG (μg) 0 50 100 200

Active wakefulness 543±15 172±107* 51±51* 13±9*
Standing/sitting motionless with
57±15 302±101 164±34 193±67
eyes open
Standing motionless with eyes
0±0 0±0 26±26 20±17
closed
Sitting motionless with head
0±0 126±87 359±44* 374±66*#
drooped (sleep-like behavior)
Total 600 600 600 600
Data are expressed as means±S.E.M. in seconds. Significant regression equations were obtained
between behavior and EGCG (μg) as follows: active wakefulness (s) = 0.025 (SE 0.006) EGCG2 –
7.604 (SE 1.306) EGCG + 529.67 (SE 49.949), R2 = 0.730, P<0.0001 and sleep-like behavior (s) =
1.868 (SE 0.402) EGCG + 45.776 (SE 48.602), R2 = 0.464, P<0.0001. * and #, Significantly
different from control and 50 μg of EGCG respectively at P<0.05. Reproduced from Adachi, N.,
Tomonaga, S., Tachibana, T., Denbow, D.M. & Furuse, M. (2006). (-)-Epigallocatechin gallate
attenuates acute stress responses through GABAergic system in the brain. Eur. J. Pharmacol., 531,
171-175, with permission from Elsevier as the authors’ right.
Figure 5. Effect of i.c.v. injection of either saline, 50 μg of EGCG, 500 ng of picrotoxin, or EGCG plus
picrotoxin on cumulative number of vocalizations in response to social separation stress for 10 min in
chicks. Data are expressed as means±S.E.M. Reproduced from Adachi, N., Tomonaga, S., Tachibana,
T., Denbow, D.M. & Furuse, M. (2006). (-)-Epigallocatechin gallate attenuates acute stress responses
through GABAergic system in the brain. Eur. J. Pharmacol., 531, 171-175, with permission from
Elsevier as the authors’ right.
The i.c.v. injection of EGCG significantly decreased plasma corticosterone release

compared with the effect of saline (Figure 4). Figure 5 shows the effect of i.c.v. injection of
EGCG with or without the GABAA receptor antagonist picrotoxin on the number of
vocalizations during the 10-min social separation stress. The suppressive effect of EGCG on
distress-induced vocalizations was attenuated by co-administered picrotoxin. Figure 6 shows
the effect of i.c.v. injection of EGCG with or without picrotoxin on plasma corticosterone
concentration during social separation stress. Picrotoxin significantly increased the plasma
corticosterone concentration compared with the effect of EGCG. Furthermore, picrotoxin
attenuated the decrease in plasma corticosterone caused by EGCG.
Figure 7 shows the effect of i.c.v. injection of EGCG with or without the GABAB
receptor antagonist CGP 54626 (3-N-(1-(3,4-dichlorophenyl)ethylamino)-2-hydroxypropyl
cyclohexylmethyl phosphinic acid hydrochloride) on the number of vocalizations during the
10-min social separation stress. Significant effects of EGCG and time were detected, but no
significant effect was observed with CGP54626. The effect of EGCG could not be attenuated
by CGP54626. These results indicate that EGCG has sedative and hypnotic effects in the
brain, acting at least partially through GABAA receptors, which consequently moderates an
acute stress response.
30
Corticosterone concentration
24
*
(ng/ml)
18
12
0
EGCG (μg) 0 0 50 50
Picrotoxin (ng) 0 500 0 500
Figure 6. Effect of i.c.v. injection of either saline, 50 μg of EGCG, 500 ng of picrotoxin, or EGCG plus
picrotoxin on plasma corticosterone concentration for 10 min in chicks. Data are expressed as
means±S.E.M. *, Significantly different from 50 μg of EGCG alone at P<0.05. Reproduced from
Adachi, N., Tomonaga, S., Tachibana, T., Denbow, D.M. & Furuse, M. (2006). (-)-Epigallocatechin
gallate attenuates acute stress responses through GABAergic system in the brain. Eur. J. Pharmacol.,
531, 171-175, with permission from Elsevier as the authors’ right.
In addition, an anti-anxiety effect of EGCG was also observed in mice (Vignes et al.,
2006). However, green tea consumption does not induce sleep even though green tea contains
abundant EGCG. This may be explained by the caffeine in green tea (Kuo et al., 2005).
Caffeine acutely stimulates the autonomic nervous system and increases wakefulness
(Quinlan et al., 2000). Consequently, it is conceivable that the hypnotic effect of EGCG may
be countered by the action of caffeine in green tea.
The 4-carbonyl group of flavonoids is important for binding to GABAA receptors
(Marder and Paladini, 2002). Apigenin, one of the major flavonoids, binds to GABAA
receptors (Dekermendjian et al., 1999). The galloyl group of EGCG contains a carbonyl
group which may substitute for the 4-carbonyl group of apigenin (Campbell et al., 2004).
Adachi et al. (2007b) investigated whether the galloyl group of EGCG is necessary to act at
GABAA receptors. The structure of (-)-epigallocatechin (EGC), one of the catechins, is
similar to that of EGCG except for the lack of the galloyl group (Figure 8). The i.c.v.
injection of EGC or EGCG clearly suppressed total spontaneous activity and the number of
vocalizations (Figure 9). Table 2 shows the effect of EGC or EGCG on various behavioral
categories of chicks during 10 min behavior observations. Significant effects of EGC or
EGCG were observed on the time for active wakefulness and the time for sleep-like behavior.
Compared with the control group, EGC and EGCG increased sleep-like behavior, but the
reverse was true for active wakefulness. The suppressive effect of EGC on increasing
distress-induced spontaneous activity was attenuated by co-administration of picrotoxin, a
GABAA receptor antagonist (Figure 10).
Figure 7. Effect of i.c.v. injection of either saline, 50 μg of EGCG, 10 ng of CGP54626, or EGCG plus
CGP54626 on the cumulative number of vocalizations in response to social separation stress for 10 min
observation in chicks. Data are expressed as means±S.E.M. Reproduced from Adachi, N., Tomonaga,
S., Tachibana, T., Denbow, D.M. & Furuse, M. (2006). (-)-Epigallocatechin gallate attenuates acute
stress responses through GABAergic system in the brain. Eur. J. Pharmacol., 531, 171-175, with
permission from Elsevier as the authors’ right.
OH
OH
HO O OH
OH
O C OH
OH
O OH
(-)-Epigallocatechin gallate (EGCG)
OH
OH
HO O
OH
OH
OH
Figure 8. Structural differences in EGCG and EGC. The broken line indicates the galloyl group.
Figure 9. Effect of i.c.v. injection of EGC (109, 218 nmol) or EGCG (109, 218 nmol) on spontaneous
activity (upper panel) and total number of distress vocalizations (lower panel) for 10 min in response to
social separation stress in chicks. Data are expressed as means±S.E.M. Groups with different letters are
significantly different (P<0.05). Reproduced from Adachi, N., Tomonaga, S., Suenaga, R., Denbow,
D.M. & Furuse, M. (2007b) Galloyl group is not necessary for a sedative effect of catechin through
GABAergic system. Lett. Drug Design Discov., 4, 163-167, with permission from Bentham Science
Publishers Ltd.
In addition, EGC suppressed the number of vocalizations, and co-administration of

picrotoxin attenuated this response (Figure 10). A significant effect of EGC was observed on
the time for active wakefulness (Table 3). On the other hand, picrotoxin did not decrease the
time of active wakefulness. Both EGC and picrotoxin significantly affected the time for sleep-
like behavior, respectively. The effects of EGC such as decrease in active wakefulness and
increase in sleep-like behavior were attenuated by picrotoxin in the CNS. It is concluded that
catechins have sedative effects acting through GABAA receptors under an acute stress
condition irrespective of the presence of the galloyl group.
200 a
Spontaneous activity
(arbitrary unit)
150
ab
100
b
50
b
0
EGC (nmol) 0 0 109 109
Picrotoxin (pmol) 0 830 0 830
a
900
a a
600
300
b
0
EGC (nmol) 0 0 109 109
Picrotoxin (pmol) 0 830 0 830
Figure 10. Effect of i.c.v. injection of EGC (109 nmol), picrotoxin (830 pmol) or both on spontaneous
activity (upper panel) and total number of distress vocalizations (lower panel) for 10 min in response to
social separation stress in chicks. Values are means±S.E.M. Groups with different letters are
significantly different (P<0.05). Reproduced from Adachi, N., Tomonaga, S., Suenaga, R., Denbow,
D.M. & Furuse, M. (2007b) Galloyl group is not necessary for a sedative effect of catechin through
GABAergic system. Lett. Drug Design Discov., 4, 163-167, with permission from Bentham Science
Publishers Ltd.
EGCG possesses two triphenolic groups in its structure. These groups are reported to be
important with respect to anticarcinogenic and antioxidant effects. To clarify the anti-
inflammatory effect of EGCG on Alzheimer's disease (AD), Kim et al. (2007) investigated
the effect of EGCG in attenuating the inflammatory response induced by interleukin (IL)-
1beta+beta-amyloid (25-35) fragment (Abeta) in human astrocytoma, U373MG cells. EGCG
significantly inhibited the IL-1beta+Abeta (25-35)-induced IL-6, IL-8, vascular endothelial
growth factor and prostaglandin E2 production (Jeong et al., 2007b, Kim et al., 2007). EGCG
also attenuated the expression of cyclooxygenase-2 (Jeong et al., 2007b, Kim et al., 2007) and
activation of nuclear factor-kappaB induced by IL-1beta+Abeta (25-35) (Kim et al., 2007).
They demonstrated that EGCG suppressed IL-1beta+Abeta (25-35)-induced phosphorylation

of the mitogen-activated protein kinase p38 and the c-Jun N-terminal kinase. In addition,
EGCG induced the expression of mitogen-activated protein kinase phosphatase-1. These
results provide the possibility for its potential therapeutic application to various
neurodegenerative diseases such as AD.
Table 2. Effect of i.c.v. injection of EGC or EGCG on various behavioral categories in

response to social separation stress for 10 min in chicks
EGC (nmol) EGCG (nmol)

Saline
109 218 109 218
Active wakefulness 412±49a 75±50b 0±0b 23±23b 42±30b
Standing/sitting motionless
176±47 232±45 99±54 97±25 265±72
with eyes open
Standing motionless with eyes
12±12 0±0 36±36 0±0 45±32
closed
Sitting motionless with head
0±0b 293±83a 465±49a 480±44a 248±105a
drooped (sleep-like behavior)
Total 600 600 600 600 600
Values are means ± S.E.M. in seconds. Groups with different letters are significantly different (P<0.05).
Reproduced with the modification from Adachi, N., Tomonaga, S., Suenaga, R., Denbow, D.M. &
Furuse, M. (2007b). Galloyl group is not necessary for a sedative effect of catechin through
GABAergic system. Lett. Drug Design Discov., 4, 163-167, with permission from Bentham
Science Publishers Ltd.
Table 3. Effect of i.c.v. injection of EGC, picrotoxin or both on various behavioral

categories in response to social separation stress for 10 min in chicks
Saline EGC (109 nmol) P values

Picrotoxin (pmol) EGC x
Picro-
0 830 0 830 EGC picro-
toxin
toxin
Active wakefulness
417 ± 51 369 ± 80 64 ± 64 295 ± 70 <0.005 NS <0.05
Standing/sitting
motionless with eyes 183 ± 51 231 ± 80 145 ± 42 305 ± 70 NS NS NS
open
Standing motionless
with eyes closed 0 0 4±4 0 NS NS NS
Sitting motionless
with head drooped 0 0 387 ± 78 0 <0.0001 <0.0001 <0.0001
(sleep-like behavior)
Total 600 600 600 600
Values are means ± S.E.M. in seconds.

Reproduced with the modification from Adachi, N., Tomonaga, S., Suenaga, R., Denbow, D.M. &
Furuse, M. (2007b). Galloyl group is not necessary for a sedative effect of catechin through
GABAergic system. Lett. Drug Design Discov., 4, 163-167, with permission from Bentham
Science Publishers Ltd.
EGCG has neuroprotective effects on oxidative stress-injured neuronal cells, especially

motorneurons. Koh et al. (2006) evaluated the effect of EGCG in the amyotrophic lateral
sclerosis (ALS) model mice with the human G93A mutated Cu/Zn-superoxide dismutase
gene. Treatment with more than 2.9 μg EGCG/g body weight significantly prolonged the
symptom onset and life span, preserved more survival signals, and attenuated death signals.
They suggested that EGCG could be a potential therapeutic candidate for ALS as a disease-
modifying agent.
Parkinson's disease (PD) is known to occur after an 80% loss of dopaminergic
nigrostriatal neurons. This loss is enhanced by oxidative stress (Mytilineou et al., 1998).
Among the oxidative stresses, nitric oxide (NO) plays a major role (Grunewald and Beak,
1999; Schulz et al., 1995a). In mice (Schulz et al., 1995b) and baboons (Hantraye et al.,
1996), inhibition of neuronal NO synthase (nNOS) prevents PD induced by 1-methyl-4-
phenyl-1,2,3,6-tetrahydropyridine (MPTP). In addition, mutant mice lacking the inducible
NOS (iNOS) gene are significantly more resistant to MPTP than their wild-type littermates
(Hantraye et al., 1996). Choi et al. (2002) examined whether EGCG attenuates MPTP-
induced PD in mice through the inhibition of NOS expression. The oral administration of
EGCG prevented the loss of tyrosine hydroxylase (TH)-positive cells in the substantia nigra
and of TH activity in the striatum. These treatments also preserved striatal levels of dopamine
and its metabolites, 3,4-dihydroxyphenylacetic acid and homovanillic acid.
Both tea and EGCG decreased expressions of nNOS in the substantia nigra. EGCG plus
MPTP treatments decreased expressions of nNOS similar to the EGCG treatment group.
Therefore, the preventive effects of tea and EGCG may be explained by the inhibition of
nNOS in the substantia nigra. EGCG also has an inhibitory effect on iNOS (Chan et al. 1997;
Soliman and Mazzio, 1998). Park et al. (2001) examined the role of iNOS in cocaine-induced
locomotor sensitization. Pretreatment with EGCG, 30 min before cocaine administration,
totally blocked the development of cocaine-induced locomotor sensitization. Dopamine
receptor binding in the nucleus accumbens showed a significant decrease in the density of D2
receptors and the affinity of D1 receptors after cocaine treatment. Pretreatment with EGCG
abolished the cocaine-induced changes in these parameters. These results suggest that iNOS
may participate in the process of locomotor sensitization through the modulation of dopamine
receptors in the nucleus accumbens.
Jeong et al. (2007a) investigated the effects of EGCG on the electrical activity of rat
substantia nigra dopaminergic neurons using whole-cell patch clamp recordings. The spike
frequency was increased, and the resting membrane potential of the cell and the amplitude of
after-hyperpolarization were decreased by EGCG. The neuronal activity of dopaminergic
neurons is closely linked to dopamine release. When neurons switch from a single-spike
firing to bursts of action potentials the release of dopamine increases. The above experimental
results suggest that EGCG increases the neuronal activity via inhibition of calcium-dependent
potassium currents underlying the after-hyperpolarization, and it could act as a facilitating
factor that elicits N-methyl-D-aspartate (NMDA)-dependent bursts of action potentials like
apamin or bicuculline methiodide.
Lin et al. (2007) reported that the protein binding of EGCG in rat plasma was 92.4±2.5%,
and EGCG may potentially penetrate through the blood-brain barrier at a lower rate. The
elimination half-life of EGCG was 62±11 and 48±13 min for intravenous (10 mg/kg) and oral
(100 mg/kg) administration, respectively. The pharmacokinetic data indicate that the oral
bioavailability of EGCG in a conscious and freely moving rat was about 4.95%.
Ullmann et al. (2003) conducted a randomized, double-blind, placebo-controlled study

assessing the safety, tolerability and plasma kinetic behavior of single oral doses of EGCG
under fasting conditions. In each group of 10 subjects, eight received oral EGCG in single
doses of 50, 100, 200, 400, 800 or 1600 mg, and two received placebo. In each dosage group,
the kinetic profile revealed rapid absorption with one plasma peak, followed by a multiphasic
decrease consisting of a distribution phase and an elimination phase. Single oral doses of
EGCG up to 1600 mg were safe and very well tolerated. Ullmann et al. (2004) further
reported the safety, tolerability, and plasma-kinetic behavior of EGCG after ten days' repeated
dosing in healthy male volunteers. Orally administered EGCG is rapidly absorbed from the
gut. Dose linearity was applied for single-dose application (day 1). After repeated dosing (day
10) dose linearity was applied between the 200 and 400 mg group. Dose escalation to 800 mg
was more than dose-proportional in rate and extent, and statistically different from the 200
mg and 400 mg group. An increase in elimination half-life and in the accumulation factor in
the 800 mg dosage group indicated dose-dependent saturation of capacity-limited excretion
routes or an increase of hepato-duodenal re-circulation. Ten days' repeated administrations of
oral dose of EGCG of up to 800 mg per day were found to be safe and very well tolerated.
CENTRAL FUNCTION OF L-THEANINE

Time-dependent changes of L-theanine in the brain of rats were investigated during the
24 h after L-theanine administration. When L-theanine was intragastrically administered to
rats, the concentrations of L-theanine reached the maximum level in the brain after 5 h
(Terashima et al., 1999). L-Theanine was incorporated into brain through the blood-brain
barrier via the leucine-preferring transport system (Yokogoshi et al., 1998a).
Intravenous administration of L-theanine affected the cortex, hippocampus and amygdala
and increased the alpha-band component of EEG in rats (Kakuda et al., 2000a). Evidence
from human EEG studies show that L-theanine has a direct effect on the brain (Juneja et al.,
1999). L-Theanine significantly increases activity in the alpha frequency band which
indicates that it relaxes the mind without inducing drowsiness. However, this effect has only
been established at higher doses than that typically found in a cup of black tea (approximately
20 mg).
Nobre et al. (2008) reported that L-theanine, at realistic dietary levels, has a significant
effect on the general state of mental alertness or arousal. L-Theanine- and theogallin-enriched
decaffeinated green tea extract is able to change the physiological pattern of electrical
hippocampus activity in a concentration dependent manner. A decrease of population spike
amplitude and attenuated long-term potentiation was observed in the presence of L-theanine
alone (Dimpfel et al., 2007a). The model Tele-Stereo-EEG (continuous recording of
intracerebral field potentials in the freely moving rat to produce an electropharmacogram) has
been used and power spectra from Fast Fourier Transformed field potential changes were
divided into six frequency bands (delta, theta, alpha1, alpha2, beta1 and beta2). Oral
administration of 30 mg/kg L-theanine led to power decreases of nearly all frequencies, being
more pronounced during the second and following hours in comparison with the first hour
(Dimpfel et al., 2007b).
Accoding to Dimpfel et al. (2007c), source density analysis of EEG recordings from 12
healthy human volunteers after ingestion of a soft drink containing green tea extract enriched
with L-theanine and theogallin revealed a general attenuation of electrical delta power under
the condition of eyes open. During a reading test increases of delta and theta power were
observed at frontal electrode sites starting with the second hour after administration,
significant at the third and fourth hour in comparison to placebo. These changes indicate a
higher level of mental performance. Increases of beta 1 power starting with the second hour
indicated a higher degree of relaxation. However, no statistical significance was reached.
Analysis of visually evoked P300 waves revealed a decrease in latency at the last hour as well
as increases of amplitudes at the electrode position Cz (from the first to the third hour,
statistically not significant). This type of result in general suggests an improvement of
attention. So-called alpha bands (8-14 Hz) can be detected during resting EEG recordings in
humans. Independently, alpha band activity has been shown to be a key component in
selective attentional processes. L-Theanine ingestion resulted in a substantial overall decrease
in background alpha levels relative to placebo while subjects were actively performing a
demanding attention task. Despite this decrease in background alpha activity, attention-related
alpha effects were significantly greater for L-theanine condition (Gomez-Ramirez et al.,
2007).
The oral administration of L-theanine to rats via gastric intubation for 2 weeks, caused a
20% lower oxidation level in the cerebral cortex as measured using thiobarbiturate reactive
substances as a marker (Nishida et al., 2008). The protein expression levels of phospholipase
C-beta 1 and -gamma 1, stress-responsible molecules, were significantly increased in the
cerebral cortex after L-theanine administration and the same tendency was observed in the
cerebellum, but not the hippocampus.
L-Theanine reduced the size of the cerebral infarct and alterations of the number of NeuN
(neuron)- and GFAP (astrocyte)-positive cell expression levels at 24 h after middle cerebral
artery occlusion in mice. This neuroprotective effect of L-theanine was prevented by
bicuculline but not 3-mercaptopropionic acid (glutamate decarboxylase inhibitor) (Egashira et
al., 2007). These results suggest that the neuroprotective effect of L-theanine is mediated, at
least in part, by GABAA receptors. L-Theanine significantly decreased the size of the cerebral
infarcts 1 day after the occlusion. In contrast, L-theanine did not affect the cerebral blood
flow, brain temperature or physiological variables in this model (Egashira et al., 2004). These
results suggest that L-theanine directly provides neuroprotection against focal cerebral
ischemia and may be clinically useful for preventing cerebral infarction. Cho et al. (2008)
investigated the protective effects of L-theanine on neurotoxicity induced by PD-related
neurotoxicants rotenone and dieldrin in a cultured human dopaminergic cell line, SH-SY5Y,
and suggested that L-theanine directly provided neuroprotection against PD-related
neurotoxicants and may be clinically useful for preventing PD symptoms.
Ischemia-induced neuronal death in the hippocampal CA1 region was significantly
prevented in a dose-dependent manner in L-theanine-pretreated groups. These findings
indicate that L-theanine might be useful clinically for preventing ischemic neuronal damage
(Kakuda et al., 2000b). L-Theanine significantly prevented the impairment of spatial memory
in rats subjected to repeated cerebral ischemia, 7 days after the second reperfusion. Moreover,
L-theanine significantly inhibited the decrease in the number of surviving cells in the
hippocampal CA1 field in the same rats. These results suggest that L-theanine prevents
memory impairment induced by repeated cerebral ischemia, in part by protecting against
neuronal cell death, and that it might be useful for preventing cerebrovascular disease
(Egashira et al., 2008).
L-Theanine did not increase the concentration of excitatory neurotransmitters in the
perfusate when injected into the rat brain striatum, but increased the concentration of glycine
in the perfusate (Yamada et al., 2009). Yamada et al. (2009) investigated the glycine and
dopamine concentrations in the perfusate, since it has been reported that L-theanine promotes
dopamine release in the rat striatum (Yamada et al., 2005; Yokogoshi et al., 1998a). Co-
injection of a glycine receptor antagonist, strychnine, reduced L-theanine-induced changes in
dopamine. Moreover, an α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA)
receptor antagonist inhibited the effects of L-theanine. L-Theanine may induce its inhibitory
effect at AMPA receptors. During brain development in infant rats, inhibitory
neurotransmission is required for mature brain function. Some neurotransmitters, including
dopamine, serotonin, glycine and GABA concentrations, increased in the infant brain, and
nerve growth factor mRNA level increased in the cerebral cortex and hippocampus when
other rats were fed L-theanine ad libitum after confinement. However, these differences were
lost by the end of nerve maturity. From these results Yamada et al. (2007) suggest that L-
theanine enhanced synthesis of nerve growth factor and neurotransmitters during a nerve
maturing period and promoted CNS maturation.
L-Theanine intake resulted in a reduction in the heart rate (HR) and salivary
immunoglobulin A (s-IgA) responses to an acute stress task relative to the placebo control
condition. Moreover, analyses of HR variability indicated that the reductions in HR and s-IgA
were likely attributable to an attenuation of sympathetic nervous activation (Kimura et al.,
2007). These facts suggested that the oral intake of L-theanine could cause anti-stress effects
via the inhibition of cortical neuron excitation.
The acute effects of L-theanine in comparison with a standard benzodiazepine anxiolytic,
alprazolam, and placebo on behavioral measures of anxiety in healthy human subjects using
the model of anticipatory anxiety were investigated (Lu et al., 2004). Sixteen healthy
volunteers received alprazolam, L-theanine or placebo in a double-blind placebo-controlled
repeated measures design. The acute effects of alprazolam and L-theanine were assessed
under a relaxed and experimentally induced anxiety condition. The results showed some
evidence for relaxing effects of L-theanine during the baseline condition on the tranquil-
troubled subscale of the visual analogue mood scale. Alprazolam did not exert any anxiolytic
effects in comparison with the placebo on any of the measures during the relaxed state.
Neither L-theanine nor alprazalam had any significant anxiolytic effects during the
experimentally induced anxiety state. The findings suggest that while L-theanine may have
some relaxing effects under resting conditions, neither L-theanine nor alprazolam
demonstrate any acute anxiolytic effects under conditions of increased anxiety in the
anticipatory anxiety model.
Kakuda et al. (2008) suggested that L-theanine would be an inhibitor of different
transporters capable of transporting glutamine (Gln) across plasma membranes toward the
modulation of the glutamate/Gln cycle required for the neurotransmitter pool of glutamate in
neurons. L-Theanine bound to three glutamate receptor subtypes (AMPA, kainate, and
NMDA glycine) in rat cortical neurons, but its IC50 of L-theanine was 80- to 30,000-fold less
than that of L-glutamate (Kakuda et al., 2002). L-Theanine and a group I metabotropic
glutamate receptor (mGluRs) agonist, DHPG, inhibited the delayed death of neurons caused
by brief exposure to glutamate, and this effect of L-theanine was abolished by group I mGluR
antagonists (Nagasawa et al., 2004). Treatment with L-theanine or DHPG alone resulted in
increased expression of phospholipase C (PLC)-beta1 and -gamma1, and the action of L-
theanine was completely abolished by group I mGluR antagonists. These findings indicate
that group I mGluRs might be involved in the neuroprotective effect of L-theanine by
increasing the expression levels of PLC-beta1 and -gamma1.
Bryan (2008) reviewed that L-theanine may interact with caffeine to enhance
performance in terms of attention switching and the ability to ignore distraction. This is likely
to be reflective of higher-level cognitive activity and may be sensitive to the detrimental
effects of overstimulation. Kakuda et al. (2000a) investigated the inhibitory action of L-
theanine on the excitation by caffeine at the concentration regularly associated with drinking
tea using EEG in rats. The stimulatory effects of caffeine were inhibited by an i.v.
administration of L-theanine and the results suggested that L-theanine has an antagonistic
effect on caffeine's stimulatory action at an almost equivalent molar concentration. On the
other hand, excitatory effects were shown in the rat i.v. administered L-theanine alone. These
results suggested two effects of L-theanine, depending on its concentration.
DIRECT COMPARISON OF EGCG AND L-THEANINE

FOR A SEDATIVE EFFECT
Although tea contains both EGCG and L-theanine that may affect sedation via the CNS,
direct comparison of their effect on the stress response is still limited. Adachi et al. (2007a)
compared the effects of L-theanine and EGCG at the same level on stress in neonatal chicks
using a social stress model. Chicks were injected i.c.v. with either saline as a control, 109
nmol of EGCG, or the same level of L-theanine. The effects of EGCG and L-theanine on total
spontaneous activity and distress vocalizations are shown in Figures 11 and 12. EGCG, but
not L-theanine, reduced total spontaneous activity and the number of vocalizations. Table 4
shows the effects of EGCG and L-theanine on four behavioral categories in chicks. Compared
with the control, EGCG significantly increased sleep-like behavior while decreasing the time
of active wakefulness. No significant effects were observed for L-theanine. The failure to
observe any effects of L-theanine at a concentration of 109 nmol on the parameters tested
should be interpreted with caution. First, it is possible that too low a concentration of L-
theanine was used to observe any stress modifying effect in chicks. When L-glutamate (up to
800 nmol) was i.c.v. administered, 400 and 800 nmol, but not 200 nmol, decreased distress
vocalization under separation-stress in chicks (Yamane et al., 2009). Second, it is possible
that the L-theanine-related relaxation found in mammals (Kimura et al., 2007) may be
mediated primarily through peripheral targets rather than via a direct action on the brain. In
this regard, no systemic administration of L-theanine was made. Finally, experimental
differences between this and other studies (Kimura et al., 2007) such as species (avian vs.
mammalian) and age (neonatal vs. adult animals) may have contributed to the failure of L-
theanine to attenuate stress.
Spontaneous activity (arbitrary unit)

a a
80
60
40
20
b
0
Control EGCG L-Theanine
Figure 11. Effect of i.c.v. injection of EGCG (109 nmol) or L-theanine (109 nmol) on total number of
spontaneous activity during a 10 min period in response to social separation stress in chicks. Data are
expressed as means±S.E.M. Groups with different letters are significantly different (P<0.05).
Reproduced from Adachi, N., Choi, Y.-H., Suenaga, R., Tomonaga, S., Denbow, D.M. & Furuse, M.
(2007a). Green tea component, (-)-epigallocatechin gallate, but not L-theanine, has sedative effects in
chick under acute stress conditions. Curr. Topics Nutraceut. Res., 5, 107-110., with permission from
New Century Health Publishers, LLC.
900 a
a
600
300
b
0
Control EGCG L-Theanine
Figure 12. Effect of i.c.v. injection of EGCG (109 nmol) or L-theanine (109 nmol) on total number of
distress vocalizations during a 10 min period in response to social separation stress in chicks. Data are
expressed as means±S.E.M. Groups with different letters are significantly different (P<0.05).
(2007a). Green tea component, (-)-epigallocatechin gallate, but not L-theanine, has sedative effects in
chick under acute stress conditions. Curr. Topics Nutraceut. Res., 5, 107-110., with permission from
New Century Health Publishers, LLC.
Table 4. Effect of i.c.v. injection of 109 nmol EGCG or L-theanine on various behaviors
in response to social separation stress for 10 min in chicks
Saline EGCG L-Theanine

Active wakefulness 480±41a 36±22b 425±70a
Standing/sitting motionless with eyes
100±29 128±37 76±23
open
Standing motionless with eyes closed 0±0 48±33 22±22
Sitting motionless with head drooped
20±20b 388±72a 77±69b
(sleep-like behavior)
Total 600 600 600
Values are means ± S.E.M. in seconds. Groups with different letters are significantly different (P<0.05).
(2007a). Green tea component, (-)-epigallocatechin gallate, but not L-theanine, has sedative effects
in chick under acute stress conditions. Curr. Topics Nutraceut. Res., 5, 107-110., with permission
from New Century Health Publishers, LLC.
CONCLUSION
Tea leaves include several ingredients, some of which are believed to act as medicinal
compounds. A feeling of relaxation can be induced by daily tea consumption. A part of this
feeling may be mediated by either EGCG, L-theanine, or both. Tea components may be
beneficial in a stressful society, and may have medicinal benefits for several mental diseases.
ACKNOWLEDGEMENTS
A part of studies described here was supported by grant-in-aid for scientific research
from Japan Society for the Promotion of Science (Nos. 16380191, 17208023 and 18208023)
to MF. This work was also supported by a Research Fellowship of the Japan Society for the
Promotion of Science for Young Scientists to ST (No. 18·9592) and HY (No. 20·03662).
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Chapter 2
GREEN TEA CATECHINS:

A CLASS OF MOLECULES WITH
ANTIMICROBIAL ACTIVITY
P. Buzzini1,*, P. Vignolini2, M. Goretti1, B. Turchetti1, E. Branda1,

E. Marchegiani1, P. Pinelli2 and A. Romani2
1
Department of Applied Biology - Microbiology, University of Perugia, Italy
2
Department of Pharmaceutical Sciences, University of Florence, Italy
ABSTRACT
A significant part of scientific interest of academy or industry is focused on
discovering novel natural antimicrobial drugs. This attention is essentially justified by the
expectation that a few of them could play a role in supporting (or even in substituting)
some antibiotics of current use. It has been estimated that, although about some tens of
novel antimicrobial drugs (either of biological or synthetic origin) are currently launched
each year, due to the increasing development of resistant microbial genotypes, their
downturn is becoming very rapid.
Taking into account these considerations, the enormous scientific and commercial
interest in discovering and developing novel classes of molecules exhibiting more or less
pronounced antimicrobial properties has oriented the work of a growing part of the
scientific community toward large-scale screening programs aimed at discovering novel
classes of bioactive molecules.
The occurrence in some plants of secondary metabolites exhibiting a more or less
pronounced antimicrobial activity is a well-known phenomenon. Among them, green tea
polyphenols represent a reservoir of molecules characterized by antioxidant, antiradical
and antimicrobial activity. In particular, catechins have proven to be effective towards
both prokaryotic and eukaryotic microorganisms. Despite the large number of studies
published so far, their actual potentialities and limitations as antimicrobial (mainly
antibacterial and antimycotic) drugs have not been critically evaluated. The present
chapter represents an overview of the recent literature on the antiviral and antimicrobial
*
Address for correspondence. E-mail: pbuzzini@unipg.it
24 P. Buzzini, P. Vignolini, M. Goretti et al.
properties exhibited by polyphenols, particularly catechins, occurring in green tea

composition.
INTRODUCTION
Tea is one of the most widely consumed beverages in the world today, second only to
water (Stagg, 1980), and its medicinal properties have been widely explored. Three billion
kilograms of tea are produced each year worldwide (Kris-Etherton and Keen, 2002). Because
of the high rates of tea consumption in the global population, it has been estimated that even
small effects in humans could have large implications for public health (Rimm and Diet,
2004). Tea is generally consumed in the forms of green, oolong, and black tea, all of which
originate from the leaves of the plant Camellia sinensis. Differences of composition between
green tea (popular in the Far East) and other teas (oolong and black teas, usually used in
Western countries) are mainly due to the oxidation steps occurring during the fermentation
process (Friedman, 2007). In particular, green tea is produced from steaming fresh leaves at
high temperatures, thereby inactivating the oxidizing enzymes and leaving the polyphenol
content intact.
Figure 1. Structures of most represented flavan-3-ols found in green tea.

Green Tea Catechins: A Class of Molecules with Antimicrobial Activity 25
The main polyphenols found in green tea are more commonly hydrolysable and
condensed tannins and include 30-40% of the extractable solids of dried green tea leaves.
Among them, catechins [e.g. epigallocatechin-3-O-gallate (EGCG), epicatechin-3-O-gallate
(ECG), gallocatechin (GC), epigallocatechin (EGC), and epicatechin (EC)] are the most
represented flavan-3-ols found in green tea (Figure 1). Enzymatic oxidation of EGC (and of
its gallate derivative) yields B-ring orto-quinones, which are dimerized spontaneously to give
deydrotheasinensis and related quinone dimers (Haslam, 2003). Theaflavins are produced by
condensation between cathecol-type and pyrogallol-type catechins. To clarify the oxidation
mechanism of green tea catechins occurring during tea fermentation, Tanaka et al. (2002)
oxidized pure catechins with a catechin-free homogenate obtained from tea leaves. Oxidation
of a mixture of (2)-EC and (2)-EGC yielded a new metabolite, named dehydrotheaflavin,
produced by the oxidation of a benzotropolone moiety of the black tea pigment theaflavin
(Figure 2). Similar oxidation of a mixture of (2)-EC and (2)-EGCG afforded a new dimer of
(2)-EGCG, which was generated by the oxidation and cycloaddition of two pyrogallol rings
(Tanaka et al., 2002).
OH
OR2
OH
HO O
OH
HO O
OH
OR1
OH
R1 R2
Theaflavin H H
Theaflavin-3-monogallate gallate H
Theaflavin-3’-monogallate H gallate
Theaflavin-3,3’-digallate gallate gallate
Figure 2. Chemical structure of theaflavins.

Concerning non-antimicrobial activities, the antioxidant properties of green tea

polyphenols are well known (Hamilton-Miller, 1995). These molecules are potent free radical
scavengers due to the hydroxyl groups in their chemical structure, which can complex (and
thus neutralize) free radicals, thus preventing the progression of the disease process due to
oxidative stress and subsequent generation of free radicals (Xu et al., 1992; Ichihashi et al.,
2000). In particular, in vivo and in vitro biological activities of tea flavan-3-ols include
inhibition of carcinogenesis, protection from cardiovascular disease, enhanced loss of body
fat, increase of bone density, protection from neurodegenerative diseases, and improvement in
type 2 diabetes (Graham, 1992; Hirose et al., 1993; Picard, 1996; Katiyar and Mukhtar, 1997;
Lee et al., 1997; Alschuler, 1998; Khafif et al., 1998; Otsuka et al., 1998; Sugiyama and
Sadzuka, 1998; Sato et al., 1999; Frei and Higdon, 2003; Cooper et al., 2005; Ramassamy,
2006; Zaveri, 2006; Friedman, 2007). However, the design and interpretation of
epidemiological and human intervention studies is limited by so far inadequate information
on bioavailability and metabolism/biotransformation of tea flavan-3-ols. Only a small
percentage of the flavan-3-ols are absorbed from the small intestine into the intestinal
epithelial cells, where they undergo conjugation to sulfated, glucuronidated, or methylated
conjugates by phase II enzymes (e.g. sulfotranferase, glucuronosyltransferase, and catechol-
O-methyltransferase). This process has been critically reviewed in detail by Lambert et al.
(2007).
Other miscellaneous non-microbiological properties possessed by green tea components
have been investigated. Some of the more interesting of these include activation of leukocytes
(Sakagami et al., 1992), antimutagenic activity (Hayatsu et al., 1992), lowering of plasma
cholesterol levels (Ikeda et al., 1992), protection from the effects of radiation (Uchida et al.,
1992), slowing the catabolism of catecholamines and strengthening of capillaries (‘‘vitamin P
effect’’) (Stagg and Millin, 1975; Min and Peigen, 1991). More recently, a few studies have
postulated a synergistic interaction between caffeine and green tea catechins. This synergy
apparently acts by extending sympathetic stimulation of thermogenesis. Green tea catechins
have also been shown to markedly inhibit digestive lipases in vitro, resulting in a decreased
lipolysis of triglycerides, which may provokes the reduction of fat digestion in humans
(Dulloo et al., 2000; Juhel et al., 2000).
As above reported, catechins are the major components of green tea, differently from
black tea where theaflavins (the oxidized form of catechins) are the major component. It is
not clear if catechins and theaflavins have equivalent antioxidant capacity (Lee et al., 2002;
Leung et al., 2001).
Green tea commercial extracts (GTEs), at different level and content in polyphenol
compounds, were currently used as food ingredients or in cosmetics preparations. A GTE,
obtained by the hot water-soluble fraction of unfermented leaves, exhibited a superior
scavenging activity toward reactive oxygen species (ROS) compared to vitamins C and E
(Zhao et al., 1989). More recently, Schroeter et al. (2001) found that GTE has the same
protective effects as 10-fold greater concentrations of vitamin C. Likewise, Jarrod et al.
(2008) tested in vivo a commercial caffeine-free GTE (Sunphenon 90DCF, Taiyo
International, Inc., USA) characterized by the following polyphenol composition:
polyphenols > 80%, catechins >80%, EGCG > 45%, caffeine < 1%). The authors observed
that physiological performances and endurance ability in mice were greatly enhanced by GTE
dietary administration. Independent of running, GTE apparently decreased serum creatine
kinase, heart and gastrocnemius lipid peroxidation and increased gastrocnemius citrate
synthase activity of rats. Based on these data, the authors suggested that the antioxidant
activity of GTE might be beneficial as therapeutic strategies to improve muscle function in
mice.
The antimicrobial activity of green tea polyphenols (particularly catechins) is well
known. Both industry and academy have been increasingly concerned with the growing
number of illness outbreaks caused by microbial pathogens. It has been estimated that,
although about some tens of novel antimicrobial drugs (either of biological or synthetic
origin) are currently launched each year, their downturn is becoming very rapid. Increasing
antibiotic resistance of some pathogens associated with over-consumption of some existing
antimicrobial drugs exacerbates this trend. Therefore, there has been an increasing interest in
studying and developing novel types of effective plant-derived antimicrobials, including those
present in green tea leaves (Friedman, 2007; Buzzini et al., 2007).
The main objective of this chapter is to unify and interpret widely scattered information
of literature on inhibitory activities of catechins occurring in green tea leaves against viruses,
bacteria, yeasts and filamentous fungi.
FUNDAMENTALS OF BIOSYNTHESIS OF GREEN TEA CATECHINS

Phenylpropanoid units derived from the shikimate pathway are common structural
elements of all flavonoid compounds and of other classes of phenylpropanoids, such as lignin,
stilbenes and cinnamate esters. The enzymes catalysing the individual steps of the
“phenylpropanoid metabolism” are: phenylalanine ammonia-lyase, cinnamate 4-hydroxylase
and 4-coumarate CoA ligase. In particular, flavan-3-ols originated from a branch pathway of
anthocyanin biosynthesis. Early studies covering enzymatic aspects of proanthocyanidins
biosynthesis have been carried out on Ginko biloba and Pseudotsuga menziesii. Such studies
reported that dihydroflavonols [(+)-dihydromyricetin (DHM) or (+)-dihydroquercetin (DHQ)]
are converted to the corresponding flavan-3,4-diols and catechin derivatives [(+)-GC, or (+)-
catechin, respectively] (Stafford and Lester, 1984, Stafford and Lester, 1985). The existence
of a relationship between the formation of 2,3-trans, catechin-derived series of flavan-3-ols
and the consecutive action of a dihydroflavonol reductase (DFR, which produces a
leucoanthocyanidin) and a leucoanthocyanidin reductase (LAR) has been postulated.
Leucocyanidin (produced by DFR via reaction with dihydroquercetin) was not observed with
tea enzyme preparations. Instead, it is immediately further converted to catechin by the LAR
present in the enzyme preparation (Punyasiria et al., 2004).
The anthocyanidin reductase (ANR) enzyme, recently isolated from Arabidopsis spp. and
Medicago spp., was shown to be present in tea leaves with very high activity. It produces EC
and EGC from their respective anthocyanidins, thus explaining the very high contents of such
compounds. The high ANR activity seems to be essential with respect to the dominance of
EC and EGC (as well as their galloyl esters) as the major flavonoid components in tea leaves.
EC and EGC, together with catechins, are also the building blocks of proanthocyanidins
reported from tea (Kiehne et al., 1997). Thus, ANR together with LAR, may be of great
importance for the biosynthesis of proanthocyanidins. The DFR/LAR two-step reaction
converted the dihydroflavonols, dihydroquercetin and dihydromyricetin to catechin and GC,
respectively, with a high activity. The pathways for the formation of many flavan 3-ol
derivatives are not known. The occurrence of a galloyl transferase has been recently reported
(Garcia et al., 2002). Catechins and EC, however, may also get hydroxylated by F3050H. In
general, in green tea is demonstrated a high activity of flavonoid 3’5’-hydroxylase (F3’5’H),
since there are more EGC and EGCG than EC and ECG in tea leaves. But also F3’H activity
must be present as B-ring dihydroxylated flavonoids like catechin, epicatechin and derivatives
are also quite prominent. Future studies will focus on the membrane-bound microsomal
enzymes F3’H and F3’5’H that are important for B-ring hydroxylation of tea flavonoids.
ANTIVIRAL ACTIVITY OF GREEN TEA CATECHINS

Among the possible modes of antiviral action of green tea polyphenols their ability to
prevent viral binding and penetration into cells, and to trigger the host cell self-defence
mechanisms are the mostly hypothesized (Friedman, 2007).
Weber et al. (2003) studied the effect of four green tea catechins on adenovirus (also
labelled as adenoidal-pharyngeal-conjuctival virus, APC) responsible for the infection of
mucous membranes of the respiratory and urinary tracts. EGCG was the most effective (IC50
= 109 μM) when added to the cells during the transition from the early to the late phase of
viral infection. The same compound exhibited a high selectivity index (SI). On the basis of
these evidences, the authors postulated that EGCG could inhibit one or more later steps of
viral infection and proposed its use for treating adenovirus infections in humans (Weber et al.,
2003).
A study by Savi et al. (2006) investigated the structure-activities relationships (SAR)
between green tea catechins and Herpes simplex virus (HSV). HSV, also known as cold sore,
causes blisters on the mouth and lips (oral and facial infections) and on genitals. The authors
observed that catechins exhibit an in vitro anti-HSV activity. The number of hydroxyl groups
on the B ring of the catechin molecules as well as the presence (or absence) of galloyl side
chains affected the SI (ranging from 1.3 to 13). Prodelphinidin B-2 3’-O-gallate, isolated from
green tea leaves, also exhibited an in vitro anti-HSV-2 activity (IC50 = 5.0 μM) (Cheng et al.,
2002). In close analogy with studies on anti-APC activity, the proposed mechanism of anti-
HSV properties of green tea catechins involves the inhibition of viral attachment and
penetration into cells. The disturbance of the late stage of viral infection has also been
postulated as an auxiliary mechanism (Friedman, 2007). Likewise, EGCG (50 μM) inhibited
the expression of lytic proteins of Epstein-Barr virus (EBV, also known as human HSV-4)
causing infectious mononucleosis. The inhibition of transcription of early genes governing the
initiation of the EBV lytic cascade has been proposed as the anti-EBV mechanisms of EGCG
(Chang et al., 2003).
The antiviral effects of green tea catechins can be targeted towards HIV (the retrovirus
causing the widespread human acquired immunodeficiency syndrome, AIDS) infection. Early
studies reported that ECG and EGCG, but not EC or EGC, are powerful antagonists of HIV
reverse transcriptase (IC50 from 10 to 20 ng/mL) (Nakane and Ono, 1990). Further studies
reported that EGCG is responsible for the observed anti-HIV-1 activity of green tea (Nakane
and Ono, 1989; Fassina et al., 2002). Several mechanisms explaining the anti-HIV properties
of EGCG have been proposed. Early hypothesis involves the inhibition of the biochemical
activity of HIV-1 reverse transcriptase, which causes the blocking of HIV-1 replication in
human cells (Nakane and Ono, 1989). More recent studies suggested a possible interference
of EGCG with HIV-1 viral infection by virion destruction and HIV-1 reverse transcriptase
inhibition (Fassina et al., 2002), whereas Yamaguchi et al. (2002) hypothesized that EGCG
could induce in vitro virion destruction by deforming phospholipids through the formation of
specific bounds with the surface of the viral envelope. Likewise, Kawai et al. (2003) reported
that EGCG apparently prevents the attachment of the HIV-1 virion (gp120), to CD4
molecules on T-helper cells. The authors found that EGCG (in concentrations from 25 to 250
μM/L) down-regulated the cell surface expression of CD4 by binding to the CD4 molecule,
presumably at a binding site recognized by gp120.
Other viruses causing different diseases have been studied as target for the antiviral
activity of green tea catechins. Preventive and curative effects of green tea extracts on
influenza virus have been claimed in a patent (Shimamura and Hara., 1991). ECGC
apparently prevents infection caused by the influenza virus by binding to the viral
hemagglutinin (HA) (Nakayama et al., 1993). So, the viral particles (bound by EGCG) cannot
attach to the target receptor cells. In vitro studies postulated that changes of viral membrane
properties could contribute to the antiviral effect of green tea catechins against the influenza
virus. EGCG and ECG were found to be 10–15 times more active against the influenza virus
than EGC. The role of 3-galloyl side chain as a factor enhancing antiviral activity of the
parent catechin molecule has been hypothesized (Song et al., 2005). As a part of a SAR study,
Song et al. (2007) screened the in vitro and in vivo antiviral activity of synthetic EGC and
(+)-catechin (C) derivatives characterized by the presence of different alkyl chain length and
aromatic ring substitutions at the 3-hydroxyl group. Pronounced activity was observed for
derivatives carrying moderate chain length (7–9 carbons) as compared to those with aromatic
rings. On the contrary, the 5’-hydroxyl group of the trihydroxy benzyl moiety did not
significantly contribute to antiviral activity. The active derivatives exerted inhibitory effects
for all influenza subtypes tested, including three major types of currently circulating human
influenza viruses (A/H1N1, A/H3N2 and B type), H2N2 and H9N2 avian influenza virus. The
observed antiviral activity appears to be mediated by the interaction of catechin derivatives
with HA and viral membrane (Song et al., 2007).
Hepatitis B virus (HBV) infection causes public health problems worldwide and is
endemic in some geographical regions (e.g. Asia). Xu et al. (2008) studied the in vitro anti-
HBV efficacy of green tea catechins and EGCG: IC50 from 5.02 to 10.76 μg/mL were
observed, whereas the 50% cytotoxic concentration (CC50) was as higher as 170 μg/mL.
Additional studies involving plant and animal viruses have been carried out. EGCG and
ECG bound to and inactivated tobacco and cucumber mosaic viruses that cause lesions in
leaves (Okada et al., 1971; Okada et al., 1978). Other studies reported that green tea catechins
prevent rotavirus and enterovirus from infecting monkey kidney cells in tissue cultures
(Mukoyama et al., 1991). This evidence was attributed to the possible interference of green
tea catechins with viral adsorption rather than a direct antiviral effect. Additionally, green tea
catechins were also proven to be active against the bovine coronavirus and rotavirus (causing
diarrhea and gastroenteritis in calves and cattle and resulting in significant losses to
agriculture) (Clark et al., 1998).
ANTIBACTERIAL ACTIVITY OF GREEN TEA CATECHINS

Green tea catechins have been tested as antimicrobial drugs against human pathogenic
bacteria (e.g. against ocular and cariogenic bacterial microflora causing minor infections).
ECGC inhibited gelatinase activity produced by a few ocular bacterial pathogens (IC50 = 200
μM) (Blanco et al., 2003). More recently, Friedman (2007) underlined that the inhibition can
delay the invasive spread of the bacteria in the eyes thriving on a gelatin substrate. Moreover,
some studies described anticariogenic effects of green tea compounds. Dental caries are
caused by a group of acid-producing species of the genus Streptococcus, in particular
Streptococcus mutans and Streptococcus sobrinus, which are reported to be the major
infective agents of human dental plaque. A key role in such process is played by salivary
amylase, which hydrolyses food starch to oligo- and monosaccharides (e.g. maltose, glucose).
The fermentation of such carbohydrates by bacterial enzymes occurring in oral cavities
provokes the formation of organic acids responsible to dental caries. Green tea components
have been seen to inhibit salivary amylase and, consequently, intra-oral hydrolysis of starch
(Zhang and Kashket, 1998). Another study showed that EGCG prevented lowering of pH
induced by cariogenic bacteria (Hirasawa et al., 2006). Likewise, an extract of green tea was
used for inhibiting growth of S. mutans. The analytical characterization of such extract
revealed that the main antibacterial components were GC, EGC and EGCG: among them, GC
was the most active component (MIC = 250 μg/mL) (Sakanaka et al., 1989).
Among pathogenic bacteria, Mycobacterium tuberculosis is a species that causes
tuberculosis in humans. Anand et al. (2006) observed that EGCG inhibited the expression of
tryptophan-aspartate containing coat protein (TACO) gene in a dose-dependent manner. This
effect was coupled with the inhibition of the survival of M. tuberculosis within host
macrophages.
The species Helicobacter pylori is a urease-producing gastric pathogen that may
contribute to the formation of ulcers and to low-grade gastric lymphoma in humans. Mabe et
al. (1999) found that EGCG displayed a strong activity against H. pylori (MIC50 = 8 μg/ml).
This compound exhibited bactericidal activity at pH 7 but not at pH < 5.0. In vivo studies
carried out on infected Mongolian gerbils, reported that H. pylori was eradicated in 10 to 36%
of the catechin-treated animals, with significant decreases in mucosal hemorrhage and erosion
(Mabe et al., 1999). Likewise, a screening carried out on green tea catechins revealed their
anti-H. pylori activity (Shin et al., 2005). Further studies reported that ECGC apparently
protects epithelial cells of gastric mucosa against H. pylori-induced apoptosis and DNA
damage (Lee et al., 2004). The authors postulated the block of activation of cellular signaling
pathways as the mechanism causing protecting activity of epithelial cells.
Legionella pneumophila causes Legionnaire disease, an infection of the lungs and other
organs. A few authors found that ECGC enhanced the in vitro resistance of alveolar
macrophages to infection caused by L. pneumophila (Yamamoto et al., 2004). Similarly,
Matsunaga et al. (2001) showed that concentrations as low as 0.5 μg/mL of EGCG inhibited
the growth of L. pneumophila in macrophages without any direct antibacterial effect. EGCG
selectively up-regulated the production of interleukin-12 (IL-12) and tumor necrosis factor
alpha (TNF-α) and down-regulated the L. pneumophila-induced production of interleukin-10
(IL-10) by macrophages. The authors sauggested that EGCG selectively leads to an enhanced
anti-L. pneumophila activity of macrophages.
Green tea catechins have been also tested as antimicrobial drugs against food-borne
pathogen and food-spoilage bacteria. In particular, food-spoilage bacteria can cause spoilage
and undesirable changes in a wide range of foods, particularly in processed, preserved, and
refrigerated food. Chou et al. (1999) used selected strains of food-borne pathogen and food-
spoilage bacteria to test the antibacterial activity of extracts from various tea products. In
general, antimicrobial activity decreased when the extents of tea fermentation increased: tea
flush and green tea extracts exerted the strongest antibacterial activity.
The activity of green tea catechins against the bacterial genera belonging to the
Bacillaceae family (Bacillus and Clostridium) was extensively studied. Bacillus cereus is a
widely distributed food-borne pathogen causing vomiting and diarrhea in mammals
(including humans). Friedman et al. (2006) demonstrated that GCG, EGCG, CG and ECG
exhibit antimicrobial activities at nano-Molar levels, whereas catechins without gallate side
chains and gallic acid were inactive. Interestingly, most compounds were more active than
commercially available antibiotics (e.g. tetracycline or vancomycin) at comparable
concentrations. In the same way, studies carried out by Hara et al. (1989; 1995) and by Ahn et
al. (1991) showed that green tea catechins strongly inhibited growth of Bacillus
stearothermophilus and Clostridium thermoaceticum in vitro. These compounds also reduced
the heat-resistance of both species growing in vending machines, which causes sour spoilage
in milk and other drinks (Sakanaka et al., 2000). Likewise, Sakanaka et al. (2000) studied the
inhibitory action of green tea catechins towards the germination of Bacillus spp. and
Clostridium spp. spores. The heat resistance of B. stearothermophilus and C. thermoaceticum
spores was reduced by the addition of EGCG. More recently, Juneja et al. (2007) found that
green tea extracts characterized by different catechin concentrations (from 141 to 697 mg of
total catechins/g of extract) determined the inhibition of Clostridium perfringens spore
germination in thawed beef, chicken, and pork. The authors underlined the catechins from
green tea can reduce the potential risk of C. perfringens spore germination and outgrowth
during abusive cooling.
Staphylococcus aureus is a highly pathogenic, toxin-producing, food-borne organism. An
early study reported that ECGC inhibits the growth of methicillin-resistant S. aureus (MRSA)
strains (Toda et al., 1991). Likewise, Ikigai et al. (1993) showed that EC was much less active
than EGCG against S. aureus strains. The MICs of EGCG and EC were 73 and 573 μg/mL,
respectively, and the bactericidal effect of EGCG was attributed to membrane perturbation
(Ikigai et al., 1993; Hamilton-Miller, 1995). The role of catechins (in particular EGC, EGCG
ECG) on the anti-MRSA strains was also confirmed by Yam et al. (1997). More recently,
Yoda et al. (2004) observed MICs from 50 to 100 μg/mL of EGCG against some species
belonging to the genus Staphylococcus (S. aureus, Staphylococcus epidermis, Staphylococcus
hominis and Staphylococcus haemolyticus). The authors postulated that the different structure
of the cell wall as well as the variable affinities of ECGC to some cell wall components (e.g.
peptidoglycans) might determine a differential activity of EGCG against such bacterial
species. Furthermore, Si et al. (2006) found that ECG and EGCG were the most active
compounds against S. aureus: EGCG exhibited the highest activity (MIC90 = 58 and 37 μg/L
for MSSA and MRSA, respectively). Scanning electron microscopy (SEM) studies showed
that both ECG and EGCG altered bacterial cell morphology, which might have resulted from
disturbed cell division. Analogously, Kim et al. (2004) found that green tea catechins
exhibited an approximately 5.0 log CFU/mL suppression of S. aureus strains (compared with
the control).
Physical factors can enhance the anti-S. aureus activity of green tea catechins. An et al.
(2004) found that irradiated (at 40 kGy) catechins extracted from green tea leaves increased
their antibacterial activities against S. aureus and S. mutans, whereas the major antioxidative
activities (e.g. electron donating, inhibition of xanthine-oxydase, metal ion chelating, and
inhibition of lipid oxidation) were unchanged after irradiation.
The strain O157:H7 of the species Escherichia coli is a food-borne, toxin-producing
enteropathogen responsible for a hemorrhagic form of colitis, bloody diarrhea, and hemolytic
uremic syndrome. Isogai et al. (1998) reported that green tea catechins protect mice against
neurologic and systemic symptoms caused by infection with E. coli O157:H7. Similar studies
found that green tea extracts exhibit a wide spectrum of activity against a series of pathogenic
bacteria, including strains of E. coli (Yam et al., 1997; Yam et al., 1998). A more recent study
(Si et al., 2006) found that green tea extracts strongly inhibit the growth of E. coli O157:H7,
Salmonella typhimurium, Listeria monocytogenes, S. aureus, and B. cereus. On the contrary,
Kim et al. (2004) found that green tea extracts suppressed the growth of only L.
monocytogenes (by approximately 3.0 log CFU/mL), whereas no influence on E. coli
O157:H7 and Salmonella enteritidis growth has been noted.
Yam et al. (1997) reported that a green tea extract inhibited the growth of several species
of food-spoilage bacteria (Proteus vulgaris, Pseudomonas aeruginosa and Serratia
marcescens) known to adversely affect the quality of some foods. More recently, Yoda et al.
(2004) found that higher levels of EGCG (MIC = 800 μg/mL) were needed to inhibit the
growth of some Gram-negative food-borne pathogenic and food-spoilage bacteria (E. coli,
Klebsiella pneumoniae, Salmonella typhi, Proteus mirabilis, P. aeruginosa, and S.
marcescens).
In close analogy with above reported data, ECG and ECGC were also found to inhibit the
growth of some phytopathogenic bacteria belonging to the genera Agrobacterium,
Clavibacter, Pseudomonas, Erwinia, and Xanthomonas (which contaminates eggplants,
grapes, cabbage, lettuce, onions, potatoes, tomatoes, etc.). MIC of about 100 μg/mL were
observed (Fukai et al., 1991).
Table 1. Some additional examples of antibacterial activity of green tea catechins
Species Active References

compound(s)
Bacillus anthracis EGCG Dell'Aica et al., 2004
Bacillis cereus catechins Hamilton-Miller, 1995; Friedman et al., 2006
Escherichia coli EGCG Taguri et al., 2004
Helicobacter pylori ECG, EGCG Setiawan et al., 2001; Yee et al., 2002; Yahiro et al., 2005
Legionella EGCG Matsunaga et al., 2002; Rogers et al., 2005
pneumophila
Staphylococcus EGCG Stapleton et al., 2004; Taguri et al., 2004
aureus
Vibrio cholerae EGC, EGCG Ikigai et al., 1990; Toda et al., 1992; Taguri et al., 2004;
Bandyopadhyay et al., 2005
In relation to the activity of green tea catechins on bacteria of technological importance,

Goto et al. (1998) reported that these compounds are able to positively affect intestinal
dysbiosis in nursing home patients by raising levels of Lactobacillus spp. and
Bifidobacterium spp. while lowering levels of Enterobacteriaceae and Bacteroidaceae.
Some additional examples of antibacterial activity of green tea catechins are reported in
Table 1.
Some hypotheses have been recently proposed for explaining the mechanism of
antibacterial action of green tea catechins. Accordingly, detailed physicochemical studies
suggested that the bactericidal activities of galloylated green tea catechins at the cell
membrane level might be due to their specific perturbations carried out over the ordered
structure of phosphatidylcholine and phosphatidylethanolamine bilayers constituting bacterial
cell wall membranes (Nakayama et al., 2000; Caturla et al., 2003). ECGC was found to be the
most effective catechin in causing leakage on E. coli-isolated membranes. Moreover, another
study suggested that EGCG could increase antibiotic susceptibility in S. aureus through the
inhibition of penicillinase produced by such bacterium (Zhao et al., 2002). In more recent
studies additional hypothesis supporting the antibacterial action of green tea catechins have
been proposed. Arakawa et al. (2004) and Hayakawa et al. (2004) suggested that the
bactericidal action of EGCG may also depend on hydrogen peroxide derived from the
reaction of EGCG with oxygen (pro-oxidative activity). Based on the above mentioned
hypotheses, Friedman (2007) concluded that the observed antimicrobial effects arise from the
interactions of catechins with oxygen, genes, cell membranes, and enzymes.
ANTI-YEAST AND ANTIFUNGAL ACTIVITY OF

GREEN TEA CATECHINS
Only a few studies have been carried out so far on the antimycotic activity of green tea
tannins. Okubo et al. (1991) early examined EGCG for their antifungal and fungicidal activity
against pathogenic filamentous fungi belonging to the species Trichophyton mentagrophytes
and Trichophyton rubrum. This compound showed fungistatic activity against both
Trichophyton species. More recently, Hirasawa and Takada (2004) have studied the
susceptibility of the opportunistic pathogenic yeast Candida albicans to various green tea
catechin under varying pH conditions. Analogously, Park et al. (2006) reported that EGCG
exhibits antimycotic activity against 21 Candida spp. isolates. Among them, the strains
belonging to the species Candida glabrata were the most susceptible. Both studies reported
that the anti-yeast activity of catechins was pH dependent. The two studies reported
contradictory results. Hirasawa and Takada (2004) observed that the MIC90 of EGCG was
2000 μg/mL at pH 6.0, 500–1000 μg/mL at pH 6.5 and 15.6–250 μg/mL at pH 7.0, whereas
Park et al. (2006) found that the MIC of EGCG increased several folds as the pH was reduced
from 7.0 to 6.0.
As a result of a large-scale screening survey on the antimycotic activity of some plant
extracts, Turchetti et al. (2005) observed that green tea extracts inhibited growth of yeasts
belonging to the species C. glabrata Cryptococcus laurentii, Clavispora lusitaniae,
Issatchenkia orientalis, Filobasidiella neoformans and Saccharomyces cerevisiae, as well as
that of yeast-like microorganisms Prototheca wickerhamii. The authors concluded that the
compounds responsible of the observed anti-yeast activity are ECG and EGCG.
The mode of action of catechins on eukaryotic microorganisms has been little studied. In
early investigations, Toyoshima et al. (1993) suggested that catechins are able to attack the
cell membrane of Trichosporon mentagrophytes causing lysis of the conidia and hyphae.
SYNERGISTIC INTERACTIONS BETWEEN

CATECHINS AND ANTIBIOTICS
Current literature reports studies describing that the combined use of antibiotics and
green tea catechins can increase the antimicrobial activity of the formers through specific
synergistic interactions. Combinations of EGCG + β-lactames antibiotics exhibited
synergistic activities against MRSA strains (Hu et al., 2002). Zhao et al. (2001) suggested that
EGCG synergistically increases the activity of β-lactam antibiotics against S. aureus by
binding to the peptidoglycan component of the bacterial cell wall. Likewise, Sudano Roccaro
et al. (2004), postulated that EGCG acts in synergy with β-lactam antibiotics by reversing
tetracycline resistance in Staphylococcus spp. isolates and by inhibiting the specific efflux
pump Tet(K). The authors observed an increased accumulation of tetracycline inside bacterial
cells as a visible effect of synergy with EGCG. On the contrary, Yanagawa et al. (2003) noted
only additive interactions between the β-lactam antibiotic amoxicillin and EGCG against non-
resistant and antibiotic-resistant isolates of H. pylori. More recently, Friedman (2007)
reported that non-galloylated catechins also potentiated the activity of oxacillin against S.
aureus.
The combined use of green tea extracts with butylated hydroxyanisole (BHA) was more
effective against bacteria and fungi than green tea alone (Simonetti et al., 2004). Glycolic
extract from green tea showed a certain activity against E. coli, but only limited activity
against S. mutans and no activity against C. albicans. Sub-inhibitory concentrations of BHA
increased the microbicidal activity of green tea extracts against S. mutans, non-susceptible E.
coli and C. albicans. In addition, green tea extracts in combination with BHA reduced the
hydrophobicity of S. mutans and greatly inhibited the formation of pseudomycelium in C.
albicans. The authors postulated that the increased antimicrobial activity of green tea extracts
(due to synergy with BHA) is related to an impairment of the barrier function in
microorganisms and a depletion of thiol groups.
Other groups of antibiotics have been studied for their synergistic interactions with green
tea catechins. Lee et al. (2005) observed that a combination of catechins and the
fluoroquinolone antibiotic ciprofloxacin acts synergistically to alleviate chronic bacterial
prostatitis in rats. Likewise, a further study showed that a green tea extract exhibited in vivo
synergy with the antibiotic levofloxacin against infection of mice caused by E. coli O157:H7
(Isogai et al., 2001).
Hirasawa and Takada (2004) reported that the addition of EGCG to sub-inhibitory
concentrations of amphotericin B (AMB, belonging to the group of polyenes) increased the
anti-yeast activity of AMB against AMB-susceptible or -resistant C. albicans. Combined
treatment with EGCG + AMB (3.12–12.5 and 0.5 μg/mL, respectively) markedly decreased
the growth of AMB-resistant C. albicans strains. Since sub-inhibitory concentrations of AMB
stimulate yeast membrane permeability, the authors suggested that the combined use of AMB
and EGCG might stimulate catechin uptake into the cell, and, consequently, the increased
intracellular catechin concentration could act as a anti-yeast agent. The same authors also
observed that when azole-susceptible C. albicans strains were treated with EGCG +
fluconazole (25–50 and 0.125–0.25 μg/mL, respectively), its growth was inhibited by 93.0–
99.4% compared with the growth observed in the presence of fluconazole alone. The
combined use of EGCG + fluconazole (12.5 and 10–50 μg/mL, respectively) also inhibited
the growth of fluconazole-resistant C. albicans by 98.5–99.7% (Hirasawa and Takada, 2004).
CONCLUDING REMARKS
Current literature herein highlight the fact that green tea polyphenols include a series of
compounds (e.g. catechins) extensively studied as potential antiviral and antimicrobial drugs.
Yet, although some promising results have been reported, most studies remains so far
confined to the laboratory scale. In other words, in relation to the commercial exploitation of
green tea catechins as antimicrobial drugs a few questions remain still open.
First of all, the lack of a rigorous analytical determination of the chemical nature of active
components of some green tea extracts is a common feature in most of the literature cited in
this chapter. Studies using purified (or partially purified) molecules (e.g. catechins) are still
very few in number. The improvement of analytical techniques will indisputably lead to a
better knowledge of the antiviral and antimicrobial properties of individual compounds
occurring in green tea composition. Hence, the high cost of commercially available pure
standards (e.g. EGCG) represents a supplementary problem, whereas synthetic strategies, so
far used only for SAR studies carried out at the laboratory-scale, are too costly and time-
consuming procedures.
Secondly, the assay protocols employed for the in vitro evaluation of antimicrobial
activity of green tea components is an additional problem. Most of the studies reporting MICs
did not apply standard CLSI guidelines (CLSI, 2002a; CLSI, 2002b) essential for monitoring
the accuracy of the method. As a result, some contradictory results could be the consequence
of their low reproducibility level. The well-documented strain-related susceptibility of
microbial genotypes is an additional problem. In this sense, the use of undetermined (or even
unidentified) strains should be discouraged.
Finally, as recently underlined by Friedman (2007), another critical aspect is to determine
whether the antimicrobial activities of green tea compounds observed in vitro can be
duplicated in vivo. It is important to remember that most of the literature cited in this chapter
reported results of in vitro investigations. Even though useful as a preliminary survey, in vivo
activity is clearly more significant. As pointed out by some authors (Kawai et al., 2003;
Nance and Shearer, 2003), the most crucial aspect of translating the observed in vitro effects
of green tea compounds to pharmacologically relevant strategies, is the requirement to
achieve physiologically relevant concentrations. Due to the well-known poor bioavailability
of some green tea compounds, most of the ingested EGCG does not get into the blood, and a
significant fraction is eliminated presystemically (Chow et al., 2001; Pisters et al., 2001; Lee
et al., 2002). So, even though the use of capsular administration of green tea or EGCG has
been proposed, some authors doubted that, in this form, the blood EGCG concentration could
remain at a low level than that used in the current in vitro studies (Kawai et al., 2003; Nance
and Shearer, 2003). Additional studies should be consequently carried out to clarify these
crucial aspects for the possible exploitation of green tea preparations for therapeutic purposes.
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Chapter 3
LIPID-SOLUBLE GREEN TEA POLYPHENOLS:

STABILIZED FOR EFFECTIVE FORMULATION
Ping Chen2, Douglas Dickinson1 and Stephen Hsu1

1
Medical College of Georgia, USA
2
Zhejiang University, China
ABSTRACT
Green tea polyphenols (GTPs), also referred to as green tea catechins, possess
properties that can provide unique health benefits to humans. As indicated in other
chapters of this book, studies using molecular, cellular, and animal models, and in human
subjects, have demonstrated that these phytochemicals from non-oxidized tea leaves have
anti-cancer, anti-oxidant, anti-microbial, and anti-inflammatory properties. Recently,
investigations in our and other laboratories indicated that topical application of GTPs
could protect the epidermis against autoimmune disorders, such as psoriasis, prevent or
repair UV-induced damage, and suppress scar tissue overgrowth. In addition, specific
gene regulation by GTPs, especially epigallocatechin-3-gallate (EGCG), promotes skin
cell differentiation, which could lead to improved homeostasis of the skin.
Based on these facts, the topical use of products containing GTPs has become more
popular, and manufacturers of cosmetic, health care, and household products are adding
GTPs or EGCG to their formulations. However, it is important to note that studies
described in this book always use “freshly prepared” GTPs or green tea, instead of “pre-
prepared” materials. This is because GTPs are potent antioxidants that react rapidly with
reactive oxygen species (ROS). As a result, GTPs in most commercially available
products have been oxidized and/or epimerized; the biological effects of the resulting
compounds are largely unknown. In addition, due to the highly water-soluble nature of
these compounds, GTPs in their original form are not lipid-soluble, and therefore not
permeable to the skin, a water-proof barrier. Another problem with formulation of GTPs
for topical application is the coloration change and precipitation caused by oxidation.
Thus, GTPs for topical application (e.g., on skin and mucous membranes) must be
prepared and used immediately prior to oxidation, coloration and precipitation. These
properties of GTPs make it difficult to formulate products containing them that have a
reasonable shelf life and maintain their activity and effectiveness. In other words, most of
the commercially available “green tea”–containing products are without the full benefits
46 Ping Chen, Douglas Dickinson and Stephen Hsu
of green tea or GTPs. Therefore, strategies to stabilize and increase the bioavailability of
GTPs are needed to provide the full benefits of GTPs to consumers or patients.
Recently, it has been shown that lipid esters of GTPs can be formed either
enzymatically or chemically. These green tea polyphenol-lipid esters, also referred to as
lipid-soluble tea polyphenols (LTPs), could significantly improve formulations of
consumer or health care products. We hypothesized that fatty acyl esterification of green
tea polyphenol would protect the hydroxyl groups from oxidation and improve skin
permeability. In the current study, we compared the activities of LTPs to GTPs for their
anti-cancer and gene regulation properties. We examined whether LTPs can be converted
into a free GTP (EGCG) in human skin keratinocyte cultures. In addition, the effects of
LTPs in a mouse model for psoriasis were evaluated. The results indicate that LTPs
effectively cause cancer cell death, induce caspase 14 gene expression both in vitro and
in vivo, and improve the skin condition in an animal model for psoriasis. Consistent with
these observations, HPLC analysis demonstrated that EGCG in its original form was
released from LTPs in situ by human epidermal keratinocytes. These results suggest that
LTPs, under appropriate conditions, function similarly to GTPs. More importantly, since
the most reactive hydroxyl group(s) is/are protected, and the lipid solubility is
dramatically increased by the fatty acyl groups, the biological activity of these
compounds can be stabilized, and their bioavailability increased significantly.
In conclusion, LTPs are a novel and more effective form of green tea polyphenols for
topical applications and other purposes, especially in formulations that require a
reasonable shelf life. In addition, LTPs can be a natural additive to consumable products
such as salad oil, fish oil, and cooking oil as antioxidants.
INTRODUCTION
Green tea polyphenols (GTPs) are under extensive study for their unique properties that
can provide protection against a number of human diseases, including neoplastic,
cardiovascular, autoimmune, and infectious diseases. During the past decade, anti-cancer,
anti-inflammatory, anti-aging and anti-photo-damaging properties of GTPs, especially
epigallocatechin-3-gallate (EGCG), have been identified [1-11]. These findings have
prompted the addition of GTPs to cosmetic and health care products for topical application.
The ingredient labels of many cosmetic and household products list various descriptions of
GTPs, such as “green tea extract”, “extract of camellia sinensis”, “green tea polyphenols”,
“green tea catechins”, “green tea leaf extracts”, and “EGCG”. In addition, due to their strong
anti-oxidant activity, this type of naturally-occurring compound can potentially be used as
natural antioxidant food additives in various products, including dietary oils.
Unfortunately, certain properties of GTPs create problems for their commercial
application. In traditional green tea-consuming countries such as China and Japan, green tea
is consumed immediately after it is brewed, i.e., “freshly prepared”. In an effort to bring green
tea’s benefits to modern populations, bottled green tea was invented, and it has become
popular even in traditionally “green tea brewing” countries. However, an aqueous
environment favors oxidation of GTPs, leading to epimerization and polymerization of the
catechin monomers. When the monomers are oxidized, their anti-oxidant and biological
activities are reduced significantly. A study of bottled tea beverages confirmed that the
majority of these drinks no longer contain significant amount of GTPs, but rather the epimer
gallocatechin gallate (GCG) and oxidized compounds [12]. In this study, dry leaves of green
Lipid-soluble Green Tea Polyphenols 47
tea had an average EGCG content of 6.87% (see Table 1 in reference 12). In most brand-
named tea beverages tested by this study, the EGCG content was at “trace” levels (<
0.00001%) to 0.00082%. One exception had a higher level (0.058%) [12]. In other words, due
to the required step of sterilization by autoclaving, the anti-oxidant and other beneficial
effects of most tea beverages have been destroyed prior to their consumption.
A second problem with GTPs is that they are highly hydrophilic, resulting in very low
solubility in dietary oils, and they form dark precipitates in these oils [13]. Formulation
experts have tried to emulsify GTPs with lipid material in order to increase their solubility in
dietary oils, but the results have not been satisfactory due to precipitation and coloration [13].
A third problem for the use of GTPs is their relatively very low bioavailability in humans
after oral administration as measured by serum concentration analysis [14]. In fact, the
majority of GTPs is excreted from the digestive and urinary system, and less than 10 µM of
GTPs concentration can be achieved in the circulation [14, 15]. This poor serum
bioavailability decreases the potential anti-cancer and anti-inflammatory effectiveness of
GTPs, which have been described in many studies that have used much higher concentrations
of GTPs. In addition, since the epidermis is not vascularized, the benefit of orally consumed
GTPs to the human epidermis is highly questionable [16]. Therefore, for the skin, topical
application with high concentrations of GTPs/EGCG has been considered as an optional route
of delivery (11, 10% EGCG). However, topical application of a high concentration of GTPs
could cause significant skin coloration, although this effect could be used for skin tanning
(US Patent 6399046). Other methods, such as “electrical assisted” methods, have been
developed to increase the skin permeability to GTPs, but they may not be a suitable strategy
to improve skin condition [17]. Taken together, the hydrophilic nature of the water-soluble
and unstable polyphenolic GTP molecules prevents them from penetrating lipid barriers such
as the stratum corneum. This compounds the fact that GTPs in these products are undergoing
oxidation, epimerization, and/or polymerization prior to reaching the customers.
Thus, the instability of GTPs in their original monomer form in an aqueous environment
at physiological pH, and their impermeability to lipid barriers and insolubility in a
hydrophobic environment, prevent their delivery at levels that provide effective benefits to
human health. Stability and bioavailability are therefore two major challenges in the
development of any effective product with a reasonable shelf life.
Green tea researchers and formulation specialists identified these limitations, and
attempts to preserve the chemical integrity and activity of GTPs began in the 1990s [13].
Esterification has been proven effective for other bio-molecules such as vitamins C (ascorbyl
palmitate) and A (retinyl palmitate), and bioactive peptides, which are widely used in today’s
consumer and cosmetic products [18, 19, and international patent WO 1992014830].
Researchers proposed that modification of GTPs with fatty acyl esterification would increase
their stability and skin permeability without significant reduction of the antioxidant property.
One group of Chinese researchers led by Professor Shuxiong Wang invented a method using
an acylation reaction (patent CN1197786A) that chemically modifies GTPs to make GTP-
esters. This method renders the final product (lipid-soluble tea polyphenols [LTPs] in a dark
brown gel-like form) soluble in oil and many hydrophobic solvents. Another method was
later developed in China by Jian Hua Zhong and Ping Chen which produces GTP-esters in a
solid powder form (CN1231277A). In Japan, similar GTP derivatives were made using the
reverse reaction of lipases from Streptomyces rochei (Japanese patent JP6279430). Recently,
Kunihiro Kaihatsu’s group in Osaka University synthesized a series of EGCG-esters by an
enzymatic method using lipase PL from Alcaligenes sp [20]. In 2002, one of us (P Chen) first
synthesized and identified the chemical structure of EGCG-4’-hexadecanate [21].
Subsequently, in 2003, the first systematic synthesis, purification, and analysis of EGCG-
acyl-derivatives in the laboratory was reported, and the purified LTP compound
epigallocatechin-3-O-4’-O-hexadecanate was obtained (referred to as EGCG-palmitate
hereafter) [22]. The antioxidant activity and stability of EGCG-palmitate was tested in dietary
oils. It was found that the antioxidant activity of EGCG-palmitate in soybean salad oil and
canola oil is greater than that of butylated hydroxyanisole (BHA) and butylated
hydroxytoluene (BHT), and similar to that of tertiary butylhydroquinone (TBHQ) [23]. In
addition, the potential toxicity of EGCG-palmitate was tested by the Zhejiang Center for
Disease Control & Prevention of China. The acute toxicology test result in mice was
LD50>5.0 g/kg, and the Ames Test result for mutagenicity was negative [23]. The anti-cancer
property of EGCG-palmitate was compared with GTPs and LTPs by determining the
inhibition of growth of the human ovarian cancer cell line HO-8910 [24]. The 50% inhibitory
concentration (IC50) for EGCG-palmitate was 78.2 µg/ml, while for GTPs it was 72.2 µg/ml,
and 88.9 µg/ml for LTP [24]. In a separate study, EGCG-myristate (epigallocatechin-3-O-4’-
O-myristate) was synthesized [25]. This is an EGCG ester with a 14 carbon fatty acyl chain.
The physical characteristics of EGCG-myristate are similar to EGCG-palmitate. It has a
whitish powder appearance and is readily soluble in lipid, but not in water. The antioxidant
activity of EGCG-myristate is almost identical to EGCG-palmitate when compared with
TBHQ, BHA and BHT (in soy bean salad oil) [25]. That is, the food industry, especially
dietary oil manufacturers, could benefit from the use of LTPs as an antioxidant.
However, it is not known if LTP enters human cells, or if it can be converted to GTPs to
induce similar biological effects. It is also not clear whether a stable LTP formulated topical
application can improve the skin condition in a mouse model for psoriasiform lesions (the
flaky skin mouse), as shown for EGCG. The current study used a powdered form of 18
carbon fatty acyl (stearoyl) and a gel-like 18 carbon monounsaturated fatty acyl (oleic acyl)-
ester of green tea polyphenols (referred to as LTPStearate and LTPOleate hereafter) to tests their
biological effects in vitro and in vivo.
MATERIAL AND METHODS
Chemicals and Antibodies
EGCG was purchased from Sigma-Aldrich (St. Louis, MO). The solid-form LTPStearate
(>90% stearoyl tea polyphenol) was purchased from Yuyao Huidelong Biological Bio-
Products, Co., Ltd, Zhejiang, China. The gel-form LTPOleate (>90% oleic acyl tea
polyphenols) was purchased from Zhejiang Cereals Oils & Foodstuffs Import & Export
Company, Ltd, Zhejiang, China. A topical formulation of LTPStearate was made of 0.1%
LTPStearate in glycerin and stored at 4o C for daily topical use (the preparation of this
formulation is not disclosed due to pending patent).
The anti-caspase 14 and anti-human actin (I-19) antibodies were obtained from Santa
Cruz Biotechnology, Santa Cruz, CA.
Cell Lines and Cell Culture
Pooled normal human primary epidermal keratinocyte (NHEK) cells were purchased
from Cambrex (East Rutherford, NJ) and sub-cultured in the specific growth media (KGM-2)
provided by the manufacturer. The OSC2 cell line was isolated from a metastatic lymph node
of a patient with oral squamous cell carcinoma [26]. The cells were cultured in Dulbecco’s
Modified Eagle’s Medium (DMEM)/Ham’s F12 50/50 MIX medium (Cellgro, Kansas City,
MO) supplemented with 10 % (v/v) fetal bovine serum, 100 I.U/ml penicillin, 100 µg/ml
streptomycin and 5 µg/ml hydrocortisone.
Morphological Analysis of Cells Treated with LTPStearate
OSC2 or NHEK cells were seeded in 24-well tissue culture plates at 2X105 cells/well and
incubated overnight. LTPStearate dissolved in vehicle (100% ethanol) at different
concentrations was added to each well. Control wells were incubated with vehicle only. At
the end of incubation, wells were washed with PBS and photographed at 100X magnification.
Western Blotting
To determine changes in the protein level of caspase 14 upon activation by EGCG or

LTPStearate, NHEK were exposed to 50 µM EGCG or LTPStearate for various times. Cell lysates
were prepared, and samples containing 30 µg protein were separated on 10 % SDS
polyacrylamide gels. Western blot analysis was performed using anti-human caspase 14 and
actin antibodies (for normalization). The method for Western analysis was previously
described [27]. The resulting bands were visualized by enhanced chemiluminescent staining
using ECL Western blotting detection reagents (Amersham Pharmacia Biotech Inc.,
Piscataway, NJ).
Animal Treatment and Immunohistochemistry
Flaky skin mice were treated daily for consecutive ten days with an LTPStearate topical
preparation (0.1% w/v in glycerin) in an area on the back of the animal. On the opposite side
of the animal, glycerin was applied topically as control. The skin samples were collected by a
biopsy puncture under anesthesia, and the animals were euthanized thereafter. Processing and
immunostaining of skin samples have been described previously [28].
HPLC Analysis of EGCG from Cell Lysates
Cultures of NHEK cells were divided into two and treated with 0.1% w/v of either
LTPOleate or LTPStearate. At 0.5 h, 1.0 h, 2.0 h, and 6.0 h, cells were washed with PBS and lysed
with 0.5 ml RIPA buffer (pH adjusted to 3.35). An equal amount of cell lysate was also
collected from NHEK treated with 100 μM EGCG for 0.5 h as a control for HPLC detection.
HPLC detection of EGCG in cell lysates was performed according to a method
previously described [29]. The Discovery C-18 reverse-phase column was purchased from
Supelco, Sigma-Aldrich (St. Louis, MO). Briefly, the column was eluted with Solution A (30
mM NaH2PO4 in 1.75% CH3CN and 0.125% tetrahydrofuran (pH 3.35) and Solution B (15
mM NaH2PO4 in 58.5% CH3CN and 6.25% tetrahydrofuran (pH 3.45), as described
previously [29].
RESULTS
LTPStearate Induces Tumor Cell Death as Efficiently as GTPs
Our previous study demonstrated that GTPs at 1 mg/ml are a powerful inducer of cell
death in OSC2 oral carcinoma cells [30, 31]. To test the effectiveness of LTPStearate, OSC2
cells were incubated with 1 mg/ml LTPStearate (0.1% w/v) for 24 hr. As shown in Figure 1,
almost all tumor cells were eliminated by LTPStearate treatment, while control OSC2 cells
treated with vehicle only remained viable. This result indicates that LTPStearate possesses the
ability of a cell death-inducer for OSC2 cells. The anti-tumor cell effect of LTPStearate was also
significant at a lower concentration (0.01% w/v), although less pronounced that at 0.1% w/v
(Figure 2), consistent with dose-dependency.
Figure 1. 0.1% LTPStearate induced massive cell death in OSC2 cells. OSC2 oral carcinoma cells were
incubated with vehicle or 0.1% LTPStearate (pre-dissolved in 100 ethanol) in cell culture medium for 24
h. The culture dishes were washed by PBS and photographed under light microscope (100 X).
Figure 2. 0.01% LTPStearate induced significant cell death in OSC2 cells. OSC2 oral carcinoma cells were
cultured until reaching confluency, and incubated with vehicle or 0.01% LTPStearate (pre-dissolved in
100 ethanol) in cell culture medium for 24 h. The culture dishes were washed by PBS and
photographed under light microscope (100 X).
Figure 3. 0.1% LTPStearate failed to induce cell death in normal human epidermal keratinocytes (NHEK).
NHEK cells were incubated with either vehicle or 0.1% LTPStearate for 24 h. The culture dishes were
washed with PBS and photographed under light microscope (100 X).
LTPStearate does not Induce Cell Death in Normal Epidermal Keratinocytes
LTPStearate at 1 mg/ml (0.1% w/v) was incubated with primary NHEK (Figure 3). In
contrast to the results with the tumor cell line OSC2, no sign of cell death or detachment was
observed, indicating that LTPStearate did not induce cytotoxicity in NHEK.
Dose-dependent Induction of Caspase 14, a Marker for Epidermal Terminal

Differentiation and Barrier Formation, by LTPStearate
The expression of caspase 14 was compared between 50 µM EGCG-treated and 0.01%

w/v LTPStearate-treated NHEK. Since 0.01% w/v of LTPStearate contains approximately
0.0025% w/v green tea polyphenols, the polyphenol content was comparable between the two
treatments. Figure 4 demonstrated that both materials induced the expression of caspase 14 at
the 20 h time point, and continued expression of caspase 14 was observed at 24 h.
Western blotting also showed that LTPStearate at 0.001% w/v to 0.01% w/v (10-100 ppm)
induced caspase 14 expression in NHEK cells in a dose-dependent manner (Figure 5).
LTPStearate Induces Caspase 14 Expression in Vivo in a Mouse Model for

Psoriasiform Lesions
Immunostaining of mouse skin treated with LTPStearate in glycerin demonstrated increased

caspase 14 expression in the supra-basal layers of the epidermis (Figure 6). This result is
consistent with our previous observation that GTPs induce and activate caspase 14 in vivo
[28].
Figure 4. Western blot results demonstrate both EGCG and LTPStearate induced caspase 14 expression in
NHEK. NHEK cells were treated with either EGCG (50 µM) or LTPStearate for 0, 2, 6, and 24 h. The cell
lysates were collected for Western blot using rabbit anti-human caspase 14 and goat anti-human actin
antibodies.
Figure 5. LTPStearate dose-dependently induced caspase 14 protein expression in NHEK cells. Various
concentrations of were prepared with DMSO, diluted with cell culture medium, and incubated with
NHEK for 24 h. Cell lysates were collected for Western blot with antibodies against human caspase 14,
and human actin as an internal control.
LTPOleate and LTPStearate are Converted to Intracellular GTPs in NHEK
HPLC analysis of cell lysates of NHEK demonstrated that intracellular EGCG appeared
in both LTPOleate and LTPStearate - treated NHEK (Table 1). However, the release time was
different between the two (Figure 7). The majority of EGCG released from LTPOleate appeared
in the cell lysate collected at 1 h after the addition of LTPOleate, while the majority of EGCG
released from LTPStearate appeared at 6 h after treatment (Figure 7).
Table 1. Retention time comparison (min) between the two types of LTP and EGCG
*Retention time of EGCG: 28.92 min.

Time of EGCG peak was recorded by HPLC using samples from cell lysates collected at different time
points after the LTPs was introduced into the cell culture medium.. ND: not determined.
Figure 6. Immunostaining of skin samples from vehicle-treated and LTPStearate -treated (flaky skin mice)
using caspase 14 antibody. Nuclear caspase 14 staining appeared in the junction of the granular layer
and the cornified layer in the LTPStearate-treated sample, while the vehicle-treated sample did not exhibit
nuclear staining of caspase 14. The supra-basal layers of the LTPStearate-treated sample showed
consistent immunostaining of caspase 14, but those of the vehicle-treated sample only showed sporadic
caspase 14 expression. The epidermal portion of the samples was photographed at a 400X
magnification.
Figure 7. EGCG release from NHEK demonstrates time differences between LTPStearate and LTPOleate.
Cell lysates were collected at indicated time points after LTPs was introduced into the cell culture
medium. HPLC analysis of the samples showed different release patterns between the two types of
LTPs. LTPS: LTPStearate. LTPO: LTPOleate.
DISCUSSION
The earliest Chinese literature indicates that the Chinese grew and used tea plants 3,000
years ago. According to legend, in 2737 B.C., the Chinese Emperor Shen-Nung placed
camellia blossom tips into a cup of boiled water and pronounced the beverage healing and
refreshing. Tea consumption became popular in China after it was said to cure the ailing
troops of General Zhu Ge Liang (181-234 A.D.). Tea was described as able to prevent
pandemics and treat patients with eye diseases. During the Tang Dynasty (618-907 A.D.), tea
became an essential component of Chinese culture. Chinese author Lu Yu (733-804 A.D.)
published the book, Cha Jing (literally, The Book of Tea), systematically chronicling the
planting, processing, preparation and consumption of tea. From then, green tea (as well as
many other types of teas derived from green tea), became the most popular beverage (second
to water) in the world.
Today, the medicinal use of green tea and its compounds is being re-visited by scientists
and clinicians, and the benefits are being unveiled almost daily. In 2006, the FDA approved
an ointment, VeregenTM (MediGene AG, Polyphenon® E Ointment), for treatment of genital
warts, an epidermal sexually transmitted disease cause by human papilloma virus (HPV). The
active ingredient(s) in this topical ointment is 10-15% Polyphenon E, a highly purified and
defined GTPs mixture developed by Dr. Yukihiko Hara and colleagues at Mistui Norin Co.
Ltd of Japan [32]. It is important to note that this ointment contains 10-15% of GTPs, far
higher than any GTP-containing beverages or topical products. Therefore, this topical
ointment treatment for this skin lesion, with an interrupted stratum corneum, still requires a
physician’s prescription due to potential side effects. However, it represents the beginning of
an era in which green tea components can be used for therapeutic purposes. It also suggests
that the anti-microbial, especially the anti-viral, effects of GTPs need to be explored further.
Recently, results from various laboratories confirmed that GTPs or EGCG, the most abundant
green tea polyphenol, possesses potent inhibitory effects on influenza A and B viruses [33,
34], hepatitis B virus [35], herpes simplex virus (HSV) [36], Epstein-Barr virus [37],
adenovirus [38], and human immunodeficiency virus (HIV) [39, 40]. In 2008, Dr. Kaihatsu at
Osaka University and colleagues reported that EGCG-palmitate is 24 times more effective
than EGCG when compared for their anti-influenza activities [20]. Their findings suggest that
EGCG-palmitate (or other types of esters) are suitable candidates for anti-viral formulations.
Due to the stability and skin permeability of LTPs in topical formulations, it would only
require less than 1% w/v to deliver similar effects in comparison to 10-15% w/v of
Polyphenon E.
We reported previously that, as measured by the MTT cell viability assay, EGCG-
palmitate possesses anti-cancer activity similar to GTPs in the human ovarian cancer cell line
HO-8910 [24]. Here we show that LTPStearate at 0.1% w/v induced massive cell death in an
oral cancer cell line OSC2 (Figure 1). When the concentration of LTPStearate was decreased to
0.01% w/v, cell death was still clearly evident (Figure 2). In contrast, no cell death was
observed in NHEK under identical conditions (Figure 3). This result is consistent with our
previous reports that GTPs or EGCG selectively induce apoptosis in cancer cells but not in
normal cells [41-47]. The significance of this finding is that the absorption of LTPs, a
lipophilic complex, is different than that of GTPs, and it could therefore target internal
cancers, such as liver cancer, with higher bioavailability by oral administration. Although the
delivery route of LTPs into the human body is still not clear, it is postulated to be via the
chylomicron pathway. If so, LTPs would be associated with lipoprotein particles only, which
significantly reduces potential binding with serum proteins, but increases the level in
lipoproteins such as LDL prior to internalization by hepatocytes. That is, in comparison to
GTPs the anti-cancer and cardiovascular benefits of LTPs could be better.
LTPStearate, even at just 0.01% w/v, significantly induced the expression of caspase 14, a
gene product specifically associated with cell differentiation and barrier formation and that
causes cell death in cancer cells [48]. The effect of LTPStearate on caspase 14 expression is
comparable to that of EGCG (Figure 4), and the induction of caspase 14 by LTPStearate is dose-
dependent (Figure 5). These results indicate that the effects of LTPStearate on the expression of
caspase 14 in epidermal keratinocytes are consistent with the results generated from EGCG
[49-51]. However, due to its water-soluble nature EGCG is not bioavailable to human skin by
oral consumption [14, 15] or topical application at 50 µM (0.0023% w/v). The concentrations
to be delivered in formulations for effectiveness are documented at 10% w/v for EGCG [11]
and 10-15% w/v for GTPs [32], which could be very costly (EGCG costs $4000 to $9000 per
Kg) and very difficult to formulate. In contrast, bioavailability can be better achieved by
LTPs through topical application due to their lipid-soluble nature (<1% w/v). This notion was
tested in vivo using a glycerin-based formula (formulation not disclosed due to pending
patents) on a mouse model for psoriasiform lesions previously described [28]. The results
demonstrated that LTPStearate effectively induced caspase 14 and improved the skin condition
in this model (Figure 6), consistent with our previous observation using 0.5% w/v GTPs
(freshly prepared immediately before use) [28, 52]. Importantly, based on coloration tests,
LTPStearate is stable in various formulas for a minimum of two years (confidential data not
shown). Therefore, formulations with LTP for topical use provide a suitable delivery vehicle
due to the stability and permeability of LTP. The LTP-containing preparations could be used
for inflammatory diseases, wound-healing, scar-treatment [53], photo-protection, and UV-
induced damage. In addition, cosmetic products could take advantage of the properties of
EGCG-palmitate, which is not only stable but also without significant coloration or
precipitation in such formulations.
A significant finding was that when LTPs (LTPOleate and LTPStearate) entered NHEK cells,
EGCG was released into the cytoplasm. The retention time of EGCG and two types of LTP
was compared and showed little difference by HPLC (Table 1). The rate of EGCG release is
apparently dependent on the type of acyl group. Figure 7 shows that LTPOleate released most
of the EGCG at the 1 h time point, while LTPStearate released the largest amount of EGCG at 6
h. This result suggests that, when the carbon number is identical, an unsaturated fatty acyl
group may promote a faster transit through the cell membrane and/or more rapid hydrolysis
by lipase/esterase, while a saturated fatty acyl group may require a longer transit time prior to
enzymatic intracellular release of EGCG. This observation could help in future formulations
aimed at different release times.
In conclusion, in addition to the food and oil industries, the physical characteristics and
biological effects of LTPs enable both health care and cosmetic industries to produce stable
formulations with the full benefits of green tea polyphenols. Potential clinical uses of LTPs
(especially EGCG-esters such as EGCG-palmitate) in stable formulations could be treatment
of skin disorders associated with: neoplasm, psoriasis, cutaneous lupus, rosacea, actinic
keratosis, seborrhreic dermatitis; skin damage caused by trauma, infection, insect bites, and
scar formation; and oral lesions due to various causes. In the cosmetic field, the anti-oxidant
and repair effects of stable formulations of LTPs could be used to protect the skin from
environmentally-caused damage and aging, such as results from exposure to ultraviolet (UV)
and other types of radiation, pollution, or toxic agents. They could also be used to promote
the homeostasis of the skin to improve the appearance. LTP-containing products could also be
used for anti-microbial purposes such as anti-viral agents to prevent and inhibit the
infection/replication of influenza viruses, herpes simplex viruses, hepatitis viruses, human
immunodeficiency virus.
Clearly, investigation by scientists and clinicians is warranted to further understand the
working mechanisms of LTPs and their metabolism in humans, and to explore their potential
for oral administration to combat many diseases such as cancer, diabetes, cardiovascular
diseases, and obesity.
ACKNOWLEDGEMENT
The authors thank Professor Xian Qiang Yang (Tea Research Institute, Zhejiang
University, China), Professor Shuxiong Wang (retired, Zhejiang University, China), and Dr.
Yukihiko Hara (Japan) for their friendship, valuable advice, and pioneer work on tea
polyphenol chemistry; Dr. Hasan Mukhtar for his pioneer work on tea polyphenol biological
effects; Professor. Richard Eckert for his interesting work on the epidermal signaling by green
tea; Dr. Santosh Katiyar and Dr. Nihal Ahmad for their work on the cutaneous
photoprotection of green tea; Dr. Douglas Walsh (Walter Reed Army Medical Center, USA),
and Joseph Wood (US Army Eisenhower Medical Center, Fort Gordon, USA) for
collaborating on the flaky skin mouse study; Professor. Chung S. Yang and Dr. Mao-Jung
Lee (Rutgers University, USA) for their pioneer physiology and pharmacology work, and
technical advice on HPLC analysis; Professor Lihong Xu (Zhejiang University, China) for her
contribution to green tea’s protective effects against environmental toxins; Professor Tokio
Osaki (Former Vice President, Kochi University, Japan) and Professor Tetsuya Yamamoto
(Kochi University, Japan) for their friendship and collaboration on the effects of green tea
against oral cancer; Dr. Kunihiro Kaihatsu (Osaka University, Japan) for his informative
advice; and Dr. Jian Hua Zhong (Zhejiang University, China), Ms. Daniel Britt, Ms. Haiyan
Qin for their technical assistance. The authors thank people at CCA Industries, Inc., USA for
their vision and pioneer formulation of effective green tea topical products; The authors also
thank the Institute of Catalysts, the Tea Research Institute, and School of Medicine of
Zhejiang University, China; the United States Army; the US Department of Veteran Affairs;
and School of Dentistry, the Institute of Molecular Medicine & Genetics of Medical College
of Georgia, USA for their support.
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Drake L., Aiken AC., Huynh CT., Das B., Osaki T., and Schuster GS.
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Aiken AC., Huynh CT., and Schuster GS. Chemoprevention of Oral Cancer by Green
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Induction of p57 Is Required for Cell Survival When Exposed to Green Tea
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3-Dependent Apoptosis. Anticancer Research. 2002. 23:1533-1540
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Dickinson D, and Schuster G. Green Tea Polyphenol Causes Differential Oxidative
Environments in Tumor versus Normal Cells. J Pharmacol Exp Ther. 2003. 307:230-6.
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Oncology. 2004. 40:461-473.
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Koehler M and Schuster G. Expression of Caspase 14 Reduces Tumorigenicity of Skin
Cancer Cells. In Vivo. 2007. 21:279-83.
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Tea Polyphenols Induce Differentiation and Proliferation in Epidermal Keratinocytes.
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[50] Hsu S. Green Tea and the Skin. Journal of the American Academy of Dermatology.
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D, Foster E, Bollag WB, Lewis J, Wataha J, Osaki T and Schuster G. Green tea
polyphenol-induced epithelial cell terminal differentiation is associated with
coordinated expression of p57/KIP2 and caspase 14. J Pharmacol Exp Ther. 2005.
312:884-90.
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Chapter 4
ASSESSMENT OF THE ANTIOXIDANT CAPACITY OF

GREEN TEAS: A CRITICAL REVIEW
Camilo López-Alarcón1,* and Eduardo Lissi2

1
Departamento de Farmacia, Facultad de Química, Pontificia Universidad Católica
de Chile, Santiago, Chile
2
Facultad de Química y Biología, Universidad de Santiago de Chile
ABSTRACT
In the last decades, the beneficial influence of green tea on human health has been
related to the antioxidant capacity (AC) of its phenolic constituents. The latter has
originated systematic studies of the AC of green tea and/or its pure antioxidants.
Different methodologies have been used with this purpose. The methods are based on:
i) Estimation of the consumption by additives of stable free radicals (DPPH,

and ABTS radical cation).
ii) Evaluation of the protection given by antioxidants to a target being oxidized
by free radicals (ORAC, TOSC, LDL oxidation assay).
iii) Estimation of the steady state of free radicals before and after addition of
additives (TAR).
iv) Estimation of the reducing power capacity of the additives (FRAP,
CUPRAC).
The assays differ in the experimental conditions and their chemistry. Therefore,
different conclusions could be obtained depending on the methodology used. For
example, green tea presents a lower AC than peumus boldus by ORAC (oxygen radical
absorbance capacity) method when fluorescein is used as target molecule. However, if
pyrogallol red is used as probe, green tea appears with an ORAC index six times higher
than peumus boldus.
In the present review, we discuss the advantages, and disadvantages of the different
methodologies employed to evaluate the AC of green tea.
*
Correspondence to C. López-Alarcón; e-mail: clopezr@uc.cl
64 Camilo López-Alarcón and Eduardo Lissi
1. INTRODUCTION
Green tea, a product made -by a non fermentative process- from leaves of Camellia
sinensis, has been considered in traditional Chinese medicine, as a healthful beverage. In the
last decades, different beneficial effects on the human health have been attributed to green
tea, including prevention of cancer, heart disease, diabetes, and neurodegenerative diseases
[1-8]. These associations can be explained, at least partially, in terms of the presence in green
tea of free radical scavengers, such as polyphenols [9-13]. Polyphenols, particularly
flavonoids, constitute the most interesting group of green tea leaf components. The main
flavonoids present in green tea include catechins (flavan-3-ols) and their gallate esters. In
addition, green tea contains gallic acid and other phenolic acids, such as chlorogenic acid and
caffeic acid, and flavonols such as kaempferol, myricetin, and quercetin [12]. The high
polyphenolic concentration present in green tea gives to this beverage their well known
antioxidant properties. This antioxidant capacity has been established in many studies using
several in vitro methodologies. In the present work, we present and discuss data of the
antioxidant ability of green tea obtained by different assays. We present the bases of each
methodology, as well as their advantages and shortcomings. In addition, the influence of the
experimental procedures employed in green tea extraction is discussed.
2. ANTIOXIDANT CAPACITY ASSAYS AND GREEN TEA

The antioxidant capacity of green tea has been established by several in vitro
methodologies. The methods involve both different concepts and experimental conditions.
The most employed methodologies are based on the evaluation of: the scavenging of stable
free radicals by additives, the protection given by antioxidants to a target being oxidized by
free radicals, and the estimation of the reducing power capacity of the additives.
Scavenging of Stable Free Radicals by Additives
These methods are based on the bleaching induced by additives of stable free radicals
such as 2,2’-azinobis-(3-ethylbenzothiazoline-6-sulphonate) radical cation (ABTSy+) and 2,2-
diphenyl-1-picrylhydrazyl radical (DPPH).
Bleaching of ABTSy+ by Additives

This assay is based on the generation of a long-lived ABTSy+ cromophore from the
reaction of ABTS with an oxidant. ABTSy+ is a water soluble free radical, characterized by
visible absorption maxima at 414, 645, 734, and 815 nm. The addition of an antioxidant or a
complex mixture to solutions containing ABTSy+, generates a decay of the original visible
bands of ABTSy+, allowing an evaluation of the antioxidant capacity of the sample
(commonly is used the decay at 734 nm). The consumption of ABTSy+ given by an additive is
compared to that of Trolox (a hydrosoluble analogue of vitamin E), being the assay named
TEAC (Trolox equivalent antioxidant capacity). TEAC values are estimated using equations
(1) and (2) for pure compounds and beverages, respectively.
Assessment of the Antioxidant Capacity of Green Teas 65
TEAC =
[ΔUAadditive ] x
[Trolox ]
(1)
[ΔUATrolox ] [ Additive ]
TEAC =
[ΔUA beverage ] x f x [Trolox ] (2)
[ΔUATrolox ]
where:
ΔUAadditive = Difference between initial absorbance (UA0 minutes) and after 6 minutes of
additive addition (UA6 minutes).
ΔUATrolox = Difference between initial absorbance (UA0 minutes) and after 6 minutes of
Trolox addition (UA6 minutes).
[Trolox] = Trolox micromolar concentration.
[Additive] = Additive micromolar concentration.
ΔUAbeverage = Difference between initial absorbance (UA0 minutes) and after 6 minutes of
beverage addition (UA6 minutes).
f= dilution factor
TEAC assay, reported first by Miller et al.[14], was based on the formation of ABTSy+ by
the reaction of ABTS with metmyoglobin/ hydrogen peroxide. Some shortcomings have been
reported for this methodology [15], being at present ABTS oxidized through manganese
dioxide, peroxodisulfate or azocompounds (AAPH) [16,17].
The consumption of ABTSy+ by green tea has been studied by several research groups
[18-22]. Pellegrini and coworkers [18] reported that the TEAC value of green tea is 1.7 times
higher than black tea. The TEAC values of green and black tea were 6.0 and 3.6 mmoles of
Trolox equivalent per liter of solution, respectively. The higher antioxidant activity of green
tea was explained in terms of a partial oxidation along the manufacturing of black tea. In this
process, oxidative polymerization by polyphenols oxidase, convert catechins or other phenols
of green tea in oxidized products [18]. This relation is according to the well known
antioxidant capacity of catechins, epicatechins and their gallate esters when they are added as
pure compounds to a ABTSy+ solution [19]. The TEAC values reported by Pellegrini et al.(6.0
mM Trolox equivalents/L)[18] are close to the TEAC values reported by Rusak et al (4.59
mM Trolox equivalents/L) [20]. However, the report published by Rusak et al.[20] showed
that experimental procedures of green tea extraction are very important, and opposite
conclusions can obtained depending of the solvent used, and the time of extraction. For
example, using water as solvent extraction, green tea appears with a lower antioxidant
capacity (TEAC) than white tea after 5 minutes of extraction. Nevertheless, after 15 or 30
minutes of extraction, the TEAC value of green tea is nearly two times higher than that of
white tea. In addition, different TEAC values have been obtained employing others extraction
procedures such as soxhlet extractions using methanol or acetone as solvents [21].
Scavenging of DPPH by Additives

DPPH is a lipophilic free radical that has been widely used to test the capacity of
compounds, plant extracts and foods as free-radical scavengers [23]. DPPH exhibit a maxima
absorption band at 517 nm in alcoholic solutions. This band has been employed to follow the
bleaching of DPPH absorption induced by additives. The antioxidant capacity of the sample
generally is expressed as the inhibitory concentration (IC) necessary to reduce the absorbance
of DPPH by 50% (IC50), or as the percentage of the DPPH consumed at an arbitrary time.
Many studies have shown the consumption of DPPH radical induced by green tea [21,24-27].
Green tea has shown a higher ability to reduce DPPH radical than black tea [24,26,28].
Furthermore, using DPPH assay, green tea has been ranked among the most potent of 52
edible plant products [25]. Bastos et al.[27] reported that water and ethanolic extracts of green
tea have a comparable DPPH scavenging activity that the extracts of both green and roasted
yerba mate (llex paraguariensis). Nevertheless, the antioxidant activity of ether extracts of
green tea showed a higher ability than yerba mate (nearly three times). These results were
explained by the authors in terms of the compounds identified by electrospray insertion mass
spectrometry (EI-MS) in the ether extract of green tea, such as caffeolylglucose,
feruloylquinic acid, methylepicatechin gallate, and 3methyl epigallocatechingallate [27].
The interaction of green tea polyphenols with DPPH included in the bilayer of liposomes
has been studied by Chen et al.[29]. The consumption of DPPH was evaluated directly by
electron spin resonance (ESR) spectroscopy. The results evidenced that green tea polyphenols
react with DPPH in the bilayer of both saturated and unsaturated phospholipids. Therefore,
green tea polyphenols not only have an antioxidant activity in aqueous media, but also could
react with free radicals involved in lipidperoxidation process.
Evaluation of the Protection Given by Antioxidants to a Target being

Oxidized by Free Radicals
Among the methodologies most used in this category are ORAC (oxygen radical
absorbance capacity) and TRAP (total radical antioxidant potential) assays. Also, the ability
of green tea to delay the oxidation of low density lipoproteins (LDL) has been frequently
employed.
ORAC Methodology
ORAC (oxygen radical absorbance capacity) index has been frequently employed in the
evaluation of the antioxidant capacity of beverages [30-32]. This methodology measures the
protection afforded by an antioxidant to a target molecule that is being oxidized by peroxyl
radicals. Trolox and gallic acid have been used as antioxidant standards, and 2,2’-Azo-bis(2-
amidinopropane) dihydrochloride (AAPH) [33] has commonly been employed as peroxyl
radical source. Assuming a simple competitive oxidation of a target molecule (probe) and a
given antioxidant (XH) by a peroxyl radical (ROOy), a kinetic model must consider reactions
(3) to (6):
AAPH + O2 ⎯⎯→ 2ROO• + N2 (3)
ROO• + probe ⎯⎯→ bleaching (4)
ROO• + XH ⎯⎯→ X• + ROOH (5)

2ROO• ⎯⎯→ non radicals products (6)
and all the self-reactions and cross-reactions of the radicals produced in steps (4) and (5). In
this scheme, and for simplicity, we are not considering the formation of alcoxyl radicals in
reaction (6).
The protection afforded by antioxidants or beverages is estimated from the changes of
area under curve of the kinetics profiles after total or 80% target molecule consumption.
ORAC values are estimated according to equation (7) and (8) for pure compounds (additives)
and beverages, respectively.
ORAC =
( AUCad − AUC0 ) [s tan dard] (7)
( AUCs tan dard − AUC0 ) [additive]
( AUC − AUC0 )
ORAC = f [s tan dard] (8)
(AUCs tandard − AUC0 )
where:
AUC = Area under curve in presence of the tested beverage.

AUCad = Area under curve in presence of the additives.
AUCº = Area under curve for the control (target molecule plus AAPH solution).
AUCstandard =Area under curve for Trolox or gallic acid.
f= Dilution factor, equal to the ratio between the total volume of the working
solution (target molecule plus AAPH plus the sample aliquot) and the
added sample volume.
[standard] = Trolox or gallic acid concentration.
[additive] = Additive concentration.
ORAC assay was proposed originally by Cao, Alessio and Cutler (1993) using β-
phycoerythrin (ORAC-PE) as target molecule [34]. Throughout ORAC-PE assay, the
antioxidant capacity of many vegetables, fruits, and beverages was studied. Cao et al.[30]
found that both, green and black tea, have higher antioxidant activity than vegetables such as
garlic, kale, spinach and Brussels sprouts. Moreover, the ORAC-PE value of green tea
correlates with catechin derivatives content [35,36], and has been reported to be lower than
that of yerba mate (llex paraguariensis) [37].
Ou et al.[38], reported that the use of β-phycoerythrin as target molecule confer to
ORAC-PE method some limitations. β-phycoerythrin shows inconsistency from lot to lot, is
not photostable, and interacts with polyphenols [38]. Thus, Ou and coworkers [38], proposed
an ORAC methodology employing fluorescein (FL) as target molecule (ORAC-FL). At
present, FL is the target molecule most employed, and have allowed to estimate the ORAC-
FL index of a large number of phenols, polyphenols, and beverages. However, ORAC-FL for
single antioxidants and/or complex mixtures including very reactive compounds is estimated
generally from kinetics profiles showing a neat induction time (lag time). Furthermore, these
lag times have been related to a repair of FL from the secondary radical [39], according to:
FL• + POH → FLH + PO • (9)
1.0
1.0
F / F0
0.5
0.0
0 400 800 1200
A / A0
Time / s
0.5
0.0
0 600 1200 1800 2400 3000
Time / s
Figure 1. Consumption of pyrogallol red and fluorescein (insert) induced by peroxyl radicals in
presence of green tea infusion. Pyrogallol red (5µM) was incubated with AAPH (10 mM) at 37ºC in
phosphate buffer 75 mM (pH 7.4) in prescence of green tea infusion: 1.5 µL/mL (); 2.5 µL/ mL ();
5 µL/mL (U). control experiment (in absence of green tea, {). Insert: Kinetic profiles of fluorescein
(70 nM) consumption AAPH (10 mM) induced in absence (z), and presence of green tea infusion (0.1
µL/mL, T).
Independently of the origin of the induction times, their generation by additives leads to
ORAC-FL indexes more related with stoichiometric factors than the reactivity of antioxidants
towards AAPH derived peroxyl radicals [40]. Recently, we have proposed a modified ORAC-
like methodology that employs pyrogallol red (PGR) as target molecule (ORAC-PGR) [40].
PGR does not produce induction times, even with very reactive polyphenols, giving ORAC-
PGR values more related to the reactivity of the antioxidants towards peroxyl radicals [40-
42]. The protective effect of green tea extract on FL and PGR bleaching induced by peroxyl
radicals is shown in Figure 1. As can be seen in this figure, green tea inhibited the
consumption of FL and PGR with different protective kinetic profiles. Green tea inhibited the
consumption of FL throughout a clear induction time, while it protected PGR without an
observable lag time. Therefore, the origin of ORAC values obtained from area under curves
employing FL or PGR is different. Also, even relative ORAC values measured for different
infusions depend upon the employed methodology [43]. For example, ORAC-FL value of
Aloysia citriodora is larger than that of green tea, while if PGR is employed as target
molecule, green tea appears as nearly nine times more efficient. Therefore, extreme care must
be given to conclusions obtained from ORAC values estimated employing a single target
molecule.
ORAC assay has been applied to determine the antioxidant capacity of a variety of foods
[44,45]. In addition, results obtained in humans indicate that the daily consumption of fruits
and vegetables correlated with ORAC values of plasma [46]. These studies allowed to
develop a rank for foods and beverages in terms of their ORAC values [45]. Also, it has been
proposed that green tea could make a significant contribution to the required total daily
antioxidant capacity intake [31]. In addition, in base of ORAC values, a minimum
consumption per day of antioxidants has been recommended (5000 ORAC units).
The consumption of tea significantly increases the antioxidant capacity of the blood,
reducing the risk of oxidative damage to important macromolecules. However, many
questions are present related to the assays used to evaluate the antioxidant capacity and the
possible effect on the human health [11]. Furthermore, is necessary to develop oxidative
damage biomarkers that represent correctly the impact of the consumption of green tea in the
human health [47].
TRAP Methodology
TRAP is one of the most employed procedures to evaluate the antioxidant status of a
biological fluids or food samples. The method, proposed originally by Wayner et al. [48], is
based on measurements of induction times in the oxidation of a lipid dispersion exposed to a
free radical source with a constant and known rate of free radical production under aerobic
conditions. Usually, AAPH is used as peroxyl radical source, and the decrease in oxygen
concentration is used to measure the oxidation rate. In addition, a based luminol TRAP
method has been proposed as a good and simple alternative to estimate the index [49]. The
TRAP index of beverages is estimated according to equation (10):
t green tea
TRAP = f [Trolox] (10)
tTrolox
Where:
t = Induction time in presence of green tea (t green tea) or Trolox (t Trolox).
f = Dilution factor.
[Trolox] = Trolox concentration.
The high antioxidant activity of green tea through a TRAP type assay has been reported
in several works [50-53]. Bunkova et al. [50] reported a TRAP value of Chinese Gunpowder
and Japanese Sencha green tea of 39 and 38,4 mM Trolox equivalents / L, respectively. In
addition, using a TRAP index, an increase of the human plasma has been observed after
intake of green and black tea infusions [51-53].
LDL Oxidation
The peroxidation of low density lipoproteins (LDL) is considered to be a major initiating
event in atherogenesis [54]. Therefore, much effort has been devoted to the development of
standardized in vitro models for assessment of oxidation resistance of LDL. The LDL
oxidation -isolated from human blood samples- can, at least in vitro, be inhibited or delayed
by antioxidants, such as phenols or polyphenols [55]. Thus, in vitro oxidation of LDL has
been developed also as a model to assess the antioxidant capacity with a more physiologically
relevant system [56]. Commonly the oxidation of LDL is initiated by the addition of copper
sulfate or AAPH [57,58]. The oxidation of LDL can be easily followed by UV spectroscopy
following the increase in absorbance at 234 nm (ε = 29.500 M-1cm-1) due to the formation of
conjugated dienes [57]. The kinetic profiles of LDL oxidation are characterized by a lag time
during which protective endogenous anti-oxidants are consumed by initiating free radical
species. After the consumption of all endogenous anti-oxidants, a lipid radical-propagated
peroxidation chain reaction begins in which the polyunsaturated fatty acids contained in the
LDL are rapidly oxidized to lipid hydroperoxides. The addition of antioxidants or their
complex mixtures to a system containing LDL plus copper sulfate or AAPH, delays the
lipoperoxidation process of LDL. Therefore, the estimation of the lag time induced by the
antioxidants added is a measure of the protective effect of the additives. The lag time
generated by the antioxidants or beverages has been employed to evaluate the antioxidant
capacity. In addition, there are several parameters, additional to the lag time, which can be
obtained from dienes vs time profiles. For example, it is possible to evaluate the time required
to reach half maximum dienes (t ½), and the maximum velocity (v) of the lipid peroxidation,
given by the peak of the first derivative [57]. Besides, the maximum diene concentration
(diene max) can be estimated from the maximum increase of the absorbance at 234 nm [57].
Green and black teas efficiently delay the in vitro LDL oxidation process [25,59].
Nevertheless, the lag times observed in the oxidation of LDL in presence of both teas depend
of the LDL sample, the experimental procedure, and the brand of tea. In some experiments
green tea appears with higher antioxidant ability than black tea, but in others the antioxidant
capacity of green tea is similar or lower than black tea. For example, Richelle et al. [59]
reported that the induction time of LDL oxidation induced by copper or AAPH is in average
248 and 163 minutes in presence of green tea, and black tea (1µL/mL), respectively.
Furthermore, a great variability of the results was observed depending of the commercial
brand used in the experiment. Green tea values of different brands fluctuated between 186-
338 minutes, while black tea values laid between 67-277 minutes [59].
Copper sulfate is a initiator of LDL oxidation commonly used in LDL based assays
[25,59]. Therefore, the protective effect of green tea or their components could be related to
the capacity of the beverage to chelate copper ions. Miller et al. [19] reported that theaflavins
are able to chelate copper ions and to inhibit copper-induced LDL oxidation. Nevertheless,
the protective effect of green tea was observed at much lower concentrations than the copper
sulfate concentration used (5 µM), implying that the inhibition of LDL oxidation by green tea
is not only due to its chelating capacity [19]. These works show that LDL susceptibility to
oxidative modification is readily inhibited, at least in vitro, by green tea extracts. However, ex
vivo studies in healthy volunteers have shown a little or no inhibition of LDL oxidation [11].
Hodgson et al. [60] reported a greater lag time of LDL oxidation for both black and green tea
compared to water. However, these changes within a healthy cohort of 20 men were either
borderline (p= 0.05 for black tea) or not significant (p = 0.17 for green tea). Miura et al. [61]
detected an increase in lag time (p = 0.05) among 22 healthy young men after they consumed
green tea extracts equivalent to seven to eight cups a day for seven days. Interestingly, also
plasma β-carotene was higher (p < 0.01) in the tea group after the intervention. Recently,
Tinahones et al. [62] reported that the consumption of green tea extract by women for five
weeks produced modifications in vascular function and an important decrease in serum
oxidizability. Nevertheless, the consumption of green and black tea extracts equivalents to six
cups per day does not affect the susceptibility of LDL to oxidation ex vivo in smokers patients
[63]. The discrepancy between the effect of tea in vitro and ex vivo on the susceptibility of
LDL to oxidation may be due to the inability to achieve concentrations in vivo as great as
those obtained with the former methods [11]. Anyway, some reports have been published
showing an inverse correlation between catechin intake or green tea intake and coronary heart
disease mortality [64,65]. Furthermore, Peters et al. [66] have provided a metanalysis that
suggested a decrease in the risk of cardiovascular diseases with increasing green tea
consumption.
Estimation of the Reducing Power Capacity of the Additives
The methods based on the evaluation of the capacity of antioxidants or their complex
mixtures to reduce metals are named FRAP (ferric reducing antioxidant powder) and
CUPRAC (cupric reducing antioxidant powder).
FRAP Method
FRAP assay was originally developed by Benzie et al.[67] to measure the reducing power
of plasma. However, FRAP assay has been used widely to study the antioxidant capacity of
pure compounds, foods and beverages [68,69]. The reaction measures the reduction of ferric
2,4,6-tripyridyl-s-triazine (TPTZ) complex to a colored product induced by polyphenols at pH
3.6. The antioxidant capacity is expressed as generated Fe2+ at a fixed time (usually 6
minutes).
The FRAP value of green and black tea has been reported by several investigators
[20,70,71]. Benzie et al. [70] reported that the FRAP index of green tea is higher than that of
black tea. However, as was shown by Benzie et al. [70], high variations of the results between
different brands were observed. Interestingly, based on FRAP results it has been proposed
that a cup of green tea have a similar antioxidant capacity than 100-200 mg of ascorbic acid.
Then, a consumption of several cups of green tea per day would offer the same antioxidant
potential (FRAP) as almost 1 gram of vitamin C.
CUPRAC Assay
CUPRAC assay, proposed originally by Apak et al. [72], is based on the reduction of the
complex copper(II)-neocuproine to copper(I)-neocuproine by the combined action of all
antioxidants (reducing agents) present in the sample. The complex copper(I)-neocuproine has
a characteristic visible band at 450 nm, being the absorption band measured to estimate the
antioxidant capacity of the sample. Apak et al. [73] reported that the CUPRAC value of green
tea was among the higher values of several herbal infusions. Furthermore, they reported that
ascorbic acid increase the antioxidant capacity of green tea, being the CUPRAC value in
presence of lemon 1.7 times higher than that of green tea alone. In this context, similar results
of ascorbic acid contribution to TEAC values have been found by Majchrzak et al. [74].
Limitations
The beneficial effects of green tea on the human health have been established in several
studies [10,11]. Nevertheless, the published data of the in vitro scavenging activity of green
tea involve both different methodologies and experimental procedures. In addition, different
values have been reported depending on the commercial brand of the green tea used [70].
Therefore, a comprehensive understanding of the in vitro antioxidant capacity of green tea is
difficult and requires an exhaustive analysis of the methodology used and the experimental
condition employed in each work. These aspects are stressed in the data of Table 1. As can be
seen in Table 1, the antioxidant activity of green tea depends on the methodology used and
the procedure of the extraction. Furthermore, within a single assay, the values are dependent
of the green tea brand, i.e. in Table 1 using ORAC-PE, LDL, and FRAP assays high
fluctuations of the antioxidant capacity values have been published for different green tea
brands. In addition, the results obtained using a particular method not always are expressed in
the same units, making difficult the comparison of results. For example, employing DPPH
test, the results have been reported in terms of IC50, DPPH consumed in percentage or in
base of epigallo-catechin-gallate (EGCG) equivalents. In addition, in the analysis of the state
of the art, it is necessary to taken into account that the methodologies provide different
information. In particular, it must be considered that a given index can be an indication of the
total amount of antioxidants (without a discrimination regarding their reactivity, TEAC,
DPPH, TRAP, ORAC-FL, FRAP), or be an indication of the quantity and reactivity of the
antioxidants present in green tea sample (ORAC-PGR). Moreover, it should be considered
that each antioxidant capability assay has their own advantages and limitations.
The simplicity to carry out procedures based on the bleaching of ABTSy+ and DPPH, is
one of the most important advantage of these assays. However, the free radicals involved
(ABTSy+ and DPPH) are very far from those free radicals relevant in oxidative stress
situations. The analysis of the kinetics obtained by ABTSy+ assay is complex [75-77] and can
be computed as antioxidant compounds that are pro-oxidant in biological systems, such as
hydrogen peroxide or organic hydroperoxides [78]. Furthermore, when the consumption of
ABTSy+ is measured at a single (long) reaction time, the result obtained is related to
stoichiometric factors and gives no information regarding the reactivity of the tested
compounds. The interpretation of data obtained by employing DPPH as the stable free radical
is less straightforward. When applied to a fixed time at a pure compound, the method
provides stoichiometric factors for highly reactive compounds and/or mixtures, and provides
reactivity and stoichiometric factors for low reactivity compounds [79,80].
Table 1. Antioxidant capacity values of green tea evaluated by different methodologies
Method Value Units Experimental conditions Reference

TEAC 6.0 mM Trolox eq /L Boiling water (5 min) [18]
4.59 mM Trolox eq /L Boiling water (5 min) [20]
6.85 mM Trolox eq /L Ethanol 70% (5min) [20]
8.0 mM Trolox eq /g Methanol (soxhlet) [21]
1.8 mM Trolox eq /g Boiling water (5 min) [22]
DPPH ≈ 25 DPPH consumed (%)a Acetone (70%) [21]
≈ 70 DPPH consumed (%)a Methanol [21]
4.14 µg/mL (IC50)b Boiling water (1 hour) [24]
194.6 µmol EGCG/g (IC50) Ethanol 70% (v/v) [25]
59.4 DPPH consumed (%)c Boiling water (5 min) [26]
88.4 DPPH consumed (%)d Water (soxhlet) [27]
92.2 DPPH consumed (%)d Ethanol (soxhlet) [27]
80 DPPH consumed (%)e Boiling water (1 h, reflux) [28]
ORAC-PE 814 µmol Trolox eq /g Boiling water (30 min) [30]
1239-1686 µmol Trolox eq /g Boiling water (3 min) [35]
1345.9 µmol Trolox eq /g Boiling water (10 min) [37]
ORAC-FL 12.3 mM Trolox eq /L Boiling water (5 min) [43]
ORAC-PGR 19.1 mM Trolox eq /L Boiling water (5 min) [43]
TRAP 38.35 mmol Trolox eq /L Boiling water (10 min) [51]
LDL 191.4 µmol EGCG/g Ethanol 70% (v/v) [25]
186-338 Minutes (lag time)f Boiling water (5 min) [60]
FRAP 272-1144 µmol /g Boiling water [71]
CUPRAC 0.94 mmol Trolox/g Boiling water (5 min) [74]
a
Estimated using a DPPH concentration of 0.1mM, and a concentration of dry green tea extract of 25
µg. b DPPH concentration = 60 µM. c DPPH concentration = 0.2 mM, and a concentration of dry
green tea extract = 0.4 mg/mL. d DPPH concentration = 20 mg/mL, and a concentration of green
tea extract = 250 µL/mL. c DPPH concentration = 0.25 mM, and a concentration of dry green tea
extract = 50 µg/mL.f Lag times obtained with a LDL concentration of 80 µg of cholesterol/mL, and
a green tea infusion concentration of 1 µL/mL.
On the other hand, procedures based on the evaluation of the protection given by
antioxidants to a target molecule being oxidized by free radicals provide information related
to stoichiometric factors and/or reactivity. The TRAP assay is based on the estimation of lag
times. Therefore, the index derived from this procedure is only determined by stoichiometric
factors. In fact, it only provides an indication of the number of radicals trapped per each
antioxidant molecule introduced into the system [81]. The ORAC methodology is a procedure
based on the estimation of the area under curve of the protective kinetic profiles. Therefore,
the index is related with the origin of the area under curve. Thus, if the target molecule is
protected totally by the antioxidants (ORAC-FL), the area under the curve and, in
consequence, the ORAC-index is mostly governed by the stoichiometry of the reaction.
However, if the area under curve is related only with a decrease in the initial rate of the target
molecule consumption (ORAC-PGR), the ORAC index would reflex the reactivity of the
additives. Similarly, protection of LDL is a complex system, and is necessary to consider the
initiator of the LDL oxidation for the analysis of the results. If copper is used, it must be
considered the possible chelating effect of the additives. If AAPH is employed as initiator, the
delay of the LDL peroxidation chain could be determined by the interaction of the
antioxidants with the primary radicals (peroxyl radicals) or with a chain-breaking antioxidant
capacity. On the other hand, a disadvantage of LDL assay is that the LDL particles are very
different depending of the subject, or even of the diet. Then, the reproducibility, and the
comparison of results is not always warranted.
Ferric and cupric reducing power assays (FRAP and CUPRAC) have the limitation that
they are only titrating all the antioxidants and those molecules with reducing power towards
ferric or cupric complexes. In addition, a shortcoming of FRAP methodology is that the pH
used is very different to the physiological value.
Regarding the validity of these indexes as indicators of the quality of a beverage and/or
as useful indicators of pathological situations, there are two questions:
• Is possible to estimate the antioxidant capacity of green tea employing only one
assay? and
• Is there any relationship between the measured index in a food or beverage and the
impact it has on the antioxidant capacity status of the organism?
As expected, there is not a clear-cut yes-or-not answer to these broad questions and it
strongly depends on the system considered. For example, using a DPPH-based method [24] it
was found that green tea was 6.5 times more powerful as an antioxidant capacity than black
tea. However, in preventing the lipid peroxidation of renal homogenates induced by hydrogen
peroxide and Fe(II), green tea was only 1.5 time more efficient. Evidently, other factors, such
as the distribution and metal chelating characteristics, preclude a quantitative relationship
between the amount of antioxidants present in a food or beverage and their biological effect.
In in vivo evaluations, the relationship between ingest and observed levels is still more
indirect, since factors such as absorption at the gut level and metabolization can be
completely different for the different antioxidants present in the samples.
Considering the aspects above discussed should be recommendable that for the
evaluation of the in vitro antioxidant capacity of foods or beverages should be used similar
experimental conditions and as many assays as possible. Recently, Seeram et al. [82] have
studied the antioxidant capacity of beverages commonly consumed in United States.
Interestingly, the study was developed considering five in vitro antioxidant capacity
measuring methodologies (DPPH, ORAC-FL, FRAP, TEAC, and LDL) and different green
tea brands. This allows obtaining a more complete profile of the antioxidant capacity, and to
correlate the results of different samples. Depending of the assay and the brand, iced green tea
showed higher or similar antioxidant ability than iced black and white teas.
3. CONCLUSION
Green tea antioxidant potential has been measured by a large number of methodologies.
All studies point to a high charge of antioxidants, mostly phenolic compounds. Comparison
with other infusions, particularly black tea, is difficult since the reported values are strongly
influenced by the employed methodology. The data discussed in the present review
emphasized two points: i) ranking of infusions or beverages can be proposed only when the
same methodology is employed, both the same extraction procedure and the assay employed
to evaluate the antioxidant capacity, and ii) to assess the antioxidant capacity of a infusion,
several methodologies should be considered, taking into account the factors that condition the
measured index (either the amount of antioxidants and/or their reactivity in the free radical
scavenging processes).
ACKNOWLEDGMENTS
This work was supported by FONDECYT n°11060323 and 1070285.
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Chapter 5
DESIGN AND ASSESSMENT OF THE IN VITRO ANTI-

OXIDANT CAPACITY OF A BEVERAGE COMPOSED OF
GREEN TEA (CAMELLIA SINENSIS L.) AND
LEMONGRASS (CYMBOPOGON CITRATUS STAP)
D. Fernando Ramos Escudero1, Luis Alberto Condezo Hoyos2,

Mónica Ramos Escudero3 and Jaime A. Yáñez4,*
1
Centro de Investigación de Bioquímica y Nutrición, Facultad de Medicina Humana,
Universidad de San Martín de Porres, Lima, Perú
2
Postdoctoral Research, Facultad de Medicina, Universidad Autónoma de Madrid,
Madrid, Spain
3
Escuela de Postgrado en Agricultura Sostenible, Universidad Nacional Agraria de la
Selva, Tingo María, Perú
4
College of Pharmacy, Department of Pharmaceutical Sciences, and Pharmacology and
Toxicology Graduate Program, Washington State University, Pullman, WA, USA
ABSTRACT
Tea is one of the most popular and widely consumed beverages in the world and it is
derived from the infusion of tea leaves (Camellia sinensis L.). Different commercial
types of tea are available, including black tea, oolong tea (semi-fermented) and green tea,
which differ on their processing and chemical composition. All these types of tea have
been reported to prevent multiple diseases such as cancer, heart conditions, among others.
On the other hand, lemongrass (Cymbopogon citratus Stap) is a rich source of essential
oils, widely employed in infusions, soaps, and perfumes, and it has been reported to
possess gastrointestinal and analgesic properties. In the present study, green tea
(Camellia sinensis L.) and lemongrass (Cymbopogon citratus Stap) leaves were collected
*
Correspondence to: Dr. Jaime A. Yáñez, Ph.D. Schering-Plough Research Institute, Pharmaceutical Sciences and
Drug Metabolism, 2015 Galloping Road, Mail Stop K15-3 3700, Kenilworth, NJ 07033, USA. Email:
jaimeayanez@gmail.com
82 D. F. Ramos Escudero, L. A. Condezo Hoyos, M. Ramos Escudero et al.
from Río Azul and Porvenir de Marona, Perú. The anti-oxidant capacity of green tea and
lemongrass extracts was evaluated using the DPPH method and it was observed that the
IC50 values for green tea was 32.4 ± 0.39 μg/mL and 1350 ± 47 μg/mL for lemongrass.
These two plants (green tea and lemongrass) were employed to design multiple infused
beverages and it was determined that an infused beverage containing 10 mg/mL total
extract (50% green tea and 50% lemongrass) reported a total catechin content of 24.4 ±
0.65 mg/100mL, a DPPH inhibition percentage of 88.6%, and exhibited the greatest
acceptance for sensory attributes such as flavor, color, and aroma (values of 6.8, 9.0, and
8.0 respectively) based on Friedman Multiple Comparisons test. The taste panel results
also indicated that the optimized acidity and sweetness were to be set at pH 3.1 and
11°Brix, while the optimum infusion time based on the total catechin content was 7
minutes. The pasteurization profile at 90°C for 5 minutes achieved mesophilic
microorganisms counts of <10 cfu/mL. The maximum shelf-life of the beverage was
achieved at 15ºC based on total catechin content and absence of browning (catechin
degradation). Furthermore, the formulated beverage was well accepted by the test panel
and presented similar anti-oxidant capacity than commercial green tea based beverages.
Keywords: Green tea, lemongrass, beverage, catechin, anti-oxidant capacity, 1,1-diphenyl-2-

picrylhydrazyl (DPPH).
INTRODUCTION
Tea is one of the most popular drinks in the world and it is recognized for its high
polyphenol content. It is derived from the infusion of tea leaves with or without a
fermentation process [1]. The fresh tea leaves are rich in catechins (a polyphenol sub-class),
which can total up to 30% of the leaves dry weight [2]. Other polyphenols include flavanols
and their glycosides, chlorogenic acid, coumarylquinic acid, theogallin (3-galloylquinic acid).
Caffeine is present on an average of 3% along very small quantities of the methylxanthines:
theobromine and theophylline and the aminoacid: theanine (N-ethyl L-glutamine) [2]. Green
tea differs from black tea in the susceptibility of the enzymes that participate in the
fermentation process (i.e. polyphenol oxidase). These enzymes are generally deactivated
during the steaming process, and independently of the steaming and/or fermentation process
green tea usually contains higher vitamin content than its counterpart (black tea) [3].
It is well recognized that the tea leaves are catechin-rich and that present a polyphenol
content between 20 to 35% based on dry matter [4]. The catechin content in green tea leaves
vary based on its origin, aging, and treatment [5]. Fresh tea leaves contain a high polyphenol
content, especially in the following catechins: (+)-catechin (C), (+)-gallocatechin (GC), (-)-
epicatechin (EC), (-)-epicatechin gallate (ECG), (-)-epigallocatechin (EGC) and (-)-
epigallocatechin gallate (EGCG) (Figure 1) [1]. While, a green tea cup (2.5 g green tea in 200
mL water), usually contains about 90 mg of (-)-epigallocatechin gallate (EGCG), about 65 mg
of (-)-epigallocatechin (EGC), and approximately 20 mg of both (-)-epicatechin gallate (ECG)
and (-)-epicatechin (EC) [6].
Design and Assessment of the in Vitro Anti-oxidant Capacity … 83
Figure 1. Structure of the main polyphenols in green tea.
The consumption of tea has been related with anti-pyretic and diuretic activities, and
multiple reports indicate that consuming significant amounts of green tea reduce the risk of
developing multiple forms of cancer [7]. Currently, multiple epidemiological and
pharmacological studies report that the components of green tea have anti-oxidant capacity,
hypocholesterolemic effect, anti-mutagenic, anti-carcinogenic, and anti-tumoral activity [3, 8,
9]. For instance, the anti-mutagenic activity of the green tea catechins has been well
correlated with their capacity to scavenge free radicals such as HO· y ROO· [9]. Furthermore,
green tea (Camellia sinensis L.) catechins have also been reported by in vitro and in vivo
studies to have anti-oxidant capacity, free radical scavenging activity [10], anti-neoplastic
properties [11], capacity to inhibit platelet aggregation [12], and anti-bacterial properties [13].
The consumption of commercial beverages based on green tea has a great demand around
the world, and most of the time these beverages remain for long time in the commercial
shelves before reaching to the final customer. However, it has been reported that the total
catechin content in tea beverages gets significantly reduced during the storage due to
progressive catechin degradation or epimerization, a phenomenom called browning [14]. For
instance, the total catechin content in fresh green tea infusions has been reported to range
between 0.9 to 75.0 mg catechin/100 mL, while the total catechin content in commercially
available green tea derived beverages range between 0.3 to 35.0 mg catechin/100 mL [14].
However, these differences in total catechin content cannot only be attributed to the
progressive catechin degradation (browning) but to the different processing treatments and/or
to the multiple other ingredients (such as ginseng, honey, and jasmine) added to the
commercial green tea derived-beverages [14].
Lemongrass (Cymbopogon citratus Stap) is a perennial grass that presents multiple
species such as: Cymbopogon citratus, Cymbopogon nardus, Cymbopogon flexuosus and
Cymbopogon martini [15], that contain characteristic aromas especially in the leaves.
Furthermore, lemongrass is a rich source of high-quality essential oils that are used in
multiple commercial beverage preparation [16]. Additionally, multiple compounds such as
monoterpenes: citronelol, geraniol, and linaldol, triterpenes, sesquiterpenes, and β-sitosterol
have been identified in lemongrass extracts [17]. Nevertheless, lemongrass is employed as a
therapeutic agent used in different countries; for instance, in the Peruvian jungle lemongrass
infusions are utilized to treat different digestive problems and as an anti-neuralgic agent [17].
Because of the attractive phytochemical content and well documented pharmacological
attributes of green tean and lemongrass, a functional beverage was designed using these two
plant species. The technological criteria to design, formulate, and assess the functional
beverage in this study include: (a) to determine the anti-oxidant capacity [inhibition of the
1,1-diphenyl-2-picrylhydrazyl (DPPH) radical] of both plant species (green tea and
lemongrass), (b) to design a beverage derived from green tea and lemongrass, considering the
ratio between green tea:lemongrass, infusion time, total catechin content, pasteurization,
browning (catechin degradation products) and anti-oxidant capacity, and (c) to assess the
stability (total catechin content, microbiological activity, and anti-oxidant capacity) of the
final beverage formulation.
MATERIALS AND METHODS
Plants
The fresh tea leaves were collected from the plantations of the Empresa Jardines del Té
S.A. (Río Azul, Pucallpa, Perú), while the lemongrass leaves were collected from the
Porvenir de Marona area (Pumahuasi, Tingo María, Perú). The tea leaves were processed
according to the previously described methodology by Varnam and Sutherland [1]; briefly,
the tea leaves were blanched at 100°C for 60 seconds to inactivate degrading enzymes
followed by air-drying at room temperature for 2 hours. Then, the leaves were hot-air dried at
100°C for 50 min and ground to 1 mm diameter particles. Finally, the ground leaves were
hot-air dried at 60°C for 40 min to achieve a final moisture between 6 to 9% [1].
The fresh lemongrass leaves were quickly washed with water, hot-air dried at 100°C for
40 min, ground to 1 mm diameter particles, and finally the ground leaves were hot-air dried at
50°C for 6 hours [18]. The final ground leaves of green tea and lemongrass were stored in
polyethylene bags and placed in a desiccator at room temperature (25 ± 2°C) to maintain the
desired moisture (6-9%).
Experimental Design
The experimental design for the functional beverage derived from green tea and
lemongrass is represented in Figure 2. The beverage design involved different stages: a)
formulation based on the following proportions of green tea: 100, 75 and 50% and the
difference of lemongrass, and a final extract concentration (green tea:lemongrass) of 5.0, 7.5
and 10.0 mg/mL. b) The two proposed acids to be used in the beverage were citric acid or
ascorbic acid. The adequate acidity and sweetness of the beverage was determined by
assessing the pH and °Brix, respectively. The assessed sweetness levels of the beverage were
7, 9 and 11°Brix, while the acidity levels studied were pH 3.1, 3.3 and 3.5. The adequate
flavor, color and aroma of the beverage were assessed by sensory evaluation. The adequate
infusion time was determined by total catechin content analysis and sensory evaluation of
color. The assessed infusion times were 3, 5 and 7 minutes. c) The adequate pasteurization
conditions (temperature/time) were assessed by microbiological analysis, anti-oxidant
capacity, and total catechin content analysis. The pasteurization conditions studied were 85,
90 and 95°C, and 5, 10 and 15 minutes. d) The effect of the light on inducing browning in the
beverage was assessed after storage for 16 days at room temperature. The first group of
bottles was stored for 16 days in light-protected bottles placed in darkness, while the second
group of bottles was stored in clear bottles exposed to regular light. e) The stability of the
beverage at different temperatures and time periods was assessed by total catechin content
analysis, browning, anti-oxidant activity, and microbiological analysis. The assessed storage
temperatures were 15, 25 and 35°C, and the time periods were 0, 4, 8, 12 and 16 days. The
adequate stability was assessed based on the degree of browning (catechin degradation),
microbiological analysis, and anti-oxidant capacity during the storage temperature/time
combination.
Commercial Green Tea Based Beverages
Commercial green tea based beverages were purchased from a local grocery and were
employed to compare their anti-oxidant capacity with our proposed beverage based in green
tea and lemongrass. The commercial beverages contain other ingredients; for instance
Arizona Green Tea contains green tea, filtered water, high fructose corn syrup, honey, citric
acid, natural flavors, ginseng extract and vitamin C. Beberash Iced Tea has as ingredients:
purified water, black tea extracts, green tea extracts, sugar, citric acid and vitamin C. Snapple
Iced Tea contains water, high fructose corn syrup, natural lime flavor with other natural
flavors, citric acid, green tea and ascorbic acid.
Figure 2. Stages of the design and evaluation of the beverage derived from green tea and lemongrass.
DPPH Radical Scavenging Capacity
The previously described method by Brand-Williams et al. [19] was employed. Briefly,
the green tea and lemongrass samples were extracted with 95% ethanol for 10 minutes under
constant agitation achieving a final stock solution of 100 mg/mL. The reaction was performed
using 50 µL of sample and 950 µL of DPPH (100 µM), and the absorbance reading at 515 nm
was performed after 1 minute [19]. The assessed extract concentrations were 3, 10, 30, and
100 μg/mL for green tea and 10, 100, 300, and 3000 μg/mL for lemongrass. Based on the
results, the necessary concentration of extract (green tea or lemongrass) needed to inhibit the
DPPH radical by 50% (IC50) was determined by model fitting using the pharmacokinetic
software WinNonlin (Ver. 5.1) (Pharsight Corporation Mountain View, CA). Catechin and
quercetin were used as standards.
The anti-oxidant capacity of the beverage formulations was determined by allowing the
reaction between 50 µL of standard or sample and 950 µL of DPPH (100 µM) for 1 minute
[19]. The results are presented in three forms: the anti-oxidant capacity (AC) represents the
DPPH radical inhibition percentage; the anti-oxidant value (AOX) represents the final
absorbance measured after the 1 minute reaction [20]; and the vitamin C equivalent anti-
oxidant capacity (VCEAC) was calculated based on a calibration curve of DPPH and vitamin
C [21].
Total Catechin Content Analysis
The content of total catechin was determined by the spectrophotometric method

described by Singh et al. [22] using the synthesized diazotized sulfanilamide. The total
catechin content analysis was determined by allowing the reaction between 1000 μL of
standard or sample and 200 μL diazotized sulfanilamide (1% w/v in acetone) and 200 μL
hydrochloric acid (30% v/v) for 1 hour at room temperature. Then, 3.6 mL of water was
added and the absorbance values were recorded at 425 nm.
Browning Reaction
The methodology to assess the browning reaction was assessed by measuring the total
catechin degradation/epimerization products by absorbance at 440 nm following the
methodology described by Bradshaw et al. [23].
Microbiological Analysis
The microbiological analysis was performed as described previously [24]. Briefly, a

sample (10 mL) was homogenized with 90 mL of sterile peptone water (0.1%) by agitation.
The samples were diluted (10-1, 10-2, 10-3) by diluting 1 mL sample with 9 mL of peptone
water, then an aliquot (1 mL) was plated in duplicate by using deep half plate count agar, and
the plates were incubated at 35 to 37°C for 48 h. Then, the colony forming units (cfu) were
counted using a colony counter and expressed as cfu/mL after correcting for the dilution
factor.
Sensory Evaluation
Representative samples of the different formulations were evaluated by the Friedman

Multiple Comparisons test described previously [25]. The sensory attributes of flavor, color,
aroma, acidity, and sweetness were assessed by 9 panelists previously trained. The panelists
were students of the quality control course of the Departamento de Ingeniería Alimentaria of
the Universidad Nacional Agraria de la Selva, Tingo María, Perú. The samples
(approximately 15 mL) were placed in disposable cups and coded with the symbols: π, θ, μ, λ,
β, α, φ, ω, η. The panelists were asked to rank order from lowest to highest based on
preference.
Statistical Analysis
Compiled data were presented as mean and standard error of the mean (mean ± SEM).
General Linear Model (GLM) Analysis of Variance (ANOVA) with Duncan’s multiple range
test with a p-value < 0.05 been statistically significant (Statistical Analysis System – SAS,
Institute Cary, NC).
RESULTS AND DISCUSSION
Anti-Oxidant Capacity of Green Tea and Lemongrass
The free radical scavenging capacity of green tea and lemongrass was assessed by the
stable DPPH radical assay. This assay reported that the green tea extracts (Figure 3A)
exhibited higher anti-oxidant capacity than the lemongrass extracts (Figure 3B) at all the
concentrations tested, exhibiting a concentration-dependent inhibitory capacity as previously
described with other compounds and extracts [19, 26].
Table 1 reports the DDPH radical IC50 values of green tea and lemongrass extracts
compared to the standards catechin and quercetin. It can be observed that the green tea extract
(IC50 = 32.4 μg/mL) is approximately 40 times more potent than the lemongrass extract (IC50
= 1350 μg/mL), while the controls (quercetin and catechin) report a more potent anti-oxidant
capacity (IC50 values of 3.35 and 4.83 μg/mL, respectively) than the green tea extract.
Previous reports indicated that the IC50 values for freeze-dried green tea, oolong tea, and
black tea samples were 4.14, 27.02 and 47.12 μg/mL, respectively [27]. The variability in
IC50 values between our study and previous reports can be attributed to differences in anti-
oxidant capacity methodology, geographical location of the green tea samples, harvesting
time, and processing/treatment of the tea as previously reported [14, 22].
Figure 3. DPPH radical scavenging capacity of the green tea (A) and lemongrass (B) extracts at
different concentrations. Data expressed as mean ± SEM, n = 3.
Table 1. DPPH radical IC50 values of green tea and lemongrass extracts compared to the
controls catechin and quercetin
Sample IC50 (µg/mL)

Green tea extract 32.4 ± 0.38a
Lemongrass extract 1350 ± 47b
Quercetin 3.35 ± 0.09c
Catechin 4.83 ± 0.13d
Data expressed as mean ± SEM, n = 3. Different letters indicate statistically significant samples
(p<0.05) based on the Duncan’s multiple range test. The DPPH radical scavenging capacity was
measured after 1 minute reaction.
It can be observed that green tea had a higher anti-oxidant capacity than lemongrass,
which might be due to the higher content of anti-oxidant compounds such as flavonoids [28]
and the flavonoid sub-class: flavanols also known as green tea catechins [29]. The
predominant green tea catechins are: (-)-epicatechin, (-)-epicatechin gallate, (-)-
epigallocatechin and (-)-epigallocatechin gallate [30].
Formulation of the Beverage Based in Green Tea and Lemongrass
The formulation of the beverage included the following stages: to determine the adequate
ratio green tea:lemongrass based on the total catechin content, DPPH radical scavenging
capacity, and sensory analysis based on flavor, color and aroma. Table 2 reports the total
catechin content of the beverages with different green tea:lemongrass proportions and total
extract concentration.
It can be observed in Table 2 that the formulations SL3P1, SL3P2 and SL3P3 exhibit a
lower total catechin content as the green tea proportion decreases, which correlates with
previous reports that indicate that the total catechin content increases proportionally with the
amount of black tea infused in 200 mL water [31].
Table 2. Total catechin content of the different beverage formulations
Formulation Total extract Proportion Total catechin

concentration Green tea (%) Lemongrass (%) content (mg/100mL)
(mg/mL)
SL3P1 10.0 100 0 67.5 ± 1.54a
SL2P1 7.50 100 0 50.2 ± 0.96b
SL3P2 10.0 75 25 47.2 ± 1.87c
SL2P2 7.50 75 25 38.0 ± 1.30d
SL1P1 5.0 100 0 34.0 ± 0.77e
SL3P3 10.0 50 50 24.4 ± 0.65f
SL1P2 5.0 75 25 23.5 ± 0.97f
SL2P3 7.50 50 50 18.2 ± 1.08g
SL1P3 5.0 50 50 12.4 ± 2.08h
(p<0.05) based on the Duncan’s multiple range test.
Table 3. Anti-oxidant capacity of the different green tea based formulations and
commercially available green tea based beverages
Formulation Total extract Proportion AC1 AOX2 VCEAC3

concentration Green tea Lemongrass (%) (A*min-1) (µmol/L)
(mg/mL) (%) (%)
SL3P1 10.0 100 0 91.97a 0.076a 37.19a
SL2P1 7.50 100 0 91.76a 0.078a 37.12a
SL3P2 10.0 75 25 90.49a 0.090a 36.63a
SL3P3 10.0 50 50 88.59b 0.108b 35.86b
SL2P2 7.50 75 25 82.25c 0.168c 33.28c
SL1P1 5.0 100 0 82.14c 0.169c 32.25c
SL2P3 7.50 50 50 79.08d 0.198d 32.03d
SL1P2 5.0 75 25 72.00e 0.265e 29.17e
SL1P3 5.0 50 50 64.50f 0.336f 26.14f
Arizona Gree NA NA NA 60.55g 0.386g 24.57g
tea
Beberash NA NA NA 91.88b 0.101b 36.30b
Iced Tea
Snapple Iced NA NA NA 89.72a 0.080a 37.17a
Tea
1
AC: anti-oxidant capacity, which represents the DPPH radical inhibition percentage.
2
AOX: anti-oxidant value, which represents the final absorbance measured after the 1 minute reaction.
3
VCEAC: vitamin C equivalent anti-oxidant capacity, which was calculated based on a calibration
curve of DPPH and vitamin C.
Table 4. Sensory analysis based on flavor, color and aroma evaluated by the Friedman
Multiple Comparisons test
Formulation Total extract Proportion Evaluation parameter

concentration Green tea Lemongrass Flavor Color Aroma
(mg/mL) (%) (%)
SL3P3 10.0 50 50 6.78a 9.00a 8.00a
SL3P2 10.0 75 25 5.78ª,b 7.44b 6.78b
SL2P3 7.5 50 50 5.44b,c 7.11b 5.89b,c
SL1P3 5.0 50 50 5.33b,c 6.22c 4.33e
SL1P2 5.0 75 25 5.22b,c 4.56d 2.11f
SL3P1 10.0 100 0 4.67b,c 4.00e 5.33c,d
SL2P1 7.50 100 0 4.47b,c 2.78f 5.33d,e
SL2P2 7.50 75 25 4.33c 2.33g 4.78e
SL1P1 5.0 100 0 2.78d 1.56h 2.44f
(p<0.05).
It can be observed in Table 3 that our green tea based formulations present similar anti-
oxidant capacity than the commercially available beverages. Table 4 reports the sensory
analysis based on flavor, color and aroma evaluated by the Friedman Multiple Comparisons
test, and it can be observed that the formulations SL3P3 and SL3P2 were statistically more
accepted by the panelists and numerically the SL3P3 formulation was superior than the
SL3P2 formulation.
Based on these results, it was determined that the formulation SL3P3 (10 mg/mL; 50%
green tea and 50% lemongrass) exhibited the greatest acceptance by the sensory analysis
panel (Table 4). Thus, this was the formulation selected for the next stage. The total extract
concentration (10 mg/mL) of green tea:lemongrass correlates with the report by Arts et al.
[31], that determined that a concentration of 10 mg/mL of tea presents an adequate total
catechin content.
Beverage Acid Selection
The selection of the acid was based on its potential in preventing browing as shown in
Figure 4. It can be observed that even though ascorbic acid prevents browning better than
citric acid up to day 3, citric acid has a higher anti-browning effect throughout one week. Our
results indicate that citric acid is more effective in preventing browning and that ascorbic acid
acts initially as an anti-oxidant, but after day 4 it appears to act as a pro-oxidant. This was
also observed previously by Podmore et al. [32] and Herbert et al. [33] that reported that the
presence of ascorbic acid as an ingredient tends to act as an anti-oxidant initially and as a pro-
oxidant in the long-term. Furthermore, Chen et al. [34] reported that ascorbic acid has a
protective effect in the short-term, and that citric acid is the ingredient of choice for long-term
storage of beverages.
Catechins in general are degraded or epimerized in neutral or slightly basic environments;
thus, it is critical to use an acid to stabilize catechins and prevent their degradation, so that
they can be absorbed from the gastrointestinal tract in their bioactive form [34]. Based on our
results, we decided to utilize citric acid to stabilize catechins for a prolonged period of time in
our formulated beverage.
Determination of the Adequate Acidity and Sweetness of the Beverage
Table 5 reports the sensory evaluation based on the acidity (pH) and sweetness (°Brix)
attributes evaluated by the Friedman Multiple Comparisons test. Based on these results, it can
be observed that the beverage adjusted to 11°Brix and pH 3.1 reported the highest acceptance
than the other formulations. Varnam and Sutherland [1] reported that beverages acidified with
citric acid are palatable at pH between 3.08 and 5.4. In the terms of sweetness, Ortega [35]
and Carmona [36] reported that an adequate sweetness in Carica candamaecensis Hook (a
papaya variety) and cocona (Solanum topiro) beverages range between 12 and 14º Brix,
respectively. Thus, the sweetness level of 11°Brix and acidity level of pH 3.1 was selected as
adequate for our green tea and lemongrass based beverage.
Figure 4. Effect of the citric acid and ascorbic acid on browning (catechin degradation) of the beverage.
Table 5. Sensory evaluation based on the acidity and sweetness parameters
Formulation Parameter Evaluation parameter

º Brix pH Sweetness Acidity
SL3P3 11.0 3.3 8.33a 4.33d
a,b
SL3P3 11.0 3.1 7.89 5.33c
b
SL3P3 11.0 3.5 7.22 2.11e
SL3P3 9.0 3.5 5.11c 2.44d,e
c
SL3P3 9.0 3.3 4.89 5.33c
d
SL3P3 9.0 3.1 4.11 6.78b
e
SL3P3 7.0 3.5 3.00 4.78d
SL3P3 7.0 3.3 2.44e,f 5.89b
f
SL3P3 7.0 3.1 2.00 8.00a
(p<0.05).
Infusion Time
Table 6 reports the total catechin content after different infusion times. Based on these
results it can be observed that longer the infusion time higher the total catechin content, this
correlates to previous reports in which black tea reaches the maximum total catechin content
after a 5 minute infusion, and that longer infusion times induce catechin degradation [31]. It
has to be noted that in Table 2 the total catechin content of the SL3P3 formulation was 24.4 ±
0.65 mg catechin/100 mL, but after adjusting the pH to 3.1 and 11°Brix the catechin content
becomes 39.7 ± 0.91 mg catechin/100 mL (Table 6). This phenomena can be explained due to
the higher stability of catechin at acidic pH [29, 34, 37] but not at almost neutral environment
(pH 6.8) [38].
Table 6. Total catechin content after different infusion times
Formulation Infusion time (minutes) Total catechin content (mg/100mL)

pH 3.1 and 11°Brix
SL3P3 7.0 39.7 ± 0.91a
SL3P3 5.0 36.2 ± 0.71b
SL3P3 3.0 33.2 ± 1.03c
Pasteurization
To determine the adequate pasteurization conditions, the total catechin content,

microbiological analysis, and anti-oxidant capacity of the formulation were assessed. Table 7
reports the total catechin content at different pasteurization conditions. It can be observed that
at higher temperature and time of the thermal treatment lower the total catechin content,
which could be related to catechin degradation during pasteurization due to longer heat
exposure. For instance, Chen et al. [14] reported that pH plays a critical role in catechin
stability of green tea beverages, in which 80% of the total catechin content gets degraded at
pH 5 to 6 but not at pH 3 to 4 after thermal treatment (120ºC/20 min).
Our results indicate that the pasteurization conditions: 85ºC/5 min, 90ºC/5 min and
85ºC/10 min reported the highest catechin retention with only a 9 to 18% catechin content
reduction based on an initial catechin content of 39.74 mg catechin/100 mL after infusion.
Thus, our results indicate that milder thermal treatments allow for a higher catechin retention,
which correlates to the report of Chen et al. [14] that demonstrated that a thermal treatment of
98ºC for 7 hours exhibited a 20% catechin loss in green tea beverages compared to a milder
thermal treatment (37ºC for 7 hours).
Table 7. Total catechin content after different pasteurization conditions
Formulation Pasteurization parameters Total catechin content (mg/100mL)

Temperature (ºC) Time (minutes)
SL3P3 85 5 35.7 ± 2.15a
SL3P3 90 5 32.8 ± 0.84b
SL3P3 85 10 32.4 ± 0.51b,c
SL3P3 90 10 30.8 ± 1.24c,d
SL3P3 85 15 29.7 ± 1.01e,d
SL3P3 95 5 29.0 ± 0.57e,d,f
SL3P3 90 15 28.6 ± 0.84e,f
SL3P3 95 10 27.2 ± 0.96f,g
SL3P3 95 15 26.4 ± 0.79g
Table 8. Microbiological analysis after different pasteurization conditions
Pasteurization parameters Dilution

Temperature (ºC) Time (minutes) 10-1 10-2 10-3
85 5 <10 cfu/mL <10 cfu/mL <10 cfu/mL
Data expressed as mean, n = 3.
Furthermore, Table 8 reports the microbiological analysis after different pasteurization

conditions. These results indicate that all the thermal treatment combinations were sufficient
to maintain the viable mesophile counts below 10 colony forming units (cfu)/mL. The low
microbiological counts might also be attributed to the anti-microbial effects of some of the
green tea bioactive compounds such as tannins [39], flavanols and theaflavins [40] that
exhibit anti-microbial activity against certain microorganisms such as Bacillus subtilis,
Bacillus stearothermophilus, Clostridium botulinum and Desulfotomaculum nigrificans [40].
Based on the low microbiological counts and high catechin content the pasteurization
conditions: 85ºC/5 min, 90ºC/5 min and 85ºC/10 min were selected and their anti-oxidant
capacity was assessed as shown in Table 9. It can be observed that there is no statistically
significant differences in anti-oxidant capacity after the three temperature/time pasteurization
combinations. Therefore, based on the no stastically significant differences in anti-oxidant
capacity and microbiological analysis between the pasteurization combinations, the
formulation pasteurized at 90°C for 5 minutes was selected due to its high total catechin
content. This formulation was selected to perform the stability tests.
Table 9. Anti-oxidant capacity after different pasteurization conditions
Formulation Pasteurization parameters Anti-oxidant capacity

Temperature (ºC) Time (minutes) AC1 (%) VCEAC2 (µmol/L)
SL3P3 85 5 94.0 ± 0.18 38.0 ± 0.07
SL3P3 90 5 93.4 ± 0.15 37.8 ± 0.06
SL3P3 85 10 93.4 ± 0.10 37.8 ± 0.04
1
AC: anti-oxidant capacity, which represents the DPPH radical inhibition percentage.
2
VCEAC: vitamin C equivalent anti-oxidant capacity, which was calculated based on a calibration
curve of DPPH and vitamin C
Light Stability
Figure 5 reports the effect of light over browning (catechin degradation) in beverage
formulation bottles protected or not from the light. It can be observed that independently of
the light protection browning occurs in similar trends indicating that light exposure is not a
critical factor in this green tea and lemongrass based beverage. However, it has to be noted
that other factos have been reported to play a critical role in browning and catechin
degradation, some of these factors include the pH of the beverage [14] and oxygen content in
the bottle [23].
Figure 5. Effect of light on browning (catechin degradation) in presence and absence of light. Data
expressed as mean ± SEM, n = 3.
Temperature Stability
The beverage formulation with all the described parameters was bottled and stored at 15,
25 and 35°C and samples were collected at day 0, 4, 8, 12 and 16 to assess total catechin
content, browning, anti-oxidant capacity and microbiological analysis. Table 10 reports the
total catechin content after storage at different temperatures, and it can be observed that the
catechin content losess were of 1, 21 and 45% after 16-day storage at 15, 25 and 35°C,
respectively. The initial total catechin content at day 0 was 32.79 ± 0.84 mg catechin/100mL.
The effect of temperature over browning (catechin degradation) is shown in Figure 6 and
it can be observed that at 35°C catechins are more instable producing more browning, while
at temperatures 15 and 25°C there was significantly less browning. However, it also can be
observed that up to day 8 there is comparable browning between all the temperatures, but
after day 8 there is a temperature-dependant increase in browning. These results correlate
with previous reports that demonstrated that browning is significantly produced after 2 days
at 45°C in a wine model with ascorbic acid and high catechin content [23]. Furthermore, it
has been reported that the stability of catechins is temperature-dependent, with increasing
stability at lower temperatures [14].
The anti-oxidant capacity of the beverage stored at different temperatures is shown in
Figure 7. It can be observed that after a 16 day-storage at 15°C presents a slightly higher anti-
oxidant capacity (higher DPPH radical percentage inhibited) than the beverage samples stored
at higher temperatures. The DPPH radical inhibition was 90.5; 88.6 and 86.4% after 16-day
storage at 15, 25 and 35°C, repectively. However, these differences in anti-oxidant capacity
between the storage temperatures are relatively small compared to the changes in total
catechin content (Table 10) and browning (catechin degradation) appearance (Figure 6),
which indicate that the degradation or epimeration sub-products of catechins also possess
certain anti-oxidant capacity (DPPH radical scavenging capacity). It has been reported that
theaflavins, which are epimerization sub-products of catechins, present similar LDL-
cholesterol protective effects in humans [41]. Furthermore, it has also been reported that the
conversion of catechins to theaflavins induced by the regular processing of black tea do not
affect the free radical scavenging capacity of black tea [41]. However, green tea also
possesses significant amounts of other more temperature-resistant compounds such as the
polyphenols that also significantly contribute to the anti-oxidant capacity of green tea [42].
One of these polyphenols is epigallocatechin gallate (EGCG) that maintains a close
relationship between its content and the anti-oxidant capacity of green tea [43].
The microbiological analysis of the beverages stored at different temperatures for 16 days
is reported in Table 11. No microbiological (mesophiles) growth was observed during the
storage time independently of the storage temperature. This might be attributed to the green
tea components that possess bacteriostatic and bacteriodical activities to inhibit bacterial,
mold and yeast growth [44]. Even though, there was catechin degradation after storage at
different temperatures no microbial growth was observed (<10cfu/mL), which could also
indicate that the epimerization products of catechins also have anti-microbial activity. For
instance, some of the catechin epimerization products that present anti-microbial activity
include gallocatechin gallate (GCG) [44], epigallocatechin (EGC), epicatechin (EC),
epicatechin gallate (ECG) and epigallocatechin gallate (EGCG) [13].
Table 10. Total catechin content after storage at different temperatures
Day 15°C 25°C 35°C

4 32.0 ± 0.75ª 30.9 ± 0.38ª 30.2 ± 0.43ª
8 31.9 ± 0.57ª 29.8 ± 0.95a,b 29.6 ± 0.57ª
12 31.9 ± 0.22ª 28.5 ± 0.57b 25.5 ± 1.42b
16 31.7 ± 0.43ª 25.8 ± 1.21c 19.6 ± 1.30c
Figure 6. Appearance of browning (catechin degradation) after storage at different temperatures. Data
expressed as mean ± SEM, n = 3. Different letters indicate statistically significant samples (p<0.05)
based on the Duncan’s multiple range test.
Table 11. Microbiological activity after storage at different temperatures
Day Dilution 15° C 25° C 35° C

cfu/mL cfu/mL cfu/mL
10-1 <10 <10 <10
0 10-2 <10 <10 <10
10-3 <10 <10 <10
10-1 <10 <10 <10
8 10-2 <10 <10 <10
10-3 <10 <10 <10
10-1 <10 <10 <10
16 10-2 <10 <10 <10
10-3 <10 <10 <10
Data expressed as mean, n = 3.
Figure 7. Anti-oxidant capacity of the beverage stored at different temperatures. Data expressed as
mean ± SEM, n = 3. Different letters indicate statistically significant samples (p<0.05) based on the
Duncan’s multiple range test.
CONCLUSION
The IC50 values for DPPH radical inhibition for green tea and lemongrass are 32.4 and
1350 μg/mL respectively. The optimized technological parameters for the beverage include a
50:50 ratio of green tea with a total extract concentration of 10 mg/mL, infustion time of 7
minutes, acidity levels of pH 3.1 using citric acid, sweetness levels of 11°Brix, pasteurization
for 5 minutes at 90°C. The beverage was stable (not significant browning and no
microbiological growth) at 15°C up to 16 days and the final beverage formulation was
reported to inhibit the DPPH radical by 93.45%. Furthermore, the formulated beverage was
well accepted by the test panel and presented similar anti-oxidant capacity than commercial
green tea based beverages.
ACKNOWLEDGMENTS
The authors would like to thank the Empresa Jardines del Te (La Divisoria, Tingo María,
Perú) for suplying the fresh green tea leaves and to Dr. Manuel Sandoval Chacón, Ph.D. for
his scientific support.
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Reviewed by:
Dr. Neal M. Davies, Ph.D. College of Pharmacy, Washington State University, Pullman,
WA 99163. ndavies@wsu.edu
Dr. Esteban I. Mejía-Meza, Ph.D. SunOpta Fruit Group, Inc. – Healthy Fruit Snacks,
Omak, WA 98841. ei_mejia@yahoo.com
Ms. Connie M. Remsberg, Pharm.D. Candidate. College of Pharmacy, Washington State
University, Pullman, WA 99163. cremsberg@wsu.edu
Chapter 6
TEAS ARE NOT ALL THE SAME: IN VITRO AND IN

VIVO ANTIOXIDANT ACTIVITY AND APPETITE
MODULATION IN RATS OF GREEN TEAS WITH
HIGH AND LOW LEVELS OF ORGANIC SELENIUM
Abdul L. Molan1, Zhuojian Liu1 and Wenhua Wei2

1
Institute of Food, Nutrition and Human Health, Massey University, Palmerston North,
New Zealand. 2 Current address: MRC Human Genetics Unit, Western General Hospital,
Crewe Road, Edinburgh EH4 2XU, UK
ABSTRACT
Green tea is a good source of various polyphenolic compounds and minerals which
are powerful antioxidants. The effects of selenium-containing green tea (Se-GTE; 1.4 mg
selenium/kg) and China green tea (CH-GTE; 0.13 mg selenium/kg) on food
consumption and body weight gain were investigated using a rat model. In addition, the
total phenolic contents (TPC), antioxidant/antiradical activities of these teas were
determined in vitro. Both teas had a satiating influence on experimental rats, as evidenced
by their ability to decrease food intake by 4.9% (CH-GTE) and 13.8% (Se-GTE),
although a statistically significant decrease over the control rats was achieved only for
Se-GTE treatment. In addition, the final body weight of rats gavaged with Se-GTE was
significantly lower (P = 0.0063) than that of the water-gavaged control rats and this
corresponds to 8.5% reduction in body weight relative to the control group. In contrast,
rats gavaged with CH-GTE showed only 1.8% reduction in the final body weight relative
to the control group. The reduction in food intake over a short period compared to a
control rats preloaded with the same volume of water suggests that the decrease in food
intake was mainly a consequence of a satiating effect, rather than a stomach distension
effect. The underlying mechanism responsible for this satiating effect was not identified
as part of this study. It is also important to mention that water intake for the groups given
the tested teas was similar to that of rats given water only and no significant differences
were observed.
Se-GTE had significantly higher TPC (P < 0.0001), higher ferric reducing
antioxidant power (FRAP) (P < 0.01), higher diphenyl-picrylhydrazyl (DPPH) free
radical scavenging activity (P < 0.05), higher ferrous-ion chelating activity (P < 0.05-
104 Abdul Lateef Molan, Zhuojian Liu and Wenhua Wei
0.01), and higher selenium contents (P < 0.0001) than CH-GTE. A strong positive
correlation was found between the TPC, and the FRAP, DPPH, and the ferrous-ion
chelating activities in both teas, indicating that the polyphenolic compounds are the major
contributors of the antioxidant/antiradical activities.
When rats were gavaged with water-soluble tea extract (10 ml/kg/day) of Se-GTE
for four consecutive days, serum FRAP increased significantly (P = 0.0002) as compared
to water-gavaged controls. The level of serum FRAP in rats gavaged with CH-GTE
increased slightly when compared with the control rats but not to a significant extent.
These results indicate that green tea may have the ability to elevate circulating
antioxidant potentials in vivo and this ability is dependent on the type of tea used.
The observed results suggest that the reduction in food intake and decrease in body
weight in experimental animals may be a consequence of antioxidant mechanisms played
by the polyphenolic compounds and the minerals found in green teas. Green tea,
especially the one with a high level of organic selenium may provide a good satiety
inducer and weight management modulator.
Keywords: Green tea; selenium; Satiety; Rats; Antioxidant; antiradical activity; FRAP
BACKGROUND
Obesity is a complex multifactorial syndrome caused by an increase in food availability,
high-fat diet, and sedentary lifestyle and is becoming a pandemic which has been rapidly
developing for three decades (Hill et al., 2003). This syndrome seems to affect all ages as
some studies have shown that in many developed countries child obesity levels have doubled
in the last two decades (Lobestein et al., 2004) and are set to double again, probably over a
shorter period. Lobstein (2006) mentioned in his review that the impending disease burden
has been described by many researchers as ‘a public health disaster waits to happen’, ‘a
massive tsunami’, and ‘a health time-bomb’, and politicians are aware that the amount of time
they have left to make their decisions is rapidly declining. Obesity is a risk factor for several
chronic disorders such as the cardiovascular diseases, hypertension, sleeping apnea,
osteoarthritis of weight-bearing joints, reduced fertility, asthma, and some cancers (Rippe,
1998; Shore and Johanson, 2005). In contrast, weight loss is known to reduce blood pressure,
lipid levels, and the incidence of type 2 diabetes mellitus (Sheard, 2003).
Unlike cardiovascular diseases and cancer, there is no functional food available for
obesity (St-Onge, 2005) and hence efficient, effective and satisfying treatments are required.
Nutritional research strategies that target aspects of the physiology of obesity are critically
important if a workable solution to this health challenge is to be found. One potential area is
to discover foods/food ingredients that can induce greater satiety through physiological
modulation, thus reduce overall food intake.
Selenium (Se) has been considered as an essential micronutrient for animals and the
human body (Navarro-Alarcon and Lopez-Martiez, 2000). Selenium deficiency is a serious
nutritional and health problem in many countries (Ge and Yang, 1993). Tinggi (2003)
reported in his review that diseases associated with Se deficiency are still a cause of concern
in many countries, and in particular in countries of low Se status such as Finland and New
Zealand. In both countries, policies to increase Se status in the human population by adding
Teas Are not All the Same 105
Se fertilizers to agricultural crops or by importation of Se-rich foods have been introduced

(Aro et al., 1995; Thompson and Robinson, 1996). Plant foods, meats and sea foods are the
major dietary sources of Se but the content of the soil where the plants are cultivated or where
the animals are raised (Toyran et al., 2008). Selenium present in most vegetables is in highly
available form, which is around 85-100% while meat products have a Se bioavailability of
approximately 15% (Navarro-Alarcon et al., 1998).
Selenium deficiency has been linked to the development of many health problems such as
heart diseases and cancer (Finley and Penland, 1998). On the other hand, some studies have
shown that Se has many biological activities such as protection against some cancers,
enhancement of neuropsycological function, and maintenance of a healthy immune system
(Clark et al., 1996; Finley and Penland, 1998; Toyran et al., 2008).
SATIETY AND SATIATION

Humans usually eat during meals until they are comfortably full (satiation), after which
they do not eat for a certain time (satiety) (Blundell et al., 1996). It is well known that internal
factors and external environmental factors determine the extent of satiety and satiation (Birch
et al., 1989). Appitite is one of the most important internal factors, which can be measured in
either with the help of objective ratings (hunger, desire to eat, prospective consumption, and
fullness) (Graaf et al., 2004), or by measuring actual food intake (the amount of food eaten
within a certain context). The expression of appetite is reflected in the relationship between
the level of psychological events and behavior, the peripheral physiology, and the central
nervous system (Bundell et al., 1996; Geliebter et al., 1996; Graaf et al., 2004).
The physiological measures that relate to subjectively rated appetite, actual food intake,
or both are defined as biomarkers of satiety and satiation (Graaf et al., 2004). According to
Graaf et al. (2004), determination of the biomarkers of satiation and satiety could be used as a
tool or index with which to measure the satiating efficiency of foods. These tools may serve
as evidence for the ability of certain food or food ingredients to enhance satiety, reduces
appetite, or both (Diplock et al., 1999). In addition, these biomarkers help to understand the
physiological mechanisms behind regulation of food intake and energy balance in humans.
Graaf et al. (2004) in their review, discussed in detail the internal biomarkers and divided
them into the following types: brain biomarkers or measures which relate to pleasantness of
food and sensory-specific satiety, physical biomarkers which relate to stomach distension and
volume of food consumed; hormonal signals (including cholecystokin (CCK), Glucagon Like
Peptide-1 (GLP-1), bombesin or gastrin-releasing peptide, ghrelin, enterostatin, glucose-
dependent insulinotropic polypeptide (GIP), pancreatic polypeptide, and somatostatin);
physiological biomarkers or measures such as body temperature and diet-induced
thermogenesis; and biochemical measures (glucose uptake, insulin and leptin levels). They
concluded that a number of physiologic measures are available that can serve as biomarkers
of satiation, satiety, or both. With respect to satiation (meal termination), physical and
chemical measures of stomach distension and blood plasma concentrations of CCK and GLP-
1 are useful. For satiety and meal initiation, glucose dynamics within a short time frame (less
than 5 min), leptin concentrations during longer-term negative energy balance (more than 2–4
days), and ghrelin concentrations at both the short-term and long-term intervals are
physiological markers (Graaf et al., 2004).
CHEMICAL COMPOSITION OF TEA

Fresh tea leaf is unusually rich in the flavanol group of polyphenols known as catechins.
The catechins may constitute up to 30% of the dry leaf weight (Graham, 1992). The other
polyphenols in tea include flavonols (quercetin, kaempferol, myricetin), and their glycosides,
and depsides such as chlorogenic acid. Caffeine is present at an average level of 3% along
with very small amounts of methylxanthines, theobromine and theophylline. Anthocyanidins
are also found in the leaf. The amino acid theanine is unique to tea. In addition to phenolic
compounds, the tea leaf contains vitamins and several minerals. Vitamin C is lost during the
processing of the fresh leaf, but carotenoids and vitamin K are present in brewed tea. Tea also
contains aluminium, potassium, fluoride and manganese (Balentine et al., 1997).
R3
OH
HO O OH
R2
R1
OH
R1 R2 R3 Monomers
H OH H Catechin (C)
OH H H Epicatechin(EC)
H OH OH Gallocatechin(GC)
OH H OH Epigallocatechin (EGC)
Figure 1. Basic Structure of green tea catechins.

The polyphenols in tea are oxidised to theaflavins and thearubigin in the presence of
polyphenoloxidase and oxygen. The oxidised constituents are unique to black tea.
Approximately 10% of the flavonoids in black tea are catechins, 10% are theaflavins, and
70% are thearubigins (Balentine et al., 1997; Higdon and Frei, 2003).
The common tea polyphenols belong to three main groups called flavan-3-ol
(flavonoids), flavonols and flavones. The chemical structure of major tea catechins (-)
epigallocatechin 3-gallate (EGCG), (-)-epigallocatechin (EGC), (-)-epicatechin (EC), (-)-
epicatechin 3-gallate (ECG) and (+)-catechin (C), are illustrated in Figure 1.
HEALTH BENEFITS OF GREEN TEA

It is well known that fruits and teas are good sources of antioxidants and have profound
effects on human health. Green tea is derived from the leaves and fine stems of the
leguminous shrub, Camellia sinensis and has been consumed as a beverage for thousands of
years (Graham, 1992).
Tea is one of the principle sources of polyphenols in the human diet because of its high
level of consumption combined with its relatively high polyphenol content compared to other
polyphenol sources (Hollman and Arts, 2000). The health promoting effects of green tea are
mainly attributed to its polyphenol content (Wolfram et al., 2006).
Recent research has demonstrated the potential of polyphenols in the prevention of
chronic diseases. In vitro and animal studies have demonstrated that flavonoids have
antioxidant and antimutagenic activities (Peterson and Dwyer, 1998), anti bacterial (Sakanaka
et al., 2000) and antiparasitic activities (Paveto et al., 2004; Molan et al., 2003, 2004). More
recent research has also shown the influence of flavonoids on metabolic reactions in vitro
(Goto et al., 1999; Anderson et al., 2002). The antioxidant effect of tea has been suggested to
have beneficial effects on Alzheimer’s disease and Parkinson’s disease (Behl et al., 1998;
Pillai et al., 1999; Checkoway et al., 2002; Tan, et al., 2003). However, the effect of tea on
such pathologies has not been fully established.
Several experimental studies indicate a strong chemo-preventive action of tea against
gastrointestinal tract and colorectal cancers (Mei et al., 2005). A case control study of 882
Japanese subjects by Ohno et al (1985) indicated a protective effect of green tea on bladder
cancer. Other studies have shown an inverse relationship between tea intake and prostate
cancer (Jain et al., 1998), breast cancer (Tavani et al., 1998), skin cancer (Zhao et al., 1999)
and mucosal Leukamia (Lee et al., 2004).
Animal studies have shown a beneficial effect of long-term feeding of tea catechins on
the development of obesity ( Murase et al., 2002; Choo, 2003; Klaus et al., 2005). It has been
concluded that the stimulation of hepatic lipid metabolism might be a factor responsible for
the anti-obesity effects of tea catechins and that long-term consumption of tea catechins is
beneficial for the suppression of diet-induced obesity. In some of these studies, It was
concluded that this suppressive effect was resulted mainly from increase in brown adipose
tissue thermogenesis through beta-adrenoceptor activation while in other studies, the
antiobesity action was attributed to the ability of green tea to modulates the glucose uptake
system in adipose tissue and skeletal muscle and to suppress the expression and/or activation
of adipogenesis–related transcription factors.
Wolfram et al. (2006) reported in their review that six studies have investigated the anti-
obesity effects of green tea and green tea catechins in humans and most of these studies
reported decreased body weight and fat mass. Some of these studies have suggested that the
body fat-lowering effects of green tea extracts/catechins may be associated with an increase
in the thermogenesis and fat oxidation. In one study, the antiobesity effect of an 80%
ethanolic dry extract from green tea (AR25) was evaluated in moderately obese patients
(Chantre and Lairon, 2002). After three months, body weight was decreased by 4.6% and
waist circumference by 4.48%. In vitro, AR25 exerted a direct inhibition of gastric and
pancreatic lipases and a stimulation of thermogenesis. The authors suggested that the green
tea extract to be a natural product for the treatment of obesity by inhibition of lipasess and
stimulation of thermogenesis.
Tea consumption has been identified as an independent factor protecting against the risk
of hip fractures in mainly women over the age of 50 (Johnell et al., 1995; Kanis et al., 1999;
Hegarty et al., 2000). Hegarty et al. (2000) found that those who drank tea had greater bone
mineral density compared to that of those who did not consume tea. Higher bone mineral
density of the lumber spine, greater trochanter and Ward’s triangle were independent of
smoking status, hormone replacement therapy and coffee drinking.
The aim of this study was to investigate the appetite suppression capacity of two types of
teas that differ in their selenium contents and also to evaluate possible correlations between
antioxidant activity and satiety effect. The total phenolic contents, the ferric reducing
antioxidant power, free radical scavenging activity and ferrous-ion chelating activity of both
CH-GTE and Se-GTE were also investigated in vitro.
MATERIAL AND METHODS
Chemicals and Standards
2, 4, 6-Tripyridyl-s-triazine (TPTZ), sodium acetate, ferric chloride and gallic acid, Folin-
Ciocalteu’s phenol reagent and ferrous sulfate were purchased from Sigma (Australia).
Preparation of Tea Extracts
China green tea (CH-GTE) was purchased from retail shops while selenium-containing
green tea (Se-GTE) was obtained from China. Se-GTE is not presently available on the
market. The aqueous extracts were made by adding 100 ml water (100 oC) to 1 g (1% extract)
tea leaves and allowed to brew for 10 minutes with stirring. Suspension was filtered through
Whatman No. 1 filter paper to retain the clear solution.
Mineral Analysis of Dry Tea Material
Dry leaves of both Se-GTE and CH-GTE were ground into fine powders and sent to the
New Zealand Laboratory Services, Ruakura Research Centre, Hamilton, New Zealand for
mineral analysis.
Total Polyphenols Contents (TPC) Determination
The amount of total phenolic content (TPC) in tea extracts was determined according to
the Folin-Ciocalteu procedure as used by Molan et al. (2008a) with some modifications.
Briefly, an aliquot of 12.5 µl of water-soluble extract was mixed with 250 µl of 2% sodium
carbonate solution in 96-well microplates and allowed to react for 5 minutes at room
temperature. Then 12.5 µl of Folin-Ciocalteu phenol reagent (50 %) was added and allowed
to stand for 30 minutes at room temperature before the absorbance of the reaction mixture
was read at 650 nm using a plate reader. Calibration was achieved with an aqueous gallic acid
solution (100-1000 µg/ml). The TPC of the extract was expressed as mg gallic acid equivalent
(GAE) per gram of tea leaves on dry basis.
Ferric Reducing Antioxidant Power Assay
The capacity to reduce ferric ions was determined using Ferric reducing antioxidant
power (FRAP) assay as described by Benzie and Strain (1996). Briefly, an aliquot of 8.5 µl of
tea (1% or 2% water extracts) was added to 275 µl of diluted FRAP reagent using micoplate
and the plates were incubated at 37 oC for 30 minutes before measuring the absorbance at 395
nm using a plate reader. The working FRAP reagent was prepared by mixing 10 volumes of
300 mmol/L acetate buffer, pH 3.6, with 1 volume of 10 mmol/L TPTZ (2,4,6-tripyridyl-s-
triazine) in 40 mmol/L hydrochloric acid and with 1 volume of 20 mmol/L ferric chloride.
Standard curve was prepared using different concentrations (200-2000 μmol/L) of
FeSO4.7H2O. The antioxidant capacity based on the ability to reduce ferric ions of the extract
was expressed as micromole FeSO4 equivalents per litre of aqueous extracts. . All solutions
were used on the day of preparation and all determinations were performed in triplicate.
Scavenging of Diphenyl-picrylhydrazyl (DPPH) Radicals
This assay detects scavenging of free radicals by the tested compound through the
scavenging activity of the stable 2, 2-diphenyl-1-picrylhydrazyl (DPPH) free radical. This
assay was performed using a previously described method (van Amsterdam et al., 1992) with
some minor modifications. Briefly, 25 μL of tea extracts was allowed to react with 250 μL of
0.2 mM DPPH in 95% ethanol in a 96-well microplate. The plate was then incubated at 37 °C
for 30 minutes after which the absorbance was measured at 550 nm using a microplate reader.
Scavenging capacity of the sample was compared to that of ascorbic acid as positive control
(0.1- 1.0 mM ascorbic acid).
The antiradical activity was calculated as a percentage of DPPH decolouration relative to

a negative control using the following equation:
Antiradical activity (%) = (absorbance of control incubation – absorbance of the tea

extract/ absorbance of control incubation) x 100.
Ferrous-ion Chelating Assay
The ferrous-ion chelating (FCA) assay used by Singh and Rajini (2004) was followed
with slight modifications. Solutions of 2 mM FeSO4 and 5 mM ferrozine were prepared.
Diluted FeSO4 (100 µl) was mixed with 100 µl of sample, followed by 100 µl of diluted
ferrozine. Assay mixtures were allowed to equilibrate for 10 minutes before measuring the
absorbance at 560 nm using a plate reader. Tea extracts were assayed in duplicate. The ability
of the tea extracts to chelate ferrous ions was calculated relative to a negative control using
the equation:
Chelating activity % = (absorbance of control incubation – absorbance of the tea extract/
absorbance of control incubation) x 100.
Animals and Housing
Thirty male Sprague-Dawley rats aged eight weeks that had been weaned onto a balanced
semi-synthetic diet were housed individually in hanging wire-mesh stainless steel cages in a
room with a temperature of 22 ± 1 0C and a 12-h light: dark cycle (light on at midnight) and
they had free access to water throughout the study. The rats were obtained from the Animal
Unit, Massey University, Palmerston North, New Zealand.
For the first seven days, the rats were habituated to a 20-h deprivation schedule whereby
a feeder containing more than one day’s expected intake of food was made available to each
rat for a total of four hours (1000 - 1400h; 2 hours at the end of the light phase, and 2 hours at
the beginning of the dark phase). This is to accommodate for the rat’s natural behaviour of
nocturnal eating’s. This regimen is in line with recent procedures reported in the literature
(Froetschel et al., 2001).
Pre-meal Administration
This study involved giving the rats a 10 ml fluid pre-meal/kg body weight, containing
either water or tea extracts. This pre-meal dosing was by pharyngeal gavage with a soft
silicon rubber tube attached to a 3-ml syringe. The rats were habituated to this procedure,
during the acclimation period. Thus, for the last three days of the acclimation period pre-
meals of 2 ml sterile water at room-temperature were gavaged daily 30 minutes before the
feeding period.
The same experimental diet was offered to all treatment groups during the feeding
period. It was formulated to meet the nutrient requirements of growing rats as per the
formulation AIN-93G (Reeves et al., 1993).
The amount of preload given to each rat was individually calculated based on body
weight. The dose was calculated based on the assumption that a 75 kg person would take 3
cups (250 ml) of 1% extract of CH-GTE or Se-GTE, which is equivalent to 10 ml per kg body
weight. In the present study, the rats were weighed at the beginning of the experiment and the
body weight (g) was multiplied by 0.01 to determine each individual pre-meal load.
For the remainder of the study (from day 8 to day 12) the rats were divided randomly into
3 equal groups (n = 10) and matched for body weight. The rats in the first group were fed the
basal diet (AIN-93G; Reeves et al, 1993) and gavaged daily with 10 ml of deionized water/kg
body weight to serve as a control group while the rats in the second and the third groups were
gavaged daily with the same volume of water soluble extracts from CH-GTE and Se-GTE,
respectively. The pre-meal was given to each rat 30 minutes before the normal meal time, and
impact of the pre-meal was determined by monitoring changes in each or rat’s total food
intake for that day. This was repeated for a period of four days. The rats were weighed at the
beginning and at the end of the experiment.
The food was presented to the rats in metal cups and food intake measured daily, as
described previously (Molan et al., 2008b). Water intake was also measured daily. At the end
of the study, the rats were euthanized by inhaling CO2 and the blood from the hearts was
collected immediately after death.
Serum Antioxidant Capacity
Serum antioxidant capacity was measured using the FRAP assay. Blood was collected
from each rat in the control group and those gavaged with CH-GTE or Se-GTE. Samples
were allowed to clot at room temperature for 25 minutes. Samples were then immediately
centrifuged (1000 g) for 15 minutes at 4 0C to recover serum. Serum was extracted and then
stored at – 80 0C for further use.
The data were recorded as means ± standard errors. One way analysis of variance
(ANOVA) and Tukey multiple comparisons were carried out to test for any significant
differences between the means. Linear regression analysis was carried out by Microsoft
Office Excel 2003. Differences between means at 5% (P < 0.05) level were considered
significant.
RESULTS
Mineral Contents
The Se-GTE and CH-GTE powdered leaves were analyzed for mineral content (Table 1).
Elemental analysis indicates that the Se-GTE leaves have a ten-fold higher mean total
selenium concentration (1.4 versus 0.13 mg of Se/kg; P < 0.0001). Moreover, Se-GTE
contains higher concentrations of phosphorus, potassium, iron, zinc and copper than CH-GTE
(Table1), but CH-GTE contains more calcium and manganese.
Table 1. The mineral contents of selenium green tea (Se-GTE) and China green tea
(CH-GTE)
Mineral Unit Value

Se-GTE CH-GTE
Phosphorus g/100g 0.46 ± 0.083 0.25 ± 0.005
Potassium g/100g 1.78 ± 0.055 1.5 ± 0.085
Calcium g/100g 0.33 ± 0.077 0.49 ± 0.015
Sodium g/100g < 0.02 < 0.02
Iron mg/kg 407 ± 13.0 305.0 ± 64
Manganese mg/kg 1159 ± 140.5 1619 ± 428.0
Zinc mg/kg 49.1 ± 11.1 43.5 ± 11.5
Copper mg/kg 25.5 ± 2.1 16.0 ± 3.8
Selenium mg/kg 1.4 ± 0.075 0.13 ± 0.002
Total Polyphenol Contents and Antioxidant Activities
Se-GTE had significantly higher (P < 0.0001) TPC than CH-GTE at both 1% and 2%
concentrations (Table 2). Aqueous extracts from Se-GTE also showed significantly higher
reducing activity as measured by FRAP (P < 0.01), higher free radical scavenging activity as
measured by DPPH assay (P < 0.05) and higher ferrous-ion chelating activity (P< 0.05- 0.01)
than CH-GTE.
The results of this study showed that the TPC values were strongly correlated with the
FRAP (R2 = 0.70 and 0.90 for CH-GTE and Se-GTE, respectively), DPPH (R2 = 0.92 and
0.76, respectively) and FCA (R2 = 0.69 and 0.71, respectively) values for both CH-GTE and
Se-GTE.
Table 2. Antioxidant activity [ferric reducing antioxidant power (FRAP; µmol/L)

values; DPPH scavenging activity and ferrous-ion chelating activity (FCA)], total
phenolic contents (TPC; mg GAE/g dry leaves) and correlation (R2) between antioxidant
values and TPC of China green tea (CH-GTE) and selenium-containing tea (Se-GTE)
TPC (mg/g DL) FRAP (µmol/L) DPPH (%) FCA (%)

CH-GTE 34.1 ± 4.2 9002.8 ± 60.6 69.4 ± 0.76 34.1 ± 0.57
Se-GTE 60.5 ± 2.9*** 9947.6 ± 37** 71.5 ± 0.6* 39.3 ± 1.3*
0.70 (CH-GTE) and 0.92 (CH-GTE) 0.69 (CH-GTE)
2
R 0.90 (Se-GTE) and 0.76 (Se- and 0.71 (Se-
versus TPC GTE) versus TPC GTE) versus
TPC
The mean of duplicate incubations of two separate experiments and standard errors are presented.
Statistical analysis based on ANOVA, * P < 0.05; ** P< 0.01 and *** P< 0.0001 compared to values
in the same column.
(A) Control CH-GTE Se-GTE
16
14
12 * *
Food intake (g/day)
*
10
0
1 2 3 4
Days after oral adminstration
(B)
% reduction in total food intake relative to
18
16 $$
14
12
control group
10
0
CH-GTE Se-GTE
Figure 2. Effects of oral administration of water extracts from two teas on food consumption (A) and %
reduction in food intake (B) in rats fasted for 20 h. Rats were divided into 3 groups of ten rats each. The
experimental rats were gavaged with ~2 ml of tea extracts daily for 4 consecutive days. The control
group was gavaged with ~2 ml of distilled water. Data are expressed as means ± SEM. * P < 0.05 versus
control group and $$ P < 0.01 versus CH-GTE.
(A)
250
**
Final body weight (g)
200
150
100
50
0
Control CH-GTE Se-GTE
(B)
12
% reduction in body weight relative to
10
$
8
control group
0
CH-GTE Se-GTE
Figure 3. Effects of oral administration of tea extracts on body weight. A: final body weight and B: %
reduction in body weight in rats. Data are expressed as means ± SEM. Significantly different from the
value in the control group: ** P < 0.01 versus control group and $ P < 0.05 versus CH-GTE.
Effect of Tea Extracts on Food Intake, Water Intake and Body Weight Gain
in Rats
The animal protocol was approved by the Massey University Animal Ethics Committee.
All rats consumed their diets as expected and demonstrated acceptable weight gains. Before
gavaging, there was no difference between rat groups in food intake. The results of this
experiment are shown in Figures 2-4. There was no difference between the rats gavaged with
water and those gavaged with CH-GTE in the daily food intake during the whole
experimental period. During the first day, there was no significant in food intake difference
between the rats gavaged with water and those gavaged with Se-GTE. However, the food
intake in rats gavaged with Se-GTE extract for 2 (13.9 ± 0.8 g/day in control vs. 11.37 ± 0.98
g/day in rats gavaged with Se-GTE; P = 0.0194), 3 (12.9 ± 0.6 g/day in control vs. 10.38 ±
0.34 g/day in rats gavaged with Se-GTE; P = 0.0204) and 4 (13.23 ± 0.22 g in control vs.
11.48 ± 0.39 g in rats gavaged with Se-GTE; P = 0.0300) days was significantly lower than
their counterparts in the control group (Figure 2A). In general, rats gavaged with CH-GTE
and Se-GTE consumed 4.9% and 13.8% less food than their counterparts in the control group,
respectively (Figure 2B).
The final body weight of rats gavaged with Se-GTE extract was significantly lower
(225.2 ± 3.7 g in controls vs. 206.0 ± 5.9 g in rats gavaged with tea; P = 0.0003) than those in
the control group (Figure 2A). This is corresponding to 8.53 % reduction in body weight
(Figure 3B). In contrast, no significant difference in final body weight was detected between
control rats and those gavaged with CH-GTE [225.2 ± 3.7 g in controls vs. 221.1 ± 1.25 g in
rats gavaged with tea; P= 0.0063; Figure 3A]. Again, Se-GTE was more effective (P =
0.0378) than CH-GTE at reducing the body weight gain (Figure 3B).
Overall, water intake in rats preloaded with CH-GTE or Se-GTE was not significantly
different when compared with the rats given water only (Data not shown).
In Vivo Antioxidant Status
Figure 4 shows the differences in serum FRAP between rats gavaged with CH-GTE, Se-
GTE and rats gavaged with water only. The FRAP value, an indicator of total antioxidant
defence, was significantly affected by gavaging with teas, especially Se-GTE [750.3 ± 65
µmol FeII/L in controls vs. 834.3 ± 29.8 in rats gavaged with CH-GTE (P > 0.05) and 1237.6
± 84.9 µmol FeII/L in rats gavaged with Se-GTE; P = 0.0002]. In addition, the FRAP value of
the serum in rats gavaged with Se-GTE was significantly higher (P = 0.0008) than that in rats
gavaged with CH-GTE.
(A)
$
1400
* *
Serum FRAP level (micromol/L)

1200
1000
800
600
400
200
0
Control CH-GTE Se-GTE
(B)
100
% increase in serum antioxidant
80 $$
60
level
40
20
0
CH-GTE Se-GTE
Figure 4. Effect of oral administration of water-soluble extracts from two teas CH-GTE and Se-GTE)
on antioxidant status (FRAP values; micromole/L) in sera collected from the rats 4 days after gavaging.
A: antioxidant activity and B: % increase in antioxidant activity relative to control group. Data are
expressed as means ± SEM. ** P < 0.01 versus control group. $ P < 0.05 and $$ P < 0.01 versus CH-
GTE.
DISCUSSION
Both teas had a satiating influence on experimental rats, as evidenced by their ability to
decrease food intake by 4.9% (CH-GTE) and 13.8% (Se-GTE), although a statistically
significant decrease over the control rats was achieved only for Se-GTE treatment. The
reduction in food intake, observed over four hours in rats given a preload of Se-GTE in
comparison to rats in a control group given the same preload volume of water, has led to the
conclusion that the decrease in food intake was mainly the consequence of a satiating effect
of the tea rather than simply a stomach distension effect.
It is well known that food intake is regulated by a variety of peripheral factors and by
central neuroendocrine systems. Various hormones, including cholecystokinin, glucagon-like
polypeptide-1, glucagon, somatostatin, and bombesin, have been reported to affect food
intake (Morley, 1987; Kalra et al., 1999). Although the precise mechanisms which underlie
the satiating effects of Se-GTE are not fully understood, it may trigger receptors for amino
acids which have been detected in the wall of the upper intestine (Mei, 1992). The afferent
fibers from these receptors may inform certain brain centres that a source of energy and/or a
specific nutrient has been ingested. Alternatively, such receptors may play a role in inducing
satiety by triggering the release of hormones such as cholecystokinin (CCK), which might act
directly at the central and/or peripheral levels to stimulate pancreatic juices and reduce gastric
emptying. Kao et al. (2000) found that epigallocatechin gallate (EGCG), but not related
catechins, significantly reduced food intake; body weight; blood levels of testosterone,
estradiol, leptin, insulin, insulin-like growth factor I, glucose, cholesterol, and triglyceride in
male rats. The authors observed similar effects in lean and obese male Zucker rats and
suggested that the effect of EGCG was independent of an intact leptin receptor. The authors
concluded that EGCG may interact specifically with a component of a leptin-independent
appetite control pathway. Further study is required to determine the mechanism by which Se-
GTE suppresses the food intake.
In addition, the final body weight of rats gavaged with Se-GTE decreased significantly
when compared with the water-gavaged control rats and this corresponds to 8.53% reduction
in body weight relative to the control group. In contrast, rats gavaged with CH-GTE showed
only 1.8% reduction in the final body weight relative to the control group. The reduction in
body weight may be due to the reduction in food intake or to other factors. Choo (2003)
reported that in Sprague-Dawley rats fed a high diet, consumption of a water extract of green
tea for 2 weeks resulted in decreased body fat accumulation. The author concluded that green
tea reduces body fat and increases energy expenditure, which is partially mediated via beta-
adreoreceptor activation.
Several animal studies have shown a beneficial effect of long-term feeding of green tea
catechins on the development of obesity (Murase et al., 2002; Choo, 2003; Klaus et al., 2005;
Kao et al., 2006). In some of these studies, it was concluded that this suppressive effect was
resulted mainly from increase in brown adipose tissue thermogenesis through beta-
adrenoceptor activation while in other studies, the anti-obesity action was attributed to the
ability of green tea to modulate the glucose uptake system in adipose tissue and skeletal
muscle and to suppress the expression and/or activation of adipogenesis-related transcription
factors.
The gavaging technique guaranteed that the rats received orally a define amount of load
and prevented several technical problems. In the present study, the load was delivered by
gavage, which eliminated the role of the orosensory cues in the feeding response. The
physiological response may differ if the orosensory factors are also taken into account (Taha
and Fields, 2005).
The time period in which rats had access to food was restricted to 4h/day in an attempt to
provide a more controlled and exaggerated appetite response at feeding (Froetschel et al.,
2001). This time interval set for meal-feeding was considered to be the minimal time that
would still allow the rats to consume enough food and grow comparably to those with 24-h
access to food (Froetschel et al., 2001). The ability of Se-GTE to reduce the food intake
coupled with the decrease in body weight gain compared with their counterparts given water
(control group), suggests that this tea may be a good satiety inducer and weight management
modulator. Recently St-Onge (2005) reported that the inclusion of foods or the replacement of
habitual foods with others that may enhance energy expenditure (EE) or improve satiety may
be a practical way to maintain a stable body weight or to assist in achieving weight loss; such
foods may act as functional foods in body weight control.
Se-GTE had significantly higher TPC, higher FRAP, higher DPPH free radical
scavenging activity, higher ferrous-ion chelating activity, and higher selenium contents than
CH-GTE. A strong positive correlation was found between the TPC, and the FRAP, DPPH,
and the ferrous-ion chelating activities in both teas, indicating that the polyphenolic
compounds are the major contributors of the antioxidant/antiradical activities. This is
consistent with previous studies which have shown that polyphenols are major contributors of
the antioxidant activity (Khokhar and Magnusdottir, 2002; Schotsmans et al., 2007; Molan et
al., 2008b).
The results of this study also demonstrated that consumption of both teas increased the
antioxidant capacity of serum in rats and Se-GTE was significantly more effective than CH-
GTE in this respect. Significant increase in FRAP was observed in rats gavaged with Se-GTE
compared to the control rats gavaged with the same volume of water. The increased
antioxidant capacity in serum (FRAP) following the oral gavage of Se-GTE indicated a direct
absorption and/or an enhanced production of antioxidants. The antioxidants responsible for
the increased antioxidant capacity following the consumption of Se-GTE are likely phenolic
compounds and selenium. Green tea leaves contain 10–30% (dry weight, DW) of
polyphenols, including catechins, flavonols, flavanones, phenolic acids, glycosides and the
aglycones of plant pigments (Pan et al., 2003). In green tea, the major flavonoids present
include catechin (flavan-3-ol) such as epicatechin, epicatechin-3-gallate, epigallocatechin and
epigallocatechin-3-gallate (EGCG; Balentine et al. 1997). It has been reported that tea leaves
are the only food products containing EGCG (Graham, 1992; Chu and Juneja, 1997). In
addition to phenolic compounds, tea leaves contain vitamins, several minerals, caffeine and
anthocyanidins, aluminum, potassium, fluoride and manganese (Chao et al., 1995). Other
studies have shown that polyphenolic compounds in green tea extracts have been found to be
potent antioxidants and free-radical scavengers due to the ability to act as hydrogen or
electron donors (Higdon and Frei, 2003; Molan et al., 2008a).
There is a possibility that the presence of selenium in the green tea sample produced a
synergistic effect with the green tea resulting in the significantly increased in the antioxidant
activity and other biological activities observed in the present study. Indeed other studies
regarding selenium and green tea have also reported on a synergistic effect between selenium
and green tea.
Hu et al., (2000) studied the physiological function of selenium (Se)-enriched tea
fertilized with sodium selenite and naturally high-Se tea in rats and found that the selenium
biological utilization rates were 65.41, 68.05 and 70.49% for sodium selenite, Se-enriched tea
and naturally high-Se tea, respectively. In another study, the effect of foliar application of Se
on increasing the antioxidant activity of tea was investigated (Xu et al., 2003) and the results
showed that the radical scavenging ability of the tea extracts were in the following order: Se-
enriched tea obtained by fertilization with selenate, Se-enriched tea obtained by fertilization
with selenite and then regular tea. The higher antioxidant activity shown by Se-GTE over that
of regular CH-GTE may be attributed to the synergistic effect of tea polyphenols and
selenium. Amantana et al. (2002) compared the antimutagenic activities of regular green tea
and selenium-enriched green tea obtained by foliar application of selenite toward the
hetrocyclic amine 2-amino-3-methylimidazol [4,5-f] quinoline (IQ) in the Salmonella assay.
The authors found that selenium-enriched green tea exhibited concentration-dependent
inhibition of IQ-induced mutagenesis and was significantly more effective than regular green
tea tested under the same conditions. They suggested an enhancing (coantimutagenic) effect
of selenium in combination (synergism) with green tea in vitro. More recently, Li et al. (2008)
found that enrichement of selenium endowed green tea with higher antioxidant and antitumor
activity.
Certainly, the presence of selenium in the diet is of major importance because selenium
deficiency was found to be associated with many defects, such as heart disease, depression,
hypothyroidism and a weakened immune system (Zimmerman and Kohrle 2002; Beck et al.,
2003). Recent studies have confirmed the fact that supplementation of selenium from natural
food materials in the form of organic selenium is safe and suitable as compared with
consumption of inorganic selenium (Hu et al., 2002).
Decreased serum antioxidant status has been suggested as a risk factor in cardiovascular
disease (Kaplan and Aviram, 1999), and cancer (Ames et al., 1995). On the other hand,
increasing the serum antioxidant status has been suggested as a possible method of reducing
the risk of many chronic degenerative disorders (Kaplan and Aviram, 1999; Georgopoulos,
1999; Vendemiale et al., 1999).
CONCLUSIONS
The current study has shown a reducing effect of Se-GTE pre-meals on subsequent meal
intake. The underlying mechanism responsible for this response was not identified as part of
this study. Se-GTE showed statistically significant satiety activity as evidenced by its ability
to reduce food intake (up to 13 %) when orally gavaged into rats compared with the food
intake of control rats given a gavage of water only. The final body weight of the rats given
Se-GTE was significantly lower than that observed in the control groups. In addition,
administration of Se-GTE was associated with a significant increase in serum antioxidant
status above the control group for the FRAP assay. The ability of Se-GTE to reduce the food
intake coupled with the decrease in body weight gain compared with their counterparts given
water (control group), suggests that this tea may be a good satiety inducer and weight
management modulator.
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Chapter 7
ANTI-OBESITY EFFECTS OF (-)-EPIGALLOCATHCHIN-

3-GALLATE AND ITS MOLECULAR MECHANISM
Cheol-Heui Yun1, Gi Rak Kim1, Min Ji Seo1,

Hyun-Seuk Moon2 and Chong-Su Cho1,*
1
Department of Agricultural Biotechnology and Research Institute for Agriculture and
Life Sciences, Seoul National University, Seoul 151-921, Republic of Korea;
2
Laboratory of Molecular Signaling, National Institute on Alcohol Abuse and
Alcoholism, National Institutes of Health, Bethesda, Maryland 20892-9410, USA
ABSTRACT
Development of obesity appears to be influenced by a complex array of genetic,
metabolic, and neural frameworks, together with one’s behavior, eating habit, and
physical activity. The incidence of obesity is significantly increasing in virtually all
societies of the world and causes important pathological consequences such as
cardiovascular diseases and type 2 diabetes mellitus. Furthermore, rates of pediatric
obesity have increased dramatically over the past decade resulting cardiovascular,
metabolic, and hepatic complications. Since ancient times, green tea has been considered
as a traditional medicine as a healthful beverage. Major components of green tea
including epigallocathchin gallate (EGCG), epigallocatechin (EGC), epicatechin gallate
(ECG) and epicatechin (EC) have been received significant scientific attention and public
awareness for its beneficial effects on prevention and therapeutic treatment of
cardiovascular diseases and cancer.
Anti-obesity effects of EGCG have been demonstrated in various in vitro and in vivo
models showing that EGCG treatment could reduce food intake, glucose uptake, blood
glucose level, and differentiation and growth activity of fat cells together with
modulation of lipolytic and lipogenic activities. In these actions, large number of
molecules including laminin receptor, fatty acid synthase, glucose-6-phosphate
dehydrogenase, cyclin-dependent kinase 2, CCAAT/enhancer binding proteins, mitogen
activated protein kinases, AMP-activated protein kinase, phosphatidylinositol-3-kinase,
*
To whom correspondence should be addressed. Tel: +82-2-880-4636; Fax: +82-2-875-2494; E-mail:
chocs@plaza.snu.ac.kr
126 Cheol-Heui Yun, Gi Rak Kim, Min Ji Seo et al.
peroxisome proliferator activated receptor, and number of transcription factors including

NF-kB were suggested to be involved. This review will focus on the effects of ECGC at
molecular level in terms of (1) receptor recognition, (2) modulation of signaling
pathways, (3) lipid metabolism and altering the lipogenic and lipolytic activities leading
to metabolic changes and apoptosis, and (4) final outcome in vitro and in vivo.
1. INTRODUCTION
Green tea is made differently during the manufacture processing compared with oolong
tea, black tea, and pu-erh tea, which allows the fresh tea leaves to wither for decrease of their
moisture content, sequentially fermenting polyphenols. This fermentation makes
polymerization of monomeric catechins, which leads to the major formation of flavins,
namely theaflavin (TF-1), theaflavin-3-gallate (TF-2a), theaflavin-3’-gallate (TF-2b), and
theaflavin-3,3’-digallate (TF-3), and thearubigins, consequently decreasing catechin content
[1, 2]. On the contrary, green tea leaves are processed under steam followed by partial
withering to prevent fermentation. The other important aspect of process is drying and rolling,
which allows yielding a dry and stable product [3]. Therefore green tea generally posses
higher content of catechins. The catechins are the main compounds in green tea, and are
polyphenolic flavonoids as phenol derivates. The four major catechins are (-)-
epigallocatechin-3-gallate (EGCG), which represents approximately 59% of the total
contents; (-)-epigallocatechin (EGC) (approximately 19%); (-)-epicatechin-3-gallate (ECG)
(approximately 13.6%); and (-)-epicatechin (EC) (approximately 6.4%) [4].
EGCG, the richest catechin in green tea, has a large number of positive health effects in
the prevention of lifestyle-related diseases including cardiovascular disease, carcinogenesis
and obesity. Obesity, characterized by increased number and size of fat cells due to the
processes of so-called mitogenesis and differentiation, has increased at an alarming rate in
recent years and it is obvious that excess body weight is now a critical health concern
worldwide. Indeed obesity became a common disease associated with risks of cancer, type 2
diabetes, hypertension, and cardiovascular disease [5]. The prevalence of obesity is reaching
epidemic proportions in many Western countries. That is the reason why development of
drugs to treat obesity or implementation of a dietary regimen to prevent obesity becomes a
public awareness and health goal. Obesity arises from the imbalance between energy intake
and its expenditure. These processes are regulated by genetic, endocrine, metabolic,
neurological, pharmacological, environmental, and nutritional factors [5, 6].
According to the requirements for treating obesity, EGCG has been focused on its
physiological effects. Simply, EGCG can reduce body weight and body fat. This contention
was further supported by experimental data where the EGCG or EGCG-containing green tea
extract not only reduces food uptake, lipid absorption, blood triglyceride, cholesterol, and
leptin levels but also stimulates energy expenditure, fat oxidation and regulates various
enzymes related to lipid anabolism and catabolism [7-10]. Moreover, EGCG appears to
modulate the mitogenic, endocrine, and metabolic functions of fat cells [11]. Accordingly, a
thorough examination of the signal element through which EGCG executes modulation of
preadipocyte mitogenesis will certainly help in the prevention and control of the development
of obesity. The mechanisms of action of EGCG seem to involve several but, certain pathways
including (1) the decrease in the energy intake, (2) the increase in energy expenditure, and (3)
Anti-obesity Effects of (-)-Epigallocathchin-3-gallate 127
the alterations in the activities of fat, liver, muscle, and intestinal cells. In this review, we
focus on the signal transduction and mechanisms which induce anti-obese effects caused by
EGCG.
2. EGCG RECEPTORS
The first step for the ligand to initiate cellular function in many cases, if not all, is to
interact with appropriate receptor(s). Recently the receptor for EGCG has been identified. It is
known as laminin receptor (LR), approximately 67 kDa accessory integrin protein, and was
first isolated from cancer cells [12]. The Kd value for the binding affinity of EGCG to LR
appears to be about 40 nM [13]. As expected, EGCG competes with laminin to bind LR [13].
It has been suggested that the LR is in association with a lipid raft of the plasma membrane
for increasing suppressive effect of EGCG on IgE receptor of B-cells [14]. Through this, the
possible interaction relationship between the EGCG receptor and other types of receptor in
particular cells has been studied. It is to note that the LR is not only found on cancer cells, but
normal cells, such as muscle cells, macrophages, neutrophils, endothelial cells, epithelial
cells, hepatocytes, interstitial cells, and neuronal cells [15]. On these normal cells, the LR
may also serve as an EGCG receptor to regulate the effects of EGCG. Basically, LR is a
heterodimeric structure, a member of the integrin family of cell adhesion receptors, and
occurs in many types on the membrane of numbers of different normal cells [13, 16, 17], such
as integrin α5 and α6 in murine adipocytes [18]. It has been clearly shown that certain types
of LRs and laminin are present in preadipocytes and adipocytes, which relate to their activity.
Activities of fat cells are regulated by their receptors on the membrane or in the cells.
Thus, EGCG or green tea may have an effect on fat cells or other types of cells through the
changes in the activation and expression of these receptors. EGCG can alter the activities or
expressions of other membrane and nuclear receptors. For example, EGCG can displace the
bound estrogen receptors α and β, each encoded by a separate gene, ESR1 and ESR2,
respectively, to 17b-estradiol [19]. To note that the estrogen receptor plays a major role as a
DNA binding transcription factor and regulates gene expression. It has been reported that
tyrosine phosphorylation of the insulin receptor in rat hepatoma cells and of IGF-I receptor in
HepG2 cells was increased when treated with EGCG [20]. Such effect of EGCG on insulin
receptor and IGF-I receptor appears to be cell type-dependent. EGCG down-regulates the
expression of the androgen receptor in LNCaP prostate cancer cells [21]. It is important to
note that MCF-7 and LNCaP cancer cells are fat-synthesizing cell lines. In addition, EGCG
can directly and indirectly inhibit the activation or gene expression of almost all receptors for
growth factors including epidermal growth factor (EGF), fibroblast growth factor (FGF),
insulin-like growth factor 1 (IGF-1), platelet-derived growth factor (PDGF), and vascular
endothelial growth factor (VEGF) receptors, and human epidermal growth factor receptor 2
(HER2/neu, also known as ErbB-2). It has been suggested that EGCG can stimulate the
apoptotic receptor CD95 (FAS/APO-1) in cancer cells [22, 23], suggesting that EGCG
potentially induce apoptosis through activating CD95 expressed on certain cancer cells.
Interestingly, some of these receptors are known to be expressed in or on adipocytes and other
normal cell types that are involved in the regulation of adipose tissues [24].
3. SIGNALING PATHWAY
Cells are equipped with a membrane that separates between inner and outer environment.
However, they are able to respond to extracellular stimuli such as mitogens and hormones,
and convert their signals into cellular processes. This conversion is usually mediated by the
binding of designated extracellular ligands to specific transmembrane receptors, which are
consequently activated to further transmit the signals of the different ligands through
intracellular signaling pathways. These pathways, operating within complex networks of
interacting or counteracting proteins, transmit the signals to various intracellular targets,
thereby regulating inducible cellular processes such as transcription, translation, proliferation,
differentiation and apoptosis. Cells receive constantly clues from their environment via
activation of surface receptors and extracellular matrix.
3.1. ERK PATHWAY

The MAPK family is an essential part of the signal transduction machinery in signal
transmissions by external stimuli, and contains three major MAPK subfamilies: Extracellular
signal-regulated kinase (ERK), p38, and c-Jun N-terminal kinase (JNK) [25, 26]. ERKs are
composed of two similar (85% sequence identity) protein kinases, originally called ERK1 and
ERK2. ERK1/2 signaling cascade is one of the key signaling pathways, which are involved in
functions including the regulation of meiosis, mitosis, and postmitotic functions in
differentiated cells [27]. A large variety of extracellular ligands can stimulate this ERK
signaling cascade and, as a consequence, the ERK1/2 signaling cascade regulates many
distinct and even opposing cellular processes. As the ERK1/2 pathway is responsive primarily
to mitogens and growth factors, and plays key roles in cell proliferation, survival, and
differentiation, this section will focus mainly on the action mechanism of ERK1/2 pathway in
relation to (pre)adipocytes.
Obesity arises from an imbalance in energy intake and energy expenditure that leads to
the pathological growth of adipocytes. Obesity is known to be induced by the hypertrophy of
adipocytes and the generation of new adipocytes from precursor cells [28]. Both two
processes are dependent on the regulation of adipocyte differentiation. The adipocyte
differentiation process at the beginning requires a precise proliferative step called mitotic
clonal expansion (MCE), which is initiated by adipogenic stimuli, such as insulin and known
to activate the ERK pathway. During MCE, a complete inhibition of ERK pathway are
required to induce complete differentiation [29], suggesting a positive role for ERK in
adipocyte differentiation in relation to the expression of the crucial adipogenic regulators,
C/EBP α, β, γ and , and PPARγ.
It has been proposed that EGCG could serve as a signal element to regulate cell growth
[30-33] and modulate the mitogenic and adipogenic signaling in preadipocytes [34-36].
EGCG induced a decrease in phosphorylated ERK1/2 in 3T3-L1 preadipocytes but did not
alter the total levels of MEK1, ERK-1, ERK-2, p38, phospho-p38, JNK, or phospho-JNK
[37], suggesting that EGCG acts specifically on the ERK. This contention is also partially
supported by the fact that exposure to EGCG induced a down-regulation in the
phosphorylated ERK1/2 of preadipocytes without altering total levels of MEK1 and ERK1/2
proteins [38]. In addition, transient amplification of phospho-ERK1/2 content by transfecting

wild type of MEK1 cDNA or its constitutively active mutant cDNA to 3T3-L1 preadipocytes
prevented EGCG-induced decreases in their cell numbers. Taken all together, these findings
demonstrate that a suppressive effect of EGCG on the proliferation of preadipocytes is likely
mediated via ERK, but not p38 and JNK, MAPK-dependent pathway and is an important
factor in preadipocyte proliferation [38].
The ERK-dependent effect of EGCG observed in 3T3-L1 preadipocytes was further
confirmed that two specific inhibitors of ERK MAPK, PD98059 and U0126, alone can inhibit
cell growth and MEK1 activity [38]. It is intriguing to note that EGCG appears to work
differently from PD98059 and U0126 in reducing levels of phosphorylated ERK1/2 proteins.
Since PD98059 is known to prevent MEK1 activation by Raf while U0126 directly protects
ERK from being phosphorylated by MEK1, one might assume that the target molecule of
EGCG could be upstream of Erk pathway. It is possible that EGCG induces a decrease in
phosphorylated ERK1/2 from preadipocytes via reducing the phosphorylation of MEK1 in
association with Raf as reported for H-Ras-transformed cells [39].
In cultured cells, ERK1/2 are phosphorylated under the regulation of a variety of factors,
including growth factors, G protein-coupled receptors, tyrosine kinase receptors, and Raf and
MEK1 kinases [27, 40]. It has been shown that EGCG significantly prevented the increase of
phosphorylated ERK1/2 by either IGF-I or IGF-II, or concomitantly reduced IGF-I receptor
activity [38, 41]. The study further proved that EGCG caused a decrease in the
phosphotyrosine-IGF-I receptor and an association of the IGF-II receptor with Giα-2 protein
[42-44]. Although the Erk-mediated EGCG effects are obvious, more detailed and thorough
studies are needed to confirm the precise target and its associated mechanisms, such as PI3K-
Akt-Bad/phosphorylated Bad pathway.
3.2. CDK2 PATHWAY

Cyclin-dependent kinases (Cdks) are known to play a role in the regulation of the
eukaryotic cell cycle and therefore, it has been implicated in the control of gene transcription,
responsible for cell division [45, 46]. The Cdks are proteins of 34~40kDa possessing Ser/Thr-
specific protein kinase activity. Cdk catalytic subunits do not act alone and the way they
trigger cell cycle events is completely dependent on associated cyclin subunits [47]. In
mammals, there are at least 10 different Cdks, Cdk1 to Cdk10. Progress of the cell cycle
appears to be controlled mainly by Cdk1 and Cdks2, 4 and 6. The role of Cdk2 in mediating
the cell-cycle inhibitory and tumor-suppressing activities of p21Cip1 and p27Kip1 appears to be
dispensable [47-49], which could be explained by compensatory activities of other kinases,
most likely Cdk1 activity through its association with Cyclin A, a partner of Cdk2.
It has been suggested that EGCG modulates cell mitogenesis and growth arrest of most
cancer cells via Cdk pathway [38, 50, 51] and can serve as the main controller of mitogenesis
and mitotic clonal expansion of preadipocytes [52]. In fact, EGCG appears to down-regulate
adipocyte differentiation through a specific type of Cdks, for instance Cdk2 signaling
pathway in preadipocytes [38]. This was further confirmed by transfection study that the
decrease of Cdk2 activity together with the increase of G1 arrest induced by EGCG in
preadipocyte were inhibited by the transfection of Cdk2 [34, 38]. Furthermore the transfection
of dnCdk2 to preadipocytes slowed down the growth of preadipocytes, as expected. These

results demonstrated that the effect of EGCG-induced preadipocyte anti-mitogenesis and
growth arrest is indeed dependent on a Cdk2 pathway and requires inactivation of the Cdk2
protein. The other hand, cyclin D1 is a G1 cyclin associated with Cdk4 and Cdk6 proteins,
which are required for cell cycle G1/S transition, and therefore, cyclin D1 protein expression
by EGCG [53, 54], suggesting the possibility of Cdk4- and Cdk6-related effects of EGCG on
preadipocyte growth arrest. However, the effect of EGCG on the reduction of cyclin D1 via
Cdk4 and/or Cdk6 has yet to be further confirmed.
Decrease in the expression of cell division cycle 2 (Cdc2) protein by EGCG [50, 51]
suggests the possibility of the action of EGCG on the G2/M phase of preadipocytes since
Cdc2 serves for the predominant Cdk in the early G2/M transition of the cell cycle [33].
Regulation of Cdk2 activity is regulated at multiple levels including the synthesis of subunits
and the association of inhibitory proteins p21 and p27 [38, 51]. Therefore all these
observations suggest that EGCG is responsible for the increased association of Cdk2 with p21
and p27, thereby leading to low Cdk2 activity and a subsequent in the percentage of G0/G1
arrest. It is also possible that EGCG acts on the CDK inhibitor (CKI) family to reduce Cdk2
activity in the particular type of preadipocyte. It would be of interest if any of CKIs, for
instance p53 [54], is involved in the action of EGCG-mediated growth arrest in
preadipocytes. Decreases in cyclin D1 protein expression of preadipocytes by EGCG could be
also involved in the association of Cdk2 with p21 and p27, since the sequestration of p21 and
p27 is mediated through the increase of synthesis rates of the induction of cyclin D1 and
cyclin D2 protein [51]. Further studies to determine whether EGCG affects the association of
cyclin D1 together with CKIs would help clarify this notion.
In smooth muscle cells, increased Cdk2 activity by endothelin is mediated via either the
activation of Erk and Cdc25A (a phosphatase) or the inactivation of WEE1 (an inhibitory
kinase) but is prevented by the inactivation of ERK and the activation of WEE1, whereas
Cdk2 activity is restored through dephosphorylation at Tyr15 by Cdc25A [55]. Therefore,
activities of WEE1 and Cdc25A are, respectively, inactivated and activated through
phosphorylation by the activation of ERK, and vice versa [38]. It is also probable that the
reduction in Cdk2 activity by EGCG in 3T3-L1 preadipocytes could be mediated by the
inactivation of ERK. Because Cdk2 activity is also regulated by the association of stimulatory
proteins, such as cyclin E [56, 57], further investigations are needed to clarify whether EGCG
can change the production of cyclin E protein in association with Cdk2. It has been suggested
that EGCG may result in altered cyclin-Cdk2 protein complexes in 3T3-L1 preadipocytes
[29] as reported in mouse liver cells [55].
These observations together with the results mentioned in the previous sections suggested
that the anti-mitogenic effect of EGCG on 3T3-L1 preadipocytes could be dependent on the
MAPK and CDK pathways and is likely mediated through decreases in their activities.
Overall, the anti-adipogenic effect of EGCG is important not only in reducing adipocyte
differentiation but also in inhibiting adipocyte proliferation, suggesting that two cell cycle
control kinases, ERK and cyclin-dependent kinase (CDK), are required for these inhibitory
effects [58]. Future studies on characterizing its oxidative stress induced by interaction of
EGCG and its receptor are also needed to elucidate the mechanisms of how EGCG signals
reduce the activities of MEK and CDK proteins.
3.3. AMP-ACTIVATED PROTEIN KINASE (AMPK)

AMP-activated protein kinase (AMPK), a metabolite-sensing protein kinase, is known to
play a major role in energy homeostasis by coordinating a number of adaptive responses in
ATP-depleting metabolic status such as ischemia/reperfusion, hypoxia, heat shock, oxidative
stress, and/or exercise [59-61]. Thus, it is certain that AMPK is sensitively regulated by the
allosteric binding of AMP under not only physiological but also pathological conditions when
ATPs are depleted [61, 62]. The prolonged and persistent activation of AMPK has a link to
p53-dependent cellular senescence, suggesting its role as an intrinsic regulator of the cell
cycle in mammalian cells [62, 63]. It has been suggested that AMPK cascades have emerged
as novel targets for the treatment of obesity and type 2 diabetes [60, 64, 65]. AMPK is also
known to be activated by aminoimidazole-4-carboxamide riboside (AICAR), which is
converted to a nucleotide that mimics the effect of AMP. Furthermore, the pro-apoptotic
potential of the activated AMPK was observed under the AMPK over-expressed conditions in
the various cells [66, 67], which was confirmed by long-term treatment with AICAR has
prevented development of diabetes in animal models [68].
EGCG treatment inducing the activation of AMPK caused inhibition of adipocyte
differentiation in 3T3-L1 cells [35], suggesting that AMPK activation is necessary for the
inhibition of adipocyte differentiation and anti-obesity effects by EGCG. Therefore, the
mechanism that affects AMPK regulation with physiological stimuli or anti-obesity agents,
for instances activation of ROS and/or using naturally occurring compounds like EGCG
treatment, appears as a promising target for the development of strategies for the treatment of
obesity [35, 69]. Generally, reactive oxygen species (ROS) have been suggested as upstream
molecules of AMPK-activated signals since AMPK cascades respond to the intracellular level
of AMP and to the AMP:ATP ratio, which is highly sensitive to oxidative stress [66], and
therefore one of the major AMPK activation mechanisms was suspected to be ROS. It is not
surprising that various therapeutic effects involving the release of ROS have been reported
[70-75]. Overall, the anti-proliferatory and lipolytic effects of EGCG have been attributed to
their ability to modulate various signaling pathways, specifically those that control the
differentiation and cell proliferation of (pre)adipocytes.
3.4. PEROXISOME PROLIFERATOR-ACTIVATED RECEPTORS (PPARS)

The peroxisome proliferator-activated receptor (PPAR) subfamily of nuclear receptors
controls many different number of target genes involved in both lipid metabolism and glucose
homeostasis [62]. By forming heterodimers with the retinoid X receptor, they regulate the
transcription of many genes in a ligand-dependent manner [2]. Three isotypes (α, γ, β/δ) of
PPAR have been identified. PPARα is predominantly expressed in tissues with a high rate of
fat catabolism (e.g. liver and muscle). A study showed that green tea including cathechins
increases PPARα expression in skeletal muscle (but not in liver) and increases PPARδ
expression in visceral and subcutaneous adipose tissues. Green tea catechins activated PPARα
in cell culture [76] and increased PPARα and PPARγ expression in a mammalian liver [35].
The expression of PPARγ is high in adipose tissue, where it triggers adipocyte differentiation
and induces the expression of genes critical for adipogenesis [77-79]. Antioxidants (via
intracellular redox state) could activate the transcription factors, NF-kB and activator protein-
1 (AP-1) [80, 81], which further regulate PPAR [82]. EGCG appears to not only promote
energy expenditure but also stimulate the oxidation of lipids by the known inhibitory effect on
catechol-O-methyltransferase, an enzyme that degrades noradrenaline [83] suggesting that
EGCG enhances lipid catabolism perhaps through counteract with the PPARδ agonist-
induced lipogenesis.
PPARγ is expressed at high levels in white and brown adipose tissues and to a lesser
extent in macrophages, colon, kidney, and liver [78, 79, 84], where it plays a major role in
many different biological processes including adipogenesis, glucose homeostasis,
atherogenesis, inflammation, and tumor susceptibility [4]. In general, the enforced expression
of PPARγ and C/EBP stimulates adipogenesis, suggesting the essential roles of these
transcription factors in regulating adipogenesis [77, 84]. EGCG-induced down-regulation of
PPARγ expression appeared to function similarly to the negative effects of EGCG on lipid
accumulation and on adipocyte differentiation [35]. The combined expression of PPARγ and
C/EBP have synergistic effects in promoting fat cell conversion in myoblasts [85], indicating
that these genes must be involved in the fat accumulation.
Both transcription factors, PPARγ and C/EBP, coordinate the expression of genes
including aP2 involved in creating and maintaining the adipocyte phenotype [86, 87]. The
aP2 is a member of the intracellular lipid binding protein family causing direct modification
of cholesterol trafficking and inflammatory responses [88, 89]. In fact, it has been
demonstrated that fat cells from insulin-resistant individuals display decreased expression of
PPARγ target genes, for instance aP2, and increased lipid partitioning together with a
subsequent enlargement of the existing adipose cells [90, 91]. PPARγ2 and C/EBP, almost
exclusively found in the adipose tissues, are linked to the adipocyte differentiation [86, 92],
indicating that they are most likely playing a crucial role both in the expression of adipose-
specific genes and in the manifestation of the mature adipose phenotype. It has been
demonstrated that the negative impact of EGCG on adipogenesis was accompanied by the
reduction of PPARγ2 protein together with the attenuation of C/EBP expression, indicating
that EGCG down-regulated the expression of maker genes during adipocyte differentiation
[91].
Binding of PPARγ2 to two specific sites in the calreticulin gene was found to stimulate
the expression of calreticulin, the major Ca2+-binding protein of the endoplasmic reticulum
lumen. In addition, PPARγ2 was found to be down-regulated in cells over-expressing
calreticulin [93, 94]. Therefore, there appears to be a negative feedback loop in which
PPARγ2 stimulates the expression of calreticulin, which, in turn, inhibits the activity and
expression of PPARγ2. It is to note that an increase in the cellular Ca2+ level induced
activation of receptors or channels [95] has been shown to inhibit the differentiation of
preadipocytes. A recent study [93] demonstrated that this process is repressed by increasing
intracellular Ca2+ and is dependent on the expression of calreticulin.
Taken all these reports together, although we have gained much insight on the action of
EGCG on adipocytes, further studies are needed to ensure certain in-depth understanding on
the mechanism of EGCG’s action in the regulation of mitogenesis of preadipocytes especially
in relation to activation of PPARs, C/EBP and aP2, and/or regulation of Ca2+ pool.
3.5. P53 AND NF-ΚB

Human prostate cancer cells, LNCaP, are fat-synthesizing cell lines and therefore, it
could be worthwhile to look into the effect of EGCG in relation to action mechanism of
LNCaP cells. The p53 is the most frequently altered tumor suppressor in human
malignancies. Indeed more than 50% of solid tumors having a loss of normal p53 expression
due to deletion or point mutation [96, 97]. It is also responsible for cell cycle arrest upon
DNA damage [98], which is also a key regulator of apoptosis. The p53-dependent apoptotic
response is well-documented as an anticancer mechanism [99].
It has been suggested that EGCG could be potentially important cancer chemopreventive
agent because of its ability to selectively induce apoptosis in cancer, but not in normal, cells.
Indeed, EGCG treatment caused a significant decrease in the percentage of viable cell along
with a concurrent induction of apoptosis while the percentage of apoptotic cells does not
coincide with the proportion of viable cells. This is probable that EGCG not only induces
apoptosis but also is responsible for G1 arrest [52]. A study showed that EGCG treatment
induces an increase in the cellular levels of p53 in LNCaP cells coincided with the
phosphorylation of p53 at serine 6, 15, 20, 37 and 392 [100, 101], suggesting that the
difference in cell viability and apoptosis caused by cell cycle arrest can be induced by
EGCG..
The importance of p53 in EGCG-mediated apoptosis was revealed in a study using PC-3
cells (null for p53), which are not very sensitive to EGCG-mediated apoptosis compared to
LNCaP cells containing wild-type p53 [101]. This notion was confirmed that PC-3 cells
became very sensitive to EGCG-mediated G1 arrest and apoptosis when wild-type p53 was
introduced into PC-3 cells [102]. EGCG-induced stabilization of p53 caused an increase in its
transcriptional activity, thereby resulting in an up-regulation of its downstream target
p21/WAF1, which further inhibits Cdk2, 4 and 6 causing cell cycle arrest [55, 103]. This was
further supported by several independent studies, which show that targeted over expression of
p21/WAF1 increases apoptosis [22, 53]. The exact mechanism by which p21 promotes the
apoptosis is not currently clear, but could be related to its ability to interact and possibly
regulate the components of DNA repair system [104, 105]. It is important to note that EGCG
treatment can induce the increase of another transcriptional target of p53, proapoptotic protein
Bax (Figure 1). Indeed, a study showed that EGCG treatment resulted in up-regulation of Bax
via p53 and a parallel down-regulation of Bcl-2, which might ultimately initiate the activation
of the casapase cascade leading to apoptosis [106].
EGCG effectively decreased NF- B transcriptional activity in LNCaP cells, which may
be related to decrease in the nuclear levels of the p65 subunit of NF- B [101]. A number of
studies suggest that NF- B activates the expression of anti-apoptotic proteins Bcl-2 and Bcl-
xL [107, 108]. EGCG-induced decrease in NF- B transcriptional activity is accompanied by
a decrease in the levels of the anti-apoptotic protein Bcl-2. It is to note that genetic
approaches using dominant-negative NF- B together with Bcl-2 reporter study will help to
understand the possible mechanism. Moreover, a direct role of p53-dependent repression of
Bcl-2, which might be independent of NF- B, cannot be ruled out. It is also important to note
that EGCG reduced the cell viability of preadipocytes, induced the appearance of DNA
fragmentation, and increased the activity of the caspase-3 [50], suggesting that the inhibition
of cell mitogenesis and induction of apoptosis could be attributable to decrease in the number
of preadipocytes by EGCG.
3.6. RESISTIN
Resistin, one of the adipokines, was initially shown to be induced during adipogenesis
and released in large amounts from white adipose tissue in mice [109, 110]. The release of
resistin was increased in obese mice and involved in insulin resistance [111, 112], suggesting
that adipocyte-derived resistin is related to obesity and diabetes. Thus, it has been proposed as
a biomarker for insulin resistance or of adipose tissue mass [113]. In fact, resistin has an
inhibitory effect on insulin-stimulated glucose uptake in differentiated 3T3-L1 adipocytes
[111]. Moreover, the increase in adipocyte number may have caused a rise in local resistin
production, inhibiting insulin action on glucose uptake in adipose tissue and, thus, preventing
further adipocyte differentiation [114, 115].
Figure 1. Signal transduction pathways mediating EGCG-induced apoptosis and cell cycle arrest (Na
and Surh, Molecular Nutrition and Food Research 2006, 50: 152 – 159; reprinted with permission).
It has been shown that intracellular resistin protein significantly decreased after EGCG
treatment, while no change was found on the extracellular level. It is probable that EGCG
could play a role to modulate the distribution of resistin between the intracellular and
extracellular compartments [116]. It has also been reported that EGCG suppressed the
expression of resistin, both mRNA and protein, in a dose- and time-dependent manner in
3T3–L1 adipocytes [116]. In particular, resistin mRNA expression in adipose tissues of obese
humans is higher than that in normal subjects [113, 117]. In humans, the expression of
resistin in adipocytes is low compared with that in rodents, but resistin mRNA is readily
detectable in circulating mononuclear cells, which suggests that human resistin may be
regulated by a different mechanism or may have a different role than that in rodents [118].
Since its discovery, resistin has been found to possess numerous physiological and
immunological actions. For instance, resistin regulates fasted blood glucose, causes
dyslipidemia, suppresses insulin-stimulated glucose uptake in adipocytes and muscle cells,
inhibits dopamine and norepinephrine release in the hypothalamus, and promotes endothelial
cell activation, smooth muscle proliferation, inflammatory responses and inflammatory bowel
diseases [109, 110, 113, 117, 119]. The expression of resistin gene appears to be regulated by
nutritional, endocrine, genetic, pharmacological, immunological and developmental factors,
and the action mechanism of resistin is emerging. For example, resistin promotes smooth
muscle cell proliferation through the activation of extracellular signal regulated kinase
(ERK1/2) and phosphatidylinositol 3-kinase (PI3K) [120, 121]. Other study showed that
over-expression with MEK1 blocked EGCG-inhibited resistin mRNA expression, suggesting
that EGCG could down-regulate the expression of resistin via MEK1-dependent pathway
[116]. Further investigations are necessary to clarify the action mode of EGCG in relation to
its target molecule(s). As discussed previously, EGCG has been reported as a
chemopreventative agent that blocks excessive body weight. Whether EGCG exerts its effects
through the control of resistin is unknown and minimal attention has given to this subject so
far, although one assumes such effect since resistin is known to cause insulin resistance and
inhibit adipocyte differentiation.
4. LIPID METABOLISM
In epidemiological studies a significant inverse relationship between tea drinking and
plasma cholesterol levels has been reported. The effect of a purified form of EGCG on lipid
metabolism has been proven through animal studies to get better understanding of the
underlying mechanism. These studies have shown that catechins inhibited cholesterol
absorption and lowered plasma cholesterol [122-126]. Chinese green tea and Jasmine tea
containing high amount of EGCG more effectively reduced cholesterol levels in rats when
compared to other teas with lower EGCG [127]. EGCG, administered by intraperitoneal
injection, did not inhibit cholesterol synthesis but oral administration of EGCG decreased
cholesterol absorption from rat intestine [124, 128]. These reports suggest that green tea or
more specifically EGCG can cause to decrease the cholesterol absorption from the gut.
Green tea catechins affect lipid metabolism by several different pathways and prevent the
appearance of atherosclerotic plaque. Among several lipid metabolic pathways, absorption of
cholesterol is considered to be one of the major pathways. The principal steps in the
absorption of cholesterol are emulsification in the stomach, hydrolysis of the ester bond by a
specific pancreatic esterase, micellar solubilization (often used as a powerful alternative for
dissolving hydrophobic drugs in aqueous environments), and absorption in the proximal
jejunum, re-esterification within the intestinal cells, and transport to the lymph by
chylomicrons [129, 130]. Due to the insolubility of cholesterol in water, solubilization of
cholesterol in mixed micelle is essentially required for its efficient absorption [131, 132]. A
micelle is polymolecular aggregates that act as a carrier and a solubilizer of cholesterol and
other lipids. A micelle is an aggregate of surfactant molecules dispersed in a liquid colloid. A
typical micelle in aqueous solution forms an aggregate with the hydrophilic head regions in
contact with surrounding solvent resulted in sequestering the hydrophobic tail regions in the
center of micelle. Once solubilized cholesterol is formed, and then transferred from the
micelle to the cell membrane of the intestinal brush border [129]. Thus, the action of EGCG
that influence cholesterol uptake could interfere with the (1) forming micelle, or (2) affinity
either of micelles for membranes or (3) of cholesterol for micelles. These alterations in the
intestinal lumen affect hepatic cholesterol metabolism and may affect synthesis and
catabolism of lipoproteins as well.
The action mechanism of EGCG on the cholesterol absorption was studied on the basis of
micelle size and cholesterol solubility. The addition of pure EGCG to mixed micelles
decreased the intermicellar cholesterol concentration by 65% [124]. These findings were
further supported by a study showing that green tea catechins reduced the cholesterol
absorption from the intestine by reducing solubility of cholesterol in mixed micelles [133]. In
addition, it was shown that pure EGCG induced an increase in the mean particle size of the
micelles [134], suggesting that the change in the micelle size could affect the solubility of
cholesterol in micelles and/or the affinity of micelles for membrane. Thus, the
hypocholesterolemic activity of EGCG appears to be due to the inhibition of intestinal
cholesterol absorption by reducing micellar solubilization of cholesterol.
Overall, intake of green tea extracts decreased the absorption of triglycerides and
cholesterol [8, 135], and, at the same time, increased a fat excretion [8]. Although number of
studies reported that green tea catechins decreased total plasma cholesterol and blood
triglyceride levels, the efficacies differ among studies [7, 124, 136-139], probably because of
the animal models used and a particular component of catechins, for instance EGCG, were
different. Green tea ingestion causes decrease in the LDL cholesterol [140, 141],
concomitantly with increase in the HDL cholesterol. The addition of EGCG to the diet
significantly lowered total plasma cholesterol and non-HDL cholesterol levels [124]. The
ratio of non-HDL: HDL-cholesterol, an indicator of atherosclerosis risk, was also positively
affected in the animals fed with a diet containing 1% of EGCG, suggesting that green tea
polyphenols exert beneficial effects on the suppression of high-fat diet-induced obesity by
modulating lipid metabolism and the risk of associated diseases, including diabetes and
coronary diseases. It has been postulated that the absence of change in body composition after
EGCG treatment, where it could trigger a shift of fat distribution from visceral to
subcutaneous adipose depots.
5. MODULATION OF LIPOLYTIC ENZYMES

The lipid substrate is either dispersed as an emulsion in aqueous medium or present as fat
droplets, and the enzyme acts at the interface between the lipid and aqueous phases. Lipases
are water-soluble enzymes that catalyze the hydrolysis of ester bonds in water–insoluble, lipid
substrates. Lipases perform essential roles in the digestion, transport and processing of dietary
lipids (e.g., triglycerides, fats, oils) in most, if not all, living organisms. Human pancreatic
lipase is the major enzyme responsible for lipid breakdown in the digestive systems, which
convert triglycerides to free fatty acids and monoglycerides [142, 143]. It is a diverse family
dependent on their substrates and the positions in substrates of the bonds that they hydrolyze
[143].
The activity of pancreatic lipase is inhibited by EGCG due to a catechin-induced lipid
emulsification process since the addition of EGCG not only dose-dependently reduces
cholesterol solubility in biliary micelles but also alters the size of the mixed
lecithin/taurocholate/cholesterol micelles [134], suggesting that the reduced lipid
emulsification and digestibility may be responsible for lowering intestinal cholesterol
absorption, total fat absorption, and serum triglyceride and cholesterol levels. It is to note that
the EGCG, but not (+)-catechin (C), appears to stimulate the activity of hormone-sensitive
lipase, which exists within adipocytes and is responsible for lipid mobilization from adipose
tissues to other peripheral tissues [134, 144]. Inhibition of adrenaline- and adrenocorticotropic
hormone-induced lipolysis by EGCG, but not C nor EC, in the primary fat cells were
confirmed by decreased release of fatty acids [35, 145]. No changes in the expression of
lipoprotein lipase, a cell surface enzyme that hydrolyzes triglycerides in lipoprotein and is
responsible for the transport of lipids from the blood to tissues, were observed in obese rats
treated with EGCG [35]. EGCG appears to affect the activity of various types of lipases
dependent on the physiological conditions of animals and the presence of the specific type of
hormones in the experimental models. Therefore clear-cut evidence together with defined
mechanism for the effects of EGCG on a variety of lipases has yet to be determined.
6. INHIBITION OF LIPOGENIC ENZYMES

Green tea catechins are known to possess anti-lipogenic activity [146] where they inhibit
the activity and/or expression of lipogenic enzymes including acetyl-CoA carboxylase
(ACC), fatty acid synthase (FAS), malic enzyme (ME), glucose-6-phosphate dehydrogenase
(G6PDH), glyceol-3-phosphate dehydrogenase (G3PDH), and stearoyl-CoA desaturase-1
(SCD1) [22,11,10,55]. ACC is the rate-limiting step in fatty acid synthesis for catalyzing the
conversion of acetyl-CoA to malonyl-CoA [68].
The green tea EGCG reveals the inhibitory activity on rat liver ACC in vitro [125] and in
vivo [134]. Interestingly neither (+)-catechin, EC, nor EGC has shown such effect. It has been
reported that when EGCG was incubated with chicken FAS, the second enzyme to catalyze
the conversion of malonyl-CoA to fatty acyl-CoA, it inhibited FAS activity [147]. In general,
EGCG-mediated inhibition of FAS activity is composed of reversible fast-binding inhibition
and irreversible slow-binding inactivation [147]. The expression of hepatic ME and G6PDH,
known to generate NADPH for fatty acid biogenesis, has decreased in obese mice treated
with EGCG [8, 139]. The activity and expression of G3PDH, the rate-limiting step in TG
biosynthesis, are also decreased by EGCG treatment [148]. Furthermore, gene expression of
SCD1, the rate-limiting enzyme in the synthesis of monounsaturated fatty acids in adipose
tissue, was suppressed in EGCG-treated obese mice [8], suggesting that EGCG or green tea
can reduce fatty acid and TG synthesis by inhibiting lipogenic enzymes.
Several cholesterol-related enzymes are also regulated by green tea catechins, supporting
its potential hypocholesterolemic effect. For instance, it has been reported that EGCG from
green tea is an inhibitor of fatty-acid synthase (FAS). Its inhibition of FAS appears to be
composed of reversible fast-binding inhibition, through which 52 μM EGCG can inhibit 50%
of the activity of FAS, and irreversible slow-binding inactivation. Therefore, the inhibitory
activity may be caused by specific binding to the enzyme or by scavenging reactive oxygen
species required for the monooxygenase reaction. EGCG also inhibited the activity of two
other cholesterol-biosynthetic enzymes, lanosterol 14α-demethylase and
oxidosqualene:lanosterol cyclase [149]. Inhibition of EGCG on cholesterol-biosynthetic
enzymes could cause the low plasma cholesterol levels, together with the inhibition of micelle
formation [124] and other steroid-related enzymes including 11β-hydroxysteroid
dehydrogenase [150], 5α-reductase [151], and aromatase [152] and the stimulation of fecal
cholesterol excretion [8]. In EGCG-treated rats, decreased activities of 5α-reductase and
aromatase could be related to weight loss of androgen-dependent organs, such as prostates
and seminal vesicles, and to low blood estrogen levels and the subsequent weight loss of
estrogen-dependent organs, such as uterus [55, 152]. These observations may explain the
beneficial use of EGCG in the prevention of prostate and breast cancers in patients [153,
154]. There are controversial observations in the green tea’s regulation of energy expenditure
and fat oxidation in humans [10, 69, 155-158]. This controversy may be attributable to the
protocols employed, the source and purity of green tea extracts, the number of times
administered, the period and dose of administration, and the method of administration
(capsules vs. tea drinking).
7. GLUCOSE TOLERANCE AND INSULIN SENSITIVITY

High level of circulating glucose (or hyperglycemia) can cause a serious problem with
diabetes mellitus type 2 (type 2 diabetes), a metabolic disorder that is primarily characterized
by insulin resistance [159]. Once chronic hyperglycemia becomes apparent, it damages
insulin target tissues and aggravates insulin resistance, forming a vicious circle that is
collectively called glucotoxicity [160, 161].
It has been suggested by epidemiological observations and laboratory studies that EGCG
has a certain anitdiabetic properties and an effect on glucose tolerance and insulin sensitivity
[7, 69]. Furthermore, EGCG is reported to lower glucose production by decrease of the
expression of genes that control gluconeogenesis [162]. Dietary supplementation with EGCG
markedly ameliorates glucose tolerance in diabetic rodents [163, 164]. It has been shown that
rats fed with standard chow with green tea had lower fasting plasma levels of glucose, insulin,
triglycerides, and free fatty acid than the control rats fed with standard chow with deionized
distilled water [164]. Some reports have also suggested that EGCG involved on the regulation
of the glucose level in blood by improved insulin secretion through rehabilitation of damaged
beta-cells [165, 166]. However, exact mechanism(s) leading to this result have not been
documented in detail yet.
Researches on the relationship between green tea and obesity-related insulin resistance
syndrome has shown that green tea enhances in vitro insulin activity [167], enhances insulin
sensitivity in human subjects [168] and rat [165], and reduces hypertriacylglycerolaemia in
mice [137]. These results indicate that EGCG indeed has insulin-potentiating activity. Indeed,
EGCG appears to mimic the performance of insulin by increasing the tyrosine
phosphorylation of both the insulin receptor and insulin receptor substrate-1 [167], which is
the first stage of insulin-stimulated glucose uptake. Some reports indicated that the
phosphorylation of IRS-1 was induced at high glucose level and subsequently repressed
hepatic glucose utilization through suppressing Akt downstream signaling [169].
Interestingly, the supplementation of EGCG alleviated this insulin signaling blockade by
improving the function of IRS-1 molecules [35]. In addition, EGCG mimics insulin actions
by inducing PI3K-sensitive phosphorylation of transcription factor FOXO1a (Forkhead box
O1a) which is sensitive to scavengers of free radicals [170].
Based on the fact that serine/threonine phosphorylation can modulate both positive and
negative signaling transmission via IRS, it has been shown that some high IRS-1 serine
phosphorylation (i. e., Ser307) leads to an insulin-signaling blockade by inhibiting insulin-
induced IRS-1 tyrosine phosphorylation [171]. In parallel with this observation, it has been
reported that EGCG treatment significantly decreases high glucose-induced IRS-1 Ser307
phosphorylation, suggesting critical role of the serine phosphorylation of IRS-1 in the
development of insulin resistance [35]. Furthermore, these EGCG effects seem dependent, at
least in part, on the cellular AMPK phosphorylation, suggesting a new molecular mechanism
for antidiabetic activities of EGCG in addition to the previously described one (see AMPK
section). It has been suggested that EGCG has indeed inhibitory role to Ser307
phosphorylation in IRS-1 and this effect was abolished by inhibition of cellular AMPK [35].
However, more studies are needed to answer the question on whether such effect was direct
interaction of EGCG with IRS-1 or AMPK or both.
It seems fair to say that EGCG indeed regulates glucose homeostasis and displays some
antidiabetic benefits. A number of studies were devoted to investigate how high glucose
condition preferentially impairs insulin downstream signaling and what role EGCG plays to
alleviate this insulin resistance state. These alterations provide an uptake for excess
circulating glucose that would be cleared by maintaining glucose homeostasis and thereby
protect individuals from glucotoxicity damages. Further studies to determine the cellular
molecule(s) responsible for direct interaction with EGCG will certainly lead to the
identification of specific molecular target(s) for the generation of therapeutic agents useful in
the management of insulin resistance disease like diabetes.
8. ANTIMITOGENIC AND APOPTOTIC EFFECTS

The mitogenic signaling in mammalian cells is carried out mainly by growth factors that
interact with receptors localized at the plasma membrane. Most of these receptors have a
tyrosine kinase activity domain that is localized at the cytoplasmic region of the molecule and
often alter the gene expression patterns and induce mitogenesis [172]. Typically, the binding
of an agonistic ligand to its cognate GPCR triggers the activation of multiple signal
transduction pathways that act in a synergistic and combinatorial manner to relay the
mitogenic signal to the nucleus and promote cell proliferation [172]. A rapid increase in the
activity of phospholipases C, D, and A2 leading to the synthesis of lipid-derived second
messengers, Ca2+ fluxes and subsequent activation of protein phosphorylation cascades,
including PKC/PKD, Raf/MEK/ERK, and Akt/mTOR/p70S6K is an important early response
to mitogenic avtivity [173, 174].
Homeostasis in multicellular organisms is maintained by a well-controlled balance

between cell proliferation and cell death. Cell death is a fundamental cellular response that
has a crucial role in shaping our bodies during development and in regulating tissue
homeostasis by eliminating unwanted cells. Several types of cell death have been described;
apoptosis (type I), cell death associated with autophagy (type II) and necrosis or oncosis (type
III) and few others (e.g., mitotic catastrophe, anoikis, excitotoxicity, Wallerian degeneration,
and cornification) [175]. Among these, apoptosis is a complex and highly regulated process
that is involved to eliminate superfluous, aged, injured or infected cells from the body in an
ordered and controlled sequence of events [176]. Once the decision to die has been made by
an individual cell, the ‘execution’ phase of apoptosis is rapid where some cell types showing
unique morphological changes as soon as 15 min after exposure to an apoptotic stimulus
[177]. All cells undergoing apoptosis show typical, well-defined morphological changes,
including plasma membrane blebbing, chromatin condensation with margination of chromatin
to the nuclear membrane, nuclear fragmentation, and finally the formation of apoptotic bodies
[177]. Apoptosis has been also characterized by several biochemical criteria, including
different kinetics of phosphatidylserine (PS) exposure on the outer leaflet of the plasma
membrane, changes in mitochondrial membrane permeability, release of intermembrane
space mitochondrial proteins, and caspase-dependent activation and nuclear translocation of a
caspase-activated DNase resulting in internucleosomal DNA cleavage, which is controlled
both positively and negatively by B-cell lymphoma protein-2 (BCL2) family members [177,
178]. Identification of these morphological and biochemical markers makes it possible to
distinguish the apoptosis from other forms of cell death.
There are two classical pathways in apoptosis [179, 180]. In the extrinsic pathway,
ligation causes cell surface receptors such as CD95/Fas to bind and oligomerize the
cytoplasmic adaptor molecule FADD. The subsequent binding of the prodomains of
procaspases 8 and/or 10 to FADD leads to presumed oligomerization of these procaspases
causing a conformational change that result in acquisition of enzymatic activity. In the
intrinsic pathway, release of cytochrome c, which is released from damaged mitochondria,
results in oligomerization of the cytoplasmic scaffolding molecule Apaf-1 (apoptotic protease
activating factor-1) caspase-9 (a member of the CED-3-like Cys protease family). Activated
caspase-9 in turn cleaves and activates downstream caspases, including caspase-3, caspase-6
and caspase-7, which carry out the execution phase of apoptosis.
There are in vitro and in vivo observations suggesting that green tea EGCG appears to
modulate the mitogenic, endocrine, and metabolic functions of fat cells. Green tea EGCG
inhibited preadipocyte proliferation [35] in dose-, time-, catechin-, and growth phase-
dependent manners suggesting anti-mitogenic effect of EGCG and different growth stages of
preadipocytes have different sensitivities and/or signals dependent on the micro-
environmental milieu such as types and sources of catechins. This contention was supported
by the light of fact that the amounts of phospho-Erk-1 and phospho-Erk-2 in log-phase, but
not latent or confluent, preadipocytes were significantly reduced by EGCG treatment [38]. In
addition to the inhibitory effect on preadipocyte proliferation, apoptosis was also enhanced by
incubation with EGCG during differentiation, suggesting that EGCG can reduce adipose
tissue mass both by inhibiting maturation and by increasing cell death.
The mechanism of EGCG in inducing apoptosis seems to be complicated. It has been
shown that treatment of 3T3-L1 preadipocytes with EGCG for 48 hours reduced the level and
activity of Cdk2 and decreased protein expression of cyclin D1 in a dose dependent manner
[50]. Furthermore, EGCG significantly increased protein levels of the cyclin kinase inhibitors
p21 and p27 and increased binding of p21 and p27 to Cdk2 [56, 181]. It has also been
suggested that EGCG can regulate other various antiapoptotic and apoptotic factors including
Bcl2, Bad, Bax, and p53, although further studies are required to determine precise
mechanisms [56]. It is worthwhile to mention that Cdk2 and caspase-3 have been found to
serve as mitogenic and apoptotic signal transducers in eukaryotic cells [50]. It is probable that
decreases in Cdk2 expression and activity of 3T3–L1 preadipocytes by EGCG could link to
the activation of caspase-3 via changing the mitochondrial transmembrane potential,
cytochrome c release, and the caspase-9 activity (an activator of caspase- 3) as reported for
the apoptotic effect of the tea polyphenol, theasinensin A [182].
Unfortunately, the Cdk2 pathway required for the mechanism of apoptotic action of
EGCG is still not clear (see Cdk pathway in this chapter). However, it is evident that Cdk2
appears to control G1 checkpoint in the cell cycle via regulating the formation of
retinoblastoma (Rb) protein and E2F, a transcriptional factor being able to interact either with
p53 or with caspase 3 to mediate apoptosis [183]. Accordingly, decreases in Cdk2 expression
and activity of 3T3-L1 preadipocytes by EGCG may cause an increase in cell death through
promoting the accumulation of the Rb and E2F complex in a p53- or caspase-3-dependent
pathway. This notion was indirectly supported by observation that p53-upregulated and
caspase-3-related Cdk inhibitors, p21 and p27, were increased when EGCG was added to
3T3-L1 preadipocytes [183, 184].
Overall, EGCG has been reported to induce growth arrest and apoptosis in 3T3-L1
preadipocytes. EGCG is able to block adipogenesis and increase apoptosis in mature
adipocytes. The apoptotic effect of EGCG on preadipocytes appears to be dependent on the
Cdk2 and caspase-3 pathways and is likely mediated through alterations in their activities.
The mechanistic results seem to offer a possible utility in the treatment of obesity using this
compound, EGCG.
9. BODY WEIGHT CONTROL

Obesity has increased at an alarming rate in recent years and is now one of the major
health problems worldwide. It is well known that obesity is the result of both increased
adipocyte size and/or increased adipocyte number. Currently interest in the role of plant
ingredients in weight control has focused their effects on interfering with the
sympathoadrenal systems [185]. The action mechanisms of green tea and EGCG involve
diverse routes including (1) the decrease in the energy intake [186], (2) the increase in energy
expenditure [157], and (3) the alterations in the activities of fat, liver, muscle, and intestinal
cells [11, 15, 137], resulting reduced body weight. The effects of long-term feeding with tea
catechins, especially EGCG, have been intensively studied, and some reports suggest a
potential role of green tea in body weight control. In vitro studies with green tea extracts
containing 25% of catechins have significantly inhibited the gastric and pancreatic lipases.
Thus, the lipolysis of long-chain triglycerides is reduced in a 37% [187]. It has been
suggested that green tea extracts interfere in the fat emulsification process before enzymes’
action, which is indispensable step for lipid intestinal absorption [124, 137]. It is important to
note that although green tea extracts are advisable for the treatment of overweight patients
whose body mass index ranges over 25 kg/m2, only when and if they do not present special
sensitiveness to xantic bases [188].
EGCG treatment in rats causes reduction of subcutaneous fat by 40–70% and abdominal
fat by 20–35%, but not epididymal fat [139]. The loss of fat in different proportion in obese
rats by EGCG treatment suggests the potential selective effect of EGCG on adipose tissues.
Decreased adipose fats may explain the observed decreases in adipose tissue mass and the
sequent hypolipidemia of animals treated with EGCG [139, 189]. It is important to note that
although the effective dose of EGCG on reducing body weight is 30–50 mg of EGCG/kg
body weight, rats gradually adapt and higher doses of EGCG are needed to reduce and/or
prevent body weight increases within 1 week [190]. As expected, the body weight loss is
reversible as animals regain the body weight when EGCG administration is stopped.
Figure 2. A proposed mechanism of the action of green tea EGCG on obesity.
Overall, pre-clinical and clinical studies have shown that body weight and fat mass of
human subjects and animals given green tea catechins decreased significantly. An elevation
of the expression of PPARδ, LPL and GLUT4 in adipose tissue without any effect on plasma
non-esterified fatty acids (NEFA) concentration seems to enhance fat depots. Further pre-
clinical and clinical studies will help us to fight against obesity and its associated diseases
including cardiovascular diseases and type 2 diabetes mellitus.
10. CONCLUSION
Obesity is closely associated with high blood cholesterol and has a high risk for
developing diabetes and cardiovascular diseases. Therefore, the management of body weight
and obesity is increasingly becoming important to maintain healthy cholesterol profiles and to
reduce cardiovascular risks. Interest in the health benefits of tea has been increased and led to
the inclusion of tea extracts in dietary supplements and functional foods. Specifically, EGCG
is known to have a beneficial effect on human health that reduced adipocyte differentiation
and decreased triglyceride levels. Recent reports on the action mechanism of anti-obesity
effects by EGCG have been proposed its use as a chemopreventive against obesity, diabetes,
cancer, neurodegenerative disorders, and cardiovascular diseases. Studies showed that
proliferation of preadipocyte was inhibited by EGCG in dose-, time-, and growth phase-
dependent manner. EGCG decreased levels of phosphor-ERK1/2, Cdk2, PPARγ, C/EBP and
cyclin D1 proteins, and increased levels of Go/G1 growth arrest. EGCG also causes dose-
dependent inhibition of lipid accumulation in maturing preadipocytes. It is interesting to note
that EGCG significantly stimulated the glucose uptake for the antiobesity action, which was
accompanied by a decrease in translocation of glucose transporter 4 (GLUT4) in adipose
tissue, while it significantly stimulated the glucose uptake with GLUT4 translocation in
skeletal muscle.
EGCG appears to effectively reduce the body weight via a decrease food uptake, lipid
absorption, and serum lipids, triglyceride, and cholesterol levels, as well as stimulating energy
expenditure, fat oxidation, high-density lipoprotein levels, and fecal lipid excretion (Figure
2). Finally, although we have gained in-depth knowledge on the action mechanism of EGCG
at cellular and molecular level in vitro and in vivo, further detailed and thorough studies will
certainly ensure better understanding of the regulatory mechanism of EGCG and its
associated signaling pathways, which will eventually, lead to define therapeutic effect of
EGCG against obesity, diabetes, and other related diseases.
ACKNOWLEDGEMENT
This work was supported by a grant of Biogreen 21 program (20080101-080-038-001-
0200), Rural Development Administration and GRCMVP for Technology Development
Program of Agriculture and Forestry, Ministry of Agriculture and Forestry, Republic of
Korea.
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Chapter 8
GREEN TEA: PROTECTIVE ACTION AGAINST

PESTICIDES AND OTHER XENOBIOTICS
PRESENT IN HUMAN DIET
Geetanjali Kaushik, Poonam Kaushik and Shivani Chaturvedi

Centre for Rural Development and Technology
Indian Institute of Technology Delhi, Hauz Khas New Delhi-110016, India
ABSTRACT
The indiscriminate usage of synthetic chemicals and pesticides has lead to a
widespread contamination of land, water and air with harmful xenobiotics. The exposure
to these toxicants results in severe health effects on organisms. Even some natural foods
contain harmless chemical species (nitrate) which however become toxic upon certain
conditions.
Hence it is pertinent to focus attention on commonly consumed plant food materials
that can potentially neutralize the toxicity damage caused by environmental agents. One
of the most important sources of antioxidants is green tea. This review focuses on the
mechanisms of oxidative damage caused by different xenobiotics and the defensive
action of green tea in mitigating the damage. It is concluded that tea polyphenols,
catechins and flavonoids scavenge reactive oxygen species (ROS) and render the hepato-
protective effect. However it is important to note that the protective effects of green tea
extract are rendered irrespective of the xenobiotic involved thereby suggesting the
involvement of a common biochemical pathway.
Key words: xenobiotics, antioxidants, reactive oxygen species, polyphenols
1.1. GREEN TEA PRODUCTION AND CONSUMPTION

The most popular beverage in the world is tea prepared from the leaves of Camellia
sinensis [1, 2]. Over 25 million kilos of tea are produced annually, with the bulk being
158 Geetanjali Kaushik, Poonam Kaushik and Shivani Chaturvedi
consumed by Asians, North Africans and Middle Easterners. From dried Camellia sinensis
leaves are produced the three principal types of tea products: Black Tea, Oolong Tea and
Green Tea. Black Tea is produced by the fermentative oxidation of green tea's astringent
phytochemicals, the catechin polyphenols, producing related compounds called theaflavins.
This process removes an appreciable amount of the bitterness characteristic of green tea,
yielding a milder, and more (subjectively) flavored beverage. Oolong tea results from a
partial oxidation of green tea polyphenols. Green tea is produced with the goal of preserving
the leaf polyphenols. After the leaves have been picked, the polyphenoldegrading enzymes
are inactivated by steaming or pan roasting and drying [3].
By tradition the Orient, North Africa and the Middle East favor green tea and the rest of
the world drinks black tea. Oolong tea production is confined to China and Taiwan. Black tea
accounts for 80% of the world tea crop [1, 2].
1.2 GREEN TEA - ACTIVE COMPONENTS

Tea leaves harbor a myriad of plant chemicals, phytochemicals, possessing a broad
spectrum of biological actions. These include antioxidant, anticancer, anti -hypertensive, anti-
cholesterolemic and antimicrobial activities.
The polyphenols are the active players in green tea, mediating both taste profile and
biological actions. From a chemical perspective, green tea polyphenols are catechins,
phytochemicals composed of several linked ring-like structures. Attached to each structure
are chemical tags called phenol groups, and because there are many phenol groups, these
catechins are called polyphenols. In native green tea, approximately 15-30% of the weight of
the leaf is composed of polyphenols. The major anti-inflammatory and anti-oxidative
polyphenols present in green tea are (−)-epigallocatechin gallate (EGCG),(−)-epigallocatechin
(EGC), (−)-epicatechin gallate (ECG), (+)-epicatechin (EC), (−)-gallocatechin gallate (GCG)
and (−)-catechin [14, 15, 16].
Over 50% of this polyphenol fraction is comprised of (-) Epigallocatechin
Gallate(EGCG), the most biologically active and influential polyphenol in green tea. Other
components include the unique amino acid theanine, carotenoids, chiorophyll and caffeine
[3].
Anthocyanidins, plant pigments are also found in green tea. Caffeine occurs in green tea
leaves at a level of 3%; brewed green tea contains approximately 35-50 mg of caffeine per
cup, contrasted to a cup of coffee, which contains between 75-95mg [3].
1.3. MECHANISM OF ACTION OF ACTIVE COMPONENTS

Green tea polyphenols, and especially EGCG, have been shown to not only protect
against undesirable prooxidant attack but also to detoxify radicals produced from the
environmental toxins paraquat. Additionally, EGCG has been shown to be over 200 times
more potent than vitamin E in protecting fats in the brain, which are exceptionally susceptible
to oxidative stress [3].
Green Tea 159
Figure 1. Chemical structure of tea catechins [50].

EGCG is a potent antioxidant, since the multiple available phenolic groups capture pro-
oxidants and free radicals, there by affording protective action against oxidative stress [3].
Hence, the antioxidative properties of green tea are due to its ability to scavenge reactive
oxygen species (ROS), to inhibit their generation and to chelate transition metal ions that can
participate in free radical transformations and in the processes of lipid peroxidation [1, 4, 5].
In addition tea polyphenols have been found to prevent inflammation, eliminate the excess
free radicals and stimulate the regeneration of damaged cells or tissues [12, 13, 16]. These are
also associated with the inhibition of redox-sensitive transcription factors, inhibition of pro-
oxidant enzymes and induction of phase II enzymes [22].
1.4. GREEN TEA: PROTECTIVE EFFECT AGAINST XENOBIOTICS

The indiscriminate usage of synthetic chemicals and pesticides has lead to a widespread
contamination of land, water and air with harmful xenobiotics. The exposure to these
toxicants results in severe health effects on organisms. Even some natural foods contain
harmless chemical species (nitrate) which however become toxic upon certain conditions.
Hence it is pertinent to focus attention on commonly consumed plant food materials
which are natural sources of relevant antioxidants such as polyphenols which can neutralize
the toxicity damage caused by environmental agents. One of the most important sources of
antioxidants is green tea. This review focuses on the mechanisms of oxidative damage caused
by different xenobiotics and the defensive action of green tea in mitigating the damage. The
xenobiotics selected include alcohol, carbon tetrachloride, pesticides, benezene,
nitrosoamines, PAHs, nitroquiunoline oxide and dopamine.
i) Alcohol
Ethanol is known to induce rapid lipid peroxidation and activation of NF-κB, which are
critical events responsible for mucosal hemorrhages and edema, in vascular smooth muscle
cells and endothelial cells [17, 18, 16].
The alcohol oxidation to acetaldehyde and acetate is accompanied by ROS formation [6,
5]. The erythrocytes are especially exposed to the action of ROS as they contain large amount
of iron and oxygen which promote free radical processes [7,5]. Both, ethanol and its
metabolites, can react with cell components, including biological membrane. Ethanol reduces
the cell membrane surface hydration and affects the membrane protein–lipid structure [8, 5].
Acetaldehyde and ROS can react with proteins and thus modify their structure and functions
[9–11, 5]. Moreover, ROS is also responsible for lipid peroxidation [6, 5]. Polyunsaturated
fatty acids, originating from membrane phospholipids, are particularly vulnerable to
peroxidation. The erythrocyte membrane, in particular, is constantly subjected to oxidative
stress due to high content of peroxidizable [11, 5]. The influence of green tea on total
antioxidant status (TAS) and on composition and electric charge of erythrocyte membrane
phospholipids in ethanol intoxicated rats was examined. It was found that ethanol
administration caused, in term, decrease in TAS and increase in the level of all phospholipids
and lipid peroxidation products. Ethanol as well significantly enhanced changes in surface
Green Tea 161
charge density of erythrocyte membrane. The ingestion of green tea partially prevented
decrease in erythrocyte antioxidant abilities observed during ethanol intoxication. Moreover,
long-term drinking of green tea protects the structure of the erythrocytes membrane disturbed
during chronic ethanol intoxication [5].
A study was designed to evaluate the efficacy of green tea polyphenols in protecting
against alcohol-induced gastric damage and to elucidate the underlying mechanisms.
Intragastric administration of ethanol to male Sprague–Dawley rats caused significant gastric
mucosal damage, which was accompanied by elevated expression of cyclooxygenase-2
(COX-2) and inducible nitric oxide synthase (iNOS) as well as transient activation of redox-
sensitive transcription factors, such as NF-κB and AP-1, and mitogen-activated protein
kinases (MAPKs). Oral administration of the green tea polyphenolic extracts (GTE)
significantly ameliorated mucosal damages induced by ethanol and also attenuated the
ethanol-induced expression of COX-2 and iNOS. Inactivation of MAPKs, especially p38 and
ERKl/2, by GTE might be responsible for inhibition of ethanol-induced expression of COX-2
and iNOS [16].
ii) Carbon-tetrachloride
Oxidative stress has been thought to be a major cause of CCl4-induced liver injury, in
which CCl4 is metabolized by cytochrome P450 in liver cells to yield the trichloromethyl free
radical. These radicals cause lipid peroxidation, which produces hepatocellular damage and
enhanced production of fibrotic tissue [21] and the depletion of antioxidant status [19, 20,
21]. In this regard, reduction of oxidative stress may be a potential and effective therapeutic
strategy for prevention and treatment of hepatic fibrosis. The effects of epigallocatechin-3-
gallate (EGCG) on hepatic fibrogenesis were studied. The rat model of carbon tetrachloride
(CCl4)-induced hepatic fibrosis was used to assess the effect of daily intraperitoneal injections
of EGCG on the indexes of fibrosis. Histological and hepatic hydroxyproline examination
revealed that EGCG significantly arrested progression of hepatic fibrosis. EGCG caused
significant amelioration of liver injury (reduced activities of serum alanine aminotransferase
and aspartate aminotransferase). The development of CCl4-induced hepatic fibrosis altered
the redox state with a decreased hepatic glutathione and increased the formation of lipid
peroxidative products, which were partially normalized by treatment with EGCG,
respectively. Moreover, EGCG markedly attenuated HSC activation as well as matrix
metalloproteinase (MMP)-2 activity. Therefore, the administration of EGCG may be an
optional therapeutic and preventive measure against oxidative stress-induced liver injury and
hepatic fibrosis [21].
iii) Pesticides
Pesticides have been used in agriculture to enhance food production by eradicating

unwanted insects and controlling disease vectors. Occupational exposure to pesticides is
becoming a common and increasingly alarming worldwide phenomenon. Approximately 3
million acute poisonings and 220,000 deaths from pesticide exposure have been reported
annually [23–25, 32]. The health effects caused by this occupational exposure are enormous.
Chlorpyriphos- O, O-diethyl-O- (3, 5, 6-trichloro-2-pyridinyl) phosphorothioate, is

classified as a moderately hazardous, Class II insecticide by the WHO [26, 32]. It belongs to
the phosphorothioate class of insecticides. Its acute toxicity varies according to the species
and route of exposure [27, 32]. The chief mechanism of action of organophosphorus
pesticides occurs by the inhibition of neuronal cholinesterase activity, a key enzyme that is
involved in neurotransmission [28, 32]. Acute and chronic exposure to chlorpyriphos results
in considerable liver damage as evidenced by changes in aspartate transaminase (AST),1 and
alanine transaminase (ALT) [29, 32]. These toxic effects probably occur through the
generation of reactive oxygen species causing damage to the various membranous
components of the cell. This is proved by marked changes to the overall histoarchitecture of
liver [30, 32]. Hepatic atrophy has also been observed when repeated doses of chlorpyriphos
are given [31, 32]. This paper reports the effect of green tea administration following
subacute toxicity caused by exposure to organophosphorus pesticide chlorpyriphos in liver of
rats. Four groups containing five male Sprague–Dawley rats each were selected. Group I
served as control. Group II rats were permitted free access to solubilised crude extract of
green tea (1.5%w/v in water) as the sole drinking fluid. Group III rats were given a single
daily oral dose of chlorpyriphos (30 mg/kg bodyweight in corn oil). Group IV rats received
oral dose of pesticide and green tea extract simultaneously. All rats were sacrificed after 15
days. Significant damage to liver was observed via increased serum levels of transaminases
and alkaline phosphatase. Lipid peroxidation showed a 5-fold increase in pesticide exposed
rats compared to control. In contrast, levels of antioxidant GSH, glutathione-dependent
enzymes like glutathione peroxidase (GPx), glutathione S-transferase (GST) and free radical
scavengers like catalase (CAT) and superoxide dismutase (SOD) were significantly lower
than those of the control group reinforcing oxidative damage. The use of green tea extract
appeared to be beneficial to rats, although not to a great extent in significantly reducing and
reversing the damage sustained by pesticide exposure and favors recovery since a decrease in
lipid peroxidation and enhancement of antioxidant GSH level is also observed following
supplementation with green tea extract. Green tea treatment may either replenish the levels of
antioxidant directly or spare the endogenous pool of GSH from being exhausted by the free
radicals generated [32].
Fenitrothion- (O,O-dimethyl-O-(3-methyl-4-nitrophenyl) phosphorothioate is an
organophosphorus insecticide widely used for controlling a wide range of insects and other
pests. Although fenitrothion exhibits low mammalian toxicity, biochemicals, morphological
and functional alterations in animal tissues have been reported. The prolonged administration
of fenitrothion increased the concentration of corticosterone and glucose in the plasma of
male rats. It also increases the weight of the adrenal gland of male rats and altered its
functions [33, 44]. Dermal, inhalation and oral exposure to fenitrothion inhibit
acetylcholinesterase enzyme (AChE)1 in plasma, erythrocytes and brain of mammals
[34,35,44], in addition to a considerable liver and kidney damage evidenced by elevation in
serum AST and ALT [36,37,44]. Another studies recorded an increase in serum cholesterol
and alternation in cell membrane fluidity and lipid content [38, 44]. Acute and chronic
exposure to fenitrothion induced ultra structural changes in liver and kidney cells of rats with
complete distortion of the nuclear membrane, total loss of nuclear intactness and abnormally
enlarged smooth endoplasmic reticulum [39, 44]. These toxic effects probably occur through
the generation of reactive oxygen species (ROS) causing damage to various membranous
components of the cell [40, 44]. The biochemical basis of oxidative damage is becoming
Green Tea 163
clearer as recent studies point to the production of ROS as a secondary means of toxicity [41,
44]. Cells succumb to oxidative damage when endogenous store of antioxidants is used up by
the oxidant exposure. The antioxidant machinery is composed of enzymes like glutathione S-
transferase (GST) [42, 44], and non-enzymatic components are primarily composed of thiols
and glutathione (GSH) [43, 44]. The ameliorative effect of daily administrated dose of green
tea extract (60 mg polyphenols/animal/day) was investigated on albino rats Rattus norvegicus
(150–180 gm) intoxicated with 1/30 and 1/60 LD50 fenitrothion organophosphate insecticide
for 28 days. Blood samples were taken at 14 and 28 days for further biochemical parameters.
Histopathological studies were carried out in the liver and kidney at the end of the
experiment. Significant inhibition in plasma cholinesterase (ChE), a biomarker of Ops, was
recorded. Damage in the liver and kidney tissues was observed and confirmed with elevation
of plasma alanine aminotransferase (ALT), aspartate aminotaransferase (AST), albumin, urea
and creatinine, as well as an elevation in the oxidative stress (OS) marker malondialdehyde
(MDA). Decrease in total glutathione (GSH) content in erythrocytes and fluctuation in
glutathione S-transferase (GST) activity in plasma was also observed. Green tea
supplementation (60 mg/animal/day) partially counteracts the toxic effect of fenitrothion on
oxidative stress parameters and repairs tissue damage in the liver and kidney, especially when
supplemented to 1/60 LD50 intoxicated animals depending on the duration. It seems that
enzyme and metabolite markers of these organs need more time to be restored to the control
level [44]. Supplementation with green tea extract to fenitrothion intoxicated animals
counteracts plasma lipid peroxidation biomarker (MDA) [45, 44]. Green tea treatment may
either replenish the levels of antioxidant directly or spare the endogenous pool of GSH from
being exhausted by the generated free radical [46, 44]. Meanwhile, green tea polyphenols
stimulate the transcription of phase II detoxifying enzymes mediated by an antioxidant-
responsive element [47, 44]. This could explain the significant elevation in the glutathione S-
transferase enzyme activity recorded in the green tea treated groups in the current study. In
spite of the previous ameliorative effect of green tea supplementation on some antioxidant
parameters, it needs more time for ameliorating membrane damage in the liver parenchymal
cells to prevent over leakage of liver enzyme markers (AIT and AST) in plasma This
hypothesis is supported by the histopathological findings in liver and kidney tissues in the
groups supplemented with green tea. Less damage was clearly noticed in liver with green tea
supplementation especially with the group of intoxicated with low dose fenitrothion and green
tea. The liver architecture was preserved withless hemorrhage and less cell degeneration;
however, inflammatory cells were noticed [44].
Paraquat - (1, 1-dimethyl-4, 4-bipyridilium; Pq) is a commonly used herbicide. Although
the exact mechanism of Pq toxicity is not completely elucidated, numerous arguments suggest
that free-radical generation by Pq plays an essential role in the mechanism of its toxicity [48,
49].
EGCg inhibited Pq-induced microsomal malondialdehyde (MDA) productions in rat liver
microsome system containing 40 mM FeSO4. Forty micromolar EGCg inhibited MDA
production significantly. EGCg may inhibit the Pq-induced MDA production by at least two
mechanisms. One may be iron-chelating activity as the inhibition disappeared when excess
amounts of FeSO4 were added to the reaction mixture, which indicated that EGCg reduced
iron driven lipid peroxidation by pulling out available irons in the reaction mixture. The other
is radical scavenging activity. EGCg scavenged DMPO-OOH spin adducts generated by the
microsome-Pq system. EGCg inhibited iron-driven lipid peroxidation presumably not only by
chelating to Fe ions but also by scavenging superoxide radicals, which are responsible for the
reduction of ferric (Fe3+) to ferrous (Fe2+) that catalyzes the Fenton reaction. Chelating and
radical scavenging activity of EGCg can be expected simultaneously in the occurrence of Pq
toxicity, which may explain the protective effects of EGCg against Pq toxicity [49].
Rotenone and dieldrin- A common denominator in the pathogenesis by pesticides
rotenone and dieldrin is the involvement of oxidative stress-mediated apoptotic process [50,
51, 52, 53]. Gamma-glutamylethylamide (L-theanine), a natural glutamate analog in green
tea, has been shown to exert strong anti-ischemic effect. The protective effects of L-theanine
on neurotoxicants, rotenone and dieldrin in cultured human dopaminergic cell line, SH-
SY5Ywere investigated. The experiments revealed that L-theanine (500 mM) attenuated both
rotenone- and dieldrin-induced morphological changes of nuclei and HO-1 elevation which is
related to overproduction of ROS [53].
Tamoxifen- A recent study has shown that tamoxifen-induced hepatotoxicity in female
rats that occurs due to oxidative stress can be reversed by green tea administration [54, 32].
iv) Nitrosoamines
Nitrite is an important additive in production of cured meat products in terms of

maintaining desirable color, texture, and especially with respect to preventing toxin formation
by Clostridium botulinum [55,71]. Recent evidence has suggested that nitrite is bactericidal
for pathogenic gastrointestinal, oral and skin bacteria when ingested and mixed with gastric
acid [56, 67]. However, significant concerns still exist because the nitrite may react with
amines and amino acids to produce N-nitrosamines, which are known to be carcinogenic,
mutagenic and teratogenic [57–59, 71]. The formation of N-nitrosamines in cured meat
products is dependent on the cooking method, residual and/or added nitrite concentration,
ascorbate or a-tocopherol concentration, nitrosamine precursors, moisture content, lean-to-
adipose tissue ratio, presence of nitrosation catalysts and inhibitors, and possibly the smoking
process [60–62, 71]. High nitrate and amine-rich food intake has been shown to increase the
risk of endogenous formation of carcinogenic N-nitroso compounds (NOCs) [63, 64, 71].
Although nitrate itself is relatively nontoxic, approximately 5% of ingested nitrate is
converted to the more toxic nitrite form in the oral cavity [65, 66, 71]. Since the discovery in
1992 that N-nitroso compounds were carcinogenic to rodents [67, 71], this class of versatile
carcinogens has been shown to induce tumors in most bodily organs of 40 animal species [68,
71]. Further, human exposure to endogenously formed NOCs has been related to increased
risk of gastric, esophageal, nasopharyngeal and bladder cancers [69, 71]. The endogenous
nitrosation occurs to a great extent when the precursors are ingested in foods. Nitrite can react
with secondary amines under the acidic conditions of the stomach to form N- nitrosamine.
Nitrosamine formation in the acidic environment of the stomach can take place when
both nitrite and secondary amines are present. Nitrite is readily protonated to nitrous acid
(HNO2), with two molecules of nitrous acid forming nitrogen trioxide (N2O3), which is the
actual nitrosating species that reacts with unprotonated amines to form nitrosamines [70,71].
In vitro experiments were performed to test inhibition of nitrite-mediated N-nitrosation by
individual catechins, green tea. The extent of inhibition was measured via nitrosamine
formation. The results show that N-dimethylamine nitrosation is partially inhibited by green
tea with or without enzymatic modification. The inhibition of nitrosation by catechins is a
Green Tea 165
result of competition between the secondary amines and catechins for nitrite in the reaction
mixture [71].
Carcinogenic N-nitroso compounds, which are easily produced under the acidic
conditions in the stomach, might cause stomach cancer [72, 76]. Soy sauce has the highest
mutagenicity when various Japanese foodstuffs are treated with nitrite and it is one of the
most important foods concerning to the relationship of Japanese food consumption and the
high gastric cancer mortality in Japan [73, 76]. Tyramine is a major mutagen precursor in soy
sauce treated with nitrite, and 1-methyl-1, 2, 3, 4- tetrahydro-b-carboline-3-carboxylic acid
(MTCCA) is a minor one [74, 76]. However, MTCCA becomes the most potent mutagen in
soy sauce when it is treated with nitrite in the presence of ethanol, while the mutagenicity
induced from nitrite-treated tyramine strongly decreases in the presence of ethanol [75, 76].
It has been shown that the mutagenicity of 1-methyl-1, 2, 3, 4-tetrahydro-b-carboline-3-
carb-oxylic acid (MTCCA), a major mutagen precursor in soy sauce on treatment with nitrite
and ethanol, was strongly decreased by the addition of hot water extracts of green, black and
oolong teas in the reaction mixture when it was treated with 50 mM nitrite at pH 3.0, 378C
for 60 min in the presence of 7.5% ethanol. The mutagenicity-decreasing activity of the teas
was scarcely decreased by washing the teas with chloroform and benzene and was partly
decreased by butanol and ethyl acetate. Typical polyphenols such as catechins were shown to
have the antimutagenicity dose dependently. The antimutagenicity and the reducing power of
tea extracts gave a positive good correlation. The results suggest that the mutagenicity of
MTCCA on treatment with nitrite in the presence of ethanol may be decreased by the mixed
fractions of lyophilic components such as polyphenols, which have high reducing power such
as catechins and the other compounds which have little reducing power including the
derivatives of the catechins and so on. Although the antimutagenicity of teas and catechins
was also considerably effective when they were added after the nitrosation, while that of
black tea and some catechins was less effective [76].
High nitrate and amine-rich food intake has been shown to result in an increased risk of
endogenous formation of carcinogenic N-nitroso compounds (NOCs) [77, 78, 79]. Vegetables
and vegetable products are an important source of nitrate in an average diet. The formation of
carcinogenic nitrosamines under simulated gastric conditions was studied during the
incubation of amine rich food and nitrate, and its possible inhibition by adding green tea
extracts. The green tea extracts were effective in reducing the formation of N-
nitrosodimethylamine (NDMA). During four weeks of test (designated EW1, EW2, EW3 and
EW4; experiment week 1, 2, 3 and 4 diets) volunteers consumed a diet of low nitrate and
amine (EW1)and consumed a fish meal rich in amines as nitrosatable precursors in
combination with intake of nitrate-containing drinking water without (EW2)or with green tea
extracts (EW4, respectively). The intake of nitrate-containing drinking water (340 mg
nitrate/100 ml) resulted in a significant rise in mean salivary nitrate and nitrite concentrations
and in mean urinary nitrate levels. Mean urinary nitrate was increased to 455.0±66.2,
334.6±67.8 and 333.4±50.7 mg/18 h after the nitrate intake of EW2, EW3 and EW4,
respectively. Significant increases in urinary dimethylamine and trimethylamine levels were
observed in consumption of diets (EW2, EW3, and EW4) rich in amine and nitrate. Green tea
extract in EW3 and EW4 enhanced the increase of urinary dimethylamine and trimethylamine
levels. Urinary excretion of N-nitrosodimethylamine in consumption of diet rich in nitrate and
amine (EW2) increased to 6504.4±2638.7 ng/18 h from 257.0±112.0 ng/18 h of low nitrate
and amine diet (EW1). Korean green tea in nitrate and amine rich diet reduced the excretion
of N-nitrosodimethylamine to 249.7±90.6 respectively, compared with 6504.4±2638.7 ng /18

h after ingestion of TD1 diet [79].
v) PAHs
Many of the polycyclic aromatic hydrocarbons (PAHs), some of which are common
environmental pollutants, are known to have carcinogenic and mutagenic effects [80, 83]. The
PAHs themselves are relatively inert biologically and essentially act as precarcinogens that
must first undergo metabolic activation by the cytochrome P-450-dependent and other
enzymes to their biologically active ultimate carcinogenic metabolites [81, 83]. The
cytochrome P-450 enzyme system is present and inducible in mouse skin [82, 83].
Benzo[a]pyrene (BP) is a prototype of PAHs that is metabolized by sequential reactions of
this enzyme system and epoxide hydrolase to the chemically reactive ultimate carcinogenic
metabolite BPDE-2 (7f3, 8a-dihydroxy- 9a, 1 Oa-epoxy- 7,8,9,1 O tetrahydrobenzo[a]pyrene)
[81,83].
The effect of pretreatment of skin of Sencar mice with topically applied green tea
polyphenols (GTP) on the skin tumor initiating activity of ( +- )- 7f3,8a-dihydroxy- 9a, 1 Oa-
epoxy- 7,8,9,1 O-tetrahydrobenzo[a]pyrene (BPDE-2) had been evaluated. The animals were
pretreated with the plant phenols (GTP 24 mg/mouse) for 7 days after which they received a
single topical application of 200 nmol of BPDE-2 as the initiating agent. Beginning 7 days
following initiation animals received twice weekly applications of 3.24 nmol of l2-0-
tetradecanoyl phorbol-13-acetate (TPA). GTP afforded significant protection against skin
tumor induction. These inhibitory effects were verified both by prolongation of the latency
period and subsequent development of tumors. Our results suggest that tannic acid and GTP
have substantial potential for protecting against the skin tumorigenic response to BPDE-2 and
the mechanism of inhibition may involve inactivation of the reactive carcinogenic moiety
[83].
vi) Benzene
Benzene is a ubiquitous environmental contaminant as well as an important industrial

chemical. It is used in the manufacture of a wide variety of consumer goods and is a
constituent of gasoline and tobacco smoke [84, 85,105]. Chronic exposure to benzene is
known to induce aplastic anemia, leukemia, and other related blood disorders [86,105].
However, the precise mechanisms by which benzene induces these effects are unknown.
Benzene itself is unlikely to be the actual toxicant but rather, is converted to bioactive
metabolites which cause myelotoxicity [87,105]. Benzene is metabolized to benzene oxide by
P450IIEl monooxygenase enzymes in the liver. Benzene oxide gives rise to phenolic
metabolites, such as phenol (PH), catechol (CT), hydroquinone (HQ) and 1,2,4-benzenetriol
(BT) and open-ring products, such as t,t muconaldehyde and t,t muconic acid [88,105]. The
phenolic metabolites can be further oxidized by peroxidases to the corresponding reactive
semiquinones and quinones, e.g. CT is converted to 1,2-benzoquinone (1,2-BQ), HQ to 1 ,4
benzoquinone (1,4- BQ) and BT to 2-hydroxy-1,4 BQ [88,105]. It has been reported that
several metabolites of benzene are capable of reacting with both DNA and protein [89,
Green Tea 167
90,105], reducing bone marrow cellularity and developing colony forming unit-erythroid in
mice [91,105]. These compounds also decrease host resistance to microorganisms and tumors
[92,105], and result in cytochrome P450 destruction [93,105]. In addition to these findings,
administration of metabolites of benzene also induces sister chromatid exchanges,
micronuclei and aneuploidy [94- 97,105], and inhibits the synthesis of DNA and RNA [98,
99,105]. They can also induce oxidative DNA damage [96,100,105] and DNA strand
breakage [l01, 102,105] in target cells. One mechanism by which benzene induces these
genotoxic effects may be mediated by the generation of one or more active oxygen species
(AOS) such as superoxide anion radical, hydrogen peroxide, hydroxyl radical and singlet
oxygen [88,105]. According to earlier studies, several polyphenolic metabolites of benzene
such as HQ, CT, BT and related polyphenol, PG, can produce AOS by autooxidation and they
are highly mutagenic [103,104,105]. Hence, especially in the presence of cuprous and iron
[96,105], benzene metabolites can increase the level of 8-hydroxy deoxyguanosine (8-
OHdG), suggesting that metabolites of benzene can induce oxidative DNA base modification
in cells [ 96,100,102,105] and may therefore play a role in benzene-induced genotoxicity,
myelotoxicity and leukemia.
Autooxidation of polyphenolic metabolites of benzene, such as hydroquionone (HQ),
catechol (CT), 1, 2, 4-benzenetriol (BT) and pyrogallol (PG), produced several kinds of active
oxygen species (AOS). BT and PG induced DNA breaks in the absence of metal ions,
especially when producing AOS such as H202, 02-, HO. or ΔgO2. HQ and CT did not result in
double-strand DNA breaks, except when ferrous ion was added, indicating the participation of
the Fenton reaction. Polyphenolic fractions isolated from green tea (GTP) exerted inhibitory
effects on the autooxidation of BT and suppressive effects on H202, or HO. generated from
phenolic metabolites of benzene in the presence of S9 or an in vivo system [105].
vii) 4-Nitroquiunoline 1-oxide
4-Nitroquiunoline 1-oxide (4-NQO), a water soluble oral carcinogen, produce papilloma

and invasive squamous cell carcinoma, resulting in clinical and histologic changes which are
similar to those observed in neoplasms of humans (106,111). 4-NQO is known to induce
multistep carcinogenesis (107,111). 4-NQO is also known to induce H-ras mutation in
chromosome 7 leading to head and neck squamous cell carcinoma in experimental murine
models (108,111). Carcinoma is preceded by a sequence of hyperplasia–papilloma/dysplasia–
carcinoma, similar to that of human oral cancer (109,111). 4-NQO is also known to cause
intracellular oxidative stress
(110,111), which leads to lipid peroxidation. 4-NQO at the concentration of 1.5mMwas
found to induce lipid peroxdation in 5% liver homogenate in phosphate buffered saline and
extent of lipid peroxidation at the different time intervals 0, 15, 30 and 45 min was studied by
assessing parameters such as hydroxyl radical production (OH), thiobarbituric acid reactants
(TBARS), reduced glutathione (GSH), superoxide dismutase (SOD) and catalase (CAT). It
was found that addition of 4- NQO caused an increase in OH and TBARS level and a
decrease in activity of SOD, CAT and the levels of GSH. Simultaneous addition of GP 10
mg/ ml significantly decreased lipid peroxidation and increased in antioxidant status. Thus, it
was concluded that green tea polyphenols, were found to nullify 4-NQO induced lipid
peroxidation in vitro and 4-NQO acts initially by causing oxidative stress and leads to
carcinogenesis (111).
viii) 6-hydroxydopamine (6-OHDA)
6-hydroxydopamine (6-OHDA), is known to induce dopaminergic neurodegeneration via

oxidative stress(112,113).
6-OHDA (350 and 50 µM) activated the iron dependent inflammatory redox sensitive
nuclear factor-κB (NF-κB) in rat pheochromocytoma (PC12) and human neuroblastoma (NB)
SH-SY5Y cells, respectively. Immunofluorescence and electromobility shift assays showed
increased nuclear translocation and binding activity of NF-κB after exposure to 6-OHDA in
NB SH-SY5Y cells, with a concomitant disappearance from the cytoplasm. Introduction of
GT extract (0.6, 3 µM total polyphenols) before 6-OHDA inhibited both NF-κB nuclear
translocation and binding activity induced by this toxin in NB SH-SY5Y cells.
Neuroprotection was attributed to the potent antioxidant and iron chelating actions of the
polyphenolic constituents of tea extracts, preventing nuclear translocation and activation of
cell death promoting NF-κB. Brain penetrating property of polyphenols may make such
compounds an important class of drugs for treatment of neurodegenerative diseases (113).
CONCLUSION
There is evidence that synthetic antioxidants cause toxicity [114; 115, 116] through
oxidative stress so it is important to focus on plant food materials that are a part of the human
diet. Green tea is a popular beverage world over and is known to be a rich source of potent
antioxidants. Hence it is important to investigate the role of green tea as a natural antioxidant
in order to prevent or retard oxidative stress from environmental agents.
It is clear from the above discussion that tea polyphenols, catechins and flavonoids
scavenge reactive oxygen species (ROS) [117, 32] and render the hepato-protective effect.
However it is important to note that the protective effects of green tea extract are rendered
irrespective of the xenobiotic involved thereby suggesting involvement of a common
biochemical pathway [118, 32].
FUTURE SCOPE
Most of these studies related to oxidative stress as a consequence of pesticide exposure
have been performed in experimental animals. And studies on oxidative stress in human
subjects exposed to different pesticides have not received much attention as yet. It is therefore
important to investigate oxidative stress, and derangement of the antioxidant defense system
in stemming from the ingestion of pesticides in human poisoning cases.
There is also need to investigate the protective effects of green tea on oxidative stress
induced by other xenobiotics and natural agents.
Green Tea 169
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Chapter 9
NEW METHOD TO IMPROVE THE FUNCTION AND

INDUSTRIAL APPLICABILITY OF GREEN TEA AND ITS
BYPRODUCTS USING IRRADIATION TECHNOLOGY
Cheorun Jo1 and Myung Woo Byun2,*

1
Department of Animal Science and Biotechnology, Chungnam National University,
Daejeon, 305-764, Republic of Korea
2
Advanced Radiation Technology Institute, Korea Atomic Energy Research Institute,
Jeongeup, 580-185, Republic of Korea
ABSTRACT
Green tea is a well-known biomaterial with various high biological functions.
Irradiation was introduced to develop a new processing method to improve color of the
extract, resulting in a higher applicability without any adverse change to the beneficial
functions such as inhibitory effects of oxidation, melanin hyperpigmentation on the skin,
and others. To investigate the application of irradiated green tea leaf extract for a real
cosmetic composition, the physiological activities of irradiated green tea leaf extract
powder dissolved in butylenes glycol and ethanol were compared to a commercial green
tea extract product. Furthermore, a cream lotion was manufactured using the powder and
the physiological activities were compared. Results showed that the irradiation of the
green tea leaf extract and the freeze-dried powder from the extract had the same
physiological activities as the commercial product in a cosmetic composition. Addition of
irradiated green tea leaf in a patty can retard lipid oxidation and add the biological
functions green tea possessed, however, the intensity of off-odor produced from green tea
is a concern. Using irradiated green tea powder, the off-odor problem can be solved.
Some research utilizing green tea byproducts were investigated. Irradiation of green tea
byproducts extract showed higher biological functions than that of non-irradiated
counterparts. Therefore, irradiation technology can be a useful method to improve
biological functions and industrial applicability of green tea and its byproducts.
*
To whom all correspondence should be addressed : Dr. Myung Woo Byun, Phone) +82-63-570-3400, Fax) +82-
63-570-3409, Email) cheorun@cnu.ac.kr
178 Cheorun Jo and Myung Woo Byun
Keywords: green tea, irradiation, biological function, application
INTRODUCTION
Separation of bioactive compounds from natural resources, which have been consumed
safely for a very long time by human beings, and their application to various fields including
the food, pharmaceutical, and cosmetic industry have been actively studied (Choi et al.,
1989). Among them, green tea has the longest history in the world and is used in about 160
countries every day as a drinking tea. Green tea is one of the 3 most popular beverages
besides coffee and cocoa (Kim, 1996). Green tea is composed of about 30% polyphenols (dry
basis) such as flavanol, flavandiol, flavonoid, and phenol acid. These polyphenols are well-
known to have various excellent biological activities, for example, the inhibition of tooth
decay (Sakanaka et al., 1989), inhibition of allergies (Yeo et al., 1995), reduction of blood
pressure (An, 1998), prevention of gout (An et al., 1996) and the inhibition of oxidation. Oak
et al. (2005) reported that the polyphenols from green tea and red wine have the antioxidant
properties mostly because of their ability to directly scavenge reactive oxygen species such as
a hydroxyl radical and a superoxide anion. Especially, the inhibition effect of green tea
polyphenol on lipid oxidation is higher than that of synthetic antioxidant, butylated
hydroxytoluene (BHT) (Wanasundara & Shahidi, 1998; Chen et al., 1996).
On the other hand, food irradiation is known to be the best method for controlling
pathogenic microorganisms and one of the best alternatives to the chemical fumigants or
preservatives usually used for a sanitation treatment for international trade (WHO 1999).
Irradiation technology has been officially adopted by international organizations
(WHO/IAEA/FAO) and experts due to its effectiveness in food, wholesomeness and
economic benefits. Besides the sanitary purposes, irradiation has been studied to reduce or
eliminate undesirable or toxic materials including food allergens (Lee et al., 2000; Lee et al.,
2001), carcinogenic volatile N-nitrosamines in sausage (Ahn et al., 2002; Ahn et al., 2003; Jo
et al., 2003e), biogenic amines (Kim et al., 2003, Kim et al., 2004), embryotoxicity of
gossypol (Jo et al., 2003c), and enhancement of the antioxidant power of phytic acid (Ahn et
al., 2004). In addition, irradiation has been shown to enhance the color of low-nitrite meat
products (Byun et al., 2000a) and to develop low-salt fermented traditional foods (Byun et al.,
2000b; Lee et al., 2002; Kim et al., 2002; Jo et al, 2004).
Byun et al. (2002) observed the breakdown of chlorophyll by irradiation, which can be
used in oil processing. Based on this result, an application for color removal of green tea leaf
extract (Jo et al., 2003a) was developed. Then, the commercial application of irradiation for
the color improvement of plants-derived products without changing their beneficial biological
activities was tested in foods or cosmetics (Jo et al., 2003b; Byun et al., 2004a).
In this chapter, some of the background research and the selected results of the related
studies are introduced and discussed for the application of irradiation technology to improve
functions and industrial applicability for the green tea industry.
New Method to Improve the Function and Industrial Applicability of Green Tea … 179
COLOR IMPROVEMENT OF GREEN TEA

LEAF EXTRACT BY IRRADIATION
Green tea leaf has been used mostly for brewing. This is mainly because of its deep dark
color and off-flavor, which makes it very difficult to apply the proper amount for the
compositions of cosmetics, medicine or foods. The feasibility of using irradiation to develop a
new processing method to obtain a light-colored material while maintaining its biological
function has been conducted (Jo et al., 2003a).
Before the irradiation was applied for color enhancement of green tea leaf extract, the
authors were working for the irradiation effect on soybean oil. The presence of green
pigments of chlorophyll in soybean oil is of interest not only for their impact on the color of
the finished product but also for their potential role in oxidative stability, especially for
photooxidation (Usuki et al., 1984; Fakourelis et al., 1987). Chlorophylls not only cause an
undesirable color change in vegetable oils but impair the hydrogenation process (Daun, 1982)
and promote oxidation in the presence of light although they may be antioxidants under dark
conditions (Abraham & deMan, 1986). A study was conducted to investigate the possibility
of chlorophyll breakdown by irradiation to inhibit the photooxidation during storage without
accelerating lipid oxidation during the irradiation process (Byun et al., 2002). Results showed
that the oil sample containing 3 ppm of chlorophyll showed no detectable chlorophyll after
being irradiated at 2 kGy either with or without N2 flush (Table 1). The non-irradiated control
sample stored in the dark to avoid a photooxidation showed no change in chlorophyll levels
during 6 hr storage. Results on peroxide values (POV) indicate that irradiation increased lipid
oxidation but the chlorophyll breakdown in the sample irradiated at 20 kGy did not induce the
photooxidation when exposed to light (Table 2). Irradiation of samples without oxygen
(treated by continuous N2-flush), did not develop lipid oxidation during the irradiation
process or photooxidation during storage under light (Byun et al., 2002). The POV value of
20 kGy-irradiated samples with N2-flushing remained 0 during the entire storage regardless of
lighting conditions, indicating that irradiation destroyed virtually all of the chlorophyll,
resulting in a complete protection from photooxidation. The results suggested that irradiation
of oils conducted in the absence of oxygen can be used to eliminate residual chlorophyll.
On the basis of chlorophyll breakdown by irradiation, a new processing method was
introduced for brighter-colored natural green tea leaf extract (GTLE) material for cosmetics,
medicine or the food industry (Jo et al., 2003a) because natural products with high functions
cannot be directly applied to the food or cosmetic composition due to their undesirable dark
color. An irradiation of GTLE (70% ethanol) showed higher Hunter color L*-value and lower
a*- and b*-values, resulting in a color change of solution to bright yellow from dark brown
(Table 3). To apply for the industry properly, this color enhancement process using irradiation
should not reduce the biological activity of GTLE, and there was no difference in the radical
scavenging (Table 4) and tyrosinase inhibition effect (the in vitro test for skin whitening
effect) by irradiation regardless of storage temperature (4 and 25°C). However, one of the
problems was the change of brighter color of the extract produced from irradiation to darker
(decreasing trend in Hunter color L*-value) during storage. The storage temperature was
important in this color change and lower storage temperature was better to minimize color
change. Also, the addition of vitamin C in the extract was effective in reducing this color
change (Jo et al., 2003a). This technology was transferred to a commercial cosmetic company
in Korea (SunBiotech Co., Ltd.) and the problem of color change was solved in the
formulation of cosmetic compositions. Similar results were obtained from a series of studies
using different natural materials with various biological functions such as persimmon leaf
(Diospyros kaki L. folium), licorice (Glycyrrhiza Uralensis Fischer) root and its stolon (Jo et
al., 2003d), Japaneses honeysuckle (Byun et al., 2004b), and root of Curcuma aromatica (Kim
et al., 2006).
Table 1. Chlorophyll content (ppm) of 20 kGy-irradiated and photooxidized linoleic acid

solution (1% in methanol) containing chlorophyll b (3 ppm) by HPLC
Irradiation Photooxidation time (hr)1

0 1 2 4 6 SEM3
Unfoiled 0 kGy 2.91a2 2.99a 2.34a 1.81b 1.51b 0.144
20 kGy nd nd nd nd nd -
20 / N24 nd nd nd nd nd -
Foiled5 0 kGy 2.88 3.05 3.02 2.85 2.73 0.108
20 kGy nd nd nd nd nd -
20 / N24 nd nd nd nd nd -
1
Light intensity was 3,300 lux at 25°C.
2
Different letters (a, b) within a row differ significantly at P<0.05.
3
SEM: Pooled standard errors of the mean (n = 10).
4
Sample was bubbled with ultra pure N2 gas during irradiation.
5
Bottles were covered by aluminum foil to avoid photooxidation.
The data was published at Byun et al. (2002), J. Am. Oil Chem. Soc. 79, 145-150.
Table 2. Peroxide value of photooxidized linoleic acid solution (1% in methanol)

containing chlorophyll b (3 ppm) by gamma irradiation
Photooxidation time (hr)1

Irradiation 0 1 2 4 6 SEM2
Unfoiled 0 kGy 0cy3,4 24.0 cy 27.5cy 49.7by 79.0ay 4.86
20 kGy 1243.7x 1251.6x 1286.3x 1253.4x 1268.8x 11.44
20 / N25 0y 0y 0z 0z 0z -
SEM6 7.81 12.90 3.82 3.38 3.03
Foiledg 0 kGy 0y 0y 0y 0y 0y -
20 kGy 1251.7x 1275.4x 1262.1x 1251.8x 1266.5x 17.40
20 / N25 0y 0y 0y 0y 0y -
SEM6 10.41 6.19 3.88 11.10 14.82
1
Light intensity was 3,300 lux at 25°C.
2
SEM: Pooled standard errors of the mean. n = 20
3
Different letters (a-c) within a row differ significantly at P<0.05.
4
Different letters (x-z) within foiled and unfoiled columns differ significantly at P<0.05.
5
Sample was bubbled with ultra pure N2 gas during 20 kGy-irradiation.
6
SEM: n = 12.
7
Bottle were covered by aluminum foil to avoid photooxidation.
The data was published at Byun et al. (2002), J. Am. Oil Chem. Soc. 79, 145-150.
Table 3. Hunter color L*-value of green tea leave extract from 70% ethanol solution
after gamma irradiation
Storage Irradiation Storage (week)

Temperature (kGy) 0 1 2 3 SEM1
4°C 0 75.37dz 75.65bz 76.21az 75.57cz 0.005
5 90.83ay 88.35by 87.50cy 86.28dx 0.013
10 93.63ax 90.14bx 88.70cw 87.12dw 0.021
20 96.22aw 91.19bw 88.19cx 86.04dy 0.008
SEM2 0.010 0.010 0.005 0.022
25°C 0 74.82az 73.27bz 72.98cz 71.99dz 0.007
5 87.33ay 81.88bx 80.21cw 80.21dw 0.011
10 89.23ax 82.16bw 79.73cx 79.23dx 0.016
20 89.98aw 80.87by 77.55cy 76.51dy 0.007
SEM2 0.008 0.012 0.015 0.015
a-d
Different letters within the same row differ significantly (P<0.05).
w-z
Different letters within the same column with the same storage temperature differ significantly
(P<0.05).
1
Standard errors of the mean (n = 12). 2(n = 12).
The data was published at Jo et al. (2003), Radiat. Phy. Chem. 66, 179-184.
Table 4. Scavenging effect of DPPH radical of green tea leave extract from 70% ethanol
solution after gamma irradiation
Storage Irradiation Storage (week)

Temperature (kGy) 0 1 2 3 SEM1
4°C 0 48.74az 30.57b 27.37b 31.66by 2.232
5 48.35az 33.20b 28.82c 36.95bx 1.078
10 51.71ay 26.74b 27.73b 30.69by 1.805
20 53.33ax 16.73b 29.70b 29.98by 3.301
SEM2 0.363 4.282 1.080 0.818
25°C 0 39.96a 33.36b 23.83c 25.58cy 0.795
5 44.85a 33.53b 28.58c 31.08cx 0.758
10 45.14a 34.85b 27.51b 29.98bx 2.450
20 45.44a 33.00b 28.57b 33.33bx 1.368
SEM2 1.087 2.445 0.975 0.982
a,b
x-z
Different letters within the same column with the same storage temperature differ significantly
(P<0.05).
1
Standard errors of the mean (n = 8). 2(n = 8).
The data was published at Jo et al. (2003), Radiat. Phy. Chem. 66, 179-184.
IRRADIATION OF THE POLYPHENOLS SEPARATED

FROM GREEN TEA LEAF
When the polyphenols of the green tea leaf were separated by Sephadex LH-20 column
and thin layer chromatography and irradiated at a higher dose (40 kGy), the major
antioxidative activities, including electron donating, inhibition of xanthine oxidase, metal ion
chelating, and inhibition of lipid oxidation were still maintained or even better after
irradiation treatment (Figures 1 and 2, An et al., 2004). The anti-microbial activities against
Staphylococcus aureus and Streptococcus mutans were higher in the irradiated green tea
polyphenols, which showed inhibition of microorganisms tested at a lower concentration than
those of the non-irradiated counterpart. Ranges of inhibition zone for growth Escherichia coli,
S. aureus, S. epidermidis, and S. mutans at 1 mg/disc were 9.3, 10.1, 22.5, and 9.3 mm in
non-irradiated control while 10.8, 11.0, 25.0, and 11.7 mm in irradiated counterpart,
respectively. Generally, the polyphenol is very stable except for thiolysis, reaction with strong
acid (McGraw et al., 1993). The irradiated polyphenol might not change its chemical structure
but some change in activity is expected of functional groups such as –OH or –COOH,
resulting in higher antimicrobial activity.
100
IP
Electron donating ability (%)
NP a a a a a
80 a a a a a
60
40
20 b b
0
1 5 10 50 100 200
Concentration (ppm)
Figure 1. Electron donating ability of irradiated polyphenol isolated from green tea. IP: irradiated
polyphenol (40 kGy), NP: non-irradiated polyphenol. Values are means of 5 replicates and those with
different alphabet letters are significantly different at P<0.01.
50
IP
NP
40 a
b
Inhibition rate (%)

30
c
d
20
e
f
10 g
gh g
h
0
1 5 10 50 100
Concentration (ppm)
Figure 2. Inhibition rate of irradiated polyphenol isolated from green tea on xanthine oxidase. IP:
irradiated polyphenol (40 kGy), NP: non-irradiated polyphenol. Values are means of 5 replicates and
those with different alphabet letters are significantly different at P<0.01.
100
IP
NP
80
a
Inhibition rate (%)
b
60
c c
40
d
20
d
e e e e e e
0
1 5 10 50 100 200
Concentration (ppm)
Figure 3. Inhibition rate of the irradiated green tea polyphenol on the collagenase activity. IP: irradiated
polyphenol (40 kGy), NP: non-irradiated polyphenol. Values are means of 3 replicates and those with
different alphabet letters are significantly different at P<0.05.
50
IP
NP
Collagen synthesis rate (%)

40
30
a
20
ab ab
bc
cd bcd
10 cd
d
0
1 5 10 50
Concentration (ppm)
Figure 4. Collagen biosynthesis enhancing activity of the irradiated green tea polyphenol on the human
fibroblast. IP: irradiated polyphenol (40 kGy), NP: non-irradiated polyphenol. Values are means of 3
replicates and those with different alphabet letters are significantly different at P<0.05.
100 a
IP a
NP
Growth inhibition rate (%)
80 b
c
60
40
d
20 de d
f ef ef ef ef
0
1 5 10 50 100 200
Concentration (ppm)
Figure 5. Growth inhibition rate of the irradiated green tea polyphenol on the malignant melanoma (SK-
MEL-2). IP: irradiated polyphenol (40 kGy), NP: non-irradiated polyphenol. Values are means of 3
replicates and those with different alphabet letters are significantly different at P<0.05.
Another study was focused on the changes of physiological activity of irradiated green
tea polyphenols on the human skin (An et al., 2005). The important factors for the human skin
wrinkles are reactive oxygen species, ultraviolet light, and the reduction of collagen
biosynthesis (Grove et al., 1989; Griffiths et al., 1992). The assessment of anti-wrinkle effect
on human skin including collagenase inhibition and collagen biosynthesis was measured form
the irradiated green tea polyphenols. Results showed that collagenase inhibition effect was
higher in the irradiated sample (65.3%) than that of the non-irradiated control (56.8%) at 200
ppm of concentration (Figure 3, An et al., 2004). Collagen biosynthesis rates using human
fibroblast were 19.4% and 16.3% in the irradiated and the non-irradiated polyphenols,
respectively (Figure 4, An et al., 2004). The tyrosinase inhibition effect, which is related to
the skin-whitening effect, showed a 45.2 and 42.9% in the irradiated and non-irradiated
polyphenols, respectively, at a 100 ppm level. A higher than 90% growth inhibition on skin
cancer cells (SK-MEL-2 and G361) was demonstrated in both the irradiated and non-
irradiated polyphenols (Figure 5, An et al., 2004). Thus, the results showed that the irradiation
of green tea polyphenol did not change and even increased its anti-wrinkle, skin-whitening,
and anticancer effects on the human skin. These results also indicate that irradiation of the
polyphenols separated from green tea leaf can be applicable in the food, pharmaceutical, and
cosmetic compositions with improved microbiological safety, already proved and
commercialized.
APPLICATION OF IRRADIATED GREEN TEA

ON MEAT PRODUCTS
Irradiation of meat to control pathogens and extend shelf-life accelerates lipid oxidation
and produces off-odors under aerobic conditions (Ahn et al., 1999). To overcome these
adverse effects of irradiation on meat products irradiated freeze-dried GTLE powder was
applied to pork patties (Jo et al., 2003b). The reason of irradiation was to obtain a bright-
colored GTLE for better appearance of the additive in pork patties. The development of lipid
oxidation of pork patties was lower and radical scavenging effect was greater in the samples
with non-irradiated or irradiated green tea extract powder than those of control samples
(without additive, Table 5). The pork patties with green tea powder had a higher Hunter color
a*-value and less cooking loss than that of control, indicating that the addition of GTLE may
improve the quality of the pork patties. Especially, sensory panelists preferred the odor of the
raw pork patties and color of the cooked pork patties added with irradiated GTLE than that
with non-irradiated GTLE (Table 6). It because the odor of native green tea adversely
affected on the sensory quality of pork patty added with GTLE and irradiation of GTLE may
reduce the native green tea odor. It has been used mainly two methods to achieve healthier
meat and meat products; avoiding undesired substances or reducing them to appropriate
limits, and increasing the levels of other substances with beneficial properties (Jimmenez-
Colmenero et al., 2001; Arihara, 2006). Currently, exogenous antioxidants including phenolic
compounds, tocopherols, plant derivatives and chelating agents are added to meat products.
From this study, it can be concluded that irradiated, freeze-dried GTLE powder can be used
for producing functionally-improved meat products with additional off-odor reduction from
plant-derived natural material in the products.
Table 5. TBARS values (mg malondialdehyde/kg meat) of raw and cooked pork patties
with added nonirradiated or irradiated freeze-dried green tea leaf extract powder
(0.1%)
Storage day
0 5 10 15 SEM1
Raw pork patties
Trt A 0.38cx 0.56bx 0.56bx 0.69ax 0.029
Trt B 0.28by 0.42ay 0.43ay 0.48ay 0.024
Trt C 0.30by 0.36az 0.35ay 0.39ay 0.016
SEM2 0.021 0.016 0.025 0.028
Cooked pork patties
Trt A 1.13bx 1.22abx 1.38ax 1.21abx 0.044
Trt B 0.48z 0.42y 0.42y 0.44y 0.042
Trt C 0.58ay 0.36by 0.44by 0.31by 0.042
SEM2 0.027 0.048 0.043 0.048
Abbreviations: Trt A, only pork patties; Trt B, patties with nonirradiated, freeze-dried green tea leaf
extract powder (0.1%); Trt C, patties with irradiated at 10 kGy, freeze-dried green tea leaf extract
powder (0.1%).
a-c
x-z
Different letters within the same column with raw and cooked pork patties differ (P<0.05).
1
Pooled standard errors of the mean (n = 6). 2(n = 8).
The data was published at Jo et al. (2003), Meat Sci. 64, 13-17.
Table 6. Sensory scores of raw and cooked pork patties with nonirradiated or irradiated
freeze-dried green tea leaf extract powder (0.1%)
Raw Cooked1
Parameter Color Odor Color Odor Taste Tenderness
Trt A 8.6 6.9b 6.78b 6.61 5.98 6.30
Trt B 8.7 5.4c 8.10a 7.41 8.16 6.67
Trt C 7.9 9.0a 8.66a 5.97 6.52 6.17
SEM2 0.47 0.90 0.47 0.80 0.75 0.69
a-c
Different letters within the same column differ (P<0.05).
1
Patties was cooked on a preheated pan for 3.5 min. The cooked patties was sliced into small pieces,
served to the panelists individually and evaluated independently in 2 different times at Day 1 and
Day 3. A 15 line-scale was provided to the panelists and scored; very undesirable (0) to very
desirable (15) for color, strong off-odor (0) to very mild odor (15) for odor, very poor taste (0) to
very tasty for taste, and very tough (0) to very tender (15) for tenderness.
2
Pooled standard errors of the mean (n = 60).
The data was published at Jo et al. (2003), Meat Sci. 64, 13-17.
Another example is shown using the pectin-based edible coating material containing
0.5% irradiated GTLE powder to pork patties (Kang et al., 2007). The edible coatings may
serve as carrier for antimicrobial compounds in order to maintain high concentrations of
presevatatives on the surface of foods (Gennadios et al., 1997) in addition to the protection of
moisture loss. During storage at 10°C for 14 days, lipid oxidation decreased (Figure 6, Kang
et al., 2007) and radical scavenging activity increased (Figure 7, Kang et al., 2007) in the pork
patties coated by the pectin-based material containing GTLE powder. In addition, the coated
patties had higher moisture content throughout the storage than control in both aerobic- and
vacuum-packaging. A number of total aerobic bacteria were significantly reduced by the
coating treatment as well as by irradiation (Figure 8, Kang et al., 2007). No difference was
detected in sensory characteristics due to the coating treatment containing irradiated GTLE
powder. The results demonstrate that the GTLE added to pectin-based coating has positive
effects on the quality of irradiated pork patty.
20 20
Vacuum (0 kGy) Control Vacuum (3 kGy)
CP
CGP
15 15
10 10
TBARS (mg malonaldehyde/kg) value

TBARS (mg malonaldehyde/kg) value
5 5
20
0 20
0
Aerobic (0 kGy) Aerobic (3 kGy)
15 15
10 10
5 5
0 0
0 7 14 0 7 14
Storage time (days)
Figure 6. 2-thiobarbituric acid reactive substance value (TBARS; mg malondialdehyde/kg sample) of

gamma-irradiated pork patty coated by a pectin-based coating material during a storage at 10°C.
APPLICATION OF IRRADIATED GREEN TEA

ON COSMETIC COMPOSITION
GTLE powder was prepared using irradiation (20 kGy) to improve its color and
antioxidative activity including electron donating and superoxide dismutase-like activities
was compared with a commercially available green tea powder produced especially for
cosmetic composition (Byun et al., 2004a). The commercial product was removed its dark
color by adsorbents through column chromatography, which is time-consuming process with
high cost. The superoxide dismutase-like activity of 1% irradiated green tea powder dissolved
in butylenes glycol showed 48.0% but that of commercial product showed only 25.1% (Table
7). This indicates that the commercial color removal process may reduce the activity of green
tea powder originally present. When 1% of the irradiated GTLE powder was diluted 400 and
800 times, the electron donating ability showed 78.0 and 51.7%, respectively, which was not
different from the commercial one. No difference in tyrosinase inhibition activity was also
found. Results showed that 1% of the irradiated green tea powder dissolved in butylenes
glycol for cosmetic composition was higher or at least had the same physiological activity
with the commercial product. After 1% of the irradiated GTLE solution dissolved in buytlene
glycol was prepared, the solution was mixed (15%) to a cosmetic composition for
manufacturing a cream lotion to conduct a real application study. The electron donating
activity of the cream lotion prepared from the solution of the irradiated green tea leaf extract
powder did not differ from the cream lotion prepared from the solution of a commercial
product. Therefore, irradiation of GTLE for cosmetic composition may be a cost-effective
method to improve industrial applicability.
40 40
CP
CGP
30 30
20 20
10 10

40
0 40
0
30 30
20 20
10 10
0 0
0 7 14 0 7 14
Storage time (days)
Figure 7. Electron donating ability (%) of gamma-irradiated pork patty coated by a pectin-based coating
material during a storage at 10°C.
12 12
CP
10 10
CGP
8 8
6 6
Total plate count (Log CFU/mg)

Total plate count (Log CFU/mg)
4 4
2 2
12
0 12
0
10 10
8 8
6 6
4 4
2 2
0 0
0 7 14 0 7 14
Storage time (day)
Figure 8. Total plate count of gamma-irradiated pork patty coated by a pectin-based coating material
during a storage at 10°C.
Table 7. Physiological activities (%) of irradiated green tea leaf extract powder (1%)
dissolved in butylene glycol prepared from laboratory and commercially available one
for a cosmetic composition
Physiological activity (%) Dilution factor Irradiated1 Commercial2

Superoxide dismutase-like 1 48.0±0.8 25.1±0.4
400 78.0±0.9 78.0±0.6
DPPH radical scavenging
800 51.7±0.6 53.7±0.9
1 81.1±1.5 82.5±0.9
Tyrosinase inhibition
5 44.0±0.8 29.0±0.3
1
Dried green tea leaf was extracted, irradiated at 20 kGy, and freeze-dried.
2
Commercially available green tea leaf extract powder for cosmetic composition.
The data was published at Byun et al. (2004), Raidat. Phy. Chem. 71, 487-489.
APPLICATION OF GREEN TEA BYPRODUCTS

USING IRRADIATION
Food processing industries create large quantities of byproducts, which are difficult to
dispose of as they have a high biological oxygen demand. Plant material wastes from these
industries sometimes contain high levels of phenolic compounds that can have an adverse
environmental impact (Kang et al., 2006). On the other hand, agricultural and industrial
byproducts are excellent sources of natural antioxidants or functional materials. Several
studies have reported that these byproducts have already been used as potential sources of
functional and nutritional materials (Lapornik et al., 2005; Esposito et al., 2005; Šližytė et al.,
2005). For instance, every year, about 50 tons of green tea byproducts are produced in
Boseong area, the biggest green tea production area in Korea, but the economic value of
green tea byproducts is much lower (about 1/100) when compared with the green tea leaf (Ki,
2004).
Green tea stem extract powder, a major byproduct of the green tea industry, was prepared
and investigated for its biological activities after a irradiation treatment for the purpose of a
brightened color. The color, total phenolic contents, antioxidative activity, tyrosinase
inhibition activity, cancer cell proliferation inhibition effect, and antimutagenicity were
measured and compared with those of a GTLE powder (Lee et al., 2006). Hunter L* values of
the irradiated GTLE and stem extracts were increased in a comparison with the non-irradiated
extracts. The content of total phenolic compounds of the GTLE and stem extract was 424.2
and 11.45 mg/g in non-irradiated sample and 335.5 and 18.65 mg/g in 20 kGy-irradiated
sample, respectively. The electron donating ability was not different between GTLE and stem
extract in both non-irradiated and irradiated conditions. Ferric reducing antioxidant power
(FRAP) of the samples showed similar results to the radical scavenging capacity. The both
GTLE and stem extract showed an inhibition of tyrosinase and also a cancer cell proliferation
inhibition effect. Especially, the antimutagenicities of the stem extracts showed a strong
inhibition effect (96.20 and 57.77%) on 3-amino-1,4-dimethyl-5H-pyrido [4,3-b]indole (Trp)
and 2-nitrofluorene (NF), respectively (Table 8).
The antioxidative and antigenotoxic activities of GTLE and stem extracts was compared
after 20 kGy of irradiation (Lee et al., 2008). In this study, in vitro antioxidative activities
including a radical scavenging, a hydrogen peroxide inhibition, the tyrosinase inhibition
activities, and the reducing power were tested. GTLE had higher antioxidative activities than
the stem extract. Irradiation of 20 kGy to GTLE showed a decreasing tendency in radical and
hydrogen peroxide scavenging activities and reducing power, while that to stem extract
showed an increasing tendency of the antioxidative activities. Ahn et al. (2004) reported that
the FRAP of phytic acid increased significantly by irradiation. Tyrosinase inhibition activity
showed no difference by irradiation in both the GTLE and stem extract. Overall, an
irradiation had positive influences on the antioxidative activity in the stem extract more than
in the GTLE including a color improvement. Antigenotoxic effect of the green tea extracts on
an oxidative DNA damage in human leucocytes by a DNA comet assay also indicated a
protective effect of GTLE (Figure 9, Jo et al., 2008). The irradiation of the GTLE began to
decrease the DNA damage significantly at 10 μg/mL which showed a higher inhibition
activity than the non-irradiated GTLE. The non-irradiated and irradiated green tea stem
extract showed similar inhibition trends and they were comparable to that of the GTLE. This
result also indicates that the green tea bypdroducts can be considered as a cost-effective
functional ingredient for industrial applications. Furthermore, irradiation of green tea
byproducts may have beneficial effects on its functional activity.
Table 8. Antimutagenicity of the green tea leaf and byproduct extract powders on S.
Typhimurium TA 981
Samples Mutagen2 Revertant Inhibition rate

colonies / plate (%)3
Green tea leaf extract Trp 51.0±1.41 96.77
Green tea leaf extract irradiated at 20 kGy 51.5±3.54 96.74
Green tea byproduct extract 60.0±4.24 96.20
Green tea byproduct extract irradiated at 20 kGy 57.0±2.83 96.39
SEM4 0.141
Green tea leaf extract NF 119.0±11.31 54.92
Green tea leaf extract irradiated at 20 kGy 110.5±6.36 58.14
Green tea byproduct extract 111.5±9.19 57.77
Green tea byproduct extract irradiated at 20 kGy 106.5±6.36 59.66
SEM4 2.295
Abbreviations: Trp, 3-amino-1,4-dimethyl-5H-pyrido[4,3-b]indole; NF, 2-nitrofluorene.
1
Concentration of samples was 10 mg per plate.
2
Concentration of Trp and NF were 1 and 3 μL per plate, respectively.
3
Means with the same letter in each column are not significantly different (P<0.05).
4
Standard error of the mean (n = 8).
The data was published at Lee et al. (2006), J. Food Sci. 71, C269-C274.
Table 9. The 50% inhibition concentration (IC50, μg/mL) of green tea leaf and stem
extract against superoxide radical and hydroxyl radical
Irradiation dose (kGy)

SEMb
0 20
Superoxide radical
Green tea leaf 2.33by a 3.25ax 0.066
Green tea stem 2.77ax 2.09by 0.107
SEM1 0.062 0.110
Hydroxy radical
Green tea leaf 1.31a 0.62by 0.013
Green tea stem 1.38a 0.95bx 0.021
SEM1 0.014 0.020
a,b
Different letters (a-b) within the same row are significantly different (P < 0.05).
x,y
Different letters (x-y) within the same column are significantly different (P < 0.05).
1
Standard error of the means (n = 6).
The data will be published at Lee et al. (2008), J. Food Biochem. 32, 782-794.
80 80
70 70
Fluorescence in tail (%)

60 60
50 50
40 40
30 30
20 20
10 10
0 0
control 0 0.1 1 10 100 control 0 0.1 1 10 100
μg /ml of sample + 200 μM H2O2 μg /ml of sample + 200μM H2O2
80 80
70

70
60 60
50 50
40 40
30 30
20 20
10 10
0 0
control 0 0.1 1 10 100 control 0 0.1 1 10 100
μg /ml of sample + 200 μM H2O2 μg /ml of sample + 200μM H2O2
Figure 9. Antigenotoxic effect of irradiated green tea leaf and stem extracts on oxidative DNA damage
in human leucocytes. A, Non-irradiated green tea leaf extract; B, Irradiated green tea leaf extract at 20
kGy; C, Non-irradiated green tea stem extracts; D, Irradiated green tea stem extract at 20 kGy.
Another study was conducted with a similar method and indicated that irradiation
decreased total phenolic contents in GTLE from 98.6 to 79.91 mg/g but that of green tea stem
extract was increased from 67.83 to 71.94 mg/g after irradiation. Total flavanol and ascorbic
acid contents also showed the same trends with the total phenolic contents. The 50%
inhibition concentration (IC50, (g/mL) of green tea leaf and stem extract against superoxide
radical and hydroxyl radical also demonstrated that green tea stem extract showed higher
activity when irradiation was applied (Table 9). However, catechins and caffeine content of
the both GTLE and stem extracts decreased by irradiation at 20 kGy (Table 10).
From the results, irradiation of green tea extract may have beneficial effects to improve
industrial applicability by enhancing the color with shorter time and lower cost than
conventional method and biological functions. In addition, the green tea byproducts, the
remainder of green tea processing, has still been considered as a potential functional
ingredient for industrial applications, in terms of an increased cost-effectiveness, when
compared with the green tea leaf. Irradiation of green tea byproducts may also have beneficial
effects on its functional activities.
Table 10. Effect of irradiation on catechins and caffeine in green tea leaf and stem
extracts. All values are on a dry basis (mg/g)
Irradiation dose (kGy) Green tea leaf Green tea stem

SEM1
0 20 0 20
Epicatechin 4.19b 4.84a 3.21c 1.35d 0.03
Epicatechingallate 1.11a 0.70b -c -c -
Epigallocatechin 10.01a 7.42b 4.20c 2.90d 0.24
Epigallocatechin gallate 8.14a 6.32b 4.57c 2.91d 0.06
Gallocatechin 0.58a 0.58a 0.30b 0.20c 0.01
Gallocatechingallate 0.07a - - - -
Caffein 8.69a 6.96b 0.54c 0.64c 0.12
Gallic acid 0.17a 0.09c 0.12b 0.09c 0.02
Total catechin 0.65 0.58 0.30 0.20
Total epicatechins 23.45 19.28 11.98 7.16
Total monomeric flavanol 24.1 19.86 12.28 7.36
a-d
Different letters within the same row are significantly different (P < 0.05).
1
Standard errors of the mean (n = 12).
The data will be published at Lee et al. (2008), J. Food Biochem. 32, 782-794.
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Chapter 10
GREEN TEA CATECHIN AS

ANGIOGENESIS INHIBITOR
Kiminori Matsubara1,2,* and Yoshiyuki Mizushina2,3

1
Department of Human Life Sciences Education, Graduate School of Education,
Hiroshima University, Higashi-Hiroshima, Hiroshima 739-8524, Japan
2
Cooperative Research Center of Life Sciences, Kobe-Gakuin University, Nishi-ku,
Kobe, Hyogo 651-2180, Japan
3
Department of Nutritional Science, Kobe-Gakuin University, Kobe,
Hyogo 651-2180, Japan
ABSTRACT
Many studies suggest that beneficial effect of green tea for human health is mainly
attributed to catechins, polyphenol compounds, having anti-oxidative activity. In the last
decade, new aspect of green tea catechin has emerged as angiogenesis inhibitor.
Angiogenesis is forming new blood vessels from a pre-existing blood vessel and involved
in various diseases including tumor growth and metastasis, diabetic retinopathy and
atherosclerosis. In addition, it has been demonstrated that obesity is prevented by
angiogenesis inhibitor and suggested that angiogenesis is closely associated with
Alzheimer’s disease. Thus, angiogenesis inhibitors in food would be expected to prevent
these diseases and give beneficial effect on our health. Interestingly, inhibitory effect of
green tea catechin on angiogenesis has been demonstrated in various models suggesting
green tea catechin could suppress cancer. Furthermore, it has been revealed that a higher
consumption of green tea catechin reduces human body fat and is associated with a lower
prevalence of cognitive impairment. These evidences suggest that anti-angiogenic
activity of green tea catechin might play important roles in human health.
*
Corresponding author. Kiminori Matsubara, Ph.D., Department of Human Life Sciences Education, Graduate
School of Education, Hiroshima University, Kagamiyama, Higashi-Hiroshima, 739-8524, Japan. Tel; +81-82-
424-6854; Fax; +81-82-4227133; E-mail; kmatsuba@hiroshima-u.ac.jp
198 Kiminori Matsubara and Yoshiyuki Mizushina
INTRODUCTION
Angiogenesis is a phenomenon forming new blood vessels from a pre-existing blood
vessel. This physiological process is necessary to supply nutrients and oxygen for
development, wound healing and reproduction [1]. On the other hand, pathological blood
vessel formation is involved in various diseases including tumor growth and metastasis,
rheumatoid and other diseases [2]. Recently, it has been demonstrated that metabolic disorder
causes various diseases such as diabetic retinopathy, atherosclerosis, and obesity, which are
associated with angiogenesis. Thus, suppression of pathological angiogenesis could prevent
these diseases. In fact, therapeutic strategy for inhibiting-angiogenesis has been developed
especially in cancer patients. This angiogenesis targeting therapy was proposed by Dr. Judah
Folkman more than 30 years ago [3]. Folkman’s group demonstrated that tumor growth
depends on angiogenesis to obtain nutrients and oxygen and to remove metabolites [4]. After
that, extensive studies have been conducted on angiogenesis inhibitors, and data obtained
from a lot of experiments and clinical trials for cancer therapy using angiogenesis inhibitors
support the usefulness of this strategy. This simple and elegant concept provides a strong
strategy for cancer therapy. In addition, as mentioned above, angiogenesis is involved in
various diseases, thus angiogenesis inhibitors could be used as drugs for various diseases.
There are many candidates for anti-angiogenesis drugs in clinical trials. One of the most
well-known angiogenesis inhibitors in cancer treatment is a humanized anti-VEGF antibody,
bevacizumab (Avastin; Genentech), which blocks the role of angiogenic factor, vascular
endothelial growth factor (VEGF). This anti-angiogenesis cancer agent validates the
usefulness of targeting angiogenesis in metastatic colorectal cancer [5]. Many other VEGF
inhibitors are in cancer clinical trials. In addition, angiogenesis inhibitors are potential drugs
in non-oncology indications, such as age-related macular degradation, rheumatoid arthritis,
psoriasis, endometriosis and cerebral oedema [5].
On the other hand, many diseases associated with angiogenesis are life-style relating
diseases. Therefore, to prevent such diseases many people would intake healthy food, from
which various biologically active substances have been isolated and characterized such as
polyphenols in red wine, green tea, herbs, vegetables. In fact, many of these polyphenols
exert pharmacological effects, and their anti-angiogenic activity is one of their attractive
effects [6]. As epidemiological studies have shown that red wine and green tea consumption
would lower the risk of coronary heart diseases and cancer [7-13], anti-angiogenic
polyphenols from red wine and green tea have attractive attention [14]. In this review, we
focus on the anti-angiogenic effect of green tea catechin and summarize the mechanisms how
green tea catechin exerts the anti-angiogenic activity and beneficial effects for human health.
GREEN TEA CATECHIN AND ANGIOGENESIS

The first report of anti-angiogenic effect of dietary polyphenol would be genistein from
soybean [15]. After this report, many research have been conducted on anti-angiogenic
activity of dietary polyphenols. In 1999, Cao and Cao, a Swedish research group, reported
that green tea catechin has anti-angiogeinc activity [16]. They demonstrated anti-angiogenic
activity of green tea catechin, especially epigallocatechin gallate (EGCG) being abundant
Green Tea Catechin as Angiogenesis Inhibitor 199
green tea catechin, by cornea and chorioallantoic membrane (CAM) assays and endothelial
cell model. Furthermore, it is notable that oral administration of green tea catechin suppressed
angiogenesis in vivo model, showing the usefulness of consumption of green tea.
The mechanisms by which green tea catechin, mainly EGCG, exerts anti-angiogenic
activity have been extensively studied. Suppressive effects of green tea catechin on in vitro
angiogenesis models have been demonstrated, and the molecular targets of green tea catechin
have been revealed; inhibition of VEGF receptor signaling [17, 18] and membrane-type I
matrix metalloproteinase [19], suppression of vascular endothelial (VE)-cadherin tyrosine
phosphorylation and Akt activation [20] and downregulation of transcription factors, Ets-1, c-
Fos, and c-Jun [21]. As reactive oxygen species (ROS) are known as stimulants for
angiogenesis [22, 23], antioxidant activity of green tea catechin may in part account for the
anti-angiogeinc activity.
As green tea catechin is considered a good model to develop angiogenesis inhibitors,
green tea catechin derivatives are expected to exert stronger anti-angiogenic activity. We
synthesized green tea catechin derivatives and their activities were compared [24, 25]. The
green tea catechin derivatives conjugated with fatty acid enhanced anti-angiogenic activity
and also strongly inhibited DNA polymerase activity. Further investigation on green tea
catechin derivatives could find more effective angiogenesis inhibitors.
GREEN TEA CATECHIN AND CANCER

Cancer is the most serious medical problem in the world. Epidemiological studies have
shown that consumption of green tea lowers the risk of cancer, and green tea catechin
suppresses tumorigenesis and tumor growth [9, 10, 12]. Green tea catechin inhibits cancer
cells proliferation at least in part by inhibiting DNA polymerases [26] and induces apoptosis
[27]. Thus, green tea catechin would directly prevent cancer cell growth. However, the
concentration of green tea catechin was relatively low than IC50 in cancer cell growth
inhibition [27]. This fact puts an idea that green catechin exerts anti-cancer effect through a
different mechanism. As the mechanism, inhibition of angiogenesis emerged, and anti-
angiogenic activity of green tea catechin was ascertained [16]. VEGF-induced angiogenesis in
cornea of mice drinking green tea was suppressed. Therefore, anti-cancer effect of green tea
catechin would be due to inhibition of cancer cell growth and angiogenesis, which is a critical
for tumor growth [2, 3]. Many cancer cells produce VEGF to induce angiogenesis. Green tea
catechin also suppresses VEGF production from many cancer cells [28-30]. Fibroblast growth
factors are known as other important pro-angiogenic factors. Green tea catechin also
suppresses fibroblast growth factor production [31]. Furthermore, green tea catechin
suppresses cellular matrix degradation enzymes such as gelatinases and matrix
metalloproteinases [32, 33]. These enzymes are involved in not only angiogenesis but also
cancer cell metastasis. Thus, green tea catechin may reduce cancer cell metastasis. However,
the relationship between green tea consumption and low mortality of cancer seems to be
controversial [34-36] due to the complex mechanisms of cancer.
GREEN TEA CATECHIN AND CARDIOVASCULAR DISEASES

Coronary heart disease has been serious medical problem in Western countries. Patients
are increasing in Asian and other countries. It is well-known that “French paradox”; red wine
consumption lowers coronary heart disease in French people [7]. Interestingly,
epidemiological studies have shown an inverse relationship between green tea consumption
and coronary heart disease [9-11]. Recent a large population-based epidemiological research
in Japan supports that green tea consumption reduces mortality due to cardiovascular disease
[34]. The formation and progression of atherosclerosis lesions are considered to be excessive
vascular remodeling and chronic inflammation resulting in lipid accumulation and complex
lesions in the arterial lumen [37, 38]. Following description of a review in which details of
atherosclerosis initiation and progression are explained [38], the revealed mechanisms of
preventive effects of green tea catechin are summarized. Initiating events of atherosclerosis
are recruitment of macrophages and their subsequent up-take of low-density lipoprotein
(LDL)-derived cholesterol. As oxidative modifications in the lipid and apolipoprotein B (apo
B) components of LDL initiate formation of atherosclerosis and induce LDL aggregation
causing macrophage degradation [39], inhibition of the oxidative component formation would
be very important. Green tea catechins prevent LDL oxidation [40] due to their anti-oxidant
property and LDL aggregation [41]. However, green tea catechins have no effect on oxidized
LDL-induced endothelial cell and monocyte THP-1 cell interaction [42], which is involved in
atherosclerosis development. Vascular smooth muscle cells (VSMCs) also play important role
in atherosclerosis. VSMCs secrete VEGF in atherosclerotic plaques inducing plaque
angiogenesis [43, 44], which is observed in atherosclerotic lesions. Angiogenesis inhibitors
reduce intimal new blood vessel formation and plaque growth [45]. As green tea catechin has
anti-angiogenic activity and inhibits platelet derived growth factor-induced VEGF expression
in VSMCs [46], regular consumption of green tea could reduce atherosclerotic plaque
development. In addition, green tea catechin induces proliferating VSMC apoptosis and
prevents VSMC invasion [47, 48].
GREEN TEA CATECHIN AND OBESITY

Obesity or overweight has become serious medical problem in United States and other
developed countries because obesity or overweight substantially increases the risk of various
diseases including type 2 diabetes mellitus, hypertension, dyslipidemia, coronary heart
disease, and colon cancer [49]. Thus, body weight control by pharmacological and
nonpharmacological treatments is very important to reduce the risks of overweight or obesity-
relating diseases. As obesity or overweight patients have excess adipose tissue, a strategy to
reduce the mass of adipose tissue seems to be a simple one. However, body weight control is
regulated by complex systems, and adipocytes have unique characteristics secreting various
hormones and differentiated from stem cells [50]. Thus, there are many targets for treatment
of overweight patients. In 2002 a new concept for body weight control emerged from U. S.
research group; adipose mass can be regulated by the vasculature [51]. Most organs in adult
body do not increase the mass however adipose tissue can increase the mass like tumor
growth. As all organs including adipose tissue need blood supply, growing adipose tissue
mass accompanies new blood vessel formation. Rupnick et al. demonstrated that angiogenesis
inhibitors could suppress adipose tissue growth [51]. The effectiveness of this strategy to
suppress overweight using angiogenesis inhibitors is confirmed by the other group [52].
These basic experiments suggest that food-derived angiogenesis inhibitors can suppress
adipose tissue growth. There are many food-derive angiogenesis inhibitors; green tea
catechins, curcumin, resveratrol, etc. Interestingly, green tea catechins have only been
demonstrated to have anti-obesity effect for humans [53, 54]. The mechanisms by which
green tea catechins suppress obesity are demonstrated to disturb the absorption of glucose and
lipids [55-57] and to stimulate lipid catabolism in the liver [58]. In addition these
mechanisms, at least in part, anti-angiogenic effect of green tea catechins would contribute to
suppression of adipose tissue growth.
GREEN TEA CATECHIN AND DEMENTIA

Dementia and Alzheimer’s disease (AD) are a public health problem in modern society
[59-61]. The number of aged population is growing, and these diseases will become more
serious concern in the world. Several risk factors for vascular dementia have been reported
[59, 62-65]. In addition, green tea catechin has been shown to have neuroprotective effect and
to ameliorate neurodegenerative diseases such as AD and Parkinson disease [66]. Molecular
mechanisms of neruoprotective and neurorescue activity of green tea catechin have been
revealed; protective effects against β-amyloid (A β)-induced neurotoxicity, modulation of
survival and cell cycle genes and promotion of neurite outgrowth activity [67-69]. On the
other hand, anti-angiogenic activity of green tea catechin may play an important role in AD
prevention because it has been suggested that drugs that reduce the risk of AD have anti-
angiogenic activity [70]. In consistent with these studies, a recent epidemiological research
shows that a higher consumption of green tea is associated with a lower prevalence of
cognitive impairment [71]. These scientific data suggest that the intake of green tea would be
useful to prevent dementia.
In summary, angiogenesis is involved in various major diseases, which are rapidly
increasing in the world as mentioned in this chapter. Consumption of green tea catechin
lowers the risks of such diseases. The anti-angiogenic activity of green tea catechin would
contribute to the lower prevalence of such diseases as observed in Asian countries. Further
investigation on green tea catechin would give clinical significance of many diseases, and
green tea would be recognized as “Asian Paradox” [72] like “French Paradox”.
ACKNOWLEDGMENTS
Our works on anti-angiogenic natural products including green tea cactechin and
derivatives are supported by “Academic Frontier” Project for Private Universities (Kobe-
Gakuin University): matching fund subsidy from Japan Ministry of Education, Culture,
Sports, Science and Technology (MEXT), 2006-2010, (K. M. and Y. M.). K. M.
acknowledges a Grant-in-Aid for Young Scientists (B) (No. 18700608) from MEXT.
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Chapter 11
NEUROPROTECTIVE EFFECT OF THEANINE

ON CEREBRAL ISCHEMIA
Nobuaki Egashira1,2,*, Kenichi Mishima1, Katsunori Iwasaki1,

Ryozo Oishi2 and Michihiro Fujiwara1
1
Department of Neuropharmacology, Faculty of Pharmaceutical Sciences, Fukuoka
University, Fukuoka 814-0180, Japan
2
Department of Pharmacy, Kyushu University Hospital, Fukuoka 812-8582, Japan
ABSTRACT
The present article introduces our study related to the neuroprotective effect of γ-
glutamylethylamide (theanine), a component Japanese green tea (Camellia sinensis), on
cerebral ischemia. Theanine (1 mg/kg) significantly decreased the size of the cerebral
infarcts in a 4 h middle cerebral artery (MCA) occlusion model in mice. However,
theanine did not affect the cerebral blood flow, brain temperature and physiological
variables (pH, pCO2, pO2 and hematocrit) in this model. Theanine also reduced the
alterations of NeuN (neuron), GFAP (astrocyte) and Iba 1 (microglia) expression levels at
24 h after MCA occlusion. This neuroprotective effect of theanine was prevented by
bicuculline, γ-aminobutyric acidA (GABAA) receptor antagonist, but not 3-
mercaptopropionic acid, glutamate decarboxylase inhibitor. Furthermore, theanine (0.3
and 1 mg/kg) significantly prevented the impairment of spatial memory in rats subjected
to twice-repeated cerebral ischemia, 7 days after the second reperfusion. In addition,
theanine (1 mg/kg) significantly inhibited the decrease in the number of surviving cells in
the hippocampal CA1 field in the same rats. These results suggest that theanine directly
provides neuroprotection against cerebral ischemia and its neuroprotective effect is
mediated, at least in part, by GABAA receptors, and that it may be clinically useful for
preventing cerebrovascular disease.
*
Address correspondence to: Nobuaki Egashira, Ph.D. Department of Pharmacy, Kyushu University Hospital, 3-1-
1 Maidashi, Higashi-ku, Fukuoka 812-8582, Japan. Telephone: 81-92-642-5920; FAX: 81-92-642-5937; E-
mail: n-egashi@pharm.med.kyushu-u.ac.jp
208 N. Egashira, K. Mishima, K. Iwasaki et al.
Keywords: γ-Gutamylethylamide; Theanine; Cerebral ischemia; Neuroprotection; Middle

cerebral artery; Cerebrovascular disease; GABAA receptor.
ABBREVIATIONS
Theanine, γ-glutamylethylamide;
MCA, middle cerebral artery;
GABA, γ-aminobutyric acid;
AMPA, α-amino-3-hydroxy-5-methyl-4-isoxazole;
NMDA, N-methy-D-aspartate;
EGCG, epigallocatechin gallate;
CBF, cerebral blood flow;
BBB, blood-brain barrier;
SHR, spontaneously hypertensive rats;
DPPH, 2,2-Diphenyl-1-picryl-hydrazyl;
CNS, central nervous system;
GAD, glutamate decarboxylase
INTRODUCTION
In general, Japanese people drink several cups of tea including Japanese green tea
(Camellia sinensis) daily to take a break or relieve stress. The physiological and
pharmacological actions of various components of Japanese green tea such as catechins,
caffeine, γ-aminobutyric acid (GABA) have been studied [1-6]. The administration of these
components has affected blood pressure and stress. Moreover, catechins such as
epigallocatechin gallate (EGCG) have antioxidative [7] and anti-ischemic effects [8].
γ-Glutamylethylamide (theanine), a component of Japanese green tea, is a natural
glutamate analog (Figure 1). While theanine has been known to have relaxing properties, the
pharmacology of theanine is relatively unknown. Several studies have shown evidence for
multiple pharmacological effects on various neurochemical systems. These pharmacological
effects include: (1) inhibition of glutamate reuptake by inhibition of the glutamate transporter
[9]; (2) increase in GABA concentrations [10]; (3) increases in dopamine release in the
striatum in rats [11]; (4) decreases or no change in brain serotonin levels in rats [12, 13]; (5)
inhibition of the binding of α-amino-3-hydroxy-5-methyl-4-isoxazole (AMPA), kainate and
N-methy-D-aspartate (NMDA) receptors [14]; and inhibition of lipid peroxidation [15]. The
present article introduces our study related to the neuroprotective effect of theanine on
cerebral ischemia.
Neuroprotective Effect of Theanine on Cerebral Ischemia 209
COOH COOH
CHNH 2 CHNH2
CH 2 CH2
CH 2 CH2
CONHC2H 5 COOH
L-theanine L-glutamic acid
Figure 1. Chemical structures of theanine and glutamic acid.
EFFECT OF THEANINE ON CEREBRAL INFARCTION IN MICE

Kakuda et al. [16] reported that global cerebral ischemia-induced delayed neuronal death
in the hippocampal CA1 region was prevented by i.c.v. injection of theanine in gerbils. We
previously reported that systemic injection of theanine (1 mg/kg, i.p.) reduced the size of
cerebral infarcts following occlusion of the middle cerebral artery (MCA) for four hours in
mice [17]. In our study, theanine significantly reduced the infarct volume when it was
injected immediately before and 3 h after the occlusion. Theanine also reduced the infarct
volume when it was injected 3 h after the occlusion. Moreover, we showed that theanine did
not affect cerebral blood flow (CBF), brain temperature or physiological variables such as
pH, pCO2, pO2 and the hematocrit of plasma. Kakuda et al [16] also reported that the i.c.v.
injection of theanine did not decrease brain temperature in gerbils. In addition, it has been
reported that a high dose (2000 mg/kg, i.p.) of theanine did not alter blood pressure in
spontaneously hypertensive rats (SHR) [18]. We also found that theanine (1-100 mg/kg, i.p.)
had no effect on the blood pressure in SHR (N Egashira et al., unpublished data). These
findings suggest that theanine provides neuroprotection directly, against focal cerebral
ischemia.
Kimura et al. [10] reported that 30 min after the i.p. injection of theanine labeled with
14
C, the amide was incorporated into the mouse brain without any metabolic change.
Yokogoshi et al. [11, 19] also reported that theanine administered orally was transported into
the brain through the blood-brain barrier (BBB). These findings suggested that theanine
passes easily through the BBB. The neuroprotective effect of systemic administration of
theanine on the cerebral ischemia provided further evidence for the permeability of the BBB
to theanine.
Maruyama and Takeda [20] suggested that theanine is a competitive antagonist of
glutamate receptors. Kakuda et al. [14] also reported that theanine bound both AMPA and
NMDA receptors in rat cortical neurons, and its binding activity for AMPA receptors was 10-
fold that for NMDA receptors. It has been reported that AMPA receptor antagonists provide
protection against ischemia induced by MCA occlusion in rats [21-24] and forebrain ischemia
in gerbils and rats [25, 26]. We also reported that a selective competitive AMPA receptor
antagonist could provide neuroprotective activity and improve the spatial memory impaired
by repeated ischemia in rats [27]. These findings suggested that there were antagonistic
effects of theanine on the AMPA receptor providing neuroprotective action. However, this
was suggested as one of the mechanisms, because the binding activity of theanine to AMPA
receptors was lower than that of L-glutamic acid [14]. Furthermore, we found that theanine
(0.1-100 μM) did not prevent the cell damage induced by glutamate (100 μM) or kainate (1
mM) in rat cultured hippocampal neurons (N Egashira et al., unpublished data).
Theanine has been reported to inhibit lipid peroxidation [15]. We reported that vitamin E
isoforms such as α-tocotrienol and γ-tocopherol, which inhibit lipid peroxidation, prevented
focal cerebral ischemia induced by MCA occlusion [28]. These isoforms were injected i.v.
immediately before and 3 h after the occlusion. These findings suggest that the period of
radical formation is a few hours after the occlusion in this model. Theanine also reduced
infarct volume when it was injected 3 h after the occlusion [17]. However, we found that
vitamin E isoforms markedly exhibit 2,2-Diphenyl-1-picryl-hydrazyl (DPPH) radical-
scavenging activity, whereas theanine did not exhibit its activity (N Egashira et al.,
unpublished data). Therefore, it is unlikely that theanine prevented the focal cerebral ischemia
through radical-scavenging activity.
INVOLVEMENT OF GABAA RECEPTORS IN NEUROPROTECTIVE

EFFECT OF THEANINE ON FOCAL CEREBRAL ISCHEMIA IN MICE
Following an i.p. injection of theanine into mice, 14C-labeled theanine was incorporated
into the brain, without metabolic change and resulted in elevation of intracerebral levels of
GABA within 30 min [10], suggesting that theanine is transported into the brain through the
BBB and alter intracerebral GABA levels.
GABA is the principal inhibitory neurotransmitter in the mammalian brain. GABAA
receptors are mainly located postsynaptically and mediate the majority of inhibitory synaptic
transmission in the central nervous system (CNS). GABA synthesis and GABAA receptor
expression is reduced following occlusion of the MCA, suggesting that these receptors play a
role in cerebral ischemia [29, 30]. It has been shown that tiagabine, a GABA reuptake
inhibitor, and muscimol, a GABAA receptor agonist, have a neuroprotective action on
cerebral ischemia in rats [31, 32]. Moreover, the anesthetics propofol and isoflurane have
protective effects on cerebral ischemia, which can be reversed by bicuculline, a GABAA
receptor antagonist [33, 34]. These findings suggest that activation of the GABAA receptor
might play a role in inhibiting neuronal death induced by cerebral ischemia.
We investigated whether the GABAA receptor was involved in neuroprotection by
theanine following ischemic brain damage in the MCA occlusion mouse model [35]. The
GABAA receptor antagonist bicuculline (10 mg/kg, i.p.) prevented the inhibition of infarct by
theanine although when administered alone, bicuculline had no effect on the size of infarct
(Figure 2). Our findings showed that the neuroprotective effect of theanine is mediated, at
least in part, by GABAA receptors (Figure 3). In our studies, theanine was effective by
injection 1 h before reperfusion in MCA model. Moreover, theanine-treatment 30 min before
reperfusion is also effective (N Egashira et al., unpublished data). However, we found that
theanine-treatment immediately after reperfusion had no effect on the focal cerebral ischemia
in same model (N Egashira et al., unpublished data). An i.p. injection of theanine was
reported to increase the intracerebral level of GABA 30 min after injection of theanine [10].
These findings suggest that the neuroprotective effect of theanine is due to act indirectly on
GABAA receptors during reperfusion. Therefore, theanine could prevent the focal cerebral
ischemia by injection 30-60 min before reperfusion. In this study, the number of NeuN
(neuron) and GFAP (astrocyte) positive cells was decreased, while the number of Iba1
(microglia) positive cells was increased in the cerebral cortex at 24 h after MCA occlusion,
and theanine (1 mg/kg, i.p.) inhibited these alterations [35]. Iba1 immunoreactive cells rapidly
appear at 3.5 h after reperfusion [36]. Therefore, the decrease of Iba1 expression levels is a
result of neuroprotection by theanine.
100 #
80
Infarct volume (mm3)
*
60 *
40
20
0
Vehicle Theanine 1 mg/kg (i.p.)
Bicuculline 10 mg/kg (i.p.)
Distance from
frontal pole
2mm
4mm
6mm
8mm
Vehicle Theanine 1 mg/kg (i.p.)

Bicuculline 10 mg/kg (i.p.)
Figure 2. Effects of theanine and bicuculline on infarct volume following MCA occlusion in mice.
Theanine and bicuculline were injected i.p. 3 h after the occlusion. Slices were immediately stained
with 2% 2,3,5-triphenyltetrazolium chloride 24 h after MCA occlusion. **P<0.01 compared with
vehicle, #P<0.05 compared with theanine alone. (This figure was reproduced from reference#35 under
permit).
Glutamatergic
neuron
GABAnergic neuron
Theanine
+ GABA
Glutamate
pre
GABAA
receptor
post
NMDA receptor AMPA receptor
Fig. 3 Neuroprotective effect
Figure 3. Theanine prevents the cerebral infarcts following occlusion of the MCA. The neuroprotective
effect of theanine is mediated, at least in part, by GABAA receptors.
In general, glutamic acid is metabolized into GABA by glutamate decarboxylase (GAD).

When theanine was administered intragastrically to rats, the concentration of theanine in the
brain was increased, however the concentration of glutamic acid in the brain remained
unchanged [19]. These findings suggest that theanine increases the intracerebral level of
GABA through a mechanism other than metabolism by GAD. We also found that GAD
inhibitor 3-MPA had no effect on the protective effect of theanine on focal cerebral ischemia
[35]. Therefore, GAD is not involved in the neuroprotective effect of theanine on focal
cerebral ischemia.
EFFECT OF THEANINE ON REPEATED ISCHEMIA-INDUCED

IMPAIRMENT OF SPATIAL MEMORY AND NEURONAL CELL DEATH
We previously reported that repeated ischemia (10 min x 2 times, 1 h interval) caused cell
death and acetylcholine dysfunction in the hippocampus, and the impairment of spatial
memory in radial maze test in rats [37]. We also reported that a AMPA receptor antagonist
YM-90K but not a NMDA receptor antagonist MK-801 prevented both this impairment of
spatial memory and cell death, suggesting that AMPA receptor may play an important role on
the impairment of the spatial memory in the rats subjected to repeated ischemia [27]. It has
been determined that the AMPA receptor has four subunits, designated GluR1 through
GluR4. AMPA receptors exist as either Ca2+-impermeable or Ca2+-permeable channels
depending on their subunit consititution; that is, the presence of the GluR2 subunit renders
heteromeric AMPA receptor assembly Ca2+-impermeable, while an AMPA receptor without
GluR2 subunit shows Ca2+-permeability [38, 39]. It has also been determined that a marked
reduction of GluR2 expression in vulnerable neurons prior to cell death following cerebral
transient ischemia, and overexpression of Ca2+-permeable via AMPA receptor lacking the
GluR2 subunit promoted delayed cell death of hippocampal CA1 neurons following the
cerebral ischemia [40]. The decrease of GluR2 mRNA is the triggers for the degeneration of
the CA1 pyramidal cell [41]. Moreover, we have reported that repeated ischemia reduced
ratios of GluR2 subunit variants to those of GluR1 [42]. AMPA receptors play an important
role in mediating cerebral ischemia [43]. Cerebral ischemia produces two main changes in the
AMPA receptors: up-regulation [44] and conformational changes characterized by decrease
of GluR2 mRNA subunit in hippocampal CA1 neurons [45]. These changes, together with
overstimulation of AMPA receptors by increased glutamate, induce excessive Ca2+ influx,
leading to intracellular Ca2+ overload which triggers mainly an apoptotic type of cell death
with a more delayed onset [43].
We found that theanine significantly prevents the impairment of spatial memory and
inhibits the decrease in the number of surviving cells in the hippocampal CA1 field in rats
subjected to repeated cerebral ischemia [46]. These results suggest that theanine prevents
memory impairment induced by repeated cerebral ischemia, at least in part, by protecting
against neuronal cell death. Maruyama and Takeda [20] suggested that theanine is a
competitive antagonist of glutamate receptors. Kakuda et al. [14] reported that theanine binds
both AMPA and NMDA receptors, and that its binding affinity for AMPA receptors is 10-
fold that for NMDA receptors. It has been reported that AMPA receptor antagonists provide
protection against ischemia induced by MCA occlusion in rats [21-23] and forebrain ischemia
in gerbils and rats [25, 26]. We also reported that a selective, competitive AMPA receptor
antagonist could provide neuroprotective activity and reduce the impairment in spatial
memory induced by repeated cerebral ischemia in rats [27]. Theanine might provide a
neuroprotective action via antagonistic effects on AMPA receptors. This mechanism was
suggested as just one of the multiple mechanisms, because the binding affinity of theanine to
AMPA receptors is lower than that of L-glutamic acid [14]. Arias et al. [47] reported that the
neuroprotective effect of a combination of AMPA and NMDA receptor antagonists was
greater than the sum of the effects of the individual antagonists in an in vitro model of
cerebral ischemia. Therefore, the neuroprotective effect of theanine may be due to its
antagonistic effects on both AMPA and NMDA receptors. It has also been reported that i.p.
injection of theanine increases the intracerebral level of GABA 30 min after injection [10].
GABA reuptake inhibitors and GABAA receptor agonists have a neuroprotective action on
cerebral ischemia in rats [31, 32]. Also, the anesthetics propofol and isoflurane have
protective effects on neuronal damage induced by cerebral ischemia, which can be reversed
by bicuculline, a GABAA receptor antagonist [33, 34]. Moreover, we previously found that
the neuroprotective effect of theanine was reversed by bicuculline in MCA occlusion mice
[35]. Therefore, theanine might prevent the impairment of spatial memory and neuronal cell
death induced by repeated cerebral ischemia not only by blocking the glutamate receptors, but
also by acting on the GABAA receptors.
This study shows that i.p. injection of theanine significantly prevents the impairment of
spatial memory and neuronal cell death induced by repeated cerebral ischemia. We previously
demonstrated that i.p. injection of theanine reduced the size of cerebral infarcts in MCA
occlusion mice [17]. These findings suggest that systemic administration of theanine provides
neuroprotection against cerebral ischemia. Kimura and Murata [10] reported that 30 min after
i.p. injection of theanine, it was incorporated into the mouse brain without any metabolic
change. In addition, Yokogoshi et al. [11, 18] reported that theanine, when administered
orally, was transported into the brain through the BBB, suggesting that theanine passes easily
through this barrier. Moreover, theanine exerts a neuroprotective action against cerebral
ischemia when administered systemically, providing further evidence for the permeability of
the BBB to theanine.
We previously showed that an effective dose of theanine did not alter CBF, brain
temperature or physiological variables such as pH, pCO2, pO2 and the hematocrit of plasma,
in MCA occlusion mice [17]. Moreover, Kakuda et al. [16] reported that i.c.v. injection of
theanine into gerbils did not decrease brain temperature. In addition, a high dose of theanine
(2000 mg/kg, i.p.) did not alter blood pressure in rats [18]. In this study, we found that
theanine did not affect body temperature in rats subjected to repeated cerebral ischemia (N.
Egashira et al., unpublished data). Thus, theanine provides neuroprotection against cerebral
ischemia without changes in CBF, body temperature, brain temperature or physiological
responses.
CONCLUSION
In conclusion, the findings of our study showed that systemic administration of theanine
protected against the focal cerebral ischemia induced by MCA occlusion without affecting
CBF and brain temperature. Our findings also show that systemic administration of theanine
prevents the impairment of spatial memory and neuronal cell death induced by repeated
cerebral ischemia. These results suggest that theanine may be clinically useful for preventing
cerebrovascular disease. Moreover, the results of this study imply that GABAA receptors are
involved in the neuroprotective mechanism of theanine following cerebral ischemia.
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Chapter 12
CHARACTERIZATION OF THE NEUROPROTECTIVE

ACTIVITY OF THE POLYPHENOL (-)-
EPIGALLOCATECHIN-3-GALLATE IN THE BRAIN
Orly Weinreb*, Tamar Amit, Moussa B. H. Youdim

and Silvia Mandel
Eve Topf and USA National Parkinson Foundation Centers of Excellence for
Neurodegenerative Diseases Research and Department of Pharmacology, Rappaport
Family Research Institute, Technion-Faculty of Medicine, Haifa, Israel
ABSTRACT
Standing after water, tea signifies the second most frequently consumed beverage
worldwide, which varies its status from a simple ancient drink and a cultural tradition to a
nutrient endowed with possible neurobiological-pharmacological actions beneficial to
human health. Accumulating evidence suggests that oxidative stress resulting in reactive
oxygen species generation plays a pivotal role in neurodegenerative diseases, supporting
the implementation of radical scavengers and metal chelating agents, such as natural tea
polyphenols for therapy. Vast epidemiology data indicate a correlation between
occurrence of neurodegenerative disorders, such as Parkinson’s and Alzheimer’s diseases
and green tea consumption. In particular, literature on the putative novel neuroprotective
mechanism of the major green tea polyphenol, (-)-epigallocatechin-3-gallate (EGCG)
strengthens the notion that diverse molecular signaling pathways, participating in the
neuroprotective activity of EGCG, thus rendering this natural compound as potential
agent to reduce the risk of various neurodegenerative diseases. In the present article, we
review the studies concerning the mechanisms of action implicated in EGCG-induced
neuroprotection in the brain and discuss the vision to translate these findings into a
lifestyle arena.
*
Correspondence should be addressed to: Dr. Orly Weinreb. Department of Pharmacology, Technion- Faculty of
Medicine; P.O.B. 9649, Haifa 31096, Israel. Tel: +972-4-8295291; Fax: +972-4-8513145; E-mail:
worly@tx.technion.ac.il
220 Orly Weinreb, Tamar Amit, Moussa B. H. Youdim et al.
ABBREVIATIONS
AD Alzheimer’s disease;
Aβ amyloid-beta peptide,
ARE antioxidant response elements;
COMT catechol-O-methyltransferase;
BBB blood-brain barrier;
DA dopamine;
EC (–)-epicatechin;
ECG epicatechin-3-gallate;
EGCG (-)-epigallocatechin-3-gallate;
EGC (–)-epigallocatechin;
MAPK extracellular mitogen-activated protein kinases;
ERK1/2 extracellular signal-regulated kinases;
GAP-43 growth associated protein-43;
6-OHDA 6-hydroxydopamine;
HSP90 heat shock protein 90;
HIF-1 hypoxia inducible factor-1;
3-HK 3-Hydroxykynurenine;
iNOS inducible nitric oxide synthase;
MEK1 mitogen-activated protein kinase 1;
MPP+ 1-methyl-4-phenylpyridinium;
MPTP N-methyl-4-phenyl-1,2,3,6-tetrahydropyridine;
OS oxidation stress;
PD Parkinson’s disease;
PKC protein kinase C;
PC12 cells rat pheochromocytoma cells;
R-APO R-apomorphine;
ROS reactive oxygen species;
GSH  reduced glutathione;
sAPPα soluble amyloid precursor protein;
TNF-α tumor necrosis-alpha,
TfR transferrin receptor;
SNPC substantia nigra pars compacta;
VEGF vascular endothelial growth factor;
UPS ubiquitin proteasome system.
Characterization of the Neuroprotective Activity … 221
Tri-hydroxyl
group (pyrogallol
structure)
B
A C
Galloyl
group
Figure 1. The chemical structure of eight polyphenolic flavanol-type compounds namely, (+)-catechin
(C), (-)-epicatechin (EC), (+)-gallocatechin (GC), (-)-epigallocatechin (EGC), (+)-catechingallate (CG),
(-)-epicatechin gallate (ECG), (+)-gallocatechin gallate (GCG) and (-)-epigallocatechin-3- gallate
(EGCG).
INTRODUCTION
The natural polyphenols present in beverages obtained from plants, fruits and vegetables
such as olive oil, red wine and teas, have been suggested to be beneficial and favorable to
human health (see reviews, [1, 2]). Flavonoids are the largest group of polyphenols, mainly
divided into anthocyanins, glycosylated derivative of anthocyanidin, present in colorful
flowers and fruits, and anthoxantins, colorless compounds which are further divided into
several monomeric compound categories including flavones, isoflavones, falvanols flavans,
and flavonols [3]. Flavonoids have a diphenypropane (C6C3C6) skeleton consisting of an
aromatic ring (Figure 1), which is condensed, to a heterocyclic ring and attached to a second
aromatic ring [3]. The abundant phenolic hydroxyl groups on the aromatic rings confer the
antioxidant and the iron chelating activities of these compounds [4].
The importance of polyphenolic flavonoids in enhancing cell resistance to oxidative
stress (OS) goes beyond the simple scavenging and iron chelating activities and is mostly
interesting in those pathologies, where OS and iron are involved. Numerous studies in the last
decade have shown that polyphenols have in-vitro and in-vivo activities in preventing and/or
reducing the deleterious effects of oxygen-derived free radicals, associated with several
chronic and stress related human diseases [5, 6]. Several lines of evidence suggest that OS
resulting in reactive oxygen species (ROS) generation, either through an enzyme or metal
catalyzed processes, play a pivotal role in clinical disorders, such as atherosclerosis,
ischemia-reperfusion injury, cancer, stroke and neurodegenerative disorders [5, 6]. Special
interest has been assigned to the therapeutic feature of antioxidants and nutritional approach
in neurodegenerative diseases, such as Parkinson’s disease (PD), Alzheimer’s disease (AD)

and amyotrophic lateral sclerosis (ALS) [7, 8]. Oxidative damage to neuronal molecules and
increased accumulation of iron in specific brain areas are considered major pathological
aspects of PD and AD [9]. Although the etiology of both disorders and their respective
dopaminergic or cholinergic neuronal degeneration remains elusive, the chemical pathology
of PD shows many similarities to AD, involving increase in iron concentration in the affected
areas, release of mitochondrial cytochrome c, protein aggregation, OS, loss of tissue reduced
glutathione (GSH), reduction in mitochondrial complex I activity and increased lipid
peroxidation [10, 11].
A large investigation on PD and cognitive impairment found a moderate risk reduction in
black (fermented), oolong (semi-fermented) and green (not-fermented) teas consumers
compared to non tea drinkers [12-14]. The favorable properties ascribed to tea consumption
are believed to rely on its bioactive flavanol class-related catechins and their derivatives,
demonstrated to act as radical scavengers and exert indirect antioxidant effects through
activation of transcription factors, signaling regulators and antioxidant enzymes (for reviews
see [1,15,16]). In line with this evidence, particular attention has been placed to study the
neuroprotective action of antioxidants, iron-chelating and anti-inflammatory agents, tea
catechins and especially, the major component of green tea, (-)-epigallocatechin-3-gallate
(EGCG) [17-19]. The revelation of novel molecular targets, possibly implicated in their
neuroprotective action include: calcium homeostasis [20], the extracellular mitogen-activated
protein kinases (MAPK) [21] and protein kinase C (PKC) signaling pathways [22, 23] and
regulation of antioxidant enzymes [24], antioxidant response element (ARE) [25], cell death
and cell survival genes and proteins associated with mitochondrial function, such as Bcl-2
family members [26-28], amyloid precursor protein (APP) pathway [22, 29] and iron
regulators and sensors encoding genes and proteins, such as transferrin receptor (TfR) and
hypoxia-inducible factor (HIF) prolyl-hydroxylases [29-31] (Figure 2).
This review aims to compile the most recent literature regarding the mechanisms of
action implicated in EGCG-induced neuroprotection in the brain, which might be a reflective
outcome of its brain permeable, antioxidant and iron chelating properties.
GREEN TEA POLYPHENOLS

Green tea is the most widely consumed beverage aside from water in Japan, China, and
other Asian nations and is becoming more popular in Western countries. Green tea belongs to
the Theacease family derived from two plant varieties, Camellia sinesis and assamica [32].
The first scientific recognition of medicinal properties of green tea was in the 16th century,
using extracts as therapeutic mean to cure fever, headache, stomach and articulation pain [33].
To date, green tea has attracted attention for its health benefits, particularly with respect to its
potential for preventing and treating cancer, cardiovascular diseases, inflammatory diseases,
aging and neurodegenerative diseases in human [34-36].
Figure 2. A schematic model of EGCG neuroprotective mechanism of action indicating potential

pathways of activity with respect to its suggested multifunctional effects in neuronal and extra-neuronal
tissues. Full explanation is discussed in the text.
Catechins account for 25-35% of the green tea dry extract and consist of eight
polyphenolic flavonoid-type compounds, namely, (+)-catechin (C), (-)-epicatechin (EC), (+)-
gallocatechin (GC), (-)-epigallocatechin (EGC), (+)-catechingallate (CG), (-)-epicatechin
gallate (ECG), (+)-gallocatechin gallate (GCG) and EGCG, (Figure 1). The most abundant
polyphenol is EGCG, thought to particularly contribute to the beneficial effects attributed to
green tea, such as its neuroprotective and antioxidant properties. It is well established that the
catechins contain free radical scavenging properties and act as biological antioxidants [18,
37]. It has been demonstrated that they can scavenge both superoxide and hydroxyl radicals,
as well as the 1,1-diphenyl-3-picrylhydrazyl radical, peroxyl radicals, nitric oxide, carbon-
center free radicals, singlet oxygen and lipid free radicals, and peroxynitrite by preventing the
nitration of tyrosine [37-39]. Additionally, catechins chelate metal ions such as copper (II)
and iron (III) to form inactive complexes and prevent the generation of potentially damaging
free radicals [40]. Catechins have been found to be more efficient radical scavengers than
vitamin E and C [18, 37]. Relative antioxidant activity among tea catechins has been found to
be EGCG=ECG >EGC>EC [41, 42]. EGCG accounts for more than 10% of the extract dry
weight, 20-35 mg per cup of green tea, followed by EGC >EC ≥ ECG [41, 42].
The metabolism of green tea catechins has been studied in various animals including
human [43, 44]. Orally catechin administration to human is absorbed, metabolized and
excreted within 24 h [45]. Study with healthy green tea consumers revealed levels of EGCG,
EGC and EC in the plasma in a dose-dependent concentration, varying between 0.2 and 2%
of the ingested amount, with maximal concentration 1.4-2.4 h after ingestion [46]. In addition,
after ingestion of 1.2 g of green tea solids (dissolved in two cups of warm water), the plasma
samples collected at 1 h from human volunteers contain 46-268 ng/ml [44]. The half-life for
EGCG is about 5 h and for EGC and EC it varies between 2.4 and 3.4 h [47, 48]. Several
reports indicated that tea polyphenols can be attained in the brain and exert neuroprotective
effect simply by drinking. Previously, it was reported that EC metabolites (epicatechin
glucuronide and 3’-O-methylated epicatechin glucuronide), formed after oral ingestion of EC
by rats, had gained entry to the brain [49]. Furthermore, study with labeled EGCG
demonstrated a wide distribution of radioactivity in mouse organs including brain, after oral
administration and small amount of [3H] EGCG excretion in the urine after direct
administration [50]. Recently, the absorption and pharmacokinetic of EGCG in various brain
regions of adult and fetal rats have been demonstrated by oral and intravenous administration,
indicating that EGCG is the most abundant catechin in brain tissue [51] and may potentially
penetrate through the blood-brain barrier (BBB) [52]. In vitro model of BBB demonstrated
that various flavonoids and some metabolites were able to traverse the BBB and that the
potential for permeation was consistent with compound lipophilicity [53].
THE BENEFICIAL EFFECTS OF EGCG IN

NEURODEGENERATIVE DISEASES
Human Epidemiology and Clinical Trials
Notwithstanding the uncertainty on the capacity of tea polyphenols to penetrate the brain
and lack of well-controlled clinical data, green tea polyphenols were suggested to inversely
correlate with the incidence of dementia and brain aging and neurodegeneration, such in PD
and AD. Previous epidemiological studies have shown a reduced risk of PD associated with
consumption of 2 cups/day or more of black tea [54]. In support, Tan et al [55] found an
inverse association between black tea and PD, based on a 12 year prospective study of over
63,000 men and women, that was due to black tea ingredients separate from its caffeine
content. In a cross-sectional study aimed at investigating an association between consumption
of green tea and cognitive function in elderly Japanese subjects, it was found that higher
consumption of green tea is associated with lower prevalence of cognitive impairment [13].
Despite the fact that the prevalence of PD is much lower in tea consumers, the association of
green tea drinking and risk of AD and other neurodegenerative diseases is not well
established. No case-control study has been accomplished that points to a beneficial effect
associated with tea consumption in AD, although treatment with the extract EGb761 derived
from Gingko biloba leaves, another natural flavonoid that does not contain catechins,
improved cognitive performance of AD patients [56]. These findings emphasize the
importance of well-designed controlled studies to assess a risk reduction of PD and AD in
consumers of green and black tea. Indeed, a randomized, double-blind and efficacy study in
Beijing China is under completion, to determine the safety, tolerability and potential
neuroprotective effects of a green tea polyphenol enriched preparation, in de novo PD patients
without taking any anti-Parkinsonism drug (sponsored by the Michael J. Fox Foundation for
Parkinson's Research).
Neuroprotection in Vitro and in Animal Models of Neurodegenerative

Diseases
Neuroprotective in-vivo studies employing the parkinsonism-inducing neurotoxin, N-

methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) have shown that both green tea extract
and EGCG possess highly potent activities in preventing mice striatal dopamine (DA)
depletion and substantia nigra (SN) dopaminergic neuron loss [24]. One possible mechanism
underlying the effectiveness of green tea and EGCG against MPTP neurotoxicity, may
involve its catechol-like structure, since it is known that catechol-containing compounds are
potent radical anti-oxidants and ferric ion chelators [39]. In agreement, the iron chelators,
radical scavengers and catechol derivative compounds R-apomorphine (R-APO), a DA
receptor agonist and its S-isomer, induced neuroprotection in animal models of PD [57, 58].
The catechol structural resemblance may account for a recently reported inhibitory effect of
green tea polyphenols on [3H] DA uptake by presynaptic transporters. This inhibition was
suggested to block the metabolic product of MPTP, the neurotoxin 1-methyl-4-
phenylpyridinium (MPP+) uptake (because of competition for the vesicular transporter)
thereby protecting DA-containing neurons against MPP+-induced injury [59]. In-vitro studies
also demonstrated inhibition of MPP+ and 6-hydroxydopamine (6-OHDA)-induced
neurotoxicity by EGCG [26]. Furthermore, EGCG, at a low IC50 concentration (0.2 μM),
inhibited the activity of the enzyme catechol-O-methyltransferase (COMT) in rat liver cytosol
homogenates [60]. DA and related catecholamines are physiological substrates of COMT.
The COMT inhibitors entacapone and tolcapone, clinically prescribed to PD-affected
individuals, dose-dependently inhibited the formation of the major metabolite of levodopa, 3-
O-methyldopa, thereby improving its bioavailability in the brain [61]. In addition, the
implication of the iron chelation property of EGCG in neuroprotection in PD animal models
has been strengthened by the observations that both MPTP and 6-OHDA significantly
increased iron in SN pars compacta (SNPC) of mice, rats and monkeys [62, 63]. Indeed, iron
accumulation in the brain has been reported in a range of neurodegenerative disorders [64]
and demonstrated to accumulate in neurons and microglia in SN of PD patients [65].
Although AD epidemiological studies did not report any established outcome relative to
green tea consumptions, in-vitro observations showed that EGCG prevents amyloid beta
peptides (Aβ)-induced neurotoxicity, [22, 66] and EC reduces nascent Aβ fibrils, elongation
of the fibrils, and destabilization of the formed assemblies [67]. In addition, EGCG was able
to regulate the proteolytic processing of APP under in-vivo and in-vitro conditions [22, 29],
suggesting that green tea polyphenols might be potentially promising therapeutic agents not
only for PD, but also for AD. EGCG promoted the non-amyloidogenic α-secretase pathway
of APP in neuronal cell cultures resulting in a consequential augment in soluble APPα
(sAPPα) [22]. This increase was inhibited by the hydroxamic acid-based metalloprotease
inhibitor Ro31-9790, indicating that this effect was mediated via α-secretase processing [22].
Also, long-term treatment of mice with EGCG resulted in decreases in cell-associated, full-
length APP levels, as well as increases in the sAPPα levels in the hippocampus [22]. New
supportive data came from a study conducted in an Alzheimer's transgenic mice model
("Swedish" mutant APP overexpressing, APPswTg) mice, showing that EGCG promoted
sAPPα generation and decreased Aβ levels and plaques via promotion of the non-
amyloidogenic α-secretase proteolytic pathway [68, 69]. Recently, long-term administration
of EGCG provided prophylactic benefits on rat spatial cognitive learning impairment caused
by Aβ cerebral ventricleinfusion [70].
Supplementary preclinical models of other neurodegenerative diseases reported
neuroprotective effects of EGCG, such as prolongation of the symptom onset and life span
and attenuation of death signals in ALS mice model expressing the human G93A mutated
Cu/Zn-superoxide dismutase (SOD1) gene [71, 72] and in a variety of cerebral ischemia
animal models [33, 73].
THE MECHANISMS UNDERLYING

EGCG-INDUCED NEUROPROTECTION
Antioxidant and Iron Chelating Mechanism of Action
The protective effect of EGCG against neurological diseases may involve its radical
scavenging and iron chelating activity and/or regulation of antioxidant protective enzymes.
The inhibition of enzymes, whose activity may promote OS or an increase of antioxidant
enzyme activities, might have beneficial significance to neuroprotection. Previous studies
reported that EGCG elevated the activity of two major antioxidant enzymes, superoxide
dismutase (SOD) and catalase in mice striatum [24] (Figure 2).
In addition, tea polyphenols were found to be potent scavengers of free radicals, such as
singlet oxygen, superoxide anions, hydroxyl radicals and peroxy radicals, in a number of in-
vitro systems [18, 74, 75]. In the majority of these studies, EGCG was shown to be more
efficient as a radical scavenger than its counterparts ECG, EC and EGC, which may be
attributed to the presence of the trihydroxyl group on the B ring (Figure 1) and the gallate
moiety at the 3’ position in the C ring [18]. EGCG attenuated hydroxykynurenine (3-HK) -
induced cell death, as well as the increase in ROS concentrations and caspase-3 activity in
neuronal culture, presumably via its antioxidant activity [76]. In rat brain tissue, green tea and
black tea extracts were shown to inhibit lipid peroxidation promoted by iron-ascorbate in
brain mitochondrial membranes [77]. A similar effect was also reported using brain
synaptosomes, in which the four major polyphenol catechins of green tea were shown to
inhibit iron-induced lipid peroxidation [39]. In this regard, it has been shown that EGCG
attenuated paraquat-induced microsomal lipid peroxidation and increased the survival rate of
paraquat-poisoned mice [78, 79]. In addition, Higuchi et al. [78] suggested that EGCG
inhibited paraquate-induced malondialdehyde production in rat liver microsome system
containing FeSO4 by two possible mechanisms: one was by scavenging of superoxide
radicals, which were responsible for the reduction of ferric to ferrous, catalyzed by the Fenton
reaction. The other was through iron-chelating activity, given that the inhibition disappeared
when excessive amount of FeSO4 were added to the reaction, indicating that EGCG inhibited
iron driven lipid peroxidation by pulling out available iron.
The ability of green tea polyphenols and EGCG in particular to chelate metal ions, such
as iron and copper, may contribute to their antioxidant/neuroprotective activity by inhibiting
transition metal-catalysed free radical formation. The two points of attachment of transition
metal ions to the flavonoids molecule are: the o-diphenolic groups in the 3’,4’-dihydroxy
positions in the B ring, and the keto structure 4-keto, 3-hyroxy or 4-keto and 5-hydroxy in the
C ring of the flavonols [4, 80]. The ability of green tea polyphenols to act as relatively potent
metal chelators [39, 81] may be of major significance for treatment of neurodegenerative
diseases, where iron accumulation has been shown in brain areas where neurodegeneration
occurs. Most importantly, green tea was reported not to affect iron absorption in healthy
human subjects [82]. The localization of iron and ferritin in PD patients is restricted to
specific brain areas [9, 83, 84] in the SNPC, but not the reticulata [83]. Similarly, AD
pathogenesis is associated with iron accumulation and is linked to the characteristic
neocortical Aβ deposition, phosphorylation of tau and tangle formation, which may be
mediated by abnormal interaction with excess of free-chelatable iron. Ionic iron can, in turn,
participate in the Fenton reaction with subsequent generation of ROS, thought to initiate the
processes of OS and inflammatory cascade resulting in production of cytotoxic cytokines
(tumor necrosis-alpha (TNF-α), interluekin-1 and -6) in the microglia and in the surrounding
neurons [85, 86] and activation of transcription factors and nuclear factor-kappa B (NF-κB)
[87, 88]. Indeed, a marked increase in NF-κB immunoreactivity was found in the nucleus of
melanized dopaminergic neurons of the Parkinsonian SNPC, compared to normal brains [89].
EGCG was found to inhibit the nuclear translocation of NF-κB in in-vitro systems:
immunofluorescence and electromobility shift assays showed that introduction of green tea
extract before 6-OHDA, inhibited both NF-κB nuclear translocation and binding activity in
human neuroblastoma SH-SY5Y cells [77, 90]. Furthermore, the reduced activity of NF-κB
by EGCG and the theaflavin-3,3'-digallate polyphenol from black tea, was associated with
inhibition of lipopolysaccharide (LPS)-induced TNF- α production [91] and the enzyme
inducible nitric oxide synthase (iNOS) [87, 92, 93], which is responsible for the production of
the short-live free radical, nitric oxide in activated macrophages.
Interestingly, recent studies have identified a novel link between iron and AD, associated
with an enhancement of endogenous APP translation and subsequent Aβ formation, via
activation of an iron responsive element (IRE-type II) in the 5' untranslated region (UTR) of
APP mRNA [94]. This finding opened a new potential therapeutic avenue aimed at reducing
amyloidosis with iron-complexing drugs that modulate APP mRNA translation. In support, a
recent in vitro study demonstrated that EGCG reduced full-length APP in SH-SY5Y cells
without altering APP mRNA levels, while exogenous iron supplementation reversed its effect
[29], suggesting a post-transcriptional action, presumably by the mechanism of chelating
intracellular iron pools (Figure 2). This is further supported by the observation that EGCG
suppressed translation of a luciferase reporter gene driven by the IRE-type II-containing
sequences of APP [29]. Furthermore, it was found that EGCG markedly reduced secreted Aβ
levels in the conditioned medium of Chinese hamster ovarian cells, overexpressing the
"Swedish" mutated APP (CHO/ΔNL) [29] and in primary neuronal cells derived from
transgenic mice bearing the APP "Swedish" mutation [69].
More recently, Friedlich et al [95] have described a putative IRE in the 5'-UTR of PD-
related α-synuclein mRNA and predicted that this RNA structure may have the potential to
function as a post-transcriptional regulator of its protein synthesis in response to iron and
redox events, resembling the pattern seen with APP and the iron-associated protein ferritin.
This finding can explain, in part, previous observation demonstrating that the iron chelating
compounds R-APO and EGCG, prevented iron-dependent up-regulation of α-synuclein in the
SNPC of MPTP-treated mice, resulting in neuroprotection of SN dopaminergic neurons [96].
Thus, the radical scavenging and free-iron complexing activities of green tea polyphenols
may directly influence aggregation and deposition of either Aβ or α-synuclein in brains of

AD and PD patients, respectively.
Regulation of Neuronal Survival PKC/MAPK Pathways Associated with

Bcl-2 Family Members
Emerging evidence suggests that the antioxidant activity of green tea polyphenols cannot
be the exclusive mechanism responsible for their neuroprotective action, but rather, their
ability to alter signaling pathways may significantly contribute to the cell survival effects.
Modulation of cellular survival and signal transduction pathways has significant biological
consequences that are important in understanding the various pharmacological and
toxicological responses of antioxidant drugs. A number of intracellular signaling pathways
have been described to play central functions in EGCG-promoted neuronal protection against
a variety of extracellular insults, such as the MAPK [31, 97, 98], PKC [23, 26, 99, 100] and
phosphatidylinositol-3-kinase (PI-3 kinase)-Akt [101-103] pathways, as described in Figure
2. Given the critical role of MAPK pathways in regulating cellular processes that are affected
in neurodegenerative diseases, the importance of MAPKs as transducers of extracellular
stimuli into a series of intracellular phosphorylation is being increasingly recognized. OS
seems to be a major stimulus for MAPK cascade, which might lead to cell survival/cell death.
Among the MAPKs, the extracellular signal-regulated kinases (ERK1/2) are mainly activated
by mitogens and growth factors [104], while p38 and c-jun-N-terminal kinase (JNK) respond
to stress stimuli [105, 106]. Previous in-vitro studies [25] demonstrated the potency of EGCG
to induce ARE-mediated defensive genes and MAPKs pathways, including the cell survival
signaling regulators, p44/42 ERK 1/2, JNK and p38 MAPK [107]. The role of ERK1/2
signaling seems to be connected to attenuation of neuronal death and cellular injury by OS
[108]. EGCG counteracted the decline in ERK1/2 induced by 6-OHDA [26] and induced
phosphorylation of ERK1/2 in serum-deprived SH-SY5Y cells [31]. Nonetheless, EGCG at
its neuroprotective concentrations (1-10 μM), did not affect the levels of ERK1/2
phosphorylation by itself, in the absence of any exogenous damage, in neuronal cell line [26].
EGCG neuroprotective activity also involves the intracellular signaling mediator PKC
[22, 23], thought to have an essential role in the regulation of cell survival and programmed
cell death [109, 110]. A rapid loss of neuronal PKC activity is a common consequence of
brain neurodegeneration [111, 112]. The induction of PKC activity in neurons by EGCG (1-
10 μM) is thought to be a prerequisite for neuroprotection against several neurotoxins, such as
Aβ [22], serum withdrawal [23, 100, 103, 113] and 6-OHDA [26] since inhibition of PKC
phosphorylation completely abolished the protection induced by EGCG and by a PKC
activator, phorbol 12-myristate 13-acetate (PMA). These in-vitro results were supported by a
previous report [22], who showed that EGCG oral administration to mice (2 mg/kg) caused a
significant increase of the PKC isozymes α end ε protein levels in the membrane and
cytosolic fractions of hippocampus. These isoforms play a crucial role in cell survival and
differentiation pathways [114] and may be involved in APP regulatory processing associated
with the pathogenesis of AD [115, 116]. Indeed, previous studies in brains of AD patients
demonstrated reduction of PKCε activity [117].
The mechanism by which PKC activation leads to neuroprotection begins to be

elucidated. Studies with extra-neuronal tissues support a role for PKCα as a kinase of the
antiapoptotic Bcl-2, probably through direct or indirect phosphorylation of this cell survival
protein [118]. Neuroprotective experimental studies demonstrated that the protective effect of
EGCG involved reduction of the apoptotic markers, cleaved caspase 3, its downstream
cleaved substrate poly-ADP-ribose-polymerase (PARP), a nuclear zinc finger DNA-binding
protein that detects and binds to DNA strand breaks and Bad, a member of a group of “BH3
domain only” proteins of the Bcl-2 family [23, 28]. This is supported by the observation that
EGCG could not overcome neuronal death under PKC pathway blockade, suggesting that this
cascade is essential for the neuroprotection and neurorescue effects of EGCG [23]. Recently,
we have identified a novel pathway in the neuroprotective mechanism of action of EGCG,
which involves a rapid PKC-mediated degradation of Bad protein by the ubiquitin proteasome
system (UPS) in SH-SY5Y cells [27]. Bad has been suggested to link survival signals to the
mitochondrial cell death machinery. Thus, the newly described role of Bad during the initial
response to EGCG-induced cell signaling, may potentially contribute to the illumination of
the EGCG mechanism of neuroprotection/neurorescue action. In addition, EGCG was shown
to induce a rapid translocation of the isoform PKCα to the membrane compartment in human
astroglioma or rat pheochromocytoma PC12 cells [23, 119], as well as upregulation of PKCε
mRNA expression and a concentration-dependent activation of PKCε in serum-deprived in
SH-SY5Y cells [100]. These findings are supported by animal studies showing that two
weeks oral consumption of EGCG prevented the extensive depletion of PKCα and
counteracted the robust increase of Bax protein in the striatum and SNPC of mice intoxicated
with MPTP [103].
A previous study in human epidermal keratinocytes indicated that EGCG promoted cell
survival by increasing the ratio of Bcl-2 to proapoptotic Bax and phosphorylation of Bad
through ERK and AKT signaling pathways [120]. Using mitogen-activated protein kinase 1
(MEK1) inhibitor (PD98059), EGCG induced only the phosphorylation of serine (Ser)136 of
Bad, and when the authors used PI-3 kinase inhibitor (LY294002), EGCG induced only the
phosphorylation of Ser112 of Bad. These results indicated that EGCG affects both the ERK
pathway, which is involved in phosphorylation of Bad at Ser112 and the PI-3 Kinase/AKT
pathway, involved in phosphorylation of Bad at Ser136 (Figure 2). Nonetheless, a study with
high concentrations with EGCG reported cell proliferation arrest of tumor cells and inhibition
of ERK1/2 and AKT phosphorylation, which was associated with reduced phosphorylation of
Bad [121]. This biphasic mode of biological activity of EGCG relies on its concentration-
dependent window of pharmacological action: EGCG exhibits pro-oxidant and pro-apoptotic
activity at high concentrations, which are responsible for its anti-cancer-cell death effect,
while lower doses exert neuroprotection against a wide spectrum of neurotoxic compounds
[16, 122]. A biphasic mode of action has been described for most of the typical radical
scavengers and antioxidants, such as ascorbic acid (vitamin C) [123] and iron chelators, such
as R-APO [124]. When SH-SY5Y cells were challenged with 6-OHDA or reduced content of
serum, a low concentration of EGCG (<10 μM) abolished the induction of proapoptotic-
related mRNAs and the decrease in Bcl-2, Bcl-w and Bcl-xL [28, 77]. The neuroprotective
effect of EGCG is thought to be mediated through down-regulation of pro-apoptotic genes, as
shown for mdm2, caspase-1, cyclin dependent kinase inhibitor p21 and TNF-related
apoptosis-inducing ligand, TRAIL rather than up-regulation of anti-apoptotic genes [28].
Theses findings support the assumption that complementary mechanisms, in addition to

antioxidant activity, are involved in the neuronal survival effect of EGCG.
Promoting of Neuronal Outgrowth
Our recent proteomic studies [31] have demonstrated that EGCG increases the level of
the binding protein, 14-3-3 gamma. By their interaction with more than 100 binding partners,
14-3-3 protein family members modulate the action of proteins that are involved in cell cycle
and transcriptional control, signal transduction, intracellular trafficking, regulation of ion
channels and expression of cytoskeletal components [125]. In this regards the
neurorescue/neuroprotective activity of EGCG may be associated with the induction of 14-3-
3 gamma, interacting with kinases of the PKC pathway and Bad and consequently preventing
neuronal death (Figure 2). Indeed, EGCG has been shown to be neuroprotective via activation
of PKC signaling, inhibition of activation of caspase-3 and its substrate, poly ADP-ribose
polymerase cleavage and induction of Bad degradation [23, 27, 31, 126]. A previous study
has demonstrated that overexpression of 14-3-3 gamma contributed to the regulation of the
dynamics of glial fibrillary acidic protein (GFAP) filaments, which may facilitate the stability
of the cytoskeleton, and thus play a specific neuroprotective role in the brain of patients with
AD [127]. In fact, recent proteomic analysis showed that EGCG dose-dependently increased
the expression levels of the stabilizer proteins of chromatin organization and DNA, histone
H1 member 4 and cytoskeletal proteins, such as the actin binding protein tropomyosin 3 and
beta-tubulin IV [31]. Since cytoskeletal proteins play a crucial role in promoting neurite
outgrowth [128], these results suggest that induction of the structural proteins by EGCG are
associated with its differentiation features including neurite extension, cell body elongation
and up-regulation of the growth associated protein-43 (GAP-43)[23, 100] in rat
pheochromocytoma PC12 cells [23] and human neuroblastoma SH-SY5Y cells [100].
Regulation of Hypoxia Inducible Factor-1 (HIF-1) Pathway
An emerging target for neuroprotection associated with iron chelation implicates the
activation of a hypoxia signal transduction pathway that culminates in the stabilization of the
transcriptional activator HIF-1 and increased transcription of genes mediating compensatory
survival processes in response to OS [129, 130]. The presence of HIF-1 within the cells is
under the strict control of a class of iron-dependent and oxygen-sensor enzymes named the
HIF prolyl-4-hydroxylases [129]. This family of enzymes hydroxylates critical proline and
asparagine residues in HIF upon high oxygen levels and iron overload, targeting it for
degradation by the UPS. This may explain the decrease in HIF-dependent cell survival genes
described in neurodegenerative diseases, such as phosphofructokinase and the angiogenic
vascular endothelial growth factor (VEGF) [131]. In this scheme, iron chelators would
stabilize HIF-1α, which in turn would heterodimerize with its partner, HIF-1β in the nucleus,
bind to an hypoxia responsive element in regulatory genes and transactivate the expression of
established protective genes, including VEGF, erythropoietin, p21waf1/cip1, glucose transporter-
1 and the glycolytic enzymes aldolase and enolase-1 [132, 133]. Indeed, EGCG and ECG
were shown to induce HIF-1α protein and HIF-1 activity and increase mRNA expression
levels of GLUT-1, VEGF, and p21waf1/cip1, whereas this effect was blocked by iron and
ascorbate, indicating that these catechins may activate HIF-1 through the chelation of iron
[134, 135]. Applying a neurorescue paradigm in neuronal culture, we have recently found that
EGCG decreased mRNA transcript and protein levels of the beta-subunit of prolyl-4-
hydroxylase and the protein levels of two molecular chaperones, which are associated with
HIF-regulation/degradation, the immunoglobulin-heavy-chain binding protein, BiP and the
heat shock protein 90 (HSP90) [31] [100](Figure 2). In support, previous finding
demonstrated that EGCG directly binds and inhibits HSP90 in mouse hepatoma cells [136].
Inhibition of HSP90 is considered a requirement for the rapid hypoxic stabilization of HIF-
1α, which otherwise might be degraded by unspecific pathway [137]. Thus, it is possible that
the protective effect of EGCG under OS/hypoxic conditions may combine the suppression of
hydroxyl radical formation via Fenton chemistry, as well as inhibition of iron-dependent
prolyl hydroxylase.
Another link between hypoxia and iron is reflected by the hypoxic-mediated positive
regulation of the iron regulatory proteins, IRP1 and IRP2 and consequential transactivation of
their target mRNAs, ferritin and transferrin receptor (TfR). Interestingly, the free iron-
induced proteasomal-mediated degradation of IRP2 involves also activation of a prolyl
hydroxylase and is inhibited by iron chelators [138-140]. Thus, it is possible that IRP2 is a
substrate for this enzyme, in a similar way as HIF, signaling it for protein degradation. Thus,
the reduction in the chelatable iron pool by EGCG may result in the inhibition of prolyl
hydroxylases and consequently, in the concerted activation of both HIF and IRP2. As HIF-1
and IRPs coordinate the expression of a wide array of genes, involved in cellular iron
homeostasis, survival and proliferation [141], their activation could be of major importance in
neurodegenerative diseases. In support, recent findings suggest the application of low
molecular weight or peptide inhibitors of the HIF prolyl 4-hydroxylases as novel neurological
therapy for neurodegenerative diseases [130].
CONCLUSIONS
Two main aspects are significantly contributing to the raising concept viewing green tea
consumption of relevance to brain health: the factors and events that influence the incidence
and progression of PD and AD are becoming better defined and understood; in parallel, the
experimental evidence documenting the neuroprotective properties of green tea catechins,
both in cell culture and animal studies is persistently increasing. It becomes evident that
syndromes, such as AD and PD will require multiple drug therapy to address the varied
pathological aspects of the disease. Therefore, the poly-pharmacological activities of green
tea catechins may be of significance for neuroprotection. Earlier viewed as a mere anti-
imflammatory and antioxidant EGCG is at the present time considered a multimodal acting
molecule, invoking various cellular neuroprotective/neurorescue mechanisms involving iron-
chelation, scavenging of oxygen and nitrogen radical species and activation of PKC signaling
pathway and pro-survival genes (Figure 2). Their non-toxic, lipophilic (and thus brain
permeable) nature is advocated for "ironing out iron" from those brain areas, where it
preferentially accumulates in neurodegenerative diseases [19]. The chelation of the reactive
free-iron pool by EGCG and consequent reduction in full-length APP translation, would
contribute to the decreased Aβ generation/fibrillization, which together with the promotion of
the non-amyloidogenic pathway and induction of neurite outgrowth, may converge in a
slowdown in the process of nerve neruonal loss in AD [113].
Future efforts in the understanding of the protective mechanism of action of EGCG must
concentrate on deciphering the cell targets, affected by this compound and other green tea
catechins. Further preclinical studies are needed to clarify if EGCG and its metabolites, at
sufficient concentrations, can reach the brain and may alter cell-signaling pathway and
whether these effects can be successfully translated into prospect human studies to affect the
progression of neurodegenerative disorders.
ACKNOWLEDGEMENT
We thank the support of Rappaport Family Research, Technion- Israel Institute of
Technology.
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Chapter 13
CARDIOVASCULAR AND METABOLIC EFFECTS

OF GREEN TEA
Kamilla Kelemen*
Department of Cardiology, Angiology and Pneumology
University of Heidelberg, INF 410, 69120 Heidelberg, Germany
ABSTRACT
Green tea (Camelia sinensis), widespread in the whole world, possesses many health
protective properties. Its polyphenolic compounds, mostly flavonols, better known as
catechins (e.g. epigallocatechin-3-gallate [EGCG]), are considered to be responsible for
the health protective effects. Tea consumption may have its strongest effect among
patients with cardiovascular disease. Recently, studies suggested that high flavonoid
intake may reduce coronary heart disease by lowering blood lipid levels and inhibiting
the oxidation of low-density lipoproteins. Trials showed that short- and long-term tea
consumption could reverse endothelial dysfunction in subjects with documented coronary
heart disease, providing one possible mechanism for an effect of tea in patients with
cardiovascular disease. Interestingly, EGCG has also been shown to have
electrophysiological effects by blocking HERG potassium channels, the most important
repolarizing potassium channel in the human ventricle that forms the α-subunit of the
rapid delayed rectifier current IKr. Inhibition of HERG channels may have profound
effects on cardiac repolarization. Furthermore, tea flavonoids have been reported to
exhibit metabolic effects in terms of anti-diabetic properties. This review summarizes the
latest studies on cardiovascular effects of green tea and discusses the possible cardiac
health benefits of green tea consumption.
INTRODUCTION
Green tea (Camelia sinensis), one of the most popular beverages in the world, especially
recommended in chinese medicine, gained increased public awareness because of many
*
Correspondence to kamilla_kelemen@web.de or kamilla.kelemen@med.uni-heidelberg.de
244 Kamilla Kelemen
health protective characteristics like anti-inflammatory, antioxidant, antihypertensive,

antidiabetic, antiobesity and antimutagenic properties.
Epidemiological evidence linking tea or flavonoid consumption with coronary heart
disease or total mortality among healthy adults is conflicting [1, 2], but tea consumption may
have its strongest effect among patients with cardiovascular disease [3, 4]. Recently, studies
suggested that high flavonoid intake may reduce coronary heart disease by lowering blood
lipid levels, inhibiting the oxidation of low-density lipoproteins and inhibiting inflammatory
reaction (4-6].
A randomized crossover trial showed that short- and long-term tea consumption could
reverse endothelial dysfunction in patients with documented coronary heart disease [3],
providing a possible mechanism for an effect of tea in patients with cardiovascular disease.
In our own study, we demonstrated for the first time that green tea flavonoid EGCG
blocks the human ether a-go-go related gene (HERG), which encodes the cardiac rapid
delayed rectifier potassium current IKr [7]. The cardiac refractory period can be prolonged by
blocking HERG. Thus, the heart might be less susceptible for cardiac arrhythmias by
blocking IKr.
Apart from its direct effects on cardiovascular disease (CVD), green tea has also been
found to affect CVD risk factors. Several animal studies and investigations in human
subjects showed a reduced risk of type 2 diabetes mellitus due to an improved insulin
sensitivity and glucose metabolism [8, 9]. Furthermore, green tea possesses hypotensive
properties, which can be partly attributed to vasodilating effects [10]. It also reduces other
CVD risk factors like obesity through higher fat oxidation and higher energy expenditure
[11-13].
Green tea contains polyphenols, flavonols, also known as catechins, which are considered
to be responsible for the health protective effect. The primary catechins in green tea are
epicatechin (EC), epicatechin-3-gallate (ECG), epigallocatechin (EGC), and epigallocatechin-
3-gallate (EGCG), which account up to 30-40% of the dry weight of green tea [14]. One cup
of green tea contains about 90 mg EGCG [14]. Pronounced cardiovascular health benefits can
be achieved by regular consumption of 5-6 or more cups of green tea per day [1].
Additionally, green tea contains also proteins, amino acids, lipids, fiber, pigments, minerals
and carbohydrates.
The content of catechins in green tea depends on the processing of the leaves. Several
studies were performed to investigate the bioavailability and biotransformation of green tea
catechins [15-17] with discrepant results for plasma EGCG levels [18, 19]. A study by Lee et
al. [16] found a mean peak plasma EGCG level of 0.17 µM in humans after drinking the
equivalent of ~ 2 cups of green tea, which was consistent with two other studies [15, 17].
However, another group found that oral intake of 525 mg EGCG could reach plasma levels of
4.4µM [18, 19]. Concentrations of EGCG up to 20 µM are achievable in the oral cavity after
drinking green tea, perhaps in the stomach and intestines, where a direct contact between
EGCG and the epithelial cells exists. The methods of green tea preparation and differences in
tea strength might affect bioavailability, plasma cetechin levels and the functional properties.
Cardiovascular and Metabolic Effects of Green Tea 245
Figure 1. Chemical formula of green tea flavonoids EGCG, EGC, ECG and EC.
1.1. EFFECTS OF GREEN TEA ON CARDIOVASCULAR DISEASE /

CORONARY HEART DISEASE
Myocardial infarction is a major cause of death in industrialized countries. The impact of
cardiovascular disease on the economic budget in industrialized countries is enormous and
increasing. For example, in Germany the costs for treatment of acute myocardial infarction in
the 90s were already in the range of 1.5 billion Euro, for coronary artery disease in general
4.45 billion Euro [20]. In the United States, expenses for cardiovascular disease were $ 393
billion for the year 2005 [21]. Reducing cardiovascular disease would have a great impact on
the economy of industrialized countries. Several studies that are discussed in this chapter
showed positive effects of green tea consumption on coronary heart disease.
A couple of Japanese and Chinese studies could prove that the positive influence of green
tea on cardiovascular health is directly correlated with increasing consumption of green tea.
For example in the Ohsaki study [22], a prospective cohort study, including 40,530 Japanese
who were followed for 11 years, the consumption of green tea resulted in an inverse
association with mortality due to all causes and due to cardiovascular disease. This
observation was especially true for women, although altogether both sexes had a benefit from
green tea intake. However, the benefit was more pronounced in women. A study by Kono et
al., including 1306 Japanese men, could show an inverse correlation between the
consumption of nine cups or more of green tea daily and the serum cholesterol level [23].
Studies by Imai et al. confirmed the latter result and expanded this observation to an inverse
246 Kamilla Kelemen
relationship between green tea consumption of more than 3 cups daily and not only serum
cholesterol levels but also triglycerides and low-density lipoproteins [24]. However, these
studies showed no effect on high density lipoprotein levels. An animal study by Yang et al.
investigated the effects of green tea in a rat model of hyperlipidemia that was induced by
administration of a high-sucrose diet [25]. Green tea consumption decreased total plasma
triglycerides and cholesterol. High density lipoprotein (HDL), which has cardioprotective
characteristics, was not changed. In liver and heart, green tea prevented the lipotrophic effects
of the diet. Green tea didn’t modify protein absorption or bile acid concentration, but
markedly reduced fat absorption.
Regarding the risk of mortality due to heart disease, another prospective study by Hertog
et al. including 12,763 and 25 follow-up years showed an inverse relationship between
mortality due to heart disease and flavonoid intake in seven countries [26].
Concerning coronary artery disease (CAD) Hirano et al. and Ohmori et al. examined 203
Japanese patients and found that green tea consumption of approximately 6 cups of green tea
per day was inversely related to the incidence of coronary artery disease during coronary
angiography [27, 28]. Another finding of this study was the observation that patients who
drank green tea on a regular basis also frequently consumed fish that is rich in omega-3-fatty
acids and vegetables and soybeans that also additionally might have contributed to the lower
incidence of CAD and myocardial infarction in these patients.
It is known that CAD has several risk factors. Among others, free radicals have been
shown to play an important role in heart disease. The free radicals of special interest in CAD
are oxygen free radicals. Oxidized free radicals are believed to cause tissue damage at the
cellular level, causing damage to the DNA, mitochondria and cell membrane. They have often
been attributed to cause aging, cancer and heart disease. Green tea is thought to antagonize
effects of excessive alcohol intake, smoking, and various chemical exposures that increase the
amount of free radicals in the body [29, 30]. To prevent free radical damage, the body has a
defense system of antioxidants. Antioxidants are a defense mechanism of the human body to
protect cells from free radical damage. They interact with free radicals and neutralize their
effects. Another study investigated moderate green tea consumption for 42 days (2 cups per
day) in 24 healthy volunteers and found an increased antioxidant capacity and decreased
plasma peroxides and reduced oxidative damage and glutathione peroxidase activity in
lymphocytes [31].
Several studies tested the effect of green tea on other cardiovascular risk factors beside
cholesterol and LDL levels like hypertension and diabetes and surprisingly found also
positive outcomes for these risk factors, which will be discussed separately below. Thus, a
reduction of cardiovascular disease needs to be seen in the context of altogether CAD risk
factors lowering characteristics of green tea.
1.2. EFFECTS OF GREEN TEA ON HYPERTENSION

The major risk factor for stroke and cardiovascular disease is hypertension, affecting
millions of people worldwide. Chinese medicine postulated hypotensive effects of green tea
for decades. However, there are only few studies examining the long-term effects of green tea
on blood pressure and the available data were conflicting until a Chinese study tested the
effect of tea (green tea and oolong tea) in detail for the past decades on the risk of newly
diagnosed hypertension in 1507 subjects. The study population was divided in habitual tea
drinkers of 120 ml/d or more for at least 1 year and nonhabitual tea drinkers.
This study could show that the daily consumption of 120-600 ml green tea per day for at
least one year reduced the risk of developing hypertension by 46%. A consumption of more
than 600 ml green tea per day showed a reduction of hypertension by 65% [32]. These results
were adjusted to socioeconomic and dietary factors and physical activity and altogether
lifestyle factors. Another study, a Norwegian epidemiological study, examined the effects of
tea consumption on blood pressure and found that the mean systolic blood pressure decreased
with increasing tea consumption [33]. Concerning these positive effects of tea on
hypertension the question arises through which possible mechanism tea might exert its
hypotensic effect. There are several animal studies showing different possible mechanisms.
One of these animal studies examined the effect of green tea extracts on angiotensin II-
induced hypertension in rats [30]. Rats were treated with drinking water with or without green
tea extracts and angiotensin II for 13 days. In the angiotensin II without green tea extracts
group blood pressure, left ventricular mass index, media- to lumen ratio and hydroperoxide
radicals, as a measure for oxidative stress, were increased. In the Green tea group
hypertension and target organ damage induced by high angiotensin II dose could be
prevented. Oxidative stress markers like plasma hydroperodxides and nitrotyrosine were
decreased by green tea. Altogether factors elevated with endothelial dysfunction like heme
oxygenase 1 (HO-1), NADPH oxidase endothelial p22phox subunit and the superoxide
dismutase SOD-1 were measured and found to be increased by angiotensin II and decreased
below baseline levels by green tea extracts.
Endothelial dysfunction plays a key role in the triggering and progression of
cardiovascular disease and predicts mortality [34, 35]. Several studies showed that green tea
flavonoids have antioxidant and endothelium-relaxing and thus vasodilatatory effects [36,
37]. Another study showing endothelial dysfunction improving effects of the green tea
flavonoid EGCG observed in vascular endothelial cells that EGCG activates endothelial nitric
oxide synthase (eNOS) and increases production of nitric oxide via a phosphaditylinositol
(PI) 3-kinase/Akt-dependent pathway [38]. The antioxidative effects of green tea were
investigated by Hakim et al. in a randomized, controlled study in 133 smokers [39].
Consumption of 4 cups of green tea compared to water for 4 months decreased urinary 8-
hydroxydeoxyguanosin, a sensitive marker of oxidative DNA damage. Another theory
concerning hypotensive characteristics of green tea concerns theanine (γ-
glutamylethylamide), which is one of the major components of amino acids in green tea and
belongs to the neurotransmitter family in the brain. Theanine administration could reduce
blood pressure through a not yet understood pathway in sponatenously hypertensive rats [40,
41].
1.3. EFFECTS OF GREEN TEA ON METABOLISM

Cardiovascular and metabolic diseases are the major causes of death in industrialized
countries. The metabolic syndrome describes a disease including several cardiovascular risk
factors like diabetes mellitus type II, obesity, hypertension, hyperlipidemia, which is
248 Kamilla Kelemen
predicted to continue to grow in the coming years in western countries. All statistics reflect an
increase in the prevalence of obesity and diabetes mellitus type II and mortality because of
successive diseases like cardiovascular disease, blindness, neuronal damage, renal failure and
diabetic foot disease. Type II diabetes mellitus is a disease that is a strong risk factor of
coronary artery disease and that was associated with 2.9 million deaths worldwide in the year
2000. Diabetic patients have a lower life-expectancy than non-diabetics [42]. The rapid
spread of diabetes mellitus II is considered to be due to increasing obesity, the major risk
factor of diabetes mellitus II. In a retrospective cohort study including 17,413 Japanese
subjects, consumption of 6 or more cups of green tea per day was found to reduce the
development of diabetes by 33% compared to consumption of less than 1 cup per week [8]. A
study with healthy young volunteers investigated the effects of green tea on glucose tolerance.
The consumption of 1.5 g green tea extract was found to significantly reduce plasma glucose
levels during the glucose tolerance test [43]. Another cross-over study investigating oolong
tea (1.5 liter with 390 mg EGCG) intake in 20 patients with diabetes mellitus II resulted in
significantly decreased plasma glucose levels [44]. A study by Shimada K. et al. [45] showed
a decreased hemoglobin A1c, increased serum LDL and adiponectin in 22 subjects with
coronary artery disease that consumed approximately 1 liter of oolong tea daily (45 mg
EGCG). Furthermore, an animal study in obese but otherwise healthy dogs observed that
consumption of green tea (80 mg/kg EGCG per day) reduced glucose and insulin levels,
suggesting that green tea improves insulin sensitivity [46].
Several animal models for diabetes exist like streptozotocin – and alloxan-induced
diabetes. In a number of studies, Babu et al .[47] examined the effects of green tea in rats with
streptozotocin-induced diabetes. They administered green tea extracts (300 mg/kg for 4
weeks) orally in rats and thus reduced lipid peroxides and activity of antioxidant enzymes as
well as increased gluthatione, an antioxidant that protects cells from toxins such as free
radicals, in the heart. In another study they observed reduced blood glucose levels, decreased
lipid peroxides, triglycerides and protein glycation in the heart of diabetic rats [48]. Lipid
peroxidation refers to the oxidative degradation of lipids resulting in cell damage. Glycation
is the non-enzymatic addition or insertion of sugar molecules into proteins that causes
damage to proteins or other molecules and is considered to be a significant contributor to
many diseases of aging. Yamabe et al. [49] tested the effects of EGCG (25, 50, 100 mg/kg for
50 days) in rats with streptozotocin-induced diabetes and subtotal nephrectomy. The authors
observed that EGCG reduced hyperglycemia, proteinuria, and lipid peroxidation. EGCG also
reduced the accumulation of renal advanced glycation end-products in the kidney. In alloxan-
induced diabetic rats administration of green tea extracts (100 mg/kg for 15 days) increased
glutathione and superoxide dismutase, which catalyzes the dismutation of superoxide into
oxygen and hydrogen peroxide and is an important antioxidant defense in nearly all cells
exposed to oxygen. Furthermore, lipid peroxidation was decreased as well as blood glucose
levels. Liver and kidney function was improved [50].
Wu et al. investigated the effects of green tea on insulin sensitivity in rats that received
green tea in their drinking water (370 mg/kg for 12 weeks). They found decreased fasting
plasma levels of glucose, insulin, free fatty acids and triglycerides [51]. Adipocytes from rats
receiving green tea showed a better capacity for glucose uptake and increased specific insulin
binding.
Another major cardiovascular and metabolic risk factor includes overweight and obesity.
A multi-center study by Chantre et al. investigated the consumption of green tea extracts (279
mg EGCG) for 12 weeks in 70 moderately overweight subjects [52]. The investigators found
a 4.6% decrease in body weight and a 4.5% reduction in waist-to hip ratio. Kovacs et al.
conducted a study that looked for the effects of green tea on the regain of body weight
following consumption of a low-energy diet for 4 weeks in 104 overweight subjects (323 mg
EGCG for 12 weeks). The authors found a continuation of loosing body fat in the group with
only low caffeine intake [53]. All other groups continued to gain weight again. An animal
model for obesity, where rats were fed a high-fat diet and then administered green tea extracts
in their drinking water for two weeks showed a decrease of body fat [54]. These effects
resulted from increased energy expenditure and a moderate reduction of food digestability.
Furthermore, the thermogenic capacity was increased. The reduction of body fat and the
increase of energy expenditure was partially mediated by β-adrenoreceptor activation.
In Zucker rats, consumption of green tea resulted in attenuated body weight gain and
decreased adipose tissue weight as well as reduced plasma cholesterol levels, whereas food
intake was not affected [55]. In another animal study with mice, consumption of EGCG, in
addition to endurance training, increased exercise capacity and β-oxidation, the process by
which fatty acids are broken down in mitochondria and/or in peroxisomes to generate Acetyl-
CoA, an important molecule in metabolism, used in many biochemical reactions . The
expression of fatty acid translocase/CD 36 mRNA was increased in skeletal muscle. This
study showed that endurance exercise along with green tea consumption can improve exercise
capacity and stimulates lipid metabolism in mice.
1.4. POTENTIAL EFFECTS OF GREEN TEA ON CARDIAC AMYLOIDOSIS

Amyloidosis is a rare disease which affects also the heart among other organs. There are
several subtypes of cardiac amyloidosis, which is characterized by infiltration of the heart
from insoluble protein deposits resulting in restrictive cardiomyopathy with heart failure and
conduction abnormalities leading to potentially life-threatening arrhythmias.
The therapeutic strategy depends on the subtype of amyloidosis and includes
chemotherapy, stem cell therapy and often requires liver transplantation.
Table 1. The different types of amyloidosis, the involved proteins and the affected
organs
Amyloidosis type Protein Affected organs

Primary Amyloidosis (AL- Light chain Heart, kidney, liver, nerval
Amyloidosis) system
Reactive Amyloidosis (AA- Amyloid A Heart, kidney, liver
Amyloidosis)
Hereditary Amyloidosis (ATTR) Mutant transthyretin Heart, nerval system,
kidney, eyes
Atrial Amyloidosis (AANF) Atrial natriuretic factor Limited to the heart
Senile systemic Amyloidosis Transthyretin Diffuse organ mnifestation
Dialysis-related Amyloidosis (β2- β2-microglobulin Bones, joints
microglobulin)
250 Kamilla Kelemen
Patients with cardiac amyloidosis predominantly die because of electromechanical

dissociation or other diagnoses that don’t benefit from an implantation of an intracardiac
defibrillator (ICD). A study by Bauer et al. tested nineteen patients with histologically proven
cardiac amyloidosis and a history of syncope and/or ventricular extra beats (Lown grade IVa
or higher) that received an ICD [56]. As a result of the study the investigators noted that only
two patients had a benefit of the implanted ICD to treat sustained ventricular tachycardia.
Most of the patients died due to electromechanical dissociation.
The study investigators concluded that only a small part of cardiac amyloidosis patients
benefit from an ICD.
So far, not a single study exists that may prove therapeutic effects of green tea in cardiac
amyloidosis. However, there is one case report published from the University of Heidelberg
[57], where one patient with cardiac amyloidosis (AL-Amyloidosis) underwent a selftest with
green tea after receiving several chemotherapies that could stabilize the disease, but were
accompanied by side effects. The patient, an emeritus professor of internal medicine and
haematology, was announced to have AL-Amyloidosis at the age of 72. He received two
different chemotherapies, the first according to the “Boston scheme” (4 mg melphalan daily
for 21 days) without success, the second according to the “Palladini scheme” (0,22 mg/kg
melphalan and 40 mg dexamethasone for 4 days every 28 days) that could stabilize the
cardiac amyloidosis. The patient then attended a lecture by Prof. E.E. Wanker on effects of
EGCG, the main phenol in green tea, on light chains and amyloid fibrils in Huntington`s
disease [58]. Since that lecture he consumed 1.5 to 2.0 liters of green tea daily without any
other treatment. Before the consumption of green tea the patient’s interventricular septum was
16.5 mm and constant for 20 months due to the success of the second chemotherapy (cardiac
amyloidosis is associated with cardiac hypertrophy). However, since the consumption of 1.5
to 2.0 liters of green tea daily, the thickness of the interventricular septum decreased month
by month to 13.2 mm. Since then the light chains didn’t increase any more and his renal
insufficiency stabilized. Also the patient’s “quality of life improved dramatically”, he says.
Now, he is almost 80 years old and the cardiac amyloidosis is greatly stabilized without any
other treatment than green tea.
Randomized trials are urgently needed to further examine the effects of green tea and
especially EGCG in cardiac amyloidosis. Possibly, treatment with EGCG might soon become
an alternative therapeutic option for cardiac amyloidosis.
1.5. EFFECTS OF TEA ON ARRHYTHMIA

The Determinants of Myocardial Infarction Onset Study (The Onset Study) was a
prospective multicenter study from 1989 to 1996, where 3882 patients with acute myocardial
infarction were enrolled in the United State [59]. Patients were asked according to a
standardized questionnaire to report about their tea and coffee drinking habits. They were
divided in non tea drinkers, moderate tea drinkers with < 14 cups per day and heavy tea
drinkers with > 14 cups per day and coffee drinkers. However, this study didn’t differentiate
between the different kinds of teas (black tea, green tea, white tea…). The first phase of the
study found that tea consumption was associated with lower mortality among patients with
acute myocardial infarction whereas coffee intake showed no effect on mortality outcome.
Figure 2. EGCG blocks HERG potassium channels, that is the molecular correlate of the cardiac rapid
delayed rectifier potassium current IKr. By blocking IKr ,which results in smaller currents, repolarization
will be prolonged.
Unexpectedly the investigators also found that tea consuming patients had a lower
prevalence of ventricular arrhythmia after myocardial infarction. However, this question was
not the focus of the study, so that no adjusted analysis was conducted and a second phase of
the Onset Study was started, where the occurrence of ventricular arrhythmia was assessed in
detail and correlated with the different tea drinker groups and the coffee drinker group with
adjustment for potentially confounding factors. The investigators found that of the 3882
patients 445 patients developed ventricular arrhythmia during hospitalization, in detail 370
patients developed ventricular tachycardia and 87 ventricular fibrillation. The results of this
second phase of the study were that moderate tea intake was associated with a lower
prevalence of ventricular arrhythmia and coffee drinkers had a higher incidence of ventricular
arrhythmia. Heavy tea drinkers had an intermediate prevalence of ventricular arrhythmia.
Possible explanations for a lower incidence of ventricular arrhythmia among tea drinkers
might be an improved gap junctional intercellular communication due to the flavonoid
content in tea. An experimental study with rat liver epithelial cells [60] showed that with
increasing levels of epicatechin, which is found in green tea, in cell culture the number of
communicating gap junctional cells increased so that gap junctional communication was
improved. Since intercellular communication in the heart is also similar to the liver, with
connexins playing the most important role, it might be assumed that cardiac intercellular gap
junctional communication might also be positively affected by flavonoids. Another
252 Kamilla Kelemen
experimental study in rat hearts [61] describes the positive minimizing effect of catechins on
ischemia-reperfusion injury and thus leading to less ischemia-triggered arrhythmia.
In our own in vitro study in xenopus laevis oocytes [7] we demonstrated for the first time,
that green tea flavonoid EGCG blocks the human ether a-go-go related gene (HERG), which
is the molecular correlate of the cardiac rapid delayed rectifier potassium current IKr.
Prolonging repolarization of the cardiac action potential is the mechanism of many
antiarrhythmic agents, e.g. dofetilide [62], a class III-antiarrhythmic agent. Characteristically,
class III-antiarrhythmic drugs prolong the cardiac refractory period and make the heart less
susceptible for cardiac arrhythmias by blocking IKr.
According to a study by Prineas et al. [63] coffee intake however is associated with a
greater prevalence of premature ventricular contractions.
CONCLUSION
Tea consumption had its origin in China almost 5,000 years ago. Green tea, a worldwide
consumed beverage, has been used as traditional medicine in areas such as China, Japan,
India and Thailand. Green tea has gained scientific attention due to its antioxidant, ant-
inflammatory, antihypertensive, antidiabetic and antimutagenic properties. Due to its high
content of polyphenolic flavonoids, mainly EGCG, green tea has especially shown exciting
cardiovascular health benefits. A number of animal as well as randomized human studies
have proven the benefits of green tea for cardiovascular and metabolic diseases concluding
that 200-300 mg of EGCG or 5-6 cups of green tea per day protects cardiovascular and
metabolic health. In times of growing cardiovascular and metabolic disease, a beverage that
has scientific evidence for its health protective properties and that is easily available for
everybody might have a tremendous impact on the health of the world population and an
increased impact on the economic budget.
A balanced diet combined with regular green tea consumption and physical activity as
well as life style changes may offer primary prevention against cardiovascular disease.
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Chapter 14
MOLECULAR BASIS FOR THE ANTI-CANCER

ACTIVITY OF EGCG IN VIVO:
MOLECULAR-TARGETING PREVENTION OF
CANCER BY GREEN TEA CATECHIN
Yoshinori Fujimura1 and Hirofumi Tachibana1,2,3,*

1
Innovation Center for Medical Redox Navigation, Kyushu University, 3-3-1 Maidashi,
Higashi-ku, Fukuoka 812-8582, Japan
2
Bio-Architecture Center, 3Department of Bioscience and Biotechnology, Faculty of
Agriculture, Kyushu University, 6-10-1 Hakozaki, Higashi-ku, Fukuoka 812-8581, Japan
ABSTRACT
For the past two decades, many researchers have been investigated the potential
cancer-preventive and therapeutic effects of green tea. (–)-Epigallocatechin-3-O-gallate
(EGCG) has been shown to be the most active and major polyphenolic compound from
green tea. The mechanisms of action of EGCG have been extensively investigated, but
the mechanisms for the cancer-preventive activity of EGCG are not completely
characterized and many features remain to be elucidated. Recently we have identified 67-
kDa laminin receptor (67LR) as a cell-surface EGCG receptor that confers EGCG
responsiveness to many cancer cells at physiological concentrations. This article reviews
some of the reported mechanisms and possible targets for the action of EGCG.
Especially, we focus the current understanding of signaling pathway for physiologically
relevant EGCG through the 67LR for cancer prevention. This information shed new light
on the molecular basis for the cancer-preventive activity of EGCG in vivo and helps in
the design of new strategies to prevent cancer.
*
Corresponding author: Hirofumi Tachibana, Department of Bioscience and Biotechnology, Faculty of Agriculture,
Kyushu University, 6-10-1 Hakozaki, Higashi-ku, Fukuoka 812-8581, Japan. E-mail address:
tatibana@agr.kyushu-u.ac.jp; Tel/Fax: +81-92-642-3008
258 Yoshinori Fujimura and Hirofumi Tachibana
1. INTRODUCTION
Tea is one of the most widely consumed beverages in the world. Green tea, black tea, and
oolong tea are all derived from the leaves of Camellia sinensis plant and contain an
assortment of compounds, the most significant components of which are polyphenols. Among
all teas consumed in the world, green tea is best studied for its health benefits. It has been
demonstrated that tea constituents exhibit various biological and pharmacological properties
such anti-carcinogenic, anti-oxidative, anti-allergic, anti-virus, anti-hypertensive, anti-
atherosclerosis, and anti-hypercholesterolemic activities [1-8]. Major principles for these
activities were shown to be a group of polyphenols, catechin. A typical green tea beverage,
prepared in a proportion of 1 g leaf to 100 ml water in a 3-min brew, usually contains 250-
350 mg tea solids, and catechins account for 30-42% of the dry weight of the solids [9].
Catechins contain a benzopyran skeleton with a phenyl group substituted at the 2-position and
a hydroxyl (or ester) function at the 3-position. Variations to catechin structure include the
stereochemistry of the 2,3-substituents and the number of hydroxyl groups in the B- and D-
ring. Belonging to the flavan-3-ol class of flavonoids, major catechins found in tea leaves are
(–)-epigallocatechin-3-O-gallate (EGCG), (–)-epigallocatechin (EGC), (–)-epicatechin-3-O-
gallate (ECG), (–)-epicatechin (EC) and their structures are shown in Figure 1. Among the
green tea catechins, EGCG is the most abundant, representing ~16.5 wt% of the water
extractable fraction of green tea leaves, and most active catechin in various kinds of
physiological activities. Because EGCG is not found to a plant except tea, EGCG is regarded
as a constituent characterizing green tea. Recently, double-blind, placebo-controlled study on
oral administration of green tea catechins (EGCG, 50%) in volunteers with high-grade
prostate intraepithelial neoplasia demonstrated that green tea catechins have potent in vivo
chemoprevention activity for human prostate cancer [10,11]. This impressive evidence has
fueled interest in the role of EGCG as chemoprevention of cancer. This chapter will discuss
the effects of green tea polyphenol EGCG on signal transduction pathways that are related to
cancer chemoprevention based on the biological importance of the target, and especially we
focused the current understanding of EGCG signaling pathway through the 67-kDa laminin
receptor (67LR) as the target molecule mediating anti-cancer effect of the physiologically
relevant EGCG
2. ANTI-OXIDANT AND PRO-OXIDANT

Tea polyphenols such as EGCG are well known for their anti-oxidant activities. They
have been reported to inhibit carcinogen-induced DNA damage and tumor promoter-induced
oxidative stress [12,13]. These results are consistent with the commonly mentioned idea that
tea prevents cancer because tea polyphenols are anti-oxidants. It is unclear, however, whether
this is a general mechanism for cancer prevention, especially in human carcinogenesis when
strong carcinogenesis and tumor promoters are not known to be involved. When carcinogen-
activation and tumor promotion were active areas of research, these events had been proposed
as the targets of tea polyphenol action. With the advancement of research on signal
transduction pathways targeted by tea polyphenols, many studies have been carried out in cell
Molecular Basis for the Anti-cancer Activity of EGCG in Vivo 259
lines. However, whether some of the phenomena observed in cell lines occur in vivo still
remains unclear.
Figure 1. Chemical structures of green tea catechins and their analogues.
There two major problems in extrapolating results observed in cell lines to animal
models. (i) The concentration used in cell line systems, for example, EGCG at 10-100 μM, or
higher concentrations, are much higher than those observed in the plasma or tissues in
experimental animals or humans after ingestion of tea or related tea preparations [12]. (ii) The
oxygen partial pressure in a cell culture system (160 mmHg) is much higher than that in the
blood or tissues (<40 mmHg). EGCG is unstable under cell culture conditions, and the half-
life, less than 2 h, can be extended several folds by the addition of superoxide dismutase
(SOD), suggesting a role for superoxide radical in the oxidation and polymerization of EGCG
[14-16]. Similar to other anti-oxidants, EGCG and other tea polyphenols may also act as pro-
oxidants. They can be oxidized to form phenolic radicals, superoxide radical and hydrogen
peroxide. These species may trigger a variety of biochemical reactions and biological
responses. In studies of EGCG and other polyphenolic compounds in cell culture, the addition
of SOD and catalase is recommend to stabilize EGCG and to avoid possible artifacts. Thus,
according to this observation, we also added those enzymes to the cell culture systems [17-
21]. It is not clear whether pro-oxidants produced by EGCG-generated reactions occur in low
oxygen partial pressure conditions in vivo in cells, which generally have strong anti-oxidative
capacity and low oxygen partial pressure. Recently, EGCG has been shown to form quinone
and dimmer quinone during the autoxidation of EGCG in vitro, but none of these oxidation
products were observed in plasma samples of mice after treatment with 50 mg/kg EGCG i.p.
daily for 3 days [22]. Therefore the roles of EGCG autoxidation in biological activities of this
compound in vivo remain to be investigated further. The difference between in vitro and in
vivo systems should be considered in studies attempting to elucidate the mechanisms of action
of EGCG.
3. BIOAVAILABILITY OF EGCG
The bioavailability and biotransformation of tea catechins following tea ingestion has
been investigated, and a Tmax (time to reach maximal concentration) in the plasma of 1.5 to
2.5 h after consumption of decaffeinated green tea solids (1.5, 3.0, and 4.5 g) [23]. The
catechins levels decreased and were not detectable by 24 h. Whereas EGCG and ECG were
not detected in the urine, 90% of the urinary EC and EGC were excreted by 8 h. Most of the
ingested EGCG apparently does not get into the blood, and absolute EGCG is preferentially
excreted through the bile to the colon [24]. EGC and EC appear to be more bioavailable, but
the fractions of these compounds that appeared in the plasma are also low, and only 3.3 and
8.9% of the ingested EGC and EC were excreted in the urine. Glucuronidation, sulfation,
methylation, and ring-fission metabolism represent the major metabolic pathways for green
tea catechins [25]. Plasma EC and EGC were present mainly in the conjugated form such as
glucuronide and sulfate conjugates, whereas 77% of the EGCG was in the free form [26].
EGC but not EC is also methylated (4’-O-methyl-EGC) in humans. EGCG has also been
shown to undergo methylation. The maximum plasma concentration of 4’,4”-di-O-methyl-
EGCG is 20% that of EGCG but the cumulative excretion of 4’,4”-di-O-methyl-EGCG is 10-
fold higher than that of EGCG over 24 h [27]. The EGCG levels in plasma, lung, and liver are
much higher than in rats when the same polyphenol preparation is given to mice [28].
Although most of published studies in cell culture systems used 10-100 μM of EGCG, the
blood level of EGCG after consuming the equivalent of 2-3 cups of green tea was 0.1-0.6 μM
and for an equivalent of 7-9 cups was still lower than 1 μM [23,29]. The rather poor
bioavailability of tea catechins needs to be considered when we extrapolate results obtained in

vitro to situations in vivo.
The biological activities of EGCG in vivo depend on its bioavailability at the specific
sites of interest. For example, EGCG is more accessible to the oral cavity and intestinal tract
due to direct contact after ingestion. The tissue levels of EGCG of internal organs depend on
the systemic bioavailability after oral ingestion. The limited systemic bioavailability is due to
its multiple phenolic groups, its rapid conversion to methylated, glucuronidated, and sulfated
metabolites, and its efflux by multiple drug resistant related proteins [12]. The peak EGCG
(including the free and conjugated forms) concentrations observed in the plasma and tissue
levels of animals or in human plasma after ingestion of tea or polyphenols are usually lower
than 1 μM [12]. When large pharmacological doses of tea polyphenols are given by oral
administration, peak plasma concentrations of 7.5 μM have been observed in human [30].
EGCG is excreted mostly through the feces, and urinary levels of EGCG are very low or
undetectable [12].
4. POSSIBLE DIRECT TARGETS FOR THE ACTION OF EGCG

Currently, there is much interest in the design and development of chemopreventive
agents that act on specific molecular and cellular targets. Searching for high-affinity proteins
that bind to EGCG is the first step to understanding the molecular and biochemical
mechanisms of the anti-cancer effects of tea polyphenols. Several proteins that can directly
bind with EGCG have been identified in vitro models and these topics were shown in Table 1
[17-19,31-56]. Some are summarized below.
4.1. Matrix Metalloproteinase
The progression of human tumors involves the matrix metalloproteinase (MMP) family.
Two particular members of this family, MMP-2 and MMP-9, seem to play an important role
in tumor invasion and metastasis. They are involved in the turnover of basement membrane
collagen under basal conditions and of other matrix proteins during angiogenesis, tissue
remodeling, and repair. EGCG exerted dose-dependent inhibition of both MMP-2 and MMP-
9. The concentrations giving 50% inhibition (IC50) were 20 and 50 μM, respectively [57]. The
invasion of HT1080 fibrosarcoma cells through a Matrigel basement membrane was inhibited
with an IC50 less than 0.1 μM EGCG. In addition to inhibitory effect of EGCG on activity of
MMPs, EGCG has also been found to inhibit the intracellular metalloproteolytic activity of
the anthrax lethal factor (LF; IC50 = 0.1 μM), which has a major role in the development of
anthrax [58]. The inhibition of LF-induced cleavage of MAPKK, which is the natural
substrate of LF, was involved in the protection of LF-induced death of macrophages by
EGCG.
Table 1. Reported inhibitory activities of EGCG and effective concentrations
4.2. Proteasome
The proteasome is a massive multicatalytic proteinase complex found in all eukaryotic

cells and is responsible for degrading most of the cellular proteins. The ubiquitin/proteasome-
dependent degradation pathway plays an essential role in up-regulation of cell proliferation,

down-regulation of cell death, and development of drug resistance in human tumor cells,
suggesting that the proteasome is a novel and promising target for cancer chemotherapy. It
has been reported that EGCG potently and specifically inhibited the chymotrypsin-like
activity of the proteasome both in cell-free systems (IC50 = 86 nM) and tumor cell lines (1-10
μM) [33]. In addition, in silico docking study suggested that a particular pose of EGCG could
lead to potential covalent modification of the N-terminal threonine (Thr 1) of the proteasome
β5 subunit in the chymotrypsin-like active site [59]. This could be accomplished via
nucleophilic attack of a pair of electrons from the hydroxyl group of Thr 1 to the ester carbon
of EGCG. Numerous substrates of the proteasome have been identified including cyclins,
cyclin-dependent kinase inhibitors, p53, Bcl-2, and IκB among others. These proteins have
various functions like regulation of the cell cycle, protection of apoptosis, and transcriptional
regulation [60]. The inhibition of the proteasome by EGCG in several tumor and transformed
cell lines results in the accumulation of two natural proteasome substrates, p27Kip1 and IκB-α,
followed by growth arrest in the G1 phase of the cell cycle.
4.3. Extracellular Signal-regulated Protein Kinase 1/2 (ERK1/2) and Akt
MAPKs have been implicated in many physiologic process, including cell proliferation,
differentiation, and death. MAPK has received increasing attention as a target molecule for
cancer prevention and therapy. There are three major types of MAPKs in mammalian cells,
the extracellular signal-regulated protein kinases (ERK), the p38 MAPKs, and c-Jun NH2-
terminal kinases (JNK). Activation of the MAPK pathways may cause the induction of phase
II detoxifying enzymes, and its inhibition may inhibit AP-1-mediated gene expression. Once
activated, MAPKs (ERK, JNK, and p38) can activate a variety of transcription factors,
including ELK and c-Jun, a component of AP-1, thus leading to changes in the expression of
genes that play critical roles in cell proliferation, migration and apoptosis. It has been shown
that treatment of H2O2 resulted in phosphorylation of ERK1/2, JNK, and p38 in human
epidermal keratinocytes. H2O2-induced phosphorylation of ERK1/2, JNK, and p38 was found
to be significantly inhibited when these cells were pretreated with EGCG. These findings
demonstrate that EGCG has the potential to inhibit oxidative stress-mediated phosphorylation
of MAPK signaling pathways [61]. EGCG inhibited the phosphorylation of ERK1/2, and p38
MAPK activity in human fibrosarcoma HT1080 cells [4]. EGCG (20 μM) inhibited the
phosphorylation of MAP/ERK kinase 1/2 (MEK1/2), ERK1/2, and ELK-1 in 30.7b Ras 12
cells (H-ras transformed JB6 mouse epidermal cell line) [62]. This study suggested that
EGCG decreased the association between RAF-1 and MEK1, and EGCG competitively
inhibited the phosphorylation of ELK-1 by ERK1/2 possibly by competing for the binding
site on ERK1/2. Epidermal growth factor receptor (EGFR) is often overexpressed in
neoplastic cells, activating signal transduction pathways that promote cell proliferation and
tumor progression [63,64]. Antagonists to EGFR are currently under intensive investigation
for cancer therapy [65]. In addition, downstream EGFR targets, including ERK1/2 and Akt,
are considered viable drug targets. EGCG inhibited epidermal growth factor-dependent
activation of EGFR, and EGFR-dependent activation of ERK1/2 and Akt at the concentration
range of 10-50 μM in the immortalized cervical cell line ECE16-1 [66]. In addition to
inhibiting EGFR activation, cell-free studies demonstrated that EGCG directly inhibits
ERK1/2 and Akt kinase activity at the concentration of more than 5 μM.
4.4. DNA Methyltransferase
Hypermethylation of DNA is a key epigenetic mechanism for the silencing of many

genes, including those for cell cycle regulation, receptors, DNA repair, and apoptosis [67-70].
In this aberrant methylation, the cytosine of the CpG island in or near the promoter region of
the newly synthesized DNA strand is methylated by 5-cytosine DNA methyltransferase
(DNMT). The methylated CpG island has higher binding affinity to methyl-CpG binding
domain proteins that recruit transcriptional corepressors such as histone deacetylases,
resulting in chromosome condensation and transcription repression [71,72]. The inhibition of
DNMT would block the hypermethylation of the newly synthesized DNA strand, resulting in
the reversal of the hypermethylation and the re-expression of the silenced genes [73-75]. The
inhibitors of DNMT have been shown to inhibit cancer cell growth, induce cancer cell
apoptosis, and reduce tumor volume in mice [76-79]. There is high potential for developing
this group of inhibitors for cancer therapy. It has been reported that EGCG can inhibit DNMT
activity and reactive methylation-silenced genes in cancer cells [37]. With nuclear extracts as
the enzyme source and polydeoxyinosine-deoxycytosine as the substrate, EGCG dose-
dependently inhibited DNMT activity, showing competitive inhibition with a Ki of 6.89 μM.
Studies with structural analogues of EGCG suggest the importance of D and B ring structures
in the inhibitory activity. Molecular modeling studies also support this conclusion, and
suggest that EGCG can form hydrogen bonds with Pro1223, Glu1265, Cys1225, Ser1229, and
Arg1309 in the catalytic pocket of DNMT. Treatment of human esophageal cancer KYSE 510
cells with 5-50 μM EGCG for 12-144 h caused a concentration- and time-dependent reversal
of hypermethylation of key tumor suppression gene p16, retinoic acid receptor β, the DNA
repair gene hMLH1, and methylguanine methyltransferase. Some of these genes were also
reactivated in colon cancer HT29 cells and prostate cancer PC3 cells. The observed effective
dose of EGCG, Ki of 6.89 μM, or IC50 of 20 μM, is achievable in the oral cavity (in the
saliva) after drinking green tea, and perhaps in the stomach, esophagus, and intestines where
there is direct contact between EGCG and the epithelial cells. This effective concentration,
however, is higher than those in the internal organs, which depend on the systemic
bioavailability of EGCG [12]. There, the extent of DNMT inhibition in vivo would depend on
the bioavailability of EGCG in a particular organ site.
4.5. Bcl-2-family Proteins
Bcl-2-family proteins are important regulators of apoptosis. Anti-apoptotic members of

this family, such as Bcl-2 and Bcl-x, contain on the surface a hydrophobic groove in which
they can bind the BH3 domain of the pro-apoptotic counterparts [80]. This binding is crucial
for the regulation of apoptosis in vivo, with pro-and anti-survival proteins neutralizing each
other’s function through dimerization. The observation that antiapoptotic Bcl-2 family
members, such as Bcl-2 and Bcl-xL, are generally overexpressed in many cancer cells [80]
has stimulated a growing interest in the discovery of small molecules targeting such proteins,
as potential anti-cancer therapeutics [81,82]. A study using a combination of nuclear magnetic
resonance binding assay, fluorescence polarization assay, and computational docking analysis
demonstrated the direct binding of EGCG to the BH3 pocket of the anti-apoptotic Bcl-2
family proteins and the inhibitory effect of EGCG on the binding of BH3 peptide to Bcl-2-
family proteins, Bcl-2 and Bcl-xL with Ki values of 335 and 490 nM, respectively [36]. On
the contrary, C, EC, and EGC did not interact with their proteins, and showed no inhibition of
the binding at 100 μM. Although the BH3 domain was recognized as one of the binding site
of EGCG in cell-free systems, the functional importance of this binding still requires more
investigation. Furthermore, whether EGCG can bind directly the intracellular Bcl-2-family
proteins in cell-culture systems or in vivo still remains unclear.
4.6. Vimentin
Vimentin, one of the type III intermediate filament (IF) proteins, is a major component of
IFs and is expressed during development in a wide range of cells, including mesenchymal
cells and in a variety of cultured cell line and tumors [83,84]. IFs are essential for structure
and mechanical integration of the cellular space and a variety of cellular functions such as
mitosis, locomotion, and organizational cell architecture, and vimentin is readily
phosphorylated by numerous protein kinases such as cycline-dependent kinases 2 (Cdc2) and
cAMP-dependent protein kinase (PKA), therefore, regulating their functions [85-87]. The
proteolytic derivatives indicate that the amino-terminal domain, but not the carboxyl-terminal
domain, has a direct effect on filament stability and polymerization [88]. Vimentin was
recently discovered as a high-affinity EGCG binding protein with a Kd of 3.3 nM [40]. The
protein was isolated from cell lysates of JB6 C141 mouse epidermal cells by EGCG-
Sepharose 4B column, and identified by two-dimensional electrophoresis and MALDI-TOF-
MS. Functional studies showed that EGCG inhibited the phosphorylation of vimentin at Ser55
and Ser50 by Cdc2 (IC50 = 17 μM) and PKA (IC50 = 2 μM), respectively. Vimentin
knockdown (by siRNA) in JB6 C141 cells resulted in a lower proliferation rate and the cells
became much less responsive to inhibition by EGCG at the concentraion of more than 15 μM.
In this study, the biological consequences of EGCG have been demonstrated, but the effective
concentration for the inhibition of cell growth is much higher. The molecular basis for this
difference needs to be investigated. Whether this is general mechanism that are responsible
for the inhibition of carcinogenesis in animal models or humans or are artifacts of the cell
culture system also needs to be determined.
4.7. Urokinase-plasminogen Activator (uPA)
uPA is primarily associated with the degradation and regeneration of the basement
membrane and extracellular matrix that leads to metastasis. It also aids in anti-thrombolytic
activities to remove blood clots and helps stimulate angiogenesis in tumor cells. uPA is a
trypsin-like protease that converts the zymogen plasminogen into active plasmin. Plasmin
facilitates the release of several proteolytic enzymes, including gelatinase, fibronectin, fibrin,
laminin, and latent forms of collagenases and stromelysins [89]. Inhibition of uPA can
decrease tumor size or even complete remission of cancers in mice [90,91]. It has been
reported that EGCG inhibited the activity of uPA [53]. With the use of molecular modeling, it
was shown that EGCG binds to urokinase, blocking His57 and Ser195 of the urokinase catalytic
triad and extending toward Arg35 from a positive charged loop of urokinase. However, the
effective concentration of needed to inhibit urokinase (2-10 mM) was at least 3 or 4 orders of
magnitude higher than the expected tissue level (close to 1 μM).
4.8. Dihydrofolate Reductase (DHFR)
DHFR catalyzes the NADPH-dependent reduction of 7,8-dihydrofolate to 5,6,7,8-

tetrahydrofolate, which acts as a coenzyme for several one-carbon group transfer reactions
that include steps in nucleotide biosynthesis. Consequently, inhibition of DHFR, resulting in
the disruption of DNA biosynthesis, is the basis of chemotherapeutic action of a range of
DHFR inhibitors, generically known as “antifolate.” Tumor cells that grow rapidly require a
higher concentration of dTTP than normal cells, and therefore are more sensitive to
antifolates. EGCG was recently reported to an inhibitor of DHFR [39]. It exhibited kinetic
characteristics of a slow tight binding inhibitor of 7,8-dihydrofolate reduction with bovine
liver DHFR (Ki = 0.11 μM), but acted as classic reversible competitive inhibitor with chicken
liver DHFR with a much larger Ki (10.3 μM). EGCG also inhibited lymphoma cell growth
(IC50 = 20 μM), G0/G1 phase arrest of the cell cycle, and the induction of apoptosis. Folate
depletion increased the sensitivity of these cell lines to the antifolate activities of EGCG. The
suggestions of this paper that EGCG may be an antifolate reagent and that tea consumption
may be related to folate deficiency are rather speculative, and further examination. If the
EGCG blood level could be maintained at 20 μM for a long term, antifolate effects might be
produced. However, this is not an achievable blood level of EGCG through oral consumption
of even a large quantity of tea. The highest peak plasma level of EGCG after the oral
administration of 1200 mg of EGCG (equivalent to ten cups of tea) is 7.5 μM, and this
amount is associated with nausea [30]. The very low Ki value observed for bovine liver
DHFR might be due to the fact that very low levels of the enzyme were used in the assay, and
the inhibition was due to the strong binding activity of EGCG to the enzyme (a slow tight
binding inhibitor, as reported), not necessarily by binding to the active site.
5. THE 67-KDA LAMININ RECEPTOR AS

A GREEN TEA CATECHIN RECEPTOR
It should be noted that most of the effects of EGCG in cell culture systems and cell-free
systems have been obtained with relatively high concentrations than observed in the plasma
or tissues of animals or in human plasma after administration of green tea or EGCG. The
pharmacokinetic studies in humans indicate that the peak plasma concentration after single
dose of EGCG is <1.0 μM. Furthermore, the intracellular levels of EGCG are much lower
than the concentrations observed in the extracellular levels. Therefore, it is not clear whether
the activities observed with high EGCG concentrations in vitro can be observed in vivo and
the proposed EGCG-binding molecules as mentioned above are responsible for in vivo
physiological activities of EGCG. A target molecule for physiological achievable
concentration (<1.0 μM) of EGCG still remained unclear.
5.1. The 67-kDa Laminin Receptor (67LR)
The 67-kDa laminin receptor (67LR) is a nonintegrin receptor for laminin, fibronectin,
and type IV collagen [92,93]. Its full-length cDNA encodes only a 37-kDa polypeptide, which
has been reported to be a ribosome-associated cytoplasmic protein, and the exact structure
and maturation mechanism by which the 67LR is synthesized from the precursor of 37-kDa
polypeptide is unclear. Expression of the 67LR has been shown to be upregulated in
neoplastic cells compared with their normal counterparts and directly correlate with an
enhanced invasive and metastatic potential in many malignancies [94,95]. The receptor has
been implicated in laminin-induced tumor cell attachment and migration, as well as in tumor
angiogenesis, invasion, and metastasis [96-98]. Surface expression of the 67LR has also been
reported to be a dominant laminin-binding protein expressed in neutrophils, macrophages,
and monocytes, which suggests that the receptor may play an important role in the regulation
of cell adherence via the basement membrane laminin [99-101]. 67LR appears to has an
important role in host defense by being expressed in cells including T lymphocytes and mast
cells, but there is relatively little information about allergy-relevant cells such as basophils,
eosinophils, and B lymphocyte [102,103]. It has been reported that the expression of 67LR in
mast cells is related to the adhesion to laminin and may contribute to the tissue distribution of
mast cells and to mast cell accumulation at sites of tissue injury and inflammation [102,104].
In the immune system, 67LR has also been shown to be able to modulate granulocyte-
macrophage colony-stimulating factor (GM-CSF) signaling by inhibiting GM-CSF-its
receptor (GMR) complex formation through its interaction with GMR [105]. Furthermore, it
has been suggested that the 67LR expression in human T cells is upregulated by stimulating
the neuropeptides GnRH-II and GnRH-I which trigger its homing to specific organs [103].
Recently, it has become clear that acting as a receptor for laminin is not the only function of
the 67LR. This protein also act as a receptor for pathogenic prion protein, cytotoxic
necrotizing factor 1 from E. coli, Sindbis virus, Venezuelan equine encephalitis virus, Dengue
virus, and adeno-associated virus [106-111].
5.2. Plasma Membrane Microdomain “Lipid Rafts”
To date, most of the research on physiological activities of EGCG has been connected its
action with anti-oxidative activity. On the other hand, it has been suggested that EGCG and
its derivatives exhibit biological activities through interactions with the cellular membranes
[112]. The study, using the liposome system, on the interaction of tea catechins with the lipid
bilayers implicates that the affinity of catechins for the lipid bilayers may be responsible for
various kinds of actions [113]. We also found that tea catechins, having potent anti-cancer
activity, can bind to the cell surface, while catechins, having lower activity did not [38].
Therefore, further investigation about the cellular interaction of EGCG may contribute to the
elucidation of the mechanism of EGCG actions. Recently, plasma membrane microdomains
referred to as ‘lipid rafts’ have received much attention as potential regulators and organizing
centers for signal transduction and membrane traffic pathways [114]. The already impressive
catalog of raft involvement in diseases, including cancer, Alzheimer disease, Parkinson
disease, prion diseases, atherosclerosis, hypertension, diabetes, allergic response, bacterial
infections, and viral infections, continues to grow (Figure 2) [115]. These microdomains have
been characterized as sphingolipid/cholesterol-rich domains in the plasma membranes. Lipid
rafts have also been shown to interact with various kinds of extracellular factors including
cytokines and growth factors, neuroproteins, and viruses [116-119]. A large number of raft
proteins and raft-associated proteins have been identified [116-118,120-141]. However, little
is known about the interaction of these microdomains with low-molecular compounds such as
tea catechins. EGCG has been found to inhibit epidermal growth factor or platelet-derived
growth factor-mediated tumor cell growth by reducing the autophosphorylation of their
receptors [52], and both of these receptors were observed in lipid rafts [116,117]. The
formation of senile plaques containing the β amyloid peptide (Aβ) derived from the amyloid
precursor protein (APP) is an invariant feature of Alzheimer’s disease. It has been
demonstrated that APP interacts with lipid rafts and which are critically involved in
regulating Aβ generation [142]. Aβ has been known to be toxic to neurons in rat primary
hippocampal cultures [143], and such neurotoxicity has been shown to be attenuated by
treatment with EGCG [144]. These circumstances have fueled interest in the role of lipid rafts
as a platform for researching the function of EGCG, but there has been no direct data proving
the relationship of EGCG with lipid rafts. To explore a potential role of lipid rafts in binding
of EGCG to the plasma membrane, a study of raft integrity was performed using MβCD, a
cholesterol-removing agent that disturbs raft function [145]. A surface plasmon resonance
(SPR) assay revealed that EGCG was able to bind to the cell surface of the human basophilic
KU812 cells, chronic myelogenous leukemia cells, and this binding was inhibited upon
treatment with MβCD [146]. In addition, we found that the disruption of lipid rafts caused a
significant reduction of EGCG activities such as the inhibition of histamine release and the
suppression of the high-affinity-IgE receptor (FcεRI) expression [146,147]. To elucidate the
interaction of EGCG with the lipid rafts, the amount of EGCG in the lipid rafts was directly
measured by sucrose gradient ultracentrifugion and high-performance liquid chromatography
(HPLC) methods. The level of EGCG in the raft fraction was much higher than the non-raft
fraction, indicating that EGCG does not homogenously bind to the membrane of the cells, but
interacts with the cells in a heterogeneous fashion [146]. This suggests that these lipid rafts
may play a possible role in the interaction of EGCG with the cells. In the study on a
disruption of lipid rafts, the treatment with MβCD clearly decreased the binding of EGCG,
and this treatment also led to a reduced amount of EGCG in the raft fraction isolated from
MβCD-treated cells [146]. These results suggest that lipid rafts is responsible for the
interaction of EGCG with the cell surface of the human chronic myelogenous leukemia
KU812 cells and plays an important role in mediating EGCG signaling. These facts raised a
possibility that the primary target of EGCG may be a membrane molecule localized in lipid
rafts on the cell surface. Therefore, we attempted to investigate the cell-surface molecule
acting as an EGCG receptor, which can mediate anti-cancer activity of EGCG.
Figure 2. Plasma membrane microdomain “lipid rafts” and its related diseases. Rafts contain proteins
attached to the outer leaflet of the bilayer by their GPI anchors, proteins binding to the inner leaflet by
acyltails (for example, the Src-family kinase). The lipid bilayer in rafts is asymmetric, with
sphingomyelin and glycosphingolipids enriched in the outer leaflet and glycerolipids (for example,
phosphatidylserine and phosphatidylethanolamine) in the inner leaflet. Cholesterol is present in both
leaflets and fills the space under the head groups of sphingolipids or extends the interdigitating fatty
acyl chain in apposing leaflet. Raft-related diseases are reviewed in ref. [115].
5.3. Discovery of 67LR as a Green Tea Catechin Receptor
To elucidate the detail molecular basis for the action of EGCG, the same as drugs, it is
necessary to identify the molecular target triggering a specific signaling of EGCG. As you
know, toll-like receptor teaches us the principal role in the lipopolysaccharide signaling [148]
and the necessity of identification of the specific receptor as a signal initiator for generating
cellular responses for understanding the specific cellular signaling of foreign or functional
substances (Figure 3). However, the molecular target for physiologically relevant EGCG that
can mediate its anti-cancer effect still remained unknown. Recently we found that all-trans-
retinoic acid (ATRA) enhances the binding of EGCG to the cell surface of cancer cells when
the binding was monitored on the basis of the increase in response units in a SPR assay [38].
To identify candidates through which EGCG inhibits cell growth, we used a subtraction
cloning strategy involving cDNA libraries constructed from cells treated or untreated with
ATRA. We isolated a single target that allows EGCG to bind to the cell surface. An analysis
of the DNA sequence identified this unknown cell surface candidate as the 67LR. In fact, the
expression of this 67LR was enhanced by ATRA treatment.
Human lung cancer A549 cells were used to assess how effectively the 67LR elicits
EGCG-mediated growth inhibition. Cells transfected with empty vector and treated with
EGCG showed no growth inhibition. However, cells transfected with the gene encoding 67LR
and treated with EGCG demonstrated considerable inhibition as compared with the cells
treated with H2O. We next tested whether the growth inhibitory activity of EGCG correlates
with the binding strength of EGCG to the cell surface. We found increased binding of EGCG
to the cell surface of cells transfected with 67LR. EGCG binding to the 67LR-transfected
cells was inhibited by treatment with an antibody to 67LR. We then measured the binding
affinity of EGCG to 67LR in equilibrium binding experiments using SPR. Kd measurements
were made with a purified recombinant 67LR protein. The predicted Kd value for the binding
of EGCG to the 67LR protein is 39.9 nM [38].
Figure 3. The importance of a primary target for triggering a specific signaling of EGCG.
Figure 4. Tumor size in C57BL/6N inoculated with 67LR-, eEF1A, or MYPT1-knockdown B16 cells.
C57BL/6N mice were subcutaneously inoculated with B16 cells stably transfected with control shRNA
or either the 67LR (A), eEF1A (B), or MYPT1 (C) shRNA expression vector. Peroral administration of
0.1% EGCG was started 1 day before the tumor cell inoculation. After 15 days, tumor size were
measured, and these data were represented as the mean±S.E. of 6 or 7 mice (*, P < 0.05 versus
untreated control).
Most of the 67LR protein was found to exist in the raft fraction rather than the non-raft
fraction [149], and this distribution pattern correlated well with the plasma membrane-
associated EGCG level after treating the cells with EGCG as previously reported [146].
Disruption of these rafts by cholesterol depletion was shown to cause a decrease in the
binding of EGCG to the cell surface [146]. A defect in the integrity of rafts induced by
MβCD treatment led to a reduced expression of 67LR on the cell surface [149]. These facts
suggest that a reduction of lipid raft-associated 67LR may be responsible for the cell-surface
binding of EGCG.
The concentrations of EGCG shown to have an effect (10-100 μM) in these previous
studies are much higher than those observed in the blood or tissues. However, the EGCG
concentrations observed in the plasma and tissue levels after ingestion of tea or polyphenols
are usually lower than 1 μM [12]. To investigate whether the 67LR can confer a sensitivity to
EGCG at physiologically relevant concentrations, we treated the 67LR-transfected A549 cells
with two concentrations of EGCG (0.1 and 1.0 μM); these concentrations are similar to the
amount of EGCG found in human plasma after drinking more than two or three cups of tea
[150]. The growth of the transfected cells was inhibited at both of these concentrations [38].
In addition, this growth-suppressive effect was completely eliminated upon treatment with
anti-67LR antibody before the addition of EGCG. Although we have identified 67LR as a cell
surface receptor for EGCG that mediates EGCG-induced cell growth inhibition [38], there is
no validation of its implication in EGCG-induced cancer prevention in vivo. We investigated
the effect of oral administration of EGCG on subcutaneous tumor growth in C57BL/6N mice
challenged with 67LR-ablated B16 cells [17]. We confirmed both silencing of 67LR by stable
RNAi in B16 cells and attenuation of the inhibitory effect of 1 μM EGCG on cell growth in
67LR-ablated B16 cells in vitro. As shown in Figure 4A, tumor growth was significantly
retarded in EGCG-administered mice implanted with the B16 cells harboring a control
shRNA, whereas tumor growth was not affected by EGCG in the mice implanted with 67LR-
ablated B16 cells, suggesting that 67LR functions as an EGCG receptor not only in vitro but
also in vivo.
Together, these observations demonstrate that the cell-surface 67LR is the receptor for
antitumor action of EGCG at the physiologically relevant concentration. Characterization of
the mechanisms by which 67LR regulates cell proliferation may provide a new approach to
cancer prevention.
5.4. The Inhibition of Cancer Cell Growth by EGCG through the Green Tea
Catechin Receptor
Phosphorylation of the myosin regulatory light chain (MRLC) at Thr18/Ser19 was shown
to increase the actin-activated Mg-ATPase activity of myosin II and the assembly of myosin
II filaments, and regulate the association between myosin II and filamentous actin (F-actin)
[151,152]. The association of myosin II with F-actin results in the formation of stress fibers in
interphase cells and the contractile ring in dividing cells (Figure 5A) [153]. During
cytokinesis an actomyosin-based contractile ring is formed at the equator of dividing cells,
then gets constricted, and finally disappears at the end of cytokinesis [154]. In higher
eukaryotes, it is well established that the state of MRLC phosphorylation is cell cycle
dependent.
To examine the effect of EGCG on cell morphology and actin cytoskeleton, we stained
HeLa cells, human cervical adenocarcinoma, for F-actin with Alexa Fluor 488 phalloidin
[19]. As shown in Figure 5B, control HeLa cells exhibited an organized network of F-actin
containing stress fibers spanning across the cell body and a thin cortical F-actin rim at the
margins. This rim was so thin that cell-cell junctions were not distinguishable. When the cells
were incubated with the indicated concentrations of EGCG for 20 min, the cells retracted and
left intercellular gaps. In addition, disappearance of the stress fibers in the central cell body
was observed upon treatment with EGCG, and the peripheral actin rim became thicker,
providing an outline of the cell-cell junctions.
Because stress fiber formation has been known to require Thr18/Ser19 phosphorylation
of MRLC [153], we examined the effect of EGCG on the MRLC phosphorylation by Western
blot analysis using phosphorylated MRLC-specific antibodies [19]. EGCG dose-dependently
decreased the diphosphorylation of MRLC at The18/Ser19. This result correlates well with
EGCG-induced actin cytoskeleton remodeling. EGCG has been reported to inhibit cancer cell
proliferation directly by affecting the signaling pathway involved in cell growth [43,155].
However, the concentration range of EGCG shown to have an effect (20-100 μM) in previous
studies is much higher than that observed in blood or tissues. To investigate whether the
physiologically relevant concentrations of EGCG can reduce the MRLC phosphorylation, we
treated HeLa cells for 24 h with three concentrations of EGCG (0.1, 1 or 10 μM). The MRLC
phosphorylation was reduced at concentrations between 1 and 10 μM.
The phosphorylation of MRLC at Thr18/Ser19 has been shown to be necessary for
formation of the contractile ring in dividing cells [153]. To examine the effect of EGCG on
contractile ring formation, HeLa cells were treated with 50 μM EGCG for 10 min and stained
for F-actin [19]. At low magnification, the contractile ring in dividing cells not treated with
EGCG exhibited spot-like aggregated F-actin (Figure 5B). However, in EGCG-treated cells
these spot-like F-actin condensations disappeared and the cell peripheral actin rim was able to
be visualized. We then double stained the dividing cells for F-actin and the phosphorylated
MRLC utilizing Alexa Fluor 488 phalloidin and the phospho-MRLC (Thr18/Ser19) specific
antibody, respectively. At high magnification it is clearly observed that EGCG inhibited the
assembly of F-actin and the phosphorylation of MRLC in the cleavage furrow of dividing
cells.
We next examined the effect of EGCG on the growth and cell cycle of HeLa cells [19].
Treatment of the cells with 50 μM EGCG inhibited cell growth and significantly reduced the
diphosphorylation of MRLC at The18/Ser19. Given that EGCG inhibited the contractile ring
formation by reducing the MRLC phosphorylation and thus inhibited the cell growth, to
determine if EGCG treatment could alter the cell cycle, we subjected EGCG-treated cells to
FACS analysis using propidium iodide staining to measure the DNA content. EGCG
treatment for 48 h significantly increased the percentage of cells in the G2/M phase. These
results suggest that the suppressive effect of EGCG on the MRLC phosphorylation continued
at least for 48 h and the increase of G2/M phase cells resulted from the effect of EGCG
Figure 5. EGCG-induced actin cytoskeleton rearrangement and cell morphology. (A) The interaction
between acitin and myosin filaments. Phosphorylation of the myosin II regulatory light chain (MRLC)
at Thr18/Ser19 was shown to increase the actin-activated Mg-ATPase activity of myosin II and the
assembly of myosin II filaments, and regulate the association between myosin II and filamentous actin
(F-actin). The association of myosin II with F-actin results in the formation of stress fibers in the
interphase cells and the contractile ring in dividing cells. (B) HeLa cells were treated with 50 μM
EGCG in DMEM. The cells were fixed and stained with Alexa Fluor 488 phalloidin.
EGCG has been known to produce H2O2 when it is added to the media of several cultured
cell lines, and H2O2 may exert biological effects on cell growth. It has been reported that 30
μM EGCG can induce apoptosis in H661 lung cancer cells, and this effect could be abolished
by the addition of catalase (50 U/ml) in the culture medium [156]. We examined whether
catalase can change the effect of EGCG on cell growth and MRLC phosphorylation [19]. The
addition of catalase could not alter the EGCG-induced reduction of the MRLC
phosphorylation level, the inhibition of the cell growth or the accumulation of the cells in
G2/M phase.
To analyze whether the suppressive effect of EGCG on the MRLC phosphorylation is
mediated by the 67LR, RNA interference (RNAi)-mediated gene silencing was utilized to
knock down the expression of the 67LR [19]. HeLa cells were transiently transfected with
three short hairpin RNA (shRNA) expression vectors for the 67LR. The 67LR silencing was
observed when each of the shRNA expression vectors was trasnsfected separately (data not
shown), however when we combined all three shRNA expression vectors, the highest
silencing was observed. We confirmed the elimination of the EGCG inhibitory effect on the
growth of cells transfected with all three shRNA expression vectors. EGCG significantly
reduced the phosphorylation of MRLC in the cells transfected with the empty vector, however
in the cells transfected with shRNA expression vectors for the 67LR, the same concentration
of EGCG only slightly reduced the phosphorylation. These results indicate that 67LR does
indeed mediate the suppressive effect of EGCG on MRLC phosphorylation. Taken together,
these findings suggest that EGCG inhibits the cancer cell growth by reducing the MRLC
phosphorylation and this effect is mediated by the 67LR.
Colorectal cancer is one of the most prevalent types of cancer in the Western world [157].
Epidemiological studies have suggested that the consumption of green tea may decrease colon
cancer risk [158]. The Wnt-pathway appears to play an important role particularly in colon
carcinogenesis [159]. The essential event in Wnt-signaling is the stabilization of β-catenin.
The resulting accumulation of β-catenin increases the pool of nuclear β-catenin bound to
transcription factor TCF/LEF in complexes that can activate certain genes, including
oncogene c-Myc. As the main binding partner of β-catenin at cell.cell junctions, E-cadherin
plays a pivotal role in β-catenin stabilization and function. E-cadherin commonly repressed in
epithelial carcinogenesis and its binding with β-catenin suppressed Wnt-signaling [160].
Previous studies have reported that EGCG-treatment downregulated the β-catenin protein
expression [41] or upregulated E-cadherin protein expression [161], suggesting that EGCG
suppressed Wnt-signaling. However, it is still not known how a physiological concentration
of EGCG induces cell growth inhibition in colorectal cancer cells. Recently, we found for the
first time that a physiologically achievable concentration of EGCG inhibited cell cycle
progression of human colon adenocarcinoma Caco-2 cells through 67LR without affecting
components of Wnt-signaling. Further, we found that EGCG at a physiological concentration
decreased the phosphorylation of MRLC at Thr-18/Ser-19 in Caco-2 cells through 67LR,
suggesting that an activation of myosin cytoskeleton is involved in anti-proliferative effect of
EGCG at a physiological concentration [18].
Caco-2 cells exhibited higher 67LR protein expression than HeLa cells. EGCG inhibited
the growth of Caco-2 cells even at the concentration of 1 μM. On the other hand, compared
with Caco-2 cells, HeLa cells were less sensitive to EGCG because its growth was inhibited
at only 10 μM EGCG. These results suggest that the expression level of 67LR may be
elevated in the cells that are sensitive to the effect of EGCG at a physiological concentration.
EGCG dose-dependently decreased the G0/G1 and S fractions while increasing the G2/M
fraction in Caco-2 cells. The inhibition of cell cycle progression was significantly observed
even at 1 μM EGCG. These results indicate that a physiologically achievable concentration of
EGCG inhibited cell growth of Caco-2 cells by accumulating the cells in G2/M phase.
We investigated the effect of EGCG on the protein expression of E-cadherin, β-catenin,
and c-Myc, which are important Wnt-signaling components, in Caco-2 cells by Western blot
analysis. E-cadherin protein expression was not affected by EGCG at 1 or 5 μM, though it
was slightly reduced by 10 μM EGCG treatment for 72 h. β-Catenin protein expression were
slightly affected by EGCG at 1 or 5 μM but dose- and time-dependency were not observed. c-
Myc protein expression was slightly reduced by 10 μM EGCG treatment for 72 h. Since these
results did not correlate with EGCG-induced cell growth inhibition as mentioned above, it is
suggested that E-cadherin, β-catenin, and c-Myc are not involved in anti-proliferative effect
of EGCG at a physiological concentration on Caco-2 cells. Although it may be proposed that
suppression of Wnt-signaling by EGCG is the mechanism for EGCG-induced cell cycle arrest
at G0/G1 phase, our results suggest that cell growth inhibition induced by a physiological
concentration of EGCG in Caco-2 cells is dependent on other mechanisms than suppression
of Wnt-signaling. We further examined the effect of EGCG on MRLC phosphorylation at
Thr-18/Ser-19 by Western blot analysis. EGCG treatment for 24 h dose-dependently reduced
the level of MRLC phosphorylation. Moreover, decrease in the phosphorylation of MRLC
was observed even at 1 μM EGCG and correlated well with EGCG-induced cell growth
inhibition.
To investigate whether the inhibitory effect of EGCG on cell cycle progression is
mediated by 67LR, we knockdown the expression of 67LR in Caco-2 cells using RNAi-
mediated gene silencing. 67LR knockdown significantly attenuated 1 μM EGCG-induced
inhibition of cell growth and accumulation of the cells in G2/M phase. Furthermore, 1 μM
EGCG-induced reduction of the phosphorylation of both MRLC (The-18/Ser-19) was also
attenuated in 67LR-shRNA expressing cells. These results suggest that 67LR mediates the
suppressive effect of EGCG at a physiological concentration on cell cycle progression and the
phosphorylation of MRLC and MYPT1.
5.5. Induction of Apoptosis by EGCG through the Green Tea Catechin

Receptor
Recently, EGCG has been shown to be able to induce growth arrest and subsequent
apoptotic cell death in multiple myeloma (MM) cells including IL-6-dependent cells and
primary patient MM cells in vitro, while having no significant effect on growth normal cells
such as peripheral blood mononuclear cells (PBMCs) and fibroblasts [162]. Treatment with
EGCG also led to significant apoptosis in human myeloma cells grown as tumors in SCID
mice. The expression of 67LR was significantly elevated in myeloma cell lines and patient
samples compared to normal PBMCs. RNAi-mediated inhibition of 67LR resulted in
abrogation of EGCG-induced apoptosis in myeloma cells, indicating that 67LR plays an
important role in mediating EGCG activity in MM while sparing PBMCs. Evaluation of
changes in gene expression profile indicates that EGCG treatment activates distinct pathways
of growth arrest and apoptosis in MM cells by inducing the expression of death-associated

protein kinase 2, the initiators and mediators of death receptor-dependent apoptosis (Fas
ligand, Fas, and caspase 4), p53-like proteins (p73, p63), positive regulators of apoptosis and
NF-kappaB activation (CARD10, CARD14), and cyclin-dependent kinase inhibitors (p16 and
p18). Expressions of related genes at the protein level were also confirmed by Western blot
analysis. These data demonstrate potent and specific anti-myeloma activity of EGCG and
provide the rationale for its clinical evaluation.
5.6. Anti-allergic Action of EGCG through the Green Tea Catechin Receptor
5.6.1. Inhibition of Histamine Release

Mast cells and basophils play a central role in immediate allergic reactions mediated by
IgE. Binding of multivalent allergens to specific IgE attached to the FcεRI on the surface of
mast cells or basophils leads to the release of both preformed and newly generated
inflammatory mediators such as histamine, cytokines, chemotactic factors, and arachidonic
acid metabolites. These mediators ultimately cause various symptoms including atopic
dermatitis, hay fever, bronchial asthma, and food allergy [163,164]. The early phase of cell
activation of mast cells and basophils includes the phosphorylation and activation of protein
tyrosine kinases and their substrates, generation of the second messengers such as inositol
trisphosphate and diacylglycerol, and elevation of intracellular Ca2+ levels [165,166]. The late
phase of the activation, which occurs after the influx of Ca2+, includes the fusion of secretory
granules with the membrane and dramatic morphological changes due to remodeling of actin
cytoskeleton, which undergo extensive membrane ruffling [167-169]. Understanding the
molecular events in this degranulation process is thought to hold promise for a therapeutic
intervention leading to the attenuation of various allergic diseases. Screening and evaluation
of anti-allergic factors based on the inhibition of histamine release has been most frequently
performed.
Recently, we reported that EGCG inhibited the calcium ionophore A23187-induced
histamine release from the human basophilic KU812 cells and could not inhibit the increase
of the intracellular Ca2+ level after stimulation with A23187 [147]. This result suggested that
the effect of EGCG on histamine release occurs after the elevation of the intracellular Ca2+
concentration. The increase of intracellular Ca2+ activates degranulation-related cytoskeleton,
and the rearrangement of cytoskeleton requires the interaction between myosin and actin
filaments [167-169]. Phosphorylation of the MRLC at Thr18/Ser19 has been shown to
regulate the association between myosin II and F-actin [151,152]. Thr18/Ser19
phosphorylation of MRLC has been reported to be temporally correlated with degranulation
in the rat basophilic RBL-2H3 cells, and the inhibition of MRLC phosphorylation has been
shown to impair the degranulation [167,170]. Although EGC, having no ability to inhibit
histamine release, showed no inhibitory effect on MRLC phosphorylation, EGCG clearly
reduced the level of phosphorylated MRLC [147]. After treatment of KU812 cells with the
anti-67LR antibody, cells were incubated with EGCG, and further challenged with A23187.
The reductive effect of EGCG on the histamine release was almost completely inhibited in
cells treated with the anti-67LR antibody. Experiment using such 67LR-downregulated cells
revealed a significant abrogation of the inhibitory effect of EGCG on degranulation.
Furthermore, the lowering effect of EGCG on the phosphorylation of MRLC was also
inhibited by either treatment with the anti-67LR antibody or 67LR-knockdown. These
findings indicate that the inhibitory effect of EGCG on degranulation was caused by a
modification of myosin cytoskeleton through the binding of EGCG to 67LR on the cell
surface.
Activation of mast cells and basophils results in extensive changes in cell morphology,
the formation of F-actin, and extensive redistribution of myosin and actin-filaments within the
cells [168,171-174]. To examine the effect of EGCG on the actin cytoskeleton, we stained F-
actin with Alexa Fluor 488 phalloidin. A wavy pattern of stained F-actin was observed in the
cells stimulated with A23187 alone, suggesting that A23187 leads to remodeling of the actin
cytoskeleton in KU812 cells. Generally, degranulation-inducing stimuli have been known to
induce dramatic change of actin cytoskeleton such as membrane ruffling, wavy form of
membrane [169,175]. Thus, the present data implicated that A23187 induced the actin
remodeling similar to membrane ruffling in KU812 cells. When the cells were stimulated
with A23187 in the presence of EGCG, membrane ruffling was inhibited and a biased F-actin
accumulation was observed. Furthermore, this EGCG-induced actin remodeling was
abolished in both anti-67LR antibody-treated cells and 67LR-knockdowned cells. Generally,
the association between F-actin and myosin II has been shown to be regulated by
phosphorylation of the MRLC at Thr18/Ser19 [151,152]. Our findings indicated that EGCG-
induced actin remodeling is caused by lowering MRLC phosphorylation mediated through the
binding of EGCG to the 67LR. Thus, these cytoskeletal modifications may have an important
role in the inhibition of histamine release by EGCG.
5.6.2. Suppression of the High-affinity IgE Receptor Expression

FcεRI plays a central role in the induction and maintenance of IgE mediated allergic
responses such as atopic dermatitis, bronchial asthma, allergic rhitis, and food allergy. The
FcεRI molecule on these cells is a tetrameric structure of one α chain, one β chain, and two
disulfide-linked γ chains. In addition, a trimeric form of FcεRI which lacks the β chain is
found in human. Among the three subunits forming FcεRI, the α chain is the specific
component of FcεRI that mostly extends out to the extracellular region and directly binds to
IgE. Analysis of α chain-deficient mice demonstrated that IgE was unable to bind to the cell
surface of mast cells, thereby inabling the induction of degranulation through IgE binding
[176]. Thus, it is expected that the downregulation of FcεRI expression in mast cells and
basophils may lead to the attenuation of the IgE-mediated allergic symptoms.
The effect of several major tea catechins, (+)-catechin (C), EC, EGC, ECG, and EGCG,
on the cell-surface expression of FcεRI in KU812 cells was studied [177]. Flow cytometric
analysis showed that only EGCG was able to decrease the cell-surface expression of FcεRI
after a 24-h treatment in a dose-dependent manner, while other catechins did not. Moreover,
immunoblot analysis revealed that the total cellular expression of the FcεRI α chain
decreased upon treatment with EGCG. KU812 cells treated with EGCG expressed lower
levels of FcεRI α and γ mRNA than nontreated cells. These results suggest that EGCG has an
ability to down-regulate FcεRI expression, and this suppressive effect may be due to the
down-regulation of FcεRI α and γ mRNA levels. We also found that EGCG has an ability to
inhibit the phosphorylation of the extracellular signal-regulated kinase1/2 (ERK1/2) [146].
This inhibition was involved in downregulation of FcεRI expression by EGCG. Moreover, the
inhibitory effect elicited by EGCG on ERK1/2 was prevented by disruption of lipid rafts.
Thus, these results suggest that the interaction between EGCG and the lipid rafts is important
for EGCG’s ability to downregulate FcεRI expression, and ERK1/2 may be involved in this
suppression signal.
We also examined the effect of the 67LR knockdown on EGCG’s abilities [149]. SPR
analysis showed a reduction of the binding of EGCG to the cells transfected with the 67LR-
shRNA vector, indicating that the EGCG’s binding to the cells lowered upon the decrease of
67LR expression. The suppressive effect of EGCG was inhibited by the knockdown of 67LR.
Furthermore, the ability of EGCG to decrease the phosphorylation of ERK1/2 was reduced in
the 67LR-knocked down cells. These results indicate that the effect of EGCG on ERK1/2
phosphorylation correlates with the expression of 67LR, which implies that the 67LR is the
molecule responsible for transducing the EGCG’s downregulatory signaling of the FcεRI.
5.6.3. Anti-allergic Effect of O-methylated Catechin through the Green Tea Catechin
The O-methylated derivative of EGCG, (–)-epigallpcatechin-3-O- (3-O-methyl)-gallate
(EGCG3”Me) and (–)-epigallpcatechin-3-O- (4-O-methyl)-gallate (EGCG4”Me), as shown in
Figure 1, which was isolated from tea leaves such as Tong-ting oolong tea or cultivars
‘Benifuuki’ and ‘Benihomare‘ (Camellia sinensis L.), has been shown to inhibit allergic
reactions [178,179]. The inhibitory effects of O-methylated EGCG on mouse type I and IV
allergies in vivo more potently than EGCG [178,180]. It is suggested that the higher anti-
allergic activity of O-methylated EGCG may be due to the higher absorption and stability in
vivo [181]. These catechins also strongly inhibited mast cell activation through the prevention
of tyrosine phosphorylation (Lyn, Syk and Btk) of cellular protein and histamine/ leukotriene
release, interleukin-2 secretion after FcεRI cross-linking [182]. Therefore, these facts may
encourage allergic patients to drink tea rich in EGCG3”Me. Indeed, a double-blind clinical
trial to treat allergic cedar pollinosis patients with ‘Benifuuki’ green tea rich in EGCG3”Me
was carried out, and promising results have been obtained by using a protocol of drinking of
1.5 g of tea powder with water twice a day for 13 weeks [181].
We have been found that EGCG3”Me can inhibit histamine release and suppress the
FcεRI expression the same as EGCG [179,183]. RNAi-mediated knockdown of 67LR
expression resulted in a decreased activity of EGCG3”Me [20]. The suppression of MRLC
phosphorylation through the cell-surface binding to the 67 LR contributes to the inhibitory
effect of EGCG3”Me on the histamine release. The 67LR also mediated the EGCG3”Me-
induced suppression of FcεRI expression by reducing ERK1/2 phosphorylation. These results
suggest that anti-allergic effects of EGCG3”Me may be triggered by the inhibition of MRLC
or ERK1/2 phosphorylation mediated through the cell-surface 67LR.
Recently, we examined the effects of methylated derivatives of EGCG, EGCG4”Me and
(–)-4’-O-methyl-epigallocatechin-3-O-(4-O-methyl) gallate (EGCG4’4”diMe) as shown in
Figure 1, on FcεRI expression and ERK1/2 phosphorylation, and each of their cell-surface
binding activities was measured [184]. EGCG4”Me, which is methylated at the 4’-position,
suppressed FcεRI expression and ERK1/2 phosphorylation, although the suppressive effects
were lower than that of EGCG. EGCG4’4”diMe, which is methylated at both the 4’- and 4”-
positions, did not demonstrate a suppressive effect. Furthermore, it was found that
EGCG4”Me could bind to the cell surface even though the binding activity was lower than
that of EGCG. EGCG4’4”diMe could not bind. These results suggest that the trihydroxyl
structure of the B ring is essential for EGCG to exert the suppressive effects and that the
hydroxyl groups on both the 4’-position in the B ring and the 4”-position in the D ring
(gallate moiety) are crucial for the cell-surface binding activity of EGCG. EGCG is known to
be unstable and is degraded easily in animal bodies. On the other hand, EGCG3”Me and
EGCG4”Me are absorbed efficiently and are more stable than EGCG in animal and human
plasma, suggesting the reason for the methylated derivatives of EGCG having potent
inhibitory activities to allergies in vivo, whereas the biological activities in vitro are lower
than those of EGCG. EGCG has been reported to undergo methylation, and EGCG4’4”diMe
has been shown to be a major metabolite of EGCG in plasma [27]. Although our studies on
methylated EGCGs may contribute to the elucidation of physiological activities of EGCG in
vivo, further investigation of the relationship between metabolites of EGCG and 67LR is
necessary for a better understanding of the molecular basis of EGCG activities in vivo.
6. IS THE 67LR A SPECIFIC RECEPTOR FOR GALLATE DERIVATIVES ?

Tea also contains other biologically active compounds such as caffeine [185]. To
compare the ability of 67LR to mediate a response to other tea constituents, we examined
caffeine and other tea polyphenols (Figure 6). None of these other compounds affected the
growth of 67LR-expressing A549 cells, nor could they bind to the cell surface [38]. EGCG is
the only gallate (gallic acid ester) we tested, suggesting that the gallate moiety may be critical
for 67 LR binding and subsequent activity.
Figure 6. The interactions between tea constituents and 67LR-transfected cells. (A) Growth inhibitory
activities of tea constituents (indicated by bars, 5 μM) on A549 cells transfected with either the gene
encoding 67LR (red) or vector only (blue) were examined. (B) The interaction between tea constituents
(5 μM) and A549 cells transfected with the 67LR vector (red line) or the empty vector (blue line) was
measured using a SPR assay.
Recently, we investigated the structure-activity relationship of major green tea catechins

and their corresponding C-2 epimeric isomers, as shown in Figure 1, on cell-surface binding
and inhibitory effect for histamine release [21]. Galloylated catechins; EGCG, (–)-
gallocatechin-3-O-gallate (GCG), ECG, and (–)-catechin-3-O-gallate (CG) showed the cell-
surface binding to the human basophilic KU812 cells by SPR analysis, but their
nongalloylated forms (EC, C, EGC, and GC) did not. The overlapped SPR sensorgram of
each catechins showed that the order of the binding strength based on the resonance unit was
EGCG > GCG > CG ECG. On the other hand, gallic acid (GA) and pyrogallol (PG), as
shown in Figure 1, which are the model molecules of galloyl group or B-ring, could not bind
to the cell surface the same as nongalloylated catechins (C, EC, GC, and EGC). These
observations indicate that only a structural moiety of galloylated catechins (CG, ECG, GCG,
and EGCG) such as basic flavan-3-ol structure (C, EC, GC, and EGC), B-ring (PG), or the
galloyl moiety (GA and PG) is not sufficient to exert the cell-surface binding. Furthermore,
these findings suggest that the combination of basic flavan-3-ol structure and galloyl group
has an essential role in cell-surface binding of tea catechins. The binding strengths of
pyrogallol-type catechins (3’,4’,5’-trihydroxyl structure of the B-ring; GCG and EGCG) were
higher than those of catechol-type catechins (3’,4’-dihydroxyl structure of B-ring; CG and
ECG). Unlike catechol-type catechins, in pyrogallol-type catechins, a remarkable difference
in binding activities was observed. These results indicate that the galloyl group and the
number of hydoxyl group on the B-ring as well as the configuration of the B-ring are
responsible for the cell-surface binding of tea catechins. These patterns were also observed in
their inhibitory effects on histamine release. RNAi-mediated downregulation of 67LR
expression caused a reduction of both activities of galloylated catechins. Among four
galloylated catechins, the maximum reduction of the binding was observed in EGCG. The
reduction rates of the binding strength of pyrogallol-type catechins (GCG and EGCG) were
significantly higher than those of catechol-type catechins (CG and ECG). Although there is a
bulky sterical difference between the C-2 epimers by the B-ring confuguration, no significant
difference in the binding was found between either pyrogallol- or catechol-type catechins.
These results suggest that the 67LR is involved in the cell-surface binding of four galloylated
catechins and that the number of the hydoxyl group on the B-ring contributes to the 67LR-
dependency of galloylated catechins on the cell-surface binding. As to the inhibitiory effect of
tea catechins on histamine release, the order of the 67LR-dependent reduction rate was EGCG
> GCG > ECG > CG. The reduction rates of inhibitory activities of pyrogallol-type catechins
(GCG and EGCG) were higher than those of catechol-type catechins (CG and ECG). This
67LR-dependency was similar to a result of the dependency on their cell-surface bindings.
The influence of different configuration of the B-ring on the 67LR-dependency was much
smaller than those of different hydroxylation of the B-ring. These results suggest that the
67LR is involved in the inhibitory effect of four galloylated catechins on histamine release,
and that the pattern of B-ring hydroxylation is a major structural determinant for the 67LR-
dependency of galloylated catechins. Taken together, our findings suggest that the
combination of galloyl group with basic flavan-3-ol structure may be necessary for the
binding of tea polyphenols to the cell-surface 67LR
7. GREEN TEA CATECHIN RECEPTOR SIGNALING MOLECULES
7.1. Genetic Suppressor Element (GSE) Methodology
In an attempt to elucidate the pathways involved in the anticancer action of EGCG, we

applied genetic suppressor element (GSE) methodology. GSEs are short cDNA fragments
encoding peptides acting as dominant inhibitors of protein function or antisense RNAs
inhibiting gene expression [186]. GSEs behave as dominant selectable markers for the
phenotype associated with the repression of the gene from which they derived, thus allowing
identification of this gene. For example, this strategy previously allowed the demonstration
that kinesin heavy chain is involved in the control of cell response to various DNA-damaging
agents [187]. For identifying genes mediating cell sensitivity to EGCG, we selected GSEs
conferring resistance to EGCG. To search for the mediators of EGCG-induced cell growth
inhibition in B16 mouse melanoma cells, we utilized a targeted genetic screen with a GSE
complementary DNA library, which was prepared from a mouse embryo. Among genetic
elements protecting cells from EGCG-induced cell growth inhibition, we isolated a GSE that
encoded the N terminus of eukaryotic translation elongation factor 1A (eEF1A) [17]. eEF1A
is an important component of the eukaryotic translation apparatus and is also known as a
multifunctional protein that is involved in a large number of cellular processes [188].
7.2. Eukaryotic Translation Elongation Factor 1A (eEF1A)
To investigate the role of eEF1A in EGCG-induced cell growth inhibition, we used stable
RNAi [189] to silence eEF1A expression in B16 cells. Remarkably, silencing of eEF1A
attenuated the inhibitory effect of 1 μM EGCG on cell growth [17]. In contrast,
overexpression of eEF1A enhanced the inhibitory effects of 1 μM EGCG on cell growth. This
concentration is similar to the amount of EGCG found in human plasma after drinking more
than two or three cups of green tea [150]. EGCG is the only known polyphenol present in
plasma in large proportion (77-90%) in a free form, although the other catechins are highly
conjugated with glucuronic acid and/or sulfate group [190]. Based on these considerations,
the activities observed at 1 μM EGCG are relevant to the in vivo situations. Given this, we
investigated the effect of oral administration of EGCG on subcutaneous tumor growth in
C57BL/6N mice challenged with eEF1A-ablated B16 cells as shown in Figure 4B [17].
Tumor growth was significantly retarded in EGCG-administered mice implanted with the
B16 cells harboring a control shRNA, whereas tumor growth was not affected by EGCG in
the mice implanted with eEF1A-ablated B16 cells, indicating that eEF1A is involved in
EGCG-induced cancer prevention.
EGCG induces growth inhibition in many cell lines; however, the efficacy of inhibition
varied, depending on the cell lines used [191]. We hypothesized that the expression level of
eEF1A in a cell line correlates to the efficacy of EGCG-induced cell growth inhibition in that
cell line. We investigated the expression levels of eEF1A in B16 cells and the following
human cancer cell lines: hepatoma HepG2, breast carcinoma MCF-7, cervical carcinoma
HeLa, and squamous cell carcinoma A431 [17]. The levels of eEF1A expression in B16 cells,
HepG2 cells, and MCF-7 cells were relatively higher than those in HeLa cells and A431 cells.
EGCG appeared to display different efficacies of growth inhibition in these cell lines, with
estimated IC50 values of 9.7 μM for MCF-7 cells, 22.7 μM for HepG2 cells, 51.6 μM for
HeLa cells, and 52.8 μM for A431 cells, respectively. The expression level of eEF1A is
elevated in cell lines that are more sensitive to the effect of EGCG. These results support our
conclusion that eEF1A serves as a mediator for EGCG-induced cancer prevention.
7.3. Myosin Phosphatase-targeting Subunit (MYPT1) and Remodeling of

Actin Cytoskeleton
MRLC phosphorylation controls the activity of myosin II, a major motor protein in
animal cells, which is involved in a wide range of processes, including muscle contraction,
cell locomotion, cell division, and receptor capping [192]. The phosphorylation of MRLC is
regulated by two classes of enzymes: MLC kinases and myosin phosphatase [193]. Myosin
light chain kinase and Rho-kinase seem to be the two major kinases that phosphorylate
MRLC in vitro as well as in vivo [193]. Myosin phosphatase is composed with three subunits:
a 37-kDa catalytic subunit, a 20-kDa subunit of unknown function, and a 110-130-kDa
myosin phosphatase-targeting subunit (MYPT1) [194]. The activity of myosin phosphatase is
known to be regulated by phosphorylation of MYPT1, and two major sites, Thr-696 and Thr-
853, have been extensively investigated and identified as an inhibitory site [194].
Previously, we reported that EGCG-induced cell growth inhibition may result from the
reduction of the phosphorylation of myosin regulatory light chain (MRLC) at Thr-18/Ser-19
through 67LR in HeLa cells [19]. The activity of myosin phosphatase is known to be
inhibited by phosphorylation of its targeting subunit MYPT1 at Thr-696 and Thr-853 [194].
We tested the effect of EGCG on the phosphorylation of MYPT1 at Thr-696 and Thr-853
[17]. Intriguingly, although the phosphorylation level at Thr-853 was unaffected by EGCG,
EGCG induced the dephosphorylation of MYPT1 at Thr-696. Further, this effect correlated
with EGCG-induced reduction of the MRLC phosphorylation, suggesting that EGCG
activates myosin phosphatase by reducing the MYPT1 phosphorylation level at Thr-696.
Next, we investigated whether MYPT1 is involved in anticancer action of EGCG in vivo. In
B16 cells, physiological concentrations of EGCG reduced the MYPT1 phosphorylation at
Thr-696 and the MRLC phosphorylation. We confirmed both silencing of MYPT1 by stable
RNAi in B16 cells and attenuation of the inhibitory effect of 1 μM EGCG on cell growth in
MYPT1-ablated B16 cells in vitro. We tested the effect of oral administration of EGCG on
subcutaneous tumor growth in C57BL/6N mice challenged with MYPT1-ablated B16 cells
(Figure 4C). Tumor growth was significantly retarded in EGCG-administered mice implanted
with the B16 cells harboring a control shRNA, whereas tumor growth was not affected by
EGCG in the mice implanted with MYPT-1-ablated B16 cells, suggesting that MYPT1 is
indispensable for EGCG-induced cancer prevention. In addition to this role of MYPT1 in
EGCG-induced inhibition of cancer cell growth, a study on RNAi-mediated knockdown of
MYPT1 expression demonstrated the importance of MYPT1 as the signaling component
mediating the inhibitory effect of EGCG on histamine release and MRLC phosphorylation on
the basis of the defect of both activities (unpublished data).
Figure 7. Model of possible EGCG signaling pathway through 67LR in vivo.
It has been reported that MYPT1 directs the action of myosin phosphatase to not only
MRLC but also other F-actin-binding proteins that influence cell contractility, morphology,
and proliferation [195-198]. We found that EGCG induces a dynamic remodeling of actin
cytoskeleton. The cells treated with EGCG exhibited a filopodial-like structure, and then after
3 h, the cell body retracted and left intracellular gaps, suggesting that the EGCG-induced
filopodial-like projections are simple residual contact sites that have not yet been released
from the substrate [17]. Together, it is suggested that EGCG-induced actin cytoskeleton
remodeling results from not only the reduction of the MRLC phosphorylation but also the
activation of myosin phosphatase.
7.4. A Hierarchy of EGCG Signaling Molecules
Further, to establish whether MYPT1 is indeed involved in the suppressive effect of

EGCG on MRLC phosphorylation and cell growth, we used stable RNAi to silence MYPT1
expression in HeLa cells [17]. Western blot analysis indicated that stable RNAi for MYPT1
specifically silenced MYPT1 protein expression in HeLa cells with no effect on the
expression of 67LR and eEF1A. Silencing of MYPT1 prevented both EGCG-induced
reduction of the MRLC phosphorylation and cell growth inhibition, suggesting that EGCG-
induced dephosphorylation of MYPT1 at Thr-696 results in the activation of myosin
phosphatase and inhibition of cell growth.
It has been reported that eEF1A binds to the ankyrin repeat of MYPT1 [198]. It is
tempting to speculate that both 67LR and eEF1A are upstream signaling components
responsible for EGCG-induced dephosphorylation of MYPT1 at Thr-696. To test this
hypothesis, we used stable RNAi to silence the expression of 67LR or eEF1A in HeLa cells.
We confirmed specific silencing of each target protein by stable RNAi in HeLa cells and
attenuation of the inhibitory effect of EGCG on cell growth in these cells [17]. In both 67LR-
ablated HeLa cells and eEF1A-ablated HeLa cells, the inhibitory effect of EGCG on both the
phosphorylation of MYPT1 at Thr-696 and the phosphorylation of MRLC was attenuated. In
addition, EGCG-induced actin cytoskeleton rearrangement was no longer observed in
MYPT1-, eEF1A-, or 67LR-ablated HeLa cells. The involvement of MYPT1 in downstream
EGCG-triggered signaling from both 67LR and eEF1A was further documented by
confirming abrogation of 1 μM EGCG-induced reduction of the MYPT1 phosphorylation
level at Thr-696 and the MRLC phosphorylation in 67LR- or eEF1A-ablated B16 cells. These
results suggest that MYPT1 is involved in downstream EGCG signaling from both 67LR and
eEF1A (Figure 7). It has been reported that MYPT1 binds to eEF1A [198], and more than
half of the total eEF1A (>60%) binds to the actin cytoskeleton [199]. Because other findings
indicate that eEF1A is also implicated in microtubule binding, bundling, or severing
[200,201], a potential role for the protein in regulating cytoskeleton organization has also
been proposed. Characterizing the mechanisms by which EGCG induces reduction of the
MYPT1 phosphorylation at Thr-696 and reorganization of actin cytoskeleton through eEF1A
should help in more precise understanding of cytoskeleton organization, although further
experiments are necessary.
8. CONCLUSION
Recent studies have highlighted the importance of genetically determined factors in
evaluating the role of green tea intake in the development of breast cancer [202-204]. In a
case-control study conducted among Asian-American women, green tea intake appeared to
reduce breast cancer risk [203]. Reduction in risk was strongest among persons who had the
low-activity catechol-O-methyltransferase (COMT) alleles, but not high-activity COMT
alleles, suggesting that these individuals were less efficient in eliminating green tea catechins
and may derive the most benefit from these compounds. Yuan et al. reported a low risk of
breast cancer among women with higher green tea intake and the low-activity genotype of
angiotensin-converting enzyme gene among Singapore Chinese women [202]. A nested case-
control study suggested protective effect of green tea against breast cancer among women
with high-activity genotypes of the methylenetetrahydrofolate reductase and thymidylate
synthase genes [204]. This effect was even stronger among those who were low consumers of
dietary folate. These observations indicate that further study to elucidate the key molecules
determining EGCG responsiveness is indispensable for a better understanding of EGCG
activity in vivo.
Chemoprevention by edible phytochemicals is now considered to be an inexpensive,
readily applicable, acceptable, and accessible approach to cancer control and management
[205]; however, little is known about the mechanism of the chemopreventive action of most
phytochemicals, including EGCG. Although previous studies have proposed various different
mechanisms for cancer-preventive action of EGCG [205-207], it remains unclear which

EGCG-induced molecular events are relevant in vivo. The activities affected by lower
concentrations of EGCG are likely to be more relevant in vivo because of the limited
bioavailability of EGCG. The essence is the identification of the primary target and the
demonstration of specific mechanisms of action in animal models and human tissues. Here we
described that each of 67LR, eEF1A, and MYPT1 is indispensable for EGCG-induced cancer
prevention in vivo, and these proteins mediate physiological concentrations of EGCG-
triggered unique signaling for cancer prevention. Our findings suggest that these proteins are
"master proteins," which determine the efficacy of cancer-preventive activity of EGCG and
have important implications for development and use of EGCG as a cancer-chemopreventive
agent. Probably, only a tumor with a high expression level of these "master proteins" has
sensitivity to physiological concentrations of EGCG, while lower expression of those
molecules causes “EGCG-resistance”. Our results not only illuminate the mechanisms for the
cancer-preventive activity of EGCG but should help in the design of new strategies to prevent
cancer and underscore the importance of tailoring cancer therapy on the basis of tumor
genotype.
The integrity of lipid rafts has been shown to be important for the pathogenesis of cancer
[115,208]. Epigenetic regulation of genes encoding raft components and its roles in cell
transformation, angiogenesis, immune escape, and metastasis has been reviewed earlier
[115,138,140,208-212]. The role of rafts in prostate cancer is intriguing and discussed
recently [213,214]. These contributions would help to best understanding current concerns in
raft-associated signaling in regulation of cancer, and will help to select strategies for better
management of neoplasm. Therefore, both orienting research toward newly proposed
interactions (EGCG-67LR-eEF1A-MYPT1-Cytoskeleton axis), as shown in Figure 7, and
investigation focused on lipid rafts as a signaling platform regulating the 67LR-mediated
action of EGCG may unravel some of the complex aspect of EGCG-induced anti-cancer
signaling.
Finally, in addition to cancer-chemopreventive and anti-allergic properties, EGCG has
been shown to possess diverse physiological activities, and we are curious to know whether
EGCG signaling through the 67LR relates to other beneficial effects of EGCG.
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Chapter 15
UTILITY OF EPIGALLOCATECHIN GALLATE IN THE

TREATMENT AND PREVENTION OF BREAST CANCER:
MOLECULAR MECHANISMS
FOR TUMOR SUPPRESSION
R. J. Rosengren*
Department of Pharmacology and Toxicology, University of Otago,
Dunedin, New Zealand
ABSTRACT
Green tea and its major constituent epigallocatechin gallate (EGCG) have been
extensively studied as a potential chemopreventative and/or treatment for a variety of
diseases including breast cancer. Experimental evidence is supported by epidemiological
studies that have shown an inverse relationship between green tea consumption and the
incidence of breast cancer. Numerous studies have demonstrated that EGCG is cytotoxic
toward both estrogen receptor-positive and estrogen receptor-negative breast cancer cell
lines. These studies have highlighted potential mechanisms for the actions of EGCG,
such as the induction of apoptosis, the alteration of the expression of cell cycle regulatory
proteins critical for cell proliferation, inhibition of the chymotrypsin-like proteasome,
inhibition of angiogenesis, as well as inhibition of cell invasion and metastasis.
Importantly, these effects occur independently of estrogen receptor expression. This
chapter will provide evidence for these events and other molecular mechanisms that
significantly contribute to the actions of EGCG in vitro. The utility of green tea extract or
EGCG as a breast cancer treatment and chemopreventative has also been extensively
investigated using various in vivo models of estrogen receptor-positive and estrogen
receptor-negative breast cancer (ie., chemical carcinogenesis and xenograft models).
Evidence for EGCG-mediated tumor suppression and the major molecular mechanisms
for this effect, such as the induction of apoptosis, the inhibition of angiogenesis and
*
Address for correspondence: Rhonda J. Rosengren. Department of Pharmacology & Toxicology. 18 Frederick
Street, Adams Building, University of Otago, Dunedin, New Zealand, 9001. E-mail:
rhonda.rosengren@otago.ac.nz; Tel: +64 3 479 9141; Fax +64 3 479 9140.
302 R. J. Rosengren
modulation of the expression of cell signaling proteins are also fully examined. These in
vivo studies have led to investigations which have focused on ways to improve the
actions of EGCG, by either using it as part of a combination therapy or by synthesizing
pro-drugs of EGCG. Both of these have been done in order to enhance the bioavailability,
stability and efficacy of EGCG. These new compounds and drug combinations have
significantly improved the tumor suppression potential of EGCG and provide an exciting
future for this multi-faceted phytochemical in the prevention and treatment of breast
cancer.
INTRODUCTION
Flavanoids are plant-derived polyphenolic compounds found in fruits, vegetables, herbs,
tea and wine [1] and are divided into several different classes based on small variations in
their structure. One such class is the flavan-3-ols, also termed the catechins, which are
differentiated by di- or tri-hydroxyl group substitutions on the B-ring and meta-5,7-dihydroxy
substitution on the A ring [2]. Catechins are particularly abundant in green tea (Camellia
sinensis), accounting for 30-40% of its dry weight [3-5]. The major catechins contained in
green tea are (-)-epigallocatechin-3-gallate (EGCG), (-)-epigallocatechin (EGC), (-)-
epicatechin gallate (ECG), (-)-epicatechin (EC) and catechin, with EGCG comprising the
greatest proportion of these catechins [4, 5].
Since tea is the second most widely consumed beverage next to water, both tea and
EGCG are generally considered to be non-toxic. However, the literature on this topic is not
consistent. For example, EGCG (500 mg/kg/d as part of the diet, for 13 weeks) did not
produce weight loss in male or female Sprague-Dawley rats [6]. In contrast, green tea extract
(GTE) (5% of the diet/d, for 90 d) caused a loss of body weight in male but not female rats
[7] and EGCG (81 mg/kg/, 8d, ip) decreased body weight in both rat sexes [8]. Even though
the LD50 of GTE in mice has been calculated to be 3.09 g/kg for females and 5 g/kg for males
[7], weight loss, hepatotoxicity and mortality have been reported following the administration
of much lower doses of EGCG. Specifically, a single dose of EGCG (150 mg/kg, ip)
produced mortality in femake mice [9] and repeated administration of EGCG (50 mg/kg/d,
7d, ip) elicited hepatotoxicity, weight loss and mortality in male and female mice [10-12].
However, all three parameters were more severe in females compared to males [10]. While
female mice were more susceptible to EGCG-mediated hepatotoxicity, this has not been
demonstrated in rats. Specifically, female Wistar rats were administered GTE (2.5 g/kg/d, po,
6 weeks or 2 g/kg/d, po, 12 weeks) and no liver injury was seen in any treatment group [13].
The conflicting evidence in rodents makes extrapolation to humans difficult. However, there
have been reports of liver injury in humans following consumption of GTE. For example, In
Europe, there have been 14 reported cases of severe hepatitis following ingestion of an
ethanolic extract of green tea, which caused the product to no longer be marketed [14]. A
further 3 cases of liver injury have been reported following the consumption of either green
tea (6 cups/day, 4 months) [15] or a micronized powder of Camellia sinensis) [16]. However,
clinical trials with high doses of GTE (2.2 g/m2, tid, 6 months) have only shown minor side-
effects most likely related to caffeine in the extract (insomnia, restlessness, gastrointestinal
complaints) [17]. Additionally, EGCG (1.6 g, as one bolus oral dose) was given to 8
volunteers without any clinical or biological adverse reactions [18]. Therefore, it is probably
Utility of Epigallocatechin Gallate … 303
safe to conclude that green tea / EGCG can be consumed without unwanted side effects in
most individuals. This is also supported by the numerous health benefits associated with
green tea / EGCG consumption. These have been consistently demonstrated in numerous
epidemiological studies which have examine the role of green tea consumption and breast
cancer risk [19-21]. In this chapter, evidence will be given for the potential application of
EGCG in the prevention and treatment of breast cancer as well as the molecular mechanisms
that underpin these actions of EGCG.
EPIDEMIOLOGICAL EVIDENCE - BREAST CANCER RISK ASSOCIATION

WITH GREEN TEA CONSUMPTION AND GENOTYPE
Epidemiological studies that have analyzed green tea consumption and the risk of
developing breast cancer have yielded conflicting results. Some studies have shown no
association with green tea drinking [19-21], while others have demonstrated a
chemopreventative effect [22] (Table 1). It is important to note that most of the published
epidemiological studies were not specifically designed to examine the relationship between
tea consumption and cancer risk and therefore the amount and frequency of tea consumption
was not always accurately determined [23]. However, the results from a meta-analysis of 4
studies (three cohort studies from Japan and one case control study from the USA) showed
that breast cancer risk was significantly reduced following green tea consumption (OR = 0.77,
95% CI = 0.61-0.97) [24]. Additionally, a decreased risk of recurrence of breast cancer has
also been shown to correlate with green tea consumption [25, 26] (Table 1). Therefore, the
overall evidence suggests a positive correlation between green tea consumption and the
development and recurrence of breast cancer.
In order to more fully understand the chemopreventative actions of green tea, studies
have genotyped breast cancer patients to determine whether genetic polymorphisms could
influence the actions of green tea. One such case-control study in Chinese-, Japanese-, and
Filipino-American women demonstrated that green tea intake was associated with a reduction
in breast cancer risk, but only in women possessing a low-activity allele of catechol-O-
methyltransferase (COMT) [22]. COMT is responsible for the rapid methylation of the
catechins in green tea and, therefore, differences in the methylation capacity between
individuals may alter the chemopreventative activity of green tea. The findings of Wu et al.
(2003) [22] suggest that chemoprevention by green tea in women possessing the low-activity
COMT allele may result from decreased metabolism and thus an increased bioavailability of
catechins. However, women with a low activity COMT would also be at a higher risk for
developing breast cancer due to a decreased production of the pro-apoptotoic and anti-
angiogenic metabolite of estradiol, 2-methoxy-estradiol [28]. Therefore, further work needs to
be conducted to determine if the reduction in risk is related to catechin metabolism or the
overall higher risk of the patients.
Other enzymes were also shown to be important as green tea consumption was also
associated with a reduced risk of breast cancer in women who expressed the high-activity, but
not the low-activity, angiotensin-converting enzyme [29]. Changes in the activity of this
enzyme support the hypothesis that a possible mechanism of chemoprevention by EGCG and
other green tea catechins involves their inhibition of reactive oxygen species via the
Table 1. Green Tea Intake and the Risk of the Development or Recurrence of Breast Cancer
Type Population Profile Risk Ratio (95% CI) Outcome Reference

Recurrence 472 Japanese women with Green tea consumption: Green tea intake was associated [25]
Stage I, II or III breast cancer Stage I and II 0.56 (0.35 - 0.91)* with a reduced risk of recurrence
Stage III 1.88 (0.79 - 4.54) of Stage I and II breast cancer
1,160 Japanese women Green tea consumption: Green tea intake was associated [26]
Stage I ≤3 cups/day 0.43 (0.22 - 0.84)* with a reduced risk of recurrence
3-5 cups/day 0.37 (0.17 - 0.80)* in Stage I breast cancer
≥6 cups/day 0.59 (0.23 - 1.52)
Stage II ≤3 cups/day 0.71 (0.35 - 1.44)
3-5 cups/day 0.80 (0.38 - 1.69)
≥6 cups/day 0.51 (0.18 - 1.46)
Stage III ≤3 cups/day 1.01 (0.50 - 2.05)
3-5 cups/day 1.06 (0.51 - 2.17)
≥6 cups/day 0.87 (0.33 - 2.27)
Development 34,759 women in Hiroshima Green tea consumption: No association [19]
& Nagasaki, Japan ≤ 1/day
2-4/day
≥ 5/day
23,667 women, members of the Life Green tea consumption: No association [20]
Span Study Cohort ≤ 1/day
2-4/day
≥ 5/day
Combined 2 cohort studies Green tea consumption: No association [21]
conducted in rural Japan 1-2 cups/day
-17,353 women cohort 1 3-4 cups/day
-24,769 women cohort 2 ≥ 5 cups/day
Chinese, Japanese and Filipino Green tea consumption: Green tea intake is associated [22]
women residing in the US 0 - 85.7 ml/day with a reduced risk of the
≥ 85.7 ml/day development of breast cancer
*Statistically significant.
Table modified from Stuart et al., (2006) [27].
angiotensin-converting enzyme. A further mechanism of chemoprevention by green tea

catechins involves the alteration of circulating steroid hormone levels. Specifically, in a study
of 130 postmenopausal Chinese women, it was shown that women who regularly consume
green tea had lower plasma levels of estrone, estradiol and androstenedione compared with
non- or irregular green tea-drinkers [30]. In this study, the changes in hormone levels were
not dependent upon the genotype of COMT. Therefore, these findings suggest that alteration
of steroid hormone levels may contribute to the chemopreventative activity of green tea.
However, in order to obtain a more thorough picture of the effect of green tea / EGCG, more
investigations must be conducted with both GTE and pure EGCG.
IN VITRO EFFECTS AND MOLECULAR MECHANISMS OF EGCG

EGCG is cytotoxic toward breast cancer cells regardless of their ER status. Specifically,
after EGCG treatment, cell number was significantly decreased from control in both ER-
positive (MCF-7, T47-D and BT474) [31-34] and ER-negative (MDA-MB-231, Hs578t,
MBA-MB-468 and BT-20) human breast cancer cells lines, [31-41]. Importantly, EGCG
displayed a greater cytotoxic potency in ER-negative breast cancer cell lines compared to ER-
positive cell lines [33, 34]. Results from in vitro ER binding and reporter gene assays as well
as in vivo functional assays demonstrated that EGCG weakly bound both ERα and ERβ, but
could not antagonize estradiol-mediated responses in vivo [11]. These results demonstrate that
EGCG is not a strong antagonist of the ER and its cytotoxic action in breast cancer cells
occurs independently of the ER. Furthermore, EGCG is cytotoxic toward breast cancer cells
that grow in response to stimulation via HER2, an isoform of the epidermal growth factor
receptor (EGFR), but are also resistant to trastuzumab immunotherapy. Specifically, EGCG
(~87-349 µM) dose-dependently decreased the proliferation of both trastuzumab-resistant
BT474 human breast cancer cells and JIMT-1 cells (derived from a patient who displayed
clinical resistance to trastuzumab immunotherapy) [42]. Therefore, EGCG is cytotoxic toward
a variety of human breast cancer cells and this effect is mediated via ER-independent
mechanisms that are critical in the regulation of cell proliferation. The major mechanisms by
which EGCG conveys its cytotoxicity toward breast cancer cells are outlined in the following
sections.
EGCG-MEDIATED INDUCTION OF APOPTOSIS AND MODULATION OF

CELL SIGNALING PATHWAYS
There is an abundance of literature obtained from research using a variety of human
cancer cell lines that has demonstrated that EGCG induces apoptosis [43-47]. It would then
be expected that EGCG would induce apoptosis in most, if not all, breast cancer cell lines.
However, the role of EGCG in breast cancer has only more recently become a focus of this
potential drug and most of this work has been conducted in ER-negative breast cancer cells.
Specifically, EGCG or green tea extract induced apoptosis in ER-negative MDA-MB-468
[38] and MDA-MB-231 cells [33, 40, 41, 48, 49], as well as trastuzumab–resistant BT474
and JINT-1 cells, but only following high concentrations in these cells (>170 µM) [42].
306 R. J. Rosengren
Mechanisms through which EGCG-induced apoptosis was mediated includes; cell cycle arrest
[31, 36, 39], changes in intracellular signaling cascades [50-53], inhibition of proteasomal
chymotrypsin-like activity [49], upregulation of p53 and changes in the ratio of Bax/Bcl-2
[38]. For example EGCG has caused ER-negative breast cancer cells to arrest in the G1 phase
of the cell cycle. Specifically, treatment of MDA-MB-231 cells with EGCG (87 µM) for 12 h
produced a 34% increase in the number of cells in the G1 phase of the cell cycle compared to
control [39], while a lower concentration of EGCG (25 µM) only increased the number of G1
phase cells by 4% [40, 41]. Therefore, the ability of EGCG to induce cell cycle arrest is
clearly dose-dependent. Further studies have demonstrated that changes in the cell cycle were
driven by EGCG-mediated alterations in the protein expression of the cyclins, cyclin
dependent kinases (cdks) and their inhibitors (CDKIs). Specifically, studies in MDA-MB-231
cells have shown that EGCG (87 µM) decreased the protein expression of Cyclin D and
Cylcin E as well as cdk4 and cdk1 by 50% [39], while the CDKIs, p21 and p27 were
increased [36]. A similar result has also been reported in MCF-7 cells where 30 µM of
EGCG-induced the expression of p21 and p27 and this correlated with an 1.5-fold increase in
the number of cells arrested in the G1 phase of the cell cycle [31].
The induction of apoptosis and alterations in the cell cycle are likely to be the result of
EGCG-mediated changes in intracellular pathways. It has been documented that EGCG alters
the phosphorylative activity of the EGFR and its downstream targets in breast cancer cells
(for an overview of EGFR and its downstream targets see Figure 1). Specifically, the
treatment of MDA-MB-231 cells with EGCG inhibited both basal and TGF-α-induced auto-
phosphorylation of the EGFR [36]. It was further established that EGCG inhibited
constitutive and TGF-α-induced AKT and STAT3 activity in these cells, but not the
expression of phosphorylated ERK. Furthermore, EGCG inhibited the activity of the HER2
and this correlated with a decrease in downstream effects of EGFR activation, such as c-fos
promoter activity [37].
Another important protein that governs cell survival is the transfactor NF-κB. NF-κB has
overlapping roles in many mitogenic signaling pathways as it is capable of promoting and
repressing the expression of proteins involved in cell growth, apoptosis, inflammation, the
stress response as well as other important physiological processes [54-56]. Therefore, it is
vital for tumor growth, and is a key player in ER-negative breast cancer as it is overexpressed
in both ER-negative breast cancer cell lines and in tumors from patients [57]. Therefore, it is
an important target in ER-negative breast cancer treatment strategies. Studies in cancer cell
lines have demonstrated that EGCG inhibits NF-κB [36, 50-53]. For example, MDA-MB-231
cells were used to demonstrate that EGCG inhibited both the basal and inducible activity of
the NF-κB complex [36]. This supported previous work showing that EGCG elicited dose-
and time-dependent inhibition of both the activation and translocation of NF-κB via the
suppression and cytoplasmic degradation of IkBa [52, 53]. Further studies demonstrated that
EGCG exhibited a concurrent effect on p53 and NF-κB, which caused a change in the ratio of
BAX/Bcl-2 and thus favored apoptosis [50]. This effect would significantly impact the
growth of many breast cancer cell lines but would not be relevant to MDA-MB-231 cell
growth, as these cells lack a functional form of p53 [58].
Figure 1. Schematic diagram of intracellular cell signaling cascades that are activated following
phosphorylation of the EGFR. Dimerization of the EGFR leads to the activation of various signaling
pathways such as PI3K/Akt and Ras/Raf/ERK. These regulate key downstream regulators of cell
growth such as NFκB and mTOR.
EGCG modulates other important cell survival pathways in MDA-MB-231 cells. One of
these is the hepatocyte growth factor (HGF)/Met signaling pathway which is involved in
proliferation, survival, motility and invasion [59]. Met is a tyrosine receptor kinase which
autophosphorylates upon activation by HGF and its downstream targets include the
PI3K/AKT and MAPK pathways [59-62]. Studies with EGCG have shown that
concentrations as low as 0.6 µM inhibited HGF-induced Met phosphorylation, and
subsequent AKT and ERK activation and this correlated with a 67% decrease in HGF-
induced invasion in MDA-MB-231 cells [59]. Importantly, the authors also demonstrated that
EGCG (5 µM) inhibited the basal invasion capabilities of MDA-MB-231 cells by 50%, which
demonstrates that EGCG decreases both basal cancer cell invasion and that induced by HGF.
Apoptosis can also be modulated in cancer cells by the chymotrypsin-like proteasome
[63]. Inhibition of this specific proteasome rapidly and selectively induces apoptosis in cancer
cells but not normal cells [64, 65]. Importantly, this effect has been shown in human cancer
308 R. J. Rosengren
cells that are resistant to numerous other anticancer agents. In MDA-MB-231 breast cancer
cells EGCG inhibited chymotrypsin-like activity by 24% and this correlated with an increase
in ubiquitinated IkBa, p27 and Bax [49]. All three of these proteins work to decrease cell
proliferation and increase apoptosis, as IkB prevents the activation of the transfactor NF-kB
[66]. Overall these studies show that, EGCG induces apoptosis in breast cancer cells by
modulating intracellular signaling pathways that control cell cycle progression, cell
motility/invasion and proteasomal degradation. Since these effects are not dependent on the
ER, EGCG is able to decrease cell growth and induce a strong apoptotic response in ER-
negative breast cancer cells. Thus EGCG has the potential to be used as a drug that will
positively impact on the lives of women with ER-negative breast cancer.
EGCG AS AN INHIBITOR OF ANGIOGENESIS

Numerous studies have demonstrated that EGCG inhibits a variety of processes involved
in angiogenesis. This action elicited by EGCG is extremely relevant to tumor suppression
since tumor growth is an angiogenic-dependent process, with new blood vessels supplying
nutrients, oxygen and growth factors that enhance tumor proliferation and expansion [67]. In
breast cancer, neovascularization has been shown to contribute to the long-term
aggressiveness of the tumor and correlates to increased metastasis [68, 69]. EGCG has been
documented to inhibit endothelial cell growth in a dose-dependent manner in vitro, which
correlated to a significant decrease in new blood vessel formation in an in vivo angiogenesis
model [70]. Furthermore, in vitro studies have shown that EGCG inhibits the production of
vascular endothelial growth factor (VEGF) and matrix metalloproteinases [71, 72]. All
isoforms of VEGF have been established as potent angiogenic agents [73] and the levels of
this growth factor are closely correlated to the induction and maintenance of the
neovasculature in breast cancer [74, 75]. VEGF initiates angiogenesis via the binding to its
receptors (VEGFR-1 and VEGFR-2) [76]. VEGFR-2 activation is responsible for most of the
mitogenic and chemotactic effects of VEGF [77] and inhibition of angiogenesis occurs
through the inhibition of both VEGF and VEGFR-2 [78, 79]. Importantly, EGCG has been
shown to inhibit VEGFR-2 phosphorylation in vitro but this study was not conducted in
human breast cancer cells [80] and thus, further work needs to be conducted to confirm this in
breast cancer models.
EGCG also decreases cancer cell invasion and metastasis [81, 82] by inhibiting cell
adhesion function via an inhibition of E-cadherin. Macrophages and other inflammatory cells
also promote angiogenesis and EGCG has decreased inflammation by suppressing the over-
expression of both cyclooxgenase [83] and nitric oxide synthatase [84]. Another potential
mechanism for EGCG is via binding to the metastatsis-associated 67-kDa laminin receptor.
While this study was conducted in lung cancer cells, the results showed that growth inhibition
occurred only in cells transfected with the 67-kDa laminin receptor and that EGCG bound to
this receptor with a Kd of 39.9 nM [85]. Interestingly, none of the other green tea catechins
were able to bind to this receptor and thus the effect was specific for EGCG. However,
epicatechin gallate was not tested and thus the gallate moiety may prove to be critical for
activity. Additionally, these experiments were not performed in breast cancer cells and thus it
has yet to be determined if this response is relevant to the antiangiogenic effect of EGCG in
breast cancer, as there are many additional facets to this process.
In breast cancer, another factor involved in angiogenesis, invasiveness and metastasis is
the oncogene Wnt. Constitutively active Wnt has been linked to increased proliferation and
invasiveness in breast cancer through the stabilization of β-catenin. In breast cancer, high β-
catenin levels correlates with poor prognosis [86-88] Additionally, the percentage of breast
cancers with high β-catenin expression is estimated to be approximately 50% [89]. Therefore,
modulation of Wnt could significantly impact on a large number of breast cancer patients.
Importantly, in MDA-MB-231 cells EGCG (25-100 µM) blocked Wnt signaling in a dose-
dependent manner, via the induction of HBP1 [90]. Induction of HBP1 occurred via an
increase in the stabilization of mRNA and not through transcriptional induction. HBP1 plays
an important role in invasive breast cancer, as a loss HBP1 is associated with invasive breast
cancer and 30% of patients with invasive disease express HBP1 mutants [89]. Importantly,
downstream effects of Wnt signaling were also modulated by EGCG, as c-mcy (a relevant
Wnt target gene) was also decreased [90]. These results correlated with a decrease in the
migration of MDA-MB-231 cells. Specifically, EGCG (50 and 100 µM) decreased the
migration of cells toward fibronectin treated medium by 50 and 75%, respectively and also
decreased the invasion of cells through Matrigel [90]. This demonstrates that EGCG has the
ability to modulate breast cancer invasiveness by increasing the stability of HBP1, which
decrease Wnt signaling, c-mcy expression and ultimately cell migration.
In breast cancer, invasion and metastasis is more extensive in breast cancer cells that lack
the alpha isoform of the ER (ERα). Breast cancer cells that are positive for ERα have a more
epithelial architecture while; ERα-negative cells have a more invasive phenotype.
Importantly, this effect is not static and the transition from an epithelial to a more
mesenchymal phenotype (EMT) has been shown to occur. During EMT, cancer cells lose the
expression of E-cadherin and γ-catenin, proteins that promote cell to cell contact and gain
markers of a mesenchymal, and thus more migratory and invasive phenotype, such as Snail,
vimentin, N-cadherin and fibronetin [91, 92]. Recently it has been shown that EMT can be
inhibited and the modulation of ERα signaling by FOXO3a (a forkhead family transcription
factor) is a key mechanistic driver of this effect. Evidence for this includes the fact that ERα
synthesis can be controled by FOXO3a, [93, 94] and FOXO3a is also greatly reduced in ERα-
negative breast cancers that are driven by HER2 [72, 93]. Importantly, EGCG repressed the
invasive phenotype of breast cancer cell growth that was driven by HER2 [95]. Specifically,
treatment of NF639 breast cancer cells with EGCG (87 µM) increased the protein expression
of E-cadherin, γ-catenin, FOXO3a and ERα and decreased the protein expression of Snail
[95]. The increase in FOXO3a by EGCG promotes ERα signaling and a less malignant
phenotype in breast cancer cells. The phenotype of these breast cancer cells changes to a more
epithelial type partly due to the inhibition of Snail, which represses the expression of E-
cadherin [92]. Further evidence for the role of FOXO3a in this phenotypic change is the fact
that FOXO3a inhibited cell migration, invasiveness in Matrigel as well as TGF-β1 stimulated
invasion [95]. Importantly, ERα signaling was required for this change toward a more
epithelial phenotype. Therefore, EGCG plays an important role in decreasing the invasiveness
of HER2 driven breast cancer cells by upregulating ERα through the stimulation of FOXO3a
gene expression. These studies provide further evidence for the clinical usefulness of EGCG,
310 R. J. Rosengren
as inactive FOXO3a expression was shown to correlate with the poor survival of breast
cancer patients [96].
EGCG AS A NEW CLASS OF CHEMICAL CHAPERONES IN VITRO

Since EGCG has a wide range of effects in a variety of cancer models, new modes of
action have been proposed. One of these is a potential role for EGCG as a new class of
chemical chaperones [97]. This theory was put forward due to the fact that both EGCG and
protein chaperones interact transiently with numerous proteins. For example, both EGCG and
chemical chaperones regulate endoplasmic reticulum stress, as the GADD153 gene is induced
by both stress [98] and EGCG [99, 100]. More evidence for this theory is the structural
similarity between the chemical chaperone trehalose and EGCG, as both have eight hydroxyl
groups. To test this theory, in silico analysis was used to assess the mobility and flexibility of
the galloyl group and the results showed that EGCG existed in variable conformations and
thus could interact with numerous molecular targets [97]. The presence of the galloyl group
also provided more possible conformations compared to the other green tea catechins. These
results support previous surface plasmon resonance studies that demonstrated that the binding
of EGCG to DNA and RNA was reversed [101, 102] in a similar manner to that of chemical
chaperones. While this theory will be strengthened by further tertiary structural analysis of the
EGCG-protein complex, the results to date provide evidence to suggest that EGCG acts as a
new class of chemical chaperones.
IN VIVO EFFECTS AND MOLECULAR MECHANISMS OF EGCG

The majority of studies investigating the effects of green tea constituents in in vivo breast
cancer models have focused on green tea mixtures rather than purified individual catechins.
The earliest studies focused on chemical-induced mammary carcinogenesis in rats and
demonstrated a protective effect of green tea compounds on tumor burden and survival [35,
103-107]. However, it is still unclear whether this protection is greater at the pre- or post-
initiation stage. Various studies using either GTE or purified EGCG have also been conducted
using breast cancer cell xenografts in mice [39, 108, 109]. For example, MDA-MB-231 breast
cancer cell xenografts were used to illustrate that tumor growth, tumor weight and endothelial
vessel density decreased following GTE (1.25 – 2.5 g/l) consumption compared to control
[108]. Delayed tumor growth onset, rate of tumor growth, tumor volume and metastasis was
also shown following the administration of a green tea polyphenol mixture in the drinking
water of BALB/c mice inoculated with 4T1 mouse mammary carcinoma cells [110]. These
effects were associated with an increase in the Bax/Bcl2 ratio and caspase-3 activation,
demonstrating that the induction of apoptosis is a major mechanism for the tumor
suppression.
There have also been studies that focused on the effects of purified EGCG. Liao et al.
(1995) [109] was the first to demonstrate that EGCG (1mg/mouse/day, i.p., 14 days) reduced
tumor size in female athymic nude mice inoculated with MCF-7 cells. More recently,
Thangapazham et al. (2007) [39] conducted a study using female athymic nude mice
inoculated with MDA-MB-231 human breast cancer cells. Mice received 3 mg of green tea
polyphenols (GTP) in the drinking water or EGCG (1 mg) by oral gavage. The treatments
began on the day of cell inoculation and continued for 10 weeks. This treatment protocol
more closely represents chemoprevention because the drug treatment began before palpable
tumors had formed. Additionally at the conclusion of the study the control tumors were one-
third the size (~30 mm3) of tumors from typical xenograft studies that begin when the tumor
volume reaches ~100 mm3. In this model, both EGCG and the green tea mixture suppressed
tumor growth (45 and 61%, respectively) and decreased incidence (10 and 20%, respectively)
and this suggests that EGCG is predominantly responsible for the chemopreventative actions
of green tea. Further examination of tumor sections demonstrated that GTP increased the
number of apoptotic cells by 3.5-fold and EGCG increased apoptotic cells by 2.6-fold [39].
PCNA staining also revealed that proliferation of tumor cells was decreased by both
treatments.
These findings have been supported by other more recent studies that followed a more
standard treatment protocol, as treatment with EGCG (25-50 mg/kg/d, 5-10 weeks) began
once palpable tumors (derived from MDA-MB-231 cell xenografts) had formed [34, 49, 111].
Results in all of these studies showed a modest but statistically significant suppression of ER-
negative breast cancer xenografts following EGCG administration. Mechanisms for this
suppression were determined and the results from both Western blotting and
immunohistochemistry demonstrated that EGCG (25 mg/kg, ip) caused a significant decrease
in Akt, bRaf, VEGF and VEGFR-1 [34, 111]. While no other markers for changes in
apoptosis or angiogenesis were determined in these studies, Akt is critical for breast cancer
cell proliferation [112] and numerous other studies have shown that EGCG inhibits
angiogenesis [71, 72, 81, 82]. Apoptosis was also linked with tumor suppression following 50
mg/kg of EGCG for 31 days, as Bax, cleaved PARP and caspase-3 activity were increased in
tumors from treated mice compared to tumors from control mice [49]. These authors also
demonstrated that chymotrypsin-like activity was decreased and this correlated with a modest
increase in IkBa protein expression. Importantly, this study also showed that apoptosis in
cancer cells could be induced by in vivo inhibition of the chymotrypsin-like proteasome and
these results were further supported by in vitro studies in MDA-MB-231 cells. Overall, recent
studies in ER-breast cancer models have shown that EGCG elicits modest tumor suppression
through an induction of apoptosis and modulation of cell signaling proteins.
IMPROVING THE EFFICACY AND BIOAVAILABILITY OF EGCG

Many of the in vitro effects elicited by EGCG are produced following the use of high
concentrations (~80-150 µM). Due to the instability of EGCG and its extensive first pass
metabolism [113], these concentrations are not achievable in vivo where plasma
concentrations are aproximately 1-10-fold lower [114, 115]. Therefore, clinical trials with
EGCG and GTP have relied on extremely high doses. While patients in these clinical studies
were relatively free from side effects, the sheer volume of the medication required in these
trials was not well tolerated [17, 115] and the maximum tolerated dose of oral, once-daily
GTE was determined to be 3 g/m2 per day [116]. Therefore, stability and bioavailability
problems, which result in the need for very high doses, are significant hurdles that EGCG
312 R. J. Rosengren
must overcome in order to make significant clinical progress. One way to circumvent this
problem would be to provide EGCG as a pro-drug. The first attempts at this involved the
introduction of peracetate groups, in order to protect the reactive hydroxyl groups of EGCG.
In cell free conditions this peracetate-EGCG was efficiently converted back to the parent
compound [117]. Further studies with this compound in MDA-MB-231 breast cancer cells
demonstrated that it was converted to EGCG, which accumulated in 2.4-fold greater amounts
compared to standard EGCG following treatment at 50 µM [49]. In all in vitro assays
examined, peracetate-EGCG was more potent than standard EGCG (namely, chymotrysin-
like activity, PARP cleavage, caspase-3 activity, and protein expression of Bax, p27 and
IkBa) [49]. Importantly, these in vitro results correlated with tumor suppression in vivo.
Specifically, peracetate-EGCG (50 mg/kg/d, 31d, sc) decreased tumor growth by 54%
compared to control, while EGCG decreased tumor growth by 23% [49]. Mechanisms which
were elicited in vitro were also shown to be relevant to tumor suppression as peractetate-
EGCG inhibited tumor proteasome activity in vivo, as IkBa, p27, and Bax proteins were all
accumulated in tumors from peracetate treated mice [49]. This study showed that there are
numerous advantages to administering EGCG in peracetate form. Specifically, peracetate-
EGCG was more stable in neutral and slightly alkaline pH than EGCG and more readily
absorbed into the tumor cells where it was subsequently hydrolyzed back to EGCG. While
these results are promising, peracetate-EGCG was parentally administered at a high dose,
namely 50 mg/kg.
Studies using a more realistic route of administration have also been performed.
Specifically, orally administered O-acyl derivatives of EGCG have also shown anti-tumor
activity in a skin cancer model. The results with these O-acyl derivatives are relevant because
they have improved bioavailability, stability and also convert back to EGCG in vivo [118]. An
important component of this work was that the O-acyl derivatives were synthesized using
green tea leaves as the starting material. Four O-acyl derivatives were synthesized with
increasing acyl side chains and one derivative contained a branched side chain. These
derivatives were administered orally at 50-53 mg/kg for a total of 20 weeks. All of the
derivatives reduced incidence, number of skin tumors and percent survival of the mice [119].
Furthermore, there was a significant decrease in the anti-tumor activity with an increase in the
size and branching of the acyl side chain. One downfall of this study was that EGCG was not
examined alongside the derivatives to demonstrate the increased potency of the derivatives.
However, the study provides important evidence for anti-tumor activity of orally administered
derivatives of EGCG. While these results are even more promising due to the fact that the
derivative was given orally, the compounds need to be examined in breast cancer models in
order to demonstrate efficacy toward this type of cancer.
Other attempts to improve the effectiveness of EGCG have involved the use of
combination therapy. These studies have used both a conventional breast cancer drug
(tamoxifen) and a natural polyphenolic compound (curcumin) in combination with EGCG.
Specifically, The combination of EGCG and curcumin was synergistically cytotoxic toward
ER-negative, but not ER-positive breast cancer cells [34]. These results correlated with a
263±16% increase in the proportion of cells in G2/M phase and a 40±4% decrease in G0/G1
phase cells compared to control following EGCG (20 µM) + curcumin (3 µM) [34]. These in
vitro results translated to in vivo efficacy as EGCG (25 mg/kg, ip) + curcumin (200 mg/kg,
po) significantly decreased tumor volume compared to all other treatment groups and 49%
compared to control in an MDA-MB-231 xenograft model of ER-negative tumorigenesis
[34]. A decrease in angiogenesis was proposed as the major mechanism for this effect as the
protein expression of VEGFR-1 was decreased 78% following treatment with EGCG +
curcumin.
Other combination studies with tamoxifen showed an even greater tumor suppression, as
EGCG (25 mg/kg,ip) + tamoxifen (75 µg/kg, po) decreased tumor volume and tumor weight
71 and 78%, respectively compared to control [111]. The mechanism for this effect is likely
to be initiated via modulation of EGFR activity, as protein levels of the EGFR and its active
phosphorylated form were both significantly reduced (78% compared to control) by EGCG +
tamoxifen treatment. The similar decrease in both the unphosphorylated and phosphorylated
forms indicates that the signaling capacity of the pathway as a whole was reduced by
approximately 78% by the combination treatment [111]. Combination treatment also caused a
similar reduction in the protein expression of mammalian target of rapamycin (mTOR). The
inhibition of mTOR is of particular significance, as this is a key regulator of protein synthesis
within the cell (Figure 1). Furthermore, studies in other cancer models have shown that a dual
suppression of EGFR and mTOR resulted in significant tumor suppression [120].
Importantly, the combination of EGCG and tamoxifen also decreased angiogenesis by
decreasing the protein expression of both VEGF and VEGFR-1 in the tumor, as shown by
both Western blotting and immunohistochemistry [111]. A final component to the mechanism
involved a decrease in the protein expression of cytochrome P5401B1 (CYP1B1), a protein
that plays a variety of roles in both the treatment and development of breast cancer.
Specifically, inhibition of CYP1B1 is critical to breast tumor formation, as CYP1B1 null
mice are protected against DMBA-induced mammary tumors compared to wild-type mice
[121]. Additionally, CYP1B1 can inactivate the cancer dug flutamide [122] and is also
induced in breast cancer cells following treatment with docetaxel [123]. Therefore, it plays an
important role in the development of resistance to chemotherapy. Since there was a 78%
decrease in the tumor protein expression of CYP1B1 following treatment with EGCG +
tamoxifen, this provides another critical component to the mechanism of action of this drug
combination.
An important aspect of this combination therapy is the fact that the doses of both drugs
were much lower than those typically used in similar studies. For example, combination
therapy with a PDK-1/Akt inhibitor has been used to sensitize MDA-MB-231 cells to the
effects of tamoxifen. In this study tamoxifen (60 mg/kg) was unable to suppress the growth of
MDA-MB-231 xenografts, but the addition of OUS-03012 (100 mg/kg) showed significant
tumor suppression of 50% compared to control [124]. This result further demonstrates that
tamoxifen can be used in combination therapies for the treatment of ER-negative breast
cancer. However, it was much more effective when combined with EGCG, as this
combination produced a greater level of tumor suppression (71%) at a much lower dose of
tamoxfien (75 µg/kg) [111]. Overall these combination studies have shown tumor suppression
following the lowest dose of EGCG used to date and thus provide another role for the use of
EGCG in ER-negative breast cancer.
The only research using a drug combination that administered green tea orally was
conducted with GTE and tamoxifen. In this study, GTE (2.5 g/l) was administered in the
drinking water and tamoxifen (20 mg) was inoculated subcutaneously as a slow-release pellet.
Treatment continued for 52 days after the establishment of MCF-7 cell xenografts in mice.
The results showed that combination treatment elicited tumor suppression that was greater
than each individual treatment and was also 81% smaller than tumors from control mice [48].
314 R. J. Rosengren
These results correlated with a significant decrease (46%) in blood vessel density in the
tumors from GTE + tamoxifen treated mice. Additionally, TUNEL analysis revealed a 2.5-
fold increase in the number of apoptotic tumor cells following combination treatment,
compared to control [48]. Importantly, the author also reported a dose-dependent increase in
the amount of three major green tea catechins (EC, ECG, EGCG) in the mammary fat pad of
mice that drank GTE (0.625 – 2.5 g/l) for 4 days. Interestingly, EGCG (2.5 g/l) accumulated
to the highest level of all the green tea catechins (45 ng/g) and this demonstrates that green
tea catechins administered orally can reach the mammary tissue. Demonstrating that the drug
reaches the target tissue is an important component of breast cancer drug design. However
another important aspect will be providing the drug at a dose that is low enough to ensure that
patient compliance remains high. This balance between high oral activity and a low oral dose
is a significant challenge for future research investigating EGCG as a treatment for breast
cancer.
CONCLUSION
EGCG induces apoptosis in ER-positive and ER-negative breast cancer cells in vitro and
the effect is not influenced by the hormone receptor status of the cell line. Many mechanisms
contribute to this effect, which cumulate in the induction of apoptosis and the inhibition of
angiogenesis. Importantly, many of the in vitro effects elicited by EGCG have also been
documented in ER-negative xenograft models following treatment with either EGCG or GTE.
This demonstrates that in vitro studies are useful for dictating / predicting in vivo results with
this multifaceted phytochemical. The latest research with EGCG indicates that there are new
roles for this compound in treatment-resistant breast cancer, including those cancers resistant
to trastuzumab (Herceptin). This new in vitro role for EGCG suggests a promising future for
this drug. Especially since in vivo studies have demonstrated that the efficacy of EGCG can
be increased by synthesizing pro-drug analogs of EGCG as well as through the use of
combination therapy with EGCG. These latest studies provide a basis for the development of
highly active, orally available analogs of EGCG that could, one day, significantly impact on
the lives of women with treatment-resistant breast cancer
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Editor: H. McKinley and M. Jamieson, pp. © 2009 Nova Science Publishers, Inc.
Chapter 16
GREEN TEA CATECHINS IN COLORECTAL CANCER
Seung Joon Baek and Mugdha Sukhthankar

Laboratory of Environmental Carcinogenesis, Department of Pathobiology, College of
Veterinary Medicine, University of Tennessee, Knoxville, TN 37996, USA
ABSTRACT
Colorectal cancer is a global problem that accounts for over 50,000 cancer-related
deaths each year in the United States. Americans have about a one in 20 lifetime risk of
developing colorectal cancer. It affects primarily those over 65, but risk starts increasing
at age 40. Colorectal cancer develops following disruptions in key cancer-causing genes
(oncogenes) like K-ras and β-catenin and tumor suppressor genes like gate keeper APC
and p53, and early detection greatly increases the chances of survival. Most cancers are
related to a combination of hereditary and environmental factors, and such factors can
either contribute to the initiation of cancer or the prevention of tumor development. There
is persuasive epidemiological and experimental evidence that a phytochemical-enriched
diet may be involved in the prevention of colon cancer. Therefore, the use of dietary
compounds for prevention and therapy of colorectal cancer would be of major importance
with potentially fewer side effects than therapeutic drugs. Green tea has received much
attention as a suitable dietary agent because of its anti-tumorigenic activity. The most
active constituents of green tea are catechins, including epigallocatechin 3-gallate
(EGCG), epigallocatechin (EGC), epicatechin-3-gallate (ECG) and epicatechin (EC).
Many laboratories, including ours, have reported preventive effects with green tea
components in cancers of the gastrointestinal tract, lung, skin, prostate, and breast. A
mechanistic study indicated that green tea decreased the total levels of early
carcinogenesis biomarkers and increased tumor suppressor proteins; in addition, reports
related to new molecular targets affected by green tea in chemoprevention study have
been increased. Since the preponderance of the data strongly indicates significant anti-
tumorigenic benefits from green tea polyphenols, this chapter will summarize the current
knowledge of molecular targets of green tea research in human colorectal cancer
prevention.
326 Seung Joon Baek and Mugdha Sukhthankar
1. INTRODUCTION
Cancer, or malignant neoplasia, is an array of diseases wherein a group of cells display
uncontrolled growth and metastasis. Cancer affects all age groups, even fetuses, but risk tends
to increase with age. According to the American Cancer Society, cancer killed 7.6 million
people in the world during 2007 [1; 2]. Cancer is caused by both external and internal factors
that may work together or in a pattern to initiate or promote carcinogenesis. The lifetime risk
(the risk of developing a disease during one’s lifetime or dying of the disease) for developing
cancer in men is 1 in 2, and in women, it is 1 in 3. Cancer is second only to heart disease as
the leading cause of death in the United States. Specifically, colorectal cancer is one of the
most prevalent causes of cancer-related mortality in the western world [3].
The process of cancer development may be divided into at least three stages: initiation,
promotion and progression [4]. Most advanced cancers are incurable, hence it is important to
prolong or block the process of carcinogenesis through chemoprevention, which turns out to
be a feasible strategy for cancer control and management [5]. Thus, further development of
therapeutic and preventive means of controlling this disease are clearly needed, particularly as
they pertain to gastrointestinal cancer [6; 7].
Epidemiological studies have suggested that nutrition plays an important role in
carcinogenesis, and dietary factors have been estimated to account for up to 80% of cancers
of the gastrointestinal tract. Approximately 30% of cancer morbidity and mortality might be
prevented with proper adjustment of diets [8; 9; 10; 11; 12]. The basic theory of
chemoprevention is to reduce the occurrence of cancer either by slowing, blocking, or
reversing the development of the disease by the administration of natural or synthetic
compounds [13]. In addition, the molecular mechanisms responsible for potential human
health benefits derived from dietary components must be studied in vitro and validated in pre-
clinical studies in animal models before strong support can be given for more extensive
clinical trials.
2. COLORECTAL CANCER
Colorectal cancer is the third most common cancer in the United States [14]. It develops
slowly over many years and usually begins as a polyp: a benign growth of tissue that starts in
the lining and grows towards the center of the colon or rectum. Early detection and removal
of polyps may prevent formation of adenocarcinomas, which account for over 95% of
colorectal cancers. Colorectal cancer is caused due to mutations in both tumor suppressors
and oncogenes [15]. Broadly, there are two types of colorectal cancers: hereditary and
sporadic.
2.1. Hereditary Cancers
Familial adenomatous polyposis (FAP) and hereditary non-polyposis colorectal cancer

(HNPCC) are the two best-known hereditary colorectal cancers.
Green Tea Catechins in Colorectal Cancer 327
Familial Adenomatous Polyposis

Familial adenomatous polyposis is very rare. It causes less than 1% of all colorectal
cancer cases. FAP is caused by mutations in the adenomatous polyposis coli (APC) gene, a
tumor suppressor. One of the many roles of APC is regulating cell replication, and hence it
earned the name “GATE KEEPER”. Around five hundred APC mutations have been
identified so far [16]. The polyps themselves are benign but because of their usual high
numbers, some of them might undergo a mutation in the normal copy of the APC gene and
trigger the development of cancer.
Hereditary Non-polyposis Colorectal Cancer

Hereditary non-polyposis colorectal cancer (HNPCC, also known as Lynch syndrome) is
caused by a mutation in one of the DNA mismatch repair (MMR) genes. The hMSH2 and
hMLH1 are most commonly mutated MMR genes in HNPCC, and the other genes involved
are hMSH6, PMS1 and PMS2 [17]. The aberrant operation of DNA mismatch repair systems
lead to Microsatellite instability (MSI) and cause naturally occurring, highly repeated short
DNA sequences, called microsatellites, to get shorter or longer than expected.
2.2. Sporadic Colorectal Cancer
In most people without an inherited mutation predisposing them to colorectal cancer or a

family history of it, a series of mutations is needed for colorectal cancer to develop (Fig. 1). It
often takes many decades for these mutations to accumulate –hence the majority of colorectal
cancer cases occur in the elderly. Mutations in APC are seen in 70 to 80% of sporadic tumors
and often occur early in the development of colorectal cancer. During early stages of
colorectal tumorigenesis, three ras genes are mutated (K-ras, N-ras, and H-ras) [18]. The p53
gene is another tumor suppressor gene that is known to be mutated. The p53-encoding gene
mutations in colorectal cancer occur in specific conserved regions of the gene and might be
present in over 50% of the colorectal cancers [19]. In addition, the DCC (deleted in colorectal
cancer) gene is mutated in 70% of colorectal cancers, and MMR genes are inactivated in
around 15% of sporadic cases.
3. DIFFERENT SIGNALING PATHWAYS INVOLVED

IN COLORECTAL CARCINOGENESIS
Many signaling pathways play a pivotal role in colorectal tumorigenesis. Specifically, the
cyclooxygenases (COX), p53, Wnt, and TGF-β pathways are important in the development of
colorectal tumorigenesis (Fig. 2). COXs, key enzymes responsible for eicosanoid production,
can profoundly influence cancer development, progression and therapeutic response. COX-2
is highly inducible by various cytokines, growth factors, and tumor promoters [20]. Elevated
COX-2 levels are associated with inflammation, neoplastic transformation and metastasis [21;
22], and increased expression of mRNA and protein is found in the great majority of
colorectal cancers [23; 24]. Mutations also commonly occur at the p53 tumor suppressor
protein locus in many forms of cancer, including colorectal cancer. Thus, it is logical when
studying colorectal cancer to consider the p53 mutation and the status of COX-2 expression.
The over-activation of the Wingless/Wnt signaling pathway also leads to the expression of
genes that favor cell growth and thus, contributes to many different cancers including
colorectal cancer. APC and β-catenin are two major genes involved in this pathway [25]. In
normal cells, APC interacts with β-catenin and forms a macromolecular complex with axin
and glycogen synthase kinase-3 β (GSK-3β), in which β-catenin is subsequently directed
towards ubiquitin-proteasomal degradation by phophorylation of GSK-3 β [26; 27; 28]. If this
complex is not formed, then β-catenin increases in cytoplasm and translocates to the nucleus
where it acts on transcription of many downstream genes. The β-catenin expression has been
shown in 90% of the sporadic colorectal cancers and in 65% of HNPCC’s [29; 30].
Transforming growth factor-β (TGF- β) is part of a superfamily of proteins known as the
transforming growth factor β superfamily. TGF- β initiates signaling by formation of
heteromeric complexes of type I and type II serine/threonine kinase receptors [31; 32; 33].
The role of TGF-β in tumorigenesis is quite complex, but it appears that TGF-β functions
both as a tumor suppressor in early tumor onset and as an oncogene during late tumor
progression [34; 35]. TGF- β receptor II somatic mutations are found in patients with
HNPCCs and in cancers that show microsatellite instability [34; 35]. De-regulation of one of
the above pathways lies at the heart of the development and progression of human colorectal
malignancies.
4. HEALTH BENEFITS OF GREEN TEA

Green tea has been known to have many health benefits. It has been shown to decrease
low-density lipoprotein and increase high-density lipoprotein [36; 37]. Subsequently, green
tea has been shown to inhibit hypertension [38] and also to prevent cardiac hypertrophy [39].
Green tea has been used as a diet drink in women as it raises metabolic rates, fastens fat
oxidation and ameliorates insulin sensitivity and glucose tolerance [40]. Green tea can even
lead to the inhibition of HIV virus binding and hence be used as a supportive therapy for HIV
patients [41] because oxalates present in tea help with HIV and general infections by cleaning
up free iron, which leaves one less task for the immune system.
5. GREEN TEA AND CANCER

Abundant experimental and epidemiological evidence accumulated mainly in the past
decade from several centers worldwide provides a convincing argument that polyphenolic
antioxidants present in green tea can reduce cancer risk in a variety of animal tumor bioassay
systems. Tea consumption also assures protection against cancers induced by chemical
carcinogens that involve the lung, forestomach, esophagus, duodenum, pancreas, liver, breast,
colon, and skin in mice, rats, and hamsters [42]. In addition, many epidemiological studies
suggest that green tea consumption shows beneficial effects on many cancers [43].
6. GREEN TEA CATECHINS

Four major polyphenol catechins in green tea include epigallocatechin gallate (EGCG),
epigallocatechin (EGC), epicatechin gallate (ECG) and, epicatechin (EC) (Fig. 3). In
experimental models, catechins show a wide range of protective effects, including
cardioprotective, chemoprotective, and antimicrobial properties. Catechin and epicatechin are
epimers, with (-)-epicatechin and (+)-catechin being the most common optical isomers found
in nature. Catechin derives its name from the plant catechu from where it was first isolated.
Epigallocatechin and gallocatechin contain an additional phenolic hydroxyl group, which
makes them stronger anti-oxidant agents compared to epicatechin and catechin, respectively,
similar to the difference in pyrogallol compared to pyrocatechol. Catechin gallates are gallic
acid esters of the catechins, such as EGCG (epigallocatechin gallate), which is the most
abundant catechin in tea. EGCG is about 25-100 times more potent than Vitamin C in its
antioxidant property [44].
7. GREEN TEA CATECHINS IN CANCER

Green tea catechins have recently gained significant acceptance as cancer preventive
agents. We and numerous other scientists have reported the cancer preventive activities of
EGCG and green tea extract: inhibition of angiogenesis and prevention of cancer metastasis,
induction of apoptosis, inhibition of inflammation, translocation of different genes, cell cycle
regulation, growth prevention of tumors in vivo, inhibiting activation of IGF-1 and alteration
of the expression of various oncogenes and tumor suppressor genes [45; 46; 47]. The role of
green tea catechins in colorectal cancers is more significant as the catechins are absorbed into
the gut [48]; however, their role in other cancers is also important. Recent studies point out
that dietary cancer chemopreventive agents modulate multiple signaling pathways that
interrupt the carcinogenic process and are also capable of extending one or more stages of
carcinogenic process [13; 49].
7.1. Effect on Angiogenesis and Cancer Metastasis
Angiogenesis is a physiological process involving the growth of new blood vessels from
pre-existing vessels in growth and development, as well as in wound healing. It is also an
important step in the progression and conversion of tumors from a dormant state to a
malignant state. Tumors induce blood vessel growth by secreting various growth factors
which can induce capillary growth into the tumor and supply required nutrients allowing for
tumor expansion [50]. There could be either mechanical or chemical stimulation of
angiogenesis. Chemical stimulation, as the name suggests, is performed by various chemical
substances or angiogenic proteins, including several growth factors like VEGF, bFGF, and
TGF-β [51]. In addition, the role of various proteases such as urokinases, MMP, and tissue
inhibitor of matrix metalloproteinases (TIMP) has also been extensively studied in tumor
angiogenesis [52]. These proteins have been linked to be a molecular target of green tea
catechins.
VEGF
The vascular endothelial growth factor (VEGF) belongs to a sub-family of growth
factors; the platelet-derived growth factor family, which are important signaling proteins
involved in both vasculogenesis (the de novo formation of the embryonic circulatory system)
and angiogenesis (the growth of blood vessels from pre-existing vasculature). VEGF has been
known to be a key mediator of the abnormal angiogenesis in many hematologic malignancies
and has been shown to be secreted by diverse types of malignant cells [53; 54; 55]. It is a
small molecule (~45 kDa) with diverse biological activities that include the regulation of
embryonic vascular development, extracellular matrix remodeling, generation of
inflammatory cytokines, and enhancement of vascular permeability, through a VEGF receptor
(VEGF-R) [56; 57]. EGCG inhibits transcriptional regulation of VEGF by decreasing the
transcript levels of VEGF [58]. This effect is mediated by the VEGF promoter region, which
contains several potential binding sites for the transcription factor activator protein-1 [59], c-
fos and c-jun [60]. ERK-1 and 2 have been reported to be important signaling cascades that
lead to over-expression of VEGF mRNA [61]; however, EGCG inhibits kinases that cause
activation of ERK-1 and 2 and hence decreases VEGF [62]. EGCG is also known to chelate
strong metal ions [63] and some receptor kinases dependent on divalent cations for their
activity, and EGCG can also inhibit the activity of receptor kinases by chelating the divalent
ions [64].
Basic Fibroblast Growth Factor

The bFGF protein (FGF-2) is a member of the fibroblast growth factor family [65]. In
normal tissue, basic fibroblast growth factor is present in basement membranes and in the sub
endothelial extracellular matrix of blood vessels. Our recent study in human colorectal cancer
cells with EGCG treatment provided insight on a new mechanism by which EGCG down-
regulates bFGF both in vitro and in vivo [46]. The bFGF protein is degraded in the presence
of EGCG through the ubiquitin/proteasome pathway. The proteasome complex includes three
activities: chymotrypsin-like, trypsin-like and caspase-like [66] and chymotrypsin-like
activity is involved in tumor survival [67]. Interestingly, our results showed that EGCG
decreases the chymotrypsin-like activity and increases the trypsin-like activity of the 20S
proteasome, subsequently degrading bFGF at the post-translational level. EGCG also

increases ubiquitination in a dose-dependent manner [46]. In addition, bFGF suppression by
EGCG is not only seen in culture but also seen in animal studies. We demonstrated the
decrease in tumor volume and number in the APCMin/+ mice fed EGCG in their drinking
water, as assessed by ELISA using the small intestinal tumor tissue lysates [46].
MMP and uPA

MMP-9 is a collagenase involved in extracellular matrix degradation during tumor
metastasis and inflammatory disorders [68]. It has been reported that EGCG inhibits MMP-9
secretion in cancer cells [69; 70]. Similary, uPA is a serine protease also called urokinase
plasminogen activator. It is present in several physiological locations, such as the blood
stream and the extracellular matrix. Plasminogen is the primary physiological substrate of
plasmin. The activation of plasmin leads to activation of a proteolysis cascade leading to
either thrombolysis or extracellular matrix degradation [71]. Swiercz et al. reported that
EGCG inhibits uPA, thereby blocking metastasis and acting as a tumor inhibitor [72].
7.2. Effect on Apoptosis and Cancer Progression
Programmed cell death in multicellular organisms is known as apoptosis. A series of

biochemical events is involved in the process of apoptosis that leads to a variety of
morphological changes, including blebbing, cell membrane asymmetry and attachment, cell
shrinkage, nuclear fragmentation, chromatin condensation, and chromosomal DNA
fragmentation. Apoptosis helps to maintain tissue integrity and function and helps to
eliminate damaged or unwanted cells [73]. EGCG induces apoptosis via different signaling
pathways such as inhibiting NF-κB activation, binding to Fas, activating tumor necrosis
factor-α, arresting cell cycle at G0/G1, and binding to and suppressing anti-apoptotic Bcl-2
family proteins [74; 75; 76]. EGCG inhibits growth of transformed cells, but not normal cells,
primarily via induction of apoptosis [74; 76; 77] and by altering several protein expressions
that are involved in apoptosis pathways.
NAG-1
NAG-1, or non-steroidal anti-inflammatory drug (NSAID)-activated gene is a pro-
apoptotic and anti tumorigenic protein [78]. It is a secreted protein belonging to the TGF-β
superfamily. NAG-1 expression causes the inhibition of cell growth of several epithelial cells
as well as primitive hematopoietic progenitors [79]. NAG-1 is also known as macrophage
inhibitory cytokine-1 (MIC-1) [80], placental transforming growth factor-β [81], prostate-
derived factor [82], growth differentiation factor 15 [83], and placental bone morphogenetic
protein [84]. It has been shown to be highly expressed in mature intestinal epithelial cells;
however, its expression is decreased in human colorectal carcinoma and neoplastic intestinal
polyps of APCMin/+ mice [85]. It has been shown that EGCG or ECG can increase NAG-1
expression in human colorectal cancer cells and that NAG-1 induction is one of the
mechanisms of catechin-induced apoptosis leading to blocking of colorectal tumorigenesis
[45]. In addition, EGCG is known to produce oxidative DNA damage at high concentrations
[86]. This DNA damage can cause induction of p53 and cause apoptosis.
p53
The p53 gene encodes the transcription factor p53, which is a very important regulator of
the cell cycle, plays a crucial role as a tumor suppressor, and hence is involved in preventing
carcinogenesis [87]. The p53 protein can cause repair of damaged DNA by activating DNA
repair proteins, and subsequently it can hold the cell cycle at the G1/S phase if DNA damage
is detected, whereas it can initiate apoptosis if the DNA damage is irreversible. EGCG
induces p53 tumor suppressor protein, followed by the induction of apoptotic related genes
[88]. Amin et al. showed that EGCG can modulate the cancer cell growth pattern in a p53-
dependent manner [89]. In addition, EGCG induces p53 proteins not only in colorectal cancer
cells, but also in other cancer cells [79].
ATF3
Activating transcription factor 3 (ATF3) belongs to the mammalian activation
transcription factor (ATF)/cAMP responsive element-binding (CREB) protein family of
transcription factors. ATF3 is known to be activated by a variety of physiological and
pathological stimuli to the cells [90]. Overexpression of ATF3 in human colorectal cancer
cells lead to apoptosis and cell cycle arrest [91]. The human colorectal cancer cells, HCT-116
(p53 wild type) and SW480 (p53 mutant), treated with 50 µM of EGCG and ECG,
respectively, caused a high induction of ATF3 [92]. This result implied that catechins can
induce ATF3 in a p53-independent manner. Furthermore, it has been shown that catechins
can act as both antioxidant and pro-oxidant agents [93], and that ECG generates oxidative
stress in the media, followed by the induction of ATF3 [92].
EGR-1
Early growth response gene-1 is an inducible zinc finger transcription factor and an
immediate-early gene induced by stress or injury, mitogens, growth factors, cytokines,
hypoxia, and differentiation factors [94]. It is mainly involved in progress of vascular
diseases. EGR-1 is also known to modulate the genes involved in growth control and
survival. Expression of EGR-1 can have a dual effect; it can cause either promotion or
inhibition of cell growth, which depends on cell type and environment. EGR-1 has been also
shown to act as a tumor suppressor gene, and its loss can lead to progression of cancer. Many
researchers have shown that EGR-1 induces apoptosis and is an important factor in neuronal
apoptosis [95]. It exhibits its pro-apoptotic function by directly binding to p53 [96], NAG-
1[97] and PTEN promoters [98]. We have previously shown that EGR-1 phosphorylation
enhances ATF3 expression in colorectal cancer cells [91], and then we demonstrated that
EGR-1 played an important role in ECG-induced ATF3 expression in the human colorectal
cancer cell line HCT-116 [92].
7.3. Effect on Cell Adhesion
Cell or cellular adhesion is an interaction of a cell to another cell or to a surface. This

interaction or adhesion is brought about by special molecules called cell adhesion molecules
(CAM), which interact with other molecules on the other cells or on the other surface. The
CAMs are transmembrane receptors and include molecules like integrins, cadherins and
selectins.
Integrins
Integrins are CAMs that interact with other cells and extracellular matrix and define
cellular shape and mobility while playing an important role in regulating the cell cycle. They
are usually attached to the cellular plasma membrane where they help to transfer a signal
from one cell to another and influence cell growth and differentiation. All these properties
make the integrins an important component in cell migration, invasion, and platelet
interaction, hence its role in tumor growth and metastasis [99]. These integrin molecules
regulate the interaction between tumor cells and other adhesion molecules such as fibronectin,
laminin and collagens [100]. The adhesive interactions between tumor cells and these
molecules lead to tumor growth, invasion and metastasis [101; 102]. EGCG has been shown
to impair the adhesion between tumor cells and proteins such as fibronectin and laminin by
binding to them [103; 104]. Suzuki et al. demonstrated the binding of EGCG to β1 integrin,
and this binding led to disruption of β1 integrin and hence impairment of cell adhesion [105].
EGCG and ECG have also been shown to bind matrix proteins and inhibit smooth muscle cell
adhesion with integrin receptor β1; EGCG can also inhibit laminin-induced smooth muscle
cell migration [106].
Cadherins
Cadherins play a crucial role in cell adhesion and do not allow cancer cells to migrate or
metastasize. However, the perturbation of cadherin function can lead to temporary or
permanent non-attachment of tumor cells and hence promote the invasion and metastasis of
such loose cells. E-cadherin is also known to bind β-catenin, which is an intracellular
anchoring protein. This adherence is important for epithelial cell homeostasis [107; 108]. In
the development of intestinal tumors in APCMin/+ mice, aberrant β-catenin signaling is a key
molecular event [109]. E-cadherin is a tumor and invasion suppressor protein that plays a
crucial suppressive role in the progression from adenoma to carcinoma in colorectal cancers
[110]. EGCG has been shown to increase E-cadherin protein levels in vitro in HT29 cells, and
this is due to translocation of β-catenin from the cytoplasm to the nuclei and then to the
plasma membrane. EGCG also increases E-cadherin protein levels by attenuating
transcriptional repression, slug/snail zinc finger protein family, or by causing
posttranscriptional modification of caveolin-1, a protein that plays an important role in the
endocytosis of E-cadherin [111; 112; 113; 114].
7.4. Effect on Cell Cycle Regulators
Cell cycle is a series of processes taking place in an eukaryotic cell that leads to its
replication. There are two major events involved: interphase, divided into 4 phases, G0, G1, S,
and G2, in which the cell grows and accumulates nutrients, and mitosis wherein the cells
divide into two daughter cells. It is very important for the cell cycle to function properly, and
certain check points are needed in order to detect and repair any kind of genetic damage so
that the cell cycle does not go unchecked leading to uncontrolled growth. The regulatory
molecules governing the ordered processes of cell cycle are cyclins and cyclin dependent
kinases (CDKs). CDKs once activated by cyclins cause phosphorylation of target proteins,
which either activates or inactivates the proteins or causes them to move into the next phase
of the cell cycle.
Cyclin D1
Cyclin D1 is produced in response to growth factors. It is an important cell cycle
regulator that is involved in G1-S transition and in regulation of proliferation and
differentiation [115]. Dysregulated degradation of cyclin D1 is associated with its increased
levels in various cancers [116]. Jeong et al. reported decreased expression of cyclin D1 in
HT-29 human colorectal cancer cell lines by EGCG [117], and this result was confirmed by
other groups, showing that EGCG and ECG decreased the expression of cyclin D1 [118]. In
addition, we have reported that ECG can significantly suppress the expression of cyclin D1 in
oral cancers [119].
GAK
GAK (Cyclin G-associated kinase) is a serine/threonine kinase that is associated with
cyclin G [120]. EGCG has been shown to down regulate the expression of GAK and hence
arrest cell cycle progression from the G1 phase to the S phase by de-activating cyclin G [121].
p21
This regulator belongs to the CIP/KIP family which are inhibitors of cyclin-dependent
kinases [122]. EGCG has been shown to increase the expression of p21 and hence lead to cell
cycle arrest [123].
8. EGCG IN OTHER CANCERS
Breast Cancer
EGCG induces apoptosis and significantly decreases invasion of breast cancer cells, and
induction of apoptosis is by increasing the pro-apoptotic agent BAX and decreasing the anti-
apoptotic agent BCL-2 [124]. EGCG also reduces invasion of cells by 24-28% on matrigel
and decreases the expression of MMP-9 [125]. EGCG has also been shown to decrease the
estrogen receptor α function, which is a pivotal pathway in breast tumorigenesis [126].
Lung Cancer
EGCG has been shown to inhibit lung tumorigenesis in several different animal model
systems. This includes lung tumorigenesis in A/J mice induced by 4-(methylnitrosamino)-1-
(3pyridyl)-1-butanone (NNK), N-nitrosodiethylamine, benzo[a]pyrene, N-nitrosomethylurea,
or cisplatin [127]. EGCG mostly accounts for decreased tumor number, size and incidence.
All these parameters are associated with the anti-proliferative, pro-apoptotic, and anti-
angiogenic activities of EGCG and inhibition of protein kinases involved in signal

transduction and cell cycle regulation.
Prostate Cancer
Prostate cancer is the second leading cause of cancer-related death in men in the United
States. Incorporating chemopreventive agents such as EGCG into the diet can delay the early
onset of prostate cancer. EGCG exerts its chemopreventive effect in the prostate via
regulation of the sex steroid receptor, growth factor-signaling, and inflammatory pathways.
Harper et al. showed that EGCG inhibited early but not late stage prostate cancer. EGCG can
also reduce cell proliferation and induce apoptosis by decreasing androgen receptor, IGF-1,
COX-2 and MAPK signaling [128].
Pancreatic Cancer
Pancreatic cancer can be a deadly disease and lead to higher mortality as it is very
difficult to diagnose. By the time it is diagnosed, it has reached the late stages. EGCG has
been shown to inhibit growth and induce apoptosis in human pancreatic cancer cells. EGCG
can effectively inhibit viability, capillary tube formation and migration of HUVEC on its own
and these effects are enhanced in presence of an ERK inhibitor in vitro. In the case of in vivo
studies, AsPC-1 xenografted tumors treated with EGCG showed significant reduction in
volume, proliferation, angiogenesis, VEGF-R2 and metastasis (MMP-2, MMP-7, MMP-9 and
MMP-12) and induction of apoptosis, caspase-3 activity and growth arrest (p21/WAF1). In
addition, there was a reduced ERK activity and enhanced p38 and JNK activities in tumor
samples from EGCG-treated mice. Hence all these results show that EGCG inhibits
pancreatic cancer growth, invasion, metastasis and angiogenesis and can be used in the
management of pancreatic cancer prevention and treatment [129].
Skin Cancer
Of all the possible causes of skin cancer, exposure to ultra violet radiation is the main
one. Sevin et al. carried out a study in 12-week-old Wistar albino rats that were exposed to
UV light and used ointment containing 2% EGCG. They found that topical application of
EGCG was effective in preventing the skin cancer before UV exposure or had a protective
effect; however, once damage was caused after exposure to UV, EGCG didn’t show any
significant recovery [130]. Another study focused on EGCG-induced caspase-14 expression
in an A431 human epidermoid cancer cell line. Caspase-14 is a member of the caspase family
associated with epithelial cell differentiation, planned cell death, and barrier formation.
Induction of caspase-14 by EGCG led to growth inhibition and reduction in tumorigenicity of
A431 cells [131]. IL-12 is an interleukin known to induce immune responses and anti-
angiogenic activities. Based on these studies, EGCG can induce IL-12 and can prevent photo-
carcinogenesis through an EGCG-induced, IL-12-dependent, DNA repair mechanism [132].
9. SUMMARY
Most of the beneficial effects of green tea are due to the major catechin EGCG, which
accounts for at least 50% of the total catechin content of green tea. EGCG has hydroxyl
groups that make it a potent anti-oxidant agent, and its activity is 25-100% more potent than
vitamin C and E.
Other catechins also play a role in blocking colorectal carcinogenesis. ECG increases
NAG-1 expression and induces apoptosis to prevent carcinogenesis.
Colorectal cancer occurs due to mutations in both tumor suppressors as well as
oncogenes. Different signaling pathways govern colorectal tumorigenesis. As shown in Fig.
4, many researchers have shown that EGCG acts on all these pathways in some way.
EGCG also acts on other cancers such as lung, pancreas, breast, prostate, and skin and
many other cancers.
Green tea and its catechins have emerged up to be very effective dietary agents that are
easily available, non- expensive, and most importantly have no side effects and can prevent
cancer growth and metastasis.
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Chapter 17
INHIBITORY EFFECT OF CATECHIN DERIVATIVES

FROM GREEN TEA ON DNA POLYMERASE ACTIVITY,
HUMAN CANCER CELL GROWTH, AND TPA (12-O-
TETRADECANOYLPHORBOL-13-ACETATE)
-INDUCED INFLAMMATION
Yuko Kumamoto-Yonezawa1, Hiromi Yoshida1,2

and Yoshiyuki Mizushina1,2,*
1
Laboratory of Food & Nutritional Sciences, Department of Nutritional Science, Kobe-
Gakuin University, Nishi-ku, Kobe, Hyogo 651-2180, Japan
2
Cooperative Research Center of Life Sciences, Kobe-Gakuin University, Nishi-ku,
Kobe, Hyogo 651-2180, Japan
ABSTRACT
Green tea obtained from the leaves of the plant, Camellia sinensis, is one of the most
popular beverages in the world. The major polyphenolic compounds in green tea are
catechin derivatives (i.e., flavan-3-ols), and their composition varies depending on the
season of harvest and the manufacturing process. The inhibitory activities against DNA
polymerases (pols) of catechin derivatives such as (+)-catechin (C), (-)-epicatechin (EC),
(-)-gallocatechin (GC), (-)-epigallocatechin (EGC), (+)-catechin gallate (Cg), (-)-
epicatechin gallate (ECg), (-)-gallocatechin gallate (GCg), and (-)-epigallocatechin
gallate (EGCg) were investigated. Among these eight catechins, several inhibited
mammalian pols with EGCg being the strongest inhibitor of pols α and λ with IC50
values of 5.1 and 3.8 μM, respectively. EGCg did not influence the activities of plant
pols, such as pols α and λ, or prokaryotic pols, and had no effect on the activities of DNA
metabolic enzymes such as calf primase of pol α, T7 RNA polymerase, T4
*
Correspondence to: Yoshiyuki Mizushina Ph.D., Laboratory of Food & Nutritional Sciences, Department of
Nutritional Science, Kobe-Gakuin University, Nishi-ku, Kobe, Hyogo 651-2180, Japan; Tel: +81-78-974-1551
(ext.3232); Fax: +81-78-974-5689; E-mail: mizushin@nutr.kobegakuin.ac.jp
348 Yuko Kumamoto-Yonezawa, Hiromi Yoshida and Yoshiyuki Mizushina
polynucleotide kinase, or bovine deoxyribonuclease I. Some tea catechins also

suppressed human cancer cell growth and/or TPA (12-O-tetradecanoylphorbol-13-
acetate)-induced inflammation, and the tendency of the pol inhibitory activity for these
compounds was the same as that of their anti-inflammatory activity rather than their anti-
cancer activity. Based on these results, the relationship between the structure of tea
catechins and their bioactivities is discussed.
Keywords: Green tea; Catechin derivatives; DNA polymerase; enzyme inhibitor; DNA
replication; Cancer cell growth inhibition; Anti-cancer activity; Anti-inflammation
ABBREVIATIONS
pol, DNA polymerase (E.C.2.7.7.7);
C, (+)-catechin;
EC, (-)-epicatechin;
GC, (-)-gallocatechin;
EGC, (-)-epigallocatechin;
Cg, (+)-catechin gallate;
ECg, (-)-epicatechin gallate; GCg, (-)-gallocatechin gallate;
EGCg, (-)-epigallocatechin gallate;
TPA, 12-O-tetradecanoylphorbol-13-acetate.
1. INTRODUCTION
Green tea, a popular beverage, is consumed worldwide. It contains an infusion of the
leaves from Camellia sinensis, which is rich in polyphenolic compounds known as catechins,
especially, (-)-epigallocatechin gallate (EGCg), (-)-epicatechin gallate (ECg), (-)-
epigallocatechin (EGC), and (-)-epicatechin (EC) [1, 2]. The catechin composition of green
tea varies depending on the season of harvest and the manufacturing process. Green tea leaves
usually contain about 1 % EC, 2-3 % EGC, 1-2 % ECg, and 5-8 % EGCg [3]; therefore, the
main constituent of catechin is EGCg.
The human genome encodes 14 pols, which conduct cellular DNA synthesis [4].
Eukaryotic cells reportedly contain three replicative types (pols α, δ and ε, mitochondrial pol
γ, and at least twelve repair types (pols β, δ, ε, ζ, η, θ, ι, κ, λ, μ, and σ, and REV1) [5]. We
have been studying selective inhibitors of each pol from natural materials including foods
such as vegetables for more than 10 years [6-13]. It is considered that food materials
containing pol inhibitory compounds could be used as bioactive functional foods with anti-
cancer, anti-virus, and immuno-suppression activities etc. Polyphenols, such as curcumin [7,
8] and anthocyanin compounds [12], were found to inhibit the activities of mammalian pols.
This review reports the inhibitory effect of catechin derivatives [11], as contained in green
tea, on bioactivities including DNA polymerase inhibition, human cancer cell growth
inhibition, and anti-inflammation, and the relationship of these bioactivities of catechin
derivatives is discussed.
EGCg Is a Potent Inhibitor of Mammalian DNA Polymerase 349
2. EFFECT OF CATECHIN DERIVATIVES ON THE ACTIVITIES OF

MAMMALIAN DNA POLYMERASES
The assay method for pol activity was described previously [15, 16]. The substrates of the
pols were poly(dA)/oligo(dT)12-18 and 2'-deoxythymidine 5'-triphosphate (dTTP) as the
DNA template-primer and nucleotide substrate (i.e., 2'-deoxynucleotide 5'-triphosphate
(dNTP)), respectively.
As described above, during searches for natural inhibitors of mammalian pols it was
found that catechin compounds from the leaves of dried green tea, Camellia sinensis,
inhibited these activities. Eight analytical grade catechin derivatives (i.e., flavan-3-ols) were
purchased commercially, including (+)-catechin (C, compound 1), (-)-epicatechin (EC,
compound 2), (-)-gallocatechin (GC, compound 3), (-)-epigallocatechin (EGC, compound 4),
(+)-catechin gallate (Cg, compound 5), (-)-epicatechin gallate (ECg, compound 6), (-)-
gallocatechin gallate (GCg, compound 7), and (-)-epigallocatechin gallate (EGCg, compound
8), and two part compounds of catechin derivatives as pyrogallol (compound 9) and gallic
acid (compound 10). The chemical structures are shown in Figure 1. This study investigated
whether compounds 1 - 10 inhibited the activities of mammalian pols α, β, and λ.
The relative activity of each pol with a set concentration (100 μM) of the test compounds
is shown in Figure 2. Immuno-affinity purified calf pol α was used as a replicative pol, and E.
coli expressed recombinant mammalian pols β and λ were prepared and used as repair related
pols. Compounds 1 (C) and 2 (EC) did not influence the activities of pols α, β or λ.
Compounds 9 (pyrogallol) and 10 (gallic acid), which are the structural parts of flavan-3-ol,
also did not inhibit these pol activities. Compounds 3 to 8 inhibited the pol activities, with the
effects on pols α and λ being stronger than that on pol β. The compounds that inhibited the
relative activity of pol α by more than 50 % were 3 (GC), 4 (EGC), 5 (Cg), 6 (ECg), 7 (GCg),
and 8 (EGCg). Compound 8 (EGCg) had the strongest inhibitory effect on the three pols of all
the catechin derivatives tested. Therefore, on the properties of EGCg were examined in the
subsequent experiments.
OH
OH
OH
OH
HO O OH
HO O
O C OH
OH OH O
OH OH
Compound 1 (C) Compound 5 (Cg)
Figure 1. (Continued)
OH
OH OH
OH
HO O OH
HO O
O C OH
OH OH O
OH OH
Compound 2 (EC) Compound 6 (ECg)
OH
OH OH
OH
HO O
HO O OH OH
OH
O C OH
OH OH O
OH OH
Compound 3 (GC) Compound 7 (GCg)
OH
OH OH
OH
HO O
HO O OH OH
OH
O C OH
OH OH O
OH OH
Compound 4 (EGC) Compound 8 (EGCg)
OH
OH
HO OH HOOC OH
OH
Compound 9 (Pyrogallol) Compound 10 (Gallic acid)
Figure 1. Structure of catechin derivatives. Compound 1, (+)-catechin (C); compound 2, (-)-epicatechin

(EC); compound 3, (-)-gallocatechin (GC); compound 4, (-)-epigallocatechin (EGC); compound 5, (+)-
catechin gallate (Cg); compound 6, (-)-epicatechin gallate (ECg); compound 7, (-)-gallocatechin gallate
(GCg); compound 8, (-)-epigallocatechin gallate (EGCg); compound 9, pyrogallol; and compound 10,
gallic acid.
Calf polƒ¿
Rat polƒÀ
Human polƒÉ
Compound 1 (C)
Compound 2 (EC)
Compound 3 (GC)
Compound 4 (EGC)
Compound 5 (Cg)
Compound 6 (ECg)
Compound 7 (GCg)
Compound 8 (EGCg)
Compound 9 (Pyrogallol)
Compound 10 (Gallic acid)
0 20 40 60 80 100
DNA polymerase activity (%)
Figure 2. Inhibitory effect of catechin derivatives on the activities of mammalian DNA polymerases.
Compounds 1 to 10 (100 μM each) were incubated with calf pol α, rat pol β and human pol λ (0.05
units each). Percent relative activity is shown. The enzymatic activities were measured as described
previously [15, 16]. Pol activity in the absence of the test compounds was taken as 100%. Data are
shown as the means ± SEM of three independent experiments.
3. EFFECTS OF EGCG ON THE ACTIVITIES OF DNA POLYMERASES

AND OTHER DNA METABOLIC ENZYMES
As shown in Table 1, EGCg inhibited the activities of all the mammalian pols tested, and
calf pol α and human pol λ were strongly inhibited with IC50 values of 5.1 and 3.8 μM,
respectively. The inhibitory effect on pols β and λ was the weakest and strongest,
respectively, of the mammalian pols tested, and the inhibition of pol β was 12.3-fold weaker
than that of pol λ, although the amino acid sequence of pol β is similar to pol λ, and these
pols are both classified as family-X pols [1, 17]. On the other hand, the activity of plant pols
such as cauliflower pol α and rice pol λ, prokaryotic pols such as the Klenow fragment of E.
coli pol I, Taq pol, and T4 pol, and DNA metabolic enzymes such as calf primase of pol α,
T7 RNA polymerase, T4 polynucleotide kinase, and bovine deoxyribonuclease I, were not
influenced by EGCg. EGCg should be classified as an inhibitor of mammalian pols.
4. EFFECT OF CATECHIN DERIVATIVES ON

CULTURED HUMAN CANCER CELL GROWTH
Catechin derivatives are of interest in developing of functional foods for cancer
chemotherapy. Replicative pols are regarded as targets of anti-cancer drugs because they play
central roles in DNA replication, which is indispensable for the proliferation of cancer cells.
Therefore, the growth effects of these compounds on a human cancer cell line (promyelocytic
leukemia cell, HL-60) were investigated in culture using MTT (3-(4,5-dimethylthiazol-2-yl)-
2,5-diphenyl tetrazolium bromide) assay [18].
Table 1. IC50 values of compound 8 (EGCg) on the activities of various DNA

polymerases and other DNA metabolic enzymes
Enzyme IC50 value (μM)

Compound 8 (EGCg)
Mammalian DNA polymerases
Calf DNA polymerase α 5.1 ± 0.3
Rat DNA polymerase β 46.8 ± 1.8
Human DNA polymerase γ 11.0 ± 0.5
Human DNA polymerase δ 17.2 ± 0.9
Human DNA polymerase ε 17.6 ± 0.9
Human DNA polymerase η 11.6 ± 0.6
Human DNA polymerase ι 12.7 ± 0.6
Human DNA polymerase κ 13.5 ± 0.7
Human DNA polymerase λ 3.8 ± 0.2
Plant DNA polymerases
Cauliflower DNA polymerase α >100
Rice DNA polymerase λ >100
Prokaryotic DNA polymerases
E. coli DNA polymerase I (Klenow fragment) >100
Taq DNA polymerase >100
T4 DNA polymerase >100
Other DNA metabolic enzymes
Calf primase of DNA polymerase α >100
T7 RNA polymerase >100
T4 polynucleotide kinase >100
Bovine deoxyribonuclease I >100
The compound was incubated with each enzyme (0.05 units). Enzymatic activity in the absence of the
compound was taken as 100 %.
Compound 1 (C)
Compound 2 (EC)
Compound 3 (GC)
Compound 4 (EGC)
Compound 5 (Cg)
Compound 6 (ECg)
Compound 7 (GCg)
Compound 8 (EGCg)

0 20 40 60 80 100
Rate of cancer cell growth (%)
Figure 3. Effects of catechin derivatives on HL-60 cancer cell growth. The compounds (100 μM each)
were incubated with the human cancer cells (promyelocytic leukemia cell line, HL-60). Percent relative
activity is shown. Cell viability was measured by MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl
tetrazolium bromide) assay [18]. The growth rate of the cancer cells in the absence of the test
compounds was taken as 100 %. Data are shown as the means ± SEM of five independent experiments.
As shown in Figure 3, 100 μM of compound 4 (EGC) had the strongest growth inhibitory
effect on this cancer cell line of the compounds tested, and the compounds that prevented
more than 50 % of cancer cell growth, were 3 (GC), 4 (EGC), 7 (GCg), and 9 (pyrogallol).
Compounds 8 (EGCg) and 10 (gallic acid) moderately suppressed cell growth, although
EGCg was the strongest inhibitor of mammalian pols (Figure 2). On the other hand,
compounds 1 (C), 2 (EC), 5 (Cg), and 6 (ECg) had no effect on cancer cell growth.
5. EFFECT OF CATECHIN DERIVATIVES

ON ANTI-INFLAMMATORY ACTIVITY
Although TPA (12-O-tetradecanoylphorbol-13-acetate) promotes tumor formation [19], it

is also known to cause inflammation and is generally used as an artificial inflammation
inducer for the screening of anti-inflammatory agents [20]. Tumor promoter-induced
inflammation can be distinguished from acute inflammation, which is exudative and
accompanied by fibroblast proliferation and granulation. Using mouse inflammatory tests, the
anti-inflammatory activities of catechin derivatives (compounds 1 – 10) were examined. The
application of TPA (0.5 μg) to a mouse ear induced edema; the weight increase of the ear disk
at 7 hr after application was 241 %. As expected, the inhibitory effect of compound 8 (EGCg)
on inflammation was the strongest among the compounds tested at an applied dose of at least
250 μg, and the level of inhibition was 65.6 % (Figure 4). Compound 7 (GCg) was the second
strongest inhibitor of inflammation with an inhibitory effect of 58.0 %. Compounds 3 (GC)

and 4 (EGC) and compounds 5 (Cg) and 6 (ECg) moderately suppressed inflammation at
levels of 30 to 32 % and 25 to 27 %, respectively. On the other hand, compounds 1 (C) and 2
(EC) and the part compounds of catechin derivatives, compounds 9 (pyrogallol) and 10 (gallic
acid), had little effect on inflammation.
These results suggested that some catechin derivatives caused a remarkable reduction in
TPA-induced inflammation, indicating that EGCg possesses anti-inflammatory activity.
Compound 1 (C)
Compound 2 (EC)
Compound 3 (GC)
Compound 4 (EGC)
Compound 5 (Cg)
Compound 6 (ECg)
Compound 7 (GCg)
Compound 8 (EGCg)

0 10 20 30 40 50 60 70
Anti-inflammatory activity (%)
Figure 4. Anti-inflammatory activity of catechin derivatives toward TPA-induced edema on mouse ear.
Compounds 1 to 10 (250 μg each) were applied individually to one ear on each mouse, and after 30 min
TPA (0.5 μg) was applied to both ears. Edema was evaluated after 7 hr. The inhibitory effect is
expressed as the percentage of edema. Data are shown as the means ± SEM of five independent
experiments.
6. RELATIONSHIP OF THE THREE BIOACTIVITIES

OF CATECHIN DERIVATIVES
To confirm if there is a relationship between mammalian pol inhibition, human cancer

cell (promyelocytic leukemia cell line, HL-60) growth inhibition, and anti-chronic
inflammation, catechin derivatives (compounds 1 - 10) were compared for their inhibitory
effects on these three bioactivities (Figure 5). Pol λ inhibition had the largest correlation
(correlation coefficient = 0.9979) with the anti-inflammatory activity of all combinations of
bioactivities (Figure 5A). On the other hand, neither pol λ inhibitory activity nor anti-
inflammatory activity was related to the inhibition of human cancer cell growth because the
correlation coefficient between these activities and the cell growth inhibitory activity was less
than 0.2 (i.e., 0.1703 in Figure 5B and 0.1851 in Figure 5C, respectively). These results led to
the speculation that TPA-induced inflammation may involve a process requiring pol λ.
R2 = 0.9979
70 8
Anti-inflammatory activity (%) 60

7
50
40
4
3
30 6
5
20
2
1
9
10
10
0
0 20 40 60 80 100
Pol λ inhibitory activity (%)
(A)
R2 = 0.1703
Cancer cell growth inhibitory activity (%)
100
4 7
80 3
9
60
8
40 10
20
1 2 6 5
0
0 20 40 60 80 100
Pol λ inhibitory activity (%)
(B)
Figure 5. (Continued)
R2 = 0.1851
70 8
Anti-inflammatory activity (%)

7
60
50
40
4
3
30 6
5
20
2
10 9
10 1
0
0 20 40 60 80 100
Cancer cell growth inhibitory activity (%) (C)
Figure 5. The relationship between pol λ inhibition, cancer cell growth inhibition, and anti-
inflammation by catechin derivatives. (A) Pol λ inhibitory activity of 100 μM of each compound versus
anti-inflammatory activity of 250 μg of the same compound. (B) Pol λ inhibitory activity of 100 μM of
each compound versus cancer cell growth inhibitory activity of 100 μM of the same compound. (C)
Cancer cell growth inhibitory activity of 100 μM of each compound versus anti-inflammatory activity
of 250 μg of the same compound. Numbers 1 to 10 in the figures indicate compounds 1 to 10,
respectively. The values of correlation coefficients are shown in each figure.
Pol λ is a pol X family polymerase [17]. Although the in vivo biochemical function of pol
λ is unclear as yet, pol λ appears to work in a similar manner to pol β. Pol β, which is widely
known to have roles in the short-patch base excision repair (BER) pathway [21-24], plays an
essential role in neural development [25]. Recently, pol λ was found to contain 5'-
deoxyribose-5-phosphate (dRP) lyase activity, but no apurinic/apyrimidinic (AP) lyase
activity [26] and to be able to substitute pol β in in vitro BER, suggesting that pol λ also
participates in BER. Northern blot analysis indicated that transcripts of pol β were abundantly
expressed in the testis, thymus, and brain in rats [27], but pol λ was efficiently transcribed
mostly in the testis [17]. Bertocci et al. reported that mice in which pol λ was knocked down
were not only viable and fertile but also displayed a normal hyper-mutation pattern [28].
TPA not only causes inflammation, but also influences cell proliferation and has
physiological effects on cells because it is a tumor promoter [29]. Therefore, anti-
inflammatory agents are expected to suppress both mammalian cell proliferation and DNA
replication / repair in nuclei related to the action of TPA. Since pol λ is a repair-related
polymerase [30], the result that the molecular target of catechin derivatives was pol λ is in
good agreement. If so, the pol λ inhibitor could be an inhibitor of chronic inflammation. A
positive relationship between anti-inflammatory and pol λ-inhibitory activities was found,
which may be useful as a new and convenient in vitro assay to screen for novel anti-chronic
inflammatory compounds.
On the other hand, the suppression of human cancer cell growth had almost the same
tendency as the inhibition of mammalian pol α among the compounds (Figure 2 and Figure
3), suggesting that the cause of the cancer cell influence might be through the activity of
replicative pols such as pols α, δ, and ε.
7. STRUCTURE AND ACTIVITY RELATIONSHIP

OF CATECHIN DERIVATIVES
Eight catechin derivatives (compounds 1 – 8) and two part compounds of them

(compounds 9 and 10) were prepared, and compound 8 (EGCg) was the strongest inhibitor of
pol λ and had the strongest anti-inflammation activity of the compounds tested. In the
structure of EGCg (Figure 6), the essential moieties of the structure for these activities were
considered as: <1> the gallic acid moiety, and <2> the hydroxyl group in the pyrogallol
moiety. Compounds 7 (GCg) and 8 (EGCg) contain these moieties; therefore, these
compounds should have the strongest activities of both pol λ inhibition and anti-
inflammation. On the other hand, catechin derivatives lacking moiety <2> (i.e., compounds 1
(C), 2 (EC), 5 (Cg), and 6 (ECg)) had no effect on the growth inhibition of human cancer
cells (promyelocytic leukemia cell line, HL-60), suggesting that this moiety must be essential
for anti-cancer activity.
OH
OH <2>
HO O
OH OH
O C OH
OH O
OH
<1>
Figure 6. Chemical structure of compound 8 (EGCg). Essential groups (i.e., <1> and <2>) for pol λ
inhibitory activity and anti-inflammatory activity in catechin derivatives are shown.
8. DISCUSSION
Previous studies demonstrated that the existence of an effective daily amount of green tea
polyphenols for cancer prevention in humans. Among green tea polyphenols comprising
catechin derivatives, compound 8 (EGCg) has cancer preventive activities in vitro, in cell
culture, and in vivo [31-36]. EGCg is able to inhibit the migration of bronchial tumor cells
and could be an attractive candidate to treat tumor invasion and cell migration [37]. EGCg
can also induce apoptosis and suppress the formation and growth of human cancers including
colorectal cancers (CRC) [38]. Furthermore, green tea polyphenols were shown to affect
several biological pathways. The anti-proliferative action of EGCg on pancreatic carcinoma is
mediated through programmed cell death or apoptosis as evident from nuclear condensation,
caspase-3 activation, and poly-ADP ribose polymerase (PARP) cleavage [39].
The biological effects of green tea and tea polyphenols, including anti-oxidative activity
toward low-density lipoproteins [40], anti-carcinogenic [41], and anti-bacterial actions [42-
44], have been examined extensively both in vitro and in vivo. Catechin derivatives have also
been receiving attention for their protective effects against cardiovascular disease and cancer
[45-49].
The amounts required are assumed to be equivalent to 10 Japanese-size cups of green tea
per day, and it contains 2.5 g green tea extract. Catechin derivatives such as EGCg have a
wide-range of target organs, such as the digestive tract, lungs, liver, pancreas, breast, bladder,
prostate, and skin [1, 2]. The human intake from the above sources would be approximately
0.2 g of EGCg per day, and this concentration in the human body (60 kg) is equivalent to 7.2
μM (3.3 μg/ml, molecular weight of EGCg is 458.37). This dose would be able to
significantly inhibit the activities of mammalian pols such as pol λ in vitro, and might give
the bioactive effects for human health in vivo.
In conclusion, green tea, containing catechin derivative, is a potential functional food for
preventing cancer and inflammation, and tea catechin derivatives, especially EGCg, could be
considered as possible candidates for anti-cancer and anti-inflammation agents.
ACKNOWLEDGEMENTS
We are grateful for the donations of calf pol α, rat pol β, human pol γ, human pols and
ε, human pols η and ι, human pol κ, and human pol λ by Dr. M. Takemura of Tokyo
University of Science (Tokyo, Japan), Dr. A. Matsukage of Japan Women's University
(Tokyo, Japan), Dr. M. Suzuki of Nagoya University (Nagoya, Japan), Dr. K. Sakaguchi of
Tokyo University of Science (Chiba, Japan), Dr. F. Hanaoka and Dr. C. Masutani of Osaka
University (Osaka, Japan), Dr. H. Ohmori of Kyoto University (Kyoto, Japan), and Dr. O.
Koiwai of Tokyo University of Science (Chiba, Japan), respectively.
This work was supported in part by a Grant-in-Aid for Kobe-Gakuin University Joint
Research (A), and the “Academic Frontier” Project for Private Universities: matching fund
subsidy from the Ministry of Education, Science, Sports, and Culture of Japan (MEXT), 2006
- 2010, (H. Y. and Y. M.). Y. M. acknowledges a Grant-in-Aid for Young Scientists (A) (No.
19680031) from MEXT, Grants-in-Aid from the Nakashima Foundation (Japan), Foundation
of Oil & Fat Industry Kaikan (Japan), and The Salt Science Research Foundation, No. 08S3
(Japan).
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Chapter 18
TELOMERASE REGULATION IN
RESPONSE TO GREEN TEA
Huaping Chen1 and Trygve O. Tollefsbol1,2,3,4,*

1
Department of Biology, University of Alabama at Birmingham, AL 35294, USA
2
Center for Aging, University of Alabama at Birmingham, AL 35294, USA
3
Comprehensive Cancer Center, University of Alabama at Birmingham, AL 35294, USA
4
Clinical Nutrition Research Center, University of Alabama at Birmingham,
AL 35294, USA
ABSTRACT
The anti-tumor effect of green tea, especially its major constituent EGCG, has been
demonstrated in several animal experiments and its ability to induce apoptosis of most of
cancer cell lines has been further documented in cell culture models. A number of
mechanisms for how green tea impacts cancer have been proposed. These mechanisms
basically include intervention of cell signal transduction pathways or changes of cell
epigenetic processes. Telomerase has been recognized as a novel target of green tea. This
important enzyme is largely localized to cancer cells, is responsible for the maintenance
of telomeres so that cancer cells can escape the replication problem due to their linear
chromosomes, and it has been shown to be reactivated in almost all tumor tissues.
Telomerase has been found to be inhibited by green tea in telomerase-positive cancer cell
lines. This analysis assesses the progress on research of the mechanisms pertaining to
how telomerase activity is regulated by green tea in cancer cells. A number of
mechanisms for how green tea works through this pathway have been proposed. Since
telomerase has been identified as a potential molecular target for cancer treatment, and
green tea has been shown to inhibit telomerase, clarifying the specific mechanisms for
how green tea functions in this pathway should shed new light on the potential to design
effective and novel preventive or anti-cancer approaches using green tea.
*
Corresponding Author. Department of Biology, 175 Campbell Hall, 1300 University Boulevard, Birmingham, AL
35294-1170, USA. Tel: +1-205-934-4573; Fax: +1-205-975-6097; Email: trygve@uab.edu
364 Huaping Chen and Trygve O. Tollefsbol
Keywords: Green tea, EGCG, Telomerase, Cancer
INTRODUCTION
Cancer still accounts for a high number of deaths worldwide. It is characterized by
unlimited cell division, invasion into adjacent tissues, and may lead to metastasis which can
spread to other tissues in the body through lymph or blood. The occurrence of cancer is a
multistage process which includes initiation, promotion, and progression of carcinogenesis.
Cancers can be caused by aberrant genetic material of the transformed cells. They are formed
due in part to the interaction between genes of humans and environmental carcinogens,
mutation in replication or inherited factors. These abnormalities include mutation of DNA
sequences and epigenetic changes of the genome. The expression levels of two sets of genes
are often changed in cancer. There are oncogenes which are responsible for the division of
cells that have been activated, and tumor suppressor genes which are often inactivated as a
result of disruption in the balance that controls proliferation of cells. Surgery, chemotherapy,
radiation therapy, immunotherapy, and monoclonal antibody therapy have been used for
treating cancer patients depending on the progress of the cancer and the health status of the
patients as well as other factors.
EGCG ECG
EC EGC
Figure 1. Molecular structures of the major polyphenol constituents of tea.

Telomerase Regulation in Response to Green Tea 365
frying or steaming Prevent oxidation of polyphenols Green tea
Fresh Partial oxidation of polyphenols Oolong tea

leaves
fermentation Oxidation of polyphenols Black tea
Figure 2. Processes by which various types of teas are prepared.
Tea is considered to be the most popular beverage worldwide next to water. Its use
originates from China, and it is regularly consumed by a large number of individuals in the
Asian countries. Tea is made from the leaves of Camellia sinensis. Several biologically active
molecules are found in its leaves and the most important group is polyphenols, although other
constituents include caffeine, flavonols such as quercetin and myricetin, and theobromine[1].
The primary polyphenols in tea are: epigallocatechin gallate (EGCG), epicatechin gallate
(ECG), epigallocatechin (EGC), epicatechin (EC), gallocatechin, and catechin (Figure 1).
EGCG is the major and most active constituent of tea. There are currently three major
types of tea: green tea, black tea, and oolong tea. They vary from each other in the procedure
by which they are prepared and correspondingly the constituents they contain. Green tea is
made by frying or steaming the fresh leaves of this plant. The oxidative enzymes in leaves
can be heat-inactivated and thus the polyphenols in leaves can be protected in this way. Black
tea is made by crushing the leaves and then letting the oxidation of polyphenols occur which
is mediated by enzymes contained in the leaves, a process known as fermentation. Oolong tea
is made by partial oxidation of the polyphenols in the leaves, and the ratio of the oxidized
polyphenols in oolong tea lies between green tea and black tea (Figure 2). Thus, the
distinguishing feature of green tea is that it is processed to prevent the oxidation of
polyphenols, while the majority of the polyphenols in black tea have been oxidized into
dimeric theaflavin molecules and polymeric thearubigins during its production, and
theaflavins render black tea’s color and taste.
Since traditional chemotherapy often produces unsatisfactory and toxic effects, non-toxic
or less cytotoxic drugs for cancer prevention or treatment are therefore warranted. The effects
of green tea have been investigated in a wide array of systems varying from animal models to
cell lines, and inhibition of tumors and apoptosis of cancer cells have been observed. A
variety of mechanisms have been proposed based on these studies. Here, we will briefly
evaluate the biological effect of green tea first, and will then focus on the telomerase
inhibition effect of green tea which has been regarded as a potential mechanism that imparts
its chemopreventive and anticancer effects.
BENEFICIAL EFFECT OF GREEN TEA

Epidemiological studies indicate that consuming green tea is associated with a decrease
in the occurrence of cancer in Asian people. In addition, research from animal models shows
that green tea can inhibit tumor formation in a number of tissues such as lung, oral cavity,
esophagus, skin, stomach, liver, prostate, small intestine, colon, breast, and pancreas. Cell
culture experiments also demonstrate that EGCG can inhibit the proliferation of cancer cells.
Here we would like to briefly review those effects.
INHIBITION OF TUMOR GROWTH IN VIVO

AND ANGIOGENESIS INHIBITION
Tea and its constituents have been shown to inhibit tumor formation in different organs in
animal models. The organs affected include but are not limited to lung, oral cavity,
esophagus, skin, stomach, liver, prostate, small intestine, colon, breast, and pancreas [2].
EGCG has been shown to block angiogenesis which is the process involving the
formation of blood vessels. This process therefore deprives the tumor cells of the nutrients
that they need to grow [3, 4]. Oral administration of GTPs (green tea polyphenols) in the
drinking water for mice can inhibit expression of angiogenic factors such as matrix
metalloproteinases (MMPs), and their natural inhibitor, TIMP1, which is upregulated in
response to GTPs [5].
PROLIFERATION INHIBITION AND INDUCTION OF APOPTOSIS OF

CANCER CELLS IN VITRO
Green tea and its major constituent, EGCG, have been shown to inhibit proliferation and
induce apoptosis of a series of tumor cell lines. For instance, It has been shown that the
exposure of human lymphoid leukemia Molt 4B cells to epigallocatechin gallate (EGCG) and
epigallocatechin (EGC) led to both growth inhibition and the induction of programmed cell
death (apoptosis) in a concentration- and time-dependent manner [6]. Green tea extract and
EGCG have also been demonstrated to inhibit growth and induce apoptosis of human
stomach cancer KATO III cells [7].
Another study has found that tea polyphenols (EGCG and EGC) can inhibit growth and
induce apoptosis in human cancer cell lines such as lung tumor cell lines H661 and H1299,
with estimated IC50 values of 22 µM, while the lung cancer cell line, H441, and colon cancer
cell line, HT-29, have weak responses to these polyphenols, with IC50 values 2- to 3-fold
higher [8]. Further work has indicated that tea polyphenol-induced apoptosis and the growth
inhibitory activities may be mediated by hydrogen peroxide generated by polyphenols [8].
EGCG has also been shown to inhibit telomerase in MCF-7 breast cancer cells leading to
suppression of cell viability and induction of apoptosis [9, 10].
OTHER BENEFICIAL EFFECTS

Besides the effects mentioned above, a series of other effects of green tea have also been
reported, including antidiabetes properties, anti-inflammatory [11], anti-arthritic [12, 13],
antibacteria, anti-viral [14, 15] and neuroprotective effects [16]. Due to the limitation of
space, these effects will not be discussed in detail here.
POTENTIAL MECHANISMS PROPOSED

To address those effects that have been observed in vivo and in vitro, a number of
mechanisms pertaining to how EGCG mediates its cancer prevention properties have been
proposed. These include the direct causes and the signal pathways that may be involved. The
direct causes may include anti-oxidatant properties, telomerase inhibition, DNMT1 inhibition,
and inhibition of the ubiquitin-proteasome and topoisomerase I [17]. Signal pathways
involved may include mediation of growth factor-mediated pathways such as those initiated
or carried out by epidermal growth factor receptor (EGFR) [18], the mitogen-activated
protein (MAP) kinase-dependent proteins [19], activator protein 1 (AP-1) [20], nuclear factor-
B (NF-kappaB) [21], matrix metalloproteinases [5] and other potential targets.
ANTI-OXIDATION AND PRO-OXIDANT ACTIVITY

Polyphenols have been shown to have anti-oxidation effects in several in vitro studies.
For example, it has been demonstrated that 40 µmol/L of EGCG can inhibit the production of
hydrogen peroxide in UVB-treated normal human keratinocytes (NHEK) by 66–80% [22].
This effect was correlated with inhibition of UVB-induced phosphorylation of ERK1/2, jun
N-terminal kinase (JNK), and p38. Similar inhibitory activity of EGCG was observed when
hydrogen peroxide was directly added to this cell culture system [22]. However, in vivo
analyses of anti-oxidative effects of green tea on tumor inhibition are very limited in number.
EGCG and theaflavin-3,3’-digallate (TFdiG) have also been shown to inhibit lipid
peroxidation in vitro [23]. Meng Q et al [24] reported that treatment of middle-aged male
Fischer 344 rats with high doses of EGCG resulted in significant decline in the concentration
of hydroxy-2'-deoxyguanosine in the plasma while maintaining a better mitochondrial
potential in the peripheral lymphocytes and preventing the deletion of the ND4 region from
mitochondrial DNA in the liver compared with low dose treatment. EGCG has been recently
shown to modulate the activity of nuclear factor erythroid 2 p45 (NF-E2)-related factor
(Nrf2), which further induces expression of glutathione S-transferase, glutathione peroxidase,
glutamate cysteine ligase, hemeoxygenase-1, and other antioxidant enzymes [25].
On the other hand, polyphenols have been demonstrated to have a pro-oxidant activity.
For instance, the polyphenols may generate superoxide radicals and hydrogen peroxide in cell
culture systems [26]. Interestingly, this is the cause for the instability of EGCG in cell culture
conditions, where its halflife is less than 2 h [27], as the radicals generated due to this activity
may cause an artificial impact on cell growth. Hong et al. [27] showed that administration of
50 µmol/L of EGCG to HT29 cells in McCoy’s 5A medium results in the production of up to
23 µmol/L of hydrogen peroxide. Moreover, adding catalase to the medium before EGCG
treatment can partially or completely block apoptosis in H661 lung cancer cells and Ras-
transformed human bronchial cells [28, 29]. These data indicate that many of the effects
observed in cell culture systems may originate from hydrogen peroxide production secondary
to polyphenol administration. However, since oxygen partial pressure in vivo (<40 mmHg) is
much lower than that in cell culture systems (152 mmHg), and there are many anti-oxidative
enzymes present in tissues, such as superoxide dismutase and glutathione peroxidase, the
probability that pro-oxidant activity of EGCG plays a role in its anticancer mechanism in vivo
is thought to be rather low.
To address issues related to the poor bioavailability of EGCG, peracetate protections
have been introduced into its hydroxyl groups to generate a more stable form which can be
converted into (–)-EGCG under cell-free conditions, and enhanced effects on proteasome
inhibition, growth suppression, and apoptosis induction in the breast cancer cell line MDA-
MB-231 have been observed compared with cells treated with unmodified EGCG [30-32].
However, other modifications of EGCG such as methylation can decrease the occurrence of
apoptosis of cancer cells [33].
RECEPTOR FOR EGCG

A 67-kDa laminin receptor (67LR) has been identified as a cell surface receptor for
EGCG that may allow cancer cells to respond to physiologically relevant concentrations of
EGCG, thereby mediating the anticancer effect of EGCG [34]. Eukaryotic translation
elongation factor 1A (eEF1A) has been further identified as a component responsible for the
anticancer activity of EGCG. By using a direct genetic screen, eEF1A can be up-regulated by
EGCG via 67LR, and eEF1A can induce the activation of myosin phosphatase by
dephosphorylation of myosin phosphatase targeting subunit 1 (MYPT1) which is a subunit of
myosin phosphatase [34]. It is proposed that the activated myosin phosphatase will then
dephosphorylate its substrates such as MRLC, which could induce the rearrangement of actin
cytoskeleton and lead to cell growth inhibition. Evidence also shows that silencing of 67LR,
eEF1A, or MYPT1 in tumor cells abolish the tumor growth inhibition effect of EGCG in vivo
[34-36]. However, due to the limited number of cell lines that has been tested in this manner,
whether or not this is a common mechanism that accounts for the anticancer effect of green
tea awaits follow up and further investigation. And it remains unresolved whether 67LR can
mediate all the effects of EGCG on cancer cells.
Moreover, caution should be taken when mechanisms pertaining to anti-cancer effects are
analyzed, since the concentration of EGCG administered in most of these in vitro studies are
impossible to be attained in humans consuming green tea, and the instability of its major
constituent, EGCG, may worsen this situation leading to artificial results. These possible
mechanisms do provide potential explanations on how green tea functions in humans,
however, they need to be further verified and corroborated.
THE SIGNIFICANCE OF CANCER PREVENTION

TARGETING TELOMERASE
Another mechanism that has been proposed for the cancer prevention effect of EGCG is
based on telomerase inhibition. Here we would like to first review some background
information about telomerase.
TELOMERE ATTRITION EFFECT

All of the ends of eukaryotic chromosomes are characterized by ribonucleoprotein
structures referred to as telomeres. A telomeric region consists of DNA sequence and many
telomere-related proteins. Some of these proteins are found exclusively in telomeres, whereas
others can be found in other sites of cells [37]. The DNA sequence in human telomeres is
composed of repeats of 5’-TTAGGG-3’, and they can form a structure called G-quadraplex.
There is a single-strand G-rich overhang of between 50 and 300 nucleotides located at the 3’
end of telomeric DNA that can fold back onto duplex telomeric DNA to form a “T-loop”
structure [38]. Telomeres have been proven to play an important role in maintaining the
stability of the whole genome and protecting chromosomes from end fusions [39]. Due to the
inability of polymerases to fully replicate the end of a duplex DNA (3’ end replication
problem of linear chromosome), a cell would lose 50-70 base pairs of its telomere sequence
with each cell division. The more times the cell divides, the shorter the telomeres become and
once the telomeres have been attenuated, DNA damage signals are generated and cell
senescence and apoptosis ensue [40].
To compensate for the loss of DNA sequence in each cell division, organisms have
developed at least two different mechanisms to circumvent this problem, one is telomerase-
dependent and the other one is telomerase-independent, which is referred to as alternative
lengthening of telomeres (ALT). Telomerase is an enzyme that can add telomeric
(TTAGGG)n hexamer nucleotide repeats to the 5’ ends of telomeric DNA to maintain the
necessary telomere length. This important enzyme has three components: telomerase reverse
transcriptase (TERT), the RNA component of telomerase (TERC), and the TERC-binding
protein dyskerin [41, 42]. TERT is the catalytic component of this enzyme, it has reverse
transcription activity, and its expression is tightly regulated. TERC functions as the template
for TERT. Although it has been recently shown that TERC expression also contributes its
share to regulate telomerase activity [43, 44], the activity of telomerase is mainly regulated by
the expression of TERT due to the tighter regulation of its transcription.
Several detailed mechanisms have been proposed pertaining to how telomerase inhibition
affects cellular functions. One theory is that once telomeres have been attenuated to a certain
length, the structure of telomeres will be changed, and a DNA damage signal will be
generated [40]. Cell lines will experience senescence thereafter, and research shows that
significant shortening of just one telomere can induce senescence of cells, while another
theory is based on telomerase’s extra-telomere effects [45, 46].
EXTRA-TELOMERE EFFECT
Besides the classic role of maintaining the minimal length of telomeres, telomerase has
been proposed to have other effects such as promoting the transformation of cells. To verify
whether telomerase has an extra-telomere effect, Stewart et al [45, 46] introduced telomerase
into the immortal ALT cell line, GM847, which is a telomerase-negative cell line. They found
that this imparts a tumorigenic phenotype to this cell line, while expression of oncogenic H-
Ras cannot lead to transformation which further indicates the potential central role of hTERT
in tumorigenesis. Additional investigation consisted of expressing a mutated telomerase that
cannot exert its telomere-maintaining role in this cell line which also resulted in
transformation, thus providing direct evidence of the extra-telomeric effect of telomerase [45,
46]. This effect can also impact many genes involved in proliferation, and the anticancer
effects via inhibition of telomerase can in some cases precede telomere shortening [47, 48].
Knocking out the expression of hTERT accompanies growth inhibition of MDA-MB-157
human mammary cancer cells, human ovarian cancer SKOV-3 cells, and human embryonic
kidney (HEK 293) cells [49]. p53 and p21 have been shown to involved in this process [49].
DIFFERENT EXPRESSION LEVEL OF TELOMERASE

IN SOMATIC AND CANCER CELLS
Telomerase is expressed in embryonic cells and adult male germline cells [50], but
exhibits barely detectable telomerase activity in normal somatic cells with the exception of
proliferating cells in tissues undergoing renewal [50-55]. In normal somatic cells, telomeric
DNA sequence will shorten after each cell division until telomere length reaches a critically
short threshold, and cell senescence will follow in response to the signal generated in the
cascade. In contrast to normal cells, telomerase activity has been detected in the early stages
of most common cancers and the higher the activity of telomerase, the poorer the prognosis of
cancer [56]. Ablation of telomerase expression leads to telomeric attrition and growth
inhibition of neoplastic cells [57, 58]. About 90% of cancer cells have been shown to have
elevated telomerase activity and these cells have no net loss of average telomere length [59].
Normal human cells are not able to undergo neoplastic transformation in the absence of
telomerase activity [60] indicating that compounds that suppress telomerase have significant
potential in cancer prevention.
Collectively, these lines of evidence have led many to the opinion that novel approaches
targeted to telomerase would be an excellent approach for cancer prevention or therapy.
Owing to its relative universality compared with other targets for cancer therapy as well as
the dependence of cancer cells on telomerase and its specificity for cancer cells, telomerase
has been considered as a promising target for treatment of cancer. This area of research has
been exponentially expanding in recent years [61-63].
Therapies targeting telomeres have also been developed in a number of laboratories.
However, considering that many population doublings may be required for cancer cells to
exhaust their telomeres through telomerase inhibition, the opportunities that drug-resistant
cancer variants may be generated are high, while telomerase’s extra-telomeric effect has also
shed a new light on strategies that targeted telomerase inhibition in chemoprevention. As a
pleiotropic molecule, EGCG can also function on multi-targets in cancer cell lines, thus
making it a promising drug against cancer.
TELOMERASE INHIBITION BY GREEN TEA

Green tea had been shown to have growth inhibition effects on all kinds of tumors and
cancer cells and several mechanisms have been proposed to address how green tea works in
this manner as discussed above. Here we would like to focus on the telomerase inhibition
effect of green tea. It has been shown that 1 µmol EGCG can induce 50% inhibition (IC50) of
telomerase in a cell free system, and that the telomerase inhibition effect of EGCG in U937
and HT29 cells can occur by treating these cells with 15 µmol EGCG for 24 h [64].
Prolonged passage of these two cell lines in the presence of nontoxic concentrations could
reduce telomere length and induce the cell lines to enter into crisis and senescence [64].
The synthesized compounds MST-312, MST-295, and MST-199, based on the structure
of EGCG, have been shown to cause human monoblastoid leukemia U937 cells to undergo
progressive telomere shortening and eventual reduction of growth rate accompanied by
induction of the senescence-associated β-galactosidase activity with a nontoxic dose. The
telomere shortening effect of MST-312 is strongest, being effective at only 1–2 µmol, which
was 15- to 20-fold lower than that of EGCG in this system [65].
EGCG and other selected polyphenols have been shown to undergo structural
rearrangements at physiologically permissible conditions that result in remarkably increased
telomerase inhibition. For instance, briefly incubating EGCG in neutral or slightly alkaline
medium or human or mouse plasma (pH 6.8) at 37° can lead to rapid chemical degradation of
the parent EGCG compound which is accompanied by a dramatic enhancement in telomerase
inhibition ( 20-fold), and the same effect is observed when other major plant polyphenols are
administered to incubate with medium [66]. It is interesting that 5 µmol EGCG can induce
marked shortening of telomeres in telomerase-positive U937 leukemia cells and eventually
lead to cellular senescence while it has no effect on the telomerase-negative Saos-2
osteosarcoma cells or normal human foreskin fibroblasts and two immortal cell lines also
with negative- and positive- telomerase activity even at 10 µmol in long-term treatment [66].
Studies from animal models have also shown the pivotal position of telomerase inhibition
by EGCG in cancer prevention. Nude mice bearing telomerase-dependent xenograft tumors
responded to prolonged oral administration of EGCG, while it has no effect on those bearing
telomerase-independent xenograft tumors cloned from a single human cancer progeny [67].
This serves as an encouraging example that EGCG and likely other structurally-related
dietary polyphenols may act as prodrug-like molecules that undergo structural changes and
further lead to inhibition of telomerase [67]. EGCG has also been shown to inhibit telomerase
and induce apoptosis in drug-resistant lung cancer cells [68]
POTENTIAL MECHANISMS THAT MEDIATE

TELOMERASE INHIBITION BY GREEN TEA
Telomerase can be regulated at different levels through multiple pathways. This includes
regulation at both the transcription level and post-transcription level. Studies show that
telomerase activity requires molecular chaperones for appropriate assembly of the
holoenzyme [69, 70]. Telomerase activity is also regulated positively by phosphorylation of
hTERT by protein kinase C and negatively by dephosphorylation by protein phosphatase 2A
[71, 72]. It has also been reported that telomerase can be regulated by its translocation from
the cytoplasm to the nucleus [73-75], and alternate splicing also plays a role in the regulation
process, where the hTERT α splice variant has been recognized as a dominant negative
inhibitor of telomerase activity [76].
Although expression level of hTERC has been indicated to be also tightly regulated,
previous research showed that copy number of the TERT and TERC telomerase subunit genes
were increased in cancer cells, and the growth advantage of the cells with more copies of
TERT and TERC have been indicated [77]. However, the expression level of hTERT is still a
major rate-limiting factor that regulates telomerase in comparison with other factors.
The hTERT gene is located at human chromosome band 5p15.33. There are several
transcriptional factor binding elements in the promoter region of hTERT, such as two
canonical E-box (CACGTG). The expression of hTERT has been shown to be tightly
regulated by various cellular factors, which can be divided into positive regulators and
negative regulators. Positive transcriptional factors include c-Myc [78], negative regulators
include the c-Myc antagonist, Mad [79], while some transcriptional factors are still
controversial such as E2F1 [80]. Several CpG islands have also been found in its promoter
region which suggests the potential epigenetic regulation mechanism involved in this process
[81].
POTENTIAL MECHANISM BEING PROPOSED
Covalent Modification of Telomerase
EGCG has been suggested to decompose to form a galloyl radical in cell-free system at
neutral pH, and the galloyl radical can covalently modify telomerase, thereby inhibiting its
activity [64], The concentration of EGCG required for this effect varies from high nanomolar
to low micromolar levels [64], and this is connected with the pro-oxidative activity of EGCG
which has already been discussed above. However, whether this mechanism functions in vivo
remains to be investigated.
Epigenetic Regulation of Telomerase Expression
The activity of telomerase has been shown to be regulated at the transcription level in
cancer cells [9, 10], and epigenetic regulation has been indicated as an important mechanism
controling gene expression in the transcriptional level. These mechanisms of epigenetic
regulation include methylation of CpG islands, histone modification and noncoding RNA.
Epigenetic regulation of transcription of hTERT has been documemted. Increasingly,
evidence has been put forward indicating that epigenetic mechanisms are also involved in the
interaction between green tea treatment and the expression of hTERT [10].
Methylation Mechanism
The methylation mechanism involves the methylation of CpG islands such that
methylation-sensitive transcription factors (methyl CpG binding proteins, MeCPs) may bind,
leading to transcriptional repression. Previous work also revealed that hypermethylation of
the hTERT CpG island accompanies its abnormal expression in cancer cells [82]. A DNA
demethylating agent, 5-aza-2-deoxycytidine (5-AZC), has been shown to induce
demethylation of the hTERT promoter and expression of the hTERT transcript in normal and
transformed cells [81, 83], while CTCF has been shown to bind to the demethylated region
and decrease the expression of hTERT [84]. Zinn et al [85] reported that using methylation-
specific PCR and bisulfite sequencing of the hTERT promoter in breast, lung, and colon
cancer cells, all cancer cell lines retain alleles with little or no methylation around the
transcription start site despite being densely methylated in a region 600 bp upstream of the
transcription start site. Both active chromatin and inactive chromatin marks have been found
in the promoter region of hTERT, indicating that active chromatin marks are associated with
unmethylated DNA while inactive chromatin marks are associated with methylated DNA in
the region around the transcription start site [85].
Other epigenetic mechanisms?

EGCG
hypomethylation
CpG island
DNMT hTERT
E2F1
hypoacetylation
at H3 lys9
E2F1
Histon
CpG island
hTERT promoter
Figure 3. Epigenetic mechanism by which the expression of hTERT is regulated in MCF-7 cells. EGCG
can bind to DNMT1 and disrupt its function, thereby causing hypomethylation of the hTERT promoter.
E2F1, which can only bind if its recognition sequence is unmethylated, would then bind to the
promoter, and transcription of hTERT would be suppressed. EGCG can also cause hypoacetylation of
the hTERT promoter at H3 lys9, which may contribute to silencing of hTERT. Other epigenetic
mechanisms remain to be investigated.
EGCG can bind to DNMT1 [86], which can disturb its function of methylation, thereby
decreasing the methylation level of the genome, including the promoter region of hTERT [10]
(Figure 3).
Investigation from our laboratory indicated that EGCG treatment can lead to
hypomethylation of the hTERT promoter region in MCF7 breast cancer cells [10]. An
increase of binding of E2F1 to the hTERT promoter has also been identified which may
suppress the transcription of the hTERT gene, and hypoacetylation at H3 lys9 has been
observed in the hTERT promoter region in response to EGCG which is connected with
inactivation of gene expression [10]. While methylation of CpG islands can lead to
transcriptional repression directly, it can also regulate chromatin structure by interacting with
other proteins [87], and modification of nucleosomal histones can also play a role in the
regulation of hTERT expression.
Histone Modification Mechanism
Histone modification has been indicated as another epigenetic mechanism that can
regulate gene expression. Adjustment of the higher structure of chromatin accompanies
activation or inactivation of genes. For example, acetyl-H3K9 and dimethyl-H3K4
accompanies gene expression, while trimethyl-H3K9 and trimethyl-H3K27 accompanies gene
inactivation [85].
As mentioned above, hypoacetylation at H3 lys9 occurs in the hTERT promoter when
EGCG is administrated to MCF-7 cells [10]. Further epigenetic analysis of cancer cells during
treatment of EGCG is currently ongoing in our laboratory. These studies will help to elucidate
how dietary green tea imparts its chemopreventive and anticancer effects and may lead to
novel approaches to cancer prevention through control of not only of dietary factors, but also
through epigenetic processes.
Relationship between Telomerase Inhibition and other Mechanisms
Several mechanisms have been proposed to explain the growth inhibition effect and
induction of apoptosis on cancer cells by green tea, however, each of these mechanisms has
their own system. For those different systems, investigators’ methods may also favor those
responses that they were ready for while omitting other possible mechanisms. Which
response is the primary response of cancer cells to polyphenols, and which is the subsequent
event that follows the first response has not yet been resolved. It is also possible that those
mechanisms may work together to cause arrest and apoptosis of cancer cells.
CONCLUSION
In comparision to other treatments targeting telomerase, investigations have shown us the
merits of green tea, such as doing no harm to normal cells, and its constituents have a variety
of beneficial effects which enhance the health of individuals. However, over consumption of
green tea may cause potential harmful effects in humans [88], such as nervousness, sleep
disorders, vomiting, headaches, epigastric pain, tachycardia caused by its caffeine content,
accumulation of too much aluminum which may cause neurodegenerative disorders [89] and
inhibition of iron bioavailability [90]. Thus, the design of drugs that possess the beneficial
effects without any harmful effects is urgent and studies on the functional mechanisms of
green tea are showing promise in this regard.
A number of mechanisms have been suggested based on previous studies of green tea and
its major constituent, EGCG, and those results provide useful insightful into understanding
how green tea may function to prevent cancer. However, current knowledge on the detailed
mechanisms through which green tea exerts its anticancer effect is still fragmentary. Results
from different studies are hard to compare due to different systems that have been used. Some
common problems with these studies is that often the concentrations of EGCG that have been
used are too high, and another issue is the limited bioavailability of EGCG since it is reported
that EGCG degrades quickly in media. It is not fully clear whether the responses observed in
cell culture systems would also occur in animals or humans following oral administration
[91]. Corresponding detailed metabolism and kinetics analysis of green tea and EGCG in each
cell culture systems as well as in vivo studies are therefore warranted, which may help us to
understand the mechanisms more clearly, and the difference between cell culture systems and
what occurs in vivo. For example, artificial results may be produced since hydrogen peroxide
may be generated when EGCG is administrated to cell culture medium; therefore future
experiments need to incorporate this information to exclude responses potentially caused by
hydrogen peroxide. Another topic worth our attention is that all the mechanisms that have
been proposed in cell culture systems should be reproduced in animal models and also
eventually in humans. Animal models and analysis of in vivo studies cannot be omitted [92],
since ultimately those responses observed in humans after consuming green tea are most
relevant to the cancer prevention effect of this beverage. Findings from animal models should
be analyzed with caution when incorporated into information from human studies due to the
discrepancies that exist in metabolism and signaling networks that can occur between humans
and other animals. Due to the huge difference between monolayer cultures of human tumor
cell lines or human xenograft tumors and tumors that exist in patients, new experimental
models urgently await to be developed [93]. In this regard, complex three-dimensional culture
systems have attracted much attention of the scientific community [94-96]. Moreover, for
different cell lines that have been tested, their genetic background should be taken into
account when we analyze the data in each treatment. Also, different cell lines have their own
metabolic patterns.
Telomerase inhibition by green tea has been regarded as an important mechanism through
which the anticancer effect of green tea works. However, as we had discussed above, the
logic linkage between telomerase inhibition and other mechanisms is still controversial,
which necessitates further investigation. To clarify the role that telomerase inhibition
conferred by green tea plays in chemoprevention, more cell lines need to be tested, and
strategies that can differentiate between the telomerase inhibition effect and other
mechanisms should be adopted. Considering that telomerase is a complex protein with several
components, additional potential mechanisms that target other components of telomerase
besides hTERT also warrant further investigation.
ACKNOWLEDGEMENT
This work was supported by a grant from the National Cancer Institute (R01 CA129415).
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Chapter 19
GREEN TEA AND CHRONIC OBSTRUCTIVE

PULMONARY DISEASE:
A CASE-CONTROL STUDY IN JAPAN
Fumi Hirayama and Andy H. Lee

School of Public Health and National Drug Research Institute, Curtin University of
Technology, Perth, WA, Australia
ABSTRACT
Background: Chronic obstructive pulmonary disease (COPD) is a common cause of
morbidity and leading cause of death in the world. Cigarette smoking has been
established as the principal risk factor for COPD. While 95% of COPD patients are, or
have been, cigarette smokers, only 20% of smokers develop COPD. Therefore, other
factors may protect against or contribute to the development of the disease.
Objective: To investigate whether green tea intake can reduce the risk of COPD.
Study design: Case-control study conducted in Aichi, Gifu and Kyoto during 2006.
Subjects: A total of 278 eligible patients (244 men and 34 women; mean age 66.5
(SD 6.7) years), with COPD diagnosed by respiratory physicians as the primary
functionally limiting illness within the past four years, were referred from the outpatient
departments of six hospitals in central Japan. During the same period, 335 age-matched
community-dwelling controls (267 men and 68 women; mean age 65.3 (SD 5.5) years)
were recruited from the same catchment areas as the cases.
Methods: Interviews were conducted face-to-face to collect information on
demographic characteristics and habitual green tea consumption using a structured
questionnaire. The reference recall period was set at 5 years before diagnosis for cases or
5 years before interview for controls. Tea drinkers were defined as persons who drunk
both Sencha, Bancha, Hojicha and Genmaicha types of green tea once a week or more
often. The effect of green tea intake on the COPD risk was assessed by multivariate
logistic regression analysis.
Results: The prevalence of regular green tea consumption was significantly higher (p
< 0.01) among controls (n = 64, 19.3%) than cases (n = 27, 9.7%). Among drinkers, life-
long exposure (years of drinking) was similar between the two groups (p = 0.70), and
about half of them drank one to three cups of green tea daily. The risk of COPD appeared
384 Fumi Hirayama and Andy H. Lee
to decrease with green tea drinking. The adjusted odds ratio was 0.45 (95% confidence
interval 0.25-0.81) for tea drinkers relative to non-drinkers after accounting for
confounding factors including age, body mass index, gender, marital status, residential
location, education level, retirement status, smoking status and alcohol consumption.
Conclusion: The preliminary evidence suggests that green tea intake may be
protective against COPD for Japanese adults. More research is required to confirm the
observed finding and to understand the biological mechanism underlying the disease
prevention role of green tea.
INTRODUCTION
Chronic Obstructive Pulmonary Disease
Chronic obstructive pulmonary disease (COPD) is the fifth most common cause of
morbidity and sixth leading cause of death in the world (Murray & Lopez 1997). It is likely to
be the third most frequent cause of death by 2020, just behind coronary and cerebrovascular
diseases (Murray & Lopez 1997). COPD is characterized by airflow limitation that is not
fully reversible, with chronic cough, sputum production, and breathlessness on exertion being
common symptoms. The disease results from the interaction of host factors and
environmental exposures (McKenzie et al. 2003; Celli & MacNee 2004; Pauwels & Rabe
2004). Host factors include genetic predisposition and airway hyper-responsiveness, while
environmental exposures include polluted air, inhaled particles and gases. Cigarette smoking
has been established as the principal cause of COPD. Although 95% of COPD patients are, or
have been, cigarette smokers, only 20% of smokers develop COPD (Madison & Irwin 1998).
Therefore, other factors may protect against or contribute to the development of the disease.
Because of the high burden and societal cost associated with COPD, new methods of
prevention are important. It is possible to reduce the incidence of COPD through an
appropriate diet (Sridhar 1995).
Green Tea
Tea is the second most common beverage in the world, just behind water (Peters, Poole
& Arab 2001). Of all tea production and consumption worldwide, 78% is black tea, 20% is
green tea and 2% is oolong tea (Jankun et al. 1997). Japanese consumed 138,000 tonnes of tea
per year in 2006, of which 73% are green tea (Iruma City Museum 2008). The common
method of preparation is to brew dry tea leaves in a teapot using hot water without milk and
sugar (Ministry of Agriculture Forestry and Fisheries 2005). Teas are made from a leaf
extract of the plant Camellia sinensis and classified into three main types, depending on the
manufacturing process. Green tea is non-fermented, oolong tea is semi-fermented, while
black tea is fermented (Regional Agricultural Administration Office 2007). The common
kinds of green tea in Japan are Sencha, Bancha, Hojicha, and Genmaicha. Sencha is harvested
in early spring and makes up 75% of all tea production in Japan. It is initially steamed for 15
to 45 seconds, knead, and then roasted (Regional Agricultural Administration Office 2007).
Bancha has lower quality and more coarse than Sencha. It is harvested from the second flush
Green Tea and Chronic Obstructive Pulmonary Disease 385
of Sencha leaf between summer and autumn, and is made from the same process as Sencha.
Both Hojicha and Genmaicha mix Sencha and Bancha tea leaves and are roasted in high
temperature, but Genmaicha also combines with roasted brown rice.
Green tea is rich in catechins, such as (-)-epigallocatechin-3-gallate (EGCG) and (-)-
epicatechin gallate (ECG), a subclass of flavonoids (Cabrera, Gimenez & Lopez 2003). The
level of catechins in green tea, which ranges from 51.5 to 84.3 mg/g, is higher than those of
black tea (5.6-47.5 mg/g) and fruit tea (8.5-13.9 mg/g) (Khokhar & Magnusdottir 2002). It
has been noted that catechins, especially EGCG, may prevent the formation of a mutated cell
(Liang et al. 2007).
Green Tea and Disease Prevention
Green tea has been shown to reduce the risk of several diseases (Crespy & Williamson
2004) including cognitive impairment (Kuriyama et al. 2006), stroke (Fraser, Mok & Lee
2007), prostate cancer (Jian et al. 2004), ovarian cancer (Zhang et al. 2004), lung cancer
(Liang et al. 2007), and can decrease mortality of cardiovascular diseases (Kuriyama et al.
2006). A review of 78 relevant articles on lung cancer concluded that high level of green tea
intake over a long period may be protective against tobacco carcinogens (Liang et al. 2007).
However, only two studies examined the association between tea consumption and
COPD. A Turkish case-control study of 40 male smokers with COPD (mean tea intake 7
glass/day) and 36 male non-smokers without COPD (mean tea intake 16 glass/day) reported
that black tea drinking was protective against COPD among smokers, with odds ratio (OR)
0.90, 95% confidence interval (CI) 0.82-0.98 (p < 0.001). The study recommended 6 to 10
cups (960-1600 ml) of tea drinking per day as a useful dietary habit (Celik & Topcu 2006).
On the other hand, a cross-sectional study of 13651 adults in the Netherlands showed no
association between tea consumption and the prevalence of COPD symptoms (Tabak et al.
2001), though the risk of COPD was not evaluated. More research is needed to ascertain the
effect of tea in view of the limited studies in the literature. Therefore, this study aims to
investigate whether green tea intake can reduce the risk of COPD for Japanese adults.
METHODS
Study Design and Subjects
A case-control study was conducted in central Japan in 2006. Three hundred COPD
patients diagnosed by respiratory physicians were recruited from the outpatient departments
of six hospitals in Aichi, Gifu and Kyoto. Patients were included in this case-control study
provided that they (i) were aged between 50 and 75 years; and (ii) had COPD as the primary
functionally limiting illness which was diagnosed within the past four years. Subjects were
excluded if they had a recent stroke, dementia or other health conditions that prohibited them
from answering the questions. Twenty-two eligible patients were subsequently excluded due
to missing or incomplete demographic and lifestyle details. The remaining 278 patients (244
men and 34 women) were available for analysis. No statistically significant differences were
found between the included and excluded cases in terms of clinical and other variables.
Permission to recruit patients and access to medical records were granted by the participating
hospitals in Japan.
During the same period, 400 community-dwelling adults, aged between 50 and 75 years,
were recruited from the same catchment areas as the cases. This convenience sample of
subjects was interviewed about their demographic and lifestyle habits when they attended
community centres or undertook health checks at hospitals. The same exclusion criteria as
cases were adopted, resulting in 335 eligible controls (267 men and 68 women). The
distribution of controls was: orthopaedic and physiotherapy clinics (n = 136, 40.6%), medical
check centres (n = 119, 35.5%), community centres (n = 46, 13.7%), and other places such as
universities and shopping malls (n = 34, 10.1%). Approval of the study protocol was obtained
from the Human Research Ethics Committee of Curtin University of Technology (approval
number HR 90/2005).
Questionnaire and Interview
A structured questionnaire was administered face-to-face by the first author to collect

information on demographic and lifestyle characteristics. Questions on habitual tea
consumption were taken from the Japan Public Health Center-based prospective study on
cancer and cardiovascular disease (Ishihara et al. 2003a). Validity and reproducibility of these
questions had been established for Japanese adults (Ishihara et al. 2003b). In our study, a
green tea drinker was defined as a person who drank Sencha, Bancha, Hojicha and
Genmaicha kinds of green tea once per week or more often. Participants were first classified
as either ‘ever’ or ‘never’ green tea drinkers. The ever drinkers were then asked about their
frequency and duration of drinking, and whether they had changed their habitual tea
consumption pattern five years ago. Demographic and lifestyle information, including age,
gender, weight (kg), height (m), marital status (single, divorced or separated; married),
education level (high school or below; college or university), residential location (urban;
rural), retirement status (working; retired), together with cigarette smoking (non-smoker;
current smoker) and alcohol consumption (non-drinker; drinker) in daily life, was obtained
from each participant. The reference recall period was set at 5 years before diagnosis for cases
or 5 years before interview for controls. For the cases, each interview was conducted in the
presence of their next-of-kin to minimize recall error, and appointment was made via their
respiratory physician. The purpose of the study was explained to each participant before
obtaining their formal written consent. Confidentiality of the information provided, and the
right to withdraw without prejudice, were ensured and maintained throughout the study. All
interviews, averaging 30 minutes in duration, took place in the hospital outpatient
departments for cases and their place of recruitment for controls.
Descriptive statistics were first applied to summarise participant characteristics. After

comparing the tea consumption pattern between cases and controls using chi-square and t
tests, multivariate logistic regression analysis was performed to assess the effect of green tea
intake on the COPD risk. Besides green tea drinking, independent variables considered in the
regression model were age, gender, body mass index (BMI = weight/height2), marital status,
education level, residential location, retirement status, smoking status and alcohol
consumption; definition of the categorical variables were given in the previous section. Data
entry, screening and statistical analyses were undertaken using the SPSS for Windows
package version 13.
RESULTS
Table 1 presents the demographic profile of the participants. The majority of subjects
were male with mean age similar between case and control groups. The mean BMI was
significantly lower for case than control subjects (p < 0.001). Most of the participants were
married, had high school or below education, resided in the rural area and retired. A
substantial proportion of cases (22.5%) continued to smoke after their diagnosis of COPD
while over half of the participants consumed alcohol on at least a monthly basis.
Table 1. Demographic profile of case and control groups
Characteristic Cases (n = 278) Controls (n = 335 )

Mean age (SD) years 66.5 (6.7) 65.3 (5.5)
Mean BMI (SD) kg/m2 21.9 (3.6) 23.4 (2.8)
Gender
Male 244 (87.8%) 267 (79.7%)
Female 34 (12.2%) 68 (20.3%)
Marital status
Single, divorced or separated 43 (15.6%) 53 (15.9%)
Married 233 (84.4%) 281 (84.1%)
Education level
High school or below 221 (80.1%) 211 (63.6%)
College or university 55 (19.9%) 121 (36.4%))
Residential location
Rural 224 (82.4%) 245 (73.8%)
Urban 48 (17.6%) 87 (26.2%)
Retirement status
Retired 153 (55.4%) 142 (42.6%)
Working 123 (44.6%) 191 (57.4%)
Smoking status
Non-smoker 214 (77.5%) 271 (81.1%)
Current smoker 62 (22.5%) 63 (18.9%)
Alcohol consumption
Non-drinker 120 (43.2%) 115 (34.3%)
Drinker 158 (56.8%) 220 (65.7%)
The prevalence of regular green tea consumption was significantly higher (p < 0.01)
among controls (n = 64, 19.3%) than cases (n = 27, 9.7%). As shown in Table 2, about half of
the tea drinkers drank one to three cups of green tea daily five years ago. Overall, the
frequency and duration of drinking were similar between the two groups of drinkers; most of
whom did not change their consumption pattern when compared with five years ago.
Results from the logistic regression analysis are given in Table 3. The risk of COPD
appeared to decrease with green tea drinking (p = 0.007). The adjusted OR was 0.45 (95% CI
0.25-0.81) for tea drinkers relative to non-drinkers, after accounting for significant
confounding factors such as gender, BMI, residential location, education level, retirement
status, smoking status and alcohol consumption.
Table 2. Consumption pattern of green tea drinkers
Cases (n = 27) Controls (n = 64) p1

Sencha/Bancha 0.346
Drink weekly 6 (22.2%) 7 (10.9%)
1-3 cups per day 13 (48.1%) 38 (59.4%)
> 3 cups per day 8 (29.6%) 19 (29.7%)
Hojicha/Genmaicha 0.776
Drink weekly 6 (22.2%) 19 (29.7%)
1-3 cups per day 15 (55.6%) 32 (50.0%)
> 3 cups per day 6 (22.2%) 13 (20.3%)
Years of drinking 49.3 (SD 16.4) 54.5 (SD 13.9) 0.704
Compared with drinking pattern 5 years ago2 0.582
No change 17 (68.0%) 49 (79.0%)
Increasing tea intake 6 (24.0%) 9 (14.5%)
Decreasing tea intake 2 (8.0%) 4 (6.5%)
1
cases versus controls.
2
Missing data present.
DISCUSSION
In this case-control study of community-dwelling Japanese adults, green tea intake was
associated with a decreased risk of COPD after accounting for significant confounders. An
extensive literature search found no published report that investigated the relationship
between green tea consumption and the development of COPD. The average duration of tea
drinking was about 50 years among drinkers. Therefore, our preliminary finding supported a
review of the protective role of green tea against lung cancer, which suggested that a long
exposure to green tea is needed in order to reduce the damage caused by tobacco carcinogens
(Liang et al. 2007).
In addition to green tea drinking, several variables were found to significantly influence
the risk of COPD. Firstly, the effect of BMI was consistent with a 10-year longitudinal study
of 458 males and 192 females in the USA, which showed that a lower BMI could increase the
risk of COPD for men (Harik-Khan, Fleg & Wise 2002). Secondly, women appeared to be
more likely to develop COPD than men with adjusted OR 2.72. However, recent reviews
indicated little difference in COPD prevalence between genders (Global Initiative for Chronic
Obstructive Lung Disease 2007; World Health Organization 2007), although two studies had
reported that females were more susceptible to severe COPD development than males
(Silverman et al. 2000; Xu et al. 1994). Thirdly, living in the rural area could elevate the risk
of COPD (OR 1.70). A similar observation was reported by a study conducted in south China
involving 3286 adults (Liu et al. 2007). Fourthly, cigarette smoking has been established as
the principal cause of COPD. It is therefore not surprising that smokers in this study were
subjected to a much higher risk of COPD than non-smokers. In terms of alcohol consumption,
however, regular drinkers seemed to have half the likelihood of developing COPD when
compared with non-drinkers. Although heavy alcohol consumption is known to have
deleterious effects on the lungs, recent and lifetime wine intake may improve pulmonary
function (Schünemann et al. 2002). Similarly, a study of 15294 American adults observed
altered pulmonary function and a reduced risk of lung restriction at modest levels of alcohol
intake (Sisson et al. 2005). Finally, retirement status and education level were found to affect
the COPD risk, despite such apparent association had not been reported in the literature.
The major strength of the present study was that information on green tea consumption
was obtained using a short validated questionnaire from the Japan Public Health Center-based
prospective study on cancer and cardiovascular disease (Ishihara et al. 2003a). A reference
recall period of five years was adopted to minimise recall error and to avoid possible changes
in tea exposure, because the diagnosis of the disease was confirmed for all patients within the
past four years. Moreover, data collection was conducted solely by the first author in order to
minimise inter-interviewer bias.
Several limitations should be considered in conjunction with the findings. The assessment
of green tea intake was based on self-report, which might lead to some misclassification of
drinking status when estimating the effect of the beverage. Although multivariate logistic
regression analysis was used to adjust for the effects of demographic and lifestyle variables,
other potential confounding factors such as habitual physical activity, dietary intake and co-
morbidities were not taken into account. In particular, data were not collected on the
consumption of foods and other beverages. The lack of detailed and specific information on
quantity of green tea intake posed another limitation on the conclusion drawn with respect to
dose-response relationship. Finally, the evidence concerning the protective effect of green tea
may be regarded as preliminary, in view of the moderate sample size especially the relatively
small number of female participants recruited into the study.
CONCLUSION
Although this case-control study has suggested a protective role of green tea against
COPD for Japanese adults, more research is required to ascertain the exact effect of green tea
intake and whether the observed finding can be generalized to other populations. In addition
to large scale case-control studies, long-term prospective cohort studies collecting detailed tea
consumption information would provide important epidemiological evidence on both
morbidity and mortality. Experimental and animal studies will also complement and provide
additional supporting evidence to understand the biological mechanism underlying the
protective role of green tea. The increasing knowledge would be beneficial towards the
prevention of this disease.
ACKNOWLEDGEMENTS
The authors are grateful to the Japanese respiratory physicians for recruitment of COPD
patients, and to all participants who volunteered their time for the project.
Table 3. Risk of COPD from logistic regression analysis
Adjusted OR 95% CI p
Green tea drinking:
Non-drinker 1.00
Drinker 0.45 0.25 – 0.81 0.007
Age 1.01 0.97 – 1.05 0.663
BMI 0.87 0.82 – 0.93 <0.001
Gender:
Male 1.00
Female 2.72 1.07 – 6.91 0.035
Marital status:
Single, divorced or separated 1.00
Married 0.87 0.47 – 1.62 0.657
Education level:
High school or below 1.00
College or university 0.59 0.37 – 0.93 0.024
Residential location:
Urban 1.00
Rural 1.70 1.04 – 2.80 0.036
Retirement status:
Working 1.00
Retired 0.62 0.40 – 0.97 0.036
Smoking status:
Non-smoker 1.00
Current smoker 75.31 23.82 – 238.05 <0.001
Alcohol consumption:
Non-drinker 1.00
Drinker 0.49 0.32 – 0.76 0.001
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Chapter 20
GREEN TEA AND DIABETES
Dongfeng Wang, Linge Wang and Li Zhang

College of Food Science and Engineering, Ocean University of China,
Qingdao 266003, China
ABSTRACT
Providing that there has been a dramatic increase in the incidence of diabetes
mellitus associated with long-term complications, it is critical to find a natural nutritional
material aimed at reducing the prevalence of diabetes which threatens human health over
the world .
Currently, green tea, only next to water, is the most widely consumed beverage in the
world and exerts beneficial bioabilities, such as anti-inflammative, anti-oxidative, anti-
mutagenic, etc. With increasing interest in the health promoting properties of tea and a
significant rise in scientific investigation, in vitro and animal studies provide more and
more strong evidence that green tea consumption has a great prophylactic and therapeutic
effect on diabetes and its complications, which is intensively associated with its mainly
bioactive components, such as tea polyphenols and tea polysaccharides.
In this article, we will summarize effect of green tea on diabetes, especially anti-
diabetic ability of tea polyphenols and polysaccharides, discuss possible mechanisms, and
make perspectives and future directions in this area.
Keywords: tea polysaccharides, tea polyphenols, green tea, diabetes, mechanism
1. INTRODUCTION
During the last century changes in human behavior and lifestyle have resulted in a
dramatic increase in the incidence of diabetes over the world. According to a widely accepted
estimation, the number of diabetic patients would reach 366 million by the year 2030 [1].
Diabetes mellitus is a disorder of metabolism caused by the relative or absolute lack of insulin
and is associated with long-term complications that affect eye, heart, blood vessel, and kidney
function.
394 Dongfeng Wang, Linge Wang and Li Zhang
Though oral hypoglycemic agents and insulin is the mainstay of treatment of diabetes,
they have prominent side effects and fail to significantly alter the course of diabetic
complications. Lifestyle modifications including appropriate diet and exercise programs have
been found to be greatly effective in the management of the disease. Diet therapy especially is
showing a bright future in the therapy of diabetes mellitus.
Green tea is a widely consumed beverage and its origins date back thousands of years in
China. The legendary Chinese emperor, Shen Nung, discovered the detoxifying and health-
maintaining properties of green tea around 2700 B.C. In recent years, green tea is being
widely studied for its beneficial effect in treatment and prevention of human diseases. It is
considered to be anti-inflammative, anti-oxidative, anti-mutagenic, and anti-carcinogenic and
can prevent cardiac disorders. What is more, it has been over 30 years since people studied
the effect of green tea on diabetes. Domestic and international researches have already
reported that green tea could be prophylactic and therapeutic against diabetes. Now we
introduce the summary on the relationship between green tea and diabetes as follows.
2. GREEN TEA CONSUMPTION AND DIABETES

Green tea, which is prepared by drying fresh tea leaves with no fermentation during
processing, retains the original color of the tea leaves and contains various bioactive
components, including polyphenols, polysaccharides, caffeine, amino acids, and other trace
compounds such as lipidsand vitamins.
Since ancient times, green tea consumption has been known to maintain and improve
health. Initial research on the benefits of green tea was carried out in China and Japan because
of the local customs. Curing diabetes with coarse green tea is a popular folk prescription in
both China and Japan. A prospective epidemiological study done in Japan found that men and
women reported drinking 6 or more cups of green tea per day had one-third less the incidence
of type 2 diabetes mellitus over 5 years [2]. But what is the reason?
On one hand, green tea extract was found to have an obvious hypoglycemic effect.
Tsuneki H et al. reported that green tea had an effect on blood glucose levels and serum
proteomic patterns in diabetic mice and promoted glucose metabolism in healthy human [3].
Many reports show the anti-hyperglycemic effect of green tea in both type 1 and type 2
diabetic animal models. This effect may be associated with the inhibitory effects on a-
glucosidase activity and glucose transport ability in small intestinal mucosa [4]. Jerzy
Ju kiewicz et al. suggested that supplementation of the diet with green tea extract
significantly reduced maltase as well as saccharase and lactase (at higher dose) activities,
which may be indicative of a reduced level of absorbable glucose and, thus, may be beneficial
in the amelioration of the diabetic state [5].
On the other hand, green tea was reported to increase insulin sensitivity. Taiwanese
investigators demonstrated recently that green tea increased insulin sensitivity in Sprague–
Dawley rats and ameliorated insulin resistance and increased glucose transporter IV content
of adipocytes isolated from the epididymal fat pads [6]. Green tea was reported to regenerate
pancreatic beta cells in streptozotocin (STZ)-induced diabetic rats to maintain normal insulin
levels [7].
Green Tea and Diabetes 395
What is more, the reduction in the risk of diabetic complications by green tea drinking
has been observed in epidemiological studies.
Diabetes leads to modification of collagen such as advanced glycation and cross-linking
which play an important role in the pathogenesis of diabetes mellitus. Pon Velayutham et al.
have investigated the effect of green tea on modification of collagen in STZ-induced diabetic
rats and shown that green tea is effective in reducing the modification of tail tendon collagen
in diabetic rats,which demonstrates that green tea may have a therapeutic effect in the
treatment of glycation induced complications of diabetes. The same scholars also reported
that green tea by ameliorating myocardial collagen characteristics may provide a therapeutic
option in the treatment of cardiovascular complications of diabetes [8]. As we all know,
Paraoxonase (PON1) is an antioxidant enzyme that protects lipoproteins against oxidative
modification [9, 10]. Lipoprotein oxidation is believed to play an essential role in the
pathogenesis of atherogenesis [11, 12]. Recently, Sibel Tasa has suggested that green tea
could slow the progression of atherogenesis by reducing oxidation of lipoproteins and
preserving paraoxonase activity [7]. The anti-hyperglycemic and antioxidant effects of green
tea may be ascribed to this activity. In addition, diabetes-induced hyperlipidemia, oxidative
stress and protein glycation impair cellular calcium and sodium homeostasis associated with
abnormal membrane-bound enzyme activities resulting in cardiac dysfunction in diabetes. To
explore the cardioprotective mechanism of green tea in diabetes, Pon Velayutham et al. have
done several investigations revealing that green tea extract indeed has a therapeutic effect and
suggesting that a possible mechanism may be associated with the attenuation of the rise in
[Ca2+] and [Na+] by ameliorating Ca2+-ATPase and Na+/K+-ATPase activities [13].
Above all, it can conclude that green tea consumption has a great prophylactic and
therapeutic effect on diabetes and its complacations, which is intensively associated with its
bioactive components, such as polyphenols, polysaccharides and other trace compounds such
as lipidsand vitamins.
3. TEA POLYSACCHARIDES AND DIABETES

It has been established that non-starch polysaccharides are likely to be associated with
less risk of diabetes. Polysaccharide is one of the main components in lower-grade green tea
which is considered to cure diabetes especially in China and Japan. With the fast development
of biological and chemical technologies during the last 20 years, great advances have been
made in the studies of tea polysaccharides on both structure and anti-diabetes activity.
3.1. Chemistry of Tea Polysaccharides
Tea polysaccharides (TPS) is a kind of macromolecular glycoprotein with a molecular

weight of approximately 1.07–1.10×105 Da. It is soluble in hot water (about 76%) of which
aqueous solutions behave as slightly acidic Newtonian fluids, but insoluble in the high
concentration solutions of such organic solvents as alcohol, ether, acetone, acetic ether etc
[14].
Wang DF et al. investigated the composition and chemical characterisitics of TPS by

methods of UV, IR, GC, and found that its polysaccharide part was composed of arabinose,
xylose, fructose, glucose and galactose with a proportion of 5.52:2.21:6.08:44.20:41.99. The
protein part was composed of 16 normal amino acids, among which Val, Ala, Gly, Glu were
the major proportion [15]. Moreover, Nie SP et al. showed that TPS was composed of seven
kinds of monosaccharides, namely ribose, rhamnose, arabinose, xylose, mannose, glucose,
galactose in molar ratio of 71:5.88:13.70:1.99:1.00:1.84:33.75, and eighteen amino acids via
HPGPC, FT-IR, GC–MS technologies [16]. Although there are many discrepancies due to
different preparation and purifying methods, the polysaccharide part of TPS is mainly
composed of arabinose, xylose, glucose and galactose obviously, and it contains many natural
amino acids, a majority of which have one amino and two carboxyls.
3.2. Method with Celerity and Availability for Assaying Content of TPS
In order to effectively valuate TPS pharmacological activities and functions,

Wang DF et al. found an available method to assay the content of TPS. Standards
of TPS is got first and then used to build a standard curve by anthrone sulfuric
acid method, then the content of TPS in tea extract is determined quantitatively
[17].
3 ,5
A490nm
3 A280nm
2 ,5
OD280nm/OD490nm
1 ,5
0 ,5
0
0 5 10 15 20 25 30 35 40 45 50 55 60
n u m b e r o f th e tu b e
Figure 1. TPS fractions separated on Sepherose Fast Flow.

The water extraction was concentrated to 1/15 volume in a rotary evaporator under
reduced pressure and subsequently precipitated by adding ethanol (1:4,V:V). The
precipitation was collected by centrifugation and desiccated in vacuum. Then the coarse TPS
had been gotten. The coarse TPS was firstly loaded onto a Sepharose Fast Flow column and
two main fractions were acquired (Figure 1). The first fraction was chosen for further
purification because of the more content of polysaccharide. It was directly loaded onto
Sephadex G100 column for further purification. And then the main fraction was collected and
named TPS-1 (Figure 2).
The purified TPS-1was white powder. Figure 3 illustrated the character of the purified
TPS-1 on HPLC. It was a single peak, which indicated the purity of TPS-1. The results
illuminated the purified TPS could be used as standards.
3.3. Anti-diabetes Activity of Tea Polysaccharides
In recent years, along with in-depth study in the functions of tea, TPS, a kind of
glycoprotein, has aroused public concern, especially in its hypoglycemic activity. A number
of studies have revealed that TPS indeed improve the status of diabetes.
0,25
A490nm
0,2
A280nm
OD280nm/OD490nm
0,15
0,1
0,05
0
0 2 4 6 8 10 12 14
number of tube
Figure 2. TPS fraction separated on SephadexG100.

Figure 3. TPS-1 performed on HPLC.
Table 1. Contents of TPP, Catechins, Caffeine, and TPS in Various Grades of Green
Tea (Grams per 100 g)
Grade*
First Second Thirst Fourth Fifth Sixth
TPP 22.03± 23.70 21.10 ± 18.55 ± 19.05 ± 17.23 ±
0.31 ±0.97 1.01 0.31 0.37 0.85
Catechins 14.07 ± 14.01 ± 12.56 ± 9.87± 0.71 9.63± 0.91 8.33± 0.79
0.97 0.78 0.51
Caffeine 4.00± 0.21 4.09 ±0.37 3.85 ± 0.11 3.25 ± 0.71 2.87 ± 0.05 3.01± 0.07
TPS 0.23± 0.10 0.29 ± 0.12 0.31 ± 0.17 0.46± 0.21 0.49 ± 0.15 0.58± 0.15
*
The first grade tea consisted of 90% one leaf and a bud and 10% two leaves and a bud; the second
grade, 60% one leaf and a bud and40% two leaves and a bud: the third grade, 30% one leaf and a
bud, 30% two leaves and a bud, and 40% three leaves and a bud; thefourth grade, 10% one leaf and
a bud, 20% two leaves and a bud, 50% three leaves and a bud, and 20% tender banjhi; the fifth
grade, 10%two leaves and a bud, 30% three leaves and a bud, 50% tender banjhi, and 10% leaves;
the sixth grade, 80% tender banjhi and 20%leaves.
Table 2. Effect of TPS on BG in Normal Mice and Model Mice (n= 10, X± SD)
Normal mice Model mice

Control 1.2 2.4 4.0 control 4.0 6.0 8.0
mg/mL mg/mL mg/mL mg/mL mg/mL mg/mL
9.6 8.8 ± 0.7 8.1± 0.4a 8.0± 0.6b 17.9 ± 16.7± 1.4 13.0± 12.1 ±
±0.6 1.3 2.1a 1.5b
a
Compared with the control group p < 0.05.
b
Compared with the control group p < 0.01.
Earlier in 1991, Isigaki K reported that the soluble polysaccharide in tea had an obvious
hypoglycemic effect. Wang DF et al. has investigated the activity of TPS for over 20 years
and gained plenty of valuable findings as follows. Treatment of diabetes with coarse tea in
both China and Japan may be related to TPS and the high content of TPS in coarse tea(Table
1), which indicates that TPS is one of the main components related to its hypoglycemic
activity. Additionally, the contents of TPP, catechin, and caffeine in the sixth-grade tea were
less than those in the first-grade tea by 20, 40, and 25%, respectively, but the content of TPS
in the sixth-grade tea was twice as high as that in the first one, which is to say that the content
of TPS increases with the age of green tea, which is why that d diabetics drink coarse tea for
curing diabetes in folk of China . When mice (7 weeks old) were injected with purified TPS,
the levels of blood glucose (BG) in normal mice and model mice with high BG were
decreased significantly by averages of 13.54%, and 22.18%, respectively (Table 2). Wang DF
et al. also reported that a 100-120 kDa fraction, essentially composed of polysaccharides
(90%) with substantial amounts of arabinogalactan proteins, contained the hypoglycemic
activity, which indicated that a soluble tea polysaccharide is the major hypoglycemic factor in
tea and that this polysaccharide may be developed to a potential natural hypoglycemic
functional ingredient [17, 18]. Ni DJ et al. has compared the hypoglycemic effect of TPS
from different teas, and found that all TPS have significant effects, especially TPS from green
tea has an obvious dose-effect relationship [19]. Juśkiewicz J et al. [5] have done further
investigation and found that the effect of green tea extract, added to the diet at 2 levels of
0.01% and 0.2% the extraction for diabetic rats, seems to be dose dependent in the case of the
following examined parameters: small intestinal saccharase and lactase activities, urinary
albumin excretion , thiobarbituric acid-reactive substances content in kidneys' tissue,
superoxide dismutase activity in the serum, and levels of total antioxidant capacity and
integral antioxidant capacity of lipophilic substances. Although the higher dose of green tea
extract did not completely protect against STZ-induced hyperglycemia and oxidative stress in
experimental rats, this study suggests that green tea extract ingested at high amounts may
prove to be a useful therapeutic option in the reversal of diabetic dysfunction.
Chen HX et al. have demonstrated that the effects of the molecular weight and protein
content of the polysaccharide conjugates on the improvement of the bioactivities appeared to
be significant, and especially a relatively low molecular weight and a high protein content
appeared to be important for the antioxidant activity [20]. Wang LY et al. have found that
TPS group showed lower incidence of diabetes, higher serum C-peptide level, decreased
lymphocytic inflammation in pancreatic islets, stronger proliferation of CD8 T subsets and
lower ratio of CD4/CD8 subgroup in splenocytes as compared with NS group, which
indicated that TPS seems to prevent the onset of type 1 diabetes in nonobese diabetes(NOD)
mice [21]. Chen JG et al. have found that TPS could significantly ease the diabetic mice’
symptoms, reduce the fasting plasma glucose, and there is a dose-response relationship. This
result indicated that TPS could decrease blood glucose and alleviate symptoms of diabetes
[22]. The other scholars have all suggested that TPS indeed have a remarkable hypoglycemic
activity in treating the diabetics.
Although TPS could decrease blood glucose and alleviate symptoms of diabetes, the
detailed mechanism has not been assured. Many scholars have investigated the mechanism as
follows.
As we all know, antioxidants are important in diabetes, with low levels of plasma
antioxidants implicates as a risk factor for the development of the disease and circulating
levels of radical scavengers impaired throughout the progression of diabetes. Many of the
complications of diabetes, including retinopathy and atherosclerotic vascular disease, the
leading cause of mortality in diabetics, have been linked to oxidative stress [23-29]. Chen HX
et al. has found that TPS exerted significant inhibitory effects on hydroxyl, superoxide
radicals and lipid peroxidation, and it could improve the activity of superoxide dismutase
(SOD) (p < 0.05). These results indicated that TPS was a potent antioxidant and there
appeared to be a direct connection between antioxidant activity and hypoglycemic activity
[30]. The same scholars have also proved that TPS extracted from low-grade green tea
exerted a significant hypoglycemic effect on both normal and experimental mice model of
high blood sugar. They proposed that TPS could enhance glucose tolerance, which was
beneficial to alleviate the symptoms of diabetic mice, through such mechanisms as weakening
the damage to the islet p cell induced by alloxan, improving the antioxidant capacity of liver,
or enhancing the activity of hepatic glucokinase [20].
In recent years, an increased number of studies have shown that the chronic, sub-clinical
and non-specific inflammation is closely related to the development of T2DM and its
complications such as artherosclerosis, diabetic nephropathy and retinopathy. According to
that inflammation can result in insulin resistance and the dysfunction of pancreatic β-cells, the
control of inflammation will become a new treatment. Wang DF et al. has found that TPS
treatment was beneficial not only for the subsequent production of interleukin (IL) 2 in spleen
cells of adjuvant arthritis (AA) rats but also because it prohibited the body from producing
too much IL-1 in AA rats [17]. These results suggested thatTPS could prevent diabetes by
controlling the body proinflammatory cytokine.
Moreover, researches have revealed that there may be a possible relationship between
TPS and insulin. Li BQ et al. has reported that treatment of TPS to model mice could reduce
the blood serum glucose and consequently improve the glycogen activity, which suggested
the effect of TPS on the glucose metabolism is similar to insulin [31]. Rui LL has also
testified TPS can not only effectively reduce blood sugar levels in type 2 diabetes mice, but
also improve glucose and lipid metabolism [32], which implies the mechanism may be related
to increased insulin sensitivity.
Quan JH et al. have shown that TPS exerts an obvious hypoglycemic effect on diabetic
rats by delaying intestinal digestion and absorption, which may be associated with the
inhibitory effects on a-glucosidase and amylase activity and glucose transport ability in small
intestinal brush border vesicles [33].
According to previous reports, there are other several main mechanisms for
polysaccharides to act on the serum glucose level, to decrease the content of liver glycogen,
to stimulate the release of insulin, or to influence the activities of metabolizing enzymes [34].
Above of all, we can conclude that TPS exerts its hypoglycemic activity through a
number of mechanisms. Indeed, TPS has more bioactivity that can benefit diabetics besides
the above, for example, the effect of protecting the body from radiation, delaying thrombosis
time, improving the blood vessel system, and decreasing lipid content.
4. TEA POLYPHENOLS AND DIABETES

Polyphenols are plant metabolites occurring widely in plant food and exhibit outstanding
antioxidant and free radical scavenging properties. Green tea is produced by inactivating the
polyphenol oxidase in the leaves of camellia sinensis which preserves abundant natural
polyphenols, such as catechins(once called vitamin P), orgallotannins, flavonols, flavandiols,
and phenolic acids.
In particular greent tea catechins and their derivatives are known to contribute beneficial
health effects ascribed to tea by their antioxidant, anti-mutagenic, and anti-carcinogenic
properties [6].The important catechins of green tea are (−)-epicatechin (EC), (−)-epicatechin-
3-gallate (ECG), (−)-epigallocatechin (EGC) and (−)-epigallocatechin- 3-gallate (EGCG),
among which EGCG is the most abundant and its chemical structure is shown as follows [35].
Since the discovery that tea polyphenols have unique chemical structures and are major
ingredients of unfermented tea, they have been found to possess widespread biological
functions and health benefits. Epidemiologic observation and laboratory studies have
indicated that polyphenolic compounds present in the tea may reduce the risk of a variety of
illnesses.
Figure 4. The structure of EC(A), EGC(B) ,ECG(C) and EGCG(D).

In recent years, more and more researches have shown that green tea polyphenols (GTP)
has anti-hyperglycemic action on both normal and diabetic animals. At the same time, GTP
also has the therapeutic effect on diabetic complications.
4.1. The Hypoglycemic Activity of Tea Polyphenols
As we all know, obesity is associated with high blood cholesterol and a high risk for
developing diabetes and cardiovascular disease. The obesity-preventive effects of green tea
and its main constituent EGCG are widely supported by results from epidemiological, cell
culture, animal, and clinical studies in the last decade. In addition, it has been shown that
dietary supplementation with EGCG could potentially contribute to nutritional strategies for
the prevention and treatment of type 2 diabetes mellitus.
Researches have shown that GTP especially EGCG injected into rats significantly
reduced food intake, body weight, blood levels of insulin, glucose, cholesterol and
triglyceride. Sabu MC et al. has found that GTP has an obvious effect on serum glucose level
in normal and diabetic mice. Administration of GTP (500 mg/kg.b.wt.) to normal rats
increased glucose tolerance significantly (P<0.05) at 60 min. GTP is also found to reduce
serum glucose level in alloxan-induced diabetic rats significantly at a dosage of 100
mg/kg.b.wt. Continued daily administration (15 days) of the extract 50, 100 mg/kg.b.wt
produce, respectively, 29% and 44% reduction in the elevated serum glucose level produced
by alloxan administration [36]. Ding RF et al. have also reported that GTP exerted an
inhibitive effect on increased blood sugar, an improvement in glucose tolerance and the level
of blood insulin in diabetic rats [37], indicating that there is a tight connection between the
hypoglycemic effect and time of GTP administration.
Nowadays, people have investigated the hypoglycemic effect of GTP only on alloxan- or
streptozocin-induced diabetic rats, and with rare reports on clinical research, and some others
do not sustain the hypoglycemic effect of GTP. Thus there is a long way to go for our
research work.
4.2. The Therapeutic Effect of Tea Polyphenols on Diabetic Complications
It is well known that diabetes is a metabolic disease which effects not only the glucose
metabolism but also lipid and protein metabolism, predisposing to markedly increased
cardiovascular mortality and nephropathy.
4.2.1. The Effect of Tea Polyphenols on Cardiovascular Complication

Atherosclerosis accounts for some 80 % of all diabetic mortality, about three-quarters of
the cardiovascular deaths from diabetes result from coronary artery disease. Researches have
reported that GTP may slow the progression of atherogenesis by reducing oxidation of
lipoproteins and preserving paraoxonase activity by its anti-hyperlipidemic and anti-oxidative
effects. Experimental and clinical studies indicate the extensive chemical modification of
collagen, an important constituent of most of the tissues, in the etiology of diabetes and
diabetic complications. The modification is due to nonenzymatic glycosylation of protein,
which results in deleterious effects on collagen. It is not the initial glucose adducts, but
subsequent oxidation products known as advanced glycation end products (AGE), particularly
the cross-linking AGE, which cause the damaging effect [38]. The enhanced myocardial
collagen content, collagen glycation and the resulting advanced glycation end products (AGE)
which exhibit the characteristics of increased cross-linking are proposed for the stiffness of
myocardium in diabetes. Pon Velayutham et al. has suggested green tea flavonoids could cure
cardiovascular complications of diabetes via ameliorating myocardial collagen characteristics,
mainly with its typical antioxidant and the anti-hyperglycemic activities [8].
In addition, previous studies have reported that supplementation with antioxidants
prevents lipid peroxidation, protein glycation and inhibition of Na+/K+-ATPase or Ca2+-
ATPase activity caused by hyperglycemia, which could lead to cardiac dysfunction in
diabetes [13]. The same scholars have measured the changes in the levels of calcium, sodium,
potassium and the activities of Na+/K+-ATPase and Ca2+-ATPase in green tea treated diabetic
rat hearts to explore the cardioprotective mechanism of green tea in diabetes, and found that
green tea exerts a great therapeutic effect on cardiac dysfunction of diabetes, with a possible
mechanism associated with the antioxidant activity of green tea catechins, which could reduce
the factors responsible for disturbed fluidity and stability of the membranes by preventing the
oxidative stress, protein glycation and hyperlipidemia.
4.2.2. The Effect of Tea Polyphenols on Diabetic Nephropathy

Diabetic nephropathy (DN) is one of the most serious chronic microvascular diabetic
complications. A great number of factors can result in DN. In particular active oxygen free
radicals and lipid peroxidation play an important role in the etiology of DN.
Renal damage is a well-known consequence of diabetes, and the early identification of
micro albuminuria is considered to be clinically relevant. A therapeutic intervention should
start early enough to be effective or to delay the development of end-stage renal disease.
Otherwise, there will be serious consequences such as renal failure accompanied by uremia
[39, 40]. Wang B et al. has studied a protective effect of tea polyphenols on diabetic rats and
found that tea polyphenols can inhibit the reduction of SOD in early experimental animal
serum and the level of lipid peroxides. Additionally, GTP exerted a reduction in albuminuria
level and an inhibitory effect on the increase of TGF-B1 mRNA and protein expression in
early DN mice, which indicated that GTP had an obvious preventive effect on the damage of
kidney in diabetes [41, 42]. Zhao QL et al. also affirmed the protective effects of tea
polyphenols on diabetic rats. They found that administration of GTP can significantly
decreased the level of albuminuria, creatinine, cholesterol and triglycerides, and mitigated the
renal damage in diabetic mice, compared with model mice without treatment [43].
There are many scholars who believe that GTP exerts its effect on DN by the typical
antioxidant activity, but it still requires further study on whether it works through direct effect
or indirect effect of regulating oxidative balance.
4.3. The Anti-Diabetic Mechanism of Green Tea Polyphenols
In all, GTP indeed has both anti-hyperglycemic action and the therapeutic effect on
diabetes and its complications. Many researchers are engaged in the prevention and treatment
of diabetes using tea polyphenols and the mechanisms of its actions are emerging based on
the various laboratory data. These mechanisms may be related to certain pathways, such as
through its antioxidant activity, inhibition of intestinal glucose transporter and related enzyme
activities, mimic insulin by decreasing the expression of genes that control gluconeogenesis,
increasing insulin sensitivity, and enhancing basal- and insulin-stimulated glucose uptake, etc.
These effects may be attributed to the anti-diabetic effect of green tea.
4.3.1. The Great Antioxidant Activity of Tea Polyphenols

The polyphenolic fraction of green tea, has been reported to have multiple
pharmacological actions, among which the antioxidant activities are the most widely
recognized [44]. GTP has been shown to possess potent antioxidant activity that is several
folds higher than that of Vitamin C and Vitamin E [45]. Different methods of evaluation the
antioxidant activity of polyphenols suggest that they exhibit scavenging activity against
superoxide radicals, free radicals and hydrogen peroxide. In addition to directly quenching
reactive oxygen species, tea flavonoids can chelate iron and copper preventing the metal-
catalyzed free radical formation [46-49]. Thus GTP can inhibit body damages induced by free
radicals.
Increased oxidation stress has been implicated in the pathogenesis of DM.
Hyperglycemia induced protein glycation generates superoxide free radicals [50-54], leading
to lipid peroxidation and formation of reactive products, which may play an important role in
the development of diabetes and its complications such as DN. Administration of GTP can
effectively improve the body antioxidant potential and increase the antioxidant enzymes such
as the activity of SOD, GSH –PX and POD which are lower in diabetes mellitus [55, 56]. In
addition, Li et al. reported that EGCG does not affect glucose-stimulated insulin secretion
under high energy conditions, and further showed that these compounds act in an allosteric
manner independent of their antioxidant activity and that the beta-cell stimulatory effects are
directly correlated with glutamine oxidation [57]. Those results strongly support the notion
that GT consumption helps maintain and improve health by increasing antioxidant defense
and cellular metabolic activities in various tissues of normal rats and provides a useful
therapeutic option in the reversal of oxidative stress induced cardiac dysfunction in diabetes
mellitus.
Hence we can conclude that the anti-diabetic activity of GTP is mainly associated with its
antioxidant activity.
4.3.2. The Inhibition of Intestinal Glucose Transporter and Related Enzyme Activities
Intestinal glucose uptake can be achieved by the sodium-dependent SGLT1. There is a
site combined with Na+ on this glucose transporter, and this modification makes transfer of
glucose easier. The glucose transportation through cell membrane depended on the effect of
SGLT1. Yoko Kobayashi et al. have found that EGCG can inhibit intestinal glucose uptake by
sodium dependent glucose transporter, SGLT1, through a competitive mechanism, which can
indicate its increase in controlling blood sugar [58].
In addition, researches have shown that GTP exert hypoglycemic effect by inhibiting the
related enzyme activities. Japanese scholars have been engaged in the related research since
1990, and found that green tea catechins have the obviously inhibitory effects on a-
glucosidase and amylase activity, which directly slow down the intestinal glucose taken [59].
This mechanism may be well ascribed for the anti-hyperglycemia activity of GTP.
4.3.3. Insulin-like Activities and Insulin-enhancing Activity of GTP

Several known compounds found in tea were testified to enhance insulin, with the
greatest activity due to EGCG, followed by ECG, tannins, and theaflavins. As support for this
result, Anderson et al. reported that tea contains in vitro insulin-enhancing activity and the
predominant active ingredient is EGCG.
The green tea flavonoid has also been shown to have insulin-like glucose-lowering
activities [60] as well as insulin-enhancing activity in mammals [61]. However, EGCG, the
principal catechin in green tea, differs from insulin in that it affects several insulin-activated
kinases with slower kinetics. Furthermore, EGCG regulates protein-tyrosine-phosphorylation
by modulating the redox state of the cell [60], and mimics insulin at least in part by repression
of gluconeogenic genes such as phosphoenolpyruvate carboxykinase, which is a key rate-
limiting enzyme in hepatic gluconeogenesis, a process which is thought to contribute to
increased glucose production in diabetes [62].What is more, other scholars have reported that
green tea catechins possess a strong hypoglycemic effect by enhancing basal- and insulin-
stimulated glucose uptake, increases insulin sensitivity and ameliorates insulin resistance in
diabetes.
5. OTHER COMPONENTS OF GREEN TEA AND DIABETES

The preventive and therapeutic effects of green tea on diabetes result from a combination
of many components. There are many other substances, apart from TPS and polyphenols,
which have positive effects on therapy of diabetes.
For example, tea pigments have a better therapeutic effect on DN by decreasing the
quantity of microspheres protein and significantly inhibiting the production of endothelin and
the activity of platelet [63, 64], and can cure diabetic cardiovascular disease through the
possible mechanism of lowing cholesterol and triglycerides and increasing high-density
lipoprotein, Vitamin C could normalize the tenacity and permeability of microvascular,
beneficial for the diabetes [65]. In addition, there are other components improving glucose
metabolism, such as Vitamin B, pantothenic acid.
6. PERSPECTIVES AND FUTURE DIRECTIONS

It is apparent that green tea is a source of a wide range of phytochemicals that are
digested, absorbed and metabolized by the body, and that tea constituents exert their anti-
diabetic effects at the cellular level. Because of several potential benefits of green tea, we
propose that this beverage may act as a supportive agent in diabetes mellitus.
For the promotion of health, there are various factors that matters like type of green tea
and preparation shown important, but so will the frequency and timing of intake as these
factors directly affect the pharmacokinetics and ultimate disposition of the polyphenols and
polysaccharides within tissues. Future research needs to define the actual magnitude of health
benefits, establish the safe range of tea consumption associated with these benefits and
elucidate potential mechanisms of action.
Further progress in the evaluation of the effects of green tea on humans depends on the
development of new experimental systems. Exploration at the cellular level allows a better
understanding of the underlying mechanisms regulating functions in normal and pathologic
states. Development of more specific and sensitive methods with more representative models
along with the development of good predictive biomarkers will give a better understanding of
how tea interacts with endogenous systems and other exogenous factors.
In order to understand the effect of green tea consumption on diabetes, additional
research on the pharmacokinetics of tea constituents as well as their mechanisms of action is
needed.
Definitive conclusions concerning the protective effect of green tea have to come from
well-designed observational epidemiological studies and intervention trials. The development
of biomarkers for tea consumption, as well as molecular markers for its biological effects,
will contribute to better future studies in this area.
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Chapter 21
GREEN TEA AND TYPE 2 DIABETES
Jae-Hyung Park, Hye-Young Sung and Dae-Kyu Song

Department of Physiology & Chronic Disease Research Center, Keimyung University
School of Medicine, 194 Dongsan-Dong, Jung-Gu, Daegu, 700-712 Korea
ABSTRACT
Green tea, which is consumed world wide as a beverage, is known to have many
beneficial effects on human health. Green tea contains various biologically-active
materials including catechins, flavonols and caffeine. Of them, catechins are the major
constituents consisting of 30% of water-extractable materials. Pharmacological
implication has been mainly made upon (-)-epigallocatechin-3-gallate (EGCG), as it is
the most abundant catechin in green tea extracts. The second biologically important
catechin is (-)-epicatechin-3-gallate (ECG), which is called one of gallated catechins as
EGCG. To date, there is no prominent evidence for green tea consumption to determine
whether be beneficial or harmful in metabolic diseases, such as type 2 diabetes and
obesity. It may be due to catechins having a variety of function in human body. This
chapter will focus on action mechanism of EGCG on ATP-sensitive potassium channels,
which manifests as actual phenotypes in cardiac and beta-cell function. Additionally,
changes by green tea or gallated catechins in insulin resistance will be reviewed.
Thereafter, a right method to use EGCG as a supportive regimen for diabetic care and
obesity will be introduced. New era should come with modifying natural products fit to
human well-being.
INTRODUCTION
Type 2 diabetes can be characterized by both fasting and postprandial hyperglycemia.
Hyperglycemia causes various tissues in the body to suffer from increased oxygen and
nitrogen free radicals, exacerbating various diabetic complications (Hong et al., 2004).
Therefore, antioxidative manipulation is a method for diabetic prevention and treatment.
Green tea catechins have chargeable moiety and specific lipophilicity according to their
412 Jae-Hyung Park, Hye-Young Sung and Dae-Kyu Song
molecular structure. Therefore, green tea catechins may act as modulators of a variety of
function in human body.
OXIDATIVE STRESS AND GREEN TEA

Green tea catechins, in particular (-)-epigallocatechin-3-gallate (EGCG) which is the
most abundant and biologically active components of green tea polyphenols, are known to
possess both antioxidant and prooxidant activity (Mandel et al., 2004; Salah et al., 1995),
which probably depends on its molecular structure or its concentration to be used. Ironically,
EGCG protects normal cells against oxygen radical insults (Xia et al., 2005), whereas it
induces apoptosis of cancer cells by generating intracellular reactive oxygen species (ROS)
(Qanungo et al., 2005). This discrepancy may be due to 1) occurrence of a specific signal
cascade in cancer cells which is targeted by catechins but not manifested in normal cells; 2)
greater susceptibility of cancer cells than normal cells to ROS, which can be more generated
in cancer cells having a higher energy need; 3) There is also discrepancy between results in in
vitro and in vivo study to evaluate the effect of EGCG on possible free radical toxicity,
suggesting that other existing factors present in vivo may modify the EGCG effect. Therefore,
it appears difficult to make an exact answer about whether EGCG is antioxidant, prooxidant,
or both in the body. Instead, it may depend on targeted tissue-specific environment that
affects the actions of EGCG (Albright et al., 2004). In other words, the action mechanism of
EGCG associated with ROS would be expressed differently according to conditions in related
tissues. In diabetes, many tissue cells, such as pancreatic beta cells and vascular endothelial
cells, suffer from increased oxidative stress mainly by prolonged hyperglycemia. This
situation is somewhat like those of cancer cells, which are also stressful owing to increased
ROS and thereby susceptible to EGCG. In normal rats, a moderate concentration of EGCG
injection (5 mg/kg, i.p.) does not induce any significant glucose intolerance during
intraperitoneal glucose tolerance test (IPGTT). However, in streptozotocin (STZ)-induced
diabetic rats which are expected to have severe oxidative stress, EGCG treatment causes
much more blood glucose elevation during IPGTT compared with non-EGCG-treated, STZ-
control rats (Yun et al., 2006; Figure 1). In histological examination, the pancreatic beta cells
in the EGCG-treated rats are more damaged than in the control (Yun et al., 2006). This result
may suggest that EGCG may act as a prooxidant rather than as an antioxidant at least in the
rat beta cells conditioned with STZ. The plasma EGCG concentration achievable by the
injection of 5 mg EGCG is nanomolar levels (Chen et al., 1997; Kao et al., 2000), which is
readily reachable in human plasma by moderate daily green tea consumption.
Green Tea and Type 2 Diabetes 413
Figure 1. Effect of EGCG on change in blood glucose level in response to high glucose loading (1.5
g/kg i.p.) in control and streptozotocin-treated diabetic rats. EGCG was intraperitoneally injected in
each diabetic rat at a dose of 0 to 50 mg/kg/day for 4 consecutive days (Yun et al., 2006; Permission
from Elsevier).
ATP-SENSITIVE POTASSIUM (KATP) CHANNELS AND GREEN TEA

Ten micromolar concentration of EGCG can be achievable in human plasma by oral
intake of 15 cups of green tea in the fasting state (Chow et al., 2001; when supposed 100 mg
EGCG per one cup). At this concentration, EGCG reduces ATP sensitivity of beta-cell ATP-
sensitive potassium (KATP) channels (Jin et al., 2007; Figure 2), which are inhibited by
increased intracellular ATP/ADP ratio. It is well known that glucose-induced KATP-channel
inhibition in beta cells triggers insulin secretion, through elevation of membrane potential and
cytosolic Ca2+ levels. The reduced ATP sensitivity of the channel by EGCG may cause
diminished insulin secretion in response to high glucose after a meal. This effect is EGCG-
specific, not demonstrated in other catechins, such as (-)-epigallocatechin (EGC), (-)-
epicatechin (EC) and ECG. The binding sites on the KATP channel for α-, β- and γ-phosphate
tails of ATP are known to be R201, K185 and R50 residues of the cytoplasmic termini on the
inwardly-rectifying potassium channel Kir6.2 (Anticliff et al., 2005). The positively-charged
residues on the channel interact with the negatively-charged phosphate tails of ATP to close
the channel. EGCG possesses many negatively-chargeable hydroxyl groups, and then hinders
the interaction of the channel with ATP. This inhibitory mechanism of EGCG is also
applicable to the binding to the channel of phosphatidylinositol polyphosphates (PIP) (Jin et
al., 2007; Figure 3), which can increase the channel open probability in contrast to ATP. The
positive residues R54 in the N-terminus, R176 and R177 in the C-terminus on Kir6.2 appear
to be PIP-binding sites (Ribalet et al., 2006). The inhibition of PIP-binding to the channel by
EGCG is more effective than of the ATP-binding because the PIP inhibition of EGCG can
occur at EGCG concentration around 1 μM, which is equivalent to 2-3 cups green tea
ingestion in humans. Therefore, there is little possibility that regular daily oral ingestion of
green tea may cause impaired glucose-stimulated insulin secretion by EGCG-mediated
decrease in the channel ATP sensitivity. Reduced PIP sensitivity by 1 μM EGCG is more
relevant, and may hinder cardiac KATP channel activation against hypoxia or ischemia.
However, it appears that cardiac-type KATP channels (Kir6.2/SUR2A) are more sensitive to
EGCG inhibiting the channel ATP sensitivity than beta-cell type KATP channels
(Kir6.2/SUR1). Hence, the inhibitory mechanism of EGCG on the channel ATP sensitivity
may be helpful for prevention of ischemic heart, by increasing the channel open probability.
Which mechanism of EGCG associated with the PIP or ATP binding is dominant in actual
cardiac tissues should be further clearly determined.
Figure 2. Effect of 10 μM EGCG on ATP-induced inhibition of KATP currents expressed in plasma

membrane of Xenopus frog oocytes (Jin et al., 2007; Permission from Elsevier).
Figure 3. Effect of 1 μM EGCG on PIP2-induced activation of KATP (Kir6.2/SUR1) currents expressed

in plasma membrane of Xenopus frog oocytes (Jin et al., 2007; Permission from Elsevier).
INTESTINAL NUTRIENT ABSORPTION AND GREEN TEA

One-hundred micromolar concentration of EGCG is difficult to be reachable in human
plasma, because it may cause severe unexpected side effects in the circulation. Nevertheless,
EGCG at this concentration inhibit the KATP channel allosterically via interacting with cellular
lipid membrane (Baek et al., 2005). ECG as well as EGCG is critical for this action.
Therefore, gallated catechins appear to perturb lipid bilayer, and thus modify the lipid
function itself as well as protein functions embedded in it. The lipid perturbation effects of
gallated catechins are useful for inhibiting micelle formation in the alimentary tract, thereby
decreasing cholesterol absorption into the circulation (Raederstorff et al., 2003). This
mechanism has been emphasized to reveal the beneficial effect of green tea consumption to
prevent obesity. This range of concentration of EGCG can also inhibit Na+-glucose co-
transporters in the alimentary tract, thereby diminishing glucose absorption as well
(Kobayashi et al., 2000). Taken together, green tea catechins, in particular gallated catechins,
seem to be effective in reducing absorption of the body energy source, over-intake of which
causes detrimental metabolic diseases.
GLUCOSE INTOLERANCE AND GREEN TEA

However, problem may arise that gallated catechins also inhibit cellular glucose uptake in
the circulation. A recent report demonstrated that ECG or EGCG inhibits glucose movement
through plasma membrane of red blood cells, competing the binding to glucose transporter
type 1 with glucose (Naftalin et al., 2003). The concentration of gallated catechins to exert
this action is around 100 nM for ECG and 1 μM for EGCG, which is readily achievable in
human plasma. Although additional observation has not been reported to date as for relation
of the gallated catechins with other glucose transporters in the body, it may be associated with
insulin resistance: Normal glucose flow into the cells is a requisite to reduce blood glucose
levels in postprandial period, which is helped by secreted insulin. In case glucose removal is
hindered by EGCG into tissues, greater secretion of insulin is needed to normalize tissue
glucose utilization as well as blood glucose level. Beta-cell overload is a well known factor to
facilitate diabetic progression. Truly, in-blood EGCG at that concentration elevates blood
glucose levels during IPGTT compared to control without EGCG (Jin et al., 2007; Figure 4).
Therefore, the luminal effect to be positive and the circulating effect to be negative of gallated
catechins against the metabolic syndromes should be put together to selectively exploit their
beneficial role. In addition, no matter how green tea is helpful for managing diabetes and
obesity, the effect may be useless at concentration of EGCG beyond its endurable blood level
in humans.
CONCLUSION
Green tea EGCG may act as a prooxidant in diabetic beta cells, which suffer from ROS
increased by chronic hyperglycemia. However, by decreasing ATP sensitivity of KATP
channels, EGCG may help the cardiac tissues with surviving against ischemic or reperfusion
injury. These effects may be relevant because it occurs at EGCG concentration around 1 μM.
Since the beta-cell channel ATP sensitivity is decreased at EGCG around 10 μM, it may not
be problem during regular normal intake of green tea. Biological meaning of PIP sensitivity
change of the KATP channel by EGCG remains to be further determined. The gallated
catechins in the alimentary tract may be helpful for controlling diabetes and obesity by
blocking glucose and cholesterol absorption, but the circulating gallated catechins may be
harmful for diabetes by blocking normal blood glucose utilization of tissues. Selecting
beneficial action of green tea catechins is needed for appropriate future usage of them in
diabetes and obesity.
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M. (2000). Green tea polyphenols inhibit the sodium-dependent glucose transporter of
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(2003). Interactions of androgens, green tea catechins and the antiandrogen flutamide
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Yun, S.Y., Kim, S.P., Song, D.K. (2006). Effects of (-)-epigallocatechin-3-gallate on
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Acknowledgments: Supported by KOSEF (R13-2002-028-03002-0)

Chapter 22
BIOCATALYTIC CONVERSION OF GREEN TEA

CATECHINS TO EPITHEAFLAGALLIN,
EPITHEAFLAGALLIN, 3-O-GALLATE, AND
THEAFLAVINS: PRODUCTION OF
PROMISING FUNCTIONAL FOODS
Nobuya Itoh1,* and Yuji Katsube2

1
Department of Biotechnology, Faculty of Engineering (Biotechnology Research Center),
Toyama Prefectural University, Kurokawa 5180, Imizu, Toyama 939-0398, Japan
2
Kracie Seiyaku, Ltd., 3-1, Kanebo-machi, Takaoka, Toyama 933-0856, Japan
ABSTRACT
World-wide tea production has reached 2.97 x 106 metric tons/year, and more than
75% of tea products are black tea. In recent years, green tea which contain catechin
derivatives such as (-)-epicatechin (EC) (1), (-)-epicatechin gallate (ECg) (2), (-)-
epigallocatechin (EGC) (3), and (-)-epigallocatechin gallate (EGCg) (4) has been
recognized as a useful functional food. However, recent development of enzymatic
processes now makes it possible to produce epitheaflagallin (5), epitheaflagallin 3-O-
gallate (6) and theaflavin (TF) (7) derivatives from catechins, which are components of
black tea. Epitheaflagallin and epitheaflagallin 3-O-gallate are preferentially synthesized
from EGC (3) and EGCg (4) in green tea extracts in the presence of laccase and gallic
acid. Takemoto and colleagues have developed a Camellia sinensis cell culture system
containing peroxidase and hydrolases of ECG (3) and EGCg (4) to produce theaflavin (7)
from tea catechins. These biocatalytic processes allow us to preferentially convert
catechin derivatives in a crude mixture of green tea into different compounds, and, thus,
to improve the composition of the catechins present in green tea.
*
Corresponding author. Tel.: +81-766-56-7500 ext. 560; fax: +81-766-56-2498; e-mail: nbito@pu-toyama.ac.jp
420 Nobuya Itoh and Yuji Katsube
Keywords: Laccase/peroxidase-catalyzed oxidation; epitheaflagallin; epitheaflagallin 3-O-

gallate; theaflavins; green tea catechins; functional food; inhibition of pancreatic lipase
1. LACCASE-MEDIATED CONVERSION OF GREEN TEA CATECHINS TO

EPITHEAFLAGALLIN AND EPITHEAFLAGALLIN 3-O-GALLATE
IN THE PRESENCE OF GALLIC ACID [1]
We recently found that laccase (EC 1.10.3.2) was able to synthesize two benzotropolone
derivatives, epitheaflagallin (5), and epitheaflagallin 3-O-gallate (6) (Figure 1), from crude
tea catechins in the presence of oxygen and gallic acid. These compounds have been
previously synthesized from EGC (3)/EGCg (4) in the presence of catechol/pyrogallol by
Takino and Imagawa. [2] Using purified compounds including (1), (2), (3), (4), we confirmed
that (5) and (6) were synthesized from (3) and (4), respectively, through laccase oxidation
reaction with gallic acid. The reaction involves the preferential oxidation of gallic acid and
the pyrogalloyl group (B ring) in (3) and (4) to the quinone intermediate galloquinone, [3]
followed by the Michael addition of the oxidized pyrogalloyl group in (3)/(4) and subsequent
carbonyl addition across the ring and decarboxylation to yield benzotropolone skeletone.
Recently, Sang et al. reported the enzymatic synthesis of tea theaflavin derivatives from
catechin derivatives in the presence of hydrogen peroxide and peroxidase (EC 1.11.1.7). [4,5]
In peroxidase oxidation, the catechoyl/pyrogalloyl/galloyl groups in EC (1)/ECg (2)/EGC
(3)/EGCg (4) are easily oxidized to form the quinone intermediates that subsequently yield
the various theaflavin-related compounds. In our oxidation system, preferential conversion of
(3) and (4) is possible, and very little EC (1) and ECG (2) reacted with the galloquinone
intermediate in the reaction mixture. Laccase-dependent preferential oxidation of gallic acid
and the pyrogalloyl group in (3) and (4) to the galloquinone intermediate makes it possible to
convert (3) and (4) in crude tea catechins to (5) and (6). The proposed mechanism for this
reaction is supported by the observation that the addition of gallic acid to the reaction mixture
accelerates the production of (5) and (6), as determined by HPLC. We hypothesized that the
low level of production of (5) and (6) in the absence of exogenously supplied gallic acid is
due to free gallic acid in the green tea extract. We also confirmed that a laccase reaction
gradually oxidizes authentic (3)/(4) in the absence of gallic acid to produce other, as yet
uncharacterized, compounds. However, these reactions were suppressed in crude green tea
extracts when gallic acid was added. Matuo et al. reported that (3) was oxidized by a Japanese
pear homogenate containing polyphenol oxidase to yield quinone dimers, one of which was
converted to theaflavin-related compounds. A minor reaction converted the quinine dimer to
(5) and to hydroxytheaflavin. [6] Therefore, laccase oxidation of (3)/(4) in the absence of
gallic acid may form these, or similar, compounds.
Biocatalytic Conversion of Green Tea Catechins … 421
OH OH HO O
HO HO HO OH
OH O O
O
O O
-O C H
OH
OH O
O -CO2
OH
gallic acid H OH
+ C
OH HO O O
O
O O
OR2
HO O -CO2 +H2O
enzymatic O OH
oxidation
OR2 HO OH
OH
OH
OH O
OH O
HO
OH
HO O
R1
OR2
OH
OR2
OH 5: R2=H
6: R2=gallate
EC 1: R1=H, R2=H
ECG 2: R1=H, R2=gallate
EGC 3: R1=OH, R2=H
EGCG 4: R1=OH, R2=gallate
Figure 1. The proposed mechanism of epitheaflagallin (5) and epitheaflagallin 3-O-gallate (6)
formation.
The reaction mixture for the production of (5) and (6) consisted of 1.0 g green tea extract
(Camellia extract 30S, Taiyo Kagaku Corp., Yokkaichi, Mie, Japan), 0.48 g gallic acid
monohydrate, and 10,000 units of laccase from Trametes sp. (DaiwaY120, Amano Enzyme
Co., Ltd., Nagoya, Japan). The reaction was allowed to proceed for 2 h at 45°C without
adjustment of the pH. Enzymatic activity was spectrophotometrically assayed at 30°C by
measuring the formation of quinonimine dye from phenol and 4-aminoantipyrine (pH 4.5) at
505 nm, according to the manufacturer’s protocol. One unit of the enzyme was defined as the
amount that increased the absorbance at 505 nm by 0.1 OD/min. The purified products (5)
(orange in color) and (6) (orange-red in color) were analyzed by MS and NMR and the
structures of them were coincided with the data from the chemically synthesized
epitheaflagallins reported by Takino et al. [7 ]and Nonaka et al. [8] The reaction was not
optimized for each substrate in the crude tea catechins, and the conversion yields of (5)/(6)
were less than 20%, although (3)/(4) in the reaction mixture was entirely consumed. These
data suggest that the reaction did not proceed stoichiometrically, but we have not yet
determined which products were synthesized in addition to (5) and (6). We hypothesized that
highly polymerized compounds of (3)/(4) were formed by oxidation in the presence of gallic
acid, since there were no additional peaks corresponding to small molecules in HPLC
chromatogram under our experimental conditions.
Table 1. Epitheaflagallin (5) and epitheaflagallin-3-O-gallate (6) content

of black tea extracts
Sample1
2
Content A B C D
(mg/g extract)
Epitheaflagallin (5) 0.35 0.46 N.D.3 0.25
Epitheaflagallin-3-O-gallate (6) 0.57 0.84 N.D. 0.47

1
FD/SD black tea extracts with different lot numbers were purchased from San-Ei Gen F.F.I., Inc.,
Osaka, Japan.
2
The amounts of (5) and (6) were calculated from the areas under the peaks on an HPLC chromatogram
using calibration curves prepared from purified (5) and (6).
3
N.D.: not detected.
To detect the presence of (5) and (6) in natural tea products, four kinds of black tea
extract (A-D) were analyzed (Table 1). We confirmed that epitheaflagallin derivatives were
minor components of all the black tea extracts tested, with the exception of one sample. The
black tea extracts contained a mean of approximately 0.1% (w/w) of (5) and (6) together. The
amount of (6) was approximately 1.8 times higher than that of (5) in each of the three
samples. These results indicate that epitheaflagallin derivatives are naturally synthesized as
minor components during the tea fermentation process. [9]
The bio-oxidation process that converts (3) and/or (4) to epitheaflagallin derivatives
could be applied to the production of useful functional food components, as described in
Section 3.
1.1. Epitheaflagallin (5)
Orange solid; 1H-NMR (CD3OD) δΗ (ppm) 2.82, 2.93 (2H, m), 4.27 (1H, m), 4.81 (1H,
s), 5.97, 5.98 (each 1H, d, J=2.4 Hz), 6.89 (1H, s), 7.33 (1H, d, J=1.0 Hz), 7.50 (1H, s), 13C-
NMR (CD3OD) δC (ppm) 29.7, 67.0, 81.5, 95.9, 96.7, 99.8, 111.9, 116.2, 117.1, 133.4, 134.6,
135.6, 135.7, 152.60, 152.65, 155.0, 156.9, 157.8, 158.1, 183.4, ESI-TOF-MS m/z 401.0869
[M+H]+ for C20H16O9, MALDI-TOF-MS m/z 401 [M+H]+, 423 [M+Na] +, E1%1cm 371 (281
nm), 446 (306 nm).
1.2. Epitheaflagallin 3-O-gallate (6)
Deep-orange solid; 1H-NMR (CD3OD) δΗ (ppm) 2.91, 3.07 (2H, m), 5.04 (1H, s), 5.67
(1H, m), 5.99 (1H, d, J=2.2 Hz), 6.02 (1H, d, J=2.2 Hz), 6.64 (1H, s), 6.89 (2H, s), 7.33 (1H,
d, J=1.2 Hz), 7.46 (1H, s), 13C-NMR (CD3OD) δC (ppm) 27.1, 69.3, 80.4, 95.9, 96.9, 99.2,
110.2, 112.0, 116.1, 116.5, 121.1, 133.5, 134.3, 134.5, 135.8, 139.9, 146.4, 152.5, 152.7,
155.1, 156.8, 158.0, 158.1, 167.4, 183.4, ESI-TOF-MS m/z 553.0993 [M+H]+ for C27H20O13,
MALDI-TOF-MS m/z 553 [M+H] +, 575 [M+Na] +, E1%1cm 494 (280 nm), 469 (302 nm).
2. PEROXIDASE-MEDIATED CONVERSION OF GREEN TEA CATECHINS

TO THEAFLAVINS IN CAMELLIA SINENSIS CELL CULTURES [5, 10]
Theaflavins are orange or orange-red in color and possess a benzotoropolone skeleton,

similar to the epitheaflagallins. Theaflavins are major pigments of black tea, which account
for 2-6% of the dry weight of black tea extracts, and consist of four major derivatives:
theaflavin (7), theaflavin 3-O-galate (8), theaflavin 3’-O-galate (9) and theaflavin 3,3’-di-O-
galate (10) (Figure 2).
In 2003, Sang et al. reported a very simple method to produce theaflavin derivatives
using horseradish peroxidase (HRP) and H2O2. [5] HRP is commercially available,
inexpensive, and is able to catalyze the oxidation of tea catechins as efficiently as the
endogenous polyphenol oxidase and peroxidase in the leaves of C. sinensis. The HRP/H2O2
system is a good mimic of pigment formation in black tea fermentation. As shown in Figure
2, the possible mechanism of formation of the benzotropolone skeleton of theaflavins is quite
similar to the formation of epitheaflagallins. In the case of peroxidase oxidation, however, not
only pyrogalloyl but also pyrocatechoyl and/or galloyl groups in the catechins are oxidized to
quinone type intermediates to form the benzotropolone skeleton from the various tea
catechins. For example, theaflavin is synthesized from EC (1) and EGC (3), theaflavin 3-O-
gallate from EC (1) and EGCg (4), theaflavin 3’-O-gallate from ECg (2) and EGC (3), and
theaflavin 3, 3’-di-O-gallate from ECg (2) and EGCg (4). The HRP/H2O2 system is a very
convenient method for obtaining standard theaflavins when purified tea catechins are
available. Although purified tea catechins are available, they are too expensive to produce
functional food components. We applied the HRP/H2O2 system to the production of
epitheaflagallins, but a large amount of purpurogallin was formed as a by-product, which is
not suitable for food materials. Peroxidase catalyzed oxidation is apparently stronger than the
laccase-mediated reaction and, therefore, is difficult to control in the conversion of tea
catechin mixtures.
Recently, Takemoto et al. of Shizuoka Prefectural University, Shizuoka, Japan,
developed a Camellia sinensis cell culture system, which possesses high peroxidase activity,
and can convert EC (1) and EGC (3) in green tea into theaflavin (TF, 7). This callus cell
culture readily converts (1) and (3) to TF when H2O2 (at a final concentration of 0.045%) is
added to reaction mixture, consisting of 6 mg (1), 6 mg (3), 1 ml acetone, 15 ml of 0.1 M
phosphate buffer (pH 6.0), 2.5 ml of C. sinensis callus cell culture broth (peroxidase, 15.5
U/ml), 2.5 ml Gamborg’s (B5) medium (total volume, 20 ml). The reaction was allowed to
proceed for 30 min and the yield of (7) was 48%. This process was the same as the method
reported by Sang et al. in terms of the peroxidase oxidation of purified catechin derivatives.
Takemoto and colleagues focused on increasing the yield of (7) from green tea extract. These
extracts usually contains many catechin derivatives, but EC (1), ECg (2), EGC (3), and EGCg
(4) make up ~10% (w/w), ~10%, >20%, ~50%, respectively, of the total catechin. Thus, the
amount of (1) and (3) in green tea extracts are not sufficient to produce high amount of (7).
Thus, Takemoto et al. developed C. sinensis callus cells, which possess not only peroxidase
activity, but also a tannase-like (EC 3.1.1.20) hydrolase activity. The latter enzyme catalyzes
the hydrolysis of ECg (2) to EC (1) and gallic acid, and EGCg (4) to EGC (3) and gallic acid
(Figure 2). As a result, production of (7) from the green tea extract increased to levels suitable
for practical production of (7) from green tea extracts. [11]
3. POTENTIAL ANTI-OBESITY BENEFITS OF EPITHEAFLAGALLIN 3-O-

GALLATE AND THEAFLAVIN 3-O-GALLATE
THAT INHIBIT PANCREATIC LIPASE [12]
There has been little physiological information regarding the effects of epitheaflagallins
on human health. Epitheaflagallin 3-O-gallate (6) inhibits some inflammation-related matrix
metallo-proteases (MMP-2,7, MT1-MMP), resulting in an anti-inflammatory effect, [13] and
also possesses growth inhibitory activity toward human colon cancer cells. [5] In contrast,
theaflavins, as well as EGCg in green tea, have attracted considerable interest because they
possess potential benefits for human health, including antioxidant, [14] and antiviral [15,16]
activities, inhibition of α-/β-glucosidase (anti-diabetic effect), [17] suppression of
cytochrome P-450 activity (anti-mutagenicity), [18] inhibition of angiotensin converting
enzyme (ACE) activity, [19] and cancer-preventing activity. [20] We have hypothesized that
epitheaflagallins, especially epitheaflagallin 3-O-gallate (6), possess beneficial effects on
human health as well, because of their structural similarity to TFs and (4). Recently, many
beneficial effects of green tea have been attributed to its abundant catechin, EGCg (4), [21]
and 3 O-gallate forms of catechins. In 2005, we initiated research to elucidate the functions of
epitheaflagallins in human health. During the course of our study, we found that (6) and TF 3-
O-gallate (8) can inhibit pancreatic lipase. Figure 3 displays the dose-dependent inhibition of
pancreatic lipase by the laccase-treated green tea catechins, the purified (6) and (8). Xenical,
an anti-obesity drug which strongly inhibits pancreatic lipase, was used as a positive control.
Although the effects of (6) (observed IC50: 1.2 mg/ml) and (8) (observed IC50: 0.4 mg/ml)
were less dramatic than Xenical (observed IC50: 0.04 mg/ml), these compounds may still be
suitable functional foods able to suppress the absorption of lipids during digestion.
Interestingly, EGCg (4) and epitheaflagallin (5) had no effect on pancreatic lipase inhibition,
indicating that the benzotropolone ring and the 3-O-gallate structure are necessary to inhibit
pancreatic lipase. Figure 4 shows the changes in triglyceride concentration in the serum of
rats after oral administration of lipid emulsions to rats that had been fasted overnight and then
given the lipids in the presence or absence of laccase-treated green tea extract or (6).
Epitheaflagallin 3-O-gallate (6) exhibited an inhibitory effect on lipid absorption in vivo in
rats. In Japan, black oolong tea is commercially available as a food for qualified health use to
suppress the absorption of dietary lipids. As epitheaflagallins and TFs have a mild, black tea-
like taste, a new tea drink rich in (6) and (8) could be popular for sale in Japan in the near
future. Our research group has also confirmed the inhibitory effects of epitheaflagallins on -
glucosidases and on metastasis of human colon cancer. The detailed results concerning the
beneficial effects of epitheaflagallins will be reported elsewhere.
O OH
OH
O OR2
OH
OH
HO O HO
OH
H 2O 2 +
OH
OH
P eroxidas e O
HO
E C (1)
OH
OH
OR1
OH O OH
O
O T F (7): R 1 =H, R 2 =H
HO
OH T F 3‐O ‐gallate: R 1 =gallate, R 2=H
OH T F 3’‐gallate: R 1=H, R 2 =gallate
OH T F 3,3’‐di‐O ‐gallate: R 1 , R 2 =gallate
OH
E G C (2)
H 2O 2 +
T annas e/internal hydrolas e P eroxidas e
E C g (3) E C (1)
E G C g (4) E G C (2)
Figure 2. Schematic diagram of the process to produce TF from green tea extract using C. sinensis cell
cultures.
Figure 3. Inhibition of pancreatic lipase by epitheaflagallin and TF derivatives.

riglyceride concentration
160
140
120
100
80
(mg/dl)
60
40
Changes in serum
20
0
-20
0 50 100 150 200 250 300 350
-40
Time after administration of lipid emulsion (min)
control epitheaflagallin 3-O-gallate (20 mg/kg)
laccase-treated green tea (500 mg/kg)
Figure 4. Inhibitory effects of epitheaflagallins on absorption of triglycerides in rats.
ACKNOWLEDGEMENTS
This work was supported by a grant for the “Toyama Medical Bio-Cluster” from The
Ministry of Education, Culture, Sports, Science and Technology (MEXT) of Japan.
REFERENCES
[1] Itoh, N.; Katsube, Y.; Yamamoto, K.; Nakajima, N.; Yoshida, K. (2007) Laccase-
catalysed conversion of green tea catechins in the presence of gallic acid to
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leaf catechins. Agric. Biol. Chem., 28, 125-130.
[3] Takino, Y.; Imagawa. H.; Aoki, Y.; Ozawa, T. (1963) Studies on the mechanism of the
oxidation of tea leaf catechins. Agric. Biol. Chem., 27, 562-568.
[4] Sang, S.; Tian, S.; Meng, X.; Stark, R.E.; Rosen, R.T.; Yang, C.S; Ho, C.-T. (2002)
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epicatechin and (−)-epigallocatechin gallate and characterized from black tea using
LC/MS/MS. Tetrahedron Lett., 43, 7129-7133.
[5] Sang, S.; Lambert, J.D.; Tian, S,; Hong, J.; Hou, Z.; Ryu, J.-H.; Stark, R.E.; Rosen,
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derivatives and their anti-inflammatory and cytotoxic activities. Bioorg. Med. Chem.,
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[6] Matsuo, Y.; Tanaka, T.; Kouno, I. (2006) A new mechanism for oxidation of
epigallocatechin and production of benzotropolone pigments. Tetrahedron, 62, 4774-
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[7] Takino, Y.; Ferretti, A.; Flanagan, V.; Gianturco, M.A.; Vogel, M. (1967) Spectral
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[10] Takemoto, M. (2007) JPN Patent Application, No. 2007-143461 (Japanese).
[11] Takemoto, M. (2007) JPN Patent Application, No. 2007-182217 (Japanese).
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Patent Application, No. 2007-285650 (Japanese).
[13] Akizawa, T.; Yahara, M.; Hashimoto, F. (2000) JPN Patent Application, No. 2000-
226329 (Japanese).
[14] Shiraki, M.; Hara, Y.; Osawa, T.; Kumon, H.; Nakayama, T.; Kawakishi, S. (1994)
Antioxidative and antimutagenic effects of theaflavins of black tea. Mutation Res., 323,
29-34.
[15] Nakayama, M.; Suzuki, K.; Toda, M.; Okubo, S.; Hara, Y.; Shimamura, T. (1993)
Inhibition of the infectivity of influenza virus by tea polyphenols. Antiviral Res., 21,
289-99.
[16] Ono, K.; Nakane, H.; Fukushima, M.; Chermann, J.C.; Barré-Sinoussi, F. (1991)
Inhibition of reverse transcriptase activity by a flavonoid compound, 5,6,7-
trihydroxyflavone. Biochem. Biophys. Res. Commun., 174, 56-62.
[17] Honda, M.; Hara, Y. (1993) Inhibition of rat small intestinal sucrose and α-glucosidase
activities by tea polyphenols. Biosci. Biotech. Biochem., 57, 123-124.
[18] Apostolides, Z.; Balentine, D.A.; Harbowy, M.E.; Hara, Y.; Weisburger, J.H. (1997)
Inhibition of PhIP mutagenicity by catechins, and by theaflavins and gallate esters.
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[19] Hara, Y.; Matsuzaki, T.; Suzuki, T. (1987) Angiotensin I converting enzyme inhibiting
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[20] Matsumoto, N.; Kori, T.; Okushio, K.; Hara, Y. (1996) Inhibitory effects of tea
catechins, black tea extract and oolong tea extract on hepatocarcinogenesis in rat. Jpn.
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[21] Liao, A; Kao, Y.-H.; Hiipakka, R. A. (2001) Green tea: biochemical and biological
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Chapter 23
PREVENTIVE EFFECTS OF
GREEN TEA CATECHINS ON DEMENTIA
Michio Hashimoto1,*, Md Abdul Haque1,

Kohinoor Begum Himi1 and Yukihiko Hara2
1
Department of Environmental Physiology, Shimane University Faculty of Medicine,
Izumo, Shimane 693-8501, Japan
2
Mitsui Norin Co., Ltd., Nishishinbashi, Minato-ku, Tokyo 105-8427, Japan
ABSTRACT
Tea is rich in polyphenols which are contained in the leaves and stems of the tea
plant. (-)-Epigallocatechin gallate (EGCG), the major and most active component of
green tea catechins, acts as an antioxidant in the biological system, and is absorbed and
distributed mainly into the mucous membranes of the small intestine and liver; more
interestingly it can cross the blood brain barrier. Oxidative stress, a condition of cellular
prooxidant-antioxidant disturbance which favors the prooxidant state, induces the
production of lipid peroxide (LPO), reactive oxygen species (ROS) and free radicals in
membrane lipids. Oxidative stress causes deterioration of a wide variety of cellular
enzymes, subsequently exacerbating neurodegenerative process.
Aging leads to a decline in memory-related learning ability. Oxidative damage to the
brain is associated with age-related cognitive dysfunction but some antioxidants are
effective in alleviating this dysfunction for Alzheimer’s disease (AD) model animals.
Moreover, a decrease in hippocampal LPO level improves spatial cognition learning in
aged rats and an increase in antioxidative activity in the hippocampus prevents or
ameliorates the impairment of learning ability in AD model rats produced by the infusion
of amyloid-β (1-40) (Aβ1-40) into their cerebral ventricle. Catechins have a protective
effect against age-related neurological diseases caused by oxidative damage.
Epidemiological studies report that a higher consumption of green tea is associated with
lower prevalence of cognitive impairment in elderly people. This chapter describes the
*
Corresponding author: Michio Hashimoto. Department of Environmental Physiology, Shimane University
Faculty of Medicine, Izumo, Shimane 693-8501, Japan; E-mail: michio1@med.shimane-u.ac.jp. Telephone:
+81 853 20 2112; Fax: +81 853 20 2110.
430 Michio Hashimoto, Md Abdul Haque, Kohinoor Begum Himi et al.
beneficial effects of green tea catechins on neuronal functions and neuronal diseases such
as dementia.
INTRODUCTION
Tea is the most widely consumed beverage in the world (mean consumption:
approximately 120 ml/day/percapita) [McKay and Blumberg, 2002]. It has only recently been
studied extensively as a health-promoting beverage to prevent a number of chronic diseases
such as cancer [Inoue et al., 1998; Mittal et al., 2004], cardiovascular diseases [Geleijnse et
al., 1999; Negishi et al., 2004], diabetes [Anderson and Polansky, 2002; Wu et al., 2004a;
2004b] and neurodegenerative diseases including Parkinson’s disease (PD) [Choi et al., 2002;
Pan et al., 2003; Weinreb et al., 2004] and Alzheimer’s disease (AD) [Levites et al., 2003;
Mandel et al., 2005; Bastianetto et al., 2006]. Oxidative damage to neuronal biomolecules
such as DNA, proteins, and lipids causes the aging process and contributes to the
pathogenesis of various neurodegenerative disorders [Halliwell, 1989] including age-related
decline in learning and memory [Foster, 2006]. Oxidative stress is clearly evident in AD brain
manifested by lipid peroxidation (LPO) and protein oxidation, along with other markers of
oxidative damages [Butterfield et al., 2007]. Most individuals with amnestic mild cognitive
impairment (MCI), an earliest phase of AD [Petersen et al., 2001; Petersen, 2004], also
demonstrate elevated oxidative stress markers in their brain [Keller et al., 2005; Wang et al.,
2006; Bufferfield et al., 2006; 2007].
Hippocampus is an indispensable part of the brain in order to form spatial memory and it
is one of the first regions to suffer oxidative damage in AD [Mortimer et al., 2004]. Spatial
cognition-related memory refers to the ability to recall previously navigated cues oriented in a
specific arrangement in an individual’s environment. Any lesion or damage in the
hippocampus has been reported to compromise the spatial learning in rats. According to
cognitive map hypothesis, an intact hippocampal function is the prerequisite for
accumulating, processing and storing spatial information of an individual’s environment
[Jeffery and Hayman, 2004]. Infusion of amyloid β peptide (1-40) (Aβ1-40) into rat brain
increases the concentrations of LPO and reactive oxygen species (ROS), and correlates with
an impairment of reference and working memory; this indicates a deficit in spatial learning
ability, a well-known characteristic of AD [Hashimoto et al., 2002; 2005a].
While tea contains a number of bioactive chemicals, it is particularly rich in polyphenolic
compounds known as catechins which contribute to the beneficial effects ascribed to tea. The
principal catechins in green tea are (-)-epicatechins (EC), (-)-epicatechin gallate (ECG), (-)-
epigallocatechin (EGC), and (-)-epigallocatechin gallate (EGCG). Among them EGCG is the
most abundant and active component of green tea. It can pass the blood brain barrier
[Suganuma et al., 1998; Nakagawa et al., 1997a; Abd EI Mohsen et al., 2002] and causes
neuroprotective activity. Among different biological properties of green tea catechins, the
potent antioxidant and radical scavenging properties have led to them a number of diseases
associated with oxidative insults. It has been demonstrated that EGCG protects neurons
against conditions such as hypoxia [Burckhardt et al., 2008], ischemia [Lee et al., 2000; Lee
et al., 2004], AD [Mandel et al., 2005], and PD [Checkoway et al., 2002; Tan et al., 2003].
Epidemiological and nutritional studies demonstrate that a higher consumption of green tea
Preventive Effects of Green Tea Catechins on Dementia 431
(two cups/day or more) lowers the prevalence of cognitive impairment in elderly people
[Kuriyama et al., 2006] and reduces risk of PD [Checkoway et al., 2002]. In animal studies,
administration of green tea catechins improves spatial cognitive learning ability in normal
young rats [Haque et al., 2006] and prevents impairment of learning ability in AD model rats
[Haque et al., 2008]. Green tea extract and EGCG also effectively prevent N-methyl-4-
phenyl-1,2,3,6-tetrahydropyridine-induced mouse striatal dopamine depletion and substantia
nigra dopaminergic neuron loss [Levites et al., 2001], and suppress cognitive impairment in
aged SAMP10 mice, a model of brain senescence with cerebral atrophy and cognitive
dysfunction [Unno et al., 2004]. We addressed the beneficial effects of the green tea catechins
on neuronal functions with special emphasis on dementia.
SOURCES, ABSORPTION AND DISTRIBUTION OF GREEN TEA

Tea belongs to the Theacease family and comes from two main varieties: Camellia
sinensis var. sinensis and Camellia sinensis var. assamicsa. Depending on the manufacturing
process, teas are classified into three major types: ‘non-fermented’ green tea (produced by
drying and steaming the fresh leaves); ‘semi-fermented’ oolong tea (produced when the fresh
leaves are subjected to a partial fermentation stage before drying); and ‘fermented’ black tea
(post-harvest fermentation stage before drying and steaming). The mean chemical
composition of green tea leaves are as follows (% of dry weight of tea leaves): 15 proteins, 4
aminoacids, 26 fibres, 7 other carbohydrates, 7 lipids, 2 pigments, 5 minerals, 30 phenolic
compounds (especially flavonoids). The main flavonoids present in green tea include
catechins (flavan-3-ols), and the four major catechins are EGCG (approximately 60% of the
total catechins), EGC (approximately 19%); ECG (approximately 13.6%) and EC
(approximately 6.4%).
In Figure 1, a possible metabolic route of EGCG is shown. When oral administration of
EGCG to rats, part of EGCG is absorbed in the intestine and enters into liver via the portal
vein [Okushio et al., 1995; 1996]. During this process most of the EGCG undergoes
conjugation in the intestinal mucosa and/or in the liver, and a part of the EGCG is further
methylated in the liver. Most of the EGCG metabolites are then excreted in the bile [Kida et
al., 2000] and a part of the EGCG enters the blood circulation, peaking at 1-2 h after
administration [Unno and Takeo, 1995; Nakagawa et al., 1997b, Chen et al., 1997]. On the
other hand, most of the unabsorbed EGCG moves into the cecum and large intestine and then
undergoes degradation by intestinal bacteria to 5-(3', 5'-dihydroxyphenyl)-γ-valerolactone
(M-1) with EGC as an intermediate. A great part of M-1 is absorbed in the body, undergoing
glucuronidation in the intestinal mucosa and/or liver, to form 5-(5'-hydroxyphenyl)-γ-
valerolactone 3'-O-β-glucuronide (M-2), which enters the blood circulation is distributed to
various tissues, and finally excreted in the urine. The occurrence of tea catechin EGCG in free
form in the various tissue organelles of rats is sown in Table 1.
intestine portal vein

blood circulation
bioavailability < 0.3%

EGCG
EGCG tissue < 0.2%
EGCG-conj
EGC+ GA bile (6%)
EGCG
M-1 M-2
EGCG-conj
Me-EGCG-conj
liver
kidney
M-1
M-2
EGCG, ECG, M-2

M-1
feces (35%) urine (32%)
Figure 1. Possible metabolic route of epigallocatechin-3-gallate (EGCG) orally administered to rats.

GA, gallic acid; M-1, 5-(3',5',-dihydroxyphenyl)-γ-valerolactone; M-2, 5-(5'-hydroxyphenyl)-γ-
valerolactone 3'-O-β-glucuronide; EGCG-conj, EGCG conjugates; Me-EGCG-conj, Methylated EGCG
conjugates. Data from Kohir et al., (2001).
Table 1. EGCG detected in blood and tissue organelles of rats
Tissue 60 min after EGCG detected c

administration a (%)
Plasma (nmol/mL) 12.3 ± 6.2 0.024 b
Liver (nmol/g) 48.4 ± 19.7 0.19 b
Brain (nmol/g) 0.5 ± 0.3 0.0003 b
Small intestine mucosa (nmol/g) 565 ± 153 0.45 b
Colon mucosa (nmol/g) 68.6 ± 40.4 0.013 b
Data are expressed as the mean ± SD of six rats. EGCG: (-)-epigallocatechin gallate, a Rats received a
single oral administration of EGCG (500 mg/Kg body weight) after 24 h of food deprivation.
b
Percentage against ingested EGCG. c Calculated from the blood mass and from the tissue weights of
rats. Data from Nakagawa and Miyazawa, (1997a).
OXIDATIVE STRESS AND NEURODEGENERATION

Oxidative stress is a condition of cellular oxidant-antioxidant imbalance in favor of
prooxidant state, i.e a relative increase in the ratio of free radicals to antioxidants [Markesbery
1997; van Rensburg et al., 2006]. Patients with neurodegenerative diseases, such as AD, PD
and amyotrophic lateral sclerosis (ALS), clearly exhibit higher oxidative stress (ROS
concentrations) in affected brain regions. The brain zones demonstrating the highest levels of
oxidative stress are typically the areas most structurally affected by diseases: hippocampus,
amygdale, parietal cortex, and other neocortical zones [Markesbery, 1997; Butterfield et al.,
2007]. Others tissues of AD patients also manifest oxidative stress. van Rensburg et al. [2006]
reported that the blood of AD patients demonstrates increased oxidative stress and abnormally
poor antioxidant status compared to healthy controls.
According to the free-radical theory of AD, an increased production of LPO and ROS,
which are produced with free radicals in membrane lipids, causes deterioration of a wide
variety of cellular enzymes, subsequently exacerbating the neurodegenerative process [Yatin
et al., 1999]. A lower LPO level in hippocampus is inversely associated with spatial cognition
learning memory both in young and aged rats [Gamoh et al., 1999; 2001]. In AD brain,
markers for lipid peroxidation such as 4-hydroxynonenal (4-HNE) and malondialdehyde, and
marker of protein oxidation such as nitrated proteins are identified in cerebral cortex and
hippocampus. 4-HNE can diffuse from the site of its production, potentially modify neuronal
organelles and changes their functions [Butterfield et al., 1997]. Approximately 2-5% of the
oxygen consumed by a cell is subsequently converted to free radicals [Floyd and Hensley,
2002; Wickens 2001]. The brain is poor in antioxidative enzymes such as catalase,
glutathione peroxidase and superoxide dismutase, compared with other organs of the body
such as the heart, liver, and kidneys [Mizuno and Ohta, 1986; Cohen, 1988]. On the other
hand, the brain alone utilizes 20% of total oxygen demand of the body [Cui et at., 2004].
Therefore, the damaging effects are dependent obviously on the balance between cellular
oxidative damage and the antioxidative defenses.
AD is histochemically characterized by the deposition of amyloid β peptide (Aβ) in the
hippocampus as well as in the cerebral cortex, and also by neuronal degeneration and loss of
cognition [Wilcock and Esiri, 1982; Selkoe, 2001]. Genetic mutations and other mechanisms
that potentially lead to increase Aβ deposition contribute to neurotoxicity. Though it is not
conclusive how exactly Aβ contributes to the disease process, oxidative stress is believed to
involve the mechanism of Aβ-induced neurotoxicity [Behl et al., 1992; 1994; Butterfield et
al., 1994; Schubert et al., 1995] and the pathogenesis of AD [Markesbery 1997; Yankner
1996]. Aβ causes lipid peroxidation in brain cell membranes [Behl et al., 1994; Harris et al.,
1995; Mark et al., 1999], alters the conformation of neuronal membrane proteins
[Subramaniam et al., 1997; Pocernich et al., 2001] and induces disorder of cellular functions.
2.00 < 3 cups/wk (reference)
Odds ratios of cognitive impairment

1.80 4-6 cups/wk or 1 cup/d
1.60 > 2 cups/d
1.40 P for trend = 0.0006
1.20
1.00
0.80 *
0.60
0.40
0.20
0.00
n=170 n=108 n=725
Figure 2. Odds ratios (ORs) for the association between different frequencies of green tea consumption
and cognitive impairment. The bars indicate adjusted ORs for the association between green tea
consumption frequencies and cognitive impairment; error bar represent the corresponding 95% CIs.
Multivariate logistic regression analysis was used to calculate ORs for cognitive impairment relative to
the consumption frequencies of green tea, with the lowest frequency category (≤3 cups/wk) treated as
the reference group. Cognitive impairment was defined as a Mini-Mental State Examination score < 26.
*P< 0.001. 1 cup = 0.1L. Data from Kuriyama et al., (2006).
GREEN TEA CATECHINS AND BRAIN FUNCTION
Influence of Green Tea Catechins on Cognitive Function
Green tea catechins currently show a profound beneficial effect on cognitive function
both in animal and humans. An epidemiological cross-sectional study involving 1003
Japanese subjects 70 years old or older, demonstrated the relationship between the
consumption of green tea and cognitive function. The study clearly showed that a higher
consumption of green tea is associated with lower prevalence of cognitive impairment in
humans [Kuriyama et al., 2006]. Drinking more than 2 cups a day of green tea slashed odds of
cognitive impairment in elderly Japanese men and women by 64% (Figure 2). The results
might partly explain the relatively lower prevalence of dementia, especially AD, in Japan
compared to Europe and North America [Ritchie and Lovestone, 2002].
A B
4 4 Control (n=8)
Reference Memory Errors

Catechins (n=9)
Working Memory Errors

3 3
2 2
a
1 b 1
a
b
0 0
0 2 4 6 8 10 0 2 4 6 8 10
Blocks of Six Trials Blocks of Six Trials
Figure 3. Reference (A) and working (B) memory-related learning ability in the radial maze task of rats
administered water alone (control, n = 8), or green tea catechins (0.5% polyphenon E; n = 9) for 26 wk.
Values are means ± SEM in each block of six trials. Groups without a common letter differ, P < 0.05.
Data from Haque et al., (2006).
In animal experiments with rats, long-term administration of green tea catechins in the
form of Polyphenon E (PE: EGCG 63%; EC 11%; EGC 6%; ECG 6%) mixed with water
(0.5% w/v) improved spatial cognition learning ability when measuring eight-arm radial maze
task (Figure 3). The radial maze estimates two types of memory function, reference and
working memory without any harmful stress to the rats. Reference memory involves utilizing
information that remains constant over time whereas working memory involves holding
information that is pertinent only within a short period of time. The lower numbers of
reference memory errors (RMEs; entry into unbaited arm within one trial) and working
memory errors (WMEs; repeated entry into arms that had already been visited within same
trial) implies a higher acquisition of spatial learning ability in rats. 0.5% PE-administered rats
had significantly lower LPO levels both in plasma and hippocampus and higher ferric
reducing antioxidant power (FRAP) levels (an indicator of plasma antioxidant status) (Table
2). A significant positive correlation between the hippocampal LPO levels and the number of
RMEs, and a negative correlation between plasma FRAP levels and the number of RMEs
were observed in block 10 of the radial maze task in controls and in 0.5% PE-administered
rats (Figure 4). These results indicate that the lower LPO and higher FRAP levels, combined
with higher acquisition of memory performance, are likely to be the effects of PE on
scavenging and/or preventing radical formation at the neuronal level. Similarly, it is reported
that dietary administration of green tea catechins prevents memory regression and DNA
oxidative damage in aged mice [Unno et al., 2007]. Thus, these findings suggest that
continued intake of green tea catechins might promote healthy ageing of the brain in older
persons.
A B
r = 0.520 r = - 0.570
3.0 p = 0.017 3.0.
p = 0.032
Reference M emory Errors

Reference M emory Errors
2.0 2.0
1.0 1.0
Control (n=8)
Catechins (n=9)
0.0 0.0
0 0.25 0.50 0.75 1 150 200 250 300 350
TBARS levels in hippocampus FRAP levels in plasma
(nmol/mg protein) ( mol/L)
Figure 4. Correlations between the numbers of reference memory errors and each of the hippocampal
TBARS (Figure 4A) and the plasma FRAP (Figure 4B) levels in controls and green tea catechins-
administered rats. (o), control rats; (●), green tea catechins (0.5% polyphenon E)-administered rats.
Data from Haque et al., (2006).
Table 2. Effects of green tea catechins (0.5% PE) administration on oxidative status of
plasma, cerebral cortex and hippocampus in rats
Plasma Cerebral cortex Hippocampus
TBARS FRAP TBARS ROS TBARS ROS
Control 4.0 ± 224 ± 1.45 ± 0.21 ± 0.63 ± 0.13 ±

(n=8) 0.1 10 0.10 0.03 0.09 0.03
Catechins 3.0 ± 271 ± 1.27 ± 0.19 ± 0.33 ± 0.05 ±

(n=9) 0.1* 11* 0.08 0.04 0.03* 0.01*
Values are means ± SEM. Rats were orally administered either water (control rats) or green tea
catechins (Polyphenon E, PE: EGCG 63%; EC 11%; EGC 6%; ECG 6%, catechins rats) for 26 weeks.
The levels of lipid peroxide were measured as TBARS (thiobarbituric acid reactive substance) indicated
in nmol malondialdehyde/mL for plasma and nmol malondialdehyde/mg protein for brain tissues.
Reactive oxygen species (ROS) is indicated in pmol/min/mg protein. The antioxidant potential of
plasma was mesured as ferric reducing antioxidation power of plasma (FRAP) is indicated in μmol/L.
*P<0.05. Data from Haque et al., (2006).
B Aβ (n=13)
A
3.5 4.0 Catechins + Aβ (n=12)
Reference Memory Errors
Working Memory Errors

3.0 3.0
a
2.5 2.0
b a
2.0 1.0
b
1.5 0.0
0 1 2 3 4 5 6 0 1 2 3 4 5 6
Blocks of Six Trials Blocks of Six Trials
Figure 5. Effects of green tea catechins (0.5% polyphenon E) preadministration to Aβ1–40-infused rats
on reference and working memory-related learning ability in the eight-arm radial maze task. Each value
represents the numbers of reference memory errors (A) and that of working memory errors (B) as the
means ± SEM in each block of six trials. Groups without a common letter for the main effects are
significantly different at P<0.05. Data from Haque et al., (2008).
Preventive Effect of Green Tea Catechins on Cognitive Impairment
We reported previously that preadministration of docosahexaenoic acid protects against

the impairment of learning ability in an animal model of AD, rats infused with Aβ1–40 into the
cerebral ventricle [Hashimoto et al., 2002]. It has been demonstrated that long-term
preadministration of PE prevents cognitive impairment in AD model rats (Figure 5). The
preventive effect of green tea is measured by an eight-arm radial maze. Randomized two-
factor (block and group) ANOVA demonstrates significant main effects of blocks of trials
and groups on the number of RMEs and that of WMEs, indicating that PE preadministration
prevents Aβ1–40-infused learning impairment of AD model rats. Chronic administration of
EGCG prevents cognitive impairment in Alzheimer’s transgenic mice [Rezai-Zadeh et al.,
2008]. As described previously, an epidemiological Japanese study also showed that a higher
consumption of green tea is associated with lower prevalence of cognitive impairment in
humans [Kuriyama et al., 2006]. Therefore, these findings suggest that green tea catechins
might be an effective prophylaxis for cognitive impairment in AD patients.
Antioxidant Effects of Green Tea Affect Brain Function
The impact of an antioxidant rich diet and specific food groups on degenerative diseases
has been widely recognized. It has been reported that a decrease in LPO level or an increase
in antioxidative defense in hippocampus prevents and/or ameliorates the impairment of
learning ability in Aβ1-40-infused AD model rats [Hashimoto et al., 2002; 2005a]. The
infusion of Aβ1-40 increases the cortico-hippocampal LPO and ROS concentrations in the AD
rats, while PE preadministration suppresses these increase of LPO and ROS concentrations
(Table 3). Regression analysis revealed inverse correlations between plasma EGCG
concentrations and hippocamapl ROS levels (Figure 6), suggesting that the antioxidative
action of EGCG is involved in reducing the Aβ-induced oxidative stress in the hippocampus.
Significantly positive correlations between the numbers of RMEs and the levels of TBARS in
plasma and hippocampus and between the numbers of WMEs and the ROS levels in
hippocampus were found (Table 4). In addition, PE preadministration increased plasma
FRAP levels and the FRAP values were negatively correlated with the numbers of WMEs
(Table 4). These results suggest that the antioxidative action of PE is involved in the
prevention of cognitive impairment in AD model rats.
Catechins
1.25 Catechins + A
1.00
(pmol/min/ mg protein)
Hippocampal ROS
0.75
0.50
r = - 0.586
0.25 P = 0.007
0.00
0 10 20 30 40 50
Plasma EGCG (ng/ml)
Figure 6. Relationship between plasma EGCG concentrations and hippocampal ROS levels after long-
term administration of green tea catechins (0.5% polyphenon E).
Table 3. Oxidative status of cerebral cortex and hippocampus of Aβ and PE+Aβ rats
TBARS ROS
(nmol/mg protein) (pmol/min/mg protein)
Cerebral cortex
A (n=13) 3.13 ± 0.23 0.223 ± 0.02
PE + A (n=12) 2.74 ± 0.22* 0.130 ± 0.02*
Hippocampus
A (n=13) 3.54 ± 0.05 0.332 ± 0.03
PE + A (n=12) 2.10 ± 0.13* 0.162 ± 0.01*
Values are means ±SEM. Rats were orally administered either water or green tea catechins (Polyphenon
E, PE). Twenty weeks after the PE administration, rats were infused with amyloid β 1-40 peptide (Aβ1-
40 ) into cerebral ventricle. Aβ, the water-preadmiistered Aβ1-40-infused rats; PE+Aβ, 0.5% PE-
preadministered Aβ1-40-infused rats. The levels of lipid peroxide were measured as TBARS
(thiobarbituric acid reactive substance). ROS, Reactive oxygen species. *P<0.05. Data from Haque et
al., (2008).
Table 4. Correlation coefficients between learning ability and oxidative stress on plasma
and brain of rats
Plasma Cerebral cortex Hippocampus
TBARS FRAP TBARS ROS TBARS ROS
RMEs + 0.324 - 0.272 - - + 0.440 + 0.280
P-value 0.023 0.058 NS NS 0.016 0.051
WMEs - - 0.296 - - + 0.245 + 0.292
P-value NS 0.039 NS NS 0.089 0.041
The numbers of reference memory errors (RMEs) and working memory errors (WMEs) in the final
session shown in Figure 5 was used as an indicator of learning ability.The data of TBARS and ROS in
cerebral cortex and hippocampus shown in Table 3 were used. Differences with P < 0.05 were
considered significant NS, not significant. Data from Haque et al., (2008).
GREEN TEA CATECHINS AND ANTIOXIDATIVE ACTION IN BRAIN

Tea catechins are powerful hydrogen-donating antioxidants and free radical scavengers
[Salah et al., 1995; Nanjo et al., 1996; Rice-Evans, 1999]. The potent radical scavenging
properties of green tea polyphenols have been attributed to the presence of the ortho-3′,4′-
dihydroxy moiety in the B ring of their molecule which participates in electron delocalization
and stabilization of the radicals. The gallocatechins possess a trihydroxyl group in the B ring
(3′, 4′, 5′-OH) and the gallates contain a galloyl moiety attached to flavan-3-ol at the 3
position (C ring), adding three more hydroxyl groups, as in the case of ECG and EGCG
(Figure 7). Both the galloyl moiety and the three hydroxyls in the B ring confer better
antioxidant activity [Nanjo et al., 1996]. In addition to their radical scavenging action, green
tea catechins possess well-established metal-chelating properties. This is likely to be mediated
through the 3′,4′-dihydroxyl group in the B ring [Hider et al., 2001] as well as via the gallate
group [Guo et al., 1996; Kumamoto et al., 2001] which neutralize ferric iron to form redox-
inactive iron and protect cells against oxidative damage. Green tea polyphenols have been
found to be more effective antioxidants than vitamins E and C on a molar basis, as indicated
by their reduction potentials (Rice-Evans et al., 1995). In reducing ferrous ion-induced lipid
peroxidation, the IC50 values of several antioxidants are as follows: 3.32 μM for EGCG, 75.65
μM for trolox, 7.63 μM for lipoic acids and 15.48 μM for melatonin (Lee et al., 2003).
OH OH
2 3 4 OH 2 3 4 OH
B B
1 6 5
HO O 1 6 5 HO 8 O
1
8 1 2 7 2
7 A C
6 A C 3
6 3
5 5 4
4
OR1 OR2
OH OH
OH R1= H: (-)- Epicatechin (EC)

R2= H: (-)- Epigallocatechin (EGC)
x = OH R1= x: (-)- Epicatechin gallate (ECG)
OH R2= x: (-)- Epigallocatechin gallate (EGCG)
Figure 7. The basic structural formulas of tea catechins.

Catechins increase the body's endogenous antioxidants to reduce oxidative damage. In

ageing, cells of the nervous system are vulnerable to oxidative damage. The antioxidative
enzymes superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx), and
glutathione reductase (GR) play an important role in preventing oxidative damage. Among
various isoforms of SOD, the mitochondrial manganese-dependent superoxide dismutase
(MnSOD) contributes a major role to detoxify superoxide radicals and prevents neuronal cell
death in the brain [Keller et al., 1998; Gonzalez-Zulueta et al., 1998; Maragos et al., 2000].
SOD neutralizes superoxide radicals to form hydrogen peroxide and molecular oxygen. The
efficacy of SOD as an antioxidant depends on cooperation with other enzymes, such as CAT
and GPx. CAT detoxifies high concentrations of H2O2 [Baud et al., 2004; Kono and
Fridorich, 1982] although its activity is much lower in the brain [Ho et al., 1997]. The activity
of GPx and GR is also important in regulating the level of H2O2 [de Haan et al., 1998;
Dringen et al., 2005]. GPx limits the production of LPO by directly catalyzing the conversion
of peroxidated lipid into lipid alcohol [Christophersen, 1968]. GR indirectly facilitates to
inactivate oxygen radicals [Huang and Philbert, 1996]. A decrease in GR activity increases
GSSG accumulation and results in a depletion in the intracellular GSH pool. This eventually
predisposes the cells towards oxidative stress. We found that six months administration of
green tea catechins to rats enhances gene expression and activity of SOD, CAT, GPx and GR
in brain (unpublished data). These findings are consistent with the report [Kishido et al.,
2007] that consumption of green tea catechin prevents the decline in GPx activity and protein
oxidative damage in ageing mouse brain. One month administration of catechin-containing
antioxidant preparation also demonstrates an increase in SOD activity in the mitochondrial
fraction of the striatum and the midbrain and a decrease in TBARS formation in the cortex
and cerebellum of aged rats [Komatsu and Hiramatsu, 2000].
Phenolic antioxidants activate the expression of some stress-response genes [Chen et al.,
2000], probably binding to the antioxidant regulatory element (ARE) present in the promoter
of their respective genes. The NF-E2-related transcription factor 2 (Nrf2) is known to activate
ARE-mediated gene expression of many antioxidants enzymes [Rushmore et al., 1991]. It has
been reported that the transcriptional activities of the stress-response genes correlate with
increased activity and nuclear binding of the transcription factors Nrf2 to the ARE sequences
contained in their promoters via activation of the MAPK pathway [Owuor and Kong, 2002].
Green tea polyphenols demonstrate the ability to affect cell signaling pathways, in particular
the MAPK signaling pathways inside brain cells which play a critical role in
neurodegenerative diseases. Therefore green tea polyphenols may also protect cells against
toxic insults through MAPK's activation of transcription factors, which provide the neuronal
cells with acquired antioxidant defense capacity to survive against oxidative stress.
The pathological accumulation of Aβ in the brain leads to oxidative damage, neuronal
destruction and, finally to the clinical syndrome of AD. Since a reduction of Aβ level in
cerebral cortex and hippocampus ameliorates the impairment of memory learning in Aβ-
infused AD model rats [Hashimoto et al., 2005b], the regulation of Aβ synthesis would be an
important target to prevent Aβ-induced cognitive impairment. EGCG decreases Aβ1–40,42
levels and attenuates Aβ plaques across the hippocampal and cortical brain regions in
TgAPPsw mice, a mouse model of AD [Rezai-Zadeh et al., 2008].
Figure 8. Possible targets of EGCG to prevent neurodegeneration. EGCG, a main green tea catechin
may increase cell survivability or prevents neuronal cell death either directly preventing oxidative stress
or indirectly modulating MAPK cascade or Aβ synthesis pathways. Long-term administration of EGCG
may down regulate β-secretase 1 (BACE 1) gene expression, up regulates transthyretin (TTR) gene
expression in brain and these combine effects may reduce Aβ levels either inhibiting Aβ production
and/or accelerating Aβ clearance and finally reduce Aβ-induced LPO and ROS levels and prevent
neuronal cell death. EGCG may also activate PKCα and ε, the two specific isoforms of PKC, increase
α-secretase cleavage activity and modulates non amyloidigenic pathway for APP processing. EGCG
may inhibit NFκb signaling pathways, a known pathway for cell death, and decreases apoptosis related-
gene expression such as bax and bad. EGCG may also activate PI3 kinase pathway which is associated
with antiapoptosis signaling in neurons. For details please see text.
The possible targets of EGCG-mediated prevention of neurodegeneration are

diagrammed in Figure 8. EGCG may increase cell survivability or prevent neuronal cell death
in neuronal diseases such as AD either by directly attenuating oxidative stress or indirectly
modulating MAPK cascade or APP processing pathways. APP is the precursor molecule of
Aβ, first cleaved by either α-secretase or β-secretase 1 (BACE1) and the resultant membrane-
attached fragments are processed by γ-secretase (Presinilin 1, 2; PS1, PS2). Up regulation of
BACE1 increases the production of Aβ. Long-term administration of green tea catechins has
been shown to decrease BACE1 gene expression in the hippocampus (unpublished data).
Alternatively, EGCG activates PKCα and ε, the two specific isoforms of PKC which increase
α-secretase cleavage activity and promotes non amyloidigenic pathway for APP processing to
produce and secretes a soluble form of APP (sAPPα) [Levites et al., 2003]. Administration of
green tea catechins up regulates the mRNA expression of transthyretin, a carrier protein of
thyroxin and retinol, in the hippocampus and the cerebral cortex (unpublished data). This
protein also acts as an Aβ scavenger, and prevents Aβ aggregation and plaque formation in
brain [Schwarzman et al., 1994]. Taken together it is suggested that EGCG administration
could reduce Aβ levels either by inhibiting Aβ production and/or accelerating Aβ clearance in
conjunction with facilitating the non-amyloidogenic α-secretase pathway, and the

combination of these effects may lead to reduced Aβ-induced oxidative stress (such as
decrease LPO and ROS levels) and prevent related neuronal cell death. Other cell signaling
pathways may also be affected by green tea to improve neuronal survivability. It has been
reported that EGCG inhibits neuronal translocation of NFκb to the nucleus [Levites et al.,
2002; Aktas et al., 2004], a known pathway for cell death. In addition EGCG decreases
apoptosis related-gene expression such as bax and bad [Weinreb et al., 2003; Kalfon et al.,
2007] and activates PI3/AKt kinase pathway [Koh et al., 2004] which are involved in
antiapoptotic signaling in neurons. From these observations, it is suggested that the potency
which neuronal cells achieve by the antioxidative action of green tea catechins may prevent
oxidative insults, Aβ-induced or otherwise at least facilitating antioxidant defense.
CONCLUSION
Green tea catechins manipulate multiple desired targets in the central nervous system.
Administration of green tea catechins reduces brain oxidative stress and improves spatial
cognitive learning ability in normal rats and prevents cognitive impairment in Aβ-infused AD
model rats. Green tea catechins decrease Aβ levels and Aβ plaques in AD model mice.
Furthermore, catechins increase the activity and gene expression of antioxidant enzymes in
rat brains. All the evidence taken together suggests that green tea catchins strengthen
antioxidant defenses in the brain against oxidative stress, Aβ-induced, and/or otherwise, and it
seems to be a therapeutic strategy in preventing neurodegenerative diseases where oxidative
stress is implicated. However, detailed long-term research is required to clarify the
mechanism of how green tea catechins contribute to the prevention of age-related dementia in
particular to cognitive impairment.
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Short Commentary
GREEN TEA AND POTENTIAL

HUMAN HEALTH EFFECTS
James E. Trosko*
Center for Integrated Toxicology, Food Safety Toxicology Center
Dept. Pediatrics/Human Development, College of Human Medicine
Michigan State University, East Lansing, Michigan 48824, USA
Keywords: Green tea; gap junctions; adult stem cells; cell-cell communication; intra-cell
signaling; anti-oxidants
INTRODUCTION: CAN GREEN TEA ASSIST IN MAINTAINING HEALTH

IN THE COMPLEX HUMAN HOMEOSTATIC –CYBERNETIC,
HIERARCHICAL “MEAT MACHINE?”
The aims of any scientific advice, bearing on whether any nutritional social policy ,
should (a) promote health or avoid harm; (b) have intended beneficiaries of the advice; and
(c) balance the strengths of the scientific evidence with the expected consequences of
scientific advice[ 1]. The intent of this short “Commentary” is directed to examine the
incomplete scientific knowledge bearing on the alleged health benefits of drinking green tea
or taking nutritional supplements of green tea chemical components.
Before one can begin to assess the growing and, in many cases, conflicting studies on the
potential role of green tea’s effect on human health, it is the fundamental assumption of this
“Commentary” that one must understand how the components of green tea might interact
with both endogenous and exogenous toxicant/toxins’ mechanisms of toxicity and how these
mechanisms contribute to the pathogeneses of various chronic diseases. To do this, a brief
*
Correspondence to be sent: James E. Trosko, Ph.D. Dept. Pediatrics/ Human Development, College of Human
Medicine, Michigan State University. East Lansing, Michigan 48824; Phone: 517-884-2053; Fax: 517-432-
6340; E-mail: james.trosko@ht.msu.edu
452 James E. Trosko
overview of philosophical concepts of the biology of human health, including how biological-
and cultural- evolutionally factors have influenced dietary practices, will be made prior to
some scientific concepts of how green tea might influence various disease- contributing
mechanisms.
From the moment of conception of the single fertilized egg, through embryonic-, fetal-,
neonatal-, adolescent- development, mature adult and geriatric periods of life, cell functions
of proliferation, differentiation, apoptosis, adaptive responses of terminally-differentiated
cells and senescence of individual cells contribute to the 100 trillion cells that make up the
multiple tissues, organs, organ systems, that lead to consciousness, behavior, and human
culture. The biological evolutionary advance that allowed the transition from colonies of
single cell organisms to the metazoan, exemplified by the human being, included the genes
that encouraged group cell adherence for the development of new phenotypes of (a) growth
control; (b) cell differentiation; (c) apoptosis; (d) cell senescence; and (e) choice of cell
“mortality” for somatic cells and for the individual or of “immortality” for the germ line for
the species and for adult or somatic stem cells for tissue growth/ repair.
From this grouping of cells that gained specialized physiological functions ( neurons;
hepatocytes; retinal cells; etc.), a delicate orchestration of molecular information transfer,
triggered by the interaction of the information coded in the individual genomes and the
specific historic environmental, dietary, social/cultural factors, had to occur, in order to
prevent disruption of development, of early diseases, and of reproductive success. This is
beautifully illustrated by Markert [2]:
“Cells interact and communicate during embryonic development and through inductive
stimuli mutually direct the divergent courses of their differentiation. Very little cell
differentiation is truly autonomous in vertebrate organisms. The myriad cell phenotypes
present in mammals, for example, must reflect a corresponding complexity in the timing,
nature, and amount of inductive interactions. Whatever the nature of inductive stimuli may be,
they emerge as a consequence of specific sequential interactions of cells during embryonic
development.
The first embryonic cells, blastomeres, of mice and other mammals are all totipotent.
During cleavage and early morphogenesis these cells come to occupy different positions in the
three-dimensional embryo. Some cells are on the outside, some inside. The different
environments of these cells cause the cells to express different patterns of metabolism in
accordance with their own developing programs of gene function. These patterns of
metabolism create new chemical environments for nearby cells and these changed
environments induce yet new programs of gene function in responding cells. Thus a
progressive series of reciprocal interactions is established between the cellular environment
and the genome of each cell. These interactions drive the cell along a specific path of
differentiation until a stable equilibrium is reached in the adult. Thereafter little change occurs
in the specialized cells and they become remarkably refractory to changes in the environment.
They seem stably locked into the terminal patterns of gene function characteristic of adult
cells. The genome seems no longer responsible to the signals that were effective earlier in
development.
Of course, changes can occur in adult cells that lead to renewed cell proliferation and
altered differentiation as seen in neoplasms, both benign and malignant, but such changes are
very rare indeed when one considers the number of cells potentially available for neoplastic
transformation. Possibly, mutations in regulatory DNA of dividing adult cells can
occasionally lead to new and highly effective programs gene function that we recognize as
neoplastic or malignant. However, most genetic changes in adult cells can probably lead to
cell death since random changes in patterns of gene activity are not likely to be beneficial.”
Clearly, this assured species survival but it could not prevent the inevitability of aging,
the diseases of aging and ultimate death of the individual. From the organization of
molecular, biochemical, cellular, tissue, organ, organ system units and the interaction of
physical/social environmental factors, the hierarchical emergence of new phenotypes
occurred [3]. Many systems had to be developed to facilitate the molecular transfer of critical
and specific information to regulate the selective expression of genes in the total genome.
This included the information from (a) the cell- matrix interaction; (b) cell-cell adhesion
molecules; (c) secreted cell communication ions and molecules; ions and selective small
molecules through gap junctional intercellular communication protein channels of coupled
cells.
Within the developing embryo, the existence of pluripotent stem cells, with unlimited
proliferation potential, gave rise to multi-potent stem cells with restricted differentiation
potential. From these stem cells, bi-polar stem cells were generated and from these uni-polar
stem cells one differentiated cell lineage was born. Progenitor cells, born from these adult
stem cells, were committed to proliferate with a finite life span, leading ultimately to terminal
differentiation, apoptosis or senescence.
THE MIS-MATCH OF BIOLOGICAL EVOLUTIONARY MEANS FOR

DIETARY SURVIVAL WITH THE RADIPLY-DEVELOPING
CULTURALLY-EVOLVED MEANS FOR DIETARY SURVIVAL
Given that the human body is hierarchical/cybernetic system of different cell types (stem,
progenitor, and differentiated) that generate tissues, organs and organ systems [4, 5], it should
be clear that biological evolution, utilizing oxygen to generate energy from dietary-supplied,
caloric/nutrient-laden foods, shaped multiple systems to deal with the by-products of
oxidative metabolism, namely the release of reactive oxygen species (ROS). [6, 7]. The early
cultural evolution of the human being shaped how the human species survived a “feast-
famine” dietary experience. With the emergence of farming practices, the first new rapidly
developing cultural dietary experience collided with the very slow biologically-evolved
mechanism, designed for “feast and famine” survival [8]
Within the last century, food preservation, food distribution, food processing and new
foods entered the continued collision of cultural dietary practices on a relatively stable
biological basis for how foods/nutrition helped to maintain health sufficient for the survival of
the human species. Clearly within a hundred or so years, the average human median life span
was about the mid- 40’s to today’s of over 70’s. As a consequence of the improvement of
agriculture, sanitation, and maybe, so medical interventions, we have the “privilege of
experiencing the effluence of our affluence”, that is, living longer, due to the reduction of
acute infectious lethal diseases, only to experience chronic diseases of both growing older and
having newer dietary practices.
Recently, in spite of the rapid increase in the population of human beings, mal-
distribution of resources, and political/economic injustices, the numbers of over-weight and
454 James E. Trosko
obese individuals over-weight individuals are increasing around the world[9,10] than .
Clearly, it seems clear that this increase in individuals’ weight could be responsible, in large
part, to the emergence of many chronic diseases (cancer, diabetes, hypertension,
cardiovascular diseases, etc.), acting together with smoking, lack of exercise, poor nutritional
diets [11]. However, what might be missed is the fact that some of the most nutritious foods
are becoming the most expensive, and the most poorly-nutritious and highly caloric-laden
foods are the least expensive. The coupling of the non-renewal fossil fuel or current energy
crisis with the energy-dependent agri-business can be seen as a major contributing factor.
What should not be ignored is that cultural evolution has also brought new eating time-
habits. With the early biological evolved biochemical systems to deal with “feast-famine”
having coincided with the diurnal rhythms, new patterns of living outside these biologically-
governed diurnal rhythms probably influenced how our dietary/nutrient patterns affect our
homeostatic regulation for a healthy life. Then, with the introduction of agriculture, a more or
less stable, if not seasonal eating pattern emerged. With cultural evolution introducing new
forms of food production, food preservation, storage, transport , processing, and preparation,
for many, eating three or more times at day challenged or biologically-evolved mechanisms to
maintain homeostatic control of a disease-free life.
EPIDEMIOLOGICAL ASSESSMENTS FOR DIETARY FACTORS

AFFECTING HUMAN HEALTH
While it can not be denied that the science of epidemiology can provide solid insights to
some factors associated with various human diseases, fundamentally, it can not be used to
understand underlying mechanisms by which any factor, might or might not affect human
health. Moreover, in any epidemiological study of human populations, what might or might
affect the individual within that affected population can not be rigorously assessed. With both
(a) genetic-, gender-, developmental state- differences that might pre-dispose or offer
resistance to diseases and (b) multiple external agents that might synergize or antagonize the
agent(s) being studied, it is very understandable why many epidemiological studies do not
agree on the alleged affect of any suspected health related agent.
In the case of dietary/nutrient epidemiological studies on human beings is the fact that
multiple decade studies, with many individuals, together with “matched” controls, and with
multiple identical or equally-rigorously-designed and executed studies must be planned. In
addition, unless basic mechanistic knowledge of how any dietary/nutrient factor might be
toxic (via mutagenic, cytotoxic, or epigenetic mechanisms), is known, unforeseen
consequences might result with the pathogenesis of any disease. This will reduce the
likelihood that the epidemiological data be used in any meaningful intervention strategy. That
was the case of the pre-mature intervention Alpha-Tocopherol Beta-Carotene (ATBC) and
Beta-Carotene and Retinol Efficacy Trial (CARET) studies, in which the objective was to
reduce the lung cancer risk in heavy cigarette smokers, but which led to the cessation of the
human trial occurred because of the higher incidence of lung cancer in the treated individuals
versus the placebo group [12].
Lastly, with the unbelievable alteration of the human diet within decades that is occurring
now by the new processing of foods, the types of foods, amounts of foods, the interaction of
what is in and on foods, the manner by which foods are preserved, prepared, transported,
stored and the effect of economic stresses, no matter what any epidemiological insights might
have been gained, the likelihood of its having rigorous scientific utility is diminished.
Moreover, even with valid information gain from these time-shaped results, the question
remains, “Will implementation of the results for the future risk reduction of diseases be
effective or even harmful?”
GREEN TEA AND HUMAN HEALTH

Without doing a rigorous evaluation of all the epidemiological studies reported, one
example will be used to illustrate the aforementioned factors. Also, what might seem obvious
in trying to assess the potential benefit of green tea, it should be remembered that drinking
green tea for centuries occurred in cultures whose caloric intact and specific dietary
components (lots of vegetables, some fruits, raw fish, plenty of soy products, and a minimum
of red meats, dairy products, sugar, etc.) are not shared by those populations in which green
tea is proposed to be used.
Human cancer is one health endpoint that has been frequently used to assess the effect of
any dietary /nutrient exposes might have on cancer frequencies. Whether caloric restriction or
caloric excess is studied or dietary factors, such as fats or red meats, as “carcinogenic”, or
fruits, vegetables, fibers, essential metals/vitamins as cancer preventive agents [13], a few of
many examples of peer-reviewed reports on green tea that state there is a positive- [[14-17] ,
negative- [ 18] , both positive and negative-effects in the same population but in different
sexes [20],or no significant side effects or adverse effects [ 21] with the agent being studies of
the health effect being measured..
In order to understand how human cancers are produced, the current understanding must
be reviewed. First, human carcinogenesis is a multi-stage, multi-mechanism process [22-24],
consisting of a single cell in the human body being irreversibly altered, in such a fashion that
it remains immortal in the first stage to become a cancer. All human beings have such
“initiated”, pre-malignant cells in our body. Three fourths of us will reach death without
having a detected malignant cancer. One fourth of us will be diagnosed with a malignant
cancer before they die. The difference between these two groups is the fact that those that get
a malignant cancer had at least one of their “initiated cells” “promoted” to a large mass ( e.g.,
polyps in the colon; papillomas in the skin, nodules in the breast) by many endogenous
(genetic/developmental stage , gender and exogenous factors - cell death, wounding, growth
factors, life style agents, such as cigarette smoke chemicals, hormones, infectious biological
factors, inflammatory physical agents-asbestos, exogenous drugs, environmental pollutants
and dietary factors.)[24].
The three fourths of us ,who are lucky to die without having to deal without treating a
cancer, have our initiated cells suppressed by endogenous factors ( genetic, gender,
developmental state) or by exogenous factors ( dietary/ medicinal, life-style, such as
exercise).
The question is how those inevitable initiated cells are either “promoted” (clonally
expanded to a large mass of abnormal, immortal cells which can eventually accrue the
hallmarks of cancer [25], the additional genetic/epigenetic changes needed to become a
456 James E. Trosko
malignant cell. This also implies that this promotion step, which could take decades in the
case of human cancers, can be interrupted or even reversed [26]. Furthermore, the promotion
phase would be the most efficacious phase of carcinogenesis to design intervention cancer
prevention strategies [27]. The fact that the “initiation” step can be prevented from increasing
(one does not have expose one’s skin to too much sun light), one can never reduce the risk to
the initiation phase to zero.
If this is a correct assumption, then it should be helpful to understand the characteristics
of the promotion and anti-promotion processes [26, 28]. Most, if not all tumor promoting
agents/conditions need “threshold” levels to cause an initiated cell to escape the mitogenic
suppressing effect of surrounding normal cells, caused by either secreted anti-mitogenic
factors or direct transfer of ions or small molecules via gap junction channels of contiguous
cells [29]. Tumor promoters also have species-, gender-, cell-type-, and organ- specificies.
Promoting conditions could occur via indirect actions of cell death or cell removal, for
instance would-healing after surgery or tissue repair after cytotoxic exposures to viruses or
cytotoxic chemicals, parasites or bacteria, such as asbestos particles, all of which induce
inflammatory agents [24]. In addition, if an initiated cell is exposed to a tumor promoter, the
exposure must be sustained for a regular and chronic period of time. Most notably, not all
tumor promoters work via the same mechanism [30], for example, phorbol esters stimulate
protein kinase C and its cascading signal transduction mechanism. However, TCDD, a
promoter of skin and liver tumors appear to work through the Aryl hydrocarbon receptor-
mediated signaling [31]. Although most, if not all of these promoting agents and conditions
are associated with altering the redox state of the cell, inducing oxidative stress –inducing
intra-cellular signaling [32-34], this might be a necessary, but insufficient, causative factor for
tumor promotion
By implication, any potential anti-tumor promoter would not be expected to be able to
ameliorate the promoting mechanisms of all the different kinds of tumor promoting
conditions [30]. Not only would one expect that the anti-promoting agent specifically
interfere with the specific mechanism of the promoter, in order to be an effective chemo-
preventive agent, but it must be there at the exact time where it might block the start of the
promoting signaling, but also at the right concentration, for the entire chronic exposure
period. One also must remember, that both the promoting agent/condition, by being organ
specific, and the anti-promoting agent must be residing in the same organ site. For example, if
the tumor promoter acts in the lung, but the anti-promoter’s active chemical does not get
distributed to the brain in sufficient time/concentration, it might not be effective.
THE “BIOLOGICAL ROSETTA STONE”: GAP JUNCTIONAL

INTERCELLULAR COMMUNICATION AND HUMAN HEALTH
It must be noted that gap junction intercellular channel, composed of a hexamer of six
transmembrane proteins, “connexin”, that form a hemi-channel , “connexon”, which unite
with the connexon of the contiguous cell, exists in all organs of the human body [35]. Twenty
connexin genes code for these proteins which are expressed in a cell type/organ-specific
manner. The function of these gap junctions can be modulated at the transcriptional,
translational and posttranslational levels[29] by both exogenous and endogenous factors,
which then can alter the intra-cellular physiology, intracellular signaling and ultimately, the
gene expression of the cells[36].While all metazoans share the common cellular structures,
such as endoplastic reticulum, nuclei, mitochondrial, receptors, spindle fibers, the gap
junction has to be singled out as absolutely critical for maintaining, directly and indirectly, the
phenotypes of growth control, differentiation and apoptosis [37 ]. Without these gap
junctions, the ability to segregate cells during development to specialize and function
independently of neighboring cells, the complex homeostatic regulation of the hierarchical
biological system of the human being could not exist.
Interference of GJIC during embryogenesis, fetal development could lead to death or
birth defects as indicated by either retinoid or thalidomide modulation of gap junction
function in human beings [38, 39]. Interference of GJIC in the mature reproductive, immune
or neuronal tissues by chemicals, such as DDT, can lead to various toxicities [40]. While it
has not been universally accepted, one cellular mechanism has been proposed, based on the
observation that most, if not all, tumor promoters and promoting conditions can reversibly
inhibit gap junctional intercellular communication , that inhibition of gap junction function,
via multiple biochemical mechanisms, allows initiated cells to escape mitotic suppression of
surround normal cells.[41]. To support this hypothesis, known tumor promoters of the skin,
liver, mammary gland, lung and bladder can reversibly inhibit gap junction function in a
threshold manner, both in vitro and in vivo. [29]. In addition many anti-tumor promoters,
such as retinoids, caretinoids, reveratrol. Even chemotherapeutic agents have been shown to
restore gap junctional communication in non-communicating cancer cells, such as with
lovastatin, SAHA, [42, 43]
Given that gap junction genes (connexins) appeared during the evolutionary-transition
from the single cell organism to the multi-cellular metazoan [3], when growth control,
differentiation and apoptosis appeared, it should not be surprising that the cancer cells, which
do not have growth control, cannot terminally-differentiate or apoptose properly, do not have
functional GJIC [44]. Having discovered that going from the normal cell to an initiated cell,
which can be expanded by the inhibition of cell-cell communication via tumor promoters
until the cell becomes malignant or tumor promoter –independent, it demonstrates the
evolutionary importance of cell-cell communication in the cybernetic regulation needed to
maintain the hierarchical nature of all the interacting units of the human body and how these
gap junctions were beautifully designed to respond to environmentally-induced redox stress
triggered signaling in order to modulate adaptive genes [45]. From this vantage point, by
understanding the fundamental role that gap junctions play in the homeostatic regulation of
all the cellular, tissue, organ and organ system, including the ultimate human brain function,
consciousness, it should not be surprising to label the gap junction function one viewed the
translation of the hieroglyphics as a “biological Rosetta Stone”.
458 James E. Trosko
GREEN TEA, GAP JUNCTIONAL INTERCELLULAR COMMUNICATION,

AND POTENTIAL PROTECTION OF GAP JUNCTIONAL INTERCELLULAR
COMMUNICATION’S ROLE IN HOMEOSTATIC REGULATION OF
HUMAN HEALTH
Given all the many contradicting studies at the molecular, in vitro, experimental animal
and epidemiological studies on green tea’s effect on prevention of various biological/health
endpoints, is there some evidence that there is or is not any potential health benefit from
drinking green tea? The answer seems potentially positive. While green tea is a solution of
many chemicals, several of which have anti-oxidant potential, it might not be unreasonable to
predict that any acute or chronic disease endpoint, correlated with oxidative stress-induced
signaling, might be prevented by the timing, threshold concentrations, chronic exposure and
linkage of specific cross-talk/interaction of the components of green tea and the
toxin/toxicants. Of course, positive chemo preventive effects at the molecular/ in vitro and
whole animal level might or might not have any practical relevance to human beings. The
former test systems can be controlled, while the green-tea drinking behavior in human beings
can not.
The major example to highlight this point in this short Commentary will be the
demonstration that green tea could ameliorate the tumor promoting effect of PCP. PCP is
known to induce oxidative stress [46]. Moreover, after rats were initiated with
pentachlorophenol (PCP), chronic exposures to PCP at a threshold and higher levels, induced
liver tumors. PCP, in vitro, is not a genomic DNA damaging agent, but an epigenetic agent
[47]. In vitro, at not cytotoxic levels, but at threshold or above levels, could inhibit GJIC. On
the other hand, pre-treatment of the in vitro cells with green tea components, prevented the
GJIC inhibitory effects of PCP. More striking, treatment of the rats exposed to both green tea
and PCP in the drinking water, ameliorated the promoting effect of the PCP on liver tumors.
While it can be argued that this example is still “correlative” not causal, it clearly is
consistent with the hypothesis that green tea has components capable of protecting the tumor
promoting ( and possibly other gap junctional-dependent health effect in other organs) of
chemicals that work like PCP. Of the several epidemiological studies of green tea, “cherry-
picking” one example , namely that of comparison of American and Japanese smokers might
serve to illustrate how green tea could be beneficial to human health [17]( Recognizing the
hazards of experimental design; cultural differences in diet/behavior; sampling errors, etc.).
However, with the experimental demonstration that green tea polyphenol, {(-)-
epigalllocatechin-3-gallate –EGCG}, suppresses cigarette smoke condensate-induced redox
intracellular signaling in normal human bronchial epithelial cells [48]
In this example, there was a correlation between the green tea drinking of the Japanese
men who smoked and their risk to lung cancers compared to American men. While it might
have been even more interesting to compare a number of American smokers who drank that
amount of green tea as the Japanese and compared them to Japanese smokers who did not
drink green tea( Unfortunately, the numbers in either category are probably too small to
obtain statistically significant results). It should be noted that if there is a demonstrated
validated health effect of green tea, there must be an underlying biological mechanism.
However, even if there might be a demonstrated mechanistic biological effect of green tea (of
which there are many), it does not necessarily imply there will be a health effect.
In summary, the scientific understanding of many biological functions involved in
maintaining human health (e.g., role of gap junctions in homeostatic control of cell behaviors;
multi-state nature of carcinogenesis; role of gap junctions in the tumor promotion step of
carcinogenesis; toxicant induced oxidative stress in many human diseases; role of oxidative
stress by tumor promoting chemicals; role of green tea components as anti-oxidants;
preventive effects of green tea components on toxicant-inhibition of gap junction function and
tumor promotion) provides some evidence that green tea could prevent some oxidative stress-
linked human diseases. However, as has been demonstrated in experimental examples, it will
be unlikely that any agent, such as green tea, will be a “silver bullet” to protect against all
toxic agents. On the other hand, it does seem it would cause “no harm” and might, in some
cases, even be beneficial.
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INDEX
acute myeloid leukemia, 341

A acute stress, 4, 5, 6, 7, 8, 9, 15, 17, 18, 19, 22, 214
acylation, 47
Aβ, 152, 438 ad hoc, 123
A1c, 248 Adams, 174, 301, 318, 379
aberrant methylation, 264 adaptation, 459
abnormalities, 249, 364 additives, xi, 46, 63, 64, 66, 67, 68, 70, 73
absorption, 13, 55, 64, 65, 71, 74, 77, 78, 118, 122, adducts, 163, 174, 403
126, 135, 136, 137, 141, 143, 151, 152, 154, 201, adenocarcinoma, 145, 150, 273, 275, 287, 299, 317,
205, 224, 227, 237, 246, 279, 340, 381, 400, 415, 377
416, 417, 424, 426 adenocarcinomas, 326
abusive, 31 adenoma, 334, 344
ACC, 137 adenosine, 298
accessibility, 291 adenovirus, 28, 42, 55, 60, 377
accounting, xviii, 302, 384, 388 adhesion, 99, 127, 169, 204, 267, 293, 294, 308, 333,
accuracy, 35 334, 339, 343, 344, 382, 453
ACE, 299, 316, 424 adhesive interaction, 334
acetaldehyde, 160, 169 adipocyte, 128, 129, 130, 131, 132, 134, 135, 141,
acetate, vi, xvii, 108, 109, 160, 165, 166, 228, 256, 143, 144, 145, 147, 148, 149
347, 348, 353, 361 adipocytes, 127, 128, 131, 132, 134, 135, 137, 141,
acetone, 65, 86, 194, 395, 423 146, 150, 200, 394
acetylation, 148, 291, 461 adipogenic, 128, 130, 148, 149
acetylcholine, 212 adiponectin, 150, 248, 255
acetylcholinesterase, 162, 170 adipose, 107, 117, 120, 127, 131, 132, 134, 136, 137,
acidic, 93, 164, 165, 395 140, 142, 143, 147, 153, 164, 200, 249, 256
acidity, xii, 82, 84, 87, 91, 92, 98 adipose tissue, 107, 117, 120, 127, 131, 132, 134,
acquired immunodeficiency syndrome, 28 137, 140, 142, 143, 147, 164, 200, 249, 256
ACS, 99 adjustment, 251, 326, 421
actin, 48, 49, 52, 53, 230, 272, 273, 274, 277, 278, adolescents, 144
284, 285, 294, 296, 297, 298, 299, 368 ADP, 229, 230, 358, 413
actinic keratosis, 56 adrenal gland, 162
action potential, 12, 252 adrenaline, 137
activators, 292 adrenocorticotropic hormone, 137
active oxygen, 43, 167, 175, 403 adriamycin, 41
active site, 263, 266 adsorption, 29
acute, 4, 5, 6, 7, 8, 9, 15, 17, 18, 19, 21, 22, 48, 161, adult, 16, 40, 200, 224, 315, 370, 446, 451, 452, 453
162, 214, 245, 250, 315, 341, 353, 453, 458 adult stem cells, 451, 453
464 Index
adults, xviii, 233, 244, 253, 255, 296, 384, 385, 386, alpha, ix, 1, 2, 13, 14, 30, 99, 145, 148, 149, 150,
388, 389, 406 151, 220, 227, 238, 240, 241, 295, 297, 298, 299,
advanced glycation end products, 403 309, 320, 340, 341, 345, 380, 443
aerobic, 69, 185, 187 alpha activity, 14
aerobic bacteria, 187 alpha-tocopherol, 340
aerodigestive tract, 344 ALT, 162, 369, 370
Africa, 158 alternative, 69, 135, 148, 154, 250, 341, 369
agar, 86 alternatives, 178
age, xvi, xvii, xix, 16, 108, 169, 198, 250, 325, 326, alters, 137, 147, 298, 306, 433, 448
338, 377, 383, 384, 386, 387, 399, 429, 430, 442, aluminium, 106
444, 453 aluminum, 118, 180, 375
ageing, 408, 435, 440, 445 alveolar macrophage, 30, 39, 42
agent, xv, xvi, xx, 12, 35, 37, 60, 83, 133, 135, 166, Alzheimer disease, 205, 268
198, 219, 252, 268, 286, 317, 322, 325, 335, 337, Alzheimer's disease, 10, 107, 197, 201, 205, 206,
373, 377, 378, 405, 454, 455, 456, 458, 459 219, 220, 222, 234, 236, 237, 240, 241, 268, 295,
agents, xiii, xx, 30, 57, 71, 131, 139, 147, 157, 160, 443, 444, 446, 448, 449
168, 174, 175, 222, 225, 252, 261, 282, 308, 321, amelioration, 161, 394, 444
322, 330, 333, 336, 337, 353, 356, 358, 394, 443, American Cancer Society, 326, 338
454, 455, 456, 457, 459 American Heart Association, 254
aggregates, 136 amide, 209
aggregation, 172, 200, 204, 222, 228, 441 amine, 119, 164, 165, 172, 173, 321
aggressiveness, 308 amines, 164, 165, 178, 195
aging, 46, 57, 82, 206, 222, 224, 234, 246, 248, 378, amino, ix, 1, 2, 15, 20, 22, 79, 106, 117, 119, 158,
430, 444, 453 164, 190, 191, 208, 215, 244, 247, 265, 351, 394,
aging process, 430 396
agonist, 15, 132, 210, 225 amino acid, ix, 1, 2, 20, 22, 79, 106, 117, 158, 164,
agricultural, 105, 190 215, 244, 247, 351, 394, 396
agricultural crop, 105 amino acids, 22, 79, 117, 164, 215, 244, 247, 394,
agriculture, 29, 161, 453, 454 396
AIDS, 28, 37, 60, 377 ammonia, 27
air, xiii, 84, 157, 160, 319, 384 AMPA, 15, 20, 208, 209, 212, 213, 215, 216, 217
AKT, 147, 229, 240, 291, 306, 307 amplitude, 12, 13
Alabama, 363 Amsterdam, 109, 122, 124
alanine, 161, 162, 163 amygdala, ix, 1, 2, 13
alanine aminotransferase, 161, 163 amylase, 30, 42, 400, 404
Alaska, 195 amyloid, xix, 10, 201, 220, 222, 225, 234, 236, 238,
albumin, 163, 399 240, 250, 268, 295, 429, 430, 433, 438, 442, 443,
albuminuria, 403 444, 446, 447, 448, 449
alcohol, xviii, 120, 160, 161, 169, 176, 202, 235, Amyloid, 233, 249, 294, 443, 447
246, 384, 386, 387, 388, 389, 395, 440, 443 amyloid beta, 225, 443, 444
alcohol consumption, xviii, 120, 235, 384, 386, 387, amyloid fibrils, 250
388, 389, 443 amyloid precursor protein, 220, 222, 234, 236, 238,
alcohols, 173 240, 268, 295
aldolase, 230 amyloid β, 433, 446
alertness, 13 amyloidosis, 227, 249, 250, 256
alkaline, 162, 312, 371 amyotrophic lateral sclerosis (ALS), 12, 20, 222,
alkaline phosphatase, 162 226, 237, 433
alkaloids, 37 anabolic, 148
alleles, 285, 303, 373 anabolism, 126
allergens, 178, 277 analgesic, xii, 81
allergic reaction, 277, 279 analog, 59, 164, 208
allergy, 267, 277, 278, 297 analysis of variance, 111
allosteric, 131, 404 analytical techniques, 35
Index 465
anaphylaxis, 297 anticancer activity, 368

androgen, 127, 138, 145, 146, 336 antidiabetic, 139, 244, 252
androgens, 417 antigen, 297
androstenedione, 305 antigenicity, 195
anesthetics, 210, 213 anti-HIV, 28, 37, 60, 377
aneuploidy, 167, 174 anti-inflammatory agents, 222, 353, 356
angiogenesis, xiv, xvi, 148, 197, 198, 199, 200, 201, antiobesity, 107, 108, 143, 244
202, 203, 206, 261, 265, 267, 286, 292, 301, 308, antioxidative, 143, 196, 343, 427, 439
309, 311, 313, 314, 319, 330, 331, 336, 339, 340, antioxidative activity, xix, 187, 190, 429
341, 345, 366, 376 antioxidative potential, 448
angiogenic, 198, 199, 201, 203, 230, 308, 331, 340, antiradical activity, 104, 110
366 antisense, 282, 319
angiography, 246 antisense RNA, 282
Angiotensin, 427 antitoxin, 37
angiotensin converting enzyme, 193, 424 antitumor, 41, 119, 122, 272, 322
angiotensin II, 247 anti-tumor, xvi, xvii, 50, 83, 312, 319, 325, 363, 456,
angiotensin-converting enzyme, 285, 303, 305 457
animal models, x, 45, 131, 136, 204, 225, 226, 248, antitumor agent, 322
259, 265, 286, 314, 326, 365, 366, 371, 375, 391, antiviral, x, 2, 23, 28, 29, 35, 37, 424
394 anxiety, 7, 15, 21
animal studies, xviii, 19, 37, 58, 107, 117, 135, 170, anxiolytic, 15
229, 231, 244, 247, 332, 389, 393, 431 aorta, 253, 255
animal tissues, 162 APC, xvi, 28, 325, 327, 329
animals, xiii, xix, 16, 30, 49, 104, 136, 137, 142, aplastic anemia, 166
163, 166, 168, 223, 260, 261, 266, 375, 402, 429 apnea, 104
ANOVA, 87, 111, 112, 437 APO, 127, 220, 225, 227, 229
antagonism, 216, 217 apoptosis pathways, 154, 332, 342
antagonist, xiv, 6, 7, 15, 124, 207, 209, 210, 212, apoptotic, 127, 131, 133, 140, 141, 146, 149, 150,
213, 216, 305, 372 164, 213, 229, 234, 240, 264, 276, 308, 311, 314,
antagonistic, 16, 38, 209, 213 332, 333, 335, 342, 448
antagonists, 16, 28, 120, 209, 213, 215, 217, 289 apoptotic cells, 133, 311
anthocyanin, 27, 348 apoptotic effect, 141, 146
anthracene, 75, 321, 322 APP, 222, 225, 227, 228, 232, 268, 441
anthrax, 37, 261, 290 appetite, 105, 108, 117, 120, 121, 122
antiangiogenic, 309 application, x, xiv, 11, 13, 40, 45, 46, 47, 48, 56, 76,
anti-angiogenic, xiv, 197, 198, 199, 200, 201, 303 118, 166, 170, 177, 178, 188, 194, 231, 239, 303,
anti-angiogenic, 336 336, 353, 360
antiapoptotic, 141, 133, 229, 234, 264, 265, 289, aqueous solution, 136, 395
335, 341, 342, 442 arabinogalactan, 399
antiarrhythmic, 252 arachidonic acid, 277, 320
antibacterial, x, 23, 30, 31, 32, 33, 36, 37, 43 argument, 329
anti-bacterial, 83, 358 Arizona, 85, 90
antibacterial properties, 36 aromatic rings, 29, 221
antibiotic, 27, 33, 34 arousal, 13
antibiotic resistance, 27 arrest, 129, 130, 133, 134, 141, 143, 146, 147, 149,
antibiotics, x, 23, 31, 34, 38, 42 150, 229, 263, 266, 276, 289, 306, 316, 317, 321,
antibodies, 48 333, 335, 336, 342, 345, 374
antibody, 54, 198, 202, 270, 272, 273, 277, 278, 364 arrhythmia, 251
anticancer, 37, 133, 146, 154, 158, 185, 282, 283, arrhythmias, 244, 249, 252
290, 308, 317, 322, 365, 368, 370, 374, 375, 378 ARS, 78
anti-cancer, x, xi, xvii, 45, 46, 47, 48, 55, 199, 229, arteries, 204
258, 261, 265, 267, 270, 286, 348, 352, 357, 358, artery, xiv, 14, 207, 208, 209, 216, 245, 246, 248,
363, 368 252, 254, 255, 402
466 Index
arthritis, 58, 376, 400 bacterial, 30, 31, 33, 34, 36, 39, 41, 42, 96, 107, 152,
articulation, 222 268
aryl hydrocarbon receptor, 241, 289 bacterial cells, 34, 41
asbestos, 455, 456 bacterial infection, 42, 268
ascorbic, 71, 72, 79, 84, 85, 91, 92, 96, 109, 192, bacteriostatic, 96
193, 229, 232 bacterium, 33, 40, 196, 361
ascorbic acid, 71, 72, 79, 84, 85, 91, 92, 96, 109, barrier, ix, x, xix, 1, 2, 12, 13, 34, 45, 56, 61, 208,
192, 193, 229, 232 209, 214, 220, 224, 235, 336, 429, 430
Asia, 2, 21, 29, 36, 151 barriers, 47
Asian, 2, 200, 201, 206, 222, 285, 299, 316, 365, 366 basal layer, 52, 54
Asian American, 316 base pair, 369
Asian Americans, 316 basement membrane, 261, 265, 267, 292, 331, 343
Asian countries, 201, 365 basic fibroblast growth factor, 331, 340
aspartate, 12, 22, 30, 161, 162, 163, 208 basophils, 267, 277, 278
assessment, 70, 76, 77, 170, 185, 194, 215, 389, 443 Bax, 133, 141, 149, 229, 306, 308, 310, 311, 312,
asthma, 104, 123, 277, 278 317
astringent, 158 BBB, 208, 209, 210, 214, 220, 224
astrocyte, xiv, 14, 21, 207, 211 B-cell, 127, 140, 292
astrocytes, 241, 445 B-cell lymphoma, 140
astrocytoma, 10, 20, 377 B-cells, 127
asymmetry, 332 bcl-2, 240, 291, 341, 342
ATF, 241, 333 Bcl-2, 133, 150, 153, 222, 228, 229, 263, 264, 289,
atherogenesis, 70, 132, 204, 395, 402 291, 306, 332
atherosclerosis, xiv, 78, 136, 149, 151, 197, 198, Bcl-xL, 133, 229, 264
200, 202, 204, 221, 252, 258, 268, 444 beef, 31, 38, 381
atherosclerotic plaque, 135, 200 behavior, xiii, 3, 5, 7, 9, 11, 13, 16, 18, 105, 125,
atherosclerotic vascular disease, 400 361, 393, 452, 458, 459, 461
atmosphere, 123, 193 Beijing, 224
atopic dermatitis, 277, 278 beneficial effect, ix, xiii, xiv, xviii, xix, 2, 47, 64, 72,
ATP, xviii, 131, 411, 413, 414, 416, 417, 418 107, 117, 125, 136, 143, 191, 192, 197, 198, 223,
ATPase, 255, 272, 274, 295, 395, 403, 407 224, 234, 286, 329, 337, 374, 394, 411, 415, 424,
atrophy, 162, 431 430, 434
attachment, 28, 29, 226, 267, 292, 293, 332, 334 benefits, x, xi, xv, xvii, 18, 37, 45, 46, 47, 55, 56,
attitudes, 338 139, 143, 155, 178, 222, 226, 232, 243, 244, 252,
atypical, 294 258, 303, 321, 325, 326, 329, 359, 394, 401, 405,
Australia, 108, 123, 383 424, 427, 451
autoimmune, x, 45, 46, 442 benign, 326, 327, 452
autoimmune disorders, x, 45 benzene, 165, 166, 167, 174, 175
autonomic nervous system, 7 benzodiazepine, 15, 19, 20
autooxidation, 167 benzoquinone, 166, 174
autophagy, 140 beta cell, 394, 412, 413, 416
avian influenza, 29 beta-carotene, 175, 459
awareness, xiii, 125, 126, 243 bevacizumab, 198, 202
Aβ, 433, 438, 440, 441, 442 beverages, xi, xvii, 24, 46, 55, 64, 66, 67, 69, 70, 71,
74, 75, 76, 77, 78, 80, 81, 83, 85, 89, 90, 91, 93,
B 96, 98, 178, 221, 243, 258, 347, 389
BH3, 229, 264
B cell, 122, 148
bias, 389
Bacillus, 31, 32, 94
bile, 151, 246, 260, 431
Bacillus subtilis, 94
bindings, 281, 321
background information, 369
bioactive compounds, 94, 178
bacteria, 27, 30, 31, 32, 33, 34, 37, 40, 41, 123, 164,
bioassay, 329
187, 431, 456
Index 467
bioavailability, xi, xvi, 12, 26, 35, 46, 47, 55, 56, body weight, ix, xii, xiii, 12, 103, 104, 108, 110, 111,
105, 171, 225, 235, 244, 260, 261, 264, 286, 288, 114, 115, 117, 118, 119, 121, 126, 135, 141, 142,
298, 302, 303, 311, 312, 322, 368, 375, 381 143, 200, 205, 249, 302, 402, 432
biocatalytic process, xix, 419 Boeing, 233
biochemistry, 40, 124 bolus, 302
biogenesis, 137 bonds, 137
biogenic amines, 178, 195 bone density, 26
biological activity, xi, 46, 179, 195, 229 bone marrow, 40, 149, 167, 174, 175, 293
biological consequences, 228, 265 borderline, 71
biological processes, 132 boreal forest, 407
biological responses, 260 Bose, 144, 344
biological systems, 72, 459 Boston, 250
biologically active compounds, 280 botulinum, 101
biomarker, 134, 163 bounds, 29
biomarkers, xvi, 69, 105, 296, 325, 406 bovine, xvii, 29, 36, 49, 266, 348, 352, 379
biomaterial, xiv, 177 brain, ix, xiv, xv, xix, 1, 2, 4, 5, 6, 7, 8, 13, 14, 15,
biomolecules, 430 16, 19, 20, 21, 22, 105, 117, 158, 162, 170, 171,
biopsy, 49 207, 208, 209, 210, 212, 214, 215, 216, 217, 219,
biosynthesis, 27, 40, 41, 137, 184, 185, 266 222, 224, 225, 226, 227, 228, 230, 231, 232, 233,
biotransformation, 26, 75, 244, 260, 287, 377 234, 235, 237, 238, 239, 240, 241, 242, 247, 255,
birth, 457 295, 356, 409, 417, 429, 430, 433, 435, 436, 439,
black tea, xi, xix, 2, 13, 24, 25, 26, 36, 39, 65, 66, 67, 440, 441, 442, 443, 444, 445, 446, 447, 448, 456
70, 71, 74, 75, 76, 78, 79, 81, 82, 85, 87, 89, 93, brain damage, 210, 237
96, 101, 107, 126, 143, 144, 158, 165, 196, 224, brain development, 15
226, 227, 238, 250, 252, 254, 258, 287, 288, 316, brain injury, 170, 409
320, 322, 361, 365, 381, 384, 385, 419, 422, 423, brain microvascular endothelial cell, 293
424, 426, 427, 431, 442 branching, 312
bladder, 2, 40, 107, 122, 153, 164, 173, 358, 457 breakdown, 136, 178, 179, 193, 194
bladder cancer, 107, 122, 153, 164, 173 breast cancer, ix, xvi, 107, 123, 138, 145, 148, 150,
bleaching, 64, 66, 68, 72 152, 203, 285, 291, 299, 301, 303, 304, 305, 306,
blindness, 248 308, 309, 310, 311, 312, 313, 314, 315, 316, 317,
blocks, 135, 198, 238, 244, 251, 252, 318, 320, 341, 318, 320, 321, 322, 323, 335, 341, 345, 366, 368,
377, 437 374, 376, 380
blood clot, 265 breast carcinoma, 145, 146, 203, 282, 289, 316, 317,
blood glucose, xiii, 125, 135, 205, 248, 253, 255, 319, 345, 376, 379, 446
394, 399, 400, 406, 409, 412, 413, 416 breathlessness, 384
blood plasma, 78, 105 broad spectrum, 158
blood pressure, 104, 153, 178, 208, 209, 214, 215, bronchial asthma, 277, 278
246, 247, 254, 255, 287, 447 bronchial epithelial cells, 361, 458, 461
blood stream, 332 Brussels, 67
blood supply, 200 budding, 294
blood vessels, xiv, 197, 198, 308, 331, 366 buffer, 50, 68, 109, 423
blood-brain barrier, ix, 1, 2, 12, 13, 208, 209, 220, building blocks, 27
224, 235 bundling, 285, 289
blot, 49, 52, 53, 273, 276, 277, 284, 356 by-products, ix, 453
blueberry, 77, 122
body composition, 136
C
body fat, xiv, 26, 108, 117, 120, 126, 197, 205, 249,
Ca2+, 132, 139, 149, 213, 240, 255, 277, 298, 395,
256
403, 407, 413
body mass index (BMI), xviii, 142, 384, 387, 388,
cabbage, 32
390
cadherin, 199, 203, 275, 276, 309, 334, 344
body temperature, 105, 214
cadherins, 334, 344
caffeic acid, 64
468 Index
caffeine, ix, xviii, 1, 2, 7, 16, 19, 20, 21, 26, 37, 118, casein, 120
120, 121, 123, 153, 158, 192, 193, 208, 224, 234, caspase, xi, 40, 46, 48, 49, 52, 53, 54, 56, 59, 60,
235, 249, 253, 280, 302, 342, 361, 365, 375, 391, 133, 140, 141, 150, 154, 226, 229, 230, 277, 310,
394, 399, 406, 411, 443 311, 312, 317, 331, 336, 345, 358, 361, 417
calcium, 12, 111, 124, 149, 217, 222, 277, 297, 338, caspase-dependent, 140, 361, 417
395, 403 caspases, 140, 233, 443
calibration, 86, 90, 95, 422 CAT, 162, 167, 440
calmodulin, 240, 296, 299 catabolic, 148
caloric restriction, 455 catabolism, 26, 126, 131, 136, 201, 205
calreticulin, 132 catalase, 59, 162, 167, 226, 260, 275, 368, 433, 440,
calvaria, 149 442, 445
CAM, 199, 333 catechol, 26, 132, 148, 166, 167, 220, 225, 236, 281,
cAMP, 255, 265, 333 285, 303, 322, 420
cancer progression, 295, 320, 322, 339 catecholamines, 26, 225
cancer treatment, xvi, xvii, 198, 301, 306, 363 category a, 66, 458
cancerous cells, 317 cation, xi, 63, 64, 76, 79
Candida, 33, 37 cattle, 29
candidates, 55, 198, 270, 358 causation, 152, 234
capacity, xi, xii, 3, 13, 26, 38, 63, 64, 65, 66, 67, 69, cavities, 30
70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 82, 83, CD95, 127, 140
84, 85, 86, 87, 88, 89, 90, 91, 93, 94, 95, 96, 98, CDK, 130, 146
100, 108, 109, 111, 118, 120, 122, 190, 224, 234, Cdk inhibitor, 141, 146, 289, 316
246, 248, 249, 260, 303, 313, 399, 400, 440 CDKIs, 306
capillary, 331, 336 CDKs, 335
carbohydrates, 30, 244, 431 cDNA, 129, 267, 270, 282
carbon, 48, 56, 160, 161, 170, 223, 263, 266 cecum, 431
carbon tetrachloride, 160, 161, 170 cell adhesion, 127, 294, 308, 333, 334, 343, 344, 453
carboxyl, 265 cell body, 230, 273, 284
carboxylic, 165, 173 cell culture, xvii, xix, 21, 50, 51, 53, 54, 131, 225,
carcinogen, 167, 173, 175, 258, 316, 321 231, 234, 251, 260, 265, 266, 289, 358, 363, 367,
carcinogenesis, xvi, xix, 26, 37, 57, 75, 126, 167, 375, 402, 419, 423, 425
168, 173, 175, 202, 235, 258, 265, 275, 287, 291, cell cycle, xvi, 38, 129, 130, 131, 133, 134, 141, 147,
296, 301, 310, 314, 315, 321, 325, 326, 333, 336, 150, 201, 206, 230, 234, 263, 264, 266, 273, 275,
337, 338, 340, 342, 361, 364, 376, 382, 455, 456, 276, 301, 306, 308, 317, 318, 330, 332, 333, 334,
459, 460, 461 335, 336, 345
carcinogenic, 83, 164, 165, 166, 172, 178, 258, 330, cell death, xi, 15, 19, 46, 50, 51, 52, 55, 56, 140, 141,
358, 394, 401, 455 154, 168, 172, 175, 212, 213, 214, 217, 222, 226,
carcinogens, 37, 164, 172, 329, 364, 385, 388 228, 229, 237, 239, 263, 276, 332, 336, 358, 366,
carcinoma, 49, 50, 51, 59, 145, 146, 147, 167, 203, 376, 440, 441, 446, 453, 455, 456
234, 282, 287, 289, 290, 292, 296, 310, 316, 317, cell differentiation, x, 45, 56, 239, 452
319, 332, 334, 341, 344, 345, 358, 376, 377, 379, cell division, 31, 129, 130, 283, 364, 369, 370
381, 446 cell growth, xvii, 128, 129, 145, 199, 264, 265, 266,
carcinomas, 175, 339 268, 270, 272, 273, 275, 276, 282, 283, 284, 285,
cardiac arrhythmia, 244, 252 288, 306, 307, 308, 309, 316, 329, 332, 333, 334,
cardiovascular disease, ix, xiii, xv, 26, 57, 71, 79, 342, 348, 353, 354, 356, 357, 367, 368, 378
104, 119, 125, 126, 142, 143, 200, 202, 204, 206, cell invasion, xvi, 205, 301, 307, 308, 341, 344, 382
222, 234, 243, 244, 245, 246, 247, 248, 252, 253, cell line, xvi, xvii, 14, 48, 49, 52, 55, 59, 127, 133,
254, 358, 361, 385, 386, 389, 391, 392, 402, 405, 145, 150, 164, 228, 239, 259, 263, 265, 266, 275,
407, 430, 454, 460 276, 282, 289, 291, 292, 296, 301, 305, 306, 314,
cardiovascular risk, 143, 246, 247, 254 317, 318, 319, 320, 333, 335, 336, 342, 343, 344,
carotene, 71, 99, 194, 460 345, 352, 353, 354, 357, 363, 365, 366, 368, 370,
carotenoids, 106, 158, 407 371, 373, 375, 376, 378, 381, 453
carrier, 136, 186, 441
Index 469
cell lines, xvi, xvii, 59, 127, 133, 259, 263, 266, 275, chicks, 4, 5, 6, 7, 8, 9, 10, 11, 16, 17, 18, 22
276, 282, 289, 291, 296, 301, 305, 306, 317, 318, children, 120, 122, 144
319, 335, 342, 343, 363, 365, 366, 368, 371, 373, Chile, 63
375, 376, 378, 381 China, xii, 2, 45, 46, 47, 48, 55, 57, 103, 108, 112,
cell membranes, 33, 293, 433 120, 158, 222, 224, 252, 365, 389, 393, 394, 395,
cell signaling, xvi, 229, 299, 302, 307, 311, 440, 442, 399, 408, 460
451 Chinese medicine, 64, 246
cell surface, 29, 137, 140, 204, 267, 270, 272, 278, Chinese women, 285, 299, 305, 316
279, 280, 281, 298, 316, 368 chloride, 108, 109, 211
cellular adhesion, 333, 339 chlorination, 175
cellular response, 445 chloroform, 165
cellular signaling pathway, 30 chlorogenic acid, 64, 82, 106
central nervous system, ix, 1, 2, 105, 208, 210, 215, chlorophyll, 178, 179, 180, 193, 194
216, 217, 232, 241, 442, 444 chlorpyriphos, 162, 170, 171
cerebellum, 14, 440 chocolate, 19, 100
cerebral amyloidosis, 237 cholecystokinin, 117, 120
cerebral blood flow, xiv, 14, 207, 208, 209 cholesterol, 26, 38, 73, 96, 117, 126, 132, 135, 136,
cerebral blood flow (CBF), 209 137, 143, 149, 151, 152, 162, 200, 205, 245, 246,
cerebral cortex, 14, 15, 211, 294, 433, 436, 438, 439, 249, 254, 268, 272, 295, 299, 340, 402, 403, 405,
440, 441 415, 416, 444
cerebral ischemia, xiv, 14, 19, 207, 208, 209, 210, cholinergic, 222
212, 213, 214, 216, 217, 226, 239, 446 cholinesterase, 162, 163, 170
cerebrospinal fluid, 238 chondrocytes, 148, 376
cerebrovascular, xv, 15, 207, 214, 254, 384 chromatid, 174
cerebrovascular disease, xv, 15, 207, 214, 384 chromatin, 140, 230, 291, 332, 344, 373, 374, 381
cerebrovascular diseases, 384 chromatograms, 100
ceruloplasmin, 409 chromatography, 59, 182, 187
cervical cancer, 345 chromosome, 167, 264, 369, 372
cervical carcinoma, 234, 282 chromosomes, xvii, 363, 369
c-Fos, 199, 203, 306, 331 chronic cough, 384
channels, xv, xviii, 132, 213, 230, 243, 251, 253, chronic disease, 107, 123, 152, 234, 430, 451, 453,
411, 413, 416, 417, 418, 453, 456 454, 458
chaperones, 231, 310, 372 chronic disorders, 104
charge density, 161 chronic myelogenous, 268
chelating agents, xv, 185, 219 chronic obstructive pulmonary disease, ix, 390, 391,
chelators, 225, 229, 230, 231, 233, 241 392
chemical degradation, 371 chymotrypsin, xvi, 263, 301, 306, 307, 311, 331
chemical properties, 194 cigarette smoke, xvii, 39, 383, 384, 454, 455, 458,
chemical structures, 41, 349, 401, 427 461
chemicals, xiii, xix, 2, 153, 157, 158, 160, 173, 430, cigarette smokers, xvii, 383, 384, 454
455, 456, 457, 458, 459 cigarette smoking, 120, 235, 386, 389, 443
chemiluminescence, 78 ciprofloxacin, 34, 39
chemoprevention, xvii, 38, 153, 154, 241, 258, 299, circulation, 13, 47, 415, 416, 431, 448
303, 305, 311, 325, 326, 338, 340, 370, 375, 447, cisplatin, 335, 409
459, 460 citrus, 195
chemopreventive agents, 147, 261, 330, 336 c-jun, 228, 331, 342, 378
chemoresistance, 291 classes, x, 23, 27, 283, 302, 360
chemotaxis, 293 classical, 140
chemotherapeutic agent, 457 cleavage, 140, 196, 230, 236, 261, 273, 312, 358,
chemotherapies, 250 441, 452
chemotherapy, 154, 249, 250, 263, 295, 313, 318, clinical disorders, 221
352, 364, 365, 459, 460, 461 clinical syndrome, 440
chicken, 31, 38, 137, 178, 266 clinical trial, 78, 154, 198, 279, 302, 311, 326
470 Index
clinical trials, 78, 198, 302, 311, 326 co-morbidities, 389

clinics, 386 competition, 165, 225
cloning, 270 complementary DNA, 282
clostridium botulinum, 37, 94, 164 complete remission, 266
cluster analysis, 123 complex systems, 200
clusters, 295 complexity, 452
c-Myc, 275, 276, 372, 380 compliance, 153, 314
CNS, ix, x, 1, 2, 3, 9, 15, 16, 208, 210, 444 complications, xiii, xviii, 125, 150, 393, 394, 395,
Co, 15, 48, 55, 100, 180, 195, 421, 429 400, 402, 403, 404, 408, 411
CO2, xiv, 111, 207 components, x, xiii, xvi, xviii, xix, 2, 18, 19, 26, 27,
coatings, 186 30, 31, 35, 39, 41, 42, 55, 64, 70, 77, 83, 96, 123,
cocaine, 12, 21 125, 133, 158, 160, 162, 165, 169, 200, 208, 215,
cocoa, 38, 78, 101, 178 230, 234, 247, 258, 275, 276, 285, 286, 295, 315,
coding, 295 325, 326, 369, 375, 378, 390, 393, 394, 395, 399,
coenzyme, 152, 266 405, 409, 412, 419, 422, 423, 427, 443, 444, 451,
coffee, 21, 108, 123, 158, 178, 250, 251, 252, 445, 455, 458, 459
448 composition, x, xii, xiv, xvii, xix, 24, 26, 35, 37, 81,
cognition, xix, 217, 429, 430, 433, 435, 444 99, 120, 121, 160, 169, 177, 179, 187, 189, 234,
cognitive, xiv, xix, 16, 197, 201, 206, 222, 224, 226, 315, 347, 348, 378, 396, 419, 431
233, 236, 237, 385, 429, 430, 431, 434, 437, 438, COMT inhibitor, 225
440, 442, 444, 446, 447 conception, 452
cognitive activity, 16 condensation, 25, 140, 264, 332, 358
cognitive deficit, 237, 444 conduction, 249
cognitive deficits, 237, 444 confidence, xviii, 384, 385
cognitive dysfunction, xix, 429, 431 confidence interval, xviii, 384, 385
cognitive function, 206, 224, 233, 434, 446 configuration, 281
cognitive impairment, xiv, xix, 197, 201, 222, 224, confinement, 15
233, 236, 385, 429, 430, 431, 434, 437, 438, 440, confounders, 388
442, 443, 445, 447, 448 conjugated dienes, 70
cognitive map, 430 conjugation, 26, 431
cognitive performance, 224 consciousness, 452, 457
cohort, 71, 245, 248, 255, 296, 303, 304, 389, 391 consensus, 120, 447
cold sore, 28 consent, 386
colitis, 32 consumer goods, 166
collaboration, 57 consumers, xi, 46, 222, 223, 224, 285
collagen, 58, 61, 185, 261, 267, 292, 344, 376, 395, contaminant, 166
402, 406, 408 contamination, xiii, 157, 160
colon, xvi, 2, 132, 145, 147, 200, 203, 260, 264, 275, content analysis, 84, 86
287, 292, 317, 319, 320, 322, 325, 326, 329, 338, contractions, 252
339, 340, 341, 344, 361, 366, 373, 377, 378, 424, control condition, 15
455, 460 control group, xii, 7, 103, 111, 113, 114, 115, 116,
colon cancer, xvi, 147, 200, 264, 275, 287, 319, 325, 117, 118, 119, 162, 387, 399
338, 339, 340, 361, 366, 373, 377, 378, 424, 460 controlled studies, 224
colon carcinogenesis, 275 conversion, 41, 96, 128, 132, 137, 145, 149, 261,
colony-stimulating factor, 267, 293 331, 420, 421, 423, 426, 440
colorectal cancer, xvi, 107, 198, 275, 296, 325, 326, cooking, xi, 46, 164, 172, 185
327, 328, 329, 330, 331, 332, 333, 334, 335, 338, cooling, 31, 38
339, 340, 358 COPD, xvii, 383, 384, 385, 387, 388, 389, 390, 391,
combination therapy, xvi, 302, 312, 313, 314, 409 392
common symptoms, 384 copper, 70, 71, 73, 78, 111, 175, 223, 226, 237, 404
communication, 99, 251, 256, 451, 453, 457, 459, corn, 85, 162
460, 461 cornea, 199, 202
community, x, xviii, 23, 375, 383, 386, 388 coronary arteries, 204
Index 471
coronary artery disease (CAD), 245, 246, 248, 252, cyclooxygenase, 10, 161, 320, 339
254, 255, 402 cyclooxygenase-2, 10, 161
coronary heart disease, xv, 71, 75, 79, 198, 200, 202, cyclooxygenases, 328
243, 244, 245, 252, 254, 362 cysteine, 367
coronavirus, 29, 36 cytochrome, 140, 141, 154, 161, 166, 167, 222, 313,
correlation, xv, 71, 112, 165, 219, 234, 245, 291, 424
354, 356, 435, 458 cytokine, 39, 40, 153, 332, 342, 400
correlation coefficient, 354, 356 cytokine response, 39
correlations, 108, 438 cytokines, 227, 268, 277, 328, 331, 333
cortex, ix, 1, 2, 13, 14, 294, 440 cytokinesis, 272, 296
cortical neurons, 15, 209, 239 cytoplasm, 56, 168, 294, 329, 334, 372
corticosterone, 5, 6, 7, 162 cytoprotective, 445
cosmetics, 26, 178, 179 cytosine, 264
Costa Rica, 99 cytoskeleton, 144, 230, 273, 274, 275, 277, 278, 284,
cost-effective, 188, 191, 192 285, 295, 368
costs, 56, 245 cytosol, 152, 225
cough, 384 cytosolic, 228, 413
coupling, 454 cytotoxic, xvi, 29, 175, 227, 267, 293, 301, 305, 312,
covalent, 263, 291 316, 365, 376, 377, 426, 454, 456, 458
covering, 27 cytotoxic action, 305
COX-2, 20, 161, 328, 336 cytotoxic agents, 175
CpG islands, 372, 373, 374 cytotoxicity, 39, 52, 99, 148, 305, 317, 360
CPI, 298 cytotoxins, 359
CRC, 120, 174, 358
creatine, 26, 449
D
creatine kinase, 26, 449
dairy, 123, 455
creatinine, 163, 403
dairy products, 455
CREB, 241, 333
Dallas, 254
crops, 105
data collection, 389
cross-linking, 279, 395, 403, 406
daughter cells, 334
cross-sectional, 206, 224, 233, 254, 385, 434, 446
de novo, 224, 331
cross-sectional study, 206, 224, 233, 254, 385, 434,
death, xvii, 12, 14, 15, 20, 50, 55, 111, 140, 209,
446
210, 212, 213, 215, 216, 217, 226, 228, 229, 230,
cross-talk, 458
234, 245, 247, 256, 261, 263, 277, 295, 326, 336,
Cryptococcus, 33
338, 383, 384, 441, 442, 443, 453, 455, 457
CSF, 267
deaths, xvi, 161, 248, 325, 364, 402
C-terminus, 414
decay, 64, 76, 178
cues, 117, 430
decisions, 104
cultural differences, 458
defects, 119, 338, 457
cultural factors, 452
defense, 124, 168, 246, 248, 267, 404, 437, 440, 442
culture, 49, 50, 51, 55, 195, 226, 231, 260, 265, 275,
defenses, 433, 442
289, 293, 316, 332, 352, 366, 367, 375, 377, 382,
defibrillator, 250, 256
423, 452
deficiency, 104, 105, 119, 120, 266
culture conditions, 260, 367
deficit, 430
culture media, 289, 377
deficits, 237, 444
curcumin, 175, 201, 312, 316, 348, 359
definition, 387
curing, 399
degenerative disease, 437
customers, 47
degradation, xii, 82, 83, 84, 85, 86, 91, 92, 93, 95,
cyclin D1, 130, 140, 143, 320, 335, 382
96, 97, 198, 199, 200, 204, 229, 230, 231, 234,
cyclin-dependent kinase inhibitor, 150, 263, 277, 345
241, 242, 248, 263, 265, 306, 308, 329, 332, 335,
cyclin-dependent kinases, 129
344, 371, 431, 445
cyclins, 263, 306, 335
degradation pathway, 263
cyclodextrins, 295
degrading, 84, 262, 332
472 Index
dehydration, 169 diet, xvi, 74, 104, 105, 107, 110, 111, 117, 119, 120,
dehydrogenase, xiii, 125, 137, 138, 152 122, 123, 136, 151, 153, 154, 165, 168, 172, 173,
delivery, 47, 56 202, 205, 246, 249, 252, 254, 256, 302, 325, 329,
delocalization, 439 336, 345, 384, 392, 394, 399, 437, 454, 458, 459
dementia, ix, xix, 201, 206, 224, 236, 385, 430, 431, dietary, xvi, 13, 19, 21, 26, 37, 39, 46, 47, 48, 75, 78,
434, 442, 448 79, 99, 100, 105, 120, 121, 123, 126, 136, 143,
demographic characteristics, xviii, 383 144, 147, 150, 153, 172, 175, 194, 198, 202, 234,
dendritic cell, 20, 40 247, 285, 299, 325, 326, 330, 337, 338, 340, 345,
dengue, 293 371, 374, 380, 385, 389, 391, 402, 424, 435, 452,
denitrification, 173 453, 454, 455
density, 12, 14, 19, 20, 26, 40, 66, 70, 78, 108, 121, dietary fat, 120, 338
143, 151, 204, 246, 253, 294, 310, 314, 319, 329, dietary fiber, 194
361, 405, 406, 407 dietary intake, 389, 391
dental caries, 30 dietary supplementation, 144, 402
dental plaque, 30, 37 dieting, 120
deoxynucleotide, 349 diets, 78, 115, 123, 165, 326, 454
deoxyribose, 356, 360 differentiated cells, 128, 452
Department of Agriculture, 75, 78, 170 differentiation, x, xiii, 45, 56, 60, 61, 125, 126, 128,
dephosphorylation, 130, 154, 283, 284, 285, 368, 129, 130, 131, 132, 134, 135, 140, 143, 144, 145,
372 147, 148, 149, 228, 230, 239, 263, 318, 332, 333,
depolarization, 361, 417 334, 335, 336, 340, 342, 379, 382, 452, 453, 457,
deposition, 227, 228, 433 460
deposits, 249 digestibility, 137
depression, 119 digestion, 26, 136, 400, 424
deprivation, 110, 120, 432 digestive tract, 358, 445
derivatives, xvii, xix, 27, 28, 29, 39, 40, 47, 58, 67, dihydroquercetin, 27, 41
165, 185, 199, 201, 203, 204, 222, 233, 265, 267, dimer, 25, 420
279, 297, 298, 312, 322, 347, 348, 349, 350, 351, dimeric, 101, 365
352, 353, 354, 356, 357, 358, 359, 361, 401, 419, dimerization, 264
420, 422, 423, 425, 426, 447 direct action, 16
dermatitis, 56 disability, 392
destruction, 29, 167, 174, 440 disaster, 104
detachment, 52 discrimination, 72, 154
detection, xvi, 49, 50, 154, 235, 325, 326, 338 disease progression, 20, 237
detoxification, 444 diseases, ix, xii, xiii, xiv, xv, xvi, xix, 29, 57, 71, 81,
detoxifying, 163, 263, 377, 394 104, 105, 120, 125, 126, 135, 142, 197, 198, 200,
developed countries, 104, 200 201, 219, 221, 226, 231, 247, 248, 252, 269, 301,
developmental factors, 135 326, 385, 429, 430, 441, 444, 453, 454, 455
dexamethasone, 250 disorder, 393, 433
diabetes, ix, xviii, 26, 57, 64, 121, 126, 131, 134, dispersion, 69
136, 138, 139, 143, 144, 150, 151, 246, 247, 248, disposition, 405
253, 255, 268, 393, 394, 395, 397, 399, 400, 402, dissociation, 250
403, 404, 405, 406, 407, 408, 409, 411, 412, 416, distilled water, 113, 138
430, 454 distraction, 16
diabetes mellitus, xviii, 121, 138, 247, 393, 394, 395, distress, 6, 7, 9, 10, 16, 17
404, 405, 407, 409 distribution, 13, 21, 74, 134, 136, 152, 224, 235, 267,
diabetic nephropathy, 255, 400, 408 272, 288, 319, 386, 417, 443, 446, 448, 453
diabetic patients, 393 disulfide, 278
diabetic retinopathy, xiv, 197, 198 diuretic, 83
diacylglycerol, 277 division, 31, 129, 130, 147, 283, 364, 369, 370
diarrhea, 29, 31, 32 DMFA, 172
dienes, 70 DNA, vi, xvii, 30, 127, 133, 140, 149, 150, 166, 167,
172, 174, 175, 190, 192, 199, 203, 229, 230, 241,
Index 473
246, 247, 255, 258, 264, 266, 270, 273, 282, 289, dysplasia, 167
290, 291, 295, 310, 321, 327, 332, 333, 336, 342, dysregulation, 146, 147
343, 345, 347, 348, 349, 351, 352, 356, 359, 360,
361, 364, 369, 370, 373, 380, 381, 430, 435, 445,
E
447, 452, 458
E. coli, 32, 33, 34, 267, 349, 352
DNA damage, 30, 133, 149, 167, 174, 175, 190, 192,
eating, xiii, 125, 454
247, 255, 258, 332, 333, 342, 369, 445
E-cadherin, 275, 276, 296, 308, 309, 334, 382
DNA polymerase, xvii, 199, 203, 347, 348, 351, 352,
edema, 160, 353, 354, 446
359, 360, 361
Education, 197, 387, 390
DNA repair, 133, 150, 264, 333, 336, 345, 360, 361
EEG, 2, 13, 14, 16, 19, 20
DNA strand breaks, 229
egg, 122, 452
DNase, 140
eicosanoid, 328
docetaxel, 313, 322
elderly, xix, 75, 121, 224, 253, 327, 429, 431, 434
docosahexaenoic acid, 437, 444
electric charge, 160
dogs, 248, 255
electrochemical detection, 235
dominance, 27
electron, 31, 32, 66, 77, 118, 182, 187, 190, 439
donations, 358
electron microscopy, 31
donors, 118
electron spin resonance, 66, 77
dopamine, 12, 15, 22, 135, 160, 176, 208, 215, 220,
electrons, 263
225, 236, 240, 431, 448
electrophoresis, 265
dopaminergic, 12, 14, 20, 21, 164, 168, 222, 225,
ELISA, 332
227, 233, 237, 238, 431, 446
elongation, 225, 230, 282, 298, 299, 368
dopaminergic neurons, 12, 20, 227, 238
embryo, 282, 452, 453
dosage, 13, 402
embryogenesis, 457
dose-response relationship, 389, 400
embryonic development, 452
dosing, 13, 22, 110, 288, 322
emulsification, 135, 137, 141
Down syndrome, 241
emulsions, 424
down-regulation, 128, 132, 133, 217, 229, 238, 263,
enantiomers, 240
278
encapsulated, 153
drinking, xviii, 16, 58, 108, 120, 121, 135, 138, 161,
encephalitis, 267, 293
162, 165, 178, 199, 202, 224, 244, 247, 248, 249,
encephalomyelitis, 442
250, 254, 264, 272, 279, 282, 286, 287, 290, 303,
encoding, 123, 222, 270, 280, 282, 286, 328
310, 311, 313, 316, 319, 332, 361, 362, 366, 383,
endocrine, 121, 126, 135, 140, 152, 315, 417
385, 386, 387, 388, 389, 390, 391, 394, 395, 451,
endocrine system, 121, 152, 315, 417
455, 458, 460
endocrinological, 152
drinking pattern, 388
endocytosis, 154, 334
drinking water, 165, 247, 248, 249, 310, 311, 313,
endometriosis, 198
332, 366, 458
endometrium, 340
drowsiness, 13
endoplasmic reticulum, 132, 154, 162, 310
drug design, 290, 314
endothelial cell, 99, 127, 135, 160, 170, 199, 200,
drug resistance, 263, 298, 322
203, 204, 247, 296, 308, 319, 340, 341, 412
drug targets, 263
endothelial cells, 99, 127, 160, 170, 203, 204, 247,
drug therapy, 231
293, 340, 341, 412
drug treatment, 311
endothelial dysfunction, xv, 243, 244, 247, 252, 254,
drug-resistant, 370, 371, 380
340
drugs, x, xvi, 23, 27, 30, 31, 35, 39, 126, 135, 168,
Endothelin, 410
198, 201, 227, 228, 236, 252, 269, 286, 302, 313,
endothelium, 247, 295, 319
325, 352, 365, 375, 455
endotoxemia, 238
dry matter, 82
end-stage renal disease, 403
drying, 84, 126, 158, 394, 431
endurance, 26, 249
duodenum, 329
energy, 105, 117, 118, 122, 126, 128, 131, 132, 138,
duration, 163, 386, 388
141, 143, 147, 153, 154, 244, 249, 253, 404, 412,
dyslipidemia, 135, 200, 255, 407
416, 453, 454
474 Index
enlargement, 132 ESR, 66, 235

enolase, 230 essential oils, xii, 81, 83
enterovirus, 29, 39 ester, 48, 59, 135, 136, 258, 263, 280, 322, 378, 417
environment, 46, 47, 93, 121, 128, 164, 333, 412, ester bonds, 136
430, 452 esterase, 56, 135, 170
environmental factors, xvi, 325 esterification, xi, 46, 47, 135
environmental impact, 190 esters, xi, 27, 46, 47, 55, 56, 64, 65, 76, 143, 240,
enzymatic, xix, 27, 48, 56, 140, 164, 351, 419, 420, 256, 330, 427, 442, 456
426 estimating, 389
enzymatic activity, 140 estradiol, 117, 127, 150, 303, 305
enzyme induction, 377 estrogen, xvi, 127, 138, 145, 150, 239, 301, 315,
enzyme inhibitors, 143 316, 317, 320, 322, 323, 335, 345
enzyme secretion, 152 estrogen receptors, 127
enzymes, xvii, xix, 24, 26, 27, 28, 30, 33, 40, 82, 84, ethanol, xiv, 37, 49, 50, 51, 73, 86, 109, 160, 161,
126, 136, 137, 152, 158, 160, 162, 163, 166, 170, 165, 169, 170, 173, 177, 179, 181, 194, 397
171, 173, 175, 199, 222, 226, 230, 241, 248, 260, ethics, 459
263, 265, 283, 303, 328, 347, 352, 365, 367, 368, ethyl acetate, 165
400, 404, 429, 433, 440, 442, 446 etiology, 120, 173, 222, 402, 403
eosinophils, 267 eukaryotes, 273
Epi, 36, 39 eukaryotic cell, 129, 141, 262, 334
epidemic, 126 Euro, 245
epidemiology, xv, 120, 202, 219, 234, 454 Europe, 120, 121, 302, 434
epidermal cells, 265 evolution, 453, 454
epidermal growth factor, 127, 147, 240, 263, 268, excess body weight, 126
287, 291, 295, 296, 305, 317, 367 excision, 150, 356, 360
epidermal growth factor receptor, 127, 147, 240, excitation, x, 1, 2, 15, 16
287, 291, 296, 305, 317, 367 excitotoxicity, 140
epidermis, x, 31, 45, 47, 52 exclusion, 386
epigenetic, xvii, 264, 291, 363, 364, 372, 373, 374, excretion, 13, 136, 138, 143, 165, 224, 260, 399
454, 455, 458, 459, 461 execution, 140
epigenetic mechanism, 264, 373, 374, 454 exercise, 131, 147, 249, 394, 454, 455
epigenetics, 291 exertion, 384
epithelial cell, 26, 30, 60, 127, 148, 244, 251, 256, exocytosis, 152
264, 292, 318, 332, 334, 336, 339, 361, 379, 409, experimental condition, xi, 63, 64, 72, 74, 287, 421
417, 458, 461 experimental design, 84, 458
epithelial cells, 26, 30, 127, 148, 244, 251, 256, 264, exploitation, 35, 36
318, 332, 339, 361, 379, 409, 417, 458, 461 exposure, xiii, xviii, 15, 57, 93, 95, 128, 140, 157,
epithelial ovarian cancer, 392 160, 161, 162, 166, 168, 170, 171, 174, 235, 336,
epoxy, 166, 173 366, 383, 388, 389, 456, 458
Epstein-Barr virus, 28, 36, 55, 60, 380 external environment, 105
equilibrium, 270, 452 extracellular matrix, 128, 265, 292, 331, 332, 334
Erk, 129, 130, 140, 205 extraction, 64, 65, 72, 75, 76, 123, 195, 381, 397,
ERK1, 128, 129, 135, 143, 147, 220, 228, 229, 240, 399, 408
263, 278, 279, 291, 367 extrapolation, 302
erosion, 30 eyes, 5, 11, 14, 18, 30, 249
erythrocyte, 160, 169, 417
erythrocytes, 152, 160, 162
F
erythroid, 167, 367
failure, 16, 153
erythropoietin, 230, 241
familial, 338
Escherichia coli, 32, 38, 99, 182, 293, 359, 361
family, 2, 31, 127, 128, 130, 137, 140, 153, 222, 229,
ESI, 76, 422, 423
230, 241, 247, 261, 264, 269, 289, 290, 291, 294,
esophageal cancer, 264, 287
309, 318, 327, 331, 332, 335, 336, 342, 351, 356,
esophagus, 2, 264, 329, 366
359, 431
Index 475
family history, 327 fish meal, 165

family members, 140, 222, 264 fish oil, xi, 46
famine, 453, 454 fission, 260
FAO, 178 flavonoid, xv, 2, 21, 27, 75, 78, 89, 176, 178, 223,
Far East, 24 224, 237, 243, 244, 246, 247, 251, 252, 253, 287,
farming, 453 405, 427
Fas, 127, 137, 140, 145, 277, 332 flavonoids, ix, xiii, xv, 1, 3, 7, 19, 20, 22, 28, 37, 38,
fasting, 13, 138, 248, 400, 411, 413 41, 64, 75, 76, 89, 99, 100, 107, 118, 120, 126,
fat, xiii, 26, 104, 108, 117, 120, 122, 125, 126, 127, 153, 157, 168, 169, 221, 224, 226, 232, 233, 235,
131, 132, 133, 136, 137, 138, 140, 141, 142, 143, 237, 243, 245, 247, 251, 252, 253, 254, 258, 289,
144, 147, 151, 153, 154, 172, 244, 246, 249, 253, 314, 340, 362, 385, 403, 404, 408, 431, 444, 447
256, 314, 329, 340, 361, 394 flavor, xii, 82, 84, 85, 87, 89, 90, 91, 179
fatigue, 144 flavors, 85
fats, 123, 136, 142, 158, 455 flexibility, 310, 341
fatty acids, 137, 142, 148, 160, 246, 248, 249, 360, flow, xiv, 14, 207, 208, 416
443 fluctuations, 72
Fatty liver, 144 fluid, 110, 162, 238
fax, 419 fluorescence, 174, 265
FDA, 55 fluorescence in situ hybridization, 174
feces, 261 fluoride, 106, 118
feedback, 132, 152, 379 flushing, 179
feeding, 107, 110, 117, 141, 287 focal cerebral ischaemia, 216
females, 253, 302, 388 folate, 266, 285, 294, 338
fermentation, 2, 24, 25, 30, 31, 36, 41, 82, 126, 195, Folate, 266
365, 394, 422, 423, 431 food additives, 46
ferric ion, 109, 225 food allergy, 277, 278
ferritin, 227, 231, 232, 236, 237 food industry, 48, 179
ferrous ion, 110, 167, 439 food intake, xii, xiii, 103, 104, 105, 111, 113, 115,
fertility, 104 116, 117, 118, 119, 120, 121, 125, 147, 152, 164,
fertilization, 118, 121 165, 249, 315, 402, 417
fertilizers, 105, 120 food production, 161, 454
fetal, 49, 224, 235, 452, 457 food products, 118
fetuses, 326 Ford, 150, 380
fever, 222 forebrain, 209, 213, 216, 217
FGF-2, 331 Forestry, 143, 384, 391
fiber, 194, 244, 273 fossil, 454
fibers, 117, 272, 273, 274, 287, 296, 378, 455, 457 fossil fuel, 454
fibrils, 225 Fourier, 13
fibrin, 265 Fox, 224
fibrinogen, 293 fragmentation, 133, 140, 172, 332
fibroblast, 127, 184, 185, 199, 203, 331, 353 free radical, xi, xii, xix, 26, 63, 64, 65, 66, 69, 70, 72,
fibroblast growth factor, 127, 199, 203, 331, 340 73, 75, 76, 77, 83, 87, 96, 100, 103, 108, 109,
fibroblast proliferation, 353 112, 118, 124, 139, 160, 161, 162, 163, 174, 175,
fibroblasts, 61, 150, 240, 276, 371, 379, 380 221, 223, 226, 232, 246, 248, 401, 403, 404, 411,
fibrogenesis, 161 412, 429, 433, 439, 443, 446, 448, 449
fibronectin, 265, 267, 293, 309, 334, 344 free radical scavenger, 26, 64, 162, 439
fibrosarcoma, 261, 263, 287, 344 free radicals, xi, xix, 26, 63, 64, 66, 72, 73, 77, 83,
fibrosis, 148, 161, 170, 376 109, 124, 139, 160, 162, 174, 221, 223, 226, 232,
filament, 265, 289, 292, 299 246, 248, 403, 404, 411, 429, 433, 446
Filipino, 303, 304 free-radical, 65, 118, 163, 433
Filobasidiella neoformans, 33 freeze-dried, xiv, 87, 177, 185, 186, 189, 194
Finland, 104, 120 fresh water, 2
fish, xi, 46, 165, 246, 455 friendship, 57
476 Index
frog, 414, 415 gene silencing, 275, 276, 291, 381

fructose, 85, 241, 396, 406, 448 generation, xv, 26, 64, 68, 100, 128, 139, 148, 160,
fruit juice, 19, 79, 100 162, 163, 167, 175, 219, 221, 223, 225, 227, 232,
fruit juices, 19, 79, 100 268, 277, 289, 331, 361
fruits, 67, 69, 99, 100, 107, 175, 221, 302, 455 genes, xvi, 28, 33, 131, 132, 138, 201, 206, 222, 228,
frying, 365 229, 230, 231, 233, 234, 236, 255, 263, 264, 275,
FTIR, 123 277, 282, 285, 286, 289, 291, 295, 298, 321, 325,
FT-IR, 396 327, 329, 330, 333, 339, 342, 343, 364, 370, 372,
FTIR spectroscopy, 123 374, 381, 404, 405, 440, 446, 448, 452, 453, 456,
fuel, 147, 153, 454 457, 460
fumigants, 178 Geneva, 196
fungi, 27, 33, 34 genistein, 198
fungicidal, 33, 40 Genistein, 147, 202
fusion, 277 genital warts, 55
genome, 348, 364, 369, 374, 452, 453
G genomes, 452
genomic, 150, 338, 458
G protein, 129, 294, 298
genomics, 148
GABA, ix, 1, 3, 15, 19, 21, 208, 210, 212, 213
genotoxic, 40, 167
GABAB, ix, 1, 6
genotype, 285, 286, 299, 305, 344
GABAergic, 4, 5, 6, 7, 8, 9, 10, 11, 18, 19, 21, 214
genotypes, x, 23, 35, 285
Gadus morhua, 196
Georgia, 45, 57, 60
galloyl, 7, 8, 9, 27, 28, 29, 143, 281, 310, 372, 420,
geriatric, 452
423, 439
germ line, 452
Gamma, 164
Germany, 36, 243, 245
gamma radiation, 194, 195
germination, 31
gamma-aminobutyric acid, 215, 216, 217
GFAP, xiv, 14, 207, 211
gas, 180
ginger, 297
gases, 384
Ginkgo biloba, 236
gasoline, 166
ginseng, 83, 85
gastric, 14, 30, 38, 39, 42, 108, 117, 120, 122, 141,
GIP, 105
150, 161, 164, 165, 169, 173, 204, 292, 341, 377,
gland, 321, 457
460
glass, 385
gastric mucosa, 30, 161, 169
glial, 230, 241, 292
gastrin, 105
glial fibrillary acidic protein, 230, 241, 292
gastritis, 41
glial fibrillary acidic protein (GFAP), 230
gastrocnemius, 26
glioblastoma, 289
gastroenteritis, 29
GLP-1, 105
gastrointestinal, xii, xvi, 81, 91, 107, 122, 164, 302,
glucagon, 117
315, 325, 326, 338, 406
gluconeogenesis, 138, 153, 404, 405
gastrointestinal tract, xvi, 91, 107, 122, 325, 326
glucose, xiii, 30, 105, 107, 117, 125, 131, 132, 134,
GC, xvii, 7, 25, 27, 30, 82, 106, 221, 223, 281, 341,
135, 137, 138, 139, 143, 145, 147, 150, 152, 153,
347, 348, 349, 350, 353, 354, 396
154, 162, 201, 205, 230, 244, 248, 253, 255, 329,
GE, 122, 145, 148, 316, 319, 320, 340, 341, 403
340, 394, 396, 399, 400, 402, 403, 404, 405, 406,
gel, 47, 48
408, 409, 412, 413, 415, 416, 417, 448
gelatin, 30
glucose metabolism, 150, 244, 255, 394, 400, 402,
gels, 49
405, 406
gender, xviii, 384, 386, 387, 388, 392, 454, 455, 456
glucose tolerance, 138, 147, 153, 248, 329, 340, 400,
gender differences, 392
402, 412
gene expression, xi, 19, 46, 127, 137, 139, 147, 148,
glucose tolerance test, 248, 412
149, 150, 151, 234, 263, 276, 282, 293, 296, 309,
glucosidases, 424
317, 342, 344, 345, 372, 374, 379, 440, 441, 442,
GLUT, 231
457, 461
GLUT4, 142, 143
gene promoter, 381
Index 477
glutamate, ix, xiv, 1, 2, 4, 14, 15, 16, 20, 164, 207, GST, 163
208, 209, 212, 213, 215, 239, 367 GTE, xii, xiii, 26, 103, 104, 108, 109, 111, 112, 113,
glutamate decarboxylase, xiv, 14, 207, 208, 212 114, 115, 116, 117, 118, 119, 161, 302, 305, 310,
glutamic acid, 22, 209, 210, 212, 213, 215 311, 313, 314
glutamine, 15, 20, 82, 404 guidelines, 35
glutathione, 21, 161, 162, 163, 167, 220, 222, 232, gut, 13, 37, 74, 135, 296, 330
246, 248, 367, 368, 433, 440, 443, 445
glutathione peroxidase, 162, 246, 367, 368, 433, 440,
H
443, 445
H. pylori, 30, 34
glycation, 248, 255, 395, 403, 404, 406, 407, 408
H1, 230
glycerin, 48, 49, 52, 56
H1N1, 29
glycerol, 152
H3N2, 29
glycine, 15
Haifa, 219
glycogen, 239, 329, 400
half-life, 12, 13, 224, 260
glycogen synthase kinase, 239, 329
hanging, 110
glycol, xiv, 177, 188, 189
harm, xx, 374, 451, 459
glycolipids, 360
harmful effects, 375
glycoprotein, 395, 397, 407
harvest, xvii, 347, 348, 431
glycosides, 82, 106, 118
harvesting, 87
glycosyl, 294
hay fever, 277
glycosylated, 221, 295
hazards, 458
glycosylation, 402
HBV, 29
GM-CSF, 267
HDL, 136, 246
gossypol, 178, 194
head and neck cancer, 175
gout, 178
headache, 222
government, iv
healing, 55, 56, 456
GPCR, 139
health care, x, xi, 45, 46, 56
GPI, 269, 294
health effects, xiii, 75, 126, 157, 160, 161, 215, 232,
GPx, 162, 440
287, 361, 391, 401
grain, 100
health problems, 105, 141
Gram-negative, 32, 42
health status, 364
granules, 277, 293
heart, xii, xv, 15, 26, 64, 71, 75, 79, 81, 105, 119,
granulocyte, 58, 267, 293
123, 198, 200, 202, 243, 244, 245, 246, 248, 249,
grapes, 32
251, 252, 254, 255, 256, 314, 326, 329, 362, 391,
grass, 83
393, 407, 414, 433
grouping, 452
heart disease, xv, 64, 71, 75, 79, 105, 119, 200, 243,
groups, xi, xii, xviii, 3, 10, 14, 26, 28, 34, 46, 65,
244, 246, 254, 314, 326
103, 107, 110, 111, 113, 115, 119, 158, 160, 162,
heart failure, 249
163, 182, 221, 226, 233, 249, 251, 258, 261, 269,
heart rate, 15
280, 310, 312, 326, 335, 337, 357, 368, 383, 387,
heart rate (HR), 15
388, 413, 420, 423, 437, 439, 455
heat, 31, 93, 101, 131, 194, 220, 231, 241, 365
growth factor, 10, 15, 117, 127, 128, 129, 139, 146,
heat shock protein, 220, 231, 241
147, 148, 170, 199, 200, 202, 203, 204, 220, 228,
heating, 381
239, 240, 263, 268, 287, 290, 291, 293, 295, 296,
height, 386
305, 307, 308, 316, 317, 319, 322, 328, 329, 331,
Helicobacter pylori, 30, 32, 39, 41, 42, 362
333, 335, 336, 340, 341, 342, 367, 381, 455
helper cells, 29
growth factors, 127, 128, 129, 139, 170, 199, 203,
hemagglutinin, 29
228, 268, 308, 328, 331, 333, 335, 455
hematocrit, xiv, 207, 209, 214
growth inhibition, 185, 199, 270, 276, 282, 284, 287,
hematologic, 331
308, 336, 340, 354, 357, 366, 368, 370, 371, 374,
hematological, 171
380
hematopoietic, 332, 340
growth rate, 353, 371
heme, 247
GSK-3, 329, 445
heme oxygenase, 247
478 Index
hemoglobin, 174, 248 histamine, 268, 277, 278, 279, 281, 283, 297
hemolytic uremic syndrome, 32 histological, 412
hemorrhage, 30, 163 histone, 148, 230, 264, 373, 461
hemorrhages, 160 HIV, 28, 37, 39, 42, 55, 60, 238, 329, 377
hepatic fibrosis, 161, 170 HIV-1, 28, 39, 42, 60, 238
hepatic necrosis, 172 HNE, 433
hepatitis, 42, 55, 57, 59, 302, 315 HO-1, 164, 247
hepatitis a, 315 holoenzyme, 372, 378
hepatitis B, 29, 42, 55, 59 homeostasis, x, 45, 57, 131, 132, 139, 222, 231, 334,
hepatitis d, 315 395, 445
hepatocarcinogenesis, 338, 427, 461 homogenized, 86
hepatocellular, 146, 161 homolog, 299
hepatocellular carcinoma, 146 homology, 234
hepatocyte, 237, 307 homovanillic acid, 12
hepatocyte growth factor, 307 Honda, 42, 60, 427
hepatocytes, 56, 127, 150, 151, 175, 452 honey, 83, 85
hepatoma, 127, 145, 231, 234, 282 hormone, 108, 137, 305, 314, 342
hepatotoxicity, 164, 170, 171, 302, 315 hormones, 117, 128, 137, 200, 455
HER2, 127, 148, 305, 306, 309, 317 hospital, 386
herbal, 72, 77, 79, 408 hospitalization, 251
herbicide, 163 hospitals, xviii, 383, 385, 386
herbs, 198, 302 host, 28, 30, 167, 174, 267, 384
herceptin, 314 hot water, 26, 165, 384, 395
hereditary non-polyposis colorectal cancer, 326 household, x, 45, 46
hERG, 253 HPLC, xi, 37, 46, 49, 50, 53, 54, 56, 57, 76, 144,
herpes, 55, 57, 59 180, 268, 397, 398, 420, 421, 422
herpes simplex, 55, 57, 59 HPV, 55
heterocycles, 36 HR, 15, 77, 147, 340, 386
heterogeneous, 268 H-ras, 167, 263, 288, 327
high blood cholesterol, 143, 402 HRP, 423
high density lipoprotein, 246, 295 HSC, 161
high fat, 151 HSP90, 220, 231
high risk, 143, 256, 402 human behavior, 393
high school, 386, 387 human brain, 293, 457
high temperature, 24, 385 human exposure, 164
high-density lipoprotein, 143, 329, 405, 406, 407 human genome, 348
high-fat, 104, 120, 136, 144, 249, 256 human immunodeficiency virus, 39, 42, 55, 57, 60,
high-performance liquid chromatography, 77, 235, 294
268 human papilloma virus, 55
high-risk, 361 human subjects, x, 1, 2, 15, 45, 138, 142, 168, 227,
high-speed, 59 244, 253, 288
high-throughput screening, 382 humans, x, 14, 20, 21, 24, 26, 28, 30, 31, 39, 45, 47,
hip, 108, 121, 249 57, 58, 69, 79, 96, 99, 105, 108, 135, 138, 145,
hip fracture, 108, 121 147, 167, 172, 201, 204, 205, 232, 244, 253, 254,
hip fractures, 108 255, 260, 265, 266, 288, 298, 302, 314, 321, 338,
hippocampal, xv, xix, 14, 207, 209, 210, 213, 215, 340, 358, 359, 362, 364, 368, 375, 406, 414, 416,
217, 236, 268, 295, 429, 430, 435, 436, 437, 438, 434, 437, 447
440, 444, 445, 446 Huntington's disease, 256
hippocampus, ix, xix, 1, 2, 13, 14, 15, 19, 212, 217, hydration, 160
225, 228, 429, 430, 433, 435, 436, 437, 438, 439, hydro, 47, 77, 136, 166, 173
440, 441 hydrocarbon, 241, 289, 456
Hippocampus, 430 hydrochloric acid, 86, 109
Hiroshima, 197, 304, 315 hydrocortisone, 49
Index 479
hydrogen, 33, 59, 65, 72, 74, 118, 167, 175, 190, ice, 12, 76, 311, 313, 361, 400
203, 248, 260, 264, 289, 366, 367, 375, 404, 420, identification, 41, 139, 172, 235, 269, 282, 286, 293,
439, 440, 442, 443 403, 443, 447
hydrogen bonds, 264 IgE, 127, 268, 277, 278, 296, 297
hydrogen peroxide, 33, 59, 65, 72, 74, 167, 175, 190, IGF, 127, 129, 146, 330, 336
203, 248, 260, 289, 366, 367, 375, 404, 420, 440, IGF-1, 127, 146, 330, 336
442, 443 IGF-I, 127, 129
hydrogenation, 179 IKr, xv, 243, 244, 251, 252
hydrolases, xix, 419 IL-1, 10, 30, 336, 400
hydrolysis, 30, 42, 56, 135, 136, 196, 424 IL-10, 30
hydrolyzed, 312 IL-6, 10, 276, 418
hydroperoxides, 70, 72, 80, 407 IL-8, 10
hydrophilic, 47, 77, 136 illumination, 229
hydrophobic, 47, 135, 264 immortal, 370, 371, 378, 379, 455
hydrophobicity, 34 immortality, 452
hydroquinone, 166, 175 immune response, 336
hydroxyl, xi, 20, 26, 28, 29, 46, 167, 175, 178, 191, immune system, 105, 119, 267, 329
192, 221, 223, 226, 231, 258, 263, 280, 302, 310, immunocytochemistry, 174
312, 330, 337, 357, 368, 400, 409, 413, 439 immunodeficient, 292
hydroxyl groups, xi, 26, 28, 46, 221, 258, 280, 310, immunofluorescence, 227
312, 337, 368, 413, 439 immunoglobulin, 15, 231, 297
hydroxylation, 28, 241, 281 immunohistochemistry, 311, 313
hydroxyproline, 161 immunological, 135
hydroxypropyl, 6 immunomodulatory, 39
hypercholesteremia, 286 immunomodulatory agent, 39
hyperglycemia, 138, 248, 399, 403, 404, 407, 411, immunoreactivity, 227
412, 416 immunotherapy, 305, 364
hyperlipidemia, 151, 246, 247, 254, 395, 403 impairments, 217
hypermethylation, 264, 291, 373 implementation, xv, 126, 219, 455
hyperplasia, 167 in situ, xi, 46, 319
hypertension, 104, 126, 145, 200, 215, 246, 247, in situ hybridization, 174
254, 268, 329, 340, 454 in utero, 235
hypertensive, 158, 208, 209, 215, 216, 247, 255, 258, inactivation, 130, 137, 138, 166, 203, 287, 322, 374
447 inactive, 31, 223, 310, 373, 380, 439
hypertrophy, 128, 145, 250, 329, 340 incidence, xiii, xvi, xviii, 104, 125, 224, 231, 246,
hypnotic, 6, 7 251, 301, 311, 312, 315, 335, 384, 393, 394, 399,
hypocholesterolemic, 83, 136, 137 454
hypomethylation, 373, 374 inclusion, 118, 143
hypotensive, 244, 246, 247 incubation, 49, 110, 140, 165
hypothalamic, 121 incurable, 326
hypothalamus, 135, 147 independent variable, 387
hypothesis, 28, 33, 163, 285, 303, 430, 446, 449, India, 157, 252
457, 458 Indian, 157
hypothyroidism, 119 indication, 72, 73
hypoxia, 131, 220, 222, 230, 231, 234, 237, 241, indicators, 74, 122
333, 414, 430, 443 Indigenous, 407
hypoxia-inducible factor, 222, 241 indirect effect, 403
hypoxia-ischemia, 237 indole, 190, 191
hypoxic, 231 inducer, xiii, 50, 104, 118, 119, 353
induction, xvi, 22, 56, 68, 69, 70, 130, 133, 134, 146,
I 153, 160, 166, 175, 203, 228, 229, 230, 232, 238,
263, 266, 278, 289, 296, 301, 306, 308, 309, 310,
IAEA, 178
311, 314, 316, 317, 319, 320, 330, 332, 333, 335,
ICD, 250
480 Index
336, 341, 342, 366, 368, 371, 374, 376, 377, 378, 281, 282, 283, 285, 317, 321, 348, 349, 351, 353,
379, 446 354, 361, 394, 400, 403, 404, 424, 458
induction time, 68, 69, 70 initiation, xvi, 28, 105, 120, 166, 200, 310, 321, 325,
industrial, ix, xiv, 166, 177, 178, 188, 190, 191, 192, 326, 338, 364, 456
195 injection, 3, 4, 5, 6, 7, 8, 9, 10, 11, 15, 17, 18, 135,
industrial application, 191, 192, 195 209, 210, 213, 214, 412
industrialized countries, 245, 247 injections, 161
industry, x, 23, 27, 48, 178, 179, 190, 194 injury, 42, 154, 161, 171, 172, 221, 225, 228, 236,
inert, 166 252, 256, 267, 302, 333, 416, 444
infarction, 14, 19, 215, 216, 245, 250 innervation, 122
infection, 28, 29, 30, 32, 34, 36, 38, 39, 41, 42, 56, inoculation, 271, 311
60, 120, 377 inorganic, 119
infections, 28, 30, 36, 39, 42, 268, 329 iNOS, 12, 161, 220, 227
infectious, 28, 46, 453, 455 inositol, 277, 295
infectious disease, 46 insecticide, 162, 171
infectious diseases, 46 insecticides, 162, 170
infectious mononucleosis, 28 insects, 161, 162
inflammation, xvii, 132, 148, 160, 200, 267, 306, insertion, 66, 248
308, 314, 328, 330, 348, 353, 354, 356, 357, 358, insight, 132, 331, 392
376, 399, 400, 424, 460 insomnia, 302
inflammatory, x, xvii, 10, 45, 46, 47, 56, 132, 135, instability, 47, 311, 327, 329, 367, 368
149, 151, 158, 163, 168, 222, 227, 244, 252, 277, insulin, xix, 105, 117, 120, 127, 128, 132, 134, 135,
308, 331, 332, 336, 342, 343, 348, 353, 354, 356, 138, 139, 144, 145, 146, 150, 151, 153, 154, 244,
357, 359, 361, 367, 424, 426, 455, 456 248, 255, 256, 329, 393, 394, 400, 402, 404, 405,
inflammatory bowel disease, 135 406, 407, 408, 409, 411, 413, 416, 442, 448
inflammatory cells, 163, 308 insulin resistance, xix, 134, 135, 138, 139, 144, 150,
inflammatory disease, 56, 222 151, 394, 400, 405, 406, 407, 411, 416, 448
inflammatory mediators, 277 insulin sensitivity, 138, 244, 248, 255, 256, 329, 394,
inflammatory response, 10, 132, 135, 361 400, 404, 405, 448
inflammatory responses, 132, 135, 361 insulin signaling, 139, 145, 151
influenza, 29, 39, 41, 55, 57, 58, 59, 427 insulin-like growth factor, 117, 127, 146
influenza a, 55 insulin-like growth factor I, 117
infusions, xii, 69, 70, 72, 74, 77, 78, 81, 83, 235, insults, 228, 412, 430, 440, 442
315, 408 integration, 265
ingest, 74 integrin, 127, 144, 334, 344
ingestion, 14, 19, 39, 58, 78, 136, 147, 161, 166, integrins, 334, 343
168, 223, 235, 253, 260, 261, 272, 288, 302, 315, integrity, 47, 174, 268, 272, 286, 332, 377
340, 414, 442 intensity, xiv, 177, 180
inhalation, 162, 170 interaction, 21, 26, 29, 66, 74, 77, 127, 130, 139,
inherited, 327, 364, 443 171, 200, 204, 217, 227, 230, 267, 274, 277, 279,
inhibitor, xiv, xvii, 14, 15, 130, 137, 145, 146, 150, 280, 295, 299, 333, 334, 344, 364, 373, 384, 413,
152, 171, 193, 197, 202, 203, 205, 207, 210, 212, 452, 453, 454, 458
225, 229, 239, 266, 291, 297, 313, 322, 323, 331, interaction effect, 217
332, 336, 347, 348, 352, 353, 354, 356, 357, 359, interaction effects, 217
360, 361, 366, 372, 378, 381 interactions, 33, 34, 37, 39, 78, 267, 280, 286, 334,
inhibitors, xiv, 36, 37, 129, 141, 143, 152, 164, 169, 343, 344, 452, 459
197, 198, 199, 200, 201, 203, 204, 213, 215, 225, interface, 136
231, 241, 264, 266, 282, 290, 291, 306, 318, 335, interference, 29, 275, 344
342, 343, 348, 349, 359, 380 interleukin, 10, 30, 40, 240, 279, 336, 345, 376, 400
inhibitory effect, xiv, 12, 15, 29, 39, 41, 55, 100, interleukin-1, 30, 40, 345, 376
130, 132, 134, 140, 144, 145, 166, 167, 174, 177, interleukin-2, 279
197, 204, 225, 261, 265, 272, 275, 276, 277, 279, internalization, 56, 293
international trade, 178
Index 481
interphase, 272, 274, 296, 334 Japanese, xiv, xviii, 38, 40, 47, 58, 69, 107, 153,
interpretation, 26, 72 165, 173, 196, 202, 204, 207, 208, 224, 245, 246,
interstitial, 127 248, 253, 254, 303, 304, 316, 358, 361, 384, 385,
interval, 117, 212 386, 388, 389, 390, 404, 406, 420, 427, 434, 437,
intervention, xvii, 26, 71, 277, 363, 403, 406, 454, 458, 460
456 Japanese women, 304
interview, xviii, 383, 386 jejunum, 135
interviews, 386 JNK, 20, 40, 128, 228, 238, 239, 263, 336, 367, 377
intestinal tract, 261 joints, 104, 249
intestine, 117, 135, 136, 195, 431 Jordan, 287, 448
intoxication, 161, 169 Jun, 11, 20, 40, 128, 144, 145, 146, 148, 149, 150,
intracellular signaling, 128, 228, 306, 308, 457, 458, 151, 154, 155, 172, 199, 203, 239, 253, 254, 255,
461 256, 263, 320
intracerebral, 13, 210, 212, 213 Jung, 20, 57, 203, 204, 236, 239, 319, 341, 377, 411,
intragastrically, 13, 212 445, 446
intraperitoneal, 135, 161, 412
intravenous, 12, 224
K
intrinsic, 131, 140, 154, 360
K+, 255, 395, 403, 407, 418
invasive, 30, 167, 267, 309, 319, 320
kappa, 170, 227, 238, 341, 442
iodine, 124
kappa B, 170, 227, 238, 442
ion channels, 230
keratinocyte, xi, 46, 49, 61, 382
ions, 70, 78, 109, 110, 160, 164, 167, 223, 226, 331,
keratinocytes, xi, 46, 51, 56, 58, 146, 229, 240, 263,
445, 453, 456
290, 318, 367, 377, 418
iron, 100, 111, 124, 160, 163, 167, 168, 221, 222,
kidney, 29, 132, 162, 171, 248, 249, 287, 370, 393,
223, 225, 226, 227, 229, 230, 231, 233, 234, 236,
403, 408, 409
237, 238, 241, 329, 375, 377, 381, 404, 439, 445,
kidneys, 433
446
killing, 296
irradiation, ix, xiv, 32, 177, 178, 179, 180, 181, 182,
kinase, xiii, xvii, 11, 40, 125, 128, 129, 130, 131,
185, 187, 190, 192, 193, 194, 195, 196, 343
135, 139, 141, 146, 147, 149, 151, 153, 206, 220,
IRS, 139
228, 229, 234, 238, 239, 240, 241, 247, 255, 256,
ischemia, xiv, 14, 19, 131, 207, 208, 209, 210, 212,
263, 265, 269, 277, 283, 286, 290, 294, 295, 296,
213, 214, 215, 216, 217, 221, 226, 234, 237, 239,
297, 298, 307, 318, 319, 321, 323, 329, 335, 341,
252, 256, 409, 414, 430, 446
343, 345, 348, 352, 367, 372, 441, 442, 445, 456
ischemic, 14, 20, 164, 208, 210, 215, 216, 217, 414,
kinase activity, 129, 139, 146, 238, 264, 318, 449
416, 445
kinases, 128, 129, 130, 146, 147, 154, 169, 220, 228,
ischemic brain injury, 445
230, 238, 239, 240, 263, 265, 277, 283, 289, 291,
island, 264, 291, 373, 381
294, 297, 298, 306, 316, 331, 335, 336, 405
isoflavones, 221
kinetic model, 66
isoforms, 210, 216, 228, 308, 315, 440, 441
kinetics, 67, 68, 72, 78, 140, 170, 235, 375, 405
isolation, 296
Kirchhoff, 360, 361
isomers, 281, 330
Kobe, 58, 197, 201, 347, 358
isozyme, 21
Korea, 125, 143, 177, 180, 190, 195, 411
isozymes, 228, 239
Korean, 165, 173, 193, 194, 195, 196
Israel, 219, 232
Italy, 23, 76 L
J
L1, 128, 129, 130, 131, 134, 140, 141, 145, 146, 149,
150, 152
JAMA, 40, 120, 204, 254
laboratory studies, 138, 401
Japan, vi, xviii, 1, 2, 18, 46, 47, 55, 57, 165, 197,
laccase, xix, 419, 420, 421, 423, 424
200, 201, 204, 207, 222, 252, 254, 257, 303, 304,
lactams, 43
315, 316, 347, 358, 359, 383, 384, 385, 386, 389,
lactase, 394, 399
391, 394, 395, 399, 408, 419, 421, 422, 423, 424,
lactation, 22
426, 429, 434, 445, 460
482 Index
Lactobacillus, 33 limitation, 74, 367, 384, 389

laminin, xiii, xv, 125, 127, 144, 257, 258, 266, 267, limitations, x, 23, 47, 67, 72, 389
287, 288, 292, 293, 295, 296, 308, 334, 344, 368, linear, xvii, 363, 369
378 linkage, 375, 458
Laminin, 266, 267, 292, 293, 343 links, 147
land, xiii, 157, 160 linoleic acid, 180, 194
large intestine, 431 lipase, 48, 56, 136, 137, 155, 420, 424, 425
large-scale, x, 23, 33 lipases, 26, 47, 108, 137, 141
larvae, 122 lipid metabolism, xiii, 107, 126, 131, 135, 136, 249,
larval, 122 254, 400
latency, 14, 166 lipid oxidation, xiv, 32, 177, 178, 179, 182, 185, 186,
LDL, xi, 56, 63, 66, 70, 72, 73, 74, 79, 96, 136, 200, 193, 195
204, 246, 248 lipid peroxidation, 26, 70, 74, 78, 160, 161, 162, 163,
leakage, 33, 163 167, 169, 170, 171, 175, 208, 210, 222, 226, 234,
learning, xix, 226, 429, 430, 431, 433, 435, 437, 439, 236, 237, 248, 255, 287, 367, 400, 403, 404, 407,
440, 442, 444 409, 430, 433, 439, 444, 445, 446, 447, 448
lecithin, 137 lipid peroxides, 248, 403, 443
left ventricular, 247 lipid profile, 254, 255
Legionella, 30, 32, 39, 40 lipid rafts, 152, 268, 269, 279, 286, 293, 294, 295,
Legionella pneumophila, 30, 32, 39, 40 299
legume, 195 lipids, xix, 132, 136, 137, 143, 151, 152, 201, 202,
lemongrass, xii, 81, 82, 83, 84, 85, 86, 87, 88, 89, 91, 244, 248, 424, 429, 430, 431, 433
95, 98 lipolysis, 26, 38, 137, 141, 152
leptin, 105, 117, 126 lipophilic, 55, 58, 59, 65, 77, 151, 231, 399
Lesion, 204 lipopolysaccharide, 40, 227, 238, 269, 294, 320
lesions, 29, 48, 56, 59, 200, 241 lipoprotein, 40, 56, 76, 78, 121, 137, 151, 200, 204,
lethal factor, 37, 261, 290 215, 246, 255, 295, 329, 361, 406, 407
lettuce, 32 lipoproteins, 56, 66, 70, 78, 136, 169, 253, 395, 402
leucine, 13 liposome, 267
leukaemia, 122 liposomes, 66, 77
leukemia, 144, 166, 174, 268, 290, 292, 297, 341, lipoxygenase, 320
352, 353, 354, 357, 366, 371, 376 liquid chromatography, 77, 235, 268
leukemia cells, 144, 268, 297, 371, 376 Listeria monocytogenes, 32
leukemic, 40 liver, ix, xix, 1, 2, 22, 55, 58, 127, 130, 131, 132,
leukemic cells, 40 137, 141, 144, 147, 150, 151, 152, 161, 162, 163,
leukocyte, 169 166, 167, 170, 171, 172, 176, 201, 205, 215, 225,
Leukocyte, 40 226, 246, 249, 251, 254, 256, 260, 266, 287, 293,
leukocytes, 26, 293 302, 315, 329, 358, 362, 366, 367, 400, 429, 431,
leukoplakia, 38 433, 456, 457, 458, 461
levodopa, 225 liver cancer, 55
life cycle, 42, 60 liver cells, 130, 161, 293
life span, 12, 226, 453 liver damage, 162
life style, 252, 455 liver disease, 58, 144, 150, 254, 362
lifespan, 379 liver transplant, 249
lifestyle, xv, 40, 58, 123, 126, 219, 247, 385, 386, liver transplantation, 249
389, 393 localization, 227, 296, 319, 341
lifestyles, 391 location, xviii, 87, 384, 386, 387, 388, 390
life-threatening, 249 locomotion, 265, 283
lifetime, xvi, 325, 326, 389 locomotor activity, 21
ligand, 127, 131, 139, 149, 229, 277, 295 locus, 328, 461
ligands, 20, 21, 128 London, 299
lignin, 27 long period, 385
likelihood, 389, 454, 455 longevity, 40
Index 483
longitudinal study, 388 malignancy, 316

long-term potentiation, 13, 217 malignant, 184, 309, 326, 331, 343, 452, 455, 456,
losses, 29 457
lovastatin, 457, 461 malignant cells, 331, 343, 455
love, 232 malignant melanoma, 184
low molecular weight, 231, 399 malondialdehyde, 163, 186, 187, 226, 433, 436
low risk, 285 malondialdehyde (MDA), 163
low-density, xv, 40, 76, 78, 200, 215, 243, 244, 246, maltose, 30
255, 294, 329, 358, 406 Mammalian, 321, 349, 352, 378
low-density lipoprotein, xv, 76, 78, 200, 215, 243, mammalian brain, 210
244, 246, 255, 329, 358, 406 mammalian cell, 131, 139, 263, 293, 298, 314, 356
low-level, 459 mammalian cells, 131, 139, 263, 293, 298, 314
LPO, xix, 429, 430, 433, 435, 437, 440, 441, 442 mammals, 16, 31, 129, 162, 217, 405, 452
LPS, 227 management, xiii, 104, 118, 119, 123, 139, 143, 215,
LSI, 35 285, 286, 326, 336, 391, 394
LTP, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 217 manganese, 65, 106, 111, 112, 118, 144, 171, 440,
luciferase, 227 445, 446
lumen, 132, 136, 200, 247 manganese superoxide, 445, 446
luminal, 295, 416 manganese superoxide dismutase, 445, 446
lung, xvi, 2, 42, 148, 154, 260, 270, 275, 287, 289, manipulation, 411
292, 295, 308, 321, 322, 325, 329, 335, 337, 341, manners, 140
345, 366, 368, 371, 373, 380, 385, 388, 389, 391, manufacturer, 49
392, 454, 456, 457, 458, 460 manufacturing, xvii, 36, 65, 188, 347, 348, 384, 431
lung cancer, 270, 275, 289, 308, 321, 322, 366, 368, MAPK, 20, 128, 129, 130, 145, 146, 220, 222, 228,
371, 380, 385, 388, 391, 454, 458, 460 233, 239, 263, 307, 336, 377, 440, 441
lungs, 30, 358, 389 MAPKs, 151, 161, 228, 233, 263, 443
lupus, 56 mapping, 20, 288
lymph, 49, 59, 135, 364 marital status, xviii, 384, 386, 387
lymph node, 49, 59 market, 108
lymphatic, 340 marrow, 174
lymphocyte, 267, 292 Maryland, 125, 443
lymphocytes, 174, 246, 367, 380 mass spectrometry, 66, 288
lymphoid, 366, 376 mast cell, 61, 267, 277, 278, 279, 293, 297
lymphoma, 30, 140, 266 mast cells, 267, 277, 278, 293, 297
lymphomas, 322 matrix, 128, 150, 161, 199, 203, 261, 265, 286, 292,
lysine, 169, 381 308, 331, 332, 334, 366, 367, 376, 424, 453
lysis, 34 matrix metalloproteinase, 150, 161, 199, 203, 261,
287, 308, 331, 366, 367, 376
M matrix protein, 261, 334
maturation, 15, 140, 267
M1, 320
Mb, 193
machinery, 128, 163, 229
MCA, xiv, 207, 208, 209, 210, 211, 212, 213, 214
machines, 31
MCI, 430
mackerel, 176
MDA, 163, 305, 306, 307, 308, 309, 310, 311, 312,
macromolecules, 69
313, 316, 317, 368, 370
macrophage, 121, 149, 200, 204, 267, 293, 294, 332,
meals, 105, 110, 119
342
mean systolic blood pressure, 247
macrophages, 30, 36, 39, 127, 132, 144, 174, 200,
measurement, 78
227, 238, 253, 261, 267
measures, 15, 66, 71, 105
Madison, 384, 391
meat, 105, 164, 172, 178, 185, 186, 194
magnetic, 265
media, 49, 66, 247, 275, 289, 333, 375, 377
maintenance, xvii, 105, 155, 256, 278, 308, 363
median, 453
males, 302, 388
mediation, 367
malic, 137
484 Index
mediators, 277, 282 metastasis, xiv, xvi, 197, 198, 199, 261, 265, 267,
medication, 311, 315 286, 287, 301, 308, 309, 310, 319, 320, 321, 326,
medicinal plants, 100, 361, 407 328, 330, 332, 334, 336, 337, 342, 345, 364, 417,
medicine, xiii, 64, 125, 179, 202, 243, 246, 250, 252, 424
295, 459 metastasize, 334
Mediterranean, 121, 253 metastatic, 49, 59, 198, 267, 299, 320
meiosis, 128, 360 metazoan, 452, 457
MEK, 130, 139, 149 metazoans, 457
melanin, xiv, 177 Methamphetamine, 446
melanoma, 154, 282, 320, 344 methanol, 65, 180, 194
melatonin, 240, 439, 448 methicillin-resistant, 31, 38, 41, 43, 99
membrane permeability, 35, 140 methylation, 148, 236, 260, 264, 280, 289, 291, 295,
membranes, 33, 36, 136, 216, 226, 267, 299, 403, 303, 322, 368, 373, 374, 381
447 Methylation, 373, 378, 381
memory, xiv, xix, 14, 19, 207, 209, 212, 213, 214, methylenetetrahydrofolate reductase, 285
216, 217, 429, 430, 433, 435, 436, 437, 439, 440, metric, xix, 419
444, 448 mGluR, 15
memory deficits, 444 mGluRs, 15, 21
memory performance, 435 micelle formation, 138, 415
men, xvii, 71, 120, 121, 150, 153, 202, 204, 224, micelles, 136, 137
245, 253, 326, 336, 383, 385, 386, 388, 394, 407, microbial, x, 23, 27, 35, 36, 45, 55, 57, 94, 96, 182,
434, 458, 460 193
mental state, 21 microcirculation, 170, 410
Merck, 36 microflora, 30
messenger RNA, 149 microglia, xiv, 207, 211, 225, 227
messengers, 139, 238, 277 microorganisms, x, xii, 23, 34, 82, 94, 167, 178, 182
meta-analysis, 79, 202, 303, 316, 392 microsatellites, 327
metabolic, xiii, xv, xvii, xviii, 107, 125, 126, 131, microscope, 50, 51
135, 138, 140, 144, 147, 148, 150, 151, 166, 198, microscopy, 31
209, 210, 214, 225, 241, 243, 247, 248, 252, 253, microsomes, 171
260, 329, 347, 352, 375, 402, 404, 411, 416, 431, microspheres, 405
432, 461 microtubule, 174, 285
metabolic disorder, 138, 198 microtubules, 299
metabolic pathways, 135, 260 microvascular, 203, 319, 403, 405
metabolic rate, 253, 329 midbrain, 440
metabolic syndrome, 144, 151, 247, 416 Middle East, 158
metabolism, xiii, 26, 27, 57, 75, 99, 100, 107, 121, middle-aged, 153, 367
124, 126, 131, 135, 136, 150, 152, 173, 174, 212, migration, 263, 267, 289, 292, 309, 334, 336, 344,
215, 223, 232, 235, 239, 244, 249, 253, 254, 255, 358
260, 287, 303, 311, 320, 322, 375, 393, 394, 400, mild cognitive impairment, 430, 443, 445, 447, 448
402, 405, 406, 442, 446, 452, 453 mild cognitive impairment (MCI), 430
metabolite, 25, 131, 163, 166, 170, 174, 225, 280, milk, 19, 31, 100, 195, 384
288, 303 minerals, xii, xiii, 103, 104, 106, 118, 244, 431
metabolites, x, 12, 23, 39, 58, 75, 78, 160, 166, 167, Mini-Mental State Examination, 434
174, 175, 198, 224, 232, 235, 253, 261, 277, 280, Ministry of Education, 201, 358, 426
288, 401, 431, 445 mitochondria, 140, 246, 249
metabolizing, 400 mitochondrial, 140, 141, 171, 222, 226, 229, 240,
metabotropic glutamate receptor, 15 348, 361, 367, 377, 417, 440, 445, 448, 457
metal chelators, 76, 227 mitochondrial DNA, 367, 448
metal ions, 160, 167, 223, 226, 331, 445 mitochondrial membrane, 140, 226, 361, 417
metalloproteinases, 199, 308, 331, 366, 367 mitogen, xiii, 11, 59, 125, 146, 161, 220, 222, 229,
metals, 71, 232, 455 233, 239, 290, 367, 377, 443
mitogen activated protein kinase, xiii, 125, 146
Index 485
mitogen activated protein kinases, xiii, 125 monomer, 47

mitogen-activated protein kinase, 11, 59, 161, 220, monomeric, 77, 126, 193, 221
222, 229, 233, 239, 290, 377, 443 monomers, 46
mitogenesis, 126, 129, 132, 134, 139, 154 mononuclear cell, 135, 276
mitogenic, 126, 128, 130, 139, 140, 141, 306, 308, mononuclear cells, 135, 276
316, 341, 456 monosaccharides, 30, 396
mitosis, 128, 147, 265, 334 monoterpenes, 83
mitotic, 128, 129, 140, 457 monounsaturated fat, 48, 137
mixing, 109 monounsaturated fatty acids, 137
MK-80, 212 mood, 15, 21
MLC, 283 Moon, 145, 150, 152, 380, 409
MMP, 161, 203, 204, 205, 261, 289, 331, 332, 335, morbidity, xvii, 326, 383, 384, 389
336, 377, 424 Morocco, 37
MMP-2, 204, 205, 261, 289, 336, 424 morphogenesis, 203, 382, 452
MMP-9, 261, 332, 335, 336, 377 morphological, 140, 162, 164, 277, 332
MMPs, 261, 366 morphology, 31, 273, 274, 278, 284
MnSOD, 440 mortality, 71, 165, 170, 199, 200, 204, 244, 245,
mobility, 310, 334 246, 247, 248, 250, 254, 296, 302, 326, 336, 385,
model fitting, 86 389, 391, 392, 400, 402, 452, 460
model system, 99, 194, 195, 335 mosaic, 29
modeling, 19, 264, 266 motorneurons, 12
models, x, xiii, xiv, xvi, xvii, 45, 70, 125, 131, 136, mouse, xi, 21, 46, 48, 52, 56, 57, 130, 166, 209, 210,
137, 167, 170, 197, 199, 204, 225, 226, 248, 259, 214, 224, 231, 235, 239, 263, 265, 279, 282, 287,
261, 265, 286, 301, 308, 310, 311, 312, 313, 314, 293, 294, 297, 310, 315, 321, 342, 344, 353, 354,
326, 330, 338, 363, 365, 366, 371, 375, 394, 406 360, 361, 371, 431, 440, 445, 448
moderates, 6 mouse model, xi, 46, 48, 56, 210, 440
modern society, 201 mouth, 28
modulation, xiii, xvi, 12, 15, 19, 104, 125, 126, 144, movement, 416
153, 201, 239, 295, 302, 309, 311, 313, 317, 343, MPA, 212
417, 443, 457 MPP, 220, 225, 236
moieties, 357 MPTP, 12, 20, 21, 220, 225, 227, 229, 236, 238
moisture, 84, 126, 164, 186 mRNA, 15, 22, 134, 135, 174, 213, 227, 229, 231,
moisture content, 126, 164, 187 249, 278, 291, 309, 328, 331, 403, 441
molar ratio, 396 MRSA, 31, 34
mold, 96 MS, 58, 66, 76, 151, 153, 155, 253, 254, 256, 265,
molecular biology, 338 317, 319, 321, 338, 396, 421, 422, 423, 426, 444,
molecular markers, 406 445, 446
molecular mechanisms, xvi, 301, 303, 326 MSI, 327
molecular medicine, 295 MTHFR, 299
molecular oxygen, 440 mucosa, 30, 320, 394, 406, 431
molecular structure, 359, 412 mucous membranes, xi, xix, 28, 45, 429
molecular weight, 231, 358, 395, 399 multicellular organisms, 140, 332, 379
molecules, x, xiii, 2, 14, 23, 26, 28, 29, 35, 47, 74, multiple myeloma, 276, 292, 296, 340
125, 131, 136, 139, 151, 164, 222, 248, 256, 265, multiple sclerosis, 241
267, 281, 285, 286, 294, 333, 334, 335, 365, 371, multivariate, xviii, 383, 386, 389
421, 453, 456 murine model, 167, 175, 238
monkeys, 225 murine models, 167
monoclonal, 319, 364 muscle, 27, 107, 117, 127, 130, 131, 135, 141, 143,
monoclonal antibodies, 319 147, 150, 151, 160, 172, 200, 205, 249, 283, 296,
monoclonal antibody, 364 298, 334, 344
monocyte, 40, 200, 204, 294 muscle cells, 127, 130, 135, 150, 160, 200, 205, 298
monocytes, 267, 293 muscle contraction, 283
monolayer, 375 mutagen, 165
486 Index
mutagenesis, 119, 175 nervous system, 239, 240, 440

mutagenic, xviii, 83, 164, 166, 167, 173, 393, 394, nervousness, 375
401, 454 Netherlands, 19, 100, 122, 314, 385
mutant, 12, 129, 225, 333 network, 123, 146, 149, 273
mutants, 309, 361 NeuN, xiv, 14, 207, 211
mutation, 133, 167, 227, 327, 328, 339, 356, 364, neural development, 356
443 neural network, 123
mutations, 39, 326, 327, 329, 337, 339, 433, 452 neurobiological, xv, 219
MYC, 381 neuroblastoma, 168, 227, 230, 234, 294, 343
Mycobacterium, 30, 36 neurodegeneration, 168, 206, 224, 227, 228, 233,
myeloid, 144, 170, 341 234, 236, 441, 443, 445, 446
myeloma, 276, 292, 296, 340 neurodegenerative, xv, xix, 11, 21, 26, 40, 64, 143,
myoblasts, 132, 148 168, 201, 206, 219, 221, 222, 224, 225, 226, 227,
myocardial infarction, 79, 245, 246, 250, 251, 252, 228, 230, 231, 232, 236, 240, 375, 417, 429, 430,
254, 256 433, 440, 442, 444, 447
myocardium, 403 neurodegenerative disease, xv, 11, 21, 26, 40, 64,
myosin, 272, 274, 275, 277, 278, 283, 284, 287, 296, 168, 201, 206, 219, 222, 224, 226, 227, 228, 230,
297, 298, 368, 378 231, 232, 240, 417, 430, 433, 440, 442, 444, 447
myricetin, 64, 106, 365 neurodegenerative disorders, xv, 143, 219, 221, 225,
232, 375, 430
N neurodegenerative processes, 236
neuroendocrine, 117
NA, 90, 149, 264, 359
neuroendocrine system, 117
Na+, 255, 395, 403, 404, 407, 415
neurogenesis, 360
NADH, 316
neurological disease, xix, 226, 429
Nash, 319
neuronal apoptosis, 217, 333
nasopharyngeal carcinoma, 381
neuronal cells, 12, 127, 227, 233, 238, 440, 442
National Academy of Sciences, 173, 318, 320, 322
neuronal death, 14, 20, 209, 210, 215, 216, 228, 229,
National Institutes of Health, 125
230
natural, x, xi, xiii, xv, xviii, xix, 19, 21, 23, 43, 46,
neuronal degeneration, 222, 433, 448
85, 99, 108, 110, 119, 145, 152, 157, 160, 164,
neuronal loss, 236
168, 178, 179, 185, 190, 194, 196, 201, 202, 208,
neuronal survival, 230
219, 221, 224, 261, 263, 312, 321, 326, 344, 348,
neurons, 12, 15, 20, 21, 40, 210, 213, 217, 225, 227,
349, 366, 376, 393, 396, 399, 401, 408, 411, 422
228, 236, 238, 239, 268, 294, 295, 430, 441, 442,
natural food, xiii, 119, 157, 160
444, 452
natural resources, 178
neuropathology, 446
nausea, 266
neuropeptides, 267, 293
neck, 167, 175, 203, 296, 317, 345
neuroprotection, xv, 14, 20, 120, 206, 207, 209, 210,
necrosis, 30, 99, 140, 154, 172, 220, 227, 238, 332,
214, 216, 217, 219, 222, 225, 226, 227, 228, 229,
380
230, 231, 233, 234, 239, 241, 442, 445, 446
nematodes, 217
neuroprotective, xiv, xv, 12, 14, 16, 19, 21, 201, 206,
neonatal, 16, 22, 452
207, 208, 209, 210, 212, 213, 214, 216, 217, 219,
neonates, 76
222, 223, 224, 226, 228, 229, 230, 231, 234, 238,
neoplasia, 258, 287, 326, 460
240, 367, 417, 430
neoplasm, 56, 286
neuroprotective drugs, 238
neoplasms, 167, 452
neurotoxic, 170, 229
neoplastic, 46, 83, 263, 267, 328, 332, 339, 370, 452
neurotoxicity, 14, 21, 171, 201, 225, 236, 268, 295,
neoplastic cells, 263, 267, 370
433, 444
neovascularization, 202, 204, 308
neurotoxins, 228
nephrectomy, 248
neurotransmission, ix, 1, 15, 22, 162
nephropathy, 402, 403
neurotransmitter, 15, 22, 210, 247
nerve, 15, 232, 239, 443
neurotransmitters, x, 2, 15
nerve cells, 239, 443
neurotrophic, 216
nerve growth factor, 15, 239
Index 487
neutrophil, 292 nuclear magnetic resonance, 265

neutrophils, 127, 267, 292 nuclear receptors, 127, 131
New York, 173, 314 nuclei, 164, 174, 334, 356, 457
New Zealand, 103, 104, 109, 110, 123, 301, 391 nucleotides, 149, 369
Newton, 239, 407 nucleus, 12, 123, 139, 227, 230, 329, 372, 442
Newtonian, 395 nucleus accumbens, 12, 123
NF-kB, xiii, 126, 132, 150, 308, 318 nursing, 33
NF-κB, 133, 160, 161, 168, 175 nursing home, 33
Ni, 197, 347, 399, 407 nutraceutical, 99, 443
Nielsen, 19, 123 nutrient, xv, 110, 117, 147, 219, 453, 454, 455
nigrostriatal, 12 nutrients, 198, 308, 331, 334, 366, 459
NIH, 148, 149 nutrition, 326, 453, 460
nitrate, xiii, 157, 160, 164, 165, 172, 173 nutritional supplements, 451
nitric oxide, 12, 19, 20, 21, 144, 161, 220, 223, 227, nuts, 123
233, 238, 247, 255, 308, 320, 443, 444
nitric oxide (NO), 12, 193
O
nitric oxide synthase (NOS), 12, 19, 20, 21, 144,
obese, 108, 117, 120, 127, 134, 137, 142, 144, 153,
161, 220, 227, 238, 247, 255, 294, 320
154, 248, 253, 255, 454
nitrite, 164, 173
obese patients, 108
nitrogen, 164, 231, 241, 411
obesity, v, xiii, xiv, xviii, 57, 104, 107, 108, 117,
nitrosamines, 164, 165, 172, 178, 193
120, 121, 122, 124, 125, 126, 131, 134, 136, 138,
nitroso compounds, 164, 165, 172, 173
141, 142, 143, 144, 145, 150, 152, 154, 155, 197,
nitrosoamines, 160
198, 200, 205, 244, 247, 248, 256, 296, 402, 409,
Nixon, 342
411, 415, 416, 424, 459
NMDA, 12, 15, 208, 209, 212, 213, 216, 217, 444
observations, xi, 7, 46, 130, 138, 140, 225, 272, 281,
NMDA receptors, 209, 213
285, 442
N-methyl-D-aspartate, 12
occlusion, xiv, 14, 207, 209, 210, 211, 212, 213, 214,
NMR, 421, 422
216
N-N, 172
occupational, 161
nodules, 455
OCs, 164, 165
non toxic, 233
odds ratio, xviii, 384, 385
non-enzymatic, 163, 248
odors, 185
non-insulin dependent diabetes, 407
oedema, 198
non-smokers, 385, 389
oil, xi, 46, 47, 56, 162, 172, 178, 179, 180, 193, 194,
nontoxic, 164, 371
221
noradrenaline, 132
oils, xii, 46, 47, 48, 76, 81, 83, 136, 179, 196
norepinephrine, 135, 215
oligodendrocytes, 442
normal, 49, 51, 55, 111, 127, 133, 135, 204, 227,
oligomeric, 122
236, 266, 267, 276, 307, 317, 318, 327, 329, 331,
oligomerization, 140
332, 339, 342, 356, 367, 370, 371, 373, 374, 394,
olive, 194, 221
396, 399, 400, 402, 404, 406, 408, 412, 416, 431,
olive oil, 194, 221
442, 456, 457, 458, 461
omega-3, 246
normalization, 49
oncogene, 275, 290, 291, 295, 309, 318, 329, 344,
North Africa, 158
380, 381
North America, 236, 407, 434
oncogenes, xvi, 325, 326, 330, 337, 364
N-ras, 327
oncogenesis, 460
Nrf2, 367, 377, 440
oncology, 60, 198, 298, 315, 317, 318, 338
N-terminal, 11, 40, 128, 228, 239, 263, 367
oncosis, 140
nuclear, 10, 54, 127, 131, 133, 140, 162, 168, 169,
online, 123
175, 227, 229, 237, 238, 264, 265, 275, 296, 318,
oocytes, 252, 414, 415
320, 332, 342, 358, 367, 380, 440, 446, 448
Opioid, 120
nuclear factor, 380
Ops, 163
nuclear factor-κB, 168
optical, 330
488 Index
optimization, 196
oral, 12, 13, 14, 15, 28, 30, 38, 40, 47, 49, 50, 51, 55,
P
56, 57, 59, 113, 114, 116, 118, 135, 152, 153,
P300, 14
162, 164, 167, 170, 172, 175, 199, 224, 228, 229,
p38, 11, 20, 128, 161, 228, 238, 239, 263, 336, 367,
235, 244, 258, 261, 264, 266, 272, 282, 283, 287,
377
288, 302, 311, 314, 315, 322, 335, 361, 366, 371,
p53, xvi, 130, 131, 133, 141, 145, 147, 149, 150,
375, 394, 413, 424, 431, 432, 442, 445, 460
154, 205, 263, 277, 306, 317, 318, 325, 327, 328,
oral cancers, 335
332, 333, 339, 342, 343, 370, 382
oral cavity, 164, 244, 261, 264, 366
pachytene, 360
oral hypoglycemic agents, 394
packaging, 187, 193
oral leukoplakia, 38
PAHs, 160, 166
oral squamous cell carcinoma, 49, 59, 175
pain, 222, 375
orchestration, 452
PAN, 40
organ, 37, 247, 249, 264, 321, 452, 453, 456, 457
pancreas, 2, 329, 337, 358, 366
organelles, 319, 431, 432, 433
pancreatic, 105, 108, 117, 135, 136, 137, 141, 147,
organic, xiii, 30, 72, 104, 119, 291, 395
148, 152, 153, 155, 336, 345, 358, 361, 394, 399,
organic solvent, 395
400, 412, 417, 418, 420, 424, 425
organic solvents, 395
pancreatic cancer, 147, 336, 345, 361, 417
organism, 31, 74, 457
Pancreatic cancer, 336
organization, 230, 285, 296, 298, 453
pancreatic islet, 399
organizations, 178
pandemic, 104
organophosphorous, 171
pantothenic acid, 405
orthopaedic, 386
paper, 100, 108, 162, 266
osteoarthritis, 104
paracrine, 341
osteosarcoma, 371
paradox, 200, 202, 206
outpatient, xviii, 383, 385, 386
parameter, 90, 92
ovarian cancer, 48, 55, 59, 341, 370, 385
paraoxonase, 121, 395, 402, 406, 407
ovariectomized, 152, 340
parasites, 456
ovariectomized rat, 152, 340
parenchymal, 163
overload, 213, 230, 340, 416
parenchymal cell, 163
overproduction, 164
parietal cortex, 433
overweight, 141, 144, 153, 200, 248, 253
Parkinson, 201, 219, 268
oxidability, 79
Parkinson disease, 201, 268
oxidants, xix, 42, 70, 225, 239, 258, 260, 447, 451,
Parkinson’s, xv, 107, 120, 171, 172, 219, 220, 222,
459
430, 443
oxidation products, 41, 260, 403
Parkinson’s disease, 107, 120, 171, 172, 220, 222,
oxidation rate, 69
430, 443
oxidative damage, xiii, xix, 69, 157, 160, 162, 170,
Parkinsonism, 20, 224, 225
237, 246, 343, 377, 409, 429, 430, 433, 435, 439,
PARP, 229, 311, 312, 358
440, 445, 446, 448
particles, 29, 56, 74, 84, 122, 384, 456
oxidative stress, xv, xix, 12, 26, 72, 130, 131, 158,
pasteurization, xii, 82, 84, 93, 94, 95, 98
160, 161, 163, 164, 167, 168, 169, 175, 203, 219,
patents, 56
221, 232, 233, 238, 239, 240, 241, 247, 255, 258,
pathogenesis, 78, 120, 164, 171, 227, 228, 232, 286,
263, 290, 333, 361, 377, 395, 399, 400, 403, 404,
379, 395, 404, 430, 433, 454
408, 409, 412, 417, 430, 433, 438, 439, 440, 441,
pathogenic, 30, 31, 32, 33, 40, 164, 178, 267
442, 443, 445, 446, 447, 456, 458, 459, 461
pathogens, 27, 30, 38, 185
oxide, 19, 20, 21, 144, 160, 161, 166, 167, 175, 220,
pathology, 169, 222, 232, 236, 447
223, 227, 233, 238, 247, 255, 308, 320, 443, 444
pathways, xvii, 27, 59, 121, 126, 128, 130, 134, 135,
oxidizability, 71
139, 140, 141, 145, 146, 151, 154, 203, 223, 228,
oxygen, xi, 33, 43, 63, 66, 69, 77, 78, 95, 107, 160,
239, 256, 258, 260, 263, 268, 276, 282, 299, 306,
167, 175, 179, 190, 198, 221, 223, 226, 230, 231,
307, 308, 317, 318, 328, 329, 332, 336, 337, 342,
232, 238, 241, 242, 246, 248, 260, 308, 368, 403,
358, 363, 367, 372, 377, 379, 404, 440, 441, 442,
411, 412, 420, 433, 436, 438, 440, 446, 453, 459
447
Index 489
patients, ix, xi, xv, xvii, 33, 40, 46, 55, 71, 120, 122, pharmacology, 2, 57, 148, 208, 216
138, 141, 150, 198, 200, 224, 225, 227, 228, 230, phenol, 108, 109, 121, 126, 158, 166, 169, 178, 196,
238, 240, 241, 243, 244, 246, 248, 250, 251, 252, 250, 391, 421
254, 255, 256, 279, 303, 306, 309, 310, 311, 315, phenolic, xi, xii, 19, 63, 64, 74, 77, 79, 80, 100, 103,
316, 322, 329, 339, 364, 375, 383, 384, 385, 389, 106, 108, 109, 112, 118, 160, 166, 167, 175, 176,
390, 391, 393, 433, 437 185, 190, 192, 221, 232, 260, 261, 315, 330, 340,
PC12 cells, 206, 220, 229, 230, 233, 239 401, 431, 443
PCP, 458, 461 phenolic acid, 64, 80, 118, 232, 315, 340, 401
PCR, 373 phenolic acids, 64, 80, 118, 232, 315, 340, 401
PDGF, 127, 204 phenolic compounds, 74, 77, 79, 106, 118, 185, 190,
PDK, 313 431
pectin, 186, 187, 188, 189, 195 phenotype, 132, 145, 282, 309, 320, 370
pediatric, xiii, 125 phenotypes, xviii, 411, 452, 453, 457
peer, 455 phenotypic, 309
penicillin, 43, 49 phenylalanine, 27
penumbra, 216 pheochromocytoma, 168, 220, 229, 230, 240
peptide, 105, 220, 231, 234, 265, 268, 399, 430, 433, philosophical, 452
438, 443, 444, 446, 449 phorbol, 166, 228, 240, 256, 456
peptides, 47, 225, 282, 290, 447 phosphate, xiii, 68, 125, 137, 152, 167, 356, 360,
performance, 14, 16, 138, 224, 235, 435, 444, 447 413, 423
peripheral blood, 276 phosphatidylcholine, 33
peripheral blood mononuclear cell, 276 phosphatidylethanolamine, 33, 153, 269
peritoneal, 174 phosphatidylserine, 140, 269
permeability, xi, 35, 46, 47, 55, 56, 140, 204, 209, phosphinic acid, 6
213, 214, 319, 331, 405 phosphoenolpyruvate, 405
permeation, 224 phospholipase C, 14, 16, 21
permit, 211 phospholipids, 29, 66, 160
peroxidation, 70, 74, 160, 162, 163, 167, 169, 210, phosphoprotein, 294
226, 248 phosphor, 143
peroxide, xix, 179, 190, 367, 368, 375, 429, 436, phosphorus, 111
438, 443, 445 phosphorylation, 11, 20, 127, 129, 130, 133, 138,
Peroxisome, 131 139, 145, 148, 199, 202, 203, 227, 228, 229, 240,
peroxisomes, 249 263, 265, 273, 275, 276, 277, 278, 279, 283, 284,
peroxynitrite, 223, 234, 445 285, 286, 288, 290, 292, 293, 295, 296, 297, 298,
personal, 338 306, 307, 308, 319, 333, 335, 342, 367, 372, 377,
Perth, 383 378, 405
perturbation, 31, 334, 415, 445 Phosphorylation, 272, 274, 277
perturbations, 33 photochemical, 124
pesticide, 161, 162, 168 photooxidation, 179, 180
pesticides, xiii, 157, 160, 161, 162, 164, 168, 172 phycoerythrin, 67
pests, 162 physical activity, xiii, 123, 125, 247, 252, 389
81, 408, 444, 447 physical exercise, 409
PGR, 68, 72, 73 physicians, xviii, 383, 385, 390
pH, xii, xiv, 30, 33, 47, 50, 68, 71, 74, 82, 84, 91, 92, physicochemical, 33
93, 95, 98, 109, 165, 207, 209, 214, 312, 371, physiological, xiv, xv, 13, 14, 20, 21, 26, 47, 74,
372, 421, 423, 445 104, 105, 117, 118, 126, 131, 135, 137, 154, 177,
phagocytosis, 154, 174 185, 188, 198, 207, 208, 209, 214, 225, 257, 258,
pharmaceutical, 178, 185 267, 275, 276, 280, 283, 286, 287, 306, 318, 319,
pharmacodynamics, 173 331, 332, 333, 356, 378, 424, 452
pharmacokinetic, 12, 36, 86, 224, 236, 266, 417 physiology, 57, 104, 105, 444, 457
pharmacokinetics, 2, 405, 406 physiotherapy, 386
pharmacological, xv, 2, 83, 126, 135, 198, 200, 208, phytochemicals, x, 45, 99, 100, 150, 158, 285, 299,
219, 228, 229, 231, 258, 261, 396, 404 340, 405
490 Index
PI3K, 129, 135, 139, 153, 307, 318 polyphenolic compounds, xii, xiii, xv, xvii, 40, 103,
pigments, 118, 158, 179, 244, 405, 423, 427, 431 104, 118, 243, 260, 302, 347, 348, 401, 430
pilot study, 153 polyphosphates, 413, 417
PKC, 220, 228, 229, 230, 231, 233, 441, 446 polyps, 326, 327, 332, 338, 455
PKD, 139 polysaccharide, 396, 397, 399, 407, 408
placebo, 13, 14, 15, 154, 236, 258, 454 polysaccharides, xviii, 393, 394, 395, 399, 400, 405,
placental, 332, 342 407, 408
plants, ix, x, xii, 1, 2, 23, 55, 82, 100, 105, 178, 221, Polysaccharides, 395, 397, 407
361, 407 polyunsaturated fat, 70
plaque, 200, 204, 441 polyunsaturated fatty acids, 70
plaques, 200, 225, 440, 442 PON1, 395
plasma, 5, 6, 7, 12, 13, 15, 26, 69, 70, 71, 78, 79, pools, 227
105, 120, 127, 135, 136, 138, 139, 140, 142, 151, poor, 35, 47, 186, 260, 309, 310, 368, 433, 454
162, 205, 209, 214, 223, 235, 244, 246, 247, 248, population, 13, 24, 58, 104, 174, 200, 201, 206, 247,
249, 253, 254, 255, 260, 261, 266, 268, 272, 280, 252, 255, 340, 370, 392, 453, 454, 455, 460
282, 288, 292, 294, 295, 298, 305, 311, 319, 334, pores, 37
340, 367, 371, 400, 407, 409, 412, 413, 414, pork, 31, 38, 185, 186, 187, 188, 189, 194, 195
415, 416, 417, 435, 436, 438, 439, 447 portal vein, 431, 447
plasma levels, 138, 244, 248, 305 positive correlation, xii, 104, 118, 303, 435, 438
plasma membrane, 15, 127, 139, 140, 268, 272, 292, positive influences, 190
294, 295, 319, 334, 414, 415, 416 positive relation, 356
plasminogen, 265, 292, 294, 332, 342 positive relationship, 356
platelet, 83, 127, 148, 200, 268, 293, 331, 334, 341, postmenopausal, 305, 316
405 post-translational, 241, 332
platelet aggregation, 83 potassium, xv, xviii, 12, 106, 111, 118, 243, 244,
platelet derived growth factor, 200 251, 252, 253, 256, 403, 411, 413, 417
platelets, 202 potassium channels, xv, xviii, 243, 251, 253, 411,
play, x, xiv, 23, 95, 117, 129, 131, 134, 167, 197, 417
200, 201, 210, 212, 221, 228, 230, 246, 261, 263, potato, 123
267, 268, 275, 277, 328, 334, 337, 352, 369, 374, potatoes, 32
395, 403, 404, 440, 457 poultry, 151, 194
PLC, 16 powder, xiv, 47, 71, 170, 177, 185, 186, 187, 189,
PMA, 228 190, 194, 195, 279, 302, 397
point mutation, 133 powders, 109, 191
poisoning, 168, 170 power, xi, xii, 13, 14, 63, 64, 71, 74, 79, 103, 108,
polarity, 147 109, 112, 165, 178, 190, 435, 436
polarization, 265 PPARγ, 131
politicians, 104 precipitation, x, 45, 47, 56, 397
pollutants, 166, 455 preclinical, 226, 232
pollution, 57 pre-clinical, 142
polyacrylamide, 49 pre-clinical, 326
polycyclic aromatic hydrocarbon, 166, 173 precursor cells, 128
polyethylene, 84 pre-existing, xiv, 197, 198, 331
polymerase, xvii, 229, 230, 347, 352, 356, 358, 359, preference, 87
360 prejudice, 386
polymerization, 46, 47, 65, 126, 260, 265, 296, 299 premature ventricular contractions, 252
polymorphism, 299, 316 preservatives, 178
polymorphisms, 303 pressure, 104, 148, 153, 178, 208, 209, 214, 215,
polymorphonuclear, 40, 293 246, 247, 254, 255, 260, 287, 340, 368, 397, 447
polynucleotide, xvii, 348, 352 presynaptic, 225
polynucleotide kinase, xvii, 348, 352 prevention, xiii, xvi, xviii, 57, 58, 64, 107, 120, 125,
polyp, 326 126, 138, 144, 147, 148, 151, 152, 161, 169, 172,
polypeptide, 105, 117, 267 173, 178, 201, 202, 203, 234, 236, 252, 257, 258,
Index 491
263, 272, 279, 282, 283, 286, 288, 289, 298, 302, prostate cancer, 107, 121, 127, 133, 145, 147, 148,
303, 321, 325, 330, 336, 338, 339, 342, 345, 358, 204, 241, 258, 264, 286, 287, 292, 299, 316, 336,
359, 361, 365, 367, 369, 370, 371, 374, 375, 345, 385, 391, 460
384, 390, 391, 394, 402, 403, 411, 414, 438, 441, prostate carcinoma, 146, 147, 317
442, 456, 458 prostatitis, 34, 39
preventive, ix, xv, xvi, xvii, xix, 12, 21, 58, 75, 107, proteases, 331, 424
145, 161, 170, 200, 232, 235, 257, 286, 289, 290, proteasome, 262
299, 325, 326, 330, 340, 358, 359, 363, 382, 402, protection, xi, 26, 46, 56, 63, 64, 66, 67, 73, 75, 95,
403, 405, 437, 448, 455, 456, 458, 459, 460 105, 166, 179, 186, 209, 213, 228, 261, 263, 310,
prion diseases, 268, 445 329, 444
probability, 368, 414 protective factors, 205
probe, xi, 63, 66, 77 protective genes, 230
processing pathways, 441 protective mechanisms, 234, 444
procyanidins, 101, 196 protective role, 388, 389, 447
prodrugs, 322, 378 protein aggregation, 222
production, xix, 10, 21, 30, 37, 40, 42, 69, 118, 130, protein binding, 12
134, 138, 154, 158, 161, 163, 164, 167, 170, 171, protein family, 132, 230, 333, 334, 342
190, 193, 199, 203, 226, 227, 232, 238, 247, 303, protein function, 282, 415
308, 317, 328, 342, 365, 367, 378, 384, 400, 405, protein kinase C, 206, 220, 222, 234, 239, 240, 295,
409, 419, 420, 421, 422, 423, 424, 427, 429, 433, 297, 372, 445, 456
440, 441, 445 protein kinase C (PKC), 222
progenitors, 332 protein kinases, xiii, 125, 128, 161, 220, 222, 233,
progeny, 371 239, 263, 265, 297, 336, 443
prognosis, 150, 309, 319, 370 protein oxidation, 169, 430, 433, 449
prognostic marker, 320 protein synthesis, 227, 313
program, 143, 154 proteinase, 262
proinflammatory, 121, 400 proteins, xiii, xvi, 28, 56, 125, 128, 129, 130, 133,
prokaryotic, x, xvii, 23, 347, 352 140, 143, 146, 153, 160, 169, 172, 174, 195, 222,
proliferation, xvi, 128, 129, 130, 131, 135, 139, 140, 229, 230, 231, 240, 241, 244, 248, 249, 261, 262,
143, 145, 148, 151, 152, 190, 199, 229, 231, 263, 264, 265, 268, 269, 277, 284, 286, 289, 291, 292,
265, 272, 273, 284, 290, 301, 305, 307, 308, 309, 294, 295, 298, 301, 306, 308, 309, 310, 311, 312,
311, 316, 317, 318, 319, 335, 336, 339, 352, 353, 325, 329, 331, 332, 333, 334, 335, 339, 341, 342,
356, 360, 364, 366, 370, 399, 452, 453 367, 369, 373, 374, 399, 430, 431, 433, 443, 448,
proliferation potential, 453 456
promote, 56, 57, 132, 139, 160, 179, 226, 263, 308, proteinuria, 248
309, 326, 334, 435, 451 proteolysis, 332
promoter, 256, 258, 264, 288, 291, 306, 331, 341, proteolytic enzyme, 265
344, 353, 356, 361, 372, 373, 374, 377, 381, 440, proteomics, 234, 239, 295, 443
456, 457, 461 protocol, 115, 279, 311, 386, 421
promoter region, 264, 331, 372, 373, 374, 381 protocols, 35, 138
promyelocytic, 352, 353, 354, 357 prototype, 145, 166
pro-oxidant, 72, 78, 91, 100, 160, 229, 240, 260, proximal, 135
333, 367 Pseudomonas, 32
property, 47, 168, 200, 225, 330, 341 Pseudomonas aeruginosa, 32
prophylactic, xviii, 226, 393, 394, 395 psoriasis, x, xi, 45, 46, 56, 198
prophylaxis, 437 Psoriasis, 60
propionic acid, 15 public, xiii, 24, 29, 104, 122, 124, 125, 126, 144,
propofol, 210, 213, 216 201, 243, 397
prostaglandin, 10, 339 public awareness, xiii, 125, 126, 243
prostate, xvi, 2, 107, 121, 127, 133, 138, 145, 146, public health, 24, 29, 104, 122, 124, 144, 201
147, 148, 204, 241, 258, 264, 286, 287, 292, 299, purification, 41, 48, 194, 397, 407
316, 317, 321, 325, 332, 336, 337, 341, 345, 358, PVA, 406, 407
361, 366, 385, 391, 460 pyramidal, 213
492 Index
pyrene, 166, 174, 335 reactive oxygen species, x, xiii, xv, xix, 26, 45, 131,
pyretic, 83 138, 148, 150, 157, 160, 162, 168, 178, 185, 199,
pyrimidine, 149 219, 220, 221, 303, 404, 408, 412, 429, 430, 453
reactive oxygen species (ROS), x, xiii, xix, 26, 45,
Q 131, 157, 160, 162, 168, 199, 221, 412, 429, 430,
453
quality control, 87
reactivity, 68, 72, 73, 75, 78
quality of life, 250
reading, 14, 86
quercetin, 64, 86, 87, 89, 106, 174, 204, 237, 289,
reagent, 108, 109, 266
343, 365
reagents, 49
Quercetin, 89
recall, xviii, 383, 386, 389, 430, 446
questionnaire, xviii, 233, 250, 383, 386, 389, 391
receptor agonist, 210, 213, 225
quinine, 420
receptor-negative, xvi, 301, 317, 320, 323
quinone, 25, 77, 174, 260, 288, 420, 423
receptor-positive, xvi, 301, 316
quinones, 25, 166, 174, 175
receptors, ix, xv, 1, 3, 6, 7, 9, 12, 14, 15, 19, 20, 22,
R 117, 127, 128, 129, 131, 132, 139, 140, 207, 208,
209, 210, 212, 213, 214, 215, 216, 217, 264, 268,
RAC, 63, 67, 72, 73 290, 292, 293, 294, 296, 308, 318, 329, 334, 340,
radiation, 26, 57, 194, 195, 336, 364, 376, 401 341, 457
Radiation, 177 reciprocal interactions, 452
radiation therapy, 364 recognition, xiii, 126, 222, 373
radical, xi, xv, 63, 64, 66, 68, 69, 70, 72, 76, 77, 78, recovery, 162, 336
79, 80, 83, 84, 86, 87, 88, 89, 90, 95, 96, 98, 100, rectum, 326
109, 118, 123, 160, 163, 167, 169, 171, 174, 175, recurrence, 303, 304, 316
178, 179, 181, 185, 186, 189, 190, 191, 192, 196, red blood cell, 237, 416
210, 219, 222, 223, 225, 226, 227, 229, 231, 233, red blood cells, 237, 416
234, 239, 246, 260, 372, 400, 404, 408, 409, 412, red meat, 455
430, 435, 439, 443, 445, 447 red wine, 178, 196, 198, 200, 202, 204, 221, 376
radical formation, 210, 226, 231, 404, 435 redistribution, 278
Ramadan, 360 redox, 132, 148, 160, 161, 168, 175, 227, 240, 257,
random, 453 405, 439, 443, 456, 457, 458, 461
range, 31, 83, 87, 89, 90, 91, 92, 93, 94, 95, 97, 98, reductases, 41, 152
162, 225, 245, 263, 265, 266, 273, 283, 310, 330, reduction, xii, xiii, 15, 22, 26, 47, 71, 93, 103, 104,
358, 405, 415 113, 114, 115, 116, 117, 130, 132, 142, 161, 164,
rapamycin, 313 178, 185, 213, 215, 222, 224, 226, 228, 229, 231,
ras, xvi, 325, 327, 461 232, 246, 247, 249, 266, 268, 272, 275, 276, 279,
rat, xii, 12, 13, 15, 16, 19, 20, 37, 39, 103, 110, 111, 281, 283, 284, 285, 286, 303, 313, 336, 341, 354,
115, 123, 127, 135, 137, 138, 147, 148, 149, 151, 371, 395, 402, 403, 439, 440, 453, 455
161, 163, 168, 170, 171, 175, 209, 215, 216, 217, refractory, 244, 252, 452
220, 225, 226, 229, 230, 235, 237, 239, 246, 251, regenerate, 394
253, 256, 268, 277, 287, 295, 297, 299, 302, 321, regeneration, 160, 265, 406
342, 344, 351, 358, 403, 406, 407, 412, 413, 427, regional, 21, 235
430, 442, 445, 446, 447, 448, 461 regression, xviii, 4, 5, 111, 321, 383, 386, 388, 389,
ratings, 105 390, 434, 435, 448
reactants, 167 regression analysis, xviii, 111, 383, 386, 388, 389,
reaction mechanism, 41, 427 390, 434
reaction time, 72 regression equation, 4, 5
reactive nitrogen, 241 regular, xviii, 85, 96, 119, 200, 244, 246, 252, 382,
reactive oxygen, x, xiii, xv, xix, 26, 45, 131, 138, 383, 388, 389, 414, 416, 456
148, 150, 157, 160, 162, 168, 175, 178, 185, 199, regulation, x, xi, 45, 46, 105, 121, 122, 127, 128,
219, 220, 221, 303, 404, 408, 412, 429, 430, 453 129, 131, 132, 133, 138, 146, 147, 148, 150, 152,
205, 213, 217, 222, 226, 227, 228, 229, 230, 231,
238, 239, 241, 263, 264, 267, 278, 286, 295, 298,
Index 493
305, 318, 319, 329, 330, 331, 335, 336, 339, 341, reticulum, 132, 154, 162, 310, 457
342, 343, 344, 345, 369, 372, 374, 379, 381, retinoblastoma, 141
418, 440, 441, 454, 457, 461 retinoic acid, 264, 270
regulators, 128, 147, 222, 228, 264, 268, 277, 307, retinoic acid receptor, 264
372 retinoids, 457
rehabilitation, 138 retinol, 441, 459
relationship, ix, xvi, xvii, 22, 27, 36, 74, 96, 105, Retinol, 454
107, 122, 127, 135, 138, 145, 165, 172, 194, 199, retinopathy, 400
200, 235, 246, 253, 254, 268, 280, 281, 288, 296, retirement, xviii, 384, 386, 387, 388, 389
301, 303, 348, 354, 356, 359, 361, 388, 389, 394, retrovirus, 28
399, 400, 406, 434 reveratrol, 457
relationships, 19, 28, 99, 175, 176, 232, 291, 340 reverse transcriptase, 28, 39, 369, 378, 380, 381, 427
relaxation, ix, 1, 2, 14, 16, 18, 20 Reynolds, 149, 232
relaxation effect, 20 rheumatoid arthritis, 198
relevance, 124, 146, 171, 172, 231, 447, 458, 459 Rho, 283, 298
remission, 266 Rho-kinase, 283, 298
remodeling, 200, 261, 273, 277, 278, 284, 331 rhythms, 454
renal, 74, 248, 250, 255, 403 ribonucleic acid, 39
renal disease, 403 ribose, 229, 230, 358, 396
renal failure, 248, 403 ribosome, 267
repair, x, 45, 57, 68, 133, 150, 261, 264, 327, 333, rice, 352, 359, 385
334, 336, 338, 345, 348, 349, 356, 360, 361, 452, right ventricle, 123
456 rings, 25
repair system, 133, 327 risk, ix, xv, xvi, xvii, 31, 42, 69, 71, 75, 79, 83, 104,
reperfusion, xv, 14, 131, 207, 210, 221, 252, 256, 108, 119, 121, 123, 136, 143, 144, 164, 165, 170,
409, 416 198, 199, 200, 201, 202, 204, 215, 219, 222, 224,
replication, xvii, 28, 39, 57, 291, 327, 334, 348, 352, 235, 244, 246, 247, 248, 252, 253, 254, 256, 275,
356, 363, 364, 369 285, 296, 299, 303, 304, 315, 316, 325, 326, 329,
repolarization, xv, 243, 251, 252 338, 340, 361, 362, 383, 385, 387, 388, 390, 391,
repression, 133, 264, 282, 334, 373, 374, 405 395, 400, 401, 402, 406, 407, 431, 445, 454, 455,
repressor, 291, 320, 344, 381 456, 458, 460
reproduction, 198 risk factors, 144, 201, 244, 246, 247, 254, 340, 390
research, ix, xiv, xvii, xviii, 18, 65, 104, 107, 177, risks, 41, 120, 126, 143, 200, 201, 235, 338, 443
178, 198, 200, 201, 258, 267, 286, 305, 313, 314, RNA, xvii, 167, 227, 275, 290, 292, 310, 321, 347,
325, 363, 366, 369, 370, 372, 384, 385, 389, 394, 352, 369, 373, 379, 380
402, 404, 405, 406, 424, 442 RNAi, 272, 275, 276, 279, 281, 282, 283, 284, 285
researchers, xv, 47, 104, 257, 333, 337, 403 rodent, 123, 321
reservation, 453 rodents, 123, 135, 138, 151, 164, 302
reservoir, x, 23 rods, 42
residential, xviii, 384, 386, 387, 388 rolling, 126, 169
residues, 169, 230, 360, 413 room temperature, 84, 85, 86, 109, 111
resistance, xix, 27, 30, 31, 34, 41, 42, 70, 134, 135, room-temperature, 110
138, 139, 144, 150, 151, 167, 221, 263, 282, 286, ROS, x, xiii, xix, 26, 45, 131, 157, 160, 162, 164,
298, 305, 313, 322, 394, 400, 405, 406, 407, 411, 168, 199, 220, 221, 226, 227, 412, 416, 429, 430,
416, 442, 448, 454 433, 436, 437, 438, 439, 441, 442, 453
resistin, 134, 135, 150, 151 rosacea, 56
resources, 178, 453 rosiglitazone, 150
respiratory, xviii, 28, 383, 385, 386, 390 rotavirus, 29, 36, 39
responsiveness, xv, 257, 285, 384 rubber, 110
restrictive cardiomyopathy, 249 rural, 304, 386, 387, 389, 391
Resveratrol, 343
retail, 108
S
retention, 56, 93
S phase, 333, 335
494 Index
Saccharomyces cerevisiae, 33 sensing, 131, 242

safety, 13, 185, 224 sensitivity, 138, 244, 248, 255, 256, 266, 272, 282,
SAHA, 457 286, 298, 329, 394, 400, 404, 405, 413, 416, 445,
saline, 6, 7, 8, 16, 167 448
saliva, 172, 264 sensitization, 12, 21
Salmonella, 32, 119, 175 sensors, 222
salt, 178, 195, 215 separation, 4, 5, 6, 8, 9, 10, 11, 16, 17, 18
salts, 151 septum, 250
sample, 54, 64, 66, 67, 70, 71, 72, 86, 109, 110, 118, sequencing, 373
179, 185, 187, 190, 386, 389, 422 series, 27, 32, 35, 47, 99, 180, 228, 327, 332, 334,
sampling, 458 366, 367, 452
sampling error, 458 serine, 133, 139, 229, 329, 332, 335
sanitation, 178, 453 serotonin, 15, 22, 208, 215, 297
SAR, 28, 29, 35 serum, ix, xiii, 1, 2, 22, 26, 47, 49, 56, 71, 104, 111,
SAS, 87 115, 118, 119, 121, 137, 143, 150, 161, 162, 171,
saturated fat, 56 206, 215, 228, 229, 233, 234, 239, 245, 248, 254,
saturation, 13 255, 341, 394, 399, 400, 402, 403, 406, 424, 448
scaffolding, 140 severity, 150, 202
Scanning electron, 31 sex, 336, 338
scar tissue, x, 45 sex steroid, 336
scavenger, 226, 441 sexually transmitted disease, 55
school, 386, 387, 390 Shanghai, 58, 296, 460
scientific community, x, 23, 375 shape, 334
scientific knowledge, 451 shaping, 140
scientific understanding, xix, 459 shock, 131, 220, 231, 241
scientists, 55, 57, 330 shoot, 100
Scomber scombrus, 176 short period, xii, 103, 435
scores, 186 short-term, 91, 106, 315
screening programs, x, 23 Shp-2, 343
SDS, 49 shrimp, 194, 195
search, 282, 388 side effects, xvi, 55, 250, 303, 311, 325, 337, 394,
searches, 349 415, 455
secrete, 200 sign, 52
secretion, x, 2, 138, 152, 153, 240, 279, 289, 297, signal transduction, xvii, 127, 128, 139, 203, 228,
332, 404, 413, 416 230, 239, 258, 263, 268, 299, 317, 336, 339, 363,
sedation, 16 377, 447, 456
sedative, ix, 1, 6, 9, 10, 11, 17, 18, 22 signalling, 343, 345
sedentary, 104 signals, 12, 105, 128, 130, 131, 140, 145, 147, 226,
sedentary lifestyle, 104 229, 290, 299, 317, 321, 369, 452
seeds, 194, 381 silicon, 110
selective attention, 14, 20 silver, xx, 459
selectivity, 28 similarity, 310, 424
selenium, ix, xii, xiii, 103, 104, 108, 111, 112, 118, Singapore, 235, 285, 299, 316
119, 120, 121, 122, 123, 124 siRNA, 265
self, 296 sister chromatid exchange, 167
self-report, 253, 389, 406 sites, 14, 132, 150, 261, 267, 283, 284, 299, 331,
SEM, 31, 87, 88, 89, 90, 92, 93, 94, 95, 97, 98, 113, 369, 413
114, 116, 180, 351, 353, 354, 435, 436, 437, 438 skeletal muscle, 107, 117, 131, 143, 147, 249
seminal vesicle, 138 skeleton, 221, 258, 297, 423
senescence, 131, 369, 370, 371, 378, 380, 431, 443, skin, x, xi, xiv, xvi, 2, 38, 40, 45, 46, 47, 48, 49, 52,
445, 448, 452, 453 54, 55, 56, 57, 58, 75, 100, 107, 120, 124, 164,
senile, 268 166, 173, 177, 179, 185, 193, 240, 312, 325, 329,
senile plaques, 268
Index 495
336, 337, 342, 345, 358, 361, 366, 376, 382, 455, speculation, 355
456, 457 speed, 59
skin cancer, 38, 40, 107, 185, 312, 336, 345, 376 spermatocyte, 360
skin disorders, 56 S-phase, 149
sleep, 3, 5, 7, 9, 11, 16, 18, 375 sphingolipids, 269
sleep disorders, 375 spin, 66, 77, 163
small intestine, xix, 26, 366, 429 spinach, 67, 360
smoke, 387 spindle, 457
smokers, xvii, 71, 79, 247, 255, 383, 384, 385, 389, spine, 108
391, 454, 458 spleen, 400
smoking, xvii, 108, 120, 123, 164, 235, 246, 338, sporadic, 54, 291, 326, 327, 329
383, 384, 386, 387, 388, 389, 392, 443, 448, 454 spore, 31, 40, 123
smooth muscle, 130, 135, 150, 151, 160, 200, 205, SPR, 268, 270, 279, 280, 281
296, 298, 334, 344 Sprague-Dawley rats, 110, 117, 256, 302, 321, 448
smooth muscle cells, 130, 150, 160, 200, 205 SPSS, 387
social environment, 453 sputum, 384
social policy, 451 squamous cell, 49, 59, 167, 175, 282, 296, 317
social stress, 16 squamous cell carcinoma, 49, 59, 167, 175, 282, 296,
socioeconomic, 247 317
SOD, 162, 167, 226, 247, 260, 400, 403, 404, 440 St. Louis, 48, 50
SOD1, 226 stability, xvi, 47, 55, 56, 84, 85, 93, 94, 96, 179, 194,
sodium, 108, 109, 118, 121, 395, 403, 404, 409, 417 230, 265, 279, 302, 309, 311, 312, 369, 403
software, 86 stabilization, 133, 230, 275, 309, 339, 439
soil, 105 stabilize, xi, 46, 91, 230, 250, 260
solid tumors, 40, 133, 315 stages, 84, 89, 140, 326, 327, 330, 336, 370
solubility, xi, 38, 46, 47, 136, 137, 205 stainless steel, 110
solvent, 65, 76, 136 standard error, 87, 111, 112, 180, 181, 186, 191, 193
solvents, 47, 65, 195 standards, 35, 66, 86, 87, 108, 396, 397
somatic cell, 370, 452 staphylococci, 41
somatic cells, 370, 452 Staphylococcus, 31, 32, 34, 38, 41, 42, 43, 99, 182
somatic mutations, 329 Staphylococcus aureus, 31, 32, 38, 41, 42, 43, 99,
somatic stem cells, 452 182
somatostatin, 105, 117 starch, 30, 42, 395
soy, 48, 165, 173, 195, 455 starch polysaccharides, 395
soybean, 48, 179, 195, 198, 246 starvation, 149, 341
Spain, 81, 99, 100 Statistical Analysis System, 87
spatial, xiv, xix, 14, 207, 209, 212, 213, 214, 217, statistics, 248, 338, 386
226, 429, 430, 431, 433, 435, 442, 444 steady state, xi, 63
spatial information, 430 steel, 110
spatial learning, 430, 435 stellate cells, 148
spatial memory, xiv, 14, 207, 209, 212, 213, 214, stem cell therapy, 249
430 stem cells, 200, 451, 452, 453, 459, 460, 461
specialized cells, 452 sterile, 86, 110
species, x, xiii, xv, xix, 16, 26, 30, 31, 32, 33, 45, 70, sterilization, 47
80, 83, 84, 131, 138, 148, 150, 157, 160, 162, steroid, 138, 305, 316, 336, 359
164, 167, 168, 175, 178, 185, 199, 219, 220, 221, steroid hormone, 305
231, 232, 241, 260, 303, 404, 408, 412, 429, 430, stiffness, 403
436, 438, 452, 453, 456 stilbenes, 27
specificity, 151, 295, 370 stimulus, 140, 228
spectrophotometric, 86, 100 stock, 86
spectrophotometric method, 86 stoichiometry, 73
spectroscopy, 66, 70 stomach, xii, 2, 41, 103, 105, 117, 120, 135, 164,
spectrum, 32, 158, 229 165, 222, 244, 264, 366, 376
496 Index
storage, 77, 83, 85, 91, 96, 97, 101, 123, 179, 181, survival, xvi, 12, 30, 36, 58, 128, 146, 154, 201, 206,
186, 187, 188, 189, 193, 194, 195, 236, 454 222, 226, 228, 229, 230, 231, 233, 234, 240, 264,
strain, 32, 35, 38, 174, 315 306, 307, 310, 312, 316, 325, 331, 333, 360, 392,
strains, 31, 32, 33, 34, 35, 42 443, 446, 453
S-transferase (GST), 162, 163 survival rate, 226
strategies, ix, xi, xvi, 27, 35, 46, 104, 131, 257, 286, survival signals, 12, 229
306, 370, 375, 402, 456 surviving, xv, 14, 207, 213, 416
strength, 244, 270, 281, 389 susceptibility, 33, 35, 40, 70, 82, 132, 174, 204, 322,
Streptomyces, 47 412, 443
striatum, 12, 15, 22, 208, 226, 229, 440 switching, 16
stroke, 151, 221, 246, 385, 391, 444, 447 symbols, 87
stromal, 149 sympathetic, 15, 26, 37
structural changes, 162, 371 symptom, 12, 226
structural protein, 230 symptoms, 14, 32, 277, 278, 384, 385, 400, 407
students, 87 synaptic transmission, 210
subacute, 162 syndrome, 104, 138, 327
subcellular, 294 synergistic, 26, 34, 118, 119, 132, 139, 317
subsidy, 201, 358 synergistic effect, 118, 119, 132
substances, ix, 1, 2, 14, 40, 185, 196, 198, 269, 331, synthesis, 15, 48, 130, 135, 136, 137, 139, 167, 174,
361, 399, 405 210, 227, 309, 313, 340, 348, 360, 420, 426, 440,
substantia nigra, 12, 20, 220, 225, 236, 237, 238, 431 441
substantia nigra pars compacta, 220 systems, 136, 141, 172, 200, 208, 226, 227, 259,
substitution, 302 260, 263, 265, 266, 327, 329, 365, 367, 374, 375,
substrates, 136, 172, 225, 263, 277, 349, 368 406, 452, 453, 454, 458, 459
subtraction, 270 systolic blood pressure, 247
sucrose, 246, 254, 268, 427
sugar, 85, 248, 384, 400, 402, 404, 455
T
suicide, 154, 345
T cells, 267, 293, 376, 377, 442
sulfate, 70, 108, 260, 282
T lymphocytes, 267
sulfuric acid, 396
T2DM, 400
summer, 385
tachycardia, 250, 251, 375
Sun, 59, 150, 170, 236, 253, 288, 289, 299, 316, 381,
Taiwan, 158
407
tamoxifen, 164, 172, 289, 312, 313, 316, 317, 322,
superoxide, 12, 162, 164, 167, 175, 178, 187, 189,
323
191, 192, 223, 226, 247, 248, 260, 361, 367, 399,
tangles, 448
400, 404, 407, 409, 433, 440, 444, 445
tannin, 42
superoxide dismutase, 12, 162, 167, 187, 226, 247,
tannins, 25, 33, 94, 100, 101, 196, 361, 405
248, 260, 368, 399, 400, 433, 440, 444, 445
TAR, xi, 63
supplements, 37, 78, 143, 234, 338, 460
target organs, 358
supply, 198, 331
targets, xv, xvii, 16, 20, 40, 75, 128, 131, 147, 148,
suppression, xvi, 32, 39, 58, 107, 108, 136, 149, 198,
169, 199, 200, 222, 232, 257, 258, 261, 263, 290,
199, 201, 203, 231, 240, 264, 268, 276, 279, 286,
294, 306, 307, 310, 321, 325, 338, 340, 343, 352,
301, 306, 308, 310, 311, 312, 313, 332, 339, 341,
367, 370, 371, 382, 441, 442, 460
345, 348, 357, 366, 368, 376, 377, 424, 445, 446,
taste, xii, 82, 158, 186, 365, 424
457
tau, 227, 236, 447
suppressor, xvi, 133, 282, 291, 298, 325, 327, 328,
tau pathology, 236, 447
329, 330, 333, 334, 343, 364
T-cell, 290
suppressors, 326, 337
T-cell receptor, 290
surfactant, 136
technology, xiv, 177, 178, 179, 194
surgery, 319, 364, 456
telomerase, ix, xvii, 363, 365, 366, 367, 369, 370,
surveillance, 338
371, 372, 374, 375, 376, 378, 379, 380, 381, 446
survivability, 441
telomere, 369, 370, 371, 378, 379, 380
telomere shortening, 370, 371, 380
Index 497
telomeres, xvii, 363, 369, 370, 371, 378, 380, 381 tocopherols, 185, 407
temperature, xiv, 14, 84, 93, 94, 96, 105, 109, 110, Tokyo, 153, 205, 286, 358, 409, 429
179, 181, 207, 209, 214, 239, 385 tolerance, 138, 147, 153, 248, 329, 340, 400, 402,
tendon, 395, 406 412
Tennessee, 325 toll-like, 269, 296
tension, 296 tomato, 32
teratogenic, 164 tonic, 36
testes, 409 total plasma, 136, 246
testis, 356 totipotent, 452
testosterone, 117 toxic, xiii, xx, 57, 157, 160, 162, 164, 178, 231, 268,
tetracycline, 31, 34, 41 302, 365, 440, 447, 454, 459
tetrahydrofuran, 50 toxic effect, 162, 365
Texas, 254 toxicities, 457
TGF, 154, 306, 309, 328, 329, 331, 332, 403, 408 toxicity, xiii, 21, 48, 123, 157, 160, 162, 163, 164,
Thailand, 252 168, 169, 170, 171, 172, 174, 206, 237, 239, 315,
thalidomide embryopathy, 461 412, 442, 443, 446, 451
theaflavin, xix, 25, 126, 146, 227, 238, 290, 365, toxicological, 228
367, 419, 420, 423, 426, 427 toxicology, 2, 48, 173, 459, 461
T-helper cell, 29 toxin, 11, 31, 32, 42, 164, 168, 458
theory, 169, 247, 310, 326, 369, 433, 448, 460 toxins, 19, 57, 158, 172, 248
therapeutic agents, 139, 225 TPA, vi, xvii, 166, 347, 348, 353, 354, 355, 356
therapeutics, 3, 236, 265, 379, 380, 444 trade, 178
therapy, xv, xvi, 108, 146, 147, 198, 219, 231, 249, tradition, xv, 158, 219
256, 263, 264, 286, 290, 291, 313, 318, 319, 325, traffic, 268
329, 340, 342, 364, 370, 394, 405, 448 training, 249
thermal treatment, 93, 94 transaminases, 162
theta, 13, 14 transcript, 231, 238, 331, 343, 373
thiobarbituric acid, 167, 187, 399, 436, 438, 446 transcriptase, 28, 39, 369, 378, 380, 381, 427
Thomson, 123, 408 transcription, xiii, 28, 107, 117, 126, 127, 128, 129,
three-dimensional, 375, 452 131, 132, 139, 148, 150, 154, 160, 161, 163, 169,
threonine, 139, 263, 329, 335 199, 203, 222, 227, 230, 238, 241, 263, 264, 275,
threshold, 370, 456, 457, 458 291, 309, 320, 329, 331, 333, 343, 344, 369, 372,
thrombosis, 401 373, 374, 381, 409, 440
thrombotic, 216 transcription factor, xiii, 107, 117, 126, 127, 132,
thymus, 356, 360 139, 154, 160, 161, 169, 199, 203, 222, 227, 238,
thyroid, 124 263, 275, 309, 320, 331, 333, 343, 344, 373, 381,
thyroxin, 441 409, 440
time frame, 105 transcription factors, xiii, 107, 117, 126, 132, 160,
time periods, 85 161, 169, 199, 222, 227, 263, 333, 373, 381, 440
timing, 405, 452, 458 transcriptional, 133, 141, 147, 227, 230, 263, 264,
TIMP, 331 309, 320, 331, 334, 344, 372, 373, 374, 381, 440,
tissue, x, 20, 21, 29, 41, 45, 49, 107, 117, 120, 131, 456
134, 137, 140, 142, 143, 147, 161, 163, 164, 200, transcriptomics, 234
205, 222, 224, 226, 235, 246, 249, 256, 261, 266, transcripts, 356
267, 272, 288, 293, 314, 326, 331, 332, 342, 361, transduction, 134, 154, 294, 297, 339
399, 412, 416, 431, 432, 448, 452, 453, 456, 457 transfection, 129, 319
tissue homeostasis, 140 transfer, 266, 334, 404, 452, 453, 456
TLR, 39 transferrin, 220, 222, 231
TNF, 30, 148, 150, 220, 227, 229, 238 transformation, 146, 286, 288, 290, 295, 328, 370,
TNF-alpha, 148, 150, 238 452, 461
tobacco, 29, 42, 152, 166, 234, 385, 388 transformations, 160
tobacco smoke, 166 transforming growth factor, 329, 332, 342
Tocopherol, 58, 454 transforming growth factor-β, 332
498 Index
transgenic, 151, 225, 227, 236, 237, 417, 437, 447 type 1 diabetes, 399
transgenic mice, 151, 225, 227, 236, 237, 417, 437, type 2 diabetes, xiii, xviii, 26, 104, 123, 125, 126,
447 131, 138, 142, 150, 153, 200, 244, 253, 255, 394,
transgenic mouse, 237 400, 402, 406, 408, 411
transition, 28, 130, 160, 226, 309, 335, 344, 452, 457 type 2 diabetes mellitus, xiii, 104, 123, 125, 142,
transition metal, 160, 226 153, 200, 244, 394, 402
transition metal ions, 160, 226 tyramine, 165
translation, 128, 227, 232, 282, 298, 368, 457 tyrosine, 12, 127, 129, 138, 139, 148, 199, 223, 234,
translational, 456 277, 279, 294, 307, 319, 343, 405
translocation, 140, 143, 154, 168, 227, 229, 238, tyrosine hydroxylase, 12
239, 306, 330, 334, 372, 380, 442
transmembrane, 128, 141, 334, 456
U
transmission, 139, 210
U.S. Department of Agriculture (USDA), 75, 78, 170
transplantation, 249
ubiquitin, 220, 229, 242, 262, 329, 331, 339, 367
transport, 13, 20, 135, 136, 137, 394, 400, 406, 454
ubiquitous, 166
transportation, 404
ultrastructure, 42
trastuzumab, 305, 314
ultraviolet, 38, 57, 75, 124, 185, 318, 345, 361, 376
trauma, 56
ultraviolet B, 318, 361
treatment-resistant, 314
ultraviolet light, 185
trend, 27, 179
uncertainty, 224
trial, 40, 59, 144, 153, 154, 236, 244, 279, 315, 435,
underlying mechanisms, 75, 161, 406, 454
454, 459
United Kingdom, 121
triggers, 131, 139, 213, 413
United States, xvi, 57, 74, 80, 200, 245, 318, 320,
triglyceride, 117, 121, 126, 136, 137, 143, 151, 402,
322, 325, 326, 336
424
universality, 370
triglycerides, 26, 38, 136, 137, 138, 141, 246, 248,
universities, 386
403, 405, 426
urea, 163
trimethylamine, 165
urease, 30
trochanter, 108
urinary, 28, 40, 47, 122, 165, 247, 253, 260, 261,
Trp, 190, 191
288, 399
trypsin, 265, 331
urinary bladder, 40, 122
tryptophan, 30
urinary bladder cancer, 122
tuberculosis, 30, 36
urinary tract, 28
tubular, 203
urine, 22, 58, 215, 224, 235, 253, 260, 288, 431
tumor cells, 50, 229, 263, 265, 291, 311, 312, 314,
urokinase, 266, 292, 294, 332, 342
334, 342, 343, 358, 366, 368
uterus, 138
tumor growth, xiv, 197, 198, 199, 200, 272, 282,
UV, x, 45, 56, 57, 70, 76, 150, 336, 396, 418
283, 306, 308, 310, 311, 312, 319, 334, 368, 417
UV exposure, 336
tumor invasion, 261, 358
UV light, 336
tumor metastasis, 332
tumor necrosis factor, 30, 99, 238, 332, 380 V
tumor progression, 263, 329, 378
tumorigenesis, 42, 199, 296, 312, 321, 327, 328, 329, vacuum, 187, 397
332, 335, 337, 338, 339, 344, 345, 361, 370, 376, validation, 77, 272
379, 461 validity, 74
tumorigenic, xvii, 166, 318, 325, 332, 370 values, xii, xvii, 11, 64, 65, 67, 68, 69, 70, 72, 73,
tumors, 40, 133, 164, 166, 167, 261, 265, 276, 306, 74, 82, 86, 87, 89, 98, 112, 116, 179, 186, 190,
311, 312, 313, 315, 321, 322, 327, 330, 331, 334, 193, 265, 283, 347, 351, 352, 356, 366, 438, 439
336, 339, 342, 365, 371, 375, 376, 456, 458 vancomycin, 31
tumour, 203, 238, 292, 319, 343, 344 variability, 15, 39, 58, 70, 87, 253, 288
tumour growth, 203, 319 variable, 31, 310, 377, 409
turnover, 261 variables, xiv, 14, 207, 209, 214, 386, 387, 388, 389
two-dimensional, 100, 265 variance, 111
Index 499
variation, 254 water, ix, x, xii, xiii, xv, xviii, 24, 37, 38, 45, 47, 48,
vascular dementia, 201 55, 56, 64, 65, 66, 71, 73, 77, 82, 84, 85, 86, 89,
vascular disease, 333, 400 103, 104, 108, 109, 110, 111, 113, 115, 116, 117,
vascular endothelial growth factor (VEGF), 10, 122, 118, 119, 135, 136, 138, 157, 160, 162, 165, 167,
127, 198, 200, 202, 203, 204, 220, 230, 234, 308, 219, 222, 223, 247, 248, 249, 258, 279, 302,
311, 313, 316, 317, 319, 331, 336, 340, 341, 376 310, 311, 313, 332, 365, 366, 384, 393, 397,
vasculature, 200, 205, 331 408, 411, 435, 436, 438, 458
vasculogenesis, 331 water-soluble, x, xiii, 38, 45, 47, 56, 104, 109, 116,
vector, 270, 271, 275, 279, 280 136
vegetable oil, 59, 179 weight control, 118, 123, 141, 200
vegetables, 67, 69, 77, 78, 100, 105, 175, 198, 221, weight gain, 115, 119, 153, 381
246, 302, 314, 348, 455 weight loss, 104, 118, 138, 142, 153, 155, 253, 256,
VEGF expression, 200, 204, 234 302
vein, 341 weight management, xiii, 104, 118, 119
velocity, 70 Weinberg, 317, 380, 381, 460
ventricle, xv, xix, 226, 243, 429, 437, 438 well-being, xix, 411
ventricular arrhythmia, 251, 256 wells, 49
ventricular fibrillation, 251 Western countries, 24, 126, 200, 222, 248
ventricular tachycardia, 250, 251 wheat, 194
vessels, 331 wild type, 129, 333
Vibrio cholerae, 32, 42 wine, 19, 96, 100, 198, 302, 389, 392
vimentin, 241, 265, 289, 292, 309 Wistar rats, 302, 315
viral envelope, 29 withdrawal, 228, 239
viral infection, 28, 29, 120, 268 Wnt signaling, 309, 320, 329
virus, 28, 29, 36, 39, 40, 41, 42, 55, 57, 58, 59, 60, women, xvii, 71, 108, 121, 153, 154, 202, 224, 245,
258, 267, 293, 294, 321, 329, 348, 380, 427 253, 285, 299, 303, 304, 305, 308, 314, 326, 329,
viruses, 27, 29, 39, 55, 57, 268, 456 383, 385, 386, 388, 394, 434, 460
visible, 34, 64, 71 working memory, 430, 435, 437, 439
vision, xv, 57, 219 World Health Organization (WHO), 162, 170, 178,
visualization, 100, 380 196, 389, 391, 392
vitamin A, 417 wound healing, 198, 331
vitamin C, 26, 71, 85, 86, 90, 95, 179, 229, 337
vitamin C, 100, 106, 240, 330, 404, 405
X
vitamin E, 64, 158, 210, 223, 407, 417, 449
xenobiotics, xiii, 157, 160, 168
vitamin K, 106
xenografts, xvi, 292, 301, 310, 311, 313, 314,
vitamins, 26, 47, 79, 106, 118, 394, 395, 439, 455
321,371, 375
vocalizations, 3, 4, 6, 7, 8, 9, 10, 16, 17
vomiting, 31, 375 Y
W
yeast, 33, 34, 37, 42, 96
yield, 161, 196, 420, 423
Wallerian degeneration, 140
Ward’s triangle, 108 Z
warts, 59
wastes, 190 ZAP-70, 290
Zinc (Zn), 12, 111, 112, 170, 171, 226, 229, 333, 334

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In: Food and Beverage Consumption and Health Series

HANDBOOK OF GREEN TEA AND

Handbook of Green Tea and Health Research

HANDBOOK OF GREEN TEA AND

Nova Science Publishers, Inc.

NOTICE TO THE READER

LIBRARY OF CONGRESS CATALOGING-IN-PUBLICATION DATA

Published by Nova Science Publishers, Inc. Ô New York

Chapter 8 Green Tea: Protective Action against Pesticides and other

Chapter 22 Biocatalytic Conversion of Green Tea Catechins to Epitheaflagallin,

CENTRAL FUNCTIONS OF GREEN TEA COMPONENTS

M. Furuse 1,2,*, N. Adachi2, S. Tomonaga2,

CENTRAL FUNCTION OF EGCG

Figure 1. Structures of tea catechins.

Figure 2. Structures of L-theanine and L-glutamate.

EGCG (μg) 0 50 100 200

The i.c.v. injection of EGCG significantly decreased plasma corticosterone release

In addition, EGC suppressed the number of vocalizations, and co-administration of

They demonstrated that EGCG suppressed IL-1beta+Abeta (25-35)-induced phosphorylation

Table 2. Effect of i.c.v. injection of EGC or EGCG on various behavioral categories in

EGC (nmol) EGCG (nmol)

Table 3. Effect of i.c.v. injection of EGC, picrotoxin or both on various behavioral

Saline EGC (109 nmol) P values

Values are means ± S.E.M. in seconds.

EGCG has neuroprotective effects on oxidative stress-injured neuronal cells, especially

Ullmann et al. (2003) conducted a randomized, double-blind, placebo-controlled study

CENTRAL FUNCTION OF L-THEANINE

DIRECT COMPARISON OF EGCG AND L-THEANINE

Spontaneous activity (arbitrary unit)

Saline EGCG L-Theanine

Terashima, T., Takido, J. &Yokogoshi, H. (1999). Time-dependent changes of amino acids in

GREEN TEA CATECHINS:

P. Buzzini1,*, P. Vignolini2, M. Goretti1, B. Turchetti1, E. Branda1,

properties exhibited by polyphenols, particularly catechins, occurring in green tea

Figure 1. Structures of most represented flavan-3-ols found in green tea.

Figure 2. Chemical structure of theaflavins.

Concerning non-antimicrobial activities, the antioxidant properties of green tea

FUNDAMENTALS OF BIOSYNTHESIS OF GREEN TEA CATECHINS

ANTIVIRAL ACTIVITY OF GREEN TEA CATECHINS

ANTIBACTERIAL ACTIVITY OF GREEN TEA CATECHINS

Table 1. Some additional examples of antibacterial activity of green tea catechins

Species Active References

In relation to the activity of green tea catechins on bacteria of technological importance,

ANTI-YEAST AND ANTIFUNGAL ACTIVITY OF

SYNERGISTIC INTERACTIONS BETWEEN

Toda, M; Okubo, S; Ikigai, H; Suzuki, T; Suzuki, Y; Hara, Y; Shimamura, T. The protective

LIPID-SOLUBLE GREEN TEA POLYPHENOLS:

Ping Chen2, Douglas Dickinson1 and Stephen Hsu1

MATERIAL AND METHODS

Chemicals and Antibodies

Cell Lines and Cell Culture

Morphological Analysis of Cells Treated with LTPStearate

To determine changes in the protein level of caspase 14 upon activation by EGCG or

Animal Treatment and Immunohistochemistry

HPLC Analysis of EGCG from Cell Lysates

LTPStearate Induces Tumor Cell Death as Efficiently as GTPs

LTPStearate does not Induce Cell Death in Normal Epidermal Keratinocytes

Dose-dependent Induction of Caspase 14, a Marker for Epidermal Terminal

The expression of caspase 14 was compared between 50 µM EGCG-treated and 0.01%

LTPStearate Induces Caspase 14 Expression in Vivo in a Mouse Model for

Immunostaining of mouse skin treated with LTPStearate in glycerin demonstrated increased

LTPOleate and LTPStearate are Converted to Intracellular GTPs in NHEK

*Retention time of EGCG: 28.92 min.