Food Chemistry 145 (2014) 145153

Contents lists available at ScienceDirect

Food Chemistry
journal homepage: www.elsevier.com/locate/foodchem

The fortication of tea with sweeteners and milk and its effect on in vitro
antioxidant potential of tea product and glutathione levels in an animal
model
M.W. Korir a, F.N. Wachira b,, J.K. Wanyoko c, R.M. Ngure a, R. Khalid c
a
b
c

Department of Biochemistry and Molecular Biology, Egerton University, P.O. Box 536, Egerton, Kenya
Association for Strengthening Agricultural Research in East and Central Africa (ASARECA), P.O. Box 765, Entebbe, Uganda
Tea Research Foundation of Kenya, P.O. Box 820-20200, Kericho, Kenya

a r t i c l e

i n f o

Article history:
Received 9 January 2013
Received in revised form 6 July 2013
Accepted 2 August 2013
Available online 11 August 2013
Keywords:
Tea
Milk
Sweeteners
Antioxidant activity
Glutathione

a b s t r a c t
Several studies have demonstrated that tea avonoids protect cells and tissues against free radicals which
have been implicated in the etiology of oxidative stress-related disease disorders. However, black tea is
commonly consumed with additives that could otherwise affect the bioavailability of the active tea molecules. In this study, the biochemical parameters of Kenyan teas were determined and the effect of added
milk and sweeteners on the antioxidant activity of Kenyan teas was investigated. The effect of tea antioxidants on glutathione (GSH) was also evaluated in vivo in a time series study using Swiss mice. Green
teas had the highest levels of total polyphenols, total and individual catechins, while black teas had high
levels of total thearubigins, total theaavins and theaavin fractions. The antioxidant activity was high in
green teas though some of the black teas were as efcacious as the green teas. The addition of milk, sugar
and honey signicantly (p < 0.05) decreased the antioxidant activity of tea in a concentration-dependent
manner. Addition of the sweetener, stevia (Stevia rebaudiana Bertoni), showed no signicant (p > 0.05)
inuence on the antioxidant activity of tea and therefore can be recommended as a preferred sweetener
for tea. Signicantly (p < 0.001) higher levels of GSH were observed in plasma than in other tissues. GSH
levels were generally highest 2 h after tea consumption, which indicates the need to repeatedly take tea
every 2 h to maximise its potential health benets.
2013 Elsevier Ltd. All rights reserved.

1. Introduction
Black (aerated) tea is the most consumed beverage globally. It is
largely produced in Africa, with Kenya being the largest producer
(Abeywickrama, Ratnasooriya, & Amarakoon, 2011; Cabrera, Reyes,
& Rafael, 2006; Economic Review of Agriculture, 2007; Kerio,
Wachira, Wanyoko, & Rotich, 2012; Mwaura & Ogise, 2007). Black
tea is processed from the tender leaves of Camellia sinensis. Fresh
tea leaves are rich in catechins which form the principal polyphenolic compounds in green (non-aerated) tea, while black teas are rich in
oxidation products of the catechins, called theaavins and thearubigins (Frei & Higdon 2003; Karori, Wachira, Wanyoko, & Ngure,
2007). Tea-derived polyphenols have been extensively studied as
promising substances in disease prevention (Cabrera et al., 2006;
Jianbo et al., 2011). For example, the chemo-preventive activity of
tea against cancer has been demonstrated in several body organs,
such as colon, stomach and lung (Sharangi, 2009; Wang et al.,
2010). Regular consumption of tea has also been linked with the
protection against harmful UV radiation, and maintenance of skin
Corresponding author. Tel.: +256 722 644279; fax: +256 414 321126.
E-mail address: f.wachira@asareca.org (F.N. Wachira).
0308-8146/$ - see front matter 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.foodchem.2013.08.016

structure and functions (Heinrich, Carolyn, Silke, Hagen, & Wilhelm,

2011). Tea catechins and theaavins have been implicated in the
protection against cardiovascular and kidney ailments through several mechanisms (Shinichi, 2008). Tea catechins also decrease glucose production and hence regulate glucose and insulin
concentrations in the blood (Wu, Juan, Hwang, Hsu, & Ho, 2004).
Tea has been demonstrated to ameliorate inammatory conditions
due to its anti-parasitic, anti-heamolytic and anti-oxidant properties (Karori, Wachira, Wanyoko, & Ngure, 2008). Regular consumption of black tea has been associated with low risks of cognitive
impairment and has been shown to delay or prevent the onset of
dementia (Chen et al., 2010; Ng, Lei, Mathew, Ee, & Keng, 2008).
Tea polyphenols with the galloyl moiety have particularly been
demonstrated to inhibit the entry of the HIV virus by binding to
the gp41, which is essential for the HIV-1 attachment to the target
cells (Shuwen et al., 2005). The uoride present in tea prevents dental decay by inhibiting the demineralization of protective coat enamel by both pathogenic microorganisms and commensal
organisms (Hamilton-Miller, 2001).
The above health effects of tea are largely ascribed to its antioxidant properties, which enable it to protect cells against oxidative
damage caused by free radicals (Gupta, Siddique, Beg, Ara, & Afzal,

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M.W. Korir et al. / Food Chemistry 145 (2014) 145153

2008). Free radicals are important in normal physiological processes but they have also been associated with the etiology of
chronic and degenerative diseases, including atherosclerosis, Alzheimers and Parkinsons disease, diabetes, cardiovascular diseases,
cancer, allergies and premature aging (Svetli, Guy, & Heidar-Ali,
2010). Endogenous antioxidant defence mechanisms are not always sufcient to fully scavenge free radicals from the body and
dietary components that have antioxidant properties, such as tea
avonoids, are therefore necessary to supplement the activity of
endogenous antioxidants. One of the major endogenous antioxidants in the body is glutathione (GSH). GSH has many functions,
one of which is the protection against oxidative damage by scavenging free radicals and peroxides produced during normal cellular
respiration, which would otherwise oxidise proteins, lipids and nucleic acids (Giustarini, Rossi, Milzani, Colombo, & Dalle-Donne,
2004; Moskaug, Harald, Myhrstad, & Rune, 2005).
Though numerous studies have been carried out to determine
the health benets associated with antioxidant properties of tea,
these efforts have largely been directed towards the biological
activity of green tea with very little effort on black tea, which is
the principle product of Africa and which is also widely consumed
in the world. Black tea is largely consumed with substantial
amounts of milk and added sweeteners, such as sugar, stevia and
honey. The effects of milk and added sweeteners on the antioxidant activity of tea have hardly been investigated. This study
aimed to establish whether addition of these secondary additives
affects the potential health benets that can be derived from the
antioxidative potency of tea. It is envisaged that evidence of the effects of these ingredients on the antioxidant activity of tea will improve preparation and consumption habits, and promote tea as a
functional beverage and contribute to its increased consumption.

2. Materials and methods

2.1. Tea samples and reagents
Leaf samples from ve tea cultivars (AHP S15/10, TRFK 303/577,
TRFK 6/8, BBK35 and TRFK 306/1) were sourced from the Tea Research Foundation of Kenya (TRFK) 026S, 3715E). Rened white
cane sugar, produced and packaged by Mumias Sugar Company,
fresh cows milk, processed and packaged by the Kenya Co-operative Creameries Ltd (KCC) and honey, processed and packaged by
Baraka Agricultural Institute, were used in this study. The milk
was ultra-heated with no additives or preservatives. Stevia powder
was bought from a local chemist shop.
2.2. Sample preparation
2.2.1. Processing of tea samples
Tea was manufactured in a miniature tea factory at the TRFK,
Kericho, Kenya. Black teas were manufactured using physical wither for 18 h to attain a moisture content of 5065%. Aeration was
carried out for 2 h at 24 C and the leaf red in a uid bed drier
at 120 C for 2025 min. Green teas were manufactured by steaming the leaf for 1 min, crushing, tearing and curling and nally ring in a uid bed drier at 120 C. The processed teas were packaged
into polythene bags and stored at room temperature prior to further analysis.
2.2.2. Determination of dry matter content
Five grammes (5 g) each of the tea products were weighed to the
nearest 0.001 g, placed in pre-weighed aluminium dishes and dried
in an oven (Oven Memmert, UND300, Germany) at 103 2 C for
16 h to constant weight. Percentage dry matter (DM) content for

each sample was calculated from the weight differences of the dried
and pre-dried tea samples.
2.3. Preparation of extracts
2.3.1. Extraction of polyphenols and catechins
Tea samples of coarse granular structure were ground to ne
powder. A sample of 0.2 0.001 g of tea was weighed into a graduated extraction tube (10 ml) and 5 ml of 70% hot water/methanol
extraction mixture added using a dispenser (Dispensette Brand
Germany), stoppered and mixed on a vortex mixer (Rotamixer,
Huck andTucker, England). The mixture was immediately placed
in a water bath at 70 C and incubated for 10 min. After incubation,
the mixture was removed from the water bath, cooled and centrifuged at 3500 rpm for 10 min (Heraeus Sepatech, Germany). The
supernatant was decanted in a graduated tube. The extraction step
was repeated and both extracts were pooled and the volume adjusted to 10 ml with cold 70% methanol.
2.3.2. Extraction of theaavins and thearubigins
Nine grammes (9 g) of coarse tea sample were weighed, and
placed in a 500 ml thermos ask and 375 ml of hot distiled water
added .The mixture was then agitated, using a mechanical shaker
for 15 min. The tea infusion was then ltered through cotton wool
into a at-bottomed beaker and allowed to cool to room
temperature.
2.4. Analysis of total polyphenols
The FolinCiocalteau reagent method was used to determine total polyphenols as per the International Organisation for Standardization (ISO) 14502-1 (ISO 14502-1, 2005). From the sample
extract, 1 ml was pipetted into a 100 ml volumetric ask and made
up to the mark with distiled water. One millilitre of the diluted
sample was complexed with 5 ml of 10% FolinCiocalteaus phenol
reagent and 4 ml of 7.5% sodium carbonate solution. The mixture
was stoppered, vortexed and the optical densities (OD) measured
after 1 h in 10 mm length cells against distiled water, using a digital grating spectrophotometer at 765 nm (model Cecil CE 393).
The total polyphenol content was expressed as gallic acid equivalents (GAE), in g/100 g.
2.5. Analysis of catechins
Catechin analysis was done (HPLC) according to ISO 14502-2,
2005. Reverse phase HPLC was employed. A Shimadzu LC AT HPLC
machine with SIL 20A auto sampler, SPD-20 UVvisible detector
with a class LC 10 chromatography work station and A Luna TM
5 lM C6, 250 mm  4.6 i.d (Phenomenex, Tolerance, CA, USA) column with a Reodyne pre-column lter 7335 model was used. One
millilitre (1 ml) of the extract (2.3.1 above) was diluted to 5 ml
with stabilizing solution (10% v/v acetonitrile with 500 lg/ml
EDTA and ascorbic acid), ltered through a 0.45 lm membrane lter and 20 ll were injected into the machine. A gradient elution
was carried out using mobile phases A and B: mobile phase A (acetonitrile/acetic acid/double distiled water-9/2/89 v/v/v), and mobile phase B (acetonitrile/acetic acid/double distiled water-80/2/
18 v/v/v). The mobile phase composition for a binary gradient condition started at 100% solvent A for 10 min, then over 15 min a linear gradient to 60% mobile phase A, 32% mobile phase B and held at
this position for 10 min. The condition was reset to 100% mobile
phase A and allowed to equilibrate for 10 min before the next sampling. The ow rate of the mobile phase was 1 ml per minute and
the temperature of the column was set at 35 0.5 C. The identication of individual catechins was carried out by comparing the
retention times and UV-absorbance of sample peaks with peaks

M.W. Korir et al. / Food Chemistry 145 (2014) 145153

obtained from the mixed known catechin standards under the

same conditions. The standards used were catechin (C), epicatechin
(EC), epigallocatechin (EGC), epicatechin gallate (ECG) and epigallocatechin gallate (EGCG) purchased from Sigma Aldrich, UK. The
quantication of catechins was performed at 278 nm and was
achieved using a caffeine standard with a calibration curve
r2 = 0.9992 in conjunction with the consensus individual catechin
relative response factor (RRF) values with respect to caffeine calculated on a dry matter basis. All determinations were done in triplicate. Total catechin, as percentage by mass, on a sample dry
matter was given on the summation of individual catechins:

% Total catechin %ECG %C %EC %EGCG %EGC

Caffeine content was quantied as follows:

%Caffeine Asample  Aintercept  RRFstd  V  d  100

Slopecaffeine  m  1000  DM

147

Total thearubigins content was determined using the method of

Roberts and Smith (1961). Six millilitres of 1% aqueous solution of
anhydrous disodium hydrogen orthophosphate were mixed with
6 ml of the cooled tea infusion prepared as described above. The
mixture was extracted with 10 ml of ethyl acetate and the aqueous
layer was drained off. Ten millilitres of the ethyl acetate extract
were diluted with 25 ml of methanol (solution A). One millilitre
of infusion was mixed with 9 ml of distiled water and the volume
was made up to 25 ml with methanol (solution B). One millilitre of
the infusion was mixed with 1 ml of aqueous 10% oxalic acid, followed by 8 ml of distiled water and the volume was made to
25 ml with methanol (solution C). Each sample was extracted in
triplicate. The absorbancies of solutions A, B and C were read at
380 nm, 460 nm and 380 nm, respectively. TFS and TRS values
were calculated using the formula;
TFs% = 2.25  A  DM%; TRs% = 7.06  (4C-B)  DM% where
DM is the dry matter of the sample (Robert & Smith, 1961).

where;
Asample Peak area of the individual component in the test
sample
Aintercept Peak area at the point of interception on Y-axis
Slopecaffeine Caffeine calibration line slope
V Sample extraction volume
d Dilution factor
m Mass in grammes of test sample
DM Dry matter content of test sample
2.6. Analysis of theaavin fractions
One millilitre (1 ml) of the infusion ((2.3.2. above) was diluted
with distiled water (1:1) and 20 ll of the diluted sample were injected into the HPLC C18 ODS column. A gradient elution was carried out, using mobile phases A (acetic acid/double distiled water
1/99 v/v) and B (acetonitrile/double distiled water 80/20 v/v).
The mobile phase composition for a binary gradient condition
started at 92% solvent A and 8% mobile phase B for 49 min, then
over a linear gradient to 69% mobile phase A and 31% mobile phase
B and held at this for 7 min. The ow rate of the mobile phase was
1.5 ml per minute; the temperatures were maintained at
35 0.5 C. The identication and quantication of the theaavin
fractions were performed at 378 nm, by comparing the retention
times and UV-absorbance of the sample peaks with peaks obtained
from a mixture of known standards of simple theaavin (TF), theaavin 3 monogallate (TFMG), theaavin 3monogallate (TF3MG)
and theaavin 33 digallate (TFDG), (Sigma Aldrich,UK) under the
same conditions.
2.7. Analysis of total theaavin (TFs) and thearubigins (TRs)
Total theaavins (TFs) and total thearubins (TRs) contents were
quantied using a digital grating spectrophotometer (Model Cecil
CE 393). Total theaavin content was determined at 625 nm, using
the Flavagnost method of Hilton (1973). Ten millilitres of the infusion were mixed with 10 ml of isobutylmethylketone-IBMK (Sigma
Aldrich, USA) and allowed to stand until the layers separated. Two
millilitres of the upper layer were transferred into labelled test
tubes in triplicate. Four millilitres of ethanol, followed by 2 ml of
Flavognost reagent were added and the absorbance read against
an IBMK/ethanol blank (1:1 v/v). The Flavagnost reagent formed
a green complex with the cis-h,i-dihydroxybenzene ring associated
with TFs. Total theaavin was calculated using the formula;
TFs (lmol/g) = A 625  479  100/DM, where A 625 is the
absorbance at 625 nm and DM is the dry matter of the sample
(Hilton, 1973).

2.8. Determination of antioxidant activity of tea, with and without

secondary additives, using DPPH radical-scavenging activity
Different tea infusions were prepared without and with different levels of secondary additives as follows: (a) green and black
tea with no additives, (b) green and black tea with milk and no
sweetener, (c) black tea with milk and sugar (d) black tea with milk
and stevia and (e) black tea with milk and honey. Each infusion
was prepared by infusing ve grammes of coarse tea samples in
100 ml of boiling distiled water in a 100 ml volumetric ask and
stirring with a magnetic stirrer on a hot plate for 10 min. Tea infusions with milk were made using 5 g of tea and different concentrations of milk (2%, 4%, 6%, 8%, 10%, 20% and 40% (v/v milk/
water). Infusions with milk and sugar were brewed with 3 g and
10 g of sugar plus the several concentrations of milk, and those
with milk and stevia (0.1 g and 0.3 g) were prepared. Similarly, teas
with several concentrations of milk plus 3 g and 10 g honey were
prepared. All the above tea infusions were strained through cotton
wool to remove the tea particles and left to cool to room temperature. The antioxidant activity of the prepared tea extracts was
determined using the 2, 20 -diphenyl-1-picryhydrazyl radical
(DPPH) method, as described by Karori et al. (2007). The antioxidant activities of sugar, stevia and honey were also assayed using
the DPPH method.

2.9. Animal studies

2.9.1. Animals
Swiss albino mice (female, 68 weeks old and weighing 2135 g
were used in this study. The animals were housed in standard mice
cages in a controlled environment and provided ad libitum with
food and water. Animal care protocols and procedures were reviewed and approved by the Institutional Animal Care and Use
Committee. The animals were treated once using 1% ivermectin
at a dose of 0.01 ml per kg/body weight during the rst week to exclude any endoparasites and ectoparasites. Twenty grammes (20 g)
of the processed tea from selected cultivars TRFK303/577 (for Black
and Green tea) and TRFK 306/1 (for Purple tea) were separately
weighed and 1 L of hot distiled water added. Infusions with milk
(2% v/v) were also prepared, using black and purple teas. The animals were randomly allocated into 5 groups, each with three mice
per group, and each group of mice being housed separately. The 5
groups represented sampling intervals (0 h; 30 min; 2 h; 4 h and
8 h) while the three mice were replications of the same treatments.
The animals were then orally fed with 0.5 ml of the prepared and
cooled tea infusions.

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M.W. Korir et al. / Food Chemistry 145 (2014) 145153

2.9.2. Sampling
The animals were anesthetised by inhalation of chloroform for
2 min. Blood, liver, brain and kidney tissues were sampled at 0,
30 min, 2 h, 4 h and at 8 h after tea consumption. The blood samples were collected using a 1 ml syringe containing ethylene diamine tetra acetic acid (EDTA) and transferred to Eppendorf tubes.
The blood samples were mixed thoroughly to prevent blood clotting and immediately centrifuged at 3000g for 4 min, using a micro-centrifuge (Fisher Scientic, 59A) to obtain plasma. The
obtained plasma samples and other tissues were collected in well
labelled and screwed cryiol vial tubes and immediately preserved
in liquid nitrogen for further analysis.
2.9.3. Glutathione (GSH) assay
A modied Rahman, Arnna, and Saibal (2007) method was used
to assay glutathione (GSH) levels in the collected animal tissues.
Tissue homogenates were prepared using Hepes buffer, pH 7.4. A
volume of 0.5 ml of protease inhibitor was added to prevent the
degradation of the proteins. Fifty ll of the tissue homogenates
were mixed with 50 ll of a solution containing sulphosalicylic acid
(5.0% w/v) and 0.25 mM EDTA and the mixture centrifuged at
8000g for 10 min at 4 C. 200 lM glutathione (GSH) standard solution was prepared in 0.5% sulphosalicylic acid (SSA) and serial dilutions prepared using the same solution (0.5% SSA) to reach nal
concentrations of 100, 50, 25, 12.5, 6.25, 3.13 and 1.56 lM. Ellmans reagent (5,5-dithiobis-2-nitrobenzoic acid) was prepared
by dissolving in 0.1 M potassium phosphate buffer and 5 mM EDTA
disodium salt, pH 7.5, to a nal concentration of 0.6 mg/ml. Volumes of 25 ll of serial standard solutions were loaded onto a 96well plate (wells AH) in columns 1, 2 and 3, followed by 25 ll
of the sample to the remaining wells in triplicate. A volume of
100 ll of freshly prepared Ellmans reagent was then added to each
well and the absorbance measured at 405 nm at intervals of 30 s,
using a multi-detection microtitre plate reader. A calibration GSH
standard curve was prepared which was used to extrapolate the
GSH levels from the biological samples.
2.10. Statistical analysis
All the above determinations were carried out in triplicate. The
data were subjected to analysis of variance and the means separated by the least signicant test, using MSTAT Version 2.10. Statistical analysis on the determination of effects of tea on GSH was

done using Graph Pad software, 2007, followed by the Bonferroni

post-hoc test to compare the obtained means.
3. Results
3.1. Flavanoid levels in the tea products
Data on the biochemical parameters assayed in green and black
tea liquors are presented in Table 1. Total polyphenol levels were
generally higher in green than in black teas. Total and individual
catechins (EGC, +C, EC, EGCG, ECG) varied signicantly (p < 0.05)
and were generally higher in green than in black teas. EGCG was
the most predominant individual catechin in the assayed tea liquors while +C was the lowest. Total thearubigins and total theaflavins were signicantly (p < 0.05) higher in black teas than in
green teas. Levels of theaavin were generally higher in black tea
than in green teas and were found to increase in the following order: theaavin 30 , 3-digallate (TFDG) > theaavin-3-monogallate
(TF-3-MG) > theaavin 30 - monogallate (TF-30 -MG) > free theaavin (TF). However, caffeine levels were found to be similar in both
black and green teas. Gallic acid levels were signicantly higher in
black teas than in green teas.
3.2. Antioxidant activity of tea
Data on antioxidant activity are presented in Table 2. The antioxidant activity of green teas was marginally higher than that of
black teas. However, black teas from some cultivars exhibited antioxidant activities that were as high as those of green teas.
When the chemical parameters were correlated with antioxidant activity, it was observed that the antioxidant activity in green
teas correlated signicantly (r = 0.91, p < 0.001) positively with the
levels of total catechins, total polyphenols (r = 0.93, p < 0.001),
EGCG (r = 0.96, p < 0.001), EGC (r = 0.78, p < 0.01), ECG (r = 0.79,
p < 0.01), and TF3MG (r = 0.53, p < 0.05). However, an inverse correlation was noted with T.TF (r = 0.07, p < 0.05), TR (r = 0.02,
p < 0.05), TF (r = 0.67, p < 0.05), and caffeine (r = 0.18, p < 0.05).
In black teas, signicant positive correlations between antioxidant
activity and the tea parameters were observed for T.TF (r = 0.73,
p < 0.01), TP (r = 0.65, p < 0.05), TR (r = 0.85, p < 0.001), TF3MG
(r = 0.91 p < 0.001), TF3MG (r = 0.93 p < 0.001), TF33DG (r = 0.95,
p < 0.001), and GA (r = 0.92, p < 0.001). Total catechins (r = 0.15,
p < 0.05), caffeine (r = 0.09, p < 0.05), epicatechin (r = -0.48,

Table 1
Phenolic composition of green and black tea liquors (%).
Tea samples

1
2
3
4
5
6
7
8
9
10
C.V (%)
LSD(p < 0.05)
G mean

T TFs lmol/g

1.26f
1.25f
1.52f
1.38f
7.84e
18.0a
17.9a
16.1b
14.6c
12.6d
9.41
1.49
9.24

Individual TFs

TC

TF

TF3MG

TF3MG

TFDG

0.58ef
0.31f
0.19f
0.46f
1.22cd
2.61b
1.62c
0.90d
1.45c
3.94a
19.9
0.44
1.28

0.33e
0.38e
0.29e
0.27e
4.87ab
5.38a
4.64ab
3.00cd
3.89c
3.84bc
26.4
1.61
2.56

0.28f
0.2f
0.31ef
0.28ef
0.77de
2.55a
2.21ab
1.55 c
1.88cd
1.74bc
27.4
0.55
1.11

0.14d
0.25d
0.64d
0.37d
0.96d
7.45b
9.44ab
10.7a
7.34b
3.07c
30.9
2.01
3.79

18.3ab
18.8a
17.5b
17.7b
6 24d
7.23c
6.11d
4.11e
5.68d
6.4cd
4.61
0.85
10.8

Individual catechins
EGC

+C

EC

EGCG

ECG

3.16c
1.99f
4.88a
1.00g
0.65h
3.35b
3.28bc
2.94d
2.81d
2.5e
3.99
0.18
2.66

0.54c
0.36d
0.13f
0.84a
0.38d
0.11fg
0.07g
0.24e
0.12fg
0.7b
11.08
0.06
0.35

1.18b
0.93c
0.33f
0.77d
0.67e
0.29f
0.19g
0.12h
0.23g
1.43a
3.81
0.05
0.62

10.6b
11.8a
10.96b
9.02c
2.17d
2.01d
1.57de
0.49f
1.24e
0.51f
7.05
0.61
5.03

2.82c
3.72b
2.81c
4.27a
2.33d
1.58e
1.48e
0.84g
1.15f
0.42h
6.56
0.24
2.14

TP

CAFF

GA

TRS

29.5a
26.9b
23.2c
27.0b
22.6c
22.2c
22.2c
20.1d
22.9c
12.3e
3.27
1.29
22.9

4.43de
4.75cd
4.75cd
4.96bc
2.06f
4.73bc
4.99bc
5.27ab
5.45a
4.26e
5.26
0.41
4.56

.79g
1.21e
0.79g
0.89f
0.68h
1.51a
1.47ab
1.37cd
1.31d
1.40bc
3.8
0.08
1.14

3.63e
2.80e
2.43e
2.63e
9.0d
10.5cd
9.1d
12.4ab
11.4bc
13.9a
11.6
1.55
7.78

Means within a column followed by the same letters are not signicantly different at p = 0.05 by the LSD test. Tea samples 15 are green tea samples (TRFK 6/8, TRFK 303/577,
AHPS15/10, TRFK BBK35, TRFK 306/1) in that order, while samples 610 are black tea samples TRFK 6/8, TRFK 303/577, S15/10, TRFK BBK35, commercial sample. T.TFs, (total
theaavins (lmol/g)); TF, (simple theaavin (%)); TF3MG, (theaavinmonogallate (%)); TF3MG, (theaavin 3 monogallate (%)); TFGD, (theaavin 3,3 digallate(%)); TC, (total
catechins(%)); EGC, (Epigallocatechin (%)); +C, (catechin (%)); EC, (Epicatechin (%)); EGCG, (Epigallocatechingallate (%)); ECG, (Epicatechingallate.(%)); TPP, (total polyphenols
(%)); CAFF, (caffeine (%)); GA, (gallic acid); TRs, (total thearubigins(%).

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M.W. Korir et al. / Food Chemistry 145 (2014) 145153

Table 2
Mean values of antioxidant activity of green and black teas with no additives.
Tea samples

Antioxidant activity (%)

92.4
92.9a
92.6ab
91.8cd
91.8cd
91.8cd
92.1bc
91.3d
90.2e
91.2d
0.44
0.69

No sweetener
0.1 g stevia/100 ml
0.3 g stevia/100 ml
3 g sugar/100 ml
10 g sugar/100 ml
3 g honey/100 ml
10 g honey/100 ml
C.V (%)
LSD

Different superscripts in the same column indicate that activities differ signicantly
at p = 0.05 by the LSD test. Tea samples Nos. 15 (Green teas), 610 (Black teas) as
described in the footnote for Table 1.

p < 0.05) and + catechin (r = 0.30, p < 0.05), showed a negative

correlation with the antioxidant activity.
3.3. Effects of added milk and sweeteners on antioxidant activity of
green and black teas
The antioxidant activity signicantly (p < 0.05) decreased with
increase in milk concentration in both green and black teas, though
the activity was generally higher in green teas than in black teas. A
signicant decrease in antioxidant activity (p < 0.05) was noted
after addition of 10% milk in both green and black teas (Table 3).
The antioxidant activities of sugar, stevia and honey were also assayed in this study. Honey had signicantly (p < 0.05) higher antioxidant activity (10.6%) than had sugar (6.77%) or stevia (4.33%)
though these were comparatively lower than those of the tea. Antioxidant activity determined on plain black tea liquors and those fortied with milk and sweeteners showed signicant variations as
shown in Table 4. However, the addition of 0.1 g and 0.3 g of stevia
had no signicant inuence on the antioxidant activity of the plain
teas or those fortied with milk. Antioxidant activity of tea liquors
with honey and sugar varied signicantly (p < 0.05) in a concentration-dependent manner. Teas fortied with honey showed less antioxidant activity than did other sweeteners.
3.4. Glutathione levels
Glutathione (GSH) levels in different tissues were measured to
investigate the effects of the tea bioactive molecules on GSH after
feeding the mice with green, black and purple teas. GSH concentra-

Table 3
Mean values of antioxidant activity of green and black with different milk
concentrations. No sweetener added.

0
2
4
6
8
10
20
40
Mean
C.V. (%)
LSD(p = 0.05)

Tea treatment

abc

1
2
3
4
5
6
7
8
9
10
C.V (%)
LSD(p = 0.05)

Milk concentrations (%)

Table 4
Mean values of antioxidant activity of sweetened plain and milk-fortied black teas.

Antioxidant activity
Green

Black

92.3a
91.7ab
91.2abc
90.5bc
90.0bc
89.5c
87.1d
79.6e
88.9
1.21
1.76

91.3a
90.6a
90.1ab
88.9abc
88.0bc
86.9c
81.4d
56.1e
84.2
1.78
2.44

Means with different letters within each column are signicantly (p < 0.05) different by LSD test.

Antioxidant activity
Plain tea

Milk fortied tea

91.6a
91.6a
90.5a
84.1b
46.5d
54.2c
37.1e
2.8
3.2

84.2a
85.0a
84.6a
75.2b
44.7d
46.5c
33.1d
3.75
4.07

Means within a column followed by the same letters are not signicantly different
at p < 0.05 by LSD test.

tions varied signicantly (p < 0.001) in different tissues, as shown

in Fig. 1(a). The highest levels of GSH were recorded in plasma after
2 h of tea consumption. The GSH levels due to various types of tea
differed signicantly (p < 0.001) in the same tissue at different time
intervals, as shown in Fig. 1(bd). Highest GSH levels were recorded for black tea fortied with 2% milk in plasma, as compared
with the other teas. However, in kidney and brain tissues, higher
GSH levels were recorded for plain black teas after 2 h and 4 h of
tea consumption, respectively. The GSH levels in the liver were
similar to those in the brain but the peak in the liver was after
30 min.

4. Discussion
The analysed tea samples showed signicant variations of tea
chemical parameters. The variations in the biochemical parameters
assayed in the tea products can be ascribed to the wide genetic variation in the tea cultivars used in the study. These variations enable
the selection of tea cultivars with specic biochemical proles. The
phenolic composition of test tea products varied greatly after aeration, demonstrating that the oxidation process during black tea
processing resulted in distinctly different polyphenol proles. Inactivation of phenol oxidases by steaming during green tea manufacture prevented oxidation of the catechins and therefore the higher
levels of total and individual catechins in green than in black teas.
Individual catechins varied signicantly, with EGCG being the most
predominant followed by EGC, ECG and nally EC in the green teas.
The results obtained in this study are similar to those reported by
others (Cabrera et al., 2006; Ferruzzi, 2010; Kerio et al., 2012). In
black tea processing, the leaf is macerated to initiate oxidation
by the enzyme polyphenol oxidase (PPO) in the presence of molecular oxygen. Quinones from the oxidation of B-ring dihydroxlated
catechins (non-gallated) condense with quinones from the trihydroxylated catechins (gallated) to form the dimeric theaavins and
polymeric thearubigins (Owuor & Obanda, 2007). Consequently,
the levels of total and individual catechins were signicantly lower
in black teas than in green teas, which conrmed that the oxidation
process during black tea manufacture determines the chemical
composition of the end-product. The gallated catechins are depleted faster than are the non-gallated ones during the aeration
process and this probably explains why the levels of EGCG and
ECG were lower in the black tea samples than in green tea when
compared to EGC, EC and (+C). These results are comparable with
those of Mendel et al. (2005) and Ferruzzi (2010). Total theaavin
levels were also signicantly higher in black teas than in green teas
conrming the oxidation process of catechins. Variations in theaflavins fractions were signicant, with theaavin-3, 30 -digallate
(TFDG) being the most predominant, followed by theaavin-3monogallate, theaavin-30 -monogallate and nally the simple

150

M.W. Korir et al. / Food Chemistry 145 (2014) 145153

15

6
5
GSH (m/l)

GSH (m/l)

12

0.0 hours
0.5 hours
2 hours
4 hours
8 hours

0.0 hours
0.5 hours
2 hours
4 hours
8 hours

4
3
2

1
0

Kidney

Brain

Plasma

0.0 hours
0.5 hours
2 hours
4 hours
8 hours

16
14

Purple

Purple m

Black

Black m

12
10

0.0 hours
0.5 hours
2 hours
4 hours
8 hours

3
GSH (m/l)

GSH (m/l)

18

Green

GSH levels due to different teas in the kidney

GSH levels in various tissues at different time intervals

20

untreated

Liver

8
1

6
4
2
0

untreated Green

Purple

Purple m Black

untreated

Green

Purple

Purple m

Black

Black m

Black m

GSH levels due to different teas in plasma

GSH levels due to different teas in mice brain

Fig. 1. (ad) GSH levels in various mice tissues after consumption of various teas at different time intervals. Untreated-Mice on water only; Purple-black tea processed from a
purple leaf coloured tea cultivar rich in anthocyanins; Purple m-black tea from a purple leaf tea cultivar fortied with 2% milk; Black m-milk (2%) fortied black tea.

theaavin fraction. These observations indicate that, indeed, the oxidation of the individual catechin precursors, which happens at different rates, determines the end-product of the oxidation process
(Obanda, Owuor, & Mangoka, 2001). EGCG and ECG, the precursors
of TFDG are signicantly higher in fresh tea leaves which results in
high levels of TFDG. Similar results were obtained by Owuor et al.
(2006) in a study to investigate the relationship between chemical
parameters and sensory evaluations for plain black tea.
Though black tea is consumed without secondary ingredients in
some countries, in the majority of cases, it is consumed with a substantial amount of milk and sweeteners, to reduce its astringency.
Studies have demonstrated that tea exerts numerous health effects
which are ascribed to its antioxidant properties (Cabrera et al.,
2006; Svetli et al., 2010). Theaavins and catechins in black and
green tea, respectively, contribute to the antioxidant characteristics of tea, which is key to the health benets associated with this
beverage. In this study, free radical-scavenging potential of black
and green tea extracts, with and without milk, sugar, honey and
stevia, was evaluated. The antioxidant activity in green teas was
signicantly higher than that in black teas. Nonetheless, some of
the black teas, such as those processed from cultivars TRFK 303/
577, and TFRK 6/8, had antioxidant activities that were comparable
to some of the green teas. This could be explained by the initial
high levels of total catechins, EGCG and ECG in the leaves of the
these cultivars which positively correlated with levels of total
theaavin, theaavin monogallates, theaavin digallate and gallic
acid, all of which, in turn, correlated positively with antioxidant
activity in the black tea products. These results were consistent
with those of Cabrera et al. (2006), Henning et al. (2006), and
Karori et al. (2007). The high antioxidant activity noted in black
teas in this study implies that black tea from appropriate tea cultivars can potentially be as efcacious in its antioxidative capacity as
green tea.

Although the antioxidant capacity was not statistically different

between teas fortied with milk at 0% (v/v) and 2% (v/v), the antioxidant activity generally decreased signicantly (p < 0.05) with increased concentrations of milk above the 2% threshold and it was
signicantly lower in black teas than in green tea. The decrease in
antioxidant activity with addition of milk reveals an interaction between tea polyphenols and milk proteins. These ndings are comparable to those obtained by Lorenz et al. (2007) who showed that the
addition of milk to black tea inhibited the biological activity of tea in
cardiovascular endothelial functions while tea without milk had positive effects on the same. The major proteins in milk are casein (acasein, b-casein, k-casein), a-lactalbumin, b-lactoglobulin, immunoglobulin and serum albumin (Etzel, 2004; William, 2004). In an
experiment to investigate the interaction of individual milk proteins
with catechins, the casein proteins were found to lower the catechin
levels though no effect was reported with the other milk proteins
(Lorenz et al., 2007). The lowering of tea antioxidant potency by
added milk may therefore be ascribed to the interaction and binding
of tea catechins by casein proteins. Catechins correlated positively
with antioxidant activity in this study and, therefore, it can be expected that the addition of casein-rich milk to tea would decrease
its antioxidant activity. The interaction and binding of polyphenols
to milk proteins depends both on the types of both polyphenol and
protein. Polyphenols with a galloyl moiety, high number of hydroxyl
groups and large molecular size have strong binding afnity to the
milk proteins (Imed et al., 2011; Jianbo et al., 2011; Svetli et al.,
2010). Binding of the tea molecules by the milk proteins reduces
their ability to donate hydrogen atoms (Svetli et al., 2010), that bind
and stabilize the free radicals and hence the decline in antioxidant
potency. The antioxidant activity correlated highly with the gallated
catechins in green teas, and TF-3-MG, TFDG, total TF and TRs in black
tea. Black teas possess high levels of TFs and TRs that have larger
molecular size, more galloyl groups and hydroxyl groups and hence

M.W. Korir et al. / Food Chemistry 145 (2014) 145153

high binding afnity to the milk proteins as compared to the catechin green teas and this could explain the lower antioxidant activity
in milk-fortied black teas than in green teas with milk. Proteins rich
in proline have particularly been shown to have a strong afnity for
the hydroxyl groups of avonoids and therefore casein proteins can
be expected to bind the tea polyphenols, unlike the other milk proteins that lack or have very low contents of proline residues (Imed
et al., 2011). Proline-rich proteins have exible secondary structures
and tend to readily form hydrogen bonds due to increased accessibility of the peptide bond and the carbonyl group of tertiary amides
(Poncet-Legrand, Gatier, Cheynier, & Imberty, 2007). These proteins
have particularly high binding afnity towards galloylated molecules, including EGCG and TFDG that signicantly contribute to
the antioxidant activity of green and black tea, respectively. Binding
of the polyphenols by milk proteins masks the active sites such that
they do not attain their optimum radical-scavenging capacity (Arts
et al., 2002; Svetli et al., 2010).
Besides milk, black tea consumers prefer to sweeten their tea.
The concentrations of the sweeteners used in this study were set
to be similar to those that are normally added to tea during consumption. Stevia, which is 300 times sweeter than sugar and
known to exhibit some antioxidant activity (Abou-Arab, Azza, &
Ferial, 2010), had no signicant inuence on the antioxidant activity of plain and milk black teas. This implies that there are either no
signicant interactions between the stevia glycosides and tea molecules in the plain teas or any interaction with the milk teas and
therefore the plain and milk black teas with different stevia concentrations were not statistically different. However, addition of
table sugar and honey to plain black teas signicantly decreased
the antioxidant activity of the latter in a concentration-dependent
manner and the decrease was more in teas with milk. These observations reveal that the antioxidant capacity of tea is not only affected by the milk proteins but also by other additives, such as
sucrose, which is a major constituent of both honey and table sugar. Complex compounds, such as pentagalloylglucose (PGG),
tetragalloylglucose (4GG) and trigalloylglucose (3GG), are likely
to be formed as glucose interacts with the gallic acid in tea where
by the glucose hydroxyl groups are serially substituted by gallic
acid (Zhang, Li, Sung-Hoon, Ann, & Junxuan, 2009). Bovine serum
albumin and other proline-rich proteins present in milk, through
hydrophobic, hydrogen and covalent bonding, ultimately interact
with these glucose complexes altering the biological activity of
tea molecules (Monthana, Ho, Wang, & Huang, 2007). This could
probably explain the decrease in antioxidant activity of black teas
after addition of table sugar and honey and also of teas with milk.
On its own, honey, though chemically similar to sugar was established to signicantly have a higher antioxidant activity than the
other sweeteners. This could be due to the processing methods
for table sugar that strip off all the vitamins, proteins and other active biomolecules that might otherwise contribute to the antioxidant activity in sugar. Though rich in sugars, honey also contains
compounds such as chrysin, pinobanksin, vitamin C, catalase, and
pinocembrin that are thought to function as antioxidants (Valentini, Miquel, & Pierre, 2010), and which could be linked to its
slightly higher antioxidant activity. Though the antioxidant activity of honey was, in this study, expected to positively affect the
antioxidant activity of tea, it was found to be the most inhibiting
sweetener in both plain and black teas with milk. The mechanism
by which sucrose inhibits the antioxidant activity in tea is still not
fully understood though, as postulated, this may involve the formation of glucosegallic complexes. There is a need for more research on this so as to elucidate the interactions between
sucrose, proteins and the bioactive molecules in tea.
The potential health effects of tea depend, not only on the
amount consumed, but on bioavailability (Joshua & Chung, 2003).
Increase in the antioxidant capacity in the plasma is indirect

151

evidence of absorption through the gut barrier after consumption

of polyphenol-rich foods (Augustin & Gary, 2000). Besides other
mechanisms, tea polyphenols are known to scavenge free radicals
directly since they are capable of donating hydrogen atoms from
the hydroxyl groups (Frei & Higdon, 2003). The ultimate antioxidant potential in vivo is dependent on the absorption, metabolism,
distribution and excretion of the bioactive compounds within the
body after ingestion (Spencer, 2003). In this study, the effect of
tea antioxidants in vivo was investigated by measuring the glutathione (GSH) levels. GSH is the most abundant intracellular thiolbased antioxidant, present in all living aerobic cells, and provides
powerful antioxidant protection to body systems heavily exposed
to reactive oxygen species (Turgut, Yasar, Bunyamin, Sebahat, &
Osman, 2006). Tea is one of the well-known antioxidant-rich and
widely-consumed beverages and its antioxidant activity after consumption is therefore expected to aid the antioxidant capacity in
the body system. The results obtained from this study clearly demonstrate the enhancing effect of tea antioxidants on GSH. Though
the specic bioactive tea molecules responsible for these effects
were not evaluated, different teas, namely, green, black and purple
teas, showed increased and signicant variations of GSH levels in
different tissues. Overall, higher levels were found in plasma while
lower levels were seen in the liver, explaining the fact that the
absorption and distribution of the tea molecules varied in the different tissues. According to Henning, Jung, and Heber (2008), gallated tea molecules, such as EGCG and ECG, mainly occur in
plasma and urine in the free form, whereas the majority of nongallated catechins, including EC, EGC, occur in the conjugated form.
Gallated tea molecules correlated signicantly with the antioxidant activity and this could have led to the higher levels of GSH
in plasma than in other tissues. Besides variation between tissues,
GSH levels also varied in the same tissues due to various types of
teas and also at different time intervals after tea consumption.
The inuence of tea consumption on GSH in the brain indicates
that indeed the tea molecules are able to cross the brain blood barrier where they can scavenge free radicals that are continuously
produced under normal physiological and pathological conditions.
Catechins, which are largely found in green tea, are rapidly
metabolised and eliminated and this could explain the lower levels
of GSH due to green tea consumption when compared to the other
types of tea (black and purple). It has been demonstrated that the
levels of EGCG, which is the most physiologically active and abundant catechin in green tea, decrease in the intestine due to the
alkaline pH that favours its oxidation, thereby reducing the antioxidant activity of green tea (Spencer, 2003). Absorption and metabolism of biomolecules of black and purple teas, that are rich in
theaavins, thearubigins and anthocyanins, respectively, are, however, not well researched. The metabolism and excretion of these
latter molecules, through the kidneys, seem to be lower than those
of the green teas, probably due to their high molecular sizes, which
could explain the high levels of GSH due to black and purple tea
consumption noted in this study. In a study to investigate the bioavailability of tea polyphenols, higher levels of theaavins than
EGCG in prostrate tissue were reported and this could also explain
the higher levels of GSH due to consumption of black teas rather
than green teas noted in this study (Henning et al., 2006). The high
levels of GSH due to black tea when compared to green tea consumption could also be due to the rapid elimination of gallated catechins via the bile to the colon, as documented by Manach, Gary,
Christine, Augustin, and Christian (2005), and McKay and Jeffrey
(2007). Results on antioxidant activity have demonstrated that
the antioxidant activity of black teas in vitro with 2% milk was similar to that of plain black tea. Consumption of black teas with milk
resulted in higher GSH levels in plasma than did consumption of
plain black teas. However, in liver, kidney and brain tissues, the
GSH levels due to consumption of plain black teas were higher

152

M.W. Korir et al. / Food Chemistry 145 (2014) 145153

than that due to black teas with milk. The binding of tea polyphenols by the milk proteins in the milk-fortied black teas could have
hindered the bioavailability of these tea molecules in these latter
tissues and therefore the low GSH levels due to black tea with milk.
The GSH levels were signicantly different at different time intervals. Although the variations were noticeable in different tissues,
overall, the GSH levels peaked at 2 h after tea consumption. This
implies that consumption of tea every 2 h would be necessary to
maximise the bioavailability of the tea molecules in the body system and hence their health benets. At 8 h after consumption of
tea, the GSH levels were low in all the tissues, meaning that almost
all the tea molecules had been metabolised and excreted. These results are consistent with those obtained by Henning et al. (2006).

5. Conclusion
The bioactive molecules assayed in black teas in this study
clearly established that Kenyan black teas possess antioxidant
properties comparable with those of green teas. Addition of secondary ingredients, especially milk and sucrose, lowers the antioxidative capacity of tea, which is associated with the positive health
effects of the beverage and therefore tea preparation and consumption habits should be rationalised so as to maximise potential
health benets. Stevia, a non-caloric sweetener, is a better alternative to sugar according to the ndings in this study. Regular consumption of tea, probably every 2 h, will ensure the bioavailability
of the bioactive tea molecules in order to maximise health effects
in the body system. Further studies are, however, required to
investigate the specic tea molecules and their optimum intake
levels, since metabolism of these compounds differs in different
body tissues. This will enable the processing of teas with specic
active biomolecules that can impact specic biological activity in
specic body organs and at the same time contribute to increased
consumption for better health.
Acknowledgement
We wish to acknowledge the Tea Research Foundation of Kenya
(TRFK) for funding this work.
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