ACID

Extraction of Antioxidants from Vegetable Biomass

A PROJECT REPORT

Submitted by





B.BHUVANA SUNDAR (Reg.No12210203006)
J.RAJKIRAN (Reg.No12210203014)



in partial fulfilment for the award of the degree of
BACHELOR OF TECHNOLOGY

IN

CHEMICAL ENGINEERING

VEL TECH HIGH TECH Dr.RANGARAJAN Dr.SAKUNTHALA
ENGINEERING COLLEGE
ANNA UNIVERSITY: CHENNAI 600 025
APRIL 2014























ACID


Extraction of Antioxidants from Vegetable Biomass
A PROJECT REPORT

Submitted by




B.BHUVANA SUNDAR (Reg.No12210203006)
J.RAJKIRAN (Reg.No12210203014)



in partial fulfilment for the award of the degree of
BACHELOR OF TECHNOLOGY

IN

CHEMICAL ENGINEERING

VEL TECH HIGH TECH Dr.RANGARAJAN Dr.SAKUNTHALA
ENGINEERING COLLEGE
ANNA UNIVERSITY: CHENNAI 600 025
APRIL 2014












BONAFIDE CERTIFICATE




Certified that this project report Extraction of Antioxidants from Vegetable Biomass
is the bonafide work of B.BHUVANA SUNDAR and J.RAJKIRAN who carried out
the project work under my supervision.































SIGNATURE
Mrs. D.PRIYADHARISANI, M.Tech
SUPERVISOR,

DEPARTMENT OF CHEMICAL ENGINEERING,
VEL TECH HIGH TECH Dr.RANGARAJAN
DR.SAKUNTHALA ENGINEERING COLLEGE,


NO: 62 AVADI-VELTECH ROAD,
CHENNAI-600062.

SIGNATURE
Mr.A.SARAVANA RAJ, M.Tech
HEAD OF THE DEPARTMENT,

DEPARTMENT OF CHEMICAL ENGINEERING,
VEL TECH HIGH TECH Dr.RANGARAJAN
DR.SAKUNTHALA ENGINEERING COLLEGE,

NO: 62 AVADI-VELTECH ROAD,
CHENNAI-600062.

CERTIFICATION OF EVALUATION



COLLEGE NAME: VEL TECH HIGH TECH Dr.RANGARAJAN
DR.SAKUNTHALA ENGINEERING COLLEGE



BRANCH : CHEMICAL ENGINEERING



SEMESTER : VIII






S.NO
NAME OF

THE STUDENT
REGISTER
NO.
TITLE OF THE
PROJECT
NAME OF THE
SUPERVISOR


1


B.BHUVANA SUNDAR


12210203006

Extraction of
Antioxidants from
Vegetable Biomass




Mrs. D.PRIYADHARISANI


2


J.RAJKIRAN


12210203014




The report of this project is submitted by the above students in partial
fulfilment for the award for the Bachelor of Technology Degree in Chemical
Engineering of Anna University are evaluated and confirmed to the report of
the work done by the above students.




INTERNAL EXAMINER EXTERNAL EXAMINER










VEL TECH HIGH TECH Dr. RANGARAJAN Dr. SAKUNTHALA
ENGINEERING COLLEGE
NO: 60, VELTECH-AVADI ROAD, CHENNAI-62
(AFFILIATED TO ANNA UNIVERSITY)







CERTIFICATE





This is to certify that the project entitled Extraction of Antioxidants from Vegetable
Biomass is a bonafide work of BHUVANA SUNDAR.B studying, final year B.Tech
Chemical Engineering (2009-2013) in VEL TECH HIGH TECH Dr.RANGARAJAN
Dr.SAKUNTHALA ENGINEERING COLLEGE during under the guidance of
project guide Mrs.D.Priyadharisani,(Assistant professor) DEPARTMENT OF
CHEMICAL ENGINEERING.




















Signature Signature


HEAD OF THE DEPARTMENT INTERNAL GUIDE















VEL TECH HIGH TECH Dr. RANGARAJAN Dr. SAKUNTHALA
ENGINEERING COLLEGE
NO: 60, VELTECH-AVADI ROAD, CHENNAI-62
(AFFILIATED TO ANNA UNIVERSITY)








CERTIFICATE





This is to certify that the project entitled Extraction of Antioxidants from Vegetable
Biomass is a bonafide work of J.RAJKIRAN studying, final year B.Tech
Chemical Engineering (2010-2014) in VEL TECH HIGH TECH Dr.RANGARAJAN
Dr.SAKUNTHALA ENGINEERING COLLEGE during 15.12.2012 to
15.2.2013 under the guidance of project guide Mrs.D.Priyadharisani,(Assistant
professor) DEPARTMENT OF CHEMICAL ENGINEERING.
















Signature Signature


HEAD OF THE DEPARTMENT INTERNAL GUIDE









ACKNOWLEDGEMENT:

We thank our beloved Chairman Prof.Col.Dr.R.Rangarajan B.E.(Elec.),
B.E. (Mech.), M.S. (Auto.), D.Sc. and Vice chancellor Dr. Sakunthala Rangarajan,
M.B.B.S, for giving us the opportunity to work on our project in the college for the
successful completion of our under graduate degree course.
Our sincere thanks to President Mr.K.V.D Kishore Kumar, B.E, M.B.A and
our Managing Trustee Mrs. Mahalakshmi Kishore kumar, B.E, M.B.A who showed
keen interest in developing our skills to perform our project work.
We extend our gratitude to Principal Dr.V.I langovan B.E., M.Tech., Ph.D.,
F.I .E and Vice Principal Mr.M.S.Durai rajan M.E., M.B.A., for monitoring and
enhancing the betterment of our process on the whole.
Our heartily thanks to our Head of the Department Mr.Saravana Raj, M.Tech.,
for helping us whenever needed and encouraging us for doing our project without whom
the project would not have been a success.
We immensely thank our Internal guide Mrs.D.Priyadharisani, M.Tech for
guiding us throughout our project and working with us for the success of our project,
without whom we would have never been able to complete the project on time and we
also thank her giving us support and encouragement for doing a research project as our
final year project.
We would like to thank Dr.S.Thenesh kumar, M.Tech, Ph.D. for creating a
thought for doing innovative projects.
We also like to thank our lab assistants, Ms.Kamini and Ms.Geetha
for helping us to work on our project in their concerned lab. I also thank our lab HOD for
permitting us to do the project in the lab, and also I thank the concerned lab in-charge for
letting us use the lab whenever needed.
We also thank our family and friends for giving us moral support and helping us to
do our project in a very smooth way.
We also thank A.C.Tech Engineering College (Anna University), Chennai for
testing our sample and giving us result as a part of completion of our project.
Finally we would like to thank the almighty for giving us this great opportunity
and for leading us in the right path with right thought and deed of excellence.
TABLE OF CONTENTS


CHAPTER NO. TITLE PAGE NO.:

ABSTRACT 1
LIST OF TABLE 3
LIST OF FIGURES 3

1. INTRODUCTION 4
1.1) ANTIOXIDANTS: 5
1.2) PHENOLIC CONTENTS 6
IN VEGETABLES:
1.3) FLAVONOIDS: 7
1.4) PHYTOCHEMICALS 8

2. LITERATURE SURVEY 10

3 . COLLECTION OF VEGETABLE
BIOMASS 33

4. PREPARATION OF VEGETABLE
BIOMASS 35

5. PREPARATION OF EXTRACT 37

6. METHODS ADOPTED FOR THE
ANALYSIS OF THE EXTRACTS 41
6.1)PROCEDURE 41
6.1.1)DETERMINATION OF TPC 42
6.1.2)REDUCING POWER CAPACITY
(OYAIZU- 1986) 43
6.1.3)DETERMINATION OF THE TFC 43
6.1.4)HYDROGEN PEROXIDE
SCAVENGING CAPACITY 44
6.1.5)PHYTOCHEMICAL SCREENING 44

7. EQUIPMENTS USED FOR THE
PREPARATION OF THE EXTRACT 46
7.1)HOT AIR OVEN 47
7.2)VACUUM DRIER 48
7.3)EQUIPMENTS USED FOR THE
ANALYSIS OF THE EXTRACT
7.3.1)UV Vis SPECTROSCOPY 51
7.3.2)DIGITAL PHOTOCOLORIMETRY 51
7.3.3)CENTRIFUGE 53

8. RESULTS AND DISCUSSION: 56
8.1)TOTAL PHENOLICS CONTENT 57
8.2)REDUCING POWER CAPACITY 59
8.3)TOTAL FLAVONOID CONTENT (TFC) 61
8.4)HYDROGEN PEROXIDE 64
SCAVENGING CAPACITY
8.5)PHYTOCHEMICAL SCREENING 66

9. CONCLUSION 67

10. REFERENCES 69













ABSTRACT










ABSTRACT:

Vegetable biomass generated from the local market by the distribution of
vegetables from wholesale vegetable complex at koyambedu, Chennai is the source
taken for the extraction of antioxidants. The main goal of the present work is to
determine the optimal conditions for the extraction of antioxidant compounds from the
vegetable biomass using aqueous solutions of methanol and to investigate the total
phenols content, total antioxidant capacity, total flavonoid content and Phytochemical
analysis of extracts made. The maximum yield for the extraction carried out under
different parameters. At first, several preliminary tests were conducted to study the
kinetics of extraction process under selected conditions (50% aqueous solution of
methanol at different temperatures and time).The maximum values showed are, Total
phenolic content : ,Ferric reducing capacity: ,Total flavonoid content:
KEYWORDS: Vegetable biomass, Antioxidants, total antioxidant capacity,
total flavonoid content, phytochemicals.
















LIST OF TABLE :

Table 8.3: Total Phenolics content in Vegetable biomass extract
Table 8.4: Reducing power capacity in Vegetable biomass extract
Table 8.5: Absorbance of Standard compound (Quercetin)
Table 8.6: Absorbance of Vegetable biomass extract for flavonoid content:
Table 8.7: Total Flavonoid contents
Table 8.8: Inhibition values


LIST OF FIGURES:

Fig 8.1: Standard curve of Gallic acid
Table 8.2: Absorbance of the vegetable biomass extract:
Fig 8.3: Total Phenolics content in Vegetable biomass extract in mg/g(GAE)
Fig 8.4: Curve for the Concentration Vs Absorbance for standard compound
(L- ascorbic acid) and extract.
Fig 8.5: Comparison of Absorbance of L-ascorbic acid Vs Extract
Fig 8.6: TPC vs. Antioxidant Capacity
Fig 8.7: Curve of Quercetin:
Fig 8.8 Curve of Vegetable biomass extract for flavonoid content:
Fig 8.9: Graph Showing TFC:
Fig 8.10 : The graph shows the variation in TPC,TFC AND Antioxidant levels
Fig 8.11:% INHIBITION OF EXTRACT vs ASCORBIC ACID




















INTRODUCTION
















1.INTRODUCTION:

Oxidation plays a major role in the production of free radicals in
food substances, chemical compounds and also in living organisms. These free
radicals have a important role in the process of food decomposing, chemical
degradation and also cause human disorder. Free radicals induce oxidative damage
in bio molecules and has a major role in the process of ageing, cardio vascular
disorders, cancer and inflammatory diseases. They are involved in depletion of
immune system. But, the antioxidants significantly prevent tissue damage that
stimulates wound healing process. Oxygen free radicals contribute to further
tissue damage in the events following skin injury and are known to impair
healing process. There are available synthetic antioxidants like butylated
hydroxy anisole (BHA), butylated hydroxy toluenes (BHT), tertiary
butylated hydroquinone and gallic acid esters, but have been suspected to cause
negative health effects. Strong restrictions have been placed on their application
and there is a trend to substitute them with naturally occurring antioxidants.
There has been an increasing interest in the therapeutic
potentials of medicinal plants as antioxidants in reducing such free radical
induced tissue injury since it is the belief that the antioxidant property is the
main contributory factor to the therapeutic benefit of many medicinal plants. As
a result, many vegetables, fruits and many other plant species have already been
exploited commercially either as antioxidant additives or a nutritional
supplements.

1.1 ANTIOXIDANTS:

An antioxidant is a molecule that inhibits the oxidation of other
molecules. Oxidation is a chemical reaction that transfers electrons or hydrogen from
a substance to an oxidizing agent. Oxidation reactions can produce free radicals. In
turn, these radicals can start chain reactions. When the chain reaction occurs in a cell,
it can cause damage or death to the cell. Antioxidants terminate these chain reactions
by removing free radical intermediates, and inhibit other oxidation reactions. They do
this by being oxidized themselves, so antioxidants are often reducing agents such as
thiols, ascorbic acid, or polyphenols.
Substituted phenols and derivatives of phenylenediamine
are common antioxidants used to inhibit gum formation in gasoline (petrol).
Although oxidation reactions are crucial for life, they can also be
damaging; plants and animals maintain complex systems of multiple types of
antioxidants, such as glutathione, vitamin C, vitamin A, and vitamin E as well as
enzymes such as catalase, superoxide dismutase and various peroxidases. Insufficient
levels of antioxidants, or inhibition of the antioxidant enzymes, cause oxidative stress
and may damage or kill cells. Oxidative stress is damage to cell structure and cell
function by overly reactive oxygen-containing molecules and chronic excessive
inflammation. Oxidative stress seems to play a significant role in many human
diseases, including cancers. The use of antioxidants in pharmacology is intensively
studied, particularly as treatments for stroke and neurodegenerative diseases. For these
reasons, oxidative stress can be considered to be both the cause and the consequence
of some diseases.
Antioxidants are widely used in dietary supplements
and have been investigated for the prevention of diseases such as cancer, coronary
heart disease and even altitude sickness. Although initial studies suggested that
antioxidant supplements might promote health, later large clinical trials with a limited
number of antioxidants detected no benefit and even suggested that excess
supplementation with certain putative antioxidants may be harmful. Antioxidants also
have many industrial uses, such as preservatives in food and cosmetics and to prevent
the degradation of rubber and gasoline.

1.2 PHENOLIC CONTENTS IN VEGETABLES:

Phenolic compounds are synthesized
industrially; they also are produced by plants and microorganisms, with variation between
and within species.
Although similar to alcohols,
phenols have unique properties and are not classified as alcohols (since the hydroxyl group
is not bonded to a saturated carbon atom). They have higher acidities due to the aromatic
ring's tight coupling with the oxygen and a relatively loose bond between the oxygen and
hydrogen. The acidity of the hydroxyl group in phenols is commonly intermediate between
that of aliphatic alcohols and carboxylic acids (their pK
a
is usually between 10 and 12).
Loss of a positive hydrogen ion
(H
+
) from the hydroxyl group of a phenol forms a corresponding negative phenolate ion or
phenoxide ion, and the corresponding salts are called phenolates or phenoxides, although the
term aryloxides is preferred according to the IUPAC Gold Book. Phenols can have two or
more hydroxy groups bonded to the aromatic ring(s) in the same molecule. The simplest
examples are the three benzenediols, each having two hydroxy groups on a benzene ring.
Organisms that synthesize phenolic
compounds do so in response to ecological pressures such as pathogen and insect attack, UV
radiation and wounding. As they are present in food consumed in human diets and in plants
used in traditional medicine of several cultures, their role in human health and disease is a
subject of research. Some phenols are germicidal and are used in formulating disinfectants.
Others possess estrogenic or endocrine disrupting activity.

1.3FLAVONOIDS:

Flavonoids (or bioflavonoids) (from the Latin word flavus
meaning yellow, their colour in nature) are a class of plant secondary metabolites.
Flavonoids were referred to as Vitamin P (probably because of the effect they had on
the permeability of vascular capillaries) from the mid-1930s to early 50s, but the term
has since fallen out of use.
According to the IUPAC nomenclature, they can be classified into:
flavonoids or bioflavonoids.
isoflavonoids, derived from 3-phenylchromen-4-one (3-phenyl-1,4-
benzopyrone) structure
neoflavonoids, derived from 4-phenylcoumarine (4-phenyl-1,2-benzopyrone)
structure.
The three flavonoid classes above are all ketone-containing compounds, and as such,
are anthoxanthins (flavones and flavonols). This class was the first to be termed
bioflavonoids. The terms flavonoid and bioflavonoid have also been more loosely used
to describe non-ketone polyhydroxy polyphenol compounds which are more
specifically termed flavanoids. The three cycle or heterocycles in the flavonoid
backbone are generally called ring A, B and C. Ring A usually shows a phloroglucinol
substitution pattern.
FUNCTIONS OF FLAVONOIDS IN PLANTS:
Flavonoids are widely distributed in plants,
fulfilling many functions. Flavonoids are the most important plant pigments for flower
coloration, producing yellow or red/blue pigmentation in petals designed to attract
pollinator animals. In higher plants, flavonoids are involved in UV filtration, symbiotic
nitrogen fixation and floral pigmentation. They may also act as chemical messengers,
physiological regulators, and cell cycle inhibitors. Flavonoids secreted by the root of
their host plant help Rhizobia in the infection stage of their symbiotic relationship with
legumes like peas, beans, clover, and soy. Rhizobia living in soil are able to sense the
flavonoids and this triggers the secretion of Nod factors, which in turn are recognized
by the host plant and can lead to root hair deformation and several cellular responses
such as ion fluxes and the formation of a root nodule. In addition, some flavonoids
have inhibitory activity against organisms that cause plant diseases, e.g. Fusarium
oxysporum.

SALUTARY EFFECTS ON HUMAN HEALTH:

Before any chemical compound can be approved
as a pharmaceutical drug or any food can be labelled with a health claim, it must
undergo extensive in vitro, in vivo, and clinical testing to confirm both safety and
efficacy. National and international regulatory authorities like the US Food and Drug
Administration (FDA) and European Food Safety Authority (EFSA) are responsible
for assessing this evidence and granting such approval. At the current time, neither the
FDA nor the EFSA has approved any health claim for flavonoids, or approved any
flavonoids as pharmaceutical drugs. Moreover, several companies have been
cautioned by the FDA over misleading health claims.



1.4 PHYTOCHEMICALS:

Phytochemicals are chemical compounds that occur naturally
in plants (phyto means "plant" in Greek). Some are responsible for color and other
organoleptic properties, such as the deep purple of blueberries and the smell of garlic.
The term is generally used to refer to those chemicals that may have biological
significance, for example antioxidants, but are not established as essential nutrients.
Scientists estimate

that there may be as many as 10,000 different phytochemicals
having the potential to affect diseases such as cancer, stroke or metabolic syndrome.

PHYTOCHEMICALS AS CANDIDATE NUTRIENTS:
Without specific knowledge of their cellular
actions or mechanisms, phytochemicals have been considered as drugs for millennia.
For example, Hippocrates may have prescribed willow tree leaves to abate fever.
Salicin, having anti-inflammatory and pain-relieving properties, was originally
extracted from the bark of the white willow tree and later synthetically produced
became the staple over-the-counter drug aspirin.
Specific phytochemicals, such as fermentable dietary fibers, are allowed limited health
claims by the US Food and Drug Administration (FDA).
Some phytochemicals with physiological properties may be elements rather than
complex organic molecules. For example, selenium, which is abundant in many fruits
and vegetables, is involved with major metabolic pathways, including thyroid
hormone metabolism and immune function. Particularly, it is an essential nutrient and
cofactor for the enzymatic synthesis of glutathione, an endogenous antioxidant.

CLINICAL TRIALS AND HEALTH CLAIM STATUS:

Lycopene from tomatoes has been tested in human studies for cardiovascular diseases
and prostate cancer. These studies, however, did not attain sufficient scientific
agreement to conclude an effect on any disease. The FDA position reads:
"Very limited and
preliminary scientific research suggests that eating one-half to one cup of tomatoes
and/or tomato sauce a week may reduce the risk of prostate cancer. The United States
Food and Drug Administration concludes that there is little scientific evidence
supporting this claim."
Phytochemical-based dietary supplements can also be purchased. According to the
American Cancer Society, "Available scientific evidence does not support claims that
taking phytochemical supplements is as good for long-term health as consuming the
fruits, vegetables, beans, and grains from which they are taken."
FOOD PROCESSING AND PHYTOCHEMICALS:

Phytochemicals in freshly harvested plant
foods may be destroyed or removed by modern processing techniques, including
cooking. For this reason, industrially processed foods likely contain fewer
phytochemicals and may thus be less beneficial than unprocessed foods. Absence or
deficiency of phytochemicals in processed foods may contribute to increased risk of
preventable diseases.
A converse example may exist in which
lycopene, a phytochemical present in tomatoes, is either unchanged in content or made
more concentrated by processing to juice or paste, maintaining good levels for
bioavailability.



















LITERATURE
SURVEY







2.LITERATURE SURVEY:
JOURNAL 1:
EXTRACTION OF ANTIOXIDANTS FROM FORESTRY BIOMASS:
KINETICS AND OPTIMIZATION OF EXTRACTION CONDITIONS
[1]

Forestry biomass, generated as result of forest operations and cleaning of the Galician
(NW Spain) mountains, was studied as a potential source of natural antioxidants. The
main goals of present work were to determine the optimal conditions for the extraction
of antioxidant compounds from the forestry biomass using aqueous solutions of
methanol and to investigate the antioxidant capacity of extracts obtained. At first,
several preliminary extraction experiments were conducted to study the kinetics of
extraction processs under selected conditions (50% aqueous solution of methanol at
25, 50 or 75 _C). The experimental results were fitted to Pelegs, Elovichs and Pages
kinetic models. The Pelegs model was proved to be the best to describe the kinetics
of extraction process. In a second stage experiments were planned according to an
incomplete 33 factorial experimental design to analyse the influence of operational
conditions on total phenols content and FRAP (Ferric Reducing Antioxidant Power),
ABTS (2,20-azino-di(3-ethylbenzothiazoline-6- sulfonic acid)) and DPPH (2,2-
diphenyl-1-picrylhydrazyl) antioxidant capacity of extracts. The examined conditions
were as follows: temperature (25-50-75C), time (5-55-105 min) and methanol
concentration (10-50-90%). The highest temperature assayed (75C), a moderate
solvent concentration (50%) and an extraction time of 55 min were Selected as the
optimum extraction conditions using the response surface methodology.The following
compounds were identified in the extract obtained under optimum
conditions:monogalloyl glucose, digalloyl glucose, (_)-gallic acid, (_)-epicatechin,
()-catechin,ellagic acid and quercetin 3-O-rhamnoside.




JOURNAL 2:
ANTIOXIDANT ACTIVITY OF CORIANDRUM SATIVUM AND
PROTECTION AGAINST DNA DAMAGE AND CANCER CELL
MIGRATION
[2]


Coriandrum sativum is a popular culinary and medicinal herb of the Apiaceae family.
Health promoting properties of this herb have been reported in pharmacognostical,
phytochemical and pharmacological studies. However, studies on C. sativum have
always focused on the aerial parts of the herb and scientific investigation on the root
is limited. The aim of this research was to
investigate the antioxidant and anticancer activities of C. sativum root, leaf and stem,
including its effect on cancer cell migration,and its protection against DNA damage,
with special focus on the roots.

JOURNAL 3:
THE EFFECT OF GRAFTING ON THE ANTIOXIDANT PROPERTIES
OF TOMATO (SOLANUM LYCOPERSICUM L.)
[3]


The use of grafted plants in vegetable crop production is now being expanded greatly.
However, few data are available on the nutritional composition of grafted vegetables
with emphasis on antioxidant properties. Therefore, the major objective of this study
was to evaluate antioxidant components of tomatoes influenced by grafting technique.
The tomato plants were grown in a greenhouse located at Krizevci, Croatia. The
cultivars Efialto, Heman, and Maxifortwere used as rootstocks, while Tamaris
was used as scion. Grafting resulted in increase of number of marketable fruits per
plant by 30%. Content of total vitamin C and total phenolics significantly decreased
after grafting. The concentration of total extractable phenolics in tomatoes ranged from
287.1 to 977.4 mg gallic acid equivalents (GAE) kg1 fresh weight, whereas lycopene
content ranged from 11.44 to 60.99 mg kg1 fresh weight. Antioxidant activities
determined by 1,1-diphenyl-2 picrylhydrazyl (DPPH) method of grafts were
significantly different compared to their respective rootstocks. The overall results
showed that tomato grafting on suitable rootstocks has positive effects on the
cultivation performance, but decreases nutritional quality of tomatoes.



JOURNAL 4:
ANTIOXIDANT AND ANTI-INFLAMMATORY ACTIVITY OF
ETHANOLIC EXTRACT OF BETA VULGARIS LINN. ROOTS
[4]

The present study deals with evaluation of antioxidant and anti-inflammatory activity
of ethanolic extract of Beta Vulgaris roots. The ethanolic extract was subjected to
screen for antioxidant activity using DPPH radical scavenging method. The anti-
inflammatory activity was carried out by using carageenan induced rat paw edema
method. The tested extract of different dilutions in range 200 g/ml to 1000 g/ml
shows activity in range of 4.34% to 18.55%. The extract shows prominent anti-
inflammatory activity as compared to that of standard (Ibuprofen gel). The extract
shows good anti-inflammatory activity on carrageenan induced rat paw edema
method.


JOURNAL 5:
ANTIOXIDANT ACTIVITY OF EXTRACTS OF WHITE CABBAGE
AND SAUERKRAUT
[5]


Phenolic compounds were extracted from white cabbage and sauerkraut using 80%
aqueous methanol. The extract of sauerkraut was characterized by a higher content of
total phenolics (8.25 mg/g) than that of white cabbage (5.72 mg/g). Phenolic
compounds present in extracts showed antioxidant and antiradical properties
investigated using the Total Antioxidant Capacity (TAC) method, DPPH radical
scavenging activity and reducing power. The total antioxidant capacity of the
sauerkraut extract (0.031 mmol Trolox/g) was stronger than that of white cabbage
(0.025 mmol Trolox/g). The extract of white cabbage exhibited a slightly stronger
ability to scavenge DPPH radical compared to that of sauerkraut. Its reducing power
was also stronger. Results of the HPLC analysis indicate the metabolism of phenolic
compounds during the fermentation of white cabbage.

JOURNAL 6:
ANTIOXIDANT ACTIVITY OF PEPPERS (CAPSICUM ANNUUM L.)
EXTRACTS AND CHARACTERIZATION OF THEIR PHENOLIC
CONSTITUENTS
[6]

The aim of this study was to characterize the phenolic constituents and evaluate the
antioxidant activity of five pepper (Capsicum annuum L.) cultivars harvested in the
same season, geographic area and climatic conditions. Phenols, flavonoids and
ascorbic acid of Anaheim, Bell, Caribe, Jalapeno and Serrano peppers were quantified,
and antioxidant activity of their extracts were evaluated by the method of radical
scavenging of DPPH and ABTS+. It was found that Serrano pepper had the highest
ascorbic acid content, followed by Bell and Caribe, whereas the lowest values were
found in Jalapeno and Anaheim. The highest contents of phenolic compounds were in
Caribe and Bell peppers. The total flavonoid contents ranged from 25.38 3.44
(Anaheim) to 60.36 9.94 mg QE/100g fw (Caribe). The Bell and Caribe extracts
showed the highest(p<0.05) stabilization of ABTS+. The highest oxidation inhibition
percentage for radical DPPH was observed in Caribe extract, coinciding with the
highest levels of gallic acid, chlorogenic acid, epicatechin, rutin, luteolin, resveratrol
(r 0.85) and ascorbic acid. In conclusion, among the pepper cultivars studied, Caribe
and Bell showed to have the best antioxidant properties and can be suggested as
preferable for human consumption.



JOURNAL 7:
ANTIOXIDANT POTENTIAL OF BELL PEPPER (CAPSICUM ANNUM L.)-A
REVIEW
[7]

The interests in the consumption of pepper fruits (Capsicum annum L.) is, to a large
extent due to its content of bioactive compounds and their importance as dietary
antioxidants. Peppers are used as a colourant, flavourant, and/or as a source of
pungency. Peppers can be used fresh, dried, fermented, or as an oleoresin extract. It
has both nutritional and nutraceutical importance. It contains an anticoagulant that
helps prevent the blood clots that can cause heart attacks. Bell Pepper is good source
of vitamin C. The benefits resulting from the use of natural products rich in bioactive
substances has promoted the growing interest of food industries. Among the
antioxidant phytochemicals, polyphenols deserve a special mention due to their free
radical scavenging properties. Antioxidant compounds and their antioxidant activity
in 4 different colored (green, yellow, orange, and red) sweet bellpeppers (Capsicum
annuum L.) were investigated.The free radical scavenging abilities of peppers
determined by the 2, 2~-diphenyl-1-picrylhydrazyl (DPPH) method. Natural
antioxidants are preferred because synthetic antioxidants are considered carcinogenic.
Antioxidants present in the (Capsicum annuum L.), protect the food or body from
oxidative damage induced by free radicals and reactive oxygen.
JOURNAL 8:
NUTRITIONAL CONTENT AND ANTIOXIDANT PROPERTIES OF PULP
WASTE FROM DAUCUS CAROTA AND BETA VULGARIS
[8]

This study reports the chemical composition and antioxidant potential of pulp waste
from two vegetables, carrot (Daucus carota) and beetroot (Beta vulgaris). Different in
vitro assays used for determining antioxidant potential of extracts of pulp wastes were:
2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging capacity, reducing power
and total antioxidant activity by hosphomolybdenum method. Total polyphenols,
tannins and antioxidative components such as vitamin C, total carotenoids and -
carotene were analysed in the samples. The moisture content of samples ranged from
79 - 84%. The protein content was high in beetroot (13.23 mg/100g) and low in carrot
(6.21mg/100g). Total polyphenols were higher in methanol extracts of samples (220-
250 mg/100g) compared to ethanol and aqueous extracts. The antioxidant activity
determined by the DPPH method exhibited 40% and 78% activity in methanol extracts
of carrot and beetroot pulp waste (20 mg) respectively. Overall, the results suggest that
carrot and beetroot pulp wastes can be exploited for their nutrients and antioxidant
components and used for value addition in food formulations. Hence, these results
pave the way for utilisation of bio-wastes from the food industry.

JOURNAL 9:

ANTIOXIDANT ACTIVITY OF PLANT EXTRACTS CONTAINING PHENOLIC
COMPOUNDS
[9]


The antioxidative activity of a total of 92 phenolic extracts from edible and nonedible
plant materials (berries, fruits, vegetables, herbs, cereals, tree materials, plant sprouts,
and seeds) was examined by autoxidation of methyl linoleate. The content of total
phenolics in the extracts was determined spectrometrically according to the Folin-
Ciocalteu procedure and calculated as gallic acid equivalents (GAE). Among edible
plant materials, remarkable high antioxidant activity and high total phenolic content
(GAE > 20 mg/g) were found in berries, especially aronia and crowberry. Apple
extracts (two varieties) showed also strong antioxidant activity even though the total
phenolic contents were low (GAE < 12.1 mg/g). Among nonedible plant materials,
high activities were found in tree materials, especially in willow bark, spruce needles,
pine bark and cork, and birch phloem, and in some medicinal plants including heather,
bog-rosemary, willow herb, and meadowsweet. In addition, potato peel and beetroot
peel extracts showed strong antioxidant effects. To utilize these significant sources of
natural antioxidants, further characterization of the phenolic composition is needed.

JOURNAL 10:

ANTIOXIDANT ACTIVITY OF CAULIFLOWER (BRASSICA OLERACEA L.)

[10]

Recently, a number of studies on the health benefits associated with fruits, vegetables,
herbs and spices demonstrated that they possess potent antioxidant, anti-inflammatory,
anti-mutagenic, and anti-carcinogenic activity. The potential antioxidant activity of
water and ethanol extracts of cauliflower (Brassica oleracea L.) were investigated to
evaluate their potential value as a natural ingredient for foods or cosmetic application.
In this study antioxidant activity was measured by 2,2'-azino-bis(3-
ethylbenzthiazoline-6-sulfonic acid) (ABTS) radical scavenging, 1,1-diphenyl-2-
picryl-hydrazyl free radical (DPPH) scavenging, N,Ndimethyl- p-phenylenediamine
dihydrochloride (DMPD) radical scavenging, superoxide anion (O2 ) radical
scavenging, totalantioxidant activity, reducing activity using Fe+3-Fe+2
transformation and CUPRAC assays, hydrogen peroxide (H2O2) scavenging, and
ferrous metal chelating activity assays. The water extract of cauliflower (WEC) and
ethanol extract of cauliflower (EEC), as antioxidants, neutralized the activity of
radicals and inhibited the peroxidation reactions of linoleic acid emulsion. Total
antioxidant activity was measured according to the ferric thiocyanate method. -
Tocopherol and trolox, a water-soluble analogue of tocopherol, were used as the
reference antioxidant compounds. WEC and EEC showed 88.6% and 80.1% inhibition
of lipid peroxidation oflinoleic acid emulsion, respectively, at the concentration of 30
g ml1. On the other hand, at the same concentration, the standard antioxidants -
tocopherol and trolox exhibited 68.1.4% and 81.3% inhibition of peroxidation of
linoleic acid emulsion, respectively. In addition, WEC and EEC had effective DPPH,
ABTS+, DMPD+, and superoxide anion radical scavenging, hydrogen peroxide
scavenging, total reducing power, and metal chelating of ferrous ion activity. Also,
those various antioxidant activities were compared to -tocopherol and trolox as
references antioxidants.

JOURNAL 11:

EFFECT OF DRYING METHOD AND EXTRACTION SOLVENT ON THE TOTAL
PHENOLICS AND ANTIOXIDANT ACTIVITY OF CAULIFLOWER (BRASSICA
OLERACEA L.) EXTRACTS
[11]


Plants, being a rich source of medicinally important compounds such as antioxidants,
have chemo-preventive role against the risk of oxidative stress-related diseases. There
has been much interest in fruits and vegetable rich diets as a natural source of
antioxidants and functional ingredients. As well as targeting plants high in antioxidant
activity it is also important to optimize extraction parameters. Four extracting solvents,
methanol, ethanol, aqueous methanol (80% v/v) and aqueous ethanol (80% v/v) were
evaluated for their efficacy to extract antioxidants from cauliflower that had undergone
different drying processes namely air-drying, sun-drying and oven-drying. There was
a significant difference (P < 0.05) in the extracting ability of each of the solvents. The
aqueous solvents were superior in their ability to extract the antioxidants and aqueous
methanol was significantly more efficient than aqueous ethanol. This result was
consistent across a number of parameters including extraction yield, total phenolic
content and antioxidant activity. Furthermore, the samples drying process prior to
extraction, also significantly influenced (P < 0.05) the extraction yield. Oven dried
(40
o
C) cauliflower had the highest yield of extractable antioxidants while air dried
(ambient, approx. 25
o
C) had the lowest. Again, there was excellent correlation
between extraction yield, antioxidant activity and total phenolic content.
JOURNAL 12:
ANTIOXIDANT AND ANTIARTHRITIC POTENTIAL OF CORIANDER
(CORIANDRUM SATIVUM L.) LEAVES
[12]

Present investigation was undertaken to assess the antioxidant and antiarthritic
activities of coriander (Coriandrum sativum L.) leaves in osteoarthritis patients.
Methods: The antioxidant and antiarthritic activities of coriander (C sativum L.) leaves
were assessed in vivo by the administration of coriander leaf powder (5 g/day) to
selected osteoarthritis patients for 60 days and by the estimation of a number of
biochemical and clinical parameters before and after the administration of coriander
leaves and by comparing with that of untreated patients.Results: Oxidative stress as
shown by increased lipid peroxidation, increased activity of catalase (CAT) in
erythrocytes, decreased serum b carotene and vitamin C observed in arthritis patients
was countered by coriander leaves in the treated group. In addition, increased activities
of erythrocyte antioxidant enzyme i.e. glutathione-S-tranferase (GST) and reduced
glutathione (GSH) content and a significantly decreased activity of alkaline
phosphatase, erythrocyte sedimentation rate (ESR) and increased serum calcium levels
observed in the treated osteoarthritis patients support the efficacy of the leaves.
Conclusions: Coriander leaves significantly influenced almost all the parameters in
arthritis patients without any detrimental effects by virtue of a number of
phytochemicals, vitamins and minerals present in the leaves having therapeutic effects.
The antioxidant and antiarthritic activities exhibited by the leaves are a result of the
synergistic action of the bioactive compounds present in the leaves.

JOURNAL 13:
ANTIOXIDANT ACTIVITY OF THE EXTRACTS OF SOME COWPEA (VIGNA
UNGUICULATA (L) WALP.) CULTIVARS COMMONLY CONSUMED IN
PAKISTAN
[13]

The present investigation has been carried out to determine the antioxidant activity of
the methanolic extracts obtained from four cultivars of cowpea (Vigna unguiculata (L)
Walp.) seeds. Phenolic compounds present in the extracts showed the antioxidant and
antiradical properties when investigated using a linoleic acid peroxidation model,
FRAP, ORAC and TRAP assays, as well as DPPH, hydroxyl, nitric oxide and
superoxide radical scavenging activity. The HPLC analysis of the cowpea extracts
showed the presence of neochlorogenic acid, chlorogenic acid and caffeic acids. The
results indicated that methanolic extract of the cowpea resembled in the
aforementioned activities those from other leguminous seeds and pulses. Phenolic
constituents contained in cowpea may have a future role as ingredients in the
development of functional foods.

JOURNAL 14:
IN VITRO ANTIOXIDANT ACTIVITY OF HYDROALCOHOLIC EXTRACT OF
CYAMOPSIS TETRAGONOLOBUS
[14]


The present investigation was conducted to investigate the in-vitro activity of hydro
alcoholic extract of seeds of Cyamopsis tetragonolobus by using DPPH radical
scavenging activity, nitric oxide radical scavenging activity, hydrogen peroxide
radical scavenging activity, hydroxyl radical and reducing power activity.The results
were expressed as IC50 value. Significant results were obtained in the estimated
parameter. Thus, indicating that the seeds of Cyamopsis tetragonolobus have
significant anti-oxidant activity.

JOURNAL 15:
PHYTOCHEMICAL SCREENING AND ANTIOXIDANT POTENTIAL OF
PRAECITRULLUS FISTULOSUS
[15]


The study of free radicals and antioxidants in biology is producing medical revolution
that promises a new age of health and disease management. The present study was
performed to evaluate antioxidant effect of petroleum ether and methanolic extract of
Praecitrullus fistulosus against free radical damage by standard method as DPPH (1,1-
diphenyl-2-picrylhydrazyl) free radical model. Results indicate that fruits possess
varying degree of antioxidant activity when compared with standard ascorbic acid.
The IC50 of pet-ether extract is 182g/ml and ethanol extract is 202g/ml.

JOURNAL 16:
ANTIOXIDANT AND FREE RADICAL SCAVENGING PROPERTIES OF
TWELVE TRADITIONALLY USED INDIAN MEDICINAL PLANTS
[16]
The methanolic crude extracts of 12 traditionally used Indian medicinal plants were
screened for their antioxidant and free radical scavenging properties using -tocopherol
and butylated hydroxy toluene (BHT) as standard antioxidants. Antioxidant activity
was measured by ferric thiocyanate (FTC) assay and compared with the thiobarbituric
acid (TBA) method. Free radical scavenging activity was evaluated using diphenyl
picryl hydrazyl (DPPH) radicals. The overall antioxidant activity of Lawsonia inermis
was the strongest, followed in descending order by Ocimum sanctum, Cichorium
intybus, Piper cubeba, Punica granatum, Allium sativum, Delonix regia, Terminalia
chebula, Terminalia bellerica, Mangifera indica, Camellia sinensis, and Trigonella
foenum-graecum.Seven plants, namely Terminalia chebula, Mangifera indica,
Terminalia bellerica, Punica granatum, Ocimum sanctum, Cichorium intybus, and
Camellia sinensis, showed strong free radical scavenging activity with the DPPH
method. Phytochemical analysis of plant extracts indicated the presence of major
phytocompounds, including phenolics, alkaloids, glycosides, flavonoids, and tannins.
The phenolic concentrations in the above plants ranged from 28.66 to 169.67 mg/g of
dry plant extract. A fair correlation between antioxidant/free radical scavenging
activity and phenolic content was observed among 9 plants; however, in 3 plants (Piper
cubeba,Lawsonia inermis and Trigonella foenum-graecum), no such relationship was
observed. The tested plant extracts showed promising antioxidant and free radical
scavenging activity, thus justifying their traditional use.

JOURNAL 17:
ANTIOXIDANT PROPERTIES AND STABILITY OF RAPHANUS SATIVUS
EXTRACTS
[17]


The present investigation was planned to analyze the antioxidant components, activity
and stability of Raphanus sativus leaves. The antioxidant activity of methanol (ME),
ethanol (EE) and water (WE) extracts was determined by DPPH radical scavenging,
reducing power and in vitro inhibition of lipid oxidation. Temperature and pH stability
of extracts was also studied. The extracts showed a dose dependent scavenging of the
free radical; DPPH. EE showed higher activity than ME and WE. The extracts also
showed varying degree of reducing capacity. Further, all the extracts inhibited the
formation of lipid peroxides and their efficacy was in the order of a-tocopherol
(80%)>EE (69%)>WE (70%)>ME (65%). The activity of WE was increased by 3%
after heat treatment. The antioxidant activity of EE at both pH 4 and 7 was higher than
WE and ME. At pH 7 all the extracts were stable and at pH 9 no activity was shown.
Data indicates the potentiality of the Raphanus sativus leaves to utilize as a natural
antioxidant.

JOURNAL 18:
ANTIOXIDANT ACTIVITIES OF THE STANDARDIZED WATER EXTRACT
FROM FRUIT OF PHYLLANTHUS EMBLICA LINN
[18]

Phyllanthus emblica Linn. is widely used in Thai traditional medicine for treatment of
various diseases. The fruit of P. emblica is known as a rich source of vitamin C, and
also contains a mixture of phenolic compounds. In this study, the tandardized water
extract of P. emblica fruit was prepared according to Thai Herbal Pharmacopoeia.
Total polyphenol contents of the extract were equivalent to 34.221.74 g gallic
acid/100g extract. Antioxidant activities of the P. emblica extract were evaluated by
several methods, including DPPH and ABTS+ radical scavenging assays and FRAP
assays. The results showed that the extract has an ability of scavenging radicals
generated by both DPPH and ABTS+. Similar to Trolox, the water extract of P.
emblica fruit also had a ferric reducing property. Additionally, the extract effectively
inhibited H2O2- induced free radical production in human myeloleukemic U937 cells
as measured by 2,7-DCF-DA. The results imply that the fruits of P. emblica are
potential sources of natural antioxidants, which have free radical scavenging activity
and might be useful for hepato-, cyto-, and radio- protection, as well as reducing
oxidative stress in many pathological conditions.

JOURNAL 19:

ANTIOXIDANT STUDY OF GARLIC AND RED ONION: A COMPARATIVE
STUDY
[19]


Garlic (Allium sativum L.) and red onion (Allium cepa L.) are among the most common
ingredients in Malaysian cuisines. These two Allium species are believed to possess
medicinal properties including antioxidants. Accordingly, the aim of this study was to
compare antioxidant level and activities (i.e. at primary and secondary levels) in both
the Allium species collected from markets around Kuantan, Pahang Darul Makmur,
Malaysia. Current results of total phenolic content (TPC) assay indicate that TPC is
higher in red onion (i.e. 53.43 1.72 mg GAE/100g) as compared to garlic (i.e. 37.60
2.31 mg GAE/100g). In addition, EC50 value of garlic is lower than that of the red
onion, showing a higher free radical scavenging activity in garlic than in red onion.
However, the primary antioxidant activities of both the samples are lower than the
standard antioxidant, BHA. Therefore, there is a poor relationship between the TPCs
and the primary antioxidant activities, indicating that the primary antioxidant activities
of both the Allium species are not solely due to the phenolic compounds. For secondary
antioxidant activity, FIC assay shows that at the highest sample concentration of 1.0
mg/mL, red onion has higher ferrous ion chelating effect (i.e. 45.00 1.73%) as
compared to garlic (i.e. 43.29 3.89%). Furthermore, both the Allium samples show
slightly higher ion chelating effect than BHA (i.e. 43.14 1.07%) but lower than
EDTA (i.e. 97.9 0.07%). Overall, the findings of the present study show a negative
relationship between the results of TPC assay, DPPH radical scavenging activity
assay, and FIC assay. To strengthen the validity of the present results and to further
assess the potential of both the Allium species as natural antioxidant sources, more
different assays need to be considered for future work.

JOURNAL 20:
ANTIOXIDANT ACTIVITIES OF DIFFERENT WILD BITTER GOURD
(MOMORDICA CHARANTIA L. VAR. ABBREVIATE SERINGE)
[20]


Antioxidant activity assays were conducted using water (H) and methanolic (M)
extracts of sixteen cultivars from indigenous wild bitter gourd (Momordica charantia
L. var. abbreviata Seringe, MCA) in Taiwan. The scavenging activities against 2,2-
diphenyl-1-picrylhydrazyl (DPPH) and hydroxyl radicals were different among MCA
cultivars and the concentrations of 50% scavenging activity (IC50) for the effective
cultivar was 181 g/mL (H) and 246 g/mL (M) in the former extract and 148 g/mL
(H) and 37 g/mL (M) in the latter. For inhibitory activities against Cu2+-induced
low-density-lipoprotein peroxidation, most MCA cultivars at 4000 g/mL (especially
for M extracts) showed protective activities and were equivalent to 0.8 mM Trolox by
thiobarbituric acid reactive substance assays. These useful data may help promote the
use and further research of MCA as an antioxidant in the health food industry.

JOURNAL 21:

ACTIVITY OF EXTRACT OF PEA AND ITS FRACTIONS OF LOW
MOLECULAR PHENOLICS AND TANNINS
[21]
Phenolic compounds were extracted from pea (Pisum sativum) seeds using 80%
aqueous acetone. Crude extract was applied onto a Sephadex LH-20 column. Fraction
I of low molecular phenolic compounds was eluted from the column by ethanol.
Fraction II of tannins was obtained using water-acetone (1:1; v/v) as mobile phases.
Phenolic compounds present in extract and its fractions showed antioxidant and
antiradical properties investigated using a -carotene-linoleate model system, Total
Antioxidant Activity (TAA) method, DPPH radical scavenging activity and reducing
power. The results of assays performed were the highest when tannins (fraction II)
were tested. For example, TAA of tannin fraction was 2.48 mol Trolox/mg, whereas
the extract and fraction I showed only 0.30 and 0.22 mol Trolox/mg, respectively.
The content of total phenolics in fraction II was found the highest (113 mg/g). The
content of tannins in this fraction determined using the vanillin method and expressed
as absorbance units at 500 nm per 1 g was 368. The HPLC analysis of pea crude extract
showed the presence of such phenolic compounds as vanillic, caffeic,p-coumaric,
ferulic and sinapic acids (after basic hydrolysis), quercetin and kaempherol,
procyanidin B2 and B3.

JOURNAL 22:

EVALUATION OF ANTIOXIDANT ACTIVITY OF BENINCASA HISPIDA FRUIT
EXTRACTS
[22]


The present study was to evaluate antioxidant activity of ethanolic and aqueous extract
of Benincasa hispida (Thunb.) Cogn. fruit for their therapeutic potential. In vitro
antioxidant activity was performed by 1, 1- diphenyl-2-picrylhydrazyl (DPPH) and
Hydrogen peroxide (H2O2). For aqueous extract the scavenging activity of DPPH is
59.7% at the concentration of 200 g/ml and the activity of H2O2 is 20.5% at
concentration of 1000 g/ml. For ethanolic extract The scavenging activity of DPPH
is 77.4% at the concentration of 250 g/ml and the activity of H2O2 is 21.3% at
concentration of 1000 g/ml. The method is compared to standard (ascorbic acid).
Presence of phytochemicals like carbohydrates, proteins and amino acids, flavonoids,
phenolic compounds might contribute to observed antioxidant activity. Benincasa
hispida fruits are potential source of natural antioxidant compounds to replace
synthetic antioxidants.

JOURNAL 23:

EVALUATION OF ANTIOXIDANT ACTIVITY OF THREE COMMON POTATO
(SOLANUM TUBEROSUM) CULTIVARS IN IRAN
[23]


Potato (Solanum tuberosum L.), as a whole food, contains high levels of vitamins and
important antioxidants including phenolic acids, carotenoids and flavonoids. The
objective of this study was to determine the total phenolic content and antioxidant
activities of three common potatoes (Solanum tuberosum) cultivars in Iran i.e.,
Savalan, Agria and Sante.

JOURNAL 24:

THE ANTIOXIDANT ACTIVITY OF TRICHOSANTHES CUCUMERINA
SEEDS
[24]


The present study was to evaluate antioxidant activity of ethanolic and aqueous extract
of Benincasa hispida (Thunb.) Cogn. fruit for their therapeutic potential. In vitro
antioxidant activity was performed by 1, 1- diphenyl-2-picrylhydrazyl (DPPH) and
Hydrogen peroxide (H2O2). For aqueous extract the scavenging activity of DPPH is
59.7% at the concentration of 200 g/ml and the activity of H2O2 is 20.5% at
concentration of 1000 g/ml. For ethanolic extract The scavenging activity of DPPH
is 77.4% at the concentration of 250 g/ml and the activity of H2O2 is 21.3% at
concentration of 1000 g/ml. The method is compared to standard (ascorbic acid).
Presence of phytochemicals like carbohydrates, proteins and amino acids, flavonoids,
phenolic compounds might contribute to observed antioxidant activity. Benincasa
hispida fruits are potential source of natural antioxidant compounds to replace
synthetic antioxidants.





JOURNAL 25:

COMPARATIVE ASSESSMENT OF MINERAL CONTENT AND
ANTIOXIDANT PROPERTIES OF SOME CABBAGE VARIETIES
AVAILABLE ON ROMANIAN MARKET
[25]


Cabbage is leafy vegetable that is currently used in human nutrition. In addition, it
contains active compounds with antifungal, antibacterial, and anticancer activity. The
present paper investigates the levels to which selected trace elements accumulate in
various cabbage varieties available on Romanian market as well as the antioxidant
properties of these vegetables. Our results revealed that purple cabbage varieties tend
to accumulate higher amounts of Fe, Pb, Zn, Cu, and Ni and display a stronger
antioxidant capacity than the green cabbage varieties.

JOURNAL 26:

REVIEW ON AMORPHOPHALLUS PAEONIIFOLIUS
[26]


Herbs have been used by people for longer than we have been keeping written record.
Originally they were found in the wild, by the gatherers and used for a lot of different
things. They were used to flavour food, as a source of nutrition, as medicines1.
Amorphophallus paeoniifolius known as Elephant foot yam is basically a crop of south
East Asian origin. In India, it is commonly known as "Suran" or "Jimmikand". It grows
in wild form in Philippines, Malaysia, Indonesia and other South East Asian countries.
This tuber is consumed by many people as a food and widely used in many Ayurvedic
preparations. In recent years the popularity of complementary medicine has increased.
Over 50% of all modern drugs are natural product origin and they play an important
role in drug development programs of the pharmaceutical industry. Epidemiological
evidence suggests that dietary factors play an important role in human health and in
the treatment of certain chronic diseases including cancer. The tuber is reported to
have antiprotease activity, CNS depressant activity, analgesic activity, and cytotoxic
activity.

JOURNAL 27:

ANTIOXIDANT ACTIVITY AND HPTLC PROFILE OF LAGENARIA SICERARIA
FRUITS

The fruits of Lagenaria siceraria Standl. (Cucurbitaceae) are widely used for
medicinal and nutritional purposes in Africa. The health promoting ability of the fruits
might be related to antioxidant properties of its constituents. In this study the
antioxidant effect of fresh and dried fruits of L. siceraria was evaluated by
Comparing the 2,2-diphenyl-1,1-picrylhydrazyl (DPPH) radical scavenging and
reducing capacity of ethyl acetate and n-butanol extracts of fresh and dried fruits. The
comparison was further emphasized by high performance thin layer chromatography
(HPTLC) analysis of the extracts so as to relate activities with their chemical profiles.
Results indicated that ethyl acetate (EA) extract of the fresh fruits exhibited higher
DPPH radical scavenging activity than other samples. At 0.01 mg/ml the order of
activity was: EA dried fruits (50.6%) < Bt (n-butanol) fresh fruits (53.3%) < Bt (n-
butanol) dried fruits (64.8%) < EA fresh fruits (68.6%) < Gallic acid (81.8%). A slight
change of activity was observed at 0.1 mg/ml, where the order was; EA dried fruits
(70%) < Bt dried fruits (71.8%) Bt fresh fruits (72%) < EA fresh fruits (81.6%) <
Gallic acid (88.5%). In the reducing capacity assay, Bt fresh fruits extract exhibited
higher reducing power than all test samples. The HPTLC chemical profiles of both
fresh and dried fruit extracts showed some slight differences. The slight differences in
antioxidant activities were justified by the HPTLC chemical profiles of the fruits.
Therefore, taking fresh or dried fruits of L. siceraria may relatively give similar
antioxidant effects. Since the fruits of this plant matures in bulky, then drying, milling
and packing the products under hygiene environment can ensure a constant supply of
antioxidant supplement.

JOURNAL 27:

THE EFFECT OF GRAFTING ON THE ANTIOXIDANT PROPERTIES OF
TOMATO (SOLANUM LYCOPERSICUM L.)
[27]


The use of grafted plants in vegetable crop production is now being expanded greatly.
However, few data are available on the nutritional composition of grafted vegetables
with emphasis on antioxidant properties. Therefore, the major objective of this study
was to evaluate antioxidant components of tomatoes influenced by grafting technique.
The tomato plants were grown in a greenhouse located at Krizevci, Croatia. The
cultivars Efialto, Heman, and Maxifort were used as rootstocks, while Tamaris
was used as scion. Grafting resulted in increase of number of marketable fruits per
plant by 30%. Content of total vitamin C and total phenolics significantly decreased
after grafting. The
concentration of total extractable phenolics in tomatoes ranged from 287.1 to 977.4
mg gallic acid equivalents (GAE) kg1 fresh weight, whereas lycopene content ranged
from 11.44 to 60.99 mg kg1 fresh weight. Antioxidant activities determined by 1,1-
diphenyl-2 picrylhydrazyl (DPPH) method of grafts were significantly different
compared to their respective rootstocks. The overall results showed that tomato
grafting on suitable rootstocks has positive effects on the cultivation performance, but
decreases nutritional quality of tomatoes.



JOURNAL 28:

COMPARATIVE ASSESSMENT OF MINERAL CONTENT AND
ANTIOXIDANT PROPERTIES OF SOME CABAGE VARIETIES AVAILABLE
ON ROMANIAN MARKET
[28]


Cabbage is leafy vegetable that is currently used in human nutrition. In addition, it
contains active compounds with antifungal, antibacterial, and anticancer activity. The
present paper investigates the levels to which selected trace elements accumulate in
various cabbage varieties available on Romanian market as well as the antioxidant
properties of these vegetables. Our results revealed that purple cabbage varieties tend
to accumulate higher amounts of Fe, Pb, Zn, Cu, and Ni and display a stronger
antioxidant capacity than the green cabbage varieties.

JOURNAL 29:

THE EFFECT OF HEATING AND FERMENTING ON ANTIOXIDANT
PROPERTIES OF WHITE CABBAGE
[29]


It is widely believed that natural antioxidants found in food are significantly lost
during processing. Nevertheless, it was recently demonstrated that processed fruits and
vegetables may retain their antioxidant activity. In the present work, the changes in
the overall antioxidant properties as a consequence of fermentation of cabbage and/or
heat treatment of cabbage juices and extracts were studied. Fermentation processes as
well as heat treatment increased the initial values of antioxidant activity. While a
decrease in the antioxidant potential of sauerkraut juice was found for short heat
treatments, a partial recovery of these properties was observed by prolonging heating
periods. The TLC analysis showed that during fermentation and thermal processes,
some substances with reactivity towards FolinCiocalteu reagent, hence with possible
antioxidant activity, were released. We demonstrated that in contrast to common
expectation, typical culinary processing of cabbage increases its antioxidant potency.
The gain in antioxidant activity of heated samples coincided with the formation of both
FC reagent reactive compounds as well as brown early Maillard reaction products.
This information may encourage the consumption of heat processed cabbage,
especially that the release of antioxidants during heating may prevent oxidation of
other food components.

JOURNAL 30:

EFFECTS OF HEAT TREATMENTS ON ANTIOXIDANT CAPACITY AND
TOTAL PHENOLIC CONTENT OF FOUR CULTIVARS OF PURPLE SKIN
EGGPLANTS
[30]


We investigated the effects of three heat treatments on antioxidant capacity and total
phenolic content in purple skinned eggplant fruits. Four cultivars of eggplant: Muang
Lek, Muang Lot Fai, Muang Kan Dam, and Muang Kan Khiao were subjected
to boiling, steaming, and microwaving for 5, 10, and 15 min in each treatment. The
results show that antioxidant capacity (DPPH and ABTS) and total phenolic content
significantly increased in all eggplant cultivars with all cooking methods compared
with those of raw fruits. Fruits microwaved for 10 and 15 min had the highest
antioxidant capacities and total phenolic content in all cultivars. Muang Lot Fai had
the highest antioxidant capacity and total phenolic content of the four cultivars
examined. A highly positive correlation for each heat treatment was found between
antioxidant capacity and total phenolic content.

JOURNAL 31:

EFFECTS OF ROOT EXUDATES AND AQUEOUS ROOT EXTRACTS OF
CUCUMBER (CUCUMIS SATIVUS) AND ALLELOCHEMICALS, ON
PHOTOSYNTHESIS AND ANTIOXIDANT ENZYMES IN CUCUMBER
[31]

The effects of root exudates and aqueous root extracts of cucumber (Cucumis sativus)
and allelochemicals on root antioxidant enzymes and leaf photosynthesis, transpiration
and stomatal conductance were investigated in cucumber. Cucumber seedlings were
incubated in solutions containing root exudates at 50 or 100 mg/l, root extracts at
1:100, 1:50, 1:25 and 1:10 (root dry weight:distilled water), and derivatives of benzoic
and cinnamic acids at 0.25 mM, respectively.Root peroxidase and superoxide
dismutase activities increased significantly after exposure to allelopathic agents.
Membrane peroxidation was also enhanced by root exudates, root extracts and some
of the tested acids. Allelopathic agents reduced leaf stomatal conductance from 1.28
mol m
-2
s
-1
to 0.020.33 mol m
-2
s
-1
. Leaf transpiration was reduced by 67% by root
exudates at 100 mg/l, by 8595% by root extracts, and by 5187% by tested acids,
respectively. Net assimilate rate was also inhibited by 29% by root exudates at 100
mg/l, by 4983% by root extracts, and by 743% by tested acids, respectively. The
intercellular CO2 concentrations for the treated seedlings were only 4789% of the
control. However, leaf temperature was increased by 24C by the tested agents.

JOURNAL 32:

EFFECTS OF HEAT TREATMENTS ON ANTIOXIDANT CAPACITY AND
TOTAL PHENOLIC CONTENT OF FOUR CULTIVARS OF PURPLE SKIN
EGGPLANTS
[32]


Phytoscreening and the effect of heat treatment on the antioxidant activity were studied
in three medicinal plant parts used mostly by nursing mothers in Southern
Nigeria;Tetrapleura tetraptera, Piper guineense and Xylopia ethiopica. Total Phenols,
Flavonoids and Vitamin C content of these spices were determined at different heating
periods (0, 30,60, and 90mins) at 100oC and the results showed significant Phenol
acumulation in tubers (1554. 54mgkg1, 1555. 01 mgkg1, and 1550. 04 mgkg1
DW) for T. tetraptera, P. guineense and X. ethiopicarespectively at zero heat treatment.
The antioxidant activity was determined by ferric ion reducing/ antioxidant power
(FRAP) assay. Results indicate a significant increases (p 0.05) in FRAP vaalues of
T. Tetraptera aqueous extracts at all monitored times.Similar trend was observed in
heat response for flavonoid content of measured spices.Increase in heat treatment
time gave statistically significant decreases in observed levels of Vitamin C for all
spices studied These suggest that heat treatment could have beneficial effect by
improving antioxidant activity in consumers of these spices but prolonged treatment
could have an inhibitory effect.The results recorded clearly indicate Vitamin C and
total Phenolic content loss due to thermal treatment. Also, high antioxidant properties
expressed by T. Tetraptera expresses a potential to inhibit possible oxidation of LDL
cholesterol.

JOURNAL 32:

IN- VITRO ANTI-OXIDANT ACTIVITY AND FREE RADICAL SCAVENGING
POTENTIAL OF ROOTS OF MALAWIAN TRICHODESMA ZEYLANICUMM
(BURM. F.)
[32]


Aim: To evaluate the antioxidant potential and free scavenging activity of T.
zeylanicum powdered root extract.
Material and Methods: The plant extract (50mg) obtained by soxhlet extraction
method was dissolved separately in 50ml of methanol and the resultant solution
serially diluted to concentrations 0.5, 0.25, 0.125 and
0.0625 mg/ml. The antioxidant potential and free radical scavenging activity were
analysed using reducing power assay and hydrogen peroxide scavenging activity
methods. Phytochemical analysis was performed on the plant extract to detect the
presence of phytoconstituents.
Results and Discussions: Phytochemical screening revealed the presence of
phenolics, alkaloids, saponins, flavonoids and tannins. The flavonoids content were
found to be 6.280.06 mg/gram of the dried extract. The reducing power assay
showed that reducing ability of the extract were significantly increased with
increasing concentration and were higher compared to the standard ascorbic acid.
Methanolic extract of T. zeylanicum also showed good scavenging ability compared
to the standard ascorbic acid. The IC values were found to be 0.122 mg/ml
compared to standard ascorbic acid
0.717mg/ml. At a concentration of 1mg/ml, the scavenging percentages were
74.82 and 48.12 for T. zeylanicum extract and standard respectively.
Conclusion: The result indicates the potential of T. zeylanicum as a source of
antioxidants relevant to wound treatment.

JOURNAL 33:

Quantitative Determination of Total Phenolic Content in Stem Bark and
Leaves Extracts of Madhuca longifolia
[33]


Total phenolic content (TPC) of Madhuca longifolia leaves and barks extracts were
evaluated using the Folin- Ciocalteu method. This phenolics component is responsible
for antioxidant activity. Gallic acid was used as a standard compound and the total
phenols were expressed as mg / g gallic acid equivalents (standard curve equation: y=
1.387x +0.055, R2=0.903). The total phenol varied from 8.290.52 to 32.540.35 mg
GAE /g. The highest concentration of phenols was measured in methanol and aqueous
extracts. Total Phenol Content in plant extracts of the species of Madhuca indica stem
bark and leavesdepends upon the type of extracts and the polarity of solvent used in
the extraction.

JOURNAL 34:

Screening of Total Phenolic and Flavonoid Content in Conventional and
Non-Conventional Species of Curcuma
[34]


The zingiberacea the largest family in zingeberales comprises generally 300 genera
and 1000 species. The present study aim at comparing the TPC and TFC in
conventional and Non-conventional species of curcuma. The TPC of all three species
ranged from 92.300.05 to 260 0.025 mg gallic acid equivalent/g and total flavonoid
content ranged from 22.520.015 to 79.360.01 mg quercetin equivalent/g. The
results of the study highlighted that conventional curcuma species had higher phenolic
and flavonoid content.

JOURNAL 35:

Hydrogen Peroxide Radical Scavenging and Total Antioxidant Activity of
Hawthorn
[35]


The aim of this research is to determine H2O2 radical scavenging and total antioxidant
activity of Crataegus monogyna (hawthorn) water and ethanol extracts of leaves,
flowers and fruits. Hawthorn leaves, flowers, and berries are used in traditional
medicine in the treatment of chronic heart failure, high blood pressure, arrhythmia,
and various digestive ailments, as well as geriatric and antiarteriosclerosis remedies.
In this study, ethanol and water extracts were prepared from powdered C.monogyna
flowers, leaves and fruits. Antioxidant activities were measured by ferric thiocyanate
method, H2O2 radical scavenging assays with UV-Vis spectrophotometer. In
conclusion, C. monogyna flowers, leaves and fruits had H2O2 radical scavenging, total
antioxidant activity, and these antioxidant activities were compared with BHA and -
tocopherol as reference antioxidants. The results of this study show that the water and
ethanol extracts of C. monogyna can be used as easily accessible source of natural
antioxidants and as a possible food supplement or in pharmaceutical industry.

JOURNAL 36:

ANTIOXIDANT ASSAY IN VIVO AND VITRO
[36]


Measurement of oxidative stress in animal tissue and human serum /plasma with help
of various methods modified time to time are presented and can be carried out in
laboratory. Present article highlights some important method of measurement of
oxidative stress, it include enzyme estimation , in vitro methods ,in vivo methods,
some assay related to oxidative damages, screening of antioxidant compounds assay
etc. .merits and demerits of each method /assay.


JOURNAL 37:

Standardized Methods for the Determination of Antioxidant Capacity and
Phenolics in Foods and Dietary Supplements
[37]


Methods available for the measurement of antioxidant capacity are reviewed,
presenting the general chemistry underlying the assays, the types of molecules
detected, and the most important advantages and shortcomings of each method. This
overview provides a basis and rationale for developing standardized antioxidant
capacity methods for the food, nutraceutical, and dietary upplement
industries. From evaluation of data presented at the First International Congress on
Antioxidant Methods in 2004 and in the literature, as well as consideration of potential
end uses of antioxidants, it is proposed that procedures and applications for three
assays be considered for standardization: the oxygen radical abs
orbance capacity (ORAC) assay, the Folin-Ciocalteu method, and possibly the Trolox
equivalent antioxidant capacity (TEAC) assay. ORAC represent a hydrogen atom
transfer (HAT) reaction mechanism, which is most relevant to human biology. The
Folin-Ciocalteu method is an electron transfer (ET) based assay and gives reducing
capacity, which has normally been expressed as phenolic contents. The TEAC assay
represents a second ET-based method. Other assays may need to be considered in the
future as more is learned about some of the other radical sources and their importance
to human biology.





JOURNAL 38:
Seasonal Variations of Phenolic Compounds and Biological Properties in
Sage (Salvia officinalis L.)
[38]


The aim was to investigate the phenolic content, antioxidant capacity, and antibacterial
activity of Dalmatian sage (Salvia officinalis L.) leaves collected during different
vegetation periods. Separation and quantification of the individual phenols were
performed by reversed-phase (RP)-HPLC coupled with a PDA (photodiode array)
detector and using an internal standard, while the contents of total phenols, flavonoids,
flavones, and flavonols were determined spectrophotometrically. The antioxidant
properties of the sage leaf extracts were evaluated using five different antioxidant
assays (FRAP, DPPH,ABTS, Briggs_Rauscher reaction, and b-carotene bleaching).
The antimicrobial activity of the extracts was tested against two Gram-positive
(Bacillus cereus and Staphylococcus aureus) and two Gramnegative (Salmonella
Infantis and Escherichia coli) bacterial reference strains. All extracts were extremely
rich in phenolic compounds, and provided good antioxidant and antibacterial
properties, but the phenophase in which the leaves were collected affected the
phenolic composition of the sage extracts and consequently their biological activity.
The May Extract, the richest in total flavonoids, showed the best antioxidant properties
and the highest antimicrobial activity. Thus, collection of the plants during May seems
the best choice for further use of them in the pharmaceutical and food industry.
JOURNAL 39:

DETERMINATION OF ANTIOXIDANT CAPACITIES OF VEGETABLE OILS
BY FERRIC-ION SPECTROPHOTOMETRIC METHODS
[39]


Two ferric-ion-based total antioxidant capacity methods: 1,10-phenanthroline (Phen)
and ferric reducing antioxidant power (FRAP) were used for determination of
antioxidant capacities (AC) of the acetonic and methanolic extracts of vegetable oils.
The obtained mean Phen and FRAP values for acetonic extracts of olive oils, rapeseed,
rice and four sunflower oils (39.3336.5 and 39.5339.6_mol Fe/100 g) were higher
than for methanolic extracts (22.8307.3 and 23.5300.1_mol Fe/100 g). However,
antioxidant capacities of methanolic extracts of corn oil, blended oils and two
sunflower oils with garden green flowers (56.5312.9 and 53.9306.5_mol Fe/100 g
for Phen and FRAP methods, respectively) were higher than for acetonic extracts of
these oils (54.2249.2 and 52.9244.7_mol Fe/100 g for Phen and FRAP methods,
respectively). There is a linear and significant correlation between these two analytical
methods (r = 0.9989 and 0.9986 for acetonic and methanolic extracts). Also, total
phenolic compounds (TPC) in the studied oils correlated with their antioxidant
capacities determined by Phen and FRAPmethods (r = 0.9012, 0.7818 and 0.8947,
0.7830 for acetonic and methanolic extracts, respectively). The comparable precision
(R.S.D. = 0.84.6%, 0.94.9% and 0.74.0%, 0.64.0% for acetonic and methanolic
extracts, respectively) and sensitivity ( = 1.27104, 1.11104 and 2.62104 dm3
mol1 cm1) for the proposed Phen and the modified FRAP methods, demonstrate
the benefit of the Phen method in the routine analysis of antioxidant capacities of
vegetable oils.












































COLLECTION OF VEGETABLE BIOMASS:



















3.COLLECTION OF VEGETABLE BIOMASS:

The vegetable biomass was collected at wholesale vegetable
complex, koyambedu, Chennai.






























PREPARATION OF
VEGETABLE
BIOMASS

















4.PREPARATION OF VEGETABLE BIOMASS:

CLEANING:
The vegetable biomass collected is cleaned well with
distilled water for the removal of dirt and other unwanted materials.


DRYING:

The well cleaned bio mass is cut to small pieces for quick
drying and subjected to dying in hot air oven at 40C for about 48 hours.

























PREPARATION OF EXTRACT




















5PREPARATION OF EXTRACT:

EXTRACTION PROCESS:

Extraction of antioxidant compounds from vegetable biomass was
carried out with methanol-water solutions in an orbital shaker. The solid - liquid ratio
was set at 1:10 (w/v) for all experiments. Temperature, time and solvent concentration
varied depending on the experimental stage. In a first stage, kinetic experiments were
performed to analyse and model the influence of extraction time (from 10 to 30 min)
and temperature (30, 40 50 and 55C) on the antioxidant extraction process. Methanol
concentration was kept constant at 50% (volume fraction), the central point of the
range analysed in the next stage (20 - 90%).The extract was filtered. The methanol
extract obtained from the extraction of dried vegetable biomass with methanol was
vacuum evaporated for the recovery of methanol from the extract- methanol mixture.
The methanol free extract was vacuum dried at temperature range of 30C and 60C
at constant pressure of 400mmHg.The extraction yield was calculated using the
formula

% Yield = (Weight of dried sample/Weight of original sample) x 100



Table 5.1: Extraction Parameters

SAMPLE
NO.
CONCENTRATION
(% volume fraction)
TIME
(mins)
TEMP
(C)
1. 20 10 30
2. 60 30 55
3. 90 10 40
4. 60 10 50
5. 20 10 50
6. 90 10 30




RECOVERY OF SOLVENT:

The recovery of the solvent (Methanol) was done by the
vacuum distillation.

VACUUM DISTILLATION:
Vacuum distillation is distillation at a reduced pressure. Since the boiling point of a
compound is lower at a lower external pressure, the compound will not have to be
heated to as high a temperature in order for it to boil. Vacuum distillation is used to
distill compounds that have a high boiling point or any compound which might
undergo decomposition on heating at atmospheric pressure. The vacuum is provided
either by a water aspirator or by a mechanical pump. You can see photos of a
fractional distillation set-up here. Always check for star cracks in the flasks before
beginning a vacuum distillation.

PROCEDURE:
A double mouth round bottom flask and another single mouth round bottom flask
were connected to the condenser mouths.
The water supply tubes to the condenser is made properly.
The feed is charged in the double mouth round bottom flask.
The heating mantle is arranged to heat the feed.
The water supply for cooling is provided.
The mantle is turned on and the feed starts to heat up.
Now the solvent (Methanol) which has the boiling point of 64.7 C is evaporated
first as the extract has the temperature high than that of methanol.
Thus the solvent is recovered from the extract.





Table 5.2: Recovery of solvent by Vacuum Distillation


STORAGE:
The methanol free extract is kept in a refrigerator at a maintained
temperature between 2C and 4C till it is used for the next process.

DRYING:



The methanol free extract is now dried by vacuum drier at temperature of 40C for
about 20 hours for increasing the concentration of the plant extract by drying all of the
residual moisture and methanol present in it.















Sample
No.
Feed
(ml)
Distillate
(ml)
Extract
(ml)
Time
(min)
Temp
(C)
1. 44 12 30 75 30
2. 22 05 12 60 30
3. 25 05 17 60 30
4. 27 09 14 60 30
5. 31 07 20 60 60
6. 50 20 27 60 60










METHODS ADOPTED
FOR THE ANALYSIS
OF THE EXTRACTS




















6.METHODS ADOPTED FOR THE ANALYSIS OF THE
EXTRACTS

PROCEDURE

6.1DETERMINATION OF TOTAL PHENOLICS CONTENTS:
The amount of total phenolics in extracts was
determined with the Folin- Ciocalteu reagent. Gallic acid was used as a standard and
the total phenolics were expressed as mg/g gallic acid equivalents (GAE).A dilute
extract of each plant extract (0.5ml of 1:5g/ml) or gallic acid is used as standard was
mixed with Folin- Ciocalteau reagent (2.5ml,1:10 diluted with distilled water) and
aqueous 7.5% of Na
2
CO
3
(2ml ,1M). The mixture was allowed to stand for 10min
and absorbance was measured by calorimetrically at 760nm the standard curve was
prepared using 0.002,0.004,0.006,0.008,0.010,0.012 mg/ml of solutions of gallic
acid in methanol- water solutions. All determination was performed in triplicate.
The Folin-Ciocalteu reagent is sensitive to reducing compounds including
polyphenols, thereby producing a blue colour chromogens upon reaction. This blue
colour is measured spectrophotometrically. Thus total phenolic content can be
determined by the formula,
C = c.V/M

C total phenolic content (mg/gm)
c Concentration of gallic acid(mg/ml)
V- volume of extract in assay(ml)
M Mass of pure plant methanolic extarce(gm)

Advantages in the usage of Folin-Ciocalteau Method:

F-C method is operationally simple,
Assay is reproducible and
Convenient for assessment of dietary antioxidant capacity since the reagent is,
commercially available,
the procedure is rather standardized,
And the absorption of the product at a long wavelength minimizes
interferences from the Sample matrix.
But, it is performed in aqueous phase, thus it is not applicable for lipophilic
compounds/matrices.



6.2REDUCING POWER CAPACITY (OYAIZU- 1986):

PRINCIPLE:
The antioxidant present in the sample reduced the oxidant probe and the respective
product interacted with some colouring agents to form a coloured complex. In this
method, the antioxidants reduced the Fe
3+
to Fe
2+
.This ion then conjugated with the
ferricyanide ion to form a Prussian blue coloured product, which is
spectrophotometrically measured at
700nm.The change in optical density is directly related to the total reducing power
of the electron donating antioxidants available in the reaction mixture.

Fe
3+
+ antioxidant Fe
2+
oxidized antioxidant
Fe
2+
+Fe(CN)6
3 -
Fe[Fe(CN)6]
-

PROCEDURE:
The extract is diluted with methanol at 1ml: 5ml. From
the above different concentrations (1, 3, 5, 7,9,11 ml) were pipetted out. 2.5 ml of 1%
potassium ferric cyanide and 2.5 ml of 0.2 M sodium phosphate buffer. The mixture
will be incubated at 50C for 20 minutes, and the reaction is terminated by the addition
of 2.5 ml of 10% (W/V) trichloroacetic acid, followed by centrifugation at 3000 rpm
for 10 minutes. 2.5 ml of the supernatant upper layer will be mixed with 2.5 ml distilled
water and 0.5 ml of 0.1% ferric chloride, which turns the reaction mixture for extract
to green colour and absorbance will be measured at 700 nm against blanks that
contained distilled water and phosphate buffer which turns Prussian blue. Increased
absorbance indicated increased reducing power of the sample. The control contained
all the reagents except the sample. Ascorbic acid is used for comparison.

6.3DETERMINATION OF THE TOTAL FLAVONOID CONTENT
(TFC):

Aluminium chloride method was used for flavonoid determination. In this method
Quercetin was used as standard and flavonoid contents were measured as Quercetin
equivalent. For this purpose, the calibration curve of Quercetin was drawn. 100l of
standard or extract solution (1, 3, 5, 7,9,11 mg/l) was taken into 10ml volumetric flask,
containing 4ml of distilled water. 300l of 5% NaNO2 added to the flask. After 5min,
100l 10% AlCl3 was added to the mixture. At the 6th min add 2ml of 1M NaOH was
added and volume made up to 10ml with distilled water. The absorbance was noted at
510nm using UV-Visible spectrophotometer.

6.4 HYDROGEN PEROXIDE SCAVENGING CAPACITY:

The scavenging activity of extract
towards hydrogen peroxide radicals was determined by the modified method of
Dehpour. Solution of hydrogen peroxide (40Mm) was prepared in phosphate buffer
pH 7.4 and its concentration was determined by measuring the absorbance at 560nm
using UV spectrophotometer. 0.1mg/ml of the extract was added to hydrogen
peroxide solution and absorbance measured at 560nm using UV spectrophotometer
against a blank solution containing phosphate buffer without hydrogen peroxide. The
percentage of hydrogen peroxide scavenging by the extract and standard compound
was calculated using the given formula:

Percentage scavenged = 1- As/Ac x100

Where,
Ac was the absorbance of the control (without extract) at 560nm;
Ac was the absorbance in the presence of the extract at 560nm. The experiment
was repeated in triplicate.

6.5 PHYTOCHEMICAL SCREENING:

The dried methanolic extract was used to analyze
qualitatively various phytoconstituents such as flavonoids, phenolic compounds and
tannin using standard procedures.

TEST FOR FLAVONOIDS:

5ml of 95% ethanol was added to aqueous methanol extract,
followed by few drops of concentrated hydrochloric acid and 0.5g magnesium
turnings. Pink colour indicated the presence of flavonoids.

TEST FOR PHENOLICS:

Neutral ferric chloride was added to the aqueous
methanol extract. Appearance of bluish green colour indicated the presence of
phenolic compounds.

KOH TEST:
1 mL of freshly prepared 10% KOH is added to 1 mL of the extract. A
dirty white precipitate indicates the presence of tannins.

FROTHING TEST FOR SAPONINS:
Water extract is obtained by boiling on a water bath.
The extract is transferred into a test tube and shaken vigorously then is left to stand for
10 minutes and the result is noted. A thick persistent froth indicates saponins.











EQUIPMENTS USED FOR THE
PREPARATION OF THE EXTRACT




















7.EQUIPMENTS USED FOR THE PREPARATION OF THE EXTRACT:

HOT AIR OVEN:

The hot oven used in our project is used to dry the cleaned vegetable
biomass. Hot air ovens are electrical devices used in sterilization. They were originally
developed by Pasteur. The oven uses dry heat to sterilize articles. Generally, they can
be operated from 50 to 300 C (122 to 572 F). There is a thermostat controlling the
temperature. These are digitally controlled to maintain the temperature. Their double
walled insulation keeps the heat in and conserves energy, the inner layer being a poor
conductor and outer layer being metallic. There is also an air filled space in between
to aid insulation. An air circulating fan helps in uniform distribution of the heat. These
are fitted with the adjustable wire mesh plated trays or aluminium trays and may have
an on/off rocker switch, as well as indicators and controls for temperature and holding
time. The capacities of these ovens vary. Power supply needs vary from country to
country, depending on the voltage and frequency used. Temperature sensitive tapes or
other devices like those using bacterial spores can be used to work as controls, to test
for the efficacy of the device in every cycle.

ADVANTAGES AND DISADVANTAGES:
They do not require water and there is not much
pressure build up within the oven, unlike an autoclave, making them safer to work
with. This also makes them more suitable to be used in a laboratory environment. They
are much smaller than autoclaves but can still be as effective. They can be more rapid
than an autoclave and higher temperatures can be reached compared to other means.
As they use dry heat instead of moist heat, some organisms like prions, may not be
killed by them every time, based on the principal of thermal inactivation by oxidation.

USAGE:
A complete cycle involves heating the oven to the required temperature,
maintaining that temperature for the proper time interval for that temperature, turning
the machine off and cooling the articles in the closed oven till they reach room
temperature. The standard settings for a hot air oven are:
1.5 to 2 hours at 160 C (320 F)
6 to 12 minutes at 190 C (374 F)
The time required to preheat the chamber before beginning the sterilization cycle. If
the door is opened before time, heat escapes and the process becomes incomplete.
Thus the cycle must be properly repeated all over.

These are widely used to sterilize articles that can withstand high temperatures and not
get burnt, like glassware and powders. Linen gets burnt and surgical sharps lose their
sharpness.

VACUUM DRIER:

Batch vacuum dryers are substantially the same as tray dryers, except
that they operate under a vacuum, and heat transfer is largely by conduction or by
radiation. The trays are enclosed in a large cabinet, which is evacuated. The water
vapour produced is generally condensed, so that the vacuum pumps have only to deal
with non-condensable gases. Another type consists of an evacuated chamber
containing a roller dryer.
Vacuum drying is a process in which materials are dried in a reduced pressure
environment, which lowers the heat needed for rapid drying. It does not take place in
a true vacuum, despite the name. Devices used for this process are known as vacuum
driers, and can vary in size from small units designed to fit on kitchen counters to
massive rooms which are used to handle things like timber products.
Drying involves reducing the moisture in an object, and is done in environments where
the air is drier than the object being dried, which encourages moisture to evaporate
out. This is often accomplished with heat to reduce the humidity of the air, but in a
vacuum drier, the temperature does not need to be as high, and the drying is often
faster. It's also possible to achieve a very high level of dryness, which may be desirable
in some cases.
One advantage to vacuum drying is that it conserves energy. Less energy is needed for
drying, cutting down on the economic and environmental costs associated with drying
a product for storage, sale, or other purposes. This process also tends to work faster
than other drying methods, cutting down on processing time, which can be important
in some facilities where products are being moved through quickly.
Another advantage of drying materials in this way is a less damaging drying process.
Some materials can experience problems at high temperatures, such as developing
hard, leathery crusts from heat exposure during the drying process. Vacuum drying
tends to retain the integrity of the original item without damaging it with heat. For
foods, this can be valuable, as other drying processes can degrade quality and make
the food less appealing.



Using vacuum drying equipment also reduces risks to workers. With other types of
drying equipment, there are vented fumes and particles which can make people sick
or which force people to wear protective garments. With a vacuum dryer, ventilation
does not occur, and workers around the drier are safer. It's also possible to recover the
precipitated moisture collected during the drying for further use or study.



































EQUIPMENTS USED FOR THE ANALYSIS OF
THE EXTRACT





















7 EQUIPMENTS USED FOR THE ANALYSIS OF THE EXTRACT:

UV Vis spectroscopy:


Ultravioletvisible spectroscopy or ultraviolet-visible spectrophotometry (UV-Vis or
UV/Vis) refers to absorption spectroscopy or reflectance spectroscopy in the
ultraviolet-visible spectral region. This means it uses light in the visible and adjacent
(near-UV and near-infrared (NIR)) ranges. The absorption or reflectance in the visible
range directly affects the perceived color of the chemicals involved. In this region of
the electromagnetic spectrum, molecules undergo electronic transitions. This
technique is complementary to fluorescence spectroscopy, in that fluorescence deals
with transitions from the excited state to the ground state, while absorption measures
transitions from the ground state to the excited state.

PRINCIPLE OF ULTRAVIOLET-VISIBLE ABSORPTION:
Molecules containing -electrons or non-bonding electrons (n-electrons)
can absorb the energy in the form of ultraviolet or visible light to excite these electrons
to higher anti-bonding molecular orbitals. The more easily excited the, the longer the
wavelength of light it can absorb.

DIGITAL PHOTOCOLORIMETRY:
A colorimeter is a device used in colorimetry. In
scientific fields the word generally refers to the device that measures the absorbance
of particular wavelengths of light by a specific solution. This device is most commonly
used to determine the concentration of a known solute in a given solution by the
application of the Beer-Lambert law, which states that the concentration of a solute is
proportional to the absorbance.


CONSTRUCTION:
Wavelength selection,
Printer button,
Concentration factor adjustment,
(UV mode selector (Deuterium lamp),
Readout,
Sample compartment,
Zero control (100% T),
Sensitivity switch,
ON/OFF switch

Essential parts of a colorimeter are:
a light source (often an ordinary low-voltage filament lamp)
an adjustable aperture
a set of coloured filters
a cuvette to hold the working solution
a detector (usually a photo resistor) to measure the transmitted light
a meter to display the output from the detector

FILTERS:
Changeable optics filters are used in the colorimeter to select the wavelength of
light which the solute absorbs the most, in order to maximize accuracy. The usual
wavelength range is from 400 to 700 nanometers (nm). If it is necessary to operate in
the ultraviolet range (below 400 nm) then some modifications to the colorimeter are
needed. In modern colorimeters the filament lamp and filters may be replaced by
several light-emitting diodes of different colors.
CUVETTES:
In a manual colorimeter the cuvettes are inserted and removed by hand.

OUTPUT:

The output from a colorimeter may be displayed by an analogue or digital meter
and may be shown as transmittance (a linear scale from 0-100%) or as absorbance (a
logarithmic scale from zero to infinity).

CENTRIFUGE:

A centrifuge is a piece of equipment, generally driven by an electric motor (or,
in some older models, by hand), that puts an object in rotation around a fixed axis,
applying a force perpendicular to the axis. A centrifuge is also used to separate the
components of blood in blood banks. The centrifuge works using the sedimentation
principle, where the centripetal acceleration causes denser substances to separate out
along the radial direction (the bottom of the tube). By



the same token lighter objects will tend to move to the top (of the tube; in the rotating
picture, move to the centre).
USES:
ISOLATING SUSPENSIONS:
Simple centrifuges are used in chemistry, biology, and biochemistry for
isolating and separating suspensions. They vary widely in speed and capacity. They
usually comprise a rotor containing two, four, six, or many more numbered wells
within which the samples, contained in centrifuge tubes, may be placed.
ISOTOPE SEPARATION:
Other centrifuges, the first being the Zippe-type centrifuge, separate
isotopes, and these kinds of centrifuges are in use in nuclear power and nuclear weapon
programs.
Gas centrifuges are used in uranium enrichment. The heavier isotope of uranium
(uranium-238) in the uranium hexafluoride gas tends to concentrate at the walls of the
centrifuge as it spins, while the desired uranium-235 isotope is extracted and
concentrated with a scoop selectively placed inside the centrifuge. It takes many
thousands of centrifugations to enrich uranium enough for use in a nuclear reactor
(around 3.5% enrichment), and many thousands more to enrich it to weapons-grade
(above 90% enrichment) for use in nuclear weapons.
AERONAUTICS AND ASTRONAUTICS:
The 20 G centrifuge at the NASA Ames Research Center
Human centrifuges are exceptionally large centrifuges that test the reactions and
tolerance of pilots and astronauts to acceleration above those experienced in the Earth's
gravity.
The US Air Force at Holloman Air Force Base, New Mexico operates a human
centrifuge. The centrifuge at Holloman AFB is operated by the aerospace physiology
department for the purpose of training and evaluating prospective fighter pilots for
high-g flight in Air Force fighter aircraft.
The use of large centrifuges to simulate a feeling of gravity has been proposed for
future long-duration space missions. Exposure to this simulated gravity would prevent
or reduce the bone decalcification and muscle atrophy that affect individuals exposed
to long periods of freefall.
The first centrifuges used for human research were used by Erasmus Darwin, the
grandfather of Charles Darwin. The first large scale human centrifuge designed for
Aeronautical training was created in Germany in 1933.
GEOTECHNICAL CENTRIFUGE MODELLING:
Geotechnical centrifuge modeling is used for
physical testing of models involving soils. Centrifuge acceleration is applied to scale
models to scale the gravitational acceleration and enable prototype scale stresses to be
obtained in scale models. Problems such as building and bridge foundations, earth
dams, tunnels, and slope stability, including effects such as blast loading and
earthquake shaking.

COMMERCIAL APPLICATIONS:
Centrifuges with a batch weight of up to 2,200 kg per charge are used in the sugar
industry to separate the sugar crystals from the mother liquor.
Standalone centrifuges for drying (hand-washed) clothes usually with a water
outlet.
Centrifuges are used in the attraction Mission: SPACE, located at Epcot in Walt
Disney World, which propels riders using a combination of a centrifuge and a
motion simulator to simulate the feeling of going into space.
In soil mechanics, centrifuges utilize centrifugal acceleration to match soil
stresses in a scale model to those found in reality.
Large industrial centrifuges are commonly used in water and wastewater
treatment to dry sludges. The resulting dry product is often termed cake, and the
water leaving a centrifuge after most of the solids have been removed is called
centrate.
Large industrial centrifuges are also used in the oil industry to remove solids from
the drilling fluid.
Disc-stack centrifuges used by some companies in Oil Sands industry to separate
small amounts of water and solids from bitumen
Centrifuges are used to separate cream (remove fat) from milk; see Separator
(milk).
















RESULTS
&
DISCUSSION:



















8.RESULTS AND DISCUSSION:

TOTAL PHENOLICS CONTENT:

The experiment for the determination of the total Phenolics content was done by the
method discussed earlier and the absorbance values and standard curve of the standard
compound (Gallic acid) is given below,

Table 8.1: Absorbance of standard Compound (Gallic Acid)

Fig 8.1:
Standard curve of Gallic acid



The absorbance curve for the extract is obtained by the determination of calculating
the concentration from equation from the standard curve, y = 62.05x - 0.0211 where
Regression value is (R
2 =
0.9995).The curve obtained for the absorbance of the extract
gives the equation of y = 0.0051x + 0.1241 and has the regression formula of R = 0.92









Table 8.2: Absorbance of the vegetable biomass extract:
y = 62.05x - 0.0211
R = 0.9995
0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0 0.002 0.004 0.006 0.008 0.01 0.012
Linear (ABSORBANCE - I
(mean))
SAMPLE NO. CONCENTRATION
(mg/ml)
ABSORBANCE (MEAN)
Max = 760nm
1 0.002 0.106
2 0.004 0.222
3 0.06 0.355
4 0.008 0.462
5 0.01 0.602
6 0.012 0.742

SAMPLE
NO.
CONCENTRATION
(mg/ml)
ABSORBANCE I
(nm)
1 0.0024 0.132
2 0.0025 0.138
3 0.0026 0.149
4 0.0027 0.152
5 0.0028 0.153
6 0.0029 0.161

Fig 8.2: Curve of obtained for the vegetable biomass extract:



Thus by calculating the total Phenolics content by the formula following values are
obtained which is expressed by mg/g equivalent of gallic acid as given in Tabe 4,which
is expressed graphically.

Table 8.3: Total Phenolics content in Vegetable biomass extract

Sample
No.
Total phenols content
(mg/g)
1. 0.00668
2. 0.01066
3. 0.00025
4. 0.01198
5. 0.04
6. 0.01331









y = 0.0051x + 0.1241
R = 0.92
0
0.05
0.1
0.15
0.2
0 2 4 6 8
ABSORBANCE - I
Linear (ABSORBANCE - I)


Fig 8.3: Total Phenolics content in Vegetable biomass extract in mg/g(GAE)



Thus the graph shows that the Total Phenolics content evaluated from sample no. 5
gives the maximum value of 0.04mg/g GAE.

REDUCING POWER CAPACITY:
The experiment for the determination of the Reducing power
capacity was done by the method discussed earlier and the absorbance values and
standard curve of the standard compound (ascorbic acid) and the extract for the
wavelength 700 nm is given below,

Table 8.4: Reducing power capacity in Vegetable biomass extract

SAMPLE
NO.
CONCENTRATION
(mg/ml)
WAVELENGTH
(nm)
ABSORBANCE(nm)
METHANOL
EXTRACT
ASCORBIC
ACID
1. 1 700 0.938 1.213
2. 3 700 2.057 1.589
3. 5 700 3.879 1.815
4. 7 700 3.329 1.919
5. 9 700 3.618 2.147
6. 11 700 3.729 2.347

At the wavelength of 700 nm, the 3
rd
sample showed the maximum absorbance of
3.879 nm.


0
0.005
0.01
0.015
0.02
0.025
0.03
0.035
0.04
0.045
1. 2. 3. 4. 5. 6.
Total phenols content (mg/g)

Fig 8.4: Curve for the Concentration Vs Absorbance for standard compound (L-
ascorbic acid) and extract.



Fig 8.6: TPC vs. Antioxidant Capacity

The above comparison shows that though the phenolic content values are not much
in the extracts, it has a high antioxidant capacity.

TOTAL FLAVONOID CONTENT (TFC):
The experiment for the determination of the total
flavonoid content was done by the aluminium chloride method which discussed
earlier and the absorbance values and standard curve of the standard compound
(ascorbic acid) and the extract for the wavelength 510 nm is given below,


y = 0.1064x + 1.1999
R = 0.9732
y = 0.2583x + 1.3749
R = 0.677
0
0.5
1
1.5
2
2.5
3
3.5
4
4.5
0 2 4 6 8 10 12
CONCENTRATION Vs ABSORBANCE
0
0.5
1
1.5
2
2.5
3
3.5
4
1 2 3 4 5 6
TPC vs. ANTIOXIDANT CAPACITY
L-ascorbic acid Extract
Table 8.5: Absorbance of Standard compound (Quercetin):











Fig 8.7: Curve of Quercetin:



Table 8.6: Absorbance of Vegetable biomass extract for flavonoid content:

Sample
No.
CONCENTRATION
mg/ml
ABSORBANCE
(nm)
1. 0.00246 0.132
2. 0.00256 0.138
3. 0.00262 0.149
4. 0.00278 0.152
5. 0.0028 0.153
6. 0.00293 0.158









y = 0.1414x + 0.0437
R = 0.9545
0
0.2
0.4
0.6
0.8
1
0 1 2 3 4 5 6 7
concentration
concentration
Linear (concentration)
Sample
No.
CONCENTRATION
mg/ml
ABSORBANCE
(nm)
1. 1 0.124
2. 3 0.335
3. 5 0.548
4. 7 0.657
5. 9 0.687
6. 11 0.881

Fig 8.8 Curve of Vegetable biomass extract for flavonoid content:



Table 8.7: Total Flavonoid contents:

Sample
No.
Total Flavonoid
content (mg/g)
1 31.52
2 51.29
3 9.33
4 27.11
5 94.25
6 34.68

Fig 8.9: Graph Showing TFC:



The above values shows that the TFC in the extract is high in sample sample with the
maximum value of 94.25 mg/g equivalent of Quercetin.

y = 0.0051x + 0.1241
R = 0.92
0
0.05
0.1
0.15
0.2
0 2 4 6 8
ABSORBANCE - I
ABSORBANCE - I
Linear (ABSORBANCE - I)
0
10
20
30
40
50
60
70
80
90
100
1 2 3 4 5 6
Total Flavonoid content(mg/g)
Fig 8.9.1: The graph shows the variation in TPC,TFC AND Antioxidant levels





HYDROGEN PEROXIDE SCAVENGING CAPACITY:

The values obtained for the absorbance of the methanolic extract and ascorbic acid is
as follows,

Table 8.7: absorbance of the methanolic extract and ascorbic acid

Sample
No.

CONCENTRATION
mg/ml
ABSORBANCE (nm)
METHANOLIC
EXTRACT
ASCORBIC
ACID
1 1 0.572 1.333
2 3 0.613 1.245
3 5 0.717 1.471
4 7 0.513 1.712
5 9 0.917 1.517
6 11 1.315 1.322

The 6
th
sample shows the maximum absorbance for methanolic extract of
1.315 nm
Total phenols content(mg/g)
Total Flavonoid content(mg/g)
0
20
40
60
80
100
1 2 3 4 5 6
TPC vs ANTIOXIDANT CAPACITY vs TFC
Total phenols content(mg/g) ANTIOXIDANT Total Flavonoid content(mg/g)



The percentage inhibition values calculated from the formula discussed earlier
Are as follows,

Table 8.8: Inhibition values

Sample
No.
Percentage inhibition of
METHANOL
EXTRACT
ASCORBIC
ACID
1 66.97 23.03
2 64.6 28.11
3 58.6 15.06
4 70.38 11.54
5 47.05 12.41
6 24.07 23.67

The 4
th
sample showed maximum percentage inhibition of 70.38 (%)

Fig 8.9.2:% INHIBITION OF EXTRACT vs ASCORBIC ACID



y = 0.0632x + 0.3954
R = 0.6194
y = 0.0143x + 1.3474
R = 0.0996
0
0.5
1
1.5
2
0 2 4 6 8 10 12
ABSORBANCE FOR EXTRACT AND STANDARD
METHANOL EXTRACT ASCORBIC ACID
Linear (METHANOL EXTRACT) Linear (ASCORBIC ACID)
METHANOL EXTRACT
ASCORBIC ACID
0
20
40
60
80
1 2 3 4 5 6
% INHIBITION OF EXTRACT vs ASCORBIC ACID
METHANOL EXTRACT ASCORBIC ACID


PHYTOCHEMICAL SCREENING:

Preliminary phytochemical screening of the methanolic extract of Vegetable biomass
revealed the presence of flavonoids, Saponins, Tannins and Phenolic compounds.













Phytocompounds Observation Inference
Flavonoids Colour changed to pink +
Tannins Colour changed to red +
Phenolic compounds Colour changed to bluish green +
Saponins Emulsion formed +





CONCLUSION

















9.CONCLUSION:

Based upon the results obtained from the current study, it is concluded that the
methanolic extract of Vegetable biomass of 10g contains total phenolic content of 5
th

sample (20% methanolic conc,10 mins,50c) is 0.04;Total flavonoids content 5
th

sample (20% methanolic conc,10 mins,50c) is 94.25mg/g ;Ferric reducing capacity
of 3
rd
sample is (90% methanolic concentration,10 mins and 40C) ;Percentage
Inhibition value for 4
th
sample (60% methanolic concentration,10mins and
50C);Hydrogen peroxide value of 6
th
sample (90% methanolic concentration,10 mins
and 30C) . It also chelates iron and has reducing power. These indicate that the extract
of vegetable biomass is a significant source of natural antioxidant, which might be
helpful in preventing the progress of various oxidative stress and treating cancer.
However, further isolation of bioactive compounds would assist to ascertain its
potency and safety as a lead candidate of antioxidant for pharmaceutical uses.











































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