Free-radical scavenging activities in Drymaria diandra Blume

Report
International Journal of Integrative Biology
A journal for biology beyond borders

ISSN 0973-8363

Free-radical scavenging activity and phytochemical analysis in the
leaf and stem of Drymaria diandra Blume

Palash Mandal
1,*
, Tarun Kumar Misra
2
, Mitali Ghosal
1


1
Department of Botany, North Bengal University, Darjeeling, WB, India
2
Institute of Plantation Science and Management, North Bengal University, Darjeeling, WB, India

Submitted: 10 May. 2009; Revised: 2 Aug. 2009; Accepted: 12 Sep. 2009


Abstract
In different parts of plain land and hill area of Darjeeling district, leaves and stems of Drymaria diandra were
evaluated for their phytochemical constituents like total phenol, ortho-dihydric phenol, flavonols, tannins and
antioxidant activity against 2,2-diphenyl-2-picrylhydrazyl (DPPH), superoxide anion, hydroxyl radical, nitric
oxide radical and anti-lipid peroxidation activity. Strong antioxidant scavenging activities were observed in both
leaf and stem in different places. Anti-oxidative efficiency to inhibit anti-lipid peroxidation of these plant
extracts in goat liver was investigated. Drymaria diandra stem showed moderate class of anti-lipid peroxidation
against thioburbituric acid but the leaves have high anti-lipid peroxidation activity. The correlation was also
drawn with antioxidants, its attributes and soil nutrients profile. This plant extract may be explored as
therapeutic agent in future.

Keywords: Drymaria diandra, free-radical scavenging, anti-lipid peroxidation, total phenols, flavonols.


INTRODUCTION
Reactive oxygen species (ROS), which consist of free
radicals such as hydroxyl(OH
-
), superoxide (O
2
-
), nitric
oxide (NO), peroxyl (RO
2
-
), lipid peroxyl (LOO
-
)
radicals and non-free radical species such as hydrogen
peroxide (H
2
O
2
), singlet oxygen(O
2
-1
), ozone (O
3
), lipid
peroxide (LOOH), are different forms of activated
oxygen (Helliwell et al., 1999; Yildirim et al., 2000;
Gulcin et al., 2002a). ROS are produced by all aerobic
organisms and can easily react with most biological
molecules including proteins, lipids, lipoproteins and
DNA. This ROS can generate oxidative stress and
produce many pathophysiological disorders such as
arthritis, diabetes, inflammation, cancer and
genotoxicity (Kourounakis et al., 1999; Gulcin et al.,
2002b).

Antioxidants can terminate or retard the oxidation
process by scavenging free radicals. These antioxidants
are considered as possible protection agents for
reducing oxidative damage of human body from ROS
and retard the progress of many chronic diseases as
well as lipid peroxidation (Peryor, 1991; Kinsella et al.,
1993; Lai et al., 2001). However, more recently the
polyphenols have found to be beneficial as strong
antioxidants (Vinson et al., 2002; Wang et al., 1997).

Natural antioxidants are presumed to be safe since they
occur in plants. Drymaria diandra is an herbaceous,
annual plant, generally found as a weed throughout
sub-Himalayan part of West Bengal. This plant is
commonly known as Abhijalo in Nepali language.
The leaves and stems of this plant are traditionally used
for curing asthma, snake bite and pneumonia diseases.

A novel anti-HIV alkaloid drymaritin and new C-
glycoside flavonoids, diandraflavone along with eight
known compounds, torosaflavone A, isovitexin,
spinasterol -D-glycoside, p-hydroxybenzoic acid, p-
hydroxybenzaldehyde, cis-p-coumarate, methyl 5-
hydroxy-4-oxopetanoate and glycerol--lignocerate
were present in D. diandra extracts (Hsieh et al., 2004).

The aim of this work is to evaluate the free-radical
scavenging properties like DPPH, nitric oxide radical,
superoxide and anti-lipid peroxidation and to quantify
phenolic constituents in methanol extracts from the
leaves and stems of D. diandra plant in plain land and
hilly area of Darjeeling. Antioxidants and its attributes
were also correlated with the soil profile of that place
for analyzing the location effect of naturally grown D.
diandra.

*
Corresponding author:
Palash Mandal, Ph.D.
Department of Botany, North Bengal University,
Darjeeling, West Bengal, 734013, India
Email: nbubotanypalash@rediffmail.com
International Journal of Integrative Biology I J I B, 2009, Vol . 7, No. 2, 80
IJIB, All rights reserved

Free-radical scavenging activities in Drymaria diandra Blume
MATERIALS AND METHODS
Plant materials
D. diandra (Abijal) was collected from three different
places of plain land: NBU (26

4269N,
88

2130.90E, 443ft from amsl), NBU Gate No. 3

(26

4242.04N, 88

2141.28E, 440ft from amsl) and

NBU Nibedeita (26

4242.88N, 88

2133.50E, 435ft
from amsl) and two hill areas of Kurseong, Darjeeling
district: Victoria I (26

5324.18N, 88

1713.80E,
5870ft from amsl) and Victoria II (26

532.61N,
88

178.94E, 5859ft from amsl), West Bengal, India.

Leaves and stems were separated and kept for drying in
oven for over night. The dried plant parts were
separately crushed in mortar pestle to form fine
powdered drug. Taxonomic position was authenticated
by the Taxonomy and Environmental Biology
Laboratory, Department of Botany, University of North
Bengal. The material has been deposited in the NBU
Herbarium recorded against the accession number
9482 dated 01-06-07.
Animal material
Goat liver, used for anti-lipid peroxidation assay, were
collected from slaughter house immediately after slay
and experiment was conducted within one hour after
collection.
Chemicals
Methanol (M), 2.2-diphenyl-1-picryl hydrazyl (DPPH),
nitro blue tetrazolium (NBT), reduced nicotinamide
adenine dinucleotide phosphate sodium salt
monohydrate (NADPH), phenazine methosulphate
(PMS), trichloro acetic acid (TCA), thiobarbituric acid
(TBA), FeSO
4
, 7 H
2
O, KOH, KH
2
PO
4,
ethylene-
diamine tetra acetic acid (EDTA), ascorbic acid,
vitamin-E, 2-deoxiribose, ferric chloride (Fecl
3
),
hydrogen peroxide (H
2
O
2
), sodium nitroprusside,
sodium carbonate (Na
2
CO
3
) were either purchased from
Sigma Chemicals (USA) or Himedia or Merck
(Germany).
Soil sampling and determination of
physicochemical properties
Soil samples were collected from five different
naturally grown areas of D. diandra mentioned above
(top and sub soil with 0-15 cm and 15-30 cm depth
respectively) and composite soil were prepared as per
the method of Misra et al., 2009. The samples were
processed for physicochemical analysis viz. pH,
electrical conductivity, moisture contents, organic
carbon, available form of nitrogen, potash as K
2
O,
phosphorus as P
2
O
5
and sulphur as SO
4
-
(Jackson,
1973).
Extraction and determination of methanol
extractive value
Powdered drug of plant parts were separately extracted
with methanol water in ratio 4:1, under Soxlet extractor
for eight hours. The refluxed samples were dried in
vacuo the extractive values were calculated on dry
weight basis from the formula given below:

100
extraction for en Weight tak
extract dry of Weight
%) (yield value extractive % =
. I
DPPH based free radical scavenging
activity
The free radical scavenging activities of each fraction
were assayed using a stable DPPH, following standard
method (Blois, 1958). The reaction mixture contained
1.8ml of 0.1mM DPPH and 0.2ml of each serial
dilution (0.5-2) of D. diandra methanol extracts.
Simultaneously, a control was prepared without sample
extracts. The reaction mixture was allowed to incubate
for 5min at room temperature in the dark and
scavenging activity of each fraction were quantified by
decolourization at 515nm. Percentage of free radical
scavenging activity was expressed as percent inhibition
from the given formula:

100
control of Abs.
sample of Abs. - control of Abs.
radical DPPH of inhibition % =
.. II
Superoxide radical scavenging assay
Measurement of superoxide radical scavenging activity
of D. diandra was done by using standard method
(Nishikimi et al., 1972) followed by slight modification.
The reaction mixture contained 1ml of NBT solution
(312 M prepared in phosphate buffer, pH-7.4), 1ml of
NADH solution (936M prepared in phosphate buffer,
pH-7.4) and standardized 50 times methanol diluted
different extracts of the sample were added. Finally,
reaction were accelerated by adding 100L PMS
solution (120M prepared in phosphate buffer, pH-7.4)
to the mixture. The reaction mixture was incubated at
25

C for 5min and absorbance at 560nm was measured
against methanol as control. Percentage inhibition was
calculated (Eq. II).
Hydroxyl radical scavenging assay
Scavenging of the hydroxyl free radical was measured
by the method of (Halliwell et al., 1989) with minor
changes. All solutions were prepared freshly. 200L of
2.8mM 2-deoxy-2-ribose, 5L methanol extracts of D.
diandra, 400L of 200M FeCl
3
, 1.04mM EDTA (1:1
V/V), 200L of H
2
O
2
(1.0mM) and 200L ascorbic
acid (1mM) was mixed to form a reaction mixture.
After an incubation period of one hour at 37
o
C the
extent of deoxyribose degradation was measured by the
International Journal of Integrative Biology I J I B, 2009, Vol . 7, No. 2, 81
IJIB, All rights reserved

Free-radical scavenging activities in Drymaria diandra Blume
TBA reaction. 1.5ml of 2.8% TCA was added in the
reaction mixture and kept for 20 min. at 100
o
C taking
Vitamin E as positive control. Percentage inhibition
was calculated (Eq. II).
Nitric oxide radical scavenging assay
Nitric oxide was generated from sodium nitroprusside
and measured by the Greiss reaction. This assay was
done by the method of (Marcocci et al., 1994). 320L
methanol extract, 360L sodium nitroprusside, 216L
Greiss reagent (1% sulfanilamide, 2% H
3
PO
4
and 0.1%
napthylethylenediamine dihydrochloride) was mixed
and incubated at 25
o
C for one hour. Lastly 2ml water
was added and absorbance was taken at 546nm.
Percentage inhibition was calculated (Eq. II).
Anti-lipid peroxidation (ALP) assay
The anti-lipid peroxidation assay in the goat liver
homogenate was measured by the standard method
(Dhalwal et al., 2005) followed by slight modification.
2.8ml of 10% goat liver homogenate, 0.1ml of 50mM
FeSO
4
and 0.1ml extract was mixed. The reaction
mixture was incubated for 30min. at 37
o
C. 1ml reaction
mixture was taken with 2ml 10%TCA-0.67%TBA in
acetic acid (50%) for stopped the reaction. Then the
mixture was boiled for 1hour at 100
o
C and centrifuged
at 10,000rpm for 5min.. Supernatant was taken for
absorbance at 535nm against a blank. This contained all
reagents except liver homogenate and extract. Identical
experiments were performed to determine the control
(without extract and FeSO
4
) and induced (without
extract) Vitamin E was used for standard. ALP
percentage was calculated using the following formula.
IC
50
values of all experiment were calculated using
different concentration of extract.

100
control of abs. on peroxidati induced + Fe2 of Abs.
sample of abs. on peroxidati induced + Fe2 of Abs.
%ALP =
... III
Estimation of total phenol content
Total phenolic compounds were determined according
to the protocol described as (Slinkerd et al., 1977). 40
L of methanolic extract of leaf and stem of D. diandra
plant were mixed separately with 1ml Folin-Ciocalteu
reagent and 2ml of 20% Na2CO3. It was mixed, boiled
for one hour, cooled, and centrifuged at 10,000rpm for
five minutes. Absorption was recorded at 575nm in
spectrophotometer.
Estimation of ortho-dihydric phenol
content
The estimation of ortho-dihydro phenol was based on
the method of Arnows (Kim et al., 2003). Made the
volume of 0.2ml of extracts upto1ml.1ml of 0.05N HCl,
1ml of Arnows reagent, 10ml water and 2ml of 1N
NaOH were mixed thoroughly with the extract.
Maintain the reagent blank similarly with out the
extract. Absorbance was measured at 515nm in
spectrophotometer.
Estimation of flavonols content
The amount of total flavonoids content for each extract
was determined by the method of (Kim et al., 2003). To
1ml sample: water (50:50, v/v) or standard solutions
quercetin (0-500 mg L
-1
) was added to 4ml H
2
O in a 10
ml volumetric flask. At zero time, 0.3 ml of 50g L
-1

NaNO
2
was added to the flask. After 5min., 0.3ml
AlCl
3
(100gL
-1
) was added. At 6min, 2ml 1mol L
-1

NaOH were added to the mixture and immediately
diluted with 204ml of water. Absorbance of the mixture
was read at 510nm vs. water blank.
Estimation of tannins content
Tannin content was measured by Folin-Denis method
(Oyaizu, 1986). 50L of extract was made upto 7.5ml
by adding double distilled water. Then 0.5ml Folin-
Denis reagent and 1ml of Na
2
CO
3
were mixed with it.
Again Volume was made upto 10ml by double distilled
water. Absorption was recorded at 700nm.
Statistical analysis
The data was pooled in triplicate and subjected to
analysis of correlation co-efficient matrix using SPSS
(Version 10.00) for drawing the relation between soil
physicochemical properties and antioxidant attributes
and MS Excel was used for comparing the antioxidant
attributes of the parts of plant collected from five
different places. Smiths Statistical Package (Version
2.5) was used for determining the IC
50
values of
antioxidants and their standard error of estimates (SEE).
RESULTS AND DISCUSSION
Fig.1 [Supplementary data] shows that the methanol
extracts of D. diandra has antiradical activity by
inhibiting DPPH radical with the IC
50
value of (0.47 to
3.28g/ml) which was comparable with quercetin
standard. IC
50
value is the effective concentration at
which the antioxidant activity is 50%. DPPH is usually
used as a substrate to evaluate anti-oxidative activity of
antioxidant. The method is based on the reduction of
methanol DPPH solution in the presence of a hydrogen
donating antioxidant due to formation of the non
radical form DPPH-H by the reaction. The extract was
able to reduce the stable radical DPPH to the yellow
colored diphenyl picrylhydrazine. It has been found
that cysteine, glutathione, ascorbic acid, tocopherol,
poly-hydroxy aromatic compounds (hydroquinone,
pyrogallol, gallic acid, etc.) reduce and decolorize
DPPH by their hydrogen donating ability (Blois, 1958).
International Journal of Integrative Biology I J I B, 2009, Vol . 7, No. 2, 82
IJIB, All rights reserved

Free-radical scavenging activities in Drymaria diandra Blume
It appears that extracts of D. diandra possess hydrogen
donating abilities to act as an antioxidant. Fig.1 shows
the highest DPPH radical scavenging effect (IC
50
value
0.47g/ml) present in the leaf extracts of hilly area
(Kurseong Victoria I and Victoria II) than the samples
of plain land (NBU Basketball Court, Gate No. 3 and
Nibedeita Hostel). The data were even superior to
standard antioxidant BHT (IC
50
value: 20.15g/ml.).

In the PMS/NADH-NBT system, superoxide anion
derived from dissolved oxygen by PMS / NADH
coupling reaction reduces NBT. The decreases of
absorbance at 560nm with antioxidants thus indicate
the consumption of superoxide anion in the reaction
mixture. Addition of various extracts of D. diandra in
above coupling reaction showed decrease in absorbance
(Fig. 2 [Supplementary data]). Maximum inhibition (IC
50
value53.85 g/ml) was found in leaf sample of hilly
area (Kurseong Victoria I). Hsieh et al. (2004)
observed that the C-glycoside flavonoids present in the
D. diandra extract have significantly selective
inhibition on superoxide anion generation from human
neutrophils stimulated by fMLP/CB with an IC
50
value
of 10.0g/ml.

The extracts with liver homogenate undergo rapid
peroxidation when incubated with FeSO
4
and produce
peroxide (Aruma, 1996) and they attack the biological
material. This leads to the formation of MDA
(malonodialdehyde) and other aldehydes, which form a
pink chromogen with TBA, absorbing at 535nm
(Kosugi et al., 1987). It was observed (Fig. 3
[Supplementary data]) that methanol extract of D. diandra
have high anti-lipid peroxidation effect against goat
liver in all samples collected from different places of
plains and hills. Addition of Fe
2+
/ascorbate to the liver,
cause increase lipid peroxidation. The extracts showed
inhibition of peroxidation effect in all concentration.
The highest anti-lipid peroxidation activity was found
(IC
50
value 74.86 g/ml) in leaf sample of hill area
(Kurseong Victoria I). In extracts inhibition value was
found to be lesser than the standard, Vitamin E (IC
50
value 14.75g/ml).

The extract was examined for its ability to act as OH
.

radical scavenging agent. Ferric EDTA was incubated
with H
2
O
2
and ascorbic acid at pH -7.4; hydroxyl
radicals were formed in free solution and were detected
by their ability to degrade 2-deoxy-2-ribose into
fragments that on heating with TBA and low pH form a
pink chromogen (Halliwell et al., 1987; Aruoma et al.,
1989). The extract and vitamin E exhibited strong
scavenging effect of hydroxyl radical which could
inhibit lipid damage in different extracts of leaf and
stems of D. diandra collected by different places of
Darjeeling. When the extracts and vitamin E were
added to the reaction mixture they removed hydroxyl
radical and prevented the degradation of 2-deoxy-2-
ribose. The maximum inhibition (IC
50
value 24.97g/ml)
was found in leaf samples of hill area (Kurseong
Victoria II). Fig. 4 [Supplementary data] shows that the
plants of hill area have more H
2
O
2
scavenging activity
than in plain land.

In addition to reactive oxygen species, nitric oxide is
also implicated in inflammation, cancer and other
pathological conditions (Moncada et al., 1991) .The
plant or plant products may have the property to
counteract the formation of nitric oxide radicals and in
turn may be of considerable interest in preventing the
ill effects of excessive nitric oxide generation in the
human body. But the extracts from this plant of both
hill and plain areas executed very less scavenging
power against NO

as documented from their higher
IC
50
values [12,305.53-8,127.33mg/ml] (Fig. 5
[Supplementary data]). Still this scavenging activity may be
considered important because the plant is consumed as
green vegetables in large quantity by local tribes.
Among these, the highest nitric oxide scavenging
activity was again showed by the samples of hilly areas
(Kurseong Victoria I).

A number of studies have focused on the biological
activities of phenolic compounds, which are potential
antioxidants and free radical scavengers (Marja et al.,
1999; Sugihara et al., 1999). Their was a wide variation
in the amount of total polyphenols in different extracts
of D. diandra tested. Among these extracts, the highest
phenol content was found (Fig. 6 [Supplementary data]) in
leaf sample of hill area (Kurseong Victoria I). From the
results, flavonol (Fig. 7 [Supplementary data]), tannin (Fig.
8 [Supplementary data]) and ortho-dihydric phenol content
(Fig. 9 [Supplementary data]) was found more in leaf
sample of plain land. Although all samples have these
phytochemicals in high amount. A new Anti-HIV C-
glycoside flavonoids-Diandraflavone have been
recently isolated from D. diandra. It is well known that
flavonoids possess a wide range of antioxidant
activities. These antioxidant properties are based on
their phenolic structures. Phenolic compounds are also
thought to be capable of regenerating endogenous -
tocopherol, in the phospholipids bi-layer or lipoproteins
particles, back to its active antioxidant form. They are
also known to inhibit various types of oxidizing
enzyme. So this flavonoids-Diandraflavone acts as
antioxidant in D. diandra. These potential mechanisms
of antioxidant action make the diverse groups of
phenolic compounds an interesting target in the search
for health beneficial phytochemicals. The methanol:
water leaf extractive value (percentage of yield) was
highest (35.00%) in sample collected from NBU
Nibedeita Hostel and lowest (7.09 %) in Victoria I (Fig.
10 [Supplementary data]).

Mineral nutritional status and physical properties
generally pH and EC of soil greatly influence the
phytochemicals constituents present in the different
parts of the plant. It is also reported that
International Journal of Integrative Biology I J I B, 2009, Vol . 7, No. 2, 83
IJIB, All rights reserved

Free-radical scavenging activities in Drymaria diandra Blume
phytochemicals constituents directly influences the
antioxidant properties of the plant extract (Misra et al.,
2008). Correlation coefficient matrix analysis in Table1
[Supplementary data] showed that antioxidant properties,
phytochemicals constituents and physicochemical
properties of soil significantly correlated with each
other at P< 0.05% level. Therefore, marked difference
of antioxidant activities as well as phytochemicals were
observed in the leaf and stem part of extract of D.
diandra collected from different places of North Bengal.
CONCLUSION
This study suggests that the D. diandra Blume plant
(leaf and stem) extract has antioxidant activity, which
might be helpful in preventing or slowing the progress
of various oxidative stress-induced diseases. The results
of the present study also indicate that the plant parts
possess many phytochemicals which could be
beneficial for human health.
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