Ijnpr 2 (2) 156-163

Indian Journal of Natural Products and Resources
Vol. 2(2), June 2011, pp. 156-163

Antioxidant and free radical scavenging activities of common wild greens
from Tiruvallur District of Tamil Nadu, India
P Jagadeesan, D Arun Prasad, P Pandikumar and S Ignacimuthu*
Division of Ethnopharmacology, Entomology Research Institute, Loyola College, Chennai 600 034, Tamil Nadu, India
Received 27 August 2010; Accepted 25 April 2011
The antioxidant and radical scavenging activities of common wild greens used by the village people in Tiruvallur
District, Tamil Nadu were evaluated. Among the plants studied, the methanol extract of Euphorbia hirta Linn. was
found to be rich in polyphenol content and had a low IC
50
value for DPPH (78.33 g) and hydroxyl radical scavenging
(662.62 g). It showed high antioxidant activity. The methanol extract of Senna auriculata (Linn.) Roxb. syn. Cassia
auriculata Linn. flowers and Eclipta prostrata Linn., leaves were also effective. The efficacies of the other plants were in
the following order: Cardiospermum helicacabum Linn.var. luridum Blume > Boerhavia diffusa Linn. > Centella asiatica
(Linn.) Urban > Cissus quadrangularis Linn. > Celosia argentea Linn. > Marsilea quadrifolia Linn. > Pisonia grandis R.
Br. syn. P. alba Span. > Solanum trilobatum Linn. These plants have been traditionally used by villagers without any
noticeable adverse effects. The present study has indicated that the isolation of some specific compounds responsible for
these activities might help in several aspects of food chemistry.
Keywords: Euphorbia hirta, Senna auriculata, Radical scavenging, Total phenolic, Nutraceuticals, Tamil Nadu.
IPC code; Int. cl. (2011.01) A61K 36/00, A61P 39/06
Introduction
Morphological, biochemical and molecular studies
undertaken in recent years revealed that oxidative
stress played a primary role in the development of
many degenerative changes in the cells and tissues.
Since all the cells and tissues are having antioxidant
enzymes like superoxide dismutase, glutathione
peroxidase, glutathione reductase and substances like
reduced glutathione, they dispose free radicals and
protect the cells and tissues from oxidative attack.
Normally a balance is maintained between the
oxidative attack of the free radicals and antioxidant
defense system prevailing in the cells and tissues.
However, if the balance is tilted more towards the
generation of free radicals, then there is damage to
cell structures, DNA, lipids and proteins. Although
the formation of free radicals is unavoidable, their
overproduction is correlated with various disease
conditions such as tumor promotion, carcinogenesis
1
,

cardiovascular
2
, and many neurodegenerative
diseases
3
. The search for natural antioxidants of
dietary, cosmetic and pharmaceutical uses has become
a major industrial and scientific challenge over the
last two decades
4
.
Synthetic antioxidants such as warferin, BHT and
BHA exert potential side-effects and their use is under
strict regulation. Antioxidants derived from fruits,
vegetables, spices and cereals are very effective and
have reduced interference with the bodys ability to
use free radicals constructively
5,6
. The presence of a
widerange of phytochemicals such as phenolics
(flavonoids, phenolic acids and alcohols, stilbenes,
tocopherols, tocotrienols), thiols and carotenoids in
plants is suggested to exert chemopreventive
7
and
cardioprotective
8
effects, thus protecting the human
body against the oxidative damage by free radicals
9
.
Many epidemiological studies have revealed that the
consumption of phenolics from plant sources appear
to be closely related to the prevention of various types
of cancer, cardiovascular, neurological and aging
related disorders
10
. Phenolics inhibit the activity of
matrix metalloproteinase and improve the endothelial
function
11
.
In recent years, the under-utilized endemic wild
species have been an interesting source for

*Correspondent author:
E-mail: entolc@hotmail.com
Phone: +91-44-28178348
Fax: +91442817 5566
JAGADEESAN et al: ANTIOXIDANT & FREE RADICAL SCAVENGING ACTIVITIES OF WILD GREENS

157
nutraceutical industries and health conscious
consumers as in the case of Euterpe oleraceae
12
. The
present study was aimed at examining the radical
scavenging and antioxidant activities of the common
wild greens used by the villagers in Tamil Nadu.

Materials and Methods
Selection of plants
The plant materials were selected by interviewing
the women and elderly people in the villages in
Tiruvallur District, Tamil Nadu. The wild plants used
frequently as greens by the villagers were collected in
the month of August, 2009 and the botanical identity
of the plants were confirmed by the taxonomist at the
Department of Botany, Loyola College, Chennai. The
plant parts used and the mode of usage were also
recorded. The voucher specimens were stored in the
herbarium of Entomology Research Institute, Loyola
College. The list of the medicinal plants with their
medicinal uses is given in Table 1 and Plate 1.

Extraction
The plant material (25 g) was macerated with
75 ml of methanol for 15 minutes and filtered through
Whatman No 1 filter paper. The filtrate was
condensed using rotary vacuum evaporator. The
extracts were stored at 4C.

Estimation of the polyphenol content of the extracts
The amount of polyphenol in the plant materials
was determined by Folin-Ciocalteu method
13
. 300 mg
of fresh plant tissue was ground in 5 ml of
methanol:water:conc. HCl (60:40:0.3). The contents
were filtered through Whatman No. 1 filter paper. The
filtrate (100 l) was mixed with 2 ml of 2% Na
2
CO
3

solution. After two minutes 100 l of 50%
Folin-phenol reagent was added. The tubes were
incubated for 30 minutes at room temperature and
read at 750 nm. Gallic acid was used as standard.
Each assay was carried out in triplicate. The results
were expressed as milligram of gallic acid equivalent
per 100 g of fresh tissue.

DPPH radical scavenging assay
The hydrogen atom or electron donor capacity of
the extracts was tested by its ability to bleach DPPH
radical according to the method of Gulcin (2007)
14
.
Various concentrations of the extracts (100-500 g)
were added to 1 ml of 0.25 mm DPPH solution in
ethanol. The tubes were incubated in dark for
30 minutes and the absorbance was read at 517 nm.
Each assay was carried out in triplicate. The
percentage scavenging was calculated as the ratio of
absorption of the sample relative to the control
without the extract. DPPH radical scavenging activity
was calculated using the following formula: DPPH
radical scavenging activity (%)=(1antioxidant OD/
control OD) 100. Antiradical OD was defined as
EC
50
and it was calculated using non-linear
regression
15
.

Determination of total antioxidant capacity of the extracts
The total antioxidant activity of the extracts was
determined by reduction of Mo (VI) to Mo (V) by the
extracts and the subsequent formation of a green
coloured Mo (V)/phosphate complex in acidic pH.
100 g of each extract in 0.3 ml of distilled water was
added to 3 ml of molybdate reagent containing
0.6 mm sulphuric acid, 28 mm sodium phosphate and
4 mm ammonium molybdate. The tubes were
incubated at 95C for 90 minutes. Then the mixture
was cooled to room temperature and the absorbance
was measured at 695 nm. The results were expressed
as equivalents of ascorbic acid and BHT
16
.

Scavenging of hydroxyl radical by deoxyribose method
Deoxyribose was oxidized by the Fenton reaction
and degraded to malondialdehyde. The inhibition of
malondialdehyde by the extracts was measured by this
reaction. 0.2 ml of 10 mm EDTA, 0.2 ml of 10 mm
FeSO
4
, 0.2 ml 10 mm deoxyribose solution, 200 l of
10 mm H
2
O
2
solution and various concentrations of
the extracts (100-500 g) were added in a screw
capped tube. The total volume of the reaction mixture
was made up to 1.8 ml with phosphate buffer
(0.1 m, pH-7.4). The reaction mixture was incubated
at 37C for 4 hours. After that 1 ml of TCA solution
(2.8%) and 1 ml of thiobarbituric acid solution (1%)
were added to the reaction mixture and kept in boiling
water bath for 10 minutes. The absorbance was
measured at 520 nm. OH scavenging activity (%)
was calculated using the following formula;
1[(Ab
s
-Ab
o
)/(Ab
c
-Ab
o
)] 100 where Ab
o
is the
absorbance with no treatment at 520 nm; Ab
c
is the
absorbance of treated control at 520 nm; Ab
s
is the
absorbance of treated sample at 520 nm.

Scavenging of superoxide radical by alkaline DMSO method
The superoxide scavenging effect of the extract was
studied using the alkaline DMSO method. The
extracts were dissolved in DMSO and 300 l of
extracts at various concentrations was added with
INDIAN J NAT PROD RESOUR, JUNE 2011

158
0.1 ml of NBT (1 mg/ml solution in DMSO) and 1 ml
of alkaline DMSO (5 mm NaOH/ml of DMSO).
Then, the change in absorbance was monitored up to
five minutes
17
.

Estimation of the pro-oxidant effect of the extracts
Many reductants such as ascorbic acid can reduce
the oxidized form of iron (Fe
3+
) to reduced form
(Fe
2+
). This reaction could enhance the liberation of
hydroxyl radical
18
. The predomination of reducing
power on ferrous ions over the radical scavenging
activity resulted in the pro-oxidant effect. The pro-
oxidant effect of the extracts was studied according to
the method of Tian and Hu (2005)
19
. 100 g of the
extracts in 500 l of water was incubated with 50 l
of 1% potassium ferricyanide solution at 50C for
20 minutes. An equal volume of 10% TCA was then
added and centrifuged at 3000 g for 10 minutes. 1.0 ml
Table 1Binomial and vernacular names, parts used and medicinal uses of the plants selected for the study

S. No Binomial name, Family
and Specimen no.
Vernacular name Parts used Distribution, Medicinal uses and References
28, 29, 30

1 Boerhavia diffusa Linn.
(Nyctaginaceae)
Specimen No: ERIP-005
Punarnava (S)
Saaranai (T)
Leaves Common weed up to 750 MSL. Used as diuretic,
expectorant, laxative, and in conditions of
inflammations, anemia and jaundice. Punarnavine
increases blood pressure and diuresis in cats.
2 Cardiospermum
helicacabum Linn. var.
luridum Blume
(Sapindaceae)
Sakralata (S)
Mudakkothan (T)
Leaves Common vines in hedges; diuretic, diaphoretic, useful
in arthritis, lumbago and neuropathy.
3 Celosia argentea Linn.
var. argentea
(Amaranthaceae)
Vitunna (S)
Thoyyak keerai (T)
Leaves Common weed in cultivated lands, locally abundant,
useful in mouth ulcers and for diseases of eyes.
4 Centella asiatica (Linn.)
Urban.
(Apiaceae)
Mandukaparani (S)
Vallarai (T)
Leaves Prostrate herb common in marshy grounds;
antileprotic, diuretic and febrifuge. A glycoside,
asiaticoside shown to be active in the treatment of
leprosy and some types of tuberculosis.
5 Cissus quadrangularis
Linn.
(Vitaceae)
Asthisrnkhala (S)
Pirrandai (T)
Tender
shoots
Common vines in scrub jungles; useful in colic,
flatulence, skin diseases, ulcers, tumors, bone
fractures, colonopathy and asthma.
6 Eclipta prostrata Linn.
(Asteraceae)
Bhrngarajah (S)
Karisalanakanni (T)
Leaves Common prostrate herb in bunds of cultivated lands
and banks; useful in hepato-spelnomeghaly,
inflammations, gastropathy, hypertension, ulcers and
jaundice. Ecliptin is isolated from this plant.
7 Euphorbia hirta Linn.
(Euphorbiaceae)
Pusitoa (S),
Ammampacharisi (T)
Leaves Common in roadsides and wastelands; useful in
bronchial affections, asthma, bowel complaints and
cough. An essential oil isolated from this plant shows a
relaxing action on smooth muscle.
8 Marsilea quadrifolia Linn.
(Marsileaceae)
Sunisannah (S),
Aarai (T)
Leaves Common mattie herb in marshy places; diuretic, for
constipating and aphrodisiac.
9 Pisonia alba Span.
(Nyctaginaceae)
Lanchamunda keerai
(T)
Leaves A small tree of the beach forests of Andaman Islands,
frequently cultivated in the gardens. Useful in
inflammations.
10 Senna auriculata (Linn.)
Roxb. syn, Cassia
auriculata Linn.
(Fabaceae)
Avarttaki (S)
Avvarai (T)
Flowers Common shrub in scrub jungles; flowers useful in
diabetes, urethrohoea and conjunctivitis.
11 Solanum trilobatum Linn.
(Solanaceae)
Alarka (S)
Thooduvalai (T)
Leaves Common vines in waysides; decoction is useful in
chronic bronchitis; solanine, an alkaloid was reported
from it.

S- Sanskrit; T- Sanskrit and Tamil vernacular names; Distribution and medicinal uses are given based on Matthew (1999)
28
,
Warrier et al (1995)
29
and Chopra et al (2002)
30
.


159

Plate 1 Common wild greens selected for the study: a- Boerhavia diffusa, b- Cardiospermum helicacabum var. luridum, c- Centella
asiatica, d- Cissus quadrangularis, e- Eclipta prostrata, f- Euphorbia hirta, g- Marsilea quadrifolia, h- Pisonia alba, i- Senna auriculata,
j- Solanum trilobatum.

160

of the upper layer of the solution was mixed with
0.2 ml of 0.1% ferric chloride solution and the
absorbance was read at 700 nm. Distilled water was
used as negative control and ascorbic acid was used as
positive control.

Statistical analysis
Results were expressed as mean SEM. Statistical
significance was calculated by oneway ANOVA
followed by Tukeys HSD and the values were
considered as significant at P 0.05. EC
50
values
were calculated using non-linear regression (Graph
Pad Prism, Version 4.0, San Diego, CA, USA).

Results and Discussion
Pharmaceutical research conducted over the past three
decades has shown that the natural products are a
potential source for novel molecules for drug
development. Targeted collection of plant materials
based on traditional uses had a higher degree of positive
results than those selected randomly. The results
obtained during present study are discussed below.

Polyphenol content of the extracts
The amount of the total polyphenol was measured
by FolinCiocalteu method and the values were
expressed as gallic acid equivalents (Table 2).
Phenolic compounds such as flavanoids, phenolic
acid and tannins possess diverse biological activities
such as anti-inflammatory, anticarcinogenic and
anti-atherosclerotic activities. These activities might
be related to their antioxidant activity
20
. In the present
study the leaves of E. hirta (1496.544.03 mg/100 g
fresh tissue) and the flowers of S. auriculata
(1409.947.00) had shown higher polyphenol content
than other plants. The water extract of S. auriculata
flowers was reported to reduce the oxidative stress
mediated complications in streptozotocin induced
diabetic rat
21
. Further, the methanol extract of
S. auriculata flowers had shown a significant alpha
glucosidase inhibitory effect which was comparable to
the reference drug, agarbose
22
. The polyphenol content
in other plants were in the following order: E. prostrata
< C. quadrangularis < C. asiatica < B. diffusa <
C. helicacabum var. luridum < M. quadrifolia
P. alba < C. argentea < S. trilobatum.
Previous works had demonstrated that the phenolic
contents in plants were correlated with their
antioxidant activity
23
. In our study also the increase in
polyphenol content was significantly correlated
(r
2
0.824; P < 0.002) with the increase in the
inhibition percentage of DPPH assay. The consumption
of polyphenol rich non-alcoholic red wine extract
(1 g/day) or quercetin (30 mg/day) reduced the ex vivo
susceptibility of LDL oxidation in a group of male
volunteers
24
. Various human intervention studies
showed consumption of fruits or fruit extracts rich in
polyphenol reduced the oxidative stress, markers of
inflammation, serum triglycerides and LDL level
25
.

DPPH radical scavenging assay
The DPPH assay constitutes a screening method
currently used to provide basic information on the
antiradical activity of the extracts. When a solution of
DPPH is mixed with a substance that can donate a
hydrogen atom, the reduced form of the radical is
generated accompanied by loss of colour and
reduction in the absorbance. In the present study, the
methanol extract of E. hirta exerted a strong antiradical
activity with least IC
50
value (73 g; r
2
0.859)
followed by the extracts of S. auriculata (198.90 g;
Table 2Polyphenol content and DPPH scavenging activity of the extracts

S. No Name of the plant Polyphenol content
(mg GAE/100 g of fresh tissue)
IC
50
values (g) for DPPH assay

1 Boerhavia diffusa 338.10 3.78
a
441.45 6.40; r
2
0.831
a

2 Cardiospermum helicacabum var. luridum 261.17 3.67
b
438.97 8.45; r
2
0.852
a

3 Celosia argentea 161.32 3.21
d
> 1000
4 Centella asiatica 355.51 02.29
e
491.32 8.96; r
2
0.874
c

5 Cissus quadrangularis 383.79 01.65
b
555.42 5.31; r
2
0.842
d

6 Eclipta prostrata 863.90 6.13
f
237.80 4.01; r
2
0.627
e

7 Euphorbia hirta 1496.54 4.03
g
78.33 9.65; r
2
0.859
f

8 Marsilea quadrifolia 225.94 11.48
h
> 1000
9 Pisonia alba 210.41 4.22
h
> 1000
10 Senna auriculata syn. Cassia auriculata 1409.94 7.00
c
193.19 7.50; r
2
0.887
b

11 Solanum trilobatum 118.79 01.43
i
> 1000

Values are mean SEM for triplicates; values carrying same alphabet did not vary significantly.


161
r
2
0.887) and E. prostrata (235.47 g; r
2
0.627)
(Table 2). The antiradical activity of the plants was in
the following order: C. helicacabum var. luridum
B. diffusa > C. asiatica > C. quadrangularis > C.
argentea > M. quadrifolia > P. alba > S. trilobatum.
The IC
50
value for the positive control ascorbic acid
was 5.23 g. In this study extracts showed higher IC
50

value than the positive control; however, the extracts
were able to scavenge the DPPH free radicals
suggesting that it may have a role in preventing free
radical mediated chain reactions.

Total antioxidant effect of the extracts
Total antioxidant activities of the extracts were
expressed as equivalents of ascorbic acid and BHT
(Table 3). The phosphomolybdenum method is
quantitative since the antioxidant activity is expressed
as the number of equivalents of ascorbic acid
26
. In the
present study, the methanol extract of E. hirta had a
high ascorbic acid equivalent followed by
S. auriculata and other extracts. The antioxidant
activities of other plants were in the following order:
E. prostrata C. asiatica > B. diffusa C.
quadrangularis M. quadrifolia C. helicacabum
var. luridum > P. alba C. argentea > S. trilobatum.

Hydroxyl radical scavenging effect of the extracts
Hydroxyl radical is the most reactive oxygen
species and short lived compared to other ROS. By
interacting with lipids, the hydroxyl radical is able to
initiate lipid peroxidation. Further it reacts with DNA
forming various types of oxidized nucelosides such as
8-OHdG and the hydroxyl radical are known to
induce conformational changes in DNA
27
. Though the
DPPH scavenging model is a useful indicator, it has
some limitations. Therefore, the extracts ability to
scavenge hydroxyl radical was assessed. The
hydroxyl radical scavenging activity of the plant
extracts were expressed as IC
50
values (Table 4). In
the present study, E. hirta, S. auriculata and
E. prostrata extracts had EC
50
values 630.23, 708.67
and 975.08 g, respectively. The hydroxyl radical
scavenging effects of the other extracts were in
following order: C. helicacabum var. luridum < C.
quadrangularis < C. asiatica C. argentea < M.
quadrifolia P. alba < B. diffusa S. trilobatum.

Superoxide scavenging effect of the extracts
Superoxide anion radical is produced in vivo and is
the first reaction product of oxygen molecule. The
conversion of superoxide and hydrogen peroxide into
more reactive species of hydroxyl radical has been
thought to be one of the unfavourable effects caused
by superoxide radicals. It directly affects the activity
of many enzymes in vivo such as catalase, peroxidase,
creatine phosphokinase and aconitase. It is also
capable of initiating lipoperoxidation and oxidation of
thiols. In the present study the extracts of E. hirta and
S. auriculata were able to reduce nitroblue
tetrazolium (NBT) much better than the other extracts
(Figure 1).

Pro-oxidant effect of the extracts
The antioxidant effect of plants is a balance of two
activities: free radical scavenging effect and reducing
power on ferrous ions
18
. Thus evaluation of this effect
is crucial because at higher concentrations the extracts
should not have any pro-oxidant activity. Many
commercially used Ayurvedic plants like Emblica
officinalis Gaertn. are having low reducing power
compared to ascorbic acid. Our results showed that
the extracts of E. hirta and S. auriculata have a low
pro-oxidant effect compared to ascorbic acid at the
same concentration (Figure 2).
Table 3Total antioxidant activity of wild greens (in g AA/100 g plant extract)

S. No Plant Name Ascorbic acid equivalent
(g AA/100 g plant extract)
BHT equivalent
(g AA/100 g plant extract)

a
12.83 0.20
a,b

2 Cardiospermum helicacabum var. luridum 14.21 0.57
a
12.49 0.59
a,b

c
07.81 0.79
d

a, d
14.89 0.21
a,e

a
13.35 0.58
a b

d
16.24 0.53
e

b
21.84 0.69
c

8 Marsilea quadrifolia 14.57 0.50
a
12.97 0.24
a,b

c
07.61 0.19
d

10 Senna auriculata syn. Cassia auriculata 24.79 0.29
b
22.72 0.20
c

e
04.10 0.06
f

Values are mean SEM for triplicates; values carrying same alphabet did not vary significantly.


162
Conclusion
Identification of wild and under-utilized edible
plants are the present target for nutraceutical,
cosmetic and healthcare industries. In the present
study the methanol extracts of E. hirta and
S. auriculata showed a potent antioxidant and radical
scavenging activity with low pro-oxidant effect.
These plants have been traditionally used by villagers
without any noticeable adverse effects. Therefore,
future studies in these plants might help in several
aspects of food chemistry.

Acknowledgments
The authors are thankful to the folklore people for
their help in collection of the plants. The authors are
thankful to Entomology Research Institute, Loyola
College for financial support.
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2 Cardiospermum
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Indian Journal of Natural Products and Resources

Vol. 2(2), June 2011, pp. 156-163

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