J. Sundari et al.

/ Journal of Pharmacy Research 2011,4(10),3743-3746

Research Article Available online through
ISSN: 0974-6943 www.jpronline.info
Antimicrobial and Antioxidant Potential of Root Bark Extracts from Jatropha curcas (Linn)
J. Sundari 1, R. Selvaraj1* and N. Rajendra Prasad2
1,1*
Department of Botany, 2Department of Biochemistry & Biotechnology,Faculty of Science,
Annamalai University, Annamalainagar - 608 002. Tamil nadu, India
Received on: 19-06-2011; Revised on: 08-07-2011; Accepted on:01-10-2011

ABSTRACT
The present study was conducted to determine the antimicrobial and antioxidant properties of Jatropha curcas root bark using different solvent extracts on
inactivation of some microorganisms i.e. gram positive bacteria Staphylococcus aureus and gram negative bacteria Klebsiella pneumoniae, Pseudomonas
aeuruginosa, Vibrio cholerae, Salmonella typhimurium and Escherichia coli, and fungal pathogens namely Candida albicans, Aspergillus flavus, A. niger, A.
fumigatus and Rhizopus sp., were determined. The filter paper disc method was used for screening of crude extracts of root bark of J. curcas for antimicrobial
activity. The antioxidant properties of J. curcas were evaluated by using different in vitro antioxidant assays. J. curcas shows scavenging effect against hydroxyl
(OHl), superoxide anion (O2l-) and 2,2-azino-bis-3-ethylbenzothiazoline-6-sulphonic acid radical cation (ABTS l+) radicals. Our results demonstrate that J. curcas
exhibits strong antimicrobial and antioxidant property in all the in vitro assays.

Key words: Jatropha curcas, antimicrobial activity, Hydroxyl, Superoxide anion, ABTS.

INTRODUCTION
Jatropha curcas (Linn) belonging to the family Euphorbiaceae is a shrub 4.5
to 8 m high. It has a smooth bark and milky latex. It is cultivated as an
ornamental plant and live fencing at an altitude of 4501300 m. The roots,
stems, leaves, seeds and fruits of the plant have been widely used in traditional
folk medicine in many parts of West Africa. The seeds have been used as
purgative, anthelmintic and abortifacient, for treating ascites, gout and skin
diseases1. Previous studies have reported the plant to exhibit pharmacological
activities against fever, mouth infections, jaundice, guinea worm sores and
joint rheumatism 2,3. Investigated on reported the anti-parasitic activity of
sap and crushed leaves of Jatropha curcas4 also reported that the crude
methanolic extract from the root of Jatropha curcas exhibited anti-diar-
rhoeal activity in mice through inhibition of prostaglandin biosynthesis and
reduction of osmotic pressure5. Nevertheless, the plant has been shown to be
a potential source of chemotherapeutic compounds6. Antimicrobial properties
of this plant have been reported 7.
Oxidative stress, the consequence of the imbalance between prooxidants and
antioxidants in an organism, is considered to play a very important role in the
pathogenesis of several degenerative diseases 8. These diseases include diabetes,
aging, cancer, cardiovascular diseases, metabolic syndrome and atherosclero-
sis. Free radicals, such as hydroxyl, singlet oxygen, nitric oxide, hydrogen
peroxide and superoxide radicals, are continuously generated in the cell, as a
result of normal human metabolism. However, they can be harmful to the
system if not properly regulated and thus may cause variety of pathological
effect such as carcinogenesis, aging, DNA damage and enzyme inactivation by
attacking biological macromolecules. The mechanisms by which free radicals
interfere with cellular functions are not yet fully understood, but one of the
most important processes seems to be the formation of lipid hydroperoxides 8.
Hence, studies on natural antioxidant have gained increasingly greater impor-
tance.
MATERIALS AND METHODS:
Collection of plant materials:
The root bark of J. curcas were collected from Cuddalore district, Tamilnadu,
India during the month of July, 2009 and they were collected and air dried at
room temperature.
Preparation of crude extracts:
The dried root bark were coarsely powdered and stored in an airtight container.
About 50 gram of root bark powder was taken in a clean sterile soxhlet
apparatus and extracted with 250 ml of different solvents such as acetone,
chloroform, ethanol and methanol. The extracts were collected and filtered
using whattman No. 1 filter paper. Then the extracts were dried in vacuum
evaporator to obtain concentrated crude extract. These extracts were used for
antimicrobial and antioxidant assays.
Test microorganisms:
The following microorganisms were used as test organisms; gram positive
bacteria Staphylococcus aureus (MTCC 098), gram negative bacteria Kleb-
siella pneumoniae (ATCC 25955), Pseudomonas auruginosa (ATCC 27853),
Salmonella typhimurium (MTCC 098) and Escherichia coli (ATCC 25922)
fungal pathogens Candida albicans, Aspergillus Flavus, A. niger, A. fumigatus
and Rhizopus sp., These bacterial and fungal strains were obtained from the
Department of Clinical Microbiology, Rajah Muthiah Medical College (RMMC)
and Hospital, Annamalai University, Tamil nadu, India.

Antioxidants can protect the human body from free radicals and reactive
oxygen species (ROS) effects9. Antioxidants prevent free radical induced cel-
lulose damage by scavenging them or promoting their decomposition. Anti-
oxidant agents are well known to retard the progress of many chronic diseases
as well as lipid peroxidation. Presently the most commonly used synthetic
antioxidants are butylated hydroxyanisole (BHA), butylated hydroxytoluene
(BHT), propylgallate and tert-butylhydroquinone. However, BHA and BHT
have been restricted by legislative rules due to doubts over their toxic and
carcinogenic effects10. Natural products are known to inhibit free radical chain
reactions and alleviate number of free radical mediated metabolic alteration.
*Corresponding author.
Dr. R. Selvaraj,
M.Sc., M.Ed., M.Phil., Ph.D.,
Professor of Botany
Annamalai University
Annamalai nagar 608 002.
E-mail: selvarajphd14@yahoo.co.in
Tel:+91-9442909910
In vitro antibacterial activity was determined by using Mueller Hinton Agar
(MHA) and Mueller Hinton Broth (MHB) for bacteria. In vitro antifungal
activity was determined by using Sabouraud Dextrose Agar (SDA) and Yeast
Nitrogen Base (YNB), for yeast fungi and Sabouraud Dextrose Broth (SDB) for
mould fungi and they were obtained from Himedia Pvt Ltd, Mumbai, India.
Antimicrobial assay:
The disc diffusion method was used to determine the antimicrobial activity of
the investigated extracts. Nutrient agar was prepared by dissolving of 25 g 1-1
in water. The sterile nutrient agar was inoculated with microbial cells (200 l of
microbial cell suspension in 20 ml agar medium) and poured into sterile Petri
dishes. Sterile filter paper discs of 6 mm in diameter were impregnated with 20
l of the extract solution (equivalent to 4 mg of the dried extract). The paper
discs were allowed to evaporate and thereafter placed on the surface of the
inoculated agar plates. Then the plates were incubated at 37C for 24 hours for
bacteria and 28C for 48 hours Yeast fungi, 2-4 days for mould fungi respec-
tively, along with standard Ciprofloxacin (bacteria) and Amphotericin-B (fungi).
The inhibition zone was measured from the edge of the disc to the inner
margin of the surrounding pathogens.

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 J. Sundari et al. / Journal of Pharmacy Research 2011,4(10),3743-3746
Free radical scavenging assay:
Hydroxyl scavenging assay:
Hydroxyl radical scavenging activity of Jatropha curcas was determined by
the method of Halliwell11. In this assay OHl is produced by reduction of H2O2 by
the transition metal (iron) in the presence of ascorbic acid. The generation of
OHl is detected by its ability to degrade to deoxyribose to form products,
which on heating with thiobarbituric acid (TBA) form a pink colour chro-
mogen. Addition of J. curcas competes with deoxyribose for OHl and dimin-
ishes the colour formation. The incubation mixture in a total volume of 1 ml
contained 0.1 ml of buffer, varying volumes of J. curcas (0.5, 10, 20, 40, 60,
80 and 100 g/ml), 0.2 ml of 500 M ferric chloride, 0.1 ml of mM ascorbic
acid 0.1 L of 1 M EDTA, 0.1 ml of 10 mM H2O2 and 0.2 ml of 2-deoxyribose.
The contents were mixed thoroughly and incubated at room temperature for
60 min. Then 1 ml of 1% TBA (1 g in 100 ml of 0.05N NaOH) and 1 ml of
28% TCA were added. All the tubes were kept in a boiling water bath for 30
min. The absorbance of the supernatant was read in a spectrophotometer at
535 nm with reagent blank containing water in place of J. curcas. The effi-
ciency of J. curcas was compared with various concentrations (0.5, 10, 20,
40, 60, 80 and 100 g/ml) of ascorbic acid as standard. Decreased absorbance
of the reaction mixture indicates increased hydroxyl radical scavenging activ-
ity. The percentage scavenging was calculated as shown below:
% of scavenging [OHl] = A0 - A1 x 100
A0
Where A0 was the absorbance of the control and A1 was the absorbance in the
presence of the sample of J. curcas or ascorbic acid as standard.
Superoxide anion scavenging assay
Superoxide anion radical scavenging activity of J. curcas was determined by
the method of Nishimiki 12 with modification. The assay was based on the
oxidation of NADH by phenazine methosulphate (PMS) to liberate PMSred.
PMSred convert oxidized nitroblue tetrazolium (NBToxi) to the reduced form
NBTred, which formed a violet coloured complex. The colour formation indi-
cated the generation of superoxide anion, which was measured spectrophoto-
metrically at 560 nm. A decrease in the formation of colour after addition of
the antioxidant was a measure of its superoxide scavenging activity. 1ml of
NBT (100 mol of NBT in 100 mM phosphate buffer, pH 7.4), 1ml of NADH
(468 mol in 100 mM phosphate buffer, pH 7.4) solution and varying vol-
umes of J. curcas (0.5, 10, 20, 40, 60, 80 and 100 g/ml) were mixed well.
The reaction was started by the addition of 100 l of PMS (60 mol/100mM
phosphate buffer, pH 7.4). The reaction mixture was incubated at 30C for 15
min. The absorbance was measured at 560 nm in a spectrophotometer. Incu-
bation without the J. curcas was used as blank. Ascorbic acid was used as a
standard for comparison. Decreased absorbance of the reaction mixture indi-
cates increased superoxide anion scavenging activity. The percentage scav-
enging was calculated as shown below:
% of scavenging [O2l-] = A0 - A1 x 100
A0
Where A0 was the absorbance of the control and
A1 was the absorbance in the presence of the
sample of J. curcas or ascorbic acid as standard.
ABTS radical scavenging assay
Zone of inhibition (mm)
This method measures the capacity of different
compounds to scavenge the 2,2-azino-bis-3-
ethylbenzothiazoline-6-sulphonic acid radical
cation (ABTS l+) Arnao13. The antioxidant activ-
ity was measured in a reaction mixture contain-
ing 0.5 ml of 15 M H2O2 0.5 ml of 7 mM ABTS l+
and 50 mM sodium phosphate buffer, pH 7.5 and
varying concentrations of J. curcas (0.5-100 g/
ml). The blank contained water in place of J.
curcas. The absorbance was read in spectropho-
tometer at 734 nm and compared with standard
ascorbic acid. IC 50 value is the concentration of
sample required to inhibit 50% of ABTSl+ produc-
tion.
l+
% of scavenging [ABTS ] = A0 - A1 x 100
A0
Where A0 was the absorbance of the control and
A1 was the absorbance in the presence of the
sample of J. curcas or ascorbic acid as standard.
Journal of Pharmacy Research Vol.4.Issue 10. October 2011
RESULTS:
Antimicrobial activity was performed by using an plant extract with four
different solvents namely acetone, chloroform, ethanol and methanol. In this
study, five bacterial and four fungal clinical pathogens viz., Staphylococcus
aureus, Klebsiella pnemoniae, Pseudomonas aeruginosa, Escherichia coli
and Salmonella typhimurium and fungal strains viz., Aspergillus fumigatus,
Aspergillus flavus, Aspergillus niger and Candida albicans have been used in
this study.
Among the five bacterial pathogens, the Staphylococcus aureus showed high
sensitivity to chloroform extract (17 mm), Klebsiella pnemoniae showed
next level to chloroform extract (15 mm) and Pseudomonas aeruginosa
showed high inhibitory zone to chloroform extract (14 mm). For all these
microorganisms, methanol, ethanol and acetone respectively stood next lev-
els to sensitivity and inhibitory activities. Escherichia coli and Salmonella
typhimurium showed moderate inhibitory zone to chloroform extracts.
Ciprofloxacin was used as control for bacterial culture. Except acetone, the
rest of the solvents showed high inhibitory activities when compared to con-
trol (Fig. 1).
Amphotroxicin was used as standard for fungal strains, all fungal pathogens
show less inhibitory zone for all the extract, when compared to the control.
Aspergillus fumigatus shows high sensitivity to chloroform extract (10 mm),
followed by methanol extract (9 mm) and ethanol extract (7 mm) respec-
tively. Candida albicans shows very less zone of inhibition for chloroform
extract (7 mm) and for ethanol extract (9 mm) and also it has not showed any
activity in acetone and methanol extract. Based on the present results, it was
observed that chloroform extract having maximum inhibitory capacity to the
fungal studied (Fig. 2).
Hydroxyl free radical scavenging activity:
The scavenging ability of acetone, chloroform ethanol and methanol extracts
(compared to ascorbic acid or vitamin C as standard) on hydroxyl radical was
shown in Fig. 3. Jatropha curcas exhibits inhibition of OH radical formation
and percentage of inhibition was higher than that of ascorbic acid at lower
concentrations (0.5, 10, 20, 40, 60, 80, 100 g/ml). The calculated IC 50 value
for acetone extract was 38.00 g/ml, chloroform IC 50 value 46.00 g/ml,
ethanol extract IC 50 value 42.00 g/ml, methanol extract IC 50 value 44.00 g/
ml and ascorbic acid 22.00 g/ml.
Superoxide anion scavenging activity
The percentage inhibition of superoxide anion by the extracts and standard
drugs is shown in (Fig. 4). All solvent extracts of J. curcas have strong super-
oxide radical scavenging activity and exhibited higher superoxide radical scav-
enging activity when compared with ascorbic acid. The concentration (0.5,
10, 20, 40, 60, 80, 100 g/ml) of inhibition of superoxide radical of acetone,
chloroform, ethanol, methanol, extracts and Ascorbic acid of J. curcas at 10
Fig. 1. Preliminary screening of antibacterial activities of
Jatropha curcas root bark
20
18
16
Acetone
14
Chloroform
12
Ethanol
10
Methanol
8
Ciprofloxacin
6
4
2
0
Staphylococcus Klebsiella Pseudomonas Escherichia Salmonella
aureus pnemoniae aeruginosa coli typhimurium
3743-3746
 J. Sundari et al. / Journal of Pharmacy Research 2011,4(10),3743-3746
and 40 g/ml was found to be 40.00 g/ml, 17.00 g/
Fig. 2. Preliminary screening of antifungal activity of ml. 12.00 g/ml, 20.00 g/ml and 19.00 g/ml respec-
tively. All activity followed a concentration depen-
Jatropha curcas root bark dent manner and compared favorably well with the
14 standard ascorbic acid.

ABTS radical scavenging activity

12 The percentage inhibition of ABTS radical by the plant
Acetone
Zone of inhibition (mm)

extracts was concentration dependent. There was in-

10 Chloroform creased ABTS radical scavenging activity with increas-
ing concentration at different solvent extraction used
Ethanol in this study (Fig. 5) at a concentration of (0.5, 10,
8 20, 40, 60, 80, 100) g/ml. J. curcas exhibited effec-
M ethanol
tive antioxidant activity at all doses. The inhibition
6 Amphotroxicin was found to be concentration. The IC 50 value of ac-
etone extract was 46 g/ml. The IC 50 value of chloro-
4 form extract was 26 g/ml. The IC 50 value of ethanol
extract was 27 g/ml. The IC 50 value of methanol ex-
2 tract was 34 g/ml and standard Ascorbic acid IC 50 value
14 g/ml respectively. This implies that the plant ex-
tract could be useful for treating radical related patho-
0 logical damages especially at higher concentrations.
Aspergillus Aspergillus Aspergillus Candida
fumigatus flavus niger albicans Effect of J. curcas different solvent extract and ascorbic
acid on superoxide anion scavenging ability values are
F i g . 3 . H y d r o x y l r a d i c a l s c ave n i n g a c ti vi t i e s o f t h e
given as mean S.D of three experiments in each
diffe r e n t e x t r a c t s of J a t r o p h a c u r c a s r o o t bar k
group.
100 F i g . 5 . A B T S r a d i c a l s c a ve n i n g a c t i vi t i e s of th e
90 di ffe r e n t e x t r a c t s o f J a t r o p h a c u r c a s r o o t b a r k
% of hydroxyl scavenging

80 120
70
60 100
% of ABTS scavenging

50
80
40
30
60
20
10 40
0
0.5 10 20 40 60 80 100 20
C o n c e n t r a t i o n ( g /m l )
0
A c e t o n e e xt ra c t I C 5 0 v a lu e = 3 8 ( g / m l) 0 .5 10 20 40 60 80 100
C h lo ro fo r m e xt r a c t I C 5 0 v a lu e = 4 6 ( g / m l)
C o n c e n t r a t io n ( g /m l)
E t h a n o l e xt r a c t I C 5 0 v a lu e = 4 2 ( g / m l)
A c e t o n e e xt r a c t I C 5 0 v a lu e = 4 6 ( g / m l)
M e t h a n o l e xt r a c t I C 5 0 v a lu e = 4 4 ( g / m l)
C h lo ro fo r m e xt r a c t I C 5 0 v a lu e = 2 6 ( g / m l)
A s c o r b ic a c id I C 5 0 v a lu e = 2 2 ( g / m l)
E t h a n o l e xt r a c t I C 5 0 v a lu e = 2 7 ( g / m l)
M e t h a n o l e xt r a c t I C 5 0 v a lu e = 3 4 ( g / m l)
F i g . 4 . S u pe r o x i de r a d i c a l s c ave n i n g a c t i vi ti e s o f th e A s c o rb ic a c id I C 5 0 v a lu e = 1 4 ( g / m l)
di f f e r e n t e x t r a c t s o f J a t r o p h a c u r c a s r o o t bar k
120
DISCUSSION:
Antimicrobial results from this study showed that acetone, chloroform, etha-
100 nol and methanol extracts of J. curcas root bark are able to inhibit the growth
% of superoxide scavenging

of both gram - positive (S. aureus) and gram negative bacteria (K. pneumo-
80 nia, P. aeruginosa, S. typhimurium and E. coli). Among the five bacterial
pathogens the S. aureus showed high sensitivity to chloroform extract (17
60 mm). The chloroform and hot water extracts obtained from Boswellia ameero,
Buxus hildebrandtii and commiphora parvifolia inhibited the growth of sta-
40 phylococcus aureus (ATCC 6538). Bacillus subtilis (ATCC 6051) and Micro-
coccus flavus 14 respectively. The present results shows that K. pneumonia
20 next level to chloroform extract (15 mm) and P. aeruginosa showed high
inhibitory zone to chloroform extract (14 mm) for all these three species.
0 Methanol, ethanol and acetone respectively stood next levels to sensitivity
0 .5 10 20 40 60 80 100 and inhibitory. The antimicrobial activity of J. curcas plant has been studied
C o n c e n t r a t i o n g /m l
earlier by many workers 15 . Root extract was found to be active against S.
aureus and E. coli16.
A c e t o n e e xt r a c t I C 5 0 v a lu e = 4 0 ( g / m l)
The presently tested microbes were also found to be inhibited by the extracts
C h lo ro fo r m e xt r a c t I C 5 0 v a lu e = 1 7 ( g / m l) of other plants like E. coli, P. aeruginosa, S. aureus and Bacillus subtillis were
E t h a n o l e xt r a c t I C 5 0 v a lu e = 1 2 ( g / m l) inhibited by aqueous and organic solvents (acetone and ethanol) of Tamarindus
M e t h a n o l e xt r a c t I C 5 0 v a lu e = 2 0 ( g / m l)
indica17 (Linn). The alcoholic extracts of many plants were found to be active
against some microbes such as methanolic extract of Palustriella commutate
A s c o rb ic a c id I C 5 0 v a lu e = 1 9 ( g / m l) showed antimicrobial activity against Micrococcus luteus (NRRL-B 1018)
Bacillus cereus (NRRL-B 3771), E. coli (ATCC 25922) and K. pneumoniae
Journal of Pharmacy Research Vol.4.Issue 10. October 2011 3743-3746
 J. Sundari et al. / Journal of Pharmacy Research 2011,4(10),3743-3746
(clinical isolate)18. Aspergillus famigatus shows high sensitivity to chloroform
extract (10 mm) followed by methanol extract (9 mm) and ethanol extract
(7 mm) respectively. Candida albicans shows very less zone of inhibitory in
ethanol extract (9 mm) and chloroform extract (7 mm) and nil activity in
acetone and methanol. Based on the results, it was observed that chloroform
extract having maximum inhibitory capacity. The ethanol extracts of J. curcas
plant has been reported to inhibit the growth of Bacillus subtillis (NCTC
8236) and Candida albicans (ATCC 10231)19 .
Antioxidants are closely related to their biofunctionalities, such as the reduc-
tion of cellular abnormalities like DNA damage, mutagenesis, carcinogenesis
and which is after associated with free radical propagation in biological sys-
tems 20 . Previous reports show that dietary phytochemicals act as excellent
free radical scavengers in different experimental systems21. Our results shows
that extract of J. curcas were capable of scavenging hydroxyl in a concentra-
tion dependent manner and have a stronger hydroxyl scavenging activity of
compared with ascorbic acid.
It was previously demonstrated that decrease of absorbance at 560 nm with
antioxidants thus indicates the consumption of superoxide anion in the reaction
mixture. Superoxide anion radical is one of the strongest reactive oxygen spe-
cies among the free radicals that are generated22. Superoxide anion is formed in
almost all aerobic cells and is a major agent in the mechanism of oxygen
toxicity. Superoxide anion produced in vivo by electron leakage from the
mitochondrial electron transport chain by activated phagocytes 10 . Compared
with other oxygen radicals, O2l- has a longer life time, can move to an aim at
a longer distance and they it induce more damaging effects to biological
molecules. Our results indicated that J. curcas had a prominent effect on
superoxide anion scavenging.
It has been previously reported that antioxidant properties of plant extracts
are due to the presence of secondary metabolites. Recent reports show the
percentage inhibition of ABTS radical by the plant extracts was concentration
dependent. There was increased ABTS radical scavenging activity with in-
creasing concentration at different solvent extract 23 . Our results correlate
with this study and J. curcas effectively scavenges ABTS l+ radical. Previous
report shows that high concentrations of the extract have been reported to be
more effective in quenching free radical in the system24.
CONCLUSION:
The extract of J. curcas root bark act against bacterial and fungal pathogens.
It can be used for agricultural applications at a low cost and safe practice. The
presence of these plant extract in J. curcas could be attributed to its pharma-
cological activities associated with free radicals. Although there could be sev-
eral mechanisms of action of its effectiveness in free radical implicated dis-
eases, the antimicrobial and antioxidant free radical scavenging properties of
this plant seem to be highly activities.
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