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

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

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

% of scavenging [O2l-] = A0 - A1 x 100


A0 Fig. 1. Preliminary screening of antibacterial activities of
Where A0 was the absorbance of the control and
Jatropha curcas root bark
A1 was the absorbance in the presence of the 20
sample of J. curcas or ascorbic acid as standard.
18
ABTS radical scavenging assay
Zone of inhibition (mm)

This method measures the capacity of different 16


compounds to scavenge the 2,2-azino-bis-3- Acetone
ethylbenzothiazoline-6-sulphonic acid radical 14
cation (ABTS l+) Arnao13. The antioxidant activ- Chloroform
ity was measured in a reaction mixture contain- 12
ing 0.5 ml of 15 M H2O2 0.5 ml of 7 mM ABTS l+ Ethanol
and 50 mM sodium phosphate buffer, pH 7.5 and 10
varying concentrations of J. curcas (0.5-100 g/ Methanol
ml). The blank contained water in place of J. 8
curcas. The absorbance was read in spectropho- Ciprofloxacin
tometer at 734 nm and compared with standard 6
ascorbic acid. IC 50 value is the concentration of
sample required to inhibit 50% of ABTSl+ produc-
4
tion. 2
l+
% of scavenging [ABTS ] = A0 - A1 x 100 0
A0
Where A0 was the absorbance of the control and Staphylococcus Klebsiella Pseudomonas Escherichia Salmonella
A1 was the absorbance in the presence of the aureus pnemoniae aeruginosa coli typhimurium
sample of J. curcas or ascorbic acid as standard.

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
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
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Source of support: Nil, Conflict of interest: None Declared

Journal of Pharmacy Research Vol.4.Issue 10. October 2011 3743-3746