Antibacterial activity of root and leaf extracts of Jatropha zeyheri Sond ( Euphorbiaceae )

Resistance of human pathogenic bacterial strains results in selective pressure against known antibiotics. Jatropha zeyheri is used by traditional medicine practitioners in the treatment of sexually transmitted and urinary tract infections. Acetone, methanol and ethyl acetate extracts of both leaves and roots of Jatropha zeyheri collected from Capricorn District, Limpopo Province, South Africa were investigated for antibacterial activity, against 14 human pathogenic bacterial strains, using disc diffusion method. Ethyl acetate extracts exhibited activity against 11 of the selected strains and showed zone of inhibition of 10.7 mm against Klebsiella pneumoniae. Largest zone of inhibition of 12 mm was obtained with the acetone leaf extract against Enterobacter cloacae and Acinetobacter calcaoceuticals and methanol extract of the leaf against Enterococccus faecalis. Acetone extract of the root exhibited minimal inhibitory concentration (MIC) of 0.39 mg/ml against Salmonella spp. followed by methanol and acetone extracts of the root (0.78 mg/ml) against Serratia marcescens. Methanol extract of the leaf exhibited MIC of 3.13 mg/ml against Staphylococcus aureus. This study validates the use of Jatropha zeyheri in the treatment of various illnesses.


INTRODUCTION
Microorganisms are frequently developing resistance to common drugs and antibiotics and this pose an enormous threat to the treatment of a wide range of serious infections (Taylor et al., 2002;Sibanda and Okoh, 2007).Since penicillin and mutation resistant strains are on the increase, there is a need to search for new compounds (that are not penicillin based) that inhibit microbial growth.Medicinal plants have been a basic source of antibiotics against a variety of illnesses over the years in most communities.South African medicinal plants extracts have been screened for antimicrobial activities (Samie et al., 2010;Buwa and Afolayan, 2009;Van Vuuren, 2008).Jatropha zeyheri, indigenously *Corresponding author.E-mail: nmongalo@pan.uzulu.ac.za.Tel: +27359026112.Fax: +27866395217.known as "Sefapabadia" amongst Sotho tribe, (root) is used by traditional medicine practitioners in the treatment of sexually transmitted infections and urinary tract infections.It is also used to treat menstrual pains, irregular periods, and to ensure a strong foetus during pregnancy (Van Wyk and Gericke, 2007).Genus Jatropha consists of approximately 172 species with significant economic importance (Bhagat and Kulkarni, 2010), distributed mainly in the tropical and subtropical regions of America and Africa and have been reported to possess a variety of biological activities.Crude aqueous extract of ground seeds of Jatropha curcus and crude extract of Jatropha elliptica were reported to possess molluscidal activity (Devappa et al., 2010), while J. curcus and J. podagrica were reported to possess antimicrobial activities (Aiyelaagbe et al., 2000;Essiett and Ajibesin, 2010;Aiyelaagbe et al., 2007).Aqueous extract of Jatropha zeyheri root combined with Warburgia salutaris bark and Pentanisia prunelloides has been reported to possess MIC of >2 mg/ml against Bacillus subtilis and Staphylococcus aureus (Jager, 2003).Genus Jatropha is known to produce diterpenes which mostly belongs to rhamnofolane, daphnane, lathyrane, tigliane, dinorditerpene, deoxy preussomerin and pimarane skeletal structures (Devappa et al., 2011).A compound, jaherin, has been isolated from Jatropha zeyheri root and has been reported to possess MIC of 8 mg/ml against Streptococcus pyogenes and 16 mg/ml against Microsporum canis, Trichophyton rubrum, Trichophyton mentagrophytes and Sporotrichum schenkii (Dekker et al., 1987).Besides antibacterial activity, dichloromethane and methane extracts of the root have been reported to possess both anti-inflammatory and mutagenic effects (Luseba et al., 2007).This paper aimed at investigating the antibacterial activity of root and leaf extracts from Jatropha zeyheri against pathogenic strains, mostly causative agents of urinary tract infections.

Chemicals used
Unless otherwise stated, all the chemicals used including solvents were of AR grade and were obtained from Sigma-Aldrich Co. Ltd.

Plant material and extraction
Roots and leaves of Jatropha zeyheri were collected on April, 2008, from the Pickum farm-Blouberg area within Capricorn District, Limpopo Province Republic of South Africa.Collected plant specimens were separately washed with distilled water to remove the adhering soil, cut into small pieces, dried in the shade and ground into thin powder (2 mm mesh) using hammer mill (Perten Instruments 3100, Sweden).Each dry powder was separately extracted (1:5w/v) with acetone, methanol, and ethyl acetate by incubating the mixture on a mechanical shaker (Merck, South Africa) at 100 rpm for 24 h at room temperature.Extracts were filtered through Whatman No1 paper and the organic solvent extracts were concentrated using rotary evaporator.Dry extracts were weighed and kept in refrigerator at 4°C until needed.

Selected bacterial strains
14 bacterial strains, seven Gram negative strains viz, E. coli ATCC 25922, P. aeruginosa ATCC 7700, E. cloacae ATCC13047, K. pneumoniae ATCC 10031, S. marscens ATCC 9986, and reference clinical isolates of Shigella flexineri and Samonella spp.and seven Gram positive strains viz, S. aureus ATCC 6538, B. cereus ATCC 10702, B. pumilus ATCC 14884, and reference clinical isolates of E. faecalis, S. epididirmis, Acinetobacter calcaoceuticals and B. subtilis were used in this study.The microorganism were obtained from the Department of Biochemistry and Microbiology, University of Zululand, and maintained on Muller-Hinton agar (MHA) (Oxoid).Clinical isolates were isolated from hospital patients of various illnesses within KwaZulu-Natal Province.

Antibacterial tests using disc diffusion method
Plant extracts were tested for antibacterial activity by the disc Mongalo et al. 477 diffusion method according to National Committee for Clinical Laboratory Standard guidelines (NCCLS, 2001).A single colony of the respective organism was aseptically transferred with an inoculating loop to a 20 ml of fresh sterile saline broth in a test tube which was vortexed thoroughly and incubated overnight at 37°C.Turbidity was then spectrophotometrically adjusted to that of 0.5 McFarland's standard.About 100 µl of the inoculum was aseptically transferred to a labeled disposable Petri-dish containing 15 ml Muller-Hinton agar and spread thoroughly using sterile glass spreader.Sterile paper discs of 5 mm (Mast Disks, UK) were impregnated with 10 µl of 5 mg/ml plant extract dissolved in 5% dimethylsulfoxide (DMSO) and gently placed individually on the seeded agar.Plates were allowed to dry for 1 h and later incubated in an inverted position at 37°C over night.Zones of inhibition were measured in millimeters, including sterile paper disc.Neomycin (10 µg/disc) was used as positive control.Negative controls were performed using paper discs loaded with 10 µl of 5% DMSO.Each experiment was repeated.

Minimal inhibitory concentrations (MIC) using microdilution assay
Extracts showing activity in Disc Diffusion were chosen to assay the minimal inhibitory concentration (Eloff, 1998) using the micro plate broth dilution assay.The 24 h old culture was diluted 1:100 (McGaw and Eloff, 2005) with saline broth.About 100 µL of extracts (50 mg/ml in 5 % DMSO) were added to multi well plate containing 100 µL of freshly prepared broth and serially diluted, yielding 12.5 mg/ml in the first well.Plates were then incubated over night at 37°C.About 40 µl of 2 mg/ml freshly prepared iodo-nitro-tetrazolium chloride were added to each well and incubated for 30 min at the same temperature.Metronidazole was used as the control.The MIC was defined as the lowest concentration of the extract to inhibit bacterial growth.

RESULTS AND DISCUSION
Results for the antibacterial activity (zones of inhibition) of Jatropha zeyheri are shown in Table 1 and ranged from 7.0 to 12 mm.Although, S. aureus continue to become a global threat to antimicrobial chemotherapy (Tacconelli, 2011;Zetola et al., 2005) while E. cloacae resisted all extracts except acetone leaf extract.E. cloacae have recently been reported to cause infections among burn victims, immunocompromised patients and patients with malignancy and may cause bacteremia, urinary tract and pulmonary infections (Musil et al., 2010), while P. aeruginosa which mainly infects the pulmonary tract and urinary tract is amongst the most difficult to treat with conventional antibiotics (Mathekga and Meyer, 1998).E. cloacae are also reported to resist expanded-spectrum cephalosporins and such resistance may be caused by stable derepression of the chromosomal Ampler class C β-lactamase (Kartali et al., 2002).Only acetone extract of the leaf and ethyl acetate extract of the root inhibited K. pneumoniae with a zone of inhibition of 10.7 mm.K. pneumoniae has been associated with nosocomial infections and may produce extended spectrum β-lactamases (ESBLs), which renders it resistant to carbapenems (Falagas et al., 2007).
Results for MIC values of Jatropha zeyheri root and leaf extracts are presented in Table 2 and ranged from 0.78 to ≥ 12.5 mg/ml.Ethyl acetate extract of the root exhibited good minimal inhibitory concentration of 1.56 mg/ml against B. pumilus, B. cereus and E. faecalis.Methanol and acetone extracts of the root exhibited activity against some gram negative bacterial strains and showed low minimal inhibitory concentration of 0.78 mg/ml against S. marcescens, which compares well to that of metronidazole (0.63 mg/ml) against similar organism.Acetone extract of the root exhibited potent MIC value of 0.39 mg/ml against Salmonella spp.According to Kruger et al. (2004), Salmonella spp producing extended-spectrum beta-lactamases are becoming prevalent in some hospitals in South Africa and are resistant to variety of expensive drugs.
Although, Luseba et al. (2007), reported the MIC of 90% methanolic extract of Jatropha zeyheri root at 0.63 mg/ml against Esherichia coli and 2.5 mg/ml against both S. aureus and P. aeruginosa, it is difficult to compare these results to our study due to differences in variables such as solvent type, extraction procedure and other environmental conditions.To our knowledge, there is no reported previous investigation on antibacterial effects of Jatropha zeyheri leaf extracts.Methanol extract of the leaf showed MIC value of 3.13 and 4.16 mg/ml against S. aureus and E. faecalis respectively.According to Aliyu et al. (2008), MIC of 3 mg/ml is of high potency.Acetone and ethyl acetate extracts of the leaf exhibited moderate activity of 6.25 mg/ml against A. calcaoceuticals.These results , in a way, validates the use of Jatropha zeyheri in the treatment of various ailments including urinary tract infections.
Traditional medicine is an important source of products for majority of developing countries in treating a variety of bacterial infections, thus mitigating many of the side effects that may be associated with synthetic antimicrobials.Bacterial strains such as S. aureus, Enterococcus, P. aeruginosa, Acinetobacter, E. coli, Klebsiella are amongst microbes causative agents of hospital acquired infections, with stethoscope being a possible vector (Randrianirina et al., 2010;Killic et al., 2011) and may cause urinary tract infections cohabiting with Proteus and Serratia species.The results of the antibacterial screening of Jatropha zeyheri root and leaf extracts against 14 bacteria species indicate the bioactivity of the plant.In general, gram negative strains were more resistant to extracts than gram positives.Plant extracts are generally known to be more effective on Gram-positive bacteria than on the Gram-negative ones (Parekh and Chanda, 2006;Chan et al., 2008) and this may be attributed to possible reasons such as permeability barrier provided by presence of cell wall with multilayer structure in Gram negative bacteria or the membrane accumulation mechanisms or presence of enzymes in periplasmic space, which are able to break down foreign molecules introduced from outside (Motamedi et al., 2010).

Conclusion
Selected bacterial strains are among the most common pathogens which cause a variety of infections in human.Activity of these extracts validates the use of Jatropha zeyheri against urinary tract infections and other human pathogens.Moreover, there is a need to screen extracts of this plant against organisms belonging to the traditional sphere of sexually transmitted infections.

Table 1 .
, it was the only strain susceptible to all the three root extracts in our current study.Largest zone of inhibition (12 mm) was exhibited by acetone extract of the leaf against E. cloacae and A. calcaoceuticals, and methanol extract of the leaf against E. faecalis.S. epidirmidis and B. subtilis were resistant to all extracts except ethyl acetate root extract while Antibacterial activity of Jatropha zeyheri leaf and root extracts (zones of inhibition in mm and n=3).
aureus, B. cereus, E. faecalis, E. coli and P. aeruginosa, while methanol extract of the root was inactive against B. cereus and E. faecalis, suggesting that leaf may be a substitute for root; a conservation effort.No zone of inhibition observed on negative control.P. aeruginosa was resistant to both acetone and ethyl acetate leaf extracts and methanol extract of the root, Results were reported as mean of three replicates;na, not active.

Table 2 .
Minimal inhibitory concentrations of Jatropha zeyheri leaf and roots extracts (n=3).Results were reported as mean of three replicates; -, not done.