Screening of plants for their potential antibacterial activity against Staphylococcus and Salmonella spp.

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1 Natural Product Radiance, Vol. 6(4), 2007, pp Research Article Screening of plants for their potential antibacterial activity against Staphylococcus and Salmonella spp. Introduction Infectious diseases are world s leading cause of premature death, killing almost 50,000 people everyday 1. Resistance to antimicrobial agents is emerging in a wide variety of pathogens and multiple drug resistance is becoming common in diverse organisms such as Staphylococcus aureus, S. epidermidis, Salmonella typhi and S. paratyphi A 2-4 ; this has necessitated a search for new antimicrobial substances from other sources including plants. Over the past 20 years, there has been a lot of investigation on plants as sources of new antimicrobial agents. But still there is an immediate need to identify novel substances active towards pathogens with high resistance 5-7. Recent studies have shown that several alcoholic extracts of various traditional medicinal plants exhibit antibacterial activity 2,8. Anti- Staphylococcal and anti-salmonella Yogesh Mahida and J S S Mohan* Department of Biosciences, Sardar Patel University Vallabh Vidyanagar , Gujarat, India *Correspondent author, jssmohan@hotmail.com Received 7 June 2006; Accepted 1 March 2007 Abstract Methanol extracts of 23 plants were screened for their antibacterial activity against multi-drug resistant bacteria, viz. Staphylococcus aureus, S. epidermidis, Salmonella typhi and S. paratyphi A to find out an alternative source of active principles/compounds. The extracts of Cryptolepis buchanani (Linn.) Roem. & Schult., Mangifera indica Linn., Manilkara hexandra (Roxb.) Dubard and Nyctanthes arbor-tristis Linn. exhibited significant antibacterial activity with MIC value of 1-8 mg/ml. Keywords: Medicinal plants, Methanol extracts, Antibacterial activity, Multi-drug resistant bacteria, MIC value. IPC code; Int. cl. 8 A61K 36/00, A61P 31/04 activities of plants have also been reported Therefore, in this context, 23 plants from 15 different families were screened against multi-drug resistant bacteria, viz. Staphylococcus aureus, S. epidermidis, Salmonella typhi and S. paratyphi A. Materials and Methods Plant materials Specimens of 23 species were collected either in the form of leaves or tubers (Curculigo orchioides Gaertn.) from different areas of Gujarat State (Table 1). All the plants were identified by Dr. A. S. Reddy, Plant Taxonomist, Department of Biosciences, Sardar Patel University. The collected plant materials were air dried under shade at room temperature and ground with a grinder into powder. Preparation of extracts Extracts were prepared by cold extraction method in which 100g of dry powdered material was soaked in methanol (5 200 ml) for 12 hours at room temperature and shaken occasionally. Each extract was filtered with Whatman filter paper No.1. The methanol filtrates were dried over water bath at 50 o C. Dried extracts were collected and stored in a refrigerator until further use. Micro-organisms In the present study, two Gram positive (Staphylococcus aureus and S. epidermidis) and two Gram negative bacteria (Salmonella typhi and S. paratyphi A) were tested which were obtained from Microbial Type Culture Collection (MTCC), Chandigarh. The bacterial cultures were grown in nutrient broth medium (HiMedia, ph 7.4) and maintained on nutrient agar slants. Antimicrobial assay Sensitivity test was performed by agar well diffusion method 12. An inoculum size of 10 8 CFU/ml of bacterium, compared with 0.5 Mc Farland turbidity standards, was used 13. About 100µl of plant extract (stock 100 mg/ml) was added carefully in a well of 10 mm diameter in a nutrient agar plate. Plates were kept in a refrigerator for prediffusion of extracts. Minimum inhibitory concentration was determined by two-fold serial broth Vol 6(4) July-August

2 dilution method. The MIC was tested in the concentration range of mg/ ml. Plates and tubes were incubated for 24 hours at 37 C in an incubator. The zone of inhibition was measured excluding the well diameter to evaluate the antimicrobial activity. Tubes showing no turbidity were recorded as MIC value. Antibiotic Ciprofloxacin at 20 µg/ml as a positive control and 100% DMSO as a negative control were used. Bioassay was carried out in duplicate and experiments were repeated twice. Results and Discussion The details of antibacterial activity of plant extracts used in this study are shown in Table 1; Figs Out of 23 plants, 11 plant species (47%, Zone of inhibition more than 5 mm) and 10 plant species (43%) were found to be effective against Staphylococcus aureus and S. epidermidis, respectively. Among different extracts, Mangifera indica Linn. and Manilkara hexandra (Roxb.) Dubard extracts were the most potent active extracts against both species of Staphylococcus (Figs. 1&2). The extracts of Adenanthera pavonina Linn., Nyctanthes arbor-tristis Linn. and Xanthium strumarium Linn. exhibited promising anti Staphylococcus aureus activity, whereas Cryptolepis buchanani (Linn.) Roem. & Schult., Pergularia daemia (Forsk.) Chiov. and Xanthium strumarium extracts Fig. 1 : Antibacterial activity of plant extracts against Staphylococcus aureus : A-35, Schrebera swietenioides; B-13, Cryptolepis buchanani;14, Curculigo orchioides; C-10, Cassia tora; 23, Mangifera indica; D-21, lpomoea obscura; 24, Manilkara hexandra; 26, Milletia ovalifolia Fig. 3 : Antibacterial activity of plant extracts against Salmonella typhi: A-34, Rauvolfia tetraphylla; 22, Leptadenia pyrotechnica; 40, Thevetia peruviana; B-1, Adenanthera pavonina; 21, lpomoea obscura; 26, Milletia ovalifolia Fig. 2 : Antibacterial activity of plant extracts against Staphylococcus epidermidis: A-1, Adenanthera pavonina; 24, Manilkara hexandra; 26, Milletia ovalifolia; B-30, Nyctanthes arbor-tristis; 31, Pergularia daemia; 44, Xanthium strumarium 302 Fig. 4 : Antibacterial activity of plant extracts against Salmonella paratyphi A: A-35, Schrebera swietenioides; 13, Cryptolepis buchanani; B-30, Nyctanthes arbor-tristis; 10, Cassia tora Natural Product Radiance

3 displayed profound antibacterial activity against Staphylococcus epidermidis. Salmonella typhi and S. paratyphi A, were inhibited by 3 plant species (16%) and 10 plant species (43%), respectively. Methanol extract of Rauvolfia tetraphylla Linn. exhibited significant antibacterial activity against both Salmonella species (Fig. 3A). The growth of Salmonella typhi was also inhibited by Schrebera swietenioides Roxb. and Wrightia tomentosa Roem. & Schult. extracts. The extracts of Hemidesmus indicus (Linn.) Schult., Mangifera indica, Wrightia tomentosa and Xanthium strumarium also showed more antibacterial activity against Salmonella paratyphi A than S. typhi. Antibacterial activity of Martynia annua Linn., Millettia ovalifolia Linn., Manilkara hexandra and Schrebera swietenioides is reported for the first time although the activity of Martynia annua and Millettia ovalifolia was not satisfactory. MIC value of active plant extracts was measured in two-fold dilution series of mg/ml. The highest level of activity against Staphylococcus aureus was noted with Cryptolepis buchanani extract showing MIC of 1mg/ml. The growth of Staphylococcus epidermidis was inhibited at MIC of 2mg/ml by Manilkara hexandra extract. Wrightia tomentosa and Xanthium strumarium extracts displayed MIC value of 2mg/ml for Salmonella paratyphi A whereas Schrebera swietenioides and Wrightia tomentosa showed MIC value of 4mg/ml for Salmonella typhi (Table 2). Medicinal plants constitute an effective source of both traditional and modern medicines. Screened plant extracts have shown effectiveness against one or more tested pathogenic strains. In this study, leaf extract of Cassia occidentalis Linn. was found to be more effective against Salmonella paratyphi A as compared to Salmonella typhi. The boiling water root extract of C. occidentalis have been found effective against Salmonella typhi 14. C. occidentalis leaf extract was effective against Staphylococcus aureus and S. epidermidis (Table 1) and similar results were obtained earlier against S. aureus 8. Leaf extract of Mangifera indica displayed good inhibitory efficacy against all pathogens with MIC value of 2-4 mg/ml. Previous study has indicated that the oil of balamani mango seed kernels was effective against S. epidermidis 15. Antifungal activity of Manilkara kauki (Linn.) Dubard has also been reported 16. However, during the present study M. hexandra extract effectively inhibited the growth of S. aureus and S. epidermidis, which is reported for the first time. Proteins isolated from leaves of Rauvolfia tetraphylla have been found to possess antifungal activity 17. R. tetraphylla leaf extract displayed significant inhibitory efficacy against both Salmonella species. The chloroform and ethyl acetate extracts of flowers of Nyctanthes arbor-tristis have shown antibacterial activity against some Gram positive and Gram negative microorganisms 18. In this study, methanol extract of leaves of N. arbor-tristis showed significant and moderate antibacterial activity against Staphylococcus epidermidis and Salmonella paratyphi A, respectively. The results obtained by Ahmad and Beg 2 differ against Staphylococcus aureus and Salmonella paratyphi A, which may be due to different extraction methods used for preparation of the extract. In the present study, methanol extract of Wrightia tinctoria R. Br. exhibited good inhibitory efficacy against both Staphylococcus and Salmonella spp. However, earlier reports have shown that water extract of air-dried leaves of W. tinctoria possess antibacterial and antifungal activity 19. Fatty oil isolated from seeds of W. tomentosa has shown potent antibacterial efficacy against Bacillus subtilis, Salmonella typhi and S. paratyphi 20, where as in the present studies, methanol extract of it exhibited significant antibacterial activity against Staphylococcus aureus and Salmonella paratyphi A. The present study conforms to the previous findings and focuses on the effect of crude extracts on the Staphylococcus aureus, S. epidermidis, Salmonella typhi and S. paratyphi A and determination of MIC values of effective extracts. Leaf extract of Xanthium strumarium was found to possess potent antibacterial efficacy against both Staphylococcus species and Salmonella paratyphi A. Other workers have also reported antibacterial activity of X. strumarium against Bacillus subtilis, Staphylococcus aureus and Proteus vulgaris but not tested against the organisms used in this study, which may be due to sesquiterpene xanthol 21, 22. The present study adds to the earlier information on antibacterial activity of more number of species. Vol 6(4) July-August

4 Table 1 : Antibacterial activity of plant extracts against Gram positive and Gram negative bacteria Plant name/family Zone of inhibition *(in mm diameter) Gram positive bacteria Gram negative bacteria Staphylococcus Staphylococcus Salmonella Salmonella epidermidis aureus typhi paratyphi A Adenanthera pavonina Linn. / Mimosaceae Caesalpinia crista Linn. / Caesalpiniaceae Cassia occidentalis Linn. / Caesalpiniaceae Cassia tora Linn. / Caesalpiniaceae Cryptolepis buchanani (Linn.) Roem.&Schult. / Asclepiadaceae Curculigo orchioides Gaertn. / Amaryllidaceae Hemidesmus indicus (Linn.) Schult. / Asclepiadaceae Ipomoea obscura (Linn.) Ker-Gawl. / Convolvulaceae Leptadenia pyrotechnica (Forsk.) Decne/ Asclepiadaceae Mangifera indica Linn. / Anacardiaceae Manilkara hexandra (Roxb.) Dubard / Sapotaceae Martynia annua Linn. / Martyniaceae Millettia ovalifolia Kurz / Fabaceae Murraya paniculata (Linn.) Jack. / Rutaceae Nyctanthes arbor-tristis Linn. / Oleaceae Pergularia daemia (Forsk.) Chiov. / Asclepiadaceae Rauvolfia tetraphylla Linn. / Apocynaceae Schrebera swietenioides Roxb. / Oleaceae Tabernaemontana divaricata (Linn.) R. Br. / Apocynaceae Thevetia peruviana (Pers.) Merrill / Apocynaceae Wrightia tinctoria R. Br. / Apocynaceae Wrightia tomentosa Roem. & Schult. /Apocynaceae Xanthium strumarium Linn. / Asteraceae * The cultures were incubated at 37 C and after 24 hours zone of inhibition was measured by excluding the diameter of well 304 Table 2 : Minimum inhibitory concentration of effective plant extracts Name of Plants Minimum inhibitory concentration (mg/ml) Gram positive bacteria Gram negative bacteria Staphylococcus Staphylococcus Salmonella Salmonella epidermidis aureus typhi paratyphi A Adenanthera pavonina Cryptolepis buchanani Mangifera indica Manilkara hexandra Nyctanthes arbor-tristis Schrebera swietenioides Wrightia tomentosa Xanthium strumarium (-) The MIC values were not tested since least activity was observed with crude extracts Natural Product Radiance

5 Conclusion Our results offer a scientific basis for the traditional use of plant extracts. Cryptolepis buchanani, Mangifera indica, Manilkara hexandra and Nyctanthes arbor-tristis extracts were found effective against Staphylococcus aureus, Staphylococcus epidermidis, Salmonella typhi and Salmonella paratyphi A which are associated with skin, respiratory diseases and enteric fever. However, in vivo studies on these medicinal plants are warranting determining toxicity of active constituents, their side effects and pharmacokinetic properties. Acknowledgements We are grateful to Microbial Type Culture Collection (MTCC), Chandigarh for providing the microbial cultures. We also thank Dr. A. S. Reddy for identification of plants used in the present study and Dr. R.P. Vaidya, Dr. V. Rama Rao and Mr. Kalpesh Ishnava for providing different plant samples. References 1. Anonymous, Centers for Disease Control and Prevention. Preventing emerging infectious diseases: A strategy for the 21 st century, 2000, pp Ahmad I and Beg AZ, Antimicrobial and phytochemical studies on 45 Indian medicinal plants against multi-drug resistant human pathogens, J Ethnopharmacol, 2001, 74, Threlfall EJ, Frost JA and Ward LR, Increasing spectrum of resistance in multi-resistant Salmonella typhimurium, Lancet, 1996, 343, Cowan MM, Plant products as antimicrobial agents, Clin Microbiol Rev, 1999, 12, Cragg GM, Newman DJ and Snader KM, Natural products in drug discovery and development, J Nat Prod, 1997, 60, Recio MC, A review of some antimicrobial compounds isolated from medicinal plants reported in the literature , Phytother Res, 1989, 3, Samy RP and Ignacimuthu S, Antibacterial activity of some folklore medicinal plants used by tribals in Western Ghats of India, J Ethnopharmacol, 2000, 69, Akinyemi KO, Oladepo O, Okawara CE, Ibe CC and Fasure KA, Screening of crude extracts of six medicinal plants used in South-West Nigerian unorthodox medicine for antimethcillin resistant Staphylococcus aureus activity, BMC Comp Alter Med, 2005, 5, Polombo EA and Semple SJ, Antibacterial activity of Australian plant extracts against methicillin resistant Staphylococcus aureus (MRSA) and Vancomycin resistant enterococi (VRE), J Basic Microbiol, 2002, 42, Rani P and Khullar N, Antimicrobial evaluation of some medicinal plants for their anti-enteric potential against multi-drug resistant Salmonella typhi, Phytother Res, 2004, 18, Perez C, Pauli M and Bazerque P, An antibiotic assay by the well agar method, Acta Biol Med Exp, 1990, 15, Perilla MJ, Ajello G, Cheryl Bopp MS, Elliott J, Facklam R, Popovic T and Wells J, Manual for the laboratory identification and antimicrobial susceptibility testing of bacterial pathogens of public health importance in the developing world, WHO, 2003, pp Perez C and Anesini C, In vitro antibacterial activity of Argentine folk medicinal plants against Salmonella typhi, J Ethnopharmacol, 1994, 44, Jain SC, Sharma RA, Jain R and Mittal C, Antimicrobial screening of Cassia occidentalis L In vivo and In vitro, Phytother Res, 1998, 13, Raman A, Nagendran S and Sundaramahalinam, Extraction, characterization and antimicrobial activity of mango seed oil, Asian J Chem, 1997, 9, Bhat N, Shivaprakasan ML and Jayarajan R, Antifungal activity of some plant extracts, Indian J For, 1994, 17, Thapliyal M, Ghosh M and Bennet SSR, Screening of six medicinal plants for their antifungal protein activity, Asian J Microbiol Biotechnol Environ Sci, 2000, 2, Khatune NA, Mosaddik MA and Haqe ME, Antibacterial and cytotoxicity of Nyctanthes arbor-tristis flowers, Fitoterapia, 2001, 72, Alam M, Joy S, Purusothaman KK, Ananden T and Veluchamy G, Studies on Wrightia tinctoria, Arogya J Health Sci, 1986, 13, Jain PP, Suri RK, Deshmukh SK and Mathus KC, Fatty acid oils from oil seeds of forest origin as antibacterial agents, Indian For, 1987, 113, Jawad ALM, Mahmoud MJ and Al-Naib A, Antimicrobial activity of Xanthium strumarium extracts, Fitoterapia, 1988, 59, Khalid S, Afza N, Rizvi HA and Badar Y, Antibacterial and phytochemical screening of some Pakistani medicinal plants of Compositae family, J Fac Pharm Gazi Uni, 1998, 15, Vol 6(4) July-August