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1 Indian Journal of Natural Products and Resources Vol. 1 (4), December 2010, pp. 481-484 Antimicrobial activity of Crotalaria burhia Buch.-Ham. root Sandeep Kataria1 *, Birendra Shrivastava1, R K Khajuria2, K A Suri2 and Piush Sharma1 1 Pharmacognosy Department, School of Pharmaceutical Sciences, Jaipur National University, Jaipur-302 025, India 2 Instrumentation Division, Indian Institute of Integrative Medicine, Jammu-180 001, India Received 30 December 2009; Accepted 18 August 2010 Crotalaria burhia Buch.-Ham. (Family- Fabaceae) is used traditionally in the treatment of eczema. Petroleum ether, chloroform, methanol and aqueous extracts of root were screened for antimicrobial activity against different strains of bacteria and fungi. The tests were carried out using agar disc diffusion method at four different concentrations of the crude extracts. All the extracts inhibited the growth of both Gram positive bacteria (Bacillus subtilis, Staphylococcus aureus, Micrococcus luteus) and Gram negative bacteria (Escherichia coli, Pseudomonas aeruginosa, Salmonella typhimurium). The Gram positive bacteria appeared to be more susceptible to the extracts than the Gram negative bacteria. Methanol extract at the concentration ranging between 250 g/ml and 1000 g/ml showed inhibitory activity against all bacteria while petroleum ether and chloroform extract showed moderate antimicrobial activity. Aqueous extract was found neutral against all bacteria. The extracts also showed significant antifungal activity against A. niger and C. albicans. All the microorganisms showed dose dependent susceptibility towards the extracts used in the study. The antibacterial and antifungal activity of the extracts and standard drugs were statistically significant. The results indicate the potential of C. burhia root in treating microbial infections, thus, justifying their traditional use in the treatment of eczema, which are of infectious origin. Keywords: Crotalaria burhia, Fabaceae, Eczema, Antimicrobial, Antibacterial, Antifungal. IPC code; Int. cl.8A61K 36/00, A61P 31/04, A61K 36/48 Introduction immunocompromised patients, who frequently develop Medicinal plants have long been the subject of opportunistic systemic and superficial mycoses13-14 human curiosity and need. Plant-derived products are such as candidiasis, dermato-mycosis, fungal present in 14 of the 15 therapeutic categories of infections, etc., have increased dramatically15-16. This is pharmaceutical preparations that are currently mainly due to the non-availability of effective recommended by medical practitioners and they form antifungal drugs for systemic fungal infections and an important part of the health-care system in the toxicity of available drugs like amphotericin-B17. Thus western world1. It is estimated that, there are about there is an increased need for the development of 2, 50,000 species of higher plants and the majority of alternative antipathogenic substances. One possible these have not been evaluated scientifically in detail for approach is to screen local medicinal plants in search their pharmacological activities. The antimicrobial of suitable chemotherapeutic antibacterial and properties of certain Indian medicinal plants were antifungal substances. The herbalists prescribed various reported based on folklore information2-9 and a few preparations of medicinal plants in treating ailments attempts have been made on inhibitory activity against such as itching, eczema, scabies and skin diseases18-20. certain pathogenic bacteria and fungi10. Infectious Crotalaria burhia Buch.-Ham. (Family-Fabaceae), diseases, particularly skin and mucosal infections are commonly known as Khip is an undershurb, fibrous common in most of the tribal inhabitants due to lack of plant, found all over the desert extensively growing on sanitation, potable water and awareness of hygienic sand dunes, common in the arid parts of West Pakistan, food habits. An important group of these skin India (Punjab, Rajasthan and Gujarat) and pathogens are the fungi, among which dermatophytes Afghanistan21-22. Fresh plant juice is applied on and Candida spp., besides certain pathogenic bacteria eczema. It is very useful in gout, hydrophobia and are the most frequent11-12. Furthermore, in the last few swelling23. Studies have shown that the plant possesses years, the numbers of immunosuppressed and anti-cancer property and roots are good coolant.21,24. __________ The present study was conducted to investigate *Correspondent author E-mail: [email protected] antimicrobial properties of different extracts of its root Phone: 09251445687 (Mob.) against wide range of bacterial and fungal species.

2 482 INDIAN J NAT PROD RESOUR, DECEMBER 2010 Materials and Methods indicate antimicrobial activity. All data are the average Plant material of quadrate analysis. Antibiotic Amikacin (10 g/ml) Root was collected during February-March 2009 and Griseofulvin (20 g/ml) were used as reference from Jaipur, Rajasthan, India. The plant material was standards as recommended by the National authenticated by the Joint Director, Botanical Survey of Committee for clinical laboratory standards25. India (BSI), Jodhpur (Rajasthan, India) and the voucher Statistical analysis specimen (JNU/JPR/PC/SK-1) has been deposited in Data are presented as the mean SD of four their herbarium. measurements. Statistical analysis was performed by The shade dried powdered root was successively students t-test26. extracted with petroleum ether, chloroform and methanol using Soxhlet apparatus. The extracts were Results and Discussion filtered and evaporated to dryness in vacuo at 40C. The percentage yield of the petroleum ether, Finally, the marc left was extracted with water under chloroform, methanol and aqueous extracts after reflux. The water phase was filtered and freeze-dried. successive extraction of root powder were 0.30, 0.20, Percentage yield of the extracts are present in results 1.92, 2.00%, respectively. It was observed that all the section. The dried extracts were dissolved in dimethyl extracts showed (Table 1) antibacterial activity sulfoxide and used for the present study. against all the microorganisms. However, no activity was observed against M. luteus, E. coli, P. aeruginosa Microorganisms and media and S. typhimurium at 100 and 250 g/ml Gram-positive bacteria: Bacillus subtilis, Staphylococcus concentration. It was also observed that all the aureus, Micrococcus luteus extracts exhibited (Table 2) antifungal activity against Gram-negative bacteria: Escherichia coli, Pseudomonas A. niger and C. albicans in a dose dependent manner. aeruginosa, Salmonella typhimurium These extracts showed a broad spectrum of activity Fungi: Aspergillus niger, Candida albicans. against the bacterial strains at the concentration of Bacteria and fungi were procured from the stock 100-1000 g/ml. Amikacin (10 g/ml) and cultures of the Institute of Microbial Technology Griseofulvin (20 g/ml) were used as positive (IMTECH), Chandigarh, India. The bacterial and fungal controls for bacteria and fungi, respectively. stock cultures were maintained on Muller Hinton agar The preliminary phytochemical screening of and Sabouraud-dextrose agar slants, respectively, which C. burhia root indicated the presence of the group were stored at 4C. Eight microorganisms maintained compounds such as alkaloids, phenols, polyphenols, on nutrient agar base were used to assess the saponins, tannins, triterpenes, anthraquinones, antimicrobial activity of the plant extracts. The fungi flavonoids and steroids. Many compounds belonging were maintained on Sabouraud-dextrose agar, which is to these secondary metabolite groups have been often used with antibiotics for the isolation of reported to their antimicrobial activities27. pathogenic fungi. In general, among the tested microbial strains, Antimicrobial screening bacteria were found to be more sensitive to many of the Agar cultures of the test microorganisms were test agents than fungi. The antibacterial activity was prepared as described by Mackeen et al 25. Three to five more pronounced on the Gram-positive bacteria similar colonies were selected and transferred to 5 ml (Staphylococcus aureus) than the Gram-negative broth with a loop and the broth cultures were incubated bacteria (Escherichia coli and Pseudomonas for 24 h at 37C. The extracts were dissolved in aeruginosa). The reason for the difference in sensitivity dimethyl sulfoxide with a magnetic stirrer. For between Gram-positive and Gram-negative bacteria screening, sterile 6 mm diam. filter paper discs were might be ascribed to the differences in morphological impregnated with 100-1000 g of different extracts and constitutions between these microorganisms, Gram- then placed in Muller Hinton agar medium. The negative bacteria having an outer phospholipidic inoculums for each organism were prepared from broth membrane carrying the structural lipopolysaccharide cultures. The concentration of culture was 1105 components. This makes the cell wall impermeable to colony forming units/ml. The results were recorded by antimicrobial chemical substances. The Gram-positive measuring the zones of growth inhibition surrounding bacteria on the other hand are more susceptible the disc. Clear inhibition zones around the discs having only an outer peptidoglycan layer which is not an

3 KATARIA et al: ANTIMICROBIAL ACTIVITY OF CROTALARIA BURHIA ROOT 483 Table 1Antibacterial activity of different extracts of Crotalaria burhia root and standard antibiotic Amikacin Samples Conc. (g/ml) Diameter of zone of inhibition (mm) BS SA ML EC PA ST Petroleum ether 100 6.4 0.14*** 6.3 0.08*** - - - - 250 6.9 0.08*** 6.4 0.14*** - - - - 500 7.2 0.17*** 7.9 0.08*** 6.4 0.14*** 8.5 0.21 7.2 0.17*** 6.9 0.08*** 1000 8.3 0.08*** 8.7 0.18*** 7.5 0.08*** 9.3 0.21 7.8 0.09*** 8.2 0.09 ** Chloroform 100 4.3 0.08*** 3.5 0.08** 4.3 0.18** 3.1 0.18** - - 250 5.2 0.14*** 4.6 0.14*** 5.9 0.21** 4.7 0.08** 5.9 0.21* - 500 6.3 0.21*** 5.9 0.08*** 7.8 0.08*** 6.7 0.08*** 6.3 0.08*** 7.9 0.08** 1000 14.5 0.21*** 13.4 0.08*** 11.7 0.16** 14.3 0.08*** 10.7 0.16*** 10.3 0.14* Methanol 100 8.1 0.26*** 7.4 0.08*** 5.6 0.08*** 8.6 0.24 - - 250 10.3 0.55*** 8.3 0.11*** 7.1 0.21** 9.3 0.08*** 8.0 1.38 8.1 0.08** 500 15.6 0.60*** 9.6 0.33*** 10.3 0.08*** 10.7 0.21 10.2 0.14*** 11.3 0.14* 1000 18.7 0.28*** 15.4 0.18*** 12.5 0.08*** 15.6 0.18* 18.3 0.08*** 17.2 0.14* Aqueous 100 - - - - - - 250 - - - - - - 500 6.7 0.14*** 6.9 0.08*** 6.6 0.08*** 6.9 0.14* - - 1000 7.3 0.14*** 7.6 0.21*** 7.7 0.21** 7.2 0.18** 6.9 0.08*** 7.0 0.08** Amikacin 10 24.2 0.6 22.8 1.0 17.4 1.0 19.3 0.6 23.4 1.4 22.3 0.6 BS- Bacillus subtilis, SA- Staphylococcus aureus, ML- Micrococcus luteus, EC- Escherichia coli, PA- Pseudomonas aeruginosa, ST- Salmonella typhimurium; -= NO inhibition zones Values are mean SD (mm) of four separate experiments Statistical value ***P

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