BIOLOGIA (PAKISTAN) 01, 8 (1&), 17-17 PK ISSN 000-09 Antibacterial activity of crude s of different parts of Butea monosperma (Lamk.) Taub. *MIAN SHAHZADA ZIA AHMAD & ZAHEER-UD-DIN KHAN Department of Botany, GC University, Lahore. ABSTRACT The antibacterial activity of leaves, bark, flowers and seeds of Butea monosperma (Lamk.) Taub. is a medicinal plant of Indo-Pak Subcontinent were investigated against five strains of bacteria viz; Staphylococcus aureus, Klebsiella pneumonia, Pseudomonas aeruginosa, Bacillus subtilis, Escherichia coli using the hole plate method. Two methods were employed for the ion of crude s, the maceration and the Soxhlet methods. The crude s in petroleum ether, chloroform, methanol and water of different parts of plants showed significant differences. The results obtained were encouraging as the methanol and aqueous s of different parts of plants exhibited comparatively higher antibacterial activity but less than one exhibited by standard antibacterial discs. The methanolic of flowers exhibited slightly better action against most of the microorganisms tested. The MIC values of the s against microorganisms tested were ranging from mg/ml to mg/ml. Keywords: Antimicrobial activity, crude s, Butea monosperma, zone of inhibition, minimum inhibitory concentration INTRODUCTION The traditional uses of plants as medicine for the treatment of microbial infections are very effective. Medicinal plants show antimicrobial activity (Chan et al., 008). Now the use of traditional medicines has expanded globally and become popular. Continuously the use of plants for primary healthcare in underprivileged countries has increased (Tadeg et al., 00). About 80% of world s population, especially the people in the rural area rely upon herbal medicine for the remedy of their ailments (WHO, 001). The continuous search for the source of new antibiotics is needed to face the problem of increasing resistant strains of bacteria (Gibbons, 199). There has been a renaissance of attention in the development of drugs from plant sources. The substances derived from plants could be found in their parts like roots, leaves, shoots, bark, flower and seeds of plants. Many plants are used to form crude s to treat common infections (Noumi & Youmi, 001).The plant selected for the present study was B. monosperma an ethnobotanically important plant of Pakistan which is used locally in the treatment of various diseases. MATERIALS AND METHODS Plant material Fresh parts of B. mmonosperma were obtained from the natural vegetation of tehsil Shakargarh, District Narowal, Punjab. *Corresponding author: ziaahmadravian@yahoo.com
18 M. S. Z. AHMAD & Z. KHAN BIOLOGIA (PAKISTAN) Preparation of plant s Collected plant materials were air dried and ground into fine powder. The crude s were obtained by using standard techniques of maceration and Soxhlet method. The filtrates were concentrated through rotary evaporator at ºC. Dried s were stored in refrigerator at ºC for further use. Antimicrobial activity The test microorganisms used in this study included five bacterial species, two Gram-positive (Staphlococcus aureus and Bacillus subtilis) and three Gram-negative (Pseudomonas aeruginosa, Bacillus subtilis and Escherichia coli). The bacteria were cultured on the nutrient agar medium. The well plate method was used for the determination of zone of inhibition. MIC was carried out according to Murray et al. (1999) using Broth dilution essay. Statistical analysis The results presented are a means of three independent measurements. Means were compared by Student s T test and differences were considered to be significant when p< 0.0. RESULTS Antimicrobial activity of crude s of leaves The results depicted in Table 1 indicated that petroleum ether of leaves obtained by maceration method had inhibitory effects against most of the test organisms except K. pneumonia while chloroform inhibited the growth of, and. However, the methanol s showed inhibition against all the microbes used. It is obvious from the results, that the aqueous s of leaves taken by Soxhlet method had inhibitory action against,,, a and. The methanol showed inhibitory action against all the test microorganisms. Antimicrobial activity of crude s of bark The petroleum ether of bark obtained by maceration method showed no inhibitory effect on the growth of most of the test organisms except, and (Table 1). The chloroform had no inhibitory effect on all the test organisms. Methanolic exhibited antibacterial action against and. Table 1 illustrates that the aqueous taken by Soxhlet method of bark had inhibitory action on K. pneumonia, P. aeruginosa and, while the methanolic exhibited antimicrobial activity against most of the test organisms except K. pneumonia and. Antimicrobial activity of crude s of flowers Petroleum ether of flowers inhibited the growth of, P. aeruginosa and (Table 1). Chloroform of the flower exhibited antimicrobial activity against,, and. The methanolic showed inhibitory action against most of the organisms tested except K. pneumonia. The of flowers showed significant inhibitory action on. Aqueous of flowers by Soxhlet method displayed antimicrobial activity against most of the microorganisms used (Table 1). Methanolic by Soxhlet method of flowers showed a significant inhibitory
VOL. 8 (1&) ANTIMICROBIAL ACTIVITY OF B. MONOSPERMA 19 activity against,, and. On the other hand, no antimicrobial activity was recorded against. Table 1: Mean Zone of Inhibition produced by crude s of parts of B. monosperma Plant part Test Microorganisms Zone of Inhibition (mm) By Maceration method By Soxhlet method Leaves Bark Flower Seeds Petroleum ether 8±0.77 10±1.1 9±0.77 1±0.77 ±0.77 8±0.77 1±0.77 1±0.77 17±1.1 11±0.77 8±0.77 ±1.1 Chloroform Methanol Aqueous 9±0.77 11±0.77 8±0.0 7±0.77 10±0.77 9±0.77 1±0.77 1±0.77 1±1.1 1±1.1 1±1.1 17±0.77 18±0.77 11±0.77 9±1.1 8±0.77 7±0.77 8±0.77 9±1.1 10±0.77 18±1.1 1±0.77 ±0.77 1±1.1 1±0.77 7±1.1 10±0.0 1±0.77 11±0.77 7±0.77 11±0.77 1±0.77 1±0.77 17±0.77 0±0.77 18±0.77 ±0.77 1±1.1 1±0.77 All the results are mean of three parallel replicates. ± indicates the standard error. Methanol 1±0.77 1±0.77 18±0.0 1±0.77 0±1.1 1±0.77 1±1.1 1±1.1 18±0.77 1±0.0 ±0.77 1±0.77 9±1.1 1±0.0 1±1.1 Antimicrobial activity of crude s of seeds Petroleum ether showed antimicrobial activity against, and E coli. Chloroform of seeds retarded the growth of S. aureus. Methanolic of seeds exhibited the antimicrobial activity against most of the microorganisms tested except K.pneumonia. Aqueous of flowers showed antimicrobial activity against most of the test microorganisms. The inhibition zone produced by the action of aqueous against was 18 mm. Methanolic of flower taken by Soxhlet method showed a significant inhibitory activity against,, and E. coli, but no activity against. Antimicrobial activity of standard discs The data in Table showed the antimicrobial activity of different standard discs against test microorganisms. The Oflaxacin µg showed significant inhibition against all the bacteria used as compared to Ampicilin 10 µg and tetracyclin 0 µg. The inhibition zone produced by Oflaxacin against P. aeruginosa was mm.
170 M. S. Z. AHMAD & Z. KHAN BIOLOGIA (PAKISTAN) Table : Antibacterial activity of the standard discs Microorganisms Zone of inhibition (mm) Ampicilin Oflaxacin Tetracyclin 1±0.77 ±1.1 ±0.77 1±0.77 8±0.77 0±0.77 10±0.0 ±0.77 ±0.77 11±1.1 ±0.77 ±0.0 1±0.77 0±1.1 8±0.77 Minimum Inhibitory Concentration (MIC) MIC of crude s of leaves The MIC value of petrolem ether of leaves against and was mg/ml, for mg/ml, for mg/ml (Table ). The chloroform of leaves showed mg/ml, mg/ml and mg/ml, MIC value against, and respectively. The MIC value of methanolic against,,, and were mg/ml, mg/ml, mg/ml, mg/ml and mg/ml, respectively. The MIC value of aqueous of leaves against and P. aeruginosa was mg/ml, for and mg/ml and for mg/ml (Table ). The MIC value of methanolic of leaves obtained by Soxhlet method against, and mg/ml, for was mg/ml and for mg/ml. MIC vale of crude s of bark The MIC value of petroleum ether of bark taken by maceration method against was 7 mg/ml and for was mg/ml (Table ). The methanolic showed mg/ml and mg/ml MIC values for P. aeruginosa and, respectively. The MIC value of the aqueous of bark against K. pneumonia, and was mg/ml and the MIC value of methanolic of bark by Soxhlet method against and was mg/ml. MIC value of crude s of flowers The petroleum ether of flowers showed mg/ml, mg/ml and mg/ml MIC value against, and respectively (Table ). The MIC value of chloroform against,, was mg/ml and for was mg/ml. The MIC value of methanol against was mg/ml, for was mg/ml and for was mg/ml. The aqueous of flowers showed mg/ml value against while mg/ml for, and E.coli (Table ).The MIC value of methanol of flowers by Soxhlet method against, and was mg/ml, for was mg/ml. MIC value of crude of seeds The petroleum ether of seeds obtained by maceration method showed mg/ml MIC value against and and MIC value of chloroform against was mg/ml (table ). The methanol showed mg/ml MIC value against, and. The aqueous of seeds showed mg/ml MIC value against, mg/ml
VOL. 8 (1&) ANTIMICROBIAL ACTIVITY OF B. MONOSPERMA 171 for while mg/ml for. The MIC value of methanol of seed against was mg/ml, for and was mg/ml. Plant part Table : The MIC value in mg/ml of crude s of parts of B. monosperma Microorganisms MIC (mg/ml) By Maceration method By Soxhlet method Leaves Bark Flowers Seeds Petroleum ether 7 Chloroform Methanol Aqueous Methanol DISCUSSION Plants provide a source of inspiration for novel drug compounds as plant derived medicines have made significant contribution towards human health. Phytomedicines can be used for the treatment of diseases as in case of Unani and Ayurvedic system of medicine or it can be the base for the development of a new medicine, as natural blueprint for the development of new drugs (Javid & Ali, 00). Present study was conducted to investigate the antimicrobial activity of ethnobotanically important plant used in Indo-Pak Subcontinent, i.e. B. monosperma. The well plate method was preferred to be used in this study since it was found to be better than the disc diffusion method (Essawi & Scour, 000). The s of different parts (i.e. leaves, bark, flowers and seeds) of the plant showed significant differences against microorganisms tested. These differences may be attributed to the fact that the cell wall of gram positive bacteria is single layered, whereas the gram negative bacterial cell wall is multilayered structure and fungus cell wall is quite complex (Yao & Moellering, 199). The s obtained by Soxhlet method showed more antimicrobial activity against the test microorganisms than the s obtained by maceration
17 M. S. Z. AHMAD & Z. KHAN BIOLOGIA (PAKISTAN) method. Therefore, it can be concluded that the Soxhlet method of ion was found more reliable as compared to the maceration method. The results obtained were encouraging as the methanolic and aqueous s of different parts of plant showed considerable antimicrobial activity. The reason of broad antimicrobial activity of methanol s could be that all of the identified components of plants, active against microorganisms are aromatic and may be containing saturated organic compounds are most often obtained through methanol or ethanol ion (Cowan, 1999). The comprehensive antimicrobial action of the B. monosperma against the microorganisms tested could be ascribed to the anionic components such as thiocynate, nitrate, chloride and sulphates besides other water soluble components which are naturally occurring in most plant materials as has been already reported by Darout et al. (00). The petroleum ether and chloroform s of different parts of B. monosperma showed mild antimicrobial action, may be due to little diffusion properties of these s in the agar. The methanolic of flowers exhibited slightly better inhibitory action against most of the microorganisms tested than the methanolic s of the other parts. On the other hand, the aqueous s of leaves and flowers showed better results for the growth inhibition of the microorganisms tested. was found more susceptible to all the types of s of different plant parts except that of the bark. Moreover, gram-positive bacteria were found to be more susceptible than gram-negative bacteria. This could be due to the fact that gram-positive bacteria lack the natural sieve effect against large molecules due to the effect against large molecules due to small pores in their cell envelops (Gould & Booker, 000). The MIC values of s were in mg/ml range, while the MIC of the standard discs as evaluated by Dulger & Gonuz (00) was in µg/ml. Thus, the s of all parts of plants were active against microorganisms tested although the activity was much lower than the standard discs. The activity of plant s against bacteria may be indicative of the presence of antibacterial compounds or simple general metabolic toxins in the plant material. After the characterization of the active ingredients in these plants, new chemical classes of antimicrobial agents as new medicine for the maintenance of human health can be developed. REFERENCES Chan, L.W., Cheah, E.L.C., Saw, C.L.L., Weng, W. & Heng, P.W.S., 008. Antimicrobial and antioxidant activities of Cortex Magnoliae offcianalis and some other medicinal plants used in South-East Asia. Chinese Med., :1. Cowan, M.M., 1999. Plant products as antimicrobial agents. Clinic Microbiol. Rev., 1: -8. Darout, I., Cristy, A., Skaug, N. & Egeberg, P., 00. Identification and quantification of some potentially antimicrobial anionic components in Miswak. Ind. J. Pharm., :11-1.
VOL. 8 (1&) ANTIMICROBIAL ACTIVITY OF B. MONOSPERMA 17 Dulger, B. and Gonuz, A., 00. Antibacterial activity of certain plants used in Turkish traditional medicines. Asian J. Plant Sci., (1): 10-107. Essawi, T. & Srour, M., 000. Screening of some Palestinian medicinal plants for antimicrobial activity. J. Ethnopharm., 70:-9. Gibbons, A., 199. Exploring new strategies to fight drug resistant microbes. Science, 7: 10-108. Gould, D. & Booker, C., 000. Applied Microbiology for Nurses. Aardvak Editorial, Mcndham, Suffolk, pp: 7-9. Javid, S. & Ali, M., 00. Antimicrobial activity of higher plants. Hamdard Medicus, XLV (): 71-7. Murray, P.R., Baron, E.J., Pfaller, M.A., Tenover, F.C. & Yolke. R.H., 1999. Manual for Clinical Microbiology. 7 th Ed. Washington: ASM, 17-9. Noumi, E. & Youmi, A., 001. Medicinal plants used for intestinal diseases in Mbalmyo region. Cameroon, 7: -. Tadeg, H., Mohammed, E., Asres, K. & Gebre-Mariam, T., 00. Antimicrobial activities of some selected traditional Ethiopian medicinal plants used in the treatment of skin disorders. J. Ethnopharm., 100: 18-17. World Health Organization, 001. General guidelines for methodologies on research and evaluation of traditional medicine.who, Geneva, Switzerland, p.1. Yao, J. & Moellering, R., 199. Antibacterial agents. In Murray, P.R., Baron, E.J., Pfaller, M.A., Tenover, F.C. & Yolke. R.H., 1999. Manual Clinical Microbiol., 7 th Ed. Washington: ASM, 181-90.