ANTIMICROBIAL ACTIVITY OF METHANOLIC AND ACETONIC EXTRACTS OF AZADIRACHTA INDICA, SARACA ASOCA AND CURCUMA LONGA
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1 International Journal of Medicine and Pharmaceutical Sciences (IJMPS) ISSN Vol. 3, Issue 2, Jun 2013, TJPRC Pvt. Ltd. ANTIMICROBIAL ACTIVITY OF METHANOLIC AND ACETONIC EXTRACTS OF AZADIRACHTA INDICA, SARACA ASOCA AND CURCUMA LONGA WINEE SURABHI LALL 1, AMIT ALEXANDER CHARAN 2 & AKHILESH BIND 3 1,2 Department of Molecular and Cellular Engineering, JSBB, SHIATS, Allahabad, Uttar Pradesh, India 3 Department of Biochemistry and Bioprocess Technology, JSBB, SHIATS, Allahabad, Uttar Pradesh, India ABSTRACT In the present work an attempt has been made to analyse the antibacterial and antifungal potential of methanolic and acetonic extracts of Azadirachta indica, Saraca asoca and Curcuma longa against E.coli and B. subtilis among bacterial strains and A. niger and A. fumigatus among fungal strains by disc diffusion method and the antibacterial and antifungal activities were determined by measuring the diameter of zone of inhibition. The methanolic extracts of Curcuma longa possessed the highest antibacterial with a zone of inhibition of 20 mm for E.coli and highest antifungal activity with a zone of inhibition of 22 mm for A. fumigatus. Acteonic extracts, exhibited lesser antibacterial activity with respect to B. subtilis 8 mm and antifungal activities with respect to A. niger being 8 mm when compared to the methanolic extracts. KEYWORDS: Antibacterial, Antifungal, s, Acteonic Extracts INTRODUCTION According to World Health Organization medicinal plants serve as the best source for a variety of drugs. Therefore, such plants are being investigated for better understanding of their medicinal properties (Alo, et al., 2012). Plants have been used since ancient times to treat common infectious diseases whether locally within the dermis or a blood infection (Ozcan et al., 2009). Resistance towards drugs developed by pathogenic microorganisms due to their indiscriminate use and side effects due to synthetic drugs, has recently drawn much attention towards plant extracts and biologically active compounds isolated from plant species used in herbal medicine. Medicinal plants represent an alternate to synthetic drugs for the treatment of several non severe infectious diseases and can also be a possible source for new potent antibiotics to which pathogen strains are not resistant. (Mathur et al., 2011). In the present study Antibacterial and antifungal activity of methanolic and acetonic extracts of Azadirachta indica, Saraca asoca and Curcuma longa has been studied. Antibacterial and antifungal properties of Azadirachta indica, Saraca asoca and Curcuma longa have previously been reported by several authors (Pradhan et al., 2009, Maragathavalli et. al., 2012 Mahesh and satish, 2008 and Pundir and Jain 2010). Leaves of Azadirachta indica possess antibacterial, antifungal, antiviral, and even antiparasitic property and therefore exhibit wide range of pharmacological activities of neem leaf which may include immunodulatory, anti-inflammatory, antihyperglycaemic, antiulcer, antimalarial, antifungal, antibacterial, antiviral, antioxidant, antimutagenic and anticarcinogenic properties. (Subapriya et al., 2005). The leaf extracts of the plant as well as exhibit antidermatophytic activity against dermatophytes such as Trichophyton ruberum, Trichophyton, Mentagrophytes, Tricophyton violaceum, Microsporum nanum and Epidermophyton floccosum.(heinrich et al., 2005). Saraca asoca exhibits antibacterial and antifungal activities against several bacterial Bacillus subtilis, E. coli, Salmonella typhosa and staphylococcus aureus and fungal Alternaria cajani, Helminthosporium sp., Curvularia lunata and Fusarium sp., species.besides this, Saraca asoca have also been reported to have Anti-cancer activity, Anti-menorrhagic activity, Antioxytoxic activity (Pradhan et al., 2009). Phytochemicals present in turmeric have been reported to have potential effects
2 80 Winee Surabhi Lall, Amit Alexander Charan & Akhilesh Bind on diseases, such as cancer, alzheimer s disease, arthiritis and diabetes (Mishra and Palanivelu, 2008 and Mahady et al., 2002). The present investigation was undertaken considering the vast potentiality of plants as sources of antibacterial and antifungal agents. Therefore, in the present work an attempt has been made to analyse the antibacterial and antifungal potential of methanolic and acetonic extracts of Azadirachta indica, Saraca asoca and Curcuma longa against selected pathogenic bacterial and fungal strains. MATERIALS AND METHODS Collection Processing of Plant Materials The fresh leaves of Azadirachta indica, Saraca asoca and Curcuma longa were collected from the Nursery of School of Forestry and Environment, SHIATS, Allahabad. The fresh leaves were surface sterilized simply by washing under tap water and distilled water and were shade dried. Preparation of Methanolic and s The completely shade dried leaves were grounded to fine powder and allowed soxhlet for further acetonic and methanolic extract preparation. The obtained extracts were then subjected to rotary evaporator and evaporated to dryness and stored in air tight bottles at 4⁰C for further use. Preparation of Methanolic and s 5gm dried leaves of Azadirachta indica, Saraca asoca and Curcuma longa were taken in separate Erlenmeyer flasks to which 50ml of required solvent i.e., methanol and acetone were added and kept in an orbital shaker for 24 h. After 24 h, it was filtered and filtrate was collected and kept in incubator at 37 0 C till all solvents completely evaporated from mixtures. The remaining powdered extract was weighed and dissolved in double amount of DMSO. It was further stored at 4 0 C till use. Microorganisms Two different bacterial strains viz., E.coli and B. subtilis and two different fungal strains viz., Aspegillus niger and Aspergillus fumigatus were used for testing antibacterial and antifungal activities. The test organisms used in this study were obtained from Microbiology department of SHIATS. The bacterial strains were cultured and maintained on nutrient agar media slants and the fungal strains were cultured and maintained on potato dextrose agar slants. The cultures were maintained by subculturing periodically and preserved at 4⁰C. Antibacterial Activity The antibacterial activity was done using methanolic and acetonic extracts of Azadirachta indica, Saraca asoca and Curcuma longa by disc diffusion method (Sardari et al., 1998). Filter paper disc (6mm in diameter) impregnated with acetonic and methanolic extracts were placed on test organism seeded plates test organism used were E.coli and B. subtilis. Autoclaved distilled water was used as control. The plates were incubated at 37 0 C for 24 h, after which the results were observed in terms of zone of inhibition. The diameters of the zones were measured in mm. Antifungal Activity The antifungal activity was done by disc diffusion method (Mahesh and Satish, 2008) using methanolic and acetonic extracts of Azadirachta indica, Saraca asoca and Curcuma longa. Potato dextrose agar plates were inoculated with each fungal culture viz. Aspergillus niger and Aspergillus fumigatus separately. Filter paper disc (6mm in diameter)
3 Antimicrobial Activity of Methanolic and s of Azadirachta indica, Saraca asoca and Curcuma longa 81 impregnated with acetonic and methanolic extracts were placed on test organism seeded plates test organism used were Aspergillus niger and Aspergillus fumigatus. Autoclaved distilled water was used as control. The plates were incubated at 28 0 C for 24 h, after which the results were observed in terms of zone of inhibition. The diameters of the zones were measured in mm. RESULTS The antibacterial and antifungal activities were estimated using methanolic and acetonic extracts of Azadirachta indica, Saraca asoca and Curcuma longa against E.coli and B. subtilis and A. niger and A. fumigatus respectively by disc diffusion method and the antibacterial and antifungal activities were determined by measuring the diameter of zone of inhibition. Solvents Bacterial Table 1: Antibacterial Activity of Azadirachta indica against E. coli and B. subtilis Distilled Water (Control) E. coli B. subtilis Figure 1: Antibacterial Activity of Azadirachta indica against E.coli and B.subtilis The results of this study reveal that Azadirachta indica, Saraca asoca and Curcuma longa posseses potential antibacterial activity against E. coli and B. subtilis. With the highest antibacterial activity against E. coli 20 mm for methanolic extract of Curcuma longa (Table 3 and Figure 3) and a minimum antibacterial activity in E. coli with a zone of inhibition of 8 mm was observed for acetonic extract of Saraca asoca (Table 2 and Figure 2). Distilled water (control) showed no zone of inhibition. Similar results were seen with ethanolic and methanolic extracts of Azadirachta indica by Maragathavalli et al. (2012) and ethanolic extracts of Saraca asoca showed similar results when tested against E. coli (Singh et al., 2009; Pradhan et al., 2009). Solvents Bacterial Table 2: Antibacterial Activity of Saraca asoca against E. coli and B. subtilis Distilled Water (Control) E. coli B. subtilis
4 82 Winee Surabhi Lall, Amit Alexander Charan & Akhilesh Bind Bacterial Figure 2: Antibacterial Activity of Saraca asoca against E. coli and B. subtilis Table 3: Antibacterial Activity of Curcuma longa against E. coli and B. subtilis Solvent in mm) Distilled Water (Control) in E. coli B. subtilis Figure 3: Antibacterial Activity of Curcuma longa against E. coli and B. subtilis Similarly, the methanolic and acetonic extracts of the three medicinal plants posseses antifungal activity against A. niger and A. fumigatus with the maximum zone of inhibition against A. niger 22 mm for methanolic extract of Curcuma longa (Table 6 and Figure 6) and the minimum inhibition was shown by acetonic extract of Azadirachta indica against A. niger 8 mm. Distilled water (control) showed no zone of inhibition. Fungal Table 4: Antifungal Activity of Azadirachta indica against A. niger and A. fumigatus Solvent Distilled Water (Control) A. niger A. fumigatus
5 Antimicrobial Activity of Methanolic and s of Azadirachta indica, Saraca asoca and Curcuma longa 83 Fungal Figure 4: Antifungal Activity of Azadirachta indica against A. niger and A. fumigatus Table 5: Antifungal Activity of Saraca asoca against A. niger and A. fumigatus Solvent in Distilled Water (Control) in A. niger A. fumigatus Fungal Solvent Figure 5: Antifungal Activity of Saraca asoca against A. niger and A. fumigatus Table 6: Antifungal Activity of Curcuma longa against A. niger and A. fumigatus in in Distilled Water (Control) A. niger A. fumigatus Figure 6: Antifungal Activity of Curcuma longa against A. niger and A. fumigatus
6 84 Winee Surabhi Lall, Amit Alexander Charan & Akhilesh Bind DISCUSSIONS Plants are becoming an important source of potentially useful structures for the developments of new chemotherapeutic agents (Mahesh and Satish, 2008).Medicinal plants have attracted the attention of several biological communities (Penecillia and Magno, 2011). From the present investigation it was observed that the leaves of three medicinal plants viz. Azadirachta indica, Saraca asoca and Curcuma longa posseses potential antibacterial activity against E. coli and B. subtilis and antifungal activity against A. niger and A. fumigatus. It has been speculated that the methanolic extracts possessed the highest antibacterial and antifungal properties against the selected bacterial and fungal cultures with Curcuma longa as the best antibacterial and antifungal agent. Acteonic extracts was capable of inhibiting the pathogenic bacterial and fungal species however, lesser antibacterial and antifungal activities were observed when compared to the methanolic extracts, with Azadirachta indica being the least effective against fungal pathogens and Saraca asoca least effective against bacterial pathogens. Similar results were obtained with methanolic and acetonic extracts of Acacia nilotica, Sida cordifolia, Tinospora cordifolia, Withania somnifera and Ziziphus mauritian when tested against several bacterial and fungal species (Mahesh and Satish, 2008). In another study, methanolic extracts of Eucalyptus camaldulensis inhibited the growth of Bacillus subtilis and Staphylococcus aureus (Babayi et al., 2004). Plants generally produce secondary metabolites which constitute an important source of antimicrobial agent and many pharmaceutical drugs (Varaprasad et al., 2009). The inhibitory effects of plants on microorganisms may be therefore, due to the presence of certain phytochemicals such as flavanoids, alkaloids, phenols and saponins (Babayi et al., 2004). Therefore, from the present investigation it is observed that leaves of Azadirachta indica, Saraca asoca and Curcuma longa have highly potent medicinal values and can therefore be utilized as the source of antimicrobial compounds.f CONCLUSIONS It can be concluded that methanolic plant extracts give the maximum inhibition against several pathogenic bacterial and fungal species, the acetonic extracts were also reported to be having antibacterial as well as antifungal properties but, to a lesser extent as compared to those of the methanolic extracts. Thus, it can be said that common plants have great potential as antimicrobial compounds; hence, drug development plant based compounds could be useful in meeting the demand of lesser side effects of synthetic drugs. They can therefore be used for the treatment of several harmful infectious diseases caused by resistant microorganisms. REFERENCES 1. Alo, M. N., Anyim, C., Igwe, J. C., Elom, M. and Uchenna, D. S. (2012). Antibacterial activity of water, ethanol and methanol extracts of Ocimum gratissimum, Vernonia amygdalina and Aframomum melegueta Advances in Applied Science Research, 3 (2): Babayi H., Kolo I., Okogun J. I. and Ijah U. J. J. (2004). The antimicrobial activities of methanolic extracts of Eucalyptus camaldulensis and Terminalia catappa against some pathogenic microorganisms BIOKEMISTRI 16(2): Heinrich, M.G. and Nesbitt, M., (2005). The Search for Biologically Active Secondary Metabolites, The cultural history of plants, London: Routledge Mahady, G.B., Pendland, S.L., Yun, G. and Lu, Z.Z. (2002). Turmeric and Curcumin Inhibit the Growth of Helicobacter pylori, a Group of Carcinogen, Anticancer Research, 22:
7 Antimicrobial Activity of Methanolic and s of Azadirachta indica, Saraca asoca and Curcuma longa Mahesh B. and Satish S. (2008). Antimicrobial Activity of Some Important Medicinal Plants Against Plant and Human Pathogens World Journal of Agricultural Sciences 4 (5) Maragathavalli, S., Brinda, S., Kaviyarasi, N.S., Annadurai, B. and Gangwar, S.K. (2012). Antimicrobial Activity in Leaf Extract of Neem, International Journal of Science and Nature, 3: Mathur Abhishek, Singh Ravinder, Yousuf Sajad, Aprajita Bhardwaj, Verma Satish K., Babu Pradeep, Gupta Vinay, Prasad GBKS and Dua V.K. (2011). Antifungal activity of some plant extracts against Clinical Pathogens. Advances in Applied Science Research, 2 (2): Mishra, S. and Palanivelu, K. (2008). The effect of Curcumin on Alzheimer s Disease: an overview, Annals of Indian Academy of Neurology,11: Ozcan Birgul, Mari Esen, M. Kemal Sangun, Arzu Coleri and Mahmut Caliskan (2010). Effective antibacterial and antioxidant properties of methanolic extract of Laurus nobilis seed oil Journal of Environmental Biology 31(5) Penecilla, Gerard L and Magno Celia P. (2011). Antibacterial Activity of Extracts of Twelve Common Medicinal plants in Philipines. Journal of Medicinal Plants Research. 5 (16) Pradhan, P., Joshep, L., Gupta, V., Chulet, R., Arya, H., Verma, R. and Bajpai, A. (2009). Saraca asoca (Ashoka): A Review, Journal of Chemical and Pharmaceutical Research, 1: Pundir Ram Kumar and Jain Pranay (2010). Comparative Studies On The Antimicrobial Activity Of Black Pepper (Piper Nigrum) and Turmeric (Curcuma Longa) Extracts International Journal of Applied Biology and Pharmaceutical Technology I (2) Singh, A., Singh, S., Sharma, B.K., Singh, U.P., Srivastava, R. and Singh, K.P. (2009). Saraca asoca (Ashoka): A Review, Journal on Alternative Medicine, 6: Subapriya, R. and Nargis, S. (2005). Medicinal Properties of Neem Leaves, A review Journal of Current med. chem. Anticancer agent, 2: Varaprasad Bobbarala, Prasanth Kumar Katikala, K. Chandrasekhar Naidu and Somasekhar Penumajji. (2009) Antifungal activity of selected plant extracts against phytopathogenic fungi Aspergillus niger F2723. Indian Journal of Science and Technology 2(4) 87-90
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