In vitro antimicrobial activity of leaf extracts of certain mangrove plants collected from Godavari estuarine of Konaseema delta, India

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1 , ISSN Vol. 1, No. 2, pp , September 2011 RESEARCH ARTICLE In vitro antimicrobial activity of leaf extracts of certain mangrove plants collected from Godavari estuarine of Konaseema delta, India Vundru Anil KUMAR 1, Kandru AMMANI 2, Busi SIDDHARDHA 3* 1 Department of Biotechnology, Acharya Nagarjuna University, Guntur, India. 2 Department of Microbiology, Acharya Nagarjuna University, Guntur, India. 3 Department of Botany and Microbiology, Gurukula Kangri Vishwavidyalaya, Haridwar , Uttarakhand, India. Article History: Received 11 th September 2011, Revised 17 th September 2011, Accepted 18 th September Abstract: Several varieties of Mangrove plants were collected from Godavari estuarine of Konaseema delta which is an extended forest of Coringa. In the present study selected mangrove plants leaf extracts were screened for their antimicrobial activity against bacteria and fungi. The mangroves, Avicennia marina (Forsk) (Avicinniaceae), Excoecaria agallocha (Linn) (Euphorbiaceae), Lumintzera racemosa (Combretaceae), Derris trifoliata (Fabaceae), Brugiera gymnorrhiza (Rhizophoraceae), Ceriops decandra (Rhizophoraceae) and Acanthus ilicifolius (Acanthaceae) were selected for this study. The crude extracts from dried leafs of the mangroves were extracted using soxhlet extraction method and obtained extracts were concentrated by using rotary evaporator. Four solvents, Chloroform, Petroleum ether, Methanol and Ethanol were used to extract the leaf material. The crude extract obtained was evaluated for antimicrobial activity against bacteria & fungi by agar well diffusion method and the zone of inhibition diameters were calculated. The extracts at a concentration of 1500 µg/ml exhibited antibacterial and antifungal activity against test microorganism with degree of variation. Out of the screened seven mangrove plants D. trifoliate extracts of four solvents showed promising antimicrobial activity with inhibition zone diameter ranging from 22-14mm. B. gymnorrhiza and C. decandra extracts showed moderate antibacterial and antifungal activity (20-14mm). Among seven mangroves plant extracts screened for antibacterial and antifungal activity, extracts of A. marina, E. agallocha, L. racemosa and A. illicifolius exhibited least activity. Keywords: Derris trifoliate; antimicrobial activity; Godavari estuarine; Mangroves; zone of inhibition. Introduction Mangrove forests are the rich source for Biodiversity. Mangrove forests are widely distributed throughout coastal areas of India, especially estuaries. In Andhra Pradesh mangroves are widely distributed at Godavari & Krishna estuarine where it associates with Bay of Bengal. In East Godavari, Konaseema is popularly known as rice bowl of Andhra Pradesh. Coringa mangrove forest is rich in biodiversity of mangrove plants where it is located near to Kakinada and extended upto Konaseema deltaic zone throughout the Godavari estuarine. Mangrove plants and their products have been extensively used in traditional medicine. These plants are well known to have diverse natural products with great pharmaceutical importance and also exhibiting antimicrobial, anti larval, anti viral and anti insecticidal activity (Premnathan et al. 1992; Premnathan et al. 1996; Kokpal et al. 1990). There is a need of development of new drugs because of the resistance developed to existing antibiotics by pathogens. Hence there is a need to search and design new alternative drugs from natural plant products to control microbial infections. Mangrove plants are the best choice to isolate bioactive natural products active against bacteria and fungi. Antibacterial activity of mangroves against fish pathogens has already been studied by many authors. Extracts of Avicennia species showed a board spectrum of antimicrobial activity against Candida albicans, Mycobacterium vaccae, Mycobacterium aurum, Mycobacterium smegmatis, Mycobacterium fortuitum, and Staphylococcus aureus (Han et al. 2007; Jun et al. 2008). The leaf and stem extracts of E. agallocha collected from Bhitarkanika mangrove forest (India) showed antibac- *Corresponding author: ( ) siddhardha.busi@gmail.com 2011 Open Access Science Research Publisher

2 terial activity against human pathogenic microorganisms. It showed antibacterial activity against both gram positive and gram negative bacteria (Konishi et al. 1998, 2000; Masuda et al. 1999; Patra et al. 2009a). The fatty acid methyl ester extract from the leaf of E. agallocha showed strong antimicrobial and antifungal activity (Agoramoorthy et al. 2007). Acanthus illicifolius, a mangrove plant, which is useful in the treatment of paralysis, asthma, rheumatic pains and possess analgesic, anti-inflammatory, antileukemic activity and Leishmanicidal activities. Clerodendron inerme, exhibits larvicidal, antiviral and uterine stimulant activity (Sampson et al. 2000). The present study is focused on the exploitation of mangrove plant leaf extracts for their antibacterial and antifungal activities. Materials and methods Collection of Sample The leaves of Avicennia marina (Forsk) (Avicinniaceae), Excoecaria agallocha Linn (Euphorbiaceae), Lumintzera racemosa (Combretaceae), Derris trifoliata (Fabaceae), Brugiera gymnorrhiza (Rhizophoraceae), Ceriops decandra (Rhizophoraceae) and Acanthus ilicifolius (Acanthaceae) were collected from Coringa Mangrove wetland forest which is extended to Konaseema deltaic zone located at 16 33' 25.32" N and 82 12' 28.49" E (Figure 1). The plant materials were taxonomically identified and stored as specimens (Voucher no: ANU BT- 01) in Biotechnology Department, Acharya Nagarjuna University, Guntur. Preparation of plant powders & extracts The collected mangrove plants leaves were dried under shade and then powdered with mechanical grinder. The obtained plant powder was used to prepare crude extracts by using Soxhlet extraction method with the organic solvents. The crude obtained was connected with rotary evaporator. Four different solvents such as chloroform, petroleum ether, methanol and ethanol were employed for extraction of each plant species. 133 Antimicrobial activity Microbial strains Bacterial and fungal cultures used in this study to evaluate the antimicrobial activity of the various mangrove plant extracts are Staphylococcus aureus (MTCC 96), Staphylococcus epidermides (MTCC 435), Escherichia coli (MTCC 443), Bacillus subtilis (MTCC 441), Pseudomonas aeruginosa (MTCC 741), Klebsiella pneumoniae (MTCC 39), Aspergillus niger (MTCC 1344), Rhizopus oryzae (MTCC 262), Candida albicans (MTCC 227) and Saccharomyces cerevisiae (MTCC 170). These cultures were procured from microbial type culture collection centre (MTCC), IMTECH, Chand i- garh. Cultures were maintained on Nutrient Agar (NA) and Potato Dextrose Agar (PDA) medium for bacteria and fungi respectively. Determination of Antimicrobial activity The crude extracts of different mangrove plants leaves were used to determine the antimicrobial activity by agar cup plate method or agar well diffusion method described Murray et al. (1995) later modified by Olurinola (1996). The four organic solvents of all plants leaves were used in this method. Sterile nutrient agar and potato dextrose agar medium were prepared as per the composition and 0.2 ml of bacterial & fungal culture was spreader on each sterile petriplates. Approximately 6mm diameter wells were punched over the agar plates using sterile borer. The crude extracts were dissolved in dimethyl sulfoxide (DMSO) to obtain specific concentration. The cups were filled with 100 µl of leaf extract at a concentration of 1500 µg/ml was allowed to diffuse over the medium for min. The plates were incubated at 37 C for 24 hrs for bacteria. The above procedure was allowed for fungal assays except the media PDA instead of NA and incubated at 25 C for 48 hrs. The zone of clearance around each well after the incubation period, confirms the antimicrobial activity of each respective leaf extract. Similar procedure was followed to the remaining plant extracts to determine the anti microbial activity. Each experiment was carried out in triplicates. DMSO is used as control. The clear zones around the well were measured and average diameter of the inhibition zone was expressed in mm.

3 134 Figure 1: Geographical location of Godavari estuarine of Konaseema delta. Results and Discussion The mangrove plants leaves powder (1 kg) was obtained by mechanical grinding of dried leaves of seven plant species. The amount of crude leaf extract obtained by Soxhlet extraction using chloroform, petroleum ether, methanol and ethanol was summarized in Table-1. The antimicrobial activity of the mangrove leaf crude extracts (chloroform, petrolium ether, meth a- nol and ethanol) of Avicennia marina (Forsk), Excoecaria agallocha Linn, Lumintzera racemosa, Derris trifoliata, Brugiera gymnorrhiza, Ceriops decandra and Acanthus illicifolius were assayed in vitro by agar well diffusion method against bacterial and fungal strains. The zone inhibition diameter (IZD) measured was summarized in Table-2. Table 1: Amount of crude extract obtained from the 1 kg of leaves powder of selected mangrove plants. Name of the Plant Name of the Organic Solvents (crude extract obtained in grams) Chloroform Petroleum ether Methanol Ethanol A. marina E. agallocha L. racemosa D. trifoliate B. gymnorrhiza C. decandra A. ilicifolius

4 135 Int. J. Med. Arom. Plants Table 2: Antimicrobial activity of organic solvent extracts of mangrove plant leaves - Zone of inhibition (1500 µg/ml). Name of the Plant A. marina E. agallocha L. racemosa D. trifoliate B. gymnorrhiza C. decandra A. ilicifolius BACTERIA C P M E C P M E C P M E C P M E C P M E C P M E C P M E Gram positive S. aureus S. epidermides B. subtilis Gram negative E. coli P. aeruginosa K. pneumonia FUNGI A. niger R. oryzae C. albicans S. cerevisiae *Where C-Chloroform, P-Petroleum ether, M-Methanol, E-Ethanol extracts of dried leaves; zone of inhibition values are in mm Chloroform and methanolic extracts of E. agallocha showed antibacterial and antifungal activity with a zone of inhibition range of mm. the chloroform extract of E. agallocha was active against S. epidermide, B. subtilis, A. niger, R. oryzae and C. albicans with a zone of inhibition diameter of 18 mm. Chloroform Extracts of A. marina showed high antimicrobial activity against C. albicans, A. niger and S. aureus with a inhibitory zone diameter of 18 mm. The extracts of L. racemosa were moderately active against test bacteria and fungi IZD with a range of mm. Chloroform extracts of D. trifoliate were highly active against S. aureus and S. epidermides with a zone of inhibition of 22 mm. A. niger and S. cerevisiae were more sensitive to chloroform extract of D. trifoliate, exhibiting an IZD value of 20 mm. D. trifoliate other solvent extracts were moderately active against test bacteria and fungi. All the four solvent extracts of B. gymnorrhiza were active against S. aureus, E. coli, P. aeruginosa, A. niger and C. albicans with a zone of inhibition diameter of 20 mm. Gram negative bacteria was more sensitive to the crude extracts of B. gymnorrhiza. The solvent extracts of C. decandra were equipotent in their activity against the bacteria and fungi. The extracts are moderately active with IZD value in between 20 to 14 mm. A. illicifolius extracts were active against S. aureus, B. subtilis, P. aeruginosa, A. niger, R. oryzae, C. albicans and S. cerevisiae with zone of inhibition diameter of 18 mm. Fungal cultures were more sensitive to A. illicifolius extracts. Among the solvent extracts, Chloroform, Petroleum ether, Methanol and Ethanol extracts of D. trifoliate and B. gymnorrhiza were significantly active against the tested bacteria and fungi. The solvent extracts of C. decandra and A. illicifolius were moderately active, whereas A. marina, E. agallocha and L. racemosa extracts were less active. Further the extracts of D. trifoliate, B. gymnorrhiza, C. decandra and A. illicifolius can be explored for the study of phytochemicals present in them and to evaluate their biological activities. Conclusion The present study is conducted to develop new chemotherapeutic agents from the mangroves. The plant extracts showed antibacterial and antifungal activity with degrees of variation. Out of the seven plant extracts tested for biological activity, Derris trifoliata extracts exhibited higher inhibitory activity against bacteria and fungi. Further studies are needed to isolate pure compounds from these plant extracts and to establish the mode of action of the isolated compounds. References Agoramoorthy, G., Chandrashekaran, M., Venkatasalu, V., Hsu, M.J Antibacterial and antifungal activities of fatty acid methyl ester of the Blind-your-eye mangrove of India. Brazilian Journal of Microbiology, 38: Han, L., Huang, X., Dahse, H.M., Moellmann, U., Fu, H., Grabley, S., Sattler, I., Lin, W

5 Unusual naphthoquinone derivatives from the Twigs of Avicennia marina. Journal of Natural Products, 70: Jun, W., Qiang, X., Jing, X., Min, Y.L., Jian, Y.P., Mei-hua, Y Natural products from true mangrove flora: source, chemistry and bioactivities. Natural Product Reports, 25: Kokpal, V., Miles, D.H., Payne, A.M., Chittawong, V Chemical constituents and bioactive compounds from mangrove plants. Studies in Natural Products Chemistry, 7: Konishi, T., Takasaki, M., Tokuda, H., Kiyosawa, S., Konoshima, T Antitumor promoting activity of diterpenes from Excoecaria agallocha. Biological and Pharmaceutical Bulletin, 21: Konishi, T., Konoshima, T., Fujiwara, Y., Kiyosawa, S Exococcarins D, E and K from Excoecaria agallocha. Journal of Natural Products, 63: Masuda, T., Yonemori, S., Oyama, Y., Takeda, Y., Takana, T., Andoh, T., Shinohara, A., Nakata, M Evaluation of the antioxidants activity of environmental plants. Journal of Agriculture Food Chemistry, 47: Murray, P.R., Baron, E.J., Pfaller, M.A., Tenover, F.C., Yolken, H.R Manual of Clinical 136 Microbiology, 6 th Edition. ASM Press, Washington, DC, pp Olurinola, P.F A laboratory manual of pharmaceutical microbiology. Idu, Abuja, Nigeria, pp Patra, J.K., Panigrahi, T.K., Rath, S.K., Dhal, N.K., Thatoi, H.N Phytochemical screening and antimicrobial assessment of leaf extracts of Excoecaria agallocha L.: a mangal species of Bhitarkanika, Orissa, India. Advances in Natural and Applied Sciences, 3(2): Premnathan, M., Chandra, K., Bajpai, S.K., Kathiresan, K A survey of some Indian marine plants for antiviral activity. Botanica Marina, 35: Premnathan, M., Nakashima, H., Kathiresan, K., Rajendra, N., Yamamoto, N In Vitro antihuman immunodeficiency virus activity of mangrove plants. Indian Journal of Medicinal Research, 103: Sampson, J.H., Phillipsin, J.D., Bowery, N.G., O Neill, M.J., Houston, J.G., Lewis, J.A Ethnomedicinally selected plants as sources of potential analgesic compounds, indication of in vitro biological activity in receptor binding assays. Phytotherapy Research, 14:

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