240 Journal of Pharmaceutical, Chemical and Biological Sciences ISSN: 2348-7658 Impact Factor (GIF): 0.615 Impact Factor (SJIF): 2.092 June-August 2015; 3(2):240-246 Available online at http://www.jpcbs.info Original Research Article Evaluation of Antifungal Activity of Crude Leaf Extracts of Indian Sacred Trees Amudha Selvi Muniyan1, Anu Swedha Anandhan2* 1 Post Graduate Department of Applied Microbiology, Justice Basheer Ahmed Sayeed College for Women, Chennai, Tamil Nadu, India. *Corresponding Author: Anu Swedha Anandhan ,Assistant Professor, Post Graduate Department of Applied Microbiology, Justice Basheer Ahmed Sayeed College for Women, Chennai, Tamil Nadu, India Received: 14 July 2015 Revised: 21 July 2015 Accepted: 28 July 2015 ABSTRACT Sacred trees are plants with a socio-economic, medicinal value which associates them with the Gods. Herbal medicines have been the basis of treatment and cure for various diseases and physiological conditions in traditional methods practiced in India such as Ayurveda, Unani and Siddha. Medicinal plants have been reported to have antimicrobial properties against many microbial organisms. Fungi are secondary invaders of an already weakened human body. Mycoses are still a critical cause of mortality second only next to bacterial diseases. Though significant advances have been made in antibacterial chemotherapy, there is a lack of serious strides in the area of antifungal drug discovery. In the present study, an attempt has been made to study and compare the antifungal efficacy of five leaf crude extracts of Indian sacred trees viz., Aegle marmelous Linn. Correa., Feronia elephantum Linn., Ficus benghalensis Linn., Ficus religiosa Linn., and Mimusops elengi Linn. The extracts which showed the highest activity were analysed and the minimum inhibitory concentration was determined. Leaf extracts of Aegle marmelos and Mimusops elengi showed good anticandidal and antifungal activity against most of the strains tested. Therefore, these medicinal trees should be conserved as sacred grooves and exploited commercially for drug development properties. Keyword: Sacred trees; mycoses; antifungal activity INTRODUCTION In India, any form of nature such as trees, containing indigenous plants and wild life. The rivers, mountains, animals are considered Pancha Bhootas such as air, land, water, fire sacred. Several villages had their own sacred (energy) and space were revered as sacred and groves which were mini biosphere reserves essential for life. Planting and nurturing trees J Pharm Chem Biol Sci, June-August 2015; 3(2):240-246 Anu et al 241 was a routine practice in ancient India. Sacred twigs and washed twice with double distilled groves are forest fragments of various sizes water and then surface sterilised using 70% which are communally protected and have a ethanol. The leaves were shade dried for 1-2 religious connection with the protecting weeks. The leaves were then ground into a community. Sacred groves are usually coarse powder form using a mixer. associated with temples, monasteries, shrines or burial grounds. Logging and hunting are Preparation of crude extract [5] prohibited strictly within these patches of trees; The crude extracts were prepared by hot and therefore they acted as a huge repository for cold method of extraction. In the hot method of various Ayurvedic medicines [1]. extraction, about 1 gram of the powdered plant Opportunistic systemic mycoses are those material was mixed with 10 ml of the solvent, infections, which are found in patients with incubated in a shaker at 37ºC for 4 hours at 250 underlying predisposing conditions whose rpm after which it was placed in a water bath at immunological defense mechanisms are 60°C for 2 hours. The supernatant was filtered weakened by endogenous causes like cancer, and dried in air at room temperature. For the leukemia or exogenous causes like cold method of extraction, about 1 gram of the immunosuppressive therapy and AIDS. of the powdered plant material was mixed with Causative agents of opportunistic mycoses 10 ml of the solvent, incubated in a shaker at include Candida albicans and different species 37°C for 4 hours at 250 rpm.The supernatant of Aspergillus. There is an emergence of drug filtered and then dried in air at room resistance among certain fungi causing temperature. The solvents used were opportunistic mycoses [2]. Medicinal plants water,ether, ethanol, methanol, chloroform, represent a rich source of antimicrobial agents. acetone and dichloromethane. The residue These plants are used in the form of extracts as obtained after drying was dissolved in the raw drugs but they are not evaluated properly appropriate solvent and used for anticandidal [3]. and antifungal screening. Today, though the number of sacred groves in different parts of India has declined, few are Microbial cultures used still protected as they serve as a biodiversity The test organisms used for screening were hotspot for the conservation of endangered Candida albicans,Aspergillus niger, Aspergillus flora and fauna. The present study is an attempt fumigatus and Aspergillus flavus.All the strains to re-establish the identity of sacred groves in were laboratory isolates of the Department of India by instilling the ethno medicinal Microbiology, J.B.A.S college for Women, importance of the trees in a sacred grove in Chennai. today’s fast paced changing scenario. Preliminary Screening using Disc Diffusion MATERIALS AND METHODS Assay [6,7] Collection and preparation of plant powder [4] Discs(6mm) prepared from Whatmann No.1 The medicinal trees included in the study were filter paper was sterilised and impregnated with Aegle marmelous (vilvam), Feronia elephantum 20µl of various crude solvent extracts (wood apple), Ficus benghalensis (banyan), (concentration:100mg/ml). Saline cultures of Ficus religiosa (peepal) and Mimusops elengi the microorganisms were prepared. The culture (Magilam).The leaves of the sacred trees were was checked for turbidity by comparing with collected from different temples in and around the McFarland Standard (4).A lawn culture of Chennai. The leaves were separated from the the organisms to be tested was made on the J Pharm Chem Biol Sci, June-August 2015; 3(2):240-246 Anu et al 242 Saboraud’s Dextrose agar media. The prepared effective. For Candida albicans, most of the discs were placed on the plate in a way such extracts showed an MIC value of 3.125 and 1.56 that each disc was at least 20mm from one mg/ml, for Aspergillus flavus and Aspergillus another. The plates were then incubated at fumigatus, all the extracts failed to grow at the 37°C for 24 hours for Candida albicans and 48 least concentration of 1.56 mg/ml, for hours for fungal molds. The inhibition zone Aspergillus niger, most of the strains showed an around each disc both in the experiment and MIC value of 6.25 and 12.5 mg/ml (table 3). the control were measured. Standard antibiotics as per the organisms tested were DISCUSSION included as positive control and respective Indian Medicinal plants are considered a vast solvents without the plant extracts were used source of several pharmacologically active as the negative control. principles and compounds, which are commonly used in home remedies against Determination of Minimum Inhibition multiple ailments [9, 10]. In the present study, Concentration/ Minimum Bactericidal for the determination of anticandidal activity, Concentration [8] the ethanol leaf extracts of Aegle marmelos and The Microbroth dilution was performed on a Mimusops elengi had shown maximum activity microtitre plate. Doubling dilutions of the crude followed by Ficus religiosa , Ficus benghalensis extract were prepared in Saboraud’s dextrose and Feronia elephantum. broth. Fungal and candidal cultures of 106 In the determination of antifungal activity, for cfu/ml dilution were prepared with McFarland Aspergillus flavus and Aspergillus fumigatus, the standard (4) and 10µl were added to each well ethanol extracts of Aegle marmelous and of the microtitre plate and mixed well. The Feronia elephantum showed maximum activity microtitre plates were incubated at 37ºC followed by Ficus benghalensis, Ficus religiosa overnight for Candida albicans and 48 hours at and Mimusops elengi. For Aspergillus niger, the room temperature for molds and a loopful of ether extracts of the medicinal trees showed the culture was streaked on to Saborauds’ maximum activity. dextrose agar plates. The plates were incubated In MIC assay to determine the MIC of the crude at the same temperature and time as before. extract, for Aspergillus niger, Mimusops elengi, The growth/no growth pattern of the organisms Feronia elephantum showed an MIC value of corresponded to the MIC /MBC of the crude 12.5mg/ml whereas Aegle marmelous and Ficus extract. benghalensis showed an MIC value of 25mg/ml and Ficus religiosa showed an MIC of RESULTS 3.25mg/ml. For Aspergillus flavus and In the preliminary screening of plant extracts Aspergillus fumigatus, the MIC value could not for anticandidal activity (table 1), the ethanol be determined, so further dilution of plant and ether extracts of the medicinal trees extract should be taken to determine the MIC showed maximum activity, followed by value. acetone, methanol and chloroform. Water Among the solvents used in the extraction extracts were least effective. In the preliminary procedures, ethanol and ether was found to be screening of plant extracts for anti-fungal highly effective in extracting the antifungal activity (table 2), the ethanol extracts of the