MATERIALS AND METHODS

Study area: The Similipal Biosphere Reserve massif lies between 20°17-22°10 N latitude and 85°57-86°47 E longitude is situated in Mayurbhanj district of northeast Orissa, India. Within the Mahanadian bio-geographic region, Similipal presents feature of four biotic provinces for which Orissa is the junction. These provinces are: Eastern Plateau, Chotanagpur Plateau, Lower Gangetic plains and the Coast Line.

Survey and collection of medicinal plants: The present work is based on explorations made in Similipal Biosphere Reserve during 2006. Field trips to Similipal Biosphere Reserve were undertaken, medicinal plants were collected and their identification was authenticated at the Post Graduate Department of Botany, North Orissa University, Baripada. Ethnomedicinal uses and medicinal properties of the plants were also collected during field trip as well as from literature survey (Table 1).

Processing: Stems, leaves and roots of plants have separately been collected during field trip from different places of Similipal Biosphere Reserve. The roots are dug out from the soil and the adhering soils were removed by shaking and washing. The leaves were plucked from the trees, washed properly and the leaves infected with fungus were discarded. After collection, the healthy leaves were dried at low temperature to maintain their green color and volatile oils, if present. The material is completely shed dried so long it does not allow for the growth of any type of fungi, molds, bacteria and other microorganisms. The dried leaves, roots and stems are powdered separately by using mortar and pestle and then they were passed through sieve so that uniform powders are maintained.

Preparation of plant extracts: Twenty grams of powder from each sample was dissolved in 100 mL of sterile distilled water separately in wide mouth bottle and were steamed for 30 min in a pressure cooker. Then all the treated samples were incubated at room temperature for 48 h. The suspension was filtered (Whatman No. 40) separately and the filtrate made up to 100 mL with sterile distilled water. The filtrate was used for studying their antibacterial properties.

Screening of antimicrobial properties
Media used: Muller Hinton Broth (MHB), Muller Hinton Agar (MHA) and Potato Dextrose Agar (PDA) procured from HI-Media, Mumbai, were used in the study and prepared as per manufacturer`s instructions.

Agar cup method: The agar cup method of Bauer et al. (1966) was followed to doubly ensure the antibacterial activity of the extracts. Over night Muller Hinton Broth culture of the test organisms were firmly seeded over the MHA plates. Wells of 0.8 mm diameter was punched over the agar plates using a sterile borer. The bottoms of the wells were sealed by pouring 50-100 μL of molten MHA into the scooped out wells. One hundred microliters of extracts were poured into the wells. The water was allowed to evaporate and the plates were incubated at 37°C for 18-24 h. A zone of clearance around the wells, after the incubation period confirms the antibacterial activity of the respective extracts. The same procedure was followed for each strain and extract. Each experiment was carried out in triplicates. The average diameter of the inhibition zone was taken for evaluating the antibacterial activity of the extracts.

RESULTS AND DISCUSSION

In total 40 medicinal plants belonging to 30 families were collected from Similipal Biosphere Reserve. The botanical and local names of the plants, the parts used along with their medicinal properties and ethnomedicinal uses are given in Table 1. Selection of the plants was based mainly on potential ethnomedicinal uses and possible antimicrobial and anticarcinogenic properties. Each of the 40 plants was prepared in the form of aqueous extracts and was tested against both gram +ve (6) and gram -ve (4) bacteria viz., Staphylococcus aureus MTCC 1144, Bacillus licheniformis MTCC 7425, Bacillus brevis MTCC 7404, Bacillus subtilis MTCC 7164, Staphylococcus epidermidis MTCC 3615, Streptococcus aureus (Lab. isolate), Pseudomonas aeruginosa MTCC 1034, Escherichia coli MTCC 1089, Vibrio cholerae (Lab. isolate), Shigella flexneri (Lab. isolate) and one fungus Candida krusei (Lab. isolate). Table 2 represents that out of the 40 medicinal plants 23 plants found to posses antimicrobial activity. Among these 14 plants such as Urginea indica (bulb), Croton roxburghii (bark),

Table 1:	Some important ethnomedicinal plants from Similipal Biosphere reserve, Orissa

Melastoma malabathricum (leaf), Diospyros melanoxylon (bark), Pterospermum acerifolium (leaf), Nyctanthes arbortristis (bark), Oroxylum indicum (bark), Agava sisalana (leaf), Clausena excavate (root) Vitex negundo (leaf), Glycyrrhiza glabra (fruit), Enhydra fluctuans (leaf) Hemidesmus indicus (leaf) and Flemingia nana (root) exhibited outstanding antimicrobial activity with inhibition zones greater than 20 mm. A total of 9 plants were found to have moderate antimicrobial activity. However, 17 medicinal plants did not have any antimicrobial activity. In the present study the results were encouraging as 23 out of 40 plants appeared to contain substances with antimicrobial property. It is clear from the present study that not all medicinal plants posses antimicrobial properties. Similar observations were also made by different workers in different parts of India (Prakash et al., 1995; Ahmad et al., 1998; Perumal Samy et al., 1998).

World Health Organization has listed over 21,000 plant species used around the world for medicinal purposes. In India, about 2, 5000 plant species belonging to more than 1000 genera, are used in indigenous system of medicine. India is tenth among the plant rich counties of the world and fourth among the Asian countries. About 250 plant species are used in regular production of Ayurvedic, Unani, Sidha and tribal medicines. Analysis of distribution shows that medicinal plants are distributed across, diverse habitats. Around 70% of India`s medicinal plants are found in tropical forests while less than 30% of the medicinal plants found are in temperate forests. As Similipal Biosphere Reserve belongs to tropical forest and harbors about 500 medicinal plants (Saxena and Brahamam, 1989), because of its diverse physiographic and climatic condition.

With regard to antimicrobial activity of gram + ve and gram -ve bacteria it is found that out of 23 medicinal plants, 21 plants showed antibacterial activity against gram positive bacteria while 20 plants have activity against gram negative bacteria. Among the microorganisms Staphylococcus aureus is more sensitive to about 17 medicinal plant extracts, Shigella flexneri is sensitive to about 15, Bacillus licheniformis, Pseudomonas aeruginosa, Bacillus brevis and Bacillus subtilis is about 12, Vibrio cholerae is sensitive to about 13, Candida krusei is about 8, Escherichia coli is 5 where as Streptococcus aureus and Staphylococcus epidermidis is sensitive to about 6 plant extracts. Medicinal plants have been widely used for treatment of many types of chronic and acute diseases in Asia and plants with antimicrobial activity have been reported by for new sources specially natural products by plants have been investigated (Cowan, 1999). There is an increase in antimicrobial resistance of some pathogens for which new types of effective and non toxic antimicrobial compounds, plant metabolites etc are being widely used in recent years as natural antimicrobial and antioxidant agents (Sobhy and El-Fefy, 2007).

Out of 40 medicinal plants studied, some plants have already been shown to have antimicrobial properties viz., Adhatoda vasica (Grange and Snell, 1996) Andrographis paniculata (Singha et al., 2003), Argyreia speciosa (Shukla et al., 1999), Casia fistula (Duraipandiyan and Ignacimuthu, 2007) and Vitex negundo (Sathiamoorthy et al., 2007). However, the extracts used and microorganisms tested in the present study may not be same as followed by earlier works. It is clear from Table 1 and 2, that the ethnomedicinal uses of some of the plants have fairly good degree of correlation with their specific antimicrobial activity. Further work is needed to isolate the active principles from the various extracts and evaluate their phytochemical properties. The preliminary results of this investigation appear to indicate that a number of medicinal plants belonging to Similipal Biosphere Reserve have high potential antimicrobial activity. It is expected that plants with high antimicrobial activity may contain anticancerious properties. The novel bioactive compounds from three plants need to be isolated and screened for their pharmaceutical and biotechnological applications for curing chronic and infectious diseases.

Table 2:	Antimicrobial activity of aqueous extracts of collected ethnomedicinal plants
***: S1: Staphylococcus aureus, S2: Shigella flexneri, S3: Bacillus licheniformis, S4: Bacillus brevis, S5: Vibrio cholerae, S6: Pseudomonas aeruginosa, S7: Streptococcus aureus, S8: Candida kruesi, S9: Staphylococcus epidermidis, S10: Bacillus subtilis, S11: Escherichia coli, ND: Not Detected

ACKNOWLEDGMENTS

The present research has been funded by the Department of Science and Technology, Government of Orissa (Grant No. 2596/17.6.2006). We are also grateful to the authorities of North Orissa University for providing necessary facilities to carry out this research.