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Pharmaceutically Relevant Metabolites from Lichens of Kodaikanal, India

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Pharmaceutically Relevant Metabolites from Lichens of Kodaikanal, India Int.J.Curr.Microbiol.App.Sci (2014) 3(11) 779-788 ISSN: 2319-7706 Volume 3 Number 11 (2014) pp. 779-788 http://www.ijcmas.com Original Research Article Pharmaceutically relevant metabolites from Lichens of Kodaikanal, India K.Ramya1 and T.Thirunalasundari2* 1Merck Millipore India Pvt. Ltd, No. 6, 6th Main Road, BDA Industrial Suburb, Near SRS Road, Peenya, Bangalore - 560 058, Karnataka, India 2Department of Industrial Biotechnology, Bharathidasan University, Tiruchirappalli - 620 024, Tamil Nadu, India *Corresponding author A B S T R A C T Lichens are a world wide spread consortium of self-supporting associations between fungi and algae. In nature they encompass a complex and diverse assemblage of life forms, which occur throughout Kodaikanal on rocks, soil and on trees. Kodaikanal is at a high altitude and has a temperate climate. The existing K e y w o r d s environment here is pollution free, which favours the growth of Lichens, hence can be rightly described as the Biomonitors. Lichens are renowned for their Lichens, extraordinary diversity of primary and secondary metabolites. Because of the Biomonitors, physiochemical environment and biological interaction, many lichen species have Macromolecules, evolved secondary metabolic pathway. Thus, promising a wealth of bioactive Antibacterial substance, many as yet unknown with novel structure and activities. Metabolic activity, activities mainly respiration and photosynthesis, frequently result in production of Phytochemicals, free radicals or reactive oxygen species (ROS). Certain antioxidant enzymes Antioxidant produced by lichens like superoxide dismutase, catalase, glutathione reductase, etc., enzymes, scavenge the effect of free radicals. Such bioactive compounds produced by lichens Secondary should be brought to the knowledge of Chemists and Pharmacologists. Having this metabolites, in mind, extracts of Ramalina sp., Usnea complanata, Usnea fischeri, Physcia Pharmaceuticals dilatata, Parmotrema austrosinensis, Parmelia andinum and Parmelia sulcata were subjected to analysis of macromolecules, phytochemicals and antibacterial activity testing on clinical isolates. Macromolecular quantification showed the presence of considerable amount of carbohydrates, proteins and fats in all the lichen species. It was found that all the extracts had antibacterial activity against one or the other clinical isolates. Introduction Whenever we discuss the probability that earth. They are diverse and ubiquitous group life exists in the harsh environment of the of lower plants, occupying 8% of the Earth planet, lichens enter the conservation, which surface. About 20,000 species of lichens occupy the most forbidding environment on e x ists worldwide with India sharing 12.25 779 Int.J.Curr.Microbiol.App.Sci (2014) 3(11) 779-788 species (Sanjeeva Nayaka et al., 2003). to treat infectious diseases. One such They do not possess roots or waxy cuticles resource being the lichens can be explored and depend mainly on the atmospheric input for the potential as producers of of water and mineral nutrients. biomedically relevant metabolites. Hence Consequently the entire thallus area of the present study was aimed to characterize lichens is susceptible to penetration and the bioactive compounds from lichens of accumulation of airborne elements, some Kodaikanal in India. essential for the proper functioning of lichen but others being toxic. These features Materials and Methods combined with their ability to grow at a wide geographical range rank lichens among Study material: Different types of Lichen the best bioindicators of air pollution (Garty, grown on plum tree. 2001). Lichens produce various groups of Collection of the study material: All metabolic byproducts. These include esters the lichen samples chosen for the study and organic acids (Julia Levy and Jack, were collected from the plum tree; 1973), which are produced by both the Prunus salcinia. partners either individually or together (Ho Nutritive value of lichens: lm Hansen, 1968). Lichen metabolites Preparation of extract: Known exert a wide variety of biological actions quantity of lichen samples were taken including antibiotic, antiinflammatory, and extracted with acetone in a pestle analgesic, antipyretic and cytotoxic effects. and mortar. The extract was centrifuged Metabolic activities mainly respiration and at 10,000 rpm for 5 mins in a centrifuge photosynthesis frequently result in and the supernatant collected was used production of reactive oxygen species for macromolecular analysis. (ROS) (Fridovich, 1999, Kohen and Nyska, Carbohydrate was estimated by DNS 2002). These are enhanced during stresses method; protein by Lowry s method such as nutrition limitation, exposure to (Lowry et al., 1951). Lipids were xenobiotics or dessication and/or analysed by making use of Cholesterol rehydration. To evade the damaging effects kit supplied by M/S Biosystems. of ROS, cells have evolved protection Antibacterial activity testing: mechanisms including antioxidant enzymes Organism chosen for antibacterial such as SOD, catalase, peroxidases and low activity testing molecular weight antioxidants. Even though Gram negative organisms like these manifold activities of lichen Escherichia coli, Proteus mirabilis, metabolites have now been recognized, their Pseudomonas aeruginosa, Klebsiella therapeutic potential has not yet been fully pneumoniae, Salmonella typhimurium explored and thus remains pharmaceutically and Gram positive organisms like unexploited (Muller, 2001). Among all the Staphylococcus aureus and lichen metabolites, usnic acid is the most Streptococcus faecalis were used for studied and used compound. After 1980 s this study. interest in such metabolites was renewed Preparation of sample because of increasing experience of Stock solutions of the samples were multidrug resistance caused by over usage of prepared by dissolving known synthetic antibiotics (Cocchietto et al., concentrations of the crude lichen 2002). Hence it is important to discover extracts in a known volume of sterile newer sources of bioactive natural products distilled water. Various concentrations 780 Int.J.Curr.Microbiol.App.Sci (2014) 3(11) 779-788 like 50 g, 100 g, 150 g and 200 g the aqueous extract and on the were taken from the stock solution and powdered samples using standard were used for antibacterial activity protocols to identify the constituents testing. as described by Sofowora (1993), Assay method. Trease and Evans (1989) and Antibacterial activity of different Harborne (1973). extracts was done by Kirby Bauyer Enzyme assay method (Bauer, 1966). Spot analysis of chemical constituents of lichens (Nylandervand Flora, 1856 The presence of two antioxidant & Asahina, 1934) enzymes; superoxide dismutase (SOD) and catalase were assayed in For the preliminary identification of the lichens, which were freshly the chemical compounds present in collected from the plum tree. lichens, spot analysis was done by SOD: Enzymatic assay of superoxide applying appropriate reagents to the dismutase was carried out by lichen fragments by means of a Winterbourn et al. (1975) and is based syringe and the color formed was on the ability of superoxide dismutase observed. The results were tabulated to inhibit the reduction of nitro-blue immediately. The test being C test tetrazolium by superoxide. (aqueous calcium hypo chlorite); K Catalase: Enzymatic assay of catalase test (10 25% aqueous potassium was performed by following the hydroxide); PD test (P method of Beer and Sizer (1952) and phenylenediamine); KC test (first K Stern (1937). test reagent and then C test reagent) and the other color tests using 5% Results and Discussion aqueous solution of chloramine T was also done. Seven lichen samples sharing different Thin layer Chromatography niches of the same tree; Prunus salcinia (Culberson, 1972) were collected from Attuvampatty campus, Thin layer chromatography was Mother Teresa Women s University, performed to identify the secondary Kodaikanal. These samples were identified metabolites. and categorized as fruticose, foliose and a. Preparation of extract: crustose (Table 1). Known quantity of lichens was extracted with acetone and the Macromolecular analysis showed the supernatant was used for TLC. presence of considerable quantity of b. Solvent used: carbohydrates, proteins and fats in the Mixture containing toluene, samples analysed. Parmelia sulcata had ethyl acetate and formic acid in high content of carbohydrate (900 g/gm of the ratio of 139:83:8 was used to sample), protein (930 g/gm of sample) and develop the chromatogram. fat (26.07 g/gm of sample) (Table 2). Phytochemical analysis of secondary To have the knowledge on secondary metabolites metabolites of lichens, which may be a Chemical tests were carried out on cause for the antibacterial activity, spot 781 Int.J.Curr.Microbiol.App.Sci (2014) 3(11) 779-788 analysis and TLC, were also performed. lichens (Table 7). PL 2 had high catalase Spot analysis of color tests revealed certain activity (10.27U/mg) followed by PL 1 & aromatic aldehydes and other related PL 7 (8.96U/mg & 8.1U/mg) and PL 2 had compounds like depsides, dibenzofuranes, high SOD activity (6.01U/mg) followed by usnic acid, etc., (Table 3). TLC data PL 1, PL 2 & PL 4 (4.8U/mg, 3.96U/mg & obtained gave a picture about the presence 3.9U/mg). Interestingly there was no of phenolic acids and other compounds in catalase activity in PL 4 & no SOD activity lichens (Table 4a,b). in PL 5 (Table 7 and Fig. 1). Screening for antibacterial activity of the In
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