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Author: search.filters.author.Paranagama, P.A.Subject: search.filters.subject.Endolichenic fungi

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    Potential Antibacterial Secondary Metabolites from an Enadolichenic Fungus Inhabiting a Lichen Collected from Negombo Lagoon, Sri Lanka
    (International Postgraduate Research Conference 2019, Faculty of Graduate Studies, University of Kelaniya, Sri Lanka, 2019) Weerasinghe, W.R.H.; Gunawardhana, M.H.A.Y.; Wickramarachchi, S.; Attanayake, R.N.; Weerakoon, G.; Paranagama, P.A.
    With the revelation of intriguing bioactive properties from natural sources, the thirst to investigate more into the mysteries of nature has immensely expanded. Endolichenic fungi (ELF) became an interesting source during the recent past in this search for novel bioactive compounds and literature sources provide evidence of many such bioactive compounds isolated from these interesting organisms. These ELF asymptomatically live inside the lichen thallus which is formed by the symbiotic relationship of fungi with an algae or a cyanobacteria. A total of 31 lichens were collected from mangrove plants in Negombo lagoon and their molecular identification revealed that they belonged 10 different species. Healthy lichen thalli were surface sterilized and were cut into small segments and plated on 2% Malt Extract Agar (MEA) medium supplemented with 0.01% streptomycin in order to obtain ELF. The obtained pure cultures of ELF were identified using molecular techniques. DNA was extracted using CTAB method and its quality and quantity were determined by agarose gel electrophoresis. DNA was diluted accordingly and was subjected to Polymerase Chain Reaction (PCR) to amplify fungal ITS rDNA region using universal primers. PCR amplification was tested using agarose gel electrophoresis and the full sequences were obtained. Ethyl acetate crude extracts of 18 such identified ELF strains were subjected to anti-bacterial assay against Escherichia coli and Staphylococcus aureus using agar well diffusion method. The species Xylaria feejeensis isolated from the lichen Graphis librata showed remarkable activity against the two bacterial strains on par with the positive control Azithromycin. The assay was carried out using 100 μl of the extract and the positive control (5 mg/ml). The inhibition zone diameters (in cm) against E. coli and S. aureus for the fungal crude were 1.9 and 2.2 respectively and for Azithromycin was 2.2 against both. In order to isolate the active compounds, a larger crude of the same was obtained and partitioned into Hexane, Chloroform and Methanol fractions based on polarity. The assay results for the three fractions revealed that only Hexane and Chloroform fractions possessed anti-bacterial potentiality. Subsequently, silica gel normal phase column chromatography was performed for further fractionation. Collected 6 fractions from the column for Chloroform fraction showed inhibition diameters of 1.9, 1.9, 1.5, 1.2, 1.3, 1.1 against E. coli comparable with 2.2 of Azithromycin and 2.4, 2.2, 1.4, 0.0, 0.7,0.0 against S. aureus comparable with 2.5 of Azithromycin. The fraction 1 and 2 showed highest activity against both bacterial strains and fraction 4 and 6 showed lowest activity against E. coli and none against S. aureus. Further isolation is being carried out for active fractions and the structures of obtaining active compounds will be elucidated using spectroscopic methods
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    Investigating the bioactive compounds of endolichenic fungus Hypoxylon anthochroum isolated from the lichen, Pseudocyphellaria sp.
    (Faculty of Graduate Studies, University of Kelaniya, 2015) Antony, K.N.L.; Samanthi, K.A.U.; Paranagama, P.A.
    It has been reported that 11 endolichenic fungal strains were isolated from lichen, Pseudocyphellaria sp. Endolichenic fungi live asymptomatically within the lichen thallus and known as a new source useful of bioactive secondary metabolites. Secondary metabolites in endolichenic fungi show distinct bioactivities including antimicrobial, anti-inflammatory, analgesic, antioxidant and cytotoxic activities. These bioactive compounds have potential importance in development of pharmaceutical drugs, nutraceuticals and agrochemicals. The present study was carried out to investigate antioxidant activities of secondary metabolites produced from Hypoxylon anthochroum inhabiting the lichen, Pseudocyphellaria sp. In this research, H. anthochroum was cultured in 50 large petridishes containing potato dextrose agar (PDA) medium and incubated at room temperature for seven days. After the incubation period, secondary metabolites were extracted into ethyl acetate in order to obtain crude extract. The crude extract was partitioned using solvents hexane, chloroform and 60% aqueous methanol. Antioxidant activities of three fractions were evaluated using micro plate DPPH assay. Since chloroform fraction showed the highest antioxidant activity (81.96 % Inhibition at 800 μg/mL) it was further subjected to silica gel column chromatography and preparative thin layer chromatography to isolate bioactive compounds. One UV active pure compound which has antioxidant activity (43.21 % Inhibition at 800 μg/mL) was isolated from chloroform fraction and its structure elucidation will be carried out using 1H NMR, 13C NMR, and 2D NMR spectral data.