Browsing by Author "Gunawardhana, M.H.A.Y."

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Antibacterial activity of the partitioned fractions of endolichenic fungus Xylaria feejeensis collected from the mangrove plant Rhizophora mucronate in Negambo Lagoon, Sri Lanka
(Proceedings of 7th Ruhuna International Science & Technology Conference, 2020) Gunawardhana, M.H.A.Y.; Wicramarachchi, S.; Weerasinghe, W.R.H.; Paranagama, P.A.
Endolichenic fungi (ELF) are known for biosynthesizing secondary metabolites with antifungal, cytotoxic, antioxidant and antibacterial activity to protect their lichen against radiation, microbial invasions and harsh surrounding environments. Mangrove plants in Negombo lagoon is a niche for a diverse range of lichens. The ELF, Xylariafeejeensis from the lichen, Graphis librata which develops on the mangrove plant, Rhizophora mucronata in Negombo lagoon was previously isolated and identified by the research group at the University of Kelaniya. The objective of this study was to determine the antibacterial activity of the secondary metabolites of X feejeensis. A pure culture Of X. feejeensis on potato dextrose agar plates (60) was incubated for 14 days and secondary metabolites were extracted to ethyl acetate by shaking overnight. The ethyl acetate crude extract (5 mg/ml, 100 gl„) was subjected to agar well diffusion anti-bacterial assay against Escherichia coli (ATCC25922), Staphylococcus aureus (ATCC25923) and Bacillus subtilis (ATCC6051) with Azithromycin positive control and it showed above 85% inhibition of growth for all three bacteria. Hence the crude extract (8.58 g) was partitioned in hexane, chloroform and 60% methanol in water (v/v) and tested for antibacterial activity. Hexane fraction showed the highest inhibition of growth of E. coli with 100% inhibition and chloroform fraction showed the highest inhibition of the groMth of S. aureus with 88.5% inhibitioh whereas methanol fraction showed no antibacterial activity. The results suggest that X. feejeensis produces secondary metabolites with high antibacterial activity. Further purification of hexane and chloroform fractions may results in antibacterial drug leads.
Exploring bioactive compounds in the endolichenic fungus, Xylaria feejeensis, inhabiting the lichen, Graphis librata, collected from Negombo lagoon, Sri Lanka
(Proceedings of the 1st International Conference on Frontiers in Chemical Technology, Institute of Chemistry Ceylon, 2020) Gunawardhana, M.H.A.Y.; Wickramarachchi, P.A.S.R.; Weerasinghe, W.R.H.; Paranagama, P.A.
The accelerating pharmaceutical problem ofbacteria growing resistant to existing antibrotics forces the scientific community to search for new antibacterial compounds for antibiotic drug development. I-iterature reveals that Sri Lankan mangrove inhabiting endolichenic fungal (ELF) population is rich in nrany such bioactive compounds- Previously isolated and cultured ELF, Xylaria feejeensis; ftom the lichen Graphis librata inhabiting in the mangrove phnt Rhizophora mucronata was culrured on 60 large Potato dextrose agar medium containing petri dishes and incubated for 14 days at room temperature. After extracting secondary metabolites to ethyl acetate (EA) the resulted crude extract was tested for its antibacterial and antioxidant activity. EA crude extract shou,ed negligible antioxidant activity in DPPH radical scavenging assay hence this assay was not proceeded further. The activity ofcrude extract (5 mg/ml) agatnstEscherichia coli (ATCC25922), Staphylococcus aureus (NCC25923) utd Bacillus subtilis (ATCC605I) was tested using agar well diffirsion antibacterial assay. An inhibition zone diameter of 1.9 cm, 2.2 cm, ar,d 2.2 cm against three bacterial strains with Azithromycin positive control (5 mg/ml) 2.2 cm,2.2 cm and 2.3 cm was observed respectively. By Partitioning of EA crude extract resulted two antibacterial active hexane and chloroform fractions and one antibacterial inactive 607o methanol in water fraction. Hexane fraction showed the highest antibacterial activity with inhibition diameter zone of 2.6 cm against E.coll with 2.6 cm diameter of inhibition zone for positive control. Further purification ofhexane fraction was performed by normal phase column chromatography and 4 fractions were resulted. The highest polar fraction showed L4, 2.landl.TcmdiameterinhibitionzonesofgroMhofE.coli,S. aureusutdB.sr.rbtillscomparablewith 2.2 cm Azithromycin. The other 3 fractions showed no antibacterial activity against these bacterial strains. Results suggest that further purification of active fraction and structure elucidation might result in new antibiotic lead compounds.
Potential Antibacterial Secondary Metabolites froe an Enadolichenic Fungus Inhabiting a Lichen Collected from Negombo Lagoon, Sri Lanka
(International Postgraduate Research Conference 2019, 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 Xylariafeejeensis isolated from the lichal Graphis librata showed remarkable activity against the two bacterial strains on par with the positive control Azithromycin. The assay was carried out using 100 gl of the extract and the positive control (5 mg/ml). The inhibition zone diameters (in cm) against E coli and S. aureus for the ftmgal 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 tvlethanol 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 fur-tha- 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, I .4, 0.0, 03,0.0 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.
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