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Item 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.Item Screening Anti-Bacterial Activity of Endoi.Ichenic Fungi Collected From Negombo Lagoon Sri Lanka(Proceedmgs of the Postgraduate Institute of Science Research Congress. Sri Lanka, 2019) Weerasinghe, W.R.H.; Attanayake, R.N.; Weerakoon, G.; Paranagama, P.A.From the ancient past, natural products have been in use to fulfill a variety of human needs, from sustenance to pharmaceutical necessities. With the discovery of Endolichenic fungi (ELF) as a novel source prominent bioactiOt1es in recent years. a new trend has been created to explore more about these fascinating organisms, which live inside the lichen thallus asymptomatically. ELF isolated from the lichens collected from mangrove plants In Negombo lagoon's Kadol kale area (Lititude: 701 Longitude: were identitied using molecular techniques and were subjected to anti-bacterual assay. Ethyl acetate crude extracts of 18 identified endolichenic fungal strains were obtained and their anti-bacterial activities were tested against aerobic bactertal species Escherichia coli, Bacillus subtilis and Staphvlococcus aureus. The assay was carried out using agar well diffusion method using HI of the extracts (5 mg/ml) with Azithromycin as the positive control. The inhibition zone diameters (in cm) against E. coli were 1.2 for Phanerothaete chnsosporium. 1.2 for Xvlaria feejecnsis, ().9 for Gelasinospora seminuda, 0.9 for Chaetomium globosum and 1.5 for Curvularia lunata while Azithromycin as the positive control showed a diameter of 2.1. The inhibition zone diameters against B. subtilis were 1.0 for Cochliobolus sp., 1.0 for P. chnsosporium, 1.8 for X. feeyeensls, 1.7 for G. seminuda, 1 2 for C. globosum, 1.5 for Hvpoxvlon lividipigmentum, 2.0 for (Pennicillium sp), 2.2 for C. lunata, 0.9 for Lasiodiplodia theobromae and 2.4 for positive control. The inhibition zone diameters against S. aureus were 1.1 for P. chrvsosporium, 22 for X'. feejeensis, 1.0 for G. seminuda, 1.0 for Chaetomium globosum, 2.0 for (Pennicillium sp). 1.8 for Curvularia lunata and 2.2 for positive control. Out of the 18 fungal strains, 9 showed significant activity against the tested bacterial species while Endomelanconiopsis endophvtica, Neofusicoccum parvum. Ncofusicoccum occulatum. Hvpoxvlon anthochroum. Cvtospora xvlocarpi, Cerrena sp., Schi:ophvllum commune, Daldinia eschscholt:i, Nodulisporium sp. fungal strains did not show any inhibition. Further studies will be conducted to isolate compounds responsible for these inhibitions.Item A NEW ANTIOXIDANT ACTIVE COMPOUND FROM THE ENDOLICHENIC FUNGUS, PENICILLIUM CITRINUM INHABITING THE LICHEN, PARMOTREMA SP(International Journal of Pharmaceutical Sciences and Research, 2019) Wickramarachchi, S.A.; Samanthi, U.; Wijeratne, K.; Paranagama, P.A.Context: Endolichenic fungi are the microorganisms living inside the thalli of the lichen and found to be a new source of bioactive secondary metabolites. This is an underutilized source of bioactive compounds and has not been studied extensively. Genus, Parmotrema is one of the foliose lichens with largely rounded lobes and occurs on rocks of rain forests, sub-montane and montane forests in Sri Lanka. Aims: The endolichenic fungi available in Sri Lanka and the chemistry of their secondary metabolites have not been explored thoroughly. This study aims at exploring novel bioactive compounds available in Penicillium citrinum inhabiting Parmotrema sp. Methods and Material: The lichen, Parmotrema sp., was collected from Hakgala Botanical Garden in Sri Lanka and its fungal strains were isolated and identified by Genomic DNA sequencing. Compounds were first extracted to ethyl acetate and subsequently subjected to bioassay-guided fractionation to isolate the bioactive compounds. The DPPH assay was used to determine the antioxidant activity. The active compound/s was separated by column chromatography, and its structure was elucidated by IR, 1D and 2D-NMR, 13C-NMR and MS. Statistical Analysis Used: The IC50 of the crude extract was calculated using Probit analysis (MINITAB® Release 14.1. Minitab Inc. 2003 Statistical Software). Results: A bioactive compound, named PP-PC-03 together with PP-PC-01 and PP-PC-02 was isolated from P. citrinum. All three compounds showed antioxidant activity in the DPPH assay with IC50 values 159.6 ± 22.3, 120.1 ± 11.7 and 68.6 ± 4.3 μg/mL respectively. Conclusion: Based on spectral analysis the structure of PP-PC-03was determined as 10-Ethylidene-2,4,9-trimethoxy-10,10a-dihydro-7,11-dioxa-benzo[b] heptalene-6,12-dione and identified as a new compound. Comparatively PP-PC-02 and PP-PC-03 had moderate antioxidant activities while PP-PC-01 showed strong radical scavenging activity.Item Molecular phylogeny and bioprospecting of Endolichenic Fungi (ELF) inhabiting in the lichens collected from a mangrove ecosystem in Sri Lanka(PLoS ONE 13(8): e0200711. https://doi.org/ 10.1371/journal.pone.0200711, 2018) Maduranga, K.; Attanayake, R.N.; Santhirasegaram, S.; Weerakoon, G.; Paranagama, P.A.Endolichenic fungi (ELF) are unexplored group of organisms as a source for the production of bioactive secondary metabolites with radical scavenging activity, antilipase and amylase inhibitory activities. Endolichenic fungi in lichens collected from mangrove or mangrove associated plants are least known for their fungal diversity and potential to produce bioactive compounds. A total of 171 ELF strains were isolated from the lichens collected from mangrove and mangrove associated plants in Puttalam lagoon. Out of this collection, 70 isolates were identified using rDNA-ITS region sequence homology to the GenBank accessions and a phylogenetic analysis was performed. Commonly isolated genera of ELF from lichens were Aspergillus, Byssochlamys, Talaromyces, Diaporthe, Phomopsis, Endomelanconiopsis, Schizophyllum, Cerrena, Trichoderma, Xylaria, Hypoxylon, Daldinia, Preussia, Sordaria, Neurospora, and Lasiodiplodia. In the present study, the effectiveness of ethyl acetate extracts of the ELF isolates were investigated against antioxidant activity, antilipase activity and α-amylase inhibition activity in in-vitro conditions. The results revealed that the extracts of Daldinia eschscholtzii, Diaporthe musigena and Sordaria sp. had the highest radical scavenging activity with smaller IC50 values (25 μg/mL to 31 μg/mL) compared to the IC50 values of BHT (76.50±1.47 μg/mL). Antilipase assay revealed that 13 extracts from ELF showed promising antiobesity activity ranged between 25% to 40%. Amylase inhibitory assay indicated that the test extracts do not contain antidiabetic secondary metabolites.Item Evaluation of anti-inflammatory and antioxidant properties of Genus Piper in Sri Lanka. International Research Symposium on Pure and Applied Sciences,(International Research Symposium on Pure and Applied Sciences, 2017 Faculty of Science, University of Kelaniya, Sri Lanka., 2017) Athapattu, R.S.K.; Jayarathna, S.P.N.C.; Paranagama, P.A.; Senanayake, S.P.In Sri Lanka, the genus Piper is represented by ten species including five wild and two cultivated species. Most of the species of the genus Piper are important as medicinal plants and used in various systems of medicine.This study aims to evaluate the bioactivity (anti-inflammatory and antioxidant properties) of the selected five Piper spp. (P. nigrum L., P. longum L., P. betle L., P. chuvya (Miq.) and P. sylvestre). Specimens of leaves and fruits were collected from their natural habitats and cultivations, and were subjected to the extraction of essential oils by steam distillation. Three replicates were done for each concentration and data were analyzed using the MINITAB 17 statistical package. Antioxidant activity was tested using 2,2-diphenyl-1- picrylhydrazyl (DPPH) free radical scavenging assay and ferric reducing antioxidant power assay. The isolated essential oils of Piper species exhibited remarkable DPPH free radical scavenging ability at different concentrations. The results revealed that the essential oil of P.betle exhibited the highest radical scavenging activity (92.8% at 1 mg/mL) and the value is higher than even of the standard, Butylated Hydroxy Toluene (BHT), (74.5% at 1 mg/mL). Further, essential oils of P. chuvya and P. longum have shown 91.8% and 63.1%inhibition percentages at 1 mg/mL. Essential oil isolated from P. betle has showed remarkable ferric reducing antioxidant power (mean absorbance, 1.89at 1 mg/mL) compared to the standard, BHT, (2.01at 1 mg/mL). Further, it was observed that its antioxidant activity increased with the increment of the concentration of essential oil (0.03-1 mg/mL). These results suggested the potential of using essential oils of leaves of P.betle, P. chuvya (1.31) and P. longum (1.28) as sources of natural antioxidants. This study also focused on evaluating anti-inflammatory activity by using Human Red Blood Cell (HRBC) membrane stabilizing method.Higher mean inhibition percentage was observed for the essential oil of P. betle leaves (79.6%at 1 mg/mL) compared to the standard (aspirin) and the activity significantly increased with higher concentrations (76.4% at 0.5 mg/mL and 79.6% at 1 mg/mL). Further, 62 % and 58.3 % of inhibitions percentages were shown by essential oils of P. chuvya and P. longum respectively. According to the findings of this study, bioactivity properties of the leaves and fruits of these Piper species can be used in future perspectives for developing novel pharmaceuticals.Item Anti-oxidant activity of selected endo lichenic fungi (ELF) in mangrove ecosystem of Puttalam lagoon.(International Research Symposium on Pure and Applied Sciences, 2017 Faculty of Science, University of Kelaniya, Sri Lanka., 2017) Maduranga, H.A.K.; Attanayake, R.N.; Amarasinghe, M.D.; Weerakoon, G.; Paranagama, P.A.Natural products based drug development has become an attractive area of research since there are limited options available to treat certain non-infectious diseases such as diabetes. Among these natural products, it has been reported that secondary metabolites of endolichenic fungi (ELF), have the ability to produce promising bioactive compounds. The objectives of this research were to isolate and identify ELF inhabiting mangroves in Puttalam lagoon, Sri Lanka using classical and DNA barcoding approach and to determine anti-oxidant activity of their secondary metabolites. Lichen hosts were collected from Puttalam lagoon in two different sites near, Athathale and around the NARA institute. The ELF were isolated following a standard procedure: a small piece of the thallus was surface sterilized, cut into pieces and dried on sterilized filter papers and then placed on malt extract agar in Petri dishes and incubated at room temperature (28 ⁰C – 30 ⁰C ) . Once pure cultures were obtained, seven isolates were randomly selected for DNA extraction following standard procedures. Quality of DNA was checked by agarose gel electrophoresis. Fungal internal transcribed spacer (ITS) region was amplified using polymerase chain reaction (PCR) with universal ITS 1 and ITS 4 primers and PCR products were sequenced using Sanger dideoxy chain-termination technology. DNA sequences were edited using BioEdit software and compared with the available sequences in the GenBank using Basic Local Sequence Alignment Search Tool (BLAST). In addition, morphological characterization of each fungal isolate was also carried out. Secondary metabolites from each isolate were extracted with ethylacetate separately and the solvent was evaporated under reduced pressure to obtain the crude extract. Free radical scavenging activity of the extracts were evaluated using 2, 2-diphenyl-1-picrylhydrdrazyl (DPPH) assay. Based on the highest sequence similarity to the GenBank sequences, isolates were identified as Diaporthe arengae (98 %), Neurospora crassa (100%), Lasiodiplodia theobromae (100 %), Schizophyllum commune (98 %), Diaporthe musigena (98 %), Hypoxylon anthochroum (98 %) and Nigrospora sphaerica (98%). IC50 values of extracts of Diaporthe arengae, Neurospora crassa and Lasiodiplodia theobromae were 375.9± 0.062μg/mL, 304.9±0.057 μg/mL and 211.2± 0.086 μg/mL respectively. Since percent inhibitions of the rest of the isolates were less than 50 % in the test doses, IC50 values were not calculated. All of the values were compared with standard Butylated Hydroxy Toluene (BHT) (IC50=108.0±0.072). Out of the seven ELF tested, L. theobromae showed the highest DPPH radical scavenging activity. Further testing of the rest of the isolates are being carried out and ELF may provide a good source of antioxidants for biotechnological applications.Item Bioactivity of leaf volatiles of Azadirachta indica A. Juss. and Murraya koenigii Spreng. against Sitophilus oryzae L.(Coleoptera: Curculionidae)(National Science Foundation of Sri Lanka., 2002) Paranagama, P.A.; Abeysekera, K.H.T.; Nugaliyadde, L.; Abeywickrama, K.Repellent activity, fumigant and contact toxicities of leaf volatiles of Azadirachta indica A. Juss. and Murraya koengii Spreng. were evaluated against Sitophilus oryzae L. In the dual choice repellency test with A indica leaf volatiles, significantly higher number of insects was repelled at doses above 100 mg, whereas volatiles of At. koenigii attracted insects at 25 mg dose and repelled at 300 mg dose. In the fumigant toxicity test 100% mortality was observed at the concentration of 32.5 mg/ml, 3 days after the treatment with 2 volatiles separately. The L C n values for fumigant toxicities were 13.5 and 22.5 mg/ml for A. indica and At. koenigii volatiles, respectively. In contact toxicity test, 100% mortality was observed immediately after 48 h contact exposure of insects at concentrations of 0375 and 0.125 mg/cm1 of A indica and At. koenigii respectively. The respective L C W values were 0.12 and 0.08 mg/cm1 for A. indica and At. koenigii leaf volatiles.Item Vacuum liquid chromatography (VLC) and gel permeation chromatography in natural product research(National Institute of Fundamental Studies, Kandy, Sri Lanka., 2016) Paranagama, P.A.Item Two new bioactive poliketides from Culvularia trifolii, an endolichenic fungus isolated from Usnea sp., in Sri Lanka(2015) Samanthi, K.A.U.; Wickramarachchi, S.; Wijeratne, E.M.K.; Paranagama, P.A.Item Bioactive constituents of wood rot extract of tea, camellia sinensis L.O. Kuntze against alates of low country live wood termite Glyptotermes dilatatus Bugnion and Popoff (Isoptera: Kalotermitidae)(2015) Senanayake, P.D.; Mohotti, K.; Paranagama, P.A.