Chemistry
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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 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 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.Item Discovering new bioactive molecules from endolichenic fungi, Curvularia trifolii and Penicillium Citrinum(2015) Paranagama, P.A.; Upamalika, S.; Wickramaarachchi, S.; Wijeratne, K.M.Endolichenic fungi are microorganisms living in the thalli of lichens that are analogous to the plant endophytic species inhabiting the intercellular spaces of the hosts. The diversity and prevalence of endolichenic fungi have not been studied extensively and there are only a few recorded studies on isolations and identifications of endolichenic fungi. The endolichenic fungi available in Sri Lanka are still an untapped source of bioactive natural products since their identity and the chemistry of their secondary metabolites have not been explored thoroughly. In our ongoing research programme on searching for new bioactive compounds from endolichenic fungi, we have investigated an endolichenic fungi, Curvularia trifolii and Penicillium citrinum isolated from the lichens, Usnea sp. and Parmotreama sp. collected in the Hakgala Botanical Garden, Sri Lanka. Ethyl acetate extracts of two endolichenic fungi, C. trifolii and P. citrinum led to isolation of four new compounds, 1- 4. The structures of the new compounds were determined on the basis of their 1D NMR, 2D NMR and FABMS spectroscopic data. The new compounds, 1 - 4 showed radical scavenging activities with IC50 values of 4.0±2.6 mg/mL, 1.3±0.2 mg/mL, 159.7±22.3 μg/mL and 68.6±4.3 μg/mL respectively in DPPH antioxidant assay and antioxidant activity of the compound 2 was comparable to the standard antioxidant compound, BHT. Moreover, the new compound, 2 were evaluated for their antiinflammatory activity and exhibited significant activity comparable to the standard antiinflammatory drug, Aspirin. The Compound 1 was evaluated for inhibition of cell proliferation in a panel of five cancer cell lines NCI-H460, MCF-7, SF-268, PC-3M, and MIA Pa Ca-2 and exhibited >90% inhibitory activity at 5 μg/mL with all the above cell lines.Item Repellency and toxicity of four essential oils to Sitophilus oryzae L. (Coleoptera: Curculionidae)(2004) Paranagama, P.A.; Abeysekera, K.H.T.; Nugaliyadde, L.; Abeywickrama, K.P.Item Physicochemical properties and antioxidant activity of peel and pulp of 'Karuthacolomban' and 'Willard' cultivars of mango (Mangifera indica L.) in Sri Lanka(Postgraduate Institute if Science, University of Peradeniya, Sri Lanka, 2015) Kuganesan, A.; Thiripuranathar, G.; Navaratne, A.N.; Paranagama, P.A.Item Arsenic and hardness in ground water from Chronic Kidney Disease of Unknown Etiology (CKDU) prevalent areas and non-CKDU prevalent areas in Sri Lanka(Postgraduate Institute of Science, University of Peradeniya, 2012) Fonseka, S.; Jayasumana, C.; Jayalath, K.; Amarasinghe, M.; Senanayake, K.; Wijewardhane, C.; Samarasinghe, D.; Dahanayake, K.; Mahamithawa, P.; Paranagama, P.A.Increasing hardness and deteriorating quality of groundwater, the primary source of potable water, has been the general observation of inhabitants of areas where chronic kidney disease of unknown etiology (CKDu) is prevalent. Present study was conducted during 2011, to determine the groundwater hardness and presence of arsenic in Padavi- Sripura, Polpithigama, Moneragala, Thanamalwila in the dry climatic zone and in Pasagoda in the wet zone. Total hardness of the water samples collected from dug wells and tube wells was measured using EDTA titration (EPA 130.2) and arsenic content was measured using GF-AAS after filtration and acid digestion (EPA 7060A). Highest average groundwater hardness (466+34 mg l-1) was observed at Padavi-Sripura (n= 28) and the values ranged from 270+54 – 820+62 mg l-1). Arsenic content in water ranged from 21.07+3.54 to more than 100.91+12.31 !" #-1. The second most hardwater was found from Polpithigama area (n= 16) which ranged 90+8 – 615+47 mg l-1. Arsenic content in water ranged 2.49+0.61 – 60.55+7.21 !" #-1. The lowest hardness in groundwater among the test sites was observed at Moneragala (n=38), where the hardness ranged 10+2 – 340+31. The arsenic content ranged 2.14+0.84 – 52.47+6.71 !"#-1. Groundwater at Thanamalwila (n=19) recorded hardness value, i.e. 279+26 mg l- 1-with a range 170+8 - 500+24 mg l-1 and the arsenic content in water ranged 39.37+5.21- >100.42+9.45 !" #-1. Groundwater at Pasgoda, the control site of this study, was not hard (60+5 mg l-1) and arsenic was not detected. Statistical analyses reveal that a positive correlation exists between total hardness of groundwater and the arsenic content in it.