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Item Antibacterial Polyketide from Lasiodiplodia theobromae and Pyrenula bahiana on Mangrove Ecosystems in Puttalam Lagoon, Sri Lanka(Asian Journal of Chemistry, 2022) Paranagama, P.A.; Santhirasegaram, S.; Wickramarachchi, S.R.; Attanayake, R.N.; Weerakoon, G.; Maduranga, K.Lasiodiplodia theobromae is one of the frequently isolated fast growing endolichenic fungus. This fungus was isolated from the lichen host, Pyrenula bahiana collected from the mangrove ecosystems in Puttlam lagoon and its identification was confirmed based on rDNA-ITS sequence homology. Secondary metabolites of L. theobromae were extracted into ethyl acetate and subjected to antibacterial assay against Escherichia coli (ATCC25922), Staphylococcus aureus (ATCC25923) and Bacillus subtilis (ATCC6051). Crude extract at a concentration of 6.8 μg/mL showed good antibacterial activity against the bacterial strain S. aureus compared with the activity of the standard azithromycin at a concentration of 5.0 μg/mL. Active crude extract was partitioned to obtain methanol, hexane and chloroform fractions. Chloroform fraction showed the highest activity to S. aureus out of three fractions. This fraction was subjected to bioassay-guided separation on silica gel column chromatography to isolate bioactive pure compounds. The bioactive pure compound was identified as (3R)-de-O-methyllasiodiplodin using LC-MS, 1D and 2D NMR spectroscopy.Item Antimicrobial Compounds Isolated from Endolichenic Fungi: A Review(Molecules 2021, 2021) Wethalawe, A.N.; Alwis, Y.V.; Udukala, D.N.; Paranagama, P.A.A lichen is a symbiotic relationship between a fungus and a photosynthetic organism, which is algae or cyanobacteria. Endolichenic fungi are a group of microfungi that resides asymptomatically within the thalli of lichens. Endolichenic fungi can be recognized as luxuriant metabolic artists that produce propitious bioactive secondary metabolites. More than any other time, there is a worldwide search for new antibiotics due to the alarming increase in microbial resistance against the currently available therapeutics. Even though a few antimicrobial compounds have been isolated from endolichenic fungi, most of them have moderate activities, implying the need for further structural optimizations. Recognizing this timely need and the significance of endolichenic fungi as a promising source of antimicrobial compounds, the activity, sources and the structures of 31 antibacterial compounds, 58 antifungal compounds, two antiviral compounds and one antiplasmodial (antimalarial) compound are summarized in this review. In addition, an overview of the common scaffolds and structural features leading to the corresponding antimicrobial properties is provided as an aid for future studies. The current challenges and major drawbacks of research related to endolichenic fungi and the remedies for them have been suggested.Item Pheromone baited biopesticide for control of Leucinodes orbonalis Guenee in brinjal plant(Frontiers in Bioscience, Elite, 2020) Nusra, M.S.F.; Paranagama, P.A.; Amarasinghe, L.D.; Udukala, D.N.Leucinodes orbonalis Guenee, Brinjal fruit and shoot borer (BFSB), is the major pest on brinjal world wide. Larvae of this pest cause the damage; which at initial stages adversely affect the shoot growth, and in later stages diminish fruit quality. Spraying of insecticides is the main pest control method. This has been absolutely ineffective due to concealed habit of the larvae. Such a phenomenon apprehends to the development of insecticides resistance which demands continuously increasing doses. Manipulating the insect behavior using semiochemicals could be an opportunity for better management of insect pest. Application of BFSB sex pheromone components (E)-11-hexadecenyl acetate (E11-16: Ac) alone or in combination with (E)-11-hexadecen-1-ol (E11-16: OH) in traps can be used to suppress the populating growth. Goal-oriented interdisciplinary research on semiochemicals for sound administration of BFSB will depend on a better understanding of the key chemical ecology stimuli of relevance to the pest.Item In vitro bioactivity of the Ayurvedic drug Ramabana Rasa in the Sri Lankan market(Faculty of Science, University of Kelaniya, Sri Lanka, 2020) Zhulfaa, Z.F.; Paranagama, P.A.; Herapathdeniya, S.Ramabana Rasa is an Ayurvedic drug which is commonly used to treat rheumatism and digestive problems. For its preparation, Hg (Parada) and S (Ghandaka) are triturated into nanoparticles of black HgS (Kajjali) and combined with various plant ingredients. Though previous studies have assessed the bioactivity of the different plant ingredients and the responsible phytochemicals separately, similar bioactive properties may not be present in the prepared drug. Lack of proper manufacturing practices in Sri Lanka may have caused significant variations in the composition and bioactivity of the drugs available in the market. The present research was carried out to investigate the in vitro bioactivity of the Ayurvedic drug Ramabana Rasa available in the market in Sri Lanka and evaluate the brand-to-brand and batch-to-batch variations. Drug samples of three different batches from three different manufacturers were purchased, powdered, shaken with methanol and water separately at 150 rpm. for 24 hours, vacuum filtered, concentrated and freeze dried. The methanol and aqueous extracts of the drugs were screened for antioxidant activity by the α,α-diphenyl-β-picrylhydrazyl (DPPH) free radical and 2,2′-azino-bis (3- ethylbenzothiazoline-6-sulfonic acid) (ABTS) cationic radical scavenging assays. Antiinflammatory activity was determined by the human red blood cell membrane stabilization assay. Antibacterial activity was explored using the agar well diffusion method against Escherichia coli, Staphylococcus aureus and Bacillus subtilis. The IC50 values were statistically analyzed using one-way analysis of variance (ANOVA) and Fisher’s comparison test. Both methanol and aqueous extracts exhibited antioxidant and anti-inflammatory activities in a dose dependent manner and the activities were found to be higher in the methanol extracts than the aqueous extracts except in a few samples at certain concentrations. The lowest IC50 reported for DPPH free radical scavenging activity was 67 ± 3 µg/ml for a methanol extract which was higher than the IC50 of the standard antioxidant 2,6-di-tert-butyl-p-cresol (BHT) (23 ± 2 µg/ml). The lowest IC50 reported for ABTS cationic radical scavenging activity was 24 ± 3 µg/ml for an aqueous extract where as BHT showed an IC50 of 14 ± 2 µg/ml. In the anti-inflammatory assay, the methanol extracts of several drug samples showed 50% inhibition at lower concentrations compared to the IC50 of the standard anti-inflammatory drug aspirin (164 ± 11 µg/ml). The lowest IC50 reported for the methanol extract was 124 ± 8 µg/ml suggesting high anti-inflammatory activity. No antibacterial activity was observed against the organisms tested. The results revealed that Ramabana Rasa drugs in the Sri Lankan market have good antioxidant activity and strong anti-inflammatory activity, while there are significant differences (ɑ = 0.05) in bioactivities between brands and batches of the same brand.Item A Novel Cytotoxic Compound From the Endolichenic Fungus, Xylaria psidii Inhabiting the Lichen, Amandinea medusulina(Natural Product Communication, 2020) Santhirasegaram, S.; Wickramarachchi, P.A.S.R.; Attanayake, R.N.; Weerakoon, G.; Samarakoon, S.; Wijeratne, K.; Paranagama, P.A.The lichen host, Amandinea medusulina, collected from mangrove habitats in Sri Lanka, and its associated endolichenic fungi were isolated and identified by rDNA-ITS sequence analysis and morphological features. One of the fungal strains frequently isolated from the lichen thalli was identified as Xylaria psidii. This study aimed at the isolation and identification of the cytotoxic compounds present in this fungus. Secondary metabolites of X. psidii were first extracted into ethyl acetate and subsequently subjected to bioassay-guided fractionation to isolate the bioactive compounds. Sulforhodamine B assay against a lung cancer (NCI-H292) cell line was used to determine the differential cytotoxic activity. Bioassay-guided fractionation led to the isolation of an active compound, SS/02/29/08, showing moderate cytotoxicity (IC50 = 27.2 µg/mL). Its structure was elucidated by IR, 1D- and 2D-NMR, and 13C-NMR spectrophotometry and MS, in combination with HRMS, 13C NMR, HSQC, HMBC, and DQF-COSY. The structure of SS/02/29/08 was determined as (Z)-3-{(3-acetyl-2-hydroxyphenyl)diazenyl}-2,4-dihydroxybenzaldehyde and identified as a new compound. This novel compound has promising differential cytotoxic activity against human lung cancer cell line (NCI-H292).Item 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.Item 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.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.