Chemistry
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Item Bioactive Properties and Metabolite Profiles of Endolichenic Fungi in Mangrove Ecosystem of Negombo Lagoon, Sri Lanka(Natural Product Communications, 2022) Weerasinghe, R. H.; Maduranga, K.; Attanayake, R. N.; Shevkar, C.; Kate, A. S.; Weerakoon, G.; Kalia, K.; Paranagama, P.Endolichenic fungi (ELF) serve as a novel source of natural products with distinctive bioactivities. A total of 58 ELF isolated from 29 lichens collected from mangrove and mangrove-associated plants of Negombo lagoon, Sri Lanka were identified using morphological and DNA barcoding techniques. Ethyl acetate extracts of 18 such identified ELF isolates were subjected to in vitro assays to determine antioxidant, anti-inflammatory, tyrosinase inhibitory and antibacterial potency. Liquid chromatography–mass spectrometry (LC–MS) dereplication was conducted on the crude extracts in order to detect the secondary metabolites present. The extracts of Daldinia eschscholtzii and Hypoxylon lividipigmentum had the highest radical scavenging activity with SC50 values 14.27 ±0.24 μg/mL and 18.34±1.37 μg/mL, respectively. D. eschscholtzii also exhibited remarkable anti-inflammatory activity (IC50 7.97 ±0.09 μg/mL). Tyrosinase inhibitory activity was highest in Cytospora xylocarpi (IC50 68.50 ±0.34 μg/mL), while the highest activity against aerobic bacterial species Escherichia coli, Bacillus subtilis, Staphylococcus aureus and the anaerobic bacterial strain Streptococcus mutans was observed in the extracts of Xylaria feegenesis and Curvularia lunata. After a thorough study of the LC–MS profiles, it was found that the chemical profiles of Neofusicoccum occulatum, H. lividipigmentum and Myramaececium rubricosum were previously poorly explored in the literature.Item Bioprospecting of an Endolichenic Fungus Phanerochaete sordida Isolated from Mangrove-Associated Lichen Bactrospora myriadea(Journal of Chemistry, 2022) Weerasinghe, R. H.; Shevkar, C. D.,; Maduranga, K.; Pandey, K. H.; Attanayake, R. N.; Kate, A. S.; Weerakoon, G.; Behera, S. K.; Kalia, K. S.; Paranagama, P. A.Bioassay-guided fractionation of the ethyl acetate extract of Phanerochaete sordida, an endolichenic fungus (ELF) isolated from the host lichen Bactrospora myriadea, collected from Negombo lagoon, Sri Lanka, led to the isolation of a bioactive compound. Following the identification of the fungus using morphological and DNA barcoding techniques, the pure compound was isolated using column chromatography, preparative TLC, and semipreparative HPLC. The structure elucidation was carried out using IR, HR-ESI-MS and 1H, 13C & 2D NMR spectroscopic methods. The in vitro bioassays conducted revealed that compound 1 has a high antioxidant activity with ABTS•+ (IC5058:91 ± 1:35 μM), moderate anti-inflammatory activity (IC50254:79 ± 1:41 μM), comparable antibacterial activity against the oral-bacterial strain Streptococcus mutans (MIC 898.79 μM and MLC 1797.58μM), moderate tyrosinase inhibition (IC501713:69 ± 8:65 μM), and moderate cytotoxicity against oral cancer (IC5013:65 ± 0:02 μM), in comparison with respective positive controls. The in silico experiments conducted for tyrosinase inhibition and cytotoxicity using Schrödinger revealed results in line with the in vitro results, thus confirming the bioactivities. The molecule also satisfies the key features of drug likeliness according to pharmacokinetic studies.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 Bioprospecting of an Endolichenic Fungus Phanerochaete sordida Isolated from Mangrove-Associated Lichen Bactrospora myriadea(Journal of Chemistry,, 2022) Weerasinghe, R. H.; Shevkar, C. D.; Maduranga, K.; Pandey, K. H.; Attanayake, R. N.; Kate, A. S.; Weerakoon, G.; Behera, S. K.; Kalia, K. S.; Paranagama, P. A.Bioassay-guided fractionation of the ethyl acetate extract of Phanerochaete sordida, an endolichenic fungus (ELF) isolated from the host lichen Bactrospora myriadea, collected from Negombo lagoon, Sri Lanka, led to the isolation of a bioactive compound. Following the identification of the fungus using morphological and DNA barcoding techniques, the pure compound was isolated using column chromatography, preparative TLC, and semipreparative HPLC. The structure elucidation was carried out using IR, HR-ESI-MS and 1H, 13C & 2D NMR spectroscopic methods. The in vitro bioassays conducted revealed that compound 1 has a high antioxidant activity with ABTS•+ (IC5058:91 ± 1:35 μM), moderate anti-inflammatory activity (IC50254:79 ± 1:41 μM), comparable antibacterial activity against the oral-bacterial strain Streptococcus mutans (MIC 898.79 μM and MLC 1797.58μM), moderate tyrosinase inhibition (IC501713:69 ± 8:65 μM), and moderate cytotoxicity against oral cancer (IC5013:65 ± 0:02 μM), in comparison with respective positive controls. The in silico experiments conducted for tyrosinase inhibition and cytotoxicity using Schrödinger revealed results in line with the in vitro results, thus confirming the bioactivities. The molecule also satisfies the key features of drug likeliness according to pharmacokinetic studies.Item Antibacterial polyketide from Lasiodiplodia theobromae, inhabiting the lichen host, Pyrenula bahiana on mangrove ecosystems in Puttalam lagoon, Sri Lanka(Asian Journal of Chemistry, 2022) Paranagama, P. A.; Santhirasegaram, S.; Fathima, M. M. A.; 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 identity 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 anti-bacterial 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,Item Identification of Novel Bioactive Compounds, Neurosporalol 1 and 2 from anEndolichenic Fungus, Neurospora ugadawe Inhabited in the Lichen Host,Graphis tsunodae Zahlbr. from Mangrove Ecosystem in Puttalam Lagoon, Sri Lanka(Asian Journal of Chemistry, 2021) Maduranga, H.; Weerasinghe, W.; Attanayake, R.; Santhirasegaram, S.; Shevkar, C.; Kate, A.S.; Weerakoon, G.; Samanthi, K.; Kalia, K.; Paranagama, P. A.Endolichenic fungi (ELF) have been recognized as a promising source of bioactive secondary metabolites. Sri Lanka, a biodiversity hotspot, harbours a remarkable diversity in mangrove ecosystems. In the present study, endolichenic fungi obtained from mangrove lichens of Puttalam lagoon were studied for their secondary metabolites. An endolichenic fungus, Neurospora ugadawe, isolated from the lichen host Graphis tsunodae Zahlbr. was identified up to the species level. Secondary metabolites of N. ugadawe were extracted into ethyl acetate and crude extract showed promising antioxidant and anti-inflammatory activities. Bioassay guided separation on silica gel column chromatography and preparative TLC were performed to isolate the bioactive pure compounds. Two novel bioactive pure compounds were identified as Neurosporalol 1 and Neurosporalol 2. Neurosporalol 1 showed the highest antioxidant activity compared with the positive control BHT. Further, this compound showed very low anti-inflammatory activity compared to that of the positive control aspirin. Neurosporalol 2 showed comparable antioxidant and moderate anti-inflammatory activities.Item A Novel Cytotoxic Compound From the Endolichenic Fungus, Xylaria psidii Inhabiting the Lichen, Amandinea medusulina(Natural Product Communications, 2020) Santhirasegaram, S.; Wickramarachchi, 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 Identification of Novel Bioactive Compounds, Neurosporalol 1 and 2 from an Endolichenic Fungus, Neurospora ugadawe Inhabited in the Lichen Host, Graphis tsunodae Zahlbr. from Mangrove Ecosystem in Puttalam Lagoon, Sri Lanka(ASSIIAN JOURNAL OF CHEMIISSTRY, 2021) Maduranga, H.; Weerasinghe, W.; Attanayake, R.; Santhirasegaram, S.; Shevkar, C.; Kate, A.; Weerakoon, G.; Samanthi, K.; Kalia, K.; Paranagama, P. A.Endolichenic fungi (ELF) have been recognized as a promising source of bioactive secondary metabolites. Sri Lanka, a biodiversity hotspot, harbours a remarkable diversity in mangrove ecosystems. In the present study, endolichenic fungi obtained from mangrove lichens of Puttalam lagoon were studied for their secondary metabolites. An endolichenic fungus, Neurospora ugadawe, isolated from the lichen host Graphis tsunodae Zahlbr. was identified up to the species level. Secondary metabolites of N. ugadawe were extracted into ethyl acetate and crude extract showed promising antioxidant and anti-inflammatory activities. Bioassay guided separation on silica gel column chromatography and preparative TLC were performed to isolate the bioactive pure compounds. Two novel bioactive pure compounds were identified as Neurosporalol 1 and Neurosporalol 2. Neurosporalol 1 showed the highest antioxidant activity compared with the positive control BHT. Further, this compound showed very low anti-inflammatory activity compared to that of the positive control aspirin. Neurosporalol 2 showed comparable antioxidant and moderate anti-inflammatory activities.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 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.