Browsing by Author "Weerakoon, G."

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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.
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.
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,
Anticancer activity of Trichoderma harzianum extract against NCI-H292 lung cancer cells.
(International Research Symposium on Pure and Applied Sciences, 2017 Faculty of Science, University of Kelaniya, Sri Lanka., 2017) Sinthujah, S.; Samarakoon, S. R.; Tennakoon, K. H.; Attanayake, R. N.; Weerakoon, G.; Gunasegara, D. S.; Paranagama, P. A.
Cancer is one of the leading causes of death worldwide. Chemotherapy has been the choice of cancer treatment for many years however, it can also affect normal cells and create many undesirable side effects and have the potential to develop resistance. Therefore, investigators must reassess their approach to translate discovery research into greater clinical success and impact aiming to find novel compounds. Endolichenic fungi (ELF) are potential source of producing many bioactive compounds. Preparations of ELFs extracts are commonly used to search for anticancer activity. Based on the fact that fungal extracts provide evidence to develop anticancer drugs, this study was conducted to evaluate the anticancer activity of an ELF, Trichoderma harzianum, (strain No: MF029755) extract against NCI-H292 lung cancer cells. Organ specific in-vitro assays are imperative in large scale screening of natural products with useful clinical activity. Among many such assays, sulforhodamine B (SRB) assay employs a protein binding aminoxanthene dye, to provide a quantitative analysis of viable cells in a culture following the introduction of the compound. Preliminary investigations revealed that crude ethylacetate extract of an endolichenic fungus, T. harzianum, and chloroform fractions of crude extract (12.5 mgL-1, 25 mgL-1, 50 mgL-1, 100 mgL-1 and 200 mgL-1) obtained by partition were positive for the SRB assay. IC50 values of crude extract and the chloroform fraction were 68.48 mgL-1 and 38.44 mgL-1 respectively. The chloroform fraction was chromatographed over silica gel column to obtain seven fractions. Cytotoxicity of the seven fractions obtained from the crude extract of the fungus was determined using SRB assay against lung cancer cell line NCI-H292 following standard protocols. The cell suspension in Dulbecco’s Modified Eagle Medium (DMEM) was aliquoted into 96-well plate. After incubation cells were treated with two concentrations (100 mgL-1 and 200 mgL-1) of fractions obtained by column chromatography. SRB dye was added to each well and acetic acid was used to remove unbound dye. Absorbance was measured at 540 nm using microplate reader. Survival percentage of the cells was calculated. If no viable cells present pink color of the medium turns colorless. In the current assay control wells and 1st fraction remained pink and all the other treatments turned pink into colorless. Seventh fraction showed the highest activity and further purification, SRB assays and structure elucidation will be carried out.
Bioactive properties and metabolite profile of an endolichenic fungus, Hypoxylon lividipigmentum
(Faculty of Science, University of Kelaniya, Sri Lanka., 2021) Weerasinghe, W. R. H.; Shevkar, C. D.; Silva, R. S. De; Attanayake, R. N.; Weerakoon, G.; Kate, A. S.; Kalia, K.; Paranagama, P. A.
Endolichenic fungi (ELF) serve as a novel source of secondary metabolites. Hypoxylon lividipigmentum is an ELF isolated from the lichen Opegrapha medusulina, collected from mangrove plant Xylocarpus granatum from Negombo lagoon, Sri Lanka. The fungus was identified to the species level using morphological and DNA barcoding techniques. Ethyl acetate extract of the fungus was subjected to in vitro assays to determine antioxidant, anti-inflammatory, tyrosinase inhibitory and antibacterial potency. Liquid Chromatography-Mass Spectrometry (LCMS) dereplication was conducted on the crude extract in order to detect the secondary metabolites present. The extract reported a IC50 value of 18.34±1.37 µg/ml on par with the positive control BHT, in DPPH radical scavenging assay. It also exhibited moderate anti-inflammatory activity with an IC50 value of 81.08±1.05 μg/ml. Tyrosinase inhibitory activity was fairly comparable with an IC50 value of 121.20±2.55 μg/ml. Agar well diffusion assay was conducted to determine antibacterial activity against aerobic bacterial species Escherichia coli, Bacillus subtilis, Staphylococcus aureus and the anaerobic bacterial species Streptococcus mutans. Suppression of growth was shown only against B. subtilis. Five major mass peaks were observed during the study of LCMS profile of the extract. After a thorough dereplication process, two masses could be presumed to be from novel scaffolds. Since none of the mass peaks could be dereplicated within the species or genus level, it could be speculated that the chemical profile of Hypoxylon lividipigmentum was previously poorly explored in literature thus making it an interesting organism to study further for novel metabolites.
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.
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.
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.
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.
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.
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.
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).
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).
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
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.