International conference on Frontiers in Chemical Technology 2020 (FCT 2020)
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Item Antibacterial activities of peptaibols, trichocellin A-I and B-II, isolated from the endophytic fungus Trichoderma reesei(Institute of Chemistry Ceylon Adamantane House, Rajagiriya, Sri Lanka., 2020) Madhushika, D. P. H.; Jayasundara, J. M. N. M.; Ratnaweera, P. B.; Williams, D. E.; de Silva, E. D.; Andersen, R. J.Antibiotics are highly used in chemotherapy for bacterial infections worldwide. However, due to misuse and overuse of antibiotics pathogenic bacteria have developed and continue to develop resistance to the existing antibiotics rendering them ineffective. Thus there is an urgent need to develop new types of antibiotics to counter the spread of these lethal, drug resistant infections. Endophytic fungi of Cyperaceae plants are a relatively unexplored but a significant resource for the discovery of new biologically active natural products. Therefore, the main objective of this study was to isolate bioactive secondary metabolites of the endophytic Trichoderma reesei (GenBank accession number MT476871) fungus isolated from Cyperus iria in Sri Lanka. Initial screening of T. reesei showed antibacterial activity against the Gram positive Bacillus cereus and Staphylococcus aureus at 400 μg/mL. T. reesei was grown in large scale and extracted into ethyl acetate following an incubation period of 13 days. Solvent-solvent partitioning of the crude extract (1005 mg) led to an active chloroform fraction (580 mg), which was chromatographed on Sephadex LH20 size exclusion column chromatography. The active fraction B (143 mg) from the six fractions (A-F) obtained from size exclusion chromatography was further purified via C18 reversed-phase high performance liquid chromatography (HPLC) to yield two peptaibols. The structures of the peptaibols were determined as trichocellin A-I and B-II by nuclear magnetic resonance (NMR) and mass spectral data. Both peptaibols A-I and B-II were active against the two Gram positive bacteria tested with minimum inhibitory concentrations (MICs) of 64 and 8 μg/mL for B. cereus and 32 and 16 μg/mL for S. aureus respectively. The positive control Gentamycin gave a MIC of 2 μg/mL for both bacteria. To the best of our knowledge this is the first study to isolate trichocellins from an endophytic T. reesei fungus. The study revealed that the isolated trichocellins from T. reesei are potent antibacterial compounds.Item Antibacterial activity of entomopathogenic fungi isolated from Vespa affinis and Apis dorsata in Sri Lanka(Institute of Chemistry Ceylon Adamantane House, Rajagiriya, Sri Lanka., 2020) Sandeepani, H. P.; Ratnaweera, P. B.Antibiotic resistance of bacteria throughout the world has caused serious health issues. Thus, there is a crucial need for the discovery and development of new antibiotics. Entomopathogenic fungi infect and eventually kill their host insects. However, entomopathogenic fungi are known to be a rich source of bioactive secondary metabolites. The main aim of the current study was to isolate and investigate the antibacterial activities of entomopathogenic fungi of two insect species Apis dorsata and Vespa affinis collected from Uva Wellassa University library premises, Badulla, Sri Lanka (810 4’ E, 60 58’N). Freshly dead insect cadavers were collected, surface sterilized, crushed and spread on antibiotic (Amoxicillin, 10 mg/mL) enriched potato dextrose agar media plates. The emerging fungi were isolated, pure cultures were obtained and extracted into ethyl acetate. The fungal crude extracts were tested for antibacterial activity against Staphylococcus aureus (ATCC 25928), Bacillus cereus (ATCC 11718), Escherichia coli (ATCC 35218) and Pseudomonas aeruginosa (ATCC 9027) using agar disc diffusion method (400 μg/disc) and bioautography. The experiments were carried out in triplicate. For the identification of fungi, genomic DNA were isolated, the ITS region of the ribosomal RNA gene was amplified using ITS1 and ITS4 primers, the obtained PCR products were sequenced and BLAST analyzed. Nine morphologically different entomopathogenic fungal strains were isolated from A. dorsata while eight were isolated from V. affinis. Thirteen fungal extracts showed antibacterial activity against at least one bacterial species tested while four extracts were inactive to all bacteria. Ten, eight and nine fungal extracts showed some activity against S. aureus, B. cereus and E. coli respectively while none of the extracts showed any activity against P. aeruginosa. Talaromyces versatilis fungal extract from A. dorsata showed the best inhibition with a mean diameter of 13.00 ± 1.00 mm against E. coli with an activity similar to the positive control (Gentamycin, 1000 ppm). The bioautography assays revealed that T. versatilis and an Aspergillus extracts were active against S. aureus while A. nomius extract was active against B. cereus. The FTIR analysis and thin layer chromatography studies exhibited the presence of many secondary metabolites in all fungal crude extracts. This study shows that entomopathogenic fungi of V. affinis and A. dorsata are potential sources for the discovery of novel antibacterial drug leads.Item Antibacterial cytochalasin B from endophytic Curvularia lunata isolated from Cyperus iria of Sri Lanka(Institute of Chemistry Ceylon Adamantane House, Rajagiriya, Sri Lanka., 2020) Kumarasinghe, K. H. T. P.; Pavalakantharasa, T.; Jayasundara, J. M. N. M.; Ratnaweera, P. B.Antibiotic resistivity of bacteria has been increasing rapidly over the last few decades and currently it has become a severe human health crisis in the world. Therefore, isolating novel antibacterial drug leads from natural sources has become an essential requirement in today’s world. Thus, the main aim of the current study was to isolate the antibacterial compounds from the endophytic fungus Curvularia lunata isolated from an underutilized plant Cyperus iria of Sri Lanka. During a preliminary research C. lunata fungal extract showed antibacterial activity against Gram positive Staphylococcus aureus and Bacillus cereus. Hence the fungus was grown in large scale in 140 Petri dishes and was extracted to ethyl acetate after 21 days of incubation. The ethyl acetate crude extract (354 mg) obtained was purified using bioassay guided fractionations using a series of solvent/ solvent fractionations, Sephadex LH20 size exclusion chromatography using methanol as the eluent, normal phase silica gel chromatography using a gradient elusion and finally by reversed phase high performance liquid chromatography to isolate the major bioactive compound. The structure elucidation using 1H, 13C and 2D (COSY, HSQC, HMBC) nuclear magnetic resonance (NMR) and mass spectroscopic data revealed the identity of the major bioactive compound as cytochalasin B (1). Cytochalasin B showed prominent antibacterial activity with a minimum inhibitory concentration (MIC) of 64 μg/mL against S. aureus while the positive control Gentamycin showed a MIC of 4 μg/ mL. Further purification of other bioactive fractions with interesting 1H NMR spectra may to lead to novel antibacterial compounds. This study reveals that endophytic C. lunata of C. iria are potential producers of antibacterial compounds.