International Research Symposium on Pure and Applied Sciences (IRSPAS)

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Author: search.filters.author.Weerakoon, G.

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    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.
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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.