Graduate Studies

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    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
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    Comparative study on Proximate and Mineral composition in Vernonia cinerea
    (International Postgraduate Research Conference 2019, Faculty of Graduate Studies, University of Kelaniya, Sri Lanka, 2019) Weerasinghe, P.; Abeysinghe, T.; Paranagama, P.A.
    Vernonia cinerea is a herb having several medicinal properties such as antidiabetic, antioxidant, antimicrobial, antiinflammatory and anticarcenogenic. This work investigated the proximate and mineral composition in different parts (leaves, roots, stem and flowers) of V. cinerea using standard procedures. Collected data of proximate and mineral analysis were analyzed using one-way ANOVA. The results of the proximate composition were found to be as follows; ash content ranged between 10.00 ± 1.32 % w/w and 20.17 ± 6.74 % w/w while both leaves and roots had the highest and flower had the least ash content which was significantly different (P<0.05). The moisture content ranged between 61.24 ± 2.17 % - 75.45 ± 4.74 % w/w in the different parts of the plant. The mean nitrogen content ranged between 1.91 ± 0.23 % w/w and 4.80 ± 0.18 % w/w. The protein content in flower, leaf, stem and roots of V. cinerea were significantly different (P<0.05) and the values ranged between 9.19 ± 1.08 % w/w - 23.07 ± 0.85 % w/w %. The plant parts were also analyzed for mineral elements such as Zn and Cu metal ions using Atomic Absorption Spectroscopy (AAS). The levels of these elements found were; Zn:5.58 ± 0.14 - 51.27 ± 0.81 mg/kg and Cu 3.29 ± 0.06 - 8.57 ± 0.10 mg/kg with few exceptions. The mineral content of flower, roots, stem and leaves were significantly different (P<0.05). Therefore, V. cinerea as a therapeutically beneficial herb has a significant amount of proximate and mineral ion composition that can be specified.
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    Identification of Volatile Compounds in Different Parts of the Brinjal Plant (Solanum melongena)
    (19th Conference on Postgraduate Research, International Postgraduate Research Conference 2018, Faculty of Graduate Studies,University of Kelaniya, Sri Lanka, 2018) Nusra, M.S.F.; Paranagama, P.A.; Amarasinghe, L.D.; Udukala, D.N.
    Plants synthesize and emit a large variety of volatile compounds, which produce a wide range of behavioral responses in insects. In some plants, these volatile organic compounds are the key compounds that are involved in attraction of insect pests. In addition, the synergism between insect pheromones and plant volatiles can increase the attraction of insect pest, offering new strategies for the development of more effective and reliable pest control programs. Volatile mediated foraging behavior is important in insect pests when they target host plants. The volatiles of brinjal may be functionally more important in host-finding behavior of their insect pest. Samples of brinjal plant leaves, fruits, shoots and flowers were collected from the unsprayed brinjal field. Steam distillation method was used to extract the volatiles from shade dried brinjal leaves, shoots, flowers and fresh fruits. The small pieces of the plant material were subjected to steam distillation for 4 hours at a distillation rate of 50 ml/hour. In this study, gas chromatography coupled with mass spectrometry technique was used for the identification of volatiles extracted from host plant brinjal. The GC oven was programmed at an initial oven temperature of 40 0C increased at a rate of 1 0C/min to 50 0C, then increased at a rate of 4 0C/min to 210 0C, and then raised at a rate of 8 0C/min to a final temperature of 230 0C. The MS data of eluted compounds were acquired and identified by the comparison of their retention times to those of authentic standards and with mass spectra from NIST library. Results indicated that brinjal plant produces 1-Methyl-5,8-dimethoxy-1,2,3,4-tetrahydro-1,4-iminonaphthalene, Benzyl alcohol, Phenylethyl Alcohol, Benzeneacetaldehyde, Butylated hydroxytoluene and Methyl salicylate from leaves; Butylated hydroxytoluene, n-Octyl formate, Butyl phthalate and 1,3-Dioxolane, 2-(methoxymethyl)-2-phenyl from fruits; Benzyl alcohol, 1,6-Octadien-3-ol, 3,7-dimethyl, Butylated hydroxytoluene, Hydroquinone and Methyl salicylate from shoots; and 1,6-Octadien-3-ol, 3,7-dimethyl, Methyl salicylate, Butylated hydroxytoluene, Hydroquinone and Benzyl alcohol from flowers as major compounds
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    Effect of the essential oil of Cymbopogon nardus on Aspergillus flavus Link isolated from Sri Lankan paddy and rice
    (Faculty of Graduate Studies, University of Kelaniya, Sri Lanka, 2002) Paranagama, P.A.; Abeysekera, K.H.; Abeywickrama, K.; Nugaliyadda, L.
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    Electroantennograme and behavioural responses of male and female Callosobruchus maculatus (F.) to essential oil of lemongrass Cymbopogon citratus (Stapf)
    (Faculty of Graduate Studies, University of Kelaniya, Sri Lanka, 2002) Paranagama, P.A.; Adhikari, A.A.C.K.; Abewickrama, K.P.; Bandara, K.A.N.P.
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    Development of Essential Oil Based Tablets from Cinnamomum Zeylanicum Leaves and Cymbopogon Nardus Against the Stored Grain Pest Insect, Sitophilus Oryzae in Traditional Rice Varieties in Sri Lanka
    (Faculty of Graduate Studies, University of Kelaniya, Sri Lanka, 2016) Paranagama, P.A.; Maduwanthi, H.M.I.
    Essential oils have been used as an alternative method to reduce postharvest losses from rice weevil, Sitophilus oryzae in traditional rice varieties cultivated in Sri Lanka. In the present study essential oils extracted from Cinnamon leaves (Cinnomomum zeylanicum) and Citronella leaves (Cymbopogon nardus) were used to control rice weevil, in three traditional rice varieties, Rathkanda al, Pachchaperumal, Suwandal along with a hybrid variety, White raw rice cultivated in Sri Lanka. Kaolin pellets treated with test essential oil mixture, C. zeylanicum and C. nardus (2: 1 v / v) were used to test the effect on rice. The major components of the essential oils of C. zeylanicum leaf oil and citronella oil were eugenol, citronellal and geraniol. Number of S. oryzae in treated rice samples throughout the test period were significantly decreased compared with the controls. A significant decrease of % seed damage was also observed during the 6 months of storage of rice in polyethylene bags. Flavor, aroma, and stickiness of cooked rice have enhanced significantly in all treated rice varieties compared to controls. The physical and organoleptic properties of cooked rice were not significantly different with controls after 6 months of treatment. Only 5 % weigh loss of kaolin tablets was revealed after completing the test. The insecticidal activity of the above essential oil mixture showed the highest effect on the rice type Suwandal.
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    Secondary Metabolites with Radical Scavenging Activity from Daldinia Eschcholzii, Isolated from The Lichen Parmotrema Sp. In Sri Lanka-Isolation and Structure Determination
    (Faculty of Graduate Studies, University of Kelaniya, Sri Lanka, 2016) Manthrirathna, M.A.T.P.; Kandiah, R.; Gunasekara, D.S.; Paranagama, P.A.
    Natural products are promising leads for novel therapeutic agents. Isolation and characterization of bioactive compounds in search for potential pharmocophores has acquired a developing interest in on-going research. Although Endolichenic fungi (EF) are a rich source of bioactive secondary metabolites, they still remain almost unexploited. The present study is focused on isolation and structure elucidation of compounds with radical scavenging activity from the EF, Daldinia eschscholzii that occur in the lichen Parmotrema sp. in Hakgala Botanical Garden, Sri Lanka. Daldinia eschscholzii cultivated on 48 large petri dishes with PDA were incubated at room temperature for one week. Mycelia were cut in to small pieces along with the medium and extracted with ethyl acetate twice. The radical scavenging activity of the crude extract was evaluated using DPPH assay. Standard antioxidant, Butylated Hydroxy Toluene (BHT) and MeOH were used as the positive control (IC50= 38.2 ± 4.0 μg/ mL) and negative control respectively. The crude extract with high radical scavenging activity (IC50 = 77.9 ± 5.1 μg/ mL), was partitioned with hexane, chloroform and aqueous methanol. All three organic extracts were then subjected to DPPH assay. Chloroform fraction with the highest activity (IC50= 63.8 ± 4.8 μg/ mL) was further fractionated using silica gel, sephadex column chromatography and preparative TLC to isolate two pure compounds. The structures of the compounds were elucidated using 1H, 13C, 2D NMR and MS data. The compounds were identified as 7-hydroxy- 2-methylchroman-4-one (1) and 5-methoxynaphthalen-1-ol (2). Compound 1 showed no activity in the assay. Compound 2 showed higher activity than the standard BHT, with IC50 value of 10.2 ± 5.8 μg/ mL.
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    Interspecific Relationships of Piper Species in Sri Lanka as Revealed by DNA Barcode ITS
    (Faculty of Graduate Studies, University of Kelaniya, Sri Lanka, 2016) Jayarathna, N.; Senanayake, S.P.; Rajapakse, S.; Jayasekera, R.; Paranagama, P.A.
    The genus Piper which is the largest genus in the family Piperaceae is economically important as it comprises with valuable crop species as well as several wild species. Although ten species of genus Piper are recorded in Sri Lanka, their interspecific relationships were not well studied. Therefore, the aim of this study was to determine interspecific relationships of Piper species in Sri Lanka using internal transcribed spacers from nuclear ribosomal DNA )ITS(. Ten varieties of Piper nigrum L., two varieties of each P. betle L.and P. longum L., P. chuvya )Miq.( C. DC., P. siriboa L., P. sylvestre Lam., P. walkeri Miq. and P. zeylanicum Miq. were used for the study. The genomic DNA was extracted from tender leaf samples using CTAB method, PCR amplified using ITS primer pair and subjected to DNA sequencing. The DNA sequence alignment analysis was carried out and a dendrogram was constructed using the multiple sequence alignment programme MUSCLE. According to the dendrogram clear formation of two major clusters can be seen and ten P. nigrum varieties were clustered together however, variety Kuching has shown a separation from the main cluster. P. longum, P. walkeri, P. sylvestre, P. siriboa, P. betle, P. chuvya and P. zeylanicum formed the other cluster in which P. siriboa has shared close similarities with P. betle, while P. sylvestre and P. walkeri displayed close relationships and clustered together with P. longum varieties. Interspecific relationships between wild species and cultivated species )P. nigrum, P. betle and P. longum( indicated the potential of using the wild species in breeding programs for crop improvement. However, further studies to reveal genetic and phenetic relatedness of those species are needed to deepen the knowledge on interspecific relationships of Piper species.
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    New Bioactive poliketide from endolichenic fungus, Curvularia trifolii, inhabiting the lichen, Usnea sp. collected from Haggala Botanical Garden
    (Faculty of Graduate Studies, University of Kelaniya, 2015) Samanthi, K.A.U.; Wickramaarachchi, S.; Wijerathne, E.M.K.; Paranagama, P.A.
    Endolichenic fungi (ELF), which live in healthy tissues of lichens, are one of the promising producers of structurally diverse novel bioactive secondary metabolites. It has been reported that endolichenic fungi are rich sources for the discovery of new compounds with significant medicinal values and this was demonstrated by isolation and identification of new anticancer, antifungal, antibacterial and antioxidant agents from endolichenic fungal extracts. In Sri Lanka, research group of University of Kelaniya initiated a research program on ELF and have isolated few interesting bioactive poliketides from ELF of three lichen species available in Sri Lanka. They have shown great potential to be a source for novel bioactive natural products. The objectives of this study were isolation and identification of bioactive compounds of the endolichenic fungus, Curvularia trifolii isolated from the lichen Usnea species. In a continuation of our chemical investigations in search of secondary metabolites of endolichenic fungi with medicinal value led to the isolation of few bioactive compounds. In this present study, C. trifolii was grown in large scale and incubated at room temperature for 10 days. The metabolites were extracted into ethyl acetate (EtOAc). Antibacterial, anticancer and insecticidal activities of the EtOAc extract were evaluated using in-vitro bioassays. The EtOAc extract was subjected to a silica gel column chromatography resulting, one major compound with high bioactivities. The isolated pure white crystalline compound was identified as 1,14-dihydroxy-6-methyl-6,7,8,9,10,10,14,14 -octahydro-1H-benzo[f][1] oxacyclododecin-4(13H)-one using 1H, 13C NMR, 2D NMR and MS data. The compound was found to possess antibacterial activity against three test pathogens, anticancer activity at (NCIH-460), (MCF-7), (SF-268), (PC3M), and (MDA-MB-231) cell lines and insecticidal activity.
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    Isolation of bioactive secondary metabolites from the endolichenic fungi, Neosartorya sp. inhabiting the lichen Parmotrema sp. in Sri Lanka
    (Faculty of Graduate Studies, University of Kelaniya, 2015) Manthrirathna, M.A.T.P.; Kandiah, R.; Gunasekara, D.S.; Paranagama, P.A.
    Natural products are a potential source of novel pharmaceutical agents. Therefore, isolation and identification of bioactive compounds from organisms adapted to various biotopes and unraveling their bioactivities in search for new pharmacophores has a mounting interest. Fungi are known to be prominent producers of useful metabolites. Endolichenic fungi (ELF) that occur asymptomatically within the lichen thalli are one of the ecological groups of fungi. ELF in Sri Lanka remain almost unexplored as a source of useful bioactive compounds. The objective of this study is to isolate bioactive secondary metabolites from ELF Neosartorya sp. isolated from Parmotrema sp. that occur in Hakgala Botanical Garden. ELF Neosartorya sp. was cultivated on 48 PDA plates and incubated at room temperature. Secondary metabolites were extracted into ethyl acetate from 9 days old cultures. Antibacterial activity of the crude extract was evaluated against Bacillus subtilis (BS) and Staphylococcus aureus (SA) using agar well diffusion method. Standard antibiotic Azithromycin was used as the positive control and Dimethyl sulfoxide as the negative control. Since the crude extract showed antibacterial activity against both BS and SA, it was partitioned with hexane, chloroform (CHCl3) and aqueous methanol. All three fractions showed activity against SA, with the CHCl3 fraction having higher activity compared to the other two fractions. Chloroform and methanol fractions showed significant activity against BS, while CHCl3 fraction showed activity comparable with Azithromycin. Chloroform fraction of Neosartorya sp. was further fractionated using bio-assay guided fractionation (silica gel column chromatography). Pure compounds were isolated using preparative TLC. One major pure compound was isolated from CHCl3 fraction and the characterization still in progress.