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Item Variation of catechin and caffeine content in exotic collection of tea [Camellia sinensis (L.) O. Kuntze] in Sri Lanka and potential implication in breeding cultivars with enhanced quality and medicinal properties(Food Chemistry Advances, 2022) Kottawa-Arachchi, J. D.; Ranatunga, M.A.B.; Amarakoon, A.M.T.; Gunasekare, M.T.K.; Attanayake, R.N.; Sharma, R.K.; Chaudhary, H.K.; Sood, V.K.; Katoch, R.; Banyal, D.K.; Piyasena, K.G.M.P.; Edirisinghe, E.N.U.Tea leaves are rich in diverse metabolites with medicinal importance. The quality of made tea is largely depended on the qualitative and quantitative attributes of key metabolites. The aim of the present study was to quantify flavan-3-ols and caffeine of exotic tea germplam and explore the medicinal properties. Fresh leaf flavan-3-ols; catechin, epicatechin (EC), epicatechin gallate (ECg), epigallocatechin (EGC) and epigallocatechin gallate (EGCg), caffeine and gallic acid of 131 accessions (87 exotic and 44 improved cultivars) were performed by High Performance Liquid Chromatography. Variations in metabolites among cultivars as well as between seasons were determined. Among the flavan-3-ols, EGCg was the most abundant followed by EGC, ECg and EC. Hierarchical clustering of 131 accessions based on metabolite diversity resulted two major clusters. Indian introductions clustered with well-known high quality cultivars indicating the potential utilization of high quality black tea production. Exotic accessions with low caffeine contents (< 20 mg g−1) were grouped separate cluster indicating the promising genetic resources for the development of low caffeine tea. Present study revealed that selected exotic tea accessions rich in bioactive compounds such as catechins and caffeine content could be utilized in producing speciality teas with enhanced quality and medicinal properties.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 Identification of the fungal pathogen causing leaf blight of Cinnamomum zeylanicum Blume and in vitro screening of Tebuconazole sensitivity(The Second National Symposium of Sri Lanka Association for Mycology and Plant Pathology (SLAMPP), 2022) Madhurangi, H. M. T. T.; Wanigatunge, R.P.; Jayasinghe, G.G.; Attanayake, R.N.Leaf Blight Disease (LBD) is a major nursery disease of cinnamon (Cinnamomum zeylanicum Blume) and the disease causes serious foliar damage in seedlings, especially under high humidity and shade conditions. It also causes considerable damage to mature cinnamon bushes through leaf necrosis, defoliation and dieback of newly emerging flush. However, not much research has been conducted on LBD in Sri Lanka despite cinnamon being one of the key export crops. Therefore, the main objectives of the present study were to identify the causative agent of the LBD in Sri Lanka and to determine the minimum inhibitory concentration of Tebuconazole, one of the commonly used fungicides, to mitigate the pathogen growth in-vitro. LBD symptomatic leaves were collected from cinnamon fields in the Matara district representing 4 categories; tender leaves with initial LBD spots, tender leaves with regular lesions, mature leaves with regular lesions, and mature leaves with irregular lesions. Infected tissues from each category were cultured on PDA and percentages of emerging fungal colonies were determined. Pure cultures of all the fungal isolates were tentatively identified using morphological traits up to the genus level. From the cultures of tender leaves, Rhizoctonia and Colletotrichum-like isolates frequently appeared in 60% and 33.3% respectively. However, in the cultures of mature leaves, Pestalotiopsis-like isolates were detected more frequently (22 – 35%). To determine the exact causal agent, Koch’s postulates were performed using representative isolates from each genus on detached tender cinnamon leaves. Actively growing mycelial plugs were inoculated on one side of the damaged leaf and the other side was inoculated with plain PDA plugs. Leaves were incubated in a moist chamber for 03 days and symptom development was observed. Typical LBD symptoms were developed when mycelial plugs of Colletotrichum-like species were introduced. Rhizoctonia and Pestalotiopsis-like species produced brown, circular or irregular lesions while Colletotrichum-like species showed a unique concentric ring pattern which is similar to the original symptoms of LBD. The potential pathogen species causing LBD was confirmed to be a Colletotrichum-like species. For species identification DNA of two Colletotrichum-like fungal isolates was extracted and ITS gene sequencing confirmed that the two isolates were C. gloeosporioides and C. horii with 100% similarity to the vouchered accessions of the GenBank. In vitro fungicide, sensitivity assay was conducted using a concentration gradient of the fungicide Tebuconazole. It was found that Tebuconazole could mitigate 100% growth of both Colletotrichum spp. at 5 ppm concentration in-vitro.Item Antioxidant, a-Amylase Inhibitory Activities and Photoprotective Properties of Peels of Nephelium Lappaceum Linn. (Malwana Special)(Oriental Journal of Chemistry, 2021) Binuwangi, A.K.D.M.; Perera, M.P.J.; Silva, A.A.G.; Attanayake, R.N.; Rajapakse, C.S.K.This study focused on evaluation of antioxidant, α-amylase inhibitory activities and photo protective properties of peels of Nephelium lappaceum Linn. (rambutan); Malwana special. Methanolic extract of peels was sequentially partitioned in hexane, dichloromethane (DCM) and aqueous methanol. The methanol extract showed a significantly (p greater then 0.05) higher DPPH radical scavenging activity than that of butylated hydroxytoluene. Among the fractions, the highest total phenolic content (TPC) was found in the aqueous methanol fraction. DCM and aqueous methanol fractions were rich in flavonoids. In vitro α-amylase inhibitory activity of the aqueous methanol fraction was also significantly higher than the standard drug, acarbose. Partially purified aqueous methanol fraction of rambutan peels exhibited UV-B absorption with a moderate solar protection factor. The results revealed that the peels of Nephelium lappaceum Linn., Malwana special can be considered as a promising source for the development of natural antioxidant, cosmeceutical sunscreen and antidiabetic agents.Item Decaying Hardwood Associated Fungi Showing Signatures of Polyethylene Degradation(BioResources, 2021) Perera, P.; Deraniyagala, A.S.; Mahawaththagea, M.P.S.; Herath, H.; Rajapakse, C.S.K.; Wijesinghe, P.; Attanayake, R.N.The involvement of wood decay fungi and the importance of their enzymes in polyethylene degradation is well documented. Therefore, decay-resistant hardwood associated fungi should be better degraders with their versatile enzymatic systems. In the current study, decaying hardwood associated fungi were isolated and their ability to degrade low-density polyethylene (LDPE) was assessed. Thirty-three isolates were identified by sequencing the internal transcribed spacer region of nuclear ribosomal DNA. Randomly selected isolates were tested for laccase producing abilities. Three species were selected to test their potentials in LDPE sheet degradation. Fungi were incubated in Czapek-Dox broth containing 20-micron LDPE sheets at room temperature for 60 days. The biodegradation signatures were assessed by analyzing the changes in structural characteristics of LDPE using Fourier transform infrared spectroscopy (FTIR), scanning electron microscopy (SEM), percent reduction of tensile properties, and weight loss. FTIR analysis revealed changes in certain functional groups compared with the control, indicating chemical changes resulting from the treatment. LDPE sheets incubated with fungi showed cracks and holes under SEM analysis, percent reduction in tensile properties, and weight loss, which are the signatures of degradation. This study revealed that the hardwood decaying basidiomycetes, Phlebiopsis flavidoalba, Schizophyllum commune, and Phanerodontia chrysosporium have the potential for in vitro LDPE degradation.Item Antioxidant activity and chemical constituents of methanolic extract of Durio zibethinus Murr. (durian) peels(MEDICINAL PLANTS - INTERNATIONAL JOURNAL OF PHYTOMEDICINES AND RELATED INDUSTRIES, 2021) Perera, P.J.; Binuwangi, A.K.D.M.; Silva, A.A.G.; Attanayake, R.N.; Wickramarachchi, S.R.; Rajapakse, C.S.K.This study aimed to determine the DPPH free radical scavenging activity, total phenolic content (TPC) and total flavonoid content (TFC) of methanolic extract of Durio zibethinus Murr. (durian) peels and its fractions. The chemical constituents of durian peels extracted into methanol by soxhlet extraction were sequentially extracted into hexane, dichloromethane and aqueous methanol. Among the fractions, the dichloromethane fraction showed the highest DPPH free radical scavenging activity (IC50 179.9 ± 6.6 μg/ml) with high TPC and TFC (85.82 ± 12.11 mg gallic acid equivalent/g of dried weight of extract and 12.66 ± 1.94 mg of quercetin equivalent/g of dried weight of extract, respectively). A very strong positive correlation (r = 0.9677) was observed between the DPPH free radical scavenging activity and the TPC of fractions and a strong positive correlation (r = 0.7858) was noticed between the DPPH free radical scavenging activity and TFC of the fractions indicating that phenolic compounds in durian peels may contribute to their strong antioxidant activity. As the dichloromethane fraction had constituents with the highest antioxidant activity, it was analyzed by Gas chromatography-Mass spectrophotometry to identify its volatile constituents. The results revealed that the dichloromethane fraction was rich in [1,2-Benzenedicarboxylic acid, bis (2-ethylhexyl) ester], [2,3-diphenylquinoxaline], [2-coumaranone], [4-((1E)-3-hydroxy-1-propenyl)-2-methoxyphenol], [7,9-di-tert-butyl-1-oxaspiro (4,5) deca-6,9-diene-2,8-dione] and [phenol, 2,4-bis(1,1-dimethylethyl)], which are known to exhibit antioxidant activity.Item Can Anaerobic Soil Disinfestation (ASD) be a Game Changer in Tropical Agriculture?(Pathogens, 2021) Priyashantha, A.K.H.; Attanayake, R.N.Anaerobic soil disinfection (ASD) has been identified as an alternative soil-borne pathogen control strategy to chemical fumigation. ASD involves the application of an easily liable carbon source followed by irrigation to field capacity and maintenance of an anaerobic condition for a certain period. A literature search undertaken on ASD found that more than 50 comprehensive research projects have been conducted since its first discovery in 2000. Most of these studies were conducted in the USA and in the Netherlands. Though the exact mechanism of ASD in pathogen control is unknown, promising results have been reported against a wide range of pathogens such as fungi, nematodes, protists, and oomycetes. However, it is interesting to note that, except for a few studies, ASD research in the developing world and in the tropical countries has lagged behind. Nevertheless, with soil quality depletion, reduction in arable lands, and exponential population growth, a drastic change to the current agricultural practices should be adapted since yield gain has reached a plateau for major staple crops. Under such circumstances, we identified the gaps and the potentials of ASD in tropical agricultural systems and proposed promising biodegradable materials.Item Optimization of DNA extraction protocol and DNA barcoding of Hedyotis quinquinervia in Sri Lanka(Faculty of Science, University of Kelaniya, Sri Lanka, 2020) Gunarathne, A.; Attanayake, R.N.; Ratnayake, R.M.C.S.Hedyotis quinquinervia, has been identified as a critically endangered possibly extinct (CR(PE)) endemic plant by the National Red List 2012 of Sri Lanka. However, in 2014 it was rediscovered from Thotupolakanda montane forests. It has a high potential as an ornamental plant due to compact, mosaic arrangement of curved shiny leaves with prominent veins. Thick leaf cuticle of this plant, hindered the extraction of good quality DNA for barcoding. Therefore, the aim of the present research was to establish an efficient DNA extraction protocol for H. quinquinervia in the absence of liquid N2. H. quinquinervia leaves were stored at -80 °C or in silica gel for 48 hours prior to the DNA extraction. Three DNA extraction protocols were tested and agarose gel electrophoresis and spectroscopic method were used for the evaluation of quality and quantity of extracted DNA. All the extracted DNA samples were subjected to Polymerase Chain Reaction (PCR) for the amplification of nuclear rDNA-ITS region for barcoding. Out of three methods, classical CTAB protocol with 0.2% β-mercaptoethanol and 2% polyvinylpyrrolidone in CTAB buffer was successful, only after removing the cuticle of the adaxial surface of the leaf. The cuticle removal was achieved simply by using a clear tape and it was confirmed by staining the clear tape with safranin and observing it under the microscope. No DNA extraction was successful with the cuticle present on the leaves. PCR amplification was successful from the extracted DNA and rDNA-ITS sequences were obtained. A sequence of 435 bp, exhibited 98% query cover and 100% identity to H. quinquinervia of the NCBI database. Sequences were deposited in the GenBank under the accession numbers MT373692 and MT373691. It was also found that with the removal of cuticle, the amount of leaves required for good quality DNA extraction was five times less than that of the leaves with cuticle. Modified CTAB buffer and the cuticle removal from fresh leaves of H. quinquinervia were quick and easy modifications to obtain good quality DNA in the absence of liquid N2. Cuticle removal method using clear tapes and the storage at -80 ℃ prior to DNA isolation could be recommended for identification of H. quinquinervia and other plant species with thick cuticle layers on the leaves.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.