International Research Symposium on Pure and Applied Sciences (IRSPAS)

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Subject: search.filters.subject.Phytochemicals

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    Isolation, purification and structure elucidation of antimicrobial and bio-active compounds of soil fungi.
    (International Research Symposium on Pure and Applied Sciences, 2017 Faculty of Science, University of Kelaniya, Sri Lanka., 2017) Wijesekara, W. A. M. A.; Wijesinghe, K.D.; Kumara, K. G. N. P.; Manage, P. M.
    Soil fungi are considered as a rich source of bioactive compounds and antibiotics. They are able to produce a great variety of secondary metabolites characterized by a broad spectrum of properties of bioactive compounds including antiviral, antibacterial, antifungal, and anticancer. Therefore, the objective of the study was isolation, purification and structure elucidation of antimicrobial and bioactive compounds of soil fungi. In the present study, soil samples were collected from the Kelani River mouth, Sri Lanka and twenty nine fungi were isolated on potato dextrose agar. The antimicrobial activity of the methanol crude extract of fungi was tested against two human pathogenic Gram-positive bacteria (Bacillus sp., Staphylococcus aureus-ATCC 2593), two human pathogenic Gram-negative bacteria (Escherichia coli-ATCC 25922, 2785 and Salmonella typhi) and two human pathogenic fungi (Candida albicans and Candida tropicalis) using agar disc diffusion method. One fungal isolate was selected based on the diameter of inhibition zone to isolate and purify antimicrobial (antifungal and antibacterial) compound. The antioxidant property in the crude extract was evaluated using DPPH (2,2-diphenyl-1-picrylhydrazyl) scavenging test (Inhibitory Concentration, IC50 = 200.42 ppm). Secondary phytochemical screening for major classes of antibacterial compounds were done using TLC visualization reagent specific for major classes of antibacterial compounds. Structural elucidation of isolated compound was carried out using Gas Chromatography Mass Spectrometry (GC-MS) and Fourier-Transform InfraRed spectroscopy (FTIR). TLC plates of the crude extract developed using different solvent system showed different number of band with antimicrobial compounds as it is revealed by bioautochromatography, solvent system 1 ( Ethyl Acetate (EA): Methanol (Me): Water (Wa) (100:13.5:10) 6 bands against Bacillus sp. solvent system 2 ( EA :Toluene (93:7) ) 2 bands against Bacillus sp. and two bands with antifungal activity was recorded against C. albicans. Band with antimicrobial compounds on Preparative Thin Layer Chromatography (PTLC) was scraped and dissolved in methanol (HPLC) to further purify using Solid Phase Extraction (SPE) and High Performance Liquid Chromatography (HPLC). Antibacterial activity of each purified compounds, was verified using bioautochromatography. According to the secondary chemical screening, the compound with the highest antimicrobial activity was found to have contain Arbutin and Anthraquinone glycoside. The results of the present study revealed that the crude extracts of soil fungi had antibacterial activity against some human pathogenic bacteria and the soil fungi is a potential candidate to utilize as a source to produce antibiotics.
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    Assessment of phytochemicals and antifungal effect of Croton aromaticus against postharvest fungal pathogens isolated from tropical fruits
    (Faculty of Science, University of Kelaniya, Sri Lanka, 2016) Wijesundara, S.A.D.T.L.; Kannangara, S.; Abeywickrama, K.
    Chemical control using synthetic chemical fungicides is still the most common method of controlling postharvest diseases of fruits. Plant extracts which are rich in antimicrobial secondary metabolites such as terpinoids, alkaloids, saponins and flavonoids could be possible alternatives for synthetic fungicides. Current study was focused on evaluating the antifungal effect of ethanolic extract of Croton aromaticus (Kappettiya) leaves in vitro against mycelial growth and the spore germination of postharvest fungal pathogens isolated from fruits of banana (Colletotrichum musae, Rhizopus sp., Lasiodiplodia theobromae) papaya (Rhizopus stolonifer, Colletotrichum gleosporioides, Lasiodiplodia theobromae) and mango (Alternaria alternata, Pestalotiopsis mangiferae, Lasiodiplodia theobromae). Surface sterilized diseased banana, papaya and mango fruit tissues were cultured on PDA plates in order to obtain pure cultures of possible fungi and they were identified by morphological and microscopic characteristics, using identification keys. Inhibitory effect of the ethanolic extract of C. aromaticus against test pathogens were investigated by well diffusion method using PDA medium, by incorporating crude extract dissolved in DMSO, ranging from 1 mg/ml up to 300 mg/ml concentrations along with the positive (Captan) and negative (DMSO) controls. Significant (P < 0.05) inhibitory effects were exhibited by the ethanolic extract of C. aromaticus leaves against all test pathogens except L. theobromae. The highest mycelial growth and spore germination inhibition of most of the pathogens were observed at 100 mg/ml. The lowest Minimum Inhibitory Concentration of the leaf extract (5 mg/ml) was observed for spore germination inhibition of C. gleosporioides and P. mangiferae. TLC analysis revealed four compounds having Rf values of 0.551, 0.672, 0.810 and 0.913. Phytochemical screening of ethanolic extract revealed the presence of alkaloids, terpenoids, quinones, phytosterols and flavonoids. Current findings indicate the potential use of ethanolic extract of C. aromaticus leaves in controlling banana, papaya and mango postharvest fungal pathogens in vitro.