ISOLATION AND CHARACTERIZATION OF BIOACTIVE COMPOUNDS FROM SELECTED MANGROVE ENDOPHYTIC FUNGI ALONG THE KENYA COAST

Abstract

Due to the global spread of resistant bacteria and fungi, antibiotics are no longer as effective as they once were, and microbial illnesses are once more a threat to human life. This situation is offering a continuous opportunity for research of alternative novel bioactive molecules to address the problem. In this study we isolated and characterized bioactive compounds from selected endophytic fungi originating from common mangroves of the Kenya coastline namely; Avicennia marina, Ceriops tagal, Rhizophora mucronata and Sonneratia alba against Staphylococcus aureus and Escherichia coli that could potentially be used for drugs development. Isolation and purification using potato dextrose agar (PDA), potato dextrose broth (PDB) gave 19 mangrove fungal endophytes (MFEs). Morphological identification resulted in 18 MFEs belonging to 5 fungal genera namely; Aspergillus, Penicillium, Fusarium, Cephalosporium and Blastomyces. Molecular identification gave 9 successfully characterized species belonging to the genus Aspergillus namely; Aspergillus flavus, Aspergillus niger, Aspergillus nomius, Aspergillus tubingensis, Aspergillus oryzae, Aspergillus awamori, Aspergillus aculeatus, Aspergillus brunneoviolaceus and Aspergillus welwitchiae. Ethyl acetate extract (EAE) and methanolic extract (ME) were produced through fermentation and extraction with ethyl acetate and ethyl acetate in 10% methanol. The extracts contained alkaloids, phenols, steroids, tannins, and terpenoids, according to preliminary phytochemical screening. Aspergillus flavus ethyl acetate extracts showed maximum activity when tested for susceptibility via disk diffusion on Muller Hinton Agar, with inhibition zone diameters (IZDs) Standard deviation (SD) of (17.1 ± 2.45) and (15.9 ± 2.45) against S. aureus and E. coli, respectively. Aspergillus flavus methanolic extracts had a lower inhibition activity (9.2 ± 0.75) and no action against E. coli at the investigated doses as compared to the positive control. The minimum inhibitory concentration of A. flavus crude extract against S. aureus and E. coli was (0.82 ± 0.05) and (0.91 ± 0.05) mg/ml, respectively. Results of one-way ANOVA test indicated no significant difference in the inhibition between the two test pathogens (p > 0.05). Results of Gas Chromatography coupled with Mass Spectrometry (GC-MS) analysis of ethyl acetate crude extracts of A. flavus revealed four known active compounds namely; Lactic acid, Isopropyl alcohol, Semi carbazone and Corydaldine. All of them were active against a broad spectrum of pathogens including S. aureus and E. coli in this study. Fractionation using Silica gel (60-120) mesh column chromatography of the antimicrobial A. flavus methanolic and ethyl acetate extract, gave 11 compounds which when characterized by TLC resulted in pure compounds with different RF values. The isolates were established to be the new δ-lactones Flavulactone B, Flavulactone C, Flavulactone D, Flavulactone E, and Flavulactone F which were all new compounds alongside the known 6-tridecyloxan-2-one now named Flavulactone A. The other oxy-compounds were the new fatty acid methyl esters; methyl (E) - octadec-3-enoate, methyl (E)- tetracos-3-enoate and Methyl (E)-3- hydroxypentadec-5-enoate, methyl (E)-5-hydroxynonadec-9-enoate and the known methyl 3-hydroxynonadecanoate and (E)-octadec-3-enoic acid. The study confirms that 24 bioactive metabolites indeed reside in endophytic fungi inhabiting selected mangroves from coastal Kenya.