Isolation, molecular and biomedical characterisation of endophytes from selected medicinal plants

Plants are primary sources of natural product drugs. However, with every new bioactive molecule reported from a plant source, there follows reports of endangered status or even extinction of a medicinally important plant due to over-harvesting. Hence, the attention turned towards fungi and bacteria namely the endophytes, which reside within plants. They have been found to produce secondary metabolites which are significant sources of novel compounds that have the potential for pharmaceutical, agricultural and biotechnological applications. The objectives in this study were to investigate the diversity and biomedical properties of endophytes in selected local medicinal plants. In efforts to bioprospect for possible antibacterial and anticancer metabolites, a total of 203 endophytic fungi were isolated from 10 medicinal plants. These selected plants were local medicinal plants indigenously used and known traditionally to have antibacterial and anticancer properties. They are Clinacanthus nutans (SK 2280/13), Strobilanthes crispus (SK 2281/13), Lonicera japonica (SK 2282/13), Senna obtusifolia (SK 2283/13), Elephantopus scaber (SK 2284/13), Leea indica (SK 2285/13), Pereskia sacharosa (SK 2286/13), Pereskia bleo (SK 2287/13), Hippobroma longiflora (SK 2288/13), and Persicaria chinensis (SK 2289/13). These endophytes were isolated and grown on 3 medium plates which are actinomycetes isolation agar (ATC), potato dextrose agar (PDA) and starch casein agar (SCA). Genetic diversity studies on the endophyte isolates done on random polymorphic DNA (RAPD) showed that the monomers (OPO 6, OPO 10 and OPO16) used for the amplification were able to cluster majority of the isolates into different parts of the plant. Antibacterial screening results showed that plant Clinacanthus nutans and Strobilanthes crispus have more potential of bioactive properties in comparison to the other 8 plants. A total of 30 fungal endophytes from these 2 plants were then further subjected to anticancer screening and molecular identification. Results from the alignment of the ITS rDNA regions of phylogenetic trees show that plant Clinacanthus nutans with 11 fungal endophytes were attributed to 8 genera whereas, plant Strobilanthes crispus with 19 fungal endophytes were attributed to 8 genera and 1 class. After much screening from the myriad of 203 fungal endophytes for antibacterial and anticancer properties, (SCA)AL3 closely identified as Neosartorya hiratsukae showed the highest significant antibacterial activity against 5 gram-positive bacteria at concentration 200 μg/disc and anticancer activity against all the 5 cancer cell lines tested with half maximal inhibitory concentration (IC50) ranging from 17.67 μg/ml to 88.33 μg/ml. As for the gas chromatography coupled with mass spectrometry (GC-MS) results, a total of 4 volatile metabolites were identified from the dichloromethane crude extract of Neosartorya hiratsukae having more than 10% abundance. It is postulated that the synergistic effects of the 4 volatile metabolites were the contribution to the antibacterial and anticancer properties of (SCA)AL3, Neosartorya hiratsukae. From a total of 203 fungal endophytes which underwent molecular studies and screened against 24 bacterial pathogens and 5 cancer cell lines, the most significant findings was (SCA)AL3, Neosartorya hiratsukae, which was the first reported fungus having medicinal properties isolated from a medicinal plant, Clinacanthus nutans, found in Malaysia.

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