Biological Activities and Bioactive Metabolites of Fungal Endophytes Isolated from the Local Medicinal Plants of Sireh (Piper betle), Mengkudu (Morinda citrifolia) and Lada Hitam (Piper nigrum) Leaves

Mohammad Norsyahir, Mohd Norazi (2019) Biological Activities and Bioactive Metabolites of Fungal Endophytes Isolated from the Local Medicinal Plants of Sireh (Piper betle), Mengkudu (Morinda citrifolia) and Lada Hitam (Piper nigrum) Leaves. Masters thesis, Universiti Malaysia Sarawak.

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Abstract

ABSTRACT Endophytic fungi have attracted great attention in the past few decades due to its ability to produce novel secondary metabolites. The production of secondary metabolites from endophytes are associated with environmental factors. Genetics exchange between endophyte with their host allow these microorganisms to produce similar bioactive compounds as their host. All 21 endophytic fungi were successfully isolated from Morinda citrifolia, Piper betle and Piper nigrum. Each host consisting of seven endophytic fungi were narrowed down to top three with the highest production of total phenolic content (TPC). Endophytic fungus Hypoxylon investiens (MC 1.3) found to contain highest concentration of TPC with 0.234 ± 0.024 GAE mg/ml. The selected endophytic fungi were successfully identified through morphological and molecular method. Selected endophytic fungi from Morinda citrifolia was identified to be Hypoxylon investiens (MC 1.3), Xylaria sp. (MC 1.4) and Curvularia sp. (MC 2.6). The endophytic fungi from Piper betle identified to be Hypoxylon investiens (PB 1.3), Phlebia acanthocystis (PB 2.3) and Hypoxylon investiens (PB 3.1). Piper nigrum’s endophytic fungi was identified to be Bipolaris cactivora (PN 1.2), Inonotus pachyphloues (PN 2.2) and Pyrrhoderma sp. (PN 3.2). Apart from total phenolic content (TPC), the selected endophytic fungi were further analyses in total flavonoid content (TFC) and DPPH free radical scavenging was tested in the antioxidant assays. In total flavonoid content (TFC), H. investiens (MC 1.3) was the highest concentration of total flavonoid content (TFC), with 0.121 ± 0.007 QUE mg/ml. The endophytic fungus H. investiens (MC 1.3) show the lowest value of EC50 with 75.23 ± 7.78 DPPH µg/ml. Endophytic fungus H. investiens (MC 1.3) was the highest production of antioxidant in the study of antioxidant assays. In antimicrobial properties, the primary screening of hydrolytic enzymes protease, chitinase, amylase and cellulase was tested. All selected endophytic fungi showed the positive result in the production of protease, amylase, chitinase and cellulase by produced halo zone around fungi colony. Antimicrobial assays were further analyses by using disc diffusion assay. In disc diffusion assay by using ethyl acetate extract of crude metabolite of endophytic fungi, the extract is more sensitive and inhibit higher in the gram-positive bacteria compare to Gram-negative bacteria. The extract from Curvularia sp. (MC 2.6) and B. cactivora (PN 1.2) showed no activity or no inhibition of Candida albicans. H. investiens (PB 3.1) found to be the largest inhibition zone towards S. aureus with diameter 15.67 ± 0.47 mm while Pyrrhoderma sp. (PN 3.2) showed the largest inhibition zone towards S. pyogenes with diameter of inhibition zone 13.67 ± 1.70 mm. In the Gram-negative bacteria tested strain of S. typhi and E. coli, H. investiens (PB 3.1) showed the largest inhibition zone 13.67 ± 1.70 mm and 13.33 ± 1.70 mm. The largest inhibition zone of C. albicans by H. investiens (PB 3.1) with 12.00 ± 0.82 mm. The study showed H. investiens (PB 3.1) found to be the highest among the selected fungi for antimicrobial properties. The major compound found in ethyl acetate extract of endophytic fungi by using GC-MS was Octadec-9-enoic acid (MC 1.3), 4-(2-Methyl-cyclohex-1-enyl)-but-3-en-2-one (MC 1.4), Cyclobutane, 1,2-diethenyl-3,4-dimethyl- (MC 2.6), 3-(n-Propylamino)-2,1-benzisothiazole (PB 1.3), 1H-Indene, 1-ethylideneoctahydro-7a-methyl-, (1E,3a. alpha.,7a. beta.) (PB 2.3), Homoserine (PB 3.1), n-hexadecanoic acid (PN 1.2), benzene, 1,1'-oxybis [4-phenoxy] (PN 2.2) and 1-Propanol, 2-methyl- (PN 3.2). In conclusion, antimicrobial activities, antioxidant activites and biological compound produce can be applied to medical, agriculture and industrial. Keywords: endophytic fungi, medicinal plant, antioxidant activity, antimicrobial activity, bioactive compounds.

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