ISOLATION, STRUCTURAL ELUCIDATION AND BIOLOGICAL ACTIVITIES OF SECONDARY METABOLITES FROM ENDOPHYTIC FUNGI OF Picralima nitida

SOURCE:

Faculty: Agriculture
Department: Pharmaceutical And Medicinal Chemistry

CONTRIBUTORS:

Nwachukwu, C. U.
Okoye, F. B. C.
Eboka, C. J.

ABSTRACT:

Fungi are promising source of bioactive compounds whose potentials are important for drug discovery programme. Endophytic fungi have raised the interest of scientists in drug discovery programme due to their immense contribution to the discovery of new bioactive compounds. This study was aimed at isolating bioactive secondary metabolites from endophytic fungi from Picralima nitida. Objectives include; isolation, purification and identification of endophytic fungi; fermentation and extraction of secondary metabolites from the endophytic fungi; screening of extracts for biological activities; isolation, structural elucidation and bioassay of compounds from the endophytic fungal extracts. Endophytic fungi were isolated from fresh and healthy leaves of Picralima nitida, by culturing the leaves on malt extract agar. The fungi were purified on malt extract agar and subjected to molecular identification. The fungi were fermentation on solid state media and metabolites were extracted using ethyl acetate. The fungal extracts were screened for antimicrobial activity using agar well diffusion method, antioxidant activity using 1,1-diphenyl-2-picryl-2-hydrazyl (DPPH) assay and cytotoxicity using 3-(4,5-dimethylthiazole-2-yl)-2,5-diphenyl tetrazolium bromide assay. The active extracts were subjected to vacuum liquid chromatography and size exclusion separation methods. The resulting fractions were subjected to high performance liquid chromatography (HPLC) coupled to diode array detector. Pure compounds were isolated using semi preparative HPLC and the structures were elucidated using mass spectroscopy and nuclear magnetic resonance spectroscopy. Two endophytic fungi were isolated from P. nitida and identified as Curvalaria sp. and Endomelanconiopsis sp. At a concentration of 10 µg/ml, the fungal crude extracts showed poor cytotoxic activity against mouse lymphoma cell lines (L5178Y), and no anti-tubercular activity against Mycobacterium tuberculosis. Results of the DPPH antioxidant assay revealed that at a concentration of 500 µg/ml, the fungal crude extracts and fractions showed varying degrees of antioxidant activities, with higher activities recorded for the fractions. Two fractions of Endomelanconiopsis sp. extract PNMR4F7 and PNMR4F15 showed good antioxidant activity with percentage inhibitions of 89.8 and 77.1 respectively. One fraction of Curvularia sp. extract, PNMR3AF13, also showed good antioxidant activity with inhibition of 76.9%. The results of the antimicrobial screening revealed that at a concentration of 10 mg/ml, Curvalaria sp. and Endomelanconiopsis sp. crude extracts showed antibacterial and antifungal activities. Some bioactive compounds were isolated from the two endophytic fungi. These compounds include acropyrone, 4-hydroxyphenylacetic acid and indole-3 acetic acid which were isolated from Curvalaria sp.; and orthosporin, tenuazoic acid, methyl 4-methoxybenzoate and methyl 2-hydoxyl-4-methylbenzoate isolated from Endomelanconiopsis sp. Results of bioassays carried out on the isolated pure compounds showed that at 500 µg/ml, orthosporin and tenuazonic acid both showed Aspergillus niger and Candida albicans.This study shows that endophytic fungi associated with P. nitida can be a potential source of antifungal agent against Aspergillus niger and Candida albicans, and antioxidant lead molecules.