Investigation Of Two Marine-Derived Fungi For Antimicrobial Secondary Metabolites

In this dissertation, 20 marine-derived fungal strains which isolated from mangrove and deep-sea sediment samples were screened under different culture conditions. The crude extracts of these fungi were comprehensively evaluated by antimicrobial assay together with TLC and HPLC chemical assay as the screen model. Two fungal strains, Nigrospora sp. MA75 (isolated from mangrove plant Pongamia pinnata) and Penicillium commune SD118 (isolated from deep-sea sediment), were chosen as the aimed strains, and were fermented on MH2 medium with shake flask culture and static flask culture respectively, for further investigation of their secondary metabolites. Most of the isolated compounds were evaluated for their bioactivities.Applying antimicrobial-guided isolation based on disk diffusion method, the bioactive compounds were isolated from the fermentation extracts by repeated column chromatography (CC) on silica gel, RP-18, Sephadex LH-20, preparative thin layer chromatography (pTLC) as well as by preparative HPLC (pHPLC) and recrystallization. The structures of these compounds were elucidated by means of spectroscopic methods including UV, IR, MS, NMR, and CD, as well as by comparison with authentic standards or with literature reports. As a result, 18 compounds were isolated and structurally determined from the extract of Nigrospora sp. MA75 fermented on shake MH2 medium, including five antimicrobial compounds (NG3, NG6, NG7, NG9, and NG10). From the extract of P. commune SD118 fermented on static MH2 medium, 17 compounds including one bioactive compound with antimicrobial and cytotoxic activity (PC1) were isolated and elucidated. In addition, the effect of different culture modes (shake flask and static flask) and culture media composition (carbon and nitrogen sources, salinity, ions) on variety of fungal secondary metabolites and yield of bioactive compounds were also studied. Other three compounds were isolated and elucidated from Nigrospora sp. MA75 fermented on static rice solid medium, including two new griseofulvin derivatives (NG1 and NG2); one cochlioquinone derivatives (NG5) were obtained from shake MH2 medium which containing 3.5% NaI and exhibited strong antibacterial and cytotoxic activities. Besides, our study also shows that the production of bioactive compound xanthocillin X(PC1)from P. commune SD118 depended on culture mode and media, and the static MH2 medium was optimal for xanthocillin X production which amounted to 361.55 mg/L. In addition, the proposed biosynthetic pathways of some bioactive compounds isolated from Nigrospora sp. MA75 were also discussed.All of the pure compounds were screened for their antimicrobial activity (5 bacteria strains and 8 plant pathogen fungal strains), and some of compounds were tested for their in vitro cytotoxic activities.In the antibacterial assay, compounds NG5 and NG10 showed strong broad spectrum antibacterial activity. Compounds NG6, NG7, and NG9 exhibited selective inhibitory activity against MRSA, Escherichia coli, and Staphylococcus epidermidis. Compound PC1 significantly inhibited the growth of Staphylococcus aureus, Escherichia coli, Bacillus subtilis, and Bacillus thuringiensis. In the antifungal assay, compound NG3 showed moderate inhibitory activity toward Valsa mali, Stemphylium solani, and Gloeosporium theae-sinensis. In the cytotoxicity assay, compound NG5 showed obvious activity against MCF-7, SW1990, SMMC7721, and MKN-28 cell lines with IC₅₀ values of 4, 5, 7, and 8μg/mL, respectively. Compounds PC1 displayed significant activity to MCF-7, HepG2, NCI-H460, HeLa, DU145, and MDA-MB-231 cell lines with IC₅₀ values of 12, 7, 10, 10, 8, and 8μg/mL, respectively. Compound PC11 showed potent activity toward DU145 cell line with an IC₅₀ value of 5μg/mL.This dissertation focused on bioassay guided isolation, structure elucidation, bioactivity evaluation and culture condition of marine-derived fungal secondary metabolites. Two fungal strains with significant antimicrobial activity, Nigrospora sp. MA75 and P. commune SD118, were systematically studied. These results may be helpful to the exploration and utilization of marine microorganisms.