Secondary Metabolites Investigation On The Endophytic Fungus Chaetomium Nigricolor F5 Harbored In The Plant Mahonia Fortunei

Endophytes,mainly including endophytic fungi and endophytic bacteria,are a class of microorganisms,which inhabit the intercellular or intracellular spaces of the healthy plants.They are an important source of small-molecule compounds with potential medicinal values.Chaetomium species are frequently found endophytic fungi that could produce structurally novel and diverse secondary metabolites.These molecules exhibit anticancer,antimicrobial,anti-inflammatory,antiviral,antioxidant,immunosuppressive,and plant growth-promoting activities,together with the prevention of agricultural pests.Previous chemical investigation on the endophytic fungus Chaetomium nigricolor F5 by our research group,led to the discovery of chamiside A with a new 6/6/5 fused tricyclic core skeleton and antibacterial activity.It suggests that C.nigricolor F5 is a potent "talented strain",worthing further exploration of cytochalasans.In addition to C.nigricolor F5 we previously obtained,44 endophytic fungi were further isolated from the plant Mahonia fortunei.All these fungal endophytes were evaluated by chemical analysis and biological activities for mining bioactive more cytochalasans.C.nigricolor F5 was finally selected as the target strain for further chemical investigation in this thesis.Based on the one strain many compounds(OSMAC)strategy,19 different culture media were selected to further induce the production of cytochalasans and other compounds in C.nigricolor F5.Rice medium with the addition of phenylalanine and potato dextrose agar(PDA)medium were successfully selected for large-scale fermentation to obtain secondary metabolites.The fermented products were isolated and purified by column chromatography and high performance liquid chromatography(HPLC).The obtained compounds were structurally elucidated by extensive spectroscopic analyses,including MS,NMR,ECD,IR,and single crystal X-ray diffraction analysis.From the rice medium of C.nigricolor F5,a total of 14 structurally diverse secondary metabolites were isolated and identified,including eight cytochalasans,chamisides B–G(1–6),and chaetoconvosins C and D(7 and 8);two steroids,demethylincisterol B(9),and ergosterol(10);one polyketide: methylpolyketide A(11);one terpenoid: granulolactone C(12);two lipids: 1-linoleoylglycerol(13),and monopalmitolein(14).Compounds 1–6 and 9–11 are new compounds.Compounds 1–3 have a new 5/6/5/5/7 fused pentacyclic skeleton,while compound 4 features a unique 6/6/5/7/5 ring system,and compounds 5 and 6 have a rare 6/6/5skeleton and a long side chain.Compounds 1–3 were hypothesized to be key biosynthetic precursors for the co-isolated compounds 4–8,updating the previously proposed biosynthetic pathway of cytochalasans.Niemann-Pick C1-like 1(NPC1L1)is an important target for the development of lipidlowering drugs.Cytochalasans were evaluated for their interactions with NPC1L1 by computer molecular docking.The results showed that the highest binding score to NPC1L1 was from compound 5.It can be well bound to the cholesterol-binding site of NPC1L1.Surface plasmon resonance(SPR)indicated that compound 5 can interact with NPC1L1 in a concentration-dependent manner,showing similar response units with the only FDA-approved NPC1L1 inhibitor ezetimibe.Further assay using cells that overexpressed NPC1L1 showed that compound 5 can inhibit cholesterol absorption,which was also similar to the positive drug ezetimibe.Extensive activity evaluation showed that the obtained cytochalasans had no obvious antibacterial,antifungal,and cytotoxic activities,and compound 1 had moderate allelopathic activity for inhibiting root elongation of Arabidopsis thaliana.Chemical investigation on the PDA media of C.nigricolor F5 afforded 16 known compounds,including two fatty acids: 9Z,12Z-octadecadienoic acid(15),and 2-dodecanedioic acid(16);two terpenoids: granulolactone(17),and myrrhlalkyldiol(18);four aromatic compounds: 2,4-dichlorobenzoic acid(19),4-(2-hydroxyethyl)pheno(20),(2R,4R)-harzialacton A(21),and(2R,4S)-harzialacton A(22);one flavonoid: daidzein(23);three dipeptides: cyclo(S-Pro-S-Ile)(24),cyclo(S-Pro-S-Leu)(25),and cyclo(D-Pro-D-Phe)(26);four bis(naphtho-gamma-pyrone)compounds: chaetochromin A(27),isochaetochromin A2(28),chaetochromin B(29),and chaetochromin C(30).In conclusion,this project explores secondary metabolites of the endophytic fungus C.nigricolor F5 harbored in the medicinal plant M.fortunei by using OSMAC strategy,mainly focusing on cytochalasans.A total of 30 compounds were isolated and identified,including six novel cytochalasans,two new steroids,and one new polyketide.Chamiside B(1)had moderate allelopathic activity on root elongation inhibition,and chamiside F(5)showed promising inhibition activity against NPC1L1 protein at the protein and cell levels,which was comparable to ezetimibe.This work enriches the structural diversity of cytochalasans,and provides the foundation for the discovery and application of bioactive cytochalasans.