Chemical And Bioactivity Investigation Of Three Coral-associated Fungi

Marine microorganisms and their host organisms have formed special metabolic pathways in the long-term co-evolution process under the special marine environment.They produce lots of secondary metabolites with significant bioactivity participating in self-defense.The possible ecological effects of these chemicals provide important clues for the discovery of active substances in drug searching.It has attracted the attention of scholars in the field of natural products.In recent years,the research on secondary metabolites of marine microorganisms has become a hotspot in marine drugs.Our group is mainly engaged in the investigation of chemical constituents of marine invertebrates and their symbiotic microorganisms.We had investigated Dongsha and Xisha of China systematically.Many novel secondary metabolites with remarkable activity were found,and some compounds showed great medicinal value.As a continued work in searching for active substances from marine microorganisms,we investigate the chemical and bioactivity of three coral-associated fungi Alternaria sp.Strain DX-FOF7?Aspergillus flavipes and Trametes hirsute.Fifteen compounds were isolated from the fermentation broth of the fungus Alternaria sp.Strain DX-FOF7 from Lobophytum sp.,these compounds are macrophorin A?A1?,?1R,5R,6R?-5-hydroxyl-4-?hydroxymethyl?-1-???1S,4aR,8aR?-5,5,8a-trimethyl-2-methylene-6-oxodecahydronaphthalen-1-yl?methyl?-7-oxabicyclo[4.1.0]hept-3-en-2-one?A2?,methyl 2-hydroxy-3-???3-hydroxyl-3-?2-??1S,4aR,6S,8aR?-6-hydroxy-5,5,8a-trimethyl-2-methylenedecahydronaphthalen-1-yl?acetyl?-5-oxocyclopent-1-en-1-yl?methyl?thio?propanoate?A3?,butyrolactone I?A4?,aspernolide A?A5?,aspterric acid?A6?,1-O-?9Z,12Z-octadecadienoyl?-glycerol?A7?,wasabidienone E?A8?,?2S,5S,7S?-3a-hydroxyl-exogonic acid?A9?,cephalosporolides E?A10?,cephalosporolides F?A11?,terrein?A12?,penicillic acid?A13?,territrems B?A14?,?9R?-territrems B?A15?Seven compounds were isolated from the fungus Aspergillus flavipes from Cladiella sp.,these compounds are trichalasin H?B1?,aspergilluchalasin?B2?,aspochalasin I?B3?,aspochalasin D?B4?,aspochalasin E?B5?,aspochalasin K?B6?,penilumamide?B7?.Compound B7 was isolated from this fungus for the first time.Nine compounds were isolated from the fungus Trametes hirsute of Cladiella sp.,these compounds are 3?-Hydroxy?22E,24R?-ergosta-5,8,22-trien-7-one?C1?,ergosterol peroxide?C2?,5?,6?-epoxy-?22E,24R?-ergosta-8,22-diene-3?,7?-diol?C3?,3?-pregn-5-ene-20-one-3-ol?C4?,proloquatifoligenin III?terpene glycoside??C5?,?3R,6E,10S?-2,6,10-trimethyl-3-hydroxydodeca-6,11-diene-2,10-diol?C6?,cepharosporolide C?C7?,Methyl 4-hydroxybenzoate?C8?,N-Acetyltyramine?C9?.The antitumor activities of compounds B1-B7 and C1-C9 were tested by CCK-8.The antitumor activities were screened by taking human non-small cell lung cancer?A549?,prostate cancer?PC-3?,liver cancer?Bel-7404?and human myeloid leukemia cells?K562?.Compounds B1,B3,B4,B6,C2 and C3 showed antitumor activity with IC₅₀<100?M.The rest IC₅₀ values were all above 100?M.Use the nuclear factor kappa B receptor activating factor ligand?RANKL?to induced bone marrow macrophages?BMMs?differentiate into mature osteoclasts The effects of compounds B1-B7 and C1-C9 on the differentiation of BMMs into osteoclasts were detected by tartrate-resistant acid phosphatase?TRAP?specific staining.The results showed that compounds B3 and B4 could significantly inhibit the differentiation of BMMs into osteoclasts,while compounds C1 and C3 could promote the differentiation of BMMs into osteoclasts.The proliferation of T lymphocyte in mice were evaluated by carboxyfluorescein diacetate,succinimidyl ester?CFSE?staining with Con A stimulation.The effects of differentiation and regulation of T lymphocyte were evaluated by APC-CD3,APC-CD4,APC-CD8 antibody labeling and flow cytometry.Some comcounds show weak activity on proliferation and differentiation of T lymphocyte in Mice.The inhibitory activity the binding of PD-1/PD-L1 protein was tested with the PD-1/PD-L1 binding assay kit.Some compounds show weak inhibitory activity on the binding of PD-1/PD-L1 protein at 100?M.In this study,we obtained 31 compounds,including two new compounds,one of which is a new skeleton.24 compounds were isolated from their respective fungi for the first time,and the chemical constituents of the fungus Trametes hirsute were systematically studied for the first time.Several activity evaluation models were used to test the activity of some compounds.Four compounds with osteoclast differentiation regulating activity were screened out.These studies have expanded the structure types and quantities of marine natural products,provided some references for bone metabolic diseases,immunomodulation and anti-cancer research targeting at PD-1/PD-L1,provided a basis for further searching for active lead compounds,and provided research data for the rational utilization of marine resources in China.