Secondary Metabolites And Bioactivity From Two Sponge-associated Fungi

Due to the high pressure,high salt,lack of oxygen and light,marine organisms have produced novel and active secondary metabolites,which are of great significance for self defense and information exchange.It has been found that there are a large number of symbiotic microorganisms attached to the surface and internal pores of invertebrates such as sponges and corals,forming a community with the host in the long evolution process,which can jointly produce active metabolites with novel structure.The research group focused on the chemical constituents and biological activities of sponge and its epiphyte from South China Sea.The fungi Nigrospora sp.and Aspergillus sclerotiorum were obtained from sponges Callyspongia sp.and Haliclona sp.,respectively.The fermentation products of fungi were isolated and purified by normal phase silica gel column chromatography,Sephadex LH-20gel column chromatography and semi preparative high efficiency liquid chromatography.The structures of the compounds were identified by NMR,high resolution mass spectrometry and modern NMR spectroscopy.Sixteen compounds were isolated,which were five sesquiterpenes,two polyketones,one alkaloid,four ergosterols,two cyclopeptides and two other compounds.Seven compounds were obtained from the fungus Nigrospora sp.associated with sponge Callyspongia sp.and identified as:ergost-7,22-dien-6?-methoxy-3?,5?-diol?A1?,punctaporonin D?A2?,punctaporonin E?A3?,punctaporonin F?A4?,desuccinoylpunctaporonin C?A5?,3?-hydroxy-5?,9?-epoxy-?22E,24R?-ergosta-7,22-dien-6-one?A6?,?20S,22E,24R?-ergosta-7,22-dien-3?,5?,6?-triol?A7?.Compound A5 was reported as a new natural product while compounds A2?A4 and A6 were reported from the fungus Nigrospora sp.for the first time.Nine compounds were obtained from fungus Aspergillus sclerotiorum associated with sponge Haliclona sp.and identified as:penicillic acid?B1?,glycerol monooleate?B2?,6?,14-dihydroxy-7?-methoxyconfertifolin?B3?,6-dihydro-6-hydroxy penicillic acid?B4?,perlolyrin?B5?,furan-2,5-diyldimethanol?B6?,sclerotides A?B7?,sclerotides B?B8?,?20S,22E,24R?-ergosta-7,22-dien-3?,5?,6?-triol?B9?.Compounds B3 and B5 were reported from the fungus Aspergillus sclerotiorum for the first time.The antitumor activity of compounds A1?A4,A6,A7,B1,B4?B6 was tested using CCK8 method with human colon cancer HCT116 cells,human liver cancer Huh-7 cells and human liver cancer Huh-7 stem cells.Preliminary screening results of in vitro activity showed that at a concentration of 50?mol/L,compounds A1,B1 and B5 had different degree of inhibition on HCT116 cells;at a concentration of 50?mol/L,compounds A1,A7and B5 had significant inhibition on Huh-7 cells;at a concentration of 25?mol/L,compound B5 had a moderate inhibitory effect on Huh-7 stem cells.Using PD-1/PD-L1 binding analysis kit,Sellake S7911 was used as a positive control drug to inhibit PD-1/PD-L1 interaction activity of compounds A1?A4,A6,A7,B1,B4?B9.Preliminary screening results showed that compounds A6 and B9 exhibited some inhibition of PD-1/PD-L1 binding activity with IC₅₀value of 22.5 and 17.9?mol/L respectively.No other compounds showed inhibitory activity.Sixteen compounds were isolated from the fungi of Nigrospora sp.and Aspergillus sclerotiorum.Compound A5 was a new natural product.Compounds A2?A4 and A6 were isolated from the fungi of Nigrospora sp.for the first time.Compounds B2,B3 and B5were isolated from Aspergillus sclerotiorum for the first time.In vitro activity test,ten compounds showed different degrees of cytotoxic activity and inhibition of PD-1/PD-L1binding inhibition activity,among which compounds A6 and B9 had PD-1/PD-L1inhibition activity,which was the first discovery that steroids had PD-1/PD-L1 binding inhibition activity;compound B5 had tumor stem cell inhibition activity.