Study On The Marine Fungus Aspergillus Fumigatu SZW01 Secondary Metabolite

Searching for natural drug candidates from marine organisms is a constant driving force for pharmaceutical scientists.Over the past few decades,the ocean has been a rich and diverse source of natural active compounds with wide application prospect.Since the early 1970s,many research groups around the world have carried out chemical research on marine organisms and achieved fruitful results.Studies have shown that there are many bioactive secondary metabolites in the ocean,including alkaloids,terpenoids,polyketones,carotenes and fatty acids and so on.These natural products are rich in chemical diversity and biological activity,and have great attraction for drug candidates.In this project,Aspergillus fumigatu SZW01,a fungus from hainiwan,Shenzhen,was used as the research object.It was fermented in rice medium,and then its secondary metabolites were extracted and identified.The isolated monomer compounds were predicted by molecular docking and their biological activities were evaluated.The strain SZW01 was identified as Aspergillus fumigatu by the combination of microbial morphology and molecular biology its sequence GenBank alignment.After large-scale fermentation in rice medium,methanol was used for ultrasonic extraction,dichloromethane and n-butanol were used for extraction,The n-butanol extract was taken as the experimental object.The secondary metabolites of fermentation were separated and purified by chromatography(TLC,silica gel column chromatography,Sephadex column chromatography,reverse ODS column chromatography and semi-preparative liquid chromatography),then eleven compounds can be acquired.Combined with modern spectroscopic techniques(1HNMR,13C-NMR),the spectra of the isolated compounds were analyzed,and the chemical structures of eleven compounds were acquired.They were Verruculogen(1)、Fumiquinazoline D(2)、Alternarosin A(3)、3-indole ethanol(4)、Cyclo(Leu-Pro)(5)、Cyclo(Val-Pro)(6)、Fumigaclavine C(7)、Cyclo(Leu-Tyr)(8)、Cyclo(Hpro-pro)(9)、Monomethylsulochrin(10)and Asperfumin(11).Among them,compounds 1,2,3,4 and 7 are alkaloids,5,6,8 and 9 are diketopiperazines,and 10-11 are polyketones.Compounds 6,8 and 9 were isolated from Aspergillus fumigatus for the first timeAccording to the structures of 11 compounds and literature search,it is speculated that compounds 1-11 may have antibacterial activity.Therefore,computer simulation of molecular docking was used to analyze the structure of the active site of lanosterol 14α-demethylase target protein,combined with molecular docking scoring to analyze the interaction mode between them,and the key residues related to catalytic activity were found to be Gln72,His392,Phe387,Ser261,His392,Arg96,Ala256,Phe387 and Phe399.Compounds 1,3,5,7,8 and 9 exhibited good binding patterns with lanosterol 14α-demethylase,and after entering the key pocket of receptor activity,they combined with key amino acid residues to exert inhibitory effect.They were excellent new antifungal lead compounds.The antibacterial activities of compounds 1-11 were further tested.The results showed that compounds 5 and 8 were resistant to Fusari.oxysporu.The MIC values of compound 5 and 8 were 16 μg/mL and 32 μg/mL,respectively.Compound 8 showed strong inhibitory activity against Fusarium oxysporum and weak inhibitory activity against Rhizoctonia solani,with MIC values of 64 μg/mL.Compound 9 showed inhibitory activity against Colletotrichum gloeosporioides with MIC value of 32 μg/mL.The conclusion obtained from the experimental verification is basically consistent with the prediction of molecular docking.Lanosterol 14α-demethylase(CYP51)can be used as a target for further study of antibacterial mechanism in the future.