Study On The Secondary Metabolites Of Wetland Fungus Aspergillus Flavipes PJ03-11 Based On OSMAC Strategy

Microbial secondary metabolites have been an important source of new drugs and their lead compounds.With the continuous development of modern genomics,it is found that in the genome of microbes,there are a large number of genes that are not expressed in the conventional culture conditions,that is,"silent gene".The existence of these silent genes has caused the waste of microbial resources.Therefore,how to activate silencing genes and produce more diverse secondary metabolites of structural types has become a hot topic of microbial research in recent years.In this paper,the wetland fungus Aspergillus flavipes PJ03-11,isolated from the Panjin Red Beach Scenic Area in Liaoning Province of China,was studied.In order to activate the potential silent gene of the strain,and obtain more active secondary metabolites with diverse structural types,we adopted the "one strain many compounds"(OSMAC)strategy.We added 1 mM NaBr and 2 mM 5-azacytidine to the rice culture medium of strain PJ03-11,respectively.Then the large scale fermentation was performed.The secondary metabolites were isolated by solvent extraction and various chromatographic methods(such as silica gel column chromatography,sephadex LH-20 column chromatography,ODS columnchromatography,and HPLC,etc.).Their structures were determined based on extensive spectroscopic analysis(such as MS,UV,IR,and NMR,etc.).35 compounds were isolated from the fermentation collected from the culture medium with 1 mM NaBr.They were elucidated as follows:2-methyl-versiquinazoline C(PA1),flavichalasine N(PA2),flavichalasine O(PA3),aspochalasin Ⅰ(PA4),aspochalasin D(PA5),aspochalasin M(PA6),aspergilluchalasin(PA7),trichalasin H(PA8),aspergillin PZ(PA9),methyl 4-(2-pyrrolyl)-1-isoquinolone-3-carboxylate(PA10),(-)-2S-N-benzoyl-L-phenylalaninol(PA11),anabellamide(PA12),butyrolactone Ⅰ(PA13),methybutyrolactone Ⅲ(PA14),2-O-methylbutyrolactone Ⅱ(PA15),7-methoxypenicillixanthone(PA16),penicillixanthone(PA17),asterric acid(PA18),methyl chloroasterrate(PA19),methyl dichloroasterrate(PA20),neogeodin hydrate(PA21),sulochrin(PA22),dihydrogeodin(PA23),(±)-geodin(PA24),questin(PA25),emodin(PA26),emodin 1-methyl ether(PA27),penipurdin A(PA28),(+)-2’S-isorhodoptilometrin(PA29),(-)-2’R-isorhodoptilometrin(PA30),(-)-methyl shikimate(PA31),(-)-methyl 4-epi-shikimate(PA32),(-)-methyl 5-epi-shikimate(PA33),methyl 2-hydroxy-4-methyl-6-methoxy-benzoate(PA34)and 3,5-dihydroxy-4-methoxy-benzoic acid(PA35).Among them,there were 4 new compounds.Compound PA1 was a new fumiquinazoline-type alkaloid;compounds PA2 and PA3 were new cytochalasans,and PA3 represented the first example of cytochalasans possessing a nitrogen-oxygen heterocycle at the macrocyclic ring part;compound PA16 was a new oxanthrone.Moreover,compounds PA17,PA24,PA28,and PA31-35 were obtained from the genus Aspergillus for the first time.17 compounds were isolated from the fermentation collected from the culture medium with 2 mM 5-azacytidine.Among them,8 compounds haven’t obtained from the above culture medium.They were elucidated as follows:flaviphenalenone D(PB1),(-)-aspergillidiene A(PB2),methyl asterrate(PB3),2,4-dichloroasterric acid(PB4),butyrolactone Ⅱ(PB5),aspulvinone Q(PB6),epicoccolides B(PB7),1,3-dihydro-7-methyl-4,5,6-trihydroxy-isobenzofuran(PB8).Among them,compounds PB1 and PB2 were new compounds.MTT method was used to test the cytotoxicity of some isolated compounds.The results showed that all the isolated cytochalasans PA2-PA9 showed different degrees of cytotoxic activity against human leukemia cell HL-60.PA2-PA5 showed potent cytotoxic activity against three cell lines(THP-1,HL-60,PC-3),and especially for cell line PC-3,PA2-PA5 showed stronger cytotoxic activity than positive control 5-fluorouracil.PAS also showed comparable cytotoxic activity with positive control against the other two cell lines(THP-1,HL-60).Meanwhile,PB2 showed potent cytotoxic activity against Caco2 and HepG2 cell lines with IC50 velues at 14.56 and 14.99μM,respectively.PA16 and PA17 showed significant cytotoxic activity against HepG2 cell line with IC50 velues at 14.56 and 14.99 pM,respectively.Compounds PA16,PA17 and PA24 exhibited moderate cytotoxic activity against Caco2 cell lines.