Secondary Metabolites From Two Deep-sea-derived Penicillium Spp

Natural compounds are well known to be an important source of innovative medicine research.According to statistics,from 1981 to 2019,a total of 1881 small molecule medicines were approved in the past 30 years,of which more than 60%came from natural products and their derivatives.It is worth mentioning that with the deepening of research,there is increasing evidence shows that most natural medicines or lead compounds are actually produced by their symbiotic and epiphyte microorganisms.The discovery of penicillin in the 1950s led to more than 70 years of of intensive research and development of terrestrial microorganisms.Up to now,terrestrial microbial secondary metabolites have been found with a repeat rate of 95%,so people have turned their attention to the ocean.The ocean accounts for about 71%of the earth’s surface area,which more than 90%are deep sea.Due to the particularity of the deep-sea environment,its microbial resources are abundant and the diversity is much richer than that of terrestrial microorganisms.The main purpose of this study is to search for active natural products from deep-sea derived fungi and expand their exploitation of marine resources while alleviating the shortage of terrestrial resources.In this study,18 deep-sea fungi were selected and cultured in 6 media using OMACS strategy to obtain a total of 108 fermented extracts.Through in vitro antitumor activity test and TLC chemical composition analysis,two strains Penicillium sp.MCCC3A00228 and Penicillium citrinum W3 with rich chemical diversity and good activity were identified as the target strains,and they were carried out systematic chemical composition study.The rice solid medium（FS1）was selected for large-scale fermentation of two target fungi,and systematic isolation and purification of secondary metabolites from crude extracts of the two fungi were performed using various modern chromatographic separation techniques,such as silica such as:silica gel column chromatography,sephadex LH-20 column chromatography,MPLC,HPLC and so on.And the structures of obtained compounds were identified by comprehensive analysis spectral data for instance:1D and 2D NMR,HR-ESI-MS,ECD and so on.Finally,a total of fifty-two compounds were obtained from two target fungi,among which five novel compounds（A1*,16*～19*）,including one glycoside（A1*）and four macrocyclic steroids（16*～19*）.Fifteen compounds were isolated and identified from Penicillium sp.MCCC3A00228,including one new glycoside compound:D-arabinitol-anofinicate（1*）;six alkaloids:aflaquinolone I（4）,3-O-methylviridicatin（5）,viridicatol（6）,dehydrocyclopeptine（7）,（–）-cyclopenol（8）,cyclo-（L-trptophyl-L-phenylalanyl）（10）;two terpenoids:piltunine A（2）,penigrisacid C（3）;six others:L-p Glu-L-Leu（9）,demethylincisterol A3（11）,ilicicolin H（12）,rugulosin A（13）,penicyrone B（14）and sarcasciol G（15）.Thirty-seven compounds were isolated and identified from the Penicillium citrinum W3,included ten steroids,four of which were unreported:23-O-methylneocyclocitrinol A（16*）,23-O-methylneocyclocitrinol B（17*）,23-O-methylneocyclocitrinol C（18*）,23-O-methylneocyclocitrinol D（19*）,neocyclocitrinol A（20）,neocyclocitrinol B（21）,neocyclocitrinol C（22）,neocyclocitrinol D（23）,12R-hydroxycyclocitrinol（24）,isocyclocitrinol B（25）;six citrinins:penicitrinol A（26）,penicitrinone C（27）,dicitrinin A（28）,penicitrinone F（29）,xerucitrinin A（30）,xerucitrinic acid B（31）;four polyketides:2’,6,6’-trihydroxy-2-methoxycarbony1-4’-methy1-benzophenone（32）,yicathin C（33）,mrlactonel（34）,coniochaetone A（35）;three nucleosides:2’-O-methyluridine（36）,thymidine（37）,uridine（38）;two anthraquinones:emodin（39）,phaseolorin I（40）and twelve other structural compounds:3,4,5-trimethyl-1,2-benzenediol（41）,phenol A（42）,latifolicinin C（43）,3,4-dihydroxyphenylacetic acid methyl ester（44）,4-hydroxy phenylacetic acid methyl ester（45）,methyl 2-（2-acetyl-3,5-dihydroxy-4,6-dimethylphenyl）acetate（46）,3,7-dihydroxy-1,9-dimethyldibenzo-furan（47）,6,8-dihydroxy-3,4,5-trimethylisochroman（48）,2,3,4-trimethyl-5,7-dihydroxy-2,3-dihydrobenzo-furan（49）,（3R,4S）-6,8-dihydroxy-3,4,5,7-tetramethylisochroman（50）,（3S,4S）-sclerotinin A（51）,trichodimerol（52）.In vitro antitumor activity test of fifty-two monomeric compounds was tested by MTT assay.Among them,the compound rugulosin A（13）showed good cytotoxic activity against lung cancer cells（H1299）,colon cancer cells（HCT116）and liver cancer cells（QGY7701）with IC₅₀ values of 17.6μM,18.2μM and 21.2μM,respectively,dicitrinin A（28）showed significant cytotoxic activity against human breast cancer cells（MCF-7）with IC₅₀ of 1.3μM.