Studies On The Secondary Metabolites Of Four Fungi Such As Alternaria Brassicicola And Their Bioactivities

This paper is divided into four chapters.The first chapter mainly discusses the secondary metabolites and biological activities of Alternaria brassicicola,which was isolated from the fresh leaves of Siegesbeckia pubescens Makino(Compositae).And plausible biogenetic pathways of the fusicoccane-derived diterpenoids with new skeleton were discussed.The second chapter introduces studies on secondary metabolites and their biological activities from the sea sediment-derived fungus Penicillium chrysogenum.In the third chapter,we investigated the secondary metabolites and their biological activities from coculture of Pestalotiopsis sp.and Penicillium bialowiezense.The fourth chapter reviews the research progress of natural fusicoccane-derived diterpenoids from 1982 to 2020.In previous studies,our group had identified a series of fusicoccane-derived diterpenoids with unprecedented carbon skeletons from Alternaria brassicicola,which was isolated from the fresh leaves of Siegesbeckia pubescens Makino(Compositae).Fusicoccanes are diterpenoids formed from Geranylgeranyl pyrophosphate(GGPP)through a series of enzyme catalyzed reactions.And its classical structural feature was described as dicyclopenta[a,d]cyclooctane(5/8/5)three-ring skeleton.Fusicoccane-derived diterpenoids not only have complex structure,but also have potent regulation of plant growth function,anticancer and antimicrobial activities.Up to now,the reported fusicoccane-derived diterpenoids of microbial origin are mainly isolated from fungi of Alternaria.To maximize the chemical diversity of secondary metabolites in this “talented” fungus and search for more new bioactive compounds.Based on the OSMAC strategy,we changed the culture method of the fungus Alternaria brassicicola and chose liquid medium to culture the fungus.A total of12 diterpenoids were isolated and identified from the liquid fermentation of this fungus,including one new skeleton compound,two new compounds and six structurally revised compounds,by means of various chromatography-separation methods,extensive NMR spectroscopic,single-crystal X-ray diffraction and quantum chemical calculations.A fusicoccane-derived diterpenoid,alterbrassicene A,bearing an unprecedented 5/9/4-fused carbocyclic skeleton was characterized for the first time,which was rated as “Hot off the Press” by the journal Natural Product Reports.In addition,we evaluated the biological activity of the fusicoccanes,and found that the new skeleton compound showed potent anti-inflammatory activity.Further pharmacological mechanism studies indicated that the new skeleton compound is a potent IKKβ inhibitor in the NF-κB signaling pathway.Secondly,we expanded the rice fermentation of Alternaria brassicicola.Thus,95 compounds were isolated and identified from the ethyl acetate extraction of the total crude,including 50 new compounds and 6 new skeleton compounds.The main components were fusicoccane-derived diterpenoids,meroterpenoids(TCAs)and α-pyranone derivatives.Four fusicoccane-derived diterpenoid dimers,a newly transformed monocyclic carbon skeleton of a highly functionalized diterpenoid and a new skeleton compound with carbon reduction and ring opening were characterized for the first time.Moreover,we evaluated the anti-inflammatory activity and cytotoxic activity of some of the compounds,and the new skeleton compound alterbrassicicene A showed strong agonistic action upon PPAR-γ.In addition,we studied the secondary metabolites of a sea sediment-derived fungus Penicillium chrysogenum.And a total of 19 natural products were isolated and described from the solid fermentation,3 new compounds included.These main compounds were cyclopiane diterpenes,alkaloids,macrolides,etc.We comfirmed a highly fused and rigid 6/5/5/5 ring system skeleton structure of cyclopiane diterpene for the first time by X-ray crystallography experiment.Furthermore,the anti-inflammatory activity of the new compounds was also evaluated,and found compound 13β-hydroxy conidiogenone C with potent anti-inflammatory activity.Further western blot and immunofluorescence experiments demonstrated its mechanism of action to be that compound inhibited the NF-κB-activated pathway.In previous studies,our group have obtained a series of compounds with cytotoxic activity from fungus Pestalotiopsis sp.,isolated from the leaves of tea tree,and other series of compounds with immunosuppressive activity from fungus Penicillium bialowiezense,isolated from the fresh soft coral.Chemical investigations of these two highly creative genus have revealed that they are a rich reservoir of structurally novel and diverse compounds,which possessed the potential for further exploration.We applied coculture strategy to the above two strains to activate the fungal gene clusters originally silenced under natural conditions,in order to explore novel bioactive secondary metabolites,based on the successful application of fungal co-culture in the study of natural products of microorganisms.Therefore,Pestalotiopsis sp.and Penicillium bialowiezense were co-cultured using rice as solid medium.Totally 40 compounds were isolated,including 14 new compounds and 1 new natural product.Additionally,some isolated compounds were evaluated for the in vitro β-glucuronidase(GUS)and butyrylcholinesterase(BChE)inhibitory activities.And several compounds showed potent inhibitory activity to provide a solid foundation for further pharmacological researches.As we all know,the secondary metabolites of fungi are one of important source of natural drugs and an irreplaceable and attractive research area in drug discovery.In this paper,we systematically studied the secondary metabolites of four strains of fungi based on the OSMAC strategy.The results showed that the fungus Alternaria brassicicola had strong genetic coding ability for novel and unique fusicoccane-derived diterpenoids.Meanwhile,on the basis of previous studies,we found a large number of novel fusicoccane diterpenoids and dimers.This study greatly enriched the skeleton diversity of fusicoccane-derived diterpenoids.At the same time,several natural small molecules with strong anti-inflammatory activity,β-glucuronidase(GUS)and butyrylcholinesterase(BChE)inhibitory activities were found in this study.