Isolation And Identification Of Endophytic Fungi From Three Euphorbia Plants And Their Secondary Metabolites Research

Plant endophytic fungi have a wide variety of species and are a substantial microbial group.They are rich in secondary metabolites,producing similar or identical active ingredients to host plants and novel products different from host plants.Therefore,they have attracted extensive attention from many scholars in recent years.As traditional medicinal plants,Euphorbia has a variety of pharmacological functions.Its milk ducts are rich and complex and contain toxic terpenoids.Therefore,the fungi that can survive in their internal environment may have unique characteristics.In addition,there are few reports on the endophytic fungi of Euphorbia at home and abroad,and its research potential is excellent.Based on this study,three species of Euphorbia were selected as materials to study their endophytic fungi and secondary metabolites to find secondary metabolites with novel structures and pharmacological activities.Based on this,three kinds of Euphorbia plants are selected as materials in this study to study their endophytic fungi and secondary metabolites,hoping to find secondary metabolites with novel structures and pharmacological activity.In this study,Euphorbia truncati L.,E.latifolia,and E.pekinensis Rupr.were selected to isolate and purify endophytic fungi from Euphorbia by tissue block method,and 36 endophytic fungi were isolated.Combining traditional morphological and molecular biology methods,they were identified into 22 genera,of which Fusarium sp.,Diaporthe sp.,Colletotrichum sp.,and Penicillium sp.were the dominant populations,accounting for 16.7%,8.3%,8.3%,and 8.3%of the total isolates.Two secondary metabolite rich strains Colletotrichum sp.S11,Cytospora sp.L2 and an endophytic fungus Epicoccum sp.DJ-F isolated from Euphorbia royleana B.were screened by small sample fermentation.The fermentation products were separated,purified,and identified by column chromatography,HPLC,HR-ESI-MS,NMR,and other methods.The results showed that 11 compounds were isolated from the fermentation products of Colletotrichum sp.S11,including 5 new sesquiterpenoids.The structural types mainly included terpenes,sterols,and benzene ring derivatives;33 compounds were isolated from the fermentation products of Cytospora sp.L2,including 2 new furans,the main structural types of furans and terpenes;10 compounds,including 4 new sterols,were isolated from the fermentation products of Epicocum sp.DJ-F.The main types were sterols and piperazine alkaloids.The separated compounds are colletotrin A（1*）,colletotrin B（2*）,colletotrin C（3*）,colletotrin D（4*）,colletotrin E（5*）,7-hydroxy-10-methoxydehydrodihy-drobotrydial（6）,terpestacin（7）,（-）-isosclerone（8）,tyrosol（9）,（22E,24R）-3β,5α,9α-trihydroxy-ergosta-7,22-dien-6-one（10）,ergosta-5,8（14）,22-trien-7-one,3-hydroxy-（3β,22E）（11）,irpexin M（12*）,irpexin N（13*）,irpexlacte B（14）,irpexlacte C（15）,irpexonjust A（16）,irpexin A（17）,5-（3-oxopentyl）-2-furancarboxaldehyde（18）,5-（4-oxopentyl）furan-2-carbaldehyde（19）,conocenolide A（20）,conocenolide B（21）,tremulenediol A（22）,（±）-epi-tremulenediol A（23）,integracin A（24）,integracin B（25）,methyl 2,5-dihydro-4-hydroxy-5-oxo-3-phenyl-2-furanpropanoate（26）,2,3-dihydroxypropyl 1H-indole-3-carboxylate（27）,1-（1,3-dihydro-4-hydroxy-1-isobenzofuranyl）butan-2,3-diol（28）,2-（2’-hydroxypropyl）-5-methyl-7-hydroxychromone（29）,（4S）-3,4-dihydro-4,8-dihydroxy-5-methoxy-1（2H）-naphthalenone（30）,rel-（2R,4S）-3,4-dihydro-2,4-dihydroxy-8-methoxy-1（2H）-naphthalenone（31）,saccharonol A（32）,3-hydroxymethyl-6,8-dimethoxycoumarin（33）,xylarinol A（34）,4-methoxy-5-methoxycarbonyl-6-propyl-2-pyrone（35）,annularin B（36）,dankasterones A（37）,6β-hydroxystigmast-4-en-3-one（38）,isocyathisterol（39）,6β-hydroxy-ergosta-4,7,22-trien-3-one（40）,（24R）-ergosta-4,6,8（14）,22-tetraene-3-one（41）,ergosta-4,6,8,22-tetraen-3-one（42）,stigmast-4-en-3-one（43）,ganodermaside E（44*）,ganodermaside F（45*）,ganodermaside G（46*）,ganodermaside H（47*）,23R-hydroxy-（20Z,24R）-ergosta-4,6,8（14）,20（22）-tetraen-3-one（48）,topsentisterol D₃（49）,eutypellazine D（50）,penicibrocazine D（51）,boydine A（52）,1,3-dihydro-5-methoxy-7-methylisobenzofuran（53）.Compounds 1-5 and 7 were evaluated for their cytotoxicities against HL-60,A549,SMMC-7721,SW480,MDA-MB-231.However,no significant activities were detected at concentrations up to 40μM.In addition,the antibacterial activity of compounds 24-26,35,37-41,and 43-49 in vitro was determined,and it was found that they had weakly inhibitory effects on Staphylococus aureus,Staphylococcus epidermidis,Pseudomonas syringae pv.Tabaci and Ralstonia solanacearum.In this thesis,the study of endophytic fungi and secondary metabolites of Euphorbia enriches the diversity of endophytic fungi and secondary metabolites of Euphorbia,providing a reference for their further development and research,but also provides a specific theoretical basis for the study of endophytic fungi of medicinal plants.