Study On Secondary Metabolites And Its Bioactivity Of Diaporthe Sp. AC1,an Endophytic Fungus From Artemisia Argyi

Endophyte is a kind of special microorganism that exists in plant tissues in the form of benign parasitism or symbiosis.It plays an important role in assisting plants to resist diseases and inducing plants to resist stress.It is also an important source of bioactive substances.In this study,the endophytic fungi of Artemisia argyi,a medicinal plant,were isolated,purified and diversified,and the secondary metabolites of an endophytic fungus with good biological activity were isolated,purified,identified and studied.（1）26 endophytic fungi were isolated from A.argyi by tissue block separation method.According to the ITS sequences identification,the 26 strains belonged to 8 genera,namely Diaporthe,Alternaria,Trichoderma,Gibberella,Colletotrichum,Chaetomium,Phoma and Fusarium.Alternaria was the largest genus,accounting for 34.62%.（2）ME medium was used for fermentation of endophytic fungi,and the fermented liquid was extracted by ethyl acetate and concentrated to obtain the crude extract,and yield of crude extract was recorded.Staphylococcus aureus,Salmonella enteritidis and Fusarium graminearum were used as indicator strains to verify the antibacterial activity of 26 strains.The results showed that a total of 25 strains had inhibitory activity against one or more pathogenic microorganism.And AC1 has not only good antibacterial activity,but also has high fermentation crude extract yield,which is used for the study of secondary metabolites.Based on morphological observation and molecular biology,AC1 was identified as Diaporthe and named Diaporthe sp.AC1.The strain AC1 was fermented with malt extract medium,and the fermentation liquid and mycelium were extracted,respectively.The fermentation liquid was extracted by Et OAc and the mycelium was extracted by Me OH/DCM（1:1）.After decompression and concentration,the 20g crude extract was obtained.（3）The fermentation product of the strain was purified by silica gel column chromatography,Sephadex LH-20 gel column chromatography as well as HPLC.Finally,a new compound phomopsolide G（1）and seven known ones phomopsolide F（2）,phomopsolide B（3）,phomopsolide A（4）,（S,E）-6-（4-hydroxy-3-oxopent-1-en-1-yl）-2H-pyran-2-o ne（5）,catenioblin A（6）,2-minaline（7）,3-hydroxybenzyl alcohol（8）were obtained,and identified by Nuclear magnetic resonance spectrum（NMR）and High resolution mass spectrometry（HR-MS）.（4）The antimicrobial activities of the eight compounds were tested using microdilution method with four pathogenic fungi（Fusarium graminearum,Fusarium moniliforme,Botrytis cinerea And Verticillium dahliae）and five pathogenic bacteria（Listeria monocytogenes,Staphylococcus aureus,Escherichia coli,Salmonella enteritidis,Pseudomonas Aeruginosa）as experimental strains.The study demonstrated that the new compound 1 had moderate antifungal activity against F.graminearum,F.moniliforme and B.cinerea,as well as weak inhibitory activity against S.aureus.Compounds 4 and 5 showed different degrees of inhibition on the nine microorganisms.Specifically,compound 5 had similar inhibition rate to positive control on F.graminearum and B.cinerea.（5）Using MTT colorimetry,cytotoxicity of the compounds against three human tumor cell lines（Hep G2,A549 and MDA-MB-231）was tested.The results showed that compounds 1,2,3,4 and 5 exhibited different degrees of cytotoxicity activity.The new compound 1 showed weak cytotoxicity against Hep G2,A549 and MDA-MB-231,with IC₅₀ values of 79.8,107.5 and54.0μM.In addition,compound 3 could inhibit the proliferation of Hep G2 cell with IC₅₀of 36.8μM.Compound 4 exhibited better inhibition activity against A549 and MAD-MB-231 with IC₅₀of 36.7 and 23.0μM,respectively.The findings provided theoretical basis for searching for drug and biologically active lead compounds from Artemisia argyi associated fungi resources.