Study On Secondary Metabolites Of Artemisia Argyi Derived Fungus Diaporthe Sp.AC1 Induced By 1-Methyl-l-tryptophan

One Strain-Many Compounds（OSMAC）strategy is to regulate the biosynthetic pathway of secondary metabolites by changing medium composition,culture conditions,co-culturing with other strains,adding enzyme inhibitors or other exogenous substances to obtain more new active metabolites.In this study,the endophytic fungus Diaporthe sp.AC1 was selected as the research object,and 5-hydroxytryptophan（5-HTP）,1-methyl-L-tryptophan（1-MT）and tryptamine were added to the culture medium for screening the best inducer.The induced secondary metabolites of Diaporthe sp.AC1 were isolated,purified,identified and studied for activity.To provide some theoretical basis for the discovery of active natural products with novel structures and the search for drug lead compounds.（1）The endophytic fungus Diaporthe sp.AC1 was fermented by adding5-hydroxytryptophan,1-methyl-L-tryptophan and tryptamine to the culture medium.The antibacterial activities of the crude extracts against five pathogenic bacteria（Staphylococcus aureus,Listeria monocytogenes,Salmonella enteritidis,Pseudomonas aeruginosa,Escherichia coli）and five pathogenic fungi（Fusarium graminearum,F.moniliforme,Bostonia cinerea,Verticillium dahlia,Candida albicans）were determined by Oxford cup method.The cytotoxicities of the crude extracts against five tumor cells（human hepatoma cell line Huh-7,human cervical cancer cell line Hela,human colorectal adenocarcinoma cell line HCT-15,human lung cancer cell line A549,human breast cancer cell line MDA-MB-231）were determined by the MTT method.The results showed that the secondary metabolites of Diaporthe sp.AC1 induced by 1-methyl-L-tryptophan had the best antibacterial activity and tumor cytotoxicity compared with the control.（2）The induction conditions of 1-methyl-L-tryptophan were optimized,and the antibacterial activities and tumor cytotoxicities of the fermentation crude extracts were determined under different induction conditions.The results showed that the best time to add1-methyl-L-tryptophan was before inoculation and the best concentration was 0.25 m M.Under this condition,Diaporthe sp.AC1 was fermented on a large scale,and the fermentation broth was extracted with ethyl acetate.After reduced pressure evaporation and concentration,about 12 g of fermentation crude extract was finally produced.（3）The fermentation crude extracts were separated and purified by silica gel column chromatography,C18 reverse phase silica gel column chromatography and Sephadex LH-20gel column chromatography.They were identified by nuclear magnetic resonance spectroscopy（NMR）and high-resolution mass spectrometry（HR-MS）.Finally nine compounds were identified:5-（2-oxo-2H-Pyran-6-yl）pentane-2,3-dione（1）,methyl（2-（1-methyl-1H-indol-3-yl）ethyl）succinate（2）,fusariumindole D（3）,N-methyl tryptophol（4）,3-（2-acetoxyethyl）-1-methylindole（5）,N-methyl-3a-hydroxyfuroindoline（6）,3,5-dimethyl-8-methoxy-3,4-dihydroisocoumarin（7）,p-hydroxyphenethyl alcohol,（8）,3-phe-nylpyrazin-2（1H）-one（9）.（4）The antibacterial activities of the compounds against five pathogenic bacteria and five pathogenic fungi were determined by microdilution method.The cytotoxicities of the compounds against five tumor cells were determined by MTT method.The results showed that the MIC value of new compound 1 against Fusarium graminearum was 128μg/m L,and the inhibitory rate against Listeria monocytogenes was above 90%at the reaction concentration of 512μg/m L.It also had significant cytotoxicity against five tumor cells with IC₅₀ values ranging from 12.26 to 52.52μM.Compound 4 also showed relatively good inhibitory effects on pathogenic bacteria,pathogenic fungi and tumor cells.The MIC value for Fusarium moniliforme,F.graminearum and Botrytis cinerea were all 128μg/m L,and above 80%for all five pathogenic bacteria at the reaction concentration of 512μg/m L.The IC₅₀ value for human cervical cancer cells Hela was 29.74μM.Compound 9 showed obvious inhibitory activity against five pathogenic bacteria at 512μg/m L,and the inhibitory rate above 90%.It also had certain inhibitory effect on pathogenic fungi and tumor cells.Other compounds also showed different degrees of inhibitory activity against pathogenic bacteria and tumor cells.