Study On The Secondary Metabolites Of Phomopsis Asparagi In A Mangrove Plant Based On Epigenetic Modification

Mangroves are woody plants that grow in the dynamic transition zone between land and ocean.Because they live on extreme tropical and subtropical coasts for a long time,in order to adapt to the unique living environment,their endophytic fungi and hosts have undergone long-term evolution,making mangrove plants’ secondary metabolites of endophytic fungi have excellent biological activity.Research reports in recent years have shown that a considerable part of the secondary metabolites with good activity comes from mangrove microorganisms.People have isolated a large number of new compounds from the metabolites of marine fungi,and used their unique biological metabolites.Activity has made a huge contribution to the research and development of new drugs.Mangrove plants are a very special marine organism,rich in endophytic fungi,and constitute the second largest marine fungus ecosystem.Related theoretical investigations have proved that less than 30% of gene clusters in fungi are normally transcribed and expressed under laboratory culture conditions.The use of epigenetic methods can greatly change the spectrum of secondary metabolites of microorganisms,not only can increase the production of known secondary metabolites,but also activate the expression of a large number of silent gene clusters to promote the production of new secondary metabolites.It has become a simple and effective method to produce new active secondary metabolites.In the previous work of our laboratory,a series of chromone compounds and cytochalasin compounds have been separated from the crude extracts of the endophytic fungus Phomopsis asparagi of the mangrove plant of mangrove,and the biological activity evaluation has proved the part.The compound has excellent immunosuppressive activity and anti-tumor biological activity.In this study,Phomopsis asparagi used chemical epigenetic regulation methods to use small molecular epigenetic reagents to change the modified form of fungal chromosomes,activate silent genes,and provide favorable conditions for further mining of secondary metabolites with excellent activity.(1)Based on the principle of chemical epigenetic modification,add DNA methyltransferase inhibitor(5-azacytidine)and histone deacetylase inhibitor(sodium butyrate)to the strain Phomopsis asparagi medium,Comparing the changes of Phomopsis asparagi colony and hyphae morphology before and after the action of small molecule epigenetic reagents,and the differences in the spectrum of its secondary metabolites,it was found that the secondary metabolites spectrum changed significantly before and after sodium butyrate stimulation,and through multiple parallel experiments Verify that the changes before and after stimulation are stable,optimize the added concentration of sodium butyrate in the medium,and carry out a large amount of fermentation.(2)The above fermentation broth is extracted with ethyl acetate to obtain the crude extract,which is separated using normal phase(silica gel column chromatography,dextran gel column)and reverse phase(reverse phase column,high performance liquid separation)separation methods.13 compounds were released,including 5 new compounds.Through one-dimensional(hydrogen spectrum,carbon spectrum,mass spectrum,etc.)and two-dimensional(HSQC,DEPT,NOESY,HMBC,COSY)spectral data,the structure of the separated compounds is analyzed and the structure is determined.Among them,compounds 5(cytochalasin)and 6(polyketides)are two monomeric compounds separated from them after adding inhibitors,and compounds 2,3,5,9,10,and 13 are cytochalasins,The rest are polyketides,indicating that cytochalasins and polyketides are the main secondary metabolites of the bacteria,and 3 new compounds(8,11,12)have been isolated from them.(3)In order to explore the drug value of new compounds,the anti-tumor and immunosuppressive activities of the isolated compounds were evaluated.The results showed that cytochalasin 5 and two polyketide compounds(1,7)showed good activity on Con A-induced T cell proliferation,compounds 8 and 11 had weaker activity,and compounds 3,5,6,and 7 had relatively low activity on tumor cells.The inhibition rate of HCT-116 is higher than 50%,which is of great significance for the development of new drugs.Based on the above research,this study used the method of chemical epigenetic modification to manipulate the endophytic fungus Phomopsis asparagi of the mangrove plant Mangrove,and successfully separated and identified 13 single species from the fermented extract of Phomopsis asparagi stimulated by sodium butyrate.Body compounds,including 6 cytochalasins and 7 polyketides,3 of which are new compounds,and 2 of which are secondary metabolites that are not found under conventional conditions,and these compounds are used for anti-tumor and Evaluation of immunosuppressive biological activity.