Research On The Chemical Constituents And Biological Activities Of Secondary Metabolites Of Endophytic Fungus Alternaria Sp. From Eucommia Ulmoides Oliv.

Endophytic fungi are organisms that live in plant tissues and organs for a period of time or at any time in their live,but do not cause significant changes in host plant tissue.The endophytic fungi can produce antibacterial,antiviral,anti-tumor,anti-oxidation,insecticide and other natural active substances,is an important microbial resources.In this study,91 strains isolated from the leaves of Eucommia ulmoides Oliv.Among them,Alternaria sp.is an endophytic fungus which has a good inhibitory effect on Bacillus cereus,Candida albicans and other pathogenic bacteria.In order to investigate the antibacterial mechanism and antimicrobial composition of Alternaria sp.,we studied the active sites,chemical constituents and bioactivities of secondary metabolites of endophytic fungi Alternaria sp..Mainly obtained the following results:The antibacterial activity of the petroleum ether,ethyl acetate and N-butanol extract of Alternaria sp.fermentation broth to 7 kinds of pathogenic bacteria were detected by disc diffusion method.The results showed that the ethyl acetate had better broad-spectrum antibacterial activity to the tested pathogenic bacteria,and the antibacterial effect was excellent.HPLC was used to detect the chemical composition of the three parts.It was found that the peak intensity of the ethyl acetate was stronger,the number of signal peaks was the majority,and the separation degree of the signal peaks was better.The results showed that the ethyl acetate fraction could be further studied as the target site of the three parts of the metabolites,the antibacterial effect and the chemical composition screening results.After expanding culture,11 compounds were isolated from the active strain by repeated column chromatography on silica gel,preparative thin layer chromatography（PTLC）and recrystallization.The structures of these compounds were elucidated by means of spectroscopic methods including(¹H-NMR,¹³C-NMR,ESI-MS,TOF-MS,etc.)and as well as chemical method.The structures were as follows:cyclo-（Pro-Ile）（1）,cyclo（Leu-Ile）（2）,cyclo-（Tyr-Pro）（3）,cyclo（Leu-hydroxypro）（4）,cyclo-（Phe-hydroxypro）（5）,cyclo-（Gly-Leu）（6）,Daucosterol（7）,L-glycero-2,3-Dihydroxypropylα-L-Fucopyranoside（8）,（D-mannitol）（9）,5-（hydroxymethyl）-6-methyl-1H-pyrimidine-2,4-dione（10）,Thymidine（11）.Cyclic dipeptides are the main secondary metabolites of Alternaria sp..These compounds are the first from the endophytic fungus Alternaria sp.In the separation found.11 compounds were evaluated for their antimicrobial activities agains 7 tested strainseleven tested strains.The antimicrobial activity showed that the 8 compounds（compounds 1-6,8,9）showed antibacterial activity against different pathogens.The MIC of Compound 1 to Escherichia coli was 9.83mg/mL,the MIC of Compound 3 against Xanthomonas Campestris was 10.31 mg/mL,and the MIC of Compound 5 against Xanthomonas Campestris,Staphylococcus aureus and Escherichia coli were 4.97mg/mL,9.50 mg/mL and 9.50 mg/mL.Based on the analysis of antibacterial effect,it is clear that the active substances of Alternaria sp.metabolites are mainly cyclic peptide compounds.The antioxidant activity showed that all of the 11 compounds had different degrees of free radical scavenging activity.6 compounds（compounds 2,3,5,9,11）showed good ability to scavenge DPPH free radicals,and 4 compounds（compounds 2,5,10,11）showed good ability to scavenge hydroxyl free radicals.In summary,this paper selected endophytic fungi Alternaria sp.antibacterial ethyl acetate good activity for further purification,11 compounds were isolated from,and these compounds were evaluated for antimicrobial activity,3 compounds have strong antibacterial activity in vitro and 6 with strong antioxidant active compounds found.Proof of the endophytic fungi Alternaria sp.is a kind of important biological resources,secondary metabolites have good biological activity,has laid a basis for further development of Eucommia and microbial resources.