Study On The Diversity,Bioactivity Of Endophytes From Aesculus Chinensis And Bioconversion Of Aescine

Aescine,which has the function of anti-tumor,anti-inflammation and anti-exudation,is the main active component of Chinese famous medicinal plant A.chinensis.It is widely used and in great demand.Aescine is mainly derived from dried seeds of A.chinensis.With the overexploitation and utilization of wild A.chinensis resources and serious habitat destruction,the wild resources of A.chinensis are becoming increasingly scarce.This study takes A.chinensis in Qinba mountain area as the test material,it aimed to analyze the diversity of its endophytic bacteria,and preliminarily establish the resources bank of endophytic fungi of A.chinensis in Qinba mountain area.The endophytes produced aescine by metabolism were screened and their biological activities were analyzed,which lay a foundation for the further preparation of aescine by microbial fermentation.Screening microbial strains with the conversion from aescine into secondary active aescine provides a new idea for improving the utilization rate of aescine.1.Isolation and bioactivity of endophytic bacteria from A.chinensisTissue homogenate method was used to isolate the endophytic bacteria from A.chinensis,a total of 124 strains of pure culture strains were obtained,and 48 strains were identified,which belong to 4 genera.The dominant group was Bacillus.The results showed that 48endophytic bacteria exhibited different antibacterial activities.Bacteriostasis test on extracts after fermentation,among them,WJ-51 has an inhibitory effect on four target bacteria;it also has an inhibitory effect on the proliferation of A₅₄₉49 cells.2.Isolation and bioactivity of endophytic fungi from A.chinensisTissue culture method was used to isolate the endophytic fungi from A.chinensis,a total of 168 strains of pure culture strains were obtained,and 44 strains were identified,which belong to 5 phylum and 20 genera.The dominant phylum was Deuteromycotina.44 strains were fermented by liquid shake flask and the bacteriostatic activity of the extract was detected.The components of aescine in the fermentation broth of the above strain were analyzed by UV-visible light and high performance liquid chromatography?HPLC?.The results showed that 44 endophytic fungi exhibited different antibacterial activities.3 strains of fungi GM43,GM66 and GM88 which had the ability to metabolize aescine were screened out.After morphological observation and ITS sequence analysis,GM43 was identified as Diospora,GM66 was identified as Aspergillus,GM88 was identified as Irpex.3.Microbial transformation of aescineGM68,a fungal strain with aescine converted into secondary saponins,was obtained by primary screening of separation medium and conversion culture medium resieve.Morphological observation and ITS sequence analysis confirmed that it was Eurotiomycetes,Eurotiales,Aspergillus.Transformed strains were fermented with aescine by liquid shake flask,aescine and their conversion products were separated by silica gel column chromatography.The qualitative analysis of aescine and their conversion products were tested by TLC and HPLC.Qualitative and quantitative analysis of sample A1 were tested by UPLC-MS,and A1 was preliminarily determined as 21?-crotonyl aescine.