Isolation And Purification Of Endophytic Bacteria From Artemisia Capillaria And Bioactivity Study Of Secondary Metabolites

Symbiotic endophytic bacteria can produce many kinds of bioactive compounds similar to the host plant,which is an important resource to search for natural active ingredients.The aim of the study is to isolate,purify and identify the endophytic bacteria of the Artemisia Capillaria,screen the active strains,then study the properties of the fermentation products,separate and purify the secondary metabolites,and finially test the activity of the secondary metabolites.The specific research contents and results are as follows:52 strains of endophytic bacteria were isolated from Artemisia Capillaria by tissue separation,the endophytic bacteria were identified by morphological observation and16Sr DNA technology.There are 29 strains of Bacillus subtilis,7 strains of Bacillus cereus,6 strains of Bacillus megaterium,4 strains of Bacillus licheniformis,2 strains of Bacillus pumilus,1 strain of Bacillus simplex,1 strain of Bacillus idriensis,1 strain of Acinetobacter calcoaceticus,1 strain of Enterococcus faecium were identified.An Acinetobacter calcoaceticus as bacteriostatic strain was screened from the endophytic bacteria by 96-well plate method and Oxford cup method.The properties of the fermentation broth of the active strain were studied,and it was determined that the fermentation product could tolerate high temperature,but could not tolerate extreme p H environment.Under the condition of 48 h fermentation time,the antibacterial activity of the fermentation product was the best,and the active component of the fermentation product wasn’t protein substances.Three kinds of solvents were selected to extract the fermentation liquid.It was found that the crude extract of ethyl acetate phase had the strongest antibacterial activity.The comparison of the antioxidant properties of different crude extracts showed that the antioxidant properties of ethyl acetate phase were higher than those of petroleum ether phase and chloroform phase.Finally,ethyl acetate was selected as the appropriate extractant.The active strains were fermented in large quantities.Silica gel column chromatography,Sephadex LH-20 gel column chromatography and preparative liquid chromatography combined with thin layer chromatography were used to separate 5monomer compounds.After testing the purity of the compounds,the structures were analyzed by modern spectroscopy(ESI-MS,¹H-NMR and ¹³C-NMR),and 1 compound were identified as dibutyl phthalate.Due to time,the identification of residual compounds will be carried out in subsequent studies.Four kinds of indicator bacteria were selected and Mic method was used to determine the antibacterial activity of the purified compounds 1-5.Five compounds have different degrees of inhibition effect on indicator bacteria.Using MTT method,MCF-7cells were selected as the model to test the antitumor activity of compound 1-5,and their IC50 values were measured.The results showed that five compounds had antitumor activity in different degrees,among which compounds 1,4 and 5 had stronger antitumor activity.In general,in the research process of secondary metabolites of plant endophytes,we found that there are many kinds of Endophytes in medicinal plants,including many active strains.Through the exploration of secondary metabolites of active strains,we can provide reference for the research and development of new high-efficiency drugs.