The Endophytic Fungi Associated With Salvia Miltiorrhiza And The Isolation,Identification Of Anti-tumor Cell Active Substances

Salvia miltiorrhiza,a valuable medicinal plant belongs to the Salvia genus and show various biological activity,such as blood circulation,lowering blood fat,anti-tumor and so on.Previous study showed that endophytic fungi was able to exudate compounds with the similar bioactivity as host.Therefore,it is important to investigate the secondary metabolites and discover new drug sources.In this study,we isolated the endophytic fungi of Salvia miltiorrhiza and identified by biological methods.The cultural condition and fermenting process of the anti-tumor strains were optimized according to literatures and the material was separated.The results as below:122 strains of endophytic fungi were isolated from Salvia miltiorrhiza using 8 kinds of selective media.The quantity of endophytic fungi from roots is more than the stems,leaves and flowers according to above isolating protocol.Morphological characteristics and ITS sequencing results indicated that the endophytic fungi belonged to 2 subphylum,8 class,10 orders,13 families and 20 genera.The cytotoxic activity of endophytic fungi isolated with Salvia miltiorrhiza.by Alamar blue method.The 5-fluorouracil(5-Fu)was used as a positive control.The results demonstrated that the HepG2 inhibited ratio of 10 strains was 50% above.The strain KLBMPSM007 was the strongest anti-tumor activity in the re-screening experiment.The medium and culture conditions of the strain KLBMPSM007 were optimized based on anti-tumor activity as the main index.The used conditions were as follows: the original pH 6.0 ~ 6.5 at 28 ℃ for 7 days and the bottling volume was 30 ~ 40% and the inoculation rate was 3%.The cytotoxic activity was increased from 84.45 ± 1.99% to 93.31 ± 2.83%,which was about 8.90% higher than previous optimization.The solid culture conditions as follows: the initial water content was 45%,the initial pH of the nutrient solution was 5.5 ~ 6.0,the material thickness was 3 cm,the inoculation rate was 8% and the fermentation time was 35 d.The cytotoxic activity was 91.31 ± 1.32%,which was about 7.60% higher than before optimization.Seven compounds were isolated and purified by preparative thin layer,silica gel column,Sephadex LH-20 gel column and high performance liquid chromatography.The compounds were identified by NMR spectra: cochlioquinone B,acetylcholine colloidone novel compounds,anhydrocochlioquinone A,palmitic acid and linoleum acid.Among them,cochlioquinone B、cochlioquinone B1 and anhydrocochlioquinone A are homologues.CoB1 is a novel compound,and anhydrocochlioquinone A exhibits strong anti-tumor activity.