Study On Accumulation Of Active Components In Salvia Miltiorrhiza And Salvia Castanea And Their Response To Methyl Jasmonate Treatment

As one of the traditional medicinal plants,Salvia miltiorrhiza Bunge has great medicinal and health value.Salvia castanea Diels f.tomentosa Stib is mainly distributed in Nyingchi,Tibet.Its medicinal value is similar to S.miltiorrhiza.It is often used as a substitute for S.miltiorrhiza in the local area.The effective ingredients in S.miltiorrhiza mainly include water-soluble phenolic acids and fat-soluble ketones,two major secondary metabolites.Previous experiments found that compared with S.castanea,the content of salvianolic acid B in S.miltiorrhiza was higher,while the content of rosmarinic acid and tanshinone were lower.The current detection methods cannot simultaneously perform qualitative and quantitative analysis of primary metabolites such as amino acids and secondary metabolites in S.miltiorrhiza.Based on the above background and the current research status of S.miltiorrhiza,this study established a quantitative nuclear magnetic resonance(qNMR)method for the determination of effective components in S.miltiorrhiza.Furthermore,through the induction of methyl jasmonate(MJ),the differences in the content of active ingredients in hairy roots between S.miltiorrhiza and S.castanea were compared,and the differences in the accumulation of secondary metabolites in hairy roots between the two and their responses to MJ were revealed.This study not only provides a fast and efficient qNMR method for the determination of active ingredients in S.miltiorrhiza,but also further deepens MJ’s understanding of the mechanism of secondary metabolism of S.miltiorrhiza and S.castanea.The specific research content of this study mainly includes the following parts:1.Taking twelve batches of S.miltiorrhiza samples from different origins as the research object,select salvianolic acid B(Sal B),lithospermic acid(LA),danshensu(DSS)and rosmarinic acid(RA)four representative phenolic acids.Through specificity and selectivity,linearity,stability,robustness and recovery test,and supplemented by high performance liquid chromatography(HPLC)for verification,a qNMR method for the determination of phenolic acids in S.miltiorrhiza was well established.2.Taking twelve batches of S.miltiorrhiza samples from different origins as the research object,four representative ketone components of tanshinone I(Tan I),tanshinone IIA(Tan IIA),dihydrotanshinone(DTS)and cryptotanshinone(CTS)were selected.Through the specificity and selectivity,linearity,stability,robustness and recovery test,and supplemented by HPLC for verification,the qNMR method for the determination of ketones in S.miltiorrhiza was well established.3.Based on the establishment of the method for the successful determination of phenolic acids in S.miltiorrhiza with qNMR,MJ was used for induction treatment,and qNMR was used to determine the content of RA and related components in S.miltiorrhiza and S.castanea at different times,and real-time fluorescence quantitative PCR(q RT-PCR)was used to determine the expression of key genes.The results show that MJ has a promoting effect on the biosynthesis of phenolic acids in and S.castanea,and the promotion effect on S.castanea is greater than that of S.miltiorrhiza.Specifically,MJ plays a role in both the phenylalanine and tyrosine branches of the RA synthesis pathway in S.castanea,and promotes the accumulation of RA by up-regulating the expression of related genes in the two b S.miltiorrhiza ranches.In the process of RA synthesis in S.miltiorrhiza,it mainly acts on the tyrosine branch,and promotes the accumulation of RA by up-regulating the expression of related genes.4.Based on the research of phenolic acids in the early stage,MJ was used for induction treatment,qNMR was used to determine the content of ketones and related components in the synthesis pathway of S.miltiorrhiza and S.castanea at different times,and q RT-PCR was used to determine the expression of key genes.The results show that MJ can promote the synthesis of ketones in S.miltiorrhiza and S.castanea,but different phenolic acids,MJ has little difference in promoting the synthesis of ketones in S.miltiorrhiza and S.castanea.Although S.miltiorrhiza and S.castanea gene expression generally have little difference in response to MJ induction,the response to MJ is different for a single gene or single component.