Study On The Changes Of Chemical Ingredients Of Cuscuta Australis And Salvia Miltiorrhiza In A Parasitic Relationship

Objective: On the basis of establishing a systematic method for detecting chemical ingredients,through parasitic experiments,the changes in chemical ingredients of Cuscuta australis and Salvia miltiorrhiza after parasitize were explored.The changes provide a theoretical basis for effectively controlling the quality of Cuscutae Semen and exploring the biological defense mechanism of secondary metabolism.Methods:1.On the basis of systematically summarizing the chemical composition of Cuscutae Semen,the HPLC-PDA and HPLC-ESI-MS/MS methods for the chemical composition analysis of Cuscutae Semen were established,the extraction conditions were optimized in combination with the response surface method,and the feasibility of analytical methods were verified.18 batches of Cuscutae Semen materials from different sources were determined a multivariate statistical variable analysis was performed.2.To establish HPLC-PDA and HPLC-ESI-MRM/MS analytical methods for simultaneous quantitative analysis of 36 chemical ingredients in C.australis and S.miltiorrhiza and conduct methodological investigation.3.Using HPLC-PDA and HPLC-ESI-MRM/MS analysis methods,the contents of chemical compounds in C.australis to the S.miltiorrhiza during growth were analyzed and assessed with multivariate statistical analysis method to explore the effect of the parasitic relationship between C.australis and S.miltiorrhiza on the active accumulation of C.australis.4.Using HPLC-PDA and HPLC-ESI-MRM/MS analysis methods,the chemical components of main metabolites in different parts(root,stem and leaves)of S.miltiorrhiza during parasitic period are analyzed.To explore the parasitic relationship affects the synthesis and accumulation of chemical components in different parts of S.miltiorrhiza,multivariate statistical analysis was performed.Results:1.Method validation results showed that the established HPLC-PDA and HPLC-ESIMS/MS methods of 12 chemical ingredients in Cuscutae Semen have good linearity,precision,high accuracy and stability.The best extraction method of Cuscutae Semen samples obtained by response surface method was: 80% methanol,extraction time 60 min,methanol-material ratio 100 m L/g.The examination of 18 batches in Cuscutae Semen had verified the practicability of the above method,and the OPLS-DA score plot had clearly shown samples was divided into three group,six chemical compounds were screened as the mark components for identification of origin.2.The results of method validation showed that the established HPLC-PDA and HPLCESI-MRM/MS methods for simultaneous determination of 36 metabolites in C.australis and S.miltiorrhiza were good in linearity,precision,accuracy,repeatability and stability.3.During the germination period,some chemical ingredients of C.australis collected from Astragalus complanatus var.complanatus were gradually synthesized and accumulated,and some of the chemical ingredients were degraded and the contents were reduced.The types,the trends of synthesis and accumulation of chemical ingredients were different.Hyperoside,isoquercitrin,quercitrin,astragalin,isorhamnetin-3-O-β-glucoside,quercetin,kaemoferol,chlorogenic acid,cryptochlorogenic acid,isochlorogenic acid B,isochlorogenic acid A and isochlorogenic acid C were both detected in C.australis at different stages.It indicated that C.australis has its own metabolic pathway and can synthesize some chemical ingredients by itself.After parasitizing S.miltiorrhiza,the presence of S.miltiorrhiza components such as danshensu,protocatechuic acid,and protocatechuic aldehyde were detected in the stem and seed of C.australis.Through multivariate statistical analysis,the six components were the different chemical components before and after parasitism in C.australis.4.Parasitism of C.australis did not affect the types of metabolites in different parts of S.miltiorrhiza,but the contents of each metabolite were significantly different.Through multivariate statistical analysis,it was possible to distinguish between parasitic and nonparasitic S.miltiorrhiza,suggesting that C.australis could affect the synthesis and accumulation of chemical ingredients in S.miltiorrhiza.Conclusion: C.australis has its own independent metabolic pathways at different stages of growth and development,and can synthesize chemical ingredients by itself.The parasitic behavior of C.australis does not affect the types of chemical ingredients in different parts of S.miltiorrhiza,but it can affect the synthetic accumulation of chemical ingredients in S.miltiorrhiza.The quality of Cuscutae Semen is affected by the host plant species.Therefore,in order to stabilize the quality of Cuscutae Semen,it is necessary to choose the host plant species and realize standardized production.