Study On The Chemical Components And Metabolism Of Corydalis Yanhusu

Yanhusuo was the dry rhizoma of Corydalis yanhusuo W.T.Wang（Papaveraceae）which with the effect of promoting blood circulation,activating qi and relieving pain.Corydalis has a long history and widespread clinical application.Drugs containing Rhizoma Corydalis,such as "Yuan Hu Zhitong tablet","Anwei tablet","dysmenorrhea pill",have been used for many years,and their safety and effectiveness have been clinically verified.However,there are few reports on the chemical composition and drug effect of the total alkaloids of the Rhizoma Corydalis,and the progress is limited.This situation is extremely unfavorable for the further and thorough study of the medicinal value of the rhizoma corydalis and guiding the clinical application of the rhizoma corydalis.In this project,we first performed a comprehensive chemical analysis of the alkaloids in Corydalis,established the chemical composition spectrum of the medicinal material,and revealed its material basis from the chemical level.Secondly,the metabolic components of Rhizoma Corydalis in vivo were analyzed on the basis of chemical composition spectrum,and the metabolite spectrum of Rhizoma Corydalis alkaloid was set up,and the pharmacodynamic basis of Corydalis Corydalis was clarified.Finally,the components with good migration and elimination rules in vivo have drug-making properties.Therefore,we explored the elimination of regularity in the drug-containing plasma of Corydalis alkaloids.This study will provide scientific basis for finding the active substance basis of total alkaloids,clarifying pharmacological effects,quality control,and guiding its clinical application.The main research contents and results mainly include the following three parts.1.Characterization of chemical constituents of alkaloids from YanhusuoTo comprehensively and rapidly screen and identify alkaloids in Yanhusuo,a target data screening strategy followed by characteristic fragment filtering by UPLC-Q-TOF/MSE was developed for rapidly and comprehensively identifying alkaloids in Corydalis yanhusuo W.T.Wang（Yanhusuo）.The proposed strategy consisted of the following four steps.（1）Fragmental patterns and characteristic fragments of various types of alkaloids were summarized based on the reference compounds and previous reference literatures.（2）Target data screening was conducted and data screening tables involving various types of alkaloids were constructed referring to the structural characteristics of alkaloids,the type and number of substituents,and characteristic fragments.This data screening table includes all possible molecular weights of various types of alkaloids.（3）The raw data detected by UPLC-Q-TOF/MSE were screened preliminarily by data screening tables,then characteristic fragments filtering was used to rapidly recognize various types of alkaloids.（4）A comparison was made with retention time,accurate mass,MS/MS fragmentation and online databases,as well as a reference to related literature to validate the data.As a result,a total of 86 compounds were identified or characterized,including 24 tetrahydroprotoberberine alkaloids,22 protoberberine alkaloids,6 protopine alkaloids,12 aporphine alkaloids and 22 other compounds.Among them,8 were potentially new compounds,and 5 components were discovered in the Yanhusuo for the first time.The method proposed in this paper was proved to be an efficient data processing approach to rapidly discover and characterize chemical constituents from complicated herbal extracts,without the help of standard substances.Furthermore,this research enriched the material basis of Yanhusuo and provided meaningful guidance for the discovery of potentially new compounds.2.In vivo metabolism study of YanhusuoThrough studying the chemical constituents of alkaloids from Corydalis,we have a comprehensive understanding of alkaloids in medicinal materials.However,it is not clear which ingredients enter into the body after oral administration.The study of chemical constituents and their metabolites in Chinese herbal medicine is of great significance for revealing the effective material basis of Chinese medicine and clarifying the metabolic characteristics of traditional Chinese medicine.However,due to the complex chemical components,diverse structure and abundant body metabolism,the identification of metabolites in vivo has become difficult.In order to detect more metabolites entering the body,the metabolites of high dose of Corydalis after gavage were analyzed.However,high dose may change the absorption process of Chinese medicine components in the body,and the clinical equivalent dose can reflect the true status of metabolism,but the content of Chinese medicine in the clinical dose is less in the body,and it is difficult to detect.In view of the above problems,this part puts forward the research strategy from monomers to medicinal materials,from high dose to clinical equivalent dose,and to analyze the metabolic profile of the alkaloid components of the clinical equivalent dose of Rhizoma Corydalis in rats,and to reflect the real metabolism in the body.In this part,First of all,the in vivo metabolism of the representative monomers in the Rhizoma Corydalis was studied,and the in vivo metabolic patterns of various alkaloids were explored,the metabolic characteristic ions were determined,and the metabolic pathways of various alkaloids were summarized.Taking the representative monomer metabolism as a reference,it provides a reference for the identification of metabolites in vivo of the same type of alkaloids.Secondly,the high dose of Corydalis alkaloids in vivo metabolism was studied.Combined with the metabolic types,metabolic characteristic ions,and 86 alkaloid compounds identified or characterized in the Rhizoma Corydalis,the compound structure was identified,and the metabolic composition spectrum of the alkaloid in the high dose of Corydalis yanhusuo was completed.Finally,based on the high-dose identification of metabolites,using the extracted ion function in MassLynx V4.1 software combined with high-dose MS/MS fragment identification and chromatographic retention behavior of metabolites at high dose to complete the study of the metabolic composition spectrum of the clinical equivalent dose in vivo.Using this strategy,metabolites in plasma,urine,bile and feces of six representative monomers and high dose and equivalent dose of Rhizoma Corydalis decoction were identified.Using this strategy,metabolites in plasma,urine,bile and feces of six representative monomers and high dose and equivalent dose of Rhizoma Corydalis decoction were identified.As a result,a total of 57 prototypes and metabolites were tentatively identified in rats after oral administration of tetrahydroberberine in feces,urine,plasma,and bile.A total of 56 prototypes and metabolites were tentatively identified in rats after oral administration of tetrahydropalmatine in feces,urine,plasma,and bile.A total of 24 prototypes and metabolites were tentatively identified in rats after oral administration of palmatine in feces,urine,plasma,and bile.A total of 30 prototypes and metabolites were tentatively identified in rats after oral administration of dehydrocorydaline in feces,urine,plasma,and bile.A total of 46 prototypes and metabolites were tentatively identified in rats after oral administration of protopine in feces,urine,plasma,and bile.A total of 49 prototypes and metabolites were tentatively identified in rats after oral administration of allocryptopine in feces,urine,plasma,and bile.The metabolic pathways of various alkaloids were summarized,such as demethylation（-14 Da）/dehydrogenation（-2 Da）,hydrogenation reduction（+2 Da）,hydroxylation（+16 Da）,combined sulfation（+80 Da）and combined with glucuronidation（+176 Da）.A total of 210 prototypes and metabolites were tentatively identified in rats after oral administration of high-dose of Rhizoma Corydalis in feces,urine,plasma,and bile.A total of 137 prototypes and metabolites were tentatively identified in rats after oral administration of equivalent dose of Rhizoma Corydalis in feces,urine,plasma,and bile.3.Analysis of components with migration and elimination in drug-containing plasma of corydalis alkaloidsIt is generally believed that the drug must be absorbed into the blood,distributed to the target organ and maintained at a certain concentration level within the corresponding target organ for a certain period of time to be able to exert the pharmacodynamic effect.Ingredients that have good migration and elimination rules in vivo can exert pharmacodynamic effects.（1）First of all,UPLC-Q-TOF/MS was used to collect data from plasma samples at different time points,and the collected data were imported into the UNIFI software.By setting certain parameters and metabolic pathways,the preliminary screening of metabolites in the plasma samples was conducted.Using the "Trendplot" function of the UNIFI metabolite solution,components with a significant content change in the parent ion are selected as components（including prototypical components and metabolites）that potentially have migration elimination rules in the plasma,and these components are further identified based on their cleavage pathway and characteristic ions.Characteristic ions and high-abundance fragment ions are selected as daughter ions.Parent ions and daughter ions are associated as MRM ion pairs.（2）Using the MRM data acquisition mode in UPLC-Q-TOF/MS,setting the corresponding MRM ion list and the corresponding collection parameters to collect the MRM data from the plasma samples of 3 rats at different time points,drawing the peak area map of the different time point ions,and obtain the real migration elimination rules in the plasma samples,which can be used to reveal the potential drug effect material base in the rhizoma corydalis.Using the MRM data acquisition mode in UPLC-Q-TOF/MS,set the corresponding MRM ion list and corresponding acquisition parameters to collect MRM data of plasma samples from 6 rats at different time points,and plot the peak area of the ion at different time points.In the figure,the composition of the plasma sample that actually has a migration elimination rule is used to reveal the underlying pharmacodynamic material basis in Corydalis yanhusuo.Using the MRM data acquisition mode in UPLC-Q-TOF/MS,set the corresponding MRM ion list and acquisition parameters to perform MRM data collection on plasma samples from 6 rats at different time points,and plot the daughter ions at different time points-Peak area map,obtained in the plasma sample really has the rule of elimination of migration,used to reveal potential drug efficacy material basis in Corydalis.Through the analysis of the trend of the daughter ion peak area and migration elimination trend chart,it was found that corydaline,protopine,tetrahydroberberine,dehydrocorydaline,tetrahydropalmatine,palmatine,DL-scoulerine,tetrahydropalmatine+C₆H₈O₆,allocryptopine-CH₂+C₆H₈O₆ and Corydalmine+C₆H₈O₆ had larger peak areas and good migration elimination rules in rat plasma.These ingredients may be components of Corydalis into the blood and the potential ingredients of the drug substance.These ingredients are more likely to be potentially potent substances in Corydalis.In this study,UPLC-Q-TOF/MSE was used to analyze the medicinal materials of Rhizoma Corydalis and metabolites in vivo.Taking the chemical composition spectrum to the metabolism spectrum,the metabolism of representative monomer to the metabolism of medicine,and from the high dose to the clinical equivalent dose as the main line,comprehensive study was conducted on alkaloids in Corydalis yanhusuo and their metabolites.It provided a scientific basis for revealing the material basis and active components of the medicinal efficacy of corydalis.