Study On The Chemical Composition Of Chaigui Granules And Its In Vivo Process

Rationales: Depression is a mental illness characterized by significant and long-lasting depression.This research group developed the traditional Chinese medicine Chaigui granules based on the classic famous formula Xiaoyao Sanhuan.It consists of 6 herbs,Atractylodis Macrocephalae Rhizoma,Bupleuri Radix,Angelicae Sinensis Radix,Glycyrrhizae Radix et Rhizoma,Radix Paeoniae albiflora and Mentha haplocalyx,which have obtained clinical approval.The chemical constituents in the traditional Chinese medicine compound are the material basis for exerting the effect.The research on the chemical composition of traditional Chinese medicine compound,and examining the components and metabolites absorbed into the body are of great significance to clarify the basis of its medicinal substances.In the previous period,this research group conducted a preliminary exploration of the chemical composition of Chaigui granules.This study intends to establish a more comprehensive and in-depth study of the chemical composition of Chaigui granules in vitro and in vivo based on LC-MS technology and its PK-PD correlation.It provides a scientific basis for the research on the basis of pharmacodynamics and clinical safety of Chaigui granules.Objective: Establish the in vitro and in vivo chemical composition spectrum of Chaigui granules,and clarify the chemical components in the compound and the components that enter the body.Methods:(1)Qualitative identification of the chromatographic peaks of Chaigui granules by UHPLC-MS chromatogram through standard control and reference data,and comprehensive identification of its chemical composition.(2)Through the eversion bowel sac experiment and one-way intestinal perfusion experiment,the absorption characteristics of Chaigui granules in the jejunum and ileum of the small intestine were investigated.The rats in the normal group and the CUMS group were used to investigate the rats' intestinal tract in different pathological conditions differences in absorption of chemical components of Chaigui granules.(3)Based on the identification of in vitro chemical constituents,serum medicinal and brain microdialysis techniques were used to study the absorption and metabolism of Chaigui granules in blood and brain,and to comprehensively investigate the absorption and metabolism of Chaigui granules in vivo.(4)Based on serum pharmacokinetics and pharmacodynamics,Compound Discoverer 3.0 software was used to calculate serum exogenous compounds and endogenous metabolites,integrate pharmacokinetics and pharmacodynamics,and study the correlation between PK-PD.Results:(1)A chemical composition analysis method for Chaigui granules was established by UHPLC-Q-orbitrap-MS/MS technology.Based on the retention time of the primary chromatographic peak and the corresponding secondary ion fragment information,combined with the retention time of the reference substance,a total of 95 chemical components were identified.It mainly includes Including saponins,flavonoids,lactones,terpenes,phthalide,organic acids,coumarins,tannins,etc.This study basically clarified the main chemical constituents in Chaigui granules,laying a foundation for the research of chemical experimental constituents in vivo.(2)The intestinal sac test and one-way intestinal perfusion test were used to study the absorption of drugs in the small intestine.Results in the eversion sac experiment,70 components were absorbed by both the jejunum and ileum of the small intestine.By comparing the absorption peak area of normal rats and CUMS rats,45 significant differences in absorption were found,among which Licochalcone B(P <0.001)and Isoliquiritigenin(P <0.05)increased nearly 21-fold and 43-fold in the ileum of CUMS rats,respectively.In the one-way intestinal perfusion experiment,85 components were absorbed by both the jejunum and ileum of the small intestine.By comparing the absorption peak areas of normal rats and CUMS rats,10 significantly different absorption components were found,namely ferulic acid,paeoniflorin,Citric acid,protocatechuic aldehyde,E-butenylphthalide,furan sesquiterpenes,Licochalcone A,saikosaponin d,saikosaponin g,and saikosaponin a.(3)Study on metabolism of Chaigui granules in vivo.28 prototype components and 38 metabolites were found in serum,and 15 prototype components were found in brain without metabolites.Based on the data analysis of Compound discoverer 3.0 software,metabolites of Paeoniflorin,5-hydroxymethyl furfural,atractylolide II,Z-ligustilide,Licoagroside,ferulic acid,Senkyunolide H,isoliquiritin,Liquiritigenin,Isoquiritigenin were found in the serum,which mainly occurred oxidation,reduction,dehydration,acetylation,glycosylation,methylation and other metabolic processes.(4)PK-PD analysis after intragastric administration of Chaigui granules to rats.As a result,62 prototype compounds and 34 exogenous metabolites were found.There were 33 identical prototype compounds and 23 identical exogenous metabolites between the normal and CUMS groups.12 prototype compounds were identified through references and secondary ion fragmentation information.Based on the HMBD database combined with the secondary fragment ion fragments,4 endogenous differential metabolites related to drug efficacy were identified.The time to reach the maximum blood concentration in the PK-PD correlation curve was 0.17-4h,and the maximum effect occurred at 12 h,indicating that there is a very obvious lag between the drug concentration and the effect.Conclusion: 1.By studying the chemical composition of Chaigui granules in vitro,95 compounds were identified to lay the foundation for the next step to study the absorption of chemical components after entering the body.2.Through the inversion of the everted gut sacs and the in situ rats single pass perfusion model experiment,it was found that most of the compounds can be absorbed by the intestine.There is a significant absorption difference between the CUMS group and the normal group.The components with significant differences may be related to antidepressant.3.Serum pharmacology and brain microdialysis technology respectively studied the components of Chaigui granules into the blood and into the brain,and found that the prototype components into the blood can be found in the intestine,and the components into the brain can also be found in the serum,indicating the drug It is absorbed into the blood after being absorbed by the small intestine,and transported from the blood to the brain to play an antidepressant effect through the blood-brain barrier.4.The PK-PD correlation study found that there is a lag in drug efficacy.Chaigui granules have a therapeutic effect on CUMS rats and have almost no effect on normal rats.This is consistent with the idea of "have no cause for death" and is Chaigui granules clinical Pre-safety and effectiveness evaluation provides PK-PD basis.