Study On The Chemical Constituents Of Huangqi Jianzhong Decoction In Vivo And Vitro

Huangqi jianzhong Tang(HQJZ),recorded originally in Zhang zhongjing "Synopsis Golden Chamber" ?Jin Gui Yao Lue?,consists of seven Herbs(Radix Astragali,Radix Paeoniae Alba,Radix Glycyrrhizae,Ramulus Cinnamomi,ginger,jujube and caramel).It was used to treat of warm tonic,relieving pain.Modern clinical practice is mostly used to treat gastrointestinal diseases,such as spleen deficiency,chronic atrophic gastritis and duodenal ulcer,which are the main compound of deficiency cold stomach disease.The composition of the chemical substance in vitro and the absorption and metabolism in the body are not clear.The efficacy of TCM can be reflected only in the presence of a certain syndrome(the body is in a specific pathological state).The identification of metabolic components in the body of the disease can reflect the material basis of HQJZ more scientifically.Therefore,in order to clarify the effect components(groups),it is necessary to study the chemical constituents in vivo and vitro,especially under the disease state.Objective: To establish a comprehensive chemical composition spectrum of HQJZ,and to analyze the metabolism of HQJZ and its mechanism of action in vivo.To explain of the chemical composition and possible metabolic mechanism of HQJZ systematically and provide a scientific basis for the quality evaluation and clinical application of the compound.Method:(1)Establishment and composition analysis of the chemical compositions of HQJZ in vitro.In this study,the methods of LC-MS combined and the method of drug analysis were first used.This study was designed to establish an UHPLC-MS method for the reliable identification the multiple chemical components in HQJZ.The UHPLC coupled with mass spectrometry method was applied to identify the chemical constituents in the HQJZ rapidly.(2)Establishment and composition analysis of the chemical composition of HQJZ in vivo.We use UHPLC-Q Exactive orbital to analysis the components in vivo after oral administration of HQJZ in control rats.After repeated administration of high dose HQJZ(36.8 g/kg)in SD rats,Anticoagulant blood of heparin sodium were collected,and plasma samples were pretreated by ethyl acetate liquid liquid extraction combined with methanol protein precipitation.Data were processed by Compound Discover 2,and the results were verified by chemical composition,retention time,precise molecular weight and characteristic fragment ions.The chemical composition of HQJZ in physiological condition was obtained.(3)Analysis the difference of blood migrating components of HQJZ between the state of control and model.Use the method established in second part to analysis four groups of C,M,C-HQJZ and M-HQJZ.Through multivariate statistical analysis,we identified the endogenous metabolites in the model rats and control rats,and identified the difference between the migrating components in the blood of the model rats and the control rats.Finally,the endogenous metabolites?the prototypes and metabolites of the HQJZ absorbed into the blood were analyzed by correlation analysis.Results:(1)Identification results of chemical components in vitro: A total of71 compounds(including saponins and flavonoidswere)were identified,which provides a scientific basis for the quality control of HQJZ.(2)Identification of chemical components in the control rats: A total of 11 prototypes and 55 related metabolites were detected and identified.The results showed that the absorbed components of HQJZ were mainly flavonoids and saponins.The transformation mainly included methylation,acetylation,sulfation and so on in vivo.(3)The C and M were compared and analyzed by S-plot and T test,and 20 endogenous metabolites(Amino acids,fatty acids and so on)werescreened and identified,which related to the pathway of amino acid metabolism,fatty acid metabolism and intestinal flora disorder.In the same way,two groups C and C-HQJZ were compared,and 13 prototypes and 21 metabolites were obtained.The prototypes of absorption include flavonoids in Astragalus and licorice and their corresponding saponins,monoterpenes in Paeonia lactiflora Pall.Metabolic reactions involve oxidation,reduction and binding reactions.The M and M-HQJZ two groups were compared and analyzed,and 11 prototypes and 13 metabolites were obtained.The absorption of the prototype component was less Cinnamic acid and Onion than that of the normal control group,and the metabolic reactions involved were basically the same.Finally,the two groups C-HQJZ and M-HQJZ were analyzed by multivariate statistical,screening and identifying 3 prototypes and 7 metabolites.Through further correlation analysis,Mudanpioside F,Methoxycinnamic acid,5-Hydroxylliquiritin,6-Shogaol,Liquiritin,Isoliquiritigenin,Gallic acid,Calycosin,Paeoniflorin,Isoliquiritin apioside,Formononetin and related metabolites are highly correlated with pathological markers of chronic atrophic gastritis,which is the potential material basis for their intervention in CAG.Conclusion: This study is based on the analysis of chemical components in vivo and in vitro and multivariate statistical analysis to study the differences of absorption and metabolism of HQJZ in control rats and model rats.Finally,we get the potential effect components(Group)of HQJZ on the prevention and treatment of chronic atrophic gastritis.This study provides a basis for identifying the biotransformation characteristics of HQJZ and its action process in vivo,and provides a reference for selecting effective components of HQJZ in the prevention and treatment of chronic atrophic gastritis.