The Chemistry Constituents And Multi-components Quantitation Study Of Honghua Xiaoyao Tablet

Honghua Xiaoyao Tablet(HHXYT)is a medicine developed by Xiaoyao San.It is first recorded in Song Dynasty,combined with the medicine experience of Yi nationality in Ailao Mountain area,Yunnan Province,China.It is prepared by the combination of Angelica sinensis(Oliv.)Diels,Paeonia lactiflora Pall.,Atractylodes macrocephala Koidz.,Poria cocos(Schw.)Wolf,Carthamus tinctorius L.,Gleditsia sinensis Lam.,Bupleurum marginatum Wall.ex DC,Mentha haplocalyx Briq.and Glycyrrhiza uralensis Fisch.HHXYT has the effects of soothing the liver,regulating qi and promoting blood circulation and mainly be used to the treatment of gynecological diseases in clinical application,such as irregular menstruation,chloasma and hyperplasia of the breast.But its chemical composition and quality control research are still lacking,which hinders the subsequent wider application of HHXYT.Objective: In this study,HHXYT with definite clinical efficacy was taken as the research object to comprehensively analyze chemistry constituents in vitro and prototype components in blood.At the same time,the multi-components quantitation study and chemometric analysis on HHXYT samples from different batches and different manufacturers were carried out to find the important quality marker(Q-marker)components in the HHXYT samples,which would be a valuable reference for the further pharmacological study and wider clinical application of HHXYT.Methods:(1)Ultra-performance liquid chromatography coupled with quadrupole time-of-flight mass(UPLC-QTof/MS)and Unifi software platform were used to collect and process the chemistry constituents’ data of HHXYT in vitro.The in-house library of HHXYT was established and imported into Unifi software,and it automatically matches the MS data collected by UPLC-QTof/MS to further fully characterize the chemical composition of HHXYT in vitro.(2)Uplc-QTof / MS was used to collect the data of plasma,and the extracted-ion chromatography(EIC)method of Masslynx workstation was combined with the automatic matching of Unifi software to analyze the prototype components of HHXT.(3)High-performance liquid chromatography coupled with triple quadrupole mass(HPLC-qqq/MS)was used to determine the content of 14 potential quality marker components in HHXYT samples.The quantitative results were analyzed through Principal component analysis(PCA)and hierarchical cluster analysis(HCA),which would be a useful reference for the further quality control study of HHXYT.Results:(1)The in-house library of HHXT contains a total of 1086 compounds.Finally,88 chemical compounds in vitro were identified,including 16 flavonoids,18 triterpenes,18 monoterpenoid glycosides,7 organic acids,3 phthalides,4 saccharides,4 coumarins and the remaining 18 other compounds.(2)A total of 22 prototype components were identified in the rat plasma samples,including 8 flavonoids,3 triterpenes,5monoterpenoid glycosides,2 organic acids,3 phthalides and 1 dihydrochromene.(3)A quantitative analysis method for simultaneous determination of 14 potential quality marker components in HHXYT samples was established.The 5 components including Z-ligustilide,senkyunolide I,formononetin,glycyrrhetinic acid and glycyrrhetinic acid3-O-glucuronide were respectively in the content ranges of 0.046-5.600 mg / g,0.004-0.153 mg / g,0.004-0.032 mg / g,0.003-0.041 mg / g and 0.008-0.072 mg / g in positive ion mode.And other 9 components within protocatechuic acid,liquiritigenin,isoliquiritigenin,licoisoflavone B,licoricone,liquiritin,isoliquiritin,paeoniflorin,glycyrrhizic acid were respectively in the content ranges of 0.054-0.362 mg / g,0.043-0.242 mg / g,0.022-0.124 mg / g,0.002-0.030 mg / g,0.001-0.012 mg / g,0.131-1.805 mg / g,0.071-0.476 mg / g,4.085-9.413 mg / g and 1.631-13.75 mg / g in the negative ion mode.The results of PCA is basically similar to HCA,So the 30 batches of samples can be separated according to different manufacturers through the analysis of 14 quality marker components.Conclusion: Based on the study of the chemistry constituents in vitro and in vivo of HHXYT,this study elucidated the composition basis of HHXYT,and clarified the original composition of blood.A rapid,sensitive and stable in vitro multi-component quantitative method for HHXYT was established.30 batches of samples from three different manufacturers were determined.The PCA and HCA were used for the quantitative results.The important quality marker components in different batches of samples were found,which provided a reference for the follow-up study and clinical application of HHXYT.