Study On Spectrum-Efficacy Relationship And The Metabolism Of Niuhuang Shangqing Tablets In Vitro

In the present research,spectrum-efficacy relationship and the metabolism in vitro of N iuhuang Shangqing tablets(N HSQ)were carried out.The models for assessing anti-endotoxin activity of NHSQ were constructed on HPLC fingerprint,so as to evaluate the NHSQ from different manufactures and batches.An ultra-high-performance liquid chromatography-quadrupole time-of-flight mass spectrometry(UPLC-Q-TOF-MS~E)method was applied to analyze the metabolism of NHSQ in rat liver microsomes(RLMs).Some chemical information could be acquired from the fingerprint of TCM(Traditional C hinese Medicine),however,biological activities are difficult to reflect.Therefore,the relationship between chemical information and biological activities were established,which could better assess the quality of TCM and would provide useful references for improving the quality evaluation system.In addition,the metabolism of NHSQ in RLMs was studied,which could further elucidate the metabolism of NHSQ in liver and provide reference for its clinical rational application.Our present research work includes the following sections:1.Fingerprint studies of NHSQ based on HPLC-PDA.HPLC fingerprint of NHSQ from different manufactures and batches was successfully developed and also validated.The results showed that the established HPLC method has good precision,repeatability and stability and could be used for the determination of the fingerprint of N HSQ.31 common peaks were obtained in the fingerprint of NHSQ,and the 20 common peaks were identified with the reference compounds,such as gallic acid,gardenoside,liquiritin,palmatine and so on.Furthermore,partial least squares-discriminant analysis(PLS-DA)and similarity evaluation were used to evaluate the quality of N HSQ from 14 manufacturers and 52batches.2.Studies on spectrum-efficacy relationship.The anti-endotoxin activities of 52 samples from 14 manufacturers were determined by dynamic turbidity method.The relationship between the normalized peak area of HPLC fingerprint and the relative anti-endotoxin activity(relative reaction time)was modeled by orthogonal partial least squares(OPLS).In this model analysis,the coefficient of cross-validation Q~2=0.571>0.5,which indicating that the model could be better predictive ability.The analysis results demonstrated that baicalin,hesperidin,liquiritin,osthole,physcion and chrysophanol might be the pharmacodynamic material basis on the anti-endotoxin activity in NHSQ.Spectrum-efficacy relationship analysis could better combine the chemical information with the efficacy information,which could provide a reference for the quality evaluation of NHSQ.3.Identification and analysis of the metabolites of NHSQ in RLMs.UPLC-Q-TOF-MS~E was applied to identify the metabolites of NHSQ in RLMs at the first time.52 metabolites were identified by Mass Lynx and UNIFI software.Among these metabolites,there are 33 flavonoids,7 anthraquinones,9 phenolic acids and 3 alkaloids.In addition,24 metabolites were underwent phase I metabolic route,28 metabolites were underwent phase II metabolic route,and phase II biotransformation pathway is predominant.Furthermore,studies on the metabolic pathways of active components in RLMs,the metabolic pathways of baicalin,liquiritin,chrysophanol,emodin and chrysin were confirmed,which showed that metabolism in RLMs is easier than metabolism in vivo.The results of metabolism of NHSQ in RLMs combined with the previous studies on metabolism in vivo,which revealed that the comprehensive metabolism of N HSQ in vitro and in vivo,and lay a foundation for the explanation of action mechanism and clinical application of NHSQ.