Pharmaceutical Constituents Investigation Of Kai-xin-san Based On Changes Of Catecholamines And The Pharmacokinetic Study Of Related Compounds

Kai-Xin-San(KXS),a famous traditional Chinese formula,was originally written in Bei Ji Qian Jin Yao Fang by Simiao Sun from Tang Dynasty.It contains four Chinese herbs,Polygalae Radix,Ginseng Radixet Rhizoma,Poria and Acori Tatarinowii Rhizoma,which was used for the treatment of amnesia,neurasthenia and AD1.To investigate pharmacodynamics in rats after oral administration of KXS.The AD model was induced by intraperitoneal injection of D-Gal and bilateral hippocampal injection of A?25-35.2 weeks after surgery,the behavioral test,brain tissue pathological biopsy and learning and memory related substances tests were carried out.The behavioral test showed that KXS group with significantly shorter escape latency in directional swimming test,with longer swimming distances in the quadrant and higher frequency across the platform in probe test compared with the model rats.Brain tissue pathological biopsy showed that the CA1 hippocampal cells of rats in KXS group were gradually returned to normal.The related substances tests showed that the rats in KXS group with higher T-SOD activity,lower MDA content and lower T-ChE activity in serum and brain tissue compared with the model rats.All experimental results show that KXS has prevention and treatment function on AD.2.The rapid and sensitive UFLC-MS/MS methods in different ionization modes for the quantification of Tyr,MHPG,VMA and HVA in rat plasma,Tyr,NME,ME,E,NE,3-MT,DA,DOPAC,DHPG,MHPG,DOMA,VMA,HVA and DOPA in rat urine without derivatization or complex sample pre-treatments were developed.After addition of the internal standard,isoproterenol,the samples were extracted by protein precipitation and separated on an Inertsil ODS-EP column at a flow of 1.0 mL/min.The intra-day and inter-day precision and accuracy of the analytes were well within acceptance criteria(±15%).The mean extraction recoveries of analytes and internal standard were all more than 60%.The methods were examined and found to be satisfied with linearity,precision,accuracy,recovery,matrix effect and stability.The methods were succefully applied in the determination of related substances of rats in blank control,model,KXS and Hup-A group.The results demonstrated that compared with the blank group,the concentrations of Tyr,HVA and VMA in model rat plasma were significantly decreased,the plasma MHPG level was significantly increased;compared with the model groups,the plasma Tyr,MHPG,VMA and HVA concentration in KXS group were recoveried to control group and were similar with Hup-A group.Compared with the blank group,the urine concentrations of Tyr,NME,ME,E,NE,3-MT,DA,DOPAC,DOMA,VMA,HVA and DOPA were significantly decreased in model group,the urine MHPG and DHPG level were significantly increased.The urine Tyr,NME,ME,E,NE,3-MT,DA,DOPAC,DHPG,MHPG,DOMA,VMA,HVA and DOPA concentrations in KXS group had also been restored and tended to be normal,were closely to Hup-A group.So KXS may have a role in prevention and treatment of AD.Moreover,as urine catecholamines concentration for example,the LASSO package in R3.1.2 program was applied to set up a logistic regression model.The mathematical regression to distinguish normal and AD rats was y=118.2-1.0931ogTyr-1.1621og3-MT-4.6771ogDA-1.011logDOPAC+1.2611ogMHPG-13.181ogHVA.This may provide a reference for the prevention and diagnosis of AD.3.GC-MS was applied to identify the volatile components in KXS.44 compounds were identified,37 from Acorus,4 from ginseng,2 from polygala and 1 from Poria cocos.UPLC-Q/TOF MS was applied to identify chemical compositions and the constituents in rat plasma after oral administration of KXS.92 compounds were identified in KXS,53 from polygala,36 from ginseng and 3 from Poria cocos,they were ginsenosides,triterpene acids,oligosaccharide esters,polygala saponins and xanthones.About the constituents in rat plasma,there were 26 compounds were identified,18 from ginseng,5 were from polygala and 5 were the metabolites.UFLC-MS/MS was applied to simultaneously determine 18 compounds in KXS.The 18 compounds were separated by Shimadzu UFLC system with Venusil MP C18(100 mm × 2.1 mm,3.0 ?m)column.The mobile phase was 0.05%formic acid water-0.05%formic acid methanol.And they were detected by AB 4000 QTRAP in MRM mode with ESI-.The method was examined and found to be satisfied with linearity,precision,accuracy,recovery and stability for all analytes.This study may be helpful for the quality control of KXS.4.A fast,sensitive and reliable ultra fast liquid chromatography-tandem mass spectrometry(UFLC-MS/MS)method has been developed and validated for simultaneous quantitation of POL,GRb1,GRd,GRe,GRg1 and TUM in rat plasma after oral administration of Kai-Xin-San.The plasma samples were extracted by liquid-liquid extraction using ethyl acetate-isopropanol(1:1,v/v).Good chromatographic separation was achieved using gradient elution with the mobile phase consisting of 0.01%acetic acid in water and methanol.The tandem mass spectrometric detection was performed in MRM mode on 4000Q UFLC-MS/MS system with turbo ion spray source in a negative and positive switching ionization mode.The lower limits of quantification were 0.2-1.5 ng/ml for all the analytes.Both intra-day and inter-day precision and accuracy of analytes were well within the acceptance criteria(15%).The mean absolute extraction recoveries of analytes and IS from rat plasma were all more than 60.0%.The validated method has been successfully applied to compare pharmacokinetic profiles of analytes in normal and AD rat plasma.The results indicated that no significant differences in pharmacokinetic parameters of GRe,GRgl and TUM were observed between the two groups,while the absorption of POL and GRd in AD group were significantly higher than those in normal group;moreover,the GRbl absorbed more rapidly in model group.