Study Of Evaluation Methods For Quality Control And Bioactivities Of Cardiotonic Pilll

Analyzing and eluating methods for chemical compositions and biological activities are important to elucidate the quality of traditional Chinese medicine.Two key issues were queried in the process of international registering of Cardiotonic Pill(CP).The first one is how to qualitatively and quantitatively clarify the mass balance of constituents,and the second one is how to develop specific methods for evaluating consistency of bioactivities.To solve these problems,CP was investigated employing pharmaceutical analysis,natural product chemistry,pharmacology,molecular biology,omics combine with bioimformatics to develop analyzing and evaluating methods in this study1.Establishment of analyzing method for chemical constituents in Cardiotonic PillAn off-line hydrophilic interaction × reversed-phase liquid chromatography-ion trap-time of flight/mass spectrometry method was developed and then employed in the deeply analyzing of phenolic acids in Salvia Miltiorrhiza and extract of CP.A total of 250 phenolic acids were tentatively identified.Furthermore,an preparative off-line hydrophilic interaction × reversed-phase liquid chromatography-mass spectrometry method was developed to targeted purifying interested compounds.Combined with nuclear magnetic resonance analysis,structures of 18 compounds were comfirmed.Among them,five compounds were firstly found in extract of CP.2.Establishment of analyzing method for bioalogical activities of Cardiotonic PillA rat acute myocardial infraction model was established to investigate the protection of CP(0.8 g/kg/day)against infraction injury.Results showed that CP could significantly improve pathological state and cardiac function,decrease infarction area and apoptosis cells in myocardial tissue,and increase ejection fraction and systolic fraction.It could also significantly decrease the levels of myocardial enzymes,oxidation products,inflammatory factor and adhesion molecule,and increase the expression of glutathione peroxidase(GSH-Px)and its substrate glutathione(GSH).A myocardial infarction network was established to investigate the mechanism of CP.The established network contain 625 different expression gnens and 3099 edges.Among them 496 genes were resisted by CP and 104 of them have a fold change that bigger than 2.CP could resist mypcardial infarction through regulating inflammation response,immune response,mitochondria and extracellular matrix matrix,and also have some improvements in carbohydrate metabolism,fatty acid metabolism and amino acid metabolism.3.Establishment of quality evaluating methods for chemical constituents in Cardiotonic PillTwo quantitative method was developed to determine chemical constituents of CP.In order to clarify chemical balance of of CP,a directly-series reversed-phase-hydrophilic interaction chromatography tandem with MS/MS method was developed to determine 30 compounds.As a result,56.4-73.0%could be determined in extract of CP,which was benefit to the elucidation of chemical mass balance.For the purpose of rapid determination of bioactive components in CP,a single standard quantification method was established to simultaneously determine danshensu,protocatechuic aldehyde,rosmarinic acid,lithospermic acid,salvianolic acid B and salvianolic acid A using ultra performance liquid chromatography.Protocatechuic aldehyde was selected as internal standard and the coversion factor of the other five compounds are 6.570,3.654,1.507,1.511 and 3.443,respectively.Specificity,precision,accuracy and stability of the estabilied method met the requirement of determination.In addition,ruggedness and robustness of conversion factors were also systematically investigated.4.Establishment of quality evaluating method for holistic bioactivity of Cardiotonic PillA gene expression-based method was established to evaluate the consistency of bioactivity of CP.HepG2 cell was firstly treated with CP for 24 h.Then gene expression array and real-time PCR was employed to screen different expression gnens with dose-dependent characteristics.Ten genes,including MMP1,CYP1 A1,EPGN,RUNX2,C8orf4,OLR1,CLMP,AKR1C1,IL24 and APOL6,were selected as markers of bioactivity of CP.Angle cosine of each genes fold change was calculated to evaluate the consistency of samples.Intra-day and inter-day precision of the established method was 0.4%and 0.6%,respectively.Six batches of CP were analyzed with similarities between 0.992-0.999,and one batch of compound Danshen Tablet was analyzed with a similarity of 0.534.Consequently,this method provided a reference for comprehensive and objective evaluation the holistic bioactivity of CP.