Characterization Of Biopharmaceutical Properties Of Chishao Terpene Glucoside Components Based On Identification Of Representative Ingredients For Pretreatment Of Myocardial Ischemia

As a main substance for the treatment of disease,Chinese medicine is often used in the form of bolus,powder,plaster and pellet.Therefore,traditional Chinese medicine preparation is the main link between medicinal materials and clinical application,and the raw material of the preparation is the traceability of the curative effect.However,selection of Chinese medicine dosage forms was affected by the physical and chemical properties of raw materials.As a basic unit of the raw materials of traditional Chinese medicine preparation,the overall characterization of the components of Chinese medicine is a key problem to be solved urgently.In this paper,Chishao terpene glucoside components(CSTGCs)was used as a model drug to identify and screen out the representative components for treatment of myocardial ischemia,and the overall biopharmaceutical properties of the CSTGCs was tentatively characterized.The main contents are as follows:1.Identification of the representative components of CSTGCs for the treatment of myocardial ischemiaCombining TCMSP database and literature reports,qualitative research was carried out on the chemical constituents of Radix Paeoniae Rubra by UPLC-TOF/MS/MS.Then,chemical constituents were molecular docked with the key receptor proteins of myocardial ischemia to preliminarily screen the anti-ischemia active ingredients.Because of the greater Libdockscore score,TGCs was confirmed as effective components in Chishao for treatment of myocardial ischemia.After that,the chemical components in plasma were analyzed after oral administration of Chishao extraction,and paeoniflorin,albiflorin,benzoyl paeoniflorin and oxidized paeoniflorin were found.In addition,paeoniflorin,albiflorin,benzoyl paeoniflorin was detected in the H9C2 cells dissociation solution after incubation with Chishao extraction.Hence,paeoniflorin,albiflorin,benzoyl paeoniflorin and oxidized paeoniflorin were the potential representative components of CSTGCs for treatment of myocardial ischemia.2.Screening of representative components for treatment of myocardial ischemia by in vitro drug efficacy experimentThe composition of CSTGCs was analyzed by HPLC,the content of paeoniflorin,albiflorin,benzoyl paeoniflorin,oxidized paeoniflorin was 33.82%,21.14%,1.91%,and 3.51%respectively,and the total mass accounts for 60.38%of the CSTGCs.The weight ratio of these 4 components is 17.69/11.14/1.0/1.83.Then,H9C2 cell hypoxia injury model was established to screen out the representative components,various groups were set up according to the original proportion of CSTGCs,and CK,LDH,AST,SOD,CAT,GSH-Px,MDA were determined and compared as evaluation index.The results showed that paeoniflorin,albiflorin and benzoyl paeoniflorin were the representative components of CSTGCs for treatment of myocardial ischemia.Considering that cell apoptosis is one of the important mechanisms of myocardial ischemia injury,the apoptosis related proteins Bax,Bcl-2,Caspase-3 and Mitochondrial function related Ndufa4,Ndufa8,Cox7a2,TFAM mRNA were analyzed to evaluate the anti-apoptosis effect.The results showed that the representative components(paeoniflorin,albiflorin and benzoyl paeoniflorin)could maintain normal mitochondria function and inhibit cells apoptosis.3.Pharmacodynamic evaluation of the representative components in vivo and determination of the contribution rate of each componentAn acute myocardial ischemia rat model was induced by ISO,myocardial pathological changes,ultrastructural changes,tunnel apoptosis index,myocardial enzyme levels(CK,LDH)and energy metabolism(ATP,glycogen)and apoptosis protein(Caspase-3,Bax,Bcl-2)were determined and compared.It was found that the representative components had a protective effect on myocardial ischemia injury,and there was no significant difference in the regulation of evaluation indexes between representative components group and CSTGCs group(300 mg/kg).Then,multiple index data normalization weighting method was used to evaluate the contribution rate of three representative components,and the contribution rates of each component are as follows:paeoniflorin was 54.66%,albiflorin was 32.58%,and benzoyl paeoniflorin was 12.76%.4.Characterization of the overall biopharmaceutical properties fitted by the contribution rate of three representative components,The equilibrium solubility and O/W partition coefficient of the representative components were determined under the single component or CSTGCs system,and the similarity degree and discrete analysis were performed.In CSTGCs system,the solubility of benzoyl paeoniflorin increased,and its Euclidean distance from paeoniflorin or albiflorin was 62.96 and 43.55 respectively,with a certain similarity.However,its Euclidean distance of O/W partition coefficient with paeoniflorin or albiflorin was around 2200.According to the theory,CSTGCs should be divided into two subcomponents.Actually,solubilized benzoylpaeoniflorin was considered as a class I agent,it does not require the exclusive process.In order to simplify the preparation,subcomponents were not defined and components still seen as a whole for characterization of the overall biopharmaceutical properties.The results showed that the equilibrium solubility of CSTGCs was 36.09?38.39 mg·mL-1,O/W partition coefficient was 1.52?1.63,Caco-2 cells apparent absorption permeability coefficient was 1.68×10-6 cm·s-1,the apparent secretion coefficient was 1.84×10-6 cm·s-1,S,suggesting that CSTGCS is a class ? drug with high solubility and low permeability.5.Improvement of oral bioavailability of CSTGCs by combining with absorption enhancerUsing in vitro absorption model of rat,effect of absorption enhancer on 2h accumulation permeation amount was investigated in 2 hours,and sodium caprate/CSTGCs(3:1)was selected for the followed pharmacokinetic test.By plotting the concentration-time curve after intragastric administration of CSTGCS(300 mg/kg)with sodium caprate or not,it was found that when combined use with sodium caprate,paeoniflorin and albiflorin AUC(0-24h)increased by 36.33%and 28.18%respectively.When concentration-time curves of each component were integrated,AUC(0-24h)of CSTGCs increased by 35.27%,indicating that sodium caprate could improve the oral bioavailability of CSTGCS,also suggesting CSTGCs is a substance with high solubility and low permeability.