Study On Material Basis For Efficacy And Mechanism Of Shenxiong Glucose Injection

Objective: Shengxiong glucose injection, a compound preparation containing Salvia miltiorrhiza and ligustrazine hydrochloride, is clinically effective and widely used for the treatment of various cardiovascular and cerebrovascular diseases. However the material basis of efficacy, ADME and action mechanism still remain unclear, which cannot provide the scientific basis for its clinical efficacy and has been preventing its achieving scaled industrialization and modernization. Thus, it is of important practical significance to launch related basic researches on the traditional Chinese medicine injection. In this thesis, the pharmacodynamic material foundations, quality control, pharmacokinetics, mechanisms and drug interactions of Shenxiong glucose injection(SGI) were studied, which is one of famous varieties of Guizhou province. Hopefully, the material basis for pharmacological functions is clear; the level of quality control is improved; and the mechanisms are illustrated. In the end, all the results can provide the scientific evidence for the safety of clinical medication.Methods: 1. To fully analysis the main ingredients of SGI by Ultra High Performance Liquid Chromatography-Diode-Array Detector-Tandem Quadrupole Time of Flight(UHPLC-DAD-Q/TOF), figure out the active ingredients of SGI by the extraction technique of myocardial cells combining with Ultra Performance Liquid Chromatography-Electrospray Ionization-Triple Quadrupole Tandem Mass Spectrometry(UPLC-ESI-MS/MS), and establish a multi-index qualitative and quantitative method based on the finger-prints using HPLC to determine the contents of several active ingredients and observe the similarities of finger-prints of the herbs, simi-finished products and injections.2. To develop a rapid and sensitive ultra-performance liquid chromatography-tandem mass spectrometry(UPLC-MS/MS) method for the simultaneousdetermination of the four major active ingredients in the rat plasma treated with SGI, including danshensu, protocatechuic aldehyde, rosmarinic acid and ligustrazine hydrochloride, investigate the dynamic feature of the extract of Salvia miltiorrhiza, ligustrazine hydrochloride and Shenxiong glucose injection in vivo and compare their pharmacokinetics parameters to reveal the rationality of the combination.3. To evaluate the inhibition effects of SGI on 6 different kinds of cytochrome P450 isoforms(CYP1A2, CYP2E1, CYP2C9, CYP2C19, CYP2D6 and CYP3A4) in human liver microsomes. In vitro, SGI were incubated with cocktail probe substrates in the human liver microsomes, using the UPLC-MS/MS to determine the concentration of drug metabolite and IC50 values and evaluate the inhibitory effects on the CYP450 enzyme. In vivo, SD rats were assigned to the experimental group and the control group randomly and were continuous administered SGI with an equal volume of saline for 7 days and 14 days by intravenous injection respectively. The mixed probe drug was administered by intravenous injection at 8 days and 15 days. The serum concentrations of drug cocktail probes were determined by UPLC-MS and their pharmacokinetic parameters were calculated for evaluation of the activity of SGI on CYP450 enzyme. In order to ensure the safety and efficacy of clinical application, the effect of SGI on 6 different cytochrome P450 isoforms was also illustrated from the different perspective in vivo and in vitro to predict the possibility of drug interactions.4. To establish the oxidative damage model by incubating cardiac H9c2 cells with H2O2 using the cell viability, SOD activity, LDH, MDA and ROS content as main indicators to determine the protective effect of SGI on H2O2-induced damage in cardiac H9c2 cells; To analysis the mitochondrial membrane potential(△ψm), the morphology change of cardiac H9c2 cells damaged by H2O2, the expression of Bcl-2, Bax and Caspase-3 detected by quantitative real-time PCR(q RT-PCR) and Western blotting to explore the anti-apoptosis effect of SGI on H9c2 cells incubated with H2O2. The expression profiling alteration caused by SGI was also examined by using Rat Genome 230 2.0 Array(Affymetrix Inc.) in order to determine the signaling pathway related to the anti-apoptosis effect of SGI.Results: 1. Through fully scanning of chemical ingredients of SGI, 13 compounds among them were determined. 8 active compounds with the protective effect on cardiac muscle cells including danshensu; Ligustrazine Hydrochloride; salvianolic acid B, C, D, I/H; lithospermic acid and rosmarinci acid were filtered out through the extraction techniques of myocardial cells, which belong to water-soluble compounds of salvianolic acid. Thus, we infer that phenolic acids are the main active ingredients of SGI. The multi-index measurement results reveal that the total content of danshensu, Ligustrazine Hydrochloride, protocatechuic aldehyde, rosmarinci acid, salvianolic acid B and salvianolic acid A were 1.295mg/m L ~ 1.387mg/m L, the average content was 1.336mg/m L and the value of RSD was 1.6%. The finger-prints of different batches of SGI show a high similarity more than 98% and the finger-prints of herbs, semi-finished products and injections also show a good relativity.2. Through the statistical moment model process, the different determined ingredients of Salvia miltiorrhiza and MRT of Ligustrazine showed the dose-dependent pharmacokinetics and in vivo metabolisms were characteristics of non-linear pharmacokinetics. The pharmacokinetics parameters of ligustrazine hydrochloride vary greatly with the different combination with extract of Salvia miltiorrhiza. Especially, AUC obviously increased. MRT, CL and V significantly decreased. The results demonstrated the existence of Salvia miltiorrhiza can cause the shorter residence time and narrower distribution of ligustrazine in vivo, which indicate that the administration dosage may become a critical issue on the clinical dose with regard to the security.3. In the human liver microsome, IC50 values of SGI on CYP1A2, CYP2E1, CYP2C9, CYP2C19, CYP2D6 and CYP3A4 were all above 15%, which indicate that SGI doesn’t show competitive inhibition on those enzymes. In the rats, compared with the control group, the major pharmacokinetics parameters of caffeine, midazolam, omeprazole, metoprolol administered continuously for 7 days and 14 days showed no significant statistic difference while the major parameters of AUC,MRT,T1/2,Cmax of tolbutamide in 7 days and 14 days group was significantly increased, CLz was significantly increased. Thus, SGI properly had an inhibition effect on CYP2C9 with the period of administration, and no inhibitory and inductive effect on CYP1A2,CYP2D6,CYP2E1,CYP2C19,CYP3A4. Therefore, attention should be paid especially when SGI is co-administered with drugs metabolized by human CYP2C9 and with narrow therapeutic range, which may result in deduced concentrations of these drugs and relevant drug-drug interaction.4. SGI can protect cardiac H9c2 cells from H2O2-induced injury due to the decrease of the release of LDH, MDA, ROS, which result in the increase of H9c2 cell viability and the mitochondrial membrane potential(△ψm) and the improvement of cell morphology. Furthermore, the expression of Bcl-2 was significantly up-regulated in SGI pre-treatment group and the expression of Caspase-3 was obviously down-regulated in H9c2 cells exposed to H2O2; Our microarray analysis found 101 genes with differential expressions, including genes involved in p53、Jak-STAT、m TOR、MAPK and PI3K/Akt pathways. Akt genes had the highest changes in SGI pre-treatment group compared with model group, which implies that the PI3K/Akt signaling pathway may play a key role in the anti-apoptosis effect of SGI.Conclusion: This thesis expounded the pharmacodynamic substance foundations of SGI by building up a method of traditional Chinese medicine’s pharmacodynamic substance foundations by UPLC/Q-TOF MS combined with the technique of target cell extraction; established a mean of systematic way to promote the quality control; and provide the powerful basis to the security on clinic application with the specific pharmacokinetic characteristics of anti- Myocardial ischemia. The research settled a strong base to make sure the SGI’s clinic application safety and effective according to the studies above, and meanwhile provided the important evidences of the Preparation’s Clinical efficacy.