Study On Pharmacokinetics And Pharmacodynamics Of The Main Effective Constituents Of Danhong Injection In Hyperlipidemia Rats

Objective To establish a stable,successful and high replications rat model of hyperlipidemia,and a HPLC-DAD method for the simultaeous quantitation of danshensu,protocatechuic aldehyde,hydroxysafflower yellow A and salvianolic acid B concentrations in hyperlipidemia rat plasma,objective to study the pharmacokinetic characteristics of orthogonal components of Danhong Injection in hyperlipidemia rats,based on the pharmacokinetics to establish PK-PD model of active ingredients in Danhong Injection anti-hyperlipidemic,anti-thrombosis effects.The aim of this study was to provide a new idea and reference for the study of pharmacokinetics and pharmacodynamics of Chinese herbal medicines.Methods 1.To establish a rat model and study on the effect of Danhong Injection on blood lipid in hyperlipidemia Rats:To establish a model of hyperlipidemia rats by high fat feed method,after adaptive feeding to continuous feeding for 6 weeks testing the plasma lipid levels(TC、TG、HDL-C、LDL-C)and included the experiment for qualified rats.The blood lipid levels were measured in 1,3 and 7 days by ELISA method after Danhong Injection intraperitoneal administration for 1 week.2.HPLC-DAD method:The hyperlipidemia rats by injection of Danhong Injection active ingredient(danshensu,protocatechuic aldehyde,hydroxysafflower yellow A,salvianolic acid B)through vena caudalis.The analysis of pretreatment plasma samples on reversed-phase column of Agilent EXTend-C18(4.6 mm x 150 mm,5 μm)and eluted with methanol and water containing 0.3%phosphate acid as mobile phase in gradient mode.The internal standard substance of rutin and column temperature is 30 ℃,the flow rate was 1 mL·min-1,Detection wavelengths were 280 nm for danshensu,protocatechuic aldehyde,salvianolic acid B and 403 nm for hydroxysafflower yellow A.3.Study on pharmacokinetics of Danhong Injection in hyperlipidemia rats:According to the orthogonal design L9(34)composed of the main effective constituents of Danhong Injection(danshensu,protocatechuic aldehyde,hydroxy safflower yellow A,salvianolic acid B),the 9 different dosage ratios combinations were formed,which were used for intravenous administration in hyperlipidemia rats to study the pharmacokinetics of effective constituents after compatibility.Using non-compartmental model of DAS 3.2.6 software to fit the pharmacokinetic parameters and using total quantum statistical moment analysis method,intuitive analysis and variance analysis to evaluate the total pharmacokinetic characteristics.4.Study on pharmacodynamics and PK—PD of Danhong injection in hyperlipidemia rats:Study on pharmacokinetics and pharmacodynamics of synchronization,and determination the pharmacodynamic index of effective components of Danhong injection on anti lipid,anti thrombosis(HMGR,PF4).The pharmacodynamic index was HMGR and PF4 as the research foundation analysis the relationship of plasma concentration and drug effect intensity.Using DAS 3.2.6 software to fit the PK-PD modeling and get the quantitative equations between Danhong Injection active components(danshensu,protocatechuic aldehyde,hydroxysafflower yellow A,salvianolic acid B)and efficacy.Result 1.Danhong injection can regulation the blood lipid levels and successful establishment of hyperlipidemia rat models:By high fat feed for 6 weeks to establish the hyperlipidemia rat model,compared with the normal group,the serum lipid levels of TC,TG,HDL-C and LDL-C were significant differences(P<0.05),and the rats in good conditions.Pharmacodynamic study showed that the Danhong Injection has a certain degree of regulation and reduce the abnormal blood lipid levels in hyperlipidemia rats,which can reduce the contents of TC,TG and LDL-C levels and increase the content of HDL-C in vivo.2.Successfully to established the qualitative and quantitative analysis HPLC-DAD method for Danhong Injection in hyperlipidemia rats:The linear ranges of danshensu,protocatechuic aldehyde,hydroxysafflower yellow A and salvianolic acid B were 0.16～40.00 mg·mL-1(R2=0.9988),0.16～10.00 mg·mL-1(R2=0.9996),0.31～40.00 mg·mL-1(R2=0.9992),0.31～40.00 mg·mL-1(R2=0.9973)respectively.The mean recoveries were between 80%and 110%,the RSD of intra-day and inter-day were less than 10%.The results of stability meet the requirements for biopharmaceutical analysis.3.Study on pharmacokinetics of Danhong injection after orthogonal test:there were different effects of the effective constituents with 9 groups orthogonal compatibility on the pharmacokinetic characteristics and the total quantum statistical moment parameters.Danshensu had the strongest effect on the total AUCt.A2B3C1D2 was the preferred dose combination for AUCt.Danshensu had the strongest effect on the total MRT and its optimal ingredient composition was A1B1C3D2.4.Study on pharmacodynamics and synchronous pharmacokinetic model of Danhong injection:The effective constituents of Danhong Injection can improved the blood lipid levels in hyperlipidemia rats,and the time delay phenomena between the drug plasma concentrations.The best models for pharmacodynamics were Sigmoid Emax models,and the fitting results are in good correlation and all P values were less than 0.05.Conclusion 1.The model of hyperlipidemia by high fat diet was successful,simple and controllable and can provide a reliable basis for the experimental study of the pharmacokinetics and pharmacodynamics.Danhong Injection can regulate and reduced the blood lipid levels it has a certain therapeutic effects on blood lipids(TC,TG,LDL-C,HDL-C).2.The HPLC-DAD method was stable,precise,rapid,and simple,which can be used to the pharmacokinetics studies on the compatibilities of danshensu,protocatechuic aldehyde,hydroxysafflower yellow A and salvianolic acid B in hyperlipidemia rats.3.Analysis of the pharmacokinetic parameters of orthogonal injection of Danhong injection(Danshensu,protocatechuic aldehyde,hydroxysafflor yellow A,salvianolic acid B)with hyperlipidemia rats,AUCt and MRTt were used to analyze the interaction between the components,and there were some differences between the 9 groups.Chinese medicinal composition analytical methods can be used to analyze the relevant research methods.4.Pharmacodynamics and PK-PD were studied by HMGR and PF4,and depth analysis the plasma concentration-drug effect-time between internal connection,and the study of PK-PD binding model has a certain effect on the lag of drug effect.Conjoint analysis for study on the whole effect of compound medicine and the mutual effect analysis and prediction has reference values.5.The pharmacokinetics and pharmacodynamics studies of Danhong Injection on hyperlipidemia rats and the PK-PD modeling,it can provide reference for the study of the correlation between the Chinese herbal medicine and compound prescription,and can be used to predict and evaluate the correlation between multi-components and effects of traditional Chinese medicine.