| ObjectiveDanshen Yin is a classical treatment for cardiovascular disease in clinic.Previous studies have found that Danshen Yin extract(DSYE)can ameliorate cell damage caused by myocardial ischemia through affecting PI3K-Akt signaling pathway.Based on pharmacokinetic studies,this study was aimed to screen the effective components in rat's plasma followed by the absorption of DSYE,establishing a stable and controllable quantitative analysis method,in order to investigate their pharmacokinetic characteristics and gastrointestinal stability.Based on this,the pharmacodynamic studies of DSYE was to be observed in myocardial ischemia rats after single and multiple doses,to further elucidate the effective substance of DSYE in treating myocardial ischemia and provide theoretical basis in guiding rational use of drugs.Methods 1.Components identification of DSYE after oral intake in ratsBased on the research of HPLC fingerprints and relevant literature research in the early stage,the chemical components of DSYE were screened.Using of Triple quadrupole mass spectrometry analysis,the blood-intake component were selected as the pharmacokinetic component of the DSYE by comparing its main effective components,drug-containing plasma,and DSYE methanol solution.2.Simultaneous quantification of tanshinone IIA,cryptotanshinone,salvianolic acid B and danshensu in rat plasma by LC/MSA method was establishing for determination of tanshinone IIA,cryptotanshinone,salvianolic acid B and danshensu in plasma by LC/MS.The separation of Tanshinone IIA and cryptotanshinone was carried out on a C18 column using a mobile phase consisting of methanol and 0.1% formic acid,the dectection was performed by positive ion multiple reaction detection mode and APCI ion source.The separation of Salvianolic acid B and Danshensu was carried out on a C18 column using a mobile phase consisting of acetonitrile and 0.1% formic acid,the dectection was performed by negative ion multiple reaction detection mode and ESI ion source.3.Pharmacokinetics study of tanshinone IIA,cryptotanshinone,salvianolic acid B and danshensu in normal and myocardial ischemia model rats after single and multiple of DSYEThe experimental animals were divided into single-dose normal group,multiple-dose normal group,single-dose model group and multiple-dose model group,in which the eyelid blood samples were drawn after 0.083,0.25,0.5,0.75,1,1.5,2,3,4,6,8 and 12 hour.The contents of tanshinone IIA,cryptotanshinone,salvianolic acid B,and danshensu were measured by LC/MS method.PK solver 2.0 software was used to calculate pharmacokinetic parameters and SPSS 20.0 software was used to compare the main pharmacokinetic parameters in normal group and myocardial ischemia model group by One-way ANOVA method.4.Pharmacodynamic study on DSYE after single and multiple-doseThe experimental animals were divided into blank group,model group,single-dose model group and multiple-dose model group,in which the eyelid blood samples were drawn after 0,0.25,0.5,1,2,4,6,8,12 and 24 hour.SOD activity and MDA contents were determined,and efficacy index over time was analyzed.5.Stability of Effective Components in DSYE in Gastrointestinal Bionic EnvironmentHPLC method,established in the early stage,was used to determine the stability of tanshinone IIA,cryptotanshinone,danshensu,and salvianolic acid B in the gastrointestinal biomimetic environment.Results 1.Component identification of DSYE after oral administration in ratsBy analyzing mass spectrometry result of the standard products,drug-contained plasma and DSYE methanol solution,we found that tanshinone IIA,cryptotanshinone,salvianolic acid B and danshensu would entrance the plasma after oral administration DSYE.Therefore,these four types of effective components were used as pharmacokinetic research indicators.2.Simultaneous quantification of tanshinone IIA,cryptotanshinone,salvianolic acid B and danshensu in rat plasma by LC/MSA determination method was successfully established to measure the content of tanshinone IIA,cryptotanshinone,salvianolic acid B and danshensu in rat plasma.The result showed that its specificialization,linearity,accuracy,precision,and stability were all satisfactory.The sample was analyzed within 13 minutes with simple procedures,which conformed to the analyse requirements in biological samples.3.Pharmacokinetics study of tanshinone IIA,cryptotanshinone,salvianolic acid B and danshensu in normal and myocardial ischemia model rats after single and multiple of DSYEThe results showed that either single or multiple dose,the pharmacokinetic behaviors of the four components in the model group is different with normal group.In the single-dose normal group the drug absorption was poor,the bioavailability of tanshinone IIA,cryptotanshinone,salvianolic acid B,and danshensu was poor.Multiple administrations increased the amount of active ingredients in the body.In the model state of myocardial ischemia,the absorption of the drug was good,the bioavailability of the four active ingredients was increased,the residence time in the body was prolonged,and multiple-dose modle grupe showed multi-peak absorption.It was suggested that in the state of myocardial ischemia,the concentration of the active ingredient in plasma increased rapidly,the time to reach the therapeutic concentration can be shortened,and the accumulation of drug can occur so as to maintain a longer period of efficacy.4.Pharmacodynamic study of DSYE after single and multiple-doseThe results of the study showed that compared with the model group,the SOD activity values in the serum were increased and the MDA contents were decreased either in single-dose group or multiple-dose group.In the state of myocardial ischemia,there is a certain delay between the efficacy and the plasma concentration after a single administration.Multiple doses can quickly reach higher efficacy index.It was showed that multiple-dose had good efficacy,Which would improve the absorption of DSYE in myocardial ischemia rat and bring into active role in therapeutical effect.5.Stability of effective components in DSYE in gastrointestinal bionic environmentThe experimental results showed that the content of tanshinone IIA,cryptotanshinone and danshensu did not change significantly in the environment of bionic gastric juice,but salvianolic acid B remained 83.61% after 6 hours.While in the environment of bionic intestinal fluid,the contents of salvianolic acid B and danshensu in intestinal fluid did not change significantly,but tanshinone IIA and cryptotanshinone remained 53.99% and 58.37% respectively after 12 hours.It showed that the water-soluble salvianolic acid B and danshensu were more stable in the gastrointestinal environment,while tanshinone IIA and cryptotanshinone were easily decomposed and transformed in the gastrointestinal environment.ConlusionIn this paper,we found that tanshinone IIA,cryptotanshinone,salvianolic acid B and danshensu would absorb into the plasma after intake by DSYE.Base on this,LC/MS was established to determine the contents of four active ingredients in plasma of biological samples in a stable,sensitive,and simple way,which provides technical means for the pharmacokinetics study of DSYE.The results showed that the pharmacokinetic behaviors of the four components in modle grupe rats were different with normal group rats.The absorption effect of the drug is much better in ischemia,and multiple dosing increases the accumulation of active ingredients in the body,which conforms to the philosophy of syndrome differentiation in Chinese medicine.Pharmacokinetics and pharmacodynamics studies have shown that DSYE has anti-ischemic effects and maintain a high efficacy index within 6 hours.There is a certain delay between the efficacy and the plasma concentration after a single administration.Multiple doses can quickly reach higher efficacy index.This study can provide pharmacokinetic basis for the development of dosage form and effective evaluation of Danshen Yin. |
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