Study On Identification Of Pharmacokinetic Markers For Guanxin Danshen Formula

At present,the safety and rationality problems of traditional Chinese medicine(TCM)clinical application receive much concerns.Studying pharmacokinetics,in order to understand the process of drug in the body,which is great significance for guiding the clinical safety and rational drug use.However,the compositions of TCM are complicated,many effective ingredients,and how to identify and select the pharmacokinetic markers to basic represent TCM whole process in the body is one of the challenges faced by the current study.Guanxin Danshen formula is composed of Salvia miltiorrhiza Bge.(Danshen),Panax notoginseng(Burk.)F.H.Chen(Sanqi),Dalbergia odorifera T.Chen(Jiangxiang),which is TCM prescription in the treatment of diseases such as coronary heart disease angina pectoris,and has wide application in clinical and obvious curative effect.But so far there are no reports concerning pharmacokinetic of Guanxin Danshen formula.This article use Guanxin Danshen formula as a carrier,to explore how to select research strategies for pharmacokinetic markers of TCM compound.Namely,proposing on the basis of clearing main ingredients of Guanxin Danshen formula,further to carry out the study of the components in vitro antioxidant activity screening(based on chemiluminescence reaction)and in vivo against myocardial ischemia effect validation,so that to find out the main efficacy composition;then,to carry out the pharmacokinetic study on the multicomponent of Guanxin Danshen formula,to clear the body process characteristics;on this basis,put forward to consider the "status" of various ingredients in this formula,explore these typical components into the blood have representative features in many aspects such as in structure,efficacy and pharmacokinetic properties as the pharmacokinetic markers,to characterize of the process in the body.In addition,we carried out the integrated PK studies.Through all above research,in order to reveal the process in vivo of Guanxin Danshen formula,at the same time,provide new ideas and reference for selecting pharmacokinetic markers of TCM compound.The main research contents and results are as follows:1.Firstly,by using HPLC-DAD to study and establish method for quantitative analysis of eight phenolic acids,five saponins and four tanshinones of Guanxin Danshen formula,and analysis these 17 constituents of Guanxin Danshen formula pharmaceutics.The results show that Guanxin Danshen dropping pills preparation mainly contains ginsenoside Rd(15.14 mg/g),Rg1(6.12 mg/g),Rb1(4.04 mg/g),tanshinone IIA(3.21 mg/g),notoginsenoside R1(1.22 mg/g)and salvianolic acid B(1.045 mg/g).This study reveals the basic compositions of Guanxin Danshen formula,which laid a foundation for further research in this paper.2.By using flow injection chemiluminescence analysis technology,the research established the screening platform for in vitro antioxidant activity based on DPPH·-luminol chemiluminescence method,and applied to evaluate the antioxidant activity of the main ingredients of Guanxin Danshen formula.According to the results,in the formula,ingredients of Danshen including dihydrogen tanshinone I,cryptotanshinone,tanshinone I and tanshinone IIA has certain ability of radical scavenging;main saponins from panax notoginseng ingredients like R1,Rg1,Rb1 and Rd has weaker ability to remove free radicals.It suggested that tanshinones have certain antioxidant effect,and saponin compounds with weaker antioxidant,so it is necessary to further use in vivo validation of these compounds against myocardial ischemia and provide reference for further research.3.By establishing model of using isopropyl adrenaline induced myocardial ischemia in mice,to study the therapeutic effects of anti-myocardial ischemia by the main active ingredient of notoginsenoside R1 and ginsenoside Rg1,Rb1,Rd,dihydrogen tanshinone I,cryptotanshinone,tanshinone I and tanshinone IIA.The results show that ginsenoside Rg1(40 mg/kg,i.v.),Rb1(40 mg/kg,i.v.)and tanshinone IIA(40mg/kg,i.v.)can effectively improve the symptoms of myocardial ischemia,the role of the other five components is not obvious.4.By using high performance liquid chromatography-mass spectrometry analysis technology,established determination method for notoginsenoside R1,ginsenoside Rg1,Rb1,Rd,dihydrogen tanshinone I,cryptotanshinone,tanshinone I and tanshinone IIA in rat plasma,and applied the way to study the pharmacokinetics of these compounds in vivo after rats intragastric administration Guanxin Danshen dropping pills.According to the results,protopanaxatriol R1,Rg1 and cryptotanshinone eliminate faster(t1/2 β <2h),protopanoxadiol Rb1,Rd,tanshinone I and tanshinone IIA with more slower eliminate(t1/2 β >2.6h);dihydrogen tanshinone I and cryptotanshinone with the lowest system exposed(AUC0-∞<30ng·h/m L).Comprehensive consideration the amount,representative structure,in vivo and in vitro activity and pharmacokinetic properties of components in Guanxin Danshen formula,eventually select ginsenoside Rg,Rb1 and tanshinone IIA as pharmacokinetic markers,to characterize pharmacokinetic behavior of mice in vivo.In addition,we carried out the integrated PK studies by using the identified PK markers according to drug concentration sum method and AUC weighting method,in order to provide references for multiple ingredients of Traditional Chinese Medicine integrated pharmacokinetic research.