Establishment Of Angiotensin Ⅱ Type Ⅰ Receptor Chromatography And Its Application In Bioactive Compounds Screening From Traditional Chinese Medicine

Cardiovascular disease is the biggest killer that endangers human health worldwide.It has the characteristics of high morbidity,high mortality,high recurrence rate,and low age.It is urgent to establish new methods and develop new technologies to ensure efficient and innovative drugs become available and to achieve effective prevention and treatment of cardiovascular diseases.Traditional Chinese medicine and its compound are the main sources for the research and development of efficient and innovative drugs.The rapid screening of their active components with high specificity is the key and bottleneck restricting drug research and development.Receptor chromatography based on immobilized proteins has potential applications and prospects in the screening of active components of traditional Chinese medicine.Preparation a highly active receptor chromatographic stationary phase is the key step for the method.In the thesis,we aim at the efficient screening of active ingredients of traditional Chinese medicine and focus on the problem of the disordered protein conformation of current functional protein immobilization methods.Inspired by the conformational stabilizing characteristics of the aptamer to the activated receptor,we took angiotensinⅡtypeⅠreceptor（AT₁R）,the main target of cardiovascular diseases,as an example,and established a highly active AT₁R chromatographic model through dual-site immobilization.The model was applied to screen the antihypertensive active components from classic compatibility of cardio-cerebrovascular diseases,Danshen-sanqi.This thesis is divided into four chapters.The main contributions of the author are as follows:1. We established a new method for the preparation of highly active AT₁R chromatographic stationary phase through dual-site immobilization.Taking AT₁R as an example,the random library of DNA was screened by SELEX technology,and the aptamer with high specificity and strong affinity to AT₁R was obtained.The aptamer and angiotensin II active peptide（AT₂（3-8））were immobilized on the surface of macroporous silica gel.Using the dual affinity of AT₁R with aptamer and AT₂（3-8）,AT₁R was captured directly from the cell lysate to prepare a dual-site immobilized receptor chromatographic stationary phase.The activity of the immobilized receptor was characterized by the specific drugs of AT₁R.The results show that the dual-site immobilization method can achieve the directional immobilization of AT₁R with high activity and high specificity,which provides a methodological reference for the rapid preparation of high activity receptor chromatographic model.2. Using valsartan,irbesartan,candesartan,azilsartan and olmesartan as probes,the interactions between the five drugs and immobilized AT₁R were studied by injection amount dependent method and nonlinear chromatography.The results showed that all the five drugs had two kinds of binding sites on the immobilized AT₁R.The affinities of the five drugs to AT₁R follow the order of azilsartan>irbesartan>valsartan>candesartan>olmesartan.The order of the dissociation rate constants of the drugs on the column is valsartan>azilsartan>irbesartan>candesartan>olmesartan.The results are consistent with the literature reported data,such as radioligand immunoassay.It is proved that the dual-site immobilized AT₁R chromatographic model can be applied to the study of drug-receptor interaction analysis and provide a new chromatographic method for screening specific active components of AT₁R.3. The active components of Danshen-sanqi were screened by the dual-site immobilized AT₁R chromatographic model and analyzed and identified by mass spectrometry.The results show that Notoginsenoside R1,ginsenoside Rg1,and rosmarinic acid are the active components targeting the AT₁R in Salvia miltiorrhiza-Panax Notoginseng pair.The study is of great significance to clarify the bioactive compounds of Danshen-sanqi herb pair for promoting blood circulation and removing blood stasis.It is proved that the established dual-site immobilized AT₁R chromatographic model can be used for the screening of active components of traditional Chinese medicine,which provides a methodological reference for the screening of active components in complex systems.