Establishment Of Endothelin A Receptor Chromatography And Screening Of Bioactive Compounds In Siwu Decoction

It is inefficient for the conventional chromatographic method to screen active compounds from tradional Chinese medicine(TCM).This study established an endothelin A receptor(ETAR)chromatographic model to analyze the interaction of drugs with ETAR and screened the active compounds in Siwu decoction,providing a methodological reference for the rapid screening of other active compounds of TCM.The dissertation is divided into four chapters and the main contributions are as follows:1.Establishment of ETAR chromatography method.The epidermal growth factor receptor(EGFR)was recombined with the ETAR,and recombinant EGFR-ETAR was expressed in E.coli.The ETAR was immobilized by covalent interaction between EGFR and substrate ibrutinib which was modified on surface of silica gel.The characterizations of surface elements composition and morphology were performed by X-ray photoelectron spectroscopy,X-ray energy dispersive spectroscopy and scanning electron microscopy,and the activity and stability of the immobilized receptors were investigated by four ligands,namely macitentan,bosentan,ambrisentan and sulfisoxazole.The results showed that the immobilized ETAR chromatography method had the good stability and ability to recognize and separate its ligands,which provides a methodological reference for the preparation of other acceptor chromatography stationary phases.2.The research method of drug and receptor interaction was established on the basis of ETAR chromatography.Using macitentan,bosentan,ambrisentan and sulfisoxazole as tools,the interaction of the drug with ETAR was investigated by the ETAR chromatography combined with AED model.The traditional frontier analysis and direct injection method were compared with AED model.The results showed that the adsorption type of the four drugs with ETAR was in accordance with the double Langmuir model,which belongs to two sites adsorption.The order of affinity of the four drugs was bosentan> macitentan > ambrisentan > sulfisoxazole.The results indicated that ETAR chromatography combined with AED model analysis could give a methodological reference for drug interaction studies.3.The active ingredients of Siwu decoction and their interaction with ETAR were cleared on the basis of ETAR chromatography.The active ingredients of Siwu decoction were seperated using ETAR chromatography and identified by HPLC-MS.The affinity parameters of active compounds with ETAR were analyzed by AED model.The molecular docking was employed to confirm the affinity of the active compounds preliminaryly.The results showed that paeoniflorin and ferulic acid were the active compounds targeting ETAR in Siwu decoction.Paeoniflorin had one kind of binding sites on immobilized ETAR and ferulic acid had two kinds of binding sites on immobilized ETAR,where the affinity was similar to antagonist.This will lay a methodological foundation for the screening of other targeted ingredients in Siwu decoction.