Establishment Of A Fishing Method And Application In Screening Target Bioactive Compounds In San-ao Decoction

Methodologies focusing on pharmacodynamical material basis are key issues for the modernization of traditional Chinese medicine.Usingβ₂-adrenoceptor（β₂-AR）as a probe,this thesis established a fast and high efficiency method for fishing of bioactive compounds that specifically bind to the magnetic nanoparticles coating withβ₂-AR（β₂-AR MPs）.The method combines the specificity of a receptor recognizing its ligand and the convenience for separating magnetic nanoparticles.Using this method,we screened and identified the bioactive compounds curing diseases like asthma and cough in San-ao decoction（SAD）.The established method plays pivotal role in separating and identifying bioactive compounds from TCMs.This whole thesis includes 4 chapters and the main contributions are as follows:1.Preparation ofβ₂-AR MPs.We immobilized his-taggedβ₂-AR on the surface of Fe₃O₄magnetic nanoparticles by a random linking method using N,N’-carbonyldiimidazole.The immobilizedβ₂-AR was characterized by dynamic light scattering,flourier transform infrared spectrometer,superconducting quantum interference device and zonal elution.The particles displayed a relatively uniform distribution with an average size of 278.3 nm and a saturation magnetization value of 14.97 emu/g,which meeting the need of magnetic based separation.The particles can be used for recognizing its specific ligands from complex matrixes and provide possibility in establishing affinity magnetic fishing based method.2.Thermodynamic study between the immobilizedβ₂-AR and its specific ligands.Usingβ₂-AR coated MPs as a tool,we investigated the interaction of the receptor and four drugs.The affinity order of the four drugs binding to the receptor was terbutaline>chlorpromazine>salbutamol>methoxyphenamine.The association constants decreased when the temperature increased from 4℃to 45℃.All the drugs had one common binding site on immobilizedβ₂-AR.Isotherm data showed that all the drugs best fit to Langmuir model and electronic force was believed to be the main driving force for the drugs binding toβ₂-AR.The result indicated that the magnetic particles had the potency in studying the interactions between the immobilized receptor and the drugs.3.Targeted bioactive compounds screening from SAD.The bioactive compounds from SAD for curing cough and asthma were fished out by immobilizedβ₂-AR MPs.Separation and identification of the compounds were performed by high performance liquid chromatography–electrospray ionization–mass spectrometry（HPLC-ESI-MS）.Molecular docking was used for further verifying the binding interaction between the bioactive compounds and the receptor.The result indicated that ephedrine,amygdalin,liquiritin and liquiritin apioside were the bioactive compounds specifically binding to the receptor.Ser 169,Ser 170 and Phe 256 of the receptor were considered as the main binding sites for the compounds.This study provides a fast and efficient alternative for screening bioactive compounds from TCMs.