Screening Bioactive Compounds From Xuanbai Chengqi Decoction Based On One-step Immobilization Strategy For Receptor Chromatography

Traditional Chinese medicine（TCM）is one of the main forms of medication for preventing and treating diseases.It is also the main source of high-efficiency innovative drug research and development in modern times.However,how to find the bioactive ingredients from TCM is the main bottleneck for drug discovery.It is urgent to develop new principles and establish new methods and techniques to rapidly analyse the bioactive ingredients from TCM.Previous studies have established a series of screening techniques toward bioactive ingredients of TCM,such as enzyme and cell models.However,these screening methods are limited in specificity and accuracy.Therefore,the development of rapid screening method is still one of the hot topics.In our previous work,by combining the specificity of drug-receptor recognition and the high separation capacity of chromatographic technology,we immobilized G protein-coupled receptors（GPCRs）on chromatographic supports,established the receptor chromatographic method and proved its capability for rapid screening of bioactive ingredients from TCM.However,the method requires a complicated process of separation and purification of receptors,thus,resulting in the loss of receptor bioactivity and the screening accuracy.Taking β₂-adrenergic receptor（β₂-AR）as an example,we developed a new one-step method to prepare immobilized receptor stationary phase and screened bioactive ingredients of Xuanbai Chengqi Decoction.We hope our method could provide a reference for the high-efficient screening from TCM.The thesis is divided into 4 chapters and the main contributions are as follows:1.Inspired by the specific covalent reaction between halogenated alkane dehalogenase（Halo-tag）and its substrates,taking β₂-AR as a probe,we developed a new one-step method for receptor immobilization.The Halo-tag was fused to the carbon-terminal of the receptors and the receptor was expressed in the recombinant E.coli.We modified 6-chlorohexanoic acid on the surface of the macroporous silica gel.The receptor immobilization was realized by the specific reaction of the Halo-tag and the 6-chlorohexanoic acid though immersing the modified gel into the cell lysate of E.coli.We characterized the elemental composition,morphology,ligand recognition activity and lipid microenvironment of the immobilized receptor by X-ray photoelectron spectroscopy,scanning electron microscopy,laser confocal microscopy and liquid chromatography-mass spectrometry,respectively.Our method avoids the purification of the receptor and covalently immobilizes the receptor on the gel surface by the formation of a homogeneous single-layer protein coating.The immobilized receptor has the characteristics of uniform orientation,high stability and strong activity.Our method provides a reference for the rapid preparation of high-activity receptor chromatographic stationary phase.2.Using the β₂-AR chromatographic stationary phase,we explored the interactions of β₂-AR with three drugs such as salbutamol,by three chromatographic methods and molecular docking.As a result,the three ligands showed only one type of binding site on the immobilized β₂-AR,and their affinities followed the order of carazolol > isoproterenol >salbutamol,which is consistent with the radioligand binding assay.Molecular docking results showed hydrogen bond was a main driving force for these drugs binding to β₂-AR.Thus,theβ₂-AR chromatographic model can be applied for the ligand-receptor interaction analysis,providing a new method for screening of bioactive ingredients that target β₂-AR.3.We used the β₂-AR chromatographic model to screen the bioactive ingredients of Xuanbai Chengqi Decoction,and identified the structure of the retained components by the liquid chromatography-mass spectrometry.We also investigated the dynamic distribution of bioactive ingredients in the lung and intestinal tissues of rats.As a result,we found amygdalin,catechin,emodin,emodin glucoside,and chrysophanol glucoside could specifically bind toβ₂-AR in lung and intestinal tissues.After oral administration of the decoction to rats for 3.0 h,we could still detect the emodin and emodin glucoside in lung and intestinal tissues.The data suggested the anti-asthma effect of Xuanbai Chengqi Decoction that through the lung and intestines may be related to the targeted ingredients of β₂-AR,including emodin and emodin glucoside.It provides a certain basis to clarify the bioactive ingredients that could treat the anti-asthma through the lung and intestines in Xuanbai Chengqi Decoction.Our work provides a methodological reference for the researches on the material basis and therapeutic principles of other TCMs.