Screening Of Targeted Components In Rhubarb Processed Products By Paper-based Affinity Chromatography With Immobilized β₂-AR

Recognition the targeted active components of traditional Chinese medicine（TCM）is key issues for the research of pharmacodynamical material in TCM.The establishment of high efficient recognition method is the core step to address this issue which need to develop new methods and technologies to realize this purpose.Using beta2-adrenoceptor（β₂-AR）as a probe,we established aβ₂-AR paper-based chromatographic platform via immobilizing the receptor on the PTFE membrane with the natural porous fibrous structure.This platform was applied to screen the active components in rhubarb.The content differences of targeted components in rhubarb processed products were analyzed to reveal the essence of processing of rhubarb and TCM.It also provides reference for rapid screening of active components in TCM.The main contents of the thesis are as follows:1.Preparation and characterization of theβ₂-AR-in-paper.The haloalkane dehalogenase（Halo）taggedβ₂-AR was expressed in genetically engineered strains and immobilized on PTFE membrane via the catalytic dehalogenation reaction of haloalkane dehalogen kinase and 6-chlorhexanoic acid.FTIR,XPS,and fluorescence analysis was used to characterize the morphology of the immobilizedβ₂-AR.Chromatographic method were used to characterize the ligand specific recognition activity of the receptor.These results showed thatβ₂-AR was immobilized on PTFE membrane as expected and had the good stability and ability to recognize its ligands.We believe such a platform sheds new light on paper chromatography for studying receptor-drug interaction and screening active components in TCM.2.Theβ₂-AR paper-based affinity chromatography platform was applied to investigate the interaction betweenβ₂-AR and drugs by frontal analysis（FA）and zonal elution（ZE）.The results of frontal analysis showed that the adsorption of the three ligands withβ₂-AR was in accordance with Langmuir model,which belongs to single site adsorption.The order of association equilibrium constant of the three ligands was salbutamol>bambuterol>terbutaline.The results of zonal elution showed that the association equilibrium constant of salbutamol ranged from 1.11 to 1.54×10⁵L/mol.There is no significant difference with the results from FA（1.51×10⁵L/mol）.The affinities of the five ligands toβ₂-AR follow the order of salbutamol>terbutaline>methoxyphenamine>isoproterenol>ephedrine,which are consistent with the literature reported data.These results indicated that theβ₂-AR paper-based affinity chromatographic platform can be used for the study of receptor-drug interactions,and provide a new method for the evaluation of activity component in TCM.3.The active components in rhubarb extract was recognized byβ₂-AR paper-based material and identified by HPLC-MS.The results showed thatβ₂-AR targeting active components in rhubarb that causes diarrhea were rhein,emodin,chrysophanol,aloe-emodin and physcione.The order of purgative intensity was rhubarb>rhubarb stir-fried with wine>cooked rhubarb taking emodin-8-glucoside and chrysophanol-8-glucoside as the index.The decrease of their content during the processing was the main factor for the reduced purgative activity.This work provides a reference for revealing the pharmacodynamical material change of purging constitution in rhubarb during processing.It also provides a methodological reference for the research of material basis of TCM in processing.