Screening Of Bioactive Compounds In Zhisou Oral Liquid By Immobilized β₂-Adrenergic Receptor Chromatography With High Accuracy

Zhisou oral liquid（ZOL）is a famous traditional Chinese medicine（TCM）produced in Shaanxi.It has made great contribution to the prevention and treatment of respiratory diseases.The medicine with complicated pharmaceutical process is composed of Radix Stemonae,Radix Platycodonis,Pericarpium Citri Reticulatae and four other herbs.Therefore,screening of the bioactive compounds has become the bottleneck for the product upgrade.Based on the holistic concept of TCM,an efficient screening method for the bioactive compounds in ZOL contributes to clarifying the material basis of its efficacy and investigating the pharmacological mechanism.It is of great significance to promote its further clinical application.Our previous work has proved that the immobilized receptor is an alternative for screening the bioactive compounds of TCM,whereas efficiency and accuracy of the method need to be improved.Herein,we investigated the effects of halide linkers on peak profile and drug-protein interaction,and developed an immobilizedβ₂-adrenergic receptor（β₂-AR）chromatography with high accuracy to screen the potential bioactive compounds in ZOL.The main contents are as follows:1.Establishment of the chromatographic method based on immobilizedβ₂-AR.In this work,the microporous silica gel was activated with 6-chlorhexanoic acid（S-1）and its four derivatives（S-2～S-5）.The receptor was rapidly captured from E.coli cell lysates and fixed to microspheres surface through the covalent bond formed between the haloalkane dehalogenase（Halo-tag）and the halide linkers on the support surface.By the immobilizedβ₂-AR,we investigated their effects on the peak profiles of five drugs and drug-protein interaction analysis.Based on the retention time changes,half-width changes for the specific drugs on theβ₂-AR columns with these linkers,we considered S-4 was the optimized halide linker to improve the accuracy of the method.To verify the superiority of the linker S-4,we used nonlinear chromatography to study the interactions between the five drugs andβ₂-AR on the columns prepared by linkers S-1 and S-4.The dissociation rate constants of salbutamol,terbutaline,methoxyphenamine,tulobuterol and bambuterol obtained on the optimized column were 60.3±0.3,76.1±1.7,34.2±1.1,37.3±0.3 and 15.6±0.2 s^-1.The method accuracy was improved by 31%,62%,61%,70%,and 60%,respectively.The strategy provided a method for screening the targeted compounds in ZOL.2.Screening of targeted compounds in ZOL.Taking ZOL and seven herbs as the research objects,we rapidly screened and identified the potential bioactive compounds by the receptor column coupled with mass spectrometry.The results indicated that the compounds in ZOL targetingβ₂-AR were glycyrrhizic acid,platycodin D,tuberostemonine and hesperidin from Radix Glycyrrhizae,Radix Platycodonis,Radix Stemonae and Pericarpium Citri Reticulatae,respectively.The study has laid the foundation for defining the bioactive compounds of ZOL.3.Assessment of potential bioactive compounds in ZOL.The antitussive effect of the four compounds was evaluated by the classical mice cough model induced by ammonia liquor.The binding mechanism of the four compounds was discussed by injection amount-dependent method and molecular docking.The results indicated that the combinational utilization of the four compounds exerted significant antitussive activity.The binding of the four compounds occurred at one type of sites on the receptor and their binding equilibrium constants were（2.59±0.12）×10⁵ for glycyrrhizic acid,（0.97±0.09）×10⁵ for platycodin D,（0.35±0.25）×10⁴for tuberostemonine and（0.21±0.13）×10⁴M^-1for hesperidin.Van der Waals’force,hydrogen bonds and static electricity were the main driving forces for the four drugs binding with the receptor.The method provided a basis for clarifying pharmacological mechanism of ZOL.