Establishment And Evaluation Of Chromatographic Method For Screening Multi-target Bioactive Compounds From Gegenqilian Decoction

As the most important sort of drug discovery,multi-target drugs have the advantage of synergistic effect.However,the screening of bioactive compounds remains open due to the complexity of traditional Chinese medicine and the lack of high throughput methodologies.GegenQinlian decoction?GQD?,one of the well-known traditional Chinese formulas,originally came from“Treatise on Febrile Diseases”compiled by Zhong-Jing Zhang.And this prescription,mainly composed of Radix Puerariae lobatae,Radix Scutellariae,Rhizoma Coptidis and Radix Glycyrrhizae.Pueraria is the main therapeutic drug and the other three drugs are supplementary property,having the effectiveness of relieving exterior syndrome and clearing away interior heat.Taking the screening of multi-target bioactive compounds in GegenQinlian decoction as target and focusing on endothelin A receptor?ETA?and angiotensin II-1 receptor?AT1?,we carried out a systematic study about the establishment and evaluation`of immobilized chromatography in this dissertation.The multi-target chromatographic method has important sense to the development of multi-target drug.The dissertation was divided into three chapters,among which the author contributed importantly in following research contents:1.Establishment of screening method for bioactive compounds in GegenQinlian decoction.We obtained two receptors of His-ETA and His-AT1 by E.coli.expression system.This work synthesized a chromatographic stationary phase through immobilizing carbonyl diimidazole to the surface of amino microspheres.Specific ligands of the two receptors(e.g.ambrisentan,sulfisoxazole,valsartan and candesartan were utilized to characterize the specificity and bioactivity of the column.Based on this study,validated application of the stationary phase was performed by screening multi-target compounds of GegenQinlian decoction using high performance affinity chromatography coupled with ESI-Q-TOF-MS.We identified an active compound with a retention time of 17.2 min as puerarin,providing a methodological reference for the screening of multi-target bioactive compounds from traditional Chinese medicine.2.Evaluation of screening method for bioactive compounds in GegenQinlian decoction.By zonal elution,we identified puerarin as a bioactive compound simultaneously binding to ETA and AT1.By injection amount dependent method,the association constants of puerarin were determined to be 2.5×10⁵ M^-1 and 1.8×10⁵ M^-1 on the ETA and AT1,respectively.The compound exhibited the binding sites of 1.0×10^-8 M?2.0×10^-8 M.Molecular docking indicated that the binding of puerarin to the ETA occurred in the form of hydrogen bonds and conjugated bonds.Hydrogen bonds binding amino acid residues are Tyr129,Cys239,Asp418,conjugated bonds binding amino acid residues are Asn137,Pro162,Gln165,and His415.The combination between puerarin and AT1 is related on Van der Waals force,the binding amino acid residues are Gly22,Arg23,Leu81,Thr88,Arg167,Val179,Cys180,Asp281.Taking together,we concluded that the column containing co-immobilized receptors has potential for screening bioactive compounds with multi-targets from complex matrices including traditional Chinese medicines.In addition,it has important sense to study the interaction between single ligand and multiple proteins.