| Objective: Natural products are abundant in resources and have diverse component structures,making them important targets for new drug research and development.However,the complexity of their components also brings great challenges to the research of effector substances.The establishment of rapid and efficient analytical methods for screening and evaluation targeted efficacy substances in natural products is a current research hotspot.Affinity chromatography is a key technology for high-throughput screening of active substances in complex systems.With the deepening of research on drug targets,new affinity models for screening natural drugs are constantly being developed.In this study,based on the principle of affinity chromatography and the rapid analysis of electrochemical analysis technology,a novel lipid raft microchromatography model was established using organic polymeric monolith column materials as solid phase support.Lipid raft microchromatography was applied to identify and screen active compounds targeting Tyrosine receptor kinase A(TrkA)by affinity extraction.The molecular docking technology was used to further explore the molecular mechanism of the interaction between small molecule ligands and TrkA,in order to provide a convenient and efficient screening strategy for the discovery of active ingredients in natural products.Methods: On the basis of the previous work of our research group,we first prepared a lipid raft silica gel packed column chromatography model and investigated its specificity.The active monomer of licorice was identified and screened.Based on the activity screening results,TrkA was used as the macromolecular receptor protein,and the active small molecule of licorice was used as the ligand for molecular docking to further explore their interaction.Based on the shortcomings of traditional biological affinity chromatography model such as time-consuming in the screening process and large consumption of biological affinity ligands,this paper attempted to construct a lipid raft chromatographic microcolumn using capillary monolith column as the solid phase carrier.We used glycidyl methacrylate(GMA)as the functional monomer,ethylene glycol dimethacrylate(EDMA)as the cross-linking agent,n-propanol /1,4-butanediol/water as the ternary pore-forming agent system,azo-diisobutyrile as the initiator.The composition and proportion of the prepolymer solution were optimized by single factor experiment,and the poly(GMA-co-EDMA)solid phase carrier material with good shape and suitable pore size was prepared by in situ polymerization.Lipid raft @poly(GMA-co-EDMA)monolith microcolumn was prepared by epoxy covalent crosslinking method,and the immobilization effect of lipid raft was observed by western blot and scanning electron microscopy.Based on the successfully constructed lipid raft monolith microcolumn,the active components targeting TrkA were separated and enriched using solid-phase extraction.The retention behavior was investigated using electrochemical analysis techniques to evaluate the selectivity of the lipid raft monolith microcolumn model,and the affinity extraction and elution conditions were optimized.Results: The active monomers of licorice,including glycyrrhetinic acid,glycyrrhizic acid,liquiritin,and isoliquiritigenin,exhibited significant retention behavior on lipid raft silica gel filled columns.Molecular docking experiments further showed that the molecular configurations of these four licorice active monomers could fit well with the active pocket of TrkA target protein,and form hydrogen bonds with the active site of multiple amino acid residues of TrkA.Capillary monolith microcolumn were prepared by in-situ polymerization method,and the effects of polymer composition and ratio on the performance of the monolith column were investigated.The optimal preparation process conditions were determined as follows:the mass ratio of monomer mixture to pore forming agent was 30:70,the initiator AIBN was 1% of the monomer mass,and the immobilized carrier matrix for rapid separation and analysis was prepared by water bath at 60 ℃ for 12 hours.Infrared spectroscopy,scanning electron microscopy,BET pore size analysis,and thermogravimetric analysis were used to characterize the mechanical stability and uniform morphology of the monolith column.Scanning electron microscopy showed that lipid rafts were coated on the surface of the monolith column.Infrared spectroscopy showed that the monolith microcolumn of lipid raft @poly(GMA-co-EDMA)had obvious characteristic peak changes.The results of Western blot showed that the monolith microcolumn of lipid raft could still be stably immobilized on the monolith column material after washing with multiple column volume buffer salt,which had good stability.The performance evaluation and preliminary application of lipid raft @poly(GMA-co-EDMA)monolith microcolumn were carried out by solid phase extraction combined with electrochemical analysis.The results showed that the TrkA inhibitor gefitinib was well retained on the lipid raft monolith column,while the non-TrkA target drugs gemcitabine and 5-fluorouracil were not significantly retained on the lipid raft monolith microcolumn,which verified the effectiveness of the screening of TrkA targets on the lipid raft monolith microcolumn.On this basis,lipid raft monolith microcolumn was applied to screen the active monomer components of glycyrrhizin,and the electrochemical content determination method of active components of glycyrrhizin was established by differential pulse voltammetry.It was confirmed that glycyrrhetinic acid,glycyrrhizic acid,liquiritin,and isoliquiritigenin had significant adsorption and retention behavior on the lipid raft monolith microcolumn.Conclusion: In this study,various aspects such as lipid raft chromatography recognition screening,molecular docking simulation,and capillary monolith columns were studied.A novel organic monolith column material was used as a solid-phase carrier to successfully prepare lipid raft monolith microcolumn,which were used as micro affinity solid-phase extraction columns and integrated with electrochemical detection technology to screen the active components of TrkA target proteins.A convenient,effective and miniaturized affinity chromatography screening method was established.It provides a reference for the construction of other new biological affinity chromatography models and the basic research of natural drugs. |
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