A New Method Of Capillary Electrophoresis To Study The Interaction Between Natural Products And Carriers

Natural products are very critical resources for new drug research and development.More than half of clinical drugs are directly or indirectly derived from natural products.However,most natural products have problems such as poor water solubility,low bioavailability,and unknown mechanism of action,so there are many limitations in clinical applications.The solubility and bioavailability of natural products can be improved by interacting with natural or synthetic carriers.Capillary electrophoresis（CE）,as an efficient separation and analysis technology,can not only realize the effective separation and quantitative analysis of biomolecules and their complexes in solution,but also allow biomolecules to maintain their natural conformations and truly reflect the interactions between molecules.Therefore,in order to improve the solubility and bioavailability of natural product molecules,CE technique was used to construct a simple and efficient analytical method to analyze the interaction between natural products and carriers.Based on the above ideas,the research content of this topic is as follows:1.We used affinity capillary electrophoresis（ACE）to study the interaction between the drug carrierβ-cyclodextrin（β-CD）and jatrorrhizine hydrochloride.First,optimize the conditions were optimized,with 20 mmol/L Tris-HCl（p H 7.4）as the background buffer,use a neutral coated capillary to reduce adsorption,and use pressure assisted（0.8 psi）to achieve rapid ACE separation analysis,and get the binding constant was（1.20×10³ L/mol,R²=0.991）.In order to explore the influence of pressure assistance on the accuracy of ACE results,the ACE method was further calibrated.The results showed that the analysis speed was fast and the results were accurate when the auxiliary pressure was not too high（<1.2psi）,which was verified by fluorescence titration experiments.Then,based on CE technology,the Taylor Dispersive Analysis（TDA）was constructed to study the interaction betweenβ-CD and folic acid.The optimal conditions were as follows:20 mmol/L Tris-HCl buffer solution（p H 7.4）,capillary length of 60 cm,and separation pressure of 3.0 psi.The binding constant（9.73×10² L/mol）and the binding ratio（1.44）ofβ-CD and folic acid were determined by the TDA method,which were basically consistent with the literature reports.At the same time,the hydrodynamic radius and diffusion coefficient before and after the combination of folic acid were also obtained.2.We established TDA method based on CE technique was used to analyze the aggregation of carrier proteinβ-lactoglobulin A（BLG-A）.The experimental conditions were optimized by using a buffer solution of 30 mmol/L disodium hydrogen phosphate-potassium dihydrogen phosphate,and the capillary length was 60 cm,and the separation pressure was2.0 psi.The influence of solution p H（5.0-9.0）on BLG-A aggregation was discussed by TDA.When the p H of the solution is 9.0,BLG-A was dominated by monomers,its diffusion coefficient is 4.23×10^-11 m²/s,and the hydrodynamic radius is 5.74 nm.When the p H is reduced from 9.0 to 5.0,the BLG-A monomer aggregates,and the hydrodynamic radius of BLG-A before and after aggregation confirms that a dimer is formed.The results of electrospray mass spectrometry（ESI-MS）and circular dichroism（CD）confirmed that the p H 5.0-6.0 was conducived to the formation of BLG-A dimers,which was consistent with the results of TDA.At the same time,the CD results found that the aggregation effect will change the secondary structure of BLG-A,increasing the relative content of spirals and random coils,while the relative content of antiparallel chains decreases.3.The interaction between BLG-A and four fatty acid ligands were studied by ESI-MS method,and the order of binding strength was obtained as follows:stearic acid>palmitic acid>myristic acid>lauric acid,indicating that the longer the carbon chain,the stronger affinity,and they all bind at a ratio of 1:1.Further use the CD method was used to explore the influence of the combination of different fatty acids on the secondary structure of BLG-A.The results showed that the effect of stearic acid（C18）was the most obvious,causing the relative content of helix in the BLG-A structure to increase from 17.56%to 21.20%.Then,a new TDA method was constructed based on the CE-MS technique.The separation voltage and auxiliary pressure at the CE end,the electrospray voltage and the ion transfer tube temperature at the MS end,and the solution conditions were optimized.Among them,the sheath fluid composition（methanol/water:50/50,v/v）and flow rate（1.0μL/min）of the interface play a key role in the stability and reliability of the MS signal.The interaction between BLG-A and stearic acid was studied by TDA method,and the ion peak height change trend of BLG-A was obtained by the mass resolution of MS.The binding constant K of the two was determined to be 1.38×10⁵ L/mol,and the binding ratio was n It is 1.45（R²=0.966）,which were basically consistent with literature reports.At the same time,the hydrodynamic radius and diffusion coefficient of BLG-A are obtained.