Study On The Interaction Between Active Components From Traditional Chinese Medicine And Serum Albumin And On Its Influences On The Binding Of Pharmaceuticals

Research on drug-protein binding is indispensable content in the study of early stage clinical pharmacokinetics of any new drugs.On one hand, it has profound effects on the pharmacological activity and the pharmacokinetics of the drug.So a thorough study on the molecular basis of drug-protein binding is of great significance in theory and practice. On the other hand,it helps to understand the basic issues of life processes, such as the binding between protein subunits,the binding between antibody and antigen,the binding between protease and inhibitor,the binding between protein and nucleic acid,and so forth.This paper discusses the binding of active components from TCM such as GA etc, which is the main component of the most commonly used traditional Chinese medicine GUF,with serum albumin.Pilot studies on its influences on the binding of pharmaceuticals(bioactive components)are also included.The thesis is mainly consisted of the following parts.1.The interaction between GA and serum albumin was studied in detail by mean of spectroscopic methods(fluorescence,CD and UV-vis spectra) and CE-FA(chapter 2).The binding constant,binding sites,binding sites number,thermodynamic parameters and the distance between GA and residue of Trp were calculated.The effects of metal ions on the binding were investigated.The binding of GA below or above the isoelectric point (pI)was compared and the results showed different binding mode.Siteâ… was one of the binding sites of GA through sites marker method.Effects of GA on the conformation of BSA were studied by synchro-fluorescence, UV-visual and CD spectra and results suggested addition of GA could enhance the hydrophobicity of the surrounding of Trp residue and decrease the percentage ofα-helix to some extent.Results showed there were two kinds of binding sites in BSA for GA,one high affinity sites(K =8.28×10⁵ L.mo1^-1)and one low affinity sites.The total binding sites numbers were 8.Percentage of binding was up to 95.2%when the ratio of protein to GA was 0.375.All these mean that high affinity,high capacity and high binding percentage characterized the binding between GA and BSA which might affect the interaction between protein and other drugs(bioactive components)and thus influenced their pharmacological and toxicological action.2.The interaction between FU/HZ and serum albumin and the displacement interaction between FU/HZ and GA were studied in detail by mean of spectroscopic methods and utracentrifugation-CE(chapter 3). The binding constant,binding sites,binding sites number,thermodynamic parameters were calculated.The effects of metal ions,GA,oleic acid and other diuretic on the binding were investigated.The binding of FU was preliminary assigned to non-specific one by mean of studying the effects of sites probe,hydrophobic probe(ANS)and the ion strength.Effects of FU/HZ on the conformation of BSA were studied by synchro-fluorescence, UV-visual and CD spectra and results suggested addition of FU didn't change the hydrophobicity of the surrounding of Trp residue in low concentration but could decrease it in high concentration.The percentage ofα-helix decrease to some extent when FU was added. Besides,several data processing methods presently used were compared.3.The interaction between 2UC/2DC and serum albumin were studied in detail by mean of spectroscopic methods(chapter 4).Data were processed by Stern-Volmer analysis and the binding force was preliminarily analyzed.The effects of metal ions,GA on the binding were investigated.Sites probe and hydrophobic probe(ANS)were applied to investigate the binding sites.Results showed the binding of 2UC/DC with serum albumin were saturable and depended on acidity,temperature and the length of alkyl chain.The binding was non-specific and the binding force was mainly hydrophobic one and adjusted by electrostatic force. Co-existence of zinc ion etc can induce biggish conformation change of BSA.4.The interaction between NF and serum albumin was studied in detail (chapter 5).The binding constant,binding sites,binding sites number, thermodynamic parameters and the distance between NF and residue of Trp were calculated.Effects of NF on the conformation of BSA were studied by synchro-fluorescence,UV-visual and CD spectra and results suggested addition of NF could enhance the hydrophobicity of the surrounding of Trp residue and inecrease the percentage ofα-helix to some extent.The effects of GA on the binding of bioactive components from traditional Chinese medicine with BSA were evaluated,such as quercetin,hyperin,rutin,quercitrin,kaempferol,rhein and emodin.5.Interaction characteristic of environmental toxic chemical Sudanâ… and BSA was preliminarily studied(chapter 6).The binding constant, binding sites number,thermodynamic parameters and the distance between Sudanâ… and residue of Trp were calculated.6.Preliminary study on the binding of FU and HSA was performed by docking method.Models were constructed for Quantitative Structure / Property(binding constant)Relationship by multivariate linear regression (chapter 7).7.A simple,rapid,sensitive and selective methods for the determination of lisinopril and hydroxyzine hydrochloride(chapter 8)in human plasma were developed,respectively.The methods were validated in the sensitivity,accuracy and reproducibility.12/18 healthy volunteers were selected for the bioequivalence study of different formulations of lisinopril and hydroxyzine hydrochloride,respectively.The significant differences of dynamics parameters between test formulations and control formulation were not observed by statistics analysis.The test drugs are bioequivalent to the control drug.The methods are of great importance for the study of in vivo drug-protein binding.8.Chemometric methods were used to analyze the components and their change in GUF,DGS,EKL,GUF-DGS and GUF-EKL, respectively.