| Cornus officinalis Sieb. et Zucc is a member of the Coranceac family, use the fruits of mature and dry as medicines, which is recorded in the "Shen Nong's Herbal Classic" for the first time. C officinalis is mainly distributed in China, such as He Nan, Zhe Jiang, Shaan Xi, Si Chuan and other provinces. As a classic Chinese herb, C. officinalis has been studied for several decades. Modern pharmaceutical studies indicated that C. officinalis not only can regulate immunities and reduce blood sugar, but also has some therapeutic effects such as anti-shock and anti-aging.Human serum albumin is a kind of protein in the people plasma. It can bond many endogenous and exogenous material, store and transport fatty acid, bile pigment, amino acids, steroid hormone, metal ions and many drug molecules, maintain blood normal osmotic pressure. Protein is used as a model to research drug interactions in the body, which has important significance in understanding the transshipment and metabolism of drug in the body.Spectrophotometry and fluorescence spectrometry which are based on monitoring the change of physicochemical properties of the drug-protein system, have been extensively used for the direct determination of drug-protein binding. The online dialysis sampling coupled with high performance liquid chromatography (DS-HPLC) system was established in this article. It had been used in the research on interactions of iridoid glycosides water-soluble extracted from C. officinalis (CIG) with HSA. It provides certain method on the study of interaction between traditional Chinese medicine and biological macromolecules, also can supply a certain theoretical information and reference for the development and application of C. officinalis.There are three parts in this thesis:Chapter one:Review.Firstly, it summarized the chemical constituents, the progress of pharmacological and prospects study about C. officinalis. And introducted the protein structure, the serum albumin structure and optical properties. It reviewed the advantages and disadvantages of the methods which research the interaction between small molecule drug and serum albumin. It included binding constant, the number of binding site, types of interaction force and thermodynamic parameters. It described the denfinition and characteristics of microdialysis. The establishment of on-line dialysis coupled with HPLC method, and application in the research of interaction between a variety of components in Chinese herb and biomacromolecule were regarded. Chapter two:The establishment of on-line dialysis sampling coupled with High Performance Liquid Chromatography system and study on dialysis characteristics of water-soluble components in C. officinalis.Firstly, the research of qualitative analysis using HPLC-ESI-MS technique in the Cornus officinalis Sieb. et Zucc. It identified the three water-soluble components:Loganin, Sweroside and Cornuside in C. officinalis, and discussed the influences:perfusion flow rate and micro-dialysis membrane length. The optimized condition as follows:perfusion flow rate is 2.0μL/min, micro-dialysis membrane length is 9cm. At last, three iridoid glycosides were quantitatively analyzed with the calibration curves obtained with standard solutions in DS-HPLC system, recoveries of dialysis sampling ranged from 73.9 to 91.7% with the RSD below 3.0%. The results showed that the DS-HPLC method has good accuracy and reliability, which can be used in the research of active ingredients of traditional Chinese medicine with serum albumin interaction.Chapter three:Study the binding characteristics on water-soluble components and human serum albumin with C. officinalis by DS-HPLC systemIn this thesis, using DS-HPLC system analysis of some parameters with CIG extracted three water-soluble components and human serum albumin. First, we investigated the interaction of C. officinalis with different concentrations human serum albumin. Then the effection of binding degree in different pH were tested. The results showed that the binding degree of loganin was 69.3%,74.5%,47.6% respective at pH 7.4,6.0,8.9 in the 0.6 mM HSA solution. But the binding degree of sweroside and cornuside was decrease at pH 6.0 and 8.9. Finally, we obtained binding constants and the number of binding site by Scatchard equation. Then calculated thermodynamic parameters and got the types of interaction force. It showed that the interaction force between loganin, sweroside and HSA was hydrophobic interaction, but the interaction force between cornuside and HSA was electrostatic force. |
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