【作者】 何梅；

【导师】 夏之宁；

【作者基本信息】 重庆大学 ， 分析化学， 2002， 硕士

【摘要】 中药现代化迫切需要现代科学技术手段来充分说明中药药效作用的物质基础、作用机理、中药药性理论和配伍等丰富的科学内涵。但目前国内中药复方的研究缺乏能解决中药复杂性和整体性的研究思路及方法，还停留在化学成分的定量表达阶段。在分离提纯、定性、定量中药及中药复方之外研究中药体系中多组分药物的相互作用将是达到中药现代化的更为重要的途径之一。本课题进一步发展了毛细管电泳相互作用（CEIA）的分析方法。从分子一对一的相互作用研究发展为多配体与受体结合的相互作用研究，建立了中药的复杂多组分与生物分子相互作用的毛细管电泳分析方法。通过单独比较实验和其它测量方法验证了建立的这种方法的正确性。该方法不仅可对具有相似分子结构的物质与同一受体作用的结合常数进行区分，还可判断各药物的结合活性，由于该方法可成功地从复杂体系中同时测得多种组分与受体间的相互作用的结合常数，从而可能成为中药活性成分筛选的有效方法。研究的主要内容和成果如下：1. 依据毛细管电泳相互作用的峰漂移法结合Scatchard方程分析，建立了复杂体系CEIA的模型。对中药大黄有效成分与β-环糊精及羟丙基β-环糊精的相互作用进行了研究，并通过单组分体系进行验证，取得了较一致的结果。这几种药物与β-环糊精的结合活性依次为大黄素＞芦荟大黄素＞大黄酚。认为是与β-环糊精的外部疏水性结合起了决定作用。这几种药物成分与羟丙基β-环糊精的结合活性依次为大黄素＞大黄酚＞芦荟大黄素＞大黄素甲醚。它们与羟丙基β-环糊精的作用是内腔包含占了主导地位，药物分子所含的疏水基团有辅助影响。羟丙基β-环糊精对大黄类药物的包结能力要强于β-环糊精。2. 在建立了复杂体系CEIA模型的基础上，将其应用于生命分子与中药多组分体系的相互作用研究，建立了具有一类结合位点的受配体结合相互作用分析方法。同时获得了大黄有效成分与牛血清白蛋白相互作用的结合常数，对具有相似分子结构的物质与同一受体作用的结合常数可进行区分，并通过紫外光谱法对结果进行了验证，结果较一致。大黄类药物与牛血清白蛋白的结合活性依次为：大黄素>芦荟大黄素>大黄酚，结合能力随其分子亲水性的增强而增强。为完善复杂体系CEIA方法，建立了具有两类结合位点的受配体结合相互作用分析方法。研究了中药乌头有效成分与人血清白蛋白的相互作用。乌头中有效成分乌头碱、次乌头碱和美沙乌头碱与人血清白蛋白结合存在两类非等价结合位点，高亲和力结合位点在结合中起决定作用。实验测得这三种药物的结合活性<WP=5>3. 依次为美沙乌头碱>乌头碱>次乌头碱，结合能力随其分子疏水性的增强而增强。4. 将建立的复杂体系CEIA方法应用于中药的活性成分筛选，研究了中药成方制剂三黄片与人血清白蛋白的相互作用，对中药筛选作了初步探索。提出了基于分子相互作用原理的复杂体系CEIA方法执行中药活性成分筛选的模型和思路。5. 在CEIA过程中，建立了一种有利于排除电渗流标记物对样品测定干扰的样品和标记物分区带进样的技术。此进样方法的进样精度RSD=6.83%，与常规进样方法的RSD=4.72%相比较，没有明显的差异。更多还原

【Abstract】 The modernization of Traditional Chinese Medicine (TCM) calls for modern scientific methods to research the pharmacology and mechanism of functions of TCM in molecular level. It is expected to screen and discover new active components from TCM. But the present research level in our country is mainly in the quantitative analysis of chemicals separated from TCM. The research of TCM need newly advanced thoughts and methods to understand the complexity and compatibility of TCM as well. One of the most important resolutions is to research the interaction between multicomponents from TCM and biomolecules, such as receptors.Capillary electrophoresis interaction analysis (CEIA) method was developed from the interaction analysis of one molecule and another to the interaction analysis of multiple ligands and one receptor. A complex system CEIA method was established, which was demonstrated to be correct by other experiments such as single component CEIA, spectroscopy and literatures. It is proved to discriminate different binding ability of drugs with similar molecular structure to receptor effectively, and it demonstrates that this method is highly desirable for the effectively screening of active components of TCM using a single-capillary CE system.The main RESEARCH and some conclusions are as below:1. Based on the peak shifting method using in CEIA and Scatchard analysis, a complex system CEIA method was established to research the interaction of effective components from TCM Rhubarb to β-cyclodextrin (β-CD) and hydroxypropyl-β-cyclodextrin (HP-β-CD). The results were validated by comparing binding constants of multicomponents to one receptor with each that of single component to the receptor. The two sets of experiment results agreed well. The binding abilities of these drugs toβ-CD rank as follow: Emodin> Aloe-emodin> Chrysophanol, and the ability lies in the external hydrophobicity ofβ-CD; The binding abilities of these drugs to HP-β-CD rank as follow: Emodin> Chrysophanol >Aloe-emodin> Emodin-monomethyl ether, and the ability mainly lies in the inner inclusion of HP-β-CD, a little partially the hydrophobicity of drugs. The inclusion abilities of HP-β-CD to effective components from Rhubarb are stronger than that of β-CD.The interaction between components from a TCM complex system and biomolecules was researched by complex system CEIA method. The analysis method of<WP=7>2. binding with homogeneous and univalent site was established to obtain simultaneously the binding constants of effective components from Rhubarb to Bovine Serum Albumin (BSA). The results agreed well with those obtained from ultraviolet spectroscopy interaction analysis. The binding ability of drugs with similar molecular structure to receptor was discriminated by this method. The binding abilities of effective components from Rhubarb to BSA rank as follow: Emodin> Aloe-emodin> Chrysophanol and this kind of binding ability is related to the hydrophilicity of drugs. 3. The analysis method of ligand-receptor binding with heterogeneous sites was established to consummate the complex system CEIA method. The interaction between effective components from Aconitum carmichaeli Debx. and Human Serum Albumin (HSA) was researched by this method. There are two populations of binding sites in the binding of Aconitine, Benzoylaconitine and Mesaconitine to HSA, and the binding ability lies in the high-affinity binding site. The binding abilities of these drugs to HSA rank as follow: Mesaconitine> Aconitine> Benzoylaconitine, and this ability is related to the hydrophobicity of drugs.4. The complex system CEIA method was applied to screen active components of TCM effectively. A primary research of TCM screening was made by studying the interaction between components from Traditional Chinese Patent Medicine "Sanhuang Pian" and HSA. The model and thought of screening active components from TCM performed by complex system CEIA method were proposed. 5. The separated zones injection technique was established in this paper to eliminate the更多还原