【作者】 胡芳弟；

【导师】 王春明； 封士兰；

【作者基本信息】 兰州大学 ， 分析化学， 2011， 博士

【摘要】 中药分析新方法的开发、中药质量标准研究以及中药体内药代动力学研究是当前中药研究领域的难点和热点。服用中药单-活性成分或中药整体用药,产生的药效可能会明显不同。中药色谱指纹图谱分析技术,从复杂中药体系的整体性和特征性出发,弱化西药研究中以化学成分分析为主的研究思维,强调中医整体用药的指导原则,对保证中药功效,提高中药工业整体水平和现代化,具有非常重要的现实意义。近年来,电化学分析方法以其不破坏样品的原有特质、灵敏度相对较高、可检测浓度范围较宽等特点,已成为中药中药活性成分分析研究新方法的又一研究热点。本论文从中药制剂质量控制、中药分析新方法学研究以及中药药代动力学研究需要出发,运用色谱技术和电化学技术,从以下几方面进行了创新性研究：(1)建立并验证了一种简单、灵敏的测定小鼠血浆及组织中木香烃内酯和去氢木香内酯的高效液相色谱方法(HPLC);研究了单次灌胃木香乙醇提取物后,木香烃内酯和去氢木香内酯在小鼠体内的药代动力学特征。(2)建立并验证了一种简单、迅速、高灵敏性和选择性的测定大鼠血浆及组织中木香烃内酯和去氢木香内酯的HPLC-MS/MS分析方法；研究并全面比较了不同给药途径下,这两种成分单一使用、协同用药及木香提取物整体用药的药动学差异,通过中药药代动力学研究结论,对其作用机理有了更好的认识,并对中药作为整体用药合理性有更深层次的理解,为该药的研究开发和临床合理应用提供科学的依据。(3)为了进一步揭示木香烃内酯和去氢木香内酯在人体内的药代动力学过程,采用平衡透析法,在体外模拟药物与人血浆蛋白结合的过程,并以高效液相色谱法进行测定。结果木香烃内酯和去氢木香内酯单独透析血浆蛋白结合率分别59.78%和65.69%,将两者混合后同时透析,血浆蛋白结合率分别为51.78%和45.83%,t检验分析结果表明同一药物,单独使用与协同用药血浆蛋白结合率有显著性差异,说明二者同时用药时,会相互影响它们的体内过程。(4)建立了一种基于多波长色谱指纹图谱相似度评价结合制剂和原料药材之间谱峰匹配质量评价模式,并应于小柴胡颗粒制剂工艺的评价。以制剂和柴胡药材的谱峰匹配结果对各制剂进行分级聚类分析,四种来源不同的小柴胡颗粒制剂被成功分为四类。进一步制剂工艺探讨研究发现基于水煎煮的小柴胡颗粒制剂工艺易于使柴胡药材中的两种重要活性成分柴胡皂苷A和D发生结构转化,推测现行小柴胡颗粒制剂工艺可能会影响到其治疗效果。该结果表明多波长指纹图谱-谱峰匹配技术不仅可以用于中药制剂的质量控制,而且可对中药制剂工艺提供有益的指导。(5)以壳聚糖作为石墨烯分散介质,在玻碳电极表面构建了石墨烯／壳聚糖复合膜，制得了壳聚糖／石墨烯修饰的玻碳电极；并探讨了芦丁在该修饰电极上的电化学行为。结果表明,于pH 4.0的PBS溶液中,循环伏安法显示芦丁分别在0.408、0.482V处有-对峰型优良的氧化还原峰；以差示脉冲伏安法(dpv)作为分析手段,使用氧化峰作为芦丁定量分析的电化学信号,与差示脉冲伏安法峰电流(ip)有良好的线性关系,其回归方程符合Y=9.9219X-0.0025,相关系数r=0.9958。本方法用于实际样品的分析,结果令人满意。更多还原

【Abstract】 Researches on analytical method, quality standard, and Pharmacokinetics are hot fields but also challenges in the study of traditional Chinese medicine(TCM).Administration of single active component or extraction of TCM, leads to change of effects. The chromatographic fingerprint analytical technique, emphasized on the overall guiding principle of TCM, inferior the thought about action of single chemical composition in western medicine, is of significance to the improvement in industrial level, modernization and globalization of TCM. Up to now, electrochemical analysis provided new idea and methods in analysis of TCM due to its advantages of high sensitivity, wide range of test concentration, less amount of sample needed and nondestructive for inhere characteristic of analytical sample.Based on the new trends in study of pharmacokinetics, quality control and new analysis methods focusing on TCM, this researches made use of chromatographic technique and elerochemical technique to carry out the several groundbreaking works as follow.1 A simple and sensitive analysis method by HPLC was established and validated to measure costunolide and dehydrocostus lactone in mouse plasma and tissue; The pharmacokinetic of costunolide and lactone dehydrogenase in mice was investigated after administration of single oral ethanol extraction of Muxiang (EEM).2 A simple, rapid, high selective and sensitive method by UPLC-ESI-MS/MS was established and validated to assay costunolide and dehydrocostus lactone in rat plasma and tissue; We studied the pharmacokinetic in rats that were treated with dehydrogenase lactone, mixed of costunolide and dehydrogenase lactone, and EEM respectively, using different ways of administration, and completely compared their pharmacokinetic parameters, which further proved that the efficacy of TCM is played by the overall ingredient rather than a single active component or partitively, and provided evidence for development and rational clinical application of this medicine.3 To further reveal the phatmacokinetic parameters of costunolide and dehydrogenase lactone in human, the binding process of drug and plasma protein was mimic using equilibrium dialysis and measured by HPLC. The plasma protein binding rate was 59.78% of costunolide and 65.69% of dehydrogenase lactone, treated with each component separately. However, the plasma protein binding rate was 51.78% of costunolide and 45.83% of dehydrogenase lactone when treated with the combination of them. There is a significant difference by t test assay. The result demonstrated treated with the combination of them will affect their own absorption, distribution, metabolism, exeretion(ADME).4 A quality evaluation system of multi-wavelength fingerprint similarity and peak-matching between preparation and its raw herbs was established and further valued preparation technology of Xiaochaihu granula. Xiaochahu granula from different companies was classified to four categories with cluster analysis utilizing the results of peak-matching. Exploitation of preparation technology revealed that boiled preparation technology in water tends to alter the structure of Saikosaponin A and D in radix bupleuri chinense. We purposed that the current preparation technology of Xiaochahu granula would have influence on the outcome of Clinical Therapy. Therefore, this system of multi-wavelength fingerprint similarity and peak-matching could be applied to quality control and optimization of TCM preparation technology.5 Chitosan/Graphene/GCE was fabricated using Chitosan solution which involved in Graphene. And electrochemical behavior of rutin on this modified electrochemical electrode was studied. Rutin effectively accumulated on the chitosan/graphene/GCE and caused a pair of redox peaks at around 408 mV and 482 mV in 0.1 M phosphate buffer solution (pH 4.0). And the anodic peak current was linear to the rutin concentration under optimized conditions. The regression equation was:Y=9.9219X-0.0025 (r=0.9918). Furthermore, this new method was successfully applied to the determination of rutin content in tablet samples with satisfactory results.更多还原