【作者】 刘军；

【导师】 郎爱东；

【作者基本信息】 山东大学 ， 药物分析学， 2007， 硕士

【摘要】 柿叶为柿树科植物柿树（Diospyros kaki L. f.）的新鲜或干燥叶片，具有较多药理活性，已经有数百年的药用历史，具有广泛的民间基础。柿叶的质量评价目前仍然依靠传统的生药学鉴别等方法，如根据药材外形或粉末特征等来评价药材内在质量，经验依赖性强、准确度较低，并未有采用现代仪器分析技术（如HPLC）对柿叶药材质量进行评价和控制的报道，现有的质量评价方法科技含量较低，难以保证药材及其制剂的质量可控、安全有效，与现代医药发展的要求不相适应。在柿叶药材及其制剂应用越来越广泛的今天，建立和完善柿叶药材的质量控制方法及评价体系，是使柿叶能够与其他中药一样进入世界药品与食品市场的一件重要的任务。中药色谱指纹图谱技术是目前国际上普遍认可的中草药及其制剂质量评价和控制的先进技术。建立柿叶药材色谱指纹图谱，用具有代表性的药效物质群体特征来综合评价柿叶药材内在质量，符合中医药理论和中药化学成分多而杂的特点，能够全面的反映柿叶药材质量的本来面貌。本研究首次对柿叶药材及其浸膏进行了指纹图谱的研究，结合系统聚类和相似度分析对柿叶药材进行了评价，并比较了药材与提取物之间的相关性；对柿叶中的药效组分黄酮类化合物总含量进行了HPLC测定，考察了药材中黄酮类成分的含量随季节和产地的变化；采用HPLC／DAD／MS技术对柿叶中黄酮类成分进行初步定性分析，推断了其中主要化合物可能的结构。主要方法及结果如下：1．高效液相色谱法（HPLC）具有灵敏度和分离效能高的优点，采用优化的液相色谱条件，能够将样品中的成分进行分离，并分别进行含量分析。柿叶中黄酮类成分主要是槲皮素和山奈酚及其糖苷。对柿叶药材中黄酮类成分进行HPLC研究并未见报道。我们首次以甲醇-0.1％磷酸溶液（55：45）为流动相，在360nm波长处进行检测，考察了不同产地柿叶药材中黄酮类成分含量的差别，以及黄酮类成分含量随季节的变化情况。实验结果表明，不同产地、品种柿叶中槲皮素和山奈酚的含量明显不同。总黄酮的含量范围在10.08～34.68mg／g，也有显著差别。其中潍坊水果柿、安徽毫州牛心柿和济南地区的柿叶黄酮含量较高，临沂次郎、山西牛心柿和青岛柿叶药材黄酮含量则相对较低。在不同月份山东济南、山东临沂和山西夏县的柿叶中，黄酮含量均以8～10月份较高，6月、7月和11月份相对偏低，故建议在8～10月份对柿叶进行采收，以保证柿叶中有较高的黄酮成分（有效成分）。2．本研究首次采用RP-HPLC法对收集的柿叶药材中的黄酮类成分指纹图谱的获取方法进行研究，采用优化的色谱条件，建立了柿叶药材黄酮类成分的高效液相色谱指纹图谱。采用聚类分析法对所收集药材进行分类。同时，我们按照柿叶制剂（脑心清片，收录于《中国药品质量标准》）质量标准中浸膏的制备方法制备并测定了浸膏的指纹图谱，比较了不同产地药材提取浸膏之间的差异，探讨了药材与浸膏之间的相关性。通过柿叶药材的指纹图谱研究发现，不同产地和品种的柿叶中黄酮类成分之间既有相关性，又有区别：共有峰的峰面积之和占总峰面积的90％以上，即其中所含化学成分比较相似；但是由于受产地、品种及采收期等因素的影响，各共有色谱峰的相对峰面积相差很大（RSD在11.14～102.95％之间），即黄酮类成分的相对含量有明显差异。12批药材样本相似度在0.78～0.99之间，其中相同产地药材的相似度均大于0.90，不同产地药材的相似度则相对较低，提示在柿叶药材的采集利用上，要注意产地对药材质量的影响。与直观比较HPLC图谱不同，用相似度结果来评价药材之间的差异，能够更确切客观的评价药材之间差异的程度。通过对柿叶浸膏指纹图谱的研究发现，12批药材的浸膏之间的相似度要更加接近，在0.87～0.99之间，没有小于0.80。这说明，与原药材比较，浸膏之间化学成分的差异小于对应的药材样本化学成分之间的差异。所以，在控制药材的同时，采用指纹图谱衡量浸膏质量的一致性将是一个控制柿叶制剂有效成分一致性和稳定性的有效途径。3．为了使本研究建立的柿叶药材色谱指纹图谱能够与柿叶化学成分相联系，在今后的实际应用中中发挥更大作用，本实验首次采用HPLC／DAD／MS联用的方法，对柿叶指纹图谱进行HPLC／MS质谱检测，在参考国内外相关文献和在线UV光谱信息的基础上，根据特征离子碎片，经过综合比较，初步推测了7个共有特征峰可能的结构类型，分别为：4号峰为金丝桃苷(槲皮素-3-葡萄呋喃糖苷，C₂₁H₂₀O₁₂)、5号峰为异槲皮素(槲皮素-3-半乳糖苷，C₂₁H₂₀O₁₂)、7号峰为甲氧基-槲皮素(M=332.2，C₁₉H₂₄O₅)己糖苷、8号和10号峰为山奈酚-3-葡萄吡喃糖苷和山奈酚-3-半乳糖苷、11号峰为槲皮素、12号峰为山奈酚。由于8号和10号峰为同分异构体，仅依靠质谱未能确定其具体化合物结构。更多还原

【Abstract】 Persimmon leaf is the fresh or dried leaves of Diospyros kaki L.f. （Ebenaceae）. It has many pharmacological activities and is widely used as medical materials for several hundred years. Persimmon leaf is employed in the treatment of hypertension, heart disease, the internal haemorrhage induced by gastric ulcer, the functional bleeding of ulterus and so on. The chemical constituents are very complex including flavonoids, triterpenoids, coumarins, phenols, etc.The quality control and evaluation of persimmon leaf is mainly by orthodox pharmacognostic methods which have lower technical contents. It’s an important task to establish a system for quality control and evaluation of persimmon leaf as the utilization is more and more population and can make it enter the world’s market as medical material today.A review on the development of research about chemical constituents and pharmacological activities of the leaves of Diospyros kaki L. f. was presented in this paper. It can give us a detailed understanding about persimmon leaf as a medical material.Our research for quality control and evaluation of the leaves of Diospyros kaki L. f. mainly include three parts.Firstly, we determined the contents of flavonoids in persimmon leaves collected by high performance liquid chromatography. Flavonoids are important components and have many pharmacodynamic actions. Early research on its total contents is mainly by UV Spectrophotometry which has low degree of accuracy. Flavonoids in persimmon leaves mainly are glycoside of quercetin and kaempferol which can hydrolyze in acidic waters. So we applied acidic waters and methanol as extracting solvent and heated at 85℃ to extracting flavonoids. HPLC separation was performed at room temperature （25℃） and a flow rate of 0.8ml/min. The mobile phase is met（?）anol and 0.1% H₃PO₄ water（55 : 45, v/v）. The wavelength is 360nm. We investigated 26 samples collected from Shandong province and other habitat and compared their differences influenced by habitats and collection time.Secondly, we studied the chromatographic fingerprints of persimmon leaves and its extract. We also compared the differences between medical materials and its extracts.Chromatographic fingerprint technique is a useful method in the identification of herbal samples or their products, and hence has been widely used for studying traditional Chinese medicines （TCM）. It is an advanced technology for quality control and evaluation of Chinese herbal medicine and is generally approved internationally.Traditionally, only a few of markers or pharmacologically active constituents were employed to assess the quality and authenticity of the complex TCM. However, as we know, the therapeutic effects of TCM are based on the complex interaction of numerous ingredients in combination, which are totally different from that of chemical drugs. Thus, many kinds of chemical fingerprint analysis methods to control the quality of TCM have gradually come into being, such as TLC, GC, HPLC, and so on. Among these methods, high-performance liquid chromatographic fingerprint has played more and more important role and was therefore introduced by the WHO as a strategy for identification and quality control of TCM.Unfortunately, although many chemical and pharmacological studies on the leaves of Diospyros kaki L.f. were already reported systematically many years ago, no chromatographic fingerprints for the quality control of the leaves of Diospyros kaki L.f. have been reported until now.In our present research, we focused on developing a simple, accurate and valid chromatographic fingerprint method to control the quality of the leaves of Diospyros kaki L.f. by high-performance liquid chromatography method. By using the professional software named " Similarity Evaluation System for Chromatographic Fingerprint of TCM " published by NPC （Version 2004A）, multi-sample batches of samples were examined to generate a mean global chromatogram as the representative standard fingerprint and the similarity of each chromatogram against the mean global chromatogram was also calculated. We also applied SPSS software for sample classifications by Hierarchical clustering analysis method. The chromatographic fingerprint method we established can be used for identifying persimmon leaves of different habitat.Thirdly, we studied seven characteristic peaks in chromatographic fingerprints by HPLC/MS method and presumed their structures, by which we can get the new message of the black box about compositions of characteristic peaks in chromatographic fingerprints.更多还原