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1、上海交通大学硕士学位论文加压毛细管电色谱法用于银杏叶的质量控制及其药物动力学研 究姓名：吴孔弦申请学位级别：硕士专业：药物分析学指导教师：闫超20081201加压毛细管电色谱法用于 银杏叶的质量控制及其药物动力学研究 摘 要 银杏叶为近年来国内外中药（植物药）研究开发的热点之一。其所含化学成分非常复杂，包括黄酮类、萜类、生物碱、多糖类、酚类、氨基酸、微量元素等。 银杏叶中黄酮类化合物含量较高，其中以槲皮素、异鼠李素和山萘酚的糖苷为主。许多实验已证实，银杏叶中黄酮类物质具有非常广泛的生理和药理活性，如扩张冠状血管、抗炎症、抗氧化、抗病毒、调节免疫功能等。本文首次采用加压毛细管电色谱法（press
2、urized capillary electrochromatography， pCEC）对银杏叶中黄酮类化合物进行分离，并以主要活性成分槲皮素和山萘酚为主要指标，研究其在大鼠体内的药物动力学。首次建立了银杏叶药材的加压毛细管电色谱指纹图谱，应用了相似度分析和主成分分析对不同产地银杏叶药材的质量进行了评价。 本论文共分四章，主要内容包括： 第一章介绍了选题背景和意义。主要介绍了银杏叶的化学成分和药理活性，综述了加压毛细管电色谱法的原理和研究进展，阐述了中药指纹图谱的重要意义。 第二章的工作，主要是建立了加压毛细管电色谱法对银杏叶中黄酮类化合物进行分离的条件。通过对检测波长、流动相、离子强度、电
3、压各主要因素的考察，最终建立的电色谱条件为：流动相：甲醇-磷酸溶液（10 mM） （55：45，v/v）等度洗脱；检测波长：360 nm；检测电压为 10 kV。并同时比较了在此相同条件下 pCEC、毛细管液相色谱（cLC） 、高效液相色谱（HPLC）的分离效果，发现 pCEC 能在最短的时间内达到最高的柱效，说明 pCEC 与 cLC、HPLC 相比，具有分析速度快，分离效率高的优势，是一个很好的微分离手段。 第三章的工作，以银杏叶中的指标性有效成分槲皮素和山萘酚为研究对象，采用加压毛细管电色谱法（pCEC）研究其在大鼠体内的药物动力学。以 SD 雄性大鼠为实验动物，灌胃给药后，用甲醇-磷酸
4、溶液（10 mM） （55：45，v/v）等度洗脱，在电压为 6 kV 下测定大鼠血浆中槲皮素和山萘酚的含量， 并绘制药时曲线。 结果显示槲皮素和山萘酚的达峰时间均为 2 h，且山萘酚吸收不规则，出现多峰现象。 第四章以 10 批不同产地的银杏叶药材为研究对象，采用填充 5 m 粒径 C18填料的毛细管色谱柱，通过对药材提取、流动相、电压各主要影响因素的考察，优化了加压毛细管电色谱银杏叶指纹图谱的条件，首次建立了银杏叶的加压毛细管电色谱指纹图谱。 采用了国家药典委员会颁布的 “中药色谱指纹图谱相似度评价系统”软件对不同产地的银杏叶进行了相似度的评价，评价的结果显示除 7 号样品的相似度为 0.
5、676 外其他九批药材的相似度均在 0.900 以上， 相似度高， 说明这九批药材的质量是相对稳定的。另外还应用了主成分分析对不同产地银杏叶进行了分类，结果与相似度评价结果都是较为一致的，为银杏叶药材的质量分析和控制提供了可靠的依据。 本文通过应用现代微分离色谱技术加压毛细管电色谱对银杏叶药材进行了中药现代化研究，并研究了其指标成分的药物动力学，首次建立了银杏叶的加压毛细管电色谱指纹图谱，并且对其质量进行了评估，发展了微量电色谱技术对银杏叶进行分离和鉴定研究，为中药分离和质量控制提供了新思路和新方法。 关键词：加压毛细管电色谱，银杏叶，指纹图谱，药物动力学 STUDY ON QUALITY C
6、ONTROL AND PHARMACOKINETICS OF GINKGO LEAVES BY PRESSURIZED CAPILLARY ELECTROCHROMATOGRAPHY Author: Kongxian Wu Advisor: Chao Yan School of Pharmacy, Shanghai Jiao Tong University, Shanghai 200240, P.R.China ABSTRACT Ginkgo biloba leaves are one of the most popular traditional Chinese medicines (phy
7、tomedicines). There are many constituents in Ginkgo biloba leaves, including flavonoids, terpene lactones, alkaloids, polysaccharides, hydroxybenzenes, amino acids and microelements. Among these components, the content of flavonoids is higher, which mainly composed of glycosides of quercetin, isorha
8、mnetin and kaempferol. Many researches have identified that quercetin possesses vasodilatation, antiflammaton, antioxidation, antivirus, immunomodulation and other important therapeutic activities. This paper is divided into four chapters as follows: Chapter 1 briefly introduced the background and s
9、ignificance of the thesis, including the chemical constituents and pharmacological activities of Ginkgo biloba leaves, the principle and advances in studies on pressurized capillary electrochromatography (pCEC) and the importance of fingerprints of traditional Chinese medicines (TCM). In Chapter 2,
10、we developed the pressurized capillary elecchromatographic condition of the separation of flavonoids. The pressurized capillary elecchromatographic condition was: Mobile phase: methanol-phosphoric acid （10 mM）（55： 45， v/v） ; Voltage: 10 kV; Detective : UV at 360 nm. Separation of the standard flavon
11、oids solution performed with pCEC, cLC, and HPLC was also compared. Higher column efficiency and shorter analysis time were obtained by pCEC. In Chapter 3, setting quercetin and kaempferol as main indexes, the pharmacokinetics of Ginkgo biloba leaves extract in rat was investigated, by using pressur
12、ized capillary electrochromatography (pCEC) method. After intragastric administrating Ginkgo biloba leaves extract to SD male rats, the concentrations of quercetin and kaempferol in rat plasma were measured at the same time, by methanol- phosphoric acid（10 mM） （55：45，v/v） detected at 360 nm. Voltage
13、: 6 kV The Concentration-Time curves were drawn. The results showed that T-max of quercetin and kaempferol were both 2 h. The absorption of kaempferol was not regular, multi-peaks appeared. In Chapter 4, the pCEC fingerprints of Ginkgo biloba leaves was established for the first time. The software o
14、f “similarity evaluation system for chromatographic fingerprint of TCM (Version 2004A)” was used to evaluate the similarity of Ginkgo biloba leaves extract. The result showed that the similarity values of 9 batches of samples were more than 0.900 except Sample 7 (0.676) indicating that the quality o
15、f the 9 batches is stable. Besides, principal component analysis were used to evaluate the quality of Ginkgo biloba leaves from different areas as well, the result was in accordance with the similarity evaluation. The study of the pCEC fingerprints of Ginkgo biloba leaves may be used as a standard f
16、or the quality control of Ginkgo biloba leaves. In conclusion, pressurized capillary electrochromatography (pCEC) was utilized for the separation and analysis of effective ingrediens in Ginkgo biloba leaves, the pCEC fingerprints of Ginkgo biloba leaves was established for the first time. The pharmacokinetics of index components in Ginkgo biloba leaves extract was also studied. The results of this thesis provided new thoughts and technolo
