中藥 術對照品製備技術及質量評價 ABSTRACT Rhizoma Curcumae (Ezhu) is the dried root of several plants belonging to the genus of Curcuma in the family of Zingiberaceae. Ezhu is rich in volatile oil which contributes to most of its pharmacological activities. In this study, four active compounds were separated and isolated from Ezhu oil and the quality control system of Ezhu was studied. This thesis is divided into four chapters: Chapter 1 is the literature review of the studies on Ezhu oil with respect to the following aspects: chemical constituents, extraction and separation skills, quality control methods, pharmacology, toxicology and drug delivery systems. Chapter 2 is about the preparation and identification of reference compounds. Four crystals were obtained from Wenyujin oil with silica gel column chromatography and preparative HPLC. The purity of each compound was checked by TLC and HPLC and found to be over 98%. By comparing the UV, MS, 'H-NMR and 13C-NMR spectra, those four crystals were identified as neocurdione, isocurcumenol, curcumol and germacrone respectively. These compounds were used as standards for both qualitative and quantitative analyses, which are very important to the establishment of multi-marker quality control system for Ezhu. Chapter 3 focuses on the establishment of HPLC/DAD and GC/MS analytical methods for the quantification of these four components in Ezhu. In HPLC/DAD method, gradient elution was applied with methanol-water solvent system to ensure the adequate separation of marker components. While in GC/MS method, target ion quantification was used after the separation by temperature gradient program. A new extraction technique, accelerated solvent extraction (ASE), was employed for the preparation of sample solutions. The efficiency of this new extraction method was compared with that of steam distillation (SD), which is a traditional extraction method for Ezhu oil. Both the HPLC/DAD and the GC/MS methods were found to be simple, accurate and reproducible. They were finally applied to the analysis of two batches of Ezhu material medica. IV 澳門大學碩士學位論文 And Chapter 4 is an overall conclusion. In conclusion, four pure compounds were isolated from Wenyujin Oil and were identified. All of these compounds were previously reported to be pharmacologically active. New HPLC/DAD and GC/MS analytical methods for quantification of these four compounds were also designed in this study. The accurate quantification may provide a very useful method for the quality control of Ezhu material medica and its related products. Keywords: Rhizoma Curcumae; volatile oil; reference compounds; quality control V

中藥莪術對照品製備技術及質量評價 摘要 我術(Rhizoma Curcumae)為薑科薑黃屬植物蓬莪術(Curcuma phaeocaulis Val.)、廣西莪術(Curcuma kwangsiensis S. G. Lee et C. F. Liang)和溫郁金(Curcuma wenyujin Y. H. Chen et C. Ling)的乾燥根莖。其味苦、辛,性溫,有破瘀行氣、消 積止痛之功效,现代研究表明我術具有抗腫瘤、抗病毒、保肝等多種藥理活性。我 術中約含1-2.5%揮發油,是其主要有效部位。本研究從溫郁金油分離出新我二酮、 異莪術烯醇、我術醇和吉馬酮四種活性成分,純度均達到定量用對照品要求。在此 基礎上,建立了可對義術中該四種成分同時定量的GC/MS 和HPLC/DAD 分析方法 本文分為四章:第一章為文獻綜述,內容包括莪術的化學成分、提取分離、質 量控制、藥理毒理及製劑五個方面。第二章為表術油對照品的分離製備及標化; 採用硅膠柱層析結合製備型高效液相色譜從市售溫郁金油中分離得到四個結晶性 化合物,經 UV光譜、MS、H-NMR、C-NMR 等確證,分別為義術醇、異莪術 烯醇、吉馬酮和新莪二酮,HPLC 檢查純度均達到98%以上,文獻報導這四種成分 均具有抗腫瘤活性和/或保肝活性。這一部分工作解決了我術缺乏化學對照品、控制 指標單一的問題,為藥材質量控制研究奠定了基礎。第三章進行了我術多指標成分 定量研究。我術藥材及製劑通常以我術醇為對照品進行鑒別及含量測定,本文以自 行分離得到的四種活性成分為指標,建立了可對四種成分同時定量的GC/MS 和 HPLC/DAD 分析方法。高效液相法以甲醇-水梯度洗脫,對各組分進行分離測定; GC/MS 通過程序升溫使各組分在毛細管柱中得到分離,並以特徵離子定量。這兩種 方法簡便快捷、準確可靠、重現性好。以這兩種方法對兩批莪術藥材進行含量測定, 兩種方法的測定結果具有一致性。此外,本文首次採用加壓溶劑提取技術(ASE) 提取義術中的藥效成分,對提取條件進行了優化,並用於樣品溶液的製備;對ASE 與傳統的水蒸氣蒸餾法進行了比較,ASE 簡便快速、提取效率高。第四章對整個研 究工作進行總結與討論。 综上所述,本研究從莪術油中分離得到四個活性成分(新 二酮、異莪術烯醇、 我術醇、吉馬酮),並進行了標準化研究。以自製對照品為指標成分,首次建立了 ASE 提取-GC/MS 和ASE 提取-HPLC/DAD 法測定莪術中四種活性成分的含量,為 莪術藥材及製劑的質量控制提供了方法學依據。 II 澳門大學碩士學位論文 關鍵字:莪術;揮發油;對照品;質量控制 ΠΙ