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研究生: 李侑穗
Yu-Sui Lee
論文名稱: 紫草成分之分析與基原之辨識
指導教授: 許順吉
Xu, Shun-Ji
學位類別: 碩士
Master
系所名稱: 化學系
Department of Chemistry
論文出版年: 2000
畢業學年度: 88
語文別: 中文
論文頁數: 132
中文關鍵詞: 紫草HPLCCE基原辨識
英文關鍵詞: Macrotomia euchroma, HPLC, CE, comparative study
論文種類: 學術論文
相關次數: 點閱:248下載:8
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  • 高效能層析(HPLC)及毛細管電泳(CE)是目前最常用來測定中藥成分量的分析方法。綜合兩者的優點、合併運用,可以擴展中藥分析的範疇。本研究以兩種儀器開發紫草藥材的分析方法,並比較CE與HPLC在實際分析中藥時的優劣。
    本研究第一部份目的在開發紫草藥材的高效能液相層析方法。紫草藥材主要的成分是含結構的化合物,分別為 shikonin(SK) 1、 acetylshikonin(ASK) 2、 teracrylshikonin(TSK) 3、 isovalerylshikonin(IVSK)4、 isobutylshikonin(IBSK) 5、β,β-dimethylacrylshikonin (BSK) 6。本實驗以醋酸的緩衝溶液為沖提系統及Cosmosil 5C18-MS分離管柱,於280 nm 的偵測波長及線性梯度沖提程式下,在55分鐘內完成紫草藥材中六個化合物的定量工作。此外,分離管柱的選擇、移動相的中醋酸的含量及有機修飾劑的比例對紫草藥材中各成分的分離效果影響都予詳盡的探討。
    此外,本研究以軟紫草(Macrotomia euchroma)及硬紫草(Lithospermum erythrorhizon)進行紫草基原的化學鑑定。研究發現軟紫草中六種成分都有明顯的吸收,硬紫草中3的含量很少,幾乎無法偵測。此外,硬紫草中除了上述的成分之外,還有兩個明顯的吸收峰,其中一個經鑑定為β-hydroxyisovalerylshikonin (HSK) 8 ,另一個標示為7未知成分A。軟紫草中的總含量為(80.28±39.75 mg/g),硬紫草中的總含量為(23.54±11.51 mg/g);軟紫草中3/2的面積比 >0.03而硬紫草係幾近於;7/2的面積比在軟紫草中係<0.1,在硬紫草中係>0.2;8/2的面積比在軟紫草為<0.1而在硬紫草中為>0.4;軟紫草中5/2的面積比值為> 0.4,硬紫草的比值為 <0.1。根據以上的數據,可作為辨識紫草基原時的參考依據。此部分最後針對紫草中成分的安定性加以評估,探討紫草在不同溫度環境下成分的變化情形。
    本研究的第二部分是開發紫草藥材的毛細管電泳的分析方法。本研究利用掃集法(sweeping),以110 mM SDS之70 mM的磷酸溶液,並加入50%的甲醇(pH = 1.85 cond.=2.40)為緩衝溶液,在40分鐘內分析出紫草中的6(BSK)、5(IBSK)、4(IVSK)及1(SK)成分,而2(ASK)與3(TSK)則會重疊在一起。
    檢討紫草藥材的CE與HPLC的分析結果,發現利用sweeping方法的偵測極限可以較HPLC低,除此之外,在基線的平穩性、再現性及各成分的分離度方面皆以HPLC為佳。

    High-performance liquid chromatography (HPLC) and capillary electrophoresis (CE) are analytical methods most commonly used presently to quantify the components of Chinese herbal medicines.
    The first section of this research, a HPLC method for Macroto- mia euchroma was established. The major components of M. euchroma are naphthaquinone derivatives, containing shikonin (SK)1, acetyl- shikonin(ASK) 2, teracrylshikonin(TSK) 3 , isovalerylshikonin (IVSK) 4 , isobutylshikonin(IBSK) 5, β,β-dimethylacrylshikonin (BSK) 6 . The RP-LC method was carried out within 55 minutes by using cosmosil C18-MS column and a gradient solvent system of acetic acid buffer-methanol-acetonitrile, and detecting at 280 nm. The effect of column selectivity, acetic acid concentraction and organic modifier concentraction of the mobile phase on the seperation of the analytes was also studied.
    In this research, Macrotomia euchroma and Lithospermum erythror- hizon was collected for analysis, it was found that the absorptions of the six cpmponents in M. euchroma were clear, but the absorption of 3 wasn't found. In addition, there were two clear absorptions in L. erythrorhizon : one was hydroxyisovalerylshikonin (marked 8 ) and the other was unknown (marked 7 ). The total amount of naphthaquinone derivatives in M. euchroma (80.28±39.75 mg/g) was higher than L. erythrorhizon(23.54±44.51 mg/g). The area ratio of 3/2 in M. euchroma was higher than 0.03, but about zero in L.erythrorhizon . In addtion, the area ratio of 7/2 was less than 0.1 for the former, and was higher than 0.2 for the latter; the ratio of 5/2 was higher than 0.4 for the former and less than 0.1 for the latter. From these datas on the chemical analysis of the herb's constituents, we can postulate the origin and quality of the herb drug.
    The second section of this research, a CE method for M. euchroma was established. We used "sweeping" as the analysis method, carried out within 40 minetes using a buffer system containing 110 mM SDS in 70 mM phosphoric acid and 50% methanol (pH = 1.85, cond.= 2.40), and detecting at 210 nm. Using this method, four components of M. euchroma were analyzed wherein ASK(2) and TSK (3) were over- lapped.
    Comparing the HPLC and CE methods for M. euchroma, we found that the detection limit in sweeping was lower than in HPLC, except the theoretical plat number and the reproducibility which were better in HPLC than in sweeping.

    目 錄 圖目錄………………………………………………………………...ii 表目錄………………………………………………………………..iv 中文摘要……………………………………………………………..vi 英文摘要……………………………………………………………viii 第一章 緒論 第一節 前言…………………………………………………..1 第二節 高效液相層析法……………………………………..4 第三節 毛細管電泳分析法…………………………………..7 第四節 分析條件參數與適宜性之評估……………………22 第二章 紫草藥材之高效能液相層析 第一節 前言…………………………………………………28 第二節 實驗部分……………………………………………32 第三節 結果與討論…………………………………………39 第三章 紫草基原的化學鑑定 第一節 前言…………………………………………………54 第二節 實驗部分……………………………………………62 第三節 結果與討論…………………………………………67 第四節 紫草安定性之評估…………………………………82 第四章 紫草藥材之毛細管電泳層析 第一節 前言…………………………………………………88 第二節 實驗部分……………………………………………92 第三節 結果與討論…………………………………………96 第五章 結論 第一節 紫草藥材HPLC與CE分析方法的比較…………110 第二節 紫草基原的比較…………………………………..116 參考文獻…………………………………………………………...118 附錄一 紫草根橫切面簡圖……………………………………...124 附錄二 紫草類根與根莖的粉末特徵圖………………………...125 附錄三 紫草的指標成分之UV吸收光譜圖……………………126 附錄四 流動相(B)的比例對紫草層析圖的影響…………….130 附錄五 紫草藥材在50℃各時間的層析圖……………………..131

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