桂皮、牡丹皮單寧之分析方法開發及牡丹皮基原鑑定研究

吳雅婷(YaTingWu)

指導教授 ： 許順吉

國立臺灣師範大學/理學院/化學系/碩士(2001年)

摘要

高效液相層析（HPLC）及毛細管電泳（CE）是目前最常用來測定中藥成分含量的分析方法。綜合兩者的優點，合併運用，可以拓展中藥化學評價的範疇。本研究以這兩種儀器開發桂皮、牡丹皮單寧酚的分析方法，並比較HPLC和CE兩分析方法的適用性及用為基原判別的工具。

單寧（tannins）為植物體內具收斂作用的物質，屬於多酚類化合物，一般指分子量500以上的類黃酮酚類化合物（flavonoid phenolic compounds）以網狀結構或氫鍵與蛋白質結合的物質，主要可分為縮合單寧（condensed tannins）與水解性單寧(hydrolysable tannins)二類。

本研究分二部分，第一部分為開發桂皮單寧的HPLC及CE分析方法。桂皮(Cinnamomum cassia Blume)為樟科植物的乾燥根皮，含1. (+) – catechin， 2. (-) – epicatechin， 3. Procyanidin B-1， 4. Procyanidin B-2，5. Arecatannin A1 ，6. Cinnantannin B2 ，7. Cinnantannin C2等七個桂皮縮合單寧成分。實驗結果顯示這七個成分可利用磷酸鹽沖提液、5C18-MS分離管柱及210nm偵測波長下，HPLC能於60分鐘內成功地分析這7個成分；用硼酸鹽、SC及異丙醇配製成緩衝溶液，在波長210nm條件下，利用MEKC分析模式，亦可在40分鐘內分析完成。在HPLC分析中，若要得到良好的基線，藥材萃取液須經適當的前處理；用CE分析時，則須加界面活性劑（SC）才有較好的解析度。

第二部分為開發牡丹皮單寧之HPLC及CE分析方法。牡丹皮是毛莨科植物（Paeonia suffruticosa Andrew）的乾燥根皮，以8個水解性丹皮單寧成分（1. 4,6-di-GG，2. 1,2,3,6-tetra -GG， 3. 1,2,3,4,6-pentra-GG， 4. 1,3,4,6-tetra-GG，5. 3,4,6-tri-GG ，6. 1,3,6-tri-GG ，7. 3,6-di-GG，8. 1,2,6-tri-GG）為指標，在磷酸鹽沖提液、5C18-MS分離管柱及以280nm為偵測波長，可在60分鐘內分析出8個成分；用硼酸鹽、SDS及異丙醇配製成緩衝溶液，在波長280nm條件下，利用MEKC分析模式，可在50min內分析完成。在HPLC分析中，樣品須經過前處理(Sep-Pak)才可得到良好的基線；用CE分析時，則須加界面活性劑（SDS）才有較好的解析度。
此外，本論文收集13批川丹皮（Paeonia suffruticosa）及10批西昌丹皮（P. delavayi），進行牡丹皮基原之化學辨識研究。結果發現西昌丹皮有八個明顯吸收峰，川丹皮的3,4,6-tri-GG(5)無法偵測；川丹皮單寧總含量為0.561 ± 0.065 mg/g，西昌丹皮則為1.390 ± 0.476 mg/g；川丹皮中PN/Pf的含量比為> 1，西昌丹皮則為< 1；川丹皮中3/2的含量比值小於11.4，西昌丹皮則大於15.2。根據以上數據，可做為辨識牡丹皮基原時的參考依據。

關鍵字

單寧 ； 基原鑑定

並列摘要

High performance liquid chromatography (HPLC) and capillary electrophoresis (CE) are currently the mostly popularly used analysis methods for assaying the constituents of Chinese herb drugs. By combining the superiorities of both methods, the scope of chemical appraisal of Chinese herb drugs can be expanded. This study is aimed to develop methods for analyzing the tannins of Cinnamomi Cortex and Moutan Cortex, whereby HPLC and CE are compared for their applicability and utility as tools in identifying botanical sources.

In plants, tannins are astringents, which belong to polyphenols, usually referring to those flavonoid phenolic compounds that have molecular weights over 500, and are bonded with protein through a reticular structure or hydrogen bonds. Tannins are chiefly divided into two categories, condensed tannins and hydrolysable tannins.

This study is divided into two parts. The first part deals with the development of HPLC and CE methods for analysis of tannins in Cinnamomi Cortex. Cinnamomi Cortex is the dried stem bark of the Lauraceous plant Cinnamomum cassia Blume, which chiefly contains seven condensed tannins, namely, 1. (+)-catechin, 2. (-)-epicatechin, 3. procyanidin B-1, 4. procyanidin B-2, 5. arecatannin A1, 6. cinnantannin B2, 7. cinnantannin C2. Experimental results show that the seven constituents can be successfully analyzed within 60 minutes, using HPLC with a phosphate eluent, a 5C18-MS column and a detection wavelength at 210 nm. The constituents can also be successfully analyzed within 40 minutes under the MEKC analysis mode with a buffer comprising borate, SC and isopropyl alcohol and a detection wavelength at 210 nm. In the HPLC analysis, the drug material has to be subjected to appropriate pretreatment in order to obtain a good baseline chromatograph. While in the CE analysis, a surfactant (SC) must be added to render good resolution.

The second part of the study deals with the development of HPLC and CE methods for analyses of the tannins in Moutan Cortex. Moutan Cortex is the dried root bark of the Ranunculaceous plant Paeonia suffruticosa Andrew. Using the eight hydrolysable tannins (1. 4,6-di-GG, 2. 1,2,3,6-tetra-GG, 3. 1,2,3,4,6-penta-GG, 4. 1,3,4,6-tetra-GG, 5. 3,4,6-tri-GG, 6. 1,3,6-tri-GG, 7. 3,6-di-GG, 8.1,2,6-tri-GG) in Moutan Cortex as the marker substances, the HPLC method with a phosphate eluent, a 5C18-MS column and a detection wavelength at 280 nm can accomplish the analysis within 60 minutes; and the MEKC method with borate, SDS, isopropyl alcohol and a detection wavelength at 280 nm can finish the analysis within 50 minutes. In the HPLC analysis, the test sample has to be subjected to appropriate pretreatment (Sep-Pak) in order to obtain a good baseline chromatograph, and in the CE analysis, a surfactant (SDS) has to be added to render a good resolution.

In addition, we have collected 13 samples of Szechuanese Moutan Cortex (from Paeonia suffruticosa) and 10 samples of Western Moutan Cortex (from P. delavayi) for chemical identification of herb sources. As a result, there are eight conspicuous peaks of corresponding tannins in Western Moutan Cortex, and there are only seven peaks in Szechuanese Moutan Cortex wherein the constituent 3,4,6-tri-GG (5) is undetectable. The total tannin content is 0.561 ± 0.065 mg/g in Szechuanese Moutan Cortex, and 1.390. ± 0.476 mg/g in Western Moutan Cortex. The greatest distinction between the two is that the PN/Pf ratio is greater than 1 in Szechuanese Moutan Cortex and less than 1 in Western Moutan Cortex; and that the 3/2 ratio is less than 11.4 in Szechuanese Moutan Cortex and larger than 15.2 in Western Moutan Cortex. The above data can be used for the identification of the herb sources of Moutan Cortex articles.

並列關鍵字

tannin

參考文獻

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