當歸藥材基原鑑定分析方法開發

Abstract

繖形科植物當歸(Angelica sinensis)為一種常用的中藥，其藥理活性包括調經，血管舒張、抗志賀氏菌及防止腦栓塞等。而日本藥典所規定的當歸(Angelica acutiloba)則與中華藥典中當歸不同，且具不同的藥理活性。由於在民間使用的廣泛性及重要性，本研究開發分析當歸中非揮發性成份與揮發性成份的方法。對非揮發性分析物以毛細管電泳法分離，以紫外光吸收偵測器分析當歸萃取物。以40%甲醇萃取當歸，研究中對影響毛細管電泳分離當歸萃取物之參數進行最佳化及探討。最佳化電泳條件為以壓力進樣，在15mM 四硼酸鈉 (pH 9.3)、20mM 硫酸月桂酸鈉、9%乙腈(v/v)為背景電解質的環境下以偵測於210nm波長下的吸光度進行分析。以 最佳化條件分析24個實際當歸樣品所得指紋圖譜，經過校準後比較其相似度可將中華當歸與日本當歸以0.5為閾值區別。本實驗開發出一個簡易快速的毛細管電泳方法，並具在實際樣品中區分中華與日本當歸的能力。 對揮發性成份以頂空固相微萃取為樣品製備方法，以二維氣相層析結合飛行時間質譜儀為分析方法。本研究對頂空固相微萃取的溫度與時間、二維氣相層析結合飛行時間質譜儀之一維、二維溫度梯度與調變器(modulator)參數進行最佳化。最佳化分析條件為以80°C萃取樣品45分鐘，所得樣品於二維氣相層析法需時36分鐘，並在其中鑑定出28個於文獻中報導的分析物。以此方法分析中華當歸與日本當歸所得之層析圖有顯著差異，顯示此分析方法有鑑定當歸基原之潛力。

Angelica is a commonly used traditional Chinese herbal medicine. Pharmacological activities of Angelica include regulating menstrual cycle, vasorelaxation, anti-Shigella and treating cerebral thrombosis. The Pharmacopeia species in Japan and Taiwan are Angelica acutiloba and Angelica sinensis, respectively. Different Angelica species show different pharmacological activities. Owing to its extensive use and importance, we developed analytical methods to analyze volatile and non-volatile components in Angelica plants. For non-volatile components, we developed a capillary electrophoresis-UV absorbance (CE-UV) method to analyze Angelica extract. The extraction solvent contains 40% methanol. We optimized CE parameters to analyze Angelica extract. The optimal CE conditions used hydrodynamic injection and the background solution was 15mM sodium tetraborate (pH was 9.3), 20mM sodium lauryl sulfate and 9% acetonitrile (v/v). The detection wavelength was 210nm. The fingerprint of 24 Angelica extract were aligned and compared the similarity. A. sinensis and A. acutiloba could be distinguished by the threshold value of 0.5. This simple and fast CE-UV method appears to be applicable to distinguish A. sinensis and A. acutiloba. For volatile components, we used headspace-microextraction (HS-SPME) as sample preparation method and the extract was analyzed by two-dimension gas chromatography-time of flight mass spectrometry (2D GC-TOF MS). We optimized the SPME extraction temperature and time. The temperature gradients in the first and second dimensional GC oven as well as the modulation period were optimized to improve the separation efficiency. The optimized condition for HS-SPME extracted the sample at 80°C for 30 minutes and the sample was analyzed by 2D- GC. The analytical time was 36 minutes, and 28 components that had been reported in Angelica were identified. Under optimal conditions, chromatograms of A. sinensis and A. acutiloba sample showed significant difference. The developed method shows potential for authentication of Angelica plant.