高效液相層析法測定當歸藥材及飲片中的游離型和可被利用的總阿魏酸的含量   全文下載

Determination of Free and Total Available Ferulic Acid in Different Types of Chinese Angelica by High Performance Liquid Chromatography

阿魏酸（ferulic acid）在當歸和其他高等植物中常以遊離型和結合型兩種形式共同存在。結合型阿魏酸很容易轉變為游離型阿魏酸，而其轉化的程度和檢測到的游離阿魏酸的含量與提取溶劑的性質和酸鹼度有關。為此，本文分別以水、70% 甲醇、甲醇- 甲酸（95:5）混液和甲醇-2% 碳酸氫鈉溶液（95:5）混液四種不同酸鹼度的溶劑探討當歸藥材中阿魏酸的提取效果。結果顯示，以中性溶劑提取、檢測到的阿魏酸的含量變化很大，而以弱酸性和弱鹼性溶劑提取、測定的阿魏酸的含量均較穩定。因此，弱酸或弱鹼性溶劑被選擇為最適宜溶劑，用於提取當歸藥材的阿魏酸；即，游離阿魏酸和可被利用的總阿魏酸，其結果穩定、再現性良好。提取物中阿魏酸的含量以下述準確、快速的高效液相層析法測定，其分離管柱為alltima C18 柱（5 μm，4.6 mm i.d. × 250 mm），連接C18 保護管柱（5 μm，4.6 mm i.d. × 250 mm）；柱溫30° C；流動相由乙?和1% 冰醋酸溶液混液組成，梯度沖提；流速1.0mL/min；檢測波長為320 nm。用此方法測定了全當歸、當歸頭、當歸尾、全當歸片、酒炙當歸和當歸炭共計20 份當歸藥材中的游離阿魏酸和可被利用的總阿魏酸的含量。結果顯示，這些樣品中可被利用的總阿魏酸的含量均大於各自的游離阿魏酸的含量，20 份樣品中前者的平均含量為後者的3.15倍（n = 20），其變化範圍為1.29-8.23 倍。本文所篩選出的提取方法可將結合型阿魏酸全部轉變成游離阿魏酸，再以高效液相層析法準確、快速地定量，可用於藥材的品質評價。

Free and conjugated forms of ferulic acid (FA) are generally available in higher plant taxa such as Chinese Angelica (CA, the roots of Angelica sinensis (Oliv.) Diels). These various forms of FA were found readily inter-convertible and the extractable level of each depended on solvent properties and acidity. Extraction efficiency using various pH solutions namely, water, 70% methanol, methanol-formic acid (95:5) and methanol-2% NaHCO3 in water (95:5) was compared. Extractable FA were found varying in samples under neutral solvents extraction, whilst relatively consistent for slightly acidic and alkali solvents which were therefore chosen as the optimal media to extract and determine the reproducible levels of free and total available FA. An accurate and rapid high performance liquid chromatographic (HPLC) analysis was conducted using an Alltima C18 column (5 μm, 4.6 mm i.d. × 250 mm) with a guard column (C18, 5 μm, 4.6 mm i.d. × 7.5 mm) at 30°C, eluted with a mixture of 1.0% acetic acid and acetonitrile in a gradient program at a flow rate of 1.0 mL/min and detected at 320 nm. Altogether 20 different types of CA samples including whole root, root head, rootlets, whole root slice, Angelica processed by Chinese yellow wine, and charred Angelica were quantified for free and total available FA. Total available FA was found more abundant than free counterpart with an average ratio of 3.15 (n = 20) in the range of 1.29 to 8.23 for these CA samples. The extraction protocol was proven reliable to quantitatively convert all conjugated FA into its free forms and thereby accurately determined by HPLC method for quality assessment.