- 學位論文
利用微胞電動層析毛細管後衍生法分離天然物有效成分及鑑定生物活性
Development of Bioanalytical System for On-Line Separation and Bio-Activity Analysis of Natural Products by Post-Micellar Electrokinetic Capillary Chromatography Derivatization
摘要
大部分天然植物中富含多種化合物，其中的酚類化合物具有不同的生化活性，例如抗氧化、抗發炎、抗凝血。而對於偵測抗氧化能力的方法，目前大多為離線測試，即利用多孔盤以分光光度計進行檢測，檢測方法如ABTS+、DPPH、ORAC等方式。這些離線測量的結果，所測得的數值範圍不大，若測量結果數值相近，再加上測量時的誤差，可能導致實驗結果無法反應真實情況。因此，利用本實驗室所研發串聯毛細管方法，能同時線上分離及量測抗氧化能力的功能性串聯毛細管電泳後衍生生化分析系統，進行天然物分離和偵測各分離物之個別抗氧化能力。實驗過程中利用串聯毛細管微胞電動層析方式(MEKC)並結合ATBS+抗氧化之延伸系統，將實驗裝置分為兩部分:系統第一部分利用微胞電動層析進行待測成分分離工作，而第二部分利用注射導入ABTS+試劑至毛細管內來偵測抗氧化物質清除自由基的能力。 本研究除了確認有58%-86%的分析物經MEKC分離後、可進入第二部份進入生化活性分析，並優化了原本連接處外管pH，發現在pH 8的條件下有較佳的分離結果，在on-line與off-line抗氧化活性相關性的實驗，caffeic acid和 gallic acid在off-line是較靈敏的，推測與單苯環化合物酚類結構有關，而具有芳香環結構的化合物在on-line會有較佳的結果。真實樣品紅酒的分析上，本系統成功地顯現出，紅酒內主要抗氧化成分及其個別的抗氧化能力。
並列摘要
The aim of this study was to optimize the conditions of on-line micellar electrokinetic chromatography (MEKC) coupling with post-capillary derivatization for antioxidative capacity determination (ACD). In this design, MEKC was employed to separate and analyze the compositions of extracts, which was followed by the second module, ACD. Separated analytes were subjected to interact with ABTS+. Reduced absorbance was then detected and area under peaks was integrated for determination of antioxidative capacity. Running conditions such as concentrations of BGE, SDS, and ABTS+, and methanol proportion of BGE were optimized. Additionally, different BGE pH was examined at the interface of these two modules to reduce post-capillary sample loss. Results indicated that BGE with pH 8 demonstrated the highest retention rate (86%) of rutin than other tested pH (7, 8.5, 9, 9.5, 10). Correlation of antioxidative capacity between off-line approach and this online setting was made. Compounds with single benzene ring such as caffeic acid and gallic acid, tends to be suitable for off-line approach, while those with aromatic rings were preferable for on-line format. Finally, the feasibility of on-line MEKC-ACD was successfully demonstrated by analyzing real samples (red wine). Taken together, this system possesses the advantages of 1) simultaneously analyzing extracts components and their antioxidative capacity, and 2) defining the proportion of components that contribute to total antioxidative capacity.
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