以離心分配層析法結合三相溶劑系統製備分離洋蔥中兩種類黃酮成分

Abstract

本研究是利用離心分配層析結合三相溶劑系統，來製備分離洋蔥中quercetin-3,4-di-O-glucoside(Q3,4G) 及quercetin-4-O-glucoside(Q4G)兩種主要類黃酮成分。 洋蔥樣品先以60%(v/v)的乙醇萃取，分離實驗中使用的溶劑系統為Hexane / Methyl tert-butyl ether / Acetonitrile / Water ( 2 : 2 : 3 : 3)的三相溶劑系統，管柱總體積為240 mL，在1000 rpm 的轉速下，固定相之滯留率為82% (其中中層相占21%、下層相79%)。首先將下層相(固定相)注入充滿管柱，再以上升法(ascending mode)注入中層相，待第一次平衡後；接著以上層相注入，達第二次平衡後可注入樣品。分離樣品過程中，首先將上層相以上升法沖堤30 分鐘後，轉換以中層相沖堤50 分鐘，最後再切換到下層相以下降法(descending mode)沖堤120 分鐘，分管收集之樣品沖提液，以電灑質譜儀分析之。洋蔥粗萃取物0.26 克，經過離心分配層析分離之後，Q3,4G 和Q4G 純度和回收量以液相層析估計為 90% (0.33 mg) and 92% (0.12 mg)。

We developed a preparative separation of two active compounds,quercetin-3,4-di-O-glucoside (Q3,4G) and quercetin-4-O-glucoside(Q4G), from onion using centrifugal partition chromatography (CPC)with a three-phase solvent system. The crude extract was obtained using a 60% (v/v) ethanol aqueous solution. The three-solvent system was composed of hexane / methyl tert-butyl ether / acetonitrile / water = 2:2:3:3 (v/v). The stationary-phase retention ratio was 82% (including 21% middle phase and 79% lower phase) in a 240-ml column under 1000 rpm rotation speed. Theseparation column was first filled with the lower phase and the middle phase was pumped into the column. After the equilibrium was reached, the upper phase was delivered until the second equilibrium was attained. We then injected the sample solution and eluted the column with the upper phase under the ascending mode for 30 min. The elution was then switched using the middle phase for 50 min. Finally, the elution was completed using the lower phase for 120 min under the descending mode. The collected fractions were analyzed using electrospray ionization mass spectrometry (ESI-Mass). The purities and recoveries, estimated using a HPLC, were 90% (0.33 mg) and 92% (0.12 mg) for quercetin-3,4-di-O-glucoside and quercetin-4-O-glucoside, starting from a 0.26 g onion crude extract.