使用離心分配層析法分離桑葉中的有效成分

Abstract

本研究利用離心分配層析來分離桑葉中的有效成分，Chlorogenic acid、Isoquercitrin、Astragalin、Quercetin 3-(6-malonylglucoside)和Kaempferol 3-(6-malonylglucoside)。 桑葉經由60% (v/v)的乙醇萃取後所獲得的有效成分先經由高效能液相層析串聯質譜進行初步鑑定，然後再使用乙酸乙酯和去離子水做批式萃取。本實驗中使用離心分配層析的溶劑系統，為三成分溶劑系統(methyl t-butyl ether /acetone/H2O =6：4：10、7：3：10、8：2：10) 和 (methyl t-butyl ether /acetone/ H2O = 6：4：10、7：3：10、8：2：10)並加入溶劑系統總體積0.6% (v/v)的formic acid、四成分溶劑系統(methyl t-butyl ether /acetone/H2O/EA =7：3：10：2) 和 (methyl t-butyl ether /acetone/ H2O/EA = 7：3：10：2)並加入溶劑系統總體積0.6% (v/v)的formic acid，都是以上層有機相當作靜相，下層水相當作動相。在900 rpm轉速下，靜相滯留於總體積240 mL管柱中的量為188mL，靜相滯留率為78%。前處理後的樣品經由離心分配層析製備分離，並使用紫外光-可見光譜儀線上偵測系統，沖提液以分管收集器每3 mL收集一管，再以高效能液相層析分析各收集管中不同成分的純度。在桑葉樣品中，所得到的純度分別為Chlorogenic acid (70.2%),Isoquercitrin (97.3%),Astragalin (97.2%),Quercetin 3-(6-malonylglucoside) (69.1%),Kaempferol 3-(6-malonylglucoside) (69.6%)。

Separations of active compounds, including Chlorogenic acid, Isoquercitrin, Astragalin, Quercetin 3-(6-malonylglucoside) and Kaempferol 3-(6-malonylglucoside) in mulberry leaves, were carried out using centrifugal partition chromatography (CPC) in this study. These five components in the crude extract by 60% (v/v) ethanol were first identified using HPLC-MS. After pre-extractions using ethyl acetate and water, we used solvent systems, (methyl t-butyl ether /acetone/H2O =6：4：10、7：3：10、8：2：10) and (methyl t-butyl ether /acetone/ H2O = 6：4：10、7：3：10、8：2：10) added with 0.6% (v/v) formic acid, four solvent systmes (methyl t-butyl ether /acetone/H2O/EA =7：3：10：2) , (methyl t-butyl ether /acetone/ H2O/EA = 7：3：10：2) added with 0.6% (v/v) formic acid, to separate sample extracts. The descending elution mode was applied, i.e. the lower aqueous layer was used as the mobile phase, and the upper organic layer as the stationary phase. Under 900 rpm, the volume of the stationary phase retained was 188 mL in the separation cells of a total volume 240 mL; stationary-phase retention ratio was 78%. The CPC effluent monitored using an on-line UV/Vis detector was fraction-collected every 3 mL, and the fractions were further analyzed using HPLC . The purities were 70.2%, 97.3%, 97.2%, 69.1% and 69.6% for Chlorogenic acid, Isoquercitrin, Astragalin, Quercetin 3-(6-malonylglucoside) and Kaempferol 3-(6-malonylglucoside), respectively, under the optimized separation.