以離心分配層析法製備分離桑葉中五種有效成分

Abstract

桑葉中的有效成分具有降低血液中總膽固醇濃度的效能，因而有助於預防動脈硬化病變。本研究是利用離心分配層析(centrifugal partition chromatography, CPC)來製備分離桑葉中的Chlorogenic acid、Isoquercitrin、Astragalin、Quercetin 3-(6-malonylglucoside)和Kaempferol 3-(6-malonylglucoside)五種成分。

離心分配層析是一種無固體靜相支持物的液相-液相層析技術，不會有固體吸附、靜相阻塞的問題，此外離心分配層析的分離管柱中比起典型的填充管柱可擁有更大量的靜相。根據上述兩種特性，因此離心分配層析很適合應用在生物分子及天然物等複雜物質的分離。桑葉經由60% (v/v)的乙醇萃取後所獲得的五種有效成分先經由電噴灑質譜進行初步鑑定，然後再使用乙酸乙酯和去離子水做批式萃取。本實驗中使用兩組離心分配層析的溶劑系統，分別是(methyl t-butyl ether (MTBE)/acetone/H2O = 6：4：10) 和 (MTBE/acetone/ H2O = 6：4：10)額外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220 mL管柱中的量為187 mL，靜相滯留率為85%。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，其沖提液以分管收集器每3 mL收集一管，以高效能液相層析分析各收集管中不同成分的純度和回收率，並以核磁共振做結構定。在20.0 g的乾燥桑葉中，這五種成分依序分別得到的純度和回收量為98% (59.2 mg)、94% (95.7 mg)、97% (56.7 mg)、93.5% (28.0 mg)、92.5% (23.0 mg)。

Active components in mulberry leaves are found effective in reducing the total cholesterol concentration in human blood, thus are able to help prevent atherosclerotic lesion. Preparative separations of five 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.

CPC is a technique of liquid-liquid chromatography which needs no solid-state support for the stationary phase. Accordingly, permanent adsorption of analytic molecules can be avoided during elution. In addition, the stationary phase volume in the separation column can be very large compared with the packed column. These two properties make the technique very suitable for preparative separations of biomolecules and natural products. These five components in the crude extract by 60% (v/v) ethanol were first identified using ESI-MS. After pre-extractions using ethyl acetate and water, we used two solvent systems, i.e. methyl t-butyl ether (MTBE) / acetone / H2O = 6:4:10 and MTBE / acetone / H2O = 6:4:10 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 187 mL in the separation cells of a total volume 220 mL. 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 and NMR. The purities and recoveries were 98% (59.2 mg)、94% (95.7 mg)、97% (56.7 mg)、93.5% (28.0 mg)、92.5% (23.0 mg), for Chlorogenic acid, Isoquercitrin, Astragalin, Quercetin 3-(6-malonylglucoside) and Kaempferol 3-(6-malonylglucoside), respectively, obtained from a 20-g dry mulberry leaf sample.