Preparative Separations of Five Active Compounds in Mulberry Leaves Using Centrifugal Partition Chromatography

• Main Authors: Chen, Ling-Yi, 陳伶宜
• Other Authors: Yu, Tiing
• Format: Others
• Language: zh-TW
• Published: 2010
• Online Access: http://ndltd.ncl.edu.tw/handle/86619337585462044663

碩士 === 國立交通大學 === 應用化學研究所 === 98 === 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.