Preparative Separation Of Glucosinolates From Radish Seeds Based On Slow Rotary Countercurrent Chromatography

Glucosinolates are secondary metabolites synthesized in the cruciferous plants. A great deal of research indicated that isothiocyanates possesses activities of preventing cancers. Thus, the separeation and purification of glucosinolates become very important. Because of the complexity of their structures, this kind of compounds is very difficult for separeation and purification. Slow rotary countercurrent chromatography (SRCCC) is a new advanced countercurrent chromatography which was less reported in separation engineering. In the present study, SRCCC was applied to preparative separation of glucosinolates from the extract of radish seeds.The crude glucosinolates about 3380.8 g were extracted from 30 kg radish seeds using boiling methanol.A SRCCC-D1000 was used for selection of separation parameters to separate glucosinolates from the extract of radish seeds.A CCC solvent system composed of butanol-acetonitrile-10% ammiaonia sulfate (0.5: 1: 2.2) was found as an excellent solvent system. Parameters of the flow rates of flow phase, rotary speeds and the sample amount were studied for the influence on the retention of the stationary phase. A flow rates of mobile phase of 3 mL/min, a rotary speed of 50 rpm and the sample loading amount of 1.5 g were selected for the separation using SRCCC-D1000 with a 1000 mL column. The extract of radish seeds of 1.5 g was separated to yield 67.6 mg of compoundâ… . Another fraction from SRCCC-D1000 separation was purified by preparative HPLC toyield 9.3 mg of compoundâ…¡and 10.4 mg of componentâ…¢.The structures of compoundsâ… andâ…¡were indentified as 4-methylsulphinyl-3-butenyl glucosinolate and allyl glucosinolate, respectively, elucidated by ESI-MS and NMR. A advanced machine of SRCCC-D30L, which was designed by Institute of Food and Biological Engineering was used for preparative separation of glucosinolates from the crude radish seeds. Using the column of 7 L, 17 L and 30 L, the sample loading was enlarged from decagram-scale to hectogram-scale. Computed according to the 4-methylsulphinyl-3-butenyl glucosinolate (GRE), the separation efficiency increased as the increase of column capacity. Using two SRCCC-D30L apparatuses connected in series a total column capacity of 60 L was successfully used for separation of kilogram-scale which meeted the primary need of industrial production. The recovery reagent from separation was repeatedly used for separation with good effect which implys that a lower product cost could be obtained with the present technology.