Combination of high-speed countercurrent chromatography and reversed phase C18 chromatography for large-scale isolation of cyanidin-3-O-β-d-glucoside from black rice bran extract

Abstract

Cyanidin-3-O-β-d-glucoside (C3G), the best known and most investigated anthocyanin, has many pharmacological properties. Black rice bran is an outstanding source for the separation of C3G due to its high C3G content and a simple anthocyanin composition. The present study described a large-scale isolation of C3G from the crude black rice bran extract by combining high-speed countercurrent chromatography (HSCCC) and reversed phase C18 column chromatography. After HSCCC enrichment with the solvent system water–n-butanol–tert-butyl-methyl-ether (TBME)–acetonitrile–trifluoroacetic acid (TFA) (5:2:2:1:0.1%), the C3G content was concentrated about 16-fold and reached to 563.23mg/g with a recovery 96.89%. Further purification was carried out using reversed phase C18 column chromatography and 1.43g of pure C3G (purity 94.38%) was obtained from 3.0g of anthocyanin-enriched extract. The method is time-saving, low risk of sample denaturation, high sample recovery, and so is suitable for large-scale preparation of C3G for further studies of bioactivities.