SEPARATION AND PURIFICATION OF FLAVONOIDS FROM BLACK CURRANT LEAVES BY HIGH-SPEED COUNTERCURRENT CHROMATOGRAPHY AND PREPARATIVE HPLC

Abstract

High-speed countercurrent chromatography (HSCCC) has been successfully used for the preparative isolation of flavonoids from the ethyl acetate extracts of black currant leaves. The HSCCC separation was performed with a two-phase solvent system composed of n-hexane/EtOAc/MeOH/H2O (1:10:1:10, v/v) at a flow rate of 1.5 mL/min. When the flow rate was increased from 1.0 to 3.0 mL/min, the retention of stationary phase decreased from 60.3% to 39.7% resulting in a loss of peak resolution, while the stationary phase retention is stable with an increase in sample size from 25 to 200 mg. From 100 mg of the crude sample, HSCCC separation yielded 4.0 mg of kaempferol 3-O-galactoside, 6.0 mg of kaempferol 3-O-glucoside, and 9.0 mg of fraction I containing a mixture of hyperoside and isoquercitrin. Then, from 18 mg of fraction I, 3.0 mg of hyperoside and 11.0 mg isoquercitrin were separated by preparative HPLC by successive sample injection at every 100 min interval. Chemical structures of all these compounds were confirmed by MS and NMR.