Preparative isolation of procyanidins from grape seed extracts by high-speed counter-current chromatography

High-speed counter-current chromatography (HSCCC) has been applied to the separation of grape seed procyanidins. The isolation of dimeric to tetrameric procyanidins is achieved after removing the polymeric compounds by solvent precipitation. An additional clean-up by solid-phase extraction on polyamide improved the purities of the isolated compounds. The solvent systems ethyl acetate/2-propanol/water (40:1:40, v/v/v), ethyl acetate/2-propanol/water (20:1:20, v/v/v), and ethyl acetate/1-butanol/water (14:1:15, v/v/v) were successfully used for the fractionation. The combination of HPLC–MS, diode array detection, and NMR analysis, as well as phloroglucinolysis, confirmed the structures of the isolated compounds: B1 [EC-(4β → 8)-C], B2 [EC-(4β → 8)-EC], B3 [C-(4α → 8)-C], B4 [C-(4α → 8)-EC], B5 [EC-4β → 6-EC], B7 [EC-(4β → 8)-C], [ECG-(4β → 8)-C], trimeric procyanidin C1 [EC-4β → 8-EC-4β → 8-EC], and the tetrameric procyanidin cinnamtannin A2 (where C: catechin, EC: epicatechin and ECG: epicatechin-3-O-gallate).