Separation of a family of antioxidants flavan-3-ol thio-conjugates from procyanidins by high-speed counter-current chromatography

A series of thiol-nucleophiles, captopril, l -cysteine, and tiopronin, were introduced to the degradation reaction of grape seed extracts and the eight new bioactive flavan-3-ol derivatives were observed. Then, these derivatives were successfully separated and prepared in large quantity by high-speed counter-current chromatography (HSCCC) and semi-preparative HPLC under the optimized separation conditions. The appropriate two solvent systems consisted of n- hexane – ethyl acetate – methanol – water (0.05:1.5:0.5:1.2, v/v/v/v) and n- hexane – ethyl acetate – water (1:10:10, v/v/v) with head – tail mode were used for HSCCC two-step separation of captopril degradation products. N- butanol – ethyl acetate – methanol – water (2:4:0.5:6, v/v/v/v) with a combination of tail – head and head – tail elution modes in HSCCC run of l- cysteine degradation products, and n- hexane – ethyl acetate – methanol – water (0.15:1.6:0.4:1, v/v/v/v) was applied with head – tail and tail – head elution modes in HSCCC separation of tiopronin degradation products. The structures of these derivatives were confirmed by MS and 1 H NMR analysis. Moreover, a systematic comparison of antioxidant activity of all the eleven degradation products was first implemented and found that most of the derivatives had stronger antioxidant activity than free catechins.