Separation and purification of four phenolic compounds from persimmon by high-speed counter-current chromatography

An efficient method was established by high-speed counter-current chromatography (HSCCC) for preparation of four phenolic compounds from the depolymerization products of persimmon tannin. Using the two solvent systems of n-hexane/ethyl acetate/water (3:17:20, v/v/v) and ethyl acetate/methanol/water (50:1:50, v/v/v), the preparative isolation was successfully performed by a two-step separation. The yields of one run (150 mg crude sample) for gallic acid, methyl gallate, and epigallocatechin-3-gallate-(4β → 8, 2β → O → 7)-epigallocatechin-3-gallate dimer (A-type EGCG dimer) were 4.7, 44.2 and 5.9 mg, respectively. In addition, 4.6 mg epicatechin-3-gallate-(4β → 8, 2β → O → 7)-epicatechin-3-gallate dimer (A-type ECG dimer) was obtained by further preparative high-performance liquid chromatography (prep-HPLC). The purities of these compounds were all above 95.0% and their structures were identified by HPLC/ESI-MS. We found that HSCCC had definite advantages for the preparation of dimeric procyanidins compared with previous methods. Furthermore, it was shown that the four phenolic compounds possessed greater antioxidant activities than Trolox.