Fractionation and characterization of high molecular weight proanthocyanidin from persimmon fruit by thiolysis-HPLC, size-exclusion chromatography, MALDI-TOF/MS, and NMR

High molecular weight proanthocyanidin (condensed tannin) from persimmon pulp was fractionated on Toyopearl TSK-HW-50-F. The crude tannin and the three fractions were characterized by thiolysis-HPLC-ESI-MS, GPC, MALDI-TOF-MS and 13C-NMR. Thiolysis-HPLC-ESI-MS showed that the proanthocyanidin terminal units were catechin and epigallocatechin gallate, and extender units were epicatechin, epigallocatechin, (epi)gallocatechin-3-O-gallate, and (epi)catechin-3-O-gallate. The crude tannin had a very high prodelphinidin content (65–80%) and a high degree of 3-O-galloylation (72%). The composition of the fractions and the unfractionated tannin was similar, but the fractions were distinguished by degree of polymerization. Thiolysis suggested that the persimmon tannin was comprised of polymers ranging from 13kD to 20kD (degree of polymerization 30–50), but sizes estimated by GPC were much smaller. MALDI-TOF-MS revealed the presence of a heteropolyflavanol series including (epi)catechin and (epi)gallocatechin repeating units, and suggested that the persimmon proanthocyanidin contained some A-type interflavan linkages. The crude material was gently chemically degraded with acid to yield products that were amenable to NMR analysis, which was used to confirm the A-type linkages.

Acknowledgements: Financial support was provided by the National Natural Science Foundation of China (No.30972398), and the Key Project of Chinese Ministry of Education (No.109115) to Huazhong Agricultural University, and by Agricultural Research Services Specific Cooperative Agreement Number 58–1932–6-634 with Miami University.