Changes in polyphenols of the seed coat during the after-darkening process in pinto beans (Phaseolus vulgaris L.).
Proanthocyanidins and flavonoids were isolated and identified from seed coats of two aged and nonaged pinto bean lines: 1533-15 and CDC Pintium. The seed coat of 1533-15 darkens slowly and never darkens to the same extent as CDC Pintium. Analysis of the overall level of proanthocyanidins using a vanillin assay demonstrated that aged and nonaged seed coats of CDC Pintium had significantly higher levels of proanthocyanidins than aged and nonaged 1533-15 seed coats. Aged and nonaged seed coats of both lines were found to contain one main flavonol monomer, kaempferol, and three minor flavonols, kaempferol 3-O-glucoside, kaempferol 3-O-glucosylxylose, and kaempferol 3-O-acetylglucoside. These compounds were identified by NMR and ESI-MS analysis (except for kaempferol 3-O-acetylglucoside, which was tentatively identified only by ESI-MS analysis) and quantified using HPLC-DAD. The combined concentrations of all the kaempferol compounds in seed coats of CDC Pintium were significantly higher than in seed coats of 1533-15, and the combined contents did not change after aging. The content of kaempferol decreased nearly by half in the seed coats of CDC Pintium after aging, whereas no significant change was observed in the seed coats of 1533-15. Proanthocyanidin fractions from both lines, aged and nonaged, were subjected to LC-MS/MS analysis and found to be composed primarily of procyanidins. Procyanidins in the seed coats were predominantly polymers with the degree of polymers higher than 10. The proportion of these polymers decreased after aging, while that of the low-molecular-weight procyanidins increased. A catechin-kaempferol adduct was tentatively identified in both lines by LC-MS/MS, and the concentration increased in the seed coats after aging.