Identification of 2″‐Galloylated Flavonol 3‐O‐Glycosides Accumulating in Developing Leaves of Persimmon

ABSTRACT Introduction Dried leaves of persimmon, traditionally consumed as a herbal tea in Japan, contain bioactive galloylated flavonol glycosides. Investigation of the seasonal compositional changes in these compounds is important for determining the optimum harvest time. Objective The three objectives were: (1) to elucidate the compositional changes of the flavonol glycosides in persimmon leaves collected in different seasons; (2) to develop an efficient method for separation of the constituents; and (3) to compare their antioxidant activities. Methodology Fresh persimmon leaves were collected at different growth stages in 2007 and 2008 in Niigata, Japan. Total flavonols were extracted with 70% aqueous acetone followed by ethyl acetate partitioning, and were analysed by high‐performance liquid chromatography. Enzymatic transformation and chromatographic fractionation was performed to isolate the individual flavonols. Two antioxidant assays were performed. Results Four nongalloylated flavonol glycosides were detected at the leaf‐shooting stage, and four additional galloylated flavonol glycosides accumulated during leaf development, resulting in a total of eight constituents. Isolation of nongalloylated and galloylated constituents was successfully achieved through enzymatic transformation of the flavonol mixture using combinations of tannase, β ‐glucosidase and β ‐galactosidase, followed by chromatographic fractionation. The gallates were identified as regiospecific 2″‐galloylated galactosides and glucosides of kaempferol and quercetin. A mixture of 2″‐galloylated flavonol glycosides had a two‐fold stronger antioxidant activity than the nongalloylated mixture. Conclusion This study showed that four 2″‐galloylated flavonol glycosides had accumulated in developing leaves of persimmon by the end of May through a rapid 2″‐galloylation of the corresponding nongalloylated flavonol glycosides. Copyright © 2011 John Wiley & Sons, Ltd.