Identification of (−)-epicatechin as the direct substrate for polyphenol oxidase from longan fruit pericarp

Postharvest browning of longan fruit results in a short life and a reduced commercial value. The experiments were conducted to separate, then purify and finally identify the polyphenol oxidase (PPO) substrates that cause longan fruit to brown. PPO and its substrates were, respectively, extracted from longan fruit pericarp tissues. The substrate for longan PPO was separated and purified using polyamide column chromatography, Sephadex LH-20 column chromatography and silica gel column chromatography, respectively. The substrate was further identified by 0.5% FeCl3 solution and enzymatic reaction with longan PPO. On the bases of UV, 1H NMR, 13C NMR, and ESI-MS data, the direct substrate for the PPO from pericarp tissues of longan fruit was identified to be (−)-epicatechin. Furthermore, the contents of (−)-epicatechin of pericarp tissues of longan fruit of two major cultivars were determined by high performance liquid chromatography (HPLC). The HPLC analysis exhibited that the contents of (−)-epicatechin of fruit pericarp of ‘Shixia’ and ‘Chuliang’ were 0.26 and 0.56 mg/g on fresh weight (FW) basis at harvest and 0.15 and 0.09 mg/g FW after 3 days of storage. The more rapid decrease in the (−)-epicatechin content of ‘Chuliang’ was due to the oxidization catalyzed by PPO, which was in agreement with the higher browning index.