BackgroundThis report represents the second part (Part 2) of a two-part report on investigating a novel hepatoprotective substance in ume extract. The heated juice concentrate of Japanese apricot (Ume, Prunus mume Sieb. et Zucc.), popularly called ume extract, is known for its health benefits. In Part 1 of this study (presented in this issue together), we reported the identification of the active substance underlying the hepatoprotective potential of ume extract for the first time. The substance, tentatively referred to unknown 1 (UK1), was shown to have the molecular formula C10H11NO9 with a molecular weight of 289. Here (in Part 2), we aimed to decipher the structure of UK1. Moreover, we show that UK1 is formed by heating the components of ume juice. MethodsThe purified UK1 (≥93.88% purity, containing 6.11% hydrate water) was subjected to nuclear magnetic resonance (NMR) spectroscopic analysis. The structure of UK1 was elucidated by one-dimension (1D)-NMR (1H, 13C, and DEPT135) and 2D-NMR (COSY, HSQC, and HMBC) experiments. Further, the artificial synthesis of UK1 was attempted by heating aqueous solutions containing citric acid and L-asparagine (L-Asn) or L-aspartic acid (L-Asp). The synthetic experiments were performed using a series of solutions of varying concentrations (1-fold, 5-fold, 10-fold, and 15-fold) of citric acid and L-Asn (molar ratio 5: 1). The molar ratio of citric acid and L-Asn (5: 1) was specified to mimic that in ume juice. ResultsThe chemical structure of UK1 was determined as 2-[3-(carboxymethyl)-3-hydroxy-2,5-dioxopyrrolidin-1-yl]butanedioic acid. More simply, UK1 was identified as an imide in which citric acid was bound to Asp. UK1 could be synthesized by heating (110 °C for 4 h) a solution containing citric acid and L-Asn, or citric acid and L-Asp. The yield of UK1 was dependent on the concentrations of citric acid and L-Asn. ConclusionsThe findings of this study further validate the conclusions made in Part 1 that UK1 is a novel hepatoprotective substance in ume extract. Based on the structure, UK1 is referred to as “ASP citrimide.” This study demonstrates that ASP citrimide is formed by the association of citric acid and L-Asn (or L-Asp) during the heat concentration of ume juice.