Identification Of A Hydroxymethylfurfural-cysteine Adduct And Its Absorption And Cytotoxicity In Caco-2 Cells

5-Hydroxymethylfurfural?HMF?is an endogenous contaminant with high content in thermally processed foods.Among the amino acids,L-cysteine?L-Cys?shows the highest activity for the elimination of HMF.In the previous study,4 adducts were found to be formed between HMF and L-Cys under thermal processing condition.However,the safety of these adducts is unknown.Inthecurrentstudy,anHMF-cysteineadductnamed1-Dicysteinethioacetal-5-hydroxymehtylfurfural?DCH?was identified as the main adduct,contributing for HMF elimination,under gastric condition.Highly purified DCH was then prepared to investigate its changes during in vitro digestion,and to test its effect toward Caco-2cells,in comparison with HMF.1.Synthesis and identification of DCH.DCH was prepared through the reaction of HMF with L-cysteine?1:8?at pH 3.0?adjusted with HCl?,90°C for 9 h.After purification using HP-20 macroporous resin and ODS,one adduct with purity 99%was obtained;which was identified?using HPLC-MS/MS and NMR?as1-dicysteinethioacetal-5-hydroxymehtylfurfural.It is a thioacetal with two molecules of L-Cys and one molecule of HMF;its molecular formula is C₁₂H₁₈N₂O₆S₂?MW=350.40?.2.Changes of DCH during in vitro digestion.In L-Cys/HMF mixture,DCH was found to be formed during in vitro digestion with high stability.The higher pH,the more HMF was eliminated.However,lower pH favored DCH formation at the stage of gastric digestion,suggesting that the elimation of HMF at higher pH in intestinal digestion might be attributed to the formation of other adducts.At the end of gastric digestion,continuous formation of DCH was suggested to be provided by the transformation from other adducts with the evidence that no further elimination of HMF was detected at this stage.3.Toxicity and absorption against Caco-2 cells.Compared with HMF,DCH displayed lower cytotoxicity against Caco-2 cells with an IC₅₀value of 31.26 mM vs 14.95 mM?HMF?.The DCH did not induce cell apoptosis,whereas HMF significantly increased the apoptosis rate after incubation at concentrations of 16,and 32mM for 72 h.DCH showed considerably lower absorption rate than HMF by Caco-2 cells.Lower absorption of DCH may result in lower toxicity against Caco-2 cells compared with HMF.The intracellular transformation of DCH has also been observed.