Identification And Cytotoxicity Of Schiff's Base Formed Between Hydroxymethylfurfural And L-Lysine

5-Hydroxymethylfurfural?HMF?is an endogenous toxin produced during the thermal processing of foods.Our previous research showed that some amino acids can react with HMF to form Schiff's bases and other products.The product may underestimate the actual exposure level of HMF in the food.With an active?-amino group,lysine is an amino acid most reactive during Maillard browning and can react with HMF.However,there are few studies on the reaction products of lysine and HMF,and their toxicity is not clear.This study identified and isolated the Schiff's base formed by the reaction of lysine?-NH₂ and HMF.A method for detecting the Schiff's base content in food was established.The toxicity of Schiff's base was tested on three cell lines:normal gastric mucosal epithelial cells?GES-1 cells?,colon cancer cells?Caco-2 cells?and human umbilical vein cells?EA.hy926 cells?.The main relults were as follows:1.Preparation and identification of Schiff's baseThe effects of four factors on the elimination rate of HMF were investigated,namely pH,reaction temperature,reaction time,and ratio of reactants.Based on the single factor experiment,Schiff's base was prepared under the following conditions:HMF and L-Lys at a molar ratio of 1:2 were reacted at pH 11,160°C for 25 min in an oil bath.Under this condition,HMF elimination rate was 74.7%.The HP-20 macroporous resin and ODS column were successively used to separate and purify the target product.The molecular formula of the compound was C₁₂H₁₈N₂O₄ and its relative molecular mass was 254.13,determined using HRMS.NMR confirmed its structure as a Schiff's base formed by Maillard reaction between carbonyl group in HMF and?-amino group of lysine.2.Detection of Schiff's base in foodA quantification method of Schiff's base in foods was established by liquid chromatography-mass spectrometry?LC-MS/MS?.The food sample was defatted by n-hexane,ultrasonically extracted in water,and concentrated by rotary evaporation to obtain sample solution,which was determined by LC-MS/MS.The quantitative ion pair and the qualitative ion pair were m/z 255.1/84.1 and m/z 255.1/130.0 respectively.The content of Schiff's base was calculated using a standard curve.In the mode of electrospray ion source positive ion multi-detection,the content of Schiff's base in five foods ranged from 0.05 to 1.90?g/kg.Its content was much lower than that of HMF?from 9.98 to 12.54 mg/kg?.3.Effect of Schiff's base on cell proliferationThree cell lines were incubated with Schiff's base and HMF for 24 h and 48 h to compare their cell toxicity.Compared with HMF,the cytotoxicity of Schiff's decreased in GES-1 cells,Caco-2 cells,EA.hy926 cells.After 48 h of culture,the IC₅₀ of Schiff's base in three cell lines increased by 25,6.3 and 108 times in comparison to HMF,respectively.4.Mechanism and absorption of Schiff's base in Caco-2 cells?1?Caco-2 cells were cultured with 10 mM,20 mM HMF and Schiff's base for 48 h to investigate the effects of HMF and Schiff's base on Caco-2 cell cycle and apoptosis.Both HMF and Schiff's base can block a large number of cells in the S phase,affecting the normal division of Caco-2 cells.HMF showed a dose-dependent manner,while Schiff's base did not.The results of apoptosis showed that Schiff's base can reduce the toxic effect on Caco-2 cells by reducing late apoptosis of cells compared with HMF.?2?Caco-2 cells were cultured with 2 mM HMF and Schiff's base for 0¹50 min to explore their absorption in Caco-2 cells.After 150 min,the absorption rate of HMF and Schiff's base by Caco-2 cells reached 8.45%and 10.52%,respectively.However,the intracellular content of two drugs in Caco-2 cells was very low.We speculated that the absorbed drugs were converted to other substances by metabolism or interact with other substances.