丙烯醛-丙氨酸加合物制备与细胞毒性

Preparation and Cytotoxicity of Acrolein-Alanine Adduct

丙烯醛是热加工食品中存在的有害物质,形成后易与氨基酸反应生成加合物,但目前对其加合物的毒性研究较少。本研究设置不同丙烯醛与丙氨酸浓度比(2:1、1:1、1:2、1:3、1:4),于37℃和50℃下分别水浴反应0.5、3、5、6 h,发现丙烯醛与丙氨酸浓度比1:2在50℃反应5 h所获得目标加合物产率最高。通过反相硅胶柱层析进行分离纯化后,经高效液相和液相色谱-质谱联用分析,结果显示该加合物最大吸收波长为220 nm,纯度达95%以上。高分辨质谱和核磁共振鉴定结果表明,该加合物相对分子质量为183.0823,是由一分子丙氨酸与两分子丙烯醛发生迈克尔加成反应后,经羟醛缩合形成的含氮六元环化合物。采用加合物培养人正常胃黏膜细胞(GES-1)24 h和48 h后发现,加合物半抑制浓度(half maximal inhibitory concentration,IC_(50))分别为3.286、0.869 mmol/L,显著降低了丙烯醛(IC_(50)分别为0.058、0.067 mmol/L)的细胞毒性,说明丙氨酸可作为食品中丙烯醛的潜在清除剂。

Acrolein(ACR)is a food toxin produced during thermal processing and shows high reactivity with amino acids to form adducts.However,the toxicity of the adducts is seldom investigated until now.In this study,ACR was reacted with alanine(Ala)at various molar ratios(2:1,1:1,1:2,1:3 and 1:4)in a water bath at 37 or 50℃for various durations(0.5,3,5 and 6 h).The highest yield of adduct was attained with an ACR to Ala ratio of 1:2 at 50℃for 5 h.After separation and purification by reversed-phase silica gel column chromatography,the purity of the adduct reached more than 95%,and it displayed maximum absorption at 220 nm as determined by high performance liquid chromatography(HPLC)and liquid chromatography-mass spectrometry.Analysis by high-resolution mass spectrometry and nuclear magnetic resonance(NMR)showed that the relative molecular mass of the adduct was 183.0823,which was a nitrogen-containing six-membered ring compound formed by the Michael addition between one molecule of Ala and two molecules of ACR followed by an aldol condensation.After incubating human normal gastric mucosal cells(GES-1)for 24 and 48 h,it was found that the cytotoxicity of the adduct(with half maximal inhibitory concentration(IC_(50))of 3.286 and 0.869 mmol/L)was significantly lower than that of its parent toxin,ACR(with IC_(50)of 0.058 and 0.067 mmol/L).This finding indicates that Ala may act as a potential scavenger of ACR.