胆固醇氧化物的鉴定、制备及其对大鼠肝细胞的毒性

Identification and Preparation of Cholesterol Oxides and Evaluation of Their Toxicity toward Buffalo Rat Liver Cells

本文用气相色谱-质谱联用仪对胆固醇高温氧化物进行鉴定,采用硅胶柱色谱法对氧化物进行分离,并由MTT法检测其对大鼠肝细胞BRL的毒性。结果显示:胆固醇在高温下的主要氧化物有5种,分别为:7α-羟基胆固醇、7β-羟基胆固醇、5,6β-环氧基胆固醇、5,6α-环氧基胆固醇、7-酮基胆固醇。单因素实验表明在160℃下加热30 min可以产生最大量的胆固醇氧化物,并在此条件下对胆固醇氧化物进行制备。粗制品经过硅胶柱色谱洗脱收集,可得纯度约为90%的胆固醇氧化物(组成为:7α-羟基胆固醇9.96%、7β-羟基胆固醇23.24%、5,6β-环氧基胆固醇15.34%、5,6α-环氧基胆固醇14.36%、7-酮基胆固醇26.77%、胆固醇3.12%、其它杂质7.21%)。MTT法实验结果表明纯化后的胆固醇氧化物在80μg/m L浓度下对大鼠肝细胞BRL表现出显著毒性作用(p<0.05),在100μg/m L时,细胞活性仅有空白对照的7.49%,胆固醇氧化物的毒性机理有待进一步研究。

Cholesterol oxides generated during high temperature were characterized by gas chromatography-mass spectrometry（GC-MS）.Silicon column chromatography was used to isolate the oxides,and their toxicity toward buffalo rat liver（BRL） cells was evaluated by MTT assay.The results indicated that there were five major cholesterol oxides generated under high temperature：7α-hydroxycholesterol,7β-hydroxycholesterol,5,6 β-epoxycholesterol,5,6α-epoxycholesterol,and 7-ketocholesterol.Single factor experiments showed that the maximum amount of cholesterol oxides were obtained after heating at 160 ℃ for 30 min;therefore,this condition was used for the preparation of cholesterol oxides in this study.The crude products were purified by silicon columns,and a cholesterol oxide product with a purity of about 90% was obtained（composition：9.96% 7α-hydroxycholesterol,23.24% 7β-hydroxycholesterol,15.34% 5,6β-epoxycholesterol,14.36% 5,6α-epoxycholesterol,26.77% 7-ketocholesterol,3.12% cholesterol,and 7.21% other impurities）.According to the result of the MTT assay,the purified cholesterol oxides showed significant（p〈0.05） toxicity to BRL cells at a concentration of 80 μg/m L,and the cell viability after a treatment at a concentration of 100 μg/m L was only 7.49% of the viability of untreated control cells.The toxicity mechanism of cholesterol oxides needs to be further explored.