高效液相色谱法测定大黄素在Caco-2细胞中的摄取  

Analysis the Absorption of Emodin in Caco-2 Cells by HPLC

作　　者：滕增辉[1] 周四元[1] 刘新友[2] 冉玉华[1] 杨润涛[1] 张邦乐[1] 杨茜[1] 刘仁旺[1] 梅其炳[1] 

机构地区：[1]第四军医大学药学系药理教研室 [2]第四军医大学唐都医院药剂科,陕西西安710032

出　　处：《现代生物医学进展》2007年第5期677-679,共3页Progress in Modern Biomedicine

基　　金：国家863项目资助课题;编号2003AA22347C

摘　　要：目的:采用反相高效液相色谱法,观察大黄素在Caco-2细胞中的摄取特点。方法:将大黄素与Caco-2细胞共同孵育,收集细胞样品,液氮反复冻融。取细胞裂解液,加入甲醇提取,提取液采用HPLC进行分析。色谱分析柱为C18柱(250mm×4．6mm,5μm,Diamonsil),流动相组成为85%乙腈及15%水(含0．1%乙酸),流速1ml·min-1,进样量20μl,柱温25℃,3D模式采集数据。结果:检测Caco-2细胞中大黄素的工作曲线的回归方程为Y=0．278x+0．148(Y=0．9996,n=5),线性范围为0．037～4．8μmol·L-1,最低检测浓度为0．018μmol·L-1。当细胞中大黄素的浓度为0．05、2和8．5μg·ml-1时,回收率分别为(101．3±7．3)%、(96．7±3．0)%和(98．7±2．1)%(n=5);相应的日内标准偏差分别为0．25%、2．9%和1．4%;相应的日间标准偏差分别为2．3%、5．6%和6．3%。大黄素在Caco-2细胞中的摄取达峰时间为10分钟,峰浓度为108．56±11．57 nmol/L·mg·protein,10分钟后Caco-2细胞中大黄素的含量迅速下降。浓度处于2-50μM之间时,Caco-2细胞对大黄素的摄取量呈线性增加,浓度达50μM后,随着剂量的增加大黄素的摄取量变化不明显。结论:大黄素可被Caco-2细胞迅速摄取,随着剂量的增加,大黄素在Caco-2细胞中的摄取存在饱和现象。Objective： To observe the absorption characteristic of emodin in Caco-2 cells by the method of RP-HPLC. Methods：Human intestinal epithelial Caco-2 cells were used to investigate the effects of time and dose on the uptake of emodin. The drug-contalning cell was freeze-thawing circulation at liquid nitrogen for three times to get the cell lysate. Finally, the absorbed substrate was extracted from the cell lysate with methanol and was analyzed by reverse phase HPLC system. The mobile phase consisted of 85% of acetonitrile and 15% of 0.1% acetic acid, and diamonsil C18 column （250×4.6 mm, 5 μm） was used. The column temperature and flow rate were 25 ℃ and 1.0ml/min, respectively. Results：The regression equation of standard curve was Y= 0.278x- 0.148 （γ=0.9996, n=5）,the linear range and lower limit of quandtadon was 0.037- 4.8·mol-L^-1 and 0.018μmol·L^- 1, respectively. At 37℃, the intracellular accumulation of emodin increased rapidly during the first 10 min and reached 108.56 ±11.57 nmol/L-mg-protein. When concentration of emodin varied from 2μM to 50 μM, the intracellular emodin increased almost linearly. However, the curve showed saturation was observed when concentration of emodin increased from 50 μM to 200 μM. Conclusions：This method with high sensitivity and good repetitiveness can be used to observe the intestinal absorption and metabolism characteristics of emodin. The emodin can be absorbed by the caco-2 cell rapidly, and the intracellular concentration show up the saturation with the increase of dosage.

关 键 词：大黄素 CACO-2细胞 高效液相色谱 

分 类 号：R91[医药卫生—药学]