新生大鼠原代肝细胞四氯化碳损伤模型与叶下珠保肝活性成分的提取与初步分离

Extraction and primary isolation of hepatoprotective active ingredients on Phyllanthus urinaria L.and carbon tetrachloride-injured models of primary hepatocytes in rats

目的:采用大鼠原代肝细胞四氯化碳化学性损伤模型,结合四甲基偶氮噻唑蓝比色法的体外药理模型,对传统中草药叶下珠的活性成分进行提取和初步分离,追踪叶下珠保肝护肝的活性单体成分。方法:实验于2006-05/12在浙江大学药学院中药开发及评价重点研究实验室完成。①制备新生大鼠原代肝细胞四氯化碳损伤的药理模型。②叶下珠提取物的制备:采用水、体积分数为0.95的乙醇对叶下珠活性成分进行回流提取。③活性成分的初步分离:依次用石油醚、氯仿、乙酸乙酯、正丁醇萃取;应用AB-8大孔吸附树脂分步洗脱法,即体积分数为0.1～0.9的乙醇洗脱。④采用四甲基偶氮噻唑蓝比色法,通过保护率及增生指数,观察回流提取物、各萃取物及AB-8大孔树脂各洗脱部位对大鼠四氯化碳损伤原代肝细胞的保护作用。结果:①正丁醇萃取物对大鼠四氯化碳损伤原代肝细胞的保护率及其增生指数高于阳性对照槲皮素及其他各萃取物(P<0.05～0.01)。②正丁醇萃取物中,体积分数为0.1,0.3,0.5的乙醇洗脱部分对大鼠四氯化碳损伤原代肝细胞具有保护作用,但体积分数为0.1的乙醇洗脱部分保护作用最强,显著高于阳性对照槲皮素及其他各处理组(P<0.01),体积分数为0.1乙醇洗脱部分在剂量为200g/L时,保护率为64.43%,增生指数达到2.952。结论:叶下珠保肝护肝的有效部位为正丁醇萃取物中AB-8大孔吸附树脂体积分数为0.1的乙醇洗脱部分。但叶下珠中保肝的有效单体成分还没有确定,有待进一步实验证明。

AIM： To extract and primarily isolate the active components from Phyllanthus urinaria L. and investigate the hepatoprotective potential of the monomer component in the pharmacological model of primary culture hepatocytes in rat following chemical injury induced by carbon tetrachloride. Hepatoprotective potential is tested by method of Methylthiazoletrazolium （MTT） colorimetry. METHODS： From May to December in 2006, an experiment was accomplished in the Key Laboratory of Traditional Chinese Medicine Development and Estimation in Zhejiang University Pharmaceutical College. （1）Newbom rat was used to prepare the pharmacological model that the hepatocytes ware injured by carbon tetrachlodde. （2） Preparation of Phyllanthus urinaria L. extracts： The active component of Phyllanthus urinaria L. was extracted by back-flow adopting water and alcohol, in which the volume fraction was 0.95. （3）Primary isolation of the active component： The extraction was performed in proper order with the petroleum ether, chloroform, ethyl acetate and n-butanol; The substep elution applied AB-8 big-hole for resin adsorptions, and adopted the alcohol of 0.1-0.9 volume fraction.（4）Using MTT colorimetry, the hepatoprotective potential of back-flow extract and eluUon by AB-8 big-hole for hepatocytes in rat injured by carbon tetrachloride was evaluated through the protection rate and the proliferation index. RESULTS： （1）The extract from n-butanol solvent had higher protection rate and the proliferation index than positive controls and other extracts （P 〈 0.05-0.01）.（2）In the extract of n-butanol, the elution by 0.1, 0.3, 0.5 volume fractions of alcohol had hepatoprotective potential, and that by 0.1 volume fraction of alcohol was the best, in which hepatoprotective potential was remarkably better than positive controls and other fractions （P 〈 0.01）. At the concentration of 200 g/L, the protective rate of elution by 0.1 volume fraction of alcohol was 64.43%, and the proliferation index was 2.952. CONCLUSION： The effective location of Phyllanthus urinaria L. to protect livers is AB-8 big-hole resin adsorptions at 0.1 volume fraction of alcohol in n-butanol extract. But the effective monomer of Phyllanthus urinaria L. does not ascertain and remains to be proved in the further experiment.