[摘要]通过观察何首乌中蒽醌类成分对HepG2细胞的细胞毒作用，并通过精密肝切片技术对细胞毒成分进行验证，探讨何首乌致肝毒性的物质基础。运用MTT法检测何首乌中游离蒽醌、结合蒽醌及柰类共11个单体成分对HepG2细胞的毒性。将有明确细胞毒的成分与大鼠肝切片共同培养6 h后制备肝组织匀浆，通过BCA 法测定匀浆液中蛋白含量，连续监测法测定并计算每1 μg蛋白中丙氨酸氨基转移酶（ALT）、天门冬氨酸氨基转移酶（AST）、γ谷氨酰氨基转肽酶（GGT）和乳酸脱氢酶（LDH）的漏出率，以考察这些成分对肝组织的毒性作用。细胞试验结果显示，只有大黄酸、大黄素、大黄素甲醚8OβD葡萄糖苷和大黄素甲醚8O（6′O乙酰基）βD葡萄糖苷显示一定的细胞毒作用，其IC50分别为7107，12562，24227，40232 μmol·L-1，而其他7个化合物的毒性很小。肝切片试验结果显示，大黄酸400 μmol·L-1组能引起肝切片ALT，AST，LDH漏出率的显著升高（P<001），100 μmol·L-1组能引起肝切片 LDH漏出率的显著升高（P<005）；随药物浓度增大，肝切片中蛋白含量显著下降（P<005），显示有一定的量毒关系。大黄素400 μmol·L-1组可引起肝切片ALT，GGT，LDH漏出率的显著升高（P<001）。大黄素甲醚8OβD葡萄糖苷800 μmol·L-1组能引起肝切片ALT，AST，LDH漏出率的显著升高（P<001或P<005），200 μmol·L-1组能引起肝切片LDH漏出率的显著升高（P<005）；且随药物浓度的增大肝切片中ALT，AST，LDH漏出率呈升高趋势，蛋白含量呈下降趋势。此外，浓度为800 μmol·L-1的大黄素甲醚8OβD葡萄糖苷还可使肝切片MTT还原能力显著下降（P<001）。结果提示，大黄酸、大黄素及大黄素甲醚8OβD葡萄糖苷只有在高浓度（≥400 μmol·L-1）时才可能对肝组织产生一定的损害作用。但是，据文献报道这3个成分的体内暴露水平都很低，要达到毒性浓度（400 μmol·L-1或800 μmol·L-1）的暴露水平，转化为健康成人至少分别需要单次口服4 898，339，5 581 g的何首乌药材，这与何首乌的临床使用剂量（生首乌3～6 g，制首乌6～12 g）相差甚远，因此，“蒽醌类成分是何首乌肝毒性成分”这一说法是缺乏科学依据的。
[关键词]何首乌；蒽醌类成分；肝细胞毒性；精密肝切片技术；肝毒性物质基础
[Abstract]By observing the cytotoxic effects of anthraquinones on HepG2 cell and using the precisioncut liver slices technique to authenticate the cytotoxic constituents， the paper aims to explore the material basis of Polygonum multiflorum root to cause liver toxicity Firstly， MTT method was used to detect the effect of 11 anthraquinone derivatives on HepG2 cell Then， the clear cytotoxic ingredients were cocultured with rat liver slices for 6h respectively， and the liver tissue homogenate was prepared BCA method was used to determine the content of protein in the homogenate and continuous monitoring method was used to monitor the leakage of alanine aminotransferase （ALT）， aspartate aminotransferase （AST）， gammaglutamine amino transpeptidase （GGT） and lactate dehydrogenase （LDH） The toxic effect of these ingredients on liver tissue was tested by calculating the leakage rate of the monitored enzymes As a result， rhein， emodin， physcion8OβDglucopyranoside and physcion8O（6′Oacetyl）βDglucopyranoside showed cytotoxic effects on HepG2 cell and their IC50 values were 7107， 12562， 24227， 40232 μmol·L-1 respectively， but the other 7 compounds are less toxic and their IC50 values can not be calculated The precisioncut liver slices tests showed that rhein group of 400 μmol·L-1 concentration significantly increased the leakage rate of ALT， AST and LDH （P<001）， and the rhein group of 100 μmol·L-1 concentration only increased the leakage rate of LDH （P<005） With the increase of rhein concentration， the protein content in liver slices decreased significantly （P<005） with a certain range of does Emodin group of 400 μmol·L-1 concentration significantly increased the leakage rate of ALT， GGT and LDH （P<001） Physcion8OβDglucopyranoside group of 800 μmol·L-1 concentration also significantly increased the leakage rate of ALT， AST and LDH （P<001 or P<005）， but the group of 200 μmol·L-1 concentration only significantly increased the LDH leakage （P<005） Along with the increase of the concentration of physcion8OβDglucopyranoside， the leakage rate of ALT， AST and LDH showed a trend of increase， but the protein content in liver slices was in decline Furthermore， MTT reduction ability of liver slices significantly decreased （P<001） in the physcion8OβDglucopyranoside group of 800 μmol·L-1 concentration The results suggested that rhein， emodin and physcion8OβDglucopyranoside at high concentrations （≥400 μmol·L-1） can produce some damage to the liver tissue However， the exposure levels of these constituents are very low， so to reach the toxic concentration （400 μmol·L-1 or 800 μmol·L-1） an adult of 65 kg body weight will need at least a single oral 4 898 g， 339 g and 5 581 g of Pmultiflorum root respectively， which is far from the statutory dose of crude P multiflorum root （36 g） or its processed product （612 g） Therefore， the conclusion that anthraquinones are the prime constituents of the hepatotoxicity of P multiflorum root are still not be proved