何首乌提取物对人正常肝细胞L02周期阻滞及凋亡的影响

目的：分析何首乌中致人肝细胞损伤物质的化学成分，初步探讨其致肝细胞损伤的机制。 方法：生、制何首乌用70%乙醇提取，提取物经AB-8型大孔树脂吸附分离，依次用水、50%乙醇和95%乙醇洗脱得到生何首乌和制何首乌水洗脱物、50%乙醇洗脱物和95%乙醇洗脱物；生、制何首乌直接水煎煮得到水提物。体外培养人正常肝细胞L02，用含何首乌洗脱物和水提物的细胞培养液与细胞共同孵育一定时间后，检测药物对L02细胞生长的影响，筛选致肝细胞损伤成分。高效液相色谱法对有明显细胞毒作用的洗脱物进行成分分析，噻唑蓝法检测其对L02细胞增殖的影响，Giemsa染色进行形态学观察，流式细胞术检测L02细胞周期和凋亡情况。 结果：生、制何首乌95%乙醇洗脱物对L02细胞有明显的生长抑制作用，其他组分对L02细胞的增殖无明显影响。成分分析显示，生、制何首乌95%乙醇洗脱物中大黄素含量最多，分别为（18.53±2.96）%和（10.28±1.34）%；何首乌95%乙醇洗脱物及大黄素可使L02细胞阻滞于S期，并诱导L02细胞凋亡。 结论：何首乌95%乙醇洗脱物是何首乌中导致肝细胞损伤的主要物质，而大黄素是导致肝细胞损伤的成分之一。

Effects of extract of Polygonum multiflorum on cell cycle arrest and apoptosis of human liver cell line L02

Objective： To analyze the chemical constituents of Polygonum multiflorum extract which may cause human liver cell damage and to explore the mechanism.
Methods： Raw and processed Polygonum multiflorum were extracted by 70% ethanol, then raw and processed Polygonum multiflorum water-eluted material （RW and PW）, 50% ethanol-eluted material （R50 and P50） and 95% ethanol-eluted material （R95 and P95） were obtained by absorbing through AB-8 macroporous resin, followed by water, 50% ethanol and 95% ethanol elution in order. The water extracts of raw and processed Polygonum multiflorum （RWE or PWE） were obtained by boiling them in water as usual. Normal human liver L02 cells were treated by different concentrations of eluted Polygonum multiflorum materials for different time, and the cell growth inhibition of each group was determined by methylthiazolyldiphenyl-tetrazolium bromide method. The chemical constituents which had a significant cytotoxicity to L02 cells were analyzed by high-performance liquid chromatography （HPLC）. Morphological changes of L02 cells were observed by Giemsa staining and cell cycle distribution was observed by flow cytometry.
Results： It was found that 95% ethanol-eluted extracts of raw and processed Polygonum multiflorum showed significant growth inhibition on normal human liver L02 cells, while the other components showed no significant inhibition on cell growth. HPLC analysis showed that the main component in 95% ethanol-eluted extract of raw and processed Polygonum multiflorum was emodin at content of （18.53±2.96）% and （10.28±1.34）% respectively. Cell cycle analysis showed that 95% ethanol-eluted material of Polygonum multiflorum and emodin had a similar significant effect of S phase arrest and all could induce L02 cell apoptosis.
Conclusion： The main part of Polygonum multiflorum causing liver cell damage is the 95% ethanol-eluted extract, and emodin is one of the important chemical constituents leading to liver cell damage.