何首乌、虎杖、大黄水提物中游离蒽醌含量的测定及对人正常肝细胞的毒性作用

Toxicity of Polygoni Multiflori Radix, Polygoni Cuspidati Rhizoma et Radix and Rhei Radix et Rhizoma in HepaRG cells

目的:测定何首乌、虎杖、大黄3种中药水提物中游离蒽醌含量,比较3种中药水提物对人肝祖细胞Hepa RG的体外细胞毒作用,探讨何首乌、虎杖、大黄中游离蒽醌含量与肝细胞毒性的关系。方法:煎煮法制备何首乌、虎杖、大黄3种受试饮片水提物,采用高效液相色谱色谱法分别同步测定3种水提物中5种主要游离蒽醌大黄素、大黄素甲醚、芦荟大黄素、大黄酸、大黄酚的含量;分别以浓度为1.0、1.5、2.0、2.5、3.0 mg/m L的3种中药水提物溶液处理Hepa RG细胞48 h后,加入噻唑兰检测其活力;分别以1.5、2.0、2.5 mg/m L的3种中药水提物溶液处理Hepa RG细胞48 h后,AnnexinⅤ/PI双染后流式细胞术检测凋亡细胞比例。结果:何首乌水提物中主要游离蒽醌含量为0.229%,虎杖水提物中为0.545%,大黄水提物中为2.593%;何首乌水提物(2.0、2.5、3.0 mg/m L)、虎杖水提物(1.5、2.0、2.5、3.0 mg/m L)、大黄水提物(1.0、1.5、2.0、2.5、3.0 mg/m L)均可抑制Hepa RG细胞活力,且抑制程度与主要游离蒽醌含量呈正相关;细胞凋亡实验结果表明,3种中药水提物(1.5、2.0、2.5 mg/m L)均明显诱导Hepa RG细胞凋亡,且凋亡率与主要游离蒽醌含量呈正相关。结论:3种中药水提物中主要游离蒽醌含量由低到高依次为何首乌、虎杖、大黄,该含量与其体外肝细胞毒性作用成正相关,提示游离蒽醌可能是3种中药产生肝细胞毒性的重要成分。

OBJECTIVE:To investigate correlations between toxicity in Hepa RG cells and free anthraquinonecontents in aqueous extracts from Polygoni Multiflori Radix(PMR),Polygoni Cuspidati Rhizoma et Radix(PCRER)and Rhei Radix et Rhizoma(RRER). METHODS:High performance liquid chromatography was used to detect thecontent of emodin, physcion, aloe emodin, rhein and chrysophanol from the three aqueous extracts. MTT assaywas employed to measure Hepa RG cell viability following treatment with theextracts in different concentrations(1.0,1.5,2.0,2.5,3.0 mg/m L) for 48 h. Hepa RG cells were also treated in concentrations of 1.5,2.0,2.5 mg/m L forthe detection of apoptosis by Annexin Ⅴ/PI dual staining for FCM. RESULTS: The contents of freeanthraquinoneins in the three extracts were 0.229%,0.545%,2.593% respectively. The survival rate of Hepa RGcells was down-regulated by PMR(2.0,2.5,3.0 mg/m L),PCRER(1.5,2.0,2.5,3.0 mg/m L) and RRER(1.0,1.5,2.0,2.5,3.0 mg/m L) in dose-dependent manners. The degree of inhibition was positively correlated with thecontent of free anthraquinones. The same trend was observed in apoptosis cell proportion in which theconcentrations of extracts were 1.5,2.0,2.5 mg/m L. CONCLUSION:The contents of free anthraquinones in thethree aqueous extracts from low to high degree were PMR, PCRER and RRER. The content was positivelycorrelated with cytotoxicity in vitro, which suggests that free anthraquinones might be the important componentinducing toxicity in these three traditional Chinese medicines.