UPLC-QTOF/MS分析芫花诱导人肝细胞L02损伤的毒性物质基础

目的: 研究芫花提取物诱导人肝细胞L02损伤的毒性物质基础。 方法: 选取人肝细胞L02作为体外实验模型,运用MTT法测定芫花提取物对细胞增殖的影响,并且采用超高效液相色谱串联四级杆飞行时间质谱(UPLC-QTOF/MS)对芫花提取物及与L02细胞亲和后胞内化学成分进行定性分析。 结果: 芫花提取物对L02细胞的生长抑制呈明显的剂量依赖关系,48 h的IC₅₀为(48.34±4.66)mg·L^-1。通过UPLC-QTOF/MS鉴定的黄酮类成分主要有:芹菜素、3'-羟基芫花素、芫花素、5,4'-二羟基-7,3'-二甲氧基黄酮;二萜原酸酯类成分主要有:芫花酯戊、genkwanine L、芫花酯丙、芫花烯、芫花酯丁、芫花酯庚、芫花酯乙、芫花酯己、芫花酯甲。在芫花提取物亲和的L02细胞中鉴定出3'-羟基芫花素、芫花素、5,4'-二羟基-7,3'-二甲氧基黄酮、芫花酯戊、芫花烯、芫花酯丁、芫花酯乙、芫花酯甲。其中二萜原酸酯类成分芫花酯甲作用L02细胞48 h的IC₅₀为(29.57±2.01) mg·L^-1,黄酮类成分芫花素(0.1～100 mg·L^-1)未发现对L02有显著的细胞毒作用。 结论: 芫花提取物对人肝细胞L02具有细胞毒作用,其中二萜原酸酯类成分与细胞有明显的亲和作用,并具显著的细胞毒作用,是芫花提取物中主要的活性物质。

Identification of Toxic Components Inducing L02 Injury in Genkwa Flos by UPLC-QTOF/MS

Objective: The aim of the present study is to identify the toxic substances in Genkwa Flos extract which could induce human liver cell line L02 injury. Method: MTT assay was applied to assess human liver cell line L02 proliferation after incubation with Genkwa Flos extract. The chemical constituents in Genkwa Flos extract and their affinity with L02 cell were measured by ultraperformance liquid chromatography-quadrupole time of flight/mass spectrometry(UPLC-QTOF/MS). Result: Genkwa Flos extract exhibited inhibitive effect on L02 cell in a dose-dependent manner. IC₅₀ value was (48.34±4.66) mg·L^-1 after 48 hours incubation with L02 cell. Four flavonoids, such as apigenin, 3'-hydroxygenkwanin, genkwanin, and velutin, were detected in Genkwa Flos extract. Nine diterpene orthoesters were also detected in Genkwa Flos extract, including yuanhuapine, genkwanine L, ynanhuafine, genkwadaphnin, yuanhuatin, yuanhuagine, yuanhuadine, yuanhuajine, and yuanhuacine. The substances in Genkwa Flos extract that had affinity with L02 were 3'-hydroxygenkwanin, genkwanin, velutin, yuanhuapine, genkwadaphnin, yuanhuatin, yuanhuadine, and yuanhuacine. Among these components, yuanhuacine inhibited the L02 proliferation in a dose-dependent manner with the IC₅₀ of (29.57±2.01) mg·L^-1 after 48 hours, while genkwanin(0.1-100 mg·L^-1) had no markedly toxic activity on L02. Conclusion: Genkwa Flos extract can inhibit the growth of human liver cell line L02 in vitro. Diterpene orthoesters exhibit cellular affinity with L02 cell and show cytotoxicity, indicating that Diterpene orthoesters are the main substances contributing to the toxicity of Genkwa Flos.