肝细胞亲和色谱法体外筛选大黄降脂活性成分研究

Screening the Lipid-lowering Active Ingredients in Rhubarb by the Hepatocyte Affinity Chromatography in Vitro

目的:评价大黄蒽醌类成分体外降脂活性及筛选其药效成分,探讨其在细胞内的成分代谢。方法:采用体外Hep G2细胞系脂肪变性模型评价大黄蒽醌类成分降脂活性,采用肝细胞亲和色谱法筛选大黄蒽醌类细胞亲和成分,以HPLC法及HPLC-Q-TOF-MS法检测鉴定亲和成分。结果:与正常组相比,建立的体外肝细胞脂肪变性模型组细胞内三酰甘油(TG)含量显著升高(P<0.05),与模型组比较,不同浓度的五种大黄蒽醌类成分作用后给药组细胞内TG含量均显著降低(P<0.05),呈剂量效应关系;肝细胞亲和色谱法筛选出大黄提取液特异性亲和色谱成分主要有4个,分别为芦荟大黄素、大黄素、大黄酚、大黄素甲醚四个化合物,而大黄酸发生了细胞内代谢。结论:大黄蒽醌类5种成分均具有体外降脂活性,芦荟大黄素、大黄素、大黄酚、大黄素甲醚以原型的形式体外肝细胞降脂,而大黄酸可能以细胞内代谢为芦荟大黄素形式降脂。本研究建立了肝细胞亲和色谱法与体外肝细胞降脂活性评价的联用技术,实现了中药成分体外降脂活性评价,药效成分体外代谢的一体化研究,为大黄蒽醌类调脂药物开发提供科学依据。

Objective： To screen and analysis of antihepatic steatosis active components and cell metabolism withinrhubarb anthraquinones in vitro. Methods： Lipid-lowering activity of liver cell affinity components in rhubarbanthraquinones was evaluated by hepatic steatosis in vitro model. Hepatocyte-specific affinity compositions werescreened by hepatocyte affinity chromatography. Hep G2 cells were incubated with rhubarb anthraquinones in vitro andwere detected by HPLC and HPLC-Q-TOF-MS method. Results： Compared with normal group, the intracellular TGcontent of model group were significantly increased（P〈0.05）. Respectively treated with different concentrations ofrhubarb anthraquinones, the intracellular TG content were significantly decreased in a dose dependent manner（P〈0.05）in comparison with model group. Compared with rhubarb solution, there are mainly four hepatocyte-specific affinitycompositions of aloe-emodin, emodin, chrysophanol and physcion, except for rhein. Conclusion： The lipid-lowering experiment in vitro verified that five rhubarb anthraquinones have significant lipid-lowering activity. This research established a biochromatography method to screen out four hepatocyte-specific affinity compositions of aloe-emodin,emodin, chrysophanol and physcion from rhubarb anthraquinones, while the conjugation sites of rhein were ambiguous.Combined hepatocyte affinity chromatography with hepatic steatosis in vitro model, it could evaluate and screen lipidlowering active ingredient in vitro and cell metabolism of Chinese traditional medicine for the drug development of lipidlowering rhubarb anthraquinones.