肝细胞亲和-HPLC法体外筛选三七总皂苷(PNS)抗肝细胞脂肪变性的活性成分

In vitro screening active components within PNS against hepatic steatosis using liver cell extraction coupled with HPLC analysis

目的通过肝细胞亲和-HPLC法体外筛选三七总皂苷(PNS)中降脂活性成分。方法采用HepG2细胞系与PNS体外共孵育,联合HPLC法检测孵育后的细胞解离液,色谱柱为Aglient-C_(18)(250 mm×4.6mm,5μm),流动相为乙腈-水,梯度洗脱(0~35 min,乙腈的体积百分比为29%;70~100 min,乙腈的体积百分比为29%→40%),流速为1.0 mL/min,检测波长为203 nm;采用体外肝细胞脂肪变性模型对PNS中亲和成分的降脂活性进行评价。结果从PNS中筛选出2个肝细胞特异性亲和成分,分别为人参皂苷Rb_1和人参皂苷Rg_1;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),不同浓度的PNS、人参皂苷Rb_1和人参皂苷Rg_1作用后给药组细胞内TG含量显著降低(P<0.05),呈剂量效应关系。结论建立肝细胞亲和-HPLC法,筛选出PNS亲和成分为人参皂苷Rb_1和人参皂苷Rg_1,经体外模型验证亲和成分具有显著降脂活性。

Objective To screen antihepatic steatosis active components within Pauax Notoginseng Saponins （PNS） using liver cell extraction with HPLC analysisin in vitro. Methods HepG2 cells were incubated with PNS in vitro, then we detected the cell dissociation solution by using HPLC method. The chromatographic condition was Aglient-C18（ 250 mm×4.6 mm, 5 microns） column, mobile phase of acetonitrile-water, gradient elution （ 0- 35 min, the volume of acetonitrile percentage is 29%; 70-100 min, aeetonitrile volume percent of 29%-40%）, flow rate of 1.0 mL/min and detection wavelength of 203 nm. Then, the hepatic steatosis in vitro model was used to evaluate liver cell affinity ingredients in PNS towards lipid-lowering activity. Results In this way, two hepatocyte-specific affinity components, ginsenoside Rb1 and Rg1 ginsenosides from PNS, were found. Compared with the normal group, the model group significantly increased intracellular TG content （P〈0.05）. Treatment with different concentrations of PNS, ginsenoside Rb- and Rgj ginsenosides, the intracellular TG content were decreased significantly in a dose dependent manner （P〈 0.05）. Conclusion This research establishes a biochromatography method to screen out the effective ingredients ginsenosides Rb1 and ginsenosides Rg1. The lipid-lowering experiment in vitro verifies that ginsenosides Rb1 and ginsenosides Rg1 have potent liDid-lowering activity.