基于多指标综合检测优选藏药砂生槐子总生物碱提取工艺及其抗肝纤维化机制研究

Optimization of Extraction Technology of Total Alkaloids from Sophora Moorcroftiana(砂生槐子)Based on Multi Index Comprehensive Detection and Its Mechanism of Anti-hepatic Fibrosis

目的:考察砂生槐子总生物碱最佳提取工艺,评价其抗肝纤维化作用。方法:采用HPLC法检测砂生槐子中氧化苦参碱、槐果碱、槐定碱和苦参碱的含量,利用L9(34)正交试验考察提取的最佳工艺。以CCl4诱导大鼠实验性肝纤维化,检测第3、6、9周空白组、模型组、秋水仙素组、砂生槐子高、中、低剂量组的ALT、AST、HA、TGF-β和Hyp指标,并对血清中miRNA-181b、miRNA-16、miRNA-200a、miRNA-126进行测定。结果:砂生槐子粉碎粒度60目,加15倍量70%乙醇加热提取2次,每次1 h,可得4种生物碱的含量分别为80.10、3.92、9.26、19.60μg/g。在实验周期内,砂生槐子高、中、低3个剂量组各检测指标均低于模型组,且具有浓度依赖性,表明总生物碱对肝纤维化有明显抑制作用,且与浓度成正相关。高剂量组miRNA-181b表达明显下降,miRNA-16、miRNA-200a、miRNA-126表达明显上升。结论:经正交试验优化提取可得最佳提取工艺,且总生物碱对CCl4诱导大鼠实验性肝纤维化有一定的治疗作用,且作用机制可能与其作用于肝星状细胞有关。

Objective： To investigate the optimum extraction technology of total alkaloids from sophora moorcroftiana and evaluate its anti-hepatic fibrosis effect. Methods： The content of oxymatrine, sophocarpine, sophoridine and matrine in sophora moorcroftiana were detected by HPLC, and the best extraction process was investigated by orthogonal test of L9（34）. The experimental liver fibrosis was induced by CCl4 in rats. The indexes of ALT, AST, HA, TGF-β and Hyp in blank group, model group, colchicine group, high, middle and low Sophora moorcroftiana dose groups were detected in the 3, 6, 9 weeks, and serum contents of miR-181 b, miR-16, miR-200 a and miR-126 were determined. Results： Sophora Moorcroftiana with particle size of 60 mesh were heated2 times in 15 volume of 70% ethanol, and the contents of 4 alkaloids were 80.10, 3.92, 9.26 and 19.60 mg/g,respectively. In the experimental period, all indexes of the high, middle and low Sophora moorcroftiana dose groups were lower than the model group with a concentration dependent manner, which showed a significant inhibitory effect of the total alkaloid on liver fibrosis. The expression of miR-181 b in high dose group decreased significantly, while the expression of miR-16, miR-200 a and miR-126 increased significantly. Conclusion： The optimum extraction process can be obtained through the optimization of orthogonal test. The total alkaloid has a certain therapeutic effect on CCl4 induced experimental hepatic fibrosis in rats and the mechanism may be related to its action on hepatic stellate cells.