江南星蕨抗HBV活性成分的定向发现及其构效关系和作用机制研究

Chronic hepatitis B (CHB) still severely does harm to human health, and screening nontoxic and effective anti-HBV drugs appears to be inevitable. Microsorium fortunei is a famous herbal of Miao, Mulao, Tujia, and other ethnic minorities. And investigation found that it is also used in the folk for the treatment of liver disease. Our pre-research showed that the extracts of Microsorium fortunei showed obvious anti-HBV activity. Guided by active tracking, an active fraction Fra.6I was obtained from the extracts by separating through solvent extraction, and chromatography with silica, sephadex LH-20 and ODS, respectively. And the inhibition ratio of Fra.6I (25 mg•L-1) to HBSAg and HBeAg of HepG2.2.15 cells could reach up to 98.2% and 99.1%, respectively. From this result, we can see clearly that the fraction Fra.6I may contain highly active anti-HBV compounds. Based on the fact, this project, in combination with active tracking method, intends to get the novel leading compound of anti-HBV activity from Microsorium fortunei through further separating, structural modifications and research of the possible mechanisms of the active compounds in vitro and in vivo experiments. The results may provide a new approach for the effective prevention and control of HBV.

乙型肝炎病毒（HBV）依然严重危害人类健康，挖掘开发高效低毒抗HBV药迫在眉睫。江南星蕨是苗族、仫佬族和土家族等多个少数民族的名药，调研发现它在民间还有治疗肝病之用。我们前期研究结果显示，江南星蕨提取物具有明显的抗HBV活性。在活性追踪指导下，该提取物用溶剂萃取后，经硅胶、凝胶和反相硅胶分别柱层析分离，我们从中得到了活性组分Fra.6I。经测定，Fra.6I在25 mg•L-1时，对HepG2.2.15细胞的HBsAg和HBeAg抑制率分别为98.2%和99.1%。这说明Fra.6I可能含有高活性抗HBV单体化合物。因此，本项目拟在此基础上进一步活性追踪分离，以明确其药效单体化合物，并通过结构修饰和体内、体外活性作用研究，初步明确其构效关系和可能作用机制，以期从江南星蕨发现新颖的抗HBV活性先导化合物，为HBV的有效防治提供新的途径。

研发新型有效的抗HBV药物依然是当前社会之急需。江南星蕨是我国多个少数民族使用的民间药，前期研究发现它的提取物具有明显的抗HBV活性。结合活性追踪，我们从中分离鉴定了约30个化合物，并筛选出其主要活性单体化合物为七叶内酯。通过结构修饰和体外活性作用评价，初步明确七叶内酯的构效关系。另外，通过研究，发现在HepG2.2.15细胞模型中，七叶内酯能抑制HBV抗原和HBV DNA的表达，且七叶内酯对乙型肝炎病毒X（HBx）蛋白表达的抑制作用呈剂量依赖性；在鸭乙型肝炎病毒（DHBV）感染的雏鸭模型中，经七叶内酯处理后，DHBV-DNA、鸭乙型肝炎病毒表面抗原（DHBsAg）、鸭乙型肝炎病毒e抗原（DHBeAg）、丙氨酸氨基转移酶（ALT）、天冬氨酸氨基转移酶（AST）水平显著下降。从而提示七叶内酯具有良好的抗HBV作用，并且该作用与通过抑制病毒的DNA合成，影响DNA复制，下调HBx蛋白表达，进而促进病毒细胞死亡有关。该研究结果，为进一步开发七叶内酯为抗HBV药物提供了科学依据。