三种鹊肾树属植物抗HBV活性苯丙素的定向发现和作用机制

Despite that some approved drugs and genetically engineered vaccines agaigst hepatitis B virus (HBV) are available for HBV patients, HBV infection is still a severe public health problem in the world. It is urgently needed to discover and develop new anti-HBV drugs. The discovery of novel anti-HBV compounds from traditional Chinese medicine by the anti-HBV activity model is an important research. Phenylpropanoids are important constituents of plants and offer great promise as potentially effective new anti-HBV drugs..Streblus genus are used as anti-HBV agents by Zhuang medicine and Yao medicine in Guangxi province, China. In our preliminary work, we found extracts of one kind of Streblus genus exhibited good anti-HBV activities. 20 phenylpropanoids including 7 new ones were got from MeOH extract of S. tonkinensis by anti-HBV-activity-guide isolation. Some compounds exhibited better anti-HBV activity than that of Lamivudine. In this project, firstly, all kinds of modern skills for separation and structural determination are employed to isolate and identify constituents of the effective fraction from S. tonkinensis, S. ilicifolius and S. macrophyllus by anti-HBV-activity-guide. Secondly, the isolated phenylpropanoids are going to be screened for anti-HBV activities in vitro. Thirdly, for the phenylpropanoids with excellent activities, molecular biological and pharmacological methods such as ELISA, PCR are going to be applied to explore their possible anti-HBV mechanism. In addition, anti-HBV effects of the active phenylpropanoids are going to be investigated in vivo using a duck model. Finally, phenylpropanoids with high anti-HBV activities are expected to be found. The aim of this study is to discover the anti-HBV phenylpropanoids from the three kinds of Streblus genus. This research may provide theoretical and experimental data for further exploiting the abundant Streblus genus resources of our country.

乙型肝炎病毒严重危害人类健康，发现高活性的抗HBV药物迫在眉睫。从传统中药中发现抗HBV新活性分子是药物研究的重要方向。苯丙素化合物是植物的重要成分，是抗HBV先导分子发现的重要来源。. 鹊肾树属植物是传统抗HBV壮药，研究发现该属植物含有丰富的抗HBV苯丙素分子，前期从该属另一种植物得到20个苯丙素化合物(有7个新化合物)，活性筛选发现新化合物ST-7具有很好的抗HBV活性，新化合物ST-1抑制HBV DNA的复制。在已有基础上，项目拟以植物亲缘关系和化合物结构相似为线索，在抗HBV活性跟踪下，利用紫外特征吸收、DAD-HPLC和HPLC-MS分析技术指导，定向快速分离鹊肾树属米扬噎、刺桑和双果桑的抗HBV活性苯丙素化合物；运用ELISA、PCR等手段探讨高活性化合物在体外和体内发挥抗HBV活性的作用机制；用动物模型初步探讨化合物的成药效果；期望发现低毒、高活性的抗HBV分子。

项目按计划进行，研究内容有米扬噎、刺桑和双果桑的总成分的提取、不同极性成分群的分离、生理活性追踪、活性部位的分离提纯、单体成分的结构测定、活性成分的测定、活性化合物体外抗HBV的可能作用机制等，以上计划都已经全部完成。具体内容如下：.①. 确定了米扬噎、刺桑和双果桑抑制乙肝病毒的的活性和有效部位：分别用石油醚、氯仿、乙酸乙酯和正丁醇对上述各植物的甲醇提取物进行萃取，对各萃取物进行抑制乙肝病毒HBsAg、HBeAg表达试验，实验结果证实米扬噎、刺桑和双果桑甲醇提取物的乙酸乙酯和正丁醇萃取物是这3种植物抑制乙肝病毒HBsAg、HBeAg表达的活性部分。.②.确定从米扬噎、刺桑和双果桑抑制乙肝病毒的活性部位分别得到的38个、54个和32个化合物。这些化合物中有30个新化合物，在余下的大部分已知化合物都是首次从这3种植物中分离得到。.③ 确定了抑制乙肝病毒活性成分：将分离得到的木脂素类、香豆素类和苯丙烯类等苯丙素类化合物进行抑制乙肝病毒HBsAg、HBeAg表达和HBV DNA复制活性筛选，发现6个化合物都具有抑制HepG2.2.15细胞肝炎病毒HBsAg和HBeAg表达的活性，它们的抑制HBsAg 的IC50值范围在40.36 µM至89.60 µM之间，抑制HBeAg 的IC50值范围在20.68 µM至56.98 µM之间。1个化合物显示有抑制肝炎病毒HBV DNA复制的活性，它的IC50值为110.58 µM。.④ 确定活性化合物体外抗HBV作用机制：选择1个活性高、量大的苯丙素化合物，利用HepG 2.2.15细胞模型，测定用药后上清液中的HBV DNA水平，以及细胞内的HBV DNA和cccDNA水平，确定化合物抑制HBV DNA复制的活性。.该课题研究完成了课题任务书约定的各项技术指标，到目前为止，发表论文11篇（10篇为SCI收录）、投稿在审论文2篇（均为SCI收录期刊）、完成3篇硕士研究生论文，培养博士研究生2人（在读）；培养硕士研究生6人。.该课题成果研究开发内容完整，系统的化学与药效研究成果明确米扬噎、刺桑和双果桑的功效和活性部位，为开发米扬噎、刺桑和双果桑抗乙肝新药源和新产品打下了理论和应用基础。