茵陈的抗乙肝病毒活性成分和作用机制研究

Hepatitis B virus (HBV) infection is a serious health problem worldwide, especially in China. The current treatment strategies (vaccines, interferons and nucleosides) are unsatisfied. Therefore, new anti-HBV drugs with unique mechanism are still needed. Traditional Chinese medicine (TCM) with hepatoprotective and antiviral activity is fascinating sources for anti-HBV drug discovery, from which many anti-hepatitis leading compounds (silymarin, schisantherin A, oxymatrine, etc.) have been obtained. Yin-Chen (Artemisia capillaris, A. scoparia), as a famous TCM documented in every edition of "Chinese Pharmacopoeia" has long been used for treating acute and chronic hepatitis. Previous investigation reveals that coumarins, flavonoids, organic acids, alky(e)nes, chromones, etc. are the main constituents of A. capillaris and A. scoparia, of which scoparone, cirsimaritin, arcapillin, quercetin, chlorogenic acid, capillarisin, capillene are recognized to possess obviously hepatoprotective and cholagogic actvity. However, the anti-HBV active constituents of Yin-Chen (A. capillaris and A. scoparia) are still unclear. Our primary study suggested that the aquous ethanol (90% and 50%) and water extracts of A. capillaris and A. scoparia both showed anti-HBV activity. The total extract of A. capillaris (A. scoparia) was obtained by combination of its respective 90%, 50% aquous ethanol and water extracts, which was further loaded on D101 macroporous resin to give 5 fractions (Frs. 1-5). Anti-HBV assay on HepG2.2.15 cell line in vitro proposed that the water, 10%, 30% and 50% aquous ethanol eluted parts (Frs. 1-4) exhibited significant activity against the secretion of HBeAg, especially for the 10% and 30% aquous ethanol eluted parts of A. capillaris and 10% aquous ethanol eluted part of A. scoparia showed obvious activity inhibiting not only HBeAg secretion but also HBV DNA replication. In order to clarify their anti-HBV constituents, our subsequent investigation on the active parts (Frs. 2-3) of A. capillaris resulted in 20 compunds, with 10 active ones, especially for caffeic acid (4) and gallic acid (5) which showed potent activity inhibiting HBV DNA replication (IC50=3.03, 6.60 μg/mL) and HBeAg secretion (IC50=12.09, 6.51 μg/mL). This is the first time to characterize the anti-HBV active constituents from Yin-Chen. Therefore, further bioassay-guided investigation on A. capillaris and A. scoparia will be very significant for clarifying their anti-HBV constituents. In this project, systematically chemical and biological study including anti-HBV constituents (inhibiting HBsAg, HBeAg, HBV DNA in vitro and DHBV DNA in vivo), chemical modification, and mechanism investigation will be conducted on the active part of A. capillaris and A. scoparia. The successful implementation of this project (if granted) will provide scientific basis not only for developing new anti-HBV leading compounds from the TCM of Yin-Chen, but also for expanding its clinical usage.

中国是乙型肝炎感染大国，现有临床治疗药物有限。基源为菊科茵陈蒿和滨蒿的茵陈具清热利湿、利胆退黄等功效，是著名抗肝炎中药，临床治疗急、慢性肝炎疗效显著。茵陈主要含有香豆素、黄酮、有机酸、炔烯等，前人已报道其保肝利胆活性成分，未见抗乙肝病毒（HBV）活性成分研究。我们首次发现茵陈蒿和滨蒿的90%、50%乙醇和水提取物均具有抗HBV活性，特别是两者总提取物经大孔树脂柱层析得到的10%和30%乙醇洗脱流分具较强抗HBV DNA和HBeAg活性。进而从茵陈蒿活性部位分离鉴定20个化合物，发现10个活性成分。在此基础上，采用天然药物化学、现代药理学和分子生物学等方法，通过在HepG2.2.15细胞模型上测定其对HBsAg、HBeAg和HBV DNA抑制作用，开展茵陈蒿和滨蒿活性成分分离、鉴定、结构修饰、体内抗鸭乙肝病毒活性和作用机制研究，为揭示茵陈抗HBV活性成分和扩大其临床应用提供化学和药理学基础。

乙型肝炎是由乙肝病毒（HBV）引起的影响人类健康的严重疾病，目前应用的抗HBV 药物不能满足治疗需要，亟需开发高效低毒、作用机制新颖的抗HBV新药。茵陈为我国传统抗肝炎中药，具有保肝利胆等活性，然而其抗HBV活性成分尚未见报道。《中国药典》收载的茵陈基源植物为菊科植物茵陈蒿（A. capillaris）和滨蒿（A. scoparia）。因此本项目主要针对两种基源植物的抗HBV活性成分开展研究。.根据研究计划，本研究对茵陈蒿和滨蒿开展了系统的抗HBV活性研究，共分离鉴定123个单体成分，发现新化合物11个，73个化合物对HBV具有抑制活性。特别是在LCMS导向下从茵陈蒿的活性部位分离得到9个绿原酸类似物，具有明显的抗HBV活性。化合物41-43和47-49对HBV DNA复制的IC50在5.5-13.7μM 之间，SI大于115.0。二咖啡酰基取代的衍生物 (47-49) 不仅对HBV DNA的复制具有较强抑制活性，对HBeAg抑制的IC50分别为71.6, 135.5和161.8。羧基酯化的三个衍生物44-46对HBV DNA的复制也具有一定的抑制作用，IC50分别为272.3，175.3以及144.7μM。两个桉烷倍半萜11 和12对HBV DNA复制具有显著的抑制作用，其IC50值分别为 19.7和12.0 μM，选择指数分别为105.5和139.2。化合物12对HBsAg 和HBeAg 的分泌也具有较强的抑制活性，IC50值分别为15.0以及9.0 μM，选择指数分别为111.3 和185.9。在LCMS的导向下，从滨蒿的活性部位分离得到1个新颖的4-吡啶酮糖苷和2个烯炔糖苷酯，其抑制HBV DNA的IC50分别为0.07（SI=23.6）和0.12（SI=17.1）mM。合成28个对羟基苯乙酮衍生物，18个化合物对HBV DNA的复制具有抑制活性，总结了其构效关系。进一步对两个活性化合物进行了作用机制研究。.本项目发表SCI论文5篇，其中学科领域Top15%论文2篇；申请专利4项，其中1项已获得授权；培养硕士研究生2名；项目申请人获资助期间晋升为副研究员，并入选中科院青年创新促进会会员（2013）和第十三批昆明市中青年学术和技术后备人选（2015），获得国家自然科学基金面上项目（2015）和中科院西部之光西部青年学者A类资助（2015)。