爵床抗H5N1禽流感病毒活性成分研究

H5N1 highly pathogenic avian influenza virus can break through species barriers and infect human beings directly, which is a serious threat to human life and health. In addition, the serious problem of few kind of antiviral agents and side effects, which make it extremely urgent to discover new efficient and low toxicity of anti H5N1 drugs. Currently, there is a growing interest to search for anti H5N1 substances with new mechanisms from natural sources. Justicia procumbens L. (Acanthaceae), known as “Jue chuang” in China, is a commonly used traditional herbal medicine embodied in Chinese Pharmacopoeia 1977 version, with clinical effects of reducing fire, removing toxic heat. The whole plant of J. procumbens mainly distributed in South China and Taiwan has long been used to treat cold, fever, cough, pain, inflammation of the throat and indigestion in children, etc. Several arylnaphthalide lignans showing cytotoxic and antivirus activities have been reported as main constituents of this plant. Preliminary tests for anti-H5N1 virus activity in vitro showed that the ethanol extract of J. procumbens exhibited obvious activity of virus-resistance, and some novel lignans and cyclopeptide alkaloids were observed. Based on the research, the herbs are going to be extracted with 60% ethanol, chemical constituents of fractions will be studied systematically and deeply to screen the anti H5N1 activity, the structure-activity relationship and mechanism of lead compounds or active fractions will also be investigated. Through this study, it will be expected to obtain fractions and lead novel compounds of significant anti H5N1 activity. The research is of great significance to clarify the chemical constituents and mechanism of J. procumbens and to develop the novel anti H5N1 drugs.

H5N1 禽流感病毒以其高致病性及高死亡率，突破了宿主的种属屏障，严重威胁了人类的生命健康。现有用于防治禽流感的药物种类较少，毒副作用较大，远不能满足临床和社会的需求，积极研究防治人禽流感的药物，尤其是从中草药中发现新作用机制的药物，已成为当前迫在眉睫的重要课题。爵床是爵床科植物爵床的干燥全草，药理研究表明具有明显抗肿瘤和抗菌抗病毒等多种生物活性。课题组前期研究发现爵床具有明显抗H5N1禽流感病毒活性，并发现了一些结构新颖的木脂素和环肽生物碱化合物，为探明爵床提取物抗H5N1禽流感病毒活性药效基础，拟对活性部位进行系统化学成分研究，并对所得化合物进行抗H5N1禽流感病毒活性筛选，阐明构效关系和作用机制。通过本研究以期能得到具有较好抗H5N1禽流感病毒活性的活性部位或结构新颖活性先导化合物，对阐明爵床抗H5N1禽流感病毒活性物质基础、作用机制及开发新型抗H5N1禽流感病毒药物都具有重要意义。

课题利用多种柱色谱和薄层色谱及物理方法对具有抗HAB/01株H5N1病毒活性的爵床三氯甲烷活性部位、乙酸乙酯活性部位和正丁醇活性部位进行了系统分离纯化工作，并利用质谱、UV、IR、1D/2D-NMR、ECD等波谱技术共完成了63个化合物的结构鉴定，其中新化合物13个，包括新的木脂素类化合物9个，新环肽生物碱4个，首次从该属植物中得到的化合物23个；首次在爵床属植物中发现环肽生物碱和倍半木脂素类化合物；探讨了芳基萘内酯类木脂素的旋转异构现象和波谱学特征。对部分可利用的爵床主要化学成分环肽生物碱和木脂素类化合物进行了抗H5N1禽流感病毒活性和体外抗肿瘤活性研究。.在爵床木脂素类抗H5N1禽流感病毒活性研究方面，课题选用犬肾传代细胞系MDCK细胞，通过CPE法初步筛选得到了两个木脂素类化合物(+)-Pinoresinol和Tuberculatin具有一定的抗H5N1禽流感病毒活性，可作为抗病毒药物研发的先导化合物。另外，课题还采用MTT法检测了爵床主要成分环肽生物碱和木脂素类化合物体外抗肿瘤活性。结果显示山荷叶素对HeLa、A549、H460细胞株的生长有较强的抑制作用，其IC 50值分别为：（3.47±0.78）、（8.79±4.95）、（16.10±6.61）µg/mL；1-苯代萘内酯型木脂素1、2、20、21、22、23、24、26对人结肠癌细胞株 LoVo 和人胃癌细胞株 BGC-823均显示强的细胞毒活性。环肽生物碱的细胞毒作用不明显。.课题进一步基于H5N1高致病性禽流感病毒药物靶点M2质子通道蛋白、血凝素蛋白、神经氨酸酶和非结构蛋白的结合位点，分别采用括Libdock、ligandfit、CDOCKER、Autodock Vina、GOLD五种对接方法对36个潜在化合物进行分子对接，并用组合了多种打分函数的“一致性”打分来评估小分子和受体的结合，旨在发现具有潜在活性的抑制剂及作用靶点。综合对接化合物结合构象、定位、结合模式以及打分一致性评价，最终筛选出一系列结合不同受体位点的化合物。对最终筛选出的新型化合物Justicianene A和H5N1神经氨酸酶复合物进行了分子动力学分析，将平均结构和晶体结构中配体构象进行叠合，得到了Justicianene A-NA相互作用方式，为新型化合物Justicianene A抗H5N1禽流感病毒抑制剂的开发提供科学依据。

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