| 题名 | 蛇莓抑制NA的活性成分鉴定及不同NA抑制剂协同作用 |
| 作者 | 王宇 |
| 学位类别 | 硕士 |
| 答辩日期 | 2015-05 |
| 授予单位 | 中国科学院研究生院 |
| 授予地点 | 中国科学院长春应用化学研究所 |
| 导师 | 徐经伟 |
| 关键词 | 蛇莓 流感 神经氨酸酶抑制剂 协同作用 |
| 中文摘要 | 流行性感冒又称流感，是由流感病毒引起的急性呼吸道感染，传染性强，传播速度快。根据世界卫生组织《急性呼吸道感染病报告》（2009年2月更新）统计，全球每年流感大流行期间流感病例约为10亿，重症病例为300万例，并有25万至50万人因此死亡。流感的危害非常严重，而流感病毒则是这一危害的罪魁祸首。 目前疗效最好的抗流感药物是神经氨酸酶抑制剂奥司他韦（oseltamivir）和扎那米韦（zanamivir），但流感病毒惊人的突变能力造成耐药病毒株的出现以及药物本身的副作用，使得扎那米韦和奥司他韦的应用遭到限制。因此，要有效遏制流感，新的抗流感病毒药物的研发和使用迫在眉睫。神经氨酸酶（NA）是存在于A型和B型流感病毒表面的一种糖蛋白，它在子代病毒释放和扩散中有着至关重要的作用，主要表现：①促进新生病毒从感染的细胞中释出；②阻止子代病毒从宿主细胞中释出后的聚集。在所有A型和B型流感病毒的NA都有一致性，即：其活性空腔均位于亚单位表面的口袋中，且催化位点的氨基酸残基均高度保守，因而可作为抗流感病毒药物的靶点。 我国有着丰富的中草药资源，在几千年的发展历史中，中草药在预防抵抗感冒方面功不可没。在对400多种传统中药的提取物进行以甲型流感病毒神经氨酸酶为靶点的系统筛选时，筛选出了五种对流感病毒神经氨酸酶抑制作用显著的中药：蛇莓、路路通、苦楝皮、夏枯草、紫草。我们选取了蛇莓为研究对象，对其具有神经氨酸酶抑制作用的物质基础进行了研究。 将蛇莓全草粉碎，利用工业酒精冷浸提取得到蛇莓提取物的总的粗浸膏。依次用石油醚、乙酸乙酯、正丁醇进行萃取，得到极性不同的石油醚部位、乙酸乙酯部位、正丁醇部位和水部位的提取物。总提取物和各分提取物对流感病毒神经 氨酸酶抑制作用的药理活性筛选结果表明，乙酸乙酯部位对神经氨酸酶的抑制作用最强。对该部位进行分离，最终得到五个有活性的单体化合物。并以槲皮素为阳性对照药，对这五种单体化合物进行了对甲型流感病毒神经氨酸酶体外抑制作用的评价。 抗流感药物的疗效在一定范围内与使用剂量成正比，但随着用药剂量的增加，因为用药而发生的毒副作用的风险也会相应的随之增加。作为抗流感药物的神经氨酸酶抑制剂亦不例外。寻找并发现有协同作用的抗流感药物可以使抗流感药物在治疗安全范围内有着更好的疗效：减少流感病毒株出现耐药性的可能性；降低用药剂量，减少因使用抗流感药物而产生的毒性。为此，本次论文选取了木犀草素等十几种不同的神经氨酸酶抑制剂，将其分别与蛇莓中分离鉴定出的对神经氨酸酶有抑制作用的活性成分混合，采用荧光测定法测试其体外对神经氨酸酶的抑制作用。实验结果表明木犀草素、黄芩素分别与委陵菜酸等蛇莓的活性成分混合后的体外抗流感作用增强，有望开发出新的抗流感药物配方。 |
| 英文摘要 | Influenza is caused by influenza virus. It’s acute respiratory tract infection, possessing strong infectivity and traveling fast. According to the World Health Organization, each year there is about 1 billion of pandemic flu cases, including 3 millions of severe cases and 250000 to 500000 people died as a result. The harm of flu is very serious and the flu virus is the culprit. At the present, the best anti-flu drugs are neuraminidase inhibitors, like oseltamivir and zanamivir. However the emergence of resistant strains caused by the influenza virus’s phenomenal ability of mutation and the side effects of the drug itself make the applications of oseltamivir and zanamivir limited. To effectively curb the flu, development for a new anti-influenza virus drug is imminent. Neuraminidase (NA) exists in type A and type B influenza virus. NA is one kind of glycoprotein on the surface of influenza virus. NA plays a vital role in the progeny virus release and diffusion, mainly in two sides: ①to promote the new virus released from infected cells ②to prevent the progeny virus from gathering when released from the host cell. All NA of type A and type B influenza virus have one consistency, namely: the active pockets are located on the surface of the cavity in the unit, and amino acid residues on the catalytic site are highly conserved, which can be used as anti flu drug targets. Our country has a rich resource of Chinese herbal medicine and during the several-thousand-year development history. Chinese herbal medicines are important in the aspect of prevention against cold. When screening on more than 400 kinds of traditional Chinese medicine extract by influenza virus neuraminidase targets system, we found that five kinds of traditional Chinese medicine (TCM) had significant neuraminidase inhibitory effect: Duchesnea indica (Andr.) Focke, Fructus Lipuidambaris Liquidambar taiwaniana Hance, Lithospermum erythrorhizon Sieb. et Zucc., Melia toosendan Sieb. et Zucc., Prunlla vulgaris L. We chose the Duchesnea indica (Andr.) Focke as the research object to study the active substance. Duchesnea indica (Andr.) Focke was shattered into pieces and extracted by industrial alcohol. Then the crude extract was extracted with petroleum ether, ethyl acetate and n-butanol in turn and we got different polar parts: petroleum ether, ethyl acetate parts, n-butanol and water area. The crude extract and the extracted parts were tested in vitro to know their influenza virus neuraminidase inhibitory effects. Screening results showed that ethyl acetate part had the strongest neuraminidase inhibitory effect. We separated this part and finally found five active monomer compounds. In the research, we evaluated the anti-NA effect of the five compounds in vitro with quercetin as the positive control drug. The curative effect of anti-influenza drugs within a certain range is proportional to the dosage. However, with the increase of dosage, the risk of the side effects of drugs will be increased accordingly. It’s the same for NA inhibitors as a kind of anti-flu drugs. To find concurrent existing synergy anti-flu drugs can make anti-flu drugs have a better curative effect in the safety range: reducing the appearence of drug-resistant flu strains, reducing the dosage of anti-flu drugs and reducing the toxicity due to use of anti-flu drugs. As a result, we selected dozens of NA inhibitors including Luteolin and texted the anti-NA effect by way of Fluorimetry when used with the active anti-NA ingredients of Duchesnea indica (Andr.) Focke. The experimental results showed that the anti-NA effects of Luteolin and Baicalein were increased when used respectively with the active anti-NA ingredients of Duchesnea indica (Andr.) Focke in vitro, which may resulted in a new anti-flu drug formulations. |
| 语种 | 中文 |
| 公开日期 | 2016-05-25 |
| 内容类型 | 学位论文 |
| 源URL | [http://ir.ciac.jl.cn/handle/322003/65049]  |
| 专题 | 长春应用化学研究所_长春应用化学研究所知识产出_学位论文 |
| 推荐引用方式 GB/T 7714 | 王宇. 蛇莓抑制NA的活性成分鉴定及不同NA抑制剂协同作用[D]. 中国科学院长春应用化学研究所. 中国科学院研究生院. 2015. |
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