一种基于神经氨酸酶固定化亲和色谱的抗流感病毒中药活性成分在线筛选平台的建立

Epidemic influenza is an acute viral respiratory tract infectious disease caused by viruses，which is a highly infective agent. The neuraminidase（NA）encoded by virus has vital role in the life cycle of the virus for its recognition, entry and release from the cells they infect, NA inhibitors have been developed and used as an effective and promising influenza drugs. The usage of current NA inhibitors in clinic is limited because of their high price, single-target mechanism and drug-resistance development and therefor，the research of novel NA inhibitors with new structure scaffold are needed urgently. Some of the compounds in Traditional Chinese Medicines have different structural backbones compared with the marketed NA inhibitors, they have shown a potential in solving drug resistance problem and in overcoming the NA subtype dependence problem of current drugs． The establish and development of a fast and accurate screening method to find the NA inhibitor from TCM is necessary for the discovery of anti-flu medication. In the research, we will establish a platform, which combines novel immobilized NA affinity chromatography with poly (HEMA-EDMA) monolithic column, RP-HPLC and mass spectrum technology, in order to search NA inhibitor in the flavonoids extracted from the aerial part of Bupleurum plants, and the inhibitor activity of the screened flavonoids will be verified with in vivo and in vitro activity test. This research can provide not only an effective tool for the discovery of novel anti flu drug, but also significant reference for researches aimed at effective utilization of resources of Bupleurum.

流行性感冒是由病毒引起的、严重危害人类健康的一种传染性疾病。神经氨酸酶（NA）在病毒传播过程中发挥了重要作用，以 NA 为药物靶点筛选抑制剂，是研究和开发抗流感病毒药物的重要途径。中药是抗流感活性成分的天然宝库，与现有的抗流感药物相比，中药价格低廉、靶点多样、且不易产生耐药性。如果能够建立一种快速、准确的活性筛选方法，寻找并确定中药中的神经氨酸酶抑制性成分，将会为抗流感药物的发现做出巨大贡献。本研究拟利用申请团队已有的亲和色谱制备经验和优势，首次尝试将NA酶固定于整体聚合 (HEMA-EDMA)毛细管柱上，构建前沿亲和色谱-反相色谱-多级质谱的二维在线筛选平台，以具有抗流感作用的柴胡地上部分黄酮类化合物为对象，进行NA高亲和成分筛选，并辅以药理学手段对筛选出的成分进行体内、外NA抑制活性验证。本研究将为寻找和发现中药抗流感成分开拓新思路，也为阐明柴胡抗病毒药效物质基础提供了有力工具。

流行性感冒是由病毒引起的、严重危害人类健康的一种传染性疾病。神经氨酸酶（NA）在病毒传播过程中发挥了重要作用，以 NA 为药物靶点筛选抑制剂，是研究和开发抗流感病毒药物的重要途径。中药是抗流感活性成分的天然宝库，与现有的抗流感药物相比， 中药价格低廉、靶点多样、且不易产生耐药性。如果能够建立一种快速、准确的活性筛选方法，寻找并确定中药中的神经氨酸酶抑制性成分，将会为抗流感药物的发现做出巨大贡献。. 本研究成功建立了神经氨酸酶抑制剂的筛选模型，分别使用了苦参碱、杨梅苷、黄芩素、山柰酚、山柰素对神经氨酸酶的活性抑制化合物筛选模型进行了验证，证明神经氨酸酶活性抑制化合物筛选模型能够用于来源于天然药物的单一化合物的筛选。并将活性筛选模型直接用于中药提取物中具有神经氨酸酶活性抑制作用的成分筛选，并成功建立了LC-MS分析检测平台。. 本研究将为寻找和发现中药抗流感成分开拓新思路，也为阐明中药抗病毒药效物质基础提供了有力工具。