基于荧光检测的流感病毒神经氨酸酶中药抑制剂快速筛选方法的建立与应用  

Establishment and Application of Rapid Screening Method for Influenza Virus Neuraminidase Inhibitors Sourced from Traditional Chinese Medicine Based on Fluorescence Detection

作　　者：谢俊杰 谭鹏 郑川[3] 张定堃[3] 尚强 何淋泷 郝露 赵军宁[2] XIE Junjie;TAN Peng;ZHENG Chuan;ZHANG Dingkun;SHANG Qiang;HE Linlong;HAO Lu;ZHAO Junning(College of Food and Biological Engineering,Chengdu University,Chengdu 610106,China;Key Laboratory of Biological Evaluation of Traditional Chinese Medicine(TCM)Quality of National Administration of TCM,Sichuan Academy of Chinese Medicine Sciences,Chengdu 610041,China;State Key Laboratory of Southwestern Specialty Chinese Medicine Resources,School of Pharmacy,Chengdu University of TCM,Chengdu 611137,China;Sichuan Guangda Pharmaceutical Co.Ltd.,Pengzhou 611930,China;Sichuan Engineering Research Center of Antiviral TCM Industrialization,Pengzhou 611930,China)

机构地区：[1]成都大学食品与生物工程学院,成都610106 [2]四川省中医药科学院国家中医药管理局中药质量生物评价重点研究室,成都610041 [3]成都中医药大学药学院,西南特色中药资源省部共建国家重点实验室,成都611137 [4]四川光大制药有限公司,四川彭州611930 [5]四川省抗病毒中药产业化工程技术研究中心,四川彭州611930

出　　处：《中国实验方剂学杂志》2023年第7期185-192,共8页Chinese Journal of Experimental Traditional Medical Formulae

基　　金：四川省科技计划资助项目(2021YFS0409,2022YFS0429,2022YFS0431);四川省中医药管理局项目(2021MS501)。

摘　　要：目的:建立一种基于荧光检测的流感病毒神经氨酸酶(NA)中药抑制剂快速筛选方法。方法:方法的构建原理是供试品和一定数量的NA作用后,部分NA的活性会被供试品抑制,加入底物后仍具有活性的NA与荧光底物结合后在特定波长下可以产生荧光,根据测得的荧光强度计算供试品对NA的抑制率,从而评估供试品对NA的体外抑制活性。利用建立的方法对来源于12味中药的49个化学成分进行体外抗NA活性评价。利用NA蛋白受体与供试品对接后的结合能和抑制常数的理论计算值来证明实测结果的准确性。将建立的方法应用于检测不同批次板蓝根颗粒和抗病毒颗粒在体外抑制NA的活性,以评价不同批次样品间的质量一致性。结果:方法学考察结果表明该方法具有较好的准确性和重复性。49个成分的筛选结果显示,其中22个成分在体外对NA的抑制活性强于帕拉米韦[半抑制浓度(IC_(50))131.2μmol·L^(-1)],如夏佛塔苷、异荭草苷、诃子酸、胡薄荷酮、异夏佛塔苷等,其余27个成分在体外对NA的抑制活性弱于帕拉米韦。分子对接结果显示,大部分化合物的结合能和抑制常数理论计算结果和实测结果基本一致。12批板蓝根颗粒对NA的抑制活性检测结果显示,样品A1、A2、B2、C1、C2、E2和F2之间的质量一致性较好。同一厂家生产的9批抗病毒颗粒对NA的抑制活性检测结果显示,样品K1~K9在质量浓度为0.02 g·mL^(-1)时对NA的抑制率分别为37.68%、36.18%、31.37%、33.98%、40.36%、33.76%、40.69%、41.08%、40.06%,平均值37.24%。结论:该研究成功建立了一种可用于快速评价中药(来源于中药的化学成分或中成药)是否具有体外抗NA活性的分析方法,可作为现有NA抑制剂筛选方法的有力补充。利用该方法从12味中药中筛选得到22个成分对NA具有较好的体外抑制活性,可为研发抗流感小分子药物提供候选化合物。Objective:To establish a rapid screening method for influenza virus neuraminidase(NA)inhibitors sourced from Chinese medicines based on fluorescence detection.Method:The method was constructed based on the principle that after the reaction of the test sample and a certain amount of NA,the activity of some NA will be inhibited by the test sample,and the NA that is still active after the addition of the substrate can generate fluorescence at a specific wavelength when combined with the fluorescent substrate,and the inhibition rate of the test sample on NA was calculated according to the measured fluorescence intensity,so as to evaluate the in vitro inhibitory activity of the test sample on NA.A total of 49 high-purity chemical components from 12 Chinese medicines were used to evaluate the in vitro anti-NA activity by the established method.The theoretical calculated values of binding energy and inhibition constant after docking between the NA protein receptor and the test sample were used to prove the accuracy of the experimental results.The established method was applied to detect the in vitro NA inhibitory activity of different batches of Banlangen granules and Kangbingdu granules,so as to evaluate the quality consistency among different batches of samples.Result:The methodological examination results showed that the method had good accuracy and repeatability.The screening results of 49 components showed that 22 of them had strong in vitro inhibitory activity against NA than peramivir[half inhibitory concentration(IC_(50))was 131.2μmol·L^(-1)],such as schaftoside,isoorientin,chebulinic acid,menthone and isoschaftoside.The inhibitory activity of the remaining 27 components was weaker than that of peramivir.The molecular docking results showed that the theoretical calculation results of binding energies and inhibition constants of most compounds were basically consistent with the experimental results.The test results of the inhibitory activity of 12 batches of Banlangen granules on NA showed that the quality cons

关 键 词：流感病毒神经氨酸酶 荧光检测 中药 分子对接 质量一致性 板蓝根颗粒 抗病毒颗粒 

分 类 号：R22[医药卫生—中医基础理论] R28[医药卫生—中医学] R931[理学—分析化学] O657[理学—化学]