抗流感病毒中药复方双黄连大鼠体内药效成分的发现及其协同作用研究

Heat-clearing and detoxifying TCM formula ShuangHuangLian, consisted of Lonicera japonica, Scutellaria baicalensis, and Forsythia suspensa three Chinese herbal medicine, is used for the prevention and treatment of influenza in clinical. It has been recommended as essential drugs in several major epidemic events due to the significant clinical efficacy. In our preliminary work, Sixty-eight components were isolated and identified from the TCM prescription ShuangHuangLian. The anti-influenza virus activities of all compounds were evaluated in vitro. However, only three of them showed moderate activities. It suggested that TCM formula ShuangHuangLian might exert the antiviral efficacy via new antiviral metabolites in vivo, or indirectly through immune regulation. Therefore, based on metabolomics methods and UPLC-Q-TOF technology, the identification of the metabolites of TCM formula ShuangHuangLian in rat plasma will be carried out in this research. And then, the metabolic processes of them will also be investigated, in order to reveal the potential efficacy ingredients in vivo. Based on structural similarity, reverse virtual screening will be employed for figuring out the possible target or metabolic enzymes interacted with the metabolites. In addition, the potential efficacy ingredients will be enriched, and its anti-influenza virus activity will be evaluated in vivo, using a restraint stressed mice model. The roles of multiple metabolites in anti-influenza virus action and the synergistic effects will be discussed. This research work will lay a solid foundation to clarify the anti-influenza virus ingredients of TCM formula ShuangHuangLian.

清热解毒中药复方双黄连由金银花，黄芩和连翘三味中药组成，用于流感的防治，临床效果显著，曾多次在重大疫情中被推荐为基本药物。申请人在前期研究中，从双黄连制剂中分离鉴定了68个化合物；然而，在体外抗流感病毒活性评价中，仅三个化合物显示出较温和的活性。这提示中药复方双黄连可能在体内发生代谢转化，形成新的抗病毒成分，或者通过对机体的免疫调节，间接发挥抗病毒的功效。为此，本研究将基于代谢物组学方法，利用UPLC-Q-TOF技术，对灌胃给中药复方双黄连提取物后，大鼠血浆中相关代谢产物进行检识，并考察其体内代谢过程，寻找潜在药效成分；基于结构相似性，反向虚拟筛选，寻找可能与其相互作用的靶点或代谢酶；采用多种方法，富集潜在药效成分，利用拘束应激小鼠流感病毒易感性模型，进行体内药效活性评价；进而探讨中药复方双黄连体内多种代谢产物在抗病毒功效中的协同作用，为阐明中药复方双黄连的抗病毒药效成分奠定坚实的基础。

清热解毒中药复方双黄连由金银花，黄芩和连翘三味中药组成，用于流感的防治，临床效果显著，曾多次在重大疫情中被推荐为基本药物。申请人在前期研究中，从双黄连制剂中分离鉴定了68个化合物；然而，在体外抗流感病毒活性评价中，仅三个化合物显示出较温和的活性。这提示中药复方双黄连可能在体内发生代谢转化，形成新的抗病毒成分，或者通过对机体的免疫调节，间接发挥抗病毒的功效。本研究利用UPLC-Q-TOF技术，从双黄连口服液中共检识170个化学成分（包括黄酮类、苯乙醇苷、环烯醚萜苷、木脂素、有机酸等五种不同母核结构）。随后，选取了母核结构不同的6个代表性单体成分，分别进行了其在大鼠体内的代谢过程研究，并在此基础上完成了实现了对双黄连口服液在大鼠体内多成分代谢轮廓的描述，共计检识相关外源物254个。采用predict FX 和Sea dock软件，对双黄连中化学成分对流感后所激活或抑制的通路的蛋白进行对接分析，发现双黄连入血成分对病毒刺激机体后的炎症和干扰素通路均有作用，从而部分揭示了双黄连通过免疫调节进而发挥抗流感的功效成分及其作用机制。