金莲花的抗病毒有效成分及作用机制研究

Trollius chinensis is a Chinese herb with the function of heat-clearing and detoxifying. It displays obvious antiviral bioactivity and is indicated for the treatment of upper respiratory tract infection, tonsillitis and pharyngitis, etc. The well-known chemical constituents of T. chinensis mainly include flavonoids, alkaloids and aromatic acids; however, its effective components and their mechanism are unclear. Among these three kinds of substances, flavonoids are the major compounds of high content, but they are difficult to be absorbed and of low bioavailability. Aromatic acids are of high bioavailability, but their bioactivity is very weak. Alkaloids are of the strongest bioactivity and of fair bioavailability, but their content is the lowest and the structure is unstable. Therefore, the antiviral effective components of T. chinensis can not be determined only based on their quantity and bioactivity in vitro due to the distinctiveness of the constituents of T. chinensis, but rather consider the absorbed bioactive components in the circulation. This project is intended to investigate the bioactive compounds in the circulation resulted from oral administration of the crude extracts, three kinds of components and their representative compounds base on ex vivo serum-pharmacological and serum-pharmacochemical methodology, and evaluate the contribution rate of three kinds of components, in order to characterize the antiviral effective constituents of T. chinensis. Meanwhile, the antiviral mechanism of T. chinensis and three kinds of components in terms of signal transduction is investigated in molecular level by using serum-pharmacological and molecular-biological methods.

金莲花为清热解毒中药，具明显的抗病毒活性，主治上呼吸道感染、扁桃体炎、咽炎等症。其化学成分比较明确，主含黄酮类、生物碱类和芳香酸类。但有效成分不确定，作用机制也不清楚。三类物质中，黄酮类含量最高，但主要为碳苷类，不利于被人体吸收，生物利用度低；芳香酸类生物利用度高，但其活性很弱；生物碱类活性最强，生物利用度尚可，但其含量最低，且结构不稳定。这些成分的特殊性决定了金莲花有效成分的确定不能只根据化学成分的含量多寡和体外活性的强弱，而需要从被吸收的血液移行成分及其活性入手。本项目采用血清药理学/血清药物化学等半体内实验与体内实验相结合的方式，研究口服总提取物、三类总成分及其代表化合物后的血液移行成分及其抗流感病毒和呼吸道合胞病毒活性，分析三类成分对金莲花抗病毒活性的贡献度，从而明确其抗病毒有效成分，并采用血清药理学和分子生物学方法，在分子水平上从信号通路角度探讨金莲花及其三类成分的抗病毒机制。

本项目建立了血清药物化学和血清药理学研究方法，采用优化的血清药物化学研究方法分别对金莲花的总提取物，总黄酮、总酚酸和总生物碱以及这三类成分的单体代表成分进行了血清药物化学研究。然后，采用血清药理学研究方法对金莲花总提取物及各类成分进行了抗病毒活性研究，结合血清药物化学研究的结果，阐明了金莲花中三类成分对金莲花抗病毒作用的贡献。最后，通过研究金莲花化学成分对Toll样受体TLR3，4和7介导的信号通路的影响探讨了金莲花总提取物及其三类成分的抗病毒作用机制。本项目的研究结果表明，金莲花中的总酚酸、总生物碱和总黄酮对金莲花的抗病毒作用都有贡献，而总酚酸和总生物碱发挥的作用更大；金莲花的抗病毒有效成分主要来源于藜芦酸、金莲花苷、原金莲酸、苯甲酸甲酯、牡荆素、荭草素、日本异当药素、槲皮素、金莲花碱及其代谢产物；金莲花提取物及其主要代表成分牡荆素、藜芦酸和金莲花碱能够下调TLR3和TLR7信号通路，减少NO、TNF-b等炎性因子的分泌并促进IFNb的蛋白表达，从而发挥保护机机体免受炎症损伤和杀病毒的双重作用。