Identification and mechanism investigation of anti-influenza virus agents from Chinese prescriptions and herbs

Ph.D.Annual influenza is estimated to cause 3 to 5 million cases of illness, and claims more than 250000 deaths every year. Many strains of influenza virus have developed resistance against the existing anti-influenza drugs, thus it is essential to develop new drugs against this virus. Traditional Chinese Medicine has its own unique advantages over modern medicine and has historically been invaluable as a source of therapeutic agents. In this study, aqueous extracts of ten single herbs and ten multi-herb formulations for antiviral activity against influenza virus were screened. As a result, prescription Sheng Jiang San (SJS) and single herb Radix Paeoniae Alba (BS) and Folium Hibisci Mutabilis (FRY) have been found to have anti-influenza activities, and their mechanisms were also investigated.Chinese prescription SJS could inhibit various of influenza viral strains in vitro. The 50% inhibitory concentration of SJS against A/WSN/33 (H1N1) (WSN) was even lower than 35 μg/ml. SJS also protected 50% of mice from A/PR/8/34 (H1N1) infection. Decline of viral titers in the lungs of mice and the lung index was obviously observed in groups administrated with SJS, compared with untreated mice. SJS was found to exert anti-influenza efficacy through targeting on the influenza neuraminidase (NA) protein and down-regulating significantly TNF-α and up-regulated IL-2 of influenza virus induced mice.BS and FRY are categorized in “heat clearing and blood cooling herb”. These two Chinese medicinal herbs were identified anti-influenza efficacy. A bioassay-guided isolation led to the discovery of three tannin compounds gallic acid (GA), methyl gallate (MG) and pentagalloyglucose (PGG) in BS aqueous extract. The IC₅₀ of these compounds against WSN virus in MDCK cells were 50, 46 and 20 μM, respectively.PGG was found to inhibit both influenza virus receptor binding and NA activity, while MG and GA inhibited NA but not hemagglutinin (HA). GA was found to target on residue R152 of NA protein by molecular docking. After generating the recombinant virus carrying single mutation R152A, the resistance of R152A virus against GA as IC₅₀ of GA was increased to 200 μM, confirming the docking site of R152 may be one of the target NA residues for GA. FRY herbal extract was further isolated to a refined fraction F3-3-2-1, the major components were characterized as flavonoid compounds by HR-MS analysis, F3-3-2-1 could suppress 100% WSN virus growth in plaque reduction assay at only 15 μg/ml. Results showed F3-3-2-1 did not act on HA or NA, but it could significantly regulate certain cytokines/chemokines TNF-α, IL-6, IL-8, MCP-1, IP-10 expression induced by virus in host cells. Fraction F3-3-2-1 may contribute to alleviate inflammatory response in lung tissue.Our work has substantiated SJS is an effective anti-influenza prescription. It is also the first in vitro study pointed out that tannin compounds and flavonoid compounds are the reason of BS and FRY aqueous extract, respectively, exerting anti-flu activity. The work enhances the confidence of using medicinal herbs to combat influenza virus and concerned herbs and prescription may be further developed by the herbal industry.每年流行性感冒被估計會導致三百萬至五百萬例的流感以及超過二十五萬例死亡。很多流感病毒株會對現有的抗流感病毒藥物逐漸產生抗性，因此開發新的抗流感病毒藥物是十分必要且刻不容緩的。從歷史經驗來看, 傳統中藥是開發新藥非常有價值的資源。經驗知識結合民族醫藥的優勢，相信我們很有可能從中藥當中找到能夠治療流感的新藥物。在此項研究中, 我們運用了一系列的生物化学試驗从十個單味中藥和十個中藥複方中筛选出具有抗流感病毒活性的药物。至此，我們已發現升降散複方，單味藥白芍和芙蓉葉顯示抗流感病毒活性。結果顯示，升降散複方對多種流感病毒株具有抑製作用，有著广譜抗病毒效果。 其中，升降散體外抗病毒株A/WSN/33 (H1N1) 的IC₅₀低於35 μg/ml。在小鼠模型中，升降散複方對50%的感染鼠肺適應流感病毒株的A/PR/8/34 (H1N1)小鼠進行死亡保護。相較於對照組，實驗組小鼠的肺指數及肺部病毒載量都顯著下降。同時，我們還探查到升降散複方體外可以抑制八成的神經氨酸酶（NA）活性，體內也能上調免疫因子TNF-α 以及下调IL-2 的表达量，減輕炎症反應。單味藥白芍和芙蓉葉屬於清熱涼血藥，我們發現二者在MDCK細胞上顯示對多種流感病毒株有抑製作用。通過生物實驗導向分離法，三個鞣質類化合物沒食子酸、沒食子甲酯以及五沒食子酰基葡萄糖最終從白芍水提物中被分離。五沒食子酰葡萄糖可同時抑制NA活性和血凝素，而沒食子酸和沒食子甲酯只對NA有抑製作用。通過分子對接模擬發現沒食子酸可能通過殘基R152與NA蛋白結合，發揮抑制效果。之後我們嘗試反向重組突變病毒R152A，發現沒食子酸抗R152A病毒的IC₅₀增至200 μM，由此驗證NA殘基R152可能是沒食子酸其中一個作用位點。而芙蓉葉水提物經過分離得到精緻黃酮組分F3-3-2-1。F3-3-2-1在15 μg/ml時可完全抑制A/WSN/33 (H1N1)病毒生長。機制研究顯示F3-3-2-1與HA, NA無明顯相互作用，但可調節宿主細胞內免疫因子TNF-α, IL-6, IL-8, MCP-1, IP-10的過量表達。由此推斷F3-3-2-1可通過免疫調節減輕肺部炎症反應。本次工作首次根據循證醫學的要求提供了有效的實驗結果，用以支持升降散在臨床上的抗流感病毒功效。同時證明鞣質類化合物和黃酮類化合物分別是白芍和芙蓉葉抗流感作用的主要有效成分，並對它們的機制進行了初步研究。我們相信這項工作有助於增強人們對傳統中藥對抗流感病毒的信心，並且幫助推進中醫藥現代化。也希望我們發現的抗流感病毒藥物將來可以被製藥行業繼續發展。Zhang, Tianbo.Thesis Ph.D. Chinese University of Hong Kong 2019.Includes bibliographical references (leaves 127-145).Abstracts also in Chinese.Title from PDF title page (viewed on 22, January, 2021)