Insights into the direct anti-influenza virus mode of action of Rhodiola rosea

•Beside flavonoids, tannins mainly including prodelphinidin gallate oligomers, mediate the strong anti-influenza virus activity of SHR-5.•Neither resistance to oseltamivir nor to M2 ion channel blockers hampered the strong activity of SHR-5 and its tannin-enriched fraction (TE) against influenza viruses of subtypes A and B.•The mode of action is based on the interaction of SHR-5 and TE with the viral envelope including also the inhibition of viral neuraminidase.•No development of SHR-5 resistance in influenza viruses was observed in vitro, which makes this multicomponent mixture an even more promising therapeutic candidate. Background: The anti-influenza A virus activities and contents of previously isolated most active flavonoids (rhodiosin and tricin) from a standardized hydro-ethanolic R. rosea root and rhizome extract (SHR-5®), did not fully explain the efficacy of SHR-5®. Moreover, the mode of antiviral action of SHR-5® is unknown. Purpose: To determine the anti-influenza viral principle of SHR-5® we evaluated i) the combined anti-influenza virus effect of rhodiosin and tricin, ii) the impact of its tannin-enriched fraction (TE), iii) its antiviral spectrum and mode of action, and iv) its propensity for resistance development in vitro. Methods: The combined anti-influenza virus effect of rhodiosin and tricin and the impact of TE were investigated with cytopathic effect (CPE)-inhibition assays in MDCK cells. A tannin-depleted fraction (TD) and TE were prepared by polyamide column chromatography and dereplicated by LC-MS. Plaque-reduction assays provided insights into the anti-influenza virus profile, the mode of action, and the propensity for resistance development of SHR-5®. Results: Our results i) did not reveal synergistic anti-influenza A virus effects of rhodiosin and tricin, but ii) proved a strong impact of TE mainly composed of prodelphinidin gallate oligomers. iii) TE inhibited the plaque-production of influenza virus A(H1N1)pdm09, A(H3N2), and B (Victoria and Yamagata) isolates (including isolates resistant to neuraminidase and/or M2 ion channel inhibitors) with 50% inhibitory concentration values between 0.12 - 0.53 µg/ml similar to SHR-5®. Mechanistic studies proved a virucidal activity, inhibition of viral adsorption, viral neuraminidase activity, and virus spread by SHR-5® and TE. iv) No resistance development was observed in vitro. Conclusion: For the first time a comprehensive analysis of the anti-influenza virus profile of a hydro-etha