Ephedrae Herba as a potential source of SARS-CoV-2 RNA-dependent RNA polymerase inhibitory activity

Introduction Maoto (麻黄湯) is a traditional Japanese Kampo formula composed of four crude drugs—Ephedrae Herba, Armeniacae Semen, Cinnamomi Cortex, and Glycyrrhizae Radix—and has been used to treat febrile viral illnesses. Among these, Ephedrae Herba has been associated with antiviral activity in previous studies. However, little is known about the effects of Ephedrae Herba on viral infection, spike (S) protein-mediated syncytium formation (cell–cell fusion), and viral enzymes, or the specific metabolites underlying these activities. In this study, we focused on Ephedrae Herba and comparatively evaluated the effects of the four crude drugs of Maoto on SARS-CoV-2 infection, S protein-mediated cell–cell fusion, and virus replication-related processes. Methods We evaluated the inhibitory effects of the four crude drugs of Maoto on SARS-CoV-2 infection in VeroE6/TMPRSS2 cells. S protein-mediated cell–cell fusion was evaluated using a split-luciferase assay in HEK293T cells. The inhibitory effects on viral enzymes, including 3C-like protease, papain-like protease, and RNA-dependent RNA polymerase (RdRp), were assessed using in vitro enzymatic assays. Results Comparative analyses of the four crude drugs showed that Ephedrae Herba, Armeniacae Semen, and Cinnamomi Cortex suppressed SARS-CoV-2 infection in VeroE6/TMPRSS2 cells, whereas Glycyrrhizae Radix inhibited S protein-mediated cell–cell fusion in HEK293T cells. In the biochemical assays, Ephedrae Herba and Cinnamomi Cortex showed inhibitory activity against SARS-CoV-2 RdRp. Further analysis of Ephedrae Herba showed that its high-polarity fraction exhibited concentration-dependent inhibitory activity against both SARS-CoV-2 and norovirus RdRp with IC 50 values of 8.6 and 16.5 μg/mL, respectively. Conclusion Ephedrae Herba contains high-polarity metabolites associated with RdRp-related inhibitory activity in biochemical assays. Nevertheless, further studies are needed to determine the extent to which this activity contributes to the effects observed in cells.