Broad-spectrum anticoronaviral activity of the traditional herbal medicine Mahuangtang by targeting the multistep viral infection process: virus entry, Mpro, PLpro, RdRp and IFN induction

Ephedra decoction, Mahuangtang (MHT), is a traditional herbal formula comprising Ephedrae sinica Stapf (ES), Prunus armeniaca L. (PA), Cinnamomum cassia (L.) J.Presl (CC), and Glycyrrhiza uralensis Fisch. (GU). MHT is used to treat colds, influenza, and other respiratory conditions. This study investigates the anticoronaviral effect of MHT and elucidates its mode of action and the active antiviral components of MHT. UHPLC-MS/MS analysis showed the holistic quality and equivalency between MHT and a mixture of four herbal components and identified nine representative compounds in MHT. The inhibitory activity of MHT and its components was examined with HCoV-OC43, SARS-CoV-2, its omicron variants, and pseudotyped lentiviruses. Time-of-addition assay, viral attachment assay, viral penetration assay, virucidal assay, and SARS-CoV-2 Mpro, PLpro, and RdRp activity assay were performed. MHT and its components, ES, CC, and GU, except PA, inhibited coronavirus infection. While MHT, and its components ES, and CC exhibited the suppression of virus entry with a virucidal activity, GU demonstrated inhibition of viral infection in the late stage. PA and its compound, amygdalin, did not inhibit coronavirus infection. Furthermore, Cinnamic aldehyde in CC and liquiritin, liquiritigenin, glycyrrhizin, and glabridin in GU were the active compounds of MHT but not ephedrine alkaloids in ES. MHT and its components inhibited the activity of Mpro and PLpro proteases and RdRp. Molecular docking analysis showed the binding affinity between RdRp and glabridin and between RBD-ACE2 and cinnamic aldehyde. Additionally, we showed that MHT induced the expression of IFN-related antiviral genes by activating the STAT1 pathway. MHT exhibited the potential to function as a multifaceted therapeutic candidate for treating broad-spectrum coronavirus infection by targeting multiple stages of viral infection.