Sichuan pepper, Zanthoxylum bungeanum Maxim., suppresses human coronavirus OC43 infection by inhibiting viral entry and impairing autolysosome accumulation

Abstract Background The dried pericarp of Zanthoxylum bungeanum Maxim. (Rutaceae), widely recognized as Sichuan pepper ( Hua Jiao ), is a prominent herb within the Traditional Chinese Medicine (TCM), boasting a long and well-documented history of ethnopharmacological application. Its traditional uses primarily involve the internal management of gastrointestinal ailments, such as stomachaches, alongside promoting systemic blood circulation. Building on this traditional foundation, modern pharmacological studies have confirmed and extended its therapeutic profile, revealing potent anti-inflammatory and antitumor biological activities. We examined the anticoronaviral effects and the mechanism of action of the 30% ethanol extract of Z. bungeanum against human coronavirus (HCoV)-OC43 infection. Methods Antiviral activity was measured using the virus-induced cytopathic effect (CPE) reduction assay, qRT-PCR was performed to calculate viral RNA copy numbers, and western blotting and immunofluorescence staining were performed to detect viral proteins. To evaluate the mode of action of Z. bungeanum , time-of-addition, attachment, penetration, and virucidal assays were performed. The effect of the extract on virus-induced autophagic flux was examined by measuring LC3 protein expression via western blotting, and autophagy, lysosomes, and lysosomal degradation were assessed using CYTO-ID® Green, LysoTracker™ Deep Red, and DQ™ Red BSA, respectively. Ultra-performance liquid chromatography quadrupole time-of-flight mass spectrometry was used to identify the active components in the extract. Results Z. bungeanum extract protected against HCoV-OC43-induced CPEs with IC 50 of 284.1 ± 44.0 µg/mL. The extract reduced the number of intracellular and extracellular viral RNA copy and viral protein expression. Z. bungeanum mainly affected the early phase of the virus life cycle by inhibiting viral entry. Additionally, Z. bungeanum inhibited HCoV-OC43 replication by inducing autolysosome accumulation, thereby blocking virus-induced autophagic flux. Hydroxy-α-sanshool and p -coumaric acid were identified as the active antiviral components. Conclusions This study suggested the potential of Z. bungeanum as a novel anticoronaviral agent. Graphical abstract