Inhibitory Effects of Citrus-Derived Flavonoids Hesperidin and Hesperetin on SARS-CoV-2 Spike-Mediated Syncytia Formation Using In Vitro Cell Model.

PURPOSE: SARS-CoV-2 infection may lead to a worse prognosis in COVID-19 patients by inducing syncytia formation which implies intercellular transmission and immune evasion. Hesperidin (HSD) and hesperetin (HST) are two citrus flavonoids that demonstrate the potential to interfere with spike/human angiotensin-converting enzyme-2 (hACE2) binding and show an inhibitory effect in the SARS-CoV-2 pseudovirus internalization model. Here, we determined the effects of HSD and HST to inhibit syncytia formation using in vitro cell models. METHODS: We confirmed spike, hACE2, and transmembrane protease, serine 2 (TMPRSS2) ectopic expressions by immunofluorescence staining (IF) after transfection using polyethylene imine (PEI) in 293T cells. Then, the cells were transfected with a set of plasmids encoding spike/hACE2/TMPRSS2 or spike/hACE2 to induce syncytia formation. Cell treatment with HSD/HST was performed 4-5 h after transfection and then incubated for another 16-18 h. Syncytia were observed using an inverted microscope or a high content screening (HCS) platform. The data obtained from syncytia formation assays were statistically analyzed using ANOVA (Bonferroni). RESULTS: We successfully observed spike, hACE2, and TMPRSS2 expression in 293T cells by IF staining. Furthermore, we showed that HSD 10 and 100 µM significantly inhibited the formation of small-to-medium-sized syncytia compared to the control cells by manual syncytia observation. In the HCS assay, 10 µM HSD showed an inhibitory effect of syncytia induced by spike WT. In contrast, 100 µM HSD, 10 and 100 µM HST, and 10 µg/mL citrus peel extract containing HSD prepared by the hydrodynamic cavitation method (HCV) inhibited syncytia formation induced by spike Omicron. CONCLUSION: HSD and HST show the potential inhibitory activity of SARS-CoV-2 intercellular transmission. Further study is needed to confirm the mechanism of action of the antiviral activity.