Active Ingredients of Reduning Injection Maintain High Potency against SARS-CoV-2 Variants

Objective: To investigate the anti-coronavirus potential and the corresponding mechanisms of the two ingredients of Reduning Injection: quercetin and luteolin. Methods: A pseudovirus system was designed to test the efficacy of quercetin and luteolin to inhibit SARS-CoV-2 infection and the corresponding cellular toxicity. Luteolin was tested for its activities against the pseudoviruses of SARS-CoV-2 and its variants. Virtual screening was performed to predict the binding sites by Autodock Vina 1.1.230 and PyMol. To validate docking results, surface plasmon resonance (SPR) was used to measure the binding affinity of the compounds with various proteins of the coronaviruses. Quercetin and luteolin were further tested for their inhibitory effects on other coronaviruses by indirect immunofluorescence assay on rhabdomyosarcoma cells infected with HCoV-OC43. Results: The inhibition of SARS-CoV-2 pseudovirus by luteolin and quercetin were strongly dose-dependent, with concentration for 50% of maximal effect (EC) of 8.817 and 52.98 µmol/L, respectively. Their cytotoxicity to BHK21-hACE2 were 177.6 and 405.1 µmol/L, respectively. In addition, luetolin significantly blocked the entry of 4 pseudoviruses of SARS-CoV-2 variants, with EC lower than 7 µmol/L. Virtual screening and SPR confirmed that luteolin binds to the S-proteins and quercetin binds to the active center of the 3CLpro, PLpro, and helicase proteins. Quercetin and luteolin showed over 99% inhibition against HCoV-OC43. Conclusions: The mechanisms were revealed of quercetin and luteolin inhibiting the infection of SARS-CoV-2 and its variants. Reduning Injection is a promising drug for COVID-19.