Outbreak of coronavirus disease 2019 occurred in Wuhan and has rapidly spread to almost all parts of world. GB-1, the herbal formula from Tian Shang Sheng Mu of Chiayi Puzi Peitian Temple, is used for the prophylaxis of SARS-CoV-2 in Taiwan. In this study, we investigated that the effect of GB-1 and the index compounds of GB-1 on the ACE2 and TMPRSS2 expression through in vitro and in vivo study. In our result, GB-1 can inhibit ACE2 and TMPRSS2 protein expression in HepG2 cells, 293T cells, and Caco-2 cells without cytotoxicity. For the mouse model, GB-1 treatment could decrease ACE2 and TMPRSS2 expression levels of the lung and kidney tissue without adverse effects, including nephrotoxicity and hepatotoxicity. In the compositions of GB-1, 0.5–1 mg/ml of Glycyrrhiza uralensis Fisch. ex DC. extract could not inhibit ACE2 mRNA and protein expression in HepG2 cells. In addition, theaflavin-3-gallate could inhibit protein expression of ACE2 and TMPRSS2 without significant cytotoxicity. Our results suggest that GB-1 and theaflavin-3-gallate could act as potential candidates for prophylaxis or treatment of SARS-CoV-2 infection through inhibiting protein expression of ACE2 and TMPRSS2 for the further study.

The ongoing COVID-19 pandemic, caused by SARS-CoV-2, continues to pose significant global health challenges. The results demonstrated that GB-2 at 200 μg/mL effectively increased the population of 293T-ACE2 cells with low RBD binding for both SARS-CoV-2 Omicron EG.5.1 and HV.1 variants by dual-color flow cytometry, indicating its ability to inhibit virus attachment. Further investigation revealed that (+)-catechin at 25 and 50 μg/mL did not significantly alter the ACE2–RBD interaction for the EG.5.1 variant. In contrast, theaflavin showed inhibitory effects at both 25 and 50 μg/mL for EG.5.1, while only the higher concentration was effective for HV.1. Notably, theaflavin 3-gallate exhibited a potent inhibition of ACE2–RBD binding for both variants at both concentrations tested. Molecular docking studies provided insight into the binding mechanisms of theaflavin and theaflavin 3-gallate with the RBD of EG.5.1 and HV.1 variants. Both compounds showed favorable docking scores, with theaflavin 3-gallate demonstrating slightly lower scores (−8 kcal/mol) compared to theaflavin (−7 kcal/mol) for both variants. These results suggest stable interactions between the compounds and key residues in the RBD, potentially explaining their inhibitory effects on virus attachment. In conclusion, GB-2, theaflavin, and theaflavin 3-gallate demonstrate significant potential as inhibitors of the ACE2–RBD interaction in Omicron variants, highlighting their therapeutic promise against COVID-19. However, these findings are primarily based on computational and in vitro studies, necessitating further in vivo research and clinical trials to confirm their efficacy and safety in humans.