Amacha, a Japanese sweet tea, inhibits infection by pseudoviruses expressing the SARS-CoV-2 spike protein

• Amacha and its compounds inhibit SARS-CoV-2 infection in Caco-2 cells • Hydrangenol and phyllodulcin inhibit ACE2–spike binding via allosteric site 1 interaction • Amacha’s bioactive content varies by harvest location and season • Alkaloids in Amacha could cause safety issues, suggesting the need for optimization • Amacha’s composition must be standardized for therapeutic use This study investigated the potential of Amacha, a traditional Japanese sweet tea made from Hydrangea macrophylla var. thunbergii and its primary bioactive compounds hydrangenol and phyllodulcin to inhibit severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. We found that both Amacha and its dihydroisocoumarin constituents significantly reduced the infection rate of pseudolentiviruses expressing the SARS-CoV-2 spike protein, including the Wuhan and Omicron variants, in Caco-2 cells. Docking studies have revealed that hydrangenol and phyllodulcin effectively bind to allosteric site 1 of angiotensin-converting enzyme 2 (ACE2), potentially preventing the spike protein from binding to ACE2, which is essential for viral entry, while maintaining the enzymatic function of ACE2. We further detected significant variability in the concentrations of these compounds in the hot-water extracts of Amacha tea leaves across different regions and seasons. However, the presence of alkaloids, including febrifugine, which exhibit cytotoxic activity, poses challenges in maintaining consistent quality and safety. Further research is needed to fully elucidate the mechanisms involved, standardize Amacha’s composition, and develop optimized products with reduced alkaloid content to ensure the safe and effective use of Amacha and its bioactive compounds to combat SARS-CoV-2 infection.