In vitro assay of TMPRSS2 serine protease activity

The serine protease activity of TMPRSS2 was estimated as previously described [12, 13]. Recombinant human TMPRSS2 (4 μg/mL final concentration) diluted with an assay buffer (50 mM Tris-HCl pH 8.0, 154 mM NaCl) and test ingredients were added to the 384 well black plate (Greiner Bio-One Japan, Tokyo, Japan). Then, Boc-Gln-Ala-Arg-MCA (10 μM final concentration) diluted with an assay buffer containing dimethyl sulfoxide (DMSO; 0.1% (w/w) final concentration) was added to induce an enzyme reaction. After incubation at room temperature for 1 h, the fluorescence intensity (FI) of fluorescent hydrolysate of Boc-Gln-Ala-Arg-MCA (7-amino-4-methylcoumarin) were read using SpectraMax M5 plate reader (Molecular Devices, San Jose, CA, USA) with excitation of 380 nm and emission of 460 nm. The inhibitory rate of ingredients was calculated as follows: Inhibition (%) = (FI of control–FI of treatment) / FI of control × 100. The dose–response relationship between inhibition (%) and test ingredient concentration was plotted and used to determine the half maximal inhibitory concentration (IC50) using the DRC package in R software program (v3.6.1), as described in [11]. In brief, the dose–response data were fitted using a four-parametric log-logistic model and used estimate the IC50.