Indigo plant leaf extract inhibits the binding of SARSCoV2 spike protein to angiotensinconverting enzyme 2

Severe acute respiratory syndrome coronavirus 2 (SARSCoV2) uses its S1 spike protein to bind to angiotensinconverting enzyme 2 (ACE2) on human cells in the first step of cell entry. Tryptanthrin, extracted from leaves of the indigo plant, Polygonum tinctorium, using dlimonene (17.3 µg/ml), is considered to inhibit ACE2mediated cell entry of another type of coronavirus, HCoVNL63. The current study examined whether this extract could inhibit the binding of the SARSCoV2 spike protein to ACE2. Binding was quantified as cellbound fluorescence intensity in live cell cultures in which canine kidney MDCK cells overexpressing ACE2 were incubated with fluoresceinlabeled S1 spike protein. When indigo extract, together with S1 protein, was added at 8,650x and 17,300x dilutions, fluorescence intensity decreased in a dose and S1 extractdependent manner, without affecting cell viability. When 4.0nM tryptanthrin was added instead of the indigo extract, fluorescence intensity also decreased, but to a lesser degree than with indigo extract. Docking simulation analyses revealed that tryptanthrin readily bound to the receptorbinding domain of the S1 protein, and identified 2 and 7amino acid sequences as the preferred binding sites. The indigo extract appeared to inhibit S1ACE2 binding at high dilutions, and evidently contained other inhibitory elements as well as tryptanthrin. This extract may be useful for the prevention or treatment of SARSCoV2 infection.