Potent antiviral activity of Agrimonia pilosa, Galla rhois, and their components against SARS-CoV-2

Abstract

Agrimonia pilosa (AP), Galla rhois (RG), and their mixture (APRG64) strongly inhibited SARS-CoV-2 by interfering with multiple steps of the viral life cycle including viral entry and replication. Furthermore, among 12 components identified in APRG64, three displayed strong antiviral activity, ursolic acid (1), quercetin (7), and 1,2,3,4,6-penta-O-galloyl-β-d-glucose (12). Molecular docking analysis showed these components to bind potently to the spike receptor-binding-domain (RBD) of the SARS-CoV-2 and its variant B.1.1.7. Taken together, these findings indicate APRG64 as a potent drug candidate to treat SARS-CoV-2 and its variants.

Keywords: APRG64; Agrimonia pilosa; Antiviral agents; COVID-19; Galla rhois; SARS-CoV-2.

Graphical abstract

Fig. 1

Anti-SARS-CoV-2 activity of Agrimonia pilosa (AP), Galla rhois (RG), and their mixture (APRG64). Vero cells were seeded 1 day before infection. (A) Cells were treated with AP (0.1 or 0.5 µg/mL), RG (0.1 or 0.5 µg/mL), or APRG64 (0.1 or 0.5 µg/mL) for 2 h and then infected with SARS-CoV-2 at 0.01 multiplicity of infection (MOI) for 1 h. After washing three times with PBS, cells were retreated under the same conditions described above. Three days later, cells were fixed and stained to visualize plaques (left panel). Plaque reduction rates are shown in the right panel. (B) Cells were infected with SARS-CoV-2 at 0.01 MOI and treated with AP (0.1 or 0.5 µg/mL), RG (0.1 or 0.5 µg/mL), or APRG64 (0.1 or 0.5 µg/mL). After incubation for 1 h, cells were washed three times with PBS. Three days later, cells were fixed and stained to visualize plaques (left panel). Plaque reduction rates are shown in the right panel. Remdesivir (Rem, 5 µM) and chloroquine phosphate (C.P, 10 µM) were used as positive controls. Data are representative of three independent experiments. *p < 0.05; ^***p < 0.001, compared with mock-treated cells (Con).

Fig. 2

Chemical structures of isolated constituents from mixture of Agrimonia pilosa (AP) leaves and Galla rhois (RG) fruits in 50% EtOH extract (APRG64).

Fig. 3

Antiviral activity of active components isolated from APRG64 against SARS-CoV-2. Vero cells were seeded 1 day before infection. (A) Cells were infected with SARS-CoV-2 at 0.01 multiplicity of infection (MOI) and treated with compounds 1–12 of APRG64 at 1, 5, or 25 µg/mL for 1 h, followed by washing three times with PBS. Three days later, cells were fixed and stained to visualize plaques. Plaque reduction rates are shown. (B) Cells were infected with SARS-CoV-2 at 0.3 MOI and treated with APRG64, compounds 1, 7, or 12, at 5 or 25 µg/mL for 1 h. After washing three times with PBS, cells were incubated for an additional 72 h. Cell supernatants were analyzed for the SARS-CoV-2 spike proteins using enzyme-linked immunosorbent assay (ELISA). (C) Cells were infected with SARS-CoV-2 at 0.3 MOI for 1 h, followed by washing three times with PBS. Cells were treated with compounds 1, 7, or 12. After 72 h, cell supernatants were analyzed for SARS-CoV-2 spike protein using ELISA. Data are representative of three independent experiments. *p < 0.05; ^**p < 0.01; ^***p < 0.001, compared with mock-treated cells (Con).

Fig. 4

Structural modeling of B.1.1.7 lineage spike receptor-binding domain (RBD). (A) 3D structures of SARS-CoV-2 spike RBD and B.1.1.7 lineage spike RBD. (B) Ramachandran plot of the B.1.1.7 lineage spike RBD structural model.

Fig. 5

Molecular binding of SARS-CoV-2 spike receptor-binding domain (RBD) and B.1.1.7 lineage spike RBD with three active components of APRG64, ursolic acid (1), quercetin (7), and 1, 2, 3, 4, 6-penta-O-galloyl-β-d-glucose (12).