Rapid Virucidal Activity of Japanese Saxifraga Species-Derived Condensed Tannins against SARS-CoV-2, Influenza A Virus, and Human Norovirus Surrogate Viruses

Abstract

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), influenza A virus (IAV), and norovirus are global threats to human health. The application of effective virucidal agents, which contribute to the inactivation of viruses on hands and environmental surfaces, is important to facilitate robust virus infection control measures. Naturally derived virucidal disinfectants have attracted attention owing to their safety and eco-friendly properties. In this study, we showed that multiple Japanese Saxifraga species-derived fractions demonstrated rapid, potent virucidal activity against the SARS-CoV-2 ancestral strain and multiple variant strains, IAV, and two human norovirus surrogates: feline calicivirus (FCV) and murine norovirus (MNV). Condensed tannins were identified as active chemical constituents that play a central role in the virucidal activities of these fractions. At a concentration of 25 μg/mL, the purified condensed tannin fraction Sst-2R induced significant reductions in the viral titers of the SARS-CoV-2 ancestral strain, IAV, and FCV (reductions of ≥3.13, ≥3.00, and 2.50 log₁₀ 50% tissue culture infective doses [TCID₅₀]/mL, respectively) within 10 s of reaction time. Furthermore, at a concentration of 100 μg/mL, Sst-2R induced a reduction of 1.75 log₁₀ TCID₅₀/mL in the viral titers of MNV within 1 min. Western blotting and transmission electron microscopy analyses revealed that Sst-2R produced structural abnormalities in viral structural proteins and envelopes, resulting in the destruction of viral particles. Furthermore, Saxifraga species-derived fraction-containing cream showed virucidal activity against multiple viruses within 10 min. Our findings indicate that Saxifraga species-derived fractions containing condensed tannins can be used as disinfectants against multiple viruses on hands and environmental surfaces. IMPORTANCE SARS-CoV-2, IAV, and norovirus are highly contagious pathogens. The use of naturally derived components as novel virucidal/antiviral agents is currently attracting attention. We showed that fractions from extracts of Saxifraga species, in the form of a solution as well as a cream, exerted potent, rapid virucidal activities against SARS-CoV-2, IAV, and surrogates of human norovirus. Condensed tannins were found to play a central role in this activity. The in vitro cytotoxicity of the purified condensed tannin fraction at a concentration that exhibited some extent of virucidal activity was lower than that of 70% ethanol or 2,000 ppm sodium hypochlorite solution, which are popular virucidal disinfectants. Our study suggests that Saxifraga species-derived fractions containing condensed tannins can be used on hands and environmental surfaces as safe virucidal agents against multiple viruses.

Keywords: SARS-CoV-2; Saxifraga; condensed tannin; influenza A virus; norovirus; virucidal activity.

FIG 1

¹³C NMR spectrum of Sst-2R [acetone-d₆-D₂O (1:1), 100 MHz].

FIG 2

TOF-MS spectra of Sst-2R. Linear (A) and spiral (B) modes.

FIG 3

Expected chemical structures of Saxifraga species-derived condensed tannins.

FIG 4

Effect of Sst-2R on viral structural proteins. SARS-CoV-2 ancestral strain, IAV, or MNV solution was mixed with DMSO or Sst-2R. The concentration of Sst-2R in the mixture was 25 μg/mL (against SARS-CoV-2 or IAV) or 100 μg/mL (against MNV). After 10 s (against SARS-CoV-2 or IAV) or 1 min (against MNV), Western blotting targeting each viral structural protein was visualized.

FIG 5

Morphology of Sst-2R-treated BCoV particles. BCoV solution was mixed with DMSO or Sst-2R. The concentration of Sst-2R in the mixture was 25 μg/mL (A and B) or 100 μg/mL (C and D). After 10 s (A and B) or 3 h (C and D), the viral particles were observed using TEM. TEM images of BCoV are shown (A and C). Black arrowheads, viral particles with normal structure; white arrowheads, viral particles with abnormal structure; black outlines, aggregated viral particles. The numbers of intact viral particles in one field of view were counted (B and D). Student’s t test was done (n = 30 fields). ***, P < 0.001; ns, not significant.

FIG 6

Morphology of Sst-2R-treated IAV particles. IAV solution was mixed with DMSO or Sst-2R. The concentration of Sst-2R in the mixture was 25 μg/mL (A and B) or 100 μg/mL (C and D). After 10 s (A and B) or 3 h (C and D), the viral particles were observed using TEM. TEM images of IAV are shown (A and C). Black arrowheads, viral particles with normal structure; white arrowheads, viral particles with abnormal structure; black outlines, aggregated viral particles. The numbers of intact viral particles in one field of view were counted (B and D). Student’s t test was done (n = 30 fields). ***, P < 0.001.

FIG 7

Morphology of Sst-2R-treated MNV particles. MNV solution was mixed with DMSO or Sst-2R. The concentration of Sst-2R in the mixture was 100 μg/mL. After 1 min (A and B) or 3 h (C and D), the viral particles were observed using TEM. TEM images of MNV are shown (A and C). Black arrowheads, viral particles with normal structure; white arrowheads, viral particles with abnormal structure; black outlines, aggregated viral particles. The numbers of intact viral particles in one field of view were counted (B and D). Student’s t test was done (n = 30 fields). ***, P < 0.001.