An Abies Extract Containing Nonvolatile Polyphenols Shows Virucidal Activity against SARS-CoV-2 That Is Enhanced in Increased pH Conditions

Abstract

Researching the beneficial health properties of wood byproducts can prevent wastage by turning them into valuable resources. In this study, the virucidal activity of two extracts from Abies sachalinensis byproducts, ASE1, and ASE2, against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) was investigated. ASE1 is rich in monoterpenoid volatile compounds, whereas ASE2 contains nonvolatile polyphenols. SARS-CoV-2 solutions were mixed with ASE1 or ASE2, and viral titer reduction was evaluated. At their original acidic pH, ASE2 showed stronger virucidal activity than ASE1. The virucidal activity of ASE2 was also significantly enhanced when pH was increased to neutral or basic, which was not the case for ASE1. At a neutral pH, ASE2 induced statistically significant viral titer reduction in 1 min. HCl and NaOH solutions, which had a pH close to that of acidic and basic ASE2 test mixtures, respectively, exhibited no virucidal activity against SARS-CoV-2. Among the SARS-CoV-2 variants, Omicron showed the highest vulnerability to ASE2. Western blotting, RT-PCR, and electron microscopic analysis revealed that neutral ASE2 interacts with SARS-CoV-2 spike proteins and moderately disrupts the SARS-CoV-2 genome and viral envelope. These findings reveal the virucidal potential of ASE2.

Keywords: Abies; COVID-19; SARS-CoV-2; conifer; phytochemicals; polyphenols; virucidal.

Figure 1

Major steps of the extraction process and analysis of ASE1 and ASE2.

Figure 2

Gas chromatography–mass spectrum of ASE1.

Figure 3

¹³C−NMR spectra of H₂O eluates (a), 50% MeOH eluates (b), and 70% acetone eluates (c) separated from ASE2.

Figure 4

Virucidal activity of ASE1 and ASE2 against SARS-CoV-2. (a–c) SARS-CoV-2 ancestral strain (a,b) and variant strains (c) were mixed with nine volumes of UPW, acidic, or neutral ASE1 (a) or nine volumes of UPW, acidic, or neutral ASE2 (b,c). The mixtures were incubated at 25 °C from 1 min to 24 h (a,b) or for 3 h (c); then, the viral titers were evaluated. The data are expressed as mean ± SD (n = 8–16 per group). One-way ANOVA followed by Tukey´s multiple comparison test or Kruskal–Wallis test with Dunn’s multiple comparison test was performed to evaluate the statistical significance among the three groups. * p < 0.05; ** p < 0.01; *** p < 0.001; ns: not significant.

Figure 5

Impact of pH on the virucidal activity of ASE2 against SARS-CoV-2. (a–c) SARS-CoV-2 ancestral strain was mixed with nine volumes of UPW, acidic, neutral, or basic ASE2 (a), nine volumes of UPW, NaOH solution (pH 10.0), or basic ASE2 (b), or nine volumes of UPW, HCl solution (pH 2.9), or acidic ASE2 (c). The estimated final pH of the mixtures containing NaOH or basic ASE2 was 8.7; it was 5.1 in the mixtures containing HCl or acidic ASE2. After 3 h (a,b) or 24 h (c) incubation at 25 °C, the viral titer in each mixture was evaluated. The data are expressed as mean ± SD (n = 8 per group). One-way ANOVA followed by Tukey´s multiple comparison test or Kruskal–Wallis test with Dunn’s multiple comparison test was performed to evaluate the statistical significance among four (a) or three (b,c) groups. * p < 0.05; ** p < 0.01; *** p < 0.001; ns: not significant.

Figure 6

Impact of ASE2 on SARS-CoV-2 structural proteins. (a,b) SARS-CoV-2 ancestral strain was mixed with nine volumes of UPW, acidic, or neutral ASE2. After 24 h incubation at 25 °C, WB targeting the S (a) and N (b) proteins of SARS-CoV-2 was performed. The experiment was repeated six times for the S protein and three times for the N protein.

Figure 7

Impact of ASE2 on the SARS-CoV-2 genome. (a,b) SARS-CoV-2 ancestral strain was mixed with nine volumes of UPW, acidic, or neutral ASE2. After 24 h incubation at 25 °C, conventional RT-PCR (a) and real-time RT-PCR (b) were performed. (a) The conventional PCR primer set amplified a 445 bp fragment in the ORF1ab region. Conventional RT-PCR was repeated five times. (b) The real-time PCR primer set amplified a 157 bp fragment in the N gene region. The Ct values are expressed as mean ± SD (n = 4 per group). One-way ANOVA followed by Tukey’s multiple comparison test was performed to evaluate the statistical significance among the three groups. ns: not significant.

Figure 8

Impact of neutral ASE2 on Betacoronavirus virions. (a–g) BCoV, a surrogate of SARS-CoV-2, was incubated with nine volumes of UPW or neutral ASE2. After 6 h incubation at 25 °C, the virions were observed using electron microscopy. (a–c) BCoV virions incubated with UPW; (d–f) BCoV virions incubated with neutral ASE2. The arrowheads indicate virions on which S protein structure could not be clearly observed. The arrows show areas of the envelope where envelope integrity was disrupted and the staining agent penetrated the virions. (g) The number of intact virions was counted within 0.52 µm² fields (20 in number) chosen randomly in three samples of each group. The results are indicated as mean ± SD (n = 3 per group). Virions with discernible S proteins, well-defined undisrupted envelope, and a uniformly clear color showing no staining agent penetration were judged as intact virions. Student’s t test was performed to evaluate the statistical significance of differences between the two groups; *** p < 0.001.