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. 2021 Jun 11;26(12):3572.
doi: 10.3390/molecules26123572.

Significant Inactivation of SARS-CoV-2 In Vitro by a Green Tea Catechin, a Catechin-Derivative, and Black Tea Galloylated Theaflavins

Eriko Ohgitani  1 , Masaharu Shin-Ya  1 , Masaki Ichitani  2 , Makoto Kobayashi  2 , Takanobu Takihara  2 , Masaya Kawamoto  1 , Hitoshi Kinugasa  2 , Osam Mazda  1
Affiliations

Significant Inactivation of SARS-CoV-2 In Vitro by a Green Tea Catechin, a Catechin-Derivative, and Black Tea Galloylated Theaflavins

Eriko Ohgitani et al. Molecules. .

Abstract

Potential effects of tea and its constituents on SARS-CoV-2 infection were assessed in vitro. Infectivity of SARS-CoV-2 was decreased to 1/100 to undetectable levels after a treatment with black tea, green tea, roasted green tea, or oolong tea for 1 min. An addition of (-) epigallocatechin gallate (EGCG) significantly inactivated SARS-CoV-2, while the same concentration of theasinensin A (TSA) and galloylated theaflavins including theaflavin 3,3'-di-O-gallate (TFDG) had more remarkable anti-viral activities. EGCG, TSA, and TFDG at 1 mM, 40 µM, and 60 µM, respectively, which are comparable to the concentrations of these compounds in tea beverages, significantly reduced infectivity of the virus, viral RNA replication in cells, and secondary virus production from the cells. EGCG, TSA, and TFDG significantly inhibited interaction between recombinant ACE2 and RBD of S protein. These results suggest potential usefulness of tea in prevention of person-to-person transmission of the novel coronavirus.

Keywords: COVID-19; catechin; novel coronavirus; polyphenol; tea; theaflavin.

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Conflict of interest statement

This study was funded by ITO EN, Ltd., Tokyo, Japan. The company also provided tea samples, sample preparations, and discussion with authors. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.

Figures

Figure A1
Figure A1
Effects of various tea beverages on infectivity of the novel coronavirus. Powdered matcha green tea, as well as leaves of green tea, roasted green tea (Hojicha), green tea (Shin-Cha, i.e., first picking of a season), black tea, Pu-erh tea, and oolong tea were purchased at a supermarket in Kyoto, and brewed in hot water according to each recipe indicated on the package. After filtrated through a 0.22 μm filter, 500 μL of each sample was mixed with 20 μL of SARS-CoV-2 suspension (5 × 105 TCID50/50 μL) and incubated at room temperature for 1 min (n = 1). Immediately, each mixture was serially diluted 10-fold into MS, and 50 μL of the samples were added to VeroE6/TMPRSS2 cells that had been cultured in 96-well-plates overnight (n = 4 wells per each dilution). Plates were incubated for 50 min with gentle shaking every 10 min. Fifty μL of MS was added to the cells, which were subsequently cultured under standard conditions for 3 days. Cells were stained with Crystal Violet, and TCID50 values were calculated by Reed–Muench method as described elsewhere (n = 4 wells per each dilution). (a) Experimental protocol is shown. Pink arrows represent the presence of tea and virus. (b) TCID50/50 μL values are shown.
Figure A2
Figure A2
Chemical formulae of the compounds used in the study.
Figure 1
Figure 1
Infectivity of SARS-CoV-2 was significantly reduced by an exposure to tea. Virus was treated with ×1 concentration of the indicated tea/DMEM for 1 min, immediately followed by a 10-fold serial dilution and infection into VeroE6/TMPRSS2 cells to measure TCID50 values as described in the Materials and Methods. Scheme of experiments (a) and virus titer of each sample (b) are shown. Values are means ± S.D. (n = 3). ** p < 0.01 vs. Control by Tukey’s multiple comparison test.
Figure 2
Figure 2
Viability of VeroE6/TMPRSS2 cells after infection with SARS-CoV-2 treated with tea. Virus was treated with each tea at the indicated dilutions for 1 min, and the virus/tea/DMEM mixture was added to VeroE6/TMPRSS2 cells at MOI = 3 for 1 h. After removal of the supernatant, cells were cultured in fresh MS for 30 h, followed by cell viability determination as described in the Materials and Methods. Scheme of experiments (a) and A450 value of each sample (b) are shown. Values are means ± S.D. (n = 3). ** p < 0.01 and *** p < 0.001 between groups by Student’s t test.
Figure 3
Figure 3
EGCG and galloylated theaflavins significantly decreased SARS-CoV-2 titer. Virus was treated with 500 μM (b) or the indicated concentrations (c) of tea catechins, a catechin-derivative, and theaflavins for 1 min, immediately followed by a 10-fold serial dilution and infection into VeroE6/TMPRSS2 cells to measure TCID50 values as in Figure 1. Scheme of experiments (a) and virus titer of each sample (b,c) are shown. Values are means ± S.D. (n = 3). * p < 0.05 and ** p < 0.01 vs. Control by Tukey’s multiple comparison test.
Figure 4
Figure 4
EGCG, TSA, and TFDG inactivated SARS-CoV-2, but did not render cells resistant to intact virus. (a,b) Virus was treated with each compound at the indicated concentrations for 1 min, and the mixture was added to VeroE6/TMPRSS2 cells at MOI = 3 for 1 h. After removal of the supernatant, cells were cultured in fresh MS for 30 h, followed by cell viability evaluation as described in the Materials and Methods section. (c,d) Cells were pretreated with the indicated concentrations of the compounds for 10 min, followed by a removal of the supernatant, wash with PBS, and an addition of virus suspension (MOI = 5). One hour later, supernatant was replaced by fresh MS, and cells were culture for 30 h until cell viability was evaluated. Values are means ± S.D. (n = 3). * p < 0.05, ** p < 0.01 and *** p < 0.001 between groups by Student’s t test.
Figure 5
Figure 5
Evaluation of viral RNA in the supernatants and the cells infected with EGCG-, TSA-, or TFDG-treated virus. Virus was treated with the indicated compounds for 1 min, and the mixture was added to VeroE6/TMPRSS2 cells at MOI = 5 for 1 h. After removal of the supernatant, cells were washed with PBS and cultured in fresh MS for 10 h (a). RNA was extracted from the supernatants and cells, and real time-RT-PCR was performed as described in the Materials and Methods section. The culture supernatants were also subjected to TCID50 assay. Means ± S.D. of relative RNA levels and TCID50 values are shown (n = 4) (b). * p < 0.05, ** p < 0.01, and *** p < 0.001 vs. Control by Tukey’s multiple comparison test.
Figure 6
Figure 6
EGCG, TSA, and TFDG interfered with the interaction between ACE2 and RBD. The indicated concentrations of each compound as well as a mixture of 40 µM TSA, 75 µM TF3G, 35 µM TF3′G, and 60 µM TFDG (cocktail) were subjected to a neutralizing assay using a SARS-CoV-2 Surrogate Virus Neutralization Test Kit (GenScript, Piscataway, NJ, USA) as described in the Materials and Methods section. % Inhibition for each sample is shown. Values are means ± S.D. (n = 3 (a) or 4 (b)). *** p < 0.001 vs. Negative control by Tukey’s multiple comparison test.
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