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Inactivation of SARS-CoV-2 by Catechins from Green Tea

Hidekazu Nishimura1*, Michiko Okamoto2*, Isolde Dapat1, Masanori Katumi3, Hitoshi

Oshitani2

1Virus Research Center, Clinical Research Division, Sendai Medical Center, Miyagino,

Sendai, Japan

2Department of Virology, Tohoku University Graduate School of Medicine, Sendai,

Japan

3Sendai City Institute for Public Health, Miyagino, Sendai, Japan.

*Equally contributed

Corresponding author: Hidekazu Nishimura, e-mail: 113-vrs.center@mail.hosp.go.jp

Keywords: COVID-19, SARS-CoV-2, inactivation, catechin mixture from green tea

Running head: Inactivation of SARS-CoV-2 by catechins

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Mailing addrcss: Virus Research Center, Clinical Research Division, Sendai Medical

Center, Miyagino 2-11 -12, Miyagino-ku, Scndai 983-8520, Japan,

Tcl: 8l-22-293-l l73. Fax: 81-22-293-1173. E-mail: hide-nishimura@mte.biglobe. ne. jp

西村秀一 1, 岡本道子 2, イソルデダパト 1， 勝見正道 3, 押谷仁 2

1宮城県仙台市宮城野区宮城野 2－11－12 国立病院機構仙台医療センター臨床

研究部ウイルスセンター

2東北大学大学院医学系研究科微生物学分野

3仙台市衛生研究所

‡責任著者連絡先

西村秀一

〒983-8520 宮城県仙台市宮城野区宮城野２－１１－１２

Tel. 022-293-1173

Fax. 022-293-1173

E-mail. 113-vrs.center@mail.hosp.go.jp

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Summary

Green tea extracts effectively inactivated SARS-CoV-2 in vitro in a dose-dependent

manner. Serially 10-fold diluted solutions of catechin mixture reagent from green tea

were mixed with the viral culture fluid at a volume ratio of nine to one, respectively,

and kept at room temperature for 5 min. The solution of 10 mg/mL catechin reagent

reduced the viral titer by 4.2 log and 1.0 mg/mL solution reduced only by one log. Pre-

infection treatment of the cells with the reagent alone did not affect the viral growth. In

addition, cells treated with only the reagent was assayed for host-cell viability using the

WST-8 system and almost no host-cell damage by the treatment was observed. These

findings suggested that the direct treatment of virus with the reagent before inoculation

decreased the viral activity and that catechins might have a potential to suppress the

SARS-CoV-2 infection.

Introduction

The pandemic of COVID-19 has circulated around the world since the emergence of

SARS-CoV-2 infection-cases in 2019, being associated with a great number of mortal

cases (1)

Trials of developing drugs and vaccines have been aggressively performed

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worldwide but none have been successful yet for general use. Any effective

pharmaceutical and non-pharmaceutical interventions are wanted. Agents that inhibit

the viral replication without adverse effects are explored extensively as candidate

materials for treatment or prophylaxis of the disease.

Catechins are a class of polyphenolic flavonoids including mainly (+)-Catechin,

(-)-Epichatechin, (EC); (-)-Epigallocatechin, (EGC), in which (+) and (-) stand for its

isomeric form. Catechins are contained particularly in tea leaves with various

concentrations among tea types, and main catechins present in the green tea are EC,

ECG, (-)Epicatechin gallate (ECg), and (-)-Epigalocatechin gallate (EGCg) (2,3).

Those from green tea were reported in the late 1990s to have a potential as anti-

influenza virus agents in several experimental studies in vitro, which showed a

decrease in the infectivity of influenza A virus in Madin–Darby canine kidney cells

(4,5), possibly by inhibiting viral entry at the host cell membrane. The catechin

compounds contained in green tea were applied for prophylaxis of influenza and the

common cold in some clinical/epidemiological studies with good results (2).

Furthermore, there was even a report that EGCg has broad-spectrum antiviral activity

against various viral families, such as Flaviviridae, Retroviridae, Hepadnaviridae,

Herpesviridae, Adenoviridae, Orthomyxoviridae, and Picornaviridae (3), and

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bunyavirus (6).

In this study, we performed inactivation experiments of the SARS-CoV-2 with a

catechin mixture extracted from green tea with a hope that it may become a good agent

for prophylactics or and treatments against the COVID-19.

Materials and metods

Virus and cells

The virus used in this study was isolated from a patient of COVID-19 in Sendai

(SARS-CoV-2/Sendai/A19-89) and propagated in Vero E6 cells cultured in Eagles’

Minimal Essential Medium (MEM) (Sigma-Aldrich, STL, USA) supplemented with

10% fetal calf serum. Its seed stocks were kept in a -80C freezer as aliquots. The culture

medium used for propagation of the virus of the isolated strain and for viral titration in

the cells was MEM with 2% calf serum (CS).

Catechin reagent

A standard catechin reagent for biochemistry (Catechin Mixture, from Green Tea,

Fujifilm-Wako Junyaku, Tokyo, Japan) was dissolved first in methanol as 100 mg/mL

solution and then diluted to working concentrations in MEM.

An infection titer assay on inhibition of viral growth

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The assay was performed using confluent Vero E6 cell layers in 96 wells microplate

(Greiner Bio-One, Kremsmünster, Austria) for cell culture. Four hundred and fifty µL

of each dilution of the catechin reagent was added to 50 µL of viral culture fluid and

left at room temperature of about 23 °C for 5 min. After the reaction, the mixture was

immediately diluted with 5mL of MEM (10-fold dilution) to stop the reaction and

minimize the possible effect of high concentrations of catechins to the cell, then

serially diluted 10-fold and subjected to an assay for active viral titer by an endpoint

dilution assay to determine the 50% tissue culture infectious dose (TCID50) in Vero

E6 cells. The medium containing a 10-fold dilution of the specimen-virus mixture in

the first line of the assay was removed after 1 hr of incubation time, cells were washed

once with MEM, and fresh MEM was added to the wells. The viral titer was

determined through cytopathic effect by the Reed-Muench method at 3 days after the

infection.

Pre-infection treatments of the cells

The cells were treated with the catechin mixture for 5 min prior to the infection with

the virus. For pre-treatment, Vero E6 cells cultured in a 24-well plate were treated with

1mg/mL of the reagent in MEM, kept for 5 min at 34 °C, washed once with MEM, and

infected with the virus at a multiplicity of infection of 0.1, followed by incubation for 1

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hr at 34°C. The cells were washed once with MEM and cultured for 3 days in MEM 2%

CS in the CO2 incubator at 34 °C. The viral growth was monitored for two days after the

inoculation by a viral RNA assay using the quantitative real-time reverse transcription-

polymerase chain reaction (rRT-PCR) assay, and the occurrence of cytopathic effect

(CPE) was checked on day 3.

Viral RNA assay

A quantitive rRT-PCR was performed for measuring viral RNA in the culture

medium of cells infected with the virus, according to the manual for the detection of

pathogen 2019-nCoV published by the National Institute of Infectious Diseases (7).

Briefly, viral RNA was extracted from 140uL of culture supernatant using QIAamp

Viral RNA Minikit (QIAGEN, Hilden, Germany) and 5uL of extracted RNA was

added to TaqMan Fast Virus 1-Step Master Mix (Thermo Fisher Scientific, MA,

USA). A Step OnePlus (Thermo Fisher Scientific, MA, USA) kit was used for the

assay.

Cell viability assay

Possible cytotoxic effects that might be caused by the specimens were measured by

the WST-8 assay (Cell Counting Kit-8, Dojindo, Tokyo, Japan) of the cellular NAD-

dependent succinate dehydrogenase activity which is proportional to the cell

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metabolic activity or viability (8).

The Vero E6 cells were mock-infected with the specimens of various concentration

without mixing with the virus and incubated for 5 min or 1 hr, followed by washing

with MEM. The assay was performed immediately, at 24 and 48 hrs after incubation,

and absorbance at 450nm was measured using a microplate reader, Model 680 (Bio-

Rad, CA, USA).

Results

Effect of the catechin reagent

The catechin mixture decreased the virus titer in a dose-dependent manner and the

virus titer decreased more than 4 Log at the concentration of 10 mg/ mL (Table 1). The

10% methanol contained in the catechin 10mg/mL solution was confrmed to have no

antiviral effect.

In order to know whether the inhibitory effects seen above were the direct effect

of the reagent to the virus or by any other effect on the host cells, experiments of pre-

infection treatment of the host cells with the reagent were performed at various reagent

concentrations. The treatments did not affect the viral growth at all. Viral RNA levels in

culture supernatants of the cells pretreated with the catechin, which were measured by

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quantitative RT-PCR as their Cq values, were almost the same with the control (data not

shown). In addition, occurrence of CPE was confirmed in the entire cell monolayer

(Table 2). Thus, the possibility of indirect effects of the reagent to the cells temporarily

caused by incubation with the reagent-virus mixture at the start of infection might be

low.

Exclusion of a possible effect of cytotoxic activity by catechins on the host cells

The inhibition of the viral growth by the catechin reagent was not due to its

cytotoxic effect on the cells. The viability was assayed by the WST-8 system on the

cells treated with the catechin reagent and it did not show a decrease at a concentration

of 1.0 mg/mL, corresponding to the first line dilution in the TCID50 assay for viral

activity inhibition test, at 5 min incubation with the cells until 48 hrs. A slight decrease

in the viability was observed at 60 min incubation and at assay at 24 hrs (Table 3).

However, it was less possible that the viral replication at the first-line wells in the

TCID50 assay was affected by the relatively lower cellular viability that was caused by

incubation with the reagent-virus mixture since the pre-infection treatment of the cells at

this concentration did not affect the viral growth (Table 2).

Discussion

In this study, we showed that a catechin mixture reagent inactivated SARS-CoV-2

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in a dose-dependent manner and suggested that some kinds of catechins have anti-viral

activity against the virus, as well as against other viruses. Limitation of this study is that

we used a reagent of catechin mixture: a crude extract from green tea, which did not

have detailed information on the kinds of catechins it contained such as EC, EGC, ECg,

EGCg and their corresponding quantities. Further investigations to know which of them

has/have the antiviral activity are awaited. In addition, determining the mechanism of

the antiviral activity was beyond the scope of the study. However, it seemed that it

might be at least different from that in severe fever with thrombocytopenia syndrome

virus (6) because the latter inhibited the viral growth by pre-treatment of the cells before

infection but the former did not. Future studies to explore the mechanism will be

welcomed.

It may be to soon to tell whether this finding is applicable to actual prophylaxis or

treatment of COVID-19, but it may have the potential to be a good agent against the

disease if some feasible application will be established, because the catechins in tea

leaves are abundant as a resource and are known to have almost no adverse effect to

humans. Exploring the anti-SARS-CoV-2 activity in green tea products to establish the

ability of catechins will be useful.

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Conflict of interest None to declare

Acknowledgement

This work was financially supported by the Clinical Research Division of Sendai

Medical Center, the Japan Agency for Medical Research and Development (AMED)

(Grant Number JPwm0125001), and the Japan Society for the Promotion of Science (JSPS)

KAKENHI (Grant Number JP19K24679).

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References

1. WHO. Novel Coronavirus (2019-nCoV) situation reports. Available at:

.

Accessed October 5, 2020.

2. Furushima D, Ide K, Yamada H. Effect of Tea Catechins on Influenza Infection and

the Common Cold with a Focus on Epidemiological/Clinical Studies. Molecules 2018;

23:1795; doi:10.3390/molecules23071795.

3. Steinmann, J.; Buer, J.; Pietschmann, T.; Steinmann, E. Anti-infective properties of

epigallocatechin-3-gallate (egcg), a component of green tea. Br. J. Pharmacol. 2013;

168: 1059–1073.

4. Nakayama M, Suzuki K, Toda M et al. Inhibition of the infectivity of influenza virus

by tea polyphenols. Antiviral Res 1993;21:289-99.

5. Tezuka M, Suzuki H, Suzuki Y, et al. Inactivation effect of tea catechins on human

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https://pubmed.ncbi.nlm.nih.gov/?term=Nakayama+M&cauthor_id=8215301https://pubmed.ncbi.nlm.nih.gov/?term=Suzuki+K&cauthor_id=8215301https://pubmed.ncbi.nlm.nih.gov/?term=Toda+M&cauthor_id=8215301

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type-A influenza virus. Jpn J. Toxical Environ. Health 1997; 43;311-315.

6. Ogawa M, Shimojima M, Saijo M et al. Several catechins and flavonols

from green tea inhibit severe fever with thrombocytopenia syndrome virus

infection in vitro. J Infect Chemother, 2020;S1341-321X(20)30275-0.

doi: 10.1016/j.jiac.2020.08.005.

7. National Institute of Infectious Diseases. Manual for the Detection of Pathogen 2019-

nCoV. Available at:

Accessed October 5, 2020.

8. Ishiyama M,Miyazono Y, Sasamoto K et al.A highly water-soluble disulfonated

tetrazoliumsalt as a chromogenic indicator for NADH as well as cell viability. Talanta,

1997; 44:1299-1305. Accep

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https://pubmed.ncbi.nlm.nih.gov/?term=Ogawa+M&cauthor_id=32868200https://pubmed.ncbi.nlm.nih.gov/?term=Shimojima+M&cauthor_id=32868200https://pubmed.ncbi.nlm.nih.gov/?term=Saijo+M&cauthor_id=32868200https://www.niid.go.jp/niid/images/epi/corona/2019

Table 1 Antiviral activity of the catechin mixture from green tea

Concentration Viral titer (log)＊

(TCID50/50μl)

0.0 mg/mL 5.50 5.75

0.1 mg/mL 4.75 NT

1.0 mg/mL 4.50 4.75

10.0 mg/mL

Table 2 Effect of pre-infection treatment of host cells with the catechin mixture on viral growth

* Viral RNA levels in supernatants were measured by quantitative RT-PCR and shown as

differences in Cq values.

** Standard deviation of Cq values.

Concentration *mean -ΔCq value of 3 tests **(SD) CPE (%)

day 0 day 1 day 2 day 3

10.0 mg 0 (0.09) 5.4 (0.24) 8.5 (0.09) 100

3.3 mg 0 (0.20) 5.6 (0.08) 8.2 (0.15) 100

1.0 mg 0 (0.18) 5.8 (0.05) 8.1 (0.08) 100

0 mg 0 (0.11) 6.9 (0.64) 8.2 (0.19) 100

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Table 3

Viability assay of cells after treatment

with the catechin mixture from green tea

Concentration Incubation time

WST-8 assay time

after mock infection

immediately 24 hs 48 hs

1.0 mg/mL 5 min 0.790 0.759 1.121

60 min 0.792 0.578 0.653

0.1 mg/mL 60 min 0.899 0.844 1.071

0.0 mg/mL 0.888 0.804 1.005

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