ORIGINAL RESEARCH
published: 29 March 2022
doi: 10.3389/fphar.2022.804103

In vitro Suppression of SARS-CoV-2
Infection by Existing Kampo Formulas
and Crude Constituent Drugs Used forTreatment of Common Cold
Respiratory Symptoms
Masaki Kakimoto 1, Toshihito Nomura 2,3, Tanuza Nazmul 3, Hiroki Kitagawa 2, Keishi Kanno 1,
Keiko Ogawa-Ochiai 1,4, Hiroki Ohge 2, Masanori Ito 1 and Takemasa Sakaguchi 3*
1
Department of General Internal Medicine, Hiroshima University Hospital, Hiroshima, Japan, 2Department of Infectious Disease,
Hiroshima University Hospital, Hiroshima, Japan, 3Department of Virology, Graduate School of Biomedical and Health Sciences,
Hiroshima University, Hiroshima, Japan, 4Kampo Clinical Center, Department of General Internal Medicine, Hiroshima University
Hospital, Hiroshima, Japan

Edited by:
Kenji Watanabe,
Yokohama College of Pharmacy,
Japan
Reviewed by:
Xuepeng Gong,
Huazhong University of Science andTechnology, China
Theresa Li-Yun Chang,
Rutgers, The State University of New
Jersey, United States*Correspondence:
Takemasa Sakaguchi
tsaka@hiroshima-u.ac.jp
Specialty section:
This article was submitted toEthnopharmacology,
a section of the journal
Frontiers in Pharmacology
Received: 28 October 2021
Accepted: 07 March 2022
Published: 29 March 2022
Citation:
Kakimoto M, Nomura T, Nazmul T,
Kitagawa H, Kanno K,
Ogawa-Ochiai K, Ohge H, Ito M and
Sakaguchi T (2022) In vitro
Suppression of SARS-CoV-2 Infection
by Existing Kampo Formulas andCrude Constituent Drugs Used forTreatment of Common Cold
Respiratory Symptoms.
Front. Pharmacol. 13:804103.
doi: 10.3389/fphar.2022.804103

Several traditional Japanese Kampo formulas are known to have inhibitory effects on
infections with viruses that cause respiratory symptoms. Although some herbs and theircomponents have been reported to suppress SARS-CoV-2 replication in vitro, it is difﬁcult
to compare effective Kampo formulas because of the different methods used in studies.
Thus, we carried out in vitro experiments on the suppression of SARS-CoV-2 infection by
Kampo formulas and crude drugs used for the common cold to compare their suppressive
effects on virus infection. After infecting VeroE6/TMPRSS2 cells with SARS-CoV-2, lysatesof the Kampo formulas and crude drugs were added, and after 24 h, the infectious titer inthe medium was measured by the TCID50 method. Maoto was the most effective among
the Kampo formulas, and Ephedrae herba was the most effective among the constituent
crude drugs. However, a comparison of the suppressive effects of Ephedrae herba andKampo formulas containing Ephedrae herba showed that the suppressive effect on virus
infection did not depend on the content of Ephedrae herba. Based on the results, we
believe that the use of Maoto among Kampo formulas is suitable as a countermeasureagainst COVID-19.
Keywords: anti-viral, COVID-19, SARS-CoV-2, herbal medicine, kampo medicine, crude drug

INTRODUCTION
In September 2021, the cumulative number of SARS-CoV-2 infection cases worldwide was morethan 200 million and the number of deaths from SARS-CoV-2 infection was more than 4.5 million.
There has been no apparent reduction in the number of new infections or deaths (World HealthOrganization, 2021). Although the efﬁcacy of vaccines has been recognized and vaccination has been
accelerated in many countries worldwide, there are still many unclear issues about vaccines as the
efﬁcacy of vaccines against emerging mutant strains (Liu et al., 2021). On the other hand, there arefew drugs that can be expected to have antiviral effects and most of the drugs are expensive (Hsu,
2020). Therefore, there is an urgent need to ﬁnd new anti-viral drugs or alternative treatments withdrug repositioning.

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was harvested and ﬁltered through a 0.45-µm ﬁlter after lowspeed centrifugation. The virus titer was determined by thestandard 50% tissue culture infectious dose (TCID50) method.
Brieﬂy, a 10-fold serial dilution of the virus was inoculated into
cells in a 96-well plate in tetraplicate or octuplicate and incubated
for 7 days to check for CPE. Based on this result, infectivity wascalculated and expressed as TCID50/ml, as described previously(Nomura et al., 2021).

Kampo medicine is a systematized medical system based on
traditional East-Asian medicine and unique drugs. Kampo
medicine originated in traditional Chinese medicine, which
was introduced to Japan around the 5th century and was
reﬁned from the 17th century to the currently used Kampo
medicine in Japan. Kampo formulas are mixtures of crude
drugs, which are extracts of herbs, insects, minerals, fungi, andother substances (Tsumura and Co., 2016). All Kampo formulas
were approved as drugs for humans by Japan’s Ministry of
Health, Labour and Welfare in 1986 and are regulated by the
National Institute of Health and Welfare (STORK, 2020). Amongthe medications certiﬁed by the Ministry of Health, Labour andWelfare, medical Kampo formulas are comparatively
inexpensive, and many of them have versatility in use. On theother hand, herbal medicines themselves are also used as folk
remedies not only in Japan but also in many other countries.
Inﬂuenza and respiratory syncytial viruses are RNA viruses
and the primary symptoms of infections with these viruses are
respiratory symptoms that are similar to the symptoms of SARSCoV-2 infection. In Japan, two Kampo prescriptions, Maoto and
Saikokeishito, have been approved for treatment of inﬂuenza
infection. There have been in vitro studies showing suppression of
infection with these viruses by Kampo formulas and crude drug
extracts (Mantani et al., 1999; Nomura et al., 2019; Hou et al.,2020). There seems to be a relationship between the clinicaleffects of Kampo formulas and inhibition of inﬂuenza virus
growth in cultured cells.
In this study, we examined the suppressive effects of Kampo
formulas on SARS-CoV-2 infection using VeroE6/TMPRSS2
cells, which are highly susceptible to SARS-CoV-2 infection(Matsuyama et al., 2020). We selected eight Kampo formulasthat have been shown to be effective against inﬂuenza virus
infections and common cold symptoms and examined theirinhibitory effects on SARS-CoV-2 infection. We also
investigated the inhibitory effects of six crude constituent
drugs constituting those Kampo formulas on SARS-CoV-2
infection.

Reagents
Extract powders of Kampo formulas including Maoto,
Saikokeishito,
Shomakakkonto, Kakkonto,
Shoseiryuto,
Senkyuchachosan, Bakumondoto, and Hochuekkito werekindly provided by Tsumura & Co. (Tokyo, Japan) (Table 1).
Crude drugs including Glycyrrhiza uralensis Fisch. ex DC[Fabaceae] (Glycyrrhizae radix), Ephedra sinica Stapf[Ephedraceae] (Ephedrae herba), Paeonia lactiﬂora Pall
[Paeoniaceae] (Paeoniae radix), Ligusticum ofﬁcinale (Makino)
Kitag [Apiaceae] (Cnidii rhizoma), Scutellaria baicalensis Georgi[Lamiaceae] (Scutellariae radix), and Bupleurum falcatum L[Apiaceae] (Bupleuri radix) were purchased from Tsumura &Co. The Kampo formulas used in this study and their constituent
crude drugs (generic name, scientiﬁc name, and percentage
included) are shown in detail in Table 1.
Solutions of the Kampo formulas for testing were prepared as
described previously (Nomura et al., 2019). The powder of each
Kampo formula was mixed with DMEM to a concentration of
20 mg/ml. The powder was dissolved at 50°C for 1 h and the
mixture was centrifuged at a low speed and then the supernatant
was ﬁlter-sterilized through a 0.22-μm ﬁlter. The crude drugs
were prepared in a similar way. For both the Kampo formulas andthe crude drugs, little insoluble material was found after lowspeed centrifugation, and the weight of the initial powder was
therefore used as the weight of the solute.

Cytotoxicity Assay
VeroE6/TMPRSS2 cells were cultured in DMEM with thespeciﬁed concentrations of reagents for 24 h, and lactate
dehydrogenase (LDH), which was released from the cells into
the medium, was assayed with a colorimetric method using the
Cytotoxicity LDH Assay Kit-WST (Dojindo Laboratories,
Kumamoto, Japan) by measuring absorbance at 490 nm in the
TriStar LB 941 plate reader (Bertohold Technologies, Wildbad,
Germany). The cytotoxicity of the reagents was calculated fromthe absorbance measurements as 100% for the high control (celllysis by surfactant) and 0% for the low control (culture mediumonly).

MATERIALS AND METHODS
Cells and Viruses
VeroE6/TMPRSS2 cells [African green monkey kidney-derived
cells expressing human TMPRSS2, purchased from JapaneseCollection of Research Bioresources (JCRB) Cell Bank, JCRB
1819] were propagated in Dulbecco’s modiﬁed Eagle’s minimum
essential medium (DMEM, Invitrogen) supplemented with 10%
fetal calf serum (FCS; Biosera, Kansas City, MO, United States),
penicillin G (100 units/ml, Meiji Seika Pharma, Tokyo, Japan),and streptomycin (100 μg/ml, Meiji Seika Pharma). The cells
were cultured at 37°C in 5% CO2. SARS-CoV-2/JP/Hiroshima46059T/2020 (Yamamotoya et al., 2021; B1.1.1, GISAID
accession ID: EPI_ISL_6289932, GenBank/DDBJ/EMBL
accession number: MZ853926) was used as the test virus.
To prepare virus suspensions, VeroE6/TMPRSS2 cells were
infected with the virus and incubated in DMEM. Whencytopathic effects were fully developed, the culture supernatantFrontiers in Pharmacology | www.frontiersin.org

Replication of SARS-CoV-2 in vitro
Conﬂuent monolayers of VeroE6/TMPRSS2 cells in a 96-wellplate were infected with 50 µl/well of the virus at an input
multiplicity of infection (m.o.i.) of 0.05 or 10. After adsorption
for 2 h, the inoculated viruses were removed, and the cells were
further cultured in 100 µl/well of DMEM containing different
concentrations of Kampo formulas or crude drugs. The
conditions of m.o.i. and virus adsorption time were based on
the conditions used in a previous study for effective infection of2

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MaotoIndications
Crude drugs(Latin name)Scientiﬁc nameRate of crude
drugs
conﬁguring
Kampo extracts(excluding
additives)
Saikokeishito
Indications
Crude drugs(Latin name)Scientiﬁc name3

Rate of crude
drugs
conﬁguring
Kampo extracts(excluding
additives)
Kakkonto
Indications
Crude drugs(Latin name)Scientiﬁc name

Prunus armeniaca var. armeniaca [Rosaceae] Prunus
armeniaca L. [Rosaceae]
32.3%

Ephedrae herbaa

Cinnamomi cortex

Glycyrrhizae radix*

Ephedra sinica Stapf [Ephedraceae]

Neolitsea cassia (L.) Kosterm.
[Lauraceae]
25.8%

Glycyrrhiza uralensis Fisch. ex DC. [Fabaceae]

Ziziphi fructus

Zingiberis rhizoma—
Zingiber ofﬁcinale Roscoe [Zingiberaceae]
—

32.3%

Common cold, Inﬂuenza, Feverish diseases such as pneumonia and pulmonary tuberculosis
Gastric ulcer, Duodenal ulcer, Cholecystitis, Gallstone, Pain in liver dysfunction and pancreatitis
Bupleuri radixa
Pinelliae Tuber Scutellariae
Glycyrrhizae
Cinnamomi
Paeoniae radixa
radixaradixacortex
Bupleurum
Pinellia ternata Scutellaria
Glycyrrhiza
Neolitsea
Paeonia lactiﬂora
falcatum L.
(Thunb.)baicalensis
uralensis Fisch. cassia (L.)
Pall.
[Apiaceae]
MakinoGeorgiex DC.
Kosterm.
[Paeoniaceae][Araceae][Lamiaceae][Fabaceae][Lauraceae]
22.7%
18.2%
9.1%
9.1%
9.1%
9.1%

Ziziphus jujuba Mill.
[Rhamnaceae]

9.1%

Ginsengradix
Panax
ginseng
C.A.Mey.
[Araliaceae]
9.1%

Common cold, Early stage of febrile disease, Inﬂammatory diseases (conjunctivitis, keratitis, otitis media, tonsillitis, mastitis, lymphadenitis)
Stiff shoulders, Neuralgia in the upper body, Urticaria
Puerariae Radix
Ziziphi fructus
Ephedrae herbaa Glycyrrhizae radixa
Cinnamomi cortex
Paeoniae radixa
Pueraria montana
var. lobata (Willd.)
Maesen &S.M.Almeida ex
Sanjappa &Predeep[Fabaceae]
22.2%

9.7%

4.6%
—

Zingiberis rhizoma

Ziziphus jujuba
Mill.
[Rhamnaceae]

Ephedra sinica i[Ephedraceae]

Glycyrrhiza uralensis
Fisch. ex DC.
[Fabaceae]

Neolitsea cassia (L.)
Kosterm.
[Lauraceae]

Paeonia lactiﬂora Pall.
[Paeoniaceae]

Zingiber ofﬁcinale Roscoe [Zingiberaceae]

16.7%

16.7%

11.1%

11.1%

11.1%

11.1%

Shomakakkonto
Indications
Common cold (early stage), Dermatitis
Crude drugs
Puerariae Radix
Paeoniae radixa(Latin name)

Cimicifugae Rhizoma

Glycyrrhizae radixa

Zingiberis rhizoma(Continued on following page)

Kampo Formulas for SARS-CoV-2

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Rate of crude
drugs
conﬁguring
Kampo extracts(excluding
additives)

Common cold, Inﬂuenza (Acute), Rheumatoid arthritis
Armeniacae semen

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TABLE 1 | The Kampo medicines used in this study and their constituent crude drugs.

Scientiﬁc nameRate of crude
drugs
conﬁguring
Kampo extracts(excluding
additives)
Shoseiryuto
Indications
Crude drugs(Latin name)Scientiﬁc name

4Rate of crude
drugs
conﬁguring
Kampo extracts(excluding
additives)

Pueraria montana var. lobata(Willd.) Maesen &S.M.Almeida ex Sanjappa &Predeep [Fabaceae]
41.7%

Paeonia lactiﬂora
Pall.
[Paeoniaceae]
25.0%

Actaea dahurica (Turcz. ex Fisch. & C.A.Mey.)
Franch. [Ranunculaceae]・ Actaea heracleifolia(Kom.) J.Compton [Ranunculaceae]・ Actaea
cimicifuga L. [Ranunculaceae]
16.7%

Common cold, Bronchitis, Bronchial asthma, Rhinitis, Allergic Rhinitis, Allergic conjunctivitis
Pinelliae tuber
Paeoniae
Zingiberid
Glycyrrhizae
Cinnamomi
radixarhizomaradixacortex
processum
Pinellia ternata
Paeonia
Zingiber
Glycyrrhiza
Neolitsea(Thunb.) Makino
lactiﬂora Pall.
ofﬁcinale Roscoe uralensis Fisch. cassia (L.)[Araceae][Paeoniaceae][Zingiberaceae]
ex DC.
Kosterm.
[Fabaceae][Lauraceae]
22.2%
11.1%
11.1%
11.1%
11.1%

Glycyrrhiza uralensis Fisch. ex DC.
[Fabaceae]

Zingiber ofﬁcinale Roscoe [Zingiberaceae]

12.5%

4.2%

Asiasari radix

Schisandrae fructus

Ephedrae herbaa

Asarumheterotropoides
F.Schmidt[Aristolochiaceae]
11.1%

Schisandra chinensis (Turcz.)
Baill. [Schisandraceae]

Ephedra sinica Stapf [Ephedraceae]

11.1%

11.1%

Bakumondoto
Indications
Crude drugs(Latin name)Coughing with a hard, Obstructive sputum, Bronchitis, and bronchial asthma
Ophiopogonis radix
Oryzae fructus
Pinelliae tuber
Ziziphi fructus

Glycyrrhizae radixa

Glycyrrhizae
radixaGlycyrrhiza
uralensis
Fisch. exDC.
[Fabaceae]

Camelliae sinensis folium—
Camellia sinensis (L.) Kuntze [Theaceae]

7.5%

7.5%

Ginseng radix(Continued on following page)

Kampo Formulas for SARS-CoV-2

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SenkyuchachosanIndications
Common cold, automatic imbalance syndrome peculiar to women resembling climacteric disturbance, and headache
Crude drugs
Cyperi rhizoma
Cnidii
Notopterygii
Schizonepetae
MenthaeAngelicae
Saposhnikoviae(Latin name)rhizomaarhizomaspica
herba
dahuricae radix
radix
Saposhnikovia
NepetaMenthaAngelica
Hansenia
Scientiﬁc name
Cyperus rotundus L.
Ligusticum
tenuifolia
canadensis
dahurica (Hoffm.) divaricata (Turcz. ex
weberbaueriana[Cyperaceae]
ofﬁcinale
Ledeb.) Schischk.
Benth.
L.
Benth. & Hook.f.
(Fedde ex(Makino)[Lamiaceae][Lamiaceae] ex Franch. & Sav. [Apiaceae]
H.Wolff)
Kitag.
[Apiaceae]
Pimenov &[Apiaceae]
Kljuykov[Apiaceae]
20.0%
15.0%
10.0%
10.0%
10.0%
10.0%
10.0%
Rate of crude
drugs
conﬁguring
Kampo extracts(excluding
additives)

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TABLE 1 | (Continued) The Kampo medicines used in this study and their constituent crude drugs.

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TABLE 1 | (Continued) The Kampo medicines used in this study and their constituent crude drugs.
Scientiﬁc nameRate of crude
drugs
conﬁguring
Kampo extracts(excluding
additives)
Hochuekkito
Indications
Crude drugs(Latin name)Scientiﬁc name5
Rate of crude
drugs
conﬁguring
Kampo extracts(excluding
additives)

Ophiopogonjaponicus (Thunb.) Ker
Gawl. [Asparagaceae]
37.0%

Oryza sativa L.
[Poaceae]
18.5%

Pinellia ternata(Thunb.) Makino[Araceae]
18.5%

Ziziphus jujuba Mill.
[Rhamnaceae]

Glycyrrhiza uralensis Fisch. ex DC.
[Fabaceae]

Panax ginseng C.A.Mey. [Araliaceae]

11.1%

7.4%

7.4%

Common cold, Summer thinness, Loss of appetite, Gastroptosis, Hemorrhoids, Anal prolapse, Drooping uterus, Pubic atrophy, Hyperhidrosis
Astragali radix
Atractylodis
Ginseng radix
Angelicae
BupleuriZiziphi fructus
Aurantii nobilis
Glycyrrhizae
Lanceaeacutilobae radix radixa
pericarpium
radixaRhizoma
Bupleurum
Ziziphus jujuba
Citrus deliciosa Ten. Glycyrrhiza
Astragalus
Atractylodes
Panax ginsengAngelica
falcatum L.
Mill.
[Rutaceae]
uralensismongholicus
lanceaC.A.Mey.
acutiloba[Apiaceae][Rhamnaceae]
Fisch. ex
Bunge [Fabaceae](Thunb.) DC.
[Araliaceae](Siebold &DC.
[Asteraceae]
Zucc.) Kitag.
[Fabaceae][Apiaceae]

16.7%

16.7%

16.7%

12.5%

8.3%

8.3%

8.3%

6.3%

Cimicifugae Rhizoma

Zingiberis
rhizomaActaea dahurica (Turcz. ex
Fisch. & C.A.Mey.) Franch.
[Ranunculaceae]・ Actaea
heracleifolia (Kom.)
J.Compton[Ranunculaceae]・ Actaea
cimicifuga L.
[Ranunculaceae]
4.2%

Zingiberofﬁcinale Roscoe[Zingiberaceae]

2.1%

Kampo medicines used in this study and their constituent crude drugs are shown with indications of the Kampo medicines. The ratio of constituents included in Kampo drugs of extract powder and crude drugs referred to the website ofTsumura & Co.
a
The crude drugs investigated are marked.

Kampo Formulas for SARS-CoV-2

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FIGURE 1 | Inhibition of SARS-CoV-2 replication by Kampo formulas. (A-H) The Kampo formula used is noted at the top of each panel. VeroE6/TMPRSS2 cells
were infected with SARS-CoV-2 at an m.o.i. of 0.05. After 2-h adsorption, the inoculum was removed and the cells were cultured in DMEM containing different
concentrations of Kampo formulas for 24 h. The viral infectivity in the medium was assayed by the TCID50 method. The left y-axis of the graph is the infectivity for eachconcentration. An exponential approximation was made by Excel to calculate the drug concentration that reduces the infectivity in the absence of a drug to 50%(IC50). The IC50 values and approximation equations are shown in the graph. If there is no decrease, the IC50 is not shown (Panel 1). VeroE6/TMPRSS2 cells wereincubated in DMEM supplemented with the designated concentrations of a Kampo formula for 24 h, and LDH values in the media were then measured to evaluate
cytotoxicity. The LDH value from detergent-treated cells was set at 100%, and the right y-axis is the percent inhibition of cytotoxicity for each concentration.

cells (Wang et al., 2020). The medium was harvested after 24 h,and viral infectivity was assayed by the TCID50 method. The
logarithm of infectivity titer and reagent concentration in themedium were plotted and an approximation straight-line wasdrawn to calculate the 50% inhibitory concentration (IC50), as
described previously (Nomura et al., 2021).
We conducted infection experiments under two conditions: m o.i.
of 0.05 and m.o.i. of 10. At m.o.i. of 10, all cells are infected at once,
allowing us to observe the process of virus entry and replication (onestep replication). When m.o.i. of 0.05, on the other hand, 20 cells are
infected with a single virus. In addition to the entry and replication of
the virus into the cell, the progeny virus is released from the cell andfurther infects the surrounding cells (multi-step replication).

method. For crude drugs, reagents at 1.25–10 mg/ml concentrations
were used considering their cytotoxicity. Phosphate-buffered saline(PBS) was used as an untreated control, and 70% (w/w) ethanol wasused as an inactivation control as described previously (Nomura et al.,2021).

RESULTS
Suppressive Effects of Kampo Formulas on
SARS-CoV-2 Infection
Eight Kampo formulas used to treat respiratory symptoms,
including respiratory symptoms of inﬂuenza and common
cold infections, were investigated for their inhibitory effects on
SARS-CoV-2 infection in vitro. Table 1 shows the clinicalindications of each Kampo formula, the crude drugs included,
and the proportions of the crude drugs.
Initially, the cytotoxicity of each Kampo formula was examined by
an LDH assay, and the values were plotted on a graph (Figure 1, △marker, right Y-axis). Twenty mg/ml of each Kampo formula ofMaoto (Figure 1A), Saikokeishito (Figure 1B), Kakkonto(Figure 1C), and Shomakakkonto (Figure 1D) showed more than

Assay for Inactivation of Viral Particles
For the Kampo formulas, the solution was mixed with 90 µl of the
drug at a concentration of 20 mg/ml and 10 µl of the virus solution at
2.0 × 109 TCID50/ml and incubated for 3 min at room temperature.
The mixture was then serially diluted 10-fold in DMEM, andadsorbed on VeroE6/TMPRSS cells for 1 h. The inoculum wasremoved, and cell culture medium was added and incubated for
7 days. The infectivity of the solution was determined by the TCID50

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the virus may be suppressed at one of the following stages:
intracellular multiplication, release from the cell, or reinfectionof neighboring cells. From the results of the experiment describedabove (Figure 2), it is unlikely that the viral particles are directly
inactivated, and intracellular proliferation or release from the cell
may therefore be impaired.

Suppressive Effects of Crude Drugs on
SARS-CoV-2 Infection
We investigated the suppressive effects on virus infection of theavailable crude drugs with high percentages of composition
among the crude drugs constituting Maoto, Senkyuchachosan,
and Saikokeishito, which had strong inhibitory effects on SARSCoV-2 infection. The crude drugs tested were Ephedrae herba,
Glycyrrhizae radix, Scutellariae radix, Paeoniae radix, Bupleuriradix, and Cnidii rhizoma (Figure 3). Ephedrae herba showed
cytotoxicity at concentration of 2.5 mg/ml and above, Bupleuriradix showed cytotoxicity at concentrations of 10 mg/ml andabove, and Scutellariae radix showed cytotoxicity atconcentrations of 5 mg/ml and above. Therefore, the effects ofthese crude drugs at lower concentrations were evaluated.
The cells were infected with the virus at an m.o.i. of 0.05, andthe infectious titer was measured by the TCID50 method(Figure 3). Ephedrae herba showed a strong inhibitory effecton virus infection, while Paeoniae radix, Scutellariae radix, andGlycyrrhizae radix showed weaker inhibitory effects (Figure 3).
To investigate the mechanisms by which the crude drugs
suppress virus infection, we conducted infection experiments
using crude drugs (Ephedrae herba, Scutellariae radix,
Paeoniae radix, Glycyrrhizae radix) at an m.o.i. of 10 so that
all of the cells would be infected (Figure 3). Ephedrae herba
showed a strong suppressive effect on virus infection even at an
m.o.i. of 10, and Paeoniae radix and Scutellariae radix also
showed suppressive effects (Figure 3). These results suggestthat Ephedrae herba, Paeoniae radix, and Scutellariae radix act
on virus-infected cells to inhibit viral replication in the cells. On
the other hand, Glycyrrhizae radix showed little inhibitory effectin the infection experiment with an m.o.i. of 10 (Figure 3),
suggesting that the antiviral effect of Glycyrrhizae radix is due toits inhibition of spread of the virus to neighboring
uninfected cells.
The inactivating effect of each of the crude drugs on virus particles
was investigated by mixing the crude drug at the highest
concentration used in the virus infection experiment andmeasuring the infectivity titer (Figure 4). Ephedrae herba
decreased the infections titer by 2.5 Log10 (TCID50/ml) comparedto the control, suggesting that Ephedrae herba may act directly on thevirus particles to inactivate them. The other crude drugs did not
reduce the infectious titer, indicating that they did not inactivate thevirus particles (Figure 4).

FIGURE 2 | Effects of Kampo formulas on virus particle inactivation. A
solution of the Kampo formula (20 mg/ml, 90 µl) and 10 µl of the virus solution
at 2.0 × 109 TCID50/ml was incubated for 3 min at room temperature. The
mixture was then serially diluted 10-fold in DMEM, and the infectivity wasdetermined by the TCID50 method. Phosphate-buffered saline (PBS) wasused as an untreated control, and ethanol [70% (w/w)] was used as aninactivation control. The dotted line indicates the detection limit (DL) of theinfectivity assay.

30% cytotoxicity. However, at concentrations below 10 mg/ml, noneof the Kampo formulas showed apparent cytotoxicity. The results at
concentrations below 10 mg/ml could be interpreted without
considering the effect of cytotoxicity, while at 20 mg/ml, theresults should be interpreted with caution.
VeroE6/TMPRSS2 cells were infected with SARS-CoV-2 at an
m.o.i. of 0.05, and each Kampo formula was added to the medium.
After 24 h, the infection titer was measured by the TCID50 method
and plotted on a graph against the concentration of the Kampo
formula (Figure 1, ○ marker, left Y-axis). In the case of Maoto, viral
replication was inhibited as the concentration was increased and was
almost completely inhibited at a concentration of 10 mg/ml(Figure 1A). An approximate line of these points was drawn, andthe equation is shown in the graph. The 50% inhibitory concentration(IC50) that was calculated from the equation is also shown in thegraph (Figure 1A). In the case of Hochuekkito, there was little changeeven when the drug concentration was increased (Figure 1H).
According to the IC50 data shown in Figure 1, both Maoto andSenkyuchachosan had strong suppressive effects on SARS-CoV-2
infection. On the other hand, no suppressive effects of Bakumondo
and Hochuekkito on virus infection were observed.
The cytotoxicity of Saikokeishito at 20 mg/ml was 44.7%, andwe cannot deny the possibility that its suppressive effect on virus
infection is due to its cytotoxicity. Since Kakkonto and Maoto
showed even higher cytotoxicity at a concentration of 20 mg/ml,
it was difﬁcult to clearly determine their suppressive effects onvirus infection at that concentration. Still, it can be consideredthat they have clear suppressive effects on virus infection atconcentrations below 10 mg/ml.
When the highest concentration (20 mg/ml) of each Kampo
formula was mixed with the virus and the infectious titer wasmeasured, none of the Kampo formulas decreased the infectious
titer. This suggests that there was no direct inactivating effect ofeach Kampo formula on the virus particles (Figure 2).
In this experiment, the virus was inoculated into cells at an
m.o.i. of 0.05. If the virus is inhibited by the Kampo formula, thenFrontiers in Pharmacology | www.frontiersin.org

Contribution of Epherae Herba in Kampo
FormulasIn this study, we found that Ephedrae herba has a strong inhibitory
effect on SARS-CoV-2 infection. Ephedrae herba is found in several

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FIGURE 3 | Inhibition of SARS-CoV-2 replication by crude drugs. (A-F) The crude drug used is noted at the top of each panel. VeroE6/TMPRSS2 cells were
infected with SARS-CoV-2 at an m.o.i. of 0.05 or 10. After 2-h adsorption, the inoculum was removed and the cells were cultured in DMEM containing different
concentrations of crude drugs for 24 h. The viral infectivity in the medium was assayed by the TCID50 method. The left y-axis of the graph is the infectivity for eachconcentration. An exponential or linear approximation was made by Excel to calculate the drug concentration that reduces the infectivity in the absence of a drug to50% (IC50). The IC50 values and approximation equations are shown in the graph. If there is little or no decrease, the IC50 is not shown (E,F). VeroE6/TMPRSS2 cells wereincubated in DMEM supplemented with the designated concentrations of a Kampo fomula for 24 h, and LDH values in the media were then measured to evaluate
cytotoxicity. The LDH value from detergent-treated cells was set at 100%, and the right y-axis is the percent inhibition of cytotoxicity for each concentration.

The IC50 value of Ephedrae herba alone was 61.1 μg/ml(Figures 3A, 5A). Maoto contains 32.3% of Ephedrae herba(Table 1), and the IC50 value was calculated by plotting theamount of Ephedrae herba in Maoto on the horizontal axis of thegraph (Figure 5B) to be 163.6 μg/ml (Figure 5B). Thus, although
it should be the same amount of Ephedrae herba, Ephedrae herba
in the form of Maoto was weakened, suggesting that othercomponents of Maoto may be inhibiting the effect Ephedrae
herba. The IC50 value of Maoto itself was calculated to be 0.5 mg/
ml (Figure 1A), being consistent with this hypothesis.
Similarly, in the case of Kakkonto containing 16.7% of Ephedrae
herba (Table 1), the IC50 value was calculated to be as high as
517.3 μg/ml based on the amount of Ephedrae herba (Figure 5C).
The IC50 value of Kakkonto itself was 2.7 mg/ml (Figure 1C).
Shoseiryuto contained 11.1% of Ephedrae herba (Table 1), andthe IC50 value of Ephedrae herba was as high as 450.4 μg/ml
based on Ephedrae herba (Figure 5D). The IC50 value of
Shoseiryuto itself was 4.9 mg/ml (Figure 1E). These results suggestthat the inhibitory effect of Ephedrae herba in the Kampo formulas
on SARS-CoV-2 infection is weakened by other components.

FIGURE 4 | Effects of crude drugs on virus particle inactivation. A
solution of a crude drug (90 µl) and 10 µl of the virus solution at 2.0 × 109
TCID50/ml was incubated for 3 min at room temperature. Reagents at
1.25–10 mg/ml concentrations were used considering their cytotoxicity.
The mixture was then serially diluted 10-fold in DMEM, and the infectivity wasdetermined by the TCID50 method. Phosphate-buffered saline (PBS) wasused as an untreated control, and ethanol [70% (w/w)] was used as aninactivation control. The dotted line indicates the detection limit (DL) of theinfectivity assay.

DISCUSSION
Kampo formulas consist of various combinations of crude drugs,
traditionally regarded as units, and are often used on the base ofancient experience (Tanaka et al., 1995; Odaguchi et al., 2019). In
Japan, Kampo formulas are covered by the national healthKampo formulas, and to verify the role of Ephedrae herba ininactivation of SARS-CoV-2 by the herbal medicines, werecalculated the effect of Ephedrae herba on SARS-CoV-2 basedon the amount of Ephedrae herba in each Kampo formula.

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FIGURE 5 | Inhibition of SARS-CoV-2 replication based on the amount of Ephedrae herba contained. (A-D) The origin of Ephedra herba is noted at the top of eachpanel. The results for growth inhibition of SARS-CoV-2 by Ephedrae herba and Kampo formulas containing Ephedrae herba (Figure 1) are re-plotted in a graph based on
the amount of Ephedrae herba contained. An exponential approximation was made by Excel to calculate the drug concentration that reduces the infectivity in theabsence of a drug to 50% (IC50). The IC50 values and approximation equations are shown in the graph.

insurance system and are prescribed by doctors under thecondition of government subsidies. Some Kampo formulas arealso available as over-the-counter drugs that can be purchased at
pharmacies without a doctor’s prescription. The extensive
experience of using Kampo formulas in Japan has led to theirsafe and easy use in medical practice. In recent years, scientiﬁcanalysis of the effects of Kampo formulas has provided evidenceof their clinical beneﬁts, and their clinical usefulness for thetreatment of COVID-19 has also been suggested (Motoo et al.,2014; Takayama et al., 2021).
Some advantages of Kampo formulas are that they can betaken orally, are relatively inexpensive, and can be taken at an
early stage. For example, Maoto, one of the Kampo formulas
examined in this study, can be used to treat common colds ininfants even before the onset of cold symptoms such as feverand nasal discharge (Kubo and Nishimura, 2007; Nagai et al.,2014). On the other hand, antivirals, as shown in clinical trials
for inﬂuenza (Muthuri et al., 2014; Dobson et al., 2015), need
to be taken after the onset of illness and before the peak of viral
replication. The clinical course of COVID-19 is longer thanthat of inﬂuenza, and viral replication in patients with
COVID-19 likely to continue for a long time. Although
existing antivirals such as remdesivir may be effective if

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treatment with the antivirals started after the diagnosis of
COVID-19, consideration should be given to the use of Kampo
formulas, which can be administered earlier.
Kampo formulas that are used for the treatment of inﬂuenza
have been reported to inhibit the growth of inﬂuenza viruses incultured cells (Mantani et al., 1999; Nomura et al., 2019; Houet al., 2020). Although the relationship between inhibition of
inﬂuenza virus replication in cultured cells and clinical efﬁcacy
is not entirely clear, the therapeutic effect of Kampo formulas
may be elicited by suppression of the ability of the virus of
replicate in the human respiratory tract. In addition, Lian hua
qing wen, which contains Ephedrae herba and is one of thetherapeutic agents used for inﬂuenza viruses in traditional
Chinese medicine, has been shown in vitro experiments to havean inhibitory effect on SARS-CoV-2 infection and has also
been sown to be effective for COVID-19 in a clinical setting,(Su et al., 2020; Hu et al., 2021). Therefore, we investigated theeffects of Kampo formulas on SARS-CoV-2 infection in vitro
experiments.
Since commercial Kampo formulas contain additives andthe types and amounts of additives are not uniform, we used
the same concentrations of Kampo formulas without additives
in this study. We found that virus infection was inhibited by

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the
Kampo formulas Maoto,
Saikokeishito,
andSenkyuchachosan but not by the Kampo formulas
Bakumondoto and Hochuekkito. We also examined theviral inhibitory effects of crude drugs as constituents of the
Kampo formulas that had strong inhibitory effects on SARSCoV-2 infection. Ephedrae herba had the strongest inhibitory
effect on SARS-CoV-2 infection. Among the six crude drugs
examined, Ephedrae herba, Paeoniae radix, Scutellariae radix,and Glycyrrhizae radix had the strongest inhibitory effects inthat order.
Ephedrae herba showed direct inactivating effects on virus
particles as well as on infected cells. Previous studies haveshown that tannins, which are components of Ephedrae herba,
inhibit inﬂuenza virus infection (Mantani et al., 1999) andthat tannins extracted from plants such as persimmon haveinactivating effects on various viruses (Ueda et al., 2013). In
the present study, it was also thought that tannins in Ephedrae
herba may have direct inhibitory effects on virus infection.
However, in the infection experiments to investigate thesuppressive effects of crude drugs, on virus infection, it was
found that Ephedra herba had a suppressive effect evenunder the condition of an m.o.i. of 10, in which all cells
were infected at once, and no virus particle formation process
was involved. Therefore, Ephedrae herba may have both adirect inactivation effect on virus particles and a suppressive
effect on infection of cells, suggesting a combined virus
suppression mechanism.
However, it was found that Kampo formulas containing
Ephedrae herba had little direct inactivation effect on virus
particles. The contents of Ephedrae herba in the Kampo
formulas used in the virus direct inactivation test (20 mg/ml)
were 6.45 mg/ml in Maoto, 3.33 mg/ml in Kakkonto, and2.22 mg/ml in Shoseiryuto. These concentrations were higher
than the concentration of Ephedrae herba tested as a crude drug
(1.25 mg/ml), which was found to be effective in the virus particle
direct inactivation test. Furthermore, when the IC50 values ofKampo formulas containing Ephedrae herba and Ephedrae herba
alone were compared on the basis of the content of Ephedrae
herba, the effects of all Kampo formulas were weaker than theeffect of Ephedrae herba alone. These results suggest that the
combination of Ephedrae herba with other crude drugs in Kampo
formulas has a weaker antiviral effect than that of Ephedrae
herba. Ephedra herba has various side effects, and Kampo
formulas are shown to contain crude drugs that alleviate theside effects of the strong Ephedra herba (Odaguchi et al., 2019).
The reduced suppressive effect of Ephedra herba on virus
infection may mean that its side effects on the human bodyare reduced.
Among the Kampo formulas that do not contain Ephedrae
herba, Saikokeishito and Senkyuchachosan showed efﬁcacy.
These Kampo formulas have been reported to inhibit
inﬂuenza virus in vitro (Shirayama et al., 2016; Nomura
et al., 2019), and the Ministry of Health, Labour, andWelfare has approved Saikokeishito for treatment of
inﬂuenza. Therefore, it would not be surprising if these
Kampo formulas also have inhibitory effects on SARSCoV-2 infection. Furthermore, viral inhibitory effects of

Frontiers in Pharmacology | www.frontiersin.org

Glycyrrhizae radix, Paeoniae radix, and Scutellariae radix
as crude drugs contained in these Ephedrae herba-free
Kampo formulas were conﬁrmed. It was shown in an
in vitro study that rhizoma had an inhibitory effect oninﬂuenza virus infecion (Nomura et al., 2019), but it had
no inhibitory effect on SARS-CoV-2 infection.
Glycyrrhizae radix is a crude drug in many Kampo
formulas. All of the Kampo formulas used in this study
contained Glycyrrhizae radix (Maoto: 9.7%, Saikokeishito:
9.1%, Kakkonto: 11%, Shomakakkonto: 12.5%, Shoseiryuto:
11%, Senkyuchachosan: 7.5%, Bakumondoto: 7.4%,
Hochuekkito: 6.3%; Table 1). Although a suppressive effectof Glycyrrhizae radix alone on SARS-CoV-2 infection wasshown in a previous study (van de Sand et al., 2021), not all of
the Kampo formulas containing Glycyrrhizae radix showed
suppressive effects on virus infection, and some Kampo
formulas showed no effect at all. Therefore, other crude
drug components in the Kampo formulas may weaken theeffect of Glycyrrhizae radix. Alternatively, the amount of
Glycyrrhizae radix in the Kampo formulas may have been
too small to reach the threshold for suppression of virus
infection.
Paeoniae radix was suggested to be effective against SARSCoV-2 infection by molecular modeling predictions (Ma et al.,2020). It was also shown to be effective against inﬂuenza virus
infection in cell infection experiments (Ho et al., 2014).
Scutellariae radix is one of the crude drugs contained in
Saikokeishito, which was effective against SARS-CoV-2
infection. In addition, Baicalin, a component of Scutellariae
radix, has been shown to have an inhibitory effect on SARSCoV-2 infection in cell and animal experiments (Song et al.,2021). In the present study, Glycyrrhizae radix, Paeoniae radix,
and Scutellariae radix showed inhibitory effects on SARS-CoV-2
infection, but their effects were inferior to the effect ofEphedrae herba.

CONCLUSION
We performed in vitro experiments to determine the inhibitory
effects on SARS-CoV-2 infection of Kampo formulas and theirconstituent crude drugs that are used to treat respiratory
symptoms including inﬂuenza and common cold symptoms.
We found that Maoto among the Kampo formulas andEphedrae herba among the crude drugs had the strongest
inhibitory effects on SARS-CoV-2 infection. Some other
Kampo formulas and crude drugs also showed suppressive
effects on virus infection. Although further analysis andevidence are needed, Kampo formulas might contribute to thetreatment of COVID-19.

DATA AVAILABILITY STATEMENTThe original contributions presented in the study are included in
the article/Supplementary Material, further inquiries can bedirected to the corresponding author.

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AUTHOR CONTRIBUTIONS(to HO and TS). This study also received funding from theSumitomo Mitsui Trust Bank COVID-19 Research Fund (toTS) and the Otsuka Toshimi Scholarship Foundation (toTNa.). The funders were not involved in the study design,collection, analysis, and interpretation of data, writing of thisarticle, or the decision to submit it for publication. All authors
declare no other competing interests.

MK, TNo and TNa. developed the study design, performed theexperiments, and analyzed the data. HK, KO-O, HO and TS
provided assistance in developing the study design andexperiments. MK, TNo, TNa and TS wrote the manuscript.
KK, KO-O, HO and MI provided advice on interpretation ofdata and revised the manuscript. All authors read and approved
the manuscript.

ACKNOWLEDGMENTS
FUNDINGWe thank Rie Hirakawa and Chika Masaki for manuscript
preparation. We also thank the staff of the Analysis Center ofLife Science, Hiroshima University for the use of theirfacilities.

This study was supported by the Research Grants for COVID-19
from AMED, Japan (to HO and TS) and a grant from theGovernment-Academia Collaboration of Hiroshima Prefecture

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Copyright © 2022 Kakimoto, Nomura, Nazmul, Kitagawa, Kanno, Ogawa-Ochiai,
Ohge, Ito and Sakaguchi. This is an open-access article distributed under theterms of the Creative Commons Attribution License (CC BY). The use,
distribution or reproduction in other forums is permitted, provided theoriginal author(s) and the copyright owner(s) are credited and that theoriginal publication in this journal is cited, in accordance with accepted
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does not comply with these terms.

Conﬂict of Interest: The authors declare that the research was conducted in theabsence of any commercial or ﬁnancial relationships that could be construed as apotential conﬂict of interest.

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