J Ethn Foods 2 (2015) 47e51

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Journal of Ethnic Foods

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Original article

Antiviral activity of Chongkukjang extracts against influenza A virus

in vitro and in vivo
Bai Wei a, *, Se-Yeoun Cha a, *, Min Kang a, Young Jin Kim b, Chang-Won Cho b,
Young Kyoung Rhee b, Hee-Do Hong b, Hyung-Kwan Jang a, *
a
Departments of Infectious Diseases and Avian Diseases, College of Veterinary Medicine and Korea Zoonosis Research Institute, Chonbuk National
University, Iksan, Jeollabuk-do, South Korea
b
Korea Food Research Institute, Songnam, Kyongki-do, South Korea

articleinfo
abstract
Article history:
Available online 5 May 2015 Background: Chongkukjang is a traditional Korean fermented product prepared from soybeans and re-
ported to have multiple biological functions, including antidiabetic, antiinflammatory, and neuro-
Keywords:
protective effects. Influenza is a respiratory disease caused by influenza viruses and continues to be a
antiviral effect worldwide threat with a high potential to cause pandemics. Besides vaccination, only two classes of
Chongkukjang extracts drugs are available for antiviral treatment against these pathogens.
influenza virus Methods: We tested the inhibitory activity of an ethyl acetate extract from Chongkukjang toward
neuraminidase inhibitory activity influenza A virus neuraminidase.
Results: All 10 compounds extracted from Chongkukjang showed neuraminidase inhibitory activity.
Extracts A3 and A8, with high neuraminidase content, had the best inhibitory activities. The in vivo
antiinfluenza virus activities of the ethyl acetate, A3, and A8 extracts as well as commercially available
genistein were evaluated using H1N1 (A/NWS/33) to test mice survivability after virus challenge. The
Chongkukjang extracts did not reduce mortality, but the A3 and A8 extracts delayed the median time to
death after influenza A virus infection of mice.
Conclusion: Our results suggest that the Chongkukjang extracts may have potential as a therapeutic
agent to treat influenza virus infection.
Copyright © 2015, Korea Food Research Institute, Published by Elsevier. This is an open access article
under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

1. Introduction
system [5,6]. Chongkukjang has multiple functions, including
antidiabetic, antiinflammatory, and neuroprotective effects [5,7].
Chongkukjang is a fermented product manufactured by short-
Influenza viruses are respiratory pathogens that affect humans
term fermentation of soybean using Bacillus subtilis that contains
and are responsible for substantial morbidity, mortality, and
many microorganisms and bioactive compounds that are absent
decreased productivity worldwide [8]. Influenza viruses contain
from unfermented soybeans (Fig. 1) [1e3]. Flavonoid glycosides are
two glycoproteins on their surface, hemagglutinin (HA) and neur-
converted into aglycones by hydrolysis during fermentation of
aminidase (NA), which recognize sialic acid in hostecell glyco-
Chongkukjang, and many of the proteins are degraded into small
conjugates. HA binds to terminal sialic acid groups on cell surface
peptides and amino acids [4]. Koreans have been consuming
glycoconjugates, leading to attachment and subsequent penetra-
Chongkukjang for hundreds of years, and most believe that
tion of virus into cells, and NA exhibits enzymatic activity that
Chongkukjang consists of proteins and minerals that promote the
removes sialic acid from glycoconjugates, facilitating the release of
generation and growth of human cells and strengthen the immune
progeny virions from infected cells and preventing the aggregation
of progeny virions [9]. Neuraminidase inhibitors (NAIs) act by
binding to the active site of the viral NA enzyme and preventing
* Corresponding author. Departments of Infectious Diseases and Avian
release and spread of progeny virions from infected cells during
Diseases, College of Veterinary Medicine and Korea Zoonosis Research Institute, the replication cycle [10]. NAIs block the neuraminidase active site
Chonbuk National University, 79 Gobong-ro, Iksan 570e752, Jeollabuk-do, South and prevent release and spread of new virions [11]. NA was chosen
Korea. as a suitable drug target because it plays a major role in the
E-mail address: hkjang@jbnu.ac.kr (H.-K. Jang).
* propagation of influenza viruses, and the amino acid residues of
B. Wei and S.-Y. Cha contributed equally to this study.
the active site

http://dx.doi.org/10.1016/j.jef.2015.04.001
2352-6181/Copyright © 2015, Korea Food Research Institute, Published by Elsevier. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/
licenses/by-nc-nd/4.0/).
4 J Ethn Foods 2015; 2: 47e51
Fig. 1. Chongkukjang is a traditional Korean fermented product prepared from soy-
bean. Chongkukjang is manufactured via the short-term fermentation of soybeans
using Bacillus subtilis.
interacting directly with the substrate or surrounding the central
active site of the enzyme are strictly conserved [12]. Treating
influenza with NAIs has become the most popular treatment
among primary care doctors. NAIs were introduced to clinical
practice in 1999, but so far only zanamivir and oseltamivir have
been licensed for use in humans [13]. Unfortunately, viruses
resistant to zanamivir and oseltamivir have been generated in vitro
and in humans recently [14]. This highlights the urgent need for
new and readily available antiinfluenza agents. In the present
study, we investigated the antiinfluenza activities of Chongkukjang
ex- tracts and demonstrated their efficacy.
2. Materials and methods
2.1. Viruses
The influenza virus used in this study (Influenza A/NWS/33;
Influenza A/chicken/Korea/MS96/96) was propagated in the allan-
toic sac of 10-day-old specific pathogen-free embryonated eggs.
○ tracts against lethal influenza virus in vivo, the mice were divided
These virus stocks were frozen at —
80 C until use. Viral HA units
were determined in 96-well microtiter plates using 1% chicken
red
blood cells.
2.2. Fluorometric neuraminidase activity assay
A fluorescence-based NA inhibition assay was used to
determine the sensitivity of the viruses to NAIs. The assay is
based on the release of a 4-methylumbelliferone fluorescent
product from the 2- (4-methylumbelliferyl)-a-d-N-
acetylneuraminic acid (MUNANA) substrate (Sigma, St. Louis,
MO, USA) as a measure of NA activity. The NA activity assay
was carried out to determine the dilution of each virus to be
used in the subsequent NA inhibition assay. Two- fold dilutions
of virus in assay buffer (32.5mM MES [2-(N-mor-
pholino)ethanesulfonic acid], pH 6.5, 4mM CaCl2 with 0.1% NP-
40, and 0.3 mg/mL bovine serum albumin) were prepared,
mixed with an equal volume (50 mL) of MUNANA substrate
(0.3mM), and
○
incubated at 37 C for 60 minutes. The reaction was terminated
by
adding 100 mL stop solution (0.14M NaOH in 83% ethanol). The
fluorometric quantification of 4-methylumbelliferone was
determined using a VICTOR X4 Multilabel Plate Reader (Perki-
nElmer Inc., Waltham, MA, USA) at an excitation wavelength of
360 nm and an emission wavelength of 448 nm. The appropriate
concentration of virus for use in the NA inhibition assay was
determined by selecting a dilution of virus in the linear portion
of the enzyme activity curve. Zanamivir was obtained from
Sigma and used as a positive control.
This assay was performed in duplicate for each isolate, and the
half-maximal inhibitory concentration (IC50) value for each virus
and for each antiviral agent was determined. The IC 50 is the anti-
viral agent concentration that inhibits 50% of neuraminidase
activity.
2.3. Isolation of an active component from Chongkukjang
Commercial Chongkukjang samples were obtained from Sun-
chang MoonOkrae Food (Sunchang, Korea), and the extracts were
prepared by the Korea Food Research Institute. Briefly, Chongkuk-
jang was extracted with ethanol, and the liquid was removed by
evaporation. The residue was suspended and successively parti-
tioned in fractions of ethyl acetate, butanol, and water. The total
crude extract was dissolved in CH 2Cl2/H2O (95:5), and the ethyl
acetate fraction was subjected to silica open-column chromatog-
raphy. The mobile phase solvent was dichloromethane/ethanol
diluted as 95:5, 90:10, 80:20, and 50:50. All 10 subfractions were
combined (A1eA10) based on thin-layer chromatography analysis.
2.4. Animal model and experimental design
Female (weight, 17e19 g) 6-week-old specific pathogen-free
BALB/c mice (Daejeon, Chungnam, Korea) were used. The mice
were quarantined 24 hours prior to use. The AIN-93M diet was
provided as feed throughout the experiment, as recommended by
the American Institute of Nutrition [15]. All animals received hu-
mane care in compliance with the National Association of Labora-
tory Animal Care and were fed in isolation (Three-Shine, Daejeon,
Korea).
To investigate the safety of the Chongkukjang extract in mice,
the mice were administered the ethyl acetate, A3, and A8 fractions,
as well as genistein (0.4 g/kg/d of body weight). Phosphate-
buffered saline was given to the normal control group. The mice
were divided into groups of 10 each, and the treatments were
carried out for 14 days. Clinical signs were recorded daily, and the
body weight was measured on Days 0, 7, 10, and 14.
To investigate the protective activity of the Chongkukjang ex-
into groups of 10 each. The inoculated mice received the ethyl ac-
etate, A3, and A8 extracts as well as genistein (0.2 and 0.4 g/kg/d),
respectively. The treatments were administered daily via gavages
for 6 days before challenge and 14 days after influenza A virus
challenge. Studies with mice were performed as described previ-
ously, and phosphate-buffered saline was used as the negative
control. The mice were anesthetized with diethyl ether and
exposed to 0.1 mL virus by intranasal instillation. A viral challenge
dose of 107.0 50% embryo infectious dose (EID 50)/0.1 mL (A/NWS/
33), which was approximately 80e90% of the lethal dose, was used.
Mice were observed daily for 14 days after infection. The protective
effect was estimated by preventing death and prolonging the me-
dian time to death (MTD).
2.5. Data analysis
Data are expressed as the mean ± standard deviation of at least
three independent experiments. Body weight was analyzed with
one-way analysis of variance [16]. Statistical analyses were
performed using the SPSS software (SPSS Inc., Chicago, IL, USA).
A p value <0.05 was considered statistically significant.
3. Results
3.1. Inhibitory effect of the Chongkukjang extracts on influenza
virus in vitro
The standard soybean isoflavones, such as daidzin, daidzein,
genistin, genistein, glycitin, and glycitein (Sigma), were applied to
the fluorometric NA activity assay, and genistein showed the best
NA inhibitory effect compared with that of the five other standard
soybean isoflavones (data not shown). Thus, genistein was used to
control the extract composition of the Chongkukjang eluent at each
step.
NA is one of the most important targets to screen antiinfluenza
virus A drugs. We first investigated whether the Chongkukjang
fractions exerted antiviral action. The ethyl acetate, butanol, and
water fractions were measured using the fluorometric NA activity
assay. The ethyl acetate fraction showed the best NA inhibitory
effect compared with that of butanol and water (data not shown).
Thus, the ethyl acetate fraction was used for further purification.
The Chongkukjang extracts were dose-dependently effective
against influenza A virus, and the calculated IC 50 values of the ex-
tracts against the influenza virus are shown in Table 1. The A3
extract showed the best NA inhibitory effect against influenza A
virus. The A8 extract also showed a high inhibitory effect. The
Chongkukjang extract seemed to be more effective against
A/NWS/ 33 (H1N1) than against A/chicken/Korea/MS96/9
(H9N2). Genistein showed similar effects against A/NWS/33
(H1N1) and A/chicken/ Korea/MS96/96 (H9N2).
3.2. Inhibitory effect of the Chongkukjang extracts on the
influenza virus in vivo
We next tested whether the Chongkukjang extract, which
inhibited NA activity, could relieve a viral infection in vivo. First,
we assessed the safety of the Chongkukjang extract in mice.
Animals treated with the Chongkukjang extract maintained a
relatively steady weight, and mean body weights of the treated
mice, except the A8 group, were higher than those of the control
group, but no significant differences were observed throughout
the study (Table 2). No clinical symptoms were observed
throughout the study.
To investigate the protective effect of the Chongkukjang extract
in vivo, groups of mice were inoculated with 107.0 EID50/0.1 mL A/
NWS/33 (H1N1). All of the mice that received the ethyl acetate, A3,
and A8 extracts as well as genistein (0.4 g/kg/d) died within the
experimental period (data not shown). The results for mice that
received a dose of 0.2 g/kg/d are shown in Fig. 2. About 10% of the
Table 1
Inhibitory effect of the Chongkukjang extracts against influenza A virus.*
No. Compound NA inhibitory effect, IC50
(mg/mL)
A/NWS/33 (H1N1) A/chicken/Korea/MS96/
96 (H9N2)
1 Ethyl 8,482.5 ± 124.3 14,264.2.5 ± 1,183.1
acetate
extract
2 A3 17,611.1 ± 3,052.7 28,242.4 ± 2,571.5
3 A8 4,565.9 ± 66.9 8,949.3 ± 974.3
4 Genistein 30,58.8 ± 218.9 2,729.6 ± 275.1
IC50, drug concentration required to inhibit half of neuraminidase activity.
*
Fluorescence-based neuraminidase (NA) inhibition assay was used to
determine the sensitivity of influenza A viruses to Chongkukjang extracts.
Table 2
Body weights (mean ± standard deviation) of the mice in each group.*
Group Body weight (g)
0 DPI 7 DPI 10 DPI 14 DPI
Negative 18.6 ± 0.5 18.6 ± 0.8 19.2 ± 0.8 19.7 ± 0.5
Ethyl acetate extract (0.4 g/kg) 17.9 ± 0.5 19.4 ± 0.6 20.3 ± 1.1 20.3 ± 1.2
A3 (0.4 g/kg) 18.0 ± 0.3 19.5 ± 1.0 20.4 ± 1.6 20.7 ± 1.9
A8 (0.4 g/kg) 18.1 ± 0.7 18.7 ± 0.5 19.2 ± 0.8 19.7 ± 0.4
Genistein (0.4 g/kg) 17.9 ± 0.4 18.7 ± 0.8 19.4 ± 1.4 20.5 ± 1.1
DPI, days post inoculation.
*
Safety of the Chongkukjang extract in mice administered with ethyl acetate, A3,
and A8 fractions, as well as genistein (0.4 g/kg/d of body weight) for consecutive 14
days.
mice from the positive control group survived until the end of
the experiment. All challenge groups, except the genistein group
(0.2 g/ kg) and the A3 group (0.2 g/kg), died by 8 days after
infection. The MTD of the mice treated with the A3 and A8 (0.2
g/kg) extracts was 5 and 6 days, which was longer than that of
the viral control group (3 days). The survival rate in the A3
group (0.2 g/kg) at 14 days post infection was 20%, which was
higher than that of the viral control group (10%).
4. Discussion
There is an urgent medical need to develop new strategies
against influenza virus infection. At the moment antiviral drugs are
the only known defense against the disease, as no vaccine is
available, which is similar to the early phase of a pandemic or
when the prediction for the seasonal influenza vaccine composition
fails. In 2010, the World Health Organization suggested the
development of novel and effective treatment strategies for
influenza virus including natural products [17]. This prompted us
to investigate the antiviral activity of traditional food extracts
against influenza virus infection. The goal of this study was to
determine whether Chongkukjang extracts could function as an
antiviral agent against influenza A virus, because Chongkukjang
has a strong record in the literature for its beneficial effects on
human health [12,18]. Our results suggest for the first time that the
Chongkukjang extracts were active against the influenza A virus.
Influenza virus NA is thought to be required to secrete newly
synthesized virus from infected cells and to aid in the movement of
the virus. NA can be administered relatively late in the viral infec-
tion and still render a significant antiviral effect [19]. Genistein
showed a better effect than the A3 and A8 extracts against
A/NWS/ 33 (H1N1) and A/chicken/Korea/MS96/96 (H9N2),
suggesting that other active compounds may be present within this
extract. This result agreed with those for the ethyl acetate fraction,
which showed less efficiency than the further purified A3 and A8
extracts. Although the Chongkukjang extracts showed less activity
than zanamivir and oseltamivir, Chongkukjang extracts showed
possible research directions as a potential candidate for zanamivir-
and oseltamivir-resistant cases in clinical practice. This provides a
good backdrop considering that after 3 years of NAI use, it has
been noted that 0.35% of isolates were found to have become
resistantdin particular, higher percentages of such resistance have
been observed in children [20,21].
Wide variations in the IC50 for oseltamivir are observed in sea-
sonal influenza A with or without the virus subtype [22]. Genistein
has a weak NA inhibitory effect of about 20 mg/mL IC50 against the
H1N1 influenza A virus [23]. Our result showed about six times
higher efficiency of the IC50 against the same NA type of influenza
A virus. An NA gene mutation (H274Y, H275Y in N1 numbering)
has been associated with NA inhibitor susceptibility [24]. The
Fig. 2. The in vivo protective activities of the Chongkukjang extracts on lethal influenza A virus-infected mice. The treatments were administered daily via gavages for 6 days before
challenge and 14 days after influenza A virus challenge. Cumulative survival curve for the indicated groups (n ¼ 10/group).
Chongkukjang extracts showed a better inhibitory effect against A/
NWS/33 (H1N1) than that against A/chicken/Korea/MS96/96
(H9N2). This result agrees with the finding that avian isolates have
significantly higher resistance to NAIs than that of human isolates
[25]. It is not apparent how accurately the IC 50 values determined
from the enzyme inhibition assay results reflect actual differences
in inhibitor potency. Once efficacy is observed against an influenza
virus, it is important to use a variety of other virus strains in
follow- up evaluations to ascertain if the antiviral effect applies to
all viral strains encountered in the clinic.
As the Chongkukjang extracts showed antiviral effects in vitro,a
mouse study was performed to determine the in vivo effects of the
Chongkukjang extracts against influenza A virus. Treatment with
the A3 and A8 (0.2 g/kg) extracts produced a modest inhibitory
effect on influenza A virus replication in mice. Influenza virus-
infected mice receiving the Chongkukjang extract treatment did
not show reduced mortality, but the A3 and A8 extracts delayed
the mice MTD after influenza A virus infection. This result shows
that the Chongkukjang extract is a potential candidate to fight
influenza A virus infection. The Chongkukjang-treated groups
(except the A8 group) maintained a higher body weight compared
with the control group, which agrees with previous reports that
soybean-related products increase body weight and lower the risk
of toxic side effects at a low concentration [14,26]. This extract may
be a potent antiviral agent against influenza virus with no host
toxicity.
We found that the A3 and A8 Chongkukjang extracts had a
potential inhibitory activity against influenza viruses. This study is
another example showing how plant extracts are capable of
inhibiting influenza A virus infection. This study addresses the
latest developments in theoretical and experimental research on
the properties of NA that are and will be driving antiinfluenza
drug development today and in the near future. Two further
research directions can be undertaken. One would be to further
characterize and identify the antiviral compounds in the extracts.
Nevertheless, it is also important to determine whether only one
compound, several closely related compounds, or the composite
activity of several different molecules is/are responsible for the
observed antiviral activity and whether the activity is selective for
influenza viruses. We clearly need to focus more attention on ethnic
foods, not only for nutrition but also for the functionality they
provide in disease control.
Conflicts of interest
The authors have no conflicts of interest to declare.
Acknowledgments
This study was supported by research grants from the Korea
Food Research Institute (E0143003528).
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