Antiviral activity of Sambucus Formosana Nakai ethanol extract and related phenolic acid constituents against human coronavirus NL63

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DOI: 10.1016/j.virusres.2019.197767
Cite this publication
Abstract
Human coronavirus NL63 (HCoV-NL63), one of the main circulating HCoVs worldwide, causes respiratory tract illnesses like runny nose, cough, bronchiolitis and pneumonia. Recently, a severe respiratory illness outbreak of HCoV-NL63 has been reported in a long-term care facility. Sambucus Formosana Nakai, a species of elderberry, is a traditional medicinal herb with anti-inflammatory and antiviral potential. The study investigated the antiviral activity of Sambucus Formosana Nakai stem ethanol extract and some phenolic acid constituents against HCoV-NL63. The extract was less cytotoxic and concentration-dependently increased anti-HCoV-NL63 activities, including cytopathicity, sub-G1 fraction, virus yield (IC50 = 1.17 μg/ml), plaque formation (IC50 = 4.67 μg/ml) and virus attachment (IC50 = 15.75 μg/ml). Among the phenolic acid constituents in Sambucus Formosana Nakai extract, caffeic acid, chlorogenic acid and gallic acid sustained the anti-HCoV-NL63 activity that was ranked in the following order of virus yield reduction: caffeic acid (IC50 = 3.54 μM) > chlorogenic acid (IC50 = 43.45 μM) > coumaric acid (IC50 = 71.48 μM). Caffeic acid significantly inhibited the replication of HCoV-NL63 in a cell- type independent manner, and specifically blocked virus attachment (IC50 = 8.1 μM). Therefore, the results revealed that Sambucus Formosana Nakai stem ethanol extract displayed the strong anti-HCoV-NL63 potential; caffeic acid could be the vital component with anti-HCoV-NL63 activity. The finding could be helpful for developing antivirals against HCoV-NL63.
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Virus Research
journal homepage: www.elsevier.com/locate/virusres
Antiviral activity of Sambucus FormosanaNakai ethanol extract and related
phenolic acid constituents against human coronavirus NL63
Jing-Ru Weng
a
, Chen-Sheng Lin
b,1
, Hsueh-Chou Lai
c,d,1
, Yu-Ping Lin
e
, Ching-Ying Wang
e
,
Yu-Chi Tsai
e
, Kun-Chang Wu
f
, Su-Hua Huang
g
, Cheng-Wen Lin
e,g,h,
a
Department of Marine Biotechnology and Resources, National Sun Yat-sen University, Kaohsiung, Taiwan
b
Division of Gastroenterology, Kuang Tien General Hospital, Taichung, Taiwan
c
School of Chinese Medicine, China Medical University, Taichung, Taiwan
d
Division of Hepato-gastroenterology, department of internal medicine, China Medical University Hospital, Taichung, Taiwan
e
Department of Medical Laboratory Science and Biotechnology, China Medical University, Taichung, Taiwan
f
School of Pharmacy, College of Pharmacy, China Medical University, Taichung, Taiwan
g
Department of Biotechnology, Asia University, Wufeng, Taichung, Taiwan
h
Chinese Medicine Research center, China Medical University, Taichung, Taiwan
ARTICLE INFO
Keywords:
Human coronavirus NL63
Sambucus FormosanaNakai
Caeic acid
Antiviral
Virus yield
Attachment inhibition
ABSTRACT
Human coronavirus NL63 (HCoV-NL63), one of the main circulating HCoVs worldwide, causes respiratory tract
illnesses like runny nose, cough, bronchiolitis and pneumonia. Recently, a severe respiratory illness outbreak of
HCoV-NL63 has been reported in a long-term care facility. Sambucus FormosanaNakai, a species of elderberry, is a
traditional medicinal herb with anti-inammatory and antiviral potential. The study investigated the antiviral
activity of Sambucus FormosanaNakai stem ethanol extract and some phenolic acid constituents against HCoV-
NL63. The extract was less cytotoxic and concentration-dependently increased anti-HCoV-NL63 activities, in-
cluding cytopathicity, sub-G1 fraction, virus yield (IC50 = 1.17 μg/ml), plaque formation (IC50 = 4.67 μg/ml)
and virus attachment (IC50 = 15.75 μg/ml). Among the phenolic acid constituents in Sambucus FormosanaNakai
extract, caeic acid, chlorogenic acid and gallic acid sustained the anti-HCoV-NL63 activity that was ranked in
the following order of virus yield reduction: caeic acid (IC
50
= 3.54 μM) > chlorogenic acid
(IC
50
= 43.45 μM) > coumaric acid (IC
50
= 71.48 μM). Caeic acid signicantly inhibited the replication of
HCoV-NL63 in a cell-type independent manner, and specically blocked virus attachment (IC
50
= 8.1 μM).
Therefore, the results revealed that Sambucus Formosana Nakai stem ethanol extract displayed the strong anti-
HCoV-NL63 potential; caeic acid could be the vital component with anti-HCoV-NL63 activity. The nding
could be helpful for developing antivirals against HCoV-NL63.
1. Introduction
Human coronavirus (HCoV) NL63, the member of the genus alpha-
coronavirus in the Coronaviridae family, is one of common human cor-
onaviruses (Li and Lin, 2013;Huang et al., 2017). HCoV-NL63 genome
is a positive-strand RNA with near 27.5 kb nucleotides containing 5
untranslated regions (UTR), ORF1a/b, spike(S), ORF3, envelope(E),
membrane(M), nucleoprotein (N), and 3UTR (Geng et al., 2012).
ORF1a/b encodes overlapping replicase 1a and 1ab via the 1ribo-
somal frameshift at the nucleotide 12,424. The papain-like (PLpro) and
3C-like (3CLpro) proteases embedded within replicase 1a and 1ab
process cis- and trans-cleavage activity to divide replicase 1a and 1ab
into nonstructural proteins (nsps) that modulate in viral RNA replica-
tion. Among common human coronaviruses like HCoV-229E, HCoV-
HKU1, and HCoV-OC43, HCoV-NL63 is one of main circulating HCoVs
in the fall and winter worldwide, causing mild upper respiratory tract
illnesses like runny nose, cough and sore throat in young children,
young adults and elderly (Cui et al., 2011;Dijkman et al., 2012). In-
terestingly, HCoV-NL63 has been frequently detected than other
HCoVs, inuenza viruses, and rhinovirus in the specimens from the
young adults with acute respiratory infection (cough and body aches or
chills or fever/feverishness) (Davis et al., 2018). Importantly, HCoV-
NL63 is also associated with lower respiratory tract illnesses, such as
pneumonia and bronchiolitis in young children and elderly (Huang
https://doi.org/10.1016/j.virusres.2019.197767
Received 20 June 2019; Received in revised form 20 September 2019; Accepted 23 September 2019
Corresponding author at: Department of Medical Laboratory Science and Biotechnology, China Medical University, 91 Hsueh-Shih Road, Taichung, 404, Taiwan.
E-mail address: cwlin@mail.cmu.edu.tw (C.-W. Lin).
1
Co-rst author.
Virus Research 273 (2019) 197767
Available online 24 September 2019
0168-1702/ © 2019 Elsevier B.V. All rights reserved.
T
et al., 2017). HCoV-NL63 infection is the high prevalence (8.4%) in
hospitalized patients with pneumonia in winter. Recently, a severe re-
spiratory illness outbreak in a long-term care facility in Louisiana has
been reported to be associated with the HCoV-NL63 infection in winter
2017 (Hand et al., 2018). Among 20 cases aged from 66 to 96, 6 pa-
tients with pneumonia have to be hospitalized and 3 patients are dead.
Moreover, screening children with acute undierentiated febrile illness
in rural Haiti indicates that HCoV-NL63 is identied in the blood
samples from four patients aged from 3 to 10 years who have no re-
spiratory symptom, but two cases have headache and the others exhibit
inuenza virus causing abdominal symptoms (Beau De Rochars et al.,
2017). Therefore, HCoV-NL63 is the notable pathogen as the etiology of
mild and severe respiratory diseases, even acute undierentiated febrile
illness.
Sambucus FormosanaNakai, also known by Sambucus chinensis Lindl
and Sambucus javanica, is a species of elderberry, belongs to family
Adoxaceae, and grows in subtropical and tropical Asian areas, including
Taiwan, China, Japan, Cambodia, India etc. (Lin and Tome, 1988;Yang
and Chiu, 1998;Hong et al., 2013). Sambucus FormosanaNakai is a
traditional medicinal herb in Taiwan for reducing inammation, en-
hancing circulation, and treating rheumatoid and low back pain,
neuritis, dermatitis, and infection diseases (Yang and Chiu, 1998). The
chemical components of S.ambucus FormosanaNakai include sambuculin
A, oleanolic acid, α-amyrin and β-amyrin, β-sitosterol, ursolic acid,
pomolic acid, lupeol palmitate, glycyrrhetic acid, phenolic acid con-
stituents, and avonoids (Chen et al., 2001,2019;Liao et al., 2006;Lin
and Tome, 1988;Yang and Lin, 2004;Zhang et al., 2010). In addition,
phenolic acid constituents of S.ambucus FormosanaNakai, including
caeic acid, caeotannic acid, chlorogenic acid, coumaric acid, ferulic
acid, and gallic acid, have been identied as the members of the most
important active components with anti-inammatory, anti-tumor and
anti-hepatotoxic activities (Chen et al., 2001;Liao et al., 2006;Yang
and Lin, 2004;Zhang et al., 2010). Active components of Sambucus
FormosanaNakai are similar to those of other Sambucus species, in-
cluding Sambucus nigra L. and Sambucus lanceolata, which process an-
tioxidant, antiradical, antiviral, antimicrobial, and anti-inammatory
activities (Barak et al., 2001;Krawitz et al., 2011;Mandrone et al.,
2014;Pinto et al., 2017;Pliszka, 2017;Porter and Bode, 2017;Turek
and Cisowski, 2007). Especially, the extract of Sambucus nigra L. exerts
the antiviral activity against inuenza A and B viruses, human im-
munodeciency virus, and the herpes simplex virus type 1 (Krawitz
et al., 2011;Serkedjieva et al., 1990;Zakay-Rones et al., 1995;Amoros
et al., 1992;Mahmood et al., 1993;Roschek et al., 2009). Sambucus
nigraphenolic acids like caeic acid show the highly inhibitory eect on
the in vitro replication of inuenza A virus (Porter and Bode, 2017;
Utsunomiya et al., 2014). Since Sambucus FormosanaNakai contains
such antiviral active components in the extract of Sambucus nigra L., the
antiviral activity of Sambucus FormosanaNakai is worthy to further in-
vestigate.
The study explored the anti-HCoV-NL63 activity of Sambucus
FormosanaNakai stem ethanol extract and some markers of its phenolic
acid constituents, like caeic acid, chlorogenic acid, coumaric acid,
ferulic acid, gallic acid. The study indicated the inhibitory activity of
Sambucus FormosanaNakai extract and its phenolic acid constituents on
HCoV-NL63 induced cytopathic eect, virus yield, and the early stage
of HCoV-NL63 replication in concentration-dependent and cell-type
independent manners.
2. Materials and methods
2.1. Viruses and cells
HCoV-NL63 provided by Dr. Lia van der Hoek (Academic Medical
Center, The Netherlands) was amplied in LLC-MK2 cells, as described
in our prior study (Huang et al., 2017). LLC-MK2 cells were cultured in
the Minimum Essential Medium (MEM) containing 2 mM L-Glutamine,
50 μg/ml penicillin, 50 μg/ml streptomycin, and 5% fetal bovine serum
(FBS) at 37 in a 5CO
2
incubator. LLC-MK2 cells were further used
to perform antiviral assays and examine antiviral mechanism. Human
airway Calu-3 cells were cultured in MEM supplemented 10% FBS and
antibiotics mentioned above, maintained at the 8090% conuent, and
then also used to conrm the antiviral activity of indicated phenolic
acid constituents.
2.2. Preparation of Sambucusformosana Nakai stem ethanol extract
Dried stems of Sambucus Formosana Nakai (Supplemental Fig. 1A)
were purchased from the medicinal herb pharmacy in Taichung, and
identied as described in Flora of Taiwan (Yang and Chiu, 1998). Dried
stem slices (Supplemental Fig. 1B) were soaked in 95% ethanol with the
sonication for 2 h; the stem ethanol extract was ltered by Whatman
No. 1 paper, then lyophilized in an IWAKI FDR-50 P freeze dryer, as
described in our previous study (Wang et al., 2012). The powder of
Sambucus Formosana Nakai stem ethanol extract was kept in the sterile
bottle; the stock solution of 10 mg/ml in dimethyl sulfoxide was pre-
pared and stored in 20 °C freezer and the test solutions of the stem
extract were diluted by the media. The phenolic acid constituents such
caeic acid, chlorogenic acid, coumaric acid, ferulic acid, and gallic
acid, were purchased from Sigma-Aldrich company.
2.3. Cell viability assay
LLC-MK2 cells (3 × 10
4
cells/well) were cultured in the 96-well
plates overnight, quintuplicate treated with Sambucus Formosana Nakai
stem ethanol extract or its phenolic acid constituents (caeic acid,
chlorogenic acid, coumaric acid, ferulic acid, and gallic acid) for 2 days,
and then incubated with 0.5 mg/ml 3-(4,5-Dimethylthiazol-2-yl)-2,5-
diphenyltetrazolium bromide (MTT) for additional 4 h. After the cell
lysis by isopropanol, yielding absorbance (OD
570-630
) at the wavelength
of 570 nm and 630 nm was assessed using a micro-ELISA reader. Cell
viability (%) and cytotoxic concentration showing 50% toxic eect
(CC
50
) were subsequently determined, as previously described (Wang
et al., 2012).
2.4. Cytopathic eect (CPE) reduction, cell cycle, and virus yield
LLC-MK2 cells (3 × 10
5
cells/well) were cultured in the 6-well
plates overnight, and then infected by 3 × 10
4
pfu (plaque formation
unit) HCoV-NL63, representing as a MOI (multiplicity of infection) of
0.1 to signicantly induce viral cytopathic eect (CPE) in LLC-MK2.
The cells were simultaneously added with HCoV NL63, and treated with
Sambucus Formosana Nakai stem ethanol extract at the concentrations of
0, 1, 10, and 50 μg/ml or the phenolic acid constituents at the con-
centrations of 0, 10, 50, and 100 μM, respectively. After the 36-h in-
cubation at 37 and 5CO
2
, HCoV NL63-induced cytopathic eect
(CPE) with cell swelling, rounding, vacuoles, and eventual detachment
was photographed using microscope, in which vacuoles in CPE from
HCoV NL63-infected LLC-MK2 cells were more predominant at 37 °C
(Lednicky et al., 2013). In the cell cycle assay, the cells were harvested
36 h post infection, stained with propidium iodide, and then examined
using ow cytometry, as described in our prior study (Wang et al.,
2018). To determine the production of progeny virions (virus yield
assay), the cultured media were collected for determined the virus titers
using plaque assay. LLC-MK2 cell monolayer in the well of 6-well plates
was added with 100 μl of serial 10-fold dilutions of above cultured
media. After a 1-h incubation, the cell monolayer was overlaid by the
medium containing 0.75% agarose (3 ml per well) for 2-day incubation
at 37 °C in a CO
2
incubator to allow the plaque formation. Finally, the
cell monolayer was stained with the solution of naphthol blue-black dye
and plaque number calculated. Fifty percent (50%) inhibitory con-
centration (IC
50
) for virus yield reduction by the stem ethanol extract
and the phenolic acid constituents was determined.
J.-R. Weng, et al. Virus Research 273 (2019) 197767
2
2.5. Plaque formation inhibition, virucidal activity, and attachment
reduction assays
In the plaque formation inhibition assays, LLC-MK2 cell monolayer
cultured in 6-well plates were infected with 200 pfu of HCoV-NL63 that
were the maximum number of plaques counted in the well of 6-well
plates. After the addition with HCoV NL63, the cells were immediately
treated with the ethanol extract at the concentrations of 0, 1, 5, and
10 μg/ml, caeic acid and chlorogenic acid at the concentrations of 0,
10, 50, and 100 μM, respectively. After a 1-h incubation at 37 °C in a 5%
CO
2
incubator, the mixtures were removed from the wells; the cell
monolayer was cultured with the medium containing 0.75% agarose
and performed by the plaque assay mentioned above. In the virucidal
assay, HCoV-NL63 (2 × 10
6
pfu) was added into in the Eppendorf tube
and directly treated with the ethanol extract, caeic acid, or chloro-
genic acid for 1 h at 37 °C. For minimizing the antiviral eect of in-
dicated agents in the cells, 100 μl (near 200 pfu HCoV-NL63) of the
10000-fold dilution from the mixtures of virus and the extract or phe-
nolic acids was added to the MK2 cell monolayer in the 6-well plate to
determining the residual viral infectivity using the plaque assay de-
scribed above. Virucidal activity was calculated based on the compar-
ison of the residual infectivity of HCoV-NL63 in the treated groups with
the un-treated group. In the HCoV-NL63 attachment assay, LLC-MK2
cell monolayer in 6-well plate was placed at 4 °C for 1 h, infected with
HCoV-NL63 (200 pfu), and then immediately added with the ethanol
extract, caeic acid or chlorogenic acid. After an additional 1-h in-
cubation at 4 °C, the mixture of virus and the extract or phenolic acids
was removed from the well; the cell monolayer was overlaid with MEM
medium containing0.75% agarose at 37 °C in a 5% CO
2
incubator, and
then executed by plaque assay, as mentioned above. The attachment
inhibition was analyzed according to the ratio of the plaque number in
treated groups to the un-treated group.
2.6. Inhibitory eect of caeic acid on viral infectivity in human airway
epithelial cells using immunouorescent staining
To measure the inhibitory activity of caeic acid on the replication
of HCoV-NL63 in human airway epithelial cells, Calu-3 cells
(1 × 10
5
cells/well) were cultured in the 6-well plates overnight, and
then infected by 5 × 10
3
pfu HCoV-NL63 (MOI = 0.05). After the ad-
dition of HCoV NL63, the cells were simultaneously treated with caeic
acid at the concentrations of 0, 10, and 50 μM. After the 36-h incuba-
tion at 32 in a 5CO
2
incubator, swelling, rounding, and eventual
de-attachment in HCoV-NL63-induced CPE were more predominant at
32 °C (Lednicky et al., 2013), and then the images were recorded by
microscope. Subsequently, the cells were xed with 4% paraformalde-
hyde solution in PBS for 30 min, incubated with the quench buer
(50 mM NH
4
CI) for 15 min, permeabilized and blocked using the cell
perforation and blocking solution containing 1% albumin bovine (Af-
fymetrix) plus triton X-100 (ThermoFisher) for 4 h at 4 °C, and then
reacted with HCoV-NL63-immunized sera in 1% BSA (1:2000) over-
night at 4 °C and secondary antibody Alexa Fluor anti-mouse IgG in 1%
BSA (1:3000) for 1 h at 4 °C (ThermoFisher). After staining with 4,6-
diamidino-2-phenylindole (DAPI, ThermoFisher) 20 min at room tem-
perature, the images of mock, infected, and infected/treated cells were
photographed using uorescent microscopy. The infectivity was
represented as the ratio of red uorescent signals (HCoV-NL63-positive
cells) to blue uorescent signals (total nucleuses) was calculated by
Image J.
2.7. Statistical analysis
The pvalue was calculated based ANOVA using SPSS program and
Student t-test with the data from three independent experiments. When
the pvalue was less than 0.05, the result of the assay was statistically
signicant.
3. Results
3.1. Antiviral activity of Sambucusformosana Nakai stem ethanol extract
against HCoV-NL63
Sambucus Formosana Nakai stem ethanol extract had a low cyto-
toxicity with a CC
50
value of 180.62 μg/ml to LLC-MK2 cells
(Supplemental Fig. 2, Table 1). Subsequently, anti-HCoV-NL63 ability
of the stem ethanol extract was assessed with cytopathicity, cell cycle
and virus yield assays (Figs. 1 and 2). Sambucus Formosana Nakai ex-
tract concentration-dependently reduced cytopathicity in HCoV-NL63
infected cells (Fig. 1A), in which vacuolation in infected cells at 37 °C
appeared more predominantly, as described in the prior report
(Lednicky et al., 2013). The extract also signicantly decreased the sub-
G1 fraction in infected cells (Fig. 1B). In addition, Sambucus For-
mosanaNakai extract inhibited the in vitro production of progeny
HCoV-NL63 by concentration-dependent manners. The plaque assay
indicated that the IC
50
value of Sambucus FormosanaNakai extract on
the virus yield was 1.17 μg/ml (Fig. 2). Remarkably, the results de-
monstrated that Sambucus FormosanaNakai stem ethanol extract served
the signicantly antiviral activity against HCoV-NL63.
3.2. Inhibitory eect of Sambucusformosana Nakai extract on the viral
plaque formation and attachment
To discover the inhibitory action of Sambucus FormosanaNakai ex-
tract on stages of HCoV-NL63 replication, plaque formation, virucidal
activity, and virus attachment assays were performed using the plaque
assays (Fig. 3,Table 1). Sambucus FormosanaNakai extract meaningfully
inhibited the plaque formation with an IC
50
value of 4.67 μg/ml
(Fig. 3A). In the virucidal assay, Sambucus FormosanaNakai extract at 1,
5, and 10 μg/ml had a slight virucidal activity with lower than 50%
inhibition to interfere with the HCoV-NL63 particle infectivity com-
pared to the mock control (Fig. 3B). In the virus attachment assay,
Sambucus FormosanaNakai extract concentration-dependently reduced
the HCoV-NL63 attachment onto LLC-MK2 cell monolayer in 6-well
plates incubated at 4 °C, which result demonstrated that the ethanol
extract had a inuentially inhibitory eect on HCoV-NL63 attachment
with an IC
50
value of 15.75 μg/ml (Fig. 3C). The results demonstrated
that Sambucus FormosanaNakai extract specically suppressed the viral
plaque formation and virus attachment during HCoV-NL63 replication.
3.3. Anti-HCoV-NL63 activity of the phenolic acid constituents
The phenolic acid constituents were rich in the extract of Sambucus
Table 1
Cytotoxicity and anti-HCoV-NL63 activity of Sambucus Formosana Nakai stem ethanol extract and the phenolic acid constituents.
Cytotoxicity (CC
50
) Virus yield (IC
50
) Plaque formation (IC
50
) Virus attachment (IC
50
)
Stem ethanol extract 180.62 ± 63.04 μg/mL 1.17 ± 0.75 μg/mL 4.67 ± 1.21 μg/mL 15.75 ± 6.65 μg/mL
Caeic acid > 500 μM 3.54 ± 0.77 μM 5.40 ± 1.20 μM 8.10 ± 0.05 μM
Chlorogenic acid > 500 μM 43.45 ± 6.01 μM 44.38 ± 3.64 μM
Gallic acid > 500 μM 71.48 ± 18.40 μM––
J.-R. Weng, et al. Virus Research 273 (2019) 197767
3
FormosanaNakai and Sambucus australis, and the presence of chloro-
genic acid, caeic acid, coumaric acid and ferulaic acid was identied
as the major compounds using LC-MS/MS analyses (Benevides Bahiense
et al., 2017;Zhang et al., 2010). Subsequently, caeic acid, chlorogenic
acid, coumaric acid, ferulic acid, and gallic acid represented the key
phenolic acid constituents in the ethanol stem extract of Sambucus
FormosanaNakai, as further assessed the cytotoxicity to LLC-MK2 cells
and the antiviral activity against HCoV-NL63 (Supplemental Figs. 2 and
4). The markers of phenolic acid constituents in Sambucus Formosana
Nakai extract were less cytotoxic to LLC-MK2 cells, in which CC
50
va-
lues of caeic acid, chlorogenic acid, and gallic acid were greater than
500 μM (Supplemental Fig. 2, Table 1). In the inhibitory assay of HCoV-
NL63 induced cytopathic eect, caeic acid, chlorogenic acid, and
gallic acid, but not coumaric acid and ferulic acid, diminished the
Fig. 1. Inhibitory eects of Sambucus Formosana Nakai extract on viral cytopathicity and Sub-G1 fraction in HCoV-NL63 infected cells. HCoV-NL63 at MOI
of 0.1 was mixed with the extract, and immediately added to LLC-MK-2 cell culture. Virus-induced cytopathic eect was photographed 36 h post-infection by
microscopy (A). Infected cells in the presence or absence of the extract were harvested 36 hpi, stained using propidium iodide, and then examined using ow
cytometry. The ow cytometry histograms (top) and the percentage of sub-G1 phase (bottom) in HCoV-NL63 infected cells was displayed (B). **, pvalue < 0.01
compared with mock-treated infected cells.
Fig. 2. Reduction of HCoV-NL63 yield in MK-2 cells
by Sambucus Formosana Nakai extract. Supernatant
of HCoV-NL63-infected cells in the presence or ab-
sence of the extract was harvested 36 hpi and HCoV-
NL63 yield in the supernatant was determined by
plaque assay (A). The rate of virus yield reduction was
calculated based on the ratio of loss particle number to
mock-treated group (B). **, pvalue < 0.01. compared
with mock-treated infected cells.
J.-R. Weng, et al. Virus Research 273 (2019) 197767
4
cytopathicity in the infected cells (Fig. 4A). The antiviral activity of the
phenolic acid constituents in the in vitro production of HCoV-NL63 was
ranked in the following order of virus yield reduction: caeic acid
(IC
50
= 3.54 μM) > chlorogenic acid (IC
50
= 43.45 μM) > coumaric
acid (IC
50
= 71.48 μM) (Fig. 4B, Table 1). The results veried that the
phenolic acid constituents, like caeic acid, chlorogenic acid, and gallic
acid exhibited the prominent antiviral activity against HCoV-NL63, as
potential anti-HCoV-NL63 components in the Sambucus For-
mosanaNakai extract.
3.4. Caeic acid, a phenolic acid constituent in Sambucus spp., potently
inhibits HCoV-NL63 attachment onto cells
To examine the antiviral mechanism of caeic acid and chlorogenic
acid against HCoV-NL63, the assays of plaque formation, virucidal ac-
tivity and virus attachment were subsequently performed (Fig. 5,
Table 1). Caeic acid had a stronger inhibitory activity on the plaque
formation than chlorogenic acid, in which the IC50 values on the
plaque formation were 5.4 μM for caeic acid and 44.38 μM for
chlorogenic acid, respectively (Fig. 5). Caeic acid, but not chlorogenic
acid, concentration-dependently served the virucidal activity
(IC
50
= 91.3 μM) and powerfully reduced HCoV-NL63 attachment to
Fig. 3. Eects of Sambucus Formosana Nakai
extract on plaque formation, virucidal ac-
tivity and virus attachment. MK-2 cell
monolayer was infected with HCoV-NL63, si-
multaneously treated with the extract for 1 h,
and then covered with the agarose overlay
medium. After 3-day incubation at 37 °C in a
CO
2
incubator, plaques were determined after
crystal violet staining. The inhibitory activity
of the extract on the plaque formation was
according to on the ratio of loss plaque number
to mock-treated group (A). In the virucidal
assay, the extract was mixed with HCoV-NL63
(10
6
pfu), then incubated at 37 °C for 1 h. The
extract/virus mixture was diluted by 1000-fold
dilution and examined for the residual infectivity by plaque assay (B). In the attachment assay, HCoV-NL63 was mixed with the extract, then immediately added onto
MK2 cell monolayer for 1 h at 4 °C. After washing, the cell monolayer was overlaid with 0.75% agarose medium for 3 days at 37 °C in CO
2
incubator. Attachment
inhibition was determined based on the residual plaques (C). *, pvalue < 0.05; **, pvalue < 0.01 compared with mock-treated cells.
Fig. 4. Inhibitory eects of the phenolic acid constituents on viral cytopathicity and virus yield in HCoV-NL63 infected cells. HCoV-NL63 at MOI of 0.1 was
added to LLC-MK-2 cell culture and then immediately treated with the phenolic acid constituents. Virus-induced cytopathic eect was photographed 36 h post-
infection by microscopy (A). Supernatant of HCoV-NL63-infected/treated cells was harvested 36 hpi; virus yield in supernatant was determined plaque assay. The
rate of virus yield reduction was calculated based on the ratio of loss particle number to mock-treated group (B). CA, caeic acid; CH, chlorogenic acid; CO, coumaric
acid; FE, ferulic acid; GA, gallic acid. **, pvalue < 0.01, ***, pvalue < 0.001 compared with mock-treated infected cells.
J.-R. Weng, et al. Virus Research 273 (2019) 197767
5
the cell surface (IC
50
of 8.1 μM), respectively (Fig. 5). In addition, in-
fectivity inhibition assay with human airway epithelia Calu-3 cells was
performed when HCoV-NL63-infected cells with a MOI of 0.05 were
treated with 10 and 50 μMcaeic acid after a 36-h incubation at 32 °C.
Microscopic photography indicated that swelling and rounding were
observed in HCoV-NL63-infected Calu-3 cells (Fig. 6A, top), as de-
scribed in the previous study (Lednicky et al., 2013). Caeic acid at 10
and 50 μM signicantly lessening viral CPE (Fig. 6A, top). Immuno-
uorescent staining demonstrated that caeic acid concentration-de-
pendently reduced HCoV-NL63 infectivity determined according to the
ratio of HCoV-NL63-positive cells to total cells stained with DAPI
(Fig. 6A, middle and bottom). The inhibitory assay with immuno-
uorescent staining on HCoV-NL63 infectivity indicated that caeic
acid had a potent antiviral activity against the replication of HCoV-
NL63 in Calu-3 cells (IC
50
= 0.2 ± 0.1 μM) (Fig. 6B). The results
revealed that caeic acid displayed the potent anti-HCoV-NL63 activity
in a cell-type independent manner, specically inhibiting on the at-
tachment stage of HCoV-NL63 replication.
4. Discussion
This study was the rst report on the antiviral ecacy of Sambucus
FormosanaNakai extract and its related phenolic acid constituents
against HCoV-NL63. Sambucus FormosanaNakai extract exhibited the
low cytotoxicity and markedly decreased cytopathic eect and sub-G1
arrest in HCoV-NL63-infected cells, in which was associated with the
virus yield reduction in a concentration-dependent manner (Figs. 1 and
2,Table 1). Moreover, Sambucus FormosanaNakai extract showed the
potent antiviral activity against HCoV-NL63 with the IC50 values in low
microg/ml ranges, such IC
50
of 1.17 μg/ml for virus yield, IC
50
of
Fig. 5. Eects of caeic acid and chloro-
genic acid on plaque formation, virucidal
activity and virus attachment. MK-2 cell
monolayer was infected with HCoV-NL63, si-
multaneously treated with the caeic acid or
chlorogenic acid for 1 h, and then covered with
the agarose overlay medium for 3-day at 37 °C
in a CO
2
incubator. After crystal violet
staining, the inhibitory activity of caeic acid
and chlorogenic acid on the plaque formation
was according to on the ratio of loss plaque
number to mock-treated group (A). In the vir-
ucidal assay, the caeic acid or chlorogenic
acid was mixed with HCoV-NL63 (10
6
pfu),
then incubated at 37 °C for 1 h. The 1000-fold
dilution of the compound/virus mixture was
examined for the residual infectivity by plaque
assay (B). In the attachment assay, MK2 cell
monolayer was infected with HCoV-NL63
(100 pfu), immediately treated with the caeic
acid or chlorogenic acid for 1 h at 4 °C, washed,
and overlaid with 0.75% agarose medium for 3
days at 37 °C in CO
2
incubator. Attachment
inhibition was determined based on the re-
sidual plaques (C). CA. caeic acid; CH,
chlorogenic acid. *, pvalue < 0.05;**, p
value < 0.01; ***, pvalue < 0.001 compared
with un-treated infected cells.
Fig. 6. Inhibition of HCoV-NL63 infectivity in human airway epithelial cells by caeic acid. Calu-3 cells were infected with HCoV-NL63 at a MOI of 0.05 and
immediately treated with caeic acid for 36 h at 32 °C. Images of virus-induced CPE eect were photographed by a light microscope (A, top). In addition, mock,
infected, and infected/treated cells were performed using immunouorescence staining anti-HCoV-NL63 immunized sera and secondary antibody Alexa Fluor anti-
mouse IgG (A, middle); total cells were stained with DAPI (A, bottom). Infectivity was determined according to the ratio of HCoV-NL63-positive cells to total cells (B).
**, pvalue < 0.01; ***, pvalue < 0.001 compared with un-treated infected cells.
J.-R. Weng, et al. Virus Research 273 (2019) 197767
6
4.67 μg/ml for plaque formation, and IC
50
of 15.75 μg/ml for virus at-
tachment. The results were consistent with the previous reports in that
Sambucus spp., such Sambucus nigra L. served the antiviral properties
against inuenza A and B viruses, and the herpes simplex virus type1
(Krawitz et al., 2011;Serkedjieva et al., 1990;Zakay-Rones et al.,
1995). In addition, Sambucus juice was the key recipe for the "Virus
Blocking Factor" that processed the in-vitro antiviral activity against
many respiratory viral infections, including inuenza A H1N1, rhino-
virus B subtype 14, respiratory syncytial virus, parainuenzavirus
subtype 3, and adenovirus C subtype 5 (Fal et al., 2016). Sambucus nigra
L. has been recognized as was a potentially safer alternative to treat
upper respiratory symptoms, such common cold and inuenza (Haw-
kins et al., 2019). Thus, Sambucus FormosanaNakai might be the alter-
native medicinal herb for caring the respiratory viral infections, espe-
cially coronavirus-associated common cold.
Among six phenolic acid constituents in the Sambucus
FormosanaNakai extract, caeic acid had the highest anti-HCoV-NL63
potency with the inhibitory eect on the virus yields (IC50 = 3.54 μM),
plaque formation (IC50 = 5.40 μM), and virus attachment
(IC50 = 8.10 μM) (Figs. 4 and 5,Table 1). Caeic acid has also been
demonstrated the antiviral activity against HCoV-NL63 in a cell-type
independent manner (Fig. 6). In addition, chlorogenic acid and gallic
acid exhibited antiviral activity against HCoV-NL63 (Figs. 4 and 5,
Table 1). The IC50 value on the inhibition of virus yield was 43.45 μM
for chlorogenic acid, and 71.48 μM for gallic acid, respectively. The
phenolic acid constituents have been identied as the major compo-
nents in the Sambucus FormosanaNakai extract and Sambucus australis
by LC-MS/MS analyses (Benevides Bahiense et al., 2017;Zhang et al.,
2010), therefore caeic acid, chlorogenic acid and gallic acid might be
the key components with anti-HCoV-NL63 activity in the Sambucus
FormosanaNakai extract.
Caeic acid has been reported to process the antiviral activity
against hepatitis B and C viruses, inuenza A virus, and herpes simplex
virus (Wang et al., 2019; Shen et al., 2018;Tanida et al., 2015;
Utsunomiya et al., 2014;Ikeda et al., 2011;Langland et al., 2018).
Caeic acid reduced the production of hepatitis C virus in vitro via the
initial stage of viral replication (Tanida et al., 2015). The antiviral
mechanism of caeic acid against hepatitis C virus was also associated
with the activation of p62-mediated Keap1/Nrf2 signaling pathway for
the HO-1-dependent production of interferon α, which signicantly
suppressed the replication of hepatitis C virus (Shen et al., 2018).
Caeic acid markedly decreased the virus yield and cytopathic eect in
inuenza A virus-infected cells, particular during the period within 3 h
post-infection, suggesting that caeic acid works at the early stages of
inuenza A virus infection (Utsunomiya et al., 2014). In addition, caf-
feic acid had no virucidal eect, but signicantly aected the produc-
tion of progeny herpes simplex virus and the binding attachment to the
heparan sulfate proteoglycans on cell surface (Ikeda et al., 2011;
Langland et al., 2018). Interestingly, HCoV-NL63 has been demon-
strated to utilize the heparan sulfate proteoglycans as the co-receptor
for attachment to target cells (Milewska et al., 2014). Caeic acid has
also been identied to exhibit a high inhibitory eect on the enzymatic
activity of angiotensin converting enzyme (ACE) (Chiou et al., 2017);
docking studies revealed that caeic acid also presented the good
docking score with the hydrogen bond interactions with the residues in
the activity site of ACE2 (Zozimus Divya Lobo et al., 2017). Thus, the
inhibitory mechanism of caeic acid on HCoV-NL63 attachment and
infection to cells might be due to that caeic acid directly target and
interfere the binding interaction of HCoV-NL63 with heparan sulfate
proteoglycans (co-receptor) and ACE2 (receptor) on cell surface.
Chlorogenic acid and gallic acid have also been demonstrated to
suppress the in vitro and in vivo replication of inuenza A virus, en-
terovirus 71 and hepatitis B and C viruses (Ding et al., 2017;Li et al.,
2013;Wang et al., 2009;You et al., 2018;Govea-Salas et al., 2016).
Chlorogenic acid specically inhibited the neuraminidase activity of
inuenza A viruses H1N1 and H3N2 that was the crucial mechanism of
chlorogenic acid for blocking the release of progeny virions from in-
fected cells (Ding et al., 2017). Gallic acid served the antioxidant ca-
pacity that linked with down-regulating the genomic RNA and proteins
expression of hepatitis C virus (Govea-Salas et al., 2016). Therefore, the
phenolic acid constituents of Sambucus Formosana Nakai extract, like
caeic acid, chlorogenic acid and gallic acid, play the key antiviral
components against HCoV-NL63 and the other viruses.
The study demonstrated the potent anti-HCoV-NL-63 activity of
Sambucus FormosanaNakai extract through the signicant reduction of
virus yield, plaque formation and virus attachment. Caeic acid,
chlorogenic acid and gallic acid were reported as the phenolic acid
constituents in the Sambucus FormosanaNakai extract, exhibiting the
antiviral capacity with reducing the production of progeny HCoV-NL63
particles in vitro. Moreover, caeic acid plays the important component
with antiviral activity, as suggested to inuence the binding of HCoV-
NL63 to the co-receptors (such heparan sulfate proteoglycans) and re-
ceptor (ACE2). Like Sambucus nigra L., Sambucus FormosanaNakai might
process the antiviral features against the broad spectrum of human
respiratory coronaviruses, as useful for developing the antiviral agents
in the future.
Declaration of Competing Interest
All authors declare no potential conict of interest.
Acknowledgements
This study was supported by China Medical University under the
Featured Areas Research Center Program within the framework of the
Higher Education Sprout Project by the Ministry of Education, Taiwan
(CHM106-6-2 and CMRC-CHM-2). This project was also funded by
grants from the Ministry of Science and Technology, Taiwan
(MOST107-2923-B-039-001-MY3, MOST105-2320-B-039-053-MY3),
China Medical University (CMU106-BC-1, CMU106-ASIA-06, CMU107-
ASIA-12, and CMU107-S-14).
Appendix A. Supplementary data
Supplementary material related to this article can be found, in the
online version, at doi:https://doi.org/10.1016/j.virusres.2019.197767.
References
Amoros, M., Simões, C.M., Girre, L., Sauvager, F., Cormier, M., 1992. Synergistic eect of
avones and avonols against herpes simplex virus type 1 in cell culture. Comparison
with the antiviral activity of propolis. J. Nat. Prod. 55 (12), 17321740.
Barak, V., Halperin, T., Kalickman, I., 2001. The eect of sambucol, a black elderberry-
based, natural product, on the production of human cytokines: I. inammatory cy-
tokines. Eur. Cytokine Networks 12 (2), 290296.
Benevides Bahiense, J., Marques, F.M., Figueira, M.M., Vargas, T.S., Kondratyuk, T.P.,
Endringer, D.C., Scherer, R., Fronza, M., 2017. Potential anti-inammatory, anti-
oxidant and antimicrobial activities of Sambucus australis. Pharm. Biol. 55 (1),
991997.
Chen, F., Liu, D.L., Wang, W., Lv, X.M., Li, W., Shao, L.D., Wang, W.J., 2019. Bioactive
triterpenoids from Sambucus javanica Blume. Nat. Prod. Res. 10 (2), 16.
Chen, W., Li, K.Q., Xiong, X.J., Zhang, J.H., 2001. Research on the eective chemical
constituents of Sumbucus chinensis Lindl. against hepatitis. J Nanchang Univ. 25 (2),
165167.
Chiou, S.Y., Sung, J.M., Huang, P.W., Lin, S.D., 2017. Antioxidant, antidiabetic, and
antihypertensive properties of echinacea purpurea ower extract and caeic acid
derivatives using in vitro models. J. Med. Food 20 (2), 171179.
Cui, L.J., Zhang, C., Zhang, T., Lu, R.J., Xie, Z.D., Zhang, L.L., Liu, C.Y., Zhou, W.M.,
Ruan, L., Ma, X.J., Tan, W.J., 2011. Human coronaviruses HCoV-NL63 and HCoV-
HKU1 in hospitalized children with acute respiratory infections in Beijing. China Adv.
Virol. 2011, 129134.
Davis, B.M., Foxman, B., Monto, A.S., Baric, R.S., Martin, E.T., Uzicanin, A., Rainey, J.J.,
Aiello, A.E., 2018. Human coronaviruses and other respiratory infections in young
adults on a university campus: prevalence, symptoms, and shedding. Inuenza Other
Respi. Viruses 12, 582590.
Hong, D., Yang, Q., Malécot, V., Bouord, D.E., 2013. Sambucus javanica. Flora of China.
Missouri Botanical Garden, St. Louis, MO & Harvard University Herbaria,
Cambridge, MA.
J.-R. Weng, et al. Virus Research 273 (2019) 197767
7
Dijkman, R., Jebbink, M.F., Gaunt, E., Rossen, J.W., Templeton, K.E., Kuijpers, T.W., van
der Hoek, L., 2012. The dominance of human coronavirus OC43 and NL63 infections
in infants. J. Clin. Virol. 53 (2), 135139.
Ding, Y., Cao, Z., Cao, L., Ding, G., Wang, Z., Xiao, W., 2017. Antiviral activity of
chlorogenic acid against inuenza A (H1N1/H3N2) virus and its inhibition of neur-
aminidase. Sci. Rep. 7, 45723.
Fal, A.M., Conrad, F., Schönknecht, K., Sievers, H., Pawińska, A., 2016. Antiviral activity
of the Virus Blocking Factor(VBF) derived i.a. From Pelargonium extract and
Sambucus juice against dierent human-pathogenic cold viruses in vitro. Wiad. Lek.
69 (3 pt 2), 499511.
Geng, H., Cui, L., Xie, Z., Lu, R., Zhao, L., Tan, W., 2012. Characterization and complete
genome sequence of human coronavirus NL63 isolated in China. J. Virol. 86 (17),
95469547.
Govea-Salas, M., Rivas-Estilla, A.M., Rodríguez-Herrera, R., Lozano-Sepúlveda, S.A.,
Aguilar-Gonzalez, C.N., Zugasti-Cruz, A., Salas-Villalobos, T.B., Morlett-Chávez, J.A.,
2016. Gallic acid decreases hepatitis C virus expression through its antioxidant ca-
pacity. Exp. Ther. Med. 11 (2), 619624.
Hand, J., Rose, E.B., Salinas, A., Lu, X., Sakthivel, S.K., Schneider, E., Watson, J.T., 2018.
Severe respiratory illness outbreak associated with human coronavirus NL63 in a
long-term care facility. Emerg. Infect. Dis. 24 (10), 19641966.
Huang, S.H., Su, M.C., Tien, N., Huang, C.J., Lan, Y.C., Lin, C.S., Chen, C.H., Lin, C.W.,
2017. Epidemiology of human coronavirus NL63 infection among hospitalized pa-
tients with pneumonia in Taiwan. J. Microbiol. Immunol. Infect. 50 (6), 763770.
Ikeda, K., Tsujimoto, K., Uozaki, M., Nishide, M., Suzuki, Y., Koyama, A.H., Yamasaki, H.,
2011. Inhibition of multiplication of herpes simplex virus by caeic acid. Int. J. Mol.
Med. 28 (4), 595598.
Krawitz, C., Mraheil, M.A., Stein, M., Imirzalioglu, C., Domann, E., Pleschka, S., Hain, T.,
2011. Inhibitory activity of a standardized elderberry liquid extract against clinically-
relevant human respiratory bacterial pathogens and inuenza A and B viruses. BMC
Complement. Altern. Med. 11, 16.
Langland, J., Jacobs, B., Wagner, C.E., Ruiz, G., Cahill, T.M., 2018. Antiviral activity of
metal chelates of caeic acid and similar compounds towards herpes simplex, VSV-
Ebola pseudotyped and vaccinia viruses. Antiviral Res. 160, 143150.
Lednicky, J.A., Waltzek, T.B., McGeehan, E., Loeb, J.C., Hamilton, S.B., Luetke, M.C.,
2013. Isolation and genetic characterization of human coronavirus NL63 in primary
human renal proximal tubular epithelial cells obtained from a commercial supplier,
and conrmation of its replication in two dierent types of human primary kidney
cells. Virol. J. 10, 213.
Li, X., Liu, Y., Hou, X., Peng, H., Zhang, L., Jiang, Q., Shi, M., Ji, Y., Wang, Y., Shi, W.,
2013. Chlorogenic acid inhibits the replication and viability of enterovirus 71 in
vitro. PLoS One 8 (9), e76007.
Li, S.W., Lin, C.W., 2013. Human coronaviruses: clinical features and phylogenetic ana-
lysis. Biomed. 3, 4350 (Taipei).
Liao, Q.F., Xie, S.P., Chen, X.H., Bi, K.S., 2006. Study on the chemical constituents of
Sumbucus chinensis Lindl. Zhong Yao Cai. 29 (9), 916918.
Lin, C.N., Tome, W.P., 1988. Antihepatotoxic principles of Sambucus formosana. Planta
Med. 54 (3), 223224.
Mahmood, N., Pizza, C., Aquino, R., De, T., Piacente, S., Colman, S., Burke, A., Hay, A.J.,
1993. Inhibition of HIV infection by avanoids. Antiviral Res. 22, 189199.
Mandrone, M., Lorenzi, B., Maggio, A., La Mantia, T., Scordino, M., Bruno, M., Poli, F.,
2014. Polyphenols pattern and correlation with antioxidant activities of berries ex-
tracts from four dierent populations of sicilian Sambucus Nigra L. Nat. Prod. Res. 28
(16), 12461253.
Milewska, A., Zarebski, M., Nowak, P., Stozek, K., Potempa, J., Pyrc, K., 2014. Human
coronavirus NL63 utilizes heparan sulfate proteoglycans for attachment to target
cells. J. Virol. 88 (22), 1322113230.
Pinto, J., Spínola, V., Llorent-Martínez, E.J., Fernández-de Córdova, M.L., L, M.-G.,
Castilho, P.C., 2017. Polyphenolic prole and antioxidant activities of Madeiran el-
derberry (Sambucus lanceolata)asaected by simulated in vitro digestion. Food Res.
Int. 100 (Pt3), 404410.
Pliszka, B., 2017. Polyphenolic content, antiradical activity, stability and microbiological
quality of elderberry (Sambucus Nigra L.) extracts. Acta Sci. Pol. Technol. Aliment. 16
(4), 393401.
Porter, R.S., Bode, R.F., 2017. A review of the antiviral properties of black elder
(Sambucus Nigra L.) products. Phytother. Res. 31 (4), 533554.
Roschek, B.Jr., Fink, R.C., McMichael, M.D., Li, D., Alberte, R.S., 2009. Elderberry a-
vonoids bind to and prevent H1N1 infection in vitro. Phytochemistry 70 (10),
12551261.
Serkedjieva, J., Manolova, N., Zgorniak-Nowosielska, I., 1990. Antiviral activity of the
infusion (SHS-174) from owers of Sambucus Nigra L., aerial parts of Hypericum
Perforatum L., and roots of Saponaria Ocinalis L. Against inuenza and herpes
simplex viruses. Phytother. Res. 4, 97100.
Shen, J., Wang, G., Zuo, J., 2018. Caeic acid inhibits HCV replication via induction of
IFNαantiviralresponse through p62-mediated Keap1/Nrf2 signaling pathway.
Antiviral Res. 154, 166173.
Tanida, I., Shirasago, Y., Suzuki, R., Abe, R., Wakita, T., Hanada, K., Fukasawa, M., 2015.
Inhibitory eects of caeic acid, a coee-related organic acid, on the propagation of
hepatitis C virus. Jpn. J. Infect. Dis. 68 (4), 268275.
Turek, S., Cisowski, W., 2007. Free and chemically bonded phenolic acids in barks of
Viburnum Opulus L. And Sambucus Nigra L. Acta Pol. Pharm. 64 (4), 377383.
Utsunomiya, H., Ichinose, M., Ikeda, K., Uozaki, M., Morishita, J., Kuwahara, T., Koyama,
A.H., Yamasaki, H., 2014. Inhibition by caeic acid of the inuenza A virus multi-
plication in vitro. Int. J. Mol. Med. 34 (4), 10201024.
Wang, C.Y., Huang, S.C., Zhang, Y., Lai, Z.R., Kung, S.H., Chang, Y.S., Lin, C.W., 2012.
Antiviral ability of Kalanchoe gracilis leaf extract against enterovirus 71 and cox-
sackievirus A16. Evid. Complement. Alternat. Med. 2012, 503165.
Wang, C.Y., Hour, M.J., Lai, H.C., Chen, C.H., Chang, P.J., Huang, S.H., Lin, C.W., 2018.
Epigallocatechin-3-gallate inhibits the early stages of Japanese encephalitis virus
infection. Virus Res. 253, 140146.
Wang, G.F., Shi, L.P., Ren, Y.D., Liu, Q.F., Liu, H.F., Zhang, R.J., Li, Z., Zhu, F.H., He, P.L.,
Tang, W., Tao, P.Z., Li, C., Zhao, W.M., Zuo, J.P., 2009. Anti-hepatitis B virus activity
of chlorogenic acid, quinic acid and caeic acid in vivo and in vitro. Antiviral Res. 83
(2), 186190.
Yang, K.C., Chiu, S.T., 1998. Caprifoliaceae. Flora of Taiwan, 2nd ed. .
Yang, Y.J., Lin, J.H., 2004. Study on the chemical constituents of Sumbucus Chinensis
Lindl. J Chin Med Mater 27 (7), 491492.
You, H.L., Huang, C.C., Chen, C.J., Chang, C.C., Liao, P.L., Huang, S.T., 2018. Anti-pan-
demic inuenza A (H1N1) virus potential of catechin and gallic acid. J. Chin. Med.
Assoc. 81 (5), 458468.
Zakay-Rones, Z., Varsano, N., Zlotnik, M., Manor, O., Regev, L., Schlesinger, M.,
Mumcuoglu, M., 1995. Inhibition of several strains of Inuenza virus in vitro and
reduction of symptoms by an elderberry extract (Sambucus Nigra L.) during an out-
break of inuenza B panama. J. Altern. Complement. Med. 1 (4), 361369.
Zhang, T., Yang, D., Wang, Y., Chen, X., Bi, K., 2010. Simultaneous determination of four
components in the herbs of Sumbucus chinensis Lindl. By high performance liquid
chromatography. Asian J. Tradit. Med. 5 (2), 7074.
Zozimus Divya Lobo, C., Syed Mohamed, A., Vedhi, C., Rajesh, S.V., Aroulmoji, V.,
Shanmugam, G., 2017. Identication of potent angiotensin converting enzyme 2
inhibitors through virtual screening and structure-based pharmacophore design. Int J
Adv Sci Eng 4 (1), 474480.
J.-R. Weng, et al. Virus Research 273 (2019) 197767
8
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  • ... Among the six components identified in the methanol extract of S. cusia leaf were tryptanthrin and indigodole B (5aRethyltryptanthrin); of which tryptanthrin exhibited the most potent antiviral activity in reducing the CPE and progeny virus production giving it the strongest antiviral potential against HCoV-NL63 infection [37]. In vitro examination by Weng and Lin (2019) of Sambucus FormosanaNakai, a species of elderberry which is a traditional medicinal herb with anti-inflammatory and antiviral activity for potential use against HCoV-NL63 found that S. FormosanaNakai interrupted the replication capabilities of the virus. The mechanism of action was thought to be the constituent phytochemicals in S. FormosanaNakai (caffeic acid, chlorogenic acid and gallic acid) of which caffeic acid most significantly inhibited replication of HCoV-NL63 in a cell-type independent manner, and specifically blocked virus attachment (IC50 = 8.1 μM) (Figure 1) [38]. ...
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    The outbreak caused by COVID-19 is causing a major challenge to clinical management and a worldwide threat to public health. So far, there is no specific anti-coronavirus therapy approved for the treatment of COVID-19. Recently, as the efficacy and safety of traditional Chinese medicine (TCM) is widely acknowledged, it has been brought to a crucial status by the public, governments, and World Health Organization (WHO). For a better popularization of TCM, governments have made several advances in regulations and policies for treatment and measures of novel coronavirus pneumonia (NCP). Therefore, on the basis of epidemiology and virology information, we reviewed relevant meta-analysis and clinical studies of anti-coronavirus therapeutics by TCM, in the aspect of mortality, symptom improvement, duration and dosage of corticosteroid, incidence of complications and the like. In addition, we also summarized preclinical rationale for anti-coronavirus activity by TCM in terms of virion assembly and release, as well as viral entry and replication, which could be a useful contribution for figuring out effective Chinese herbal medicine (CHM) for coronavirus, including ingredients from single monomeric compounds, Chinese herbs, Chinese herb extracts and Chinese herb formulas, or potential targets for medicine. We would like to see these relevant studies, ranging from basic researches to clinical application, could provide some idea on effects of CHM to combat COVID-19 or other coronaviruses, and also offer new thinking for the exploration of therapeutic strategies under the guidance of TCM.
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    Caffeic acid is one of the pharmacologically active compounds of many medicinal herbs including Echinacea purpurea which was recently recommended as prophylactic treatment for all coronaviruses, including newly occurring strains, such as SARS-CoV-2. Cell-surface heparan sulfate proteoglycans (HSPGs) provide the binding sites for SARS-CoV invasion at the early attachment phase. In addition to ACE2, HSPGs are essential cell-surface molecules involved in SARS-CoV cell entry. Caffeic acid might be a potent molecule for the treatment of new SARS-CoV-2 virus.
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    Organic compounds with a caffeoyl moiety (e.g. caffeic acid, rosmarinic acid, chicoric acid, etc.) have antiviral properties towards herpes simplex (HSV), influenza and immunodeficiency viruses (HIV). This study evaluated the HSV antiviral properties of caffeic acid when paired with a variety of metal and other inorganic ions. The results demonstrated that the antiviral activity of caffeic acid increased upwards of 100-fold by the addition of cations, such as Fe³⁺, and anionic molecules, such as molybdate and phosphate. Cellular toxicity tests of the caffeic acid chelates showed that they have low toxicities with selectivity indices (TD50/EC50) for Fe³⁺, MoO4²⁻, and PO4³⁻ chelates being 1700, >540, and >30, respectively. Caffeic acid paired with Fe³⁺ was tested against eight strains of viruses, including those from different families. The caffeic acid chelates were mostly effective against HSV1 and HSV2, but they also had moderate activity against vaccinia virus and a VSV-Ebola pseudotyped virus. All the viruses that were strongly impacted by the caffeic chelates require heparan sulfate proteoglycans for cellular attachment, so it is likely that caffeic chelates target and interfere with this mechanism. Since the caffeic acid chelates target an extra-cellular process, they might be able to be combined with existing medications, such as acyclovir, that target an intracellular process to achieve greater viral control.
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    We describe an outbreak of severe respiratory illness associated with human coronavirus NL63 in a long-term care facility in Louisiana in November 2017. Six of 20 case-patients were hospitalized with pneumonia, and 3 of 20 died. Clinicians should consider human coronavirus NL63 for patients in similar settings with respiratory disease. © 2018, Centers for Disease Control and Prevention (CDC). All rights reserved.
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    Epigallocatechin-3-gallate (EGCG), a green tea catechin, shows broad sepectrum antiviral activity against many RNA and DNA viruses. This study investigated the antiviral efficacy of EGCG against Japanese encephalitis virus (JEV), a zoonotic flavivirus in Southeast Asia and the Western Pacific region. EGCG concentration-dependently reduced CPE, sub-G1 phase, and virus yield of infected cells with different JEV strains at different MOIs. The antiviral activity of EGCG against JEV in different assays declined in the following order: virus yield (IC50 of 7.0 μM) > virus attachment (IC50 of 7.9 μM) > virus entry (IC50 of 9.4 μM) > receptor binding and post-entry. However, EGCG had no virucidal effect on the infectivity of JEV particles. The results indicated that antiviral mechanism of EGCG against JEV was associated with blocking the early steps of JEV infection. The study suggests EGCG as a lead compound for developing broad-spectrum antiviral agents.
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    Background The prevalence, symptom course, and shedding in persons infected with the four most common human coronaviruses (HCoV) ‐229E, HKU1, NL63 and OC43 are poorly described Objectives We estimate their prevalence and associated symptoms among college students identified via a social network study design. Patients/Methods We collected 1‐3 samples (n=250 specimens) from 176 participants between October 2012 and January 17, 2013: participants with acute respiratory infection (ARI) (cough and body aches or chills or fever/feverishness) and their social contacts. Virus was detected using RT‐PCR. Results 30.4% (76/250) of specimens tested positive for any virus tested and 4.8% (12/250) were positive for two or more viruses. Human coronaviruses (HCoVs [22.0%; 55/250]), rhinovirus (7.6%; 19/250), and influenza A (6.4%; 16/250) were most prevalent. Symptoms changed significantly over time among ARI participants with HCoV: the prevalence of cough and chills decreased over 6 days (p=0.04, and p=0.01, respectively), while runny nose increased over the same period (p=0.02). HCoV‐NL63 was the most frequent virus detected 6 days following symptom onset (8.9%), followed by rhinovirus (6.7%). Conclusions During a 3‐month period covering a single season, HCoVs were common, even among social contacts without respiratory symptoms; specific symptoms may change over the course of HCoV‐associated illness and were similar to symptoms from influenza and rhinovirus. This article is protected by copyright. All rights reserved.
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    Hepatitis C virus (HCV) infection and its related liver disease have constituted a heavy burden worldwide. It had been reported that Drinking coffee could decrease mortality risk of HCV infected patients. Caffeic Acid (CA), the Coffee-related organic acid could inhibit HCV replication, however, the detailed mechanism of CA against HCV is unclear. In this study, we showed that CA could notably inhibit HCV replication. Mechanism study demonstrated that CA could induce HO-1 expression, which would trigger the IFNα antiviral response, and the antiviral effect of CA was attenuated when HO-1 activity was inhibited by SnPP (an HO-1 inhibitor). CA could also increase erythroid 2-related factor 2 (Nrf2) expression. When Nrf2 was knocked down by specific siRNA, HO-1 expression was concomitantly decreased while HCV expression was restored. Further study indicated that kelch-like ECH-associated protein 1 (Keap1) expression was decreased by CA in a p62/Sequestosome1 (p62)-dependent way, which would lead to the stabilization and accumulation of Nrf2. The decrease of Keap1 was restored when p62 was silenced by specific p62 siRNA, suggesting p62 was required for CA-mediated Keap1 downregulation. Taken together, the results demonstrated that CA could modulate Keap1/Nrf2 interaction via increasing p62 expression, which will lead to stabilization of Nrf2 and HO-1 induction and elicit IFNα antiviral response to suppress HCV replication.
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    Background. The pharmaceutical and food industries expect detailed knowledge on the physicochemical properties of elderberry fruit extracts, their stability and microbiological quality, as well as the polyphenol content in elderberry cultivars. The characteristics of the extracts might be additionally modified by citric acid, which improves the stability of anthocyanins and protects processed fruits and syrups from pathogenic microorganisms. The choice of the method with citric acid was a consequence of the physicochemical characteristics of elderberry pigments, which are not stable under the effect of light in alcoholic solutions. The aim of study was to analyze the properties of elderberry fruit extracts regarding polyphenol content and antiradical activity, as well as their stability and microbiological quality. Material and methods. The plant material consisted of fruit from four cultivars (Alleso, Korsor, Sampo, Samyl) of black elderberry (Sambucus nigra L.). The following were determined in fruit extracts: polyphenolic content (HPLC), antiradical activity (ABTS and DPPH) and stability and microbiological quality. Results. The HPLC analysis of polyphenols demonstrated that the extracts from fruits collected from cv. Samyl had the highest 3-sambubioside cyanidin content and those from cv. Korsor contained the highest quantity of 3-glucoside cyanidin. The extracts from cv. Sampo fruit had a dominant 3-sambubioside-5-glucoside cyanidin and 3,5-diglucoside cyanidin content. The highest quercetin (5.92 mg 100 mg⁻¹ of extract) and caffeic acid (1.21 mg 100 mg⁻¹ of extract) content was found in fruit extracts from cv. Alleso. The cultivars Samyl and Korsor had a higher level of anthocyanins and higher antiradical activity (ABTS) in fruit extracts than cv. Alleso and Sampo. The antiradical activity (DPPH) of fruit extracts from elderberry cultivars assessed in this research was similar. The degradation index for all fruit extracts was similar (DI = 1.035). The microbiological species detected in extracts were classified as moulds (Penicillum sp., Aspergillus sp.) and yeasts (Rhodotorula sp., Torulopsis sp., Trichosporon sp., Saccharomyces sp.). Conclusions. The research findings may support the selection of certain cultivars for industrial applications. The high stability of anthocyanins and low level of microbiological impurities in elderberry extracts ensure the high quality of such a raw material in food and pharmaceutical processing.
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    Background: The pandemic influenza A (H1N1) virus has spread worldwide and infected a large proportion of the human population. Discovery of new and effective drugs for the treatment of influenza is a crucial issue for the global medical community. According to our previous study, TSL-1, a fraction of the aqueous extract from the tender leaf of Toonasinensis, has demonstrated antiviral activities against pandemic influenza A (H1N1) through the down-regulation of adhesion molecules and chemokine to prevent viral attachment. Methods: The aim of the present study was to identify the active compounds in TSL-1 which exert anti-influenza A (H1N1) virus effects. XTT assay was used to detect the cell viability. Meanwhile, the inhibitory effect on the pandemic influenza A (H1N1) virus was analyzed by observing plaque formation, qRT-PCR, neuraminidase activity, and immunofluorescence staining of influenza A-specific glycoprotein. Results: Both catechin and gallic acid were found to be potent inhibitors in terms of influenza virus mRNA replication and MDCK plaque formation. Additionally, both compounds inhibited neuraminidase activities and viral glycoprotein. The 50% effective inhibition concentration (EC50) of catechin and gallic acid for the influenza A (H1N1) virus were 18.4 μg/mL and 2.6 μg/mL, respectively; whereas the 50% cytotoxic concentrations (CC50) of catechin and gallic acid were >100 μg/mL and 22.1 μg/mL, respectively. Thus, the selectivity indexes (SI) of catechin and gallic acid were >5.6 and 22.1, respectively. Conclusion: The present study demonstrates that catechin might be a safe reagent for long-term use to prevent influenza A (H1N1) virus infection; whereas gallic acid might be a sensitive reagent to inhibit influenza virus infection. We conclude that these two phyto-chemicals in TSL-1 are responsible for exerting anti-pandemic influenza A (H1N1) virus effects.
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    Background: The pharmaceutical and food industries expect detailed knowledge on the physicochemical properties of elderberry fruit extracts, their stability and microbiological quality, as well as the polyphenol content in elderberry cultivars. The characteristics of the extracts might be additionally modified by citric acid, which improves the stability of anthocyanins and protects processed fruits and syrups from pathogenic microorganisms. The choice of the method with citric acid was a consequence of the physicochemical charac teristics of elderberry pigments, which are not stable under the effect of light in alcoholic solutions. The aim of study was to analyze the properties of elderberry fruit extracts regarding polyphenol content and antiradical activity, as well as their stability and microbiological quality. Methods: The plant material consisted of fruit from four cultivars (Alleso, Korsor, Sampo, Samyl) of black elderberry (Sambucus nigra L.). The following were determined in fruit extracts: polyphe- nolic content (HPLC), antiradical activity (ABTS and DPPH) and stability and microbiological quality. Results: The HPLC analysis of polyphenols demonstrated that the extracts from fruits collected from cv. Samyl had the highest 3-sambubioside cyanidin content and those from cv. Korsor contained the highest quantity of 3-glucoside cyanidin. The extracts from cv. Sampo fruit had a dominant 3-sambubioside-5-gluco- side cyanidin and 3,5-diglucoside cyanidin content. The highest quercetin (5.92 mg 100 mg-1 of extract) and caffeic acid (1.21 mg 100 mg-1 of extract) content was found in fruit extracts from cv. Alleso. The cultivars Samyl and Korsor had a higher level of anthocyanins and higher antiradical activity (ABTS) in fruit extracts than cv. Alleso and Sampo. The antiradical activity (DPPH) of fruit extracts from elderberry cultivars as- sessed in this research was similar. The degradation index for all fruit extracts was similar (DI = 1.035). The microbiological species detected in extracts were classified as moulds (Penicillum sp., Aspergillus sp.) and yeasts (Rhodotorula sp., Torulopsis sp., Trichosporon sp., Saccharomyces sp.). Conclusions: The research findings may support the selection of certain cultivars for industrial applications. The high stability of anthocyanins and low level of microbiological impurities in elderberry extracts ensure the high quality of such a raw material in food and pharmaceutical processing.
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    Angiotensin Converting Enzyme (ACE), a metallo-peptidase is the best known important drug target in the treatment of hypertension and responds to broad range ACE inhibitors such as Captopril. Whilst, many phytochemical compounds including alkaloids and flavonoids were also reported with anti-hypertensive activity. On the other hand, ACE2 is considered as an interesting new cardio-renal disease target as it is close and unique ACE homologue. In this scenario, the anti-hypertensive activities of 17 phytochemical compounds were analyzed through docking studies with ACE2. Also, the other ACE inhibitors with reported IC50 values were considered for docking interactions and used as training set. Further, the best docked phytochemical compound Rosemarinic acid and the training set compounds with ACE inhibitor activity were used to design the pharmacophore and validated. The generated 3D pharmacophore is subjected to screen the compounds with the significant chemical features against May bridged database consisting of more than one lakh compounds and subsequently, the hit compounds were screened using various filters such as estimated activity, Lipinski's rule of five, and ADMET properties and resulted Eight compounds. The anti-hypertensive activities of these 5 compounds with good fit values were selected for further docking studies with ACE2. The five compounds PD 00533, CD 01374, CD 04888, CD 01278 and BTB 04932 exhibited the best docking scores and also favors the necessary hydrogen bond interactions with in the activity site of ACE and thus identified as novel leads with anti-hypertensive activity.