Natural Sulforaphane From Broccoli Seeds Against Influenza A Virus Replication in MDCK Cells

Abstract

We explored the potential application of sulforaphane against influenza A virus and elucidated the underlying cytopathic effect (CPE) and cytotoxicity. In the present study, 2 sulforaphane products were used to investigate the CPEs on influenza A virus replication in Madin-Darby canine kidney cells and to conduct a cytotoxicity test. One was the standard sample and the other was extracted from broccoli seeds. The 2 products of sulforaphane were each diluted to different concentrations. The results indicated that sulforaphane possessed antiviral activity against influenza A/WSN/33 (H1N1) virus, and the standard sulforaphane sample showed biological activity against influenza virus with low cytotoxicity at concentrations of 6.25 to 12.5 μM. The same phenomenon was observed with a broccoli seed extract concentration of sulforaphane of 6.25 μM. Both samples displayed higher cytotoxicity at 50 μM of sulforaphane, and the extract samples showed stronger cytotoxicity at sulforaphane concentrations of 12.5 to 100 μM compared with the standard, particularly at 100 μM. In conclusion, natural sulforaphane from broccoli seeds showed potential as an agent against influenza A virus infection, and the CPE after treatment with sulforaphane was concentration dependent; a suitable sulforaphane concentration of 6.25 μM is suggested and was demonstrated as effective, with high antiviral activity and low cytotoxicity.

Full text

1Institute of Vegetables and Flowers, Chinese Academy of Agricultural
Sciences, Beijing, P.R. China
2Key Laboratory of Biology and Genetic Improvement of Horticultural
Crops, Ministry of Agriculture, Beijing, P.R. China
Corresponding Author:
Zhansheng Li, Key Laboratory of Biology and Genetic Improvement of
Horticultural Crops, Ministry of Agriculture, Beijing, P.R. China.
Email: lizhansheng@ caas. cn
Original Article
Natural Product Communications
June 2019: 1– 8
© The Author(s) 2019
Article reuse guidelines:
sagepub. com/ journals- permissions
DOI: 10.1177/1934578X19858221
journals. sagepub. com/ home/ npx
Creative Commons Non Commercial CC BY-NC: This article is distributed under the terms of the Creative Commons Attribution-NonCommercial 4.0
License (http://www. creativecommons. org/ licenses/ by- nc/ 4. 0/) which permits non-commercial use, reproduction and distribution of the work without
further permission provided the original work is attributed as specified on the SAGE and Open Access pages (https:// us. sagepub. com/ en- us/ nam/ open-
access- at- sage).
Natural Sulforaphane From Broccoli
Seeds Against Influenza A Virus
Replication in MDCK Cells
Zhansheng Li1,2, Yumei Liu1,2, Zhiyuan Fang1,2, Limei Yang1,2, Mu Zhuang1,2,
Yangyong Zhang1,2, and Honghao Lv1,2
Abstract
We explored the potential application of sulforaphane against influenza A virus and elucidated the underlying cytopathic ef-
fect (CPE) and cytotoxicity. In the present study, 2 sulforaphane products were used to investigate the CPEs on influenza A
virus replication in Madin-Darby canine kidney cells and to conduct a cytotoxicity test. One was the standard sample and the
other was extracted from broccoli seeds. The 2 products of sulforaphane were each diluted to different concentrations. The
results indicated that sulforaphane possessed antiviral activity against influenza A/WSN/33 (H1N1) virus, and the standard
sulforaphane sample showed biological activity against influenza virus with low cytotoxicity at concentrations of 6.25 to 12.5
μM. The same phenomenon was observed with a broccoli seed extract concentration of sulforaphane of 6.25 μM. Both
samples displayed higher cytotoxicity at 50 μM of sulforaphane, and the extract samples showed stronger cytotoxicity at
sulforaphane concentrations of 12.5 to 100 μM compared with the standard, particularly at 100 μM. In conclusion, natural
sulforaphane from broccoli seeds showed potential as an agent against influenza A virus infection, and the CPE after treat-
ment with sulforaphane was concentration dependent; a suitable sulforaphane concentration of 6.25 μM is suggested and was
demonstrated as effective, with high antiviral activity and low cytotoxicity.
Keywords
sulforaphane, broccoli, cytotoxicity, influenza virus
Received: December 11th, 2018; Accepted: March 20th, 2019.
Inuenza A virus, a member of the Orthomyxoviridae fam-
ily, is a major human pathogen that typically causes annual
epidemics and occasional pandemics.1-3 Furthermore, the
recent emergence of highly pathogenic avian and swine u
has become a global issue for humans.4,5 In the past 100
years, 5 inuenza epidemics and pandemics have caused
increasing morbidity and mortality worldwide, including
H1N1 in 1918, H2N2 in 1957, H3N2 in 1968, H5N1 in 2009,
and H7N9 in 2014. Particularly, inuenza A (H7N9) causes
high mortality in China.2,6,7 Inuenza A virus is an RNA
virus with a rough spherical shape. These viruses are nega-
tive sense, single-stranded, and segmented with 7 or 8 pieces,
and each piece encodes 1 or 2 genes. Two large glycopro-
teins, hemagglutinin (HA) and neuraminidase (NA), have
been identied in inuenza A viruses of dierent subtypes on
the surface of the viral envelope. Thus far, 18 types of HA
and 11 types of NA have been identied among dierent
inuenza viruses.8,9
HA is a glycol protein expressed on the viral surface along
with NA, and these proteins are responsible for the
attachment of the viral particle to the host cell through cell
surface sialic acid receptors.3,10 HA subsequently completes
the fusion of the viral envelope with the host membrane to
release the viral genome into the target cells, initiating infec-
tion. NA is an enzyme that cleaves the sialic acid residue
tethering the progeny virus and detaches it from infected
cells, accomplishing virus propagation.10,11 Currently, 2
main strategies, using vaccination and anti-inuenza drugs,
are widely employed. However, eective vaccination may
require sustained reproduction to match the antigenic

Natural Product Communications2
changes of viruses, implying that it is almost impossible to
produce ecient and timely vaccines to control inuenza
outbreaks.12,13 Currently, 2 classes of anti-inuenza drugs,
amantadine and rimantadine, have been developed for the
interruption of certain processes of inuenza infection by
targeting either the M2 channel or NA.3,12 Additionally, osel-
tamivir (OSV) and zanamivir target NA protein by the inhi-
bition of its enzymatic activity, rendering the tethered
progeny virus unable to escape from the host cells.14
However, resistant variants have continued to emerge from
patients after treatment, regardless of both classes of drugs
and other drugs, making it urgent to identify novel anti-inu-
enza drugs with safe and eective activity.3,15 Sulforaphane
[1-isothiocyanato-4-(methylsulnyl) butane] is an isothiocy-
anate produced by the hydrolysis of glucoraphanin-rich
broccoli (Brassica oleracea var. italica).16-19 Epidemiological
studies have shown that sulforaphane exhibits anticancer
activity, cardiovascular disease prevention, hypotensive
eects, and myopia prevention.20,21 The anticancer activities
of sulforaphane have been widely demonstrated and studied
in many cancers, including those of the lung,22 stomach,19
liver,23 colon,24 breast,25 prostate,20 and bladder.26 This
mechanism of sulforaphane is attributed to its induction of
phase II detoxication enzymes and prevention of the gener-
ation of phase I detoxication enzymes and
mutagenesis.27,28
Many studies have reported that sulforaphane plays a key
role in sulforaphane preconditioning of the Nrf2 defense
pathway, which protects cerebral vasculature against blood-
brain barrier disruption, neurological decits in stroke, and
tumor growth and spread.29 Additionally, in the past 30 years,
studies have shown that sulforaphane plays an important role
in regulation and as an inducer of Nrf2 signaling and ecacy
as an inhibitor of carcinogenesis, as well as preventing infec-
tious disease, cardiovascular disease, and recently new med-
ical areas.30 Measures of the Nrf2 pathway response and
function serve as guideposts for the optimization of dose,
schedule, and formulation as clinical trials with sulforaphane
and broccoli-based preparations have become more com-
monplace and more rigorous in design and implementa-
tion.3,12 Thus, it is necessary to investigate the response of
standard sulforaphane and the broccoli extract sulforaphane
against HA cells, which can aid in new drug research and
support human health via the consumption of broccoli.19,22,31
Thus far, few reports have shown whether sulforaphane
could prevent inuenza virus infection; therefore, it is neces-
sary to explore this issue. In the present study, inuenza A/
WSN/33(H1N1) virus was selected to examine sulforaphane
in cytopathic eect (CPE) reduction assays and cytotoxicity
tests, which are benecial for research on inuenza preven-
tion, the development of new drugs, and epidemiology
studies.
In the present study, we selected broccoli seeds with high
sulforaphane content as material for extracting sulforaphane.
The sulforaphane content in broccoli seeds was 5512.63
mg/L DW, as detected by HPLC, which is a high content in
Brassica.32,33 The chromatography of sulforaphane in the
standard and extracted samples showed that the system was
eective and stable. The linearity was good within the range
of 5.0 to 300.0 mg/L, and the linear equation was Y = 2.76 ×
10-4X −0.73 (R2 = 0.9998), where the X-axis represents the
peak area and the Y-axis represents the concentration (mg/L).
The HPLC method, with low-temperature working condi-
tions and good stability, has been widely used for the deter-
mination of sulforaphane and glucosinolates and their
hydrolysis products.33,34 Sulforaphane, the second product
derived from glucoraphanin, is not stable and easily changes
into either nitrile material or other compounds at high tem-
peratures or under dierent chemical conditions.33 Thus, GC
and GC-MS are not suitable for the determination of sulfora-
phane or glucosinolates. Most studies have successfully
detected sulforaphane in cruciferous vegetables, particularly
in B. oleracea, with HPLC and HPLC-MS methods.
Sulforaphane is particularly rich in broccoli; the ripe seed
contains the highest levels, followed by seedlings, buds, o-
rets and stems, and leaves, and almost no sulforaphane has
been detected in the roots.33 Thus, HPLC and HPLC-MS
have become typical and simple determination methods for
sulforaphane.33,35
Initially, we examined the cytotoxicity of sulforaphane in
Madin-Darby canine kidney (MDCK) cells by using the
CellTiter-Glo® assay. Culture medium containing 0.5%
dimethyl sulfoxide (DMSO) served as a negative control.
The compound showed cytotoxicity in MDCK cells at con-
centrations of more than 25 µM, while the compounds did
not show signicant cytotoxicity to MDCK cells at concen-
trations of less than 12.5 µM (Figure 1). As shown in
Figure 1. The induction of viral resistance was examined
by continuous sulforaphane treatment. Plaque formation was
observed at different concentrations of sulforaphane (6.25, 12.5,
25.0, 50.0, and 100 μM) by microscopy. (a) Standard, (b) the
broccoli seed extract. Madin-Darby canine kidney cells were
infected with influenza A/WSN/33 (MOI = 0.01) and treated with
sulforaphane and dimethyl sulfoxide. At 40 hours postinfection,
the supernatants were collected and used for infection in the next
round of investigation. Virus yields of mock-treated cells were set
at 100%.

Li et al. 3
Figure 1, concentrations of 6.25 and 12.5 µM of sulfora-
phane showed signicant bioactivity against inuenza A
virus, and the cytotoxicity in MDCK cells was not signi-
cantly dierent from that of the control.
CPE screening, an assay for measuring the damage to
host cells during virus invasion, was utilized to screen and
identify compounds that display a reduction in the CPE on
inuenza A/WSN/33 virus.2 We found that 6.25 and 12.5 µM
concentrations of standard sulforaphane showed signicant
bioactivity against inuenza A virus (Figure 1a), and the
cytotoxicity to MDCK cells was not signicantly dierent
from that of the control. The reduction in CPE was conrmed
by direct microscopic observation, which detected far less
CPE than in the DMSO control (Figure 1). In addition, this
compound exerts a well-dened dose-dependent response
against the A/WSN/33 virus based on plaque formation
(Figure 1). This is an alternative assay for the evaluation of
potency, with an EC50 of approximately 6.25 µM, which is
almost 2-fold lower than that of OSV.3,36 The same eect
was observed at a concentration of 6.25 µM of sulforaphane
seed extract (Figure 1b), consistent with that of the standard
sulforaphane.
According to microscope observations, there were dier-
ent reections of MDCK cells alone and those infected with
virus to dierent gradient concentrations of sulforaphane
diluted with DMSO based on the same magnication
(Figure 1). MDCK cells with virus showed a greater reduc-
tion in CPE at sulforaphane concentrations of 100 and 50
µM than that of standard with more transparent cells. The
extract from broccoli seed showed higher CPE reduction at
sulforaphane concentrations of 100, 50, and 25 µM, and both
samples demonstrated that sulforaphane could prevent inu-
enza A virus, especially at low concentrations of 12.5 and
6.25 µM, compared with the control without virus (DMSO).
The most characteristic feature of sulforaphane is its high
chemical reactivity due to the electrophilicity of the central
carbon of the isothiocyanate (-N=C=S) group,37 which read-
ily reacts with sulfur-, nitrogen-, and oxygen-centered nucle-
ophiles.38 Chemical modication of the sensor cysteine of
KEAP1 by sulforaphane impairs its substrate adaptor func-
tion, leading to Nrf2 accumulation and the enhanced tran-
scription of Nrf2-dependent genes.38 These genes have
antioxidant response elements (AREs) in their upstream reg-
ulatory regions, which are the sites of binding Nrf2 as a het-
erodimer with a small Maf transcription factor.39
Nrf2-dependent genes encode multiple functionally
diverse enzymes and other proteins with cytoprotective
activities.29,30 Several studies have indicated that there is an
inverse relationship between the levels of Nrf2 expression
and the viral entry and replication, and an attractive thera-
peutic intervention demonstrated that supplementation with
Nrf2-activating antioxidants inhibits viral replication in
human NEC.29,40 Sulforaphane could increase the accumula-
tion of Nrf2; therefore, additional studies are needed to
explore the relation between the virus and standard sulfora-
phane or its extract from broccoli.21,40
Studies have reported that glucoraphanin is also taken up
from the gut to the liver where it is interconverted to its
reduced glucosinolate analog, glucoerucin, while sulfora-
phane is converted to its corresponding reduced isothiocya-
nate analog, erucin [1-isothiocyanato-4-(methylthio)
butane].41,42 Glucoerucin and glucoraphanin have poor bio-
availability; thus, the bioavailability found in humans or
other mammals is typically due to sulforaphane in a specic
test.43 These ndings suggested that sulforaphane at concen-
trations of 6.25 and 12.5 µM were good for living cells and
eective against inuenza A virus. Higher cytotoxicity was
observed at concentrations higher than 25.0 µM in both the
standard and extracted samples (Figure 1). Moreover, the
extract contained more toxic compounds from the extraction
process than the standard, which should be investigated in
future research.
We examined the inhibitory activity of the test compounds
against virus replication in MDCK cells using the inuenza
A/WSN/33 (H1N1 subtype) virus strain. The results are
shown in Figure 2, including OSV as a positive control. The
compounds showed signicant anti-inuenza A/WSN/33
virus activity at 25, 12.5, and 6.25 µM of the standard sul-
foraphane, and 6.25 µM of the sulforaphane from broccoli
seed extract (Figure 2) (P < 0.05).
The MDCK cells without the inuenza A virus (control)
indicated that DMSO and OSV showed no signicant cyto-
toxic activity on MDCK cells (Figure 2), whereas 6.25 µM
Figure 2. The cytotoxicity toward influenza A virus in Madin-
Darby canine kidney cells treated with different concentrations
of sulforaphane from the standard and extract samples. The
capital letters show significant differences at the level of 0.01
based on one-way ANOVA. Capital letters shown in red and
black represent parallel comparisons of black bars and red bars,
respectively

Natural Product Communications4
sulforaphane of the standard slightly reduced the activity of
MDCK cells maintaining 90.2% of initial cell numbers
(DMSO). Notably, 12.5 µM standard sulforaphane and 6.25
µM sulforaphane in the extract from broccoli seed showed
the same level of activity, maintaining 81.7% and 77.0% of
initial cell numbers, respectively. A concentration of 25.0
µM standard sulforaphane maintained 67.0% of initial cell
numbers, and both 100 µM standard sulforaphane and 12.5
µM sulforaphane in the extract from broccoli seed showed
the same level of activity (57.5% and 55.3%, respectively,
responding to initial cell numbers). This result suggested that
sulforaphane is eective against inuenza (EC50 > 12.5 µM).
The concentration of 50.0 µM sulforaphane in the extract of
broccoli seed was better than 50.0 µM sulforaphane standard
and 25.0 µM sulforaphane in the extract of broccoli seed
with regard to cytotoxic activity for MDCK cells. The 100.0
µM concentration of sulforaphane in the extract from broc-
coli seed showed strong cytotoxic activity toward MDCK
cells, maintaining only 6.5% of initial cell numbers.
The positive OSV control treatments showed that the
numbers of MDCK cells infected with inuenza A virus and
treated with the OSV positive control (Figure 2), 6.25 µM
sulforaphane standard and in the extract samples, and 12.5
µM sulforaphane standard were higher than the numbers of
MDCK cells treated with DMSO (negative control), suggest-
ing that sulforaphane at a concentration of 6.25 µM from
both standard and broccoli extract showed better bioactivity
against inuenza A virus in MDCK cells. Standard and
extract samples showed 19.2% and 17.8% increased cell
numbers, respectively, compared with DMSO-treated cells,
and the positive control increased cell numbers by 40.0%.
The same function against virus was also found at 12.5 and
6.25 µM sulforaphane, while 25 µM sulforaphane standard
had no signicant eect on MDCK cells infected with inu-
enza A virus compared with the negative control. Additionally,
100 µM of sulforaphane standard and 12.5 µM sulforaphane
in the extract from broccoli seed showed the same eect,
maintaining 84.0% and 81.4% of initial cells (DMSO),
respectively. Moreover, 50 µM sulforaphane standard or in
the extract samples maintained 65.6% and 63.9% of the ini-
tial cells (DMSO), respectively, and 25.0 µM sulforaphane
standard could maintain 55.0% of the initial cells, whereas
100 µM sulforaphane in the extract only maintained 7% of
the initial cells, suggesting strong cytotoxicity to MDCK
cells.
During the hydrolysis of glucoraphanin, more sulfora-
phane product is catalyzed by myrosinase at neutral or high
pH, in the presence of Zn2+ at low temperature. In contrast,
at acidic pH, in the presence of Fe2+ or Cu2+, and at high
temperature, nitrile will be favored.44,45 According to the
observed eects of the sulforaphane standard on MDCK
cells, we concluded that sulforaphane was the main compo-
nent, but there might be a small amount of nitrile or residual
product(s) from broccoli seeds in the extract, which could be
present in samples of a higher concentration, causing
toxicity to MDCK cells (Figure 2). Moreover, many studies
have demonstrated and validated that sulforaphane is readily
absorbed in humans or mammals and is rapidly eliminated,
and more than 70% of the administered dose of sulforaphane
can be recovered as thiol conjugates in the urine with a bio-
logical half-life of only a few hours, providing further evi-
dence of sulforaphane with no signicant toxicity to animals
and mammals.46 Additionally, according to the cytotoxicity
test activity of inuenza A virus treated with dierent con-
centrations of sulforaphane from the standard and extract
samples, except for the 100 µM concentration of sulfora-
phane derived from the extract with higher cytotoxicity to
MDCK cells, low concentrations of sulforaphane (6.25–50
µM) from the standard and the extract showed lower cyto-
toxicity to MDCK cells (C and C+V). Moreover, some addi-
tional chemical compounds in the sulforaphane extract
contributed to the cytotoxicity to MDCK cells.
This result suggested that the cytotoxicity test activity of
inuenza A virus treated with dierent concentrations of sul-
foraphane in MDCK cells demonstrated concentration
dependence, and the concentrations of 6.25 to 12.5 µM based
on the standard were eective for MDCK cell activity with
no obvious cytotoxicity. Additionally, the purication meth-
ods, such as preparative liquid chromatography and DEAE-
Sephacel, could be used for better purication of sulforaphane
before application in food engineering.44,45
HA represents an attractive target for discovering new
anti-inuenza agents because of its popular role in host cell
attachment and fusion processes.10 Recent reports reveal that
several compounds with small molecules can interfere with
the HA functions by hindering one or both functions. The
major function is binding to the receptor-binding site and
competing with sialic acids, such as triterpenoids,2,12 and
sialic acid mimetic peptides.8 Recently, arbidol has been
commercialized in China,4,47 and stachyin derivatives,
CL-61917, polyphenols, BMY 27709, and some others have
recently been explored.48
The Nrf2 signaling pathway can regulate N600 genes, of
which N200 encodes cytoprotective proteins that are also
associated with inammation, cancer, neurodegenerative
diseases, and other major diseases. Recently, Nrf2 expres-
sion was shown to modify inuenza A entry and replication
in nasal epithelial cells, which provided good cross evidence
for exploring the function of sulforaphane in anti-inuenza A
virus.21,29 Recently, several studies have focused on extract-
ing sulforaphane from broccoli seeds and sprouts to examine
its anti-cancer activity, showing that sulforaphane and sul-
foraphane broccoli extract have the same or similar functions
against cancers.49
Since the recognition of the bioactivity of sulforaphane in
1992,2,12 several studies have examined its action in cells,
animals, and humans. Additionally, increasing evidence has
shown that broccoli, particularly as seeds and young sprouts,
is a rich source of sulforaphane, and broccoli-based prepara-
tions are now used in clinical studies probing their ecacy in

Li et al. 5
health preservation and disease mitigation.23 The transcrip-
tion factor Nrf2 is a master regulator of cell survival
responses to endogenous and exogenous stressors.50 Studies
have revealed that many putative cellular targets are aected
by sulforaphane, although only KEAP1-Nrf2 signaling can
be considered a validated target at this time.21,29 Thus, we
propose that sulforaphane plays a role in preventing HA by
interfering in the Nrf2 signaling pathway, which provided
the premise for the present study. In addition, sulforaphane
has therapeutic eects on inammatory diseases through the
Nrf2 signaling pathway.51,52
Currently, sappanone, an anti-inuenza, antiallergic, and
neuroprotective medication, is widely distributed in
Southeast Asia.53 Bixin extracted from the seeds of Bixa
orellana is used to treat infectious and inammatory diseases
in Mexico and South America,54 and both sappanone and
bixin play medical roles via Nrf2-dependent mechanisms.
According to the above-mentioned studies and other recent
reports, sulforaphane has activities against cancer, inamma-
tion, cardiovascular disease, and neurological diseases and
improves immunity.27,37 Thus, sulforaphane might play an
important role as an anti-inuenza virus by increasing the
accumulation of Nrf2 factors and decreasing the replication
of the virus. These plant compounds activate the Nrf2 signal-
ing pathway mainly in the form of electrophilic materials
that modify the cysteine residues of KEAP1, leading to free
nuclear Nrf2 binding with the ARE, resulting in Nrf2 accu-
mulation and the activation of the transcription of the corre-
sponding genes.2,55,56
Experimental
Cells and Materials
MDCK cells were grown in Dulbecco’s modied Eagle
medium (DMEM) (Gibco BRL, Inc., Gaithersburg, MD,
USA) supplemented with 10% fetal bovine serum (FBS)
(PAA Laboratories, Linz, Austria) at 37°C under 5% CO2.
Inuenza A/WSN/33 (H1N1) virus, provided by HAKE
Genetics Co., Ltd., was used in the present study. “WSN” is
the acronym for the inuenza A/Wilson Smith/1933 (H1N1)
neurotropic variant, which was deliberately selected by
repeatedly passaging its parent virus, inuenza A/Wilson
Smith/1933 (H1N1) virus (WS), in mouse brain. The WS
virus was isolated in 1933 by Wilson Smith and colleagues
from human inuenza by inoculating ferrets.2 Broccoli
“B61” seeds were cultured at the Institute of Vegetables and
Flowers, Chinese Academy of Agricultural Sciences, and
subsequently collected for extracting the bioactive com-
pound sulforaphane.
Chemicals
Sulforaphane standard was purchased from LKT Labs (LKT
Laboratories, Inc., St Paul, MN, USA), and the purity was
more than 98% (HPLC grade). Methanol, ethyl acetate, and
DMSO were obtained from Sigma (Sigma Chemical
Company, St Louis, MO, USA). Phosphates were purchased
from Beijing Chemical Company (Beijing, China). The stan-
dard samples were dissolved in 10 mL of DMSO (Sigma
Chemical Company) with a concentration of 1.0 g/L and
then serially diluted to concentrations of 6.25, 12.5, 25, 50,
and 100 µM for CPE assays and cytotoxicity tests; another
concentration gradient was used to determine the linearity of
sulforaphane.
HPLC Conditions
The Shimadzu LC-20A HPLC system was equipped with an
SPD-20 UV detector and a reverse-phase C18 column (250 ×
4.6 mm, 5 µm, Shiseido, Japan). The gradient mobile phase
consisted of 5% tetrahydrofuran for pump A and 100% meth-
anol for pump B. The solvent for pump B was initially set at
40%, then linearly changed to 60% by the 10th minute, and
subsequently returned to full methanol (100%) after an addi-
tional 10 minutes, maintained at 100% for 15 minutes at a
ow rate of 0.80 mL/min, and nally returned to the initial
condition. The absorbance value was 254 nm, and the col-
umn oven temperature 32°C. A total of 10 mg of the sulfora-
phane standard was dissolved in 10 mL of methanol to
generate a dilution series: 5.0, 50.0, 100.0, 200.0, and 300.0
mg/L. The precision of the system was measured by standard
peak areas (n = 6, 100 mg/L), and the recovery was dened
by adding standard samples (100 mg/L) at known concentra-
tions (5.51, 12.55, 20.43, 45.51, 60.27, 80.69 mg/L) (n = 6).
The determination method was performed as previously
described,57 and this method is popularly used for the deter-
mination of sulforaphane in plants.32
Extraction of Sulforaphane
The extraction method was performed according to Liang,44
with some modications, as detailed in previous studies.32,33
A total of 0.5 g of seed powder was homogenized in 15 mL
of neutral phosphate buer (0.1 M and pH 7.0). The homog-
enate was then transferred to a beaker with stirring for 2
hours, after which 30 mL ethyl acetate was injected. Thirty
minutes later, the mixture was centrifuged for 10 minutes at
6000 × g (Thermo, MA, USA). The supernatant in the tubes
was collected, and the remaining mixture was transferred to
a fresh beaker. Then, 30 mL of ethyl acetate was added to the
mixture and treated by the initial process. The same opera-
tion was repeated again. Finally, the 3 supernatants were
evaporated in a rotavapor (RII, BÜCHI TM, Switzerland) at
35°C. The residue was then dissolved in 10 mL of methanol
and ltered through a 0.22 µm (D 13 mm) nylon lter paper
(Agela, China). The solution was stored at -20°C until HPLC
analysis.
CPE Reduction Assay
This assay was performed as previously described,2 with
some modications. MDCK cells were seeded onto 96-well

Natural Product Communications6
plates, incubated overnight, and infected with inuenza virus
(MOI = 0.1) suspended in DMEM supplemented with 1%
FBS, containing test compound and 2 mg/L TPCK-treated
trypsin, with a nal DMSO concentration of 1% in each
well. After 40 hours of incubation, CellTiter-Glo reagent
(Promega Corp., Madison, WI, USA) was added, and the
plates were read using a plate reader (Tecan Innite M2000
PRO; Tecan Group Ltd, Mannedorf, Switzerland). The CPE
in virus-infected cells was observed through microscopy.
MDCK cells were infected with inuenza A/WSN/33 (MOI
= 0.01) and treated with dierent concentrations of sulfora-
phane from the standard and extract samples diluted with
DMSO to 6.25, 12.5, 25, 50, and 100 µM. Each treatment
was designed in triplicate (n = 3). The postinfection superna-
tants were collected and used for infection in the next round
of investigation.3
Cytotoxicity Test
Cells grown on 96-well plates overnight were cultured in 1%
FBS with increasing amounts of the test compounds for 40
hours, with OSV as a positive control. Cytotoxicity was
assessed with the CellTiter-Glo assay as described above.
The cells were treated with dierent concentrations of sul-
foraphane from the standard and extract samples after 40
hours. The cytotoxicity was measured by the neutral red
uptake assay (Tecan Innite M2000 PRO; Tecan Group Ltd).
The cytotoxicity was computed by comparisons to normal
cells of wells containing compounds with wells containing
DMSO.
Data Analysis
All statistical analyses were performed by using SPSS 12.0.
The results are expressed as the means ± standard deviation
(SD) from experiments performed in triplicate. The statisti-
cal signicance between 2 groups was analyzed by Student’s
t test, and one-way ANOVA with Duncan multiple compari-
sons was used in the present study. A P value of <0.05 was
regarded as statistically signicant.
Declaration of Conflicting Interests
The author(s) declared no potential conicts of interest with respect
to the research, authorship, and/or publication of this article.
Funding
The author(s) disclosed receipt of the following nancial support
for the research, authorship, and/or publication of this article:
The present study was funded by grants from the National Key
Research and Development Program of China (2017YFD0101805),
the National Nature Science Foundation (31501761), the China
Agriculture Research System (CARS-23-A8), and the Science
and Technology Innovation Program of the Chinese Academy of
Agricultural Sciences (CAAS-ASTIP-IVFCAAS).
References
1. Reading PC, Hartley CA, Ezekowitz RA, Anders EM. A serum
mannose-binding lectin mediates complement-dependent lysis
of inuenza virus-infected cells. Biochem Biophys Res Com-
mun. 1995;217(3):1128-1136.
2. Yu M, Si L, Wang Y, et al. Discovery of pentacyclic triterpe-
noids as potential entry inhibitors of inuenza viruses. J Med
Chem. 2014;57(23):10058-10071.
3. Li R, Liu T, Liu M, Chen F, Liu S, Yang J. Anti-inuenza A
virus activity of dendrobine and its mechanism of action. J
Agric Food Chem. 2017;65(18):3665-3674.
4. Zhao K, Gu M, Zhong L, et al. Characterization of three H5N5
and one H5N8 highly pathogenic avian inuenza viruses in
China. Vet Microbiol. 2013;163(3-4):351-357.
5. Lee Y-J, Kang H-M, Lee E-K, et al. Novel Reassortant Inu-
enza A(H5N8) Viruses, South Korea, 2014. Emerg Infect Dis.
2014;20(6):1086-1089.
6. Poole DS, Yu S, Caì Y, et al. Inuenza A virus polymerase is a
site for adaptive changes during experimental evolution in bat
cells. J Virol. 2014;88(21):12572-12585.
7. Li Q, Zhou L, Zhou M, et al. Epidemiology of human infec-
tions with avian inuenza A(H7N9) virus in China. N Engl J
Med. 2014;370(6):520-532.
8. Tong S, Zhu X, Li Y, et al. New World bats harbor diverse
inuenza A viruses. PLoS Pathog. 2013;9(10):e1003657.
9. Chen Y, Shang S, Tang Y, Zhang C, Tong M, Dai Y. Clinical
features of severe inuenza A (H1N1) virus infection. Indian J
Pediatr. 2013;80(2):97-101.
10. Hartshorn KL, Liou LS, White MR, Kazhdan MM, Tauber JL,
Tauber AI. Neutrophil deactivation by inuenza-a virus. Role
of hemagglutinin binding to specic sialic acid-bearing cellular
proteins. J Immunol. 1995;154(8):3952-3960.
11. Rudneva I, Ignatieva A, Timofeeva T, et al. Escape mutants
of pandemic inuenza A/H1N1 2009 virus: variations in anti-
genic specicity and receptor anity of the hemagglutinin.
Virus Res. 2012;166(1-2):61-67.
12. Tang R-B, Chen H-L. An overview of the recent outbreaks of
the avian-origin inuenza A (H7N9) virus in the human. J Chin
Med Assoc. 2013;76(5):245-248.
13. Lafond KE, Tam JS, Bresee JS, Widdowson MA. International
Meeting on inuenza vaccine eectiveness, 3-4 December
2012, Geneva, Switzerland. Vaccine. 2014;32:6591-6595.
14. Kim CU, Lew W, Williams MA, et al. Inuenza neuraminidase
inhibitors possessing a novel hydrophobic interaction in the
enzyme active site: design, synthesis, and structural analysis
of carbocyclic sialic acid analogues with potent anti-inuenza
activity. J Am Chem Soc. 1997;119(4):681-690.
15. Amarelle L, Lecuona E, Sznajder JI. Anti-inuenza treatment:
drugs currently used and under development. Arch Bronconeu-
mol. 2017;53(1):19-26.
16. Ares AM, Bernal J, Martín MT, Bernal JL, Nozal MJ. Opti-
mized formation, extraction, and determination of sulfora-
phane in broccoli by liquid chromatography with diode array
detection. Food Anal Methods. 2014;7(3):730-740.

Li et al. 7
17. Baenas N, Silván JM, Medina S, de Pascual-Teresa S, García-
Viguera C, Moreno DA. Metabolism and antiproliferative
eects of sulforaphane and broccoli sprouts in human intes-
tinal (Caco-2) and hepatic (HepG2) cells. Phytochem Rev.
2015;14(6):1035-1044.
18. Bricker GV, Riedl KM, Ralston RA, Tober KL, Oberyszyn
TM, Schwartz SJ. Isothiocyanate metabolism, distribution, and
interconversion in mice following consumption of thermally
processed broccoli sprouts or puried sulforaphane. Mol Nutr
Food Res. 2014;58(10):1991-2000.
19. Fahey JW, Haristoy X, Dolan PM, et al. Sulforaphane
inhibits extracellular, intracellular, and antibiotic-resist-
ant strains of Helicobacter pylori and prevents benzo[a]
pyrene-induced stomach tumors. Proc Natl Acad Sci U S A.
2002;99(11):7610-7615.
20. Abbas A, Hall JA, Patterson WL, et al. Sulforaphane modu-
lates telomerase activity via epigenetic regulation in prostate
cancer cell lines. Biochem Cell Biol. 2016;94(1):71-81.
21. Alumkal JJ, Slottke R, Schwartzman J, et al. A phase II study
of sulforaphane-rich broccoli sprout extracts in men with recur-
rent prostate cancer. Invest New Drugs. 2015;33(2):480-489.
22. Abdull Razis AF, Iori R, Ioannides C. The natural chemopreven-
tive phytochemical R-sulforaphane is a far more potent inducer
of the carcinogen-detoxifying enzyme systems in rat liver and
lung than the S-isomer. Int J Cancer. 2011;128(12):2775-2782.
23. Perocco P, Bronzetti G, Canistro D, et al. Glucoraphanin, the
bioprecursor of the widely extolled chemopreventive agent
sulforaphane found in broccoli, induces phase-I xenobiotic
metabolizing enzymes and increases free radical generation in
rat liver. Mutat Res. 2006;595(1-2):125-136.
24. Chung FL, Conaway CC, Rao CV, Reddy BS. Chemopre-
vention of colonic aberrant crypt foci in Fischer rats by sul-
foraphane and phenethyl isothiocyanate. Carcinogenesis.
2000;21(12):2287-2291.
25. Agyeman AS, Chaerkady R, Shaw PG, et al. Transcrip-
tomic and proteomic proling of Keap1 disrupted and sul-
foraphane-treated human breast epithelial cells reveals
common expression proles. Breast Cancer Res Treat.
2012;132(1):175-187.
26. Shan Y, Wu K, Wang W, et al. Sulforaphane down-regulates
COX-2 expression by activating p38 and inhibiting NF-kap-
paB-DNA-binding activity in human bladder T24 cells. Int J
Oncol. 2009;34(4):1129-1134.
27. Stewart KE, Jeery EH. Transcriptional regulation of phase II
detoxication enzymes by the glucosinolate metabolites sulfo-
raphane and crambene. Faseb J. 2003;17:A1164-A1165.
28. Munday R, Munday CM. Induction of phase II detoxication
enzymes in rats by plant-derived isothiocyanates: comparison
of allyl isothiocyanate with sulforaphane and related com-
pounds. J Agric Food Chem. 2004;52(7):1867-1871.
29. Aleri A, Srivastava S, Siow RCM, et al. Sulforaphane pre-
conditioning of the Nrf2/HO-1 defense pathway protects the
cerebral vasculature against blood-brain barrier disruption
and neurological decits in stroke. Free Radic Biol Med.
2013;65:1012-1022.
30. Kensler TW, Egner PA, Agyeman AS, et al. Keap1-Nrf2 sign-
aling: a target for cancer prevention by sulforaphane. Top Curr
Chem. 2013;329:163-177.
31. Abel EL, Boulware S, Fields T, et al. Sulforaphane induces
phase II detoxication enzymes in mouse skin and prevents
mutagenesis induced by a mustard gas analog. Toxicol Appl
Pharmacol. 2013;266(3):439-442.
32. ZS L, Liu YM, Fang ZY, et al. Development and identication
of anti-cancer component of sulforaphane in developmental
stages of broccoli (Brassica oleracea var. italica). J Food Nutr
Res. 2016;4:490-497.
33. Liang H, Yuan QP, Dong HR, Liu YM. Determination of sul-
foraphane in broccoli and cabbage by high-performance liquid
chromatography. J Food Compost Anal. 2006;19(5):473-476.
34. ZS L, Liu YM, Fang ZY, et al. Variation of sulforaphane lev-
els in broccoli (Brassica oleracea var. italica) during ower
development and the role of gene AOP2. J Liq Chromatogr
Relat Technol. 2014;37:1199-1211.
35. Delonga K, Smit Z, Dragovic-Uzelac V, Mrkic V, Vorka-
pic-Furac J. Hydrolysis products of glucosinolates from white
cabbage (Brassica oleracea L. var capitata) and cauliower
(Brassica oleracea L. var. botrytis) analyzed by HPLC and
GC/MS. Biol Act Phytochem Food. 2001;269:213-216.
36. Moscona A. Global transmission of oseltamivir-resistant inu-
enza. N Engl J Med. 2009;360(10):953-956.
37. Shawky NM, Segar L. Sulforaphane inhibits platelet-derived
growth factor-induced vascular smooth muscle cell prolifera-
tion by targeting mTOR/p70S6 kinase signaling independent
of Nrf2 activation. Pharmacol Res. 2017;119:251-264.
38. Bai Y, Zheng Y, Chen Q, et al. Prevention by sulforaphane of
diabetic cardiomyopathy is associated with Nrf2 up-regulation
and activation. Diabetes. 2012;61:A120-A120.
39. Higgins LG, Kelleher MO, Eggleston IM, Itoh K, Yamamoto
M, Hayes JD. Transcription factor Nrf2 mediates an adap-
tive response to sulforaphane that protects broblasts in
vitro against the cytotoxic eects of electrophiles, perox-
ides and redox-cycling agents. Toxicol Appl Pharmacol.
2009;237(3):267-280.
40. Kim H-J, Barajas B, Wang M, Nel AE. Nrf2 activation by
sulforaphane restores the age-related decrease of T(H)1
immunity: role of dendritic cells. J Allergy Clin Immunol.
2008;121(5):1255-1261.
41. Harris KE, Jeery EH. Sulforaphane and erucin increase the
expression of multidrug resistance protein 1 in A549 human
lung carcinoma cells. J Nutr. 2007;137:289s-289s.
42. Cramer JM, Jeery EH. A comparison of the bioavailability of
sulforaphane from broccoli sprouts and a semi-puried broc-
coli powder rich in glucoraphanin in healthy human males.
Faseb J. 2009;23(1):LB1-1039.5.
43. Harris KE, Jeery EH. Sulforaphane and erucin increase
MRP1 and MRP2 in human carcinoma cell lines. J Nutr Bio-
chem. 2008;19(4):246-254.
44. Liang H, Yuan Q, Xiao Q. Eects of metal ions on myrosinase
activity and the formation of sulforaphane in broccoli seed. J
Mol Catal B Enzym. 2006;43(1-4):19-22.

Natural Product Communications8
45. Sarvan I, Kramer E, Bouwmeester H, Dekker M, Verkerk R.
Sulforaphane formation and bioaccessibility are more aected
by steaming time than meal composition during in vitro diges-
tion of broccoli. Food Chem. 2017;214:580-586.
46. Fahey JW, Wade KL, Wehage SL, et al. Stabilized sulfora-
phane for clinical use: phytochemical delivery eciency. Mol
Nutr Food Res. 2017;61(4):1600766.
47. Boriskin YS, Leneva IA, Pécheur E-I, Polyak SJ. Arbidol: a
broad-spectrum antiviral compound that blocks viral fusion.
Curr Med Chem. 2008;15(10):997-1005.
48. Leneva IA, Russell RJ, Boriskin YS, Hay AJ. Characteristics
of arbidol-resistant mutants of inuenza virus: implications for
the mechanism of anti-inuenza action of arbidol. Antiviral
Res. 2009;81(2):132-140.
49. CF L, Liang H, Yuan QP, Hou XD. Optimization of sulfora-
phane separation from broccoli seeds by macroporous resins.
Separation Sci Technol. 2008;43:609-623.
50. Lan A, Li W, Liu Y, et al. Chemoprevention of oxida-
tive stress-associated oral carcinogenesis by sulforaphane
depends on Nrf2 and the isothiocyanate moiety. Oncotarget.
2016;7(33):53502-53514.
51. Boyanapalli SSS, Paredes-Gonzalez X, Fuentes F, et al.
Nrf2 knockout attenuates the anti-inammatory eects of
phenethyl isothiocyanate and curcumin. Chem Res Toxicol.
2014;27(12):2036-2043.
52. Li C-zhen, Jin H-hong, Sun H-xin, CZ L, Jin HH, et al. Eri-
odictyol attenuates cisplatin-induced kidney injury by inhib-
iting oxidative stress and inammation. Eur J Pharmacol.
2016;772:124-130.
53. Lee S, Choi S-Y, Choo Y-Y, et al. Sappanone A exhibits anti-in-
ammatory eects via modulation of Nrf2 and NF-κB. Int
Immunopharmacol. 2015;28(1):328-336.
54. Tao S, Rojo de la Vega M, Quijada H, et al. Bixin protects mice
against ventilation-induced lung injury in an Nrf2-dependent
manner. Sci Rep. 2016;6(1):18760.
55. Agamennone M, Pietrantoni A, Superti F. Identication of
small molecules acting against H1N1 inuenza A virus. Virol-
ogy. 2016;488:249-258.
56. Dinkova-Kostova AT, Fahey JW, Kostov RV, Kensler TW.
Keap1 and done? Targeting the Nrf2 pathway with sulfora-
phane. Trends Food Sci Technol. 2017;69(Pt B):257-269.
57. Mahn A, Martin C, Reyes A, Saavedra A. Evolution of sulfora-
phane content in sulforaphane-enriched broccoli during TraY
drying. J Food Eng. 2016;186:27-33.