Antiviral activity of Isatidis Radix derived glucosinolate isomers and their breakdown products against influenza A in vitro/ovo and mechanism of action

Article abstract of DOI:10.1016/j.jep.2020.112550

Ethnopharmacological relevance: Isatidis Radix, the sun-dried roots of Isatis indigotica Fortune ex Lindl., is one of the most usually used traditional Chinese medicines. For centuries, the herb has been employed in clinical practice for treatment of virus infection and inflammation. However, its active ingredients remain unclear. Aim of the study: In the present study, the anti-influenza virus activity of epiprogoitrin, progoitrin, epigoitrin and goitrin, the Isatidis Radix derived glucosinolate isomers and their breakdown products, was firstly evaluated in vitro and in ovo and their mechanism of action was investigated. Materials and methods: Epiprogoitrin, progoitrin, epigoitrin and goitrin were isolated from Isatidis Radix by chiral separation. In vitro and in ovo evaluations were performed on Madin-Darby canine kidney (MDCK) cells and embryonated eggs respectively, both using protocols including prevention, treatment and virus neutralization. Hemagglutination (HA) and neuraminidase (NA) inhibition assays were performed for further understanding of the antiviral mechanism. Results: Isatidis Radix derived glucosinolate isomers and their breakdown products all exhibited dose-dependent inhibition effect against influenza A virus (H1N1) without toxicity. The antiviral potency of the components was in the order of progoitrin > goitrin > epigoitrin > epiprogoitrin. The attachment of the constituents to the viral envelope conduced to the mechanism of their antiviral action without disturbing viral adsorption or budding. Conclusion: Taken together, these results are promising for further development of Isatidis Radix and may contribute an adjunct to pharmacotherapy for influenza virus infection.

Full text of DOI:10.1016/j.jep.2020.112550

Journal Pre-proof
Antiviral activity of Isatidis Radix derived glucosinolate isomers and their breakdown
products against influenza A in vitro/ovo and mechanism of action
Li-xing Nie, Yan-lin Wu, Zhong Dai, Shuang-cheng Ma
PII:
S0378-8741(19)33356-2
https://doi.org/10.1016/j.jep.2020.112550
JEP 112550
DOI:
Reference:
To appear in: Journal of Ethnopharmacology
Received Date: 27 August 2019
Revised Date: 24 October 2019
Accepted Date: 4 January 2020
Please cite this article as: Nie, L.-x., Wu, Y.-l., Dai, Z., Ma, S.-c., Antiviral activity of Isatidis Radix
derived glucosinolate isomers and their breakdown products against influenza A in vitro/ovo
and mechanism of action, Journal of Ethnopharmacology (2020), doi: https://doi.org/10.1016/
j.jep.2020.112550.
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Antiviral activity of Isatidis Radix derived glucosinolate isomers and their
breakdown products against influenza A in vitro/ovo and mechanism of action
a,b
a,**
a
a,b *
Li-xing Nie , Yan-lin Wu , Zhong Dai , Shuang-cheng Ma
a
Chinese Academy of Medical Science & Peking Union Medical College, Beijing, PR
China
b
National Institutes for Food and Drug Control, National Medical Products
Administration, Beijing, PR China
*
Corresponding author. National Institutes for Food and Drug Control, No.2,
Tiantan Xili, Dongcheng District, Beijing, 10050, PR China. Tel: +86 1067095272.
Fax:+86 1067023650.
** Corresponding author. National Institutes for Food and Drug Control, No.2,
Tiantan Xili, Dongcheng District, Beijing, 10050, PR China. Tel: +86 1067095282.
Fax:+86 1067023650.
E-mail addresses: nielixing@163.com (L.-X. Nie), wyljoe@126.com (Y.-L. Wu),
daizhong88@sina.com (Z. Dai), masc@nifdc.org.cn (S.-C. Ma)

Abbreviations
AAF
amnio-allantoic fluid
CCK8
CPE
Cell Counting Kit-8
cytopathic effect
CRBCs
DMEM
DMSO
EID50
FBS
chicken red blood cells
Dulbecco's Modified Eagle Medium
dimethyl sulfoxide
50% egg infective dose
fetal bovine serum
GAP
good agricultural practice
hemagglutination
HA
HAI
hemagglutination inhibition
high performance liquid chromatography
50% inhibition concentration
Madin-Darby canine kidney
mass spectroscopy
HPLC
IC50
MDCK
MS
NA
neuraminidase
NMR
NPLC
PBS
nuclear magnetic resonance
normal phase liquid chromatography
phosphate buffered saline
ribonucleic acid
RNA
SARS
SFC
severe acute respiratory syndrome
supercritical fluid chromatography
specific pathogen free
SPF
TCID50
TPCK
50% tissue culture infective dose
N-p-Tosyl-L-phenylalanine chloromethyl ketone

Abstract:
Ethnopharmacological relevance: Isatidis Radix, the sun-dried roots of Isatis
indigotica Fortune ex Lindl., is one of the most usually used traditional Chinese
medicines. For centuries, the herb has been employed in clinical practice for treatment
of virus infection and inflammation. However, its active ingredients remain unclear.
Aim of the study: In the present study, the anti-influenza virus activity of epiprogoitrin,
progoitrin, epigoitrin and goitrin, the Isatidis Radix derived glucosinolate isomers and
their breakdown products, was firstly evaluated in vitro and in ovo and their
mechanism of action was investigated.
Materials and Methods: Epiprogoitrin, progoitrin, epigoitrin and goitrin were isolated
from Isatidis Radix by chiral separation. In vitro and in ovo evaluations were
performed on Madin-Darby canine kidney (MDCK) cells and embryonated eggs
respectively, both using protocols including prevention, treatment and virus
neutralization. Hemagglutination (HA) and neuraminidase (NA) inhibition assays
were performed for further understanding of the antiviral mechanism.
Results: Isatidis Radix derived glucosinolate isomers and their breakdown products all
exhibited dose-dependent inhibition effect against influenza A virus (H1N1) without
toxicity. The antiviral potency of the components was in the order of progoitrin >
goitrin > epigoitrin > epiprogoitrin. The attachment of the constituents to the viral
envelope conduced to the mechanism of their antiviral action without disturbing viral
adsorption or budding.
Conclusion: Taken together, these results are promising for further development of
Isatidis Radix and may contribute an adjunct to pharmacotherapy for influenza virus
infection.
Keywords: Influenza; Antivirals; Glucosinolate; Isomer; Isatidis Radix

1
. Introduction
Influenza viruses are enveloped RNA viruses which can cause morbidity or even
mortality in humans by seasonal or pandemic infections. Annually, influenza
epidemics lead to 200-500,000 fatal cases globally (Courtin et al., 2017). Currently,
therapeutic strategies to lower the mortality rates mainly rely on vaccines and
chemical drugs. But usage of vaccines has been often challenged by frequent mutation
of the antigen and narrow spectrum (Tao et al., 2017; Zarubaev et al., 2015). On the
other side, viral resistance and adverse effects restrict the clinic application of
currently available anti-influenza virus drugs including neuraminidase inhibitors, M2
channel blockers and RNA synthesis inhibitors (Hayden and De Jong, 2011; Samson
et al., 2013; Correia et al., 2015). Hence, alternative drugs with low toxicity, broad
spectrum and less resistance remain to be developed.
Ethnic medicines are valuable heritage and commercially important resources in
the prevention and treatment of viral infectious diseases, a growing number of which
are accumulating evidence of experimental and/or clinical efficacy (Li and Peng, 2017;
Li et al., 2019). Among them, Isatidis Radix (“Ban-Lan-Gen” in Chinese, the
sun-dried root of Isatis indigotica Fortune ex Lindl.) is well-known for its antiviral
activity. In ethnic medical practice, Isatidis Radix has been prescribed for treating
fever, pestilence, sore throat, seasonal toxin, papule caused by warm toxin, macula,
scarlatina, mumps, swelling abscess, erysipelas and erysipelas facialis (Chinese
Pharmacopoeia Commission, 2015). As officially approved medicinal material and
products in China, Isatidis Radix and its formulations have been used as the major
herbal remedies in prevention and treatment against a wide range of viral infections,
such as seasonal epidemics, severe acute respiratory syndrome (SARS) in 2003 and
H1N1 flu outbreaks in 2009 (Lin et al., 2005; Deng et al., 2013). Regardless of the

various subtypes of viruses and their rapid mutations, Isatidis Radix was found to be
effective in clinic against infections caused by influenza viruses with different strains
and subtypes (Ke et al., 2012). Notwithstanding the anti- influenza efficacy of Isatidis
Radix and its preparations is well acknowledged, their active constituents and
pharmacological actions have not been elucidated. So far, a few antiviral components
have been identified in Isatidis Radix, including (3H)-quinazolinone (Li et al., 1993),
glycoprotein (Yamada, 1999), epigoitrin, 2,4(1H,3H)-quinazolinedione (Xu et al.,
2
005), indirubin (Mak et al., 2004), fructopyrano-(1→4)-glucopyranose (Zhang et
al., 2013), clemastanin (Yang et al., 2013),
lariciresinol-4-O-β-D-glucopyranoside (Li et al., 2015), polysaccharides (Li et al.,
017), isatithioetherin B and isatithioetherin D (Guo et al., 2018). Among these, the
B
2
antiviral monomer epigoitrin has received much attention and it was chosen as the
marker of antiviral efficacy in Isatidis Radix when drafting the 2010 edition of the
Chinese Pharmacopoeia.
In previous study of the authors, the so-called ‘epigoitrin’ used in former reports
(Xu et al., 2005; Ye et al., 2011; Zhang et al., 2013 ) was found to be mixtures of
unequal amounts of epigoitrin ((R)–goitrin, (R)-5-vinyloxazolidine-2-thione) and
goitrin ( (S)–goitrin, (S)-5-vinyloxazolidine-2-thione) in fact, and the isomers could
not be separated by regular C column. Consequently, the assay marker for Isatidis
1
8
Radix was changed to (R, S)-goitrin in 2010 and 2015 editions of the Chinese
Pharmacopoeia. In addition, (R, S)-goitrin was first resolved by the authors (Nie et al.,
2
010) using normal phase liquid chromatography (NPLC). Later, novel supercritical
fluid chromatography (SFC) and high performance liquid chromatography (HPLC)
methods were developed for high-throughput chiral separation and stereospecific assay,
respectively (Nie et al., 2016, 2017). On the other hand, epigoitrin and goitrin were

verified as breakdown products of their glucosinolate prototypes, epiprogoitrin
(
(R)-hydroxy-3-butenylglucosinolate)
(S)-hydroxy-3-butenylglucosinolate), on the effect of myrosinase (Fig. 1) (Nie et al.,
011; Xie et al., 2011). As well as being isomers, pure enantiomeric forms of
and
progoitrin
(
2
epiprogoitrin and progoitrin were extracted and resolved by the authors from Isatidis
Radix. In spite of the chemical structures and the biotransformation of the
glucosinolates and their breakdown products are illuminated (Xie et al., 2011), the
antiviral activity of the monomers and their mode of action remain elusive.
Fig. 1. Breakdown pathway of epiprogoitrin and progoitrin to epigoitrin and goitrin,
respectively.
The current study aims to go further into the comparative investigation of the
anti-influenza activity of the individual glucosinolates and their breakdown products in

vitro and in ovo. Moreover, mechanistic studies were conducted to find their antiviral
target and mode of action.
2
. Materials and methods
2
.1. Plant material and preparation of epiprogoitrin, progoitrin, epigoitrin and
goitrin
Isatidis Radix (“Ban-Lan-Gen” in Chinese and “Isatidis root” in English) is the
sun-dried root of Isatis indigotica Fortune ex Lindl. The scientific name of the plant
has been checked with http://www.theplantlist.org on 26, August, 2019. The plant
material, grown in good agricultural practice (GAP) base in Yutian, He-Bei Province,
was obtained from Beijing Tongrentang Co., Ltd. The origin was authenticated by
Associate Professor Shuai Kang, Curator of the Traditional Chinese Medicine
Herbarium of National Institutes for Food and Drug Control, using macroscopic and
microscopic analysis according to the Chinese pharmacopoeia (Chinese
Pharmacopoeia Commission, 2015). For future reference, its specimen (No. B-1) was
deposited at the Traditional Chinese Medicine Herbarium, National Institutes of Food
and Drug Control (Beijing, PR China). Epiprogoitrin and progoitrin were extracted
and isolated form Isatidis Radix based on a similar strategy from a previous report
(Xie et al., 2011). First, the pulverized Isatidis Radix was extracted with methanol;
then the extraction was deproteinized with ammonium acetate and purified with
granular activated carbon to obtain the crude extracts of glucosinolates; and, finally,
epiprogoitrin and progoitrin were separated from the crude extracts by preparative
ion-pair chromatography. Epigoitrin and goitrin were separated from (R, S)-goitrin
reference standard (National Institutes for Food and Drug Control, Lot No.
111753-201706) by SFC chiral resolution on a Chiralpak IC column with acetonitrile
and carbon dioxide as the mobile phase (Nie et al., 2016). The structures of the

1
13
purified compound were confirmed by H and C NMR (Bruker DRX-500), MS
(Waters SYNAPT G2) and optical rotation (Rudolph Autopol IV). The purities of the
prepared compounds were all determined to be > 98% by HPLC (Xie et al., 2011) and
chiral SFC (Nie et al., 2016) methods. Contents of epiprogoitrin, progoitrin, epigoitrin
and goitrin in the plant material were determined to be 0.19%, 0.10%, 0.10% and 0.05%
respectively by HPLC methods (Nie et al., 2017; Xie et al., 2011). Epiprogoitrin,
progoitrin, epigoitrin and goitrin were dissolved in 1 mL of dimethyl sulfoxide
(DMSO) and diluted with sodium chloride injection to get test solutions at
concentrations. The solutions were kept at 4 °C in a sterilized container.
2
.2. Virus, cell and animal
Influenza virus strain A/California/7/2009 (H1N1) NYMC X-179A was from
Shanghai Institute of Biological Products Co., Ltd. Continuous Madin-Darby canine
kidney (MDCK) cells were maintained in fetal bovine serum (FBS) free Dulbecco's
Modified Eagle Medium (DMEM, GIBCO) with TPCK-treated trypsin (8 µg/mL).
Ten-day-old specific pathogen free (SPF) embryonated chicken eggs were from
Beijing Merial Laboratory Animal Technology Co., Ltd. The clean eggs were
fertilized and incubated at 37.2 ± 0.1 °C and 60 ± 5% relative humidity and candled
every day to eliminate infertile eggs and dead embryos.
2
.3. Reagents
Peramivir (Guangzhou Nanxin Pharmaceutical Co., Ltd.) was used as the
reference compound. The reagents used in the study were as follows: sodium chloride
injection (Shijiazhuang Siyao Co., Ltd.), phosphate buffered saline (PBS, Hyclone),
dimethyl sulfoxide (DMSO, Sigma), Alsever's solution (pH 6.1) (an isotonic solution
of 2.05% glucose, 0.8% trisodium citrate, 0.42% sodium chloride and 0.055% citric
acid in double-distilled water. 0.6% suspension of Leghorn chicken red blood cells

(CRBCs) was prepared with equal volume of chicken (male) blood and Alsever's
solution (pH 6.1). Then the mixture was washed with PBS for 3 times. The
supernatant was discarded and an appropriate amount of blood erythrocytes was
diluted with PBS to prepare 0.6% erythrocyte suspension. Cell Counting Kit-8 (CCK8)
was from Dojindo. Neuraminidase Inhibitors Screen Kit was from Beyotime Institute
of Biotechnology Co., Ltd., which contains 1 mL of neuraminidase, 10 mL of buffer,
1
mL of fluorescent substrate and 1.2 mL of water. Ammonium acetate, granular
activated carbon, acetonitrile, glucose, sodium chloride, trisodium citrate and citric
acid were from Sinopharm Chemical Reagent Co., Ltd. Carbon dioxide was from
Chengweifeng Inc. De-ionized water was purified by a Milli-Q system (Millipore,
Bedford, MA, USA).
2
.3. Determination of cytotoxicity
Cytotoxic effect of epiprogoitrin, progoitrin, epigoitrin and goitrin was examined
by Cell Counting Kit-8 (CCK8) method. MDCK cells were cultured in a 96-well plate
3
(
3 × 10 cells/well) for 24 h. The culture supernatant was replaced with solutions
-3
-7
containing each of the four components at various concentrations (10 -10 mol/L).
Six duplicates were used for each concentration. After incubation for 72 h, the culture
supernatant was discarded, and 190 µL of DMEM and 10 µL of CCK8 were added to
each well. The plates were incubated at 37 °C for 3 h. The fluorescence intensity was
recorded by a microplate spectrophotometer (Gemini EM, Molecular Device, USA) at
4
50 nm. The cell survival rate was calculated, representing the cell viability.
.4. Cytopathic effect (CPE) inhibition assay
2
MDCK cells were inoculated with influenza virus A/California/7/2009 (H1N1).
The 50% tissue culture infective dose (TCID ) determined according to the
5
0
-5
Reed-Muench method (Reed, 1938) was 10 . Assay of antiviral activity of

epiprogoitrin, progoitrin, epigoitrin and goitrin in vitro were performed using the CPE
4
and CCK8 methods. MDCK cells were grown in a 96-well culture plate (1 × 10
cells/well) for 24 h. To identify the probably affected viral life cycle, cells were
treated with 100 µL of virus at 100 TCID and 100 µL of sample solutions at various
5
0
-3
-7
concentrations (10 mol/L-10 mol/L) using three different protocols including
prevention, treatment and virus neutralization. For protocol 1 (prevention), cells were
pre-incubated at 37 °C with sample solutions for 2 h before viral adsorption. Then the
cells were washed with PBS and inoculated with virus for 2 h without the compounds
and further grown for 72 h. For protocol 2 (treatment), cells were first inoculated at
3
7 °C with the virus for 2 h, then washed with PBS and cultured with the compounds
for 72 h. For protocol 3 (virus neutralization), sample solutions were mixed with the
virus and incubated at room temperature for 30 min, and the mixtures were added to
cells and cultured for 72 h. After 3 days’ incubation, the solution was discarded and
the cells were washed with PBS, then 190 µL of DMEM and 10 µL of CCK8 were
added to each well. The plates were incubated in the dark for 3 h at 37 °C. The
absorbance was read at 450/630 nm by a microplate spectrophotometer (Gemini EM,
Molecular Device, USA). Six duplicates were used for each dilution ratio. Cell
controls with/without the sample solutions and virus controls were included. The
inhibition ratio was calculated using the following formula: Inhibition activity (%) =
(ODsample – ODvirus)/(ODcellular control – ODvirus) × 100%, where ODsample is
the optical density of the tested sample at a certain concentration, ODvirus is the
optical density of the influenza virus control, and ODcellular control is the optical
density of normal cells. The 50% inhibition concentration (IC ) was determined by
5
0
linear extrapolation of the results from serious doses tested.
.5. Assessment of antiviral potency in embryonated eggs
2

2
.5.1. Virus titration
The 10 day embryonated eggs were inoculated with influenza virus strain
A/California/7/2009 (H1N1). The 50% egg infective dose (EID ) calculated using the
5
0
-9
Reed-Muench method (Reed, 1938) was 10 . Likewise, the in ovo evaluation was
performed using three different protocols. First, the eggs were pre-treated with 0.2 mL
of sample solution and incubated at 35 °C for 2 h before administration of 0.1 mL of
virus (50 EID ). Second, the eggs were inoculated with 0.1 mL of virus (50 EID ) at
5
0
50
3
5 °C for 2 h before administration of 0.2 mL of sample solution. Third, 0.1 mL of
virus (50 EID ) and 0.2 mL of sample solution were incubated for 30 minutes at
5
0
room temperature. Then the mixture was administered to the eggs and incubated at
5 °C for 2 h. Before harvesting, all the eggs were stored at 35 °C for 50 h to facilitate
3
the harvest procedure. The eggs were decapped and the allantoic membrane was
carefully moved to sides to pipette out the upper allantoic fluid. After harvesting, the
allantoic fluid was subjected to hemagglutination (HA) test.
2
.5.2. Hemagglutination (HA) test
A U-bottom 96 well microtiter plate was labeled horizontally 1:2 to 1:128
dilutions. Using a multi-channel pipette, 50 µL of PBS was added to all the wells. 50
µL of allantoic fluid was added to the first well of each row and only PBS is added to
the last row. Two-fold dilutions were made by transferring 50 µL from the first well of
each column. This was done until the last column and the remaining 50 µL was
discarded. 50 µL of 0.6% Leghorn chicken red blood cells (CRBCs) were added to all
the wells and were mixed by manual agitation to the plates thoroughly. The plates
were incubated for 60 min at room temperature. If the CRBCs formed a button or a
ring at the bottom of the wells, it was recorded as negative using a “-” symbol. If
hemagglutination occurred, CRBCs were agglutinated and immobilized, it was

considered positive and recorded using a “+” symbol. The highest dilution of virus
that caused complete hemagglutination was considered as the end-point in HA
titration. The results were recorded according to the HA effect of the well at 1:16
dilution. Eggs that still alive were scored as survival and eggs showing no HA activity
were scored as negative.
2
.5.3. Experiment design
Epiprogoitrin, progoitrin, epigoitrin and goitrin were assessed at various
concentrations at four dose levels (0.625, 1.25, 2.5 and 5 mg/mL) in different
experiments with virus concentration of 50 EID . 3 mg/mL of peramivir was used as
5
0
the positive control and sodium chloride injection was used as the negative control.
Equal dose of virus and DMSO were set as virus control and dilute control,
respectively. Sham-operated group was performed as well. 8 eggs were inoculated in
each group.
2
.6. Hemagglutination inhibition (HAI) assay
To elucidate the mode of their antiviral action, the inhibition effect of
epiprogoitrin, progoitrin, epigoitrin and goitrin on two glycoproteins of the virus were
evaluated. First, HAI assay was conducted to investigate the inhibition effect of the
components on hemagglutinin.
For hemagglutination (HA) titration, six embryonated chicken eggs were
inoculated with 0.1 mL of influenza virus strain A/California/7/2009 (H1N1) (50
EID ) and incubated at 35 °C for 4 days. The amnio-allantoic fluid (AAF) was
5
0
0
11
harvested from the live embryos and diluted with PBS from 2 to 2 for virus
replication by the HA assay. Each diluted viral solution was mixed with an equal
volume (25 µL) of 0.6% suspension of Leghorn chicken red blood cells (CRBCs) and
incubated at 4 °C. 25 µL of undiluted virus and PBS were used as the positive and the

negative control, respectively. After 60 min, hemagglutination titer was determined.
The highest dilution to cause complete agglutination in the HA assay was 128, which
was labeled as the hemagglutination titer of the virus.
For the hemagglutination inhibition (HAI) assay, 4 times of the hemagglutination
titer (1:32) was used as the standardized hemagglutinin viral solution. 1:1 to 1:128
dilutions of the 5 mg/mL stock solutions of epiprogoitrin, progoitrin, epigoitrin and
goitrin were prepared with PBS, respectively. Equal volumes (25 µL) of the
standardized hemagglutinin viral solution and the sample solutions were mixed gently
and incubated at room temperature for 30 min. 25 µL of the standardized
hemagglutinin viral solution and PBS were used as the positive and the negative
control, respectively. Then, 25 µL of 0.6% suspension of CRBCs was added to the
mixtures and incubated at 4 °C for 60 min. Finally, hemagglutination inhibition effect
of the components was observed. The experiment was performed in duplicate for each
dilution ratio.
2
.7. Neuraminidase inhibition (NA) assay
To evaluate the inhibition effect of epiprogoitrin, progoitrin, epigoitrin and
goitrin on neuraminidase, the NA inhibition assay was performed in a 96-well
microplate reader using the Neuraminidase Inhibitors Screen Kit. For each well, 70
µL of the reaction buffer, 10 µL of NA and 10 µL of sample solution were added. The
reaction mixture was vibrated for 1 min and incubated at 37 °C for 2 min for full
interaction. Then 10 µL of the fluorescent substrate was added and the entire mixture
was vibrated for 1 min and incubated at 37 °C for 1 h. Epiprogoitrin, progoitrin,
epigoitrin and goitrin were diluted with water to prepare sample solution at different
concentrations (0.625, 1.25, 2.5 and 5 mg/mL). Peramivir at different concentrations
(0.375, 0.75, 1.5 and 3 mg/mL) were used as the positive control and water was used

as the negative control. Triplicates were used for each dilution ratio. The fluorescence
was recorded by a microplate spectrophotometer (Gemini EM, Molecular Device,
USA) and the excitation wavelength and the emission wavelength were set at 322 nm
and 450 nm, respectively. The inhibition rate (%) was calculated as follows: NA
inhibitory activity (%) = [1 – (F –F )/(F -F )] × 100%, where F is the fluorescence
s
0
m
0
s
intensity of the sample, F is the absorbance of the background, and F is the
0
m
absorbance of the negative control.
2
.8. Statistical analysis
All experiments were carried out in triplicate and the data are presented as means
standard deviations. The values of TCID , EID and IC were calculated using
±
5
0
50
50
SPSS 16.0 software.
3
. Results
3
.1. Cellular toxicity of epiprogoitrin, progoitrin, epigoitrin and goitrin
The results of the cytotoxicity test indicated that epiprogoitrin, progoitrin,
epigoitrin and goitrin at all investigated concentrations showed no influence on cell
viability. The maximum non-toxic dose of Isatidis Radix derived glucosinolate
-3
isomers and their breakdown products on MDCK cells was 10 mol/L and the CPE
assay was performed at this dose and below.
3
.2. Anti-influenza activity of epiprogoitrin, progoitrin, epigoitrin and goitrin in vitro
Epiprogoitrin, progoitrin, epigoitrin and goitrin were evaluated for their antiviral
activity against the influenza virus strain A/California/7/2009 (H1N1) on MDCK cells
via cytopathic effect (CPE) inhibition assay. The mode of action of the components
was studied. When the cells were pre-treated with epiprogoitrin, progoitrin, epigoitrin
and goitrin before viral adsorption or the cells were treated with the components after
virus incubation, no obvious protective effect was observed. However, when the cells

were treated with pre-reacted virus and component, a pronounced CPE inhibition
effect was detected. Epiprogoitrin, progoitrin, epigoitrin and goitrin exhibited
dose-dependent anti-influenza virus activity with an IC value of 0.44 ± 0.03, 0.19 ±
5
0
0
.01, 0.36 ± 0.02 and 0.19 ± 0.02 µM, respectively (Fig. 2).
Fig. 2. Inhibitory effect of epiprogoitrin, progoitrin, epigoitrin and goitrin on
influenza virus A (H1N1) in Madin-Darby canine kidney (MDCK) cells (n = 3). 100
µL of sample solutions at different concentrations were mixed with 100 µL of virus at
1
00 TCID . After incubation at room temperature for 30 min, the mixtures were
50
added to the cells. After incubation at 37 °C in 5% CO for 72 h, the antiviral activity
2
was investigated by cytopathic effect (CPE) inhibition assay and Cell Counting Kit-8
(CCK8) method.
3
.3. Anti-influenza activity of epiprogoitrin, progoitrin, epigoitrin and goitrin in ovo
In agreement with results of in vitro test, epiprogoitrin, progoitrin, epigoitrin and
goitrin failed to exhibit prevention or treatment effect against influenza virus A (H1N1)

in ovo. However, when the chicken embryos were treated with the pre-mixed
solutions of the samples at a concentration of 5 mg/mL and the virus, their allantoic
fluid showed no HA effect on CRBCs. As shown in Table 1, Isatidis Radix derived
glucosinolate isomers and their breakdown products were all found to neutralize
influenza virus A (H1N1) in a dose dependent manner. The minimum concentrations
for epiprogoitrin, progoitrin, epigoitrin and goitrin exhibiting significant inhibition
(100% inhibition rate) were 5.0, 0.625, 1.25 and 1.25 mg/mL, respectively. At the
minimum investigated concentration of 0.625 mg/mL, the inhibition rate for
epiprogoitrin, progoitrin, epigoitrin and goitrin were 0, 100%, 25% and 50%,
respectively. At all the concentrations, the components were found to be safe.
Table 1
Neutralization effect of epiprogoitrin, progoitrin, epigoitrin and goitrin on influenza
virus A (H1N1) in embryonated eggs (n = 3).
Compound
Inhibition
rate, %
Group
Survival Negative
concentration, mg/mL
Sham-operated
group
N/A
N/A
N/A
8
8
8
8
0
8
N/A
N/A
N/A
Virus control group
Dilute control
group
Negative control
group
N/A
8
8
N/A
Positive control
group
3
.0
8
8
8
0
100
0
Epiprogoitrin
0.625

1
.25
8
8
8
8
8
8
8
8
8
8
8
8
8
8
8
2 ± 1
25
2
.5
4 ± 1
50
5
.0
8
100
100
100
100
100
25
0
0
0
.625
8
1
.25
8
Progoitrin
Epigoitrin
Goitrin
2
.5
8
5
.0
8
.625
.25
2 ± 1
1
8
100
100
100
50
2
.5
8
5
.0
8
4 ± 1
8
.625
.25
1
100
100
100
2
.5
8
5
.0
8
3
.4. Effect of epiprogoitrin, progoitrin, epigoitrin and goitrin on hemagglutinin
The inhibitory effect of epiprogoitrin, progoitrin, epigoitrin and goitrin on
hemagglutinin was examined to characterize the mode of their antiviral action. As
shown in Fig. 3, hemagglutinin was not inhibited in the reaction solution used as
positive control, nor in the presence of epiprogoitrin, progoitrin, epigoitrin or goitrin,
even at the highest concentration (5 mg/mL) which exhibited antiviral activity.

Fig. 3. Effect of epiprogoitrin, progoitrin, epigoitrin and goitrin on hemagglutinin.
Effect of epiprogoitrin, progoitrin, epigoitrin and goitrin on hemagglutinin was
evaluated by hemagglutination inhibition (HAI) assay. Epiprogoitrin, progoitrin,
epigoitrin and goitrin at a concentration of 5 mg/mL was serially diluted 2-fold and
added to the 96-well plate. The standardized hemagglutinin viral solution was added
to the 96-well plate. Then, 0.6% chicken red blood cells suspension was added to the
mixtures as described in the Material and Methods.
3
.5. Effect of epiprogoitrin, progoitrin, epigoitrin and goitrin on neuraminidase
NA has been regarded as one of the most important target of the anti-influenza
virus activity of the drugs. As shown in Table 2, peramivir presented dose-dependent
NA inhibitory activity with IC50 value of 0.69 ± 0.05 mg/mL. But no apparent
inhibition effect of epiprogoitrin, progoitrin, epigoitrin or goitrin on NA was observed,
even at the highest concentration (5 mg/mL).

Table 2
Inhibitory effect of epiprogoitrin, progoitrin, epigoitrin and goitrin on neuraminidase
(n = 3).
Inhibition
Group
Compound concentration, mg/mL
rate, %
0
0
0
0
0
.375
35.80 ± 1.25
0
.75
50.25 ± 2.64
Positive control
1
.5
.0
71.53 ± 4.72
3
79.43 ± 3.81
.625
.25
0
1
0
Epiprogoitrin
Progoitrin
Epigoitrin
Goitrin
2
.5
.0
0
5
0
.625
.25
5.95 ± 0.68
1
0.12 ± 0.04
2
.5
.0
0
5
0
.625
.25
0
1
0
2
.5
.0
0
5
0
.625
.25
9.67 ± 1.77
3.85 ± 0.70
2.13 ± 0.56
0.50 ± 0.07
1
2
.5
.0
5

4
. Discussion
As a highly contagious disease, influenza causes high morbidity and mortality in
epidemics each year. Due to adverse effect and viral resistance, development of novel
natural anti-influenza drugs becomes necessary and urgent. Isatidis Radix is used for
ages to cure symptoms similar to influenza and has been reported to show satisfactory
efficacy on respiratory viruses, but the mechanism remains unilluminated till now (Xu
et al., 2019). Different types of compounds have been isolated from this traditional
medicine including alkaloids, lignans and flavonoids, etc. For the first time, antiviral
effect and mode of action of glucosinolates and their biotransformed products from
Isatidis Radix were discussed in this study.
Two pairs of enantiomers, namely epiprogoitrin, progoitrin, epigoitrin and goitrin,
were prepared from Isatidis Radix by chiral separation. First, CPE inhibition assays
were designed in three different ways to examine their inhibitory effect against
influenza virus strain A/California/7/2009 (H1N1) in vitro, and to determine at which
stage they act in the viral replication cycle. The results indicated that the antiviral
activity of epiprogoitrin, progoitrin, epigoitrin and goitrin was mainly attributed to
their virucidal effect, but not action on host cell protection or post-virus-adsorption
interruption. Virus, rather than cell, was more probably to be the action target of the
components. Hence, further insight into the antiviral activity in ovo was performed by
hemagglutination test on the allantoic fluid of embryonated eggs. In agreement with
previous observations, the four components were all found to significantly reduce
viral titer in chicken embryos when mixed with virus before infection. Taken together,
it was concluded that epiprogoitrin, progoitrin, epigoitrin and goitrin showed obvious
virucidal effect on influenza virus A (H1N1) in vitro and in ovo.
For a deeper understanding of the mechanism of the antiviral action, the effect of

epiprogoitrin, progoitrin, epigoitrin and goitrin on hemagglutinin and neuraminidase
was evaluated by hemagglutination inhibition (HAI) assay and neuraminidase
inhibition (NA) assay. Influenza virus is an enveloped RNA virus with hemagglutinin
(HA) and neuraminidase (NA) embedded in the lipid envelope. Viral adsorption to
cells is the initial step of influenza infection, which is performed via the binding
between sialic acid on the cell surface and hemagglutinin of the virus (Skehel and
Wiley, 2000). Results of HAI assay showed that exposure to epiprogoitrin, progoitrin,
epigoitrin or goitrin did not affect the hemagglutinin activity of H1N1 virus. The
components were indicated not to interfere with the binding of sialic acid and
hemagglutinin. On the other hand, for the release of the influenza virus from the cell,
NA functions as an enzyme cleaving sialic acid groups from the host glycoproteins
(Huang et al., 2008). Epiprogoitrin, progoitrin, epigoitrin and goitrin were found to
exert low inhibition rates on neuraminidase by a NA inhibition assay. The components
were indicated not able to prevent budding of progeny virus from the cell membrane.
Therefore, it is implied that epiprogoitrin, progoitrin, epigoitrin and goitrin play their
antiviral activity by acting as virus neutralizers, which associate with some
component on the viral envelope without interfering with viral adsorption or budding.
Based on the IC values obtained from CPE inhibition assay, the activity of the
5
0
compounds in vitro was in the order of progoitrin = goitrin > epigoitrin >
epiprogoitrin. Comprehensive comparison of the minimum concentrations exhibiting
1
0
00% inhibition and the inhibition rate at a minimum investigated concentration of
.625 mg/mL obtained from HA assay on embryonated eggs, the activity of the
compounds in ovo was in the similar order of progoitrin > goitrin > epigoitrin >
epiprogoitrin. When comparing the potency of the antiviral activity between the
monomers, no remarkable difference was found between the precursor and the

myrosinase-derived product (epiprogoitrin vs. epigoitrin and progoitrin vs. goitrin).
On the contrary, considerable discrepancy was observed between the enantiomers
(epiprogoitrin vs. progoitrin and epigoitrin vs. goitrin). The gap between the antiviral
potency of progoitrin and epiprogoitrin was larger than that between goitrin and
epigoitrin. S-enantiomers (progoitrin and goitrin) claimed higher activity than
R-enantiomers (epiprogoitrin and epigoitrin). It is implied that the heteroaromatic
chain/ring is the main functional group of the Isatidis Radix derived glucosinolates
isomers and their breakdown products. And cyclization of the heteroaromatic chain to
the oxazoline ring has no obvious influence on the function. The stereogenic center
(chiral carbon atom) in the heteroaromatic chain of epiprogoitrin and progoitrin and in
the oxazoline ring of epigoitrin and goitrin is an important structural feature from both
chemical and biological points of view. Lucky enough, all the components were found
to be safe both to the cells and the embryos.
5
. Conclusions
In conclusion, experimental results indicated that epiprogoitrin, progoitrin,
epigoitrin and goitrin, the Isatidis Radix derived glucosinolates isomers and their
breakdown products, could neutralize the influenza virus strain A/California/7/2009
(H1N1), thus exhibiting anti-viral efficacy in vitro and in ovo. The finding enriched
the pharmacology and the mechanism for research and development of Isatidis Radix,
the most common traditional medicine used for treating influenza. Further
mechanistic studies implied that the above components had no inhibitory effect on
hemagglutinin and neuraminidase. Thus, the attachment to the viral envelope without
disturbing viral adsorption or budding to host cells was suggested to contribute
critically to the mode of antiviral action of the components. Potential activity on other
influenza virus targets like endonuclease, viral polymerase, nucleoprotein or M2

protein needs future exploration. With accurate anti-viral efficacy and low toxicity,
epiprogoitrin, progoitrin, epigoitrin and goitrin from Isatidis Radix are expected to be
used as promising antiviral drug resources. Consequently, further validation including
in vivo evaluation and clinical trials are required before introducing them from the
research into practical application. However, whether the glucosinolates and their
enzymatic hydrolysates could inhibit other influenza viruses or not is unknown and
the molecular mechanism still needs to be clarified by further investigation.
Author contributions
Lixing Nie and Yanlin Wu designed and carried out the experiments, analyzed the data,
and wrote the manuscript. Zhong Dai checked the data. Shuangcheng Ma edited the article
.
All authors approved and reviewed the manuscript.
Conflicts of interest
None.
Acknowledgments
This work was supported by the National Natural Science Foundation of China
grant No. 81303194) and the Academic Leader Foundation of National Institutes for
(
Food and Drug Control (grant No. 2017X1).
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2

Fig. 1. Breakdown pathway of epiprogoitrin and progoitrin to epigoitrin and goitrin,
respectively.
Fig. 2. Inhibitory effect of epiprogoitrin, progoitrin, epigoitrin and goitrin on
influenza virus A (H1N1) in Madin-Darby canine kidney (MDCK) cells (n = 3). 100
µL of sample solution at different concentrations were mixed with 100 µL of virus at
1
00 TCID . After incubation at room temperature for 30 min, the mixtures were
50
added to the cells. After incubation at 37 °C in 5% CO for 72 h, the antiviral activity
2
was investigated by cytopathic effect (CPE) inhibition assay and Cell Counting Kit-8
(
CCK8) method.
Fig. 3. Effect of epiprogoitrin, progoitrin, epigoitrin and goitrin on hemagglutinin (n =
). Effect of epiprogoitrin, progoitrin, epigoitrin and goitrin on hemagglutinin was
3
evaluated by hemagglutination inhibition (HAI) assay. Epiprogoitrin, progoitrin,
epigoitrin and goitrin at a concentration of 5 mg/mL was serially diluted 2-fold and
added to the 96-well plate. The standardized hemagglutinin viral solution was added
to the 96-well plate. Then, 0.6% chicken red blood cells suspension was added to the
mixtures as described in the Material and Methods.