Baicalein triazole prevents respiratory tract infection by RSV through suppression of oxidative damage

Article abstract of DOI:10.1016/j.micpath.2019.03.026

Baicalein, an isolate of secutellaria baicalensis is known for its anti-inflammatory activity. In the present study, 12-triazole derivatives of baicalein were synthesized and evaluated against RSV infected BEAS-2B cells in vitro and in mice model in vivo.

Full text of DOI:10.1016/j.micpath.2019.03.026

Accepted Manuscript
Baicalein triazole prevents respiratory tract infection by RSV through suppression of
oxidative damage
Cuicui Zhang, Na Li, Fang Niu
PII:
S0882-4010(19)30093-2
DOI:
https://doi.org/10.1016/j.micpath.2019.03.026
YMPAT 3457
Reference:
To appear in: Microbial Pathogenesis
Received Date: 16 January 2019
Revised Date: 20 March 2019
Accepted Date: 20 March 2019
Please cite this article as: Zhang C, Li N, Niu F, Baicalein triazole prevents respiratory tract infection
by RSV through suppression of oxidative damage, Microbial Pathogenesis (2019), doi: https://
doi.org/10.1016/j.micpath.2019.03.026.
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to
our customers we are providing this early version of the manuscript. The manuscript will undergo
copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please
note that during the production process errors may be discovered which could affect the content, and all
legal disclaimers that apply to the journal pertain.

ACCEPTED MANUSCRIPT
Baicalein triazole prevents respiratory tract infection by RSV through suppression of
oxidative damage
1
, 2
3
1,2
Cuicui Zhang , Na Li , Fang Niu *
1
. Department of Critical Care Medicine, Affiliated Hospital of Jining Medical University,
Jining, Shandong, China-272029
2
. Intensive Medical, Jining Medical University, Jining, Shandong, China-272029
. Caocheng community health service center, Heze, Shandong, China-274400
3
*
Corresponding author: Fang Niu, Department of Critical Care Medicine, Affiliated Hospital
of Jining Medical University, Jining, Shandong, China-272029; Tel/Fex: 0086-0537-
903399; Email: NormaMannzky@yahoo.com.
2

ACCEPTED MANUSCRIPT
Abstract
Baicalein, an isolate of secutellaria baicalensis is known for its anti-inflammatory activity. In
the present study, 12-triazole derivatives of baicalein were synthesized and evaluated against
RSV infected BEAS-2B cells in vitro and in mice model in vivo. The preventive effect of
most active compound 5f against RSV infection was studied in detail. The compound 5f
treatment increased IFN-β1 expression in BEAS-2B cells infected with RSV. In BEAS-2B
cells treatment with compound 5f inhibited RSV-induced secretion of interleukin-6 and -8
cytokines. It decreased RSV-induced nitric oxide & malondialdehyde production and
inhibited the RSV-mediated activation of NF-κB, COX-2, Stat3 and MAPK. The p38
phosphorylation was enhanced significantly in RSV infected cells by compound 5f pre-
treatment. RT-qPCR showed that compound 5f treatment of the RSV-infected mice
significantly (P<0.05) decreased viral load through reduction in the viral replication. In the
mice model of RSV-infection compound 5f treatment decreased interleukin-6, -8 and tumor
necrosis factor-α expression. The level of MPO, nitric oxide and malondialdehyde was also
decreased significantly by compound 5f in the RSV infected mice BALF. It also reduced the
infiltration of neutrophils and lymphocytes in the BALF of RVS-infected mice. In summary,
compound 5f inhibits RSV-infection and prevents pulmonary airway inflammation through
the activation of IFN signalling pathway.
Running title: Compound 5f prevents respiratory tract infection
Keywords: Pneumonia, bronchiolitis, anti-inflammatory, tumor necrosis
Introduction

ACCEPTED MANUSCRIPT
The respiratory syncytial virus (RSV), a member of the Paramyxoviridae family infects
children to cause epidemic bronchiolitis and pneumonia. Infection by RSV in children is
responsible for bronchiolitis in 40-60% cases and pneumonia in 15-25% patients [1a-d].
During early stage of life RVS infection leads to asthma and its severe infection increases
mortality rate of infants throughout the world [2]. RVS infection has been found to induce
inflammation of pulmonary airways through enhancement of cytokine level, increased mucus
secretion and recruitment of inflammatory cells [3]. Down-regulation of viral replication and
prevention of inflammation of the pulmonary airways constitute main target for RSV
infection treatment [3]. There is no vaccine available for RSV infection and therefore,
identification of the molecule which can prevent its replication is of great importance.
Scutellaria baicalensis also known as Huang qin or Scutellariae radix belongs to
the Labiatae family [4]. It is commonly used for the treatment of diseases of cardiovascular
system [4], bleeding disorders like hematemesis, hematuria, and metrorrhagia [5] in the
Traditional Chinese system of medicine (TCM). The plant is also used for the treatment of
various diseases as a constituent of combination therapies [6-8]. Photochemical investigation
of Scutellaria baicalensis has led to the isolation and characterisation of a large number of
compounds including baicalin [9,10], its aglycone baicalein and other glycosides [11].
The ethanolic extract of Scutellaria baicalensis possess anti-inflammatory property on the
macrophage activation induced by LPS through reduction of nitric oxide generation [9]. The
compounds isolated from Scutellaria baicalensis such as baicalein, wogonin and oroxylin A
also inhibit LPS-induced macrophage activation through down-regulation of nuclear factor-
κB (NF-κB) pathway [12,13]. Mechanistic study has revealed activation of estrogen receptor
on the surface of macrophages by baicalein [14]. The baicalein-induced activation of estrogen
receptor on the surface of macrophages leads to anti-inflammatory effect through inhibition
of NF-kB activation [15]. The blocking of estrogen signalling pathway inhibits the anti-
inflammatory effect of baicalein [14]. The baicalin alone or after chelation with zinc metal
inhibits replication of HIV [16]. In the present study a library of baicalin triazoles was
synthesized and evaluated against RSV infection. The study identified compound 5f as the
most active molecule for inhibition of RSV replication. Further investigation showed that
compound 5f inhibits replication of RSV through the activation of IFN signalling pathway.
The compound 5f prevented inflammation of pulmonary airways through down-regulation of
reactive oxygen species generation and pro-inflammatory cytokine expression.

ACCEPTED MANUSCRIPT
Materials and methods
1
13
General. H and C NMR spectra of all the synthesized compounds were recorded in
DMSO-d solvent using a Bruker AC-300P spectrometer. The internal standard was taken as
6
tetramethylsilane (TMS). The chemical shift (δ values) and coupling constants (J values),
respectively are expressed in ppm and Hz. HRMS were measured using the Bruker Daltonics
electro spray ionization apparatus. For thin layer chromatography commercially available
silica gel plates GF254 aluminium plates were used. TLC plates were visualized using ceric
sulfate reagent with ultraviolet light at 254 nm. All the chemicals and solvents were
purchased from Sigma-Aldrich (Sigma, St. Louis, MO, USA). Column chromatography was
carried out on silica gel (Qualigens, 60-120 mesh). All the chemicals and solvents were
purchased from Sigma-Aldrich (Sigma, St. Louis, MO, USA). Column chromatography was
carried out on silica gel (Qualigens, 60-120 mesh).
Preparation of aryl Azides. To a solution of 2,5-dimethyl aniline (500 mg, 4.13 mmol) in 1,4-
dioxane at -15 °C, 2 M sulphuric acid (10.3 mL, 20.6 mmol) was added in small instalments
while stirring. After 5 min, 3 M aqueous sodium nitrite solution (2.75 mL, 8.26 mmol) was
added drop wise followed by careful addition of 3 M sodium azide solution (4.13 mL, 12.39
mmol). After the addition was over, the reaction mixture was allowed to attain room
temperature and extracted with diethyl ether (3 × 50 mL). The combined organic layer was
washed with saturated sodium bicarbonate solution, dried over anhydrous sodium sulfate, and
concentrated under reduced pressure to yield aromatic azide that was used in the next reaction
without purification.
Cell lines and culture. The BEAS-2B bronchial epithelial cells were obtained from the
American Type Culture Collection (ATCC; Rockville, MD, USA). The cell cultures were
performed in the Dulbecco's modified Eagle's medium (DMEM; Gibco Life Technologies,
Carlsbad, CA, USA) containing 10% fetal calf serum (FCS), L-glutamine (2 mM), penicillin
(
100 U/ml) and streptomycin (100 mg/ml) under humidified atmosphere of 5% CO at 37˚C.
2
The transgenic RSV viral strain was grown in the HEp-2 cells (ATCC).
4
Treatment. BEAS-2B cells at a density of 5 x 10 cells/ml were plated and grown for 48 h
before experimentation into 96-well plates. The RSV titers used for in vitro induction of
infection was determined using immunoplaque assay. The cells were treated with various
indicated concentrations of compound 5f to analyse changes in the viral replication.

ACCEPTED MANUSCRIPT
Analysis of cytotoxic effect of compound 5f. Determination of cell viability was performed by
non-radioactive method (MTS-PMS assay) employing the cell proliferation kit, CellTiter 96
Aqueous One Solution (Promega, Madison, WI, USA). The measurement of absorbance was
carried out using microplate reader (SpectraFluor; Tecan Austria Gmbh, Salzburg, Austria) at
a wavelength of 570 nm. The effect of compound 5f on the viability of epithelial cells of lung
airways was determined. Treatment with 0.5 to 3.0 µM concentrations of compound 5f didn’t
induce any cytotoxicity in BEAS-2B cell cultures.
Animal and treatment. Forty female BALB/c mice (8-week old) were obtained from the
Capital Medical University (Beijing, China), Animal Centre. The mice were maintained
under standard and controlled laboratory conditions, with a temperature of 23±2˚C, relative
humidity 62±8% and under photoperiods of 12-h light/dark. The experiments on mice were
performed in accordance with the guidelines from the Committee for the Control and
Supervision on Experiments on Animals. All the mice were fed on a standard pellet diet and
sterilized water provided ad libitum. The mice were acclimated one week prior to the start of
experiment to the laboratory environment. The experimental protocols for the mice were
approved by the ethics committee of the Capital Medical University (Beijing, China). Thirty
5
mice were subjected to intranasal administration of RSV at 5 x 10 PFU in the 100 µl of
phosphate-buffered saline (PBS) and ten in the negative control group PBS alone using
isoflurane anesthesia. The confirmation of the establishment of RSV infection mice model
was performed the known ex vivo plaque assay. The mice in the two treatment groups
received 1 and 2 mg/kg doses of compound 5f intraperitoneally one day prior to the
administration of RSV.
Histopathological analysis. Five mice from each of the group were sacrificed at 48 h of the
RVS administration using pentobarbital anaesthesia. From each of the mice pulmonary
tissues, serum and Bronchoalveolar lavage fluid (BALF) was collected for histopathological
examination. The commercially available kits were used for the detection of interleukin-6, -8,
nitric oxide and malondialdehyde in accordance with the manual protocol. The rest of the
mice in each group were sacrificed on the day 5 of RSV infection. The BALF was obtained
by penetrating cannula through the tracheal tube and passing 3 x 0.5 ml ice-cold PBS aliquots
into the lungs. Inflammation of the pulmonary airways was determined by measuring
macrophage and neutrophil accumulation in the in the mice BALF. The hemocytometer
(XB.K.25; Shanghai Quijing Bohao Biotech Co., Ltd., Shanghai, China) was used for the

ACCEPTED MANUSCRIPT
calculation of the total macrophages and neutrophils in the BALF. Distinction between the
neutrophils and lymphocytes in the BALF was performed on the basis of presence of cell
surface marker GR-1 and CD3 using the fluorescence-activated cell sorting and cytospin
staining.
Reverse transcription-quantitative polymerase chain reaction (RT-qPCR). From lung tissues
and BEAS-2B cells total RNA was isolated using the commercially available RNA extraction
kit supplied by the BioTeke Corp., (Beijing, China). The quantification of total RNA,
subsequently reverse transcription of the 2 µg samples into cDNA and amplification was
performed using the TaqMan Gene reverse transcriptase (Takara Co., Ltd. Japan). The
amplification and quantification of the cDNA was carried out using Applied Biosystems®
StepOnePlus™ Real-Time PCR systems. The following sense and antisense primer
sequences were used for the amplification of RIG-I, MDA-5, IFN-α and IFN-β1 mRNA. The
conditions used for PCR cycling were: 40 sec at 95˚C, 50 sec at 59˚C, 35 sec at 70˚C (30
cycles) followed by a 5-min extension at 72˚C. The analysis of the reaction products was
carried out on 1.0% agarose gel and visualization by ethidium bromide. GAPDH was taken as
internal loading control.
Western blot analysis. BEAS-2B cells after treatment with RSV and/compound 5f (0.5, 1.0,
1
.5, 2.0, 2.5 and 3.0 µM concentrations) were collected and washed with PBS. The cells were
subsequently subjected to lysis with RIPA buffer containing 1% solution of
phenylmethylsulfonyl fluoride. The cell lysates were subjected to centrifugation at 12,000 x g
for 20 min at 4˚C temperature to isolate the insoluble debris. The concentration of proteins
was measured using the commercially available BCA kit (Pierce Chemical Co., Rockford, IL,
USA) and bicinchoninic acid assay. The 20 µg protein samples were separated by
electrophoresis using the 10% sodium dodecyl sulfate-polyacrylamide gel. The proteins were
subsequently transferred to a nitrocellulose membrane. The membranes were incubated for 2
h with non-fat milk powder in Tris-buffered saline mixed with Tween-20 (0.05%) at room
temperature for blocking the non-specific sites. Incubation of the membranes with primary
antibodies was carried out at 4˚C for overnight. The membranes were then washed with PBS
three times and subsequently incubated with secondary HRP-conjugated antibodies for 1 h at
the room temperature. The band visualization was performed by enhanced chemilumines-
cence. The following antibodies were used: COX-2 (dilution 1:1,000; catalog number
#
D5H5), p-38 (dilution 1:1,000; catalog number #D13E1), p-p-38 (dilution 1:1,000; catalog

ACCEPTED MANUSCRIPT
number #D3F9), ERK (dilution 1:1,000; catalog number #137F5), p-ERK (dilution 1:1,000;
catalog number #D13.14.4E), p-JNK (dilution 1:1,000; catalog number #81E11), IκBα
(dilution 1:1,000; catalog number #44D4), Stat3 (dilution 1:1,000; catalog number #D3Z2G)
and p-Stat3 (dilution 1:1,000; catalog number #D3A7). All the antibodies were obtained from
the Cell Signaling Technology, Inc. (Beverly, MA, USA).
Statistical analysis. The presented data are the mean ± SD of three independent experiments.
The comparison were made between different groups using Student's t-test and one-way
ANOVA. The Statistical Package for Social Sciences (SPSS for Windows, version 17.0;
SPSS, Inc., Chicago, IL, USA) was used for processing of all the data. The differences are
considered statistically significant at P<0.05.
Results
Synthesis of the compound library. Taking into account the importance of triazoles in the
development of drugs the present study was designed to synthesize the triazolyl derivatives of
baicalin 1. The aromatic azides required were synthesized from the anilines by diazotization
reaction with sodium nitrite under acidic conditions and subsequently reacted in situ with
sodium azide (Scheme 1).
Scheme 1. Preparation of aromatic triazoles.
A library of 12 compounds (5a-5l) was synthesized for evaluation against RSV infection. The
baicalin 1 was treated with dimethoxy benzaldehyde in acidic medium for two hours to
protect the two hydroxyl groups (Scheme 2). The third –OH group adjacent to the carbonyl
group remained unprotected probably because of its H-binding with O-atom rendering it
unavailable for reaction with di-methoxybenzaldehyde. The hydroxyl protected compound 2
was obtained in quantitative yield. The compound 2 on reaction with propargyl bromide for
one hour in THF using caesium carbonate as base delivered O-propargylated compound 3 in
9
8% yield. Deprotection of the hydroxyl groups was performed using ceric ammonium
nitrate in THF as solvent for 45 min to deliver compound 4 in 94% yield. Reaction of the
compound 4 with aryl azide, sodium ascorbate and copper sulphate in tertiary butanol and
water solvent system (2:1) afforded the corresponding triazole derivatives 5a-5l in excellent
yield over three hours (Table 1). The compounds were purified by column chromatography

ACCEPTED MANUSCRIPT
I
using ethyl acetate: hexane (20:80). All the synthesized compounds characterised using H
1
3
NMR, C NMR and HRMS spectral techniques.
Scheme 2: Synthesis of baicalin triazolyl derivatives.
Table 1: Library of the compounds synthesized.

ACCEPTED MANUSCRIPT
Screening of the synthesized compounds against RSV infection. The effect of synthesized
compound library (5a-5l) on RSV replication was examined in BEAS-2B cells infected with
RSV using viral plaque assay. The cells were pre-treated with the synthesized compounds
(
5a-5l) for 24 h prior to RSV-infection. The minimal inhibitory concentration (at which no
visible growth was observed MICs) of the standard anti-RSV drug, ribavirin was found to be
.7 µM. Though most of the synthesized compounds exhibited some activity against RSV but
2
this activity was not significant enough to proceed with them for further RSV drug discovery
as their MIC values ranged between 50–100 µM, which was significantly higher than the
standard drugs. However, the MIC values of the four molecules 5a, 5f, 5g and 5h were close
to that of the standard drug. The MIC values of the compounds 5a, 5f, 5g and 5h,

ACCEPTED MANUSCRIPT
respectively were 3.0, 2.5, 3.25 and 3.12 µM. Among the four molecules, compound 5f
exhibited MIC value slightly lower than that of the standard drug, ribavirin.
Compound 5f enhances RIG-I and IFN-β1 expression. BEAS-2B cells were pre-treated with
0
.5, 1.0, 1.5, 2.0 and 2.5 µM concentrations of compound 5f for 24 h prior to RSV-infection.
The viral plaque assay showed that compound 5f treatment caused a significant (P<0.05)
decrease in the RSV infection in BEAS-2B cells infected with RSV (Figure 1A). Treatment
with compound 5f at 2.5 µM concentration lead to almost complete disappearance of viral
plaques in BEAS-2B cells infected with RSV. In the RSV-infected BEAS-2B cells a
significant (P<0.05) increase in the expression of RIG-I gene was observed on treatment with
compound 5f (Figure 1B). The level of IFN-β1 expression was also enhanced in BEAS-2B
cells infected with RSV by treatment with compound 5f.
Figure 1. Compound 5f inhibits replication and increases IFN activation in RSV infected
BEAS-2B cells. (A) The RSV infected cells were pre-treated with compound 5f and
thensubjected to immunostaining. (B) The RSV infected cells after compound 5f pre-

ACCEPTED MANUSCRIPT
treatment were assessed for expression of RIG-I and IFN-β1 mRNA expression by RT-qPCR.
The represented data are means ± SD. *P<0.05 and **P<0.02 verses RSV infected cells.
Effect of compound 5f on release of inflammatory cytokine and level of oxidative stress.
BEAS-2B cells infected with RSV secreted considerably higher level of interleukin-6 and -8
as revealed by western blot assay (Figure 2A,B). In BEAS-2B cells pre-treatment with
compound 5f inhibited RSV-induced secretion of both interleukin-6 and -8. The cells were
pre-treated with 0.5, 1.0, 1.5, 2.0 and 2.5 µM concentrations of compound 5f for 24 h prior to
RSV-infection. The decrease in secretion of interleukin-6 and -8 was significant (P<0.05)
from 1.0 µM concentration of compound 5f. The level of nitric oxide and malondialdehyde in
BEAS-2B cells infected with RSV was found to be considerably higher (Figure 2C). Pre-
treatment of BEAS-2B cells with compound 5f significantly decreased the level of RSV-
induced nitric oxide and malondialdehyde. The suppression of RSV-induced nitric oxide and
malondialdehyde level was significant (P<0.05) on treatment with 1.0 µM concentration of
compound 5f.
Figure 2. Compound 5f inhibits pro-inflammatory cytokine level and oxidative stress
molecule expression in RSV-infected BEAS-2B cells. Production of interleukin-6 and -8 in
the RSV infected cells pre-treated with compound 5f was measured by ELISA (A) and
western blot assay (B). The level of malondialdehyde and nitric monoxide was determined
using commercially available kits (C). The data are representative of the means ± SD.
*
P<0.05, **P<0.02 and ***P<0.01 verses untreated cells.

ACCEPTED MANUSCRIPT
Effect of compound 5f on NF-κB, COX-2, Stat3 and MAPK signaling pathways. RSV
infection of BEAS-2B cells lead to the activation of NF-κB, COX-2, Stat3 and MAPK
pathways (Figure 3). The activation of NF-κB in BEAS-2B cells was observed through the
degradation of IκBα after RSV-infection. Pre-treatment of BEAS-2B cells with compound 5f
inhibited the RSV-mediated activation of NF-κB, COX-2, Stat3 and MAPK. The inhibition
was significant on pre-treatment with compound 5f from 1.0 µM concentration. In RSV-
infected BEAS-2B cells compound 5f pre-treatment caused a significant increase in the p38
phosphorylation. Pre-treatment with compound 5f did not change the phosphorylation level
of ERK and JNK in the RSV infected cells.
Figure 3. Effect of compound 5f on pro-inflammatory cytokine and oxidative stress molecule
expression in RSV-infected BEAS-2B cells. The RSV infected cells pre-treated with
compound 5f were subjected to western blotting. The level of GAPDH was taken as loading
control. STAT3; signal transducers and activators of transcription 3, MAPK;
mitogen-activated protein kinase.
Effect of compound 5f on in vivo RSV infection in the mice model. The effect of compound 5f
on RSV replication was also investigated in vivo in the mouse model. The RT-qPCR showed
that compound 5f treatment of the RSV-infected mice significantly (P<0.05) decreased the
viral load through reduction in the viral replication. In compound 5f treated mice with RSV-
infection, the expression of RSV L gene was also inhibited significantly than those of
untreated mice (Figure 4A). In addition, the production of malondialdehyde was also higher
in the BALF of RSV-infected mice (Figure 4B). The level of malondialdehyde was
decreased significantly in the BALF on compound 5f treated mice.
BALF examination of RSV infected mice using enzyme-linked immunosorbent assay
(
ELISA) assay showed a markedly higher level of interleukin-6 and tumor necrosis factor-α
Figure 4C). In the mice model of RSV-infection compound 5f treatment significantly
(

ACCEPTED MANUSCRIPT
decreased the level of both interleukin-6 and tumor necrosis factor-α. The BALF cells and the
nd
sections of lungs of the mice harvested on the 2 day of RSV infection showed increased
level of MPO enzyme. Moreover, the secretion of nitric oxide was also higher in the BALF of
RSV-infected mice (Figure 4C). The level of MPO and nitric oxide was decreased
significantly in the BALF on compound 5f treated mice. The infiltration of neutrophils and
lymphocytes was promoted in the BALF of the mice by RVS infection (Figure 4). The
infiltration of neutrophils and lymphocytes was promoted in the BALF of the mice by RVS
infection. Treatment of the RVS-infected mice with compound 5f lead to a marked reduction
in the infiltration of neutrophils and lymphocytes in BALF.
Figure 4. Compound 5f inhibits replication and prevents inflammation in RSV-infected mice.
The mice were sacrificed at 48 h after RSV infection to collect the BALF, blood samples and
lung tissues. (A) The expression of RSV-L gene in lung tissues was measured by RT-qPCR.
(B) Production of oxidative stress molecule, malondialdehyde was analysed using detection
kits available commercially. (C) ELISA and western blotting were used for analysis of pro-
inflammatory cytokine expression in BALF. *P<0.05 and **P<0.02 verses negative control.

ACCEPTED MANUSCRIPT
Compound 5f downregulated inflammatory-related signaling pathways and upregulate IFN-
related signaling pathways during in vivo RSV infection. The expression of IκBα was
markedly decreased whereas that of COX-2 increased in the epithelial cells of lung of mice
infected with RVS. This shows that RVS infection activates NF-κB and cyclooxygenase-2 in
the mice (Figure 5). The activation of NF-κB and cyclooxygenase-2 in the mice by RVS
infection was inhibited on treatment with compound 5f. Compound 5f treatment of the RVS
infected mice enhanced IFN-α and IFN-β1 expression markedly compared to the untreated
group (Figure 5). These results provide evidence for the involvement of IFN-I signaling
pathway in the anti-viral effect of compound 5f.
Figure 5. Effect of compound 5f on IFN-I, NF-κB and COX-2 in mice model of RSV
infection. Lungs of the mice were harvested and subjected to assessment for IκBα and COX-2
expression by RT-qPCR. The data are representative of three experiments performed
independently. *P<0.05 and **P<0.02 verses negative control mice.
Discussion
RSV infection is characterised by different symptoms ranging from mild cold signs requiring
no specific treatment to serious illnesses such as bronchiolitis and pneumonia in which
patient may need hospitalization [17]. In the present study a library of baicalein triazole
derivatives was synthesized and evaluated against RSV infection in vitro in BEAS-2B cells
and in vivo in mice model. From the library of 12-compounds synthesized and characterised,
4
-compounds showed significant activity against RSV infection in the MIC range of the
standard drug, ribavirin. The antiviral effect of the most active molecule, compound 5f
against RSV infection was investigated in detail. The pathway which plays an important role

ACCEPTED MANUSCRIPT
in combating viral infection is IFN-α/β signalling but it is regulated tightly in the cells. The
impairment in the process of induction of IFN-stimulated gene transcription has been found
to inhibit antiviral effect [18]. It is reported that promotion of IFNLR1expression in the non-
responsive cells enhances expression of receptor and restores sensitivity to IFN-λ, which
consequently increases viral pathogen resistance [19]. In the present study pre-treatment of
BEAS-2B cells with compound 5f prior to RSV-infection caused a significant decrease in the
RSV infection as was evidenced by decrease in the viral plaques. The compound 5f treatment
activated IFN-α/β signalling pathway in BEAS-2B cells through up-regulation of RIG-I gene
and IFN-β1 expression. Inhibition of inflammatory reactions in the cells is of significance for
the treatment of allergic asthma [20]. The butyrate molecule prevented lipopolysaccharide-
induced lung injury in the murine model through anti-inflammatory mechanism [21]. In the
present study in vitro as well as in vivo approaches were used to elucidate the function of
compound 5f in the regulation of airway inflammation. In BEAS-2B cells infection with RSV
led to the secretion of considerably higher level of interleukin-6 and -8. However, pre-
treatment with compound 5f inhibited RSV-induced secretion of both interleukin-6 and -8 in
vitro in BEAS-2B cells and in vivo in mice BALF. RVS-infection of BEAS-2B cells also
increased the level of nitric oxide and malondialdehyde. The compound 5f pre-treatment
alleviated the RSV-induced up-regulation of nitric oxide and malondialdehyde in vitro in
BEAS-2B cells and in vivo in mice BALF. The increase in interleukin-6 expression is
associated with the suppression of Treg function which clearly explains correlation of the
RSV infection with the asthma sensitization [22]. The chemotaxis of neutrophils into the
lungs is mediated by the expression of interleukin-8 [22]. In the present study compound 5f
treatment inhibited RSV-mediated up-regulation of interleukin-6 and -8. The inhibition of
interleukin-6 and -8 expression by compound 5f explains how it prevents infiltration of
neutrophils and lymphocytes into the mice BALF after RSV-infection.
Compound 5f treatment also caused a significant decrease in the phosphorylation of pathways
associated with inflammation such as COX-2, NK-κB, Stat3 and MAPKs. These findings
suggest that compound 5f treatment partly regulates RSV-mediated inflammation through
targeting inflammatory signalling pathway activation. The pathogenesis of bronchiolitis
induced by the RSV infection is associated with the oxidative tissue damage. Under the
conditions of oxidative stress, endothelial cells of the lung airways secrete many molecules
which damage the membrane lipids, proteins and to the nucleic acids [23]. RSV infection has
been reported to generate high content of reactive oxygen species which is linked with the

ACCEPTED MANUSCRIPT
expression of chemokine gene and activation of transcription factor IRF [24]. In the present
study RVS infection increased the generation of nitric oxide, malondialdehyde and
myeloperoxidase both in vitro and in vivo. Treatment with compound 5f significantly
inhibited the RVS-induced production of nitric oxide, malondialdehyde and
myeloperoxidase. The compound 5f therefore, prevents RVS infection through inhibition of
oxidative damage and lung airway inflammation. Structure-Activity-Relationship (SAR)
analysis revealed that all the four compounds i.e., 5a, 5f, 5g and 5h which demonstrated good
activity against RSV induced infection contained substituents in the ortho-position. The
substituents present in the ortho-position of compounds 5a, 5f, 5g and 5h are floro,
trifloromethyl, nitrile and bromo, respectively. It appears that these compounds exhibit
inhibitory effect on RSV infection by interacting with the proteins containing active site that
can accommodate only ortho substituted compounds.
Conclusion
In summary, the present study lead to the identification of compound 5f as a candidate for
inhibition of RSV-infection and for prevention of pulmonary airway inflammation. The
compound 5f inhibits replication of RSV through the activation of IFN signalling pathway. It
prevented inflammation of pulmonary airways through down-regulation of reactive oxygen
species generation and pro-inflammatory cytokine expression. Thus the anti-viral effect of
compound 5f may be partly due to inhibition of RSV replication and partly because of
improvement of host defence mechanism. Thus compound 5f can be of therapeutic
significance for treatment of respiratory infection but further studies need to be performed in
this direction.
Conflict of interests
The authors declare no conflict of interests.
References
1
. (a) Tahirah K, Malik AW, Ali ML and Qazi PH. Oscimum sanctum extract inhibits
growth of Gram positive and Gram negative bacterial strains. Microbial Pathogenesis
2
018; 118: 211-213; (b) Muneer AB, Rayees AM, Poonam P and Ali ML. Preparation
and evaluation of antibacterial potential of Pithecellobium dulce root extract against
Gram positive and Gram negative bacteria. Microbial Pathogenesis, 2018; 116: 49-53; (c)
Ali ML, Muzafar AR, Muneer AB, Zubair SB, Irfan QT, Poonam P. Synthesis and in
vitro evaluation of 2-(((2-ether)amino)methylene)-dimedone derivatives as potential

ACCEPTED MANUSCRIPT
antimicrobial agents. Microbial Pathogenesis, 2018; 114: 431-435; (d) Shay DK, Holman
RC, Newman RD, Liu LL, Stout JW and Anderson LJ. Bronchiolitis-associated
hospitalizations among US children, 1980-1996. JAMA 1999; 282: 1440-1446.
2
. Monick M, Staber J, Thomas K and Hunninghake G. Respiratory syncytial virus infection
results in activation of multiple protein kinase C isoforms leading to activation of
mitogen-activated protein kinase. J Immunol 2001; 166: 2681-2687.
3
. Rudd BD, Burstein E, Duckett CS, Li X and Lukacs NW. Differential role for TLR3 in
respiratory syncytial virus-induced chemokine expression. J Virol 2005; 79: 3350-3357.
. Chen Z, Nihei K, Tanaka H, Uda Y and Kabuyama Y. Identification of a nitric oxide
generation-stimulative principle in Scutellariae radix. Biosci Biotechnol Biochem. 2013;
4
7
7(3): 657-9.
5
. Yoon SB, Lee YJ, Park SK, Kim HC, Bae H, Kim HM, Ko SG, Choi HY, Oh MS
and Park W. Anti-inflammatory effects of Scutellaria baicalensis water extract on LPS-
activated RAW 264.7 macrophages. J Ethnopharmacol. 2009; 125(2): 286-90.
. Ho FM, Liao YH, Yang AJ, Lee Chao PD, Hou YC, Huang CT, Lin SR, Lee KR, Huang
KC and Lin WW. Anti-atherosclerotic action of Ger-Gen-Chyn-Lian-Tang and AMPK-
dependent lipid lowering effect in hepatocytes. J Ethnopharmacol. 2012; 142(1): 175-87.
. Lee JJ, Kim T, Cho WK and Ma JY. Antithrombotic and antiplatelet activities of
Soshiho-tang extract. BMC Complement Altern Med. 2013; 13: 137.
6
7
8
. Zhang H, Chen Q, Zhou W, Gao S, Lin H, Ye S, Xu Y and Cai J. Chinese medicine
injection shuanghuanglian for treatment of acute upper respiratory tract infection: a
systematic review of randomized controlled trials. Evid Based Complement Alternat
Med. 2013; 2013: 987326.
9
. Yue GG, Chan BC, Kwok HF, To MH, Hon KL, Fung KP, Lau CB and Leung
PC. Screening for anti-inflammatory and bronchorelaxant activities of 12 commonly
used Chinese herbal medicines. Phytother Res. 2012; 26(6): 915-25.
1
0. Huang WH, Chien PY, Yang CH and Lee AR. Novel synthesis of flavonoids of
Scutellaria baicalensis Georgi. Chem Pharm Bull (Tokyo). 2003; 51(3): 339-40.
1. Yu C, Qu F, Mao Y, Li D, Zhen Z, Nass R, Calway T, Wang Y, Yuan CS and Wang CZ.
Different extraction pretreatments significantly change the flavonoid contents of
Scutellaria baicalensis. Pharm Biol. 2013; 51(10): 1228-35.
1
1
2. Tseng TL, Chen MF, Tsai MJ, Hsu YH, Chen CP and Lee TJ. Oroxylin-A rescues LPS-
induced acute lung injury via regulation of NF-κB signaling pathway in rodents. PLoS
One. 2012; 7(10): e47403.

ACCEPTED MANUSCRIPT
1
1
3. Yao J, Hu R, Sun J, Lin B, Zhao L, Sha Y, Zhu B, You QD, Yan T and Guo QL.
Oroxylin A prevents inflammation-related tumor through down-regulation of
inflammatory gene expression by inhibiting NF-κB signaling. Mol Carcinog. 2012; 53(2):
1
45-58.
4. Fan GW, Zhang Y, Jiang X, Zhu Y, Wang B, Su L, Cao W, Zhang H and Gao X. Anti-
inflammatory Activity of Baicalein in LPS-Stimulated RAW264.7 Macrophages via
Estrogen Receptor and NF-κB-Dependent Pathways. Inflammation. 2013; 36(6): 1584-
9
1.
1
1
1
1
5. Cutolo M, Sulli A, Seriolo B, Accardo S and Masi AT. Estrogens, the immune response
and autoimmunity. Clin Exp Rheumatol. 1995; 13(2): 217-26.
6. Wang Q, Wang YT, Pu SP and Zheng YT. Zinc coupling potentiates anti-HIV-1 activity
of baicalin. Biochem Biophys Res Commun. 2004; 324(2): 605-10.
7. Hall CB, Geiman JM, Biggar R, Kotok DI, Hogan PM and Douglas GR Jr. Respiratory
syncytial virus infections within families. N Engl J Med 1976; 294: 414-419.
8. Chang H-M, Paulson M, Holko M, Rice CM, Williams BR, Marié I and Levy DE.
Induction of interferon-stimulated gene expression and antiviral responses require protein
deacetylase activity. Proc Natl Acad Sci USA 2004; 101: 9578-9583.
1
9. Ding S, Khoury-Hanold W, Iwasaki A and Robek MD. Epigenetic reprogramming of the
type III interferon response potentiates antiviral activity and suppresses tumor growth.
PLoS Biol 2014; 12: e1001758.
2
0. Toki S, Goleniewska K, Reiss S, Zhou W, Newcomb DC, Bloodworth MH, Stier MT,
Boyd KL, Polosukhin VV, Subramaniam S and Peebles RS Jr. The histone deacetylase
inhibitor trichostatin A suppresses murine innate allergic inflammation by blocking group
2
innate lymphoid cell (ILC2) activation. Thorax 2016; 71: 633-645.
2
2
2
1. Ni YF, Wang J, Yan XL, Tian F, Zhao JB, Wang YJ and Jiang T. Histone deacetylase
inhibitor, butyrate, attenuates lipopolysaccharide-induced acute lung injury in
mice.Respir Res 2010; 11: 33.
2. Cacalano G, Lee J, Kikly K, Ryan AM, Pitts-Meek S, Hultgren B, Wood WI and Moore
MW. Neutrophil and B cell expansion in mice that lack the murine IL-8 receptor
homolog. Science 1994; 265: 682-684.
3. Hosakote YM, Komaravelli N, Mautemps N, Liu T, Garofalo RP and Casola A.
Antioxidant mimetics modulate oxidative stress and cellular signaling in airway epithelial
cells infected with respiratory syncytial virus. Am J Physiol Lung Cell Mol Physiol 2012;
3
03: L991-L1000.

ACCEPTED MANUSCRIPT
2
4. Jamaluddin M, Tian B, Boldogh I, Garofalo RP and Brasier AR. Respiratory syncytial
virus infection induces a reactive oxygen species-MSK1-phospho-Ser-276 RelA pathway
required for cytokine expression. J Virol 2009; 83: 10605-10615.

ACCEPTED MANUSCRIPT
High lights
‹
‹
‹
‹
‹
‹
‹
Twelve-triazole derivatives of baicalein were synthesized.
Four compounds showed MIC in the range of standard drug, ribavirin.
Compound 5f treatment significantly decreased RSV infection.
It increased IFN-β1 expression in BEAS-2B cells infected with RSV.
It inhibited RSV-induced secretion of both interleukin-6 and -8 cytokines.
It inhibited the RSV-mediated activation of NF-κB, COX-2, Stat3 and MAPK.
It enhanced p38 phosphorylation.