molecules

Article
Anti-Inflammatory Effect of Quercetin on RAW
264.7 Mouse Macrophages Induced with
Polyinosinic-Polycytidylic Acid
Young-Jin Kim and Wansu Park *
Department of Pathology, College of Korean Medicine, Gachon University, Seong-nam 13120, Korea;
godsentry@naver.com
* Correspondence: pws98@gachon.ac.kr; Tel.: +82-31-750-8821

Academic Editors: Norbert Latruffe and Derek J. McPhee

Received: 3 February 2016; Accepted: 29 March 2016; Published: 4 April 2016
Abstract: Quercetin (3,31 ,41 ,5,6-pentahydroxyflavone) is a well-known antioxidant and a flavonol
found in many fruits, leaves, and vegetables. Quercetin also has known anti-inflammatory effects
on lipopolysaccharide-induced macrophages. However, the effects of quercetin on virus-induced
macrophages have not been fully reported. In this study, the anti-inflammatory effect of
quercetin on double-stranded RNA (dsRNA)-induced macrophages was examined. Quercetin
at concentrations up to 50 µM significantly inhibited the production of NO, IL-6, MCP-1, IP-10,
RANTES, GM-CSF, G-CSF, TNF-α, LIF, LIX, and VEGF as well as calcium release in dsRNA
(50 µg/mL of polyinosinic-polycytidylic acid)-induced RAW 264.7 mouse macrophages (p < 0.05).
Quercetin at concentrations up to 50 µM also significantly inhibited mRNA expression of signal
transducer and activated transcription 1 (STAT1) and STAT3 in dsRNA-induced RAW 264.7 cells
(p < 0.05). In conclusion, quercetin had alleviating effects on viral inflammation based on inhibition
of NO, cytokines, chemokines, and growth factors in dsRNA-induced macrophages via the
calcium-STAT pathway.

Keywords: quercetin; dsRNA; inflammation; macrophages; nitric oxide; cytokine; calcium; STAT

1. Introduction
Immunity is essential for life and innate immunity provides the first line of response to invading
pathogens [1]. Inflammation is a primordial response that protects against infection and restores
damaged tissue to its normal physiological functioning [2]. A recent study reported that it is becoming
more important to maintain inflammatory homeostasis and to regulate the immune system [3].
Inflammation is an innate immune response by various immune cells, such as macrophages, to
protect against harmful stimuli, such as viruses and bacteria [4]. During the inflammatory reaction,
macrophages produce inflammatory mediators, such as nitric oxide (NO), cytokines, and growth
factors. Calcium is released by macrophages during inflammatory processes. Therefore, modulating
macrophage-mediated inflammatory responses is important for creating a new therapeutic approach
against inflammatory diseases [5].
Viruses can induce tissue necrosis and inflammation and viral infections activate the immune
response, trigger inflammatory diseases, and promote cancer growth [6]. dsRNA, which is recognized
by Toll-like receptor-3, induces the production of inflammatory mediators, such as NO, interleukin
(IL)-6, and tumor necrosis factor (TNF)-α, in macrophages; polyinosinic-polycytidylic acid (poly(I:C))
is considered a synthetic analog of dsRNA [7]. dsRNA activates nuclear factor kappa beta and the
calcium signaling pathway [8].
Quercetin (3,31 ,41 ,5,6-pentahydroxyflavone, Figure 1) is a flavonol found in many fruits, vegetables,
and leaves [9]. The biological activities of quercetin have been extensively demonstrated in various

Molecules 2016, 21, 450; doi:10.3390/molecules21040450 www.mdpi.com/journal/molecules

Molecules 2016, 21, 450 2 of 10
Molecules 2016, 21, 450  2 of 9 

in  various  experimental 

experimental models. For example,models. LiaoFor and
example, 
Lin have Liao  and  that
reported Lin  quercetin
have  reported  that  quercetin 
significantly increased
significantly  increased 
IL-10 secretions IL‐10 macrophages
by peritoneal secretions  by  peritoneal 
of the macrophages 
lipopolysaccharide of  the  lipopolysaccharide 
(LPS)-induced septic mice [10];
(LPS)‐induced 
Gardi et al. have septic  mice that
reported [10];  Gardi  et lowered
quercetin al.  have levels
reported  that  quercetin 
of IL-1β, C-reactivelowered 
protein,levels  of  IL‐1β, 
and monocyte
C‐reactive protein, and monocyte chemotactic protein‐1 (MCP‐1) in a rat model of adjuvant arthritis 
chemotactic protein-1 (MCP-1) in a rat model of adjuvant arthritis [11]; Guazelli et al. have reported
[11];  Guazelli 
that the et  al.  have  reported 
oral administration that  the 
of quercetin oral  administration 
-loaded microcapsulesof decreases
quercetin neutrophil
‐loaded  microcapsules 
recruitment,
decreases  neutrophil  recruitment,  attenuates  histological  alterations,  and 
attenuates histological alterations, and reduces macroscopical damage, edema, and IL-1β and IL-33 reduces  macroscopical 
damage,  edema, 
production and  IL‐1β 
in a mouse model and  IL‐33 acid
of acetic production 
-induced in colitis
a  mouse 
[12].model 
Recently,of  acetic 
Chiowacid 
et al.‐induced  colitis 
have reported
[12]. 
that quercetin could inhibit both mouse hepatitis virus and dengue virus (type 2) [13]. Wovirus 
Recently,  Chiow  et  al.  have  reported  that  quercetin  could  inhibit  both  mouse  hepatitis  et al.
and dengue virus (type 2) [13]. Wo et al. have reported that quercetin may exert its antiviral activity 
have reported that quercetin may exert its antiviral activity against different influenza virus strains
against  different 
(including H1N1 and influenza 
H3N2)virus  strains  (including 
via interaction with viralH1N1  and  H3N2) 
hemagglutinin via  interaction 
protein and then inhibitwith virus
viral 
hemagglutinin  protein  and  then  inhibit  virus  entry  into  the  cell  [14].  However, 
entry into the cell [14]. However, the effects of quercetin on poly(I:C)-induced macrophages have not the  effects  of 
quercetin on poly(I:C)‐induced macrophages have not been fully reported. 
been fully reported.

 
Figure 1. Chemical structure of quercetin. 
Figure 1. Chemical structure of quercetin.

In  the  present  study,  quercetin  at  concentrations  up  to  50  μM  significantly  inhibited  the 
In the present study, quercetin at concentrations up to 50 µM significantly inhibited the production
production of various factors, as well as calcium release and mRNA expression of signal transducer 
of various factors, as well as calcium release and mRNA expression of signal transducer and activated
and  activated  transcription  1  (STAT1)  and  STAT3  in  poly(I:C)‐induced  RAW  264.7  mouse 
transcription 1 (STAT1) and STAT3 in poly(I:C)-induced RAW 264.7 mouse macrophages.
macrophages. 
2. Results
2. Results 
2.1. Effect of Quercetin on NO Production and Intracellular Calcium Release
2.1. Effect of Quercetin on NO Production and Intracellular Calcium Release 
NO is a major inflammatory mediator during the immuno-inflammatory reaction. NO
NO  is  a inmajor 
concentration cultureinflammatory 
medium canmediator 
be determinedduring bythe 
theimmuno‐inflammatory 
Griess reaction. In thisreaction. 
study, RAW NO 
concentration in culture medium can be determined by the Griess reaction. In this study, RAW 264.7 
264.7 mouse macrophages were incubated with poly(I:C) and/or quercetin for 24 h, and 100 µL of
mouse  macrophages 
supernatant from eachwere well wasincubated  with 100
mixed with poly(I:C) 
µL Griess and/or 
reagentquercetin  for  plate
in a 96-well 24  h, toand  100  μL NO
determine of 
supernatant from each well was mixed with 100 μL Griess reagent in a 96‐well plate to determine 
concentration. The results show that quercetin significantly inhibited excessive production of NO in the
NO 
cells concentration. 
at concentrations The 
ofresults  show 
5, 10, 25, and that 
50 µM quercetin  significantly 
(Figure 2). inhibited 
No association excessive 
was found production 
between of 
quercetin
NO  in  the  cells 
concentration andat NO
concentrations  of  5,  10, 
level. The inhibitory 25,  of
effect and 
50 50 
µMμM  (Figure 
quercetin 2).  No than
stronger association 
that at 5 was 
and 10found 
µM.
between  quercetin 
Therefore, quercetinconcentration 
concentrationsand  NO 
of 10, 25,level. 
and 50The 
µM inhibitory 
were choseneffect 
forof  50 multiplex
the μM  quercetin 
cytokinestronger 
assay,
than that at 5 and 10 μM. Therefore, quercetin concentrations of 10, 25, and 50 μM were chosen for 
and concentrations of 25 and 50 µM were chosen for the STAT mRNA expression evaluation.
the multiplex cytokine assay, and concentrations of 25 and 50 μM were chosen for the STAT mRNA 
Intracellular calcium release is related with many cellular processes including the inflammatory
expression evaluation. 
reaction. Calcium release from RAW 264.7 mouse macrophages can be determined by the Fluo-4 assay.
Intracellular calcium release is related with many cellular processes including the inflammatory 
Because calcium is involved in a signaling pathway, the cells were incubated with molecules for 18 h in
reaction. 
this study.Calcium  release  from 
After incubating RAW 
the cells with264.7  mouse 
poly(I:C) macrophages 
and/or quercetin can  be 
for 18 h,determined 
the mediumby  wasthe  Fluo‐4 
removed,
assay. Because calcium is involved in a signaling pathway, the cells were incubated with molecules 
and the cells were incubated with the Fluo-4 dye loading solution. After incubation, fluorescence
for 18 h in this study. After incubating the cells with poly(I:C) and/or quercetin for 18 h, the medium 
intensity of each well was determined spectrofluorometrically with excitation and emission filters of
was removed, and the cells were incubated with the Fluo‐4 dye loading solution. After incubation, 
485 nm and 535 nm, respectively. The data show that quercetin inhibited significantly calcium release
fluorescence  intensity  of 
from poly(I:C)-induced RAWeach 264.7
well mouse
was  determined 
macrophages spectrofluorometrically 
at concentrations of 5, with  excitation 
10, 25, and 50 and 
µM
emission 
(Figure 2).filters  of  485  nm 
No significant and  535 was
association nm, found
respectively. 
betweenThe  data  show 
quercetin that  quercetin 
concentration inhibited 
and intracellular
significantly 
calcium level. calcium  release  from  poly(I:C)‐induced  RAW  264.7  mouse  macrophages  at 
concentrations  of  5,  10,  25, 
The NO production andand  50  μM 
calcium (Figure 
release 2). are
results No shown
significant  association relative
as a percentage was  found 
to thebetween 
control
quercetin concentration and intracellular calcium level. 
group. Indomethacin was used as a positive control.
The  NO  production  and  calcium  release  results  are  shown  as  a  percentage  relative  to  the 
control group. Indomethacin was used as a positive control. 
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10

A B
120 120
A
110
B
110 *
100 *** 100
120 120
90
110 *** 90
110 **
80 *** *
100 80
100
70 *** *** 70
*** ***
90 *** 90 **
60
80 60
80
50
70 *** *** 50
70
*** ***
40
60 40
60
30
50 30
50
20
40 20
10
40
30 10
30
20 0 20 0
10 Nor Con 5 10 25 50 IN Nor Con 5 10 25 50 IN
10
0 0
Quercetin M) IN
Nor Con 5 10 25 50 Nor Con 5 Quercetin
10 25 M) IN
50
Quercetin
poly(I:C) (50 M)
g/mL) Quercetin
poly(I:C) (50 M)
g/mL)
poly(I:C) (50 g/mL) poly(I:C) (50 g/mL)
 
Figure 
Figure 2. 2. Effect
Effect ofof quercetin
quercetin on on nitric
nitric oxide
oxide (NO)
(NO) production
production (A) (A) and
and  calcium 
calcium release 
release
  byby 
(B)(B) 
Figure 
dsRNA-induced2.  Effect 
dsRNA‐induced  RAWof  quercetin 
RAW 264.7264.7 
mouse on  nitric macrophages. 
mouse  oxide  (NO) 
macrophages. After 24 production 
After  24  h (A) 
h treatment, and  calcium 
treatment, 
NO release 
NO  production 
production was (B) was 
measured by 
by measured by the Griess reaction assay. Calcium release was measured with the Fluo‐4 calcium assay 
dsRNA‐induced 
the Griess reaction RAW  264.7 
assay. mouse release
Calcium macrophages. 
was measured After  with
24  h the treatment,  NO  production 
Fluo-4 calcium assay after was 
an
after  an  18  h  The
h treatment. treatment. 
normalThe groupnormal 
(Nor)group  (Nor)  was 
was treated withtreated 
18measured by the Griess reaction assay. Calcium release was measured with the Fluo‐4 calcium assay 
media only.with  media  only. group
The control The  control  group 
(Con) was
(Con) 
after  was 
an  18  treated 
h  with 
treatment.  50 
The  μg/mL 
normal  of  the 
group  dsRNA 
(Nor)  synthetic 
was  treated  analog 
with  polyinosinic‐polycytidylic 
media 
treated with 50 µg/mL of the dsRNA synthetic analog polyinosinic-polycytidylic acid (poly(I:C)) alone. only.  The  control  acid 
group 
IN, (poly(I:C)) 
(Con) 
indomethacin alone. 
was  treated  IN, 
(0.5with indomethacin 
µM). 50  μg/mL 
Values areof  (0.5 dsRNA 
the 
mean ˘μM).  Values 
standard are analog 
synthetic 
deviation mean  ±  standard  deviation 
polyinosinic‐polycytidylic 
of three independent of  three 
experiments.acid 
independent 
* p(poly(I:C))  experiments. 
< 0.05 vs. alone. 
Con; **IN,  0.01;* p 
***< p0.05 vs. 
p <indomethacin  (0.5 Con; 
< 0.001. μM).  p  <  0.01; 
** Values 
Significant *** mean 
are  p  <  0.001. 
differences were Significant 
±  standard 
examined differences 
deviation 
using one-waywere 
of  three 
examined using one‐way analysis of variance test followed by Tukey’s multiple comparison test. 
independent 
analysis experiments. 
of variance * p  < by
test followed 0.05 vs. 
Tukey’sCon;  **  p  <  comparison
multiple 0.01;  ***  p  < test.
0.001.  Significant  differences  were 
examined using one‐way analysis of variance test followed by Tukey’s multiple comparison test. 
2.2. Effect of Quercetin on Cytokine Production 
2.2. Effect of Quercetin on Cytokine Production
2.2. Effect of Quercetin on Cytokine Production 
Cytokines 
Cytokines areare  a  distinct 
a distinct category category 
of smallof  small 
(5–20 kDa) (5–20  kDa) 
proteins thatproteins  that  are 
are important important  in 
in inflammation
and Cytokines 
inflammation 
cell signaling. are 
and  a  distinct 
cell 
Cytokine signaling.  category 
production Cytokine of production 
in culturesmall 
medium(5–20 in 
was kDa) 
culture proteins 
medium 
determined by that  are  important 
was multiplex
the determined  in 
by  the 
cytokine
inflammation 
multiplex  and 
cytokine  cell  signaling. 
assay.  After  Cytokine 
incubating  production 
the  cells  in 
with  culture  medium 
poly(I:C)  and/or 
assay. After incubating the cells with poly(I:C) and/or quercetin for 24 h, the cytokines released was  determined 
quercetin  for  by 
24  the 
h,  the 
multiplex 
cytokines 
from cytokine 
the cellsreleased  assay. 
from  the 
were measured After  incubating 
incells 
cell were 
culture the  cells 
measured  in with 
supernatants cell  poly(I:C) 
culture 
using and/or  assay
quercetin 
supernatants 
the Luminex using 
based for 
on24 
the  h,  the 
Luminex 
xMAP
cytokines 
technology. released 
assay  based Theon  from 
xMAP 
result show the that
cells 
technology.  were  measured 
The 
quercetin in  cell 
result  show 
significantly culture 
that 
reduced supernatants 
quercetin 
excess using 
significantly 
production the  Luminex 
ofreduced 
IL-6, excess 
TNF-α,
assay  based  on  xMAP  technology.  The  result  show  that  quercetin  significantly 
MCP-1, interferon gamma-induced protein-10 (IP-10), regulated on activation, normal T cell expressedon 
production  of  IL‐6,  TNF‐α,  MCP‐1,  interferon  gamma‐induced  protein‐10  reduced 
(IP‐10),  excess 
regulated 
production 
activation, 
and of  IL‐6,  T 
normal 
secreted (RANTES), TNF‐α,  MCP‐1, 
cell  expressed 
leukemia interferon 
and factor
inhibitory gamma‐induced 
secreted 
(LIF),(RANTES),  protein‐10 inhibitory 
leukemia 
lipopolysaccharide-induced (IP‐10), 
CXCregulated  on 
factor  (LIF), 
chemokine
activation,  normal  T  cell 
lipopolysaccharide‐induced 
(LIX), granulocyte-colony expressed  and  secreted 
(G-CSF), (RANTES), 
granulocyteleukemia  inhibitory  stimulating
CXC  chemokine (LIX), granulocyte‐colony stimulating factor (G‐CSF), 
stimulating factor macrophage-colony factor  (LIF), 
lipopolysaccharide‐induced  CXC  chemokine (LIX), granulocyte‐colony stimulating factor (G‐CSF), 
factor (GM-CSF), and vascular endothelial growth factor (VEGF) in poly(I:C)-induced RAW growth 
granulocyte  macrophage‐colony  stimulating  factor  (GM‐CSF),  and  vascular  endothelial  264.7
granulocyte  macrophage‐colony  stimulating  factor  (GM‐CSF),  and  vascular  endothelial  growth 
factor (VEGF) in poly(I:C)‐induced RAW 264.7 mouse macrophages (Figure 3). 
mouse macrophages (Figure 3).
factor (VEGF) in poly(I:C)‐induced RAW 264.7 mouse macrophages (Figure 3). 

 
Figure 3. Cont.
 
Figure 3. Cont. 
Figure 3. Cont. 
Molecules 2016, 21, 450 4 of 10
Molecules 2016, 21, 450  4 of 9 

 
Figure 3. Effect of quercetin on production of cytokines, such as interleukin (IL)-6 (A); tumor necrosis
Figure 3. Effect of quercetin on production of cytokines, such as interleukin (IL)‐6 (A); tumor necrosis 
factor (TNF)-α (B); monocyte chemoattractant protein-1 (MCP-1) (C); interferon gamma-induced
protein-10(TNF)‐α 
factor  (B); RANTES
(IP-10) (D); monocyte  (E),chemoattractant 
leukemia inhibitory protein‐1  (MCP‐1) 
factor (LIF) (F);(C);  interferon  gamma‐induced 
lipopolysaccharide-induced
CXC protein‐10 (IP‐10) (D); RANTES (E), leukemia inhibitory factor (LIF) (F); lipopolysaccharide‐induced 
chemokine (LIX) (G); granulocyte-colony stimulating factor (G-CSF) (H); granulocyte
CXC  chemokine  stimulating
macrophage-colony (LIX)  (G); factorgranulocyte‐colony 
(GM-CSF) (I); and stimulating  factor  (G‐CSF) 
vascular endothelial growth(H);  granulocyte 
factor (VEGF)
macrophage‐colony stimulating factor (GM‐CSF) (I); and vascular endothelial growth factor (VEGF) 
(J) in dsRNA-induced RAW 264.7 mouse macrophages. Flourescene intensity of each cytokine in the
(J) in dsRNA‐induced RAW 264.7 mouse macrophages. Flourescene intensity of each cytokine in the 
culture medium was measured by a multiplex bead-based cytokine assay after the 24 h incubation. The
culture medium was measured by a multiplex bead‐based cytokine assay after the 24 h incubation. 
normal group (Nor) was treated with media only. The control group (Con) was treated with 50 µg/mL
of The  normal synthetic
the dsRNA group  (Nor) analogwas polyinosinic-polycytidylic
treated  with  media  only. acid The  (poly(I:C))
control  group  (Con) 
alone. IN,was  treated  with   
indomethacin
(0.550  μg/mL 
µM). of  are
Values the mean
dsRNA  synthetic 
˘ standard analog  polyinosinic‐polycytidylic 
deviation of three independent experiments.acid  (poly(I:C)) 
* p < 0.05 alone. 
vs. Con;IN, 
indomethacin  (0.5  μM).  Values  are  mean  ±  standard  deviation  of  three  independent 
** p < 0.01; *** p < 0.001. Significant differences were examined using one-way analysis of variance test experiments.   
*  p  <  0.05 vs. 
followed Con; multiple
by Tukey’s **  p  <  0.01;  ***  p  <  0.001. 
comparison test. Significant  differences  were  examined  using  one‐way 
analysis of variance test followed by Tukey’s multiple comparison test. 
2.3. Effect of Quercetin on STAT1 and STAT3 mRNA Expression
2.3. Effect of Quercetin on STAT1 and STAT3 mRNA Expression 
Because STAT proteins are involved in a signaling pathway that activates macrophages, the cells
Because with
were incubated STAT proteins are involved 
molecules for 18 h. Afterin  a  signaling 
incubating the pathway 
cells with that  activates 
poly(I:C) macrophages, 
and/or quercetin forthe 
cells  were  incubated  with  molecules  for  18  h.  After  incubating  the  cells  with 
18 h, real-time reverse-transcription polymerase chain reaction (RT-PCR) was performed to determine poly(I:C)  and/or 
quercetin 
STAT1 for  18 
(GenBank: h,  real‐time 
NM_009283) andreverse‐transcription 
STAT3 (GenBank: NM_213659)polymerase mRNA chain  reaction  The
expression. (RT‐PCR) 
GAPDH was 
performed to determine STAT1 (GenBank: NM_009283) and STAT3 (GenBank: NM_213659) mRNA 
gene (GenBank: NM_001001303) was used for RNA normalization. The results show that quercetin
expression. inhibited
significantly The  GAPDH 
STAT1gene 
and (GenBank: 
STAT3 mRNA NM_001001303) 
expression inwas  used  for  RNA RAW
poly(I:C)-induced normalization.  The 
264.7 mouse
macrophages at concentrations of 25 and 50 µM (Figure 4), indicating that quercetin modulates in 
results  show  that  quercetin  significantly  inhibited  STAT1  and  STAT3  mRNA  expression 
poly(I:C)‐induced 
inflammatory RAW 
reactions 264.7  mouse  macrophages 
in poly(I:C)-induced macrophages at  concentrations  of  25  and 
via the calcium-STAT 50  μM  (Figure  4), 
pathway.
indicating that quercetin modulates inflammatory reactions in poly(I:C)‐induced macrophages via 
3. the calcium‐STAT pathway. 
Discussion
Inflammation in the immune system is essential to fight an infection and is a general term used to
3. Discussion 
describe many diverse processes that tissues employ in response to infections by pathogens (such as
Inflammation in the immune system is essential to fight an infection and is a general term used 
bacteria, viruses, parasites, and fungi) and injuries caused by toxic molecules or physical damage such
as to describe many diverse processes that tissues employ in response to infections by pathogens (such 
burns or cuts [15].
as bacteria, viruses, parasites, and fungi) and injuries caused by toxic molecules or physical damage 
such as burns or cuts [15]. 
Molecules 2016, 21, 450
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10

Figure 4. Effect of quercetin on signal transducer and activated transcription 1 (STAT1) (A) and STAT3
 
Figure expression
mRNA 4.  Effect  of 
(B)quercetin  on  signal  transducer 
in dsRNA-induced RAW 264.7and 
mouseactivated  transcription 
macrophages. After 1 
an(STAT1)  (A)  and 
18 h treatment,
STAT3  mRNA  expression  (B)  in  dsRNA‐induced  RAW  264.7  mouse  macrophages. 
STAT1 and STAT3 mRNA expression was measured by real-time reverse-transcription polymerase After  an  18  h 
treatment,  STAT1  and  STAT3  mRNA  expression  was  measured  by  real‐time 
chain reaction assay. STAT1 and STAT3 mRNA were normalized to GAPDH mRNA. The normal group reverse‐transcription 
polymerase 
(Nor) was treatedchain  reaction 
with media assay.  STAT1 
only. The and 
control STAT3 
group mRNA 
(Con) were  normalized 
was treated to  GAPDH 
with 50 µg/mL mRNA. 
of the dsRNA
The normal group (Nor) was treated with media only. The control group (Con) was treated with 50 
synthetic analog polyinosinic-polycytidylic acid (poly(I:C)) alone. IN, indomethacin (0.5 µM). Values
μg/mL 
are of  ˘
the mean the  dsRNA deviation
standard synthetic  analog 
of three polyinosinic‐polycytidylic 
independent experiments. ** p acid 
< 0.01(poly(I:C)) 
vs. Con; *** alone.  IN, 
p < 0.001.
indomethacin (0.5 μM). Values are the mean ± standard deviation of three independent experiments. 
Significant differences were examined using one-way analysis of variance test followed by Tukey’s
** p < 0.01 vs. Con; *** p < 0.001. 
multiple comparison test. Significant  differences  were  examined  using  one‐way  analysis  of 
variance test followed by Tukey’s multiple comparison test. 
Viral infections trigger anti-viral type I interferon responses by stimulating endosomal and
Viral  infections  trigger  anti‐viral  type  I  interferon  responses  by  stimulating  endosomal  and 
cytosolic pattern recognition receptors, including Toll-like receptors, which are key initiators of
cytosolic  pattern  recognition  receptors,  including  Toll‐like  receptors,  which  are  key  initiators  of 
inflammation [16]. Immunomodulatory approaches are being considered to combat a vast range
inflammation [16]. Immunomodulatory approaches are being considered to combat a vast range of 
of human and animal diseases including incurable viral diseases, cancers, autoimmune diseases,
human and animal diseases including incurable viral diseases, cancers, autoimmune diseases, and 
and inflammatory conditions [17]. Viral and bacterial infections exacerbate the immune system and
inflammatory  conditions  [17].  Viral  and  bacterial  infections  exacerbate  the  immune  system  and 
increase morbidity of patients with chronic airways diseases; viral infections trigger an innate immune
increase  morbidity  of  patients  with  chronic  airways  diseases;  viral  infections  trigger  an  innate 
response through host detection of viral dsRNA via several different mechanisms, including activation
immune  response  through  host  detection  of  viral  dsRNA  via  several  different  mechanisms, 
of a member of the class of pattern recognition receptors, Toll-like receptor 3; lung inflammation
including activation of a member of the class of pattern recognition receptors, Toll‐like receptor 3; 
associated with viral infections includes robust increase in cytokines, such as IL-1β, IL-6, IL-8, TNF-α,
lung  inflammation  associated  with  viral  infections  includes  robust  increase  in  cytokines,  such  as 
GM-CSF, IP-10, RANTES, and MIP-1α [18].
IL‐1β, IL‐6, IL‐8, TNF‐α, GM‐CSF, IP‐10, RANTES, and MIP‐1α [18]. 
Among the many immuno-inflammatory leukocytes, macrophages and monocytes are of great
Among the many immuno‐inflammatory leukocytes, macrophages and monocytes are of great 
importance [19]. Macrophages are found throughout all tissues and are a key component of the immune
importance  [19].  Macrophages  are  found  throughout  all  tissues  and  are  a  key  component  of  the 
system. Recruitment of macrophages to specific sites is important for normal host defense [20], as
immune system. Recruitment of macrophages to specific sites is important for normal host defense 
they play an important role in inflammatory disease by releasing factors, such as NO, reactive oxygen
[20],  as  they  play  an  important  role  in  inflammatory  disease  by  releasing  factors,  such  as  NO, 
species, inflammatory cytokines, chemokines, growth factors, and prostaglandin mediators involved
reactive  oxygen  species,  inflammatory  cytokines,  chemokines,  growth  factors,  and  prostaglandin 
in the immune response [21].
mediators involved in the immune response [21]. 
NO is a membrane-permeable signaling molecule involved in a broad array of biologic
NO  is  a  membrane‐permeable  signaling  molecule  involved  in  a  broad  array  of  biologic 
processes through its ability to modify proteins, lipids, and DNA and alter their functions and
processes  through  its  ability  to  modify  proteins,  lipids,  and  DNA  and  alter  their  functions  and 
immunogenicity [22]. Appropriate levels of NO assist in mounting an effective defense against
immunogenicity  [22].  Appropriate  levels  of  NO  assist  in  mounting  an  effective  defense  against 
invading microbes, whereas the inability to generate NO results in serious, even fatal, susceptibility
invading microbes, whereas the inability to generate NO results in serious, even fatal, susceptibility 
to infections. Furthermore, dysregulation or overproduction of NO has been implicated in the
to  infections.  Furthermore,  dysregulation  or  overproduction  of  NO  has  been  implicated  in  the 
pathogenesis of many disorders, including atherosclerosis, neurodegenerative diseases, inflammatory
pathogenesis  of  many  disorders,  including  atherosclerosis,  neurodegenerative  diseases, 
autoimmune diseases, and cancers; therefore, the potential exists for NO to behave like a
inflammatory  autoimmune  diseases,  and  cancers;  therefore,  the  potential  exists  for  NO  to  behave 
“double-edged” biological sword depending on it level. Thus, it is crucial to understand regulation of
like  a  “double‐edged”  biological  sword  depending  on  it  level.  Thus,  it  is  crucial  to  understand 
NO [23].
regulation of NO [23]. 
Some studies show that the regulation of inflammation is important for maintaining
Some  studies  show  that  the  regulation  of  inflammation  is  important  for  maintaining 
homeostasis [3]. Inflammation plays a critical defensive role in the human body, however, uncontrolled
homeostasis  [3].  Inflammation  plays  a  critical  defensive  role  in  the  human  body,  however, 
or aberrant inflammatory responses contribute to various acute and chronic inflammatory diseases [24].
uncontrolled  or  aberrant  inflammatory  responses  contribute  to  various  acute  and  chronic 
Inflammation helps restore homeostasis after an insult but can be more damaging than the insult itself
inflammatory diseases [24]. Inflammation helps restore homeostasis after an insult but can be more 
if uncontrolled, excessive, or prolonged [25]. For example, dysregulation of the inflammatory/immune
damaging  than  the  insult  itself  if  uncontrolled,  excessive,  or  prolonged  [25].  For  example, 
dysregulation  of  the  inflammatory/immune  responses  is  responsible  for  multiple  organ  failure  in 
Molecules 2016, 21, 450 6 of 10

responses is responsible for multiple organ failure in patients with sepsis, for which over expression
of proinflammatory cytokines is a major mechanism [26]. It is well known that viral and bacterial
infections contribute to the pathogenesis of severe sepsis, which is characterized by an overwhelming
production of NO and proinflammatory cytokines, such as IL-6 [27]. Despite antibiotics, sepsis remains
a clinical challenge, with high mortality rates and increasing prevalence [28]. The expression level
of RANTES increases in lung tissue infected with influenza virus [29]. The robust, uncontrolled
cytokine production, otherwise known as hypercytokinemia or a “cytokine storm”, is thought to
initiate the severe systemic inflammatory response in various infections, such as those caused by
influenza virus, cytomegalovirus, variola virus, severe acute respiratory syndrome coronavirus,
and avian H5N1 influenza virus [30]. Thus, it is important to focus on therapeutics to modulate
virus-induced hyperinflammation.
The poly(I:C)-induced activation of macrophages is regarded as an experimental model of viral
inflammation in vitro. Although many studies on biological effects of quercetin have been reported,
the effect of quercetin on poly(I:C)-induced macrophages has not been fully reported. The present
study shows that quercetin may alleviate poly(I:C)-induced inflammation by decreasing levels of
inflammatory mediators, such as NO, cytokines, chemokines, and growth factors, in RAW 264.7
mouse macrophages; quercetin is a candidate for modulating virus-hyperinflammation such as a
cytokine storm.
Oxidative stress reduces endoplasmic reticulum (ER) calcium stores and increases intracellular
calcium concentrations, resulting in ER stress-mediated STAT1 activation. Preapoptotic ER stress
itself has proinflammatory effects in macrophages [31]. In the present study, quercetin significantly
inhibited calcium release and STAT1 and STAT3 mRNA expression in poly(I:C)-induced RAW 264.7
mouse macrophages. Thus, quercetin might modulate poly(I:C)-induced macrophage activation via
the calcium-STAT pathway.
Finally, the present study demonstrated that quercetin has anti-inflammatory effects related with
inhibiting NO, IL-6, TNF-α, MCP-1, IP-10, RANTES, LIF, LIX, G-CSF, GM-CSF, and VEGF production
in poly(I:C)-induced macrophages via the calcium-STAT pathway. Further study is needed to evaluate
the clinical utility of quercetin for viral inflammation.

4. Materials and Methods

4.1. Materials
DMEM, fetal bovine serum, penicillin, streptomycin, PBS, and other tissue culture reagents were
purchased from Gibco BRL (Grand Island, NY, USA). Quercetin, indomethacin, Griess reagent, and all
other chemicals were purchased from Sigma-Aldrich (St. Louis, MO, USA).

4.2. Quantification of NO Production and Intracellular Calcium Release

NO concentration in culture medium was determined by the Griess reaction. Specifically, after
incubating the cells with poly(I:C) and/or quercetin for 24 h, 100 µL of supernatant from each well was
mixed with 100 µL Griess reagent in a 96-well plate. After a 15 min incubation at room temperature,
optical density was determined at 540 nm with a microplate reader (Bio-Rad, Hercules, CA, USA) [32].
Calcium release from RAW 264.7 mouse macrophages was determined by the Fluo-4 assay. RAW
264.7 mouse macrophages in 96-well plates were incubated with poly(I:C) and/or quercetin for 18 h at
37 ˝ C. Thereafter, the medium was removed, and the cells were incubated with 100 µL of the Fluo-4
dye loading solution (Molecular Probes, Eugene, OR, USA) for 30 min at 37 ˝ C. After the incubation,
fluorescence intensity in each well was determined spectrofluorometrically (Dynex, West Sussex, UK)
with excitation and emission filters of 485 nm and 535 nm, respectively [33].
Molecules 2016, 21, 450 7 of 10

4.3. Multiplex Bead-Based Cytokine Assay

After 24 h treatment with poly(I:C) and/or quercetin, the cytokines released from treated cells
were measured in cell culture supernatants using a Luminex assay based on xMAP technology.
This assay was performed with Milliplex kits (Millipore, Billerica, MA, USA) and the Bio-Plex 200
suspension array system (Bio-Rad) as described previously [34–36]. Standard curves for each cytokine
were generated using the kit-supplied reference cytokine samples.

4.4. RNA Isolation and Real Time RT-PCR Analysis

At the end of the 18 h incubation with poly(I:C) and/or quercetin, RAW 264.7 mouse macrophages
were lysed and total RNA was isolated using the NucleoSpin RNA kit (Macherey-Nagel, Duren,
Germany) according to the manufacturer’s instructions. RNA quantity and quality were confirmed
using the Experion RNA StdSens Analysis kit (Bio-Rad) and Experion Automatic Electrophoresis
System (Bio-Rad). cDNA was synthesized from 1 µg total RNA using the iScript cDNA Synthesis
kit (Bio-Rad). Real-time RT-PCR was performed using the iQ SYBR Green Supermix (Bio-Rad).
The GAPDH gene was used for RNA normalization. The analysis was performed on a Bio-Rad
CFX 96 Real-Time PCR Detection System (Bio-Rad). Relative changes in gene expression were
calculated using the comparative threshold cycle (Ct) method (Bio-Rad) [12]. The following primers
were used: STAT1 (GenBank: NM_009283), sense: 51 -TGAGATGTCCCGGATAGTGG-31 , antisense:
51 -CGCCAGAGAGAAATTCGTGT-31 ; STAT3 (GenBank: NM_213659), sense: 51 -GTCTGCAGAGT
TCAAGCACCT-31 , antisense: 51 -TCCTCAGTCACGATCAAGGAG-31 ; GAPDH (GenBank: NM_0010
01303), sense: 51 -AACCTGCCAAGTATGATGAC-31 , antisense: 51 -GGGAGTTGCTGTTGAAGT-31 .

4.5. Statistical Analysis

The results shown are summarized from three independent experiments and represent mean
˘ standard deviation. Significant differences were examined using one-way analysis of variance
test followed by Tukey’s multiple comparison test with SPSS 11.0 software (SPSS Inc., Chicago,
IL, USA). In addition, a correlation analysis was conducted to examine the relationships between
quercetin concentration and the levels of NO and/or intracellular calcium. A p-value < 0.05 was
considered significant.

5. Conclusions
In conclusion, this study demonstrated that quercetin has anti-inflammatory effects related with
inhibiting NO, IL-6, TNF-α, MCP-1, IP-10, RANTES, LIF, LIX, G-CSF, GM-CSF, and VEGF production
in poly(I:C)-induced macrophages via the calcium-STAT pathway. Further study is needed to evaluate
the clinical utility of quercetin for viral inflammation.

Acknowledgments: The authors thank Ji Young Lee and Hyun Joo Kim (College of Korean Medicine,
Gachon University) for their technical assistance.
Author Contributions: Y.K. and W.P. conceived and designed the experiments; Y.K. performed the experiments;
W.P. analyzed the data; and W.P. wrote the manuscript.
Conflicts of Interest: The authors declare no conflicts of interest.

Abbreviations
The following abbreviations are used in this manuscript:
NO nitric oxide
dsRNA double-stranded RNA
TLR Toll like receptor
poly(I:C) polyinosinic-polycytidylic acid
LPS lipopolysaccharide
Molecules 2016, 21, 450 8 of 10

IL interleukin
TNF tumor necrosis factor
MCP monocyte chemotactic protein
IP-10 interferon inducible protein-10
LIX lipopolysaccharide-induced CXC chemokine
LIF leukemia inhibitory factor
G-CSF granulocyte colony-stimulating factor
GM-CSF granulocyte macrophage colony-stimulating factor
VEGF vascular endothelial growth factor
STAT1 signal transducers and activators of transcription 1
STAT3 signal transducers and activators of transcription 3
Nor normal
Con control
ER endoplasmic reticulum

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Sample Availability: Quercetin samples are available from the authors.

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(CC-BY) license (http://creativecommons.org/licenses/by/4.0/).