Journal of Functional Foods 19 (2015) 495504

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Quercetin, a main flavonoid in onion, inhibits

the PGF2-induced uterine contraction in vitro
and in vivo

Chi-Hao Wu a,*, Tzong-Ming Shieh b, Kai-Lee Wang a,

Tsui-Chin Huang c, Shih-Min Hsia a,**
a
School of Nutrition and Health Science, Taipei Medical University, Taipei, Taiwan
b
Department of Dental Hygiene, College of Health Care, China Medical University, Taichung, Taiwan
c
PhD Program for Cancer Biology and Drug Discovery, College of Medical Science and Technology, Taipei
Medical University and Academia Sinica, Taipei, Taiwan

A R T I C L E I N F O A B S T R A C T

Article history: Dysmenorrhoea is considered to be caused by excessive levels of prostaglandins, which stimu-
Received 18 June 2015 late abnormal uterine contractions. This study aimed to investigate the modulatory effects
Received in revised form 31 August of quercetin that is a main flavonoid in onion on uterine contractility and its possible un-
2015 derlying mechanisms. In vitro and in vivo contractile activities of the uteri were determined
Accepted 11 September 2015 using uterine horns isolated from adult rats. The results indicated that (1) for contractions
Available online 26 October 2015 induced by PGF2-, oxytocin-, and carbachol, quercetin showed the most potent suppress-
ing effect among the tested flavonoids; (2) Ca2+-dependent uterine contractions were inhibited
Keywords: by quercetin; (3) quercetin reduced the PGF2-elicited Ca2+ responses in human uterine smooth
Onion muscle cells; (4) quercetin inhibited uterine contractions stimulated by Ca2+ channel acti-
Dysmenorrhoea vator and depolarisation in response to high K+; (5) quercetin was able to block Ca2+ influx
Flavonoids through voltage-operated Ca2+ channels in plasma membrane; and (6) quercetin effec-
PGF2 tively reduced PGF2-induced contractions through the administration of increasing doses
Quercetin of 10, 25, and 50 mg/kg in rats. The present findings suggest that onions major active com-
Uterine contractions pound, quercetin, may be a potential adjuvant for treating uterine disorders.
2015 Elsevier Ltd. All rights reserved.

* Corresponding author. School of Nutrition and Health Science, Taipei Medical University, Taipei, Taiwan. Tel.: +886 2 7361661 6554; fax:
+886 2 27373112.
E-mail address: wch@tmu.edu.tw (C.-H. Wu).
** Corresponding author. School of Nutrition and Health Science, Taipei Medical University, Taipei, Taiwan. Tel.: +886 2 7361661 6558; fax:
+886 2 27373112.
E-mail address: bryanhsia@tmu.edu.tw (S.-M. Hsia).
Abbreviations: ANOVA, analysis of variances; [Ca2+]i, intracellular calcium concentrations; DMEM, Dulbeccos modified Eagles medium;
DMSO, dimethyl sulphoxide; EDTA, ethylenediaminetetraacetic acid; EGTA, ethylene glycol bis(3-aminoethyl ether)-N,N,N,N-tetraacetic
acid; Fura 2-AM, fura-2/acetoxymethyl ester; FP, PGF2 receptor; HutSMCs, human uterine smooth muscle cells; MLC20, myosin light chain;
MLCK, myosin light chain kinase; MLCP, myosin light chain phosphatase; NSAIDs, non-steroidal anti-inflammatory drugs; PMSF,
phenylmethylsulfonyl fluoride; PGF2, prostaglandin F2; PVDF, polyvinylidene difluoride; ROCs, receptor-operated channels; SD, Sprague-
Dawley; SDS-PAGE, SDS-polyacrylamide gel electrophoresis; VOCs, voltage-operated channels
http://dx.doi.org/10.1016/j.jff.2015.09.028
1756-4646/ 2015 Elsevier Ltd. All rights reserved.
496 Journal of Functional Foods 19 (2015) 495504
1. Introduction
Flavonoids are widely occurring polyphenols that are found in
vegetables, fruits, wine, and tea. They are recognised for their
diverse physiological activity, including antioxidation, anti-
cancer, anti-inflammatory, anticarcinogenesis, antidiabetic,
antiallergic, acrylamide reduction and anti-vascular smooth
muscle contractions (Cheng, Chen, Zhao, & Zhang, 2015; Fu,
Chen, Li, Zheng, & Li, 2013; Park et al., 2012; Plaza et al., 2014;
Xu et al., 2013). Flavonoids based on different structures have
been identified as flavones, flavanones, flavanols, flavonols,
anthocyanidins, and isoflavonoids (Agati, Azzarello, Pollastri,
& Tattini, 2012; Friedman, 2007; Terahara, 2015). The relaxant
activity of flavonoids has been reported in different organs
showing spontaneous rhythmicity, including bladder (Dambros
et al., 2005) and intestine (Amira, Rotondo, & Mule, 2008).
Onion is a common vegetable and ingredient in the tradi-
tional medicine and dietary culture all over the world. Research
in modern medicine has confirmed that onion possesses many
positive health effects, including anti-tumour, anti-
inflammatory, and prevention and treatment of cardiovascular
diseases (Albishi, John, Al-Khalifa, & Shahidi, 2013a; Jakubowski,
2003; Terahara, 2015). Onions are known to contain a large
amount of flavonoids, including free forms (quercetin and
kaempferol) and glucose derivatives forms; the majority is
glucose derivative of quercetin and kaempferol (Albishi, John,
Al-Khalifa, & Shahidi, 2013b). Quercetin is a flavonoid found
in vegetables, nut, fruits and onion (Albishi et al., 2013a, 2013b;
John & Shahidi, 2010). This compound possesses physiologi-
cal activity, including the ability to induce glucose uptake in
L6 myotubes under oxidative stress (Dhanya et al., 2014). In a
previous study, we provided evidence that flavonoids (quer-
cetin and naringenin) have the most potent inhibitory effect
on uterine contractions in rats (Hsia, Kuo, Chiang, & Wang,
2008). However, phenolic acids (ferulic acid, vanillic acid, caffic
acid, gallic acid, syringic acid, and p-coumaric acid) did not
exhibit inhibitory effect on uterine contractions. Our previ-
ous study also showed that resveratrol had spasmolytic activity
on uterine contractions in rats (Hsia, Wang, & Wang, 2011).
These results demonstrated that flavonoids could be consid-
ered as a therapeutic agent for dysmenorrhoea.
Dysmenorrhoea or painful periods is a medical term for
cramps during menstruation (Latthe & Champaneria, 2014). The
prevalence of dysmenorrhoea is about 25%, and it usually de-
creases with age. According to a survey done on adolescent
females, the prevalence varies from 67.2 to 90% (Al-Jefout et al.,
2015; Ju, Jones, & Mishra, 2014; Kazama, Maruyama, &
Nakamura, 2015). In clinical practice, there are two types of dys-
menorrhoea: primary and secondary. Primary dysmenorrhoea
is a more common gynaecological disorder than secondary dys-
menorrhoea (Dawood, 2006; Latthe & Champaneria, 2014).
Secondary dysmenorrhoea is mainly due to pelvic lesions such
as endometriosis. Primary dysmenorrhoea has been reported
to cause elevation in prostaglandin production, such as pros-
taglandin F2 (PGF2), which may result in the contraction of
blood vessels and myometrium and insufficient blood flow to
the endometrium and cause pain in women (Bottcher et al.,
2014). Many treatments are available to alleviate the symp-
toms, such as the use of non-steroidal anti-inflammatory drugs
(NSAIDs). Most women choose to take NSAIDs as the first thera-
peutic option for dysmenorrhoea (Rainsford, 2006). Although
NSAIDs are effective and rapid in relieving menstrual pain,
however, it has many side effects that can affect the hepatic,
digestive, cardiac and renal systems (Hayes & Rock, 2002). There-
fore, traditional Chinese medicine may be a feasible alternative
to improve dysmenorrhoea (Mirabi, Alamolhoda, Esmaeilzadeh,
& Mojab, 2014). In this study, we investigated the effect of quer-
cetin on PGF2-induced uterine smooth muscle contraction both
in vitro and in vivo.
2. Materials and methods
2.1. Materials and chemicals
The analytic standards used in this research were purchased
from Sigma Chemical (St. Louis, MO, USA) except for luteolin
(ChromaDex, Irvine, CA, USA). Onion extract (#7-178A) that con-
tains 2% quercetin was purchased from a herbal company in
Taiwan (BIOMED HERBAL RESEARCH CO., LTD., Taichung,
Taiwan).
2.2. Uterine preparations and measurement of uterine
contraction
Female Sprague-Dawley (SD) rats weighing 200~300g were
housed in a temperature-controlled room (22 1 C) with
14 h/day artificial illumination (0600~2000), and food and water
provided ad libitum. The use of the animals was approved by
the Institutional Animal Care and Use Committee of the Taipei
Medical University (No. LAC-101-0236). All animals received
humane care in compliance with the Principles of Laboratory
Animal Care and the Guide for the Care and Use of Labora-
tory Animals, published by the National Science Council, Taiwan.
In the experiment, rats at estrus stage, which was confirmed
by microscopic examination of a vaginal smear, were decapi-
tated; both uterine horns were surgically removed and placed
in a Petri dish containing Krebss solution (113 mM NaCl, 4.8 mM
KCl, 2.5 mM CaCl2, 18 mM NaHCO3, 1.2 mM KH2PO4, 1.2 mM
MgSO4, 5.5 mM glucose, 30 mM mannitol, and pH adjusted to
7.4). After the adherent fat and mesenteric attachments were
removed, each uterine horn was cut into segments of equal
length (10 mm); these were used for measuring uterine oscil-
latory contraction. The segments were placed in isolated organ
baths containing physical solution at 37 C with 95% O2~5% CO2
supply. Each station was equilibrated using 1 g preload for at
least 60 min. Contractions were recorded with force displace-
ment transducers (PowerLab recorder ML785; Castle Hill, NSW,
Australia) by using Chart 5 software (Castle Hill, NSW, Austra-
lia) (Fig. 2A).
2.3. Human uterine smooth muscle cell (HutSMCs)
culture
Human uterine smooth muscle cells (HutSMCs) were pur-
chased from PromoCell Co. (PromoCell, Heidelberg, Germany).
HutSMCs were cultured in smooth muscle cell growth
medium-2 containing 5% (v/v) foetal calf serum, 0.5 ng/ml
 Journal of Functional Foods 19 (2015) 495504 497

epidermal growth factor, 2 ng/ml basic fibroblast growth factor,

and 5 g/mL insulin (PromoCell); seeded in a 24-well plate; and
incubated at 37 C in Dulbeccos modified Eagles medium
(DMEM)-F12 containing 100 U/mL penicillin and 100 g/mL strep-
tomycin. After incubation for approximately 24 h, the uterine
smooth muscle cells were collected and intracellular calcium
mobilisation was measured.

2.4. Measurement of [Ca2+]i

HutSMCs were treated with 10, 25, 50, 75, or 100 M quercetin
and 200 nM PGF2 for 24 h. They were then harvested using the
culture medium and washed twice with the same medium. A
cell suspension (1 106 cells/mL) was loaded with 5 mg of fura-
2/acetoxymethyl ester (Fura 2-AM; Fluka Chemical Corporation,
Milwaukee, WI, USA) dissolved in 5 mL of DMSO. A fluores-
cent probe was used for monitoring the intracellular calcium
concentrations ([Ca2+]i). The cells were incubated in the dark
for 30 min at 37 C. After extensive washing, 1 106 cells were
resuspended in 2.5 mL loading buffer (NaCl, 152 mM; MgCl2,
1.2 mM; CaCl2, 2.2 mM; KCl, 4.98 mM; and HEPES, 10 mM). Fluo-
rescence emission at 505 nm was monitored at 37 C by a dual-
wavelength spectrometer system, with excitation at 340 and
380 nm. Free [Ca2+]i was calculated using the method devel-
oped by Grynkiewicz (Grynkiewicz, Poenie, & Tsien, 1985) by
using the ratio of fluorescence intensities obtained every second
with a dissociation constant (Kd) of 135 nM. The dye was con-
sidered saturated after lysis with digitonin at the final
concentration of 0.16 mM. Minimum fluorescence was deter-
mined by adding 0.5 mL ethylene glycol bis(3-aminoethyl ether)-
N,N,N,N-tetraacetic acid (EGTA; Sigma Chemical Co.) to obtain
a final concentration of 8 mM.

2.5. Western blotting for MLC20 and p-ser19-MLC20

After uterine muscle strips treatment with vehicle (dimethyl
sulphoxide, DMSO) and quercetin for 20 min at 37 C, the treat-
ment uterine strips were collected and frozen immediately
in liquid nitrogen. The strips were homogenised in a
homogenisation buffer (pH 8.0) containing 1.5% sodium-
lauroylsarcosine, 1 103 M ethylenediaminetetraacetic acid
(EDTA), 2.5 103 M Tris-base, 0.68% phenylmethylsulfonyl fluo-
ride (PMSF), and 2% proteinase inhibitor cocktail, and then
disrupted by ground-glass homogeniser in ice-cold buffer. Tissue
extracts were centrifuged at 13,500 g for 10 min. The super-
natant fluid was collected and the protein concentration was
determined. Extracted proteins were denatured by boiling for
5 min in SDS buffer (0.125 M Tris-base, 4% SDS, 0.001% bro-
mophenol blue, 12% sucrose, and 0.15 M dithiothreitol). The
proteins (20 g) in the samples were separated on 12.5% SDS-
polyacrylamide gel electrophoresis (SDS-PAGE) at 50 V for 30 min
and then at 90 V for 90 min using a running buffer. The pro-
teins were transferred to polyvinylidene difluoride (PVDF)
membranes (NEN Life Science Products, Inc., Boston, MA, USA)
using a Trans-Blot SD semi-dry transfer cell (170-3940, Bio-
Rad, Hercules, CA, USA) at 64 mA (for 8 mm 10 mm membrane)
for 45 min in a blotting solution. ECL detection reagent
(PerkinElmer Life Sciences Boston, MA, USA) was used to
visualise the immunoreactive proteins on PVDF membranes
after transfer. The quantification software was Multi-Gauge V3.0.
Fig. 1 Effect of onion extract on PGF2-induced uterine
contractions in the rats. (A) Representative recordings of
PGF2 (106 M)-induced contractions treated with vehicle
(PBS) only, and the effects of cumulative additions of onion
extract (0.11 mg/mL) are shown. (B) Rat uterine segments
were treated PGF2 (106 M), and exposure of rat uterine
smooth muscles to vehicle or onion extract (0.1, 0.125, 0.25,
0.5 and 1 mg/mL). Recovery test about onion extract on
PGF2-induced uterine smooth muscle contractions in the
rats. 1st: First PGF2-treated; 2nd: second PGF2-treated after
wash. PGF2-induced contractions before the addition of
quercetin were considered as the control (100%, quercetin:
0 mg/ml group). Data represent mean SEM from four
independent experiments. *p < 0.05, **p < 0.01 when
compared with control at stimulated with PGF2.
2.6. Measurement of uterine contraction in vivo
In the experiment, the rats that were confirmed to be in the
estrous stage by microscopic examination of a vaginal smear
were used. They were anaesthetised with pentobarbitone (18 mg
in 0.3 mL, i.p). A small mid-portion of a uterine horn with as-
sociated mesometrium was obtained through a ventral incision
made in the skin and body wall. A 1- to 2-mm-long incision
498 Journal of Functional Foods 19 (2015) 495504

Fig. 2 Effect of flavonoids on PGF2-induced uterine contractions in the rats. (A) The scheme of rat uterine preparations and
measurement of uterine contraction in vitro. (B) Rat uterine segments were treated PGF2 (106 M), and exposure of rat
uterine smooth muscles to vehicle or flavonoids (apigenin, naringenin, luteolin, quercetin and kaempferol) (10, 25, 50, 75
and 100 M). (C) Representative recordings of PGF2 (106 M), oxytocin (106 M) or carbachol (105 M)-induced contractions
treated with vehicle (DMSO) only, and the effects of cumulative additions of quercetin (10100 M) are shown.
(D) Dose-dependent effects of quercetin on the mean peak amplitude. PGF2-, oxytocin- or carbachol-induced contractions
before the addition of quercetin were considered as the control (100%, quercetin: 0 M group). Data represent mean SEM
from six independent experiments. *p < 0.05, **p < 0.01 when compared with control stimulated with PGF2, oxytocin or
carbachol.
 Journal of Functional Foods 19 (2015) 495504 499

was made at the distal end of the exposed uterus, and a thin,
finger-shaped latex balloon was attached to a polyethylene cath-
eter (Becton-Dickinson, Franklin Lakes, NJ, USA), which was a
modification of a method described in a previous study (Hsia
et al., 2011; Hsia, Yeh, Kuo, Wang, & Chiang, 2007). The cath-
eter was connected to a transducer (PowerLab recorder ML785;
Castle Hill). Then, the rats were catheterised via the right jugular
vein and injected with (PGF2 0.2 mg/kg) or PGF2 plus querce-
tin (10, 25, or 50 mg/kg) via the jugular catheter, and the
contractions were recorded by this transducer with Chart 5.1
software (Castle Hill, NSW, Australia) (Fig. 5A).
2.7. Statistical analysis
Data were presented as the mean standard deviation (SD).
Differences between the means were analysed by one-way
analysis of variance (ANOVA) using the SPSS system, version
11.0 (SPSS, Chicago, IL, USA). Group means were compared using
a one-way ANOVA and Duncans multiple-range test. For com-
parison of two groups, Students t-test was used. The difference
between two means was considered statistically significant
when p < 0.05 and highly significant when p < 0.01.
3. Results and discussion
3.1. Effect of onion extract on PGF2-induced contraction
An increase in the [Ca2+]i in the uterine smooth muscles induces
muscles to contract. Previous studies have demonstrated that
[Ca2+]i is regulated via two different Ca2+ channels receptor-

Fig. 3 Inhibitory actions of quercetin on Ca2+-dependent contractile responses. (A) Muscle segments were initially
pretreated in a Ca2+-free medium containing the vehicle (0.2% DMSO) or quercetin (100 g/mL) for 60 min, and calcium
(0.055 mM) was then cumulatively applied to trigger muscle contraction. (B) Inhibition of PGF2-induced increases in [Ca2+]i
by quercetin in HutSMCs. HutSMCs were treated with vehicle (0.2% DMSO) or quercetin (10, 25, 50 and 100 g/mL) along
with PGF2 (200 nM). (C) Inhibition of PGF2-induced increases in MLC20 phosphation by quercetin in rat uterine smooth
muscle. Rat uterine segments were treated with vehicle (DMSO) or quercetin (10, 25, 50 and 100 g/mL) along with PGF2
(106 M). **P < 0.01 vs. PGF2-treated group, assessed by Duncans multiple-range test. #P < 0.05 vs. vehicle-treated group,
assessed by Students t test. Each column represents the mean SEM.
500 Journal of Functional Foods 19 (2015) 495504

and voltage-operated channels (ROCs and VOCs) in the mem-

brane of uterine smooth muscle cells (Berridge, 2008; Bolton,
1979). For ROCs, when uterotonic compounds (PGF2, oxyto-
cin or carbachol) bind to uterine smooth muscle cell membrane
G-protein coupled receptors to elicit uterine smooth muscle
contractions, the [Ca2+]i increases via both the influx of extra-
cellular Ca2+ through Ca2+ channels and by release of intracellular
stored Ca2+. To investigate the potential inhibition of PGF2-
induced uterine contraction by onion extract in rats, we first
examined the effect of flavonoids on PGF2-induced uterine con-
traction. PGF2 is a major factor for inducing uterine contractions
during dysmenorrhoea (Bottcher et al., 2014). PGF2 connects
with PGF2 receptor (FP) on the spiral arterioles to increase
uterine contractility, causing ischaemia pain. PGF2 increases
the intracellular calcium concentration by stimulating the
release of stored calcium, which produces a phasic contrac-
tion (Rosenwaks et al., 1981). When PGF 2 was added to
spontaneously contracting uteri, a significant increase in the
amplitude was observed (Dawood, 2006; Rosenwaks et al., 1981).
In this study, onion extract was effective in inhibiting
PGF2-induced uterine contraction (Fig. 1A and B). We also as-
sessed tissue viability using a recovery study by removing the
inhibitory substance and washing the tissue with Krebss so-
lution for 20 min, then re-analysing the tissue in the presence
of a stimulus (PGF2, 106 M). From our results, we found that
PGF2 could reverse the inhibition after removing the resveratrol
(Fig. 1C). Our previous study showed that adlay (Coix lachryma-
jobi L. var. ma-yuen Stapf.) hull extract could inhibit
PGF2-induced uterine smooth muscle contraction both in vitro
and in vivo (Hsia et al., 2008). Thus, adlay hull may be consid-
ered as a potential alternative therapeutic agent for
dysmenorrhoea. Adlay is a plant used both as a medicine and Fig. 4 Effects of quercetin on high K+ (KCl) or Bay K 8644-
food. Research has confirmed that adlay possesses many posi- induced uterine contractions in the rats. (A) Representative
tive health effects, including the ability to regulate blood sugar, recordings of high K+ (KCl 50 mM) or Bay K 8644
blood lipids, and blood pressure; to improve gastrointestinal (106 M)-induced contractions and the effects of cumulative
physiology; to regulate immune and having anti-inflammatory, additions of quercetin (10100 M) are shown. (B) Rat
antitumour effects; and to regulate reproductive endocrine hor- uterine segments were treated high K+ (KCl 50 mM) or Bay
mones (Hsia, Chiang, Kuo, & Wang, 2006; Hsia et al., 2007, 2008, K 8644 (106 M), and exposure of rat uterine smooth
2009; Hsu, Lin, Lin, Kuo, & Chiang, 2003; Huang, Chung, Kuo, muscles to quercetin (10, 25, 50, 75 and 100 M). High K+
Lin, & Chiang, 2009; Huang et al., 2014; Kuo et al., 2002; Wu (KCl) or Bay K 8644-induced contractions before the
et al., 2014). We also provided evidence that flavonoids (quer- addition of quercetin were considered as the control (100%,
cetin and naringenin) have the most potent inhibitory effect quercetin: 0 M group).
on uterine contractions in rats (Hsia et al., 2008). However, phe-
nolic acid (ferulic acid, vanillic acid, caffic acid, gallic acid,
syringic acid, and p-coumaric acid) did not exhibit inhibitory
effect on uterine contractions. Our previous study also showed hormone responsible for uterine and mammary gland con-
that resveratrol had spasmolytic activity on uterine contrac- tractions. Carbachol also has the ability to increase [Ca2+]i in
tions in the rats (Hsia et al., 2011). Thus, these flavonoids could the uterine smooth muscles to induce uterine contractions. In
be considered as a therapeutic agent for dysmenorrhoea. our study, PGF2 (106 M), oxytocin (107 M) and carbachol (106 M)
caused rhythmic contractions of the isolated rat uterus. Quer-
3.2. Effect of flavonoids on PGF2-, oxytocin-, and cetin (10100 M) showed a significant inhibitory effect on the
carbachol-induced contraction amplitude of PGF2-, oxytocin-, and carbachol-induced con-
traction in a concentration-dependent manner (Fig. 2C and D).
To investigate the potential inhibition of PGF2-induced uterine All contractions were ceased when the highest concentra-
contraction by flavonoids (apigenin, naringenin, luteolin, tion of quercetin (100 M) was added. Quercetin and kaempferol
kaempferol and quercetin) in rats, we first examined the effect are well-known antioxidative agents (Albishi et al., 2013b). Also,
of flavonoids on PGF2-induced uterine contraction. In this study, quercetin is a major active compound in onion skin extract.
quercetin was more effective in inhibiting PGF2-induced uterine A previous study has shown that quercetin could interfere with
contraction compared to other flavonoids (apigenin, naringenin, vascular smooth muscle contraction (Hou, Liu, Niu, Cui, &
luteolin and kaempferol) (Fig. 2B). Oxytocin is a nonapeptide Zhang, 2014). In the present study, quercetin was found to block
 Journal of Functional Foods 19 (2015) 495504 501

ROCs and being able to cause uterine contraction. These results
suggest that quercetin acts on the downstream receptors for
the muscle relaxation effect.
Owing to quercetin as a major flavonoid component present
in onions, and the different components present in onions prob-
ably have an additive action, the commercial onion extracts
that contain 2% quercetin were used in this study. We esti-
mated that 50, 125, 250, 500, 1000, and 2000 g/mL onion extracts
contain 1, 2.5, 5, 10, 20, and 40 g/mL quercetin, respectively.
Therefore, quercetin and the same quercetin content of onion
extracts were used to examine the anti-contraction capacity.
The present results show that quercetin incorporated either
as a purified compound or as component in onion extracts at
the same concentrations could similarly attenuate PGF2-
induced uterine contraction in SD rats, suggesting no observable
matrix effects and the anti-contraction activity of onion is likely
attributable to its higher levels of quercetin (Supplemental
Fig. S1).
3.3. Effect of quercetin on Ca2+-dependent contractions
External Ca2+ is necessary for spontaneous contraction. PGF2??
could increase external Ca2+ influx into cell through calcium
channels and induce uterine contraction. To investigate whether
the inhibition of uterine contractions by quercetin is due to
blocking the external Ca2+ influx, we performed the following
experiments in a Ca2+-free Krebss solution. In the absence of
external Ca2+, the spontaneous contractions were abolished.
Further, when the Ca2+-free Krebss solution was supplied with
increasing concentrations of Ca2+ from 0.05 to 5 mM, the spon-
taneous contractions were restored. However, when 100 g/
mL quercetin was added to the Ca2+-containing Krebss solution,
the Ca2+-induced uterine contractions were not observed
(Fig. 3A). Thus, this inhibitory effect of quercetin was due to
the blockage of external Ca2+ influx. In our previous study, we
also found that resveratrol and adlay extract could block ex-
ternal Ca2+ influx into smooth muscle cell.

Fig. 5 Effects of quercetin on PGF2-induced uterine contractions in vivo. (A) The scheme of rat uterine preparations and
measurement of uterine contraction in vivo. The rats were catheterised via the right jugular vein and injected with (PGF2
0.2 mg/kg) or PGF2 plus quercetin (10, 25, or 50 mg/kg) via the jugular catheter, and the contractions were recorded.
Representative recordings of PGF2-induced contractions and the effects of cumulative additions of quercetin (10100 M)
are shown. (B) Dose-dependent effects of quercetin on the mean peak amplitude. These results are representative of the
records of 6 rats (n = 6). **P < 0.01 vs. PGF2-treated group, assessed by Duncans multiple-range test. #P < 0.05 vs. vehicle-
treated group, assessed by Students t test. Each column represents the mean SEM.
502 Journal of Functional Foods 19 (2015) 495504

3.4. Effects of quercetin on [Ca2+]i and myosin light chain
(MLC20) phosphorylation
In order to study whether quercetin inhibits [Ca2+]i and affects
muscular contraction, the HutSMCs were treated with differ-
ent concentrations of quercetin (10, 25, 50, and 100 M) along
with PGF2 (200 nM). Quercetin (25 to 100 mM) significantly
reduced the PGF2-induced [Ca2+]i (Fig. 3B). In contrast, admin-
istration of quercetin (10~100 M) had no effect on HutSMCs
number (data not shown). This result implies that the de-
crease in [Ca 2+]i was not attributed to the cytotoxicity of
quercetin on smooth muscle cells. In addition, in order to study
whether quercetin inhibits the MLC20 phosphorylation and
leads to smooth muscle relaxation, the rat uterine smooth
muscle was treated with quercetin (10, 25, 50, and 100 M) along
with PGF2 (106 M). Quercetin treatment (50100 M) signifi- Phillippe, 1996). Both Bay K 8644 and high K+ induced contrac-
cantly reduced the PGF2-induced MLC20 phosphorylation
(Fig. 3C and D). Myosin light chain (MLC20), which is phos-
phorylated at Thr18 and Ser19 by myosin light chain kinase
(MLCK) in a Ca2+/calmodulin-dependent manner, is one of the
proteins involved in uterine smooth muscle contraction. The
phosphorylation of MLC20 could interact with -actin fila-
ments, resulting in uterine smooth muscle contraction.
Conversely, when MLC20 is dephosphorylated by myosin light
chain phosphatase (MLCP), it leads to relaxation (Ikebe &
Hartshorne, 1985; Ito, Nakano, Erdodi, & Hartshorne, 2004). Here,
we demonstrated that quercetin inhibited smooth muscle con-
traction by affecting MLC20 phosphorylation.
3.5. Effect of quercetin on KCl- and BayK8644-induced
contraction
Addition of KCl (50 mM) caused tonic contraction and maximum
contraction of the uterine strips. Bay K8644 also caused
rhythmic contractions of the isolated rat uterus. However, in
the presence of quercetin (10100 M), the amplitude of KCl-
and Bay K8644-induced contraction was significantly de-
creased in a concentration-dependent manner (Fig. 4A and B).
High K+ (>30mM) is known to cause smooth muscle contrac-
tion through the opening of voltage-dependent L-type Ca2+
channels, thus allowing an influx of extracellular Ca2+ causing
a contractile effect (Bolton, 1979). In the present study, quer-
cetin was able to reduce the contraction produced by KCl on
isolated uterus tissues, showing its ability in blocking Ca2+ influx.
Bay K8644 is an L-type Ca2+-channel activator that can in-
crease intracellular calcium [Ca 2+ ]i and activate the
phosphatidylinositol-signalling pathway and cytosolic calcium
oscillation-like phenomena, thereby resulting in the genera-
tion of phasic myometrial contractions (Chien, Saunders, &
tion were also abolished by quercetin administration. Moreover,
the spontaneous contractions stimulated by elevated extra-
cellular Ca2+ were also abolished by quercetin treatment. In
addition, PGF2 increased [Ca2+]i concentration, which was also
reduced by quercetin treatment in HutSMCs. This result dem-
onstrated that quercetin is capable of inhibiting Ca2+ influx.
3.6. Effects of quercetin on isolated uterus in rat (in vivo)
Flavonoids are widely found in plants and food and exhibit nu-
merous pharmacological and biological effects, including
vascular protection. Previous studies revealed that flavonoids
could inhibit vascular smooth muscle contraction (Sun et al.,
2013; Wang et al., 2014). Results from this study showed that
quercetin at pharmacologically relevant concentration (100 M)
was able to inhibit PGF2-induced uterine contraction in vivo
(Fig. 5B and C). To the best of our knowledge, this study is
the first to investigate the effect of quercetin in inhibiting

Fig. 6 The mechanism of quercetin inhibits PGF2-induced uterine contractions. (1) Ca2+-dependent uterine contractions
were inhibited by quercetin; (2) quercetin reduced the PGF2-elicited Ca2+ responses in human uterine smooth muscle cells;
(3) quercetin inhibited uterine contractions stimulated by Ca2+ channel activator and depolarisation in response to high K+;
and (4) quercetin was able to block Ca2+ influx through voltage-operated Ca2+ channels in plasma membrane.
 Journal of Functional Foods 19 (2015) 495504
PGF2-induced uterine contraction in rats. In summary, the
present study demonstrates that the effect of quercetin on
uterine contractions could be due to its interference with ROCs
or/and VOCs. Our present study demonstrated that quercetin
could inhibit [Ca2+]i induced by PGF2, oxytocin, carbachol, KCl,
and Bay K 8644, and block the Ca2+ influx through ROCs and
VOCs (Fig. 6).
4. Conclusions
The present study demonstrated that quercetin could inhibit
PGF2-induced uterine smooth muscle contraction both in vitro
and in vivo. The inhibition of uterine smooth muscle contrac-
tion is, in part, due to the blockage of ROCs and VOCs in rats.
Therefore, quercetin may potentially be used in the treat-
ment of dysmenorrhoea. However, additional clinical
experiments are required to confirm this finding.
Acknowledgements
This study was supported by grants [MOST103-2313-B-038-
003-MY3, MOST103-2313-B-038-001-MY3, MOST102-2314-B-
039-015-MY3 and NSC102-2313-B-038-001-] from the Ministry
of Science and Technology, Taiwan, Republic of China.
Appendix: Supplementary material
Supplementary data to this article can be found online at
doi:10.1016/j.jff.2015.09.028.
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