Quercetin affects uterine smooth muscle contractile activity in gilts.

Quercetin is one of the natural flavonoids occurring in fruits (e.g. apple, blueberries, black currant, orange), vegetables (e.g. onion, broccoli, spinach, cabbage), flowers (e.g. hawthorn, chestnut, black lilac) and herbaceous plants (e.g. horsetail, rue, chamomile) [ 1 ]. It is also present in tea, wine [ 2 ] and honey [ 3 ]. After oral administration, quercetin is deglycosylated in the lumen of the small intestine in the brush border membrane by lactose-frisine hydrolase, from where the detached aglycons are absorbed by the intestinal wall through passive diffusion. Quercetin is also deglycosylated in enterocytes, from where the aglycones are absorbed and metabolized in the small intestine, colon, liver and kidneys through active transport by the Na + / glucose transporter. Subsequently, in liver epithelial cells, quercetin is metabolized by phase II metabolism proteins. The resulting metabolites are then transported through the portal vein to the liver where they are further degraded. Metabolites formed in the liver travel with the blood to the tissues, where they show different biological activity. In turn, metabolites resistant to the action of intestinal hydrolases and not absorbed in the small intestine pass into the large intestine, where they are transformed with the participation of enzymes secreted by enterobacteria [ 4 ]. Quercetin has strong antioxidant properties because it has the ability to scavenge free radicals and chelating transition metal ions [ 5 ]. It is one of the most potent scavengers of reactive oxygen species [ 6 ]. Quercetin not only prevents the propagation of lipid peroxidation but also increases glutathione levels which contribute to preventing free radical formation [ 7 ]. The antioxidant activity of quercetin includes the inhibition of lipid peroxidation of cell membranes, protects against the oxidation of low-density lipoproteins (LDL) and increases high-density lipoproteins [ 8 ]. Because of antioxidant properties, quercetin can reduce the occurrence of neurodegenerative processes occurring, e.g. in Alzheimer’s and Parkinson’s diseases [ 9 ]. Moreover, the consumption of flavonoids showed a reduction in coronary heart disease [ 10 ]. In vitro studies have been demonstrated that quercetin exerts endothelium-independent vasodilator effects, protective effect on nitric oxide and endothelial function under conditions of oxidative stress, platelet antiaggregant effects, inhibition of LDL oxidation, reduction of adhesion molecules and other inflammatory markers and prevention of neuronal oxidative and inflammatory damage [ 11 ]. Moreover, it has anti-cancer properties through its antioxidative effects as well as kinase and cell cycle inhibition and induced apoptosis [ 12 ]. However, the pro-oxidant activity of quercetin has also been observed [ 13 ]. The available research results also indicate that quercetin significantly affect reproductive tract function. Both in vitro and in vivo results demonstrated by Park et al. [ 14 ] suggest that quercetin can be used as a natural therapeutic to reduce and/or treat endometriosis. Moreover, quercetin improved polycystic ovary syndrome in rats through its phytoestrogenic effects and mimicking oestrogen’s function, expressed as an increase in the adiponectin level and a decrease in both the expression of aromatase and the oestradiol level [ 15 ]. It has also been demonstrated that the addition of quercetin to feed significantly increased the secretion of oestradiol, progesterone, follicle-stimulating hormone and luteinizing hormone in laying hens [ 16 ]. It has been demonstrated that quercetin has an anti-inflammatory activity which is associated with inhibition of arachidonic acid metabolism and production of inflammatory mediators such as prostaglandins and leukotriens [ 17 ]. However, it is well-documented that both prostaglandins and leukotrienes are involved in the regulation of uterine contractile activity [ 18 – 20 ]. Moreover, it was shown that quercetin relaxes the tonic contraction in the rat uterus induced by prostaglandin F 2α (PGF 2α ), oxytocin (OT) and carbachol [ 21 ]. Quercetin is a naturally occurring selective phosphodiesterase-4 (PDE 4 ) inhibitor [ 22 ]. It has been demonstrated that two PDE 4 inhibitors (citomilast and rolipram) inhibit smooth muscle contractions in the human uterus and these changes were strongly expressed in pregnant than nonpregnant myometrium [ 23 ]. Moreover, it has also been shown that phosphodiesterase activity increased from proestrus to a peak in late metestrus and then decreased until the next proestrus period during the oestrous cycle in rats [ 24 ]. At present, there are no data on the role of quercetin in the regulation of the uterine contractile activity in pigs. Therefore, this study aimed to determine the effect of quercetin on the contractility of the myometrium in females with different physiological status, i.e. immature, cyclic and early pregnant gilts.

Eighteen crossbreed gilts (Large White x Polish Landrace) came from private breeding farm (L. Wisniewski breeding farm, Krolikowo, Poland) were assigned into three experimental groups (n = 6 in each). In the first group (immature), the uteri of gilts at the age of 4 to 5 months with an average body weight of 50–55 kg were used. In the second group (cyclic), the uteri of gilts on days 12–14 of the oestrous cycle at the age of 7 to 8 months with an average body weight of 120–130 kg were collected; the phase of the oestrous cycle was confirmed based on the morphology of the ovaries [ 25 ]. In the third group (pregnant), the uteri were collected from gilts on days 12–16 of gestation (implantation period); about 7–8 months old, weighing 120–130 kg; pregnancy was confirmed by washing the horns of the uterus with 10 mL PBS, pH = 7.4 in the presence of embryos. The procedure of selection and insemination of the gilts in this group was described previously [ 26 ]. Uteri from all animals were collected immediately after slaughter in meat processing plant ("TOMUS" Tomasz Reihs, Krolikowo, Poland) and then transported on ice to the laboratory within 0.5 h. All studies were conducted in accordance with the ethical standards of the Local Ethics Committee of the University of Warmia and Mazury in Olsztyn, which approved all procedures and granted consent (No. 91/2011 / DTN). The uterine strips used in the study were prepared as previously described [ 27 , 28 ]. Briefly, after slaughter uteri were placed on ice and strips (3 × 5 mm) of myometrium (comprising longitudinal and circular layer) were collected from the middle part of the horns. Thereafter, the strips were washed in saline and mounted between two stainless steel hooks in an organ bath (Schuler Organ bath type 809; Hugo Sachs Electronic, Germany) under a resting tension of 10 mN. The strips were kept in 5 mL of the Krebs-Ringer solution with the following composition (mM/l): NaCl—120.3, KCl—5.9, CaCl 2 −2.5, MgCl 2 −1.2, NaHCO 3 −15.5, glucose—11.5, temp. 37°C and pH 7.4. During the experiment, the solution was continuously saturated with a mixture of 95% O 2 and 5% CO 2 . For measuring the contractile activity of myometrial strips, a force transducer (HSE F-30 type 372) with a type 570 bridge coupler was used. A graphic recording was made on a recorder (Hugo Sachs Elektronik) with the HSE-ACAD/W software. The recording was started after a 60–90 min equilibration period. To check the viability of tissues and their usefulness for the study the strips were stimulated with increasing (10 −6 –10 −4 M) concentrations of ACh [ 29 ]. After washing, the strips were incubated with increasing (10 −13 –10 −1 M) concentrations of quercetin (Que) added at 15-minute intervals. Thereafter, the organ bath was washed and ACh at concentrations of 10 −6 –10 −4 M was administered. Only those results were differences in response to ACh stimulation at the beginning and the end of the experiment were less than 20% were included in the statistical analysis [ 29 ]. A sigmoid dose-response model relationship between examined effects (amplitude, frequency and tension) and quercetin concentration was analysed by nonlinear regression analysis with automatic outlier elimination (ROUT method, Q = 1%). All calculations were performed using GraphPad Prism version 6.07 (Graphpad Software, San Diego, CA, USA). In the first phase, exploratory dose-response analyses were conducted using various models. Finally, a log (inhibitor) vs. response model (variable-slope) model without constants was selected. The selected parameters were calculated: E max −maximal effect value, E 0 –no effect value (baseline), Slope–value of Hill slope of the model, and LogIC 50 –log-transformed concentration of quercetin that gives a half-maximal response. Tension (resting/baseline tension expressed in mN), frequency (the number of observed peaks) and amplitude (the difference between the minimum and maximum value for a single contraction expressed in mN) as parameters of spontaneous myometrial strips contractile activity were calculated for 10 min before quercetin administration (pre-treatment period) and accepted as 100%. The results calculated for 10 min periods after the administration of each concentration of quercetin were expressed as a percentage of the tension, frequency and amplitude measured in the pre-treatment period. The statistical significance of the differences between pre-treatment and post-treatment periods as well as between three examined groups of animals were assessed by one-way analysis of variance ANOVA (Graphpad Prism 6.07, Graphpad Software, San Diego, CA, USA) followed by Bonferroni’s multiple comparison test. The differences with a P-value less than 0.05 were considered significant. A separate statistical analysis was performed to assess the quality of pharmacodynamic models. The models were qualified for raw data analysis based on Akaike information criterion (AIC) and after evaluation, the standard error of the estimate by robust standard deviation of the residuals, calculation (RSDR) of the best model was selected.

Typical diagrams showing contractile activity before and after quercetin administration in all examined groups are presented in Fig 1 . Quercetin significantly (P<0.05–0.01) decreased the tension only in the cyclic group at concentrations of 10 −6 –10 −1 M as compared to the pre-treatment period but it did not alter this parameter in immature or early pregnant groups ( Fig 2 ). The frequency of contractions decreased in all examined groups and these changes were significant after quercetin administration in concentrations of 10 −5 –10 −1 M (P<0.01) in the immature group, in concentrations of 10 −11 –10 −1 M (P<0.05–0.001) in the cyclic group and in concentrations of 10 −7 –10 −1 M (P<0.01–0.001) in the early pregnant group compared to the pre-treatment period ( Fig 2 ). Quercetin significantly (P<0.01–0.001) inhibited the amplitude of contractions in concentrations of 10 −11 –10 −1 M in the cyclic group and in concentrations of 10 −10 –10 −1 M the early pregnant group but did not cause significant changes in the immature group compared to the pre-treatment period ( Fig 2 ). Diagrams showing the contractile activity of the porcine myometrial strips collected from sexually immature gilts (A), on days 12–14 of the oestrous cycle (B) and on days 12–16 (C) before (15 min) and after administration of concentration (Que; 10 −13 –10 −1 M). Effect of quercetin (Que) on tension (A), frequency (B) and amplitude (C) of contractions of myometrium in immature pigs (-●-; n = 6) and on days 12–14 of the oestrous cycle (-▲-; n = 6) and 12–16 of pregnancy (-○-; n = 6). The results calculated for a 15 min period after treatment with each quercetin concentration were expressed as a percentage (mean ± SD) of the amplitude, frequency and tension determined for a 15 min period before treatment (Bt). *p<0.05, **p<0.01,***p<0.001 –significant differences as compared to the contractile activity before the treatment. Calculated pharmacodynamic parameters and statistical parameters describing PD model goodness-of-fit are presented in Table 1 . In all groups (immature, cyclic, pregnant), the effect of quercetin on model parameters was noted. Both in case tension and frequency, the LogIC 50 values were highest for the immature group and the lowest for cyclic (LogIC 50 value: cyclic<pregnant<immature). Regarding amplitude, the LogIC 50 values were highest in the immature group and lowest for pregnant pigs (LogIC 50 value: pregnant<cyclic<immature). Dose-response value (shown as mean with 90% confidence interval) of calculated parameters based on changes in tension and amplitude and frequency of contractions of the uterine myometrial strips collected from gilts immature (n = 6), on days 12–14 of the oestrous cycle (cyclic; n = 6) and 12–16 of pregnancy (pregnant; n = 6) and treated with increasing (10 −13 –10 −1 M) concentrations of quercetin (Que). E max −maximal effect value, E 0 –no effect value (baseline), Slope–value of Hill’s slope of the model, LogIC 50 –log-transformed concentration of quercetin that gives half-maximal response, AIC–Akaike information criterion, RSDR–robust standard deviation of the residuals.

In conclusion, the present study shows that quercetin causes relaxation of the myometrium in both cyclic and early pregnant pigs under progesterone dominance. However, the exact mechanism of this action has yet to be determined.

All the inorganic salts (NaCl, KCl, CaCl 2 , MgCl 2 , NaHCO 3 ) needed to prepare the Krebs-Ringer buffer, ethanol and glucose were purchased from Chempur (Piekary Śląskie, Poland). Acetylcholine chloride (ACh) and quercetin (Que) were purchased from Sigma (St. Louis, MO, USA). A stock solution of quercetin (10 −1 M) was prepared in ethanol and series of dilutions were made with deionized water on the day of experimentation.