Polyphenol supplementation inhibits physiological increase of ... - plefa

Prostaglandins, Leukotrienes and Essential Fatty Acids 136 (2018) 77–83

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Prostaglandins, Leukotrienes and Essential Fatty Acids journal homepage: www.elsevier.com/locate/plefa

Polyphenol supplementation inhibits physiological increase of prostaglandin E2 during reproductive period – A randomized clinical trial☆

T

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A.M. Zilioa, , P. Zielinskya,b, I. Viana, K. Lamperta, D. Rauppa, C. Weschenfeldera, C. Bruma, A. Arnta, A. Piccoli Jr.a, L.H. Nicolosoa, M.I. Schauna, M. Markoskia a b

Fetal Cardiology Unit, Institute of Cardiology, Porto Alegre, Rio Grande do Sul – Brazil Universidade Federal do Rio Grande do Sul – UFRGS, Porto Alegre, Rio Grande do Sul – Brazil

A R T I C L E I N F O

A B S T R A C T

Keywords: Polyphenols Prostaglandin E2 Hormonal contraceptives Supplementation Inflammation

Anti-inflammatory property of polyphenols and their effect on the metabolism of prostaglandins is not established in healthy humans. This study aimed to evaluate the effect of polyphenol supplementation in plasma levels of prostaglandin E2 and other markers of inflammation and oxidative stress in women using contraceptives. In this randomized double-blind clinical trial, women aged 25–35 years were selected. Participants received capsules containing polyphenols or placebo, to be consumed for fifteen days. From 40 women randomized, 28 completed the study. Control group showed a significant increase in the levels of PGE2 (p=0.01) while the polyphenols group showed no change in these levels (p=0.79). There was an increase in hsCRP (p < 0.01) and F2-isoprostane (p=0.04) in the control group. The GSSG to GSH ratio significantly reduced in the polyphenols group (p=0.02). Supplementation with polyphenol capsules inhibited the increase in markers of inflammation and oxidative stress in women of childbearing age using combined hormonal contraceptives.

1. Introduction The growing evidence of the antioxidant and anti-inflammatory effects of polyphenols and their potential to prevent degenerative diseases [1], has interested researchers and the general public, leading to an increase in the intake of polyphenol-rich foods as well as the consumption of these substances in the form of supplements [2]. Despite the potential benefits, the effect of high consumption of polyphenols during the gestational period is still controversial [3]. The consumption of polyphenol-rich foods after the third trimester of pregnancy has been shown to influence the fetal dynamics of the ductus arteriosus [4,5], and maternal restriction of polyphenols can lead to reversal of fetal ductal constriction [3,6]. This functional damage has high prevalence (abstract in Arquivos Brasileiros de Cardiologia) and may cause severe fetal and neonatal complications such as heart failure, fetal hydrops, persistent pulmonary hypertension of the newborn and even death [7]. The patency of the fetal ductus arteriosus is maintained by

prostaglandin E2 (PGE2), an eicosanoid derived from arachidonic acid (AA) which is produced via cyclooxygenase (COX) during the inflammatory response and stimulated on oxidative stress [8] and is also physiologically released after the third trimester of pregnancy [9,10]. It is believed that excessive consumption of polyphenols in the third trimester of pregnancy can lower the levels of circulating PGE2 by inhibition of COX, similar to the effects to nonsteroidal anti-inflammatory drugs (NSAIDs), and therefore change the dynamics of the fetal ductus arteriosus. Although experimental studies have already demonstrated the influence of polyphenols on inflammatory responses and levels of prostaglandins, clinical studies with healthy individuals are inconclusive and, in most cases, evaluate only clinical outcomes [2]. Therefore, the role of polyphenols on inflammatory responses, inducing alteration in plasma concentration of PGE2 in women of childbearing age using combined hormonal contraceptives, population chosen here to approach the gestational period, due to the pro-inflammatory effect of hormones, is not clarified.

Abbreviations: 8-iso-PGF2a, F2-isoprostane; AA, arachidonic acid; ANCOVA, analysis of covariance; BMI, body mass index; CG, control group; COX, cyclooxygenase; FFQ, Food Frequency Questionnaire; GEE, Generalized Estimating Equation; GSSG/GSH, Glutathione disulphide to Glutathione ratio; hs-CRP, sensitivity C-reactive protein; IPAQ, International Physical Activity Questionnaire; mg GAE/g creatinine, milligrams of gallic acid equivalents per gram of creatinine; mg, milligrams; mg/dL, milligram per deciliter; NSAIDs, nonsteroidal anti-inflammatory drugs; PG, polyphenols group; pg/mL, picogram per milliliter; PGE2, prostaglandin E2; S.P.S.S., Statistical Package for Social Science Program; WHO, World Health Organization ☆ This trial is registered at clinicaltrials.gov as NCT02567617. ⁎ Correspondence to: Fetal Cardiology Unit, Princesa Isabel Avenue, 370 Santana Porto Alegre, CEP: 90, 620-001 RS, Brazil. E-mail addresses: [email protected], [email protected] (A.M. Zilio). http://dx.doi.org/10.1016/j.plefa.2017.04.001 Received 27 January 2017; Received in revised form 30 March 2017; Accepted 3 April 2017

0952-3278/ © 2017 Elsevier Ltd. All rights reserved.


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Table 1 Composition of polyphenol's capsules.

Substance name Scientific name Class Subclass Chemical structure Dose / capsule% Manufacturer Total weighta Part used Characteristics Date of manufacture / validity a

Substance 1

Substance 2

Substance 3

Substance 4

Substance 5

Resveratrol Polygonum cuspidatum Stilbene

Chlorogenic acid Coffea arabica L.

Cyanidin Pinus pinaster

Quercetin Dimorphandra mollis

Catechin Camellia sinensis

Phenolic acids Hydroxycinnamic acids

Flavonoids Anthocyanins

Flavonoids Flavonols

Flavonoids Flavones

C16H18O9 19.4% Gama 612 mg Seed of Green Coffee Yellowish brown powder with characteristic odor and taste Man:08/2013 Val:08/2015

C15H11O6+ 19.6% Fagron 618 mg Endoderm of plant Reddish brown powder with characteristic odor Man:10/2012 Val:10/2015

C15H10O7 20% Galena 630 mg Pods of plant Crystalline fine powder, orange staining, odorless Man:09/2013 Val:09/2016

C15H14O6 22% Pharmanostra 690 mg Green tea leaf Brown powder

C14H12O3 19% Fagron 600 mg Grape seed White crystalline powder Man:10/2012 Val:10/2014

Man:08/2012 Val:08/ 2014

Considering dose/day (10 capsules).

polyphenol concentration three times higher than the median observed in the study of validation of the Food Frequency Questionnaire (FFQ) [11], totaling 3000 mg of polyphenols per day. This dose was defined to evaluate the effect of polyphenols in PGE2 levels, i.e., observe the dose effect of this supplementation. A higher dosage was chosen in order to reduce the risk of food ingestion of control group (CG) to overcome the dosage of polyphenol supplementation in the PG. The polyphenol capsules contained extracts with resveratrol, catechin, quercetin, chlorogenic acid and cyanidin, as detailed in Table 1. The placebo capsules, containing only corn starch, were manufactured by Company Delaware in 10/09/2013 with validity until 10/09/2015. The starch presented a thin, white to slightly yellowish aspect, was odorless and tasteless. The substances were included in white and bordeaux capsules, similar to the control ones, by the compounding pharmacy Pharmacus (Porto Alegre, Brazil). The final weight, appearance and smell of the capsules were identical. The participants were advised to ingest the capsules without opening them. Participants of both the polyphenol and control groups were oriented to consume 10 (ten) capsules per day for 15 (fifteen) days. The last capsule was ingested 12 h before the second sample collection, because this is the estimated time for excretion of polyphenols [12]. The 15-day supplementation period was established based on reversal of fetal ductal constriction after suspension of maternal intake of polyphenol-rich foods [6].

The aim of this study was to evaluate the effect of polyphenol supplementation on the plasma level of PGE2 in women during reproductive period using combined hormonal contraceptives. Secondary objectives included the evaluation of markers of systemic inflammation and oxidative stress, as well as total polyphenols in urine excretion as an indicative of polyphenol metabolizing. 2. Materials and methods 2.1. Subjects and study design In this double-blind randomized clinical trial, criteria for inclusion were women aged between 25 and 35 years, using combined hormonal contraceptives with synthetic derivatives of estrogen and progesterone. The participants were on the same contraceptive period, between the fifth and the seventh pill. Exclusion criteria were pregnancy, use of antiinflammatory drugs, omega-3 supplementation, BMI ≥30 kg/m2, previous diagnosis of diabetes mellitus, hypertension, dyslipidemia, cancer or infection and smoking. The interviews and collection of blood and urine samples were performed at the Fetal Cardiology Unit of the Institute of Cardiology of Rio Grande do Sul. This study was approved by the Research Ethics Committee of the Institute of Cardiology of Rio Grande do Sul. After having been fully informed of the purpose of the project, all the participants signed an informed consent form. The study followed the guidance of the Resolution 466/12 of the National Health Council, which establishes rules for research with human beings, preserving the anonymity and privacy of participants. The interviews and guidelines were made by two blinded investigators. Participants were blinded throughout the study process. The randomization and the allocation of the participants were performed by a third investigator, who did not participate in the interviews or analyzes.

2.4. Polyphenols and omega 3 consumption The usual consumption of total polyphenols was evaluated through a FFQ [11]. The amount of polyphenols in the foods listed was obtained from a French database [13]. The consumption of total polyphenols on the food survey was described in milligrams (mg). Omega 3 food intake data were collected for the purpose of characterizing the initial sample. It was used the quantitative FFQ applied for consumption of polyphenols, adapting to these foods sources of omega 3: olive oil, canola oil, sunflower oil, corn oil, soybean oil, brazilian nuts, walnuts, flaxseed, tuna, salmon and sardines.

2.2. Blood and urine collection Blood and urine samples were collected before and after the intervention. The collections were made in the morning, with the same pattern of time before and after the period studied for each participant. Peripheral blood was collected by venipuncture in Vacutainer® tubes (BD Diagnostics, Plymouth, United Kingdom) containing EDTA or heparin, depending on the analysis. Urine samples were collected in sterile vials and protected from light. The plasma was centrifuged and aliquots were stored at −80 °C until analysis.

2.5. Assessment of nutritional status and level of physical activity Initially, anthropometric measurements of height and weight were conducted with an anthropometric digital scale. The body mass index (BMI) was used for diagnosis of the nutritional status, according to the classifications proposed by the World Health Organization (WHO). The usual level of physical activity of the participants was determined using the International Physical Activity Questionnaire (IPAQ) - Short Form [14].

2.3. Composition of capsules Participants in the polyphenols group (PG) received capsules with a 78


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2.6. Levels of Prostaglandin E2 and F2-isoprostane (8-iso-PGF2a)

2.9. Urinary excretion of total polyphenols

PGE2 levels were determined in plasma samples with EDTA. Levels of 8-iso-PGF2a were determined in urine. The levels were quantified by capture ELISA method (kits eBiosciences and Cusabio Biotech). The assays were performed following the manufacturer's instructions. The optical density (absorbance) were measured using a spectrophotometer (Spectramax M2e, Molecular Devices) at a temperature of 25 °C, with reduction of background. The measurements were obtained by linear regression of 4 parameters (Excel, Microsoft). Data were expressed in picograms per milliliter (pg/mL).

The urinary excretion of total polyphenols was determined as previously reported [16]. Urine samples stored at −80 °C were thawed and centrifuged at 4 °C for 10 min. Excretion values were expressed in milligrams of gallic acid equivalents (GAE) per gram of creatinine (mg GAE/g creatinine). 2.10. Sample size determination Sample size was calculated based on the mean and standard deviation of PGE2 levels presented by half of the final sample of this study (79.65 ± 25.30), on a pilot study. An expected difference of 40% in the levels of PGE2 was defined, based in a previous study which showed this reduction in PGE2 levels after induction of inflammation in human plasma previously treated with curcumin, a polyphenol extracted from roots of turmeric long [17]. Considering a 90% power, with absolute error margin of 5%, a total of 13 patients in each group would be necessary in the present study. Taking into account the possible losses due to the intervention proposed in the study, 20 women were randomly assigned to each group.

2.7. Glutathione disulphide to Glutathione ratio (GSSG/GSH) Circulating eritrocytes were prepared and assayed for GSH and GSSG contents, as described in Kolberg et al. [15]. For GSH and GSSG measurements, erythrocytes extracted from whole blood samples after centrifugation (2000 rpm) were disrupted in 1600 mL of 5% (w/v) metaphosphoric acid (MPA). After centrifugation (5000 rpm, 5 min at room temperature), cell lysates were spectrophotometrically (415 nm) assayed on a microplate reader (SpectraMax M2, Molecular Devices, Sunnyvale, CA, USA) by modification of the 5,5′-dithiobis(2-nitrobenzoic acid) [DTNB]/GSSG reductase (Sigma-Aldrich, Saint Louis, Missouri, USA) recycling method, using the N-ethylmaleimide (NEM, Sigma-Aldrich, Saint Louis, Missouri, USA) conjugating technique for GSSG sample preparation.

2.11. Statistical analysis The subjects were randomized by blocks of twenty participants through randomization software. Continuous variables with normal distribution were compared before and after the intervention with the Student's t-test for paired samples, and the Student's t-test for independent samples was applied for comparisons between groups. The Generalized Estimating Equation (GEE) was used for intra-group analyses, and the analysis of covariance (ANCOVA) for analyses between the groups, with adjustments for baseline. Correlations between PGE2 levels and the food frequency questionnaire or hs-PCR were assessed with the Spearman correlation test. The significance level considered was 5%. The Statistical Package for Social Science Program (S.P.S.S.) version 19.0 was used for data analysis. The analysis by

2.8. Levels of high-sensitivity C-reactive protein (hs-CRP) Plasma hs-CRP levels were measured by immunoturbidimetry, using a commercial kit from Roche Diagnostics Corporation (Mannheim, Germany), following the manufacturer's instructions. The results were expressed as milligram per deciliter (mg/dL).

Fig. 1. Flowchart of randomization.

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intention to treat showed no significant difference between the initial analyzed samples (p=0.28 in the polyphenol group; p=0.03 in the control group) with the results after the losses. It was presented analysis per protocol, in which only women who have completed the intervention time would be analyzed.

polyphenols group, however there was no significant difference between the groups.

3. Results

The markers of inflammation and oxidative stress had similar patterns, as shown by the levels of PGE2, 8-iso-PGF2a and hs-CRP in the polyphenols and control groups (Fig. 2). In the control group, significant increases were observed in the levels of PGE2 (mean ± standard deviation: 70.6 ± 14.8 pg/mL before intervention; 81.7 ± 14.4 pg/mL after the intervention, p=0.01), 8-iso-PGF2a (median/interquartile range: 563/473–712 pg/mL before intervention; 795/ 481–1345 pg/mL after the intervention, p=0.04) and hs-CRP (median/ interquartile range: 0.12/0.09–0.26 mg/dL before intervention; 0.4/ 0.12–0.55 mg/dL after the intervention, p=0.005). No differences, however, were observed in the polyphenols group before and after the intervention. PGE2 levels were 81.8 ± 24.5 pg/mL before and 82.7 ± 22.7 pg/mL after intervention (p=0.79). 8-isoPGF2a levels, presented as median/interquartile range, were 586/ 401–887 pg/mL before and 610/381–1064 pg/mL after the intervention (p=0.71). hs-CRP levels, also presented as median/interquartile range, were 0.24/0.12–0.32 mg/dL before and 0.26/0.11–0.33 mg/dL after the intervention (p=0.84). There was no significant difference between the groups after the intervention in any of the markers (PGE2 p=0.24; 8-iso-PGF2a p=0.14; hs-CRP p=0.12). The reduced glutathione was preserved in the polyphenols group (416.23 ± 208.79 nmolml−1 before intervention and 827.26± 330.34 nmolml−1 after intervention, p < 0.05). Control group showed no difference (p=0.14). Fig. 2 shows the results of GSSG to GSH ratio of control and polyphenols groups. The polyphenols group presented as median / interquartile range, 0.97/0.69 to 2.2 before and 0.51/0.45 to 0.94 after intervention (p=0.02). The control group presented at baseline 0.71/0.62 to 0.81 and in the end, 0.55/0.32 to 0.71 (p=0.42). A positive correlation was observed between hs-CRP and PGE2 levels in the PG at the end of the study (r=0.64 p=0.01). PGE2 levels in the PG after ingestion of the capsules was inversely correlated with the amount of polyphenols estimated in the FFQ (r=0.5 p=0.05). Fig. 2 also presents the excretion of polyphenols in the urine of participants. As expected, the polyphenols group showed an increased excretion of polyphenols, with significant difference between the groups (p=0.001).

3.2. Effect of polyphenol supplementation on markers of inflammation and oxidative damage

3.1. Randomization and sample characteristics Fig. 1 shows the sequence of activities with selection of the participants who were randomized, losses, exclusions and the number of women included. Forty women were randomized, of whom 15 in polyphenols group and 13 in the control group finished the study. Data were collected from May to August 2014. The side effects reported by some participants were nausea, vomiting and constipation. Women who presented vomiting were excluded. All the participants were in use of combined hormonal contraceptives with synthetic derivatives of estrogen and progesterone for at least one year. The following combinations of types and dosages were reported: Gestodene (00.06 mg to 0.075 mg) + ethinyl estradiol (0.015 mg to 0.03 mg) −12 women; Drospirenone (3 mg) + ethinyl estradiol (3 mg) −5 women; Cyproterone acetate (2 mg) + ethinyl estradiol (0.035 mg): 4 women; Levonorgestrel (0.1 mg to 0.15 mg) + ethinyl estradiol (0.02 mg to 0.03 mg) −4 women; Norethindrone enanthate (50 mg) + estradiol valerate (5 mg) −2 women; Desogestrel (0.15 mg) + ethinyl estradiol (0.02 mg) −1 woman. The characteristics of the sample are described in Table 2. No significant differences were observed between the groups. The reported consumption of polyphenols and omega-3 is described in Table 3. The control group showed a median food consumption greater than the Table 2 Baseline characteristics of the study population.

Age (years) Scholarity (years) Weight (kg) Height (m) BMIa (kg/m2) Nutritional status (n) Eutrophic Overweight IPAQb classification (n) Sedentary lifestyle Irregularly active Active Very active

Polyphenol group (n=15)

Control group (n=13)

p

28.4 ± 2.8 14.3 ± 3.2 65.2 ± 9.4 1.63 ± 0.04 24.4 ± 3.09

29.7 ± 2.9 12.3 ± 2.4 64.4 ± 9.4 1.64 ± 0.06 23.7 ± 2.8

0.26 0.07 0.81 0.49 0.52 0.68

11 (73.3) 4 (26.7)

8 (61.5) 5 (38.5)

3 (20) 10 (66.7) 1 (6.7) 1 (6.7)

3 (15.4) 8 (61.5) 3 (23.1) 0

0.41

4. Discussion This study aimed to evaluate the effect of polyphenol supplementation in the plasma levels of PGE2 in women of childbearing age using combined hormonal contraceptives, in order to assess the possible antiinflammatory action of the polyphenols. The results showed that the control group presented the expected increase in PGE2 levels, but remained unchanged in the polyphenols group. This finding suggests

Values expressed as mean ± standard deviation or n (%). a Body mass index. b International physical activity questionnaire.

Table 3 Dietary consumption of polyphenols and omega 3. p

Polyphenol group (n=15)

FFQ (mg) Omega 3 (g)

Control group

p

pa

pb

0.71 NA

0.48 0.78

0.89 NA

(n=13)

Before

After

855.3(680.6/5538) 0.08(0.01/0.26)

850.5(680.6/5566) NA

0.21 NA

Before

After

2199.6(793.8/3850.5) 0.15(0/0.49)

1634.5(748.3/3924.4) NA

Values expressed in median (P25/P75). FFQ: Food Frequency Questionnaire. NA: not analyzed. a Comparison between groups before intervention. b Comparison between groups after intervention

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Fig. 2. Results in polyphenol group and control group after the period studied in markers of inflammation, redox state and polyphenol urinary excretion. Significant increases were observed in the control group on levels of PGE2 (p=0.01), 8-iso-PGF2a (p=0.04) and hs-CRP (p=0.01), while GSSG to GSH ratio showed no changes. No changes where observed in polyphenols group on levels of PGE2 (p=0.79), 8-iso-PGF2a (p=0.71) and PCRus (p=0.84). However, GSSG to GSH ratio showed significant reduce in polyphenols group (p=0.02). Polyphenols group showed an increased excretion of polyphenols, with significant difference between the groups (p=0.001). BT, before treatment/basal: AT, after treatment. Analyses carried out with GEE. *p < 0.05 indicates statistical significance the between baseline and after a 15 days intervention period.

healthy individuals showed that curcumin, a polyphenol derived from turmeric and used as a natural dye for foods, inhibited PGE2 formation [17]. Supporting the previous studies, the results presented here show the influence of these substances on inflammation, in healthy women using hormonal contraceptives. It is known that the use of combined oral contraceptives induces inflammation such as, for example, increasing concentrations of Creactive protein, an important predictor of cardiovascular disease [19]. Our results agree with studies that evaluate the anti-inflammatory

that the use of polyphenols during this period inhibited the increase in PGE2 levels in the treated group. The same pattern, i.e. an increase in the CG and no modification in the PG, was observed for the markers of systemic oxidative stress F2-isoprostane and of inflammation, hs-CRP. The GSSG to GSH ratio was significantly reduced in GP, supporting the other results. Clinical studies with polyphenols effects usually evaluate raw outcomes or other markers of inflammation, instead of PGE2 [18]. A study with lipopolysaccharide-stimulated peripheral blood cells from 81


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of interpersonal characteristics, level of physical activity, and exclusion of pro-inflammatory diseases. In addition, the pro-inflammatory effect of hormone contraceptives was also considered, to verify the influence of polyphenols on the levels of PGE2. To this end, all samples were collected during the same period of contraceptive pills. The option for analyzing women using hormones was linked to the objective of this work, which was to consider the use of hormones as a pro-inflammatory factor and to evaluate the action of polyphenols in the inflammatory cascade by analysis of PGE2 levels in women in the reproductive period, considering that this situation is closer to the end of the gestation period, also considered pro-inflammatory.

effect of polyphenols on the levels of hs-CRP [20]. Increased levels of estrogens and progesterone stimulate the production of PGE2 [21]. The level of PGE2 and lipids appears to be sensitive to hormonal changes in women using hormonal contraceptives, and changes in the metabolism of these substances may represent a risk for cardiovascular diseases, although the findings are controversial [22]. PGE2 is the main prostaglandin mediating inflammatory processes of the cardiovascular system [10]. The main result of this study confirms other reports with women using hormonal contraceptives, who presented increased levels of PGE2 [22]. This was the reason to choose this specific population in the present study, where the hormones used for contraception induce a pro-inflammatory state, similarly to what occurs during pregnancy [2,22]. In this study, polyphenol intake was higher than the average estimated consumption in Europe, 1.193 mg/day, and the 75 percentile of intake of a population of pregnant women in Southern Brazil, 1089 mg/day [11]. This may be related to consumption of mate, an infusion widely consumed regardless of the socio-economic level, including pregnant women, and that represents the largest source of polyphenols in this geographical region. The activity of polyphenols depends on individual absorption and excretion rates and on the bioavailability and amount of polyphenols present, which vary depending on the source of the food [23]. The feeding habits of the population may also have influence on the consumption of polyphenols. Although dietary consumption of polyphenols observed in the control group was greater than in the polyphenols group, the total amount of polyphenols ingested considering capsules was significantly lower comparing to polyphenols group. Among participants of the polyphenols group, an inverse correlation was observed between the consumption of polyphenols estimated by the FFQ with PGE2 levels at the end of the study. This finding had not yet been previously described in the literature and reinforces the hypothesis of the present study regarding the action of polyphenols in reducing PGE2 levels. The liver is the main organ involved in the metabolism of polyphenols, and metabolites are secreted in bile and urine. Excretion of polyphenols in participants of the PG was significantly higher than in the CG, confirming the effective ingestion of capsules and absorption of compounds, which can vary depending on the amount ingested, the chemical structure of the substance and the intestinal flora of the subjects [12]. The quantification of isoprostanes can not only evaluate the antioxidant activity as well as a pro-oxidant activity of the polyphenols, since these substances are produced in vivo by peroxidation of polyunsaturated fatty acids of phospholipids, being released in free form due to the action of phospholipases. F2-isoprostane has been referred to as the gold standard of in vivo lipid peroxidation and oxidative stress [24]. It is suggested that isoprostanes regulate the vascular tone by their excitatory or inhibitory action, since they may bind to prostaglandins and thromboxanes receptors [24]. Clinical studies have shown that consumption of flavonoids reduces the levels of F2-isoprostane both in healthy individuals as in oxidative stress situations [25]. The present study demonstrated that the use of polyphenols prevented the increase in levels of this marker, even in non-stressed women. A similar study with healthy women in pre- and postmenopause period showed that consumption of 36 g of freeze-dried grape for four weeks was able to reduce the levels of F2-isoprostanes [26]. Similarly as F2 isoprostane, the reduction of GSSG to GSH ratio in PG has demonstrated that supplementation had effect on the preservation of the redox state, saving reduced glutathione. This result is consistent with other findings in the literature [27]. The concentration of prostaglandins depends on the ratio between synthesis and catabolism. They are labile compounds, and vary according to the inflammatory status. Studies with induction of inflammatory conditions usually show more important changes in PGE2 levels after interventions [17]. In the present work, this limitation was controlled by decreasing the within-group heterogeneity in terms

5. Conclusion The increase in biomarkers of inflammation and oxidative stress observed in the present study were possibly caused by the use of hormonal contraceptives, as verified in the CG, and this change was not observed in the group that used polyphenols. Therefore, the results of this polyphenol supplementation showed that the antioxidant and antiinflammatory effects observed in the studied population is due to the reduction in plasma levels of PGE2, supporting the conceptual hypothesis, by its action on the inflammatory cascade, probably by COX inhibition. Conflict of interest There is no conflict of interest. Author's Contributions The authors’ responsibilities were as follows — A. M. Zilio designed the project, trained and supervised the team to collect data, analyzed data, performed statistical analysis and wrote this manuscript. P. Zielinsky and I. Vian were involved in all stages of the project including the writing of this manuscript. M. Schaun and M. Markoski developed the analysis of markers of inflammation and oxidative stress and participated of the discussion and review of the manuscript. K. Lampert, D. Raupp, C. Weschenfelder, C. Brum and A. Arnt participated of the data collection and collaborated with statistical analysis and calculations of questionnaires. A. Piccoli and L. H. Nicoloso participated of the discussion and review of the manuscript. This manuscript was reviewed and approved by all the authors, who also agreed with the submission of the manuscript to this journal. Acknowledgments The present work was supported by FAPICC (Fundo de Apoio do Instituto de Cardiologia/FUC à Ciência e Cultura), FAPERGS (Fundação de Amparo à Pesquisa do Estado do Rio Grande do Sul) and funding of awards and publications of the Fetal Cardiology Group of the Institute of Cardiology of the RS. References [1] A. Scalbert, C. Manach, C. Morand, C. Rémésy, L. Jiménez, Dietary polyphenols and the prevention of diseases, Crit. Rev. Food Sci. Nutr. 45 (4) (2005) 287–306. [2] C. Ly, J. Yockell-Lelievre, Z.M. Ferraro, J.T. Arnason, J. Ferrier, A. Gruslin, The effects of dietary polyphenols on reproductive health and early development, Hum. Reprod. Update 21 (2) (2014) 228–248. [3] P. Zielinsky, A. Piccoli, J.L. Manica, L.H. Nicoloso, H. Menezes, A. Busato, M.R. Moraes, J. Silva, L. Bender, P. Pizzato, et al., Maternal consumption of polyphenol-rich foods in late pregnancy and fetal ductus arteriosus flow dynamics, J. Perinatol. Nat. Publ. Group 30 (1) (2010) 17–21. [4] P. Zielinsky, J.L. Manica, A. Piccoli, L.H. Nicoloso, M. Barra, M.M. Alievi, I. Vian, A.M. Zilio, P.E. Pizzato, J.S. Silva, et al., Fetal ductal constriction caused by maternal ingestion of green tea in late pregnancy: an experimental study, Prenat. Diagn. 32 (10) (2012) 921–926. [5] G.B. Bubols, P. Zielinsky, A. Piccoli, L.H. Nicoloso, I. Vian, A.M. Moro, M.F. Charão, N. Brucker, R.P. Bulcão, S.N. Nascimento, et al., Nitric oxide and reactive species

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[6]

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