Pomegranate Extract Improves Maximal Performance of Trained Cyclists after an Exhausting Endurance Trial: A Randomised Controlled Trial

cy of pomegranate (Punica granatum) extract (PE) for improving performance and post-exercise recovery in an active population was equivocal in previous studies. In this study, a randomised, double-blinded, placebo-controlled, balanced, cross-over trial with two arms was conducted. Eligibility criteria for participants were as follows: male, amateur cyclist, with a training routine of 2 to 4 sessions per week (at least one hour per session). The cyclists (n= 26) were divided into treatment (PE) and placebo (PLA) groups for a period of 15 days. After physical tests, the groups were exchanged after a 14-day washout period. Exercise tests consisted of endurance bouts (square-wave endurance exercise test followed by an incremental exercise test to exhaustion) and an eccentric exercise drill. The objective was to assess the ef cacy of PE in performance outcomes and post-exercise muscular recovery and force restoration after a prolonged submaximal effort. Twenty-six participants were included for statistical analysis. There was a statistically signi cant difference in total time to exhaustion (TTE)(17.66–170.94 s,p< 0.02) and the time to reach ventilatory threshold 2 (VT2)(26.98–82.55 s,p< 0.001), with greater values for the PE compared to the PLA group. No signi cant results were obtained for force restoration in the isokinetic unilateral low limb test. PE, after a prolonged submaximal effort, may be effective in improving performance outcomes at maximal effort and might help to restore force in the damaged muscles. Keywords: pomegranate; exercise performance; sports nutrition; delayed onset muscle soreness; muscle recovery; antioxidants 1. Introduction Polyphenols are natural occurring antioxidants and represent one of the most numerous and widely distributed groups of substances in the plant kingdom; as much as 8000 phenolic

effective in improving performance outcomes at maximal effort and might help to restore force in the damaged muscles. Keywords: pomegranate; exercise performance; sports nutrition; delayed onset muscle soreness; muscle recovery; antioxidants 1. Introduction Polyphenols are natural occurring antioxidants and represent one of the most numerous and widely distributed groups of substances in the plant kingdom; as much as 8000 phenolic structures are currently categorized into four groups ( avonoids, stilbenes, lignans and phenolic acids) [1]. Polyphenols are widely found in foods including wine, green tea and red-coloured fruits such as pomegranate [2,3]. Some clinical intervention studies support the hypothesis of some cardiovascular bene ts arising from polyphenol-rich beverages (red wine, tea and cocoa) [4,5]. In fact, epidemiological evidence suggests that polyphenols, at least in part, might explain the cardiovascular bene ts from increased fruit and vegetable intake [6]. The pomegranate fruit includes bioactive substances such as hydrolysable tannins (gallotannins and ellagitannins), ellagic acid and its derivatives, gallic acid, anthocyanins/anthocyanidins, proanthocyanidins, avonoids, vitamins, as well as sterols, lignans, saccharides, fatty acids, organic Nutrients2019,11, 721; doi:10.3390/nu11040721

Nutrients2019,11, 721 2 of 16 acids, terpenes and terpenoids, among others [7]. A study on different varieties of pomegranate showed that their total polyphenol content (TPC) and antioxidant activity vary depending on the part used and cultivar [8,9]; the TPC can be 20 times higher in the whole fruit than in arils [10]. Pomegranate juice is a source of polyphenols such as anthocyanins, avanols and some ellagitannins (especially punicalagin), revealing a potent antioxidant activity that is three times higher than the well-known antioxidant properties of red wine or green tea [11]. The most abundant of these polyphenols in pomegranate juice is punicalagin [12], belonging to the ellagitannin subgroup (hydrolysable tannins), implicated as the bioactive constituent responsible for more than 50% of the juice's potent antioxidant activity [13] but the content of polyphenols in commercial pomegranate juices varies according to variety and industrial manufacturing process with considerable variability in the punicalagin content [11]. It seems that most of the studies carried out with pomegranate extracts were juices with a high polyphenol content without a standardized punicallangin content. Pomegranate juice has shown many bene cial effects on markers of cardiovascular health including: A)Blood pressure: Systolic blood pressure (SBP) and diastolic blood pressure (DBP) lowering, as showed in a meta-analysis (n= 322) expressed as the weighed mean difference (WMD) [SBP (WMD: 4.96 mmHg, 95% CI: 7.67 to 2.25,p< 0.001) and DBP (WMD: 2.01 mmHg, 95% CI: 3.71 to 0.31,p= 0.021)] [14]; and through decreased activity of related enzymes: serum angiotensin-converting enzyme (36% decrease) [15]; and possibly 11 -hydroxysteroid dehydrogenase type 1 enzyme activity [16]. B)Improved lipid metabolism: by a diminished low-density lipoproteins aggregation and an increased serum paraoxonase activity up to 20% [17] (an enzyme possessing atheroprotective properties [18]); and decreased glucose conversion to fat (by inhibition of basal glucose incorporation into lipids in human adipocytes [19]). C)Reduced markers of oxidative stress: by diminished lipid peroxidation in overweighed and obese humans after physical exercise [20] and against smoking (in rats), also showing increased levels of antioxidant enzymes [21]. In sport, polyphenols exert physiological effects that can increase by 1.90% [95% con dence interval

to fat (by inhibition of basal glucose incorporation into lipids in human adipocytes [19]). C)Reduced markers of oxidative stress: by diminished lipid peroxidation in overweighed and obese humans after physical exercise [20] and against smoking (in rats), also showing increased levels of antioxidant enzymes [21]. In sport, polyphenols exert physiological effects that can increase by 1.90% [95% con dence interval (CI) 0.40–3.39] a diversity of athletic performance parameters – such as exercise time to fatigue, distance covered in a pre-selected time period, time to complete a certain distance and maximum power output – in both untrained but predominantly trained males [22]. Moreover, there are a number of both narrative and systematic reviews supporting the role of polyphenol supplementation in endurance performance [23–26] showing decreased rate of perceived exertion (RPE), increased maximal oxygen uptake and a faster recovery of muscle capacity (with a parallel trend to a faster decrease of in ammatory markers [27]). Polyphenols have been purported to improve aerobic metabolism through stimulation of mitochondrial biogenesis (by increasing expression of genes encoding cytoprotective proteins [28] and activation of sirtuins [29] mediated by speci c polyphenols such as catechins, resveratrol, quercetin and curcumin [30]). On the other hand, antioxidant supplementation may impair muscle performance by decreasing force production (by blocking oxygen delivery from blood to myocytes [31] and by modifying basal cellular redox state [32]) and training adaptations derived from physical stress [23–26]. The purpose of this study was (1) to test the hypothesis that pomegranate extract (PE), in dietary doses, can bene t endurance capacity (sub-maximal and maximal) after an extenuating bout and (2) the contribution of PE to post-exercise strength recovery after an exercise induced muscular damage. 2. Materials and Methods 2.1. Subjects Thirty amateur endurance-trained male athletes (age: 34.9 10.0 years; weight: 74.8 11.3 kg; height: 1.75 0.05 m; body mass index (BMI): 24.5 3.0 kg/m 2 ; maximal oxygen consumption (VO2max): 54.4 9.0 mL/min/kg) volunteered to participate in the study. Inclusion criteria were as follows: (1) male aged between 18–55 years old; (2) amateur cyclist, with a training routine of 2 to 4

34.9 10.0 years; weight: 74.8 11.3 kg; height: 1.75 0.05 m; body mass index (BMI): 24.5 3.0 kg/m 2 ; maximal oxygen consumption (VO2max): 54.4 9.0 mL/min/kg) volunteered to participate in the study. Inclusion criteria were as follows: (1) male aged between 18–55 years old; (2) amateur cyclist, with a training routine of 2 to 4

Nutrients2019,11, 721 3 of 16 sessions per week, for at least one hour per session. Exclusion criteria were as follows: (1) allergy to pomegranate or any of its by-products; (2) serious clinical pathology or antecedents; (3) regular smoker; and (4) supplementation with ergogenic aids in the last 3 months. Participants were informed (verbally and written) of the purpose of this study, the characteristics of the product used for supplementation, its effects, as well as any possible risk and side effects resulting from the supplement and the procedures of the study. Subject were informed of their right to quit the study at any time, without the need to provide any reason. Participants gave written consent before the study was started. The study protocol and informed consent were approved by the Ethics Committee of the Catholic University of Murcia (UCAM) and were in agreement with the Declaration of Helsinki. 2.2. Trial Design A double-blind, placebo-controlled, randomised, balanced, crossover design with two different study arms was used to test the effect of pomegranate extract (PE) or placebo (PLA) supplementation. Randomization was performed by a scientist not participating in the study, using software (Epidat 4.2, 2016) that generated random codes which were assigned to participants. An initial incremental exercise test to exhaustion (IETE) was carried out to make an initial assessment of the physical condition of each participant. Then, supplementation protocol of each group (PE or PLA) commenced ( rst allocation round), for a period of 15 days, after which cyclists underwent the exercise protocol (endurance and strength) to measure intervention effect. Afterwards, supplementation was discontinued 14 days for washout and the same procedure was repeated in the second allocation round (crossover design). 2.3. Supplementation Protocol Participants ingested two capsules of PE (composition per capsule: 375 mg of POMANOX ® P30 with 30% punicalagins; total amount of punicalagins + per capsule: 112.5 mg) per day, immediately after breakfast; that is, a total dose of 225 mg punicalagins/day, for 15 days of treatment per study arm (or PLA (placebo): 15 days/15 days of experimental product), with 14 days of washout between them. Both products, PE

capsule: 375 mg of POMANOX ® P30 with 30% punicalagins; total amount of punicalagins + per capsule: 112.5 mg) per day, immediately after breakfast; that is, a total dose of 225 mg punicalagins/day, for 15 days of treatment per study arm (or PLA (placebo): 15 days/15 days of experimental product), with 14 days of washout between them. Both products, PE (POMANOX ® P30, EUROMED S.A., Barcelona, Spain) and PLA (maltodextrin) were identical in appearance: hard, orange-coloured capsules sealed in a 15-capsule aluminium blister pack, inside a cardboard box (3 blisters per box), properly labelled, randomized and identi ed. Storage instructions stated to store the product in a cool dry place, away from sunlight and intense odours. Identi cation and quanti cation tests of the nal product were performed by high-performance liquid chromatography (HPLC) according to the supplier reference standard, by a validated method of analysis (SOP No. HPLC-757). Compliance with the identi cation and quanti cation of the active ingredient, providing at least 30% of punicalagins + , was checked with the certi cate of analysis provided by the manufacturer to ensure proper nal product speci cations. 2.3.1. Compliance and Follow-Up To ensure compliance and ful lment, participants were given an extra blister pack provided as a backup (in case of accidental loss) and were asked to return empty blister packs and spare capsules after each intervention. For the follow-up, participants were reminded verbally and through e-mail communication to consume the experimental supplements. 2.3.2. Dietary Assessment and Control The dietary habits of the participants were recorded using a validated food questionnaire. Subjects did not change their usual diet during the study period. On the day before and the same day as any performance test, volunteers had to comply with a previously detailed diet developed by a nutritionist, until the test was performed. This included refraining from taking caffeine and any other ergogenic aids or drugs that could affect performance measures. This measure was taken to ensure that the observations made were only due to the supplement and were not in uenced by other modi cations in

previously detailed diet developed by a nutritionist, until the test was performed. This included refraining from taking caffeine and any other ergogenic aids or drugs that could affect performance measures. This measure was taken to ensure that the observations made were only due to the supplement and were not in uenced by other modi cations in

Nutrients2019,11, 721 4 of 16 the diet. Additionally, volunteers were asked for their meal intake when they arrived at the laboratory to check diet compliance. Any variation in diet was written in a control table by the nutritionist to keep track of diet modi cations. 2.4. Exercise Tests At every supplementation completion, a square-wave endurance exercise test (SWEET), followed by an IETE and a subsequent eccentric exercise drill were performed. The purpose of the rst two tests (endurance tests) was to assess performance outcomes. The purpose of the eccentric protocol was to evoke exercise induced muscle damage (EIMD) to assess the contribution of the supplement to post-exercise biomarkers; therefore, no performance data were collected for this drill. All three tests were conducted sequentially in the same session after each allocation round, separated by a 29-day lapse (14 days for washout and 15 days in the other crossover supplementation arm). Athletes did not change their physical activity habits during the study and were told to avoid physical exercise the day before they performed the tests. Environmental conditions (room temperature and humidity) were replicated in every exercise test for optimal conditions using room air conditioning system and were additionally measured during the tests. 2.4.1. Initial Physical Assessment: Aerobic and Health Assessment A preliminary IETE test was performed to assess the training status of volunteers at baseline, 7 days prior to commencement of the exercise tests. The purposes of this test were to (1) familiarize volunteers with the testing procedures and subjective feelings of the exercise tests; (2) determine submaximal external workload for each of the exercise tests (set at 60% and 70% of the VO2max); and (3) establishventilatory thresholds of participants (corresponding the anaerobic ventilatory threshold to the ventilatory threshold 2 (VT2)). Every participant used their own bicycle placed on the rear wheel, so repeatability was controlled by this corrective measures: 1)Front–rear slope-ratio was corrected to zero (using a front wheel riser) during the trial; 2)Bike con guration (gear set, saddle and handlebars) should be kept during the study; 3)Bike tting (seat-post height and angle, handlebar reach, height and grip position) should

Every participant used their own bicycle placed on the rear wheel, so repeatability was controlled by this corrective measures: 1)Front–rear slope-ratio was corrected to zero (using a front wheel riser) during the trial; 2)Bike con guration (gear set, saddle and handlebars) should be kept during the study; 3)Bike tting (seat-post height and angle, handlebar reach, height and grip position) should be the same and; 4)Preferred pedalling system (use of cycling shoes and type of clip/cleat) should be consistent. Test consisted of a 3-min warm-up at a self-paced intensity, followed by an IETE (initial load: 50 Watts (W), with a 35-W step increment every minute) on an electronically braked cycle ergometer (Cyclus2, RBM elektronik-automation GmbH, Leipzig, Germany) at a self-selected cadence between 60–100 revolutions per minute (RPM) on a xed gear selected at the beginning of the test. Volunteers were verbally encouraged by the staff to exert maximal effort. Exhaustion was deemed to occur when the subject decided to stop, when pedal cadence dropped 20 RPM below the minimum cadence established (i.e., 40 RPM) or when power output could not be maintained. Heart rate was monitored continuously using an electrocardiograph and oxygen consumption (VO2) was collected continuously during this test using an automated breath-by-breath system (Jaeger Oxycon Pro TM , CareFusion, Höchberg, Germany) calibrated before each test. All measures were analysed using software (LABManager 5.3.0.4, VIASYS Healthcare GmbH, Höchberg, Germany) and were stored in a personal computer for later recall. Maximal criteria were interpreted according to [33], de ned as a plateau of VO2, respiratory quotient (RQ) above 1.10 and heart rate (HR) above 95% of the theoretical maximum HR. Ventilatory aerobic and anaerobic threshold were plotted in a graph by using previously mentioned software and interpreted according to the three-phase model [34] by ventilatory equivalents (VE) [35]. VT2 was set as the intersection point between the carbon dioxide ventilatory equivalent (VE/VCO2) and the oxygen ventilatory equivalent (VE/VO2) against time - de ned as the point in

model [34] by ventilatory equivalents (VE) [35]. VT2 was set as the intersection point between the carbon dioxide ventilatory equivalent (VE/VCO2) and the oxygen ventilatory equivalent (VE/VO2) against time - de ned as the point in

Nutrients2019,11, 721 5 of 16 which pulmonary ventilation during exercise (VE) starts to increase at a faster rate than oxygen uptake (VO2). Time values to reach VT2 were provided by the same software when a vertical line was placed on this intersection point. After completion of the initial IETE, subjects were familiarized with the eccentric drill and isokinetic test. 2.4.2. Exercise Tests: Endurance Test and Strength Protocol Once the supplementation protocol was completed, sets of different exercise tests were performed on the same day, which are summarised as follows: 1. a. b. 2. c. Endurance Tests (SWEET and IETE) a. on the same electronically braked ergometer in same conditions. Subjects were instructed to complete a self-paced 10-min warm-up, without reaching initial load, followed by 90 min of SWEET with an individual load in watts, corresponding to 70% of VO2maxas calculated after a preliminary test. HR was continuously monitored using a pulsometer (Polar RS800CX, Polar Electro Oy, Finland) to double-check that athletes remained under VT2 at the given intensity, by screening the heart rate variability which showed signi cant correlation with VT2 in previous work [36]. To ensure proper performance, cyclists followed a hydration protocol, which was measured during the trial [37]. Subjects were asked to estimate their rate of perceived exertion (RPE) using the Borg scale [38] (scale from 1 to 20) after warm-up (10 min after commencement) and after 30, 50, 70 and 90 min (end of the test). b. Incremental exercise test to exhaustion (IETE): Once the SWEET was completed, the maximal incremental cycling test was performed without interruption. Following 3 min of recovery at a self-selected intensity (never above the initial load), subjects performed a progressive incremental cycling test (initial load: 60% of VO2max) with the same equipment and conditions as the preliminary test. The difference now was that every step consisted of 3 min instead of one (i.e.,: 35-W increase every 3 min). Lactate samples were collected 1 min 40 s after completing the test by lancing the left ring- nger pad and were immediately analysed by a blood gas analyser (ABL90FLEX, Radiometer Medical APS, Copenhagen,

This study assesses the effects of pomegranate extract on performance and recovery in trained cyclists.