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 (flavonoids, stilbenes, lignans and phenolic acids) [1
]. Polyphenols are widely found in foods including wine, green tea and red-coloured fruits such as pomegranate [2
]. Some clinical intervention studies support the hypothesis of some cardiovascular benefits arising from polyphenol-rich beverages (red wine, tea and cocoa) [4
]. In fact, epidemiological evidence suggests that polyphenols, at least in part, might explain the cardiovascular benefits 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, flavonoids, vitamins, as well as sterols, lignans, saccharides, fatty acids, organic 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
]; 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, flavanols 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 beneficial 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 11β-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% confidence 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
] 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 inflammatory 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 specific 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
The purpose of this study was (1) to test the hypothesis that pomegranate extract (PE), in dietary doses, can benefit 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
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/m2; 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 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 (first 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 α + β 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 identified. Storage instructions stated to store the product in a cool dry place, away from sunlight and intense odours.
Identification and quantification tests of the final 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 identification and quantification of the active ingredient, providing at least 30% of punicalagins α + β, was checked with the certificate of analysis provided by the manufacturer to ensure proper final product specifications.
2.3.1. Compliance and Follow-Up
To ensure compliance and fulfilment, 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 influenced by other modifications in 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 modifications.
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 first 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) establish ventilatory 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 configuration (gear set, saddle and handlebars) should be kept during the study; 3)Bike fitting (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 fixed 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 ProTM
, CareFusion, Höchberg, Germany) calibrated before each test. All measures were analysed using software (LABManager 220.127.116.11, VIASYS Healthcare GmbH, Höchberg, Germany) and were stored in a personal computer for later recall. Maximal criteria were interpreted according to [33
], defined 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 - defined as the point in 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:
Square-wave endurance exercise test (SWEET), followed immediately by
Incremental exercise test to exhaustion (IETE), followed by 5 min of rest.
Eccentric exercise drill.
Endurance Tests (SWEET and IETE)
Square-wave endurance exercise test (SWEET): A constant intensity cycling test was performed 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 VO2max
as 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 significant 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).
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-finger pad and were immediately analysed by a blood gas analyser (ABL90FLEX, Radiometer Medical APS, Copenhagen, Denmark). Subjects were then asked again to estimate their RPE.
Strength Protocol (Eccentric Drill)
Subjects were given 5 min of transition time before performing the eccentric exercise test in another room with same conditions. The whole drill consisted of 15 repetitions, for a total of 6 sets per leg (15 × 6 = 90 repetitions per leg) performed at a specific cadence (1:4). The exercise sequence is depicted in Scheme 1
A single-leg barbell step-up onto a bench, with no load and locked upper body (straight spine), was employed as described in Reference [39
]. Subjects were given a plastic tube (200 cm long), which they had to place resting on their shoulders behind their head (with prone grip), forming a 45° angle between the forearm and arm. A fitness bench was placed in front of them, creating a 90° angle between the thigh and lower leg of the raised leg. Individual angles were measured by a goniometer prior to test, for bench height positioning. Subjects had to extend the leg placed on the bench, settling the other leg next to it (finishing the movement with both legs together and both knees extended) in 1 s, while reverting to initial position had to be completed in 4 s (consisting the full sequence of 5 s). Cadence compliance was facilitated by playing a free metronome app (Mobile Metronome 1.2.4F (2012), Gabriel Simoes, Google Play-Google LLC) at a speed of 60 beats per minute (bpm) in a 5/4 measure (with a different sound for the first beat of every measure) corresponding every beat to 1 s. One researcher supervised proper positioning and cadence of subjects during the drill.
2.5. Variables and Measurements
The following variables were measured: Initial physical assessment (IETE): Total time to exhaustion (TTE), oxygen consumption (VO2) and maximum oxygen consumption (VO2max). Endurance tests: Constant intensity endurance test (SWEET): Rate of perceived exertion (RPE). Maximal test (IETE): TTE, time to reach VT2 (time to VT2), VO2max, oxygen consumption at VT2 (VO2 at VT2) and lactate blood concentration (mmol/L) at the end of the test.
Post-exercise force recovery was measured by an isokinetic unilateral leg test (with the dominant limb) performed 2, 24, 48 and 72 h after the maximal IETE by a specially designed device (SYSTEM 3 PRO, Biodex Medical Systems Inc., Shirley, NY, USA) consisting of a dynamometer, chair and belts. Subjects had to perform maximal force output during a fixed angular velocity (60° per second) for both knee flexion and extension. The isokinetic variables measured were: Peak torque (N × m/Kg), relative work (J/Kg), work fatigue (%) and average power (W).
Evolution of muscular damage and inflammation biomarkers (creatine kinase (CK) and C-reactive protein (CRP), respectively) were measured by blood collection, 10 min before the first isokinetic test and subsequently upon each strength assessment completion (2, 24, 48 and 72 h later). Samples were divided and placed in vacuum tubes for either freezing or centrifugation and were immediately sent for laboratory analysis performed by an automatic analyser (IL Ilab 600, Chema diagnostica, Monsano AN, Italy).
A summary of the experimental design is depicted in Scheme 2
2.6. Statistical Analysis
Quantitative variables are described as the mean, standard deviation and 95% confidence interval. This description was made for the total sample and was stratified by the randomized treatment arm. Qualitative variables are presented in tabular form, including the relative and absolute frequencies for the treatment groups and the global sample. Data were checked prior to analysis; in all cases, the Kolmogorov–Smirnov test was applied to test for a normal distribution and Levene’s test was used to test for homoscedasticity.
The evolution of these quantitative variables was analysed by parametric tests: a repeated measures t-test for the obtained variables from the IETE endurance exercise test and a two-way repeated measures ANOVA test with one within-subject factor (product) and one between-subject factor (time) for the variables obtained in the SWEET endurance exercise test and from the strength protocol. For the post hoc group comparison, the Bonferroni test was employed.
Statistical analysis was performed using SPSS software (version 21.0, Chicago, IL, USA) and p values are reported for every group and group × time interaction; p < 0.05 is considered statistically significant.
3.1. Participant Flow Diagram and Baseline Characteristics
The participant flow diagram is depicted in Figure 1
A total of 37 subjects were initially recruited, of which seven were excluded due to non-compliance with the inclusion criteria (n
= 5) or unwillingness to participate (n
= 2). Thirty subjects met the initial screening criteria and were randomized. During the study, four of them (two from each arm) dropped out because of poor adherence to supplementation protocol. Finally, 26 subjects (13 subjects in each arm) received treatment successfully and were included for statistical analysis. All participants were cyclists competing at the regional level (Region of Murcia, Spain). The descriptive analysis at baseline is summarized in Table 1
No significant differences were found for intra-subject values in any of the studied measures before each allocation. Therefore, we assumed that: (1) Participants started each intervention in same conditions; (2) Washout was successful in restoring values to baseline.
3.2. Conditions during Exercise Tests
No statistical difference was found for any of the measures in any group for temperature (p = 0.52), relative humidity (p = 0.97) or hydration (p = 0.77). During the PE tests, temperature was 22.12 ± 1.56 °C with a relative humidity of 57 ± 0.94%, whereas PLA temperature and relative humidity were 22.31 ± 1.57 °C and 57 ± 0.96%, respectively. Average water consumption was 1171.7 ± 304.8 mL for PE and 1156.3 ± 295.8 mL for PLA. Therefore, we assumed homogeneity of conditions among the performance trials for both groups.
3.3. RPE during and after the Square-Wave Test (SWEET)
The Borg’s scale results are presented in Table 2
There was no statistically significant interaction effect between the effects of treatment and time on perceived effort. There was a statistically significant change in RPE for both groups from the beginning of the test (after the 10-min warm up), without a statistical inter-group difference. The average RPE reported for both groups was 13, corresponding in Borg’s scale to “somewhat hard”.
3.4. Incremental Exercise Test to Exhaustion (IETE)
There was a statistically significant difference in TTE and the time to reach VT2, with greater values for the PE compared to the PLA group. There was no statistically significant difference for VO2max, VO2 at VT2 or lactate.
3.5. Strength Protocol: Eccentric Exercise and Isokinetic Force
All subjects completed the eccentric exercise drill successfully. The isokinetic force results are shown in Table 4
There was no statistical significance for the group × time values of any of the variables. There were greater values in PE for peak torque and relative work, both in extension and flexion values at 72 h. Work fatigue was lower for PE than for PLA, whereas average power was greater for PE in flexion and for PLA in extension.
3.6. Post-Exercise Muscular Damage and Inflammation
The evolution of muscular markers is presented in Table 5
Baseline conditions were similar for both groups for CK and CRP. There was a significant change from baseline conditions for both groups for the time value but there was no statistical significance for CK and CRP between groups. Both CK and CRP were lower for PE than for PLA 72 h after the trial, especially CRP (27.96% lower than that observed in PLA).