Intensive physical exercise may cause increase oxidative stress and muscular injury in elite football athletes. The aim of this study was to exploit the effect of cocoa polyphenols on oxidative stress and muscular injuries induced by intensive physical exercise in elite football players. Oxidant/antioxidant status and markers of muscle damage were evaluated in 24 elite football players and 15 controls. Furthermore, the 24 elite football players were randomly assigned to either a dark chocolate (>85% cocoa) intake (
Intensive physical exercise may increase oxidative stress and cause muscular injury in elite athletes [
The generation of reactive oxygen species (ROS) is a fundamental and physiological process of normal human biology. However, when ROS production and endogenous antioxidant ability are imbalanced, a maladaptive biological response occurs leading to both oxidative stress and inflammation [
Many studies have identified the potential antioxidant effect of polyphenols, a large group of natural compounds found in food and beverages [
Accordingly, the purpose of this study was to exploit the effect of chronic dark chocolate supplementation on muscular injury and oxidative stress during training exercise in elite football players.
The study was performed on 24 young (17.2 ± 0.7 years) elite male football players during the first month of the regular season and 15 physically active male subjects who did not practice football but practice aerobic sports such as running, swimming, or gymnastics (engaging in at least 3-day·week-1 of moderate-to-intense physical activity, ranging from 3.0 to 6.0 METs and/or >6.0 METs) (24.8 ± 3.5 years) (Table
Baseline characteristics of controls and elite football players.
Controls ( |
Football elite players ( |
||
---|---|---|---|
Age (years) | 24.8 ± 3.5 | 17.2 ± 0.7 | <0.001 |
Gender (male/female) | 15/0 | 24/0 | 1 |
WBC (×103 |
7.1 ± 1.4 | 5.6 ± 1.3 | 0.001 |
PLT (×103 |
233.8 ± 48.4 | 228 ± 39.7 | 0.357 |
RBC (×106 |
5.3 ± 0.3 | 5.8 ± 0.3 | 0.559 |
Cholesterol (mg/dl) | 185.1 ± 30.8 | 172.3 ± 29.4 | 0.130 |
BMI | 24.3 ± 1.4 | 22.5 ± 1.5 | <0.01 |
Glycaemia (mg/dl) | 89.0 ± 28.8 | 83.5 ± 15.2 | 0.276 |
LDH (U/l) | 179.5 ± 23.5 | 387.0 ± 50.7 | <0.01 |
CK (U/l) | 192.3 ± 28.7 | 342.6 ± 70.2 | <0.01 |
Myoglobin (ng/ml) | 50.6 ± 11.3 | 100.1 ± 42.9 | <0.01 |
sNox2-dp (pg/ml) | 13.8 ± 7.7 | 19.5 ± 6.9 | <0.01 |
H2O2 ( |
22.6 ± 13.2 | 38.8 ± 7.3 | <0.0001 |
HBA (%) | 52.9 ± 23.0 | 37.5 ± 11.4 | <0.0001 |
Training per week (h) | 7.2 ± 1.5 | 18 ± 2 | <0.0001 |
Football practice (yrs.) | 0 | 10 ± 1.2 | <0.0001 |
WBC: white blood cells; PLT: platelet; RBC: red blood cells; BMI: body mass index; LDH: lactate dehydrogenase; CK: creatine kinase; HBA: hydrogen peroxide (H2O2) breakdown activity.
All participants and the head coach were explained the study’s purposes, risks, and benefits; they were familiarized with the study’s protocol during the pre-season screening; and they gave a written informed consent. The institutional review board approved this study (C.E. 4662), and the randomized controlled trial was registered on ClinicalTrials.gov (Identifier:
Elite male athlete volunteers, aged between 18 and 35 years, were included in the study. Subjects were excluded if they suffer from an allergy to cocoa or any of the ingredients contained within either of the chocolate bars; they have a low platelet count (<170 × 1009/L); they are taking aspirin or aspirin-containing drugs, other anti-inflammatory drugs, or any drugs or herbal medicines known to alter platelet function or the haemostatic system in general (without a minimum washout period of one month); they are taking fish oils or evening primrose oil, or fat-soluble vitamin supplements within the last 4 weeks; they have unsuitable veins for blood sampling and/or cannulation; they have a BMI below 18 or above 35 kg/m2; they are taking any medicine known to affect lipid and/or glucose metabolism; they are suffering from alcohol or any other substance abuse or are having eating disorders; they have any known clinical signs of diabetes, hypertension, renal, hepatic, and haematological diseases, gastrointestinal disorders, endocrine disorders, coronary heart disease, infection, or cancer.
In the first phase, we performed a cross-sectional study to compare oxidative stress, as assessed by blood levels of soluble Nox2 (sNox2-dp), H2O2 production, H2O2 breakdown activity (HBA) which is a method evaluating the antioxidant capacity of serum, and markers of muscle damage such as creatine kinase (CK), lactate dehydrogenase (LDH), and myoglobin in 24 young well-trained male elite football athletes and 15 sex-matched amateur controls. In the second phase, we performed a randomized controlled trial in elite football athletes to investigate the effect of daily supplementation with normal diet and 40 g of dark chocolate (20 g every 12 h) vs normal diet, for 30 days, on markers of oxidative stress and muscle damage.
Elite football athletes were randomly allocated to a treatment sequence with normal diet plus 40 g/day of commercially available dark chocolate in tablet (cocoa solids >85%, cocoa mass, fat-reduced cocoa, cocoa butter, sugar, and vanilla) or normal diet for 30 days. The content of total polyphenols in the dark chocolate employed in our study was 799
Blood levels of CK, LDH, myoglobin, total polyphenols, and oxidative stress biomarkers were assessed at baseline and at 30 days after the last ingestion of chocolate. During the trial, participants were required to follow a diet adjusted according to their anthropometric and clinical characteristics and to the amount of calories coming from chocolate intake; furthermore, participants were restrained by having foods with high polyphenol content (blueberry, sweet cherry, strawberry, blackberry, red raspberry, chestnut, black tea, green tea, pure apple juice, hazelnut, red wine, and pomegranate juice) and/or additional chocolate.
Blood samples were collected in the morning (between 08 : 00 and 09 : 00 hours) after a fasting period of 8 h at baseline and 30 days after the last ingestion of chocolate.
An individual not involved in the study assigned codes to the study treatments, randomly allocated the participants to a treatment sequence with normal diet plus dark chocolate or normal diet, and kept the key in a sealed envelope. The randomisation was carried out by a procedure based on a random numeric sequence. The authors and laboratory technicians were unaware of the treatment allocation.
Sample size calculation was computed with G∗Power [
One g of chocolate was weighted, and fat was removed by using 1 ml of n-hexane. Polyphenols were extracted from the defatted pellet using a total volume of 3 ml (1 × 3 ml) with 80% (
Total polyphenol content in extracted phenolic fraction from chocolate was evaluated by a modified Folin–Ciocalteu colorimetric method [
Catechin and epicatechin were extracted and quantified according to Gottumukkala et al. [
Plasma samples were extracted by the method described by Spadafranca et al. [
The HPLC analysis was performed using an HPLC system (Agilent 1200 Infinity Series HPLC system, Santa Clara, USA). Separations were carried out at a flow rate of 1.5 ml/min with an isocratic mobile phase of 85% Na2PO4 10 mol/l, pH 3, and 15% acetonitrile. Chromatograms were recorded at 279 nm, and plasma epicatechin identification was made by comparison of retention times with those of commercially available authentic (-)-epicatechin (Sigma, St. Louis, MO) through the same procedures as the plasma samples
Serum hydrogen peroxide (H2O2) breakdown activity (HBA) was measured with HBA assay kit (Aurogene, code HPSA-50). The % of HBA was calculated according to the following formula: % of
Serum Nox2 was measured as soluble Nox2-derived peptide (sNox2dp) with an ELISA method, according to Pignatelli et al. [
Hydrogen peroxide (H2O2) was evaluated by a Colorimetric Detection Kit (Arbor Assays) and expressed as
Serum creatine kinase (CK), lactate dehydrogenase (LDH), and myoglobin levels were analyzed using a commercial ELISA kit (Antibodies, Germany; EIAab, China; DRG Instruments GmbH, Germany); the intra- and interassay coefficients were <10%.
The murine myoblast cell line C2C12 was cultured in DMEM/20% heat-inactivated foetal bovine serum (FBS), 2 mM glutamine, and 1% antibiotics (all Gibco) for expansion and maintenance of the undifferentiating state. When cultures reached 80% confluence, myogenic differentiation was induced by replacing the expansion media with DMEM/0.2% FBS. Afterwards, cells were stimulated with H2O2 (5 mM, Solarbio, Beijing, China) alone or in combination with cocoa-derived polyphenols (50, 100, and 150
Continuous variables are reported as
Clinical characteristics of elite football players and controls are reported in Table
Linear correlation between sNox2-dp and creatine kinase (a), between sNox2-dp and LDH (b), and between sNox2-dp and myoglobin (c) in 15 controls (circle empty mark) and 24 elite football players (circle full mark).
Total polyphenol, catechin, and epicatechin contents of dark chocolate are reported in Table
Total polyphenol content in dark chocolate.
Compounds | Dark chocolate |
---|---|
Total polyphenols ( |
799 |
Epicatechin (mg/g) | 0.65 |
Catechin (mg/g) | 0.26 |
No significant differences between clinical characteristics and biochemical parameters were found at baseline in the elite athlete groups allocated to dark chocolate intake and no dark chocolate intake (Table
Baseline characteristics of elite football players before treatment.
No dark chocolate ( |
Dark chocolate ( |
||
---|---|---|---|
Age (years) | 17.0 ± 0.9 | 17.4 ± 0.5 | 0.859 |
Gender (male/female) | 10/0 | 10/0 | 1 |
WBC (×103 ml) | 5.0 ± 1.3 | 6.2 ± 1.1 | 0.983 |
PLT (×103 ml) | 226.6 ± 55.4 | 230.0 ± 12.8 | 0.580 |
RBC (×106 ml) | 5.3 ± 0.3 | 5.5 ± 0.3 | 0.950 |
LDL (mg/dl) | 82.9 ± 18.3 | 84.3 ± 22.2 | 0.566 |
Glycaemia (mg/dl) | 84.0 ± 11.2 | 83.1 ± 19.1 | 0.448 |
LDH (U/l) | 389.7 ± 59.9 | 384.2 ± 42.2 | 0.398 |
CK (U/l) | 363 ± 21.83 | 341.1 ± 20.13 | 0.880 |
Myoglobin (ng/ml) | 97.4 ± 40.8 | 105.8 ± 41.9 | 0.579 |
hs-PCR (mg/l) | 0.6 ± 0.4 | 0.7 ± 0.6 | 0.759 |
sNox2-dp (pg/ml) | 18.9 ± 7.0 | 19.5 ± 6.7 | 0.844 |
H2O2 ( |
38.7 ± 10.1 | 37.7 ± 6.6 | 0.746 |
HBA (%) | 37.8 ± 27.8 | 34.9 ± 29.8 | 0.775 |
WBC: white blood cells; PLT: platelet; RBC: red blood cells; LDL: low-density lipoprotein; LDH: lactate dehydrogenase; CK: creatine kinase; HBA: hydrogen peroxide (H2O2) breakdown activity.
sNox2-dp levels (a), H2O2 production (b), hydrogen peroxide breakdown activity (HBA) (c), and total polyphenol (d) before and 30 days after daily intake of 40 g of dark chocolate (grey line) or without dark chocolate (black line) in elite football players.
Myoglobin (a), creatine kinase (CK) (b), and lactate dehydrogenase (LDH) (c) concentration before and 30 days after daily intake of 40 g of dark chocolate (grey line) or without dark chocolate (black line) in elite football players.
After 30 days of training, the control group showed increased levels of sNox2-dp and H2O2 compared to baseline (from 18.9 ± 7.0 pg/ml to 34.6 ± 7.5 pg/ml,
A significant difference between the two treatments (no dark chocolate vs dark chocolate) was found regarding sNox2-dp (34.6 ± 7.5 pg/ml vs 23.55 ± 5.6 pg/ml,
Serum
To assess the adherence to the protocol, we analyzed the levels of epicatechin which is a major component of dark chocolate. The results showed a significant increase of epicatechin in the dark chocolate group compared to the control group (189.8 ± 54.0 ng/ml vs <10 ng/ml,
A sensitivity analysis was then conducted by using generalized estimating equations (GLM), and point estimates of effect, 95% confidence intervals, and corresponding
No significant correlation was found between baseline blood parameters and days of unavailability days of FKT and occurrence of muscular and joint lesions (all
No significant effect of chocolate intake was found on days of unavailability, days of FKT, and occurrence of muscular and joint lesions (all
In order to corroborate the clinical effects of cocoa-derived polyphenols on muscle redox state, we performed an in vitro study with a polyphenol extract at concentrations (50-150
sNox2-dp levels (a) and H2O2 production (b) in murine myoblast cell line C2C12 stimulated with H2O2 (5 mM), alone or in combination with cocoa-derived polyphenols (50, 100, and 150
This study showed that (1) oxidative stress and markers of muscle damage are significantly increased in elite football players compared to controls and (2) chronic intake of dark chocolate is able to reduce oxidative stress and muscle damage biomarkers during elite football players’ training session.
The novel finding of the present study is the improvement of oxidative stress and muscle damage enzymes after 30 days by ingestion of dark chocolate in elite football athletes during intensive physical exercise. The effect of dark chocolate supports the hypothesis that polyphenol content, in particular epicatechin, may be responsible for this effect. Accordingly, total polyphenol content and epicatechin plasma levels were increased in the group of athletes randomized for dark chocolate intake.
The scientific background of our research was based on the evidence that intensive physical exercise implies a wide range of multifaceted biological activities challenging the physiological homeostasis of the body. The relationship between exercise and oxidative stress is extremely complex and mainly depends on mode, frequency, intensity, and duration of exercise. On the one hand, several experimental and epidemiological evidences have underlined the key role of physical exercise (PE) in decreasing oxidative stress, especially if associated to aging, and to prevent and positively modulate cardiovascular-associated risk factors [
According to this premise, professional training programs including those for elite football players could indirectly and physiologically induce oxidative stress in athletes and significantly influence biological antioxidant patterns. Accordingly, in our cross-sectional study, we found an increase in Nox2 activation and H2O2 production with reduced antioxidant property as indicated by decreased HBA in elite football players compared to controls. Moreover, at the same time, we observed an increase in muscle damage markers such as CK, LDH, and myoglobin.
These aspects represent a serious issue since a recent study showed that levels of oxidative stress markers are directly correlated with markers of muscular damage in elite football players playing in the Italian Serie A league [
For this reason, dietary regimens including antioxidant supplementation are now considered important interventions able to counteract the hazardous effects of free radicals by increasing the antioxidant profile and regulating the equilibrium between oxidant and antioxidant species [
We planned to use this body of evidence as a benchmark for the development of new strategies in the setting of elite athlete training programs. In this interventional study, we thus demonstrated for the first time that polyphenol-rich nutrient supplementation of dark chocolate reduces exercise-induced oxidative stress and muscular injury biomarkers in elite football athletes.
The antioxidative effects induced by cocoa-derived polyphenols were also confirmed on skeletal muscle cells
In conclusion, the present study provides the first direct relationship between cocoa-based polyphenol-rich nutrient supplementation and the effect of high-intensity training on elite athletes’ antioxidant status. Based on our results, the development and improvement of training techniques focusing also on new nutrition strategies may help to reduce muscular damage in elite football players.
The data used to support the findings of this study are available from the corresponding author upon request.
The institutional review board approved this study (C.E. 4662), and the randomized controlled trial was registered on ClinicalTrials.gov (Identifier:
The authors declare that they have no conflict of interest.
Giacomo Frati, Roberto Carnevale, Elena Cavarretta, and Mariangela Peruzzi have equal contribution to this work.
This work was partially supported by an unrestricted grant from Villa Stuart Sport Clinic, FIFA Medical Centre of Excellence, and Sapienza University of Rome (grant number 2016/43780) to Elena Cavarretta. The authors are grateful to Dr. Vincenzo Costa, MD, for his invaluable help in the athletes’ follow-up.