Effects of antioxidant supplementation on oxidative stress balance in young footballers: a randomized controlled trial

The intensive physical exercise in which athletes take part in competitive sports can negatively affect the pro-oxidative–antioxidant balance. The use of compounds with high antioxidant potential, which certainly should include chokeberry, can prevent these adverse changes. The study was conducted on a group of football players aged 16–17 years, who underwent 7 weeks of supplementation with 200 ml chokeberry juice per day. Before and after supplementation, the participants performed an intensive physical exercise test (beep test). At rest, immediately after exercise and after 3 and 24 h of rest, venous blood was taken from the subjects, in which levels of thiobarbituric acid reactive products (TBARS), hydroxy-2'-deoxyguanosine (8-OHdG), total antioxidant capacity (TAC), iron (Fe), hepcidin, ferritin, myoglobin, albumin and morphological parameters were examined.


Conclusions
The supplementation applied to footballers showed no effects under the in uence of the applied exercise stress test. Such results may be the result of both the players' adaptation to the applied exercise loads and the insu cient antioxidant capacity of the supplement used.

Background
Increased metabolism during physical exercise is accompanied by increased generation of reactive oxygen species, which may also cause disorders in the functioning of the immune system [1,2]. This applies primarily to physical exercise of high intensity or long duration. The mechanism of this process is not fully understood. It is believed that excessive production of free oxygen radicals leads, among other things, to damage to erythrocytes (as a consequence of lipid peroxidation), thus increasing their sensitivity to degradation. As a result of increased haemolysis, there is a signi cant increase in redoxactive iron in the circulation. Fenton's reaction under the in uence of iron is the main cause of Fe toxicity in the body. High reactivity and low speci city •OH might facilitate destructive process of cell components and body uids Under conditions of oxidative stress, activation of the immune system and in ammation are also observed, which is an early defence response of the body. Probably in this way, oxidative stress is 'sustained' also during post-workout restitution. The increase in ionized iron concentration in the blood serum, which is a consequence of this process, can contribute both to the intensi cation of free radical reactions on the one hand [3], and to weakening of the immune system on the other, thereby increasing susceptibility to infection [4][5][6]. Acute post-exercise depression of the immune system may result not only in increased frequency of infections in competitors, but also a higher percentage of cases (especially for upper respiratory tract diseases -URTI) and a much longer duration of URTI [7].
Chokeberry contains a wide range of biologically active compounds, including polyphenols such as anthocyanin, avonoids and phenolic acids [8,9]. Analysis of literature data indicates that compounds contained in chokeberry can have a positive effect on health [10]. Particular importance is attributed to anthocyanins, in which chokeberry fruit is rich. The advantage of these compounds is their comprehensive impact on both the immune system and reduction of oxidative stress, including the ability to chelate iron ions, which seems to be a key element not only for iron management. Anthocyanin supplementation might lead to reduced post-exercise muscle soreness [11] and improvement of performance parameters.
Analysis of the results available in the scienti c databases conducted on athletes, as well as the results of numerous scienti c reports based on research conducted on non-training people and animals, leads to the conclusion that the endogenous defence of an organism subjected to intense exercise load is insu cient. It seems that preparations rich in anthocyanins may be an important factor in alleviating the adverse effects of extreme exercise loads. Thus, it seems advisable to introduce to the diet of competitors plants rich in anthocyanins, which not only show the ability to form stable complexes with transition metals, but also increase the body's antioxidant potential. Such supplementation can both reduce oxidative stress, signi cantly reducing post-exercise in ammatory processes, and contribute to an increase in ergogenic potential.
The aim of the study was to analyse the effect of 7-week supplementation with chokeberry juice on the parameters of pro-oxidative-antioxidant balance and iron levels in footballers during the football season.

Research material
Twenty footballers from the MUKS Zawisza Bydgoszcz club participating in the Central Junior League competitions took part in the research. Basic data regarding the study group are included in Table 1. Football players receiving chokeberry juice and a placebo implemented a uniform training load scheme.
Training loads in the week preceding and ending the experiment are shown in Table 2. All subjects were informed about the purpose of the research and the procedures used, and voluntarily agreed to   Legend: min -minutes, * -training load scale where 1 means training at the lowest intensity and 10 means training at the highest intensity Experiment/supplementation The participants were randomly divided into two groups: supplemented (N = 12), which received 200 ml of chokeberry juice (100 ml twice a day in the morning and evening) for 7 weeks, and control (n = 8) which received a placebo at the same time. The placebo was identical to chokeberry juice in appearance and taste. Both the chokeberry juice and placebo were produced by MLB Biotrade Sp. z o.o., Poland. Juice and placebo were placed in identical dark coded bottles. The label codes were decoded after the test. In addition, the antioxidant capacity of chokeberry juice was determined using the DPPH and ABTS methods, which amounted to 8.83 and 7.62 mg/ml, respectively (tests were carried out by the Lubuskie Centre for Innovation and Agricultural Implementation of the University of Zielona Góra). The players participating in the study were informed during the experiment not to take any additional supplements or medications or to change their daily diet. Before and after supplementation, all competitors performed the Maximal Multistage 20 m Shuttle Run Test [12]. The exercise test ('beep test') was carried out in a fullsize sports hall with a classic surface. During the test, the air temperature was 19.1 °C and humidity was 51%. All tested players were informed about the test procedures and additionally motivated by the trainer to make maximum effort.

Material Collection And Examination
During the control exercise tests (beep test) carried out at the beginning and end of the experiment, blood was taken from the competitors four times. Blood was drawn before the exercise test, immediately after the end of the exercise test, and 3 and 24 h after the end of the exercise. Blood was drawn from the vein of the elbow into 9 ml polyethylene tubes (to obtain serum). The resulting material was then centrifuged (3000 rpm/10 min) and stored at − 80 °C until further examination. In addition, venous blood was also collected from the competitors in 5 ml tubes that contained EDTAK2 anticoagulant. This blood was used to determine the morphological parameters of the blood (RBC, HGB, HCT, MCV, MCH, MCHC). The examinations were made using ow cytometry using Sysmex XS-1000i apparatus.

The iron (Fe) level was determined in plasma taken from lithium heparin and determined by in vitro IRON
2 test for the quantitative determination of iron in human serum and plasma in a Roche/Hitachi Cobas c. system using a Cobas c 501 analyser.
Lactic acid (LA) was measured in capillary blood collected from the earlobe before and immediately after the test. The measurement was made using a Dr. Lange Plus LP20 biochemical analyser.

Statistical analysis
Sample size calculation was done based on previous results on the effects of supplementation on TBARS in males [13], as the variable of primary interest in the above study, using a calculator available online [http://powerandsamplesize.com/Calculators/Compare-2-Means/2-Sample-1-Sided]. As in previous research [13], it was decided to increase the sample size in the intervention group by setting the sampling ration as 1.5. Power was set to 0.8, while the Type I error rate was 5%. The calculated sample size in intervention group was n = 12. The Shapiro-Wilk W test was used to test the assumption of normality.
Two-factor ANOVA with group coe cient (supplemented group/placebo group) and time (trial/trial II) was selected for the analysis of physical tness variables when all assumptions were met. In the case where the normality assumption was violated, aligned rank transform for nonparametric factorial ANOVA was used, analysed using R [14]. The post-hoc test for differences of differences was done using the R package phia [15]. To assess dynamics of biochemical parameters in response to the physical exercise test, a linear mixed model t by REML with t-tests using Satterthwaite's method was applied with the R statistical packages lme4 and lmerTest. Subject and time (before vs just after vs 3 h after vs 24 h after physical exercise test in the case of biochemical parameters; and before vs 3 h after in the case of blood morphometry parameters) factors were set as random effects. Group (placebo vs supplemented) and intervention (before vs after physical exercise programme) were set as xed effects. Interaction between xed effects was calculated as well as the con dence interval (95%) for estimating interaction. As posthoc tests, a series of t-tests with Benjamini-Hochberg adjusted p value was applied to control for false discovery rate (FDR) using an online calculator (https://tools.carbocation.com/FDR). Mean value and standard deviation (SD) are reported and the alpha level was set to 0.05.

Results
There was no signi cant interaction between time × group and VO2max (58.82 ml/kg/min before vs 60.35 after in the juice group, 58.48 ml/kg/min before vs 60.  (Table 3a)). The results of post-hoc tests were statistically insigni cant.   (Table 3a)). The results of post-hoc tests were statistically insigni cant.
Biochemical analysis of the remaining selected parameters of in ammation did not show a signi cant interaction in the supplemented or placebo groups (Table 3a).
A signi cant effect of the intervention period on TBARS dynamics was observed under the in uence of the physical exercise test in the studied groups (estimate = 4.77 (CI of estimate = 2.62; 6.92), t = 4.33, p = 0.00003 (Table 3b)). The results of post-hoc tests were statistically insigni cant.
A signi cant effect of the intervention period on 8-OHdG dynamics was observed under the in uence of the physical exercise test in the studied groups (estimate = 0.79 (CI of estimate = 0.36; 1.22), t = 3.56, p = 0.0005 (Table 3b)). The results of post-hoc tests were statistically insigni cant.
Biochemical analysis of the remaining selected parameters of pro-oxidative-antioxidant balance did not show any signi cant interaction in the supplemented group or the placebo group (Table 3b) No signi cant effect of the chokeberry supplementation period was observed on the blood morphotic elements tested (Table 4). Legend: SD -standard deviation, WBC -white blood cells, RBC -red blood cells, Hb -haemoglobin, Hct -haematocrit, Fe -iron, LA -lactate acid, before -before the test, after -180 s after the test, 3 h after -3 h after the test As Table 5 shows, no signi cant changes in body weight, BMI or adipose tissue were observed after supplementation in any of the groups.

Discussion
The physical exercise in which athletes take part in competitive sport may result in a disturbance of homeostasis of the body, which in turn leads to much worse sports performance, as well as a deterioration of health. Analysis of the available literature indicates that the compounds contained in chokeberry have strong antioxidant activity. In this respect, a key role is attributed to anthocyanins, which prevent the excessive formation of free radicals, namely superoxide, hydroxyl, nitrite and chlorine radicals [16,17]. The anti-radical activity of these compounds is increased by the number of hydroxyl groups on the B ring and the arylation of sugar residues with phenolic acids. As demonstrated in the studies of van Acker et al., anthocyanins have 100 times higher activity in nitric oxide radical (•NO) removal than the endogenous antioxidant glutathione [18]. Due to the presence of hydroxyl groups in the C ring, these compounds are able to chelate transition metal ions (e.g. iron and copper) [19]. Another important feature of anthocyanins from the point of view of health is their ability to inhibit lipid peroxidation [20], which can be of great importance in reducing haemolysis induced by intense physical exertion.
Research shows that the antioxidant capacity of chokeberry measured by total antioxidant capacity (TAC) as well as the strength of reduction of Fe 3+ to Fe 2+ is very high and depends on weather conditions; the presence of active compounds is in uenced by both the average air temperature and hours of sunshine from May to September [21].
The antioxidant potential of the chokeberry juice given to football players was measured using two methods, DPPH and ABTS, as 8.83 mg/ml and 7.62 mg/ml, respectively (relative to the activity of the Trolox reference compound), which indicates that it was relatively low. This can probably be explained by the lack of statistically signi cant differences demonstrated not only in the level of TAC, but also in other biochemical, morphological and performance parameters in football players (Tables 3a, 3b, 4 and 5).
Petrovic et al. used chokeberry juice supplementation in handball players and showed that 100 ml/day supplementation of chokeberry contributed to small changes in the lipid pro le and reduced TBARS levels; however, these changes were observed only in men [22]. In our research, the chokeberry juice dose was twice as high, which had no effect on the reduction of free radical damage measured with both TBARS and 8-OHdG levels (Table 3b). García-Flores et al. combined chokeberry extract with citrus juice (200 ml of drink was 95% fresh citrus juice and 5% chokeberry extract); this combination of ingredients signi cantly reduced post-exercise changes in the level of DNA damage markers measured in both the plasma and urine of triathlon riders [23].
Analysis of the available literature indicates that the advantage of compounds derived from chokeberry is their comprehensive effect on both the immune system and reduction of oxidative stress, including the ability to chelate iron ions, which seems to be a key element not only for iron management. For this reason, we expected it to reduce markers of oxidative stress. However, the lower (statistically insigni cant) average values of the tested markers of oxidative stress, obtained in the second test period (after supplementation), concerned both the supplemented and control groups, which may be a result of the players' adaptation to the applied exercise loads. Zügel et al. analysed the cumulative effect of training stress in highly quali ed athletes practising rowing on the level of hepcidin and its impact on parameters related to iron management. They showed that the levels of hepcidin and ferritin as acutephase proteins were a sensitive indicator of changes in training loads (increase in volume and intensity of exercise). In their own research, football players were subjected to the same training loads throughout the entire study period (Table 2), which probably explains the lack of statistically signi cant differences in the levels of hepcidin and ferritin (Table 3a) [24]. In other studies conducted by the team of Villaño et al., the effect of physical exercise and supplementation with juice high in polyphenols (the juice also contained chokeberry extract) on the level of hepcidin was analysed in a group of triathletes of both sexes. The study did not show a signi cant impact of the supplement on this parameter, while its reduction was associated with adaptation of the players' bodies to the applied exercise loads [25].
In our study of footballers, interesting trends related to iron levels were observed in the second study period; namely, the level of this parameter after 3 h of rest in the supplemented group decreased, while in the control group it increased (Tables 3a and 4). Similar changes in iron levels were observed by Punduk et al. in volunteers who received intensive platelet-rich plasma therapy during intense exercise. This therapy aimed to improve muscle regeneration, which was damaged by the use of high-intensity exercises (exercise-induced muscle damage, EIMD) [26]. It can be assumed that the ability to chelate iron ions, through the active compounds contained in chokeberry, can also counteract damage to muscle bres. Con rmation of this thesis can be observed from the changes in myoglobin level in subjects, although they were not statistically signi cant (Table 3a). Myoglobin is a marker of muscle bre damage; in the group supplemented with chokeberry it showed a downward trend, while in the control group the level of this parameter increased.
In the in ammatory process, the role of anthocyanins can result from both the ability to sequestrate iron [27] and from their regulatory action on various components of the immune system involved in the development of in ammation [28]. Research conducted by Ohgami et al. on animal models has shown that chokeberry extract has a strong anti-in ammatory effect on endotoxin-induced uveitis in rats. The authors observed that the number of in ammatory cells, the protein concentration, and the levels of NO, pyrogenic prostaglandin E2 (PGE2) and TNF α in the aqueous humour in the groups treated with aronia crude extract were signi cantly reduced, and effect strength depends on the dose used [29]. For this reason, the standardization of chokeberry products for the presence of anthocyanin compounds, which play a key role in health protection, may be of great importance.
Summing up the research results presented above, it can be stated that the use of chokeberry products in the diet did not cause signi cant changes in the parameters analysed. The reason could be both good adaptation of the examined players to their physical exercise, and the use of juice with low antioxidant capacity. Therefore, it seems reasonable to consider the use of chokeberry extracts standardized for the presence of anthocyanins. Another issue that should be explored is to understand the mechanisms of how and what compounds contained in chokeberry may be responsible for improving the parameters studied.