The study was designed as a double-blind randomised controlled trial with parallel groups. After screening with respect to the inclusion and exclusion criteria by laboratory assistants, 20 young male semi-professional footballers (15.8 ± 0.7-years-old) from Międzyszkolny Uczniowski Klub Sportowy (MUKS) Zawisza Bydgoszcz club (Bydgoszcz, Poland), participating in the Central Junior League competitions, took part in the study. The subjects were randomly assigned to the supplemented (n = 12; FP-S) or the placebo group (n = 8; FP-C). Each group was similar in terms of the anthropometric data and the position on the football field (Table 1). Basic characteristics of the study group are summarized in Table 1. The participants receiving chokeberry juice or a placebo followed a uniform training load scheme. Training loads for the entire experimental period (microcycle) are shown in Table 2. Load time intensity from the beginning of the season until the end of the experiment is summarized in Table 3.
All subjects were informed about the purpose of the research and the procedures, and voluntarily agreed to participate in the study. The research was conducted according to the Declaration of Helsinki and was approved by the local Bioethics Committee at Collegium Medicum in Bydgoszcz (approval no. KB 382/2017). All players were assessed with respect to the inclusion and exclusion criteria, and were asked not to use any supplements (vitamins, ergogenic supplements, herbal extracts, caffeine, theine, etc.) 2 weeks before and during the experiment. One week before the exercise test and during the experiment, the participants adopted similar eating habits. They were asked to eat balanced meals prepared based of the daily energy requirements in relation to age and physical activity. Substances that could interfere with the test results, containing large amounts of anthocyanins, phytosterols, and antioxidants were excluded from the meals. All meals were prepared according to the guidelines of professional sports nutrition by a sport nutritionist, as recommended by the Polish Football Association .
The participants were randomly divided into two groups: the supplemented group (n = 12), which received 200 ml of chokeberry juice (100 ml twice a day, in the morning and in the evening) for 7 weeks; and the control group (n = 8), which received a placebo at the corresponding times, according to published guidelines . In previous studies, the average duration of chokeberry supplementation tested was 6 to 8 weeks . The research protocol scheme is presented in Fig. 1.
Physical exercise program
During the entire experimental period, all subjects followed their regular physical exercise program. The physical exercise program was planned by the main coach of the team, and was the same for both groups. The training program microcycle (presented in Table 2) consisted of a uniform pattern of tasks performed during the game season, during which the research was conducted, with the intensity level of a given training unit expressed on a scale from 1 to 10 (the training loads scale).
The anthocyanin content was determined to be 165.3 mg/100 ml of juice. Briefly, the anthocyanin pigment content was analysed by high-performance liquid chromatography, as described by Oszmański and Sapis . For the analysis, LC Agilent Technologies 1200 Rapid Resolution (Waldbronn, Germany) system equipped with a UV–Vis detector (DAD 1260, Waldbronn, Germany) and Zorbax SB-C18 column (4.6 × 150 mm, 5 μm) (Agilent, Wilmington, Delaware, USA) were used. Separation was achieved using a reversed-phase system with gradient elution. Chromatographic conditions were as follows: injection volume, 20 μm; flow rate, 1.0 ml/min; solvent A, 10% formic acid in water; solvent B, 10% formic acid, 30% acetonitrile, 60% water. The following gradient was used: 0–8 min 20–40% B, 8–15 min 40–50% B, 15–16 min 50–100% B, 16–20 min 100% B (isocratic), 20–23 min 100–20% B. Chromatographic data were acquired at 400 to 600 nm, and integrated at 520 nm for anthocyanins. The results are expressed as cyanidin-3-O-glucoside (external standard) (LGC Standards, Bury, UK) (mg/100 g or %). Cyanidin-3-O-glucoside was dissolved in water, and the chokeberry juice was diluted 10 times in redistilled water and filtered through 0.45-μm filter prior to analysis.
Subjects in the control group were given the placebo containing 6.6% solution of betaine [(CH3+)3 N+ · CH2 COO−] and 1% solution of citric acid. The placebo was identical in appearance and taste to chokeberry juice, and both were given in 200-ml numbered sintered glass bottles. The label codes were decoded after the examination of all biochemical factors after intervention completion. The participant play position or volume of competition play (starters vs. non-starters) was not considered in the randomization. Both the chokeberry juice and placebo were produced by MLB Biotrade Sp. z o.o., Poland (Poznan, Poland). The players and researchers were blinded to group assignment.
Antioxidant capacity of chokeberry juice
The antioxidant capacity of chokeberry juice was determined using 2,20-azinobis (3-ethylbenzthiazoline-6-sulfonic acid) (ABTS) and 2,2-di-phenyl-1-picrylhydrazyl radical (DPPH) methods at the Lubuskie Centre for Innovation and Agricultural Implementation of the University of Zielona Góra (Sulechów, Poland). ABTS, DPPH, and other reagents were purchased from Sigma Aldrich (St. Louis, MO, USA). The juice contained 8.83 mg/ml ABTS and 7.62 mg/ml DPPH.
Physical exercise test
Before and after the 7-week supplementation period, all players performed the maximal multistage 20-m shuttle run test (the ‘beep test’) . The test was carried out in a full-size sports hall with a classic surface, from 9:30 AM to 10:30 AM. The participants were asked to eat a light meal approximately 2 h prior to the test. They were instructed not to consume alcohol, caffeine, theine, or taurine on the test day. VO2max was calculated indirectly based on the results of the physical exercise test, as described elsewhere , on the assumption that retroextrapolated VO2max is not substantially different from VO2max measured directly . The physical exercise test took place on a Tuesday instead of the planned training session. The supplementation began on the following Monday and ended after 7 weeks on a Sunday. After the supplementation period, the test was repeated on the following Tuesday instead of the planned training session. During the test, the air temperature was 19.1 °C and humidity was 51%. All the tested players were informed about the test procedures and were additionally motivated by the trainer to make maximum effort.
Blood sampling and analysis
Blood samples were taken for analysis at four time points at the beginning and at the end of the supplementation period: before, immediately after, and 3 and 24 h after the beep test. These time points were selected because the levels of hepcidin and related parameters (interleukin, IL, 6) achieve a maximum 3 h after exercise [30, 31]. Further, blood sampling after 24 h allows determination whether the tested parameters have returned to the resting values. That is important because the training program consisted of daily physical exercise sessions in the examined subjects. Blood for serum analysis was collected from the ulnar vein into 9-ml serum tubes containing a coagulant (Sarstedt, Germany). The blood was centrifuged (3000 rpm, 10 min), and the serum was aliquoted, frozen in liquid nitrogen, and stored at − 80 °C until analysis.
To determine the morphological blood parameters (red blood cells, RBC; haemoglobin, HGB; haematocrit, HCT; mean corpuscular volume, MCV; mean corpuscular haemoglobin, MCH; and mean corpuscular haemoglobin concentration, MCHC), venous blood was collected into 5-ml tubes containing EDTAK2 as the anticoagulant. Morphological examinations were performed using flow cytometry on Sysmex XS-1000i apparatus (Kobe, Japan).
Iron levels were 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, using Roche/Hitachi Cobas c. system and a Cobas c 501 analyser (Cobas, Rotkreuz, Switzerland).
Lactic acid (LA) levels were measured in capillary blood collected from the earlobe before and immediately after the beep test, using a Dr. Lange Plus LP20 biochemical analyser (Dr. Lange, Berlin, Germany).
For detailed analysis of changes in the body’s iron management, total antioxidant levels, and the inflammatory cell response, the following enzyme-linked immunosorbent assay (ELISA) kits were used, according to the manufacturers’ instructions: ferritin ELISA kit EIA-1872, IL-6 ELISA kit EIA-4640, myoglobin ELISA kit EIA-3955, and hepcidin 25 (bioactive) HS ELISA kit EIA-5782, from DRG International, Inc. (Springfield, New Jersey USA); human thiobarbituric acid reactive substances (TBARS) ELISA kit (catalogue no. 201–12-7298) and human 8-oxo-2′-deoxyguanosine (8-OHdG) ELISA kit (catalogue no. 201–12-1437), from Shanghai SunRed Biological Technology Co. Ltd. (Shanghai, China); human albumin ELISA kit (catalogue no. EA2201–1) from Assaypro LLC (St. Charles, MO, USA); and TAC Fast Track DM P-4100 from LDN Labor Diagnostika Nord GmbH & Co. KG (Nordhorn, Germany). Thermo Scientific Multiscan GO microplate spectrophotometer produced by Fisher Scientific Finland (Vantaa, Finland) was used for the analyses.
Sample size calculation was done based on previous results on the effects of chokeberry supplementation on TBARS levels in males , as the variable of primary interest in the study, using a calculator available online [https://powerandsamplesize.com/Calculators/Compare-2-Means/2-Sample-1-Sided]. As in the previous study , sample size was increased in the intervention group by setting the sampling ratio as 1.5. The power was set to 0.8, with the type I error rate of 5%. The calculated sample size in the intervention group was n = 12. Shapiro–Wilk W test and visual histogram assessment were used to test the assumption of normality.
Two-factor analysis of variance (ANOVA) with group coefficient (supplemented group/placebo group) and time (before/after supplementation) was selected for the analysis of physical fitness variables using aligned rank transform for nonparametric factorial ANOVA with ARTool package for R . Post-hoc test for differences of differences was done using the R package phia . Partial eta-squared was calculated to assess the effect size of interaction in two-way ANOVA. To assess the dynamics of biochemical parameters in response to the physical exercise test, a linear mixed model fit by REML with t-tests using Satterthwaite’s method was implemented in the R statistical packages lme4 and lmerTest [35, 36]. Subject factor was set as a random effect. Time (before vs. just after vs. 3 h after vs. 24 h after the physical exercise test in the case of biochemical parameters; and before vs. 3 h after the physical exercise test in the case of blood morphometry parameters), group (placebo vs. supplemented), and intervention (before vs. after the physical exercise programme) were set as fixed effects. Interaction between fixed effects and the confidence interval (CI, 95%) for determining the interaction were calculated. Mean values and standard deviation (SD) are reported. Alpha level was set to 0.05.