In this study, we verified the hypothesis that supplementation with chokeberry juice (50 mg three times a day for 6 weeks) may prevent or at least attenuate the consequences of inflammation associated with intensive physical exercise, and exerts beneficial effects on the parameters of iron metabolism. The hereby documented favorable changes after supplementation with the chokeberry juice likely reflected chemical composition of the latter and resultant pleotropic, antioxidative and anti-inflammatory, effects.
The ergometric exercise test performed by our rowers was reflected by a significant post-exercise decrease in the TAC of the plasma, which was observed both prior to and after supplementation (Figure 2A). Previous studies also showed that exhaustive physical exercise can lead to reduction of the plasma TAC [11] and a resultant increase in the concentration of insufficiently neutralized free radicals, which may induce peroxidation of polyunsaturated fatty acids in erythrocyte membranes. Fiorani et al. [12], revealed that human erythrocytes can uptake flavonoids via a passive diffusion mechanism, and, therefore, constitute a specific reservoir. While the vast majority of flavonoids (up to 85%) are accumulated in the cytosol, they are also incorporated into the erythrocyte membrane. According to Arora et al. [13] and Erlejman et al. [14], the flavonoids accumulate at a lipid bilayer-aqueous phase interface, similar to cholesterol and alpha-tocopherol. Due to this intracellular location, flavonoids play vital roles in the stabilization of biological membranes, which become less fluid and thus more resistant to oxidation [15]. It is also worth highlighting the interactions of flavonoids, alpha-tocopherol, and ascorbic acid.
Flavonoids were shown to prevent intracellular oxidation of alpha-tocopherol and convert oxidized alpha-tocopherol back to its radical form (similar to vitamin C). Moreover, flavonoids protect ascorbic acid against oxidative injury and vice versa; thus, the protective effect of flavonoids is enhanced by vitamin C [16],[17]. According to Heidi et al. [18], the phenolic compounds present in chokeberry juice are more efficient in regenerating and protecting alpha-tocopherol than ascorbic acid and the phenolic compounds of blackcurrant. These differences were attributed to high concentrations of two anthocyanins, cyanidin-3-arabinoside and cyanidin-3-galactoside, in chokeberry juice and the lack of these compounds in blackcurrants. In turn, Hwang et al. [19], suggested that the strong antioxidant and radical-scavenging activities of black chokeberry extract can be associated with its high levels of antioxidants (total phenolics, total flavonoids, and proanthocyanidin contents), which protect against damage from reactive oxygen radicals. Our study also showed favorable changes in the TAC of athletes who supplemented with chokeberry juice. Compared to the respective pre-supplementation values, a significant increase in the TAC was documented in the supplemented group during the recovery period; furthermore, the post-supplementation TAC determined during the recovery period was significantly higher in the supplemented group than in the controls (Figure 2A).
Braakhuis et al. [20], documented an inverse association between the antioxidant biomarker, the TAC, of rowing athletes and the chronic training dose on a performance test. A similar relationship was also reported by Margonis et al. [21]. The changes in the plasma level of antioxidants, observed after exhaustive physical exercise, are probably associated with a transfer of some of these compounds from tissues to plasma. Previous in vivo studies identified uric acid, an end-product of purine metabolism, as a major plasma antioxidant [22]. According to Wayner et al. [23], the uric acid contribution to the TAC of the plasma is about 35-65%. Our athletes showed a significant increase in uric acid concentrations during the recovery periods after the exercise tests performed at Trials I and II (Table 6). However, as mentioned above, the concomitant increase in the TAC was observed solely in the supplemented group.
A relative balance between oxidized, reduced, and radical forms of antioxidants is maintained by flavonoids and constitutes an important element of protection against increased concentrations of reactive oxygen species. However, the role of flavonoids in the chelation of iron ions seems even more important, as this prevents formation of a highly reactive hydroxyl radical, a potent inductor of peroxidation of polyunsaturated fatty acids and polymerization of proteins, which are both present in erythrocyte membranes at high concentrations.
Previous studies [24],[25] revealed that structural alterations of erythrocyte membranes, resulting from enhanced generation of free radicals in response to exhaustive or long-term physical exercise, may lead to severe post-exercise hemolysis, which results in an increase in the plasma level of free iron. Under physiological conditions, the iron of heme proteins (hemoglobin, myoglobin, cytochromes) is protected inside a cell. However, it can be released as a result of cellular injury. Therefore, control of free Fe ions represents an important aspect of iron metabolism. Two important biological mechanisms are postulated to be involved in this process. The first is associated with preventing formation of highly toxic reactive oxygen species via the control of free or weakly-bound iron ions, and the second is aimed at protection of iron resources against bacterial degradation [26],[27]. A number of bacterial species (e.g. Mycobacterium tuberculosis, Salmonella spp., and Yersinia spp.) require Fe3+/Fe2+ ions for their growth. The decrease in pH, associated with exhaustive physical exercise and post-exercise inflammation (secretion of LA by activated granulocytes) promotes release of iron from hemoglobin, ferritin, and transferrin. Therefore, binding Fe+2 and other intermediate metals seems to be of vital importance. Anthocyanins, the major component of chokeberry, can chelate iron due to their specific chemical structure (presence of hydroxyl group in the C-ring) [28]. We showed that the same exercise test lead to different effects on the serum concentration of iron. There was an insignificant increase in this parameter at Trial I; while, the concentration slightly decrease at Trial II (Figure 3B). This suggests that the dynamics of serum iron are determined by a phase of training, rather than by the supplementation. We also did not document significant effects of chokeberry juice supplementation on the remaining parameters of iron metabolism, namely the levels of ferritin, TIBC and UIBC (Table 6). Although ferritin is considered an acute phase protein, its level in our rowers did not change significantly after the ergometric test. Similar findings were previously reported by Antosiewicz et al. [29], who found that high-intensity interval exercise (triple Wingate anaerobic test) did not induce statistically significant changes in the levels of ferritin, iron and TIBC of highly trained judo athletes.
The competitive phase of a training cycle (i.e. the period corresponding to the end of our experiment) was characterized by a markedly greater proportion of high-intensity training (Table 4) and a higher severity of post-exercise inflammation. This was also reflected by a post-exercise increase in the TNF-alpha level (Figure 1B). Other authors [30],[31] also observed elevated TNF-alpha levels in rowing athletes exposed to intensive training loads.
At the end of our experiment, the levels of iron determined during the recovery period were significantly higher in the supplemented rowers than in the controls (Figure 3B). In addition, we documented a significantly lower pre-exercise level of TNF-alpha in the supplemented group when compared to the pre-supplementation level. Furthermore, the TNF-alpha level at recovery turned out to be significantly lower in the supplemented group than in the controls (Figure 1B). We also observed an inverse correlation between the post-supplementation levels of iron and TNF-alpha (-0.476; p < 0.05). According to a prior report, anthocyanins can attenuate the activity of major inflammatory enzymes, and prevent adhesion of leukocytes and their interaction with vascular endothelial cells via inactivation with TNF-alpha [32]. The administration of blackcurrant extract (equivalent to 48 g of blackcurrants) to individuals performing 30-minutes of exercise on a rowing ergometer with an intensity corresponding to 80% VO2max was reflected by a markedly less pronounced post-exercise increase in TNF-alpha and IL-6 levels [33]. We did not document a significant influence of chokeberry juice on the level of IL-6 (Figure 1A). However, the concentration of this cytokine proved to be significantly modulated by physical exercise, which caused an increase in this parameter, irrespective of the analyzed group and trial. The level of IL-6 was positively correlated with the hepcidin level, both prior to (0.737; p < 0.05) and after the supplementation (0.506; p < 0.05). Hepcidin is considered an acute phase protein, as its synthesis in hepatocytes is induced by IL-6 [34]. This hormone is postulated to be an important mediator of post-exercise iron deficiency, which is observed in response to a number of physiological processes, such as inflammation, hypoxia, and an elevated concentration of Fe resulting from enhanced hemolysis, e.g. due to oxidative injury of erythrocyte membranes [3],[35]. We observed a post-exercise increase in the activity of hepcidin solely at Trial II, i.e. after supplementation (Figure 3A). Perhaps, this was the reason behind the post-exercise decreases in the serum level of iron observed after supplementation.
We are well aware of potential limitations of the study. While we measured the parameters of iron metabolism, also determination of the markers of post-exercise hemolysis and its severity, such as bilirubin, haptoglobin, methemalbumin and free hemoglobin, would add considerably to our knowledge of beneficial effects of chokeberry juice in elite athletes.
