The impact of supplementation with pomegranate fruit (Punica granatum L.) juice on selected antioxidant parameters and markers of iron metabolism in rowers

In this study, supplementation with pomegranate fruit juice boosted the antioxidant potential of rowers, as expressed by TAC. The level of this parameter in the supplemented group was significantly higher during the restitution period than in the placebo group (Fig. 1a). The increase in antioxidant potential did not exert a significant effect on other study parameters, however. Previous studies [23] have demonstrated that pomegranate fruit juice has three-fold greater antioxidant activity than other food products widely recognised for their antioxidant properties, such as red wine and green tea. The antioxidant potential of pomegranate fruit juice results from its high content of polyphenols, especially proanthocyanidins [24]. An increase in TAC after a two week supplementation with pomegranate fruit juice has also been reported by other authors [25].

Prior to the supplementation (at baseline), intense physical exercise resulted in a significant decrease in TAC in the study athletes (Fig. 1). Free radicals that are accumulated in excess and inadequately inactivated may, inter alia, initiate the peroxidation of polyunsaturated fatty acids of erythrocyte membranes, and thus enhance post-exercise haemolysis [26, 27]. This hypothesis might also be supported by the observation that prior to supplementation, our rowers showed greater post-exercise increases in iron concentration (Fig. 2c). Manthou et al. [28] demonstrated that healthy subjects supplemented for 14 days with pomegranate fruit juice had increased RBC count, haemoglobin concentration and haematocrit levels. According to those authors, these favourable changes might result from more effective prevention of RBC degradation among other thinks. Fiorani et al. [29] demonstrated that human erythrocytes can absorb extracellular flavonoids via passive diffusion, and constitute a reservoir of these compounds. While most flavonoids (according to the authors, up to 85%) reach the cytosol, some are incorporated into cell membrane. Studies [30, 31] have shown that, similar to cholesterol and alpha-tocopherol, intracellular flavonoids are localised in close proximity to the cell membrane, between the lipid bilayer and aqueous phase. As a result of this location, flavonoids play a vital role in the cell, stabilising plasma membranes that become less fluid, and thus, more resistant to oxidation [32]. Another key issue is cooperation between flavonoids, alpha-tocopherol and ascorbic acid. Flavonoids were shown to inhibit the oxidation of intracellular alpha-tocopherol and to regenerate (as does vitamin C) oxidised alpha-tocopherol to its radical. Ascorbic acid, also protected by flavonoids against oxidation, can in turn inhibit oxidative changes in flavonoids, prolonging their protective effect [33, 34]. Flavonoids therefore maintain a relative balance between oxidised and reduced forms of antioxidants and their radicals, and therefore provide another protective mechanism against elevated concentrations of reactive oxygen species.

Although only athletes from the supplemented group presented with enhanced antioxidant potential during the ergometric test conducted at the end of the follow-up period, physical exercise did not induce significant changes in TAC in either study group (Fig. 1a). Uric acid, the final product of purine metabolism, which proved to be an important antioxidant of blood plasma during in vivo studies [35], did not contribute to changes in TAC levels, although our rowers presented with elevated concentrations during the restitution period (Fig. 1b). Braakhuis et al. [36] demonstrated that the result of a 30 min rowing-ergometer test correlated positively with years and hours of training and the antioxidant status of the blood in elite rowers. According to those authors, these factors had a greater impact on TAC than the dietary intake of antioxidants. The results of our present study suggest that another modulator of TAC may be the phase of the training cycle. The second ergometric test took place during the competitive period when the organism of a well-trained athlete should be characterised by so-called “readiness for competition”, that is be fully adapted to an exercise load specific for a given discipline. It should be stressed that during rowing competitions, athletes participate in qualification and final races, and sometimes need to cover a 2000 m distance twice in a single day. The adaptation of our rowers to this type of exercise load was confirmed by other parameters analysed: a lack of statistically significant changes in IL-6 concentration (Fig. 4) and post-exercise increases in iron levels (Fig. 2c). A study of elite male rowers conducted prior to the Rowing World Championships showed a significant association between the level of proinflammatory cytokines, such as IL-1β, TNF-α and IL-6, and measures of depressed mood, sleep disturbances and fatigue [37]. The lack of statistically significant post-exercise changes in concentrations of proinflammatory cytokines may thus provide important information about the readiness of athletes for competition.

Irrespective of the testing period, our athletes did not show statistically significant changes in hepcidin, myoglobin or CK levels (Fig.

2a

,

b

, Fig.

5

). To the best of our knowledge, CK activity has rarely been studied in POM-supplemented subjects. We found only one report documenting a significant increase in CK activity in a group of recreationally active males receiving either POM or a placebo for a period of nine days; this effect was probably a consequence of myocyte damage in both study groups [

38

]. The lack of significant changes in hepcidin, myoglobin and CK levels in our study subjects could perhaps be explained by their good adaptation to large training loads; this issue seems to be an interesting topic for future research. Nevertheless, athletes from both groups showed a significant post-exercise increase in serum concentration iron at baseline measurements (Fig.

2c

).

Fig. 5

Data represents the mean (SD) values for creatine kinase (CK) levels during exercise tests performed before and after the supplementation (mean ± SD). Note: CK = creatine kinase; – SUPL = supplemented group; – PLA = placebo group; B = baseline; Ex = immediately after the exercise; R = after a 1 – day recovery

Both supplemented athletes and controls showed a significant post-exercise increase in UIBC during the follow-up test, which persisted after a 24 h recovery (Fig. 3a). Similarly, the post-exercise changes in TIBC seemed to be supplementation-independent, since a significant post-exercise decrease in this parameter was observed post-intervention regardless of the study group, both immediately after the ergometric test and following a 24 h recovery (Fig. 3b). Monitoring of sTfR and body iron has previously shown to be a reliable tool for the determination of Fe metabolism and successful prevention of its deficiency [39]. In our present study, ergometric tests conducted at the baseline contributed to a significant increase in sTfR level in the supplemented group and to a significant decrease in this parameter in the controls. Noticeably, athletes from the supplemented group presented with significantly higher pre-exercise levels of sTfR than the controls during the post-intervention ergometric test (Fig. 3d). Neither supplementation with POM nor physical exercise had a significant effect on serum ferritin levels in our study subjects (Fig. 3c), which is consistent with the results of other studies [40, 41].