How can chokeberry ( Aronia ) ( poly)phenol-rich supplementation help athletes? A systematic review of human clinical trials †

Athletes are increasingly consuming (poly)phenol supplements to modify oxidative stress and/or exercise-induced in ﬂ ammation, in the hope that this will enhance exercise performance. Chokeberries are rich in (poly)phenols and may therefore in ﬂ uence the health and performance of athletes. The objective of this systematic review was to comprehensively explore the e ﬀ ects of chokeberry supplementation on performance and exercise-induced biomarkers of oxidative stress, in ﬂ ammation, and haematology in the athletic population. A search was conducted in PubMed, Web of Science, and SCOPUS. Studies were included if the participants were athletes, supplemented with chokeberry or chokeberry-based products, and evaluated sports-related outcomes. A total of ten articles were included in the study. The participants of all the studies were athletes and included rowers, football players, handball players, triathletes, and runners. A qualitative comprehensive summary of the applications of chokeberry supplementation targeting the athletic population has been evaluated. This included the e ﬀ ect of chokeberry supplementation on redox status, exercise-induced in ﬂ ammation, haematology, iron metabolism, platelet aggregation, metabolic markers, body composition, and exercise performance. Chokeberry (poly)phenol-rich supplementation may be e ﬀ ective in enhancing the redox balance of athletes, yet more evidence is required to provide solid conclusions on its e ﬀ ect on in ﬂ ammation, platelet function, iron metabolism and exercise performance.


Introduction
Black chokeberry (Aronia melanocarpa L.) is a perennial shrub belonging to the Rosaceae family and native to the eastern regions of North America. 1 Chokeberries are gaining popularity among consumers due to their high amount of ( poly) phenols which are regarded as crucial for chokeberry fruits' high level of bioactivity. 2 Chokeberries are among the best sources of ( poly)phenols, rich in anthocyanins, flavonols, flavanols, proanthocyanidins, and phenolic acids. 3 The ( poly) phenol content of chokeberry fruit is reported to range from 6.3 g per 100 g dry material (DM) 4 to 7.8 g per 100 g DM, 5 which is approximately 3-8-fold that of red raspberry (Rubus idaeus) and 10-times that of strawberry (Fragaria ananassa). 6 The dark blue colour of chokeberry fruit is due to the high concentration of anthocyanins, which represent ∼25% of the total ( poly)phenol content. 7 Interestingly, it has been reported that almost 40% of the antioxidant activity of chokeberries is related to proanthocyanidins. 8 In terms of bioavailability, it has been reported that chokeberry anthocyanins are recovered in blood and urine in nanomolar concentrations, and glucuronidation and methylation are two of the common metabolism pathways of chokeberry anthocyanins. 9 Generally, the bioavailability of anthocyanins is affected by the extensive gut microbial metabolism in the colon, leading to the breaking down of the anthocyanin heterocycle which results in a wide range of low molecular weight phenolic metabolites which are more bioavailable and have higher absorption rates. 10 (Poly)phenols are increasingly researched for their pleiotropic effects on human health. The common ( poly)phenol intake in our diet could reach up to 1 g day −1 , this is about 10-100 times more than the intake of other antioxidants. 11 Most of the health benefits of ( poly)phenols are ascribed to their antioxidant and anti-inflammatory activities. Such effects have led to interest from athletic populations. Indeed, antioxidant and anti-inflammatory supplements are purported to enhance exercise and/or accelerate recovery after strenuous exercise. 12,13 However, their use by athletes remains controversial; while ( poly)phenols regulate various mechanisms associated with exercise performance, antioxidants have also been suggested to blunt training adaptations. 14,15 While exerciseinduced increases in reactive oxygen species (ROS) production may be crucial for training adaptations, 16 when ROS production exceeds the body's antioxidant capacity, the resulting oxidative stress may have a deleterious effect on recovery, performance, and general health. 17 Growing evidence suggests that ( poly)phenols can upregulate endogenous antioxidant capacity via the nuclear respiratory factor 2 (NRF2) pathway. 18 NRF2, plays an important role in mitochondrial biogenesis, and variants of the NRF2 gene have also been associated with endurance performance. 19 Another effect of oxidative damage is reduced vasodilatory capacity and blood flow. 20 (Poly)phenols have been shown to enhance flow-mediated dilatation and endothelial function in humans by promoting endothelial nitric oxide (NO) synthesis. 21 Furthermore, ( poly)phenols have shown to reduce the formation of peroxynitrite by inhibiting NADPH oxidase, one of the key sources of superoxide production. 22 At the same time, this increases endogenous antioxidant capacity and preserves NO bioavailability. In fact, it has been proposed that ( poly)phenols can modulate gene expression in general by increasing the activity of transcription factors, but also by affecting the expression of microRNAs. 23 Consequently, ( poly) phenol supplementation may counteract fatigue and improve performance by improving the perfusion of the exercising muscle. 24 In practice it has been suggested that acute supplementation (300 mg ( poly)phenols 1-2 h before exercise) may exhibit ergogenic properties during endurance and repeated sprint exercise. 24 On the other hand, chronic supplementation (more than 3 days with >1000 mg ( poly)phenols prior to and following exercise) could be helpful to enhance recovery after muscle damage.
Due to chokeberry's high ( poly)phenol content several studies have investigated the effects of supplementation with chokeberry fruits or derivates on biomarkers associated with sports and exercise performance and recovery, such as inflammatory status, 25 oxidative stress, [26][27][28] and body composition. 29 Indeed, oxidative stress and inflammation are important factors closely related to muscle catabolism. 30 Athletes performing exhausting exercises, involving heavy training loads, may be affected by sports anaemia due to depletion of iron reserves. 31 This is caused by haemolysis, haematuria, and increase in plasma volume as a result of intensive exercise. 32 (Poly)phenols may positively impact the cardiovascular status of athletes by inhibiting of platelet function 33 and iron metabolism. 25 This is achieved by targeting specific thrombogenic pathways. 33 Indeed, chokeberry ( poly)phenols have been evaluated for their effect on haematology, iron metabolism and platelet aggregation in athletes. 26,28,29 Chokeberry fruit and chokeberry-based supplements are principally of interest to athletes due to their high ( poly) phenol content in comparison to other sources of ( poly) phenols, and therefore potential use as an antioxidant and anti-inflammatory. However, as chokeberry fruit may also impact haematology and iron metabolism, supplementation could alter several aspects of athlete's health status and ultimately enhance their performance. To our knowledge, the effects of black chokeberan ry-based supplementation in healthy people engaged in exercise, and athletes, have not been reviewed comprehensively. Therefore, the aim of this systematic review is to evaluate the biological effects of chokeberry fruit in athletic populations; specifically, the effects on inflammation, oxidative stress, iron metabolism, haematological markers, and performance.

Methods
The systematic review was conducted based on the recommendation of The Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA). 34 The protocol of this systematic review was registered at the Open Science Framework (https://doi.org/10.17605/OSF.IO/Y832W).

Literature search
Three electronic databases (PubMed, Web of Science, and SCOPUS) were searched for relevant studies on 17 th November 2022 and updated on 14 th May 2023. The search strategy was based on including ("chokeberry" OR "Aronia") AND ("sport" OR "performance" OR "athlete" OR "exercise"). An example of the full electronic search strategy is provided in the ESI. † Two of the reviewers (RZ and AAR) were responsible for independently screening each article's title, abstract, and full text. A third reviewer (GC) arbitrated when needed.

Study selection
The inclusion criteria of this systematic review were: (i) clinical trial (human study), (ii) participants supplemented with chokeberry or chokeberry-based products, (iii) participants were athletes and performed a type of sports activity as a part of a sports team or as independent professional/semi-professional/ recreational athletes, or participants who were physically active performing at least 1 h of exercise per day for at least 3 days a week, (iv) study investigated sports and athletic-related outcomes that are linked with exercise performance and biomarkers, and (v) studies were peer-reviewed and written in English. For eligible studies, the PICO considered was as follows: population = athletes or physically active individuals, intervention = chokeberry supplement, comparison = not applicable, and outcomes = inflammation, oxidative stress, iron metabolism, haematological markers, and performancerelated outcomes. The search strategy excluded (i) non-clinical studies (including in vitro and in vivo (animals)), (ii) clinical studies that did not use chokeberry-based supplements (iii) clinical studies with inactive or unhealthy participants, (iv) studies evaluating outcomes not relevant to exercise performance and athletic metabolic status or biomarkers, (v) non-English studies, and (vi) comments, editorials, or reviews.

Data extraction
Two of the reviewers (RZ and AAR) independently extracted the data. The characteristics of the included studies are summarized in Table 1. The following information was extracted: background (name of the first author and year of publication), study design, focus topic, characteristics of athletes (number, sports performed, sex, and age), supplementation intervention (number of participants, supplementation form, and supplementation dosage), placebo intervention (number of participants, placebo form, and placebo dosage), study duration, experimental design, and key findings. We did not plan to conduct a meta-analysis due the heterogeneity in study designs, outcomes, and limited studies on this topic.

Risk of bias assessment
Two of the reviewers (RZ and AAR) independently assessed the risk of bias for each included study according to the revised Cochrane tool for assessing the risk of bias in randomised trials (RoB 2 tool), 35 and non-randomised studies of interventions (ROBINS-I tool). 36 A third reviewer (GC) arbitrated when needed. For randomised studies, the assessment was based on (1) bias arising from the randomisation process, (2) bias due to deviations from the intended intervention, (3) bias due to missing outcome data, (4) bias in the measurement of the outcome, and (5) bias in the selection of the reported results. For crossover studies, the risk of bias arising from period and carryover effects was also considered. Each domain was assessed and assigned either a low risk of bias, high risk of bias or some concerns with respect to risk of bias. For non-randomised studies, the assessment also evaluated the bias due to confounding in addition to the domains stated for randomised studies. Visualising of the risk of bias assessment was performed using robvis online tool. 37

Study selection
The review identified 401 records by searching the three databases, from which 352 non-clinical articles were identified and removed before the screening. After removing duplicates, 23 articles remained and were screened by title, abstract, and keywords by two of the reviewers (RZ and AAR) independently. A third reviewer (GC) arbitrated when needed. Thirteen articles were excluded for different reasons: supplementation was not based on chokeberry (n = 3), the study did not target exercise performance-related variables (targeted diseases such as cancer (n = 3), hypercholesterolemia (n = 1), bowel disease (n = 1), intermittent claudication (n = 1)), the study did not target athletes or active individuals (n = 3), or the study evaluated the bioavailability of chokeberry supplementation rather than bioactivity (n = 1). Ten articles were assessed for eligibility, and all ten were eligible and included in the qualitative synthesis of the current systematic review. The details of the study selection process are shown in Fig. 1.

Characteristics of the included studies
Among the included studies, five studies were randomised double-blind placebo-controlled trials, [25][26][27][28]38 three studies were randomised double-blind placebo-controlled crossover trials, [39][40][41] one study was single-blind placebo-controlled crossover trial, 42 and one study was a non-randomised non-blind trial. 29 The participants of all the studies were athletes and this included rowers (n = 38), 25,26 football players (n = 42), 28,38 handball players (n = 48), 27,29 triathletes (n = 48), [39][40][41] and runners (n = 10). 42 In terms of supplementation form and dosage of the chronic studies four of nine chronic studies used pure chokeberry juice 25-28 for a period of 4-8 weeks. Three of these studies quantified the anthocyanin content of the juice such that the dose of chokeberry anthocyanins was 330-3600 mg day −1 . 25,26,28 Three studies used a two-ingredient fruit juice containing chokeberry (5%) and citrus (95%). [39][40][41] The total anthocyanin content of this juice was 50 mg day −1 . Two studies used extracts, of which, one used dry chokeberry extract 38 and another one used aqueous chokeberry extract 29 the anthocyanin content of these extracts was not determined. The only acute study in this review used a chokeberry juice with a ( poly)phenol dose of 2600 mg.

General findings
The comprehensive search revealed that chokeberries in the field of sports science has mostly been evaluated for its effects on oxidative stress, inflammation, iron metabolism, and platelet aggregation.
3.3.1. Effect of chokeberry on redox status and exerciseinduced inflammation. Eight studies measured the effects of chokeberry on markers of oxidative stress, i.e., lipid and DNA oxidation. [26][27][28][29]38,39,41 Three studies found that chokeberry had no effect on thiobarbituric acid reactive substances (TBARS), [26][27][28] while in one study TBARS was significantly reduced, as were nitrites. 29 In three studies by the same author, chokeberry was shown to reduce other urinary biomarkers of lipid peroxidation. [39][40][41] In one study, 8-8-hydroxydeoxyguanosine was reduced after supplementation. 38 In two studies chokeberry increased total antioxidant capacity (TAC), 25,38 but not in another. 28 Two studies measured endogenous antioxidants; in one, superoxide dismutase was reduced 24 hours-and glutathione peroxidase immediatelypost exercise compared to a control. 26 In another non-controlled study, superoxide dismutase, reduced glutathione, and catalase activity were measured pre-and post-exercise following 6 and 12 weeks of supplementation with chokeberry. 29 At 6 weeks, compared to baseline, reduced glutathione was reduced and SOD was lower post-exercise only. Relative to baseline, at  thiobarbituric acid reactive substances. a Training load quantification was performed using the Objective Load Scale (ECOs).

Food & Function Review
This journal is © The Royal Society of Chemistry 2023 Food Funct. 12 weeks, catalase was increased, and reduced glutathione was decreased, both pre-and post-exercise. Two measured inflammatory markers. 25,38 In one tumour necrosis factor (TNF)alpha was lower 24-hour post-exercise following 8-week chokeberry supplementation compared to a placebo. 25 In the other interleukin (I)L-6 was lower and IL-10 was higher after supplementation. 38 3.3.2. Effect of chokeberry on haematology, iron metabolism and platelet aggregation. Three studies measured the effects of chokeberry on haematology. 26,28,29 One study reported increased levels of HGB and RBC and decreased WBC levels after 6 weeks of supplementation compared to baseline. However, two placebo-controlled studies found no effect of chokeberry on haemoglobin (HGB) 26,28 and one in red blood cell (RBC), haematocrit (HCT), mean corpuscular volume (MCV), mean corpuscular haemoglobin (MCH), and mean cor-puscular haemoglobin concentration (MCHC). 28 Three studies analysed markers of iron metabolism. 25,28,38 Eight weeks of chokeberry supplementation was found to increase iron levels 24 hours, but not immediately following a 2000 m row versus a control. 25 The same authors found no effect on other markers (hepcidin, ferritin, myoglobin, total iron-binding capacity, unsaturated iron-binding capacity). In the other studies, chokeberry did not affect iron, hepcidin, or ferritin at any point (immediately, post or 24 h) following an exercise endurance test. 28,38 One study investigated the effects of acute chokeberry supplementation on platelet aggregation; 42 compared to a placebo, chokeberry decreased the expression of platelet activation markers (P-selectin and glycoprotein IIb/IIIa) but did not influence platelet aggregation following a simulated half-marathon race.
3.3.3. Effect of chokeberry on other outcomes. Other outcomes studied included blood markers; albumin, 28 creatine kinase, 26 creatinine, 29 fatty acids, 27 glucose, 27,29 lactate, 25,26 lactic acid, 25,28 lipid profiles, 27,29 urea, 29 and uric acid. 25 In addition, four studies measured the effect of chokeberry supplementation on body composition [27][28][29]38 and three on performance parameters. 25,29,38 In one study distance covered in a multistage 20 m shuttle test was longer and post-exercise lactate was higher after 90-day supplementation with chokeberry. 38 One study found decreased lactate and CK immediately post-exercise with chokeberry but not placebo. 26 One 12-week uncontrolled study found that chokeberry decreased body fat and body fat percentage and increased the levels of high-density lipoprotein, glucose, creatinine, and urea relative to baseline. 29 Additionally, Petrovic and colleagues 27 reported decreased levels of C18:1n-9 and C18:3n-3 in men, but not in females.

Risk of bias assessment
A total of eight out of ten studies were randomised controlled trials, while two were non-randomised. Most of the randomised controlled trials were associated with a low risk of bias (Fig. 2), except one study 28 which was associated with some concerns with respect to bias in measurement of outcome. No clear information was provided regarding the awareness of outcome assessors of the intervention received, yet it was probably not influenced by knowledge of intervention since researchers were blinded to group assignment. The two nonrandomised studies 29,42 were associated with a moderate risk of bias due to moderate bias in the classification of interventions and measurement of outcomes (Fig. 3). The bias in the classification of intervention was linked with the lack of infor-

Discussion
This is the first study to systematically evaluate the impact of chokeberry (Aronia) supplementation on inflammation, oxidative stress, iron metabolism, and platelet activation in athletes. The current review identified 10 human studies that fit the criteria; there was some evidence that chokeberry could alter redox status, however, there were limited studies on the other outcomes precluding any firm conclusions to be drawn from the available evidence.
The studies in the current review were conducted on athletes from several different disciplines; rowers, footballers, handballers, triathletes, and runners. It is well documented that high-intensity and/or prolonged exercise evoke a pro-oxidative and proinflammatory response. 30,43,44 Although the generation of ROS and reactive nitrogen species (RNS) are important signalling molecules in physiological processes, excess generation of the most volatile ROS and RNS (e.g., peroxynitrite), as is common after high strenuous exercise, can overwhelm the endogenous antioxidant defence, causing oxidative damage. 45 As oxidative damage to proteins, lipids, and DNA can affect force production and exercise performance, there is a significant interest in whether dietary supplements with antioxidant properties can enhance performance. 30 Chokeberries contain bioactive compounds that act as natural antioxidants (e.g., vitamin C and carotenoids 46 ), although their antioxidative potential is predominantly attributed to their ( poly)phenolics. 47 In accordance, several studies included in this review demonstrated that chokeberry supplementation reduced markers of exercise-induced lipid peroxidation, even in those with vitamin C-matched placebos, 29,38-41 and increased TAC following exercise. 25,38 In addition, chokeberry supplementation modified antioxidant enzymes. In one study, GPx activity immediately and SOD activity 24 h following an incremental rowing exercise test were lower compared to a placebo after 4 weeks. 26 In another study, following 12 weeks of supplementation catalase was increased and GSH was decreased immediately and 10 min after a physical load test relative to baseline. 29 Although not included in the current review due to the non-athletic population Chung et al., (2023) also reported the ability of chokeberry to modulate the glutathione defence system by increasing GSH availability and GPx activity immediately and 30 minutes post-exercise. 48 These data could suggest (i) less reliance on the endogenous antioxidant defence systems due to the antioxidative capacity of the chokeberry or (ii) upregulation of endogenous antioxidant production via the NRF2 antioxidant response element pathway after longer-term chokeberry administration. 49 For example, chokeberry supplementation has been

Review
Food & Function shown to activate NRF2 by degrading its repressor, Kelch-like ECH-associated protein 1, leading to an increase in expression of antioxidant enzymes in mice. 50 However, due to the limitations of the markers used to assess redox status in vivo 51 and the non-controlled study design of ref. 29, these findings should be approached with caution. Nevertheless, based on available studies, these findings suggest chokeberries have the potential to influence redox status and therefore performance during exercise that are negatively affected by the excess generation of ROS and RNS.
After an intense exercise bout, the increase in RONS is generally accompanied by further secondary inflammatory-mediated damage. 24,52,53 In addition to antioxidant activities, the antiinflammatory properties of (poly)phenols are well documented. 54 Specifically, (poly)phenols have been shown to interact with cellular enzymes and signalling pathways involved in the inflammatory process. 55 However, as compared to other (poly)phenol-rich fruits the anti-inflammatory effects of chokeberry in exercise paradigms have been examined to a much lesser extent. 56 In the current review, only two studies included inflammatory markers. 25,38 Both studies found that supplementation with chokeberry reduced markers of inflammation (TNF-α and IL-6) and Stankiewicz et al., 2023 found an increase in anti-inflammatory cytokine IL-10. 38 While more studies are needed to corroborate the findings of ref. 25, the potential anti-inflammatory actions of chokeberries could have applications in exercise recovery by counteracting any inflammatory-related damage to skeletal muscle. As the oxidative and inflammatory response to exercise is now widely accepted as playing a role in driving training adaptations, athletes should be cautious of interfering with these processes when adaptations are a priority (e.g., during pre-season). Notwithstanding, there is limited evidence that polyphenol supplements disrupt training adaptations. 24,52,53,57 Additionally, exercise-induced oxidative stress and inflammation can have deleterious physiological effects such as enhanced platelet activation and anaemia. 58,59 In one study, acute chokeberry supplementation was shown to decrease the expression of platelet activation markers (P-selectin and glycoprotein IIb/IIIa) but did not influence platelet aggregation following a simulated half-marathon race. 42 This may be beneficial to those unaccustomed to prolonged exercise in which acute stress can increase platelet hyperactivity and have negative consequences on the cardiovascular system. 60 In addition, anthocyanins, a major chokeberry (poly)phenol, have been shown to protect erythrocytes from oxidative damage 61 and regulate iron metabolism by inhibiting the expression of hepcidin. 62 The role of chokeberries in iron metabolism after exercise was reported in three studies with one showing increased iron levels 24 hours, but not immediately following, a 2000 m row 25 and the other no effect after a beep test. 28 However, the two studies 25,38 demonstrated no effect on levels of hepcidin and the authors suggested that the dynamics of serum iron are determined by a phase of training rather than supplementation, thus the effects of chokeberry on iron metabolism are not well evidenced.
A key strength of this study is that it is the first review to systematically and comprehensively review studies supplementing chokeberry in athletes, according to PRISMA guidelines. Nevertheless, there a several limitations that should be acknowledged. Firstly, we were not able to conduct a meta-analysis due the heterogeneity in study design, population, training status/types and supplementation regimes. Secondly, we only found a small number of studies for each of the study outcomes, which limits our interpretation of the findings due to conflicting findings and the ability to draw evidence-based recommendations for athletes based on the current literature. For example, it is not possible to suggest the recommended dose of chokeberry supplementation. Although fruit juice was the most common form of chokeberry supplementation, the amount of anthocyanins in all the pure juice supplements was not analysed or reported. Future studies should conduct an analysis of the anthocyanin and polyphenol content of the study supplement batch to help aid with critical analysis of the study outcomes and recommended dose of chokeberry for athletes. Finally, a limitation of the evidence included in this systematic review was the mixed quality, including methodological rigour due to non-randomised or single-blinded studies being included. Although this was done to gain a better insight into the current literature, the findings should be interpreted cautiously as there is a need for further well-controlled investigations in this area.

Conclusion
Taken together, the findings from the 10 studies in this review suggest that chokeberry supplementation could alter redox balance in athletes. However, there were insufficient studies examining the effects of chokeberry supplements on other outcomes of interest (inflammation, platelet function, iron metabolism and performance) to draw any firm conclusions. Further large-scale, well-controlled investigations are needed to clarify the potential benefits of chokeberry supplementation on these biological markers in athletes.

Author contributions
RZ and AAR: concept, planning, design, literature screening and assessment of risk of bias. RZ, RK, AAR: data analysis and interpretation. RZ, RK, AAR, GC: writing the manuscript. RZ, RK, AAR and TC: revision of the manuscript.

Conflicts of interest
All authors declare that they have no conflict of interest relevant to the content of this review.