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Effects of supplementation with acai (Euterpe oleracea Mart.) berry-based juice blend on the blood antioxidant defence capacity and lipid profile in junior hurdlers. A pilot study

The purpose of this pilot study was to examine whether regular consumption of an acai berry-based juice blend would affect sprint performance and improve blood antioxidant status and lipid profile in junior athletes. Seven junior hurdlers (17.5±1.2 years) taking part in a pre-season conditioning camp were supplemented once a day, for six weeks, with 100 ml of the juice blend. At the start and the end of the camp the athletes performed a 300-m sprint running test on an outdoor track. Blood samples were taken before and immediately after the test and after 1 h of recovery. Blood antioxidant status was evaluated based on activities of antioxidant enzymes (superoxide dismutase [SOD], catalase [CAT], glutathione peroxidase [GSH-Px], glutathione reductase [GR]), concentrations of non-enzymatic antioxidants (reduced glutathione [GSH], uric acid), total plasma polyphenols, ferric reducing ability of plasma (FRAP), thiobarbituric acid reactive substances (TBARS) and activities of creatine kinase (CK) and lactate dehydrogenase (LDH) as muscle damage markers. In order to evaluate potential health benefits of the acai berry, the post-treatment changes in lipid profile parameters (triglycerides, cholesterol and its fractions) were analysed. Six weeks’ consumption of acai berry-based juice blend had no effect on sprint performance, but it led to a marked increase in the total antioxidant capacity of plasma, attenuation of the exercise-induced muscle damage, and a substantial improvement of serum lipid profile. These findings strongly support the view of the health benefits of supplementation with the acai berry-based juice blend, mainly attributed to its high total polyphenol content and the related high in vivo antioxidant and hypocholesterolaemic activities of this supplement.


INTRODUCTION
It is well established that strenuous muscular exercise is associated with substantially enhanced production of free radicals and other reactive oxygen and nitrogen species (ROS/RNS) [1], widely regarded as factors that may contribute to exercise-induced muscle damage. This increases requirements for exogenous antioxidant nutrients to strengthen the antioxidant defence system in heavily exercising humans. Studies have generally shown that antioxidant supplements do not improve performance, but there is a lot of evidence that supplementation with natural antioxidants may limit or even prevent exercise-induced tissue injury, as well as help the athletes to recover from the oxidative damage induced by free radicals [2,3]. On the other hand, the reactive oxygen and nitrogen species (ROS/RNS) generated during muscle contraction play an important signalling role in muscle adaptation to exercise [1,4].

Effects of supplementation with acai (Euterpe oleracea Mart.) berry-based juice blend on the blood antioxidant defence capacity and lipid profile in junior hurdlers. A pilot study
and metabolized. Indirect antioxidant activities of dietary polyphenols are associated with activation of the nuclear factor erythroid-2-related factor 2 (Nrf2), which stimulates expression of antioxidant enzyme genes, such as those for superoxide dismutase (SOD), glutathione peroxidase (GSH-Px) or glutathione S-transferase (GST), and haem oxygenase-1 (HO-1) [7].
Paradoxically, many polyphenols are known to have pro-oxidant activities both in vitro and in vivo [9]. As antioxidants, polyphenols enhance cell survival, while as pro-oxidants, they may induce apoptosis and prevent tumour growth. Among the most promising sources of natural antioxidants are fruits of the Amazonian palm tree Euterpe oleracea Mart., called acai, used to prepare acai pulp, then combined with different amounts of other fruit juices to produce several commercial beverages [10]. The product chosen for this study, mainly because of its proven non-toxicity, was the MonaVie Active juice blend [11]. MonaVie Active is a mixture of the Amazonian palm fruit acai (Euterpe oleracea Mart.), as a predominant ingredient, and of lesser amounts of some other processed fruits, including wolfberry, pomegranate, camu camu, passion fruit, aronia, acerola, bilberry, known for their antioxidant properties, and some common fruits, including white and purple grapes, nashi pear, cranberry, apricot, prune, kiwi, blueberry, lychee, banana and capuacu, and glucosamine hydrochloride as a functional ingredient, recognized as safe by the U.S. Food and Drug Administration [6,12]. The acai fruit is famous not only for its powerful antioxidant and anti-inflammatory properties [6,8,5,12], but also for its hypocholesterolaemic activity, evidenced by favourable changes in the cholesterol profile in the circulatory system [13,14,15,16]. To our knowledge, however, there is no information as to how consumption of the combination of these ingredients affects sports performance and metabolic response to exercise in junior athletes. Therefore, the aim of this pilot study was to examine whether 6 weeks' daily consumption of this acai berry-based juice blend would affect sprint performance and improve blood antioxidant status and lipid profile in junior hurdlers.

Study protocol
In this study, conducted during the early outdoor season, the athletes were tested before and after 6 weeks' training, during which they were supplemented daily with 100 ml of the MonaVie Active (Mo-naVie, Salt Lake City, Utah, USA) juice blend. During this time, all subjects were engaged in common hurdle training activities designed for the general preparation phase (GPP), including special endurance, technical and rhythm training. The weekly conditioning programme consisted of 6 to 7 training sessions, lasting approximately 90 minutes each. Before starting and after completion of a six-week training programme and daily supplementation with MonaVie Active, the athletes performed a 300-m sprint test, on an outdoor track, after an approximately 20-min warm-up. During this 300-m sprint run test, used for evaluation of the athletes' general endurance capacity during the GPP [17], blood samples were drawn from the antecubital vein into test tubes anticoagulated with heparin and/or into serum separator tubes (SST) at rest (before the warm-up), immediately following the run, and after 1 hour of passive recovery.
The activities of all antioxidant enzymes were measured at 37°C and expressed per 1 g of haemoglobin as assayed by a standard cyanmethemoglobin method using a diagnostic kit (HG980, Randox, UK). To evaluate risk for vascular disease, the lipid ratios (T-C/HDL-C, LDL-C/HDL-C, TG/HDL-C) and the atherogenic index of plasma [AIP=log 10 (TG/HDL) with TG and HDL-C expressed in molar concentrations] [21] were calculated. Assessment of lipid peroxidation was done using the thiobarbituric acid (TBARS) reaction according to Buege and Aust [22] by reading the absorption at λ=532 nm TABLE 2. Changes in plasma lactate and activities of CK and LDH induced by the dietary treatment using a multi-mode microplate reader (Synergy 2 SIAFRT, BioTek, USA). Standard curves were prepared using water solutions of 1,1,3,3-tetramethoxypropane (TMP) as standard.
The total antioxidant capacity of the MonaVie juice blend and plasma was assessed using the ferric reducing ability of plasma (FRAP) assay according to Benzie and Strain [23]. FRAP values were calculated from changes in absorbance at 593 nm at 37° to quantify the amount of coloured ferrous tripyridyltriazine complex formed as a result of ferric to ferrous ion reduction at pH=3.6. The final results were expressed in Trolox equivalents per ml of juice (µmol TE · mL -1 ) or per 1 L of plasma (µmol TE · L -1 ).
The total polyphenolic content of the MonaVie Active and plasma total polyphenols were determined using the colorimetric method with phosphomolybdic-phosphotungstic acid reagent (Folin-Ciocalteu) adapted from Singleton and Rossi [24]. The absorbance was read at 765 nm on a multi-mode microplate reader (Synergy 2 SIAFRT, BioTek, USA), and the results are expressed as mg gallic acid equivalents per ml of juice (mg GAE · mL -1 ) or per l L of plasma (mg GAE · L -1 ).
Total anthocyanin content was measured according to Niketic-Aleksic and Hrazdina [25]. Absorbance of juice dissolved in 0. Colorimetric assays of glucose and total protein contents in the MonaVie juice blend were performed using commercial assay kits from PZ Cormay S.A. (Poland). The absorbance was read on a multimode microplate reader (Synergy 2 SIAFRT, BioTek, USA) at 500 nm and 546 nm, respectively. The results of the phytochemical analysis of the MonaVie juice blend are presented in Table 1.

Statistical analysis
All data are reported as means (SD). A two-way ANOVA with exercise and supplementation as the main factors, followed, when appropriate, by the Newman-Keuls post hoc test, was used to test the significance of between-group differences. With the aim of interpreting the results by indicating the relative degree to which the variance found in the ANOVA is associated with each of the main effects and their interaction, the Eta squared (η 2 ), as a measure of the effect size in ANOVA, was calculated using the following formula: η 2 = SS effect /SS total , where SS effect is the sum of squares for whatever effect is of interest, and SS total is the total sum of squares for all effects, interactions, and errors in the ANOVA [26,27]. Spearman's rank order correlation coefficients were computed to reveal relationships between the variables. In all cases, P<0.05 was considered significant. All statistical analyses were performed with STATISTICA 9.0 (StatSoft, Inc. 2009, USA) software.

RESULTS
Mean time of running 300 m distance on an outdoor track achieved by the athletes at the start of the study (38.68 ± 0.88 s) was very close to that recorded after 6 weeks' consumption of acai berry-based juice blend (38.60 ± 0.36 s). Acute exercise-induced changes in the activity of CK and LDH in plasma are presented in Table 2. In both trials, marked increases in the activities of CK and LDH, observed immediately post-run, were followed by a tendency toward lower levels after 1 h of recovery.
What is noteworthy, as revealed by two-way ANOVA, the effect of exercise appeared to be significant, although significant (P<0.05) post-run increases in CK and LDH activities were observed only in the first trial (baseline) performed before starting supplementation.
Compared to the resting and post-recovery values, plasma lactate concentration was significantly higher immediately post-exercise (Table 2), but it was only moderately affected by supplementation.
In this study, neither exercise nor supplementation markedly affected the activity of SOD. More evident, but opposite in direction, were post-exercise changes in activities of GSH-Px and GR ( Table 3).
Consumption of a diet supplemented with acai berry-based juice blend resulted in a borderline increase in activities of CAT and a significant rise in resting blood glutathione (GSH) level, followed by a marked post-exercise decline observed in both trials (Table 4).
There was a significant increase in plasma UA level in response to exercise and during the recovery (Table 4), but obviously unaffected by supplementation. Six weeks' daily consumption of acai berry-based juice blend induced a slight enhancement of the total antioxidant potential of plasma determined by the FRAP assay, as well as an increase in plasma total polyphenol content and, as shown by 2-way ANOVA, in both cases the effect of supplementation appeared to be significant (    (Table 4).

Importantly, this dietary intervention led to beneficial changes in lipid profile, such as a reduction in T-C, LDL-C and triglycerides,
and an increase in HDL-C. This was associated with favourable changes in common lipid ratios (T-C/HDL-C, LDL-C/HDL-C, TG/ HDL-C) and a significant decline in the atherogenic index of plasma (AIP), and in all these cases the effect of supplementation was significant (Table 5).
To identify the relationships between the biochemical variables, the Spearman rank correlation coefficients were computed, and the identified statistically significant associations are presented in Table 6.
The most evident were negative associations of total plasma polyphenols with lipid profile measures, lipid ratios, muscle damage and lipid peroxidation markers, and a positive association with GSH.

DISCUSSION
This study attempted to evaluate whether 6 weeks' supplementation with acai berry-based juice blend (MonaVie Active) would enhance sprint performance, improve pro/antioxidant status and affect cholesterol homeostasis in junior hurdlers. We demonstrated that regu-lar consumption of this flavonoid-rich juice blend at a daily dose of 100 ml had no effect on sprint performance of junior hurdlers, as   that acai berry-based juice blend is non-toxic, and no adverse events related to oral consumption of this beverage were reported [11]. Our results on the in vitro antioxidant activity of this juice measured by the FRAP method, as well as total polyphenol content (see Table 1), are very close to those reported previously by Jensen et al. [6], namely 22.2 µmol TE · mL -1 as assessed by oxygen radical absorbance capacity (ORAC) and 1.48 mg GAE · mL -1 of juice, respectively. These and other authors [5] reported that the acai pulp, the main component of this juice blend, is a rich source of high concentrations of polyphenol compounds including anthocyanins (cyanidin 3-glucoside and cyanidin 3-rutinoside being the most predominant), proanthocyanidins, and phenolic acids, substances having a high antioxidant capacity, as well as high anti-inflammatory bioactivity [5,6].
It should be emphasized, however, that the bioefficacy of dietary polyphenolics depends on their bioavailability [28]. It has appeared that chronic or long-term ingestion of polyphenol-rich food does not always result in marked increases in their plasma concentrations.
Intestinal absorption of polyphenols is highly variable, often slow and largely incomplete, as in the case of high-molecular-weight proanthocyanidins [29]. The maximal human plasma concentrations of them were reached usually between 0.75 and 4 hours after consumption of food rich in polyphenolic compounds [8,28]. Their half-lives in plasma are short, usually in the range of a few hours, which may restrict the capacity of dietary polyphenols to act as antioxidants in plasma in vivo [7]. In contrast to this view, our study showed that chronic consumption of acai juice blend resulted in a significant rise in plasma total polyphenols concentration and a significant tendency toward higher total plasma antioxidant capacity, assessed by the FRAP method. Interestingly, opposite tendencies toward lower plasma total polyphenols and higher FRAP scores were observed immediately after both 300-m sprint running tests. A post-exercise decline in plasma polyphenol content may imply that they have been used as scavengers of free radicals generated by contracting muscles [1], whereas a post-exercise rise in FRAP scores may, most likely, be attributed to concurrent increases in plasma urate con-tent [30], although the association between FRAP and plasma urate in our study did not reach significance.
Several studies have reported that acute anaerobic exercise serves as a sufficient stimulus to elicit enhanced formation of ROS and RNS, and thus to increase oxidative stress [1,4,31]. However, unlike aerobic exercise, where mitochondrial leakage of electrons to molecular oxygen is the main source of ROS, the increased radical production during high intensity anaerobic exercise is mainly mediated by xanthine and NADPH oxidases [1,4]. The most widely used method for quantifying exercise-induced oxidative stress consists in measuring the concentration of thiobarbituric acid-reactive substances (TBARS), a commonly used marker reflecting the degree of free radical-induced oxidative damage to lipids [32]. There are no unequivocal reports of plasma TBARS concentrations after exercise.
Some authors have reported an increase or no changes in post-exercise plasma TBARS level [1,3]. In the present study none of the factors tested, neither the supplementation nor acute physical load, had a significant impact on plasma TBARS concentration; however, a slight decrease in TBARS levels was evidenced during the second trial performed after 6 weeks' supplementation with acai juice blend.
Moreover, plasma CK and LDH activities were significantly higher immediately after the 300-m sprint running test, but only when performed at baseline before starting supplementation. What is noteworthy, the relative post-exercise changes in CK and LDH activities, expressed as percentage increases, were markedly lower after 6 weeks of daily juice consumption, namely 175 vs. 158% and 134 vs. 112%, for CK and LDH respectively. We observed a significant inverse correlation between plasma TBARS and total polyphenols, as well as between CK and total polyphenols (see Table 6). It may be presumed that the trend toward lower TBARS and smaller postexercise increases in CK and LDH activities after supplementation with acai juice blend might be related to the antioxidant properties of its constituents. What is more, Heim et al. [33] previously re- Supplementation with acai berry-based juice blend resulted in moderate increases in the activities of CAT and GR, in the latter case this was associated with a significant increase in blood GSH contents.
Opposite post-exercise changes were recorded in the activities of GSH-Px and CAT, which tended to lower levels, and GR, which tended to increase following the sprint running tests, although me-  [15,28,36]. In most of these studies, polyphenol ingestion resulted in a decrease in T-C, LDL-C, triglycerides, apolipoprotein B or lipoprotein(a), and in an increase in the level of HDL-C, apolipoprotein A-I and up-regulation of LDL receptor activity.
In our study, a significant improvement, compared to baseline, in lipid profile parameters was observed in all athletes after six weeks' supplementation with acai-based juice blend. Taking into consideration the young age of our hurdlers, all lipid profile parameters and common lipid ratios were within the respective normal ranges already at the baseline, but their further improvement following supplementation was observed in each case (see Table 5). Of note, a two-way ANOVA with follow-up post hoc tests demonstrated larger effects of supplementation with the acai juice blend on all studied lipid profile markers (0.20 ≥ η 2 ≤ 0.30). In addition, the negative values of AIP recorded in all athletes at both endpoints, and especially at the end of the study (post-treatment), which was reported to be inversely correlated with particle size of LDL and HDL [7], and an inverse correlation of AIP with plasma total polyphenols, strongly support the view of their high anti-atherogenic activity. The beneficial effect of polyphenolic compounds on lowering the incidence of coronary heart diseases is well documented in the literature [5,16,28]. Importantly, polyphenols are potent inhibitors of LDL oxidation, considered a key mechanism of atherosclerotic plaque formation [37].
On the other hand, it may be hypothesized that the observed improvement of lipid profile and higher blood antioxidant defence capacity at the endpoint of the study, compared to baseline, may also be due to the concurrent exercise training [38]. However, higher η 2 levels for supplementation-induced changes in individual lipid profile parameters, compared to those induced by 6 weeks of exercise training, indicate a larger impact of this dietary treatment on cholesterol homeostasis. Moreover, significant inverse correlations between plasma total polyphenols and total cholesterol, triglycerides, TG/ HDL-C ratio and AIP, nearly significant negative associations with LDL-C and T-C/HDL-C, as well as inverse relationships between plasma total polyphenols and muscle damage (CK) and lipid peroxidation (TBARS) markers, seem to strongly support our presumption that the beneficial changes in lipid profile and the blood antioxidant defence status of our junior athletes were mostly due to 6 weeks' intake of the polyphenol-rich fruit and berry juice blend.
The main limitations of our study, designed to explore whether regular consumption of an acai berry-based juice blend would affect sprint performance and improve blood antioxidant status and lipid profile in junior athletes, is its open-label nature and the lack of a placebo-control group. This was due to the small number of participants recruited from the elite, national level junior hurdlers taking part in a pre-season training camp held in a special training centre, far away from their home locations.

CONCLUSIONS
Six weeks' consumption of the acai berry-based juice blend had no effect on sprint performance of junior hurdlers, but it caused a marked increase in the total antioxidant capacity of plasma, a substantial improvement of lipid profile, and moderate attenuation of the exerciseinduced muscle damage. These findings strongly support the view of the health benefits of supplementation with acai berry-based juice blend, mainly attributed to its high total polyphenol content and the related high in vivo antioxidant and hypocholesterolaemic activities of this supplement.