Journal of Dietary Supplements, Early Online:1–15, 2014  C 2014 by Informa Healthcare USA, Inc. Available online at www.informahealthcare.com/jds DOI: 10.3109/19390211.2014.952864

ARTICLE

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Black Currant Nectar Reduces Muscle Damage and Inflammation Following a Bout of High-Intensity Eccentric Contractions Alexander T Hutchison1 , Emily B Flieller2 , Kimber J Dillon3 , & Betsy D Leverett4 1

University of the Incarnate Word, School of Math, Science, & Engineering, Department of Biology, San Antonio, Texas, USA, 2 University of the Incarnate Word, School of Nursing & Health Professions, Department of Athletic Training, San Antonio, Texas, USA, 3 University of the Incarnate Word, School of Math, Science, & Engineering, Department of Nutrition, San Antonio, Texas, USA, 4 University of the Incarnate Word, School of Math, Science, & Engineering, Department of Chemistry, San Antonio, Texas, USA

ABSTRACT. This investigation determined the efficacy of black currant nectar (BCN) in reducing symptoms of exercise-induced muscle damage (EIMD). Sixteen college students were randomly assigned to drink either 16 oz of BCN or a placebo (PLA) twice a day for eight consecutive days. A bout of eccentric knee extensions (3 × 10 sets @ 115% of 1RM) was performed on the fourth day. Outcome measures included muscle soreness (subjective scale from 0 to 10) and blood markers of muscle damage (creatine kinase, CK), inflammation (interleukin-6, IL-6), and oxygen radical absorbance capacity (ORAC). Although there were no differences in reported soreness between groups, consumption of BCN reduced CK levels at both 48 (PLA = 82.13% vs. BCN = −6.71%, p = .042) and 96 h post exercise (PLA = 74.96% vs. BCN = −12.11%, p = .030). The change in IL-6 was higher in the PLA group (PLA = 8.84% vs. BCN = −6.54%, p = .023) at 24 h post exercise. The change in ORAC levels was higher in the treatment group (BCN = 2.68% vs. PLA = −6.02%, p = .039) at 48 h post exercise. Our results demonstrate that consumption of BCN prior to and after a bout of eccentric exercise attenuates muscle damage and inflammation. KEYWORDS. damage

anthocyanins, antioxidants, inflammation, muscle damage, oxidative

INTRODUCTION High-intensity eccentric muscular contractions are common in many athletic events, particularly those involving ballistic motions such as rapid changes in Address correspondence to: Dr Alexander Torrealba Hutchison, PhD, University of the Incarnate Word, Biology, 4301 Broadway, San Antonio, TX 78209, USA (E-mail: [email protected]). AT Hutchison is a recipient of a professional development award from the University of the Incarnate Word. E Flieller is a recipient of an undergraduate research award from the University of the Incarnate Word.

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direction and speed, including running, jumping, and resistance training. Depending on the intensity, volume, and duration of the activity, events requiring eccentric contractions can result in varying degrees of damage to skeletal muscle (Aoi et al., 2004; Baird et al., 2012; Powers, Nelson, & Hudson 2011). Exercise-induced muscle damage (EIMD) is often associated with reduced contractile force, leakage of myofibril contents, localized edema, inflammation, and pain (Beaton et al., 2002; Baird et al., 2012; Powers et al., 2011). In well-trained athletes, impaired muscle function and pain can reduce athletic performance during successive bouts, or prevent training on successive days, inhibiting the adaptations that result in athletic improvement (Krentz & Farthing, 2002; Schoenfeld, 2012). These effects tend to be worse in the untrained and can discourage further participation in exercise activities (Krentz & Farthing 2002). Although the mechanisms resulting in EIMD have not been fully elucidated it is believed that following an initial phase of mechanical disruption to the myofibrilar structure, i.e. extracellular matrix, sarcolemma, and contractile proteins, there is a secondary phase of damage resulting from a loss of calcium homeostasis. The secondary phase involves the production and release of reactive oxygen species (ROS) at the site of injury by both the disrupted myofibers and phagocytic cells including neutrophils and macrophages that are recruited to repair the damage (Charge & Rudnicki, 2004; Gabay, 2006; Kosmidou, 2002; Tidball, 2005). ROS are chemically reactive molecules that, under normal circumstances, play critical roles in cell signaling and homeostasis (Powers et al., 2011; Powers & Jackson, 2008). However, when released in excess as a result of environmental stresses (e.g., EIMD), ROS overwhelm the innate cellular defense mechanisms, (e.g., endogenous antioxidant molecules including glutathione, catalase, and superoxide dismutase) damage critical components of cellular structure and function including DNA, phospholipids, and enzymes (Aoi et al., 2004; Powers et al., 2011; Cooke et al., 2003), and stimulate the production of inflammatory cytokines including IL-6, which subsequently stimulate nociceptors to signal pain (Murphy et al., 1999; Xu et al., 1997) Past research focusing on the efficacy of exogenous antioxidant preparations including ascorbic acid (vitamins C), and tocopherols (vitamin E) to hasten recovery following EIMD has consistently reported minimal benefits (McGinley, Shafat, & Donnelly, 2009; Peternelj & Coombes 2011; Beaton et al., 2002). By contrast, studies utilizing whole fruits (specifically tart cherries, blueberries, and pomegranates) prior to, and/or following bouts of eccentric exercise have reported favorable results, e.g. reduced inflammation and faster strength recovery (Bowtell et al., 2011; Connolly et al., 2006; Howatson et al., 2010; Kuehl et al., 2010; Trombold et al., 2010; Trombold et al., 2011). However, to our knowledge only two studies have reported reduced soreness following EIMD (Connolly et al., 2006; Kuehl et al., 2010). One study involved well-trained runners, who were less susceptible to EIMDinduced soreness (Kuehl et al., 2010), while the other observed only an overall treatment by time interaction effect with no significant differences observed between groups at any time point post-EIMD (Connolly et al., 2006). Black currants contain high concentrations of anthocyanins, pigments with potent antioxidant and anti-inflammatory properties (Rubinskiene et al., 2005). Black currants have been characterized as “super fruit” because of their capacity to attenuate the effects of oxidative stress (Lister et al., 2002). In relation to EIMD, consumption of black

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currant nectar (BCN) before and after a bout of rowing has been shown to reduce some symptoms of eccentric damage (Lyall et al., 2009). However, post-EIMD soreness was not assessed. These findings suggest that consumption of BCN may attenuate the oxidative stress associated with the secondary phase of EIMD and reduce soreness. Thus, the aim of the present study was to investigate the effects of BCN consumption on symptoms of EIMD after a bout of strenuous (115% of 1 repetition maximum, 1RM) eccentric exercise in a group of untrained participants.

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METHODOLOGY Experimental Approach to the Problem The present study utilized a double blind, placebo controlled design. Participants were randomly assigned to either the experimental group that received BCN or the control group that received an isocaloric drink of similar color and taste (PLA). An independent third party who had no direct contact with either the investigators or participants during the study completed the randomization of participants using the random number generation function in Excel (Microsoft, Redmond, WA). Four days prior to the eccentric exercise session, participants arrived to the testing facility to provide a baseline blood draw and leg soreness measure. The 1RM for squatting was also assessed at this time. Participants were asked to keep separate food and activity logs for the duration of the study. Additionally, participants were asked to maintain their normal diet and were provided a list of antioxidantrich foods to avoid changing in their diet. At the end of the session the participants were given their assigned drinks. For eight consecutive days the participants drank two, 16 oz. bottles of their assigned drink, one in the morning with breakfast and one at night with dinner. On the fifth day of the protocol, participants returned to the testing facility and performed three sets of super-maximal (115% of 1RM), eccentric squatting contractions of the quadriceps muscles. For the next four days, participants returned at the same time of day to be assessed for leg soreness (all four days) and provide a blood sample at 24, 48, and 96 h post eccentric exercise. Institutional policy allowed for only three blood draws in a one-week period, precluding sampling on all four days post-exercise. The 24 and 48 h time points were chosen because these are when it was anticipated that circulating CK values would be highest. The 96 h time point was chosen because it was the last day of the protocol. Participants Twenty-four healthy participants (six males and 18 females) were recruited to take part in this study (Table 1). Inclusion criteria included being untrained, moderately active, between the ages of 18–40 yrs. The participants were undergraduate students at the University of the Incarnate Word (UIW). Untrained was defined as having not participated in regular resistance training for the six consecutive months prior to the study. Exclusion criteria included recent history of ankle, knee, hip, or back pain that precluded squatting exercises, and the use of anti-inflammatory or analgesic drugs that would reduce pain. Participants were asked to avoid exercise for the duration of the protocol and any treatment of muscle soreness or pain (e.g.

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TABLE 1. Participant Characteristics

Age (yr) Gender F (M) Weight (kg) Height (cm)

BCN

PLA

19.5 ± 0.3 7 (1) 64.8 ± 4.8 167.6 ± 1.9

20.9 ± 0.9 6 (2) 63.6 ± 5.2 163.9 ± 4.1

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Values are means ± SEM.

pain medication, massage, ice, heat). The UIW institutional review board approved the study, and all Participants provided written informed consent. Assessment of 1RM A 45 lb. bar was placed on the safety arms in a position that required the participants to squat with the knees at 90◦ in order place the bar on the shoulders (Figure 1A). The participants then extended the quadriceps muscles of the thigh, standing upright (Figure 1B & 1C). Once the bar was raised, one spotter on either side of the bar guided the participants forward to safely rack the weight (Figure 1D). The participants stepped back out of the squat rack. The spotters then lowered the bar to its original position. During the course of the study, eight participants withdrew due to illnesses (four) and injuries (three) unrelated to the treatment or exercise protocol, and one was removed after it was determined that he failed to report his participation in

FIGURE 1. Assessment of one-repetition maximum (1RM) for concentric (upward only) leg exercise. Participants began in the squatting position (A). The participants extended the knees by shortening the quadriceps muscle and standing up (B & C). Spotters on either side of the participants guided weighted bar into its safely racked position (D).

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TABLE 2. Caloric and Antioxidant Contents of BCN and PLA Drinks (BCN Measures are Per 100 g of Black Currents)

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Calories Vitamin A (% DV) Vitamin C (% DV) Anthocyanin (mg) Malvidin glucosides Cyanidin glucosides TEAC (μmol trolox equ)

BCN

PLA

63 5 302

63 0 10

193.25 175.69 7340

0 0 3.32

%DV, percentage of recommended daily value. TEAC, trolox equivalent antioxidant capacity.

resistance training immediately prior to the study. The remaining 16 participants (eight in each group) completed the study. Drink Preparation The commercially available BCN, CurrantC©, was provided by CropPharms (Staatsburg, NY). Each 16 oz bottle contained approximately 100 grams of fruit. The placebo drink was produced by mixing black cherry Kool-aid powder (Kraft, Ryerbrook, New York, USA) with water as per the manufacturers recommendations. Sucrose was added to match the carbohydrate composition of the BCN. CropPharms heat pasteurized and bottled the placebo drink using the same process as that used for the CurrantC©. The labels were removed from the non-descript plastic bottles, so the participants and testers were unaware as to which drink had been assigned. The nutritional information (provided by the manufacturers), anthocyanin content, and total antioxidant capacity (as measured by our lab) for both drinks is provided in Table 2. Antioxidant and Anthocyanin Content The total antioxidant capacity of the BCN was determined using the 2,2 -azinobis 3-ethylbenzothiazoline-6-sulphonic acid (ABTS) cation radical decolorization assay as described by Re et al. (1999) and using some of the modifications of Valyova et al. (2012). An ABTS radical cation stock solution was prepared by mixing a 7.0 mM solution of ABTS with 2.45 mM potassium persulfate and allowing the mixture to stand in dark at room temperature for 12 h. This ABTS•+ solution was diluted with ethanol to give an absorbance of 0.7 ± 0.05 at 734 nm. Trolox standard solutions in ethanol (10 mL volumes for 2–16 mM assay concentrations) or BCN samples diluted with deionized water (10 mL of 1:10, 1:20, and 1:100 diluted sample) were added to 90 mL of ABTS•+ solution and the absorbance was measured at 734 nm at one minute intervals for six minutes. The quenching of the ABTS radical was observed as the decrease in absorbance at six minutes relative to an unquenched control sample containing the ABTS solution and water (for BCN samples) or ethanol (for Trolox standards). A standard curve was prepared using the quenching of ABTS•+ observed for Trolox standards. Using this curve, the quenching observed for the BCN sample was expressed as a Trolox equivalent antioxidant capacity (TEAC) value from which a mean value, standard deviation (SD,

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n = 3) and coefficient of variation (CV) were calculated. The average coefficient of variation for replicates ranged between 1.7 and 3.7%. The anthocyanin content of the BCN was determined using the Association of Official Agricultural Chemists (AOAC) Official pH Differential Method presented by Lee, Durst, & Wrolstad (2005), and including both cyanidin-3-glucoside and malvidin-3-glucoside as reference anthocyanins (Lee, Rennaker & Wrolstad,2008). The method is based on the reversible color change (observed at 520 nm) undergone by monomeric anthocyanins from pH 1 (colored oxonium form) to pH 4.5 (colorless hemiketal form). The magnitude of the color change observed for an anthocyanin is proportional to its concentration. The anthocyanin standards (10 mL, 100–2500 mg/L assay concentrations) and undiluted BCN samples (10 mL) were added to 90 mL of potassium chloride buffer (pH 1) and separately to 90 mL of sodium acetate buffer (pH 4.5). The absorbance of each mixture was observed at 520 nm and 700 nm, and the absorbances compared as described by Lee et al. (Powers et al., 2011). A standard curve was prepared for each of the anthocyanin standards, and the pigment content for the BCN was expressed in milligrams per liter of cyanidin-3-glucoside equivalents and malvidin-3-glucoside equivalents. The standards and BCN samples were each determined in triplicate, and a mean, standard deviation (SD), and coefficient of variation (CV) calculated for each. The average coefficient of variation for replicates ranged between 3.0 and 7.7%. Eccentric Contractions On the fifth day of the protocol, the participants reported to the testing facility for the eccentric squatting exercise session. Following a brief warm-up, the participants performed three sets of ten repetitions of eccentric contractions (down only) using a bar weighted with 115% of the respective 1RM weight (assessed at baseline). For each repetition, the participants stepped into the squat rack with the bar set at shoulder height (Figure 2A). The participants placed the bar onto their shoulders and stepped back from the rack to complete the squat (Figure 2B). The participants bent at the knees and hips until the knee joint reaches 90◦ (Figure 2C and 3D). The safety arms were placed so the participants were able to displace the bar from the shoulders and step out of the rack once the knees reached 90◦ (Figure 2E). There were spotters on either side of the participants to guide them down and replace the bar at the top of the rack after each repetition (Figure 2F). There was a two min rest period between sets of ten repetitions. Between each repetition, the rest period was at least ten sec. Procedures for Outcome Measures Leg soreness was assessed during a full range squat with no external weight. Each participant started in the standing position, squatted until the knee was at 90◦ , and stood back up. Immediately afterwards, they were asked to look at the series of faces on the Wong-Baker faces scale and verbally rate their perceived level of discomfort on a scale of 0–10 with zero indicating no soreness and ten representing extreme discomfort (Hockenberry & Wilson 2009). Venous blood samples (10 ml) were collected from a peripheral arm vein into evacuated tubes coated with sodium heparin (BD Vacutainer) at four time points

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FIGURE 2. Eccentric contraction protocol (downward only). The bar was loaded with a super-maximal load (115% of 1RM). Participants began in the erect position (A), loading the weighted bar onto the shoulders (B). With spotters on either side of the weighted bar, the participants slowly lowered the bar on the safety arms until the knee was at a 90◦ angle (C & D). The participants then stepped out of the rack (E), and the spotters replaced the weighted bar to the starting position (F).

(baseline, 24, 48, and 96 h post exercise). Blood samples were spun in a refrigerated (4◦ C) centrifuge at 1500g for 15 min. Plasma supernatants were aliquoted and stored at −80◦ C for later analysis. Plasma samples were analyzed for CK activity, IL-6 concentration, and oxygen radical antioxidant capacity (ORAC). Plasma CK activity was measured using a plate-based, colorimetric reaction (Bioo Scientific, Austin, TX, USA). Plasma IL6 concentration was determined by ELISA (Thermo Fischer Scientific, Rockford,

FIGURE 3. Subjective pain scores in the legs after eccentric exercise (0–10 scale). a (p < 0.05) represents significant difference from baseline score. Values are means ± SEM.

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IL, USA). Plasma ORAC was measured using a fluorescent, plate-based assay (Cell Biolabs, Inc., San Diego, CA, USA). All assays were measured with an Infite M200Pro on Magellan software (Tecan Systems, Inc. San Jose, CA, USA). The average coefficient of variation ± SEM between duplicates for the assays were CK (3.32 ± 0.22%), IL-6 (4.32 ± 0.65%), and ORAC (3.055 ± 0.34%), respectively.

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Statistical Analyses All effect size calculations were made using G-power (v.3.1.2; Bonn, Germany). A search of relevant literature revealed that the comparison of self-reported pain in a group given tart cherry juice (similar in antioxidant content to currant juice) or placebo after eccentric damage to the leg muscles appeared to be most applicable to the present project (Kuehl et al., 2010). The Cohen d value (1.33; the portion of the variance accounted for by the effect) was calculated by using the mean values for reported pain (Tart Cherry vs. Placebo) and the group variances. In order to achieve 80% power, it was estimated that we would require a minimum of 11 participants per group (total N = 22) to detect a significant group difference in perceived pain (effect size = 1.33, power = 0.80). In order to account for possible loss of participants as a result of attrition, we recruited 12 participants per group for a total of 24 participants in the study. Statistical analysis on both the raw and normalized data was completed using SPSS v18.0 (SPSS, Chicago, IL). Because a two-group design was utilized we felt it appropriate to focus on the normalized data (i.e., percent change from baseline) as our primary outcome measure. The raw values (Table 3) for all circulating markers were analyzed by using 2 [(group; BCN and PLA) × 3 (treatment time: 24, 48, and 96 h post-exercise)] factorial ANCOVA’s with repeated measures on the second factor and baseline values as the covariate. The normalized values for all circulating markers were analyzed by using 2 [(group) × 4 (treatment time)] factorial ANOVA’s with repeated measures on the second factor. Leg soreness was analyzed by using a 2 [(group) × 5 (treatment time: Baseline, 24, 48, 72, and 96 h post-exercise] factorial ANOVA’s with repeated measures on the second factor. Significance was set at p < .05 and when found, a Student’s t-test with Bonferroni correction for multiple comparisons was used to determine the location of significance. Figures represent values as mean percent change relative to baseline ± SEM.

TABLE 3. Plasma Markers of Muscle Damage, Inflammation, and Antioxidant Capacity Measure

Group

CK activty (IU/L)

BCN PLA BCN PLA BCN PLA

IL-6 (pg/ml) ORAC (μM TE)

Baseline

24 h

48 h

96 h

393.75 ± 81.94 359.32 ± 46.70 3.59 ± 0.49 4.29 ± 0.68 82.30 ± 2.48 87.54 ± 1.50

577.65 ± 114.68a 694.60 ± 104.17a 3.25 ± 0.35 4.54 ± 0.56b 81.03 ± 1.96 82.40 ± 1.50

320.52 ± 54.05 673.01 ± 171.94b 3.48 ± 0.41b 4.39 ± 0.61 84.10 ± 1.18 82.27 ± 2.42a

300.58 ± 23.65 586.67 ± 110.61b 3.36 ± 0.40 4.67 ± 0.71 77.80 ± 2.63 80.90 ± 2.49a

BCN, Treatment with CurrantC; PLZ, Placebo; CK, creatine kinase; IL-6, interleukin-6; ORAC, oxygen radical absorbance capacity. a Significantly different than Baseline measure, (p < .05). b Significantly different than BCN, (p < .05); values are means ± SEM (n = 8 per goup).

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RESULTS BCN Supplementation Did Not Reduce Leg Soreness After EIMD

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Self-reported leg soreness peaked at 48 h post-exercise in both groups, returning to baseline levels by 96 h (Figure 3). Leg soreness in the PLA group was significantly higher than baseline (0.38 ± 0.26) at 24 h (3.0 ± 0.63, p = .002, η2 = 0.772), 48 h (3.38 ± 0.75, p = .003, η2 = 0.735), and 72 h (1.63 ± 0.46, p = .019, η2 = 0.568). In the BCN group, leg soreness was significantly higher than baseline (0.0 ± 0.0) at 24 h (2.44 ± 0.65, p = .007, η2 = 0.667), and 48 h (2.75 ± 0.86, p = .015, η2 = 0.782) post-exercise. By 72 h, there was no difference relative to baseline (1.38 ± 0.71, p = .92). There was no time by treatment interaction effect for pain scores between groups (p = .859). BCN Supplementation Reduced CK Activity After EIMD The percent change in CK activity peaked at 24 h post-exercise and was significantly different than baseline in both groups, (PLA, 101.57 ± 24.69%, p = .004, η2 = 0.707) and (BCN, 71.08 ± 30.35%, p = .052, η2 = 0.439, Figure 4). While CK activity remained elevated in the PLA group at 48 h (82.13 ± 37.14%, p = .063) and 96 h post-exercise (74.96 ± 31.10% p = .047, η2 = 0.454), consumption of BCN reduced CK activity below baseline levels at both 48 h (−6.71 ± 14.10%, p = .648) and 96 h (−12.11 ± 13.91%, p = .286). The differences in plasma CK activity between groups were significant at both 48 h (p = .042, η2 = 0.263) and 96 h post-exercise (p = .030, η2 = 0.353). When controlling for differences in baseline measures, BCN supplementation reduced absolute CK activity at both 48 h (320.52 ± 54.05 vs. 678.01 ± 171.94 IU/L, p = .039, η2 = 0.29) and 96 h (300.58 ± 23.65 vs. 586.67 ± 110.61 IU/L, p = .013, η2 = 0.39, Table 3).

FIGURE 4. Percent change in plasma creatine kinase (CK) activity after eccentric exercise. a (p < 0.05) represents significant difference from baseline values. b (p < 0.05) represents significant difference between groups. Values are means ± SEM.

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FIGURE 5. Percent change in plasma interleukin-6 (IL-6) concentration after eccentric exercise. b (p < 0.05) represents significant difference between groups. Values are means ± SEM.

BCN Supplementation Attenuated IL-6 Response at 24 h Post-EIMD Although it approached significance at 24 h (8.84 ± 4.15%, p = .071, Figure 5), there were no significant differences in the percent change of plasma IL-6 concentrations at any time points in the PLA group. Consumption of BCN resulted in non-significant reductions in the percent change of plasma IL-6 concentrations below baseline at all time points. At 24 h there was a significant difference in the percent change in IL-6 concentration between groups (BCN, −6.54 ± 4.39% vs. PLA, 8.84 ± 4.15%, p = .023, η2 = 0.316), but not 48 h or 96 h post-exercise. BCN Supplementation Maintained Plasma ORAC After EIMD There were significant decreases in the percent change of plasma ORAC scores below baseline at 24 h (−5.88 ± 2.20%, p = .048, η2 = 0.450) and 96 h (−7.57 ± 2.42%, p = .027, η2 = 0.528) post-exercise in the PLA group (Figure 6). The value at 48 h approached significance (−6.20 ± 2.38%, p = .058, η2 = 0.422). By contrast, in the BCN group, the percent change in plasma ORAC was not different than baseline at any time point. There was a significant treatment × time interaction at 48 h post-exercise (p = .039, η2 = 0.563). DISCUSSION AND CONCLUSIONS In partial support of our primary hypotheses, we found that consumption of BCN for four days before and three days after a bout of eccentric leg exercise significantly reduced circulating markers of muscle damage while maintaining circulating antioxidant capacity. Although pain scores in the BCN group returned to baseline a day earlier than the PLA group, there were no significant differences observed

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FIGURE 6. Percent change in plasma oxygen radical absorbance capacity (ORAC) after eccentric exercise. a (p < 0.05) represents significant difference from baseline values. b (p < 0.05) represents significant difference between groups. Values are means ± SEM.

between groups at any time point after exercise. To our knowledge, this is the first study to examine the effects of consumption of BCN on symptoms of EIMD in untrained, healthy participants. Both groups experienced elevated circulating CK levels at 24 h post exercise. However, only the PLA group was significantly different than baseline. Thereafter, CK values in the BCN group returned to baseline while those in the PLA group remained elevated. These results suggest that even at 24 h post-EIMD, consumption of BCN offered a protective effect against the damage that often results in release of CK following EIMD. The mechanism of protection was likely mediated by neutralization of ROS in the damaged myofibers, attenuating further disruption of the sarcolemma by oxidative reactions, thus preventing additional leakage of CK. A search of the relevant literature revealed no other study with a similar design that reported reduced markers of sarcolemmal disruption (circulating CK), following antioxidant supplementation. Specifically, recent studies by three groups found that consumption of different antioxidant-rich foods or supplements did not significantly reduce circulating CK levels relative to the respective control conditions following EIMD (Beaton et al., 2002; Bowtell et al., 2011; McLeay et al., 2012). However, there are critical differences in experimental design between these three studies and the present study that may account the different outcomes in the dependent variable. Although the daily dosage of antioxidants was similar in all studies, the dosage pattern varied widely. Beaton et al. supplemented 1200 IU•d−1 of vitamin E for 30 d prior to a bout of eccentric knee extension/flexion contractions. Unlike the present study, Beaton et al. did not provide post-exercise antioxidant supplementation. Although there was no interaction effect at 3 d post-exercise, only the placebo group

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experienced CK levels that were significantly higher than baseline. This trend disappeared by 7 d post-exercise when both groups had CK levels significantly higher than baseline. Being that the half-life of vitamin E is approximately 48 h in vivo, this may account for the CK values being higher at 7 d than at 3 d post-exercise (ZZZ 2000). Perhaps continuing supplementation for the duration of the study would have reversed EIMD and reduced CK levels post-exercise. McLeay et al. reported that consumption of New Zealand blueberries reduced CK levels to near baseline values at 60 h after a bout of eccentric quadriceps contractions. By contrast, CK levels during the placebo trial were significantly higher than baseline (∼150%) at the 60 h time point. In this study, the participants were given four blueberry smoothies at 10 and five hours before, and 12 and 36 h after exercise. It is reasonable to suggest that more doses before and/or after exercise may have provided enough antioxidants to hasten the healing process and reduce CK levels. Bowtell et al. tested the effectiveness of Montmorency cherry juice to reduce symptoms of EIMD following a bout of single-leg knee extensions at 80% 1RM. Serum CK levels peaked at 24 h post-exercise (treatment 108.3%, placebo 127.6%), and decreased by 48 h (treatment 38.2%, placebo 73.7%). Although the differences between conditions were not significant, the pattern of change was similar to that observed in the present study. Interestingly, the dosage schedule used was also similar to ours. Cherry juice was provided for seven days prior to, and two days postexercise. When observed together with the aforementioned studies, our results suggest that the critical window during which supplementation is most effective in reducing symptoms of EIMD is immediately after eccentric exercise. This is when the circulating concentration of ROS is rapidly increasing, spilling from the disrupted myofibers (along with CK). The ROS oxidize sarcolemmal phospholipids, further destabilizing the membrane in a positive feedback loop that ends when the flow of ROS is stopped. Although supplementation of antioxidants before exercise appears to reduce the initial flood of ROS, as evidenced by a blunted increase in CK activity at 24 h, continued supplementation with antioxidants after eccentric exercise appears to rapidly reverse EIMD by as early as 48 h post-exercise. The present study used three sets of ten (30 total) repetitions of eccentric phase contractions at 115% of concentric 1RM to induce EIMD. By contrast, McLeay et al., Beaton et al., and Bowtell et al. induced muscle damage with bouts of 300, 240, and 100 eccentric repetitions, respectively. It is possible that the severity of EMID generated in these studies was greater than ours and thus required more antioxidant supplementation in order to observe protection. Indeed, McLeay et al. observed a >200% change in circulating CK levels at 24 h post-exercise in both groups. We chose our protocol because we thought it more closely approximated a single bout of leg exercises performed by an inexperienced weight lifter beginning a new fitness routine. Finally, the training status of our participants was different than that of the participants recruited by McLeay et al. and Bowtell et al., who were actively involved in resistance training activities. It is well established that even a single bout of eccentric contractions can impart protection on muscles for up to six months, reducing symptoms of EIMD following subsequent bouts (McHugh 2003; Nosaka & Newton

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2002). It is possible that by using trained populations, McLeay et al. and Bowtell et al. reduced their statistical power, masking the true protective effects of their respective treatments. Although consumption of BCN reduced plasma IL-6 by 6.15% at 24 h postexercise, while the PLA group experienced an 8.84% increase relative to baseline, the changes in the absolute values were minimal and consistent between the two groups. Previous studies have shown that IL-6 levels peak between one and 24 h post-exercise (Howatson et al., 2010; Phillips et al., 2003). Because of the aforementioned institutional policies, we were limited to sampling blood at three time points post exercise and likely missed the peak changes in IL-6 immediately after EIMD was induced. This is an acknowledged limitation to our study design that we hope to rectify in the future. Although there were no significant differences in self-reported pain observed at any time point, the BCN group returned to baseline values a full day before the PLA group. There was also a clear trend toward lower self-reported soreness in the BCN group at all time points. Our data are in agreement with two studies that reported reduced post-EIMD pain/soreness following antioxidant supplementation. Kuehl et al. observed lower pain scores in runners given either tart cherry juice or a placebo for seven days prior to, and on the day of a 26 k race. Similarly, Connolly et al. reported that the pain values of the treatment group (tart cherry juice), averaged over four days post-exercise were lower than that of the placebo group. Interestingly, the present study followed a similar experimental design with regard to the eccentric exercise, dosing schedule, pain scale used, and order of events, and observed similar changes in reported pain (0–3.5). However, the present study is novel in the fact that neither of the previous studies assessed markers of muscle damage or inflammation, so it is impossible to determine how well self-reported pain correlated with other measures of EIMD. The withdrawal of eight participants during the study may have reduced our statistical power, making it less likely to observe differences between groups when they actually existed, i.e. a type II error. The fact that we found significant differences between groups in three of four outcome measures in spite of our reduced statistical power implies that BCN supplementation is more effective in reducing the signs of EIMD than were able to observe in the current study. In addition, the majority of our participants were female. Although estrogen has been shown to stabilize plasma membrane integrity, (Kerksick et al., 2008) and fluctuating levels during estrus could have altered our observed results, participants were randomly and evenly distributed between the groups, thus reducing the likely effect of this confounder. Finally, due to technical issues with the assay we were unable to measure ROS in our samples. We hope to repeat the study using adequate sample numbers and a crossover design to improve our statistical power, while including plasma estrogen levels as a statistical covariate. When compared with a placebo, BCN taken for four days before and after a bout of eccentric contractions of the quadriceps was an effective means for reducing signs of EIMD, most notably the circulating levels of CK. Consumption of BCN may represent a natural food alternative to taking analgesic and anti-inflammatory drugs following high-intensity, ballistic exercises.

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Hutchison et al.

Declaration of interest: The authors report no conflict of interest. The authors alone are responsible for the content and writing of this paper.

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ABOUT THE AUTHORS Alexander T. Hutchison is an assistant professor of Biology at the University of the Incarnate Word, San Antonio, Texas, USA. Emily B. Flieller is an undergraduate student of athletic training at the University of the Incarnate Word, San Antonio, Texas, USA. Kimber J. Dillon is a graduate student of Nutrition at the University of the Incarnate Word, San Antonio, Texas, USA. Betsy D. Leverett is an assistant professor of Chemistry at the University of the Incarnate Word, San Antonio, Texas, USA. REFERENCES Anonymous. Dietary Reference Intakes for Vitamnin C, Vitamin E, Selenium, and Carotenoids. 2000, Institute of Medicine:Washington, D. C. Aoi W, Naito, Y, Takanami Y, Kawai Y, Sakuma K, Ichikawa H. et al. Oxidative stress and delayed-onset muscle damage after exercise. Free Radic Biol Med. 2004;37(4):480–7. Baird MF, Graham SM, Baker JS, Bickerstaff GF. Creatine-kinase- and exercise-related muscle damage implications for muscle performance and recovery. J Nutr Metab. 2012;2012:960363. Beaton LJ, Allan DA, Tarnopolsky MA, Tiidus PM, Phillips SM. Contraction-induced muscle damage is unaffected by vitamin E supplementation. Med Sci Sports Exerc. 2002;34(5):798–805. Bowtell JL, Sumners DP, Dyer A, Fox P, Mileva KN. Montmorency cherry juice reduces muscle damage caused by intensive strength exercise. Med Sci Sports Exerc. 2011;43(8):1544–51. Charge SB, Rudnicki MA. Cellular and molecular regulation of muscle regeneration. Physiol Rev. 2004;84(1):209–38. Connolly DA, McHugh MP, Padilla-Zakour OI, Carlson L, Sayers SP. Efficacy of a tart cherry juice blend in preventing the symptoms of muscle damage. Br J Sports Med. 2006;40(8):679–83; discussion 683. Cooke MS, Evans MD, Dizdaroglu M, Lunec J. Oxidative DNA damage: mechanisms, mutation, and disease. FASEB J. 2003;17(10):1195–214. Gabay C. Interleukin-6 and chronic inflammation. Arthritis Res Ther. 2006;8 Suppl 2:S3. Howatson G, McHugh MP, Hill JA, Brouner J, Jewell AP, van Someren KA, et al. Influence of tart cherry juice on indices of recovery following marathon running. Scand J Med Sci Sports. 2010;20(6):843–52. Kerksick C, Taylor Lt, Harvey A, Willoughby D. Gender-related differences in muscle injury, oxidative stress, and apoptosis. Med Sci Sports Exerc. 2008;40(10):1772–80. Kosmidou I, Vassilakopoulos T, Xagorari A, Zakynthinos S, Papapetropoulos A, Roussos C. Production of interleukin-6 by skeletal myotubes: role of reactive oxygen species. Am J Respir Cell Mol Biol. 2002;26(5):587–93. Krentz JR, Farthing JP. Neural and morphological changes in response to a 20-day intense eccentric training protocol. Eur J Appl Physiol. 2002;110(2):333–40. Kuehl KS, Perrier ET, Elliot DL, Chesnutt JC. Efficacy of tart cherry juice in reducing muscle pain during running: a randomized controlled trial. J Int Soc Sports Nutr. 2010;7(17):1–6. Lee J, Durst RW, Wrolstad RE. Determination of total monomeric anthocyanin pigment content of fruit juices, beverages, natural colorants, and wines by the pH differential method: collaborative study. J AOAC Int. 2005;88(5):1269–78. Lee J, Rennaker C, Wrolstad RE. Correlation of two anthocyanin quantification methods: HPLC and spectrophotometric methods. Food Chemistry. 2008;110(3):782–786. Lister C, Wilson P, Sutton K, Morrison S. Understanding the health benefits of blackcurrants. Proc 8th Inter Rubus and Ribes Sym. 2002;1-2:443–449.

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Black Currant Nectar Reduces Muscle Damage and Inflammation Following a Bout of High-Intensity Eccentric Contractions.

This investigation determined the efficacy of black currant nectar (BCN) in reducing symptoms of exercise-induced muscle damage (EIMD). Sixteen colleg...
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