Effect of New Zealand Blackcurrant Extract on Performance during the Running Based Anaerobic Sprint Test in Trained Youth and Recreationally Active Male Football Players

It was observed previously that New Zealand blackcurrant (NZBC) extract reduced slowing of the maximal 15 m sprint speed during the Loughborough Intermittent Shuttle Test. We examined the effect of NZBC extract on the performance of the Running Based Anaerobic Sprint Test (RAST, 6 35-m sprints with 10 seconds passive recovery) in trained youth and recreationally active football players. Fifteen recreationally active (University team) (age: 20 1 years, height: 174 19 cm, body mass: 80 13 kg) and nine trained youth players (English professional club) (age:17 0 years, height: 178 8 cm, body mass: 69 9 kg, mean SD) participated in three testing sessions. Prior to the RASTs, participants consumed two capsules of NZBC extract (600 mg day 1 CurraNZ ® ) or placebo (P) for 7 days (double blind, randomised, cross-over design, wash-out at least 14 days). Ability difference between groups was shown by sprint 1 time. In the placebo condition, trained youth players had faster times for sprint 1 (5.00 0.05 s) than recreationally active players (5.42 0.08 s ) (p< 0.01). In trained youth players, there was a trend for an effect of NZBC extract (p= 0.10) on the slowing of the sprint 1 time. NZBC extract reduced slowing of the sprint 5 time (P:0.56 0.22 s ; NZBC:0.35 0.25 ,p= 0.02) and this was not observed in recreationally active players (P:0.57 0.48 s ; NZBC: 0.56 0.33,p= 0.90). For fatigue index, expressed as a % change in fastest sprint time, there was a strong trend to be lower in both trained

sprint 1 time. NZBC extract reduced slowing of the sprint 5 time (P:0.56 0.22 s ; NZBC:0.35 0.25 ,p= 0.02) and this was not observed in recreationally active players (P:0.57 0.48 s ; NZBC: 0.56 0.33,p= 0.90). For fatigue index, expressed as a % change in fastest sprint time, there was a strong trend to be lower in both trained youth and recreationally active players combined by NZBC extract (P: 13 7%; NZBC: 11 6%,p= 0.06) with 12 participants ( ve trained youth) experiencing less fatigue. New Zealand blackcurrant extract seems to bene t repeated sprint performance only in trained football players. Keywords:football; running sprints; fatigue; elite athletes; anthocyanins; polyphenols 1. Introduction Blackcurrant contains primarily the anthocyanins delphinidin-3-glucoside, delphinidin-3- rutinoside, cyanidin-3-glucoside and cyanidin-3-rutinoside [1]. Anthocyanins contribute to a wide range of human health bene ts including suppression of proliferation in tumor cell lines [2], improved LDL cholesterol in diseased patients [3], improved vision in patients with normal tension glaucoma [4], increased cognitive function in healthy young adults [5] and may enhance exercise-induced immune function [6]. Anthocyanins have also been associated with anti-in ammatory [7] and antioxidant activity [6,8] and may therefore counteract negative physiological effects that may occur during (e.g., fatigue) and after exercise (e.g., in ammation). Sports2017,5, 69; doi:10.3390/sports5030069

Sports2017,5, 69 2 of 10 Anthocyanins act on the vascular endothelium [9] and increase endothelial nitric oxide synthase activity with production of nitric oxide to vasodilate blood vessels in skeletal muscle [10]. Therefore, anthocyanins increase peripheral blood ow during exercise. For example, Matsumoto et al. [11] observed that peripheral forearm blood ow increased by 22% and reduced fatigue during typing work in humans three hours after blackcurrant intake. Furthermore, intake of blackcurrant extract increased the femoral artery diameter during submaximal sustained isometric exercise [12]. In general, an increase in blood ow during exercise may enhance performance. Cook et al. [13] observed in trained cyclists an enhanced performance by 2.4% for a 16.1 km cycling time trial. Bene cial effects of blackcurrant intake on performance were also observed during repeated high-intensity running exercise. For example, Perkins et al. [14] observed that New Zealand blackcurrant intake increased repeated high-intensity treadmill running by 10.8%. In the study by Perkins et al. [14], the treadmill running protocol that was used correlated with the YoYo IR2 test reported by Krustrup et al. [15]. In addition, better maintenance of maximal sprinting ability was also observed during the Loughborough Intermittent Shuttle Test [16]. Therefore, it seems that blackcurrant may be ergogenic for activities that are typically part of team sports such as football. Performance analysis has revealed that sprinting constitutes approximately 10% of the total distance covered during a football match [17]. In recent years, there has been an 80% increase in the number of sprints performed in the premier league [18]. The ability to perform repeated sprints has been associated with football match-related physical performance [19] and is usually assessed by coaches and applied sports scientists in the eld. The ability to repeatedly perform maximal sprints ( 10 s) with short recovery periods ( 10) has been termed repeated sprint ability [20]. The running anaerobic sprint test has been popular to quantify anaerobic power and fatigue in a non-laboratory testing environment [21]. The test is highly reliable and correlates with the Wingate test (peak powerr= 0.46; mean powerr= 0.53; fatigue indexr= 0.63) and 35, 50, 100,

( 10 s) with short recovery periods ( 10) has been termed repeated sprint ability [20]. The running anaerobic sprint test has been popular to quantify anaerobic power and fatigue in a non-laboratory testing environment [21]. The test is highly reliable and correlates with the Wingate test (peak powerr= 0.46; mean powerr= 0.53; fatigue indexr= 0.63) and 35, 50, 100, 200 and 400 m sprint performance [22]. Ergogenic effects of New Zealand blackcurrant on maximal repeated sprint ability during the running anaerobic sprint test are not known. Recently, some studies have examined whether training status affects the ergogenic properties of performance-enhancing supplementation. For caffeine, it was reported that trained subjects only experienced bene cial effects during morning testing. However, for beta-alanine, the ergogenic effects were bene cial for untrained and trained cyclists for lower-body Wingate tests [23]. It is not known whether a potential performance enhancing effect by New Zealand blackcurrant extract for repeated maximal running sprints depends on training status. Therefore, the aims of the present study were to examine the effects of 7-day intake of a New Zealand blackcurrant extract on the performance during the running anaerobic sprint test and whether trained youth and recreationally active football players would show different performance responses. 2. Materials and Methods 2.1. Participants Nine healthy trained youth and fteen recreationally male football players volunteered. After explanation of the experimental protocol and procedures, potential risks and bene ts, participants completed a health history questionnaire and provided written informed consent. Trained youth football players were recruited from a team of an English professional club. Recreationally active football players were recruited from a University team. Approval for the study was granted by the University of Chichester Research Ethics Committee (Code: 1516_39) with experimental protocols and procedures conforming to the 2013 Declaration of Helsinki. Participants did not receive payment for participation.

Sports2017,5, 69 3 of 10 2.2. Experimental Design The study consisted of three testing sessions within ve weeks. In the rst visit, participants performed a multistage tness test to exhaustion to establish predicted . V O2max(maximum oxygen uptake). After a 20-min rest, participants were familiarised with the running anaerobic sprint test. Following familiarisation, participants were randomly assigned in a double-blind, placebo controlled, cross-over design and consumed two capsules of New Zealand blackcurrant extract or identical looking placebo capsules every morning with breakfast for 7 days. On the nal day of supplementation, participants performed testing at the same time in the morning to limit any circadian rhythm variation and approximately 2 h after breakfast. Testing sessions two and three were separated by a period of at least 21 days which allowed a minimum 14-day washout period and a second supplementation period of 7 days. 2.3. Anthropometry Characteristics Anthropometric parameters were recorded in the rst testing session. Body height and body mass were measured using a portable stadiometer (Leicester Height Measure MKII, Invicta Plastics, Leicester, UK) and digital scales (Seca Model 876, Seca Ltd, Birmingham, UK). To estimate adiposity, skinfold thickness was measured at four sites on the right side of the body (i.e., biceps, triceps, subscapular and suprailiac skinfolds) [24]. Skinfold thickness was measured using a Harpenden skinfold calliper (Bodycare products, Southam, Warwickshire, UK) to the nearest 0.2 mm in accordance with the International Society for the Advancement of Kinanthropometry (ISAK). Sum of skinfolds and body fat percentage were calculated using the equations described by Durnin and Womersley [24]. 2.4. Dietary Standardisation Each participant recorded their dietary intake in a written food diary for the 48 h prior to the rst running anaerobic sprint test and were instructed to replicate the dietary intake before the second running anaerobic sprint test. Participants were instructed not to perform vigorous exercise, not to consume alcohol 48 h before each testing session, be well-rested and hydrated on arrival. Participants were also instructed not to consume other supplementation during the study that may add further nutritional value to their diet. 2.5. Independent Physical Activity Questionnaire Participants

dietary intake before the second running anaerobic sprint test. Participants were instructed not to perform vigorous exercise, not to consume alcohol 48 h before each testing session, be well-rested and hydrated on arrival. Participants were also instructed not to consume other supplementation during the study that may add further nutritional value to their diet. 2.5. Independent Physical Activity Questionnaire Participants were instructed to keep their weekly exercise schedule as consistent as possible throughout the study. Prior to the rst running anaerobic sprint test, each participant completed an international physical activity questionnaire to determine the total amount of physical activity performed for the previous 7 days. Speci cally, the international physical activity questionnaire assessed physical activity performed across a comprehensive set of domains including leisure time, domestic gardening, work-related and transport-related activity. The total metabolic equivalent for the 7-day period was calculated. 2.6. Anthocyanin Content Prior to the rst running anaerobic sprint test, each participant completed a food frequency questionnaire specifying an average consumption for each fruit, vegetable and drink during the past year (i.e., never, less than 1 per month, 1 per month, 2–3 per month, once a week, 3–4 per week, 5–6 per week or every day). In addition, each participant selected a typical serving size (i.e., small, medium or large). The total daily anthocyanin intake amount for each fruit, vegetable and drink could be calculated individually using the following equation: serving size anthocyanin content of fruit, vegetable or drink, using Phenol Explorer (http://phenol-explorer.eu/). Once calculated, the total amount of anthocyanins consumed during the past year was determined by adding together the total

Sports2017,5, 69 4 of 10 daily anthocyanin intake for each fruit, vegetable and drink. The estimated daily anthocyanin intake is presented in Table. 2.7. Estimation of Maximal Oxygen Uptake Participants performed a 20-m multistage tness test to exhaustion to establish predicted . V O2max. The multistage tness test required participants to perform shuttle runs back and forth between two lines 20-m apart at a progressively increased speed controlled by an audio signal from a CD player. The test began at an initial running velocity of 8.5 km hr 1 and increased incrementally by 1 km hr 1 each level. When the participant failed twice to reach the nishing line in time or could no longer run at the imposed running velocity, heart rate (Polar Heart Rate Monitors, F1, Polar Electro UK Ltd., Warwick, UK), total distance, and the nal stage completed were recorded. Participants ran in groups of no more than four to ensure maximal effort. Verbal encouragement was provided during the test. Predicted . V O2maxfor each participant was calculated using an equation described by Leger et al. [25]. Participant characteristics are presented in Table. Table 1.Participant characteristics. Parameter Trained Youth Football Players (n= 9) Recreationally Active Football Players (n= 15) Age (years) 17 0 20 1 Height (cm) 178 8 174 19 Body mass (kg) 69 9 80 13 Body fat (%) 12.1 2.1 15.9 2.9 Sum of skinfolds (mm) 28 5 40 10 . VO 2max(mL kg min 1 ) 45 5 44 5 HRmax(beats min 1 ) 200 4 185 10 Total MET (min week 1 ) 12,393 5375 7187 4441 Anthocyanin intake (mg day 1 ) 19 23 11 14 . V O2max, predicted maximum oxygen uptake; HR, heart rate; MET, metabolic equivalent. Data reported as mean SD. 2.8. Sprint Criterion Threshold Prior to establishment of the sprint criterion threshold in each sprint session, participants performed a standardised warm up consisting of dynamic stretches and submaximal intensity running. Following a 2-min rest period, each participant performed a 35-m maximal individual sprint to determine their sprint criterion threshold. The sprint criterion threshold was recorded to prevent

as mean SD. 2.8. Sprint Criterion Threshold Prior to establishment of the sprint criterion threshold in each sprint session, participants performed a standardised warm up consisting of dynamic stretches and submaximal intensity running. Following a 2-min rest period, each participant performed a 35-m maximal individual sprint to determine their sprint criterion threshold. The sprint criterion threshold was recorded to prevent a pacing strategy during the running anaerobic sprint test [26]. Once the sprint criterion threshold was determined, participants were instructed to have a 5-min recovery period before starting the running anaerobic sprint test. During the rst sprint of the running anaerobic sprint test, participants were required to achieve at least 95% of their sprint criterion threshold. If the time of the rst sprint time was less than 95% of the individuals' sprint criterion threshold, participants were instructed to have a 5-min recovery period and recommence the running anaerobic sprint test. However, every participant achieved at least 95% of their sprint criterion threshold during the rst sprint. 2.9. Running Anaerobic Sprint Test (RAST) The running anaerobic sprint test was performed on an arti cial football pitch for the trained youth football players and University athletics track for the recreationally active football players. Environmental conditions were recorded (environmental temperature: 11 1 C; humidity:65 3% ; air pressure: 1015 4 mbar, wind speed 7 4 km h 1 ). The test consisted of 6 35-m maximal sprints interspersed by 10-second passive recovery periods. Each sprint was recorded using a photocell system (Fusion Smartspeed lightgates, Hab International, Warwickshire, UK) which were positioned on the start line and nish line at a height of 75 cm. Each sprint began from a standing start position.

Sports2017,5, 69 5 of 10 The standing start position was adopted 30 cm behind the start line to avoid breaking the infrared beam produced by the photocell system. Sprint time was recorded and fatigue index (FI) calculated using the following equation. Fatigue index(%)= f astest sprint time slowest sprint time f astest sprint time 100% (1) After each sprint, the rating of perceived exertion (RPE) [27] was recorded. 2.10. Supplementation Prior to the running anaerobic sprint test, participants consumed two capsules of concentrated New Zealand blackcurrant extract (one capsule of 300 mg contains 35–50% delphinidin-3-rutinoside, 5–20% delphinidin-3-glucoside, 30–45% cyanidin-3-rutinoside, and 3–10% cyanidin-3-glucoside, CurraNZ ® , Health Currancy Ltd, Surrey, UK) or two identical looking placebo capsules (one capsule contains 300 mg microcrystalline cellulose M102, Lifesource Supplements, Ripon, UK) every morning with breakfast for 7 days. 2.11. Statistical Analysis Statistical analyses were conducted on SPSS version 20.0 for Windows (SPSS Inc., Chicago, IL, USA). Data normality assumptions were assessed using the Kolmogorov–Smirnov test. For the data of both trained youth and recreationally active football players, a two-way repeated-measures ANOVA (analysis of variance) was conducted to analyse differences between the supplement conditions (New Zealand blackcurrant vs. placebo) by sprint time and RPE during the running anaerobic sprint test. Within each group, a two-way repeated-measures ANOVA was conducted to analyse the change in sprint time for sprints 2–6 between the placebo and blackcurrant condition with post-hoct-tests when appropriate. Mauchly's test of sphericity was conducted to test for homogeneity of data and if violated (p< 0.05), the Greenhouse-Geisser adjustment value was used. For both groups combined, the fatigue index was analysed with paired samplest-test. To determine the effect sizes, Cohen'sdwas calculated [28]. Data were presented as mean ( SD) and statistical signi cance was set at an alpha level ofp 0.05. Interpretation of 0.05 >p 0.1 was according to guidelines by Curran-Everett and Benos [29]. 3. Results 3.1. New Zealand Blackcurrant vs. Placebo In both conditions, for all participants combined, there was an increase in sprint time and RPE (p< 0.05) (Table) during the running anaerobic sprint test. However, there were no differences between

set at an alpha level ofp 0.05. Interpretation of 0.05 >p 0.1 was according to guidelines by Curran-Everett and Benos [29]. 3. Results 3.1. New Zealand Blackcurrant vs. Placebo In both conditions, for all participants combined, there was an increase in sprint time and RPE (p< 0.05) (Table) during the running anaerobic sprint test. However, there were no differences between conditions for sprint time (p= 0.25) and RPE (p= 0.63). In addition, there were no interaction effects between conditions for sprint time (p= 0.75) and RPE (p= 0.63). Table 2. Sprint time and rating of perceived exertion (RPE) for each sprint during the running anaerobic sprint test (i.e., 6 35-m sprints) for trained youth and recreationally active football players. Parameter One Two Three Four Five Six Sprint time (s) Placebo 5.26 0.32 5.38 0.30 *5.59 0.29 * ,$ 5.68 0.36 * ,$ 5.82 0.42 * ,$ 5.77 0.37 * ,$,# NZBC extract 5.25 0.33 5.36 0.30 *5.51 0.36 * ,$ 5.62 0.42 * ,$,# 5.73 0.43 * ,$,#,£ 5.69 0.40 * ,$,# RPE Placebo 8 3 9 3 * 11 3 * ,$ 13 3 * ,$,# 15 3 * ,$,#,£ 16 3 * ,$,#,£,@ NZBC extract 7 2 9 2 * 11 2 * ,$ 13 2 * ,$,# 15 3 * ,$,#,£ 16 3 * ,$,#,£,@ Data reported as mean SD from 24 participants. * Difference with rst sprint; $ difference with second sprint; # difference with third sprint; £ difference with fourth sprint; @ difference with fth sprint (p< 0.05). NZBC = New Zealand blackcurrant extract; RPE = rating of perceived exertion.

Sports2017,5, 69 6 of 10 For both young trained and recreationally active football players, there was a strong trend for the fatigue index to be 12% lower following New Zealand blackcurrant extract compared to placebo (PLA: 13 7 vs. NZBC: 11 6%, range: 68%–26%, 12 participants showed a decrease and 12 no change) (p= 0.06) (d= 0.3) (Figure).Sports 2017, 5, 69 6 of 10 placebo NZBC extract -20 -15 -10 -5 0 *Fatigue index (%) Figure 1. Fatigue index of the running anaerobic sprint test for trained youth and recreationally active football players for the placebo and New Zealand blackcurrant (NZBC) extract condition. * Denotes a strong trend for a difference between placebo and New Zealand blackcurrant extract (p = 0.06). 3.2. Sprint Performance New Zealand blackcurrant extract had no effect on maximum sprint time (PLA: 5.61 ± 0.14 vs. NZBC: 5.54 ± 0.24 s, p = 0.35), minimum sprint time (PLA: 4.99 ± 0.14 vs. NZBC: 5.05 ± 0.30 s, p = 0.59) and mean sprint time (PLA: 5.34 ± 0.12 vs. NZBC: 5.29 ± 0.23 s, p =0.52) in trained youth football players. Similarly, New Zealand blackcurrant extract had no effect on maximum sprint time (PLA: 6.10 ± 0.42 vs. NZBC: 5.96 ± 0.41 s, p = 0.13), minimum sprint time (PLA: 5.38 ± 0.30 vs. NZBC: 5.33 ± 0.28 s, p = 0.39) and mean sprint time (PLA: 5.73 ± 0.27 vs. NZBC: 5.67 ± 0.32 s, p = 0.38) in recreationally active football players. 3.3. Change in Sprint Performance Compared to Baseline Within each group separately, a two-way ANOVA was conducted to analyse the changes in sprint times that occurred during the RAST in placebo and NZBC conditions. In recreationally active football players, New Zealand blackcurrant extract had no effect on the change in sprint times compared to the fastest sprint time of sprint 1 in sprint 2 (PLA: 0.08 ± 0.15 vs. NZBC: 0.14 ± 0.21 s, p = 0.37), sprint 3 (PLA: 0.29 ± 0.21 vs. NZBC: 0.29 ± 0.29, p = 0.94), sprint 4 (PLA: 0.42 ± 0.28 vs. NZBC: 0.44 ± 0.33,

extract had no effect on the change in sprint times compared to the fastest sprint time of sprint 1 in sprint 2 (PLA: 0.08 ± 0.15 vs. NZBC: 0.14 ± 0.21 s, p = 0.37), sprint 3 (PLA: 0.29 ± 0.21 vs. NZBC: 0.29 ± 0.29, p = 0.94), sprint 4 (PLA: 0.42 ± 0.28 vs. NZBC: 0.44 ± 0.33, p = 0.84), sprint 5 (PLA: 0.57 ± 0.48 vs. NZBC: 0.56 ± 0.33, p = 0.90) and sprint 6 (PLA: 0.52 ± 0.36 vs. NZBC: 0.50 ± 0.38 s, p = 0.86) (Figure 2a). In trained youth football players, there was a trend for a condition effect (p = 0.10). New Zealand blackcurrant extract reduced slowing of the fastest time of sprint 1 in sprint 5 (PLA: 0.56 ± 0.22 vs. NZBC: 0.34 ± 0.34 s, p = 0.02, d = −1) but not in sprint 2 (PLA: 0.19 ± 0.20 vs. NZBC: 0.07 ± 0.20 s, p = 0.15, sprint 3 (PLA: 0.40 ± 0.22 vs. NZBC: 0.21 ± 0.23 s, p = 0.16), sprint 4 (PLA: 0.41 ± 0.26 vs. NZBC: 0.27 ± 0.25 s, p = 0.29) and sprint 6 (PLA: 0.49 ± 0.23 vs. NZBC: 0.34 ± 0.34 s, p = 0.26) (Figure 2b). Figure 1. Fatigue index of the running anaerobic sprint test for trained youth and recreationally active football players for the placebo and New Zealand blackcurrant (NZBC) extract condition. * Denotes a strong trend for a difference between placebo and New Zealand blackcurrant extract (p= 0.06). 3.2. Sprint Performance New Zealand blackcurrant extract had no effect on maximum sprint time (PLA: 5.61 0.14 vs. NZBC: 5.54 0.24 s,p= 0.35), minimum sprint time (PLA: 4.99 0.14 vs. NZBC: 5.05 0.30 s,p= 0.59) and mean sprint time (PLA: 5.34 0.12 vs. NZBC: 5.29 0.23 s,p=0.52) in trained youth football players. Similarly, New Zealand blackcurrant extract had no effect on maximum sprint time (PLA: 6.10 0.42 vs. NZBC: 5.96 0.41 s,p= 0.13), minimum sprint time (PLA: 5.38 0.30 vs. NZBC: 5.33 0.28 s,p= 0.39) and mean sprint time (PLA: 5.73 0.27 vs. NZBC: 5.67 0.32

The study investigates the impact of New Zealand blackcurrant extract on sprint performance.