Abstract

Introduction

During exercise, the demand for oxygen and energy substrates is elevated in active skeletal muscle. To meet increased demand, blood flow to working musculature is increased in a process known as exercise hyperemia (Hellsten et al. 2012). Exercise hyperemia is accomplished by a number of central and peripheral cardiovascular adjustments affecting blood flow; nitric oxide (NO) has been identified as an important contributor to the vasodilation observed with exercise (Hellsten et al. 2012).

The nitric oxide synthase (NOS)-dependent pathway of NO production includes a series of reactions oxidizing L-arginine to L-citrulline and NO (Bailey et al. 2011). Reactions in this pathway are catalyzed by NOS enzymes, and require the presence of a number of substrates and cofactors including oxygen and L-arginine (Bailey et al. 2011). Given their importance in the NOS-dependent pathway, a number of studies have investigated the effects of arginine and citrulline-based supplements on exercise performance (Bescos et al. 2012; Camic et al. 2010; Hickner et al. 2006). An alternative, NOS-independent pathway of NO production has been identified, in which nitrate (NO₃) is reduced to nitrite (NO₂), and further reduced to NO (Bailey et al. 2011). The NOS-independent pathway can function anaerobically and is stimulated by hypoxia and hydrogen ions, potentially indicating a significant role in NO formation during exercise (Bailey et al. 2011). Previous studies on nitrate supplementation have reported reduced blood pressure (Siervo et al. 2013), oxygen uptake (Bailey et al. 2011), and blood lactate (Ferguson et al. 2013); enhanced exercise tolerance (Bailey et al. 2009), exercise performance (Bescos et al. 2012), and mitochondrial efficiency (Larsen et al. 2011); and increased oxygenation and blood flow to skeletal muscle (Ferguson et al. 2013). Several exogenous food sources have been purported to naturally augment this pathway. Beetroot juice has recently been shown to be an ergogenic source of supplemental nitrate, and pomegranate extract (PE) is another viable source, with very high nitrate and polyphenol concentrations and research supporting similar cardiovascular effects (Aviram and Dornfeld 2001; Aviram et al. 2004; Sumner et al. 2005). Aside from their dietary nitrate content, the effects observed in previous pomegranate and beetroot research may also be attributed, in part, to polyphenol content. Supplementation with polyphenols has been shown to increase blood flow and vessel dilation (Barona et al. 2012). These results indicate potential for an acute ergogenic effect, and may be explained by enhanced endothelial NOS expression and NO production (Leikert et al. 2002).

The purpose of the current study was to investigate the effects of acute PE ingestion on vessel diameter, blood flow, and exercise performance. Previous studies have required participants to consume a nitrate-rich supplement chronically or multiple hours before exercise (Bailey et al. 2011; Bescos et al. 2012), but the high nitrate and polyphenol concentration of PE may allow for a more convenient timing protocol. The current study aimed to investigate the effects of acute PE ingestion 30 minutes prior to an exercise bout, utilizing highly active participants. It was hypothesized that ingestion of the PE supplement would enhance vessel diameter and blood flow before and after exercise. It was also hypothesized that PE ingestion would enhance submaximal aerobic exercise performance.

Materials and methods

Results

Table 1 presents change in time to exhaustion (sec) after ingestion of the PE and PL. Ingestion of the PE was found to significantly augment TTE at 90% (387.9 ± 199.2 vs. 346 ± 162.5 sec, p = 0.009) and 100% PV (170.8 ± 66.3 vs. 159.3 ± 62.3, p = 0.027) (Table 1). However, PE did not produce a significant effect on CV (p = 0.096) or ARC (p = 0.106). The visual analog (pain) scale was not significantly different between treatments. However, on the vitality scale, the following statement, “At this moment I feel alive and vital” was found to be significantly greater (p = 0.037) 30 min following ingestion PE compared to PL.

Vessel diameter was significantly increased (p = 0.036) 30min post-exercise with the consumption of PE (VD = 0.42 ± 0.07 cm) in comparison to PL (VD = 0.39 ± 0.07 cm) (Table 2). Blood flow was significantly augmented (p = 0.033) 30min post-ingestion of PE (BF =40.6 ± 24.8 ml/min) in comparison to PL (BF = 29.6 ± 24.9 ml/min) (Table 3). No other time points were significantly different as a result of PE ingestion.

Discussion

Compared to PL, acute ingestion of PE resulted in a significant increase in TTE at 90% and 100% PV. Vessel diameter 30minPEx and blood flow 30minPI were significantly augmented following ingestion of PE. Participants also reported significantly greater feelings of vitality after PE compared to PL, which may have important implications for exercise tolerance. Previous research has indicated that supplementation with dietary nitrate (Ferguson et al. 2013) and polyphenols (Barona et al. 2012) may increase blood flow and vessel dilation, which could contribute to the ergogenic effect of PE.

Improvements in TTE at 90% (Δ = 41.9 ± 58.4; p = 0.009) and 100% (Δ = 11.6 ± 21.0; p = 0.027) of PV suggest an ergogenic effect from PE. This finding is consistent with previous studies reporting improvements in high-intensity exercise as a result of nitrate supplementation (Bailey et al. 2011; Bescos et al. 2012), and supports the hypothesis that acute ingestion of 1000 mg PE would be ergogenic in trained participants. Supplementation with dietary nitrate has been shown to enhance exercise efficiency by decreasing oxygen consumption during exercise without limiting performance (Bailey et al. 2011), increasing blood lactate (Ferguson et al. 2013), or increasing energy contribution from glycolysis or the phosphagen system (Bailey et al. 2010). This enhanced efficiency may contribute to the increased TTE observed in the present study. It has been previously demonstrated that NO plays a role in modulating mitochondrial respiration (Castello et al. 2006), and that inhibition of endogenous NO production increases oxygen consumption at rest (Shen et al. 1994). Bailey et al. (Bailey et al. 2010) showed that nitrate supplementation prior to exercise decreased total ATP turnover, indicating that the reduced oxygen cost of exercise may be attributed to a reduction in the ATP cost of force production. Larsen et al. (Larsen et al. 2011) concluded that increased mitochondrial efficiency may reduce oxygen cost of exercise following nitrate supplementation. Despite improvements in TTE at 90% and 100% of PV, no improvement was observed at 110%. Given the potential ergogenic mechanisms of pomegranate extract involving oxygen delivery and mitochondrial efficiency, it is possible that PE supplementation preferentially enhances aerobic exercise capacity compared to higher-intensity anaerobic activities; more research is needed to confirm this observation.

Many studies have investigated the effects of various NO-related supplements on exercise performance, targeting both pathways of NO production. L-arginine is the direct precursor to NO in the NOS-dependent pathway, prompting researchers to investigate a potential ergogenic effect of L-arginine-based supplements. Although some evidence has indicated an ergogenic benefit from L-arginine supplementation, this effect appears to be training status-dependent. While positive findings have been reported in untrained and moderately-trained participants, an ergogenic effect is not likely in well-trained athletes (Bescos et al. 2012).

Although L-arginine is the direct NOS-dependent precursor to NO, L-citrulline supplementation increases plasma L-arginine concentrations more effectively than L-arginine supplementation (Schwedhelm et al. 2008). This has led to speculation that L-citrulline-based supplements may improve performance more effectively than L-arginine. Although Perez-Guisado and Jakeman (Perez-Guisado and Jakeman 2010) showed that an acute dose of citrulline malate increased work capacity in moderately-trained participants completing a resistance training protocol, this effect cannot be conclusively attributed to NO-related mechanisms. Conversely, an acute dose of L-citrulline was shown to have an ergolytic effect by decreasing TTE on an incremental treadmill test in moderately-trained participants (Hickner et al. 2006). More research is needed to evaluate the effects of L-citrulline supplementation on blood flow and exercise performance.

Initial research in nitrate-rich ingredients like beetroot and pomegranate has demonstrated ergogenic effects. Beetroot has been shown to improve performance and reduce the oxygen cost of exercise (Bailey et al. 2011; Bescos et al. 2012). Further, the ergogenic effect of beetroot does not appear to apply exclusively to untrained populations; Lansley et al. (Lansley et al. 2011) reported increased power output and enhanced time trial performance following a single acute dose in competitive cyclists. While previous studies with pomegranate juice have documented improved recovery and decreased soreness following eccentric exercise in recreationally active (Trombold et al. 2010) and resistance-trained (Trombold et al. 2011) males, the current study supports the hypothesis of an ergogenic effect from PE, and features a convenient protocol of acute ingestion 30 minutes prior to an exercise bout. Past research with beetroot has required participants to follow somewhat inconvenient dosing protocols, consuming the product chronically, or 2.5 hours before exercise (Bailey et al. 2011; Bescos et al. 2012). Pomegranate extract is a highly concentrated source of dietary nitrate and polyphenols, which may allow for a significantly shorter time between supplement ingestion and an observable ergogenic effect. The current study also utilized a high-intensity, intermittent exercise protocol that may simulate a number of sports more effectively than a single time trial.

Beetroot juice supplementation has been shown to increase limb blood flow in exercising rats (Ferguson et al. 2013). Our results support previous research demonstrating enhanced vasodilation and blood flow in response to nitrate and polyphenols. Thirty minutes after exercise, vessel diameter was significantly greater with PE consumption in comparison to PL (0.42 ± 0.07 cm vs. 0.39 ± 0.07 cm; p = 0.036). Further, blood flow measured 30 minutes post-ingestion was significantly greater in the PE condition compared to PL (40.6 ± 24.8 ml/min vs. 29.6 ± 24.9 ml/min; p = 0.033). The vasodilatory effect of PE supplementation may contribute to the ergogenic effect observed in the present study. Enhanced vasodilation is likely to improve metabolic control and oxidative function by increasing oxygen delivery relative to consumption, increasing the oxygen driving pressure from the capillary to the myocyte (Ferguson et al. 2013). While increases in vessel diameter and blood flow were not found uniformly at every time point, it is possible that the hyperemic response to exercise concealed any significant changes to blood flow or vessel diameter in the measurements taken immediately after each run to exhaustion. Conversely, significant effects on flow and vessel diameter were observed at rest, both 30 minutes after PE ingestion and 30 minutes after exercise. While research in beetroot juice has shown enhanced blood flow to exercising skeletal muscle (Ferguson et al. 2013), the high concentration of nitrate and polyphenol content in PE may further augment the delivery of blood, oxygen, and energy substrates to active musculature.

The effect of PE on vasodilation and blood flow may also have significant implications for recovery from exercise. Vessel diameter in the PE condition was significantly greater than PL 30 minutes post-exercise, which could aid in exercise recovery by enhancing nutrient delivery to skeletal muscle. This may be a plausible mechanism for the improved recovery observed in previous studies utilizing pomegranate juice (Trombold et al. 2010; Trombold et al. 2011). Aside from the well-documented effects of nitrate on blood flow, this effect may also be attributed to the polyphenol content of PE. Evidence suggests that polyphenols increase flow-mediated vasodilation (Barona et al. 2012), likely via increased endothelial NOS expression and NO production following use of polyphenols (Leikert et al. 2002). Previous studies have suggested that polyphenol-rich ingredients may enhance recovery from damaging exercise (Malaguti et al. 2013). While research with pomegranate juice has indicated improved recovery from exercise (Trombold et al. 2010; Trombold et al. 2011), more research is needed to investigate the effects of PE on exercise recovery and the underlying mechanisms. In the present study, participants in the PE condition also reported higher scores on a subjective vitality scale (p = 0.037), suggesting a potential value to individuals engaged in rigorous training programs or clinical populations with a low tolerance for exercise.

Results of the present study indicate that PE enhances vessel diameter and blood flow with a concomitant increase in TTE, thus implicating PE as a promising ergogenic aid in the context of high-intensity intermittent running. Results of the current study indicate that further research on PE is warranted. Research regarding optimal dosing and timing strategies may enhance the effects of PE on blood flow and performance. Future research may also help to determine if the ergogenic effect of PE supplementation is applicable to other exercise modalities and intensities, and if chronic PE supplementation confers more pronounced effects. Previous research has shown pomegranate juice to decrease blood pressure (Aviram and Dornfeld 2001; Aviram et al. 2004) and increase myocardial perfusion (Sumner et al. 2005), while beetroot has been shown to improve exercise tolerance in peripheral artery disease (Kenjale et al. 2011). More research is needed to determine if the results of the present study translate to a significant improvement in exercise tolerance, blood pressure, and tissue perfusion in clinical populations.

In conclusion, ingestion of PE in close proximity to an exercise bout led to enhanced vessel diameter, blood flow, and delayed fatigue in highly active participants. These effects are likely attributed to PE’s high nitrate and polyphenol content, which have been shown to increase nitric oxide production and enhance exercise efficiency. Whereas previous research has required participants to ingest sources of dietary nitrate chronically or multiple hours before exercise, PE ingestion resulted in an ergogenic effect when ingested 30 minutes prior to the bout. Results of the current study indicate that PE is an efficacious ergogenic aid for sports involving intermittent running, eliciting beneficial effects on blood flow and exercise performance as few as 30 minutes after ingestion. More research is needed to evaluate the effects of chronic PE supplementation and potential clinical applications.

Acknowledgments

References