Abstract
Background and Aims:
Watermelon juice is a rich food source of cardioprotective compounds such as arginine, citrulline, and lycopene. Preventative interventions are warranted as risk of cardiovascular disease increases among women after menopause, and age alone is an independent risk factor for vascular dysfunction. Thus, this study evaluated the effects of 100% watermelon juice on measures of vascular function.
Methods and Results:
In this randomized, double-blind, placebo-controlled, crossover trial, 21 healthy postmenopausal women were randomized to consume two 360 mL servings of 100% watermelon juice per day or an isocaloric placebo for four weeks. Following a two-week washout period, they consumed the other beverage for an additional four weeks. Before and after each treatment arm, a fasting blood sample was taken for measurement of serum arginine, citrulline, lycopene, glucose, and insulin. Assessments of vascular function included pulse pressure, pulse wave velocity, 24-hour ambulatory blood pressure, and flow-mediated dilation. General linear mixed models with intent-to-treat analyses were used to examine the effects of the intervention.
Despite a significant treatment effect for circulating lycopene (p=0.002), no changes in arginine, citrulline, or any vascular measures were observed. Although the juice intervention resulted in a slight but significant increase in fasting serum glucose (p=0.001), changes in glucose homeostasis were not clinically significant.
Conclusion:
In contrast to findings from previous studies in younger adults and those with pre-existing hypertension, measures of vascular function in this cohort of healthy postmenopausal women were not impacted by supplemental watermelon juice.
Keywords: watermelon juice, blood pressure, lycopene, arginine, citrulline
Introduction
Cardiovascular diseases (CVD) are the leading cause of death among women in the United States [1]. Impaired vascular function is a precursor to CVD [2], and arterial stiffness is an early predictor of future cardiac events [3]. Arterial stiffness and endothelial dysfunction intensify with age due mainly to increased oxidative stress [4] and decreased bioavailability of nitric oxide, the principal vasodilatory molecule in the vascular endothelium [5–7]. However, endothelial dysfunction occurs very early in the process of atherosclerosis at a stage when CVD is potentially reversible [8]. Hence, food-first interventions that aim to optimize the vascular function of postmenopausal women before frank CVD ensues are indicated.
Previous studies support the effectiveness of watermelon extracts for improvement of vascular function in middle-aged men and women [9–13]. In a previous randomized placebo-controlled trial specifically examining postmenopausal women, six-week supplementation with watermelon extract also resulted in reduced arterial stiffness [14]. However, these studies investigated effects of the intervention in people with pre-existing hypertension and obesity. Watermelon interventions in normotensive adults have produced equivocal results [15]. Because age and menopause are independent risk factors for vascular dysfunction [16, 17], the present study aimed to investigate effects of watermelon juice supplementation on clinical measures of vascular health.
Protective effects of watermelon on vascular function have been attributed to its high content of arginine and citrulline as these two amino acids lead to production of the vasodilator molecule nitric oxide [15]. Age-related decreases in nitric oxide have been directly implicated in declines of vascular endothelial function [18]. This may be even more pronounced in postmenopausal women as previous studies have reported decreases in plasma arginine and vascular endothelial function with estrogen deficiency after menopause [17, 19]. Although less studied, asymmetric dimethylarginine (ADMA) is an endogenously produced metabolite that acts as a competitive inhibitor of arginine, thereby reducing nitric oxide synthesis [5].
Circulating ADMA levels increase with age [20, 21], and it has been suggested that decreases in estrogen with menopause may contribute to age-related increases in ADMA [22, 23]. However, dietary citrulline has been shown to improve both arginine/ADMA ratios and vascular endothelial function [24].
The high lycopene content of watermelon may also confer cardioprotective properties [25]. Watermelon is among the richest food sources of lycopene [26], and circulating lycopene levels have been associated with improved cardiovascular health [27]. A recent study among overweight/obese postmenopausal women found that six-week supplementation with watermelon puree increased circulating arginine and lycopene while decreasing plasma sVCAM-1, a biomarker of atherosclerosis [28]. However, effects of supplementation on clinical measures of non-obese postmenopausal women remains unknown.
Although watermelon is purported to be a functional food for vascular health, pragmatic approaches for real-world dietary interventions are lacking. The watermelon extracts used in previous studies are not commercially available, and it may be impractical for older adults to regularly consume fresh watermelon fruit due to its seasonality and the inconvenience of cutting and preparing fresh fruit [29]. Thus, we examined 100% watermelon juice as a convenient, shelf-stable vehicle for conferring the cardiovascular benefits of watermelon.
Health benefits versus risks of 100% fruit juice have been debated in recent years. Some experts recommend limiting juice consumption, citing its sugar content and lack of fiber that may provide excess kilocalories without concomitant satiety [30]. So, any intervention study should also consider effects of juice on body composition and glucose homeostasis.
Acknowledging the potential promise of watermelon as a cardioprotective intervention along with current gaps in the literature, the primary aim of this study was to investigate the effects of 100% watermelon juice supplementation on vascular health of postmenopausal women. As secondary aims, we examined effects of supplementation on circulating levels of arginine, citrulline, lycopene, glucose, and insulin as well as weight and percent body fat.
Materials and Methods
In a double-blind crossover design [31], participants were randomized to two 360 mL servings of 100% watermelon juice per day or an isocaloric placebo for four weeks each with a two-week washout period in between.
Participants
Participants were community-dwelling postmenopausal women ages 55–70 y with BMI <30 kg/m2 (non-obese). Postmenopausal status was confirmed by self-report of no menstrual period for at least 12 months. Participants were recruited by word of mouth and fliers placed in local community organizations, and data were collected October 2016 – June 2018. Exclusion criteria included blood pressure readings at screening ≥ 140/90 mm Hg or history of previous cardiac events, renal disease, diabetes, or diagnosis of any inborn error of metabolism. Women were also excluded if they reported tobacco use, change in weight of > 4.5kg over the past year, or use of antihypertensive or cholesterol-lowering medications, vasodilatory dietary supplements (garlic, fish oil), or dietary supplements containing lycopene, ascorbic acid, L-glutamine, Larginine, or L-citrulline. The University of Alabama Institutional Review Board approved the study protocol, and all participants provided written informed consent.
Protocol
In a double-blind crossover design, participants were randomized to supplement their diets twice daily for four weeks with either 360 mL of 100% watermelon juice or an isocaloric placebo that was matched for sugar content. Details of the placebo beverage were previously described [32]. Briefly, the placebo consisted of water, sucrose, non-nutritive watermelon flavoring, malic acid, pectin, cellulose, and food coloring. Two 360mL servings of 100% watermelon juice provided daily doses of 14.4 ± 0.34 mg lycopene, 1.15 ± 0.0013g arginine, and 1.63 ± 0.101g citrulline. Each treatment arm was separated by a two-week washout period. Physiological doses of watermelon juice and the washout period between arms were determined by previous watermelon studies [33, 34]. A blocked randomization was generated using PROC PLAN in SAS Version 9.4, and a closed envelope method was used for participant assignment to either juice or placebo for the first arm. The principal investigators, participants, and all study staff responsible for data collection and analysis were blinded to treatment order. Three members of the study staff who were unblinded packaged juice and placebo to provide two 360mL servings per day. Because lycopene is fat-soluble [25], participants were asked to consume one dose in the morning and another in the evening with a mixed macronutrient meal to maximize its bioavailability. Pasteurized watermelon juice for this study was supplied by Frey Farms (Keenes, IL) from Estrella variety melons. To ensure that all juice contained similar concentrations of the bioactive food compounds of interest, each batch was tested in the Food and Nutrition Research Laboratory at the University of Alabama for lycopene, citrulline, and arginine content.
Participants were asked to maintain their usual physical activity patterns for the duration of the study. Two weeks prior to the intervention, during each intervention arm, and during the washout period, participants consumed their typical diet except for foods high in lycopene. At screening, a list of lycopene-containing foods was provided, and participants were instructed to limit these foods to two or fewer servings per day for the duration of the study. Dietary intake was closely monitored by three-day food diaries submitted during the two-week run-in period as well as by unannounced 24-hour diet recalls during the washout and treatment periods. Diet recalls were administered using a multi-pass method that has been validated for use in older adults (Nutrition Data System for Research Software Version 2015) [35]. Adherence to supplement and placebo was assessed by log forms with check-off boxes for each dose.
Before and after each treatment arm, participants came to the laboratory after an overnight fast of at least eight hours to complete the assessments detailed below. Participants were also instructed to refrain from physical activity on the mornings of testing.
Clinical assessments of vascular function
Blood pressure.
Office measurements of blood pressure were measured using a calibrated, automated monitor (Mobil-O-Graph, IEM, Stolberg, Germany) according to standard guidelines [36]. After at least five minutes of rest with the non-dominant arm elevated at heart level, a minimum of two readings were taken with at least one minute in between. In case of a difference in readings greater than five mmHg, a third reading was taken, and the two readings were averaged.
Pulse pressure.
Pulse pressure was calculated as the difference between office systolic blood pressure (SBP) and diastolic blood pressure (DBP). As arteries become increasingly stiff, SBP increases without a concomitant increase in DBP. This results in increased pulse pressure which has been identified as an independent risk factor for cardiac events [4].
Pulse pressure amplification.
The Mobil-O-Graph system was also used for measurements of central arterial pressure. Pulse pressure amplification is the ratio of peripheral to central pulse pressure [37]. After menopause, central blood pressure tends to be higher, so pulse pressure amplification is lower [38]. Thus, higher pulse pressure but lower pulse pressure amplification reflects elevated CVD risk [39].
Pulse wave velocity (PWV).
PWV, the rate at which a pulse propagates through a vessel, is considered a gold standard assessment of arterial stiffness [3]. The cuff-based Mobil-O-Graph system was used to measure brachial oscillometric pressure waveforms and generate central pressure curves by propriety algorithms. PWV estimates from the Mobil-O-Graph system have been validated against direct intra-arterial measurements via catheterization [40].
24-hour ambulatory blood pressure monitoring (ABPM).
In addition to office measurements, the Mobil-O-Graph system was used to obtain 24-hour ambulatory blood pressure measurements. Criteria for valid ABPM were at least 75% of planned readings without error during waking hours and at least seven error-free sleep readings [41, 42].
Flow-mediated dilation (FMD).
Endothelial-dependent vasodilation was assessed by FMD. A well-established method for determining endothelial function [43], FMD uses ultrasound technology to quantify changes in brachial artery diameter in response to reactive hyperemia. According to evidence-based recommendations [44], a blood pressure cuff was placed on the right forearm distal to the brachial artery with the participant supine and rested in a thermoneutral room. Pre-inflation diameter was recorded for one minute, and the cuff was inflated to 50 mmHg above resting SBP for five minutes using a 7.5 × 40 cm straight cuff and aneroid sphygmomanometer (D. E. Hokanson, Inc., Bellevue, WA). To capture peak diameter after deflation, images were recorded for 30 seconds before and 120 seconds after deflation. Images were taken with high-resolution B-mode ultrasound with a 9–12 MHz linear-array probes (depending on the size of each participant’s arm) and isonization angle < 60 degrees. Peak diameter was determined as an average of the five highest measurements over five seconds post-deflation [45] using edge detection software (Vascular Research Tools 6 Software, Medical Imaging Applications, LLC, Coralville, IA). FMD% was expressed as the percentage increase in peak diameter from baseline:
Analysis of biological samples
A fasting blood sample was obtained to quantify serum levels of lycopene, citrulline, arginine, and ADMA. Serum was stored at −80 degrees C until time of analysis at which time it was thawed and handled under dim light. Proteins were precipitated according to a published method using methanol/acetonitrile/acetone (1:1:1, v/v/v) added to serum in a ratio of 1:4 (v/v) [46]. Extraction and quantitation of lycopene was conducted according to a previously described method by Maurer et al, 2014 [47]. Lycopene and β-carotene were sourced from Sigma-Aldrich (St. Louis, MO), and β-carotene served as the internal standard for quantitation. The chromatographic separation was carried out using an ACQUITY ultra-high-performance liquid chromatography (UPLC) system with a photodiode array (PDA) detector and ACQUITY BEH Shield RP18 2.1 × 100mm, 1.7μum (Waters, Milford, MA). The limit of quantitation for lycopene was 0.391 μmol/L.
Assessment of serum citrulline and arginine was carried out according to a standardized method for quantitation of amino acids in biological samples [48]. Each amino acid as well as glutamine-d5 were sourced from Sigma-Aldrich and glutamine-d5 served as the internal standard for quantitation. The chromatographic separation was carried out using Waters Xevo TQ-S mass spectrometer interfaced with ACQUITY UPLC system and ACQUITY HSS T3 2.1 × 100mm, 1.8μum. The limit of quantitation for each amino acid was 0.2 μmol/L.
ADMA was quantified using an ELISA kit (Enzo Life Sciences, Farmingdale, NY) with absorbance measured at 450nm against a 620nm reference and a limit of detection of 0.05umol. Using this data, the ratio of arginine/ADMA was calculated.
Other relevant clinical assessments
Height was measured at each visit with a stadiometer, and weight was measured at each visit with a calibrated scale. Body mass index (BMI) was calculated as kg/m2. At each visit, percent body fat was also estimated by bioelectrical impedance analysis (BIA) (RJL Systems Inc., Clinton Township, MI).
Fasting serum glucose was measured by a glucose oxidase method (Stanbio Sirrus analyzer; Stanbio Laboratory, Boerne, TX). This analysis had a mean intra-assay coefficient of variation (CV) of 1.28%, and a mean inter-assay CV of 4.48%. Serum insulin was measured by immunofluorescence (TOSOH AIA-II analyzer, TOSOH Corporation, South San Francisco, CA; mean intra-assay CV 1.49%; mean inter-assay CV 3.95%). Fasting insulin and glucose values in μU/mL and mg/dL, respectively, were used to calculate HOMA-IR as (glucose × insulin)/405 [49]. Although HOMA-IR provides only a proxy indicator of insulin resistance, it is widely used in research and has been validated with the gold-standard hyperinsulinemic clamp in young adults without diabetes [50, 51].
Statistical analysis
Sample size was based on the ability to detect changes in lycopene. Assuming an average change in serum lycopene of 0.1μM and a standard deviation of 0.025 [52], 15 participants would allow for 80% power to detect changes in lycopene at a significance level of 5%. General linear mixed models with intent-to-treat analysis were used to model the effect of treatment (i.e., randomization to watermelon juice versus placebo) on the outcomes of interest. Hommel-adjusted p-values and false discovery rate were calculated to account for multiple testing. Given the modest sample size, p-values based on permutation tests were also calculated. To gain insight about the clinical relevance of these findings, effect sizes for all outcomes were also calculated.
Results
Twenty-one women were randomized to treatment or placebo arms, and seventeen participants completed the study (Figure 1). Baseline characteristics and demographics for participants who completed the study are shown in Table 1. Vascular and serum measures before and after each treatment arm are displayed in Table 2. Aside from the prescribed beverages, dietary intake analysis revealed no significant differences in intake of lycopene, arginine, protein, or fat between the two treatment arms (data not shown). The juice regimen was well-tolerated with the exception of one participant who dropped out after developing a skin rash during the juice arm. The rash resolved without treatment.
Figure 1.
CONSORT Flow Diagram
Table 1.
Participant Demographics and Baseline Characteristics
| Demographics | Mean ± SD |
|---|---|
| Age (y) | 60.00 ± 4.30 |
| Weight (kg) | 65.31 ± 10.16 |
| BMI (kg/m2) | 25.05 ± 3.56 |
| Percent Body Fat | 36.71 ± 5.05 |
| Serum Glucose (mg/dL) | 94.81 ± 8.45 |
| Serum Insulin (μU/mL) | 5.05 ± 2.84 |
| HOMA-IR | 1.20 ± 0.70 |
| Serum Lycopene (μM) | 1.45 ± 0.81 |
| Serum Arginine (μM) | 61.54 ± 19.44 |
| Serum Citrulline (μM) | 27.96 ± 6.17 |
| Serum ADMA (μM) | 0.33 ± 0.07 |
BMI = body mass index; HOMA-IR = homeostasis model assessment-estimated insulin resistance = (fasting insulin × fasting glucose) / 405; ADMA = asymmetric dimethyl arginine
Table 2.
Vascular and Serum Measures Before and After each Treatment Arm (Mean ± SD)
| 100% Watermelon Juice | Placebo Beverage | ||||
|---|---|---|---|---|---|
| Measure | Before | After | Before | After | *P-value |
| Office SBP (mmHg) | 116.00 ± 9.96 | 115.12 ± 10.69 | 119.88 ± 8.63 | 115.47 ± 12.85 | 0.286 |
| Office DBP (mmHg) | 74.35 ± 7.25 | 73.71 ± 8.55 | 76.59 ± 8.43 | 74.18 ± 7.58 | 0.241 |
| Office Pulse Pressure (mmHg) | 41.65 ± 7.52 | 41.41 ± 10.15 | 43.29 ± 8.93 | 41.29 ± 10.60 | 0.598 |
| Office Pulse Pressure Amplification | 1.31 ± 0.09 | 1.35 ± 0.09 | 1.31 ± 0.11 | 1.39 ± 0.36 | 0.975 |
| PWV (m/s) | 8.18 ± 0.70 | 8.15 ± 0.77 | 8.39 ± 0.67 | 8.20 ± 0.79 | 0.367 |
| 24-Hour ABPM SBP (mmHg) | 115.18 ± 7.47 | 115.41 ± 7.62 | 113.94 ± 7.00 | 112.71 ± 8.46 | 0.515 |
| 24-Hour ABPM DBP (mmHg) | 71.00 ± 7.66 | 70.47 ± 7.25 | 69.76 ± 6.89 | 69.41 ± 7.13 | 0.906 |
| FMD% | 8.17 ± 5.68 | 11.02 ± 6.15 | 9.30 ± 5.53 | 11.47 ± 6.96 | 0.816 |
| Lycopene (μM) | 1.72 ± 0.68 | 8.63 ± 7.48 | 1.64 ± 1.00 | 4.73 ± 4.9 | 0.135 |
| Arginine (μM) | 67.89 ± 25.22 | 72.17 ± 24.40 | 64.21 ± 27.33 | 76.83 ± 18.70 | 0.542 |
| Citrulline (μM) | 28.64 ± 7.92 | 27.26 ± 6.69 | 26.29 ± 11.33 | 27.51 ± 7.94 | 0.520 |
| Glucose (mg/dL) | 92.31 ± 9.48 | 95.65 ± 7.79 | 94.35 ± 8.23 | 92.5 ± 8.55 | 0.003 |
| Insulin (μU/mL) | 5.44 ± 3.63 | 6.48 ± 2.76 | 5.03 ± 2.64 | 5.13 ± 2.40 | 0.405 |
| Weight (kg) | 64.94 ± 9.82 | 65.05 ± 10.19 | 65.41 ± 10.39 | 65.14 ± 9.89 | 0.342 |
| BMI (kg/m2) | 24.91 ± 3.46 | 24.94 ± 3.51 | 25.08 ± 3.62 | 24.98 ± 3.42 | 0.389 |
| Percent Fat | 36.27 ± 4.87 | 35.99 ± 5.47 | 36.15 ± 5.68 | 35.56 ± 6.79 | 0.723 |
SBP = systolic blood pressure, DBP = diastolic blood pressure, PVW = pulse wave velocity, m/s = meters per second, ABPM = ambulatory blood pressure monitoring, FMD = flow-mediated dilation, BMI = body mass index
n =17; (n = 16 for serum measures due to insufficient blood sampling for one participant)
P-values for delta variables from juice and placebo arms (paired t-tests and Wilcoxin signed ranks tests)
As previously reported [53], results from mixed models revealed that compared to placebo, watermelon juice supplementation significantly increased circulating lycopene levels (Beta coefficient = 0.548, 95% CI = (0.204,0.891), p = 0.002) (Table 3). Changes in circulating arginine, citrulline, ADMA, and arginine/ADMA ratio were not significant. Despite the increase in serum lycopene, no significant changes in any of the vascular outcomes were observed (Table 3).
Table 3.
Effects of the Watermelon Juice Intervention on Vascular Measures and Circulating Levels of Bioactive Food Compounds
| Variable | Point Estimate | SEM | P-Value | Confidence Interval | Adjusted P-Value* | Effect Size |
|---|---|---|---|---|---|---|
| Serum Lycopene (μM) | 0.548 | 0.175 | 0.002 | (0.204,0.891) | 0.068 | 0.795 |
| Serum Citrulline (μM) | −0.018 | 0.089 | 0.839 | (−0.192, 0.156) | 0.941 | −0.115 |
| Serum Arginine (μM) | −0.117 | 0.118 | 0.321 | (−0.349, 0.114) | 0.846 | −0.274 |
| Serum ADMA (μM) | −0.018 | 0.041 | 0.660 | (−0.099, 0.062) | 0.914 | −0.321 |
| Serum Arginine/ADMA Ratio | −0.099 | 0.139 | 0.478 | (−0.372, 0.174) | 0.846 | −0.106 |
| Serum Glucose (mg/dL) | 0.053 | 0.015 | 0.001 | (0.023,0.084) | 0.011 | 0.496 |
| Serum Insulin (μU/mL) | 0.195 | 0.115 | 0.088 | (−0.029,0.420) | 0.547 | 0.262 |
| HOMA-IR | 0.237 | 0.120 | 0.050 | (0.0003,0.4726) | 0.613 | 0.556 |
| Office SBP (mmHg) | 0.007 | 0.019 | 0.728 | (−0.031, 0.045) | 0.914 | 0.311 |
| Office DBP (mmHg) | 0.006 | 0.015 | 0.716 | (−0.024, 0.036) | 0.914 | 0.301 |
| Office Pulse Pressure (mmHg) | 0.008 | 0.072 | 0.911 | (−0.132, 0.148) | 0.957 | −0.085 |
| Office Pulse Pressure Amplification | −0.012 | 0.044 | 0.791 | (−0.098, 0.075) | 0.914 | 0.207 |
| PWV (m/s) | 0.011 | 0.017 | 0.517 | (−0.022, 0.044) | 0.846 | 0.152 |
| 24-Hour ABPM SBP (mmHg) | 0.015 | 0.017 | 0.397 | (−0.019, 0.048) | 0.846 | 0.272 |
| 24-Hour ABPM DBP (mmHg) | 0.013 | 0.020 | 0.526 | (−0.027, 0.052) | 0.846 | 0.238 |
| FMD% | 0.070 | 0.219 | 0.749 | (−0.359, 0.500) | 0.914 | 0.634 |
ADMA = asymmetric dimethyl arginine; HOMA-IR = homeostasis model assessment-estimated insulin resistance = (fasting insulin × fasting glucose) / 405; SBP = systolic blood pressure; DBP = diastolic blood pressure; PVW = pulse wave velocity; ABPM = ambulatory blood pressure monitoring; FMD = flow-mediated dilation
n =17; (n = 16 for serum measures due to insufficient blood sampling for one participant)
P-Value adjusted for multiple comparisons by the false discovery rate method
Supplementation did not appreciably affect BMI or percent body fat (p = 0.444 and p = 0.773 from mixed models, respectively); however, compared to the placebo, watermelon juice resulted in a statistically significant increase in fasting serum glucose [Beta coefficient = 0.053, 95% CI = (0.023, 0.084), p = 0.001]. Although change in HOMA-IR was in the same direction, it was not statistically significant (Table 3). Maximum serum glucose did not meet the World Health Organization’s definition of impaired fasting glucose (110 mg/dL) [54].
Discussion
CVD is the leading cause of death among women in the United States [1]. Because vascular dysfunction precedes overt CVD, it is important to intervene early before frank disease ensues [5]. Considering age as an independent risk factor for vascular dysfunction [16], previous studies supporting cardiovascular benefits of watermelon extracts [9, 11–14], and the popularity of watermelon juice as a functional food [27], we sought to examine the effects of a physiological dose of 100% watermelon juice on measures of vascular function and glucose homeostasis in postmenopausal women. We also examined effects of the intervention on circulating levels of the key bioactive food compounds in watermelon: arginine, citrulline, and lycopene. Although four-week supplementation with 100% watermelon juice did result in increased fasting serum lycopene, no effects on circulating amino acids or biomarkers of vascular function were observed. Compared to an isocaloric placebo beverage, juice supplementation resulted in a slight but significant increase in fasting blood glucose; however, impact on glucose homeostasis was not clinically significant.
Supplementation with watermelon juice did not appreciably affect measures of vascular function. This was contrary to our hypothesis because previous randomized controlled trials demonstrated improvement in blood pressure [9, 11–13], pulse pressure [10], and PWV [14] with six-week supplementation of watermelon extract. These studies attributed the effects of watermelon on vascular function to the arginine and citrulline. It is well-known that the endothelial dysfunction and arterial stiffness that worsen with age are related to decreased bioavailability of nitric oxide and increased oxidative stress [5, 55]. Nitric oxide, the most prominent endogenous vasodilator [56], is produced by endothelial cells from the substrate arginine [57]. In addition to its high arginine content, watermelon is the richest food source of citrulline [58], a non-essential amino acid that is readily converted to arginine in the kidneys [59]. While arginine undergoes considerable intestinal and hepatic metabolism before reaching circulation, citrulline escapes first-pass splanchnic extraction. Thus, supplemental citrulline has been shown to increase blood levels of arginine even more than arginine itself [15]. In the present study, although watermelon juice provided an extra 1.15 ± 0.0013g of dietary arginine and 1.63 ± 0.101g of citrulline daily, supplementation did not appreciably increase fasting serum levels of these amino acids. Previous pharmacokinetic studies that have shown increases in circulating citrulline and arginine after acute citrulline ingestion [60] as well as studies examining citrulline supplementation for days or weeks [15] have used dosages of 2g and higher. Prior watermelon studies with similar dosages of citrulline likewise reported no changes in plasma citrulline [28, 33]. However, a previous randomized crossover study reported significant increases in circulating arginine among healthy men and women after three weeks of supplementation with 720mL of watermelon juice [33], and a study among overweight/obese postmenopausal women showed increases in plasma arginine after six weeks of supplementation with 710mL of watermelon puree [28]. The arginine content of our juice intervention exceeded that of these previous studies, so we hypothesized that supplementation would likewise increase fasting serum levels among our cohort. Conversely, the dose of 720 mL/day of 100% watermelon juice was insufficient to produce changes in serum arginine among the lean, normotensive postmenopausal women in our cohort. This observation is intriguing considering multiple previous studies that have shown increases in circulating arginine after supplementation with 100% watermelon juice. Discordant results may relate to differences in processing as most previous studies have used watermelon juice that was not pasteurized [33, 61–63]. However, a recent study described the thermal decomposition of arginine at high temperatures [64]. Although the temperatures applied in this thermal stability study exceeded those typically used for food pasteurization, future research is warranted to describe the stability of bioactive compounds in watermelon with different methods of processing. Future pharmacokinetic studies are also indicated to assess the bioavailability of watermelon’s bioactive compounds among individuals of different ages and body composition.
Four-week supplementation with 100% watermelon juice did, however, significantly increase fasting serum lycopene. Unlike arginine and citrulline, lycopene cannot be synthesized endogenously, so it must be consumed in the diet [65]. Moreover, the thermal processing of pasteurization increases lycopene bioavailability through isomerization [66, 67]. Because of the many conjugated double bonds in its structure, lycopene has strong antioxidant activity [68]. Considering that oxidative stress underlies age-associated endothelial dysfunction [5], we anticipated that increases in the antioxidant lycopene would translate into improved vascular function. However, despite increases in serum lycopene, we did not observe changes in any vascular measures. Results contrast previous studies that reported independent inverse associations with circulating lycopene and PWV [67, 69], atherosclerosis [70], risk of stroke [71]. Prior studies also reported antihypertensive effects [72] and improved endothelial-dependent vasodilation with supplemental lycopene [73]. However, in those studies, lycopene’s beneficial effects on endothelial function were seen in people with pre-existing CVD [67, 73] and a 2013 meta-analysis reported that supplemental lycopene was effective for reducing SBP among hypertensive participants [72]. Similarly, previous studies showing improvements in vascular measures with watermelon supplementation have included overweight and hypertensive individuals [9–14, 28]. Null results for clinical outcomes in this study may reflect the vascular health of our participants at baseline. By study design, our cohort was non-obese with no evidence of overt CVD. Because age alone is a risk factor for vascular dysfunction [55] and because a previous study of younger healthy adults did show significant decreases in blood pressure after a week of citrulline supplementation [74], the present study aimed to examine effects of watermelon supplementation among postmenopausal women who did not yet present with diagnostic vascular impairment. However, taken in the context of other studies, it is likely that bioactive compounds in watermelon may be more effective for those with pre-existing comorbidities.
The health benefits versus risks of 100% fruit juice have been debated in recent years. Possible drawbacks of juice consumption relate to its concentrated sugar content and concerns that it may provide excess kilocalories without concomitant satiety [30]. Because the glycemic index of fruit juice is higher than that of whole fruit, there is also concern that juice may negatively impact glucose homeostasis [75]. The juice intervention resulted in a statistically significant increase in fasting serum glucose; however, increases were not clinically significant as levels were well below diagnostic for impaired fasting glucose [54]. Similarly, in a prior study, Shanely et al reported no changes in fasting blood glucose, insulin, or HOMA-IR among overweight and obese postmenopausal women after six-week supplementation of watermelon puree [28]. So, while it is possible that unfavorable effects on glucose and insulin response offset positive effects of the intervention on vascular function, it is unlikely.
This study provides valuable insight regarding the potential of 100% watermelon juice as a preventative food-first intervention to affect vascular health of postmenopausal women. However, our selection of a relatively healthy cohort of women with good vascular function at baseline presents a limitation as it is likely more challenging to see changes in health outcomes compared to women with vascular dysfunction. Hormone replacement therapy was also not considered among the inclusion/exclusion criteria, and this could be considered a limitation. Although participants were asked to maintain their usual physical activity levels for the duration of the study, physical activity was not closely monitored. Future studies examining synergistic effects of watermelon and exercise on vascular outcomes would be of interest. Because of the relatively homogenous cohort of Caucasian women from the southeastern United States, generalizability of results is also limited. Additionally, based on official guidelines from the Brachial Artery Reactivity Task Force; a sample size of 20–30 participants is recommended to detect changes in FMD with a crossover design [76]. So, although this study was powered to detect changes in other outcomes, a larger cohort may be necessary for the outcome measure of FMD, especially considering the large inter-individual variation observed for this outcome. Nevertheless, this study was strengthened by its robust design, laboratory assays, clinical assessments of vascular function, and statistical analysis.
Conclusions
In summary, this study investigated effects of four-week supplementation with pasteurized 100% watermelon juice on vascular function of healthy postmenopausal women. Supplementation was effective for increasing circulating lycopene, but no effects on circulating arginine, citrulline, or any clinical measure of vascular function were observed. Healthy participants for this study were selected in an attempt to examine early intervention before CVD onset, but we did not observe the same changes in vascular measures seen in previous studies among men and women with pre-existing risk factors [15].
Highlights.
Two 360 mL servings of 100% watermelon juice per day increased circulating lycopene.
The four-week intervention did not affect blood pressure or measures of arterial stiffness.
Juice supplementation resulted in a slight but significant increase in fasting blood glucose.
Acknowledgements
This work was supported by the American Heart Association Mentored Clinical and Population Research Award #16MCPRP27260233, National Institute on Aging Translational Nutrition and Aging Research Academic Career Leadership Award K07AG043588, and National Institute of Diabetes and Digestive and Kidney Diseases Center Core Grant P30DK056336.
Amy Ellis, PhD, RD and Kristi Crowe-White, PhD, RD are Co-Principal Investigators who contributed equally to the design and management of this study. As such, they should be considered co-anchor authors on this article.
Footnotes
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Conflicts of Interest: The authors have no conflicts of interest to disclose.
ClinicalTrials.gov Identifier: NCT03626168
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