Citrulline and Arginine Content of Taxa of Cucurbitaceae

Watermelon is the most significant, natural plant source of L-citrulline, a non-proteinaceous amino acid that benefits cardiovascular health and increases vasodilation in many tissues of the body. Watermelon is a member of the Cucurbitaceae, which includes squash, melon, pumpkin, and cucumber. It is possible that other cucurbits could be good sources of citrulline or of arginine, its direct precursor. Twenty-one cultigens were evaluated in triplicate at two locations in North Carolina to estimate citrulline and arginine amounts and variation due to cultigen, replication, and environment. Cultigens containing the highest amount of citrulline (based on LS means) in g/kg fresh weight were ’Crimson Sweet’ watermelon (2.85), ’Dixielee’ watermelon (2.43), casaba-type melon (0.86), mouse melon (0.64), and horned melon rind (0.45). Additionally, mouse melon, horned melon, and bitter gourd (arils) may be interesting sources of arginine-family amino acids, perhaps because of their large seed and aril content relative to mesocarp.


Introduction
Watermelon [Citrullus lanatus (Thunb.)Matasum, and Nakai] is a natural plant source of lycopene, citrulline, and arginine, which are phytochemicals associated with reduced risk to several cancers and increase of vasodilation in many tissues of the body [1].Citrulline, a non-proteinaceous amino acid, is unusually abundant in watermelon, more than any other plant taxon recorded to date [2].Citrulline has a high nitrogen to carbon ratio, having roles in both plant and human metabolism.In human health, citrulline offers many health benefits, especially in exercise supplementation for muscle performance and recovery [3][4][5].While dietary uses have received the most attention, citrulline has been investigated in other human-health fields, including pharmacology [6,7], immunology [8], and neurology [9].Citrulline is a precursor to the essential amino acid arginine; pharmacological study shows that in diseases related to arginine deficiencies, supplementation with citrulline can be more effective than arginine itself.This effect is due to its more targeted metabolism, which contributes to increased bioavailability, better absorption into the bloodstream and reduced side-effects when administered orally [3,[10][11][12][13].
The production and role of citrulline in plants is both less studied and more complex than in human systems.Several studies that examined physiological and environmental variables in watermelon suggest potential influences on citrulline abundance.Citrulline accumulates dramatically in the leaves of Cucumis melo (melon) and some Citrullus species in response to stresses of drought and high light intensity, suggesting a role in osmotic adjustment, radical oxygen species scavenging, We were unable to collect reliable data from all winter squash and some horned melon due to susceptibility to downy mildew, so winter squash and several horned melon plots were excluded from the analysis.
Cultigens were evaluated in 2016 at two locations, the Horticultural Crops Research Station in Clinton, NC and the Cunningham Research Station in Kinston, NC.The experiment was a randomized complete block design with three replications.Plants were grown using five-plant plots with 0.6 m spacing in 3.6 m long plots and 1.5 m row spacing.The vines were turned until the initiation of fruit set to prevent them from growing into adjacent plots.Plots were grown using horticultural practices recommended by the North Carolina Extension Service, also including drip irrigation and black plastic mulch [34].Cucurbits were harvested by groups: Summer squash, winter squash, and all other cultigens.

Sample Collection
Fruit Harvest and Processing Watermelon fruit were harvested when ripe (brown tendril, yellow ground spot, largest fruit, full seeds, and red flesh).Squash were harvested unripe, before seed development, according to market guidelines.Cantaloupe were harvested when rind began to yellow and at half-slip or later, according to market guidelines.Pickling melons (Cucumis melo Conomon Group) were harvested at approximately a 7.5 cm diameter.Cucumbers were harvested at a 5 cm diameter, and mouse melons when easily detached from the peduncle and fading spots were observed.Horned melons were harvested when the skin turned completely orange.Bitter gourds were harvested at an unripe, green stage before seeds matured, and again when the fruit ripened, and the end of the fruit began to dehisce, exposing the inner, bright-red arils.
All samples were collected and stored individually in zip-seal polyethylene bags.Samples were immediately transferred to coolers and stored on ice (up to 6 hr) before being transferred to a −18 • C freezer (up to 5 months) until blending.The portion of fruit sampled varied with cucurbit taxon and crop and ranged from flesh only to flesh and peel, as described in Table 1.
Ripe watermelon and melon samples were partially thawed at room temperature for 30 min, while whole-fruit samples were partially thawed at 4 • C. Variations or details for the specific fruit type are described in Table 1.Depending on cucurbit taxon and crop, the fruit were blended individually or combined by weight or size (using the middle third of fruit).For fruit considered ripe at the marketable stage (harvest time), SSC (soluble solids content), and pH data were taken to ensure only ripe fruit were sampled and combined (watermelon, most melons).Samples were blended for 45-90 s until homogenized into a consistent slurry, using stainless steel blender cups (50-250 mL) and a Waring Laboratory 7010S 1L 2 Speed Blender w/Timer.Variations or details for the specific cucurbit taxon and crop are described in Table 1.

Extraction
Frozen puree samples were thawed at room temperature and weighed as 0.2 g +/− 0.01 g aliquots.Phosphoric acid (1.2 mL, 0.03 M) was added to samples before vortexing for 1 min.Samples were sonicated (30 min), left at room temperature (10 min), and then centrifuged for 20 min at 4 • C, 5700× g; (Eppendorf, Model 5417R).A 1 mL aliquot of supernatant was filtered (17 mm nylon syringe filter, F2513-2, Thermo Scientific) into amber HPLC vials and held at −80 • C until HPLC analysis.z NC BL = North Carolina Breeding Line.y M1: Fruit were sampled by cutting transversely and scooping flesh from the innermost portion; samples were combined by weight.M2: Fruit were sampled by cutting a section of flesh approximately 2in x 2in opposite the groundspot.Rind was removed before bagging and freezing; samples were combined by weight.M3: Whole fruit were harvested and frozen.After thawing, middle thirds of the fruit were sampled, peels were removed, and samples were combined by weight (±0.1 g).M4: Whole fruit were sampled and frozen.After thawing, samples were blended whole and combined by weight (±0.1 g, all but mouse melons and bitter gourd arils).M5: Flesh was blended to loosen seeds from arils; samples were centrifuged to remove seed debris from the sample.Supernatant was tested.M6: All ripe arils were quickly removed from the seeds while still frozen.
x After freezing, outer cuticle was removed.

Quantification by HPLC
Citrulline and arginine concentrations were determined using the method of Jayaprakasha et al. [35] with modifications.

Data Analysis
LS means (least squares means) and standard deviations were generated and simultaneously compared via the procedure for general linear models (PROC GLMMIX) using SAS (SAS Institute, Cary, NC, USA).Locations, replications, and genotypes were all analyzed as random effects.Analysis of variance (ANOVA) was employed to determine the significance of each variance component and their interactions for each of the five traits (soluble solids content, lycopene content, citrulline content, and arginine content, and their combination).Correlations among quality traits were calculated using PROC CORR.

Discussion
Several cucurbits have significant bioactive and nutrient profiles.For example, watermelon has a high concentration of citrulline and lycopene, which impart human health benefits.Citrulline benefits include increased vasodilation, cardiovascular health, and reduced risks for stroke and several cancers [1].Citrulline is also thought to benefit the plants that synthesize it, by mediating drought and salt stress in plants, while also serving as a radical oxygen species scavenger [14,35,36].
Our data indicate that, among the cucurbits sampled, watermelon is 7-41 fold higher in citrulline than other cucurbits (Table 2).These results echo the smaller study done by Fish (2012).We were not able to see clear trends among cucurbit species.Within Cucumis melo types, citrulline ranged from 0.16 to 0.86 gkg −1 fresh weight (Table 2).Citrulline content also differed with tissue type in cucurbits.Citrulline content was much higher in horned melon rind than in flesh (Table 2), yet rind and aril contents were similar in ripe bitter gourd.In contrast, the arginine content of bitter gourd arils was much higher than rind.It may be that conversion of arginine to citrulline in bitter melon is inhibited and/or catabolism of citrulline in rind is enhanced [28].In red-fleshed watermelon, citrulline was reported to be lower in rind than flesh on a fresh (0.8 and 2.0 g/100 g) or dry weight basis (1.6 and 1.8 g/kg) [22].The difference was greater in fresh than in dry tissue, perhaps because rind has only half of the sugar content of flesh.
Amounts of citrulline in watermelon relative to other cucurbits were similar between Fish (2012) and this study with watermelon having ~10 times more citrulline than cucumbers, six-nine times more than cantaloupe, and 41 times more than squash (in this study).Casaba-type melon was highest in citrulline among the other cucurbits.Mouse melon and horned melon may also be interesting sources of the arginine family of amino acids, perhaps because of their large seed (or aril) content relative to mesocarp.In watermelon, arginine but not citrulline was found in seeds [31].Several studies on seeds and seedlings indicate that arginine's plethora of roles includes reserves of easily mobilized nitrogen, not as a source of arginine for NOS [19].Investigation of the physiology of these fruits could be interesting for seed biologists in germination efficacy and plant ecologists for dispersal studies.
The negative correlations of SSC to citrulline and cit+arg across cucurbits contradicts the moderate positive correlations reported previously in a watermelon heritability study [37].Given the wide array of cultigens, correlations here may be affected by our choice of cucurbit species to test.Investigation of specific genera, species, and types may be warranted to pursue meaningful relationships between the arginine family amino acids and ripeness parameters.
Citrulline and arginine concentrations in cucurbits can be influenced by extraction methods, ripeness, and water activity in fresh fruit and value-added products.In our study, the extraction method employed for all cucurbits was optimized for extraction using watermelon tissue, which is low in cellulose and easy to homogenize.Citrulline is both water and lipid soluble and the best extraction may depend on obtaining the smallest particle size during homogenization.Tissues like horned melon rind, bitter gourd, and zucchini may be difficult to process due to firm flesh and rind, though our study reports relatively high citrulline content in horned melon rind compared to flesh.However, percent recovery could not be estimated because spiked samples were not included, and no base-line level of citrulline is known in this fruit.Additionally, environmental components (e.g., drought stress, light stress) seem to be impactful in citrulline and arginine content in cucurbitaceous crops, but not considered in detail in this study.Physiological ripeness is also an important factor to incorporate, since in watermelon, citrulline content peaks at physiological ripeness [26,27,30].Apart from bitter gourd, all cucurbits were studied only at marketable stages, which do not always correlate with physiological ripeness (cucumber, squash, and other immature fruits eaten as culinary vegetables).These limitations warrant further investigation, as they may provide a significant barrier to understanding variation and correlation within and among traits of different cucurbit species.Citrulline has been proposed as the central organic nitrogen carrier in Cucurbitaceous species.This idea, coupled with the hypothesized decrease in citrulline catabolism in the fruit with ripening, may help uncover mechanisms of amino acid transport within the plant.

Conclusions
In cucurbit fruit sampled at a consumer maturity stage, citrulline content was the highest in watermelon.While citrulline was detected in other cucurbits sampled, content was <1%-14% that of watermelon.Arginine content was highest in watermelon and in the arils of ripe bitter gourd.These results show that citrulline is present in many cucurbits, but only watermelon contains significant amounts, while mouse melon, horned melon, and bitter gourd may be interesting sources of arginine-family amino acids.

Table 1 .
Cucurbits evaluated, including seed source and blending method.

Table 2 .
Analysis of variance for four quality traits studied in 2 locations (loc), 3 replications (rep), and 24 cultigens (clt); all factors were considered to be random.
z Degrees of freedom.y Combined concentrations of citrulline and arginine.*Indicates significant F value at P = 0.05.

Table 3 .
Means and standard deviations for five fruit quality traits from 21 cucurbit taxa.Data points (DP) represent number of plots, whose values are averaged (ct.= count of total fruit sampled over all plots).y Combined citrulline and arginine concentrations. z

Table 4 .
LS means for citrulline and arginine content of 21 cucurbit taxa, grouped based on significant differences (P ≤ 0.05) between LS means.

Table 5 .
Pearson correlation coefficients of the five compositional components tested across all 21 cucurbit taxa.Combined citrulline and arginine concentrations.y Soluble solids content.* Indicates significant at P = 0.05. z