Comparison of Free Citrulline and Arginine in Watermelon Seeds and Flesh

Citrulline, a non-essential amino acid found in watermelon, helps promote vasodilation in humans by stimulating the nitric oxide system.  Arginine is a primary amino acid used in the nitric oxide system of mammals. Citrulline has been reported in both flesh and rind of watermelon, and reports vary widely regarding relative content in fruit grafted or seeded or seedless watermelon cultivars.  Little is known about watermelon seeds.  In this study, watermelon seeds (embryos) of various types and cultivars were analyzed for free citrulline and arginine and compared to amounts found in grafted and not grafted seedless watermelons.  Samples were extracted with 0.03M phosphoric acid and supernatant injected onto a Hitachi HPLC equipped with DAD detector (195 nm), a Gemini 3u C18 110A 250x4.6 mm column, and using a mobile phase of 0.015M phosphoric acid with flow rate of 0.5 ml/min at 25 °C for 30 min.  A subset of samples was run by amino analyzer to verify results. Free amino analysis was done using 0.5 ml supernatant diluted with 0.02N HCl to a final volume of 1 ml and analyzed using a Hitachi Model L-8900 Analyzer, 570 and 440 nm and an analytical column (model 2622SC PF; 40-mm length, 6.0-mm ID). Separation was done by using a borate buffers and a 30 to 70°C gradient buffer.  Ninhydrin was used for instrumental postcolumn derivatization. Amino acid standard curves of 1 to 5 nM were made with serial dilutions of an amino acid standard mixture containing 2.5 pmol of 18 amino acids in 0.02 N HCl. Non-treated watermelon seeds of heirloom, hybrid seeded, or seedless types were purchased from seed companies.  Grafted and not grafted seedless watermelons (‘Fascination’ with ‘Carnivor’ rootstock) were obtained from field plantings in SC.  The citrulline and arginine contents of watermelon flesh was much higher than that of seeds (2-2.5 vs 0.03 to 0.1 mg/g fresh weight), and 7-9 vs 0.3 to 1 mg/g fwt, respectively.  The ratio of citrulline to arginine was much less in seeds (0.04 to 0.33) compared to flesh (2 to 4).  The different citrulline and arginine ratios between seeds and placental tissue most likely reflect the very different roles these tissues play in the reproduction system of watermelon.  Citrulline can act as a storage mechanism for nitrogen while arginine is a nitrogen storage form in seeds. Citrulline content of flesh was less from grafted watermelons than from fruit of ungrafted plants.