Pumpkin Seeds Harbor Hidden Agonists: Adenosine-Mediated A1 Receptor Activation and Antioxidant Activity

Hydroethanolic Cucurbita pepo seed extracts are traditionally used for alleviating lower urinary tract symptoms (LUTS), yet their mechanisms remain unclear. Adenosine, a purine nucleoside involved in neuromodulation and smooth muscle relaxation, was recently identified in C. pepo seeds. Since A1 adenosine receptors (A1AR) suppress parasympathetic bladder overactivity by inhibiting acetylcholine (ACh) release, we investigated to which extent purines from pumpkin seed extracts contribute to A1AR activation. Complementary antioxidant capacity was assessed using the 2,2-diphenyl-1-picrylhydrazyl (DPPH) assay. Three hydrophilic seed extracts containing different adenosine levels (0.60–1.18 mg/g dw) were evaluated for agonist activity using a cAMP inhibition assay. The most active extract showed an EC50 of 40.22 µg/mL. Selective removal of adenosine shifted the dose–response curve rightward, while further elimination of an adenosine derivative increased the EC50 to 212.10 µg/mL, confirming adenosine as the principal active compound. Guanosine and inosine did not exhibit A1AR agonist or allosteric effects. All samples exhibited measurable but weak antioxidant activity (IC50 = 1.02–4.19 mg/mL), consistent with their low total phenolic content. These findings underscore the importance of accounting for naturally occurring agonists in plant extracts to avoid overestimating receptor-mediated effects in vitro which are not translatable in vivo.