Comprehensive evaluation of the anti-inflammatory potential of cucurbita maxima leaf extract. LC–MS phytochemical profiling coupled with in vitro, in vivo, and in silico approaches

Ethnopharmacological relevance: Cucurbita maxima (pumpkin) leaves have been traditionally used in folk medicine for the treatment of inflammation, fever, and oxidative stress–related disorders. However, scientific validation of these claims remains limited. This study aimed to evaluate the anti-inflammatory, antioxidant, and protective effects of C. maxima leaf aqueous extract and to elucidate its phytochemical composition and molecular mechanisms of action. Materials and methods: The phytochemical profile of the aqueous extract was determined using UPLC–ESI–MS/MS analysis. In vivo anti-inflammatory activity was assessed in a benzylthiouracil-induced inflammation model by measuring white blood cell (WBC) counts, C-reactive protein (CRP) levels, and histopathological changes in liver and kidney tissues. In vitro anti-inflammatory potential was evaluated through the protein denaturation inhibition assay, using diclofenac as a reference. Pharmacokinetic and toxicity properties of major compounds were predicted using ADMET tools, while molecular docking studies were performed to evaluate interactions with COX-1 and COX-2 enzymes. Results: UPLC–ESI–MS/MS analysis revealed a complex mixture of polyphenols, with caffeic acid (56.04%), rutin (9.25%), ferulic acid (8.79%), and myricetin-3-rhamnose (8.62%) as the main constituents. The extract significantly reduced inflammation by normalizing WBC counts (from 7.2 to 4.5 × 109/L), lowering CRP levels (from 630 to 220 mg/L), and protecting liver and kidney tissues. The extract also inhibited protein denaturation in vitro (IC50 = 2.976 mg/mL) compared to diclofenac (IC50 = 0.718 mg/mL). Molecular docking revealed that major flavonoids exhibited strong binding affinities toward COX-1 and COX-2, often surpassing diclofenac, supported by stable hydrogen bonding and hydrophobic interactions. ADMET and toxicity predictions indicated good intestinal absorption for several major compounds, absence of predicted hepatotoxicity or skin sensitization, and generally low acute toxicity, with high predicted LD50 values and no mutagenic risk for most constituents. Conclusion: These findings support the traditional use of C. maxima leaves in folk medicine for the management of inflammation-related conditions, fever, and associated oxidative stress. Further isolation of the key active constituents and clinical evaluation are recommended to confirm their therapeutic efficacy.