Phytochemical Characterization and Integrated In Vitro Cellular Evaluation of Immunomodulatory, Cytotoxic, Genotoxic Activities and In Vivo Assessment of the Antioxidant and Anti-Arthritic Activities of Urtica urens L. Ethanolic Extract

Rheumatoid arthritis (RA) is a chronic autoimmune disease characterized by persistent synovial inflammation, oxidative stress, and progressive joint destruction. Given the limitations and adverse effects of current therapies, medicinal plants represent a valuable source of novel anti-inflammatory agents. This study evaluated the phytochemical composition, antioxidant, immunomodulatory, and anti-arthritic activities of an ethanolic extract of Urtica urens (UUEE). LC-ESI-HRMS analysis identified forty metabolites, mainly hydroxycinnamic acid derivatives, flavonoids, terpenoid glycosides, and oxidized fatty acids. Antioxidant activity was assessed using DPPH, ABTS, and CUPRAC assays together with total phenolic and flavonoid contents. Anti-arthritic activity was investigated by inhibition of heat-induced bovine serum albumin denaturation, while cytotoxicity and genotoxicity were evaluated in HeLa and HaCaT cells. Immunomodulatory activity was assessed by analyzing IL-6 and IL-1β expression in zymosan-stimulated HaCaT cells. Therapeutic efficacy was validated in a Freund’s complete adjuvant-induced arthritis model in Wistar rats. UUEE showed strong antioxidant activity, concentration-dependent inhibition of protein denaturation, low cytotoxicity up to 100 μg/mL, downregulation of pro-inflammatory cytokines, and significant in vivo anti-arthritic effects, reducing oxidative stress, histopathological damage, and arthritis severity while improving hematological and biochemical parameters. These findings support U. urens as a promising source of bioactive compounds for RA management.