In vitro anti-inflammatory potential and in vivo anti-arthritis activities of Ximenia caffra extract on antigen-induced arthritis in rats

Abstract The current study estimated the in vitro anti-inflammatory activity and in vivo anti-arthritic activities of the aqueous ethanolic extract of Ximenia caffra ( X. caffra ) seeds extract. It was hypothesized that X. caffra seeds extract, rich in phytochemicals that could modulate inflammatory pathways and protect joint tissues in an antigen-induced arthritis rat model. The chemical composition of X. caffra seeds extract was examined using liquid chromatography high-resolution mass spectrometry (LC-HRMS). Ximenia caffra seeds extract showed promising in vitro anti-inflammatory activity with an IC 50  = 26.01 ± 0.85 µg/ml. To evaluate in vivo efficacy, antigen-induced arthritis was established in rats using Complete Freund’s Adjuvant (CFA), followed by subcutaneous administration of X. caffra extract at doses of 26, 50, and 100 mg/kg body weight, alongside a standard drug control [Methotrexate (MTX), 0.3 mg/kg] in separate groups of animals. Anti-arthritic effects were assessed by measuring joint diameter, arthritic score, body weight, and through histopathological and ultrastructural analyses of joint and muscle tissues as well as osteoclast assessment, cytokine analyses, renal and kidney functions. The optimal dose (26 mg/kg) significantly alleviated arthritis symptoms, restoring joint and muscle morphology toward normal architecture. Ex vivo osteoclast evaluation and flow cytometric apoptosis analysis indicated that X. caffra extract promoted cellular recovery and reduced inflammatory damage. Furthermore, cytokine profiling demonstrated that treatment with X. caffra shifted pro-inflammatory mediators (IL-1β, IL-6, IL-17, IFN-γ) toward an anti-inflammatory balance by elevating IL-4 and regulating IgG1a/IgG2a ratios. Collectively, these results support the hypothesis that X. caffra seeds extract had potent anti-inflammatory and anti-arthritic effects by modulating immune responses and preserving joint integrity, suggesting its potential as a natural therapeutic alternative for rheumatoid arthritis management after further validation.