Dual evaluation of Citrullus colocynthis fruit extract for anti-rheumatic activity: integrating in silico docking and in vivo murine RA model analysis

Rheumatoid arthritis (RA) is a chronic degenerative autoimmune disease, which is characterized by synovial inflammation and cartilage erosion. The present study aimed to explore the therapeutic potential of methanolic extract of Citrullus colocynthis (C. colocynthis) against RA using both in vivo and in silico methods. 36 experimentally adjuvant-induced arthritic mice were divided into six groups (n = 6): negative control, positive control, methotrexate (0.5 mg/kg), and C. colocynthis extract-treated groups (50, 100, and 200 mg/kg). Paw edema, hematological and biochemical parameters, rheumatoid factor, C-reactive protein, pro-inflammatory cytokine expression (TNF-α, IL-1β, IL-6), and joint histopathology were assessed. One-way, two-way, and non-parametric ANOVA were used in RStudio to compare the parameters among the groups. In parallel, GC–MS-identified phytoconstituents were screened for drug-likeness and binding affinity to key inflammatory targets via molecular docking, molecular dynamics simulations, post-simulation MM-GBSA, principal component analysis, and ADMET profiling. The extract at 200 mg/kg significantly reduced FCA-induced paw thickness (1.23 ± 0.04 mm vs. 2.85 ± 0.07 mm in arthritic controls, p < 0.001), lowered C-reactive protein (14.3 ± 0.8 ng/mL), and rheumatoid factor (48.9 ± 2.7 IU/mL), also significantly downregulated TNF-α, IL-1β, and IL-6 expression that was comparable to methotrexate. Results of histopathology revealed the preserved joint architecture with minimal pannus formation. In silico studies showed that trehalose and 3,5-dihydroxybiphenyl were the key molecules with strong binding affinities to TNF-α and dihydrofolate reductase, suggesting their cytokine-modulating potential. This study demonstrated C. colocynthis methanolic extract as a promising, multi-targeted natural therapy for RA.