Metabolomic and network Pharmacology insights into the anti-inflammatory and anti-urolithiatic potential of Ficus religiosa seeds

Urolithiasis remains a persistent global health challenge with limited therapeutic options and high recurrence rates. The present study investigated the anti-inflammatory and anti-urolithiatic potential of Ficus religiosa seed extracts through a comprehensive experimental and computational framework. In vitro assays demonstrated that the ethyl acetate extract significantly inhibited red blood cell hemolysis (IC₅₀: 346.63 ± 1.303 µg/ml) and protein denaturation (IC₅₀: 524.10 ± 1.29 µg/ml), highlighting strong anti-inflammatory effects comparable to diclofenac and acetylsalicylic acid. In the semipermeable membrane model, the ethyl acetate extract exhibited dose-dependent calcium oxalate dissolution, achieving 31.61 ± 0.06% to 84.56 ± 0.03% from 100 to 500 µg/ml with an IC₅₀ of 179.99 ± 0.34 µg/ml, demonstrating efficacy comparable to that of the reference drug Cystone and surpassing it at higher concentrations. Metabolomic profiling by GC-MS and LC-MS identified 151 phytoconstituents, including flavonoids (quercetin 3'-methyl ether, apigenin), phytosterols (4-cholestenone), and fatty acid esters. Multivariate analyses confirmed solvent-specific clustering, with flavonoid-rich extracts showing superior activity. Network pharmacology integrated these findings, revealing 173 overlapping targets between the identified phytoconstituents and urolithiasis and/or inflammation-associated genes. Functional enrichment analysis highlighted the PI3K-AKT, MAPK, NF-κB, and calcium signaling pathways as key modulatory axes. Molecular docking further validated the high binding affinities of flavonoids and phytosterols with central regulatory proteins such as PI3K, AKT1, IKKβ, MMP-9, and CaMKII, supporting their roles in inflammatory suppression and extracellular matrix remodeling. Overall, this study validates the ethnomedicinal use of Ficus religiosa seeds for the treatment of urinary and inflammatory disorders and highlights their bioactive, multitarget therapeutic potential for managing urolithiasis, warranting further preclinical and clinical evaluation.