Activity‐Guided Isolation, Characterization, and Antibacterial Evaluation of <i>Ziziphus Oxyphylla</i> Leaves Extract: Molecular Docking, MD Simulations, and ADMET Studies

Abstract The emergence of antibiotic‐resistant bacteria has necessitated the discovery of new antimicrobial agents. The methanol extract of Z. oxyphylla leaves was fractionated ( n ‐hexane, chloroform, ethyl acetate, n ‐butanol, and aqueous), and the ethyl acetate subfractions (EAF) were screened for antibacterial activity using the agar well diffusion method. Ethyl acetate sub‐fractions (EAF‐1 to EAF‐5), EAF‐4 (caffeic acid) and EAF‐5 (ursolic acid) already known pure compounds, showed significant zones of inhibition (ZOI) against S. aureus (24.0 and 25.0 mm), P. aeruginosa (23.06 and 24.05 mm), and B. bronchiseptica (23.0 and 24.0 mm), comparable to cefepime. Caffeic acid and ursolic acid displayed prominent bacteriostatic (MIC = 0.390 and 0.195 µg/mL) and bactericidal (MBC = 0.781 and 0.390 µg/mL) effects. Molecular docking revealed that caffeic acid exhibited strong binding with DNA topoisomerase II (−8.27 kcal/mol). MD simulation (100 ns) confirmed the stability of the protein‐ligand complex through RMSD, RMSF, Rg, SASA, and H‐bond analyses. Post‐simulation binding free energies were −10.61 kcal/mol (MM/PBSA) and −21.75 kcal/mol (MM/GBSA), confirming thermodynamic stability. ADMET analysis indicated Lipinski compliance; however, ursolic acid exhibited toxicity, necessitating further validation. Overall, caffeic acid demonstrated significant antibacterial potential and stable binding to DNA topoisomerase II, supporting its role as a promising antibacterial agent.