Membrane-disrupting antibacterial activity of Artocarpus lacucha bark extract: a mechanistic study through...

Artocarpus lacucha Buch. -Ham. (syn. Artocarpus lakoocha) (A. lacucha) has been traditionally used in various ethnomedicinal approaches for treating infectious diseases, gastrointestinal ailments, and skin disorders. Particularly, its bark has been employed to manage microbial infections and wounds. Despite its widespread traditional use, the antibacterial potential and the possible mechanism of action of A. lacucha bark remain underexplored and scientifically undocumented.

This study aimed to evaluate the antibacterial activity of the hexane extract of A. lacucha bark obtained through different extraction methods and to investigate the underlying mechanism of action of the most potent extract, focusing on membrane-targeted bactericidal effects using both biological and computational approaches.

The bark of A. lacucha was extracted using three techniques: maceration (AL-MAC), ultrasound-assisted extraction (AL-UAE), and Soxhlet extraction (AL-SOX) with n-hexane as the solvent. Phytochemical characterization of the extracts was carried out using GC-MS. Antibacterial activity was assessed against E. coli, P. aeruginosa, S. aureus, and B. subtilis through agar disc diffusion, antibacterial susceptibility determination, and time-kill kinetics. Mechanistic studies of membrane disruption included outer and inner membrane permeability assays, viz., 1-N-phenylnaphthylamine (NPN) and o-Nitrophenyl-β-D-galactopyranoside (ONPG), protein leakage analysis, flow cytometry with propidium iodide (PI) staining, and field emission scanning electron microscopy (FESEM) imaging. Phytochemicals from AL-SOX, identified by GC-MS, with maximum yields and drug-likeness were further validated for their molecular mechanism to disrupt the bacterial membrane through target prediction, molecular docking, and molecular dynamics (MD) simulation.

AL-SOX exhibited the highest extraction yield (10.25 %) and contained a diverse array of bioactive compounds, including α-amyrin acetate, taraxasterol acetate, and vasicinolone. Among all extracts, AL-SOX demonstrated the most potent antibacterial activity, particularly against B. subtilis (MIC: 15.62 μg/mL, MBC: 31.25 μg/mL). Time-kill studies revealed a dose- and time-dependent bactericidal effect, with complete eradication of B. subtilis at 2 × MIC. NPN and ONPG assays confirmed outer and inner membrane disruption in Gram-negative bacteria, while protein leakage assays showed significantly higher cytoplasmic leakage in B. subtilis. Flow cytometry analysis confirmed membrane compromise with a marked increase (20.8 %) in PI-positive cells. FESEM analysis revealed morphological damage in B. subtilis cells treated with AL-SOX, validating the membrane-disrupting action of AL-SOX. In silico analyses revealed stable binding of vasicinolone and α-amyrin acetate to FabH, insinuating its role as a potential molecular target underlying the antibacterial activity of AL-SOX.

This integrative study demonstrates that the hexane extract of A. lacucha bark exhibits promising antibacterial action, and its putative mechanism could be direct membrane disruption, along with possible FabH inhibition, thereby validating its traditional use. The findings highlight the potential of A. lacucha as a promising source of natural antibacterial agents and underscore the importance of integrating ethnopharmacological knowledge with modern mechanistic insights to address the growing threat of antimicrobial resistance.