Anti-typhoid-like salmonellosis activity of Phyllanthus amarus Schum. & Thonn. (Phyllanthaceae) leaves extract: UHPLC-ESI-DAD-MS profiling, in vitro and in vivo efficacy assessment, and molecular dynamics simulations targeting SseK3

Phyllanthus amarus Schum. & Thonn. (Phyllanthaceae) has been traditionally employed for its antibacterial properties. The emergence of multidrug-resistant Salmonella strains has necessitated the urgent development of alternative therapeutic interventions. This study was designed to investigate the anti-typhoid-like salmonellosis activity of P. amarus leaf extract. Compounds were isolated using column chromatography and fully structurally characterized using modern NMR and HRMS data, while some of them were identified by UHPLC-ESI-DAD-MS analysis. The activity of crude extract against Salmonella strains was performed in vitro , and the curative effect was evaluated in vivo against typhoid-like salmonellosis in infected rats. The invasion gene ( invA ) was detected by singleplex PCR pre- and post-treatment. Molecular docking and molecular dynamics simulations were employed to predict binding interactions with the Salmonella effector protein SseK3 (PDB ID: 6EYT). Isolated compounds (1 – 13) comprised triterpenoids (lupeol, α-amyrin, friedelin, 3α-friedelanol, simiarenol, betulinic acid, oleanolic acid, ursolic acid, maslinic acid), phytosterols (stigmasterol, β-sitosterol and their glucosides), a coumarin (isoscopoletin), and a flavonoid (kaempferol) and eleven compounds were tentatively identified by UHPLC-ESI-DAD-MS included diterpene, sesquiterpene and triterpenoid. The extract exhibited potent antibacterial activity with minimum inhibitory concentrations (MIC) of 31.25–62.5 μg.mL -1 against S. typhi , S. typhimurium , and S. enteritidis . In vivo administration significantly ameliorated hepatorenal dysfunction and restored normal histological architecture in liver, kidney, jejunum, and splenic tissues. In silico analyses revealed favourable binding affinities between key bioactive compounds and SseK3. P. amarus leaf extract contains multiple bioactive phytochemicals demonstrating significant anti-typhoid-like salmonellosis activity through multi-target mechanisms, providing scientific validation for its ethnomedicinal application and supporting its potential development as a complementary therapeutic agent against typhoid-like salmonellosis. • P. amarus extract and its compounds inhibited Salmonella spp. in vitro • P. amarus extract demonstrated anti-typhoid-like salmonellosis efficacy in vivo • P. amarus extract repaired the damages caused by S. typhimurium in the infected rat model • Kaempferol, cyanidenon, and quercetin showed binding affinities to SseK3 • SseK3 could be a P. amarus extract therapeutic target in treating Salmonellosis