LC-MS/MS Profiling and Evaluation of Antibacterial, Antivirulence, Antioxidant, and Anti-Inflammatory Activities of...

Pinus halepensis Mill. is one of the most common Mediterranean trees, known for its ecological and medicinal importance. In this study we investigated the phenolic composition of P. halepensis methanolic and aqueous bark extracts and evaluated their antioxidant, anti-inflammatory, antibacterial, and antivirulence activities. LC-MS/MS analysis revealed quinic acid and protocatechuic acid as the predominant compounds. Both extracts showed strong antioxidant activity, with IC₅₀ values ranging from 1.45 to 2.00 µg/mL, while the methanolic extract demonstrated greater anti-inflammatory effect (86.15%) compared to the aqueous extract (65.28%). Antibacterial assays demonstrated significant inhibitory effects, with MIC values reaching 7.81 µg/mL for the methanolic extract and 15.62 µg/mL for the aqueous extract. Furthermore, both extracts reduced biofilm formation by at least 50% and significantly attenuated quorum sensing-regulated virulence traits in Pseudomonas aeruginosa strains, including pyocyanin production and swimming/swarming motility, with inhibition rates of up to 70% and 50%, respectively. Molecular docking revealed strong binding affinity of all tested compounds to P. aeruginosa flagellin FliC, with quercetin exhibiting the highest binding stability and protocatechuic aldehyde displaying the strongest hydrogen bond energy, suggesting their potential as FliC inhibitors. These findings highlight P. halepensis Mill. as a promising bioactive source compounds for therapeutic agents against antibiotic-resistant bacteria.