Integrated Phytochemical Profiling, GC-MS Characterization, and In Silico, In Vitro Evaluation of Synergistic Antimicrobial, Antioxidant, and Anti-Inflammatory Activities of Morus alba Bark and Pinus densiflora Extracts with Methyl Gallate

The growing challenge of antibiotic resistance and inflammation-related disorders calls for safe, multi-target therapeutic strategies. <i>Morus alba</i> (MOAL) and <i>Pinus densiflora</i> (PIDE) are known for their medicinal properties, yet their combined potential with methyl gallate (MG) has not been fully explored. In this study, the phytochemical composition of MOAL and PIDE was characterized using GC-MS, and their combined antimicrobial, antioxidant, and anti-inflammatory activities were evaluated. Hydroethanolic extracts were prepared and assessed for antioxidant activity (DPPH assay), antibacterial activity (disk diffusion, MIC, time kill), and nitric oxide (NO) suppression in Lipopolysaccharide (LPS)-stimulated macrophages, alongside MTT cytotoxicity screening. MOAL exhibited a higher extraction efficiency, reaching 500 mg/mL at 4 h, whereas <i>Pinus</i> achieved 450 mg/mL at the same time point. Both exhibited a diverse and abundant phytochemical profile. The optimized blend (MOAL:PIDE:MG, 1:1:0.1) demonstrated significantly enhanced bioactivity, with over 90% DPPH scavenging with the low IC₅₀ value (66.62 mg/mL), potent inhibition of both Gram-positive and Gram-negative bacteria, and the strongest effect against <i>Staphylococcus aureus</i> (264 μg/mL). Time-kill assays confirmed rapid bactericidal action, and NO production was reduced by approximately 75% without cytotoxicity. Molecular docking identified a lead multi-target compound exhibiting strong binding affinities to COX-2, TNF-α, and Keap1, supporting its observed anti-inflammatory and antioxidant potential. These findings highlight the promise of synergistic phytochemical formulations as broad-spectrum, multifunctional therapeutic candidates, supporting further in vivo and clinical validation.