Antioxidant and antibacterial potency of Pleurotus citrinopileatus: by GC-MS characterization, bioactive compound profiling, synergy analysis, and In-Silico validation of microbial target interactions.

The rapid development of antimicrobial resistance has intensified the demand for safe, multifunctional, and mechanistically validated natural antimicrobials beyond conventional antibiotics. Edible mushrooms, particularly Pleurotus species, represent an underexplored reservoir of bioactive metabolites; however, most studies remain limited to preliminary bioactivity screening without mechanistic confirmation. In this study, the antibacterial and antioxidant potential of Pleurotus citrinopileatus was systematically assessed through integrated experimental and in-silico methods. Aqueous (AEPC), ethanolic (EEPC), and methanolic (MEPC) extracts were subjected to GC-MS profiling, antioxidant assays, antibacterial activity, synergistic interaction study of Mushroom with plants, molecular docking, and 100-ns molecular dynamics (MD) simulations. GC-MS analysis identified 9, 19, and 25 bioactive compounds in AEPC, EEPC, and MEPC, respectively, with sterols such as ergosterol and cholesta-8,24-dien-3-ol predominating in MEPC. Methanolic extract exhibited the strongest antioxidant activity (91.18% DPPH scavenging; IC₅₀ ≈ 8 µL/mL), while EEPC demonstrated superior antibacterial efficacy, producing pronounced inhibition zones against Proteus vulgaris, Pseudomonas aeruginosa, and Staphylococcus aureus. All extracts showed bactericidal activity with MIC values ranging from 25-150 mg/mL and MBC/MIC ratios ≤ 2. Synergistic combinations with neem and tulsi enhanced antibacterial zones by 5-7 mm and increased antioxidant activity up to 92.28%. Molecular docking revealed strong binding affinities (- 7.2 to - 11.9 kcal/mol) of key sterols against multiple bacterial targets, comparable to ciprofloxacin, with ergosterol exhibiting the highest affinity toward P. aeruginosa alginate lyase. MD simulations confirmed the stability of protein-ligand complexes, supported by low RMSD, stable radius of gyration, and persistent hydrogen-bond interactions. Overall, P. citrinopileatus emerges as a promising natural reservoir of antibacterial and antioxidant agents, offering a synergistically enhanced, mechanistically validated strategy for combating pathogenic bacteria. These findings support the potential of Pleurotus citrinopileatus as a natural source of antioxidant and antibacterial agents, warranting further toxicological and in vivo investigations for therapeutic applications.