Sequential soxhlet fractionation of Cistus ladanifer L. reveals a polarity-activity gradient: GC-MS profiling, multimechanistic bioactivity, and in silico identification of antifungal and antibacterial lead compounds.

INTRODUCTION: AMR and oxidative stress are among the most pressing biomedical challenges, requiring structurally novel multi-target bioactive molecules. Cistus ladanifer L. is an ethnobotanical resource whose polarity-resolved phytochemical profile remains largely incomplete. METHODS: C. ladanifer leaves (Rmilate forest, Tangier) were fractionated by sequential Soxhlet extraction (n-hexane, chloroform, ethanol, water) and evaluated by phytochemical quantification, FTIR, GC-MS, four antioxidant assays (DPPH, ABTS, FRAP, ORAC), antibacterial well-diffusion/microdilution (S. aureus, B. subtilis, E. coli, P. aeruginosa), antifungal assays (T. rubrum, M. canis, B. cinerea, F. oxysporum), and ADMET/docking of four marker compounds (EOMCP, DHMAQ, esculin, diosgenin). RESULTS: A strict polarity-dependent gradient was observed, with the aqueous fraction showing the highest phenolic (140 mg GAE/g DW), flavonoid (148 mg QE/g DW), and tannin (210 mg TAE/g DW) content; strongest antioxidant activity (DPPH IC50 = 0.09 mg/mL; FRAP = 65.25 mg TE/g DW); lowest MIC (0.25 mg/mL, bactericidal, MBC/MIC ≤4); and highest antifungal inhibition (86.0% against T. rubrum at 4 mg/mL). Diosgenin showed the strongest docking affinity against CYP51B (5FRB, -12.7 kcal/mol), exceeding the reference ligand. DISCUSSION: These findings establish a polarity-activity framework supporting C. ladanifer's valorization as an antioxidant, antibacterial, and antifungal resource, pending cytocompatibility and in vivo evaluation.