Optimization of Hot and Cold‐Water Extractions of <i>Cistus salviifolius</i> L. for Enhanced Phenolic Content and Antimicrobial Efficacy

ABSTRACT This study optimized cold‐ and hot‐water extraction methods for Cistus salviifolius aerial parts using response surface methodology, based on three‐level and three‐factor Box–Behnken design, to maximize phenolic content and antibacterial activity against Staphylococcus aureus and Pseudomonas aeruginosa . The optimization process systematically evaluated the effects of extraction time, temperature, and water‐to‐plant material ratio on total phenolic, flavonoid, and tannin content, as well as bacterial growth inhibition. The extraction method critically influenced the resulting phytochemical profiles. Hot‐water extract demonstrated superior efficiency for overall polyphenol recovery, characterized by a higher flavonol content likely derived from the thermal hydrolysis of ellagitannins. Conversely, cold‐water extract yielded a distinct chemical profile rich in ellagitannin isomers, including punicalagin and cistusin. These compositional variations led to divergent antibacterial efficacies; hot‐water extract was more potent against P. aeruginosa , whereas cold‐water extract exhibited superior bactericidal activity against S. aureus . Significantly, cold‐water extract displayed a remarkable, previously undocumented capacity to disaggregate mature biofilms. The study concludes that while hot water extraction offers a more comprehensive recovery of bioactive compounds, cold‐water extraction can be specifically tailored to yield extracts with potent, targeted activity against S. aureus and its biofilms, highlighting the importance of selecting the appropriate extraction method for desired therapeutic outcomes.