Vladic, JelenaRadman, SanjaBesu, IrinaStanojkovic, TatjanaZloh, MireJerkovic, IgorKaradzic Banjac, MilicaIvkovic, MilenaPereira, HugoGouveia, Luisa2026-02-032026-02-032025-11-282168-0485PURE: 151571987PURE UUID: 999cca69-4043-46a6-9107-d65870fc6763Scopus: 105024874320WOS: 001628242600001http://hdl.handle.net/10362/199948Publisher Copyright: © 2025 American Chemical SocietyThis study explores the impact of enzymatic (ENZ), microwave (MW), and ultrasound (US) pretreatments on supercritical CO2 (scCO2) extraction efficiency, chemical composition, and cytotoxic activity of Tetraselmis sp., Tetradesmus obliquus, and Chlorococcum sp. Pretreatments significantly enhanced extraction yields, with ENZ being most effective for Tetraselmis and Chlorococcum, and MW for T. obliquus. UPLC-HRMS profiling revealed species- and pretreatment-specific shifts: ENZ and US improved pigment recovery in Tetraselmis, while MW enriched carotenoids and chlorophyll derivatives. In Chlorococcum, MW boosted pigment diversity, whereas ENZ and US favored fatty acid derivatives in the extracts. Multivariate analysis confirmed significant compositional changes, particularly after ENZ and MW pretreatments. Tetraselmis extracts, especially those pretreated with MW, exhibited the strongest cytotoxic activity and highest selectivity indices against HeLa and MDA-MB-453 cancer cell lines. Correlation analysis identified compounds such as 2,3-dihydroxypropyl stearate, fucoxanthin, and (3β)-3-hydroxystigmast-5-en-7-one as strongly linked to cytotoxicity. Molecular docking further showed that abundant compounds in Tetraselmis extracts have high predicted affinities for cancer-related targets (e.g., BCL2, EGFR, PDK1). The results suggest that cytotoxic effects arise from both specific bioactive compounds and their synergistic interactions. These findings show that pretreatments can purposefully tune scCO2 extracts and provide a data-driven basis for designing more sustainable microalgal extraction workflows.181898059engChlorococcumCytotoxicityPretreatmentssupercritical COTetradesmusTetraselmisUHPLCGeneral ChemistryEnvironmental ChemistryGeneral Chemical EngineeringRenewable Energy, Sustainability and the EnvironmentSDG 3 - Good Health and Well-beingSDG 7 - Affordable and Clean Energyjournal article10.1021/acssuschemeng.5c09964https://www.scopus.com/pages/publications/105024874320https://www.webofscience.com/wos/woscc/full-record/WOS:001628242600001