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Brown seaweeds produce useful bioactive substances with high cosmetic and pharmacological values due to the presence of antioxidant derivatives, mainly phlorotannins (PHT), which are of particular interest. A central composite rotatable design (CCRD) with three variables (extraction time, dry material-to-solvent ratio and ethanol concentration) and two responses was performed to optimize the microwave-assisted extraction (MAE) of phlorotannins from Cystoseira sedoides using response surface methodology (RSM). Results showed that the independent variables significantly affected both phlorotannin content and the scavenging capacity. The optimum operating conditions were extraction time, 101.74 sec; dry material-to-solvent ratio, 1:10 g/mL; and ethanol extraction, 50%. Under these conditions, the predicted values of PHT content and radical scavenging activity-IC50 were close to the observed values and were 383.887 µg PGE/g Dm and 18.353 µg/mL, respectively. Characterization of the phlorotannin-rich fraction was conducted by high-performance liquid chromatography and Fourier transform infrared spectroscopy. The evaluation of the anticancer activity against MCF-7 breast cancer cell line showed a potent activity to trigger apoptotic death in more than a half of the MCF-7 cells, with an estimated IC50 value of 78 μg/mL. In addition, this fraction induced a notable growth regression effect on 3D spheroids model in a concentration-dependent manner, with a growth rate of about 1.17, at 200 µg/mL. Abbreviations: CCRD: Central composite rotatable design; Dm: Dry material; DMBA: 2,4-dimethoxy benzaldehyde; DMSO: Dimethyl sulfoxide; DPPH: 1,1-diphenyl-2-picrylhydrazyl; EDTA: Ethylene diamine tetraacetic acid; FBS: Fetal bovine serum; FT-IR: Fourier transform infrared; HPLC: High-performance liquid chromatography; IC50: Half-maximal inhibitory concentration; MAE: Microwave-assisted extraction; Min: minutes; PBS: Phosphate buffered saline; PGE: Phloroglucinol equivalent; PHT: Phlorotannins; PHT-SED: phlorotannins derived from Cystoseira sedoides; PI: Propidium iodide; RSA: Radical scavenging activity; RSM: Response surface methodology; Sec: seconds [ABSTRACT FROM AUTHOR]