Echinacea species is provided as dietary supplements for various infectious and immune related disorders and has a potential role in cancer prevention. The aim of this study was to optimize the extraction of total flavonoids using different extraction methods and investigate the cytotoxic effects on various cancer cell lines (CaCo-2, MCF-7, A549, U87MG, and HeLa) and VERO (African green monkey) as a non-cancerous cell line. Box-Behnken statistical design was used to evaluate the effect of pressure (100-200 bar), temperature (40-80 degrees C) and ethanol as co-solvent (6-20 wt%) at a flow rate of 15 g/min for 60 min in supercritical CO2 extraction and the effect of temperature (60-100 degrees C), time (5-15 min) and power (300-900W) in microwave-assisted extraction. Optimum extraction conditions were elicited as 300 bar, 80 degrees C and 13% co-solvent yielding 0.472 mg rutin equivalent total flavonoids/g extract in SC-CO2 extraction, whereas 60 degrees C, 10 min and 300W yielded the highest (0.202 mg rutin equivalent) total flavonoids in microwave-assisted extraction. Additional trials with subcritical water (0.022 mg/g) and Soxhlet extraction with methanol (0.238 mg/g) yielded lower flavonoid contents. The exposures upto 50 mu g/ml of extracts revealed no significant inhibition on the proliferation of both tested cancer cells and healthy VERO cells. (C) 2014 Elsevier B.V. All rights reserved.