Abstract
The main objectives of this study were to develop an optimized green extraction process to obtain high contents of oleuropein and flavonoids from olive leaves. A deep eutectic solvent (DES) combined with wall-breaking extraction (WBE) was employed. A DES composed of choline chloride and ethylene glycol in a 1:2 molar ratio with 30% moisture content outperformed lactic acid and methanol as extraction solvents. The optimal conditions, determined by response surface methodology, were 30% moisture content, 140 s of wall-breaking time, and a 230 mL/g liquid-solid ratio. Under these conditions, 88.87 mg/g DM oleuropein, 4.57 mg/g DM luteolin-7-O-glucoside, and 114.31 mg RE/g total flavonoids were obtained. Among three olive varieties (Arbosana, Arbequina, and Picholine) cultivated in China, young Picholine leaves exhibited the highest contents. The Picholine-enriched extract demonstrated higher antioxidant activity (ABTS(•+) 155.10 mg/mL, DPPH(•) 44.58 mg/mL) compared to other DES-based extracts, although it was lower than that of purified compounds. Furthermore, the CCK-8 assay revealed significant inhibition of Eca-109 human esophageal cells by the Picholine-enriched extract at 25 µg/mL for 24 h, compared to Het-1A cells. This process effectively recovers bioactive compounds from olive by-product, and shows potential for applications in nutritional supplements, cosmetics, and the food industry.