Deep Eutectic Solvents and Wall-Breaking Technique: A New Frontier in the Extraction of Oleuropein and Flavonoids from Olive Leaves with Superior Antioxidant and Antitumor Potential.

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.