| Summary: | Olive leaves are a rich source of polyphenols that have beneficial antihypertensive, hypocholesterolemic, cardioprotective, and anti-inflammatory effects. The aim of this study was to compare the efficiency of conventional extraction (CE), microwave-assisted extraction (MWE), and microwave–ultrasound-assisted extraction (MWUE) for the extraction of bioactive molecules from olive leaves using water as a solvent and to define the optimal extraction conditions for all three methods used. CE conditions (temperature, time, magnetic stirrer rotational rate and particle diameter) and MWE extraction and MWUE conditions (microwave power, time, particle diameter, and temperature) were optimized using response surface methodology (RSM) based on the Box–Behnken experimental design. The total polyphenol content and antioxidant activity of all prepared extracts was analyzed and compared. The results showed that MWUE provided the highest amount of total polyphenols (Total Polyphenolic Content (TPC) = 273.779 ± 4.968 mg<sub>GAE</sub> g<sub>d.m.</sub><sup>−1</sup>) and the highest antioxidant activity, which was about 3.1 times higher than CE. Optimal extraction conditions were determined to be 80 °C, 15 min, 200 μm, and 750 min-1 for CE, 700 W, 7.5 min, 300 μm, and 80 °C for MWE, and 800 W, 5 min, 100 μm, and 60 °C for MWUE. Considering the maximum amount of total polyphenols extracted, the results suggest that MWUE is the most effective green extraction process that extracted the highest amount of polyphenols and could be used by the food industry for commercial exploitation of currently unprofitable plant bioactive sources.
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