2.3. Ultrasound-Assisted Extraction

Firstly, the solid–liquid extraction of EOP was performed in an ultrasonic bath (Ultrasonic, J.P. Selecta, Barcelona, Spain), which was operated in continuous mode at a power of 100 W and a frequency of 40 kHz. The milled EOP was added to different acetone–water solutions in 250 mL ISO flasks (POBEL, Madrid, Spain). The total extraction yield, TPC, TFC, and antioxidant activity were evaluated. The EOP acetone extraction was performed according to a Box–Behnken experimental design (BBD) with 17 experiments in random order, including five central points, which allowed for the determination of the optimal extraction conditions based on the desirability function. As operational variables, the effect of the solid loading (2–15%, w/v), extraction time (10–60 min), and acetone concentration (20–80%, v/v) were studied (Table 1). The natural and coded values of these factors are presented in Table 1.

Uncoded and coded values of the factors studied by a Box–Behnken design and a two-level factorial design using bath (B)- and probe (P)-type ultrasound-assisted extraction (UAE), respectively.

¹ Maximum value allowed by the device and ultrasound probe.

In addition to the control of the conditions at room temperature (without the ultrasonic action and without agitation) was carried out to check the efficiency of the ultrasonic extraction at the conditions of the central points (8.5% solid loading, 35 min, and 50% acetone).

The samples were not cooled; therefore, although the experiments were initiated in the bath at room temperature (26 ± 4 °C), the temperature increased during extraction due to the effects of sonication (up to 46 °C). Accordingly, the temperature reached at the end of each assay was measured. After each extraction, the samples were vacuum-filtered, and around 76% of the volume was recovered. An aliquot of the extracts was filtered with a syringe filter (nylon; 0.45 μm pore size) (SinerLab Group, Madrid, Spain) and stored at −20 °C until analysis. A portion of the extracts was dried at around 45 °C in an oven (Memmert, Schwabach, Germany) for 24 h, and another one was directly frozen and freeze-dried till room temperature using a Noxair freeze-drier (Barcelona, Spain). The remaining powder was redissolved in a 40% acetone solution using the same volume evaporated before analysis.

Additionally, to obtain the extraction yield, another portion of 1 mL of each extract was dried at 105 °C to constant weight. All samples were measured in triplicate, and the extraction yields were expressed as g of extract/100 g of EOP.

Moreover, based on the optimal conditions obtained by the BBD and to shorten the extraction time, a two-level factorial design (FD) was performed on a probe-type ultrasound (Branson SFX150, Ultrasonics Corporation, Brookfield, CT, USA) (power: 150 W; frequency: 40 kHz) working in a continuous mode. For the extraction, 250 mL ISO bottles were used as before, and a 3.17 mm diameter microtip was immersed 1 cm deep into the sample. For this purpose, the solid loading and the acetone percentage were set at the optimal conditions and the amplitude (30–70%) and extraction time (2–12 min) were varied. The temperature at the beginning and the end of the assays was also measured, with a maximum increment of 26 °C (Table S1). The extracts were filtered as before for further analyses, and a mean volume of around 75% was recovered.