0.2 μm ptfe syringe filters

The aerial parts of M. officinalis, O. basilicum, and T. vulgaris were supplied by Nutraceutical SRL (Brazov, Romania). The raw material was packed in sealed plastic bags and stored in the dark at room temperature until analysis. EOs were obtained by solvent-free microwave extraction (SFME) with a NEOS^® apparatus (Milestone, Sorisole, Italy) using methodology previously employed by Moro et al. [29 (link)]. In total, 150 g of the plant was placed in the NEOS reactor with 250 mL of Milli-Q water to wet the dry plant sample. As its name implies, the technique does not use a solvent, but the plant must contain the water that drags the essential oils when heated by microwaves, the principle with which this equipment works. Exhaustive extraction of EOs was then performed (35 min): the extraction power was set at 600 W (5 min) and then at 250 W (30 min), and the temperature was monitored with an infrared sensor for avoiding overheating (95 ± 5 °C). The oil was collected in the device with graduation marks available to the equipment itself for this purpose. For the antimicrobial activity test, the EOs were filtered using 0.2-μm PTFE syringe filters (Millipore, Madrid, Spain) to ensure the absence of microorganisms before use.

The SFE-CO₂ experiments were performed as previously described using a Waters Prep Supercritical Fluid Extraction system (SFE-100) [19 (link)]. For the extraction, 5 g of SC stem powder was mixed with sea sand at a ratio of 1:2. The SFE step was carried out in dynamic mode at 40 °C using a total flow rate of 22 g/min of CO₂ plus 15% ethanol. The extraction pressure was set at 150 bar during the 1 h process. The collected extract was concentrated in a water bath at 40 °C using a rotary evaporator, and was reconstituted in ethanol (co-solvent extraction) up to a concentration of 1000 mg/L. The extract was filtered through 0.2 μm PTFE syringe filters (Millipore, Bedford, MA, USA) and then stored at −20 °C until analysis.