3.4. Procedure for Microwave-Assisted Extraction (MAE)

MAE of bioactive compounds was carried out following the experimental plan given in Table 6 with 50% aqueous ethanol solution as a solvent. Extraction was performed on a Milestone flexiWAVE Microwave apparatus using a closed-vessel system (Milestone Srl, Sorisole (BG), Italy) and setting the maximum microwave power at 700 W. In order to maintain the constant temperature, the power was not applied constantly during the experiment. Pre-heating time was set for 2 min and cooling time for 10 min. A light yellow to a dark brown solution was obtained after MAE. Then the vessels were cooled down to room temperature, evaporated on SpeedVac (SPD1030, Thermo Scientific, Waltham, MA, USA) and dry extracts were stored in the +4 °C until analyzed. The extraction yield was calculated using Equation (9) where W₁ represents the mass of the obtained extract after solvent evaporation and W₂ represent the mass of plant material (tobacco leaves and waste) used for the extraction process.