2.3. Extraction of Phenolic Compounds

Plants from the first four subcultures were combined and separated into aerial parts and roots. All samples were frozen at −20 °C, lyophilized and homogenized to get a fine powder. Four different solvents were used to perform phenolic extraction, based on different MeOH:water (v/v) ratios, i.e., 40% MeOH (M40), 60% MeOH (M60), 80% MeOH (M80) and absolute MeOH (M100). Briefly, 100 mg of dry weight (DW) of plant materials was mixed with 10 mL of each solvent, vortexed, and subjected to solvent extraction at 60 °C in a water bath for 10 min. Samples were then cooled down to room temperature and sonicated for 30 min, prior to their centrifugation at 3500 rpm for 10 min. The supernatants were then separated, cooled down to room temperature, and filtered through 0.45 µm PTFE membrane filters to obtain phenolic extracts. Extracts were stored at 4 °C until use. The extraction procedure was carried out in triplicate.