Soxhlet Extraction of U. barbata Lichen Compounds

In a Soxhlet continuous reflux system, the dried lichen was ground into a powder; 20 g of dried lichen was extracted for 8 h with 150 mL solvent: ethyl acetate, methanol (Chemical Company, Iasi, Romania), acetone, ethanol (Chimreactiv S.R.L., Bucharest, Romania), and water [46 (link)]. The extraction temperature was different for each extract, being around the boiling point of each solvent (Table 1). After filtration with filter paper, UBW was concentrated on a Rotavapor R-215 with a vacuum controller V-850 (BÜCHI Labortechnik AG, Flawil, Switzerland), and lyophilized with a freeze-dryer Christ Alpha 1-2L (Martin Christ Gefriertrocknungsanlagen GmbH, Osterode am Harz, Germany) connected to a vacuum pump RZ 2.5 (VACUUBRAND GmbH, Wertheim, Germany) [46 (link)]. For UBEA, UBA, UBE, and UBM, the rotary evaporator TurboVap 500 (Caliper Life Sciences Inc, Hopkinton, MA, USA) evaporated the correspondent solvents [41 (link)]. Next, each extract was kept for 16 h in a chemical exhaust hood for optimal solvent evaporation. All obtained U. barbata dry extracts were transferred to sealed-glass containers and stored in the freezer (Sirge^® Elettrodomestici—S.A.C. Rappresentanze, Torino, Avigliana (TO) Italy) at −24 °C until processing [46 (link)].