Heizbad hei vap

In order to obtain the samples for LC-MS analysis, liquid extracts (the samples obtained under the optimal extraction conditions in every employed extraction technique for achieving the highest polyphenol content) were dried. Additionally, the samples for FT-IR spectroscopy, antimicrobial, and skin regeneration potential analyses were lyophilized. The ethanol from the extracts was evaporated using Heizbad Hei-VAP (Heidolph, Schwabach, Germany) at 40–50 °C, a pressure of 50 mbar, and a rotation speed of 150 rpm. Subsequently, the sample was frozen in the freezer, at −80 °C for 1 h, and freeze-dried at −75 °C and pressure of 0.011 mbar for 24 h and at −65 °C and pressure of 0.054 mbar for one additional hour (Alpha 2-4 LSCplus, Christ, Osterode am Harz, Germany).

Five milliliters of each subfraction was concentrated and freeze-dried in a Heidolph Rotary Evaporator (Heizbad Hei-VAP, Heidolph Instruments GmbH & CO. KG, Germany) and Biocool Freeze Dryer (Biocool FD-1C-50, Boyikang, Beijing), respectively. The obtained powders were weighed and added to the RPMI 1640 culture media with 1% DMSO, and the final concentration of each solution was 200 μg/mL.
Exponentially growing RBL-2H3 cells in 6-well dishes were washed with PBS 3 times. Two milliliters of the tested supernatant was added to the corresponding wells, including the negative control group (RPMI 1640 culture media with 1% DMSO) and the positive control group (C 48/80; 20 μg/mL). Each group was made in triplicate. The 6-well dishes were incubated in an incubator (37°C, 5% CO₂) for 30 min before the supernatants were discarded. The dishes were placed under an inverted microscope (Nikon Eclipse TS100-F, Japan) for observation. The groups with high degranulation rates were considered to have the potential constituents of the anaphylactoid reaction.

In order to evaporate ethanol from liquid extracts, the samples were placed in rotary evaporator Heizbad Hei-VAP (Heidolph, Berlin, Germany) at 40–50 °C, pressure of 50 mbar and rotation speed of 150 rpm. Subsequently, 50 mL of liquid extract without ethanol was mixed with 2.5 g of gelatin and the mixture was stirred on the magnetic stir plate, RTC basic (IKA, Königswinter, Germany), at 40 °C for approximately 15 min.
Before the lyophilization, the samples (dissolved gelatin, pure extracts and extracts with 5% w/w of gelatin) were frozen in freezer, LAB11/EL19LT (Elcold, Hobro, Denmark), at −80 °C for 1 h. In freeze dryer Beta 2–8 LD plus (Christ, Osterode am Harz, Germany), main drying was carried out at −75 °C and pressure of 0.011 mbar for 24 h and final drying at −65 °C and pressure of 0.054 mbar for 1 h.
Spray drying of dissolved gelatin, pure extracts and extracts with 5% w/w of gelatin was performed in Mini Spray Dryer B-290 (Büchi, Flawil, Switzerland) equipped with nozzle of 0.7 mm. The inlet temperature was 140 °C, while outlet temperature was 72–75 °C. The air flow rate was 45 mm and the rate of liquid feed was 30%.