Rotavapor r 205

A 0.1% (w/v) HA solution was prepared by dissolving 100 mg sodium hyaluronate in 100 mL deionized water for 12 h at room temperature, followed by 2 h of sonication using a 30 W ultrasonic processor (Sonics VC130PB, Sonics and Materials Inc., Newtown, CT, USA). A 0.1% (w/v) CS solution was dissolved for 2 h in deionized water adjusted to pH = 3 with 1 M HCl. Insulin (pI = 5.3) was dissolved in the acidified CS solution. 2 mL of the dissolved mixture was then added to 10 mL HA solution in a 25 mL glass beaker while using a rate of ~4 mL/2 s with stirring, which yielded an MR of 1:5 for CS: HA. Other MRs were achieved by adapting the volume of CS solution accordingly. PECs were immediately formed, yielding two theoretical insulin concentrations of 100 µg/mL or 500 µg/mL. The dispersion was left to stir for 10 min allowing for stabilisation. In order to provide a coating around complexes, EL100 (6 mg/mL) was dissolved in ethanol solution (97% ethanol/3% water (v/v)) and it was added to the dispersion of complexes dropwise at a rate of 4 mL over 8 min in a 1:1 (v/v) ratio to yield a final concentration of EL100 in the PEC dispersion of 3 mg/mL. The ethanol was removed under vacuum by rota-evaporation using a Büchi Rotavapor R-205. For cytotoxicity studies, PEC dispersions were lyophilised with 3% (w/v) trehalose as a cytoprotectant [32 (link)].

Extraction of rose fruits with carbon dioxide in a supercritical state was carried out on a semi-automatic, multi-station Supercritical fluid extraction (SFE) system (MV-10 ASFE System, Waters, and Milford, MA, USA). The process was automatically controlled by a computer using Chromo ScopeTM software. As the modifier, 96% ethanol was used. Extractions were carried out in the following conditions: temperature—60 °C; pressure—280 bar; time—30 min (static time); 10 min (dynamic time); CO₂ flow—4 mL/min; and modifier flow—1 mL/min. Next, the extracts were concentrated in a rotary evaporator (Rotavapor R-205, Büchi, New Castle, DE, USA). The following temperatures were used: bath heating 60 °C, condensate 40 °C, cooling water 20 °C. The concentrated extract was frozen to −80 °C and then lyophilized for 72 h (Alpha 1-4, Christ, Osterode am Harz, Germany).

The leaves were chopped with a knife in a dark place, weighed by electronic digital balance, and put into an Erlenmeyer flask containing 80% methanol. Enough volume of solvent was added so as it covers the crushed plant material in the flask; then, the flask was sealed with aluminum foil and frequently agitated using a rotary shaker at 120 rpm for 24 h. It was then filtered using a Whatman No. 1 filter paper (90 mm diameter, Whatman Ltd, England) and macerated 3x for a total of 72 h and filtered. The combined filtrates were concentrated using a rotary evaporator (BUCHI Rotavapor R-205, Switzerland) at a temperature not exceeding 40°C. The concentrated extract was then transferred into a freeze-drying flask and lyophilized (OPERAN Lyophilizer, Korea). The dried extract was kept in a tightly sealed container at -20°C until use.

A total of 1 kg of the ground raspberry seeds were placed in a 3 L glass vessel and filled with hexane. The extraction is carried out for 24 h at a temperature of 25 ± 2 °C in dark with stirring. The solvent was removed by vacuum filtration, and the sample was extracted twice. After the last filtration, the extract was pooled, hexane was removed with a vacuum rotary evaporator Rotavapor R-205 (BÜCHI Labortechnik AG, Flawil, Switzerland) at 35 ± 2 °C and 170 mbar pressure, purged with nitrogen, and stored at −18 °C until analysis.

The dried Moldavian Balm plant was extracted using maceration method with ethanol 50% (v/v), plant to solvent ratio of 1/10 (w/v), and an extraction temperature of 25°C. After 48 hr, the extract was filtered using a filter paper. Then, the extract was concentrated in a rotary evaporator (Buchi Rotavapor R‐205, Switzerland) at 38 ± 2°C and 100 rpm to obtain concentrate of 4% (40 g/L). Afterwards, carriers were added to the extract in different levels (1:10, 2:10, 3:10 (g:g); apple pectin: Maltodextrins, with dextrose equivalent of 16). The carriers purchased from Sigma‐Aldrich. The phytochemical compounds of extract were measured before drying, and the following results were obtained: total phenol content: 6.45 mg GAE/g DW, total flavonoid content: 0.41 mg QUE/g DW and antioxidant activity: 60%.