Model 600

The dried active extract (EtOAc) was adsorbed on silica gel (70 to 230 mesh) and loaded on a silica gel column (600 mm height × 55 mm diameter), which was eluted (Waters, model 600) with a step gradient increasing in polarity- hexane: dichloromethane, 9.5:0.5, 9:1, 8.5:0.5, 8:2, 7:3; 6:4, 5:5, 4:6, 3:7, 2:8, 1:9, and 0:10. The column was then eluted with 100% dichloromethane, then 100% EtOAc, followed by a mixture of EtOAc and methanol (9.5:0.5, 9:1, 8:2, 7:3, 6:4, 5:5, 4:6, 3:7, 2:8, and 1:9). Finally, the column was eluted with 100% methanol (Supplementary Figure S1). In each step, ten tubes of 40 mL fractions were collected, and the solvent was evaporated. The whole separation was monitored by a Dual λ model 2487 absorbance detector at 280 nm and 254 nm.

Two milliliters of preheated PBS (37 °C) was added to the film substrates for in vitro release testing. Films were incubated under gentle agitation on an orbital shaker (150 rpm) at 37 °C. After each predetermined time interval, 1 mL of PBS was withdrawn from the vials and replaced with fresh fluid. This investigation was conducted in triplicate. DEX quantification in the samples was performed based on HPLC analysis using a Waters HPLC equipped with a pump and controller (Model 600), an autosampler (Model 717plus) and a UV absorbance detector (Model 486) (Waters, Milford, U.S.). The mobile phase consisted of water and acetonitrile (60:40 (v/v)), and an Equisil ODS C18 column with a precolumn (5 µm C18, 125 × 4 mm; Techlab, Braunschweig, Germany) was chosen as the stationary phase. The flow rate was 1 mL/min, and the detection wavelength was set at 238 nm. Twenty microliters of the samples were injected, and the DEX retention time was found to be ~3 min. Calibration was performed in the range from 0.2 to 20 µg/mL (r² > 0.999). Furthermore, the DEX degradation product 17-oxo-dexamethasone (17-oxo-DEX) was quantified. The peak-retention time for this compound was ~5 min, and calibration was conducted in the span between 0.2 and 26 µg/mL (r² > 0.999). Peak integration and evaluation were carried out with Clarity^TM 8.0 software (DataApex, Prague, Czech Republic).

HPLC analysis was performed using a quaternary pump (model 600, Waters, Milford, MA, USA) coupled to an autosampler (Varian ProStar, Palo Alto, CA, USA, model 420). The column was ACE 3 C18-PFP (4.6 × 150 mm, Symta, Madrid, Spain). The temperature of the column was kept constant at 40 °C. The injection volume was 20 µL. The detection of peaks was carried out using a photodiode array detector (ProStar, Varian, Palo Alto, CA, USA) and peaks were analyzed using the Varian Star LC workstation 6.41. Quantification was performed at 284 nm. The mobile phase was acetonitrile/water 60:40 (v/v) at 0.7 mL/min during 25 min. Both solvents contained 0.1% (v/v) of formic acid.