Cary uv vis spectrophotometer

The solubility of prednisolone (PRD) was assessed in different oils (OA, IPM, and LC), surfactants (TW-20 and LB), cosurfactants (ethanol, PG, PEG 400, and TC-P), and water, as previously described [16 (link)]. Briefly, PRD was added in excess to 2 mL of oils, surfactants, cosurfactants, and water in screwed test tubes. Samples were vortexed and kept to dissolve in an isothermal shaker maintained at 37 ± 1 °C for 24 h to equilibrate. Then, they were removed from the shaker, centrifuged for 15 min at 3500 rpm, and the supernatant was diluted suitably with methanol. The concentration of PRD was determined using UV spectrophotometry (Varian, Cary UV/VIS spectrophotometer) at 240 nm using a calibration curve created by preparing a set of PRD standard solutions.

The solubility of CIP.HCl was determined in nanoemulsion components as follows: OA and IPM as oil phases; TW-20 and Labrasol^® as surfactants; PG, PEG 400, and ethanol as cosurfactants; and 5% acetic acid and water as aqueous phases, as described in [49 (link)]. Briefly, an excess amount of CIP.HCl was dissolved in 3 mL of tested samples placed in 10 mL screwed-on test tubes. Samples were mixed using a vortex mixer and placed in a water bath at 37 °C for 24 h. Then samples were centrifuged at 3500× g for 15 min (Hermle Z326K centrifuge, Wehingen, Germany), and the supernatants were appropriately diluted with 0.1 N HCl. The concentration of CIP.HCl was assessed by UV-Vis spectrophotometry (Varian, Cary UV/Vis spectrophotometer) at a wavelength (λ_max) of 277 nm, using a standard calibration curve of CIP.HCl prepared in 0.1 N HCl.

The extraction of polyphenols from EVOOs, necessary for the chromatographic analyses, was performed using hexane (1:1 w/v), following the method of Fratianni et al. [15 (link)]. The mixture was then charged onto cartridges SPE C₁₈, and eluted three times with methanol. The three residues were pooled, dried, re-suspended in 1 mL of methanol and filtered through a 0.20 mm filter before the analysis. Total phenolic (TP) content was determined using the Folin-Ciocalteau reagent [16 ]. The absorbance at λ = 760 nm was determined (Cary UV/Vis spectrophotometer, Varian, Palo Alto, CA, USA) at room temperature. A standard curve generated using gallic acid as standard was used to quantify total polyphenols.

Total polyphenols (TP) of the extracts were determined using the Folin-Ciocalteu colorimetric method, as described by Singleton and Rossi Jr. [11 ]. Briefly, 50 μL of extract was added to 50 μL of Folin-Ciocalteu reagent in 800 μL of distilled water. The reaction was neutralized with a sodium carbonate solution (20 g/100 mL). After incubation for 2 h at room temperature, the absorbance at λ = 760 nm was determined using a Cary UV/Vis spectrophotometer (Varian, Palo Alto, CA, USA). Quantification was based on a standard curve generated using gallic acid. The results were expressed as μg of gallic acid equivalents (GAE)/g of DW samples ± standard deviation (SD).

The amount of anthocyanins was determined by the differential pH method [13 ]. Absorbance was measured in a Cary UV/Vis spectrophotometer (Varian, USA), simultaneously at λ = 510 nm and λ = 700 nm in buffers of pH 1.0 and 4.5, using the formula A = (A510 − A700)_pH1.0 − (A510 − A700)_pH4.5. A molar absorption of 26,900 mol/cm was used for cyanidin-3-glucoside (molecular weight of 449.2 g/mol). Results were expressed as micrograms of cyanidin-3-glucoside equivalents/g of DW samples ± standard deviation (SD).

A solution of methanol:acetic acid 99%:1% v/v was added to 10 g of a sample in a 2:1 v/w ratio (28 (link)). Samples were perfectly pounded and left for 24 h in the dark at 4°C. Then, they were centrifuged at 4 °C at 11,600 g (Biofuge, Beckman, Cassina de Pecchi, Italy) for 10 min. The removed, filtered, and dried supernatant was then resuspended in 10 ml of 80% ethanol, and ethyl acetate was added. This last step was repeated three times. The organic fraction obtained was then treated with sodium sulphate anhydrous, filtered, and dried again. The residue was resuspended in 1 ml of 50% methanol and stored at −26°C until the analysis. The colorimetric analysis of total phenolics followed the method of Singleton and Rossi (29 (link)) with Folin–Ciocalteu phenol as a reagent. The absorbance at λ = 760 nm was determined at room temperature with a Cary UV/Vis spectrophotometer (Varian, Palo Alto, CA, USA). Quantification was based on a standard curve created using gallic acid (y = 0.497 x + 0.0239 R² = 0.9908). The results are expressed as mg of gallic acid equivalents (GAE)/100 g of fresh weight (FW) of the product ± SD.