Chromafil syringe filter

For quantification of the identified compounds, the HPLC system Dionex UltiMate^® 3000, equipped with a quaternary pump and an UltiMate^® diode array detector (Dionex, California, USA) was used. The HPLC separation column and the elution program are described in the previous paragraph 3.3.6. The dry extracts were dissolved in deionized water (10 mg/mL), filtered through a Chromafil syringe filter, pore-size 0.45 μm (Macherey-Nagel GmbH & Co., Duren, Germany), and 50 µL were loaded onto the column. For the identified phenolic compounds for which a commercial standard was not available, the quantification was performed through the calibration curve of the most similar available standards. Results were expressed as μg of phenolic compound/g DE.

RP-HPLC analysis was performed, as reported in Squillaci et al. [17 (link)]. The samples were dissolved in PBS at concentration of 10 mg/mL, filtered through a CHROMAFIL syringe filter (pore size 0.45 μm, Macherey-Nagel GmbH and Co., Duren, Germany), and injected into the column (50 μL). The peak elution was monitored at 280 nm. Phenolic compounds were identified by comparing the retention times with those of pure commercial standards, and by co-injection with the corresponding standards. Quantification was performed by calibration curves obtained by injecting increasing quantities of the pure compound, and the results were expressed as mg/g CSDE.

In vitro release study was carried out by diffusion using dialysis bag. The dialysis membrane (Sigma, MWCO 12,000 Da) was cut into equal pieces (6 × 2.5 cm²) and soaked in distilled water for 24 h before use. An accurately weighed amount of nanoparticles (equivalent to 10 mg BZ) was dispersed in 2 mL of PBS buffer (pH = 7.4) and transferred into the dialysis bag. Each bag was placed into a beaker containing 20 mL dissolution media (PBS buffer, pH 7.4) and kept on an electromagnetic stirrer at 50 rpm and 37 ± 0.5 °C. Samples of 2 mL were taken at predetermined time intervals and replaced with equivalent volume of fresh media. The samples were then filtered (0.45 μm Chromafil^® syringe filter, Macherey-Nagel, Düren, Germany) and analyzed for drug content as mentioned above. Mean results of triplicate measurements and standard deviation were reported.