Cas 117 39 5

The total flavonoid content was determined based on the aluminum chloride colorimetric method in which quercetin (Sigma-Aldrich, CAS 117-39-5, ≥95% purity by HPLC) was used as a reference compound [21]. Dissolving 1.0 mg quercetin in 10 mL methanol (Merck) by which the stock solution was made, then by serial dilutions, including using methanol, the standard quercetin solutions were prepared. Briefly, 1 mL of aluminum chloride (Merck) (2% w/v) was added to 1 mL diluted extract or standard quercetin solutions separately, and with methanol, the mixture was made up to 10 mL in quantity. Then, the solution was mixed and incubated for 15 min at room temperature. The absorbance of the mixtures was calibrated at 416 nm with a UV-Vis spectrometer. The measurements were carried out in triplicate. The calibration curve was plotted using standard quercetin. The total flavonoids content was estimated from the calibration plot and expressed as milligram of quercetin equivalent (QE) per gram of dry mass.

The following reagents were used in the experimental part: quercetin (Quercetin ≥95% (HPLC), CAS 117-39-5, Sigma-Aldrich, Saint Louis, MO, USA), methylcellulose (MC) (CAS 9004-67-5, Sigma-Aldrich, Saint Louis, MO, USA), polyacrylic acid (Carbomer, CARBOPOL^® 980 NF POLYMER, CAS 9007-20-9, Lubrizol, Wickliffe, OH, USA), ethyl alcohol 96% (ethanol 96%, pure CAS 64-17-5, Chempur, Piekary Śląskie, Poland), sodium hydroxide (CAS 1310-73-2, Sigma-Aldrich, Saint Louis, MO, USA), and glycerol (CAS 56-81-5, Sigma-Aldrich, Saint Louis, MO, USA).

The methodology proposed by Chang et al. [23 ] was used, with some modifications. A total of 500 µL of the extract, 1.5 mL of 80% ethanol (v/v), 100 µL of 10% hexahydrate aluminum chloride solution, 100 µL of 1 M potassium acetate, and 2.8 mL of distilled water were placed, stirred and incubated for 30 min at room temperature.
Subsequently, an absorbance at 415 nm was read on a UV-Vis spectrophotometer (7305 Jenway Staffordshire, UK). A quercetin calibration curve (Sigma Aldrich, CAS 117-39-5, Germany) was used at a concentration of 0 to 0.1 g/L. The results were expressed in mg quercetin equivalents per g substrate on dry weight (mg QE/g substrate dw). These analyzes were performed in triplicate.