Aluminum chloride

Egyptian raw honey bee’s propolis was collected from Delta region Qaluiobia Governorate (PQG) and kept in dark sterile glass containers at room temperature until further use. All chemicals and reagents i.e. Folin–Ciocalteu reagent, 2,2-diphenyl-1-picryl hydrazyl radical (DPPH), Tween 20 (T₂₀), Ethanol, Aluminum chloride, Potassium acetate, linoleic acid, Gallic acid, Sodium alginate, Butylated hydroxyltoluene (BHT), Sodium Thiocyanate, Ascorbic acid, Quercetin, Methanol, Phosphate buffer, Ammonium thiocyanate, Ferrous chloride, Potassium ferricyanide, Trichloroacetic acid, Ferric chloride, Sodium carbonate, Aluminum chloride, Formic acid, Acetonitrile, Phosphotungestic acid, Chloroform, Glacial acetic acid, Sodium thiosulphate were of analytical grade and purchased from Sigma Aldrich Chemical (Merck KGaA, Darmstadt, Germany).
The work does not involve humans or conduct experiments on live animals.

Sulfuric acid, 2,2-Diphenyl-1-picrylhydrazyl (DPPH), sodium thiosulphate, Folin–Ciocalteu reagent (2N), aluminum chloride, sodium hydroxide, sodium acetate, gallic acid potassium chloride, aluminum chloride, sodium hydroxide, ferric chloride heptahydrate, potassium carbonate, sodium acetate, methanol, pectin, whey protein isolates, and sodium caseinate were obtained from Sigma Aldrich (St. Luis, MO, USA).

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.