Uv 2500 ultraviolet visible spectrophotometer

Contents of flavonoid in propolis extracts were determined according to the method of Kaijv et al. [20 ], with minor modifications. A standard curve was built with chrysin solution. Aliquots ranging from 0 to 12 mL of standard solution (100 μg/mL) were pipetted into 25 mL volumetric flasks containing 1.0 mL aliquots of 5% NaNO₂ (w/v) solution. After 6 min, 1.0 mL 10% AL(NO₃)₃ (w/v) solution was added and thoroughly mixed. 10 mL 4.3% NaOH (w/v) solution was added after 6 min and the volume was made up with ethanol. The blank was prepared by using ethanol instead of standard solution or sample solutions. After 15 min, the absorbance was measured at 510 nm by using UV-2500/Ultraviolet visible spectrophotometer with UVProbe software (Shimadzu Co., Ltd., Tokyo, Japan). For determination of the total flavonoid contents in propolis extracts, 0.5 mL of aqueous solution at a concentration of 1 mg/mL was used. Total flavonoid contents were expressed as milligrams chrysin equivalents per gram extract (CE).

Ferric reducing/antioxidant power (FRAP) assay was carried out according to the method described by Benzie and Strain with slight modifications [17 (link)]. Briefly, 200 μL propolis extracts of various concentrations (0.1–1 mg/mL) were mixed with 2.5 mL PBS buffer (0.2 M, pH 6.6) and 2.5 mL potassium ferricyanide (1.0%). Then the mixture was incubated for 20 min at 50°C. After 1.0 mL 10% trichloroacetic acid was added, the mixture was centrifuged at 2500 ×g for 10 min. Then 2.5 mL supernatant was mixed with 2.5 mL distilled water and 0.5 mL 0.1% ferric chloride. Absorbance of the final mixture was measured at 700 nm using UV-2500/Ultraviolet visible spectrophotometer (Shimadzu Co., Ltd., Tokyo, Japan). Aqueous solutions of Trolox concentrations in the range of 100 to 200 μg/mL were used for the calibration, and the FRAP values were expressed as microgram of Trolox equivalents per milligram of propolis (mg Trolox/mg).

DPPH radical-scavenging activity was measured according to the method [15 (link)] with slight modifications. Briefly, 200 μL of various concentrations of propolis extracts (0.1–1.0 mg/mL) and 1.8 mL of corresponding extraction solvents were added to 2 mL of 0.2 mM ethanol solution of DPPH. The resulting solution was thoroughly mixed and incubated in the dark for 20 min at room temperature. Absorbance of the solution was measured at 517 nm using UV-2500/Ultraviolet visible spectrophotometer (Shimadzu Co., Ltd., Tokyo, Japan). The blank sample only contained the same volume of corresponding extraction solvents. Trolox was used as antioxidant standard. DPPH radical-scavenging activity was expressed as IC₅₀ (concentration of total phenolics able to scavenger 50% of DPPH free radical).