150 uv 1800 spectrophotometer

The total phenolic content (TPC) of samples was evaluated using Folin–Ciocalteu (FC) method (Singleton et al., 1999 (link)). 25 μL of diluted PE were mixed with FC reagent, Na₂CO₃ solution (20%), and ultra‐pure water and incubated for 1 h at room temperature, and the absorbance was measured against the blank at 760 nm (Shimadzu 150 UV‐1800 spectrophotometer, Kyoto, Japan). The results were given as mg gallic acid equivalent (GAE)/g raw propolis with a linear range of 0.2–2 mg/mL (R² = .999). The total flavonoid content (TFC) of samples was evaluated by the method of Andrade et al. (2017 (link)). The diluted PE was mixed with 0.1 mL of AlCl₃ and CH₃COONa solution, the mixture was incubated at room temperature for 30 min, and the absorbance was read against blank at 415 nm. The results were given as mg quercetin (QE)/g raw propolis with a linear range of 0.05–0.5 mg/mL (R² = .999).

Free and bound phenolic compounds are determined and the total phenolic content is calculated by summing these values.
The free and bound phenolic contents were determined by the Folin-Ciocalteu method with some modification. 100 µL of extracts in methanol was mixed with 500 µL of Folin-Ciocalteu reagent (2 N) and 1.5 mL of Na₂CO₃ solution (200 g/L) and 7.9 mL distilled water. For the blank sample, 100 µL of methanol was added instead of extract. After 120 min standing in dark, it is centrifuged at 4000× g for 5 min. Then, the absorbance was measured at 760 nm by Shimadzu 150 UV-1800 spectrophotometer (Kyoto, Japan). The total phenolic contents were calculated based on the calibration curve of gallic acid and expressed as gallic acid equivalents (GAE), in ppm.

The antioxidant capacity was performed as described by Singh et al. [36 (link)] with the DPPH radical scavenging activity method. In this method, 4.9 mL of fresh 1,1-diphenyl-2-picrylhydrazil (DPPH) solution was added to 100 µL of the wheat extract. The absorbance value of the solution was measured at 515 nm by a Shimadzu 150 UV-1800 spectrophotometer, following incubation at 30 °C for 60 min (Kyoto, Japan). The results are reported as mg TE/100 g ground wheat sample.