A modified FRAP assay was used to assess the ferric reducing capacity of plant extracts [65 (link)]. The reduction of ferric iron (Fe3+) to ferrous iron (Fe2+) by antioxidants present in the samples is how the assay determines the antioxidant potential. Blue coloration results from the conversion of ferric iron (Fe3+) to ferrous iron (Fe2+).
Equal amounts of 0.1 g dry extracts were dissolved in 100 mL 50% ethanol for every plant extract used in our study. Eight corresponding volumes of each obtained solution were brought into volumetric flasks and adjusted to 10 mL by adding the same solvent as above. An amount of 2.5 mL of each diluted solution was mixed with phosphate buffer (pH 6.6, Sigma–Aldrich, Hamburg, Germany) and 2.5 mL K3(FeCN)6 1% (Sigma–Aldrich, Hamburg, Germany) before being heated to 50 °C for 20 min. 2.5 mL trichloroacetic acid (Sigma–Aldrich, Hamburg, Germany) was added to each sample. Furthermore, 2.5 mL of distilled water and 0.5 mL FeCl3 0.1% (Sigma–Aldrich, Hamburg, Germany) were added to 2.5 mL of each of the resulting solutions, the samples being left thereafter idle for 10 min. The change in the absorbance at 700 nm was measured relative to a blank sample obtained by mixing 5 mL distilled water with 0.5 mL FeCl3 0.1%.
The antioxidant capacity was calculated using the IC50 (half of the antioxidant effect—IC—effective concentration) value (mg/mL), which represents the solution concentration for which the absorbance has a value of 0.5.
Different extract volumes were tested in order to reach the absorbance value of 0.5, due to the variability of plant characteristics and the nonuniformity of phytochemical profiles of plant extracts (experimental values closer to the target value result in more accurate approximation—IC50 for y = 0.5). The optimized values have been set as mentioned above in order to conduct an appropriate comparative study within the same technique and between other methods of assessing the antioxidant activity.
Equal amounts of 0.1 g dry extracts were dissolved in 100 mL 50% ethanol for every plant extract used in our study. Eight corresponding volumes of each obtained solution were brought into volumetric flasks and adjusted to 10 mL by adding the same solvent as above. An amount of 2.5 mL of each diluted solution was mixed with phosphate buffer (pH 6.6, Sigma–Aldrich, Hamburg, Germany) and 2.5 mL K3(FeCN)6 1% (Sigma–Aldrich, Hamburg, Germany) before being heated to 50 °C for 20 min. 2.5 mL trichloroacetic acid (Sigma–Aldrich, Hamburg, Germany) was added to each sample. Furthermore, 2.5 mL of distilled water and 0.5 mL FeCl3 0.1% (Sigma–Aldrich, Hamburg, Germany) were added to 2.5 mL of each of the resulting solutions, the samples being left thereafter idle for 10 min. The change in the absorbance at 700 nm was measured relative to a blank sample obtained by mixing 5 mL distilled water with 0.5 mL FeCl3 0.1%.
The antioxidant capacity was calculated using the IC50 (half of the antioxidant effect—IC—effective concentration) value (mg/mL), which represents the solution concentration for which the absorbance has a value of 0.5.
Different extract volumes were tested in order to reach the absorbance value of 0.5, due to the variability of plant characteristics and the nonuniformity of phytochemical profiles of plant extracts (experimental values closer to the target value result in more accurate approximation—IC50 for y = 0.5). The optimized values have been set as mentioned above in order to conduct an appropriate comparative study within the same technique and between other methods of assessing the antioxidant activity.
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