Once the methods were optimized and validated, their application was evaluated with eight methanol plant extracts.
The DPPH free radical reduction method was carried out by mixing 100 μL of the plant extract sample at different concentrations (250–0.25 μg/mL ethanol) with 100 μL of DPPH (280 μM in ethanol). A negative control was included (corresponding to 100% DPPH) in which ethanol was added instead of the sample. The mixture was incubated in the dark for 15 min at room temperature, and then the absorbance was measured at 540 nm with a Multiskan FC microplate reader.
For the ABTS free radical reduction method, a 7 mM solution of ABTS in 2.45 mM aqueous potassium persulfate was prepared. The solution was incubated for 12–16 h in the dark to produce the ABTS+ free radical. This solution was diluted with ethanol to adjust the absorbance value to 0.7. To evaluate antiradical activity, 60 μL of a plant extract sample solution (150–0.25 μg/mL ethanol) was mixed with 140 μL of the ABTS+ solution (A=0.7). A negative control was included (corresponding to 100% ABTS) in which ethanol was added instead of the sample. The mixture was incubated in the dark for 6 min at room temperature and the absorbance was measured at 750 nm with a Multiskan FC microplate reader.
For both DPPH and ABTS, the calculations used to obtain the percentage reduction of the light-absorbing species were those used for the optimization and validation of the method (Eq. (1)). A linear regression plot was constructed using the respective percentage reduction from each sample. The EC50 was assessed by curve interpolation for each of the plant extract samples. The results were also expressed as Quercetin equivalents for DPPH (μmol QE/g of Fresh Weight) and Trolox equivalents for ABTS (μmol TE/g of Fresh Weight) using standard curves. Quercetin standard solutions were prepared at concentrations from 1.65 to 26.47 μM and the t concentration of Trolox standard solutions ranged from 1 to 23.97 μM.
The DPPH free radical reduction method was carried out by mixing 100 μL of the plant extract sample at different concentrations (250–0.25 μg/mL ethanol) with 100 μL of DPPH (280 μM in ethanol). A negative control was included (corresponding to 100% DPPH) in which ethanol was added instead of the sample. The mixture was incubated in the dark for 15 min at room temperature, and then the absorbance was measured at 540 nm with a Multiskan FC microplate reader.
For the ABTS free radical reduction method, a 7 mM solution of ABTS in 2.45 mM aqueous potassium persulfate was prepared. The solution was incubated for 12–16 h in the dark to produce the ABTS+ free radical. This solution was diluted with ethanol to adjust the absorbance value to 0.7. To evaluate antiradical activity, 60 μL of a plant extract sample solution (150–0.25 μg/mL ethanol) was mixed with 140 μL of the ABTS+ solution (A=0.7). A negative control was included (corresponding to 100% ABTS) in which ethanol was added instead of the sample. The mixture was incubated in the dark for 6 min at room temperature and the absorbance was measured at 750 nm with a Multiskan FC microplate reader.
For both DPPH and ABTS, the calculations used to obtain the percentage reduction of the light-absorbing species were those used for the optimization and validation of the method (Eq. (1)). A linear regression plot was constructed using the respective percentage reduction from each sample. The EC50 was assessed by curve interpolation for each of the plant extract samples. The results were also expressed as Quercetin equivalents for DPPH (μmol QE/g of Fresh Weight) and Trolox equivalents for ABTS (μmol TE/g of Fresh Weight) using standard curves. Quercetin standard solutions were prepared at concentrations from 1.65 to 26.47 μM and the t concentration of Trolox standard solutions ranged from 1 to 23.97 μM.