2 2 diphenyl 1 picrylhydrazyl (dpph)

Free radical scavenging activity was assessed using a modified version of the method proposed by Cheng, et al. [54 (link)] (n = 4–6 per group). In brief, 100 μL of sample extracts (4, 20, 100, 500, 2500 μg/mL) or isolated compounds (10, 25, 50, 100, 200 μM) dissolved in 50% ethanol were added to 100 μL of 0.2 mM DPPH (Fisher Scientific, Leicestershire, UK) solution, reaching a final concentration of 0.1 mM DPPH. Each reaction mixture was shaken for five seconds and left to stand for 30 min in the dark at 25 °C. The absorbance of each mixture was measured at 515 nm against a blank (50% ethanol) using a SpectraMax i3x Microplate spectrophotometer (Molecular devices). The inhibition rate of DPPH radicals was calculated as follows:

For the 2,2′-diphenylpicrylhydrazyl (DPPH) assay, antioxidant compounds were dissolved in ethanol with a stock concentration of 4 mM and stirred for 24 h. DPPH (Alfa Aesar) was also dissolved in ethanol at 0.127 mM. Different compound solutions were prepared by serial dilutions of each stock solution (4, 2, 1, 0.5, 0.25, 0.125, 0.063, 0.031, 0.015, and 0.007 M). Resveratrol was prepared using the same protocol and used as antioxidant control. Amounts of 80 µL of each solution and 80 µL of DPPH were added in a 96-well plate. The absorbance was measured at different times (10, 20 and 30 min and 1 and 2 h) by a Multi-Detection Microplate Reader Synergy HT (λ_MAX = 515 nm). Radical scavenging activity (RSA, %) was calculated using Equation (1)
$\mathrm{RSA}(\%)=\frac{{\mathrm{A}}_{\mathrm{<a\; class="product"\; href="/product/lCPhCZIBPBHhf-iFnQYw/"\; target="\_blank">DPPH</a>}}-{\mathrm{A}}_{\mathrm{EXTRACT}}}{{\mathrm{A}}_{\mathrm{<a\; class="product"\; href="/product/lCPhCZIBPBHhf-iFnQYw/"\; target="\_blank">DPPH</a>}}}·100$
where A_EXTRACT and A_DPPH correspond to the absorbance of DPPH with and without the extracts, respectively. Eight replicates were used for each compound and results were expressed as mean value ± standard deviation. The RSA half-maximal inhibitory concentration (IC₅₀) values were calculated from the relationship curve of RSA versus concentrations by a non-linear fit using the software GraphPad Prism 7.