3.7. The DPPH Scavenging Assay

DPPH radical scavenging assay was performed according to the procedure described by Matejic et al. [58], with modifications. 50 μL of appropriately diluted extract (1.2 mg/mL, 0.6 mg/mL, 0.3 mg/mL, 0.15 mg/mL, 0.075 mg/mL, 0.0375 mg/mL, 0.01875 mg/mL, 0.00938 mg/mL, 0.00469 mg/mL, 0.00234 mg/mL, 0.00117 mg/mL and 0.00059 mg/mL) were mixed with 50 μL DPPH working solution (25 mM in 99.9% methanol; A₅₄₀ ≈ 1). 50 μL of solvent mixed with 50 μL DPPH served as a control sample. Following 10 min incubation at room temperature in darkness, the absorbance of the samples was measured at λ = 540 nm using FilterMax F5 microplate reader (Molecular Devices, San Jose, CA, USA). The measured values of measurements were corrected by the absorbance value of the sample without DPPH. The percentage of DPPH radical neutralization was calculated using the following Equation

where Abs(S)-the corrected absorbance of the extract, Abs(C)-the corrected absorbance of the control sample (DPPH + solvent).

The analysis was conducted in three independent repetitions, using vitamin C as a reference compound. The calibration curve (y = −0.1241x + 0.24; R² = 0.9974) was prepared using 0–1 mg/mL trolox. The content of total antioxidants in each sample was calculated as miligrams of trolox equivalents per gram of dried extract weight (TE/g dw).