Red-fleshed apples: old autochthonous fruits as a novel source of anthocyanin antioxidants
Supplementary Material
Methods applied for the antioxidant assessment in cell-free systems
Determination of DPPH radical scavenging capacity
Extracts or Trolox® were dissolved in a DPPH• methanol solution (9.4 × 10−5 M; 1.0 mL) at room
temperature. After 30 min, the absorption at 515 nm was measured in reference to a blank. The
sample-mediated initial DPPH• absorption decrease (%) was calculated.
Determination of ABTS radical cation scavenging capacity
Investigated extracts or Trolox® were dissolved in 1.0 mL of diluted ABTS•+ solution, previously
generated by reacting ABTS (7.0 mM) and potassium persulfate (2.45 mM). After 6 min, the
absorption at 734 nm was measured in reference to a blank. The sample-mediated initial ABTS•+
absorption decrease (%) was calculated.
Determination of oxygen radical absorbance capacity (ORAC)
Five dose levels of each extract (20 µL each) and fluorescein (FL, 120.0 µL; 70 nM, final
concentration) were pre-incubated for 15 min at 37°C in 75 mM phosphate buffer (pH 7.4). Then,
AAPH solution (60.0 µL; 12 mM, final concentration) was rapidly added. In parallel with the test
samples, a blank (FL + AAPH) and Trolox® were properly prepared. The fluorescence decay (λex =
485 nm, λem = 525 nm) was recorded every minute for 120 min. The antioxidant activity (ORAC
value) was calculated for each sample by using the Trolox® calibration curve.
Determination of Superoxide Anion Radical (O2) Scavenging Capacity
Investigated apple extracts or Trolox® were dissolved at room temperature in 3.0 mL of a reaction
mixture containing sodium phosphate buffer (50.0 mM, pH 7.8), methionine (13.0 mM), riboflavin
(2.0 M), EDTA (100.0 M) and NBT (75.0 M). The O2 production was followed by monitoring
the increase in absorbance at 560 nm after 10 min illumination with a fluorescent lamp.
Determination of Hydroxyl Radical (OH) Scavenging Capacity
To the reagent mixture (H2O2 (100.0 µM), EDTA (100.0 µM), FeCl3 (100.0 µM), and ascorbic acid
(100.0 µM) in KH2PO4/KOH buffer (10 mM, pH 7.4)), previously incubated at 37 °C for 1 h, 2deoxyribose (28.0 mM) was added and allowed to stand at 37 °C for 5 min. Thus, 1.0 mL of diluted
reagent mixture was added into tubes containing test samples or Trolox® and incubated at 37 °C for
1 h. To each tube 1.0 mL of a 1.0% TBA (thiobarbituric acid) in 50 mM NaOH and 1.0 mL of a
solution at 2.8% of TCA (trichloroacetic acid) were added and placed in a water bath at 90°C for 30
min, before reading at 532 nm.