Thermo evolution 260 bio spectrophotometer

The phenol reagent of Folin–Ciocalteu was diluted 10-fold in distilled water and mixed with the phenol compound 5:1, v/v. After 10 min, Na₂CO₃ (7.8%) was added to obtain a blue complex between the Folin–Ciocalteu reagent and the phenols in the alkaline medium [33 ]. After 1 h, the absorbance value of the blue complex was measured with a Thermo Evolution 260 Bio spectrophotometer (Thermo Fischer Scientific, Waltham, MA, USA) at 766 nm, which was proportional to the concentration of the phenolic compounds present in the sample. The values obtained are expressed in relation to gallic acid, which is considered the reference polyphenol. Therefore, it was necessary to establish the calibration curve for gallic acid (linear range: 0.01–0.1 mg mL⁻¹).

The Trolox equivalent test for antioxidant capacity requires the use of ABTS·⁺, a radical cation produced by oxidation of ABTS (7 × 10⁻³ M) in water with K₂S₂O₈ (2.5 × 10⁻³ M). The ABTS·⁺ is a long-lived radical that stabilises after at least 20 h at room temperature in the dark. The working solution is prepared by diluting the stock solution to 1:40 v/v and then mixing with the sample and water prior to the measurements at a ratio of 25:1:4, ABTS·⁺/sample/solvent, v/v/v. The protocol is based on absorbance measurement at 735 nm using a Thermo Evolution 260 Bio Spectrophotometer (Thermo Fischer Scientific, Waltham, MA, USA). In the presence of an antioxidant compound, the measured absorbance for the radical cation decreases in direct proportion to the antiradical capacity. The results were expressed in relation to Trolox equivalent (8.5 × 10⁻⁶ M) [31 ], a compound that is considered a reference antioxidant.

The protocol requires the use of DPPH^• (2.5 × 10⁻⁴ M), a stable radical obtained by dissolving DPPH in a suitable solvent (ethanol). The stock solution is mixed with the sample in a volumetric ratio of 1:0.1:1.9, DPPH^•/sample/solvent, v/v/v. The protocol is based on the measurement of the decrease in DPPH absorption maximum at 515 nm using a Thermo Evolution 260 Bio Spectrophotometer (Thermo Fischer Scientific, Waltham, MA, USA). In the presence of an antioxidant compound, the absorption of DPPH- decreases in direct proportion to the antiradical capacity. The results were expressed as Trolox (8.5 × 10⁻⁶ M) equivalent [32 (link)].