5.1. Methods Used to Determine the Antioxidant Potential

The TEAC is a free radicals scavenging method. It evaluates the ability to scavenge the ABTS radical [86]. It is possible to use two different oxidizing agents to obtain the goals: metmyoglobin-H₂O₂ or potassium persulfate. Both agents oxidize the ABTS, making ABTS^•+ (colored), then the addition of antioxidants causes a loss of the green color spectrophotometrically evaluable (λ 734 nm) [78,85]. This method detects the antioxidant potential of lipophilic and hydrophilic extracts and is not affected by ionic strength [85]. Briefly, K₂S₂O₈ (3 mM) react for 16 h with ABTS dissolved in distilled water (8 mM) in the dark at room temperature. Then, the ABTS^•+ solution is diluted in phosphate buffer solution (pH 7.4) and NaCl (in PBS 150 mM). The absorbance of 1.5 at 730 nm is read. Reaction kinetics are performed by taking readings every 15 min over a 2 h period. The reaction time is determined (generally 30 min.). Standards (100 μm) and samples (100 μm) are reacted with ABTS^•+ (2900 μm) for the reaction time previously determined [85]. The antioxidant potential was expressed as Trolox equivalents [85].

The DPPH detects the ability of a compound to transfer one electron [79]. The antioxidants reduce DPPH radical to DPPH-H [79]. The decrease of the absorbance value at λ 515 nm (DPPH absorbance) indicates the antioxidant potential. This test overestimates antioxidants with many phenol groups as flavonols [86]. Briefly, samples (20 μL) are added to 3 mL of DPPH solution (6 × 10⁻⁵ mol/L), and the spectrophotometric analysis is performed. The absorbance is read at λ 517 nm every 5 min until the steady state. The calibration curve is made using 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid (Trolox). The results were expressed as mmol Trolox equivalent (TE) kg⁻¹ FW [87].

The FRAP assay measures antioxidants’ ability to reduce a ferric tripyridyltriazine (Fe³⁺-TPTZ) to the ferrous (Fe²⁺-TPTZ). The antioxidant’s power is positively related to absorbance absorption at λ 593 nm. [87]. FRAP cannot detect proteins and thiols which have radical-quenching abilities. This test work at pH 3.6 [79]. Briefly, a solution of TPTZ (10 mmol/L) is added in HCl (40 mmol/L), ferric chloride (12 mmol/L), and sodium acetate buffer (300 mmol/L, pH 3.6) at a ratio of 1:1:10. Samples and standard antioxidant solutions (both 1 mmol/L) are added to the FRAP solution (3 mL). They must react for 90 min at 37 °C before taking the spectrophotometric reading at λ 593 nm [87].

The CUPRAC assay measures antioxidants’ ability to reduce Cu(II)-neocuproine (Nc) at λ 450 nm after 30 min. [88]. This test works at pH 7, detects the antioxidant potential of both lipophilic and hydrophilic antioxidants [88], and determines the reducing power of thiol-type antioxidants [89]. Briefly, sample (0.1 mL;) is mixed with distilled water (1 mL) copper chloride (0.4262 g dissolved in H₂O and diluted to 250 mL with additional water), neocuproine (7.5 × 10⁻³ M), and ammonium acetate buffer solution (19.27 g in water and diluting to 250 mL; pH 7) at 1:1:1 to obtain total reaction mixture of 4.1 mL. They must react 30 min at room temperature before taking the spectrophotometric reading at λ 450 nm. Results were expressed as μM Trolox equivalents [89].

The DCFH test assay measures antioxidants’ ability to prevent the oxidation of dichlorofluorecin into dichlorofluorescein (DCF) by 2,2’-azobis (2-amidinopropane) (ABAP)-generated peroxyl radicals in human hepatocarcinoma cells (HepG2 cells). Antioxidant power is negatively related to cellular fluorescence growth (λexc = 485 nm, λem = 538 nm) [90]. Briefly, myelomonocytic cells (HL-60, 1 × 10⁶ cells/mL) are suspended in Roswell Park Memorial Institute (RPMI 1640) medium with 10% fetal bovine serum (FBS) and antibiotics in 5% CO₂: 95% air at 37 °C. The cell suspension (125 μL) is added to the plates, treated for 30 min with the test material, and stimulated with phorbol 12-myristate 13-acetate (PMA,100 ng/mL) 30 min. Then, the cells are added to molecular probes (5 μg/mL DCFH-DA) and incubated for 15 min. DCFH-DA is a nonfluorescent probe that diffuses into cells. The levels of DCF are measured using a fluorescence measurement system [90].

The detection of lipid peroxidation can evaluate in skin keratinocytes cells (HaCaT cells). Epinephrine is used to induce lipid peroxidation. Antioxidant ability is negatively related to cellular fluorescence growth (λexc = 510 nm, λem = 580 nm) [16]. Briefly, HaCaT (1.8 × 10⁴) are seeded in 96-well plates and then incubated for 24 h with samples or beta-blocker (ICI-118,551). Successively, the cells are washed in phosphate-buffered saline (PBS) and then incubated for 30 min with the lipid peroxidation sensor (dyeC11-Bodipy) at 37 °C. Finally, epinephrine (50 μM) is used to make lipid peroxidation. The levels of peroxidized lipids are measured using a fluorescence measurement system [16].

The carbonylated protein levels can be evaluated in a spontaneously transformed aneuploid immortal keratinocyte cell line from adult human skin (HaCaT cells) via enzyme-linked immunosorbent assay (ELISA) using a specific antibody against 2,4-dinitrophenol (DNP). Epinephrine is used to induce protein carbonylation. The ability of antioxidants is negatively related to the growth in cellular fluorescence [16]. Briefly, HaCaT (1.5 × 10⁴) cells were put in 96-well plates and incubated for 24 h with the samples or ICI-118,551 and epinephrine (50 μM). Successively, the cells were washed in PBS and fixed in 4% paraformaldehyde (PFA). Then, cells were washed with PBS and 0.05% Polyethylene glycol sorbitan monolaurate (Tween 20) and incubated for 1 h with 2,4-Dinitrophenyl-hydrazine (DNPH; 5 mM) in 2 N HCl at room temperature. The carbonylated products were detected using a specific antibody against DNP (sc69697) by using the ELISA method. The skin punches were incubated for 24 h with the sample and epinephrine (56 nM). Successively, the punches were fixed for 6 h with PFA, washed in PBS, incubated in sucrose (15% and 30%), fixed in optimal cutting temperature (OCT) medium, frozen, and stored −80 °C. Cryosections (10 μm) were incubated with DNPH (5 mM) in 2N HCl for 1 h at room temperature, washed in PBS/EtOH (1:1), and PBS/Tween 20. The slides were incubated for 30 min in BSA, washed with PBS/Tween 20, incubated with antibody anti-DNP (1:50 dilution), and then mixed with the conjugated antibody Alexa Fluor 488. The signals were measured using fluorescent microscopy [16].