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. 2020 Jul 29;9(8):1018. doi: 10.3390/foods9081018

Table 3.

Assays employed for the evaluation of antioxidant activity.

Assay Methodology Refs.
EVALUATION OF THE CAPACITY TO SCAVENGE FREE RADICALS AND OXIDANT SPECIES
Scavenging effect on hydrogen peroxide (H₂O₂) radicals Measurement of the reduction in the absorbance of a H₂O₂ solution at 230 nm after incubation with potential antioxidants. [20,31,48]
Scavenging effect on ABTS (2,2’-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) radicals ABTS radicals that absorb at 734 nm are produced by the reaction of ABTS with potassium persulfate. The method evaluates the reduction in the absorbance of ABTS radicals due to the presence of potential antioxidants. [3,11,14,16,17,20,24,25,28,31,33,34,35,36,37,41,42,45,46,49,51,52,54,55,58,62,65]
Scavenging effect on nitric oxide (NO) radicals Nitric oxide radicals are formed from nitroprusside and the incubation of formed nitric oxide radicals with a Griess reagent (1% sulphanilamide, 2% H3PO4, and 0.1% naphthylethylene diamine dihydrochloride) results in nitrite ions. Nitrite ions can be measured by the formation of a compound that absorbs at 546 nm. The scavenging of nitric oxide radicals by potential antioxidants reduces nitrite ion formation and their absorbance at 546 nm. [20,31,49]
Scavenging effect on DPPH (1,1-diphenyl-2-picrylhydrazyl) radicals Measurement of the decrease in the absorption of DPPH radicals at 515–517 nm when potential antioxidants are added. [14,17,20,21,26,27,30,31,34,36,37,38,39,40,41,42,43,44,45,46,47,48,49,51,52,53,54,55,56,59,60,61,63,65]
EVALUATION OF THE CAPACITY TO INHIBIT OXIDATION REACTIONS
Inhibition of formation of superoxide (O²⁻) radicals The assay measures the rate of pyrogallol autooxidation in presence and absence of potential antioxidants at 320–420 nm. [50,55]
Inhibition of formation of hydroxyl (OH•) radicals Hydroxyl radicals are generated by the oxidation of Fe2+ to Fe3+ in the presence of H₂O₂. The presence of Fe2+ is monitored by the formation of a complex with 1,10-phenanthroline that absorbs at 536 nm. The presence of potential antioxidants inhibits the oxidation of Fe2+ and results in an absorbance increase. [3,11,14,16,17,25,27,28,33,35,47,55,65]
Oxygen radical antioxidant capacity (ORAC) The method is based on the oxidation of fluorescein by reactive oxygen species (ROS) resulting from the radical initiator 2,2’-azobis(2-methylpropionamidine) dihydrochloride. The inhibition of fluorescein oxidation by the presence of potential antioxidants is measured from the increase in fluorescence intensity. [51,65]
EVALUATION OF THE REDUCING POWER
Ferric reducing antioxidant power (FRAP) Measures the ability of potential antioxidants to reduce Fe³⁺ from the ferricyanide complex to Fe²⁺-complex. Formation of Fe²⁺-complex is measured at 700 nm. [14,16,20,27,28,30,31,33,35,38,39,40,45,46,47,49,50,52,56,58,62,63,65]
Ammonium phosphomolybdenum The method evaluates the capacity of potential antioxidants to reduce Mo⁶⁺ to Mo⁵⁺. Presence of Mo⁵⁺ is monitored by the subsequent formation of a green phosphor/Mo⁵⁺ complex that absorbs at 695–65 nm. [20,21,30,31,59]
EVALUATION OF THE METAL QUELATION ACTIVITY
Ferrous ion chelation activity (FICA) Ferrozine reacts with Fe²⁺ to form a complex that absorbs at 562 nm. In the presence of chelating agents, the complex is disrupted, resulting in a decrease in absorption at 562 nm. [45,46,52,53,59,61,63,65]
Cuprous ion chelation activity (CICA) Reaction of pyrocatechol and Cu²⁺ results in a substance that absorbs at 632 nm. The presence of a metal chelator disrupts this molecule and reduces the absorbance. [47]
EVALUATION OF THE CAPACITY TO INHIBIT LIPIDS AND LIPOPROTEINS OXIDATION
Ferric thiocyanate Primary products resulting from the oxidation of linoleic acid are incubated with EtOH, ammonium thiocyanate, and FeCl₂, leading to the formation of Fe(SCN)²⁺ that absorbs at 500 nm. Presence of potential antioxidants results in the inhibition of linoleic acid oxidation and the reduction of absorption. [14,16,17,28,33,35,44,49,56,63]
Thiobarbituric acid reactive substances (TBARS) The presence of secondary oxidation products formed during oxidation of linoleic acid is measured by the reaction of one of them, the malondialdehyde, with SDS, acetic acid, and TBA at 532 nm. The presence of potential antioxidants reduces this absorbance. [20,23,31,32,44,53,57,66]
β-carotene linoleate It measures the ability of potential antioxidants to decrease the oxidative bleaching of β-carotene in an oil-in-water emulsion. The reaction is monitored by measuring the absorbance at 470 nm immediately after the addition of a potential antioxidant. [66]
Inhibition of Cu²⁺-induced low-density lipoprotein (LDL) peroxidation This assay measures the peroxidation induced by cupric sulfate in LDL. Presence of potential antioxidants results in the inhibition of the oxidation and the reduction of the absorbance of conjugated dienes at 344 nm. [66]
EVALUATION OF THE CAPACITY TO INHIBIT DNA OXIDATION
Supercoiled-to-Nicked-Circular-Conversion (SNCC) Oxidation of supercoiled DNA into nicked circular DNA in the presence of Cu²⁺ and H₂O₂ is monitored by measuring the fluorescent intensity of ethidium-stained nicked circular DNA. The presence of a potential antioxidant inhibits this reaction, and the signal corresponding to the oxidized form of DNA decreases. [30]
Inhibition of peroxyl and hydroxyl radical-induced supercoiled strands scission Strand scission of supercoiled DNA is measured in the presence of peroxyl and hydroxyl radicals. After incubation, DNA is separated by gel electrophoresis, and the intensity of supercoiled DNA bands in the presence and absence of potential antioxidant are compared. [66]
EVALUATION OF THE CAPACITY TO INHIBIT OXIDATIVE DAMAGE INDUCED IN CELLS
2’, 7’-dichloro-dihydro-fluorescein diacetate (DCFH-DA) fluorescent probe Oxidative stress in cells is induced by the addition of a strong oxidant (H₂O₂ or other peroxide). DCFH-DA fluorescence probe, added to cell culture, reacts with ROS to produce fluorescent DCF that is measured at an λexcitation of 488 nm and an λemission of 585 and 530 nm. The presence of a potential antioxidant inhibits ROS generation and DCF signal decreases. [16,51]
Intracellular concentration of Ca²⁺ determination Intracellular Ca²⁺ is measured with fluorescent dye Fura-2 AM. Fura-2AM is cleaved by intracellular esterase, and the resulting Fura-2 can bind to Ca²⁺ and cause strong fluorescence under a 330–350 nm excitation light. Fluorescence intensity decreases in H₂O₂-damaged cells treated with potential antioxidants. [51]
Acridine orange/ethidium bromide (AO/EB) fluorescent staining Cell membrane damage is measured by evaluating the staining of DNA with EB or AO using an inverted fluorescence microscope. The presence of potential antioxidants will reduce the number of red cells resulting from the staining with EB and will increase the number of green cells resulting from the staining with AO. [51]