Analytical Methods Used in Determining Antioxidant Activity: A Review

Abstract

The study of antioxidants and their implications in various fields, from food engineering to medicine and pharmacy, is of major interest to the scientific community. The present paper is a critical presentation of the most important tests used to determine the antioxidant activity, detection mechanism, applicability, advantages and disadvantages of these methods. Out of the tests based on the transfer of a hydrogen atom, the following were presented: the Oxygen Radical Absorption Capacity (ORAC) test, the Hydroxyl Radical Antioxidant Capacity (HORAC) test, the Total Peroxyl Radical Trapping Antioxidant Parameter (TRAP) test, and the Total Oxyradical Scavenging Capacity (TOSC) test. The tests based on the transfer of one electron include the Cupric Reducing Antioxidant Power (CUPRAC) test, the Ferric Reducing Antioxidant Power (FRAP) test, the Folin-Ciocalteu test. Mixed tests, including the transfer of both a hydrogen atom and an electron, include the 2,2'-Azinobis-(3-ethylbenzothiazoline-6-sulfonic acid (ABTS) test, and the [2,2-di(4-tert-octylphenyl)-1-picrylhydrazyl] (DPPH) test. All these assays are based on chemical reactions and assessing the kinetics or reaching the equilibrium state relies on spectrophotometry, presupposing the occurrence of characteristic colours or the discolouration of the solutions to be analysed, which are processes monitored by specific wavelength adsorption. These assays were successfully applied in antioxidant analysis or the determination of the antioxidant capacity of complex samples. As a complementary method in such studies, one may use methods based on electrochemical (bio)sensors, requiring stages of calibration and validation. The use of chemical methods together with electrochemical methods may result in clarification of the operating mechanisms and kinetics of the processes involving several antioxidants.

Keywords: antioxidant activity; reactive oxygen species (ROS); superoxyde dismutase (SOD).

Figure 1

Classification of antioxidants [6].

Figure 2

Application scope of antioxidants [7].

Figure 3

Reaction schemes involved in Oxygen Radical Absorption Capacity (ORAC) assay for the detection of hydroxyl and peroxyl radicals.

Figure 4

(1) Dihydrofluorescein diacetate; (2) Luminol.

Figure 5

Reaction scheme involved in Cupric Reducing Antioxidant Power (CUPRAC) assay. HA represents an antioxidant molecule and A⁺ an oxidised antioxidant molecule (a); Colour change in the assay (b).

Figure 6

Ferric reducing antioxidant power (FRAP) reaction mechanism. ArOH represents a phenolic antioxidant and [ArOH]⁺ an oxidised phenolic antioxidant (a); Chemical structure of the complexes involved in the chemical reaction and the colour change (b).

Figure 6

Ferric reducing antioxidant power (FRAP) reaction mechanism. ArOH represents a phenolic antioxidant and [ArOH]⁺ an oxidised phenolic antioxidant (a); Chemical structure of the complexes involved in the chemical reaction and the colour change (b).

Figure 7

Reaction between the phenolic compounds and the derivatives of the phosphotungstic and phosphomolybdic acids in an alkaline environment, resulting in the formation of a blue colour by the Folin–Ciocalteu method (a); Colour variation observed in the assay (b).

Figure 8

(A) The α-Keggin structure of the anionic derivative [PM₁₂O₄₀]³⁻, where M stands for molybdenum (Mo) or tungsten (W); (B) the big wheel structure of the blue complex [Mo₁₂₆⁶⁺Mo₂₈⁵⁺O₄₆₂H₁₄(H₂O)₇₀]¹⁴⁻.

Figure 9

Colour variation in ABTS assay (a); Reaction scheme involved in 2,2′-Azinobis-(3-ethylbenzothiazoline-6-sulfonic acid (ABTS) radical cation scavenging activity assay (b).

Figure 10

DPPH scavenging mechanisms by an antioxidant (AH).