**7.7 Method of inhibition of the (2,2′-azinobis-(3-ethylbenozothiazoline-6 sulfonate)) radical cation (ABTS•+)**

ABTS is a target molecule used to evaluate the reactivity of antioxidant samples in the presence of peroxides. The ABTS initially is subjected to an oxidation reaction with potassium permanganate, potassium persulfate or 2,2′-azo-bis (2-amidinopropane), producing the radical cation of the ABTS (ABTS•+) with a blue greenish color that absorbs at wavelengths of 415, 645, 734, and 815 nm [100–102]. The ABTS•+ is stable for several minutes. The ABTS•+ is subjected to the antioxidant sample causing the reduction of ABTS•+ and consequently the discoloration of the reaction mixture (**Figure 14**). Therefore, the degree of discoloration can be expressed as the inhibition percentage of ABTS•+, which is determined as a function of antioxidant concentration and time. This method can be used at different pH and is useful to study the effect of pH on antioxidant activity. ABTS is soluble in both aqueous and organic solvents and consequently is useful for evaluating the antioxidant activity of samples in different media and is commonly used in solutions that simulate an ionic serum (pH 7.4) based on a phosphate buffer (PBS) containing 150 mM NaCl. When a medium of PBS is used, the samples react in a time interval of approximately 30 min, while in alcohol, they require longer reaction times [103]. The level of peroxide is determined by the absorbance at some of the above-mentioned wavelengths. The IC50 is calculated by plotting the percentage of inhibition against different

**41**

*Antioxidant Compounds and Their Antioxidant Mechanism*

concentrations of the antioxidant sample [104]. The IC50 values indicate the sample concentration required to eliminate 50% of the ABTS•+. Low IC50 values indicate high radical uptake activity. The antioxidant activity against ABTS•+ can also be evaluated through the unit of antioxidant activity (TAA), which expresses the equivalents of

The inhibition of ABTS•+ activity in an antioxidant sample has a strong correla-

TAC is defined as the ability of a compound to inhibit the oxidative degradation of lipids [66]. Lipid peroxidation involves the oxidative deterioration of lipids with unsaturation. This peroxidation, called the initiation process, begins with the formation of conjugated dienes and trienes, known as primary oxidation products due to the abstraction of a hydrogen atom. Subsequently, a propagation process is carried out that consists of the reaction of the deprotonated species derived from

energy of free radicals promotes the abstraction of hydrogen atoms from neighboring fatty acids. This leads to the formation of hydroperoxides that promotes the

stable molecules of the R-R and ROOR type [107]. To encourage the antioxidant activity of a chemical compound, it is necessary to inhibit the peroxidation of a fatty acid emulsion; linoleic acid is generally used as a model. The hydroperoxides derived from linoleic acid subsequently react with Fe2+, causing the oxidation of this ion to produce Fe3+. The Fe3+ ions form a complex with thiocyanate (SCN<sup>−</sup>), and this complex has a maximum absorbance at 500 nm [108]. This complex is used to

The ferric thiocyanate method is used to measure the peroxide value in edible oils. To avoid errors in the determination of the peroxide value, it is important to avoid the presence of oxygen in the reaction medium and this can be achieved by bubbling nitrogen [109]. These authors found that the results of the thiocyanate assay also depend on the solvent, reducing agent and type of hydroperoxides pres-

The reaction mechanisms involved in the antioxidant activity/capacity of different groups of compounds depend on several factors. Among these factors are the chemical structure of these compounds, the nature of the solvent, the temperature

radicals. The latter radicals react with each other to produce

because both radicals have the

). The high

from the antioxidant compounds present in the

trolox in μmol with respect to each gram of sample extract in dry base.

the lipids with O2, leading to the formation of peroxyl radicals (ROO•

tion with the radical scavenging capacity DPPH•

capacity to accept electrons and H•

*Reaction of ABTS•+ with antioxidant compounds.*

**7.8 Total antioxidant capacity (TAC)**

samples [105, 106].

**Figure 14.**

formation of new R•

measure the peroxide value.

ent in the sample.

**8. Conclusions**

*DOI: http://dx.doi.org/10.5772/intechopen.85270*

**Figure 13.** *Reaction mechanism for the FRAP assay in the presence of an antioxidant [55].*

*Antioxidant Compounds and Their Antioxidant Mechanism DOI: http://dx.doi.org/10.5772/intechopen.85270*

#### **Figure 14.**

*Antioxidants*

Fe2+, forming a blue complex. A high absorption at a wavelength of 700 nm indicates a high reduction power of the chemical compound or extract [66]. The value of FRAP has been used to determine the antioxidant activity of red wines [97]. The work of Schleisier et al. [98] was designed to determine the antioxidant activity in tea extracts and juices expressed in Fe2+ equivalents. The absolute initial index of the reduction of ferrylmyoglobin determined by spectroscopy in the visible region has been suggested to characterize the antioxidant activity of individual flavonoids [99]. There are several trials to evaluate FRAP; one of them is to evaluate the power of a compound or extract to reduce the complex of 2,4,6-tripyridyl-s-triazine-Fe2+ (TPTZ-Fe2+). An antioxidant reduces the ferric ion (Fe3+) to ferrous ion (Fe2+) in the TPTZ complex; the latter forms a blue complex (Fe2+/TPTZ), which absorbs at a wavelength of 590 nm (**Figure 13**). The reaction must be carried out under acidic conditions (pH 3.6) to preserve the solubility of Fe. The reducing power is related to the degree

The FRAP assay has an incubation time of 4 min at 37°C for the antioxidant activity of most samples. This is done because the redox reactions, involved in the assay, occur within the incubation period. However, it has been shown that FRAP values can vary significantly, depending on the time scale of analysis [55, 96].

**7.7 Method of inhibition of the (2,2′-azinobis-(3-ethylbenozothiazoline-6-**

ABTS is a target molecule used to evaluate the reactivity of antioxidant samples in the presence of peroxides. The ABTS initially is subjected to an oxidation reaction with potassium permanganate, potassium persulfate or 2,2′-azo-bis (2-amidinopropane), producing the radical cation of the ABTS (ABTS•+) with a blue greenish color that absorbs at wavelengths of 415, 645, 734, and 815 nm [100–102]. The ABTS•+ is stable for several minutes. The ABTS•+ is subjected to the antioxidant sample causing the reduction of ABTS•+ and consequently the discoloration of the reaction mixture (**Figure 14**). Therefore, the degree of discoloration can be expressed as the inhibition percentage of ABTS•+, which is determined as a function of antioxidant concentration and time. This method can be used at different pH and is useful to study the effect of pH on antioxidant activity. ABTS is soluble in both aqueous and organic solvents and consequently is useful for evaluating the antioxidant activity of samples in different media and is commonly used in solutions that simulate an ionic serum (pH 7.4) based on a phosphate buffer (PBS) containing 150 mM NaCl. When a medium of PBS is used, the samples react in a time interval of approximately 30 min, while in alcohol, they require longer reaction times [103]. The level of peroxide is determined by the absorbance at some of the above-mentioned wavelengths. The IC50 is calculated by plotting the percentage of inhibition against different

of hydroxylation and the conjugation in the phenols [55].

*Reaction mechanism for the FRAP assay in the presence of an antioxidant [55].*

**sulfonate)) radical cation (ABTS•+)**

**40**

**Figure 13.**

*Reaction of ABTS•+ with antioxidant compounds.*

concentrations of the antioxidant sample [104]. The IC50 values indicate the sample concentration required to eliminate 50% of the ABTS•+. Low IC50 values indicate high radical uptake activity. The antioxidant activity against ABTS•+ can also be evaluated through the unit of antioxidant activity (TAA), which expresses the equivalents of trolox in μmol with respect to each gram of sample extract in dry base.

The inhibition of ABTS•+ activity in an antioxidant sample has a strong correlation with the radical scavenging capacity DPPH• because both radicals have the capacity to accept electrons and H• from the antioxidant compounds present in the samples [105, 106].

#### **7.8 Total antioxidant capacity (TAC)**

TAC is defined as the ability of a compound to inhibit the oxidative degradation of lipids [66]. Lipid peroxidation involves the oxidative deterioration of lipids with unsaturation. This peroxidation, called the initiation process, begins with the formation of conjugated dienes and trienes, known as primary oxidation products due to the abstraction of a hydrogen atom. Subsequently, a propagation process is carried out that consists of the reaction of the deprotonated species derived from the lipids with O2, leading to the formation of peroxyl radicals (ROO• ). The high energy of free radicals promotes the abstraction of hydrogen atoms from neighboring fatty acids. This leads to the formation of hydroperoxides that promotes the formation of new R• radicals. The latter radicals react with each other to produce stable molecules of the R-R and ROOR type [107]. To encourage the antioxidant activity of a chemical compound, it is necessary to inhibit the peroxidation of a fatty acid emulsion; linoleic acid is generally used as a model. The hydroperoxides derived from linoleic acid subsequently react with Fe2+, causing the oxidation of this ion to produce Fe3+. The Fe3+ ions form a complex with thiocyanate (SCN<sup>−</sup>), and this complex has a maximum absorbance at 500 nm [108]. This complex is used to measure the peroxide value.

The ferric thiocyanate method is used to measure the peroxide value in edible oils. To avoid errors in the determination of the peroxide value, it is important to avoid the presence of oxygen in the reaction medium and this can be achieved by bubbling nitrogen [109]. These authors found that the results of the thiocyanate assay also depend on the solvent, reducing agent and type of hydroperoxides present in the sample.

#### **8. Conclusions**

The reaction mechanisms involved in the antioxidant activity/capacity of different groups of compounds depend on several factors. Among these factors are the chemical structure of these compounds, the nature of the solvent, the temperature

and pH, as well as the reactivity and chemical structure of free radicals. All these factors can also influence the reaction rate. Consequently, it is very important that, for studies of antioxidant properties, at least three evaluation methods are selected: one to exclusively evaluate the HAT, another the SET, and a combined method, HAT/SET. Also, it is important to perform reaction kinetics. In addition to this, it is essential to consider that in mixtures of antioxidant compounds, possible synergistic effects are present and can enhance the activity/capacity or even modify their reaction mechanisms.
