**5. Mechanisms of action of antioxidants**

A compound that reduces *in vitro* radicals does not necessarily behave as an antioxidant in an *in vivo* system. This is because FR diffuse and spread easily. Some have extremely short life spans, on the order of nanoseconds, so it is difficult for the antioxidant to be present at the time and place where oxidative damage is being generated. Additionally, the reactions between antioxidants and FR are second order reactions. Therefore, they not only depend on the concentration of antioxidants and free radicals but are also dependent on factors related to the chemical structure of both reagents, the medium and the reaction conditions.

#### **5.1 Phenolic compounds**

The phenolic compounds constitute a wide group of chemical substances, with diverse chemical structures and different biological activities, encompassing more than 8000 different compounds which are a significant part of the human and animal diet [24]. The phenolic compounds are important components in the mechanism of signaling and defense of plants. These compounds combat the stress brought about by pathogenic organisms and predators. The function of these compounds in plants is diverse: they are found as precursors of compounds of greater complexity or the intervention in the processes of regulation and control of plant growth, as well as the defensive medium of plants. Phenolic compounds have the capacity to act as hydrogen donors or to chelate metal ions such as iron and copper, by inhibiting the oxidation of low-density lipoproteins (LDL). These characteristics in the phenolic compounds are associated with a decrease in risks of neurodegenerative diseases, such as cardiovascular diseases [25], gastrointestinal cancers [26], colon [27], breast and ovarian cancers [28], and leukemia [29–31]. Phenolic compounds also have vasorelaxation and anti-allergenic activity [32]. The phenolic compounds inhibit the oxidation of *in vitro* LDL [33].

**31**

*Antioxidant Compounds and Their Antioxidant Mechanism*

Phenolic compounds reduce or inhibit free radicals by transfer of a hydrogen atom, from its hydroxyl group. The reaction mechanism of a phenolic compound

from the phenol to the radical, forming a transition state of an H-O bond with one electron. The antioxidant capacity of the phenolic compounds is strongly reduced when the reaction medium consists of a solvent prone to the formation of hydrogen bonds with the phenolic compounds. For example, alcohols have a double effect on the reaction rate between the phenol and the peroxyl radical. On the one hand, the alcohols act as acceptors of hydrogen bonds. On the other hand, they favor the ionization of the phenols to anion phenoxides, which can react rapidly with the peroxyl radicals, through an electron transfer. The overall effect of the solvent on the antioxidant activity of the phenolic compounds depends to a great extent on the degree of ionization of the last compounds [34]. Leopoldini et al. [35] conducted a theoretical study to determine the dissociation energy of OH bonds and the adiabatic ionization potentials of phenolic compounds of varied structure and polarity, among them tyrosol, hydroxytyrosol, and gallic and caffeic acids. These studies were performed simulating solvated and vacuum conditions. The results showed a clear difference in the behavior of these phenolic compounds. The compounds most likely to undergo a HAT were tocopherol, followed by hydroxytyrosol, gallic acid, caffeic acid, and epicatechin (**Figure 6**), while the phenolic compounds, which were better able to SET, were kaempferol and resveratrol (**Figure 7**). This undoubtedly gives us an indication that phenolic compounds can suffer both HAT and SET and that this depends mainly on the chemical structure of the phenolic compounds. The method based on the Folin-Ciocalteu reagent is commonly used to determine and quantify total phenols. This method evaluates the ability of phenols to react with oxidizing agents. The Folin-Ciocalteu reagent contains sodium molybdate and tungstate, which react with any type of phenol [36]. The transfer of electrons at basic pH reduces the sodium molybdate and tungstate in oxides of tungsten (W8O23) and molybdenum (Mo8O23), which have a bright blue color in solution. This color intensity is proportional to the number of hydroxyl groups of

Carotenoids are found in virtually all plants, animals, and microorganisms, and more than 700 carotenoids have been identified and characterized [38]. Most carotenoids have a characteristic symmetrical tetraterpene skeleton. The linear hydrocarbon skeleton is made up of 40 carbons and is susceptible to various structural modifications. These structural characteristics are related to degree of hydrogenation, *cis*-*trans* isomerization, presence of cycles at one or both ends of the linear skeleton, or the addition of side groups (which often contain oxygen) with their subsequent glycosylation. The most complex changes are related to the shortening or elongation of the resulting tetraterpene skeleton, to form carotenoids with chains of 50 carbons. It is also possible to find carotenoids with tetraterpene skeletons of 30 carbons, from the condensation of two units of farnesyl [39]. These compounds, in addition to conferring pigmentation on biological systems, fulfill other important functions. The most recent studies of these compounds are focused mainly on evaluating their function as antioxidants. The structural base fragment of the carotenoids is a conjugated polyunsaturated chain. This fragment is primarily responsible for the ability of these compounds to inhibit free radicals. Variations in the polyunsaturated chain from one carotenoid to another, together with the presence of hydroxyl groups, substantially modify the reactivity of the carotenoids. The reactivity of these compounds is also affected by the environmental conditions where they are

) involves a concerted transfer of the hydrogen cation

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

with a peroxyl radical (ROO•

the molecule [37].

**5.2 Carotenoids**

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

*Antioxidants*

examples include systems of DNA repair enzymes (polymerases, glycosylases, and nucleases) and proteolytic enzymes (proteinases, proteases, and peptidases) found in both the cytosol and the mitochondria of mammalian cells. Specific examples of these enzymes are GPx, glutathione reductase (GR), and methionine sulfoxide reductase (MSR). These enzymes act as intermediaries in the repair process of the oxidative damage caused by the attack of excess ROS. Any environmental factor that inhibits or modifies a regular biological activity becomes a condition that favors

The main characteristic of a compound or antioxidant system is the prevention or detection of a chain of oxidative propagation, by stabilizing the generated radical, thus helping to reduce oxidative damage in the human body [21]. Gordon [22] provided a classification of antioxidants, mentioning that characteristic. There are two main types of antioxidants, the primary (breaking the chain reaction, free radical scavengers) and the secondary or preventive. The secondary antioxidant mechanisms may include the deactivation of metals, inhibition of lipid hydroperoxides by interrupting the production of undesirable volatiles, the regeneration of primary antioxidants, and the elimination of singlet oxygen. Therefore, antioxidants can be defined as "those substances that, in low quantities, act by preventing or greatly retarding the oxidation of easily oxidizable materials such as fats" [23].

A compound that reduces *in vitro* radicals does not necessarily behave as an antioxidant in an *in vivo* system. This is because FR diffuse and spread easily. Some have extremely short life spans, on the order of nanoseconds, so it is difficult for the antioxidant to be present at the time and place where oxidative damage is being generated. Additionally, the reactions between antioxidants and FR are second order reactions. Therefore, they not only depend on the concentration of antioxidants and free radicals but are also dependent on factors related to the chemical structure of

The phenolic compounds constitute a wide group of chemical substances, with diverse chemical structures and different biological activities, encompassing more than 8000 different compounds which are a significant part of the human and animal diet [24]. The phenolic compounds are important components in the mechanism of signaling and defense of plants. These compounds combat the stress brought about by pathogenic organisms and predators. The function of these compounds in plants is diverse: they are found as precursors of compounds of greater complexity or the intervention in the processes of regulation and control of plant growth, as well as the defensive medium of plants. Phenolic compounds have the capacity to act as hydrogen donors or to chelate metal ions such as iron and copper, by inhibiting the oxidation of low-density lipoproteins (LDL). These characteristics in the phenolic compounds are associated with a decrease in risks of neurodegenerative diseases, such as cardiovascular diseases [25], gastrointestinal cancers [26], colon [27], breast and ovarian cancers [28], and leukemia [29–31]. Phenolic compounds also have vasorelaxation and anti-allergenic activity [32]. The phenolic compounds inhibit the oxidation of *in vitro* LDL [33].

the appearance or reinforcement of oxidative stress.

**4. Characteristics of antioxidants**

**5. Mechanisms of action of antioxidants**

both reagents, the medium and the reaction conditions.

**5.1 Phenolic compounds**

**30**

Phenolic compounds reduce or inhibit free radicals by transfer of a hydrogen atom, from its hydroxyl group. The reaction mechanism of a phenolic compound with a peroxyl radical (ROO• ) involves a concerted transfer of the hydrogen cation from the phenol to the radical, forming a transition state of an H-O bond with one electron. The antioxidant capacity of the phenolic compounds is strongly reduced when the reaction medium consists of a solvent prone to the formation of hydrogen bonds with the phenolic compounds. For example, alcohols have a double effect on the reaction rate between the phenol and the peroxyl radical. On the one hand, the alcohols act as acceptors of hydrogen bonds. On the other hand, they favor the ionization of the phenols to anion phenoxides, which can react rapidly with the peroxyl radicals, through an electron transfer. The overall effect of the solvent on the antioxidant activity of the phenolic compounds depends to a great extent on the degree of ionization of the last compounds [34]. Leopoldini et al. [35] conducted a theoretical study to determine the dissociation energy of OH bonds and the adiabatic ionization potentials of phenolic compounds of varied structure and polarity, among them tyrosol, hydroxytyrosol, and gallic and caffeic acids. These studies were performed simulating solvated and vacuum conditions. The results showed a clear difference in the behavior of these phenolic compounds. The compounds most likely to undergo a HAT were tocopherol, followed by hydroxytyrosol, gallic acid, caffeic acid, and epicatechin (**Figure 6**), while the phenolic compounds, which were better able to SET, were kaempferol and resveratrol (**Figure 7**). This undoubtedly gives us an indication that phenolic compounds can suffer both HAT and SET and that this depends mainly on the chemical structure of the phenolic compounds.

The method based on the Folin-Ciocalteu reagent is commonly used to determine and quantify total phenols. This method evaluates the ability of phenols to react with oxidizing agents. The Folin-Ciocalteu reagent contains sodium molybdate and tungstate, which react with any type of phenol [36]. The transfer of electrons at basic pH reduces the sodium molybdate and tungstate in oxides of tungsten (W8O23) and molybdenum (Mo8O23), which have a bright blue color in solution. This color intensity is proportional to the number of hydroxyl groups of the molecule [37].

#### **5.2 Carotenoids**

Carotenoids are found in virtually all plants, animals, and microorganisms, and more than 700 carotenoids have been identified and characterized [38]. Most carotenoids have a characteristic symmetrical tetraterpene skeleton. The linear hydrocarbon skeleton is made up of 40 carbons and is susceptible to various structural modifications. These structural characteristics are related to degree of hydrogenation, *cis*-*trans* isomerization, presence of cycles at one or both ends of the linear skeleton, or the addition of side groups (which often contain oxygen) with their subsequent glycosylation. The most complex changes are related to the shortening or elongation of the resulting tetraterpene skeleton, to form carotenoids with chains of 50 carbons. It is also possible to find carotenoids with tetraterpene skeletons of 30 carbons, from the condensation of two units of farnesyl [39]. These compounds, in addition to conferring pigmentation on biological systems, fulfill other important functions. The most recent studies of these compounds are focused mainly on evaluating their function as antioxidants. The structural base fragment of the carotenoids is a conjugated polyunsaturated chain. This fragment is primarily responsible for the ability of these compounds to inhibit free radicals. Variations in the polyunsaturated chain from one carotenoid to another, together with the presence of hydroxyl groups, substantially modify the reactivity of the carotenoids. The reactivity of these compounds is also affected by the environmental conditions where they are

#### *Antioxidants*

**Figure 6.** *Phenolic compounds with ability to HAT.*

**Figure 7.** *Phenolic compounds with ability to SET.*

found. For example, Edge and Truscott [40] found that carotenoids switch the antioxidant behavior to the prooxidant as a function of oxygen concentration. The study used a system that emulates a cell, to observe the protection effect induced by lycopene when exposing the system to high-energy radiation. Total protection is achieved in the absence of O2, but in the presence of 100% O2, protection is completely lost. Carotenoids are characterized as excellent peroxyl radical scavengers. The polyunsaturated chains that make up the base structure of carotenoids give these compounds a lipophilic character. Carotenoids play an important role in the protection of cell membranes and lipoproteins against peroxyl radicals.

The carotenoids react as antioxidant agents through three mechanisms: the first is the SET, the second from the formation of one adduct, and the third by HAT. In general, the antioxidant properties of carotenoids are related to their high capacity for electron donation. Everett et al. [41] found that β-carotene reacts with NO2 • via SET. Carotenoid reactivity studies have also been carried out in the presence of the benzyl peroxyl radical, which has low reactivity, and it was concluded that in this case, the reaction mechanisms involved the formation of an adduct, while reactions by HAT are of little relevance [42].

Other studies have evaluated the effect of the chemical structure of carotenoids on the reactivity toward FR. One of these studies found that carotenoids substituted with electrons are more susceptible to oxidation than carotenoids with withdrawn electron groups. A study of carotenoid reactivity with phenoxy radicals shows the

**33**

2 × 105

(SET/HAT) [52].

*Antioxidant Compounds and Their Antioxidant Mechanism*

adducts takes place first and then the SET [44].

**6. Methods to evaluate antioxidant activity**

order of reactivity to be lycopene > β-carotene > zeaxanthin > lutein > echinenone >

The effect of the solvent on the reactivity of carotenoids in the presence of FR has also been evaluated, and it was found that in nonpolar solvents, the reactions are promoted via adduct formation; while in polar solvents, the formation of

Vitamin C refers to a group of ascorbic acid analogs that can be both synthetic and natural molecules. Ascorbic acid is a water-soluble ketolactone with two ionizable hydroxyl groups. Anion ascorbate is the dominant form at physiological pH (**Figure 8**). Ascorbate is a potent reducing agent and undergoes two subsequent losses of an electron, to form an ascorbate radical and dehydroascorbic acid. The ascorbate radical is relatively stable because the unpaired electron is delocalized by resonance. The ascorbate concentration in plasma of healthy humans is around 10 μg/mL. At these concentrations, the ascorbate is a co-antioxidant with vitamin E to protect LDL from peroxyl radicals [45]. The ascorbate radical is poorly reactive and can be reduced to ascorbate by reductase-dependent NADH and NADPH [46]. The ascorbate radical can alternatively undergo a disproportionation reaction that depends on pH, resulting in the formation of ascorbate and

Vitamin C is chemically capable of reacting with most of the physiologically important ROS and acts as a hydrosoluble antioxidant. The antioxidant reaction mechanisms of vitamin C are based on the HAT to peroxyl radicals, the inactivation of singlet oxygen, and the elimination of molecular oxygen [48, 49]. For example, ascorbic acid can donate a hydrogen atom to a tocopheroxyl radical at the rate of

 mol/s [50]. Also, it has been proven that ascorbate can produce reactions with oxidizing agents through SET [51] or a concerted transfer of electron/protons

The antioxidant activity of a compound can be evaluated *in vitro* or *in vivo* by means of simple experiments, and at the same time, the possible prooxidant effect

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

astaxanthin [43].

*Chemical species related to vitamin C.*

**Figure 8.**

**5.3 Vitamin C**

dehydroascorbic acid [47].

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

**Figure 8.** *Chemical species related to vitamin C.*

order of reactivity to be lycopene > β-carotene > zeaxanthin > lutein > echinenone > astaxanthin [43].

The effect of the solvent on the reactivity of carotenoids in the presence of FR has also been evaluated, and it was found that in nonpolar solvents, the reactions are promoted via adduct formation; while in polar solvents, the formation of adducts takes place first and then the SET [44].

#### **5.3 Vitamin C**

*Antioxidants*

**Figure 6.**

**Figure 7.**

*Phenolic compounds with ability to HAT.*

*Phenolic compounds with ability to SET.*

**32**

by HAT are of little relevance [42].

found. For example, Edge and Truscott [40] found that carotenoids switch the antioxidant behavior to the prooxidant as a function of oxygen concentration. The study used a system that emulates a cell, to observe the protection effect induced by lycopene when exposing the system to high-energy radiation. Total protection is achieved in the absence of O2, but in the presence of 100% O2, protection is completely lost. Carotenoids are characterized as excellent peroxyl radical scavengers. The polyunsaturated chains that make up the base structure of carotenoids give these compounds a lipophilic character. Carotenoids play an important role in the

protection of cell membranes and lipoproteins against peroxyl radicals.

The carotenoids react as antioxidant agents through three mechanisms: the first is the SET, the second from the formation of one adduct, and the third by HAT. In general, the antioxidant properties of carotenoids are related to their high capacity for electron donation. Everett et al. [41] found that β-carotene reacts with NO2

SET. Carotenoid reactivity studies have also been carried out in the presence of the benzyl peroxyl radical, which has low reactivity, and it was concluded that in this case, the reaction mechanisms involved the formation of an adduct, while reactions

Other studies have evaluated the effect of the chemical structure of carotenoids on the reactivity toward FR. One of these studies found that carotenoids substituted with electrons are more susceptible to oxidation than carotenoids with withdrawn electron groups. A study of carotenoid reactivity with phenoxy radicals shows the

• via

Vitamin C refers to a group of ascorbic acid analogs that can be both synthetic and natural molecules. Ascorbic acid is a water-soluble ketolactone with two ionizable hydroxyl groups. Anion ascorbate is the dominant form at physiological pH (**Figure 8**). Ascorbate is a potent reducing agent and undergoes two subsequent losses of an electron, to form an ascorbate radical and dehydroascorbic acid. The ascorbate radical is relatively stable because the unpaired electron is delocalized by resonance. The ascorbate concentration in plasma of healthy humans is around 10 μg/mL. At these concentrations, the ascorbate is a co-antioxidant with vitamin E to protect LDL from peroxyl radicals [45]. The ascorbate radical is poorly reactive and can be reduced to ascorbate by reductase-dependent NADH and NADPH [46]. The ascorbate radical can alternatively undergo a disproportionation reaction that depends on pH, resulting in the formation of ascorbate and dehydroascorbic acid [47].

Vitamin C is chemically capable of reacting with most of the physiologically important ROS and acts as a hydrosoluble antioxidant. The antioxidant reaction mechanisms of vitamin C are based on the HAT to peroxyl radicals, the inactivation of singlet oxygen, and the elimination of molecular oxygen [48, 49]. For example, ascorbic acid can donate a hydrogen atom to a tocopheroxyl radical at the rate of 2 × 105 mol/s [50]. Also, it has been proven that ascorbate can produce reactions with oxidizing agents through SET [51] or a concerted transfer of electron/protons (SET/HAT) [52].

#### **6. Methods to evaluate antioxidant activity**

The antioxidant activity of a compound can be evaluated *in vitro* or *in vivo* by means of simple experiments, and at the same time, the possible prooxidant effect on different molecules can be evaluated. Antioxidant activity cannot be measured directly but is determined by the effects of the antioxidant to control the degree of oxidation. There are a variety of methods to evaluate antioxidant activity. Some methods involve a different oxidation step followed by the measurement of the response, which depends on the method used to evaluate the activity.

When the antioxidant activity of a sample is studied, it is necessary to consider the source of ROS as well as the target substrate. An antioxidant can protect lipids against oxidative damage, while, on the other hand, it can promote the oxidation of other biological molecules [53].

Most assays of antioxidant activity involve inducing accelerated oxidation in the presence of a promoter and controlling one or more variables in the test system, for example, temperature, antioxidant concentration, pH, etc. However, the oxidation mechanisms can change when modifications are carried out on some of these variables. Therefore, it is important to evaluate the intervals in which the quantification of the antioxidant activity is done to generate reliable results.


**35**

*Antioxidant Compounds and Their Antioxidant Mechanism*

The methods to determine the antioxidant capacity are divided into two general groups. This division is based on the reaction mechanisms involved in the RF reduction process. The first group of methods is based on the SET and the second group is based on the HAT. The result is the same: the inactivation of free radicals; however, the kinetics and secondary reactions involved in the process are different. The methods based on SET detect the capacity of a potential antioxidant for the transmission of a chemical species, including metals, carbonyls, and radicals. SET is shown through a change in color as the oxidant is reduced by antioxidant [54]. The group of methods based on HAT measures the ability of an antioxidant to inactivate FR through the donation of a hydrogen atom. HAT reactions are theoretically independent of solvent nature and pH. These reactions are rapid and occur in no more than a few minutes. The presence of other reducing agents in samples, in addition to the antioxidants under study, makes HAT testing difficult and can lead to significant errors [55]. **Table 2** shows the methods

The methods of evaluation of antioxidant activity must be fast, reproducible, and require small amounts of the chemical compounds to be analyzed, in addition to not being influenced by the physical properties of said compounds [56]. The results of *in vitro* assays can be used as a direct indicator of antioxidant activity *in vivo*; a compound that is ineffective *in vitro* will not be better *in vivo* [53]. These tests can also serve as warnings of possible harmful effects of chemical compounds. Because many factors can affect oxidation, including temperature, the concentration of oxygen in the reaction medium, and metal catalysts, the results may vary depending on the oxidation conditions employed. Tests that measure substrates or

The TRAP is used to determine the status of a secondary antioxidant in plasma. The

at a constant rate (**Figure 9**). This starts with the

The test is based on the measurement of O2 uptake during a controlled peroxidation reaction, promoted by the thermal decomposition of 2,2′-azobis-(2-amidopropane)

addition of ABAP to human plasma; the parameter to be evaluated is the "delay time" of the O2 absorption in plasma induced by the antioxidant compounds present in the medium. The delay time is measured from the O2 concentration data in plasma diluted in a buffer solution monitored with an electrode. In addition to ABAP, other free radical initiators have been used, such as the ABTS [67], dichlorofluorescein diacetate [68],

One of the main disadvantages of the TRAP method is the possibility of an error in the detection of the end point caused by the instability of the O2 electrode, because this point can take 2 h to reach. To minimize this problem, the electrochemical detection of O2 can be performed with a chemiluminescent detection based on

This method is based on the evaluation of antioxidant activity in the gas phase, which consists of exposing α-keto-γ-methylthiobutyric acid (KMBA) to powerful

trapped per liter of plasma [58].

products can also give variable results depending on their specificity [57].

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

of evaluation of the antioxidant activity *in vitro*.

These methods are briefly described below.

results (TRAP value) are expressed as μmol of ROO•

the use of luminol and horseradish peroxidase [71].

**7.2 Total oxyradical scavenging capacity assay (TOSCA)**

(ABAP), which produces ROO•

phycoerythrin [69], and luminol [70].

**7.1 Total radical-trapping antioxidant parameter (TRAP)**

**7. Antioxidant capacity/activity** *in vitro* **evaluation**

### **Table 2.**

*Methods most commonly used to evaluate antioxidant capacity/activity in vitro.*

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

*Antioxidants*

other biological molecules [53].

**Method Reaction** 

Total radical-trapping antioxidant parameter

Total oxyradical scavenging capacity total assay (TOSCA)

Crocin-bleaching assays

Oxygen radical absorbance capacity (ORAC)

Inhibition of 2,2-diphenyl-1-picrylhydracyl radical

Inhibition of 2,2′-azino-bis- (3-ethylbenzothiazoline-6 sulphonic acid) (ABTS•+)

Total phenols assay by Folin-

Ferric-reducing antioxidant

Total antioxidant capacity

(TRAP)

(CBAs)

(DPPH• )

cation radical

Ciocalteu reagent

power (FRAP)

(TAC)

**Table 2.**

on different molecules can be evaluated. Antioxidant activity cannot be measured directly but is determined by the effects of the antioxidant to control the degree of oxidation. There are a variety of methods to evaluate antioxidant activity. Some methods involve a different oxidation step followed by the measurement of the

When the antioxidant activity of a sample is studied, it is necessary to consider the source of ROS as well as the target substrate. An antioxidant can protect lipids against oxidative damage, while, on the other hand, it can promote the oxidation of

Most assays of antioxidant activity involve inducing accelerated oxidation in the presence of a promoter and controlling one or more variables in the test system, for example, temperature, antioxidant concentration, pH, etc. However, the oxidation mechanisms can change when modifications are carried out on some of these variables. Therefore, it is important to evaluate the intervals in which the quantification

HAT TRAP assay involves the initiation of lipid

HAT Evaluates inhibition oxidation of α-keto-γ-

**Characteristics Reference**

and

[58]

[59]

[60]

[61]

[62]

[63]

[64]

[65]

[66]

peroxidation by generating water-soluble ROO•

methiolbutyric acid (KMBA) by ROS. The antioxidant activity is measured through ethylene concentration, generated during decomposition of KMBA, relative to a control reaction monitored by headspace gas chromatography (HS-GC)

HAT CBA is based on the abstraction of hydrogen atoms

system accounting for crocin bleaching

HAT ORAC assay is based upon the inhibition of peroxyl radical induced oxidation initiated by thermal decomposition of azo compounds such as AAPH

SET or HAT Colorimetric method based on the measurement

SET or HAT Colorimetric method to evaluate the decay of ABTS•+ in the presence of an antioxidant agent

SET Colorimetric method that evaluates the reduction

SET This method is used to measure the peroxide level

form a complex with thiocyanate

SET A mixture of phosphomolybdate and

phenolic compounds

DPPH•

*Methods most commonly used to evaluate antioxidant capacity/activity in vitro.*

is sensitive to all known chain-breaking antioxidants

and/or addition of radical to the polyene structure of crocin and results in a disruption of the conjugated

of the scavenging capacity of antioxidants towards

phosphotungstate in highly basic medium oxidized

of Fe3+-tripyridyltriazine complex (Fe3+-TPTZ) by turning it into a ferrous form (Fe2+-TPTZ)

during the initial stage of lipid oxidation. Peroxides are formed during the linoleic acid oxidation, which reacts with Fe2+ to form Fe3+ and later these ions

response, which depends on the method used to evaluate the activity.

of the antioxidant activity is done to generate reliable results.

**mechanism**

**34**

The methods to determine the antioxidant capacity are divided into two general groups. This division is based on the reaction mechanisms involved in the RF reduction process. The first group of methods is based on the SET and the second group is based on the HAT. The result is the same: the inactivation of free radicals; however, the kinetics and secondary reactions involved in the process are different. The methods based on SET detect the capacity of a potential antioxidant for the transmission of a chemical species, including metals, carbonyls, and radicals. SET is shown through a change in color as the oxidant is reduced by antioxidant [54]. The group of methods based on HAT measures the ability of an antioxidant to inactivate FR through the donation of a hydrogen atom. HAT reactions are theoretically independent of solvent nature and pH. These reactions are rapid and occur in no more than a few minutes. The presence of other reducing agents in samples, in addition to the antioxidants under study, makes HAT testing difficult and can lead to significant errors [55]. **Table 2** shows the methods of evaluation of the antioxidant activity *in vitro*.
