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

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

These methods are briefly described below.

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

The TRAP is used to determine the status of a secondary antioxidant in plasma. The results (TRAP value) are expressed as μmol of ROO• trapped per liter of plasma [58]. 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) (ABAP), which produces ROO• at a constant rate (**Figure 9**). This starts with the 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], phycoerythrin [69], and luminol [70].

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 the use of luminol and horseradish peroxidase [71].

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

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

#### *Antioxidants*

oxidizing agents, such as • OH, ROO• , and ONOO<sup>−</sup> [59] (**Figure 10**). These oxidizing agents induce a transformation of KMBA to ethylene. To evaluate the effect of antioxidants, the ethylene formation is evaluated and compared to a control reaction by the use of headspace gas chromatography (HS-GC). The TOSCA assay is based on the inhibition of ethylene formation in the presence of antioxidant compounds that compete with KMBA for ROS.

The TOSCA method is not suitable for a high performance analysis because multiple injections of each sample are required to measure ethylene production [55]. The reaction kinetics of this method do not allow a linear relationship between the percentage of inhibition of KMBA oxidation and the concentration of antioxidants [72], which is a serious limitation.

#### **7.3 Crocin-bleaching assay (CBA)**

The crocin bleaching test (CBA) is a method originally proposed to evaluate the inhibition of alkoxyl radicals produced photolytically. This is done by measuring the protective effect exerted by antioxidant compounds on crocin, a carotenoid that presents an intense red color, under the effect of alkoxyl radicals [60] (**Figure 11**). To achieve this, reaction kinetics are carried out in a UV-Vis spectrophotometer, measuring the absorbance at a wavelength of 440 nm to obtain the relative velocity constants. These constants present a good correlation with the known antioxidant activity of reference compounds. The absolute bleaching velocity of crocin depends strongly on the type of radical that attacks the polyene structure of crocin. Crocin exhibits a high selectivity toward the alkoxyl radicals produced during the photolysis of hydroperoxides, as well as peroxyl radicals produced after the thermolysis of azo initiators. Ordoudi and Tsimidou [73] carried out a detailed evaluation of the CBA, and among the factors, they considered the crocin probe, the antioxidant compound to be evaluated, the peroxyl radical generation conditions, and the monitoring of the reaction. As a result of this, they found that any commercial saffron could be used as a source of crocin for the preparation of the probe, because it is possible to eliminate interferences, such as tocopherols. They also found that the concentration of the working solution could be adjusted and that changes in the

**Figure 9.** *Formation of peroxyl radical from ABAP.*

**37**

β-cyclodextrins [78].

*Antioxidant Compounds and Their Antioxidant Mechanism*

stock solution of the probe can occur during storage. Ordoudi and Tsimidou [74] also evaluated a group of 39 phenolic compounds of diverse structures, including hydroxybenzoic, hydroxyphenylacetic, hydroxyphenylpropanoic, and hydroxycinnamic acids. The results of that study showed that the activity depends strongly on the position of -COOH groups in relation to the position of the -OH groups. Therefore, the CBA allows evaluation of the effect of the position of functional

The ORAC method is based on the inhibition of oxidation induced by peroxyl radicals and simultaneously evaluates the time effect and the inhibition degree. The ORAC test is based on hydrogen atom transfer (HAT) and uses a reaction mechanism that competes between antioxidants and a fluorescence probe (fluorescein) for a radical [61]. The test begins with the thermal decomposition of azo compounds, such as [2,2′-azobis-(2-amidino-propane)dihydrochloride (AAPH)], which is the source of free radicals that promotes the degradation of fluorescein. The antioxidant to be evaluated promotes the elimination of the peroxyl radicals, protecting the fluorescein from degradation. The decay in fluorescence due to the attack of the radicals and the protection by the antioxidants results in a curve. The antioxidant capacity is calculated from the area under the fluorescence decrease curve (AUC). This assay uses trolox as a standard; therefore, generally the antioxidant activity in this assay is expressed in terms of trolox equivalents. The ORAC method has been widely used to measure the antioxidant capacity of beverages [75], supplements

There are modifications to this assay that include the use of fluorescein as a probe, adaptation to a high performance format, and the ability to measure the

The ORAC assay is carried out at pH 7.4, adjusted with a phosphate buffer, in the presence of the antioxidant, AAPH, and fluorescein at a constant temperature of 37°C. Fluorescence is monitored at 1 min intervals for 35 min at an excitation

radicals by modifying the initiators. In addition, the method has been modified for the detection of lipophilic antioxidants, encapsulating these compounds in

as a stable free radical because pi electrons of the aromatic systems present in the

most other free radicals do. The delocalization of the electron also gives rise to a

 **method**

OH and other

) (**Figure 12**) is characterized

does not dimerize, as

lipophilic, hydrophilic, and total antioxidant capacity of a substance.

wavelength of 485 nm and an emission wavelength of 520 nm [77]. The ORAC method can also be used for the detection of •

The 1,1-diphenyl-2-picrylhydrazyl radical (DPPH•

molecule can compensate for the lack of an electron. DPPH•

groups that cause antioxidant activity in a chemical compound.

**7.4 Oxygen radical absorbance capacity (ORAC) method**

[55], and vegetables and fruits [55, 76].

**7.5 Radical scavenging capacity DPPH•**

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

**Figure 11.**

*Chemical structure of crocin.*

**Figure 10.** *Reaction between ROO• and KMBA.*

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

**Figure 11.** *Chemical structure of crocin.*

*Antioxidants*

oxidizing agents, such as •

compete with KMBA for ROS.

**7.3 Crocin-bleaching assay (CBA)**

antioxidants [72], which is a serious limitation.

OH, ROO•

agents induce a transformation of KMBA to ethylene. To evaluate the effect of antioxidants, the ethylene formation is evaluated and compared to a control reaction by the use of headspace gas chromatography (HS-GC). The TOSCA assay is based on the inhibition of ethylene formation in the presence of antioxidant compounds that

The TOSCA method is not suitable for a high performance analysis because multiple injections of each sample are required to measure ethylene production [55]. The reaction kinetics of this method do not allow a linear relationship between the percentage of inhibition of KMBA oxidation and the concentration of

The crocin bleaching test (CBA) is a method originally proposed to evaluate the inhibition of alkoxyl radicals produced photolytically. This is done by measuring the protective effect exerted by antioxidant compounds on crocin, a carotenoid that presents an intense red color, under the effect of alkoxyl radicals [60] (**Figure 11**). To achieve this, reaction kinetics are carried out in a UV-Vis spectrophotometer, measuring the absorbance at a wavelength of 440 nm to obtain the relative velocity constants. These constants present a good correlation with the known antioxidant activity of reference compounds. The absolute bleaching velocity of crocin depends strongly on the type of radical that attacks the polyene structure of crocin. Crocin exhibits a high selectivity toward the alkoxyl radicals produced during the photolysis of hydroperoxides, as well as peroxyl radicals produced after the thermolysis of azo initiators. Ordoudi and Tsimidou [73] carried out a detailed evaluation of the CBA, and among the factors, they considered the crocin probe, the antioxidant compound to be evaluated, the peroxyl radical generation conditions, and the monitoring of the reaction. As a result of this, they found that any commercial saffron could be used as a source of crocin for the preparation of the probe, because it is possible to eliminate interferences, such as tocopherols. They also found that the concentration of the working solution could be adjusted and that changes in the

, and ONOO<sup>−</sup> [59] (**Figure 10**). These oxidizing

**36**

**Figure 10.**

**Figure 9.**

*Reaction between ROO•*

*Formation of peroxyl radical from ABAP.*

 *and KMBA.*

stock solution of the probe can occur during storage. Ordoudi and Tsimidou [74] also evaluated a group of 39 phenolic compounds of diverse structures, including hydroxybenzoic, hydroxyphenylacetic, hydroxyphenylpropanoic, and hydroxycinnamic acids. The results of that study showed that the activity depends strongly on the position of -COOH groups in relation to the position of the -OH groups. Therefore, the CBA allows evaluation of the effect of the position of functional groups that cause antioxidant activity in a chemical compound.

## **7.4 Oxygen radical absorbance capacity (ORAC) method**

The ORAC method is based on the inhibition of oxidation induced by peroxyl radicals and simultaneously evaluates the time effect and the inhibition degree. The ORAC test is based on hydrogen atom transfer (HAT) and uses a reaction mechanism that competes between antioxidants and a fluorescence probe (fluorescein) for a radical [61]. The test begins with the thermal decomposition of azo compounds, such as [2,2′-azobis-(2-amidino-propane)dihydrochloride (AAPH)], which is the source of free radicals that promotes the degradation of fluorescein. The antioxidant to be evaluated promotes the elimination of the peroxyl radicals, protecting the fluorescein from degradation. The decay in fluorescence due to the attack of the radicals and the protection by the antioxidants results in a curve. The antioxidant capacity is calculated from the area under the fluorescence decrease curve (AUC). This assay uses trolox as a standard; therefore, generally the antioxidant activity in this assay is expressed in terms of trolox equivalents. The ORAC method has been widely used to measure the antioxidant capacity of beverages [75], supplements [55], and vegetables and fruits [55, 76].

There are modifications to this assay that include the use of fluorescein as a probe, adaptation to a high performance format, and the ability to measure the lipophilic, hydrophilic, and total antioxidant capacity of a substance.

The ORAC assay is carried out at pH 7.4, adjusted with a phosphate buffer, in the presence of the antioxidant, AAPH, and fluorescein at a constant temperature of 37°C. Fluorescence is monitored at 1 min intervals for 35 min at an excitation wavelength of 485 nm and an emission wavelength of 520 nm [77].

The ORAC method can also be used for the detection of • OH and other radicals by modifying the initiators. In addition, the method has been modified for the detection of lipophilic antioxidants, encapsulating these compounds in β-cyclodextrins [78].

#### **7.5 Radical scavenging capacity DPPH• method**

The 1,1-diphenyl-2-picrylhydrazyl radical (DPPH• ) (**Figure 12**) is characterized as a stable free radical because pi electrons of the aromatic systems present in the molecule can compensate for the lack of an electron. DPPH• does not dimerize, as most other free radicals do. The delocalization of the electron also gives rise to a

**Figure 12.** *DPPH• reduction by an antioxidant.*

deep violet color, characterized by absorption in solution at around 517 nm. Brand-Williams et al. [62] evaluated the activity of specific compounds or extracts using DPPH• in solution. When a solution of DPPH• is in contact with a substance that can donate a hydrogen atom or with another radical (R• ), the reduced form DPPH-H or DPPH-R is produced with the consequent loss of color and therefore the decrease or loss of absorbance (**Figure 8**). Consequently, the reduction of DPPH• provides an index to estimate the ability of the test compound to trap radicals. The alcoholic solutions of 0.5 mM are densely colored, and in this concentration, the law of Lambert-Beer is fulfilled in the useful absorption interval [79].

ArOH is an antioxidant that acts by donating hydrogen atoms, to obtain radicals with stable molecular structures that will stop the chain reaction. The new radical (ArO• ) can interact with another radical to form stable molecules (DPPH-OAr, ArO-OAr). The reaction between DPPH• and an antioxidant compound depends on the structural conformation of the same, so quantitative comparisons are not always appropriate.

The basis of this methodology is focused on measuring the reduction of free radicals by antioxidant compounds. Different concentrations and the time of the reaction are measured (30 min or until the steady state is reached). So far, there are no reports about the existence of a mathematical kinetic model that helps to understand the behavior of antioxidants [80].

The experimental models use the percentage of DPPH• remaining to obtain the necessary quantities that are required to reduce the initial concentration to 50% (EC50). In addition, kinetics is performed to determine the amount of time needed for the steady state to reach EC50 from the curves. EC50 and effective concentration 50 (TEC50) are used to calculate antiradical efficiency (AE). Low values of EC50 and TEC50 show a high antioxidant strength, and a rapid decrease in absorption is observed during the reaction [81]. The antiradical efficiency can be estimated based on the scale contained in **Table 3**.

It is a fast, simple, inexpensive, and widely used method to measure the ability of compounds to act as free radical scavengers or hydrogen donors. It can also be used to quantify antioxidants in complex biological systems, for solid or liquid samples. The method is applied to measure the overall antioxidant capacity [82] and the activity of eliminating free radicals from fruit and vegetable juices [83]. It has been successfully used to investigate the antioxidant properties of wheat grain and bran, vegetables, oils, and flours in various solvents, including ethanol, aqueous acetone, methanol, and benzene [84–87].

The radical scavenging DPPH• method allows for a reaction with almost any type of antioxidant due to the stability of DPPH• . This means there is sufficient

**39**

**Table 4.**

*Antioxidant Compounds and Their Antioxidant Mechanism*

time for even weak antioxidants to react with DPPH•

concentration ratio of the antioxidant/DPPH•

Because the radical scavenging DPPH•

AE = 1 × 10<sup>−</sup><sup>3</sup> Low

*Scale of antiradical efficiency (AE) against DPPH•*

/DPPH

Flow injection analysis (FIA) by high performance liquid chromatography (HPLC)

PC-controlled sequential injection analysis (SIA)

Electrochemical selective determination of antioxidant activity based on DPPH•

Relative DPPH radical scavenging capacity (RDSC)

High performance thin layer chromatography (TLC)-DPPH•

Hyphenated high speed counter current chromatography (HSCCC)-DPPH•

< AE = 5 × 10<sup>−</sup><sup>3</sup> Medium

samples

DPPH•

antioxidant reactions

wavelengths

*Automated modes to evaluate radical scavenging capacity DPPH•*

The plates are scanned before DPPH•

on-line radical-scavenging detection

 < AE = 10 × 10<sup>−</sup><sup>3</sup> High AE ≫ 10 × 10<sup>−</sup><sup>3</sup> Very high

dissolved oxygen [88]. The absorbance of DPPH•

**7.6 Ferric reducing/antioxidant power (FRAP) method**

with both polar and nonpolar organic solvents to evaluate hydrophilic and lipophilic

other radicals and consequently the time to reach the stable state is not linear to the

ous fields of chemistry, automated assays combined with analytical techniques have

The FRAP analysis was introduced by [65, 96] to measure total antioxidant activity and is based on the ability of samples to reduce ferric ion Fe3+ to ferrous ion

 *[81].*

**Automation Characteristics References**

after reaction with the antioxidant

Bioassay-guided fractionation of natural products or food

SIA is a FIA technique modified by using a pump to continuously draw sample and reagent solutions into different lines of tubing

Current intensity is proportional to the residual concentration of

The RDSC uses the area under the curve, expressed as trolox equivalents. These approaches take into account both the kinetic and the thermodynamic measurements of the radical-

Post-chromatographic derivatization is carried out with DPPH•

derivatization in absorption-reflection mode at optimized

After the HSCCC separation, the effluent is split into two streams by use of an adjustable high-pressure stream splitter. One portion is sent through the detector and the fraction collector, while the second portion is sent to a secondary coil for

*.*

**Range Antiradical efficiency classification**

be affected by solvents with properties of a Lewis base, as well as the presence of

The method has some disadvantages, among which is that DPPH•

[82]. This method can be used

[62, 80]. The stability of DPPH•

method is quite simple and used in vari-

in methanol and acetone is lower

can react with

[90]

[91]

[92]

[93]

[94]

[95]

.

and 30 min after DPPH

can

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

antioxidants [55].

than with other solvents [89].

been developed (**Table 4**).

1 × 10<sup>−</sup><sup>3</sup>

5 × 10<sup>−</sup><sup>3</sup>

**Table 3.**

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

time for even weak antioxidants to react with DPPH• [82]. This method can be used with both polar and nonpolar organic solvents to evaluate hydrophilic and lipophilic antioxidants [55].

The method has some disadvantages, among which is that DPPH• can react with other radicals and consequently the time to reach the stable state is not linear to the concentration ratio of the antioxidant/DPPH• [62, 80]. The stability of DPPH• can be affected by solvents with properties of a Lewis base, as well as the presence of dissolved oxygen [88]. The absorbance of DPPH• in methanol and acetone is lower than with other solvents [89].

Because the radical scavenging DPPH• method is quite simple and used in various fields of chemistry, automated assays combined with analytical techniques have been developed (**Table 4**).

### **7.6 Ferric reducing/antioxidant power (FRAP) method**

The FRAP analysis was introduced by [65, 96] to measure total antioxidant activity and is based on the ability of samples to reduce ferric ion Fe3+ to ferrous ion


#### **Table 3.**

*Antioxidants*

DPPH•

**Figure 12.** *DPPH•*

(ArO•

appropriate.

deep violet color, characterized by absorption in solution at around 517 nm. Brand-Williams et al. [62] evaluated the activity of specific compounds or extracts using

DPPH-R is produced with the consequent loss of color and therefore the decrease

an index to estimate the ability of the test compound to trap radicals. The alcoholic solutions of 0.5 mM are densely colored, and in this concentration, the law of

ArOH is an antioxidant that acts by donating hydrogen atoms, to obtain radicals with stable molecular structures that will stop the chain reaction. The new radical

) can interact with another radical to form stable molecules (DPPH-OAr,

the structural conformation of the same, so quantitative comparisons are not always

The basis of this methodology is focused on measuring the reduction of free radicals by antioxidant compounds. Different concentrations and the time of the reaction are measured (30 min or until the steady state is reached). So far, there are no reports about the existence of a mathematical kinetic model that helps to

necessary quantities that are required to reduce the initial concentration to 50% (EC50). In addition, kinetics is performed to determine the amount of time needed for the steady state to reach EC50 from the curves. EC50 and effective concentration 50 (TEC50) are used to calculate antiradical efficiency (AE). Low values of EC50 and TEC50 show a high antioxidant strength, and a rapid decrease in absorption is observed during the reaction [81]. The antiradical efficiency can be estimated based

It is a fast, simple, inexpensive, and widely used method to measure the ability of compounds to act as free radical scavengers or hydrogen donors. It can also be used to quantify antioxidants in complex biological systems, for solid or liquid samples. The method is applied to measure the overall antioxidant capacity [82] and the activity of eliminating free radicals from fruit and vegetable juices [83]. It has been successfully used to investigate the antioxidant properties of wheat grain and bran, vegetables, oils, and flours in various solvents, including ethanol, aqueous

or loss of absorbance (**Figure 8**). Consequently, the reduction of DPPH•

Lambert-Beer is fulfilled in the useful absorption interval [79].

The experimental models use the percentage of DPPH•

is in contact with a substance that can

and an antioxidant compound depends on

method allows for a reaction with almost any

. This means there is sufficient

remaining to obtain the

), the reduced form DPPH-H or

provides

in solution. When a solution of DPPH•

donate a hydrogen atom or with another radical (R•

ArO-OAr). The reaction between DPPH•

 *reduction by an antioxidant.*

understand the behavior of antioxidants [80].

on the scale contained in **Table 3**.

acetone, methanol, and benzene [84–87]. The radical scavenging DPPH•

type of antioxidant due to the stability of DPPH•

**38**

*Scale of antiradical efficiency (AE) against DPPH• [81].*


#### **Table 4.**

*Automated modes to evaluate radical scavenging capacity DPPH• .* 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 of hydroxylation and the conjugation in the phenols [55].

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].
