**5. Antioxidants: mechanisms of action**

Generally, the antioxidants defend against free-radicals-induced oxidative damage by various mechanisms as discussed in below sections.

#### **5.1 Preventive antioxidants**

ROS such as H2O2, O2 • , and OH• are produced irreversibly during metabolism. Therefore, methods have been extensively studied to reduce the damage enhanced by oxidative stress. Intracellular antioxidant enzymes produced in the cell are an essential protective mechanism against free radicals formation. SOD, CAT, GPx, GR, GST, thioredoxin reductase, and hemeoxygenase are the most important antioxidants enzymes. SODs convert O2 • into H2O2, which is then converted into water by CAT, GPx, and Fenton reaction. Thus, two toxic species are converted into a harmless product (**Figure 3**) [5].

During metabolism, peroxides are formed and then eliminated via both GST and GPX. GRd regulates the equivalent of GSH and oxidized glutathione (GSSG), and the ratio of GSH/GSSG is a known index of oxidative stress [60]. The action of GRd plays an imperative role in increasing GSH concentration, which maintains the oxido-redox condition in the organism [14]. Consequently, the oxidative stress role has been reported in the progress and clinical symptom of autism. Recently, a comparison study between autism and control individuals showed decrease in GSH/GSSG ratio and increase in free radical generation in autism compared to control cells [60]. In addition, GPx is presented throughout the cell, while CAT is frequently limited to peroxisomes. In the brain, which is very sensitive to free radical damage, it has seven times more GPx activity than CAT activity. Moreover, CAT's highest levels are found in the liver, kidney, and erythrocytes, where it decomposes the most of H2O2 [61].

#### **5.2 Free radical scavengers**

#### *5.2.1 Scavenging superoxide and other ROS*

Superoxide (O2 •¯), a predominant cellular free radical, is contributed in a huge number of deleterious alterations often linked to a low concentration of antioxidants

**15**

OH•

**Figure 3.**

*Antioxidant enzyme system, O2*

activity as OH•

ing of OH•

*Antioxidant Categories and Mode of Action DOI: http://dx.doi.org/10.5772/intechopen.83544*

and associated with a raise in peroxidative processes. Though O2

*H2O2 into water. By Fenton reaction, H2O2 is also converted to the highly reactive OH•*

*5.2.2 Scavenging hydroxyl radical and other ROS*

*•*

*through oxidation of Fe2+ to Fe3+. Adapted from Pandey and Rizvi [5].*

*water by CAT and GPx. In this way, two toxic species, O2*

is considered to be formed from the Fe2+ or Cu+

Hydroxyl radical (OH•

nontoxic compounds [3].

form extremely reactive OH•

H2O2 to produce OH•

*5.2.3 Metal ion (Fe2+, Fe3+, Cu2+, and Cu+*

H2O2 forms more singlet oxygen than OH•

to biomolecules, it assists in production of stronger OH•, and ONOO¯.O2

in large quantities, in phagocytes via NADPH oxidase enzyme during pathogenkilling process. In addition, it is a byproduct of mitochondrial respiration [3].

*•*

*water. GPx neutralized H2O2 via taking hydrogen from two GSH molecules forming two molecules of water and GSSG. GR then regenerates GSH from GSSG. CAT, the essential part of enzymatic defense, neutralizes* 

molecules such as DNA, lipids, and proteins than other radical species. In general,

through incubation FeSO4 and H2O2 in aqueous solution. Therefore, antioxidants

scavenger can be accomplished by direct scavenging or prohibit-

generation by the chelation of free metal ions or altering H2O2 to other

*) chelating*

. Fe2+ and Cu+

radical in the presence of vitamin C (Eq. (3)). OH•

Even though trace minerals are essential dietary components, they can function

Cu2+, respectively. Cellular reductant such as NADH and oxidized metal ions Fe3+, and Cu2+ are reduced and permit the recycling to react with another molecule of

as prooxidants (through enhancing formation of free radicals). Fe2+ and Cu+

with H2O2, which is a product produced by the dismutation of the O2

) is an extremely active and more toxic radical on biologic

*, is dismutaed toH2O2 by SOD enzyme. The resulted H2O2 is converted into* 

 *and H2O2, are converted into the harmless product* 

(Eq. (2)). Dissimilarly, iron and copper's reaction with

•¯ itself is not reactive

 *and then to water* 

/H2O2 Fenton reaction system,

are oxidized to Fe3+ and

•¯ is formed

react

•¯ via SOD, to

is strongly

*Antioxidant Categories and Mode of Action DOI: http://dx.doi.org/10.5772/intechopen.83544*

#### **Figure 3.**

*Antioxidants*

*4.2.9 Tannins*

*4.2.9.1 Application*

Tannins are relatively high-molecular compounds, which comprise the third essential group of phenolics and can be divided into condensed and hydrolysable tannins. Condensed tannins are produced by the polymerization of flavonoid units. The mainly studied condensed tannins are based on flavan-3-ols: (−)-epicatechin and (+)-catechin. Hydrolysable tannins are heterogeneous polymers containing phenolic acids, in particular, gallic acid (3,4,5 trihydroxyl benzoic acid) and simple sugar [42, 55].

Because of tannin features, such as being the potential metal ion chelators, protein-precipitating agents, and biological antioxidants, tannins have different effects on biological systems. As a consequence of the diverse tannins biological roles and structural variation, it has been difficult to modify models that would let a precise prediction of their effects in any system. Therefore, the tannin structure modification

and activity relationship are important to predict their biological effect [42].

damage by various mechanisms as discussed in below sections.

, and OH•

Generally, the antioxidants defend against free-radicals-induced oxidative

Therefore, methods have been extensively studied to reduce the damage enhanced by oxidative stress. Intracellular antioxidant enzymes produced in the cell are an essential protective mechanism against free radicals formation. SOD, CAT, GPx, GR, GST, thioredoxin reductase, and hemeoxygenase are the most important

•

water by CAT, GPx, and Fenton reaction. Thus, two toxic species are converted into

During metabolism, peroxides are formed and then eliminated via both GST and GPX. GRd regulates the equivalent of GSH and oxidized glutathione (GSSG), and the ratio of GSH/GSSG is a known index of oxidative stress [60]. The action of GRd plays an imperative role in increasing GSH concentration, which maintains the oxido-redox condition in the organism [14]. Consequently, the oxidative stress role has been reported in the progress and clinical symptom of autism. Recently, a comparison study between autism and control individuals showed decrease in GSH/GSSG ratio and increase in free radical generation in autism compared to control cells [60]. In addition, GPx is presented throughout the cell, while CAT is frequently limited to peroxisomes. In the brain, which is very sensitive to free radical damage, it has seven times more GPx activity than CAT activity. Moreover, CAT's highest levels are found in the liver, kidney, and erythrocytes, where it decomposes the most of H2O2 [61].

•¯), a predominant cellular free radical, is contributed in a huge

number of deleterious alterations often linked to a low concentration of antioxidants

are produced irreversibly during metabolism.

into H2O2, which is then converted into

**5. Antioxidants: mechanisms of action**

•

antioxidants enzymes. SODs convert O2

a harmless product (**Figure 3**) [5].

**5.2 Free radical scavengers**

Superoxide (O2

*5.2.1 Scavenging superoxide and other ROS*

**5.1 Preventive antioxidants**

ROS such as H2O2, O2

**14**

*Antioxidant enzyme system, O2 • , is dismutaed toH2O2 by SOD enzyme. The resulted H2O2 is converted into water by CAT and GPx. In this way, two toxic species, O2 • and H2O2, are converted into the harmless product water. GPx neutralized H2O2 via taking hydrogen from two GSH molecules forming two molecules of water and GSSG. GR then regenerates GSH from GSSG. CAT, the essential part of enzymatic defense, neutralizes H2O2 into water. By Fenton reaction, H2O2 is also converted to the highly reactive OH• and then to water through oxidation of Fe2+ to Fe3+. Adapted from Pandey and Rizvi [5].*

and associated with a raise in peroxidative processes. Though O2 •¯ itself is not reactive to biomolecules, it assists in production of stronger OH•, and ONOO¯.O2 •¯ is formed in large quantities, in phagocytes via NADPH oxidase enzyme during pathogenkilling process. In addition, it is a byproduct of mitochondrial respiration [3].

#### *5.2.2 Scavenging hydroxyl radical and other ROS*

Hydroxyl radical (OH• ) is an extremely active and more toxic radical on biologic molecules such as DNA, lipids, and proteins than other radical species. In general, OH• is considered to be formed from the Fe2+ or Cu+ /H2O2 Fenton reaction system, through incubation FeSO4 and H2O2 in aqueous solution. Therefore, antioxidants activity as OH• scavenger can be accomplished by direct scavenging or prohibiting of OH• generation by the chelation of free metal ions or altering H2O2 to other nontoxic compounds [3].

#### *5.2.3 Metal ion (Fe2+, Fe3+, Cu2+, and Cu+ ) chelating*

Even though trace minerals are essential dietary components, they can function as prooxidants (through enhancing formation of free radicals). Fe2+ and Cu+ react with H2O2, which is a product produced by the dismutation of the O2 •¯ via SOD, to form extremely reactive OH• (Eq. (2)). Dissimilarly, iron and copper's reaction with H2O2 forms more singlet oxygen than OH• . Fe2+ and Cu+ are oxidized to Fe3+ and Cu2+, respectively. Cellular reductant such as NADH and oxidized metal ions Fe3+, and Cu2+ are reduced and permit the recycling to react with another molecule of H2O2 to produce OH• radical in the presence of vitamin C (Eq. (3)). OH• is strongly

reactive and can directly react with proteins and lipids to produce carbonyls (aldehydes and ketones), cross linking, and lipid peroxidation. Chelating metal ions are able to decrease their action, thus reducing the ROS formation.

$$\text{Fe}^{2+} \text{(or Cu}^{\ast}\text{)} \star \text{H}\_2\text{O}\_2 \text{Fe}^{3+} \text{(or Cu}^{2+}\text{)} \star \text{OH}^{\bullet} \text{+ OH}^{\cdot} \tag{2}$$

$$\text{Fe}^{3+} \text{(or Cu}^{2+}\text{)} \text{ + } \text{vit} \text{ C H}^{-} \text{ Fe}^{2+} \text{(or Cu}^{+}\text{)} \text{ + } \text{vit} \text{amin} \text{ C}^{-\*} \text{ + H}^{+} \tag{3}$$

Studies showed that Se antioxidant is able to chelate Cu<sup>+</sup> (formed in situ with Cu2+/ascorbic acid) extremely efficiently and prevent the damage of DNA by OH• radical (formed via Cu<sup>+</sup> /H2O2) [3].

#### **5.3 Free radical generating enzyme inhibitors**

It has been reported that the main sources of free radicals in different physiological and pathological conditions is associated with a number of enzymes. NADPH oxidases are a type of plasma membrane linked enzymes that have an ability to transfer one electron from the cytosolic donor NADPH to a molecule of extracellular oxygen, forming O2 •¯ [62]. Uric acid is formed by xanthin oxidase enzyme through catalyzing the oxidation of hypoxanthine and xanthin to uric acid yielding O2 •¯ and H2O2 and increase the oxidation level in an organism [63]. In addition, O2 •¯ is also formed as a by-product of mitochondrial respiration as well as several other enzymes, for example NADH oxidase, monooxygenases and cyclooxygenases. O2 •¯ is biologically quite toxic and is produced in significant amounts by the enzyme NADPH oxidase to be used in oxygen dependent killing mechanisms for invading pathogens. During the respiratory burst, it is an important control of reactive oxygen derivatives production for the defense of an organism against invading microorganisms, without causing an important loss of tissue functions [3]. Nonetheless, excessive ROS enhance oxidative stress such as low density lipoprotein (LDL) oxidation. A direct link between elevated phagocytic NADPH oxidase activities and increased circulating oxidized LDL in metabolic syndrome patients has been found. As a result, both modulation of NADPH oxidase to prohibit ROS overproduction and antioxidants supplementation have been reported as active strategies to prevent the deleterious effect of oxidative stress in hemodialysis patients [64]. In recent years, many natural antioxidants have revealed potential to inhibit enzymes that promote O2 •¯ generation as well as the development of new therapeutic agents for oxidative stress-related diseases [3].

## **5.4 Prevention of lipid peroxidation**

Lipid peroxidation is defined as oxidative deterioration of lipids composed of C-C double bonds such as unsaturated fatty acids, glycolipids, cholesterol, cholesterol ester, phospholipids. ROS damage the unsaturated fatty acids, which include numerous double bonds and the methylene-CH2-groups with particularly reactive hydrogen atoms, and begin the radical peroxidation chain reactions [65]. Antioxidants are able to directly react and quench peroxide radicals to stop the chain reaction. Lipid peroxidation and DNA damage are related to different chronic diseases, such as cancer, and atherosclerosis. Antioxidants can scavenge ROS and peroxide radicals, therefore prohibiting or treating certain pathogenic situations. Scientific attention has been concentrated in lipid peroxidation for recognizing natural antioxidants and studying their mechanism of action. Researches on antioxidants such as vitamins, polyphenols and flavones against free radical enhanced lipid peroxidation have been assumed in

**17**

*Antioxidant Categories and Mode of Action DOI: http://dx.doi.org/10.5772/intechopen.83544*

**5.5 Prevention of DNA damage**

*In vivo*, the OH•

damage caused by Cu+

O═N─O─O¯

**6. Conclusion**

with the free radicals.

tion of O2

situ with ascorbic acid. The OH•

**5.6 Prevention of protein modification**

ONOO¯ is a much stronger oxidizing agent than O2

antioxidants, like polyphenols, may scavenge O2

and the microenvironment of the reaction medium [3].

induced OH•

causing the ordinarily supercoiled plasmid DNA to unwind [3].

range of different molecules such as DNA and proteins. ONOO¯

many systems such as lipid, red blood cells and LDL. The antioxidant activity of these polyphenols depends considerably on molecules structure, the initiation conditions

react directly with plasmid DNA macromolecules to cleave one DNA strand, leading to oxidative DNA damage. Cell death and mutation as a result of DNA damage are associated with neurodegenerative and heart diseases, cancer and aging. Consequently, DNA or plasmid damage has received attention and been utilized as models for the study and identification of antioxidants [66]. A study has been progressed include DNA

Cu2+, ascorbic acid and H2O2 at pH 7. The reaction includes reduction of Cu2+ to Cu+

Besides lipid peroxidation and DNA damage, protein modification through nitration or chloration of amino acids also is caused by ROS. *In vivo*, peroxynitrite,

form peroxynitrous acids (ONOOH) are capable of exerting direct oxidative modifications during one or two electron oxidation processes [67]. *In vivo*, ONOO¯ reacts nucleophically with CO2 to produce nitrosoperoxy carbonate, which is the predominant pathway for ONOO¯. These modifications often cause the alteration of protein function or structure, in addition to enzyme activities inhibition. Proteins containing nitrotyrosine residues have been detected in various pathogenic conditions, such as diabetes, hypertension, and atherosclerosis, all linked with promoted oxidative stress, including increased formation of ONOO¯. Antioxidants and antioxidant enzyme are utilized to prevent the protein modification of ONOO¯. Antioxidants or enzyme such as CAT is able to remove H2O2 and also inhibit HOCl formation; similarly, SOD or

This chapter briefly summarized types of antioxidants, and their mode of action. The harmful products formed during normal cellular functions are oxygen radical derivatives that are the most important free radical in the biological system. For normal physiological functioning, it is important to maintain a tolerated antioxidant status by increasing intake of natural antioxidants. Studies have shown that different types of antioxidants, including natural and synthetic antioxidants, can help in disease prevention. The antioxidant compounds may directly react with the reactive radicals to destroy them via accepting or donating electron(s) to directly remove the unpaired status of the radical. Moreover, they may indirectly reduce the production of free radicals by inhibiting the efficacy or expressions of free radical creating enzymes or by stimulating the activities and expressions of other antioxidant enzymes. Thus, it is essential to know the antioxidant mechanisms of action

, is a powerful oxidant and nitrating agent formed through the reac-

•¯ with free radical nitric oxide via a diffusion-controlled reaction. In cells,

radical formed via Cu+

and ONOO¯ radicals produced from nitric oxide and O2

, through metal-free plasmid DNA mixed with

/H2O2 cleaves one DNA strand,

•¯ and is able to damage a wide

•¯ and inhibit ONOO¯ formation [3].

and its protonated

•¯ are able to

in

many systems such as lipid, red blood cells and LDL. The antioxidant activity of these polyphenols depends considerably on molecules structure, the initiation conditions and the microenvironment of the reaction medium [3].
