Antioxidant Compounds and Their Antioxidant Mechanism

*Norma Francenia Santos-Sánchez, Raúl Salas-Coronado, Claudia Villanueva-Cañongo and Beatriz Hernández-Carlos*

## **Abstract**

An antioxidant is a substance that at low concentrations delays or prevents oxidation of a substrate. Antioxidant compounds act through several chemical mechanisms: hydrogen atom transfer (HAT), single electron transfer (SET), and the ability to chelate transition metals. The importance of antioxidant mechanisms is to understand the biological meaning of antioxidants, their possible uses, their production by organic synthesis or biotechnological methods, or for the standardization of the determination of antioxidant activity. In general, antioxidant molecules can react either by multiple mechanisms or by a predominant mechanism. The chemical structure of the antioxidant substance allows understanding of the antioxidant reaction mechanism. This chapter reviews the *in vitro* antioxidant reaction mechanisms of organic compounds polyphenols, carotenoids, and vitamins C against free radicals (FR) and prooxidant compounds under diverse conditions, as well as the most commonly used methods to evaluate the antioxidant activity of these compounds according to the mechanism involved in the reaction with free radicals and the methods of *in vitro* antioxidant evaluation that are used frequently depending on the reaction mechanism of the antioxidant.

**Keywords:** antioxidants, oxidative stress, reactive oxygen species, free radical, hydrogen atom transfer, single electron transfer

## **1. Introduction**

Oxidative stress in biological systems is a complex process that is characterized by an imbalance between the production of free radicals (FR) and the ability of the body to eliminate these reactive species through the use of endogenous and exogenous antioxidants. During the metabolic processes, a great variety of reactions take place, where the promoters are the reactive oxygen species (ROS), such as hydrogen peroxide (H2O2) and the superoxide radical anion (O2 •<sup>−</sup>), among others. A biological system in the presence of an excess of ROS can present different pathologies, from cardiovascular diseases to the promotion of cancer. Biological systems have antioxidant mechanisms to control damage of enzymatic and nonenzymatic natures that allow ROS to be inactivated. The endogenous antioxidants are enzymes, such as superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase, or non-enzymatic compounds, such as bilirubin and albumin. When an organism is exposed to a high concentration of ROS, the endogenous antioxidant system is compromised and, consequently, it fails to guarantee complete protection of the organism. To compensate this deficit

of antioxidants, the body can use exogenous antioxidants supplied through food, nutritional supplements, or pharmaceuticals. Among the most important exogenous antioxidants are phenolic compounds carotenoids and vitamins C and some minerals such as selenium and zinc.

In the study of antioxidant compounds and the mechanisms involved, it is important to distinguish between the concepts of antioxidant activity and capacity. These terms are often used interchangeably. However, antioxidant activity refers to the rate constant of a reaction between an antioxidant and an oxidant. The antioxidant capacity is a measure of the amount of a certain free radical captured by an antioxidant sample [1]. Therefore, during the selection of a method, the response parameter must be considered to evaluate the antioxidant properties of a sample, which may be a function of the concentration of the substrate or concentration and the time required to inhibit a defined concentration of the ROS.

The reaction mechanisms of the antioxidant compounds are closely related to the reactivity and chemical structure of FR as well as the environment in which these reactive species are found. Therefore, it is very important to describe the ROS and, to a lesser degree, the reactive nitrogen species (RNS), which include both precursors and free radicals.

In the literature, there are many *in vitro* methods to evaluate the effectiveness of antioxidant compounds present in a variety of matrices (plant extracts, blood serum, etc.) using lipophilic, hydrophilic, and amphiphilic media (emulsions). The *in vitro* methods can be divided into two main groups: (1) hydrogen atom transfer (HAT) reactions and (2) transfer reactions of a single electron (SET). These methods are widely used because of their high speed and sensitivity. When carrying out a study related to the antioxidant properties of a sample, more than one method is usually used to evaluate the antioxidant capacity/activity [2]. This chapter describes the methods of *in vitro* antioxidant evaluation that are used frequently depending on the reaction mechanism of the antioxidant.

## **2. Oxidative stress**

Oxygen is associated with aerobic life conditions [3], representing the driving force for the maintenance of cell metabolism and viability and at the same time involving a potential danger due to its paramagnetic characteristics. These characteristics promote the formation of partially oxidized intermediates with a high reactivity. These compounds are known as reactive oxygen species (ROS). ROS are free radicals (FR) or radical precursors. In stable neutral molecules, the electrons are paired in their respective molecular orbitals, known as maximum natural stability. Therefore, if there are unpaired electrons in an orbital, highly reactive, molecular species are generated that tend to trap an electron from any other molecule to compensate for its electron deficiency. The oxygen triplet is the main free radical, since it has two unpaired electrons. The reaction rate of triplet oxygen in biological systems is slow. However, it can become highly toxic because it metabolically transforms into one or more highly reactive intermediates that can react with cellular components. This metabolic activation is favored in biological systems, because the reduction of O2 to H2O in the electron transport chain occurs by the transfer of an electron to form FR or ROS [4].

Free radicals in a biological system can be produced by exogenous factors such as solar radiation, due to the presence of ultraviolet rays. Ultraviolet radiation causes the homolytic breakdown of bonds in molecules. FR also occur during the course of a disease. In a heart attack, for example, when the supply of oxygen and glucose to the heart muscle is suspended, many FR are produced. Another exogenous factor is

**25**

(NO2 •

*Antioxidant Compounds and Their Antioxidant Mechanism*

•<sup>−</sup>). O2

sion when consuming O2. The radical O2

As a defense mechanism cells generate •

on intracellular arginine. The combination of O2 with •

additives, pesticides, etc., in food can also become a source of FR.

chemical intoxication, which promotes the formation of FR. The organism, because it requires the conversion of toxic substances to less dangerous substances, promotes the release of FR. The toxicity of many drugs is actually due to their conversion into free radicals or their effect on the formation of FR. The presence of contaminants,

Inflammatory processes are due to endogenous factors that promote the presence of FR in the system. These FR, present inside the cleansing cells of the immune system, have the function of killing pathogenic microorganisms. Tissue damage is caused when FR are excessive during this process. Phagocytic cells (neutrophils, monocytes, or macrophages) use the NADPH oxidase system directly generating

with other molecules through enzymatic processes or catalyzed by metals gener-

from the irradiation of molecular oxygen with UV rays, photolysis of water, and by exposure of O2 to organic radicals formed in aerobic cells such as NAD•

semiquinone radicals, cation radical pyridinium or by hemoproteins. Likewise, it is produced by phagocytic leukocytes as the initial product of the respiratory explo-

marked way in autoimmune diseases such as rheumatoid arthritis, systemic lupus erythematosus, primary biliary cirrhosis, type 1 diabetes, celiac disease, Graves' disease, Hashimoto's disease, inflammatory bowel disease, scleroderma, multiple

FR are necessarily present during metabolic processes because many of the chemical reactions involved require these chemical species. For example, the reactions of polymerization of amino acids to form proteins or the reactions of polymerization of glucose to form glycogen involve the participation of FR. FR are also involved in the catalytic activation of various enzymes of intermediary metabolism, such as hypoxanthine, xanthine oxidase, aldehyde oxidase, monoamine oxidase, cyclooxygenase, and lipoxygenase [5]. Generally, antioxidant enzymes efficiently

Another generating source of ROS is the structural alteration of essential macromolecules of the cell (DNA, protein, and lipids) by irreversible chemical reactions. These reactions generate derivatives, such as malonaldehyde and hydroperoxides

), as well as peroxynitrite (ONOO<sup>−</sup>), nitrosoperoxycarbonate (ONOOCO2

and dinitrogen trioxide (N2O3). These species are generated in small amounts during normal cellular processes such as cell signaling, neurotransmission, muscle relaxation, peristalsis, platelet aggregation, blood pressure modulation, immune system control, phagocytosis, production of cellular energy, and regulation of cell growth [6]. **Table 1** shows the most representative FR present during the process of

), and the neutral species, peroxynitrous acid (ONOOH)

Additionally, there are also RNS, such as nitric oxide (NO•

There are many ROS that act as biological oxidants, but the O2

oxidant; the simple addition of a proton leads to the formation of HO2

very active oxidizing agent. These transformations are summarized in **Figure 1**.

+

energy production in aerobic biological systems.

**2.1 Oxidative damage to biomolecules**

•<sup>−</sup> is protonated to produce H2O2 and HO2

, which induces lipid peroxidation in lipoproteins. This happens in a very

•<sup>−</sup> is considered the primary ROS and when reacting

• . O2

•<sup>−</sup> does not react directly with polypeptides,

NO by the action of nitric oxide-synthase

NO results in the formation

), nitrogen dioxide

•<sup>−</sup> is the largest

, becoming a

•

<sup>−</sup>),

•<sup>−</sup> is produced

, FpH• ,

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

the superoxide ion (O2

ates secondary ROS. O2

sugars, or nucleic acids.

control these radicals.

and nitronium ions (NO2

sclerosis, psoriasis, and vitiligo.

that propagate oxidative damage.

of ONOO•

*Antioxidants*

such as selenium and zinc.

precursors and free radicals.

**2. Oxidative stress**

form FR or ROS [4].

on the reaction mechanism of the antioxidant.

of antioxidants, the body can use exogenous antioxidants supplied through food, nutritional supplements, or pharmaceuticals. Among the most important exogenous antioxidants are phenolic compounds carotenoids and vitamins C and some minerals

In the study of antioxidant compounds and the mechanisms involved, it is important to distinguish between the concepts of antioxidant activity and capacity. These terms are often used interchangeably. However, antioxidant activity refers to the rate constant of a reaction between an antioxidant and an oxidant. The antioxidant capacity is a measure of the amount of a certain free radical captured by an antioxidant sample [1]. Therefore, during the selection of a method, the response parameter must be considered to evaluate the antioxidant properties of a sample, which may be a function of the concentration of the substrate or concentration and

The reaction mechanisms of the antioxidant compounds are closely related to the reactivity and chemical structure of FR as well as the environment in which these reactive species are found. Therefore, it is very important to describe the ROS and, to a lesser degree, the reactive nitrogen species (RNS), which include both

In the literature, there are many *in vitro* methods to evaluate the effectiveness of antioxidant compounds present in a variety of matrices (plant extracts, blood serum, etc.) using lipophilic, hydrophilic, and amphiphilic media (emulsions). The *in vitro* methods can be divided into two main groups: (1) hydrogen atom transfer (HAT) reactions and (2) transfer reactions of a single electron (SET). These methods are widely used because of their high speed and sensitivity. When carrying out a study related to the antioxidant properties of a sample, more than one method is usually used to evaluate the antioxidant capacity/activity [2]. This chapter describes the methods of *in vitro* antioxidant evaluation that are used frequently depending

Oxygen is associated with aerobic life conditions [3], representing the driving force for the maintenance of cell metabolism and viability and at the same time involving a potential danger due to its paramagnetic characteristics. These characteristics promote the formation of partially oxidized intermediates with a high reactivity. These compounds are known as reactive oxygen species (ROS). ROS are free radicals (FR) or radical precursors. In stable neutral molecules, the electrons are paired in their respective molecular orbitals, known as maximum natural stability. Therefore, if there are unpaired electrons in an orbital, highly reactive, molecular species are generated that tend to trap an electron from any other molecule to compensate for its electron deficiency. The oxygen triplet is the main free radical, since it has two unpaired electrons. The reaction rate of triplet oxygen in biological systems is slow. However, it can become highly toxic because it metabolically transforms into one or more highly reactive intermediates that can react with cellular components. This metabolic activation is favored in biological systems, because the reduction of O2 to H2O in the electron transport chain occurs by the transfer of an electron to

Free radicals in a biological system can be produced by exogenous factors such as solar radiation, due to the presence of ultraviolet rays. Ultraviolet radiation causes the homolytic breakdown of bonds in molecules. FR also occur during the course of a disease. In a heart attack, for example, when the supply of oxygen and glucose to the heart muscle is suspended, many FR are produced. Another exogenous factor is

the time required to inhibit a defined concentration of the ROS.

**24**

chemical intoxication, which promotes the formation of FR. The organism, because it requires the conversion of toxic substances to less dangerous substances, promotes the release of FR. The toxicity of many drugs is actually due to their conversion into free radicals or their effect on the formation of FR. The presence of contaminants, additives, pesticides, etc., in food can also become a source of FR.

Inflammatory processes are due to endogenous factors that promote the presence of FR in the system. These FR, present inside the cleansing cells of the immune system, have the function of killing pathogenic microorganisms. Tissue damage is caused when FR are excessive during this process. Phagocytic cells (neutrophils, monocytes, or macrophages) use the NADPH oxidase system directly generating the superoxide ion (O2 •<sup>−</sup>). O2 •<sup>−</sup> is considered the primary ROS and when reacting with other molecules through enzymatic processes or catalyzed by metals generates secondary ROS. O2 •<sup>−</sup> is protonated to produce H2O2 and HO2 • . O2 •<sup>−</sup> is produced from the irradiation of molecular oxygen with UV rays, photolysis of water, and by exposure of O2 to organic radicals formed in aerobic cells such as NAD• , FpH• , semiquinone radicals, cation radical pyridinium or by hemoproteins. Likewise, it is produced by phagocytic leukocytes as the initial product of the respiratory explosion when consuming O2. The radical O2 •<sup>−</sup> does not react directly with polypeptides, sugars, or nucleic acids.

As a defense mechanism cells generate • NO by the action of nitric oxide-synthase on intracellular arginine. The combination of O2 with • NO results in the formation of ONOO• , which induces lipid peroxidation in lipoproteins. This happens in a very marked way in autoimmune diseases such as rheumatoid arthritis, systemic lupus erythematosus, primary biliary cirrhosis, type 1 diabetes, celiac disease, Graves' disease, Hashimoto's disease, inflammatory bowel disease, scleroderma, multiple sclerosis, psoriasis, and vitiligo.

FR are necessarily present during metabolic processes because many of the chemical reactions involved require these chemical species. For example, the reactions of polymerization of amino acids to form proteins or the reactions of polymerization of glucose to form glycogen involve the participation of FR. FR are also involved in the catalytic activation of various enzymes of intermediary metabolism, such as hypoxanthine, xanthine oxidase, aldehyde oxidase, monoamine oxidase, cyclooxygenase, and lipoxygenase [5]. Generally, antioxidant enzymes efficiently control these radicals.

Another generating source of ROS is the structural alteration of essential macromolecules of the cell (DNA, protein, and lipids) by irreversible chemical reactions. These reactions generate derivatives, such as malonaldehyde and hydroperoxides that propagate oxidative damage.

Additionally, there are also RNS, such as nitric oxide (NO• ), nitrogen dioxide (NO2 • ), as well as peroxynitrite (ONOO<sup>−</sup>), nitrosoperoxycarbonate (ONOOCO2 <sup>−</sup>), and nitronium ions (NO2 + ), and the neutral species, peroxynitrous acid (ONOOH) and dinitrogen trioxide (N2O3). These species are generated in small amounts during normal cellular processes such as cell signaling, neurotransmission, muscle relaxation, peristalsis, platelet aggregation, blood pressure modulation, immune system control, phagocytosis, production of cellular energy, and regulation of cell growth [6]. **Table 1** shows the most representative FR present during the process of energy production in aerobic biological systems.

#### **2.1 Oxidative damage to biomolecules**

There are many ROS that act as biological oxidants, but the O2 •<sup>−</sup> is the largest oxidant; the simple addition of a proton leads to the formation of HO2 • , becoming a very active oxidizing agent. These transformations are summarized in **Figure 1**.


#### **Table 1.**

*Free radicals (FR) generated in biological systems.*

Free radicals produce diverse actions on the metabolism of immediate principles, which can be the origin of cell damage [7]:

