**2.3. Generation and chemical reactions of free radicals**

Free radicals are generated through various physiological processes in living organisms. Once generated, they can react with other biomolecules to attain stability.

**Superoxide (O• 2 )** is generally produced when a single electron is added unto oxygen. In living systems, superoxide can be generated through several mechanisms [10]. Several molecules such as flavine nucleotides, adrenaline, thiol compounds, glucose, etc. can be oxidized in the presence of oxygen to generate superoxide and these reactions are greatly accelerated by the presence of transition metals such as iron or copper. During the electron transport chain in the inner mitochondrial membrane, oxygen is reduced to water thereby producing free radical intermediates that subsequently reacts with free electrons to produce superoxide [11]. Certain reactions by enzymes such as cytochrome p450 oxidase in the liver releases free electrons that can react with oxygen to produce superoxide. Other enzymes can neutralize nitric oxide thereby producing superoxide [12]. Also, phagocytic cells during respiratory burst can generate superoxide [13].

**Hydrogen peroxide (H2 O2 ):** Hydrogen peroxide is mostly produced from the spontaneous dismutation reaction of superoxide in biological systems. Also, several enzymatic reactions including those catalyzed by D-amino acid and glycolate oxidases can directly produce H<sup>2</sup> O2 [14]. Generally, H<sup>2</sup> O2 is not a free radical but it is considered as a reactive oxygen species (ROS) because it can be transformed to other free radicals such as hydroxyl radical which mediate most of the toxic effects ascribed to H<sup>2</sup> O2 . Myeloperoxidase can decompose H2 O2 into singlet oxygen and hypochlorous acid, a mechanism which phagocytes utilize to kill bacteria [15]. However, H2 O2 is a weak oxidizing agent that might directly damage enzymes and proteins which contain reactive thiol groups. One of the most vital properties of H2 O2 over superoxide is its ability to freely traverse cell membranes [16].

**Hydroxyl radical (OH•)** is one of the most important free radicals as it is extremely reactive with almost all type of biomolecules including amino acids, sugars, lipids and nucleotides. Most ROS are usually converted to hydroxyl radical. Thus, it is usually the final mediator of most free radical induced tissue damage [17]. Hydroxyl radical is generated by various mechanisms but the most important is the in vivo mechanism due to decomposition of superoxide and hydrogen peroxide catalyzed by transition metals [18]. Transition metals generally contain one or more unpaired electrons and thus are capable to transfer a single electron. Iron and copper are the most common transition metals capable of generating free radicals and much implicated in human diseases. As shown by Fenton [19], hydrogen peroxide can react with iron II (or copper I) to generate hydroxyl radical:

$$\rm Fe^{2+} + H\_{2}O\_{2} \rightarrow \rm Fe^{3+} + OH^{\*} + OH^{-} \tag{1}$$

At physiological pH, iron is usually oxidized to Fe3+ and chelates to biological molecules. Thus, for Fenton reaction to occur, iron must be converted to its reduced form Fe2+. Superoxide radicals can reduce Fe3+ to Fe2+ ions thereby enabling the Fenton reaction.

$$\mathrm{Fe^{3+}} + \mathrm{O\_{2}}^{-} \rightarrow \mathrm{Fe^{2+}} + \mathrm{O\_{2}}.\tag{2}$$

net reaction (Haber-Weiss reaction):

cytochrome P450 system, phagocytosis and prostaglandin synthesis. Some of these endogenous sources of free radicals generation include reactions in the mitochondria, phagocytes, inflammation, arachidonate pathways, etc. Also, reactions involving iron and other transition metals, peroxisomes, xanthine oxidase, etc. are also endogenous sources of free radicals.

**Physiological sources:** Certain physiological state or processes like stress, emotion, aging, etc. mental status and disease conditions are also responsible for the formation of free radicals. For example, hyperglycemia is a major source of free radicals in diabetes patients through various metabolic pathways which include increase flux of glucose through the polyol pathway, increase formation of advanced glycation end-products (AGEs) and activation of their receptors, activation of protein kinase C (PKC) isoforms, activation of overactivity of hexos-

Free radicals are generated through various physiological processes in living organisms.

ing systems, superoxide can be generated through several mechanisms [10]. Several molecules such as flavine nucleotides, adrenaline, thiol compounds, glucose, etc. can be oxidized in the presence of oxygen to generate superoxide and these reactions are greatly accelerated by the presence of transition metals such as iron or copper. During the electron transport chain in the inner mitochondrial membrane, oxygen is reduced to water thereby producing free radical intermediates that subsequently reacts with free electrons to produce superoxide [11]. Certain reactions by enzymes such as cytochrome p450 oxidase in the liver releases free electrons that can react with oxygen to produce superoxide. Other enzymes can neutralize nitric oxide thereby producing superoxide [12]. Also, phagocytic cells during respiratory

mutation reaction of superoxide in biological systems. Also, several enzymatic reactions includ-

because it can be transformed to other free radicals such as hydroxyl radical which mediate

oxygen and hypochlorous acid, a mechanism which phagocytes utilize to kill bacteria [15].

**Hydroxyl radical (OH•)** is one of the most important free radicals as it is extremely reactive with almost all type of biomolecules including amino acids, sugars, lipids and nucleotides. Most ROS are usually converted to hydroxyl radical. Thus, it is usually the final mediator of most free radical induced tissue damage [17]. Hydroxyl radical is generated by various mechanisms but the most important is the in vivo mechanism due to decomposition of superoxide

ing those catalyzed by D-amino acid and glycolate oxidases can directly produce H<sup>2</sup>

O2

which contain reactive thiol groups. One of the most vital properties of H2

**)** is generally produced when a single electron is added unto oxygen. In liv-

**):** Hydrogen peroxide is mostly produced from the spontaneous dis-

. Myeloperoxidase can decompose H2

is not a free radical but it is considered as a reactive oxygen species (ROS)

is a weak oxidizing agent that might directly damage enzymes and proteins

O2 [14].

into singlet

over superoxide

O2

O2

Once generated, they can react with other biomolecules to attain stability.

amine pathway and decrease antioxidant defense [9].

52 Phytochemicals - Source of Antioxidants and Role in Disease Prevention

**Superoxide (O•**

**2**

burst can generate superoxide [13].

**O2**

is its ability to freely traverse cell membranes [16].

**Hydrogen peroxide (H2**

O2

O2

most of the toxic effects ascribed to H<sup>2</sup>

Generally, H<sup>2</sup>

However, H2

**2.3. Generation and chemical reactions of free radicals**

$$\rm O\_2^- + H\_2O\_2 \to OH^- + OH^\* + O\_2 \tag{3}$$

**Figure 1.** Reactive oxygen species (ROS)-induced oxidative damage. Source: Kohen and Nyska [21].

**Nitric oxide (NO•)** otherwise known as nitrogen monoxide is a radical produced by the oxidation of one of the terminal guanido nitrogen atoms of L-arginine catalyzed by the enzyme nitric oxide synthase (NOS) [6]. L-arginine and L-citrulline are both converted to nitric oxide. Nitric oxide can further react with superoxide to form peroxynitrite.

$$\mathrm{NO}^\* + \mathrm{O}^\*\mathrm{I}\_2^- \to \mathrm{ONOO}^-. \tag{4}$$

LOOH → LO• + LOO• + aldehydes (8)

Free Radicals and the Role of Plant Phytochemicals as Antioxidants Against Oxidative Stress-Related Diseases

http://dx.doi.org/10.5772/intechopen.76719

55

LOO• + OH• → LOOH + O• (9)

LOO• + α − tocopherol − OH → LOOH + α − tocopherol − O• (10)

**Proteins:** Proteins are major targets for attack by ROS predominantly by the OH•, RO• and nitrogen-reactive radicals causing damage. Hydrogen peroxide and superoxide radicals have weak effects on proteins except for proteins containing SH groups. Following interaction with ROS, proteins can undergo direct damages such as damaging specific amino acid residues and changing their tertiary structures and indirect damages such as peroxidation, degradation and fragmentation. The consequences of protein damage include loss of enzymatic activity and altered cellular functions. Protein oxidation products are usually keto, aldehydes and carbonyls compounds. Oxidation of tyrosine by ONOO• and other nitrogen reactive radicals leads to the formation of 3-nitrotyrosine which is a detectable marker for protein oxidation. Oxidation of proline and glutamate by OH• radicals usually leads to the formation of hydroxyproline and glutamyl semialdehyde. Following protein oxidation, proteins are susceptible to many changes in their function which include inactivation, chemical fragmenta-

**Nucleic acid:** Though DNA is a stable molecule, ROS can interact with it to cause several types of damages which include double- and single- DNA breaks, modification of DNA bases, loss of purines (apurinic sites), DNA-protein cross-linkage, damage to the deoxyribose sugar and damage to the DNA repair system. Hydroxyl radical is the most detrimental ROS that affects nucleic acids [25]. For example, OH• can attack guanine and adenine to yield an oxidation product, 8-hydroxydeoxyguanosine [26] and hydroxyadenine respectively. Also, hydroxyl radicals can attack pyrimidines leading to the formation of thymine peroxide, thy-

do not have direct interaction with DNA and hence do not lead to damage at their physiological concentrations. Transition metals such as iron that have high-binding affinity to DNA sites

When the concentration of ROS exceeds those of antioxidant neutralizing species, a condition known as oxidative stress occurs. As reviewed from Rahman et al. [27], oxidative stress has been implicated in several diseases including atherosclerosis, cancer, malaria, rheumatoid arthritis, chronic fatigue syndrome, and neurodegenerative diseases such as Alzheimer's disease, Parkinson's disease and Huntington's disease [28]. Evidence via monitoring biomarkers

2 and H2

O2

mine glycols, 5-(hydroxymethyl) uracyl, and other such products. ROS such as O•

can catalyze the production of OH• which in turns attack DNA.

**4. Oxidative stress and human diseases**

Termination by another radical:

Termination by an antioxidant:

tion and increased proteolytic degradation [24].

Protonated form of peroxynitrite (ONOOH) acts as a powerful oxidizing agent to sulfhydryl (SH) groups thereby causing oxidation of many molecules and proteins leading to cellular damage [20]. It can also cause DNA damage such as breaks, protein oxidation and nitration of aromatic amino acid residues in proteins. Reactive oxygen species and their oxidative stress induced damaged is summarized in **Figure 1**.
