**3. ROS induced oxidative damage**

Continual influx and generation of ROS from endogenous and exogenous sources lead to oxidative damage of cellular components and may impair many cellular functions [22]. The most vulnerable biological targets to oxidative damage include proteins, enzymes, lipidic membranes and DNA [5].

**Lipids:** All cellular membranes are generally vulnerable to oxidative damage since they are highly rich in unsaturated fatty acid. The lipid damage due to ROS usually known as lipid peroxidation occurs in three stages [23]. The first stage, known as initiation involves the attack of a reactive oxygen metabolite capable of abstracting a hydrogen atom from a methylene group in the lipid due to the presence of a weak double bond. As such, the remaining fatty acid radical retains one electron and stabilizes by rearrangement of the molecular structure to form a conjugated diene. In the propagation stage, the fatty acid radical reacts with oxygen to form ROO•. The ROO• is capable of abstracting another hydrogen atom from a neighboring fatty acid molecule, which again leads to the production of fatty acid radicals. These propagation reactions occur repeatedly leading to the peroxidation of several unsaturated lipid in the membrane. The ROO• becomes a lipid hydroperoxide which can further be decomposed to an aldehyde or form cyclic endoperoxide, isoprotans, and hydrocarbons. The last stage which is chain termination occurs following interaction of one ROO• with another radical or antioxidants.

Initiation:

$$\text{RH} + \text{R}^\* \rightarrow \text{L}^\* + \text{RH} \tag{5}$$

Propagation:

$$\mathsf{R}^\* \mathsf{+O}\_2 \to \mathsf{LCO}^\* \tag{6}$$

$$\text{LCO}^\* + \text{LH} \rightarrow \text{LCOH} + \text{L}^\* \tag{7}$$

Free Radicals and the Role of Plant Phytochemicals as Antioxidants Against Oxidative Stress-Related Diseases http://dx.doi.org/10.5772/intechopen.76719 55

$$\text{LCOH} \rightarrow \text{LO}^\* + \text{LCO}^\* + \text{aldehydes} \tag{8}$$

Termination by another radical:

**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.

2

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

Continual influx and generation of ROS from endogenous and exogenous sources lead to oxidative damage of cellular components and may impair many cellular functions [22]. The most vulnerable biological targets to oxidative damage include proteins, enzymes, lipidic

**Lipids:** All cellular membranes are generally vulnerable to oxidative damage since they are highly rich in unsaturated fatty acid. The lipid damage due to ROS usually known as lipid peroxidation occurs in three stages [23]. The first stage, known as initiation involves the attack of a reactive oxygen metabolite capable of abstracting a hydrogen atom from a methylene group in the lipid due to the presence of a weak double bond. As such, the remaining fatty acid radical retains one electron and stabilizes by rearrangement of the molecular structure to form a conjugated diene. In the propagation stage, the fatty acid radical reacts with oxygen to form ROO•. The ROO• is capable of abstracting another hydrogen atom from a neighboring fatty acid molecule, which again leads to the production of fatty acid radicals. These propagation reactions occur repeatedly leading to the peroxidation of several unsaturated lipid in the membrane. The ROO• becomes a lipid hydroperoxide which can further be decomposed to an aldehyde or form cyclic endoperoxide, isoprotans, and hydrocarbons. The last stage which is chain termi-

nation occurs following interaction of one ROO• with another radical or antioxidants.

RH + R• → L• + RH (5)

R• + O2 → LOO• (6)

LOO• + LH → LOOH + L• (7)

<sup>−</sup> → ONOO<sup>−</sup>

. (4)

Nitric oxide can further react with superoxide to form peroxynitrite.

NO• + O•

54 Phytochemicals - Source of Antioxidants and Role in Disease Prevention

induced damaged is summarized in **Figure 1**.

**3. ROS induced oxidative damage**

membranes and DNA [5].

Initiation:

Propagation:

$$\mathrm{LCO^{\*}} + \mathrm{OH^{\*}} \rightarrow \mathrm{LCOOH} + \mathrm{O^{\*}} \tag{9}$$

Termination by an antioxidant:

$$\text{LCO}^\* + a \text{ -tocopherol} \text{ -OH} \rightarrow \text{LCOH} + a \text{ -tocopherol} \text{ -O}^\* \tag{10}$$

**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 fragmentation and increased proteolytic degradation [24].

**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, thymine glycols, 5-(hydroxymethyl) uracyl, and other such products. ROS such as O• 2 and H2 O2 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 can catalyze the production of OH• which in turns attack DNA.

#### **4. Oxidative stress and human diseases**

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 such as the presence of ROS and RNS as well as antioxidant defense has indicated oxidative damage may be implicated in the pathogenesis of these diseases [29]. Elevated levels of free radicals such as 4-hydroxy-2,3-nonenal (HNE), acrolein, malondialdehyde (MDA) and F2-isoprostanes have been observed in Alzheimer's disease [30, 31]. Oxidative stress also contributes to tissue injury following hyperoxia and irradiation. Evidence from studies have shown oxidative stress to play an important role in the pathogenesis and development of metabolic syndrome related disorders such as obesity, hypertension, diabetes, dyslipidemia etc. as well as in cardiovascular related diseases such as myocardial infarction, aortic valve stenosis, angina pectoris, atherosclerosis and heart failure [32–35]. Cancer is another disease associated with ROS as ROS have been suggested to stimulate oncogenes such as Jun and Fos whose overexpression is directly associated with lung cancer [36]. In lung cancers, p53 can be mutated by ROS thereby losing its function of apoptosis and functioning as an oncogene [37]. Also, the development of gastric cancer has been thought to be due to increase production of ROS and RNS by *Helicobacter pylori* infection in human stomach [29]. Excess ROS in human kidney leads to urolithiasis [29]. ROS have also been reported to damage cellular components in cartilage leading to osteoarthritis [38] and has been shown to be involved in damaging the islets cells of the pancreas [39]. More so, hyperglycemia triggers ROS production in both tubular and mesangial cells of human kidney, making functional and structural changes in glomeruli causing diabetic nephropathy [40].

**5.2. Direct defense mechanism**

*5.2.1. Enzymatic antioxidants*

hydrogen peroxide:

O2

ide to water and oxygen in two stages:

Stage 1: Catalase–Fe(III) <sup>+</sup> H2 O2 <sup>→</sup> compound <sup>I</sup>

Stage 2: Compound <sup>I</sup> <sup>+</sup> H2 O2 <sup>→</sup> catalase–Fe(III) <sup>+</sup> 2H2 <sup>O</sup> <sup>+</sup> O2

enzymatic and non-enzymatic antioxidants.

tase (GRx) and glutathione peroxidase (GPx).

This category of defense system which constitutes the antioxidant system is the most important because they directly act on free radicals either by decomposing, scavenging or converting free radicals to less reactive forms. This defense mechanism constitute two groups; the

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

The enzymatic antioxidants include superoxide dismutase (SOD), catalase, glutathione reduc-

**Superoxide dismutase (SOD):** SOD is an enzymatic antioxidant that exists in three forms in mammalian tissues and differs on their cofactor, subcellular location and tissue distribution. 1. Copper zinc superoxide dismutase (CuZnSOD) is present in the cytoplasm and organelles of almost all mammalian cells [43]. This enzyme has a molecular mass of about 32,000 kDa with two protein subunits, each containing a catalytically active copper and zinc atom. 2. Manganese superoxide dismutase (MnSOD) has a molecular mass of 40,000 kDa and is found in the mitochondria of almost all cells [44]. It consists of four protein subunits, each containing a single manganese atom. 3. Extracellular superoxide dismutase (ECSOD) is a secretory copper and zinc containing SOD which is different from CuZnSOD [45]. It is synthesized only in fibroblasts and endothelial cells and expressed on the cell surface where it binds to heparan sulfates. Following its release from heparin, it is secreted into extracellular fluids and enters into the circulation. Superoxide dismutase catalyzes the dismutation of superoxide to

<sup>−</sup> + 2H+ → H2 O2 + O2

**Catalase:** Catalase was the first antioxidant enzyme to be characterized. It is located mostly within the peroxisomes of cells which contain most of the enzymes capable of generating hydrogen peroxide. It consists of four protein subunits, each containing a haem group and a molecule of NADPH [46]. Catalase is mostly present in liver and erythrocytes showing the greatest activities but is found in other tissues. It catalyzes the conversion of hydrogen perox-

**Glutathione peroxidases (GPx):** Glutathione peroxidase is an enzyme which is synthesized mainly in the kidney and found in almost all tissues although it is highly found in the liver [47]. Its subcellular location is usually the cytosol and mitochondria. Selenium serves as its cofactor located at the active site of the enzyme and deficiency of selenium greatly affects the activity of the enzyme [48]. Glutathione peroxidases catalyze the oxidation of reduced glutathione (GSH) decomposing hydrogen peroxide or another species such as a lipid hydroperoxide:

. (11)

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

57

<sup>−</sup> + O2

The hydrogen peroxide can then be removed by catalase or glutathione peroxidase.
