**5. Regulation of free radicals with nonenzymatic small natural exogenous antioxidants**

#### **5.1 Vitamins**

*Free Radical Medicine and Biology*

**4.2 Free radical damage to DNA bases**

*2,5-dideoxypentose-4-ulose as an end group of a broken DNA strand.*

Oxidation of the C1′, C2′, and C5′ radicals yields products such as 2-deoxypentonic

Besides reacting with the sugar moiety of DNA, the highly reactive hydroxyl radical (**·**OH) reacts with the heterocyclic bases guanine (**Figure 3**), thymine, and cytosine, causing free radical-induced DNA damage by several different pathways. Guanine, however, possesses the lowest reduction potential (1.29 V) among the four DNA bases, rendering the motif the best electron donor and prone to preferential oxidization [24]. The hydroxyl radical reacts with the C4-, C5-, and C8-positions of guanine and to a lesser extent with the C2-position, generating a plethora of products. Interestingly, the HO-adduct radicals generated from the addition reactions

 may exhibit reducing or oxidizing properties (redox ambivalence), yielding the relevant products accordingly. Hence, while the C5-OH– and the C8-OH–adduct radicals are reducing, the C4-OH–adduct radical is predominantly oxidizing. The last two adduct radicals form in yields of 17% and 65–70%, respectively, whereas the yield of the C5-OH– adduct radical is lower than 10% [25]. Although formed in relatively lower yields, the C8-OH–adduct radical produces the major byproducts of guanine reactions (**Figure 3**). Thus, as shown in **Figure 3** and following reaction of the hydroxyl radical with the C-8 position of guanine, one-electron oxidation of the resulting C8-OH–adduct radical yields the enol form of 8-hydroxyguanine which undergoes tautomerization to generate the predominant keto form [21]. The latter may also form via a pathway involving 1,2-hydride-shift and subsequent oxidation of the C8-OH–adduct radical. The 1,2-hydride-shift radical product may also undergo single electron reduction, followed by ring opening reaction to form 2,6-diamino-4-hydroxy-5- formamidopyrimidine. The preceding radical damage to DNA has been directly correlated to several disease states such as genetic mutation,

atherosclerosis, Alzheimer's disease, and the aging process [26, 27].

*Oxidative and reductive product formation from reactions of the C8-OH–adduct radical of guanine.*

acid lactone, erythrose, 2-deoxytetradialdose, and 5′ aldehyde [22].

*Mechanism of product formation from reactions of the C4*′*-radical of 2*′*-deoxyribose, leading to* 

**98**

**Figure 3.**

of HO.

**Figure 2.**

#### *5.1.1 Vitamin E*

Vitamin E is a collection of optically active methylated phenolic compounds comprising four tocopherols and four tocotrienols [29] where α-tocopherol is the most common and biologically active species (**Figure 5**) [30]. The structures feature two primary parts: a densely substituted polar chromanol aromatic ring and a lipophilic long polyprenyl side chain. The main chemical structural difference between different forms of Vitamin E is that tocotrienols feature unsaturated isoprenoid hydrocarbon side chains with three carbon-carbon double bonds versus saturated isoprenoid side chains for tocopherols. Within each group, the vitamers are differentiated by the number and positions of the methyls in the chromate ring. The polyprenyl precursor for the biosynthesis of tocopherols and tocotrienols is phytyl pyrophosphate (PPP) and geranylgeranyl pyrophosphate (GGPP), respectively [31]. Vitamin E is biosynthesized though the shikimate pathway, and while α-tocopherol and α-tocotrienol are considered structurally unique, the remaining compounds in each class are constitutional isomers. The presence of three stereogenic centers (position C2 of the chromate ring, position C4 and C8 of the phytyl side chain) produces 8 different stereoisomers (four pairs of enantiomers) depending on the position and orientation of the groups in each of the chiral centers. Since the discovery of vitamin E in 1920, it has been shown to be the most powerful membrane-bound antioxidant utilized by cells to scavenge reactive nitrogen and oxygen species with consequent disruption of oxidative damage to cell membrane phospholipids during cellular lipid peroxidation of the polyunsaturated fatty acids (PFA) and low-density lipoprotein (LDL) [32]. The antioxidant is liposoluble and localized to cell membranes. Vitamin E functions by reducing lipid peroxyl radicals (LOO**.** ) by transferring the phenolic hydrogen atom of the chroman ring (**Figure 5**), resulting in a relatively stable and unreactive resonance-stabilized tocopheroxyl radical which is unable to trigger

#### *Free Radical Medicine and Biology*

further lipid peroxidation itself. The α-tocopherol radical can be reduced back to the original active α-tocopherol form by ascorbic acid or coenzyme Q10 [33, 34]. Alternatively, it may quench a second peroxyl radical where the resulting tocopheryl peroxide eliminates a peroxide leaving group, forms a hemiketal after reacting with water, and lastly hydrolyses to the tocopherolquinone. This is an essential foundation and benchmark of a good antioxidant. The synergistic antioxidation interactions between vitamin E and the ascorbate ion of vitamin C position the former at the forefront of the anti-radical defense system. Vitamin E is exogenous and hence is essential and must be obtained through diet in small amounts since the organism cannot synthesize it. Its biosynthesis is restricted to plants, photosynthetic algae, and certain cyanobacteria. Although vitamin A deficiency is rare, the most frequent manifestations of its lack comprise a number of disorders and disease states which include encephalomalacia, exudative diathesis, muscular dystrophy, and ceroid pigmentation. α-Tocopherol exhibits the highest bioactivity (100%), with the relative activities of β-, γ-, and δ-tocopherols being 50, 10, and 3%, respectively [35].

**Figure 5.**

*Chemical structures of the tocopherols and tocotrienols that comprise vitamin E and termination of lipid peroxidation with α-tocopherol.*

#### *5.1.2 Vitamin A*

Vitamin A, just like vitamin E, is a term that designates a family of unsaturated liposoluble organic compounds that include retinol, retinal, retinoic acid, and retinyl palmitate, and many provitamin A carotenoids such as beta-carotene (**Figure 6**). All forms share a beta-ionone ring to which an isoprenoid tether known as retinyl group is attached. It is noteworthy that both features are essential for vitamin A activity. The common chemical structure is a diterpene (C20H32) where the various molecular forms differ by the terminal side chain functional group. Thus, retinol contains a hydroxyl group, retinal contains an aldehyde function, retinoic acid has a terminal carboxylic acid group, and retinyl palmitate bears an ester moiety. The discovery of the antioxidant activity of vitamin A dates back to 1932 when Schmitt and Monaghan reported that vitamin A prevents lipid rancidity [36]. Several reviews outlining the antioxidant role and metabolic functions of vitamin A have appeared in the literature [37, 38]. Besides eliminating free radicals, it plays a major role in maintaining good vision. The aldehyde form of vitamin E is required by the retina to form the light-absorbing molecule rhodopsin necessary for both color and scotopic vision [39]. On the other hand, the fully irreversibly oxidized form of retinol functions in a very different way as a growth factor for epithelial and other types of cells [38]. As an antioxidant, vitamin A scavenges lipid peroxyl radicals (LOO**.** ) according to the mechanism shown in **Figure 6**. Thus, by trapping the peroxyl radical through an addition reaction to the

**101**

*5.1.3 Vitamin C*

(AscH−

**Figure 6.**

*Nonenzymatic Exogenous and Endogenous Antioxidants DOI: http://dx.doi.org/10.5772/intechopen.87778*

may eliminate LO radical and oxidizes to 5,6-retinol epoxide [15].

*Chemical structure of vitamin A and termination of lipid peroxidation with retinol.*

beta-ionone ring of retinol, the resultant tertiary and highly conjugated trans-retinol carbon radical intermediate is relatively stable and under normal conditions is not reactive enough to induce further lipid peroxidation itself. However, the intermediate may continue reacting with lipid peroxyl radicals or molecular oxygen to produce a bis-peroxyl adduct or retinol-derived peroxyl radical, respectively. Alternatively, it

Vitamin C (L-ascorbic acid) is an optically-active hydrosoluble free radical scavenger that bears a highly acidic hydroxyl group (pKa = 4.2) known to be completely ionized at neutral pH [35, 40]. Thus, the acidic vitamin readily loses a proton from the 3-hydroxyl group affording a resonance-stabilized ascorbate anion

of two conjugated double bonds which stabilize the deprotonated monoanionic conjugate base. Furthermore, these same electronic factors impart stability to the radical form of vitamin C when it undergoes one electron oxidation by lipid radicals to generate the ascorbate radical (**Figure 7**), a much less reactive species than most other free radicals. As such, vitamin C is able to assume the role of a free-radical scavenger. The low standard 1-electron reduction potential (282 mV) renders vitamin C an excellent electron donor. As well, at low ascorbate concentrations, it may function as a pro-oxidant reducing agent and is able to reduce redox-active copper and iron metals. Vitamin C is therefore required as a cofactor for a number of metabolic processes that mediate essential biological functions in all animals and plants [41]. The structure features a chiral 3,4-dihydroxyfuran-2(5*H*)-one ring and a 1,2-dihydroxyethyl tether containing another stereogenic center. The 6-carbon ketolactone is structurally related to glucose. Although four stereoisomers are expected depending on the position of the substituents around the stereogenic centers, only the L-enantiomer exhibits antioxidant capacity in biological systems, both in vitro and in vivo. While vitamin C is biosynthesized by nearly all animals, humans comprise a notable exception. Consequently, it is an essential nutrient and must be obtained through dietary means. In biological species, the vitamin exists in the protonated form at low pH, but in media with pH above 5, it is found in the dissociated ascorbate form [42]. This species is a 2-electron donor and gets oxidized to a molecule of dehydroascorbate (DHA) which does not have any antioxidant capacity. However, regeneration of the ascorbate from DHA is possible by the addition of two electrons and has been proposed to be carried out by oxidoreductase [43]. In animals, the biosynthesis of ascorbic acid is carried out by several enzymes in the liver from glucose [42], by a synthetic route which initially involves oxidation to D-glucuronic acid via uridine diphosphate (UDP) derivatives. Subsequent reduction of the open-chain aldehyde form of D-glucuronic acid to the primary

) (**Figure 7**). The unusual acidity of the alcohol is related to the presence

*Nonenzymatic Exogenous and Endogenous Antioxidants DOI: http://dx.doi.org/10.5772/intechopen.87778*

beta-ionone ring of retinol, the resultant tertiary and highly conjugated trans-retinol carbon radical intermediate is relatively stable and under normal conditions is not reactive enough to induce further lipid peroxidation itself. However, the intermediate may continue reacting with lipid peroxyl radicals or molecular oxygen to produce a bis-peroxyl adduct or retinol-derived peroxyl radical, respectively. Alternatively, it may eliminate LO radical and oxidizes to 5,6-retinol epoxide [15].

#### **Figure 6.**

*Free Radical Medicine and Biology*

further lipid peroxidation itself. The α-tocopherol radical can be reduced back to the original active α-tocopherol form by ascorbic acid or coenzyme Q10 [33, 34]. Alternatively, it may quench a second peroxyl radical where the resulting tocopheryl peroxide eliminates a peroxide leaving group, forms a hemiketal after reacting with water, and lastly hydrolyses to the tocopherolquinone. This is an essential foundation and benchmark of a good antioxidant. The synergistic antioxidation interactions between vitamin E and the ascorbate ion of vitamin C position the former at the forefront of the anti-radical defense system. Vitamin E is exogenous and hence is essential and must be obtained through diet in small amounts since the organism cannot synthesize it. Its biosynthesis is restricted to plants, photosynthetic algae, and certain cyanobacteria. Although vitamin A deficiency is rare, the most frequent manifestations of its lack comprise a number of disorders and disease states which include encephalomalacia, exudative diathesis, muscular dystrophy, and ceroid pigmentation. α-Tocopherol exhibits the highest bioactivity (100%), with the relative activities of β-, γ-, and δ-tocopherols being 50, 10, and 3%, respectively [35].

Vitamin A, just like vitamin E, is a term that designates a family of unsaturated liposoluble organic compounds that include retinol, retinal, retinoic acid, and retinyl palmitate, and many provitamin A carotenoids such as beta-carotene (**Figure 6**). All forms share a beta-ionone ring to which an isoprenoid tether known as retinyl group is attached. It is noteworthy that both features are essential for vitamin A activity. The common chemical structure is a diterpene (C20H32) where the various molecular forms differ by the terminal side chain functional group. Thus, retinol contains a hydroxyl group, retinal contains an aldehyde function, retinoic acid has a terminal carboxylic acid group, and retinyl palmitate bears an ester moiety. The discovery of the antioxidant activity of vitamin A dates back to 1932 when Schmitt and Monaghan reported that vitamin A prevents lipid rancidity [36]. Several reviews outlining the antioxidant role and metabolic functions of vitamin A have appeared in the literature [37, 38]. Besides eliminating free radicals, it plays a major role in maintaining good vision. The aldehyde form of vitamin E is required by the retina to form the light-absorbing molecule rhodopsin necessary for both color and scotopic vision [39]. On the other hand, the fully irreversibly oxidized form of retinol functions in a very different way as a growth factor for epithelial and other types of cells [38]. As an antioxidant,

*Chemical structures of the tocopherols and tocotrienols that comprise vitamin E and termination of lipid* 

in **Figure 6**. Thus, by trapping the peroxyl radical through an addition reaction to the

) according to the mechanism shown

**100**

vitamin A scavenges lipid peroxyl radicals (LOO**.**

*5.1.2 Vitamin A*

*peroxidation with α-tocopherol.*

**Figure 5.**

*Chemical structure of vitamin A and termination of lipid peroxidation with retinol.*

#### *5.1.3 Vitamin C*

Vitamin C (L-ascorbic acid) is an optically-active hydrosoluble free radical scavenger that bears a highly acidic hydroxyl group (pKa = 4.2) known to be completely ionized at neutral pH [35, 40]. Thus, the acidic vitamin readily loses a proton from the 3-hydroxyl group affording a resonance-stabilized ascorbate anion (AscH− ) (**Figure 7**). The unusual acidity of the alcohol is related to the presence of two conjugated double bonds which stabilize the deprotonated monoanionic conjugate base. Furthermore, these same electronic factors impart stability to the radical form of vitamin C when it undergoes one electron oxidation by lipid radicals to generate the ascorbate radical (**Figure 7**), a much less reactive species than most other free radicals. As such, vitamin C is able to assume the role of a free-radical scavenger. The low standard 1-electron reduction potential (282 mV) renders vitamin C an excellent electron donor. As well, at low ascorbate concentrations, it may function as a pro-oxidant reducing agent and is able to reduce redox-active copper and iron metals. Vitamin C is therefore required as a cofactor for a number of metabolic processes that mediate essential biological functions in all animals and plants [41]. The structure features a chiral 3,4-dihydroxyfuran-2(5*H*)-one ring and a 1,2-dihydroxyethyl tether containing another stereogenic center. The 6-carbon ketolactone is structurally related to glucose. Although four stereoisomers are expected depending on the position of the substituents around the stereogenic centers, only the L-enantiomer exhibits antioxidant capacity in biological systems, both in vitro and in vivo. While vitamin C is biosynthesized by nearly all animals, humans comprise a notable exception. Consequently, it is an essential nutrient and must be obtained through dietary means. In biological species, the vitamin exists in the protonated form at low pH, but in media with pH above 5, it is found in the dissociated ascorbate form [42]. This species is a 2-electron donor and gets oxidized to a molecule of dehydroascorbate (DHA) which does not have any antioxidant capacity. However, regeneration of the ascorbate from DHA is possible by the addition of two electrons and has been proposed to be carried out by oxidoreductase [43]. In animals, the biosynthesis of ascorbic acid is carried out by several enzymes in the liver from glucose [42], by a synthetic route which initially involves oxidation to D-glucuronic acid via uridine diphosphate (UDP) derivatives. Subsequent reduction of the open-chain aldehyde form of D-glucuronic acid to the primary

alcohol (L-gulonic acid), lactone formation between the carboxyl and 4-hydroxyl group, oxidation of the secondary hydroxyl function to a carbonyl, and subsequent enolization result in L-ascorbic acid. The latter, specifically in the ascorbate form, acts as a reducing agent, donating electrons to lipid radicals in order to terminate the lipid peroxidation chain reaction (**Figure 7**). Another main function of Vitamin C as an antioxidant is to regenerate vitamin E (HO-tocopherol) from its oxidized form (**.**O-tocopherol) back to its active state by reducing vitamin E radicals formed when vitamin E scavenges oxygen radicals. The recycling of vitamin E is carried out in cell membranes in conjunction with glutathione (GSH) or other sacrificial reductants [33, 34]. Likewise, vitamin C acts as an antioxidant and reducing agent by donating electrons to various enzymatic and nonenzymatic reactions. It reduces the transition metal ions of several biosynthetic enzymes, thus preventing biological oxidation of macromolecules. In plants, vitamin C is a substrate for the enzyme ascorbate peroxidase which catalyzes the reduction of toxic hydrogen peroxide (H2O2) to water (H2O) [44]. Currently, this vitamin is the most widely employed vitamin in drugs, premedication, and dietary supplements worldwide.

**Figure 7.**

*Biosynthesis, chemical structure, and reduction mechanism of ascorbic acid.*
