**5.2 Flavonoids**

Flavonoids are exogenous antioxidants displaying rich structural diversity and are ubiquitous in plants and certain photosynthetic organisms. More than 8000 of these benzo-γ-pyran derivatives have been identified and characterized [45, 46]. The general structure features a C6-C3-C6 15-carbon flavone skeleton, which comprises two phenyl rings (A and B) linked by a heterocyclic ring (C) (**Figure 8**). Flavonoids have been classified into flavones, flavanones, flavanols, flavonols, and anthocyanins. While flavones have a double bond between C2 and C3, flavanones have a saturated C2–C3 bond. Compared to flavones, the corresponding flavonols have an additional hydroxyl group at the C3 position while flavonols are C2-C3 saturated analogs of flavonols. Flavonoid groups are differentiated based on the number of hydroxyl and other substituents on the phenyl rings [47].

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radical (**Figure 10**).

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

otides brought about by H2O2, HO**.**

metal ions and high pH [47].

**5.3 Carotenoids**

**Figure 9.**

Quercetin (3,5,7,3′,4′–pentahydroxyflavone) (**Figure 9**) is the most ubiquitous polyphenolic flavonoid known to prevent oxidative damage to DNA oligonucle-

**.−**. On the other hand, anthocyanidin is

, and O2

a strong inhibitor of lipid oxidations. Thus, as shown in **Figure 9**, the antioxidant mechanism of lipid peroxyl radicals scavenging capability of anthocyanidin is based on its hydrogen radical donation ability from the *p*-hydroxyl group of ring B to generate a resonance-stabilized anthocyanidin radical incapable of participating in other radical reactions. In addition, the effectiveness of anthocyanidin in inhibiting lipid peroxidation has been correlated to their metal-ion chelating power [48, 49]. In particular, the ortho-dihydroxy groups in the B-ring confer upon this class of compounds antiperoxidative properties [50]. However, phenolic compounds can also act as prooxidants if present in high concentrations with

Carotenoids, also known as tetraterpenoids, are a group of phytonutrients produced by plants and algae, as well as some bacteria and fungi [51]. The long unsaturated hydrocarbon alkyl chain renders carotenoids highly liposoluble. Hence, they play a key role in the protection of lipoproteins and cellular membranes from lipid peroxidation and exhibit particularly efficient scavenging capacity against peroxyl radicals as compared to any other ROS and they are known to be the most common lipid-soluble antioxidants [52, 53]. Over 1100 carotenoids have been identified and classified primarily into two groups: the oxygen-containing xanthophylls and those that are purely hydrocarbons, carotenes (**Figure 10**). Biosynthetically, all carotenoids are tetraterpenes

*Structure of quercetin and mechanism of radical scavenging activity of anthocyanidin.*

comprising 40 carbon atoms which are produced from eight isoprene units. The structural backbone consists of isoprenoid units biosynthesized either by headto-tail or by tail to-tail process. The basic building blocks of carotenoids are isopentyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP) which produce the major carotenoid precursor geranylgeranyl pyrophosphate (GGPP) [54]. GGPP undergoes several different reactions within the carotenoid biosynthetic pathway to afford carotenes or xanthophylls. Carotenoids reduce the peroxyl radicals to form a resonance-stabilized carbon-centered radical product. Lycopene and carotene are the most prominent and potent carotenoid antioxidants. The former is notably a strong singlet oxygen quencher due to the high number of conjugated *trans*-configuration double bonds present in the structure. In general, the extended conjugated system in carotenoids is strongly-reducing, facilitating abstraction of hydrogen atoms from the allylic positions to this conjugation, as well allowing free-radical addition reactions to proceed with ease. Lycopene for instance reduces peroxyl radicals through electron transfer to afford an unreactive resonance stabilized carbon-centered

**Figure 8.** *General skeletal structure of the various flavonoid classes.*

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

Quercetin (3,5,7,3′,4′–pentahydroxyflavone) (**Figure 9**) is the most ubiquitous polyphenolic flavonoid known to prevent oxidative damage to DNA oligonucleotides brought about by H2O2, HO**.** , and O2 **.−**. On the other hand, anthocyanidin is a strong inhibitor of lipid oxidations. Thus, as shown in **Figure 9**, the antioxidant mechanism of lipid peroxyl radicals scavenging capability of anthocyanidin is based on its hydrogen radical donation ability from the *p*-hydroxyl group of ring B to generate a resonance-stabilized anthocyanidin radical incapable of participating in other radical reactions. In addition, the effectiveness of anthocyanidin in inhibiting lipid peroxidation has been correlated to their metal-ion chelating power [48, 49]. In particular, the ortho-dihydroxy groups in the B-ring confer upon this class of compounds antiperoxidative properties [50]. However, phenolic compounds can also act as prooxidants if present in high concentrations with metal ions and high pH [47].

**Figure 9.** *Structure of quercetin and mechanism of radical scavenging activity of anthocyanidin.*

### **5.3 Carotenoids**

*Free Radical Medicine and Biology*

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.

Flavonoids are exogenous antioxidants displaying rich structural diversity and are ubiquitous in plants and certain photosynthetic organisms. More than 8000 of these benzo-γ-pyran derivatives have been identified and characterized [45, 46]. The general structure features a C6-C3-C6 15-carbon flavone skeleton, which comprises two phenyl rings (A and B) linked by a heterocyclic ring (C) (**Figure 8**). Flavonoids have been classified into flavones, flavanones, flavanols, flavonols, and anthocyanins. While flavones have a double bond between C2 and C3, flavanones have a saturated C2–C3 bond. Compared to flavones, the corresponding flavonols have an additional hydroxyl group at the C3 position while flavonols are C2-C3 saturated analogs of flavonols. Flavonoid groups are differentiated based on the number of

**102**

**Figure 8.**

**5.2 Flavonoids**

**Figure 7.**

*General skeletal structure of the various flavonoid classes.*

hydroxyl and other substituents on the phenyl rings [47].

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

Carotenoids, also known as tetraterpenoids, are a group of phytonutrients produced by plants and algae, as well as some bacteria and fungi [51]. The long unsaturated hydrocarbon alkyl chain renders carotenoids highly liposoluble. Hence, they play a key role in the protection of lipoproteins and cellular membranes from lipid peroxidation and exhibit particularly efficient scavenging capacity against peroxyl radicals as compared to any other ROS and they are known to be the most common lipid-soluble antioxidants [52, 53]. Over 1100 carotenoids have been identified and classified primarily into two groups: the oxygen-containing xanthophylls and those that are purely hydrocarbons, carotenes (**Figure 10**). Biosynthetically, all carotenoids are tetraterpenes comprising 40 carbon atoms which are produced from eight isoprene units. The structural backbone consists of isoprenoid units biosynthesized either by headto-tail or by tail to-tail process. The basic building blocks of carotenoids are isopentyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP) which produce the major carotenoid precursor geranylgeranyl pyrophosphate (GGPP) [54]. GGPP undergoes several different reactions within the carotenoid biosynthetic pathway to afford carotenes or xanthophylls. Carotenoids reduce the peroxyl radicals to form a resonance-stabilized carbon-centered radical product. Lycopene and carotene are the most prominent and potent carotenoid antioxidants. The former is notably a strong singlet oxygen quencher due to the high number of conjugated *trans*-configuration double bonds present in the structure. In general, the extended conjugated system in carotenoids is strongly-reducing, facilitating abstraction of hydrogen atoms from the allylic positions to this conjugation, as well allowing free-radical addition reactions to proceed with ease. Lycopene for instance reduces peroxyl radicals through electron transfer to afford an unreactive resonance stabilized carbon-centered radical (**Figure 10**).

**Figure 10.**

*Structure and radical scavenging mechanism of some prominent xanthophyll and carotene carotenoids.*

## **5.4 Hydroxycinnamic acids**

Hydroxycinnamic acids (hydroxycinnamates) are a class of phenylpropanoids possessing a C6–C3 skeleton. These compounds are hydroxy derivatives of cinnamic acid which is their common biosynthetic precursor. Mechanistically, bimolecular elimination of ammonia from the side chain of L-Phenylalanine generates *trans-* (*E)-*cinnamic acid (**Figure 11**). A subsequent cytochrome P-450-dependent direct hydroxylation reaction of cinnamic acid mediated by cinnamate 4-hydroxylase enzyme (E2) produces the first member of this class, *p*-coumaric acid. The substitution patterns of the remaining cinnamic acids are constructed sequentially by further hydroxylation and methylation reactions, which is typical of shikimate pathway metabolites. Hence, direct hydroxylation of *p*-coumaric acid mediated by *p*-coumarate 3-hydroxylase enzyme (E3) generates caffeic acid. Subsequent methylation of the latter by caffeic acid *O*-methyltransferase (E4) produces ferulic acid. Hydroxylation of ferulic acid by ferulate-5-hydroxylase (E5), a cytochrome P450 dependent monooxygenase enzyme, followed by methylation with SAM produces the last member, sinapic acid. As chain-breaking antioxidants, hydroxycinnamic acids prevent oxidation of LDL, although in varying efficiencies, depending on their standard one-electron reduction potential, hydrogen or electron donating ability, and the capacity to delocalize and stabilize the resulting phenoxyl radical within their structural framework [55, 56]. The antioxidant activity of the derivatives is correlated with the methylation and hydoxylation substitution pattern of the benzene ring. Thus, the antioxidant efficiency of the hydroxycinnamate conjugates on human LDL oxidation has been found to increase in the order of *p*-coumaric acid, ferulic acid, sinapic acid, and caffeic acid [57]. The general mechanism of free radical scavenging by which these antioxidants act involves donation of a *p*-hydroxyl hydrogen atom to ROS and generation of resonance stabilized carbonbased radical. Additionally, the presence of *ortho*-dihydroxyl groups allows metalion chelation much like flavonoids and enhances their antioxidant capacity against lipid peroxidation.

**105**

**6.1 Uric acid**

**Figure 12.**

radical (O2

carbonate ions (CO3

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

**5.5 Other natural exogenous antioxidants (allyl sulfides & curcumin)**

Allicin (diallyl thiosulfinate), a compound mainly found in garlic, and curcumin are biologically active compounds possessing antioxidative properties. The active form responsible for the antioxidant activity of allicin is 2-propenesulfenic acid [58], formed via a cope elimination reaction of the former precursor (**Figure 12**) [59, 60]. The radical-scavenging mechanism of allicin involves H-atom abstraction by a peroxyl radical from the sulfenic acid residue [61, 62]. The bis-α,β-unsaturated β-diketone, curcumin, is a liposoluble free radical scavenger that displays remarkable chain breaking ability similar to that of vitamin E [63]. As shown in **Figure 11**, the methylene group of the β-diketone residue and the phenolic hydroxyl (OH) function are sites that can transfer electrons or H-atoms to quench free radicals and generate extended resonance-stabilized carbon- or oxygen-centered radicals. The phenoxyl radical, which has been credited for the antioxidative properties of curcumin [64], generates a quinone methide as it moves through the carbon framework and reacts with molecular oxygen to produce a peroxyl radical. Subsequent reduction of the peroxyl radical and dehydration of the resulting hydroperoxide, followed by rearrangement into a spiro-epoxide and hydrolysis, give the final bis-cyclopentadione product.

**6. Regulation of free radicals with nonenzymatic small endogenous** 

Uric acid (UA) is a hydrophilic antioxidant generated during the metabolism of purine nucleotides and accounts nearly for 66% of the total oxygen scavenging activity in the blood serum. Mammals and humans are capable of producing UA, making it the most predominant aqueous antioxidant present in humans [65, 66] with an approximate blood level of 3.5–7.5 mg/dL. UA is a strong electron donor

ascorbic acid and thiols in its cycle for complete scavenging of such species [67, 68]. Peroxynitrite is formed by the reaction between nitric oxide (·NO) and superoxide

scavenging peroxynitrite, UA reacts with hydroxyl radicals, singlet oxygen, lipid peroxides, and hypochlorous acid, itself getting converted to innocuous chemical species like urea and allantoin. Furthermore, it has been implicated in scavenging

copper and iron ions, resulting in the inhibition of deleterious free radical reactions like the Fenton and the Haber-Weiss reactions [65]. Some have suggested that UA

**.−**) and nitrogen dioxide (NO2

**.−**) (**Figure 1**) and has been implicated in many pathologies. Besides

**.**

), requiring the participation of

) [69], and in complexation with

**(synthetic/physiological) antioxidants**

*Structures and radical-scavenging activities of curcumin and allicin.*

and a selective scavenger of peroxynitrite (ONOO−

**Figure 11.** *Structures, biosynthesis, and free radical scavenging mechanism of hydroxycinnamic acids.*

## **5.5 Other natural exogenous antioxidants (allyl sulfides & curcumin)**

Allicin (diallyl thiosulfinate), a compound mainly found in garlic, and curcumin are biologically active compounds possessing antioxidative properties. The active form responsible for the antioxidant activity of allicin is 2-propenesulfenic acid [58], formed via a cope elimination reaction of the former precursor (**Figure 12**) [59, 60]. The radical-scavenging mechanism of allicin involves H-atom abstraction by a peroxyl radical from the sulfenic acid residue [61, 62]. The bis-α,β-unsaturated β-diketone, curcumin, is a liposoluble free radical scavenger that displays remarkable chain breaking ability similar to that of vitamin E [63]. As shown in **Figure 11**, the methylene group of the β-diketone residue and the phenolic hydroxyl (OH) function are sites that can transfer electrons or H-atoms to quench free radicals and generate extended resonance-stabilized carbon- or oxygen-centered radicals. The phenoxyl radical, which has been credited for the antioxidative properties of curcumin [64], generates a quinone methide as it moves through the carbon framework and reacts with molecular oxygen to produce a peroxyl radical. Subsequent reduction of the peroxyl radical and dehydration of the resulting hydroperoxide, followed by rearrangement into a spiro-epoxide and hydrolysis, give the final bis-cyclopentadione product.

**Figure 12.**

*Free Radical Medicine and Biology*

**5.4 Hydroxycinnamic acids**

**Figure 10.**

Hydroxycinnamic acids (hydroxycinnamates) are a class of phenylpropanoids possessing a C6–C3 skeleton. These compounds are hydroxy derivatives of cinnamic acid which is their common biosynthetic precursor. Mechanistically, bimolecular elimination of ammonia from the side chain of L-Phenylalanine generates *trans-* (*E)-*cinnamic acid (**Figure 11**). A subsequent cytochrome P-450-dependent direct hydroxylation reaction of cinnamic acid mediated by cinnamate 4-hydroxylase enzyme (E2) produces the first member of this class, *p*-coumaric acid. The substitution patterns of the remaining cinnamic acids are constructed sequentially by further hydroxylation and methylation reactions, which is typical of shikimate pathway metabolites. Hence, direct hydroxylation of *p*-coumaric acid mediated by *p*-coumarate 3-hydroxylase enzyme (E3) generates caffeic acid. Subsequent methylation of the latter by caffeic acid *O*-methyltransferase (E4) produces ferulic acid. Hydroxylation of ferulic acid by ferulate-5-hydroxylase (E5), a cytochrome P450 dependent monooxygenase enzyme, followed by methylation with SAM produces the last member, sinapic acid. As chain-breaking antioxidants, hydroxycinnamic acids prevent oxidation of LDL, although in varying efficiencies, depending on their standard one-electron reduction potential, hydrogen or electron donating ability, and the capacity to delocalize and stabilize the resulting phenoxyl radical within their structural framework [55, 56]. The antioxidant activity of the derivatives is correlated with the methylation and hydoxylation substitution pattern of the benzene ring. Thus, the antioxidant efficiency of the hydroxycinnamate conjugates on human LDL oxidation has been found to increase in the order of *p*-coumaric acid, ferulic acid, sinapic acid, and caffeic acid [57]. The general mechanism of free radical scavenging by which these antioxidants act involves donation of a *p*-hydroxyl hydrogen atom to ROS and generation of resonance stabilized carbonbased radical. Additionally, the presence of *ortho*-dihydroxyl groups allows metalion chelation much like flavonoids and enhances their antioxidant capacity against

*Structure and radical scavenging mechanism of some prominent xanthophyll and carotene carotenoids.*

*Structures, biosynthesis, and free radical scavenging mechanism of hydroxycinnamic acids.*

**104**

**Figure 11.**

lipid peroxidation.

*Structures and radical-scavenging activities of curcumin and allicin.*
