Free radical scavenging
Antioxidants scavenge free radicals of foods by donating hydrogen to them, and they produce relatively stable antioxidant radicals with low standard reduction potential, less than 500 mV (Choe and Min 2005). Rates of hydrogen abstraction from lipids and antioxidants are in the order of 10°/M/s and 105 to 106/M/s, respectively (Burton and others 1985; Mukai and others 1993; Amorati and others 2007). The higher stability of antioxidant radicals than that of food radicals is due to resonance delocalization throughout the phenolic ring structure (Choe and Min 2006). Examples of antioxidants to scavenge free radicals are phenolic compounds (tocopherols, butylated hydroxytoluene (BHT), butylated hydroxyanisole (BHA), tert-butylhydroquinone (TBHQ), propyl gallate (PG), lignans, flavonoids, and phenolic acids), ubiquinone (coenzyme Q), carotenoids, ascorbic acids, and amino acids. Thiacremonone (Figure 7) extracted from heated garlic at 130 °C has higher radical scavenging activity than ascorbic acid, α-tocopherol, or BHA (Hwang and others 2007).
The effectiveness of antioxidants to scavenge free radicals of foods depends on the bond dissociation energy between oxygen and a phenolic hydrogen, pH related to the acid dissociation constant, and reduction potential and delocalization of the antioxidant radicals (Litwinienko and Ingold 2003; Choe and Min 2006; Cao and others 2007). Hydrogen transfer from antioxidants to the peroxy or alkyl radicals of foods is more thermodynamically favorable when the bond dissociation energy for O–H in the antioxidants is low (Cao and others 2007). Bond dissociation energy for O–H of phenolic antioxidants corresponds to 70 to 80 kcal/mol (Berkowits and others 1994; Lucarini and others 1996; Wright and others 2001), and decreases in the order of δ > γ > β > α-tocopherol (Wright and others 2001). Bond dissociation energy for O–H of phenolic antioxidants is affected by surrounding solvents; it is higher in polar solvents such as acetonitrile and tert-butyl alcohol than nonpolar benzene (Lucarini and others 2002; Zhang and Wang 2005). Thus, polar solvents decrease the radical scavenging activity of the antioxidants due to the intermolecular hydrogen bonding between oxygen or nitrogen in a polar solvent and OH group in phenolic antioxidants (Amorati and others 2007).
The bond dissociation energy for O–H of the phenolic antioxidants also predicts the stabilization of antioxidant radicals. The lower the bond dissociation energy for the O–H group of the antioxidants, the more stable the antioxidant radical. The antioxidants with low bond dissociation energy are thus more efficient hydrogen donors and better antioxidants. The O–H bond strength of phenolic antioxidants is affected by substitution of hydrogen in a benzene ring. The antioxidant activity of the phenolic antioxidants is dependent on the balance between the electron-donating effect of the substituents and the steric crowding around the phenolic OH groups which is related to the position of the substituents (Amorati and others 2007). Any substituent destabilizing the ground-state phenolic antioxidants, and/or stabilizing the phenoxy radical form of the antioxidants, reduces the O–H bond strength. Substituents such as an alkyl or a 2nd hydroxy group improve stabilization of the antioxidant radicals and increase radical scavenging activity (Shahidi and Wanasundara 1992). A single substitution of methyl, tert-methyl, or methoxy group at the ortho-position decreased the O–H bond strength by 1.75, 1.75, and 0.2 kcal/mol, and the O–H bond strength decrease by the same substituent at the meta-position was about 0.5 kcal/mol (Brigati and others 2002).
An intramolecular hydrogen bond between phenolic hydrogen and the oxygen-containing substituent, such as a methoxy group at the ortho-position, stabilizes ground-state phenol to cancel the O–H bond strength decrease by the methoxy group, and there is a negligible change in the bond dissociation energy (0.2 kcal/mol decrease; Brigati and others 2002). Double substitution interactively (additively or synergistically) contributes to the O–H bond strength. Electron-withdrawing substituents such as COOR and COOH at the para-position stabilize the phenol form of antioxidants, and destabilize the phenoxy radical form of the antioxidants, to increase the O–H bond strength and make the antioxidants less efficient (Rice-Evans and others 1996). However, the substituent such as methyl, tert-butyl, methoxy, or phenyl group decreases the O–H bond strength (Brigati and others 2002). When the substituent at the para-position is an unsaturated hydrocarbon in which the unpaired electron is highly delocalized, the phenoxy radical is strongly stabilized and the bond dissociation energy for the O–H is decreased (Brigati and others 2002). The hydrogen-donating ability decreases in the order of hydroxytyrosol, oleuropein, caffeic acid, chlorogenic acid, and ferulic acid in olive oil (Roche and others 2005).
The antioxidant activity of phenolic acids such as caffeic, protocatechuic, and chlorogenic acids is dependent on the pH; they are not efficient radical scavengers under acidic pH, but very good scavengers above pH 7 to 8 (Mukai and others 1997; Amorati and others 2006). At the basic pH, phenolic acids are ionized to a phenolated form. The phenolated antioxidant has a higher electron-donating capacity than the parent species and activates the phenolic group to give higher free radical scavenging activity (Amorati and others 2006). The higher radical scavenging activity of the phenolated form of phenolic acids was suggested to be due to a rapid electron transfer to lipid peroxy radicals from the anion of the phenolic acids (Amorati and others 2006).
The reduction potential of antioxidant radicals can predict the ease of a compound to donate hydrogen to food radicals; the lower the reduction potential of the antioxidant radicals, the greater the hydrogen donating ability of the antioxidants (Choe and Min 2005). Any compound whose radical has a reduction potential lower than food radicals or oxygen-related radicals can donate hydrogen to them, and can act as an antioxidant (Choe and Min 2005). The reduction potentials of hydroxy, alkyl, alkoxy, alkyl peroxy, and superoxide anion radicals are approximately 2300, 600, 1600, 1000, and 940 mV (Choe and Min 2005), respectively. Tocopherol, ascorbic acid, and quercetin radicals have reduction potentials of 500, 330, and 330 mV (Steenken and Neta 1982; Jovanovic and others 1996), respectively, which are lower than peroxy, alkoxy, and alkyl radicals. This enables for tocopherol and ascorbic acid to donate hydrogen to the peroxy, alkoxy, and alkyl radicals to slow down the formation of food radicals. Phenolic compounds can donate hydrogen to alkyl peroxy radicals and the resulting phenolic radicals do not catalyze the oxidation of other molecules due to the low reduction potential (Shahidi and Wanasundara 1992). The phenolic radicals react with each other to form hydroquinone with regeneration of phenolic antioxidants or to form phenolic dimers. The phenolic radical can react with lipid peroxy radicals to form phenolic-peroxy species adducts that undergo the degradation reactions (Reische and others 2002).
α-Tocopherol reacts with alkyl peroxy radicals more rapidly than alkyl radicals since the difference in reduction potential between tocopherol radicals and alkyl peroxy radicals (500 mV) is higher than that between tocopherol radicals and alkyl radicals (100 mV). Tocopherol donates hydrogen at the 6-hydroxy group on a chromanol ring to alkyl peroxy radical, and alkyl hydroperoxide and tocopherol radical are formed. Tocopherol radical is relatively stable due to a resonance structure (Figure 8). Tocopherol radical can react with lipid peroxy radical to produce tocopherol semiquinone having no vitamin E activity, or react with each other for the formation of tocopherol dimer (Reische and others 2002). Reaction rates of peroxy radical of unsaturated fatty acids with α-tocopherol are 1.85 × 106/M/s (Kamal-Eldin and others 2008). Tocopherols slowly and irreversibly react with superoxide anion radicals in organic solvents and produce tocopherol radical, but the reaction is insignificant in aqueous solution (Arudi and others 1983; Halliwell and Gutteridge 2001).
Tocopherol radical sometimes reacts with lipid peroxy radicals at their very high concentration and produces tocopherol peroxide. Tocopherol peroxide produces 2 isomers of epoxy-8α-hydroperoxytocopherone by elimination of an alkoxy radical followed by oxygen addition and hydrogen abstraction. Epoxy-8α-hydroperoxytocopherone becomes epoxyquinones upon hydrolysis (Liebler and others 1990). This reaction produces alkoxy radicals, instead of peroxy radicals, and loses only tocopherol. Since there is no net decrease in free radicals in the system, tocopherol does not act as an antioxidant; however, reducing agents such as ascorbic acid can regenerate tocopherols from tocopherylquinone.
Tocopherol radical at high concentration sometimes abstracts hydrogen from lipids having very low concentration of peroxy radical and produces tocopherol and lipid radical; however, the rate is very low (Kamal-Eldin and others 2008). The resulting lipid radical can increase the lipid oxidation by reacting with triplet oxygen, and tocopherol acts as prooxidant instead of antioxidant (Bowry and Stocker 1993; Yamamoto 2001). Tocopherol-mediated peroxidation is prevented by ascorbic acid since ascorbic acid quickly reduces tocopherol radicals to tocopherols (Yamamoto 2001).
Tyrosol and hydroxytyrosol in olive oil (Chimi and others 1991) and sesamol and sesaminol in sesame oil (Dachtler and others 2003; Suja and others 2005) scavenge free radicals by a similar mechanism as tocopherols due to the presence of phenolic hydrogen. Phenolic hydrogens of tyrosol and hydroxytyrosol are transferred to food radicals with the production of semiquinone radicals. The semiquinone radical of tyrosol or hydroxytyrosol may scavenge another radical to give a quinone, disproportionate with another semiquinone radical to give the parent compound and quinone, or react with oxygen to produce quinone and hydroperoxy radical (Niki and Noguchi 2000).
Flavonoids should have special structural features for scavenging free radicals as shown in Figure 9: the ortho-dihydroxy or catechol group in the B-ring, the conjugation of the B-ring to the 4-oxo group (Rice-Evans and others 1995; Van Acker and others 1996; Pietta 2000; Silva and others 2002). Quercetin, rutin, and luteolin satisfy the requirements and are known as some of the most efficient radical scavengers among the nonvitamin plant phenols (Rice-Evans and others 1995). Catechin, an efficient radical scavenger, does not have a 2,3-double bond and 4-carbonyl group, but it has many hydroxy groups to donate hydrogen (Rice-Evans and others 1996). Catechol-structured flavonoids scavenge lipid peroxy radicals by donating hydrogen and become more stable phenoxy radicals. Phenoxy radicals undergo disproportionation and produce phenolic quinone and a dihydroxy phenolic compound, as shown in Figure 10 (Shahidi and Wanasundara 1992).
Carotenoids can give electrons and then donate hydrogen as shown in Figure 11. Two electrons rather than 1 are transferred per carotenoid with 2 reduction potentials, E1 and E2. Ease of electron donation of carotenoids depends on the nature of substituents on the carotenoids (Jeevarajan and Kispert 1996). Reduction potential for sequential transferring 2 electrons are different in canthaxanthin and astaxanthin, generally E1 < E2, while lycopene, β-carotene, and zeaxanthin have similar E1 and E2 values (Jeevarajan and Kispert 1996; Liu and others 2000). Electron donation of carotenoids containing terminal electron acceptor group is difficult and the 2nd electron donation occurs at quite a different potential to the 1st oxidation step. As the electron-accepting strength of the end groups decreases, ΔE (E1 – E2) decreases or cation radical can be reduced to carotenoid radical with a reduction potential E3 which is generally much lower than E1 (Jeevarajan and Kispert 1996). The standard reduction potential of carotenoid radical cation (700 to 1000 mV; Jeevarajan and Kispert 1996; Liu and others 2000; Niedzwiedzki and others 2005; Han and others 2006) is not low enough that carotenoid cation donates hydrogen to alkyl (E°′= 600 mV) or peroxy radicals of polyunsaturated fatty acids (E°′= 770 to 1440 mV). It is easier for carotenoids to give hydrogen to hydroxy radicals having a high reduction potential (2310 mV) than to alkyl peroxy radicals. The energy required to remove hydrogen from carbons in carotene cation is about 65 kcal/mol (Zhou and others 2000). Lycopene radical cation has the lowest reduction potential (748 mV) followed by the radical cations of β-carotene (780 mV), zeaxanthin (812 mV), and canthaxanthin (930 mV) (Jeevarajan and Kispert 1996). Astaxanthin is a weaker antioxidant than zeaxanthin (Mortensen and Skibsted 1997a; Edge and others 1998).
β-Carotene may donate hydrogen to lipid peroxy radical with some limitations and produce carotene radical (Edge and others 1998). Carotene radical is a fairly stable species due to delocalization of unpaired electrons in its conjugated polyene, and has enough lifetime for a reaction with lipid peroxy radicals at low oxygen concentration and forms nonradical carotene peroxides (Burton and Ingold 1984; Beutner and others 2001). Carotene radical can also undergo oxygen addition, and subsequent reaction with another carotene molecule, and produce carotene epoxides and carbonyl compounds of carotene (Beutner and others 2001) as shown in Figure 12.
In addition to the radical scavenging activity of carotenoids by donating hydrogen to lipid peroxy radicals, carotenoids can enhance lipid oxidation (Lee and others 2003). Lipid peroxy radicals (ROO •) from the oxidation of oils may be added to β-carotene (Car) and produce carotene peroxy radical (ROO–Car •), especially at oxygen pressure higher than 150 mm Hg (Burton and Ingold 1984). β-Carotene peroxy radical reacts with triplet oxygen to form peroxy radical of carotene peroxide (ROO–Car–OO •), which then abstracts hydrogen from another lipid molecule and produces lipid radicals (R′ •). The resulting lipid radicals propagate the chain reaction of lipid oxidation (Iannone and others 1998), thus β-carotene acts as a prooxidant:
β-Carotene may donate electrons to free radicals and become β-carotene radical cation (Liebler 1993; Mortensen and others 2001). β-Carotene radical cation is stable due to resonance, and the reaction rate with oxygen is very low (Edge and Truscott 2000; Decker 2002). However, β-carotene radical cation can easily oxidize tocopherols and ubiquinones (Liebler 1993) as well as tyrosine and cystein (Burke and others 2001). Hydrogen or electron transfers from carotenoids to food radicals depend on the reduction potentials of food radicals and chemical structures of carotenoids, especially the presence of oxygen-containing functional groups (Edge and others 1997). Electron-transfer reaction from carotenoids to free radicals is favored when the alkyl peroxy radicals contain electron withdrawing R groups (Edge and others 1998).
Ascorbic acid and glutathione scavenge free radicals by donating hydrogen to food radicals, producing more stable ascorbic acid and glutathione radicals than food radicals (Buettner 1993). Ascorbic acid radicals become dehydroascorbic acid by loss of proton (Decker 2002). Amino acids containing sulfhydryl or hydroxy groups such as cystein, tyrosine, phenylalanine, and proline also inactivate free radicals (Gebicki and Gebicki 1993). Inactivation of food radicals by proteinaceous compounds might be a result of competition between proteinaceous compounds and lipid for high-energy food radicals, rather than an actual chain breaker (Decker 2002).
Metals reduce the activation energy of the oxidation, especially in the initiation step, to accelerate oil oxidation (Jadhav and others 1996). The activation energies for the autoxidation of refined bleached and deodorized soybean, sunflower, and olive oils were 17.6, 19.0, and 12.5 kcal/mol, respectively (Lee and others 2007). Metals catalyze food radical formation by abstracting hydrogen. They also produce hydroxy radicals by catalyzing decomposition of hydrogen peroxide (Andersson 1998) or hydroperoxides (Benjelloun and others 1991). Ferric ions decrease the oxidative stability of olive oil by decomposing phenolic antioxidants such as caffeic acid (Keceli and Gordon 2002).
Crude oil contains transition metals such as iron or copper, often existing in chelated form rather than in a free form (Decker 2002). Oil refining decreases metal contents. Edible oils manufactured without refining, such as extra virgin olive oil (9.8 ppb copper and 0.73 ppm iron) and roasted sesame oil (16 ppb copper and 1.16 ppm iron), contain relatively high amounts of transition metals (Choe and others 2005).
Metal chelators decrease oxidation by preventing metal redox cycling, forming insoluble metal complexes, or providing steric hindrance between metals and food components or their oxidation intermediates (Graf and Eaton 1990). EDTA and citric acid are the most common metal chelators in foods. Most chelators are water-soluble, but citric acid can be dissolved in oils with some limitation to chelate metals in the oil system. Phospholipids also act as metal chelators (Koidis and Boskou 2006). Flavonoids can also bind the metal ions (Rice-Evans and others 1996) and the activity is closely related with the structural features: 3′, 4′-dihydroxy group in the B ring, the 4-carbonyl and 3-hydroxy group in the C ring, or the 4-carbonyl group in the C ring together with the 5-hydroxy group in the A ring (Hudson and Lewis 1983; Feralli and others 1997). Lignans, polyphenols, ascorbic acid, and amino acids such as carnosine and histidine can also chelate metals (Decker and others 2001).
Singlet oxygen quenching
Singlet oxygen having high energy of 93.6 kJ above the ground state triplet oxygen (Korycka-Dahl and Richardson 1978; Girotti 1998) reacts with lipids at a higher rate than triplet oxygen. Tocopherols, carotenoids, curcumin, phenolics, urate, and ascorbate can quench singlet oxygen (Lee and Min 1992; Das and Das 2002; Choe and Min 2005). Singlet oxygen quenching includes both physical and chemical quenching. Physical quenching leads to deactivation of singlet oxygen to the ground state triplet oxygen by energy transfer or charge transfer (Min and others 1989). There is neither oxygen consumption nor product formation. Singlet oxygen quenching by energy transfer occurs when the energy level of a quencher (Q) is very near or below that of singlet oxygen:
Carotenoids with 9 or more conjugated double bonds are good singlet oxygen quenchers by energy transfer. The singlet oxygen quenching activity of carotenoids depends on the number of conjugated double bonds in the structure (Beutner and others 2001; Min and Boff 2002; Foss and others 2004) and the substituents in the β-ionone ring (Di Mascio and others 1989). β-Carotene and lycopene which have 11 conjugated double bonds are more effective singlet oxygen quenchers than lutein which has 10 conjugated double bonds (Viljanen and others 2002). The presence of oxo and conjugated keto groups, or cyclopentane ring in the structure increases the singlet oxygen quenching ability (Di Mascio and others 1989); however, β-ionone ring substituted with hydroxy, epoxy, or methoxy groups is less effective (Viljanen and others 2002). The rate constants for singlet oxygen quenching by canthaxanthin, β-apo-8′-carotenal, all trans β-carotene, and ethyl β-apo-8′-carotenate are 1.45 × 1010, 1.38 × 1010, 1.25 × 1010, and 1.20 × 1010/M/s, respectively (Min and others 1989).
When a quencher has high reduction potential and low triplet energy, it quenches singlet oxygen by a charge transfer mechanism. These types of quenchers are amines, phenols (including tocopherols), sulfides, iodide, and azides, which all have many electrons (Min and others 1989). The quencher donates electron to singlet oxygen to form a singlet state charge transfer complex and then changes the complex to the triplet state by intersystem crossing. Finally, the triplet state charge transfer complex is dissociated into triplet oxygen and a quencher:
Chemical quenching of singlet oxygen is a reaction involving the oxidation of a quencher rather than a quenching, thus producing breakdown or oxidation products of a quencher. β-Carotene, tocopherols, ascorbic acid, amino acids (such as histidine, tryptophan, cysteine, and methionine), peptides, and phenolics are oxidized by singlet oxygen, and they are all chemical quenchers of singlet oxygen (Foote 1976; Michaeli and Feitelson 1994; Halliwell and Gutteridge 2001). β-Carotene reacts with singlet oxygen at a rate of 5.0 × 109/M/s (Devasagayam and others 1992) and produces 5,8-endoperoxides of β-carotene (Stratton and others 1993). Reaction of ascorbic acid with singlet oxygen produces an unstable hydroperoxide of ascorbic acid as shown in Figure 13. Tocopherol reacts irreversibly with singlet oxygen and produces tocopherol hydroperoxydienone, tocopherylquinone, and quinone epoxide (Decker 2002). The reaction rates of tocopherols with singlet oxygen are different among isomers: α-tocopherol shows the highest reaction rate of 2.1 × 108/M/s, followed by β- tocopherol with 1.5 × 108/M/s, γ-tocopherol with 1.4 × 108/M/s, and δ-tocopherol with 5.3 × 107/M/s (Mukai and others 1991).