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Mechanisms of Antioxidants in the Oxidation of Foods
Author Choe is with Dept. of Food and Nutrition, Inha Univ., Incheon, Korea. Author Min is with Dept. of Food Science and Technology, The Ohio State Univ., 2015 Fyffe Rd., Columbus, Ohio, U.S.A. Direct inquiries to author Min (E-mail: Min.firstname.lastname@example.org).
Author Choe is with Dept. of Food and Nutrition, Inha Univ., Incheon, Korea. Author Min is with Dept. of Food Science and Technology, The Ohio State Univ., 2015 Fyffe Rd., Columbus, Ohio, U.S.A. Direct inquiries to author Min (E-mail: Min.email@example.com).
ABSTRACT: Antioxidants delay or inhibit lipid oxidation at low concentration. Tocopherols, ascorbic acid, carotenoids, flavonoids, amino acids, phospholipids, and sterols are natural antioxidants in foods. Antioxidants inhibit the oxidation of foods by scavenging free radicals, chelating prooxidative metals, quenching singlet oxygen and photosensitizers, and inactivating lipoxygenase. Antioxidants show interactions, such as synergism (tocopherols and ascorbic acids), antagonism (α-tocopherol and caffeic acid), and simple addition. Synergism occurs when one antioxidant is regenerated by others, when one antioxidant protects another antioxidant by its sacrificial oxidation, and when 2 or more antioxidants show different antioxidant mechanisms.
Oxidation decreases consumer acceptability of foods by producing low-molecular-weight off-flavor compounds, as well as by destroying essential nutrients, and it produces toxic compounds and dimers or polymers of lipids and proteins (Aruoma 1998). Oxidation of foods can be minimized by removing prooxidants such as free fatty acids, metals, and oxidized compounds, and by protecting foods from light. Air evacuation by reduced pressure or adding oxygen scavengers can also reduce oxidation. Since it is very difficult to completely remove all the prooxidants and air, antioxidants are now increasingly added to foods to slow down the process of oxidation.
Antioxidants significantly delay or inhibit oxidation of oxidizable substrates at low concentration, compared to the higher contents of lipids and proteins in foods (Halliwell and Gutteridge 2001). Antioxidants in foods do not necessarily protect biological tissues from free radical oxidative damage because they have to be converted into usable forms in tissues and interact with other substances, in addition to effective concentration differences, and they must display difficulty in absorption from the diet (Azzi and others 2004). The antioxidants are naturally present in foods, or can be added or formed during processing. Antioxidants for foods should be reasonable in cost, nontoxic, stable, effective at low concentration, have carry-through, and should not change flavor, color, and texture of the food matrix (Schuler 1990). The effects of antioxidants on the oxidation of foods are dependent on their concentration (Frankel and others 1996), polarity, and the medium (Cuvelier and others 2000; Samotyja and Malecka 2007), and also the presence of other antioxidants (Decker 2002). The objective of this article was to discuss the reaction mechanisms of antioxidants by focusing on their thermodynamic and kinetic characteristics depending on their surroundings during the oxidation of foods.
Major Antioxidants in Foods
Extensive research has been done on the isolation, purification, and identification of the various antioxidants. Phenolic compounds and ascorbic acid are the most important natural antioxidants. Carotenoids, protein-related compounds, Maillard reaction products, phospholipids, and sterols also show natural antioxidant activities in foods.
Tocopherols Tocopherols are monophenolic compounds and derivatives of chromanol as shown in Figure 1. They are very soluble in oil and thus are the most important antioxidants in edible fats and oils. Tocopherols are more frequently found in vegetable oils than animal fats, especially soybean, canola, sunflower, corn, and palm oils. Most vegetable oils contain tocopherols at concentrations higher than 500 ppm; beef tallow and lard contain less than 40 ppm (Choe and others 2005). Palm oil contains tocopherols at 100 to 150 ppm, and also 620 to 650 ppm tocotrienols (Al-Saqer and others 2004). The refining process, especially deodorization, reduces tocopherol contents in oils (Jung and others 1989; Reische and others 2002; Eidhin and others 2003). Tocopherols in crude soybean oil (1670 ppm) were decreased to 1138 ppm during deodorization (Jung and others 1989).
Flavonoids are major plant polyphenols and are derivatives of diphenylpropanes and a heterocyclic 6-membered ring with oxygen. They include flavanols (catechins, naringin), flavanones (hesperidin, naringenin), flavones (apigenin, luteolin), flavonols (kaempferol, quercitrin, myricetin, quercetin), anthocyanins, and leucoanthocyanidins. The glycosylation of flavonoids results in lower antioxidant activity than the corresponding aglycons (Shahidi and Wanasundara 1992). The solubility of flavonoids in fats and oils is very low and their role in the oxidation of oil is not significant; however, they can contribute to decreasing the oxidation of oil in food emulsions (Zhou and others 2005).
Phenolic acids Phenolic acids are closely related to flavonoids. They include hydroxycinnamic acids (coumaric, ferulic, caffeic, chlorogenic, and sinapic acids), hydroxycoumarin (scopoletin), and hydroxybenzoic acids (ellagic, gallic, gentisic, salicylic, and vanillic acids). Chlorogenic and caffeic acids are present in sunflower oil, and sinapic and ferulic acids are present in rapeseed (Leonardis and others 2003) and defatted rice bran oils (Devi and others 2007), respectively. Olive oil contains vanillic, syringic, caffeic, and cinnamic acids (Servili and Montedoro 2002). Phenolic acids as antioxidants in oils are also limited due to solubility problems.
Lignans Lignans are phenylpropanoids derived from phenylalanine as shown in Figure 2. They include sesamol, sesamin, sesamolin, sesaminol, sesamolinol, pinoresinol, and secoisolariciresinol. The major lignans in unroasted sesame oil are sesamin (474 ppm), sesamolin (159 ppm), and sesamol (<7 ppm) (Fukuda and others 1986; Dachtler and others 2003). Concentration of sesamol is increased to higher than 36 ppm by roasting the sesame seeds due to hydrolysis of sesamolin to sesamol (Kim and Choe 2005). Sesamin and sesamolin extracted from roasted sesame oil and sesaminol in bleached sesame oil are more heat-resistant than α-tocopherol (Fukuda and others 1986; Lee and others 2007). Secoisolariciresinol and secoisolariciresinol diglucoside (14.1 to 30.9 mg/g, dry basis) are found in flaxseed (Eliasson and others 2003).
Ascorbic acid, sodium ascorbate, and calcium ascorbate are water soluble and have a limitation as antioxidants for fats and oils. Ascorbyl palmitate is used in fat-containing foods to decrease their oxidation.
Carotenoids are polyenoic terpenoids having conjugated trans double bonds. They include carotenes (β-carotene and lycopene), which are polyene hydrocarbons, and xanthophylls (lutein, zeaxanthin, capsanthin, canthaxanthin, astaxanthin, and violaxanthin) having oxygen in the form of hydroxy, oxo, or epoxy groups (Figure 3). Carotenoids are fat soluble and play an important role in the oxidation of fats and oils.
Carotene is the major carotenoid in oils, and β-carotene is the most studied. Palm oil is one of the richest sources of carotenoids. Crude palm oil and red palm olein contain 500 to 700 ppm carotenoids (Bonnie and Choo 2000), but refined plam oil is not a good source of carotenoids. Virgin olive oil contains 1.0 to 2.7 ppm β-carotene, as well as 0.9 to 2.3 ppm lutein (Psomiadou and Tsimidou 2002). Corn, soybean, and peanut oils contain lower amounts of β-carotene at 1.2, 0.28, and 0.13 ppm, respectively (Parry and others 2006).
Hypoxanthine, xanthine, glycine, methionine, histidine, tryptophan, proline, lysine, ferritin, transferritin, and carnosine show their antioxidant activities in the oxidation of lipid-containing foods (Reische and others 2002). Enzymes such as glucose oxidase, superoxide dismutase, catalase, and glutathione peroxidase are known to decrease the oxidation of foods (Yuan and Kitts 1997). Application of enzymes and proteins as antioxidants is limited to unprocessed oil because oil processing denatures the enzymes and proteins.
Maillard reaction products
Maillard reaction products from amines and reducing sugars or carbonyl compounds from lipid oxidation slow down lipid oxidation (Kumari and Waller 1987; Saito and Ishihara 1997). There are a number of Maillard reaction products, but the responsible compounds for the antioxidant activity have not been clearly determined to date.
Crude oil contains phospholipids such as phosphatidylethanolamine, phosphatidylcholine, phosphatidylinositol, and phosphatidylserine, but most of them are removed by oil processing such as degumming (Jung and others 1989). Oils that are consumed without refining contain higher amounts of phospholipids. Crude soybean oil contains phosphatidylcholine and phosphatidylethanolamine at 501 and 214 ppm, respectively; however, RBD soybean oil contains only 0.86 and 0.12 ppm phosphatidylcholine and phosphatidylethanolamine, respectively (Yoon and others 1987). Unroasted sesame oil contains 690 ppm phospholipids (Yen 1990). Extra virgin olive oil contains 34 to 156 ppm phospholipids and filtration of the oil lowers the contents to 21 to 124 ppm (Koidis and Boskou 2006).
Phosphatidylcholine decreased the oxidation of docosahexaenoic acid (DHA) and soybean oil in the dark (Koo and Kim 2005; Lyberg and others 2005). Egg yolk phospholipids at 0.031% to 0.097% decreased the autoxidation of DHA-rich oil and squalene, and the antioxidant activity of egg yolk phosphatidylethanolamine was higher than that of phosphatidylcholine (Sugino and others 1997). Although phospholipids are generally known as antioxidants, they can increase lipid oxidation depending on the environment such as presence of iron (Yoon and Min 1987). Lee (2007) reported that phosphatidylcholine and phosphatidylethanolamine increased the oxidation of tocopherol-stripped canola oil with added chlorophyll b under light.
Sterols are steroid alcohols with an aliphatic hydrocarbon side chain of 8 to 10 carbons at the C17-position and a hydroxy group at the C3-position (Figure 4). β-Sitosterol, stigmasterol, and sitostanol are present in edible oils, with the highest amount of β-sitosterol. Corn and rapeseed oils have 8000 ppm sterols, and palm and coconut oils have 600 to 1000 ppm sterols (Verhe and others 2006). Virgin and refined olive oils contain β-sitosterol at 667 and 898 ppm, respectively (Canabate-Diaz and others 2007). Antioxidant activity of β-sitosterol was lower than those of ferulic acid and tocopherol in the autoxidation of soybean oil (Devi and others 2007). Solubility of plant sterols in corn oil is 2% to 3% at 25 °C (Vaikousi and others 2007).
Oxidation Mechanisms of Fats and Oils
Different chemical mechanisms are responsible for the oxidation of fats and oils during processing, storage, and cooking. Two types of oxygen, atmospheric triplet oxygen and singlet oxygen, can react with fats and oils. Triplet oxygen, having a radical character, reacts with radicals and causes autoxidation. The nonradical electrophilic singlet oxygen does not require radicals to react with; it directly reacts with the double bonds of unsaturated fats and oils with high electron densities, which is called type II photosensitized oxidation (Choe and Min 2005).
Fats and oils should be in radical forms to react with triplet oxygen in autoxidation. Lipids are normally in nonradical singlet state and heat, metals, or light accelerates their radical formation. Allylic hydrogen, especially hydrogen attached to the carbon between 2 double bonds, is easily removed due to low bond dissociation energy (Min and Boff 2002; Choe and Min 2005). The carbon and hydrogen dissociation energies are the lowest at the bis-allylic methylene position (Wagner and others 1994). Bis-allylic hydrogen at C11 of linoleic acid is removed at 75 to 80 kcal/mol. The energy required to remove allylic hydrogen in C8 or C14 of linoleic acid is 88 kcal/mol, and 101 kcal/mol is necessary to remove alkyl hydrogen from C17 or C18 (Wagner and others 1994; Min and Boff 2002; Choe and Min 2005). Upon formation of lipid radicals by hydrogen removal, the double bond adjacent to the carbon radical in linoleic and linolenic acids shifts to the more stable next carbon, resulting in conjugated diene structures. The shifted double bond mostly takes the more thermodynamically stable trans form.
The lipid radical reacts with triplet oxygen very quickly at normal oxygen pressure (2 to 8 × 109/M/s; Zhu and Sevilla 1990) and forms lipid peroxy radical. The lipid peroxy radical abstracts hydrogen from other lipid molecules to form lipid hydroperoxide and another lipid radical. The radicals automatically catalyze the reaction and the autoxidation is called free radical chain reaction. When radicals react with each other, nonradical species are produced to stop the reaction.
Light accelerates lipid oxidation, especially in the presence of photosensitizers such as chlorophylls. Chlorophylls in singlet state become excited upon absorption of light energy in pico second (Choe and Min 2006). Excited singlet state chlorophylls become excited triplet state via intersystem crossing (k = 1 to 20 × 108/s; Min and Boff 2002). Excited triplet state chlorophylls react with triplet oxygen and produce singlet oxygen by energy transfer, returning to their ground singlet state. Singlet oxygen is able to diffuse over larger distances, about 270 nm (Skovsen and others 2005), to react with electron-rich compounds. Since singlet oxygen is electrophilic due to a completely vacant 2 pπ orbital, it directly reacts with high-electron-density double bonds via 6-membered ring without lipid radical formation (Gollnick 1978; Choe and Min 2005). The resulting hydroperoxides by singlet oxygen are both conjugated and nonconjugated (Frankel 1985; Figure 5). Production of nonconjugated hydroperoxides does not occur in autoxidation. The oxidation of linoleic acid by singlet oxygen produces C9- and C13-hydroperoxides, as well as C10- and C12-hydroperoxides (Frankel 1985).
The reaction rate of lipid with singlet oxygen is much higher than that with triplet oxygen; the reaction rates of linoleic acid with singlet oxygen and triplet oxygen are 1.3 × 105 and 8.9 × 101/M/s, respectively (Rawls and Van Santen 1970).
Heating of oil produces various chemical changes including oxidation. The chemical mechanism of thermal oxidation is basically the same as the autoxidation mechanism. The rate of thermal oxidation is faster than the autoxidation, and the unstable primary oxidation products, hydroperoxides, are decomposed rapidly into secondary oxidation products such as aldehydes and ketones (Choe and Min 2007). Specific and detailed scientific information and comparisons of the oxidation rates between thermal oxidation and autoxidation are not yet available.
Thermal oxidation of oil produces many volatiles and nonvolatiles. Volatiles such as aldehydes, ketones, short-chain hydrocarbons, lactones, alcohols, and esters are produced from decomposition of hydroperoxides by the same mechanisms as the autoxidation. Many nonvolatile polar compounds and triacylglycerol dimers and polymers are produced in thermally oxidized oil by radical reactions. Dimerization and polymerization are major reactions in the thermal oxidation in oil. Dimers and polymers are large molecules with a molecular weight range of 692 to 1600 Daltons and formed by a combination of –C–C–, –C–O–C–, and –C–O–O–C– bonds (Kim and others 1999). Polymerization occurs more easily in oil with high linoleic acid than in high oleic acid oil contents (Bastida and Sanchez-Muniz 2001). C–C bonds are formed between 2 acyl groups to produce acyclic dimers in heated oil under low oxygen (Nawar 1996). The Diels-Alder reaction produces cyclic dimers of tetrasusbtituted cyclohexene, and radical reactions within or between triacylglycerols also produce cyclic polymers (Choe and Min 2007). Polymers are rich in oxygen and highly conjugated dienes and produce a brown, resin-like residue (Moreira and others 1999).
Lipid oxidation is catalyzed by lipoxygenase in a nonradical mechanism (Niki 2004). Lipoxygenase is an iron-bound enzyme with Fe in its active center. Lipoxygenase oxidizes unsaturated fatty acids having a 1-cis, 4-cis-pentadiene system resulting in oil deterioration (Engeseth and others 1987), and oils containing linoleic, linolenic, and arachidonic acids are favored substrates (Hsieh and Kinsella 1986). Eicosapentaenoic acid (EPA) and DHA can also be oxidized by lipoxygenase (Wang and others 1991).
Lipoxygenase with iron in the ferric state (LOX-Fe3+) forms a stereospecific complex with the unsaturated fatty acid having a 1,4-pentadienyl system (RH), and it abstracts hydrogens from interrupted methylenes in the fatty acids (Figure 6). It binds to pentadienyl radical which is rearranged into a conjugated diene system, followed by the reaction with oxygen to produce lipid peroxy radicals (ROO •). The iron in the enzyme is reduced to the ferrous state (LOX-Fe2+). Lipid peroxy radicals are reduced to ROO− by lipoxygenase with iron in a ferric state again, and the attachment of a proton, which is produced by the oxidation of hydrogen abstracted from fats and oils by lipoxygenase, results in release of hydroperoxides (Belitz and Grosch 1999).
Mechanisms of Antioxidants in the Oxidation of Foods
Antioxidants slow down the oxidation rates of foods by a combination of scavenging free radicals, chelating prooxidative metals, quenching singlet oxygen and photosensitizers, and inactivating lipoxygenase.
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).
Foods contain sensitizers such as chlorophylls and riboflavin (Jung and others 1989; Salvador and others 2001), which are activated by light. Photoactivated sensitizers transfer the energy to triplet atmospheric oxygen to form singlet oxygen, or transfer an electron to the triplet oxygen to form a superoxide anion radical, and these reactive oxygen species react with food components to produce free radicals (Min and Lee 1988). Carotenoids having fewer than 9 conjugated double bonds prefer the inactivation of photosensitizers instead of singlet oxygen quenching; singlet oxygen quenching is preferable by carotenoids with 9 or more conjugated double bonds (Viljanen and others 2002). Energy of the photosensitizer is transferred to the singlet state of carotenoids to become a triplet state of carotenoids, which is changed to the singlet state by transferring the energy to the surrounding or emitting phosphorescence (Stahl and Sies 1992). The edge-to-edge distance for a direct quenching of triplet state of chlorophyll by carotenoids must be less than the van der Waals distance (0.36 nm), which enables some overlap between electron orbitals of these 2 pigments (Edge and Truscott 1999).
Lipoxygenase is a catalytic enzyme in the oxidation of lipids and is inactivated by tempering, which is heat treatment with moisture. Steaming of ground soybeans at 100 °C for 2 min or 116 °C under 44.5 N for 1 min decreases the lipoxygenase activity by 80% to 100%, with a decrease in peroxide values, which improves the sensory quality of crude soybean oil (Engeseth and others 1987).
Interactions of Antioxidants in the Oxidation of Foods
Interactions among antioxidants can be synergistic, antagonistic, or merely additive. Synergism is a phenomenon in which a net interactive antioxidant effect is higher than the sum of the individual effects. A typical example of antioxidant synergism is between α-tocopherol and ascorbic acid in autoxidation (Liebler 1993) and photooxidation of lipids (Van Aardt and others 2005). Antagonism is a phenomenon in which a net interactive antioxidant effect is lower than the sum of the individual antioxidant effects, and the additive interaction means that a net interactive antioxidant effect is the same as the sum of individual effects. Polyphenolic compounds such as epigallocatechin gallate, quercetin, epicatechin gallate, epicatechin, and cyanidin showed additive effects on free radical scavenging activity with ascorbic acid or α-tocopherol (Murakami and others 2003).
Several mechanisms are involved in synergism among antioxidants: a combination of 2 or more different free radical scavengers in which one antioxidant is regenerated by others, a sacrificial oxidation of an antioxidant to protect another antioxidant, and a combination of 2 or more antioxidants whose antioxidant mechanisms are different (Decker 2002). Regeneration of a more effective free radical scavenger (primary antioxidant) by a less effective free radical scavenger (coantioxidant, synergist) occurs mostly when one free radical scavenger has a higher reduction potential than the other. The free radical scavenger having a higher reduction potential acts as a primary antioxidant. Regeneration of primary antioxidants contributes to a higher net interactive antioxidant effect than the simple sum of individual effects (Decker 2002). The antioxidant system of ascorbic acid and tocopherols is an example, in which tocopherols (E°= 500 mV) are primary antioxidants and ascorbic acid (E°= 330 mV) is a synergist (Liebler 1993). Tocopherols (TH) act as antioxidant by donating hydrogen to alkyl (R •) or alkyl peroxy (ROO •) radicals in foods and become tocopherol radical (T •) which does not have antioxidant activity. Ascorbic acid (AsH) gives hydrogen to tocopherol radical to regenerate tocopherols and it becomes semihydroascorbyl radical (As •), and then dehydroascorbic acid (DHAs; Buettner 1993):
Two antioxidants whose bond dissociation energy difference is high exert a synergistic antioxidant effect (Decker 2002). Regeneration of the antioxidant is fast when a synergist has a higher bond dissociation energy than the primary antioxidant (Pedrielli and Skibsted 2002). Also, the primary antioxidant can be regenerated when the rate constant for regeneration of the primary antioxidant is at least 103/M/s and the reaction constant of alkyl peroxy radicals with that of the antioxidant radicals is similar (Amorati and others 2002a, 2002b). Regeneration of the antioxidant can be accomplished by electron transfer from a synergist to a primary antioxidant (Jovanovic and others 1995).
Synergistic antioxidant effects can be achieved by the protective action of one antioxidant by means of its sacrificial oxidation (Decker 2002). The less effective antioxidant traps alkyl or alkyl peroxy radicals in foods, resulting in protecting more an effective antioxidant from the oxidation due to antioxidant action. Or the antioxidant radical produced from the oxidation of the less effective antioxidant competes with more effective antioxidant for trapping alkyl peroxy radicals to decrease the oxidation of the more effective antioxidant. The interaction between tocopherols and carotenoids partly results from this mechanism (Haila and others 1996).
When there are 2 or more antioxidants whose antioxidant mechanisms are different, the antioxidation can also show a synergism (Decker 2002). A combination of metal chelators and free radical scavengers is a good example. They show synergism in inhibiting the oxidation of food components, mainly due to the sparing action of free radical scavengers by chelators. Metal chelators such as phospholipids inhibit metal-catalyzed oxidation (Koidis and Boskou 2006), producing lower levels of radicals to be reduced by the antioxidants acting as free radical scavengers. Metal chelators mainly act during the initiation step of lipid oxidation and free radical scavengers do so at the propagation step (Choe and Min 2006). Phosphatidylinositol acts as a synergist with tocopherols in decreasing lipid oxidation, mainly by inactive complex formation with prooxidative metals (Servili and Montedoro 2002). Quercetin and α-tocopherol show a synergism in decreasing the oxidation of lard by the mechanism in which α-tocopherol acts as a free radical scavenger while quercetin acts as a metal chelator (Hudson and Lewis 1983).
Antagonism among antioxidants in the oxidation of food components can arise by regeneration of the less effective antioxidant by the more effective antioxidant (Peyrat-Maillard and others 2003), oxidation of the more effective antioxidant by the radicals of the less effective antioxidant, competition between formation of antioxidant radical adducts and regeneration of the antioxidant (Mortensen and Skibsted 1997a, 1997c), and alteration of microenvironment of one antioxidant by another antioxidant. Antagonism of antioxidants in the oxidation of foods has not yet been described in detail.
Reaction mechanisms and the type of natural antioxidants in foods, tocopherols, ascorbic acid, carotenoids, flavonoids, amino acids, phospholipids, and sterols were reviewed kinetically and thermodynamically. They inhibit the oxidation of useful food components by inactivating free radicals, chelating prooxidative metals, and quenching singlet oxygen and photosensitizers. When there are 2 or more antioxidants together, interaction occurs such as synergism, antagonism, and simple addition.