phenolic antioxidants critical reviews in food science...
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Phenolic antioxidantsFereidoon Shahidi a , P. K. Janitha a & P. D. Wanasundara aa Department of Biochemistry, Memorial University of Newfoundland, St. John's, NF, A1B3X9,Canada
Available online: 29 Sep 2009
To cite this article: Fereidoon Shahidi, P. K. Janitha & P. D. Wanasundara (1992): Phenolic antioxidants, Critical Reviews inFood Science and Nutrition, 32:1, 67-103
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Critical Reviews in Food Science and Nutrition, 32(l):67-103 (1992)
Phenolic Antioxidants
Fereidoon Shahidi and P. K. Janitha P. D. WanasundaraDepartment of Biochemistry, Memorial University of Newfoundland, St. John's, NF, CanadaA1B3X9
KEY WORDS: antioxidants, phenolic, synthetic, natural.
I. INTRODUCTION
One of the major changes that occurs duringprocessing, distribution, and final preparation offood is oxidation. Oxidation of lipids initiatesother changes in the food system that affect itsnutritional quality, wholesomeness, safety, color,flavor, and texture. Antioxidants are one of theprincipal ingredients that protect food quality bypreventing oxidative deterioration of lipids.
Autoxidation of polyunsaturated lipids of foodinvolves a free radical chain reaction that is mostfrequently initiated by exposing lipids to light,heat, ionizing radiation, metal ions, or metallo-protein catalysts. Enzyme lipoxygenäse can alsoinitiate oxidation. The classic route of autoxi-dation includes initiation (production of lipid freeradicals), propagation, and termination (produc-tion of nonradical products) reactions. Antioxi-dants can interfere with the oxidation process byreacting with free radicals, chelating catalyticmetals, and also by acting as oxygen scavengers.Phenolic antioxidants function as free radical ter-minators and sometimes also as metal chelators.Phenolic compounds and some of their deriva-tives are very efficient in preventing autoxida-tion; however, only a few phenolic compoundsare currently permissible by law as food antiox-idants. The major considerations for acceptabilityof such antioxidants are their activity and poten-tial toxicity. The approved phenolic antioxidantshave been studied extensively, but the toxicologyof their degradation products is still not clear.Some plant phenolic compounds have been con-
sidered recently as antioxidants and are beingproduced commercially. Flavonoids, the mostpotent antioxidative compounds of plant pheno-lics, need further investigation for their feasibilityof use in foods as well as their toxicologicalimplications.
In this review, phenolic antioxidants of bothsynthetic and natural origin, their mode of action,and the active components of naturally occurringplant foods are discussed thoroughly.
II. AUTOXIDATION — GENERALCONSIDERATIONS
Autoxidation is a natural process that takesplace between molecular oxygen and unsaturatedlipids in the environment. In food systems, au-toxidation of unsaturated fatty acid moieties bringsabout deterioration of food lipids. Several foodand feed quality parameters can be severely af-fected by these reactions. Quality attributes suchas the aroma derived from formation or modifi-cation of volatile odoriferous compounds,27 thetaste formed by hydroxyacids, the color fromMaillard-type reactions between reducing sub-stances originating from lipids and proteins,78 thetexture created by cross-linking reactions of pro-teins, the nutritive value from decreasing the es-sential fatty acids (EFAs), as well as the destruc-tion of fat-soluble vitamins have been studiedthoroughly. The safety of food, for instance, ox-idation of cholesterol,27 also was investigated re-cently. The key reactions leading to quality
1040-8398/92/$.50© 1992 by CRC Press, Inc.
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changes in foodstuffs, feeds, and various factorsthat enhance or inhibit them are summarized inFigure 1.
III. MECHANISM OF LIPIDAUTOXIDATION
Much of the basic understanding of the mech-anism of autoxidation has been provided fromstudies reported by Bateman and co-workers.89
These investigations were carried out largely inmodel systems and have established that hydro-peroxides are the primary products of autoxida-tion. Studies carried out by Farmer et al.,28 Bol-land and Gee," and Bateman9 have furtherestablished that autoxidation is a free radical chainreaction and could be described in terms of ini-tiation, propagation, and termination processes(Reactions 1 to 4).
In type 2, molecular oxygen rather than thesubstrate is presumably the species that reactswith the sensitizer upon light absorption (Reac-tions 7 and 8).
Sens + O2 + hv » Intermediate-2 (7)
Intermediate-2 + A > Products + Sens (8)
Several substances are commonly found infat-containing foods that can act as a photosen-sitizer to produce 'O2. These include natural pig-ments that are generally present in foods such aschlorophyll a, pheophytin a, myoglobin, hema-toprophyrin, flavin, and riboflavin.47 The syn-thetic colorant, erythrosine, may also act as anactive photosensitizer,73 and metal ions could beinvolved in activating molecular oxygen to pro-duce singlet oxygen (Reaction 9).
InitiationPropagation
Termination
RH-R• + O2
ROO + RH
R + R-]R + ROO
ROO + ROO
R + H'ROOR + ROOH
nonradical products
(1)(2)(3)
(4)
Since Reaction 1 is thermodynamically difficult(activation energy of about 35 kcal/mol), pro-duction of the first few radicals is necessary tobegin the propagation reaction.73 The reaction ofa lipid with molecular oxygen in its excited sin-glet state ('O2), or by metal catalysis, or by ex-posure to light, can form lipid peroxy radicals.The conversion of triplet oxygen to singlet ox-ygen may occur in many ways; the most impor-tant is via photosensitization of the natural pig-ments present in foods. Two pathways have beenproposed for photosensitized oxidation.15 In type1, the sensitizer presumably reacts after absorp-tion of light with substrate (A) to form inter-mediates that may then react with ground state(triplet) oxygen to yield the oxidation products(Reactions 5 and 6).
Mn + + O,
Sens + A + h v
Intermediate-1 + O2
Intermediate-1 (5)
Products + Sens (6)
+
— e"
+ HH
'O2
(9)
Lipid radicals are highly reactive and canreadily undergo propagation reactions either byabstraction of a hydrogen atom from the a po-sition adjacent to a double bond or by reactionwith molecular oxygen in its ground state (Re-action 2). The oxygénation reaction is very rapid,having almost a zero activation energy; therefore,the concentration of the peroxy radical (ROO*)is much higher than that of the alkyl radical (R*)in food systems in which oxygen is present. Theperoxy radical ROO' can take part in all typicalradical-mediated reactions such as that in path-way 3. The sequence of Reactions 2 and 3 are
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RH
Initialion
Propagation
H abstraction
Initiators (uv light, O metal catalysts, heat, etc.)
R'flipid free radicals)
ROO-
ROOH (HydroperoxidBS)
Termination
Oxidation of pigments,
flavors and vitamins
Breakdown products
such as ketones, aldehydes,alcohols, hydrocarbons, acids,epoxides
(including rancid off-flavorcompounds)
Polymerization products
(dark color, possibly toxic)
Insolubilization of proteins
(changes of functionality
and texture)
FIGURE 1. General scheme for autoxidation of polyunsaturated fatty acids of lipids and theirconsequences.
repeated. Due to the resonance stabilization ofR" species, the reaction pathway is usually ac-companied by a shift in the position of doublebonds, thus resulting in the formation of isomerichydroperoxides that often contain conjugateddiene groups.73
The combination of two radicals is a processwith a low enthalpy of activation; however, itsoccurrence is limited both by low concentrationof radicals as well as steric factors when radicalsare required to colloid with a specific orientation.Termination reactions may become important inedible oils heated at elevated temperatures, suchas when large amounts of polymers are formedin frying oils. Hydroperoxides decompose read-ily and spontaneously at 160°C17 and the peroxyradical concentration can become relatively highunder such conditions, thus leading to the for-mation of polymers.30 Hydroperoxides may alsodecompose to produce alcohols, aldehydes, alkylformates, ketones, hydrocarbons, etc.73 A gen-
eralized scheme for autoxidation of lipids is givenin Figure 1.
IV. PREVENTION OF AUTOXIDATIONAND USE OF ANTIOXIDANTS
Vacuum (or sous vide) packaging or pack-aging under an inert gas (i.e., modified atmos-phere packaging) to exclude oxygen, as well asrefrigeration or freezing can reduce the rate ofautoxidation.21 However, these means are not al-ways practical because very little oxygen is neededto initiate and maintain the oxidation process. Itis neither economical nor practical to removetraces of oxygen from foods. Therefore, it is quitecommon to combine such methods with the useof antioxidants. The main justification for using(an) antioxidant(s) is to extend the shelf life offoodstuffs and to reduce wastage and nutritionallosses by inhibiting and delaying oxidation. An-
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tioxidants cannot, however, improve the qualityof an already oxidized food product.21-96
Antioxidants are regarded as compounds ca-pable of delaying, retarding, or preventing au-toxidation processes. According to the USDACode of Federal Regulations [21 CFR 170.3 (0)(3)], "antioxidants are substances used to pre-serve food by retarding deterioration, rancidityor discoloration due to oxidation."24 Synergistsare substances that enhance the activity of an-tioxidants without having their own antioxidantactivity.73
It has been suggested21 that an ideal food-grade antioxidant should be safe; not impart color,odor, or flavor; be effective at low concentra-tions; be easy to incorporate; survive after pro-cessing; and be stable in the finished product(carry-through) as well as being available at alow cost.
V. ESTIMATION OF ANTIOXIDANTACTIVITY
The activity of an antioxidant can be esti-mated by quantitatively determining primary orsecondary products of autoxidation of lipids orby monitoring other variables. Generally, thisoccurs via the delay in hydroperoxide formationor production of secondary products of autoxi-dation by chemical or sensory methods. Theseprocedures can be applied to either intact foods,their extracts, or to model systems. Studies onfoods can be performed under normal storageconditions or under accelerated oxidation such asthe active oxygen method (AOM), Schaal oventest, oxygen uptake/absorption, oxygen bomb ca-lorimetry, or by using a fully automated Ranci-mat apparatus.45-46 The extension of the inductionperiod through the addition of an antioxidant hasbeen related to antioxidant efficacy that is some-times expressed as antioxidant index or protec-tion factor.
The formation of hydroperoxides is measuredby an iodometric titration of released iodine byhydroperoxides and is generally expressed as theperoxide value (PV). Of the available methodsfor measuring hydroperoxide decompositionproducts, determination of aldehydic compounds(e.g., spectrophotometric determination of ma-
lonaldehyde using the 2-thiobarbituric acid (TBA)test or p-anisidine test), measurement of totalcarbonyls or selected carbonyl compounds, andassessment of off-flavors and off-odors due tothe formation of volatile decomposition productsof hydroperoxides by objective and subjectivemeans are used extensively. Sensory evaluationprovides information on the overall acceptabilityof foods in addition to the results provided bychemical determinations.45
Model systems for testing the antioxidant ac-tivity of food components and additives have beenused extensively. Peroxidation catalyzed by met-myoglobin or a Fe2+-EDTA system as well asAOM tests may be performed on linoleic acidinstead of food in evaluating the activity of an-tioxidants. However, model systems are at a dis-advantage in that intact foods also contain naturalcompounds that may possess antioxidant or syn-ergistic properties.
VI. TYPES OF ANTIOXIDANTS
According to their mode of action, antioxi-dants can be classified as free radical terminators,chelators of metal ions capable of catalyzing lipidoxidation, or as oxygen scavengers that react withoxygen in closed systems. Thus, primary antiox-idants react with high-energy lipid radicals toconvert them to thermodynamically more stableproducts. Secondary antioxidants, also known aspreventive antioxidants, function by retarding therate of chain initiation by breaking down hydro-peroxides. Examples of these secondary antiox-idants include dilauryl thiodipropionate andthiodipropionic acid.2430 Phenolic antioxidants(AH) are included in the category of free radicalterminators. Since the main emphasis is on AH,the mechanism of action of free radical termi-nators is discussed here in detail.
A. Mechanism of Action of PhenolicAntioxidants
The first detailed kinetic study of antioxidantactivity was conducted by Boland and ten-Have,12
who postulated Reactions 10 and 11 for free rad-ical terminators. AH interfere with lipid oxida-
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tion by rapid donation of a hydrogen atom tolipid radicals (Reactions 10 and 11). The latterreactions compete with chain propagation reac-tions 3 and 14.
ROO- + AH
RO + AH
ROO + A'
RO- + A-
ROO' + RH
ROOH + A" (10)
ROH + A" (11)
ROO A (12)
ROA (13)
ROOH + R- (14)
The above reactions are exothermic in nature.The activation energy increases with increasingA-H and R-H bond dissociation energy. There-fore, the efficiency of the AH increases with de-creasing A-H bond strength. The resulting phen-oxy radical itself must not initiate a new freeradical or be subject to rapid oxidation by a chainreaction. In this respect, phenolic antioxidantsare excellent hydrogen or electron donors and,in addition, their radical intermediates are rela-tively stable due to resonance delocalization andlack of suitable sites for attack by molecularoxygen.10-73-95
The phenoxy radical formed by reaction ofa phenol with a lipid radical is stabilized by de-localization of unpaired electrons around the ar-omatic ring, as indicated by the valence bondisomers (Reaction 15).
(15)
However, phenol itself is inactive as an antiox-idant. Substitution of the hydrogen atoms in theortho- and para-positions with alkyl groups in-creases the electron density of the OH moiety by
an inductive effect and thus enhances its reactiv-ity toward lipid radicals. Substitution at the para-position with an ethyl or «-butyl group rather thana methyl group improves the activity of the AH;however, the presence of chain or branched alkylgroups in this position decreases the antioxidantactivity.30
The stability of the phenoxy radical is in-creased by bulky groups at the or/Ao-position,for example, in 2,6-di-tertiary-butyl,4-methoxy-phenol, or butylated hydroxyanisole (BHA).62
Since these substituents increase the steric hin-drance in the region of the radicals, they furtherreduce the rate of possible propagation reactionsthat may occur (Reactions 16, 17, and 18) in-volving antioxidant free radicals.30
A- + O2
AOO- + RH
A" + RH
AOO- (16)
AOOH + R- (17)
AH + R- (18)
The introduction of a second hydroxy groupat the ortho- or />ara-position of the hydroxy groupof a phenol increases its antioxidant activity. Theeffectiveness of a 1,2-dihydroxybenzene deriv-ative is increased by the stabilization of the phen-oxy radical through an intramolecular hydrogenbond (Reaction 19).
(19)
The antioxidant activity of dihydroxybenzene de-rivatives is partly due to the fact that the initiallyproduced semiquinoid radical can be further ox-idized to a quinone by reaction with another lipidradical. It can also form into a quinone or hy-droquinone molecule (Reaction 20).
ROO ROOH
OH
0OH
ROOT ROOH
(20)
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The antioxidant activity of 2-methoxyphenolis much lower than that of catechol, which pos-sesses two free hydroxy groups,91 because 2-methoxyphenols are unable to stabilize the phen-oxy radical by hydrogen bonding, as in Reaction19.30
The effect of antioxidant concentration onautoxidation rates depends on many factors, in-cluding the structure of the antioxidant, oxidationconditions, and the nature of the sample beingoxidized. Often AH lose their activity at highconcentrations and behave as pro-oxidants20 viainvolvement in initiation reactions (see Reactions16, 17, and 18).30 Antioxidative activity by do-nation of a hydrogen atom is unlikely to be lim-ited to phenols. Endo et al.26 have suggested thatthe antioxidant effect of chlorophyll in the darkoccurs by the same mechanism as AH. AH aremore effective in extending the induction periodwhen added to an oil that has not deteriorated toany great extent; however, they are ineffectivein retarding decomposition of already deterio-rated lipids.58 Thus, antioxidants should be addedto foodstuffs as early as possible to achieve max-imum protection against oxidation.21
B. Synthetic Food Phenolic Antioxidants
The application of antioxidants to foods isgoverned by federal regulations. Food and DrugAdministration (FDA) regulations require thatantioxidants and their carriers be declared on theingredient labels of products and should be fol-lowed by an explanation of their intended pur-
1. Butylated Hydroxyanisole (BHA) andButylated Hydroxytoluene (BHT)
BHA and BHT are monohydric phenolic an-tioxidants (Figure 2) that, prior to their intro-duction and acceptance in the food industry, wereused to protect petroleum against oxidative de-gumming.79 Chemically, BHA is a mixture oftwo isomers, 3-tertiary-butyl-4-hydroxyanisole(90%) and 2-tertiary-butyl-4-hydroxyanisole(10%). BHA is commercially available as whitewaxy flakes and BHT is available as a whitecrystalline compound. Both are extremely solu-ble in fats and are insoluble in water (Table 1).Furthermore, both assert a good carry-througheffect, although BHA is slightly better than BHTin this respect.24 BHT is, however, more effec-tive in suppressing oxidation of animal fats thanvegetable oils. Among its multiple applications,BHA is particularly useful in protecting the flavorand color of essential oils and is considered themost effective of all food-approved antioxidantsfor this application.97 BHA is particularly effec-tive in controlling the oxidation of short-chainfatty acids such as those found in coconut andpalm kernel oils that are used typically in cerealand confectionery products.24
As a monophenol, BHT can produce radicalintermediates with moderate resonance delocal-ization. The tertiary-butyl groups of BHT do notgenerally allow the involvement of the radicalformed from it after hydrogen abstraction in otherreactions. Thus, a lipid peroxy radical may jointhe molecule of BHT, as given in Reaction 21.24
(21)
pose.24 Tables 1 and 2 summarize the permissiblefood AH and some of their properties and amountsof their allowable usage. Synthetic food antiox-idants currently permitted for use in foods arebutylated hydroxytoluene (BHT), butylated hy-droxyanisole BHA, propyl gállate (PG), dodecylgállate (DG), and tertiary-butylhydroquinone(TBHQ).
Due to their volatile nature, both BHA andBHT are important additives used in packagingmaterials because they are able to migrate intofoods. To this purpose, these antioxidants areeither added directly to the wax used in makinginnerliners or applied to the packaging board asan emulsion.24-79 A synergistic effect has beenshown to exist when BHT and BHA were used
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TABLE 1Synthetic Food Antioxidants and Their Properties2124
Gallates
Characteristic
Melting point (°C)Carry-throughproperties
Synergism
Solubility (w/w%) inWaterPropyleneglycol
LardCorn oilGlycerolMethyl
linoleate
BHA
50-52Very good
BHT andgallates
050
30-40301Very
soluble
BHT
69-70Fair-good
BHA
00
50400Verysoluble
Propyl
146-148Poor
BHA
<16.5
10251
Dodecyl
95-98Fair-good
BHA
<14
—0—1
TBHQ
126-128Good
—
<130
5-1010<1>10
TABLE 2Maximum Usage Levels Permitted by U.S. FDA inSpecific Application of Antioxidants, from Code ofFederal Regulations24
Max usage levels (ppm)
Food
Dehydrated potato shredsActive dry yeast
Beverages preparedfrom dry mixes
Dry breakfast cerealsDry diced glazed fruits
Dry mixes for bever-ages and deserts
Emulsion stabilizers forshortenings
Potato flakesPotato granulesSweet potato flakesPoultry products9
Dry sausage"Fresh sausage"Dried meat"
" 21 CFR 172.110." Given levels are for totalc 21 CFR 172.115.d 21 CFR 184.1660.• 21 CFR 172.185.1 BHA only.9 9 CFR 381.147(f) (3).h CFR 318.7 (c) (4).1 BHT only.
BHA'b
501000'
2'
50'32'50
200
501050
100'30'
100'100'
BHT and
BHT«
50——
50——
200
501050
100*30¡
100'100*
BHA.
PGd
——
———
—
———10030
100100
TBHQ«
———
———
—
———.10030
100100
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<CH3>3C
OCH,
OH
CH,
C(CH3)3
C(CH3)3
BHA
3-tertiary-butyl-4-hydroxy anisóle
2-tertiary-butyl-4-methoxyphenol
BHT
3,5-d¡-tert¡ary-butyl-4-hydroxytoluene
2,6-d¡-tert¡ary-butyl-4-methylphenol
FIGURE 2. Structures of BHA and BHT.
in combination. As an example, the oxidativereactions of nut and nut products are very re-sponsive to the combination of these twoantioxidants.24
2. Tertiary-Butylhydroquinone (TBHQ)
Tertiary-butylhydroquinone (Figure 3) is re-garded as the best antioxidant for protecting fryingoils against oxidation. It provides good carry-through protection similar to that of BHA andBHT. The Eastman Chemical Company, Inc. hasdescribed TBHQ as an alternative or supplementto oil hydrogénation for increasing oxidative sta-bility.24 TBHQ is adequately soluble in fats anddoes not complex with iron or copper, as wasobserved for PG; therefore, it does not discolorthe treated products. TBHQ is available as a beige-colored powder to be used alone or in combi-nation with BHA or BHT at a maximum amount
of 0.02% or 200 ppm, based on the fat contentof foods, including essential oils. TBHQ is notpermitted in combination with PG,24 and has re-portedly been shown to be a good stabilizer ofcrude oils.21
Chelating agents such as citric acid and mon-oglyceride citrate can further enhance the lipid-stabilizing properties of TBHQ. This combina-tion is used primarily in vegetable oils and short-enings but not extensively for animal fats. Con-fectioneries, including nuts and candies, alsobenefit from the addition of TBHQ or its mix-tures.13 As a diphenolic antioxidant, TBHQ reactswith peroxy radicals to form a semiquinone res-onance hybrid. The semiquinone radical inter-mediates may undergo different reactions to formmore stable products; they can react with oneanother to produce dimers, dismutate, and re-generation of the semiquinone; and they can reactwith another peroxy radical, as summarized inReactions 22, 23, and 24.
ROO ROOH +
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C(CH3)3
TBHQ
tertiary-butylhydroquinone
philic nature of PG makes it a very effectiveantioxidant for dry vegetable oils. Gallates havelower volatility and thus have less phenolic odorthan monohydric phenols such as BHA andBHT.24
FIGURE 3. Chemical structure of TBHQ.
ROO (24)
OOR
3. Propyl Gállate (PG)
PG (Figure 4) is commercially prepared byesterification of gallic acid with propyl alcoholfollowed by distillation to remove the excess al-cohol. PG is available as a white crystalline pow-der and is sparingly soluble in water. It functionsparticularly well in stabilizing animal fats andvegetable oils. With a melting point of 148°C,PG loses its effectiveness during heat processingand is therefore not suitable in frying applicationsthat involve temperatures exceeding 190°C. PGchelates iron ions and forms an unappealing blue-black complex. Hence, PG is always used withchelators such as citric acid to eliminate the pro-oxidative iron and copper catalysts. Good syn-ergism is obtained with BHA and BHT; however,application with TBHQ is not permitted.l3
PG may be used to inhibit the oxidation ofvegetable oils, animal fats, meat products in-cluding fresh and frozen sausages, and snacks.Its usage has been permitted in chewing gum baseat <0.1% and with BHA and/or BHT at a totalconcentration of <0.1%. Moreover, the amphi-
PG
3,4,5-trihydroxypropylbenzoate
COOC3H7
FIGURE 4. Structure of PG.
4. Nordihydroguaiaretic Acid (NDGA)
NDGA (Figure 5) is a grayish-white crys-talline compound that was widely used as an an-tioxidant in animal fats in the 1950s and 1960s.It possesses phenolic properties similar to gal-lates, including their advantages and disadvan-tages. Besides the isolation of natural material(resinous exúdate of creosote bush), NDGA hasalso been chemically synthesized. Due to unfa-vorable toxicological findings, NDGA is no longerof practical importance in the food industry.31-96
C. Degradation Products of PhenolicAntioxidants and Their AntioxidantActivity
The mono-, di-, and triphenolic antioxidantsthat occur during the oxidation of fats and oilsundergo degradation. Generally antioxidant di-mers are the most common breakdown products.These dimers may be produced by the formationof phenoxy radicals followed by radical rear-rangement and a coupling reaction with anotherradical.49 Moreover, most oxidation products ofAH retain antioxidant activity, which may influ-ence the activity of the parent antioxidant duringthe course of its degradation.49
The degradation products of BHT in autox-idized soybean oil (irradiated by UV light) havebeen isolated and identified by Japanese research-ers (Figure 6). The degradation products 1 to 5seen in the figure are formed from BHT. Theantioxidant activities of these breakdown prod-ucts were determined as the ratio of the inductionperiod of the oil containing these compounds tothat of the oil containing the parent antioxidant.It was concluded that 3,5-di-/-butyl-4-hydroxy-benzaldehyde (BHT-ald) and 3,5,3',5'-tetra-r-butyl-4,4'-dihydroxy-l ,2-diphenylethane (BE)were active and thus, together with the remainingBHT, may protect the oil against autoxidation.49
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HO
NDGA
4,4l-(2,3-dimethyl-1,4-
butanediyl)bis[1,2-ben2enediol]
FIGURE 5. Structure of NDGA.
BHT
CH2OH
BHT-alc (2)
C H 2 — CH2
BE (4)
R = tertiafy butyl
(1)3,5-ditert-butyl-4-hydroxybenzaldehyde
(2)3,5-ditert-butyl-4-hydraxybenzyIalcohol
(3) 2,6-d"rtert-butyl-ben2oquinone
(4) 3,5,3',5'-tetra-tert-butyl-4,4'-dihydroxy-1,2-diphenylethane
(5)3,5,3",5'-tetra-tert-butyl-stübenequinone
FIGURE 6. Degradation products of BHT.49
The degradation of BHA may result in theformation of Compounds 7 and 8 (see Figure 7).Kikugawa et al.49 reported that the antioxidantactivity of these breakdown products was lessthan that of BHA and was in the order of BHA> (8) > (7).
The mechanism and breakdown products ofTBHQ due to irradiation were reported by Ku-rechi et al.53 and Kurechi and KunugiS4>55 (Figure
8). All the oxidation products of TBHQ retainedsome antioxidant effect; however, their activitydepended on the substrate oil. Furthermore,Compounds 10 and 12 exhibited antioxidant ac-tivities greater than that of TBHQ itself, but thisalso depended on the nature of the substrateoil.54-55
Irradiation of PG in ethanol produced ellagicacid [2,3,7,8-tetra-hydroxy-(l)benzopyrano-
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OCH,
-H
- H ,
OCH-
(7)
.OCH0
(8)
Ft = tertiary butyl
(7) 2,2'-d¡hydroxy-5,5'-d¡methoxy-3,3'-di-tert-butyl biprienyl
(8) 21,3-d¡tert-butyl-2-hydroxy-4',5-d¡methoxy biphenyl ether
FIGURE 7. Degradation products of BHA.49
(5,4,3-cde)(l)benzopyran-5,10-dione] (Figure 9,Compound 14) that had excellent antioxidantactivity.49
D. Degradation Products of AntioxidantMixtures and Their Antioxidant Activity
Degradation of mixtures of BHA, BHT, andPG (simulated synergists) produced heterodimersbetween different antioxidant components. Irra-diation of a mixture of BHT and BHA in benzeneafforded a new product (Figure 10, Compound15).51-52 Products 1, 4, 5, 7, 8, and BHA werealso present. In a mixture of BHA and PG, Com-pounds 7, 8, 14, 16, and 17 were found as deg-radation products in addition to the original BHAand PG. The heterodimers (16) and (17) (Figure10) exhibited activities comparable to that of PGitself.49
VII. NATURAL ANTIOXIDANTS
Antioxidants in foods may originate from
compounds that occur naturally in the foodstuffor from substances formed during its processing.Natural antioxidants are primarily plant poly-phenolic compounds that may occur in all partsof the plant. Plant phenolics are multifunctionaland can act as reducing agents (free radical ter-minators), metal chelators, and singlet oxygenquenchers. Examples of common plant phenolicantioxidants include flavonoid compounds, cin-namic acid derivatives, coumarins, tocopherols,and polyfunctional organic acids.86 Several stud-ies have been carried out in order to identifynatural phenolics that possess antioxidant activ-ity. Some natural antioxidants have already beenextracted from plant sources and are producedcommercially.96
A. Tocopherols
Tocopherols occur widely in nature and aremonophenolic antioxidants that help to stabilizemost of the oils derived from plants. Tocopherols
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C(CH3)3
.C(CH3)3
Ç(CH3)2
OH
(13)
(9) 2-tert-butyl-p-benzoquinone
(10) 2,2'-dimethyl-5-hydroxy-2,3-dihydrobenzol6]furan
(11) 2-[2-(3"-tert-butyl-4l-hydroxy-phenoxy-2-methyl-1-propyl] hydroquinone
(12) 2-(2-hydroxy-2-methyl-1-propyl)hydroquinone
(13) 2-tert-butyl-4-ethoxyphenol
FIGURE 8. Degradation products of TBHQ.49
are composed of eight different compounds be-longing to two families, tocols and tocotrienols,having the prefix a, ß, "y, or 8, depending onthe number and position of methyl groups at-tached to the chromane rings. Tocopherols alsopossess vitamin E activity. In tocols, the sidechain is saturated, while in tocotrienols it is un-saturated (Table 3). With respect to vitamin E
activity, a-tocopherol is the most potent memberof this family. The antioxidant activity decreasesfrom 5 t o a (Figure II).24
Vegetable foods contain considerableamounts of different tocopherols and tocotrienolsin their lipid fraction (Table 4). Cereals and cer-eal products, oilseeds, nuts, and vegetables arerich sources of tocopherols (Table 5). In the an-
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(14) ellagic acid
FIGURE 9. Degradation products of PG.49
imal kingdom, tocopherols are only found in tracequantities. During manufacturing of oils 30 to40% of tocols and tocotrienols are lost.24-96
Tocopherols are important biological antiox-idants. a-Tocopherol or vitamin E prevents ox-
idation of body lipids, including polyunsaturatedfatty acids and lipid components of cells and or-ganelle membranes. Tocopherols are producedcommercially and are used as food antioxidants.
1. Mechanism of Antioxidant Activity ofTocopherol
The antioxidant activity of tocopherol is basedmainly on the tocopherol-tocopheryl quinone re-dox system (Figure 12). Tocopherols (AH2) areradical scavengers and quench lipid radicals (R'),thus regenerating RH molecules as well as pro-ducing a tocopheryl semiquinone radical. Twotocopheryl semiquinone radicals (AH; Figure 13)may form a molecule of tocopheryl quinone (A)and a regenerated molecule of tocopherol (Re-actions 25 and 26).
OCH'3
(15)
diphenylmethane
propyl-S.S-dihydroxy-^a'-hydroxy-S-methoxy-
3'-tertiary butyl phenoxy) benzoate
OCH0
COOC3H7
propyl-3,4-dihydroxy-5-(2'-hydroxy-
5'-methoxy-3Mertiary butyl phenoxy)
benzoate
(17)R = tertiary butyl
FIGURE 10. Degradation products of a mixture of BHA and PG.49
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TABLE 3Structure of Vitamin E and Related Compounds96
Tocols
Compound
5,7.8-Trimethyl tocol (a-tocopherol) CH3 CH3 CH3
7,8-Dimethyl tocol (ß-tocopherol) H CH3
5,8-Dimethyl tocol (7-tocopherol) CH3 H8-Methyl tocol (S-tocopherol) H H
Tocotrienols
CH3
CH3
CH,
5,7,8-Trimethyl tocotrienoi (a-tocotrienol) CH3 CH3 CH3
7,8-Dimethyl tocotrienoi (ß-tocotrienol) H CH3 CH3
5,8-Dimethyl tocotrienoi (7-tocotrienol) CH3 H CH3
8-Methyl tocotrienoi (8-tocotrienol) H H CH3
a-tocopherol [16]
7-formyl-p- tocopherol
5-tormyl- Y -tocopherol [4]
5-formyl- Y - tocopherol-3-ene
Y-tocopherol [33]
radical
a. - tocopherolether dimer [33]
5-tocopherol [35]
5-formyl-
5- tocopherol
Y-tocopherol-
biphenyl dimer [22]
Y - tocopherolether dimer [32]
5- tocopherolether dimer [28]
FIGURE 11 . Antioxidant activity of some tocopherols and their decomposition products."12
Length of induction period in days are given in brackets (control without antioxidants, 2 d).
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TABLE 4Approximate Content of Tocopherol and Tocotrienol Found in Vegetable Oils (mg/kg)96
CoconutCottonseedMaize, grainMaize, germOlivePalmPeanutRapeseed/canolaSafflowerSoybeanSunflowerWalnutWheat germ
a
5-1040-56060-260
300-4301-240
180-26080-330
180-280340-45030-120
350-700560
560-1200
Tocopherols
ß
—0
1-200
Trace———
0-20—
20-40660-810
7
5270-410400-900450-790
0320
130-590380-59070-190
250-93010-50
590260
8
50
1-505-60
070
10-2010-20
230-24050-450
1-10450270
a
5————
120-150———0
——
20-90
Tocotrienols
ß
Trace—0——
20-40———0
——
80-190
y
1-20—
0-240——
260-300———0———
5
—0
——70———————
TABLESTocopherol Content of Cereal Grains37
Tocopherols (mg/100 g)
Product
Whole yellow cornYellow corn mealWhole wheatWheat flourWhole oatsOat mealWhole riceMilled rice
a
1.50.40.90.11.51.30.40.1
ß
—2.11.2———
7
5.10.9——0.050.20.40.3
a-Tocotrienol
0.5—0.1—0.30.5—
H2O
C16H33 H 2 °
2[H]
a - tocopherylquinone
(stable)
FIGURE 12. a-Tocopherol a-tocopheryl quinone redox system.96
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C16H33 3 C16H33
a - tocopheryl semiquinone
(a)
C16H33
methyl-tocopheryl quinone
(b)
FIGURE 13. Structure of (a) a-tocopheryl semiquinone and (b)methyl-tocopherylquinone.96
R + AH,
2AH
RH + AH
A + AH,
(25)
(26)
The mechanism of oxidation of a-tocopherolwith linoleate hydroperoxides has been studiedin detail.101 After releasing one H atom, theformed a-tocopheryl radical releases another Hatom to produce methyl tocopheryl quinone (Fig-ure 13). Methyl tocopheryl quinone is unstableand gives rise to a-tocopheryl quinone as its mainproduct (Figure 12).
The reaction between two semiquinoid rad-icals may also lead to the formation of an a-tocopherol dimer, which possesses antioxidantproperties.96 The antioxidant effect of the prod-ucts of tocopherols has been described by Ishi-kawa and Yuki42 as oxidized a-, 7-, and 8-to-copherol with trimethylamine oxide. Some of theoxidation products thus formed were isolated andtested for their antioxidant activity. The resultsare summarized in Figure 11.
2. Extraction of Tocopherols fromNatural Sources
Tocopherols are commercially extracted fromsludge obtained in the deodorization of vegetableoils. Various tocols and tocotrienols of such ex-tracts contain sterols, esters of sterols, free fattyacids (FFA), and triglycérides. The separation oftocopherols from other compounds is possible viaesterification with a lower alcohol, washing andvacuum distillation, or by saponification or frac-tional liquid-liquid extraction. Further purifica-tion may be achieved using molecular distilla-tion, extraction, crystallization, or a combinationof these processes.96 The total tocopherol contentof the extracts is usually between 30 and 80%,and is higher in 7- and 8-tocopherols. To obtainstable a-tocopheryl acetate, the mixture is meth-ylated and subsequently acetylated. The a-to-copheryl acetate is the commercially availableform of vitamin E, which is not an antioxidantbecause its active OH group is blocked. How-
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ever, under acidic aqueous conditions, toco-pherol is released by hydrolysis. The releasedtocopherol may then act as an antioxidant.96
The commercial synthesis of a-tocopherolinvolves the condensation of 2,3,5-trimethyl hy-droquinone with phytol, isophytol, or phytal hal-ogenides. The crude tocopherol product is puri-fied by vacuum distillation. Since the use ofisophytol is preferred, the distillation produces aracemic mixture of all possible tocopherol iso-mers,96 as summarized in Table 3. Products in-tended for antioxidant applications are generallymarketed in oil forms. Pure, all racemic a-to-copherol, mixed tocopherols having various con-tents of a-, y-, and/or 8-tocopherols, synergisticmixtures composed of tocopherols, ascorbyl pal-mitate or other antioxidants, and synergists suchas lecithin, citric acid, and carriers are alsoavailable.96
B. Phenolic Acids and Flavonoids
Flavonoids occur in living cells as glycosidesand may break down to their respective aglyconeand sugar by enzymes or acid-heat treatments.Pratt and co-workers8a-82 have considered fla-vonoids as primary antioxidants. Many of theflavonoid and related phenolic acids have shownmarked antioxidant characteristics61 (Tables 6,7,and 8). Structures of these flavonoid and relatedcompounds are given in Figure 14.
C. Structure-Activity Relationships
Flavonoids and cinnamic acids are known asprimary antioxidants and act as free radical ac-ceptors and chain breakers. Flavonols are knownto chelate metal ions at the 3-hydroxy-4-ketogroup and/or the 5-hydroxy-4-keto group (whenthe A ring is hydroxylated at position 5). An o-quinol group at the B ring can also demonstratemetal chelating activity.86
It has been established that the position andthe degree of hydroxylation is of primary im-portance in determining the antioxidant activityof flavonoids. The o-dihydroxylation of the Bring contributes to the antioxidant activity. The
p-quinol structure of the B ring has been shownto impart an even greater activity than o-quinol;however, p- and m-hydroxylation of the B ringdo not occur naturally.86 All flavonoids with 3',4'-dihydroxy configuration possess antioxidantactivity.23
Robinetin and myricetin have an additionalhydroxy group at their 5' position, thus leadingto enhanced antioxidant activities over those oftheir corresponding flavones that do not possessthe 5' hydroxy group (fisetin and quercetin) (Fig-ure 15 and Table 6). Two flavanones, naringeninand hesperitin, have only one hydroxy group onthe B ring and possess little antioxidant activity(Figure 15 and Table 7). Hydroxylation of the Bring is the major consideration for antioxidantactivity.86 Other important features include a car-bonyl group at position 4 and a free hydroxygroup at position 3 and/or 5.23
The importance of other sites of hydroxyl-ation has been investigated by Uri.103 It has beenshown that the o-dihydroxy grouping on one ringand the p-dihydroxy grouping on the other ring(e.g., 3,5,8,3',4'- and 3,7,8,2',5'-pentahydroxyflavones) produce very potent antioxidants, while5,7-hydroxylation of the A ring apparently haslittle influence on the antioxidant activity of thecompounds.86 Thus, quercetin and fisetin havealmost the same activity, while myricetin pos-sesses an activity similar to that of robinetin (Fig-ure 15 and Table 6). The 3-glycosylation of fla-vonoids with monosaccharides/disaccharidesreduces their activity when compared with thecorresponding aglycones (e.g., rutin is less activethan quercetin; Figure 15).
The ability of flavonoids to form complexeswith a cupric ion (Figure 16) has also been dem-onstrated by UV spectral studies. Such complex-ations may contribute to the antioxidative actionof flavonoids.40 Chelation of metal ions rendersthem catalytically inactive.
Chalcones, the natural precursors of flavonesand flavanones, are readily cyclized under acidicconditions (Figure 15) and have been shown topossess potent antioxidant activity. The 3,4-dih-ydroxychalcones are particularly effective andchalcones are more effective than their corre-sponding flavanones. Effectiveness of the 3,4-dihydroxychalcones, butein and okanin (Figure
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TABLE 6Antioxidant Activity of Flavones86
Compound
ControlStripped corn oilLard
AglyconesQuercetin
(3,5,7,3',4'-pentahydroxy)Fisetin
(3,7,3',4'-tetrahydroxy)Lueteolin
(5,7,3',4'-tetrahydroxy)Myricetin
(3,5,7,3',4',5'-hexahydroxy)Robinetin
(3,7,3',4',5'-pentahydroxy)Rhamnetein(3,5,3',4'-tetrahydroxy-7-methoxy)
GlycosidesQuercitrin (3-rhamnoside)Rutin (3-rhamnoglucoside)
• 5 x 10-* Min stripped corn oil." 2.3 x 10- 4Minlard.
TABLE 7
Time to reach*peroxide
value of 50 (h)
105—
475
450
—
552
750
375
475195
Antioxidant Activity of Fiavanones86
Compound
ControlStripped corn oilLard
AglyconesTaxifolin (dihydroquercetin)
(3,5,7,3',4'-pentahydroxy)Fustin
(3,7,3',4'-tetrahydroxy)Eriodictyol
(3,7,3',4'-tetrahydroxy)Naringenin
(5,7,4'-trihydroxy)Hesperitin
(5,7,3'-trihydroy-4 methoxy)Glycosides
Hesperidin (7-rhamnoglucoside)Neohesperidin (7-glucoside)
Time to reach*peroxide
value of 50 (h)
105—
470
—
—
198
125
125135
Induction periodby Rancimat (h)
—1.4
7.1
8.5
4.3
—
—
—
1.9—
Induction period"by Rancimat (h)
—1.4
8.2
6.7
6.7
—
—
——
5 x 10~4 M in stripped com oil.2.3 x 10-« M in lard.
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TABLE 8Antioxidant Activity of Some Fiavonoid-Related Compounds8
Compound
ControlStripped com oilLard
IsoflavonesDaidzein (7,4'-dihydroxy)Genistein (5,7,4'-trihydroxy)
ChalconesButein (2',4',3,4-tetrahydroxy)Okanin (2',3',4',3,4-pentahydroxy)
Phenolic acidsProtocatechuic acid
(3,4-dihydroxybenzoic acid)Gallic acid (3,4,5-trihydroxybenzoic acid)Coumaric acid (p-hydroxycinnamic acid)Ferulic acid
(4-hydroxy-3-methoxycinnamic acid)Caffeic acid (3,4-dihydroxycinnamic
acid)Dihydrocaffeic acid
(3,4-dihydroxyphenylpropionic acid)Chlorogenic acid (caffeoyl quinic ester)Quinic acid
Phenolic esterPropyl gállate
MiscellaneousD-CatechinHesperidin methylchalconeAsculetin (6,7-dihydroxycoumarin)
a 5 x 10-* M in stripped corn oil." 0.05% of test compound in lard, batch A or B.
Time to reach*peroxide value
of 50 (h)
110
—
—
—
E—————
Induction period"by Rancimat (h)
A1.3
1.42.6
94.097.0
—
120145
495
— -
505105
435
410135
B0.35
—
—
4.8
28.60.82.0
23.3
31.4
—
21.8
—
15.5
15 and Table 8), depends on the formation ofresonance-stabilized free radicals23 (Figure 17).
In the isoflavone, it is clear that both hydroxygroups in the 4' and 5 positions are needed forsignificant antioxidant activity; an example isgenistein (Figure 18). Even 6,7,4'-trihydroxyi-soflavone is marginally active when comparedwith the analogous flavone, apigenin (Figure 15),which is inactive as an antioxidant. Genistein isparticularly active. The resonance-stabilized qui-noid structures show that for isoflavone the car-bonyl group at position 4 remains intact and caninteract with the 5 hydroxy group, if present;however, in flavone, the carbonyl group at po-sition 4 loses its functionality. This may explainthe superior antioxidant activity of genistein whencompared with apigenin.22
Naturally occurring isoflavones are foundmainly in the Leguminoseae family and are muchless widespread than flavones. Pronounced syn-ergism was observed when isoflavones were sup-plemented with phospholipids such asphosphatidylethanolamine.22
The antioxidant activity of phenolic acids andtheir esters depends on the number of hydroxygroups in the molecule, which would be strength-ened by steric hindrance.23 Hydroxylated cin-namic acids were found to be more effective thantheir benzoic acid counterparts.
At least two, and even three, neighboringphenolic hydroxy groups (catechol or pyrogallolstructure) and a carbonyl group in the form of anaromatic ester or lactone or a chalcone, flava-
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OH
OH O
Flavones (luteolin)
OH O
Isoflavones (daidzein)
OH O
Flavanones (eriodictyol) Chalcones (butein)
HO,
HO 'i
Coumarins (aesculetin) Cinnamic acids (caffeic acid)
FIGURE 14. Structure of flavonoids and related compounds.
none, or flavone are the essential molecular fea-tures required to achieve a high level of antiox-idant activity.23
VIII. NATURAL SOURCES OF PLANTANTIOXIDANTS
A. Soybean
Soybean (Glycene max L.) ingredients areused advantageously in many food products fornutritional and/or functional reasons. Soy flourhas shown antioxidant properties in various foodproducts (Table 9). In soybean oil, the activeantioxidant is tocopherol, mainly a-tocopherol,and to a lesser extent, 8-tocopherol.96 Soybeanflour and other soybean derivatives are sources
of a large variety of antioxidant compounds (Ta-ble 10).
The antioxidant components of soybean flourhave been shown to be isoflavone glycosides andtheir derivatives, phospholipids, tocopherols,amino acids, and peptides.32-34-36-63-64-65-66 Isolatedisoflavone glycosides from soy flour (Figure 18)were genistein, daidzein, and glycitein and 7,4'-dihydroxy-6-methylisoflavone.6487
It has been reported that 99% of the isofla-vones are present as glycosides, 64% of whichare genistein, 23% daidzein, and 13% glycitein7-O-ß-glycoside.64 Murakani et al.63 have shownthat in tempeh, a fermented soybean product, theisoflavones are liberated from glycosides by acidsformed during the fermentation. The main an-tioxidants in tempeh were determined to be daid-zein and genistein, while glycitein had only a
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Flavones
Apigenin : 5,7,3'-trihydroxy
Quercetin : 3,5,7,3',4-pentahydroxy
Rsetin : 3,7,3',4'-tetrahydroxy
Taxifolin : 3,5,7,3',4'-pentahydroxy
Robinetin : 3,7,3'¿".S-pentahydroxy
Rufin : ciueteetin-3-rhamnosylglucoside
Flavanones
Naringenin : 3,5,3'-trihydroxy
Hesperitin : 5,7.3'-trihydroxy-4-methoxy
5 Butein : 2,4,3'A'-tetíaty&oxy
Okanin : 2,3,4,3',4'-pentahydroxy
FIGURE 15. Structure of antioxidative flavone, flavanone, and chalcone.
3-hydroxyflavone
CiT
5-hydroxyflavone
3-hydroxyflavanone 5-hydroxyflavanone
FIGURE 16. Forms of copper complexes with flavones andflavanones.40
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OH oOH O OH O"
FIGURE 17. Resonance-stabilized free radical formation of S'^'-dihydrochalcone.*3
*1
H
CH3
H
H
R2
H
H
H
CH3
R3
H
H
H
H
R 4
H
H
OHOH
- Daidzein
- Formononetin- Genistein
- Prunetin
H H OH H - ^.e'.y-trihydroxyisoflavone
H Gluc OCH3 H - glycitein (7-O-glucoside)
FIGURE 18. Structure of soybean isoflavone glycosides.2383
TABLE 9Effective Antioxidative Levels of Soybean in Various Products86
Product
LardPremier jus
Ghee (buffalo butter)Frozen pastry
Raw pastry mixes andbaked pastry
Ration biscuits
Dehydrated pork-cornmeal scrapple
Frozen, raw ground porkand frozen, precookedground pork
Degermed, uncookedcorn meal-soy flourblend
Degermed, uncookedcom meal-soy flourblend plus ferroussulfate
Instant, fully cookedcorn meal-soy flour-milk blend (plus 5%soybean oil)
Effective level (% of flour)
5-102-6 (unspecified type)
0.5-1.0 (full fat)5-20 (full fat)
10 (low fat)
4-20 (defatted)
2.8 (full fat)
2.5-7.5 (full fat)
15, 20 (toasted, defatted) or(commercial-process full fat)or (extrusion-cooked full fat)
15-25 (toasted, defatted)
27.5 (toasted, defatted)
Comment
Original condition of fatstrongly influenced soyeffectiveness
About equally effectiveAll concentrations were
about equally effective50°C storage
Progressively lower peroxidevalues with increasingcone.
No significant difference inperoxide values with cone.
No flavor difference between15 and 20% products
No rancidity after storage
Low peroxide values afterstorage
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TABLE 10Probable Principle Antioxidant Compounds of SoybeanProducts and Derivatives33-34-63-82-84
DerivativeAqueous extract
Organic solvent extractSoy protein concentrate/isolateProtein hydrolysateTextured vegetable proteins
Antioxidant componentIsoflavone glycosides, their aglycones,phenolic acids
Flavonoids, tocopherols, phospholipidsIsoflavone glycosides, their aglyconesAmino acids and peptidesPhospholipids
slight antioxidant activity. The reported isofla-vone and isoflavone glycosides in soybean mealare summarized in Table 11.
Hayes et al.34 reported that aglycones hadsuperior antioxidant activity compared to the par-ent isoflavone, glycoside. As discussed earlier,it was shown that the antioxidant activity of is-oflavones was not as pronounced as their cor-responding flavones that possessed dihydroxysubstitutes in either the A or B ring. Antioxidantactivity of soybean isoflavones, as determinedspectrophotometrically by the coupled oxidationof ß-carotene and linoleate, is given in Figure19.
Phenolic acids possessing antioxidative ac-tivity have also been found in soybean and soy-bean products.5i33-83 At least nine phenolic acids,including syringic, vanillic, caffeic, ferulic, p-coumaric, and p-hydroxybenzoic acids, have beenidentified and isolated from soybean and defattedflour.5 Among polyphenolic antioxidants ex-tracted by methanol or water from dried and freshsoybean, chlorogenic, isochlorogenic, caffeic,
and ferulic acids were identified in addition toisoflavones.83 The antioxidant activity of thesephenolic acids in ß-carotene/linoleate model sys-tems are assembled in Figure 19.
B. Peanut and Cottonseed
The methanolic extracts of protein ingredi-ents of peanut (Arachis hypogea, variety: Span-ish) and glandless cottonseed (Gossypium hir-sutum, variety: McNair) have demonstratedantioxidant activity in lipid peroxidation modelsystems (catalyzed by metmyoglobin and Fe2+-EDTA) and in fresh beef homogenate.92 The sameprotein ingredients in beef patties (replaced beefup to 10%) were able to retard rancidity devel-opment when cooked and refrigerated.110 Cot-tonseed protein ingredients have been shown tohave a higher activity in retarding developmentof rancidity than peanut meals.
Pratt and Miller85 have identified the dihy-droquercetin, taxifolin (Figure 15), as an antiox-
TABLE 11Isoflavone and Isoflavone Glycoside Content ofSoybean Meal (mg/100 g)zs
Component
DaidzinGlycitin-Z-ß-glucosideGenisteinDaidzeinGlyciteinGenistein
Full fat flakes(fat-free basis)
118.50.9
204.12.01.04.4
Defatted flakes
114.50.8
188.52.51.2 .4.4
Total 330.9 311.4
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0.60 -
15 30 45 60TIME IN MINUTES
75 90 105
FIGURE 19. The antioxidant activity of isoflavones andcinnamic and derivatives of soybean and quercetin, (1)control, (2) glycitein, (3) diadzein, (4) genistein, (5) quer-cetin, (6) 6,7,4'-trihydroxy isoflavone, (7) p-coumaric acid,and (8) ferulic acid.83
idant flavonol in hot methanolic extracts of pea-nut (variety: Spanish). A study carried out byWhiltern et al.107 on the methanolic extracts ofthe delinted cottonseed identified quercetin andrutin (Figure 15) as the major flavonoids present.Further investigation into these compounds con-firmed that quercetin derivatives were the mainflavonoids in methanolic extracts of cottonseed.Rutin was found to be one of the major quercetinglycosides. Both quercetin and rutin possess po-tent antioxidant activity; however, rutin is com-paratively inferior.108
C. Mustard and Rapeseed
Mustard and rapeseeds, including canola,contain phenolic compounds. Their phenolics oc-cur in the form of phenolic acids, and/or theirderivatives, and condensed tannins.48 Phenolicacids identified in rapeseed flour include sali-cylic, cinnamic, p-hydroxybenzoic, veratric,vanillic, gentisic, protocatechuic, syringic, caf-feic, sinapic, and ferulic acids. The main con-densed tannins of rapeseed hulls and flour are
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cyanidin, pelargonidin, and kaempferol-7-glu-coside-3-sophoroside.
The antioxidant activity of phenolics of ra-peseed and mustard has been reported in modelsystems of ß-carotene/Iinoleate and musclefoods.88-93-100 Both canola and mustard flour assuch, or their extracts, possess strong antioxi-dative activity. The effectiveness of an enzyme-deactivated mustard flour, referred to as deheatedmustard flour (DMF), at a 1 to 2% level of ad-dition in retarding meat flavor deterioration wassuperior to that of BHT at 200 ppm. Extracts ofDMF, however, were less effective. Further-more, the activity of these extracts was propor-tional to the individual total content of phenolics.93
D. Rice
The methanolic extracts of rice hulls (Oryzasativa Linn.) from long- (Katakutara) and short-lived (Kusabue) varieties exhibit a superior an-tioxidant activity when compared with a-toco-pherol.90 The high-performance liquid chroma-tography (HPLC) separation of methanol-water(50:50) extracts of both varieties showed that theshort-life variety includes compounds similar toa-tocopherol. The long-life variety had a strongerantioxidant activity and contained compoundsother than a-tocopherol. The active compoundhas been characterized as isovitexin, a C-glycosylflavonoid90 (Figure 20).
It has been observed and established that thestorability and longevity of rice grains is related
HO- OHOH O
Isovitexin
FIGURE 20. Isovitexin.90
to the antioxidative activity of the seed coat orhusk.75-89 Presence of isovitexin has confirmedthe antioxidative activity of long-life rice.90
E. Sesame Seed
The oil from the sesame seed (Sesamum in-dicum L.) has a superior oxidative stability whencompared with other vegetable oils. It has beensuggested that this special property is largely dueto the presence of sesamol in sesame seeds.57
Sesamol is usually present in trace amounts, butmay also be released from sesamolin by hydro-génation, bleaching earth, or other conditions ofprocessing. Sesame seed contained 0.4 to 1.1%sesamin, 0.3 to 0.65% sesamolin, and traceamounts of sesamol (Figure 21). Sesamol is afree 3,4-methylenediphenoxy phenol. Sesamolinis an acetal-type derivative of sesamol and se-samin. The 3,4-methylenedioxy phenol is di-rectly attached to the 2,7-dioxabicyclo-(3,3,0)-octane nucleus. Sesamol was found to be as ef-fective as BHT and BHA and more effective thanPG.57 Fukuda et al.29 have reported that acetoneextract of sesame seed produced a strong antiox-idant activity. The active compounds were iden-tified as bisepoxylignan or sesamolinol,76 as seenin Figure 22. Sesamol is readily oxidized to se-samol dimers and then to the sesamol dimer se-miquinone by treatment with H2O2/horseradishperoxidase.49
F. Canary Seeds
Canary seed (Phalaris canariensis) is a pop-ular bird feed. Takagi and Iida100 have shownthat extracts (i.e., successive extraction of seedswith hexane, ether, and methanol) of canary seedsexhibit excellent antioxidant activity in lard andsardine oil. The active components were identi-fied by gas chromatography (GC) to be esters ofcaffeic acid as well as cycloartenol, gramisterol,sitosterol, and campesterol, and lesser quantitiesof 24-methylenecycloartanol, obtusifoliol, bras-sicasterol, and A7-stigmasterol.100 These sterols
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Sesamolin
FIGURE 21. Structures of sesamol and sesamolin.29
Sesamolinol
FIGURE 22. Structure of sesamolinol.
and triterpene alcohol esters of caffeic acid arelipid soluble and have higher melting points. Lipidsolubility and lack of bitter taste may enhancethe potential use of this source of naturalantioxidant.
G. Tea
The presence of catechin and its derivativesin tea has been well documented. Matsazaki andHara60 have reported on the antioxidative effi-ciency of isolated catechins from green tea leaves.The extracts included (-)-epicatechin (EC), ( - ) -epigallocatechin (EGC), (-)-epicatechin gállate(ECg), and ( - )-epigallocatechin gállate (EGCg)(Figure 23). The activity of catechins in modelsystems was on the order of
EC < ECg < EGC < EGCg
At similar molar concentrations, the activity ofthese compounds was superior to those of BHAand DL-a-tocopherol in lard.60-72
H. Herbs and Spices
Herbs and spices have been used for manycenturies to improve flavor and to extend theshelf life of various foods. Chipault et ai.18-19
have investigated antioxidant activity of spicesin various fats. In general, alcoholic and etherextracts of spices are less active than the nativespices themselves (Tables 12 and 13). Allspice,clove, sage, orégano, rosemary, and thyme wereshown to possess antioxidant activity in all typesof fats examined. Clove appeared to be the mostactive antioxidant in vegetable oils; however, ex-tracts of rosemary and sage were the most effec-tive. l8-19 Spice extracts have attracted a great dealof interest in recent years because they can beeasily added to fats and oils in bulk. Many ex-tracts possess a strong odor and bitter taste andthus are of limited use in many food products.Rosemary and sage are found to provide the mostpotent antioxidant spice extracts with little odorof the original spice.
I. Rosemary and Sage
Chang et al.'6 were able to prepare an odor-less and flavorless natural antioxidant from rose-mary (Rosmarinasefficinalis L.) and sage (Salviaofficinalis L.). These antioxidants can be suc-cessfully extracted into different organic solventssuch as benzene, chloroform, diethylether, andmethanol. The diethylether extract of rosemarywas purified and evaluated for its antioxidant ac-tivity (PV) at 0.02% (w/w) in potato chips, sun-flower oil, and com oil. It showed a very low
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0R2
EC (Epicatechin) : R^H, R2=H
EGC (Epigallocatechin) : R^OH, R2=H
ECg (Epicatechin gállate) : R^H, R2 = — O
OH
-OH
OH.OH
EGCg (Epigallocatechin gállate) : R^OH, R2= — -OH
OH
FIGURE 23. Structural formula of catechins from green tea.
TABLE 12Antioxidant Activity of Spices in Oils and Fats- (AOM h)18-19
Spice/antioxidant(30 ppm)
FennelGingerCapsicumCloveGarlicTurmericBHA (20 mg/100 g)Control
Antioxidant index
Soybean Linseed Olive Sesame
2.31.92.05.12.32.01.19.5
Time for treated sample to reach PV of 100Time for control to reach PV of 100
2.22.62.66.02.22.11.13.7
8.07.87.3
23.86.68.52.5
16.0
5.13.85.48.57.03.31.18.7
PV and provided excellent flavor stability to theproducts tested.16
The extracts of rosemary leaves contained aphenolic diterpene, carnosol.38 Furthermore, ros-manol, another phenolic diterpene, which has a
structure that is closely related to carnosol, androsmaridiphenol were also identified in rosemaryleaves (Figure 24). Rosmaridiphenol (0.02%) hasshown antioxidant activity similar to BHT at thesame level in prime steam lard.38 Camosic and
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TABLE 13Antioxidative Activity of Ground Spices, Distilled Water-Soluble Fraction, and Ethanol-Soluble Fraction from theSpices in Lard (AOM h)106
Spice
ControlAllspiceBlack pepperCapsicumCloveGingerMaceNutmegRosemarySageTurmeric
Ground SDice(0.25%)
5.711.87.78.0
19.614.724.021.767.054.816.8
Distilled watersoluble
a
5.79.75.86.8
13.96.76.36.18.26.97.0
fraction
b
21.932.828.931.733.430.827.127.329.726.627.7
Ethanolsolublefraction
a
5.78.76.47.0
18.911.721.718.558.542.112.4
b
21.915.524.721.827.524.734.621.758.142.724.5
• 0.25% ground spice equivalent.b Soluble fraction + 100 ppm of a-tocopherol.
OH
O^\*^K^CH(CH3>2C
H3C CH3
Rosmanol Rosmaridiphenol
OH
CH(CH3)2
H3C CH3
Carnosic acid Rosmaric acid
FIGURE 24. Structure of antioxidative compounds in rosemary.3856
rosmaric acids were reported to be the most activeantioxidant constituents of rosemary. Rosmaricacid has been shown to possess an activity com-parable to that of caffeic acid.96 In animal fats,carnosic acid has been described as the most ac-
tive antioxidative constituent of rosemary.96
Commercial antioxidant extracts (molecular orvacuum distilled) from rosemary are available asa fine powder. Depending on their content ofactive antioxidants, they are recommended for
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use at concentrations ranging between 200 to 1000ppm of the processed product. However, purecarnosic acid is not available as a food antioxi-dant.96 It has also been observed that ascorbicacid (500 ppm ascorbic acid + 200 ppm rose-mary extract) enhances the antioxidant activityof rosemary extract in lard.16
J. Oregano
Oregano {Origanum vulgäre L.) belongs tothe same family (Lamiaceae, formerly Labiatae)as rosemary. The dried leaves of orégano weresuccessively extracted with dichloromethane andmethanol and their antioxidative compounds wereisolated as reported for rosemary. The main an-tioxidative compound of orégano was identifiedas a phenolic glycoside.70 Further investigationshave suggested that the compound is 2-caffeoyl-oxy-3[2-(4-hydroxybenzoyl)-4,5-dihydroxy]-phenyl propionic acid50 (Figure 25).
K. Mace
Papua mace (Myristica argéntea) contains 2-allylphenols and a number of lignans.68 Thesecompounds were found to possess strong antiox-idative effects. Capsicin is a pungent antioxidantcomponent of (Capsicum frutescens,) and a newstrong antioxidant (Figure 26) that has no pun-gency has also been isolated from this spice.71
L. Black Pepper
Black pepper (Riper nigum) contains fivephenolic acid amides with antioxidative proper-
ties. Ferulic acid amide of tyramine (Figure 26)and piperine-related compounds with an openmethylenedioxy ring showed a stronger antioxi-dant activity than tocopherol. These compoundsare fat soluble, odorless, and tasteless.69
M. Turmeric
Turmeric (Curcuma longa L.) is generallyused in foods as a colorant. It also includes te-trahydrocurcumin (Figure 26), which is a col-orless, heat-resistant, antioxidative compound.The structural skeleton of this compound is bas-ically the same as curcumin and possesses bothphenol and ß-diketo groups.77
IX. OTHER SOURCES OFANTIOXIDANTS
A. Olive
Olives and their leaves contain polyphenoliccompounds. Sheabar and Neeman94 extractedthem into methanol and ethyl acetate, succes-sively, and evaluated their antioxidant activity inolive and soybean oils. Fractions of this poly-phenolic extract were eluted on thin-layer chro-matography (TLC), and the compounds havingthe highest o-diphenol concentration were sepa-rated and evaluated for their inhibition of oxi-dative deterioration of vegetable oils. Oxidativedeterioration of soybean and olive oils was in-hibited when the extracts were used at a concen-tration of 100 ppm. Nergiz and Unal74 have re-ported that virgin olive oil contains high amountsof p-coumaric acid (0.73 to 10.37 |xg/g) whencompared with syringic, vanillic, and ferulic
HO
Antioxidative glycoside from Oregano
FIGURE 25. Chemical structure of antioxidative compound inorégano.50
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H3CO,
OH (a)
(c)
FIGURE 26. Antioxidative compounds from (a) blackpepper, (b) capsicum, and (c) turmeric.72
acids. Caffeic and4-O-caffeoyl quinic acids werealso identified. The effective antioxidant activityof sweet potato was proposed to be due mainlyto the synergistic effect of these phenolic com-pounds with amino acids present.35
D. Oats
Oat (Avena sativa L.) products have beensuggested as potential food antioxidants. Com-mercially germinated oat grains or their aqueousextracts are used in some countries at a level of1.5% in fatty products and may also be used toimpregnate wrapping papers. The antioxidant ac-tivity of oat is supposedly based on its high con-tent of dihydrocaffeic acid and phospholipids.96
acids, which were also present. The active an-tioxidant compounds in olive have not been stud-ied in detail.
B. Onion
Varieties of onion (Allium cepa L.) with col-ored skins have exceptionally higher flavonolcontent than those with white skins.36 It has beenshown that the outer dry skin of colored onionscontains 2.5 to 6.5% quercetin (as aglycone).Akaranta and Odozi2 have shown that acetoneextracts of red onion skin (0.3%) reduce the PVof oils as effectively as the commercial antioxi-dant l,2-dihydro-2,2,4-trimethylquinoline at a0.1% level of addition.
C. Sweet Potato
The waste fluid from the starch and ethanolmanufacturing process of sweet potato (Iopomeabatatas) contains various phenolic compounds.35
The antioxidative activity of a 70% methanol ex-tract of sweet potato was evaluated in a linoleateaqueous system and was shown to have strongactivity. Major phenolic components containedin 70% methanolic extract were identified byHPLC as being chlorogenic and isochlorogenic
E. Guaiac Gum
Guaiac gum has been used to stabilize refinedanimal fats in combination with phosphoric acidderivatives, which act as synergists. Guaiac resinis obtained from the Guajaucum officinale L.tree. Its use is limited to certain countries and itsactivity is due to a- and ß-guaiaconic acids.96
F. Creosote Bush Exudate/Leaf Wax
NDGA is the major constituent of the resi-nous exúdate of the creosote bush, Larrea di-varicata. The exúdate is used to isolate the strongantioxidant NDGA, as described previously.96 Suand co-workers98 have studied the potential an-tioxidant activity of Taiwanese medicinal plantsand found that some of them possess strong an-tioxidant activity. They also reported that Os-beckia chinensis L. contains kaempferol, quer-cetin, and its glucosides, ellagic acid, gallic acid,and methyl gállate as its antioxidativecomponents.99
G. Fungi
Flavoglaucin (Figure 27), a phenolic com-pound isolated from the mycelial mat of Eurotium
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CHO
Flavoglaucin
FIGURE 27. Flavoglaucin.
chevalieri, was found to be an excellent antiox-idant in vegetable oils at a 0.05% (w/w) level ofaddition.43 It stabilized lard when used in com-bination with a-tocopherol at a 0.04% concen-tration. However, this fungal antioxidative com-pound has not shown mutagenic activity toSalmonella typhimurium, TA100, andTA98, andseems to have limited potential use in foods.43
Aoyamà et al.4 screened 750 strains of fila-mentous fungi from soil for their antioxidant ac-tivity against linoleate. Of these, 14 strains werepositive for the test and the 2 superior strainswere studied for their microbial products. Cur-vulic acid (Figure 28), produced by these twofungi strains, possesses significant antioxidantactivity. Furthermore, they were able to find pro-tocatechuic acid and citrinin (Figure 28) as otherantioxidative components present. The antioxi-dative activity of these compounds was comparedwith BHA and a-tocopherol and the authors4 con-cluded that citrinin was as effective as a-toco-pherol, while curvulic acid and protocatechuicacid showed an activity between a-tocopheroland BHA. Citrinin is a known mycotoxin andhas a limited potential in foods. Protocatechuicacid is a known antioxidant and curvulic acid hasshown low toxicity at 150 mg/kg in rats.4
H. Wood Smoke
Wood smoke is a complex system consistingof dispersed and particulate phases. Absorptionof vapor (dispersed phase) by foodstuffs resultsin the characteristic color, flavor, and preserva-tive properties of the smoked foods. It has beenshown that certain levels and types of specificwood smoke have antioxidative properties.59
Wood smoke can retard oxidative rancidity insmoked foods.
Wood smoke can be fractionated into acidic,basic, and neutral components. The neutral frac-tion, which contains most of the phenols, hasbeen shown to possess the highest antioxidativeproperties, while the basic fraction actually pro-moted lipid oxidation.59 Recent work on woodsmoke antioxidants has confirmed that smolder-ing smoke generation results in strong antioxidantproperties.
The three major constituents of wood are cel-lulose, hemicellulose, and lignin, and occur at aratio of 2:1:1, respectively. During pyrolysis,these constituents produce the characteristic com-pounds of wood smoke. Pyrolysis of celluloseproduces small amounts of furans and phenols,while pyrolysis of hemicellulose yields furan andits derivatives together with a range of aliphaticcarboxylic acids. Moreover, pyrolysis of ligninproduces the most important compounds insmoke, phenols and phenol ethers, typified byguaiacol (2-methoxyphenol) and syringol (2,6-dimethoxyphenol) and their homologous deriv-atives. Under pyrolytic conditions, the aromaticring is relatively stable and may possibly yieldguaiacol and its homologs. Soft woods form pre-dominately guaiacols, while hard woods producea mixture of guaiacols and syringols.59
HOOC
OH
CH3 CH3
Citrinin
COOH
CH2COOH
Curvulic acid
OH
Protocatechuic acid
FIGURE 28. Structure of curvulic acid, citrinin, and protocatechuic acid.
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It is interesting to note that lignin itself hasantioxidant properties. The inclusion of 1 to 10%lignin in the rat diet resulted in increased depo-sition of retinal when compared with cellulose-fed controls.14 Studies on lignin have shown thepresence of phenolic compounds such as ferulic,vanillic, syringic, andp-hydroxybenzoic acids.14
These compounds may contribute to the antiox-idant properties of lignin.
Miller and Quackenbush62 demonstrated thatphenols are the antioxidant-related components,primarily associated with smoke. Phenols with ahigh boiling point are the key compounds re-sponsible for antioxidant properties, while low-boiling phenols show weak antioxidant activ-ity.7-59 Antioxidative phenolic compounds ofwood smoke are given in Figure 29.
X. TOXICOLOGY OF PHENOLICANTIOXIDANTS USED AS FOODADDITIVES
The toxicological properties and safety of an-tioxidants must be verified by long-term animalstudies prior to their approval for food use. TheJoint FAO/WHO Expert Committee on Food Ad-ditives (JECFA), the European Commission, andthe Scientific Committee for Food (SCF) haveestablished acceptable daily intake (ADI) guide-lines for antioxidants. ADI is defined as theamount of chemical, expressed on a body weightbasis, which is considered as being consumeddaily over a lifetime without causing harm.6
A. Tocopherol
JECFA has allocated an ADI of 0.15 to 2mg/kg b.w.3 Extracts of naturally occurring mixedtocopherols and a-, 7-, and 8-tocopherols arepermitted for use as antioxidants. Higher levelsof a-tocopheryl acetate (400 to 500 mg) mayproduce adverse effects due to increased iodineuptake by the thyroid gland.1 Even at high oraldoses, no severe hepatotoxicity was observed inrats. However, excessive supplements of vitaminE — up to 1000 mg/d — are potentially toxic.
B. BHA
The only toxicological problem found in BHAis formation of lesions in the rat forestomach.High doses of BHA have a light proliferativeeffect on the esophagi of pig and monkey.44
JECFA has established an ADI of 0 to 0.5 mg/kg b.w. for BHA.3
C. BHT
An ADI of 0 to 0.125 mg/kg b.w. is allocatedfor BHT.3 A number of studies have shown thatBHT may cause internal and external hemor-rhaging at high doses that is severe enough tocause death in some strains of mice and guineapigs. This effect is due to the ability of BHT toreduce vitamin K-depending blood-clottingfactors.44
D. TBHQ
While TBHQ is allowed as a food antioxidantin the U.S., it is not permitted in the EuropeanEconomic Community countries and Canada dueto the lack of adequate toxicological informationacceptable to those countries.6102 This antioxi-dant has shown mutagenic activity in vivo.104 AnADI of 0 to 0.2 mg/kg b.w. is allocated forTBHQ.3
E. PG
No positive mutagenic or carcinogenic activ-ity has been shown for PG;105 however, gallates,as a group, are reported to cause contact der-matitis in bakers and other workers handling them.An ADI of 0 to 2.5 mg/kg b.w. was allocatedfor PG.3
F. Flavonoids
Flavonoids have been shown to exert anti-mutagenic activity39 in their inhibition of lipox-
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COOH
OCH,
Guaiacol
OH
2-Hydroxybenzoicacid Hydroquinone
COOH CHO
OCH-
OH
4-Hydroxybenzoic acid
CH 2 CH=CH 2
Isoeugenol Salicylaldehyde
FIGURE 29. Phenolic compounds of smoke with proven antioxidantactivity.
ygenase and tumor-promotion activities.67 Morestudies with toxicology of flavonoids arewarranted.
XI. RESEARCH NEEDS
Many sources of naturally occurring antiox-idants from plants have been discovered in recentyears. Full structural identification of the activecomponents of antioxidant components of plantfoods is therefore required. These novel antiox-idants must undergo toxicological studies, ofcourse, at reasonable concentrations, such as thosepresent in their natural environment. The activityof these individual antioxidants must be tested in
different food systems. Furthermore, breakdownproducts of these novel antioxidants, under dif-ferent conditions, and their antioxidant and tox-icological properties must be investigated.
REFERENCES
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2. Akaranta, O. and Odozi, T. O., Antioxidant prop-erties of red onion skin tannin extracts, Agric. Wastes,18, 299, 1986.
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3. Anon., Joint FAO/WHO Expert Committee on FoodAdditives. Tech. Rep. Ser. 751-18. World HealthOrganization, Geneva, 1987.
4. Aoyama, T., Nakakita, Y., Nakagawa, M., andSakai, H., Screening for antioxidants of microbiolorigin, Agric. Biol. Chem., 46, 2369, 1982.
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