6-antioxidant measurement and applications-an overview

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Chapter 1

AntioxidantMeasurementandApplications:AnOverview

FereidoonShahidi1 and Chi-Tang Ho21Department ofBiochemistry,MemorialUniversityofNewfoundland,

St.John s, NewfoundlandA1 3X9, Canada2Department of FoodScience,Rutgers,TheStateUniversityof New Jersey,65DudleyRoad, NewBrunswick,NJ 89 1

Antioxidants are added to fats, oils and fatty foods to preventtheir oxidative deterioration. Antioxidants also protect theintegrity of cellular structures and macromolecules fromdamage due to free radicals. Carotenoids and phenoliccompounds are major dietary antioxidants. Because of theimportance of antioxidant potential in foods and dietarysupplements, it is necessary to have good and reliable methodsformeasuring antioxidantactivity.Variousin vitroandinvivomethods for assessing antioxidant activities are discussed.

IntroductionAutoxidation occurs widely in fats, oils and lipid-containing foods, and

causes food quality deterioration with concomitant generation of loss ofnutrients, unpleasant flavors, and even potentially toxic substances. Among themethods for preventing oxidation, addition of antioxidants is the most effective,convenient and economical one (7).Antioxidants are also important to human health. Antioxidant protectionfrom damage due to free radicals is vital for the integrity of cellular structures

2 2007American ChemicalSociety

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3and macromolecules 2,3).As we age, the systemwhichutilizes antioxidants forour defense and protection also declines, and can be aggravated by the presenceo fvarious oxidativestressescaused bypollution,exercise, smoke exposure andradiation. This defense system operates through a series complex networksbetween vitamins C and E, carotenoids,zinc,copper, selenium, and magnesium-dependent enzyme antioxidants as we l l as other phytonutrients, which togetherperform highly involved recycling and regeneration reactions to optimize freeradical protection. Deficiencies in any of the mentioned necessary componentscouldpotentially lead to a severely compromised defense system 4,5).Owingtothe incomplete efficiency of our endogeneous defense systems, dietaryantioxidants are needed to overcome the oxidative damage 5).

Dietary AntioxidantsCarotenoids and phenolic compounds are major dietary antioxidants. Both

o fthesegroups of compounds contain hundred of members 6).Carotenoids are natural, fat-soluble pigmentsthatprovide bright coloration

to plants and animals and act as antioxidants, which include the possibilityofproviding vitamin A activity. One defining characteristic of carotenoids is thechemical structure of what is often considered their backbone molecule, a 40-carbon polyene chain. The polyene backbone consists of a pattern of conjugateddouble bonds,whichallows the carotenoids to take up excess energy from othermolecules (7).Thischaracteristic may be responsible for the antioxidant activityseen in biological carotenoids. In addition to scavenging free radicals, otherhealth benefits related to this observed antioxidative activity include protectionfrom sunburn and inhibition of the development of certain types of cancers (


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4lutein and zeaxanthin contribute to health and delay age related maculardegeneration of the eyes and, to a lesser extent, cancers andheartdiseases (72-14). The evidence for the role of lutein and zeaxanthin in eye health is verystrong because of their exclusive presence in the ocular tissues and the highnumbers of epidemiological studies that have been conducted. Wi th a highaccumulation in the macula of the eye, the area of highest visualacuity, luteinand zeaxanthin are proposed to have the abilityto filter out harmful blue light,while at the same time acting as antioxidants to quench potentially damagingreactive oxygen species ROS ;75).

Lycopene, a carotenoid found in tomatoes, watermelon, papaya, apricot,orange and pink grapefruit, exhibits antioxidant and anticancer activities (76).About 80 of lycopene is consumed through tomatoes and tomato-relatedproducts. Numerous studies have suggested reduced risk ofprostatecancer fromthe consumption of processed tomato products (77).Although, thesebeneficialhealth effects of lycopene are thought to be due to its antioxidant properties,evidence is accumulating to suggest other mechanisms of action like hormoneand immune system modulation 18).Lycopene is the most abundant carotenoidinhuman plasma,whichmayimplyits elevated levelof importance in the humanbody comparedwithother carotenoids, such as-caroteneand lutein 19).

Phenolic compounds occurring commonly in foods may be classified intosimplephenols, hydroxybenzoic and hydrocinnamicacidderivatives, flavonoids,stibenes, lignans and hydrolysable as we l l as condensed tannins 20).Phenolicsinfoods may occur in the free, esterified, etherified and insoluble-bound forms.

The most abundant phenolic compounds in food are flavonoids. Flavonoidsare present in edible fruits, leafy vegetables, roots, tuber bulbs, herbs, spices,legumes, tea, coffee, cocoa, chocolate and red wine. They can be classified intoseven groups: flavones, flavanones, flavonols, flavanonols, isoflavones,flavanols (catechins) and anthocyanidins. In general, the leaves, flowers andfruits or the plant itself contain flavonoid glycosides, woody tissues containaglycones, andseedsmay contain both 20).

A sa result of their ubiquity in plants, flavonoids are an integralpartof thehuman diet. It is estimated that the average American's daily intake of theconsumption offlavonols is close to 20-25 mg/day 21).

Almost all flavonoids possess several common biological and chemicalproperties: (a) antioxidant activity, (b) the ability to scavenge RO S, (c) theability to scavenge electrophiles, (d) the ability to inhibit nitrosation, (e) theability to chelate metals, (f) the potential to produce hydrogen peroxide in thepresence of certain metals and (g) the capability to modulate certain cellularenzyme activities 22). It appears that diets rich in flavonoids may protectagainst cardiovascular diseases, neurodegenerative disorders and some forms ofcancer.

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AntioxidantMeasurement5

The need to measure antioxidant activity is we l l documented; these arecarried out for meaningful comparison of foods or commercial products and forprovisionofqualitystandardsfor regulatory issues and health claims(23). Thereare numerous methods for measureing antioxidant activity; these may beclassified into two categories. The first category measures the ability ofantioxidants in inhibiting oxidation in a model system by monitoring theassociated changes using physical, chemical or instrumental means. Radicalscavengingassays include methods based on hydrogen atom transfer H A T ) orelectron transfer (ET) mechanisms. Oxygen radical absorbance capacity O RAC ) ,total radical trapping antioxidant parameter T R A P ) andcrocinbleachingassaysare the major methods that measure HAT whileTrolox equivalent antioxidantcapacity T E A C ) , ferric reducing antioxidant antioxidant power F R A P )and 2,2-diphenyl-l-picrglhydrozyl D P PH ) assays represent ET-based methods.Extensiverelevant reviews are provided in the literature(23-25)as we l las in thisvolume (26).

It is interesting tonotethatD P P H radical is used totestantioxidant activityby its ability toabstracthydrogen atoms from polyphenols (27). Another stableradical,tris(2,4,6-trichloro-3,5-dinitrophenyl)methyl radical, was developed as agood sensor to test the activity of polyphenols measuring their capacity toparticipate in electron transfer reactions (27).Antioxidant activity of a compound can also be evaluated in different cellculture assay for the prevention of carcinogenesis. Because oxidative DNAdamage is considered to be relevant in carcinogenic process, one can evaluatethe possible anticarcinogenic effect of dietary antioxidants by determining theireffect on 12-Otetradecanoylphorbol 13-acetate(TPA)-inducingROS generation,H 2 0 2 scavenging, H202-induced apoptosis, xanthine oxidase activity, andlipopolysaccharide(LPS)-inducing NO generation. Details are discussed in thisvolume (28).

BioavailabilityofDietaryAntioxidantsBioavailabilityis thedegreetowhicha drug, nutrient, dietary supplement or

nutraceutical is available to the body.Bioavailability is influenced by how mucho f a substance is absorbed and circulated in the human body. Problems withbioavailabilityarise when trying to elucidate exactly what dose brings about thedesiredphysiologicalresponse. Manufactures producing 500 mg vitamin C pillscannot claimthat500 mg of the vitamin are taken in and used by the body.

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6There are variations between different human subjects and their uptakeof

certain food-based chemicals. Thismeansthattwo people taking the samedosecould actually absorb different amountsof the samecompound. One might onlysee the effect of 200 mg of a 500 mg pi l l while the other might see the effect of100 mg. This disparity is due to variability in absorption, distribution,metabolism and excretion of the bioactives abbreviated as A D M E . In addition,it is sometimes the case that the ingested chemical is not the final bioactiveagent.Manymoleculesenterthe digestive system in one form only to be brokendown into smaller metabolites that interact through absorption. Science has yetto identify many ofthese breakdown reactions sufficiently to understand howthesereactions affect the bioavailabilityof compounds.

Studies have shown that 11 of caffeic acid and trace amounts ofchlorogenic acid,presentin coffee, are found in urine indicatingthatthey failedto be fullyabsorbed through the gut barrier(29).The exact fateof the remainingcaffeic acid is unknown. This is the problem ofbioavailabilityand ittranslatestonearly everyaspectof nutraceuticals.

Whileit isdifficultto say forsurehow much of a compound w i l lbe taken inand used by an organism,thereis some strong evidence showing how low someo f the uptakes can be. In a study using chlorogenic acid, only 1.7 of it wasrecovered unchanged in the urine. Inthesecases, the colon could play a largerrole in the metabolism of polyphenols (30).

Polyphenols reaching the colon can be broken into smaller metabolites bycolonicmicrobiota, the bacteria found in the colon(30). These bacteria are ableto break down phenols, allowing absorption ofthesesmaller metabolites by thekidneys, liver and other organs. Later,thesesmaller metabolites may find theirway into the urine. Without a clear understanding of their chemicalnature, wecannot screen for them in the urine. Thesamestudy also concludedthata largepart of the ingested polyphenols w i l l probably never enter the peripheralcirculationas smaller metabolites (30).

The problem of reduced antioxidant activity found in smaller metabolites oflarger parent compounds increases the uncertainty ofbioavailabilitystudies. Anorganism is not likelyto absorb an entire dose, and it is likelythatthe compoundwil l be broken down into smaller, unidentified compounds. Further studies arewarranted to identify these compounds. Consideringthese variables, it is verydifficult to predict the total effect of an antioxidant onhostcells.

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Hoboken,N J ,2005, pp. 431-489.2. De la Fuente, M .Eur. J. Clin Nutr.2002 56Suppl.3,S5-S8.

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73. Grimble,R . F .New Horiz. 1994 2,175-185.4. Calder,P . C . ;Kew, S.Br.J.Nutr.2002 88(Suppl.2),S165-S177.5. Pietta, P . G .J. Nat. Prod.2000 63, 1035-1042.6. Packer, L . ; Hiramatsu,M.;Yoshikawa, T.Antioxidant FoodSupplementsin

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6-antioxidant measurement and applications-an overview

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