Antioxidant Activity of Resveratrol Compared with Common Food

379

Journalof Food Protection, Vol. 64, No. 3, 2001, Pages 379– 384 Copyright q,International Association forFood Protection

AntioxidantActivity ofResveratrol Compared withCommon FoodAdditives

M.ANTONIA MURCIA* AND MAGDALENA MARTI´NEZ-TOME´

Departmentof Food Science, Veterinary Faculty, Campus of Espinardo,University ofMurcia,Apartado de Correos 4021, E-30080-Murcia, Spain

MS00-238:Received 24July 2000/ Accepted 2October2000

ABSTRACT Downloaded from http://meridian.allenpress.com/jfp/article-pdf/64/3/379/2007778/0362-028x-64_3_379.pdf by guest on 29 September 2021 Resveratrolis a phenoliccompound of thestilbene family present in wines and various parts of the grape, including the skin.In this study, the antioxidant and prooxidant activities of resveratrol were compared with other antioxidants (butylated hydroxytoluene[BHT], butylatedhydroxyacetone [BHA], phenol,propyl gallate [PG], sodium tripolyphosphate [TPP], a- tocopherol,and vanillin) widely used in foods. The ability of these compounds to inhibit lipid peroxidation was as follows: BHA . resveratrol . PG . tripolyphosphate . vanillin . phenol . BHT . a-tocopherol,the rstthree inhibiting the peroxidationin a concentration-dependentmanner .Theorder of OH scavengeractivity of the tested compounds was BHA . TPP . BHT.Resveratroland vanillin produced between 10 to 7% and 16 to 10% inhibition of the deoxyribose attack, respectively,but they do not scavenge OH .Neitherthe resveratrol analyzed nor PG orthe rest of compounds reacted with H2O2 andmust be considered inef cient in catalyzing any subsequent oxidation. The ability to scavenge HOCl was,in decreasingorder, PG . resveratrol . a-tocopherol . phenol.The other compounds did not scavenge HOCl.

Anantioxidant may be de ned as ‘ ‘asubstancethat beenproposed to have antioxidant capacities include tea, whenpresent at low concentrations compared with those of olives,grapes, apple, clove, Vanillaplanifolia, among oth- anoxidizablesubstrate such as fats,proteins, carbohydrates, ers.The avonoidsand other polyphenols found in some orDNA, signicantly delays or prevents the oxidation of ofthese extracts have been widely discussed as potential thesubstrate’ ’ (1). antioxidantprophylactics (3). Therole of avonoidsin the Antioxidantsare of interestto food scientists, polymer dietis receiving increasing interest and the biological ac- chemists,radiation chemists, and food manufacturers (5). tivityof these polyphenolic components is of great impor- Oxidationof thelipid components in food by the free rad- tancefor understandingtheir mode of action in vivo. In- icalchain reaction of lipidperoxidation is amajorproblem teresthas focused on the health bene ts derived from adiet for foodmanufacturers. The extent to which oxidation of richin avonoids (3). fattyacids and their esters occurs in foods depends on the Amongthe most frequently used antioxidants in food chemicalstructure of thefatty acid, the nature of foodpro- issodium tripolyphosphate (TPP ,E-452)that is used as a cessing,and the temperature at which the foods are stored preservative,metal ion sequestrant, and texturizer in meat and/orcooked, and the minor constituent antioxidants (2). and shproducts (26, 27). BHA(E-320)and BHT (E-321) Manyfood antio xidants(butyl atedhydrox yanisole havebeen used as foodantioxidants because the end-prod- [BHA], butylatedhydroxytoluene [BHT], andpropyl gal- uctsof lipid peroxidation are toxic and can impart a ran- late[PG]) arechain-breaking inhibitors of lipid peroxida- cidityor off- avor to foods like cakes, cookies, vegetable tionthat act through a mechanismthat produces a reaction oils,and dehydrated soups (5). Propylgallate (E-310) has betweenthe radicals and proteins or fatty-acid side-chains beenused as a foodantioxidant, e.g., in vegetableoils, mar- (21). Theuse of these compounds is increasingly limited garine,instant mashed potato, and breakfast cereals. V anil- outof safety considerations, and their replacement by nat- linis also a widelyused avoringagent, e.g., in icecream, uralantioxidants is widely advocated (1). Also,food man- cakes,syrups, and chocolate (4, 11). a-Tocopherol(E-307) ufacturersare very interested in natural antioxidants to act isthemost important natural antioxidant found in vegetable asreplacements for thesynthetic antioxidants currently oil-derivedfoods. It has a widelyextended use in fats and usedby the industry to combat the formation of off-avors (23). andrancidity, particularly in fat-based products (1, 15). oils a-Tocopherolcontains a phenolicstructure that Thereis an emergingconsensus that the micronutrients scavengeslipid and oxygen radicals (28). andnonnutrient components of fruits and vegetables play Resveratrol(3,4 9,5-trihydroxystilbene) isa phenolic apreventiverole in the development of chronic diseases. compoundof the stilbene family together with other hy- Epidemiologicalstudies have found that diets high in fruits, droxylatecompounds. Resveratrol is found in winesand in vegetables,herbs, and spices correlate with low incidences variousparts of the grape, including the skin (38). Wines ofcancerand heart disease (29, 30). Plantextracts that have from variousparts of the world have been shown to vary widelyin their resveratrol content, on account of thein u- *Authorfor correspondence. E-mail: [email protected]. enceof soil and climate factors (12). Anumberof research- 380 MURCIA AND MARTI´NEZ-TOME´ J.FoodProt., Vol. 64, No. 3 ers havemeasured the resveratrol concentration of wines incubationperiod, 0.1 ml of 2%(wt/vol)BHT wasadded to each (9, 13) anddiscussed the differences in termsof the various mixturefollowed by addition of 1 mleach of 1% (wt/ vol)thio- grapevarieties used (14,19, 34). Thephenolic content of barbituricacid (TBA) and2.8% (wt/ vol)trichloroacetic acid. The redwine is about 20 to 50 times higher than that of white solutionswere heated in a waterbath at 80 8Cfor20 min to de- wine (22). velopthe malondialdehyde thiobarbituric adduct ([TBA] 2-MDA). Theaddition of BHTtothe reaction mixtures minimizes erroneous Inrecent years, many papers have been published on increasesin color due to iron ion-dependent hydroperoxide de- resveratrolin grapes and wines, re ecting the widespread compositionduring the acid heating stage (29). The (TBA)2-MDA interestthis compound has aroused in connection with chromogenwas extracted into 2 mlof butan-1-ol and the extent pharmacologicalproperties that would seem to help prevent ofperoxidation measured in the organic layer as absorbance at cardiovasculardisease. Resveratrol would seem to have an- 532 nm. tiplateletaggregating properties and facilitate an increasein ThisTBA testmeasures not only peroxidation occurring in high-densitylipoprotein cholesterol. Moreover some au- theexperiment itself but also peroxidation taking place during the thorshave recently reported that resveratrol delays the onset acid-heatingstage of theassay. In order to avoidany interference, oftumors in animals. It is known that the moderate con- theTBA testwas performed in the presence of the antioxidant Downloaded from http://meridian.allenpress.com/jfp/article-pdf/64/3/379/2007778/0362-028x-64_3_379.pdf by guest on 29 September 2021 sumptionof redwine reduces ADP-induced platelet aggre- BHT toinhibit peroxidation during the assay itself (6). gation (12). Frankeland coworkers (18) andEctor et al. Hydroxylradical scavenging. Ina nalvolume of 1.2 ml, (16) attributedthe positive bene ts ofredwine to theability reactionmixtures contained the following reagents at the nal ofphenolic substances to prevent oxidation of low-density concentrationstated of 10 mM KH 2PO4-KOH buffer(pH 7.4),2.8 lipoprotein,a criticalevent in the process of atherogenesis mM H2O2,2.8mM deoxyribose(where used), 50 mM FeCl3 pre- (22). Inaddition, investigators concluded that the bene cial mixedwith 100 mMEDTA beforeaddition to the reaction mix- effectsof the beverages, such as beer and red wine, are ture,and 0.05, 0.125, 0.25, and 0.5 mM ofresveratroland the rest partiallydue to components other than the alcohol (37). ofthe compounds tested dissolved in water .Ascorbate(100 mM), whereused, was added to start the reaction. The tubes were in- Neverthelessit should always be takeninto account that the cubatedat 37 8Cfor1 h.The products of hydroxyl radical (OH ) problemsof alcoholism might be a greaterproblem than attackupon deoxyribose were measured as described in Aruoma anyof the bene ts derived from thementioned alcoholic et al. (7). beverages. Theresults of epidemiologicalstudies based on W orld Scavengingof hydrogen peroxide. Resveratrol,PG, andthe HealthOrganization data indicate that the consumption of differentcomponents to be tested for their reaction with H 2O2 wereincubated at 0.05, 0.125, 0.25, and 0.5 mM with6.72 mM wine,especially red wine, may reduce the risk of coronary H O for10 min at 25 8C.Aliquotsof thesecompounds were then heartdisease by 40% (33). 2 2 takenand assayed for remaining H 2O2 byusing the peroxidase Resveratrolis a naturalproduct that has received at- system (6). Theremaining H 2O2 wasmeasured as the formation tentionfor useas a possiblefood antioxidant. The aim of ofa browncolor (recorded at 436 nm) in reaction mixtures con- thisstudy is to compare the in vitro effect of resveratrol taining,in a nalvolume of 1 ml,0.15 M KH 2PO4-KOH buffer, withother widely used antioxidants, such as BHT ,BHA, pH 7.4, 50 mlguaiacolsolution (made by adding 100 ml of pure phenol,PG, TPP, a-tocopherol,and vanillin to better un- liquidto 100 ml water), and 10 mlofSigma type IV horseradish derstandthe nature of the bene cial effect of resveratrol. peroxidase(5 mg/ mlin the same phosphate buffer). MATERIALS AND METHODS Reactionswith hypochlorousacid. Thehypochlorous acid (HOCl)reaction was studied using the elastase assay, essentially Chemicalswere of the highest quality available and were asdescribed by Aruoma et al. (7). Forthe assay, 68 mM HOCl purchasedfrom Sigma Chemical Co. (Poole, Dorset). Resveratrol, (producedimmediately before use by adjusting NaOCl topH 6.2 BHT,BHA, phenol,PG, TPP, a-tocopherol,and vanillin were also withdilute H 2SO4)andthe compounds to be tested (0.5 mM) fromSigma Chemical Co. wereincubated for 20 min in a nalvolume of 1.0 ml in phosphate-bufferedsaline, pH 7.4,containing 140 mM NaCl,2.7 mM Peroxidationof phospholipid liposomes. Theability of KCl, 16 mM Na2HPO4,and2.9 mM KH 2PO4. a1-Antiproteinase compoundsto inhibitlipid peroxidation at pH 7.4was tested using (2mg/ ml)were added to the reaction mixture. This allows any oxbrain phospholipid liposomes, essentially as describedin Quin- HOCl remainingto inactivate a1-antiproteinase.After a further lan et al. (32) andin Aruoma et al. (8). Theexperiments were 20-minincubation, 0.05 ml of2.5mg/ mlelastase was added. The conductedin phosphate-buffere dsaline(3.4 mM Na 2HPO4- mixturewas allowed to stand for 30 minmore before the 2 mlof NaH2PO4,0.15M NaCl),pH 7.4. In a nalvolume of 1 mlthe phosphate-bufferedsaline was added. The remaining elastase ac- assaymixtures were made up with phosphate-buffered saline, 0.5 tivitywas then measured by adding elastase substrate (5 mg/ ml, mg/mlphospholipid liposomes, 100 mM FeCl3,varyingconcen- N-succinyltriala- p-nitroanilide),which is hydrolyzed by elastase, trationsof resveratrol, and the rest of the compounds analyzed resultingin an increase in A 410. (BHT,BHA, phenol,PG, TPP, a-tocopherol,and vanillin) at 0.05, 0.125,0.25, and 0.5 mM dissolvedeither in water or in ethanol Dataanalysis. Theresulting data were analyzed using Sta- orin methanol (neither ethanol nor methanol affect the outcome tisticalPackage for the Social Sciences Windows 9.0. An analysis ofthe lipid peroxidation assay; BHT and a-tocopherolare not ofvariance was carried out after triplicate experiments, calculating fullysoluble in aqueous solution, and their emulsions are not ho- thesigni cance level by using a proportionstest. mogeneous).T oovercomethis problem it was necessary to use RESULTS ANDDISCUSSION deionizedwater, with conductivity not more than 4 mS/cm, to dissolvethem (35), and 100 mMascorbate(added last to startthe Inhibitionof phospholipid peroxidation. Lipid per- reaction).Incubations were at 37 8Cfor60 min. At theend of this oxidationis sometimes a majormechanism of cell injury J.FoodProt., Vol. 64, No. 3 ANTIOXIDANT ACTIVITY OFRESVERATROL 381

TABLE 1. Inhibitionof peroxidation by resveratrol in the lipid nillin,phenol, and BHT workedbest at 25% inhibition. a- systemusing ox brain phospholipids compared with the activity Tocopherolwas lesseffective than PG withpoor inhibition ofdifferent compounds frequently used as food additives a capacityof peroxidation. BHT showedincreasing antioxi- Addition to dantactivity in ethanol or in methanol. However , a-to- reaction Concentration copheroldid not show a signicant difference ( P # 0.05) mixturesb (mM) %inhibition in% peroxylradical inhibition by change in solvent. Manyof the tested compounds are organic. In fact Resveratrol 0.5 62 0.25 35 someof them like resveratrol (20) and a-tocopherol (36, 0.125 24 40) havebeen reported to incorporate into model mem- 0.05 22 branesand biomembranes. The experiment was carriedout PG 0.5 51 withthe compounds dissolved in ethanol or methanol, and 0.25 47 theresults were almostidentical. Also, these compounds 0.125 39 were observedto have a similarantioxidant capacity when 0.05 20 theywere dissolvedin water .Theresults showed a gradual Downloaded from http://meridian.allenpress.com/jfp/article-pdf/64/3/379/2007778/0362-028x-64_3_379.pdf by guest on 29 September 2021 BHA 0.5 71 increasein lipid peroxidation inhibition with increasing 0.25 62 concentrationsof resveratrol, PG, andBHA ( P # 0.05). 0.125 36 Weobservedthat resveratrol dissolved in ethanolproduced 0.05 26 BHT 0.5 22 aninhibition that varied from 57%to 3% at the concentra- 0.25 24 tionstested; the BHA resultsvaried from 70to 17% inhi- 0.125 22 bitionfor concentrationsof 0.5 to 0.05 mM. PG showed 0.05 24 valuesfrom 57to 3% inhibition and BHT inethanol Phenol 0.5 25 showeda considerableincrease in itsinhibitory activity that 0.25 21 variedfrom 64to 1% inhibition. The results obtained for 0.125 20 phenol,vanillin, and a-tocopheroldissolved in ethanol 0.05 18 were similarto those obtained in aqueous medium. These TPP 0.5 26 compoundscould be used as preserversof fator oilfoods. 0.25 25 TPPwas apoorinhibitor of peroxidation in ethanol. 0.125 22 Our resultsare in accord with Aruoma et al. (4), who 0.05 21 Vanillin 0.5 26 showedthat BHT ,BHA, PG, andvanillin dissolved in eth- 0.25 22 anolinhibited lipid peroxidation, although rat liver micro- 0.125 21 someswere usedin this experiment. Several reports have 0.05 20 demonstratedthe protective action of a-tocopherolagainst a-Tocopherol 0.5 15 lipidperoxidation (17). Also,Decker and Xu (15) showed 0.25 13 thatBHA, BHT,andPG inhibitlipid oxidation by free rad- 0.125 10 icalscavenging. 0.05 6 Brand-Williamset al. (10) determinedthe ef ciency of a Statisticaldifferences were analyzed by analysis of variance ( P monophenols(phenol, vanillin) and polyphenols as anti- # 0.05). radicals.Camire and Dougherty (11) concludedthat vanillin b Compoundsin aqueous medium. protectsagainst lipid oxidation better than BHT insnacks- typefoods. Free radicaltermination, rather than iron bind- ing,is the probable mechanism for thisretardation of oxi- inorganismssubjected to oxidativestress, although it isby dation. nomeansthe only mechanism of injury (6). Oxbrain phos- pholipidliposomes undergo rapid nonenzymatic peroxida- Assessingthe antioxidantaction of resveratrol in tionwhen incubated with FeCl 3 andascorbic acid at pH the deoxyriboseassay. Thedeoxyribose method evaluates 7.4 (1). At lowconcentrations, ascorbate accelerates lipid theability to damage carbohydrates. Hydroxyl radicals peroxidationthrough its ability to reduce iron into the ac- damagethe sugar deoxyribose. Highly reactive hydroxyl tiveferrous state while, at high concentrations, ascorbate radicals(OH )aregenerated by a mixtureof ascorbate and inhibitslipid oxidation by inactivating free radicals (15). FeCl3-EDTA atpH 7.4 (1). Thedeoxyribose is broken Theability of resveratrol,BHT ,BHA, phenol,PG, TPP, a- downinto fragments and, on heating with TBA atlowpH, tocopherol,and vanillin to inhibit phospholipid liposome generatesa pinkchromogen. The addition of ascorbic acid peroxidationwas tested.All these compounds scavenge the greatlyincreases the rate of OH generationby reducing peroxylradical when they are dissolved in water .Thein- ironand maintaining the supply of Fe 21 (2). hibitionpercentage of peroxidation recorded was inthefol- Theeffects of increasing concentrations of resveratrol lowingdecreasing order: BHA . resveratrol . PG . TPP onthe deoxyribose attack by OH radicalcompared with . vanillin . phenol . BHT . a-tocopherol( P # 0.05) differentcompounds frequently used as food additives (Table1). Resveratrol, BHA, andPG inhibitedperoxidation (BHA, BHT,phenol,PG, TPP,vanillin,and a-tocopherol) inaconcentration-dependentmanner .BHA, resveratrol,and areshown in T able2. The results can be divided into sev- PGperformedthe best % inhibitionup to 50%. TPP ,va- eralclasses. The rst,comprised of those compounds that 382 MURCIA AND MARTI´NEZ-TOME´ J.FoodProt., Vol. 64, No. 3

TABLE 2. Desoxyriboseattack by OH radicalin the presence of resveratrol compared with the activity of different compounds fre- quentlyused as food additives a

c Damage todeoxyribose A 532nm Addition to reaction % mixturesb mM RM 1 DR inhibition Omit ASC

Resveratrol 0.5 1.21 10 0.51 0.25 1.22 9 0.50 0.125 1.24 8 0.49 0.05 1.24 7 0.48 PG 0.5 1.71 — 0.83 0.25 1.56 — 0.76 0.125 1.42 — 0.70

0.05 1.36 — 0.65 Downloaded from http://meridian.allenpress.com/jfp/article-pdf/64/3/379/2007778/0362-028x-64_3_379.pdf by guest on 29 September 2021 BHA 0.5 1.00 25 0.09 0.25 1.10 18 0.10 0.125 1.12 16 0.12 0.05 1.19 11 0.14 BHT 0.5 1.22 9 0.25 0.25 1.30 3 0.27 0.125 1.31 2 0.32 0.05 1.33 1 0.36 Phenol 0.5 1.58 — 0.16 0.25 1.43 — 0.18 0.125 1.33 — 0.22 0.05 1.25 7 0.27 TPP 0.5 1.18 12 0.25 0.25 1.20 10 0.35 0.125 1.26 6 0.24 0.05 1.35 — 0.33 Vanillin 0.5 1.13 16 0.55 0.25 1.15 14 0.45 0.125 1.19 11 0.34 0.05 1.20 10 0.26 a-Tocopherol 0.5 1.31 2 0.38 0.25 1.30 3 0.46 0.125 1.26 6 0.52 0.05 1.11 17 0.82 a Statisticaldifferences were analyzed by analysis of variance ( P # 0.05). b Compoundsin aqueous medium. c Whendeoxyribose is omitted the range of values oscilated from 0.001 to 0.006 absorbance units. RM, reactionmixtures; DR, deoxyribose;ASC, ascorbate;— ,no% inhibitiondetected. showmoderate ( ,25%)inhibition as adirectresult of OH oxyribosedamage, whereas a prooxidantmolecule will ac- scavengingactivity of the antioxidants, was BHA . TPP celeratethe damage (4). . BHT (P # 0.05)(T able2). Only BHA increasesuntil Thelast class is comprised of compounds that scav- 25%inhibition on deoxyriboseattack, and the % inhibition engelow levels of OH radicals.The results also show that decreasedwith the concentration. a-tocopheroland phenol, in thepresence of ascorbate,may Thesecond, comprised of those that react with ascor- onlyact as antioxidant at low and medium concentrations. bate,resveratrol, and vanillin, produced similar % inhibi- Thesecompounds were shownto have at least a potential tionto TPP andBHT .Butthey did not scavenge OH , be- asantioxidants within a certainrange of concentrations.As causewhen ascorbate was omitted,the level of the pink theirconcentration increases, the antioxidative effect reach- chromogenexceeded that of the control. Probably these esa maximumand may, at higher concentrations, be re- compoundsreact with ascorbate, decreasing the amount of versedinto a prooxidativeeffect (25). OH generated. Accordingto our results, other authors have described Anotherclass is comprised of those that show proox- phenolas a poorantiradical compound for hydroxylgroups idativeactivity. PG was prooxidantat the concentrations usingthe free radical2,2-diphenyl-1-picr ylhydrazyl.Poly- testedbecause it has a synergiceffect with ascorbate and phenolshave a higherantioxidant activity than monophen- stimulatesdeoxyribose degradation, as Aruoma et al. (7) olsdue to effective hydrogen donation accompanied by the found.This property has been adopted as one measure of effectivedelocalization of anunpairedelectron. The double prooxidantcapacity (24). An OH scavengerwill inhibit de- bondin the C-ring in conjugation with the carbonyl im- J.FoodProt., Vol. 64, No. 3 ANTIOXIDANT ACTIVITY OFRESVERATROL 383 proveselectron delocalization that stabilizes the antioxidant TABLE 3. Scavengingof HOC1 byresveratrolusing the elastase radical.The poor ef ciency of phenolis dueto theabsence assaycompared with the capacity of different compounds fre- ofany electron-donating group (10, 28). quentlyused as food additives a BHAandphenol are very good scavengers of OH rad- Elastase activityin icalsin theabsence of addedascorbic acid (T able2). When nalreaction ascorbatewas omittedfrom thedeoxyribose reaction mix- mixture b ture,absorbance fell substantially because of the slow rate Additionto rst reactionmixture (D A410/min) of OH generation.BHT ,TPP,and a-tocopherolshowed low Elastaseonly 1.174 scavengingability. In fact, Aruoma et al. (4) explainedthat a1-AP 0.022 a-tocopheroland other phenolic compounds are capable of HOC1 1 a1-AP 0.597 bindingiron (III) andreducing it toiron(II) thatmay result HOC1 1 0.5 mM PG 1 a1-AP 0.162 ina prooxidantaction under certain circumstances. HOC1 1 0.5mM Resveratrol 1 a1-AP 0.213 Thisassay was onlydeveloped with the compounds HOC1 1 0.5 mM BHA 1 a1AP 1.759 dissolvedin waterbecause organic solvents such as ethanol HOC1 1 0.5 mM BHT 1 a1-AP 1.488 Downloaded from http://meridian.allenpress.com/jfp/article-pdf/64/3/379/2007778/0362-028x-64_3_379.pdf by guest on 29 September 2021 arethemselves good scavengers of OH (4). HOC1 1 0.5mM Phenol 1 a1-AP 0.834 HOC1 1 0.5 mM TP 1 a1-AP 1.856 Hydrogenperoxide scavenging. Thegeneration of HOC1 1 0.5 mM a-tocopherol 1 a1-AP 0.649 hydrogenperoxide by activated phagocytes is known to HOC1 1 0.5mM vanillin 1 a1-AP 1.257 playan important part in the killing of severalbacterial and a Statisticaldifferences were analyzed by analysis of variance ( P fungalstrains. Additionally, hydrogen peroxide is generated # 0.05). invivo by several oxidase enzymes (21). Thereis increas- b Compoundsin aqueous medium. ingevidence that H 2O2, directlyor indirectlyvia its reduc- tionproduct OH ,actsas amessengermolecule in the syn- thesisand activation of in ammatory mediators (39). vanillin,and TPP showeda greatercapacity to scavengein Thescavenging of hydrogenperoxide activity is easily HOCl thanin water ,51,48, 44, and 27% inhibition, re- andsensitively measured by using peroxidase-based assay spectively.The phenol (83% inhibition) was abetterHOCl systems,when one looks for adecreasein the absorption scavengerin ethanol. The a-tocopherol(48% inhibition) spectrumafter the compound is added to peroxidase-H 2O2 showedsimilar abilities in bothwater and ethanol. None of mixtures.In our study, neither the resveratrol analyzed nor thecompounds tested interfered with the ability of a1-an- PGorthe rest of the compounds reacted with H 2O2 and tiproteinaseto inhibit elastase nor did they inhibit elastase mustbe considered inef cient catalysts of any subsequent directlyunder our assay conditions. oxidation. Animportant observation from thepresent work is that thedifferent compounds analyzed have different antioxi- Scavengingof HOCl. HOCl isproduced by the neu- dantproperties in the different assays used. Each assay trophil-derivedenzyme, myeloperoxidase, at in ammation measureda differentparameter .Resveratrolextract is sitesand when activated neutrophils in ltrate reoxygenated shownto be a goodnatural antioxidant that could easily be tissue.One of the important targets attacked by HOCl in incorporatedin the human diet. vivo is a -antiproteinase,the major circulating inhibitor of 1 Theconsumption of vine products (e.g., wine, un l- proteolyticenzymes such as elastase (5). Thus,a goodtest teredjuice, and whole berries without seeds, and especially, for physiologicallyrelevant HOCl scavengingactivity by a productsmade with pomace puree) could be a meansof compoundis to see whether that compound, in theconcen- incorporatinga signicant quantity of resveratrolin the diet trationsachieved in vivo, can protect a -antiproteinase 1 (16). againstinactivation by HOCl (31). InT able3 thescavenging of HOCl byresveratrol using ACKNOWLEDGMENTS theelastase assay is compared with the capacity of different Thiswork was fundedby the FEDER project, Ministerio de Edu- compoundsfrequently used as food additives. a1-Antipro- caciońyCultura,Spain, 1FD97-0021 and the Fundacio ńSeńeca project, teinaseinhibited the activity of elastase in vitro. After in- Consejer´õ adeCulturay Educacioń,Murcia(Spain), 00263/ /CV/97.M.M. cubationof 68 mM HOCl with a1-antiproteinase,which is is arecipientof a fellowshipfrom the University of Murcia (Spain). veryrapidly inactivated by HOCl, a -antiproteinaseloses 1 REFERENCES itselastase-inhibitory capacity. 1.Aeschbach, R.,J. Lo¨liger,B.C.Scott,A. Murcia,J. Butler ,B.Hal- Resveratrolscavenges HOCl, so that a1-antiproteinase actsby inhibiting elastase activity. The ability to scavenge liwell,and O. I.Aruoma.1994. Antioxidant actions of thymol,car- vacrol,6-gingerol, zingerone and hydroxytyrosol. Food Chem. 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