1412

Journalof FoodProtection, Vol. 64, No. 9, 2001, Pages 1412– 1419 Copyright ,International Association forFood Protection

AntioxidantProperties ofMediterranean Spices Compared with Common FoodAdditives

MAGDALENA MARTI´NEZ-TOME´ ,1 ANTONIA M.JIME´ NEZ,1 SILVERIO RUGGIERI, 2 NATALE FREGA, 2 ROSANNA STRABBIOLI, 2 AND M.ANTONIA MURCIA 1*

1Departmentof Food Science, Veterinary Faculty, Campus de Espinardo, University ofMurcia, Apartado de Correos 4021, E-30008-Murcia, Spain; and 2Dipartimentodi Biotecnologie Agrarie ed Ambientali, Universita ´diAncona,Via Brecce Bianche,60131-Ancona, Italy

MS00-458:Received 20December 2000/Accepted 20February 2001 Downloaded from http://meridian.allenpress.com/jfp/article-pdf/64/9/1412/1674653/0362-028x-64_9_1412.pdf by guest on 28 September 2021 ABSTRACT

Inthis study, the antioxidant properties of Mediterranean food spices (annatto, cumin, oregano, sweet and hot paprika, rosemary,and saffron) at 5% concentration and of common food additives (butylated hydroxyanisole [BHA], butylatedhy- droxytoluene[BHT], andpropyl gallate) at 100 g/garecompared. The ability of thesecompounds to inhibit lipid peroxidation was,in decreasing order, rosemary oregano propylgallate annatto BHA sweetpaprika cumin hot paprika saffron BHT.Deoxyribosedamage is partially inhibited in the presence of cumin extract that exhibits the strongest protectiveaction. The rest of thespices also protect deoxyribose better than the BHA andBHT usedin theassay. Finally, the resultsobtained in the assay point to the prooxidant effect of propylgallate. Hydrogen peroxide scavenging activity is measured byusing peroxidase-based assay systems. In aqueous medium, the spice extracts show lower antioxidant activity than propyl gallate,the decreasing order being cumin oregano annatto rosemary hot paprika sweetpaprika. BHA andBHT didnot scavenge H 2O2. Spicesare able to scavenge HOCl andprotect 1-antiproteinase.The results indicate that rosemary andoregano are more effective HOCl scavengersthan the other substances analyzed, which, in decreasingorder ,werepropyl gallate,annatto, sweet and hot paprika, saffron, and cumin. The effect of Mediterraneanfood spices on theoxidative stability ofreŽ ned olive oil tested by theRancimat method was compared with common food additives during storage (72 h, 2, 4,and 6months)at room temperature. The results showed that the spice extracts analyzed have signiŽ cant stabilizing effects ( P , 0.05).

Inthe preparation and commercialization of foodstuffs extractsare being marketed as antioxidants for usein the orprepared foods, the aim is always to obtaina productof foodindustry (29). maximalquality that is aswell preserved as possible before Leavesand extract of rosemary( RosmarinusofŽ cinalis reachingthe consumer .Normalpractice is toadd substances L.) havean agreeable odor and a slightlycamphoraceous thatprevent degradation (stabilizers, antioxidants), improve taste.The volatile oil is used in medicine and as food  a- avor(sweeteners), increase Ž rmness(thickeners, agglutin- voring,especially in foods containing animal fats (meats, ants),color maintenance, or antimicrobials (36). sausages),soups, sauces, vegetable oils, and other food Despitethe widespread use of antioxidants, lipid oxi- products (23, 39). dationis still a majorcause of food quality deterioration Oregano (Origanumvulgare L.)iswidelyused in med- duringthe storage of oils, fats, and fat-containing foods. icineand food  avoring,mainly in stews and Italian foods Themost widely used synthetic antioxidants, butylated hy- likepizza, macaroni, and spaghetti. The essential oils of droxyanisole(BHA; E-320),butyla tedhydrox ytoluene oreganohave expectorant, antispasmodic, tonic, antiseptic, (BHT;E-321),and propyl gallate (E-310), are chain-break- analgesic,antimicr obial,antifung icidal,and germicid al inginhibitors of lipid peroxidation, acting through a mech- properties (46). anismthat produces a reactionbetween the radicals and Rosemaryand oregano show high antioxidant activity proteinslike albumin or fatty-acid side chains (24). How- intheir ground form andas extracts, in which a numberof ever,BHAandBHT arequite volatile and easily decom- phenoliccompounds with strong antioxidant activity have poseat high temperatures (1). Theuse of spices as anti- beenidentiŽ ed. Flavonoids and other phenolics are thought oxidantsin processed foods is a promisingalternative to toplay a preventiverole in the development of cancerand theuse of synthetic antioxidants, especially because of in- heartdisease (25). creasingconsumer interest in natural food additives (30). Thereis increasing interest in the spice paprika ( Cap- Traditionally,spices have been added to different types sicumannuum L.),which is an excellent source of natural (28). offood to improve the  avor Currently,there is a color (33), dueto carotenoid pigments (34). Carotenoids growingawareness that spices also improve the oxidative actas a naturalantioxidant that quenches singlet oxygen, stabilityof processedproducts and, as aconsequence,spice actingas electron donors. Certain carotenoids have been *Authorfor correspondence. Tel: 34 68 364792; Fax: 34 68 364147; consideredas anticancer agents, antiulcer agents through E-mail: [email protected] theirprotection of the gastric mucosa, and positive regu- J.FoodProt., Vol. 64, No. 9 ANTIOXIDANT PROPERTIESOF SPICES 1413 latorsof theimmune response (11). Theyalso contain vital powderedusing a mortarand pestle. Every powdered spice (5 g) micronutrients,such as vitamins C andE, thathave been wasextracted for 1 hbystirring at room temperature with 100 conŽrmed by manyepidemiological studies as reducing the mlwater or ethanoland centrifuged at 3,000 rpm for 10 min. The riskof cancer and cardiovascular disease when taken daily 5%spice extracts were used in the different assays. BHA, BHT, inadequate amounts (31). andpropyl gallate were used at the permitted commercial con- centrationof 100 g/g (19). Propylgallate was also tested at 5% Annatto (Bixaorellana L.)extractsare listed among concentration,except for the Rancimat test. thecolors that may be used singly or in combination with additiveslike emulsifying salts (sodium citrate and diso- Peroxidationof phospholipid liposomes. Theability of diumphosphate) (44), inthe cream and butter industries compoundsto inhibit lipid peroxidation at pH7.4was tested using (43) andin certain foods like cheese, where a pinkish- oxbrain phospholipid liposomes, as described in Aruoma et al. brownor salmon color develops during manufacture (45). (10). Theexperiments were conducted in phosphate-buffere dsa- line(3.4 mM Na HPO -NaH PO ,0.15M NaCl),pH 7.4. In a From ancienttimes to the present, saffron ( Crocus sa- 2 4 2 4 Žnalvolume of 1ml,the assay mixtures were made up withphos- tivus L.) hashad diverse uses in medicine in preventing

phate-bufferedsaline, 0.5 mg/ mlphospholipidliposomes, 100 M Downloaded from http://meridian.allenpress.com/jfp/article-pdf/64/9/1412/1674653/0362-028x-64_9_1412.pdf by guest on 28 September 2021 cardiovasculardisease (15). As aspiceit has been used in FeCl3, and 100 lofthetested samples (5% extract) dissolved in foodrecipes, including rice, bread, buns, cakes, and soups eitherwater or ethanol,and 100 Mascorbate(added last to start (15) inall oriental civilizations and in the Mediterranean thereaction). Ethanol does not affect the outcome of the lipid region,where it is widely used in typical dishes like paella peroxidationassay, while BHT isnot fully soluble in aqueous inSpain. Saffron isvalued both for itscoloring and  a- solution,and its emulsion is not homogeneous. In order to dis- voringproperties (38). solveit, deionized water ,witha conductivityof not more than 4 The seeds of Cuminumcyminum L.areknown as cum- S/cmwasused. Incubations were at 37 8Cfor60 min. At theend in,a productof the Mediterranean Basin. It has been used ofthis incubation period, 0.1 ml of 2% (wt/ vol)BHT wasadded asafoodspice  avoringfoods, especially bakery products toeach mixture followed by 1 mleach of 1% (wt/ vol)thiobar- bituricacid (TBA) and2.8% (wt/ vol)trichloroacetic acid. The for sprinklingon bread and cheese (26). Recently,many solutionswere heated in a waterbath at 80 8Cfor20 min to de- medicalproperties have been attributed to the cumin seed velopthe malondialdehyde thiobarbituric adduct ([TBA] -MDA). and/oritsoil, including antineoplasic, antibacterial, antifun- 2 The (TBA)2-MDA chromogenwas extracted into 2 mlof butan- gal,and antihelminthic effects (48). 1-ol,and the extent of peroxidation was measured in the organic Since1993 it has been accepted in the United States, layeras absorbance at 532 nm. thatcertain foods (legumes, vegetables, fruits, spices) re- ThisTBA testmeasures not only peroxidation occurring in ducethe risk of disease.The Food and Drug Administration theexperiment itself but also the peroxidation taking place during hasassessed scientiŽ cally the correlation between the pres- theacid-heating stage of the assay. In order to avoid any inter- enceof thesecompounds, nutrients, or foodsin the diet and ference,the TBA testwas performed in the presence of the anti- thelevel of riskto certaindiseases. Both the Food and Drug oxidantBHT toinhibit peroxidation during the assay itself. Administrationand the ScientiŽ c Committeeon Food for Hydroxylradical scavenging. Ina Žnalvolume of 1.2 ml, NovelFoods and Novel Foods Processes in Europe,on the thereaction mixtures contained the following reagents: 10 mM basisof scientiŽc informationavailable and on thebasis of KH2PO4-KOH buffer(pH 7.4), 2.8 mM H 2O2,2.8mM deoxyri- agreementbetween experts, provide health claims as acon- bose(where used), 50 M FeCl3 premixedwith 100 M EDTA sequenceof availablescientiŽ c evidence (12, 40) for use in beforeaddition to the reaction mixture, and 100 lofthe tested thelabeling of thesespecial foods called functional food, a samples(5% spice extracts) and additives dissolved in water. typeof Novel Foods, clariŽ ed by Diplock et al. (18), em- Ascorbate(100 M),whereused, was added to startthe reaction. Thetubes were incubated at 37 8Cfor1 h.The products of the phasizingthe presence of components, nutrient or nonnu- hydroxylradical (OH )attackon deoxyribose were measured as trient,that help to maintainthe health claims and to reduce describedin Aruoma et al. (9). diseaserisks. Theaim of this study is to characterize some spices Scavengingof hydrogen peroxide. Thesamples to be tested frequentlyused in the Mediterranean diet, in several sys- with H2O2 (dissolvedin either water or ethanol) were incubated tems,in order to assess their antioxidant activity compared with0.84 mM H 2O2 for10 min at 25 8C.Aliquotsof these com- poundswere then taken and assayed for remaining H O by using withcommon food additives (BHA, BHT,andpropyl gal- 2 2 theperoxidase system (8). Theremaining H 2O2 wasmeasured as late).In addition, the activity of thesespices is studieddur- theformation of a chromophorrecorded at 436 nm in reaction ingstorage at room temperature for theiruse as natural mixturescontaining, in a Žnalvolume of 1ml,0.15 M KH 2PO4- additivesand for thedesign of new functional foods with KOH buffer,pH7.4, 50 lguaiacolsolution (made by adding highantioxidant properties. 100 lofpure guaiacol liquid to 100 ml water), and 10 l of Sigmatype IV horseradishperoxidase (5 mg/mlin the same phos- MATERIALS AND METHODS phatebuffer). Samplepreparation. Propylgallate, BHA, BHT,andthe Reactionswith hypochlorousacid. Thehypochlorous acid chemicalsused were of the highest quality available and were (HOCl)reaction was studied using the elastase assay, essentially purchasedfrom Sigma Chemical Co. (Poole, Dorset, UK). The asdescribed by Aruoma et al. (9). Forthe assay, 68 M HOCl Mediterraneanfood spices like annatto ( B. orellana L.), cumin (C. (producedimmediately before use by adjusting NaOCl topH 6.2 cyminum L.),oregano ( O. vulgare L.),sweet and hot paprika ( C. withdilute H 2SO4)andthe tested samples were incubated for 20 annuum L.),rosemary ( R.ofŽcinalis L.),and saffron ( C. sativus minin a Žnalvolume of 1.0 ml in phosphate-buffere dsaline,pH L.)were purchased from a supermarket.The dried spices were 7.4,containing 140 mM NaCl,2.7 mM KCl, 16mM Na 2HPO4, 1414 MARTI´NEZ-TOME´ ET AL. J.FoodProt., Vol. 64, No. 9

and 2.9 mM KH2PO4. 1-Antiproteinase(2 mg/ml)was added to TABLE 1. Inhibitionof peroxidationin the lipid system using ox thereaction mixture. This allows any HOCl remainingto inacti- brainphospholipids by Mediterraneanfood spices compared with vate 1-antiproteinase.After a further20-min incubation, 0.05 ml theactivity of different compounds frequently used as food addi- of2.5 mg/ mlelastase was added. This mixture was allowed to tivesa stand30 min more before the 2 mlof phosphate-buffer edsaline Addedto reaction mixtures b %inhibition wasadded. The remaining elastase activity was then measured by addingelastase substrate (5 mg/ ml, N-succinyltriala- p-nitroani- None(control) —e lide),which is hydrolyzed by elastase, resulting in an increase in Annattoc 78.46 A410. Cuminc 63.08 Oreganoc 93.09 Rancimattest for oxidative stability. Samplepreparation Paprika,hot c 59.26 inthe Rancimat test consisted of macerationof thedifferent spices Paprika,sweet c 65.78 andantioxidants with 100 g reŽned olive oil (provided by the Rosemaryc 94.81 manufacturingcompany and free of added antioxidants or preser- Saffronc 55.13 vatives).The spices were used at 5%(wt/wt)concentration while Downloaded from http://meridian.allenpress.com/jfp/article-pdf/64/9/1412/1674653/0362-028x-64_9_1412.pdf by guest on 28 September 2021 Propylgallate c 83.07 thewidely used antioxidant additives, BHA, BHT,andpropyl gal- Propylgallate d 51.69 late,were used at the permitted commercial concentration of 100 BHAd 71.05 g/g (19). BHTd 22.96 Alloxidative stability measurements were performed with a Rancimatapparatus (Metrohm model 743; Herisan, Switzerland) a Statisticaldifferences were analyzed by analysis of variance ( P bymeasuring the induction period of reŽ ned olive oil with or , 0.05). withoutthe addition of the tested compounds, using the Auto- b Compoundsin aqueous medium. matedSwift T est.Determination of theinduction period, at 110 8C, c Concentration5%. wasbased on thedetection of volatileacids. The induction period d Concentration100 g/g. isthe time elapsed until the in ection point of the conductivity e No% inhibitiondetected. versustime curve recorded by the Rancimat (14). Allsamples were prepared on the same day (day 0) and di- videdinto four batches before being stored at room temperature. Thesewere analyzed after 72 h andafter 2, 4, and 6 monthsof inhibitionthan BHA at100 g/gbutlower antioxidant ac- macerationusing 5 gofsample and an air  owrate set of 20 tivitywith respect to rosemary,oregano, and propyl gallate. liters/h.The relative activity of the antioxidants is expressed by Sweetpaprika, cumin, hot paprika, and saffron, al- theprotection factor ,oxidativestability, or antioxidant index, thoughless effective scavengers than propyl gallate at 5% whichis calculated by dividing the induction period of oil with concentration,showed inhibition percentages higher than addedantioxidants by the induction period of the control (olive oil alone) (10). 50%.With the exception of saffron, all the tested spices Thistechnique has been questioned by some authors (20), showeda betterantioxidant capacity than propyl gallate but,in agreement with Bonilla et al. (13), wedecided to apply it whenthis synthetic additive was analyzedat the permitted inthis work because it is acommonlyused procedure in thefood concentration(100 g/g) (19). Atthisconcentration, propyl industryand governmental analytic laboratories. gallatedid not show signiŽ cant differences with respect to saffron.BHT showedthe lowest peroxyl radical scavenging Dataanalysis. Thedata were analyzed using the Statistical capacityof all the compounds tested. Packagefor Social Sciences Windows 9.0. The analyses of vari- ancewere carried out after triplicate experiments, calculating the Someof the synthetic compounds tested in this study signiŽcance level by using the least signiŽ cant difference multiple areorganic structures, and some food additives like -to- range test. copherol (41, 49) havebeen reported to be incorporatedin modelmembranes and biomembranes. Thus, the experi- RESULTS ANDDISCUSSION mentwas carriedout with the compounds dissolved in eth- Inhibitionof phospholipid peroxidation. Lipid per- anol.When this was done,spices and food additives oxidationis sometimes a majormechanism of cell injury showedsimilar results ( P , 0.05)to those observed in an inorganisms subjected to oxidative stress, although it is by aqueousmedium. In the ethanol medium, there were no nomeans the only mechanism of injury (8). Oxbrain phos- signiŽcant differences between rosemary, oregano, propyl pholipidliposomes undergo rapid nonenzymatic peroxida- gallate,and annatto extracts at 5%concentration,the results showing90, 82, 80, and 79% inhibition, respectively. tionwhen incubated with FeCl 3 andascorbic acid at pH 7.4 (2). At lowconcentrations, ascorbate accelerates lipid Again,rosemary and oregano exhibited higher antioxidant peroxidationthrough its ability to reduce iron into the ac- activitythan propyl gallate, while the rest of the spices tiveferrous state, while, at high concentrations, ascorbic showedlower efŽ cacy. inhibitslipid oxidation by inactivating free radicals (17). Whenthe inhibition of lipid peroxidation by commer- Table1 showsthe inhibition of lipid peroxidation in cialfood additives, BHA, BHT,andpropyl gallate (100 g/ thepresence of some Mediterranean food spices and com- g)was analyzedand compared with cumin, hot paprika, monfood additives. Rosemary, oregano, and propyl gallate andsweet paprika (5% extracts), the resulting percent in- were themost effective scavengers of peroxyl radicals at hibitionwas 70,64, 58, 64, 61, and 48%, respectively. Saf- thesame concentration (5%) ( P , 0.05).Another very fronextract showed the lowest antioxidant activity (42% goodantioxidant was annatto,which showed a higher% lipidperoxidation inhibition). BHT showeda considerable J.FoodProt., Vol. 64, No. 9 ANTIOXIDANT PROPERTIESOF SPICES 1415 increasein inhibitoryactivity when analyzed in ethanolme- TABLE 2. Deoxyribosedamage by OH· radical in the presence dium. ofMediterranean food spices compared with the activity of dif- a Theantioxidant activity shown by oregano and rose- ferentcompounds frequently used as food additives maryin this study was asexpected according to previous Damage todeoxyribose c reports (10). Theseauthors analyzed a spicecocktail that (A532) includedoregano, rosemary, thyme, and sage and conclud- Addedto reaction mixturesb RM 1 DRd %inhibitionOmit ASC d edthat the plant extracts were capableof minimizing li- posomallipid peroxidation. None(control) 2.466 —g 0.855 Rosemaryreportedly reduces lipid peroxidation in Annattoe 1.118 54.66 0.530 meatproducts (23), invegetable oil, and Ž shoil (21), and Cumine 0.828 66.42 0.299 itsantioxidant effects have also been in peanut oil (16). Oreganoe 1.712 30.58 1.687 e Similarstudies with oregano have found that when added Paprika,hot 1.218 50.61 0.626 Paprika,sweet e 1.519 38.40 1.152 topotato chips (27), Frenchdressing, and mayonnaise, it Rosemarye 1.603 35.00 1.511 Downloaded from http://meridian.allenpress.com/jfp/article-pdf/64/9/1412/1674653/0362-028x-64_9_1412.pdf by guest on 28 September 2021 retardsoxidative deterioration (30). Onthe other hand, Ab- Saffrone 1.570 36.33 1.679 dallaand Roozen (1) observedthat the antioxidant activity Propylgallate e 2.723 —g 2.767 oforeganoextracts was moreeffective in sunower oilthan Propylgallate f 2.902 —g 1.867 ina 20%oil-in-water emulsion. BHAf 1.839 25.42 0.201 Also,the results obtained with sweet and hot paprika BHTf 2.245 8.95 0.559 were inaccordance with those of Aguirreza ´balet al. (3), a Statisticaldifferences were analyzed by analysis of variance ( P who,by measuring the TBA reactivesubstances content, , 0.05). establishedthat these spices are effective in inhibiting lipid b Compoundsin aqueous medium. c oxidationin dry sausages. Matsufuji et al. (33) studied the A532,absorbancevalues recorded at 532 nm; when deoxyribose antioxidantability of paprikacomponents by measuring the wasomitted the values ranged from 0.001 to 0.006 absorbance free-radicaloxidation of methyl linoleate and found effec- units. tiveantioxidant activity. Other authors have detected good d RM,reactionmixtures; DR, deoxyribose;ASC, ascorbate. e levelsof antioxidant vitamins like ascorbic acid and to- Concentration5%. f copherolsin paprika (31). Concentration100 g/g. g No% inhibitiondetected. Assessingthe antioxidantaction of Mediterranean foodspices by the deoxyriboseassay. Thedeoxyribose asynergisticeffect with ascorbate and stimulates deoxyri- methodevaluates the ability to damage carbohydrates. Hy- bosedegradation. droxylradicals damage sugar deoxyribose. Highly reactive Madsenet al. (29), usingan electron spin resonance hydroxylradicals (OH )aregenerated by a mixtureof spintrapping method, have shown that phenolic-rich ex- ascorbateand FeCl 3-EDTA atpH 7.4 (2). Thedeoxyribose tracts,such as oregano and dittany (a memberof the La- isbroken down into fragments and, on heating with TBA biataefamily like rosemary and oregano), are able to scav- atlow pH, generates a pinkchromogen. The addition of engethe hydroxyl radicals generated in theFenton reaction. ascorbicacid greatly increases the rate of OH generation Table2 alsoshows that when ascorbate was omitted, byreducing iron and maintaining the supply of Fe 21 (4). oregano,saffron, rosemary, and sweet paprika ( P , 0.05) Table2 showsthe results of the deoxyribose damage exhibitedprooxidant activity because the level of the pink by OH radical,in thepresence of Mediterranean food spic- chromogenexceeded that of the control. These spices esandcommon food additives, studied in aqueousmedium. showedpoor antioxidant activity in the full assay (RM 1 Theresults are expressed as a percentinhibition of deoxy- DR), probablybecause they do not act as hydroxyl scav- riboseattack, where 100% attack is deŽ ned as the absor- engersbut react with ascorbate to decrease the amount of bancelevels resulting for deoxyribosewithout the addition OH generated (35). Inthis sense, other investigators ob- ofthe tested compounds (control). servedthat extracts from Cassia tora L.(Chineseherb Jue- Thisattack is partially inhibited in the presence of the ming-zi)inhibited the oxidation of deoxyriboseinduced by 31 compoundstested. Cumin exhibited the strongest protective Fe -EDTA/H2O2/ascorbicacid but acted as prooxidant in action(more than60% inhibition) ( P , 0.05),while the theabsence of ascorbate (50). restof the spices exhibited decreasing degrees of protec- Thisassay was onlydeveloped with the compounds tion:annatto and hot paprika more than 50% inhibition ( P dissolvedin water because organic solvents, such as etha- , 0.05);sweet paprika, saffron, rosemary, and oregano nol,may themselves act as good scavengers of OH (5). about30 to 40% inhibition. There were nosigniŽ cant dif- Hydrogenperoxide scavenging. Thegeneration of ferencesbetween oregano, rosemary, and saffron inhibition hydrogenperoxide by activated phagocytes is known to (P , 0.05). playan important role in the killing of severalbacterial and TheseŽ ndingsshow that the spices scavenge OH rad- fungalstrains. Additionally, hydrogen peroxide is generated icalsand protect deoxyribose better than BHA andBHT . invivo by several oxidase enzymes (24). Thereis increas- Theresults also show the prooxidant effect of propyl gal- ingevidence that H 2O2, eitherdirectly or indirectly via its late.According to Aruomaet al. (9), propylgallate exhibits reductionproduct OH canact as a messengermolecule in 1416 MARTI´NEZ-TOME´ ET AL. J.FoodProt., Vol. 64, No. 9

TABLE 3. Scavengingof hydrogen peroxide by different Medi- TABLE 4. Scavengingof HOCl byMediterranean food spices terraneanfood spices compared with the activity of different com- usingthe elastase assay compared with the activity of different poundsfrequently used as food additives using peroxidase-based compoundsfrequently used as food additives a a assays b Addedto reaction mixtures Absorbance( A410 nm) b Addedto reaction mixtures Absorbance( A436 nm) None(control) 0.802 None(control) 0.723 Annattoc 0.130 Annattoc 0.401 Cuminc 0.253 Cuminc 0.227 Oreganoc 0.052 Oreganoc 0.305 Paprika,hot c 0.187 Paprika,hot c 0.529 Paprika,sweet c 0.185 Paprika,sweet c 0.614 Rosemaryc 0.020 Rosemaryc 0.478 Saffronc 0.214 Saffronc,d —f Propylgallate c 0.084 Downloaded from http://meridian.allenpress.com/jfp/article-pdf/64/9/1412/1674653/0362-028x-64_9_1412.pdf by guest on 28 September 2021 Propylgallate c 0.073 Propylgallate d 0.116 Propylgallate e 0.705 BHAd 1.202 BHAe 0.896 BHTd 1.016 BHTe 0.814 a Statisticaldifferences were analyzed by analysis of variance ( P a Statisticaldifferences were analyzed by analysis of variance ( P , 0.05). , 0.05). b Compoundsin aqueous medium. b Compoundsin aqueous medium. c Concentration5%. c Concentration5%. d Concentration100 g/g. d Thiscompound shows intense color ,from390 to 490 nm, that interferedwith the hydrogen peroxide generated signal. e Concentration100 g/g. spicesto show inhibition (48 and 24% inhibition, respec- f No% inhibitiondetected. tively),though both lower than the corresponding results in theaqueous medium described above. Thesespices show high levels of phenoliccompounds thesynthesis and activation of several in ammatory me- (25) thatcan react with hydrogen peroxide and act as sub- diators (47). stratesfor peroxidases (7). Thescavenging of hydrogenperoxide activity is easily andsensitively measured by using peroxidase-based assay Scavengingof HOCl. HOCl isproduced by the neu- systems,when one looks for adecreasein the absorption trophil-derivedenzyme, myeloperoxidase, at in ammation spectrumafter the compound is added to peroxidase-H 2O2 sitesand when activated neutrophils inŽ ltrate reoxygenated mixtures. tissues.One of themost important targets attacked by HOCl Table3 showsthe scavenging of hydrogenperoxide by in vivo is 1-antiproteinase,the major circulating inhibitor theMediterranean food spices and food additives studied. ofproteolytic enzymes such as elastase (7). Thus, a good Inaqueous medium, the spice extracts show lower an- testfor physiologicallyrelevant HOCl scavengingactivity tioxidantactivity ( P , 0.05)than propyl gallate (5%). bya compoundis to analyze whether that compound, at Cuminand oregano, with 69 and 58% inhibition, respec- theconcentrations achieved in vivo, can protect 1-antipro- tively,did not show signiŽ cant differences ( P , 0.05) and teinaseagainst inactivation by HOCl (37). exhibitedhigher antioxidant activity than annatto and rose- InT able4 thescavenging of HOCl byMediterranean mary,with 44 and34% inhibition, respectively, hot paprika foodspices is compared with the capacity of differentcom- andsweet paprika showed a moderateinhibitory effect of poundsfrequently used as food additives. After incubation about20% inhibition at this concentration. On the other of HOCl with 1-antiproteinase,which is very rapidly in- hand,the intensive color of saffron extract in aqueous me- activatedby HOCl, 1-antiproteinaseloses its elastase-in- diuminterfered with the scavenging activity signal of hy- hibitorycapacity. drogenperoxide. BHA andBHT didnot react with H 2O2 Hence,when compounds are dissolved in aqueousme- andmust be considered inefŽ cient. dium,spices are able to scavenge HOCl andprotect 1- Whendissolved in ethanol medium, annatto, sweet and antiproteinase.Our resultsindicate that rosemary and oreg- hotpaprika, and saffron extracts showed greater absorbance anoextracts were moreeffective HOCl scavengers( P , thanthe control, perhaps because interfering materials have 0.05)(97 and 93%, respectively) than the rest of samples maximumabsorption wavelengths near 410 nm. Propyl gal- analyzed,which, in decreasing order ,were propylgallate, late(5%), which exhibited similar results to thoseobtained annatto,sweet and hot paprika, saffron, and cumin (90, 84, inaqueous medium, showed signiŽ cant differences ( P , 77,77, 73, and 68% inhibition, respectively) ( P , 0.05). 0.05)from therest of the spices tested. However ,propyl Theantioxidant activity exhibited by propylgallate did not gallatedid not show this scavenging capacity at the per- varysigniŽ catly ( P , 0.05)when comparing the concen- mittedconcentration in foods (100 g/g).BHA andBHT trationsused in thisassay, 100 g/g,whichis thepermitted dissolvedin ethanolmedium are inefŽ cient catalysts of any commercialconcentration (19) andat 5%, i.e., at the same subsequentoxidation because they did not react with H 2O2. concentrationas thespices. This last concentration was too Oreganoand rosemary ethanol extracts were theonly two highand it guaranteesthe importance of theeffect produced J.FoodProt., Vol. 64, No. 9 ANTIOXIDANT PROPERTIESOF SPICES 1417

centration(100 g/goil)by European law for BHA, BHT, andpropyl gallate (20). Propylgallate was asigniŽcantly ( P , 0.05)more ef- fectiveantioxidant than all the other compounds tested. Amongthe spices tested, rosemary conferred a signiŽcantly (P , 0.05)greater protection, followed by annatto. The differencebetween oregano and sweet paprika was too smallto be signiŽ cant statistically, although the level of protectionwas higherthan that obtained with the widely usedBHA andBHT .Gordonand Kourimska ´ (22), who usedthe Rancimat test, observed that BHA andBHT had noantioxidant activity, probably re ecting the volatility of theseadditives. After 2months’storage with or without added spices Downloaded from http://meridian.allenpress.com/jfp/article-pdf/64/9/1412/1674653/0362-028x-64_9_1412.pdf by guest on 28 September 2021 orfood additives, oil rancidity was evaluatedin order to ascertaintheir effectiveness in retardingthe development of rancidity.The short induction time of the control olive oil FIGURE 1. Oxidativestability during storage at room tempera- sample(7.9 h) isapparentlydue to theincreased formation turefor Mediterranean food spices, compared with common food ofchain-initiating radical forms (10), whilelonger induc- additives.BHA, butylatedhydroxyanisole; BHT, butylatedhy- tionperiods point to the strong activity of the added anti- droxytoluene;P, paprika;PG, propylgallate. oxidants (42). Allthe compounds tested showed antioxidant activity(Fig. 1) in the following decreasing order: propyl gallate,rosemary, hot paprika, cumin, annatto, oregano, byrosemary and oregano and their traditional uses as me- sweetpaprika, and saffron. Propyl gallate exhibited a much dicinalherbs (23,39, 46). BHAandBHT were unableto longerinduction period despite its much lower concentra- scavengeHOCl inaqueous medium. tion.After 2months’maceration, all the tested spices Whenthe compounds were dissolvedin ethanol me- showeda higherantioxidant index than after 72 h macer- dium,saffron and cumin extracts exhibited stronger anti- ation. oxidantactivity than in aqueousmedium, with 90 and72% After 4months’maceration, the Rancimat test revealed inhibition.The rest of the spices, and propyl gallate, thatprolonged storage increases the susceptibility of reŽ ned showedlower antioxidant activity than in aqueousmedium, oliveoil to oxidation (5.8 h). Figure 1 showsthe increased asdescribed above. In decreasing order ,propylgallate oxidativestability of reŽned olive oil macerated with spices (5%),annatto, propyl gallate (100 g/g),hot paprika, sweet comparedwith the results for 2months.Also at this time paprikaand oregano, exhibited 81, 72, 62, 61, 59, and 54% (4months) rosemary was seento have increased its pro- inhibition,respectively. Finally, BHA andBHT showedthe tectionfactor more than the other spices ( P , 0.05). lowestantioxidant activity in ethanolmedium with percents After 6months’maceration, the olive oil exhibited a inhibitionof 51 and 48%, respectively. shorterinduction time (4.7 h), and the antioxidant activity Accordingto Aruoma et al. (9), propylgallate effec- ofthe tested compounds were propylgallate rosemary paprika hot oregano annatto cumin paprika tivelyprotects 1-antiproteinaseactivity against HOCl. Othercompounds, too, have been considered as scavengers sweet saffron BHT BHA(Fig.1), with rosemary ofHOCl,including synthetic antioxidants like BHT (inor- showinga similarstabilizing effect to that obtained after 4 ganicsolvent) (32). months.However ,annatto,oregano, and saffron showed higherstabilizing effects than after 4 months. Rancimatresults. ReŽned olive oil alone (control) Theincreased induction time after more prolonged startsthe radical chain reactions of the propagation phase macerationwith the spices (2, 4, and 6 months)can be ofautoxidation after 8.9 h. The time required for thefor- explainedby atime-dependentincrease in the extraction of mationof a sufŽcient concentration of initiating radicals activecomponents from thespices (10). (initiationphase) was slightlygreater when the spices or Theseresults are in agreement with those obtained by foodadditives were added,delaying the time of onset of Schwarzet al. (42) for samplesof rosemary,using the Ran- thepropagation phase of the radical chain reaction. cimattest. Also, Aruoma and Halliwell (6) andAruoma et Figure1 showsthe effect of the food spices and ad- al. (10), againworking with Rancimat, reported good an- ditiveson the oxidative stability of reŽ ned olive oil tested tioxidantactivity for oreganoand rosemary in different bythe Rancimat method during storage at room tempera- typesof fats. These compounds can act as chain-breaking ture.After 72h, the following order of stability was ob- antioxidantsand can react with peroxyl radicals, introduc- tainedas a resultof the addition of the different samples: inga lagperiod into the peroxidation process that corre- propylgallate rosemary annatto oregano sweet spondswith the time taken for theantioxidant to be con- paprika hot paprika cumin saffron BHA BHT. sumed. Allthe spices were usedat 5% concentration, while the Inconclusion, the above results indicate that the Med- foodadditives were testedat thepermitted commercial con- iterraneanspices, like annatto, cumin, oregano, sweet and 1418 MARTI´NEZ-TOME´ ET AL. J.FoodProt., Vol. 64, No. 9 hotpaprika, rosemary, and saffron, that are traditionally M.Roberfroid.1999. ScientiŽ c conceptsof functional foods in Eu- usedfor theiraromatic properties in the preparation of rope:consensus document. Br .J.Nutr.81:S1. 19.FAO/ WHO.1992. Codex alimentarius. Rome, Italy. Mediterraneanfood, exhibit good antioxidant activity as 20.Frankel, E. N.1993.In search ofbettermethods to evaluate natural scavengersof several reactive oxygen species. This fact antioxidantsand oxidative stability in foodslipids. Trends Food Sci. supportsthe replacement of the synthetic antioxidant by Technol.4:220 –225. suchnatural spice extracts that because of theircontribution 21.Frankel, E. N.,and S. W.Huang.1996. 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