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Journal of Cereal Science 56 (2012) 214e222

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Journal of Cereal Science

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Dissection of antioxidant activity of durum wheat (Triticum durum Desf.) grainsas evaluated by the new LOX/RNO method

Maura Nicoletta Laus a,b, Damiana Tozzi a,b, Mario Soccio a,b, Alessandra Fratianni c, Gianfranco Panfili c,Donato Pastore a,b,*

aDipartimento di Scienze Agro-ambientali, Chimica e Difesa Vegetale, Facoltà di Agraria, Università degli Studi di Foggia, Via Napoli 25, 71122 Foggia, ItalybCentro di Ricerca Interdipartimentale BIOAGROMED, Università degli Studi di Foggia, Via Napoli 52, 71122 Foggia, ItalycDipartimento di Scienze e Tecnologie Agro-alimentari, Ambientali e Microbiologiche, Facoltà di Agraria, Università degli Studi del Molise, Via de Sanctis, 86100 Campobasso, Italy

a r t i c l e i n f o

Article history:Received 31 August 2011Received in revised form30 January 2012Accepted 29 March 2012

Keywords:Antioxidant activityDurum wheat grainsLOX/RNO methodSynergism

Abbreviations: AA, antioxidant activity; AAPH, 2, 2ABTS, 2, 20-azino-bis-(3-ethylbenzothiazoline-6-sulfocurve; BSA, bovine serum albumin; DMPD, 4-amino-N2-diphenyl-1-picrylhydrazyl; d.w., dry weight; EU,substrate transformed/min); fluorescein, 30 , 60-dih[3H], 90[9H]-xanthen]-3-one; LOX, lipoxygenase (linolEC 1.13.11.12); ORAC, Oxygen Radical Absorbance Cadimethylaniline; TEAC, Trolox Equivalent Antioxidanradical Scavenging Capacity; Trolox, (�)-6-hydroxy-2,2-carboxylic acid.* Corresponding author. Dipartimento di Scienz

e Difesa Vegetale, Facoltà di Agraria, Università degli S71122 Foggia, Italy. Tel.: þ39 (0) 881589427/32; fax:

E-mail addresses: m.laus@unifg.it (M.N. Laus),m.soccio@unifg.it (M. Soccio), fratianni@unimol.it (A(G. Panfili), d.pastore@unifg.it (D. Pastore).

0733-5210/$ e see front matter � 2012 Elsevier Ltd.doi:10.1016/j.jcs.2012.03.003

a b s t r a c t

Antioxidant activity (AA) of durum wheat (Triticum durum Desf.) grains was studied using the innovativeLOX/RNO method, able to simultaneously detect different antioxidant mechanisms, and the TEAC assay,one of the most widely used assays. Insoluble-bound and free-soluble phenols, hydrophilic and lipophiliccompounds were extracted from eight different whole flour samples; extracts were analyzed for AA andtheir content in several antioxidants. The LOX/RNO method measured very high AA values, with thehighest ones [850e1500 mmol Trolox eq./g whole flour (dry weight)] for insoluble-bound phenolicextracts, highly correlated to total phenolic (r ¼ 0.761, P < 0.001) and ferulic acid (r ¼ 0.816, P < 0.001)contents. Hydrophilic and lipophilic extracts showed lower AA [70-140 and 40e60 mmol Trolox eq./g (dryweight), respectively], highly correlated to flavonoid (r ¼ 0.583, P < 0.01) and protein (r ¼ 0.602,P < 0.01), as well as b-tocotrienol (r ¼ 0.684, P < 0.05) contents, respectively. Interestingly, the LOX/RNOmethod suggests that insoluble-bound phenolic compounds may exert very strong synergistic interac-tions within the extract. Contrarily, the TEAC assay did not correlate to any antioxidant content, resultedunable to highlight differences among samples, measured much lower AA values and did not suggestsynergism. The use of the LOX/RNO method is useful to unearth new properties of phytochemicals fromdurum wheat grains, potentially giving health benefits.

� 2012 Elsevier Ltd. All rights reserved.

0-azobis(2-amidinopropane);nic acid); AUC, area under, N-dimethylaniline; DPPH, 2,Enzymatic Units (mmol of

ydroxyspiro[isobenzofuran-1eate: oxygen oxidoreductase,pacity; RNO, 4-nitroso-N, N-t Capacity; TOSC, Total Oxy-5, 7, 8-tetramethylchromane-

e Agro-ambientali, Chimicatudi di Foggia, Via Napoli 25,þ39 (0) 881587108.d.tozzi@unifg.it (D. Tozzi),

. Fratianni), panfili@unimol.it

All rights reserved.

1. Introduction

Wheat is one of the major cereal crops in the temperate zonesand represents an important component of the human diet becauseof the universal use of its grains for production of flour andsemolina, the basic ingredients of bread and other bakery productsand pasta (Adom et al., 2003 and refs. therein). Besides starch,proteins, dietary fiber and minerals, whole wheat grains are knownfor their unique healthy value due to their high and peculiarcontent in non-nutrient biologically active compounds, known asphytochemicals. They include awide variety of both water- and fat-soluble compounds: phenolic acids (belonging to the benzoic andcinnamic acid families), flavonoids, anthocyanidins, lignans, carot-enoids, tocotrienols, tocopherols, phytosterols (Liu, 2007). Antiox-idant properties of whole grain phytochemicals have beensuggested to be strongly related to health-beneficial properties ofhigh whole grain consumption, such as reduced total mortality andreduced incidence of degenerative diseases (Liu, 2007 and refs.therein). Therefore, current interest in health benefits of whole

M.N. Laus et al. / Journal of Cereal Science 56 (2012) 214e222 215

grain dietary intake has promoted many studies aimed at evalu-ating antioxidant composition of wheat grains with particularattention given to the effect of genotype, growing environment andmilling process (Adom et al., 2003; Fratianni et al., 2005; Heimleret al., 2010; Liyana-Pathirana and Shahidi, 2007a, 2007b and refs.therein; Moore et al., 2006 and refs. therein; Mpofu et al., 2006;Panfili et al., 2003, 2004; Yu et al., 2002).

Generally, a central point in these kind of studies is the difficultyto relate analytical determinations of individual dietary antioxi-dants to the true whole antioxidant activity (AA) in food, since thecomplex mixture of phytochemicals acts through a combination ofadditive and/or synergistic effects (Liu, 2007 and refs. therein).Therefore, the direct determination of AA of food samples hasgained an increasing interest as a tool in exploring the putative roleof antioxidant-rich products in enhancing human health (Serafiniet al., 2002 and refs. therein). Moreover, the more the AA assess-ment is able to highlight synergistic interactions of dietary anti-oxidants, the more biologically relevant information about thehealth promoting potential of foods may be provided.

In this regard, we have recently developed an innovative andadvanced assay for in vitro AA assessment of food extracts, thelipoxygenase/4-nitroso-N,N-dimethylaniline (LOX/RNO) method(Pastore et al., 2009). It uses the RNO bleaching reaction due tosome radical species, namely alkoxyl, peroxyl and hydroxyl radi-cals, as well as, in the presence of imidazole, singlet oxygen. Thesereactive species are generated by anaerobic reactions catalyzed bysoybean LOX-1 isoenzyme, occurring when the main reaction ofpolyunsaturated fatty acid hydroperoxidation has consumedoxygen (Pastore et al., 2000a). With respect to the majority of AAassays, the peculiarity of the LOX/RNOmethod is to simultaneouslydetect the food scavenging capacity towards some radical specieshaving relevant cellular significance, by means of a simple andrapid experimental protocol, as well as under experimentalconditions resembling the physiological ones. Moreover, the newmethod may also highlight other important mechanisms of dietaryantioxidant protection, including activity of both metal chelationand reduction (because an oxidized iron ion is essential for LOXcatalysis), as well as direct inhibition of LOX apo-enzymeaccounting for an antiperoxidative activity. In the light of this, theLOX/RNO method may provide a more integrated and compre-hensive information about AA of food extracts. Above all, it hasbeen proposed that the new method may highlight, better thanother well-known AA assays, the synergistic effects of complexmixtures containing different antioxidant compounds (Pastoreet al., 2009).

In this study, the new LOX/RNO method has been used to focusAA of durum wheat (Triticum durum Desf.) grains, since, to date,only few data concerning this species are available (Adom et al.,2003; Heimler et al., 2010; Liyana-Pathirana and Shahidi, 2007a,2007b). In these studies, AA of grains from different varieties andyears of production (Adom et al., 2003; Heimler et al., 2010) and ofdifferent milling fractions (Liyana-Pathirana and Shahidi, 2007a,2007b) was evaluated, by using assays able to measure each timeone possiblemechanism of antioxidant protection against oxidativedamage: mainly a scavenging activity or a reducing capacitytowards single radical species or a metal chelating activity. By usingthese assays it may be more difficult to highlight synergisticinteractions among phytochemicals. Moreover, some of theseassays used radical species and experimental conditions that arestrongly non-physiological; above all, these assays often measuredAA values not clearly related to any antioxidant compound(Heimler et al., 2010). The availability of the LOX/RNO method, ableto give useful information from a physiological point of view, mayunearth new properties of phytochemicals from durum wheatgrains, potentially related to health benefits.

In this study, the performances of the LOX/RNO method werecompared with that of the widely used Trolox Equivalent Antioxi-dant Capacity (TEAC) assay (Re et al., 1999), that measures thecapacity of antioxidants to induce the bleaching of the non-physiological 2,20-azino-bis-(3-ethylbenzothiazoline-6-sulfonicacid) (ABTS) radical cation. This assay is generally classified asbased on direct reduction via single electron transfer (SET) reaction,however, neutralization of ABTS�þ radical cation by radicalquenching via hydrogen atom transfer (HAT) reaction has been alsoreported (Prior et al., 2005 and refs. therein). Hydrophilic,insoluble-bound and free-soluble phenolic, as well as lipophilicextracts, were prepared from eight different whole wheat floursamples and then analyzed for AA and for their content in severalantioxidant compounds. An investigation of possible synergisticinteractions among insoluble-bound phenols, the most activeantioxidant compounds in cereal whole grains (Adom and Liu,2002; Liu, 2007 and refs. therein), was also carried out by meansof the LOX/RNO method. As a comparison, different methods wereused: the TEAC assay, as well as the Oxygen Radical AbsorbanceCapacity (ORAC) protocol (Ou et al., 2001) and the 4-amino-N,N-dimethylaniline (DMPD)-based method (Fogliano et al., 1999),measuring the chain-breaking antioxidant capacity against peroxylradicals and the capability of antioxidants to induce the bleachingof the DMPD�þ radical cation, respectively.

2. Material and methods

2.1. Chemicals

All reagents at the highest commercially available purity werepurchased from SIGMA Chemical Co. (St. Louis, MO, USA). RNO wasdissolved in 80 mM sodium borate buffer pH 9.00; catechin, gallicacid, bovine serum albumin (BSA), ABTS, potassium persulfate,DMPD and ferric chloride were dissolved in deionized water; 30,60-dihydroxyspiro[isobenzofuran-1[3H], 90[9H]-xanthen]-3-one(fluorescein) and 2,20-azobis(2-amidinopropane) (AAPH) in75 mM sodium phosphate buffer pH 7.40. Ferulic acid and Trolox[(�)-6-hydroxy-2,5,7,8-tetramethylchromane-2-carboxylic acid]were dissolved in different media depending on the AA assay used.An ammonium sulfate suspension of soybean LOX type V (LOX-1isoenzyme) was used, properly diluted with 80 mM sodium boratebuffer pH 9.00.

2.2. Plant material

Plant material consisted of eight different durum wheat (T.durum Desf.) whole grain samples, kindly provided by Prof. Z.Flagella (University of Foggia), deriving from an agronomic trial, inwhich two cultivars (Simeto ¼ S and Ofanto ¼ O) were subjected totwo levels of sulfur fertilization (0 and 33 kg/ha) and two levels ofwatering [irrigated¼ I (two irrigation treatments of 720m3/ha) andnon-irrigated ¼ NI]. Samples were indicated by Arabic numbers asfollows: 1 ¼ S0I; 2 ¼ S33I; 3 ¼ S0NI; 4 ¼ S33NI; 5 ¼ O0I; 6 ¼ O33I;7 ¼ O0NI; 8 ¼ O33NI.

Whole grain samples were stored as vacuum-packaged at 4 �Cfor no longer than 3 months; before use whole grains were dailymilled by means of a Cyclotec 1093 Sample Mill (1 mm sieve).

2.3. Preparation of aqueous solutions of linoleate

The sodium linoleate solution was prepared as described inPastore et al. (2009) and the exact linoleate concentration wasdetermined bymeans of the LOX assay (Pastore et al., 2000a, 2000band refs. therein), by using a PerkineElmer l45 UVeVis Spectro-photometer (PerkineElmer, Wellesley, MA).

M.N. Laus et al. / Journal of Cereal Science 56 (2012) 214e222216

2.4. Extraction of hydrophilic, phenolic and lipophilic compoundsfrom daily milled whole flour

2.4.1. Hydrophilic extractsHydrophilic extracts from whole grain flour were prepared as

already described in Pastore et al. (2009), by suspending thesamples in deionized water at a (w/v) ratio equal to 1 g/3 ml. Thesuspensionwas placed in an ice-water bath for 1 h, stirred 1 min at15 min intervals, and then centrifuged twice at 18700 � g for20 min at 4 �C. The final supernatant represents the hydrophilicextract.

2.4.1.1. Determination of endogenous LOX activity in hydrophilicextracts. The occurrence of an endogenous LOX activity in hydro-philic extracts was checked by measuring linoleate hydro-peroxidation activity of the extract as reported in Pastore et al.(2000b, 2009). Briefly, hydroperoxide generation was spectropho-tometrically determined at 25 �C by monitoring absorbanceincrease at 234 nm, in an assay mixture (2 ml) containing 80 mMsodium borate buffer pH 9.00, 0.4 mM sodium linoleate, 1.5 mlTween 20/mmol linoleate; the reaction was started by adding anappropriate amount of water extract.

2.4.2. Phenolic extractsInsoluble-bound and free-soluble phenolic compounds were

extracted from whole flour samples according to the proceduredescribed in Sosulski et al. (1982), modified as described in Pastoreet al. (2009).

2.4.2.1. Free-soluble phenols. Wholewheat flour (1 g) samples wereextracted twice with 10 ml of 80% (v/v) ethanol for 10 min at roomtemperature and centrifuged at 5000 � g for 10 min at 20 �C. Thesupernatants were collected and combined; the combined super-natants were evaporated under vacuum at 40 �C using a Buchievaporator to remove ethanol and concentrated to approximately2ml; then, theywere diluted to 4mlwithwater, acidified to pH 2e3using 1 M HCl and centrifuged at 5000 � g for 10 min at 20 �C. Theresultant supernatant was extracted twice with n-hexane (at a n-hexane/waterphase ratio equal to1:1) to remove free fatty acids andother lipid contaminants; then, thewater phaseswere collected andcombined and three extractions in ethyl acetate (at an ethyl acetate/water phase ratio equal to 1:1) were carried out. The ethyl acetatefractions were combined and evaporated to dryness under vacuumat 40 �C; the dry residue was reconstituted in 1.5 ml of water.

2.4.2.2. Insoluble-bound phenols. The residue from ethanol extrac-tionwas digested with 20 ml of 2 M NaOH at room temperature for1 h under nitrogen. The resultant hydrolysate was acidified to pH5e6 with acetic acid, centrifuged at 5000 � g for 15 min at 20 �Cand the supernatant was retained; the residue was washed twicewith 10ml of water and centrifuged at 5000� g for 10 min at 20 �C.The combined supernatants were concentrated under vacuum at40 �C to 15 ml, adjusted to pH 2e3 with 6 M HCl, and extractedfirstly with n-hexane and then with ethyl acetate, as describedabove. The ethyl acetate fraction was evaporated to dryness undervacuum at 40 �C and the dry residue was reconstituted in 2 ml ofwater.

2.4.3. Lipophilic extractsLipophilic compounds were extracted from whole wheat flour

according to the procedure described by Fratianni et al. (2005) andPanfili et al. (2003, 2004), modified as in Pastore et al. (2009).Briefly, whole wheat flour (2 g) was saponified with 60% (w/v)KOH under nitrogen at 70 �C for 45 min and then the suspensionwas extracted twice with 15 ml of n-hexane/ethyl acetate (9:1, v/

v). The organic phases were collected, combined and partitionedin two equal volumes that were separately evaporated to drynessunder vacuum at 40 �C. For AA assessment by the LOX/RNOmethod, the first dry residue was reconstituted in 10 ml of 80 mMsodium borate buffer pH 9.00 containing 2 mM sodium linoleateand 1.5 ml Tween 20/mmol linoleate; for AA measurement by theTEAC method, the second residue was reconstituted in 1 ml ofethanol.

2.5. Spectrophotometric determination of total phenolic, flavonoidand protein contents

2.5.1. Total phenolic contentThe phenolic content of phenolic extracts was determined

according to the colorimetric Folin-Ciocalteu method described bySingleton et al. (1999). Briefly, 125 ml of an appropriate dilution ofextracts were mixed with 500 ml of deionized water and 125 ml ofFolin-Ciocalteu reagent. After 6 min, 1.25 ml of a 7.5% (w/v) sodiumcarbonate solution and 1 ml of water were added. The sampleswere mixed and allowed to stand at room temperature for 90 min.The absorbance of the resulting blue solution was measured at760 nm. Phenolic content was determined by means of a propercalibration curve prepared using gallic (or ferulic) acid andexpressed as mmol gallic (or ferulic) acid eq./100 g of whole flour(dry weight, d.w.). A multiplication factor equal to 1.36may be usedto convert data expressed as gallic acid eq. into ferulic acid eq.

2.5.2. Total flavonoid contentFlavonoid content in hydrophilic extracts was determined by

the colorimetric method described by Dewanto et al. (2002).Briefly, 0.25 ml of the extract was mixed with 1.25 ml of deionizedwater and 75 ml of a 5% (w/v) sodium nitrite solution. After 6 min,150 ml of a 10% (w/v) AlCl3$6H2O solutionwas added and allowed tostand for 5 min before 0.5 ml of 1 M NaOH was added. The mixturewas brought to 2.5 ml with deionized water and well mixed. Theabsorbance was measured immediately at 510 nm. Flavonoidcontent was quantified by means of a proper calibration curveprepared using (þ)-catechin standard solution and expressed asmmol catechin eq./100 g (d.w.).

2.5.3. Protein contentProtein content of hydrophilic extracts was determined

according to the method described by Waddel and Hill (1956) withslight modifications. Briefly, 20 ml of water extracts were mixedwith 3.3 mM NaOH (final volume equal to 3 ml). The absorbancewas measured at both 215 and 225 nm and the absorbance differ-ence DA215e225 was calculated. Protein content was determined bymeans of a proper calibration curve prepared using a BSA standardsolution and expressed as mg BSA eq./g (d.w.).

2.6. HPLC analysis of ferulic acid, carotenoid and tocol contents

Ferulic acid content in insoluble-bound phenolic extracts wasquantified by HPLC according to the procedure described by Miglioet al. (2008), modified as described in Pastore et al. (2009). Briefly,20 ml of phenolic extract was analyzed by an HPLC system (Shi-madzu LC10, Japan) equipped with a diode array detector (SPD-M10A Shimadzu, Japan) and a Prodigy column (5 mm ODS3 100A,250 � 4.60 mm; Phenomenex, Torrance, CA) at a flow rate of 1 ml/min. The mobile phase was a mixture of water/formic acid (95:5, v/v) and methanol. Peak identification in the HPLC chromatogram ofthe extracts was obtained by comparing the retention times ofeluted compounds with those of pure standards at 325 nm andquantified on the basis of a calibration curve of standard solutions.

M.N. Laus et al. / Journal of Cereal Science 56 (2012) 214e222 217

Carotenoid, tocotrienol and tocopherol content in lipophilicextracts was quantified by HPLC according to the proceduredescribed by Fratianni et al. (2005) and Panfili et al. (2003, 2004).Samples were analyzed under normal phase conditions, usinga 250 mm � 4.6 mm i.d., 5 mm particle size, Kromasil PhenomenexSi column (Torrance, CA). An HPLC Dionex (Sunnyvale, CA) analyt-ical system consisting of a P680 solvent delivery system and a 20 mlinjector loop (Rheodyne, Cotati) was used. Spectrophotometricdetection for carotenoids (l ¼ 450 nm) and fluorimetric detectionfor tocols (lex ¼ 290 nm, lem ¼ 330 nm) were achieved by meansof UVD170 spectrophotometer and an RF 2000 spectrofluorimeter(Dionex, Sunnyvale), respectively. For tocol determination themobile phase was n-hexane/ethyl acetate/acetic acid (97.3:1.8:0.9,v/v/v) at a flow rate of 1.6 ml/min (Panfili et al., 2003). For carot-enoid determination, the mobile phase was n-hexane/isopropylalcohol (5%) at a flow rate of 1.5 ml/min (Panfili et al., 2004).Compounds were identified by comparison of their retention timeswith those of known available standard solutions and quantified onthe basis of calibration curves of standard solutions. Data werestored and processed by a Dionex Chromeleon Version 6.6 chro-matography system (Sunnyvale, CA).

2.7. Determination of antioxidant activity (AA) of hydrophilic,phenolic and lipophilic extracts from durum wheat whole flour bymeans of the LOX/RNO, TEAC, ORAC and DMPD methods

All determinations were carried out in triplicate and at leastthree different amounts of extract or Trolox concentrations wereanalyzed.

2.7.1. LOX/RNO methodThe LOX/RNO reaction was spectrophotometrically monitored,

as described in Pastore et al. (2000a, 2009), by measuring the RNOabsorbance decrease at 440 nm and at 25 �C in a reaction mixture(2 ml) containing 80 mM sodium borate buffer pH 9.00, 1 mMsodium linoleate, 1.5 ml Tween 20/mmol linoleate and 15 mM RNO.The reaction was started by adding 0.3 Enzymatic Units (EU) ofsoybean LOX-1. The LOX/RNO reaction was carried out both in theabsence (control) and in the presence of the extract (or Trolox asstandard antioxidant, dissolved in sodium borate buffer pH 9.00).The (%) decrease of the rate of RNO bleaching measured in thepresence of the extract (or Trolox) with respect to the control wasused to quantify AA. This was made by means of a doseeresponsecurve derived for Trolox by plotting the (%) decrease of the rate ofRNO bleaching as a function of the standard antioxidantconcentration.

The control was made so that, under experimental conditions ofthe LOX/RNO assay, the small amounts of water extracts usuallytested [20e80 mg (d.w.)] exhibited a negligible endogenous LOXactivity (1e5%) in respect to that of commercial soybean LOX usedin the assay. Moreover, control was made to verify that absorbanceat 440 nm due to carotenoid pigments of lipophilic extracts had noeffect on the LOX/RNO assay: the usually tested amounts of lipo-philic extracts [50e150 mg (d.w.)], showing a carotenoid contentranging from about 0.15 to 0.4 mM,were found to induce an increaseof RNO absorbance at 440 nm not exceeding 5%, so were unable toaffect the measurement of the LOX/RNO reaction rate.

2.7.2. TEAC methodThe TEAC assay reported by Re et al. (1999) was used with slight

modifications. The colored radical cation ABTS�þ was produced byadding 2.45 mM (final concentration) potassium persulfate solu-tion to 7 mM ABTS solution and allowing the mixture to stand inthe dark at 4 �C preferably for 12e16 h, but at least for 6 h. Beforeuse, the ABTS�þ solution was diluted with either 5 mM sodium

phosphate buffer pH 7.40 (for AA determination of both hydrophilicand phenolic extracts) or ethanol (for AA determination of lipo-philic extracts) in order to obtain an absorbance at 734 nm and at25 �C (A734) of 0.70 � 0.20. Then, 1.0 ml of diluted ABTS�þ solutionwas added with the extract (or Trolox as standard antioxidant,dissolved in either sodium phosphate buffer pH 7.40 or ethanol)and an appropriate volume of sodium phosphate buffer pH 7.40 (orethanol) to obtain a final volume of assay mixture equal to 1.1 ml;A734 was measured exactly 5 min (in the case of hydrophilic andphenolic extracts) or 2.5 min (for lipophilic extracts) after theextract (or Trolox) addition. The (%) decrease of A734 measured 5 or2.5 min after the extract (or Trolox) addition with respect to A734 ofthe uninhibited radical cation solution (blank) was calculated andused to quantify AA by means of a proper concentration-responsecurve prepared with Trolox by plotting the (%) decrease of A734 asa function of the standard antioxidant concentration.

2.7.3. ORAC methodThe ORAC protocol described in Ou et al. (2001) was applied

with somemodifications. The assay mixture (2 ml), preincubated at37 �C for 15 min, contained 75 mM sodium phosphate buffer pH7.40 and 6.3 nM fluorescein; the reaction was started by adding38 mM AAPH. Fluorescence intensity decay due to fluoresceinoxidation by peroxyl radicals generated by AAPH thermal decom-position was continuously monitored by using time drive applica-tion (collecting fluorescence data every 0.1 s) at 37 �C at excitationand emission wavelengths of 485 and 515 nm, respectively, bymeans of a Perkin Elmer LS 55 spectrofluorimeter. Measurementswere carried out in the absence (blank) and presence of the sample(either different ferulic acid concentrations or different amounts ofphenolic extract). The sample fluorescence decay curves were firstnormalized to the blank curve by multiplying original data by thefactor fluorescenceblank, t ¼ 0/fluorescencesample, t ¼ 0. Then, the areasunder the curves (AUC), corresponding to the sample (AUCsample)and to the blank (AUCblank), were calculated and the AUCnet wasobtained by subtracting the AUCblank from the AUCsample. Then, theAUCnet was plotted as a function of either the extract amount or theferulic acid concentration.

2.7.4. DMPD methodThe DMPD method described by Fogliano et al. (1999) was used

with minor modifications. Firstly, 100 mM DMPD solution wasdiluted 1:100 with 0.1 M sodium acetate buffer pH 5.25 and thecolored radical cation DMPD�þ was prepared by reacting the DMPDdiluted solution with 0.1 mM (final concentration) ferric chloride;this solution was allowed to stand for 10 min at 4 �C in the darkbefore verifying an absorbance at 505 nm and at 25 �C (A505)ranging from 0.80 to 1.00. Then, 2 ml of radical cation DMPD�þ

solutionwere addedwith phenolic extract (or pure ferulic acid) andan appropriate volume of water to obtain a final volume of assaymixture equal to 2.2. ml; A505 was measured exactly 10 min afterthe extract (or ferulic acid) addition. The (%) decrease of A505measured 10 min after the extract (or ferulic acid) addition withrespect to A505 of the uninhibited radical cation solution (blank)was calculated and plotted as a function of either the extractamount or the ferulic acid concentration.

2.8. Statistical analysis

Regression analysis of data reported in Fig. 1 was obtained bymeans of GRAFIT 5.0 (ERITHACUS) software. Data of Fig. 2 weresubmitted to a “one-factor” analysis of variance (ANOVA) modelusing a completely randomized block design and the mean sepa-ration was tested by the Duncan’s test at 0.01 P levels of signifi-cance. ANOVA, as well as correlation analysis of Table 2, were

Fig. 1. Inhibition of the LOX/RNO reaction by hydrophilic (A), insoluble-boundphenolic (B) and lipophilic (C) extracts from durum wheat whole grains. Extractswere from the sample 6. The numbers alongside the traces represent the rates of RNObleaching. In the insets, the (%) inhibition of the rate vs flour amount is reported.

M.N. Laus et al. / Journal of Cereal Science 56 (2012) 214e222218

performed using the MSTAT-C statistical package (version 2.1, 1991;Crop and Soil Sciences Department, Michigan State University, EastLansing, MI, USA).

3. Results and discussion

In this study, AA of durumwheat grains was evaluated bymeansof the recently developed LOX/RNOmethod (Pastore et al., 2009) incomparison with the widely used TEAC (Re et al., 1999) assay. Forthis purpose, hydrophilic antioxidants, insoluble-bound and free-soluble phenols, as well as lipophilic compounds, extracted fromeight different whole flour samples, were used.

The LOX/RNO reaction was photometrically monitored bycontinuously measuring the RNO absorbance decrease at 440 nm.In Fig. 1, the typical inhibition of the LOX/RNO reaction due toantioxidant compounds present in the hydrophilic extract (A), aswell as in the insoluble-bound phenolic (B) and lipophilic extracts(C), obtained from the whole flour sample 6 (see plant material,2.2), is shown. As reported in Pastore et al. (2009), the LOX/RNOreaction consists of (i) a lag phase, representing the time occurringto consume the oxygen in the reaction mixture due to the primaryLOX-1 reaction of linoleate hydroperoxidation, and (ii) an RNObleaching phase, due to the LOX-1 mediated generation of physi-ologically relevant radical species (mainly alkoxyl, peroxyl andhydroxyl radicals) occurring when anaerobiosis is reached in theassay mixture. Hydrophilic compounds were found to inhibit therate of the RNO bleaching, without significantly affecting the lagphase (Fig. 1A; see also Pastore et al., 2009). Insoluble-boundphenolic compounds also inhibited mainly the reaction rate, soshowing a radical scavenging activity, although an effect on the lagphase was also observed (Fig. 1B). On the other hand, the lipophilicextract induced both a decrease of the reaction rate and an evidentincrease of the lag phase (Fig. 1C). The different behavior of thedifferent classes of antioxidants from durum wheat whole flourwith respect to the lag phase and the RNO bleaching rate stronglysuggests different antioxidant actions of phytochemicals: a mainradical scavenging activity of the hydrophilic compounds anda high antiperoxidative action of the lipophilic ones. This last pointis in agreement with the inhibition of LOXes from different sourcesby pure fat-soluble antioxidants (Pastore et al., 2009 and refs.therein). Interestingly, the phenolic extract showed an interme-diate behavior, so suggesting both types of actions. For each type ofextract, an increasing inhibition of the reaction rate with increasingamounts of extract was observed. In particular, a linear dependenceof the inhibition on the amount of hydrophilic, phenolic or lipo-philic extract was obtained in the range 25e85 mg (d.w.),2.5e10 mg (d.w.) and 50e150 mg (d.w.), respectively (insets ofFig. 1AeC, respectively). On the basis of a proper calibration curveobtained by using Trolox as standard antioxidant, by plotting the(%) decrease of the rate of RNO bleaching as a function of Troloxconcentration, AA values may be calculated from data of Fig. 1. Theywere about 140, 1400 and 60 mmol Trolox eq./g (d.w.), for hydro-philic, phenolic and lipophilic extracts, respectively. To validateresults, the control was made so that under our experimentalconditions (i) each type of extract did not significantly affect theRNO spectral properties, as well as the LOX activity by altering pH ofthe assay medium, (ii) the analyzed amounts of hydrophilic extractdid not show significant endogenous LOX activity, and (iii) thecontent of carotenoids (that show absorbance properties at 440 nmand are co-oxidized by LOX) of the usually tested amounts oflipophilic extract did not affect the measurement of the rate of theLOX/RNO reaction and AA calculation (see Materials and Methods).

In Fig. 2AeC, AA values of hydrophilic, phenolic and lipophilicextracts obtained from the eight different whole grain samples arereported, respectively, measured by using the LOX/RNO protocol. Asa comparison, AAwas also evaluated by means of the TEAC method(Fig. 2A0eC0). The LOX/RNO method measured AA values due towater-soluble antioxidant compounds of wheat grains rangingbetween about 70 and 145 mmol Trolox eq./g (d.w.) (Fig. 2A),28e44-fold higher than those obtained with the TEAC assay [about2.5e3.3 mmol Trolox eq./g (d.w.)] (Fig. 2A0). As regards phenolicextracts, very high AA values were obtained with the LOX/RNOmethod for the insoluble-bound phenolic component of grains,ranging from about 850 to 1500 mmol Trolox eq./g (d.w.) (Fig. 2B).For the same type of extract, the TEAC values [about 5e7 mmolTrolox eq./g (d.w.), Fig. 2B0] resulted about 170e210-fold lower,distributed according to a different ranking list. Since free-soluble

Fig. 2. Antioxidant activity (AA), evaluated by means of the LOX/RNO (A, B, C) and TEAC (A0 , B0 , C0) methods, of hydrophilic (A, A0), phenolic (B, B0) and lipophilic extracts (C, C0) fromdurum wheat whole grains. Different capital letters indicate significant differences at 0.01 P level, according to Duncan’s test. In Fig. 2B, italic letters indicate a separate statisticalanalysis relative to free-soluble phenols.

M.N. Laus et al. / Journal of Cereal Science 56 (2012) 214e222 219

phenols represent minor antioxidant components in cereal grains(Adom and Liu, 2002), they were analyzed only with the LOX/RNOmethod; values obtained ranged from about 25 to 400 mmol Troloxeq./g (d.w.) (Fig. 2B). As regards the lipophilic antioxidant-enrichedextracts, AA values, measured by the LOX/RNO method, rangedbetween about 40 and 60 mmol Trolox eq./g (d.w.) (Fig. 2C), about130e150-fold higher than the ones measured by the TEAC protocol[about 0.3e0.4 mmol Trolox eq./g (d.w.)] (Fig. 2C0). On thewhole, theLOX/RNO method was able to measure very high AA values ofwhole flour extracts, also highlighting significant differencesamong different flour samples and different classes of phyto-chemicals, with the highest AA exerted by the bound phenoliccompounds. This result confirms the phenolic compounds ininsoluble-bound form as the major contributors to AA of wheatgrains (see also Adom and Liu, 2002; Liu, 2007 and refs. therein). Onthe other hand, the TEAC protocol pointed out lower differencesamong grain samples and measured much lower AA values. This

result is in general accordance with TEAC performances, exceptwhen AA measurement of cereal products is carried out accordingto Serpen et al. (2008) by directly mixing cereal flour with ABTS�þ

solution, thus giving higher AA values. The much higher AA valuesobtained with the LOX/RNOmethod with respect to the TEAC assaymay be attributed to the capability of the new method to simul-taneously detect more antioxidant functions, as stated in theIntroduction.

In order to gain a first insight on the antioxidant compoundsresponsible for AA measured by the LOX/RNO, the content inseveral hydrophilic, phenolic and lipophilic antioxidantcompounds of the eight whole flour samples was determined. Theresults are reported in Table 1. The hydrophilic extract from durumwheat whole flour was characterized by a flavonoid concentrationranging from about 16 to 37 mmol catechin eq./100 g (d.w.) anda high protein content [about 20e26 mg BSA eq./g (d.w.)]. Unfor-tunately, proteins interfere with the measurement of phenols by

Table 1Durum wheat whole grain content in several hydrophilic, phenolic and lipophilic compounds.

Compounds Samples

1 2 3 4 5 6 7 8

Hydrophilic extractFlavonoidsa 21.5 � 0.50* 21.0 � 1.70 16.8 � 1.10 20.0 � 0.90 20.8 � 0.40 36.4 � 1.50 23.1 � 0.60 25.0 � 0.40Proteinsb 21.7 � 0.56 23.7 � 0.77 19.3 � 0.59 20.6 � 0.67 20.3 � 3.06 25.9 � 1.12 22.1 � 0.94 22.3 � 0.91Phenolic extractFree-soluble phenolsc 22.9 � 1.40 14.6 � 0.60 74.5 � 4.40 23.9 � 2.40 68.1 � 1.20 11.5 � 0.90 17.6 � 1.00 52.8 � 1.60Insoluble-bound phenolsc 302 � 8 171 � 6 174 � 8 216 � 10 260 � 9 181 � 8 351 � 15 191 � 5Insoluble-bound ferulic acidd 143 � 5 77 � 2 61 � 6 120 � 8 161 � 4 102 � 2 185 � 3 111 � 9Lipophilic extractb-carotenee 14 � 0.3 16 � 0.5 13 � 0.1 13 � 0.6 13 � 0.4 18 � 0.1 14 � 0.2 13 � 0.1Luteine 268 � 9 300 � 9 317 � 2 323 � 13 250 � 5 294 � 3 265 � 5 270 � 25Zeaxanthine 21 � 0.9 24 � 1.6 24 � 0.7 25 � 0.9 19 � 0.6 19 � 0.4 24 � 1.6 20 � 4.0n.d. 1e,f 17 � 1.3 16 � 0.5 17 � 0.6 18 � 0.1 15 � 0.4 15 � 0.3 14 � 0.3 15 � 2.2a-tocopherole 1108 � 15 995 � 33 1074 � 61 1097 � 32 1109 � 49 946 � 42 1053 � 43 975 � 26b-tocopherole 214 � 5 201 � 6 252 � 11 248 � 7 302 � 14 262 � 9 320 � 12 302 � 7a-tocotrienole 575 � 9 535 � 8 573 � 36 632 � 12 505 � 16 542 � 32 556 � 20 518 � 11b-tocotrienole 2725 � 87 2691 � 18 3045 � 179 3196 � 38 3549 � 86 3548 � 191 3768 � 107 3641 � 89

* mean value � SD (n ¼ 3).a Data are expressed as mmol catechin eq./100 g (d.w.).b mg BSA eq./g (d.w.).c mmol gallic acid eq./100 g (d.w.).d mmol/100 g (d.w.).e mg/100 g (d.w.).f n.d. 1 ¼ non identified peak (probable lutein isomer).

M.N. Laus et al. / Journal of Cereal Science 56 (2012) 214e222220

the Folin-Ciocalteu method, so preventing correct quantification oftotal phenolic content in this extract. In accordance with the liter-ature data (Adom and Liu, 2002), insoluble-bound phenoliccompounds from the eight whole flour samples resulted higherthan those of the free-soluble ones. Ferulic acid resulted the mostabundant compound in the insoluble-bound phenolic fraction ofdurum wheat grains (see also Liu, 2007 and refs. therein), rangingfrom 60 to185 mmol/100 g (d.w.). HPLC analysis of lipophilic extractshowed a high carotenoid content, with lutein as the most abun-dant one, followed by zeaxanthin and b-carotene. Tocols resultedthe major lipophilic compounds in durum wheat grains, b-toco-trienol being the most abundant one [about 2700e3800 mg/100 g(d.w.)], in agreement with literature data (see Panfili et al., 2003).

Table 2Correlation analysis of antioxidant activity (AA) of durum wheat grain extractsevaluated by means of the LOX/RNO and TEAC methods and content of antioxidantcompounds.

Correlation coefficient (r)

Hydrophilic extractAALOX/RNO Flavonoid content 0.583**AALOX/RNO Protein content 0.602**AATEAC Flavonoid content �0.504*AATEAC Protein content �0.378n.s.

AALOX/RNO AATEAC �0.219n.s.

Insoluble-bound phenolic extractPhenolic content Ferulic acid content 0.910***AALOX/RNO Phenolic content 0.761*** a

AALOX/RNO Ferulic acid content 0.816***AATEAC Phenolic content 0.663*AATEAC Ferulic acid content 0.491n.s.

AALOX/RNO AATEAC 0.484n.s.

Lipophilic extractAALOX/RNO b-tocotrienol content 0.684*AATEAC b-tocotrienol content 0.435n.s.

AALOX/RNO AATEAC 0.597n.s.

*P � 0.05, **P � 0.01, ***P � 0.001, P represents the probability level.n.s.: not significant.

a when free-soluble phenolic compounds are considered, correlation coefficient is0.509**; when both the insoluble-bound and free-soluble phenolic extracts areconsidered, correlation coefficient is 0.952***.

A goal of this study was to evaluate the relationship between AAmeasured by in vitro assays and the content in antioxidantcompounds fromwhole flour. The correlation analysis of AA valuesof hydrophilic, phenolic and lipophilic extracts of Fig. 2 and thecontent of some antioxidant compounds of Table 1 is reported inTable 2.

As for the hydrophilic extract, a statistically significant positivecorrelation was found between AA values determined by the LOX/RNOmethod and both flavonoid and protein contents, This result isconsistent with the well-known antioxidant properties of flavo-noids and also of proteins: AA of some soy andwheat gluten proteinhydrolysates has been reported, as well as AA of casein, maize zein,and peptides of gelatin, elastin and egg white proteins (Park et al.,2008 and refs. therein). On the contrary, TEAC values were unre-lated to proteins and even inversely related to flavonoid content.

Regarding phenolic extracts, as expected, a statistically highpositive correlation between total phenols and ferulic acid contentwas found. AA of insoluble-bound phenolic extracts measured bythe LOX/RNO method was found to be highly correlated to bothferulic acid content and total phenols. Interestingly, the LOX/RNOmethod gave AA values significantly correlated to phenolic contentalso in the case of free-soluble phenolic extracts (r ¼ 0.509,P < 0.01), so that the correlation between total (bound þ free) AAand total phenols increased to 0.952 (P < 0.001). On the contrary,the TEAC assay showed only a low positive correlation withinsoluble-bound phenols, but it was even unrelated to feruliccontent. The positive correlation between AA values measured bythe LOX RNO method for the bound phenolic component and totalphenolic/ferulic acid contents is in agreement with literature data,in which other methods, such as the Total Oxyradical ScavengingCapacity (TOSC) method (Adom and Liu, 2002; Adom et al., 2003)and the 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavengingactivity assay (Li et al., 2005), were used. Also the correlationrelative to free-soluble phenolic extracts is consistent with resultsof other studies, in which AA assessment of this phenolic compo-nent was performed by using the L-a-phosphatidylcholine lipo-some system (Zieli�nski and Koz1owska, 2000), ORAC protocol(Moore et al., 2006) and ABTS�þ (Moore et al., 2006; Zieli�nski andKoz1owska, 2000) and DPPH� (Moore et al., 2006; Mpofu et al.,

M.N. Laus et al. / Journal of Cereal Science 56 (2012) 214e222 221

2006) scavenging capacity assays. On the other hand, in otherstudies no correlationwas found between free-soluble phenols andAAmeasured by TOSC (Adom and Liu, 2002), DPPH and ABTS-based(Yu et al., 2002) methods. Also, in a recent study properly investi-gating durum wheat grains (Heimler et al., 2010), the DPPH assayresulted unable to correlate AA of free-soluble phenolic extractswith phenolic content (see Introduction). These literature data,together with some results obtained in the present study, suggestthat TOSC, DPPH and ABTS-based assays may be sometimes unfit tocorrelate with phenolic content.

From a methodological point of view, our comparison betweenthe LOX/RNO and the TEAC methods remarkably shows that theformer is able to correlate with antioxidant content, thus appearingmore reliable, under the same experimental conditions in whichthe latter fails. Consistently, correlation was never found betweenAA values measured with the LOX/RNO method and that obtainedwith the TEAC assay (Table 2).

In the case of the lipophilic extracts, AA evaluated by the LOX/RNO method, but not with the TEAC protocol, resulted positivelycorrelated to b-tocotrienol content. This is the first study, to the bestof our knowledge, reporting a significant positive correlationbetween AA and b-tocotrienol content, the major lipophiliccompound in durum wheat grains (Panfili et al., 2003).

Since the regression coefficient may represent a measure of the“strength” of the relationship between AA and antioxidant content,the high correlation coefficients between AA according to the LOX/RNOmethod and flavonoids, proteins, total phenols/ferulic acid andb-tocotrienol strongly suggest a high antioxidant efficiency of theseantioxidant compounds in durum wheat grains.

As insoluble-bound phenolic extract is associated with thehighest AA values, the occurrence of a possible synergistic interac-tion among phenolic compounds was evaluated (Fig. 3). The capa-bility of the LOX/RNO method, compared to the TEAC and ORACassays, to highlight synergism has been recently demonstrated bycomparing the mathematical sum of AA values of three differenttypes (hydrophilic, phenolic and lipophilic) of extracts with AAvalues obtained after mixing the extracts (Pastore et al., 2009).Under the same experimental conditions, the LOX/RNO methodshowed more than doubled activity due to mixing compounds,while ORAC showed lower effectiveness in revealing synergism andTEACwas ineffective. In the same study, the existence of very strongsynergistic interactions among phenols within the same type of

Fig. 3. Evaluation of possible synergism among phenolic compounds from durumwheat whole flour by using the LOX/RNO, ORAC, TEAC and DMPD methods. IC50 valueswere calculated as the concentrations able to induce a 50% inhibition of the LOX/RNOreaction, a 100% increase of AUCnet for ORAC measurements and a 50% absorbancedecrease in the case of the TEAC and DMPD assays. For each method the ratio betweenthe IC50 values of ferulic acid and that of phenolic extract is reported. Data areexpressed as mean value � SD (n ¼ 3). Extract was from the sample 2.

extract was also supposed, but it was evaluated by using only theLOX/RNO reaction. Therefore, in the present paper, the study wastaken up and completed by comparing the response of the LOX/RNOmethod with that of other AA assays. In particular, insoluble-boundphenolic compounds were extracted from a durum wheat wholeflour sample. Then, the inhibition of the LOX/RNO reaction, inducedby the bound phenolic mixture, was measured and compared withthat exerted by a high purity preparation of ferulic acid (the mostabundant phenolic acid as measured in Table 2). In particular,phenolic extract was found to induce a 50% inhibition of the LOX/RNO reaction at a phenolic concentration of 0.022mM (mmol ferulicacid eq./ml), while, on the other hand, the same concentration ofpure ferulic acid preparation did not affect the LOX/RNO reaction. Toobtain a 50% inhibition using pure ferulic acid, a concentration of9 mM was required. So, phenolic mixture resulted much more effi-cient than the single pure phenolic compound in inhibiting the LOX/RNO reaction. Inhibition effectiveness of the extract (IEextract) wasquantified in terms of the ratio between the concentration of ferulicacid able to induce a 50% inhibition of the reaction rate (IC50 ferulic

acid) and that of phenolicmixture (IC50 phenolic extract). In this case, theIC50 ferulic acid/IC50 phenolic extract ratio was 9 mM/0.022 mM ¼ 410,indicating an IEextract about 400-fold higher than the single purecompound. The same comparison was carried out using the ORAC,TEAC andDMPDmethods (Fig. 3). The IC50 ferulic acid/IC50 phenolic extractratios and IEextract values were: 0.42 mM/0.15 mM ¼ 2.8, 4.9 mM/5.5 mM¼ 0.89 and 9.3 mM/104 mM¼ 0.09, respectively. These resultssuggest the capability of the LOX/RNO method to point out a verystrong synergistic effect of phenolic compounds within this extract.A synergistic interaction, althoughwith amuch lower effectiveness,seems to be highlighted also by the ORAC method, the most widelyused assay for assessment of peroxyl radical scavenging capacity.Also in this case, the better performance of the LOX/RNOmethod islikely to be dependent on the capability to simultaneously evaluatedifferent antioxidant functions. The TEAC and DMPD assays do notsuggest synergism, showing IEextract values < 1.

These results also suggest that synergistic effects may stronglylower phenolic concentration able to exert AA in vivo. An inter-esting hypothesis is that the remarkable increase in activity due tosynergy could partly compensate for the very low phenolicbioavailability, roughly ranging from 4 to 0.005%, as can be inferredfrom data reported in Fernandez-Panchon et al. (2008) regardingphenolic concentration values detected in plasma after phenolicdose consumption.

4. Conclusions

The results of this study indicate that the new LOX/RNOmethod isadvisable to carry out analysis of AA of durum wheat whole flourextracts. In particular, this assay, showing the advantage to evaluatedifferent antioxidant mechanisms under more physiological condi-tions than other methods, is able to measure high AA values and toeasily discriminate among samples, also suggesting strong synergisticeffects amongphenols. This last point is of particular interest in light ofthe remarkable contribution of both phenolic compounds and syner-gistic interactions in foods in promoting health.

On the whole, the LOX/RNO method is able to unearth antioxi-dant properties of durumwheat grains, potentially related to healthbenefits; therefore, its use in further studies regarding other cerealspecies and food samples is worthwhile.

Acknowledgements

This work was supported by the Research Project of theMinistryof Agriculture: “Miglioramento delle proprietà igienico-sanitarie,salutistiche e funzionali di commodity per l’alimentazione

M.N. Laus et al. / Journal of Cereal Science 56 (2012) 214e222222

dell’uomo e/o degli animali (ALISAL)”. We gratefully acknowledgethe skilful cooperation of Dr. A. Gagliardi who participated asa student in this work.

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