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Journal of Cereal Science 57 (2013) 162e169

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Journal of Cereal Science

journal homepage: www.elsevier .com/locate/ jcs

Influence of genotype and environment on the content of 5-n alkylresorcinols,total phenols and on the antiradical activity of whole durum wheat grains

Silvia Bellato a,b, Roberto Ciccoritti a,b, Viviana Del Frate a, Daniela Sgrulletta a, Katya Carbone a,*

aConsiglio per la ricerca e la sperimentazione in agricoltura, Via Cassia 176, 00191 Rome, ItalybCampus Bio-Medico Rome-University, Via Alvaro del Portillo 21, 00128 Rome, Italy

a r t i c l e i n f o

Article history:Received 4 April 2012Received in revised form20 November 2012Accepted 21 November 2012

Keywords:Durum wheatTotal soluble phenolic compoundsAlkylresorcinolsDPPH�

Abbreviations: AA, antiradical activity; ANOVA, analkylresorcinols; CTE, catechin equivalents; DM, drypicrylhydrazyl radical; E, environment; G, genotypegrain (on DM basis) required to obtain 50% DPPHcomponent analysis; TMCS, trimethylchlorosilane;content.* Corresponding author. CRA-QCE, Via Cassia 176, 0

3295705; fax: þ39 06 36306022.E-mail address: katya.carbone@entecra.it (K. Carb

0733-5210/$ e see front matter � 2012 Elsevier Ltd.http://dx.doi.org/10.1016/j.jcs.2012.11.003

a b s t r a c t

The 5-n-alkylresorcinol (AR) contents of thirty different cultivars of durum wheat grown in two years(2009 and 2010) in two Italian locations were determined and related to the total soluble phenoliccontent (TPC) and antiradical activity (AA). On average, AR and TPC ranged from 161.3 to 405.8 mg/g (drymatter, DM) and from 19.0 to 192.4 mg/g (DM), respectively. AA (EC50 values) ranged from 70.9 to289.2 mg of dry whole milled wheat grain (DM). The results showed that the environment (E) and thegenotype (G), as well as their interactions (G � E), significantly influenced the phytochemical profiles ofthe samples. The contribution of G � E to the total variance was much lower than that due to the separateeffects (G and E). Principal component analysis identified genotypes that were richer in ARs and morestable across environments. There were significant negative correlations between ARs and TPC (p < 0.05)and between TPC and AA (p < 0.01), but not between ARs and AA. Graphical representation was efficientin summarizing the overall antiradical profiles of the durum wheat grain in each environment.

� 2012 Elsevier Ltd. All rights reserved.

1. Introduction

The demand for natural antioxidants is increasing because of theneed to improve the quality of food and its stability and to the factthat theymay also act as nutraceuticals, providing additional healthbenefits to consumers (Liu, 2007). There is strong epidemiologicalevidence that a regular whole grain intake may protect againstvarious types of chronic diseases. This protective effect could beattributed to the presence of significant amounts of bioactivephytochemicals including antioxidants such as phenoliccompounds (Adom et al., 2003; Liu, 2007). A specific class ofphenolics present in cereal grain is 5-n-alkylresorcinols (ARs),which are a group of phenolic lipids synthesized as secondarymetabolites in the outer parts of grain of some cereals such aswheat, rye, triticale, barley, millet andmaize. From a chemical point

alysis of variance; ARs, 5-n-basis; DPPH�, 1,1-diphenyl-2-; EC50, mg of wheat milledscavenging; PCA, principal

TPC, total soluble phenolic

0191 Rome, Italy. Tel.: þ39 06

one).

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of view, ARs are 1,3-dihydroxybenzene derivatives with an oddnumbered alk(en)yl chain at position 5 of the benzene ring andcarbon chains of different lengths (i.e., C15:0, C17:0, C19:0, C21:0,C25:0) (Andersson et al., 2008). In the plant, they may act asprotective agents against microbes including fungi (Zarnowski andKozubek, 2002). Recent reports have also described their antioxi-dant potential and their ability to prevent oxidation of lipids innatural membranes (Kamal-Eldin et al., 2000; Parikka et al., 2006).Other authors have reported a range of other biological properties(i.e. antimicrobial activity, interaction with proteins, biologicalmembranes and enzymatic activities), which may be associatedwith their amphiphilic structure (Gasiorowski et al., 1996; Kozubekand Demel, 1981; Kozubek and Tyman, 1999; Ross et al., 2004).

In recent years, there has been increasing interest in the rolethat these compounds may have in the development of functionalfoods using wheat-based ingredients (Andersson et al., 2008, 2010;Ross et al., 2003). After rye grain, whose AR content ranges between500 and 1300 mg/g DM (Nystrom et al., 2008; Ross, 2012), wholewheat grain is considered to be one of the main dietary sources ofARs, with contents ranging from 399 (mean value of ten durumwheat varieties) to 605 mg/g DM in five varieties of spelt (Anderssonet al., 2008).

Durumwheat (Triticumturgidum L., ssp.durum) is oneof themostimportant crops in Italy, accounting for about 7e10% of the globalwheat production (FAOSTAT Data, 2006). However, limited

Table 1Environmental conditions at the growing locations.

Location Temperature (�C)a Total rainfall (mm)b

Min Max NoveJun ApreJun

Jesi 2009 7.0 � 4.9 17.7 � 7.1 616 239Jesi 2010 6.7 � 4.7 16.7 � 6.7 702 310Foggia 2009 7.0 � 4.6 17.0 � 7.0 749 180Foggia 2010 6.6 � 4.1 16.5 � 6.1 440 105

a Averages � Standard deviation per location from November (sowing time) toJune (harvest).

b Total amount from November (sowing time) or from Apr (flowering) to June(harvest).

S. Bellato et al. / Journal of Cereal Science 57 (2013) 162e169 163

information is available about its content of potentially beneficialcomponents, such as 5-n-alkylresorcinols (ARs), and their antioxi-dant capacities. Similarly there have been few studies of the effect ofvariety and growth conditions on the concentration and composi-tion of AR in durum wheat. Recently, Menga et al. (2010) reportedthat location had the greatest impact on the total polyphenoliccontent (TPC) of a set of durumwheat varietieswhile Yu et al. (2004)reported significant effects both of variety and growing location onantioxidant properties and TPC of hard winter wheat. Mpofu et al.(2006) observed a greater impact of environment (E) than of geno-type (G) on the antioxidant activity and phenolic content of hardwinter wheat, with the G � E interactions scarcely significant. Theeffects of G and E on theAR content ofwinter and springwheat grainwere recently studied byAndersson et al. (2010) and byShewryet al.(2010). Significant variation was observed among different loca-tions, years andvarieties, suggesting that genetic andenvironmentalfactors could affect AR accumulation.

The aim of the present study was to determine the AR content ofthirty different cultivars of durum wheat grown in two Italianlocations in 2009 and in 2010 and to relate them to total phenolsand antiradical activity. Emphasis is placed on the effects of geno-type, environmental factors and G � E interactions on theseproperties, in order to explore the potential for exploiting thehealth benefits for producing functional foods.

2. Materials and methods

2.1. Chemicals

All reagents were of analytical spectrophotometric grade (CarloErba, Rome, Italy). FolineCiocalteu reagent and all organic solventswere purchased from Carlo Erba (Italy). (þ) catechin, 1,1-diphenyl-2-picrylhydrazyl radical (DPPH�), Fast blue B salt and methylbehenate (C22:0, methyl docosanoate) were purchased fromSigmaeAldrich (Milan, Italy). Trimethylchlorosilane (TMCS), whichwas used to prepare the trimethylsilyl ether derivates of ARextracts, was purchased from Restek (Milan, Italy). Standards 5-n-pentadecylresorcinol (C15:0), 5-n-heptadecylresorcinol (C17:0), 5-n-nonadecylresorcinol (C19:0), 5-n-heneicosylresorcinol (C21:0),5-n-tricosylresorcinol (C23:0) and 5-n-pentacosylresorcinol(C25:0) were purchased from ReseaLife Chem. Science (Burgdorf,Switzerland).

2.2. Wheat samples

Thirty Italian commercial varieties of durumwheat (T. turgidum,L., ssp. durum) was grown in two different geographical areas, Jesiand Foggia located in Central and Southern Italy, respectively, intwo successive years (2008e2009 and 2009e2010). Each varietywas grown in 10 m2 plots in randomized blocks with three repli-cates. The agronomic practices were typical for durum wheatproduction in the selected areas. Table 1 shows the average envi-ronmental conditions for the growing areas, from sowing time toharvest. Other details of the characteristics of the locations arereported previously (Ciccoritti et al., 2011). Nineteen varietiescommon to all environments were considered to determine E, Gand G � E effects.

2.3. Sample pretreatment

Immediately after harvest, combined samples of grain from thethree replicates were milled using a laboratory cyclone mill(Cyclotec 1093, Foss, Italy) to pass a 0.5 mm screen, to producewholemeal flour which was stored at 4 �C. The wholemeal flourswere thoroughly mixed to ensure uniformity and all analyses were

carried out in triplicate on two independent aliquots of eachcomposite sample. All values are reported on a dry matter (DM)basis. Moisture content was determined at 120 �C with a thermo-balance (Sartorius MA 40, Gottingen, Germany).

2.4. Extraction of bioactive compounds

Samples (1 g each) were extracted to determine AR, TPC, and AAcontent. They were placed in 50-mL tubes and extracted with40 mL acetone, for 48 h, by continuous mechanical shaking at roomtemperature. The extracts were then filtered through Whatman n.42 filter paper and evaporated to dryness at 60 �C in a rotaryevaporator (Buchi R-114, Switzerland). The dry residues from all theextractions were then dissolved in pure methanol (1 mL) andimmediately analyzed.

2.5. Determination of 5-n alkylresorcinol (AR) content

AR fractions were analyzed by a rapid colorimetric method basedon the use of Fast Blue B salt, according to Gajda et al. (2008), withoutmodifications. The total AR contentwas calculated from a calibrationcurve, using 5-n-pentadecylresorcinol (C15:0) as standard.

2.6. Determination of total soluble phenolic compounds

The TPC of extracts was determined using the FolineCiocalteu(FeC) method as reported by Moore and Yu (2008). TPC wascalculated from a calibration curve, using (þ) catechin as a stan-dard. Results are expressed as micrograms of catechin equivalents(CTE) per g of whole milled grain (DM).

2.7. Gas chromatographyemass spectrometry (GCeMS) analysis

Total AR content in whole grain extracts were determined byGCeMS, according to Landberg et al. (2009) using methyl behenateas internal standard (IS). AR extracts (10 mL) were dried undernitrogen and silylated with a mixture (400 mL) of pyridine andtrimethylchlorosilane (TMCS; 9:1 v/v). The mixture was thenshaken and heated at 70 �c for 60 min. GCeMS analysis was per-formed with a Perkin Elmer gas chromatograph GC Clarus 600series coupled to a mass spectrometer Clarus 580D (Perkin Elmer,Milan, Italy) equipped with a split/splitless injector, a RTX-5MScolumn (0.25 mm 30 m, 0.25 mm film thickness, Restek, Milan,Italy) and a quadrupole mass spectrometer (Clarus 580D, PerkinElmer, Milan, Italy) operating in electronic impact (EI) ionizationmode (70 eV). Heliumwas used as the carrier gas (1 mLmin�1). Thecolumn temperature was programmed from 250 �C to 320 �C (hold2 min) at 3.5 �C min�1 and then to 330 �C at 1.1 �C min�1 heatingrate. The ion source and detector temperatures were 250 and350 �C, respectively. The injector temperature was 325 �C. The totalion count (TIC) mode was used to record the positive ion massspectra of the samples in the range betweenm/z 50 and 650. The AR

Table 2Mean values, standard deviation (SD) and mean square of genotype, environmentand their interaction for 5-n-alkylresorcinol (AR), total phenolic (TP) contents (mg/g)and antiradical activity (AA) in the examined 19 durum wheat varieties grown infour environments.

Source of variation FDa AR TP AAb

Genotype (G) 18 8674.2*** 1440.5*** 1515.5**Error 18 205.5 123.8 381.1Environment (E) 3 37522.6*** 5273.7*** 12153.9***G � E 54 2352.1*** 914.3*** 1283.3***Error 57 276.6 143.3 424.7Mean value (%DM)

� SD264.0 � 32.9 122.1 � 13.4 128.5 � 13.8

a Freedom degrees.b Antiradical activity was expressed in terms of EC50 ¼ mg of milled grain (DM)

required to obtain 50% DPPH� scavenging. **p < 0.01; ***p < 0.001.

S. Bellato et al. / Journal of Cereal Science 57 (2013) 162e169164

content was determined by comparing the relative retention timeswith those obtained for a mix of AR homologue standards: C17:0,C19:0, C21:0, C23:0, C25:0. The chromatographic peak areas of ARhomologues in each samplewere summed to yield total AR content.

2.8. Antiradical activity (AA) measurements

The DPPH� quenching capacity of wholemilled grain extracts wasdetermined spectrophotometrically as reported by Carbone et al.(2011). EC50 was calculated according to Sanchez-Moreno et al.(1998), from a calibration curve obtained with different amounts ofmethanolic extracts. Results are expressed as mg of wheat milledgrain (on DM basis) required to obtain 50% DPPH� scavenging (EC50).

2.9. Statistical analysis

As reported by Li et al. (2009), two independent aliquots ofmilled whole grains were considered as statistical replicates of eachcomposite grain sample. The combinations of years (2009 and2010) and locations (Foggia and Jesi) were treated as four envi-ronments. Analysis of variance (ANOVA) was performed with theMSTATC program (Michigan State University, East Lausing, MI)using a factorial model (mod.9) with G, E (locality and year) andG � E interaction. Genotype means were separated using Duncan’smultiple range test (p � 0.05) by combining the results acrossenvironments. Principal component analysis (PCA), performedwithMATLAB software (R2010a version, MathWorks Inc., USA), wasused to study the variation associated with the genotype and theenvironment. Pearson’s correlation coefficients (r) were alsocalculated. A correspondence analysis was performed between AR,TPC and AA arbitrary category and environment variables. Box-plots were used to display descriptive statistics (the median, theupper and lower quartiles and the minimum and maximum datavalues) for comparisons among varieties and environments.

3. Results and discussion

3.1. General aspects

Thirty Italian durumwheat varieties, grown in two Italian areasdedicated to the cultivation of durum wheat in two consecutiveyears, were analyzed to determine their AR content, TPC and AA.The samples showed a normal range of variation in grain physicalcharacteristics (Ciccoritti et al., 2011). The AR contents ranged from161.3 to 405.8 mg/g (DM) which is consistent with other studies(Andersson et al., 2008; Zarnowski et al., 2004), although Landberget al. (2009) reported higher AR values, applying a correction factorto the values obtained from a C15:0 calibration curve. The latterreported an average AR content of 452 � 138 mg/g (DM) for 14durum wheat samples. The TPC values determined in the presentstudy ranged from 19.0 to 192.4 mg/g (DM), which is slightly lowerthan the values reported for durumwheat by Adom et al. (2003). Itis worth noting that when gallic acid is used as a standard forquantification of TPC the resulting values are slightly higher thanwhen catechin is used (Imeh and Khokhar, 2002). Moreover, it mustbe borne in mind that wheat is a rich source of phenolics, presentboth in free (or soluble) and bound (insoluble) forms (Adom et al.,2003). We only analyzed the free phenolic fraction, which wasextracted with acetone and referred to it as “total soluble phenoliccontent”. It is known that in grains up to 95% of the total phenoliccompounds are localized in the bran tissue (Serpen et al., 2008).These are largely bound to cell wall polysaccharides, constitutingthe dietary fiber-phenolic compounds discussed by Vitaglione et al.(2008). The bound fraction must be hydrolyzed to be extracted andquantified. In the present study, we used acetone as a solvent to

extract the ARs and then quantified the total phenols present inthese extracts. This method does not include hydrolysis, whichmayaccount for the low TPC levels determined.

Analyses were also carried out to determine the relationshipbetween the GCeMS and colorimetric methods for determinationof ARs. Twelve of the genotypes used in the present work wereobtained from Montelibretti, an area near Rome (Central Italy),from 2010. On average, the colorimetric assay gave an AR content of305.0 � 46.2 (range 219.3e414.3 mg/g DM), compared to223.0 � 39.2 (range 168.6e319.9 mg/g DM) by GCeMS analysis. Thevalues from the two types of analysis were highly correlated (r:0.91; p < 0.001), in agreement with other reports (Andersson et al.,2008; Landberg et al., 2009). This result therefore justified ourchoice to use a simple spectrophotometric assay to quantify the ARcontent of a large number of samples.

Previous studies have compared the antioxidant properties ofdifferent grain samples, such as einkorn, emmer, spring and durumwheat (Lachman et al., 2011; Okarte et al., 2010). We thereforedetermined the antiradical capacity of the samples, using thescavenging capacity of the free radical DPPH. The results areexpressed indirectly, by measuring the quantity of antioxidantnecessary to reduce the initial DPPH� concentration by 50%, whichis a value generally defined as EC50 and defined in the present studyas the AA index. Lower EC50 values indicate stronger ability of theextract to act as DPPH� scavenger, while higher EC50 values indi-cates lower scavenging activity. On average, the EC50 values ofsamples ranged between 70.9 and 289.2 mg of wholemeal flour(DM), showing significant differences in ability to scavenge theDPPH� radical exhibited by different genotypes. This trend isconfirmed by literature data on the antioxidant properties ofdifferent cereal grains (Di Silvestro et al., 2012; Yu et al., 2002) but itis difficult tomake a precise comparison between reports due to thedifferent units of measurement used to express the AA (i.e. Troloxequivalents, mmol of vitamin C equivalent, mg extracts/mL, etc.). Wefound a significant but low statistical relationship between theantiradical activity of the samples and their soluble phenoliccontents (r: �0.36; p < 0.01). Correlations between TPC and AA arenot well established in the literature with the coefficients beingeither high or low depending on the study (Yu et al., 2002; Zielinskiand Kozlowska, 2000). The correlation between ARs and TPC wasalso significant but low (r: �0.18; p < 0.05), while no correlationwas found between ARs and AA.

3.2. Effects of genotype and environment

Data for 19 durum wheat cultivars grown in the four environ-ments were analyzed by ANOVA (Table 2). This showed that both Gand E had highly significant effects on AR content and TPC(p< 0.001) as well as on AA (p< 0.01), with highly significant G� E

Fig. 1. Dataset box plots: evaluation of E effect. Box plot explanation: right edge of the box, 75th centile; left edge, 25th centile; vertical bar within box, median; right vertical baroutside box, maximum value; left vertical outside box, minimum value. Points outside the box are outliers or suspected outliers. A. Alkylresorcinol content (mg/g DM); B. Totalphenolic content (mg/g DM); C. Antiradical activity (EC50 mg DM). Different letters indicate that averages are significantly different from each other (p < 0.05).

S. Bellato et al. / Journal of Cereal Science 57 (2013) 162e169 165

S. Bellato et al. / Journal of Cereal Science 57 (2013) 162e169166

interactions. However, the contribution of G � E interactions to thetotal variability was lower than that of the genotype or environ-ment alone. The analysis showed that E was the main factorcontributing to the total variation in the parameters that weremeasured. This is consistent with Menga et al. (2010), whoobserved a strong impact of environment on TPC of durum wheat.The results are also in agreement with another study of springwheat (Mpofu et al., 2006). Moore and Yu (2008) observed effectsof both E and G on the TPC and AA of wheat bran, which werestronger than the G � E interaction. Yu et al. (2004) reported thatthe DPPH� radical scavenging activity and phenolic content ofwheat flour samples were significantly influenced by the variety(G) and growing location (E). Andersson et al. (2010) also reporteda strong influence of genotype as well as of environment on thecontent and composition of ARs in bread wheat.

3.2.1. Effects of the environmentBox plots were used to compare the variation in the measured

parameters within and between environments (Fig.1). Between theenvironments, the AR content (Fig. 1A) showed a significantlyhigher mean value in Foggia 2010 (310.4 mg/g DM.) with a rangebetween 194.2 and 392.6 mg/g DM. On average, the lowest ARcontent (240.9 mg/g DM) was recorded in Foggia in 2009 and thehighest in Foggia in 2010, which is the location where the lowestamount of precipitation occurred. Andersson et al. (2010) reportedgreat variability in AR content of bread wheat between year andlocation, with highest contents in an environment characterized byhot dry conditions during grain filling. In the present study thelowest mean value for TPC was recorded in Foggia in 2010 (99.0 mg/g DM) and the highest in Foggia in 2009 (129.4 mg/g DM) and in Jesiin 2010 (132.2 mg/g DM) (Fig. 1B). It should be noted that in thepresent study high TPCs and low ARs were recorded for the envi-ronments characterized by high precipitation during the growingseason. The means for AA (Fig. 1C) significantly varied betweenenvironments, from 109.3 (Foggia 2009) to 149.9 (Jesi 2010) mgwholemeal flour. A significant influence of the growing conditionson the antiradical properties of wheat flour was reported by Yuet al. (2004), who did not detect any relationships with specificenvironmental factors, including total solar radiation, daily averagesolar radiation or the hours exceeding 32 �C. We found a significant

Fig. 2. Scatter plot of the correspondence analysis performed between the ARs (5-n-alkyenvironment variables. The range of the arbitrary categories are the follow: ARs, low � 200high � 150.1; AA, low � 90.0, medium � 90.1 � 180.0, high � 180.1.

influence of E on the scavenging properties, but no relationshipbetween AA and total precipitation.

3.2.2. Correspondence analysisThe whole data sets (30 durum wheat varieties � four environ-

ments) for ARs, TPC andAAwere converted to a frequency tablewiththe environment as the first variable and three arbitrary categoriesfor each trait: low, medium, high (second variable). The arbitrarycategory limits, based on the mean values and standard deviations,are shown in Fig. 2, which also shows graphical representations ofthe frequency table. In this representation the columns are theenvironments and the rows the categories of the traits (ARs, TPC andAA). The distances between the points that represent the environ-ments are a measure of the similarity among the environment-frequency profiles. Each trait-category point will lie close to theenvironment for which the trait-category is prominent. Corre-spondence analysis revealed three main environments. Foggia isa Southern Italy location with Mediterranean climatic conditionsand the 2009 season was characterized by an exceptionally highamountof total rainfall. This environment is graphically locatednearLowAAand close to LowARs andHigh TPC,which are the prominentcategories of this environment. The closeness of the AR, TPC and AAmedium categories to Jesi 2009 and Jesi 2010, which are locatedrelatively close to each other, indicates that these categories are“strongly” associatedwith these environments and that themediumcategory is prominent for all of the parameters. Unlike Foggia, Jesi(Central Northern-Italy, altitude 97 m) has a continental climatewith a cold humid winter and sultry humid summer. The closenessofHighARs, LowTPCandHighAAtoFoggia2010 indicates thehigherfrequency of these categories in this environment, while High ARsand Low TPC determine the position of Foggia 2010 on the plot. Thefact that Foggia 2010 is located relatively far from the other envi-ronments indicates that the climatic conditions recorded duringgrainfilling andharvesting in this environment stronglyaffected thephytochemical profile of the grain.

3.2.3. Effect of genotypeBox plots of the 19 varieties grown in all of the environments (1),

as well as the variation in the measurement within a single varietyare shown in Fig. 3. The genotypes differed considerably in their AR

lresorcinols), TP (Total phenols) and AA (antiradical activity) arbitrary category and.0, medium � 200.1 � 300.0, high � 300.1; TP, low � 100.0, medium � 100.1 � 150.0,

Fig. 3. Dataset box plots: evaluation of G effect. Box plot explanation: upper horizontal line of box, 75th centile; lower horizontal line, 25th centile; horizontal bar within box,median; upper horizontal bar outside box, maximum value; lower bar outside box, minimum value. Points outside the box are outliers or suspected outliers. A: alkylresorcinolcontent (mg/g DM), B: total phenolic content (mg/g DM); C: antiradical activity (EC50 mg DM). Different letters indicate that averages are significantly different from each other(p < 0.05).

S. Bellato et al. / Journal of Cereal Science 57 (2013) 162e169 167

Fig. 4. Genotype � Environment biplot from PCA for AR (5-n-alkylresorcinol) content of 19 durum wheat varieties grown in four environments.

S. Bellato et al. / Journal of Cereal Science 57 (2013) 162e169168

contents (Fig. 3A). On average, the values ranged from 189.1 (Nor-manno) to 329.6 mg/g DM (Iride). The box plots show limitedvariation in some of the genotypes (Creso, Iride, Minosse, Nor-manno, Simeto, Tirex), underlying the contributions of genotype tothe AR content. The TPC values (Fig. 3B) ranged from 98.6 (Minosse)to 144.9 mg/g DM (Trionfo). A broad range of variationwas observedfor AncoMarzio, Imhopet and Simeto. The AA (EC50) means calcu-lated across replicates and environments ranged from 100.1 (Tirex)to 156.7 mg DM (in Alemanno). Most of the genotypes, except forAlemanno, showed a broad range of variation in AA content due tothe genotype and environment.

3.2.4. Effects of genotype � environment interactionsThe variance associated with G � E interactions was significant

for all the traits (Table 2). The effect of the environment on ARcontent can be clearly seen by PCA of the varieties and environ-ments (Fig. 4). The environments are in similar positions on thepositive side of PC1 (explained variance ¼ 66%) and only Foggia2010 is located on the negative side for PC2 (explainedvariance ¼ 30%). The varieties with positive scores for PC1 and PC2generally had AR contents slightly above the average. Among them,only Iride (positive value for PC1) showed higher and more stablecontents of AR across environments (AR range: 303.6e351.6 mg/gDM). In addition, Normanno (AR range: 173.9e204.2 mg/g DM)and Simeto (AR range: 192.3e227.2 mg/g DM) appeared to be lessaffected by the environment but had low AR contents. Seven vari-eties with positive scores for PC1 showed very strong interactionswith the environment and had the highest contents of ARs inFoggia 2010. Within this environment, the varieties had ARcontents between 328.2 (Duilio) and 392.6 mg/g DM (Imhopet).

4. Conclusions

This paper focuses on bioactive components of durum wheatwhole grain: 5-n alkylresorcinols and free phenolics. Dietaryphenolics exert high antiradical activity with potential healthbenefits. Previous reports showed significant differences betweenthe phenolic contents and antioxidant properties of wheat culti-vars. Those studies generally showed that the most abundantphenolic compounds in cereals are phenolic acids and that about95% of grain phenolic compounds are linked to cell wall poly-saccharides (Vitaglione et al., 2008). In the present study, the

samples showed considerable differences among cultivars in thecontents of free phenolics (generally referred as TPC). Moreover, therapid colorimetric method used in this study was found to besensitive and in good agreement with the GCeMS method and istherefore a valid method for the determination of total AR contentin cereal grains, particularly in screening large numbers of samples.Genotype and environment are the main factors that influence thephytochemical content of whole grain. In the present study,significant E and G effects were found for all analyzed traits(p< 0.001 and p< 0.01). In addition the contribution of G� E to thetotal variance was much lower than that due to the separate effects(G and E), and E accounted for the highest proportion of the vari-ation. The range of variation in environmental conditions, espe-cially in precipitation, allowed us to identify the contribution of thisenvironmental factor to the AR content. In fact, the highest ARconcentrations were obtained at lowest mean temperatures andrainfall, while the opposite was observed for TPC and high wateravailability during grain filling appeared to increase the accumu-lation of free phenols. The results emphasized the importance of Gin determining the phytochemical components of durum wheatand also allow us to identify some promising varieties (Iride, Tirex,Trionfo), which are potentially capable of producing high AR or TPCin whole grain with greater stability under different environmentalconditions. In particular, the PCA allowed us to identify the geno-types which are richer in ARs and stable across environments. Thegraphical representation of the correspondence analysis alsoprovided a good summary of the overall phytochemical profiles ineach of the environments.

The results of this study will provide useful information todurum wheat scientists, regarding the relative importance ofgenotype and environment on the accumulation in the grains of 5-n-alkylresorcinols, total phenols and their antiradical activity.Moreover, our results clearly show that the effects of E and G� E onthe levels of AR and TPC should be taken into account during thebreeding programs aimed at improving the health benefit of wheat.

Acknowledgments

This research was financially supported by “AGER-AGRO-ALIMENTARE e RICERCA” Project “From seed to pasta: An inte-grated research network for the production of high-quality durumwheat”, website: www.progettoager.it.

S. Bellato et al. / Journal of Cereal Science 57 (2013) 162e169 169

Appendix A. Supplementary data

Supplementary data related to this article can be found at http://dx.doi.org/10.1016/j.jcs.2012.11.003.

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