Journal of Cereal Science 58 (2013) 123e131

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Journal of Cereal Science

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

Phenolic acids composition, total polyphenols content and antioxidant

activity of Triticum monococcum, Triticum turgidum and Triticum
aestivum: A two-years evaluation
Andrea Brandolini a, Paolo Castoldi b, Luca Plizzari a, Alyssa Hidalgo b, *
a
Consiglio per la Ricerca e la Sperimentazione in Agricoltura, Unità di Ricerca per la Selezione dei Cereali e la Valorizzazione delle Varietà Vegetali
(CRA-SCV), Via Forlani 3, 26866 S. Angelo Lodigiano (LO), Italy
b
Department of Food, Environmental and Nutritional Sciences (DeFENS), Università degli Studi di Milano, Via Celoria 2, 20133 Milano, Italy

a r t i c l e i n f o a b s t r a c t

Article history: Wheat is a good source of polyphenols, plant metabolytes with beneficial effects on human health.
Received 21 December 2012 However, little information is available on phenolic acid composition and concentration in different
Received in revised form Triticum species, as well as on possible environmental effects. To shed some light on this issue, thirty-
15 March 2013
nine wheat accessions cropped for two years and belonging to different Triticum species (Triticum
Accepted 21 March 2013
monococcum, Triticum turgidum ssp. dicoccum, turanicum and durum, Triticum aestivum ssp. spelta and
aestivum) were assessed for phenolic acids (ferulic, p-coumaric, vanillic, syringic, p-hydroxybenzoic,
Keywords:
caffeic acids and syringaldehyde), total polyphenols and antioxidant activity in soluble conjugated and
Ferulic acid
Kernel fractions
insoluble bound extracts. Ferulic acid was the most abundant compound in both extracts. Insoluble
Wheat bound phenolic acids represented >90% of total phenolic acids. Einkorn showed the maximum con-
Cropping year centration of conjugated phenolic acids (50.5 mg/kg DM), while durum and bread wheats presented the
highest content of bound phenolic acids (651.8 and 629.2 mg/kg DM, respectively). Cropping year
influenced the concentration of conjugated but not of bound phenolic acids. A survey of phenolic acid
distribution in the kernel showed that they are rare in endosperm, but abundant in germ and bran. Total
polyphenols and antioxidant activity were highly correlated to phenolic acid content.
Ó 2013 Elsevier Ltd. All rights reserved.

1. Introduction The phenolic compounds contain one or more aromatic ring

with one or more hydroxyl groups and have structures that vary
The phenolic compounds, secondary metabolites synthesized from simple molecules to complex polymers. This relevant struc-
during plant development and in response to stress conditions, are tural diversity influences their difference in bioavailability: simple
excellent oxygen radical scavengers, with an electron reduction phenolic acids (e.g. caffeic acid) more easily cross the intestinal
potential lower than the oxygen radicals. Additionally, the phe- barrier, while complex molecules (e.g. proanthocyanins) are
noxyl radicals are less reactive than the oxygen radicals and scav- scarcely absorbed (Scalbert et al., 2002).
enge reactive oxygen intermediates without promoting further Cereals are a good source of plant phenolics and, as staple food
oxidative reactions. Therefore, environmental stresses that induce of humankind, may provide a relevant share of these molecules.
oxidative damage often promote the synthesis of phenolic metab- Phenolic acids, the most common type of phenolic compounds in
olites (Ainsworth and Gillespie, 2007), which act as phytoalexins, cereals (Li et al., 2008), are present in three forms: soluble free,
have antioxidant and antinutritional properties, protect against UV soluble conjugated, esterified to sugars and other low molecular
radiations and safeguard cell wall integrity. This strong antioxidant weight components, and insoluble bound, linked to cell wall con-
activity leads to beneficial anti-inflammatory, anti-microbial, anti- stituents such as polysaccharides, protein, lignin, cutin or suberin
thrombotic, anti-atherogenic, anti-thrombotic, vasodilatatory and (Naczk and Shahidi, 2004). In wheat, the most abundant fraction is
cardio-protective effects on human health (Quiñones et al., 2013). the insoluble bound (77%), followed by the soluble conjugated
(22%) and the soluble free (<0.5e1%) (Li et al., 2008).
Phenolic acids are often scarce in commercial wheat flours,
because their highest concentrations are in the aleuronic and hya-
* Corresponding author. Tel.: þ39 02 51309189; fax: þ39 02 50319190. line layers, the germ and the seed coat (Barron et al., 2007), which
E-mail address: [email protected] (A. Hidalgo). are usually eliminated during milling. In whole meal flours, the

0733-5210/$ e see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.jcs.2013.03.011
124 A. Brandolini et al. / Journal of Cereal Science 58 (2013) 123e131

amount of polyphenols is highly variable and is mostly related to of whole meal flour, and bran and flour. The germ-to-seed ratio was
species and variety (Adom et al., 2005). Einkorn is a high- determined by manually dissecting germs from four 50-seed
nutritional-value cereal, with high protein (Brandolini et al., samples: after moistening the kernels to 40% humidity, the germs
2008), carotenoid and tocol (Hidalgo et al., 2006) content. Howev- were enucleated with a scalpel, and the adhering pericarp layers as
er, a significant gap remains in its phenolic acid composition. Li et al. well as any endosperm remains were carefully removed.
(2008) recorded a total phenolic acid content similar to winter,
spring and durum wheats, slightly higher than spelt but marginally 2.2. Analytical methods
lower than emmer. Serpen et al. (2008) measured a higher con-
centration of bound ferulic acid in emmer than in einkorn. However, Dry matter content was determined following AACC method 44-
Abdel-Aal and Rabalski (2008) reported more phenolic acids in 15A (1995).
einkorn than in several primitive and modern wheats.
Phenolic acid variation is also affected by the cropping envi- 2.2.1. Phenolic acid extraction
ronment, but the information available in literature is limited. An The phenolic extracts were prepared as outlined in Abdel-Aal
influence of the cultivation year on total polyphenols was recorded et al. (2001), with some modifications. Exactly 0.5 g of whole
by Heimler et al. (2010) in their survey of 9 Triticum durum and 17 meal flour were mixed with 15 mL of a methanol/acetone/water
Triticum aestivum cultivars, sampled in the same site over two (7:7:6) solution. After 15 min in an ice bath under discontinuous
years. Similarly, Lachman et al. (2011) reported significant differ- vortexing and after centrifugation (11,178 g, 10 min, 8  C) with a
ences between cropping years in their two-year study involving Centrikon K24 centrifuge (Kontron Instruments, Bletchley, UK), the
two Triticum dicoccum, two Triticum monococcum and three supernatant was recovered; the extraction from the sediment was
T. aestivum accessions. Likewise, the phenolic acids of one repeated twice more, and the three extracts were pooled.
T. aestivum genotype cultivated under organic and conventional
cropping systems varied significantly from year to year, with 2.2.1.1. Soluble conjugated phenolic compounds. The supernatant
changes up to 55% (Stracke et al., 2009). Environmental differences was evaporated under vacuum at 35  C for 18 min with a rotator
were recorded also by Mpofu et al. (2006), who analysed phenolic evaporator Laborota 4000 (Heidolph, Milan, Italy), and nitrogen
acids, total polyphenols and antioxidant activity of six T. aestivum flux for 1 min. The samples were then digested with 15 mL of 4 M
genotypes from four different locations. NaOH under nitrogen for 4 h at room temperature and continuous
The aim of this research was to explore differences in the shaking, brought to pH 1.5e2 with 6 M HCL and extracted twice
phenolic acid content and in antioxidant activity of different Triti- with 20 mL of diethyl ether/ethyl ether (1:1 v/v). The extracts were
cum species and subspecies, as well as any changes prompted by then clarified with sodium sulphate, filtered with glass fibre 110 mm
different cropping years. Additionally, the distribution of phenolic (Whatman, Maidstone, England), evaporated as previously out-
acids in the germ, bran and flour fractions of einkorn kernel was lined, resuspended in 2 mL of methanol:water (1:1 v/v) and filtered
assessed. Therefore, phenolic acid composition, total polyphenol with a 0.22 mm PTFE membrane (Millipore, Carrigtwohill Co., Cork,
concentration and antioxidant activity were analysed in soluble Ireland).
conjugated and insoluble bound extracts of the different accessions.
2.2.1.2. Insoluble bound phenolic compounds. The sediment was
2. Experimental digested with 15 mL of 4 M NaOH under nitrogen for 4 h at room
temperature and discontinuous vortexing, brought to pH 1.5e2
2.1. Materials with 6 M HCL and extracted twice with 20 mL of diethyl ether/ethyl
acetate (1:1, v/v). After centrifugation (11,178 g, 10 min, 8  C), the
Thirteen accessions of einkorn (T. monococcum ssp. mono- supernatants were clarified with sodium sulphate, filtered, evapo-
coccum), 13 accessions of tetraploid wheats (emmer, KamutÒ and rated as previously outlined, resuspended in 2 mL of methanol-
durum wheat, i.e. T. turgidum ssp. dicoccum, turanicum and durum, water (1:1 v/v) and filtered with a 0.22 mm PTFE membrane (Mil-
respectively) and 13 accessions of hexaploid wheats (spelt and lipore, Carrigtwohill Co., Cork, Ireland).
bread wheat, i.e. T. aestivum ssp. spelta and aestivum, respectively) All extractions were performed in duplicate under dim light; the
were cropped during the 2007e2008 and the 2010e2011 growing extraction tubes were wrapped with aluminium foil, to avoid
seasons at S. Angelo Lodigiano (Italy), in single rows 2 m long, sample degradation by photo-oxidation.
adopting standard cultural practices. A complete list of the acces-
sions is reported in Supplementary Table 1. After manual harvest- 2.2.2. Phenolic acid quantification by HPLC
ing, the samples were stored at 5  C. Just before analysis, the Twenty mL of each soluble conjugated and insoluble bound
kernels of T. monococcum, T. turgidum ssp. dicoccum and T. aestivum extract were analysed by RP-HPLC according to Mpofu et al. (2006),
ssp. spelta were de-hulled with a M3B micro-thresher (Co.Mi.L, slightly modified. The following operating conditions were adop-
Rome, Italy); dehulling was not necessary for the other wheats, all ted: column Altima C18 5 mm 4.6 mm  250 mm (Grace Davison
free-threshing. The samples were ground with a Cyclotec 1093 lab Discovery Sciences, Deerfield, IL, USA), precolumn Altima C18 5 mm
mill (FOSS Tecator, Denmark) to particle size < 200 mm; the flour 4.6 mm  10 mm (Grace Davison Discovery Sciences, Deerfield, IL,
was stored under vacuum at 20  C until analysis. USA) thermostated at 30  C; pump L-2130 Elite La Chrom (VWR,
The whole meal flour of bread wheat cultivar Serio, harvested in Hitachi, Japan), column oven L-2300 Elite La Chrom (VWR, Hitachi,
2006, was used to evaluate method repeatability, as well as ferulic Japan); mobile phase: A) 1% (v/v) acetic acid in water, B) methanol;
and p-coumaric acid recovery. flow rate 1.5 mL/min. The gradient, in terms of eluent B, was: at
To assess phenolic acid content in kernel fractions, bran and time 0, 15%; at 10 min, 20%; at 16 min, 23%; at 24e28, 27%; at 30e
flour were prepared from 200 g of manually degerminated seeds 34, 15%. The HPLC system was controlled by the software EZChrom
(ca 1/3 of each seed, including germ and scutellum, was removed Client/Server version 3.1.7. The compounds were detected at
with a scalpel) of T. monococcum ssp. monococcum cv Monlis, milled 280 nm with a Diode Array Detector L2450 Elite La Chrom (Merck,
with a Bona-GBR lab mill (Bona, Monza, Italy). The bran was care- Hitachi, Japan).
fully sieved using an 18 mesh screen, to separate any adhering flour. For peak quantification, calibration curves of the following
Germ composition was computed as difference between the results compounds using standards from SigmaeAldrich (St. Louis, MO,
 A. Brandolini et al. / Journal of Cereal Science 58 (2013) 123e131 125

USA) were constructed: caffeic acid (between 0 and 7.29 mg/L), 30 min; at this time, the absorbance value reached a plateau, cor-
ferulic acid (0e200.38 mg/L), p-coumaric acid (0e9.93 mg/L), p- responding to the residual DPPH percentage. The radical reduction
hydroxybenzoic acid (0e26.48 mg/L), syringaldehyde (0e11.44 mg/ by the antioxidants was monitored by measuring its absorbance at
L), syringic acid (0e19.62 mg/L), vanillic acid (0e19.58 mg/L). The 517 nm, using a DU-62 spectrophotometer (Beckman, Brea, CA,
calibration curves were linear in the concentration intervals USA). For each sample, two extracts were analysed, each with five
assessed. On the basis of the calibration curves, the detection limits different dilutions.
in the standard solutions were 0.05, 1.18, 0.09, 0.14, 0.09, 0.11, A doseeresponse line was computed for each sample and the
0.16 mg/L, respectively, corresponding to 0.22, 5.36, 0.41, 0.62, 0.39, whole meal flour quantity needed to scavenge 50% of the radical
0.48, 0.73 mg/kg DM in whole meal flour containing 88 g/100 g DM. (I50) was determined. A regression line was also computed for the
The identity of phenolic acids was confirmed by congruence of reference antioxidant Trolox (SigmaeAldrich, St. Louis, MO, USA),
retention times and UV/Vis spectra with those of pure authentic with concentrations between 3 and 50 mM. The antioxidant activity
standards. The analyses were performed twice; the results are was expressed as the ratio between I50 of Trolox and I50 of the
expressed as mg/kg of whole meal flour on a dry matter (DM) basis. sample, i.e. millimoles of Trolox equivalent (TE)/kg DM. The
Method repeatability was performed by extracting six and seven repeatability of the method, expressed as mean  standard error
times, respectively, from the very same whole meal sample (bread and coefficient of variation, was 0.50  0.010 mmol TE/kg DM and
wheat cv Serio). The repeatability for ferulic acid, p-coumaric, 4.8% for conjugated, and 2.80  0.060 and 5.7% for bound phenols.
syringic acid, p-hydroxybenzoic acid, and vanillic acid, expressed as
mean  standard error (mg/kg DM) and coefficient of variation, 2.3. Statistical analysis
was: for the soluble extract 19.89  0.225 and 2.8%, 1.32  0.027
and 5.1%, 6.77  0.125 and 4.5%, 3.28  0.078 and 5.8%, 5.27  0.083 A two-way analysis of variance (ANOVA) was performed,
and 3.8%, respectively; for the insoluble extract 711.8  11.152 and considering as factors the years and the species or the subspecies;
4.1%, 20.16  0.276 and 3.6%, 8.67  0.180 and 5.5%, 3.05  0.055 the interaction between the two factors was also assessed. When
and 4.8%, 4.71  0.081 and 4.53, respectively. significant differences (p  0.05) were detected, Fisher’s least sig-
To assess ferulic and p-coumaric acid recovery, the same sam- nificant difference (LSD) was computed. Both ANOVA and LSD were
ple used in the repeatability test was spiked with 50 mL of ferulic determined using the statistical software STATGRAPHICS plus,
acid (200.38 mg/L) and 100 mL of p-coumaric acid (9.93 mg/L) for version 4 (Statpoint Technologies, INC., Warrenton, VA, USA).
soluble conjugated phenolics, and with 1000 mL of ferulic acid Pearson’s correlations of the means were calculated using the
(200.38 mg/L) and 1000 mL of p-coumaric acid (9.93 mg/L) for SYSTAT for Windows (version 5) software.
insoluble bound phenolics. The recoveries were 91.7  1.0% and
79.2  2.9% in the soluble extract, and 88.5  0.7% and 80.9  4.7% 3. Results and discussion
in the insoluble extract for ferulic and p-coumaric acids, respec-
tively. These values compare favourably with the results of Adom 3.1. Phenolic acid composition
et al. (2005), which describe an 89.3 recovery for ferulic acid,
and of Stracke et al. (2009), which report recoveries from 72 to 87% The soluble conjugated and the insoluble bound phenolic acids,
for phenolic acids. as well as the total phenolic acids, total polyphenols and antioxi-
dant activity of the 39 different T. monococcum, T. turgidum and
2.2.3. Total phenols content in the soluble conjugated and insoluble T. aestivum accessions in 2008 and 2011 are presented in
bound extracts Supplementary Tables 1, 2 and 3. The HPLC analysis detected seven
Total phenols content analysis was achieved with the Foline phenolic acids across the wheat samples investigated. Ranking
Ciocalteu method as reported by Lavelli et al. (2009), using ferulic them over species from most to least abundant (two-year mean;
acid as a standard. The reaction mixture contained 0.2 mL of extract sum of conjugated and bound fractions), the phenolic acids iden-
(for soluble conjugated phenols) or 0.1 mL of extract þ 0.1 mL tified were: ferulic (566  8.8 mg/kg DM), p-coumaric (25.1  1.10),
distilled water (for insoluble bound phenols) and 0.4 mL of 1:10 vanillic (9.7  0.20), syringic (7.5  0.26), p-hydroxibenzoic
diluted Folin-Ciocalteu reagent (SigmaeAldrich, St. Louis, MO, (4.6  0.23), caffeic (3.1  0.29), and syringaldehide (1.2  0.04).
USA). After vortexing and resting for 5 min, 1.4 mL of 7% Na2CO2 Ferulic acid was by far the most abundant compound, both in the
were added, mixed and kept in darkness at 40  C for 30 min. The conjugated and in the bound fraction (Fig. 1), as already described
samples were then read against a blank at 765 nm, using a Du-62 by Moore et al. (2005), Okarter et al. (2010) and Serpen et al. (2008).
Beckman spectrophotometer (Beckman Coulter, Nyon, VD, Li et al. (2008) reported similar results for the bound fraction, but
Switzerland). The analyses were performed twice. measured a prevalence of sinapic and 2,4 dihydroxybenzoic acids in
The total phenols content, in mg ferulic acid equivalent (FAE)/kg the conjugated fraction.
DM, was computed from a reference curve obtained from six ferulic The concentration of ferulic acid in the bound form was similar
acid (SigmaeAldrich, St. Louis, MO, USA) concentrations, in the among species (93.0e94.9% of total phenolic acids), but some dif-
range 0e150 mg/L. The repeatability of the method, expressed as ferences existed in the conjugated form, which in einkorn repre-
mean  standard error and coefficient of variation, was sented 69.5% of conjugated phenolic acids, in the tetraploid wheats
142.0  2.128 mg FAE/kg DM and 3.7% for conjugated, and 59.6% and in the hexaploid wheats as low as 56.5%. Overall, the
1176.3  16.647 mg FAE/kg DM and 3.7% for bound phenolic acids. contribution of ferulic acid to the total phenolic acid content in the
three different species was similar, ranging from 91.1%
2.2.4. Radical DPPH scavenging capacity (T. monococcum) to 92.4% (T. aestivum); these results are similar to
The antioxidant activity was evaluated according to Lavelli et al. those (83.5e89.5%, 85.3e89.3% and 72.5e90%, respectively)
(2009), with slight changes. The radical scavenging capacity of the described by Moore et al. (2005), Serpen et al. (2008), and
soluble and insoluble extracts was assessed with the stable 2,2- Zuchowski et al. (2011), while are higher than those (72e85% and
diphenyl-1-(2,4,6-trinitrophenyl)hydrazyl radical (DPPH; Sigmae 49.7e69.8, respectively) reported by Li et al. (2008) and Mpofu et al.
Aldrich, St. Louis, MO, USA). To 2 mL of DPPH in methanol (2006).
(6.35  105 M), 1 mL of extraction solvent with different extract The conjugated fraction represented only a small portion of the
dilutions was added. The reaction was performed at 25  C for total phenolic acids, ranging from 6.5% in T. aestivum to 8.3% in
126 A. Brandolini et al. / Journal of Cereal Science 58 (2013) 123e131

Fig. 1. Percentage of phenolic acids in soluble conjugated and insoluble bound extracts from whole meal flours of T. monococcum (A), T. turgidum (B) and T. aestivum (C).

T. monococcum; these values are lower than those reported by (p  0.01) for all conjugated and bound phenolic acids, except
Sosulski et al. (1982) for white flour (16%), and by Li et al. (2008) for bound caffeic acid. A significant effect of the cropping year (p  0.01
whole meal flour (22%). or p  0.05) was recorded for the conjugated phenolic acids (except
for p-hydroxybenzoic acid), while the species  year interaction
3.2. Phenolic acid content was significant (p  0.05) only for p-coumaric acid. In 2008 on
average, the total conjugated phenolic acids were 42.2  1.25 mg/kg
The conjugated and bound phenolic acids recorded in the DM, while in 2011 they reached 48.5  1.31 mg/kg DM; the increase
different species/subspecies are depicted in Table 1. The total con- was more pronounced among the T. turgidum than the T. aestivum
jugated phenolic acids (Table 2) ranged between 36.0 and 52.6 mg/ and T. monococcum samples. The higher concentration of conju-
kg DM, a distribution that partially overlaps the values (44.7e gated phenolic compounds in 2011 might be due to drought stress
60.2 mg/kg) reported by Moore et al. (2005) in eight selected (in 2011 the rainfall during the heading and maturing stages of
Maryland-grown soft wheat varieties but is inferior to the con- wheat development was minimal and led to early ripening) more
centrations (76e416 mg/kg DM) described by Li et al. (2008) in a than to heat shock (the temperatures were very similar in both
group of 175 wheats belonging to different Triticum species. The years), as evident from Fig. 2. Mallick et al. (2011) reported an in-
total bound phenolic acids (Table 2), instead, varied between 441 crease of total polyphenols (13.7e33.2%) in fresh leaf samples of
and 715 mg/kg DM, perfectly fitting within the results (208e eight bread wheat genotypes under drought stress. On the contrary,
964 mg/kg DM) reported by Li et al. (2008). the bound phenolic acids generally did not change considerably
The ANOVAs (not shown), performed considering species and from year to year, with the exception of caffeic, p-coumaric and
years as factors, showed significant differences among species syringic acids, and of antioxidant activity; significant year  species
 A. Brandolini et al. / Journal of Cereal Science 58 (2013) 123e131 127

Table 1
Mean (standard error) of soluble conjugated and insoluble bound phenolic acids (mg/kg DM) in whole meal flour of diploid T. monococcum ssp. monococcum (13 samples),
tetraploid T. turgidum ssp. dicoccum (7 samples), T. turgidum ssp. turanicum (2 samples), T. turgidum ssp. durum (4 samples), and hexaploid T. aestivum ssp. spelta (9 samples)
and T. aestivum ssp. aestivum (4 samples) accessions cropped in two different years.

p-Hydroxybenzoic Vanillic Syringic Syringaldehyde p-Coumaric Ferulic

2008 2011 2008 2011 2008 2011 2008 2011 2008 2011 2008 2011

Soluble conjugated phenolic acids

T. monococcum 2.2  0.34 1.6  0.11 5.1  0.29 6.0  0.29 3.5  0.13 4.8  0.19 1.4  0.14 1.4  0.12 2.2  0.16 2.6  0.17 34.1  1.69 36.1  1.82
T. turgidum 3.4  0.39 3.4  0.35 6.1  0.31 7.4  0.22 2.8  0.16 4.5  0.11 1.1  0.09 1.3  0.10 2.2  0.31 3.5  0.48 23.6  1.31 28.9  1.10
ssp. dicoccum 3.4  0.64 3.4  0.62 5.6  0.19 7.4  0.33 2.7  0.29 4.6  0.18 1.1  0.13 1.5  0.12 1.5  0.13 2.8  0.61 26.4  1.66 30.1  1.58
ssp. turanicum 3.2  0.20 3.7  0.29 4.8  0.04 6.7  0.56 2.8  0.22 4.4  0.32 0.8  0.08 0.9  0.04 1.7  0.02 3.7  0.63 22.7  1.24 26.8  2.79
ssp. durum 3.4  0.72 3.1  0.43 7.5  0.25 7.6  0.28 2.8  0.14 4.4  0.06 1.2  0.14 1.1  0.07 3.7  0.34 4.6  0.96 19.2  1.07 27.7  1.88
T. aestivum 3.8  0.32 3.5  0.36 6.2  0.21 6.6  0.32 4.4  0.19 5.8  0.63 1.0  0.06 1.2  0.08 1.7  0.35 1.8  0.22 21.7  1.06 25.2  1.40
ssp. spelta 3.8  0.40 3.8  0.47 5.8  0.10 6.1  0.18 4.6  0.23 5.1  0.66 1.0  0.07 1.2  0.11 1.2  0.19 1.6  0.25 22.0  1.22 25.0  1.81
ssp. aestivum 4.0  0.59 2.8  0.39 7.0  0.45 7.5  0.81 4.1  0.31 7.2  1.23 1.2  0.11 1.3  0.05 2.9  0.80 2.4  0.33 21.0  2.34 25.7  2.38

p-Hydroxybenzoic Vanillic Syringic Caffeic p-Coumaric Ferulic

2008 2011 2008 2011 2008 2011 2008 2011 2008 2011 2008 2011

Insoluble bound phenolic acids

T. monococcum 1.0  0.08 1.0  0.07 3.1  0.15 3.6  0.17 3.0  0.22 2.9  0.29 1.4  0.40 5.2  0.54 24.4  1.52 32.8  2.27 507  18.6 538  16.1
T. turgidum 1.3  0.20 1.5  0.17 3.1  0.26 3.1  0.20 2.8  0.18 2.9  0.55 1.1  0.35 4.0  0.89 22.4  3.08 22.4  2.97 525  35.2 521  17.6
ssp. dicoccum 1.1  0.16 1.3  0.20 2.4  0.22 2.6  0.20 3.0  0.19 1.7  0.44 1.5  0.61 5.8  1.28 21.8  2.34 28.8  4.10 468  25.6 506  28.6
ssp. turanicum 2.8  0.10 1.2  0.10 3.2  0.15 3.3  0.26 1.9  0.10 3.2  0.01 0.3  0.05 1.1  0.19 13.7  0.91 14.0  0.39 420  8.0 495  18.5
ssp. durum 1.1  0.09 1.9  0.38 4.2  0.18 3.8  0.08 2.9  0.36 4.9  1.01 0.8  0.29 2.4  0.62 27.9  9.00 15.4  1.86 679  46.3 560  18.9
T. aestivum 2.4  0.21 2.3  0.22 4.0  0.35 4.0  0.32 4.6  0.25 2.9  0.68 2.0  0.23 4.8  0.62 16.2  1.55 18.4  0.82 569  21.4 567  9.9
ssp. spelta 2.4  0.30 2.4  0.30 3.2  0.12 3.4  0.12 4.5  0.21 3.1  0.70 1.9  0.27 5.1  0.77 13.8  0.91 17.8  0.94 551  22.6 562  5.1
ssp. aestivum 2.4  0.25 2.1  0.27 5.6  0.42 5.4  0.67 4.9  0.71 2.4  1.74 2.4  0.39 4.2  1.14 21.4  3.60 19.8  1.59 610  45.8 578  32.5

interactions (p  0.05) were noticed only for syringic and p-cou- only in p-coumaric acid, but had lower concentrations of p-
maric acids. hydroxybenzoic, vanillic and, in 2008 only, of syringic and caffeic
A similar picture was observed when the subspecies were acids. The total bound phenolic acid levels were slightly lower in
considered (ANOVAs not shown): the conjugated phenolic acids einkorn and T. turgidum than in T. aestivum (Table 2; 540  18.9 vs.
were almost always influenced by the cropping year, while the 556  37.6 vs. 598  22.9 mg/kg DM in 2008; 584  17.0 vs.
bound phenolic acids were quite stable, with some variation due to 555  19.6 vs. 600  10.2 mg/kg DM in 2011, respectively).
minor year  subspecies interactions. Considering the different subspecies, a better understanding of the
The influence of the cropping year on phenolic acid composition results was possible: T. turgidum ssp. turanicum and ssp. dicoccum
and concentration has been scantily studied. Stracke et al. (2009), showed low contents of some bound phenolic acids, whereas
analysing total (free þ conjugated þ bound) phenolic acids of one durum and bread wheat had the highest bound ferulic and total
bread wheat cultivar cropped over three different years following bound phenolic acids concentrations.
standard and organic techniques, did not detect differences be- When the sum of soluble and insoluble extracts was considered,
tween managements but reported changes up to 55% among significant differences were detected between species as well as
cultivation years, thus deducing that climatic factors have greater between subspecies (ANOVA not presented). In both years,
impact on phytochemical concentrations than the production T. turgidum ssp. durum and T. aestivum ssp. aestivum showed the
method. The bound phenolic acid variations between different highest total phenolic acids (Table 3), while T. turgidum ssp. tur-
cropping locations reported by Mpofu et al. (2006) were more anicum constantly displayed the poorest contents. Li et al. (2008)
relevant than the genetic differences between six Canadian bread recorded the highest phenolic acids in emmer (779  109 mg/kg
wheats; however, the changes were not correlated to temperature DM), durum (699  51) and bread wheat (664  15); nevertheless,
or rainfall from heading time to maturity. these values did not differ significantly from those of spelt
The comparison of the conjugated compounds means (Table 1) (579  57) and einkorn (615  74). On the other hand, Serpen et al.
demonstrated that einkorn was poorer than the polyploid wheats (2008) stated that emmer has more ferulic acid (and consequently
in p-hydroxybenzoic and vanillic acids, but was richer in syringal- more total phenolic acids) than einkorn and two bread wheat
dehyde and ferulic acid. Furthermore, the overall conjugated acids controls.
content (Table 2) of einkorn (48.5  2.31 and 52.6  2.33 mg/kg DM
in 2008 and 2011, respectively) was significantly higher than that of 3.3. Total polyphenol content
T. turgidum (39.1  1.38 and 48.9  1.29 mg/kg DM) and T. aestivum
(38.9  1.61 and 44.1  2.47 mg/kg DM). The concentration values The total polyphenol content, determined spectrophotometri-
found for the different subspecies belonging to T. turgidum and cally by reduction of the FolineCiocalteu reagent, was expressed as
T. aestivum (Table 1) confirmed these results, also allowing an mg FAE/kg DM: ferulic acid was used as reference, instead of gallic
enhanced discrimination: for example, durum wheat showed the acid (often found in literature), because it is by far the most
highest p-coumaric acid content. The literature comparing conju- representative phenolic acid in wheat. The values obtained with
gated phenolic acid content in different wheat species is very this method were generally higher than those determined by direct
scarce; however, Li et al. (2008) measured lower concentrations in HPLC quantification of the different phenolic acids (Table 2),
emmer (172  18 mg/kg DM), spelt (138  17) and bread wheat particularly in the case of the conjugated fraction. This discrepancy
(162  4), than in durum wheat (267  20) and einkorn is possibly due to the presence of other reducing agents in the
(229  35 mg/g DM). extracts (Everette et al., 2010); additionally, some phenolic acids
The bound compound means (Table 1) showed that may escape HPLC determination, being unknown or in low con-
T. monococcum and T. turgidum had higher content than T. aestivum centration. The total polyphenol content was closely correlated to
128 A. Brandolini et al. / Journal of Cereal Science 58 (2013) 123e131

30

Mean (standard error) of phenolic acids (mg/kg DM), total polyphenols (mg ferulic acid equivalent/kg DM) and antioxidant activity (mmol Trolox equivalent/kg DM) in whole meal flour of diploid T. monococcum ssp.
monococcum (13 samples), tetraploid T. turgidum ssp. dicoccum (7 samples), T. turgidum ssp. turanicum (2 samples), T. turgidum ssp. durum (4 samples), and hexaploid T. aestivum ssp. spelta (9 samples) and T. aestivum ssp.

 0.04
 0.08
 0.08
 0.07

 0.06

 0.06
 0.12

 0.03
A

2011
25

Antioxidant activity

2.6
2.6
2.4
2.4

2.2

2.8
2.3

2.6

Temperature (°C)
20

0.09
0.17
0.13
0.06

0.06

0.15
0.08

0.11
2.5 
2.9 
2.3 
2.1 

2.2 

2.7 
2.0 

2.4 
15
2008

10

 20.3
 53.6
 41.3
 30.8

 20.7

 16.0
 62.1

 14.9
5
1020
941

1043
893

838

1108
2011

912

980 0
Total polyphenols

1/4 8/4 15/4 22/4 29/4 6/5 13/5 20/5 27/5 3/6 10/6 17/6 24/6 1/7 8/7 15/7
 42.0
 60.3
 54.6
 27.6

 15.3

 62.2
 38.1

 41.4

50
45 B
1001
957

1205
844

881

1136
2008

836

941

40
35

Rainfall (mm)
 33.2

 10.2
 20.4
 19.6
 17.0

 18.0

 32.8
 5.7

30
Insoluble bound fraction

2011

25
546

600
588
555
584

518

612
594

20
Phenolic acids

15
 27.8

 22.9
 55.1
 37.6
 18.9

 49.6
 23.1
 7.2

10
2008

498

598
715
556
540

441

647
577

5
0
1/4 8/4 15/4 22/4 29/4 6/5 13/5 20/5 27/5 3/6 10/6 17/6 24/6 1/7 8/7 15/7
 0.07

 0.04
 0.05
 0.05
 0.03

 0.02

 0.07
 0.04

Date
2011
Antioxidant activity

0.7

0.5
0.5
0.6
0.8

0.6

0.4
0.6

2008 2011

Fig. 2. Mean temperature (A) and rainfall (B) from May 1 to July 15 2008 and from May
 0.05
 0.07

 0.04
 0.06
 0.05
 0.04

 0.01

 0.02

1 to July 15 2011 at S. Angelo Lodigiano (LO), Italy.

2008

0.6
0.6

0.5
0.4
0.5
0.8

0.6

0.4

the phenolic acid quantification for both conjugated (r ¼ 0.77;

p  0.01) and bound (r ¼ 0.85; p  0.01) fractions, as well as for
 11.5
 22.4

 10.2
 16.2
 16.8
 13.8

 36.6

 23.8

their sum (r ¼ 0.83; p  0.01). In good agreement with the phenolic

acid results, only a small proportion of total polyphenols, ranging
2011

205
276

204
211
244
273

193

201

from 15.4% in T. aestivum to 22.8% in T. monococcum, was due to the

Total polyphenols

conjugated fraction.
The ANOVAs (not presented) confirmed the existence of sizeable
11.8
15.2

9.6
7.8
7.3
8.7

8.0

7.0

differences among species, and einkorn showed a higher concen-









tration of conjugated polyphenols (Table 2; on average, 252  10.9

2008
aestivum (4 samples) accessions cropped in two different years.

173
205

164
169
187
231

159

144

vs. 215  10.9 vs. 184  7.5 mg FAE/kg DM, respectively) and a lower
content of bound polyphenols (868  20.8 vs. 949  33.6 vs.
1010  22.9 mg FAE/kg DM) than T. turgidum and T. aestivum. With
 3.09
 1.53

 2.47
 3.13
 1.29
 2.33

 3.41

 4.24
Soluble conjugated fraction

regard to total (i.e. conjugated þ bound) polyphenols, einkorn

showed the lowest value among species. Nevertheless, when the
2011

42.8
49.9

44.1
48.6
48.9
52.6

46.3

47.0

subspecies were taken into account, it became evident that einkorn

(1120  27.5 mg FAE/kg DM), emmer (1115  42.8 mg FAE/kg DM),
Phenolic acids

KamutÒ (1035  23.7 mg FAE/kg DM) and spelt (1149  23.3 mg

 2.00
 2.23

 1.61
 1.77
 1.38
 2.31

 1.76

 2.98

FAE/kg DM) had similar contents, while durum wheat

(1314  58.1 mg FAE/kg DM) and bread wheat (1294  31.3 mg FAE/
2008

38.3
40.7

38.9
37.9
39.1
48.5

36.0

40.1

kg DM) showed the highest concentrations. Serpen et al. (2008)

stated that emmer (6.33  0.98 mmol gallic acid equivalent/g) had
a higher concentration of these compounds than einkorn and the
ssp. turanicum
ssp. dicoccum

ssp. aestivum
T. monococcum

bread wheat controls (3.37  0.76 and 4.36  1.55 mmol gallic acid
ssp. durum

ssp. spelta
T. turgidum

T. aestivum

equivalent/g, respectively). On the other hand, Abdel-Aal and

Rabalski (2008) reported that einkorn (2319e2355 mg FAE/g),
Table 2

emmer (2323 mg FAE/g) and spelt (1121e2382 mg FAE/g) had similar

values, significantly higher than those of KamutÒ (1851e1917 mg
 A. Brandolini et al. / Journal of Cereal Science 58 (2013) 123e131 129

Table 3
Mean (standard error) of total phenolic acids (mg/kg DM), total polyphenols (mg ferulic acid eqivalent/kg DM) and antioxidant activity (mmol Trolox equivalent/kg DM) in
whole meal flour of diploid T. monococcum ssp. monococcum (13 samples), tetraploid T. turgidum ssp. dicoccum (7 samples), T. turgidum ssp. turanicum (2 samples), T. turgidum
ssp. durum (4 samples), and hexaploid T. aestivum ssp. spelta (9 samples) and T. aestivum ssp. aestivum (4 samples) accessions cropped in two different years.

Soluble conjugated þ insoluble bound fractions

Phenolic acids Polyphenols Antioxidant activity

2008 2011 2008 2011 2008 2011

T. monococcum 588  20.4 636  18.1 1075  36.7 1166  38.0 2.9  0.09 3.2  0.09
T. turgidum 595  37.4 604  19.6 1143  50.1 1184  44.4 2.9  0.10 3.0  0.07
ssp. dicoccum 539  27.1 596  32.8 1041  34.3 1188  70.4 2.6  0.09 3.0  0.11
ssp. turanicum 477  5.5 564  21.4 1040  7.3 1030  57.2 2.8  0.05 2.8  0.09
ssp. durum 753  55.6 637  22.3 1374  53.6 1254  46.5 3.3  0.12 3.1  0.10
T. aestivum 637  23.5 644  10.9 1164  40.6 1223  19.8 3.1  0.10 3.1  0.05
ssp. spelta 615  23.3 637  6.3 1113  42.0 1185  14.5 3.0  0.13 3.1  0.05
ssp. aestivum 687  52.3 659  34.8 1279  66.7 1309  16.5 3.2  0.17 3.2  0.12

FAE/g), durum wheat (881 mg FAE/g) and bread wheat (1144e (2.58  0.052 and 2.37  0.075 vs. 2.25  0.054 mmol TE/kg DM,
2190 mg FAE/g). respectively); the overall antioxidant activity (sum of conjugated
As formerly stated for phenolic acids, the year had significant and bound activities) of the three species was similar (Table 3).
effect only on total polyphenols of the conjugated fraction. The Direct comparison of these results to those reported in literature is
influence of the environment on free total polyphenols using the difficult, because of different combinations of the time/tempera-
FolineCiocalteu method has been tested by few researchers. ture/concentration parameters or diverse methods to evaluate
Heimler et al. (2010), surveying 9 T. turgidum and 17 T. aestivum antioxidant activity. However, in general Abdel-Aal and Rabalski
accessions, detected differences for free total polyphenols between (2008) recorded few differences in scavenging capacities among
two cultivation years. Lachman et al. (2011) recorded significant selected primitive and modern wheat varieties of different ploidy
changes between two cropping years in their study of two emmer, levels; Serpen et al. (2008), instead, noticed that the antioxidant
two einkorn and three bread wheats, and concluded that the su- capacity of emmer was about 1.2-fold higher than that of einkorn
perior free total polyphenol contents were a consequence of lower and about 1.7-fold more than that of two bread wheat controls.
rainfall and higher temperatures during the ripening stages of the
cereals. 3.5. Polyphenol distribution in the kernel

3.4. Antioxidant activity The HPLC analysis carried out on the different milling fractions
of the einkorn cv. Monlis (Table 4) showed that the conjugated
The beneficial properties of polyphenols are due to their strong phenolic acids are extremely scarce in the endosperm
antioxidant properties against free radicals and other reactive ox- (1.3  0.72 mg/kg DM), while they are more abundant in the bran
ygen species. In this experiment, the antioxidant capacities of the (151  2.36 mg/kg DM), and very concentrated in the germ
different soluble conjugated and insoluble bound extracts were (1039  33.97 mg/kg DM). A similar distribution was observed for
challenged using the DPPH method and the reactive radical Trolox. the conjugated polyphenols (82.9  0.69, 635.0  1.13 and
The results were coherent with the phenolic acid and poly- 2921.4  215.61 mg FAE/kg, respectively). These data confirmed the
phenol data (Table 2 and Supplementary Table 3). The ANOVAs (not results reported in literature, which state the paucity of phenolic
presented) confirmed the existence of differences among species/ acids in the endosperm and their abundance in the bran (Jiang et al.,
subspecies and years. The conjugated extracts of einkorn showed a 2011; Kim et al., 2006).
higher antioxidant activity than those of the tetraploid and hexa- The endosperm was almost bound phenolic acid-free too: in
ploid wheats (0.80  0.027 vs. 0.57  0.034 vs. 0.53  0.029 mmol fact, only p-coumaric acid (1.7  0.05 mg/kg DM) and ferulic acid
TE/kg DM, respectively), while for the bound extracts such activity (35.5  0.50 mg/kg DM) were detected. As a consequence, bound
was highest in T. aestivum ssp. aestivum and T. turgidum ssp. durum total phenolic acids (37.2  0.45 mg/kg) and bound total

Table 4
Mean (standard error) of selected soluble conjugated, insoluble bound and conjugated þ bound phenolic acids (mg/kg DM), and total polyphenols (mg ferulic acid equivalent/
kg DM) in endosperm, bran and germ of Triticum monococcum ssp. monococcum cv Monlis.

p-Hydroxybenzoic Vanillic Syringic p-coumaric Ferulic Total phenolic acids Total polyphenols

Conjugated
Endosperm 0.00 0.4  0.39 0.3  0.30 0.7  0.03 0.00 1.3  0.72 82.9  0.69
Bran 3.5  0.07 10.9  0.15 4.5  0.06 9.5  0.34 122.5  2.31 151.0  2.36 635.0  1.13
Germ 41.7  16.26 157.4  7.97 97.0  4.12 25.3  1.10 717.7  28.70 1039.0  33.97 2921.4  215.61
Bound
Endosperm 0.00 0.00 0.00 1.7  0.05 35.5  0.50 37.2  0.45 58.9  1.72
Bran 8.5  0.45 21.4  0.74 11.8  0.39 97.8  2.30 2479.5  7.51 2619.0  10.61 4260.7  42.96
Germ 0.00 0.00 18.3  3.66 0.00 2692.6  50.43 2710.9  54.09 4190.5  54.09
Conjugated þ Bound
Endosperm 0.00 0.4  0.39 0.3  0.30 2.3  0.02 35.5  0.50 38.5  1.17 141.7  2.41
Bran 12.0  0.53 32.4  0.89 16.3  0.33 107.3  1.95 2601.9  5.21 2770.0  8.25 4895.7  44.09
Germ 41.7  16.26 157.4  7.97 25.3  7.78 25.3  1.10 3410.3  21.74 3749.9  20.13 7111.9  20.13
130 A. Brandolini et al. / Journal of Cereal Science 58 (2013) 123e131

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