LWT - Food Science and Technology 54 (2013) 353e359

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LWT - Food Science and Technology

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

Phytochemical composition of extracts from wheat grain fractions

obtained by tangential abrasive dehullingq
Yongfen Chen a, Nurhan Turgut Dunford a, b, *, Carla Goad c
a

Department of Biosystems and Agricultural Engineering, Oklahoma State University, USA

Robert M. Kerr Food & Agricultural Products Center, Oklahoma State University, USA
c
Department of Statistics, Oklahoma State University, USA
b

a r t i c l e i n f o

a b s t r a c t

Article history:
Received 2 March 2013
Received in revised form
4 June 2013
Accepted 5 July 2013

This study examined the phytochemical content and composition of extracts from wheat bran fractions
obtained by abrasive dehulling.
Wheat grain was fractionated using a Tangential Abrasive Dehulling Device (TADD). The aqueous
ethanol extracts of whole wheat, bran, TADD and commercial aleurone samples were analyzed for their
total phenolic content (TPC), oxygen radical absorbance capacity (ORAC) and 2,2-diphenyl-1-picrylhydrazyl radical scavenging capacity (DPPH). The fractions with the highest antioxidant capacity
were further analyzed for their tocopherol, phenolic acid, carotenoid and organic acid contents. The
correlations between chemical composition and antioxidant properties of wheat extracts were
developed.
All TADD samples had higher tocopherol contents than the bran from a quadrumat senior mill. Lutein
was the primary carotenoid in all the samples. Ferulic and caffeic acids were the strongest contributor to
DPPH and TPC of the extracts, respectively. Correlation between ORAC and tocols content of the samples
was positive and strong (r 0.75793, p < 0.0001). This study demonstrated that TADD was more
effective than quadrumat senior mill to obtain wheat bran fractions enriched in health benecial phytochemicals. The correlations between chemical composition and antioxidant properties of TADD bran
extracts developed in this study are helpful to formulate products with desired efcacy.
2013 Elsevier Ltd. All rights reserved.

Keywords:
Tangential abrasive dehulling
Phytochemicals
Wheat
Antioxidants

1. Introduction
Diverse health benets of wheat including reducing cardiovascular disease, diabetes and some cancers are attributed to its high
concentration of bioactive components with antioxidant properties
(Liese et al., 2003; Liu, 2007; McCullough et al., 2003). Bioactive
wheat components are concentrated in the outer layers of the
grain. It has been shown that antioxidant capacity of bran is more
than two times higher than that of the rened our (Yu, 2008).
Similarly, Liyana-Pathirana and Shahidi (2007) reported higher
antioxidant capacity in bran than our and shorts. Tocopherols,
tocotrienols, carotenoids, phenolic acids and organic acids
contribute to the antioxidant properties of wheat.

q Published with approval of the Director, Oklahoma Agricultural Experiment

Station.
* Corresponding author. Robert M. Kerr Food & Agricultural Products Center,
FAPC Room 103, Stillwater, OK 74078-6055, USA. Tel.: 1 405 744 7062; fax: 1 405
744 6313.
E-mail address: Nurhan.Dunford@okstate.edu (N.T. Dunford).
0023-6438/$ e see front matter 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.lwt.2013.07.007

Wheat is a good source of tocopherols and tocotrienols which

are together referred to as tocols. These compounds are not evenly
distributed in the kernel. Total tocols content in wheat bran is
almost two-fold higher than that of whole wheat our (Khan &
Shewry, 2009). Wheat germ contains specically high contents of
a-tocopherol, 256 mg/g, and b-tocopherol, 114 mg/g (Morrison,
Coventry, & Barnes, 1982; Piironen, Syvaoja, Varo, Salminen, &
Koivistoinen, 1986).
Total phenolic acid content in wheat bran (4527 mg/g) is
signicantly higher than that of whole wheat our (1342 mg/g)
(Mattila, Pihlava, & Hellstrom, 2005). Ferulic, vanillic, caffeic,
syringic, hydroxybenzoic, and p-coumaric acids are commonly
found in wheat (Kim, Tsao, Yang, & Cui, 2006; Okarter, Liu, Sorrells,
& Liu, 2010). Ferulic acid is the major cinnamic acid in cereals, and
is the most abundant phenolic acid in wheat grain. Ferulic acid is
concentrated in the wheat aleurone, 7.22e9.06 mg/mg (Antoine
et al., 2003). The ferulic acid content of bran and germ is 52e70
fold higher than the endosperm (Adom, Sorrells, & Liu, 2005).
Vanilic, syringic and p-coumaric acids are also found in wheat
samples but in lesser amounts than ferulic acid (Moore et al.,
2005).

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Y. Chen et al. / LWT - Food Science and Technology 54 (2013) 353e359

Even though carotenoid content in wheat grain is not very high,

wheat could provide signicant amounts of carotenoids as wheat is
readily found in most daily diets. b-Carotene, zeaxanthin, cryptoxanthin and lutein are the major carotenoids and lutein is the
primary xanthophyll found in wheat (Khan & Shewry, 2009). Moore
et al. (2005) reported that total carotenoid contents in eight soft
wheat varieties grown in Maryland ranged from 1.30 to 1.68 mg/g
and lutein was the most abundant carotenoid, 0.82e1.14 mg/g, followed by zeaxanthin and b-carotene. In another study lutein was
also found to be the predominant carotenoid, 0.97e2.43 mg/g, in
hard red winter wheat. Signicant amounts of zeaxanthin, bcryptoxanthin and b-carotene were detected in wheat samples as
well (Zhou, Yin, & Yu, 2005). Carotenoid content of bran and germ
mixture was shown to be almost two-fold higher than that of the
wheat endosperm (Adom et al., 2005).
Information on organic acid content in wheat grain is limited.
Malic, aconitic and citric acids are the major organic acids in wheat
(Nelson & Hasselbring, 1931). Seven varieties of two and four weekold wheat grain samples were examined for their organic acid
content (Burke, Hilliard, Watkins, Russ, & Scott, 1985). Succinic,
malic, aconitic and citric acids were detected in the two weeks old
wheat while no succinic acid was detected in the four weeks old
samples indicating that compositional changes occur during grain
development.
Abrasive decortication or dehulling is a relatively inexpensive
process that has been used to separate bran and abraded grain in
small scale production facilities. Although bran fraction from
abrasive dehulling operation is expected to be rich in health
benecial phytochemicals, its potential for utilization in functional
foods has not been examined. Interest in plant extracts as rich
sources of health benecial bioactive compounds and antioxidants
continue to grow. Plant extracts are very complex mixtures of a
number of compounds that may interact synergistically. An understanding of the correlations between chemical composition and
antioxidant capacity of wheat grain fractions is critical for
designing efcient separation techniques to produce antioxidant
rich products. Furthermore, formulation of nutraceutical products
for specic health conditions entails the knowledge of relationship
between chemical composition and the function and effectiveness
of the product ingredients.
In a previous study we have reported that tangential abrasive
dehulling is an effective technique to obtain wheat grain fractions
with enhanced antioxidant properties (Chen, Dunford, & Goad,
2013a). Wheat grain fractions obtained by using a tangential
abrasive dehulling device (TADD) were characterized for their
proximate composition and antioxidant capacity. Phytosterol and
policosanols contents of the TADD fractions have also been reported elsewhere (Chen, Dunford, & Goad, 2013b) The objective of
this study is to characterize the phytochemical content and
composition (phenolic compounds, tocols and organic acids) of
aqueous ethanol extracts of TADD fractions. Correlations between
antioxidant capacity and the chemical compositions of the wheat
bran fractions are also developed.
2. Materials and methods
2.1. Sample preparation
Intrada wheat was collected from Oklahoma State University
(OSU) variety testing program plots at Balko (100 070 W, 36 060 N),
Goodwell (101060 W, 36 060 N), and Alva (36 480 700 N, 98 390 5700 W)
in Oklahoma. Plots had eight 15 cm wide and 12 m long rows. Seeds
were sown into a Ulysses silt loam (ne-silty, mixed, superactive,
mesic Aridic Haplustolls) at Balko; into a Richeld silt loam (ne,
smectitic, mesic Aridic Argiustolls) at Goodwell; and into a Grant

silt loam at Alva. All elds were managed under grain only
practices. Detailed information on plant growth conditions were
reported elsewhere (Chen, 2011). Whole wheat grain samples were
collected after normal harvest. All the Intrada samples from
different locations were mixed prior to the experiments. A commercial aleurone sample from Cargill Co. (Wayzata, MN, U.S.A.) was
included as a reference. Samples were stored in paper bags and
kept in a freezer at 20  C after being received in our laboratory
until the testing began within six months.
2.2. Grain milling and extraction
Wheat grain fractions obtained by using a quadrumat senior
mill (C.W. Brabender Instrument, INC, South Hackensack, NJ,
USA) and a sieve tester (Gilson Company, Inc, Lewis Center, OH,
USA) (particle size over 500 mm) have been referred to as bran
in this study. Grain dehulling by TADD was described in detail
elsewhere (Chen et al., 2013a). Dehulling experiments were carried out at 1, 3 and 5 min abrasion time and three grain moisture
levels, 15, 20 and 11% (the original wheat moisture level) by using
a TADD (Venables Tangential Abrasive Dehulling Device, Model
no. 4E-10/220, Venables Machine Works, Ltd, Saskatoon, SK,
Canada). The distance between the abrasive plate (Coarse disc,
Type 4, Perten Instruments, Huddinge, Sweden) and the bottom
edge of the sample cups was set at 0.042 inch. Bran fractions
obtained from TADD processing were referred to as TADD
samples throughout this paper. Whole grain sample was prepared by grinding the wheat kernels using a coffee grinder (Black
& Decker CBG5, Miami, FL, USA) at medium speed 3 times in 30 s
intervals.
The antioxidant extraction method used in this study was
adapted from literature (Adom & Liu, 2002; Naczka & Shahidi,
2004). Ten grams of TADD, bran, aleurone, and whole wheat samples were extracted separately with 100 mL of ethanol at 80%, v/v,
in water for 16 h in the dark and under nitrogen at room temperature (22  C). Extraction was repeated twice and extracts were
combined. Extract concentration was determined by evaporating
the solvent from 25 mL of extract under vacuum (35  C, 210e
240 mbar) and reported as mg solvent free extract/mL solvent. The
protocols used for ORAC, TPC and DPPH measurements were discussed in detail elsewhere (Chen et al., 2013a).
2.3. Analytical tests
2.3.1. Tocopherol and tocotrienols
Tocopherol and tocotrienol contents of the samples were
analyzed by using an HPLC system (Alliance 2690, Waters Corp.,
Milford, MA, USA) which consisted of a separations module (Model
2695) and a Photodiode Array Detector (PDA) (Model 2996, Waters). Two microliter of sample was injected into a normal phase
HPLC column, Zorbax RX-SIL (5 mm particle size, 4.6  250 mm,
Agilent Technologies, Santa Clara, CA, USA) and separation was
achieved by using a mobile phase consisting of hexane (HPLC
Grade, Fisher Scientist, Fairlawn, NJ, USA) and isopropanol (HPLC
grade, Pharmco Co., Brookeld, CT, USA) at a ratio of 99:1 (v/v).
Isocratic ow rate was 1.3 mL/min. Column temperature was set at
35  C (Katsanidis & Addis, 1999). Seven individual tocol standards,
a-tocopherol (a-T), b-tocopherol (b-T), d-tocopherol (d-T), gtocopherol (g-T), a-tocotrienol (a-T3), b-tocotrienol (b-T3), d-tocotrienol (d-T3) and g-tocotrienol (g-T3) were purchased from Sigma
(SigmaeAldrich Corporation, St. Louis, MO, USA) and used for peak
identication. The data collection and analysis were managed using
Waters Pro Empower software (Version 5.00.00.00, Waters Corp.)
running on a PC (DELL, XP-Professional, Round Rock, TX, USA).

Y. Chen et al. / LWT - Food Science and Technology 54 (2013) 353e359

2.3.2. Phenolic compounds

Phenolic acids in samples were analyzed by a Waters 2695 series
HPLC (Alliance 2690, Waters Corp.) equipped with a PDA (Model
2996, Waters Corp.) and a SunFire C18 (250  4.60 mm, 5 mm, Phenomenex, Torrance, CA, USA) column. After ltered through a
0.45 mm syringe lter, 10 mL of sample was injected to the HPLC. A
mobile phase consisting of acetonitrile (solvent A) (HPLC grade,
VWR, West Chester, PA, USA) and 2 mL acetic acid in 100 mL water
(solvent B) was used at a ow rate of 1.0 mL/min for a total run time of
70 min. The solvent gradient program was as follows: 100 mL/100 mL
B to 85 mL/100 mL B in 30 min, 85 mL/100 mL B to 50 mL/100 mL B in
20 min, 50 mL/100 mL B to 0 mL/100 mL B in 5 min and 0 mL/100 mL B
to 100 mL/100 mL B in 15 min. Benzoic and cinnamic acids were
monitored at wavelengths, 280 and 320 nm, respectively. Gallic,
benzoic, vanillic, caffeic, syringic, p-coumaric and ferulic acids from
Sigma were used as standards. Data signals were acquired and processed on a PC running the Waters Pro Empower software.
2.3.3. Carotenoids
The carotenoid analysis was conducted by using a Waters HPLC
system equipped with a Waters 2487 dual-wavelength absorbance
detector, a Waters 600S controller, a Waters 616 pump, an inline
degasser, and a Waters 717 autosampler (Waters Corp.). Mobile
phase consisted of HPLC grade methanol (Fisher Scientic, Pittsburgh, PA, USA) and deionized water from a Millipore water purication system (Millipore Corporation, Molsheim, France) at a ratio
of 95:5 (v/v) as solvent A and HPLC grade Methyl tert-butyl ether
(MTBE) (Mallinckrodt Baker, Inc. Phillipsburg, NJ, USA) as solvent B.
Isocratic elution was achieved with 75 mL/100 mL solvent A and
25% solvent B. Sample (10 mL) was injected to YMC Carotenoid S-3
column (250  4.6 mm, 3 mm column, Waters Corp.) at a ow rate of
1.9 mL/min. Column temperature was set at 35  C. The individual
carotenoid standards, lutein, zeaxanthin and b-carotene from
Sigma were detected at 450 nm. Data signals were acquired and
processed on a PC (running the Waters Empower2 software
(Version 6.20.00.00, Waters Corp.)).
2.3.4. Organic acids
The organic acids analysis was performed by using an Agilent
1200 Series HPLC system. The mobile phase, 0.005 M sulfuric acid,
was degassed by a Degasser (G1322A, Agilent, Santa Clara, CA, USA)
by using a Quad Pump (G1311A, Agilent). A Bio-rad Aminex HPX87H column (30  7.8 mm, Bio-rad, Hercules, CA, USA) equipped
with a guard column (30  4.6 mm, Bio-rad) were used for the
analysis. Sample, 5 mL, was injected by an autosampler (ASL,
G1329A, Agilent). Flow rate was 0.6 mL/min. Column temperature
was 35  C. A dual-wavelength absorbance detector (G1315D, Agilent) was used to measure the absorbance of organic acids at
210 nm. Citric, ascorbic, malic, succinic, lactic and fumaric acids
from SigmaeAldrich were used as standards. Data signals were
acquired and processed on a PC running the ChemStation for LC 3D
software (Rev. B.04.0x. Agilent).
3. Experimental design and statistical analysis
For TADD experiments a 3  3 factorial (1, 3 and 5 min abrasion
time and 15, 20 and 11 g/100 g moisture levels) design was used.
Three references (aleurone, Intrada bran, and whole wheat) were
also analyzed. The antioxidant capacity of TADD samples were
compared to the aleurone sample by using Dunnetts multiple
comparisons method. Samples having signicantly higher or similar
antioxidant capacity as the aleurone were further analyzed for their
bioactive compounds. All the milling tests were carried out at least
in duplicate and in randomized order with the mean values being
reported. Analytical tests were performed in triplicate. The means

355

were compared using Tukeys adjustment. Experimental data were

assessed for non-normality and heterogeneity of variances. Appropriate linear and generalized linear mixed models were analyzed
using version 9 SAS/GLM, SAS/MIXED and SAS/GLIMMIX procedures
(Software Version 9.2. SAS Institute Inc., Cary, NC). All statistical tests
were performed at the 0.05 level of signicance.
4. Results and discussion
The bran fractions obtained by using a TADD at various grain
moisture contents and abrasion times were labeled as follows:
TADD e Moisture content of the sample e abrasion time. For
example TADD-20-3 refers to the TADD sample obtained at 20 g/
100 g grain moisture content and 3 min TADD abrasion time. A
statistical analysis of the results obtained from three antioxidant
capacity tests, TPC, DPPH and ORAC, indicated that TADD-11-1,
TADD-15-1, TADD-15-3, TADD-20-3 and TADD-20-5 had higher
antioxidant capacity than the other TADD samples (Chen et al.,
2013a). Therefore, these samples were further analyzed for their
chemical composition focusing on the health benecial bioactive
compounds with antioxidant properties.
Although the same amounts of sample and solvent were used to
obtain all the extracts there were signicant differences in the
extract concentrations. Commercial aleurone resulted in the highest extract concentration and consequently highest extract amount
(Table 1). This is due to the very low starch content in the aleurone
sample and very low amount of aqueous ethanol soluble compounds present in starch. Hence, extract yield varied with starch,
bran and oil content of the samples (Chen et al., 2013a). There were
signicant differences among the ORAC, TPC and DPPH of the extracts (Table 1). Although aleurone gave the highest extract yield,
the extract did not result in the highest ORAC value indicating that
the extract contained more material that did not possess oxygen
radical absorbance capacity than that in the TADD-11-1 and TADD20-5 extracts. Hence, phytochemical content and composition of
the extracts were examined to evaluate the effect of extract
composition on antioxidant capacity as measured by different
methods, ORAC, TPC and DPPH.
4.1. Tocopherols and tocotrienols
Whole wheat had the lowest tocols content, 3.02 mg/g sample
(Table 2). Aleurone (12.20 mg/g sample), TADD-20-3 (11.02 mg/g
sample), and TADD-15-1 (10.47 mg/g sample) contained signicantly

Table 1
ORAC, TPC, DPPH and concentration of wheat extracts.
Samplea

ORAC (mmol TE/g TPC (GE mg/g DPPH (%

Extract
inhibition)
extract,
concentration of extract,
dry basis)
(mg/mL)
dry basis)

TADD-11-1
TADD-15-1
TADD-15-3
TADD-20-3
TADD-20-5
Bran
Whole wheat
Aleurone

10.1
10.3
8.4
9.2
7.7
10.1
5.3
11.3










0.04B
0.5B
0.4D
0.2C
0.03E
0.1B
0.3F
0.1A

478.7
256.6
276.6
472.7
276.8
205.4
159.3
411.6










21.8A
11.7E
12.8D
22.0B
13.8D
9.6F
7.7G
20.9C

22.8
22.1
26.4
23.0
26.2
19.7
19.7
22.1










0.3B
0.9B
1.1A
0.8B
1.1A
0.5C
1.0C
1.1B

42.6
41.2
40.8
33.0
13.0
30.0
9.8
42.4










0.4A
0.4AB
0.3B
0.4C
0.2E
0.3D
0.1F
0.2A

ABCDEF
Sample means within a column that have the same letter are not signicantly
different (a 0.05).
The data is reported as mean  standard deviation.
a
For TADD samples: TADD e moisture content e abrasion time, i.e. TADD-11-1
refers to bran obtained by using TADD at 11% (original) moisture level, 1 min
abrasion time. Whole wheat refers to the whole Intrada wheat sample. Bran and
aleurone refer to the bran from quadrumat mill and commercial samples,
respectively.

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Y. Chen et al. / LWT - Food Science and Technology 54 (2013) 353e359

higher tocols than the other samples. Dehulling time and grain
moisture interaction had a signicant effect on the tocopherol
content of the samples (p < 0.0001, F 224.91). All TADD samples
had higher tocols content than the bran indicating higher efciency
of TADD than quadrumat senior mill for tocol enrichment in wheat
fractions. The tocols content of bran sample reported in this study
was in agreement with other studies published earlier (Zhou et al.,
2005; Zhou & Yu, 2004). Our results also support previous reports
that have established concentration of tocols in the outer layers of
wheat grain (Hidalgo & Brandolini, 2008).
HPLC method used in this study did not separate a-tocopherol
from a-tocotrienol; hence the result was expressed as atocopherol a-tocotrienol. Similarly g-tocopherol and g-tocotrienol peaks overlapped on the sample chromatograms; therefore,
results for these compounds were reported as g-tocopherol gtocotrienol. a-Tocopherol a-tocotrienol and g-tocopherol gtocotrienol were detected in all TADD and reference samples except
whole wheat (Table 2). Only a-tocopherol a-tocotrienol peak was
identied in the whole wheat. The only sample that contained btocopherol and d-tocotrienols was aleurone. In a study of
Maryland-grown soft wheat bran, only a-tocopherol, ranging from
3.4 to 10.1 mg/g, was reported (Moore et al., 2005). Zhou et al.
(2005) examined tocopherol content and compositions in hard
red winter wheat bran and reported the presence of a-tocopherol
(4.10e6.51 mg/g), d-tocopherol (0.16e0.38 mg/g) and g-tocopherol
(3.68e5.59 mg/g). The latter results are in agreement with the data
presented in this study. TADD-15-1 and TADD-20-3 had signicantly higher a-tocopherol a-tocotrienol as well as gtocopherol g-tocotrienol content than the other samples. Tocols
are lipid soluble antioxidants that are concentrated in bran and
germ. TADD-20-3 and aleurone contained signicant amount of
lipid (Chen et al., 2013a).
4.2. Carotenoids
Total carotenoid content of the samples ranged from 1.89 to
3.83 mg/g sample (Table 3). Whole wheat had lower carotenoid
content than the other samples due to the presence of large
quantity of endosperm in the sample (Chen et al., 2013a). Effect of
grain moisture and abrasion time interaction had a signicant effect on the total carotenoid content of the samples (p < 0.0001,
F 174.33). Total carotenoids and lutein contents of TADD-15-1,
TADD-15-3, TADD-20-3, and TADD-20-5 were higher than the aleurone. Lutein, followed by zeaxanthin is reported to be the major
carotenoids in cereal grains (Konopka, Czaplicki, & Rotkiewicz,
2006). In this study, only lutein and zeaxanthin were detected in

Table 2
Tocols content and compositions of wheat samples (mg/g sample, dry basis).
Sample

a-T a-T3a b-Ta

TADD-11-1
TADD-15-1
TADD-15-3
TADD-20-3
TADD-20-5
Bran
Whole
wheat
Aleurone

5.4
6.6
4.4
7.7
5.3
3.8
3.0









0.2B
0.2A
0.1CD
0.3A
0.2BC
0.05D
0.04E

6.6  0.2A

n.d.b
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.

g-T g-T3a d-Ta

2.4 
3.8 
2.2 
3.3 
2.3 
1.9 
n.d.

0.07B
0.1A
0.07BC
0.1A
0.09BC
0.06C

2.4  0.09 1.5  0.04D

n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.

Total tocols
content
7.8
10.5
6.7
11.0
7.5
5.8
3.0









0.3B
0.3A
0.2BC
0.3A
0.1B
0.08C
0.04D

1.70  0.05 12.2  0.4A

ABCDE
Sample means within a column that have the same letter are not signicantly
different (a 0.05).
a
See Table 1 for the sample abbreviations. Other abbreviations are as following:
a-T a-T3: a-tocopherol a-tocotrienol; b-T: b-tocopherol; g-T g-T3: gtocopherol g-tocotrienol; d-T: d-tocopherol.
b
n.d.: Not detected.

Table 3
Carotenoids content and compositions of wheat samplesa (mg/g sample, dry basis).
Sample

Lutein

TADD-11-1
TADD-15-1
TADD-15-3
TADD-20-3
TADD-20-5
Bran
Whole wheat
Aleurone

2.6
2.7
2.8
2.9
2.8
2.6
1.4
2.4










0.02BC
0.02AB
0.04AB
0.05A
0.03AB
0.006B
0.01D
0.02C

Zeaxanthin
0.9
0.9
1.0
0.9
1.0
0.9
0.5
0.9










0.004B
0.005AB
0.03A
0.007AB
0.005A
0.003AB
0.001C
0.007AB

Total carotenoids content

3.5
3.6
3.8
3.8
3.8
3.5
1.9
3.3










0.02CD
0.02ABC
0.06AB
0.05A
0.03AB
0.007BCD
0.01E
0.03D

ABCD
Sample means within a column that have the same letter are not signicantly
different (a 0.05).
a
Refer to Table 1 for sample abbreviations.

all the samples. Lutein was the primary carotenoid in all samples
ranging from 1.41 to 2.92 mg/g sample. This range is comparable to
the values reported in earlier studies (Hidalgo, Brandolini, Pompei,
& Piscozzi, 2006; Zhou & Yu, 2004). Amount of lutein in the
samples was almost two to three times higher than that of zeaxanthin (0.48e0.95 mg/g sample). Similar results were also reported
by other research groups (Moore et al., 2005; Zhou et al., 2005;
Zhou & Yu, 2004). Similar to the ndings of this study, Adom and
Liu (2002) reported higher carotenoids content in wheat bran
than endosperm.
4.3. Phenolic acids
In cereal grains, phenolic acids are present in three forms: free,
soluble-conjugated and insoluble-bound. Most phenolic acids exist
in a bound form. Only free phenolic acids can be extracted by
aqueous ethanol (Adom & Liu, 2002). Whole wheat had signicantly lower total phenolic acids (16.33 mg/g sample) than other
samples mainly due to the presence of endosperm (Table 4). TADD20-3, TADD-11-1, TADD-15-3 and aleurone had the highest total
phenolic acids content among the samples (41.21e47.23 mg/g
sample). TADD-11-1, TADD-15-3, and TADD-20-3 had signicantly
higher phenolic acids contents than bran (39.51 mg/g sample), but
not signicant from the commercial aleurone (45.11 mg/g sample).
Presence of free phenolic acids in wheat germ, the main source of
lipids is known (King, 1962). TADD-11-1, TADD-15-3, and TADD-203 contained signicantly higher amount of lipid than bran indicating that latter TADD samples contained some germ fraction
(Chen et al., 2013a). Hence, three TADD fractions with high oil
content also had higher phenolic acids than bran. Effect of grain
moisture content and abrasion time interaction had a signicant
effect on the total phenolic acid content of the samples.
Benzoic, vanillic, caffeic, syringic, p-coumaric, and ferulic acids
were found in all the samples (Table 4). Ferulic, benzoic, syringic,
and vanillic were the major phenolic acids extracted from wheat
samples. TADD-11-1 and aleurone contained the highest ferulic
acid contents, 14.98 and 12.87 mg/g sample, respectively. There was
no signicant difference between TADD-11-1 and aleurone
(p 0.3007), but TADD-11-1 showed signicantly higher ferulic
acid content than the rest of the samples. Kim et al. (2006)
examined four types of wheat bran and found that free phenolic
acid content in the samples ranged from 3.87 to 31.13 mg/g bran. It
was also reported that ferulic, vanillic and syringic acids were the
major free phenolic acids in bran. These results are comparable to
our study. Adom and Liu (2002) reported lower free ferulic acid
content in wheat sample, 1.11 mg/g grain, than that found in our
samples. It is important to note that Adom and Liu (2002) used
dehulled grain in their study. Free phenolic acids are a small
portion of the total phenolic acids in wheat (Sosulski, Krygier, &
Hogge, 1982). Hence, it is expected that actual total phenolic acid

Y. Chen et al. / LWT - Food Science and Technology 54 (2013) 353e359

357

Table 4
Phenolic acids content and compositions of wheat samplesa (mg/g sample, dry basis).
Sample

Benzoic acid

TADD-11-1
TADD-15-1
TADD-15-3
TADD-20-3
TADD-20-5
Bran
Whole wheat
Aleurone

6.1
6.2
7.8
7.2
6.5
5.6
3.4
6.4










Vanillic acid

0.2CD
0.09CD
0.2A
0.2AB
0.1BC
0.2D
0.01E
0.08BCD

5.8
5.8
8.3
10.3
6.9
6.2
3.3
9.6










Caffeic acid

0.1D
0.08D
0.2B
0.2A
0.2C
0.1CD
0.02E
0.3AB

4.4 
4.6 
5.0 
4.9 
4.8 
4.8 
n.d.b
5.4 

0.1B
0.05B
0.08AB
0.1AB
0.1AB
0.09AB
0.2A

Syringic acid
4.7
4.4
7.6
7.1
5.0
7.2
2.5
6.2










p-coumaric acid

0.2C
0.03C
0.3A
0.1AB
0.1C
0.1AB
0.04D
0.2B

5.2
4.5
5.8
6.1
5.6
4.8
2.4
4.7










Ferulic acid

0.2ABCD
0.03D
0.2AB
0.1A
0.2ABC
0.1BCD
0.1E
0.2CD

15.0
8.6
10.5
11.5
8.7
10.8
4.8
12.9










0.6A
0.3C
0.3BC
0.3B
0.3C
0.5B
0.2D
0.2AB

Total phenolic acids content

41.2
34.2
45.0
47.2
37.5
39.5
16.3
45.1










0.9BC
0.3D
1.0AB
0.6A
0.8CD
0.7C
0.1E
0.9AB

Sample means within a column that have the same letter are not signicantly different (a 0.05).
Refer to Table 1 for sample abbreviations.
n.d.: Not detected.

ABCD
a
b

content (free bound conjugated) of the samples examined in

this study would be much higher than the amounts we report here.
4.4. Organic acids
Whole grain had the lowest organic acid content (29.79 mmol/g
sample). TADD-15-1 (82.50 mmol/g sample) and bran (76.64 mmol/g
sample) had higher amount of organic acids than other samples
(Table 5). Higher organic acid content in all TADD samples than the
aleurone indicates that organic acids are concentrated in the bran
fractions. Effect of grain moisture content and abrasion time
interaction on total organic acid content of the samples was signicant (p < 0.0001, F 226.86).
HPLC method used to analyze organic acids could not separate
ascorbic and malic acids. Hence results were reported as
ascorbic malic acids. Citric, ascorbic malic, succinic, and
fumaric acids were found in all the samples (Table 5). Succinic,
citric, and fumaric acids were the most abundant organic acids in
the samples. In aleurone, the succinic acid comprised approximately 73% of the total organic acids. Literature on organic acids
contents and compositions of wheat components are limited. Most
of the studies on organic acids were carried out on wheat leaves
(Burke et al., 1985; Clark, 1969). It has been reported that wheat
kernel had much greater organic acids contents than those found
in wheat leaves (Lohaus, Blos, & Rudiger, 1983). Malic, citric, succinic and fumaric acids were the major organic acids found in
wheat grain. The latter study reported higher amounts of organic
acids in wheat as compared to our study possibly due to the
different extraction method used.
4.5. Correlations between chemical composition and antioxidant
capacity of the samples
Correlations between ORAC-DPPH [r 0.48288, where r refers
to Pearson Correlation (Coefcient)], ORAC-TPC (r 0.45253) and
TPC-DPPH (r 0.71478) were all signicant (p < 0.0001) and
positive. The strongest correlation was found between TPC and

DPPH, r 0.71478. Similar ndings have been reported by other

research groups (Zhou, Su, & Yu, 2004; Zhou & Yu, 2004).
A positive and signicant correlation was found between DPPH
and total tocols (r 0.62777), phenolic acids (r 0.685000), carotenoids (r 0.52381) and organic acids (r 0.38060). DPPH has
been widely used to evaluate radical scavenging ability of phenolic
compounds which are strong quenchers of the DPPH radicals
(Fauconneau et al., 1997; Villao, Fernndez-Pachn, Moy,
Troncoso, & Garca-Parrilla, 2007). Phenolic acids have both
hydrogen donating and electron transfer ability (Rice-Evans, Miller,
& Paganga, 1996, 1997). DPPH radicals react with phenolic compounds via two different mechanisms, hydrogen atom transfer
(HAT) or the single electron transfer (ET). In these reactions solvent
type plays a key role. The HAT mechanism is dominant when the
reaction takes place in apolar solvent while polar solvents such as
methanol or ethanol leads to ET mechanism (Foti, Daquino, &
Geraci, 2004). In our study, ET mechanism is expected to be
dominant since aqueous ethanol was used for the extraction and
DPPH tests. Tocols, carotenoids, and phenolic acids were shown to
have a signicant positive correlation with DPPH assay (Prior, Wu,
& Schaich, 2005). Tocols and carotenoids can stop oxidation by
donating a hydrogen atom to a free radical (Gurney et al., 1996;
Niki, Saito, Kawakami, & Kamiya, 1984; Terao, 1989).
Contribution of individual compounds to antioxidant capacity
was calculated by using r2 selection method. Ferulic acid
(r2 0.5497) was the strongest contributor to DPPH radical inhibition properties of the extracts (Table 6).
As expected phenolic acid content was positively correlated
with TPC (r 0.83516, r < 0.0001). Caffeic acid was the most
important compound affecting the TPC result (r2 0.8750)
(Table 6).
Benzoic, vanilic, syringic and coumaric acids also had signicant
and positive contributions to TPC.
Strong correlation between ORAC and tocols content of the
samples (r 0.75793, p < 0.0001) (Table 6) supports the peroxyl
radical scavenger properties of the wheat bran extracts. Total
phenolic acid (r 0.65813) and carotenoid (r 0.42861) contents
also had positive and signicant correlation with ORAC. Phenolic

Table 5
Organic acids content and compositions of wheat samplesa (mmol/g sample, dry basis).
Sample
TADD-11-1
TADD-15-1
TADD-15-3
TADD-20-3
TADD-20-5
Bran
Whole wheat
Aleurone

17.9
25.2
22.7
24.4
18.3
23.2
10.4
1.1










0.2
0.3A
0.4A
0.4A
0.4B
0.3A
0.2C
0.03D

1.5
1.5
1.4
1.1
1.0
0.6
0.3
0.4










0.03
0.04A
0.05A
0.04B
0.03C
0.01D
0.005F
0.01E

Succinic acid
25.0
37.7
21.2
19.8
28.8
32.0
13.1
34.1










0.7
0.7A
0.8E
0.6E
0.5CD
0.4BC
0.3F
0.5AB

Sample means within a column that have the same letter are not signicantly different (a 0.05).
Refer to Table 1 for sample abbreviations.

ABCD
a

Ascorbic acid malic acid

Citric acid

Fumaric acid
11.0
18.1
10.8
10.9
14.0
20.9
6.1
11.0










0.2
0.2B
0.2D
0.2D
0.2C
0.3A
0.06E
0.2D

Total organic acids content

55.3
82.5
56.0
56.2
61.9
76.6
29.8
46.6










0.9C
0.9A
1.0BC
0.8BC
0.7B
0.5A
0.4E
0.6D

358

Y. Chen et al. / LWT - Food Science and Technology 54 (2013) 353e359

Table 6
Contribution of bioactive compounds to ORAC, DPPH and TPC assay as determined
by R-square selection method.
Compounds

ORAC

DPPH

TPC

Ferulic acid
a-Tocopherol a-tocotrienol
Vanillic acid
p-Coumaric acid
Benzoic acid
Caffeic acid
g-Tocopherol g-tocotrienol
Lutein
Zeaxanthin
b-Tocopherol
d-Tocopherol
Ascorbic acid malic acid
Syringic acid
Fumaric acid
Citric acid
Succinic acid

0.6282
0.5904
0.3673
0.2693
0.2533
0.2369
0.2342
0.1857
0.1635
0.1615
0.1573
0.1269
0.0866
0.0397
0.0177
0.0167

0.5497
0.2402
0.2156
0.2136
0.3540
0.4293
0.2402
0.2718
0.2986
0.0995
0.1000
0.2874
0.2513
0.0602
0.0161
0.2093

0.3800
e
0.4197
0.5851
0.7008
0.8750
e
e
e
e
e
e
0.3404
e
e
e

acids are effective peroxyl radical scavengers because of their

hydrogen atom donation ability (Yeh & Yen, 2003). Correlation
between total organic acid content and ORAC was not signicant
(p 0.7312). Both ferulic acid-ORAC and a-tocopherol a-tocotrienol-ORAC correlations were positive. Among the bioactive
compounds, ferulic acid was the highest contributor to the ORAC
values (r2 0.6282). Ferulic acid is a potent antioxidant due to its
phenolic nucleus and unsaturated side chain (Castelluccio, Bolwell,
Gerrish, & Rice-Evans, 1996). The eCOOH substitution on the
phenol ring makes it easier for the ferulic acid to donate its
hydrogen atom (Nenadis, Zhang, & Tsimidou, 2003).
5. Conclusions
This study demonstrated that TADD was more effective than
quadrumat senior mill to obtain wheat bran fractions enriched in
health benecial phytochemicals. Effect of grain moisture content
and abrasion time on chemical composition of TADD samples was
signicant (p < 0.001). Considering that abrasive dehulling is a
relatively inexpensive technique and not difcult to operate, it can
be suitable for small scale local production of specialty products.
TADD bran fractions can be utilized as ingredients in functional
food formulations. Extraction of TADD products with aqueous
ethanol would further concentrate phytochemicals with antioxidant properties in the nal product. These extracts could be
formulated into functional foods and nutraceuticals. The correlations between chemical composition and antioxidant properties of
TADD bran extracts developed in this study are helpful to formulate
products with desired efcacy.
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