Research Article

Received: 9 April 2014 Revised: 24 June 2014 Accepted article published: 25 July 2014 Published online in Wiley Online Library:

(wileyonlinelibrary.com) DOI 10.1002/jsfa.6840

Optimisation of ultrasound-assisted extraction

of natural antioxidants from mustard seed
cultivars
Aleksandra Szydłowska-Czerniak,a* Agnieszka Tułodziecka,a György
Karlovitsb and Edward Szłyka

Abstract
BACKGROUND: Modified mustard varieties can produce edible oil with reduced amounts of erucic acid and glucosinolates
and enhanced antioxidant potential. Therefore, this work focused on the optimisation of the ultrasound-assisted extraction of
compounds with high antioxidant capacity from three white mustard seed cultivars using response surface methodology.

RESULTS: The predicted optimum solvent polarity (57.2, 56.5 and 57.6) and ultrasound power-to-sonication time ratio (4.5, 4.8
and 4.3 W min−1 ) resulted in antioxidant capacities determined by the ferric-reducing antioxidant power (FRAP) assay [54.37,
65.75 and 68.55 mmol Trolox equivalent (TE) kg−1 ] and the 2,2′ -diphenyl-1-picrylhydrazyl (DPPH) method (141.65, 175.26 and
185.10 mmol TE kg−1 ) and total phenolics content (23.70, 27.16 and 11.29 mg sinapic acid g−1 ) for extracts obtained from one
traditional and two modified mustard seed varieties. The highest FRAP and DPPH values (69.51 and 197.73 mmol TE kg−1 )
revealed 50% methanolic extract prepared from modified mustard seed cultivar without erucic acid and glucosinolates treated
with ultrasound for 30 min (ultrasound power/ultrasound time = 4 W min−1 ).

CONCLUSION: Ultrasound-assisted extraction was found to be a more rapid, convenient and appropriate extraction method with
higher yield of antioxidants, shorter time and lower solvent consumption in comparison to conventional extraction.
© 2014 Society of Chemical Industry

Keywords: mustard seed; antioxidant capacity; ultrasound-assisted extraction; optimisation

INTRODUCTION mustard samples.2,9,10 Phenolic compounds are an important class

Mustard (Sinapis alba) and rapeseed (Brassica napus) are annual
plants of the family Brassicaceae. However, traditional mustard
seed oil contains high amounts of erucic acid (25–35%) and glu-
cosinolates (250–350 μmol g−1 of defatted seed meal), which lim-
its its application as an edible oil in the European Union (EU). This
is the reason that seed breeder institutions in Poland developed a
new double-zero (00) variety of white mustard seed with contents
of erucic acid and glucosinolates lower than 2% and 3 μmol g−1 ,
respectively. This variety fulfills the EU limit concerning glucosi-
nolates level.1 Moreover, mustard seed is a rich source of pheno-
lic antioxidants such as sinapic acid and sinapine.2 – 4 Sinapic acid
constitutes over 73% of free phenolic acids and about 80–99% of
the total phenolic acids, mainly occurring as an ester of sinapic
acid, sinapine and glucosides.
Phenolic compounds in mustard varieties were determined by
different analytical methods. Among them, a spectrophotomet-
ric method using the Folin–Ciocalteu reagent has often been
proposed for assaying total phenolics content (2.62–36.5 mg g−1 )
in mustard samples.3,5 – 8 However, for separation, identification
and quantification of the individual phenolic compounds chro-
matographic techniques are required.2,4,9,10 High-performance liq-
uid chromatography (HPLC) analysis revealed about 62% sinapine
and about 16% sinapic acid in mustard meal extracts, whereas
only 2.1–211.0 μg g−1 of sinapic acid was determined in various
of natural components in mustard seed cultivars with high antiox-
idant capacity. It has been know that the level of antioxidants in
mustard seed depends on breeding factors such as cultivation
techniques, cultivar type, growing conditions, ripening process, as
well as stress conditions: UV radiation, infection by pathogens and
parasites, wounding, air pollution and exposure to extreme tem-
peratures. However, by combining genetic engineering with classi-
cal plant breeding types of mustard varieties, the functional edible
mustard oil with reduced amounts of erucic acid and glucosino-
lates and enhanced antioxidant potential will be produced.
Only few methods, such as 2,2′ -diphenyl-1-picrylhydrazyl
(DPPH), ferric-reducing antioxidant power (FRAP), 2,2′ -azino-
bis(3-ethylbenzothiazoline)-6-sulfonic acid (ABTS) and oxy-
gen radical absorbance capacity (ORAC) for determination
∗ Correspondence to: Aleksandra Szydłowska-Czerniak, Faculty of Chemistry,
Nicolaus Copernicus University, ul. Gagarina 7, 87-100 Toruń, Poland. E-mail:
olasz@umk.pl
a Faculty of Chemistry, Nicolaus Copernicus University, ul. Gagarina 7, 87-100,
Toruń, Poland
b Bunge Europe Research and Development Center, ul. Niepodległości 42, 88-150,
Kruszwica, Poland

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of antioxidant capacity of mustard samples have been
employed.2,3,5,8,9,11 – 14
One of the most important steps during analysis of total antiox-
idant potential is sample preparation. In general, the traditional
solid–liquid extraction has been proposed before determination
of antioxidant capacity by spectrophotometric assays. However,
this methodology requires very tedious and time-consuming pro-
cedures. Therefore, the use of ultrasound-assisted extraction (UAE)
methodology for extraction of antioxidants from the matrix could
be applied to facilitate their analytical determination, and for
the development of faster and less expensive procedures that
can operate at room temperature and atmospheric pressure and
reduce large amounts of solvents and samples, and hence con-
tribute to the achievement of the green principles in analytical
chemistry. Ultrasonic cavitation creates shear forces that break
cell walls mechanically and improve material transfer, whereas
high-frequency sound disrupts the plant cell wall thereby enhanc-
ing solvent penetration into the plant material and facilitating
the release of analytes. In addition, the energy consumption of
the UAE could be effectively decreased by employing a lower
processing temperature and shorter extraction times.15 However,
degradation of antioxidants and lipids present in fat-rich products
after sonication was reported, therefore flavour and composition
of these foods deteriorates.16,17 Recently, the UAE processes have
often been applied for the isolation of antioxidant compounds
from various plant seeds by liquid solvents.16,18 – 21 Also, Wu et al.,8
Halvorsen et al.,13 Fang et al.,9 Harbaum et al.5 and Dubie et al.3
extracted antioxidants from mustard seed, freeze-dried pot-herb
mustard powder, leaf mustard cultivars and defatted mustard
meal, respectively, using ultrasonication before determination of
phenolic compounds and antioxidant activity.
However, to the best of our knowledge, the impact of the UAE
conditions such as solvent polarity and ratio of ultrasound power
to sonication time on the antioxidant capacity and total phenolics
content (TPC) in extracts of mustard seed varieties has not been
reported.
Therefore, this work focused on the optimisation of the
ultrasound-assisted extraction of compounds with high antiox-
idant capacity from three white mustard seed cultivars: the
traditional S1 variety with a high content of erucic acid and glu-
cosinolates, the S2(0) variety with reduced levels of erucic acid,
and the S3(00) variety without erucic acid and glucosinolates.
Response surface methodology was used for evaluation of the
effects of two independent variables (X 1 , solvent polarity, and X 2 ,
the ratio of ultrasound power to ultrasound time) and their interac-
tions on the response variables: antioxidant capacity determined
by the modified FRAP and DPPH assays, and total phenolics con-
tent analysed by the Folin–Ciocalteu method. Moreover, princi-
pal component analysis was applied for grouping the analysed
extracts, their characterisation, and detection of differences.
MATERIALS AND METHODS
Materials
Three cultivars of white mustard seed (Sinapis alba L.) – S1, the
traditional variety, which contains erucic acid (40%) and glucosino-
lates (160 μmol g−1 ); S2(0), a variety without erucic acid, but with
high amounts of glucosinolates (130 μmol g−1 ); and the S3(00)
(double low) variety, without erucic acid and with a reduced
content of glucosinolates (3 μmol g−1 ) – were kindly donated by
Professor Iwona Bartkowiak-Broda from the Plant Breeding and
Acclimatization Institute in Poznań (Poland). The S1, S2(0) and
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A Szydłowska-Czerniak et al.
S3(00) cultivars are listed in the Polish National List of the Research
Centre for Cultivar Testing (COBORU) and were assigned the num-
bers R38, R1716 and R2383, respectively. Mustard seed samples in
the original packing were stored in the dark at ambient tempera-
ture, until treatment and further analysis.
Reagents
All reagents were of analytical or HPLC grade. 2,4,6-tris(2-Pyridyl)-
s-triazine (TPTZ, 99%), 6-hydroxy-2,5,7,8-tetramethylchromane-2-
carboxylic acid [Trolox (TE), 97%], 2,2′ -diphenyl-1-picrylhydrazyl
radical (DPPH, 95%), Folin–Ciocalteu reagent (1 mol L−1 ), sinapic
acid (SA, 98%), iron(III) chloride hexahydrate, sodium acetate,
sodium carbonate, acetic acid, hydrochloric acid, methanol
(99.8%) were purchased from Sigma–Aldrich (Poznań, Poland).
Redistilled water was used for preparation of solutions.
Extraction procedures
Conventional extraction
Mustard samples were ground using a Retsch grinder and sieved
to particle size of 0.50 mm. Each sample was extracted in tripli-
cate. A portion (2.0 g) of ground mustard seed and 20 mL × 3 of
solvent (methanol, methanol/water (1:1 v/v) or redistilled water;
solvent-to-material ratio = 60 mL 2 g-1 = 30 mL g-1 ) with increas-
ing polarity (dielectric constant = 32.6, 56.3 and 78.4) were trans-
ferred into three conical flasks, respectively. The mixtures were
shaken using a shaker SHKA 2508-1CE (Labo Plus, Warsaw, Poland)
at room temperature for 20 min × 3. To set this time (60 min) and
solvent-to-solid ratio (30 mL g−1 ), previous experiments were per-
formed for rapeseed.22 Contact time was evaluated up to 90 min
for total antioxidants extraction and the antioxidant capacity of
extracts did not significantly increase after 60 min. The resid-
ual mustard flour was separated by centrifugation at 3120 × g,
15 min (centrifuge MPW-310, Labo-Mix, Łódź, Poland). The pooled
extracts were filtered and stored in a refrigerator at 4 ∘ C prior to
analysis.
Ultrasound-assisted extraction
A portion (2.0 g) of grounded mustard seed (particle size of
0.50 mm) and 10 mL of solvent (methanol, methanol/water
(1:1 v/v) or redistilled water) with increasing polarity (dielec-
tric constant = 32.6, 56.3 and 78.4) were transferred into three
beakers and placed exactly at the centre (the maximum inten-
sity was obtained in this region) of the ultrasonic cleaning bath,
300 × 240 × 150 mm (5200DTD; Chemland, Stargard Szczeciński,
Poland,) with a frequency of 40 kHz, ultrasound input power of
120 W and heating power of 800 W, equipped with digital timer
and temperature controller. The sample and solvent level in the
beaker was 3.5 cm below the water surface in the ultrasonic bath.
The height of the bottom surface of the beaker from the bottom
surface of the tank (face of transducers) was 4.5 cm and this posi-
tion was kept constant throughout the experiments. The UAE was
performed for 5 and 10 min, respectively, and the temperature
was kept constant at 25 ± 0.3 ∘ C. The same mustard sample was
extracted in triplicate, and the residual mustard flour was sepa-
rated by centrifugation at 3120 × g, 15 min (centrifuge MPW-310,
Labo-Mix). The application of an ultrasonic bath for the UAE is
preferred to that of a probe, because a bath is cheaper, offers
fewer degrading conditions and there is no limit to the extractant
concentration and composition, whereas the probe is a powerful
system for solid–liquid extraction of analytes, but they can be
degraded. Moreover, the temperature in the ultrasound-treated
J Sci Food Agric (2014)
Ultrasound-assisted extraction of antioxidants from mustard seed
sample rapidly increases in the system of probe. Therefore oil in
mustard seed can be oxidised at high temperatures. Also metal
particles of the probe can initiate the oxidation reaction.16 – 18 The
pooled extracts were filtered and stored in a refrigerator at 4 ∘ C
prior to analysis.
Analytical methods
Ferric-reducing antioxidant power method
The modified FRAP method was used for antioxidant capacity
determination of each mustard extract.22 Freshly prepared FRAP
reagent (2.5 mL of a 10 mmol L−1 TPTZ solution in 40 mmol L−1
HCl, 2.5 mL of 20 mmol L−1 FeCl3 , and 25 mL of 0.1 mol L−1
acetate buffer, pH 3.6) was incubated at 40 ∘ C for 15 min. Then,
0.02–0.1 mL of seed extracts and 2 mL of FRAP reagent were
transferred into a 10 mL volumetric flask and made up to volume
with redistilled water. The blue solutions obtained were kept at
room temperature for 20 min. The resulting absorbance was mea-
sured at 593 nm against a reagent blank (2 mL of FRAP reagent
made up to 10 mL with redistilled water) using a 1 cm quartz cell
in a Shimadzu UV-1601 spectrophotometer (Shimadzu, Kyoto,
Japan).
The 2,2′ -diphenyl-1-picrylhydrazyl method
Antioxidant capacity of each extract from mustard seed cul-
tivars was determined by spectrophotometric DPPH method
with some modifications as previously described in detail.22
In brief, 0.01–0.10 mL of the extracts obtained were added to
1.99–1.90 mL of methanol and 0.5 mL of DPPH methanolic solu-
tion (304.0 μmol L−1 ). The mixture was shaken vigorously and
left in darkness for 15 min. The absorbance was measured at
517 nm against a reagent blank (2 mL of methanol + 0.5 mL of
DPPH methanolic solution) using a 1 cm quartz cell in a Shimadzu
UV-1601 spectrophotometer.
Determination of total phenolics content
TPC was determined spectrophotometrically using the
Folin–Ciocalteu phenol reagent, according to a procedure
described previously.22 In brief, 0.05 or 0.1 mL of the prepared
extracts and 0.5 mL of Folin–Ciocalteu reagent were transferred
into a 10 mL calibrated flask and shaken for 3 min. Then, 1 mL
of saturated sodium carbonate solution was added and made
up to the mark with redistilled water. After 1 h, solutions were
centrifuged at 3120 × g, 5 min (centrifuge MPW-310, Labo-Mix)
and the absorbance at 725 nm was measured against a reagent
blank using a Shimadzu UV-1601 spectrophotometer.
Calibration curves
Calibration curves were prepared using working solu-
tions of Trolox in methanol between 1.00 × 10−3 and
1.80 × 10−2 μmol mL−1 for the FRAP method and between
2.00 × 10−2 and 1.00 × 10−1 μmol mL−1 for the DPPH method,
whereas solutions of sinapic acid in the concentration range
0.88–13.20 μg mL−1 were prepared for the Folin–Ciocalteu assay.
Five calibration curves for each method were plotted on the same
day. The least-squares method was applied to calculate the line’s
equations:
• For FRAP, A593 = (44.44 ± 0.38)cTE + (0.011 ± 0.004)
• For DPPH, the %DPPH = (688.6 ± 8.1)cTE + (1.14 ± 0.53)
• For the Folin–Ciocalteu method, A725 = (0.062 ± 0.002)cSA +
(0.098 ± 0.017)
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where cTE and cSA are concentration of Trolox and concentration
of sinapic acid, respectively. Values for the resulting determination
coefficients were 0.9998, 0.9995 and 0.9969, respectively. The
relative standard deviations (RSDs, n = 5) of the slopes ranged
between 0.11% and 4.92%. The within-day precision of each
method was tested by analysing five replicate samples containing
8.00 × 10−3 , 6.00 × 10−2 μmol TE mL−1 for the FRAP and DPPH
assays and 6.60 μg SA mL−1 for the Folin–Ciocalteu method.
The obtained values of RSD (2.49%, 4.30% and 1.84% for FRAP,
DPPH and Folin–Ciocalteu methods, respectively) indicate rea-
sonable repeatability of these methods. Moreover, the calculated
detection limits (4.10 × 10−4 μmol TE mL−1 , 2.70 × 10−3 μmol TE
mL−1 and 1.17 μg SA mL−1 for FRAP, DPPH and Folin–Ciocalteu
methods, respectively) and quantification limits (1.37 × 10−3 μmol
TE mL−1 , 9.00 × 10−3 μmol TE mL−1 and 3.90 μg SA mL−1 for
FRAP, DPPH and Folin–Ciocalteu methods, respectively), confirm
linearity concentrations ranges for antioxidant capacity deter-
mination by the modified FRAP and DPPH assays as well as TPC
analysis by Folin–Ciocalteu method. The proposed analytical
methods appeared to be sensitive (molar extinction coeffi-
cients = 4.80 × 104 L mol−1 cm−1 , 2.40 × 103 L mol−1 cm−1 and
2.00 × 104 L mol−1 cm−1 for the FRAP, DPPH and Folin–Ciocalteu
methods, respectively).
Experimental design and statistical analysis
The results of antioxidant capacity and TPC in the studied extracts
of three mustard seed cultivars were determined (five portions of
each extract analysed within 1 day) by the modified FRAP, DPPH
and Folin–Ciocalteu methods, respectively. The results obtained
were presented as mean (c) ± standard deviation (SD). Mean dif-
ferences were considered significant at the P < 0.05 level. One-way
analysis of variance (ANOVA), followed by the Duncan test, was
performed to analyse the significant differences between data
(P < 0.05).
Response surface methodology was used to study the simul-
taneous effects of the solvent polarity and ultrasound power to
sonication time ratio (US power/US time) on antioxidant capacity
determined by FRAP and DPPH methods and TPC in mustard seed
extracts. This US power/US time = 0 W 60 min−1 = 0 W min−1
for the conventional extraction (without ultrasonication),
whereas US power/US time = 120 W 15 min−1 = 8 W min−1
and 120 W 30 min−1 = 4 W min−1 , respectively for the UAE
procedures.
The experimental design applied for the analysis was a central
composite design with three factors and three levels. In this
experimental design, there were three coded factor levels: −1,
0, +1 where, −1 corresponds to the low level of each factor,
+1 to the high level and 0 to the mid-level. The factors and
respective coded and uncoded levels are given in Table 1, Table 2
and Table 3.
The experiments consisted of 11 runs with two factors and
two replicates of the central point for the estimation of pure
error. The effect of the two independent variables (X 1 , sol-
vent polarity, and X 2 , US power/US time) on the responses
(Yn , Y 1 , FRAP, Y 2 , DPPH and Y 3 , TPC) was modelled using
a polynomial response surface. The second-order response
function for the experiments was predicted by the following
equation:
( )2 ( )2
Yn = 𝛽0 + 𝛽1 X1 + 𝛽2 X2 + 𝛽11 X1 + 𝛽22 X2 + 𝛽12 X1 X2
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Table 1. Central composite design and experimental results of antioxidant capacity response determined by the FRAP method for three mustard
seed varieties

Dependent variables
S1, FRAP∗ ± SD S2(0), FRAP∗ ± SD S3(00), FRAP∗ ± SD
Coded level Independent variables (mmol TE kg−1 ) (mmol TE kg−1 ) (mmol TE kg−1 )
Solvent US power/US time
Exp. X1 X2 polarity (W min−1 ) Experimental Predicted Experimental Predicted Experimental Predicted

1 −1 −1 32.6 0 13.34 ± 0.29a 13.36 16.52 ± 0.51a 17.26 16.50 ± 0.52a 18.36
2 −1 0 32.6 4 20.68 ± 0.40c 20.06 21.35 ± 0.31c,d 21.07 27.78 ± 0.92b 24.13
3 −1 +1 32.6 8 17.35 ± 0.44b 17.95 18.43 ± 0.37b 17.97 17.15 ± 0.30a 18.94
4 0 −1 56.3 0 48.75 ± 1.54f 49.19 60.55 ± 1.79f 60.78 62.31 ± 1.48d 62.47
5 0 0 56.3 4 54.27 ± 1.34h 54.35 66.11 ± 1.96g 65.62 67.73 ± 1.78e 68.58
6 0 +1 56.3 8 51.47 ± 1.92g 50.70 60.82 ± 1.19f 63.54 63.42 ± 1.34d 63.74
7 +1 −1 78.4 0 23.62 ± 0.51d 23.16 20.50 ± 0.38c 19.53 28.24 ± 0.83b 26.22
8 +1 0 78.4 4 26.59 ± 0.34e 26.88 22.08 ± 0.30d 25.31 28.52 ± 0.61b 32.65
9 +1 +1 78.4 8 21.63 ± 0.47c 21.80 26.45 ± 0.46e 24.19 30.23 ± 0.51c 28.13
10 0 0 56.3 4 54.44 ± 0.75h 54.35 66.03 ± 1.02g 65.62 68.99 ± 0.63f 68.58
11 0 0 56.3 4 54.01 ± 0.53h 54.35 67.67 ± 1.27h 65.62 69.51 ± 0.77f 68.58
∗ n = 5; SD, standard deviation; probability, P = 0.05.
Different letters within the same column indicate significant differences between FRAP results of the mustard extracts (one-way ANOVA and Duncan
test, P < 0.05).
FRAP, ferric reducing antioxidant power; TE, Trolox equivalents; US, ultrasound.

Table 2. Central composite design and experimental results of antioxidant capacity response determined by the DPPH method for three mustard
seed varieties

Dependent variables
S1, DPPH∗ ± SD S2(0), DPPH∗ ± SD S3(00), DPPH∗ ± SD
Coded level Independent variables (mmol TE kg−1 ) (mmol TE kg−1 ) (mmol TE kg−1 )
Solvent US power/US time
Exp. X 1 X2 polarity (W min−1 ) Experimental Predicted Experimental Predicted Experimental Predicted

1 −1 −1 32.6 0 19.84 ± 0.79a 17.11 21.68 ± 0.58a 25.46 36.37 ± 0.74a 29.96
2 −1 0 32.6 4 44.97 ± 1.37c 57.83 54.63 ± 2.62c 44.90 65.60 ± 1.27c 85.95
3 −1 +1 32.6 8 36.29 ± 0.51b 26.16 37.60 ± 1.53b 43.56 38.04 ± 1.75a 24.10
4 0 −1 56.3 0 102.89 ± 2.83g 101.98 156.40 ± 2.93f 150.65 123.92 ± 4.31f 128.19
5 0 0 56.3 4 149.73 ± 5.64i 141.31 168.09 ± 6.85g 173.18 197.73 ± 1.45h 185.42
6 0 +1 56.3 8 93.83 ± 2.41f 108.25 185.19 ± 8.81h 174.92 104.90 ± 4.27d 124.79
7 +1 −1 78.4 0 53.19 ± 1.79d 56.83 40.11 ± 1.94b 42.09 55.56 ± 1.71b 57.69
8 +1 0 78.4 4 94.21 ± 4.31f 94.86 73.78 ± 3.25e 67.49 112.25 ± 5.19e 116.07
9 +1 +1 78.4 8 64.80 ± 1.76e 60.51 67.80 ± 2.68d 72.11 62.54 ± 1.64c 56.59
10 0 0 56.3 4 141.30 ± 6.83h 141.31 164.61 ± 2.72g 173.18 187.45 ± 4.23g 185.42
11 0 0 56.3 4 146.40 ± 4.21i 141.31 170.81 ± 8.00g 173.18 195.24 ± 1.21h 185.42
∗ n = 5; SD, standard deviation; probability, P = 0.05.
Different letters within the same column indicate significant differences between DPPH results of the mustard extracts (one-way ANOVA and Duncan
test, P < 0.05).
DPPH, 2,2′ -diphenyl-1-picrylhydrazyl; US, ultrasound; TE, Trolox equivalents.

where Yn is one of the nine responses; X 1 and X 2 represent the
independent variables; 𝛽 0 is the constant; 𝛽 1 , 𝛽 2 are the linear-term
coefficients; 𝛽 11 , 𝛽 22 are the quadratic-term coefficients; and 𝛽 12 is
the cross-term coefficient. The fitness of the model was evaluated
by the determination coefficient, R2 , the fraction of the variation
explained by the model, and analysis of variance (ANOVA). The
F-test was applied to confirm whether the variance explained by
the regression model was significantly larger than the variance of
the residual and to evaluate the model lack-of-fit (model error). The
effects of two factors and their interactions on FRAP, DPPH and TPC
in extracts of mustard seed cultivars were displayed in surface and
contour plots.
Also, principal component analysis was employed to study
clustering and differentiation of mustard seed extracts on the basis
of the FRAP, DPPH and TPC results. The scores and loadings of the
data analysed by principal component analysis were displayed as a
bi-plot. The chemometric analyses were performed using the Sta-
tistica (Windows software package, version 8.0).

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Table 3. Central composite design and experimental results of total phenolics response determined by the Folin–Ciocalteu method for three
mustard seed varieties

Dependent variables
S1, TPC∗ ± SD S2(0), TPC∗ ± SD S3(00), TPC∗ ± SD
Coded level Independent variables (mg SA g−1 ) (mg SA g−1 ) (mg SA g−1 )
Solvent US power/US time
Exp. X1 X2 polarity (W min−1 ) Experimental Predicted Experimental Predicted Experimental Predicted

1 −1 −1 32.6 0 6.43 ± 0.17a 6.54 6.68 ± 0.24a 6.41 1.67 ± 0.03a 1.53
2 −1 0 32.6 4 10.03 ± 0.07c 9.89 8.76 ± 0.33b 9.38 2.86 ± 0.09b 2.95
3 −1 +1 32.6 8 8.05 ± 0.23b 8.08 6.89 ± 0.20a 6.53 1.84 ± 0.05a 1.90
4 0 −1 56.3 0 19.76 ± 0.86f 19.57 23.24 ± 0.21e 24.17 8.99 ± 0.31f 8.95
5 0 0 56.3 4 23.47 ± 0.52h,i 23.57 27.52 ± 0.60f 27.24 11.04 ± 0.48g,h 11.16
6 0 +1 56.3 8 22.45 ± 0.95g 22.41 23.39 ± 0.67e 24.50 11.34 ± 0.18h 10.89
7 +1 −1 78.4 0 7.57 ± 0.29b 7.66 10.08 ± 0.31c 9.42 3.29 ± 0.12c 3.47
8 +1 0 78.4 4 12.37 ± 0.34e 12.27 11.18 ± 0.25d 12.60 6.98 ± 0.11e 6.40
9 +1 +1 78.4 8 11.71 ± 0.36d 11.72 10.72 ± 0.41d 9.96 6.46 ± 0.15d 6.86
10 0 0 56.3 4 23.89 ± 0.47i 23.57 27.80 ± 0.55f 27.24 11.09 ± 0.14g,h 11.16
11 0 0 56.3 4 23.13 ± 0.50h 23.57 28.45 ± 0.58g 27.24 10.85 ± 0.35g 11.16
∗ n = 5; SD, standard deviation; probability, P = 0.05.
Different letters within the same column indicate significant differences between TPC results of the mustard extracts (one-way ANOVA and Duncan
test, P < 0.05).
TPC, total phenolics content, SA, sinapic acid; US, ultrasound.

RESULTS AND DISCUSSION
Antioxidant capacity and total phenolics content in mustard
seed extracts
The experimental and predicted values of FRAP, DPPH and TPC in
extracts obtained from three mustard seed cultivars without and
after ultrasonication treatment are listed in Tables 1–3. It can be
noted that FRAP results ranged between 13.34 and 62.31 mmol TE
kg−1 , 17.15 and 63.42 mmol TE kg−1 and 20.68 and 69.51 mmol TE
kg−1 for extracts from mustard samples without ultrasound treat-
ment and sonicated during 15 and 30 min, respectively. However,
DPPH values (19.84–156.40 mmol TE kg−1 , 36.29–185.19 mmol TE
kg−1 and 44.97–197.73 mmol TE kg−1 ) of mustard extracts pre-
pared by the traditional stirring (US power/US time = 0 W min−1 )
and the UAE (US power/US time = 8 and 4 W min−1 , respectively)
procedures are up to three times higher than antioxidant capac-
ity determined by FRAP method. This fact can be explained by the
fact that FRAP and DPPH assays are a single electron-transfer based
reactions; however, the FRAP method measures the ability of mus-
tard antioxidants to transfer one electron to reduce iron ions,
which form the blue complex with TPTZ, whereas DPPH method
is based on the measurement of the reducing ability of mustard
antioxidants towards the red radical (DPPH• ). Furthermore, the
FRAP assay does not detect antioxidant compounds containing
functional group, which reduction potential is lower than that of
the Fe3+/ Fe2+ half-reaction.
Similar FRAP results (14.17–105.27 mmol TE kg−1 ) for yellow
mustard seed were obtained by Halvorsen et al.13 Besides, a signif-
icantly lower DPPH values for two leaf mustard varieties cultivated
in Germany and China (19.41–39.64 mmol TE kg−1 ), mustard
seed (3.50 mmol epigallocatechin-3-gallate kg−1 ) and meal of
different mustard seed cultivars (8.30–38.80 mmol SA kg−1 ) were
reported by Harbaum et al.5 , Katsube et al.11 and Dubie et al.3 ,
respectively. In contrast, DPPH values (5745.17–8739.54 mmol TE
kg−1 ) of five traditional mustard (Sinapis alba L.) genotypes and
variety with low erucic acid content were two orders higher than
our results.12
However, total phenolics contents in samples obtained after the
conventional solid-liquid extraction and the UAE (US power/US
time = 8 and 4 W min−1 ) ranged between 1.67 and 23.24 mg SA
g−1 , 1.84–23.39 mg SA g−1 and 2.86–28.45 mg SA g−1 , respec-
tively.
The highest FRAP and DPPH values (69.51 and 197.73 mmol TE
kg−1 ) revealed 50% methanolic extract prepared from doble low
mustard seed cultivar S3(00) treated with ultrasound for 30 min
(US power/US time = 4 W min−1 ), whereas FRAP (13.34 mmol
TE kg−1 ) and DPPH (19.84 mmol TE kg−1 ) were the lowest for
the methanolic extract of traditional mustard seed cultivar S1
obtained by the conventional extraction (without ultrasound
treatment, Tables 1 and 2).
However, a surprisingly low mean levels of TPC (1.67–11.34 mg
SA g−1 ) in extracts of the modified S3(00) cultivar were anal-
ysed (Table 3). The results obtained suggest that glucosinolates
present in high amounts in S1 and S2(0) samples might react with
Folin–Ciocalteu reagent. Cabello-Hurtado et al.23 reported that
glucosinolates are powerful electrophiles through their -N=C=S
group and their direct antioxidant action might be controver-
sial, but sulfur from methylthio group of the aliphatic glucosi-
nolates side chain can act as an electron donor. Therefore, the
Folin–Ciocalteu method detects not only phenolics, but also other
non-phenolic compounds that have reducing power such as glu-
cosinolates, amino acids, carbohydrates, aromatic amines, sulfur
dioxide and others, which are all naturally present in mustard
seed. The Folin–Ciocalteu reagent is not specific to phenolic com-
pounds, because the interfering compounds may be implicated
in oxidative–reduction reaction resulting in products which may
contribute to the absorbance measurement by Folin–Ciocalteu
method. It is noteworthy that TPC values (6.43–23.89 mg SA
g−1 ) for the traditional mustard cultivar S1 with high amounts

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 www.soci.org
of glucosinolates and erucic acid were very similar in compari-
son to TPC results (6.68–28.45 mg SA g−1 ) for S2(0) variety with
high glucosinolates content and about two and four times higher
than TPC values (1.67–11.34 mg SA g−1 ) for double low mustard
seed variety S3(00) with reduced levels of glucosinolates and eru-
cic acid (Table 3). Total phenolics amounts in the studied mus-
tard seed varieties determined by the modified Folin–Ciocalteu
method were comparable to results (2.72–46.91 mg gallic acid
g−1 ) reported by others.5,6,8,12
The results obtained for antioxidant capacity and TPC indi-
cated that the methanol–water mixture (1:1 v/v, dielectric
constant = 56.3) was more efficient solvent than methanol (dielec-
tric constant = 32.6), and water (dielectric constant = 78.4) for
extraction of antioxidants from the studied mustard seed culti-
vars (Tables 1–3). This suggests that FRAP, DPPH and TPC values
in the resulting extracts did not increase with a line in solvent
polarity. This fact indicated that mustard seed varieties are sources
of hydrophilic and lipophilic compounds that need the use
of solvents with different polarity to obtain high yield of total
antioxidants. A similar effect of solvent polarity on antioxidant
capacity determined by DPPH (20.47–25.47 mmol SA kg−1 for
water extracts, and 25.51–32.74 mmol SA kg−1 for ethanolic
extracts) and ABTS (25.52–35.81 mmol SA kg−1 for water extracts,
and 23.69–26.67 mmol SA kg−1 for ethanolic extracts) methods,
as well as TPC (7.83–13.79 mg SA g−1 for water extracts, and
8.00–8.81 mg SA g−1 for ethanolic extracts) in extracts from B.
juncea seed meal was observed by Dubie et al.3 It can be noted
that antioxidant capacity and TPC in the studied mustard seed
extracts differ significantly from each other (P < 0.05, Duncan test).
This variability can be explained by the influence of extraction
conditions and analytical parameters of the applied methods,
which would affect the total level of antioxidants. Although,
methanolic (US power/US time = 4 and 8 W min−1 ) and water
(US power/US time = 8 and 0 W min−1 ) as well as 50% methano-
lic (US power/US time = 8 W min−1 ) and water (US power/US
time = 4 W min−1 ) extracts of traditional mustard seed cultivar
revealed similar values of FRAP, TPC and DPPH (Tables 1–3).
Also, antioxidant capacity of aqueous and methanolic extracts of
S2(0) obtained by the conventional solid-liquid extraction and
after sonication for 15 and 30 min did not differ significantly.
In addition, there were no significant differences (Duncan test,
P > 0.05) in FRAP and DPPH for methanolic and aqueous extracts
of double low S3(00) variety obtained after the UAE and the
classical extraction. Similar results of antioxidant capacity and
TPC in some mustard seed extracts obtained by the UAE and
the conventional extraction can be explained by the fact that
ultrasound treatment also lead to degradation and oxidation
of antioxidants by radicals formed during cavitation.16,17 It is
noteworthy that there are an insignificant differences in total
phenolics for water extracts of S2(0) and 50% methanolic extracts
of S3(00) cultivars treated by ultrasounds during 15 and 30 min,
respectively. Obviously, similar results (Duncan test, P > 0.05) of
antioxidant capacities and TPC were observed between mustard
seed extracts obtained under the same extraction conditions
(solvent polarity = 56.3, and US power/US time = 4 W min−1 ,
Tables 1–3).
The values of RSD ranged between 1.0–3.7%, 0.6–4.8% and
0.7–4.4%, respectively, indicating reasonable repeatability of the
FRAP, DPPH and TPC determinations for the studied mustard seed
extracts. For comparison, higher RSD values of DPPH (0.2–25.3%)
and TPC (0.5–8.5%) in different mustard seed varieties were
reported by others.3,11,12
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A Szydłowska-Czerniak et al.
Fitting the models
Response surface analysis was applied and the second-order poly-
nomial response surface models were fitted to response variables:
FRAP, DPPH and TPC in extracts obtained from three white mustard
seed cultivars without sonication and after ultrasound treatments
(Tables 1–3). Multiple regression coefficients were determined by
the least-squares technique to predict quadratic polynomial mod-
els for the studied response variables. The following regression
equations were obtained:
FRAPS1 = −143.7 + 6.73X1 + 3.31X2 − 0.059X12
− 0.28X22 − 0.016X1 X2
FRAPS2(0) = −191.0 + 9.03X1 + 1.47X2 − 0.081X12
− 0.22X22 + 0.011X1 X2
FRAPS3(00) = −182.6 + 8.66X1 + 2.69X2 − 0.076X12
− 0.34X22 + 0.0040X1 X2
DPPHS1 = −325.0 + 14.50X1 + 19.71X2 − 0.12X12
− 2.26X22 − 0.015X1 X2
DPPHS2(0) = −555.3 + 25.07X1 + 6.40X2 − 0.22X12
− 0.65X22 + 0.033X1 X2
DPPHS3(00) = −399.1 + 18.38X1 + 28.30X2 − 0.16X12
− 3.68X22 + 0.013X1 X2
TPCS1 = −55.01 + 2.66X1 + 1.26X2 − 0.024X12
− 0.16X22 + 0.0069X1 X2
TPCS2(0) = −74.76 + 3.50X1 + 1.43X2 − 0.031X12
− 0.18X22 + 0.0012X1 X2
TPCS3(00) = −31.20 + 1.40X1 + 0.40X2 − 0.012X12
− 0.077X22 + 0.0082X1 X2
in which the FRAP and DPPH concentrations are in mmol TE
kg−1 , and the TPC concentrations are in mg SA g−1 . The ANOVA
results for the predicted response quadratic models are listed
in Table 4. ANOVA test revealed, that the quadratic polynomial
models adequately represent responses of FRAP, DPPH and TPC
in various extracts of S1, S2(0) and S3(00) with the coefficients of
determination, R2 ranged between 0.9648 and 0.9993 (Table 4).
The calculated R2 and the adjusted R2 values for the studied
response variables were higher than 0.90, hence there is a close
agreement between the experimental results and the theoretical
values predicted by the proposed models.
The model adequacy was tested using the lack-of-fit F-test,
which was not significant for P > 0.05. The ANOVA results of
responses revealed insignificant lack-of-fit (F ranged between 0.2
and 18.2, P > 0.05) (Table 4). Therefore, these models were ade-
quate for prediction within the range of variables employed.
Moreover, the linear and quadratic effects of the independent
variables (X 1 , solvent polarity and X 2 , US power/US time) and their
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Ultrasound-assisted extraction of antioxidants from mustard seed www.soci.org

Table 4. ANOVA results for the responses: FRAP, DPPH and TPC in three mustard seed cultivars

S1 S2(0) S3(00)
Model Degrees Sum of Mean Sum of Mean Sum of Mean
parameters of freedom squares square F value squares square F value squares square F value

Antioxidant capacity (FRAP)

Regression 5 2 527.4 505.5 10 777.9∗ 4 613.7 922.7 1 079.3∗ 4 248.1 849.6 1 014.2∗
Residual 5 2.0 0.4 – 29.4 5.9 – 47.5 9.5 –
Lack-of-fit 3 1.9 0.6 13.5 27.7 9.2 10.8 45.8 15.3 18.2
Pure error 2 0.1 0.05 – 1.7 0.85 – 1.7 0.85 –
Total 10 2 529.4 – – 4 643.1 – – 4 295.6 – –
R2 , Adjusted R2 0.9993, 0.9987 0.9944, 0.9887 0.9905, 0.9809
Antioxidant capacity (DPPH)
Regression 5 15 917.1 3 183.4 176.6∗ 36 360.2 7 272.0 752.9∗ 27 995.8 5 599.2 194.7∗
Residual 5 613.2 122.6 – 449.9 90.0 – 1 370.4 274.1 –
Lack-of-fit 3 577.1 192.4 10.7 430.6 143.5 14.9 1 312.9 437.6 15.2
Pure error 2 36.1 18.05 – 19.3 9.65 – 57.5 28.75 –
Total 10 16 530.3 – – 36 810.1 – – 29 366.2 – –
R2 , Adjusted R2 0.9713, 0.9423 0.9892, 0.9783 0.9648, 0.9295
Total phenolics content (TPC)
Regression 5 431.2 86.2 595.0∗ 701.4 140.3 616.2∗ 133.8 26.8 1 668.4∗
Residual 5 0.4 0.08 – 7.6 1.5 – 0.93 0.2 –
Lack-of-fit 3 0.1 0.03 0.2 7.1 2.4 10.4 0.9 0.3 17.6
Pure error 2 0.3 0.15 – 0.5 0.25 – 0.03 0.015 –
Total 10 431.6 – – 709.0 – – 134.7 – –
R2 , Adjusted R2 0.9992, 0.9985 0.9910, 0.9819 0.9944, 0.9888
∗ Significant at the P < 0.05 level.

interactions on the response variables (FRAP, DPPH and TPC) were
analysed by ANOVA.
The model is highly significant when the computed F-value is
greater than the tabulated value and the probability value is low
(P < 0.0001). All linear (X 1 , X 2 ), quadratic (X 1 2 , X 2 2 ) and interaction
(X 1 × X 2 ) parameters of the models were statistically significant
(F = 83.4–51080, P < 0.05) for FRAP of traditional mustard seed
S1 extract and TPC in sample of mustard seed cultivar S3(00)
without erucic acid content and reduced level of glucosinolates.
Only interaction X 1 × X 2 term produced an insignificant effect on
TPC in S1 and DPPH of S2(0) extracts. Moreover, both quadratic
(X 1 2 , X 2 2 ) and linear (X 1 ) parameters of the models significantly
influenced (F values ranged between 31.6 and 5312, P < 0.05) on
FRAP of S2(0) and S3(00), DPPH of S1 and S3(00), as well as TPC in
S2(0) extracts. It is noteworthy that the variable with the largest
negative effect (F = 581.0–51080) on FRAP, DPPH and TPC in all
studied mustard seed extracts was the quadratic term of solvent
polarity.
Polarity of the solvent employed for extracts preparation and
ratio of ultrasound power to sonication treatment time nega-
tively affected the antioxidant capacity and total phenolics in the
resultant extracts of all analysed mustard seed varieties. Thus,
the studied samples have higher total antioxidant potential when
extracted with less polar solvent (lower dielectric constant) from
longer ultrasonic treatment mustard seed cultivars (lower US
power/US time ratio). The effect of solvent polarity (X 1 2 ) on FRAP,
DPPH and TPC in extracts of S1 and S3(00) was about seven, two
and four times greater, respectively, than the effect of US power/US
time ratio (X 2 2 ). However, the quadratic term of US power/US time
ratio exhibits about 12 and six times lower effect on antioxidant
capacities and total phenolics in S2(0) extracts, respectively.
Analysis of response surfaces
The effects of the two independent variables (solvent polarity and
US power/US time ratio) on FRAP, DPPH and TPC in the obtained
mustard seed extracts were illustrated using surface response and
contour plots of the quadratic polynomial models (Fig. 1).
The parabolic shapes of the presented response surfaces were
caused by the negative values of the quadratic terms of solvent
polarity and US power/US time ratio. It is evident that the antiox-
idants content in the prepared mustard seed extracts increased
with the increase of solvent polarity up to 56.3 and further increase
in dielectric constant leads to deceleration of antioxidants extrac-
tion. Figure 1 indicates that the highest FRAP, DPPH and TPC values
in the studied extracts occurred when 50% methanol (dielectric
constant = 56.3) was used for extraction of antioxidants from three
white mustard seed varieties. The solvent polarity has a significant
effect on measured FRAP, DPPH and TPC values in the studied mus-
tard seed extracts, whereas ratio of ultrasound power to sonica-
tion time only slightly influences these responses (Fig. 1). However,
the response surface and contour plot of DPPH results of S3(00)
cultivar (Fig. 1b) depicted a maximum at an intermediate values
of polarity of the used solvent (dielectric constant = 56.3) and US
power/US time ratio (4 W min−1 ).
The proposed mathematical model allows on determination of
the optimum parameters of total antioxidants extraction from
ultrasound treatment mustard seed varieties. The optimum sol-
vent polarity and US power/US time for extract preparation of
traditional S1 variety before determination its antioxidant capac-
ity and total phenolics were as follows: dielectric constant = 57.2,
US power/US time = 4.5 W min−1 , under which FRAP = 54.37 mmol
TE kg−1 , DPPH = 141.65 mmol TE kg−1 and TPC = 23.70 mg SA
g−1 were predicted. However, the optimum levels of solvent

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 www.soci.org A Szydłowska-Czerniak et al.

2.5

2.0 A2

1.5 S3-MA-4 S3-MA-4

S3-MA-4

PC2: 12.63%
S3MA-0
1.0
S3-MA-8 S3-M-4
S3-A-4
S3-A-0
0.5 S3-M-0
DPPH S3-A-8
S3-M-8
FRAP
0.0 S2-A-8 S1-A-0 A1
S2-MA-8 S2MA-0
S2-M-8S2-M-0
S1-A-4 S2-M-4
S2-MA-4 S1-MA-4
S1-MA-4 S2-A-4 S1-M-8 S1-M-0
–0.5 TPC S1MA-0 S1-A-8S1-M-4S2-A-0
S2-MA-4 S2-MA-4S1-MA-4
S1-MA-8
–1.0

–1.5

–2.0
–4 –3 –2 –1 0 1 2 3 4
PC1: 85.31%

Figure 2. Biplot of scores and loadings of data obtained from antioxidant

capacities determined by the modified FRAP and DPPH methods and total
phenolics (TPC) in extracts of three white mustard seed varieties.

polarity and ratio of ultrasound power to time for extraction total

antioxidants from modified mustard seed cultivars were 56.5 and
4.8 W min−1 for S2(0) and 57.6 and 4.3 W min−1 for S3(00). Using
these optimal conditions, the FRAP = 65.75 and 68.55 mmol TE
kg−1 , DPPH = 175.26 and 185.10 mmol TE kg−1 and TPC = 27.16
and 11.29 mg SA g−1 in extracts of S2(0) and S3(00) cultivars,
respectively were predicted.

Principal component analysis

Figure 1. Response surfaces and contour plots for the effect of two
independent variables: solvent polarity and US power/US time on FRAP of
S2(0) extracts (a), DPPH of S3(00) extracts (b) and total phenolics content
(TPC) in S1 extracts (c).
Principal component analysis was applied to observe any possible
groups within the analysed extracts of three mustard seed vari-
eties. A set of three orthogonal variables (PCs) was generated by
principal component analysis. The first principal component (PC1)
had the highest eigenvalue of 2.56 and accounted for 85.31% of
the variability in the data set. The remaining two generated PCs
(PC2 and PC3) yielded progressively lower eigenvalues (<1; 0.38
and 0.062, respectively) and did not explain the variability in the
data (<14.69% total). It can be noted that the PC1 inversely corre-
lated with all variables: FRAP (−0.9588), DPPH (−0.9543) and TPC
(−0.8539), whereas PC2 was highly contributed by TPC (−0.5204).
Evidently, PC1 is generally more correlated with the variables than
PC2. The distribution of the most significant variables (antioxidant
capacities determined by FRAP, DPPH methods and TPC) along
the two first principal components and the groupings and/or
the differences among mustard extracts were presented in the
bi-plot (Fig. 2).
The principal component analysis graph depicted that FRAP
and DPPH of the mustard seed extracts were the variables with
negative loadings on PC1 and positive loadings on PC2, whereas,
TPC was the feature with negative loadings on PC1 and PC2.
It is noteworthy that the methanolic–aqueous (1:1 v/v) extracts
of the studied mustard seed cultivars with high antioxidants con-
tent were located to the left in the score bi-plot and had negative
values for PC1, whereas methanolic and aqueous extracts of three
mustard seed varieties with lower FRAP, DPPH and TPC values were
situated to the right in the diagram and had positive values for PC1.
Moreover, all extracts of white mustard seed cultivar S3(00) with-
out erucic acid and glucosinolates with high antioxidant capaci-
ties and low amount of total phenolics were situated in the upper
side of the scores plot. However, samples of mustard seed varieties

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Ultrasound-assisted extraction of antioxidants from mustard seed
[S1 and S2(0)] with high TPC and low FRAP and DPPH results were
located under the A1 axis.
It can be noted that 50% methanolic extracts of S3(00)
cultivar obtained in longer sonication time (US power/US
time = 4 W min−1 ) with the longest distance from other extracts
revealed the highest values of FRAP and DPPH.
Thus, there are differences in the total amount of antioxidants
extracted by different solvents for various extraction times from
the studied mustard samples.
CONCLUSIONS
The response surface methodology appeared to be useful for
studying the influence of the UAE conditions, such as solvent polar-
ity and ratio of ultrasound power to ultrasound time on antioxi-
dant capacity and total phenolics content in the resulting extracts
of three mustard seed varieties. The second-order polynomial
model can be applied to optimise the parameters of extraction to
obtain extracts of mustard seed cultivars with high antioxidants
amount. The UAE of mustard seed varieties produced higher recov-
eries of total antioxidants in comparison with the conventional
solid–liquid extraction.
The proposed FRAP and DPPH methods are relatively sim-
ple, precise, and convenient for the determination of antioxidant
capacity of mustard seed cultivars, and response surface method-
ology can be employed for preparation mustard seed samples with
high content of total antioxidants. The proposed analytical meth-
ods can offer useful information for mustard crop and further utili-
sation of mustard oil and meal. The obtained results could be very
interesting for industrial purposes, since the UAE would make it
possible to improve process rates and consequently reduce pro-
cessing times and costs.
ACKNOWLEDGEMENTS
The authors wish to thank the Polish Ministry of Science
and Higher Education and National Science Center for the
financial support: Grant No. N N312 465740 and Grant No.
DEC-2013/09/N/NZ9/01451.
REFERENCES
1 Karlovits G and Kozakiewicz E, Characteristics Of New (“Double Zero”)
White Mustard Seed, Bunge Interim Report. Bunge Europe Research
and Development Center, Kruszwica, Poland (2013).
2 Das R, Bhattacherjee Ch and Ghosh S, Preparation of mustard (Brassica
juncea L.) protein isolate and recovery of phenolic compounds by
ultrafiltration. Ind Eng Chem Res 48:4939–4947 (2009).
3 Dubie J, Stancik A, Morra M and Nindo C, Antioxidant extraction from
mustard (Brassica juncea) seed meal using high-intensity ultrasound.
J Food Sci 78:542–548 (2013).
4 Shahidi F, Wanasundara UN and Amarowicz R, Natural antioxidants
from low-pungency mustard flour. Food Res Int 27:489–493 (1994).
5 Harbaum B, Hubbermann EM, Zhu Z and Schwarz K, Impact of fer-
mentation on phenolic compounds in leaves of pak choi (Brassica
J Sci Food Agric (2014) © 2014 Society of Chemical Industry
www.soci.org
campestris L. ssp. chinensis var. communis) and Chinese leaf mustard
(Brassica juncea Coss). J Agric Food Chem 56:148–157 (2008).
6 Matthaüs B, Antioxidant activity of extracts obtained from residues of
different oilseeds. J Agric Food Chem 50:3444–3452 (2002).
7 Singh P, Agrawal M and Agrawal SB, Differences in ozone sensitivity at
different NPK levels of three tropical varieties of mustard (Brassica
campestris L.): Photosynthetic pigments, metabolites, and antioxi-
dants. Water Air Soil Pollut 214:435–450 (2011).
8 Wu X, Beecher GR, Holden JM, Haytowitz DB, Gebhardt SE and Prior RL,
Lipophilic and hydrophilic antioxidant capacities of common foods
in the United States. J Agric Food Chem 52:4026–4037 (2004)
9 Fang Z, Hu Y, Liu D, Chen J and Ye X, Changes of phenolic acids and
antioxidant activities during potherb mustard (Brassica juncea, Coss.)
pickling. Food Chem 108:811–817 (2008).
10 Harbaum B, Hubbermann EM, Zhu Z and Schwarz K, Free and bound
phenolic compounds in leaves of pak choi (Brassica campestris L. ssp.
chinensis var. communis) and Chinese leaf mustard (Brassica juncea
Coss). Food Chem 110:838–846 (2008).
11 Katsube T, Tabata H, Ohta Y, Yamasaki Y, Anuurad E, Shiwaku K,
et al., Screening for antioxidant activity in edible plant products:
comparison of low-density lipoprotein oxidation assay, DPPH radical
scavenging assay, and Folin–Ciocalteu assay. J Agric Food Chem
52:2391–2396 (2004).
12 Schuster-Gajzágó I, Kiszter AK, Tóth-Márkus M, Baráth A,
Márkus-Bednarik Z and Czukor B, The effect of radio frequency
heat treatment on nutritional and colloid-chemical properties of
different white mustard (Sinapis alba L.) varieties. Innov Food Sci
Emerg Technol 7:74–79 (2006).
13 Halvorsen BL, Carlsen MH, Phillips KM, Bøhn SK, Holte K, Jacobs Jr DR,
et al., Content of redox-active compounds (ie, antioxidants) in foods
consumed in the United States. Am J Clin Nutr 84:95–135 (2006).
14 Szőllősi R, Varga ISz, Erdei L and Mihalik E, Cadmium-induced oxidative
stress and antioxidative mechanisms in germinating Indian mustard
(Brassica juncea L.) seeds. Ecotox Environ Safe 72:1337–1342 (2009).
15 Esclapez MD, García-Pérez JV, Mulet A and Cárcel JA,
Ultrasound-assisted extraction of natural products. Food Eng
Rev 3:108–120 (2011).
16 Pingret D, Fabiano-Tixier A-S and Chemat F, Degradation during appli-
cation of ultrasound in food processing: A review. Food Control
31:593–606 (2013).
17 Pingret D, Durand G, Fabiano-Tixier A-S, Rockenbauer A, Ginies Ch
and Chemat F, Degradation of edible oil during food processing
by ultrasound: electron paramagnetic resonance, physicochemical,
and sensory appreciation. J Agric Food Chem 60:7761–7768 (2012).
18 Chemat F, Zill-e-Huma and Khan MK, Applications of ultrasound in
food technology: Processing, preservation and extraction. Ultrason
Sonochem 18:813–835 (2011).
19 Pan G, Yu G, Zhu Ch and Qiao J, Optimization of ultrasound-assisted
extraction (UAE) of flavonoids compounds (FC) from hawthorn seed
(HS). Ultrason Sonochem 19:486–490 (2012).
20 Da Porto C, Porretto E and Decorti D, Comparison of
ultrasound-assisted extraction with conventional extraction meth-
ods of oil and polyphenols from grape (Vitis vinifera L.) seeds.
Ultrason Sonochem 20:1076–1080 (2013).
21 Wen L, Yang B, Cui Ch, You L and Zhao M, Ultrasound-assisted extrac-
tion of phenolics from Longan (Dimocarpus longan Lour.) fruit seed
with artificial neural network and their antioxidant activity. Food
Anal Method 5:1244–1251 (2012).
22 Szydłowska-Czerniak A, Tułodziecka A and Szłyk E, A silver nanoparticle
based method for determination of antioxidant capacity of rapeseed
and its products. Analyst 137:3750–3759 (2012).
23 Cabello-Hurtado F, Gicquel M and Esnault M-A, Evaluation of the
antioxidant potential of cauliflower (Brassica oleracea) from a glu-
cosinolate content perspective. Food Chem 132:1003–1009 (2012).
wileyonlinelibrary.com/jsfa