Industrial Crops & Products 135 (2019) 294–300

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Industrial Crops & Products

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

Biologically active compounds from white and black mustard grains: An T

optimization study for recovery and identification of phenolic antioxidants
⁎
Gabriela Boscariol Raseraa, , Marina Hermenegildo Hilknera, Severino Matias de Alencarb,
Ruann Janser Soares de Castroa
a
Department of Food Science, School of Food Engineering, University of Campinas, 80 Rua Monteiro Lobato, Campinas, São Paulo, Brazil
b
Department of Agri-Food Industry, Food and Nutrition, ‘Luiz de Queiroz’ College of Agriculture, University of São Paulo, Piracicaba, Brazil

A R T I C LE I N FO A B S T R A C T

Keywords: This work aimed to determine the best solvent mixture for extraction of antioxidant compounds from two
Mustard grain varieties of mustard grains (white - Sinapsis alba and black - Brassica nigra) using a simplex centroid mixture
Antioxidant properties design. For this, the experiments were performed using pure, binary or ternary solvent mixtures containing
Statistical mixture design water, acetone and methanol. All extracts were analyzed for total phenolic compounds (TPC), DPPH- and ABTS-
Identification
radical scavenging activities. The binary mixture of water and acetone, in equal proportions, was the best solvent
combination to obtain an extract with higher TPC content and antioxidant properties. The extraction of anti-
oxidant compounds with water/acetone resulted in increases of 23-folds for TPC, 48-folds for ABTS and 25-folds
for DPPH compared to pure acetone. For black mustard, the extract obtained with this solvent combination was
19-, 31- and 27-folds higher than that produced with pure acetone for TPC, ABTS and DPPH, respectively.
Additionally, the bioactive compounds from the mustard extracts were identified by UHPLC-MS/MS, as follows:
3,4-di-hydroxybenzoic acid, ferulic acid and sinapic acid in white mustard and 3,4-di-hydroxybenzoic acid,
ferulic acid, sinapic acid and rutin in black mustard. This work reported for the first time an optimization study
for recovery of phenolic compounds from mustard grains in order to obtain extracts with better antioxidant
properties.

1. Introduction oxidative stress. Known as secondary plant metabolism, phenolic

Consumer interest in spices is present in humanity since ancient
times. They are also widely used in nutraceuticals, pharmaceuticals,
toiletry, perfumery, cosmetics industries and for centuries in Indian
traditional medicinal systems (Aruna and Baskaran, 2010). Mustard, in
special, is known for centuries as an important commercially condiment
in all of the world with manifold uses and applications (Fahey, 2016).
Mustard grains are classified in three different species: Brassica nigra,
Sinapis alba and Brassica junceae, popular know as black mustard, white
mustard and yellow mustard, respectively (Sforza and Prandi, 2016). In
general, the Brassica vegetables are sources of vitamin C, glucosinolates,
flavonoids, carotenoids and tocopherols that have antioxidant and an-
ticancer properties (Li et al., 2017). Mustard grains are also rich in
minerals, Omega-3 fatty acid, essential oils, vitamins B and E
(Divakaran and Babu, 2016).
Fruits, legumes, spices, herbs and grains are the major sources of
phenolic compounds. The high intake of them is associated to lower
risks of chronic and degenerative disease’s development caused by
compounds are the front-line defense of plants and can be divided into
several sub-groups according to their structural characteristics. The
three main sub-groups most commonly found in plant food are phenolic
acids, flavonoids and non-flavonoids. In addition, the antioxidant
properties of these compounds are strictly related with their molecular
structure. Thus, some characteristics showed recognized relevance in
their antioxidant properties, such as: the number and the position of
hydroxyl groups in the molecule, the hydroxylation degree, the distance
between carbonyl group and the aromatic ring besides the number of
aromatic rings (Zhang and Tsao, 2016).
The extract yield of phenolic compounds can be affected by several
factors as the solvent chemical nature, extraction time, solvent:solid
ratio, solvent concentration, particle size and temperature of the plant
material. Besides that, pure solvents cannot extract all the antioxidants
with their different polarities and structures (Garcia-Salas et al., 2010).
Therefore, it is convenient the use of a solvent’s mixture, that can be
binary, ternary or even multicomponent mixture. In this system, it is
possible to observe synergistic effects between the solvents which may

⁎
Corresponding author.
E-mail address: gabi_boscariol@hotmail.com (G. Boscariol Rasera).

https://doi.org/10.1016/j.indcrop.2019.04.059
Received 11 March 2019; Received in revised form 16 April 2019; Accepted 27 April 2019
Available online 02 May 2019
0926-6690/ © 2019 Elsevier B.V. All rights reserved.
G. Boscariol Rasera, et al. Industrial Crops & Products 135 (2019) 294–300

result in the extraction of compounds with different chemical char- the regression coefficients for each linear effect term, the binary and
acteristics (Marcus, 2002). In addition, it is already known the im- ternary interaction effect terms, respectively. The experimental mixture
portance of that, solvent optimization to obtain extracts rich in anti- design, data analysis and model building were performed using the
oxidant compounds from mustard grains is not found in literature yet. software Statistica® 13.3 from TIBCO (Palo Alto, California, USA).
In this work, a simplex centroid mixture design was used to study Three additional assays were performed under the most adequate
the most adequate solvent mixture for extraction of antioxidant com- condition determined in the mixture design to confirm the validity of
pounds from two varieties of mustard grains (Brassica nigra and Sinapsis the models. The experimental values obtained in validation tests were
alba). The total phenolic compounds and the antioxidant properties compared with the predicted values by the models at 95% confidence
evaluated by DPPH and ABTS-radical scavenging were used as re- interval.
sponses to select the best extractor. Additionally, the phenolic com- The recovering of antioxidant compounds was performed according
pounds from the mustard extracts were identified by UHPLC-MS/MS. to each assay showed in the Table 1. For this, 25 mg of samples were
mixed with 1 mL of solvent and maintained under stirring (150 rpm) for
2. Material and methods 20 min at 25 °C (Yeo and Shahidi, 2017). After extraction, the solution
was centrifuged (17 000 x g) for 15 min, and the supernatant was
2.1. Material collected and stored at −18 °C for further analysis. Ten extracts were
obtained and analyzed to their total phenolic compounds (TPC) content
Samples of two varieties of mustard grains, namely black (Brassica and antioxidant properties using two methods (ABTS- and DPPH-ra-
nigra) and white (Sinapsis alba), were purchased in a local market from dical scavenging).
Piracicaba (Sao Paulo, Brazil). The Folin and Ciocalteau’s phenol re-
agent, sodium carbonate, 2,2'-azino-bis(3-ethylbenzothiazoline-6-sul- 2.3. Total phenolic content (TPC)
phonic acid (ABTS), potassium persulphate, 6-hydroxy-2,5,7,8-tetra-
methylchroman-2-carboxylic acid (Trolox), 2,2-diphenyl-1- The total phenolics were estimated according to the method of
picrylhydrazyl (DPPH) and the UPLC standards (chlorogenic acid, Swain and Hillis (1959) with a modified version as described by de
protocatechuic acid, myricetin, caffeic acid, vanillic acid, gallic acid, Camargo et al. (2012). The Folin and Ciocalteau’s phenol reagent
3,4-di-hydroxybenzoic acid, quercetin, sinapic acid, ferulic acid, rutin (0.5 mL) was incorporated with 0.5 mL of extracts and 4 mL of distilled
and coumaric acid) were purchased from Sigma-Aldrich (Steinheim, water. After 3 min of incubation, 1 mL of saturated sodium carbonate
Germany). All other chemicals were purchased in the grade commer- solution (0.3 g mL1) was added to each tube. The reaction mixtures
cially available. were allowed to stand for 2 h at room temperature in the dark. The
absorbance was read at 760 nm. The results were expressed as mg of
2.2. Simplex centroid mixture design gallic acid equivalents (GAE) per gram dry weight of defatted sample
(mg GAE g−1).
A simplex centroid mixture design was applied to obtain the op-
timum mixture of the different solvents for maximum extraction of 2.4. ABTS radical cation scavenging activity
phenolic compounds, antioxidant properties and to identify the pre-
sence of synergistic or antagonistic effects between the components The ABTS assay was performed using a microplate reader Spectra
(solvents). Each solvent of the system was studied at six levels ac- Max M3 (Molecular Devices, LLC, Sunnyvale, CA, USA) according to the
cording to the 10 runs presented in the matrix of the experimental method described by Al-Duais et al. (2009). The ABTS [2,2′-azino-bis(3-
design (Table 1). ethylbenzothiazoline-6-sulphonic acid)] radical cation was made in
Quadratic or special cubic regression models were adjusted in 75 mM potassium phosphate buffer saline solution (PBS) (pH 7.4). The
function of significant effects for the variations of all the responses (p ≤ working solution of ABTS radical cation (potassium persulphate
0.10), considering acceptable determination coefficients above 70% (140 mM, 0.088 mL) and ABTS (7 mM, 10 mL) in PBS was prepared at
(R² > 0.70). Eq. 1 represents these models as follows: the time of analysis, by diluting its stock solution in PBS to reach an
q q q
absorbance value of 0.70 ± 0.20 (734 nm).
Yi = ∑ βiXi + ∑ ∑ βijXiXj + ∑ ∑ ∑ βijkXiXjXk Mustard extracts were diluted in PBS until reach 2.5 mg mL−1 as a
i=1 i<j i<j<k (1) final concentration. Aliquots of 20 μL of each extract were added to
220 μL of ABTS radical cation solution and the absorbance was read
where ‘Yi’ represents the predicted response by the model; ‘q’ is the after 6 min at 734 nm. The control assay was made with distilled water
number of independent variables (components) in the mixture; ‘Xi, Xj, in place of the samples. A standard curve was prepared using Trolox at
Xk’ indicate the coded independent variables; ‘βi’, ‘βij’ and ‘βijk’ represent different concentrations (2.5–200 μM) and the results were expressed as
μmol of Trolox equivalents per g of sample (μmol TE g−1).
Table 1
Matrix of the simplex centroid mixture design for extraction of antioxidant
2.5. DPPH radical scavenging activity
compounds from mustard grains using different solvents and their mixtures.
Run Independent variables DPPH radical scavenging activity was carried out according to the
method described by Al-Duais et al. (2009). Briefly, 134 μL of 150 μM
Water Acetone Methanol
x1 x2 x3 DPPH radical solution, which was freshly made in ethanol, was added
to 66 μL of diluted extracts (2.5 mg mL1) or standards. After 45 min of
1 1 0 0 incubation in the dark at room temperature, the absorbance was mea-
2 0 1 0
sured using a microplate reader Spectra Max M3 at 517 nm (Molecular
3 0 0 1
4 1/2 1/2 0 Devices, LLC, Sunnyvale, CA, USA). Ethanol was used as blank. A
5 1/2 0 1/2 standard curve was prepared with different concentrations of Trolox
6 0 1/2 1/2 (20–140 μM) and the results were expressed as μmol TE g−1.
7 2/3 1/6 1/6
8 1/6 2/3 1/6
2.6. Identification of phenolic compounds by UHPLC-MS/MS
9 1/6 1/6 2/3
10 1/3 1/3 1/3
Identification of phenolic compounds was carried out according to

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Xavier et al. (2017) with slight modifications. For that, it was used an scavenging ranging from 0.95 to 26.24 μmol TE g−1.
ACQUITY Ultra Performance LC™ system (Waters, Milford, MA, USA) It is important to note that this great variation observed between the
linked simultaneously to Micromass Quattro micro™ API benchtop values shows the importance of the study to determinate the best sol-
tandem quadrupole mass spectrometer (Waters MS Technologies, vent combination to extract antioxidant compounds. Further, choose
Manchester, UK), coupled with an electrospray ionization interface the most appropriated solvent for each plant material is one of the most
(ESI) source in a negative mode (3 kv of capillary voltage, 150 °C of important factors to obtain a higher bioactive compounds content
source temperature and 400 °C of desolvation gas temperature, nitrogen (González-Montelongo et al., 2010).
flow rates of 800 and 100 L/h for the desolvation and cone gases, re- The proposed models showed high significance at a 95% confidence
spectively). level since the analysis of variance (ANOVA) test indicated that the p-
An Acquity BEH C18 column (50 mm x 2.1 mm) with a 1.7 μm par- values for the responses were less than 0.05. The variability of the ex-
ticle size (Waters MS Technologies, Manchester, UK) were used for perimental data was checked by the coefficient of determination value
chromatographic separations. The mobile fase components were ultra- (R2) and values of R2 ranged from 0.71 to 0.98 indicated that the
pure water containing 0.1% formic acid (A) and acetonitrile (B) at a models were able to explain 71–98% of the experimental data varia-
flow rate of 0.2 mL/min. Aliquots of 10 μL of samples were injected, bility.
under the linear gradient starting at 3% B, increasing to 40% B in The statistical significance of the models can also be proven by the
15 min. The multiple reaction monitoring (MRM) mode as well as m/z F-test, in which the calculated F-values for regressions were greater
transitions of the precursor and product ions were employed to identify than the tabulated F- values. Equations in Table 3 represent the models
and confirm the presence of phenolic compounds in the sample. with significant factors for experimental data. Statistical analysis
Phenolic standards (12) monitored were: chlorogenic acid, proto- showed that the models were predictive for TPC, ABTS and DPPH-ra-
catechuic acid, myricetin, caffeic acid, vanillic acid, gallic acid, 3,4-di- dical scavenging of the mustard extracts.
hydroxybenzoic acid, quercetin, sinapic acid, ferulic acid, rutin and The variations in the TPC and antioxidant properties of the extracts
coumaric acid. obtained from black and white mustards were also depicted using
mixture contour plots (Figs. 1 and 2).
2.7. Calculations and statistics Contour plot evaluation indicated that the best extraction solvent
was the binary combination between water and acetone for both, white
The results were expressed as the arithmetic mean and the software and black mustard. The highest efficiency of the water:acetone mixture
Minitab® 18 from Minitab Inc. (USA) was used to verify if there was in the phenolic compounds extraction was also observed for others
statistical difference (p-value ≤ 0.05) between the values when ana- authors (González-Montelongo et al., 2010; Meneses et al., 2013). Be-
lyzed by the Tukey test. sides that, the use of ethanol and acetone is recommended, mainly
because they are less toxic than other organic solvents as methanol and
3. Results and discussion have high extraction efficiency (Socaci et al., 2018).
TPC, ABTS- and DPPH-radical scavenging contour plots interpreta-
The results for each assay of the mixture design to TPC, DPPH- and tion for both mustard varieties showed similar profiles for each solvent
ABTS-radical scavenging of black and white mustard extracts are pre- and mixtures of them, indicating that the mixture of water and acetone
sented in Table 2. The extracts obtained from white mustard showed was the most appropriate combination to recover antioxidant com-
TPC ranging from 0.82 to 20 mg g−1, ABTS-radical scavenging ranging pounds. In addition, it is possible to observe a clear relationship be-
from 2.25–109.25 μmol TE g−1 and DPPH-radical scavenging ranging tween the content of phenolic compounds and the antioxidant proper-
from 2.01–58.7 μmol TE g−1. Extracts from black mustard showed va- ties of the extracts (Figs. 1 and 2).
lues of TPC ranging from 0.65 to 12.16 mg g−1, ABTS-radical scaven- Validation essays were done, in genuine triplicate, to confirm the
ging ranging from 1.14–36.22 μmol TE g−1 and DPPH-radical predictive capacity of proposed models and the results are showed in
Table 3. The experimental values agreed with the values predicted by
Table 2 the models within a 95% confidence interval, confirming the model’s
Responses for TPC, ABTS- and DPPH-radical scavenging from the mixture de- validity for the evaluated responses (Table 4).
sign used to study the most adequate solvent to obtain extracts rich in phenolic In general, phytochemicals compounds with higher polar character
compounds and high antioxidant properties from white and black mustard can be easily extracted with water, while highly hydroxylated phenolic
grains. compounds, such as catechins, are more soluble in alcohols like ethanol
Run TPC (mg GAE g−1) ABTS (μmol TE g−1) DPPH (μmol TE g−1) and methanol (Arts and Hollman, 1998). Acetone, as a pure solvent,
White mustard grain was inefficient for the recovering of the interest compounds. Thus,
1 12.48 ± 0.65 72.24 ± 4.18 43.89 ± 0.32 knowing that less polar solvents, like ethyl acetate, acetone and
2 0.82 ± 0.27 2.25 ± 0.18 2.02 ± 0.14
chloroform have more affinity towards compounds with low polarity
3 10.48 ± 0.29 61.75 ± 3.55 30.91 ± 1.09
4 19.39 ± 0.66 109.25 ± 4.61 51.25 ± 0.08 (Lafka et al., 2007), it is assumed that a few compounds with these
5 18.49 ± 0.61 105.86 ± 0.68 50.18 ± 0.71 chemical characteristics were found in the mustard extracts evaluated.
6 7.89 ± 0.48 74.79 ± 1.67 44.78 ± 0.08 On the other hand, the combination of different solvents resulted in
7 20.00 ± 0.33 97.13 ± 5.14 47.69 ± 1.03 interesting synergistic effects. Although the binary combination of
8 19.52 ± 0.61 99.52 ± 1.76 57.44 ± 0.55
water and methanol had an important synergistic effect, considering a
9 19.41 ± 0.65 93.97 ± 4.25 53.14 ± 0.31
10 16.84 ± 0.02 108.05 ± 1.45 58.70 ± 0.63 common condition of extraction for the two varieties of mustard, the
Black mustard grain mixture of water and acetone, in equal proportions, resulted in the
1 5.32 ± 0.08 6.21 ± 0.82 8.88 ± 0.08 highest values for TPC and antioxidant properties, an indicative that the
2 0.65 ± 0.00 1.14 ± 0.03 0.95 ± 0.18
majority antioxidant compounds of white and black mustard have high
3 4.56 ± 0.02 13.30 ± 1.00 15.40 ± 0.62
4 12.16 ± 0.71 36.22 ± 1.10 26.24 ± 0.28 affinity for these two solvents. According to some authors, the mixture
5 8.02 ± 0.03 20.61 ± 1.80 17.24 ± 0.83 of water and organic solvents is able to create a more polar condition,
6 3.05 ± 0.04 8.62 ± 0.50 7.47 ± 0.36 which can facilitate the water and/or organic solvents extraction of
7 9.60 ± 0.54 25.09 ± 0.59 20.17 ± 0.85 soluble phenolic compounds (Do et al., 2014; Meneses et al., 2013).
8 10.00 ± 0.15 24.63 ± 0.38 24.00 ± 2.60
Socaci et al. (2018) elucidated that extraction yield is reduced because
9 6.21 ± 0.13 14.15 ± 0.25 11.16 ± 0.04
10 7.84 ± 0.33 16.73 ± 1.23 15.05 ± 0.75 phenolic compounds in general are more soluble in organic solvents less
polar than water, and, that’s the reason why suitable proportion study

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G. Boscariol Rasera, et al. Industrial Crops & Products 135 (2019) 294–300

Table 3
ANOVA and codified mathematic models for total phenolic compounds (TPC and antioxidant activity (ABTS and DPPH) from white and black mustard extracts with
different solvents.
Response Model Equations Fcalculated Ftabulated R2 p-value

White mustard
TPC Quadratic 14.73x1+3.29x2+15.02x3+58.06 x1x2 4.92 3.37 0.71 0.04
ABTS Quadratic 87.76x1+5.2x2+78.69x3+272.33x1x2+168.30 x2x3 6.07 3.48 0.82 0.03
DPPH Quadratic 48.22x1+3.96x2+37.35x3+113.68x1x2+117.94x2x3 6.34 3.48 0.83 0.03
Black mustard
TPC Quadratic 5.21x1+0.68x2+5.06x3+36.97x1x2+13.52x1x3 75.85 3.48 0.98 < 0.001
ABTS Quadratic 6.71x1+0.16x2+13.34x3+123.58x1x2+38.85x1x3 43.18 3.48 0.97 < 0.001
DPPH Quadratic 12.14 x1-0.67x2+16.77x3+85.70x1x2 21.19 3.37 0.91 0.001

The coded values in model equations represent the independent variables and their interactions: x1 = water; x2 = acetone and x3 = methanol.

Fig. 1. Contour plots for total phenolic compound (TPC) and antioxidant activity evaluated by ABTS and DPPH methods, respectively, for white mustard extracts.

Fig. 2. Contour plots for total phenolic compound (TPC) and antioxidant activities evaluated by ABTS and DPPH methods, respectively, for black.
mustard extracts.

Table 4
Validation tests performed to determine the adequacy of the polynomial models obtained for the for TPC, ABTS and DPPH for white and black mustard extracts.
Response Independent variables (validation assays) Predicted values Experimental values

x1 (water) x2 (acetone) x3 (methanol)

White mustard

TPC 0.5 0.5 0 23.51a 23.03 ± 4.22a

ABTS 0.5 0.5 0 114.56a 123.28 ± 12.73a
DPPH 0.5 0.5 0 54.49a 57.82 ± 4.79a
Black mustard
TPC 0.5 0.5 0 12.18a 11.21 ± 1.74a
ABTS 0.5 0.5 0 34.32a 34.78 ± 0.98a
DPPH 0.5 0.5 0 27.16a 26.22 ± 0.60a

Results are presented as the mean (n = 3) ± SD, and those with different letters are significantly different, with p < 0.05. Comparisons were made between the
observed and predict values for each correspondent response.

between water and an organic solvent is a decisive factor.
Similar results were observed in other studies. The use of a binary
mixture of water (1/3) and acetone (2/3) resulted in the highest re-
covery of phenolic compounds from seeds of chia (Alcântara et al.,
2019). A solution of 70:30% acetone:water (v:v) was also effective in
the extraction of phenolic compounds from eggplant by-product when
compared to 70:30% methanol:water (v:v) and 70:30% ethanol:water
(v:v) (Boulekbache-Makhlouf et al., 2013). Besides, phenolic com-
pounds from brewer’s spent grains were better extracted with a solution
60:40% acetone:water (v:v) and resulted in the highest values for total

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Fig. 3. Phenolic compounds identification of the extract from white mustard.
phenolic compounds and antioxidant capacity measured by FRAP and
DPPH methods (Meneses et al., 2013).
The identification of phenolic compounds was performed using the
mustard extract obtained with the binary mixture of 50:50% water:-
acetone (v:v). The bioactive compounds 3,4-di-hydroxybenzoic acid,
ferulic acid and sinapic acid were identified in white mustard samples
(Fig. 3). For black mustard, 3,4-di-hydroxybenzoic acid, ferulic acid,
sinapic acid and rutin were found (Fig. 4).
Many of the protective effect of phenolic compounds is due their
antioxidant, anticarcinogenic, antimicrobial, antimutagenic and anti-
inflammatory properties (Shahidi and Ambigaipalan, 2015) and the
results obtained in this work indicated the bioactive potential of mus-
tard.
The majority of phenolic compounds found are phenolic acids, ex-
cept rutin, that is a flavonoid. Phenolic acids are present as hydro-
xybenzoic acids (p-hydroxybenzoic, protecatechuic, vanillic, siringic
and gallic acids) and hydroxycinnamic acids (p-coumaric, caffeic,
ferulic and sinapic acid) and this second group is the most abundant in
plant kingdom. Those compounds behave as antioxidant due their re-
activity with its phenol portion, that act mostly donating hydrogen
atom (Shahidi and Ambigaipalan, 2015).
Studies on phenolic profile of mustard seeds have been reported
since 1983, but with few works until nowadays. In the most of re-
searches, the main objective was the study of the oil fraction of mus-
tard, mainly rapeseed mustard, used to biofuel. For identification, dif-
ferent solvents were used as noted in the sequence. Kozłowska et al.
(1983b) identified and quantified p-hydroxybenzoic, vanillic, gentisic,
o-coumaric, syringic, p-coumaric, ferulic, caffeic and sinapic acids in
soluble extracts (80% methanol) of white mustard flour. p-hydro-
xybenzoic was the most abundant phenolic acid. In another study of the
same research group, Kozlowska et al. (1983a) identified and quantified
a similar phenolic compounds profile in white mustard compared to
rapeseed. Shahidi et al. (1994) encouraged the use of mustard seeds as a
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Industrial Crops & Products 135 (2019) 294–300
protective ingredient against lipid oxidation, confirmed with the anti-
oxidative properties of ethanolic extracts of mustard flour when com-
pared to synthetic antioxidants such as BHA. Engels et al. (2012) found
sinapic acid and several sinapic acids conjugates in acidified methanol
or acetone extracts of coarsely ground Oriental mustard seed meal
(Brassica junceae) by UHPLC-DAD-ESI-MS. All those results observed in
literature, report the absence of optimization of phenolic compounds
solvent extraction in mustard seeds, an important differential of the
present study.
Thiyam-Holländer et al. (2014) confirmed that seeds, oil, flour and
meal from mustard contain significant amounts of phenolic compounds,
specially sinapic acid derivates. More recently, Sharma et al. (2017)
showed a similar profile of phenolic compounds in defatted rapeseed
mustard (Brassica junceae), in which were identified sinapic acid, galic
acid, ferulic acid, caffeic acid, 4-hydroxybenzoic acid, and p-coumaric
acid.
Hydroxybenzoic acid, ferulic acid and rutin were already identified
in chia seeds (Salvia hispanica L.) and it already known the importance
of water to extraction of hydroxybenzoic acids (Alcântara et al., 2019),
what is in agreement with our results. Ferulic acid, a hydroxycinnamic
acid derivative, was found in finger millets (Xiang et al., 2019) and at
seven millet grain samples (Chandrasekara and Shahidi, 2011).
4. Conclusions
Considering the diversity of composition of each grain variety in
nature, individually designed and optimized studies of solvent extrac-
tion of bioactive compounds are extremely necessary. In this case, the
extraction of antioxidant compounds from mustard grains showed that
the combination between water and acetone, in equal proportions, was
the most adequate solution. At this condition, the bioactive compounds
3,4-di-hydroxybenzoic acid, ferulic acid and sinapic acid were identi-
fied in white mustard and 3,4-di-hydroxybenzoic acid, ferulic acid,
G. Boscariol Rasera, et al.
Fig. 4. Phenolic compounds identification of the extract from black mustard.
sinapic acid and rutin in the black mustard. These results can support
the future researches with mustard grains, since this is the first study
that reported the optimization of the extraction of mustard antioxidant
compounds. Besides, it encourages mustard grains consumption, since
they presented antioxidant activity and potential biological function.
Acknowledgements
This study was financed in part by the National Council for
Scientific and Technological Development - Brazil (CNPq) and
Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - Brazil
(CAPES) - Finance Code 001 (PROEX process number 23038.000795/
2018-61).
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