INTRODUCTION
Lipid oxidation is one of the major chemical
changes that occur during food processing, storage, and
preparation. Lipid molecules are rapidly oxidized in the
presence of oxygen, especially in the case of unsaturated
fatty acids [1]. Antioxidants are widely used today to
reduce the rate of oxidation reaction of fats in foods.
Antioxidants are molecules or compounds that
act against free radicals which damage to molecules,
resulting in the loss of their function. Antioxidants
provide a primary defense against such oxidative
degradations [2]. In industrial processes, synthetic
antioxidants – such as butyl hydroxy toluene and butyl
hydroxy anisol – are mainly used to increase the food’s
shelf life. In this regard, nutritionists have found that these
compounds can have adverse effects on the body [3].
Therefore, it is necessary to use strong antioxidants
with lower toxicity and greater efficacy. In recent
decades, natural antioxidants have drawn the attention of
food researchers due to their safety in food formulation.
These are extracts and essential oils of various plants
that produce positive effects on nutrient oxidation
reactions.
Pre-extraction seed treatment is one of the most
essential steps to ensure high quality extraction. One
of the treatment methods is the use of a pulsed electric
field. It is an important non-thermal method of treating
foodstuffs by placing them in a chamber between two
187
Arab Shirazi S.H. et al. Foods and Raw Materials, 2020, vol. 8, no. 1, pp. 186–195
electrodes and subjecting to high-voltage pulses for
a short time. A pulsed electric field focuses mainly on
the microscopic scale so that pores are created in the
cell membrane, accelerating the exit of intercellular
compounds. This process preserves qualitative,
nutritional, and energy consumption properties, as well
as increases productivity in food production [4].
Most importantly, a pulsed electric field destroys
the cell wall and its membrane and increases the mass
transfer rate. Indeed, when a living cell is affected by
such a field, the cell wall and its membrane are naturally
damaged. The inside material is easily removed and
the surrounding material enters the cell, resulting in its
destruction. With increased permeability of plant and
animal cells, their intracellular material is extracted
more easily and quickly. Therefore, this treatment can
be used as a pre-processing step in the extraction of
valuable cellular materials [5, 6].
Salvia leriifolia L. is one of the plants that contain
antioxidant compounds. It is a native species of
Lamiaceae family to Khorasan and Semnan provinces,
Iran [7]. It grows in cold and semi-arid or arid regions at
altitudes between 900 and 1650 meters, with an average
rainfall of 80 mm. A special shape of its leathery leaves,
especially white villi on both sides, and a wide growth
on the surface of the soil make this plant resistant to
harsh winter winds or severe heat [8].
Various studies have reported therapeutic properties
of Salvia leriifolia. For example, its aqueous and
alcoholic root extracts have neuroprotective properties
against topical anemia in the rat brain [9]. The analgesic
and sedative activity of Salvia leriifolia leaf extract
in the amount of 500 mg/kg is comparable to that of
diazepam in the amount of 5 mg/kg [8]. In treating
chronic inflammation, the plant’s extract is similar to
diclofenac [10]. Its aqueous and alcoholic leaf extracts
were found to prevent gastric ulcers in rats similarly to
Sucralfate [11].
In addition, the plant’s root and leaf extracts
showed considerable antimicrobial activity [9]. They
also have strong antioxidant properties that prevent
the oxidation of oils. This property is competitive
with that of antioxidants commonly used in the food
industry, such as butylated hydroxy toluene and alphatocopherol.
It is due to the presence of a secondary
metabolite of chalcones, called butin, in this plant.
Finally, Salvia leriifolia is of industrial importance. In
this regard, researchers have found that its seeds contain
26% yellow oil, with a very low peroxide index and a
high antioxidant index, which increases its shelf life
compared to other oils [12].
Another plant with antioxidant properties is Linum
usitalissmum L. It is a one-year-old plant of Linaceae
family that grows in bushes. This plant has over 200
species but only Linum usitalissmum has economic
importance. In addition, its seeds have several powerful
antioxidants, including lignans. 100 g of Linum
usitalissmum contains about 9.2 mg of vitamin E, mainly
in the form of gamatocopherol [13].
The most common method for extracting compounds
from plant tissues uses aqueous and ethanol solvents.
Therefore, we aimed to evaluate effects of an electrical
pulse pre-treatment and to compare the aqueous and
alcoholic extracts of Linum usitalissmum and Salvia
leriifolia seeds.
STUDY OBJECTS AND METHODS
Preparation of raw materials. For this study, Linum
usitalissmum L. seeds and Salvia leriifolia L. aerial
limbs, leaves, and stems were obtained from a certified
apothecary. We also used chemicals produced by Merck
(Germany).
Extraction of aqueous and alcohol extracts
from Linum usitalissmum and Salvia leriifolia seeds
pretreated with a pulsed electric field. Initially,
Linum usitalissmum and Salvia leriifolia seeds were
cleaned and the external materials and impurities were
separated and dried in an oven at 45°C. The samples
were powdered in a household mill (Fama Model Cs,
Germany) and passed through a 40-mesh sieve. Finally,
they were packed in air- and water-proof packages and
kept in a freezer at –18°C until further experiments
to preserve the extract’s antioxidant and functional
properties.
The aqueous and alcoholic extracts were made using
the Kabiri and Seyyedlangi method [14, 15]. For this,
the prepared powders were mixed with a water solvent
(aqueous extract) or 80% methanol (alcoholic extract) at
the ratio of 50:1.
Subsequently, to apply a pulsed electric field
pretreatment, each of the extracts was subjected
to an alternating electric field with zero (without
pretreatment), 3, and 6 kV·cm–1 intensity and a constant
pulse number of 30 (Table 1). The linear electric current
in this device is transmitted to a series of capacitors and
the energy stored in the capacitors is discharged to the
chamber containing two electrodes with a pulse switch.
The discharge chamber is made of Plexiglass 1 and the
distance between the two electrodes is 4 cm. These
waves were applied to facilitate the extraction.
Evaluation of antioxidant properties of Linum
usitalissmum and Salvia leriifolia aqueous and
alcoholic extracts. Total phenolic compounds. The
amount of total phenolic compounds was measured by
the Folin-Ciocalteu method according to Oardoz et al.
[16]. For this purpose, 10 g of extracts was first extracted
with 200 mL of methanol for 24 h at room temperature
using a magnetic stirrer. The extract was filtered with
Whatman Paper No. 1 and the sediment was extracted
again under the same conditions. The solvent was then
removed by a vacuum evaporator at less than 40°C and
concentrated as far as possible. Then, 0.5 mL of the
extract was mixed with 2.5 mL of 0.2N Folin-Ciocalteu
reagent and 2 mL of 7.5% sodium carbonate solution.
188
Arab Shirazi S.H. et al. Foods and Raw Materials, 2020, vol. 8, no. 1, pp. 186–195
The mixture was kept at room temperature for 120
min. The absorbance rate of the solution was then read
by a spectrophotometer at 760 nm. The total content of
phenolic compounds was expressed in mg/g of extract
using the line equation drawn on the basis of gallic acid.
The calibration curve was plotted as follows.
Different concentrations of gallic acid were first
prepared and 0.5 mL of each was mixed with 2.5 mL
of 10% Folin-Ciocalteu reagent (v/v) and 2 mL of 7.5%
sodium carbonate for half to 8 min (w/v). The samples
were stored at room temperature for 30 minutes and then
absorbed at 760 nm [17]. Distilled water was used as a
control.
Antioxidant activity by DPPH method. To extract
antioxidant compounds, 10 g of aqueous and alcoholic
extracts with 100 mL methanol was stirred with a
magnetic stirrer at a speed of 100 rpm at 25°C for 24 h
and finally filtered with Whatman filter paper. The
solution was then transferred to a freezing dryer for
methanol removal and, finally, the dried extract was
stored at –20°C [18]. The antioxidant activity of the
samples was further evaluated by the method of Brand
Williams et al. [19]. 3.9 mL of DPPH stock was poured
into the cell and read by a spectrophotometer at 515 nm.
Then, 0.1 mL of each extract was added to the DPPH
stock solution and after 90 minutes of incubation, the
absorbance of the samples was read at 515 nm. The
inhibition percentage of DPPH radical was calculated
using Eqs. (1) and (2).
I(%)= 100 × (A0−As)/A0 (1)
where A0 is control absorption and As is sample absorption.
The results were then expressed as IC50 (the amount
of antioxidant required to reach 50% of the initial DPPH
concentration). To draw a standard curve, we used a
Trolox solution with a concentration of 1000–100 μmol.
First, the percentage of radical neutralization activity
was obtained for each sample. Then, we calculated the
antioxidant activity of the samples using a standard
curve in μmol of Trolox per gram dry weight (μmol/g).
Antioxidant activity by TEACI method. To extract
antioxidant compounds, 10 g of the milled sample with
100 mL of methanol was mixed with a magnetic stirrer
at 100 rpm and 25°C for 24 h and then filtered with a
Whatman filter. Then, the methanol was transferred to a
freezing dryer and, finally, the dried extract was stored
at –20°C [18]. The antioxidant activity of the samples
was further evaluated by the method of Yu et al. [20].
First, we made an aqueous solution of ABTSII at
a concentration of 1 mM to prepare the radical ABTS.
Potassium persulfate was then added to this solution to
reach a final concentration of 2.45 mM. The resulting
solution was incubated at room temperature and
darkness for 2 h. During this time, the ABTS molecule
produced the ABTS•+ cation radical. Then, 4 μL of the
samples was taken with a Peptide and mixed with 4 mL
of the ABTS•+ solution in the cell. Its absorption at a
734 nm wavelength was verified at 6 min after mixing
(for 30 s). A standard curve was plotted, corresponding
to the reaction of 40 μL of Trolox (at concentrations of
50, 100, 250, 500, 750, and 1000 μM) to 4 mL of the
ABTS•+ solution. The inhibition percentage of ABTS•+
of the samples was calculated according to Eq. (2). Also,
the ABTS•+ radical inhibition activity was expressed
based on the standard Trolox curve as the Trolox
solution equivalent antioxidant capacity (mM TEAC).
Statistical design and analysis of results. The
results of our study were evaluated with SPSS 16
software.
To extract the essential oil, we used a completely
randomized design with a three-factor arrangement.
In particular, the three factors were a plant source
(Salvia leriifolia and Linum usitalissmum), a type of
pretreatment (pulsed electric field at the intensity of zero
(no pretreatment), 3 and 6 kV·cm–1), and a type of solvent
(aqueous and alcoholic).
The samples were obtained in three replications
and the means were compared by the Duncan test at a
significant level of 5% (P < 0.05). Finally, Excel software
was used to plot the diagrams.
AA%= [Ablank−Asample/Ablank] × 100 (2)
where Ablank is the absorption of a control sample without
the active compound and Asample is the absorption of a sample
containing a distilled extract).
RESULTS AND DISCUSSION
Total phenolic compounds. Fig. 1 presents
independent effects of the factors, Fig. 2 shows their
binary effect, while Table 3 indicates the interaction
between the three factors in their effect on the content
of phenolic compounds in the extracts. We found that
a higher intensity of a pulsed electric field and the use
of an alcoholic solvent significantly increased total
I Trolox equivalent antioxidant capacity
II 2,2’-Azino-Bis(3-ethylbenzothiazoline-6-Sulphonic Acid)
Table 1 Treatments investigated in the study
Extraction
method
Intensity of pulsed Herbal source
electric field pretreatment,
kV·cm–1
Treatment
code
Linum aqueous
usitatissimum
1 0
2 alcoholic
3 3 aqueous
4 alcoholic
5 6 aqueous
6 alcoholic
7 0 Salvia leriifolia aqueous
8 alcoholic
9 3 aqueous
10 alcoholic
11 6 aqueous
12 alcoholic
189
Arab Shirazi S.H. et al. Foods and Raw Materials, 2020, vol. 8, no. 1, pp. 186–195
b
a
0
10
20
30
40
50
Linum Usitatissimum Salvia leriifolia
Total phenolic compounds,
mg Galic acid/g
Herbal source
c
b
a
0
10
20
30
40
50
0 3 6
Total phenolic compounds,
mg Galic acid/g
Intensity of electric pulse field, kV·cm1
b
a
0
10
20
30
40
50
aqueous alcoholic
Total phenolic compounds,
mg Galic acid/g
Solvent type
b
a
b
a
0
15
30
45
Linum Usitatissimum Salvia leriifolia
Total phenolic compounds,
mg Galic acid/g
Herbal source
aqueous alcoholic
Solvent type
b
a
b
a
b
a
0
10
20
30
40
50
Linum Usitatissimum Salvia leriifolia
Total phenolic compounds,
mg Galic acid/g
Herbal source
0 3 6
Intensity of electric pulse field, kV·cm1
d
cd
c c
b
a
0
15
30
45
0 3 6
Total phenolic compounds,
mg Galic acid/g
Intensity of electric pulse field, kV·cm1
aqueous alcoholic
Solvent type
a
b
0
100
200
300
Linum Usitatissimum Salvia leriifolia
DPPH IC50,
μg/mL
Herbal source
a
b
c
0
100
200
300
0 3 6
DPPH IC50,
μg/mL
Intensity of electric pulse field, kV·cm1
a
b
0
100
200
300
aqueous alcoholic
DPPH IC50,
μg/mL Solvent type
a
d
b
d
c
d
0
100
200
300
Linum Usitatissimum Salvia leriifolia
DPPH IC50,
μg/mL
Herbal source
0 3 6
Intensity of electric pulse field, kV·cm1 m
a
c
b
c
100
200
300
DPPH IC50,
μgr/mL
Solvent type
a
b
c
b
cd
d
100
200
300
DPPH IC50,
μg/mL
Solvent type
phenolic compounds at a 95% confidence level. On the
other hand, the content of total phenolic compounds was
higher in the Salvia leriifolia L. extract, compared to
Linum usitalissmum L. (P < 0.05).
Various factors, such as plant variety, harvest area,
and harvest time, appear to affect the content of phenolic
compounds. Different studies have found different
amounts of total phenolic compounds in the Salvia
leriifolia plant. For example, Hamrouni-Sellami et al.,
Ahmadi et al., Najafi et al., Abadi et al., and Bahadori
et al. reported total phenolic compounds of 0.399–
2.37, 40.47–61.32, 11.28–23.88, 12.68–83.85, and 17.3–
294.9 mg of gallic acid per gram of extract, respectively
[21–25]. In our study, this value reached 33.24–63.98
mg of gallic acid per gram of extract, depending on
Figure 1 Independent effects of herbal source (a), pretreatment
(b), and solvent type (c) on total phenolic compounds
(P < 0.05)
b
a
0
10
20
30
40
50
Linum Usitatissimum Salvia leriifolia
Total phenolic compounds,
mg Galic acid/g
Herbal source
c
b
a
0
10
20
30
40
50
0 3 6
Total phenolic compounds,
mg Galic acid/g
Intensity of electric pulse field, kV·cm1
b
a
0
10
20
30
40
50
aqueous alcoholic
Total phenolic compounds,
mg Galic acid/g
Solvent type
b
a
b
a
0
15
30
45
Linum Usitatissimum Salvia leriifolia
Total phenolic compounds,
mg Galic acid/g
Herbal source
aqueous alcoholic
Solvent type
b
a
b
a
b
a
0
10
20
30
40
50
Linum Usitatissimum Salvia leriifolia
Total phenolic compounds,
mg Galic acid/g
Herbal source
0 3 6
Intensity of electric pulse field, kV·cm1
d
cd
c c
b
a
0
15
30
45
0 3 6
Total phenolic compounds,
mg Galic acid/g
Intensity of electric pulse field, kV·cm1
aqueous alcoholic
Solvent type
a
b
0
100
200
300
Linum Usitatissimum Salvia leriifolia
DPPH IC50,
μg/mL
Herbal source
a
b
c
0
100
200
300
0 3 6
DPPH IC50,
μg/mL
Intensity of electric pulse field, kV·cm1
300
a
b
300
Intensity of electric pulse field, kV·cm1 m
a
Salvia leriifolia
c
b
a
0
10
20
30
40
50
0 3 6
Total phenolic compounds,
mg Galic acid/g
Intensity of electric pulse field, kV·cm1
a
alcoholic
b
a
b
a
0
15
30
45
Linum Usitatissimum Salvia leriifolia
Total phenolic compounds,
mg Galic acid/g
Herbal source
aqueous alcoholic
Solvent type
a
a
a
Salvia leriifolia
Herbal source
6
pulse field, kV·cm1
d
cd
c c
b
a
0
15
30
45
0 3 6
Total phenolic compounds,
mg Galic acid/g
Intensity of electric pulse field, kV·cm1
aqueous alcoholic
Solvent type
b
Salvia leriifolia
a
b
c
0
100
200
300
0 3 6
DPPH IC50,
μg/mL
Intensity of electric pulse field, kV·cm1
a
d
b
d
c
100
200
300
DPPH IC50,
μg/mL
Intensity of electric pulse field, kV·cm1 m
(а)
(c)
(b)
Figure 2 Binary effects of herbal source and pre-treatment (a),
herbal source and solvent (b), and pre-treatment and solvent (c)
on total phenolic compounds (P < 0.05)
b
0
10
20
Linum Usitatissimum Salvia leriifolia
Total phenolic mg Galic Herbal source
c
0
10
20
0 3 6
Total phenolic mg Galic Intensity of electric pulse field, kV·cm1
b
a
0
10
20
30
40
50
aqueous alcoholic
Total phenolic compounds,
mg Galic acid/g
Solvent type
b
a
b
a
0
15
30
45
Linum Usitatissimum Salvia leriifolia
Total phenolic compounds,
mg Galic acid/g
Herbal source
aqueous alcoholic
Solvent type
b
a
b
a
b
a
0
10
20
30
40
50
Linum Usitatissimum Salvia leriifolia
Total phenolic compounds,
mg Galic acid/g
Herbal source
0 3 6
Intensity of electric pulse field, kV·cm1
d
cd
c c
b
a
0
15
30
45
0 3 6
Total phenolic compounds,
mg Galic acid/g
Intensity of electric pulse field, kV·cm1
aqueous alcoholic
Solvent type
a
b
0
100
200
300
Linum Usitatissimum Salvia leriifolia
DPPH IC50,
μg/mL
Herbal source
a
b
c
0
100
200
300
0 3 6
DPPH IC50,
μg/mL
Intensity of electric pulse field, kV·cm1
a
b
0
100
200
300
aqueous alcoholic
DPPH IC50,
μg/mL
Solvent type
a
d
b
d
c
d
0
100
200
300
Linum Usitatissimum Salvia leriifolia DPPH IC50,
μg/mL
Herbal source
0 3 6
Intensity of electric pulse field, kV·cm1 m
a
c
b
c
0
100
200
300
Linum Usitatissimum Salvia leriifolia
DPPH IC50,
μgr/mL
Herbal source
aqueous alcoholic
Solvent type
a
b
c
b
cd
d
0
100
200
300
0 3 6
DPPH IC50,
μg/mL
Intensity of electric pulse field, kV·cm1
aqueous alcoholic
Solvent type
(а)
(c)
(b)
b
0
10
20
30
Linum Usitatissimum Salvia leriifolia
Total phenolic compounds,
mg Galic acid/Herbal source
c
0
10
20
30
0 Total phenolic compounds,
mg Galic acid/Intensity of electric b
a
0
10
20
30
40
50
aqueous alcoholic
Total phenolic compounds,
mg Galic acid/g
Solvent type
b
b
0
15
30
45
Linum Usitatissimum Total phenolic compounds,
mg Galic acid/g
aqueous Solvent b
a
b
a
b
a
0
10
20
30
40
50
Linum Usitatissimum Salvia leriifolia
Total phenolic compounds,
mg Galic acid/g
Herbal source
0 3 6
Intensity of electric pulse field, kV·cm1
0
15
30
45
Total phenolic compounds,
mg Galic acid/g
a
b
0
100
200
300
Linum Usitatissimum Salvia leriifolia
DPPH IC50,
μg/mL
Herbal source
a
b
0
100
200
300
0 3 DPPH IC50,
μg/mL
Intensity of electric pulse a
b
0
100
200
300
aqueous alcoholic
DPPH IC50,
μg/mL
Solvent type
a
b
c
0
100
200
300
Linum Usitatissimum DPPH IC50,
μg/mL
0 Intensity of electric a
c
b
c
0
100
200
300
Linum Usitatissimum Salvia leriifolia
DPPH IC50,
μgr/mL
Herbal source
aqueous alcoholic
Solvent type
0
100
200
300
DPPH IC50,
μg/mL
b
a
0
10
20
30
40
50
Linum Usitatissimum Salvia leriifolia
Total phenolic compounds,
mg Galic acid/g
Herbal source
c
b
a
0
10
20
30
40
50
0 3 6
Total phenolic compounds,
mg Galic acid/g
Intensity of electric pulse field, kV·cm1
b
a
0
10
20
30
40
50
aqueous alcoholic
Total phenolic compounds,
mg Galic acid/g
Solvent type
b
a
b
a
0
15
30
45
Linum Usitatissimum Salvia leriifolia
Total phenolic compounds,
mg Galic acid/g
Herbal source
aqueous alcoholic
Solvent type
b
a
b
a
b
a
0
10
20
30
40
50
Linum Usitatissimum Salvia leriifolia
Total phenolic compounds,
mg Galic acid/g
Herbal source
0 3 6
Intensity of electric pulse field, kV·cm1
d
cd
c c
b
a
0
15
30
45
0 3 6
Total phenolic compounds,
mg Galic acid/g
Intensity of electric pulse field, kV·cm1
aqueous alcoholic
Solvent type
a
b
0
100
200
300
Linum Usitatissimum Salvia leriifolia
DPPH IC50,
μg/mL
Herbal source
a
b
c
0
100
200
300
0 3 6
DPPH IC50,
μg/mL
Intensity of electric pulse field, kV·cm1
a
b
0
100
200
300
aqueous alcoholic
DPPH IC50,
μg/mL
Solvent type
a
d
b
d
c
d
0
100
200
300
Linum Usitatissimum Salvia leriifolia
DPPH IC50,
μg/mL
Herbal source
0 3 6
Intensity of electric pulse field, kV·cm1 m
a
c
b
c
0
100
200
300
Linum Usitatissimum Salvia leriifolia
DPPH IC50,
μgr/mL
Herbal source
aqueous alcoholic
Solvent type
a
b
c
b
cd
d
0
100
200
300
0 3 6
DPPH IC50,
μg/mL
Intensity of electric pulse field, kV·cm1
aqueous alcoholic
Solvent type
Table 2 Interaction between herbal source, pre-treatment,
and solvent type in their effects on total phenolic compounds
Total phenolic
compounds (mg
of galic acid/g)
Intensity of pulsed
electric field pretreatment,
kV·cm–1
Type of
solvent
Herbal
source
Linum aqueous 0 4.28 ± 0.21c
usitatissimum
alcoholic 8.11 ± 0.54c
aqueous 3 5.33 ± 0.37c
alcoholic 8.49 ± 0.181c
aqueous 6 5.73 ± 0.33c
alcoholic 10.37 ± 0.81c
Salvia aqueous 0 33.24 ± 0.37b
leriifolia alcoholic 51.75 ± 1.63ab
aqueous 3 38.63 ± 0.76b
alcoholic 54.81 ± 1.30ab
aqueous 6 44.19 ± 0.63b
alcoholic 63.98 ± 1.11a
P < 0.05
Linum usitatissimum Salvia leriifolia
Linum usitatissimum Salvia leriifolia
Linum usitatissimum Salvia leriifolia
190
Arab Shirazi S.H. et al. Foods and Raw Materials, 2020, vol. 8, no. 1, pp. 186–195
Figure 4 Binary effects of herbal source and pre-treatment (a),
herbal source and solvent type (b), and pre-treatment
and solvent type (c) on antioxidant activity by DPPH method
(P ˂ 0.05)
the solvent type and the use of a pulsed electric field
pretreatment.
Total phenolic compounds in Linum usitalissmum
oilseed have been reported by Oomah et al., Brodowska
et al., and Russo and Reggiani at 8–10 mg of caffeic
acid per gram of extract), 0.988 mg of catechin per gram
of extract, and 4.64–9.40 mg caffeic acid per gram of
extract, respectively [26–28]. In our study, their amount
ranged from 4.28 to 10.37 mg gallic acid per gram of
extract, depending on the solvent type and the use of a
pulsed electric field pretreatment.
The studies showed that the amount of extracted
phenolic compounds increased with a higher intensity
of a pulsed electric field, reaching their maximum at a
6 kV·cm–1 pre-treatment. Schroeder et al. attributed this
b
0
100
Linum Usitatissimum Salvia leriifolia
DPPH μg/Herbal source
c
0 3 6
DPPH IC50,
μg/mL
Intensity of electric pulse field, kV·cm1
a
0
100
200
300
aqueous alcoholic
DPPH IC50,
μg/mL
Solvent type
a
d
b
d
c
d
0
100
200
300
Linum Usitatissimum Salvia leriifolia
DPPH IC50,
μg/mL
Herbal source
0 3 6
Intensity of electric pulse field, kV·cm1 m
a
c
b
c
0
100
200
300
Linum Usitatissimum Salvia leriifolia
DPPH IC50,
μgr/mL
Herbal source
aqueous alcoholic
Solvent type
a
b
c
b
cd
d
0
100
200
300
0 3 6
DPPH IC50,
μg/mL
Intensity of electric pulse field, kV·cm1
aqueous alcoholic
Solvent type
b
0
100
Linum Usitatissimum Salvia leriifolia
DPPH μg/Herbal source
0
100
0 3 DPPH IC50,
μg/mL
Intensity of electric pulse a
b
0
100
200
300
aqueous alcoholic
DPPH IC50,
μg/mL
Solvent type
a
b
c
0
100
200
300
Linum Usitatissimum DPPH IC50,
μg/mL
0 Intensity of electric a
c
b
c
0
100
200
300
Linum Usitatissimum Salvia leriifolia
DPPH IC50,
μgr/mL
Herbal source
aqueous alcoholic
Solvent type
0
100
200
300
DPPH IC50,
μg/mL
(а)
(c)
(b)
b
b
b
0
10
20
30
Linum Usitatissimum Salvia leriifolia
Total phenolic mg Galic Herbal source
0 3 6
d
cd
c c
b
0
15
30
0 3 6
Total phenolic compounds,
mg Galic acid/Intensity of electric pulse field, kV·cm1
aqueous alcoholic
a
b
0
100
200
300
Linum Usitatissimum Salvia leriifolia
DPPH IC50,
μg/mL
Herbal source
a
b
c
0
100
200
300
0 3 6
DPPH IC50,
μg/mL
Intensity of electric pulse field, kV·cm1
a
b
0
100
200
300
aqueous alcoholic
DPPH IC50,
μg/mL
Solvent type
a
d
b
d
c
d
0
100
200
300
Linum Usitatissimum Salvia leriifolia
DPPH IC50,
μg/mL
Herbal source
0 3 6
Intensity of electric pulse field, kV·cm1 m
a
c
b
c
0
100
200
300
Linum Usitatissimum Salvia leriifolia
DPPH IC50,
μgr/mL
Herbal source
aqueous alcoholic
Solvent type
a
b
c
b
cd
d
0
100
200
300
0 3 6
DPPH IC50,
μg/mL
Intensity of electric pulse field, kV·cm1
aqueous alcoholic
Solvent type
b
b
b
0
10
20
30
Linum Usitatissimum Salvia leriifolia
Total phenolic mg Galic Herbal source
0 3 6
d
cd
c c
b
0
15
30
0 3 6
Total phenolic compounds,
mg Galic acid/Intensity of electric pulse field, kV·cm1
aqueous alcoholic
a
b
0
100
200
300
Linum Usitatissimum Salvia leriifolia
DPPH IC50,
μg/mL
Herbal source
a
b
c
0
100
200
300
0 3 6
DPPH IC50,
μg/mL
Intensity of electric pulse field, kV·cm1
a
b
0
100
200
300
aqueous alcoholic
DPPH IC50,
μg/mL
Solvent type
a
d
b
d
c
d
0
100
200
300
Linum Usitatissimum Salvia leriifolia
DPPH IC50,
μg/mL
Herbal source
0 3 6
Intensity of electric pulse field, kV·cm1 m
a
c
b
c
0
100
200
300
Linum Usitatissimum Salvia leriifolia
DPPH IC50,
μgr/mL
Herbal source
aqueous alcoholic
Solvent type
a
b
c
b
cd
d
0
100
200
300
0 3 6
DPPH IC50,
μg/mL
Intensity of electric pulse field, kV·cm1
aqueous alcoholic
Solvent type
to the electrical degradation of cells and their increased
permeability due to the use of a pulsed electric field [29].
In this regard, Bozinou et al. investigated the extraction
of phenolic compounds and antioxidant activity of
dried oak leaves with a 7 kV·cm–1 pulse electric field
pretreatment [30]. They stated that the highest amount of
phenolic compounds was obtained with a pulse time of
20 ms, a pulsing duration of 40 min, and a pulse interval
of 100 ms.
Liu et al. examined the enhancement of extracted
phenolic compounds in onion pre-treated with a
pulsed electric field [31]. They stated that the optimum
conditions for this purpose were a pulsed electric field
of 2.5 kV, 90 pulses, and a temperature of 45°C. In
these conditions, the amounts of extracted phenolic
and flavonoid compounds were 86.82 mg of gallic
acid per 100 g and 37.58 mg of quencherine per 100 g,
respectively. These values were 2.2 times and 2.7 times
as high as those in the control samples, respectively.
Figure 3 Independent effects of herbal source (a), pretreatment
(b), and solvent type (c) on antioxidant activity by
DPPH method (P ˂ 0.05)
b
b
b
0
10
Linum Usitatissimum Salvia leriifolia
Total phenolic mg Herbal source
0 3 6
d
0
15
0 3 6
Total phenolic mg Intensity of electric pulse field, kV·cm1
aqueous alcoholic
a
b
0
100
200
300
Linum Usitatissimum Salvia leriifolia
DPPH IC50,
μg/mL
Herbal source
a
b
0
100
200
300
0 3 6
DPPH IC50,
μg/mL
Intensity of electric pulse field, kV·cm1
a
b
0
100
200
300
aqueous alcoholic
DPPH IC50,
μg/mL
Solvent type
a
d
b
d
c
d
0
100
200
300
Linum Usitatissimum Salvia leriifolia
DPPH IC50,
μg/mL
Herbal source
0 3 6
Intensity of electric pulse field, kV·cm1 m
a
c
b
c
0
100
200
300
Linum Usitatissimum Salvia leriifolia
DPPH IC50,
μgr/mL
Herbal source
aqueous alcoholic
Solvent type
a
b
c
b
cd
d
0
100
200
300
0 3 6
DPPH IC50,
μg/mL Intensity of electric pulse field, kV·
cm
1
aqueous alcoholic
Solvent type
(а)
(c)
(b)
alcoholic
b
b
0
15
Linum Usitatissimum Salvia leriifolia
Total phenolic mg Galic Herbal source
aqueous alcoholic
a
a
a
Salvia leriifolia
Herbal source
6
pulse field, kV·cm1
d
cd
c c
b
a
0
15
30
45
0 3 6
Total phenolic compounds,
mg Galic acid/g
Intensity of electric pulse field, kV·cm1
aqueous alcoholic
Solvent type
b
Salvia leriifolia
source
a
b
c
0
100
200
300
0 3 6
DPPH IC50,
μg/mL
Intensity of electric pulse field, kV·cm1
b
alcoholic
type
a
d
b
d
c
d
0
100
200
300
Linum Usitatissimum Salvia leriifolia
DPPH IC50,
μg/mL
Herbal source
0 3 6
Intensity of electric pulse field, kV·cm1 m
c
c
Salvia leriifolia
Herbal source
alcoholic
Solvent type
a
b
c
b
cd
d
0
100
200
300
0 3 6
DPPH IC50,
μg/mL
Intensity of electric pulse field, kV·cm1
aqueous alcoholic
Solvent type
0
10
aqueous alcoholic
Total phenolic mg Solvent type
b
b
0
15
Linum Usitatissimum Salvia leriifolia
Total phenolic mg Herbal source
aqueous alcoholic
b
a
b
a
b
a
0
10
20
30
40
50
Linum Usitatissimum Salvia leriifolia
Total phenolic compounds,
mg Galic acid/g
Herbal source
0 3 6
Intensity of electric pulse field, kV·cm1
d
cd
c c
b
0
15
30
45
0 3 6
Total phenolic compounds,
mg Galic acid/g
Intensity of electric pulse field, kV·cm1
aqueous alcoholic
Solvent type
a
b
0
100
200
300
Linum Usitatissimum Salvia leriifolia
DPPH IC50,
μg/mL
Herbal source
a
b
c
0
100
200
300
0 3 6
DPPH IC50,
μg/mL
Intensity of electric pulse field, kV·cm1
a
b
0
100
200
300
aqueous alcoholic
DPPH IC50,
μg/mL
Solvent type
a
d
b
d
c
d
0
100
200
300
Linum Usitatissimum Salvia leriifolia
DPPH IC50,
μg/mL
Herbal source
0 3 6
Intensity of electric pulse field, kV·cm1 m
a
c
b
c
0
100
200
300
Linum Usitatissimum Salvia leriifolia
DPPH IC50,
μgr/mL
Herbal source
aqueous alcoholic
Solvent type
a
b
c
b
cd
d
0
100
200
300
0 3 6
DPPH IC50,
μg/mL
Intensity of electric pulse field, kV·cm1
aqueous alcoholic
Solvent type
Linum usitatissimum Salvia leriifolia
Linum usitatissimum Salvia leriifolia
Linum usitatissimum Salvia leriifolia
IC50,
IC50IC , 50,
IC50,
IC50IC , 50,
191
Arab Shirazi S.H. et al. Foods and Raw Materials, 2020, vol. 8, no. 1, pp. 186–195
Antioxidant activity by DPPH method. Fig. 3
presents independent effects of the agents, Fig. 4
shows their binary effects, and Table 3 indicates their
interaction in relation to the antioxidant activity of the
extracts derived with the DPPH method. As we can see,
their antioxidant activity significantly increased, at a
95% confidence level, with a higher intensity of a pulsed
electric field and the use of an alcoholic solvent. At the
same time, we found that the antioxidant activity of
Salvia leriifolia extracts was higher than that of Linum
usitalissmum (P < 0.05).
The content of phenolic compounds is not an
accurate measure of antioxidant activity. Since the
Folin-Ciocalteu reagent nonspecifically reacts with
phenolic and other compounds, such as organic acids,
sugars are also able to reduce this reagent. Therefore, it
is also necessary to measure antioxidant activity in other
ways, for example, by the DPPH method, which we used
in our study [32].
Phenolic compounds donated hydrogen or electron to
the groups exposed to oxidation [33]. Thus, the content
of phenolic compounds can be used as an important
indicator of antioxidant activity. As noted above, various
factors, such as plant variety, harvest area, and harvest
time, appear to affect the amount of phenolic compounds
and, subsequently, antioxidant activity.
In our study, the antioxidant activity of extracts
(IC50) from Salvia leriifolia plant extracted by the
DPPH method ranged between 25.28 and 41.38 μg/mL,
depending on the type of solvent and the intensity of a
pulsed electric field.
We also investigated various sources of antioxidant
activity (IC50) in Linum usitalissmum. This value was
reported by Brodowska et al. and Alachaher et al. to
reach 299.00 and 220.05 μg/mL of extract, respectively
[27, 34]. In our study, the amount of total phenolic
compounds extracted from Linum usitalissmum varied
from 157.37 to 312.51 μg/mL, depending on the solvent
type and the use of a pulsed electric field pretreatment.
On the other hand, we found that the extracts’
antioxidant activity increased with a higher pulsed
electric field intensity. The highest values were observed
in the samples with a 6 kV·cm–1 pretreatment. This was
quite predictable from the measurement of total phenolic
compounds, whose content also increased with a higher
intensity of the applied electric field. In this regard,
Bozinou et al. investigated the extraction of phenolic
compounds and antioxidant activity of dried oak leaves
with a 7 kV·cm–1 pulse electric field pretreatment
[30]. They stated that the antioxidant activity was
proportional to the content of total phenolic compounds:
the higher the amount of phenolic compounds, the
higher the antioxidant activity. In their study, phenolic
compounds were highest with a pulse time of 20 ms,
Table 3 Interaction between herbal source, pre-treatment,
and solvent type in their effects on antioxidant activity by
DPPH method
DPPH IC50,
μg/mL
Intensity of pulsed
electric field pretreatment,
kV·cm–1
Solvent
type
Herbal
source
Linum aqueous 0 312.51 ± 12.10a
usitatissimum
alcoholic 220.22 ± 8.06b
aqueous 3 304.31 ± 10.11a
alcoholic 207.63 ± 12.10b
aqueous 6 267.81 ± 5.81ab
alcoholic 157.32 ± 4.16bc
Salvia aqueous 0 41.38 ± 3.17c
leriifolia alcoholic 33.54 ± 2.87c
aqueous 3 40.14 ± 1.36c
alcoholic 31.67 ± 2.23c
aqueous 6 36.91 ± 5.17c
alcoholic 25.28 ± 1.10c
P < 0.05
Figure 5 Independent effects of herbal source (a), pretreatment
(b), and solvent type (c) on antioxidant activity by
TEAC method (P < 0.05)
(а)
(c)
(b)
Linum usitatissimum Salvia leriifolia
192
Arab Shirazi S.H. et al. Foods and Raw Materials, 2020, vol. 8, no. 1, pp. 186–195
a pulsing duration of 40 min, and a pulse interval of
100 ms. Under these conditions, the sample’s antioxidant
activity was maximum.
Liu et al. studied the enhancement of extracted
phenolic compounds in onion subjected to a pulsed
electric field [31]. They stated that the optimum
conditions for this purpose were a pulsed electric field
of 2.5 kV, 90 pulses, and a temperature of 45°C. The
researchers also found that the extract’s antioxidant
activity increased with a higher pulse electric field
intensity and a larger number of pulses applied.
Their finding also proved the correlation between
the antioxidant activity and the amount of phenolic
compounds.
Lopez Giral et al. also investigated a pulsed electric
field pretreatment to improve the extraction of phenolic
compounds from three different grape varieties
(Graciano, Tempranillo, and Grenache) during two
production periods [35]. The pretreatment conditions
included a pulsed electric field of 7.4 kV·cm–1, a pulse
width of 20 ms, and a frequency of 400 Hz. They stated
that using a pulsed electric field increased the color
intensity, total phenol index, anthocyanin index, and
total antioxidant power. These researchers therefore
introduced a pulsed electric field pretreatment as a
suitable technology for extracting phenolic compounds.
However, they acknowledged that the ability of the
method depended on the type of grape and the initial
amount of phenolic compounds.
Similarly, Minussi et al. demonstrated a positive
relationship between antioxidant power and the content
of total polyphenolic compounds in grape juice,
particularly compounds such as gallic acid, catechin,
and epi-catechin [36].
Antioxidant activity by TEAC method.
Independent, binary, and combined effects of the agents
on the antioxidant activity of the extracts extracted
with the Trolox method are presented in Fig. 5, Fig. 6,
and Table 4. As observed, a higher intensity of a pulsed
electric field pretreatment and the use of an alcoholic
solvent significantly increased the TEAC number of the
extracts at a 95% confidence level. On the other hand,
the number of TEAC extracts of Salvia leriifolia was
higher than that of Linum usitalissmum (P < 0.05).
As noted earlier, the amount of phenolic compounds
alone is not a precise measure for antioxidant activity.
Since the Folin-Ciocalteu reagent nonspecifically reacts
with phenolic and other compounds such as organic
acids, sugars also can reduce this reagent. Therefore, it is
also necessary to measure antioxidant activity with other
methods [32]. Therefore, we used the Trolox Equivalent
Antioxidant Capacity (TEAC) method to measure
antioxidant activity.
There was a significant correlation between the
values of antioxidant activity measured by the DPPH
Figure 6 Binary effects of herbal source and pre-treatment
(a), herbal source and solvent type (b), and pre-treatment and
solvent type (c) on antioxidant activity by TEAC method
(P ˂ 0.05)
(а)
(c)
(b)
Table 4 Interaction between herbal source, pre-treatment,
and solvent type in their effects on antioxidant activity by
TEAC method
TEAC (micromole
Trolox per g
dry herb weight)
Intensity of pulsed
electric field pretreatment,
kV·cm–1
Solvent
type
Herbal
source
Linum aqueous 0 0.89 ± 0.18c
usitatissimum
alcoholic 12.44 ± 0.21c
aqueous 3 1.32 ± 0.01c
alcoholic 13.96 ± 1.45c
aqueous 6 6.92 ± 0.07c
alcoholic 21.48 ± 1.23c
Salvia aqueous 0 108.54 ± 2.82bc
leriifolia alcoholic 188.59 ± 2.37ab
aqueous 3 132.43 ± 1.06b
alcoholic 196.93 ± 2.24ab
aqueous 6 156.76 ± 1.14b
alcoholic 235.87 ± 2.87a
P < 0.05
Linum usitatissimum Salvia leriifolia
Linum usitatissimum Salvia leriifolia
193
Arab Shirazi S.H. et al. Foods and Raw Materials, 2020, vol. 8, no. 1, pp. 186–195
of the applied electric field. In this regard, Bozinou et al.
investigated the extraction of phenolic compounds and
antioxidant activity of dried oak leaves by using a pulsed
electric field pretreatment at 7 kV·cm–1 [30]. They stated
that the level of antioxidant activity was proportional
to the amount of total phenolic compounds, so a higher
content of phenolic compounds increased the antioxidant
activity. In their study, the highest amount of phenolic
compounds was associated with a pulse time of 20 ms,
pulse duration of 40 min, and pulse interval of 100 ms.
Under these conditions, the level of antioxidant activity
was also maximum.
CONCLUSION
Our study aimed to compare the antioxidant activity
of aqueous and alcoholic extracts derived from Salvia
leriifolia L. and Linum usitalissmum L. subjected to a
pulsed electric field at the intensities of zero (without
pre-treatment), 3 and 6 kV·cm–1 with a constant pulse of
30. We investigated such parameters as total phenolic
compounds and antioxidant activity. According to our
results, the Salvia leriifolia extract had more phenolic
compounds and higher antioxidant activity than the
Linum usitalissmum extract under the same conditions.
On the other hand, a pulsed electric field
pretreatment and the use of an alcoholic solvent
(methanol) for extraction increased the content of
phenolic compounds and the extract’s antioxidant
activity. In fact, the solubility of phenolic compounds
depended on the type of solvent and their interaction.
Finally, the extract derived from Salvia leriifolia with an
alcoholic solvent and a pulsed electric field pretreatment
(at 6 kV·cm–1 with 30 pulses) was selected as possessing
desirable antioxidant properties.
CONTRIBUTION
The authors equally participated in the research and
preparation of manuscript.
CONFLICT OF INTEREST
The authors declare that there is no conflict of
interests regarding the publication of this article.
method and the TEAC method (P < 0.01 and r = 0.932).
The Trolox equivalent antioxidant capacity test and
diphenyl picryl hydrazyl are both synthetic free radicals
with similar application. However, the Trolox equivalent
antioxidant potential can be used to measure antioxidant
activity of polar and nonpolar compounds [37].
The ABTS•+ cation radical is more active than
the DPPH radical and is therefore widely used in
the measurement of antioxidant activity. In this test,
ABTS oxidation first occurred following the reaction
with potassium persulfate. The ABTS•+ cation
radical subsequently reacted with antioxidants or
other hydrogen donating radicals and transformed in
a reduced form [37]. Consequently, the antioxidant
inhibition percentage can be measured by determining
the absorption reduction rate. The radical inhibition
activity in this test was reported based on the Trolox
equivalent antioxidant capacity.
As noted above, various factors (plant variety,
harvest area and time) appear to affect the amount of
phenolic compounds and, subsequently, antioxidant
activity.
We found that the antioxidant activity of Salvia
leriifolia extracts measured with the TEAC method
ranged between 108.54 and 235.87 μmol of Trolox per g
dry plant weight, depending on the type of solvent and
the intensity of pulsed electric field pretreatment.
Some studies evaluated the antioxidant activity of
Linum usitalissmum with the TEAC method. Russo
and Ragiani and Deng et al. reported the value of 560–
860 (for oilseed Linum usitalissmum) and 22 000 μmol
Trolox/g dry weight, respectively [28, 28]. In our study,
the amount of total phenolic compounds extracted from
Linum usitalissmum ranged from 0.89 to 21.48 μmol
Trolox/g dry weight, depending on the solvent type and
the intensity of pulsed electric field pretreatment.
On the other hand, the antioxidant activity of the
extracts increased with a higher pulsed electric field
intensity. A pre-treatment of 6 kV·cm–1 provided
the highest amount of compounds. This result was
predictable from the measurement of total phenolic
compounds, which also increased with a higher intensity

1. Angelo AJ, Vercelotti J, Jacks T, Legendre M. Lipid oxidation in foods. Critical Reviews in Food Science and Nutrition. 1996;36(3):175-224. DOI: https://doi.org/10.1080/10408399609527723.

2. Valko M, Rhodes CJ, Moncol J, Izakovic M, Mazur M. Free radicals, metals and antioxidants in oxidative stressinduced cancer. Chemico-Biological Interactions. 2006;160(1):1-40. DOI: https://doi.org/10.1016/j.cbi.2005.12.009.

3. Gao J-J, Igalashi K, Nukina M. Radical scavenging activity of phenylpropanoid glycosides in Caryopteris incana. Bioscience Biotechnology and Biochemistry. 1999;63(6):983-988. DOI: https://doi.org/10.1271/bbb.63.983.

4. Asavasanti S, Ristenpart W, Stroeve P, Barrett DM. Permeabilization of plant tissue by monopolar pulsed electric field: effect of frequency. Journal of Food Science. 2011;76(1):E98-E111. DOI: https://doi.org/10.1111/j.1750-3841.2010.01940.x.

5. Sale AJH, Hamilton WA. Effects of high electric fields on microorganisms: I. killing of bacteria and yeast. BBA - General Subjects. 1967;148(3):781-788. DOI: https://doi.org/10.1016/0304-4165(67)90052-9.

6. Hui SW. Effect of pulse length and strength on electroporation efficiency. Method in molecular biology. 1995. 48:29-40. DOI: https://doi.org/10.1385/0-89603-328-7:29.

7. Abdel-Samie MAS, Wan JJ, Huang WN, Chung OK, Xu BC. Effects of cumin and ginger as antioxidants on dough mixing properties and cookie quality. Cereal Chemistry. 2010;87(5):454-460. DOI: https://doi.org/10.1094/CCHEM-01-10-0012.

8. Haralick RM, Dinstein I, Shanmugam K. Textural features for image classification. IEEE Transactions on Systems, Man and Cybernetics. 1973;3(6):610-621. DOI: https://doi.org/10.1109/TSMC.1973.4309314.

9. Sadeghnia HR, Nassiri Asl M, Haddad Khodaparast MH, Hosseinzadeh H. The effect of Salvia leriifolia Benth root extracts on lipid peroxidation during global ischemic-reperfusion in rats. Journal of Medicinal Plants. 2003;3(7):19-28.

10. Hosseinzadeh H, Yavary M. Anti-inflammatory effect of Salvia leriifolia Benth. leaf extract in mice and rat. Pharmaceutical and Pharmacological Letters. 1999;9(2):60-61.

11. Hosseinzadeh H, Lary P. Effect of Salvia leriifolia leaf extracts on morphine dependence in mice. Phytotherapy Research. 2000;14(5):384-387. DOI: https://doi.org/10.1002/1099-1573(200008)14:5<384::AID- TR641>3.0.CO;2-F.

12. Hadad Khodaparast MH, Haghdoost A, Elhami-Rad AH, Movahhed G, Karazhiyan H. Antioxidant activity and thermal Properties of Salvia leriifolia (Norozak) root extract. Proceedings of the international conference on Innovations in Food and Bioprocess Technologies; 2006; Pathumthani. Pathumthani: AIT; 2006. p. 378.

13. Martinchik AN, Baturin AK, Zubtsov VV, Molofeev VY. Nutritional value and functional properties of flaxseed. Problems of Nutrition. 2012l;81(3):4-10. (In Russ.).

14. Kabiri S, Sayyed-Alangi SZ. Comparison of antioxidant effect of different extracts from Melissa officinalis leaves with immersion and microwave-assisted extractions and its oxidative stability on soybean oil. Journal of Innovative Food Technologies. 2015;2(4):23-38. DOI: https://doi.org/10.22104/jift.2015.201.

15. Farhoosh R, Purazrang H, Khodaparast MHH, Rahimizadeh M, Seyedi SM. Extraction and separation of antioxidative compounds from Salvia leriifolia leaves. Journal of Agricultural Science and Technology. 2004;6:57-62.

16. Ordoeez AAL, Gomez JD, Vattuone MA, Isla MI. Antioxidant activities of Sechium edule (Jacq) Swartz extracts. Food Chemistry. 2006;97(3):452-458. DOI: https://doi.org/10.1016/j.foodchem.2005.05.024.

17. Shahidi F, Naczk M. Phenolic in food and nutraceuticals. CRC press; 2004. 558 p.

18. Pag AI, Radu DG, Draganescu D, Popa MI, Sirghie C. Flaxseed cake - a sustainable source of antioxidant and antibacterial extracts. Cellulose Chemistry and Technology. 2014;48(3-4):265-273.

19. Brand-Williams W, Cuvelier ME, Berset C. Use of a free radical method to evaluate Antioxidant activity. Food Science and Technology. 1995;28(1):25-30.

20. You LJ, Zhao M, Regenstein JM, Ren JY. Changes in the antioxidant activity of loach (Misgurnus anguillicaudatus) protein hydrolysates during a simulated gastrointestinal digestion. Food Chemistry. 2010;120(3):810-816. DOI: https://doi.org/10.1016/j.foodchem.2009.11.018.

21. Hamrouni-Sellami I, Rahali FZ, Rebey IB, Bourgou S, Limam F, Marzouk B. Total phenolics, flavonoids, and antioxidant activity of sage (Salvia officinalis L.) plants as affected by different drying methods. Food and Bioprocess Technology. 2013;6(3):806-817. DOI: https://doi.org/10.1007/s11947-012-0877-7.

22. Ahmadi F, Sabzalian MR, Mirlohi A. Impacts of planting dates on essential oil, phenolic compounds and some morphological traits in Nuruozak. 3rd national congress on medicinal plants; 2014; Mashhad. Mashhad: National network of research and technology of medicinal plants; 2014. p. 478.

23. Najafi S, Mir N, Shafeghat M. Antioxidant and antibacterial activities of six medicinally important species of the genus Salvia from north east of Iran. Journal of Genetic Resources. 2016;2(1):41-47. DOI: https://doi.org/10.22080/JGR.2016.1479.

24. Abadi ZHM, Mahdavi B, Rezaei-Seresht E. Contents of aerial parts of Salvia leriifolia benth. Journal of Chemical Health Risks. 2016;6(3):185-194. DOI: https://doi.org/10.22034/JCHR.2016.544146.

25. Bahadori MB, Asghari B, Dinparast L, Zengin G, Sarikurkcu C, Abbas-Mohammadi M, et al. Salvia nemorosa L.: A novel source of bioactive agents with functional connections. LWT - Food Science and Technology. 2017;75: 42-50. DOI: https://doi.org/10.1016/j.lwt.2016.08.048.

26. Oomah BD, Kenaschuk EO, Mazza G. Phenolic acids in flaxseed. Journal of Agricultural and Food Chemistry. 1995;43(8):2016-2019. DOI: https://doi.org/10.1021/jf00056a011.

27. Brodowska K, Catthoor R, Brodowska AJ, Symonowicz M, Łodyga-Chruścińska E. A comparison of antioxidant properties of extracts from defatted and non-defatted flax (Linum usitatissimum) seeds. Albanian Journal of Agricultural Science. 2014;13(2):16-23.

28. Russo R, Reggiani R. Phenolics and antioxidant activity in flax varieties with different productive attitude. International Food Research Journal. 2015;22(4):1736-1739.

29. Schroeder S, Buckow R, Knoerzer K. Numerical simulation of pulsed electric field (pef) processing for chamber design and optimization. 7th international conference on CFD in the minerals and process industries; 2009; Melbourne. Melbourne: CSIRO; 2009.

30. Bozinou E, Karageorgou I, Batra G, Dourtoglou VG, Lalas SI. Pulsed electric field extraction and antioxidant activity determination of Moringa oleifera dry leaves: A comparative study with other extraction techniques. Beverages. 2019;5(1). DOI: https://doi.org/10.3390/beverages5010008.

31. Liu ZW, Zeng XA, Ngadi M. Enhanced extraction of phenolic compounds from onion by pulsed electric field (PEF). Journal of Food Processing and Preservation. 2018;42(9). DOI: https://doi.org/10.1111/jfpp.13755.

32. Mohsen SM, Ammar ASM. Total phenolic contents and antioxidant activity of corn tassel extracts. Food Chemistry. 2009;112(3):595-598. DOI: https://doi.org/10.1016/j.foodchem.2008.06.014.

33. Cuvelier M-E, Richard H, Berset C. Comparison of the antioxidative activity of some acid-phenols: structure-activity relationship. Bioscience, Biotechnology and Biochemistry. 1992;56(2):324-325. DOI: https://doi.org/10.1271/bbb.56.324.

34. Alachaher FZ, Dali S, Dida N, Krouf D. Comparison of phytochemical and antioxidant properties of extracts from flaxseed (Linum usitatissimum) using different solvents. International Food Research Journal. 2018;25(1):75-82.

35. López-Giral N, González-Arenzana L, González-Ferrero C, López R, Santamaría P, López-Alfaro I, et al. Pulsed electric field treatment to improve the phenolic compound extraction from Graciano, Tempranillo and Grenache grape varieties during two vintages. Innovative Food Science and Emerging Technologies. 2015;28:31-39. DOI: https://doi.org/10.1016/j.ifset.2015.01.003.

36. Minussi RC, Rossi M, Bologna L, Cordi L, Rotilio D, Pastore GM, et al. Phenolic compounds and total antioxidant potential of commercial wines. Food Chemistry. 2003;82(3):409-416. DOI: https://doi.org/10.1016/s0308-8146(02)00590-3.

37. Arnao MB. Some methodological problems in the determination of antioxidant activity using chromogen radicals: a practical case. Trends Food Science and Technology. 2000;11(1):419-421. DOI: https://doi.org/10.1016/S0924-2244(01)00027-9.

38. Deng QC, Yu X, Ma FL, Xu JQ, Huang FH, Huang QD, et al. Comparative analysis of the in-vitro antioxidant activity and bioactive compounds of flaxseed in China according to variety and geographical origin. International Journal of Food Properties. 2018;20:S2708-S2722. DOI: https://doi.org/10.1080/10942912.2017.1402029.