Journal of Applied Research on Medicinal and Aromatic Plants 32 (2023) 100449

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Journal of Applied Research on Medicinal and

Aromatic Plants
journal homepage: www.elsevier.com/locate/jarmap

Optimization of ultrasonic-assisted extraction of phenolic compounds and

antioxidant activities From Pistacia atlantica Desf. galls using response
surface methodology
Fatiha Hefied *, 1, Ziyad B Ahmed, Mohamed Yousfi
Laboratoire des Sciences Fondamentales, Université Amar Telidj, BP 37G, Route de Ghardaia, 03000 Laghouat, Algeria

A R T I C L E I N F O A B S T R A C T

Keywords: In the present study, the response surface methodology (RSM) using Box-Behnken design (BBD) was employed to
Pistacia atlantica Desf. galls optimize conditions for the extraction of phenolic compounds from Pistacia atlantica Desf. leaf galls. Three
Box–Behnken design variables were evaluated at three levels (15 experimental runs): the extraction time (40 min, 45 min, and 50
Response surface methodology
min), the solid-to-liquid ratio (1:10 g/ml, 1: 20 g/ml, and 1:30 g/ml) and temperature (40 ◦ C, 50 ◦ C, and 60 ◦ C).
Phenolic compounds
Antioxidant activity
According to the three-dimensional response surface, the optimum extraction phenolic compounds were found at
40 min extraction time, 1:20 g/ml solid-liquid ratio, and 60 ◦ C extraction temperature. In these conditions, the
content of TPC, TFC, CTC, and antioxidant activity (DPPH and iron chelation) were 139.88 mg GAE/g DW, 2.43
mg QE/g DW, 121.32 mg CE/g DW, 65.94 mg AAE/g DW, and 501.38 mg TE/g DW, respectively. The results
indicate an agreement between the prediction values and experimental ones, which points the success of the
optimization. Moreover, they indicate that P. atlantica galls produce a high amount of antioxidants and anti­
radicals under optimized conditions determined by RSM. Actually, the used method is a simple, reliable one that
serves as an alternative to various other extraction techniques.

1. Introduction Bedouin populations dominating the semi-arid steppe that stretches

The Pistacia belongs to the Anacardiaceae (Therebinthaceae) family
(Chirinos et al., 2007). This genus is widespread in the Northern
Hemisphere with about seven species in the Mediterranean region
(Mediterranean Eurasia and the adjacent Africa), two or three species in
eastern Asia, and one or two species in the southwestern USA and
Mexico (Xie et al., 2014). Several studies indicated that Pistacia species
play a vital role in the nutrition and agricultural economy of many
communities living in arid and semi-arid regions (Rauf et al., 2017).
Pistacia atlantica Desf. (wild pistachio) occurs from the Mediter­
ranean basin to central Asia, especially in Iran, Turkey, Iraq, and Saudi
Arabia (Ben Ahmed et al., 2022). It is one of the few tree species which
still natively occur in semi-arid and arid areas of North Africa, and even
in the Sahara (Monjauze, 1980). The vernacular name of P. atlantica is
"Betoum" (Wu et al., 2015) or "Tismelal" in the Berber language (Nassiri
et al., 2011). It is an endemic species among the non-cultivated plants
protected in Algeria (Chirinos et al., 2007). Different parts of P. atlantica
such as leaves, stem, bark, galls, and fruits have found different uses.
from Morocco to Egypt through Algeria, Tunisia, Sudan, and Mauritania
use the wild pistachios as a support for grapevines, while others collect
the fruits of wild pistachios, roast, and consume them like other nuts
(Limane et al., 2014). They are traditionally used as a stimulant, for its
diuretic properties, and to treat hypertension, coughs, sore throats,
eczema, stomach aches, kidney stones, jaundice (Hosseini et al., 2013),
dyspepsia, and peptic ulcer (Benhammou et al., 2008). P. atlantica is
known as an excellent source of natural antioxidants. Important phy­
tochemicals, such as terpenes, flavonoids, tannins, steroids, carotenoid,
fatty acids, essential oils, and other compounds are present in the fruit,
leaf, stem-bark, root bark and mastic of P. atlantica (Ben Ahmed et al.,
2021).
P. atlantica galls result from abnormal leaves growth induced by
various parasitic organisms, especially aphids and chalcid wasps
(Mehrnejad, 2010). They are considered neoformed plant organs
developed from cellular hypertrophy, tissue hyperplasia and cellular
redifferentiation of the host tissues (Oliveira et al., 2016). Furthermore,
the galls were found to contain higher levels of defensive secondary

* Corresponding author.
E-mail address: f.hefied@lagh-univ.dz (F. Hefied).
1
ORCID ID:0000-0001-5006-4764

https://doi.org/10.1016/j.jarmap.2022.100449
Received 11 July 2022; Received in revised form 28 October 2022; Accepted 28 November 2022
Available online 30 November 2022
2214-7861/© 2022 Elsevier GmbH. All rights reserved.
F. Hefied et al.
metabolites than ungalled plant tissues. This accumulation of such me­
tabolites in the galls provides multilevel protection against variable
natural enemies (Yoram and Inbar, 2011).
The therapeutic virtues of P. atlantica can be largely attributed to
phenolic compounds, which display antioxidant capacity (Zaddem,
2014). These phenolic compounds include hydrolysable tannins (gal­
loylquinic acids and ellagitannins), phenolic acids, and flavonoids (3,5)
(Ben Ahmed et al., 2016). However, The quantity and quality of the
phenolic compounds depend on the extraction method. The extraction is
influenced by several factors, such as the chemical nature of the com­
pounds (simple or complex), the extraction method used (solvent
extraction, ultrasound-assisted extraction, etc.), the storage conditions
(Chirinos et al., 2007), the temperature, extraction time, and the
solid-to-liquid ratio (Prasad et al., 2011). Ultrasound-assisted extraction
accelerates solvent extraction of bioactive compounds from plants. In
addition, it offers many advantages, such as the reduction of solvents
consumption, temperature, and extraction time, which is very beneficial
for the extraction of thermolabile and unstable compounds (Yang et al.,
2010).
There are several statistical tools available to optimize the extraction
process (Tariq et al., 2018). Traditionally, optimization is based on the
one-factor-at-a-time approach, in which only one factor is variable, and
the others are kept constant. This method is laborious, costly,
time-consuming and fails to evaluate the interactions between variables
(Prasad et al., 2011), so it does not represent the complete effects of the
parameters on the response (Nassiri et al., 2011). The response surface
method (RSM) can overcome these difficulties (Prasad et al., 2011). In
addition, RSM requires a reduced number of experiments and less work
time. it has been successfully used to model and optimize the extraction
of phenolic compounds from different sources (Heydari Majd et al.,
2014). This model uses a limited number of experiments to account for
the linear, quadratic, and interactive effects of various factors on the
response (Tariq et al., 2018). Since no studies have been undertaken on
optimising extraction of phenolic compounds from P. atlantica leaf galls,
the objective of this study was to optimize ultrasonic-assisted extraction
parameters such as time, solid-to-liquid ratio, and extraction tempera­
ture from P. atlantica galls using RSM, as well as to measure the content
of (total phenolic contents, flavonoids and tannins) and the antioxidant
activity (DPPH and iron chelation) from the extracts obtained.
2. Materials and methods
2.1. Plant material
About 1000 g of galls were randomly collected from leaves of
P. atlantica trees in September 2010 (tree height is approximately 10 m)
from the region of Laghouat, located at 400 km south of Algiers, Algeria
(Fig. 1). The location of Laghouat (latitude 33◦47_(N); longitude 02◦52_
Fig. 1. Pistacia atlantica galls.
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Journal of Applied Research on Medicinal and Aromatic Plants 32 (2023) 100449
(E); altitude 750 m) is characterized by an annual precipitation of 18
mm and an average summer temperature of 41.4 ◦ C. This location is
characterized by a clay soil type. The plant material was spread out on
paper and shade dried at room temperature for a week. Once dried, the
galls were dry cleaned to remove insects and eggs, crushed, sieved,
stored in paper bags, and kept in refrigerator at + 4 ◦ C until use. The
identity of the gall samples was confirmed by Prof. M. Yousfi (Labo­
ratoire des Sciences Fondamentales, Université Amar Telidji, Laghouat,
Algeria).
2.2. Chemicals and reagents
Methanol, Folin-Ciocalteu reagent, gallic acid, 2,2-diphenyl-1-picryl­
hydrazyl (DPPH), vanillin, quercetin, catechin, iron chloride (FeCl3),
and Orthophenanthroline were obtained from Sigma-Aldrich. In addi­
tion, sodium carbonate (Na2CO3), vitamin E (α-tocopherol), vitamin C
(ascorbic acid), and hydrochloric acid (HCl) were purchased from Fluka.
2.3. Preliminary study of extraction conditions
The preliminary study of extraction conditions was to select from an
interval of each factor the value giving the maximum yield of total
phenolic compounds by varying one of the three parameters studied and
fixing the two others. The extraction procedure was carried out using an
ultrasonic bath (220 V 50 Hz Joident Ultrasonic-Cleaner, METO 5,
Ningbo, China).
Extraction time. When the extraction time was varied (5–60 min),
the other extraction conditions were fixed at the raw material to pure
methanol ratio of 1:25 (g/ml) and temperature at 25 ◦ C.
Sample to solvent ratio. the sample-to- solvent ratio was set at a
range of 1 g/(10− 90) ml “pure methanol”, while other variables were
kept constant (extraction time was 45 min at 50◦C).
Extraction temperature. The samples were extracted at numerous
temperatures ranging from 20◦ to 65◦ C, for 45 min with the solid-to-
liquid ratio of 25:1 ml/g.
After achieving the extraction process, the extracts were filtered, and
the methanol was removed using rotary evaporation at 45 ◦ C. In each
experiment, total phenolic content (TPC) were measured in order to find
out the suitable parameters of time, sample to solvent ratio and tem­
perature (Fig. 2).
2.4. Total phenolic content (TPC)
The total phenolic content (TPC) was determined using the Folin-
Ciocalteu reagent, according to the colorimetric method of (Boizot and
Charpentier, 2006) with some modifications. In a test tube, 100 μl of
diluted sample was mixed with 500 μl Folin-Ciocalteu reagent
(10-fold-diluted in water). After 3 min, 2 ml of Na2CO3 sodium
F. Hefied et al.
Fig. 2. Effect of extraction time (a), solid-to-liquid ratio (b) and temperature (c) on the total phenolic content. Values marked by the same letter are not significantly
different (p > 0.05) according to Tukey’s post hoc test.
carbonate (2%) was added and stirred. Then, the solution was allowed to
stand for 30 min in the dark and at room temperature. After that, the
absorbance was measured at 760 nm using a UV-Vis spectrophotometer
(UV-1601, Shimadzu, Kyoto, Japan). The total phenolic content of the
sample was determined using the standard gallic acid calibration curve
and expressed as mg gallic acid equivalents per g dry weight (mg GAE/g
DW).
2.5. Total flavonoid content
The total flavonoid content was carried out using an aluminum
chloride colorimetric method (Vongsak et al., 2013). First, 500 μl of the
sample was mixed with 500 μl of aluminum chloride solution (2%).
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Journal of Applied Research on Medicinal and Aromatic Plants 32 (2023) 100449
Then, after incubation at room temperature for 15 min, the absorbance
was measured at 430 nm using a UV-Vis spectrophotometer. A calibra­
tion curve was obtained with quercetin, and the results were expressed
as mg quercetin equivalents per g dry weight (mg QE/g DW).
2.6. Condensed tannin content (CTC)
The condensed tannin content was determined according to the
vanillin method described by (Chupin et al., 2013) with some modifi­
cations. First, (1%) vanillin was mixed with 8% (v / v) HCl and kept at
30 ◦ C. Next, 1 ml of vanillin solution was added to 200 μl of the sample.
The mixture was allowed to stand for 15 min at 30 ◦ C. Then, the
absorbance was measured at 500 nm. Finally, the results were expressed
F. Hefied et al. Journal of Applied Research on Medicinal and Aromatic Plants 32 (2023) 100449

as mg catechin equivalent per g dry weight (mg CE/g DW). Table 1

Box- Behnken design (BBD), Factors and levels (coded and uncoded) used for
2.7. Antioxidant activities determination optimization.
Factors
2.7.1. DPPH• (2,2-diphenyl-1-picrylhydrazyl) assay Run X1 X2 X3
The DPPH• radical scavenging activity was performed according to
1 0 0 0
the method reported by (Du et al., 2014) with minor modifications. A 2 -1 0 +1
quantity of 1 ml of methanolic DPPH• solution (250 μM) was mixed with 3 +1 -1 0
1 ml of diluted extract. Then, the preparation was agitated in a vortex 4 0 -1 -1
and incubated for 30 min in the dark at room temperature. The decrease 5 -1 -1 0
6 +1 0 +1
in absorbance was read at 517 nm. Finally, the antiradical capacity was 7 0 +1 -1
defined using a calibration curve obtained with ascorbic acid and 8 0 0 0
expressed as mg of ascorbic acid equivalents (AAE) per g dry weight. The 9 +1 +1 0
antioxidant capacity (AC) was calculated using the following equation: 10 +1 0 -1
11 -1 +1 0
AC =[ (Acont – Asample)/ α)] x D x V x (1/Wsample) 12 0 -1 +1
13 -1 0 -1
Where Acont the absorbance of the DPPH• solution (without the 14 0 +1 +1
15 0 0 0
extract), Asample the absorbance of extract solution, α the slope of the
Codedlevels
standard curve, D the dilution factor of the extract, V the total extract Factors Symbol -1 0 +1
volume (10 ml) and Wsample the weight of the sample used for extraction Time (min) X1 40 45 50
(fixed for all samples, approximately 1 g). Solid-liquid ratio (g/ml) X2 1:10 1:20 1:30
Temperature (◦ C) X3 40 50 60

2.7.2. Iron chelation assay

The iron-chelating capacity of the extracts was measured to follow were used to verify the importance of the regression coefficient. Finally,
the inhibition of the formation of Fe (II)-phenanthroline complex the adequacy of the model was determined by assessing the lack of fit.
described by (Qureshi et al., 2009) with some modifications. First,
500 μl of extract diluted was mixed with 500 μl of
2.10. Verification of model
ethanolic-phenanthroline solution (0.4%). Then, 250 μl of ferric chlo­
ride solution (FeCl3) (0.12% in ethanol) was added. The mixture was
In order to verify the predictive power of the model used for
allowed to stand for 4 min at room temperature. The absorbance was
obtaining optimal conditions for the extraction of phenolic compounds
measured at 510 nm. The Iron chelating activity was expressed as mg
from Pistacia atlantica galls, the predicted data were compared with the
tocopherol equivalents (TE) per g dry weight (DW).
experimental data.

2.8. Experimental design

3. Results and discussion

The experimental design is based on the Box-Behnken design, which

includes three variables and three factorial levels (− 1, 0 and 1), making
it possible to construct a response surface and to determine the true
optimal extraction conditions. The independent variables used in the
present study are extraction time (X1), solid-liquid ratio (X2), and tem­
perature (X3), while responses are TPC, TFC, CTC, and antioxidant ac­
tivity (DDPH assay and iron chelation). A total of 15 experiment runs
with three replicates at the center points were carried out. The experi­
ment design and levels of independent variables are given in Table 1. A
second-order polynomial equation used in the response surface analysis
is shown in Eq. (1) (Ben Ahmed et al., 2016):
Y= β0 + β1X1 + β2X2 + β3X3 + β12 X1X2 + β13 X1X3 + β23 X2X3 + β11X21 +
β22 X22 + β33 X23 (1)
Where Y is the predicted response; β0 is the intercept; β1, β2, and β3
are the linear coefficients; β11, β22, and β33 are the quadratic coefficients;
β12, β13, and β23 are the interaction coefficients and X1, X2, and X3 are the
independent variables.
2.9. Statistical analysis
All experiment data were carried out in triplicates, and the results
were expressed as means ± SD. The results in the single factor ( pre­
liminary) experiments were evaluated by an analysis of variance
(ANOVA) with the Tukey’s test, using Minitab 18.1 software (Minitab
Inc., United States) to detect significant differences (p < 0.05) between
the means. The JMP (Version 10. SAS, USA) software was used to
determine the polynomial coefficients for each response. Besides, anal­
ysis of variance (ANOVA) was used in order to test the significance of the
model coefficients (p < 0.05). Moreover, The F-test and the p-value
3.1. Determination of the experimental ranges of independent variables
3.1.1. Extraction time
The extraction time is one of the important parameters because it has
an influence on the contact between the solvent and the solid matter
(Yang et al., 2010). The effect of extraction time on the extraction yield
of phenolic compounds is shown in Fig. 2a. the independent variable of
time was varied from 5 to 60 min, while the other extraction conditions
were set as follows: temperature (25 ◦ C) and ratio (S/L) (1:25 g/ml). The
results revealed that running the extraction for 45 min yielded the
highest amount of TPC. The Tukey’s post-hoc test indicated 2 groups (a)
and (b). From 50–60 min, the decrease of TPC may be due to the longer
extraction times that increase the phenolics oxidation (Hachani et al.,
2020).
3.1.2. Sample to solvent ratio
The effect of solid-to-liquid ratio on the extraction of phenolic
compounds was evaluated using eleven ratios (from 1:10–1:90 g/ml)
over an extraction period of 45 min, at 50 ◦ C (Fig. 2b). This ratio plays a
significant role in the mass transfer of extracts between materials and
extraction solvents (Pham et al., 2017). As shown in Fig. 2b, when the
solid-to-liquid ratio increased from 1:10–1:20 /ml, the extraction yields
increased significantly. This could be explained by the fact that a higher
ratio of solvent increases the concentration gradient, leading to an in­
crease diffusion rate of polyphenols from the extracted raw material into
the solvent (Hachani). This result is similar with the studies carried out
by (Tabaraki and Nateghi, 2011) on rice bran, (Prasad et al., 2011) on
the bark of the fruit Mangifera pajang Kosterm and (Jennan et al., 2015)
on T. hyemalis. However, a decrease in extraction yield was observed
when the ratio exceeds 1:20, which indicating that the excessive dilution

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F. Hefied et al. Journal of Applied Research on Medicinal and Aromatic Plants 32 (2023) 100449

of the target material may not increase the TPC under the same inves­
tigated conditions (Bamba et al., 2018).
3.1.3. Extraction temperature
Fig. 2c shows the effect of the extraction temperature on TPC (from
20◦ to 65◦ C). The other extraction conditions were fixed as follows: time
(45 min) and solid-to-liquid ratio (1:25 g/ml). A one-way analysis of
variance showed a significant difference among the yields at the
extraction temperatures studied. The results indicated that the TPC
increased significantly when the temperature increased from 20◦ to
50◦ C and reached the highest TPC value at 50 ◦ C. This result is in
agreement with the study carried out by (Bouafia et al., 2021) on
Centaurea sp. reported that the highest TPC was obtained at 50◦C.
Elevated temperature enhances the solubility of phenolic compounds,
increases the rate of diffusion and reduces the viscosity of solvents
(Prasad et al., 2011). Nevertheless, a certain temperature limit must be
considered to avoid the degradation of heat-sensitive polyphenols
(Heydari Majd et al., 2014). This could explain why TPC content
decreased when temperature exceeded 50 ◦ C.
Thus, the extraction time (40–50 min), the sample to the solvent
ratio (1: 10–1: 30 g/ml) and the extraction temperature (40–60 ◦ C) were
used in the study to optimize the extraction of phenolic compounds.
3.2. Optimization of extraction conditions
The response surface method allowed us to determine an approxi­
mation relationship between the set independent variables (time, solid-
to-liquid ratio, and temperature) and the response. This relation was
formulated by a second-order polynomial that can optimize the extrac­
tion parameters to achieve desirable responses. To optimize the
extraction process of TPC, TFC, CTC, and antioxidant capacity (DPPH
and Iron chelation) from Pistacia atlantica galls, an extraction time of
45 min, a solid-to-liquid ratio of 1: 20 (g/ml), and a temperature of
50 ◦ C were chosen as the central condition of Box-Behnken design
(BBD). Table 1 presents the experimental conditions for the BBD. The
responses to the experimental design are shown in Table 2, which in­
cludes the experimental and the predicted values. TPC, TFC, CTC, DPPH
test, and iron chelation test values ranged from 52.48 to 139.88 mg
GAE/g DW, 1.08–2.43 mg QE/g DW, 45.90–121.32 mg CE/g DW,
32.12–65.94 mg AAE/g DW, and 240.35–501.38 mg TE/g DW, respec­
tively. The highest values (TPC, TFC, and CTC) and the best antioxidant
activities (DPPH and iron chelation) were recorded for an extraction
time of 40 min, a solid-to-liquid ratio of 1: 20 (g/ml), and an extraction
temperature of 60 ◦ C. Comparing our results with those of (Benarfa
et al., 2020), who chose the same variables and carried out
ultrasound-assisted extraction from D. scoparia flowers, the time and
temperature that gave high TPC were 45 min and 50 ◦ C, both close to
our results. However, the solid-to-liquid ratio (1: 70 g/ml) was very
different from ours.
The following multiple regression equations were obtained from the
model (Eq. 01) by integrating the independent variables (X1, X2, and X3)
and their coefficients:
TPC= 123⋅1– 4⋅2X1+ 5⋅5X2+ 23⋅1X3+ 11⋅4X1X2 – 8⋅5X1X3 – 1⋅0 X2X3 –
5⋅1X21– 27⋅8X22– 13⋅6X23 (2)
TFC= 2⋅3 – 0⋅14X1+ 0⋅08X2 + 0⋅48X3 + 0⋅05X1X2 – 0⋅03X1X3 – 0⋅04X2X3 –
0⋅15X21 – 0⋅37X22 – 0⋅17X23 (3)
CTC= 106⋅8– 6⋅3X1+ 5⋅4X2+ 23⋅2X3+ 10⋅3X1X2 – 3⋅3X1X3+ 0⋅5X2X3–
5⋅3X21– 23⋅1X22– 8⋅5X23 (4)
DPPH= 60⋅5– 3⋅4X1+ 2⋅8X2 + 7⋅5X3+ 7⋅7X1X2 – 5⋅4X1X3 + 0⋅6X2X3 –
1⋅9X21 – 10⋅8X22 – 7⋅2X23 (5)
Iron chelation= 441⋅1– 2⋅1X1 – 12⋅2X2 + 46⋅4X3 +62⋅9X1X2 – 44⋅1 X1X3 +
4⋅9 X2X3 – 41⋅9 X21 – 66⋅8X22– 34⋅7X23 (6)
The more reliable way to evaluate the adapted model quality is the
application of the analysis of variance (ANOVA). ANOVA consists of
knowing the effects of several factors on response and deciding whether
to consider this effect. The analysis of the variance of the second-order
polynomial models for the content of TPC, TFC, CTC, DPPH, and iron
chelation test from P. atlantica galls is summarized in Table 3.
The coefficient of determination (R2) measures the adequacy be­
tween the multiple regression model and the experimental data
observed (Yang et al., 2010). They were 0.98, 0.93, 0.98, 0.98, and 0.82,
which means that only 2%, 7%, 2%, 2%, and 18% of the variations were
not explained by the models for TPC, TFC, CTC, DPPH, and iron chela­
tion test, respectively. The closer R2 is to 1, the more the experimental
model corresponds to the real data. However, if the value of R2 is low,
that indicates some important variables have not been taken into ac­
count in the model (Heydari Majd et al., 2014). The adjusted R2 is a
modified version of R2. It is adjusted for the number of variables in the
model. The variance analysis of TPC, TFC, CTC, DPPH, and iron chela­
tion test gave a R2adj values of 0.98, 0.81, 0.96, 0.96, and 0.49,
respectively. Thus, R2 adj increases only if the variables enhance the
model more than expected, and decreases if the variables enhance the
model less than expected. Nevertheless, it is always lower than R2.
The p-value was used to check the significance of the applied model.
It is less than 0.05; which indicates that these models are significant and
that the regression equation fits very well with the actual situation,
reflecting the relationship between the extraction of phenolic com­
pounds and extraction conditions (time, solid-to-liquid ratio, and

Table 2
The average of experimental and predicted values (n = 3) for total phenolic content (TPC), total flavonoids content (TFC), condensed tannin content (CTC) and
antioxidant activities by DPPH and Iron chelation expressed in mg E/ g DW.
Experimental Predicted

Run TPC TFC CTC DPPH Ironchelation TPC TFC CTC DPPH Ironchelation

1 127.30 2.24 109.00 60.50 427.42 123.10 2.26 106.83 60.48 441.13
2 139.90 2.43 121.32 65.94 501.38 140.30 2.60 125.75 67.60 457.20
3 68.31 1.30 53.22 32.12 282.95 70.00 1.45 56.45 33.85 279.73
4 52.50 1.08 45.90 32.93 351.28 52.10 1.10 47.11 32.85 310.31
5 102.02 1.90 93.13 56.30 404.41 100.32 1.85 89.65 56.10 409.65
6 117.00 2.22 109.00 50.31 400.43 114.87 2.25 106.71 50.04 364.70
7 66.41 1.16 55.91 35.72 315.01 65.10 1.35 56.86 37.18 276.06
8 121.71 2.18 105.42 61.42 472.90 123.10 2.26 106.83 60.50 441.13
9 101.24 1.74 84.47 54.67 386.23 102.94 1.72 87.95 54.90 381.00
10 86.02 1.50 71.17 47.50 315.94 85.62 1.32 66.73 45.83 360.14
11 89.24 2.07 83.06 47.91 256.27 88.45 1.92 79.83 46.20 259.50
12 99.10 2.35 93.46 48.07 354.40 100.41 2.16 92.51 46.61 393.35
13 74.84 1.61 70.50 41.57 240.35 76.94 1.57 72.75 41.84 276.10
14 109.00 2.26 105.65 53.30 337.71 109.36 2.24 104.44 53.40 378.70
15 120.29 2.35 106.07 59.53 423.05 123.10 2.26 106.83 60.50 441.13

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F. Hefied et al. Journal of Applied Research on Medicinal and Aromatic Plants 32 (2023) 100449

Table 3
Analysis of variance for the quadratic model.
F-value of
Response R2 R2 (adj) F-value of model P-value of model Lack of fit P-value of Lack of fit

Y TFC 0.99 0.98 103.81 ˂.0001* 0.48 0.72

Y TFC 0.93 0.81 7.90 0.0174* 8.14 0.11
Y CTC 0.98 0.96 39.04 0.0004* 9.12 0.10
Y DPPH 0.98 0.96 48.19 0.0003* 5.96 0.14
Y Ironchelation 0.82 0.49 2.55 0.1574 5.65 0.15

temperature) (Yang et al., 2010), with the exception of the iron chela­
tion test which is 0.1574. In practice, this value is always significant; Table 4
Regression coefficients and their statistical significance.
otherwise, the model is absolutely useless (Ben Ahmed et al., 2016). In
addition, the non-significant lack of fit (p > 0.05) also increases the Term Coefficient Std. error t Ratio p-value
reliability and adequacy of the models (Tabaraki and Nateghi, 2011) and TPC
which means that a few unknown factors were interfering with the test β0 123.1 1.8 69.3 ˂.0001*
result (Yang et al., 2010). The p-values of lack of fit were 0.72, 0.11, β1 -4.2 1.1 -3.8 0.012*
β2 5.5 1.1 5.1 0.004*
0.10, 0.14, and 0.15 for TPC, TFC, CTC, DPPH, and the iron chelation β3 23.1 1.1 21.3 ˂.0001*
test, respectively, which implies that the fitting of these models is β12 11.4 1.5 7.4 0.0007*
adequate to describe experimental data. β13 -8.5 1.5 -5.5 0.003*
The significance of each coefficient was also determined. The results β23 -1.0 1.5 -0.7 0.54
-5.1 1.6 -3.2 0.024*
are given in Table 4. According to the p-values of each model term, the β11
β22 -27.8 1.6 -17.4 ˂.0001*
coefficients that were the most significant for the responses are the β33 -13.6 1.6 -8.5 0.0004*
linear coefficient of the extraction temperature (β3) (positive linear ef­ TFC
fect) and quadratic solid-to-liquid ratio (β22) (negative quadratic ef­ β0 2.3 0.11 19.71 ˂.0001*
fects). In addition, the interaction between the extraction time and the β1 -0.14 0.07 -2.12 0.09
0.08 0.07 1.18 0.29
solid-to-liquid ratio (β 12), the quadratic term of the extraction tem­
β2
β3 0.48 0.07 6.97 0.0009*
perature (β33), and the linear term of the extraction time (β1) tended to β12 0.05 0.09 0.53 0.62
be significant for most responses. The negative effects of the regression β13 -0.03 0.09 -0.25 0.81
coefficients cause a decrease in the response (Ben Ahmed et al., 2016), β23 -0.04 0.09 -0.43 0.69
-0.15 0.10 -1.41 0.22
which confirms the deceleration in the extraction of phenolic com­ β11
β22 -0. 37 0.10 -3.62 0.015*
pounds (Heydari Majd et al., 2014). β33 -0.17 0.10 -1.66 0.16
CTC
β0 106.8 2.7 40.0 < .0001*
3.3. Analysis of response surface plots β1 -6.3 1.6 -3.8 0.01*
β2 5.4 1.6 3.3 0.02*
β3 23.2 1.6 14.2 < .0001*
The surface plots of the quadratic models were obtained by keeping a β12 10.3 2.3 4.5 0.007*
constant variable at its central level and varying two others within the β13 -3.3 2.3 -1.4 0.22
experimental range. They illustrated the linear, quadratic, and interac­ β23 0.5 2.3 0.2 0.82
tive effects on the response (Zaddem, 2014). β11 -5.3 2.4 -2.2 0.08
-23.1 2.4 -9.6 0.0002*
The three-dimensional response surface plots (Fig. 3a, b, c, d, and e)
β22
β33 -8.5 2.4 -3.6 0.02*
illustrate the relationship between the response (TPC, TFC, CTC, DPPH, DPPH
and iron chelation) of P. atlantica galls and the two experimental vari­ β0 60.5 1.0 55.0 < .0001*
ables (extraction time and solid-to-liquid ratio) at fixed extraction β1 -3.4 0.6 -5.0 0.004*
2.8 0.6 4.1 0.009*
temperature of 50 ◦ C. Although the solid-to-liquid ratio showed an β2
β3 7.5 0.6 11.2 < .0001*
explicit influence on the content (TPC, TFC, CTC, DPPH, and iron che­ β12 7.7 0.9 8.2 0.0004*
lation) in a quadratic manner (Table 4), the extraction time had a β13 -5.4 0.9 -5.7 0.002*
slightly longer positive effect. A higher amount was produced at a solid- β23 0.6 0.9 0.6 0.55
to-liquid ratio between 1: 20–1: 25 (g/ml) and an extraction time be­ β11 -1.9 0.9 -1.9 0.10
-10.8 0.9 -10.9 0.0001*
tween 43 and 47 min. This behavior is consistent with the study con­
β22
β33 -7.2 0.9 -7.3 0.0007 *
ducted by (Jennan et al., 2015), who explained that when the Ironchelation
solid-to-liquid ratio increases, the solvent is introduced into the cells β0 441.1 31.0 14.2 < .0001*
allowing the phenolic compounds to be released in the extract. In β1 -2.1 19.0 -0.1 0.92
-12.2 19.0 -0.6 0.55
addition, the solid-to-liquid ratio indicates that the solubility of different β2
β3 46.4 19.0 2.4 0.06
molecules depends on the intermolecular forces between solute and β12 62.9 26.9 2.3 0.07
solvent (Ngamkhae et al., 2022). On the other hand, the influence of the β13 -44.1 26.9 -1.6 0.16
extraction time is limited, which also confirms the results obtained by β23 4.9 26.9 0.2 0.86
(Ben Ahmed et al., 2016). β11 -41.9 27.9 -1.5 0.19
-66.8 27.9 -2.4 0.06
The effects of extraction time and temperature on TPC, TFC, CTC,
β22
β33 -34.7 27.9 -1.2 0.27
DPPH, and iron chelation of P. atlantica galls at a constant solid-to-liquid
ratio of 1: 20 (g/ml) are shown in Fig. 4 (plots a, b, c, d, and e, Significance with p < 0.05. (*) p < 0.01.
respectively). Both variables had an independent influence on the re­
sponses. However, the extraction temperature had an effect much more (Table 4). The highest levels were observed at extraction times between
important than the extraction time. This is mainly due to the linear term 43 and 45 min and temperatures between 50 and 60 ◦ C. This has also
of the temperature (β3), and secondarily the linear term of extraction been observed by (Bachir bey et al., 2014), who found that the extrac­
time (β1), and the quadratic effect of the extraction temperature (β 33) tion of phenolic compounds was related to time and temperature. The

6
F. Hefied et al.
Fig. 3. Response surface plots showing the effect of extraction time (min) and solid-to-liquid ratio (g/ml) on TPC (a), TFC (b), CTC (c), DPPH (d) and Iron chelation
(e) from Pistacia atlantica galls at the temperature of 50 ◦ C.
mass transfer increased with time until maximum extraction was ach­
ieved and temperature accelerated diffusion. Alternatively, the study by
(Tabaraki and Nateghi, 2011) on the rice bran extracts indicates that the
TPC has increased with increasing temperature up to 54 ◦ C followed by a
slight decrease. The heating of the extract softens the plant tissues,
causing the weakening of the cell walls, the hydrolysis of the bonds of
the phenolic compounds bound to other compounds (phenol-protein or
phenol-polysaccharide), and the improvement of the phenolic com­
pounds solubility, which subsequently allows a better distribution in the
7
Journal of Applied Research on Medicinal and Aromatic Plants 32 (2023) 100449
solvent. Moreover, the same observation was underlined by the previous
study (Bouafia et al., 2021) on Centaurea sp. reporting that relatively
higher temperatures enhanced both the diffusion and solubility rates of
the different components, leading to a higher rate of extraction of
phenolic compounds, and hence, most often higher activity of the ob­
tained extracts.
The influence of the solid-to-liquid ratio (X2) and the extraction
temperature (X3) on the TPC, TFC, CTC, DPPH, and iron chelation at a
fixed extraction time of 45 min is presented in Fig. 5 (plots a, b, c, d, and
F. Hefied et al. Journal of Applied Research on Medicinal and Aromatic Plants 32 (2023) 100449

Fig. 4. Response surface plots showing the effect of extraction time (min) and temperature (◦ C) on TPC (a), TFC (b), CTC (c), DPPH (d) and Iron chelation (e) from
Pistacia atlantica galls at the solid-to-liquid ratio of 1:20 g/ml.

e, respectively). Thus, both variables strongly affected the polyphenols
and the two activities studied, which explains the shape of the response
surfaces that is mainly due to the linear and quadratic terms of the
extraction temperature (β3, β33) and the quadratic term (β22) of the solid-
to-liquid ratio. The highest levels were observed at intermediate solid-
liquid ratios and at 50–60 ◦ C extraction temperatures. Our results
agreed with previous studies (Hachani et al., 2020) reporting that total
phenolic content from Date cultivar of Phoenix dactylifera L increased
with the time. The same effect was found by varying liquid-to-solid ratio
and temperature, this effect has a positive relationship with TPC due to
the significant positive effect of the quadratic term (β22).
From the foregoing, the extraction temperature played a most
important role in the extraction of phenolic compounds (TPC, TFC, and
CTC) and the antioxidant activity (DPPH and iron chelation), followed
by the solid-to-liquid ratio, and slightly the extraction time.
3.4. Verification of predictive models
In this part, the objective was to verify the predictive capacity of the
model and to determine the best combination of extraction conditions

8
F. Hefied et al.
Fig. 5. Response surface plots showing the effect of solid-to-liquid ratio (g/ml) and temperature (◦ C) on TPC (a), TFC (b), CTC (c), DPPH (d) and Iron chelation (e)
from Pistacia atlantica galls at the extraction time of 45 min.
for the phenolic compounds from Pistacia atlantica galls. The values
predicted were estimated using the desirability value by JMP, ranging
from 0 to 1, with perfect values when d = 1 and acceptable values when
d > 0.7 (Zaddem, 2014).
The optimal conditions for TPC, TFC, CTC, DPPH, and iron chelation
are as follows, an extraction time of [40–49] min, a solid-to-liquid ratio
of 1: 20 (g/ml), and a temperature of 60 ◦ C. Under the optimal condi­
tions, the predicted responses were: TPC = 140.71 (mg GAE/g DW), TFC
9
Journal of Applied Research on Medicinal and Aromatic Plants 32 (2023) 100449
= 2.26 (mg QE/g DW), CTC = 125.96 (mg CE/g DW), DPPH = 68.12
(mg AAE/g DW), and iron chelation= 475.80 (mg TE / g DW) with a
desirability of 0.89, 0.99, 0.93, 0.93, and 0.77, respectively. Experi­
mental results are close with the predicted values corresponding 139.88
(mg GAE/g DW), 2.43 (mg QE/g DW), 121.32 (mg CE/g DW), 65.94 (mg
AAE/g DW), and 501.38 (mg TE/g DW), this accordance demonstrated
the adequacy and predictability of the statistical model used in this
optimization study.
F. Hefied et al.
In the final step, when compared, the amount of phenolic compounds
in P. atlantica galls (139.88 mg GAE/g DW) with the reported amounts
in the works of (Hachani et al., 2020), (Bouafia et al., 2021), and
(Bouafia et al., 2021) who investigated on Phoenix dactylifera L,
D. scoparia, and Centaurea sp., respectively, and adopted the RMS
method under the same extraction conditions, their phenolic compound
contents were: 1.705 mg GAE/g DW, 9.46 mg GAE/g DW, and 46.44
± 0.53 mg GAE/g DW, respectively. This comparison shows clearly the
richness of the galls in total phenolic compounds.
4. Conclusion
An Ultrasonic-assisted extraction process was successfully applied to
optimize the conditions for the extraction of antioxidant compounds
from P. atlantica galls collected in Algeria. The RSM with BBD technique
was used to optimize the experimental variables. In addition, a second-
order model was used to estimate the content of TPC, TFC, and CTC and
to evaluate the antioxidant activity by both tests (DPPH and iron­
chelation). Results of maximized responses revealed that extraction for
40 min at 60 ◦ C and with solid-to-liquid ratio of 1: 20 (g/ml) is the best
combination to optimize the extraction. It was found that both predicted
and experimental values were in close agreement, which clearly dem­
onstrates the suitability of the model. The extraction temperature was
the most significant parameter, due to its positive influence on the
extraction performance. This method can be utilized for further
screening of active fraction/compounds from P. atlantica galls.
Declaration of Competing Interest
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influence
the work reported in this paper.
Acknowledgements
The authors wish to thank the General Directorate of Scientific
Research and Technological Development (DGRSDT) and the University
of Amar Telidji, Laghouat - Algeria for their financial support.
References
Bachir bey, M., Meziant, L., Benchikh, Y., Louaileche, H., 2014. Deployment of response
surface methodology to optimize recovery of dark fresh fig (Ficus carica L., var.
Azenjar) total phenolic compounds and antioxidant activity. Food Chemistry 162,
277–282. 〈https://doi.org/10.1016/j.foodchem.2014.04.054〉.
Bamba, B.S.B., Shi, J., Tranchant, C.C., Xue, S.J., Forney, C.F., Lim, L.-T., Xu, W., Xu, G.,
2018. Coencapsulation of polyphenols and anthocyanins from blueberry pomace by
double emulsion stabilized by whey proteins: effect of homogenization parameters.
Molecules 23, 2525. https://doi.org/10.3390/molecules23102525.
Ben Ahmed, Z., Yousfi, M., Viaene, J., Dejaegher, B., Demeyer, K., Mangelings, D.,
Heyden, Y.V., 2016. Determination of optimal extraction conditions for phenolic
compounds from Pistacia atlantica leaves using the response surface methodology.
Analytical Methods 8, 6107–6114. https://doi.org/10.1039/C6AY01739H.
Ben Ahmed, Z., Yousfi, M., Viaene, J., Dejaegher, B., Demeyer, K., Heyden, Y.V., 2021.
Four Pistacia atlantica subspecies (atlantica, cabulica, kurdica and mutica): a review
of their botany, ethnobotany, phytochemistry and pharmacology. Journal of
Ethnopharmacology 265, 113329. https://doi.org/10.1016/j.jep.2020.113329.
Ben Ahmed, Z., Hefied, F., Yousfi, M., Demeyer, K., Vander Heyden, Y., 2022. Study of
the antioxidant activity of Pistacia atlantica Desf. Gall extracts and evaluation of the
responsible compounds. Biochemical Systematics and Ecology 100, 104358. https://
doi.org/10.1016/j.bse.2021.104358.
Benarfa, A., Gourine, N., Hachani, S., Harrat, M., Yousfi, M., 2020. Optimization of
ultrasound-assisted extraction of antioxidative phenolic compounds from Deverra
scoparia Coss. & Durieu (flowers) using response surface methodology. Journal of
Food Processing and Preservation 44, e14514. https://doi.org/10.1111/jfpp.14514.
Benhammou, N., Atik Bekkara, F., Kadifkova Panovska, T., 2008. Antioxidant and
antimicrobial activities of the Pistacia lentiscus and Pistacia atlantica extracts.
African Journal of Pharmacy and Pharmacology 2, 022–028.
Boizot, N., Charpentier, J.-P.J.-P., 2006. Méthode rapide d′ évaluation du contenu en
composés phénoliques des organes d′ un arbre forestier. Cahier des Techniques
Délelő tt l′ INRA 79.
Bouafia, M., Colak, N., Ayaz, F.A., Benarfa, A., Harrat, M., Gourine, N., Yousfi, M., 2021.
The optimization of ultrasonic-assisted extraction of Centaurea sp. antioxidative
10
Journal of Applied Research on Medicinal and Aromatic Plants 32 (2023) 100449
phenolic compounds using response surface methodology. Journal of Applied
Research on Medicinal and Aromatic Plants 25, 100330. 〈https://doi.org/10.1016/j.
jarmap.2021.100330〉.
Chirinos, R., Rogez, H., Campos, D., Pedreschi, R., Larondelle, Y., 2007. Optimization of
extraction conditions of antioxidant phenolic compounds from mashua (Tropaeolum
tuberosum Ruíz & Pavón) tubers. Separation and Purification Technology 55,
217–225. https://doi.org/10.1016/j.seppur.2006.12.005.
Chupin, L., Motillon, C., Charrier-El Bouhtoury, F., Pizzi, A., Charrier, B., 2013.
Characterisation of maritime pine (Pinus pinaster) bark tannins extracted under
different conditions by spectroscopic methods, FTIR and HPLC. Industrial Crops and
Products 49, 897–903. https://doi.org/10.1016/j.indcrop.2013.06.045.
Du, L., Shen, Y., Zhang, X., Prinyawiwatkul, W., Xu, Z., 2014. Antioxidant-rich
phytochemicals in miracle berry (Synsepalum dulcificum) and antioxidant activity of
its extracts. Food Chemistry 153, 279–284. https://doi.org/10.1016/j.
foodchem.2013.12.072.
Hachani, S., Boukhalkhal, S., Ahmed, Z.B., Harrat, M., Silva, A.M., Yousfi, M., 2020.
Exploiting response surface methodoloy (RSM) as a novel approach for the
optimization of phenolics and antioxidant activity of date palm Fruit. Chemistry and
Chemical Technology 14, 572–582.
Heydari Majd, M., Rajaei, A., Salar Bashi, D., Mortazavi, S.A., Bolourian, S., 2014.
Optimization of ultrasonic-assisted extraction of phenolic compounds from bovine
pennyroyal (Phlomidoschema parviflorum) leaves using response surface
methodology. Industrial Crops and Products 57, 195–202. https://doi.org/10.1016/
j.indcrop.2014. 03.031.
Hosseini, F., Adlgostar, A., Sharifnia, F., 2013. Antibacterial activity of Pistacia atlantica
extracts on Streptococcus mutans biofilm. International Research. Journal of
Biological Sciences 2, 1–7.
Jennan, S., Farah, A., Mahjoubi, F., 2015. Optimisation of ultrasound assisted extraction
of T.hyemalis using the response surface methodology 6. Journal Material
Environment 6.
Limane, A., Smail-Saadoun, N., Thomas, G., 2014. Root architecture of Atlas pistachio in
relation to underlying soil properties under arid conditions. African Journal of
Agricultural Research 9, 620–626. https://doi.org/10.5897/AJAR2013.7291.
Mehrnejad, M.R., 2010. Potential biological control agents of the common pistachio
psylla, Agonoscena pistaciae, a review. Entomofauna 31, 317–340.
Monjauze, A., 1980. Connaissance du bétoum Pistacia atlantica Desf. Revue forestière
française 32, 356–363.
Nassiri, H., Arabi, H., Hakim, S., Bolandi, S., 2011. Polymerization of propylene with
Ziegler–Natta catalyst: optimization of operating conditions by response surface
methodology (RSM. Polymer Bulletin 67, 1393–1411. https://doi.org/10.1007/
s00289-011-0568-y.
Ngamkhae, N., Monthakantirat, O., Chulikhit, Y., Boonyarat, C., Maneenet, J.,
Khamphukdee, C., Kwankhao, P., Pitiporn, S., Daodee, S., 2022. Optimization of
extraction method for Kleeb Bua Daeng formula and comparison between
ultrasound-assisted and microwave-assisted extraction. Journal of Applied Research
on Medicinal and Aromatic Plants 28, 100369. https://doi.org/10.1016/j.
jarmap.2022.100369.
Oliveira, D.C., Isaias, R.M.S., Fernandes, G.W., Ferreira, B.G., Carneiro, R.G.S.,
Fuzaro, L., 2016. Manipulation of host plant cells and tissues by gall-inducing insects
and adaptive strategies used by different feeding guilds. Journal of Insect
Physiology, Plant-reprogramming Insects: from Effector Molecules to Ecosystem
Engineering 84, 103–113. https://doi.org/10.1016/j.jinsphys.2015.11.012.
Pham, H.N.T., Vuong, Q.V., Bowyer, M.C., Scarlett, C.J., 2017. Optimization of
ultrasound-assisted extraction of Helicteres hirsuta Lour. for enhanced total phenolic
compound and antioxidant yield. Journal of Applied Research on Medicinal and
Aromatic Plants 7, 113–123. https://doi.org/10.1016/j.jarmap.2017.07.002.
Prasad, K.N., Hassan, F.A., Yang, B., Kong, K.W., Ramanan, R.N., Azlan, A., Ismail, A.,
2011. Response surface optimisation for the extraction of phenolic compounds and
antioxidant capacities of underutilised Mangifera pajang Kosterm. peels. Food
Chemistry 128, 1121–1127. https://doi.org/10.1016/j.foodchem.2011.03.105.
Qureshi, N.N., Kuchekar, B.S., Logade, N.A., Haleem, M.A., 2009. Antioxidant and
hepatoprotective activity of Cordia macleodii leaves. Saudi Pharmaceutical Journal
17, 299–302. https://doi.org/10.1016/j.jsps.2009.10.007.
Rauf, A., Patel, S., Uddin, G., Siddiqui, B.S., Ahmad, B., Muhammad, N., Mabkhot, Y.N.,
Hadda, T.B., 2017. Phytochemical, ethnomedicinal uses and pharmacological profile
of genus Pistacia. Biomedicine & Pharmacotherapy 86, 393–404. https://doi.org/
10.1016/j.biopha.2016.12.017.
Tabaraki, R., Nateghi, A., 2011. Optimization of ultrasonic-assisted extraction of natural
antioxidants from rice bran using response surface methodology. Ultrasonics
Sonochemistry 18, 1279–1286. https://doi.org/10.1016/j.ultsonch.2011.05.004.
Tariq, M., Tarun, B., Indra, D.B., Veena, P., Shyamal, K.N., 2018. Polyphenolics in leaves
of Paris polyphylla: An important high value Himalayan medicinal herb. Industrial
Crops and Products 117, 66–74. https://doi.org/10.1016/j.indcrop.2018.02.071.
Vongsak, B., Sithisarn, P., Mangmool, S., Thongpraditchote, S., Wongkrajang, Y.,
Gritsanapan, W., 2013. Maximizing total phenolics, total flavonoids contents and
antioxidant activity of Moringa oleifera leaf extract by the appropriate extraction
method. Industrial Crops and Products 44, 566–571. 〈https://doi.org/10.1016/j.in
dcrop.2012.09.021〉.
Wu, J., Yu, D., Sun, H., Zhang, Y., Zhang, W., Meng, F., Du, X., 2015. Optimizing the
extraction of anti-tumor alkaloids from the stem of Berberis amurensis by response
surface methodology. Industrial Crops and Products 69, 68–75. https://doi.org/
10.1016/j.indcrop.2015.01.053.
Xie, L., Yang, Z.-Y., Wen, J., Li, D.-Z., Yi, T.-S., 2014. Biogeographic history of Pistacia
(Anacardiaceae), emphasizing the evolution of the Madrean-Tethyan and the eastern
Asian-Tethyan disjunctions. Molecular Phylogenetics and Evolution 77, 136–146.
https://doi.org/10.1016/j.ympev.2014.04.006.
F. Hefied et al. Journal of Applied Research on Medicinal and Aromatic Plants 32 (2023) 100449

Yang, L., Cao, Y.-L., Jiang, J.-G., Lin, Q.-S., Chen, J., Zhu, L., 2010. Response surface Zaddem, M., 2014. Application de la méthode des surfaces de réponse pour
optimization of ultrasound-assisted flavonoids extraction from the flower of Citrus l′ optimisation du blanchiment du son de blé par du peroxyde d′ hydrogène et son
aurantium L. var. amara Engl. Journal of Separation Science 33, 1349–1355. https:// incorporation dans une farine de pain. Université Laval, Québec, Canada. 〈http://h
doi.org/10.1002/jssc.200900776. dl.handle.net/20.500.11794/24876〉.
Yoram, G., Inbar, M., 2011. Distinct antimicrobial activities in aphid galls on Pistacia
atlantica. Plant Signaling & Behavior 6, 2008–2012. https://doi.org/10.4161/
psb.6.12.18031.

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