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Impact and Optimization of the Conditions of Extraction of Phenolic

Compounds and Antioxidant Activity of Olive Leaves (Moroccan picholine)
Using Response Surface Methodology

Article in Separations · May 2023

DOI: 10.3390/separations10060326

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 separations

Article
Impact and Optimization of the Conditions of Extraction of
Phenolic Compounds and Antioxidant Activity of Olive Leaves
(Moroccan picholine) Using Response Surface Methodology
El Mustapha El Adnany 1 , Najat Elhadiri 1 , Ayoub Mourjane 2 , Mourad Ouhammou 1, *, Nadia Hidar 1 ,
Abderrahim Jaouad 1 , Khalid Bitar 3 and Mostafa Mahrouz 1

1 Laboratory of Material Sciences and Process Optimization, Faculty of Sciences Semallaia, Cadi Ayyad
University, Marrakesh 40000, Morocco; [email protected] (E.M.E.A.);
[email protected] (N.E.); [email protected] (N.H.); [email protected] (A.J.);
[email protected] (M.M.)
2 Laboratory of Bioprocesses and Bio Interfaces, Sciences and Technologies Faculty, University Sultan Moulay
Slimane, Beni Mella 23000, Morocco; [email protected]
3 IRis COSmetologie, ZI Al-Massar, Marrakesh 40000, Morocco; [email protected]
* Correspondence: [email protected]

Abstract: The Moroccan picholine tree’s leaves contain phenolic compounds that benefit human health.
However, the amount and type of these compounds can vary based on factors such as the extrac-
tion method and conditions. This study aimed to improve phenolic compounds’ extraction while
minimising harmful chemicals’ use. It has been found that using ethanol as a solvent with ultra-
sonic extraction is the most effective and environmentally friendly technique. Several parameters,
such as the extraction time, solid/solvent ratio, and ethanol concentration as independent vari-
ables, were evaluated using a surface response method (RSM) based on the Box–Behnken design
(BBD) to optimize the extraction conditions. The experimental data were fitted to a second-order
Citation: El Adnany, E.M.; Elhadiri,
polynomial equation using multiple regression analysis and also examined using the appropri-
N.; Mourjane, A.; Ouhammou, M.; ate statistical methods. In optimal conditions, the ultrasonic time, the ratio (solvent/solid) and
Hidar, N.; Jaouad, A.; Bitar, K.; the concentration (ethanol/water), the content of total polyphenols (TPC), total flavonoids (TFC),
Mahrouz, M. Impact and and antioxidant activity (by DPPH, ABTS, FRAP) were, respectively, 74.45 ± 1.22 mg EAG/g DM,
Optimization of the Conditions of 17.08 ± 1.85 mg EC/g DM, 83.45 ± 0.89% 82.85 ± 1.52%, and 85.01 ± 2.35%. The identification of
Extraction of Phenolic Compounds phenolic compounds by chromatography coupled with mass spectrum (HPLC-MS) under optimal
and Antioxidant Activity of Olive conditions with two successive extractions showed the presence of hydroxytyrosol, catechin, caffeic
Leaves (Moroccan picholine) Using acid, vanillin, naringin, oleuropein, quercetin, and kaempferol at high concentrations.
Response Surface Methodology.
Separations 2023, 10, 326. https://
Keywords: olive leaves; extraction; optimization; ultrasound; polyphenols; flavonoids; antioxidant
doi.org/10.3390/separations10060326

Academic Editor: Faiyaz Shakeel

Received: 15 April 2023

1. Introduction
Revised: 17 May 2023
Accepted: 19 May 2023 The food and pharmaceutical industries are interested in agricultural wastes due
Published: 25 May 2023 to their high content of phenolic bioactive compounds, carbohydrates, oils, and other
biochemical molecules [1].
The olive tree is commonly found in the Mediterranean region and is widely spread
throughout Morocco, covering 65% of the national tree area. The regions of Fez-Meknes
Copyright: © 2023 by the authors. and Marrakech-Safi have the highest concentration of olive-growing areas, covering 54%
Licensee MDPI, Basel, Switzerland. of the total area and meeting 19% of the demand for edible oils. The olive transformation
This article is an open access article by-products, including the skin, pulp, pits, and leaves, have caught the attention of the
distributed under the terms and
food and pharmaceutical industries due to the presence of phenolic compounds.
conditions of the Creative Commons
Studies have shown that phenolic compounds found in olive tree leaves have benefi-
Attribution (CC BY) license (https://
cial properties, such as antioxidants [2], anticancer, antimicrobial [3], and hypolipidemic
creativecommons.org/licenses/by/
activities [4]. However, the amount of phenolic compounds present can vary based on
4.0/).

Separations 2023, 10, 326. https://doi.org/10.3390/separations10060326 https://www.mdpi.com/journal/separations

Separations 2023, 10, 326 2 of 17

climate, moisture, plant age and variety [5], and extraction methods [6]. Traditional extrac-
tion methods, such as maceration and Soxhlet extraction, are slow and yield low amounts
of bioactive products [1]. The ultrasonic method, a newer extraction technique, has been
developed to efficiently extract organic bioactive compounds from plants [7]. This step is
critical in the production of bioactive.
Ultrasonic-assisted extraction (UAE) is considered the greenest extraction process
compared to microwave-assisted extraction (MAE), meeting the requirements of the green
extraction method [8] as it reduces the temperature, time, and solvent usage [9–11]. This
method has been widely used to extract valuable bioactive compounds from various
plant materials. One of the food and pharmaceutical industries’ dilemmas is improving
extraction efficiency while reducing costs, which can be achieved by optimizing extraction
conditions [12]. In addition, this technique is usually performed to study some independent
factors, requiring more experiments, leading to increased cost and time [13].
Response surface methodology (RSM) is a powerful statistical tool that optimizes
complex processes. It has gained popularity for its effectiveness in extracting methods,
identifying optimal variable combinations, and simplifying experiment interpretation.
This tool has been widely used in various fields [14]. The objective of this study was
to examine the effect of certain independent factors of extraction (time, solid/solvent
ratio, ethanol (%)) of bioactive compounds (TPC, TFC) and antioxidant activity (DPPH,
ABTS, FRAP) with the ultrasonic-assisted extraction (UAE) method using response surface
methodology (RSM).

2. Materials and Methods

2.1. Preparation of the Powder
Olive leaves of the Moroccan picholine variety were harvested in Marrakech, Morocco.
The leaves were rinsed with water and dried in a ventilated oven (OVEN 19L DRYING AND
STERILIZATION DIGITHEAT J.P.SELECTA) with a thickness of 1 cm at 80 ◦ C (according
to previous studies [15]) for 5 h (stable weight), then ground using a propeller mill (Mill
Grinder For Spices And Professional Coffee, 1 kg). The leaf powder obtained was sieved
(digital vibrating laboratory analysis sieve/GKM Siebtechnik GmbH) into four fractions
(>125 µm, (125 µm; 50 µm), (50 µm, 25 µm), and <25 µm). The particle size was set at
25–50 µm. The leaf powder was stored at 4 ◦ C in plastic bags.

2.2. Chemicals
The reagents used were pure ethanol, methanol (HPLC grade), Folin–Ciocalteu’s,
Sodium carbonate (Na2 CO3 ), 2,2-diphenyl-1-picrylhydrazyl (DPPH), 2,20 -azino-bis
(3-ethylbenzothiazoline-6-sulphonic acid) (ABTS), aluminum trichloride (AlCl3 ), potassium
persulfate (K2 S2 O8 ), tripyridyltriazine complex (TPTZ), sodium acetate buffer (C2 H3 NaO2
3H2 O and C2 H4 O2 ), and hydrochloric acid (HCl).

2.3. Experimental Design and Statistical Analysis

The Box–Behnken design was used to determine the best combination of extraction
variables for organic bioactive compounds based on the results of the preliminary single-
factor test. Different variables such as extraction time and temperature, sample/solvent
ratio, solvent percentage, and pH influence the determination of the content of phyto-
chemicals [16]. Extraction time (min, X1), sample/solvent ratio (g/mL, X2), and ethanol
concentration (v/v, X3) were chosen as independent variables, and their coded and uncoded
(real) levels of independent variables are shown in Table 1.
Separations 2023, 10, 326 3 of 17

Table 1. Coded and actual values for Box–Behnken design (BBD).

Level
Code Symbols Independent Variables
−1 0 +1
X1 Time (min) 30 45 60
X2 Ratio (mL/g) 5 12.5 20
X3 Ethanol (%) 20 60 100

The variation of the response values (Y), with respect to the three variables was fitted
into a response surface model and presented in the form of the second-order polynomial
equation, is as follows:
k k k
Yi = β0 + ∑i=1 βiXi + ∑i=1 βiiXi2 + ∑ j=1 ∑<i=2 βijXiXj + ε,

where Yi are the experiment responses; β0 represents the theoretical mean value of the
response; βi, βj are the coefficients of the linear terms; βii, are the coefficients of the
quadratic terms; βij are the coefficients of the interaction terms, and ε the error term.

2.4. Ultrasound-Assisted Extraction of Bioactive Compounds

Extraction with organic solvents has economic and environmental drawbacks. The
“green chemistry” concept encourages developing and using less hazardous processes and
materials without reducing efficiency [17]. Consequently, solvent extraction of bioactive
compounds must be optimized for maximum response using fewer organic solvents. Thus,
water was chosen to be studied in combination with ethanol. Ethanol was chosen instead
of methanol as the extraction solvent due to the high toxicity of methanol in the human
body [18]. Ethanol has the highest affinity for phenolic compounds; therefore, it the first
choice for extracting phenolic compounds from fruit and vegetable wastes [19]. We mixed
1 g of olive leaf powder with ethanol/water. The extraction process was performed using
a typical ultrasonic apparatus (Heating cleaning bath “Ultrasound HD”-Model 3000866),
and the extract was filtered to collect the supernatant. A UV-T80 spectrophotometer was
used to analyse the polyphenols, flavonoids, and total antioxidant activity in the samples.

2.5. Total Phenolic Content (TPC) and Total Flavonoid Content (TFC)
The determination of total polyphenols by the method using the Folin–Ciocalteu
reagent is described by Singleton et al. [20]. A total of 0.25 mL of leaf extract was mixed
with 0.25 mL of Folin–Ciocalteu and 2 mL of distilled water; the mixture was vortexed.
After 3 min, 0.25 mL of sodium carbonate (20%) was added; the mixture was stirred and
then incubated for 30 min in the dark at room temperature. The absorbance was measured
at 750 nm using a UV/VIS spectrophotometer “T80-PG Instruments. The calibration curve
for gallic acid was performed, and the results are expressed as mg gallic acid per g dry
matter (mg GAE/g DW).
Flavonoid content was determined based on the formation of a flavonoid–aluminum
complex that absorbs at 430 nm. The flavonoid assay was performed according to the
protocol described by Djeridane et al. (2006) [21]. A total of 1.5 mL of the olive leaf extract
was added with 1.5 mL of aluminum trichloride (AlCl3: 2%). After 30 min incubation
at room temperature, the absorbance of the reaction mixture was read at 430 nm using a
UV/VIS spectrophotometer (T80-PG Instruments). The flavonoid content in the extracts
was calculated by reference to a calibration curve established with catechin. Results are
expressed as mg catechin equivalent per 1 g dry matter (mg EC/g DW).

2.6. In Vitro Antioxidant Activity

2.6.1. DPPH Radical Reduction Test
For the anti-radical activity of the different extracts of the leaves dried at different
temperatures, we used the method based on DPPH (1,1-diphenyl-2-picrylhydrazyl) as a
Separations 2023, 10, 326 4 of 17

relatively stable radical, according to the protocol described by Abdel Hameed et al. [22].
Briefly, 1 mL of leaf extract was added to 1 mL of DPPH solution (prepared by solubi-
lizing 4 mg of DPPH in 100 mL of ethanol). The mixtures were incubated in the dark
for 30 min at room temperature. The decolorization compared to the negative control
containing only DPPH solution measured at 517 nm using a UV/visible spectropho-
tometer type T80. The radical-scavenging activity of DPPH was calculated as follows:
%(AA) = ((A517 control − A517 sample)/A517 control) × 100. A517 control is the ab-
sorbance of DPPH solution (without sample extract), and A517 sample is the absorbance of
the sample with DPPH solution.

2.6.2. ABTS Radical Test

The ABTS•+ radical cation decolorization test also evaluated the anti-radical activity
according to the method used by Aadesariya et al. [23]. The ABTS+- radical was generated
by the reaction of 7 mM ABTS+ and 2.45 mM potassium persulfate. An equal mixture
volume was incubated in the dark for 12–16 h. The ABTS+-solution was diluted with
methanol to an absorbance of 0.700 ± 0.02 at 734 nm before use. Then, 1 mL of ABTS+-
solution was mixed with 10 µL of leaf extract. The mixture was incubated for 30 min at
30 ◦ C, and the absorbance was measured at 734 nm. The radical scavenging activity was
expressed as the percentage of free radical inhibition by the sample and was calculated by
the following formula:

ABTS scavenged (%) = [(A734 control − A734 sample)/A517 control] × 100.

A734 control is the absorbance of the control reaction, and A 734 test is the absorbance
in the presence of the sample extracts.

2.6.3. Ferric Reducing Antioxidant Power (FRAP) Test

The FRAP (ferric reducing antioxidant power) method is based on the reduction
of ferric ions (Fe3+ ) to ferrous ions (Fe2+ ). This method evaluates the declining power
of compounds at low pH [24]. The ferrous tripyridyltriazine (TPTZ) complex has an
intense blue color measured by a spectrophotometer at 593 nm. The FRAP assay was
performed according to the protocol of [25]. The FRAP reagent was prepared by mixing
300 mM sodium acetate buffer (3.1 g C2 H3 NaO2 3H2 O and 16 mL C2 H4 O2 ), pH: 3.6;
10 mM solution of TPTZ in 40 mM HCl; and 20 mM FeCl3 at a ratio of 10:01:01 (v/v/v).
One hundred microliters of each extract were added to 3 mL of FRAP reagent and 300 µL of
H2 O. After incubation at 37 ◦ C for 30 min; the absorbance was measured at 593 nm against
the blank [26].

2.7. Model Verification

The extraction conditions were numerically optimized for maximum TPC and TFC
content with high antioxidant activities based on regression analysis and 3D surface curves
of independent variables. Responses were determined according to the recommended
extraction conditions.

2.8. Qualitative and Quantitative Analysis by HPLC-MS

Identification and quantification of phenolic compounds by HPLC-MS were performed
according to the method used by Puigventos et al. (2015) [27]. The injection volume of each
sample was 10 µL with separation between solvent A (0.1% aqueous formic acid solution)
and solvent B (methanol) as follows: 0–3 min, linear gradient from 5 to 25% B; 3–6 min,
at 25% B; 6–9 min, from 25 to 37% B; 9–13 min, at 37% B; 13–18 min, from 37 to 54% B;
18–22 min, at 54% B; 22–26 min, from 54 to 95% B; 26–29 min, at 95% B; 29–29. 15 min, back
to initial conditions at 5% B; and 29.15 to 36 min, at 5% B. The mobile phase flow rate was
1 mL/min. Ion transfer tube temperature was set at 350◦ and the full scan MS acquisition
mode to m/z 50–1000. The polyphenolic compounds were obtained at 31 min.
Separations 2023, 10, 326 5 of 17

2.9. Statistical Analysis

Analysis of variance (ANOVA) and multiple regression analysis were performed to fit
the mathematical model using Design Expert 13 software. Significant terms (p < 0.05) in the
model for each response were found by analysis of variance, and significance was judged by
the F statistic calculated from the data. The experimental data were evaluated with various
descriptive statistical analyses such as p-value, F-value, sum of squares (SS), degrees of
freedom (DF), coefficient variation (CV), the mean sum of squares (MSS), coefficient of
determination (R2), and adjusted coefficient determination (Radj2); this is to obtain the
statistical significance of the developed quadratic mathematical model.

3. Results and Discussion

3.1. Evaluation and Optimization of Extraction Conditions
This study determined the relationship between response functions and process vari-
ables using a three-factor based on the Box–Behnken (BBD) design. The goal was to
optimize the extraction conditions for bioactive compounds such as total polyphenols
(TPC), total flavonoids (TFC), their corresponding antioxidant activities. Similar scientific
studies were used as a reference. [28].
The outcomes of the conducted responses are reported in Table 2. The total polyphenol
(TPC) content ranged from 48.69 to 72.98 mg EAG/g DM. The total flavonoid (TFC) content
ranged from 10.45 to 16.36 mg EC/g DM. The entire content of TPC and TFC was obtained
for trials 13, 14, and 15 under the experimental conditions of X1 = 45 min; X2 = 12.5 mL/g;
and X3 = 60%. Regarding DPPH radical scavenging capacity, ABTS and FRAP ranged
from 70.95% to 85.69%, 74.67 to 85.41%, and 71.92 to 86.95%, respectively. The highest
antioxidant activity was obtained for tests 2 and 13 under X1 = 60 min; X2 = 5 mL/g;
X3 = 60% and X1 = 45 min; X2 = 5 mL/g; and X3 = 60%, respectively. Based on these data,
the extraction process was optimized to achieve the maximum desirable response.

Table 2. The experimental run from Box–Behnken design (BBD).

TPC
TFC DPPH ABTS FRAP
Time Ratio Concentration (mg EAG/g
N◦ Exp (mg EC/g DM; Y2) (%; Y3) (%; Y4) (%; Y5)
(min; X1 ) (mL/g; X2 ) (%; X3 ) DM; Y1)
Reel Predicts Reel Predicts Reel Predicts Reel Predicts Reel Predicts
1 30(−1) 5(−1) 60(0) 59.23 58.65 11.12 11.14 76.12 74.97 76.34 76.89 73.65 72.21
2 60(1) 5(−1) 60(0) 66.36 63.65 13.96 14.05 85.69 84.59 85.41 84.05 76.55 79.15
3 30(−1) 20(1) 60(0) 52.98 55.69 11.85 11.77 76.25 77.35 79.69 81.05 82.65 80.05
4 60(1) 20(1) 60(0) 53.31 53.90 11.96 11.95 75.36 76.51 76.23 75.68 73.76 75.20
5 30(−1) 12.5(0) 20(−1) 56.96 56.02 11.08 10.88 77.36 76.46 75.66 74.58 72.36 74.92
6 60(1) 12.5(0) 20(−1) 55.25 56.43 12.15 11.88 78.85 81.84 79.84 81.22 76.02 74.53
7 30(−1) 12.5(0) 100(1) 48.69 47.51 10.69 10.96 70.95 80.99 83.46 82.08 74.12 75.61
8 60(1) 12.5(0) 100(1) 49.36 50.31 12.85 13.05 78.36 79.26 77.24 77.77 80.65 78.09
9 45(0) 5(−1) 20(−1) 60.26 61.79 10.96 11.15 75.28 77.34 76.08 76.06 75.14 74.03
10 45(0) 20(1) 20(−1) 56.45 54.69 10.45 10.74 76.52 76.33 74.67 73.84 71.92 71.96
11 45(0) 5(−1) 100(1) 51.96 50.31 12.39 12.10 77.39 79.58 76.87 77.70 72.18 72.14
12 45(0) 20(1) 100(1) 49.65 48.12 11.23 11.04 74.96 72.90 75.69 75.71 76.98 78.10
13 45(0) 12.5(0) 60(0) 72.98 72.41 15.99 16.34 82.25 81.65 82.96 82.65 86.95 86.28
14 45(0) 12.5(0) 60(0) 72.26 72.41 16.98 16.34 81.12 81.65 83.23 82.65 86.45 86.28
15 45(0) 12.5(0) 60(0) 71.99 72.41 16.06 16.34 81.59 76.99 81.75 82.65 85.45 86.28

Pearson’s test showed a strong positive correlation, r = 0.8- 0.85, between TPC and
TFC and between TFC and FRAP. This implies that these answers evolve proportionally.
The positive average correlations r = 0.5–0.75 appear between the other answers.

3.2. Fitting the Model and Analysis of Variance

The extraction process was optimised by applying the second-order polynomial model
fit. The results are presented in Table 3. The model shows a high significance level and
a good fit, with the experimental data of TPC and TFC contents showing less variation
around the mean (R2 values 0.969 and 0.991), respectively.
Separations 2023, 10, 326 6 of 17

Table 3. Experimental design of the surface response and statistical table of results.

Sum of Estimation of Degree of Medium

Source Value F Value p Remarks
Squares Coefficients Freedom Square
TPC
Model 990.43 9 11005 17.53 0.0028 significant
Intercept, X0 72.41 *
Linear
X1 5.15 0.8025 1 5.15 0.8209 0.4065
X2 80.77 −3.18 * 1 80.77 12.87 0.0157
X3 107.02 −3.66 * 1 107.02 17.05 0.0091
Interaction
X1 X2 11.56 −1.70 1 11.56 1.84 0.2328
X1 X3 1.42 0.5950 1 1.42 0.2256 0.6548
X2 X3 0.5625 0.3750 1 0.5625 0.0896 0.7767
Quadratic
X2 1 249.94 −8.23 * 1 249.94 39.83 0.0015
X2 2 142.51 −6.21 * 1 142.51 22.71 0.0050
X2 3 498.34 −11.62 * 1 498.34 79.40 0.0003
Residual 31.38 5 6.28
Lack of fit 30.86 3 10.29 39.27 0.0249 significant
Error 0.5238 2 0.2619
Total 1021.81 14
Accuracy
12.17
Adequacy
CV% 4.28
R2 0.969
R2Ajust 0.91
Average 58.51
TFC
Model 61.99 9 6.89 31.55 0.0007 significant
Intercept, X0 16.34 *
Linear
X1 4.77 0.7725 * 1 4.77 21.87 0.0055
X2 1.08 −0.3675 1 1.08 4.95 0.0767
X3 0.7938 0.3150 1 0.7938 3.64 0.1148
Interaction
X1 X2 1.86 −0.6825 * 1 1.86 8.54 0.0330
X1 X3 0.2970 0.2725 1 0.2970 1.36 0.2960
X2 X3 0.1056 −0.1625 1 0.1056 0.4838 0.5177
Quadratic
X2 1 12.54 −1.84 * 1 12.54 57.44 0.0006
X2 2 19.16 −2.28 * 1 19.16 87.76 0.0002
X2 3 29.11 −2.81 * 1 29.11 133.35 <0.0001
Residual 1.09 5 0.2183
Lack of fit 0.4811 3 0.1604 0.5253 0.7074 Not significant
Error 0.6105 2 0.3052
Total 63.08 14
Accuracy
14.69
Adequacy
CV% 3.69
R2 0.98
R2Ajust 0.95
Average 12.65
DPPH
Model 165.56 9 18.40 5.19 0.0423 significant
Intercept, X0 81.65 *
Linear
X1 38.63 2.20 * 1 38.63 10.89 0.0215
X2 16.22 −1.42 1 16.22 4.57 0.0855
X3 5.04 −0.7937 1 5.04 1.42 0.2867
Interaction
X1 X2 27.35 −2.61 * 1 27.35 7.71 0.0390
X1 X3 8.76 1.48 1 8.76 2.47 0.1768
X2 X3 3.37 −0.9175 1 3.37 0.9493 0.3747
Quadratic
X2 1 8.06 −1.48 1 8.06 2.27 0.1920
X2 2 12.24 −1.82 1 12.24 3.45 0.1224
Separations 2023, 10, 326 7 of 17

Table 3. Cont.

Sum of Estimation of Degree of Medium

Source Value F Value p Remarks
Squares Coefficients Freedom Square
X2 3 53.19 −3.80 * 1 53.19 14.99 0.0117
Residual 17.74 5 3.55
Lack of fit 17.09 3 5.70 17.68 0.0540 Not significant
Error 0.6445 2 0.3222
Total 183.30 14
Accuracy
8.2473
Adequacy
CV% 2.42
R2 0.9032
R2Ajust 0.73
Average 77.87
ABTS
Model 163.76 9 18.20 8.05 0.0168 significant
Intercept, X0 82.65 *
Linear
X1 1.59 0.4462 1 1.59 0.7045 0.4395
X2 8.86 −1.05 1 8.86 3.92 0.1046
X3 6.14 0.8762 1 6.14 2.72 0.1602
Interaction
X1 X2 39.25 −3.13 * 1 39.25 17.36 0.0088
X1 X3 27.04 −2.60 * 1 27.04 11.96 0.0181
X2 X3 0.0132 0.0575 1 0.0132 0.0058 0.9420
Quadratic
X2 1 0.0000 −0.0033 1 0.0000 0.0000 0.9968
X2 2 38.42 −3.23 * 1 38.42 16.99 0.0092
X2 3 47.68 −3.59 * 1 47.68 21.08 0.0059
Residual 11.31 5 2.26
Lack of fit 10.06 3 3.35 5.40 0.1602 Not significant
Error 1.24 2 0.6212
Total 175.07 14
Accuracy
8.31
Adequacy
CV% 1.90
R2 0.94
R2Ajust 0.82
Average 79.01
FRAP
Model 364.85 9 40.54 5.21 0.0418 significant
Intercept, X0 86.28 *
Linear
X1 2.20 0.5250 1 2.20 0.2836 0.6171
X2 7.59 0.9737 1 7.59 0.9757 0.3686
X3 9.01 1.06 1 9.01 1.16 0.3309
Interaction 0.5250
X1 X2 34.75 −2.95 1 34.75 4.47 0.0881
X1 X3 2.06 0.7175 1 2.06 0.2649 0.6287
X2 X3 16.08 2.00 1 16.08 2.07 0.2099
Quadratic
X2 1 98.21 −3.95 * 1 57.58 7.41 0.0417
X2 2 275.63 −5.68 * 1 119.19 15.33 0.0112
X2 3 101.96 −6.55 * 1 158.25 20.36 0.0063
Residual 57.58 5 7.77
Lack of fit 119.19 3 12.57 21.55 0.0447 significant
Error 158.25 2 0.5833
Total 38.87 14
Accuracy
6.29
Adequacy
CV% 3.59
R2 0.90
R2Ajust 0.73
Average 77.79
* Significant (p < 0.05).
Separations 2023, 10, 326 8 of 17

The antioxidant activity (DPPH, ABTS, and FRAP) shows that the model is significant,
and the polynomial equation fit using the coefficient of determination (R2) 0.90, 0.93, and
0.90, respectively. The regression coefficients for the dependent variables were obtained by
Separations 2023, 10, x FOR PEER REVIEW
multiple linear regressions, as shown in Table 3. 9 of 19

 The linear effect of extraction time (X1 ) was significant for TFC and DPPH;
 The solvent/solid ratio (X2 ) was significant for TPC;
  The The X1 X2 interaction
concentration effect
(X3 ) was also significantly
significant for the TPC;impacted TFC, DPPH, and ABTS;
  The The X1 X3 interaction
quadratic was significant
effect of solvent for ABTS.
concentration (X3 ) and ratio (X2 ) was significant for all
responses except DPPH;
The ANOVA result for each variable response indicates that at least one of the model
The X1 X2can
 parameters interaction effect
explain the also significantly
experimental impacted
variation TFC, DPPH,
of the response and ABTS;
variables (Table 3).
 The X X interaction
The1corresponding
3 was significant for ABTS.
variables would be more significant if the F-value becomes larger
andThetheANOVA
p-valueresult
becomes smaller
for each [29]. response
variable The p-value < 0.05that
indicates showed that
at least onethe
of model terms
the model
were significant. In terms of coefficients of variation (CV), the
parameters can explain the experimental variation of the response variables (Table 3).models recorded a CV for
CPT,
TheCFT, DPPH, ABTS,
corresponding and FRAP
variables would of be
4.24%,
more3.69%, 2.42%,if1.9%,
significant and 3.59%,
the F-value respectively.
becomes larger
and theGenerally, the acceptable
p-value becomes smallercoefficient
[29]. The of variation
p-value (CV)
< 0.05 value should
showed that thebemodel
less than 20%.
terms
Thesignificant.
were diagnosticIndiagram
terms ofascoefficients
the predicted versus actual
of variation (CV), the values (Figure
models 1) evaluates
recorded a CV forthe
relationship
CPT, CFT, DPPH, and model
ABTS, andsatisfaction
FRAP of 4.24%, between
3.69%, the experimental
2.42%, 1.9%, and and 3.59%,predicted values
respectively.
obtained from the developed models. From Figure 1, it is observed
Generally, the acceptable coefficient of variation (CV) value should be less than 20%. that the data points
Theare located near
diagnostic the straight
diagram line, which
as the predicted means
versus a high
actual correlation
values (Figure 1) between
evaluatesexperimental
the rela-
and predicted
tionship and model datasatisfaction
obtained for TFC and
between thea experimental
medium correlation betweenvalues
and predicted experimental
obtainedand
predicted
from data ofmodels.
the developed TPC and Fromantioxidant
Figure 1, itactivity (DPPH,
is observed that ABTS,
the dataand FRAP)
points from the
are located
near the straight line, which means a high correlation between experimental and predicted
models.
data obtained for TFC and a medium correlation between experimental and predicted data
of TPC and antioxidant activity (DPPH, ABTS, and FRAP) from the models.

Predicted vs. Actual Predicted vs. Actual

TPC TFC
75 17

Color points by value of Color points by value of

TPC: TFC:
16

48,69 72,98 70 10,45 16,98

15

65

14
Predicted
Predicted

60

13

55
12

50 11

10
45

10 11 12 13 14 15 16 17
45 50 55 60 65 70 75

Actual
Actual

Figure 1. Cont.
 Separations 2023, 10, x FOR PEER REVIEW 10 of 19

Separations 2023, 10, 326 9 of 17

Predicted vs. Actual Predicted vs. Actual

DPPH FRAP
90 90

Color points by value of Color points by value of

DPPH: FRAP:
70,95 85,69 71,92 86,95

85 85
Predicted

Predicted
80 80

75 75

70 70

70 75 80 85 90 70 75 80 85 90

Actual Actual

Predicted vs. Actual

ABTS
86

Color points by value of

ABTS:
84
74,67 85,41

82

80
Predicted

78

76

74

72

72 74 76 78 80 82 84 86

Actual

Figure
Figure 1. 1.Diagnosis
Diagnosisbetween
betweenexperimental
experimental and
and predicted
predicted values
values for
for TPC,
TPC,TFC,
TFC,DPPH,
DPPH,ABTS,
ABTS,and
FRAP.
and FRAP.

3.3.3.3.
Development
Development of Second Order
of Second Polynomial
Order PolynomialModels
Models
A statistical analysis
A statistical was
analysis conducted
was conducted using a second-order
using a second-order polynomial
polynomialequation
equationwith
with
interaction
interaction terms to model
terms the connection
to model betweenbetween
the connection three process
threevariables
processand the efficiency
variables and the
of ultrasonic
efficiency extraction.
of ultrasonic This model can
extraction. be utilized
This to anticipate
model can be utilizedthe
toefficiency
anticipateofthe
ultrasonic
efficiency
extraction for varying combinations of process variables.
of ultrasonic extraction for varying combinations of process variables.
Five models
Five modelswere developed
were developedfromfrom
this study to have
this study to the
haveultrasound extraction
the ultrasound effi-
extraction
ciency of TPC,ofTFC,
efficiency TPC,DPPH,
TFC, ABTS,
DPPH,and FRAPand
ABTS, from olive from
FRAP leaves.olive
The leaves.
second-order equations
The second-order
of the responses
equations in terms
of the of coded
responses factors
in terms are given
of coded below:
factors are given below:
2 − 6.21X22 − 211.62X233 2
YTPC =Y72.41 − 3.18X
TPC = 72.41 2 −2 3.66X
− 3.18X 3 3−
− 3.66X 8.23X211
− 8.23X 11 − 6.21X 2 − 11.62X 33

YTFC =Y16.34 + 0.77X1 − 0.68X1 X2 − 1.84X2 11 − 2.28X2 22 − 2.81X2 33

TFC = 16.34 + 0.77X1 − 0.68X1X2 − 1.84X211 − 2.28X222 − 2.81X233

YDPPH = 81.65 + 2.2X1 − 2.61X1 X2 − 3.8X2 3

YDPPH = 81.65X+ 2.2X
− 3.13X 1 − 2.61X1X2 − 3.8X 23 2 2
YABTS = 82.65 1 2 − 2.60X1 X3 − 3.23X 2 − 3.59X 33

Y FRAP = 86.28 − 3.95X2 − 5.68X2 − 6.55X 2 .

1 2 3
YABTS = 82.65 − 3.13X1X2 − 2.60X1X3 − 3.23X22 − 3.59X233

3.4. Effect of Process Variables

YFRAP = 86.28 − 3.95X 1 − 5.68X 2 − 6.55X3 . 2 2 2
This study analyzed the impact of process variables on the extraction of bioactive
compounds (TPC, TFC, and free radical antioxidants) from olive leaves using a two-level
Box–Behnken design with three factors: extraction time, solid/solvent ratio, and ethanol
concentration. The results were presented using a three-dimensional response surface,
Separations 2023, 10, 326 10 of 17

Separations 2023, 10, x FOR PEER REVIEW 11 of 18

demonstrating the relationship between the independent and dependent variables. By

holding two factors constant and varying the third, the response surface curves display
the main effects
particularly and interactions
impactful of the
for flavonoids andindependent
DPPH. The variables on/with
linear effect the dependent
of extraction time was
variables
significant [1].
(p <These
0.05) graphs
for theseprovide insight but
two variables intonot
how forthe
thedifferent variables affect
other independent the
variables.
extraction
This effectprocess.
is likely due to the extended time the plant matrix is exposed to the solvent,
improving the solubility of the leaves’ constituents. Essentially, the duration of the solvent
3.4.1. Effectfacilitates
exposure of Extraction Time
the migration of chemical compounds into the solution [30].
Based
Studies onhave
the results
shownobtained,
that the alonger
longerthecontact time between
extraction time, the thehigher
sample and
the solvent
content of
leads to a higher
polyphenols andtransfer rate of bioactive
flavonoids. However,compounds, ultimately
it is essential to noteresulting in better extrac-
that excessively long
tion efficiency.
extraction times Figure 2 illustrates
can lead that the extraction
to the degradation time of 30–45
of the bioactive min was
compounds, asparticularly
indicated in
impactful
[31]. On the other hand, some studies found that extraction time was not asignificant
for flavonoids and DPPH. The linear effect of extraction time was significant
(pfactor
< 0.05)infor
thethese two variables butextraction
ultrasound‐assisted not for theofother independent
phenolic compounds variables.
[32,33].ThisIn effect
other
isstudies,
likely due to the extended time the plant matrix is exposed to the solvent,
such as those on Genipap berry pulp, blueberries, and carob pulp, the extraction improving
the
timesolubility of thecompounds
for bioactive leaves’ constituents. Essentially, the duration
using ultrasonic‐assisted extraction of(UAE)
the solvent exposure
was around 49,
facilitates the migration of chemical
50, and 57 min, respectively [34–36]. compounds into the solution [30].

Figure2.2.Response
Figure Responsesurface
surfaceplot
plotshowing
showingthe
thevariation
variationofofresponses
responsesasasaafunction
functionofofextraction
extractiontime.
time.

3.4.2.Studies have
Effect of shown that
Solid–Liquid the longer the extraction time, the higher the content of
Ratio
polyphenols and flavonoids. However,
The amount of solvent used in it isorganic
essentialbioactive
to note that excessivelyand
compounds longantioxidant
extraction
times
extraction is a crucial factor. When evaluating its impact on the extraction of [31].
can lead to the degradation of the bioactive compounds, as indicated in On
phenolic
the other hand,the
compounds, some studies
results, found that
presented extraction
in Figure time was
3, indicate notincreasing
that a significant
thefactor in the
solid‐liquid
ultrasound-assisted extraction of phenolic compounds [32,33]. In other
ratio up to 12.5 mL/g resulted in an increase in polyphenol and flavonoid content andstudies, such as
those on Genipap berry pulp, blueberries, and carob pulp, the extraction time for bioactive
antioxidant activity. This indicates that the volume of solvent used plays a significant role
compounds using ultrasonic-assisted extraction (UAE) was around 49, 50, and 57 min,
in achieving good infusion and easy release of bioactive compounds into the surrounding
respectively [34–36].
environment [37]. However, according to Zakaria Fazila (2021) [38], a high solvent‐to‐
solid ratio (between
3.4.2. Effect 10–30 Ratio
of Solid–Liquid mL/g) can lead to a decrease in phenolic compound content.
The most effective ratio for maximum phenolic compound extraction was found to be 10
The amount of solvent used in organic bioactive compounds and antioxidant extraction
mL/g.
is a crucial factor. When evaluating its impact on the extraction of phenolic compounds,
This research found that once the solid–liquid ratio surpasses 12.5 mL/g, the solution
the results, presented in Figure 3, indicate that increasing the solid-liquid ratio up to
becomes oversaturated with solute. This can lead to a reduction in the rate of mass transfer
12.5 mL/g resulted in an increase in polyphenol and flavonoid content and antioxi-
and a hindrance in the penetration of organic bioactive compounds into the solution, ul‐
dant activity. This indicates that the volume of solvent used plays a significant role in
timately resulting in a decrease in the yield of the extraction process.
Separations 2023, 10, 326 11 of 17

achieving good infusion and easy release of bioactive compounds into the surrounding
environment
Separations 2023, 10, x FOR PEER REVIEW [37]. However, according to Zakaria Fazila (2021) [38], a high solvent-to-solid
12 of 18
ratio (between 10–30 mL/g) can lead to a decrease in phenolic compound content. The
most effective ratio for maximum phenolic compound extraction was found to be 10 mL/g.

Figure 3. Response
Responsesurface plot
surface showing
plot the the
showing variation of responses
variation as a function
of responses of solid/solvent
as a function of solid/
ratio. ratio.
solvent

3.4.3.This
Effect of Solvent
research Concentration
found that once the solid–liquid ratio surpasses 12.5 mL/g, the solution
becomes oversaturated with
The solubility of organic solute. This can
bioactive lead to a reduction
compounds in the rate of
can be enhanced bymass transfer
varying the
and a hindrance in the penetration of organic bioactive compounds into
concentration of the solvent [39]. Olive leaves were extracted using different ethanolthe solution,
ultimately resulting
concentrations at noin a decrease
more than 50in°Cthe
foryield of the
60 min extraction
under process.
ultrasonic conditions. The results,
shown in Figure 4, indicate that the samples containing 60% ethanol had the highest TPC,
3.4.3. Effect of Solvent Concentration
TFC, DPPH, ABTS, and FRAP values. This is because the solubility of polyphenol
The solubility
compounds of organic
increases bioactive compounds
with increasing can be enhanced
ethanol concentrations. This by
mayvarying thewhy
explain concen-
the
tration of the solvent [39]. Olive leaves were extracted using different ethanol concentrations
presence of water ◦in ethanol improves the swelling of the plant material, while ethanol
at no more
disrupts than
the 50 C for
binding 60 minthe
between under ultrasonic
solute and theconditions. Thestudies
plant. Other results,[40,41]
shownhave
in Figure
found4,
indicate that the samples containing 60% ethanol had the highest TPC, TFC,
that the recovery of organic bioactive compounds increased and peaked at an ethanol DPPH, ABTS,
and FRAP values. This is because the solubility of polyphenol compounds increases with
concentration of around 70% before slightly decreasing. Studies have shown that
increasing ethanol concentrations. This may explain why the presence of water in ethanol
increasing the ethanol concentration can enhance the yield of phenolic compounds up to
improves the swelling of the plant material, while ethanol disrupts the binding between the
an average concentration of 40% ethanol [42]; however, Caldas et al. [43] observed that
solute and the plant. Other studies [40,41] have found that the recovery of organic bioactive
the highest phenolic compound content was achieved at an average concentration of 60%
compounds increased and peaked at an ethanol concentration of around 70% before slightly
ethanol, possibly due to the different polarities of organic bioactive compounds in grape
decreasing. Studies have shown that increasing the ethanol concentration can enhance the
skin. For Triticum aestivum, the maximum yield of phenolic compounds was obtained
yield of phenolic compounds up to an average concentration of 40% ethanol [42]; however,
using an ethanol concentration of 56% [44]. Similarly, studies on various plants such as
Caldas et al. [43] observed that the highest phenolic compound content was achieved at an
Jaboticaba bark, blueberry, and apple pulp have found that the concentration of ethanol
average concentration of 60% ethanol, possibly due to the different polarities of organic
required to extract the maximum amount of phenolic compounds ranges from 40–80%
bioactive compounds in grape skin. For Triticum aestivum, the maximum yield of phenolic
(V/V) [35–46].
compounds was obtained using an ethanol concentration of 56% [44]. Similarly, studies
on various plants such as Jaboticaba bark, blueberry, and apple pulp have found that the
concentration of ethanol required to extsract the maximum amount of phenolic compounds
ranges from 40–80% (V/V) [35–46].
Separations 2023,
Separations 2023, 10,
10, 326
x FOR PEER REVIEW 13
12 of 18
of 17

responses with
Figure 4. Response surface plot showing the variation of responses with ethanol
ethanol concentration.
concentration.

3.5.
3.5. Determination
Determination andand Validation
Validation of
of Optimal
Optimal Conditions
Conditions
This study aimed to determine
This study aimed to determine the bestthe best experimental
experimentalconditions for extracting
conditions phe-
for extracting
nolic compounds that benefit human health, such as antioxidant activity.
phenolic compounds that benefit human health, such as antioxidant activity. This This involves
determining the ideal sonication
involves determining the idealtime, solid/liquid
sonication time,ratio, and ethanol
solid/liquid concentration
ratio, and ethanol for
maximum extraction yield. The software Design Expert Version 13 was
concentration for maximum extraction yield. The software Design Expert Version 13 was used to find the
composite
used to find optimum for achieving
the composite optimumthe highest bioactivethe
for achieving compounds extraction
highest bioactive yield and
compounds
the most significant antioxidant activity.
extraction yield and the most significant antioxidant activity.
The optimization process involved assigning the response’s desirability values be-
The optimization process involved assigning the response’s desirability values
tween 0 and 1. Figure 5 displays a desirability value of 0.8735 and the predicted optimal
between 0 and 1. Figure 5 displays a desirability value of 0.8735 and the predicted optimal
values. Experiments were conducted under optimal triplicate conditions to compare the
values. Experiments were conducted under optimal triplicate conditions to compare the
experimental and predicted values of the responses. The mean values are presented in
experimental and predicted values of the responses. The mean values are presented in
Table 4. The optimal conditions for the experiments were 53.52 min, a solid/liquid ra-
Table 4. The optimal conditions for the experiments were 53.52 min, a solid/liquid ratio of
tio of 9.83 mL/g, and an ethanol concentration of 59.7% (V/V). The experimental val-
9.83 mL/g, and an ethanol concentration of 59.7% (V/V). The experimental values for TPC
ues for TPC and TFC bioactive compounds were 74.45 ± 1.22 mg EAG/g DM and
and TFC bioactive compounds were 74.45 ± 1.22 mg EAG/g DM and 17.08 ± 1.85 mg EC/g
17.08 ± 1.85 mg EC/g DM, respectively, while the antioxidant activity DPPH, ABTS, and
DM, respectively, while the antioxidant activity DPPH, ABTS, and FRAP were 83.45 ±
FRAP were 83.45 ± 0.89%, 82.85 ± 1.52%, and 87.01 ± 2.35%, respectively. These experimen-
0.89%, 82.85
tal results were± 1.52%,
found toand
be 87.01
similar± to
2.35%, respectively.
the predicted model These experimental
for TPC results DM),
(72.40 mg EAG/g were
found
TFC to bemg
(16.42 similar
EC/gtoDM),
the predicted model for
DPPH (82.58%), TPC
ABTS (72.40 mg
(83.06%), andEAG/g
FRAPDM), TFC Therefore,
(85.79%). (16.42 mg
EC/g DM), DPPH (82.58%), ABTS (83.06%), and FRAP (85.79%).
there is a synergy between the results found and the Box–Behnken design. Therefore, there is a
synergy between the results found and the Box–Behnken design.
Table 4. Optimal conditions and predicted and actual response values of olive leaf extract.
Table 4. Optimal conditions and predicted and actual response values of olive leaf extract.
The Optimal Conditions The Answers
The Optimal
X1 X2 X3 TPC TFC The Answers
DPPH ABTS FRAP
Conditions
(min) (mL/g) (%) (mg GAE/g DM) (mg EC/g DM) (%) (%) (%)
X1
53.5 X2
9.83 X3
59.7 Reel TPC Predict Reel TFC Predict Reel DPPH Predict ReelABTS Predict Reel FRAP Predict
Objective(ml/g) (%)
(min) Maximum
(mg GAE/g DM) Maximum
(mg EC/g DM) Maximum(%) Maximum
(%) Maximum
(%)
Optimized values 74.45 ± 1.22 72.40 17.08 ± 1.85 16.42 83.45 ± 0.89 82.58 82.85 ± 1.52 83.06 87.01 ± 2.35 85.79
53.5 9.83 59.7 Reel Predict Reel Predict Reel Predict Reel Predict Reel Predict
Objective Maximum Maximum Maximum Maximum Maximum
17.08 ± 83.45 ± 82.85 ± 87.01 ±
Optimized values 74.45 ± 1.22 72.40 16.42 82.58 83.06 85.79
1.85 0.89 1.52 2.35
Separations 2023, 10, 326 13 of 17
Separations 2023, 10, x FOR PEER REVIEW 14 of 18

Figure 5. The predicted optimal values and desirability of olive leaf optimization.
Figure 5. The predicted optimal values and desirability of olive leaf optimization.
3.6. HPLC‐MS Analysis
3.6. HPLC-MS
FigureAnalysis
6 displays the phenolic compounds identified in olive leaf extracts through
ultrasound in the first and second extractions. The peaks on the graph correspond to these
Figure 6 displays the phenolic compounds identified in olive leaf extracts through
compounds. The results indicate that the first extraction in optimal conditions (Figure 6a)
ultrasound resultedininthe
a highfirst andofsecond
release phenolic extractions. The peaks
compounds. In contrast, on the
the second graph(Fig‐
extraction correspond to
these compounds.
ure 6b) had fewer The results
bioactive indicate This
compounds. thatsuggests
the firstthatextraction in extraction
the ultrasonic optimal conditions
(Figuremethod 6a) resulted
is effective.inEight
a high release
phenolic of phenolic
compounds compounds.
were identified: In contrast,
hydroxytyrosol, catechin, the second
extraction caffeic(Figure
acid, vanillin,
6b) had naringin,
feweroleuropein, quercetin,
bioactive and kaempferol.
compounds. As shownthat
This suggests in Table
the ultrasonic
5, oleuropein was the most abundant compound, with a concentration of 114.10 mg/g DM,
extraction method is effective. Eight phenolic compounds were identified:
followed by hydroxytyrosol, caffeic acid, and kaempferol. The optimized ultrasound‐as‐
hydroxyty-
rosol, catechin, caffeic acid, vanillin, naringin, oleuropein, quercetin,
sisted extraction method was likely responsible for the high amount of phenolic com‐ and kaempferol. As
shownpounds in Table 5, first
in the oleuropein
extraction.was the most abundant compound, with a concentration of
114.10 mg/g DM, followed by hydroxytyrosol, caffeic acid, and kaempferol. The opti-
mized
Separations 2023, 10, x ultrasound-assisted
FOR PEER REVIEW extraction method was likely responsible for the high amount 15 of 18of
phenolic compounds in the first extraction.

Figure 6. HPLC chromatograms

Figure 6. HPLC chromatograms of polyphenols of polyphenols of olive
of olive leaf leaf extracts
extracts fromfrom
thethe First
First UEAUEA under
under the
the
optimal conditions (a) and the Second UEA under the same optimal conditions (b).
optimal conditions (a) and the Second UEA under the same optimal conditions (b).
Table 5. Concentration of phenolic compounds identified in olive leaf extracts under the optimal
conditions of the first and second ultrasound extraction (mg/g DM).

Chemical For‐ Concentration of the Concentration of Sec‐

N° PIC TR (min) Phenolic Compounds
mulas First Extraction (mg/g) ond Extraction (mg/g)
1 7.09 Hydroxytyrosol C8H10O3 45.40 ± 1.2 10.02 ± 2.12
2 10.52 Catechin C15H14O6 12.90 ± 1.35 2.21 ± 1.36
Separations 2023, 10, 326 14 of 17

Table 5. Concentration of phenolic compounds identified in olive leaf extracts under the optimal
conditions of the first and second ultrasound extraction (mg/g DM).

Concentration of the First Concentration of Second

N◦ PIC TR (min) Phenolic Compounds Chemical Formulas
Extraction (mg/g) Extraction (mg/g)
1 7.09 Hydroxytyrosol C8 H10 O3 45.40 ± 1.2 10.02 ± 2.12
2 10.52 Catechin C15 H14 O6 12.90 ± 1.35 2.21 ± 1.36
3 12.72 Caffeic acid C9 H8 O4 79.50 ± 1.25 15.32 ± 3.36
4 15.23 Vanillin C8 H8 O3 12.70 ± 1.36 0.98 ± 2.36
5 20.52 Naringin C9 H8 O3 45.40 ± 2.45 32.23 ± 1.25
6 21.30 Oleuropein C25 H32 O13 114.10 ± 3.42 40.23 ± 2.78
7 26.48 Quercetin C15 H10 O7 23.00 ± 2.38 12.21 ± 1.45
8 27.44 Kaempferol C15 H10 O6 29.00 ± 1.96 9.36 ± 1.69

3.7. Scanning Electron Microscopy

After being dried and ground, the leaf powder was examined under a scanning
electron microscope. There was a visible difference between the untreated sample and
the one treated with EWM and UAE (as shown in Figure 7). The untreated sample was
densely compacted, while the treated sample showed structural changes. UAE caused
more damage and formation of cracks than maceration, possibly due to the cavitation
effects of the ultrasound [47]. During extraction, high ultrasound intensities can enhance
solvent penetration and destroy cell membranes [48], releasing more bioactive compounds
Separations 2023, 10, x FOR PEER REVIEW 16 of 18
from the sample matrix.

Figure 7. SEM images of olive leaf powder before extraction (RM), leaf powder after ultrasound‐
Figure 7. SEM images of olive leaf powder before extraction (RM), leaf powder after ultrasound-
assisted extraction (UAE) under optimal conditions, and leaf powder treated by ethanol water mac‐
assisted extraction (UAE) under optimal conditions, and leaf powder treated by ethanol water
eration (EWM).
maceration (EWM).
4. Conclusions
4. Conclusions
The Box–Behnken
The Box–Behnken design design(BBD)
(BBD)method,
method,along
alongwith
withthethe
surface response
surface responsedesign ap‐
design
proach (RSM), was used to study the impact of ultrasonic‐assisted extraction
approach (RSM), was used to study the impact of ultrasonic-assisted extraction (UAE) (UAE) pro‐
cess parameters
process parameters ononthethe
content
contentofofpolyphenolic
polyphenoliccompounds
compoundsin in Moroccan
Moroccan picholine olive
picholine olive
leaves. The
leaves. The results
results showed
showed that
that this
this eco-friendly
eco‐friendly technique
technique isis beneficial
beneficial in
in optimizing
optimizing thethe
conditions for extracting
conditions extracting phenolic compounds (TPC, TFC). To achieve achieve a high
high yield,
yield, it
it is
is
recommended to use
recommended use an
anextraction
extractiontime
timeofof53.5
53.5min,
min,a solvent/solid
a solvent/solid ratio of 9.95
ratio mL/g,
of 9.95 and
mL/g,
an ethanol
and concentration
an ethanol of 59.7%.
concentration The content
of 59.7%. of TPC
The content ofand
TPCTFC andare
TFC74.45
are ±74.45
1.22 mg EAG/g
± 1.22 mg
DM and 17.08 ± 1.85 mg EC/g DM, respectively, while the antioxidant
EAG/g DM and 17.08 ± 1.85 mg EC/g DM, respectively, while the antioxidant activity of activity of DPPH,
ABTS, and
DPPH, ABTS,FRAP are 83.45
and FRAP ± 0.89%,
are 83.45 82.85 82.85
± 0.89%, ± 1.52%, and 87.01
± 1.52%, ± 2.35%
and 87.01 respectively.
± 2.35% The
respectively.
presence
The presenceof certain
of certainphenolic
phenolicbioactive
bioactivecompounds
compoundsininhighhigh concentrations,
concentrations, specifically
oleuropein and
oleuropein and hydroxytyrosol,
hydroxytyrosol,was wasconfirmed
confirmedthrough
throughanalysis
analysisby byHPLC-MS.
HPLC‐MS.
To better
To better understand
understand the extraction and and optimization
optimizationphenomena,
phenomena,ititwouldwouldbe bebene‐
ben-
eficial
ficial totostudy
studyadditional
additionalparameters
parameters such
such as as sonication
sonication temperature,
temperature, frequency,
frequency, andand
sol‐
solvent nature.
vent nature.

Author Contributions: E.M.E.A.: doctoral student, practical work, and main leader of the operative
manipulations; A.M.: doctoral assistant of operations and preparation of raw material; N.E.
(FSSM/UCA), A.J. (FSSM/UCA) and M.M. (Emeritus FSSM/UCA): professors who initiated the fol‐
low‐up and planning of the thesis work; M.O. and N.H.: assistants doctors of analysis; K.B., doctor,
pharmacist, and CEO of the company, Iris Cosmétologie (IRCOS) Laboratoires, our industrial part‐
Separations 2023, 10, 326 15 of 17

Author Contributions: E.M.E.A.: doctoral student, practical work, and main leader of the op-
erative manipulations; A.M.: doctoral assistant of operations and preparation of raw material;
N.E. (FSSM/UCA), A.J. (FSSM/UCA) and M.M. (Emeritus FSSM/UCA): professors who initiated
the follow-up and planning of the thesis work; M.O. and N.H.: assistants doctors of analysis; K.B.,
doctor, pharmacist, and CEO of the company, Iris Cosmétologie (IRCOS) Laboratoires, our industrial
partner: availability of raw materials, logistics and production machines. All authors have read and
agreed to the published version of the manuscript.
Funding: This research was funded by the National Center for Scientific and Technical Research
(CNRST) and the Ministry of Higher Education, Scientific Research and Vocational Training of
Morocco, “PPR-BR2BINOV-Mahrouz-FS-UCA-Marrakesh”.
Data Availability Statement: Not applicable.
Acknowledgments: The authors thank the City of Innovation of Marrakech and the company, Iris
Cosmétologie (IRCOS) Laboratoires.
Conflicts of Interest: The authors declare no conflict of interest.

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