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Optimization of ultrasound-assisted extraction of phenolic compounds and

antioxidant activity from carob pulp (Ceratonia siliqua L.) by using response
surface methodology

Article in Annals of the University Dunarea de Jos of Galati · January 2018

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The Annals of the University Dunarea de Jos of Galati
Fascicle VI – Food Technology (2018), 42(1), 26-39

ORIGINAL RESEARCH PAPER

OPTIMIZATION OF ULTRASOUND-ASSISTED EXTRACTION OF

PHENOLIC COMPOUNDS AND ANTIOXIDANT ACTIVITY FROM
CAROB PULP (CERATONIA SILIQUA L.) BY USING RESPONSE
SURFACE METHODOLOGY

FAIROUZ SACI1, YASSINE BENCHIKH*1,2, HAYETTE LOUAILECHE1, MOSTAPHA BACHIR

BEY1

1
Laboratoire de Biochimie Appliquée, Faculté des Sciences de la Nature et de la Vie, Université
de Bejaia, 06000 Bejaia, Algeria.
2
Département de Biotechnologie, Institut de la Nutrition, de l'Alimentation et des Technologies
Agro-Alimentaires (I.N.A.T.A.A.), Université Frères Mentouri – Constantine 1, 25000
Constantine, Algeria.
*
Corresponding author: [Link]@[Link]; Phone: +213 793390091

Received on 25th September 2017

Revised on 10th November 2017

In this study, the ultrasonic-assisted extraction for phenolic compound extraction

from the carob pulp (Ceratonia siliqua L.) was applied. The Box-Behnken design
and response surface methodology (RSM) were used to study and optimize the
effect of processing variables, acetone concentration (60-80%), extraction
amplitude (30-100%) and extraction time (10-60 min) on the total phenolic
content (TPC) and the antioxidant activity. The optimal values for the TPC yield
and for the antioxidant activity were at 59.30% and 61.70% acetone concentration,
85.86% and 90.12% extraction amplitude, and 53.24 min and 56.82 min of
extraction time, respectively. The experimental values were in accordance with
those predicted by the model, which indicated the suitability of response surface
methodology in optimizing the antioxidants extraction from carob pulp by
ultrasonic-assisted extraction.

Keywords: optimization; carob pulp; phenolic compounds; antioxidant activity;

ultrasonic-assisted extraction; response surface methodology

Introduction
Carob fruits are among the most important tree fruit crops in the Mediterranean
countries and their production and consumption have increased considerably in
recent years (FAO, 2014). The composition and quantification of polyphenols in
carob fruit have been elucidated, as well as their antioxidant activity (Benchikh et
al., 2014; Benchikh et al., 2016).
 Saci et al. / AUDJG – Food Technology (2018), 42(1), 26-39 27

Phenolic compounds are a group of very diverse chemicals that includes simple
phenols, benzoic and cinnamic acid derivatives and flavonoids (Moraes et al.,
2013). Many of these compounds have been shown to present useful traits that
support their use as bioactive compounds for human health (Sanmukhani et al.,
2013; Vajic et al., 2015).
Many methodologies have been used for the extraction of phenolic compounds
from carob (Benchikh and Louaileche, 2014; Mulet et al., 2015). Recently, new
extraction techniques have been developed for the extraction of target compounds
from different materials including ultrasound- and microwave-assisted,
supercritical fluid and accelerated solvent extraction (Hammi et al., 2015; Siddiqui
and Aeri, 2017). Among these, ultrasound-assisted extraction (UAE) is one of the
most rapid methods and efficient alternatives to conventional extraction techniques
(Heydari Majd et al., 2014). UAE has been applied to extract bioactive compounds
from different materials owing to its high reproducibility, simplified manipulation,
and significant reduction in solvent consumption (Liu et al., 2013). In fact, the
ultrasound induces a swelling of the cells, solvent uptake and an enlargement of the
pores of the cell walls allowing higher diffusivity of the solvent across the cell
walls. The ultrasound could even cause a breakdown of the cell walls and
facilitates the washing out of the cell content (Talmaciu et al., 2015). In recent
studies, UAE has been applied to obtain bioactive compounds from different
materials such Tecomellaun dulata Bark (Siddiqui and Aeri, 2017).
Many factors including solvent concentration, extraction amplitude and extraction
time affect the efficiency of the extraction process. In order to optimize the
operational conditions, the response surface methodology (RSM) has been widely
used (Bachir bey et al., 2014; Ilaiyaraja et al., 2015; Siddiqui and Aeri, 2017).
RSM is an effective statistical technique for modeling and optimizing the complex
processes (Siddiqui and Aeri, 2017). Compared with the classical methods, as the
one-factor-one method, the RSM is more efficient, requires fewer experiments, and
provides interaction effects on the responses besides factor effects (Bernardo-Gil et
al., 2011).
In the present study, optimization of solvent concentration, ultrasound amplitude,
and extraction time for extraction of total phenolic compounds from Ceratonia
siliqua L. pulp by using UAE and evaluation of antioxidant activity of the extract
was carried out according to RSM.

Materials and methods

Plant material
The carob mature fruits were randomly harvested from the same region (Bejaia,
Algeria). The carob cloves were chosen for uniformity in shape and colour. The
seeds were manually separated from carob, and the rest of the fruit (carob pulp)
was oven-dried at 45°C for 5 days, then the dried pulp was ground with a crusher
(IKA, A 11 basic, Staufen, Germany), and passed through a 500 μm sieve before
analyses.
28 Saci et al. / AUDJG – Food Technology (2018), 42(1), 26-39

Chemicals and reagents

Folin–Ciocalteu reagent, methanol, and acetone were purchased from Biochem,
Chemopharma (Montreal, Quebec), sodium carbonate anhydrous was from Sigma
Chemical (Sigma–Aldrich GmbH, Switzerland), gallic acid was from Prolabo
(Montreuil, France), and 1,1-diphenyl-2-picrylhydrazyl (DPPH) was from Sigma
Chemical (Sigma–Aldrich GmbH, Germany).
Ultrasound assisted extraction
Ultrasonic equipment (Sonics Vibra cell. VCX 130 PB, USA) was used for UAE
with working frequency fixed at 20 kHz. The energy input was controlled by
setting the amplitude of the Sonicator Probe. For extraction, 25 mg of samples
were homogenized in 15 mL of acetone. The obtained suspension was exposed to
acoustic waves for varying acetone concentration (40-80%), amplitude (30-100%),
and extraction time (10-60 min) (Table 1). The temperature was fixed at 25°C. The
extract was centrifuged at 1700 × g (Nüve NF 200, Ankara, Turkey) for 20 min and
filtered.
Analytical methodology
Determination of total phenolic content
Total phenolic content was estimated using Folin–Ciocalteu method (Singleton and
Rossi, 1965). A volume of 100 μL of the extracts was added to 1 mL of Folin-
Ciocalteu solution (diluted 10-fold with distilled water). Subsequently, 800 µL of
sodium carbonate (7.5%) were added. After 30 min of incubation in the dark at
room temperature, the absorbance at 765 nm was measured (Uviline 9400 UV-
visible spectrophotometer, Secomam, France). Total phenolic contents were
expressed as mg gallic acid equivalent per 100 g dry matter (mg GAE/100g DM).
Antioxidant activity
Free radical scavenging activity of the extracts was evaluated using the 2,2-
diphenyl-1-picrylhydrazyl (DPPH) assay (Brand-Williams et al., 1995). A volume
of 50 μL of extract was added to 1 mL of DPPH solution (60µM). The decrease in
absorbance was determined at 517 nm, after incubation for 30 min in the dark.
Gallic acid was used as a standard, and the antioxidant activity was expressed as
mg gallic acid equivalents per 100 g dry matter (mg GAE/100g DM).
Statistical analysis
Experimental design
Response Surface Methodology (RSM) was used to investigate the optimal
conditions for antioxidants extraction from carob pulp. A Box–Behnken design
was applied to evaluate the effects of the solvent concentration (x1, acetone/water
%, v/v), ultrasound amplitude (x2, %), and time (x3, min) on total phenolic content
and antioxidant activity. As indicated in Table 1, the three independent variables
were analysed at 3 levels, coded -1, 0, and +1 for lowest, central, and highest value,
respectively.
 Saci et al. / AUDJG – Food Technology (2018), 42(1), 26-39 29

Table 1. Experimental values and coded levels of the independent variables used for the
Box-Behnken design
Uncoded levels
Coded
Acetone concentration, Ultrasound amplitude, Extraction time,
levels
% % min
-1 40 30 10
0 60 65 35
+1 80 100 60

Data analysis
The experimental results of the response surface design were analyzed using JMP
10 (statistical analysis system Inc., SAS) software. All experiments were conducted
in triplicate and the mean was reported. The complete design consisted of 15
experimental runs, including three replicates of the central point to evaluate the
experimental error measurement (Table 2).
A second-degree polynomial regression model was used to correlate the
relationship between independent variables and responses (TPC and antioxidant
activity), and the second-degree polynomial model was as follows equation (1).
3 3 2 3
2
Y= ∝0 + ∑ ∝i xi + ∑ ∝ii xi + ∑ ∑ ∝ij xi xj (1)
i=1 i=1 i=1 j=i+1
Where Y was the response variables; α 0, α i, α ii, and α ij are the intercept, linear,
quadratic, and interactive coefficients, respectively; xi and xj are the levels of the
independent variables (i≠j).
Student’s t-test permitted the checking of the statistical significance of the
regression coefficient and Fisher’s F-test determined the second-order model
equation at a probability (p) of 0.001, 0.01 or 0.05. Model adequacy was evaluated
using the lack of fit, the coefficient of determination (R2) and the F-test value
obtained from the analysis of variance (ANOVA). Regression analysis and three
dimensional response surface plots were plotted to determine optimum conditions
for TPC and antioxidant activity. The test of statistical significance was based on
the total error criteria with a confidence level of 95.0%.

Table 2. Box-Behnken design and responses values for total phenolic compounds (TPC)
extraction and antioxidant activity
Run Variable levels a TPCb Antioxidant activityb
x1 x2 x3 Observed Predicted Observed Predicted
1 60(0) 65(0) 35(0) 28.41 28.68 20.31 20.52
2 60(0) 30(-1) 10(-1) 16.80 17.31 11.63 11.49
3 60(0) 65(0) 35(0) 28.72 28.68 20.35 20.52
4 60(0) 100(+1) 10(-1) 22.00 22.38 16.94 16.62
5 80(+1) 30(-1) 35(0) 16.30 16.40 12.85 12.93
30 Saci et al. / AUDJG – Food Technology (2018), 42(1), 26-39

Run Variable levels a TPCb Antioxidant activityb

x1 x2 x3 Observed Predicted Observed Predicted
6 40(-1) 65(0) 10(-1) 20.04 19.76 14.78 15.18
7 60(0) 30(-1) 60(+1) 21.91 21.54 16.10 16.42
8 40(-1) 100(+1) 35(0) 24.69 24.59 19.40 19.32
9 80(+1) 100(+1) 35(0) 24.21 24.44 19.14 19.40
10 60(0) 65(0) 35(0) 28.92 28.68 20.89 20.52
11 60(0) 100(+1) 60(+1) 30.69 30.18 22.32 22.46
12 80(+1) 65(0) 10(-1) 18.96 18.36 13.40 13.46
13 80(+1) 65(0) 60(+1) 24.16 24.44 20.17 19.77
14 40(-1) 30(-1) 35(0) 19.15 18.92 14.88 14.62
15 40(-1) 65(0) 60(+1) 25.11 25.71 19.72 19.66
ax ,
1 Solvent concentration (%); x2, Ultrasoundamplitude (%); x3, Extraction time (min).
bTPC and antioxidant activity were expressed in mg GAE/g DM of carob pulp.

Results and discussion

Model fitting
A RSM approach was conducted in order to determine the correlative effect of the
solvent concentration, amplitude and extraction time on both TPC and antioxidant
activity. In accordance with the experimental design, the observed and predicted
response values were indicated in Table 2. Overall, a close relationship between the
experimental values and the predicted ones was observed attesting satisfactory of
developed models. In addition, it was indicated that the TPC and the antioxidant
activity ranged from 16.30 to 30.69 mg GAE/g, and from 11.63 to 22.32 mg
GAE/g, respectively. The regression coefficients of the intercept, linear, quadratic
and interaction terms of the models were calculated using the least squares
technique and were displayed in Table 3.
Table 4 shows the results of fitting quadratic models of data. Analysis of variance
(ANOVA) indicates that the contribution of quadratic effect was significant. The
fitted quadratic models for TPC and antioxidant activity (AA) were given in Eqs. 2
and 3, respectively.

TPC = 28.6833 - 0.6700x1 + 3.4288x2 + 3.0088x3 + 0.8950x2 x3 - 4.1892x21 - 3.4067x22 - 2.4267x23 (2)
AA = 20.5167 - 0.4025x1 + 2.7925x2 + 2.6950x3 - 1.8396x21 - 2.1096x22 - 1.6596x23 (3)

The fitness of model was evaluated by lack of fit, which was insignificant for TPC
(0.101) and antioxidant activity (0.316), indicating the goodness of the both
models.
P-values obtained for TPC and antioxidant activity indicated the suitability of
models to accurately predict responses (Table 4). The quality of the fit model can
be determined by the coefficient of determination (R2) which was 0.993 and 0.994
for TPC and antioxidant activity, respectively.
 Saci et al. / AUDJG – Food Technology (2018), 42(1), 26-39 31

The corresponding variables would be more significant if the absolute F-value

becomes greater and the P-value becomes smaller. The F-value of the models was
82.66 in case of the TPC (P< 0.0001) and 93.29 for antioxidant activity (P<
0.0001). The P-value of the models was less than 0.0001, which indicates that the
model was significant and could work well for the prediction of TPC and
antioxidant activity of carob pulp.

Table 3. Regression coefficient, standard (Std.) error, and Student’s t-test results of
response surface for TPC and antioxidant activity
Parameter Estimate Std. error t Ratio Prob> |t|
TPC
Intercept 28.6833 0.3593 79.8395 <0.0001*
x1 -0.6700 0.2200 -3.0454 0.0286*
x2 3.4288 0.2200 15.5851 <0.0001*
x3 3.0088 0.2200 13.6760 <0.0001*
x1-x2 0.5925 0.3111 1.9043 0.1152
x1-x3 0.0325 0.3111 0.1045 0.9209
x2-x3 0.8950 0.3111 2.8766 0.0347*
x1-x1 -4.1892 0.3238 -12.9361 <0.0001*
x2-x2 -3.4067 0.3238 -10.5198 0.0001*
x3-x3 -2.4267 0.3238 -7.4935 0.0007*
DPPH assay
Intercept 20.5167 0.2501 82,0309 <0.0001*
x1 -0.4025 0.1532 -2,628 0.0466*
x2 2.7925 0.1532 18,2326 <0.0001*
x3 2.6950 0.1532 17,596 <0.0001*
x1-x2 0.4425 0.2166 2.0429 0.0965
x1-x3 0.4575 0.2166 2,1122 0.0884
x2-x3 0.2275 0.2166 1,0503 0.3417
x1-x1 -1.8396 0.2254 -8,1598 0.0004*
x2-x2 -2.1096 0.2254 -9,3574 0.0002*
x3-x3 -1.6596 0.2254 -7,3614 0.0007*
x1, solvent concentration; x2, ultrasound amplitude; x3, extraction time
*Values statistically significant at P < 0.05.

The results showed that amplitude and extraction time had a positive linear effect
on both TPC extraction and antioxidant activity as well as interaction term between
amplitude and time for TPC. However, interaction terms between solvent
concentration-amplitude and solvent concentration-time were found to have no
effects.
32 Saci et al. / AUDJG – Food Technology (2018), 42(1), 26-39

Table 4. ANOVA table for the effect of acetone concentration, amplitude and time on TPC
extraction and antioxidant activity (mg GAE/g DM)
Source DF Sum of squares F value P value
TPC
x1 1 3.5912 9.2746 0.0286*
x2 1 94.0506 242.8941 <0.0001*
x3 1 72.4206 187.0327 <0.0001*
x1-x2 1 1.4042 3.6265 0.1152
x1-x3 1 0.0042 0.0109 0.9209
x2-x3 1 3.2041 8.2749 0.0347*
x1-x1 1 64.7967 167.3434 <0.0001*
x2-x2 1 42.8506 110.6656 0.0001*
x3-x3 1 21.7429 56.1531 0.0007*
Model 9 288.0610 82.6604 <0.0001*
Error 5 1.9360
Total 4 289.9970
2
R 0.993
2
Adj. R 0.981
Lack of fit 3 1.8039 9.1064 0.101
DPPH
x1 1 1.2961 6.9063 0.0466*
x2 1 62.3845 332.4275 <0.0001*
x3 1 58.1042 309.6194 <0.0001*
x1-x2 1 0.7832 4.1736 0.0965
x1-x3 1 0.8372 4.4613 0.0884
x2-x3 1 0.2070 1.1032 0.3417
x1-x1 1 12.4950 66.5821 0.0004*
x2-x2 1 16.4320 87.5612 0.0002*
x3-x3 1 10.1694 54.1897 0.0007*
Model 9 157.5778 93.2982 <0.0001
Error 5 0.9383
Total 14 158.5161
R2 0.994
Adj. R2 0.983
Lack ok fit 3 0.7284 2.314 0.316
x1, solvent concentration; x2, amplitude; x3, time; DF: Degree of freedom.
* Values statistically significant at P < 0.05.
 Saci et al. / AUDJG – Food Technology (2018), 42(1), 26-39 33

Analysis of response surfaces

Interpretation of response surface plots
To illustrate the correlations between the independent and dependent variables
considered, three-dimensional response surface plots were built on the basis of the
model equations mentioned above. The type of plots showed the effects of two out
of the three independent variables on the response factor, keeping the third one at
level-coded zero (center value of the testing ranges). The profile of the response
surface plots showed a strong interaction between the tested variables. From these
three-dimensional profiles, it is easy to see the interaction effects between any two
independent factors.
Response surface analysis of total phenolic contents
The effect results of extraction factors studied were shown in Figures 1 and 2. Each
variable contributes significantly to affect the quantity of extractable phenolic
compounds. Amongst them, the polarity of the solvent plays an important role
which is due to its ability to extract these molecules by solubilisation. Since
phenolic compounds have a wide spectrum of solubility; a combination of acetone
and water may be desirable and effective for separation than any single solvents
(Benchikh and Louaileche, 2014).
In our study, the influencing factors on TPC were determined by the significant
coefficient of the second-order polynomial regression equation. The results
indicated that the first-order linear effect was significant for acetone concentration
(x1), extraction amplitude (x2) and extraction time (x3); the second-order quadratic
effect was significant for all the factors; the interactive effect was significant for
ultrasound amplitude and time (x2-x3).
In our results, Figure 1A showed that when the extraction time was fixed at central
levels, the phenolic compounds extraction increased slightly by increasing the
acetone concentration and reached the maximum value at 59.30%. As shown in our
previous study using the one-factor-one method for extraction of phenolic
compounds from carob pulp, 70% acetone was the best extracting solvent
(Benchikh and Louaileche, 2014), but in this work, 59.30% acetone was the
optimum parameter which indicates that the combination of acetone with a bit
more water was better than 70% acetone. The binary-solvent system was more
efficient in the extraction of phenolic compounds from plant samples compared
with a mono-solvent system (Chew et al., 2011). These results can be explained by
the principle of “like dissolves like”; any given solvent would only extract
compounds that have a similar polarity (Zhang et al., 2007). Furthermore, adding
water to organic solvent can enhance the extraction efficiency of phytochemicals
by causing the plant material to swell, allowing the solvent to penetrate more easily
into the solid particles of the sample matrix (Gertenbach, 2001; Luthria, 2012). A
linear increase of TPC was noticed with increasing amplitude, and a maximum of
TPC was obtained with 85.86% amplitude, and thereafter, this content decreased.
The ultrasound induces a swelling of cells, a solvent uptake and an enlargement of
the pores of the cell walls allowing higher diffusivity of the solvent across the cell
34 Saci et al. / AUDJG – Food Technology (2018), 42(1), 26-39

walls. The ultrasound could even cause a breakdown of the cell walls and facilitate
the washing out of the cell content (Talmaciu et al., 2015).
Figure 1B showed the interaction between acetone concentration and extraction
time on total phenolic extraction. An increase in the total phenolic contents was
observed as increasing extraction time up to 53.24 min, but decreased thereafter.
Also as shown in our previous study, 90 min was the best extraction time
(Benchikh and Louaileche, 2014), but in the present results, we have gained 36.76
min because the model from Box-Behnken design takes into consideration the all
the parameters in order to determine the optimum one. The decreasing of phenolic
compounds after 53 min could be due to a phenolic compound oxidation occurring
by prolonging exposure to environmental factors, such as light and oxygen (Chan
et al., 2009). The results obtained were explained by Fick’s second law of
diffusion, which predicts that after a certain time, there will be a final equilibrium
between the solute concentration in the solid matrix (plant sample) and in the bulk
solution (extraction solvent) (Silva et al., 2007). Hence, excessive extraction time
was not useful in extracting more phenolic compounds from carob pulps. Figure
1C reflects the effects of amplitude and extraction time on the ultrasonic-assisted
extraction of TPC from carob pulp. The highest total phenolic content was
observed at a higher amplitude and higher extraction time.
Response surface analysis of antioxidant activity
The relationship between the antioxidant activity and extraction factors was
investigated by response surface plots in Figure 2. Figure 2A shows the effect of
acetone concentration, amplitude and their mutual interaction on the antioxidant
activity measured by DPPH radical scavenging assay. The concentration of acetone
influenced significantly the antioxidant activity in first order linear (p<0.05) and
second order quadratic (p<0.05). The regression coefficient for this factor was a
negative value indicating that an increase in acetone concentration caused a
decrease of the antioxidant activity. The highest antioxidant activity was observed
at 61.70% of acetone concentration and 90.12% of amplitude extraction. The effect
of extraction time, acetone concentration and their mutual interaction on the
antioxidant activity was illustrated in Figure 2B. Generally, antioxidant activity
was increased with increasing extraction time. An increase in the antioxidant
activity was observed with the increasing of extraction time and the trend was
reserved when the extraction time reached to approximately 56min, and then
antioxidant activity began to decrease. As mentioned above, this decreasing of the
antioxidant activity was probably due to the oxidation of the antioxidants. Figure
2C represents the effect of extraction amplitude, extraction time and their mutual
interaction on the antioxidant activity. The highest antioxidant capacity was
observed at higher extraction amplitude and higher extraction time.
Optimization of extraction parameters and validation of the model
After determining the optimum conditions and predicting the responses under these
conditions, a new set of experiment was designed and conducted with the selected
optimal conditions of the process parameters to predict and verify the accuracy of
the mathematical model. The results were shown in Table 5.
 Saci et al. / AUDJG – Food Technology (2018), 42(1), 26-39 35

The predicted results matched well with the experimental results obtained using
optimum extraction conditions which validated the response surface methodology
model with a significant correlation. As a result, the model from Box-Behnken
design was considered to be accurate and reliable for predicting total phenolic
content and antioxidant activity of carob pulp for ultrasonic assisted extraction.

Figure 1. Response surface plots of the total phenolic content (TPC) of carob pulp as
affected by (a) acetone concentration and extraction amplitude; (b) acetone concentration
and extraction time; (c) extraction amplitude and extraction time following UAE
36 Saci et al. / AUDJG – Food Technology (2018), 42(1), 26-39

Figure 2. Response surface plots of the antioxidant activity of carob pulp as affected by (a)
acetone concentration and extraction amplitude; (b) acetone concentration and extraction
time; (c) extraction amplitude and extraction time following UAE
 Saci et al. / AUDJG – Food Technology (2018), 42(1), 26-39 37

Table 5. Optimum conditions and predicting the responses under extraction conditions
Responses Optimum UAE conditions Maximum values
Acetone Extraction Extraction
Experimental Predicted
concentration, amplitude time,
values Values
% % min
TPC, mg
59.30 85.86 53.24 30.75 ± 9.82 30.81
GAE/g DM
Antioxidant
activity, mg 61.70 90.12 56.82 22.96 ± 6.35 22.67
GAE/g DM

Conclusions
An ultrasonic-assisted extraction technique was applied for the extraction of
phenolic compounds from carob pulp and optimized by response surface
methodology. The high correlation of the models exhibited that the second-order
polynomial model could be successfully used for optimizing the extraction
parameters. The results showed that the extraction conditions including acetone
concentration, extraction amplitude and extraction time markedly influenced the
total phenolic content and antioxidant activity of carob pulp. The optimum
ultrasonic assisted extraction conditions for TPC and antioxidant activity were
respectively 59.30-61.70% of acetone concentration using extraction amplitude of
85.86–90.12% during 53.24–56.82min.

Acknowledgements
The authors are grateful to the Algerian Ministry of Higher Education and
Scientific Research for the financial support.

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