Received: 29 March 2021 | Revised: 2 August 2021 | Accepted: 5 August 2021

DOI: 10.1111/jfpp.15870

ORIGINAL ARTICLE

Optimization conditions of ultrasound-­assisted extraction of

phenolic compounds from orange peels using response surface
methodology

Mohamed G. Shehata1 | Nourhan M. Abd El Aziz1 | Mohamed M. Youssef2 |

Sobhy A. El-­Sohaimy1,3

1
Food Technology Department, Arid Lands
Cultivation Research Institute (ALCRI),
City of Scientific Research and Technology
Applications (SRTA-­City), Alexandria, 21934,
Egypt
2
Food Science and Technology Department,
Faculty of Agriculture, Alexandria University,
Alexandria, 21526, Egypt
3
Department of Technology and
Organization of Public Catering, Institute of
Sport, Tourism and Service, South Ural State
University, Chelyabinsk, 454080, Russia
Correspondence
Sobhy A. El-­Sohaimy, Food Technology
Department, Arid Lands Cultivation
Research Institute (ALCRI), City of Scientific
Research and Technology Applications
(SRTA-­City), New Borg El-­Arab City,
Alexandria, Egypt.
Email: elsohaimys@gmail.com
Abstract
This study focused on optimizing the extraction conditions of bioactive compounds
from orange peels using response surface methodology (RSM) to decrease the num-
ber of experimental trials and increase the yield of bioactive compounds. Ultrasonic
extraction time, extraction temperature, and ethanol concentration have been opti-
mized. Twenty experimental trials have been carried out to optimize the extraction
conditions. The RSM results showed that the extraction temperature and ethanol
concentration had a remarkable effect on the total phenolic content (TPC), total fla-
vonoid content (TFC), and antioxidant activity. The optimal ultrasound-­assisted ex-
traction conditions were 44 min extraction time, 50℃ extraction temperature, and
57.7% ethanol concentration. Under these optimal conditions, TPC and TFC were
292.158 µg GAE/g and 191.144 µg catechol/g, respectively, whereas antioxidant ac-
tivities were 5.199 and 2.96 µg/ml for 2,2-­diphenyl-­1-­picrylhydrazyl and 2,20-­azin
o-­bis(3-­ethylbenzothiazoline-­60-­sulfonic acid) diammonium salt, respectively. RSM
saves the extraction time and effective for optimizing extraction conditions with the
highest efficiency and yield.
Novelty impact statement: The findings of the current work emphasized usefulness
of ultrasound assisted extraction for elevation the extraction efficiency with de-
creasing the extraction time. The extraction temperature and ethanol concentration
are the main effective factors of extraction efficiency. The optimal extraction time
was 44 min at 50℃ extraction temperature, and the optimal ethanol concentration
was 57.7%. These optimal conditions were giving the highest yield of TPC and TFC
that were 292.158 µg GAE/g and 191.144 µg catechol/g. These novel optimized ex-
traction conditions could be useful in different food and pharmaceutical applications.

1 | I NTRO D U C TI O N
Orange juice production is progressively increasing worldwide
every year, except in 2020 due to the coronavirus disease 2019
pandemic. The global orange juice production in 2019 was about
2.2 million metric tons (Citrus: World Markets and Trade, July
2020; The European Fruit Juice Association). This huge produc-
tion of orange juice worldwide produces a large amount of waste
of peels, membranes, and seeds. This waste is mainly used as ani-
mal feed or discarded in the environment (Abd El-­A ziz et al., 2021;
Badr et al., 2020; Garcia-­C astello et al., 2015; Sharma et al., 2017;
Shehata, Awad, et al., 2021). New research adds value to this waste

J Food Process Preserv. 2021;00:e15870. wileyonlinelibrary.com/journal/jfpp © 2021 Wiley Periodicals LLC. | 1 of 10

https://doi.org/10.1111/jfpp.15870
2 of 10 | SHEHATA et al.
to extract valuable bioactive compounds for food and pharma-
ceutical purposes (Abdel-­Razek et al., 2020; Awad et al., 2020).
Notwithstanding, citrus peels are an excellent source of phenolic
acids and flavonoids, vitamin C, folate, and potassium with sev-
eral biological benefits (anti-­
inflammatory, anticarcinogenic, an-
tidegenerative, antioxidant, and antimicrobial properties; Oboh &
Ademosun, 2012; Rafiq et al., 2018). Conventional extraction meth-
ods, such as cold pressing, heating reflux, and Soxhlet, are time-­
consuming and have safety hazards and high energy input (Contini
et al., 2008; Mandal et al., 2007). To increase the exploitation of
citrus peels, an ecofriendly extraction method can be applied to re-
cover bioactive compounds from citrus waste. “Green” extraction
technology can decrease solvents, shorten the extraction time,
lower energy, and decrease environmental pollution and uses safe
thermolabile constituents. Green extraction techniques, such as
ultrasound-­assisted extraction (UAE), supercritical extraction, and
microwave-­
assisted extraction, have specific applications (Kate
et al., 2016; Liović et al., 2021; Viell et al., 2020). Ultrasound waves
(from 20 kHz to 100 MHz) during extraction can damage and disrupt
cell membranes, release cell contents, and enhance the extraction
yield (Chemat et al., 2009; Liović et al., 2021). The main advantages
of UAE are reduced extraction time, saving energy, and solvent use,
faster energy transfer, reduced extraction temperature, reduced
equipment size, faster response to the process, and quick start-­up
(Chemat et al., 2004; Viell et al., 2020).
Response surface methodology (RSM) enables the evaluation
of the effects of many factors and their interactions on response
variables. The main advantage of RSM is the reduced number of
experimental trials needed to evaluate multiple parameters and
their interactions; therefore, it is less laborious and time-­consuming
than other approaches required to optimize a process (Aybastıer &
Demir, 2010; Bezerra et al., 2008; Lee et al., 2000). The most com-
mon designs (i.e., central composite design [CCD] and Box-­Behnken
design) of RSM have been widely used in various experiments. CCD,
a spherical and revolving design, has been applied to optimize chem-
ical and physical processes due to its reasoning design and excellent
outcomes (Aybastıer & Demir, 2010). RSM was used in this study to
manipulate and optimize the critical factors for ecofriendly UAE to
recover antioxidant compounds from orange peels.
2 | M ATE R I A L S A N D M E TH O DS
2.1 | Plant materials and chemicals
Orange fruits (Citrus sinensis) obtained from a local market in
Alexandria, Egypt in 2019, were washed and manually treated. The
peels were cut into small pieces (20 × 10 mm) and lyophilized for
48 hr at −56℃ in a Dura-­Dry MP freeze dryer (FTS Process, USA;
0.04 Mbar). Freeze-­dried peels were crushed to fine powder using a
grinder (Waring Commercial) and stored at −20℃ for further analy-
sis. All analytical-­grade chemicals were obtained from Sigma–­Aldrich
(St. Louis, MO, USA).
TA B L E 1 Coded and uncoded factors and their range level using
in central composite design
Independent variables Coded and uncoded levels
X1: ultrasonic time (min) 40 (−1) 50 (0) 60 (+1)
X2: Extraction temperature (°C) 30 (−1) 40 (0) 50 (+1)
X3: Ethanol concentration (%) 40 (−1) 50 (0) 60 (+1)
2.2 | Optimization procedure
2.2.1 | Single-­factor experiments
Extraction temperature (30–­70℃), extraction time (10–­8 0 min), and
ethanol-­to-­water ratio (30%–­8 0%) were investigated as parameters
that influence the extraction yield in the experimental design. The
experimental results of a single-­factor and the impact of extraction
parameters and their upper and lower levels on the RSM design were
applied.
2.2.2 | Experimental design
CCD was used to evaluate responses and the optimum combina-
tion of UOD variables, including three extraction parameters at
three levels (Table 1). Extraction temperature (℃), extraction time
(min), and ethanol concentration (%) were the three independent
variables. Total phenolic content (TPC), total flavonoid content
(TFC), 2,2-­diphenyl-­1-­picrylhydrazyl (DPPH), and 2,20-­azino-­bis(
3-­ethylbenzothiazoline-­6 0-­sulfonic acid) diammonium salt (ABTS)
results of the dependent parameters were included. Twenty ex-
perimental trials were given by the complete design (Table 2). The
RSM polynomial equation of second degree (Equation [1]) was
used as follows:
Y = b0 + b1 X1 + b2 X2 + b3 X3 + b11 X12 + b22 X22 + b33 X32 + b12 X1 X2 + b13 X1 X3 + b23 X2 X3
(1)
The intercept and linear, quadratic, and interaction coefficients
are b 0, b1, b2, b3, b11, b22, …; the noncoded ethanol concentration
values, temperature, time, and interaction terms are X1, X 2, and X3.
Analysis of variance (ANOVA) was used to assess the lack of fit and
effects of optimization on TPC, TFC, and antioxidant activity of lin-
ear, quadratic, and interaction variables. Design-­E xpert version 12
(Stat-­Ease, Inc., Minneapolis, MN, USA) was used to analyze the data.
2.3 | Ultrasonic assisted extraction (UAE)
Dried powder (10 g) was applied to 100 ml ethanol (1:10, wt/vol)
and carried out in a tunable cold ultrasonic bath (4℃) for 30 s for
phenolic extraction. The extract was centrifuged at 12,000×g for
15 min at 20℃ and filtered with Whatman No. 1 filter paper. A
rotary vacuum evaporator (IKAÔ RV05 Basic) was used to remove
SHEHATA et al. | 3 of 10

TA B L E 2 Total phenolic content, total flavonoids content and antioxidant activity by 2,2-­diphenyl-­1-­picrylhydrazyl and 2,20-­azino-­
bis(3-­ethylbenzothiazoline-­60-­sulfonic acid) diammonium salt of the extract of citrus peel under different conditions of ultrasonic-­assisted
extraction based on a central composite design for response surface analysis

Factors Response

Run X1 (min) X2 (℃) X3 (%) TPC TFC DPPH (IC50) (µg/ml) ABTS (IC50) (µg/ml)

1 50 40 50 270 170 8.05 6.2

2 40 30 40 195 106 17.3 14.9
3 50 40 50 290 190 5.08 2.9
4 60 30 40 230 130 13.5 12.15
5 60 50 40 287 187 6.5 4.1
6 40 30 60 265 165 9.5 6.9
7 60 30 60 280 180 7.2 4.88
8 40 50 60 290 190 5.08 2.9
9 50 40 50 255 155 10.5 7.7
10 50 40 50 293 193 4.95 2.8
11 40 50 40 260 160 9.8 7.2
12 60 50 60 274 174 9.2 6.6
13 66.8179 40 50 276 163 9.6 6.6
14 50 23.1821 50 260 160 9.8 7.2
15 50 40 66.8179 290 190 5.08 2.9
16 50 40 50 285 185 6.9 4.72
17 33.1821 40 50 275 175 9.5 6.2
18 50 40 50 270 170 8.05 6.2
19 50 40 33.1821 240 140 12.8 11.8
20 50 56.8179 50 295 195 4.7 2.65

excess solvent at 40℃. The extract was lyophilized and stored at and mixed. The solution mixture was incubated at room temperature
−20℃ before analysis. for another 6 min. The absorbance of the solution mixture at 510 nm
was measured immediately using a spectrophotometer after mixing
with 2 ml NaOH (4%). TFC was expressed as catechol equivalents
2.4 | Total phenolic content (TPC) (µg CE/g DW).

TPC was determined as described by Singleton et al. (1999) and

Shehata, Abu-­Serie, et al. (2021) using the standard curve of gallic
acid. TPC was expressed as µg gallic acid equivalent/g dry extract.
The sample was prepared by mixing 0.1 ml orange peel extract with
7.9 ml deionized water and 0.5 ml FCR. The mixture was left in the
dark for 5 to 8 min before 1.5 ml of 20% sodium carbonate solution
was added to the sample. The absorbance value of the sample was
then read using an ultraviolet-­visible spectrophotometer at a wave-
length of 765 nm.
2.5 | Total flavonoids content (TFC)
2.6 | DPPH radical-­scavenging activity
The antiradical activities of the extracts were determined based on
a reaction with stable DPPH radicals dissolved in absolute ethanol
(Shehata, Ahmad, et al., 2020; Shehata, Darwish, et al., 2020). The
reaction requires preparing a mixture of 500 μl extracts at various
concentrations with 375 μl ethanol and 125 μl DPPH solution (0.02%
prepared in ethanol). A control containing 875 μl ethanol and 125 μl
DPPH solution was also prepared. After incubation for 60 min in the
dark, the absorbance at 517 nm was measured. The antiradical activ-
ity was determined using the following formula:

TFC was determined according to the method of Dewanto Abs control − Abs sample
Inhibition of DPPH radical ( % ) = × 100
et al. (2002) with some modifications using catechol as a standard. Abs sample
Briefly, the properly diluted sample (0.5 ml) was mixed with 2.2 ml
deionized water and 150 μl NaNO2 (5%) and incubated at room tem- Notably, a lower absorbance by the reaction mixture indicated a
perature for 6 min. After incubation, 150 μl AlCl3 (10%) was added higher DPPH radical-­scavenging activity.
4 of 10 | SHEHATA et al.

2.7 | ABTS radical cation

The free radical-­scavenging activity was determined by the ABTS

radical cation decolorization assay described by Re et al. (1999) and
Hashem and Shehata (2021). ABTS was dissolved in water to a con-
centration of 7 μM. ABTS radical cations (ABTS+) were generated
by reacting the ABTS stock solution with 2.45 μM potassium per-
sulfate (final concentration), and the mixture was kept in the dark at
room temperature for 12 to 16 hr before use. The radical was stable
in this form for more than 2 days when stored in the dark at room
temperature. To study the radical-­scavenging ability of the prepared
solutions, the samples containing ABTS solution were diluted with
distilled water to an absorbance of 0.700 ± 0.02 at 734 nm and
equilibrated at 30℃. A reagent blank reading was taken (A0). After
adding 1 ml diluted ABTS+ solution (A734 nm = 0.700 ± 0.02) to 1 ml
extract, the absorbance was read exactly 6 min after the initial mix-
ing (At):

Abs control − Abs sample

Inhibition of ABTS radical ( % ) = × 100
Abs sample

3 | R E S U LT S A N D D I S CU S S I O N

3.1 | Single-­factor experimental analysis

The design of the experiment was done to determine the contribu-

tion of each ultrasonic extraction parameter (time, temperature,
and ethanol concentration) to the extraction efficiency of phenolic
content. Figure 1 shows the influence of each extraction factor
on TPC from orange peels. The effect of time from 10 to 80 min
on total polyphenol was explored as a single-­variable extraction
factor (Figure 1a). In this RSM investigation, the total polyphenol
yield multiplied as the extraction time was increased from 10 to
50 min and then decreased as the extraction time increased for
more than 50 min. This might be due to the structural destruc-
tion of polyphenolic compounds (Carrera et al., 2012; Liović
et al., 2021; Sun et al., 2011). The optimum ultrasonic time for
polyphenol extraction from orange peels was from 30 to 60 min.
The influence of temperature on phenolic compound extraction
is shown in Figure 1b. As extraction temperature increased, TPC
slowly increased and reached the maximum at 40℃ and then
slightly decreased at above 40℃. The obtained results agreed
with previous findings, which showed that Euryale ferox seed
shells had the same response to polyphenol extraction tempera-
ture (Liu et al., 2013). Extraction of different types and forms of F I G U R E 1 Effect of ultrasonic time (a), extraction temperature
polyphenols was directly correlated with extraction temperature (b), ethanol concentrations (c) on the yields of total phenolic
(Ahmad-­Qasem et al., 2013). In contrast, the effect of ethanol-­to-­ content in single factor experiments
water ratio on polyphenol extraction was investigated from 30%
to 80% (Figure 1c). TPC increased with 50% ethanol concentra- the polarity of phenolic compounds in the raw materials (Şahin &
tion, emphasizing that 50% ethanol is the optimum concentration Şamlı, 2013). Therefore, ethanol concentration might differ based
for maximum polyphenol extraction from orange peels. The water-­ on the raw materials and their phenolic compound content.
to-­ethanol ratio affected the polarity of the extraction solvent, This RSM investigation revealed that, in UAE, extraction time,
thus affecting the extraction efficiency of the solvent according to extraction temperature, and ethanol concentration are the crucial
SHEHATA et al.
and effective parameters of phenolic compound extraction effi-
ciency (Table 1).
3.2 | Fitting the model
Table 1 lists the values coded and decoded and the responses of
each independent variable. TPC of orange peel extracts ranged from
195 to 295 µg/g dried extract. TFC ranged from 106 to 195 µg/g
extract. Table 1 also shows the antioxidant potential as IC50, which
ranged from 4.7 to 17.3 µg/ml for DPPH and 2.65 to 14.9 µg/ml for
ABTS. Equations (2)‒­(5) show the independent variables and re-
sponse correlation:
Y1 = 278.71 + 4.59X1 + 14.63X2 + 16.19X3 − 4.88X1 X2 − 7.87X1 X3
− 12.88X2 X3 − 3.32X12 − 2.61X22 − 7.03X32 (2)
Y2 = 178.26 + 2.18X1 + 13.83X2 + 15.38X3 − 3.50X1 X2 − 6.50X1 X3
− 11.50X2 X3 − 4.80X12 − 1.79X22 − 6.21X32 (3)
Y3 = 7.13 − 0.37X1 − 1.87X2 − 2.13X3 + 0.86X1 X2 + 1.11X1 X3 + 1.51X2 X3
+ 1.05X12 + 0.24X22 + 0.83X32
(4)
Y4 = 4.96 − 0.25X1 − 1.88X2 − 2.35X3 − 0.67X1 X2 − 0.94X1 X3
+ 1.68X2 X3 + 0.71X12 + 0.19X22 + 1.06X32 (5)
where Y1 is TPC (µg GAE/g DW), Y2 is TFC (µg catechol/g DW), Y3 is
DPPH (IC50; µg/ml), Y4 is ABTS (IC50; µg/ml), X1 is UAE time (min), X2 is
UAE temperature (℃), and X3 is ethanol concentration (%).
In the analysis, a good model of R 2 values was obtained from
the equations: 0.85, 0.82, 0.84, and 0.85, for TPC, TFC, DPPH, and
ABTS, respectively. These values indicated that the variation in pa-
2
rameters by models is representable. When R > 0.75, the model is
more suitable according to Le Man et al. (2010). At the same time,
ANOVA showed a highly significant p value between .047 and
<.0012 (Table 2). Furthermore, the lack of fit statistics for all param-
eters that calculate the models’ fitness was not significant (p > .05).
High F values (5.94–­21.64) confirmed the reliability of the models
within the process conditions studied.
3.3 | Response surface analysis of TPC
The effects of extraction factors X1 (ultrasonic time), X 2 (extraction
temperature), and X3 (ethanol concentrations) were studied. The
coefficient significance was indicated by F and p values. The cor-
relation between independent variables and the responses could be
presented as follows:
Y1 (𝜇g GAE/g DW) = 278.71 + 4.59X1 + 14.63X2 + 16.19X3 − 4.88X1 X2
− 7.87X1 X3 − 12.88X2 X3 − 3.32X12 − 2.61X22 − 7.03X32
(6)
| 5 of 10
The results showed a strong coefficient of regression between
TPC and extraction factors (R 2 = 0.855). Higher F value with lower
p value led to a greater correspondence between the different inde-
pendent variables. With p < .05, X 2, X3, X 2 X3, and X32 were significant.
However, X1, X1 X 2, X1 X3, X12, and X22 were not significant due to a higher
p value. Figure 1 shows the complex interaction of ultrasonic time,
extraction temperature, and ethanol concentration. A significant (p
> .05) high total phenolic yield was obtained at higher extraction
temperature (50℃) and lower ultrasonic time (44 min; Figure 2a).
However, fixed ultrasonic time (44 min) with increased extraction
temperature (50℃) led to a significant increase in total phenolic yield
(p > .05) and reached optimum at the shortest tested ultrasonic time
(44 min). Figure 2b shows the relationship between ethanol concen-
tration and extraction time, which affected the extraction efficiency
and consequently TPC. This optimization experiment emphasized
that phenolic content significantly (p > .05) increased with increased
ethanol concentration up to 58% and thereafter dropped. A signif-
icant increase in TPC was obtained at high extraction temperature
(50℃) and increasing ethanol-­to-­water ratio up to 57.7% (Figure 1c).
The extraction temperature up to 50℃ may increase and support
the solubility and diffusion of phenolic compounds in the extraction
solvent (ethanol). However, an increased temperature of >60℃ may
destruct polyphenolic compounds, causing a decrease in the antioxi-
dant activity (Dorta et al., 2012; Ling et al., 2009). Based on the RSM
results, ethanol concentration, followed by extraction temperature,
plays a crucial role in the efficiency of phenolic compound extraction
based on linear and quadratic coefficients (Table 3). These findings
suggested the optimum conditions for UAE methodology as an ecof-
riendly method for extracting polyphenols from different sources
(Feng et al., 2015; Ho et al., 2014; Viell et al., 2020).
3.4 | Response surface analysis of TFC
The acceptable regression value for TFC (R 2 = 0.82) and the cor-
relation between total flavonoid yield and extraction factors are re-
ported in Table 3. Quadratic polynomial equations are shown below
with significant terms of TFC of orange peel extracts that neglect
insignificant terms:
Y2 (mg catechol/g DW) = 178.26 + 2.18X1 + 13.83X2 + 15.38X3
(7)
− 3.50X1 X2 − 6.50X1 X3 − 11.50X2 X3 − 4.80X12 − 1.79X22 − 6.21X32
X2, X3, and X2 X3 are the most effective extraction parameters of
TFC of orange peels. In contrast, X1, X1 X2, X1 X3, X12, X22, and X32 had less
effect on TFC. Figure 3 shows the correlation between TFC and ex-
traction parameters. Figure 3a shows the effect of ultrasonic time, ex-
traction temperature, and their mutual interaction on TFC. There was
a significant increase (p > .05) in TFC with increasing ethanol concen-
tration (58%) at a lower extraction time (43 min), demonstrating the
effect of different ethanol concentrations, extraction time, and their
mutual interaction on TFC (Figure 3b). Thus, the maximum total fla-
vonoid yield was obtained at 58% ethanol concentration and 43 min
6 of 10 | SHEHATA et al.

TA B L E 3 Analysis of variance (ANOVA) for the fitted quadratic

polynomial model for optimization of extraction parameters

p-­Value

Source TPC TFC DPPH ABTS

Model 0.0070 0.0142 0.0094 0.0079

X1 0.2388 0.5725 0.4756 0.6238
X2 0.0030 0.0048 0.0048 0.0047
X3 0.0016 0.0026 0.0022 0.0012
X1 X2 0.3319 0.4906 0.2205 0.3349
X1 X3 0.1320 0.2148 0.1239 0.1869
X 2 X3 0.0241 0.0425 0.0471 0.0309
X12 0.3735 0.2188 0.0597 0.1769

X22 0.4800 0.6329 0.6342 0.6962

X32 0.0785 0.1211 0.1209 0.0601

Lack of fit 0.8191 0.7815 0.8666 0.7588
R2 0.85 0.82 0.84 0.85

with a higher ethanol concentration (58%) increased the total flavonoid

yield. The extraction time affected the solute-­solvent contact, leading
to increased extraction yield (Ghafoor et al., 2009). The experimental
trial generated by RSM in this study showed that the highest TFC yield
(191.144 mg CE/g DW) was observed at 58% ethanol concentration
and 43 min extraction time at 50℃ extraction temperature.

3.5 | Response surface analysis of

antioxidant activity

Table 3 shows the remarkable regression value for DDPH and ABTS
inhibition (R 2 = 0.84 and 0.85, respectively) and the correlation be-
tween antioxidant activity and extraction time, temperature, and
ethanol concentration. Quadratic polynomial equations are shown
below with significant terms of antioxidant activity of orange peel
extracts that neglect insignificant terms:

[ ]
Y3 DPPH − IC50 (𝜇g∕ml) = 7.13 − 0.37X1 − 1.87X2 − 2.13X3
+ 0.86X1 X2 + 1.11X1 X3 + 1.51X2 X3 + 1.05X12 + 0.24X22 + 0.83X32 (8)

[ ]
Y4 ABTS − IC50 (𝜇g∕ml) = 4.96 − 0.25X1 − 1.88X2 − 2.35X3
− 0.67X1 X2 − 0.94X1 X3 + 1.68X2 X3 + 0.71X12 + 0.19X22 + 1.06X32 (9)

F I G U R E 2 Response surface plots for the effect of (a)

temperature and time, (b) ethanol concentration and time, (c)
ethanol concentration and temperature on the total phenolic
content
extraction time. The separate effects of ethanol concentration, ex-
traction temperature, and their correlation on total flavonoid yield are
shown in Figure 3c. The increase of extraction temperature up to 50℃
X 2, X3, and X 2 X3 are the most significant parameters that af-
fected the antioxidant activity of orange peel extraction. X1, X1 X 2,
X1 X3, X12, X22, and X32 had little influence on antioxidant activity.
Figure 2 summarizes the relationship between antioxidant poten-
tial and extraction parameters. Extraction temperature and ethanol
concentration had remarkable effects on DPPH and ABTS values
of orange peel extracts. Optimization assessment emphasized that
SHEHATA et al. | 7 of 10

F I G U R E 3 Response surface plots for the effect of (a)

temperature and time, (b) ethanol concentration and time, (c) ethanol
concentration and temperature on the total flavonoids content

F I G U R E 4 Response surface plots for the effect of (a) temperature

ethanol-­to-­water ratio was the most effective parameter, followed and time, (b) ethanol concentration and time, (c) ethanol concentration
by extraction temperature and then extraction time (Figure 3 and and temperature on the antioxidant activity 2,20-­azino-­bis(3-­
Table 2). Extraction temperature and ethanol concentration showed ethylbenzothiazoline-­60-­sulfonic acid) diammonium salt
8 of 10 | SHEHATA et al.
a significant linear interaction and quadratic effect in the extraction
process. Figure 2a shows the different extraction time, tempera-
ture, and their mutual effect on antioxidant activity. Antioxidant
F I G U R E 5 Response surface plots for the effect of (a)
temperature and time, (b) ethanol concentration and time, (c)
ethanol concentration and temperature on the antioxidant activity
2,2-­diphenyl-­1-­picrylhydrazyl
potential increased when extraction temperature increased at a
shorter extraction time (43 min). Maximum antioxidant activity was
obtained at an extraction temperature of 50℃ and extraction time
of 43 min. The effects of ethanol concentration, ultrasonic time,
and their influence on antioxidant potential are shown in Figure 2b.
Based on obtained results, increasing ethanol concentrations up to
58% with a shorter ultrasonic time (43 min) increased antioxidant
activity, whereas increased temperature above 50℃ always eva-
nesced the activity. This might be due to the effect of high tem-
perature on the nature and structure of the phenolic compounds.
The antioxidant activity peak could be traced at an extraction tem-
perature of 50℃ and an ethanol concentration of 58% (Figure 2c).
Figure 3c shows that the highest antioxidant activity (lowest IC 50
values of DPPH = 5.198 μg/ml) significantly (p > .05) increased with
increased UAE temperature and ethanol concentration, reaching a
maximum value at 50℃ and 58.5% (Table 3). The quadratic effects
of extraction temperature and ethanol concentration on ABTS were
significant, indicating that these factors played an important role in
antioxidant activity as measured by ABTS (IC50 μg/ml) (Figure 5).
Figure 4a shows that the highest antioxidant activity by ABTS (low-
est IC50 values of ABTS = 2.985 μg/ml extract) was observed at
50℃, 58% ethanol, and 43 min extraction conditions. Meanwhile,
repulsive solute-­solvent interaction can be increased as extraction
temperature increases, leading to decreased antioxidant activity.
There was a strong correlation between TPC, TFC, and antioxidant
potentials of orange peel extracts, as reported previously (Abd El-­
Aziz et al., 2021; Li et al., 2016).
4 | CO N C LU S I O N S
This study focused on optimizing the Ultrasound Assisted Extraction
(UAE) of phenolic compounds from orange peels using RSM and a CCD
method for manipulating the most critical factors, such as extraction
time, extraction temperature, and ethanol concentration. The results
revealed that the extraction temperature and ethanol concentration
are the main effective factors of extraction efficiency. Ultrasound
Assisted Extraction (UAE) is an effective ecofriendly method for ex-
tracting phenolic compounds from orange peels with a considerable
yield and saving the extraction time. This optimized extraction method
could be useful in different food and pharmaceutical applications.
CO N S E NT FO R PU B LI C ATI O N
All authors have read and agreed to publish the current version of
the manuscript in the Journal of Food Processing and Preservation.
SHEHATA et al.
C O N FL I C T S O F I N T E R E S T
The author declares that there is no conflict of interest that could be
perceived as prejudicing the impartiality of the research reported.
E T H I C A L S TAT E M E N T
The work of current manuscript did not include animal or human
experiments.
AU T H O R C O N T R I B U T I O N S
review & editing: Sobhy
Supervision; Validation; Visualization; Writing-­
A EL-­Sohaimy. Conceptualization; Data curation; Formal analysis;
Investigation; Methodology; Software; Visualization; Writing-­original draft:
Mohammed Gamal Shehata. Methodology; Visualization; Writing-­original
draft: Nourhan M Abd El Aziz. Supervision; Validation; Visualization;
Writing-­review & editing: Mohammed Mahmoud Youssef.
DATA AVA I L A B I L I T Y S TAT E M E N T
Data and material of the current work is available with the corre-
sponding author upon request.
ORCID
Mohamed G. Shehata https://orcid.org/0000-0003-0760-5945
Mohamed M. Youssef https://orcid.org/0000-0002-7700-737X
Sobhy A. El-­Sohaimy https://orcid.org/0000-0002-1657-5162
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How to cite this article: Shehata, M. G., Abd El Aziz, N. M.,
Youssef, M. M., & El-­Sohaimy, S. A. (2021). Optimization
conditions of ultrasound-­assisted extraction of phenolic
compounds from orange peels using response surface
methodology. Journal of Food Processing and Preservation, 00,
e15870. https://doi.org/10.1111/jfpp.15870