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), Abstract
City of Scientific Research and Technology This study focused on optimizing the extraction conditions of bioactive compounds
Applications (SRTA-­City), Alexandria, 21934,
Egypt from orange peels using response surface methodology (RSM) to decrease the num-
2
Food Science and Technology Department, ber of experimental trials and increase the yield of bioactive compounds. Ultrasonic
Faculty of Agriculture, Alexandria University,
extraction time, extraction temperature, and ethanol concentration have been opti-
Alexandria, 21526, Egypt
3
Department of Technology and
mized. Twenty experimental trials have been carried out to optimize the extraction
Organization of Public Catering, Institute of conditions. The RSM results showed that the extraction temperature and ethanol
Sport, Tourism and Service, South Ural State
University, Chelyabinsk, 454080, Russia
concentration had a remarkable effect on the total phenolic content (TPC), total fla-
vonoid content (TFC), and antioxidant activity. The optimal ultrasound-­assisted ex-
Correspondence
Sobhy A. El-­Sohaimy, Food Technology
traction conditions were 44 min extraction time, 50℃ extraction temperature, and
Department, Arid Lands Cultivation 57.7% ethanol concentration. Under these optimal conditions, TPC and TFC were
Research Institute (ALCRI), City of Scientific
Research and Technology Applications
292.158 µg GAE/g and 191.144 µg catechol/g, respectively, whereas antioxidant ac-
(SRTA-­City), New Borg El-­Arab City, tivities were 5.199 and 2.96 µg/ml for 2,2-­diphenyl-­1-­picrylhydrazyl and 2,20-­azin
Alexandria, Egypt.
Email: elsohaimys@gmail.com
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 2020; The European Fruit Juice Association). This huge produc-
tion of orange juice worldwide produces a large amount of waste
Orange juice production is progressively increasing worldwide of peels, membranes, and seeds. This waste is mainly used as ani-
every year, except in 2020 due to the coronavirus disease 2019 mal feed or discarded in the environment (Abd El-­A ziz et al., 2021;
pandemic. The global orange juice production in 2019 was about Badr et al., 2020; Garcia-­C astello et al., 2015; Sharma et al., 2017;
2.2 million metric tons (Citrus: World Markets and Trade, July 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- TA B L E 1 Coded and uncoded factors and their range level using
ceutical purposes (Abdel-­Razek et al., 2020; Awad et al., 2020). in central composite design

Notwithstanding, citrus peels are an excellent source of phenolic Independent variables Coded and uncoded levels
acids and flavonoids, vitamin C, folate, and potassium with sev-
X1: ultrasonic time (min) 40 (−1) 50 (0) 60 (+1)
eral biological benefits (anti-­
inflammatory, anticarcinogenic, an-
X2: Extraction temperature (°C) 30 (−1) 40 (0) 50 (+1)
tidegenerative, antioxidant, and antimicrobial properties; Oboh &
X3: Ethanol concentration (%) 40 (−1) 50 (0) 60 (+1)
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 2.2 | Optimization procedure
et al., 2008; Mandal et al., 2007). To increase the exploitation of
citrus peels, an ecofriendly extraction method can be applied to re- 2.2.1 | Single-­factor experiments
cover bioactive compounds from citrus waste. “Green” extraction
technology can decrease solvents, shorten the extraction time, Extraction temperature (30–­70℃), extraction time (10–­8 0 min), and
lower energy, and decrease environmental pollution and uses safe ethanol-­to-­water ratio (30%–­8 0%) were investigated as parameters
thermolabile constituents. Green extraction techniques, such as that influence the extraction yield in the experimental design. The
ultrasound-­assisted extraction (UAE), supercritical extraction, and experimental results of a single-­factor and the impact of extraction
microwave-­
assisted extraction, have specific applications (Kate parameters and their upper and lower levels on the RSM design were
et al., 2016; Liović et al., 2021; Viell et al., 2020). Ultrasound waves applied.
(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 2.2.2 | Experimental design
of UAE are reduced extraction time, saving energy, and solvent use,
faster energy transfer, reduced extraction temperature, reduced CCD was used to evaluate responses and the optimum combina-
equipment size, faster response to the process, and quick start-­up tion of UOD variables, including three extraction parameters at
(Chemat et al., 2004; Viell et al., 2020). three levels (Table 1). Extraction temperature (℃), extraction time
Response surface methodology (RSM) enables the evaluation (min), and ethanol concentration (%) were the three independent
of the effects of many factors and their interactions on response variables. Total phenolic content (TPC), total flavonoid content
variables. The main advantage of RSM is the reduced number of (TFC), 2,2-­diphenyl-­1-­picrylhydrazyl (DPPH), and 2,20-­azino-­bis(
experimental trials needed to evaluate multiple parameters and 3-­ethylbenzothiazoline-­6 0-­sulfonic acid) diammonium salt (ABTS)
their interactions; therefore, it is less laborious and time-­consuming results of the dependent parameters were included. Twenty ex-
than other approaches required to optimize a process (Aybastıer & perimental trials were given by the complete design (Table 2). The
Demir, 2010; Bezerra et al., 2008; Lee et al., 2000). The most com- RSM polynomial equation of second degree (Equation [1]) was
mon designs (i.e., central composite design [CCD] and Box-­Behnken used as follows:
design) of RSM have been widely used in various experiments. CCD,
a spherical and revolving design, has been applied to optimize chem- Y = b0 + b1 X1 + b2 X2 + b3 X3 + b11 X12 + b22 X22 + b33 X32 + b12 X1 X2 + b13 X1 X3 + b23 X2 X3

ical and physical processes due to its reasoning design and excellent (1)
outcomes (Aybastıer & Demir, 2010). RSM was used in this study to
manipulate and optimize the critical factors for ecofriendly UAE to The intercept and linear, quadratic, and interaction coefficients
recover antioxidant compounds from orange peels. 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
2 | M ATE R I A L S A N D M E TH O DS effects of optimization on TPC, TFC, and antioxidant activity of lin-
ear, quadratic, and interaction variables. Design-­E xpert version 12
2.1 | Plant materials and chemicals (Stat-­Ease, Inc., Minneapolis, MN, USA) was used to analyze the data.

Orange fruits (Citrus sinensis) obtained from a local market in

Alexandria, Egypt in 2019, were washed and manually treated. The 2.3 | Ultrasonic assisted extraction (UAE)
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; Dried powder (10 g) was applied to 100 ml ethanol (1:10, wt/vol)
0.04 Mbar). Freeze-­dried peels were crushed to fine powder using a and carried out in a tunable cold ultrasonic bath (4℃) for 30 s for
grinder (Waring Commercial) and stored at −20℃ for further analy- phenolic extraction. The extract was centrifuged at 12,000×g for
sis. All analytical-­grade chemicals were obtained from Sigma–­Aldrich 15 min at 20℃ and filtered with Whatman No. 1 filter paper. A
(St. Louis, MO, USA). 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 2.6 | DPPH radical-­scavenging activity
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 The antiradical activities of the extracts were determined based on
7.9 ml deionized water and 0.5 ml FCR. The mixture was left in the a reaction with stable DPPH radicals dissolved in absolute ethanol
dark for 5 to 8 min before 1.5 ml of 20% sodium carbonate solution (Shehata, Ahmad, et al., 2020; Shehata, Darwish, et al., 2020). The
was added to the sample. The absorbance value of the sample was reaction requires preparing a mixture of 500 μl extracts at various
then read using an ultraviolet-­visible spectrophotometer at a wave- concentrations with 375 μl ethanol and 125 μl DPPH solution (0.02%
length of 765 nm. 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-
2.5 | Total flavonoids content (TFC) 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. | 5 of 10

and effective parameters of phenolic compound extraction effi- The results showed a strong coefficient of regression between
ciency (Table 1). 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.
3.2 | Fitting the model 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,
Table 1 lists the values coded and decoded and the responses of extraction temperature, and ethanol concentration. A significant (p
each independent variable. TPC of orange peel extracts ranged from > .05) high total phenolic yield was obtained at higher extraction
195 to 295 µg/g dried extract. TFC ranged from 106 to 195 µg/g temperature (50℃) and lower ultrasonic time (44 min; Figure 2a).
extract. Table 1 also shows the antioxidant potential as IC50, which However, fixed ultrasonic time (44 min) with increased extraction
ranged from 4.7 to 17.3 µg/ml for DPPH and 2.65 to 14.9 µg/ml for temperature (50℃) led to a significant increase in total phenolic yield
ABTS. Equations (2)‒­(5) show the independent variables and re- (p > .05) and reached optimum at the shortest tested ultrasonic time
sponse correlation: (44 min). Figure 2b shows the relationship between ethanol concen-
tration and extraction time, which affected the extraction efficiency
Y1 = 278.71 + 4.59X1 + 14.63X2 + 16.19X3 − 4.88X1 X2 − 7.87X1 X3 and consequently TPC. This optimization experiment emphasized
− 12.88X2 X3 − 3.32X12 − 2.61X22 − 7.03X32 (2) that phenolic content significantly (p > .05) increased with increased
ethanol concentration up to 58% and thereafter dropped. A signif-
Y2 = 178.26 + 2.18X1 + 13.83X2 + 15.38X3 − 3.50X1 X2 − 6.50X1 X3 icant increase in TPC was obtained at high extraction temperature
− 11.50X2 X3 − 4.80X12 − 1.79X22 − 6.21X32 (3) (50℃) and increasing ethanol-­to-­water ratio up to 57.7% (Figure 1c).
The extraction temperature up to 50℃ may increase and support
Y3 = 7.13 − 0.37X1 − 1.87X2 − 2.13X3 + 0.86X1 X2 + 1.11X1 X3 + 1.51X2 X3 the solubility and diffusion of phenolic compounds in the extraction

+ 1.05X12 + 0.24X22 + 0.83X32 solvent (ethanol). However, an increased temperature of >60℃ may
(4)
destruct polyphenolic compounds, causing a decrease in the antioxi-
dant activity (Dorta et al., 2012; Ling et al., 2009). Based on the RSM
Y4 = 4.96 − 0.25X1 − 1.88X2 − 2.35X3 − 0.67X1 X2 − 0.94X1 X3
results, ethanol concentration, followed by extraction temperature,
+ 1.68X2 X3 + 0.71X12 + 0.19X22 + 1.06X32 (5)
plays a crucial role in the efficiency of phenolic compound extraction
based on linear and quadratic coefficients (Table 3). These findings
where Y1 is TPC (µg GAE/g DW), Y2 is TFC (µg catechol/g DW), Y3 is suggested the optimum conditions for UAE methodology as an ecof-
DPPH (IC50; µg/ml), Y4 is ABTS (IC50; µg/ml), X1 is UAE time (min), X2 is riendly method for extracting polyphenols from different sources
UAE temperature (℃), and X3 is ethanol concentration (%). (Feng et al., 2015; Ho et al., 2014; Viell et al., 2020).
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- 3.4 | Response surface analysis of TFC
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, The acceptable regression value for TFC (R 2 = 0.82) and the cor-
ANOVA showed a highly significant p value between .047 and relation between total flavonoid yield and extraction factors are re-
<.0012 (Table 2). Furthermore, the lack of fit statistics for all param- ported in Table 3. Quadratic polynomial equations are shown below
eters that calculate the models’ fitness was not significant (p > .05). with significant terms of TFC of orange peel extracts that neglect
High F values (5.94–­21.64) confirmed the reliability of the models insignificant terms:
within the process conditions studied.
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

3.3 | Response surface analysis of TPC

X2, X3, and X2 X3 are the most effective extraction parameters of
The effects of extraction factors X1 (ultrasonic time), X 2 (extraction TFC of orange peels. In contrast, X1, X1 X2, X1 X3, X12, X22, and X32 had less
temperature), and X3 (ethanol concentrations) were studied. The effect on TFC. Figure 3 shows the correlation between TFC and ex-
coefficient significance was indicated by F and p values. The cor- traction parameters. Figure 3a shows the effect of ultrasonic time, ex-
relation between independent variables and the responses could be traction temperature, and their mutual interaction on TFC. There was
presented as follows: a significant increase (p > .05) in TFC with increasing ethanol concen-
tration (58%) at a lower extraction time (43 min), demonstrating the
Y1 (𝜇g GAE/g DW) = 278.71 + 4.59X1 + 14.63X2 + 16.19X3 − 4.88X1 X2
effect of different ethanol concentrations, extraction time, and their
− 7.87X1 X3 − 12.88X2 X3 − 3.32X12 − 2.61X22 − 7.03X32
mutual interaction on TFC (Figure 3b). Thus, the maximum total fla-
(6)
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) X 2, X3, and X 2 X3 are the most significant parameters that af-
ethanol concentration and temperature on the total phenolic fected the antioxidant activity of orange peel extraction. X1, X1 X 2,
content X1 X3, X12, X22, and X32 had little influence on antioxidant activity.
Figure 2 summarizes the relationship between antioxidant poten-
extraction time. The separate effects of ethanol concentration, ex- tial and extraction parameters. Extraction temperature and ethanol
traction temperature, and their correlation on total flavonoid yield are concentration had remarkable effects on DPPH and ABTS values
shown in Figure 3c. The increase of extraction temperature up to 50℃ 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 F I G U R E 5 Response surface plots for the effect of (a)
process. Figure 2a shows the different extraction time, tempera- temperature and time, (b) ethanol concentration and time, (c)
ethanol concentration and temperature on the antioxidant activity
ture, and their mutual effect on antioxidant activity. Antioxidant
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. | 9 of 10

C O N FL I C T S O F I N T E R E S T Chemat, S., Lagha, A., AitAmar, H., Bartels, P. V., & Chemat, F. (2004).
Comparison of conventional and ultrasound-­assisted extraction of
The author declares that there is no conflict of interest that could be
carvone and limonene from caraway seeds. Flavour and Fragrance
perceived as prejudicing the impartiality of the research reported. Journal, 19, 188–­195.
Contini, M., Baccelloni, S., Massantini, R., & Anelli, G. (2008). Extraction
E T H I C A L S TAT E M E N T of natural antioxidants from hazelnut (Corylus avellana L.) shell and
The work of current manuscript did not include animal or human skin wastes by long maceration at room temperature. Food Chemistry,
110, 659–­669.
experiments.
Dewanto, V., Wu, X., Adom, K. K., & Liu, R. H. (2002). Thermal process-
ing enhances the nutritional value of tomatoes by increasing total
AU T H O R C O N T R I B U T I O N S antioxidant activity. Journal of Agriculture and Food Chemistry, 50(10),
review & editing: Sobhy
Supervision; Validation; Visualization; Writing-­ 3010–­3 014.
Dorta, E., Lobo, M. G., & Gonzalez, M. (2012). Reutilization of mango
A EL-­Sohaimy. Conceptualization; Data curation; Formal analysis;
by-­products: Study of the effect of extraction solvent and tempera-
Investigation; Methodology; Software; Visualization; Writing-­original draft: ture on their antioxidant properties. Journal of Food Science, 77(1),
Mohammed Gamal Shehata. Methodology; Visualization; Writing-­original C80–­C88.
draft: Nourhan M Abd El Aziz. Supervision; Validation; Visualization; Feng, S. M., Luo, Z. S., Tao, B. P., & Chen, C. (2015). Ultrasonic-­assisted
extraction and purification of phenolic compounds from sugarcane
Writing-­review & editing: Mohammed Mahmoud Youssef.
(Saccharum officinarum L.) rinds. LWT, 60(2), 970–­976.
Garcia-­C astello, E. M., Rodriguez-­Lopez, A. D., Mayor, L., Ballesteros, R.,
DATA AVA I L A B I L I T Y S TAT E M E N T Conidi, C., & Cassano, A. (2015). Optimization of conventional and
Data and material of the current work is available with the corre- ultrasound assisted extraction of flavonoids from grapefruit (Citrus
paradisi L.) solid wastes. LWT, 64, 1114–­1122.
sponding author upon request.
Ghafoor, K., Choi, Y. H., Jeon, J. Y., & Jo, I. H. (2009). Optimization of
ultrasound-­ assisted extraction of phenolic compounds, antioxi-
ORCID dants, and antho-­c yanins from grape (Vitis vinifera) seeds. Journal of
Mohamed G. Shehata https://orcid.org/0000-0003-0760-5945 Agriculture and Food Chemistry, 57(11), 4988–­4994.
Hashem, N. M., & Shehata, M. G. (2021). Antioxidant and antimicro-
Mohamed M. Youssef https://orcid.org/0000-0002-7700-737X
bial activity of Cleome droserifolia (Forssk.) Del. and its biological
Sobhy A. El-­Sohaimy https://orcid.org/0000-0002-1657-5162 effects on redox status, immunity, and gut microflora. Animals, 11,
1929.
REFERENCES Ho, S. K., Tan, C. P., Thoo, Y. Y., Abas, F., & Ho, C. W. (2014). Ultrasound-­
Abd El-­A ziz, N. M., Awad, O. M., Shehata, M. G., & El-­Sohaimy, S. A. assisted extraction of antioxidants in Misai Kucing (Orthosiphon
(2021). Antioxidant and anti-­acetylcholinesterase potential of arti- stamineus). Molecules, 19(8), 12640–­12659.
choke phenolic compounds. Food Bioscience, 41, 101006. https://doi. Kate, A. E., Singh, A., Shahi, N. C., Pandey, J. P., Om, P., & Singh, T. P.
org/10.1016/j.fbio.2021.101006 (2016). Novel eco-­friendly techniques for extraction of food based
Abdel-­Razek, A. G., Shehata, M. G., Badr, A. N., Gromadzka, K., & Stepin, lipophilic compounds from biological materials. Natural Products
L. (2020). The effect of chemical composition of wild Opuntia Ficus Chemistry and Research, 4, 231.
Indica by-­products on its nutritional quality, antioxidant and antifun- Le Man, H., Behera, S. K., & Park, H. S. (2010). Optimization of operational
gal efficacy. Egyptian Journal of Chemistry, 63(7), 1–­15. parameters for ethanol production from Korean food waste leachate.
Ahmad-­Q asem, M. H., Cánovas, J., Barrajón-­C atalán, E., Micol, V., Cárcel, International Journal of Environmental Science and Technology, 7(1),
J. A., & García-­Pérez, J. V. (2013). Kinetic and compositional study of 157–­164.
phenolic extraction from olive leaves (var. Serrana) by using power Lee, J., Ye, L., William, O. L., Eitenmiller, J. R., & Ronaldo, R. (2000).
ultrasound. Innovative Food Science and Emerging Technologies, 17, Optimization of an extraction procedure for the quantification of vi-
120–­129. tamin E in tomato and broccoli using response surface methodology.
Awad, O. M., El-­Sohaimy, S. A., Ghareeb, D. A., Aboul-­enein, A. M., Saleh, Journal of Food Composition and Analysis, 13, 45–­57.
S. R., & Abd El-­A ziz, N. M. (2020). Phytochemical analysis and toxicity Li, H., Zhang, Z., Xue, J., Cui, L., Hou, T., Li, X., & Chen, T. (2016).
assessment of artichoke by-­product extract. PJBS, 23, 81–­91. Optimization of ultrasound-­ assisted extraction of phenolic com-
Aybastıer, O., & Demir, C. (2010). Optimization of immobilization condi- pounds, antioxidants and rosmarinic acid from perilla leaves using
tions of Thermomyces lanuginosus lipase on styrene–­divinylbenzene response surface methodology. Food Science and Technology
copolymer using response surface methodology. Journal of Molecular (Campinas), 36(4), 686–­693.
Catalysis, 63, 170–­178. Ling, L. T., Yap, S. A., Radhakrishnan, A. K., Subramaniam, T., Cheng, H.
Badr, A. N., Ali, H. S., Abdel-­Razek, A. G., Shehata, M. G., & Albaridi, N. A. M., & Palanisamy, U. D. (2009). Standardised Mangifera indica extract
(2020). Bioactive components of pomegranate oil and their influence is an ideal antioxidant. Food Chemistry, 113(4), 1154–­1159.
on mycotoxin secretion. Toxins, 12, 748. https://doi.org/10.3390/ Liović, N., Bratanić, A., Zorić, Z., Pedisić, S., Režek Jambrak, A., Krešić,
toxin​s1212​0748 G., & Bilušić, T. (2021). The effect of freeze-­drying, pasteurisation
Bezerra, M. A., Santelli, R. E., Oliveira, E. P., Villar, L. S., & Escaleira, L. A. and high-­intensity ultrasound on gastrointestinal stability and anti-
(2008). Response surface methodology (RSM) as a tool for optimiza- oxidant activity of blueberry phenolics. International Journal of Food
tion in analytical chemistry. Talanta, 76, 965–­977. Science & Technology, 56, 1996–­2008.
Carrera, C., Ruiz-­ Rodríguez, A., Palma, M., & Barroso, C. G. (2012). Liu, Y., Wei, S., & Liao, M. (2013). Optimization of ultrasonic extraction of
Ultrasound assisted extraction of phenolic compounds from grapes. phenolic compounds from Euryale ferox seed shells using response
Analytica Chimica Acta, 732, 100–­104. surface methodology. Industrial Crops and Products, 49, 837–­8 43.
Chemat, F., Tomao, V., & Virot, M. (2009). Ultrasound-­assisted extraction Mandal, V., Mohan, Y., & Hemalatha, S. (2007). Microwave assisted ex-
in food analysis. In S. Otles (Ed.), Handbook of food analysis instru- traction—­An innovative and promising extraction tool for medicinal
ments (1st ed., pp. 85‒­94). CRC Press. ISBN 9781420045666. plant research. Pharmacognosy Reviews, 1, 7–­18.
10 of 10 | SHEHATA et al.

Oboh, G., & Ademosun, A. O. (2012). Characterization of the antioxidant Shehata, M. G., Darwish, A. M. G., & El-­ Sohaimy, S. A. (2020).
properties of phenolic extracts from some citrus peels. Journal of Physicochemical, structural and functional properties of water-­
Food Science and Technology, 49(6), 729–­736. soluble polysaccharides extracted from Egyptian agricultural by-­
Rafiq, S., Kaul, R., Sofi, S. A., Bashir, N., Nazir, F., & Ahmad Nayik, G. products. Annals of Agricultural Sciences, 65, 21–­27.
(2018). Citrus peel as a source of functional ingredient: A review. Singleton, V. L., Orthofer, R., & Lamuela-­Ravent, R. M. (1999). Analysis of
Journal of the Saudi Society of Agricultural Sciences, 17, 351–­358. total phenols and other oxidation substrates and antioxidants by means
https://doi.org/10.1016/j.jssas.2016.07.006 of folin-­ciocalteu reagent. Methods in Enzymology, 299, 152–­178.
Re, R., Pellegrini, N., Proteggente, A., Pannala, A., Yang, M., & Rice-­Evans, C. Sun, Y., Xu, W., Zhang, W., Hu, Q., & Zeng, X. (2011). Optimizing the
(1999). Antioxidant activity applying an improved ABTS radical cation extraction of phenolic antioxidants from kudingcha made from
decolorization assay. Free Radical Biology and Medicine, 26, 1231–­1237. Ilex kudingcha C.J. Tseng by using response surface methodology.
Şahin, S., & Şamlı, R. (2013). Optimization of olive leaf extract obtained Separation and Purification Technology, 78, 311–­320.
by ultrasound-­assisted extraction with response surface methodol- Viell, F. L. G., Madeira, T. B., Nixdorf, S. L., Gomes, S. T. M., Bona, E., &
ogy. Ultrasonics Sonochemistry, 20, 595–­602. Matsushita, M. (2020). Comparison between ultra-­homogenisation
Sharma, K., Mahato, N., Cho, M. H., & Lee, Y. R. (2017). Converting citrus and ultrasound for extraction of phenolic compounds from teff
wastes into value-­added products: Economic and environmentally (Eragrostis tef (Zucc.)). International Journal of Food Science &
friendly approaches. Nutrition, 34, 29–­46. Technology, 55, 2700–­2709.
Shehata, M. G., Abu-­Serie, M. M., Abd El-­A ziz, N. M., & El-­Sohaimy, S. A.
(2021). Nutritional, phytochemical, and in vitro anticancer potential
of sugar apple (Annona squamosa) fruits. Scientific Reports, 11, 6224.
How to cite this article: Shehata, M. G., Abd El Aziz, N. M.,
Shehata, M. G., Ahmad, F. T., Badr, A. N., Masry, S. H., & El-­Sohaimy, S.
A. (2020). Chemical analysis, antioxidant, cytotoxic and antimicrobial
Youssef, M. M., & El-­Sohaimy, S. A. (2021). Optimization
properties of propolis from different geographic regions. Annals of conditions of ultrasound-­assisted extraction of phenolic
Agricultural Sciences, 65, 209–­217. compounds from orange peels using response surface
Shehata, M. G., Awad, T. S., Asker, D., El Sohaimy, S. A., Abd El-­Aziz, N. methodology. Journal of Food Processing and Preservation, 00,
M., & Youssef, M. M. (2021). Antioxidant and antimicrobial activities
e15870. https://doi.org/10.1111/jfpp.15870
and UPLC-­ESI-­MS/MS polyphenolic profile of sweet orange peel
extracts. Current Research in Nutrition and Food Science, 4, 326–­335.