Optimization of ultrasonic-assisted extraction of pomegranate (Punica granatum L.) peel antioxidants by response surface methodology

Optimization of ultrasonic-assisted extraction of pomegranate (Punica granatum L.) peel antioxidants by response surface methodology

Separation and Purification Technology 98 (2012) 16–23 Contents lists available at SciVerse ScienceDirect Separation and Purification Technology journ...

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Separation and Purification Technology 98 (2012) 16–23

Contents lists available at SciVerse ScienceDirect

Separation and Purification Technology journal homepage: www.elsevier.com/locate/seppur

Optimization of ultrasonic-assisted extraction of pomegranate (Punica granatum L.) peel antioxidants by response surface methodology Reza Tabaraki a,⇑, Elham Heidarizadi a, Ali Benvidi b a b

Department of Chemistry, Faculty of Science, Ilam University, Ilam, Iran Department of Chemistry, Faculty of Science, Yazd University, Yazd, Iran

a r t i c l e

i n f o

Article history: Received 19 September 2011 Received in revised form 13 June 2012 Accepted 27 June 2012 Available online 5 July 2012 Keywords: Ultrasonic Antioxidants Pomegranate peel Response surface methodology

a b s t r a c t The carcinogenic effects of synthetic antioxidants in foods have led to increased interest in natural sources of antioxidants. Food industries produce substantial quantities of phenolics-rich by-products, which have gained much attention due to their antioxidant properties. Ultrasound assisted extraction (UAE) was applied for the extraction of polyphenol and antioxidants from pomegranate (Punica granatum L.) peel using ethanol–water mixture as a food grade solvent. A central composite design (CCD) and response surface methodology (RSM) were used to optimize experimental conditions for extraction of polyphenol and antioxidants. The independent processing variables were solvent type, solvent to solid ratio, particle size, ethanol concentration (% v:v), temperature (°C) and time (min). The dependent variables were total phenolic content (TPC), ferric reducing antioxidant power (FRAP), scavenging activity of 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical and yield. TPC varied from 5506.42 to 8923.24 mg gallic acid equivalent/100 g of dry weight. FRAP and DPPH values varied from 24.30 to 63.37 mmol Fe2+/100 g dry weight and 60.12–83.52% inhibition, respectively. Extraction yields ranged from 29.78% to 45.38%. The extract can be used as substitute of synthetic antioxidants for food products, color and oxidative stabilization. Therefore, the highest yield is recommended for industrial applications. The optimal conditions for this aim were 70% ethanol–water mixture as solvent, temperature of 60 °C and extraction time of 30 min. The experimental values agreed with those predicted by RSM models, thus indicating suitability of the model employed and the success of RSM in optimizing the extraction conditions. Ó 2012 Elsevier B.V. All rights reserved.

1. Introduction Reactive oxygen species (ROS) exist in the aerobic metabolism. The reactive oxygen species are known to play a major role in either the initiation or progression of carcinogenesis by inducing oxidative stress. Since free radicals are toxic to cells, body has developed a sophisticated antioxidant system that essentially relies upon antioxidant nutrients in order to protect our health from oxidative stress. The oxidative stress can damage cellular lipids, proteins or DNA. Antioxidants counter act free radicals and prevent the damage by crumbling oxidants, preventing chain reactions or preventing the activation of oxygen to highly reactive products [1]. Along the past two decades, several methods such as the ABTS (2,2-azinobis-(3-ethylbenzobis-(3-ethylbenzothiazoline-6-sulfonate))) radical cation assay [2], the DPPH (2,2-diphenyl-1-picrylhydrazyl radical) assay [3], the ferric reducing antioxidant power (FRAP) assay [4], oxygen radical absorbance capacity (ORAC) assay [5] and the electrochemical estimation of ⇑ Corresponding author. Tel./fax: +98 841 2227022. E-mail addresses: [email protected], [email protected] (R. Tabaraki). 1383-5866/$ - see front matter Ó 2012 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.seppur.2012.06.038

total reducing capacity [6] have been proposed for assessment of total antioxidant capacity. Pomegranate (Punica granatum L.) is known as one of the healthiest fruits due to its high antioxidant activity [7,8]. The pomegranate means a seeded or granular apple (from the Latin words pomus and granatus) and has been cultivated for thousands of years [9]. The total production of pomegranate in Iran was 700,000 tons in 2011 [10]. The pharmacological functions of pomegranate include anti-inflammatory, anti-tumor, anti-hepatotoxicity, anti-lipoperoxidation and anti-bacteria properties [11]. Potent antioxidant properties were attributed to its high content of polyphenols including ellagitannins, ellagic acid, and other flavonoids (quercetin, kaempferol, and luteolin glycosides). The most abundant of these polyphenols is punicalagin, an ellagitannin implicated as the bioactive constituent responsible for potent antioxidant activity [12]. Nutrient values of raw pomegranate per 100 g fruit are: moisture, 77.93 g; protein, 1.67 g; total lipid, 1.17 g; carbohydrate 18.70 g; fiber 4.0 g; sugars, 13.67 g; vitamin C, 10.2 mg; vitamin E, 0.60 mg; vitamin K, 16.4 lg; niacin, 0.293 mg [13]. Total phenolics (gallic acid equivalents) of all pomegranate fruit parts were reported by Aviram et al [14].

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R. Tabaraki et al. / Separation and Purification Technology 98 (2012) 16–23 Table 1 Experimental details of reported studies of extraction of antioxidants from pomegranate peel. Method

Solvent

T (°C)

t (min)

Magnetic stirrer Soxhlet extraction Centrifugation Solvent extraction SFE Solvent extraction Ultrasonic Solvent extraction Ultrasonic Ultrasonic

EtOAc, MeOH, water EtOAc, MeOH, water Water Acetone, MeOH, EtOH MeOH Acetone Acetone Water MeOH Water

30 – – – 40 Room Room 25 – 25

60 240 10 – 10 360 30 2–90 120 2–90

Variable optimization

Ref.

No No No No Yes No No One-at-a-time No One-at-a-time

[22] [23] [24] [25] [26] [26] [26] [27] [28] [29]

T, extraction temperature; t, time; SFE, supercritical fluid extraction; MeOH, methanol; EtOH, ethanol; EtOAC, ethylacetate.

In recent years, there has been an increasing interest in extraction of antioxidants from agricultural and industrial by-products [15]. The extraction of bioactive compounds under ultrasound irradiation (20–100 kHz) is one of the upcoming extraction techniques that can offer shorter operation times, simplified manipulation, reduced solvent consumption and temperature and lower energy input. Hence, ultrasound-assisted extraction (UAE) can be called an ‘‘environment-friendly’’ or ‘‘green’’ technique [16]. The main advantages of ultrasound-assisted extraction over conventional Soxhlet extraction are: (a) cavitation increases the polarity of the system and increases the extraction efficiency, (b) allows the extraction of thermolabile analytes and (c) operating time is invariably shorter. Disadvantages of ultrasound-assisted extraction with respect to conventional Soxhlet extraction are: (a) the solvent cannot be renewed during process, so its efficiency is a function of its partitioning coefficient, (b) the need for filtering and rinsing after extraction increases solvent consumption and risk of losses or contamination of the extract and (c) Soxhelt extraction is usually more reproducible [17]. The ultrasound-based technique surpasses supercritical fluid extraction (SFE) in the following respects: (a) the equipment is much simpler, so the overall cost of the extraction is much lower and (b) allows the extraction of wide variety of compound with different polarity. However, SFE surpasses ultrasound-assisted extraction in following respects: (a) the SFE technique is simpler and faster, (b) supercritical CO2 is not environmentally hazardous and (c) usually, SFE methods are more precise [17]. Ultrasound-assisted extraction provides advantages over microwave-assisted extraction as follows: (a) it is sometimes faster and (b) in many cases, the ultrasonic procedure is simpler. Disadvantages of ultrasound-assisted extraction when compared to microwave-assisted extraction are: (a) it is usually less robust and (b) particle size is a critical factor in ultrasound-assisted applications [17]. The enhancement of extraction efficiency of organic compounds by ultrasound is attributed to the phenomenon of cavitation produced in the solvent by the passage of an ultrasonic wave. Cavitation bubbles are produced and compressed during the application of ultrasound. The increase in the pressure and temperature caused by the compression leads to the collapse of the bubble [18]. Shock waves and microjets generated during inertial cavitations are responsible for the transdermal permeability enhancement. Shock waves are generated in the process of a spherical collapse of bubbles, which in turn yields high pressure cores that emit shock waves with amplitudes exceeding 10 kbar. Microjets are formed when a flat solid surface causes the bubble to involute and develop a high-speed liquid microjet towards the solid surface. Because most of the available energy is transferred to the accelerating microjet, rather than the bubble wall itself, this microjet can reach velocities of hundreds of meters per second [19]. Very high effec-

tive temperatures (which increase solubility and diffusivity) and pressure (which favor penetration and transport) at the interface between solution subjected to ultrasonic energy and a solid matrix result in high extractive power [17]. Response surface methodology (RSM) has been used to optimize food processing operations in recent years [20]. Response surface methodology is a collection of mathematical and statistical techniques based on the fit of a polynomial equation to the experimental data, which must describe the behavior of a data set with statistical previsions. It can be well applied when a response or a set of responses of interest are influenced by several variables. The objective is simultaneously optimization of these variables to attain the best system performance. The main advantage of RSM is a reduced number of experimental trails needed to evaluate multiple parameters and their interactions. Therefore it is less laborious and time-consuming than other approaches required to optimize a process [21]. High extraction efficiency is advantage for an industrial process. Many factors influence the extraction efficiency such as extraction methods, solvent type, solvent concentration, extraction temperature and time. The extraction of antioxidants from pomegranate peel has been reported [22–29]. Experimental details of these reports are shown in Table 1. In the most of these works, methanol and acetone came up as suitable extraction solvents to reach good yields. However, environmentally benign and non-toxic food grade organic solvents like ethanol, n-butanol is recommended by the US Food and Drug Administration for extraction purposes [30]. Although studies have been published on the ultrasonic-assisted extraction of antioxidants from pomegranate peel, these studies have not evaluated the interaction of the experimental variables. Therefore, due to restriction of synthetic antioxidants use in foods and also due to the large annual production of pomegranate peels as a by-product of the juice industries, the objective of this study was to optimize the ultrasonic-assisted extraction of natural antioxidants from pomegranate peel by response surface methodology for preparation of food grade extracts. 2. Materials and methods 2.1. Plant material Pomegranates were purchased from local markets. Fruits were manually peeled and collected peels were then rinsed with distilled water. The peels were dried in an oven (Memmert, GmbH+Co. KG, DIN 40050, Germany) with air circulation at 40 °C, and they were finely ground in a laboratory grinder (Pars Co., Iran). The ground sample was fractioned by a series of sieves (0.85, 0.425, 0.25 and 0.18 mm, Damavand Co., Iran) to obtain the particle size distribution. The dry sample was then stored at 20 °C.

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Table 2 Coded and uncoded levels of the independent variables. Independent variables

Code units

Ethanol concentration (%) Temperature (°C) Time (min)

X1 X2 X3

Coded levels 1

0

1

30 30 10

50 45 20

70 60 30

2.2. Chemicals 2,4,6-tris (2-pyridyl)-s-triazine (TPTZ), Folin–Ciocalteu (FC) reagent and gallic acid were purchased from Merck. 2,2-diphenyl-1-picrylhydrazyl (DPPH) was purchased from Sigma–Aldrich. 2.3. Extraction procedure The process of extraction from pomegranate peel powder by ultrasonic was performed in an ultrasonic bath RK103H (BANDELIN SONOREX, Germany) with a maximum capacity of 4 L (35 kHz, 140 W). Pomegranate peel powder (0.2 g) was sonicated in the solvent (10 ml) for different times at required temperature. Then, the pomegranate peel extract was centrifuged at 4500 rpm for 10 min. The extracts were concentrated by rotary evaporation at 40 °C under vacuum to dryness and the yield of extraction was determined. 2.4. Experimental design A central composite design (CCD) was used to identify the relationship between three independent factors and the dependent variables (responses). The independent variables were ethanol concentration (X1), extraction temperature (X2) and extraction time (X3) and each factor was set at three separate coded levels (Table 2). The total phenolic content (Y1), FRAP value (Y2), DPPH scavenging activity (Y3) and extraction yield (Y4) were chosen as the dependent variables. Experimental data were fitted to the following second order polynomial model and regression coefficients were obtained. The generalized second-order polynomial model proposed for the response surface analysis was given as follows:

Y ¼ b0 þ

3 3 2 X 3 X X X bi X i þ bii X 2i þ bij X i Y j i¼1

i

i¼1 j¼iþ1

where b0, bi, bii, bij are regression coefficients for intercept, linear, quadratic and interaction terms, respectively. Xi and Xj are coded value of the independent variables while k equals to the number of the tested factors (k = 3). All of the experiments were carried out in triplicates and the experimental results were expressed as mean ± standard deviations. Statistical analysis was performed by using the Minitab 15.1 (Minitab Inc., State College, PA, USA) software and fitted to a second-order polynomial regression model containing the coefficient of linear, quadratic and interaction terms. An analysis of variance (ANOVA) was then carried out for each response variable in order to test the model significance and suitability. The significances of all terms in the polynomial were statistically analyzed by computing the F-value at a probability (p) of 0.001, 0.01 or 0.05. 2.5. Total phenolic contents Total phenolic contents of the pomegranate peel extracts were determined using Folin–Ciocalteu (FC) reagent assay which was described by Singleton and Rossi [31]. 40 ll of properly diluted

pomegranate peel extract solution were mixed with 1.8 ml of FC reagent. The reagent was pre-diluted, 10 times, with distilled water. After standing for 5 min at room temperature, 1.2 ml of (7.5% w/v) sodium carbonate solution were added. The solution were mixed and allowed to stand for 1 h at room temperature. Finally, the absorbance was measured at 765 nm [32], using a UV–Vis spectrophotometer. A calibration graph [DA = 503.88  x(M) + 0.0208, concentration range: 0.0004–0.004 M, r2 = 0.9958] was constructed by plotting absorbance difference against the gallic acid concentration at nine concentration levels. The results of total phenolic content were expressed as mg gallic acid equivalents per 100 g dry weight of pomegranate peel. 2.6. FRAP assay The FRAP assay was done according to Benzie and Strain [4] with some modifications. The stock solutions included 300 mM acetate buffer (pH 3.6), 10 mM TPTZ solution in 40 mM HCl and 20 mM FeCl3 solution in proportions of 10:1:1 (v/v), respectively. The fresh FRAP reagent was prepared daily. Pomegranate peel extracts (10 lL) in 1 ml distilled water was allowed to react with 1.8 ml of the FRAP solution for 10 min at 37 °C. The absorbance of the reaction mixture was then recorded at 593 nm [32]. Results were expressed in mmol Fe+2/100 g dry weight of pomegranate peel. 2.7. DPPH radical-scavenging activity Scavenging activity of DPPH radical of pomegranate peel extract was determined according to the method reported by Brand-Williams et al. [3] with some modification. An aliquot of 0.5 ml of sample solution was mixed with 2.5 ml of a 0.5 mM methanolic solution of DPPH. The mixture was shaken vigorously and incubated for 30 min in the dark at room temperature. The absorbance was measured at 517 nm against a blank, using a UV–Vis spectrophotometer. Results were expressed as percentage of inhibition of the DPPH radical. Percentage of inhibition of the DPPH radical was calculated according to the following equation:

% Inhibition ¼ ½ðAc  As Þ=Ac   100 where As is the absorbance of the sample and Ac is the absorbance of the control. 3. Results and discussion 3.1. Effect of solvent Before the development of response surface models, effects of solvent type, solvent to solid ratio and particle size on TPC, FRAP value, DPPH scavenging activity and extraction yield were investigated. Results were expressed as means ± standard deviations of triplicate measurements. In this study, several extraction solvents such as methanol, ethanol, water, acetone and ethyl acetate were used due to the wide range of polarity of antioxidants over a 20 min extraction period at 45 °C, solvent to solid ratio 40:1 and particle size 0.425 mm. The selection of solvent can have a significant effect on the performance of antioxidant extraction from complex samples. The results showed that methanolic extracts had the highest TPC, FRAP and DPPH values (Fig. 1). However, the use of organic solvents in the manufacturing process of food ingredients is strictly regulated. For example, methanol is not a food-grade solvent. Moreover, some environmental aspects should be taken into account related to the use of methanol as an extraction solvent. Environmentally benign and non-toxic food grade organic solvents like ethanol and n-buta-

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Fig. 1. Effect of solvent type on the extraction of total phenolics and antioxidants from pomegranate peel. TPC (mg GA/gdw), FRAP (mmol Fe2+/100 gdw) and DPPH (% Inhibition).

Fig. 3. Effect of the particle size on the responses. TPC (mg GA/gdw), FRAP (mmol Fe2+/100 gdw), DPPH (% Inhibition) and yield (%).

Fig. 2. Effect of the solvent to solid ratio (v:w) on the responses. TPC (mg GA/gdw), FRAP (mmol Fe2+/100 gdw), DPPH (% Inhibition) and yield (%).

0.18 mm). The results showed that smaller particle size increases the antioxidant extraction and yield over a 20 min extraction period with a 50% ethanol solution at 45 °C and solvent to solid ratio 50:1 (Fig. 3). In general, smaller particle size was preferred for industrial process to shorten the extraction time even though the energy used for grinding needs to be considered. The increase of the extraction yield for the small particles is due to larger surface area per mass unit. In addition, the migration rate of the analyte through the pores of the solid matrix is also increased with the decrease in particle size [34]. The ANOVA indicated that difference between 0.18 and 0.25 mm particles on the extraction of antioxidants were not significant except for total phenolic content (TPC: p0.18, 0.25 = 0.002; FRAP: p0.18, DPPH: p0.18, 0.25 = 0.692; yield: p50,60 = 0.616; 0.25 = 0.882; p P 0.05: difference is not significant). Therefore, particle size of 0.25 mm was used for next experiments.

nol are recommended by the US Food and Drug Administration for extraction purposes [30]. So ethanol–water mixture was chosen as the extraction solvent for the next experiments. 3.2. Effect of solvent to solid ratio The effect of solvent to solid ratio on the extraction of antioxidants was also studied with five ratios (20:1, 30:1, 40:1, 50:1 and 60:1; v:w) over a 20 min extraction period, with a 50% ethanol solution, at 45 °C. The results showed that antioxidant extraction and yield increased with the increase of the solvent to solid ratio (Fig. 2). This is consistent with mass transfer principles; the driving force during mass transfer is the concentration gradient between the solid and the liquid, which is greater when a higher solvent to solid ratio used [33]. However, higher solvent to solid ratio may mean more solvent usage in extraction and energy consumption for concentration in a later processing stage. The ANOVA indicated that difference between 50:1 and 60:1 solvent to solid ratios on the extraction of antioxidants were not significant except for total phenolic content (TPC: p50, 60 = 0.046; FRAP: p50, 60 = 0.531; DPPH: p50, 60 = 0.285; yield: p50, 60 = 0.276; p P 0.05: difference is not significant). Therefore, the solvent to solid ratio of 50:1(v:w) was used for further experiments. 3.3. Effect of particle size Pomegranate peel powder was passed through four different standard-size sieves (pore sizes of 0.85, 0.425, 0.25 and

3.4. Central composite design results The responses (total phenolic content, antioxidant activities and yield) of each run of the experimental design were presented in Table 3. The coded, decoded values of independent variables for each experiment are also presented. Total phenolic content of pomegranate peel extract varied from 4936.58 to 8923.24 mg gallic acid/100 g dry weight. As shown in Table 3, activity values varied from 24.30 to 63.37 mmol Fe2+/100 g of dry weight, 60.12–83.52% for FRAP and DPPH assays, respectively. Extraction yields ranged from 29.78% to 45.38%. ANOVA was used to estimate the statistical significance of the factors and interactions between them. Regression coefficient and analysis of variance of the second-order polynomial models for total phenolic content, antioxidant activity of pomegranate peel extracts and yield are summarized in Table 4. As shown, the regression parameters of the surface response analysis of the models, the linear, quadratic and interaction terms have significant effects (p 6 0.001, p 6 0.01 or p 6 0.05). The large values of the R2 and R2adj reveal that the models adequately represent the experimental results. The absence of any lack of fit (p > 0.05) also strengthened the reliability of all models. The second-order polynomial equations of the response surfaces are as follow:

Y 1 ðmg GA=100 gdwÞ ¼ 483:42 þ 235:71E  16:83T  137:12t  1:52E2 þ 0:39T 2 þ 6:98t2  0:37ET  1:99Et þ 1:36Tt

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Table 3 Central composite design of three variables with their observed responses. Exp.

X1

X2

X3

E (%)

T (°C)

t (min)

TPC (mg GA/100 gdw)

FRAP (mmol Fe2+/100 gdw)

DPPH (%)

Yield (%)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16

1 0 1 1 0 1 1 0 0 0 1 1 1 1 1 0

1 0 1 1 1 1 1 0 0 0 1 0 1 0 1 1

1 0 1 1 0 1 1 1 1 0 1 0 1 0 1 0

70 50 70 30 50 30 70 50 50 50 30 30 70 70 30 50

60 45 30 30 30 60 30 45 45 45 60 45 60 45 30 60

30 20 30 10 20 30 10 10 30 20 10 20 10 20 30 20

8677.83 6946.70 7622.17 4936.58 6321.11 8553.17 7094.62 6373.98 8923.24 6887.61 5618.27 5506.42 6908.76 7175.87 7485.83 7756.29

63.37 34.86 62.44 36.74 37.05 48.00 52.45 43.80 54.55 34.41 41.92 24.30 56.35 38.26 53.34 40.32

68.66 83.52 60.55 64.76 74.48 69.25 60.12 80.90 81.39 82.61 66.74 73.44 65.85 62.36 75.69 79.94

45.38 35.33 41.33 29.78 34.76 39.00 30.93 31.36 40.79 39.91 33.84 32.31 42.58 41.54 31.20 41.90

Exp., experiments; X1–X3: coded factors in central composite design for solvent percentage, temperature and time, respectively; E, ethanol percentage; T, temperature; t, time; TPC, total phenolic content; FRAP, ferric reducing antioxidant power; DPPH, scavenging activity of 2,2-diphenyl-1-picrylhydrazyl radical; GA, gallic acid; dw, dry weight.

Table 4 Regression coefficients of predicted polynomial models for the investigated responses from pomegranate peel extracts. Coefficient

b0 b1 b2 b3 b11 b22 b33 b12 b13 b23 Model Linear Quadratic Crossproduct Lack of fit R2 R2adj

Response TPC

FRAP

DPPH

Yield

483.42 235.71** 16.83 137.12 1.52* 0.39 6.98* 0.37 1.99* 1.36

84.73*** 0.96** 1.77** 4.95*** 0.64  102,* 0.21  101,*** 0.15*** 0.21  102 0. 35  102 0.11  101,**

16.98 2.76*** 1.11 0.44 0.31  101,*** 0.14  101 0.70  102 0.76  102,* 0.64  102 0.50  102

5.83 0.45* 0.33 0.21 0.43  102,* 0.13  102 0.78  103 0.16  102 0.41  102 0.32  102

***

***

**

**

*

***

**

*

***

***

ns

ns

ns

ns ns ns

ns 0.96 0.91

ns 0.99 0.99

ns 0.95 0.88

ns 0.94 0.86

TPC, total phenolic content; FRAP, ferric reducing antioxidant power; DPPH, scavenging activity of 2,2-diphenyl-1-picrylhydrazyl radical; b, regression coefficient. ns, Not significant at p > 0.05. * Significant at p 6 0.05. ** Significant at p 6 0.01. *** Significant at p 6 0.001.

þ2

Y 2 ðmmol Fe =100 gdwÞ ¼ 84:73 þ 0:96E  1:77T  4:95t  0:64  102 E2 þ 0:22  101 T 2 þ 0:15t2 þ 0:21  102 ET  0:35  102 Et  0:11  101 Tt Y 3 ð% InhibitionÞ ¼ 16:98 þ 2:76E þ 1:11T þ 0:44t  0:31  101 E2  0:14    102 T 2 þ 0:70  102 t 2 þ 0:76  102 ET  0:64  102 Et  0:50  102 Tt Y 4 ð%Þ ¼ 5:83 þ 0:45E þ 0:33T þ 0:21t  0:42  102 E2  0:13  102 T 2  0:78  102 t 2 þ 0:16  102 ET þ 0:41  102 Et  0:32  102 Tt

where E, T and t are ethanol concentration (%), temperature (°C) and time (min), respectively. The visualization of the predicted model equation can be obtained by the surface response plot. This graphical representation is an n-dimensional surface in the (n + 1)-dimensional space. Usually, a two-dimensional representation of a three-dimensional plot can be drawn. Thus, if there are three or more variables, the plot visualization is possible only if one or more variables are set to a constant value. Figs. 4–7 illustrates response surfaces for dependent variables (TPC, FRAP, DPPH and yield) in function of two factors when the third factor was kept constant at middle level (Table 1). These surfaces were drawn by Minitab software. 3.5. Effect of process variables Analysis of the experimental results showed that the ethanol concentration had the greatest effect on TPC, FRAP value, DPPH scavenging activity and extraction yield. Ethanol concentration demonstrated a pronounced influence on responses in linear and quadratic manner. This effect may be attributed to the change of solvent polarity with change in ethanol concentration. In general, the polarity of ethanol–water mixture would increase continuously with the addition of water to ethanol. More polar phenolic compounds may be extracted according to ‘‘like dissolves like’’ principle. It has been reported that the polarity of solvent used in extraction directly affects not only the quantity of total phenolics, but also the composition of phenolic compounds [35]. The effect of ethanol concentration on TPC, FRAP, DPPH and extraction yield at constant time and temperature are shown in Figs. 4–7a and b, respectively. The extraction temperature had significant effect on the extraction of bioactive compounds from pomegranate peel. The highest effect was on the FRAP value. The effect of changing the extraction temperature was not statistically significant for TPC, DPPH scavenging activity and yield extraction. The interaction between extraction temperature and ethanol concentration was statistically significant for DPPH scavenging activity. The effect of temperature on TPC, FRAP, DPPH and extraction yield at constant time and ethanol concentration are shown in Figs. 4–7a and c, respectively. Mild heating (temperatures between 52 °C and 67 °C for a treatment time of few seconds to about half an hour [36]) might soften the plant tissue, weaken the cell wall integrity, hydrolyze the bonds of bound phenolic compounds (phenol–protein or phenol– polysaccharide) as well as enhance phenolics solubility, thus more phenolics would distribute to the solvent.

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Fig. 4. Responses surface plots for the effect of (a) EtOH/temperature, (b) EtOH/ time and (c) temperature/time on total phenolic content (TPC).

Extraction time significantly affected FRAP value. The interaction between extraction time and solvent composition influenced the TP content. The effect of time on TPC, FRAP, DPPH and extraction yield at constant temperature and concentration ethanol are shown in Figs. 4–7b and c, respectively. As shown, longer extraction time had positive effect on all responses. The optimum extraction times for all responses were 30 min. 3.6. Optimal conditions The optimum UAE conditions for the response variables from pomegranate peel extract as obtained by RSM are presented in Table 5. The predictive ability of the models was examined by extractions at optimal conditions. The predicted results matched well with the experimental results obtained using optimum extraction conditions which validated the RSM models with good correlations. The extract can be used as substitute of synthetic antioxidants for food products, color and oxidative stabilization. Therefore, the highest yield is recommended for industrial applications. The optimal conditions for this aim were solvent percentage of 70% ethanol, temperature of 60 °C and extraction time of 30 min. Each antioxidant assay (TPC, FRAP, DPPH, etc.) only provides an estimate of antioxidant capacity that is subjective to its conditions,

Fig. 5. Responses surface plots for the effect of (a) EtOH/temperature, (b) EtOH/ time and (c) temperature/time on the antioxidant activity assay (FRAP).

reagents and different classes of antioxidants. Therefore, the use of different antioxidant assays help to identify variations in the response of the compounds extracted from the samples. These natural antioxidant compounds can be separated by HPLC from the extract. 4. Conclusions Increased concern over the safety of synthetic antioxidants like butylated hydroxylanisole (BHA) and butylated hydroxyltoluene (BHT) has lead to an increased interest in exploration of effective and economical natural antioxidants. By-products of food processing industries assume significance because of their acceptability, non-toxicity and availability in large quantities. Pomegranate peel is one of these by-products. Pomegranate peel could be a good commercial source of anthocyanins, gallotannins, ellagitannins,

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Fig. 6. Responses surface plots for the effect of (a) EtOH/temperature, (b) EtOH/ time and (c) temperature/time on the antioxidant activity assay (DPPH).

ferulic acid, coumaric acid, gallic acid, caffeic acid and dihydroflavonols and they can be separated and concentrated through extraction process [37]. The extract can be used as substitute of synthetic antioxidants for food products, color and oxidative stabilization of raw ground meat during refrigerated storage [38], stabilization of sunflower oil [39], preparation of dietary supplements [40] and preparation of ingredients in the prevention of UV induced damage in human skin [41]. In this study, the response surface methodology was successfully employed to optimize the antioxidant extraction from pomegranate peel. Extracts rich in antioxidants can be efficiently obtained from pomegranate peel by adequately selecting experimental conditions. The factor that had the greatest effect on the TPC, FRAP value, DPPH scavenging activity and the extraction yield was the solvent concentration. Therefore, ultrasound-assisted extraction of antioxidants from pomegranate peel is a green

Fig. 7. Responses surface plots for the effect of (a) EtOH/temperature, (b) EtOH/ time and (c) temperature/time on the extraction yield.

Table 5 Estimated optimum conditions, predicted and experimental values of responses under these conditions. Response variables

TPC (mg GA/100 gr dw) FRAP(mmol Fe2+/ 100 gdw) DPPH (%) Yield (%)

Optimum UAE conditions

Maximum values

Ethanol (%)

T (°C)

t (min)

Predicted

Actual

50 70

60 30

30 30

9365.70 63.10

9198.60 62.44

46 70

46 60

30 30

84.01 46.98

82.55 45.40

UAE, ultrasonic-assisted-extraction; T, temperature; t, time; TPC, total phenolic content; FRAP, ferric reducing antioxidant power; DPPH, scavenging activity of 2,2diphenyl-1-picrylhydrazyl radical; GA, gallic acid; dw, dry weight.

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process for the preparation of extracts rich in natural antioxidants aimed at replacing synthetic antioxidants. Experimental details of previous studies of antioxidant extraction from pomegranate peel reports are summarized in Table 1. Comparison of results of these works and our work is as follow: (1) In the most of these works [22,23,25,26,28], methanol and acetone came up as suitable extraction solvents. However, environmentally benign and non-toxic food grade organic solvents like water and ethanol are recommended by the US Food and Drug Administration for extraction purposes [30]. (2) Yield of extraction with water–ethanol as solvent (45.4%, this work) was four times better than water (11–14%, [27,29]). Yield of extraction with water–ethanol as solvent (45.4%, this work) was also better than methanol (29–35.5%, [25,28]). (3) Although studies have been published on the ultrasonicassisted extraction of antioxidants from pomegranate peel [26,28,29], these studies have not simultaneously evaluated the interaction of the experimental variables.

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