Optimization of pulsed ultrasound-assisted technique for extraction of phenolics from pomegranate peel of Malas variety: Punicalagin and hydroxybenzoic acids

Accepted Manuscript Optimization of Pulsed Ultrasound-Assisted Technique for Extraction of Phenolics from Pomegranate Peel of Malas Variety: Punicalagin and Hydroxybenzoic Acids Milad Kazemi, Roselina Karim, Hamed Mirhosseini, Azizah Abdul Hamid PII: DOI: Reference:

S0308-8146(16)30359-4 http://dx.doi.org/10.1016/j.foodchem.2016.03.017 FOCH 18901

To appear in:

Food Chemistry

Received Date: Revised Date: Accepted Date:

13 September 2015 18 February 2016 7 March 2016

Please cite this article as: Kazemi, M., Karim, R., Mirhosseini, H., Hamid, A.A., Optimization of Pulsed UltrasoundAssisted Technique for Extraction of Phenolics from Pomegranate Peel of Malas Variety: Punicalagin and Hydroxybenzoic Acids, Food Chemistry (2016), doi: http://dx.doi.org/10.1016/j.foodchem.2016.03.017

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1

Optimization of Pulsed Ultrasound-Assisted Technique for Extraction of Phenolics

2

from Pomegranate Peel of Malas Variety: Punicalagin and Hydroxybenzoic Acids

3 4 5 Milad Kazemi1, *Roselina Karim1, Hamed Mirhosseini1 , and Azizah Abdul Hamid2

6 7 8 9

1

10

Department of Food Technology, Faculty of Food Science and Technology,

11

Universiti Putra Malaysia, 43400, UPM Serdang, Selangor, Malaysia 2

12 13

Department of Food Science, Faculty of Food Science and Technology, Universiti Putra Malaysia, 43400, UPM Serdang, Selangor, Malaysia

14 15 16 17 18 19 20 21 22 *

Corresponding author.

E-mail address: [email protected], Tel: 006038946 8372, Fax: 006038942 3552

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23

Abstract

24 25

Pomegranate peel is a rich source of phenolic compounds (such as punicalagin and

26

hydroxybenzoic acids). However, the content of such bioactive compounds in the peel

27

extract can be affected by extraction type and condition. It was hypothesized that the

28

optimization of a pulsed ultrasound-assisted extraction (PUAE) technique could result in

29

the pomegranate peel extract with higher yield and antioxidant activity. The main goal

30

was to optimize PUAE condition resulting in the highest yield and antioxidant activity as

31

well as the highest contents of punicalagin and hydroxybenzoic acids. The operation at the

32

intensity level of 105 W/cm2 and duty cycle of 50% for a short time (10 min) had a high

33

efficiency for extraction of phenolics from pomegranate peel. The application of such

34

short extraction can save the energy and cost of the production. Punicalagin and ellagic

35

acid were the most predominant phenolic compounds quantified in the pomegranate peel

36

extract (PPE) from Malas variety. PPE contained a minor content of gallic acid.

37 38

Keywords: Pomegranate peel extract (PPE); Punicalagin; hydroxybenzoic acids;

39

Antioxidant activity; Pulsed ultrasound-assisted extraction

40 41 42 43 44 45 46 47

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1. Introduction

49 50

Pomegranate (Punica granatum L.) is one of the healthiest fruits originated from Iran

51

(Mousavinejad, Emam-Djomeh, Rezaei, & Khodaparast, 2009). Pomegranate contains a

52

substantial amount of phenolic compounds including ellagitannins, phenolic acids (mainly

53

hydroxybenzoic acids) and flavonoids (anthocyanins and other complex flavonoids). Such

54

phenolic compounds are in different parts of the pomegranate fruit (Çam & Hışıl, 2010;

55

Mousavinejad et al., 2009). They have many functional properties such as

56

anticarcinogenic, antimutagenic, antitumoral, antidiabetic, and antioxidant properties

57

(Viuda‐Martos, Fernández‐López, & Pérez‐Álvarez, 2010). Pomegranate peel is an

58

agricultural biomass waste containing higher antioxidant activity than the edible portion

59

(Akhtar, Ismail, Fraternale, & Sestili, 2015). It comprises ~40-50% of the total fruit

60

weight. It is a rich source of phenolic compounds especially ellagitannins and

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hydroxybenzoic acids (Fischer, Carle, & Kammerer, 2011; Viuda‐Martos et al., 2010).

62 63

Ellagitannins (hydrolyzable tannins) are a group of tannins defined as the esters of

64

hexahydroxydiphenic acid and a polyol, usually glucose or quinic acid (Clifford &

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Scalbert, 2000). Punicalagin (C48H28O30) is a water soluble phenolic compound with a

66

high molecular weight (MW = 1108). It is the most predominant polyphenol compound in

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the pomegranate peel (Fischer et al., 2011). It is naturally found in the forms of two

68

reversible α and β anomers. However, they are often mentioned in the singular

69

punicalagin. As stated by Clifford and Scalbert (2000), it is difficult to quantify

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punicalagins separately. Hydroxybenzoic acids are a type of phenolic acids which

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naturally exist in different fruits and plants such as berries, persimmons, green and black

72

tea, currant, and several certain red fruits including pomegranate. Some of hydroxybenzoic

3

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acids (e.g. gallic acid, ellagic acid, syringic acid, and vanillic acid) are found in a simple

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form in the fruits. However, hydroxybenzoic acids commonly exist in plants in a

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conjugated form with glycosides or esters (such as ellagic acid glucoside, ellagic acid

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arabinoside, galloyl glucose and gallic acid ethyl ester) (Tomás-Barberán, Ferreres, & Gil,

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2000). Ellagic acid (C14H6O8) is a dimeric derivative of gallic acid. It is considered as one

78

of the most important functional compounds in pomegranate peel. Gallic acid (C7H6O5) is

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also one of the phenolic compounds in pomegranate peel. It shows high free radical-

80

scavenging activity as reported by Fischer et al. (2011).

81 82

Although pomegranate peel contains a notable amounts of phenolics such as punicalagin

83

and ellagic acid, it is not being commercially utilized. In fact, the majority of pomegranate

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peel is thrown away as a waste of the food industry. As reported by Qu et al. (2009), 1 ton

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of fresh pomegranate generate 669 kg pomegranate marc including 78% peel and 22%

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seeds. Therefore, it is necessary to conduct more studies on such valuable waste and

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attempt to make a zero waste strategy in supporting the green technology campaign.

88 89

Pulsed ultrasound-assisted extraction (PUAE) is one of the most effective extraction

90

techniques. The application of an efficient technique such techniqe for extraction of

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natural antioxidants (like phenolic compounds) from pomegranate peel can be very useful

92

and efficient. PUAE can provide higher efficiency than the convectional extraction

93

methods. It requires shorter time, lower energy, chemicals and solvents than the

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conventional methods. Moreover, the PUAE technique has more advantages than the

95

continuous

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technological problems (such as depreciation of the equipment and erosion of the tip) than

97

CUAE (Dey & Rathod, 2013; Pan, Qu, Ma, Atungulu, & McHugh, 2011; Vilkhu,

ultrasound-assisted

extraction

4

(CUAE)

technique.

It

causes

lower

98

Mawson, Simons, & Bates, 2008). It should be noted that the ultrasound processor is

99

turned on and off intermittently during pulsed extraction, thus making lower heat

100

generation than CUAE. In this condition, PUAE can be more suitable than CUAE for the

101

extraction of thermo-sensitive bioactive compounds such as polyphenols (Awad,

102

Moharram, Shaltout, Asker, & Youssef, 2012). Pan et al. (2011) compared the efficiency

103

of CUAE and PUAE techniques as well as conventional solvent extraction for the

104

extraction of phenolic compounds from dry pomegranate peel. They reported that the

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application of PUAE led to save the extraction time up to 87% and increase the

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antioxidant yield by 22% as compared to the conventional solvent extraction. Pan et al.

107

(2011) also revealed the superiority of PUAE over CUAE because of 50% energy saving.

108

The intensity level, duty cycle of ultrasonic waves and exposure time are some of the most

109

important variables affecting the yield and antioxidant activity of the extract (Pan et al.,

110

2011).

111 112

This study was undertaken to optimize PUAE conditions for extraction of phenolic

113

compounds from the pomegranate peel of Malas variety. The main goal was to determine

114

the optimum extraction condition resulting in the highest extraction yield and antioxidant

115

activity and the highest content of punicalagin, ellagic acid and gallic acid. Malas variety

116

is one of the most popular commercial Iranian pomegranate varieties, which was chosen

117

for this research. To the best of our knowledge, there is no published report on the

118

optimization of PUAE conditions for extraction of phenolic compounds from pomegranate

119

peel of Malas variety.

120 121 122 123 5

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2. Materials and Methods

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2.1. Chemicals and Materials

126 127

Ripened pomegranate fruits (Punica granatum L. var. Malas) were collected from a

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research center of Agricultural Science and Natural Resources (Isfahan, Iran). In term of

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maturity stage, the collected fruits were ready to be used for the juice industry. Pure

130

standards of punicalagin (≥ 97%) and ellagic acid (≥ 97%) were purchased from

131

ChromaDex, Inc. (Irvine, CA, USA). Folin-Ciocalteau’s phenol reagent, 2,2-diphenyl-1-

132

1picrylhydrazyl (DPPH), gallic acid (98%), and sodium carbonate were supplied by

133

Merck Company (Darmstadt, Germany). Ethanol, acetonitrile, methanol, phosphoric acid

134

and hydrochloric acid (analytical and HPLC grade) were purchased from Fisher Scientific

135

(Leicestershire, UK).

136 137

2.2. Pulsed ultrasound-assisted extraction of phenolic compounds from pomegranate

138

peel

139 140

The pomegranate fruits (Punica granatum L. var. Malas) were washed and the peels were

141

manually separated from the arils and adhering materials. Approximately 18 kg Fresh

142

peels were immediately air-dried for approximately 7 days at 20 ± 2 ˚C (during autumn).

143

Then, the dried peels (~8 kg) were put in the moisture proof bottles, then covered with

144

parafilm. Finally, the bottles were closed and kept at -20 ± 2 °C prior to extraction. The

145

dried pomegranate peels were ground by a grinder (Panasonic, MX-798S, Selangor,

146

Malaysia), then sieved (180 µm mesh size). The extraction was performed by means of an

147

ultrasound (LABSONIC ® P, Gottingen, Germany) at a constant frequency of 24 kHz. The

148

ultrasound processor was attached to a probe with the area of 1.53 cm2 at different

6

149

intensity levels up to 105 W/cm2. The operation was carried out under the the pulsed

150

mode. In order to apply PUAE, 10.0 g of pomegranate peel powder was mixed with 100

151

ml of the ethanol (70%). Phenolic compounds were extracted from pomegranate peel by

152

using different solvents such as methanol, acetone, water and ethanol-water (Li, Guo,

153

Yang, Wei, Xu, & Cheng, 2006; Tabaraki, Heidarizadi, & Benvidi, 2012). However, the

154

USA Food and Drug Administration (FDA) recommended non-toxic food grade solvents

155

like ethanol for extraction purpose (Bartnick, Mohler, & Houlihan, 2006). The sample

156

container was covered with an aluminum-foil sheet to avoid exposure to light. The

157

extraction was carried out under the following experimental condition: Extraction time

158

(2,6, and 10 min); Duty cycle (50, 70, and 90%); Intensity level (53, 79, and 105 W/cm2).

159

Several preliminary trials were carried out to find the most suitable biomass to solvent

160

ratio, solvent concentration, and extraction conditions ranges.

161 162

2.3. Determination of extraction yield

163 164

Extraction yield was determined according to the method reported by Khan, Abert-Vian,

165

Fabiano-Tixier, Dangles, and Chemat (2010). A rotary evaporator (N-1001S-W; Eyela,

166

Tokyo, Japan) was used to remove ethanol from the extracts at 40°C. Then, the samples

167

were put in a -86 ºC freezer for one day. After that, the frozen samples were lyophilized

168

by a freeze dryer (Labconco Freezone 18, Model 77550, MO, USA). Finally, the yield

169

(%)was calculated based on equation 1.

170

 % =

        ×        

171 172 7

(1)

173

2.4. Determination of antioxidant activity

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2.4.1. Determination of total phenolic content

175 176

The phenolic content of the pomegranate peel extract was measured according to the

177

method reported by Jayaprakasha, Singh, and Sakariah (2001). In this study, 5 mg

178

pomegranate peel extract was dissolved in 20 ml methanol (0.25 mg/ml). Then, 1 ml of

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extract-methanol mixture was mixed with 5.0 ml of 10-fold diluted Folin-Ciocalteu

180

reagent and 4.0 ml sodium carbonate solution (7.5%). The mixture was left for 30 min at

181

room temperature (25 ± 2 ˚C), then its absorbance was measured at 765 nm by means of a

182

UV-visible spectrophotometer (Shimadzu UV-1650 PC, Tokyo, Japan). The total phenolic

183

compounds was determined in triplicate for each sample and the results were expressed as

184

gallic acid equivalents.

185 186

2.4.2. DPPH radical scavenging assay

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The radical scavenging activity of pomegranate peel extract was determined using 2,2-

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Diphenyil-1-picrylhydrazyl (DPPH) according to the method described by Brand-

190

Williams, Cuvelier, and Berset (1995) with minor modifications. The DPPH solution of

191

0.1 mM was prepared in methanol. The different concentrations of pomegranate peel

192

extracts (0.05, 0.1, 0.25, 0.5 and 1 mg/ml) were prepared by dissolving the specific

193

amount of pomegranate peel extract in methanol. Then, 0.1 ml of each pomegranate peel

194

extract solution was added to 3.9 ml methanolic DPPH solution (0.1 mM). Then, the

195

mixture was shaken vigorously and left in a dark chamber at room temperature for 30 min.

196

The changes in color from dark violet to light yellow was determined at 517 nm by a UV-

197

visible spectrophotometer (Shimadzu UV-1650 PC, Tokyo, Japan). A control was

8

198

prepared by adding a blank (methanol) to the DPPH solution. The diminution in

199

absorbance was then converted to the antiradical activity (AA) percentage for each

200

concentration of pomegranate peel extract based on Equation 2.

%  =

−" ×  

(2)

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where, A is the absorbance of DPPH minus absorbance of blank solution (without the

202

pomegranate peel extract). B is the absorbance of DPPH solution containing the peel

203

extract. The antiradical activity of the pomegranate peel extract is represented in the form

204

of IC50. IC50 is defined as the concentration of the extract (test materials) required to cause

205

50% reduction in the primary DPPH concentration. It was calculated through interpolation

206

of a linear regression. The determination of the antiradical activity of the pomegranate

207

peel extract was done in triplicate and the results were expressed as µg/ml.

208 209

2.5. Quantification of phenolic compounds

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Quantification of phenolic compounds was performed according to the official method of

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International Olive Council (IOC, 2009) with minor modifications. Quantitative analysis

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of phenolic compounds in pomegranate peel extract was carried out by an

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Agilent 1200 HPLC system (Agilent Technologies, Waldbronn, Germany), equipped with

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a quaternary pump (Model Quat pump-G1311A) and a UV- diode array detector (Model

216

DAD G1315D). The system was also equipped with an auto sampler (Model

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ALSG1329A), a column oven (Model TCC-G1316A) and a degasser system (Model

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Degasser-G1322A). Before injection, each sample (150 mg) was dissolved in 5 ml

219

methanol (80%), then the supernatant was allowed to pass through a 0.2 µm nylon syringe

220

filter. Injection volume was 10 µl and the column temperature was maintained at 40 ºC

221

during analysis. A Hypercil gold column C18 (5 µm, 250 x 4.6 mm, Thermo Scientific, 9

222

Waltham, MA, USA) was employed for analysis. The mobile phase consisted of solvent A

223

(0.2% v/v, solution of phosphoric acid in water) and solvent B (50:50 v/v, methanol-

224

acetonitrile). A flow rate of 0.6 ml/min was considered for HPLC analysis. The gradient

225

profile was 96% A at 0-25 min, 83% A at 25 min, 60% A at 35 min, 60% A at 40 min and

226

96% A at 45-50 min. Chromatograms were recorded at 280 nm. The individual phenolic

227

compound was quantified by comparing the peak area versus the standard peak area for

228

each reference compound. Four different concentrations of ellagic acid (0.40, 0.60, 0.80,

229

1.00 mg/ml), punicalagin (0.30, 0.35, 0.40, 0.45 mg/ml) and gallic acid (0.05, 0.10, 0.15,

230

0.20 mg/ml) were injected to draw the standard calibration curves. The contents of

231

punicalagin, ellagic acid and gallic acid were reported in g of pomegranate peel extract.

232

The experiment was carried out in triplicate for each sample.

233 234

2.6. Experimental design and data analysis

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A three-factor Box-Behnken design with 3 repeated center points was designed to

237

optimize the PUAE conditions for obtaining the highest extraction yield, antioxidant

238

activity, total phenolic content, and punicalagin, ellagic acid and gallic acid contents. The

239

independent variables were extraction time (x1), duty cycle (x2), and intensity level (x3).

240

Fig. 1 displays the overall methodology including the matrix of Box-Behnken design for

241

the pulsed ultrasound-assisted extraction of phenolic compounds from the pomegranate

242

peel. The general equation for the empiric second order polynomial model including three

243

different factors is as follows:

 = # + #   + #%  % + #& & + # % + #%%  %% + #&&  %& + #%   % + #&   & + #%&  % &

10

(3)

244

In Equation (3), Yi indicates the response variable, β0 is the constant term; β1, β2 and β3 are

245

the regression coefficients of the single effects, β11, β22 and β33 are the quadratic effects

246

coefficients, β12, β13, β23 are the interactions coefficients and x1, x2 and x3 display the

247

independent variables including time, duty cycle, and intensity level, respectively. In this

248

study, the interaction effects (x1x2, x1x3 and x2x3) and quadratic (x12, x22 and x32) effects of

249

all ultrasound variables are determined along with their single effects (x1, x2 and x3).

250

Analysis of variance (ANOVA) was employed to evaluate the data and revealed the

251

significant terms (p ≤ 0.05) in the model. In order to analyze RSM model, the terms

252

statistically determined as insignificant (p > 0.05) were eliminated from the initial model.

253

Then, the empirical data were re-fitted to only significant (p ≤ 0.05) terms for obtaining a

254

final reduced model (Mirhosseini & Tan, 2009). Minitab software (version 16, Minitab

255

Inc., PA, USA) was used for running the experimental design and data analysis.

256 257

2.7. Optimization and validation process

258 259

The optimization process was carried out to determine the optimum PUAE condition for

260

obtaining the extract with the most desirable properties by using the graphical and

261

numerical optimization procedures. Graphical optimization was done by drawing three-

262

dimensional (3D) response surface plot for visualizing the significant (p ≤ 0.05)

263

interaction effects of PUAE variables on target responses. Simultaneously, an overlaid

264

contour plot was applied to demonstrate how the yield, antioxidant activity and phenolic

265

compound contents were affected by the interaction effects of two ultrasound variables,

266

while the remaining independent variable was kept constant at the middle level. The

267

validation process was done to determine the adequacy of the final reduced model and

268

recommended optimum conditions (Mirhosseini & Tan, 2009). Then, the predicted

11

269

optimum extraction condition was practically applied to obtain the most desirable

270

pomegranate peel extract. Finally, the experimental and predicted values of each response

271

were compared by T-test. The insignificant difference (p > 0.05) observed between the

272

experimental data and predicted values confirmed the validity of the final reduced model

273

(Mirhosseini & Tan, 2009).

274 275

3. Results and Discussion

276

3.1. Effect of pulsed ultrasound-assisted extraction variables on extraction yield

277 278

The results indicated that the extraction yield (Y1) was most significantly affected by the

279

main effect of extraction time. However, the quadratic effect of intensity level has also

280

affected the yield (Table 1). Table 2 displays the predicted regression coefficients, R2, p-

281

values of regression and lack of fit for all response surface models. High R2 value (R2 =

282

0.994) of the final reduced model illustrated that the model could explain 99% of the

283

variations in the yield of pomegranate peel extract. The extraction yield was improved by

284

simultaneously prolonging extraction and increasing the duty cycle at the intensity level of

285

64 W/cm2 (Fig. 2a). This finding confirmed the significant positive effect of time on the

286

extraction yield. The diffusion of bioactive mass from the sample matrix into the solvent

287

can be facilitated by prolonging the extraction, thereby increasing the yield (Corrales,

288

García, Butz, & Tauscher, 2009).

289 290

Fig. 2a shows that the increment in intensity level of ultrasonic waves had both positive

291

and negative (or dual) effects on the extraction yield. More precisely, the enhancement of

292

intensity level of the ultrasonic waves up to 64 W/cm2 led to induce a negligible increase

293

in the amount of the extract. On the other hand, the extraction yield was decreased from

12

294

42.5% to 39.8% by a further increment of the intensity level. This could be attributed to

295

the simultaneous effects of the high intensity level and duty cycle on the yield, thereby

296

increasing the temperature. In fact, a continuous radiation of high-intensity ultrasonic

297

waves could create several temporary hot spots through the collapse of the cavitation

298

bubbles (Flint & Suslick, 1991). It should be noted that the pomegranate peel contains

299

different fibres such as cellulose and hemicellulose (Hasnaoui, Wathelet, & Jiménez-

300

Araujo, 2014) and complex polysaccharides such as pectin (Hasnaoui et al., 2014;

301

Moorthy, Maran, Muneeswari, Naganyashree, & Shivamathi, 2015). As stated by Sun,

302

Liu, Chen, Ye and Yu (2011), the high-intensity level of ultrasonic waves can lead to

303

aggregation of the polysaccharide molecules. The application of higher extraction

304

temperature might result in more swelling of fibres in the pomegranate cell wall

305

(Mantanis, Young, & Rowell, 1995), thus reducing the leakage of the aggregated

306

polysaccharides and macromolecules from the cell matrix to the solvent.

307 308

In this study, the highest and lowest experimental yields were 41.6% and 26.8%,

309

respectively. Fig. 2a shows that the highest possible predicted yield (~ 42.5%) would be

310

achieved by applying the intensity level of ~ 64 W/cm2 for 10 min at the duty cycle of

311

90%. Tabaraki et al. (2012) reported that the application of the continuous ultrasound-

312

assisted extraction for 30 min resulted in relatively high yield (45.4%) for pomegranate

313

peel extract; while the current study revealed that the application of 10 min extraction

314

under the pulsed mode also resulted in the high yield (41.6%). In comparison with

315

Tabaraki et al. (2012), the extraction yield did not show a significant difference (45.4% 10

316

min extraction under the pulsed mode resulted in the almost similar yield (41.6%). Despite

317

the reduction in the extraction time from 30 min to 10 min, the extraction yield did not

318

show a noticeable decline (45.4% and 41.6%). This might confirm the efficiency of PUAE

13

319

as compared to CUAE. This might also prove that the lower energy is required to extract

320

the phenolic compounds from the pomegranate peel by applying PUAE. Besides, the ratio

321

of sample to solvent (ethanol 70%) in this study was five times lower than the ratio

322

applied by Tabaraki et al. (2012). As a main outcome of this study, the reduction of

323

solvent used for extraction can result in lower harmful side effects on the human health

324

and lower energy consumption for the separation of solvent residue from the extract.

325 326

The extraction yield was substantially increased by extending extraction at a fixed

327

intensity level and the maximum yield was acheived by applying the possible longest

328

extraction (Fig. 2b). In fact, the longer extraction can provide more sufficient time for

329

further disruption of the cell walls during ultrasonic extraction under the constant

330

intensity. In this condition, the higher amount of the solvent can penetrate into the cells,

331

resulting in more efficient dissolution of compounds into the solvent and consequently

332

higher extraction yield (Balachandran, Kentish, Mawson, & Ashokkumar, 2006).

333 334

On the other hand, the extraction yield was increased by increasing the intensity level at a

335

fixed extraction time (Fig. 2b). When the intensity level is increased, more microscopic

336

bubbles might be generated in the solvent as a result of cavitation phenomenon. Implosive

337

collapses of such microscopic bubbles resulted in the generation of more microjets and

338

shock waves. These microjets move towards the surfaces of the cell wall with the

339

velocities of hundred meters per second, thus resulting in the formation of more pores in

340

the peel cell walls and facilitating the extraction of bioactive compounds from the

341

pomegranate peel (Suslick, Eddingsaas, Flannigan, Hopkins, & Xu, 2011). This indicated

342

that the extraction time plays more significant role than the ultrasonic intensity level in the

343

extraction yield.

14

344 345

At a low duty cycle, the extraction yield can be improved by increasing the intensity level

346

(Fig. 2c). For instance, the extraction yield was increased from 34.3% to 43.4% by

347

increasing the intensity level from 52.5 to 105 W/cm2 at the duty cycle of 50%. At duty

348

cycle 90%, the increase in the intensity level led to induce a dual effect on the extraction

349

yield. When the intensity level was increased from 52.5 to ~75 W/cm2, the yield of

350

extraction was improved from 38.9% to 39.6%; however, a downward trend was observed

351

(39.6% to 38.1%) at higher intensity level up to 105W/cm2. Consequently, the intensity

352

level of ultrasonic waves had both positive and negative effects on the extraction yield.

353 354

3.2. Effect of pulsed ultrasound-assisted extraction variables on total phenolic

355

content and antioxidant activity of pomegranate peel extract

356 357

The results indicated that the main effects of the extraction time and interaction of time

358

and intensity level had the highest- and lowest significant effect on the total phenolic

359

content (Y2), respectively (Table 1). The maximum and minimum phenolic contents were

360

320.26 (mg GAE/g) and 272.05 (mg GAE/g), respectively. Fig. 2d showed that the

361

phenolic content in the pomegranate peel extract was increased by prolonging the

362

extraction. This was in accordance with the finding reported by

363

Martínez-Ávila, Wong-Paz, Belmares-Cerda, Rodríguez-Herrera, and Aguilar (2013).

364

They indicated a positive effect of the sonication time on the extraction yield of

365

polyphenols from Laurus nobilis. Numerical optimization also showed that the total

366

phenolic content was increased by increasing the intensity level (Fig. 2d). This might be

367

explained by the cavitation phenomena, which generates stable bubbles (vapor cavities) in

368

the solvent by the diffusion of the ultrasonic waves (Raso & Barbosa-Cánovas, 2003).

15

Muñiz-Márquez,

369

These bubbles were compressed and the pressure and temperature inside them were

370

gradually increased during process. The microscopic bubbles will collapse in the vicinity

371

of the plants cell wall, thus resulting in the generatation of microjets. This facilitates the

372

disruption of the plant cell wall and consequently enhances the release rate of plant

373

components from the sample matrix to the solvent (Rostagno, Palma, & Barroso, 2003).

374 375

Fig. 2e shows the interaction effect of the extraction time and intensity level on the total

376

phenolic content of the pomegranate peel extract at 75% duty cycle. The result indicated

377

that the extraction time had a more significant effect than the intensity level on the

378

phenolic content. For instance, as the intensity level was increased from 52.5 to 105.0

379

W/cm2 at a fixed extraction time of 10 min, the total phenolic content was increased from

380

308.00 to 318.00 mg GAE/g; while the higher increment of total phenolic compound

381

(from 276.00 to 318.00 mg GAE/g) was observed by prolonging the extraction process

382

from 2 to 10 min at a fixed intensity level of 105 W/cm2. This observation might be

383

explained by the fact that the formation of micro-bubbles is fascilitated by extending the

384

extraction, thereby inducing more cell wall damage (Naziri, Mantzouridou, & Tsimidou,

385

2012; Vilkhu et al., 2008). In this condition, it is expected that the phenolic compounds is

386

released much easier from the damaged cells to the solvent.

387 388

The radical scavenging activity (IC50, Y3) of the pomegranate peel extract was

389

significantly (p ≤ 0.05) affected by the extraction time and intensity level (Table 1). The

390

single effect of extraction time had the most significant (p ≤ 0.05) positive effect on

391

DPPH. As reported by Pan et al. (2011), the application of ultrasound assisted extraction

392

at the intensity level of 59.2 W/cm2 for 60 min could not significantly (p > 0.05) increase

393

DPPH radical scavenging activity of the pomegranate peel extract. In this study, the

16

394

operation of PUAE for the short time (10 min) at the optimum intensity level (105 W/cm2)

395

led to induce the significant (p < 0.05) improvement in the radical scavenging activity of

396

the pomegranate peel extract.

397 398

As shown in Fig. 2f, the reduction of IC50 might confirm that the DPPH radical scavenging

399

activity of pomegranate peel extract was substantially increased by extending the

400

extraction and increasing the intensity level. The results also showed that the duty cycle

401

did not significantly (p > 0.05) affect the antioxidant activity of the peel extract.

402

Therefore, the lowest duty cycle (50%) was recommended for the extraction of the

403

phenolic compounds from the pomegranate peel. This is because the minimum energy is

404

required by applying extraction at the lowest duty cycle. The extraction for 10 min using

405

the intensity level of 105 W/cm2 resulted in the lowest IC50 (or the highest DPPH radical

406

scavenging activity, 5.51 µg/ml). Masci, Coccia, Lendaro, Mosca, Paolicelli, and Cesa

407

(2016) applied very long process (24) for the extraction of the phenolic compounds from

408

pomegranate peel. They reported slightly stronger DPPH radical scavenging activity for

409

the pomegranate peel extract from two different varieties (Israeli and Italian). Masci et al.

410

(2016) reported IC50 values of 3.13 µg/ml and 3.56 µg/ml for the extracts obtained from

411

Israeli and Italian pomegranate variety, respectively. In contrast, Panichayupakarananta,

412

Issuriya, Sirikatitham, and Wang (2010) reported a negligibly lower DPPH radical

413

scavenging activity (5.8 µg/ml) for the pomegranate peel extract after refluxing in ethyl

414

acetate for 1 h. Such differences between the antioxidant activity of different pomegranate

415

peel extracts might be due different varieties, solvents and extraction methods and

416

conditions.

417

17

418

3.3. Effect of pulsed ultrasound-assisted extraction variables on hydroxybenzoic

419

acids and punicalagin of pomegranate peel extract

420 421

Fig. 3a shows the HPLC chromatogram of the peel extract at the wavelength of 280 nm. It

422

was observed that gallic acid, α-punicalagin, β-punicalagin, and ellagic acid were eluted at

423

retention times of 9.71, 15.81, 21.31 and 37.96 min, respectively. In the current research,

424

the punicalagin content (α + β) varied from 128.02 to 146.61 mg/g depending on the

425

ultrasound extraction condition. The extract with the highest punicalagin content (146.61

426

mg/g) was achieved when the extraction was carried out for 10 min at intensity level of

427

105 W/cm2. Lu, Ding, and Yuan (2008) determined the punicalagin content in the husk of

428

16 different varieties of pomegranate peel. They obtained pomegranate husk extract by

429

applying ultrasound-assisted extraction in aqueous ethanol for 30 min twice. The highest

430

and lowest contents of punicalagin were 121.5 and 39.8 mg/g dry basis, respectively. The

431

results of their study obviously showed that the punicalagin content was significantly

432

different among the peels with different varieties. In another study, pomegranate peel

433

extract was obtained from nine different Turkish cultivars by applying pressurized water

434

extraction for 10 min. The punicalagin content was 116.6 mg/g on dry matter basis (Çam

435

& Hışıl, 2010).

436 437

Based on statistical analysis, the punicalagin content was significantly (p ≤ 0.05) affected

438

by the single effect of time and interaction effect of time and intensity level; while the

439

single effect of intensity level and duty cycle depicted insignificant (p > 0.05) effects on

440

the punicalagin content (Table 1). A high R2 value (0.986) represented that the final

441

reduced model could accurately predict the changes of punicalagin content as a function of

442

ultrasound variables (Table 2). Fig. 3b shows the positive interaction effect of extraction

18

443

time and ultrasonic wave intensity on the content of punicalagin. The punicalagin content

444

was gradually increased with increasing of intensity level at a fixed extraction time; while

445

the punicalagin content was greatly increased by extending the extraction time at a fixed

446

intensity level. For instance, 6 mg/g was added to punicalagin content (from ~140 to 146)

447

when the intensity level was increased from 52.5 W/cm2 to 105 W/cm2 during 10 min

448

extraction. However, extending the extraction time from 2 to 10 min at intensity level of

449

105 W/cm2 led to a 16 mg/g increment in the punicalagin content (from ~130 to 146

450

mg/g). Numerical optimization also represented higher positive effect of extraction time

451

than intensity level on the punicalagin content (Fig. 3c).

452 453

Optimization of the extraction process, in order to obtain the highest content of ellagic

454

acid from pomegranate peel, is an important contribution to the food and pharmaceutical

455

industries. The result of this study also indicated that ellagic acid content varied from

456

10.12 to 22.53 mg/g at different PUAE conditions. The lowest ellagic acid content (10.12

457

mg/g) was quantified after 2 min extraction, whereas the highest content was obtained

458

when the extraction was carried out for 10 min. The ellagic acid content obtained under

459

optimum PUAE condition in this study was considerably higher compared to the results of

460

Çam and Hışıl (2010) who reported 1.25 mg/g ellagic acid in the extract of pomegranate

461

peel which was prepared by applying pressurized water extraction after 10 min. The

462

quantified value of ellagic acid in this study was also higher than the previous findings by

463

Masci et al. (2016), who reported 11.85 mg/g of ellagic acid in Israeli pomegranate peel

464

extract obtained by stirring in ethanol for 24 h. This results might be due to different

465

extraction methods and conditions as well as different varieties of pomegranates.

466

19

467

As displayed in Fig. 3d, the ellagic acid content was increased by prolonging extraction

468

process. The single effect of extraction time was the only significant (p ≤ 0.05) factor

469

affecting the content of ellagic acid in pomegranate peel extract (Table 1). In fact, the

470

extraction of ellagic acid was noticeably time dependent (F-ratio = 71.84). This

471

observation was in agreement with the findings reported by Jerman, Trebše, and Mozetič

472

Vodopivec (2010). Theese researchers reported that the higher content of phenolic

473

compounds from olive fruit was obtained by prolonging the ultrasound extraction.

474

In this study, a very low content of gallic acid was quantified in pomegranate peel extract.

475

In fact, the highest content of gallic acid in the extract was 0.051 mg/g which was much

476

lower than punicalagin and ellagic acid contents. Elfalleh et al. (2011) quantified gallic

477

acid content in 6 different types of Tunisian pomegranate peel after extraction by stirring

478

in methanol for one night twice. They reported that the gallic acid contents were between

479

1.09 and 1.31 mg/g which is extremely higher than the gallic acid content (0.051 mg/g)

480

which is determined in the current study. Table 1 showed that the interaction effect of

481

extraction time and intensity level significantly (p ≤ 0.05) influenced the gallic acid

482

content; while the other terms did not noticeably affect the gallic acid content. The final

483

reduced model showed high R2 value (0.926) when it was fitted based on gallic acid

484

content (Table 2). Accordingly, about 92% of the variation in the gallic acid content could

485

be explained as a function of significant PUAE variables. In order to visualize the

486

interaction effect of time and intensity level on gallic acid content, a response surface plot

487

was constructed (Fig. 3e).

488

The results illustrated that the gallic acid content was substantially increased by

489

simultaneously extending the extraction time and increasing the intensity level. The

490

maximum gallic acid content (0.051 mg/g) was obtained when the highest intensity level

491

(105 W/cm2) and longest time (10 min) were applied for extraction (Fig. 3e). Fig. 3f

20

492

showed that time had a more positive significant effect than intensity level on the recovery

493

of gallic acid. The punicalagin and gallic acid contents were considerably affected by

494

interaction effect of extraction time and intensity level. The most probable reason might

495

be attributed to the application of ultrasound energy. In fact, the application of high-

496

intensity level at a longer extraction time led to the generation of more bubbles with

497

higher energy. The collapse of cavitation bubbles releases an enormous amount of energy

498

in the solvent (McNamara, Didenko, & Suslick, 1999). Higher level of energy produces

499

macro-turbulence which leads to the increase in the collision of micro particles in the

500

biomass (Ji, Lu, Cai, & Xu, 2006). This phenomenon enhances the diffusion and solubility

501

of phenolic compounds in the solvent.

502 503

None of the quantified bioactive compounds were significantly affected by duty cycle

504

within the selected experimental range. In fact, there is no noticeable difference between

505

high and low duty cycle on the extraction efficiency of aforementioned phenolic

506

compounds. Therefore, this finding prioritizes the usage of low duty cycle value (50%) for

507

extraction of phenolic compounds from pomegranate peel. It can be explained by the fact

508

that at lower duty cycle, the operating time of sonicator is reduced, thus lowering the

509

energy consumption and sonicator depreciation. Consequently, the highest punicalagin

510

(146.58 mg/g), ellagic acid (20.66 mg/g) and gallic acid (0.053 mg/g) contents were

511

predicted to be achieved by the ultrasound extraction in 10 min at intensity level of 105

512

W/cm2 and duty cycle of 50%.

513 514

HPLC analysis revealed that punicalagin, ellagic acid and gallic acid constitute almost

515

168.55 mg/g of the peel extract. In fact, punicalagin was the most abundant compound

516

among all quantified polyphenols in the pomegranate peel extract. In this study, the

21

517

maximum punicalagin, ellagic acid and gallic acid contents along with the highest

518

phenolic content and antioxidant activity were obtained within 10 min of extraction using

519

105 W/cm2 intensity level. This finding also indicated that the antioxidant activity of the

520

peel extract is directly correlated to the contents of the important phenolic compounds

521

(such as punicalagin and ellagic acid) which are quantified in the extract.

522 523 524 525

3.4. Optimization and validation procedures

526 527

In this study, the optimum PUAE conditions would result in the peel extract with the

528

highest extraction yield, total phenolic content, antioxidant activity, punicalagin, ellagic

529

acid and gallic acid contents. Multiple graphical and numerical optimizations were carried

530

out to obtain the overall optimum PUAE condition. The favorable functional properties

531

were achieved by PUAE under the predicted optimum condition. The significant (p ≤

532

0.05) interaction effects of PUAE variables on extraction yield, total phenolic content,

533

punicalagin and gallic acid contents are indicated by the multiple overlaid contour plot

534

(Fig. 4a). The white area on the plot represents the suitable range of PUAE conditions

535

which lead to desirable response variables. This would imply that if the independent

536

variables are set at the levels demonstrated in the white regions, the dependent variables

537

will fall within the target ranges.

538 539

In order to achieve the exact optimum point of each PUAE variable the response optimizer

540

plot was drawn (Fig. 4b). The multiple numerical optimization predicted that the most

541

desirable pomegranate peel extract can be obtained by extraction for 10 min at 50% duty

22

542

cycle and intensity level of 105 W/cm2. Under the suggested optimum ultrasound

543

extraction condition, the following predicted values were expected to be achieved: 41.14%

544

of extraction yield, 318.71 mg GAE/g total phenolic content, 5.50 µg/ml DPPH radical

545

scavenging activity, 146.58 mg/g punicalagin, 20.66 mg/g ellagic acid and 0.053 mg/g

546

gallic acid.

547 548 549 550

3.5. Verification of the final reduced models

551 552

The appropriateness of the response surface models was studied by comparing the

553

experimental values with those predicted by the final reduced models. For this purpose,

554

the linear regressions were fitted between empirical data and the predicted values. High R2

555

values ranging from 0.84 to 0.99 showed the closeness between the experimental and

556

predicted values, thereby verifying the accuracy of the final reduced models. An

557

experimental validation was also applied by running PUAE under the optimum condition.

558

Then, the optimum pomegranate peel extract was subjected to all analytical tests

559

mentioned earlier. No significant (p > 0.05) differences were observed between the

560

experimental data and the predicted values using one-sample T-test. This finding

561

confirmed that the efficiency of the final reduced models was accurately authenticated.

562 563

4. Conclusions

564 565

The current research reveals that the optimization of PUAE led to achieve a high

566

experimental amount of crude extract (41.10%) in a short extraction time of 10 min. In

23

567

fact, when PUAE was applied at the duty cycle of 50%, the operating time of the sonicator

568

was reduced to 5 min. The application of PUAE under optimum conditions would be

569

practical for large-scale food industry production as it enhances both the extraction yield

570

and rate. Furthermore, under the optimum condition of PUAE, a considerable

571

experimental amounts of punicalagin (146.55 mg/g) and ellagic acid (20.65 mg/g) were

572

quantified in the pomegranate peel extract of Malas variety, however, gallic acid content

573

was very low (0.051 mg/g). Therefore, the application of PUAE as an efficient extraction

574

technique can provide the extract with higher contents of punicalagin and ellagic acid.

575

Such active compound have the potential to be used in the formulation of functional foods

576

and nutritional supplements. The current study revealed that Malas variety has relatively

577

lower galic acid than other varieties tested earlier. PUAE of phenolic compounds from

578

pomegranate peel was performed using ethanol 70% as a food grade solvent. The current

579

study revealed that PUAE can be considered as a high-efficiency, safe and emerging

580

technique which reduce the extraction time and energy consumption.

581 582

References

583 584 585

Akhtar, S., Ismail, T., Fraternale, D., & Sestili, P. (2015). Pomegranate peel and peel extracts: Chemistry and food features. Food Chemistry, 174, 417-425.

586

Awad, T., Moharram, H., Shaltout, O., Asker, D., & Youssef, M. (2012). Applications of

587

ultrasound in analysis, processing and quality control of food: A review. Food

588

Research International, 48(2), 410-427.

589

Balachandran, S., Kentish, S., Mawson, R., & Ashokkumar, M. (2006). Ultrasonic

590

enhancement

of

the

supercritical

591

Sonochemistry, 13(6), 471-479.

24

extraction

from

ginger.

Ultrasonics

592 593 594 595 596 597

Bartnick, D. D., Mohler, C. M., & Houlihan, M. (2006). U.S. Patent No. 20,060,088,627. Washington, DC: U.S. Patent and Trademark Office. Brand-Williams, W., Cuvelier, M., & Berset, C. (1995). Use of a free radical method to evaluate antioxidant activity. LWT-Food Science and Technology, 28(1), 25-30. Çam, M., & Hışıl, Y. (2010). Pressurised water extraction of polyphenols from pomegranate peels. Food Chemistry, 123(3), 878-885.

598

Clifford, M. N., & Scalbert, A. (2000). Ellagitannins–nature, occurrence and dietary

599

burden. Journal of the Science of Food and Agriculture, 80(7), 1118-1125.

600

Corrales, M., García, A. F., Butz, P., & Tauscher, B. (2009). Extraction of anthocyanins

601

from grape skins assisted by high hydrostatic pressure. Journal of Food

602

Engineering, 90(4), 415-421.

603 604

Dey, S., & Rathod, V. K. (2013). Ultrasound assisted extraction of β-carotene from Spirulina platensis. Ultrasonics Sonochemistry, 20(1), 271-276.

605

Elfalleh, W., Tlili, N., Nasri, N., Yahia, Y., Hannachi, H., Chaira, N., Ying, M., &

606

Ferchichi, A. (2011). Antioxidant capacities of phenolic compounds and

607

tocopherols from Tunisian pomegranate (Punica granatum) fruits. Journal of Food

608

Science, 76(5), 707-713.

609

Fischer, U. A., Carle, R., & Kammerer, D. R. (2011). Identification and quantification of

610

phenolic compounds from pomegranate (Punica granatum L.) peel, mesocarp, aril

611

and differently produced juices by HPLC-DAD–ESI/MS. Food Chemistry, 127(2),

612

807-821.

613 614

Flint, E. B., & Suslick, K. S. (1991). The temperature of cavitation. Science, 253(5026), 1397-1399.

615

Gil, M. I., Tomás-Barberán, F. A., Hess-Pierce, B., & Kader, A. A. (2002). Antioxidant

616

capacities, phenolic compounds, carotenoids, and vitamin C contents of nectarine,

25

617

peach, and plum cultivars from California. Journal of Agricultural and Food

618

Chemistry, 50(17), 4976-4982.

619

Hasnaoui, N., Wathelet, B., & Jiménez-Araujo, A. (2014). Valorization of pomegranate

620

peel from 12 cultivars: Dietary fibre composition, antioxidant capacity and

621

functional properties. Food Chemistry, 160, 196-203.

622 623

IOC. (2009). Determination of biophenols in olive oils by HPLC. COI/T.20/Doc. No 29. International Olive Oil Council, Madrid, Spain.

624

Jayaprakasha, G., Singh, R., & Sakariah, K. (2001). Antioxidant activity of grape seed

625

(Vitis vinifera) extracts on peroxidation models in vitro. Food Chemistry, 73(3),

626

285-290.

627

Jerman, T., Trebše, P., & Mozetič Vodopivec, B. (2010). Ultrasound-assisted solid liquid

628

extraction (USLE) of olive fruit (Olea europaea) phenolic compounds. Food

629

Chemistry, 123(1), 175-182.

630 631

Ji, J. b., Lu, X. h., Cai, M. q., & Xu, Z. c. (2006). Improvement of leaching process of Geniposide with ultrasound. Ultrasonics Sonochemistry, 13(5), 455-462.

632

Khan, M. K., Abert-Vian, M., Fabiano-Tixier, A. S., Dangles, O., & Chemat, F. (2010).

633

Ultrasound-assisted extraction of polyphenols (flavanone glycosides) from orange

634

(Citrus sinensis L.) peel. Food Chemistry, 119(2), 851-858.

635

Li, Y., Guo, C., Yang, J., Wei, J., Xu, J., & Cheng, S. (2006). Evaluation of antioxidant

636

properties of pomegranate peel extract in comparison with pomegranate pulp

637

extract. Food Chemistry, 96(2), 254-260.

638 639 640 641

Lu, J., Ding, K., & Yuan, Q. (2008). Determination of punicalagin isomers in pomegranate husk. Chromatographia, 68(3-4), 303-306. Mantanis, G., Young, R., & Rowell, R. (1995). Swelling of compressed cellulose fiber webs in organic liquids. Cellulose, 2(1), 1-22.

26

642

Masci, A., Coccia, A. E., Lendaro, E., Mosca, L., Paolicelli, P., & Cesa, S. (2016).

643

Evaluation of different extraction methods from pomegranate whole fruit or peels

644

and the antioxidant and antiproliferative activity of the polyphenolic fraction. Food

645

Chemistry. doi: http://dx.doi.org/10.1016/j.foodchem.2016.01.106

646 647

McNamara, W. B., Didenko, Y. T., & Suslick, K. S. (1999). Sonoluminescence temperatures during multi-bubble cavitation. Nature, 401(6755), 772-775.

648

Mirhosseini, H., & Tan, C. P. (2009). Response surface methodology and multivariate

649

analysis of equilibrium headspace concentration of orange beverage emulsion as

650

function of emulsion composition and structure. Food Chemistry, 115(1), 324-333.

651

Moorthy, I. G., Maran, J. P., Muneeswari, S., Naganyashree, S., & Shivamathi, C. (2015).

652

Response surface optimization of ultrasound assisted extraction of pectin from

653

pomegranate peel. International Journal of Biological Macromolecules, 72, 1323-

654

1328.

655

Mousavinejad, G., Emam-Djomeh, Z., Rezaei, K., & Khodaparast, M. H. H. (2009).

656

Identification and quantification of phenolic compounds and their effects on

657

antioxidant activity in pomegranate juices of eight Iranian cultivars. Food

658

Chemistry, 115(4), 1274-1278.

659

Muñiz-Márquez, D. B., Martínez-Ávila, G. C., Wong-Paz, J. E., Belmares-Cerda, R.,

660

Rodríguez-Herrera, R., & Aguilar, C. N. (2013). Ultrasound-assisted extraction of

661

phenolic compounds from Laurus nobilis L. and their antioxidant activity.

662

Ultrasonics Sonochemistry, 20(5), 1149-1154.

663

Naziri, E., Mantzouridou, F., & Tsimidou, M. Z. (2012). Recovery of squalene from wine

664

lees using ultrasound assisted extraction- A feasibility study. Journal of

665

Agricultural and Food Chemistry, 60(36), 9195-9201.

27

666

Pan, Z., Qu, W., Ma, H., Atungulu, G. G., & McHugh, T. H. (2011). Continuous and

667

pulsed ultrasound-assisted extractions of antioxidants from pomegranate peel.

668

Ultrasonics Sonochemistry, 18(5), 1249-1257.

669

Panichayupakarananta, P., Issuriya, A., Sirikatitham, A., & Wang, W. (2010). Antioxidant

670

assay-guided purification and LC determination of ellagic acid in pomegranate

671

peel. Journal of Chromatographic Science, 48(6), 456-459.

672

Qu, W., Pan, Z., Zhang, R., Ma, H., Chen, X., Zhu, B., Wang, Z., & Atungulu, G. (2009).

673

Integrated extraction and anaerobic digestion process for recovery of nutraceuticals

674

and biogas from pomegranate marc. Transactions of the ASABE, 52(6), 1997-2006.

675

Raso, J., & Barbosa-Cánovas, G. V. (2003). Nonthermal preservation of foods using

676

combined processing techniques. Critical Reviews in Food Science and Nutrition,

677

43(3), 265-285.

678 679

Rostagno, M. A., Palma, M., & Barroso, C. G. (2003). Ultrasound-assisted extraction of soy isoflavones. Journal of Chromatography A, 1012(2), 119-128.

680

Sun, Y., Liu, D., Chen, J., Ye, X., & Yu, D. (2011). Effects of different factors of

681

ultrasound treatment on the extraction yield of the all-trans-β-carotene from citrus

682

peels. Ultrasonics Sonochemistry, 18(1), 243-249.

683

Suslick, K. S., Eddingsaas, N. C., Flannigan, D. J., Hopkins, S. D., & Xu, H. (2011).

684

Extreme conditions during multibubble cavitation: Sonoluminescence as a

685

spectroscopic probe. Ultrasonics Sonochemistry, 18(4), 842-846.

686

Tabaraki, R., Heidarizadi, E., & Benvidi, A. (2012). Optimization of ultrasonic-assisted

687

extraction of pomegranate ( Punica granatum L.) peel antioxidants by response

688

surface methodology. Separation and Purification Technology, 98, 16-23.

28

689

Tomás-Barberán, F., Ferreres, F., & Gil, M. (2000). Antioxidant phenolic metabolites

690

from fruit and vegetables and changes during postharvest storage and processing.

691

Studies in Natural Products Chemistry, 23, 739-795.

692

Vilkhu, K., Mawson, R., Simons, L., & Bates, D. (2008). Applications and opportunities

693

for ultrasound assisted extraction in the food industry—A review. Innovative Food

694

Science & Emerging Technologies, 9(2), 161-169.

695

Viuda-Martos, M., Fernández-López, J., & Pérez-Álvarez, J. (2010). Pomegranate and its

696

many functional components as related to human health: a review. Comprehensive

697

Reviews in Food Science and Food Safety, 9(6), 635-654.

698

Yolmeh, M., Habibi Najafi, M. B., & Farhoosh, R. (2014). Optimisation of ultrasound-

699

assisted extraction of natural pigment from annatto seeds by response surface

700

methodology (RSM). Food Chemistry, 155, 319-324.

701 702 703 704 705 706 707 708 709 710 711 712 713

29

Figure 1. Overview of pulsed ultrasound-assisted extraction of phenolic compounds and antioxidants from pomegranate peel extract. PUAE, PPE, HPLC, TPC, and DPPH refer to the pulsed ultrasound-assisted extraction, pomegranate peel extract, high performance liquid chromatography, total phenolic content, and 2,2-diphenyl-1-picrylhydrazyl, respectively

30

Figure 2. Interaction effects of PUAE variables on yield (a-c), total phenolics content (TPC) (d,e) and DPPH antioxidant activity (f) of pomegranate peel extract (PPE)

31

Figure 3. Chromatogram of individual phenolic compounds from pomegranate peel extract (a) and interaction effects of PUAE variables on punicalagin, ellagic acid and gallic acid contents (b-f)

32

Figure 4. Graphical (a) and numerical (b) multiple optimization plots demonstrating the optimum pulsed ultrasound extraction of phenolic compounds from pomegranate peel

714 715 716 717 718 719 720

33

Table 1. The p-value and F-ratio of pulsed ultrasound-assisted extraction (PUAE) variables in the final reduced models fitted for phenolic compounds from pomegranate peel Response

Main effects x1

x2

x3

Quadratic effects x1

2

x2

2

x3

2

Interaction effects x1 x2

x1 x3

x2 x3

Yield (Y1, %)

p-value F-ratio

0.000 0.992* 0.000 182.87 0.000 53.89

0.000 90.52

0.035 7.37

0.006 16.77

-

0.017 10.64

0.001 30.88

TPC (Y2, mg/g)

p-value F-ratio

0.000 123.20

-

0.011 10.14

0.000 114.78

-

0.010 10.53

-

0.02 8.03

-

IC50 (Y3, µ g/ml)

p-value F-ratio

0.000 36.67

-

0.000 32.92

0.012 9.08

-

-

-

-

-

Punicalagin (Y5, mg/g)

p-value F-ratio

0.003 14.25

-

0.203* 1.83

-

-

-

-

0.031 6.12

-

EA content (Y6, mg/g )

p-value F-ratio

0.000 71.84

-

-

-

-

-

-

-

-

GA content (Y7, mg/g)

p-value F-ratio

0.974* 0.00

-

0.248* 1.49

-

-

-

-

0.023 6.91

-

Note: *= Insignificant at p > 0.05; TPC= total phenolic content; IC50= inhibitory concentration of the extract that reduce 50% of stable DPPH radical; FRAP= ferric reducing antioxidant power; x1= time; x2 = duty cycle; x3= intensity level of ultrasound; x1, x2 and x3 = represent the single effects of variables; x12, x22 and x32 = represent the quadratic effects of variables; x1 x2 , x1 x3 and x2 x3 = represent the interactions between variables.

Table 2. Regression coefficients, R2 and lack of fit of the final reduced models fitted for phenolic compounds from pomegranate peel by PUAE Regression coefficient b

Yield (Y1, %) - 2.00739

TPC (Y2, mg/g) 277.250

b

3.91967

9.651

- 0.00107

b b b b b b b b

0 1

2

3 2 1 2 2 2 3 12 13 23 2

R P- value (Regression) P- value (lack of fit)

IC50 (Y3, µg/ml) 7.293

Punicalagin (Y5, mg/g) 123.018

EA (Y6, mg/g ) 9.123

3.12158

-0.191

1.092

1.154

0.00003

-

-

-

-

-

0.54303

- 0.627

-0.006

0.032

-

- 0.00009

- 0.16355

- 0.552

0.007

-

-

-

0.00187

-

-

-

-

-

- 0.00163

0.004

-

-

-

-

-

-

-

-

-

-

- 0.00821

0.021

-

0.009

-

0.00003

- 0.00280

-

-

-

-

-

0.994

0.993

0.956

0.986

0.846

0.924

0.000*

0.000*

0.000*

0.000

0.000*

0.000*

0.238

0.445

0.516

0.756

0.102

0.925

GA (Y7, mg/g)

Note: *= Significant at p ≤ 0.05; TPC= total phenolic content; IC50= inhibitory concentration of the extract that reduce 50% of stable DPPH radical; FRAP= ferric reducing antioxidant power; b1, b2 and b3 are the regression coefficients of the main or single effect of time, duty cycle and intensity level, respectively; bi, bii and bij= the estimated regression coefficient for the single, quadratic and interaction effects, respectively

721 722 723 724 725 726 727 728 729 730 731 732 733 734 735 736 737 738 739 740 741

Highlights

•

Pulsed ultrasound-assisted extraction conditions were optimized for recovery of the extract from pomegranate peel.

•

The extraction for 10 min, intensity level of 105 W/cm2 and duty cycle of 50% was the optimum extraction condition.

•

Punicalagin, ellagic and gallic acids were the predominant phenolic compound in pomegranate peel extract.

•

The extraction time and duty cycle were the most and least significant ultrasound extraction variable, respectively.

•

This study demonstrated that the pomegranate peel extract from Malas variety had high antioxidant activity.

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