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
1
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
2
48
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
61
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 &
65
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
67
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
70
punicalagins separately. Hydroxybenzoic acids are a type of phenolic acids which
71
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
73
acids (e.g. gallic acid, ellagic acid, syringic acid, and vanillic acid) are found in a simple
74
form in the fruits. However, hydroxybenzoic acids commonly exist in plants in a
75
conjugated form with glycosides or esters (such as ellagic acid glucoside, ellagic acid
76
arabinoside, galloyl glucose and gallic acid ethyl ester) (Tomás-Barberán, Ferreres, & Gil,
77
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
79
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
84
peel is thrown away as a waste of the food industry. As reported by Qu et al. (2009), 1 ton
85
of fresh pomegranate generate 669 kg pomegranate marc including 78% peel and 22%
86
seeds. Therefore, it is necessary to conduct more studies on such valuable waste and
87
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
91
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
94
conventional methods. Moreover, the PUAE technique has more advantages than the
95
continuous
96
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
105
application of PUAE led to save the extraction time up to 87% and increase the
106
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
124
2. Materials and Methods
125
2.1. Chemicals and Materials
126 127
Ripened pomegranate fruits (Punica granatum L. var. Malas) were collected from a
128
research center of Agricultural Science and Natural Resources (Isfahan, Iran). In term of
129
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
174
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
179
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
187 188
The radical scavenging activity of pomegranate peel extract was determined using 2,2-
189
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)
201
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
210 211
Quantification of phenolic compounds was performed according to the official method of
212
International Olive Council (IOC, 2009) with minor modifications. Quantitative analysis
213
of phenolic compounds in pomegranate peel extract was carried out by an
214
Agilent 1200 HPLC system (Agilent Technologies, Waldbronn, Germany), equipped with
215
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
217
ALSG1329A), a column oven (Model TCC-G1316A) and a degasser system (Model
218
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
235 236
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
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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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