Optimization of ultrasound-assisted extraction of polyphenolic compounds from pomegranate peel using response surface methodology

Optimization of ultrasound-assisted extraction of polyphenolic compounds from pomegranate peel using response surface methodology

Separation and Purification Technology 194 (2018) 40–47 Contents lists available at ScienceDirect Separation and Purification Technology journal home...

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Separation and Purification Technology 194 (2018) 40–47

Contents lists available at ScienceDirect

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

Optimization of ultrasound-assisted extraction of polyphenolic compounds from pomegranate peel using response surface methodology

T



Jelena Živković , Katarina Šavikin, Teodora Janković, Nada Ćujić, Nebojša Menković Institute for Medicinal Plant Research “Dr. Josif Pančić”, Tadeuša Košćuška 1, 11000 Belgrade, Serbia

A R T I C L E I N F O

A B S T R A C T

Keywords: Extraction optimization Pomegranate Punicalagin Punicalin

The pomegranate peel is discarded as waste although it contains high amount of biologically active phenolics and has high value for recycling. In this study we have determined optimal conditions for ultrasound-assisted extraction (UAE) of polyphenols from pomegranate peel using response surface methodology (RSM). The influence of extraction time (X1: 10–60 min), ethanol concentration (X2: 10–90%), solid to solvent ratio (X3: 1:10–1:50) and extraction temperature (X4: 20–80 °C) on total polyphenols content (TPC) as well on content of individual polyphenolic compounds (ellagic acid, gallic acid, punicalagin and punicalin) was investigated. The optimal extraction process conditions were as follows: extraction time of 25 min, ethanol concentration of 59%, solid to solvent ratio of 1:44, and extraction temperature of 80 °C. Experimentally obtained values were in agreement with those predicted by RSM model, indicating suitability of the employed model and the success of RSM in optimizing the extraction conditions.

1. Introduction Pomegranate (Punica granatum L., Punicaceae), native to many tropical and subtropical regions, is one of the oldest edible fruits [1]. It is mainly consumed as a fresh fruit, but in the last ten years there are a lot pomegranate products on the market, predominantly juices. The peel of pomegranate fruit, which constitutes about 50% of the total weight, is usually discarded as waste [2]. On the other hand, it was shown that pomegranate peel contains ellagic acid and its derivatives, ellagitannins such as punicalin and punicalagin, and proanthocyanidins and flavonoids [3,4] in a higher level compared to juice [5,6]. Due to such chemical composition, a lot of biological activities were reported for pomegranate peel extracts such as anti-inflammatory, antioxidant, antimicrobial, anti-mutagenic and apoptotic [5,7–9]. Moreover, low toxicity and high safety were also showed using in vitro and in vivo studies [10–12]. Heber et al. [13] reported safety of pomegranate ellagitannin-enriched polyphenol capsules in normal and obese individuals in doses up to 1420 mg per day for four weeks. Moreover, it was shown that ellagic acid and its derivatives possesses antiviral, antimicrobial, antihepatotoxic, anticarcinogenic, and, antifibrogenic properties [14,15]. Recent results of Sun et al. [16], showed that ellagic acid, punicalin, and punicalagin had significant in vitro antioxidant activities while ellagic acid was more effective than other two compounds in protecting against oxidative injury in vivo. Diverse activities of gallic acid and its derivatives has also been presented including



antiproliferative, chemopreventive, antibacterial, antifungal, and antiviral [17]. Therefore, it is not surprising that the pomegranate peel has a long ethnobotanical history. It is traditionally recommended in the case of diarrhoea, dysentery, colitis, headache, aphthae, ulcers and lot of other conditions [3,18]. Still, even though its use in ethnomedicine and marked content of polyphenolic compounds, it does not have wide commercial use. Currently, pomegranate peel extracts have been utilized only in various dietary supplements such as tablets or classic and soft gels capsules [3]. In that view, the additional research that can promote its application is needed. Various extraction techniques can be applied for polyphenol recovery from plant material, and the selection of extraction method is mainly based on the extraction yield and production cost. Application of ultrasound waves shorten the extraction time and enhance “environment-friendly” aspects of the process in terms of reagents consumption and energy requirements in comparison to conventional extraction techniques. The quality of the extract depends on the employed extraction procedure [19]. The food grade solvents such as water [20], ethanol [21], or their mixtures [22], are dominantly used for preparation of peel extracts. Other parameters such as number of extractions, extraction time, temperature, solid-to-solvent ratio, particle size may also influence the yield and recovery of various compounds. Therefore, it is necessary to optimize the extraction conditions which enables good yield, and high level of bioactive compounds.

Corresponding author. E-mail address: [email protected] (J. Živković).

https://doi.org/10.1016/j.seppur.2017.11.032 Received 11 July 2017; Received in revised form 14 October 2017; Accepted 12 November 2017 Available online 13 November 2017 1383-5866/ © 2017 Elsevier B.V. All rights reserved.

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respectively. Optimal extraction conditions were determined considering TPC, and ellagic acid, gallic acid, punicalin and punicalagin content as responses. The software STATGRAPHIC Centurion XVII (Statpoint Technologies, Inc., USA) was used for experiment design, data analysis and determination of optimal conditions. ANOVA was used for evaluation of the significance of independent variables’ influence and interactions (the differences were statistically significant at p < .05). Standardized Pareto charts were applied to obtain the significance of impact of tested variables on mentioned responses. The adequacy of the model was evaluated by the coefficient of determination (R2), and p values for the model and lack-of-fit testing. The correctness of the model was verified by performing UAE at obtained optimal conditions (extraction time, ethanol concentration, solid to solvent ratio and temperature) in order to obtain maximal TPC, ellagic acid, gallic acid, punicalagin and punicalin content.

According to the literature data, there are few studies dealing with optimization of extraction of pomegranate peel and all of them recorded total polyphenols or flavonoid content as final responses [22,24,25]. Different experimental design methods are applied for the optimization of the extraction procedures [26–28], but two most commonly used are the single-factor experiments and RSM. In our study, we have applied RSM in order to determine the impact of extraction temperature, extraction time, solid to solvent ratio and ethanol concentration on TPC, and on content of individual bioactive compounds (ellagic acid, gallic acid, punicalin and punicalagin) in pomegranate peel extract. 2. Methodology 2.1. Plant material, standards and reagents

2.3. Extraction process

Pomegranate fruits were collected at natural locality in village Do, Bosnia and Herzegovina during November 2015. Peels were manually separated from the seeds, air-dried at room temperature for 4–6 days and grounded using a laboratory mill. Particles 0.75–2 mm size were obtained using sieves according to Yugoslav Pharmacopoeia [29]. Following standards and reagents were used: punicalagin and punicalin (Sigma-Aldrich, Steinheim, Germany), gallic and ellagic acid (Extrasynthese, Cedex, France). All reagents used for analytical procedures were analytical grade: Folin–Ciocalteu phenol reagent (Sigma-Aldrich, Steinheim, Germany), sodium carbonate, methanol, formic acid, orthophosphoric acid were purchased from Sigma–Aldrich Chemie GmbH, Munich, Germany while acetonitrile (HPLC grade) was Merck, Germany.

Different quantities of powdered pomegranate peel (0.5–2.5 g) were mixed with 25 mL of different concentrations of ethanol (10–90%) using different solid to solvent ratios (1:10–1:50). These samples were extracted in ultrasonic bath (Bandelin, Sonorex) at various temperatures (20–80 °C) for varying time periods (5–65 min). Extraction time and bath temperature were controlled from the panel of the instrument. After extraction, samples were filtered through filter paper and stored at −18 °C until further analysis. 2.4. Determination of total polyphenols TPC in pomegranate peel extracts was determined spectrophotometrically using Folin-Ciocalteu method [31]. Gallic acid (0–100 mg/L) was used for calibration of a standard curve. The results were expressed as milligrams of gallic acid equivalents per gram of dry weight of fraction (mg GAE/g dw). All experiments were repeated at three times.

2.2. Experimental design and statistical model In this study we have used central composite design (CCD) for optimization of UAE of pomegranate peel. Compared to some other designs, it provides better prediction [30], and can be used in two-step sequential response surface methods. The design consisted of 30 randomized runs with five replicates at the central point. As the responses in the designed experiment we have selected four variables (extraction time, ethanol concentration, solid to solvent ratio and extraction temperature), and each variable was tested at five different levels. The real and coded values of independent variables used in CCD are given in Table 1. For statistical calculations the variables were coded by the equation:

2.5. HPLC analysis Analyses were carried out on Agilent 1200 RR HPLC instrument (Agilent, Waldbronn, Germany), equipped with binary pump SL, autosampler, column temperature controller, and diode-array detector working in the range of 190–550 nm. The chromatographic data was recorded and processed with Agilent ChemStation software. The samples were separated with DAD detector, on a reverse phase Zorbax SBC18 (Agilent) analytical column (150 mm × 4.6 mm i.d.; 5 µm particle size) according to previously published procedure [32]. The mobile phase A was 1% v/v solution of orthophosphoric acid in water, mobile phase B was acetonitrile. Gradient elution was according to the following scheme: 0–5 min, 98–90% A; 5–15 min, 90% A; 15–20 min, 90–85% A; 20–25 min, 85–70% A; 25–30 min, 70–40% A; 30–34 min, 40–0% A, with post-time of 2 min. Detection wavelengths were set at 260, 280, 320, 360 and 380 nm, and the flow rate was 1 mL/min. The injection volume was 3 μL and the column temperature was maintained at 25 °C. Standards of punicalin and punicalagin were purchased from Sigma-Aldrich (Steinheim, Germany) while ellagic and gallic acids were Extrasynthese (Cedex, France). Identification of the compounds was achieved by comparing their UV spectra and retention time with those from authentic substances. The amounts of the compounds were calculated using calibration curves. The results are presented as milligrams per gram of dry weight (mg/g dw).

Xi = (xi−x 0)/Δxi where Xi is a coded value of the independent variable, xi is the actual value of the independent variable, x0 is the actual value of the independent variable in the center of the domain and Δxi is the step change value. The model proposed for each response of Y was:

Y= β0 + β1 X1 + β2 X2 + β3 X3 + β4 X 4 + β11 X12 + β22 X22 + β33 X32 + β44 X24 + β12 X1X2 + β13 X1X3 + β14 X1X 4 + β23 X2X3 + β24 X2X 4 where Y is response, X1 – extraction time, X2 – ethanol concentration, X3 – the solid to solvent ratio, X4 – extraction temperature, βo – intercept, β1, β2, β3 and β4 – linear, β11, β22, β33 and β44 – quadratic and β12, β13, β14, β23 and β24 – interaction regression coefficient terms, Table 1 Real and coded values of UAE parameters. Independent variable Extraction time – X1 (min) Ethanol concentration – X2 (%) Solid to solvent ratio – X3 (1:X3) Extraction temperature – X4 (°C)

−2 5 10 10 20

−1 20 30 20 35

0 35 50 30 50

1 50 70 40 65

3. Results and discussion

2 65 90 50 80

RSM is a compilation of statistical and mathematical techniques that are established on the fit of polynomial equation to the experimental data [33]. It describes well the behaviour of data set aiming to make 41

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276.35 mg GAE/g of dry sample. In our study, the highest TPC was obtained by application of following parameters: X1: 50 min, X2: 30% ethanol, X3: 1:40 and X4: 65 °C. On the other hand, the lowest TPC was achieved using following conditions: X1: 35 min, X2: 90% ethanol, X3: 1:30 and X4: 50 °C. In Fig. 2 we have presented the influence of tested extraction parameters on TPC. These graphs were prepared by fixing two extraction variables at central design values and varying the resting two. According to the results of the statistical analysis, it is evident that the most dominant factor influencing extraction of total polyphenols was ethanol concentration, and this factor affected TPC negatively (Fig. 2). Accordingly, TPC in pomegranate peel extracts initially increased with increase in ethanol concentration and reached a maximum level (∼40% ethanol) after which it started to decrease with further increase in ethanol concentration. This is in accordance with previous results for phenolic compounds. Sharmila et al. [40], showed that higher water content in aqueous methanol improved the extraction of phenolic compounds due to its polarity. In a more polar medium, breaking of hydrogen bonds, and consequent extraction of phenolic compounds are easier [41]. The addition of water into ethanol (or other organic solvents) enhances swelling of the plant material that way increasing the contact area between solid material and solvent. Also, high water content can improve extraction of highly hydrophilic fraction of the glycosylated phenolic compounds [38]. Solid to solvent ratio, extraction time and temperature had lower positive, but still significant impact on TPC. As shown in Fig. 2. TPC sharply increased with the increase of solid to solvent ratio and temperature. The influence of temperature increase on better extraction rate of bioactive compounds was previously described in the literature. Sood and Gupta [22] showed that TPC in pomegranate peel was under direct influence of solid to solvent ratio. This effect may be the consequence of higher amount of solvent that can penetrates into plant cells. Kazemi et al. [42], showed that during UAE, longer extraction time can contribute to additional disruption of cell walls and better penetration of solvent into cells. At these conditions phenolic compounds are released at higher level from impaired cells. Temperature has notable influence on mass transfer process, leading to reduction of solvent viscosity and improvement of solvent penetration [43]. At higher temperatures, the level of plant matrix and cellular structures degradation is higher which makes cells more permeable [41]. Also, the weakening of interactions among phenolic compounds and proteins, and among phenolic compounds and polysaccharides occurs at higher temperatures, increasing the diffusion rate of phenolics in this way [38]. Pomegranate peel contains high amount of complex polysaccharide pectin as phenolic binder. According to results obtained in our study, we can conclude that phenolics present in pomegranate peel are generally stable at high temperature conditions. Similar trend

statistical previsions. It is more favorable than the traditionally used single parameter optimization, since it reduces time, space and raw material usage [34]. Scientific information dealing with optimization of UAE process of pomegranate peel extracts are very limited. Zhu et al. [35] and Moorthy et al. [36] performed optimization of UAE of polysaccharides and pectin from this plant matrix, respectively. Sood and Gupta [22], applied mentioned technique to obtain pomegranate peel extracts, but as dependent variables they selected TPC, ferric reducing antioxidant power (FRAP), scavenging activity of 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical and extract yield. According to our knowledge, this is the first paper dealing with optimization of UAE based on individual biologically active polyphenols as dependent variables. Application of the efficient extraction procedure should achieve maximal recovery of target biologically active compounds and their minimal degradation [37]. Different extraction conditions promote higher extraction of different groups of compounds. Moreover, extraction level of phenolic compounds from natural matrix depends on their structural diversity, and interaction with other cellular components [38]. In this research we have applied a CCD with four variables tested at five levels, in order to obtain pomegranate peel extract with the highest content of punicalin, punicalagin, ellagic acid and gallic acid, since they were major compounds of the obtained extracts characterised by HPLC-DAD (Fig. 1). TPC was also followed as one of the responses. Experimental and predicted results of 30 randomized runs for selected responses obtained under different experimental conditions are presented in Table 2. Experimentally obtained values of TPC, and individual phenolic compounds have been fitted to quadratic polynomial model while ANOVA was applied to assess effects of studied variables, interactions between them and statistical significance of the model. P values of regression coefficients for each of the investigated responses are summarized in Table 3. Based on statistically significant p values, it could be concluded that quadratic polynomial model represented good approximation for investigated responses. This has been confirmed with reasonably high coefficients of multiple determinations (R2) which values were 0.90, 0.76, 0.92, 0.68 and 0.75 for TPC, gallic acid, ellagic acid, punicalagin and punicalin content, respectively. Insignificant lack of fit (p > .05) was also the evidence for the good adequacy of the model.

3.1. Effect of extraction parameters on TPC TPC in extracts obtained using UAE technique varied between 118.01 and 190.94 mg GAE/g dw. These results were in the upper part of previously reported ones for pomegranate peel. Namely, according to Carbone et al. [39], TPC in pomegranate peels ranges from 13.98 to

Fig. 1. HPLC chromatogram of pomegranate peel extract recorded at 260 nm. Peak identification: (1) gallic acid, (2) punicalin, (3) punicalagin (α + β), (4) ellagic acid.

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Table 2 Central composite design with coded parameters of UAE, and experimentally observed responses in the obtained extracts. Run

Extraction time – X1 (min)

Ethanol concentration – X2 (%)

Solid to solvent ratio – X3 (1:X3)

Extraction temperature – X4 (°C)

TPC (mg GAE/g dw)

Ellagic acid content (mg/g dw)

Gallic acid content (mg/g dw)

Punicalin content (mg/g dw)

Punicalagin content (mg/g dw)

1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26. 27. 28. 29. 30.

1 −1 −1 −1 1 0 −1 1 0 1 −1 1 0 1 1 −1 −1 −1 0 1 0 0 0 0 2 0 −2 0 0 0

1 −1 1 1 −1 0 −1 −1 0 1 1 −1 0 1 −1 −1 −1 1 0 1 0 0 −2 0 0 0 0 0 0 2

1 −1 −1 1 −1 0 1 1 0 −1 1 −1 0 −1 1 −1 1 −1 0 1 0 0 0 −2 0 0 0 2 0 0

−1 1 −1 1 −1 0 −1 1 0 1 −1 1 0 −1 −1 −1 1 1 0 1 0 −2 0 0 0 2 0 0 0 0

149.66 161.20 122.87 121.84 147.55 162.68 154.47 190.94 175.66 139.02 134.58 159.11 162.04 118.01 158.79 127.47 183.43 126.12 160.82 168.54 148.59 158.74 153.24 127.43 172.07 161.70 147.67 180.14 169.30 81.61

8.85 5.46 5.55 11.24 6.35 5.26 5.66 4.77 6.58 8.85 6.78 4.54 6.48 6.22 5.35 4.05 4.90 9.21 5.67 12.54 5.15 5.46 5.98 4.09 6.33 6.21 6.32 9.07 6.71 11.61

2.01 2.61 2.37 3.00 2.66 2.80 1.13 1.95 3.58 2.58 2.01 2.67 2.70 2.38 2.24 1.35 2.54 2.64 2.79 2.12 2.35 1.24 2.47 2.42 3.23 3.01 2.65 2.71 3.15 1.77

54.53 61.92 49.65 61.07 56.86 55.35 36.69 59.13 65.67 52.97 55.55 56.15 60.38 49.70 55.40 35.99 63.90 58.25 52.99 57.98 52.86 34.04 56.15 50.73 62.01 59.78 59.53 63.75 64.24 28.38

14.81 15.47 14.01 17.43 9.94 17.16 27.47 16.01 18.61 15.22 15.55 15.09 17.49 14.72 12.60 26.11 15.93 16.12 16.61 14.13 13.64 35.05 7.04 11.62 17.32 13.87 17.25 17.95 19.22 10.21

TPC – total polyphenols content; dw – dry weight.

and respective responses [22]. The final predictive equation for describing the efficacy of extraction for achieving the maximum TPC from pomegranate peel, having in mind only significant parameters is as follows:

Table 3 Estimated regression coefficients and analysis of the variance of the fitted second-order polynomial models for the investigated parameters. TPC

Source Model X1-extraction time X2-ethanol concentration X3-solid to solvent ratio X4-temperature X1X2 X1X3 X1X4 X2X3 X2X4 X3X4 X12 X22 X32 X42 Lack of fit

Ellagic acid content

Gallic acid content

Punicalin content

Punicalagin content

TPC (mg GAE/g dw) = 59.2653 + 0.0412333X1 + 2.03379X2 + 1.10967X3 + 0.396444X 4−0.0275375X22

p-value* 0.0001 0.0000 0.0167 0.3164 0.0000 0.0000

0.0001 0.3165 0.7890

0.0244 0.4867 0.2457

0.0000 0.1335 0.6500

0.0003

0.0007

0.4303

0.1799

0.3850

0.0205 0.3816 0.3010 0.5034 0.6006 0.1090 0.9593 0.6952 0.0001 0.2821 0.7253 0.4665

0.0071 0.4678 0.7352 0.2063 0.0240 0.0007 0.8051 0.6184 0.0013 0.3960 0.8497 0.5505

0.0029 0.1182 0.3322 0.0368 0.7247 0.6333 0.7770 0.8303 0.0158 0.1859 0.0158 0.6646

0.0025 0.1955 0.9982 0.0616 0.6180 0.2312 0.9218 0.4992 0.0165 0.9708 0.0817 0.5569

0.0348 0.1577 0.9093 0.1655 0.8473 0.3522 0.8262 0.9634 0.0286 0.5109 0.0533 0.1253

Results obtained in our study exhibited that optimal conditions for extraction of maximal TPC are extraction time 65 min, ethanol concentration 31%, solid to solvent ratio 1:50 and extraction temperature 80 °C. 3.2. Effect of extraction parameters on ellagic acid content The content of ellagic acid in pomegranate peel extracts obtained using UAE varied from 4.05 to 12.54 mg/g dw. Our results were two fold lower compared to previous results for ellagic acid content in pomegranate peel extracts obtained using pulsed UAE technique [42]. On the other hand, our results were in accordance with those previously obtained for Israeli pomegranate peel extract obtained by stirring in the ethanol for 24 h [44]. According to our results, the highest content of ellagic acid was achieved using 70% ethanol, temperature of 65 °C, 1:40 solid to solvent ratio and extraction time of 50 min. On the other hand, the lowest content was obtained in extract prepared using 30% ethanol, 1:20 solid to solvent ratio, extraction temperature of 35 °C and 20 min extraction time. Except time, all investigated extraction parameters showed significant positive impact on final content of ellagic acid (Fig. 2II). As in the case of TPC, ethanol concentration showed the highest impact on ellagic acid content in extracts, but this time the influence was positive. Panichayupakarananta et al. [45] investigated the influence of different methanol concentrations on the content of ellagic acid in pomegranate peel extracts. According to their results,

* p values lower than 0.05 are statistically significant.

of higher extraction of TPC with increase in temperature has been observed by other authors [27,38]. In our study, the quadratic level of ethanol concentration also significantly affected TPC, while other parameters were insignificant. Nonsignificant interactions (p > .05) were also obtained between factors. Using regression analysis second-order polynomial equation was developed in order to study the relations between input process variables

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Fig. 2. Response surface plots for the effect of (a) time/ethanol concentration and (b) solid to solvent ratio/temperature on TPC (I), ellagic acid content (II) and gallic acid content (III).

90% (v/v) methanol gave significantly higher content of ellagic acid compared to other analyzed percentage of methanol (80%, 85%, 95% and 100%). Rodrigues et al. [37] also showed that during UAE of jabuticaba tree, the best results for ellagic acid were achieved at high ethanol concentration and high extraction time. Zhang et al. [46] showed that among studied extraction parameters, ultrasonic extraction temperature was the most significant one in affecting extraction yield of ellagic acid from infructescence of Platycarya strobilacea. According to this study, extraction temperature of 70 °C was the optimal one for UAE of ellagic acid. Di et al. [47] previously reported that the optimal temperature for UAE of ellagic acid from pomegranate peel was 60 °C. Qu et al. [48] suggested that the increase in ellagic acid content after thermal treatment, during sterilization of pomegranate peel liquid extracts, might be a consequence of the degradation of ellagitannins or ellagic acid-glycosides. According to Kazemi et al. [42], during pulsed UAE the content of ellagic acid in pomegranate peel extracts increased with the increasing of extraction time. This is in contradiction with our results, as well as with those previously reported by Ya-Qin et al. [49]. They reported that exposure of phenolic acids (such as caffeic acid, pcoumaric acid, ferulic acid and p-hydroxybenzoic acid) to long ultrasonic treatment may cause their loss due to degradation. In our study, the quadratic level of ethanol concentration, and interactions among ethanol concentration and solid to solvent ratio, and ethanol concentration and temperature also significantly affected ellagic acid content, while other parameters were insignificant. The final predictive equation for describing the efficacy of extraction for achieving the maximal ellagic acid content from pomegranate peel, having in mind only significant parameters is as follows:

Ellagic acid content (mg/g dw) = 14.6667−0.344608X2−0.0626494X3 −0.129107X 4 + 0.0017108X22 + 0.00290358X2X3 + 0.00337113X2X 4 Our study showed that optimal conditions for extraction of maximal content of ellagic acid from pomegranate peel are as follows: extraction time 5 min, ethanol concentration 88%, solid to solvent ratio 1:50 and extraction temperature 80 °C. 3.3. Effect of extraction parameters on gallic acid content The content of gallic acid in pomegranate peel extracts obtained using UAE varied from 1.13 to 3.58 mg/g dw. Our results were significantly higher compared to study of Kazemi et al. [42], where the highest content of gallic acid in pomegranate peel extracts obtained using pulsed ultrasound assisted technique was 0.05 mg/g. On the other hand, Elfalleh et al. [50], reported that the gallic acid content in six varieties of Tunisian pomegranate peel after methanol extraction overnight varied from 1.09 to 1.31 mg/g. In our study, the extract with the highest gallic acid content was obtained using follow parameters: time 35 min, ethanol concentration 50%, solid to solvent ratio 1:30 and temperature 50 °C. However, the lowest content was obtained in extract prepared using 30% ethanol, 1:40 solid to solvent ratio, extraction temperature of 35 °C and 20 min extraction time. ANOVA showed that the most dominant factor influencing extraction of gallic acid was temperature, and this factor affected gallic acid content positively (Fig. 2III). The quadratic levels of ethanol concentration and temperature, and interactions among extraction time and temperature also significantly changed gallic acid content, while other parameters were 44

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insignificant. The final predictive equation for describing the efficacy of extraction for achieving the maximal gallic acid content from pomegranate peel, having in mind only significant parameters is as follows:

time 5 min, ethanol concentration 45%, solid to solvent ratio 1:35 and extraction temperature 80 °C.

Gallic acid content (mg/g dw) = −3.73115 + 0.0540525X1 + 0.0513287X2

Punicalagin is found in nature in two reversible anomer forms α and β. Since it is difficult to quantify them individually, they are often mentioned in the singular punicalagin [42]. According to literature, punicalagin contents in pomegranate peel ranging from 10.50 to 98.00 mg/g [39]. In our study, depending on applied extraction conditions, punicalagin content varied from 7.04 to 35.05 mg/g dw. The extract with the highest punicalagin content was obtained using following parameters: time 35 min, ethanol concentration 50%, solid to solvent ratio 1:30 and temperature 20 °C. On the other hand, the lowest content was obtained in extract prepared using 10% Ethanol, 1:30 solid to solvent ratio, extraction temperature of 50 °C and 35 min extraction time. ANOVA showed that the most dominant factor influencing extraction of punicalagin was temperature, and this factor affected punicalagin content negatively probably due to degradation. High temperatures during longer extraction time may induce hydrolysis of the galloyl moiety attached to the glucose and also release of ellagic acid from ellagitannins [52]. The quadratic level of ethanol concentration was also with significant influence, while other parameters, and interactions among them were insignificant. The final predictive equation for describing the efficacy of extraction for achieving the maximal punicalagin content from pomegranate peel, having in mind only significant parameters is as follows:

3.5. Effect of extraction parameters on punicalagin (α + β) content

+ 0.149891X 4−0.00108105X1X 4 −0.000513287X22−0.000912171X24 Optimal conditions for UAE of maximal content of gallic acid from pomegranate peel are as follows: extraction time 5 min, ethanol concentration 66%, solid to solvent ratio 1:40 and extraction temperature 77 °C. Regarding temperature and ethanol concentration, our results were in accordance with those previously obtained by Pawar and Surana [51]. As optimal conditions for extraction of gallic acid from Caesalpina decapetala wood they recorded 70% ethanol and 65–70 °C as extraction temperature. 3.4. Effect of extraction parameters on punicalin content In the current research punicalin content varied from 28.38 to 65.67 mg/g dw. In our study, based on the experimental data, the extract with the highest punicalin content was obtained using follow parameters: time 35 min, ethanol concentration 50%, solid to solvent ratio 1:30 and temperature 50 °C. On the other hand, the lowest content was obtained in extract prepared using 90% ethanol, 1:30 solid to solvent ratio, extraction temperature of 50 °C and 35 min extraction time. ANOVA showed that the most dominant factor influencing extraction of punicalin was temperature, and this factor affected punicalin content positively. As shown in Fig. 3I punicalin content sharply increased with the increase in extraction temperature. The quadratic level of ethanol concentration also exhibited significant effect on this response. Other parameters, and interactions among them were insignificant. The final predictive equation for describing the efficacy of extraction for achieving the maximal punicalin content from pomegranate peel, having in mind only significant parameters is as follows:

Punicalagin content (mg/g dw) = 11.3473 + 0.566806X2−0.144969X 4 −0.00566806X22 Our study showed that optimal conditions for extraction of maximal content of punicalagin from pomegranate peel are as follows: extraction time – 5 min, ethanol concentration – 26%, solid to solvent ratio – 1:47 and extraction temperature – 20 °C. 3.6. Optimization of extraction parameters and model validation

Punicalin content (mg/g dw) = 17.3522 + 0.880252X2 + 0.356918X 4 Determination of predicted values and optimal conditions was established on desirability function which was in the range from 0.95 to 1, where 1 represents the most desirable response (Table 4). After applying the desirability function method for all of the investigated

−0.00880252X22 Our study showed that optimal conditions for extraction of maximal content of punicalin from pomegranate peel are as follows: extraction

Fig. 3. Response surface plots for the effect of (a) time/ethanol concentration and (b) solid to solvent ratio/temperature on punicalin content (I) and punicalagin content (II).

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Table 4 Comparison between predicted and experimentally obtained values for investigated responses. Response values (mg/g dw)

Predicted value

Experimental value

TPC Ellagic acid content Gallic acid content Punicalin content Punicalagin content

157.35 11.01 2.77 65.00 17.99

149.12 ± 7.46 11.65 ± 0.42 2.87 ± 0.11 61.93 ± 2.41 18.05 ± 0.62

[6]

[7]

[8]

dw – dry weight; TPC – total polyphenols content.

[9]

responses (total polyphenols, ellagic acid, gallic acid, punicalagin and punicalin content) it can be concluded that optimal conditions for extraction of their maximal content from pomegranate peel are as follows: extraction time of 25 min, ethanol concentration of 59%, solid to solvent ratio of 1:44, and extraction temperature of 80 °C. Predicted response values obtained by models under optimum conditions were checked experimentally using same extraction conditions. Results obtained during validation of optimized conditions were close to predicted values (Table 3). This confirms that selected RSM model was successfully applied for UAE of pomegranate peel in order to obtain extracts with maximal TPC, and content of individual bioactive compounds (ellagic acid, gallic acid, punicalagin, punicalin).

[10] [11]

[12]

[13]

[14] [15] [16]

4. Conclusion

[17]

Pomegranate peel, although mainly considered as a waste of juice industry, attracts increasing interest due to the complex chemical composition and confirmed biological activities. In the present study, the effects of ethanol concentration, extraction time, solid to solvent ratio and extraction temperature on UAE of pomegranate peel were evaluated using response surface methodology. A multi-response optimization study based on central composite design enables us the prediction of optimal conditions for high rate extraction of total polyphenols, ellagic acid, gallic acid, punicalin and punicalagin from pomegranate peel. Extraction time of 25 min, ethanol concentration of 59%, solid to solvent ratio of 1:44, and extraction temperature of 80 °C were prove to be optimal in the case of the pomegranate peel phenolics. Further, investigation of in vitro and in vivo biological activities of the extract obtained under optimal conditions are in progress in our laboratory.

[18]

[19]

[20]

[21]

[22] [24] [25]

Conflict of interest [26]

The authors declare that there are no conflicts of interest.

[27]

Acknowledgements This work was supported by the Ministry of Education, Science and Technological Development of the Republic of Serbia, project number 46013.

[28]

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