Evaluation of different extraction methods from pomegranate whole fruit or peels and the antioxidant and antiproliferative activity of the polyphenolic fraction

Accepted Manuscript Evaluation of different extraction methods from pomegranate whole fruit or peels and the antioxidant and antiproliferative activity of the polyphenolic fraction Alessandra Masci, Andrea Coccia, Eugenio Lendaro, Luciana Mosca, Patrizia Paolicelli, Stefania Cesa PII: DOI: Reference:

S0308-8146(16)30105-4 http://dx.doi.org/10.1016/j.foodchem.2016.01.106 FOCH 18680

To appear in:

Food Chemistry

Received Date: Revised Date: Accepted Date:

31 July 2015 17 December 2015 26 January 2016

Please cite this article as: Masci, A., Coccia, A., Lendaro, E., Mosca, L., Paolicelli, P., Cesa, S., Evaluation of different extraction methods from pomegranate whole fruit or peels and the antioxidant and antiproliferative activity of the polyphenolic fraction, Food Chemistry (2016), doi: http://dx.doi.org/10.1016/j.foodchem.2016.01.106

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Evaluation of different extraction methods from pomegranate whole fruit or peels and the antioxidant and antiproliferative activity of the polyphenolic fraction

Alessandra Mascia*, Andrea Cocciab, Eugenio Lendarob, Luciana Moscac, Patrizia Paolicellid, Stefania Cesad

a

Dipartimento di Medicina Sperimentale, Unità di Ricerca di Scienza dell’Alimentazione e

Nutrizione Umana, Università degli Studi di Roma “La Sapienza”, viale del Policlinico, 155 00161 Roma, Italy b

Dipartimento di Scienze e Biotecnologie Medico-Chirurgiche, Università degli Studi di Roma “La

Sapienza”, corso della Repubblica, 79 - 04100 Latina, Italy c

Dipartimento di Scienze Biochimiche, Università degli Studi di Roma “La Sapienza”, piazzale

Aldo Moro, 5 - 00185 Roma, Italy d

Dipartimento di Chimica e Tecnologie del Farmaco, Università degli Studi di Roma “La

Sapienza”, piazzale Aldo Moro, 5 - 00185 Roma, Italy

*Corresponding author: Alessandra Masci Address: Dipartimento di Medicina Sperimentale, Unità di Ricerca di Scienza dell’Alimentazione e Nutrizione Umana, Università degli Studi di Roma “La Sapienza”, viale del Policlinico, 155 00161 Roma, Italy Fax: +390649910699 e-mail: [email protected]

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Abstract

Pomegranate is a functional food of great interest, due to its multiple beneficial effects on human health. This fruit is rich in anthocyanins and ellagitannins, which exert a protective role towards degenerative diseases. The aim of the present work was to optimize the extraction procedure, from different parts of the fruit, to obtain extracts enriched in selected polyphenols while retaining biological activity. Whole fruits or peels of pomegranate cultivars, with different geographic origin, were subjected to several extraction methods. The obtained extracts were analyzed for polyphenolic content, evaluated for antioxidant capacity and tested for antiproliferative activity on human bladder cancer T24 cells. Two different extraction procedures, employing ethyl acetate as a solvent, were useful in obtaining extracts enriched in ellagic acid and/or punicalagins. Antioxidative and antiproliferative assays demonstrated that the antioxidant capability is directly related to the phenolic content, whereas the antiproliferative activity is to be mainly attributed to ellagic acid.

Keywords Pomegranate; Ellagic acid; HPLC-DAD; Antioxidant capacity; Antiproliferative activity; Human bladder cancer T24 cells.

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1. Introduction

Plant-based diets have long been associated with increased life expectancy and a reduced risk of chronic and degenerative diseases. Many juices, obtained from fruits or vegetables, are considered of interest as nutraceuticals for their high content in bioactive phytochemicals. Among phytochemicals, polyphenols, and anthocyanins in particular, attract a huge scientific interest. They are secondary metabolites that plants produce as a defense from different types of environmental stresses, such as UV irradiation and the attack by pathogens, parasites and herbivores, playing an important role as antioxidant and protective substances (Pandey & Rizvi, 2009). Due to their properties, the intake of polyphenols is associated with a lower incidence of several human diseases and lower mortality rates (Akhtar, Ismail, Fraternale & Sestili, 2015; Ferrari et al., 2011; TresserraRimbau et al., 2014). Among polyphenol-rich plants, pomegranate has a long history of use in folk and Ayurvedic medicine (Li, Guo, Yang, Wei, Xu & Cheng, 2006). Pomegranate fruits come from a shrub or a small tree (Punica granatum L.) belonging to the family of Punicaceae. This plant is a native of the tropical and subtropical regions of Middle East and India, but the cultivation of pomegranate is nowadays widely distributed in the temperate regions of China, Mediterranean countries, the United States and Mexico (Lansky & Newman, 2007; Mena, Vegara, Marti, Garcia-Viguera, Saura & Valero, 2013). Pomegranate fruits comprise of four parts: the not edible exocarp and mesocarp (the peel), and also the edible endocarp which contains the seed, forming the arils. All of these contain interesting bioactive molecules, such as anthocyanins in the arils, hydrolysable tannins in the peel and punicic acid in the seeds, hence pomegranate whole fruit extracts are also very interesting as dietary supplements and nutraceuticals (Akhtar, et al., 2015; Lansky et al., 2007). Anthocyanins are the main pomegranate pigments, responsible for the deep red colour of the pulp and belong to a complex group of glycosides represented by only six aglycones, i.e. flavylium

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cations substituted in various positions by different sugars. Only 3-glucosides and 3,5-diglucosides of cyanidin, delphinidin and pelargonidin are present in pomegranate pulp, however the anthocyanins concentration in pomegranate is very high compared to other fruits (Can, Arli & Atkosar, 2012; Gomez-Caravaca, Verardo, Toselli, Segura-Carretero, Fernandez-Gutierrez & Caboni, 2013; Sentandreu, Cerdan-Calero & Sendra, 2013). These molecules, in particular cyanidin-3-O-β-glucoside, are the most represented anthocyanin in pomegranate, and are considered to be responsible for the protective effect towards cancer and tumor migration and invasion (Chen, Chu, Chiou, Kuo, Chiang & Hsieh, 2006; Domitrovic, 2011). Polyphenols, mainly ellagitannins and ellagic acid, are an important class of compounds well represented in pomegranate. These molecules are particularly abundant in pomegranate peels and are responsible for the antioxidant, anti-mutagenic, anti-cancer, anti-inflammatory, anti-diabetic and health promoting role of this fruit. They act as radical scavengers, both in preventing the degradation of food and in protecting human body towards reactive oxygen species (ROS), whose role in the pathogenesis of human diseases is widely recognized (Fischer, Jaksch, Carle & Kammerer, 2013; Gil, Tomas-Barberan, Hess-Pierce, Holcroft & Kader, 2000; Negi, Jayaprakasha & Jena, 2003; Usta, Ozdemir, Schiariti & Puddu, 2013). These components also display an antiatherogenic effect correlated with the high antioxidant potential of the pomegranate extracts. Indeed, it was demonstrated that pomegranate juice consumption in humans decreases lipid peroxidation and LDL susceptibility to aggregation (Aviram et al., 2000). Ellagic acid in its free and bound forms is one of the most abundant polyphenols in pomegranate (Gil et al., 2000). In the last few years, many studies were devoted to evaluate the protection afforded by ellagic acid as a key species in scavenging a wide variety of free radicals in aqueous solutions and at physiological pH (Galano, Marquez & Perez-Gonzalez, 2014). These studies also demonstrated that ellagic acid exerts an efficient and continuous protection against oxidative stress at low concentrations, through regeneration after the scavenging of two radical molecules. Ellagic acid also displays a wide

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spectrum of antitumor properties through multiple pathways (Zhang, Zhao, Li, Xu, Chen & Tao, 2014). In recent studies, ellagic acid has shown anti-invasive effects on androgen-independent human (PC-3) and rat (PLS10) prostate cancer cell lines, and it also decreases the secretion of MMP-2 from both cells (Pitchakarn et al., 2013). Moreover, a recent study has indicated that ellagic acid at low concentrations (0.5-3 µM) triggers apoptosis and inhibits the proliferation of the human pancreatic cancer MIA PaCa-2 and HPAF-II cells (Edderkaoui et al., 2013). Given the wide spectrum of health promoting activities exerted by pomegranate and the enormous interest that bioactive components isolated from this fruit have raised in the scientific community, we focused our efforts in optimizing the extraction procedure of bioactive compounds in order to obtain extracts enriched in polyphenols while retaining antioxidant and antiproliferative activities. We also aimed at evaluating the differences in polyphenolic composition of the extracts prepared from separated peels or pomegranate whole fruit in order to optimize the content in bioactive compounds by minimizing possible interferences due to other constituents. Our attention was mainly focused on the ellagitannin components of pomegranate, i.e. punicalagins and ellagic acid, that are concentrated in the husks, without neglecting other fruit parts that are rich in bioactive constituents and that could represent a good source for substitutes of synthetic food additives, nutraceuticals and chemopreventive agents (Akhtar et al., 2015). We selected two subvarieties of an Italian selected cultivar, the Dente di Cavallo, and for comparison we purchased, from a local market, two other cultivars which were generically from Israel or Italy. Several extraction methods were employed and the obtained extracts were submitted to qualitative-quantitative HPLC-DAD and spectrophotometric analyses. The biological activity of the extracts was also evaluated in terms of in vitro antioxidant capacity and antiproliferative activity towards the human bladder cancer T24 cells.

2. Materials and methods

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2.1. Materials

96% ethanol, methanol, 85% formic acid and bidistilled water were purchased from Carlo Erba (Milan, Italy), glacial acetic acid and ethyl acetate were purchased from Fluka (Milan, Italy), acetonitrile RS for HPLC and dimethyl sulfoxide (DMSO) were purchased from Sigma-Aldrich Chemistry (Milan, Italy). Punicalagin (≥98%) and ellagic acid (≥95%) were purchased from SigmaAldrich Chemistry. All the other reagents were analytical grade reagents from Sigma-Aldrich Chemistry. Pomegranate fruits of Israeli (Isr) and Italian (Ita) origin were purchased from local markets; some pomegranate fruits of Italian origin were a generous gift from a local producer (Azienda Biologica Giovomel, Avellino, Italy) and were from two different subvarieties of cv. Dente di Cavallo (DC1 and DC2), with about two weeks difference in the harvest time.

2.2. Sample preparation

Pomegranate fruits were used as whole fruit or separated in two parts: arils and peels (exocarp and mesocarp). Whole fruits were washed, blended in a mixer for 30 sec and extracted as described. Peels manually separated from washed fruit were blended in a mixer for 30 sec and extracted as described.

2.3. Extraction methods

2.3.1. Whole fruit or peel extraction with ethanol or methanol, or ethanol and acidified water 100 g of the blended whole fruit or the manually separated peels were extracted with 400 ml ethanol or methanol or with 400 ml ethanol:acidified water (10% acetic acid) in 1:1 or 3:1 ratios. Samples

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were stirred for 24 h at room temperature in the dark. The extraction mixture was well decanted and filtered on paper, then evaporated at 40 °C in the dark obtaining a purple-red sticky residue from whole fruit, or a yellow-brown residue from peels (about 10 g) that were immediately analyzed.

2.3.2. Repartition in ethyl acetate The dried extract obtained from the previous extraction were dissolved in 100 ml of water and extracted three times in a separating funnel with 100 ml ethyl acetate. The aqueous solution was freeze-dried, whereas the ethyl acetate solution was dried on sodium sulfate and evaporated at 40 °C in the dark. The obtained dried extract, respectively from ethyl acetate and aqueous fractions, were immediately analyzed or stored at –18 °C.

2.3.3. Peel extraction in Soxhlet apparatus 100 g of the blended peels were extracted with 400 ml ethyl acetate in a Soxhlet extractor for 3, 6 and 15 h in the dark (4-5 extraction cycles per hour) as described by Negi et al. (2003). The obtained solution was evaporated at 40 °C in the dark to a dry yellow-brown residue that was immediately analyzed or stored at –18 °C.

2.4. Determination of phenols, flavonoids and anthocyanins

2.4.1. Total phenols Total phenols were determined by the Folin-Ciocalteu assay, as described by Singleton & Rossi Jr (1965). Briefly, the reaction solution was prepared by mixing 10 µl of blank, gallic acid standard or sample (200 µg/ml in 1:50 (v/v) DMSO:H2O solution) with 790 µl of distilled water. After addition of 50 µl Folin-Ciocalteu reagent the reaction mixture was incubated for 3 min at room temperature

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and then 150 µl of a 20% (w/v) Na2CO3 aqueous solution was added. After 2 h of incubation, the absorbance at 760 nm was measured on a Hitachi U2000 spectrophotometer (Hitachi, Tokyo, Japan). The results were expressed as millimoles of gallic acid equivalents (GAE) per gram of dry extract.

2.4.2. Total flavonoids The total flavonoid content of the extracts was determined using the aluminium trichloride assay as described by Dewanto, Wu, Adom & Liu (2002). The assay was carried out in a 96-well plate and in each well the following solutions were added: 20 µl rutin hydrate standard (half serial dilutions of 4 mg/ml) in DMSO or 20 µl dry extract solution (1.5 mg/ml) in DMSO or blank, 20 µl sodium nitrate solution (3%, w/v), 20 µl aluminium trichloride solution (1%, w/v) in methanol and 100 µl sodium hydroxide solution (0.5 M). Absorbance was measured at 450 nm with an Appliskan microplate reader (Thermo Scientific, Vantaa, Finland). Results were expressed as millimoles of rutin equivalents (RE) per gram of dry extract.

2.4.3. Total anthocyanins Total anthocyanins quantification was performed by the pH-differential method as described by Giusti & Wrolstad (2001). An aliquot of 70 µl of a 10 mg/ml dry extract in DMSO was diluted in 1 ml of a pH 1.0 solution (0.1 M HCl, 25 mM KCl) or 1 ml of a pH 4.5 solution (0.4 M CH3COONa). The absorbance of the mixtures was then measured at 535 and 700 nm against distilled water. The value (Abs535 – Abs700)pH1.0 – (Abs535 – Abs700)pH4.5 corresponds to the absorbance due to the anthocyanins. Calculation of the anthocyanins concentration was based on a cyanidin-3-Oglucoside molar extinction coefficient of 25,965 M-1 × cm-1 and a molecular mass of 449.2 g/mol. Results were expressed as micromoles cyanidin-3-O-glucoside equivalents (CGE) per gram of dry extract.

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2.4.4. HPLC analyses Dried extracts from the previous described extractions were weighed, dissolved in DMSO or water and filtered before injection. HPLC analyses were performed with a Perkin Elmer apparatus consisting of a Serie 200 LC pump, a Serie 200 DAD and a Serie 200 autosampler, using the Totalchrom Perkin Elmer software for the data acquisition. Chromatography was performed on a LiChrosorb RP18 column (250 × 4.6 mm i.d., 5 µm) using a mobile phase composed of acetonitrile (solvent A) and water containing 5% formic acid (solvent B), which in 40 min changed from 5% A and 95% B to 20% A and 80% B, with a flow rate of 1 ml/min, at λ 360 nm (Gomez-Caravaca et al., 2013; Sentandreu et al., 2013). Punicalagin anomers α and β and ellagic acid were identified in the samples by comparison of the retention times and UV spectra of pure external standards. The quantitative analyses were performed by calibration curves ranging from 1-100 mg/g (y=15.3x+26.8, with correlation coefficient R2=0.9986) for punicalagins, and from 0.2-80 mg/g (y=39.6x+49.5, with correlation coefficient R2=0.9919) for ellagic acid, separately.

2.5. Antioxidant activity

2.5.1. NBT assay The effect of pomegranate extracts as a superoxide anion scavenger were assayed by the inhibition of nitroblue tetrazolium chloride (NBT) reduction by β-nicotinamide adenine dinucleotide reduced form (βNADH) in the presence of phenazine methosulfate (PM) as described by Yuting, Rongliang, Zhongjian & Yong (1990). Reaction mixtures contained 73 µM βNADH, 15 µM PM, 50 µM NBT and 0-200 µg/ml of each extract in 1 ml of 0.02 M Tris-HCl buffer, pH 8.0. Absorbance variations (∆Abs/min) were determined at 560 nm, by measuring the initial rate of superoxide anion-induced NBT reduction. The percentage inhibition of ∆Abs560 nm/min after 15 sec at 25 °C was calculated

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and plotted as a function of concentration of antioxidants. Stock solutions (1 mM) were freshly prepared every day dissolving PM in ethanol, NBT in water and βNADH in 0.05 M phosphate buffer, pH 7.4.

2.5.2. ABTS assay The 2,2′-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS) was dissolved in water to a 7 mM final concentration. ABTS radical cation (ABTS•+) was produced by reacting ABTS stock solution with 2.45 mM potassium persulfate for 16 h in the dark at room temperature (Re, Pellegrini, Proteggente, Pannala, Yang & Rice-Evans, 1999). ABTS•+ was diluted with ethanol, to obtain an absorbance of 0.70 (±0.02) at 734 nm. An aliquot of 10 µl of diluted extract in DMSO (0-40 µg/ml final concentration) was added to 1 ml of the working ABTS•+ solution. The percentage inhibition of absorbance at 734 nm after 6 min at room temperature was calculated and plotted as a function of antioxidant concentration.

2.5.3. DPPH assay An aliquot of 10 µl of diluted extracts in DMSO (0-50 µg/ml final concentration) was added to 1 ml of 30 µM ethanolic solution of stable nitrogen centered free radical 2,2-diphenyl-1-picrylhydrazyl (DPPH•) and the absorbance was monitored spectrophotometrically at 517 nm after 15 min at room temperature. Radical DPPH• scavenging capacity was estimated from the difference in absorbance with or without antioxidants and expressed as percent DPPH• disappearance as a function of the sample concentration.

2.5.4. ORAC assay For oxygen radical absorbance capacity (ORAC) assay, the method of Cao & Prior (1999) was used. The assay was carried out in a black-walled 96-well plate and each well contained a final

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volume of 200 µl. Pomegranate extracts were diluted with 75 mM phosphate buffer (pH 7.0) to a final concentration of 1.25 µg/ml. The 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid (Trolox) was used as a standard to a final concentration of 5 µM. The reaction solution also contained 3.4 µg/ml of R-phycoerythrin, and the plate was incubated at 37 °C for 15 min. Then 2,2′azobis(2-methylpropionamidine) dihydrochloride (ABAP) was added to each well to a final concentration of 16 mM, and fluorescence intensity was estimated every 5 min for a total of 120 min using an excitation filter of 492/10 nm and an emission filter of 570/10 nm. Results were calculated on the basis of the differences in area under the curve between the standard and the sample.

2.6. Antiproliferative activity

2.6.1. Cell cultures T-24 cells (human urinary bladder carcinoma) were obtained from CLS (Cell Lines Service) and cultured in DMEM medium supplemented with 10% FBS, 2 mM glutamine, 100 U/ml penicillin and 100 µg/ml streptomycin. All cell culture products were purchased from Invitrogen (Carlsberg, CA, USA) and certified as endotoxin-free.

2.6.2. Cytotoxicity assay Cell viability was assessed by using the tetrazolium compound [3-(4,5-dimethylthiazol-2-yl)-5-(3carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt; MTS] and an electron coupling reagent (phenazine ethosulfate; PES). The assay is based on the ability of living cells to convert MTS into an insoluble purple-coloured formazan, whose amount is proportional to the number of living cells. Cells seeded in 96-well plates at a density of 5,000 cells/well were exposed to various extracts at a concentration ranging between 200 to 25 µg/ml. After 48 h, the media was

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discarded and the cells were incubated for 2 h with 100 µl of media containing 20 µl of MTS solution (Promega). After the treatment the absorbance was read at 492 nm using a microplate reader.

2.7. Statistical analysis

Each assay was replicated at least three times, and statistical significance was determined using Graphpad Prism 4 statistical software package (Graphpad, San Diego, CA, USA). Data are expressed as mean ± SEM. The correlation values between experimental data was evaluated by the Pearson coefficient. In the antiproliferative experiments, comparison of the groups was made by one-way ANOVA followed by Bonferroni’s post hoc test. Statistical significance was defined as P<0.05.

3. Results

3.1. Polyphenols extraction

Extraction yields (% w/w) from whole fruits or peels achieved with the above described conditions from the four groups of selected samples (Isr, Ita, DC1 and DC2), are reported in Table 1. Experiments 1-8 were performed on whole fruits, whereas experiments 9-24 were performed on manually separated peels. The use of ethanol or methanol at room temperature on whole fruits (exp. 1-8) gave yields between 9.5% and 14.2%, without significant differences between the two solvents (on average 12.0% and 12.6% respectively) except in the case of the Isr sample. At the same yield, the less safe use made the methanol an undesirable solvent, so the subsequent experiments on peels samples (exp. 9-20) were all performed with ethanol. Pomegranate peels were submitted to 12

extraction by ethanol alone (average yield 10.5%) or by mixture with acidified water (10% acetic acid) in the ratio 3:1 (average yield 12.9%) or 1:1 (average yield 11.5%). When ethanol was used in the mixture, the extraction yield was generally slightly increased, in particular in the ratio 3:1, but these procedures are less practical in the removal of the aqueous phase by evaporation. The direct extraction of peels in Soxhlet apparatus (exp. 21-24) employing ethyl acetate, without any addition of water, and at the ebullioscopic temperature of this solvent (77 °C), were performed at 3, 6 and 15 h without significant differences of yield in relation to the adopted extraction times (data not shown). The results of the extractions conducted for 6 h gave yields between 0.66%1.09%. The dried extracts obtained from experiments 1-20 were extracted again in experiments 25-44 using a repartition system of water and ethyl acetate. The yields of ethyl acetate extracts, ranging between 2.2%-7.9%, combined with the first extraction yields, gave overall yields between 0.21%-1.10%, that should be compared with the direct extraction of peels with ethyl acetate by using the Soxhlet extractor. These results are partially comparable.

3.2. Qualitative-quantitative analysis of the polyphenolic fraction

Some of the previous obtained extracts (experiments 1, 25 aqueous and 25 ethyl acetate fraction from Israeli whole fruit; experiments 9, 33 aqueous, 33 ethyl acetate fraction and 21 from Israeli peels; experiments 12, 36 ethyl acetate fraction and 24 from DC2 peels) were submitted to qualitative-quantitative analysis of the polyphenolic fraction (Table 2). The total phenolic content of freeze-dried pomegranate extracts, as measured by the Folin-Ciocalteu method and expressed as mmol GAE/g, is reported in Table 2. Results show that extracts prepared starting from the peel have a phenolics content two to three times higher than the corresponding extracts obtained from the whole fruit; the ethanol extract and the aqueous fraction show a 3-fold

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increase (P<0.001), and the ethyl acetate fraction a 2-fold increase (P<0.01) in polyphenolic content. The content of total polyphenols in the Isr peel was similar to that found in the DC2 peel. The fraction in ethyl acetate is the one that showed the greatest concentration of phenolic compounds compared to the ethanol extract and the aqueous fraction that were comparable to each other (Isr whole fruit: 4-fold increase, P<0.001; Isr peel: 3-fold increase, P<0.01; DC2 peel: 2-fold increase, P<0.001). The direct extraction using Soxhlet apparatus for the Isr sample showed a 0.3fold decrease (P<0.01) compared to the repartition with ethyl acetate, while the DC2 sample was the same as obtained in ethyl acetate fraction. Table 2 also reports data regarding flavonoid and anthocyanin concentrations in the selected extracts. Comparing the same extraction procedure, similarly to what already observed for the total polyphenols, the content of flavonoids was always higher in pomegranate peel extracts with respect to whole fruit; the ethanol extract and the aqueous fraction show a 3-fold increase (P<0.001) whereas the ethyl acetate fraction a 1.5-fold increase (P<0.001). Comparably to total polyphenols, the content of flavonoids in the Isr peel was substantially similar to that of the DC2 peel except for the fraction in ethyl acetate (DC2 respect to Isr peel: 0.5-fold increase, P<0.05). Once again the higher content of flavonoids is obtained in the repartition with ethyl acetate compared to the ethanolic extract, which was similar to that of the aqueous fraction (Isr whole fruit: a 4.4-fold increase, P<0.001; Isr peel: a 2.0-fold increase, P<0.001; DC2 peel: a 2.5-fold increase, P<0.05). The extraction of pomegranate peel with ethyl acetate using Soxhlet apparatus showed a slight decrease of flavonoid concentration compared to the ethyl acetate fraction (Isr peel: a 0.2-fold decrease, P<0.01; DC2 peel: a 0.4-fold decrease, P<0.05). All the different types of extract obtained from the whole fruit showed a 30% increase in content of flavonoids with respect to total polyphenols. For the peels, the content in flavonoids was more variable, ranging from 25 to 40%. Regarding anthocyanins, results indicate that the amount in the two ethanolic extracts from whole fruit or peel of the Isr sample were comparable. The anthocyanins were almost completely

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transferred in the aqueous fraction, whereas they were not found in the ethyl acetate fraction. From Isr peel, the content of anthocyanins in the Soxhlet extract was comparable to that of the ethanolic extract. Regarding DC2, it was much less colourful than the Isr sample and indeed only the extraction of peels by Soxhlet was able to highlight the presence of traces of anthocyanins in a 10fold lower amount compared to the Soxhlet extract of Isr peel. Fig. 1 reports representative chromatographic analyses of the phenolic fraction. The chromatograms were similar to those already reported in the literature (Seeram, Lee, Hardy & Heber, 2005; Zhang, Wang, Lee, Henning & Heber, 2009) and demonstrated the predominant presence of punicalagin α and β anomers and ellagic acid that were quantified in all the selected extracts. HPLC-DAD results of polyphenolic analyses are reported in Table 2. The Isr sample showed a punicalagins content of 8.96 mg/g and an ellagic acid content of 0.61 mg/g in the ethanolic extract from whole fruit, which increased 3-fold and 20-fold, respectively, when the extract was obtained from peels. If we consider the repartition in ethyl acetate from whole fruit, a 2.4-fold increase for punicalagins concentration and even a 40-fold increase in the ellagic acid concentration. The repartition in ethyl acetate from peel gave a comparable punicalagins enrichment with respect to ethanolic extract, both in the Isr and in the DC2 samples, whereas the ellagic acid had a 4-fold increase for the Isr sample and a 9fold increase for the DC2 sample. The extracts in ethyl acetate from peels showed the highest amount of punicalagins and ellagic acid in total, although with an inverted ratio. The highest content of ellagic acid was instead achieved in the two Soxhlet extracts, with a certain variability between the two different samples.

3.3. Antioxidant activity

The antioxidant activity of pomegranate extracts was evaluated by four different methods, each directed towards a specific free radical (Table 3). In the ORAC assay the Trolox was used as

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reference standard and the results were expressed as µM Trolox equivalents per milligram of freezedried extract. Instead, in the NBT, ABTS and DPPH assays the antioxidant activity was calculated as the concentration (µg/ml) of freeze-dried extract able to induce a variation of 50% (IC50) in absorbance of the free radical target or of its chromogenic substrate. When measuring the antioxidant activity of pomegranate extracts by the NBT method, a particularly low antioxidant activity was observed compared to the ABTS and DPPH methods (statistical significance variable from P<0.05 to P<0.001, data not shown in Table 3). Substantially, all analytical methods indicated a higher antioxidant activity in extracts prepared from peel rather than whole fruit. Comparing the extracts obtained from the peel of the two different samples, Isr and DC2, data on antioxidant activity were rather similar for all assays examined, except for a greater (P<0.05) scavenging activity toward superoxide anions highlighted by the NBT method in the Soxhlet extract for DC2 peel compared to the same extract prepared from Isr peel. For all the samples examined, the ethyl acetate fraction showed the greatest antioxidant activity, compared to that from the ethanol extract and the aqueous fraction that were almost always comparable to each other. The direct extraction of peel by Soxhlet gave a free radical scavenging activity quite comparable to that of the ethyl acetate with one exception, as already observed before, for the NBT assay, and in any case the antioxidant activity appears to be higher than that of the ethanol extract as well as the aqueous fraction. As inferable from the data on the correlation analysis shown in Table 4, the antioxidant potential measured by these assays, essentially follow the differences in content of total polyphenols, that is, the increase of the concentration of polyphenolic compounds corresponds to an increase of the antioxidant capacity of the extracts (ORAC: ρ = 0.97; NBT: ρ = –0.94; ABTS: ρ = –0.86; DPPH: ρ = –0.74). A good correlation with the antioxidant activity of pomegranate extracts was also appreciable with respect to total flavonoids (ORAC: ρ = 0.92; NBT: ρ = –0.82; ABTS: ρ = –0.82; DPPH: ρ = –0.73) and, although less relevant in some analyses, to ellagic acid concentration (ORAC: ρ = 0.85; NBT: ρ = –0.71; ABTS: ρ = –0.68; DPPH: ρ = –0.58).

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3.4. Antiproliferative activity

The antiproliferative activity was evaluated by measuring the percent growth inhibition of T24 cells after 48 h of incubation in the presence of 50 µg/ml of the various extracts (Fig. 2 A). The most efficacious was the Soxhlet extract from Isr peel, with a 54.3% inhibition of cell proliferation and the highest ellagic acid content of 63.61 mg/g (Table 2), whereas the less efficacious was the aqueous extract from Isr whole fruit, exerting a negligible inhibition on cell proliferation (data not shown) and showing the lowest ellagic acid content (0.26 mg/g, Table 2). The statistical analysis (Table 4) reveals a strikingly high correlation (ρ = 0.94) between percent growth inhibition and ellagic acid amount, less relevant for total polyphenols (ρ = 0.73), whereas no correlation was evidenced for punicalagins (ρ = –0.11). In order to verify whether the biological activity of the extract could be mainly attributed to ellagic acid, we also performed viability experiments with purified polyphenol. Fig. 2B shows a comparison between the dose-response curve (0-25 µM) of ellagic acid when the polyphenol is present alone or in the extract. Interestingly, our data indicate that purified ellagic acid activity was significantly lower (P<0.001) than that exerted when the same amount of polyphenol was present in the extract. The percent inhibition at 10 µM purified ellagic acid was about 25% after 48 h of treatment, whereas it was more than 50% for the same amount as extract.

4. Discussion The aim of this work was to develop the best extraction method for either total polyphenols or specific polyphenolic compounds (i.e. ellagitannins) from pomegranate whole fruits or peels, and evaluate these fractions in terms of antioxidant and antiproliferative capacity. In particular, the peels

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generally represent a waste of pomegranate juice production, although they are an abundant source of these interesting class of bioactive compounds. The polar nature of polyphenols makes them widely soluble in alcoholic and hydroalchoholic solvents, but is also well known that ellagic acid, peculiar polyphenol of pomegranate, is poorly soluble in polar solvents. So in our experimental planning the conventional extraction methods were combined with others based on the use of less polar solvents. Between the alcoholic solvents used for the first extraction from whole fruit (Rababah, Banat, Rababah, Ereifej & Yang, 2010), ethanol was preferred over methanol, both for safety reasons and because it favours the water fruit content removal during the evaporation step. The extractions performed with ethanol on the peels gave yields slightly lower with respect to those from whole fruits, but a higher amount of polyphenols was obtained, as their presence is highest in this part of the fruit (Tzulker, Glazer, Bar-Ilan, Holland, Aviram & Amir, 2007), as demonstrated by qualitative-quantitative analyses. When ethanol was used with acidified water (Fischer, Carle & Kammerer, 2011) in the ratio 3:1 or 1:1 for the peels extraction, the use of a 3:1 ratio allowed the highest extraction yield and simplified the next freeze-drying passage, in any case necessary. However, this last step of lyophilization makes the procedure more complex and more expensive, hence the simple ethanolic extraction was preferred. On the bases of the aforementioned reasons, the dried extracts from the first procedure were submitted to repartition with an organic medium-polarity solvent, such as ethyl acetate. After repartition in water:ethyl acetate (Panichayupakarananta, Issuriya, Sirikatitham & Wang, 2010) the dried residue from the first extraction was distributed 9:1 between the two phases. Considering the overall yields obtained from the first extraction and the subsequent repartition in ethyl acetate, in the case of the peels, the results were almost comparable with those obtained from direct extraction with ethyl acetate in a Soxhlet apparatus (Negi et al., 2003). While developing the Soxhlet extraction method, different attempts were made to optimize the procedure, testing different times

18

of extraction, up to 24 h, but our data indicate that within 6 h the maximum extraction is obtained. Our findings on total polyphenol content detected by the Folin-Ciocalteau assay in the analyzed extracts can be cautiously compared with the data available in the literature, bearing in mind the unavoidable differences between the samples and the partial overlap of their processing conditions. In order to compare our results with those already reported in the literature we recalculated the total polyphenol content in ethanol extracts obtained from Isr and DC2 peel samples as mg GAE/g of fresh weight of peel, thus obtaining 26.6 mg GAE/g and 21.1 mg GAE/g, respectively. These data are respectively 9.3-fold and 7.7-fold higher with respect to the average 2.7 mg GAE/g reported by Gozlekci, Saracoglu, Onursal & Ozgen (2011) for their 50% (v/v) ethanol:water extraction of sundried peels of 4 different Turkish pomegranate cultivars. Another useful point of comparison for our ethanol extracts is the study of Rababah et al. (2010) where extraction with 70% (v/v) ethanol:water at 20 °C for 60 min was carried out both on sweet and sour pomegranate types, both from whole fruit and peels. Indeed, our total phenolics content values, also, for this comparison, expressed as mg GAE/g of fresh weight of vegetable matrix, resulted in 9.8 mg GAE/g in Isr whole fruit and 26.6 mg GAE/g in Isr peel, which are comparable with the range defined by these authors, ranging from 7.1 to 17.0 mg GAE/g for whole fruit and from 11.8 to 20.5 mg GAE/g for peels, which, refer to the dry weight of the whole fruit or peel. Recently, the review by Akhtar et al. (2015) reported the ultrasonic assisted extraction with 70% (v/v) ethanol:water at 60 °C for 30 min as one of the best extraction procedures in terms of pomegranate peel phenolics, yielding 86.7 mg GAE/g of dry weight which, compared to our data on ethanol extracts from fresh weight, is about 3-fold more concentrated. As example of Soxhlet extraction for 4 h with ethyl acetate from sun-dried and powdered pomegranate peels, the total polyphenols content data reported by Negi et al. (2003), 16.5% with respect to dry extract and expressed as catechin equivalents, was lower compared to our results, 41.9% and 54.9% for Soxhlet extracts respectively from Isr and DC2 samples, always respect to dry extract even though expressed as gallic acid equivalents. In conclusion, considering

19

what has been reported, our first extraction procedures by 24 h blending in 96% ethanol and through a Soxhlet apparatus with ethyl acetate as extractant for 6 h, have proved adequate to obtain good yields for the polyphenolic fraction of pomegranate peels. Furthermore, it seems useful to emphasize that the use of a fresh matrix, rather than a dehydrated one, can be a practice capable of ensuring a processing time-saving, and reducing the risk of loss of temperature sensitive bioactive molecules. In line with previous data, reported by Sestili et al. (2007), our findings indicate a higher total polyphenols content in the peel than in the pomegranate whole fruit. By comparing the polyphenol yields obtained with the different extraction procedures, ethyl acetate revealed to be the best extraction medium, particularly on peels. As regards the high similarity of the phenolics amount observed between Isr and DC2 peel samples of pomegranate, it may represent a general characteristic of this fruit, as appreciable for example in the study of Li et al. (2015), where the total polyphenol content of aril juices obtained from 10 different cultivars from 4 different regions of China was almost overlapping. The results of chemical characterization of pomegranate extracts suggest that the extraction capacity of the different employed procedures is fairly well comparable between total phenolic compounds and their flavonoid subfraction. The latter represents almost always 30% of the total polyphenols in all the analyzed extracts. Our data confirm the observations made by Li et al. (2006) who reported a 24% of flavonoids compared to total polyphenols in the pomegranate peel, regardless for the particular extractor solvent employed. Regarding anthocyanins, our results confirmed the greater affinity of these polar and positively charged molecules towards hydrophilic phases, with a their total repartition in the aqueous fraction of the ethanol extract. The generally recognized major abundance of these pigments in the arils (Gil et al., 2000) is consistent with our findings of a lower content in the peel than in the whole fruit, considering the quantitative analysis of total anthocyanin content in ethanol extracts, 1.9 mg/100 g

20

of fresh weight for Isr peel and 2.6 mg/100 g of fresh weight for Isr whole fruit, respectively. These data are comparable to those reported by Rababah et al. (2010) for sweet and sour pomegranate peel (1.3-1.7 mg CGE/100 g dry weight), but higher with respect to whole fruit (0.3-0.4 mg CGE/100 g dry weight sample). On the other hand, unexpectedly, these authors reported a yield of anthocyanins from the peel greater with respect to the whole fruit. HPLC qualitative-quantitative analysis of punicalagin anomers and ellagic acid, demonstrated a selective extraction of these two compounds in the different extracts. Taking the sum of punicalagins and ellagic acid as 100%, punicalagins represent 95% of the ethanolic extract of Isr whole fruit, whereas ellagic acid represents the remaining 5%. Regarding the comparison between Isr whole fruit and peels, in the ethanolic extract from peels we observed a 3-fold increase for punicalagins and a 20-fold increase for ellagic acid, with ellagic acid representing a 30% of the total amount. By subsequent water:ethyl acetate repartition, both in Isr whole fruit and peels the punicalagins are distributed between the two phases, whereas ellagic acid is mainly concentrated in the organic one, rising to about 55% in whole fruit and about 60% in peel extracts, respectively. If we compare the ethyl acetate extracts obtained from Isr and DC2 peels, we observe an inversion in the ratio punicalagins vs ellagic acid, representing a 60 and 40% of DC2 extract. In the Soxhlet procedure, in which peels are extracted in the absence of water, the ellagic acid is selectively extracted with respect to punicalagins and represents the main component, being 90% of Isr peel and 84% of DC2 peel, always considering the sum of ellagic acid and punicalagins. The absolute amount of ellagic acid in the two samples was 63.61 mg/g in Isr and 37.71 mg/g in DC2 (see Tab. 2). These results demonstrated that the temperature adopted in the Soxhlet extraction (ethyl acetate Teb. 77 °C) is compatible with a selective extraction of ellagic acid, as confirmed by tests on the biological activity. Only limited data are available regarding the absolute quantities of punicalagins and even less of ellagic acid in alcoholic extracts from pomegranate peels. On the whole, our data indicate a 3-6 fold

21

higher amount of punicalagins and a 1.5-4 fold of ellagic acid with respect to the data, directly comparable as they are reported in mg/g of dried extract, by Kam et al. (2013). Other results in literature, show contents of punicalagins between 39.8 and 121.5 mg/g of dried peels (Lu, Ding & Yuan, 2008). Taking into account our extraction yields from the matrices and a mean moisture content of about 70-75%, we could calculate punicalagins contents of about 10-12 and 15-18 mg, respectively, in the ethanolic extracts from peels. Such different results could be justified by considering the used extracting methods, only partially overlapping, and the peculiarity of each single sample. Cam & Hisil (2010) reported punicalagin contents of 116.6 mg/g in dry matter but this data is not directly comparable with our results. Regarding the repartition in ethyl acetate of alcoholic extracts from peels, our data indicate an amount of ellagic acid which is less than half of that reported by Panichayupakarananta et al. (2010), however it must be emphasized that these authors used an extraction procedure with methanol under reflux which provided a higher starting ellagic acid yield instead of ethanol at room temperature. All these data are well confirmed by the results from the antioxidant capacity and those from the antiproliferative activity. In agreement with several previous reports (Gil et al., 2000; Li et al., 2015; Singh, Jha, Kumar, Hettiarachchy, Rai & Sharma, 2014), our study reveals that a higher phenolic content in extracts corresponds to a greater antioxidant activity. Moreover, for the first time the present study also shows a good positive correlation of the antioxidant potential based on the content of total flavonoids of the different extracts examined. In addition, between major polyphenols identified in pomegranate extracts using HPLC-DAD analysis, only the ellagic acid showed its higher concentration related to a greater antioxidant potential, as also observed by Singh et al. (2014) considering their DPPH assay data. The attempt to define the single contribution of other subfractions of total phenolic compounds (e.g. anthocyanins or punicalagins) is rather difficult,

22

probably in view of different, specific values of antioxidant activity of each of these groups of molecules (Gil et al., 2000). Comparing the IC50 values, from NBT, ABTS and DPPH antioxidant assays applied in the present study, it is appreciable that prevalent constituents of the extracts were forms preferentially neutralizing the ABTS and DPPH free radicals rather than superoxide anion (analyzed by the NBT assay). On the other hand, a general strong correlation between scavenging activity and polyphenol content, as well as their flavonoid subgroup concentration, was evidenced in the order of ORAC ≥ NBT > ABTS > DPPH. In particular, the NBT assay resulted the most sensitive method to highlight variations in phenolics content in the samples. Indeed, this antioxidant capacity assay was the only one to evidence the higher total polyphenol content in the Soxhlet extract from DC2 peel compared to Isr peel. As a result of differences in the content of total polyphenols evidenced in our extracts, and based on the correlation of this parameter with the antioxidant potential, all our analytical methods indicate a higher antioxidant activity of the extracts prepared from peel rather than from the whole fruit, confirming previous results in literature (Gil et al., 2000). For the same reason, the fraction in ethyl acetate as well as the extract by Soxhlet, show an antioxidant activity comparable to each other (with the exception of the NBT assay) but higher than ethanolic or aqueous fraction. A large body of evidence in the literature demonstrates that pomegranate bioactive molecules can exert anti-cancer activities by inhibiting tumor growth, progression and angiogenesis by modulating intracellular pathways mediated by NF-κB, MAP kinases and mTOR, and these properties have been mainly attributed to its high content in antioxidant tannins and flavonoids (Syed, Chamcheu, Adhami & Mukhtar, 2013). As expected, the data revealed that the most efficacious extracts were those in which the polyphenolic content was enriched, and most notably the antiproliferative effect was strongly correlated with the ellagic acid content of the extract. These results support previous findings indicating that ellagic acid is the main molecule responsible for the anticancer activity of pomegranate (Turrini, Ferruzzi & Fimognari, 2015), and indicate that the different extraction

23

procedures do not affect the biological activity of the extract. The evidence that complex pomegranate extracts possess greater bioactivity than purified ellagic acid indicate a multifactorial effect and chemical synergy of the action for multiple compounds compared to single purified active components.

5. Conclusions In the present work, for the first time, extractions were performed on blended pomegranate whole fruit with respect to conventional squeezing and juice’s analysis. The several extraction procedures, applied on whole fruit and peels, provided the selection of the polyphenolic fraction with a different distribution of its components. A higher content of polyphenols, flavonoids, punicalagins and ellagic acid were found in the peel, when compared to whole fruit, and the highest polyphenol concentration was gained in the repartition in ethyl acetate. Ellagic acid and punicalagins in Soxhlet extracts from pomegranate peels were detected, where ellagic acid was the predominant component of the mixture. A highly significant correlation was shown between total polyphenols and antioxidant capacity and between ellagic acid content and antiproliferative activity on human bladder cancer T24 cells. However, a two-fold enhanced antiproliferative activity was observed when extracts were compared to the purified ellagic acid used as reference. This evidence may indicate a synergistic effect among the polyphenolic compounds in the complex mixture.

Acknowledgements This work was financially supported by funding from the Sapienza University of Roma, Scientific Research Programs 2013 and 2014.

24

Caption to the Figures: Fig. 1. HPLC chromatographic profiles at 360 nm of the phenolic compounds present in Isr pomegranate peel extracts. (A) ethanolic extract; (B) aqueous fraction after repartition from ethanolic extract; (C) ethyl acetate fraction after repartition from ethanolic extract; (D) Soxhlet extract with ethyl acetate. P-α, punicalagin α anomer; P-β, punicalagin β anomer; EA, ellagic acid.

Fig. 2. Pomegranate extracts inhibit human bladder cancer T24 cells proliferation. (A) Growth inhibition after 48 h of exposure to 50 µg/ml of extracts obtained from different extraction methods applied to various matrices derived from the pomegranate. The data are presented as mean ± SEM (n = 3). The pairs of treatment groups compared with each other are indicated by a square bracket when they present a significant difference in their values (#, P<0.01; §, P<0.001). (B) Doseresponse curves obtained after 48 h of exposure to purified ellagic acid or Isr peel pomegranate extracts containing the same amount (1.5-25 µM) of ellagic acid. The data are presented as mean ± SEM (n = 3). §, P<0.001 compared to purified ellagic acid treated cells.

25

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Table 1 Yield of different extraction methods from whole fruit or peel of pomegranates of Italian or Israeli origin A. 2nd Ext. yieldC Exp. N. 1st Ext. yieldB Exp. N. st Sample 1 Ext. method Ethyl acetate (1st ext.) (% w/w) (2nd ext.) (% w/w)

Overall yieldD (% w/w)

WHOLE FRUIT Isr Ita DC1 DC2 Isr Ita DC1 DC2

1 2 3 4 5 6 7 8

EtOH EtOH EtOH EtOH MetOH MetOH MetOH MetOH

12.0±0.6 14.0±0.4 12.4±1.5 9.5±0.6 14.2±0.4 14.2±0.5 11.4±0.1 10.5±0.2

a*

a*

25 26 27 28 29 30 31 32

2.2±0.1 2.8±0.2 4.2±0.1 6.2±1.0 3.5±0.3 2.7±0.1 4.2±0.2 5.4±0.2

0.26±0.01 0.39±0.01 0.53±0.01 0.59±0.01 0.50±0.02 0.38±0.01 0.48±0.01 0.56±0.01

#

e

f* #

e

f*

PEEL §# # b* 3.9±0.4 Isr 9 EtOH 10.4±0.6 33 0.41±0.01 gjp § § # EtOH 7.9±0.2 Ita 10 13.9±1.3 34 1.10±0.01 hkq # § § # EtOH 2.3±0.1 DC1 11 9.2±0.5 35 0.21±0.01 c ilr DC2 12 EtOH 8.4±1.6 36 7.9±0.4 0.66±0.01 s* § § b*d* 4.6±0.5 Isr 13 EtOH / CH3COOH 3:1 14.7±0.9 37 0.68±0.01 gm § # 3.0±0.2 Ita 14 EtOH / CH3COOH 3:1 11.3±2.0 38 0.34±0.01 h n*t § # 3.0±0.3 DC1 15 EtOH / CH3COOH 3:1 14.6±2.0 39 0.44±0.01 i o u* DC2 16 EtOH / CH3COOH 3:1 10.9±2.0 40 7.2±1.4 0.79±0.03 v* # § # d* 3.0±0.2 Isr 17 EtOH / CH3COOH 1:1 10.7±0.7 41 0.32±0.01 jmw § # 3.7±0.4 Ita 18 EtOH / CH3COOH 1:1 11.0±0.6 42 0.41±0.01 k n*x # § # # 2.4±0.2 DC1 19 EtOH / CH3COOH 1:1 14.8±0.7 43 0.36±0.01 c loy DC2 20 EtOH / CH3COOH 1:1 9.5±0.5 44 7.4±0.3 0.70±0.01 z* # # Isr 21 Soxhlet 0.79±0.04 pw ## # Ita 22 Soxhlet 0.77±0.03 qtx # # DC1 23 Soxhlet 0.66±0.03 r u*y DC2 24 Soxhlet 1.09±0.05 s*v*z* Values with the same letters are significantly different (*, P<0.05; #, P<0.01; §, P<0.001). A All data are expressed as mean ± SEM of at least three replicates for each sample. B st 1 Ext. yield (% w/w) = percentage ratio between the weight of dry residue of 1 st extraction and weight of the starting matrix. C nd 2 Ext. yield (% w/w) = percentage ratio between the weight of dry residue of 2 nd extraction and weight of the dry residue of 1 st extraction. D Overall yield (% w/w) = percentage ratio between the weight of dry residue of 2nd or Soxhlet extraction and weight of the starting matrix.

Table 2 Qualitative-quantitative analyses of the polyphenolic fraction in selected extracts A. WHOLE FRUIT

PEEL

Isr Exp. N. Total polyphenols (mmol GAEB/g) Total flavonoids (mmol REC/g)

EtOH 1

Aqueous 25

Isr Ethyl acetate 25

0.479±0.011 0.406±0.003 1.924±0.022 §§ § § § § # a*b l a*c m bcn 0.134±0.001 0.122±0.002 0.561±0.008 § § § § § § § a*b m a*c n bco

Total anthocyanins 0.476±0.048 0.458±0.036 (μmol CGED/g)

NDE

EtOH 9

Aqueous 33

DC2

Ethyl acetate 33

Soxhlet 21

1.503±0.014 1.227±0.007 3.750±0.094 2.462±0.021 § # §§ § # § § # ## # § §# defl dghm egin fhi 0.471±0.002 0.387±0.004 0.881±0.011 0.676±0.001 § § § § § § § § § § §# § § §# defmp dghn e g i o q* fhi 0.404±0.019 0.400±0.006

ND

0.421±0.102

8.96±0.19 13.03±0.06 21.66±0.39 28.45±0.77 41.36±0.03 32.68±0.39 7.39±0.27 § §§ § # § § # § # §§ # # # § § #§ § § § §§ abl acm bcn d e*f l o dghm e*g i n p fhi 0.61±0.01 0.26±0.01 24.97±0.37 11.85±0.37 1.37±0.01 48.55±0.24 63.61±1.21 Ellagic acid § § # § § § § § § # § § # § # § § § § §# § # § §# # (mg/g) abm acn bco defmp dghn egioq fhir Values with the same letters are significantly different (*, P<0.05; #, P<0.01; §, P<0.001). A All data refer to 1 g of dry extract and are expressed as mean ± SEM of at least three replicates for each sample. B GAE = gallic acid equivalents; C RE = rutin equivalents; D CGE = cyanidin-3-O-glucoside. E ND = not detectable. Punicalagins (mg/g)

EtOH 12

Ethyl acetate 36

Soxhlet 24

1.476±0.014 3.234±0.050 3.228±0.051 § § § § jk j k 0.517±0.002 1.298±0.096 0.710±0.016 # § # j*k p k l* j*l*q* ND 54.35±1.87 # # jo 3.79±0.16 § § § jkp

ND 48.91±0.60 § § kp 32.83±1.02 § # j l*q

0.056±0.001 6.80±0.17 # § jk 37.71±0.26 § # k l*r

Table 3 Antioxidant activity in selected extractsA. WHOLE FRUIT

PEEL

Isr Exp. N. ORAC assay (μmol TE/mg)

Isr

DC2

EtOH 1

Aqueous 25

Ethyl acetate 25

EtOH 9

Aqueous 33

Ethyl acetate 33

Soxhlet 21

EtOH 12

0.960±0.066 a*h*

0.888±0.110 b*

3.733±0.486 a*b*

3.145±0.412 c*h*

2.217±0.401 # d*e

5.427±0.865 d*

5.201±0.484 # c*e

2.980±0.261 # f*g

NBT assay IC50 (μg/ml)

63.477±0.015 45.140±0.785 32.770±4.146 # # a b*i* a b*j*

ABTS assay IC50 (μg/ml)

13.043±0.163 14.093±0.210 §§ § § a*b j a*c k

5.013±0.402 § d*e*j

Soxhlet 24

6.171±0.499 5.728±0.098 # g f*

34.833±2.455 16.500±1.607 8.530±0.087 # # # # fg f h* g h*k*

5.490±0.059 § § f g*k

1.725±0.026 §§ d*f l

3.077±0.341 e*g*

4.413±0.058 §§ hi

1.643±0.059 1.888±0.007 § § h i

17.703±0.008 19.140±0.062 4.715±0.015 3.132±0.092 4.020±0.164 # § § # § § § § § # § § § # # § § § abm acn bco defmp dghn Values with the same letters are significantly different (*, P<0.05; #, P<0.01; §, P<0.001). A All data are expressed as mean ± SEM of at least three replicates for each sample.

2.430±0.031 § § § # e g i*o q

2.273±0.024 § § § f h i*r

3.567±0.012 § § # jkp

2.120±0.023 2.950±0.015 §§ # §§ § jlq klr

DPPH assay IC50 (μg/ml)

3.017±0.058 § §§ bcl

42.000±3.215 38.500±2.179 12.067±0.526 29.750±3.301 # # # # c i* d c d e*j* e*k*

Ethyl acetate 36

Table 4 Correlation between polyphenol compositions, antioxidant capacities and antiproliferative activity. Total Total Total ORAC NBT polyphenols flavonoids anthocyanins assay assay Total polyphenols 1.00 0.89 –0.70 0.97 –0.94 Total flavonoids –0.89 1.00 –0.69 0.92 –0.82 Total anthocyanins –0.70 –0.69 1.00 –0.65 0.73 ORAC assay 0.97 0.92 –0.65 1.00 –0.91 NBT assay –0.94 –0.82 0.73 –0.91 1.00 ABTS assay –0.86 –0.82 0.71 –0.89 0.83 DPPH assay –0.74 –0.73 0.57 –0.80 0.72 Punicalagins 0.14 0.41 –0.47 0.13 –0.19 Ellagic acid 0.83 0.67 –0.33 0.85 –0.71 Antiproliferative activity 0.73 0.60 –0.20 0.75 –0.57 The results were espresse as Pearson correlation coefficients (ρ value).

ABTS assay –0.86 –0.82 0.71 –0.89 0.83 1.00 0.97 –0.36 –0.68 –0.62

DPPH assay –0.74 –0.73 0.57 –0.80 0.72 0.97 1.00 –0.44 –0.58 –0.59

Punicalagins

Ellagic acid

Antiproliferative activity

0.14 0.41 –0.47 0.13 –0.19 –0.36 –0.44 1.00 –0.24 –0.12

0.83 0.67 –0.33 0.85 –0.71 –0.68 –0.58 –0.24 1.00 0.94

0.73 0.60 –0.20 0.75 –0.57 –0.62 –0.59 –0.12 0.94 1.00

Highlights 1) Optimization of extraction procedures for ellagitannin components of pomegranate 2) HPLC-DAD and spectrophotometric analysis of polyphenolic fraction 3) Evaluation of antioxidant capacity of the selected extracts 4) Evaluation of antiproliferative activity on T24 human cancer bladder cells

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