Polyphenolic compounds, anthocyanins and antioxidant activity of nineteen pomegranate fruits: A rich source of bioactive compounds

Journal of Functional Foods 23 (2016) 628–636

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Polyphenolic compounds, anthocyanins and antioxidant activity of nineteen pomegranate fruits: A rich source of bioactive compounds Pilar Legua a,*, María Ángeles Forner-Giner b, Nallely Nuncio-Jáuregui c, Francisca Hernández a a

Plant Science and Microbiology Department, Research Group in Plant Production and Technology, Miguel Hernandez de Elche University, Ctra. Beniel, km 3.2, Orihuela, Alicante 03312, Spain b Centro de Citricultura y Producción Vegetal, Instituto Valenciano de Investigaciones Agrarias (IVIA), Apartado Oficial, Moncada, Valencia 46113, Spain c Food Technology Department, Research Group in Food Quality and Safety, Miguel Hernandez de Elche University, Ctra. Beniel, km 3.2, Orihuela 03312, Alicante, Spain

A R T I C L E

I N F O

A B S T R A C T

Article history:

The chemical composition of 19 pomegranate cultivars was analysed to demonstrate the

Received 14 July 2015

wide diversity among pomegranate juices. The individual phenolic compounds and antho-

Received in revised form 27 January

cyanins were measured by HPLC-DAD-MS/MS-ESI. The antioxidant activity, total phenolics,

2016

organic acid, sugar profile and colour were also measured. Ten phenolic compounds (mainly

Accepted 29 January 2016

hydrolysable tannins and ellagic acid derivatives) and six anthocyanins (especially cyani-

Available online

din 3-O-diglucoside and cyanidin 3,5-O-diglucoside) were identified and quantified. Cultivars HIZ and WOND had the highest a* (6.50 and 6.52, respectively) value which corresponded

Keywords:

to the highest total concentration of anthocyanins (259 and 217 µM of cyanidin 3-O-

Punica granatum L.

glucoside, respectively) and antioxidant activity in the hydrophilic fraction (172 and 149 mg

Antioxidant activity

eq Trolox/100 mL, respectively). Total phenolic content ranged from 90 to 145 mg GAE/

Phenolic profile

100 mL. The WOND cultivar presented the highest values in total organic acids (3.09 g/

Anthocyanins

100 mL) and ME13 cultivar in sugars (15.3 g/100 mL).

Organic acids

© 2016 Elsevier Ltd. All rights reserved.

Colour

1.

Introduction

Nowadays, growing interest in the chemical composition and antioxidant properties of fruits is being shown. Consumers are becoming increasingly aware about buying foods that are rich in bioactive compounds. However, not all fruits have the same composition and properties. Regarding this aspect, pomegran-

ate (Punica granatum L.) fruit has been consumed and used as a medicinal food in the Middle East for thousands of years. The potential capabilities of pomegranate that have been listed on web sites and research papers include its use as an antioxidant, anti-inflammatory, antiviral, antibacterial, and antifungal (Johanningsmeier & Harris, 2011). Also, pomegranate juice contains high levels of antioxidants, higher than other juices and beverages, e.g. red wine, grape, blueberry, blackberry,

* Corresponding author. Plant Science and Microbiology Department, Research Group in Plant Production and Technology, Miguel Hernandez de Elche University, Ctra. Beniel, km 3.2, Orihuela, Alicante 03312, Spain. Tel.: 0034 966749669; fax: 0034 966749693. E-mail address: [email protected] (P. Legua). http://dx.doi.org/10.1016/j.jff.2016.01.043 1756-4646/© 2016 Elsevier Ltd. All rights reserved.

Journal of Functional Foods 23 (2016) 628–636

cranberry and apple juice (Nuncio-Jáuregui et al., 2015a; Seeram et al., 2008). Pomegranate polyphenols include flavonoids, condensed and hydrolysable tannins, which together with organic and phenolic acids, comprise most of the known bioactive compounds (Elfalleh et al., 2011). Numerous methods for determining phenolic compounds in pomegranate fruits exist (Fischer, Carle, & Kammerer, 2011). However, high-performance liquid chromatography (HPLC) is one of the most convenient techniques for the separation and characterization of phenolic compounds in plant material (Gavrilova, Kajdzanoska, Gjamovski, & Stefova, 2011). The HPLC technique was used to analyse the most popular pomegranate cultivars in the Mediterranean region of Spain, these being “Mollar de Elche” and “Valenciana”, which offer attractive sensory characteristics for Spanish consumers. The “Wonderful” cultivar, one of the most cultivated varieties known worldwide, is characterized by its intense uniform red colour, although other Spanish cultivars have interesting chemical properties and phenolic compositions. Therefore, the aim of this study was to analyse the chemical composition, and to characterize the individual polyphenols and anthocyanins, of 19 Spanish pomegranate cultivars by HPLC DAD and mass spectrometry (MSn), as well as antioxidant activity, total phenolic content, organic acid, sugar profile and colour. The obtained data can provide insights into the variety and quantity of phenolic compounds in Spanish pomegranate cultivars. This information can be used for further studies which aim to introduce promising varieties for cultivation, and to use them as supplements in food or pharmaceutical industries.

2.

Materials and methods

2.1.

Plant material and sample processing

Nineteen different pomegranate cultivars were harvested at the end of October of 2014. Sixteen cultivars belonged to one of the most important European pomegranate gene banks, which is located at the experimental field station of the Miguel Hernandez University in the province of Alicante, Spain (02°03′50″E, 38°03′50″N, and 25 masl). These cultivars were: Piñón Tierno de Ojós 5 (PTO5), Piñón Tierno de Ojós 8 (PTO8); Mollar de Elche 13 (ME13), Mollar de Elche 14 (ME14), Mollar de Elche 17 (ME17); Mollar de Orihuela 5 (MO5), Mollar de Orihuela 6 (MO6); Mollar de Albatera 3 (MA3), Mollar de Albatera 4 (MA4); Valenciana 1 (VA1), Valenciana 6 (VA6), Valenciana 7 (VA7), Valenciana 11 (VA11); Hizcaznar (HIZ); Agridulce de Beniel (ADBE1); and Piñón duro de Albatera (PDA1).The other three cultivars were purchased in the farmer’s market of the area: Wonderful (WOND), Valenciana (VA) and Mollar de Elche (ME). Fully mature fruits were manually harvested and immediately transported to the Miguel Hernandez de Elche University (UMH) laboratories. For each cultivar, 10 fruits were randomly picked and each husk was carefully cut at the equatorial zone with a sharpened knife, and then arils were manually extracted and pressurized inside a nylon mesh. Chemical composition was immediately determined on the juice obtained by squeezing the arils.

2.2.

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Determination of instrumental colour

Colour was measured in pomegranate juice according to Manera et al. (2013), using a Minolta C-300 Chroma Meter (Minolta Corp., Osaka, Japan) coupled to a Minolta DP-301 data processor. This colorimeter uses an illuminant D65 and a 10° observer as references. Colour was assessed according to the Commission Internationale de l’Éclairage (CIE) and expressed as L*, a*, b*. Coordinate L* indicates lightness, taking values within the range 0–100 (black–white, respectively), and a* and b* are the chromatic coordinates, green–red and blue–yellow coordinates, respectively. The a* value represents red colour when positive and green when negative, whereas positive b* is for yellow colour and negative one for blue colour. Colour results (mean ± standard error) were the mean of 3 determinations for each sample.

2.3.

Analysis of organic acids and sugar profile

Individual organic acids and sugar profile were determined according to Legua, Melgarejo, Martínez, Martínez, and Hernández (2012a). Twenty millilitres of pomegranate juice obtained by squeezing the arils were centrifuged at 10,000 g for 20 min (Sigma 3–18K, Osterode and Harz, Germany). The supernatant was filtered through a cellulose nitrate membrane filter (0.45 µm pore size). Then, samples were injected (10 µL) into a Hewlett-Packard HPLC series 1100 (Wilmington DE, USA) with an autosampler and an UV detector, set at 210 nm, coupled with a refractive index detector (HP 1100, G1362A). The elution system consisted of 0.1% phosphoric acid with a flow rate of 0.5 mL min−1. Organic acids were isolated using a Supelco column [Supelcogel TM C-610H column (30 cm × 7.8 mm), i.d., Supelco, Bellefonte, PA, USA] and Supelguard C610H column (5 cm × 4.6 mm, Supelco, Inc.). The absorbance was measured at 210 nm using a diode-array detector (DAD). These same HPLC conditions (elution buffer, flow rate and column) were used for the analysis of sugars. The detection was conducted using a refractive index detector (RID). Standard curves of pure organic acids (oxalic, citric, tartaric, malic, quinic, shikimic, and fumaric acids) and sugars (glucose, fructose and sucrose) were used for quantification. Sugar and organic acid standards were obtained from Sigma (Poole, Dorset, UK). Results were expressed as g/100 mL.

2.4.

Total phenolic content (TPC)

The TPC were quantified using Folin–Ciocalteu reagent (Singleton, Orthofer, & Lamuela-Raventos, 1999). Briefly, for each sample, 5 mL of pomegranate juice was mixed with 5 mL of MeOH/water (80:20 v/v) containing 2 mM NaF and then centrifuged at 15,000 g for 15 min. Later, 50 µL of sample was mixed with 2.5 mL of Folin–Ciocalteu reagent (1:10 v/v), 450 µL of phosphate buffer (pH 7.8) and 2 mL of sodium carbonate (75 g/ L). The samples were left in a water bath at 50 °C for 5 min. Absorption was measured at 760 nm using a spectrophotometer (ThermoSpectronic Heγios γ, England). Results (mean ± standard error) were expressed as milligram of gallic acid equivalent per 100 mL of juice (mg GAE/100 mL).

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2.5.

Journal of Functional Foods 23 (2016) 628–636

Antioxidant activity (AA)

The AA was quantified according to Legua et al. (2012b). This procedure allowed determining both the hydrophilic and lipophilic AA in the same extraction. Briefly, 5 mL of pomegranate juice were homogenized in 5 mL of 50 mM phosphate buffer pH = 7.8 and 3 mL of ethyl acetate, then centrifuged at 10,000 g for 15 min at 4 °C. The upper fraction was used for AA due to lipophilic compounds (L-AA) and the lower fraction for AA due to hydrophilic compounds (H-AA). In both cases, AA was determined in triplicate in each extract using the enzymatic system composed of the chromophore 2,2′-azino-bis-(3-ethylbenzothiazoline-6-sulphonic acid) diammonium salt (ABTS) (Sigma–Aldrich; Steinheim, Germany), peroxidase enzyme and its oxidant substrate (hydrogen peroxide), in which ABTS• + are generated and monitored at 730 nm. The decrease in absorbance after adding the extract was proportional to AA of the sample. A calibration curve was performed with Trolox and results were expressed as mg eq Trolox/100 mL.

3.

Results and discussion

3.1.

Instrumental colour

Pomegranate juice colour is an important quality attribute in pomegranate marketing and processing. While pomegranate fruits are ripening, colour coordinates evolve (Manera et al., 2013). The increase in the green–red coordinate, a*, is doubtlessly related to increased biosynthesis and the accumulation of anthocyanin pigments, which are responsible for the intense red colour of ripe pomegranate fruits. Presence of anthocyanins in pomegranate juice contributes mainly to chronic disease prevention (Mena et al., 2011; CORDIS, 2014). Significant differences (p < 0.001) were observed in colour coordinates among cultivars (Table 1). Cultivars WOND and HIZ presented the highest a* values (6.52 and 6.50, respectively). The characteristic garnet colour of pomegranate juice is presented with high a* and C* values and low b* and H* values. This trend was shown by cultivars HIZ, WOND, ME14 and MA4.

3.2. 2.6. HPLC-DAD-ESI-MSn analysis, identification and quantification of phenolic compounds Pomegranate juice (5 mL) was mixed with 5 mL of MeOH and then mixing was carried out by vortex for 1 min, and the extraction was performed in an ultrasonic bath for 10 min at room temperature. The extract was centrifuged at 4000 g for 4 min and passed through a 0.45 µm PTFE filter (Waters, Milford, USA) prior to injection into the chromatographic system. Chromatographic analyses were performed on an Agilent 1100 series HPLC-ESI-DAD-MSn Ion Trap (Agilent Technologies, Waldbronn, Germany). A reversed-phase Agilent Pursuit XRs 5 C18 column (250 × 4.6 mm i.d. and particle size 5 µm, Phenomenex, Macclesfield, UK). The mobile phase consisted of two solvents: (1) waterformic acid (95:5, v/v) and (2) acetonitrile, with a flow rate of 0.8 mL/min. The gradient started with 5% of solvent B, reaching 60% solvent B at 37 min, and 98% at 40 min, which was maintained up to 2 min. The injection volume was 10 µL. The identification of the compounds was carried out by their fragmentation patterns obtained from mass spectra (MSn). Data provided by reference standards and literature information were also employed for the comprehensive evaluation of samples. Ellagitannins were monitored and quantified at 360 nm and anthocyanins at 520 nm. Results were expressed as µM of ellagic acid and µM of cyanidin 3-O-glucoside (Extrasynthese, Genay, France).

2.7.

Statistical analyses

One way analysis of variance (ANOVA) and multiple-range tests were used to evaluate the significance of differences among pomegranate samples composition. The method used to discriminate among the means (Multiple Range Test) was Fisher’s Least Significant Difference (LSD) procedure. Differences were considered statistically significant at p ≤ 0.05. Statistical analyses were performed using SPSS 22.0 for Windows (SPSS Science, Chicago, IL, USA).

Organic acids and sugar profile

Table 2 shows the main organic acids and sugar profiles in pomegranate juice, for which significant differences (p < 0.001) were found among the cultivars. Organic acids are considered compounds that may enhance antioxidant action by acting as a “synergistic antioxidant” (Hernández Rodríguez & Sastre Gallego, 1999). These organic acids include citric, malic, tartaric, lactic, and others. The results showed that quinic and malic acids predominated over citric and tartaric acids in most cultivars. Although quinic and malic acids showed the highest concentrations in total acids (especially in sweet cultivars), citric was the major acid in the sour-sweet and sour cultivars. Fruits from sour cultivars obtained the highest value of total acids (2.64 g/100 mL), followed by the sour-sweet (2.06 g/100 mL) and sweet ones (1.30 g/100 mL). In general, citric acid is considered the main acid in pomegranate fruits (Beaulieu et al., 2015). However, the relative amounts of individual organic acids and sugar profiles differ among cultivars, and also according to location, ripening stage, extraction technique, etc. Regarding the sugar profile, glucose and fructose were the main sugars in pomegranate juices, and content in total sugars ranged from 9.41 g/100 mL (WOND) to 15.3 g/100 mL (ME13). These two sugar types are known as main sources of energy and sweetness (Tezcan, Gültekin-Özgüven, Diken, Özçelik, & Erim, 2009).

3.3.

Total phenolic content (TPC)

TPC in pomegranate juice is presented in Fig. 1. Significant differences (p < 0.001) were observed among cultivars, with TPC values ranging from 90 to 145 mg GAE/100 mL. Different methods are available to measure total antioxidant activity. The mechanism of the Folin–Ciocalteu assay is based on the formation of a blue chromophore constituted by a phosphotungstic-phosphomolybdenum complex, where the maximum absorption of chromophores depends on the alkaline solution and the concentration of phenolic compounds (Blainski, Lopes, & Palazzo de Mello, 2013). Thus the analysis of phenols provides valuable information for selecting

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Journal of Functional Foods 23 (2016) 628–636

Table 1 – Colour coordinates in pomegranate juice. Cultivar

L*

VA VA1 VA6 VA7 VA11 ME ME13 ME14 ME17 MO5 MO6 MA3 MA4 PTO5 PTO8 WOND HIZ ADBE1 PDA1

a

a b

24.3 ± 0.3 j 25.2 ± 0.1 i 28.5 ± 0.4 b 27.1 ± 0.2 c 25.7 ± 0.1 ghi 25.6 ± 0.1 ghi 29.4 ± 0.1 a 28.2 ± 0.4 b 26.4 ± 0.5 def 25.8 ± 0.1 ghi 26.0 ± 0.2 efg 25.8 ± 0.3 ghi 26.6 ± 0.1 cde 25.4 ± 0.1 hi 26.1 ± 0.20 defg 26.7 ± 0.1 cd 26.4 ± b0.1 def 23.7 ± 0.3 j 25.9 ± 0.1 fgh b

a*

b*

C*

HUE

2.26 ± 0.58 g 3.18 ± 0.09 ef 3.93 ± 0.11 cd 3.87 ± 0.01 d 3.90 ± 0.30 cd 4.44 ± 0.18 c 3.02 ± 0.01 f 3.94 ± 0.18 cd 3.96 ± 0.09 cd 3.98 ± 0.05 cd 3.69 ± 0.08 de 4.11 ± 0.10 cd 4.17 ± 0.06 cd 5.35 ± 0.19 b 3.64 ± 0.11de 6.52 ± 0.01 a 6.50 ± 0.48 a 1.15 ± 0.06 h 5.75 ± 0.50 b

0.58 ± 0.05 c 0.03 ± 0.07 ef −0.22 ± 0.09 fg −0.65 ± 0.03 hi −0.41 ± 0.06 gh 0.62 ± 0.14 c 1.00 ± 0.01 ab −0.85 ± 0.03 i −0.33 ± 0.07 g −0.38 ± 0.03 gh −0.19 ± 0.08 fg −0.32 ± 0.24 g −0.75 ± 0.02 i 0.97 ± 0.09 ab 1.10 ± 0.04 a 0.56 ± 0.06 cd 1.05 ± 0.31 ab 0.27 ± 0.06 de 0.75 ± 0.50 ab

2.33 ± 0.57 f 3.18 ± 0.08 e 3.94 ± 0.10 cd 3.92 ± 0.02 cd 3.92 ± 0.03 cd 4.50 ± 0.20 c 3.19 ± 0.01 e 4.03 ± 0.17 cd 3.98 ± 0.08 cd 3.99 ± 0.05 cd 3.69 ± 0.08 de 4.14 ± 0.11 cd 4.24 ± 0.06 cd 5.44 ± 0.20 b 3.81 ± 0.12 d 6.54 ± 0.01 a 6.60 ± 0.52 a 1.19 ± 0.07 g 5.80 ± 0.05 b

15.6 ± 0.2 ab 0.73 ± 0.37 f −3.25 ± 0.30 fg −9.57 ± 0.42 hi −6.01 ± 0.84 gh 7.81 ± 0.45 de 18.3 ± 0.2 a −12.2 ± 0.8 i −4.84 ± 1.11 g −5.46 ± 0.46 g −2.94 ± 1.19 fg −4.36 ± 0.27 g −10.2 ± 0.1 i 10.2 ± 0.8 cd 16.8 ± 0.2 ab 4.90 ± 0.48 e 8.90 ± 0.91 de 13.2 ± 0.8 bc 7.43 ± 0.30 de

Values are the mean of 3 replications (±standard error). Values followed by different letters (a, b, c, etc.) within the same column are statistically different according to Fisher’s least significant difference (LSD) test. All were significant at p < 0.001.

varieties with a higher antioxidant potential. In line with this, the cultivars with higher TPC were HIZ, ME13 and VA1, with TPC values of 145, 132 and 125 mg GAE/100 mL, respectively. For pomegranate juice, Gil, Tomas-Barberán, Hess-Pierce, Holcroft, and Kader (2000) reported a TPC value of 211 mg/ 100 mL; Nuncio-Jáuregui et al. (2015b) a value of 245 mg GAE/ 100 mL; and Cam, Hısıl, and Durmaz (2009) indicated values that ranged from 208 to 343 mg catechin equivalents /100 mL of pomegranate juice. Comparing these values with those re-

ported in other fruit juices such as grape juice (70.5–117 mg/ 100 mL), apple juice (25.4 mg/100 mL) and Spanish blueberry juice (128 mg/mL) (Calín-Sánchez et al., 2013; Dávalos, Bartolome, & Gómez-Cordoves, 2005; Sharma, Kori, & Parmar, 2015) show that the TPC in pomegranate juice can be attributed to the fact that pomegranate fruit is one of the sources with a high phenolic content in nature which provides antioxidants with an anti-free radical mechanism to prevent the cellular oxidation process (Pedreño López, 2012).

Table 2 – Organic acids and sugar profile (g/100 mL) in pomegranate juice. Cultivar

Citric

Tartaric

Malic

Quinic

Total acids

Glucose

Fructose

Total sugars

0.28 ± 0.01 f 0.21 ± 0.01 g 0.35 ± 0.02 e 0.13 ± 0.03 i 0.06 ± 0.01 j 0.07 ± 0.02 j 0.75 ± 0.02 a 0.41 ± 0.01 d 0.02 ± 0.01 k 0.18 ± 0.02 gh 0.16 ± 0.02 hi 0.02 ± 0.02 k 0.07 ± 0.02 j 0.56 ± 0.03 b 0.45 ± 0.03 c 0.01 ± 0.02 k 0.14 ± 0.02 i 0.08 ± 0.01 j 0.01 ± 0.01 k

0.40 ± 0.02 def 0.29 ± 0.01 ijk 0.35 ± 0.01 fghi 0.51 ± 0.02 b 0.43 ± 0.05 de 0.40 ± 0.1 ef 0.38 ± 0.04 efg 0.33 ± 0.04 ghij 0.58 ± 0.01 a 0.54 ± 0.04 ab 0.33 ± 0.04 ghij 0.52 ± 0.01 b 0.49 ± 0.03 bc 0.26 ± 0.01 k 0.31 ± 0.02 hijk 0.29 ± 0.03 jk 0.45 ± 0.01 cd 0.29 ± 0.01 jk 0.36 ± 0.03 fgh

0.87 ± 0.01 bc 0.51 ± 0.01 hijk 0.51 ± 0.01 hijk 0.40 ± 0.02 l 0.61 ± 0.07 gh 0.60 ± 0.03 ghi 0.73 ± 0.09 ef 0.53 ± 0.01 hij 0.50 ± 0.01 ijk 0.76 ± 0.01 de 0.52 ± 0.01 hij 0.49 ± 0.01 jkl 0.72 ± 0.01 ef 0.68 ± 0.04 efg 0.41 ± 0.02 kl 1.31 ± 0.70 a 0.83 ± 0.04 cd 0.65 ± 0.03 fg 0.94 ± 0.02 b

1.59 ± 0.17 e 1.06 ± 0.09 i 1.23 ± 0.10 ghi 1.10 ± 0.11 i 1.13 ± 0.14 i 1.14 ± 0.13 i 1.92 ± 0.17 d 1.38 ± 0.09 fg 1.20 ± 0.14 hi 1.57 ± 0.16 e 1.09 ± 0.10 i 1.11 ± 0.13 i 1.35 ± 0.16 fgh 1.78 ± 0.11 d 1.44 ± 0.04 ef 3.09 ± 0.37 a 2.19 ± 0.16 c 2.20 ± 0.16 c 2.84 ± 0.33 b

4.82 ± 0.02 cde 4.22 ± 0.02 i 4.72 ± 0.01 de 5.22 ± 0.02 b 4.25 ± 0.48 f 4.21 ± 0.01 fg 5.67 ± 0.01 i 3.92 ± 0.02 gh 5.28 ± 0.01 b 5.25 ± 0.01 b 5.06 ± 0.07 bc 4.70 ± 0.02 e 5.19 ± 0.01 b 3.79 ± 0.02 h 3.80 ± 0.01 h 3.31 ± 0.01 fg 3.42 ± 0.01 a 3.9 ± 0.01 fgh 5.05 ± 0.01 bcd

8.26 ± 0.03 c 7.22 ± 0.02 fg 7.42 ± 0.01 d 9.65 ± 0.06 a 8.22 ± 0.64 c 7.33 ± 0.01 d 9.63 ± 0.01 ef 7.11 ± 0.01 d 9.42 ± 0.01 ab 9.26 ± 0.01 ab 9.08 ± 0.05 b 8.38 ± 0.05 c 9.17 ± 0.01 b 5.93 ± 0.05 g 6.63 ± 0.01 e 6.10 ± 0.01 d 6.53 ± 0.01 a 6.54 ± 0.01 ef 8.54 ± 0.03 c

13.1 ± 0.1 de 11.4 ± 0.1 k 12.1 ± 0.1 fg 14.9 ± 0.1 ab 12.5 ± 0.1 ef 11.5 ± 0.1 gh 15.3 ± 0.1 a 11.0 ± 0.1 hi 14.7 ± 0.1 ab 14.5 ± 0.1 b 14.1 ± 0.1 bc 13.0 ± 0.2 de 14.4 ± 0.1 bc 9.72 ± 0.10 jk 10.4 ± 0.1 ij 9.41 ± 0.01 gh 9.95 ± 0.01 jk 10.5 ± 0.1 ij 13.6 ± 0.1 cd

(g/100 mL) VA VA1 VA6 VA7 VA11 ME ME13 ME14 ME17 MO5 MO6 MA3 MA4 PTO5 PTO8 WOND HIZ ADBE1 PDA1 a b

0.04 ± b0.01 fg 0.05 ± 0.01 efg 0.02 ± 0.02 g 0.06 ± 0.01 efg 0.04 ± 0.04 fg 0.08 ± 0.01 ef 0.05 ± 0.01 efg 0.10 ± 0.01 e 0.10 ± 0.01 e 0.08 ± 0.01 ef 0.07 ± 0.01 ef 0.08 ± 0.01 ef 0.07 ± 0.01 ef 0.27 ± 0.02 d 0.27 ± 0.02 d 1.49 ± 0.07 a 0.77 ± 0.01 c 1.17 v 0.02 b 1.54 ± 0.02 a

a

Values are the mean of 3 replications (±standard error). Values followed by different letters (a, b, c, etc.) within the same column are statistically different according to Fisher’s least significant difference (LSD) test. All were significant at p < 0.001.

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160

Total polyphenol content (mg GAE / 100 mL)

ANOVA *** 140

120

100

80

VA

VA1

VA6

VA7 VA11

ME ME13 ME14 ME17 MO5 MO6 MA3 MA4 PTO5 PTO8 WOND HIZ ABDE1 PDA1

Pomegranate cultivars Fig. 1 – Total polyphenol content (mg GAE/100 mL) in pomegranate juice.

3.4. Hydrophilic and lipophilic antioxidant activity (H-AA and L-AA) Most methods used to measure antioxidant activity are based on the analysis of compounds of a hydrophilic nature due to the reactive species and oxidizable substrates employed. However, the ABTS method is used to measure lipophilic and hydrophilic antioxidants (Alcolea, Cano, Acosta, & Arnao, 2002).

Fig. 2 shows the H-AA and L-AA of pomegranate juice, for which significant differences (p < 0.001) were observed among cultivars. The H-AA values ranged from 70 to 172 mg eq Trolox/ 100 mL and from 16 to 29 mg eq Trolox/100 mL for L-AA. The cultivars with the highest H-AA values were HIZ, WOND, VA11 and VA7, and they were WOND, ABDE1 and PDA1 for L-AA. The difference for antioxidant activity among cultivars has been previously reported by Nuncio-Jáuregui et al. (2015b) and Mena

Fig. 2 – Hydrophilic (H-AA) and lipophilic (L-AA) antioxidant activity in pomegranate juice. The samples were statistically different according to Fisher’s least significant difference (LSD) test. All were significant at p < 0.001.

633

c

Values are the mean of 3 replications (±standard error). Values followed by different letters (a, b, c, etc.) within the same column are statistically different according to Fisher’s least significant difference (LSD) test. All were significant at p < 0.001. n.d., not detected. b

3.07 ± b0.23 d 3.30 ± 0.12 d 11.3 ± 0.5 a 1.65 ± 0.1 f 2.60 ± 0.18 e 1.89 ± 0.27 f 8.73 ± 0.16 b 1.65 ± 0.90 f 2.12 ± 0.33 e 2.83 ± 0.12 e 1.18 ± 0.34 f 1.89 ± 0.09 f 1.65 ± 0.11 f 3.30 ± 0.10 d 2.60 ± 0.20 e 12.7 ± 0.4 a 3.54 ± 0.09 d 4.01 ± 0.30 c 1.42 ± 0.50 f a

VA VA1 VA6 VA7 VA11 ME ME13 ME14 ME17 MO5 MO6 MA3 MA4 PTO5 PTO8 WOND HIZ ADBE1 PDA1

a

2.83 ± 0.07 h 3.07 ± 0.10 g 8.73 ± 0.11 c 7.79 ± 0.07 d 4.72 ± 0.31 g 1.42 ± 0.09 h 3.54 ± 0.08 g 4.72 ± 0.76 f 1.42 ± 0.13 h 5.19 ± 0.23 e 1.89 ± 0.07 h 1.18 ± 0.45 h 1.42 ± 0.20 h 8.26 ± 0.21 c 19.6 ± 1.3 a 12.5 ± 0.8 b 7.08 ± 0.15 d 5.66 ± 1.34 e 4.01 ± 0.12 f 6.37 ± 0.03 e 6.84 ± 0.07 e 8.26 ± 0.87 d 7.55 ± 0.33 d 10.9 ± 0.2 cd 5.90 ± 0.02 e 7.79 ± 0.01 d 5.90 ± 0.03 e 8.50 ± 0.09 d 11.6 ± 0.3 bcd 6.37 ± 0.32 e 6.61 ± 0.43 e 5.90 ± 0.21 e 17.9 ± 0.1 b 34.2 ± 0.1 a 11.1 ± 0.3 bcd 12.3 ± 0.9 bc 12.7 ± 0.2 bc 13.0 ± 0.9 bc 1.18 ± 0.20 b n.d. n.d. 1.18 ± 0.02 b 1.65 ± 0.03 b 1.18 ± 0.01 b 1.89 ± 0.31 b 0.71 ± 0.01 c n.d. 1.42 ± 0.05 b 1.18 ± 0.10 b n.d. n.d. 1.18 ± 0.11 b 1.65 ± 0.09 b 1.42 ± 0.70 b 2.60 ± 0.31 a n.d. 2.36 ± 0.07 a 9.44 ± 0.81 e 6.37 ± 0.02 f 17.9 ± 0.7 cd 9.20 ± 0.51 e 6.37 ± 0.84 f 4.96 ± 0.31 gh 9.20 ± 0.50 e 7.32 ± 0.23 f 4.25 ± 0.90 h 3.07 ± 0.34 i 3.07 ± 0.18 i 5.19 ± 0.03 g 3.30 ± 0.01 i 17.0 ± 0.9 cd 16.0 ± 0.5 cd 37.8 ± 0.2 a 14.6 ± 0.1 d 18.6 ± 1.1 c 22.9 ± 1.2 b n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. 4.25 ± 0.45 7.79 ± 0.60 fg n.d. 9.68 ± 0.90 e n.d. 12.3 ± 1.0 cd 9.44 ± 0.80 e 7.55 ± 0.90 fg n.d. 11.1 ± 2.0 d 13.9 ± 0.1 c 8.26 ± 0.31 e 6.61 ± 0.51 g 7.08 ± 0.70 fg 12.0 ± 1.2 cd 13.9 ± 1.4 c 5.43 ± 0.91 h 21.9 ± 1.1 a 17.2 ± 0.8 b 14.2 ± 0.5 c n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. 13.4 ± 0.5 n.d. n.d. n.d. n.d. n.d. 26.9 ± 0.3 a n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. 21.9 ± 0.5 b n.d. n.d. 16.3 ± 0.9 a n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. 14.2 ± 0.5 b

Granatin B Gallolyl-HHDP hexoside Ellagic acid hexoside β-Punicalag α-Punicalag Punicalin Cultivar

Table 3 – Individual phenolic compounds (µM ellagic acid) in pomegranate juice at 360 nm.

Tables 3 and 4 show the pomegranate polyphenols, which mainly include flavonoids and hydrolysable tannins. Pomegranate juice is rich in hydrolysable tannins like ellagitannins and their derivatives. These compounds have been found to perform high antioxidant and free radical-scavenging activities (Aviram, Kaplan, Rosenblat, & Fuhrman, 2005). In their structure, ellagitannins have a group known as HHDP acid (hexahidroxidifenic acid). This compound is hydrolysed in the presence of strong acids or bases to release ellagitannins HHDP, and subsequently, ellagic acid molecules originate (Cruz-Atonio et al., 2010). Ten phenolic compounds were found in the pomegranate juice of 19 different cultivars: punicalin isomer (Rt = 4.95 min, [M − H]− at m/z 781), α-punicalagin (Rt = 7.49 min, [M − H]− at m/z 1083) β-punicalagin (Rt = 9.05 min, [M − H]− at m/z 1083), ellagic acid hexoside (Rt = 11.47 min, [M − H]− at m/z 463), gallagylHHDP-hexoside (Rt = 10.84 min, [M − H]− at m/z 633), granatin B (Rt = 14.07 min, [M − H]− at m/z 951), ellagic acid glucoside (Rt = 13.21 min, [M − H]− at m/z 463), ellagic acid pentoside (Rt = 16.03 min, [M − H]− at m/z 433), ellagic acid deoxyhexoside (R t = 16.38 min, [M − H] − at m/z 447) and ellagic acid (Rt = 18.92 min, [M − H]− at m/z 301). Fischer et al. (2011) identified these compounds in pomegranate juice, and most showed the typical ellagic acid fragments (m/z 301). These authors reported the presence of ellagic acid derivatives, such as ellagic acid hexoside pentoside and deoxyhexoside. Table 3 and Fig. 4 presents the concentration of individual phenolic compounds. The cultivars with the highest concentration were: VA6 (99.1 µM ellagic acid), PDA1 (98.2 µM ellagic acid), PTO8 (87.9 µM ellagic acid) and WOND (80.9 µM ellagic acid). The concentration of the other 15 cultivars fell between 19.3 and 62.0 µM ellagic acid. As the phenolic compounds identified in pomegranate juice were mostly hydrolysable tannins and ellagic acid-derived, they may be responsible for high antioxidant activity in the hydrophilic phase. Fig. 3 shows the six most abundant anthocyanins in pomegranate juice: cyanidin 3-O-diglucoside (peak 4), cyanidin 3,5-

Ellagic acid glucoside

3.5. Quantification of individual polyphenols and anthocyanins by HPLC

c

Ellagic acid pentoside

Ellagic acid deoxyhexoside

Ellagic acid

Total

et al. (2011), and it depends on many factors, such as cultivar, maturity index, geographical source, irrigation regime, etc. In their study about the stability of the antioxidant capacity of different fruit juices subjected to in vitro digestion, Ryan and Prescott (2010) concluded that of all the analysed samples, pomegranate juice was the most potent antioxidant that fruit beverages contained. Seeram et al. (2008) concluded that antioxidant capacity in beverages rich in polyphenols followed this order: pomegranate juice > red wine > grape juice > blueberry juice > blackberry juice = cranberry juice > orange juice = iced tea beverages = apple juice. This is why pomegranate juice is considered a food that can potentially be used as an antioxidant. Some human clinical trials have shown positive effects of pomegranate juice consumption on prostate cancer prevention, cardiovascular health and arthritis (Ahmed, Wang, Hafeez, Cheruvu, & Haqqi, 2005; Aviram & Dornfeld, 2001; Pantuck et al., 2006). Therefore, the antioxidant activity of pomegranate juice appears to have some relationship to the human body’s ability to ward off oxidative stress.

30.7 ± 0.3 d 19.6 ± 0.1 ef 99.1 ± 1.2 a 27.4 ± 0.8 e 38.5 ± 0.4 d 24.8 ± 0.8 e 38.7 ± 1.2 d 20.3 ± 0.5 ef 27.4 ± 1.3 e 38.0 ± 0.9 d 21.9 ± 0.9 e 21-5 ± 0.8 e 19.3 ± 0.5 f 59.6 ± 1.3 cd 87.9 ± 2.1 ab 80.9 ± 1.9 b 62.0 ± 2.3 c 58.2 ± 0.9 cd 98.2 ± 0.7 a

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Table 4 – Individual anthocyanins (µM of cyanidin 3-O-glucoside) in pomegranate juice at 520 nm. Cultivar

Delp-3,5-diglc

Cya-3,5-diglc

Pg-3,5-diglc

Delp, 3-glc

Cya-3-glc

Pg-3-glc

Cya-pent

Total

VA VA1 VA6 VA7 VA11 ME ME13 ME14 ME17 MO5 MO6 MA3 MA4 PTO5 PTO8 WOND HIZ ADBE1 PDA1

3.94 ± 0.05 cd 3.25 ± 0.06 d 9.58 ± 0.11 b c n.d. 5.27 ± 0.11 c n.d. 1.49 ± 0.02 e 3.19 ± 0.07 d n.d. 8.41 ± 0.12 b 0.59 ± 0.01 f n.d. n.d. 4.63 ± 0.09 c n.d. 37.3 ± 0.9 a 8.78 ± 0.20 b 3.83 ± 0.09 d 4.52 ± 0.11 c

19.2 ± 0.3 f 53.9 ± 0.3 c 25.7 ± 0.2 e 28.2 ± 0.2 e 63.7 ± 0.2 b 25.8 ± 0.1 e 8.20 ± 0.09 21.7 ± 0.1 e 23.9 ± 0.1 e 56.8 ± 0.2 c 31.5 ± 0.2 d 8.57 ± 0.11 36.9 ± 0.3 d 35.7 ± 0.3 d 16.7 ± 0.5 g 57.4 ± 0.9 bc 100 ± 1 a 21.2 ± 0.1 e 21.0 ± 0.1 e

n.d. n.d. 13.3 ± 0.2 ab 5.16 ± 0.11e 10.7 ± 0.2 b 3.46 ± 0.21 g 2.71 ± 0.08 h 7.56 ± 0.09 c 2.98 ± 0.10 h 14.9 ± 0.1 a 4.74 ± 0.05 f 3.41 ± 0.05 g 5.48 ± 0.10 e 6.55 ± 0.13 d 2.66 ± 0.10 h n.d. n.d. n.d. n.d.

6.12 ± 0.20 d 10.8 ± 0.5 c n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. 47.1 ± 0.5 a 15.4 ± 0.4 b 2.13 ± 0.11 f 5.86 ± 0.10 de

23.8 ± 1.1 ef 33.3 ± 1.0 de 31.5 ± 0.9 def 39.8 ± 1.3 de 35.2 ± 0.1 de 29.4 ± 0.1 ef 8.89 ± 0.09 g 39.5 ± 0.5 de 22.1 ± 1.0 f 61.3 ± 1.1 bc 39.8 ± 0.1 de 19.7 ± 0.5 f 43.9 ± 0.1 cd 21.7 ± 0.9 f 112 ± 1 a 70.3 ± 0.1 b 119 ± 2 a 19.7 ± 1.0 f 26.9 ± 0.9 ef

5.27 ± 0.11 11.9 ± 0.9 bc 10.3 ± 0.7 c 11.5 ± 0.5 bc 11.8 ± 0.5 bc 7.98 ± 0.05 de 2.24 ± 0.01 f 9.69 ± 0.01 de 5.64 ± 0.05 e 22.2 ± 0.1 a 10.6 ± 0.2 c 5.43 ± 0.11 e 12.8 ± 0.4 b 4.79 ± 0.05 e 2.87 ± 0.01 f 5.00 ± 0.01 12.6 ± 0.1 b 1.86 ± 0.01 g 2.18 ± 0.03 f

n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. 2.82 ± 0.10 n.d. n.d.

58.4 ± 0.3 f 113 ± 1 b 90.4 ± 0.7 c 84.7 ± 0.5 cd 127 ± 2 b 66.6 ± 0.3 e 23.5 ± 0.1 i 81.6 ± 0.3 cd 54.6 ± 0.1 f 164 ± 1 ab 87.2 ± 0.5 cd 37.1 ± 0.1 h 99.0 ± 0.4 c 73.4 ± 0.3 de 134 ± 2 b 217 ± 1 a 259 ± 1 a 48.7 ± 0.9 g 60.5 ± 0.7 e

a b

c

a

b

Values are the mean of 3 replications (±standard error). Values followed by different letters (a, b, c, etc.) within the same column are statistically different according to Fisher’s least significant difference (LSD) test. All were significant at p < 0.001. n.d., not detected.

O-diglucoside (peak 2), pelargonidin 3-O-diglucoside (peak 5), delphinidin 3-O-diglucoside (peak 3), delphinidin 3,5-Odigluside (peak 1) and cyanidin pentoside (peak 6). At an optimum harvest time, cyanidin 3-O-diglucoside is the main pigment in pomegranate juice (Hernández Rodríguez & Sastre Gallego, 1999). Anthocyanins are responsible for the bright red colour of pomegranate juice, possess known pharmacological properties and are used by humans for therapeutic purposes (Elfalleh et al., 2011). Table 4 indicates the concentration of the individual anthocyanins for each pomegranate cultivar. Cultivars HIZ and WOND gave the highest anthocyannins

concentrations (259 and 217 µM of cyanidin 3-O-glucoside, respectively), especially in cyanidin 3-O-diglucoside and cyanidin 3,5-O-diglucoside. Vázquez-Araújo et al. (2014) reported that cultivars HIZ and WOND were characterized by an intense red arils colour and are appropriate for juice manufacturing because the heat treatments involved in processing lighten juice colour. They also reported that the VA cultivar is lighter in colour and is suitable for fresh consumption. This study shows that cultivars HIZ and WOND, with the higher values for coordinate a* of colour and parallel, presented the highest total concentration of anthocyanins and

Fig. 3 – Model chromatogram of anthocyanins (HIZ cultivar). Peak: (1) Delphinidin 3,5-O-diglucoside; (2) Cyanidin 3,5-Odiglucoside; (3) Delphinidin 3-O-diglucoside,(4) Cyanidin 3-O-diglucoside, (5) Pelargonidin 3-O-diglucoside; (6) Cyanidin pentoside.

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Fig. 4 – Model chromatogram of individual phenolic compounds (PDA1cultivar) at 360 nm. Peak: (1) Punicalin, (2) α-punicalagin, (3) β-punicalagin, (4) Gallolyl-HHDP hexoside, (5) Ellagic acid hexoside, (6) Ellagic acid glucoside, (7) Granatin B, (8) Ellagic acid pentoside, (9) Ellagic acid deoxyhexoside, (10) Ellagic acid.

antioxidant activity of the hydrophilic fraction. Conversely, cultivars VA and ADBE1 obtained the lowest values for these parameters.

4.

Conclusions

The results showed the difference in chemical composition, individual phenolic compounds and anthocyanins, as well as antioxidant activity, among pomegranate cultivars. Cultivars HIZ and WOND obtained the highest values for colour (a*), anthocyanins, antioxidant activity in the hydrophilic phase and organic acids. In general, the “Mollar de Elche” cultivars presented the highest sugar content values. The analysis and characterization of compounds such as polyphenols, organic acids and antioxidant properties combined with continued research on the specific bioactive components in pomegranate, are promising for the products (current and future) that can deliver health benefits.

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