Phenolic Compounds and Antioxidant Activity of Juices from Ten Iranian Pomegranate Cultivars Depend on Extraction

1Department of Food Science and Technology, Faculty of Agriculture, Shahid Bahonar University of Kerman, P.O. Box 76169-133, Kerman, Iran 2Department of Food Science and Technology, Faculty of Agriculture, Tarbiat Modares University, P.O. Box 14115-336, Tehran, Iran 3Institut Prof. Dr. Georg Kurz GmbH, Stöckheimer Weg 1, 50829 Köln, Germany 4Department of Nutritional and Food Sciences, University of Bonn, Römerstraße 164, 53117 Bonn, Germany


Introduction
Pomegranate fruit (Punica granatum L.) and its products are rich sources of bioactive compounds such as flavonoids, ellagitannins, mainly punicalagins, ellagic acid, and punicalins that also affect visual appearance and flavour of pomegranate juices [1,2].These compounds are discussed as health promoting, but excessive amounts of polyphenols can form undesirable astringent taste.This fruit is one of the most important commercial fruits in Iran and its total production in 2013 was ∼910,000 tons [3].The pomegranate is consumed worldwide as fresh fruit, juice, jam, and jelly, and nutritional supplements.
Thus, the major phenolic compounds of pomegranate juices have been documented, but no information is available about identification and quantification of phenolic compounds and antioxidant activities of juices obtained from arils only and from whole Iranian pomegranate cultivars.This study can help to select pomegranate cultivars for commercial use.Therefore, the aims of this study were (1) to quantify individual phenolic compounds, (2) to determine total phenolic content as well as antioxidant activity, and (3) to study correlation of these parameters in pomegranate juices obtained from arils and whole pomegranates of different cultivars.
The trees were grown under the same environmental conditions, basin irrigation (∼2-3 times per month during growing season from March to September 2011), and routine cultural practices suitable for commercial fruit production.The trees were planted with an average of 6 m within rows and 3 m between them.Yazd province is located in a desert environment with an annual precipitation of <100 mm.The average temperature, the amount of rainfall, and relative humidity in growing season of 2011 were 25.0 ∘ C, 14.0 mm, and 23.6%, respectively.The soil texture is sandy-loam, with EC = 4.12 dSm −1 and soil pH = 7.21.Ten fruits (approximately 2.5 kg) from four trees (2-3 fruits from each tree) were harvested when fully matured according to commercial practice.It should be noted that the pomegranate fruits in Yazd province were fully matured and harvested between 15th and 30th September [12].

Preparation of Pomegranate
Juice.Each pomegranate cultivar was washed in cold tap water and drained.The fruits were pressed using a self-made press consisting of a movable car lifter and a stainless steel pot creating a pressure of 67.4 kp/cm 2 .The supernatant juice was decanted and frozen at −20 ∘ C until analysis.Juices were filtered through disposable membrane filters (regenerated cellulose, 0.2 m pore size, Macherey + Nagel, Düren, Germany) and analysed without any further sample pretreatment.Suffixes A and W (such as MYA and MYW) at each abbreviation of pomegranate cultivars indicate the juices obtained from arils (i.e., without the exocarp and the endocarp) and whole pomegranate juices, respectively.Approximately 2.5 kg of pomegranate fruits was cut in two halves, in order to obtain aril juice and whole pomegranate juice from the same fruits.For the aril juices, the arils were separated manually and the inner white parts of the fruit were removed.
Anthocyanins were separated using an Acquity HSS-T3 RP18 column (100 mm × 2.1 mm, 1.8 m particle size) from Waters with a guard column (5 mm × 2.1 mm).Eluent A was water/1% formic acid; eluent B was acetonitrile/0.1% formic acid.The gradient program at a flow rate of 0.45 mL/min was as follows: 0-0.2 min, 0% B; 0.6 min, 5% B; 20 min, 12% B; 21-24 min, 95% B; and 25-27 min, 0% B. The mass spectrometer was tuned using a solution of cyanidin 3-Oglucoside.The following parameters were obtained: capillary voltage 1.6 kV, cone voltage 32 V, extractor voltage 3.0 V, RF voltage 1.3 V, source temperature 150 ∘ C, desolvation temperature 450 ∘ C, cone gas (nitrogen) flow 50 L/h, and desolvation gas (nitrogen) flow 800 L/h.The collision gas (argon) flow used in tandem mass spectrometry experiments was 0.3 mL/min.MS detection was used for confirmation of peak identity in selected reaction monitoring (SRM) mode.For quantification, an external calibration of cyanidin 3-Oglucoside was used (detection wavelength of 500 nm).Each sample was analyzed once.All compounds were identified by MS (SRM) and quantified by UV-Vis.
The mass spectrometer was tuned using a solution of punicalagin, resulting in the following parameters: capillary voltage −2.0 kV, cone voltage 34 V, extractor voltage 2.0 V, RF voltage 0.3 V, source temperature 150 ∘ C, desolvation temperature 450 ∘ C, cone gas (nitrogen) flow 50 L/h, and desolvation gas (nitrogen) flow 900 L/h.The collision gas (argon) flow used in tandem mass spectrometry experiments was 0.3 mL/min.For quantification, an external calibration of punicalagin and ellagic acid was used (detection wavelength of 360 nm).All compounds were identified by MS (SRM) and quantified by UV.

Determination of Total Phenolic Content (TPC) and
Antioxidant Activity of Juices.Total phenolic content was measured using Folin-Ciocalteu method described by Tezcan et al. [8] using gallic acid as reference.The antioxidant capacity of pomegranate juices was evaluated based on free

Punicalagins and Ellagic
Acids.In this study, the major individual phenolic compounds of ten pomegranate cultivars were identified by HPLC/PDA/MS 2 , namely, punicalagin A, punicalagin B, and ellagic acid (Table 1), besides anthocyanins (see the next paragraph).These results are in accordance with previously reports on other pomegranate cultivars [1,2,4,11,16].Gallic acid, gallagic acid, and galloyl glucose were found in minor amounts only and therefore not considered in the following evaluation.A typical chromatogram of the detected phenolic compounds of pomegranate juice (MYW cultivar) is shown in Figure 1.
The individual and total phenolic contents of all studied cultivars were significantly different ( < 0.01).The concentrations of the main phenolics showed wide ranges: punicalagin B 25.9-884 mg/L, ellagic acid 17.4-928 mg/L, and punicalagin A 5.40-285 mg/L, which is in agreement with previous findings of juices from other pomegranate cultivars [1,4,13,14].Pearson's correlation coefficients were high (>0.98) between punicalagin A and punicalagin B at same juices (arils or whole pomegranate), but ellagic acid value was not significantly correlated with the other phenolic compounds.In each cultivar, the total individual phenolic content of whole pomegranate juices was increased by 2.5-1136% compared to pomegranate aril juices.In this regard, the MY and JPGRT had increased 2.5 and 35%, respectively, but other samples showed higher increases (>100%).The highest increase (1136%) was awarded to GSY cultivar.The highest punicalagin contents among aril and whole fruit juices were found in JPGRTA (607 mg/L) and GSYW (1169 mg/L), respectively (Table 1).Punicalagin is the most characteristic ellagitannin, because it is found almost exclusively in pomegranate.However, pomegranate arils contain low or no punicalagins, but pomegranate rind parts are an abundant source.The higher punicalagin content of commercial pomegranate juices was due to the entrance of these compounds from rind parts of the fruit during processing [1,11].The phenolic content of four types of "Wonderful" pomegranate juices including juices from fresh arils, frozen arils, and whole pomegranates and concentrate were evaluated [1].Punicalagin A in four types of pomegranate juices was 12.7, 14.4, 421, and 435 mg/L; punicalagin B was 10.1, 11.1, 839, and 918 mg/L, and total ellagic derivatives were 33.2, 26.5, 121, and 264 mg/L, respectively.The four major hydrolyzable tannins of punicalagin, ellagic acid, punicalin, and gallagic acid were detected in 29 different pomegranate accessions by Tzulker et al. [11].The contents of these compounds significantly differed in the whole fruit homogenates.The content of punicalagin was about 10-fold higher than the punicalin and gallagic acid contents and 100-fold higher than the content of ellagic acid.Although punicalagin, punicalin, and gallagic acid were also found in aril juices (ellagic acid was below the detection level), these contents were very low in comparison to the whole fruit homogenates [11].
In literature, punicalagins content was reported to be lower in pomegranate arils than in whole pomegranate fruit or even absent in the former [1,2,11].But in the current study, the cultivar JPGRT had such a high punicalagin content that the juice made from the arils of JPGRT had a higher content (607 mg/L) than the juice made from whole pomegranate of certain cultivars (MYW, TMLW, SRABW, and PSYW).Therefore, the content (or lack) of punicalagins in pomegranate juice is not only affected by extraction method, but also-and even more-by the pomegranate cultivar.
Among different species or even cultivars of the same species, the anthocyanin content varies considerably, affected by genetic make-up, light, temperature, and agronomic factors [21].Intrinsic properties of the product and the process such as pH, temperature, light, oxygen, enzymes, ascorbic acids, sugars, metal ions, and copigmentation (intermolecular and intramolecular complex formations, self-association, and metal complexation) as well as chemical structure and concentration of anthocyanins can affect anthocyanin stability [22].
The variation of anthocyanin content in juices from arils and whole pomegranate can be affected by the entrance of anthocyanins from the peel into the juices.Therefore, the amount of anthocyanins of the same cultivar varied with extraction methods.
The antioxidant properties were measured by ABTS, FRAP, and DPPH assay (Table 3).The results showed considerable antioxidant activity of juice samples.Antioxidant activities of juices from whole pomegranate fruit were significantly higher than juices from pomegranate arils that indicated the importance of the juice extraction methods.These results are in agreement with previously reported findings [1,11].The antioxidant capacity of pomegranate juice (and other fruit juices) depends on cultivar, growing region, fruit maturation, and agricultural factors.The technology used for juice processing may affect antioxidant capacity of pomegranate juice [7].In this regard, the antioxidant activity of commercial pomegranate juice was 50% higher than juices from pomegranate arils, due to the entrance of phenolic compounds from the peel into juices [1].Also, Tzulker et al. [11] reported that antioxidant activity of whole fruit homogenates is about 20-fold higher than that found in the arils.These results indicated that hydrolysable tannins including punicalagin were the main compounds influencing the antioxidant capacity of pomegranate juices [1,11].This compound shows a high antioxidant activity due to the 16 phenolic hydroxyl groups in the molecule [1].
Moreover, a good correlation was observed between total phenolic content and ABTS and FRAP methods in all pomegranate juices (>0.90).Correlations between total phenolic and DPPH in juices of arils and whole pomegranate were 0.92 and 0.86, respectively.However, Pearson correlation showed that the correlation of three measurement antioxidant methods is affected by pomegranate cultivars, as mentioned in previous research [7,11].In this regard, Tzulker et al. [11] reported that the antioxidant activity (FRAP and DPPH methods) of juices from whole pomegranate homogenate and arils alone significantly correlated with the polyphenol level, but it did not correlate with anthocyanins level.The highest correlation value was obtained between antioxidant activity and punicalagin in the whole pomegranate homogenate.The FRAP method compared to DPPH method gave the highest correlation with total polyphenol content.

Conclusion
The individual and total phenolic contents of all studied cultivars were significantly different.In this study, among nine major and minor anthocyanins, the cyanidinpentoside, cyanidin-pentoside-hexoside, and delphinidinpentoside were identified for the first time in Iranian pomegranate cultivars.The phenolic contents and antioxidant activity of whole pomegranate juice were significantly higher than those of juices from pomegranate arils from the same cultivar, due to the entrance of phenolic compounds from the rind parts of pomegranate to the juices.The variation between the cultivars is very wide; therefore, the contents of ellagitannins and anthocyanins in the juice from whole pomegranates of the low-polyphenol cultivars (as MY) are lower than those in the juices made from arils only of the high-polyphenol cultivars (as JPGRT).In other words, the cultivar has a higher influence on the phenolic content of the juice than the extraction method.

Figure 1 :
Figure 1: Typical chromatogram of the nonanthocyanidin phenolics in pomegranate juice (MYW) as detected by MS in selected ion monitoring (SIM) mode.Punicalagins appear as doubly charged ions.Ions with m/z = 301 other than ellagic acid are in-source fragments of other ellagitannins.

Table 1 :
Contents of individual phenolic compounds (mg/L) in pomegranate juices (PJ).Different letters in the same column present significant difference at  < 0.05.
2.6.Statistical Analysis.The data were subjected to analyses of variance (ANOVA) using SPSS 17.0.For the comparison of significance between cultivars, Duncan's multiple range test ( < 0.05) was used as post hoc test.Results are the means ± SD of two determinations for each juice.

Table 2 :
Contents of individual anthocyanins (mg/L) in pomegranate juices.