Comparative study of phenolic compounds and their antioxidant attributes of eighteen pomegranate (Punica granatum L.) cultivars grown in Morocco

2.1 Biological material

The study was performed in a pomegranate (P. granatum L.) collection with 18 cutlivars (Table 1) at the INRA (National Institute for Agricultural Research) Experimental Station, Meknes-Morocco (altitude 500 m), which has a semi–arid climate. There is about 400 mm of rainfall per year. The soil is calcareous with a high percentage of clay. Trees are planted at 5 × 3 m spacing and irrigated at 3500 m³ per year supplied from May to October. The cutlivars are cultivated under the same geographical conditions and with the same applied agronomic practices.

Twenty fruits of each cultivar are collected at harvest maturity in the beginning of October 2009, which is the normal ripening period for the pomegranate. Five fruits were harvested randomly from each of the four orientations of the tree, and were immediately taken to the laboratory for analysis.

The fruits were peeled and the skins covering the seeds were removed manually. The juice was obtained from pomegranate arils by mechanical press, and was stored frozen (−20 °C) until analyzed. Three replicates were maintained for each analysis.

2.2 Chemicals and reagents

Folin–Ciocalteu’s phenol reagent, sodium acetate, aluminum chloride, ferrous sulfate, ferric chloride, sodium carbonate, sodium hydroxide and methanol were purchased from R&M Chemicals (Essex. UK). 2,4,6-Tris(1-pyridyl)-5-triazine (TPTZ), and 1,1-diphenyl-2-picrylhydrazyl (DPPH) were purchased from the Fluka company (Switzerland). Trolox (6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid), which is a hydrophilic analogue of vitamin E, ABTS (2,2′-azinobis-3-ethylbenzthiazoline-6-sulfonic acid) and the polyphenols standards were supplied by Sigma–Aldrich (St. Louis, MO, USA).

2.3 Juice yield, titrable acidity, pH, total soluble solids and maturity index of pomegranate juice

The titrable acidity (TA) was determined by titration to pH 8.1 with 0.1 M NaOH solution and expressed as g of citric acid per 100 ml of juice. The pH measurements were performed using a digital pH meter (Thermo Orion 3 star) at 21 °C. The total soluble solids (TSS) were determined with a digital refractometer (Metteler-Toledo Gmbh, 30 PX, Switzerland, calibrated using distilled water). Results were reported as °Brix at 21 °C. The yield of juice, was obtained from extraction of juice from the five fruits of each cultivar taken at random and expressed as volume of juice per 100 g of fruits. The maturity index (MI) was calculated by dividing the total soluble solid with titrable acidity.

2.4 Total phenolics (TP)

The TP of PJ was determined by using the Folin–Ciocalteu method (Singleton et al., 1965). 300 μL of diluted pomegranate juice in the ratio of 1:100 with methanol: water (6:4) was mixed with 1.5 mL of 10-fold-diluted Folin–Ciocalteu reagent and 1.2 mL of 7.5% sodium carbonate. The mixture was allowed to stand for 90 min at room temperature before the absorbance was measured by a Safas UV–Visible spectrophotometer at 760 nm. Gallic acid was used as a standard. The results were expressed as mg gallic acid equivalent in a liter of fruit juice (mg GAE/L of juice).

2.5 Total flavonoids (TF)

The total flavonoid content in juices was determined spectrophotometrically according to the method of Lamaison and Carnat (1990), using a method based on the formation of a complex flavonoid-aluminum, having the abosorbtivity maximum at 430 nm. Rutin was used to make the calibration curve. 1 ml of dilued sample was separately mixed with 1 ml of 2% aluminum chloride methanolic solution. After incubation at room temperature for 15 min, the absorbance of the reaction mixture was measured at 430 nm with a Safas UV–Visible spectrophotometer and the flavonoid content was expressed as mg of rutin equivalent per L of juice.

2.6 Total anthocyanins (TA)

The TA was estimated by pH differential method using two buffer systems: potassium chloride buffer pH 1.0 (25 mM) and sodium acetate buffer pH 4.5 (0.4 M) (Ozgen et al., 2008). Briefly, 0.4 mL of pomegranate juice sample was mixed with 3.6 mL of corresponding buffers and read against water as a blank at 510 and 700 nm. $$\text{Absorbance}(\text{A})\text{was calculated as}:\text{A}=(A_{510\text{nm}}-A_{700\text{nm}}){\text{pH}}_{1.0}-(A_{510\text{nm}}-A_{700\text{nm}}){\text{pH}}_{4.5}$$

The TA of samples (mg cyanidin-3-glucoside/L of PJ) was calculated by the following equation: $$\text{TA}=[\text{A}\times \text{MW}\times \text{DF}\times 100]\times 1/\text{MA}$$ where A: absorbance; MW: molecular weight (449.2 g/moL); DF: dilution factor (10); MA: molar absorptivity coefficient of cyanidin-3-glucoside (26.900).

2.7 Identification and quantification of phenolic compounds by HPLC analysis

The sample of the juice was analyzed using an Agilent HPLC System (Agilent Technologies, Waldbronn, Germany). Separation was performed on a reverse phase Nucleosyl LC-18 column. Column temperature was maintained at 30 °C. Eluent (A) was composed of water and formic acid (95:5, v/v) while eluent (B) was composed of acetonitrile, water and formic acid, (80:15:5, v/v/v) at a flow rate of 1 mL/min. The elution program of the solvent (B) used was as follows: 0–19 min, 3%; 19–30 min, 13%; 38–55 min, 14%; 55–65 min, 30%; 65–68 min, 35%. The chromatogram was monitored simultaneously at 280, 320 and 360 nm, with spectra taken continuously throughout the elution. The UV spectra of the different compounds were recorded with a diode array detector. Calculation of concentrations was based on the external standard method. Dilutions 1:0, 1:1, 1:2 and 1:4 of an aqueous solution containing 30 mg/L of each of the phenolic standards (gallic, chlorogenic, caffeic, ferulic, ellagic acids, catechin, epicatechin, phloridzin, quercetin and rutin) were used to fit a standard curve (peak area versus concentration in mg/L) with linear regression for each individual compound.

2.8 Determination of antioxidant activities

2.9 Statistical analysis

All the analyses were performed by Statistical Analysis System (SAS) software 917 SAS Istitute Cay N.C. (USA). Using analysis of variance (ANOVA) the differences among means were determined for significance at P < 0.05 using the PROC GLM procedure.

3.1 Juice yield, titrable acidity, pH, total soluble solids and maturity index

The results for juice yield, titrable acidity (TA), pH and total soluble solids (TSS) from the different cultivars are presented in Table 1. Significant differences (P < 0.05) were revealed among the pomegranate cultivars for all parameters.

The juice percentage varied from 29.73% L1 to 54.42% F1, which agree with the results (26.95–46.55%) reported by Tehranifar et al. (2010). The greatest volume of juice per 100 g of fruits observed was for the cultivars F1, F2, F6, L6 and L9. These cultivars can be interesting for juice production industries. The highest total soluble solid content was in L10 (17.07 °Brix) and the lowest was in L2 (12.33 °Brix). Our results were lower than the values observed (16–19 °Brix) by Poyrazoglu et al. (2002), while our results were in agreement with values (10–16.5 °Brix) reported by Fadavi et al. (2005). The pH values ranged between 2.85 L4 and 4.22 L8. The pH values obtained in the current study are greater than those reported by Tehranifar et al. (2010) on pomegranate cultivars grown in Iran (3.16–4.09). The titrable acidity content varied from 0.19 L4 to 2.31 g/100 ml F2. Similar results were also reported by Fadavi et al. (2005), whereas the values reported by Legua et al. (2012) for ten cultivars grown in different regions of Morocco are relatively higher than the results obtained in this work (0.24–3.7 g/L). According to the results, cultivar type plays an important role in terms of their total soluble solids, pH and titrable acidity of the pomegranate juice.

Within the current classification for Spanish cultivars established by Melgarejo (1993) are: Sweet cultivars (MI = 31–98), sour–sweet cultivars (MI = 17–24), and sour cultivars (MI = 5–7). The maturity index (TSS/TA) of the evaluated cultivars presented in Table 1 shows that all cultivars belong to the first category with the exception of L8 which belongs to the second category and F2 which belongs to the third category.

3.2 Total phenolic (TP), total flavonoid (TF) and total anthocyanin (TA) content

Fig. 1 shows the TP, TF and TA of eighteen PJ. A significant variation in TP concentration was found among the 18 PJ and the values ranged from 1385 to 9476 mg GAE/L of local cultivars and foreign cultivars ranged from 1284 to 8295 mg GAE/L. The hierarchy for the values observed was L1 > L3 > F1 > F5 > L4 > F4 > F2 > L8 > L7 > F3 > L10 > L2 > L9 > L6 > F6 > F7 > L5 > F8. The highest composition of TP was observed for the cultivars L1 (9476 ± 102 mg/L), L3 (8805 ± 65 mg/L), and F1 (8295 ± 127 mg/L). Gil et al. (2000) reported the TP of pomegranate juice from fresh arils produced from Wonderful cultivar harvested in California as 2117 ± 95 mg/L and for a commercial PJ as 2566 ± 131 mg/L and TP of eight pomegranate arils widely grown in Turkey are between 2083 and 3436 mg/L (Çam et al., 2009), their results were in agreement with our results. The TP of PJ were more than that of the other juices such as turnip juice (772 mg/L), red grape juices (1728 mg/L) and red wine (1869 mg/L) (Gatti et al., 2011).

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Figure 1 Total Phenolic content (A), total flavonoid content (B) and total anthocyanin content (C) of eighteen pomegranate juices. The data presented represent the mean ± standard error of three replicates from accession. ANOVA was used to determine the statistically significant difference at p < 0.05 as identified by different letters.

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As shown in Fig. 1B, a great variation in terms of TF was observed among the pomegranate cultivars (14,446–56,989 mg RE/L) and the differences were statistically significant (P < 0.05). The hierarchy for the values observed was L5 > L3 > L6 > L1 > L2 > L8 > F8 > F3 > F7 > F1 > F6 > F5 > L4 > L10 > L9 > L7 > F4 > F2. Guo et al. (2008) reported that the value obtained for flavonoid content in the pomegranate juice was 174 mg/L while for the apple juice was 92 mg/L, in the same research they concluded that daily consumption of pomegranate juice is potentially better than apple juice in improving the antioxidant function in the elderly.

The TA are water-soluble pigments primarily responsible for the attractive red–purple color of many fruits, including pomegranate juice, and they are well known for their antioxidant capacity (Seeram and Nair, 2002). As shown in Fig. 1C a great variation in terms of TA was observed among the pomegranate cultivars and the differences were statistically significant (P < 0.05). TA of local clones varied from 64.16 (L3) to 188.7 mg/L (L4) and of foreign cultivars ranged between 56.58 (F1) and 178.79 mg/L (F3). The hierarchy for the values observed was L4 > F3 > L6 > L1 > L10 > L9 > L8 > L2 > F6 > F5 > L5 > F2 > F7 > L3 > L7 > F8 > F4 > F1. Our results were lower than the results published for eight pomegranate cultivars widely grown in Turkey, with anthocyanin values between 81 and 369 mg/L of juice extracted from seeds by press (Çam et al., 2009). These results are very important because they can select cultivars with the high anthocyanin composition (L4, F3 and L6) to extract and use them in pharmaceutical industry or as food additive.

3.3 Identification and quantification of phenolic compounds by HPLC analysis

The chromatogram illustrating the phenolic compounds in the PJ is shown in Fig. 2, whereas Table 2 gives the concentrations of individual phenolic compounds identified in pomegranate samples of different varieties. Intervarietal differences in the phenolic compound compositions of pomegranate samples were distinct and the differences were statistically significant (P < 0.05). A total of 10 phenolic compounds, which were hydroxybenzoic acids such as gallic and ellagic acids, hydroxycinnamic acids such as chlorogenic, caffeic and ferulic acids, flavan-3-ols such as catechin and epicatechin, dihydrochalcones such as phloridzin, flavonols such as quercetin and flavonol, glycosides such as rutin were identified in the pomegranate juices. Each phenolic compound identified in pomegranate juices was found in minor quantities except ellagic and gallic acids. In our study, the concentrations of phenolic compounds were as follows: gallic acid (12.42 ‘F4’–88.51 mg/L ‘F6’), catechin (1.31 ‘F5’–6.84 mg/L ‘F3’), epicatechin (1.14 ‘F5’–13.88 mg/L ‘L9’), ellagic acid (23.43 ‘L9’–95.02 mg/L ‘L3’), chlorogenic acid (0.9 ‘L9’–2.63 mg/L ‘F1’), caffeic acid (0.16 ‘F5’–1.64 mg/L ‘F8’), ferulic acid (0.41 ‘F4’–4.78 mg/L ‘L8’), quercetin (0.60 ‘L7’–5.61 mg/L ‘L10’), phloridzin (0.07 ‘F2’–0.62 mg/L ‘L7’), and rutin (0.68 ‘F2’–3.12 mg/L ‘L5’). The concentrations reported in this work represent only the free forms of phenolic compounds since no hydrolysis was applied to the samples before HPLC analysis. In the other study repoted by Poyrazoglu et al. (2002), the concentrations of phenolic compounds were as follows: gallic acid 4.55 ± 8.55 mg/L, catechin 3.72 ± 2.29 mg/L, chlorogenic acid 1.24 ± 1.42 mg/L, caffeic acid 0.78 ± 0.79 mg/L, ferulic acid 0.01 ± 0.02 mg/L, phloridzin 0.99 ± 1.47 mg/L, and quercetin 2.50 ± 1.96 mg/L. The ferulic acid was not detected in the cultivar F1, while the cultivar F4 did not contain quercetin and rutin. The presence of gallic acid, quercetin, catechin, chlorogenic acid and o-coumaric acid in pomegranate juices has also been reported previously by Artik et al. (1998).

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Figure 2 Chromatogram of phenolic compounds in pomegranate juice (L5) grown in Morocco. Peaks: 1 (Gallic acid), 2 (Catechin), 3 (Epicatechin), 4 (Ellagic acid), 5 (Phloridzin), 6 (Chlorogenic acid), 7 (Caffeic acid), 8 (Ferulic acid), 9 (Rutin), and 10 (Quercetin).

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In the current study, all cultivars contain ellagic acid, without exception, and the highest concentrations were observed for the cultivars L3 (95 mg/L), L1 (74.3 mg/L) and L4 (70.4 mg/L), so these cultivars should have a beneficial effect on human health because this compound has been found to have antimutagenic, antiviral and antioxidative properties (Bhargava and Westfall, 1968). Amakura et al. (2000) found that the ellagic acid is an important phenolic acid with high antioxidant capacity, it has been reported in some fruit juices. And was also detected in the pomegranate juices from arils in the others study such as Mousavinejad et al. (2009) (7–160 mg/L) and Gil et al. (2000) (15.3 mg/L). The content of ellagic acid in dietary supplements has been selected as the method for assuring that supplements contain genuine pomegranate fruit extract (Zhang et al., 2009).

3.4 Antioxidant activities

The results for antioxidant activities measured by three different methods DPPH, FRAP and ABTS assay analysis from the different cultivars are displayed in Fig. 3. The DPPH radical scavenging assay is commonly employed to evaluate the ability of antioxidant to scavenge free radicals. The degree of discoloration indicates the scavenging potentials of the antioxidant extract. In this study, the differences in antioxidant capacity among the pomegranate cultivars were statistically significant and the values ranged from 31.16% to 66.82% for local cultivars and for foreign cultivars from 45.65% and 76.3%. When the value is high, the total antioxidant capacity is high. The hierarchy for antioxidant capacity with respect to their DPPH was F2 > F4 > L2 > L5 > F5 > F1 > L8 > L6 > F6 > L1 > F7 > L10 > F3 > L7 > F8 > L3 > L4 > L9. These values were in agreement with the values (10.37–67.46%) reported by Tezcan et al. (2009) on seven commercial pomegranate juices from Turkey, all marks contain 100% pomegranate juices and without added ingredients, while our results were higher than the values reported by Tehranifar et al. (2010) on twenty pomegranate juices extracted from arils in Iran (15.59–40.72%).

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Figure 3 Antioxidant capacity of 18 pomegranate juices measured by three methods: DPPH (A), FRAP (B) and ABTS (C). The data presented represent the mean ± standard error of three replicates from accession. ANOVA was used to determine the statistically significant difference at p < 0.05 as identified by different letters.

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FRAP assay is commonly used to study the antioxidant capacity of plant materials. The antioxidant capacity of fruit extracts is determined by the ability of the antioxidants in these extracts to reduce ferric iron to ferrous in FRAP reagent, which consists of 2,4,6-tris(1-pyridyl)-5-triazine (TPTZ) prepared in sodium acetate buffer, pH 3.6. The reduction of ferric iron in FRAP reagent will result in the formation of a blue product (Ferrous-TPTZ complex) whose absorbance can be read at 593 nm. This method showed (Fig. 3B) the values ranged from 18.49 to 47.1 Mm/L Fe²⁺ for local cultivars and from 17.65 and 33.81 Mm/L Fe²⁺ for foreign cultivars. The hierarchy for antioxidant capacity with respect to their FRAP was L1 > L2 > L10 > F4 > L8 > F6 > F1 > L6 > F5 > L5 > L3 > F3 > L4 > F7 > F8 > L9 > L7 > F2. All PJ exhibited the highest antioxidant activity measured by the FRAP method. The results published for commercial pomegranate juices in Turkey (18.34–121.80 Mm/L Fe²⁺) are greater than our values because the commercial pomegranate juice is extracted from the whole fruit (Tezcan et al., 2009). Similarly, Gil et al. (2000) reported that the antioxidant capacity was higher in commercial juices produced from Wonderful pomegranates harvested in California than the experimental ones obtained in the laboratory by hand pressing the arils and they suggested that punicalagin originating from the peels is one of the major phytochemicals contributing to the total antioxidant capacity of pomegranate juice.

The ABTS method based on the capacity of a sample to inhibit the ABTS radical (ABTS^•+) was compared with a reference antioxidant standard (Trolox®). ABTS radical scavenging capacities of PJ were expressed as TEAC, an ET based method (Huang et al., 2005). While the values ranged from 2648 (L9) to 4577 (L2) mg/L for local cultivars and among 2923 (F3) and 4653 (F1) mg/L for foreign cultivars.The hierarchy for antioxidant capacity with respect to their TEAC was F1 > L2 > L8 > L5 > L7 > L10 > F2 > F5 > L6 > F6 > F4 > F8 > L4 > L1 > L3 > F7 > F3 > L9. Our results were in agreement with values (2212–4183 mg/L) reported by Çam et al. (2009).

In the current study, the correlation value found among TP and FRAP was 0.308, but no significant correlation was found between the ABTS and the concentration of phenolics and among DPPH and phenolics content. Nevertheless, these results must be interpreted with caution as the Folin–Ciocalteu method used over estimates the concentration of phenolic juice containing compounds such as ascorbic acids and vitamins could interfere during TP evaluation and that do not give significant correlation. The correlations between FRAP and total phenolic may be explained in numerous ways, in fact, the concentration of polyphenols is very high in fruits of pomegranate. In addition, the synergism between the antioxidants in the mixture makes the antioxidant capacity not only dependant on the concentration, but also on the structure and the interaction between the antioxidants. However, Zhuang et al. (2011) found significant correlations between polyphenol content and 1,1-diphenyl-2-picrylhydrazine (DPPH) and between total phenolic content and ferric reducing/antioxidant power (FRAP).

Whereas, the correlation among TAC and DPPH, ABTS, and FRAP are, respectively 0.157, 0.251 and 0.351. So the correlation between anthocyanin and phenolic composition is correlated with the methods used for the measurement of antioxidant capacity. The correlation values between the flavonoid content and antioxidant capacity of the pomegranate juice (DPPH, FRAP and ABTS values) were, respectively 0.931, 0.668 and 0.660. These correlations show that the flavonoids were among the microconstituents contributing in the antioxidant activities of pomegranate fruit.

Finally the correlation between DPPH and ABTS (0.585), FRAP and ABTS (0.415) and DPPH and FRAP (0.304) (significant at P < 0.05 level). This correlation could be due to the same mechanism that DPPH, FRAP and ABTS methods rely on. This mechanism concerns the ability of the antioxidants to reduce certain radicals (DPPH radical, ferric iron and ABTS radical).