Total Phenols and Antioxidant Capacity in 10 Moroccan Pomegranate Varieties

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Pilar Legua, Pablo Melgarejo, Haddioui Abdelmajid, Juan Jos Mart e nez, Rafael Mart nez, Hmid Ilham, Hanine Hada, and Francisca Hernndez a

Abstract: The study focused on characterizing the 10 major Moroccan cultivars to acknowledge the quality and bioactive compounds of the fruits. The following determinations were assessed: pomegranate fruit maturity index, which truly denes juice taste, as well as organic acids and sugar contents, total phenols, and antioxidant activity of pomegranate juice. The cultivar per se (genotype) behaved as the most inuencing factor conditioning pomegranate sugar and organic acids proles, antioxidant activity, and total phenolics. The assessment of pomegranate chemical compositions implies the great potential of Moroccan cultivars for both fresh market and fruit processing. In fact, the "Hamde sour cv. seems particularly suitable for juice production because of its high phenolics content. Additionally, the presence of bioactive compounds in pomegranate juices may encourage their consumption for potential health benets. Keywords: antioxidant activity, organic acids, phenolics, pomegranate, sugars

Practical Application: The cultivar per se (genotype) behaved as the most inuencing factor conditioning pomegranate

sugar and organic acids proles, antioxidant activity, and total phenolics. Given the divergence observed on bioactive compounds concentrations and antioxidant activity among evaluated cultivars, the genotype factor should be considered as the most inuencing factor in future breeding programs to enhance the synthesis of benecial bioactive compounds.

Introduction
Pomegranate (Punica granatum L.) is an important fruit tree of tropical and subtropical world regions, where it is highly appreciated for its delicious edible fruit (Badizadegan and Khabbazian, 1977). It is cultivated in Iran, Afghanistan, India, Mediterranean basin (Morocco, Spain, Turkey, Tunisia, and Egypt), and MiddleEast countries, the worlds leading producers (Jbir and others 2008; Melgarejo and others 2009). This fruit tree (2n = 16) is a monoecious and preferably allogamous species (Mars and Marrakchi 2004; Jbir and others 2008). Pomegranate varieties are mainly local cultivars well adapted to diverse environmental conditions; they are selected by farmers for their fruit qualities and clonally propagated by cuttings (Mars and Marrakchi 1998). There has been recently an explosion of interest in pomegranate fruit worldwide. Though freshly consumed, they are also used for processing: fresh juice, canned beverages, alcoholic beverages, jellies, avored and colored drinks. It is considered as well a functional product of great benet for human diet since it contains several groups of substances useful for disease risk reduction (Martnez and others 2006; Cam and others 2009a).

MS 20110866 Submitted 7/19/2011, Accepted 10/17/2011. Authors Legua, Melgarejo, JJ Martnez, R Martnez, and Hern ndez are with Dept. of a Plant Science and Microbiology, EPSO, Univ. Miguel Hern ndez, Ctra. a Beniel km. 3.2, 03312, Orihuela, Alicante, Spain. Authors Abdelmajid, Ilham, and Hada are with Laboratoire de Gestion et Valorisation des Ressources Naturelles, Equipe de Gntique et Biotechnologie Vgtale, Univ. Sultan Moulay Slimane, Face e ee ult des Sciences et Techniques, B.P 523, Bni Mellal, Maroc. Direct inquiries to author e e Hern ndez (E-mail: francisca.hernandez@umh.es). a

The edible parts of pomegranate fruit are named arils. They contain large amounts of organic acids, sugars, minerals, vitamins, and polyphenols, showing high antioxidant activity and several medical benets (Schubert and others 1999; Gil and others 2000; Al-Maiman and Dilshad 2002; Poyrazoglu and others 2002; Gonzalez-Molina and others 2009; Caligiani and others 2010; Kasimsetty and others 2010; Panichayupakaranant and others 2010; Ryan and Prescott 2010; Tehranifar and others 2010). The fruit contains anticarcinogenic (Bell and Hawthorne 2008), antimicrobial (Reddy and others 2007), and antiviral compounds (Kotwal, 2007). Recent biological studies have proven that certain compounds contained in pomegranate juice, which has been shown to reduce blood pressure, are antiatherosclerotic and significantly reduce low density lipoprotein (LDL) oxidation (Aviram and others 2004). Many researchers have shown that preparations containing the pomegranate peel extract can be used to prevent and/or cure atherosclerosis, diarrhea, gastric ulcer, venereal disease, and estrogen-related diseases (Reddy and others 2007). Moroccan pomegranate production exceeded 58000 tons in 2009 with a total cultivated area of 4000 ha. Beni Mellal is largely the leading producing region in Morocco with 30000 tons and 1460 ha (MAPM, 2010). Though many different pomegranate cultivars are grown all over Moroccan agricultural areas, hardly any literature is available regarding cultivar characterization and fruit properties. Therefore, the current research focused on characterizing the 10 major Moroccan cultivars to acknowledge the quality and bioactive compounds of the fruits. The following determinations were assessed: pomegranate fruit maturity index (MI), which truly denes juice taste, as well as organic acids and sugar contents, total phenols, and antioxidant activity of pomegranate juice.

C 2011 Institute of Food Technologists R doi: 10.1111/j.1750-3841.2011.02516.x

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Further reproduction without permission is prohibited

Bioactive compounds in pomegranate . . .

Material and Methods

Plant material Ten pomegranate cultivars were selected from 4 provinces in Morocco (Table 1; Figure 1). Fruit samples of 15 kg per commercial variety were harvested at commercial ripening and placed in refrigerated containers to subsequently take them to the laboratory. Once all samples were gathered, those fruits showing any damage as cracking, sunburns, and bruises were rejected. Soon afterward, pomegranates were sent to Spain by air courier to perform there the lab characterization of Moroccan cultivars.

Juice extraction Samples of 10 pomegranates per selected cultivar were gathered, and then divided into 3 subsamples of 3 ripe fruits each. They were hand-peeled and arils removed and squeezed using a commercial blender. The extracted juices (3 subsamples per cultivar) were later Determination of sugars and organic acids sieved and centrifuged at 15000 rpm for 20 min (Sigma 318K, One milliliter of the centrifuged juice was passed through a Osterode and Harz, Germany). 0.45-m Millipore lter and then injected into a Hewlett-Packard series 1100 (Wilmington Del., U.S.A.) high-performance liquid chromatography (HPLC). The elution system consisted of 0.1% Table 1Geographic origin of 10 Moroccan pomegranate phosphoric acid with a ow rate of 0.5 mL/min. Organic acids varieties. were separated on a Supelcogel TM C-610H column (30 cm 7.8 Geographic Latitude Longitude Altitude Rainfall mm i.d., Supelco, Bellefonte, Pa., U.S.A.) and Supelguard column Genotypes origin North West (m) (mm) (5 cm 4.6 mm, Supelco, Inc.), and detected by absorbance at 210 Sefri Ouled Abdellah 32 32 6 27 480 400 nm. For sugar determinations, the same HPLC, elution system, (Beni Mellal) ow rate, and columns were used. The detection of sugars was Ounk Ouled Abdellah 32 32 6 27 480 400 obtained with a refractive index detector (HP 1100, G1362A). Hmam (Beni Mellal) Standard curves were established to quantify organic acids and Ruby Ouled Abdellah 32 32 6 27 480 400 sugars proles of pomegranate juices (Table 3 and Figure 2). (Beni Mellal)
Grenade Rouge Bou adime a Grenade Jaune Meski Mesri Kharaji Hamde Al Ouidane (Marrakech) Bzou Al Ouidane (Marrakech) Ouled Abdellah (Beni Mellal) Mekn` s e Bzou Bzou 31 41 32 06 31 41 32 32 33 57 32 06 32 06

Descriptive analyses Total soluble solids contents (TSSs) were assessed by triplicate with a digital refractometer Atago N1 (Atago Co. Ltd., Tokyo, Japan) at 20 C, and expressed as percentage ( Brix). Total acidity (TA) was also determined by triplicate using an automatic titration device (877 Titrino plus, Metrohm ion analyses CH9101, Herisau, Switzerland) with 0.1 N NaOH up to pH 8.1, and results expressed as gram per liter of citric acid. Results were shown as mean values SE (Table 2). Once the TSS and TA contents were assessed, the MIs (TSS/TA) of the evaluated commercial varieties were determined. Based on their MI, Moroccan pomegranates were categorized according to the current classication for Spanish cultivars established by Melgarejo (1993): Sweet varieties (MI = 31 to 98), soursweet varieties (MI = 17 to 24), and sour varieties (MI = 5 to 7).

C: Food Chemistry

7 41 7 02 7 41 6 27 5 33 7 02 7 02

521 400 521 480 500 400 400

250 350 250 400 580 350 350 Table 2 Descriptive analyses of pomegranate juices from evaluated Moroccan cultivars. Results expressed as mean values of total soluble solids (TSSs), titratable acidity (TA), and MI. Those values within the same columns and not followed by the same letter were signicantly different (P < 0.05). Cultivars Sefri Ounk Hmam Ruby Grenade Rouge Bou adime a Grenade Jaune Meski Mesri Kharaji Hamde TSS ( Brix) TA (gram citric acid/L) MI (TSS/TA) 15.5c 16.6abc 16.4abc 15.7c 17.6a 15.2c 15.7c 17.2ab 15.3c 16.2bc 2.44b 2.74b 3.8b 3.05b 4.85b 3.74b 3.87b 4.18b 3.31b 37.51a 64.29a 61.64ab 43.63c 51.94bc 36.32c 41.19c 40.81c 41.78c 46.74c 4.48d

Table 3Major organic acids of pomegranates (g/100 g). Each value represented the mean of 3 replicates; nd, undetected. Those values within the same columns and not followed by the same letter were signicantly different (P < 0.05). Cultivars Sefri Ounk Hmam Ruby Grenade Rouge Bou adime a Grenade Jaune Meski Mesri Kharaji Hamde Oxalic acid 0.032cd 0.037c 0.031cd 0.021de 0.110a 0.085b 0.011e 0.036cd 0.030cd 0.042c Citric acid nd nd 0.018b nd 0.241 0.182b 0.204b 0.227b nd 3.223 Malic acid 0.584cd 0.643c 0.554cd 0.432de 1.563a 1.244b 0.313e 0.621cd 0.553cd 0.7c Quinic acid 0.934b 0.853b 1.194a 0.046f 0.516c 0.376d 0.231e 0.144ef 0.063f 0.444cd Succinic acid 0.063a 0.052a 0.033a 0.032a 0.369a 0.268c 0.164d 0.102e 0.044a 0.318b

Figure 1Map of localization.

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Bioactive compounds in pomegranate . . .

The following pure organic acids and sugars, respectively, were purchased from Sigma (Poole, Dorset, U.K.): oxalic, citric, tartaric, malic, acetic, fumaric, succinic, shikimic, quinic, and ascorbic acids, as well as glucose, maltose, fructose, sucrose, and sorbitol. Results for both proles were expressed as gram per 100 g (%). Sugars and organic acids were quantied by triplicate for every evaluated cultivar. triplicate for every extracted solution using the FolinCiocalteu reagent. Absorption was measured at 760 nm by spectrophotometry (ThermoSpectronic Heios , Cambridge, England), and results were expressed as milligram of gallic acid equivalent (GAE) per 100 g of fresh weight (mean values SE).

Total antioxidant activity (TAA) determination TAA was quantied according to Daz-Mula and others (2008). This procedure allowed determining both the hydrophilic and lipophilic TAA in the same extraction. A quantity of 5 g of arils from every subsample was homogenized in 5 mL of 50 mM phosphate buffer (pH = 7.8) and 3 mL of ethyl acetate, and then centrifuged at 10000 g for 15 min at 4 C. The upper fraction was used for quantifying the TAA due to lipophilic compounds (L-TAA), and the lower one for the TAA due to hydrophilic compounds (H-TAA). All juice extracts were subjected to enzymatic digestion for TAA determination (by triplicate). The following enzymatic system was added: the chromophore 2,2 azino-bis-(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS), the horse radish peroxidase enzyme, and its oxidant substrate (hydrogen peroxide), in which ABTS+ radicals were generated and monitored at 730 nm. After adding the extract, the decrease recorded in absorbance was proportional to the TAA of the sample. A calibration curve was performed with Trolox ((R)-(+)-6-hydroxy-2,5,7,8-tetramethyl-croman-2-carboxylic acid) (0 to 20 nmol) from Sigma (Madrid, Spain). And, results were nally expressed as milligram of Trolox equivalent per 100 g(mean values SE). Determination of total phenolics (TPs) TPs were extracted following the procedure established by Tom s- Barber n and Espin (2001). A dilution of 5 mL of waa a ter:methanol (2:8), containing 2 mM NaF, was added to 5 g of arils from every subsample. TPs quantication was performed by
25

Statistical analysis Statistical analyses were performed using the SPSS software package for Windows version 18. All data were subjected to basic descriptive statistics, and then evaluated by an analysis of variance for mean comparisons. Fishers Least Signicant Difference test (LSD at 95.0% of condence level) was applied for mean values discrimination (Multiple Range Test).

Results and Discussion

Fruit quality parameters The Moroccan pomegranate cultivars evaluated on this study signicantly differed in taste. While the cultivars "Sefri," "Ounk Hman," and "Grenade Rouge" showed strong sweet tastes, "Hamde" exhibited a powerful sour one. Table 2 showed pomegranate qualitative traits. TSS contents signicantly differed among the evaluated varieties, ranging from 15.2 Brix to 17.6 Brix for "Grenade Jaune" and "Boua dime," a respectively. This TSS interval range agreed with those reported from other collections grown in different regions around the world (Chace and others 1981; Khodade and others 1990; Barone and others 1998, 2001; Fadavi and others 2005; Martnez and others 2006; Caln-S nchez and others 2010; Dafny-Yalin and others a 2010; Tehranifar and others 2010; Cristofori and others 2011). TSS assessment is not only important for juice quality evaluation, but for determining also the suitability of cultivars for pomegranate winemaking (Seser and others 2007). Regarding TA contents, remarkable differences were found among Moroccan cultivars. "Hamde" scored the highest content by far (37.5 g/L of citric acid), whereas all the others just ranged from 2.4 to 4.8 g/L. Moroccan TA contents were similar
Figure 2Sugar contents of pomegranates (g/100 g). Bars (mean value SE) with different letters showed signicant differences among pomegranate cultivars (P < 0.05).

20 bc Sugar content (g / 100 g) 15

ab

ab

ab ab

ab

a bc

Glucose Fructose Total ab

ab 10 b b ab a

ab

ab ab a

ab c b

ab ab a

a b

ab ab b

0 Ounk Hman Ruby Grenade Rouge Bouadime Meski Grenade Jaune Kharaji Hamde Sefri Mesri Varieties

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Bioactive compounds in pomegranate . . .

to those showed by Spanish, Italian, and Iranian cultivars (Martnez and others 2006; Tehranifar and others 2010; Cristofori and others 2011). Only "Hamde" differed from the rest of Moroccan cv., scoring a TA content similar to those yielded by the Iranian "TSK" (23.1 g/L) and the Italian "Tordimonte A" (23.7 g/L) (Tehranifar and others 2010; Cristofori and others 2011). The acidity content denitively plays an important role in the perception of fruit quality. Data from other collections around the world suggested that the TA content and pomegranate taste depend on climate and growing conditions (Dafny-Yalin and others 2010). The taste and avor of pomegranates clearly rely on the MI (Martnez and others 2006; Cam and others 2009b). The MI sig nicantly varied among Moroccan cultivars (Table 2). Whereas "Sefri" showed a MI of 64.29, "Hamde" MI was 4.48. According to Martnez and others (2006), Moroccan pomegranate varieties were grouped as sweet ("Sefri," "Ounk Hmam," "Ruby," "Meski," "Grenade Rouge," "Grenade Jaune," "Bou adime," "Kharaji," and a "Mesri") and sour ("Hamde"). Likewise, previous studies reported variable ranges of MIs (Viswanath and others 1999; Martnez and others 2006; Cam and others 2009b; Sarkhosh and oth ers 2009). So, it can be stated that pomegranate juice MI was basically conditioned by the cultivar factor. Since the evaluated Moroccan varieties showed high TSS contents, they are suitable for both fresh market and juice processing, and "Hamde" only appropriate for the industry because of its high acidity content. rmed the predominance of fructose and glucose as the main pomegranate sugars, which indeed agreed with previous literature. However, results disagreed regarding pomegranate main sugar (fructose or glucose). For instance, Al-Maiman and Dilshad (2002), Mirdehghan and others (2006), Ozgen and others (2008), and Cam and others (2009b) stated that glucose prevailed over fructose. Whereas Melgarejo and others (2000), Poyrazoglu and others (2002), Tezcan and others (2009), Hasnaoui and others (2011) found fructose as the predominant pomegranate sugar. To some researchers, the divergence noticed on sugar contents might be due to diverse agro-climatic conditions. To others in contrast, the cultivar factor per se (genotype) mostly inuences pomegranate sugars and organic acids proles (Hasnaoui and others 2011).

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Organic acids and sugars The organic acid prole determines the sensory attributes of pomegranate juice and contributes to potential health benets as well (Aarabi and others 2008). Besides, it regulates microorganism incidence in fruit and byproducts and therefore controls juice quality and shelf life (Dumlu and G rkan 2007). HPLC results u (Table 3) revealed malic as the main pomegranate organic acid (ranging from 0.31 to 1.56 g/100 g) followed by quinic acid (ranged from 0.063 to 1.19 g/100 g). Oxalic, citric, and succinic acids were also detected at much lower concentrations (from 0.011 to 0.11, 0.018 to 3.22, and 0.032 to 0.36 g/100 g, respectively). And only traces of fumaric acid were detected (data not shown). Similar results were reported by Legua and others (2000) and Mirdehghan and others (2006). Unlike the results mentioned above, citric was the predominant organic acid found in Iranian pomegranates (Aarabi and others 2008) as well as in 6 Georgian accessions (Pande and Akoh 2009). Furthermore, citric acid also predominated in some Spanish accessions (Melgarejo and others 2000), in 10 Turkish pomegranates (Poyrazoglu and others 2002), in accessions grown in Israel (Dafny-Yalin and others 2010) and in 10 Tunisians cultivars (Hasnaoui and others 2011). Additionally, a high fumaric level was detected in Iranian accessions (Aarabi and others 2008); tartaric was the 2nd most abundant organic acid in the Georgian collection (Pande and Akoh 2009); and the quinic acid was also present in Turkish pomegranates (Poyrazoglu and others 2002). In fact, pomegranate fruit showed a great diversity of organic acid proles. About pomegranate sugars (Figure 2), fructose and glucose were the predominant ones (7.8 to 10.4 g/100 g and 6.9 to 8.6 g/100 g, respectively). Sucrose and sorbitol occurred at traces or were even undetectable (data not shown). The total sugar content of Moroccan pomegranates varied from 16.1 g/100 g for "Mesri" to 19.3 g/100 g for "Sefri." These results conC118 Journal of Food Science r Vol. 71, Nr. 1, 2012

Total antioxidant activity (TAA) The antioxidant activity results (H-TAA and L-TAA) are shown in Figure 3. Lipophilic and hydrophilic fractions considerably differed among Moroccan cultivars. The hydrophilic fraction (H-TAA) was signicantly higher than the lipophilic one (LTAA). The cultivar Ounk Hman yielded the highest H-TAA (143.8 mg/100 g) while Boua dime the lowest one (39.8 a mg/100 g). Regarding lipophillic fractions, the sour Hamde scored the highest L-TAA (36.5 mg/100 g) whereas Meski the lowest L-TAA (6.5 mg/100 g). The antioxidant activity of commercial pomegranate juices was reported for Turkish and Iranian cultivars (Mousavinejad and others 2009; Tezcan and others 2009; Tehranifar and others 2010). Their TAA results disagreed with those for the Moroccan cultivars Sefri, Ounk Hman, Ruby, Mesri, Kharaji, and Hamde. Factors such as pomegranate genotypes and sample extraction protocols might certainly account for the divergence observed. Moreover, pomegranate antioxidant activity uctuated depending on the fruit portion processed. Tzulker and others (2007) reported that homogenates from the whole fruit exhibited an antioxidant activity of approximately 20-fold higher than those from aril juices. Overall, pomegranates showed a much higher antioxidant capacity than most commonly consumed fruits and juices. Total phenolics (TPs) Figure 4 showed the TPs results for the Moroccan pomegranates. TPs concentrations signicantly varied among the 10 evaluated cultivars (from 41.01 to 83.43 mg/100 g). The sour cv. "Hamde" gave the highest TPs concentration while "Mesri" the lowest one (Figure 4). The reported levels of pomegranate TPs widely ranged from 23.7 up to 930.4 mg/100 g (Gil and others 2000; Vardin and Fenercioglu 2003; Ozgen and others 2008; Cam and others 2009a; Mousavinejad and others 2009; Tezcan and others 2009). Moroccan pomegranates yielded TPs concentrations that fell within previous literature TPs ranges. Nevertheless, the broad interval range of TPs concentrations must obey to differences among cultivars (genotypes), growing seasons, agricultural practices, and determination assays (Cam and others 2009a; Tehranifar and others 2010). Furthermore, Gil and others (2000) reported TPs concentrations of 2117 95 and 2566 131 mg/L for pomegranate juice from fresh arils and for a commercial pomegranate juice, respectively. Finally, and since all evaluated Moroccan pomegranates were similarly grown, the genetic variability by itself led to differences in TPs concentrations by modifying the biosynthesis path of secondary phenolics metabolites in these varieties.

Bioactive compounds in pomegranate . . .

160 a 140 c H -TAA plus L-TAA (mg / 100 g) 120 e 100 80 60 40 20 f 0 Ounk Hman Ruby Grenade Rouge Meski Mesri Sefri Kharaji Bouaadime Grenade Jaune Hamde Varieties b f H-TAA L-TAA d b

Figure 3Pomegranate total antioxidant activity, hydrophilic (H-TAA, black bars), and lipophilic (L-TAA, gray bars) fractions. Bars (mean value SE) with different letters showed signicant differences among pomegranate cultivars (P < 0.05).

g h cd bcd cd bcd

g d e bc a

100

abc 80 Total Phenolics (mg / 100 g) cde 60 f g 40 ab bcd de e abc

Figure 4TPs concentrations of pomegranates. Bars (mean value SE) with different letters showed signicant differences among cultivars (P < 0.05).

20

0 Ounk Hman Ruby Grenade Rouge Bouadime Meski Grenade Jaune Kharaji Hamde Sefri Mesri Varieties

Conclusion
Statistically signicant differences were observed among the evaluated Moroccan pomegranates. The cultivar per se (genotype) behaved as the most inuencing factor conditioning pomegranate sugar and organic acids proles, antioxidant activity, and TPs. The assessment of pomegranate chemical compositions implies the great potential of Moroccan cultivars for both fresh market and fruit processing. In fact, the "Hamde" sour cv. seems particularly suitable for juice production because of its high phenolics content. Additionally, the presence of bioactive compounds in pomegranate juices may encourage their consumption for potential health benets.

Acknowledgments
This study was supported by the projects A/017483/08 and A/023796/09, held within the SpanishMoroccan Interuniversity Cooperation Program (Escuela Polit cnica Superior de Orihuela, e Miguel Hern ndez Univ., Spain and Faculty of Sciences and Techa niques, Univ. of Sultan Moulay Slimane, Morocco, respectively). Both projects were funded by the Spanish Agency for Intl. Cooperation and Development (AECID).

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