Assessment of Minerals, Bioactive Compounds, and Antioxidant Activity of Ten Moroccan Pomegranate Cultivars

Laboratory of Virology Microbiology Quality and Biotechnology ETB, Faculty of Science and Techniques in Mohammedia, Hassan II University, P.O. Box 146 Yasmina City, Mohammedia 20650, Morocco Laboratory of Agri-Food Technology and Quality, Research Unit of Plant Improvement and Quality, Regional Center for Agricultural Research in Marrakesh, National Institute for Agricultural Research (INRA), P.O. Box 533, Marrakesh 40000, Morocco Analysis and Characterization Center (CAC), Faculty of Sciences Semlalia, Cadi Ayyad University, P.O. Box 2390, Marrakesh 40000, Morocco Polyvalent Laboratory of Research and Development, Polydisciplinary Faculty, SultanMoulay Slimane University, P.O. Box 592, Beni Mellal 23000, Morocco


Introduction
e pomegranate (Punica granatum L.) is one of the ancient fruit trees of the Punicaceae family that has been cultivated for centuries and was highly appreciated for its delicious fruit flavour [1]. Pomegranate is consumed as fresh or in other processed forms such as juice, jam, and jelly [2,3] or used as a source of nutraceutical substances [4]. Pomegranate consumption has gradually increased due to its nutritional value and bioactive compound content. is fruit is rich in polysaccharides, sugars, proteins, minerals, and organic acids [5] and comprises significant amount of vitamin C and phenolic compounds with high antioxidant activity [6]. Accordingly, its consumption has several medical benefits such as the treatment of cardiovascular diseases, Alzheimer disease, cancer, diabetes, arthritis, and colitis [7]. In Morocco, despite its internationally recognized benefits, the pomegranate remains under exploited compared to other producing countries. Exports are very low, not exceeding 0.5% of total production. e main products are locally marketed with low processing and valorisation. In addition, the great variability of the plant material generates heterogeneity of production, and the characteristics of the majority of cultivars are not known [8]. In this context, this study has focused on cultivars of pomegranates not yet characterized, identified by the Ministry of Agriculture, Fisheries, Rural Development, Water, and Forests as terroir products.
ese cultivars are belonging to the terroirs of Tmassine, Sidi Abdellah, Ain Lahjar and Sour Laaz [9], in addition to cultivars of Lalla Takerkoust and Beni Meskine regions known for their delicious fruits, and the cultivar "Sefri" of Ouled Abdellah, which is labeled as protected geographical indication [10]. e characterization analyses aim to assess the variability in terms of mineral content and biochemical properties of ten pomegranate cultivars so as to better know their fruit nutritional and functional potential.
e information generated will be useful in selecting the best pomegranate cultivars and determining their suitability for fresh fruit consumption, processing, producing nutraceutical substances and possibly contributing in their labelling as quality linked to origin.

Ash and Minerals.
Pomegranate juice was incinerated at 600°C for 7 hours ( ermolyne Type 1400 Oven) and ash content (g/100 g of fresh weight (FW)) was determined. For mineral determination, the ash was digested with nitric acid and diluted with ultrapure water. e determination of concentration of macro-and microelements was carried out using an inductively coupled plasma mass spectrometry ( ermo Scientific ICAP RQ, ICP-MS) calibrated with different concentration of multielement standards provided by TECHLAB. Ultrapure water and nitric acid (69%, analytic grade) were used as solvents and Q-tegra was the reading software. e measurements were realized in triplicate for each cultivar. Results were expressed in mg/100 g of fresh weight (FW).

Organic Acid
Profile. Organic acids were quantified according to Alcaraz-Mármol et al. [11] by Dionex Ultimate 3000 ultrahigh-performance liquid chromatography (UHPLC) equipped with a diode array detector (PDA) and Xcalibur software for reading and processing results. A Vertex Plus C18 reversed phase column (250 × 4.6 mm, Eurospher II 100-5) was used. Merck (Darmstadt, Germany) provided solvents and standards. An isocratic mode of solvents was used with 95% of water acidified with orthophosphoric acid at 0.1% and 5% of acetonitrile. Results were expressed as g/100 mL of pomegranate juice. Data were obtained in triplicate. (TP). TP was determined by the Folin-Ciocalteu method, based on the condition optimized by Georgé et al. [12]. TP was expressed as mg gallic acid equivalent (GAE)/kg of pomegranate juice. Calibration curves at concentrations between 0 and 500 g GAE/L showing good linearity (the coefficient of determination R 2 ≥ 0.997) were used to quantify TP. (TA). TA was determined by pH differential method according to Hmid et al. [13] using two buffers systems: sodium acetate buffer pH 4.5 (0.4 M) and potassium chloride buffer pH 1.0 (0.025 M). TA was expressed as mg/L of pomegranate juice. e measurements were repeated three times for each cultivar.

Phenolic Monomers.
Pomegranate juice was first diluted with a mixture of methanol/water (6 : 4) and centrifuged at 10000 rpm at 4°C for 10 min. Polyphenolic profile of pomegranate juice was determined for the ten cultivars by ultrahighperformance liquid chromatography tandem mass spectrometry according to Puigventós et al. [14]. e UHPLC chain (Dionex Ultimate 3000) was equipped with a diode array detector coupled to a TSQ Endura tandem mass spectrometer ( ermo Fisher Scientific). A Vertex Plus C18 reversed phase column (250 × 4.6 mm, Eurospher II 100-5) was used. Merck (Darmstadt, Germany) provided solvents and standards. Xcalibur was the reading software. Detection was done within a mass range of 50-1100 m/z. e measurements were repeated three times for each genotype juice. Results were expressed in terms of the pick percentages over the two years.

Antioxidant Activity (AA)
. AA was measured in terms of radical scavenging ability using the stable free radical 2,2- Journal of Food Quality diphenyl-1-picrylhydrazyl (DPPH) according to the procedure described by Moktan et al. [15]. Calibration curves at concentrations between 0 and 500 g of ascorbic acid equivalent (AAE/L), which showed good linearity (the coefficient of determination R 2 ≥ 0.999), were used to quantify antioxidant activity. e AA was expressed in mg AAE/kg of fresh juice.

Statistical
Analysis. e data obtained were statistically analysed using SPSS 22.0 statistical database for Windows. One-way analysis of variance (ANOVA) for the comparison of means was performed after a basic descriptive statistical analysis. e significance level was taken at α � 0.01. Tukey's HSD test was used to perform multiple comparisons among means. Correlation coefficients (r) were determined using the Pearson correlation matrix method to reveal possible relationships between traits. e XLSTAT Addinsoft TM software (XLSTAT, 2016) was used to perform the principal component analysis (PCA) and the cluster analysis (CA).

Ash and Minerals.
Ash content and mineral composition of pomegranate juice of ten Moroccan cultivars are presented in Table 2. Ash content varied from 0.19% for "Sefri 6" to 0.39% for "Lhamdha." ese results agreed with Al-Maiman and Ahmad [5] who found an ash content of 0.32% for full ripe pomegranate juice from Saudi Arabia. e results of mineral composition showed clearly that iodine (I) was the most abundant macroelement in pomegranate juice, followed by phosphorus (P) and sulphur (S). ese results were not in agreement with Ekşi andÖzhamamc [16] and Alcaraz-Mármol et al. [11] who found that potassium (K) was the main macroelement in pomegranate fruits for Turkish and Spanish cultivars, respectively. Iron (Fe) and aluminium (Al) were the main microelements which agreed with the results found by Fawole and Opara [17] for South African cultivars and Alcaraz-Mármol et al. [11] for Spanish cultivars. e studied cultivars had higher concentrations of minerals compared to cultivars from Saudi Arabia [5], Spain [11], Turkey [16], and South Africa [17] mainly of iron, zinc, magnesium, phosphorus, sulphur, calcium, and aluminium. e variation of the concentration of pomegranate minerals could be indorsed to differences in cultivar type, plant nutrition, climate, and soil conditions [11,17].

Organic Acid
Profile. e organic acid profile contributes to potential health benefits and defines the sensory qualities of pomegranate juice [18]. e UHPLC results of organic acid contents showed significant differences (P < 0.01) among the ten studied cultivars (Table 3). e most dominant organic acid was citric acid with concentrations ranging from 0.11 g/100 mL for the cultivar "Bzeq Tir" to 2.92 g/100 mL for the sour cultivar "Lhamdha" (which means acidic). ese concentrations were consistent with those found by Legua et al. [19] for other Moroccan cultivars. Oxalic and malic acids were found in lower concentrations, respectively, from 0.04 g/100 mL for the cultivar "Lhamdha" to 0.10 g/100 mL for "Sefri 4" and from 0.09 g/100 mL for "Sefri 3" to 0.17 g/100 mL for "Sefri 6" and "Lhamdha." e concentrations of malic acid agreed with the results found by Garima and Akoh [20] and Nuncio-Jáuregui et al. [21] for American and Spanish cultivars, respectively. Oxalic acid was mostly detected in sweet cultivars, especially "Bzeq Tir," "Sefri 2," and "Sefri 4" and the results found were in agreement with Legua et al. [19]. Succinic, ascorbic, and fumaric acids were detected in very low concentrations (from 0.01 to 0.07, 0.01 to 0.02, and 0.00 to 0.01 g/100 mL, respectively). Pomegranate juices showed a great diversity of organic acid concentrations [1] that could be endorsed to the cultivar factor and agroclimatic conditions [22].

Total Polyphenols Content (TP).
Polyphenols are the most abundant compounds found in all parts of the pomegranate fruits [23]. e antioxidant activity of pomegranate is mainly linked to the concentration of these compounds [7,24]. e results of total polyphenol concentrations showed significant differences among the ten studied cultivars (Table 4). e sour cultivar "Lhamdha" presented the highest TP concentration with 1168.29 mg GAE/kg while the cultivar "Sefri 1" presented the lowest one with 632.84 mg GAE/kg. e TP data were in agreement with the results obtained for other Moroccan cultivars (410.1-834.3 mg/kg) [1], Iranian cultivars (220-1266.8 mg/ L), [25] and Indian cultivars (876.2-1536.2 mg/kg) [26]. e broad interval range of TP concentrations is related to  genetic variability, agricultural practices, geographical and climate effects, growing seasons, and determination assays [27,28].

Total Anthocyanins Content (TA).
e attractive red colour of pomegranate arils is accredited to the biosynthesis and accumulation of red anthocyanins in the fruit [29]. is appealing red colour is an important index for juice quality and is highly requested by pomegranate juice makers, as long as the heat treatment during the production process reduces significantly the colour of the juice [3]. TA contents of the ten studied pomegranate cultivars are presented in Table 4. A significant diversity (P < 0.01) was revealed among these cultivars. e highest TA content was observed for cultivars "Lahmer" (which means red) and "Marrakchia" with, respectively, 204.09 and 212.76 mg/L, while the lowest concentrations were shown for cultivars "Sefri 1" with 51.38 mg/ L and "Sefri 3" with 59.85 mg/L. ese contents are higher than those reported by Hmid et al. [30] for other Moroccan cultivars and by Li et al. [7] for Chinese cultivars. Although, these total anthocyanins contents are lower than those reported by Tehranifar et al. [28] for Iranian cultivars (ranging between 5.56 and 30.11 mg/100 g). e differences in pomegranate anthocyanin content around the world are endorsed to the differences in cultivars, agroclimatic conditions, and ripening stages. e cultivars "Sefri 6," "Lahmer," and "Marrakchia" showed the highest anthocyanin content and could then be of interest for production of fresh pomegranate juice with a high potential of health benefit.

Phenolic Monomers.
e phenolic compounds identified in the ten studied pomegranate cultivars are presented in Table 5. Seventeen polyphenolic compounds were recognized from the 30 major peaks based on the combined information from PDA (280 nm), MS, and literature data.
e second important class of phenolic compounds was anthocyanins with peak area percentages ranging from 2.65% for "Lhamdha" to 10.44% for "Sefri 6." Phenolic acids came in the third place, represented mainly by vanillic acid, dihexoside, and coumaric acid hexoside, with percentages ranging from 2.61% for "Lhamdha" to 6.46% for "Sefri 1." Lastly comes the group of flavonoids represented by syringetin hexoside. e results obtained differed from those reported by the previous studies [31,32,35] who stated the richness of pomegranate juice with anthocyanin compounds. Russo et al. [32] reported an anthocyanin percentage around 40% of the total percentages of identified phenolic compounds in pomegranate juice. e divergences in phenolic profile could be attributed to the differences in extracting methods [31]. Table 4 shows the results of AA of the ten studied pomegranate cultivars. e differences in AA among these cultivars were statistically significant. e sour cultivar "Lhamdha" and the sweet cultivar "Sefri 6" presented the highest antioxidant activity with 1112.39 and 1059.85 mg AAE/kg, respectively, while the cultivar "Sefri 1" presented the lowest AA with 597.83 mg AAE/kg. e results found for the AA agreed with those of Li et al. [7] for Chinese cultivars. ese results indicated that sour pomegranate had no automatically higher antioxidant activity compared with sweet pomegranates. Higher antioxidant potential was although attributed to higher total polyphenols content present in pomegranate and other fruits [7,27].

Correlation Analysis.
Pearson correlation was used to examine the dependence among the studied variables at P < 0.01. Significant positive correlation was found between antioxidant activity and total phenol contents (r � 0.792).

Principal Component Analysis (PCA).
PCA was applied to the results to achieve better understanding of the relationships and trends between variables for the different pomegranate cultivars. e first two principal components explained 68.34% of the total variation ( Figure 2). e proportion of variation for the first and the second components was 48.74% and 19.60%, respectively. e first component F1 representing 48.74% of the total variation was positively linked to total polyphenols contents, antioxidant activity, ash, fumaric, malic, citric, and succinic acids, sulphur (S), ellagitannins, and gallagyl esters while negatively linked to potassium (K), sodium (Na), calcium (Ca), gallotannins, phenolic acids, and flavonoids. e second component F2 accounted for 19.60% of the variance and was positively correlated with anthocyanin, oxalic, and ascorbic acids, whereas negatively correlated with iron (Fe) and iodine (I) minerals. Based on the plot of the principal component scores, the cultivar "Lhamdha" showed high positive score on F1. is cultivar presented a rich source in organic acids (fumaric, succinic, and citric acids), gallagyl esters, and ellagitannins and showed high antioxidant activity, total polyphenol, and ash contents. is sour cultivar can be used as a functional source in medical or cosmetic industry as it showed high content of beneficial compounds. On the other hand, the cultivar "Sefri 1" presented large negative score on F1 and F2 showing high content of minerals (I, Na, Ca, and Fe), gallotannins, flavonoids, and phenolic acids. e cultivars "Sefri 2," "Sefri 3," "Sefri 4," and  Journal of Food Quality "Bzeq Tir" were relatively close to each other along the x-axis and presented a low negative score on F1 and F2. ey were characterized also by the same characteristics as the cultivar "Sefri 1." Lastly, "Lahmer," "Marrakchia," "Sefri 5," and "Sefri 6" were close to each other along the y-axis and showed a positive score on F1. ey had red juices rich in anthocyanins and oxalic and ascorbic acids with high antioxidant activity and total polyphenols contents and then could be used for production of fresh pomegranate juice with high health benefits.
3.9. Cluster Analysis. Figure 3 shows the results obtained from cluster analysis for the ten studied Moroccan pomegranate cultivars. Four main groups were clustered based on the mineral and biochemical parameters. e first and second groups were composed by one cultivar: "Lhamdha" and "Sefri 1," respectively. e third group consisted of the cultivars "Bzeq Tir," "Sefri 2," "Sefri 3," and "Sefri 4," while the last group included "Sefri 5," "Sefri 6," "Lahmer," and "Marrakchia." is cluster analysis revealed the divergence of cultivars with the same appellation "Sefri" confirming the problem of homonymy or synonymy exposed by Ajal et al. [38] in the pomegranate cultivars appellation. PCA and cluster analysis indicated that there were great differences among cultivars in terms of physicochemical and biochemical attributes.

Conclusions
Statistically significant differences were found between the ten studied pomegranate cultivars within the determined physicochemical and biochemical parameters. e sour cultivar "Lhamdha" showed promising characteristics and could be used as a source of nutraceutical substances while the cultivars "Sefri 1" or "Sefri" of Lalla Takerkoust, "Sefri 2" or "Sefri" of Sour Laaz, and "Sefri 3" or "Sefri" of Tmassine have showed interesting content of minerals. e cultivars "Sefri 5" or "Sefri" of Ouled Abdellah, "Sefri 6" or "Sefri" of Beni Meskine, "Lahmer," and "Marrakchia" are suitable for the production of fresh pomegranate juice with high potential of health benefit, while the cultivars "Sefri" of Sour Laaz, "Sefri" of Tmassine, "Sefri 4" or "Sefri" of Sidi Abdellah, and "Bzeq Tir" could be intended for fresh fruit consumption or for commercialization of ready-to-eat arils. Cluster analysis revealed the divergence of cultivars with the same appellation "Sefri" confirming the problem of homonymy or synonymy in the pomegranate cultivars appellation.
is study clearly demonstrates the potential of the studied pomegranate cultivars and the importance of their valorisation, especially for food and pharmaceutical industries.

Data Availability
e data used to support the findings of this study are available from the corresponding author upon request.

Disclosure
is research did not receive specific funding. It was performed at the Regional Center for Agricultural Research in Marrakesh belonging to the National Institute for Agricultural Research in the framework of the research activities of the Laboratory of Agri-Food Technology and Quality (INRA, Morocco), at the Laboratory of Virology, Microbiology, Quality, and Biotechnology/ETB, Faculty of Science and Techniques in Mohammedia, Hassan II University (Casablanca, Morocco), and at the Analysis and Characterization Center (CAC) of Cadi Ayyad University, Faculty of Science Semlalia (Marrakesh, Morocco).

Conflicts of Interest
e authors declare that they have no conflicts of interest regarding the publication of this paper.