Oil Content, Fatty AcidComposition, Physicochemical Properties, and Antioxidant Activity of Seed Oils of Ten Moroccan Pomegranate Cultivars

Laboratory of Virology Microbiology Quality and Biotechnology/ETB, Faculty of Sciences and Techniques in Mohammedia, Hassan II University, P.O. Box 146, Yasmina City, Mohammedia 20650, Morocco Laboratory of Agri-Food Technology and Quality, Regional Center for Agricultural Research in Marrakesh, Regional Center for Agricultural Research Marrakesh, National Institute for Agricultural Research (INRA), P.O. Box 533, Marrakesh 40000, Morocco


Introduction
Pomegranate (Punica granatum L.) is one of the ancient fruit trees of the Punicaceae family that has been cultivated for centuries and highly appreciated for its delicious fruit taste [1]. e pomegranate seeds are quantitatively and qualitatively a relevant agri-food by-product rich in compounds beneficial to human health. e oil contained in pomegranate seeds has attracted increasing interests due to the abundance of conjugated fatty acids; the most important of them is punicic acid, a positional and geometric isomer of α-linolenic acid [2,3]. e pomegranate seed oil (PSO) is also a rich source of health-beneficial compounds. It showed high content of phytosterols [3,4], tocopherols [5], especially c-tocopherol [3,5,6], and phenolic compounds [7]. Phytosterols have the ability to inhibit cholesterol absorption, while tocopherols act as natural preservatives and their presence in seed oils is habitually linked with the relative abundance of unsaturated fatty acids [8]. PSO is shown to acquire antioxidant, antitumor, and anti-inflammatory properties [9]; it also modulates lipid metabolism and has antiobesity properties [10].
anks to its medicinal and nutritional properties, the PSO has been considered as a functional ingredient in the food industry [11][12][13]. In Morocco, in spite of the evolution of its production, pomegranate is less valued compared to other producer countries.
e major production part is marketed locally with low processing and exploitation of by-products [14]. To this end, many efforts are being made to promote this fruit. In the Beni-Mellal/Khenifra region, the "Sefri Ouled Abdellah" cultivar has been labelled as "Protected Geographical Indication (IGP)" within the Green Morocco Plan [15]. Moreover, the pomegranates from four other regions "Ain Lahjar," "Tmassine," "Skhour Rhamna," and "Sour Laaz" have been named as flagship local products [16]. In this context, this study aims to valorize the by-products from the main cultivars in these regions. To the best of our knowledge, the majority of these cultivars were characterized for the first time, especially "Lhamdha," "Bzeq Tir," "Marrakchia," "Lahmer," "Sefri 1," "Sefri 2," "Sefri 3," "Sefri 4," and "Sefri 6." e characteristics of pomegranate seeds and seed oils were evaluated and compared in terms of physical and physicochemical criteria, fatty acid composition, and antioxidant activity. e obtained results will allow us to better evaluate the potential of the PSO to be used as functional or nutraceutical food ingredients.

Biometric and Physical Parameters of Pomegranate Arils and Seeds.
irty fruits per cultivar were analysed. Each pomegranate fruit was cut in the equatorial zone and peeled and the arils were detached manually. e fruit (FW, g) and arils weights (AsW, g) were evaluated. e seed yield was calculated as the ratio between acquired grams of seed per 100 g of arils sample, and the seed yield per fruit was then deducted (Syf, g/100 g fruit). Twenty-five arils of each cultivar were randomly selected from a homogeneous sample and evaluated individually for the following parameters: aril weight (Aw, g), seed weight (Sw, g), maximum seed length (Ls, mm), and seed width (Ws, mm). Arils and seeds were weighed with a precision balance. Seed length and width were measured with a digital electronic caliper with an accuracy of 10 −2 mm. e proportion of the seeds, or seed index (Si), was calculated as follows [20]: A panel of eight experts from the Regional Center for Agricultural Research in Marrakech evaluated independently the hardness of the seeds using sensory analysis. A numerical scale with 0 represents none and ten represents extremely hard was used [21]. e sensory analysis was carried out in the Sensory Analysis Laboratory of Food Technology and Quality in the INRA, Marrakesh, established in accordance with the general guidelines for premises of sensory evaluation ISO 8589 [22].

Seed Oil Extraction.
Seeds were obtained by pressing arils with a manual press, which keeps them intact. e seeds were washed, air-dried, and ground to a powder by an electric mill (FRITSCH PULVERISETTE 14). Oil was extracted with 250 mL of n-hexane from 40 g of seed powder using a Soxhlet extraction system for 6 hours. At the end of the extraction, the solvent was evaporated in a rotary evaporator under vacuum at 30°C. e obtained oils were then flushed with nitrogen to remove the residual traces of hexane and stored in the dark at 4°C. Oil content was expressed in g/100 g of dry matter of seed powder. e analysis was performed in triplicate.

Extinction Coefficients.
e specific extinction coefficients K 232 and K 270 were determined according to the IOC standard method [24] using a UV visible spectrophotometer (Varian Cary 50 Bio). e absorption at the wavelengths 232 nm and 270 nm is due to the presence, respectively, of conjugated diene and triene compounds resulting from the oxidation processes. e absorptions at these two wavelengths are expressed as specific extinctions E1% (the extinction of 1% of oil solution in the prescribed solvent in a 1 cm cell) conventionally indicated by K [24].

Chlorophyll and Pheophytin Contents.
e chlorophyll and pheophytin contents (expressed in ppm) were determined by the methods described by Wolff [25] for chlorophyll and by Psomiadou and Tsimidou [26] for α-pheophytin. e fractions of pheophytin and chlorophyll were quantified at the wavelengths 630, 670, and 710 nm using a UV visible spectrophotometer (Varian Cary 50 Bio).

Antioxidant Activity (AA).
e antioxidant activity was measured in terms of radical scavenging ability by the free radical 2,2-diphenyl-1-picrylhydrazyl (DPPH) according to Moktan et al. [27]. Elfalleh et al. advocated that this test is the most confined to successfully determine the antioxidant activity of the conjugated linoleic acid isomers [28]. Briefly, pomegranate seed oil was diluted in n-butanol to concentrations ranging from 0 to 100 mg/mL. One hundred μL of the diluted PSO was added to 3 mL of a solution of DPPH. e mixture was shaken instantly and allowed to stand in the dark at room temperature. After 60 min of reaction, the decrease in absorbance was measured at 517 nm with a UV visible spectrophotometer (Varian Cary 50 Bio). e required amount of antioxidant to reduce the DPPH concentration by 50% was calculated (IC50).

Fatty Acid Composition.
e fatty acid composition was determined according to the analytical methods described in the IOC standard [29]. Briefly, 0.1 mL of a methanolic solution of potassium hydroxide (2 N) was added to the oil solution (0.1 g) purified in 1 mL of n-heptane to prepare the fatty acid methyl esters (FAME). e mixture was shaken vigorously and let to stand until the upper part became clear. e methyl ester mixture was analysed by gas chromatography performed on a Varian CP 3380 with a flame ionization detector equipped with a capillary column (CP-Wax 52 CB: L � 30 m; Φ � 0.25 mm; Ft � 0.20 μm). For the oven, it was applied a temperature programming from 170 to 180°C at 3°C/min and from 180 to 190°C at 1°C/min and then held isothermally for 25 min. e flame ionization detector was maintained at 230°C, while the injector temperature was 220°C. Nitrogen was used as the carrier gas. e fatty acid identification was attained using control fatty acids. For fatty acids quantification, the percentage of each peak (FAx (%)) was calculated as follows: where Ax is the individual peak area of each FAME and AT is the total area of all FAME peaks that appear in the chromatogram from C14: 0 to C22: 0.

Statistical
Analysis. e data obtained were statistically analysed using the SPSS 22.0 statistical database for windows. One-way analysis of variance (ANOVA) for the comparison of means was executed 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).

Physical and Biometric Parameters of Pomegranate Seeds.
e physical and biometric characteristics of pomegranate fruits, arils, and seeds are described in Table 2. e weight means of the fruits and the arils were, respectively, between  Table  2: Mean values of physical and biometric parameters of fruits, arils, and seeds of pomegranate fruits of ten Moroccan cultivars (1,2) .  Journal of Food Quality 246.90 g and 131.30 g for the cultivar "Lhamdha" (lowest) and between 589.57 g and 420.62 g for the cultivar "Sefri 4" (highest). ese results agreed with the fruit weight presented by other Moroccan cultivars (between 430.8 and 535.1 g) [20], Croatian cultivars (between 189.4 and 595.9 g) [30], and Italian cultivars (169-575 g) [31]. Differences in aril and fruit weights are influenced by cultivar and agro-climatic conditions within regions [23]. e seed yield per fruit varied from 11.92% for "Marrakchia" to 18.11% for "Lhamdha." ese results agreed with those reported by previous studies [31,32].
Concerning the parameters of individual arils and seeds, the weight of one aril ranged from 0.227 g for the "Lhamdha" cultivar to 0.479 g for the "Sefri 4" cultivar. e cultivars "Sefri 1," "Sefri 4," and "Sefri 5" had significantly higher aril weight (Aw > 0.46 g) compared to other cultivars. e range found for aril weight agreed with the results reported for Moroccan, Spanish, Turkish, and Italian cultivars ranging from 0.24 to 0.68 g [20,23,31,[33][34][35][36]. e length, width, and weight of seeds were, respectively, within the range of 6.51 mm for "Marrakchia" and 7.82 mm for "Sefri 1," 2.52 mm for "Sefri 6" and 3.00 mm for "Sefri 1," and 0.028 g for "Sefri 6" and 0.043 g for "Sefri 1." e results presented for pomegranate seeds agreed with those reported for Spanish and Moroccan cultivars [20,33,35]. e seed index (Si) which indicated the proportion of the seed compared to the weight of the aril [23] varied from 7.03% for "Sefri 6" to 9.75% for "Sefri 2." However, the cultivar "Lhamdha" showed significantly higher Si (17.10%) compared to other cultivars. e cultivar "Lhamdha" also presented higher Si compared with the results reported for Spanish cultivars (4.75-15.3%) [23,35,36], Italian cultivars (3.7-12.2%) [31], and Moroccan cultivars (6.1-10.7%) [20]. In general, cultivars with the lowest values in Si and Sw and simultaneously high values in Aw can be considered ideal for fresh consumption [21]. e results of seed hardness varied significantly among the ten cultivars. Except for the cultivar "Lhamdha" that had the hardest seeds, all cultivars had tender or semihard seeds and then could be intended for fresh consumption. e oil contents in pomegranate seeds showed a great variability among the studied cultivars. e oil yield ranged from 17.59% to 24.69%. In particular, "Sefri 3," "Sefri 4," "Sefri 6," and "Lahmer" were the cultivars richest in oil content with values higher than 22%, indicating a promising convenience to extract oil from their seeds. ese results were consistent with the results reported by Ferrara et al. [31] for Italian cultivars, 4.9-26.8%. However, they were higher than the data reported for Spanish cultivars, 4.44-13.70% [37], and Iranian cultivars, 6.63-19.3% [38]. It has been reported that the quantity of oil in pomegranate seeds depends upon the geographical location and maturity of cultivated pomegranate fruits [39].

Colour Coordinates.
Colour coordinates of pomegranate seed oil of ten studied Moroccan cultivars are presented in Table 3. e results showed significant differences between the cultivars. Oils revealed a clear yellow e studied oils presented a more intense yellow colour than seed oils extracted from Turkish cultivars (L * � 58.91, a * � −5.64, and b * � 22.60) [40]. e cultivars "Sefri 6" and "Lahmer" showed the most intense and saturated yellow colour with values of chroma (C * ) higher than 100 and hue angle (H * ) inferior (−84°).

Extinction Coefficients.
e specific extinction coefficients K 232 and K 270 characterize primary oxidation (conjugated dienes) and secondary oxidation (conjugated trienes) products, respectively. ey are directly associated with the amount of peroxide in vegetable oils [41]. e determination of these two extinction coefficients can present information on the quality of the oil and its state of preservation [24]. e specific extinction coefficients values K 232 and K 270 for the studied oils are shown in Table 3. e results found for K 232 ranged from 4.06 for "Bzeq Tir" to 5.34 for "Lahmer" and 5.39 for "Sefri 5," while the results for K 270 ranged between 3.45 for "Sefri 6" and 3.75 for "Marrakchia," without showing significant differences among the studied cultivars. ese data are close to those reported for Tunisian cultivars: 4.15 for K 232 and 3.95 for K 270 [41] and Turkish cultivars: 3.83 for K 232 and 4.00 for K 270 [42].

Chlorophyll and Pheophytin Contents.
Chlorophylls are responsible for green colour of oils. In the absence of light, chlorophylls may act as weak antioxidants, while in the presence of light, it is recognized that chlorophylls act as robust oxidation promoters [43]. Acidification and thermal processing cause the conversion of chlorophyll to pheophytin, resulting in noticeable discoloration of oils from green to brown [44]. Chlorophyll and pheophytin contents of pomegranate seed oils of the ten studied pomegranate cultivars are shown in Table 3. Oils contained chlorophyll and pheophytin that ranged, respectively, from 0.12 mg/kg and 0.39 m/kg for cultivar "Lhamdha" to 1.87 mg/kg for cultivar "Sefri 6" and 3.87 mg/kg for cultivar "Lahmer." e results found for chlorophyll were within the range reported by Amri et al. [41] and Alfekaik and Al-Hilfi [45] for Tunisian and Iraqi cultivars, respectively. e level of chlorophylls depends on genetic factors, extraction technology, and degree of fruit ripening. e level declines as the fruit ripens [43].

Antioxidant Activity.
According to the European legislation on food additives, antioxidants are defined as "substances which prolong the shelf life of foods by protecting them against deterioration caused by oxidation, such as fat rancidity and colour changes" [46]. PSO with higher antioxidant activity could then be used as additives in food industry. Table 3 shows the antioxidant activity results obtained for the studied cultivars. PSO presented high antioxidant activity with IC50 that ranged from 0.69 mg/mL for "Marrakchia" to 1.80 mg/mL for "Lhamdha." Antioxidant activity was within the range found by Amri et al. [41]   Journal of Food Quality Journal of Food Quality 7 (IC50 � 0.37 mg/mL) and by Melo et al. [3] (IC50 � 3.77 mg/ mL) for, respectively, Tunisian and Brazilian cultivars. In fact, the antioxidant activity of the PSO was found to be significantly superior to those of green tea, red wine, and the synthetic antioxidant butylated hydroxyl anisole (BHA) [9]. A significant correlation was found between the antioxidant activity of PSO and the total content of tocopherols [3,5] suggesting the contribution of tocopherols to the antioxidant properties of this oil. e divergence in antioxidant activity among pomegranate cultivars may due to the genotypes, growing region, and to different antioxidant activity evaluations employed by other studies such as ABTS and FRAP assays [47].

Fatty Acid Composition.
e profile of seed oil fatty acids among cultivars is important information in assessing seed oil stability against oxidation and food fortification. Cultivars with PSO high in essential fatty acids could be used to improve the accessibility of these fatty acids in other foods [47]. e fatty acid profile of the studied PSO is presented in Table 4. Total saturated fatty acids (SFAs) were in the range of 6.29 to 7.33% of total fatty acids, with the unsaturated/ saturated ratio varying between 12.65 and 14.91. ese results were in agreement with those reported by Fernandes et al. [37] for Spanish cultivars. e most important saturated fatty acids were palmitic acid (C16: 0) and stearic acid (C18: 0) ranging, respectively, from 3.96% to 1.74% for "Sefri 4" to 4.67 % and 2.08 % for "Bzeq Tir." In addition, the extracted pomegranate seed oils presented high contents of unsaturated fatty acids (92.86-93.89%). ese results were in agreement with those reported for Spanish cultivars (from 91.8 to 94.2%) [37]. Monounsaturated fatty acids (MUFAs) ranged between 4.32 and 7.05%. e predominant monounsaturated fatty acid was oleic acid (C18: 1 c 9) which was in the range of 3.87% for "Sefri 1" and 6.53% for "Marrakchia." Polyunsaturated fatty acids (PUFAs) presented the major fatty acid class, ranging between 85.98 and 89.03% of total fatty acids. Linoleic acid (C18: 2n 6c, 9c) was detected with values ranging from 5.29 for "Sefri 1" to 7.70% for "Marrakchia" which agreed with values reported by Verardo et al. [2]. e major polyunsaturated fatty acid was punicic acid (C18: 3 c9, t11, c13) whose content ranged from 73.74% for "Bzeq Tir" to 80.67% for "Sefri 1." In particular, "Sefri 1," "Sefri 4," and "Sefri 5" were the cultivars richest in punicic acid with values higher than 80%. ese results were in agreement with those reported by Costa et al. [6]. However, they were lower than values reported by Fernandes et al. [37], Ferrara et al. [31], and Verardo et al. [2] (72.4-84.1%). Punicic acid is the principal conjugated linolenic acid that endorses the potential biological and health benefits of pomegranate seed oil [48]. ree other conjugated linolenic acid isomers were also identified, α-eleostearic acid, catalpic acid, and β-eleostearic acid, which were, respectively, in the range of 1.66-3.12%, 0.34-1.59%, and 0.06-2.03%. e cultivar "Bzeq Tir" demonstrated the highest content of these three fatty acids. PSO is, therefore, a rich source of the bioactive conjugated linolenic isomers, representing approximately 84% of the oils fatty acids (especially the cultivars "Sefri 1," "Sefri 4," and "Sefri 5"). is result agreed with that of Costa et al. [6]. Lastly, among minor fatty acids, the saturated arachidic acid (C20: 0) and behenic acid (C22: 0) and unsaturated gadoleic acid (C20: 1) and linolenic acid (C18: 3n3c, 6c, 9c) were detected with average values less than 1%. e PSO presented significant differences among the studied cultivars (P < 0.01); its composition could be influenced by the pomegranate variety, climate conditions, and ripening stage at harvest [22,41].

Correlation Analysis.
Pearson's correlation analyses were used to examine the interaction among the studied parameters at P < 0.01 (Table 5).
e oil content of the pomegranate seeds was positively correlated with aril weight, chlorophyll, and pheophytin contents with correlation coefficients of 0.541, 0.586, and 0.664, respectively, while it was negatively correlated with seed index (r � −0.541) and seed hardness (r � −0.472) indicating that tender pomegranate seeds provide high oil content. Highly significant positive correlations were found between chlorophyll and pheophytin contents and the colour coordinates. e coefficients of correlation were, respectively, 0.945 and 0.847 with a * , 0.603 and 0.656 with b * , and 0.592 and 0.648 with C * . is result confirmed that chlorophyll was the main contributor to the PSO colour. e IC50 was positively correlated with seed hardness (r � 0.566), which indicated that oils extracted from tender seeds showed higher antioxidant activity compared to oils extracted from hard seeds. e correlation between the studied variables and environmental conditions showed that favorable environmental conditions help the production of larger edible proportion. Aw was positively correlated with maximum temperature (r � 0.609), altitude (r � 0.607), and longitude west (r � 0.613) while negatively correlated with minimum temperature (r � −0.668) ( Table 5), which explained the presence of heavy aril weight in cultivars cultivated in the Center of Morocco compared with other cultivars. Tapia-Campos et al. [49] recorded the same observation for Mexican cultivars. e altitude favors the production of PSO rich in PUFA, especially punicic acid. A significant correlation was found between altitude, PUFA, and punicic acid (r � 0.598 and r � 0.653 respectively). Minimum temperature is favorable for the production of MUFA (r � 0.832) which explained the high content of MUFA in PSO of cultivars cultivated in the coastal town (Essaouira).

Principal Component Analysis (PCA).
e first two principal components explained 74.14% of the total variation ( Figure 1). e proportion of variation for the first and the second factors was 43.55% and 30.59%, respectively. e first component F1 was positively linked to seed index, seed hardness, antioxidant activity (IC50), and MUFA and negatively linked to oil yield, UFA/SFA, and PUFA. e second component F2 accounted for 30.59% of the variance and was positively correlated to punicic acid. Based on the plot of the principal component scores, the studied cultivars were separated into distinctive groups based on their seed and oil characteristics. e cultivar "Lhamdha" showed a high positive Table  5: Pearson correlation of environmental conditions, morphological, physicochemical, and biochemical characteristics of seeds and pomegranate seed oils of ten Moroccan pomegranate cultivars harvested in 2018. score on F1 and F2. is cultivar was characterized by high seed index and seed hardness and low antioxidant activity (high IC50). e cultivars "Bzeq Tir," "Marrakchia," and "Sefri 6" presented a positive score on F1 and negative score on F2. ey showed important amounts of saturated and monounsaturated fatty acids. e cultivars "Sefri 1," "Sefri 2," "Sefri 4," and "Sefri 5" were relatively close to each other along the x-axis and presented a negative score on F1 and positive score on F2. ey were characterized by their high contents of punicic acid, UFA, and PUFA, with tender or semihard seeds. On the other hand, "Lahmer" and "Sefri 3" showed negative scores on F1 and on F2. ey provided high yields of oil with relatively high antioxidant activity (low IC50).

Cluster Analysis.
e results obtained from cluster analysis for the ten Moroccan pomegranate cultivars are shown in Figure 2. Four principal groups were clustered based on the studied parameters. e first group included "Sefri 1," "Sefri 2," "Sefri 4," and "Sefri 5." e second group consisted only of cultivar "Lhamdha." e third group was formed by the cultivars "Bzeq Tir" and "Marrakchia," while the last group included cultivar "Sefri 6," "Sefri 3," and "Lahmer." Cluster analysis revealed the divergence of cultivars with the same appellation "Sefri." In fact, "Sefri 6" and "Sefri 3" were different from "Sefri 1," "Sefri 2," "Sefri 4," and "Sefri 5." is confirmed the problem of homonymy or synonymy in the Moroccan cultivar appellation reported by Ajal et al. [50] in their study which was based on the assessment of genetic diversity.

Conclusions
is study aimed to assess and compare, for the first time, the characteristics of pomegranate seeds and seed oils of ten cultivars grown in the Center of Morocco. Pomegranate seeds of the studied cultivars presented an important oil yield that ranged from 17.59% to 24.69%. e cultivars "Lahmer" and "Sefri 3" provided the highest oil yield, with results exceeding 24%. Oils showed an intense yellow colour with a significant presence of chlorophyll and pheophytin pigments. e studied oils were rich sources of polyunsaturated fatty acids (85.98-89.03%) with high contents of punicic acid that represented 80% of the total fatty acids. Polyunsaturated fatty acids and punicic acid were highly correlated with altitude. e oils also showed high levels of antioxidant activity (IC50: 0.69-1.80 mg/mL). e cultivar "Marrakchia" was rich in monounsaturated fatty acids, while the cultivars "Sefri 1," "Sefri 4," and "Sefri 5" presented high contents of polyunsaturated fatty acids. ese four cultivars were the most promising, also showing higher antioxidant activities. Based on their fatty acid profile and antioxidant activity, the studied oils could be useful in the formulation of novel foods or used as preservatives and functional components in food, cosmetic, and pharmaceutical industries. Further studies are scheduled for a better characterization and exploitation of this resource, especially the determination of biologically active compounds such as tocochromanols, carotenoids, phytosterols, and phenolic compounds.

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

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