Oil Characterization and Lipids Class Composition of Pomegranate Seeds

This study aims to investigate the physicochemical characteristics, phenolics content, and oil composition of pomegranate oil seeds (PSO). Quality indices, pigments, phenolics content, and antioxidant activity were determined. PSO was fractioned into polar lipids: glycolipids (GL) and phospholipids (PL). Sterols profile and fatty acids composition of total lipids (TL), GL, and PL were determined by GC/FID. The free acidity, the peroxide value, and the specific extinction coefficients were, respectively, 1.69%, 3.42 in milliequivalents of active oxygen per kilogram of oil, 4.15, and 3.95. PSO is rich in phenols (93.42 mg/Kg) but poor in pigments. The sterols markers were β-sitosterol (77.94%), Δ5-avenasterol (7.45%), and campesterol (6.35%). Oil content was 12.2%, wherein 23.9% were GL and 24.35% were PL. TL were rich in unsaturated fatty acids (63.17%), while saturated fatty acids were more present in PL and GL (71.97% and 66.29%, resp.). Conjugated fatty acids were about 13.30%, 2.03%, and 4.91%, respectively, in TL, PL, and GL. The cis/trans ratio of TL, PL, and GL was, respectively, 49.82%, 42.91%, and 27.39%. Monounsaturated fatty acids were more bound in PL, whereas polyunsaturated fatty acids were more bound in GL. PSO is a good source of essential fatty acids, phenolics compounds, phytosterols, and lipid-soluble fractions.


Introduction
Several studies have reported that consumed oils have enormous effects on human physiology, including lipid metabolism, development of chronic disease, and well-being [1]. No oil from a single source has been found to be suitable for all purposes because oils from different sources generally differ in their composition [2]. So interest in new sources of edible oils has recently grown. In this regard, plant seeds are known to be a good source of oils of nutritional, industrial, and pharmaceutical importance. Although conventional edible oils such as soybean, corn, and canola have their own importance, there are more rare and unfamiliar oils having unique characteristics and health-promoting traits. Pomegranate seeds oil (PSO) is such oil. It is considered a powerful health-benefiting agent due to its antioxidative, anticarcinogenic, and antilipidemic properties [3][4][5]. The composition of the fatty acids of PSO has been reported [1, [6][7][8][9], while little is known about the oil constitution, specially its minor compounds such as phenols and polar lipids. In addition, natural fats and oils contain, apart from glycerides, a number of lipophilic materials. Among the most interesting are the glycolipids, phospholipids, sterols, fatsoluble vitamins, and phenols. So the study of PSO for its minor constituents, however, can be useful in order to use both oil and the minor constituents effectively. For example, phenolic compounds have been reported to be present in all vegetable oils as secondary metabolites and they are important for the oxidative stability of the PUFA of these oils [10]. Furthermore, commercial antioxidants such as butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), and tert-butylhydroquinone (TBHQ) [11] were usually added to food in many manufacturers to prevent quality deterioration and to maintain the nutritional value of different food products including oils and products containing oil [12]. In this work, physicochemical properties, phenolic content, pigment content, sterols composition, and fatty acids profile of PSO and its lipid classes have been analyzed. The results 2 BioMed Research International will be important as an indication of the potential economical utility of PSO as a new source of edible oils. Besides, to our knowledge, no study about phenolic content and lipid classes of PSO has been carried out previously.

Plant Materiel.
Fruits sample were collected at full maturity from pomegranate trees of Tounsi variety in governorate of Mahdia, Tunisia, in October 2015. The grains were manually separated from the pulp, carefully washed, and dried in the sun until constant weight. Then, the grains were crushed and sieved to obtain fine powders.

Oil Extraction.
Oil was extracted by the method of Soxhlet as described previously by Nasri and Triki (2004) [13]. About 30 g seeds were extracted with 200 ml of hexane at room temperature for 6 h. The solvent was removed by evaporation at 40 ∘ C and the oil was flushed with nitrogen stream and stored at −20 ∘ C in sealed tubes.  Kiritsakis (1998) [15]. Absorbance was measured at 630, 670, and 710 nm and carbon tetrachloride was used as blank. The calculation of the total chlorophyll content is as follows:

Determination of Quality
where is the absorbance of the oil at the respective wavelength and is the cell thickness (cm).

Beta-Carotene Content.
Beta-carotene was measured according to the method described by   [16] and the content was expressed using the following equation: For O-diphenols content, 100 l of combined fractions was mixed with 1 ml of HCL solution (0.5 N), 1 ml of solution of a mixture of NaNO 2 (10 g) and MaMoO 4 ⋅2H 2 O (10 g) in 100 ml H 2 O and finally 1 ml of NaOH solution (1 N). After 30 min of incubation, the content of O-diphenols was measured at 500 nm and expressed as milligrams of gallic acid equivalents per kg of oil.  [19]. One ml of extract or standard solutions of catechin was mixed with 4 ml of distiller water. Then 0.3 ml of NaNO 2 (5%, w/v) was added. After 5 min, 0.3 ml of AlCl 3 (10% w/v) was added and 2 ml of NaOH solution (1 M) was added after 6 min. Finally, 2.4 ml of distilled water was added to adjust final volume to 10 ml. After vigorous shaking, the absorbance was read at 510 nm. Content of flavonoids was expressed as mg catechin equivalents (CEQ)/g of sample.
2.6. DPPH Free Radical Scavenging Activity. The capacity of PSO to scavenge the free radical 2,2-diphenyl-1picrylhydrazyl (DPPH) was measured according to the method described by Bouaziz et al. (2005) [20]. 0.25 ml of phenolic fraction of PSO was mixed with 0.5 ml of methanolic BioMed Research International 3 solution containing DPPH radicals (6 × 10 −6 M). The mixture was shaken vigorously and incubated for 30 min in the dark at room temperature and absorbance was measured at 517 nm. DPPH scavenging effect was calculated as the percentage of DPPH discoloration using the following equation: where is the absorbance of the solution when the sample extract is added at a particular level, and is the absorbance of the DPPH solution. The extract concentration providing 50% inhibition (IC50) was calculated from the graph of scavenging effect percentage against extract concentration in the solution.

Lipid Class Separation and Fatty Acid Methyl Ester
(FAME) Analysis. Total lipids, glycolipids, and phospholipids from grounded seeds were extracted according to Bligh and Dyer (1959) [22]. For the analysis of glycolipids and phospholipids from seed, the lipids were fractionated on silicic acid columns into neutral lipids, glycolipids, and phospholipids by elution with chloroform, acetone, and methanol, respectively. For total fatty acids, glycolipids fatty acids, and phospholipids fatty acids analysis, the lipid extract was directly transesterified by reaction with 14% boron-trifluoride in methanol at 65 ∘ C for 30 minutes, after which it was reextracted using hexane and subjected to gas chromatography (GC) analysis. FAMEs analysis was carried out according to the European Union Commission modified Regulation EEC 2568/91 (13) on a Hewlett-Packard gas chromatograph (Hewlett-Packard, Palo Alto, CA), fitted with a flame ionization detector and a split-splitless injector, set at 270 ∘ C. The carrier gas was nitrogen (1 mL/min), and elution was performed with a fused silica Agilent DB23 capillary column (60 m length, 0.32 mm inner diameter, and 0.25 m film thickness). Conditions were as follows: injector temperature, 270 ∘ C; flame ionization detector, 280 ∘ C; injector split ratio, 1 : 50; the initial column temperature, 130 ∘ C; step 1, 6.5 ∘ C/min to 170 ∘ C; step 2,

Results and Discussion
3.1. Quality Indexes. The physicochemical properties of pomegranate oil are presented in Table 1. Free acidity is an important quality factor and has been extensively used as a traditional criterion for classifying olive oil in various commercial grades. The free acidity value of PSO is 1.69, significantly lower than the results found by Dadashi et al. (2013) [9] in Iranian varieties (3.78 to 8.36). This acidity is higher than that found in some edible oil such as linseeds oil and sunflower oil indicating that PSO would need refining to make it suitable for edible purposes and suggest that some hydrolytic reactions occur during the extraction [24]. The oxidative state of oils is determined using the peroxide value and specific extinction at 232 and 270 nm, respectively. The peroxide value PV of oil is a valuable index to determine oil quality. If the peroxide value becomes higher than 9 meqO 2 /kg oil, it indicates oxidative corruption in oil [25]. As seen in Table 1, the amount of PV in studied variety is 3.42 ± 0.68 which represents good extraction and maintenance condition. This result indicates that pomegranate seeds oil can be stored for a long times without deterioration, since oils become rancid when the peroxide value ranges from 20 to 40 meqO 2 /Kg oil [26]. Peroxide value of PSO is significantly lower than that of some seeds oils like linseed oil (11.28 meqO 2 /Kg) and sunflower oil (12.87 meqO 2 /Kg) [27].
The specific extinction coefficients at 232 nm and 270 nm are related, respectively, to the degree of primary and secondary oxidation of the oils and thus directly correlate to the amount of peroxide [24,28].
The values of 232 (4.15) and 270 (3.95) are relatively higher than that found in another plant oils such as soybean oil (2.78 and 0.73) [26], sunflower oil (3.83 and 3.65), and olive oil (2.52 and 0.2) [27]. This result confirms that PSO is much oxidized than these oils. Table 1, results show lower content of chlorophylls (0.02) and -carotene (3.17). These results correlate with yellow color of oil.

Pigment Content. As shown in
The level of pigments, however, depends on the stage of fruit ripeness, the extraction process, and storage conditions. Thus, oils extracted from older fruits may contain more carotene pigment or oils from younger fruits contain more chlorophyll pigment [29]. Our fruits are collected in full maturity, which confirms these results.

Total Phenols and Flavonoids.
Phenolic content is primary parameter for vegetables quality evaluation and directly involved in the prevention of oxidation and oil preservation. Seeds oils generally contain polyphenols preventing their oxidation [30]. As shown in Table 1, the amounts of total phenols, O-diphenols, and flavonoids in PSO are 93.42, 30.1, and 59.46, respectively. The content of polyphenols in Tounsi variety is lower than that found by Schubert et al. (1999) [3] (15 mg/100 g) in pomegranate cold pressed seed oil. The content of O-diphenols is lower than that found in comparative study of four Iranian pomegranate varieties where the content of O-diphenols can be attained as 58 mg/g [9]. As reported by different studies, the amount of phenolic compounds in olive oil depends on several factors such as cultivar degree of maturation, climate, oil production, and storage [31,32].

Antioxidant Activity.
The antioxidant activity of the PSO was measured by DPPH test. Table 1 shows that the IC50 is 370 g/ml. Compared to commercial synthetic antioxidant such as BHT (IC50 = 9.12 g/ml), we concluded that PSO has strong antioxidant activity. This high antioxidant activity can be attributed to the phenolic compounds mainly to richness in O-diphenols. Phenolic compounds have been reported to be present in all vegetable oils, which is very important for the oxidative stability of the polyunsaturated fatty acids of these oils. In fact, linear relationship exists between the phenol content and the oxidative stability of the extra virgin olive oil [33] and the O-diphenols family could be identified as the main source of the overall antioxidant activity and sensorial proprieties of extra virgin oil [32,34].

Sterols Composition.
Sterols are an important nonacylglycerol constituents of vegetable oil because they relate to the quality of the oil and are widely used to check genuineness while it can be used to determine adulteration of an olive oil, and it has recently been suggested that it may be used to classify virgin olive oils according to their fruit variety. Table 2 shows the sterols composition in PSO. 11 compounds were postulated for wherein the sterol marker was -sitosterol constituting about 77.94% of the total sterols content. The next major components were Δ 5 -avenasterol (7.45%) and campesterol (6.35%). These are followed by stigmasterol (3.21%). All other sterols are present with amounts lower than 3%. Clinical studies have demonstrated that directly intake of phytosterols as a part of the normal diet, or as a supplement, contributes to the reduction of cholesterol levels and the prevention of many diseases and various type of cancers [32]. Recently, phytosterols have been added to vegetable oils as an example of a successful functional food [35]. Our results are slightly different to that found by previous study [1].
The predominance of linoleic acid was confirmed in almost PSO studies but its amount was lower than that found in our study. For example, Mekni et al. (2014) [6] reported a level of 7 to 5% of linoleic acid in three Tunisian pomegranate varieties.
Omega 3 PUFAs were represented by the -linolenic acid (C18:3 w3) accounting for 1.02% and eicosatrienoic acid (C20:3 w3) with 0.11%. Compared to others varieties, the amount of -linolenic acid is the highest. For example, Dadashi et al., 2013, reported a level of 0.1 to 0.4%. Others omega families were found like omega 9 and omega 7 and omega 8 but with very few amounts.
In our study, the MUFA/PUFA ratio was 0.36% which indicates the PUFA richness of PSO so the health-benefiting potential.

Lipids Class and Its Fatty Acids Composition.
The total lipids isolated from PSO were fractioned into neutral lipids and polar lipids (namely, also bound lipids) which represented by glycolipids GL and phospholipids PL. Then PL and GL fractions were taken for fatty acid composition study. The results were illustrated in Table 3. Typical chromatogram of studied samples was illustrated in Figure 1. Compared to total lipids, we found that polar lipids were richer in SFA. The amounts of SFA in PL and GL were, respectively, 71.97% and 66.29% wherein palmitic acid and stearic acid were the major SFAs which together comprised more than 91% of total identified SFA. So, it concludes that SFAs were more bound in nature and it might be complexed chemically or physically with carbohydrates or proteins. Besides, it was reported that the ability to stabilize lipids was also affected by the chain length and degree of UFAs. In fact, PL with longer chain length and PL containing more SFA were the most effective antioxidants [42]. Others SFAs like C12:0, C22:0, and C20:0 were detected but in lesser contents. For example, GL contained more amount of C12:0 and C22:0, while PL were richer in C20:0. The ratio of USFA to SFA was lower in polar lipids than in total lipids. Its values were 1.82, 0.39, and 0.45, respectively, in TL, PL, and GL. It was reported that a high ratio of USFA/SFA is regarded favorable for the reduction of serum CT and atherosclerosis and prevention of heart diseases [43].
USFA amounts in GL and PL did not differ significantly from each other. In fact, GL resemble PL in their contents of MUFA in which oleic acid (C18:1 w9 (cis)) was the major MUFA accounting for 8.88% and 7.74%, respectively, in PL and GL fractions.
Concerning PUFA, the amount is slightly higher in GL than in PL. Linoleic acid, the main PUFA, was found to be in similar amount in the two lipid classes, similar to linolenic acid, the next major PUFA. Compared to TL, the ratio of MUFA to PUFA was much higher in polar lipids.
The corresponding values for this ratio in PL and GL were, respectively, 0.73% and 0.57%. This indicates that MUFA were more bound in PL than in GL. Conjugated PUFA amount in GL were found to be twofold of that in PL and they were represented mainly by punicic acid and -eleostearic acid.

Conclusion
In conclusion of this investigation, it is clear that pomegranate seeds give a considerable yield of oil and the oil seems to be a good source of essential fatty acids, phenolics compounds, and phytosterols. Furthermore, the high percentage of PUFA, sterols, and the considerable amount of phenols make it desirable in terms of nutrition and new nonconventional supply for edible purposes and pharmaceutical industries. This work could also serve for developing quality characteristics of PSO.

Disclosure
This work was presented in "5`e me Congrès de l' Association Tunisienne de Physiologie & de Bio-Surveillance de l'Environnement."

Conflicts of Interest
The authors declare that there are no conflicts of interest regarding the publication of this article.