Oil content, lipid profiling and oxidative stability of “Sefri” Moroccan pomegranate (Punica granatum L.) seed oil

Ahmed Hajib, Issmail Nounah, Hicham Harhar, Said Gharby, Badreddine Kartah, Bertrand Matthäus, Khalid Bougrin and Zoubida Charrouf 1 Equipe de Chimie des Plantes et de Synthèse Organique et Bioorganique, URAC23, Faculty of Science, B.P. 1014, Geophysics, Natural Patrimony and Green Chemistry (GEOPAC) Research Center, Mohammed V University, Rabat, Morocco 2 Laboratory of Nanotechnology, Materials and Environment, Department of Chemistry, Faculty of Science, University Mohammed V, Av. Ibn Batouta, BP. 1014, Rabat, Morocco 3 Laboratory Biotechnology, Materials and Environment (LBME), Faculty Polydisciplinary of Taroudant, University Ibn Zohr, Agadir, Morocco 4 Max Rubner-Institut, Federal Research Institute for Nutrition and Food, Working Group for Lipid Research, Schützenberg 12, D-32756 Detmold, Germany


Introduction
Pomegranate (Punica granatum L., Punicaceae) is an ancient, beloved plant and delicious fruit consumed worldwide (Jing et al., 2012). They are widely grown in the Mediterranean regions and India, but sparsely cultivated in the USA, China, Japan and Russia (Fadavi et al., 2006). The edible parts of pomegranate fruits are consumed fresh and they are also used for the preparation of fresh juice (Fadavi et al., 2006). The fruits contain considerable amounts of seeds (40-100 g/kg fruit weight) (Syed et al., 2007), that are usually waste products from the fruit processing. Pomegranate seeds a rich source of lipids, which approximately 12 to 20% of total seeds weight, varies according to cultivars (Ky`ralan et al., 2009). Many studies have indicated the pharmaceutical importance of pomegranate seed oil (PSO). For instance, they have been reported to promote epidermal tissue regeneration, boost the immune system in vivo, reduce the accumulation of hepatic triglycerides and display chemopreventive activity against hormone-related (prostate and breast) and colon cancers (Melo et al., 2014).
Currently, pomegranate plant is produced throughout the world in tropical and sub-tropical areas. The Mediterranean countries, India, Iran and Californian are considered as the main producers. Argentina, Brazil, Peru, Chile and South Africa are the other important producer countries (Kahramanoglu and Usanmaz, 2016). In 2014, the total production of pomegranate in the world was estimated to be around three million tons. Morocco is one of the biggest producers, the cultivation of pomegranate covers an area of about 4000 ha which gives an estimated production of 46 000 T, an average yield of 12 T/ha (Haddioui, 2012).
Many studies indicate a quantitative and qualitative difference in the chemical composition of pomegranate species oil, growing in different regions. To our knowledge, no study has been done to analyse Moroccan pomegranate seed oil. The aim of this study is to determine the lipid profile and evaluate the oxidative stability of the pomegranate seed oil growing in Morocco.

Sample collection
Pomegranate seeds and press oil samples, from "Sefri" Moroccan variety, analysed to determine the chemical composition and oxidative stability, were collected and prepared in 2017 from Flora cooperative (Boujad city, Morocco, 32°46'10"N 6°23'49"W). The annual average temperature in this region was 29°C, while the annual average precipitation recorded between October and April was 1 mm.

Oil content
The oil content was determined according to the method ISO standard (ISO 659, 1998). About 2 g of the seeds were ground in a ball mill and extracted with petroleum ether in a Twisselmann apparatus for 6 h. The solvent was removed by a rotary evaporator at 40°C and 25 Torr. The oil was dried by a stream of nitrogen and stored at À20°C until used.

Fatty acid composition
The fatty acid composition was determined following the ISO standard (ISO 12966-2, 2017). Fatty acids (FAs) were converted into fatty acid methyl esters (FAMEs) by shaking a solution of 60 mg oil and 3 mL of hexane with 0.3 mL of 2 N methanolic potassium hydroxide for 25 min. The fatty acid composition was determined as their corresponding methyl esters by gas chromatography (Varian 5890) coupled with a flame ionization detector (GC-FID). The capillary column CP-Sil 88 (100 m long, 0.25 mm ID, film thickness 0.2 mm) was used. The carrier gas was helium and the total gas flow rate was 1 mL/min. The temperature program was as follows: from 155°C; heated to 220°C (1.5°C/min), 10 min isotherm; injector 250°C, detector 250°C; carrier gas 36 cm/s hydrogen; split ratio 1:50; detector gas 30 mL/min hydrogen; 300 mL/min air and 30 mL/min nitrogen; manual injection volume less than 1 ml. The peak areas were computed by the integration software, and percentages of fatty acid methyl esters (FAME) were obtained as weight percentage by direct internal normalization method.

Tocopherol composition
For determination of tocopherols, a solution of 250 mg of press oil in 25 mL of n-heptane was directly used for the HPLC as descripted in Hajib et al., (2018) work. The HPLC analysis was conducted using a Merck-Hitachi low-pressure gradient system, fitted with a L-6000 pump, a Merck-Hitachi F-1000 fluorescence spectrophotometer (detector wavelengths for excitation 295 nm, for emission 330 nm), and a D-2500 integration system. The samples in the amount of 20 ml were injected by a Merck 655-A40 autosampler onto a Diol phase HPLC column 25 cm Â 4.6 mm ID (Merck, Darmstadt, Germany) used with a flow rate of 1.3 mL/min. The mobile phase used was n-heptane/tert-butyl methyl ether (99:1, v/v).

Sterol composition
The sterol composition of the press oil was determined following the ISO standard (ISO 12228-1, 2014). In brief, 250 mg of press oil was saponified with a solution of ethanolic potassium hydroxide (2 N) by boiling under reflux. The unsaponifiable matter was isolated by solid-phase extraction on an aluminum oxide column (Merck, Darmstadt, Germany) on which fatty acid anions were retained and sterols passed through. The sterol fraction was separated from unsaponifiable matter by thin-layer chromatography (Merck, Darmstadt, Germany), re-extracted from the TLC material, and afterward, the composition of the sterol fraction was determined by GLC using betulin as internal standard. The compounds were separated on a SE 54 CB (Macherey-Nagel, Düren, Germany; 50 m long, 0.32 mm ID, 0.25 mm film thickness). Further parameters were as follows: helium (1 mL/min) as a carrier gas, split ratio 1:20, injection and detection temperature adjusted to 320°C, temperature program, 245°C to 260°C at 5°C/min. Peaks were identified either by standard compounds (b-sitosterol, campesterol, stigmasterol) by a mixture of sterols isolated from rapeseed oil (brassicasterol) or by a mixture of sterols isolated from sunflower oil (D-7-avenasterol, D-7-stigmasterol, and D-7-campesterol). All other sterols were identified by GC-MS for the first time and afterwards by comparison of the retention time.

Oxidative stability
The oxidative stability of the press oil was determined by the Rancimat method, according to Gharby et al., (2012) work. All experiments were carried out with a 743 Rancimat (Methrom AG, Herisau, Switzerland). In brief, 3.6 g press oil were weighed into the reaction vessel, which was placed into the heating block kept at 120°C. Air flow was set at 20 L/h for all determinations. Volatile compounds released during the degradation process were collected in a receiving flask filled with 60 mL of distilled water. The conductivity of this solution was measured and recorded. The resulting curves were evaluated automatically by the software of the Rancimat. All determinations were carried out in triplicate.
Punicic acid was first isolated from pomegranate seed oil by Toyama and Tsuchiya, (1935). This fact was later confirmed by a reinvestigation of the oil by Farmer and Van den Heuvel (1936) and also by other authors (Ahlers et al., 1954). According to Sassano et al., (2009) in nature, CLnAs are not found to any great extent in animal fat, but they are found in many seed oils as either C18 trienes or C18 tetraenes. The most commonly CLnAs known isomers found in seed oils from important plants are a-eleostearic acid (Vernicia fordii), punicic acid (Punica granatum L.), calendic acid (Calendula officinalis L.), jacaric acid (Jacaranda mimosifolia), catalpic acid (Catalpa ovata), b-calendic (Calendula officinalis L.) and a-parinaric acid (Parinarium laurinum) (Sassano et al., 2009;Melo et al., 2014). In general, CLnAs are synthesized from linoleic acid through a specific conjugase enzyme, but they Iran (Fadavi et al., 2006) Oil content 22.63 ± 0.54 13. 95-24.13 5.89-21.58 11.4-14.9 4.44-13.7 6.63-19.3 Values are given as means of three replicates ± SD. may also be produced during the processing of vegetable oils, as a result of isomerization and dehydration of secondary oxidation products of linoleic and a-linolenic acids (Koba et al., 2007b;Hennessy et al., 2011).

Sterol composition
Sterols are very useful parameters for detecting adulterations or to check authenticity, since it can be considered as a fingerprint (Gharby et al., 2017. Besides, their determination is of major interest due to their antioxidant activity and impact on health. Table 3 lists the sterol levels obtained from Moroccan pomegranate seed oil. The total sterol contents of pomegranate seed oil were 494.61 mg/100g, which was found to be in the range of previously published values for pomegranate seed oil from other countries reported in the literature (408.9-620.5 mg/100g) (Kaufman and Wiesman, 2007). In pomegranate seed oil, b-sitosterol was also the most abundant sterol which constituted about 404.59 mg/100g. This sterol is also abundantly found in Table 2. Comparison of fatty acid composition (g/100g) of "Sefri" Moroccan pomegranate seed oil with literature.

Compound
Our results (g/100g oil) (Jing et al.,  Values are given as means of three replicates ± SD. sesame, cactus and olive oil (Gharby et al., 2012(Gharby et al., , 2017. Among the different sterols, b-sitosterol has been most intensively investigated with respect to its beneficial and physiological effects on human health. Besides, b-sitosterol lowers cholesterol levels, enhances immunity, and has anti-inflammatory, antipyretic and anti-carcinogenic effects (prostate essentially) (Gupta et al., 1980;Villaseñor et al., 2002). The next major component was campesterol where it reaches about 38.18 mg/ 100g of the total sterols. D5-avenasterol and stigmasterol accounted for about 17.86 and 15.53 mg/100g respectively in this oil. Minor sterols were also detected (D7-stigmasterol and cholesterol). The sterol content of pomegranate seed oil from Morocco was similar to that from Georgia and Spain, as reported by Pande andAkoh, (2009) andFernandes et al., (2015), respectively.

Tocopherol composition
In addition to the fatty acid composition and sterol profile, the composition of vitamin E active compounds is an important characteristic feature used to describe the identity of vegetable oils. These compounds have some nutritional importance because they are known to have an antioxidative activity, which protects the polyunsaturated fatty acids against oxidative deterioration; additionally, a biological activity exists, which protects cells against oxidative stress (Bieri and Evarts, 1974;Blumberg and Block, 1994;Tucker and Townsend, 2005).
Our tocopherol profile was similar to the tocopherol composition reported by Fernandes et al., (2015), who revealed that the major tocopherol in pomegranate seed oil is g-tocopherol (123.0-449.7 mg/100g). However, this result contrasts with that reported by Pande and Akoh, (2009) study, where the a-tocopherol was the major tocopherol in pomegranate seeds oil (161.2-173.7 mg/100g). In addition, Jing et al., (2012) found that the main tocopherol in pomegranate seed oil is the d-tocopherol (141.42-351.32 mg/100g).

Oxidative stability
The preservation of edible or cosmetic oil is an important economic parameter (Matthäus et al., 2010). In fact, oxidation of lipid is a major cause of deterioration in the quality of oils. It is the cause of important deteriorative changes in their chemical, sensory and nutritional properties (Gray, 1978;Frankel, 1980). The oxidative stability of pomegranate seed oil is expressed as the induction period determined by the Rancimat method at 120°C. The induction time of pomegranate seed oil, as evaluated by the Rancimat accelerated method, was found to be 3.6 ± 0.93 h at 120°C. At the same temperature, the Rancimat induction time is 6.1 and 5.5 h for argan and olive, respectively (Mateos et al., 2006;Matthäus and Brühl, 2015). Therefore, the oxidation sensitivity of pomegranate seed oil, that is much higher than that of argan and olive oils, could be likely attributed to high content of CLnAs; molecules that do oxidize easily (Tab. 5).

Conclusion
The study of pomegranate (Punica granatum L.) seed oil growing in Morocco revealed high oil content, with conjugated linolenic acids (punicic acid, catalpic acid) as the predominant fatty acid, beside considerable amounts of tocopherols and sterols. This study shows also that Moroccan pomegranate seed oil is particularly sentitive to oxidation, which could be explain by the high content of CLnAs. Thus, special care such as refrigeration should be considered for oil prolonged storage. If enough precautions are taken, pomegranate seed oil deserve Table 4. Comparison of tocopherol composition (mg/100g) of "Sefri" Moroccan pomegranate seed oil with literature.

Compounds
Our results (mg/100g oil) (Fernandes et al., 2015 Values are given as means of three replicates ± SD. Table 5. Comparison of oxidative stability (h) of "Sefri" Moroccan pomegranate with argan and olive seed oils.
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