Analysis of polar lipid fraction of Pinus halepensis Mill. seeds from North Algeria

Industrial Crops and Products 51 (2013) 116–122

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Analysis of polar lipid fraction of Pinus halepensis Mill. seeds from North Algeria Nabil Kadri a,b,∗ , Bachra Khettal b , Rachida Yahiaoui-zaidi b , Veronique Barragan-Montero a , Jean-Louis Montero a a University of Montpellier II, Institute of Biomolecules Max Mousseron UMR 4257 - Glycochemistry and Molecular Recognition, 34296 Montpellier Cedex 5, France b University of Abderrahmane MIRA, Faculty of Natural Sciences and Life, Laboratory of Plant Biotechnologie and Ethnobotany, 06000 Bejaia, Algeria

a r t i c l e

i n f o

Article history: Received 15 May 2013 Received in revised form 21 August 2013 Accepted 23 August 2013 Keywords: Pinus halepensis Mill LC/MS Glycolipids Phospholipids Fatty acids

a b s t r a c t Lipid fraction from Pinus halepensis Mill. seeds was extacted and separated by column chromatography (CC). The seeds were found to be rich in lipids (35.89% of the crude seed). Different classes of glycolipids (GLs) and phospholipids (PLs) were then separated and identified by liquid chromatography-mass spectrometry (LC/MS). A relatively high level of GLs was found compared to the PLs content. Four classes of glycolipids were detected: Esterified steryl glucosides (ESG), monogalactosyldiacylglycerols (MGDG), cerebrosides (Cer), and digalactosyldiacylglycerols (DGDG). Six classes of phospholipids were also identified: Phosphatidic acid (PA), phosphatidyl ethanolamine (PE), phosphatidyl inositol (PI), phosphatidyl serine (PS), phosphatidyl glycerol (PG) and diphosphatidyl glycerol (DPG). The method of quantitative determination of the sugars that make up the classes of glycolipids is described. The maximum ratio of sugars was observed in digalactosyldiacylglycerols (DGDG). The main unsaturated fatty acids for both lipids classes are oleic acid and linoleic acid, while the main saturated fatty acids are arachidic, palmatic and stearic acids in various proportions. © 2013 Elsevier B.V. All rights reserved.

1. Introduction Pinus halepensis Mill., a species belonging to the Pineaceae family, is naturally distributed in the Mediterranean region where it covers more than 25.000km2 and dominates forest formations in semi-arid and arid regions. It is also invading some areas of the southern hemisphere. Pinus halepensis Mill. has been widely planted in the western Mediterranean basin during this century, but in North Africa it predominates, especially in Algeria and Tunisia. It comprises one of the most important crops because it is a pioneer species resistant against drought (Maestre et al., 2003; Nahal, 1986). This species is often used in cosmetics for its thorns, rich in aromatic essential oils. However, the seeds of this species are also used in traditional medicine and in many kitchens where they adorn the traditional breads, salads, rice and fish. In fact, they are very high in fat 34.63-48.12% and have many virtues, both therapeutic and culinary (Nasri et al., 2005). Nevertheless, a study of lipids components of these oilseeds is necessary for the use of these oils effectively. Oils and natural fats, known for their important composition of neutral lipids, contain

∗ Corresponding author. Tél.: +33 4 67 14 43 43; fax: +33 4 67 14 43 43. E-mail addresses: [email protected], [email protected] (N. Kadri). 0926-6690/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.indcrop.2013.08.071

also a number of chemically diversified lipophilic materials. Among the most important; polar lipids, including glycolipids (GLs) and phospholipids (PLs) (Ramadan et al., 2006). The different families known of glycolipids and phospholipids of plant species were esterified steryl glycoside (ESG), monogalactosyldiacylglycérol (MGDG), steryl glucoside (SG), sulfoquinovosyldiacylglycerol (SQDG), cerebroside (Cer) and digalactosyldiacylglycerol (DGDG), for the glycolipids and phosphatidyl inositol (PI), phosphatidyl serine (PS), phasphatidyl ethanolamine (PE), phosphatidyl glycerol (PG), phosphatidyl choline (PC), diphosphatidyls glycerol (DPG) and phasphatidic acid (PA), for the phospholipids with the amount of each varying from one species to another (Ramadan et al., 2006; Pacetti et al., 2007; Changhu et al., 2002; Rosário et al., 2008; Carrier et al., 2000; Narvaez-Rivas et al., 2011). Edible plant polar lipids are thought to be nutrients in the human diet, but little is known about their intestinal digestion, absorption in mammals and others biological activities (Andersson et al., 1995). The aim of the present work is the isolation of polar lipids group of Algerian Pinus halepensis Mill. seeds and to make an effort to identify and characterize these compounds and their chemical constituents as a starting point for the development of new applications in the cosmetic, nutraceutical and pharmaceutical industry. High performance liquid chromatography (HPLC) coupled online with ion-trap mass spectrometry (MS) was used

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because it considered as a very useful approach to increase the specificity of the GLs and PLs analysis. For the development of this method, the identification was comforted by GC analysis (flame ionization detection, FID) of the fatty acids of the total lipids, neutral lipids and polar lipids group. 2. Material and methods 2.1. Plant materials The seeds of Aleppo pine (Pinus halepensis Mill.) were obtained from Djurdjura National Park located in Tikjda, Bouira province of northern Algeria. They were cleaned; dried in an oven at 40 ◦ C for 2 days, then finely crushed using an electric grinder (KIKA Labortechnik M20) until it became a fine powder which was stored at 4◦ C for lipid extraction. 2.2. Methods 2.2.1. Extraction of total lipid (TL) Total lipids, which comprised the finely crushed seeds, were extracted with a soxlhet using solvent mixture of chloroform/methanol (2:1, v/v). A volume of 0.2 ml of an aqueous solution of sodium chloride (0.75%) was added. The whole was carefully mixed without magnetic stirring. The phases were separated and the chloroform layer was retrieved. The lipid extracts were collected in a flask and then treated with sodium sulphate to remove traces of water. After filtration, the extract was taken to dryness on a rotary evaporator at 40 ◦ C (Ramadan and Moersel, 2003). 2.2.2. Isolation of neutral lipids, glycolipids and phospholipids by Column chromatography (CC) Lipid extract was loaded on a silica gel column (1.5 × 30 cm) containing silica gel 60 (0.035–0.070 mm) (1/30). Neutral lipids, glycolipids and phospholipids were successively eluted with chloroform (60ml), acetone/methanol (9:1 v/v, 90ml) and methanol (60ml) for four times. Each elution fraction was collected separately and then weighed after solvent evaporation. Lipids were detected by anthrone reagent for glycolipids and molybdenum blue reagent for phospholipids (Liengprayoon et al., 2011). 2.2.3. Analysis of glycolipids and phospholipids of Pinus halepensis Mill. seeds by LC/MS 2.2.3.1. LC separation of glycolipids classes. Glycolipid fraction obtained after CC were dissolved in CH3 OH-H2 O (95:5, v/v), molecular species were separated using a reversed-phase HPLC column (Hypersil C18, 3 ␮m, 50 x 2.1 mm thermo). The High Performance liquid chromatography (HPLC) was performed on an Alliance 2790 (Waters). Liquid system with a UV-VIS variable detector, wavelength (205 nm) and coupled to Waters Micromass Q-TOF spectrometer combined with ESI probe. The elution system for glycolipids was CH3 OH-H2 O (95:5, v/v) with a flow rate of 3.0 ml/min (Changhu et al., 2002). 2.2.3.2. LC separation of phospholipids classes. The phospholipid fraction was analysed by HPLC, the systeme consisted of an Agilent HP 1100 liquid chromatograph, composed with the following units: a solvent delivery module, an autosampler with variable injection volume (0-100 ␮L), a controller module, a column oven and Waters Micromass Q-TOF spectrometer combined with ESI probe. The analysis was performed on Hypersil silica column 10.0 cm x 0.46 cm I.D., 5 ␮m particle size. The temperature of the column was held at 25 ◦ C. A gradient elution was carried out using different ratios of solutions A (chloroform: methanol: ammonia solution, 80:19.5:0.5, v/v/v) and B (chloroform: methanol: triethylamine: water, 69.53:25.58:0.49:4.40, v/v/v/v). A better separation

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was obtained using the following gradient: from 0 to 5 min, B was increased from 0% to 40%; from 5 to 7 min, B was kept constant at 40%; from 7 to 13 min, B was increased from 40% to 100%; from 13 to 20 min, B was kept constant at 100%; from 20 to 30 min B, was decreased from 100% to 0%; a time post-run of 5 min was done to equilibrate the column before the next injection. The flow rate was supported at 1 mL.min-1 during 30 min and the injection volume was 50 ␮L. The evaporative light scattering detector used nitrogen as nebulising gas (Narvaez-Rivas et al., 2011). Each fraction of glycolipids and phospholipids classes was identified as an HPLC peak. The mass spectrometer was operated in positive ESI mode (capillary voltage 3000 V, cone voltage 20 V, source temperature 150 ◦ C, desolvation temperature 250 ◦ C, desolvation nitrogen flow 260 l/h cone nitrogen flow 30 l/h. The complete analysis of the mass spectrum was acquired in the range m/z 01500. 2.2.4. Separation and quantification of glycolipids classes by column chromatography (CC) Different classes of glycolipids were separated by column chromatography on silica gel 60 A (0.035-0.070mm) using their eluent properties. Each fraction was purified four times. MGDG, DGDG, ESG and Cer were purified by column chromatography with additional elution solvents, CHCl3 -CH3 OH-CH3 COCH3 -HAc (73:1.5:25:0.5, v/v/v/v), CH3 COCH3 -C6 H6 -H2 O (91:30:4, v/v/v), CHCl3 -CH3 OH (50:50,v/v), CHCl3 -CH3 COH3 (50:50,v/v) respectively and were identified by TLC and Fourier transform infrared (FTIR) compared with the literature and quantified gravimetrically. The glycolipids fractions were detected by the anthron reagent (Changhu et al., 2002). 2.2.5. Enzymatic hydrolysis of glycolipids fractions Glycolipids fractions collected were dispersed by probe ultrasounication in 0.5 ml buffer (50 mM KH2 PO4 , 0.5% of Triton X-100, pH ajusted to 7.2 with KOH) and incubated for 60 min at 37◦ C with 40.000 U of lipase Type II Porcine pencrea from Aldrich), After isopropanol (4 ml) was added, the mixture taken to dryness (rotary evaporator) and redissolved in a small volume of chloroform/methanol (1:1 v/v). The hydrolysis products were separated by TLC in chloroform/methanol/ammonia (25% w/v) (65:35:5 v/v) and zones containing glycosidic compounds were recovered for analysis (Diehl et al., 1995). 2.2.6. Determination of sugar content of glycolipids fractions after hydrolysis The determination of the sugar content was determined by the phenol-sulfuric acid method. It is based on the absorption at 490 nm of a colored complex formed between the aromatic phenol and carbohydrates. The amount of sugars was determined by comparison with a standard curve using a spectrophotometer (Dubois et al., 1956). 2.2.7. Gas chromatography analysis of fatty acid methyl ester The fatty acids of neutral lipids (NL), total glycolipids (TGL), total phospholipids (TPL) and glycolipids classes (GL) were converted to methyl esters (FAME) by heating in 10% BF3 -methanol, according to the procedure reported by Metcalfe et al., 1966. FAME were identified on a Shimadzu GC-14A equipped with FID and C-R4AX chromatopac integrator (Kyoto, Japan). The carrier gas (helium) had a flow rate of 20 ml/min, split 1:40. A sample of 1 ml was injected on a 30 m × 0.25 mm × 0.2 ␮m film thickness Supelco SPTM -2380 (Bellefonte, PA, USA) capillary column. The injector and FID temperatures were set at 250 ◦ C. The initial column temperature was 100 ◦ C, programmed by 5◦ C/min until 175 ◦ C and kept for 10 min at 175 ◦ C, then 8◦ C/min until 220 ◦ C and kept for 10 min at 220 ◦ C. A

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Table 1 Total lipids classes content, neutral lipids, glycolipids and phospholipids extracted from Pinus halepensis Mill. seeds. Fractions

g/100g

Rf

Total lipids Neutral lipids Glycolipids Phospholipids

35.89a ± 0.198 83.87b ± 0.777 02.68b ± 0.0169 01.78b ± 0.065

/ 0.75 0.4 0.22

a: g/100g of seeds. b: g/100g of total lipids.

comparision of the retention times of the samples with those of coinjected authentic standards was made to facilitate identification (Ramadan and Moersel, 2003). 3. Results and discussion 3.1. General Nutritional and therapeutic properties of Aleppo pine seeds (Pinus halepensis Mill.) are known. They are used by the Algerian population in food and especially in traditional medicine. Its large geographical distribution around the Mediterranean basin, and particullary its high oil content that reaches the 36% of the crude seed, can constitute a major asset for the food and pharmaceutical industries. 3.2. Levels of total lipids extract and its classes A chromatographic procedure on Silica gel was used to obtain major lipids classes of Pinus halepensis Mill. seed oils. The amounts of total lipids and its classes presented in Aleppo pine seed oil as well as Rf values of these classes are shown in Table 1. Total lipids (TL) amount to 35.89%. Among the TL present in the seeds, the level of neutral lipids (NL) is highest (83.87% of TL), followed by glycolipids (GL) (2.68% of TL) and phospholipids (PL) (1.78% of TL), respectively. The amounts of polar lipids are lower than those of neutral lipids. These results are similar to those reported for oleaginous plant fruits as Olea europea (Ranalli et al., 2002), Hippophae rhamnoide (Yang and Kallio, 2002) and Vibrinum opulus (Karimova et al., 2004) and seeds as Guizotia abyssinica (Ramadan and Moersel, 2003), Bauhinia purpurea L. (Ramadan et al., 2006), Cuminum cyminum (Shahinaz et al., 2004) and Pistacia vera (Chahed et al., 2008).

Fig. 1. LC Chromatogram obtained for glycolipids classes (GL) of Aleppo pine (Pinus halepensis Mill.). Peaks: 1, ESG (Rt = 1.47 min); 2, MGDG (Rt = 3.59 min); 3, Cer (5.42min); 4, DGDG (Rt = 7.36-7.74 min).

(6.39min), PS (6.87 min), PG (7.32min) and DPG (8.16min) according to their degree of polarity. Therfore, we can observe in these figures a good separation of different glycolipids and phospholipids classes obtained in relation to the choice of mobile phases used. Although, these methods allow obtaining excellent separation in less than 20 min, since the peak widths are lower than that indicated in the references (Avalli and Contarini, 2005; Caboni et al., 1994), where 1 to 2 columns of 250 mm were used. 3.3.2. ESI-MS Identification The pseudomolecular mass peaks of different classes of glycolipids and phospholipids were detected using positive ions full-scan ESI-MS. The masses detected are listed in (Tables 2 and 3). Mass spectra were acquired by chromatograms peaks obtained from positive ions reconstructed in time provided for the elution of each molecular species. To achieve the characterization of each class of glycolipids, fragments of positive ions formed after collisionactivated dissociation (CAD) were studied. Fragmentations were compared with those obtained from known standards and data in the previous literature. The second column of the table shows the possible molecular species corresponding to the ions masses with the fatty acid and the sterol composition of the total glycolipids and phospholipids fractions of P. Halepensis Mill. seeds mentioned above.

3.3. LC/MS analysis of glycolipids and phospholipids classes of Pinus halpensis Mill. seeds 3.3.1. LC Separation The glycolipids and phospholipids classes of P. Halepensis Mill. were separated by HPLC and identified by ESI-MS coupled to the latter. The chromatograms shown (Fig. 1) are assumed to correspond to four common peaks, ESG (1.47 min), MGDG (3.59 min), Cer (5.42min) and DGDG (7.36-7.74 min). Regarding the polarity of eluting solvents which were used, it should be noted that ESG is more polar than MGDG, Cer and DGDG in most solvents tested and were separated as a function of the length and the degree of unsaturation of fatty acid portion (FA). Thus, the retention times of unsaturated ESG are shorter than its saturated counterparts. At a relatively high level of total glycolipids, these classes were found in all seed oils studied. However, we observed a lack of sulfoquinovosyldiacylglycerol (SQD) class which was highlighted by Ramadan and Moersel, 2003 in seeds of Nigella sativa L., Coriandrum sativum and Guizotia abyssinica Cass.). Regarding chromatograms shown in (Fig. 2), they are assumed to correspond to six common peaks PA (4.58 min), PE (5.74 min), PI

Fig. 2. LC Chromatogram obtained for phospholipids classes (PL) of Aleppo pine (Pinus halepensis Mill.). Peaks: 1, PA (Rt = 4.58 min); 2, PE (Rt = 5.74 min); 3, PI (Rt = 6.39 min); 4, PS (Rt = 6.87 min); 5, PG (Rt = 7.32 min); 6, DPG (Rt = 8.16 min).

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Table 2 Phospholipid seed classes of Pinus halepensis Mill. identified from molecular species detected by ESI-MS of each family. The assignment of the structure corresponding to each detected ion was obtained by the mass of fatty acids in the total phospholipid fraction. Compounds

(M+H)+

(M-2H)+

Phosphatidic Acid (PA) C4 H10 O6 P C20 H41 O8 P (16:0) C20 H39 O8 P (16:1) C38 H69 O10 P (C16:1/C18:2),(C16:2/C18:1) Phosphatidyl Ethanolamine (PE) C22 H46 NO8 P+ (C16:0) C22 H46 NO8 P+ (C16:0) C40 H79 NO10 P+ (C16:1/C18:0) (C16:0/C18:1)

(M+Na+H)+

(M+NH4 )+

(M+K)+

203 463 739 501 522 765 163 445 446 365 366 387 620 642 289 527 528

Phosphatidyl Glycerol (PG) C3 H8 O3 C3 H8 O6 P C23 H46 O10 P (C16:0) C23 H46 O10 P (C16:0) Unknown Unknown Unknown Diphosphatidyl Glycerol (DPG) C6 H12 O9 P2 C8 H15 O10 P2 C8 H12 O10 P2 C8 H18 O10 P2 C10 H22 O10 P2 C10 H23 O10 P2 C45 H82 O15 P2 (C16:1/C18:0),(C16:0/C18:1) C45 H82 O15 P2 (C16:1/C18:0),(C16:0/C18:1) C45 H87 O15 P2 (C16:0/C18:0),(C16:0/C18:0) C45 H87 O15 P2 (C16:1/C18:0),(C16:0/C18:1)

(M+2Na)+

462

Phosphatidyl Inositol (PI) C5 H9 O6 C20 H38 O7 P (C16:1) C20 H40 O7 P (C16:0) C9 H16 O12 P C9 H17 O12 P C9 H17 O12 P C25 H47 O14 P (C16:1) C25 H48 O14 P (C16:0) Phosphatidyl Serine (PS) C7 H14 NO8 P C23 H45 NO10 P (C16:0) C23 H46 NO10 P (C16:0)

(M+Na)+

115 217 536 537 538 539 540

313 331

3.3.2.1. Phospholipid molecular species. Phospholipid is an amphiphilic lipid constituted by a “head” polar (hydrophilic) and two aliphatic tails (hydrophobic). Most phospholipids are phosphoglycerides whose head is organized around a glycerol3-phosphate residue esterified with a polar molecule, both tails representing aliphatic chains of two fatty acids. Other phospholipids are sphingomyelin, which are structurally derived from sphingosine and not glycerol. Sphingosine constitutes one of the two aliphatic tails. The first phospholipids isolated from living tissue were identified in 1847 by French chemist Nicolas Theodore Gobley from egg yolk lecithin; it was specifically phosphatidylcholines (Gobley, 1850). A tentative identification of phospholipids classes of Pinus halepensis Mill. seeds was conducted. The results obtained were given in Fig. 2 and Table 2. 3.3.2.1.1. Phosphatidic acid (PA). The targeted analysis of molecules contained in the phospholipid fraction of Aleppo pine lipid extract has revealed some number of phosphatidic acid ions (PA). This molecular species was detected with its adduct products which are sodium ([M+Na]+ ), ([M+Na+H]+ ) and ammonium ([M+NH4)]+ ). Four ions of phosphatidic acid were detected in this sample which correspond to the basic structure of phosphatidic acid without fatty acids [C4 H10 O6 P] at m/z 203 and a single fatty

353 359 381 382 947 948 951 952

acid fragment (Sn-1) [C20 H41 O8 P] (C16:0) at m/z 462, [C20 H39 O8 P] m/z 463, [C38 H69 O10 P] (C16:1/C18:1) m/z 739 (Table 2). The fragments detected were derived from the loss of different groups, such as (a) the loss of two fatty acid fragments (C:16 and C: 18) on both positions Sn-1 and Sn-2 and addition of molecular ion of ammonium (NH4 + ) leading to the formation of m/z 203 [M+NH4 (C16/C18)]+ and (b) fatty acid fragments (C:18) in a single position Sn-1 with the addition of sodium leading to the formation of m/z 462 and 463 [M+Na-(C18)]+ . The presence of the whole molecule of PA with a combination of unsaturated fatty acids (C16:1/C18:2) or (C16:2/C18:1) on the positions Sn-1 and Sn-2 besides, the addition of sodium as molecular ion leads to the formation of m/z 739. 3.3.2.1.2. Phosphatidyl ethanolamine (PE). Under a positive ionization mode, this class showed more fragmentation than the previous phospholipid class. The addition of potassium molecular ion ([M+K)] + ) was observed as major ion, ammonium ([M+NH4)]+ ) and hydrogen ([M+H]+ ) were also detected. Fragments m/z 522 and 501 corresponds to two molecules of PE [C22 H46 NO8 P+ ] with a single fragment of fatty acid (C16:0) in Sn-1 position and loss of an other in Sn-2 position with addition of molecular ions of potassium and ammonium respectively [M+K-(C16/C18)]+ and [M+NH4 -(C16/C18)].+ The m/z 765 corresponds to the whole

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Table 3 Glycolipid seed classes of Pinus halepensis Mill. identified from molecular species detected by ESI-MS of each family. The assignment of the structure corresponding to each detected ion was obtained by the mass of fatty acids and sterols present in the total glycolipid fraction total. Compounds

(M+H)+

Esterified Steryl Glycosides (ESG) [b-Sitosteryl glucoside/C18:3], [Stigmasteryl glucoside/C18:2], [Avenasteryl glucoside/C18:2] [b-Sitosteryl glucoside/C18:3], [Stigmasteryl glucoside/C18:2], [Avenasteryl glucoside/C18:2] [b-Sitosteryl glucoside/C18:2], [Stigmasteryl glucoside/C18:1], [Avenasteryl glucoside/C18:1] [b-Sitosteryl glucoside/FuFA] Monogalactoside Diacyl Glycerol (MGDG) [C16:0/C 18:2] [C16:0/C18: 0] [C16:0/18:0] [C18:2/C18:2], [C18:1/C18:3] [C18:1/18:2], [C18:0/C18:3] [FuFA/C16:1], [FuFA/C18:2]

(M+Na)+

(M+NH4 )+

(M+Na+H)+

(M+NH4 +H)+

(M+NH4 +2H)+

859 860 862 882 756 783 784 799 803 829

Cerebroside (Cer) [C18:2/C16:0] [C18:2/C16:0] [C18:2/C16:0] Unknown Digalactoside Diacyl Glycerol (DGDG) [C16:0/C18:0] [C16:0/C18:2], [C16:1/C18:1]

molecule [C40 H79 NO10 P+ ] in the presence of two fatty acid fragments in position Sn-1 and Sn-2 (C16:1/C18:0) (C16:0/C18:1) with addition of hydrogen as molecular ion (Table 2). 3.3.2.1.3. Phosphatidyl inositol (PI). The mass spectra of the phsphatidyl inositol (PI) molecular species present in the lipid fraction of P. Halepensis Mill. seeds grown in North of Algeria under positive ionization mode are complexed; this is probably due to the presence of substituted mixture of phosphatidyl inositol. There are four fragmentation pathways for this molecuar species under this ionization type with ([M-2H]+ ), sodium ([M+Na]+ ), ([M+Na+H]+ ), ammonium ([M+NH4)]+ ) and potassium ([M+K)]+ ) as adduct ions. 50% of ions that were detected for this species are derived from the loss of the fatty acids in position Sn-1 and Sn-2 [M-(C16/C18)], [C5 H9 O6 ], m/z 199[M-2H-(C4 H8 O7 P)]+ , m/z 365 [C9 H16 O12 P], m/z 387 [C9 H17 O12 P], m/z 366 [C9 H17 O12 P] and the other half resulting from the combination with a single fatty acid fragment in Sn-1 position and the loss of another fragment in Sn-2 position, at m/z 642 [C25 H48 O14 P] (C16:0), m/z 620, [C25 H47 O14 P] (C16:1), m/z 446 [C20 H40 O7 P] (C16:0) et m/z 445 [C20 H38 O7 P] (C16:1) (Table 2). 3.3.2.1.4. Phosphatidyl serine (PS). The fragmentation pathway of phosphatidyl serine under positive ionization mode differs from other phospholipids classes. The main product ions result from the loss of fatty acids in one position Sn-1 or in dual position Sn1and Sn-2. They are mainly produced with the molecular adduct of hydrogen ion ([M+H]+ ) and ammonium ([M+NH4)] + ). The ion fragment m/z 289 is derived from the [C7 H14 NO8 P] group which composed the PS [M+NH4 -(C16/C18)] + . The peaks at m/z 527 derived from C23 H45 NO10 P and C23 H46 NO10 P of the PS in combination to a single fatty acid (C16: 0) on one position Sn-1 (Table 2). 3.3.2.1.5. Phosphatidyl glycérol (PG). The majority of ion fragments in the positive ionization mode includes sodium ions ([M+Na]+ ), ([M+2Na]+ ) and ([M+Na+H]+ ). The base peaks of m/z 115 and m/z 217 which represent C3 H8 O and C3 H8 O6 P, respectively, belong to skeleton of phosphatidyl glycerol. These latter derive from the loss of important fragments [M+Na-(C4 H7 O6 P)]+ and [M+2Na-(C4 H7 O3 )]+ combined with fatty acids in the Sn-1 and Sn-2 position (C16/C18). The fragments of ions m/z 536 and m/z 537 derived from glycerol with one fatty acid C23 H46 O10 P (C16:0) on position Sn-1 and the loss of (C16/C18) on position Sn-2 with addition of molecular ions ([M+Na]+ ) and ([M+Na+H]+ ) respectively.

731 732 714 763 938 939

These ions can specifically inform us about the presence of the glycerol group, and therefore the class of phospholipids. (Table 2).The m/z 538, m/z 539 and m/z 540 which belong to this class have not been identified. 3.3.2.1.6. Diphosphatidyl glycerol (DPG). ESI-MS spectra in positive ionization mode of diphosphatidyl glycerol of P. Halepensis Mill. seeds showed group of ion fragments. These groups containing ions that are derived from the basic skeleton of DPG having lost important fragments combined with fatty acids, namely, m/z 313, 326, 331, 353, 359, 381 and 382 deriving respectively from [C6 H12 O9 P2 ], [C8 H15 O10 P2 ], [C7 H13 O9 P2 ], [C8 H12 O10 P2 ], [C8 H18 O10 P2 ], [C10 H22 O10 P2 ] and [C10 H23 O10 P2 ] and ions which derived from the loss of small fragments of skeleton DPG but in the probable presence of fatty acids combination on dual position Sn-1 and Sn-2, m/z 947, 948, 951 and 952, deriving respectively from [C45 H82 O15 P2 ](C16:1/C18:0), (C16:0/C18:1), [C45 H82 O15 P2 ] (C16:1/C18:0), (C16:0/C18:1), [C45 H87 O15 P2 ] (C16:0/C18:0),(C16:0/C18:0) and [C45 H87 O15 P2] (C16:1/C18:0), (C16:0/C18:1) with addition of sodium molecular ions ([M+Na]+ ), ([M+Na+H]+ ) and ([M-2H]+ ) (Table 2). In conclusion, the qualitative composition of phospholipids classes and their fatty acids are similar to that found in the oil of several plants namely, Kachnar (Bauhinia purpurea L.) (Ramadan et al., 2006) and the fruits of Myrtus communis var. Italica. (Aidi Wannes et al., 2010). 3.3.2.2. Glycolipid molecular species. Glycolipids or saccharolipides result mainly from the esterification or amidation of fatty acids by amino sugars or monosaccharides (Kobata and Takasaki, 1992). Glycolipids can be divided into glyceroglycolipides abundant in bacteria and plants and glycosphingolipids (GSL), major glycolipids in animals. Many studies suggest that glycolipids are intermediate of recognition and cell signaling involved in both tumor processes and in normal development (Hurtley and Service, 2001). 3.3.2.2.1. Esterified steryls glycosides (ESG). ESG molecular species were detected as their adducts, with sodium ([M+Na]+ ) ([M+Na+H]+ ) and ammonium ([M+NH4 +2H]+ ). Four major ions were detected in samples of P. Halepensis Mill. oil seeds, corresponding to b-sitosteryl, Stigmasteryl or glucoside Avenasteryl which was in accordance with the sterols composition of

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glycolipids fractions found in (Table 3). At m/z 859 (C18:3/18:2/18:2), m/z 860 (C18:3/C18:2/C18:2), m/z 862 (C18:2/C18:1/C18:1) and m/z 882 (FUFA) respectively. Generally, sterols exist in plants as free sterols, acylated sterol, acylated steryl glycoside and steryl glycoside (Moreau et al., 2002; Phillips et al., 2002). Sugar often reported in most SG is glucose, although galactose, mannose and gentiobiose were also found. Indeed, a study of some oilseed indicated that the glucose was attached to the ESG and SG fractions (Ramadan and Moersel, 2003). ESG biosynthesis in plants is done by transfer of the phospholipids or galactolipids acyl group to SG by microsomal enzyme (Potocka and Zimowski, 2008). The fatty acid found in ESG of P. Halepensis Mill. seeds is mainly linoleic acid C18: 1, C18: 2, C18: 3. However, Furan fatty acid (FUFA) was also detected (Table 3). Furan fatty acids are omnipresent in living organisms but appear to be synthesized by plants, where they are usually found in trace amounts, and by some microorganisms (Spiteller, 2005), this one was not detected in phospholipids, it could move towards the hypothesis that the ESG biosynthesis in these seeds comes from a transfer of acyl groups from galactolipids since it is found in MGDG. 3.3.2.2.2. Monogalactosyldiacylglycerols (MGDG). The most abundant ion in MGDG family which found in P. Halepensis Mill. seed oils is m/z 783. It was detected with their adduct molecules, sodium ([M+Na] + ), ([M+Na+H] + ) and hydrogen ([M+H]+ ) (Table 3). The tandem mass spectrum with pseudo-molecular ions at m/z 756, 783, 784, 799 and 803 result from the loss of a oleic fragment, linoleic or palmitic acid, respectively, while the fragment at m/z 829 result from the simultaneous loss of a FUFA group (furan fatty acid) or a linoleic group, oleic or palmitic group. In these glycolipids classes, we found a predominance of unsaturated fatty acids, C16:1, C18:1, C18:2 and C18:3 which gives to the seed the beneficial dietary properties because it is known that unsaturated fatty acids can affect the physical properties of the membrane such as fluidity and permeability (Bruckert, 2001). These results are in perfect agreement with the studies conducted on the fatty acids composition of the majority MGDG of plants and seeds (Changhu et al., 2002; Ramadan and Moersel, 2003). Nevertheless, we indicate the presence of a furan fatty acid (FUFA) in small quantity of this glycolipid class which was also characterized in glycolipids of Hevea brasiliensis latex (Liengprayoon et al., 2011). 3.3.2.2.3. Cerebrosides (Cer) and Digalactosyldiacylglycerols (DGDG). The mass spectra of the Cer LC peak were found with their adduct ions ammonium ([M+NH4 ] + ), ([M+NH4 +H] + ) and sodium ([M+Na]+ ) ([M+Na+H]+ ) (Table 3). The linoleic/oleic (18:2/16:0) combination was found as the most abundant ion in

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Table 4 Glycolipids classes content extracted from Pinus halepensis Mill. seeds. Glycolipids classes

glycolipidsa

Rf

Sugar b

MGDG DGDG ESG Cer

76.44 ± 0.359 12.25 ± 0.226 8.75 ± 0.814 2.62 ± 0.226

0.65 0.19 0.75 0.45

14.04 ± 0.545 27.32 ± 0.344 11.24 ± 0.203 5.96 ± 0.149

a: g/100g of total glycolipids. b: mg/g of glycolipids classes.

the P. Halepensis seed oils (m/z 731, 714 and 732). However, m/z 763 had no correspondence in Cer fraction. The combination of fatty acids for the DGDG class was found almost similar to that of Cer (Table 3). The peaks of the mass spectra were detected as their additional products, sodium ([M+Na]+ ) and ammonium ([M+NH4 ] + ), the possible structure of m/z 938 is (C16:0/C18:0) and m/z 939 is (C16:0/C18:2) or (C16:1/C18:1). These combinations are very essential because they confer to the seed pharmacological properties in the blood clotting, nerve cell construction and cardiovascular prevention (Nergiz and Domnez, 2004). This combination being almost identical with the fatty acid composition of the Cer class of Avoca fruit (Persea americana Mill.) (Pacetti et al., 2007) and Nigella seeds, coriander and Niger (Ramadan and Moersel, 2003). 3.4. Separation and quantification of glycolipids classes by Column chromatography (CC) An appropriate combination of chromatographic methods on silica gel was used to separate glycolipids classes of Pinus helapensis Mill. seed oils grown in North of Algeria. The proportions of classes contained in the Aleppo pine seed oils determined by gravimetry, the Rf of these classes and their sugar content were determined after enzymatic hydrolysis of each glycolipid classe (Table 4). GLs contain four classes, namely MGDG with a high rate (76.44% of total glycolipids), followed by DGDG (12.25% of total glycolipids) then ESG (8.75% of total glycolipids) and finally Cer (2.62% of total glycolipids). These results are different from those found in the black cumin seed oils, where the abundance was noted in DGDG fraction with a rate of 55.6% of total glycolipids (Ramadan and Moersel, 2003). But they are consistent with studies conducted by Benning and Ohta, 2004, where they found that MGDG is the major fraction in majority plants. The sugar contents that make up each glycolipid class of Aleppo pine seeds are important enough, they are mostly abundant in the DGDG (27.32% of DGDG total fraction) followed by MGDG (14.04% of MGDG total fraction) then ESG (11.24%

Table 5 Fatty acid composition of different glycerolipids classes extracted from Pinus halepensis Mill. seeds. Fatty acids

NL

TGL

TPL

MGDG

DGDG

ESG

Cer

C 12:0 C14:0 C16:0 C18:0 C18:1n9c+18:1n7c C18:2n-6c C18:3n-6c C 20:0 C20:3n-6 C22:2n-6  SFA  UNFA  SFA/UNFA

ND 0.14 ± 0.01 0.1 ± 0.2 6.43 ± 0.2 48.41 ± 1.00 31.06 ± 0.8 ND 6.78 ± 2.3 6.48 ± 0.6 0.39 ± 1.3 13.45 ± 0.6 86.34 ± 1.2 0.15 ± 0.9

2.49 ± 0.1 0.64 ± 0.8 8.32 ± 0.38 9.10 ± 0.14 33.94 ± 0.39 29.09 ± 0.71 ND 10.56 ± 1.8 2.08 ± 1.2 2.46 ± 0.5 31.11 ± 0.7 67.57 ± 0.4 0.46 ± 0.5

1.27 ± 0.1 0.42 ± 0.29 1.18 ± 0.62 11.37 ± 0.72 27.95 ± 0.47 22.40 ± 0.5 1.24 ± 0.18 12.29 ± 0.54 3.32 ± 0.9 9.84 ± 1.23 26.53 ± 0.45 64.75 ± 0.6 0.41 ± 0.3

1.54 ± 0.23 0.99 ± 0.78 1.22 ± 0.3 6.92 ± 0.9 41.71 ± 0.82 33.74 ± 0.4 ND 8.08 ± 0.7 3.24 ± 1.6 1.63 ± 0.2 17.53 ± 0.5 80.32 ± 0.5 0.22 ± 0.5

1.25 ± 0.3 0.72 ± 0.1 3.89 ± 0.12 12.58 ± 0.7 25.80 ± 0.42 25.38 ± 0.65 1.08 ± 0.1 15.45 ± 0.35 1.09 ± 0.5 1.57 ± 0.66 33.89 ± 0.3 54.92 ± 0.4 0.61 ± 0.1

3.33 ± 0.44 0.90 ± 0.65 1.36 ± 0.45 3.27 ± 0.3 16.57 ± 0.6 48.21 ± 0.1 ND 4.15 ± 0.95 18.73 ± 0.8 1.03 ± 0.9 11.65 ± 0.4 84.54 ± 0.1 0.13 ± 0.2

0.99 ± 0.6 1.22 ± 0.44 2.79 ± 0.18 1.51 ± 0.00 61.14 ± 0.2 9.16 ± 0.87 3.27 ± 0.49 12.30 ± 0.55 ND 1.48 ± 0.3 18.81 ± 0.3 75.05 ± 0.2 0.25 ± 0.1

Results are given as the average of triplicate estimations (ND = Not detected). C12:0: lauric acid; C14:0: myristic acid; C16:0: palmitic acid; C18:0: stearic acid; C18:1n9c+18:1n7c: oleic acid; C18:2n-6c: linoleic acid; C18:3n-6c: ␥- linoleic acid; C20:0: arachidic acid; C20:3n-6: dihomo- ␥- linoleic acid; C22:2n-6: eicosadienoïc acid; SFA: Saturated fatty acid; UNFA: Unsaturated fatty acid; NL: neutral lipid; TGL: Total glycolipid; TPL: Total phospholipid; MGDG: Monogalactosyldiacylglycerol; DGDG: Digalactosyldiacylglycerols; ESG: Esterified steryl glycoside; Cer: Cerebroside.

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of ESG total fraction) and finally Cer (5.96% of Cer total fraction), which confers to the seed oils nutritional and energy properties and can also play a role in molecular recognition at cell membranes. 3.5. Fatty acid composition of different glycerolipid classes extracted from Pinus halepensis Mill. seeds The fatty acid composition of different glycerolipids classes as well as individual GL classes, obtained from Pinus halepensis Mill. seed oils, is presented in Table 5. As expected the fatty acid composition were characterized by the predominance of unsaturated fatty acids in all lipids classes with 86.34% of TFA in NL, 67.57% of TFA in TGL, 64.75% of TFA in TPL, 80.32% of TFA in MGDG, 54.92% of TFA in DGDG, 84.54% of TFA in ESG and 75.05% of TFA in Cer. Oleic acid was the major unsaturated fatty acid with 48.41% of TFA in NL, 33.94% of TFA in TGL, 27.95% of TFA in TPL, 41.71% of TFA in MGDG, 25.80% of TFA in DGDG, 16.57% of TFA in ESG and 61.14% of TFA in Cer. The second unsaturated fatty acid in both lipids classes was linoleic acid with 31.06% of TFA in NL, 29.09% of TFA in TGL, 22.04% of TFA in TPL, 33.74% of TFA in MGDG, 25.38% of TFA in DGDG, 48.21% of TFA in ESG and 9.16% of TFA in Cer. In both lipids classes, arachidic acid was the major saturated fatty acid ranging 4.15-12.30% of TFA, while the second major saturated fatty acid was stearic acid (6.43-12.58% of TFA) excepted in ESG and Cer when the lauric acid and palmitic acid were the second major saturated fatty acid with 3.33% of TFA and 2.79% of TFA respectively. In addition to oleic, linoleic, arachidic acid, others fatty acids were also detected in all or some lipids classes (Table 5) as lauric acid, myristic acid, palmitic, stearic acid, ␥- linoleic acid, dihomo- ␥linoleic acid and eicosadienoïque acid belong to the minor fraction. Generally, both seed oils, were characterized by high level of unsaturated fatty acids especially, oleic and linoleic acid (Ramadan and Moersel, 2003; Nergiz and Domnez, 2004, Cheikh-Rouhou et al., 2006), which is in perfect agreement with the results reported in this investigation. Linoleic and oleic acid present in large quantities in the lipids fractions of P. Halepensis Mill. seeds are essential for healthy growth of human skin and in the construction of the nerve cells (Bruckert, 2001). They can be changed by the organism into a set of closely related compounds of prostaglandins and so have a key role in preventing cardiovascular diseases (Nergiz and Domnez, 2004). 4. Conclusion Oil seeds lipids fractions (neutral lipids, glycolipids and phospholipids) were separated, identified and analyzed via the experimental conditions described. Very significant levels in polar lipophilic materials (GLs and PLs) could render these oils useful in the food industry for exploiting their nutritional value and to the pharmaceutical industry for exploiting their bioactivities. References Aidi Wannes, W., Mhamdi, B., Sriti, J., Marzouk, B., 2010. Glycerolipid and fatty acid distribution in pericarp, seed and whole fruit oils of Myrtus communis Var. italic. Ind Crop. Prod. 31, 77–83. Andersson, L., Bratt, C., Arnoldsson, K.C., Herslof, B., Olsson, N.U., Sternby, B., Nilsson, A., 1995. Hydrolysis of galactolipids by human pancreatic lipolytic enzymes and duodenal contents. J. Lipid Res. 36, 1392–1400. Avalli, A., Contarini, G., 2005. Determination of phospholipids in dairy products by SPE/HPLC/ELSD. J. Chromatogr. A. 1071, 185–190. Benning, C., Ohta, H., 2004. Three enzyme systems for galactoglycerolipid biosynthesis are coordinately regulated in plants. J. Biol. Chem. 280, 2397–2400.

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