Chemical Composition of Iran ’ s Pistacia atlantica Cold-Pressed Oil

e lipid fraction of Pistacia atlantica seeds was extracted for the �rst time by means of cold-press technique and analyzed for its chemical composition. e fatty acids, sterols, triacylglycerols (TAG), tocopherols, polyphenols, and pigments were identi�ed and their concentrations were determined by means of reversed-phase high-performance liquid chromatography (RP-HPLC) and gas chromatography (GC). Because of its high content of unsaturated fatty acids, it might prove to be of value in diets and it may be used as edible cooking or salad oils or for margarine manufacture. Pistacia atlantica seed oil has the unique sterols and tocopherols content providing source of natural antioxidants. e main triacylglycerols were SLL + PLO, SOL + POO, OOLn + PLL, OOO, and SOO. is paper examined the phenolic fraction of Pistacia atlantica seed oil. Moreover, caffeic acid followed by cinnamic acid, pinoresinol, vanillin, p-Coumaric acid, ferulic acid, and o-Coumaric acid was also determined. is paper presents the �rst investigation of chlorophyll�s and carotene�s composition in Pistacia atlantica seed oil. Furthermore, pheophytin a was the major component, followed by luteoxanthin, neoxanthin, violaxanthin, lutein, lutein isomers, chlorophyll a, chlorophyll a , and pheophytin a were also determined.


Introduction
e genus of Pistacia which contains 13 or more species belongs to Anacardiaceae family [1].One of the most widely distributed species of Pistacia is Pistacia atlantica which is called "Baneh" in Iran and is the most economically important tree species in many rural areas.e resin of wild pistachio, called Saqez, is used for a variety of industrial and traditional uses, including food and medicine [2].e fruit of wild pistachio is used by natives as �avor in food a�er grinding it and it is used for its oil, although the fruit is small and not commercially valuable.
Tree nuts and their oils contain several bioactive and health-promoting components.Dietary consumption of tree nut oils may have even more bene�cial health effects than consumption of whole tree nuts, possibly due to the replacement of dietary carbohydrates with unsaturated lipids and/or other components present in the oil extracts [3].�nre�ned oils contain a number of minor components such as tocopherols, carotenoids, and phenols.Tocopherols and carotenoids improve oil stability and thus oils naturally rich in these constituents are preferred [4,5].Tocopherols and phenols can act as antioxidants and reduce the risk of cancer and cardiovascular diseases [6][7][8].Phytosterols have been used as blood cholesterol-lowering agents for the last half century.ey have been shown to be effective and safe [9].
Conventional vegetable oil extraction is carried out by pressing or solvent extraction.Solvent oil extraction is the most efficient method; however, its application presents some industrial disadvantages such as emissions of volatile organic compounds into the atmosphere, high operation costs, and poor quality products caused by high processing temperatures [10].
In this work, oil was extracted from the fruit of Pistacia atlantica by cold press technique and the fatty acid, sterol, triacylglycerol, tocopherol, phenolic, and pigment composition of the oil of Pistacia atlantica was determined.Although some work has been performed before to determine the sterol and triacylglycerol composition of Pistacia atlantica fruit oil from Algeria [11][12][13] and total phenol and total tocopherol of Pistacia atlantica from Iran [14], it is the �rst time Pistacia atlantica cold press oil from Iran was investigated and phenol, pigment, and tocopherol fractions of Pistacia atlantica seed oil were determined.

Experimental
2.1.Samples.Pistacia atlantica seeds were collected in September from three locations in Iran including Fars, Isfahan, and Kohkeloye Boyerahmad province.Samples were mixed equally and the outer skin was manually peeled and then dried in the shade.

Reagents and Standards.
All chemicals and solvents used in this study were of analytical grade and were purchased from Sigma, Fluka, BDH and Merck chemical companies.

Oil Extraction.
Oil was obtained by pressing 5 kilograms of Pistacia atlantica seed by means of cold press machine (PR500, Germany).is operation was carried out three times and then �ne particles in the expressed oil were separated by �ltration; before each analysis, these �ltered crude oils were centrifuged in a centrifuge Kokusan (model H-11n, Tokyo, Japan) at 4000 rpm during 15 minutes.Experiments were carried out in triplicate aer oil extraction as soon as possible; otherwise extracted oil samples were stored at 3 ∘ C for a short time.e yield of seed oil was calculated as 15.32 ± 0.6% in dry weight.

TGA Analysis.
TAGs were separated by RP-HPLC (model Young-Lin Acme 9000).Oil samples were dissolved in HPLC-grade acetone (5%) and aer �ltration by a syringe �lter (0.22 μ), 20 μL were injected into C18 Column (25 cm × 4.6 mm i.d., 5 μm particle size, Tracer Excel 120 ODSA) and were eluted with acetonitrile and acetone (40 : 60) at a �ow rate of 1.5 mL/min.e column was equilibrated at room temperature and the effluent monitored with a refractive index detector.e TAGs were identi�ed by comparing retention times to pure standards (Lardon AB Limhamnsgardens Alle 16 Malmo, Sweden).

Sterol Analysis.
For determination of sterols, 5 g of sample were saponi�ed with 50 mL of 2 N ethanolic KOH at 85 ∘ C for 30 min (the solution becomes clear) in the presence of 2 mg -cholestanol as internal standard.Aer addition of 50 mL of water, unsaponi�ables were extracted three times with 100 mL diethyl ether.e pooled extracts were washed three times with 50 mL of deionized water and the solvent was removed under reduced pressure with a rotary evaporator.e unsaponi�able was diluted in chloroform and then submitted to thin layer chromatography (TLC) on silica gel previously immersed in the 0.2 N ethanolic potassium hydroxide solution and then placed in stove at 100 ∘ C for 1 hour.e developing solvent was hexane/diethyl ether (65 : 35, (v/v)).Aer drying, the spots were identi�ed by spraying 2,7-dichloro�uorescein (0.1% in ethanol).Sterol band was observed under ultraviolet (UV) light at 232 nm by comparing its rate of �ow (RF) value with standard.e sterol fraction was then extracted from the silica gel with hot pure chloroform.Aer solvent evaporation sterol derivatives (trimethylsilyl ethers, TMS) were synthesized at 90 ∘ C for 30 min in 10 mL anhydrous pyridine with 10 mL of a mixture of hexamethyldisilane-trimethylchlorosilane (99 : 1, v/v).e reaction mixture was �nally evaporated to dryness and the residue was diluted in 1 mL chloroform prior to GC analyses.e silanized sterols were analyzed by gas chromatography using Young-Lin gas chromatograph (Model 6000, South Korea) supplied with �ame-ionization detector (FID) and capillary column (60 m × 0.32 mm i.d.; �lm thickness was 0.25 μm).e carrier gas was hydrogen at 1 mL/min column �ow and 1 : 20 split ratio.Injector and detector temperatures were 300 and 320 ∘ C, respectively.Oven temperature was at 250 ∘ C. Peak identi�cation was carried out by comparing with the retention times of the standards (cholesterol (99%), stigmasterol (95%), b-sitosterol (95%), campesterol (98%), and sitostanol (96.7%) were purchased from Sigma Chemical Co.(St.Louis, MO, USA)).

Tocopherol Analysis.
Tocopherols were measured by RP-HPLC and UV-detection at 295 nm.extracted oil was diluted with acetone (1 : 10) and 20 microliter was injected to HPLC aer �ltration by a syringe �lter (0.22 μ) with a C-18 lichrospher RP-100 (250 mm × 4.6 mm, 5 μm) column and guard column (4.6 × 1.5 mm).e mobile phase was acetonitrile, methanol, and water (47.5, 47.5, 5 v/v) at a �ow rate of 1 mL/min.e amounts of each tocopherol were calculated by comparing with standards purchased from Sigma.
2.9.Pigment Analysis.Oil samples were extracted by liquidphase distribution between N,N-dimethyl-formamide (DMF) and hexane.25 g of Pistacia atlantica oil samples were dissolved in 150 mL DMF and treated with �ve 50 mL portions of hexane in decanting funnel.e DMF phase contained xanthophylls, chlorophylls and chlorophyllic derivatives was treated with a 2% Na 2 SO 4 solution at 0 ∘ C and transferred to 100 mL of a mixture of hexane/ethyl ether (1 : 1; v/v).e aqueous phase was eliminated polyphenols and other water soluble compounds.e ether was evaporated in a rotary evaporator at reduced pressure at 30 ∘ C. e dry residue was dissolved in methanol, and analyzed by HPLC.Separation was performed using C18 Column (25 cm × 4.6 mm i.d., 5 μm particle size, Tracer Excel 120 ODSA) and elution was performed at a �ow rate of 1.0 mL/min at room temperature.e mobile phase was a mixture of methanol and water (8 : 2, v/v) containing 0.025% ammonium acetate and 0.05% triethylamine as phase A and methanol and acetone (1 : 1, v/v) as phase B. e pigments were eluted according to solvent gradient as follow: 0-10 min, 75% A, 25% B, 10-14 min, 50% A, 50% B, 14-21 min 20% A, 80% B, 21-40 min, 0% A, 100% B, 40-50 min 75% A, 25% B. Identi�cation of compounds was achieved by comparing with standards.1 shows the fatty acid composition of Pistacia atlantica seed oil.e most predominant fatty acid (FA) was oleic acid, with a mean value of ∼51%.In addition to oleic acid, seed oil of Pistacia atlantica contained high amount of linoleic acid (∼30%).e seed oil also contains saturated fatty acid especially palmitic and stearic acids.e level of palmitic acid was ∼13% and higher than the amount of stearic acid 3%.e polyunsaturated (PU) fatty acids of the oil amounted to ∼30% of the total fatty acid, while the monounsaturated (MU) and saturated (SA) fatty acids amounted to 53% and 17%, respectively.e ratio of unsaturated/saturated fatty acid of Pistacia atlantica seed oil was 5.1.Oleic acid has an important role in food industry.Foods prepared with oleic acid will remain safe to eat for longer periods, even without refrigeration.Oleic acid is also used as a cleaning agent in the manufacturing of soaps and detergents and as an emollient or soening agent, in creams, lotions, lipsticks, and skin products [16].It has been found to be fungistatic against a wide spectrum of saprophytic moulds and yeasts.

Fatty Acid Composition. Table
According to the results of this study, Pistacia atlantica seed oil is regarded as oleiclinoleic oil because oleic acid is most abundant, followed by linoleic acid and it may be used as edible cooking or salad oils or for margarine manufacture [17]. 2 shows sterol composition of seed oil of Pistacia atlantica.Results from the quantitative analysis of sterols from Pistacia atlantica (expressed in mg/100 g of oil) showed that the major sterol of the oil is betasitosterol (189.9 mg/100 g oil), which amounted to ∼87% of the total amount of sterols.Campesterol (9.4 mg/100 g oil), and Δ 5 -avenasterol (4.9 mg/100 g oil) were presented, with about 4 and 2% of the total sterols, respectively.
Phytosterol has good effectiveness in decreasing serum low-density lipoprotein (LDL) cholesterol levels that could be effective in protecting against cardiovascular diseases, thus it can be used to improve the functional foods.In nuts (walnuts, almonds, peanuts, hazelnuts, and the macadamia nuts), beta-sitosterol is the most abundant sterol, the total sterol contents ranging from 99.12 to 207.17 mg/100 g oil [18].Pistacia atlantica has the high level of beta-sitosterol (189.9 mg/100 g oil).
3.3.TGA Composition.e fatty acid composition can be used to evaluate the stability and nutritional quality of fats and oils, but not always their functional properties.e type and the amounts of the various TAG species in the oil are too important.It is the TAG compositions that determine the �nal physical and functional properties of the oils.Table 3 shows the TAG composition of Pistacia atlantica.e most predominant TGA species are SLL + PLO (21.82%) and then SOL + POO (16.56%),OOLn + PLL (15.68%),OOO (14.07), and SOO (13.72).Other TGAs are minor (OLL + PoOL, PLnP, POS, SLS, LLL, PPP, OLLn, SOS).Where P, palmitic; S, stearic; O, oleic; L, linoleic; Ln, linolenic; Po, palmitoleic.TGAs in Pistacia atlantica with ECN of 48 were dominant (30.63%), followed by triacylglycerols with ECN of 46 (27.65%).

Tocopherol Composition.
Because of the critical role of the tocopherols in nutrition and their relative instability, qualitative and quantitative analyses are very important.To the authors� knowledge it is the �rst time that tocopherol composition of cold press Pistacia atlantica fruit oil has been evaluated.Table 4 shows the tocopherol content of Pistacia atlantica seed oil.High level of tocopherols in Pistacia atlantica cold press oil (409.97mg/kg oil) was determined.-tocopherol was in highest concentration.It was 379.68 mg/kg.( + )-tocopherol and -tocopherol were 20.70 and 9.59 mg/kg oil, respectively.
Radical-chain breaking antioxidant in membranes, lipoproteins and foods is the main function of -tocopherol [20].-tocopherol ability to act as an antioxidant and various functions at the molecular level reduce the risk of cancer and cardiovascular diseases [6,7].A direct relationship has been found between the content of total phenolics and antioxidant capacity of plants [8].Phenolic acids have been widely investigated as potential models for the development of new primary antioxidants, which can prevent or delay in vitro and/or in vivo oxidation processes [21] (see Table 5).

Phenol
3.6.Pigment Content.is work contains the �rst qualitative quantitative investigation of the chlorophyll and carotenoid pigments composition of Pistacia atlantica kernel oil.As Table 6 shows, pheophytin a was the major component (12.02 mg/kg), followed by luteoxanthin (10.41 mg/kg).Chlorophyll and carotenoid play key roles in photosynthesis.Animals cannot synthesize chlorophylls and carotenoids, thus they must obtain them from foods.Several reports have demonstrated that plant pigments play important roles in health [22,23].Carotenoids have antioxidant activity, which protects cells and tissues from free radicals and singlet oxygen.Lutein have a fundamental role in the protection of the macula region of the retina and in the prevention of the cataracts� other bene�cial actions of carotenoids include enhancement of the immune response, protection against solar radiation, inhibition of some cancers and prevention of degenerative and cardiovascular diseases [24,25].

Conclusion
e cold-pressing procedure involves neither heat nor chemical treatments, and it is becoming an interesting substitute for conventional practices because of consumers' desire for natural and safe food products.e consumption of new and improved products such as cold pressed oils may improve human health and may prevent certain diseases.Improved knowledge on the composition of Pistacia atlantica seed oil would assist in efforts to achieve industrial application of this plant.Data about cold pressed Pistacia atlantica seed oil are very few.In concluding this investigation, it is clear that the performed chromatographic techniques constituted a �exible analytical system, which gave valuable information about the structure of the seed oil.Pistacia atlantica seeds give a considerable yield of oil and the cold press oil seems to be a good source of fatty acids and lipid-soluble bioactives.e high linoleic and oleic acid content makes the oil nutritionally valuable.Tocopherols and sterols, at the level estimated, may be of nutritional importance in the application of the seed oil.
Content.is research examined the phenolic fraction of Pistacia atlantica seed oil.As Table4shows, T 3: Triacylglycerol composition of Pistacia atlantica seed oil.