Chemical composition of oils from wild almond ( Prunus scoparia ) and wild pistachio ( Pistacia atlantica )

El objetivo de este estudio fue determinar la composición en ácidos grasos, esteroles, triglicéridos, así como tocoferoles, fenoles totales y pigmentos de aceites de almendras y pistachos silvestres prensados en frío. Triglicéridos (TAG), tocoferoles y pigmentos se analizaron mediante HPLC, los ácidos grasos y esteroles mediante cromatografía de gases, y los fenoles totales espectrofotométricamente. Los principales ácidos grasos de ambas especies fueron los ácidos oleico, linoleico y palmítico. Las especies de TAG predominantes son SLL + OLP (21,83%) en el pistacho silvestre y OOO (47,27%) en almendras silvestre. Feofitina a es un pigmento importante en los aceites de pistacho silvestre. No se detectó pigmentos en los aceites de almendras silvestres. Los fenoles totales fueron 57,6 mg kg y 45,3 mg kg en los aceites de pistacho silvestre y de almendra silvestre respectivamente.


INTRODUCTION
A large quantity of oils and fats, whether for human consumption or for industrial purposes, is presently derived from plant sources and because of this, interest in newer sources of edible oils has recently grown.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.Tree nuts and their oils contain several bioactive and health promoting components.Dietary consumption of tree nut oils may have even more beneficial health effects than the consumption of whole tree nuts, possibly due to the suppressing of dietary carbohydrates and more a concentrated content of unsaturated lipids and/or other components present in the oil extracts.(Hu and Stampfer, 1999).Vegetable oils are mainly constituted by triacylglycerol (95-98%) and complex mixtures of minor compounds (2-5%) of a wide range of chemical nature (Aluyor et al., 2009).The minor components include mono and diglycerides, free fatty acids, tocopherols, sterols, carotenoids and polyphenols.Tocopherols act as antioxidants and are required to protect cells against oxidative damage by free radicals (Foster et al., 2009).Tocopherols and carotenoids improve oil stability and thus oils naturally rich in these constituents are preferred (Warner and Frankel, 1987;Frankel, 1991).Phenols can act as antioxidants and reduce the risk of cancer and cardiovascular diseases (Burton, 1994;Burton and Traber, 1990;Ferreira et al., 2007).Sterols are essential components of cell membranes that play a role in controlling membrane fluidity and permeability.These compounds have a similar structure to cholesterol.When they are present in foods, they can inhibit the absorption of cholesterol resulting in a reduction in low-density lipoprotein (LDL).This improves the cholesterol profile and can help to reduce the risk of developing coronary heart diseases (CHD) (Warner and Frankel, 1987).
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 (Uquiche et al., 2008).Cold pressing is simple, ecological and does not require much M.H. GIVIANRAD, M. SABER-TEHRANI AND S.A. JAFARI MOHAMMADI extracted with 600 mL hexane at room temperature with vigorous shaking for 3 h in a flask covered with aluminum foil.Then, the mixture was filtrated under a vacuum.The solvent was removed by rotary evaporation at 35 o C. Extraction was carried out in triplicate.
The oil of wild pistachio seeds was obtained by pressing 5 kilograms of seeds by means of a cold press machine (PR500, Germany).The screw speed was at 35 rpm.This operation was carried out three consecutive times and fine particles were removed from the oil by filtration.Additionally, before each analysis, these filtered crude oils were centrifuged in a centrifuge Kokusan (model H-11n, Tokyo, Japan) at 4000 rpm for 15 minutes.
Experiments were carried out in triplicate immediately after oil extraction; otherwise, extracted oil samples were stored at 3 o C for one week.

Analytical methods
The analysis was carried out in triplicate.The values of different parameters were expressed as the mean ± standard deviation

Sterol analysis
The unsaponifiable fractions were extracted after an alkaline hydrolysis of wild pistachio and wild almond oil samples and the sterols were isolated from the other constituents by thinlayer chromatography according to the method of Givianrad et al., (2011).Trimethylsilyl ether derivatives (TMSE) of sterols were synthesized by adding anhydrous pyridine with a mixture of hexamethyldisilane-trimethylchlorosilane (99:1, v/v) to each dried sample; afterwards, the composition of the sterol fraction was determined using Young-Lin gas chromatograph (Model 6000 South Korea) supplied with a flame ionization detector (FID) and capillary column (60 m × 0.32 mm i.d.; 0.25 µm film thickness).The carrier gas was hydrogen at 1 mL energy (Álvarez-Ortí et al., 2012).The cold-pressing procedure involves neither heat nor chemical treatments.The consumption of cold-pressed oils may improve human health and may prevent certain diseases (Siger et al., 2008).
Almonds and pistachios are grown as orchard crops, are highly nutritious and hav sites and have evolved throughout southwest and Central Asia from Turkey and Syria into the Caucasus Mountains, through Iran, and into the deserts of the Tian-Shan and Hindu Kush Mountains of Tajikistan, Uzbekistan, and Afghanistan (Browicz and Zohary, 1996).Iran is optimally situated for growing almond trees.Nearly 20 of the wild species have been reported from Iran (Gorttapeh et al., 2006).Prunus Scoparia is one of them, which grows in the Kerman Province of Iran.The fruits of wild almond and pistachio trees are used by natives for food after peeling and grinding them.In spite of the wide distribution of wild almond and pistachio trees with fruits of high nutritional value and low cost, they have not been used for industrial applications.Although numerous studies have been reported on the chemical characteristics of commercial almond and pistachio species, there are few which deal with the chemical composition of wild almond and pistachio species.Furthermore, this is the first time that a complete chemical analysis of cold pressed wild pistachio oil and wild almond oil (solvent extracted) is determined including sterols, TAGs, tocopherols, fatty acids, phenols and pigment assessment (Yousfi et al., 2002;Yousfi et al., 2005;Benhassaini et al., 2007;Farhoosh and Tavakoli., 2008;Ozcan et al., 2011).

Samples
Wild pistachio (Pistacia atlantica) seeds were gathered in September from three locations in Iran including the provinces of Fars, Isfahan and Kohkeloye Boyerahmad and were mixed equally.Wild almond (Prunus Scoparia) seeds were collected in October from the Kerman Province in Iran.The outer skin was manually peeled and then the nuts were dried at room temperature in the shade for 1 week for both samples.The moisture content of the air-dried nuts at room temperature was calculated by the difference in weight of about 5 g kernel samples before and after further drying at 105 °C for 3 h.

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

Oil extraction
Oil was obtained from wild almond by solvent extraction.Powdered wild almond (200 g) was CHEMICAL COMPOSITION OF OILS FROM WILD ALMOND (PRUNUS SCOPARIA) AND WILD PISTACHIO (PISTACIA ATLANTICA) the solution was allowed to stand for 2 h at ambient temperature (about 22 o C).The total phenol content was determined colorimetrically at 750 nm, and expressed as gallic acid equivalents (Zengin et al., 2011).

Fatty acid composition
Oil yield was 15.32± 0.6% and 45.34± 0.95% for wild pistachio and wild almond oil, respectively, on a dry weight basis.The moisture of the samples was 15.2 ± 1.5% and 8.9 ± 0.9 for wild pistachio oil and 11.1 ±1.1 and 6.3 ± 2.1 for wild almond oil before and after drying, respectively.
Table 1 shows the fatty acid composition of wild pistachio and wild almond seed oils.The main fatty acids from the studied samples were oleic, linoleic and palmitic acids.In both studied edible oils, the most abundant fatty acid was oleic acid.It represented 51% in wild pistachio and 67% in wild almond seed oils.The seed oils of wild pistachios and wild almonds contained high percentages of linoleic acid (about 30% for wild pistachio and about 22% for wild almond oil).They also contain saturated fatty acids especially palmitic and stearic acids.The latter was poorly represented with about 3% for both oils.Indeed, this fatty acid is constantly min -1 and 1:20 split ratio.The injector and detector temperatures were 300 and 320 o C, respectively and Oven Temperature was 250 o C. Peak identification was carried out by comparing retention times to those of standards.

Triacylglycerols (TAG) analysis
TAGs were separated by RP-HPLC (model Young Lin Acme 9000).Wild pistachio and wild almond oil samples were dissolved in HPLC-grade acetone, filtred by a syringe filter and injected into a C18 Column (25 cm ×4.6 mm i.d., 5 µm particle size, Tracer excel 120 ODSA).Elution was conducted at a flow rate of 1.5 mL/min with acetonitrile and acetone (40:60).The column was equilibrated at room temperature and the effluent monitored with a refractive index detector.

Total Phenolic Contents
Phenolic compounds were extracted from the oils in a hexane solution with water and methanol (40/60, v/v) 3 times.The three extracts were combined, and evaporated to dryness in a rotary evaporator.Samples were dissolved in 1 mL methanol and placed in a 25 mL test tube followed by the addition 1 mL of Folin-Ciocalteu reagent and 2 mL Na 2 CO 3 (7.5%)respectively.The final volume was brought up to 7 mL with deionized water, and the different phytosterols, β-sitosterol has been most intensively investigated with respect to its beneficial and physiological effects on health.β-sitosterol lowers cholesterol levels (Pegel, 1997), enhances immunity (Nieman, 1994), and has anti-inflammatory, antipyretic and anti-carcinogenic effects (prostate essentially) (Klippell, 1997;Kritchevsky and Shirley, 2005).
The next major components were campesterol in wild pistachio seed oil and ∆ 5 -avenasterol in wild almond seed oil, where they reach about 4% of the total sterols.∆ 5 -avenasterol accounted for about 2% in pistachio oil.Each of the ∆ 5,24 stigmastadienol, ∆ 7 -avenasterol and stigmasterol amounted to about 1% of the total amount of sterols in wild pistachio oil and about 0.3%, 0.3% and 0.4%, respectively in wild almond seed oil.Cholesterol, which is specific to animal lipids, is present at low levels in most vegetable oils.It was found at about 0.4% for wild pistachio oil and 0.2% for wild almond oil.
The total sterol contents in wild pistachio and wild almond seed oils were 2164.5 mg kg -1 and 2084.5 mg kg -1 oil, respectively.These values were higher than those determind by Miraliakbari and Shahidi (2008) and Abidi (2001) for the solventextracted oils of almonds, Brazil nuts, hazelnuts, pecans, pine nuts, pistachios and walnuts (800 to 1600 mg Kg -1 oil).

TAG composition
The fatty acid composition can be used to evaluate the stability and the nutritional quality of fats and oils, but not always their functional properties.The type and the amounts of the various TAG species in the oil are important as well because they determine the final physical and functional properties of the oils (Jahaniaval et al., 2000).Table 3 shows the TAG composition of wild pistachio and wild almond oils.The most predominant TAG species are SLL+PLO (21.83%) and then SOL+POO (16.56%),OOLn+ PLL (15.68%),OOO (14.07%) and SOO (13.72%) in wild pistachio oil and OOO (47.27%),POO+SOL (26.25%),OOL+PLnP (10.67%) in wild almond oil.Where the P, Palmitic; S, Stearic; O, Oleic; L, Linoleic; Ln, Linolenic; Po, Palmitoleic TAG profiles determined in wild almond oil present good similarity with that of olive oil (Abaza et al., 2002).The Studied oils are clearly distinguishable from sunflower oil because of its poor levels of LLL (Noor Lida et al., 2002).The internal position is occupied either by oleic acid (both oils) or linoleic acid (pistachio oil), making these two fatty acids biologically easily available.Oleic acid is an important element in reducing cardiovascular diseases and linoleic acid constitutes an essential fatty acid that helps prevent physiological problems (López Alonso and García Maroto, 2000).In both seed oils TAGs with ECN of 48 were dominant (30.63% in wild pistachio and 76.32% in wild almond), followed by triacylglycerols with ECN of 46 (27.65% in wild pistachio and 15.36% in wild almond).solicited for the biosynthesis of oleic acid.C17:0 and C17:1 fatty acids presented in wild pistachios are known to be found in animals, but they can only be detected in plants in very small amounts (Satil et al., 2003).
The polyunsaturated (PU) and monounsaturated (MU) and saturated (SA) fatty acids amounted to about 30%, 53% and 17%, respectively for wild pistachio seed oil and about 22%, 67% and 10% for wild almond seed oil.The ratio of unsaturated / saturated fatty acids of wild pistachio seed oil was 5.1 and 8.8 for wild almond seed oil.Results of this study on oleic acid levels in wild pistachios are comparable to those reported by Tsantili et al., (2010) (52%) and Arena et al., (2007) (55%) for commercial pistachios from Iran.The oleic acid contents of wild almonds from the current study was comparable to those reported by Moayedi et al. (2011) (68%), Maguire et al. (2004) (69.2%) and Miraliakbari and shahidi (2008) (69.9%) for commercial and domestic almonds.Low contents of saturated fatty acids and high contents of monounsaturated oleic acid are highly favorable for human nutrition.Monounsaturated fatty acid-rich oils prevent cardiovascular diseases and they are less exposed to oxidization (Kocyigit et al., 2006).According to the results of this study, wild pistachio and wild almond seed oils are regarded as oleiclinoleic oil because oleic acid is most abundant, followed by linoleic acid and it may be used as edible cooking or salad oil.

Sterol composition
Phytosterols are of a great interest due to their antioxidant activity and impact on health (Maestro-Durán and Borja-Padilla, 1993).Table 2 shows the sterol composition of the seed oils of wild pistachios and wild almonds.In these oils, the marker was β-sitosterol, which constituted about 88% and 91% in wild pistachio and wild almond seed oils, respectively.In most plants such as nuts (walnuts, almonds, peanuts, hazelnuts and the macadamia nuts), β-sitosterol is also the most abundant sterol.Among oil for almond, respectively.The tocopherol contents in nuts (Brazil nuts, hazelnuts, pecans, pine nuts and walnuts) were obtained from 106.8 to 321.9 mg kg -1 oil (Miraliakbari et al., 2008).α-tocopherol was found in its highest concentration in wild pistachio and wild almond fruit oils.It was 379.68 mg kg -1 oil for wild pistachio and 446.92 mg kg -1 oil for wild almond.α-tocopherol was not detected in commercial pistachios (Kornsteiner et al., 2006), but Maguire et al., (2004) mentioned that α-tocopherol was the most dominant tocopherol in almonds, peanuts, hazelnuts and macadamias.It ranged from 9.4 (peanuts) to 186.4 µg g -1 oil (almonds).α-tocopherol acts as a radical-chain breaking antioxidant in membranes, lipoproteins and foods as the main function of α-tocopherol (Tsantili et al., 2010).This ability allows for reducing the risk of cancer and cardiovascular diseases (Burton, 1994;Burton and Traber, 1990).However, not only α-tocopherol but also other tocopherol forms are recently considered to be of biological importance (Wagner et al., 2004).(γ + β)-tocopherol and δ-tocopherol amounts were 20.70 and 9.59 mg kg -1 oil in wild pistachio, and 35.40 and 5.60 mg kg -1 oil in wild almond seed oils, respectively.

Tocopherol composition
Tocopherols and tocotrienols are naturally occurring constituents found in vegetable oils in varying amounts.The presence of these compounds is important in relation to oil stability and nutritional labeling.Besides, consumption of these oils is recommended thanks to their beneficial effects on health (Gliszczynska-Swigło and Sikorska, 2004).Because of the critical role of tocopherols in nutrition and their relative instability, qualitative and quantitative analysis is very important.To the authors' knowledge, this is the first time that the tocopherol compositions of wild almond and cold press wild pistachio oils have been evaluated.Table 4 shows the tocopherol contents of wild pistachio and wild almond seed oils.Relatively high level of tocopherols in wild pistachio cold press oil (409.97mg kg -1 oil) and wild almond oil (487.92mg kg -1 oil) were determined.The total tocopherols were more than the amounts of those Miraliakbari et al., (2008) and Kornsteiner et al., (2006) reported for commercial pistachio and almond when the oil was extracted with different solvents such as 218.5 to 298 mg kg -1 for pistachio and 170.6 to 273 mg kg -1  and prevention of degenerative and cardiovascular diseases (Miraliakbari and Shahidi, 2008).

Total phenolic content
The total phenol contents of wild pistachio and wild almond oils are expressed as their equivalent gallic acid in mg per kg oil.They reached 57.6 mg kg -1 oil for wild pistachio and 45.3 mg kg -1 oil for wild almond oil.Moayedi et al., (2011) reported for regular almond a value of 37.7 mg kg -1 oil, which is slightly inferior to ours, whereas Miraliakbari and Shahidi (2008) mentioned a total phenol content of 158 mg kg -1 oil for commercial pistachio, obviously lighter than what we put in evidence in this research.
A direct relationship has been found between the content of total phenolics and the antioxidant capacity of plants (Ferreira et al., 2007).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 (Siquet et al., 2006).

CONCLUSIONS
A large quantity of oils and fats is presently derived from plant sources and because of this, interest in newer sources of edible oils has recently grown.Nuts contain a diverse group of compounds that enhance the nutritional value of the human diet.Almond and pistachio nuts and their oils contain several bioactive and health-promoting components but they remain expensive.The fruits of wild almonds and pistachios are used by natives for food.In spite of awide distribution of wild almonds and pistachios with high nutritional value and low cost, they have not been used for industrial applications.Improved knowledge of the composition of wild pistachio and wild almond seed oils would assist in efforts to achieve industrial applications of these plants.Data about wild almond and cold pressed wild pistachio seed oils are very few.Tocopherols and sterols in these seed oils may be of nutritional importance for possible applications in food.More studies are required to evaluate a possible risk of antinutritional components from these sources, which was outside the scope of this study.
Lutein and chlorophyl values are significantly lower than those in pistachio nuts (Pistacia vera) of different geographic regions (Bellomo and Fallico, 2007).
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 (Maguire et al., 2004).Carotenoids have antioxidant activity, which protects cells and tissues from free radicals and singlet oxygen.Other beneficial actions of carotenoids include enhancement of the immune response, protection against solar radiation, inhibition of some cancers

Table 1 Fatty acid compositions of wild pistachio and wild almond seed oils (mean ± standard error (SE), %) Fatty acids Pistacia atlantica Wild almond
a SFA, saturated fatty acid b MUFA, monounsaturated fatty acid c PUFA, polyunsaturated fatty acid M.H. GIVIANRAD, M. SABER-TEHRANI AND S.A. JAFARI MOHAMMADI

Table 2 Sterol composition of wild pistachio and wild almond seed oil (mean ± standard error (SE), %)
CHEMICAL COMPOSITION OF OILS FROM WILD ALMOND (PRUNUS SCOPARIA) AND WILD PISTACHIO (PISTACIA ATLANTICA)