Fatty acids and triacylglycerols composition from Tunisian Acacia species seed oil

2.1 Seed materials

Seeds were collected in June 2010 at random from at least three wild trees of three Acacia species (A. cyclops, A. ligulata and A. salicina) located in Tunisia. Mature collected seeds were directly mixed and placed in an oven for 48 h at 60 °C until the difference between successive weight (before and after putting the samples in the oven) is less than or equal to 5 mg. Tables 1 and 2 provide more information about their geographical origin and climatic conditions.

2.2 Reagent and standards

Triacylglycerols standards: glyceryltripalmitate (⩾99%) was purchased from Sigma Aldrich (St. Louis), and chloroform was purchased from Fisher Scientific Company (Ottawa, Canada).

Methanol, and n-hexane, solvents of LC grade were purchased from Panreac Quimica SA. (Barcelona, Spain). Isopropanol and acetic acid were from Fisher Scientific SA (Loughborough, Spain). Petroleum ether was purchased from Sigma Aldrich. Sodium chloride was purchased from Panreac Quimica SA (Barcelona, Spain).

2.3 Oil extraction

The oil from seeds was extracted by a Soxhlet extractor as reported previously (Nasri et al., 2013) following ISO method 659:1998. The solvent was evaporated under reduced pressure, using a rotary evaporator at 50 °C, flushing with nitrogen to blanket the oil during storage. Oil was weighed and stored at −20 °C. All the analyses were conducted in triplicate.

2.4 Fatty acids analysis

Fatty acids analysis was performed using the method published by Youzbachi et al. (2012). Fats were transmethylated using boron trifluoride in methanol. The fatty acid methyl esters formed by transmethylation were analyzed in an HP 5890 gas chromatograph (Hewlett Packard, Palo Alto, CA, USA) fitted with a CPSIL-88 column 100 m × 0.25 mm i.d., film thickness 0.20 μm (Varian, Les Ulis, France). Hydrogen was used as a carrier gas (inlet pressure 210 kPa). The oven temperature was held at 60 °C for 15 min, increased to 85 °C at a rate of 3 °C/min and then to 190 °C at a rate of 20 °C/min, finally being held at 190 °C. The injector and flame ionization detector were maintained at 250 °C and 280 °C, respectively. Fatty acid methyl esters were identified by comparison with standards. The data were computed using the Galaxie software (Varian, Les Ulis, France) and reported as a percentage of total fatty acids. All analyses were performed in triplicate.

2.5 Triacylglycerols determination

Triacylglycerols containing sodium adducts (+23 m/z) were detected using Waters QTOF-1 mass spectrometer electrospray ionization (operating in positive ion mode).

50 μl of unmodified plant oil weighed and diluted in 950 μl of chloroform. Five μl of this solution (depending on observed intensities) was mixed with 1 ml of a 90:10 methanol/chloroform solution containing 5.75 × 10⁻⁵ of both PPP standards. The final solution was then spiked with 50 μl of a 3 mg/ml NaCl solution. The TAG-containing solution was injected into a 15 μl sample loop and carried by a methanol phase with a flow of 60 μl/min to the electrospray capillary, which was held at 3 kV. The source temperature was 80 °C and the desolvation temperature was set to 150 °C. The capillary voltage was 3 kV and the cone voltage was 45 V. ESI-MS was focused on mass ranges of 700–1100 m/z. Isotopic overlap between species differing by 2 Da (1 double bond) was accounted for using the theoretical natural abundances of Carbon, Oxygen and Hydrogen isotopes and in-house software.

To quantify the different TAG species a total mass spectral intensity were used. As an example, the sodium adducted PPL and POP will have an m/z of 853.7 and 855.7, respectively. Theoretical percentage of PPL containing two C₁₃ isotopes (855.7 m/z), relative to PPL that containing no C₁₃ isotopes, is 18.4%. This directly corresponds to the m/z of PPO. The experimental intensities occurring from PPL (853.7) and PPL-1C₁₃ (854.7 m/z) were summed with the theoretical intensities of PPL-2C₁₃ (855.7 m/z) and PPL-3C₁₃ (856.7 m/z) to give the total molecular species intensity.

After that the corrected intensity of POP (855.7 m/z) was calculated by subtracting the theoretical intensity of PPL-2C₁₃ (855.7 m/z) from the experimental intensity at that m/z. The same was done for POP-1C₁₃; experimental intensity 856.7 less the theoretical intensity of PPL-3C₁₃ to give corrected intensity of POP-1C₁₃. The obtained corrected intensities were used to calculate the theoretical intensities of their 2C₁₃ and 3C₁₃ counterparts, as reported by Tlili et al. (2011) and Trabelsi et al. (2014).

Quantification was achieved by comparison with the total ion count of the internal standard (Tripalmitin) with intensity corrections made for isotopic peak overlap according to the following equation: $$[\text{TAG}]={\text{TAG}}_{\text{intensity}}\frac{[\text{STD}]}{\text{STD intensity}}$$ where TAG: Triacylglycerols; STD: Internal Standards.

2.6 Statistical analysis

The experimental data were analyzed using the analysis of variance (ANOVA) and the Statistical Analysis System (XLSTAT 2008). Differences at p ⩽ 0.05 were considered statistically significant by Duncan’s new multiple range test.

3.1 Oil content

Table 3 shows the seed-oil content of the three Acacia species. The mean content of total lipids was 9.35 ± 2.68% on a dry weight basis (dw%), and varied between Acacia species. A. cyclops contained about 6.83%, A. ligulata contained 9.04% and A. salicina contained the highest value (12.18%). Indeed, Youzbachi et al. (2012) have reported that Acacia cyanophylla in Tunisia contains about 10% of oil. Other previous studies have reported that the oil content in Acacia species in India is between 4% and 10% (Sundar Rao et al., 1983; Maity and Mandal, 1990; Khan et al., 2012), and in 20 species of Acacia in Australia is between 3% and 22% (Brown et al., 1987). These differences were probably due to genetic factor and also to location affect since the crops chemical composition can vary with the crop varieties, soil and climatic conditions of the area (Breene et al., 2007).

3.2 Fatty acids composition

The results of the analysis of the fatty acid fraction of Acacia seed oil are summarized in Table 3. Thirteen fatty acids were identified (C12 to C22).

The most abundant saturated fatty acid was palmitic acid followed by stearic acid. A difference in palmitic acid contents was noted within species; Acacia cyclpos contained the highest level (10.25%) followed by A. ligulata (7.71%) than A. salicina (5.36%). The highest value of staric acid was detected once again in A. cyclpos (2.10%) while the amounts in A. ligulata and A. salicina were 1.61% and 1.39%, respectively. Lauric acid was detected only in A. cyclops with a low level (0.09%).

Acacia seed oil can be regarded as linoleic–oleic oil because of the abundance of linoleic acid, precursor of ω6, followed by oleic acid. A. ligulata contained the highest level of linoleic acid (67.75%) while the levels in A. salicina and A. cyclops were 54.31% and 52.39%, respectively. A. cyclops presented the high value of oleic acid (27.13%) while the values in A. ligulata and A. salicina were 17.95% and 15.06%, respectively.

The observed differences could be due to genetic and/or environmental factors. In fact, our samples were from different locations in Tunisia. These regions are characterized by different environmental conditions, such as temperature and rainfall (Tables 1 and 2). Indeed, previous studies on numerous plant-seeds have suggested the effect of the environmental conditions (precipitation and temperature) on the composition of the fatty acids. Aslam et al. (2009), Schulte et al. (2013) and BenLajnef et al. (2015) have reported the effect of temperature and precipitation on palmitic acid, oleic acid and linoleic acid composition, which support our findings.

Youzbachi et al. (2012) have suggested that in 12 Tunisian A. cyanophylla, linoleic acid (61.11–65.45%) and oleic acid (19.67–22.85%) are the major fatty acids. Moreover, previous study on 20 species of edible Australian Acacia seeds has reported that the major detected fatty acids are linoleic (12–71%), oleic (12–56%) and plamitic (7–35%) (Brown et al., 1987). Furthermore, other authors have reported that in Indian Acacia species linoleic acid and oleic acid are the main fatty acids (Khan et al., 2012; Maity and Mandal, 1990; Banerji et al., 1988).

Acacia seed oil of this study contained more than 70% of unsaturated fatty acids and A. ligulata showed the highest level with 88.86% (Table 3). These high values were essentially due to the high values of polyunsaturated fatty acids (ca. 68%, ca. 54% and ca. 52% for A. ligulata, A. salicina and A. cyclops, respectively). The monounsaturated fatty acids were between 16.54 (A. salicina) and 28.87 (A. cyclops). Youzbachi et al. (2012) have reported that the unsaturated fatty acids content in A. cyanophylla is about 86% (23% and 63% for monounsaturated and polyunsaturated, respectively). Khan et al. (2012) have suggested that the polyunsaturated content of Acacia tortilis is about 83%. In Acacia mollissima the amount is 85.6% (Banerji et al., 1988).

The level of saturated fatty acids was between 8.06% (A. salicina) and 14.29% (A. cyclops). The values reported for A. mollissima, A. cyanophylla and A. tortilis are 12%, 12.68%, and 16.95%, respectively (Khan et al., 2012; Youzbachi et al., 2012; Banerji et al., 1988).

The detected unsaturated fatty acids confer a dietary and an industrial importance to Acacia seeds. Indeed, it is known that polyunsaturated fatty acids can influence some physical properties of the cellular membranes such as fluidity and permeability (Ward and Singh, 2005). Moreover, the benefits of polyunsaturated fatty acids in some diseases, such as cardiovascular diseases and autoimmune disorders, have been reported (Reiffel and Mcdonald, 2006).

Results showed that the polyunsaturated/saturated ratio of Acacia seed oil was between 3.69 (A. cyclops) and 6.77 (A. salicina). The obtained values were similar to those reported by Khan et al. (2012) for 15 Acacia species from India (1.98–7.48). This ratio was slightly high than the value reported by Nehdi (2011) for soybean (2.68) and close to some fenugreek cultivars as reported by Skakovskii et al. (2013). Indeed, a high polyunsaturated/saturated ratio is regarded favorable for reduction of serum cholesterol and prevention of heart diseases (Oomah et al., 2002).

The ω3 and ω6 polyunsaturated fatty acids are essential components of the human diet because humans are unable to synthesize these fats, both of which are considered to be the precursor of eicosanoids. Moreover, linoleic fatty acid is indispensable for the healthy growth of human skin (Bruckert, 2001). Results clearly showed that Acacia seeds oil contained more than 52% of linoleic acid (ω6), while α-linolenic acid (ω3) did not exceed 0.74%. These findings were similar to other reports for other Acacia species (Khan et al., 2012; Youzbachi et al., 2012; Banerji et al., 1988).

Therefore, Acacia seed oil could be used as promising source of oil with high value for pharmaceutical and cosmetic sectors; nevertheless its safety must be tested before use. Previous work has shown that Acacia species contain cyanogenic glycosides (Maslin et al., 2003). In fact, cyanogenic glycoside, especially in the presence of β-glycosidases, is potentially poisonous. Indeed, Seigler (2003) has reported that among the known cyanogenic plants, some species have been described that have cyanogenic glycosides and not the enzymes, other plants have the enzymes and not the glycosides, and other species have both; and this last category is potentially toxic to livestock.

3.3 Triacylglycerols composition

Fig. 1 shows a typical TAG chromatogram. Total mass spectral intensities (Fig. 2) were used to quantify the different TAG species. Fourteen molecular species of TAG were detected (Table 4). The TAG composition was different between the three Acacia species. The predominant molecular species of triacylglycerols were those containing fatty acids 18:2 and 18:1, which was in agreement with the above results showing that the major fatty acids were 18:2n-6 and 18:1n-9. Results showed that the species PLL, PLO, LnLO, OLL, OOL, and OOO were the major forms in the all Acacia seed oils.

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Figure 1 Chromatogram of TAG from Acacia species.

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Figure 2 Spectrum of the different molecular species of TAG from Acacia species.

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It can be concluded that the main forms of TAG in A. cyclops were LnLO (19.25%), followed by SSL (16.36%), OOO (12.88%) and OLL (12.14%). In A. ligulata, the major fraction was LnLO (43.66%) followed by OLL (21.55%) and PLL (11.23%). The dominant fraction in A. salicina was LnLO (37.7%), followed by OLL (24.59%) and PLL (12.26%). PPS, PSO, SSL and SSO were identified only in A. cyclops (1.17%, 0.85%, 16.36 and 2.03%, respectively). These quantitative variations observed in the major TAG composition of the three Acacia species may be due to differences in the levels of major fatty acids: oleic, linoleic and palmitic acids. Indeed, A. cyclops presented the high values of C16:0, C18:0 and C18:1, which could explain the high level of OOO, PPS, PSO, SSL and SSO in this species compared to A. ligulata and A. salicina. This ascertainment was in agreement with those of Jellum (1970) and Harrabi et al. (2010) for corn kernel. Moreover, the separation and the identification of TAG from vegetable oil seem to be a hard task due to the presence of numerous TAG species with similar physicochemical properties (Trabelsi et al., 2014).

The obtained results were slightly different to that of some common seed oils in which the linoleic acid is the major fatty acid such as soybean oil (16% OLL, 7% LLLn, 2% OOO, and 8% OOL) and Pinus pinea seed oil which has 24.38% LLO, 17.82% LOO, 11.31% PLO, and 7.5% POO (Padley, 1994; Nasri et al., 2009). Our results agreed with other authors who reported that the TAG content can vary among populations and also can vary among species (Crane et al., 2005; Nasri et al., 2009).

Spady and Dietschy (1985) have suggested that saturated dietary TAG inhibit the activity of hepatic LDL receptors in the Hamster. Other studies have reported the benefic effect of triacylglycerol on glucose homeostasis and on the prevention of obesity and atherosclerosis (Kojima et al., 2010; Pegorier et al., 1985). Acacia seed oil could be used as potential source of TAG with high industrial value; however its safety must be proved.