Lipid composition of seed oils of diﬀerent pomegranate ( Punica granatum L.) cultivars from Spain

Invited paper from the International


Sampling and extraction of seed oils
Nine cultivars harvested in Valencia (Spain), in the crop season of 2013, were selected, namely: CG8, Cis 127, Mollar de Elche, Parfianka, Katirbasi, Valenciana, White, Wonderful 1 and Wonderful 2 (Figure 1).All of them were collected when fully ripe.For each variety, 15 grams of seeds were crushed in a mortar with a pestle, and dehydrated by addition of anhydrous sodium sulphate.The lipids were extracted in a Soxhlet device with petroleum ether (with 0.01% BHT -2,6-di-tert-butyl-4-methylphenol) for a 4 h period, in triplicate for each cultivar.(Melgarejo & Artés, 2000;Pande & Akoh, 2009;Elfalleh, Ying, et al., 2011) .For quantification of total fatty acid content in the oil, an internal standard (triundecanoin) was used.

Vitamin E
An accurate amount of oil was weighed, the internal standard added (tocol purchased from Matreya, USA), dissolved in n-hexane, centrifuged at 13,000 rpm and transferred to the injection vials.An HPLC chromatograph (Jasco) equipped with a pump (PU-980 model), mixing chamber (HG 980-30) and an autosampler (AS2057 Plus model) was used for tocochromanol separation.Detection was performed by fluorescence (FP2020 Plus model at 290 nm (excitation) and 330 nm (emission) wavelengths).

Statistical analysis
The statistical software SPSS, version 18.0 (SPSS Inc., Chicago, IL), was used for the statistical treatment of the data.The influence of the cultivar over fatty acid and vitamin E compositions was evaluated using the one-way analysis of variance (one-way ANOVA) (p<0.05),followed by the Tukey's HSD post hoc test, when variances of the groups were identical.On the other hand, when variances were not identical, the Games-Howell's test coupled with Welch's statistic was applied.The variance homogeneity was evaluated by the Levene's test.
A Principal Component Analysis (PCA) was also performed for the results for the fatty acids and tocopherols of the pomegranate cultivars.
The PCA score plot was used to differentiate pomegranate cultivars through their chemical compositions.
3 Results and Discussion

Fatty Acid Composition
The total lipid content and fatty acid composition of pomegranate seed oils are given in Table 1 and Figure 2 .The total lipids ranged between 4.44 -13.70% for Valenciana and Katirbasi cultivars, respectively.This range was in agreement with Melgarejo and Artés (2000), with 6.2 to 12.2% for the Piñón Tierno de Ojos -PTO4 and Piñonenca de Blanca -PB1 varieties, respectively.Moreover, our maximums were identical to those found by Jing et al. (2012) for the Suanshiliu and Sanbaitian varieties of 11.4 and 14.8%, respectively.On contrary, our results were lower than Pande and Akoh (2009)

Tocopherols and Tocotrienols
In the pomegranate cultivars investigated, the tocopherols identified were α-, γ-and δ-tocopherol, while β-tocopherol and tocotrienols were not detected.The total amount of tocopherols varied from 174.5 to 627.3 mg/100g of extracted oil for the White and Katirbasi cultivars, with a mean value of 445.4 mg/100g of oil (Figure 3).The predominant tocopherol was γ-tocopherol, with contents ranging from 159.7 and 586.2 mg/100g for the White and Katirbasi cultivars, respectively, and a mean value of 416.7 mg/100g (93.5%).αtocopherol concentration varied between 8.9 and 26.1 mg/100g of oil for White and Katirbasi cultivars, respectively, with a mean value of 16.6 mg/100g of oil (3.7% of total tocopherols).δtocopherol was found in slight lower amounts, varying between 6.0 and 15.2 mg/100g of oil for the White and Parfianka cultivars, respectively, with a mean value of 12.2 mg/100g of oil (2.7% of total tocopherols).Moreover, the contents of tocopherols differed significantly between cultivars.Cultivar Katirbasi had the highest levels of total tocopherols, α-and γ-tocopherol.The content of δ-tocopherol in the Katirbasi cultivar did not differ from that in Parfianka cultivar which had the highest δ-tocopherol level among the cultivars investigated.Our γ-tocopherol contents (159.7 to 586.2 mg/100 g of oil) were similar to those described by Liu et al. (2012), whose values varied between 120.62 and 672.56 mg/100 g oil.On the other hand, our α-tocopherol values (13.0 to 20.6 mg/100g) were lower than those obtained by Pande and Akoh (2009) (between 161.2 and 173.7 mg/100g).In the present work the following order was found: γ-tocopherol > α-tocopherol > δtocopherol; this was different to that reported by Jing et al. (2012) (δ-tocopherol > α-tocopherol > γ-tocopherol) and Elfalleh, Tlili, et al. (2011) (αtocopherol > γ-tocopherol > δ-tocopherol).Furthermore, in the present work β-tocopherol was not detected unlike Caligiani et al. (2010), who found high amounts of this compound in all the samples analyzed.These differences might be a consequence of the chromatographic separation mode used, in our particular case normal-phase, enabling full separation of all tocopherols and tocotrienols compounds.

Principal Component Analysis
PCA was done to differentiate the nine pomegranate cultivars using fatty acid and tocopherol contents.content.When applying PCA to the fatty acids and tocopherols composition, significant variability between some of pomegranate cultivars was found.These components could be used for a better population profiling.Moreover, the differences found on tocopherols content show that it is possible to increase the levels of healthy compounds by the appropriate choice of cultivars.Advanced plant breeding could further improve the plant material and produce superior cultivars for use, e.g., in the fortification of pomegranate juices.In summary, this study provided valuable information for the cultivar selection and for developing value-added utilization of pomegranate seeds or seed fractions, such as oil, as nutraceuticals or functional food ingredients.

Table 1 :
Total oil content, total SFA, MUFA and PUFA (%) of the seed oils extracted from nine pomegranate cultivars IJFSApril 2015 Volume 4 pages 95-103 Figure 2: The four most abundant fatty acids (%) in the seed oils extracted from nine pomegranate cultivars