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High tocopherol and triacylglycerol contentsin Pinus pinea L. seeds
NIZAR NASRI1, NIZAR TLILI1, KAMEL BEN AMMAR2,
ABDELHAMID KHALDI3, BRUNO FADY4 & SAIDA TRIKI1
1Laboratoire de Biochimie, Departement de Biologie, Faculte des Sciences de Tunis, Universite
Tunis El-Manar, Tunis, Tunisia, 2Office National de l’Huile, Tunis, Tunisia, 3Unite de Recherche
Gestion et Valorisation des Ressources Forestieres, INRGREF, Tunisia, and 4INRA, UR 629,
Recherches Forestieres Mediterraneennes, Domaine St Paul, Site Agroparc, Avignon, France
AbstractOleaginous seeds are among the functional foods most recognized for their tocopherols andtriacylglycerols because of their role in lipid metabolism. In this paper, the tocopherol andtriacylglycerol contents in seeds of several Pinus pinea L. populations around the MediterraneanBasin were investigated. Lipids were extracted from fully ripen seeds with petroleum ether. Thetocopherol (a-tocopherol, g-tocopherol, and d-tocopherol) contents were, respectively, 15.3493.75 ppm, 1,681.759404.03 ppm and 41.8799.79 ppm. Lipids (mainly triacylglycerols) inP. pinea seeds averaged 48% on a dry weight basis. Triacylglycerols with an equivalent carbonnumber of 44 (32.27%) and of 46 (30.91%) were dominant. The major triacylglycerol was LLO(24.06%). Tocopherols and triacylglycerols were present at remarkably high levels, thus makingP. pinea oil a valuable source of antioxidants and unsaturated fatty acids with varying levelsacross the geographical range of P. pinea.
Keywords: Mediterranean stone pine, seeds, triacylglycerols, tocopherols, Mediterranean diet
Introduction
The use of functional foods to treat and prevent chronic diseases is very attractive. These
products are both economical and efficient, as well as widely tolerated by the general
public as alternatives to synthetic drugs (Kanu et al. 2007; Niva 2007). Oleaginous
seeds may serve as such functional foods as they are present in the Mediterranean diet
and include foods such as olives or stone pine nuts. Many epidemiological studies have
concluded that a Mediterranean diet could significantly decrease chronic disease rates
and increase adult life expectancy (Local Food-Nutraceuticals Consortium 2005;
Mackenbach 2007; Schroder 2007). Oleaginous seeds are often valuable for their
tocopherols and unsaturated fatty acids, among others.
Tocopherols are known as vitamin E and represent an important class of antioxidants.
Structurally, all known tocopherols consist of a chromanol head group attached to a
phytyl tail, and differ only in the number and position of methyl groups on the
chromanol rings (Dellapenna and Pogson 2006). They are commonly known to reduce
Correspondence: Nizar Nasri, Laboratoire de Biochimie, Departement de Biologie, Faculte des Sciences
de Tunis, Universite Tunis El-Manar, Tunis 2092, Tunisia Tel: 216 97 35 31 97. E-mail: Nizar.
ISSN 0963-7486 print/ISSN 1465-3478 online # 2009 Informa UK Ltd
DOI: 10.1080/09637480802577854
International Journal of Food Sciences and Nutrition,
August 2009; 60(S1): 161�169
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lipid peroxidation. Recent studies report that these metabolites (as well as phenols)
prevent free-radical-induced cells by preventing the formation of radicals, scavenging
them, or by promoting their decomposition (Xu et al. 2001; Youn and Woodside 2001).
Four classes of lipids are habitually found in vegetable oils: triacylglycerols (TAGs),
diacylglycerols, polar lipids and free fatty acids. A previous study on Pinus pinea lipid
class contents (Nasri and Triki 2004) confirmed that TAGs constitute the greatest
part of lipids with 96.28% of the total content, while diacylglycerols, polar lipids and
free fatty acids are in lesser proportions*respectively 1.95%, 0.84% and 0.93% of the
total content.
TAGs are natural compounds consisting of saturated and unsaturated fatty acids
differing in their acyl chain lengths and the number and positions of double bonds.
Saturated and unsaturated fatty acids play an important role in biological processes,
and in the construction of biological structures (such as cell membranes).
Recently, there has been strong consumer concern about unsaturated fatty acid
contents in foods. Several epidemiological studies have also pointed out the relation-
ship between unsaturated fatty acid intake and coronary heart disease (Bruckert 2001;
Ochoa et al. 2002) as well as their potential adverse effects on the metabolism of
essential fatty acids (i.e. a-linolenic acid and linoleic acid).
The main lipid compounds have not been extensively studied in all oleaginous
seeds, despite their health potential. Most of the available literature is related to
studies on olives. Stone pine (P. pinea L.) is a widely distributed Mediterranean pine
that has been part of the Mediterranean diet for a very long time.
To our knowledge, a comprehensive study regarding the tocopherol levels of stone
pine seeds has not yet been reported. The objective of this study was to complete two
previous studies on lipids of stone pine (Nasri et al. 2005, 2007) by assessing their
tocopherol and triacylglycerol contents for the first time. Further, as this species is
known for its genetic uniformity (Fallour et al. 1997; Vendramin et al. 2008), we
compared the tocopherol and triacylglycerol contents with population geographical
structuring, analysing the potential use of these compounds for population profiling.
Materials and methods
Materials
The seeds used were drawn from a bulk seed collection made from three or four cones
per tree, collected from 20�30 trees per population in six forest stands*Bechateur
(Tunisia), Cordillera Central (Spain), Saint-Aygulf (France), Agios Nikolaos
(Greece), Feniglia (Italy), and Izmir (Turkey)*as presented in Table I. Seeds were
stored at 48C until use.
Oil extraction
Lipids were extracted by the method of reference (Folch et al. 1957) as described
previously in Nasri et al. (2005). All solvents were of reagent grade purchased from
Merck Chimie S.A.S. (Fontenay-sous-Bois, France) and were used without any
further purification. A total of 30 ml methanol was added to 5 g ground seeds and was
then mixed for at least 30 min at room temperature. Then, 60 ml chloroform was
added and the solution was stirred for 30 min. Extracts were centrifuged and solvents
162 N. Nasri et al.
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were removed using a rotary evaporator, and lipid fractions were stored in chloroform
at �208C for tocopherol and TAG analysis.
Determination of tocopherol contents
Tocopherols were determined by high-performance liquid chromatography (HPLC)
with fluorescence detection according to reference (Pocklington and Dieffenbacher
1988). The HPLC system consisted of a Lichrospher 100 RP-18 silica column (5 mm,
250�4 mm) equipped with a Rheodyne 7125 sample injector fitted with a LC-7A
pump, and a fluorescence detector with the excitation and emission wavelength set at
290 nm and 330 nm, respectively, and an integrator C-R6A Chromatopac (Shimadzu,
Touzart et Matignon, France). The chromatograms were recorded automatically by a
chromate-integrator computer. The isocratic mobile phase used was hexane/propane-
2-ol (99.5:0.05, vol/vol) at a flow rate of 2 ml/min. Tocopherol peaks were identified
and quantified with the help of an in-house reference solution of a-tocopherol, g-
tocopherol, and d-tocopherol purchased from Sigma-Aldrich (St Louis, MO, USA).
b-Tocopherol levels were not measured as fruits and vegetables have been reported to
contain little or no b-tocopherols (Chun et al. 2006).
Determination of TAG contents
TAG analysis was performed according to equivalent carbon number (ECNs) of
TAGs given by the reference method (IUPAC 1990). The instrument used was a
Hewlett Packard Model 1100 liquid chromatograph (Hewlett Packard, USA)
equipped with refractive index detector. HPLC separation of TAGs was conducted
on a Hypersil ODS column (5 mm, 150�4.6 mm; Hewlett Packard, USA) with a
mobile phase of acetone/acetonitrile (70:30), and elution was at a flow rate of 1.5 ml/
min. Separated TAGs, as a function of ECNs, were identified using reference TAGs
purchased from Sigma-Aldrich, and their relative percentages were computed
automatically.
Statistical and chemometric methods
Each population value is a duplicate gas chromatography analysis of the same seed
sample. Values of different parameters were expressed as the mean9standard
deviation. Population values for each compound were compared with the mean of
all populations by calculating a confidence interval. An analysis of variance was used
to compare populations grouped according to their geographical origin (eastern versus
western Mediterranean Basin) and thus identify possible regional trends.
Table I. Location of the P. pinea populations studied.
Population Code Country Latitude Longitude
Bechateur BE Tunisia 37814?N 9856?ECordillera Central E2 Spain 40830?N 4820?WSaint-Aygulf F2 France 43827?N 6841?EAgios Nikolaos G2 Greece 40814?N 23834?EFeniglia I Italy 42825?N 11817?EIzmir T2 Turkey 39812?N 26857?E
Tocopherol and triacylglycerol contents in P. pinea L. seeds 163
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Results and discussion
Total lipid contents of P. pinea L. seeds
Table II presents some differences in the total lipid content of P. pinea L. seeds of the
six Mediterranean populations studied. Mean population values fluctuated between
44.44% (G2) and 51.87% (BE) and were not included in the 5% confidence interval
around the overall mean of 48.7292.29%. The Bechateur population (BE) is richer
than other populations. These values are in the higher range of those found for
oleaginous species, such as sunflower (40%) or rapeseed (43%) (Gunstone and Norris
1983).
Tocopherol contents of P. pinea L. seed oil
The most frequent form of tocopherol found in stone pine seeds was g-tocopherol
(ca. 1,700 ppm), followed by d-tocopherol (ca. 41 ppm) and a-tocopherol (ca.
15 ppm), as shown in Table III. Figure 1 shows a typical P. pinea chromatogram in
which the peaks of tocopherols can be observed. In other seeds such as rapeseed
and linum, the major tocopherol is also g-tocopherol, with contents of 699 ppm
and 773 ppm, respectively (Sivaoja et al. 1986). However, in sunflower and olive seed
oils, a-tocopherol is the major compound, with 622 ppm and 119 ppm contents,
respectively.
When grouping populations according to their geographic origin (e.g. eastern
versus western Mediterranean Basin) and comparing their compound contents using
analysis of variance, no significant differences appeared among groups. However,
the population from Spain had significantly higher, and the population from
Turkey significantly lower, tocopherol contents than all other populations tested,
which could be used further if improving the tocopherol content was a goal in the
stone pine.
Table II. Total lipid contentsa of P. pinea L. seeds.
BE E2 F2 G2 I T2 Mean9standard deviation (n�6)
Total lipid content (%) 51.87b 48.8 49.8 44.44b 47.99 49.47 48.7292.47
aContents were determined by Soxhlet apparatus.bValues are significantly higher than the mean at p�0.05.
Table III. Tocopherol composition (ppma) of P. pinea L. seed oil.
Population BE E2 F2 G2 I T2
Mean9standard
deviation (n�6)
a-Tocopherol 16.19 25.58b 12.19 13.74 15.53 8.86b 15.3493.75
g-Tocopherol 1,956.29 2,619.32b 1,570.38 1,547.28 1,483.59 913.67b 1,681.759404.03
d-Tocopherol 33.97 58.78b 48.19 32.56 48.03 29.7b 41.8799.79
Total tocopherols
(ppm)
2,006.45 2,703.68 1,630.76 1,593.58 1,547.15 952.23 1,738.979410.72
aEach population value is a duplicate gas chromatography analysis of the same seed sample. bAll values are
significantly higher than the mean at P�0.05.
164 N. Nasri et al.
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Triacylglycerol composition of P. pinea L. seed oil
The distribution of TAGs (by carbon number), with equivalent carbon number,
determined by HPLC, is presented in Table IV. Only fatty acids having 16�20 atoms
of carbon were considered. Figure 2 shows a typical P. pinea chromatogram in which
the peaks of TAGs can be observed.
The TAG contents followed the same trends in all P. pinea populations. TAGs with
an ECN of 44 were dominant (32.27%), followed by TAGs with an ECN of 46
(30.91%). The most frequent TAGs were LLO (24.06%), LOO (18.75%), LLL
(12.96%) and PLO (11.07%) followed by POO and OOO (7�8% each). All other
Figure 1. Typical chromatogram of tocopherols prepared from extracted oils from P. pinea seeds from Izmir,
Turkey (code T2, Table I). Analysis was performed on a Lichrospher 100 RP-18 silica column (5 mm, 250�4 mm) equipped with a Rheodyne 7125 sample injector fitted with a LC-7A pump, and a fluorescence
detector. Peak 1, d-tocopherol; peak 2, g-tocopherol; peak 3, a-tocopherol.
Tocopherol and triacylglycerol contents in P. pinea L. seeds 165
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Table IV. Triacylglycerol compositiona of P. pinea L. seed oils.
Population
Triacylglycerol ECN
Retention
time (min) BE E2 F2 G2 I T2
Mean9standard
deviation (n�6)
LLSi 40 9.9 0.34 0.3 0.34 0.38 0.35 0.28b 0.3390.03
PLnLn 40 10.1 0.61 0.57 0.49 0.54 0.57 0.51 0.5490.02
LLLn 40 10.8 0.66 0.4 0.41 0.55 0.55 0.56 0.5290.07
LLL 42 14.1 14.8 13.3 11.13 11.42 13.56 13.58 12.9691.05
OLnL 42 13.6 1.79 1.79 2.34 2.6b 2.16 1.93 2.1090.24
PLnL 42 12.9 0.59 0.59 0.73 0.74 0.69 0.59 0.6590.06
LLO 44 14.9 24.38 24.63 23.5 23.63 24.22 24.05 24.0690.35
OLnO 44 15.5 7.93b 7.11b 7.49 7.33 7.4 7.51 7.4690.11
PLL 44 16.2 0.63 0.72 0.89 0.87 0.72 0.68 0.7590.08
LOO 46 20.2 17.82 19.2 19.4 19.38 18.32 18.39 18.7590.46
PLO 46 21.1 11.31 10.86 11.45 10.84 10.75 11.22 11.0790.24
LPP 46 22.7 1.06 1.12 0.89b 1.15 1.11 1.22 1.0990.08
OOO 48 26.2 6.74 7.12 8.03 8.29 7.59 7.31 7.5190.39
POO 48 27.3 7.53 7.61 8.04b 7.74 7.7 7.8 7.7390.11
OPP 48 29.1 0.93 0.92 0.95 0.95 0.99 1.05b 0.9690.03
SLS 50 34.1 0.21 0.3 0.38 0.37 0.28 0.24 0.2990.04
SOO 50 36.5 2.13 2.55 2.68b 2.45 2.31 2.27 2.3990.13
POS 50 38.6 0.48 0.65 0.77 0.69 0.64 0.61 0.6490.04
P, palmitic; S, stearic; O, oleic; L, linoleic; Ln, linolenic. ECN�carbon number � 2�number of double bonds. aEach population value is a duplicate gas chromatography
analysis of the same seed sample. bAll values are significantly higher than the mean at P�0.05.
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TAGs were present at lesser than 1%. TAGs that contain unusual polymethylene-
interrupted unsaturated fatty acids with a cis-5 ethylenic bond were not identified, as
more selective methods would have been needed.
Nergiz and Donmez (2004) reported that pine nuts from Izmir (Turkey) contained
six ECN TAG families (from ECN 42 to ECN 52). TAGs with an ECN of 44 were
Figure 2. Typical chromatogram of TAGs prepared from extracted oils from P. pinea seeds from Saint-
Aygulf, France (code F2, Table I). Analysis was performed on a Hypersil ODS column (150�4.6 mm) with
a mobile phase of acetone/acetonitrile (70:30). Peak 1, LLSi; peak 2, PLnLn; peak 3, LLLn; peak 4, LLL;
peak 5, OLnL; peak 6, PLnL; peak 7, LLO; peak 8, LnOO; peak 9, PLL; peak 10, LOO; peak 11, PLO;
peak 12, LPP; peak 13, OOO; peak 14, POO; peak 15, OPP; peak 16, SLS; peak 17, SOO; peak 18, POS.
Tocopherol and triacylglycerol contents in P. pinea L. seeds 167
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also dominant (48.7%) in their sample, but followed by TAGs with an ECN of 48
(19.8%). Their most frequent TAG was OLnO (23.5%), followed by LLO (18.6%),
LLL (10.8%) and OOO (10.3%). This indicates that the TAG content can vary
among populations, even those geographically close (our T2 population and their
Izmir population). However, these differences might be due to experimental
differences more than genetic factors, as those TAGs were not significantly different
among the wide-ranging populations in the samples examined.
Further, the most frequent TAGs were not discriminating factors among popula-
tions; and when the analysis of variance was used to test for the presence of a
geographic structure, PLL and SLS appeared the most discriminative TAGs,
opposing the eastern populations from Greece (G2), Italy (I), and Turkey (T2)
from the others (P values were respectively 0.007 and 0.008).
Tocopherols are present in high amounts (1,738 ppm) in P. pinea. The most
dominant tocopherols were g-tocopherol (ca. 1,700 ppm), followed by d-tocopherol
(ca. 40 ppm) and a-tocopherol (ca. 15 ppm). TAGs with an ECN of 44 were
dominant (32.27%), followed by TAGs with an ECN of 46 (30.91%). The TAG most
commonly found was OLnO (24.06%).
Triacylglycerols (as compounds of glycerol and three fatty acids) and tocopherols
(as antioxidants) are recognized to improve resistance to lipids. Therefore, our analysis
and previous studies confirm that stone pine seeds can be considered a health food
from their lipid composition point of view.
Although we were able to identify some populations with significantly higher
tocopherol content (e.g. in Spain), we were not able to derive a convincing geographic
structure out of the wide range of samples of populations used. Therefore, our study
confirms that the overall homogeneity found ranges wide in this species using genetic
and biochemical markers, and does not suggest that either tocopherols or TAGs could
be used efficiently for population profiling.
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