Phytosterol, Squalene, Tocopherol Content and Fatty Acid Profile of Selected Seeds, Grains, and Legumes

Abstract

The unsaponifiable lipid fraction of plant-based foods is a potential source of bioactive components such as phytosterols, squalene, and tocopherols. The objective of the present study was to determine the levels of phytosterols, and squalene, as well as tocopherols (alpha and beta + gamma) in selected grains, seeds, and legumes. The method comprised acid hydrolysis and lipid extraction followed by alkaline saponification, prior to analysis by HPLC. In addition, the fatty acid profile of the foods was determined via total lipid extraction, fatty acid derivitisation and GC analysis. In general, beta-sitosterol was the most prevalent phytosterol, ranging in concentration from 24.9 mg/100 g in pumpkin seed to 191.4 mg/100 g in peas. Squalene identified in all foods examined in this study, was particularly abundant in pumpkin seed (89.0 mg/100 g). The sum of alpha- and beta+ gamma-tocopherols ranged from 0.1 mg/100 g in rye to 15.9 mg/100 g in pumpkin seeds. Total oil content ranged from 0.9% (w/w) in butter beans to 42.3% (w/w) in pumpkin seed and the type of fat, in all foods examined, was predominantly unsaturated. In conclusion, seeds, grains, and legumes are a rich natural source of phytosterols. Additionally, they contain noticeable amounts of squalene and tocopherols, and in general, their fatty acid profile is favorable.

Full text

ORIGINAL PAPER
Phytosterol, Squalene, Tocopherol Content and Fatty Acid
Profile of Selected Seeds, Grains, and Legumes
E. Ryan & K. Galvin & T. P. O’Connor & A. R. Maguire &
N. M. O’Brien
Published online: 27 June 2007
#
Springer Science + Business Media, LLC 2007
Abstract The unsaponifiable lipid fraction of plant-based
foods is a potential source of bioactive components such as
phytosterols, squalene, and tocopherols. The objective of
the present study was to determine the levels of phytoster-
ols, and squalene, as well as tocopherols (α and β + γ)in
selected grains, seeds, and legumes. The met hod comprised
acid hydrolysis and lipid extraction followed by alkaline
saponification, prior to analysis by HPL C. In addition, the
fatty acid profile of the foods was determined via total lipid
extraction, fatty acid derivitisation and GC analysis. In
general, β-sitosterol was the most prevalent phytosterol,
ranging in concentration from 24.9 mg/100 g in pumpkin
seed to 191.4 mg/100 g in peas. Squalene identified in all
foods examined in this study, was particularly abundant in
pumpkin seed (89.0 mg/100 g). The sum of α- and β+ γ-
tocopherols ranged from 0.1 mg/100 g in rye to 15.9 mg/
100 g in pumpkin seeds. Total oil content ranged from 0.9%
(w/w) in butter beans to 42.3% (w/w) in pumpkin seed and
the type of fat, in all foods examined, was predominantly
unsaturated. In conclusion, seeds, grains, and legumes are a
rich natural source of phytosterols. Additionally, they
contain noticeable amounts of squalene and tocopherols,
and in general, their fatty acid profile is favorable.
Keywords Phytosterols
.
Squalene
.
Tocopherols
.
Seeds
.
Legumes
.
Cereals
Abbreviations
CHD coronary heart disease
FAME fatty acid methyl esters
GC gas chrom atography
HPLC high performance liquid chromatography
LDL low-density lipoprotein
MUFA monounsaturated fatty acids
ND not detected
NMK nitrosaminoketone
PUFA polyunsaturated fatty acids
SCE sister chromatid exchange
SFA saturated fatty acids
Tr trace
Introduction
Phytosterols, squalene, and tocopherols are components
present in the unsaponifiable lipid fraction of foods.
Phytosterols, primarily β-sitosterol, campesterol, and stig-
masterol are integral natural components of plant cell
membranes that are abundant in vegetable oils, nuts, seeds,
and grains, [1] and added components in enriched margarines
[2]. Whilst phytosterols are proposed to have a wide
spectrum of biological effects including anti-inflammatory,
anti-oxidative, and anticarcinogenic activities [3, 4], their
cholesterol-lowering capacity has been the most extensively
researched. Several studies have shown that plant sterols
inhibit the intestinal absorption of cholesterol, thereby
lowering total plasma cholesterol and low-density lipoprotein
(LDL) levels [3].
Squalene, a 30 carbon isoprenoid, is a key intermediate
in cholesterol biosynthesis and is abundant in shark liver oil
(Squaluss spp.) and olive oil. Several studies have indicated
Plant Foods Hum Nutr (2007) 62:85–91
DOI 10.1007/s11130-007-0046-8
E. Ryan
:
K. Galvin
:
T. P. O’Connor
:
N. M. O’Brien (*)
Department of Food and Nutritional Sciences, University College,
Cork, Ireland
e-mail: nob@ucc.ie
A. R. Maguire
Department of Chemistry and School of Pharmacy,
Analytical and Biological Chemistry Research Facility,
University College,
Cork, Ireland

that squalene is an important dietary cancer chemopreven-
tive agent [5]. More recently, squalene has been shown to
act as an antidote to reduce accidental drug-induced toxicities
[6, 7]. The protective effect of squalene may be attributed to
its ability to serve as an antioxidant. It has been demonstrat-
ed to be a potent quencher of singlet oxygen [8] and protects
against H
2
O
2
-induced sister chromatid exchange (SCE) in
Chinese hamster V79 cells [9].
Tocopherols, the major vitamers of vitamin E, are fat-
soluble antioxidants that function as scavengers of lipid
peroxyl radicals. Knekt et al. [10] and Kushi et al. [11]
demonstrated that the tocopherol content in food is
inversely associated with mortality from cardiovascular
disease. In addition, tocopherols, due to their capacity to
quench free radical damage, play a putative role in
prevention of Alzheimer’s disease and cancer [12].
In the past few years the beneficial health effects attributed
to both phytosterols and tocopherols and to a lesser extent
squalene have prompted interest in quantifying these com-
pounds in different foods. Current food databases contain
limited or dated compositional data with respect to these
components. Therefore, the present study attempted to
determine the composition of plant foods with respect to
phytosterol, tocopherol, and squalene content. Additionally,
current dietary guidelines emphasize a diet rich in plant foods
including whole grains, seeds, and legumes. Total lipid and
fatty acid profile of these foods was also analysed in this study.
Materials and Methods
Samples
Five types of seed; Linum usitatissimum (linseed), Brassica
nigra (mustard), Papaver sonniferum (poppy), Cucurbita
spp. (pumpkin), and Sesamum indicum (sesame) seeds,
seven types of grain; Hordeum vulgare (barley), Fagopyrum
esculentum (buckwheat), Zea mays (maize), Pennisetum
americanum (millet), Che nopodium quinoa (quinoa),
Secale cereale (rye), and Triticum spelta (spelt), and five
types of legumes; Phaseol us lunatus (butter beans), Cicer
arietinum (chick peas), Phaseolus vulgaris (kidney beans),
Lens culinaris (lentils), and Pisum sativum (peas-marrow-
fat) were analysed in this study. The items were bought
from a local health food store in Cork, Ireland. Solvents
[high-performance liquid chromatography (HPLC) grade]
were obtai ned from JT Baker (London, UK).
Acid Hydr olysis for Sterol, Tocopherol and Squalene
Analysis
Samples were finely ground (1.0 mm mesh size) using a
Moulinex Optiblend 2000 and 1 g of each sample was
weighed into a 25×150 mm Pyrex culture tube with Teflon-
lined screw cap. Samples were spiked with 2.5 ml internal
standard (50 μg 6-ketocholesterol dissolved in 2.5 ml
ethanol). Samples were hydrolysed under acidic conditions
by a modification of a procedure previously described by
Toivo et al. [13]. Briefly, 1 ml of absolute ethanol and 5 ml
of 6 M HCl were added to each tube and samples were
shaken vigorously. Tubes were then kept at 80°C for 1 h in
a water bath, during which tubes were shaken every
10 min. The tubes were then cooled on ice and 5 ml
ethanol, 10 ml hexane/diethyl-ether (1:1, v/v) were added to
each sample. Tubes were vortexed for 1 min and then
centrifuged at 1,000 rpm for 10 min. The upper solvent
layer was removed and the extraction repeated with a
further 10 ml hexane/diethyl-ether. The combined extracts
were dried under nitrogen and stored in a refrigerator until
saponified.
Saponification for Sterol, Tocopherol and Squalene
Analysis
Samples were s aponified by a procedure previously
described by Maguire et al. [
14]. Briefly, the dried extract
was mixed thoroughly with 300 μl of 50% KOH (w/v) and
2 ml of 1% ethanolic pyrogallol (w/v) in screw-top tubes
fitted with Teflon-lined screw-caps. The tubes were kept for
30 min at 70°C in a water bath. The tubes were cooled on
ice and 1 ml water and 4 ml hexane were added. The tubes
were shaken vigorously and then centrifuged at 2,000 rpm
for 10 min. The hexane layer was removed and the
extraction repeated with a further 2 ml hexane. The
combined extracts were dried under nitrogen. The extract
was redissolved in 200 μl ethanol, transferred to a plastic
insert in a HPLC vial and stored at −20°C until further
analysis by HPLC.
Analysis of Phytosterols, Squalene and Tocopherols
by HPLC
The HPLC syst em consisted of a Waters 510 pump and a
Waters 717 plus autosampler (Waters Corporation, Milford,
Massachusetts, USA). For phytosterol analyses , 20 μl
sample was injected onto a Luna C8 (2) column (250×
4.6 mm i.d.; Phenomenex, Cheshire, UK). Detection was
done by a Waters 995 photodiode array detector. The
mobile phase was 80% acetonitrile and 20% water at a
flow rate of 1.6 ml/min. Column temperature was main-
tained at 50°C. The HPLC system used for squalene and
tocopherol analysis was the same, except the column used
was a Supelcosil LC-18-DB (250×4.6 mm i.d.; Supelco,
Bellefonte, Pennsylvania, USA). The mobile phase was
99% methanol and 1% water at a flow rate of 1.2 ml/mi n.
Column temperature was maintained at 25°C. Peak areas
86 Plant Foods Hum Nutr (2007) 62:85–91

were recorded using Millennium 32 Chromatography Man-
ager software (Waters Corporation, Milford, Massachusetts,
USA). For phytosterol, squalene and tocopherol analysis,
chromatograms wer e measured at 205, 215 and 292 nm,
respectively. Concerning tocopherol analysis, reverse phase
chromatography does not distinguish between the β and γ-
isomers of tocopherol, thus the sum of these isomers is
shown throughout as β + γ-tocopherol.
Lipid Extraction for the Determination of % Oi l
and Fatty Acid Profile
Samples (2 g) were finely ground (1.0 mm mesh size) using
a Moulinex Optiblend 2000. The oil from the finely ground
samples was extracted by a modification of a procedure
previously described [15]. Briefly, oil was extracted with
6 ml hexane/isopropanol (3:2, v/v) at room temperature
under vigorous stirring for 1 h in glass beakers to facilitate
homogenisation of the food. The food preparations were
filtered through a vacuum, the residues were washed twice
with 4 ml hexane/isop ropanol solvent. Thereafter, 7 ml of
6.7% sodium sulphate (w/v) were added and the samples
were vortexed for 30 s and centrifuged at 2,000 rpm for
10 min. The solvent layer was removed, dried under
nitrogen and the pure oil was weighed to calculate the
percentage yield.
Preparation of Fatty Acid Methyl Esters
Fatty acid methyl esters (FAME) were prepared from
extracted oil by the method of Slover and Lanza [16].
Briefly, approximately 40 mg extracted oil were treated
with 1 ml methanolic NaOH at 100°C for 15 min in 25×
150 mm Pyrex culture tube with Teflon-lined screw cap.
The tubes were cooled on ice, 2 ml boron trifluoride were
added and the tubes were boiled for a further 15 min. The
tubes were cooled on ice, then 1 ml isooctane and 2 ml
saturated sodium chloride were added, shaken vigorously
and left to stand to allow the layers to separate. The upper
hexane layer containing the FAME was transferred to a
small tube and stored at − 20°C until further analysis by gas
chromatography (GC).
FAME Analysis by GC
For FAME analysis, a DB-WAX capillary column (30 m×
0.32 mm i.d.; J and W Scientific, Folsom, California, USA)
was used. The column was connected to a Shimadzu GC-
14A (Kyoto, Japan) gas chromatograph equipped with a
flame-ionization detector. Nitrogen was used as the carrier
gas. The temperature programme was as follows: initial
temperature 50°C; increase to 200°C at 10°C/min, hold for
25 min; and increase to 230°C at 10°C/min, hold for
20 min. Injector and detector temperatures were 250°C.
Chromatograms were recorded using Millennium 32 chro-
matography manager software (Waters Corporation, Milford,
Massachusetts, USA).
Results and Discussion
In the last decade, few functional food ingredients have
created more interest than phytosterols. When consumed in
enriched products, these bioactive plant components have
been shown to significantly reduce LDL cholesterol [3].
Whereas most clinical studies have involved relatively high
doses of phytosterols (2–7 g/day) using enriched foods,
research by Ostlun d et al. [17] and Andersson et al. [18] has
suggested that much lower levels of phytosterols, such as
those that occur naturally in diets rich in plant foods, may
be effective i n reducing cholesterol absorption. More
research is needed to clarify this association. For this task,
reliable food composition data is warranted. Some data
exist on phytosterol content in nuts and seeds [19],
vegetables, fruits, and berries [20], and cereals [21].
However, data on certain foods are limited or dated. For
instance, the only existing data on phytosterol content in
legumes are those reported by Weihrauch and Gardner [1].
In the present study the phytosterol contents of various
seeds, grains, and legumes were analysed. Both free sterols
and sterols bound to conjugates (esters and glycosides),
were measured via a combination of both acid and alkaline
hydrolysis. The levels of phytosterols (β-sitosterol, cam-
pesterol, and stigmasterols) in seeds ranged from 33.3 mg/
100 g (pumpkin seed) to 202 mg/100 g (sesame seed) with
β-sitosterol been the most abundant. Pumpkin seed was
unusual, insofar as the β-sitosterol content was quite low
(24.9 mg/100 g). Similarly, Philips et al. [19] indicate that
pumpkin seed kernel contains 13.1 mg/100 g β-sitosterol.
In addition, the latter study noted that whilst β-sitosterol is
the predominant sterol in virtually all plant foods, pumpkin
seed was found to contain 241 mg/100 g (>90%) of other
sterols (identified as any peak in the gas chromatography-
flame ionization detection (GC-FID) chromatograms that
had a retention time in the sterol region). Phytosterol
content in grains ranged from 43.6 mg/100 g (maize) to
106.5 mg/100 g (buckwheat). Normen et al. [21] reported
similar levels for millet, maize, rye, and buckwheat. Again
β-sitosterol was found to be the main sterol. Whilst cereals
generally contain lesser amounts of phytos terols than seeds
or legumes, nonetheless, they represent a very important
dietary source of phytosterols. In three European studies,
cereals and cereal products have been found to be the main
contributors to phytosterol intake [22–24]. Total phytoster-
ol content detected in the legumes ranged from 134 mg/
100 g (kidney beans) to 242 mg/100 g (peas). Whilst
Plant Foods Hum Nutr (2007) 62:85–91 87

Weihrauch and Gardner [1] reported similar phytosterol
levels for kidney beans at 127 mg/100 g, they reported a
much lower concentration of phytosterols for chick peas,
35 mg/100 g as opposed to 205 mg/100 g in the present
study. Butter beans and kidney beans contained high levels
of stigmasterol (86.2 mg/100 g an d 41.4 mg/100 g ,
respectively). In this regard, legumes seem to have a very
different phytosterol profile to other food groups, Table 1.
Squalene, a biosynthetic precursor to all steroids both in
plant and animal cells, also exists with phytosterols and
tocopherols in the unsaponifiable fraction of foods. There is
an obvious scarcity of data on squalene content in foods.
Squalene was identified in all foods employed in the
present study; levels were notably high in pumpkin seed
(89.0 mg/100 g) and quinoa (58.4 mg/100 g). Among plant
foods, amaranth, a pseudo cereal grain, contains relatively
high amoun ts of squalene, approximately 132 mg/100 g to
424 mg/100 g [25]. Research indicates that amaranth oil
may have significant benefit for patients with CHD, this
effect may be due, in part, to its high content of squalene
[26]. Another exceptionally rich source of squalene is olive
oil, which is reported to contain 2,000 to 7,000 μg/g oil
[27]. An inverse relationship between olive oil consumption
and cancer risk has been observed, and may, in part be due
to the presence of squalene. Experimental studies have
shown that squalene can inhibit chemically induced colon,
lung and skin tumorigenesis in rodents [5]. In addition,
several experimental studies demonstrated the detoxifying
activities of squalene against a wide range of chemicals
such as arsenic, hexachlorobenzene and phenobarbital.
Therefore, it is suggested that squalene may act as a sink for
highly lipophilic xenobiotics, assisting in their elimination
from the body [28–30]. In addition, it is reported that
squalene exhibits protective activity against several carci-
nogens, including azoxymethane induced colon cancer [31]
and nicotine derived nitrosaminoketone (NMK) induced
lung carcinogenesis [32]. Whilst the squalene content of
the foods employed in the present study is lower than that of
the squalene content reported for amaranth grain and olive
oil, the abundance of plant foods in our diet suggest that they
represent a significant source of squalene.
Most plant-derived foods contain low to moderate levels
of vitamin E activity. However, owing to the abundance of
plant-derived foods in our diets, they provide a significant
and consistent source of vitamin E [33]. In the present
study, α- and β + γ-tocopherol content of the selected
foods was also measured. Pumpkin seeds were found to
have the greatest content of tocopherols (16 mg/100 g) with
β + γ-tocopherol being predominant over α-tocopherol.
Murkovic et al. [34] reported similar levels in pumpkin
seeds and γ-tocopherol was the primary vitamer identified.
Generally, tocopherol content was higher in seeds and
legumes than cer eals. Nonetheless, cereal grains are
considered to be a good source of tocopherols in the diet.
Piironen et al. [35]reportedthatupto30%ofthe
recommended dietary allowance of α-tocopherol equiva-
lents comes from cereal products in Finland whilst Wyatt et
al. [36] reported that corn tortillas contributed 17% of the
dietary intake of vitamin E in Mexican diets. In the present
study, the content of β + γ-tocopherol was greater than α-
tocopherol in most foods, with levels rangi ng from 0.1 mg/
100 g in rye to 14.8 mg/100 g in pumpkin seed. However,
peas contained greater amounts of α than β + γ-tocopherol
(10.4 mg/100 g and 5.7 mg/100 g, respectively) and chick
peas contained similar levels of α- and β + γ-tocopherol
(6.9 mg/100 g and 5.5 mg/100 g, respectively). Whilst α-
tocopherol may, in theory, be a more potent chain breaking
anti-oxidant, a preparation of mixed tocopherols has been
shown to have better antioxidant and anti-inflammatory
effectsinanimalmodelsandalimitednumberof
preliminary clinical studies [37], Table 2.
In order to maximize the content of phytosterols, squalene
and tocopherols in plant foods, it is important to consider
factors such as processing conditions, cultivar, growing
season and planting location [25, 38–43]. In addition,
enhanced contents of these bioactive components may be
achieved through crop engineering [44–46].
The total oil content of the foods analysed in the present
study ranged from 0.9 to 42.3% with pumpkin seed
yielding the greatest percentage of oil (Table 3). Butter
beans, barley and buckwheat were found to have the
Table 1 β-Sitosterol, campesterol, and stigmasterol content (mg/100 g)
of selected seeds, grains, and legumes
Sample β-Sitosterol
(mg/100 g)
Campesterol
(mg/100 g)
Stigmasterol
(mg/100 g)
Linseed 57.4±2.4 19.0±0.7 21.8±0.8
Mustard 74.4±3.4 26.5±1.3 2.5±0.3
Poppy 58.3±1.0 9.8±0.4 5.7±0.6
Pumkin 24.9±1.4 ND 8.4±0.3
Sesame 139.0±7.4 22.3±1.3 41.5±2.1
Barley 38.1±1.0 12.0±1.0 0.3±0.1
Buckwheat 94.5±4.1 10.4±0.4 1.6±0.2
Maize 34.1±1.1 9.1±0.5 0.4±0.0
Millet 48.3±5.5 8.7±2.4 0.8±0.3
Quinoa 63.7±4.0 15.6±8.7 3.2±0.1
Rye 58.4±5.6 16.8±1.7 0.7±0.1
Spelt 53.3±2.7 15.1±3.4 0.4±0.0
Butter beans 85.1±7.3 15.2±2.9 86.2±5.7
Chick peas 159.8±7.1 21.4±0.7 23.4±0.7
Kidney beans 86.5±2.6 6.5±0.8 41.4±1.6
Lentils 123.4±4.1 15.0±0.4 20.0±0.6
Peas 191.4±0.4 25.0±6.9 26.0±0.6
Results are the mean value ± standard error of the mean for at least
three independent experiments
ND Not detected
88 Plant Foods Hum Nutr (2007) 62:85–91

greatest % saturated fatty acids (28.7, 22.5 and 21.9%,
respectively). The levels of total unsaturated fatty acids
ranged from 71.4% in butter beans to 93.7% in mustard
seed. The major MUFA present in all foods was oleic acid
(C18:1) which was particula rly high in buckwheat and
sesame seed. Linoleic acid (C18:2) was the most abundant
polyunsaturated fatty acid (PUFA) identified in most foods.
PUFA (n -6) ha ve n um erou s ben ef icia l effects on cardio-
vascular disease including improved blood lipid profile
[47], improved insulin sensitivity [48], lower incidence of
type 2 diabetes [49] and anti-arrhythmic effects [50].
Exceptionally, in linseed and kidney beans, the main
PUFA was the essential fatty acid, linolenic acid (n-3
PUFA). Prospective cohort studies and secondary inter-
vention trials have provided strong evidence that an
increasing intake of n-3 fatty acids from fish o r plant
sources s ubstantially lowers risk of cardiovascu lar morta l-
ity [ 51]. Certainly, whether it is n-3/n-6 PUFA, or MUFA,
there is strong evidence that replacing saturated with
unsaturated fat is far more effective in lowering risk of
CHD than simply reducing total fat consumption. Among
the foods a nalysed, mustard seed h ad a different fatty acid
profile, ins ofar as the fat was p rimar ily monounsaturated
due to its exceptionally high content of erucic acid
(C22:1). There is some s uggestion that erucic acid-rich
mustard may bear a cardiotoxic or pro-oxidant substrate
[52].
In conclusion, the present study indicates that seeds,
legumes, and cereal grains are good natural sources of
phytosterols. Additionally, they contain appreciable amounts
of squalene, α-andβ + γ-tocopherol, and generally, their
fatty acid profile is favorable from a cardio-protective
perspective.
Table 2 Squalene, α-Tocopherol and β + γ-T ocopherol content (mg/100 g)
of selected seeds, grains, and legumes
Sample Squalene
(mg/100 g)
α-Tocopherol
(mg/100 g)
β + γ-Tocopherol
(mg/100 g)
Linseed 1.0±0.04 0.1±0.02 8.2±0.41
Mustard 0.5±0.05 0.6±0.02 6.3±0.30
Poppy 0.6±0.01 0.2±0.02 4.7±0.13
Pumpkin 89.0±8.70 0.9±0.06 14.8±0.78
Sesame 0.6±0.04 Tr 10.0±0.26
Barley 0.2±0.08 1.5±0.06 0.1±0.01
Buckwheat 1.9±0.58 0.1±0.04 4.5±0.28
Maize 1.6±0.60 0.2±0.03 1.1±0.02
Millet 8.8±0.80 0.2±0.05 2.4± 0.20
Quinoa 58.4±0.69 2.1±0.22 3.1±0.07
Rye 0.3±0.05 Tr 0.1±0.01
Spelt 2.0±0.68 0.6±0.09 0.5±0.06
Butter beans 0.4±0.02 0.7±0.18 4.7±0.40
Chick peas 0.5±0.03 6.9±0.04 5.5±0.72
Kidney beans 0.7±0.05 1.2±0.16 2.6±0.13
Lentils 0.7±0.15 1.6±0.43 4.5±0.11
Peas 1.0±0.07 10.4±0.09 5.7±0.64
Results are the mean value ± standard error of the mean for at least
three independent experiments
Tr Trace amounts (<0.1 mg/100 g)
Table 3 Total oil content (g/100 g) and fatty acid composition (% of total) of various seeds, grains, and legumes
Sample Total oil Fatty acid
16:0 16:1 17:0 18:0 18:1 18:2 18:3 20:0 20:1 22:0 22:1 SFA MUFA PUFA
Linseed 29.3 6.59 0.08 1.25 4.11 23.99 19.90 43.27 0.39 0.23 0.19 ND 12.5 24.3 36.2
Mustard 15.2 4.15 0.11 ND 1.40 26.28 10.68 8.16 0.53 9.68 0.53 38.76 4.9 74.8 18.8
Poppy 39.5 12.20 0.27 0.76 2.30 22.19 59.87 1.30 0.67 0.16 ND 0.29 13.7 22.9 61.2
Pumpkin 42.3 14.00 0.16 0.11 6.93 35.80 40.70 0.34 1.43 0.21 0.17 0.26 22.7 36.4 41.0
Sesame 40.5 8.62 0.11 ND 5.43 39.09 40.39 0.69 1.77 3.77 0.12 0.29 15.9 43.3 41.1
Barley 1.3 20.45 0.07 ND 1.28 14.88 58.01 4.37 0.24 0.21 0.49 ND 22.5 15.2 62.4
Buckwheat 2.7 16.96 0.15 ND 2.00 40.91 34.43 1.76 0.83 0.12 2.08 0.73 21.9 41.9 36.2
Maize 1.6 12.48 0.26 0.11 1.96 29.26 52.99 1.62 0.57 0.50 0.20 ND 15.4 30.0 54.6
Millet 4.0 8.54 0.20 ND 1.37 23.16 64.40 1.06 0.57 0.36 0.35 ND 10.8 23.7 65.5
Quinoa 6.3 9.18 0.27 0.06 0.59 29.49 48.07 7.99 0.52 1.57 0.71 1.43 11.2 32.8 56.1
Rye 1.3 14.98 0.08 ND 0.79 17.36 58.71 6.80 0.61 0.49 ND 0.18 16.4 18.1 65.5
Spelt 2.0 15.30 0.15 ND 0.97 21.51 57.33 3.45 0.71 0.49 0.19 ND 17.2 22.2 60.8
Butter Bean 0.9 23.68 0.20 0.37 3.62 10.35 42.43 18.34 ND ND 0.30 ND 28.7 10.5 60.8
Chick Peas 5.0 10.87 0.23 0.06 1.85 33.51 49.74 2.41 0.60 0.39 0.21 Tr 13.7 34.2 52.1
Kidney Bean 1.2 14.20 0.16 0.22 1.30 11.97 26.04 45.69 0.24 ND 0.51 ND 16.5 12.1 71.7
Lentils 1.4 14.57 0.09 0.13 1.24 22.95 47.17 11.67 0.44 0.70 0.28 ND 16.7 23.7 58.8
Peas 1.5 10.65 0.07 0.19 3.11 28.15 47.59 9.29 0.22 0.21 ND ND 14.7 28.4 56.9
Results are the mean for at least three independent experiments
SFA Saturated fatty acids, MUFA monounsaturated fatty acids, PUFA polyunsaturated fatty acids, ND not detected, Tr trace amounts (<0.05)
Plant Foods Hum Nutr (2007) 62:85–91 89

Acknowledgements This work has been supported by Enterprise
Ireland Basic Research Grant.
References
1. Weihrauch JL, Gardner JM (1978) Sterol content of foods of plant
origin. J Am Diet Assoc 73:39–44
2. Moreau RA, Whitaker BD, Hicks KB (2002) Phytosterols,
phytostanols, and their conjugates in foods: structural diversity,
quantitative analysis, and health promoting uses. Prog Lipid Res
41:457–500
3. de Jong N, Plat J, Mensink RP (2003) Metabolic effects of plant
sterols and stanols. J Nutr Biochem 4:362–369
4. Berger A, Jones PJH, Abumweis SS (2004) Plant sterols: factors
affecting their efficacy and safety as functional food ingredients. This
article is available from: http://www.lipidworld.com/content/3/1/5
5. Smith TJ (2000) Squalene: potential chemopreventive agent.
Expert Opin Invest Drugs 9:1841–1848
6. Aguilera Y, Dorado ME, Prada FA, Martinez JJ, Quesada A, Ruiz-
Gutierrez V (2005) The protective role of squalene in alcohol
damage in the chick embryo retina. Exp Eye Res 80:535–543
7. Senthilkumar S, Devaki T, Manohar BM, Babu MS (2006) Effect
of squalene on cyclophosphamide-induced toxicity. Clin Chim
Acta 364:335–342
8. Kohno Y, Egawa Y, Itoh S, Nagaoka S, Takahashi M, Mukai K
(1995) Kinetic study of quenching reaction of singlet oxygen and
scavenging reaction of free radicals by squalene in n-butanol.
Biochem Biophys Acta 1256:52–56
9. O’Sullivan L, Woods JA, O’Brien NM (2002) Squalene but not n-
3 fatty acids protect against hydrogen peroxide-induced sister
chromatid exchanges in Chinese hamster V79 cells. Nutr Res
22:847–857
10. Knekt P, Reunanen A, Jarvinen R, Seppanen R, Heliovaara M,
Aromaa A (199 4) Antioxidant vitamin i ntake and coronary
mortality in a longitudinal population study. Am J Epidemiol
139:1180–1189
11. Kushi LH, Folsom AR, Prineas RJ, Mink PJ, Wu Y, Bostick RM
(1996) Dietary antioxidant vitamins and death from coronary heart
disease in postmenopausal women. N Engl J Med 330:1029–1035
12. Tucker JM, Townsend DM (2005) Alpha-tocopherol: roles in
prevention and therapy of human disease. Biomed Pharmacother
59:380–387
13. Toivo J, Philips K, Lampi AM, Piironen V (2001) Determination
of sterols in foods: Recovery of free, esterified, and glycosidic
sterols. J Food Comp Anal 14:631–643
14. Maguire LS, O’Sullivan SM, Galvin K, O’Connor TP, O’Brien
NM (2004) Fatty acid profile, tocopherol, squalene and phytos-
terol content of walnuts, almonds, peanuts, hazelnuts and the
macadamia nut. Int J Food Sci Nutr 55:171–178
15. Savage GP, McNeill DL, Dutta PC (1997) Lipid composition and
oxidative stability of oils in hazelnuts (Corulus avellana L.)
grown in New Zealand. J Am Oil Chem Soc 74:755–759
16. Slover HT, Lanza E (1979) Quantitative analysis of food fatty
acids by capillary gas chromatography. J Am Oil Chem Soc
56:933–943
17. Ostlund RE, Racette SB, Okeke A (2002) Phytosterols that are
naturally present in commercial corn oil significantly reduce
cholesterol absorption in humans. Am J Clin Nutr 75:1000–1004
18. Andersson SW, Skinner J, Ellegard L, Welch AA, Bingham A,
Mulligan A, et al. (2004) Intake of dietary plant sterols is
inversely related to serum cholesterol concentration in men and
women in the EPIC Norfolk population: a cross-sectional study.
Eur J Clin Nutr 58:1378–1385
19. Philips KM, Ruggio DM, Ashraf-Khorassani M (2005) Phytos-
terol composition of nuts and seeds commonly consumed in the
United States. J Agric Food Chem 53:9436–9445
20. Piironen V, Toivo J, Puupponen-Pimia R, Lampi AM (2003) Plant
sterols in vegetables, fruit and berries. J Food Sci Agric 83:330–
337
21. Normen L, Bryngelsson S, Johnsson M, Evheden P, Ellegard L,
Brants H, et al. (2002) The phytosterol content of some cereal
foods commonly consumed in Sweden and in the Netherlands. J
Food Comp Anal 15:693–704
22. Morton G, Lee S, Buss D, Lawrance P (1995) Intakes and major
dietary sources of cholesterol and phytosterols in the British diet. J
Hum Nutr Diet 8:429–440
23. Normen AL, Brants HA, Voorrips LE, Andersson HA, van den
Brandt PA, Goldbohm RA (2001) Phytosterol intakes and
colorectal cancer risk in the Netherlands cohort study on diet
and cancer. Am J Clin Nutr 74:141–148
24. Valsta LM, Lemstrom A, Ovaskainen M-L, Lampi AM, Toivo J,
Korhonen T, et al . (2004) Es timation of plant st erol and
cholesterol intake in Finland: quality of new values and their
effect on intake. Br J Nutr 92:671–678
25. Berganza BE, Moran AW, Rodriguez G, Coto NM, Santamaria M,
Bressani R (2003) Effect of variety and location on the total fat,
fatty acids and squalene content of Amaranth. Plant Food Hum
Nutr 58:1–6
26. Martirosyan DM, Miroshnichenko LA, Kulakova SN, Pogojeva
AV, Zoloedov VI (2007) Amaranth oil application for coronary
heart disease and hypertension. Lipids Health Dis 6:1
27. Liu GCK, Ahrens EH, Schreibman PH, Crouse JR (1976)
Measurement of squalene in human tissues and plasma: validation
and application. J Lipid Res 17:38–45
28. Fan S, Ho I, Yeoh FL, Lin C, Lee T (1996) Squalene inhibits
sodium arsenite-induced sister chromatid exchanges and micro-
nuclei in Chinese hamster ovary-K1 cells. Mutat Res 368:165–169
29. Kamimara H, Koga N, Oguri K, Yoshimura H (1992) Enhanced
elimination of theophylline, phenobarbital and strychnine from the
bodies of rats and mice by squalene treatment. J Pharmacobiodyn
15:215–221
30. Richter E, Schafer SG (1982) Effect of squalene on hexachlor-
obenzene (HCB) concentrations in tissues of mice. J Environ Sci
Health B 17:195–203
31. Rao CV, Newmark HL, Reddy BS (1998) Chemopreventive effect
of squalene on colon cancer. Carcinogenesis 19:287–290
32. Smith TJ, Yang GY, Seril DN, Liao J, Kim S (1998) Inhibition of
4-(methylnitrosamine)-1-(3-pyridyl)-1-butanone-induced tumoro-
genesis by dietary olive oil and squalene. Carcinogenesis
19:703–706
33. Eitenmiller RR, Lee J (2004) Vitamin E: food chemistry,
composition and analysis. Marcel Decker, New York
34. Murkovic M, Piironen V, Lampi AM, Kraushofer T, Sontag G
(2004) Changes in the chemical composition of pumpkin seeds
during the roasting process for production of pumpkin seed oil.
Food Chem 84:359–365
35. Piironen V, Syvaoja EL, Varo P, Salminen K, Koivistoinen P
(1986) Tocopherols and tocotrienols in cereal products from
Finland. Cereal Chem 63:78–81
36. Wyatt CJ, Carballido SP, Mendez RO (1998) α- and γ-Tocopherol
content of selected foods in the Mexican diet. J Agric Food Chem
46:4657–4661
37. Saldeen K, Saldeen T (2005) Importance of tocopherols beyond
α-tocopherol: evidence from animal and human studies. Nutr Res
25:877–889
90 Plant Foods Hum Nutr (2007) 62:85–91

38. Chung TY, Nwokolo EN, Sim JS (1989) Compositional and
digestibility changes in sprouted barley and canola seeds. Plant
Food Hum Nutr 39:267–278
39. Maatta K, Lampi AM, Petterson J, Fogelfors BM, Piironen V,
Kamal-Eldin A (1999) Phytosterol content in seven oat cultivars
grown at three locations in Sweden. J Food Sci Agric 79:1021–
1027
40. Marcone MF, Kakuda Y, Yada RY (2004) Amaranth as a rich
dietary source of β-sitosterol and other phytosterols. Plant Food
Hum Nutr 58:207–211
41. Zangenberg M, Hansen HB, Jorgensen JR, Hellgren LI (2004)
Cultivar and year to year variation of phytosterol content in rye
(Secale cereale L). J Agric Food Chem 52:2593–2597
42. Kalinova J, Triska J, Vrchotova N (2006) Distribution of vitamin
E, squalene, epicatechin, and rutin in common buckwheat plants
(Fagopyrum esculentum Moench). J Agric Food Chem 54:5330–
5335
43. Zhang H, Vasanthan T, Wettasinghe M (2007) Enrichment of
tocopherols and phytosterol in canola oil during seed germination.
J Agric Food Chem 55:355–359
44. Karunanandaa B, Post-Beittenmiller M, Venkatramesh M,
Kishore GM, Thorne GM, LeDeaux JR (2004) Transgenic
plants cont aining altered lev els of steroid compounds. US patent
6,882,214,2
45. Karunanandaa B, Qi Q, Hao M, Baszis SR, Jensen PK, Wong YH
(2005) Metabolically engineered oilseed crops with enhanced seed
tocopherol. Metab Eng 7:384–400
46. Hey SJ, Powers SJ, Beale MH, Hawkins ND, Ward JL, Halford
NG (2006) Enhanced seed phytosterol accumulation through
expression of a modified HMG-CoA reductase. Plant Biotechnol
J 4:219–229
47. Keys A, Parlin RW (1966) Serum-cholesterol response to changes
in dietary lipids. Am J Clin Nutr 19:175–181
48. Lovejoy JC (1999) Dietary fatty acids and insulin resistance. Curr
Atheroscler Rep 1:215–220
49. Hu F, Salmeron J, Manson J, Stampfer M, Colditz G, Rimm E,
Willet W (1999) Dietary fat and risk of type 2 diabetes in women.
Am J Epidemiol 149:S1
50. Abeywardena MY, McLeannan PL, Charnock JS (1991) Differ-
ential effects of dietary fish oil on myocardial prostaglandin 12
and thromboxane A2 production. Am J Physiol 260:379–385
51. Hu FB, Manson JE, Willett WC (2001) Types of dietary fat and
risk of coronary heart disease: a critical review. J Am Coll Nutr
20:5–19
52. Watkins TR, Lenz PH, Siderits R, Struck M, Bierenbaum ML
(1995) Dietary mustard, rape seed oils and selenium exert distinct
effects on serum Se, lipid, peroxidation products and platelet
aggregability. J Am Coll Nutr 14(2):176–183
Plant Foods Hum Nutr (2007) 62:85–91 91