Eur. J. Lipid Sci. Technol.

2012, 000, 0000–0000 1

Research Article
Steryl glycosides and acylated steryl glycosides in plant
foods reflect unique sterol patterns

Laura Nyström1, Aline Schär1 and Anna-Maija Lampi2

1
Institute of Food, Nutrition and Health, ETH Zurich, Schmelzbergstrasse, Zurich, Switzerland
2
Department of Food and Environmental Sciences, University of Helsinki, Latokartanonkaari, Helsinki, Finland

Glycosylated plant sterols (steryl glycosides, SG and acylated steryl glycosides, ASG) are minor
glycolipids found in a variety of plants at different levels and in different compositions. They have
until now been far less studied than other sterol conjugates, though they possess at least the same
potential health benefits as free or esterified plant sterols. Additionally, due to the more hydrophilic
conjugated part, they may possess further beneficial properties in forming water-soluble structures. We
studied the content and sterol profiles of glycosylated sterols in various plant foods to be able to better
understand their levels in human diets, as well as to identify sources with high levels and/or unique sterol
profile for possible extraction for ingredients. Glycosylated sterols contributed up to almost 60% of total
sterols in e.g. potatoes, indicating that they indeed are an important group of sterols in plants. The ratio of
ASG and SG varied from 0.4 to 3.6, indicating significant differences in the proportions of these two
conjugates in various plants. Furthermore, extremely large differences were observed in the sterol profiles
not only between but also within different plants, which indicates that the sterol profile of the glycosylated
sterols does not always reflect the total sterol composition in the same plant tissue.

Practical applications: Glycosylated sterols have hydrophilic properties owing to the carbohydrate
moiety of the conjugate. As a consequence, they could potentially be introduced to water-based func-
tional foods (unlike the hydrophobic free sterols and sterol esters in current applications). Various plant
materials or side streams of food processes were shown to be good sources of these glycosylated plant
sterols, and could be used as sources for extraction of glycosylated sterols for food ingredients.
Furthermore, the detailed compositional information reported here will provide a basis to select also
the desired sterol composition for the product. Finally, the data presented in this paper is invaluable for
estimating the contribution of different foods as sources of glycosylated sterols in the diet.

Keywords: Acylated steryl glycoside / Phytosterol / Plant food / Plant sterol / Steryl glycoside

Received: January 21, 2012 / Revised: February 28, 2012 / Accepted: March 7, 2012
DOI: 10.1002/ejlt.201200033

1 Introduction
Steryl glycosides (SGs) and acylated steryl glycosides (ASGs)
are secondary plant metabolites found in a range of plant
tissues, microorganisms, and fungi [1]. These glycosylated
sterol conjugates are much less studied than the free (non-
Correspondence: Professor Laura Nyström, Institute of Food, Nutrition
and Health, ETH Zurich, Schmelzbergstrasse 9, CH-8092 Zurich,
Switzerland
E-mail: laura.nystroem@hest.ethz.ch
Fax: þ41-44-632-1123
Abbreviations: ASG, acylated steryl glycoside; bp, base peak; dm, dry
matter; fm, fresh matter; SG, steryl glycoside
conjugated) sterols and their fatty acid esters. In steryl glyco-
sides (SGs) the hydroxyl group of C-3 in the sterol body is
bound to sugar, most commonly glucose, with a glycosidic
bond (Fig. 1). The sugar moiety can further be acylated with a
fatty acid in the C-6 hydroxyl group thus forming an ASG.
Grille et al. recently reviewed the data on glycosylated plant
sterols, and concluded that compared to other glycolipids or
the other sterol conjugates, surprisingly little is known about
the functions of glycosylated sterols in plants, as well as poten-
tial effects as minor food components [1]. However, the genes
responsible for the biosynthesis of glycosylated sterols have
been recognized recently, and so more ideas on their role in
plants are available and studied in more details.
The positive health benefits of plant sterols in general are
well identified and widely acknowledged. They are added to

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2 L. Nyström et al.
CH2 OH CH 2 OH
O O
H H
H H
OH H OH
OH H OH
1.
H OH H OH
CH 2 O
O O
H
H
OH H
OH H
3.
H OH
Figure 1. Examples of the structural formulas of common ASG and SG: (1) spinasteryl glucoside, (2) stigmasteryl glucoside, and
(3) sitosteryl palmitoyl glucoside.
several functional foods as fatty acid esters or as free, non-
conjugated sterols to help in maintaining healthy cholesterol
levels. Health claims for plant sterols are accepted by both the
European Food Safety Authority (EFSA) (sterol esters and free
sterols) and the Food and Drug Administration (FDA) in the
United States (currently only sterol esters, free sterols in con-
sideration) [2, 3]. It is generally accepted that a daily dose of 1–
3 g of plant sterols significantly decreases the total cholesterol
level by 10–15%, and also decreases the LDL-cholesterol, thus
improving the ratio between cholesterol in HDLs and LDLs.
Recent studies have demonstrated that not only added
plant sterols from enriched functional foods, but also natu-
rally occurring sterols have a positive effect on cholesterol
levels in humans. A recent epidemiologic study by Klingberg
et al. [4] demonstrated a strong and significant inverse
relation between serum cholesterol levels and natural dietary
intake of plant sterols. The association was shown to be a true
link between dietary phytosterol intake and serum cholesterol
level, not merely decreased cholesterol levels associated to
high fiber and low fat diets, which are also rich in plant sterols.
The natural daily intake of plant sterols varies from about
170–440 mg/day, which is significantly lower than the dose
from enriched foods. However, doses as low as 150 mg in one
meal have been shown to significantly decrease the intestinal
absorption of cholesterol [5].
Until recently the health benefits of glycosylated sterols
have been questioned, as their metabolism in humans and
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Eur. J. Lipid Sci. Technol. 2012, 000, 0000–0000
O O
H
H 2.
other effects have been more or less unknown. However,
the recent studies of Lin et al. have demonstrated that glyco-
sylated sterols are also potential health benefiting dietary com-
ponents [6, 7]. SGs were able to decrease the absorption of
cholesterol from the gut in human subjects by 37.6%, which
was comparable to the decrease obtained with the better-
known steryl esters (30.6% decrease) [7]. A more recent study
of the same group focused on the effects of ASGs in mice, and
demonstrated similar decreases in cholesterol absorption (45
and 40% with ASGs and steryl esters, respectively) [6].
Further, it was shown that the acyl group was cleaved from
the ASG (thus forming SG), but the glycosidic bond remained
intact in the digestive tract. These studies give evidence to
believe that both ASG and SG are also effective dietary com-
ponents in cholesterol lowering, and that their intake can in
part help in decreasing the risk of cardiovascular diseases.
Literature on the occurrence, contents, and sterol com-
position of ASG and SG in plant foods is still relatively scarce.
Many of the commonly applied methods for the analysis of
total plant sterols are based on procedures for cholesterol
analysis. In these analyses sterols are hydrolyzed to non-
conjugated free sterols before analysis. However, as choles-
terol is commonly not found as a glycoside, these procedures
do not include acid hydrolysis required to hydrolyze the
glycosidic bond of SG, and therefore the glycosylated sterols
are not included in the analysis and the total sterol content.
Phillips et al. demonstrated that exclusion of the glycosylated
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Eur. J. Lipid Sci. Technol. 2012, 000, 0000–0000
sterols could cause an underestimation of 37% in the content
of total sterol content in nuts and seeds [8]. Furthermore,
even less information is available on the contents of ASG and
SG separately, or on the composition of the different mol-
ecular species of sterols found as glycosides. From a dietary
perspective, it is also important to know the content of sterols
contributed by the ASG and SG, not the mass of the overall
compound, which may vary significantly depending on the
conjugated carbohydrate (monosaccharide, disaccharide)
and the possible presence of a fatty acid (present in ASG,
absent in SG). In fact, of the molecular formula of ASG, the
contribution of the sterol is only about 50%, and so for the
calculations of sterol contents in intake estimations, one
should use a conversion factor of 0.5 to convert the content
of ASG to reflect the sterol content (and similarly a factor of
about 0.7 to SG). These kinds of conversion factors will
help in estimating the sterol content contributed by the
glycosylated sterols, but will never give an accurate content.
Another unique characteristic to glycosylated sterols com-
pared to the free or esterified sterols is the polar nature of the
carbohydrate group that gives a more hydrophilic character
and better solubility in more polar solvents. This polarity
difference between the two parts of the molecule (hydrophobic
sterol vs. hydrophilic carbohydrate) gives the glycosylated
sterols also unique amphiphilic characters in solutions. In a
recent study by Sánchez-Ferrer et al. the glycosylated sterols
were shown to form various chiral nanostructures by self-
assembly [9]. These structures are soluble in aqueous environ-
ments, and could hence be applied to foods with higher water
contents than the common sterol-enriched functional foods
(margarine, yoghurt, etc.), and thus potentially rich sources of
glycosylated sterols need to be identified.
The aim of this study was to gather detailed information
on the content and sterol composition of glycosylated sterols
in plant tissues. Further focus was put on the study of poten-
tial differences in sterol profile (composition of the different
sterol species) found in ASG and SG compared to the total
sterol profile of the same plant materials. Samples were
selected based on the current literature by choosing plant
materials (mostly such that are used as foods), in which
glycosylated sterols have been reported to occur, and
additional samples were chosen from the same botanical
families. With these data it is possible to better estimate
the intakes of glycosylated plant sterols, and to find potential
sources for their extraction.
2 Materials and methods
2.1 Samples
All samples were bought from local grocery stores. Plant
foods, in which glycosylated sterols have been reported pre-
viously, were used as a basis for sampling, and the selection
was expanded by including other plants from the same bot-
anical families. The fresh samples such as cucumber, banana,
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Glycosylated sterols in plant foods 3
banana leaves, potato (peeled), potato peel (peeled using a
peeling knife), cauliflower, aubergine, zucchini, broccoli,
melon, tomato, spinach, peppers, and beetroot were cut in
small pieces and freeze dried (458C, 0.05 mBar for 48–
72 h). The freeze-dried samples were then ground using a
Retsch Grindomix GM200. The dry samples such as adzuki-
beans (country of origin Turkey, TU), almonds with or with-
out peel (USA), amaranth (Germany), black beans (China,
CH), cashew nuts (India), chickpeas (TU), black eye beans
(TU), flaxseed (CH), hazel nut, lentils (yellow, orange and
large green TU, small green Canada), peanut (Egypt), pecan,
pine nut (Italy), pumpkin seeds (Hungary), quinoa (Bolivia),
red beans (CH), black and white sesame seeds (CH), soy
beans (Canada), sunflower seeds (CH), walnut (France),
white beans medium and small (TU) were ground directly.
The samples chestnut flour (Switzerland), lecithin, chocolate
powder, mustard seeds, and the wheat flour were analyzed
directly without any preparation. The water content was deter-
mined gravimetrically by drying overnight in an oven at 1208C.
2.2 Analysis of total sterol content
The analysis of total sterol content was performed as
described by Nyström et al. [10] with minor modifications.
Shortly, dihydrocholesterol (5a-cholestan-3b-ol, Sigma–
Aldrich, ca. 95%) in ethanol (Sigma–Aldrich, puriss p.a.)
(0.02 mg/mL) was used as internal standard. To the sample
(0.5 g) weighed in a 30 mL glass tube 2 mL (40 mg) of the
internal standard and 2.5 mL of 6 M hydrochloric acid
(Fluka) for acid hydrolysis were added. After shaking on a
vortex the sample was hydrolyzed for 60 min at 858C in a
shaking water bath, with additional shaking every tenth
minute by hand. After cooling to RT, lipids were extracted
with 10 mL of a solvent mixture heptane (Fluka, Sigma–
Aldrich, >99%) – diethylether (Sigma–Aldrich, water free,
99%) (1:1) by mixing for 10 min in a test tube rotator. The
organic layer was then transferred to a round-bottom flask
and evaporated to dryness under vacuum at 508C in a rotary
evaporator. To remove saponifiable lipids the residue was
dissolved in 4 mL ethanol and transferred again to a 30 mL
glass tube with of 0.25 mL saturated aqueous potassium
hydroxide, KOH (Sigma–Aldrich), and hydrolyzed in a shak-
ing water bath at 858C for 30 min. After cooling to RT the
unsaponifiables were extracted by adding 6 mL of water and
10 mL of cyclohexane (Sigma–Aldrich, >99.5%) and mixing
for 10 min in a test tube rotator. The organic phase was then
collected and evaporated to dryness under vacuum at 508C in
a rotary evaporator. The residue was dissolved in 2 mL mix of
heptane–diethylether (9:1).
To purify the unsaponifiable fraction LC-Si SPE car-
tridges with a bead load of 500 mg (Supelco, USA) were
activated with 5 mL heptane before the sample was loaded to
the cartridge. After sample addition, the cartridges were
washed with 5 mL heptane and 5 mL heptane–diethylether
(9:1). After this the free sterols were eluted with 5 mL of
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4 L. Nyström et al.
heptane–diethylether (1:1). The collected sample was evap-
orated to dryness under nitrogen at 508C. The residue was
dissolved in 0.6 mL dichloromethane (Sigma–Aldrich,
99.9%) from which 200 mL were transferred to a vial
and silylated and analyzed by GC as described below.
2.3 Analysis of glycosylated sterols (ASG and SG)
To extract the glycosylated sterols an accelerated solvent
extractor (ASE 350 Dionex) was used. Each cell was loaded
with 2 g sample. The temperature was set to 1208C and
the pressure to 1500 psi. The static time was 10 min, the
purge 60 s, and the flush 60%. One extraction consisted of
three runs and the solvent used was acetone (Sigma–Aldrich,
99.8%). The extract was then transferred to a round-
bottom flask and evaporated to dryness under vacuum at
508C, the residue was dissolved in 2.5 mL mix heptane–
isopropanol (Fluka, 99%) at a ratio 95:5.
To separate the SG and ASG and eliminate other sterols a
fractionation by SPE was performed. The correct separation
was verified by TLC (plates: silica gel 60, F245, Merck; eluent:
chloroform, methanol, 9:1). The Diol SPE-cartridge (GL
Science, InertSep1, 500 mg, 3 mL) was activated with
5 mL of heptane, before the sample was loaded to the cartridge.
After washing the column with 5 mL of heptane and 5 mL mix
of heptane and isopropanol (97:3), the ASG were eluted with
7.5 mL of heptane–isopropanol (92:8). Finally the SG were
eluted with 7.5 mL of heptane–isopropanol (85:15). The sol-
vents were evaporated under nitrogen at 508C. The further
steps were performed in parallel for the SG and ASG fractions
separately. The residue was dissolved in 2 mL of internal
standard solution as described above and transferred to a
30 mL glass tube. The acid hydrolysis and the saponification
were performed as described above for total sterols with the
exceptions that 1 mL of milk (UHT, full fat) was added to the
extract before acid hydrolysis to protect the sterols from
destruction by acid. Further, the time for acid hydrolysis
was reduced to 30 min, and after the saponification the residue
was dissolved in 1 mL dichloromethane instead of 0.6 mL.
Finally 200 mL of this solution was transferred to a vial for
TMS-derivatization.
2.4 GC-analysis
The dichloromethane was evaporated under nitrogen at 508C
and the residue dissolved in 100 mL pyridine (Sigma–
Aldrich, 99.8, water free) and 100 mL silylation reagent
by mixing thoroughly. The silylation reagent consisted of
BSTFA (Sigma–Aldrich, >98%) and TMCA (Sigma–
Aldrich, 98%) at a ratio 99:1. The sample was derivatized
at 608C for 30 min in an oven before it was evaporated to
dryness under nitrogen at 508C. The residue was dissolved in
200 mL heptane and transferred to a GC vial.
A Trace GC 2000 gas chromatograph from
ThermoQuest, CE Instruments equipped with an
ß 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
Eur. J. Lipid Sci. Technol. 2012, 000, 0000–0000
autosampler, on column injector and an FID (3008C) was
used. The column was an RTX-5 fused-silica capillary
column (diphenyl and dimethyl polysiloxane [5:95];
60 m  0.32 mm id, 0.1 mm film, Restek Corp.,
Bellefonte, PA, USA). As carrier gas helium was used with
a constant pressure of 130 kPa. The oven program was
set the following: initial temperature 708C for 1 min then
an increase to 2458C with a speed of 608C/min a further
increase to 2758C with 38C/min at which the temperature was
held for 32 min. The quantification was based on internal
standard (dihydrocholesterol) and a ratio of 1:100 or
higher in the peak areas (Sample: ISTD) was required for
quantification.
2.5 Identification of sterols with GC-MS
GC-MS was used to verify the identification of sterols and
with special emphasis to characterize D7-sterols. Aliquots of
ca 200 mL of the dichloromethane solutions of total sterol,
ASG and SG fractions were taken for TMS-derivatization.
Derivatization was done as described above, and the residue
was dissolved in 100–200 mL heptane and transferred to a
GC vial. GC-MS analyses were done according to Soupas
et al. [11] with minor modifications. Hewlett-Packard 6890
GC (Wilmington, PA, USA) equipped with an Agilent 5973
MS (Palo Alto, CA, USA) was used. The column was other-
wise similar than used in the GC-FID analysis except for the
column internal diameter (60 m  0.25 mm id, 0.10 mm
film). The injector temperature was 2758C, and splitless
injection was used. Helium was used as carrier gas with a
constant flow of 1.2 mL/min. The oven temperature program
was as follows: initial temperature 1508C for 1 min, an
increase to 2458C with a speed of 608C/min and a 1 min
hold, and a further increase to 2808C with a speed of 38C/min
at which the temperature was held for 35 min. MS conditions
were as follows: interface temperature 2808C, ion source
2308C, electron impact ionization at 70 eV. Identification
of sterols was done in full-scan mode (m/z 100–600). The MS
obtained were compared to those presented by, e.g., Rahier
and Benveniste [12] and Goad and Akihisa [13].
3 Results and discussion
Glycosylated sterols (ASG and SG) were analyzed from
various plant food sources (Table 1), and significant differ-
ences were observed in the ASG and SG contents, as well as
their sterol profiles. The results are expressed as the content
of free sterols (contributed by ASG and SG) without the mass
of the conjugated part (sugar or sugar þ fatty acid). To
compare our results to the values in the literature on the
entire molecules, we have used a conversion factor of
0.503 and 0.720 for ASG and SG, respectively. These cor-
respond to the molecular proportion of the sterol moiety in
sitosteryl palmitoyl glucoside and sitosteryl glucoside, which
are abundantly found glycosylated sterols in plants.
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Eur. J. Lipid Sci. Technol. 2012, 000, 0000–0000 Glycosylated sterols in plant foods 5

Table 1. Contents of total sterols, ASGs, and SGs mg/g dm in various plants; contents are expressed as sterol contents without the
contribution of the conjugated sugars and fatty acids.

(A) Samples dominated with D5-sterols

Sitosterol Campesterol Stigmasterol Stanols Other sterols Total

ASG/SG-
Moisture n Mean SD Mean SD Mean SD Mean SD Mean SD Mean SD % ratio

Fabaceae
Adzukibeans Total 14.2% 3 172.1 4.2 12.5 0.5 279.8 1.6 17.1 5.9 126.5 15.7 608.1 9.7 2.75
ASG 3 39.5 0.6 2.1 0.2 61.1 3.2 0.3 0.02 17.4 1.7 120.5 23.9 19.8%
SG 3 15.7 0.9 1.1 0.3 20.5 1.4 0.3 0.01 6.2 0.6 43.8 3.2 7.2%
Black beans Total 10.0% 3 325.0 16.3 116.0 6.5 87.0 4.8 7.4 0.5 32.3 7.6 567.6 27.8 1.79
ASG 3 63.5 5.1 23.7 1.6 14.0 0.9 0.5 0.02 2.7 0.3 104.3 19.2 18.4%
SG 3 35.9 0.8 13.7 0.4 7.0 0.3 0.3 0.01 1.6 0.2 58.4 6.0 10.3%
Chickpeas Total 9.5% 3 496.8 19.4 82.8 3.3 59.1 2.1 27.8 1.4 86.8 11.8 753.2 26.4 1.05
ASG 3 40.1 3.4 5.2 0.6 2.6 0.1 0.2 0.2 2.2 0.3 50.4 3.4 6.7%
SG 3 38.1 6.7 5.1 1.0 2.5 0.3 0.3 0.04 2.1 0.3 48.1 3.1 6.4%
Black eye beans Total 10.5% 3 180.2 9.9 44.1 2.1 280.1 5.4 2.6 0.6 87.8 19.6 594.8 17.6 2.79
ASG 3 30.9 1.1 4.8 0.3 85.5 6.5 0.1 0.02 7.1 0.6 128.4 27.7 21.6%
SG 3 15.3 0.7 2.2 0.2 25.5 2.1 0.3 0.1 2.8 0.3 46.0 3.6 7.7%
Lentils green small Total 10.9% 3 584.5 32.7 70.3 4.6 93.1 5.9 36.7 4.8 59.2 10.8 843.8 43.8 0.85
ASG 3 36.4 6.6 3.3 0.1 3.6 0.3 0.2 0.01 2.7 1.1 46.2 2.5 5.5%
SG 3 43.0 2.0 4.2 0.2 5.0 0.1 0.4 0.03 2.2 0.1 54.7 3.5 6.5%
Lentils green large Total 11.6% 3 625.5 13.5 91.3 5.6 83.2 3.2 36.8 2.5 22.0 1.6 858.7 25.2 1.00
ASG 3 53.1 7.4 5.5 1.1 5.1 1.0 0.4 0.3 2.6 0.3 66.6 5.2 7.8%
SG 3 54.2 9.9 5.3 1.0 5.2 1.0 0.3 0.1 1.7 0.3 66.7 5.2 7.8%
Lentils orange Total 11.1% 3 543.6 23.5 77.6 2.7 67.8 2.3 29.5 4.5 46.6 10.8 765.1 28.2 1.19
ASG 3 46.6 1.5 4.7 0.02 4.4 0.2 0.1 0.1 1.6 0.1 57.4 4.3 7.5%
SG 3 39.0 3.6 4.5 0.2 3.2 0.1 0.1 0.1 1.6 0.5 48.4 3.1 6.3%
Lentils yellow Total 9.1% 3 535.4 31.1 42.4 1.6 27.0 2.0 20.7 1.3 83.4 20.5 708.8 46.4 1.22
ASG 3 48.2 3.4 3.2 0.04 1.7 0.3 0.1 0.03 4.0 0.7 57.3 4.6 8.1%
SG 3 41.0 0.6 2.6 0.2 1.5 0.1 0.1 0.02 1.9 0.5 47.1 3.1 6.6%
Peanuts Total 2.2% 3 716.7 56.6 127.0 10.8 102.7 16.5 11.5 2.7 145.5 29.2 1103.4 109.9 1.74
ASG 3 52.2 7.3 8.4 1.3 10.3 1.3 0.2 0.03 5.6 1.2 76.7 5.3 7.0%
SG 3 30.7 2.8 5.7 0.4 4.7 0.8 0.2 0.02 2.8 0.4 44.1 1.8 4.0%
Red beans Total 10.9% 5 378.1 38.2 26.2 2.3 226.8 20.7 3.1 0.3 73.7 10.5 708.0 87.7 2.40
ASG 3 46.5 2.2 2.1 0.1 40.0 1.8 0.1 0.01 3.1 0.3 91.8 11.9 13.0%
SG 3 21.0 1.1 1.1 0.1 14.4 0.8 0.1 0.02 1.7 0.2 38.2 2.1 5.4%
Soy beans Total 9.8% 3 288.4 9.0 128.9 2.2 94.7 1.7 8.0 1.2 83.7 12.9 603.7 17.3 1.17
ASG 3 57.2 1.5 27.3 2.8 16.2 0.9 0.6 0.1 3.2 0.5 104.5 18.1 17.3%
SG 3 50.0 5.7 23.4 1.3 13.5 1.3 0.5 0.02 1.8 0.1 89.2 13.2 14.8%
White beans small Total 11.3% 3 296.5 13.5 30.5 2.1 234.9 9.1 8.4 0.8 87.2 12.2 657.5 34.7 1.85
ASG 3 51.3 1.7 3.5 0.1 47.3 2.0 0.1 0.04 5.8 0.4 108.0 17.7 16.4%
SG 3 28.7 5.7 2.3 0.4 24.0 5.5 0.2 0.02 3.0 0.5 58.2 5.2 8.9%
White beans large Total 15.3% 4 343.9 21.3 34.0 2.8 221.4 9.4 7.7 0.9 90.7 17.6 697.7 37.9 3.62
ASG 3 48.9 1.4 3.3 0.05 43.8 0.9 0.3 0.1 4.2 0.2 100.5 14.5 14.4%
SG 3 14.3 1.4 1.2 0.05 10.5 0.9 0.2 0.1 1.5 0.2 27.7 1.1 4.0%
Brassicaceae
Broccoli Total 91.7% 3 1836.1 27.5 365.2 6.1 59.6 4.7 21.8 1.1 110.8 25.9 2393.4 23.5 1.13
ASG 3 103.2 32.3 18.5 7.2 3.6 1.2 0.6 0.3 2.7 0.8 128.5 6.9 5.4%
SG 3 92.2 13.9 16.4 3.7 2.1 0.5 0.4 0.2 2.2 0.7 113.4 5.4 4.7%
Cauliflower Total 93.1% 3 2854.2 111.6 935.5 27.5 260.2 10.0 52.6 36.4 46.0 11.5 4148.6 156.4 1.73
ASG 3 233.1 27.2 58.5 4.8 12.6 2.0 0.4 0.1 6.4 1.3 310.9 23.3 7.5%
SG 3 133.2 5.1 34.0 2.1 7.1 0.4 0.5 0.4 4.8 0.9 179.6 7.8 4.3%
Mustard seeds Total 7.6% 3 1356.6 41.6 626.0 16.1 11.3 1.4 12.7 4.0 341.0 11.8 2347.5 60.4 1.23
ASG 3 48.4 4.0 15.7 1.3 0.3 0.1 0.7 0.3 7.2 0.6 72.2 2.2 3.1%
SG 3 39.0 1.0 13.4 0.7 0.1 0.1 0.4 0.1 5.6 0.1 58.6 1.5 2.5%

(Continued)

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6 L. Nyström et al. Eur. J. Lipid Sci. Technol. 2012, 000, 0000–0000

Table 1. Continued

(A) Samples dominated with D5-sterols

Sitosterol Campesterol Stigmasterol Stanols Other sterols Total

ASG/SG-
Moisture n Mean SD Mean SD Mean SD Mean SD Mean SD Mean SD % ratio

Solanaceae
Aubergine Total 93.2% 3 687.0 33.9 82.4 5.0 315.0 15.6 5.1 1.5 83.4 8.2 1173.0 56.2 2.46
ASG 3 116.3 6.5 10.7 0.9 33.6 1.8 0.3 0.1 5.3 0.9 166.1 23.5 14.2%
SG 3 48.3 4.2 4.1 0.3 12.7 1.4 0.3 0.03 2.2 0.2 67.6 3.9 5.8%
Peppers Total 93.2% 3 518.2 7.6 217.0 0.4 34.8 0.3 21.3 3.1 51.3 4.7 842.6 12.3 1.26
ASG 3 70.2 12.2 30.1 3.4 2.5 0.3 0.4 0.2 3.4 0.4 106.7 13.5 12.7%
SG 3 56.4 6.7 22.0 2.4 2.1 0.4 0.5 0.2 3.3 0.4 84.4 8.5 10.0%
Potato Total 77.4% 3 109.8 7.2 1.3 0.1 9.4 0.5 0.8 0.1 44.6 3.4 165.8 9.7 1.12
ASG 3 32.8 2.2 0.7 0.2 2.5 0.3 0.3 0.1 13.1 1.4 49.4 14.7 29.8%
SG 3 28.6 8.4 0.5 0.1 1.9 0.8 0.3 0.2 12.7 4.3 44.0 11.6 26.5%
Potato peel Total 80.3% 3 218.6 17.3 3.0 0.3 22.6 1.8 6.8 0.3 67.4 5.3 318.4 22.3 2.58
ASG 3 91.5 3.8 1.1 0.1 8.6 0.4 2.1 0.3 28.0 1.4 131.3 54.1 41.2%
SG 3 35.4 1.2 0.6 0.1 3.1 0.01 1.7 0.1 10.1 0.2 50.9 8.1 16.0%
Tomato Total 95.1% 4 248.6 17.2 61.0 3.1 199.9 17.2 29.4 3.4 70.0 7.9 608.8 49.6 1.97
ASG 3 42.8 8.8 11.4 0.5 32.7 2.6 3.2 0.3 9.4 1.9 99.5 16.3 16.3%
SG 3 20.2 1.2 6.0 0.4 17.1 0.7 1.9 0.1 5.2 0.9 50.5 4.2 8.3%
Rosaceae
Almonds with peel Total 4.4% 4 1070.2 160.8 39.3 10.7 32.0 2.2 13.8 4.9 174.0 28.4 1329.3 140.8 1.25
ASG 3 70.2 1.0 2.2 1.3 1.8 0.9 0.7 0.1 5.8 0.7 80.9 4.9 6.1%
SG 3 56.3 8.9 1.5 0.7 1.3 0.4 0.6 0.1 4.9 0.7 64.4 3.1 4.8%
Almonds without peel Total 5.6% 3 920.5 33.4 24.6 0.4 4.6 2.1 10.1 0.1 149.8 16.1 1109.6 32.9 1.34
ASG 3 72.5 7.0 1.5 0.2 0.2 0.1 0.5 0.1 5.7 1.0 80.5 5.8 7.3%
SG 3 53.7 2.2 1.1 0.01 0.3 0.1 0.4 0.03 4.6 0.3 60.1 3.3 5.4%
Juglandaceae
Pecan Total 3.2% 5 921.0 171.5 57.1 36.7 18.8 2.8 12.2 3.3 204.3 66.8 1213.4 268.1 0.93
ASG 3 43.5 2.6 1.3 0.1 0.6 0.1 0.1 0.04 3.3 0.4 48.8 2.0 4.0%
SG 2 47.7 6.5 1.4 0.1 0.7 0.1 0.2 0.02 2.4 0.2 52.3 2.3 4.3%
Walnut Total 4.7% 4 985.5 69.8 47.4 3.1 3.4 1.1 5.3 1.2 231.5 35.7 1273.1 79.1 0.87
ASG 3 32.6 1.6 1.3 0.1 0.3 0.1 0.1 0.02 2.1 0.3 36.5 1.0 2.9%
SG 3 38.2 9.4 1.5 0.3 0.3 0.2 0.1 0.03 1.8 0.4 41.8 1.4 3.3%
Musacacea
Banana Total 77.1% 3 416.7 14.0 55.0 0.4 79.9 1.3 n.d. 57.2 7.1 608.8 19.9 0.79
ASG 3 33.1 5.5 5.0 1.6 3.0 1.2 n.d. 2.6 0.4 43.5 3.1 7.2%
SG 3 42.2 6.1 5.2 0.9 4.0 0.7 n.d. 3.7 0.7 55.2 5.0 9.1%
Banana leaves Total 78.5% 3 825.4 67.8 230.5 13.4 524.4 22.5 n.d. 181.5 15.3 1761.8 101.9 1.62
ASG 3 99.8 7.6 18.9 1.8 20.5 5.6 0.2 0.05 9.3 6.2 148.7 12.6 8.4%
SG 3 61.7 10.2 12.7 1.9 13.7 2.3 0.1 0.02 3.3 0.5 91.5 4.8 5.2%
Lamiales
Sesam blacka) Total 4.1% 57.6% 18.1% 7.5% 0.2% 16.5% 1.37
ASG 3 75.5 4.7 10.1 0.9 5.5 0.4 0.2 0.1 6.4 0.8 97.7 27.0
SG 3 55.5 15.2 8.1 2.2 2.6 0.5 0.2 0.04 4.9 1.1 71.4 14.4
Sesam whitea) Total 3.6% 3 62.7% 15.2% 5.9% 0.3% 16.0% 0.61
ASG 3 33.4 1.4 5.8 0.6 3.8 0.4 0.2 0.1 3.8 0.8 47.0 11.3
SG 3 57.8 5.2 9.9 1.0 4.5 0.9 0.2 0.02 5.0 0.5 77.4 30.8
Cashew nut Total 3.3% 3 849.2 95.6 66.0 5.7 1.3 0.5 5.5 1.6 202.9 42.3 1124.9 128.3 1.51
Anacardiaceae ASG 3 95.5 1.7 5.7 0.2 0.9 0.7 0.2 0.1 5.9 0.5 108.2 10.4 9.6%
SG 3 62.5 19.2 3.8 1.2 0.2 0.2 0.1 0.03 5.2 1.3 71.9 4.6 6.4%
Chestnut flour Total 8.8% 3 240.8 9.9 37.8 1.9 101.7 6.1 n.d. 45.9 13.7 426.2 19.6 1.72
Fagacea ASG 3 77.5 15.2 13.0 2.9 29.6 6.4 0.3 0.2 8.1 1.9 128.4 38.7 30.1%
SG 3 44.8 2.7 7.8 0.7 16.9 0.7 0.1 0.04 5.0 0.6 74.5 13.0 17.5%
Chocolate powder Total 6.1% 3 408.6 22.6 48.5 2.2 134.5 6.7 2.4 0.6 17.4 1.7 611.4 33.0 1.54
Malvaceae ASG 3 92.6 8.8 8.6 0.6 22.6 1.8 0.5 0.3 1.6 0.3 125.8 25.9 20.6%
SG 3 63.2 2.8 4.8 0.5 12.3 1.0 0.6 0.1 1.1 0.1 82.0 11.0 13.4%

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Eur. J. Lipid Sci. Technol. 2012, 000, 0000–0000 Glycosylated sterols in plant foods 7

Flaxseed Total 6.9% 3 653.9 22.8 329.1 13.4 91.9 2.2 6.2 0.8 513.1 15.0 1594.3 27.0 0.67
Linaceae ASG 3 19.4 3.6 9.6 1.4 1.8 0.4 0.2 0.1 6.1 1.2 37.1 0.9 2.3%
SG 3 32.7 4.6 16.3 2.2 3.1 0.6 0.1 0.02 2.9 0.6 55.2 1.9 3.5%
Hazel nut Total 4.7% 3 734.6 8.0 32.0 0.8 11.2 0.8 21.6 0.2 44.7 3.4 844.2 7.3 0.75
Betulaceae ASG 3 44.0 19.5 2.0 0.8 0.5 0.3 0.9 0.4 3.1 1.7 50.6 3.0 6.0%
SG 3 59.0 2.6 2.5 0.4 0.9 0.4 1.9 0.3 2.8 0.6 67.1 5.3 8.0%
Pine nut Total 5.4% 3 1858.4 34.6 337.6 9.6 4.5 2.9 16.0 3.2 145.1 15.4 2361.7 32.2 2.17
Pinaceae ASG 3 95.4 10.6 10.3 1.0 n.d. 0.2 0.2 8.9 5.0 114.7 5.6 4.9%
SG 3 45.1 11.1 5.5 3.1 0.3 0.1 0.05 0.02 2.0 1.4 52.9 1.2 2.2%
Sunflower seeds Total 6.0% 3 1197.8 52.2 200.3 6.5 159.7 6.0 n.d. 296.6 22.8 1854.4 70.9 0.67
Asteraceae ASG 3 37.2 9.0 4.5 1.4 3.3 1.0 0.2 0.1 3.6 1.1 48.7 1.3 2.6%
SG 3 56.4 5.3 6.5 0.4 4.1 0.2 0.2 0.02 5.5 0.7 72.8 2.9 3.9%
Wheat flour Total 10.4% 15 344.2 20.6 92.4 5.2 12.8 1.1 114.5 10.6 30.4 6.4 594.3 33.0 0.94
Poaceae ASG 12 19.7 3.4 5.0 0.7 1.0 0.8 2.8 0.6 2.3 2.0 30.8 1.6 5.2%
SG 11 22.8 4.3 5.6 0.9 0.8 0.3 2.3 0.6 1.3 0.3 32.7 1.8 5.5%

(B) Samples dominated with D7-sterols

Unidentified
Spinasterol Campesterol delta-7-sterol Stanols Other sterols Total
ASG/SG-
Mean SD Mean SD Mean SD Mean SD Mean SD Mean SD % ratio

Amarantaceae
Amaranth Total 11.2% 5 401.0 83.2 30.0 5.1 186.4 20.6 4.8 2.1 509.5 137.9 1131.6 222.1 0.43
ASG 3 10.2 1.0 1.8 0.1 23.5 3.1 0.3 0.02 2.6 0.2 38.4 1.3 3.4%
SG 3 20.4 2.2 4.4 0.6 60.6 7.6 0.7 0.1 3.4 0.4 89.4 7.1 7.9%
Beetroot Total 86.3% 3 325.1 12.4 17.5 1.1 132.8 7.1 1.0 0.1 177.8 4.4 654.2 22.8 0.87
ASG 3 36.9 6.3 2.8 1.0 11.8 1.7 0.4 0.3 10.3 1.6 62.2 5.9 9.5%
SG 3 45.5 1.5 2.8 0.1 15.6 1.0 0.2 0.1 7.1 0.4 71.3 7.8 10.9%
Quinoa Total 9.1% 3 231.1 6.2 18.8 0.4 53.9 2.1 11.8 0.4 346.9 4.4 662.4 11.5 0.91
ASG 3 24.9 3.6 1.8 0.2 7.0 0.7 2.0 0.4 4.6 0.6 40.2 2.4 6.1%
SG 3 28.9 0.3 2.2 0.1 8.6 0.2 0.5 0.1 3.9 0.4 44.1 2.9 6.7%
Spinach Total 94.9% 3 916.4 28.0 32.0 1.0 315.0 38.7 9.0 1.8 381.2 14.4 1653.6 61.8 1.85
ASG 3 45.5 2.8 3.5 0.4 44.9 4.3 0.9 0.1 21.2 1.4 116.0 8.1 7.0%
SG 3 22.3 1.9 2.0 0.2 31.2 2.1 0.5 0.2 7.0 0.9 62.9 2.4 3.8%
Cucurbitaceae
Cucumber Total 95.6% 3 755.2 13.5 17.2 2.2 188.4 20.1 3.9 0.1 701.1 30.3 1665.8 30.8 0.82
ASG 3 51.6 20.0 2.4 0.9 17.2 7.2 n.d. 26.4 6.3 97.6 5.7 5.9%
SG 3 64.3 14.7 2.4 0.6 22.5 6.2 n.d. 29.8 10.1 119.1 8.5 7.1%
Melon Total 90.3% 3 268.7 3.6 18.6 1.3 103.1 5.2 53.2 3.1 192.9 11.5 636.5 9.8 1.13
ASG 3 41.8 0.8 2.9 0.1 15.5 0.2 0.1 0.1 12.2 1.0 72.5 8.3 11.4%
SG 3 38.7 2.9 2.7 0.3 13.2 0.8 0.2 0.1 9.2 1.3 64.0 6.4 10.1%
Pumpkin Seeds Total 4.7% 3 495.8 26.2 15.8 0.4 66.5 3.6 38.3 4.4 553.0 24.6 1169.4 41.0 1.50
ASG 3 10.2 0.8 0.4 0.1 9.7 0.6 0.6 0.1 50.1 6.5 71.1 4.3 6.1%
SG 3 10.7 2.7 15.0 2.7 9.3 1.2 n.d. 12.3 5.0 47.3 1.9 4.0%
Zuccini Total 94.1% 4 1715.5 145.2 22.8 4.0 370.3 50.4 3.0 1.1 1599.7 201.7 3711.4 354.2 1.00
ASG 3 123.4 9.1 3.5 0.4 43.3 3.1 1.5 0.3 59.4 4.4 231.1 14.4 6.2%
SG 3 118.9 11.9 2.9 0.5 49.8 4.4 0.9 0.3 58.5 6.7 231.0 14.4 6.2%
a)
The total sterols of the sesame seed samples could not be quantified due to an impurity that co-eluted with the internal standard, and thus
the relative proportions of different sterols are expressed as percentages.

3.1 Contents of glycosylated plant sterols in foods
The highest contents of glycosylated plant sterols, on fresh
matter (fm) basis, were observed in cacao powder, chestnuts,
soybeans, and cashew nuts (175–195 mg/g fm). Based on dry
matter (dm), on the other hand, higher contents than these
were observed in cauliflower, zucchini, broccoli, banana
leaves, eggplants, and cucumber (490, 462, 241, 240, 234,
and 217 mg/g dm, respectively). However, due to the high
water content of these plant tissues (78% in banana leaves,
>90% in others), the content of glycosylated sterols in these
plants is much diluted in the actual foods that are consumed.
Further, banana leaves were not included as an edible plant
sample, but rather as a potential waste tissue, from which
glycosylated sterols could be potentially extracted from, but it
seems that the contents of glycosylated sterols are not so high

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8 L. Nyström et al.
that extraction would be economically feasible. From a mere
yield point of view (if the sterol composition is not import-
ant), the best source as extraction raw material for ASG and
SG would be lecithin from, e.g., soybeans or rapeseed, which
has been reported to have a content of glycosylated sterols in
the range of 30 000–40 000 mg/g [14].
Glycosylated sterol contents were analyzed from similar
samples in the study of Sugawara and Miyazawa, who per-
formed the analysis with an HPLC-method to separate differ-
ent glycolipid classes, which were then quantified using
evaporative light scattering detector [15]. Most of the values
obtained in their study are very much higher than the ones
reported here. For example, they reported the contents of
glycosylated sterols in adzuki beans, spinach, broccoli,
cucumber, eggplant, and tomato to be 694, 215, 135, 89,
109, and 51 mg/g fm, respectively, whereas in our study the
values for the corresponding samples had contents of 141, 9,
20, 10, 16, and 7 mg/g fm. On the other hand, the results for
potatoes and bananas were on a comparable level, being 31
and 15 mg/g fm by Sugawara and Miyazawa, and 21 and
23 mg/g fm in our study, respectively. This suggests that
the difference may not be only due to the analytical pro-
cedure, but also on the variability in the plant tissues. Another
study by Takakuwa et al. reported the contents of glycosy-
lated sterols (after saponification, i.e., ASG also converted to
SG) from various plant and plant parts [16]. Their values for
glycosylated sterols in potato tuber (101 mg/g dm) was in
agreement with our result (93 mg/g dm), but the value for
amaranth grains in their study was 3.5-fold (460 mg/g dm)
compared to ours (128 mg/g dm).
As the studies of Sugawara and Miyazawa and Takakuwa
et al. [15, 16] were carried out on intact molecules (ASG and/
or SG), and converted now for comparison to sterol contents
by the theoretical factors, there is the possibility of a mis-
calculation if the conjugated parts of the molecules are very
different from the expected (glucose and palmitoyl-
glucoside). This explanation is most feasible with the adzuki
bean sample, were also di-, tri-, and even tetra-glycosylated
sterols have been identified [17]. To be able to evaluate the
correctness of the conversion factors used, one should have a
much deeper knowledge about the different conjugated com-
pounds and their relative abundances than is available from
the current literature.
The content of glycosylated sterols in whole-wheat flour
was the lowest of all samples 64 mg/g dm. This value is in
good agreement with our earlier study (75 mg/g dm) [18], and
the value reported by Phillips et al. (66 mg/g dm) [8].
Somewhat higher contents for glycosylated sterols in wheat
samples were reported by Ruibal-Mendieta and coworkers,
whose study showed a glycosylated sterol range of 93–
127 mg/g. The difference may lay in the analytical differences:
whereas our study and the one of Phillips et al. were con-
ducted using acid and alkaline hydrolysis followed by gas
chromatographic analysis, Ruibal-Mendieta et al. applied
an LC-MS-method, which may have a better sensitivity to
ß 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
Eur. J. Lipid Sci. Technol. 2012, 000, 0000–0000
detect also minor sterols better. Whole-wheat flour was
also used as an in-house reference sample, and was
analyzed in each analytical batch of samples. During the
period of analysis, the whole-wheat flour was analyzed
more than 11 times, and the average content of ASG
was 31  1.6 mg/g dm (mean  SD, n ¼ 12), and SG
33  1.8 mg/g dm (n ¼ 11), showing that the day-to-day
variation of the method was very low (5%).
Most beans and lentils (which all belong to the botanical
family of Fabaceae) had a comparable content glycosylated
sterols ranging between 100 and 150 mg/g dm, and the con-
tent on the fm basis was only some 10–15% lower. In these
samples, the content of glycosylated sterols in the final edible
food product will be reduced, as the samples are soaked and
cooked in large volumes of water. On the other hand, gener-
ally they may be eaten in relatively large quantities as a side
dish, and therefore are likely to be among the important
dietary sources of glycosylated sterols.
In most of the nut samples the contents of glycosylated
sterols were in a moderate range (80–120 mg/g dm), and
additionally the content is not even diluted with added water
during food preparation. Highest contents of glycosylated
sterols in nuts were analyzed in cashew and pine nuts (180
and 168 mg/g dm), respectively. The content of glycosylated
sterols in pine nuts are comparable with the results of Phillips
et al., who reported a content of 209 mg/g [8]. As stated
above, also their result for the whole-wheat flour was almost
identical to ours, but on the other common samples the values
for glycosylated sterols were somewhat higher in the study
of Phillips et al. Their values for flaxseed, soybeans, and
almonds were 350, 379, and 392 mg/g, whereas the respective
contents of glycosylated sterols in our samples were 92, 194,
and 145 mg/g dm. As the values for a part of the samples are
on the same level, one can assume that the difference is not
primarily caused by analytical factors, but rather on true
differences between the samples, which may be caused by
varietal and environmental factors.
3.2 Proportion of glycosylated sterols of total plant
sterols
In the plant foods analyzed in this study, the glycosylated
sterols (ASG þ SG) contributed about 5–60% of the total
sterols (Table 1). The proportion was the highest in potato
and potato peel (56 and 57%), chestnut flour (48%), and
cacao powder (34%). In earlier literature, glycosylated sterols
have been demonstrated to be the major sterol class in pota-
toes, where ASG contributed about 56–72% of sterol lipids
[19]. SG on the other hand accounted for 12–16% of sterol
lipids. However, these percentages are calculated on the
weight of the entire molecule, and so the percentage of sterols
contributed by ASG may be somewhat lower. For other
samples only a few comparisons to literature are possible,
as there are only very few studies that report the total sterol
content in addition to the glycosylated sterols. Soybeans,
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Eur. J. Lipid Sci. Technol. 2012, 000, 0000–0000
almonds, flaxseed, and pine nuts were included in the sample
portfolio of Phillips et al. [8] and their analysis estimated a
glycosylated sterol percentage (of total sterols) to be 36, 22,
21, and 11%, respectively. This is in good agreement with our
results on soybeans and pine nuts, whereas our study suggests
lower percentage of glycosylated sterols for almonds and
flaxseed (respective values 32, 13, 7, and 6%). The study
by Phillips et al. concluded that the exclusion of glycosylated
sterols in the total sterol content could lead to an under-
estimation of about 37% in nuts and seeds [20], which is in
line with our observations. Further, our results show that in
the case of potatoes the underestimation is even larger.
Of the two classes of glycosylated sterols, ASG and SG,
the acylated form was generally more abundant than the
glycoside without the fatty acid tail. The opposite ratio
(SG>ASG) was the clearest in amaranth seeds, where the
contents of ASG and SG were 38 and 89 mg/g, giving an
ASG/SG-ratio of 0.43. The ratio was below 1 (indicating
more SG than ASG) also in beetroot, quinoa, cucumber,
walnut, pecan, banana, white sesame seeds, sunflower seeds,
flaxseed, hazelnut, and wheat flour. It is noteworthy here, that
the proportion of ASG was generally quite low in the samples
belonging to the families of Amarantaceae or Cucurbitaceae,
which are dominated by D7-sterols (see the next section for
more discussion about D5 vs. D7-sterols.)
The ratio of ASG and SG in potato peels was very high
(2.5), indicating that the ASG is the main sterol lipid in
potatoes. Also in the peeled potatoes ASG were the dominant
species (30% of sterols), but the contribution of the SG was
almost comparable. In potatoes the ASG/SG-ratio was 1.12,
indicating that the contents of the two glycosylated sterol
groups are closer to each other, and suggest that the ASG
are more concentrated in the outer part of the tuber (peel). In
fact the ASG/SG-ratio was relatively high in all samples that
belong to the Solanaceae family (potato, pepper, tomato, egg-
plant). Further, excluding the lentils the ASG/SG-ratio also
the Fabaceae family was characterized by a moderate or high
ASG/SG-ratio. For the lentils the ratio ranged from 0.84 to
1.19, but in the pea, bean, and nuts samples in the ASG/SG-
ratio was >1.5. The highest proportion of ASG in all samples
was observed in small white beans (ASG/SG-ratio 3.6).
The relative proportions of ASG and SG have mostly
been studied on a qualitative level, or by comparing the actual
masses of the compounds, not by the sterol contents con-
tributed by each of the classes. Eichenberger and Grob,
Whitaker as well as Sugawara and Miyakawa have also before
reported the dominance of ASG in the Solanaceae family.
Whitaker reported the concentrations (nmol/g of sample) of
ASG and SG in tomatoes during storage, and observed a
ASG/SG-ratio at a range of 2.0–4.2. The higher ASG/SG-
ratios were observed generally in earlier time points,
suggesting that changes between the sterol classes occur
during storage [21]. Our value for ASG/SG-ratio of tomato,
1.97, is in good agreement with the results of Whitaker. For
the other studies, one needs again to convert the analyzed
ß 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
Glycosylated sterols in plant foods 9
contents of the entire molecules of ASG and SG to the
contents of sterols contributed by each class. After using
the theoretical conversion factors (discussed above), some
more comparisons on the ASG/SG-ratios to the current lit-
erature can be made. As stated above, ASG contributed the
most of the sterol lipids in potato peels, whereas in peeled
potato tubers the ASG/SG-ratio was more equal (2.5 vs. 1.12,
respectively). The dominance of ASG in potatoes is sup-
ported by earlier literature, the estimated ASG/SG-ratios
of 2.5 from the results of Eichenberger and Grob, as well
as the 5.2 from the results of Sugawara and Miyakawa [15,
22]. For some of the similar samples included in our study
and in the one of Sugawara and Miyakawa the ASG/SG-
ratios were well in line. For example, the ASG/SG-ratios of
spinach, cucumber, eggplant, and banana were 1.8, 0.8, 2.5,
and 0.8 in our study, and the respective values of 1.4, 1.5, 3.4,
and 0.5 were reported by Sugawara and Miyakawa. However,
their study suggested in general higher ASG/SG-ratios, e.g.,
9.6 for adzuki beans, 8.8 for milled rice, and 6.0 for leek,
which indicates either differences caused by the analytical
methods, or the deviation caused by the over simplification in
establishing the conversion factor. On the other hand,
this shows that for a better understanding of the levels of
glycosylated sterols in foods (for intake estimations, etc.)
much more detailed and comparable data are needed.
3.3 Sterol profiles of glycosylated sterols in plant
foods
The sterol profiles, i.e., the composition of different sterols in
a sterol class, is also of very much interest. It is generally
considered, that the sterol profile of glycosylated sterols in
any plant material reflects the total sterol profile in the same
plant [1]. However, as 4-monomethyl- and 4,40 -dimethyl-
sterols are poor substrates for the glycosylating enzymes, they
are unlikely found as glycosides and hence the profile must be
somewhat different. This also suggests that different sterol
conjugates have unequal affinities toward different enzymes,
and may therefore be affected differently by enzymatic reac-
tions during food processing and storage.
A major difference in the sterol composition of the plants
analyzed in this paper is the dominance of D5- and D7-sterols,
i.e., sterols in which the double bond of the ring structure is
between C-5 and C-6 or C-7 and C-8, respectively. The D7-
sterols are characteristic to the families of Amarantaceae and
Cucurbitaceae, and will be discussed separately. The D5-ster-
ols are the most common sterols found in the nature and also
dominant sterols in the other plant families of this study.
Their identification was verified by the GC-MS analysis: the
D5-sterol TMS derivatives gave a characteristic m/z 129 ion as
a base peak (bp), major ions at M-90 and M-129, and a small
molecular ion [13, 23]. Sitosterol, stigmasterol, and campes-
terol were the most common sterols detected in the various
plant materials analyzed in this study, but their relative pro-
portions differed between the samples significantly (Table 2).
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10 L. Nyström et al. Eur. J. Lipid Sci. Technol. 2012, 000, 0000–0000

Table 2. The sterol composition (%) of total sterols, ASGs, and SGs

Other Proportion of
Sitosterol Campesterol Stigmasterol Stanols sterols total sterols

Fabaceae
Adzukibeans Total 28.3 2.1 46.0 2.8 20.8
ASG 32.8 1.7 50.8 0.3 14.4 19.8
SG 35.9 2.6 46.8 0.7 14.1 7.2
Black beans Total 57.3 20.4 15.3 1.3 5.7
ASG 60.8 22.7 13.4 0.5 2.5 18.4
SG 61.4 23.4 12.0 0.5 2.7 10.3
Chickpeas Total 65.9 11.0 7.8 3.7 11.5
ASG 79.7 10.3 5.3 0.4 4.3 6.7
SG 79.2 10.7 5.2 0.6 4.4 6.4
Black eye beans Total 30.3 7.4 47.1 0.4 14.8
ASG 24.0 3.7 66.6 0.1 5.5 21.6
SG 33.2 4.7 55.4 0.6 6.0 7.7
Lentils green small Total 69.3 8.3 11.0 4.3 7.0
ASG 78.7 7.2 7.7 0.5 5.7 5.5
SG 78.6 7.6 9.1 0.7 4.0 6.5
Lentils green large Total 72.8 10.6 9.7 4.3 2.6
ASG 79.7 8.2 7.7 0.6 3.8 7.8
SG 81.3 7.9 7.8 0.5 2.5 7.8
Lentils orange Total 71.1 10.1 8.9 3.9 6.1
ASG 81.2 8.2 7.7 0.3 2.7 7.5
SG 80.6 9.3 6.6 0.3 3.3 6.3
Lentils yellow Total 75.5 6.0 3.8 2.9 11.8
ASG 84.2 5.6 3.0 0.2 7.0 8.1
SG 87.0 5.5 3.1 0.3 4.1 6.6
Peanuts Total 65.0 11.5 9.3 1.0 13.2
ASG 68.1 10.9 13.4 0.3 7.2 7.0
SG 69.7 12.9 10.7 0.5 6.3 4.0
Red beans Total 53.4 3.7 32.0 0.4 10.4
ASG 50.7 2.3 43.5 0.1 3.4 13.0
SG 54.9 2.9 37.6 0.2 4.4 5.4
Soy beans Total 47.8 21.4 15.7 1.3 13.9
ASG 54.7 26.1 15.6 0.6 3.0 17.3
SG 56.1 26.2 15.1 0.6 2.0 14.8
White beans small Total 45.1 4.6 35.7 1.3 13.3
ASG 47.5 3.2 43.8 0.1 5.3 16.4
SG 49.3 4.0 41.2 0.3 5.2 8.9
White beans large Total 49.3 4.9 31.7 1.1 13.0
ASG 48.7 3.3 43.5 0.3 4.2 14.4
SG 51.6 4.3 38.0 0.6 5.5 4.0
Brassicaceae
Broccoli Total 76.7 15.3 2.5 0.9 4.6
ASG 80.3 14.4 2.8 0.4 2.1 5.4
SG 81.4 14.5 1.9 0.4 1.9 4.7
Cauliflower Total 68.8 22.5 6.3 1.3 1.1
ASG 75.0 18.8 4.0 0.1 2.0 7.5
SG 74.2 18.9 4.0 0.3 2.7 4.3
Mustard seeds Total 57.8 26.7 0.5 0.5 14.5
ASG 67.0 21.8 0.4 1.0 10.0 3.1
SG 66.7 23.0 0.2 0.6 9.5 2.5

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Eur. J. Lipid Sci. Technol. 2012, 000, 0000–0000 Glycosylated sterols in plant foods 11

Solanaceae
Aubergine Total 58.6 7.0 26.9 0.4 7.1
ASG 70.0 6.4 20.2 0.2 3.2 14.2
SG 71.4 6.1 18.9 0.4 3.3 5.8
Peppers Total 61.5 25.8 4.1 2.5 6.1
ASG 65.8 28.2 2.4 0.4 3.2 12.7
SG 66.9 26.0 2.5 0.6 4.0 10.0
Potato Total 66.2 0.8 5.6 0.5 26.9
ASG 66.4 1.4 5.1 0.6 26.6 29.8
SG 65.0 1.0 4.4 0.7 29.0 26.5
Potato peel Total 68.7 0.9 7.1 2.1 21.2
ASG 69.7 0.8 6.6 1.6 21.4 41.2
SG 69.5 1.2 6.1 3.3 19.9 16.0
Tomato Total 40.8 10.0 32.8 4.8 11.5
ASG 43.0 11.4 32.9 3.2 9.5 16.3
SG 39.9 11.9 33.9 3.9 10.4 8.3
Rosaceae
Almonds with peel Total 80.5 3.0 2.4 1.0 13.1
ASG 86.9 2.8 2.2 0.9 7.2 6.1
SG 87.4 2.3 2.0 0.9 7.5 4.8
Almonds without peel Total 83.0 2.2 0.4 0.9 13.5
ASG 90.1 1.8 0.3 0.6 7.1 7.3
SG 89.4 1.8 0.5 0.6 7.7 5.4
Juglandaceae
Pecan Total 75.9 4.7 1.6 1.0 16.8
ASG 89.2 2.7 1.2 0.2 6.7 4.0
SG 91.2 2.7 1.3 0.3 4.6 4.3
Walnut Total 77.4 3.7 0.3 0.4 18.2
ASG 89.4 3.6 1.0 0.3 5.8 2.9
SG 91.2 3.5 0.7 0.3 4.3 3.3
Musacacea
Banana Total 68.5 9.0 13.1 0.0 9.4
ASG 75.9 11.4 6.8 0.0 5.9 7.2
SG 76.5 9.5 7.3 0.0 6.7 9.1
Banana leaves Total 46.8 13.1 29.8 0.0 10.3
ASG 67.1 12.7 13.8 0.1 6.2 8.4
SG 67.4 13.9 15.0 0.1 3.6 5.2
Lamiales
Sesam blacka) Total 57.6 18.1 7.5 0.2 16.5
ASG 77.3 10.3 5.7 0.2 6.6 27.6
SG 77.7 11.4 3.7 0.3 6.8 20.2
Sesam whitea) Total 62.7 15.2 5.9 0.3 16.0
ASG 71.0 12.2 8.2 0.5 8.1 24.2
SG 74.7 12.8 5.8 0.3 6.4 39.8
Cashew nut Total 75.5 5.9 0.1 0.5 18.0
Anacardiaceae ASG 88.3 5.3 0.9 0.2 5.4 9.6
SG 87.0 5.3 0.3 0.1 7.2 6.4
Chestnut flour Total 56.5 8.9 23.9 0.0 10.8
Fagacea ASG 60.3 10.1 23.0 0.2 6.3 30.1
SG 60.1 10.4 22.7 0.1 6.7 17.5
Chocolate powder Total 66.8 7.9 22.0 0.4 2.9
Malvaceae ASG 73.6 6.8 17.9 0.4 1.2 20.6
SG 77.1 5.9 15.0 0.7 1.3 13.4
Flaxseed Total 41.0 20.6 5.8 0.4 32.2
Linaceae ASG 52.2 26.0 4.9 0.6 16.4 2.3
SG 59.3 29.5 5.6 0.2 5.3 3.5
Hazel nut Total 87.0 3.8 1.3 2.6 5.3
Betulaceae ASG 86.9 4.0 1.1 1.9 6.2 6.0
SG 87.9 3.7 1.3 2.9 4.2 8.0
(Continued)

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12 L. Nyström et al. Eur. J. Lipid Sci. Technol. 2012, 000, 0000–0000

Table 2. Continued

Other Proportion of
Sitosterol Campesterol Stigmasterol Stanols sterols total sterols

Pine nut Total 78.7 14.3 0.2 0.7 6.1

Pinaceae ASG 83.1 9.0 0.0 0.2 7.7 4.9
SG 85.2 10.5 0.6 0.1 3.7 2.2
Sunflower seeds Total 64.6 10.8 8.6 0.0 16.0
Asteraceae ASG 76.3 9.2 6.8 0.3 7.4 2.6
SG 77.6 9.0 5.7 0.3 7.5 3.9
Wheat flour Total 57.9 15.5 2.2 19.3 5.1
Poaceae ASG 64.0 16.2 3.1 9.2 7.5 5.2
SG 69.7 17.1 2.4 7.0 3.9 5.5

Unidentified Other Proportion of

Spinasterol Campesterol delta-7-sterol Stanols sterols total sterols

Amarantaceae
Amaranth Total 35.4 2.6 16.5 0.4 45.0
ASG 26.6 4.7 61.2 0.9 6.6 3.4
SG 22.8 4.9 67.8 0.7 3.8 7.9
Beetroot Total 49.9 2.7 20.4 0.1 27.3
ASG 59.3 4.6 18.9 0.7 16.5 9.5
SG 63.9 3.9 21.9 0.3 10.0 10.9
Quinoa Total 34.9 2.8 8.1 1.8 52.4
ASG 61.9 4.5 17.3 4.9 11.3 6.1
SG 65.6 4.9 19.5 1.2 8.8 6.7
Spinach Total 55.4 1.9 19.1 0.5 23.1
ASG 39.2 3.0 38.7 0.8 18.2 7.0
SG 35.5 3.1 49.6 0.8 11.1 3.8
Cucurbitaceae
Cucumber Total 45.3 1.0 11.3 0.2 42.1
ASG 52.9 2.5 17.6 0.0 27.1 5.9
SG 54.0 2.0 18.9 0.0 25.0 7.1
Melon Total 42.2 2.9 16.2 8.4 30.3
ASG 57.7 4.0 21.4 0.1 16.8 11.4
SG 60.4 4.2 20.7 0.3 14.4 10.1
Pumpkin seeds Total 42.4 1.4 5.7 3.3 47.3
ASG 14.4 0.6 13.7 0.8 70.5 6.1
SG 22.7 31.6 19.7 0.0 26.0 4.0
Zuccini Total 46.2 0.6 10.0 0.1 43.1
ASG 53.4 1.5 18.8 0.6 25.7 6.2
SG 51.5 1.3 21.6 0.4 25.3 6.2

In the family of Fabaceae, lentils and beans showed quite a
different sterol composition: whereas lentils were all charac-
teristically high in sitosterol as the main glycosylated sterol
(79–87%), the percentage of sitosterol in beans ranged from
24 to 61%. In the bean samples, the percentage of stigma-
sterol was respectively higher, ranging from 12 to 66%, com-
pared to 3–9% in lentils. Significant variation between the
sterol profiles in the Fabaceae family were also observed in the
proportions of campesterol, which was remarkably high in
soybeans and black beans (>23%), but very low in other
beans and lentils. Adzuki beans and black eyebeans differed
perhaps the most in their sterol composition from the other
samples of the Fabaceae family: highest absolute contents of
stigmasterol as glycosides were observed in these 61.1 and
85.5 mg/g dm as ASG, and 20.5 and 25.5 mg/g dm as SG in
adzuki beans and black eyebeans, respectively.
In the family of Brassicaceae, campesterol was also high-
lighted in the sterol profile, with the expense of stigmasterol,

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Eur. J. Lipid Sci. Technol. 2012, 000, 0000–0000
that contributed less than 3% of the glycosylated sterols.
The highest absolute content of glycosylated campesterol
in all samples was seen in cauliflower, which had 58.5 and
34.0 mg/g dm of campesterol as ASG and SG, respectively. In
addition to the high percentage of glycosylated campesterol
(22–23%), mustard seeds had a unique trait in the sterol
profile, as it contained significant amounts of glycosylated
brassicasterol (included in the ‘‘Other sterols’’-column of
Table 2). Brassicasterol is naturally very abundant in the
plants of the Brassicaceae family, but has not to our knowledge
been reported before in the glycosylated form.
The plants of the Solanaceae family, which is well known
for its high percentage of glycosylated sterols, also showed
significant differences in their sterol profiles. Though sitos-
terol was again the most abundant sterol found as glycoside
(40–70%), also campesterol in peppers (26–28%), and stig-
masterol in aubergine (19–20%) and tomato (33–34%) were
characteristic to these samples. Even more unique, though,
was the high level of stigmast-5,24(25)-dienol in potatoes and
potato peels. It contributed the majority of the other sterols.
The sterol profile of different steryl conjugates in tomatoes
has been shown to vary during storage and depending on
storage conditions. The sterol profile of tomatoes reported in
the studies of Whitaker [21, 24] mostly agrees with our
profiles in the percentage of sitosterol (40–43% in our study,
39–60% reported by Whitaker). However, in our study the
proportion of stigmasterol is higher and the miscellaneous
minor sterols smaller than in the study of Whitaker. He also
reported that the stigmasterol/sitosterol-ratio changed during
storage and dependently on storage conditions. The stigmas-
terol/sitosterol-ratios observed in our samples were 0.77 and
0.84 for ASG and SG, respectively, which based on the data
by Whitaker would suggest that the tomatoes were harvested
8–12 days earlier and stored at a moderate temperature.
The differences in the total sterol profile vs. the sterol
profile of the glycosylated sterols seemed to be the highest in
black eyebeans, aubergine, flaxseed, sunflower seeds, and
wheat flours. In fact, in black eyebeans the sterol profile of
SG was relatively similar to total sterol profile, whereas the
one of ASG was characterized with a lower percentage of
sitosterol and campesterol, but a higher level of stigmasterol
and other sterols. In aubergine, flaxseed, sunflower seeds, and
wheat flour, the total sterol profile was distinctively different
from the sterol profiles of the glycosylated sterols. Most
importantly, in wheat flower, the difference between the
sterol profiles was observed in the percentages of stanols,
which have a saturated ring structure and which are particu-
larly abundant in cereal grains [18]. They contributed about
19% of total sterols in whole-wheat flour, but less than 10% of
the glycosylated sterols, which is in accordance with the value
in our previous study [18].
The sterol profiles of the D7-sterols need to be taken only
as indicative, due to analytical challenges. The sterol profile
of the D7-sterols seems to be remarkably different in the total
sterols versus glycosylated sterols in a way that the spinasterol
ß 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
Glycosylated sterols in plant foods 13
would be the predominant in the total sterols, but the uniden-
tified D7-sterols in the glycosylated sterols. However, this
alteration in the sterol profile is not caused by the sample
itself, but rather by an artifact from the acid hydrolysis, which
is performed to liberate the sterols to their free form for
subsequent GC-analysis. This phenomenon was verified by
comparing the total sterol profiles of the samples after acid
and alkaline hydrolyses versus alkaline hydrolysis alone. In
pumpkin seeds and spinach, e.g., the proportion spinasterol
of total sterols was 43 and 94% higher than without acid
hydrolysis, indicating that a vast majority of spinasterol may
isomerize to other sterols in the acid hydrolysis. Similar
isomerization is reported, e.g., for D5-avenasterol, which is
converted to stigmastadienol during acid hydrolysis [25].
This is especially relevant for various nuts and seeds, where
D5-avenasterol occurs abundantly. (In this study both
D5-avenasterol and stigmastadienol are included in the sum
of other sterols.) The isomerization pathway of the unidenti-
fied D7-sterol is, however, still unknown. This compound
eluting at 33.3 min (RRT to internal standard dihydro-
cholesterol 1.18) was tentatively characterized as a D7- or a
D8-sterol TMS derivative with one double bond in the
side chain by MS (characteristic ions for D7- and D8-sterols
at m/z 229 and 255, a strong molecular ion at m/z 484 and
ions at m/z 343–345) [12, 13]. Phillips et al. [20] also found
that an unidentified D7-sterol with comparable retention
and similarities is MS was formed during acid hydrolysis.
Furthermore, the GC-MS-analysis revealed that the spi-
nasterol peak, which is a major peak in the D7-sterol chro-
matograms, is in fact an overlay of two different sterols. For
example, in the glycosylated sterols of cucumber, zucchini,
melon, and beetroot, it seems that the majority of this peak is
actually not spinasterol, but some other, still unidentified
compound. The mass spectra of this compound (which elutes
mostly in the early part of the peak), gave us the data to
tentatively identify it as a D8-sterol with a saturated side chain
(characteristic ions at m/z 255, 345, 381, and 486 (bp)). In
addition, GC-MS data indicated the presence of D7-avenas-
terol (stigmasta-7,24(28)-dien-3b-ol) (characteristic ions at
m/z 343 (bp), 386, 469, and 484), D7-stigmastenol (stigmast-
7-en-3b-ol) (characteristic ions at m/z 255, 345, 471, and 486
(bp)), and stigmasta-7,23-dien-3b-ol (characteristic ions at
m/z 255, 283, 343 (bp), 379, and 484) that were included in
the other sterols. Nevertheless, it may be concluded that
the D7-sterols contain spinasterol and other sterols in
their glycosylated forms, and further studies without acid
hydrolysis need to be performed to verify the accurate sterol
composition of these plants.
4 Conclusions
The contents of SGs and ASGs, as well as the sterol profile
found in the glycosylated form, is heavily dependent on the
plant sample. This may also be significantly different
from the total sterol profile in the same tissue, indicating
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14 L. Nyström et al.
preferential glycosylation of sterols. There is still very little
data on the contents of glycosylated sterols in plant foods
available, and the comparison of the data is extremely difficult
due to analytical differences and the differences in the expres-
sion of the data (contents of the sterols with or without the
conjugated sugar and possible fatty acid). However, taken
the health benefits and special amphiphilic nature of the
glycosylated sterols, it is important to know the sources of
glycosylated sterols (not only those of free sterol alcohols
and fatty acid esters). The data presented in this paper
demonstrates wide variability in the sterol profiles, but further
studies are definitely needed to better understand also the
nature of the conjugated compounds (identity of the sugars),
as well as the sterol composition (for D7-sterols analysis with-
out artifact forming acid hydrolysis, either after enzymatic
hydrolysis or with an LC-MS analysis).
The authors have declared no conflict of interest.
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