A new method for the analysis of seed phytosterols in sunflower breeding
programs
Fernández-Cuesta Álvaro1, Fernández-Martínez José M.1, Velasco Leonardo1
1
Instituto de Agricultura Sostenible (IAS-CSIC), Alameda del Obispo s/n, 14004 Córdoba, Spain,
lvelasco@ias.csic.es
ABSTRACT
 Phytosterols or plant sterols are natural constituents of plants. Ingestion of phytosterols prevents
intestinal absorption of cholesterol in humans, resulting in a lowering of serum cholesterol. For
this reason, phytosterol derivatives have become important ingredients of a broad spectrum of
functional foods marketed for their lowering-cholesterol properties. Sunflower seeds and oils are
important sources of phytosterols. Despite the great value of phytosterols for human health,
practically no research has been conducted to characterize the variation for phytosterol content
and profile in sunflower germplasm and to design breeding strategies to improve the phytosterol
fraction in sunflower seeds and oils. The main reason for this has been the lack of appropriate
analytical methods matching sunflower breeder's requirements, i.e. they should be fast, costeffective, non-destructive, and based on small samples. Conventional methods for the analysis of
phytosterols are based on the analysis of the extracted oils, which makes them useful only in the
final steps of a breeding program. The aim of this research was to develop and test a new method
for quantitative analysis of phytosterols in sunflower half seeds that can be used at any step of
breeding.
 The method comprises half-seed excision and crushing, a simultaneous step of phytosterol
extraction and derivatization from crushed half seeds, and finally separation and quantification
by gas chromatography.
 The new method allows the quantification of individual phytosterols as well as total phytosterol
content in sunflower half seeds. The method is non-destructive, fast, accurate, and repeatable.
The accuracy of the method was checked by comparing the results with those obtained with a
conventional method on extracted oils, which resulted in a correlation coefficient of 0.85. The
repeatability of the method was evaluated by calculating the standard error in the measurement
of 48 replicates of two different ground samples, which revealed that the average coefficient of
variation attributable to the analytical method was only 4.1%.
 Thus far, sunflower breeders had no efficient tools for screening breeding materials for
phytosterol content and profile, as conventional methods required the analysis of the extracted
oils. The present research provided a new methodology that will allow the incorporation of
phytosterols as target traits into sunflower breeding programs.
 The possibility of analysing phytosterols in half seeds opens up new perspectives for screening
large germplasm collections and segregating populations and lays the basis for breeding for
phytosterol content and profile in sunflower.
Key words: Analytical method - breeding - oil quality - phytosterols - plant sterols - seed quality
INTRODUCTION
Phytosterols or plant sterols are natural constituents of plants that are similar to cholesterol, both in their
chemical structure and biological function. They play essential functions in plant cells in a similar manner
to cholesterol in mammalian cell membranes (Hartmann, 1998). Because of their structural similarity to
cholesterol, dietary phytosterols compete with cholesterol for absorption in the intestine, thus contributing
to reduce serum cholesterol levels (Plat and Mensink, 2005). Vegetable oils and oil-based products are the
richest dietary sources of phytosterols, followed by cereal grains, cereal-based products, and nuts
(Piironen et al., 2000). Because of their cholesterol-lowering activity, phytosterols and related compounds
are increasingly being used as functional food ingredients (Berger et al., 2004). Additionally, several
studies have shown the beneficial effect of phytosterols as natural components of regular diet (Andersson
et al., 2004).
Sunflower seeds and oils are rich sources of phytosterols (Phillips et al., 2002, 2005). Because of
the health-promoting effects of these compounds, further increasing phytosterol levels in sunflower seeds
through breeding strategies emerges as a challenging objective for sunflower breeders. However, little
research has been conducted in this direction thus far. One of the bottlenecks to conduct breeding research
on phytosterols in sunflower is the lack of analytical methods suitable for plant breeding programmes,
particularly at their initial steps when seed is produced in low amounts and is too valuable to be destroyed
for chemical analyses. Common methods for analysis of phytosterols require an initial step of extraction
of the oil from the seeds. Then, the unsaponifiable fraction is extracted and the phytosterols are
derivatised and analysed by gas chromatography (Toivo et al., 2000). However, analysis of phytosterols
in the extracted oil is unsuitable for plant breeding. Any method for the analysis of seed phytosterols in a
sunflower breeding laboratory has to be based on the direct analysis of seeds, preferably on a small
amount of seeds, more preferably on a small amount of half seeds to make the analysis non-destructive,
and even more preferably on a single half seed to expedite selection if the inheritance of the trait is totally
or partially gametophytic, i.e. the trait is totally or partially determined by the genotype of the developing
embryo, as it is the case of fatty acid and tocopherol profiles in sunflower seeds (Fernández-Martínez et
al., 2009).
Amar et al. (2008) developed a method for the direct analysis of phytosterols on small amounts
of rapeseed seeds, without previous oil extraction. Roche et al. (2010) studied the evolution of
phytosterols during seed development in sunflower analysing phytosterols directly on sunflower seeds.
Fernández-Cuesta et al. (2012) used a similar method, with small variations, to evaluate the correlation
between the analysis of phytosterols in seeds and the analysis in the extracted oil, using a set of 87
sunflower inbred lines. In that study, analyses were conducted on samples consisting of six hulled
achenes. The results revealed a high correlation between both methods, which validated the approach of
analysing sunflower seeds instead of sunflower oils. The objective of the present research was to evaluate
the applicability of the method reported by Fernández-Cuesta et al. (2012) to the analysis of single
sunflower half seeds.
MATERIALS AND METHODS
The method of analysis of phytosterols in single sunflower half seeds was adapted from previously
published methods on the analysis of phytosterols in several types of seeds (Bruni et al., 2002; Amar et
al., 2008; Roche et al., 2010). The method consists of the following steps:
1.
2.
3.
4.
5.
Excise a half seed, i.e. a small seed portion from the seed part distal to the embryo (around one
fifth of the seed length) and discard the hull.
Weight the excised seed part and place it in a 10-ml propylene tube. Place the embryocontaining seed part in a 96-well plate.
Add 200 µL of internal standard solution to the tube. The internal standard solution is prepared
by dissolving cholesterol (99% purity, reference C8667, Sigma-Aldrich, St. Louis, MO, USA) in
hexane-ethanol (3:2) solution at a concentration of 0.1%.
For alkaline hydrolysis, add 2 mL of a solution of potassium hydroxide dissolved in ethanol at a
concentration of 2%.
Crush finely the half seed within the potassium hydroxide solution using a stainless-steel rod. In
our lab we use a homogenizer (Heidolph RC 500, Kelheim, Germany) equipped with a stainlesssteel rod of 8 mm diameter at a speed of 5,000 rpm for about 15 sec.
6.
7.
8.
9.
10.
11.
12.
13.
Wash the rod with 1 mL of the ethanolic solution of potassium hydroxide and collect it in the
tube.
Place the tube in a water bath at 80 ºC for 15 min, vortexing every five minutes.
For phytosterol extraction, add 1 mL hexane and 1.5 mL water and vortex thoroughly.
Centrifugue at 3,500 rpm for 5 min.
Transfer the upper hexane layer to a 2-mL glass vial and maintain it in the oven at 37.5 ºC
overnight.
Add
50
µL
n-heptane
and
50
µL
silylating
mixture
composed
of
pyridine:hexamethyldisilazane:trimethylchlorosilane 9:3:1 by vol (Silan-Sterol-1, reference
355650.0922, Panreac Química, Barcelona, Spain) to the dried pellet and leave the vial at room
temperature for 15 min.
Transfer the solution to a 2-mL glass vial containing a 200 µL insert and centrifuge at 4,000 rpm
for 10 min.
Cap the vial and place it in the autosampler of the gas chromatograph or, if necessary, conserve
it at -20ºC till analysis.
Gas chromatographic equipment and analytical conditions: Perkin Elmer Clarus 600 GC
equipped with a ZB-5 capillary column (id = 0.25 mm, length = 30 m, film thickness = 0.10 µm;
Phenomenex, Torrance, CA, USA). Injected sample: 1 µL. Carrier gas: hydrogen at a pressure of
125 KPa. Split ratio: 1:25. Temperature of split injector and flame ionization detector: 320 ºC.
Oven thermal regime: initial temperature of 240 ºC, increased at 5 ºC min-1 to final temperature
of 265 ºC and held for 10 min. Total analytical time: 15 min.
The accuracy of the method of analysis of phytosterols for different amounts of sunflower seed was
determined by calculating the standard error of the analysis of 48 replicates of two different samples
following the procedure reported by Fernández-Cuesta et al. (2012).
RESULTS AND DISCUSSION
The standardized methods for analysis of phytosterols in vegetable oils are based in the analysis of 2.5 g
oil (Fernández-Cuesta et al., 2012). Initial studies on the development of methods for direct analysis of
seeds faced a significant reduction of sample size. Bruni et al. (2002) reduced the sample weight to 1 g of
Theobroma subincanum seed embryos. Amar et al. (2008) analysed phytosterols on samples of 200 mg
rape seeds, whereas Roche et al. (2010) reported the analysis of ground sunflower seed samples of 250
mg. The method described in the present research was initially conceived for the screening of sunflower
germplasm by analysing six hulled achenes per accession or per single plant (Fernández-Cuesta et al.,
2012). In that study, the weight of six hulled achenes ranged from 77 to 695 mg. The accuracy of the
method was checked by comparing the results with those obtained with a conventional method on
extracted oils, which resulted in a correlation coefficient of 0.85. Additionally, the repeatability of the
method was evaluated by calculating the standard error in the measurement of 48 replicates of two
different ground samples, which revealed that the average coefficient of variation attributable to the
analytical method was only 4.1% (Fernández-Cuesta et al., 2012).
In a further approach, the method was evaluated for the analysis of six half seeds instead of six
whole seeds. This modification is important for evaluating germplasm with poor seed set under bagging
conditions, such as most of the germplasm of open pollinated populations. In these cases the seeds have to
be analysed in a non-destructive way, which is achieved by analysing only a small piece of each seed. By
using half seeds instead of whole seeds, the sample weight was reduced by around 80%. The accuracy of
the method was evaluated on two inbred lines in which the average weight of six half seeds were 51 and
23 mg, respectively and the average seed phytosterol content were 2,634 and 4,430 mg kg-1, respectively.
The standard error of the analysis of 48 replicates of each line was 138.5 mg kg -1, indicating that the
average coefficient of variation attributable to the analytical method assuming complete homogeneity of
the seed samples was 3.9%, similar to that obtained with six whole seeds. The results suggested no loss of
accuracy by reducing the sample size to six half seeds.
The analysis of quality traits in single half seeds contributes to expedite selection for traits
controlled by the genotype of the developing embryo. In this case, seeds from the same head having
different genotypes will have different values for the trait, and such differences can be detected by
analysing individual half seeds. This is the case for fatty acid and tocopherol profiles, for which the use of
the single half-seed analysis has contributed significantly to produce a broad variation in sunflower
(Fernández-Martínez et al., 2009). After having identified variation for phytosterol profile in sunflower
germplasm (Fernández-Cuesta et al., 2012), we have observed that the trait is controlled by the genotype
of the developing embryo in a similar manner as fatty acid and tocopherol profiles. Therefore, selection
for modified phytosterol content can be conducted at the single-seed level provided that the analytical
method is accurate enough for such a huge sample size reduction. The accuracy of the analysis of
phytosterol content in single half seeds of sunflower was evaluated by analysing replicates of half seeds
as described above. In this case, the standard error of the analysis was 229.0 mg kg -1, which represented a
coefficient of variation of 6.5% compared to 3.9% for six half-seeds. However, it is important to note that
the average weight of half seeds in this study was 8.9 mg, compared to an average weight of 37 mg in the
samples of six half seeds. Therefore, it was concluded that the reduction of the sample weight by around
75% had only a moderate negative impact on the analytical accuracy, which is still good enough for
selection purposes.
Apart from the advantages of analysing total phytosterol content at the single-seed level, the
half-seed technique is particularly useful for identifying differences for the phytosterol profile. The
phytosterol fraction of sunflower seeds is dominated by beta-sitosterol, which represents around 60% of
the total phytosterols. Other phytosterols present in lower amounts are campesterol (8%), stigmasterol
(8%), delta-5-avenasterol (4%), delta-7-stigmastenol (15%), and delta-7-avenasterol (4%). Other
phytosterols are usually present in amounts below 1% of the total phytosterols (Padley et al., 1994). In the
evaluation of sunflower germplasm for seed phytosterols, we have identified material with increased
levels of individual phytosterols such as campesterol and delta-7-stigmastenol and selection for these
traits is currently being conducted. Chromatograms of two half seeds with contrasting phytosterol profiles
are shown in Fig. 1. Peak areas are very small due to the minute amount of seed used, but differences for
phytosterol profile are clearly observed.
Fig. 1. Chromatograms of phytosterols in single half-seeds of sunflower with contrasting phytosterol
profiles. A: 2.5% campesterol (CAMP), 12.4% stigmasterol (STIG), 28.6% beta-sitosterol (BSITO), 4.2%
delta-5-avenasterol (D5AV), 43.0% delta-7-stigmastenol (D7STIG), and 7.3% delta-7-avenasterol
(D7AV). B: 34.9% campesterol, 6.6% stigmasterol, 43.8% beta-sitosterol, 10.7% delta-5-avenasterol,
1.6% delta-7-stigmastenol, and 1.8% delta-7-avenasterol. INT STD=internal standard.
Extensive use of the half-seed analysis in sunflower has allowed the isolation of many variants for fatty
acid and tocopherol profiles through germplasm evaluation and mutagenesis, and has facilitated their
introgression into elite cultivars. For fatty acids, these variants include low saturated fatty acid content
(Vick et al., 2002), high saturated fatty acid content (Osorio et al., 1995), mid oleic acid content (Vick
and Miller, 1996), and high oleic acid content (Soldatov, 1976; Fernández-Martínez et al., 1993). Variants
with modified tocopherol profiles include mid beta-tocopherol content (Demurin, 1993; Velasco and
Fernández-Martínez, 2003), high beta-tocopherol content (Velasco et al., 2004), high gamma-tocopherol
content (Demurin, 1993; Velasco and Fernández-Martínez, 2003; Velasco et al., 2004), and high deltatocopherol content (Velasco et al., 2004). Our results revealed that there is also variation for seed
phytosterol profile in sunflower germplasm and that the half-seed technique is suitable for identifying and
isolating variants and for introgressing them into elite germplasm.
There is little information on the nutritional properties of individual phytosterols and accordingly
on the advantages of partially replacing the predominant beta-sitosterol by other phytosterols such as
those shown in Fig. 1 for which variation is already available. The availability of sunflower inbred lines
with stable levels of modified phytosterols will provide a unique opportunity for evaluating nutritional
differences associated with modified phytosterol profiles. Also, it will facilitate the study of genetic,
biochemical and physiological aspects related to plant sterol biosynthesis and function. Additionally,
phytosterols may also play an important role in the technological properties of sunflower oil. Whereas
most phytosterols have no antioxidant effect, those containing an ethylidene group in the side chain such
as delta-5-avenasterol and delta-7-avenasterol are effective antioxidants at high temperatures, as they
protect the oil against polymerisation and also retard the loss of tocopherols (Rossell, 2001). Accordingly,
selection for increased levels of these phytosterols coupled with combination with other traits related to
thermoxidative stability such as high saturated fatty acid, high oleic acid, high gamma-tocopherol and
high delta-tocopherol content might result in a sunflower germplasm producing a unique seed oil for
high-temperature uses such as deep frying.
In summary, this research showed for the first time the feasibility of analysing total phytosterol
content and profile in single sunflower half seeds. The possibility of analysing phytosterols
nondestructively at the single seed level opens up new opportunities for breeding for these traits of great
nutritional and commercial value.
ACKNOLEDGEMENTS
This work was supported by research project P07-AGR-03011 from Junta de Andalucía and EU FEDER
funds. The authors wish to thank Dr. C. Möllers, University of Göttingen, Germany and Dr. M.V. RuizMéndez, Instituto de la Grasa (CSIC), Spain for kind collaboration in the development of the method of
analysis of phytosterols in sunflower seeds.
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