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Optimization of secoisolariciresinol diglucoside

extraction from flaxseed (Linum usitatissimum L.)
and isolation by a simple HPLC-UV method

Claudia Fuentealba, Fernando Figuerola, Ana María Estévez, Adrian

González-Muñoz & Ociel Muñoz

To cite this article: Claudia Fuentealba, Fernando Figuerola, Ana María Estévez, Adrian
González-Muñoz & Ociel Muñoz (2015) Optimization of secoisolariciresinol diglucoside extraction
from flaxseed (Linum usitatissimum L.) and isolation by a simple HPLC-UV method, CyTA - Journal
of Food, 13:2, 273-281, DOI: 10.1080/19476337.2014.953209

To link to this article: http://dx.doi.org/10.1080/19476337.2014.953209

© 2014 Taylor & Francis Published online: 25 Sep 2014.

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 CyTA – Journal of Food, 2015
Vol. 13, No. 2, 273–281, http://dx.doi.org/10.1080/19476337.2014.953209

Optimization of secoisolariciresinol diglucoside extraction from flaxseed (Linum usitatissimum L.)

and isolation by a simple HPLC-UV method
Optimización de la extracción de secoisolariciresinol diglucósido desde linaza (Linum
usitatissimum L.) y su purificación a través de un método simple HPLC-UV
Claudia Fuentealbaa,b, Fernando Figuerolaa, Ana María Estévezc, Adrian González-Muñozd and Ociel Muñoza*
a
Institute of Food Science and Technology (ICYTAL), Faculty of Agricultural Sciences, Universidad Austral de Chile. Campus Isla Teja s/n,
Valdivia, Chile; bEscuela de Alimentos, Facultad de Recursos Naturales, Pontificia Universidad Católica de Valparaíso. Avenida
Waddington 716, Valparaíso, Chile; cDepartment of Agribusiness and Enology, Faculty of Agricultural Sciences, Universidad de Chile.
Avenida Santa Rosa 11315 La Pintana, Santiago, Chile; dDepartment of Food Science and Technology, Universidad de Santiago de Chile,
Obispo Umaña 050, Estación Central, Santiago, Chile
(Received 5 March 2014; final version received 3 August 2014)
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This research was conducted to develop and optimize a methodology to quantify the content of secoisolariciresinol diglucoside (SDG), the
main vegetable lignan present in flaxseed. However, at the time of this work, commercial SDG was not available for standard use. Therefore,
an effective method to obtain purified SDG from flaxseed was developed using chromatographic techniques. The SDG obtained was of
approximately 97% purity. The methanol extraction time, alkali concentrations, and reaction temperature were optimized through a response
surface methodology (RSM), and the optimal conditions established for the extractions were 47°C, 58 mmol L−1 sodium methoxide, and 24 h.
This methodology was then applied to study the variation of SDG content in flaxseed from different cultivars. The SDG content in different
varieties of flaxseed ranged from 10.8 to 17.9 mg g−1 in defatted flaxseed flour and from 6.0 to 10.9 mg g−1 in whole flaxseed.
Keywords: flaxseed; lignan; secoisolariciresinol diglucoside; RSM

El objetivo de esta investigación fue desarrollar y optimizar una metodología para cuantificar el contenido de secoisolariciresinol
diglucósido (SDG), el cual es el principal lignano vegetal presente en linaza. Sin embargo, al realizar este trabajo no existía SDG comercial
para ser utilizado como estándar externo. Por lo tanto, se desarrolló un método efectivo para obtener SDG purificado usando técnicas
cromatográficas. El SDG obtenido fue de 97% de pureza. Se optimizó el tiempo de extracción metanólica, concentración de alcali y
temperatura de reacción a través de un diseño de superficie respuesta. Las condiciones óptimas establecidas fueron: 47°C, 58 mmol L−1 de
metóxido de sodio y 24 h. Esta metodología luego fue aplicada para estudiar la variación del contenido de SDG en linaza de diferentes
cultivares. El contenido de SDG en diferentes variedades de linaza se encontró en el rango de 10,8 a 17,9 mg g−1 en harina desgrasada de
linaza y de 6,0 a 10,9 mg g−1 en linaza entera.
Palabras claves: linaza; lignano; secoisolariciresinol diglucósido; superficie respuesta

Introduction Doeswijk, Voragen, & Gruppen, 2009). Their formation occurs in

Flaxseed (Linum usitatissimum L.) is an important source of both
insoluble and soluble fiber and is highly rich in polyunsaturated
fatty acids, especially α-linolenic acid (Muir & Wescott, 2003).
In addition, flaxseed is also known for its high content of
lignans, a plant secondary metabolite, particularly secoisolaricir-
esinol diglucoside (SDG), compared to those found in other
grains, legumes, fruits, and vegetables (Meagher & Beecher,
2000; Thompson, Boucher, Liu, Cotterchio, & Kreiger, 2006).
Carob seed flour has high contents of isolariciresinol and pinor-
esinol (Durazzo et al., 2014); isolariciresinol is the predominant
lignan in pomegranate (Fischer, Jaksch, Carle, & Kammerer,
2012) and mataisolariciresinol is the main lignan in cereals like
wheat, rye (Hosseinian & Mazza, 2009), and legumes (Konar,
2013). Also, the vegetables green bean, carrot, cauliflower, white
cabbage, iceberg lettuce, and artichoke have important contents
of secoisolariciresinol (Konar, Poyrazoğlu, Demir,&Artik, 2012).
SDG is an esterified ester polymer that is composed of 3-
hydroxy-3-methyl glutaric acid (HMGA) and other phenolic com-
pounds such as glycosides of p-coumaric acid and ferulic acid
(Johnsson et al., 2002; Kamal-Eldin et al., 2001; Struijs, Vincken,
the outer layer of the seed; therefore, the greatest concentration of
SDG is found in the hulls of flaxseeds (Hano et al., 2006). In
addition to being esterified into an oligomer, SDG is complexed
with insoluble fiber, gums, polysaccharides, and mucilage asso-
ciated with the hull (Thompson, Robb, Serraino, & Cheung,
1991). SDG is the main precursor of the mammalian lignans
enterodiol (ED) and enterolactone (EL), which are formed by
intestinal mammalian bacteria (Clavel, Borrmann, Braune, Dore,
& Blaut, 2006; Eeckhaut et al., 2008; Wang, Meselhy, Li, Qin, &
Hattori, 2000). The enterolignans ED and EL have phytoestro-
genic effects due to their similarity to 17β-estradiol, which reg-
ulates sex-specific functions, and may be involved in preventing
some cancers (Muir & Wescott, 2003). SDG and its metabolites
are known for their potential benefits to human health because
they act as antioxidants (Hu, Yuan, & Kitts, 2007; Kitts, Yuan,
Wijewickreme, & Thompson, 1999), reducing serum cholesterol
levels and inhibiting the development of type I and II diabetes
(Prasad, 2000; Zhang et al., 2008), and it has been proven that
lignans can reduce the incidence of breast cancer and prostate
cancer through modulation of the synthesis, bioavailability, and

*Corresponding author. Email: ocielmunoz@uach.cl

© 2014 Taylor & Francis

 274 C. Fuentealba et al.

Table 1. Flaxseed lignan extraction methods.

Tabla 1. Métodos de extracción de lignanos de linaza.

Extraction methods Concentration Temperature Time Reference

Alcoholic extraction 1,4-dioxane/95% ethanol 60°C 16 h Johnsson et al. (2000)

Alkaline hydrolysis 0.3 mol L−1 NaOH Environment 48 h

Direct alkaline hydrolysis 1 NaOH 20°C 1h Eliasson et al. (2003)

Precipitation 60% ethanol
Alcoholic extraction 70% methanol Environment 4h Li, Yuan, Xu, Wang, and Liu (2008)
Alkaline hydrolysis 20 mmol L−1 NaOH 50°C 15 min
Alcoholic extraction 60% methanol 40°C 1h Chen et al. (2007)
Alkaline hydrolysis 1 mol L−1 NaOH
Alcoholic extraction 1,4-dioxane/ethanol 40°C 2h Coran, Giannellini, and Bambagiotti-Alberti (2004)
Alkaline hydrolysis 0.1 mol L−1 NaOH
Alcoholic extraction 70% ethanol 40°C 28 h Zhang et al. (2007)
Alkaline hydrolysis 1 mol L−1 NaOH Environment 12 h
Microwave-assisted extraction 70% methanol 50 W 3 min Beejmohun et al. (2007)
1 mol L−1 NaOH
Microwave-assisted extraction 40.9% ethanol 130 W 90.5 s Zhang and Xu (2007)
Alkaline hydrolysis 0.25 mol L−1 NaOH 25°C 2h
Alcoholic extraction 70% methanol Environment 2h Degenhardt, Habben, and Winterhalter (2002)
Alkaline hydrolysis 1 mol L−1 NaOH 3h
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activity of steroid hormones by binding to low levels of estrogen
receptors (Muir & Wescott, 2003; Touillaud et al., 2007).
In a first instance, for the extraction of lignans from flaxseed,
an alcoholic extraction and alkaline hydrolysis are usually used
to release SDG from its oligomer. Table 1 shows the extraction
methods used by several authors showing that mainly are differ-
entiated according to the solvent, temperature, and time of
extraction conditions. Hydrolysis of ester and glycosidic bonds
is performed to simplify the chromatographic analysis of the
SDG–oligomer-containing extracts. On the one hand, strong
acid hydrolysis breaks both bonds, resulting in addition in a
destruction of some lignans and cause transformation in other
lignans; on the other hand, alkaline hydrolysis breaks only the
ester bonds (Smeds et al., 2007). On this basis, Chen, Liu, Shi,
and Ma (2007) developed a high performance liquid chromato-
graphy (HPLC) method with ultraviolet (UV) detection for the
analysis of SDG in flaxseed, which involves extraction from
defatted flaxseed flour (DFF) with methanol and basic hydrolysis
with aqueous sodium hydroxide. To simplify this extraction
methodology, Eliasson, Kamal-Eldin, Andersson, and Åman
(2003) eliminated the alcohol extraction step by using aqueous
alkaline hydrolysis directly, resulting in a higher yield than that
obtained by the hydrolysis of alcoholic extracts (Johnsson,
Kamal-Eldin, Lundgren, & Åman, 2000). Other technique
includes the use of supercritical CO2 for lignan extraction from
flaxseed which results in lower SDG extraction compared to the
traditional solvent extractions (Comin, Temelli, & Saldaña,
2011). However, supercritical water is suitable for SDG extrac-
tion from flaxseed (Kanmaz & Ova, 2013).
To determine the conditions that optimize an extraction of
SDG from flaxseed, response surface methodology (RSM) is a
useful tool. Zhang et al. (2007) optimized with RSM the etha-
nol–water extraction of lignans from flaxseed using ethanol
concentration, temperature, and extraction time as factors. RSM
allows determining the levels of each factor and their interactions
that result in maximum yield (Myers, Montgomery, & Anderson-
Cook, 2009).
To quantify SDG, many authors have purified SDG by using
an external standard because this compound was not available at
the time of analysis. Johnsson et al. (2000) have purified SDG
from flaxseed using chromatographic methods. Starting with an
extraction with 1,4-dioxane/ethanol and alkaline hydrolysis, they
were able to purify SDG by reverse-phase chromatography using
silica gel, and they were finally able to confirm the identity of the
compound by nuclear magnetic resonance (NMR). This same
technique was used by other authors, who conducted similar
studies for the quantification of SDG (Eliasson et al., 2003).
Afterward, Li et al. (2008) and Yuan, Li, Xu, Wang, and Liu
(2008) purified SDG with chromatographic techniques using
solid-phase extraction (SPE), silica gel columns, and thin-layer
chromatography prior to the extraction with methanol. An effec-
tive method has also been developed for the purification of SDG
from flaxseed, which includes a series of extractions, involving
microwave and ion-exchange chromatography and reverse-phase
SDG, and produces over 95% purity (Stasevich, Mikhalenok, &
Kurchenko, 2009). By contrast, Bravi, Perretti, Marconi, Patrizi,
and Fantozzi (2011) used the aglycone secoisolariciresinol
(SECO) as a SDG standard, considering it as an equivalent
compound for the calibration curve and subsequent quantifica-
tion in a study of lignan determination in flaxseed oil.
Currently, there are patented purification techniques, among
them Westcott and Muir (1998) created a process of lignan SDG
extraction from defatted flaxseed, which has also been performed
by other researchers (Beejmohun et al., 2007; Zhang et al.,
2007). This method uses methanol extraction, alkaline hydroly-
sis, anion exchange chromatography, and finally separation by
reverse-phase HPLC and yields SDG in more than 90% purity.
Thus, a process for isolating and purifying SDG extractions,
mainly using supercritical CO2 and chromatographic separation,
was also patented (Pihlava, Hyvarinen, Ryhanen, & Hietantemi,
2004). The methods most commonly used for lignan determina-
tion in flaxseed are based on HPLC coupled with different
detector systems such as UV (Herchi et al., 2014).
The aim of this work was to develop and optimize a lignan
extraction from flaxseed for the quantification of SDG in differ-
ent flaxseed cultivars by HPLC/UV. To develop the methodol-
ogy, the methanol extraction, alkali concentrations, reaction
temperature, and chromatographic conditions were optimized
by RSM. At the time of this work, no commercial standard
was available for SDG. Therefore, it was necessary to purify

SDG from flaxseed to be used for quantification of SDG by
HPLC.
Materials and methods
Materials
Flaxseeds for extraction, optimization, and purification of SDG
were of the Celestina variety, year 2007, and obtained from
Seeds Baer SA (Araucanía Region, Chile). The flax varieties
used to study the contents of SDG consisted of two Chilean
varieties (Celestina and Carmine) and two cultivars from Canada
(Bethune and Sorrel), all obtained in 2008. Subsequently, the
same varieties were harvested in 2009 from two locations: Cajón
and Gorbea, both located in La Araucania, Chile. Samples were
kept refrigerated at 4°C before being analyzed by HPLC.
All reagents were of analytical and HPLC grade and were
purchased from Merck (Darmstadt, Germany). SECO standard
and the enzyme β-glucosidase from almonds (5.2 units mg−1
solid) were purchased from Sigma-Aldrich (Saint Louis,
MO, USA).
Downloaded by [180.253.103.200] at 09:21 10 September 2017
Purification of SDG
SDG extraction
Flaxseeds were ground in a coffee grinder to obtain fine flour.
DFF was prepared by the Soxhlet method for 2 h at 70°C with n-
hexane, as described by Johnsson et al. (2000) with some mod-
ifications. DFF (5 g) was mixed with 100 mL of 0.1 mmol L−1
sodium methoxide and sonicated for 30 min. The mixture was
incubated at 60°C for 24 h with agitation (100 rpm). The solu-
tion was filtered through Whatman paper Nº1 to remove the solid
waste and then neutralized with 12 mmol L−1 HCl. Twenty
milliliters of 0.1 mmol L−1 sodium acetate at pH 5.0 was
added, and the mixture was centrifuged for 15 min at 4200 g,
after which the supernatant was filtered. The solvent was evapo-
rated at 40°C, and 60 mL of 0.1 mmol L−1 sodium acetate at pH
5.0 was added to the resulting aqueous extract. The solution was
centrifuged again for 15 min at 4200 g and filtered. The lignans
were cleaned by SPE using 6 mL Sep-Pack® Vac C18 cartridges
(500 mg capacity) (Waters, Milford, MA, USA). The columns
were activated by successive washing with methanol and deio-
nized water. Then, 5 mL of sample was loaded and eluted with
2 mL of methanol. The procedure was performed in duplicate,
after which 4 mL of lignan extract (containing SDG) was
obtained and subsequently purified by HPLC.
Purification by HPLC
A pump model constaMetric 3200, a UV-Visible detector
model SM4000 (LDC Analytical, Riviera Beach, FL, USA)
and a manual injector model 7161 (Rheodyne, Oak Harbor,
WA, USA) were used. Separation was achieved on a Luna C8
column (250 × 4.6 mm id, 5 µm particle size; Phenomenex,
Torrance, CA, USA) operated at 20°C. The detection was
made at a wavelength of 280 nm. The chromatograms were
analyzed using Clarity software®, and the injection volume
was 100 µL. The mobile phase consisted of 1% aqueous
acetic acid/acetonitrile (85:15 v/v) with a flow rate of
1 mL min−1 (Chen et al., 2007; Chimichi, Bambagiotti-
Alberti, Coran, Giannellini, & Biddau, 1999). A fraction col-
lector model 2212 Helirac (LKB Bromma, Sweden) was used,
which fractionates every 30 s. The process was repeated 20
CyTA – Journal of Food 275
times to obtain approximately 9 mL in each fraction. The
fractions, corresponding to the peaks observed at 280 nm,
were measured in a T70 UV/Vis spectrophotometer (Pharma
Test, Hainburg, Germany) and were concentrated by Sep-
Pack® SPE C18 cartridges, Vac 6 cc (500 mg) (Waters,
IADET, Santiago, Chile), following the same procedure
described above. Samples were eluted with 2 mL of methanol.
The solvent was evaporated to dryness at 40°C in a forced-air
oven model ZRD-A-5030 (Zhicheng®) to gravimetrically
obtain the amount of solids in the sample.
Enzymatic confirmation
One milliliter of 0.1 mmol L−1 sodium acetate at pH 5.0 and
0.25 mg of β-glucosidase enzyme was added to the dried frac-
tions corresponding to each of the peaks detected. Enzymatic
hydrolysis was performed at 40°C for 12 h, according to
Renouard et al. (2010), with modifications. β-glucosidase
enzyme is efficient to remove the glycosyls groups from the
SDG and release the aglycone (SECO). All fractions were cen-
trifuged at 4200 g for 5 min, and the supernatants were then
separated by Sep-Pack® SPE C18 cartridges, Vac 6 cc (500 mg)
(Waters), following the same methodology described above. The
hydrolyzed fractions were analyzed by HPLC and compared
with the standard of SECO, and the chromatographic conditions
included a Luna C8 column, 250 × 4.6 mm id, and 5 µm particle
size (Phenomenex). The mobile phase consisted of 1% aqueous
acetic acid/acetonitrile (65:35 v/v) with a flow rate of
1 mL min−1 and detected at 280 nm. Therefore, the SECO-
hydrolyzed fraction identified corresponds to the fraction
of SDG.
Optimization and quantification of SDG from flaxseed
To optimize the SDG extraction from flaxseed, a response
surface methodology through the Box–Behnken design was
developed. The extraction method to assess the concentration
of sodium methoxide (50 and 150 mmol L−1), the reaction
times (12 and 24 h), and the temperature (40 and 60°C) were
used, which involved 15 experimental runs including three
central points. The optimal condition of extraction for quanti-
fication of SDG in flaxseed cultivars was used. The procedure
was performed in triplicate, and samples were kept refriger-
ated at 4°C before being analyzed by HPLC. For HPLC
measurements, the same equipment described above was
used. The injection volume was 20 µL, and a Luna C8
column with 250 × 4.6 mm id and a 5 µm particle size
(Phenomenex) was used at 20°C. The mobile phase consisted
of 1% aqueous acetic acid/acetonitrile (85:15 v/v) with a flow
rate of 1 mL min−1.
Validation of the analytical methodology
Optimal conditions of lignan extraction from flaxseed were
obtained using the second-order polynomial model of RSM.
The experimental and predicted SDG contents were compared
in order to determine the validity of the model.
To ensure the reliability of the results, the limit of detection
(LOD) and limit of quantification (LOQ) for SDG were deter-
mined by performing five calibration curves with five concentra-
tion levels. The accuracy and precision were evaluated by
spiking the standard and by calculating the coefficient of varia-
tion (CV), respectively.
 276 C. Fuentealba et al.

Statistical analysis
Multifactorial analysis of variance (ANOVA) to determine sta-
tistically significant differences (p < 0.05) between cultivars
(Bethune, Carmine, Celestina, and Sorrel), between the crop
years (2008 and 2009), and between the location of cultivation
(Cajón and Gorbea) was performed. The statistical analyses and
RSM were performed using Statgraphics Centurion XV
(StatPoint, Rockville, MD, USA).
Results and discussion
Purification of SDG
The release of the SDG molecule from the oligomer by an
alkaline hydrolysis using methanol as a solvent was performed.
Using this methanolic base hydrolysis method to hydrolyze, the
ester bond between SDG and HMGA was achievable. With this
technique, the union in a single step of the alcoholic extraction
and aqueous base hydrolysis was possible, thus shortening the
entire process of lignan extraction. After the methanolic base
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hydrolysis, the extract was neutralized using 12 mmol L−1 HCl
to prevent the ionization of carboxylic and phenolic groups. The
methanol extract was centrifuged in order to precipitate high-
molecular-weight molecules, such as cellulose and gums present
in flaxseed, permitting working with a supernatant composed by
lignan fractions. The solvent was evaporated and then reconsti-
tuted in an aqueous solution at pH 5.0, and the aqueous extract
was subsequently centrifuged and filtered again.
SPE to separate the lignans from other water-soluble
compounds, including some proteins that may interfere with
HPLC analysis, was performed. In this HPLC methodology, a
C8 column instead of a C18 column was used, but almost the
same retention time was obtained using the same mobile
phase as used by Chen et al. (2007). Figure 1 shows a
chromatogram of the lignan extract, and one of the peaks
observed corresponds to SDG. To identify and purify SDG,
each peak was collected. The fractions were subjected to
enzymatic hydrolysis with β-glucosidase to produce SECO
from SDG (Figure 2) and then compared with the external
SECO standard. Figure 3 shows a comparison of the peak

[mV]
4.9
5.5

12
2.8

10
18.1

8
Voltage

17.0

6
7.7

9.6

4
22.2
2.5

8.4
3.8
4.0
4.2

2
29.2
14.4

19.8

21.0

0 5 10 15 20 25 30
Time [min.]

Figure 1. HPLC chromatogram of lignan SDG and other compounds extracted from flaxseed recorded at 280 nm. The injection volume was 100 µL and
the mobile phase consisted of 1% aqueous acetic acid/acetonitrile (85:15 v/v) with a flow rate of 1 mL min−1.
Figura 1. Cromatograma HPLC del lignano SDG y otros compuestos extraídos desde linaza registrado a 280 nm. El volumen de inyección fue 100 µL y
la fase móvil consistió de 1% de ácido acético acuoso/acetonitrilo (85:15 v/v) con flujo de 1 mL min−1.

Figure 2. Enzymatic hydrolysis with β-glucosidase to produce SECO from SDG.

Figura 2. Hidrolisis enzimatica con β-glucosidasa para producir SECO desde SDG.
 CyTA – Journal of Food 277
[mV]

–99.40

SDG + β-Glucosidase

–99.45
Voltage

–99.50

SECO

2 4 6 8 10 12 14 [min.]
Time
Downloaded by [180.253.103.200] at 09:21 10 September 2017

Figure 3. HPLC chromatogram for the enzymatic test recorded at 280 nm. The injection volume was 20 µL and the mobile phase consisted of 1%
aqueous acetic acid/acetonitrile (65:35 v/v) with a flow rate of 1 mL min−1.
Figura 3. Cromatograma HPLC para la confirmación enzimática registrado a 280 nm. El volumen de inyección fue 20 µL y la fase móvil consistió de 1%
de ácido acético acuoso/acetonitrilo (65:35 v/v) con flujo de 1 mL min−1.

[µV]
600

500

400

300
Voltage

200

100

–100

0 2 4 6 8 10 12 14 [min.]
Time

Figure 4. HPLC chromatogram of lignan SDG purified by collecting fractions, recorded at 280 nm. The injection volume was 20 µL and the mobile
phase consisted of 1% aqueous acetic acid/acetonitrile (85:15 v/v) with a flow rate of 1 mL min−1.
Figura 4. Cromatograma HPLC del lignano SDG purificado a través de recolector de fracciones registrado a 280 nm. El volumen de inyección fue 20 µL
y la fase móvil consistió de 1% de ácido acético acuoso/acetonitrilo (85:15 v/v) con flujo de 1 mL min−1.

retention time of 9.6 min from the enzymatic hydrolysis with
the respective SECO standard, thus verifying that the 9.6 min
peak from the corresponding lignan extract corresponds to
SDG. The SDG obtained was approximately of 97% purity
(Figure 4). Once the purified compound was obtained, SPE to
change the solvent in which it was dissolved (mobile phase)
and to leave only the metabolite dissolved in methanol was
performed. Then, it was possible to evaporate the solvent at
40°C and gravimetrically determine the amount of SDG stan-
dard. Approximately, 10 mg of the compound was obtained,
which was then dissolved in methanol at a concentration of
1 mg mL−1.
Optimization of SDG extraction from flaxseed by RSM
A Box–Behnken design was performed to determine the optimal
conditions of temperature (X1), sodium methoxide concentration
(X2), and time (X3) for the SDG extraction from flaxseed. The
values of the three independent variables and SDG yield (Y) are
 278 C. Fuentealba et al.

Table 2. Box–Behnken design and its results.

Tabla 2. Diseño Box–Behnken y sus resultados. 9.2

SDG (mg g–1 flaxseed)

8.7
Sodium SDG yield
methoxide (mg g−1 8.2

Experimental concentration Temperature Time whole 7.7

run (mmol L−1) (ºC) (h) flaxseed) 7.2 150
130
6.7 110
90
1 50 40 18 8.455 40 44 70
48 52 56 50 Concentration (mMol L–1)
60
2 150 40 18 7.351 Temperature (°C)
3 50 60 18 7.702
4 150 60 18 7.085 Figure 5. Estimated response surface after 18 h of the extraction
5 50 50 12 8.910 process of flaxseed lignans.
6 150 50 12 8.497
7 50 50 24 9.100 Figura 5. Superficie respuesta estimada de extracción de lignanos de
8 150 50 24 7.602 linaza con 18 h de tiempo de reacción.
9 100 40 12 8.844
10 100 60 12 6.994
11 100 40 24 9.109
12 100 60 24 7.682 the maximum SDG, which features the following
13 100 50 18 8.562
14 100 50 18 9.211 conditions as optimal for the extraction process: 47°ºC,
15 100 50 18 9.031 58 mmol L−1 sodium methoxide, and 24 h. At this point,
9.4 mg of SDG per gram of whole flaxseed was obtained as
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predicted value.

shown in Table 2. A second-order polynomial equation for SDG

was obtained as follows:
Y ¼  8:47 þ 0:718433  X1 þ 0:0316767  X2  0:0433333  X3
 0:00828208  X12 þ 0:0002435  X1 X2 þ 0:0017625  X1 X3
 0:000183283X22  0:000904167X2 X3 þ 0:00141088X32
The results of the ANOVA for the fitted model are summar-
ized in Table 3. The adjusted model has a coefficient of
correlation (R2 = 0.862). Since the p-value of the Durbin–
Watson test was not significant (p > 0.05), there is no indica-
tion of autocorrelation in the residuals at the 5% significant
level. The non-significant lack of fit indicates that the model
is adequate for the observed data at 95% confidence level.
The ANOVA indicated that the concentration of sodium
methoxide has a statistically significant effect (p < 0.05) on
the SDG yield, in addition with the quadratic term of tem-
perature (p < 0.05). The predicted model, through the esti-
mated response surface (Figure 5), was used to obtain
Validation of the methodology
The suitability of the predicted model equation for the opti-
mum SDG yield was tested. The experimental conditions were
evaluated in the same variety in which the optimization was
made (Celestina), i.e. a comparison of SDG experimental
values from the predicted ones of Celestina harvested in
2008 and 2009 (Cajón and Gorbea) was performed. The
SDG contents were 7.64 ± 0.51, 10.91 ± 0.72, and
8.83 ± 0.60 mg g−1 whole flaxseed, respectively. The experi-
mental values were found to be close to the predicted one
(9.4 mg g−1 whole flaxseed).
The accuracy of the method by calculating the CV in three
replicates of each variety of flaxseed studied was verified. The
CV was between 0.7% and 6.7%. The accuracy of the method
was measured by adding a known amount of SDG standard to
the samples, and the average recovery rate was 104.5% ± 8.8%.
The LOD of the method was 1.8 µg g−1 and the LOQ was
5.4 µg g−1.

Table 3. Analysis of variance for the fitted model.

Tabla 3. Análisis de varianza para el modelo ajustado.

Source Sum of squares Degrees of freedom Mean squares F-value P-value

X1 (concentration, mmol L−1) 1.6489 1 1.6489 14.69 0.0618

X2 (temperature, °C) 2.3070 1 2.3070 20.55 0.0454
X3 (Time, min) 0.0077 1 0.0077 0.07 0.8180
X12 0.7752 1 0.7752 6.91 0.1194
X22 0.0593 1 0.0593 0.53 0.5429
X32 0.2943 1 0.2943 2.62 0.2468
X1 X2 2.5327 1 2.5327 22.56 0.0416
X1 X3 0.0447 1 0.0447 0.40 0.5924
X2 X3 0.0095 1 0.0095 0.08 0.7983
Lack of fit 0.9855 3 0.3285 2.93 0.2650
Pure error 0.2245 2 0.1123
Total (corr.) 8.7586 14
R2 = 0.8619 Standard error of Est. = 0.4919 Durbin–Watson statistic = 2.1887 (p = 0.4205)
Adjusted R2 = 0.6132 Mean absolute error = 0.2555

Figure 6. SDG content of flaxseed in four varieties according to their
location of cultivation (Cajón and Gorbea, harvested in 2009) and year of
harvest (2008). Bar graphs with the same letter are not significantly
different (p < 0.05).
Downloaded by [180.253.103.200] at 09:21 10 September 2017
Figura 6. Contenido de SDG de linaza en cuatro variedades de acuerdo
a su localidad de cultivo (Cajón y Gorbea, cosechada en 2009) y año de
cosecha (2008). Gráficos de barras con la misma letra no muestra
diferencias significativas (p < 0.05).
SDG content in flaxseed cultivars
The method for the determination of SDG to four varieties of
flax (Celestina, Carmine, Bethune, and Sorrel) was applied. The
first two correspond to cultivars of Chilean origin, and the last
two were of Canadian origin. The compound was identified by
comparing retention times with the respective standards, and
the amount of SDG was in the range of 11.3–13 µg g−1 in DFF
and 6.9 and 7.6 µg g−1 in whole flaxseeds (Figure 6). These
values were similar to those obtained by other authors. Kanmaz
and Ova (2013) reported 12.9 mg g−1 of SDG dry weight (d.
w.). Chen et al. (2007) observed between 5.7 and 13 mg g−1 of
SDG in flaxseed from the Gansu province, China. Yuan et al.
(2008) found SDG in DFF in the range of 6–29 mg g−1 and Li
et al. (2008) reported a SDG content of 15.4 mg g−1 in flax-
seed. In addition, Johnsson et al. (2000) obtained SDG in the
range of 11.7–24.1 mg g−1 in DFF and 6.1–13.3 mg g−1 in
whole flaxseed from cultivars of Denmark and Sweden. Hao
and Beta (2012) reported that the SDG content in flaxseed hulls
varied from 16.4 to 33.9 mg g−1. The SDG content found in
this research was higher than that found in other food sources,
i.e. total lignans in carob seed flour ranged from 1.6 to
17 µg g−1 d.w. (Durazzo et al., 2014), 45.8 µg g−1 d.w. in
pomegranate twigs (Fischer et al., 2012), 67 µg g−1 in rye
(Smeds, Jauhiainen, Tuomola, & Peltonen-Sainio, 2009), and
7.36 and 3.10 mg g−1 d.w. in white and black sesame seeds,
respectively (Kim et al., 2014).
In this study, the highest content of SDG was found in the
Celestina variety, followed by Sorrel, while lower levels were
found in the Bethune and Carmine varieties. No significant
differences were found between the Canadian strains; however,
the differences between the Chilean varieties (Celestina and
Carmine) were statistically significant (p < 0.05), as they cor-
respond to varieties with higher and lower SDG contents,
respectively. All the varieties described above were obtained
in 2008.
The content of SDG was also measured in the 2009 harvest
of the same varieties from two different areas, Cajón and
CyTA – Journal of Food 279
Gorbea, both in the region of La Araucanía. The highest con-
tent of SDG was observed in the cultivars from Celestine,
where the contents of lignans in Cajón and Gorbea were 10.9
and 8.8 mg g−1 of whole flaxseed, respectively. These results
showed a similar pattern to that from crop year 2008, where the
Chilean variety (Celestine) presented a significantly higher
SDG content than other cultivars. The Sorrel and Bethune
Canadian cultivars showed the lowest levels of SDG for both
areas; in Cajón, the SDG content reached 7.1 and 6.0 mg g−1 of
whole flaxseed, respectively. Figure 6 shows the SDG content
of the different varieties of flax studied in various locations of
cultivation and harvest years. In regard to the location of
cultivation, the SDG content varied significantly between the
cultivars from Gorbea and Cajón, and the highest content was
found in the latter location. However, no significant differences
were found for the cultivar from Sorrel when grown in both
locations. Significantly different amounts of flax were obtained
from Gorbea and Cajón because of the drastic difference in
their soil types. Gorbea is composed of trumao soils (volcanic
soils) and Cajón is mainly composed of transition soils
(between trumao and clay soil). Another difference between
these two locations was that, at the time of cultivation, there
were three irrigation systems in Gorbea, while no irrigation
systems existed in Cajón. These conditions can explain the
difference in performance and content of SDG. The varieties
of flax from the 2008 harvest were different from the Gorbea
and Cajón crops in 2009 (p < 0.05), specifically in the Chilean
varieties (Celestina and Carmine); however, harvesting these
varieties in Cajón significantly increased their performance. In
contrast, there was no significant increase in yield at harvest in
Cajón for the Canadian variety Bethune. However, there was a
reduction in the harvest yield in Gorbea. These results indicate
that agronomic variations significantly influence the amount of
lignans found in flaxseed.
Conclusions
A method for the purification of SDG from flaxseed using
chromatographic techniques as well as HPLC/UV was devel-
oped. This method involves the extraction from DFF with
sodium methoxide, SPE, and purification of the compound by
HPLC. The process of lignan extraction from flaxseed by com-
bining the alcohol extraction and the alkaline hydrolysis in a
single step, through a methanolysis with 58 mmol L−1 sodium
methoxide at 47°C for 24 h, was optimized. This method was
validated and applied for the quantification of SDG and was
found that different cultivars of Canadian and Chilean varieties
of flaxseed have significantly different SDG contents. In addi-
tion, differences in SDG content were observed between harvests
of 2008 and 2009, as well as between the location of crops
(Cajón and Gorbea). The content of SDG in flaxseed ranged
between 6.0 and 10.9 mg g−1 in whole flaxseeds. This work
could be used in an easy way for characterizing SDG among
flaxseed.
Acknowledgments
The authors wish to thank Mr Fernando Asenjo for his technical
assistance. This research was supported by the project
FONDECYT 1080277, for which the authors are deeply
indebted. C. Fuentealba obtained the CONICYT doctoral fellow-
ship in 2009.
 280 C. Fuentealba et al.

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