J.

of Supercritical Fluids 75 (2013) 94–100

Contents lists available at SciVerse ScienceDirect

The Journal of Supercritical Fluids

journal homepage: www.elsevier.com/locate/supflu

Supercritical antisolvent fractionation of lignans from the ethanol

extract of flaxseed
Giuseppe Perretti, Claudia Virgili, Antonio Troilo, Ombretta Marconi,
Gian Franco Regnicoli ∗ , Paolo Fantozzi
Department of Economic and Food Science, via S. Costanzo n.c.n., University of Perugia, 06126 Perugia, Italy

a r t i c l e i n f o a b s t r a c t

Article history: Supercritical antisolvent fractionation (SAF) was evaluated in the fractionation and concentration of a
Received 7 November 2012 family of beneficial plant compounds called lignans from the ethanol extract of flaxseed. The amount of
Received in revised form lignans obtained in different fractions was studied under varying pressures (10–30 MPa), CO2 flow rates
15 December 2012
(5–15 kg h−1 ) and times of treatment (60–180 min) in a three-stage fractionation column with constant
Accepted 16 December 2012
temperature fixed at 313, 323 and 333 K. The determination of lignan content was performed by HPLC cou-
pled with a coulometric array detector. The effects of each individual variable as well as their interactions
Keywords:
were investigated using a full factorial design with three factors and two levels and the optimal condi-
Supercritical carbon dioxide
Antisolvent
tions were calculated through response surface methodology. A statistically significant increase in lignan
Fractionation content was obtained after the SAF process; from an average initial lignan content of 1.66 ± 0.13 g L−1 it
Flaxseed was possible to obtain a total lignan content ranging from 3.42 to 12.96 g L−1 . We conclude that SAF is an
Lignans appropriate technique for the isolation of lignans from flaxseed.
Ethanol extract © 2013 Elsevier B.V. All rights reserved.

1. Introduction dimerized into pinoresinol (PINO). Subsequently, reduction, oxida-

Flax (Linum usitatissimum L.) is a plant known for its commer-
cial utility for the production of fiber and oil. The fiber is primarily
found in the long stems of flax while the oil is expressed from the
large seeds which containing 15–40% oil by weight [1]. Although the
flaxseed oil has important industrial applications, it also plays a role
in the food supply. As stated by Oomah [2], flaxseed oil is a promi-
nent functional food due to its content of ␻-3 fatty acids; in fact,
the essential fatty acid ␣-linolenic acid represents approximately
52% of the total fatty acid content of flaxseed oil [2]. Flaxseeds also
contain phenolic compounds such as lignans in very high concen-
trations (>600 mg kg−1 ) compared to other plants and plant parts
[3].
Lignans are characterized by having two propyl-benzene
molecules (coniferyl alcohols) that are linked by a bond between
the 8 and 8 positions. Lignans are biosynthesized through
the phenylpropanoid pathway and they are stereospecifically
Abbreviations: HYDMA, idrossimataresinol; ISO, isolariciresinol; LARI, lar-
iciresinol; MATA, matairesinol; PINO, pinoresinol; RF, residual fraction; RSM,
response surface methodology; SECO, secoisolariciresinol; SDG, secoisolariciresinol
diglucoside; SF, separator fraction; SAF, supercritical antisolvent fractionation;
SC-CO2 , supercritical carbon dioxide.
∗ Corresponding author. Tel.: +39 075 585 7923; fax: +39 075 585 7939.
E-mail address: gianregnicoli@libero.it (G.F. Regnicoli).
tion, dehydrogenation and addition reactions lead to the formation
of a broad range of lignans which are present in both aglyconic
and glycosylated forms. The lignans found in the highest concen-
trations in flaxseed are secoisolariciresinol (SECO), matairesinol
(MATA), pinoresinol (PINO), lariciresinol (LARI), idrossimataresinol
(HYDMA), isolariciresinol (ISO) and secoisolariciresinol diglucoside
(SDG) [4]. Lignans are stored in plants predominantly as glycosides,
and they are converted by intestinal bacteria into metabolites with
estrogen-like activity like equol, enterodiol and enterolactone [3].
There is evidence of health benefits attributable to lignans, and
enterolactone and enterodiol are considered partially responsible
for the prevention/inhibition of human prostate, colon and skin
cancers as well as reduction of menopausal symptoms and post-
prandial blood glucose and protection of the cardiovascular system,
fertility and thyroid function [5–10].
To promote lignan consumption, SDG extracts can be incorpo-
rated as ingredients into foods and drinks; in fact some patents
and applications already exist. For example, SDG can be isolated
from flaxseed and dried into powder by standard techniques such
as vacuum concentration, spray drying or freeze drying. These SDG-
rich products can be used directly as a powder or as ingredients
in the production of foods such as soy flour or in other various
foods and drinks [11]. Typical examples include spreads, dress-
ing, mayonnaise, ice creams, cream alternatives, health bars, health
drinks, sports drinks, confectionery, bakery products, soups, cere-
als, sauces, fillings and coatings [12]. Products such as these can be

0896-8446/$ – see front matter © 2013 Elsevier B.V. All rights reserved.
http://dx.doi.org/10.1016/j.supflu.2012.12.028
 G. Perretti et al. / J. of Supercritical Fluids 75 (2013) 94–100
developed to contain 0.1–90% SDG by weight depending on serv-
ing size. For products with high water content that are consumed
in large amounts such as drinks and soups, the SDG content can
be appropriately decreased to a range 0.001–10% by weight, for
example [11].
After the first studies of SDG by Bakke and Klosterman (1956)
[13], some methods for the analysis of lignans and other phenolic
compounds from flaxseeds have been reported [14,15]. Literature
shows that lignans can be extracted from flaxseed and other matri-
ces, using dioxane, methanol [13], ethanol or enzymatic hydrolysis,
followed by different purification systems [13–17]. The extraction
methods vary in relation with the sample matrix and the target
compound [14]. The method of sample preparation described in
this article shows, after experimental tests, a high content of lig-
nans, the use of non-toxic solvents and the use of a short time
compared to the methods shown in the literature [14].
Our laboratory has studied the application of supercritical fluids
extraction to valorize flaxseed oil. We found extraction from ground
flaxseed yields 19.5 ± 0.9% oil by weight using neat SC-CO2 (super-
critical carbon dioxide), and 23.0 ± 2.2% oil by weight with the use
of ethanol as a co-solvent. The quality of the extracted oils was ana-
lyzed by common parameters (e.g., free acidity, peroxide number)
and functional aspects (e.g., polyphenol content, lignan content).
We found the oils obtained by supercritical fluid extraction were
generally comparable to oils produced by traditional extraction
technologies, with an increase in total polyphenol extraction but
without an increase in lignan extraction [18–20]. The same authors
already studied the behavior of flax seed oil fractionation by neat
supercritical carbon dioxide under pressures from 10 to 33 MPa
and temperatures of 313, 323, and 333 K, even if in this specific
case a low efficiency was observed (no statistical differences for
p < 0.05) [18]. Additionally, we also studied the shelf life of flaxseed
oil and SDG content [21]. In all those experiments, the concentra-
tion of lignans in flaxseed oil was very low (2–4 mg kg−1 ), probably
due to the polar and polymeric nature of lignans and their pres-
ence as glycosides. It is known that when polar compounds have
to be concentrated and fractionated from an organic solution, the
hydrophobic character of CO2 can be an advantage. In this con-
text, the supercritical antisolvent fractionation (SAF) process could
improve lignan extraction because it involves the continuous con-
tact between the SC-CO2 and the liquid mixture in a pressurized
vessel during concentration and fractionation [22].
The aim of this study was to assess the efficiency of SAF for the
concentration of flaxseed lignans using an ethanol extraction of
lignans obtained from defatted flaxseed flour.
2. Materials and methods
2.1. Samples and reagents
Flaxseeds (2011 harvest, Barbara variety) were purchased from
Terra Bio Soc. Coop. (Urbino, Italy). The flaxseed moisture deter-
mined (8.65% ± 0.05) is comprised in the common range of flaxseed
moisture reported in literature [1].
Standard preparations of the isolated lignans SECO, MATA, PINO,
LARI, HYDMA and ISO were purchased from Arbonova (Turku,
Finland). SDG was purchased from Chemos GmbH (Reenstauf,
Germany). Methanol, ethanol, acetonitrile, petroleum ether, and
glacial acetic acid were purchased from Sigma–Aldrich Chemie
GmbH (Steinhein, Germany).
2.2. Sample preparation
The ethanol extraction of lignans from flaxseeds was performed
following the method of Westcott and Muir (1998) [17] modified
95
as follows: once removed from cold storage, 120 g of fresh seeds
were ground in a laboratory grinder (Osterizer Sunbeam Model No.
4153-50) at maximum speed for 30 s. Solvent defatting was per-
formed over 2 h using 500 mL of petroleum ether (25:6, v:w) under
magnetic stirring at room temperature; the mixture was filtered
with a 22.09 cm2 steel mesh (0.5 mm pore size) and the resid-
ual petroleum ether was evaporated for 1 h at room temperature
under a constant stream of air. The defatted seeds, now weighing
approximately 100 g, were stirred for 2 h at room temperature in
600 mL of ethanol, water and 1 M NaOH at a ratio of 4:1:1 (v:v:v).
After extraction, this solution was filtered with the above described
steel mesh, neutralized with glacial acetic acid to pH 6 and cen-
trifuged at 6000 × g for 20 min to precipitate and remove water
soluble polysaccharides and proteins. For each run, the preparation
of fresh ethanolic extract was repeated.
2.3. Supercritical antisolvent fractionation
The SAF experiments were conducted in triplicate, using a
Muller Extract Company GmbH (Koburg, Germany) pilot plant
(Fig. 1), equipped with a three-stage fractionation column (total
length 3 m, diameter 3 cm) with an internal volume of 2 L and
packed with stainless steel Raschig rings (10 mm × 10 mm); each
column stage was individually thermostated. The separation sec-
tion of the plant consisted of a 1 L cylindrical separator followed by
two cyclonic separators, both set at 303 K and 6 MPa. Each exper-
iment was conducted in one batch with a feed of 350 mL. The
three-stage column temperatures were fixed at 313, 323 and 333 K,
from the bottom to the top. These temperatures were chosen fol-
lowing some suggestion reported in the literature of supercritical
fluids fractionation [23,24] to preserve the ethanol evaporation,
to save energy and to explore the behavior at the related den-
sities. This determined a density gradient in the solvent stream,
with SC-CO2 densities varying along the column. The feed was
introduced into the column from the bottom. At the end run, the
residual fraction (RF) was recovered from the bottom of the column
and measured. The separator fraction (SF) was recovered in the
separator.
2.4. Experimental design and statistical software
The effects of three variables affecting the concentration of lig-
nans (time of treatment (X1 ), CO2 flow rate (X2 ) and pressure (X3 ))
were studied following a full factorial design (23 ) and response sur-
face methodology. Two factor levels were chosen for each variable
considering the limits of the experimental apparatus and the order
of the runs was randomized to avoid systematic errors. The mea-
sured response (Y%) was the enrichment (E%) of lignans after SAF.
Table 1 shows the experimental matrix for the 23 factorial design
(3 factors, each run at the two levels).
The influence of the different parameters on lignan content (per-
centage ratio between the RF lignan content and the initial lignan
content) was determined using the following interactions regres-
sion equation (Eq. (1)).
Y = b0 − b1 X1 − b2 X2 − b3 X3 + b4 X1 X2 + b5 X1 X3 + b6 X2 X3 (1)
where Y is the enrichment (%), b0 is the constant term, b1 , b2 and b3
are the coefficients of the individual variables X1 , X2 , X3 and b4 , b5 ,
b6 are the coefficients of the pairs of interactions between the vari-
ables X1 X2 , X1 X3 and X2 X3 . All computations involving regression
models, response surface plots and ANOVA tests were performed
using the Statistic-Toolbox 7 with MATLAB (The Mathworks Inc.,
Natick, USA).
96 G. Perretti et al. / J. of Supercritical Fluids 75 (2013) 94–100

Fig. 1. Fractionation pilot plant scheme.

Table 1 shown; the r2 levels are above 0.9991 for all the analytes (data not
Experimental parameters under the full factorial design.
reported).
Exp. Time (min) CO2 flow (kg h−1 ) Pressure (MPa) X1 X2 X3 The lignans in the ethanol extract of flaxseeds were identified
1 60 5 10 −1 −1 −1 by comparison of their retention times with those of the standards
2 60 15 10 −1 1 −1 and they were quantified by using their calibration curves in the
3 60 5 30 −1 −1 1 linear range 50–1050 ␮g L−1 .
4 60 15 30 −1 1 1
5 180 5 10 1 −1 −1
6 180 15 10 1 1 −1 2.6. Ethanol quantification
7 180 5 30 1 −1 1
8 180 15 30 1 1 1 The ethanol contents of the extracts, RFs and SFs, were deter-
X1 , time; X2 , CO2 flow rate; X3 , pressure. mined by the OIV-MA-AS312-01A: R2009 4. C. method.

2.7. Moisture quantification

2.5. HPLC analysis of the extracts
The moisture content of the flaxseed was determined by the
In order to quantify the concentration of lignans obtained by AOAC (1984, XIV edition) 14.004 method.
SAF, the fractions obtained from each experiment were analyzed
by high performance liquid chromatography coupled with coulo-
3. Results and discussion
metric detector array (HPLC-ECD). Two Jasco PU-1580 pumps
connected to a gradient solvent system and a Basic Marathon
3.1. Lignan enrichment
“Spark” autosampler (Erkerode, The Netherlands) with a 100 ␮L
loop were used. An Inertsil ODS-3 V (C18 250 mm × 4.6 mm Ø; par-
Both total lignan content and SDG content in the RFs differed sig-
ticle size 5 ␮m) column, a CoulArray detector (ESA Inc., Chelmsford,
nificantly (ANOVA, p < 0.05) and their values under each condition
USA) with eight electrode potentials set to 100–905 mV at incre-
are reported in Table 3.
ments of 115 mV were used. Mobile phase A was 0.05 M KH2 PO4
The SAF process was effective in concentrating lignan content in
and 0.05 ␮M sodium lauryl sulphate (SLS) and mobile phase B was
the RF; from an average initial lignan content of 1.66 ± 0.13 g L−1 the
phase A/CH3 –OH/CH3 CN, 30:20:50 (v:v:v) and 0.05 ␮M SLS. The
RF total lignan content increased to a range 3.42–12.96 g L−1 , which
mobile phases were adjusted to pH 3.35 with 85% orthophosphoric
corresponds to enrichment values from 201 to 753%, as reported in
acid and were filtered with a 0.22 ␮m membrane filter (Millipore,
Table 3. Very low concentrations of lignans (0.03 ± 0.02 g L−1 ) were
Bedford, USA, for aqueous solvents; MSI, USA, for organic solvents).
detected in the SF. In all experiments the major lignan present
The voltammetric data were collected and analyzed by CoulArray
in the RF was SDG. Of the other lignans analyzed in this study,
software. In Table 2, the calibration results of the standards are
HYDMA was present at a low concentration both in the initial
extract (0.04 ± 0.01 g L−1 ) and in the RF (0.02–0.12 g L−1 ). ISO was
Table 2 also detected at low levels, with an initial extract concentration of
Calibration data. 0.006 ± 0.002 g L−1 and 0.02–0.06 g L−1 in the RF. SECO, LARI, PINO
and MATA were observed below the limit of quantification of the
Name RT (min) Calibration range (␮g L−1 ) LOD (␮g L−1 ) LOQ (␮g L−1 )
analytical method in both the initial extract and in the RF. The spe-
SDG 15.25 50–1050 26.96 28.65 cific enrichment in SDG shows that there is also a potential slight
ISO 18.73 50–1050 21.27 27.18
HYDMA 20.53 50–1050 27.87 33.13
fractionation of the different lignans when SDG enrichment % is
SECO 22.03 50–1050 42.58 49.35 different than the total enrichment %. Furthermore the trace lignan
LARI 22.55 75–1050 53.57 58.74 content in the SF (0.03 ± 0.02 g L−1 ) confirms the strong selective
PINO 26.94 50–750 25.45 32.31 action of SC-CO2 by extracting mainly ethanol from the column.
MATA 30.97 50–1050 23.73 28.17
The mathematical models for total lignan enrichment and
RT, retention time; LOD, limit of detection; LOQ, limit of quantification. SDG enrichment (Eqs. (2) and (3), respectively) provide a good
 G. Perretti et al. / J. of Supercritical Fluids 75 (2013) 94–100 97

Table 3
Total lignan content and SDG content (g L−1 ) and enrichment % (E) in the RFs.

Exp. Time (min) CO2 flow (kg h−1 ) Pressure (MPa) Total lignans* SDG*
−1
(g L ) E (%) (g L−1 ) E (%)

1 60 5 10 3.42ab +201ab 3.41ab +201ab

2 60 15 10 3.81ab +283ab 3.81abc +283abc
3 60 5 30 6.15abc +353abc 6.03abc +355abc
4 60 15 30 6.63bcd +388bcd 6.34bcd +392bcd
5 180 5 10 3.79ab +240ab 3.60abc +236abc
6 180 15 10 5.24abc +323abc 5.22abc +322abc
7 180 5 30 9.11cd +560cd 8.85cd +565cd
8 180 15 30 12.96e +753e 12.87e +753e
*
Means in the same column not sharing a common superscript letter were significantly different (one way ANOVA; p < 0.05, n = 3).

representation of the real trend of the experiments.
%Etot = 232.375 − 1.36041 XT − 1.3125 XP − 1.275 XF
+ 0.1027 XTP + 0.066250 XTF + 0.1575 XPF
2
r = 0.9935; RMSE = 19.6250%
is probably due to the changed solubility of the residual ethanol
at the low concentrations in the RF, where the ethanol solubility
in water is still evident (the RF is mainly composed of water and
polar moieties). On the other hand, increasing the flow rate of the
shorter treatment time had a positive effect on the concentration of
(2) lignans. In fact, with 60 min of treatment at 300 bar and flow rate at

%ESDG = 227.625 − 1.3729 XT − 1.0625 XP − 0.775 XF a) Interaction Regression

800
+ 0.1035 XTP + 0.1425 XPF
700 exp8
r 2 = 0.9943; RMSE = 18.3750% (3) Enrichment % (Lignans) Predicted data

XT , XP , and XF represent independent variables of time of treatment, 600 exp7

pressure and CO2 flow rate, respectively while XTP , XTF , and XPF rep-
resent the interactions among the three variables. The regression 500
coefficient is represented by r2 and RMSE is the root mean square
exp4
error.
400
The graphs in Fig. 2 show the actual and predicted data disper- exp6
sion around the bisector. The equation line almost coincides with exp3
300
the bisector, therefore it is completely covered and is not visible. exp2
exp1
The dispersion of the 8 experiments is linear and in agreement with exp5
the model as confirmed by the high regression coefficient (0.9935 200
for total lignans and 0.9943 for SDG) and by the low root mean
square error (19.6250% for total lignans and 18.3750% for SDG). 100
Taking into account the suitability of the regression method, it
is possible to describe the relationship between the different vari-
100 200 300 400 500 600 700 800
ables and enrichment in lignans and SDG using response surface Enrichment % (Lignans) Experimental data
model (RSM) methodology. The quadratic response surface (Fig. 3)
graphically reveals the results from the model. The highest levels of
b) Interaction Regression
lignan enrichment were obtained at the highest levels of the three 800
variables (30 Mpa, 15 kg h−1 , 180 min) confirming the evidence of
the results reported in Table 2. exp8
700
The same behavior was observed for SDG (Fig. 4).
Enrichment % (SDG) Predicted data

The response surfaces for pairs of variables are given in Fig. 5.

exp7
The time–pressure interaction (Fig. 5a) indicates a major role for 600
pressure in influencing lignan enrichment. Likewise the time–CO2
flow rate interaction (Fig. 5b) indicates a major role for CO2 flow rate
500
in determining the separation yield. Lastly, the pressure–CO2 flow
rate interaction (Fig. 5c) indicates another major role for pressure exp4
in the determination of the separation yield. 400
exp6
At the lowest pressure level, neither the stability of the sepa-
exp3
ration (Fig. 5a) nor the solvent flow rate (Fig. 5c) was influenced, 300
and the same behavior was observed for SDG (response surfaces exp2
exp1
not reported).
200 exp5
In order to explore the validity of the mathematical model
for higher flow rates, times and pressures additional trials were
conducted. The experiments at 300 min of treatment, flow rate 100
of 15 kg h−1 and pressure of 30 or 33 MPa and also 180 min of 100 200 300 400 500 600 700 800
Enrichment % (SDG) Experimental data
treatment with flow rate of 15 kg h−1 did not influence the concen-
tration of lignans except for in experiment 8. This indicates a limit Fig. 2. Regression model of actual values–predicted values for total lignans (a) and
of the mathematical model for the higher pressure and time. This SDG (b).
98 G. Perretti et al. / J. of Supercritical Fluids 75 (2013) 94–100

Fig. 3. Quadratic response surface model for lignan enrichment.

24 kg h−1 it was possible to obtain higher enrichment values than

achieved in trials 1–6, as predicted by the mathematical model.
The comparisons of the different enrichments in lignans
obtained with the use of similar CO2 mass (experiments 2 vs. 5 and
4 vs. 7) were conducted. These comparisons show a higher content
of lignans (variations not statistically significant) for the tests with
a higher CO2 superficial velocity, respectively the experiments 2
and 4.

3.2. Ethanol extraction

The initial content of ethanol was 66.67% of the total volume

and decreased in the RF to a range 5.44–26.67%, which correspond
to a decrease in value (D%) from 60 to 92% as reported in Table 4.
The SF contained 100% ethanol by volume.
The ethanol content model (Eq. (4)) provided a good represen-
tation of the real trends of the experimental conditions.

% Dethanol = −34.7603 + 0.2530 XT + 0.9181 XP + 1.5077 XF

− 0.0021 XTP − 0.0114 XTF − 0.0014 XPF

2
r = 0.9791; RMSE = 2.000% (4)

Equation (4) represents the interaction regression equation for the

percent of ethanol decrease, where XT , XP , and XF are the indepen-
dent variables of time of treatment, pressure and CO2 flow rate,

Fig. 5. Response surfaces for pairs of variables. Time–pressure (a), Time–CO2 flow
rate (b), and Pressure–CO2 flow rate (c).
Fig. 4. Quadratic response surface model for SDG enrichment.
 G. Perretti et al. / J. of Supercritical Fluids 75 (2013) 94–100 99

Table 4
Ethanol contents of the RFs in the different experiments and the decrease in ethanol
content from the initial sample.

Exp. Time (min) CO2 flow (kg h−1 ) Pressure (MPa) Ethanol*

(Vol %) D (%)
a
1 60 5 10 26.67 60a
2 60 15 10 18.68b 72b
3 60 5 30 13.58cd 80cd
4 60 15 30 10.95ce 84ce
5 180 5 10 10.05e 85e
6 180 15 10 16.36bd 75bd
7 180 5 30 5.44f 92f
8 180 15 30 6.75f 90f
*
Means in the same column not sharing a common superscript letter were sig-
nificantly different (one way ANOVA; p < 0.05, n = 3).

respectively. XTP , XTF , XPF represent the interactions between pairs

of variables. The regression coefficient is represented by r2 and Fig. 7. Ethanol quadratic response surface model.
RMSE is the root mean square error.
The graph of actual values versus predicted values in Fig. 6 shows
the actual and predicted data dispersion around the bisector. The
equation line almost coincides with the bisector and it is therefore
completely covered and not visible. The dispersion of the 8 experi-
ments is linear and in good agreement with the model as confirmed
by the high regression coefficient (0.9791) and by the low root mean
square error (2.0000%).
As for total lignan and SDG contents, RSM methodology can also
be applied to the change in ethanol content. The quadratic sur-
face of the model is presented in Fig. 7. The maximum decrease in
ethanol was obtained in experiment 7 (30 Mpa, 5 kg h−1 , 180 min),
in agreement with the results reported in Table 4.
The response surfaces for pairs of variables reported in Fig. 8
show a different behavior for the decreasing ethanol content at
different times of treatment.
The reduction of ethanol after 60 min of treatment (experiments
1–4) was greatest at a flow rate of 15 kg h−1 , the same as was
observed for lignan enrichment. However, under the condition of
180 min of treatment (experiments 5–8) ethanol reduction was
greatest at a flow rate of 5 kg h−1 . The comparisons of the differ-
ent ethanol reductions obtained with the use of similar CO2 mass
(experiments 2 vs. 5 and 4 vs. 7) were conducted. These compar-
isons show a statistically significant ethanol reduction in the RF

Interaction Regression
100

95 exp7
Decreasing % (Ethanol) Predicted data

90
exp4 exp8
85
exp5
80 exp6
exp3
75

70 exp2

65 exp1

60

55

50
50 60 70 80 90 100
Decreasing % (Ethanol) Experimental data Fig. 8. Response surface models for pressure and CO2 flow rate at 60 min (a) and
180 min (b) of treatment.
Fig. 6. Regression model of actual values of experiments (black line) and predicted
values (gray line) for ethanol.
100 G. Perretti et al. / J. of Supercritical Fluids 75 (2013) 94–100
higher for the tests with lower CO2 surface velocity, respectively
the experiments 5 and 7. This behavior is probably due to the dif-
ferent transport coefficients of the matter, which reflect upon the
different balances between CO2 , ethanol and water that are estab-
lished within the column in a treatment time of 180 min compared
with a treatment time of 60 min.
Alternative concentration methods to remove ethanol from the
extract could be vacuum separation, distillation and membrane
techniques (reverse osmosis and perevaporation), but unfortu-
nately, in this case, some components could be denatured or
removed by these processes [21].
4. Conclusions
SAF presents a useful technique for the concentration of lignans
from flaxseed.
The regression models and response surface models revealed
that SAF produced the greatest lignan content under the maximum
levels of the variables studied (treatment time 180 min, pressure
30 MPa and CO2 flow rate 15 kg h−1 ). Furthermore, pressure plays
the greatest role, confirming that as the pressure increases, the
solvent power of SC-CO2 increases too.
After the verification of the nearly complete elimination of the
residual ethanol, the characterization of the composition of the RF
at the end of the process, and after the study of suitable supple-
mentation formulas, the purified lignans could be scaled up to
increase their production and could be proposed for commercial
use as ingredients in functional foods.
Finally, the verification of the antioxidant activity in the RF and
the study of the residual panels as useful by-products of the flaxseed
industry can add further value to the proposed process and product.
Acknowledgments
The authors thank the Italian Ministry of Agricultural Food,
Forestry and Fishery Policies, for their support through the grant
“CERSUOM”, and Dr. Valeria Sileoni for her collaboration in per-
forming the statistical analyses.
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