Separation and Purification Technology 57 (2007) 17–24

Optimization of ethanol–water extraction of lignans from flaxseed

Zhen-Shan Zhang a , Dong Li a,∗ , Li-Jun Wang b , Necati Ozkan c ,
Xiao Dong Chen a,d , Zhi-Huai Mao a , Hong-Zhi Yang a
a College of Engineering, China Agricultural University, 17 Qinghua Donglu, Beijing 100083, PR China
b College of Food Science and Nutritional Engineering, China Agricultural University, 17 Qinghua Donglu, Beijing 100083, PR China
c Central Laboratory, Middle East Technical University, Ankara 06531, Turkey
d Department of Chemical Engineering, Monash University, Clayton Campus, Victoria, Australia

Received 30 January 2007; received in revised form 7 March 2007; accepted 7 March 2007

Abstract
The optimal conditions for the extraction of lignans from flaxseed were determined. A second-order regression for rotation-orthogonal composite
design was used to investigate the effects of three independent variables, namely ethanol concentration (%), extraction temperature (◦ C), and
extraction time (h) on the response, the acquired ratio of lignans. The independent variables were coded at five levels and their actual values
selected on the basis of results of single-factor experiment. The second-order regression for rotation-orthogonal composite design consisted of 23
experimental points and 9 replications at the central point. Data were analyzed using MATLAB software. The results indicated that acquired ratio
of lignans might be beyond 8% at a probability of 95% in the range of ethanol concentration: 56.6–83.4%, extraction time: 26.7–27.9 h, extraction
temperature: 38.7–40.1 ◦ C. And the optimal extraction conditions were ethanol concentration of 70%, extraction time of 28 h and extraction
temperature of 40 ◦ C according to the analysis of response surface. In this condition, the experimental acquired ratio of lignans was 8.975% (w/w;
lignans/defatted flaxseed powder), which is close to the predicted value (9.316%).
© 2007 Elsevier B.V. All rights reserved.

Keywords: Flaxseed; Acquired ratio of lignans; Response surface; Optimization; Ethanol–water extraction

1. Introduction
Secoisolariciresinol diglycoside (SDG), a kind of phytoestro-
gen, is the main component of flaxseed lignans [1]. SDG is
the precursor of the major mammalian lignans enterodiol and
enterolactone found in the urine and faeces of humans after
flaxseed intake [2,3]. Previous researches indicated that SDG
could prevent mammary, colonic, and prostate cancer [4–8] and
have a beneficial effect on tumour [9,10] and atherosclerosis
[11]. Positive results were also found on lupus nephritis [12,13],
hypertension [14,15], diabetes [16,17] and other diseases after
SDG intake. Although SDG can be found in many food prod-
ucts, flaxseed appears to be the richest source, with values more
than 100 times higher than those of the other foods. The content
of SDG in flaxseed is 0.2–0.3%, and about 1–4% in defatted
flaxseed powder [2,18,19].
∗ Corresponding author at: College of Engineering, China Agricultural Uni-
versity, P.O. Box 50, 17 Qinghua Donglu, Beijing 100083, PR China.
Fax: +86 10 62737351.
E-mail address: wlj@cau.edu.cn (D. Li).
Flaxseeds are the seeds of the herb flax, which belong to
flaxen section living one or more years. Flaxseeds are flat, oval,
4–6 mm long with a smooth, shiny surface, brown to light brown
in color. Seed weight is about 5 ± 1 g/1000, and contains about
40% oil, 30% dietary fiber, 20% protein, 4% ash, and 6% mois-
ture [20,21]. At present, flaxseed was mainly used to produce
oil for a variety of industrial purposes (manufacturing of paints,
varnishes, linoleum, printing inks, cosmetics, etc.) and oil-free
meal for livestock [22,23]. Phytoestrogen-rich flaxseed is also
a popular ingredient for use in various functional foods such
as cereals, pancakes, muffins, pizza, and bread [24]. The first
report of SDG occurring in flaxseed was done by Bakke and
Klostermann [2].
Many extraction solvents have been utilized to extract
flaxseed lignans from flaxseed. In 1956, Bakke and Kloster-
mann described a laboratory process for extracting SDG from
defatted flaxseed powder (DFP) using equal parts of 95% ethanol
and 1,4-dioxane [18]. Degenhardt and Charlet obtained flaxseed
lignans using methanol–water in their experiments [3,25]. And
a solvent comprising acetone and water was adopted by Dob-
binsan et al. in their American patent [18]. Complex solvent of

1383-5866/$ – see front matter © 2007 Elsevier B.V. All rights reserved.
doi:10.1016/j.seppur.2007.03.006
18 Z.-S. Zhang et al. / Separation and Purification Technology 57 (2007) 17–24
ethanol and water was introduced into the extraction of lignans
by Westcott, but he did not investigate the optimal extraction
conditions. Complex solvent of ethanol and water is a prefer-
able solvent from considerations of safety to health and handling
[26].
The acquired ratio of flaxseed lignans is affected by various
factors, such as extraction time, extraction temperature, solvent
concentration, solid to liquid ratio, and particle size of flaxseed.
The traditional one factor at a time approach to process optimiza-
tion is time consuming and neglects the interaction of the factors.
Response surface methodology (RSM), which takes interaction
into consideration, can reduce the times of experiments [27,28].
So it is an effective method to optimize the conditions of lignans
extraction from flaxseed.
The purpose of present work was to investigate the effect
of ethanol concentration, extraction temperature, and extraction
time on the acquired ratio of lignans by single factor experiment,
respectively. A second-order polynomial model was set up to
predict the acquired ratio of lignans using RSM. The interaction
among ethanol concentration, extraction time, and extraction
temperature was investigated by the response surface analysis.
Finally, the optimal condition was obtained through solving the
predicted equation.
2. Materials and methods
2.1. Materials
The flaxseed was planted in Lanxi County of Heilongjiang
province, China. Its species is Neiya 3. Analytical grade ethanol,
sodium hydroxide (NaOH) and hydrochloric acid (HCL) were
purchased from Beijing Beihua Chemical Company.
2.2. Preparation of defatted flaxseed powder
Flaxseeds were ground in a coffee grinder to obtain a fine
powder. The DFP samples used in this study were defatted by
blending the ground material with hexane (1:6, w/v, 12 h) at
ambient temperature (about 23 ◦ C). The DFP was air dried for
12 h and stored in a sealed container for later use.
2.3. Process of extracting lignans
Lignans was extracted using a method, which was slightly
modified adaptation of a previously reported technique [26]. Two
hundred grams of DFP (M0 ) was blended with 1.2 L complex sol-
vent of ethanol and water. The values of solvent concentration,
extraction temperature, and extraction time were set accord-
ing to the requirement of experiment. The extract was filtered
using a sand core funnel, and then it was concentrated at 50 ◦ C
with a rotary evaporator (RE-2S, Beijing Jing Zhi Jie laboratory
apparatus Co., Ltd., China) at 90 rpm. Light yellow syrup was
obtained. The syrup was hydrolyzed with 1 M NaOH at room
temperature for 12 h. The hydrolyzed syrup was acidified with
0.5 M HCL to pH 6, which was measured using a pH meter (PB-
20, Sartorius AG, Germany). The solution was cooled down to
15 ◦ C, and then it was centrifuged with a high-speed centrifuge
(LG10-2.4A, Beijing Jing Li Centrifuge Co., Ltd., China) at
2000 rpm for 10 min to precipitate and remove water-soluble
polysaccharides and proteins after filtering the salt with a sand
core funnel [29]. After freeze-drying process, the weight of lig-
nans (M) was determined and the acquired ratio of lignans was
calculated using the following equation:
acquired ratio of lignans (%)
weight of freeze-dried lignans M
= × 100 = × 100
weight of defatted flaxseed powder M0
(1)
2.4. Experimental design
2.4.1. Single factor experiments
(1) Lignans were extracted using complex solvent of ethanol
and water at room temperature for 24 h. The proportion of
ethanol in the solvent varied in the range of 50–100% (v/v).
The best solvent concentration was selected according to
the acquired ratio of lignans.
(2) Lignans were extracted using the best solvent selected in the
previous step. The extraction temperature varied from 20 to
60 ◦ C while fixing the extraction time constant at 24 h.
(3) Using the best ethanol concentration selected in first step,
lignans were extracted during various extraction time course
ranges from 4 to 28 h at the optimum temperature deter-
mined in the second step.
The ranges of three factors were determined for RSM accord-
ing to the results of experiment.
2.4.2. Experiment of RSM
Optimization of extracting lignans from flaxseed was car-
ried out using RSM, which is used as a generic means for
optimization. A three-factor and five-level second-order regres-
sion for rotation-orthogonal composite designs consisting of 23
experimental runs was employed including 9 replicates at the
center point. Nine replicate runs at the central of the design
were performed to allow the estimate of pure error [30]. Three
independent variables of the design were ethanol concentra-
tion (X1 , % (v/v, ethanol/water)), extraction time (X2 , h) and
extraction temperature (X3 , ◦ C) while the response variable was
the acquired ratio of lignans. In the optimization process the
response could be related to the selected variables by a second-
order polynomial model. The generalized model proposed for
the response was given in the following equation:

3 
3 
2 
3
Y = β0 + βi xi + βii xi2 + βij xi xj (2)
i=1 i=1 i=1 j=i+1
where Y is the response, β0 the constant coefficient, βI ’s are the
linear coefficients, βii ’s are the quadratic coefficients, βij ’s are
the interaction coefficients, and xi and xj are the coded value
of independent variables. MATLAB® software (Version 6.5.1)
was used to generate the response surfaces and contour plots
while holding one variable constant in the second-order poly-
 Z.-S. Zhang et al. / Separation and Purification Technology 57 (2007) 17–24
nomial model. Frequency analysis was applied to determine the
optimum ranges of extraction conditions [31].
2.5. Verification of the optimal conditions
Optimal conditions and the maximum predicted acquired
ratio for lignans extraction were obtained using the second-order
polynomial model of RSM. The practical acquired ratio was
obtained under the optimal conditions. The experimental and
predicted acquired ratios of lignans were compared in order to
determine the validity of the model.
3. Results and analysis
3.1. Results of single factor experiment
3.1.1. Influence of ethanol concentration on acquired ratio
of lignans
As can be seen from Fig. 1, the acquired ratio of lignans as a
function of ethanol concentration follows a parabola shape. The
acquired ratio of lignans increased with increasing of the ethanol
proportion in the extraction medium up to 70% and then began
to decline with the further increase of ethanol proportion in the
extraction medium. Ethanol is a low-polar solvent while water is
a strong polar solvent, and they can be blended with each other
in any proportion. With the addition of water to ethanol, the
polarity of complex solvent will increase continuously. Lignans
molecules are also polar ones [32]. So the acquired ratio of lig-
nans increased with increasing water content according to “like
dissolves like” principle. When the water content of the solvent
exceeds 30%, the acquired ratio of lignans was reduced. It can
be explained by the same principle as above. Lignans are organic
matter and ethanol is organic solvent, while water is inorganic
solvent. Organic matter prefers to dissolve in organic solvent. So
the acquired ratio of lignans decreased with excessive increase
of water in the complex solvent. The proportion of ethanol in
the extraction solvent was varied between 50 and 90% for the
optimization design. The solvent with 70% ethanol content was
chosen for the determination of optimal extraction temperature
and time.
Fig. 1. Influence of ethanol concentration on the acquired ratio of lignans.
19
Fig. 2. Influence of extraction temperature on the acquired ratio of lignans.
3.1.2. Influence of extraction temperature on acquired ratio
of lignans
As it was shown in Fig. 2, the acquired ratio of lignans was
increased slightly with increasing of the extraction temperature.
This observation suggests that the extraction temperature had no
significant effect on the acquired ratio of lignans.
The extraction of lignans was increased slightly with increas-
ing of the extraction temperature by enhancing the solubility
of lignans in solvent. But very high extraction temperatures
will decrease the activity of lignans and increase the consump-
tion of solvent sharply, because the boiling point of ethanol is
as low as 78.3 ◦ C. Manipulation steps and extraction costs are
expected to increase with increasing of the extraction tempera-
ture. Thus the extraction temperatures ranging from 20 to 40 ◦ C
were selected as the extraction temperatures for the optimiza-
tion design. Experiments for selecting the optimal extraction
time were conducted at room temperature.
3.1.3. Influence of extraction time course on acquired ratio
of lignans
As can be seen from Fig. 3 the acquired ratios of lignans
were increased gradually with increasing of the extraction time,
the maximum acquired ratio of 7.798% was obtained when
the extraction time was 24 h. No increase was observed in the
acquired ratio of lignans further increasing of the extraction
Fig. 3. Influence of extraction time on the acquired ratio of lignans.
20 Z.-S. Zhang et al. / Separation and Purification Technology 57 (2007) 17–24

time beyond 24 h. It was obvious that the extraction time had Table 1
an important influence on the acquired ratio of lignans. There- Independent variables and their coded and actual values used for the optimization
fore, the extraction times ranging from 20 to 28 h was selected Independent variable Units Symbol Coded levels
for the optimization. −α −1 0 1 α

Ethanol concentration % (v/v) X1 50 58.1 70 81.9 90

3.2. Results of RSM experiments
Extraction time h X2 20 21.6 24 26.4 28
Extraction temperature ◦C X3 20 24.1 30 36.0 40
3.2.1. Fitting the model
Based on the single-factor experiments, the following values
were selected as the ranges of independent variables, influencing significant (F = 6.493 > F7,15,0.01 = 4.14). Thus the fitted model
the acquired ratio of lignans: (i) the ethanol concentration of the was appropriate for describing of the response surface. Taking
extraction solvent: 50–90%, (ii) the extraction time: 20–28 h, the coefficients into the generalized model (Eq. (2)), a second-
and (iii) the extraction temperature: 20–40 ◦ C. order polynomial equation for the acquired ratio of lignans was
The relationship between the natural variables (Xi ) and coded obtained for the coded unit as shown in the following equation:
variables (xi ) is defined as in the following equation:
Xi − [Xmax + Xmin ]/2 Y = 6.790 + 0.022x1 + 0.384x2 + 0.171x3 + 0.161x1 x2
xi = (3)
[Xmax − Xmin ]/2a + 0.156x1 x3 + 0.370x2 x3 − 0.560x12 + 0.028x22
where xi ’s are the dimensionless coded values of the natural vari- + 0.193x32 (4)
able Xi , while the Xmax and Xmin are the maximum and minimum
values of the natural variable, and star point a = 1.682. The F-test and p value in Table 3 indicated that the quadratic
The level of factors and the results of coding were shown in term of ethanol concentration (x12 ) had the largest effect on the
Table 1. The design arrangement and the experimental results acquired ratio of lignans, and followed by the extraction time
of the optimization design were shown in Table 2. Multiple (x2 ) and the interaction of the extraction time and the extraction
regression coefficients were calculated according to the method temperature (x2 x3 ).
described by Yuan and Zhou [31] and were summarized in The predicted model was rearranged by eliminating the terms,
Table 3. As shown in Table 3, all the coefficients are significant which were not significant in the second-order polynomial
at different levels except β1 , β1 β2 , β1 β3 and β22 . The F0.05 value model. Finally the model was reduced to the following equation:
for lack of fit (1.709) did not exceed the tabulated value of 3.50
Y = 6.790 + 0.384x2 + 0.171x3 + 0.370x2 x3
(7, 8, 3.50) indicating that the lack of fit was not significant. The
F-test value (p < 0.01) indicated that the regression equation was − 0.560x12 + 0.193x32 (5)

Table 2
The arrangement and response of the three-factor, five-level second-order regression for rotation-orthogonal composite design
Trial Arrangement of the design Responses

X0 X1 X2 X3 X1 X2 X1 X3 X2 X3 X1 X2 X3 Y

1 1 −1 −1 −1 1 1 1 0.406 0.406 0.406 6.000

2 1 −1 −1 1 1 −1 −1 0.406 0.406 0.406 5.812
3 1 −1 1 −1 −1 1 −1 0.406 0.406 0.406 5.906
4 1 −1 1 1 −1 −1 1 0.406 0.406 0.406 6.855
5 1 1 −1 −1 −1 −1 1 0.406 0.406 0.406 5.618
6 1 1 −1 1 −1 1 −1 0.406 0.406 0.406 5.712
7 1 1 1 −1 1 −1 −1 0.406 0.406 0.406 5.823
8 1 1 1 1 1 1 1 0.406 0.406 0.406 7.740
9 1 −1.682 0 0 0 0 0 2.234 −0.594 −0.594 5.550
10 1 1.682 0 0 0 0 0 2.234 −0.594 −0.594 5.542
11 1 0 −1.682 0 0 0 0 −0.594 2.234 −0.594 6.594
12 1 0 1.682 0 0 0 0 −0.594 2.234 −0.594 7.821
13 1 0 0 −1.682 0 0 0 −0.594 −0.594 2.234 7.798
14 1 0 0 1.682 0 0 0 −0.594 −0.594 2.234 7.542
15 1 0 0 0 0 0 0 −0.594 −0.594 −0.594 6.960
16 1 0 0 0 0 0 0 −0.594 −0.594 −0.594 7.171
17 1 0 0 0 0 0 0 −0.594 −0.594 −0.594 6.890
18 1 0 0 0 0 0 0 −0.594 −0.594 −0.594 6.635
19 1 0 0 0 0 0 0 −0.594 −0.594 −0.594 6.012
20 1 0 0 0 0 0 0 −0.594 −0.594 −0.594 7.280
21 1 0 0 0 0 0 0 −0.594 −0.594 −0.594 6.760
22 1 0 0 0 0 0 0 −0.594 −0.594 −0.594 6.325
23 1 0 0 0 0 0 0 −0.594 −0.594 −0.594 6.800
 Z.-S. Zhang et al. / Separation and Purification Technology 57 (2007) 17–24 21

Table 3
Regression coefficient of the predicted second-order polynomial model for the response of acquired ratio of lignans (Y)
β0 β1 β2 β3 β1 β2 β1 β3 β2 β3 β12 β22 β32

βi 6.790 0.022 0.384 0.171 0.161 0.156 0.370 −0.560 0.028 0.193
Fi 0.029 8.372** 1.666* 0.856 0.810 4.549** 20.931*** 0.054 2.460*
* Significant at 0.1.
** Significant at 0.05.
*** Significant at 0.01.

Afterwards, the interaction analysis was carried out with the
natural variables and the following equation was obtained.
Y = 6.3644 + 0.5545X1 − 0.6225X2 − 0.925X3
+ 0.0261X2 X3 − 0.0040X12 + 0.005521
This predicted polynomial model was used to obtain the response
surfaces and contour plots for all the interaction among the
ethanol concentration of the extraction solvent, the extraction
time, and the extraction temperature.
Fig. 5 denoted the effects of extraction time and temperature
on the acquired ratio of lignans at fixed ethanol concentration of
70%. The acquired ratio of lignans was found to be a function of
the linear and quadratic effects of extraction temperature. The
(6) linear and quadratic effects were both positive, which resulted
in a curvilinear increase with the extraction temperature for
all the extraction times employed (Fig. 5). Other factors that
contributed to the acquired ratio of lignans included the linear
effect of the extraction time and its interaction effect with the

3.2.2. Analysis of response surface

Three-dimensional response surfaces of the ethanol concen-
tration, the extraction temperature, and the extraction time were
obtained by keeping one of the variables constant. The con-
stant was equal to the natural value of zero level. The response
surfaces are shown in Figs. 4–6.
Fig. 4 showed the effect of the ethanol concentration of the
extraction solvent and the extraction time on the acquired ratio
of lignans at 30 ◦ C. As shown in Table 3, the acquired ratio of lig-
nans mainly depend on the ethanol concentration as its quadratic
effects (p < 0.01) was significant, which result in a curvilinear
increase in acquired ratio of lignans for all the extraction times
employed (Fig. 4). However, the linear and interaction effects
with ethanol concentration were insignificant. The acquired ratio
of lignans was linearly related to the extraction time and the
quadratic term was found to be not significant. For all ethanol
Fig. 5. Response surface showing the effect of extraction time and extraction
concentrations the acquired ratio of lignans was increased lin- temperature on acquired ratio of lignans.
early with increasing of the extraction time (Fig. 4).

Fig. 4. Response surface showing the effect of the ethanol concentration and Fig. 6. Response surfaces showing the effect of ethanol concentration and
the extraction time on the acquired ratio of lignans. extraction temperature on acquired ratio of lignans.
22 Z.-S. Zhang et al. / Separation and Purification Technology 57 (2007) 17–24

Fig. 8. Contour plot showing the effect of extraction time and extraction tem-
Fig. 7. Contour plot showing the effect of ethanol concentration and extraction
perature on acquired ratio of lignans.
time on acquired ratio of lignans.

short (20–23 h). But when the extraction time is long (25–28 h),
extraction temperature (Table 3). The acquired ratio of lignans
the acquired ratio of lignans was increased with increasing of the
was linearly related to the extraction time, and the quadratic
extraction temperature. The maximum acquired ratio of lignans
effect was insignificant, which resulted in a linear increase in
was obtained at 28 h and 40 ◦ C.
the acquired ratio of lignans with the extraction time for all
Contour plot for the response of ethanol concentration and
extraction temperatures (Fig. 5).
the extraction temperature to the acquired ratios of lignans were
In Fig. 6, the effects of ethanol concentration and the extrac-
presented in Fig. 9. As shown in Fig. 9, an increase in the
tion temperature on the acquired ratio of lignans were presented
ethanol concentration increased the acquired ratio of lignans
at the extraction time of 24 h. Ethanol concentration had a
up to 70%, beyond which acquired ratio of lignans decreased
pronounced effect on the acquired ratio of lignans at all extrac-
with the increase of ethanol concentration. On the contrary,
tion temperatures. The linear effect was insignificant while
the acquired ratio of lignans decreased with the increase of
the quadratic was significant (p < 0.01), which explained the
extraction temperature up to 28 ◦ C, and then increased with
observed nature of the curve as shown in Fig. 6. The acquired
increasing extraction temperature. The ranges of ethanol con-
ratio of lignans was found to be a function of the linear and
centration 65–75% and extraction temperature from 36 to 40 ◦ C
quadratic effects of extraction temperature, and both of the
were considered to be the most suitable for lignans extraction.
effects were positive, which resulted in a curvilinear increase
In the limits of experimental conditions, the optimal condi-
in acquired ratio of lignans for all the ethanol concentration
tions, obtained using MATLAB software, were as follows: the
employed (Fig. 6). The interaction effect between ethanol con-
ethanol concentration of 70%, the extraction time of 28 h, and
centration and extraction temperature was insignificant.
the extraction temperature of 40 ◦ C. The model showed that the
maximum acquired ratio of lignans was 9.316% at this point.
3.2.3. Optimization of extraction conditions
Figs. 7–9 show the optimum conditions of the extraction pro-
cess to yield maximum acquired ratio of lignans by locating
extraction temperature at 30 ◦ C, ethanol concentration at 70%
and the extraction time of 24 h, respectively.
Fig. 7 shows the contour plots for the response of ethanol con-
centration and extraction time to the acquired ratio of lignans.
The contours indicated that both high ethanol concentration and
long extraction time could increase the acquired ratio of lig-
nans. However, when the ethanol concentration exceeded 70%,
the acquired ratio of lignans started to decrease. In other words,
it was not advisable to use very high ethanol concentration when
extracting lignans from flaxseed. The optimum extraction con-
dition for the acquired ratio of lignans was 28 h and the ethanol
concentration was between 65 and 75%.
Fig. 8 shows the contour plots for the response of extraction
time and extraction temperature to the acquired ratio of lignans.
It was observed that very high extraction temperature did not Fig. 9. Contour plot showing the effect of ethanol concentration and extraction
increase the acquired ratio of lignans when the extraction time is temperature on acquired ratio of lignans.
 Z.-S. Zhang et al. / Separation and Purification Technology 57 (2007) 17–24
Table 4
The quantity and frequency of the acquired ratio of lignans beyond 8%
Factors
Frequency of ethanol
concentration (X1 )
Coding
−1.682 0 0
−1 2 0
0 3
1 2
1.682 0 4
Average value (x̄) 0 1.390
95% confidence interval (xi ) −1.12–1.12
Parameter interval (Xi ) 56.6–83.4
The higher acquired ratio of lignans might be obtained at
certain point or area beyond limitative ranges, such as under the
condition that extraction temperature exceeded 40 ◦ C, extraction
time exceeded 28 h, and both extraction temperature and time
exceeded them maximum values (>40 ◦ C, >28 h). However, In
the light of practical factors, such as the acquired ratio of lignans,
economic costs, safety and credibility, product quality and so
on, it was unadvisable to increase extraction temperature and
prolong extraction time excessively.
3.3. Frequency analysis
A three-factor and five-level experiment design can require
53 = 125 arrangements. According to the method presented in
literature [31], frequency analysis could be carried out in the
arrangements, which the acquired ratio of lignans were beyond
8%. Seven arrangements were up to the demand by statistics,
which occupied 5.6% in all arrangements. The results of fre-
quency analysis were showed in Table 4.
Lignans were extracted under the conditions listed in the last
row of Table 4. The conditions were the ethanol concentration
ranging from 56.6 to 83.4%, the extraction times ranging from
26.7 to 27.9 h, and the extraction temperatures ranging from 38.7
to 40.1 ◦ C and the acquired ratio of lignans might be beyond 8%
at a probability of 95% in these ranges.
3.4. Verification of the results
The suitability of the model equation for predicting the opti-
mum response values was tested using the recommended optimal
conditions. The set of optimal conditions, determined using
the RSM optimization approach, were tested experimentally in
order to the model equation. The experimental value (8.975%)
was found to be close to the predicted one.
4. Conclusion
An adequate quadratic polynomial model for predicting the
values of acquired ratio of lignans was determined according to
the optimization designs. Three independent variables involved
in the model were ethanol concentration, extraction time and
23
Frequency of Frequency of extraction
extraction time (X2 ) temperature (X3 )
0
0
0 0
3 1
6
1.585
1.166–1.613 1.473–1.696
26.7–27.9 38.7–40.1
extraction temperature. The F-test and p value indicated that
the quadratic term of ethanol concentration (x12 ) had the largest
effect on the acquired ratio of lignans, and followed by the
extraction time (x2 ) and the interaction of the extraction time
and the extraction temperature (x2 x3 ). Frequency analysis indi-
cated that the acquired ratio of lignans might be beyond 8%
at a probability of 95% in the range of ethanol concentration:
56.6–83.4%, extraction time: 26.7–27.9 h, extraction tempera-
ture: 38.7–40.1 ◦ C. And the optimal extraction conditions were
obtained at the ethanol concentration of 70%, the extraction time
of 28 h, and extraction temperature of 40 ◦ C using the RSM. In
this condition, the experimental acquired ratio of lignans was
8.975%, which was close to the predicted value (9.316%).
Acknowledgements
Research support was provided by the Funding System
for Scientific Research Projects of Doctor Subject of Chi-
nese Advanced University (No. 20050019029), the Key Project
of Chinese Ministry of Education (No. 105014) and Funding
System for Scientific Research Projects of China Agricultural
University (No. 2004010).
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