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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 Flaxseeds are the seeds of the herb flax, which belong to
flaxen section living one or more years. Flaxseeds are flat, oval,
Secoisolariciresinol diglycoside (SDG), a kind of phytoestro- 4–6 mm long with a smooth, shiny surface, brown to light brown
gen, is the main component of flaxseed lignans [1]. SDG is in color. Seed weight is about 5 ± 1 g/1000, and contains about
the precursor of the major mammalian lignans enterodiol and 40% oil, 30% dietary fiber, 20% protein, 4% ash, and 6% mois-
enterolactone found in the urine and faeces of humans after ture [20,21]. At present, flaxseed was mainly used to produce
flaxseed intake [2,3]. Previous researches indicated that SDG oil for a variety of industrial purposes (manufacturing of paints,
could prevent mammary, colonic, and prostate cancer [4–8] and varnishes, linoleum, printing inks, cosmetics, etc.) and oil-free
have a beneficial effect on tumour [9,10] and atherosclerosis meal for livestock [22,23]. Phytoestrogen-rich flaxseed is also
[11]. Positive results were also found on lupus nephritis [12,13], a popular ingredient for use in various functional foods such
hypertension [14,15], diabetes [16,17] and other diseases after as cereals, pancakes, muffins, pizza, and bread [24]. The first
SDG intake. Although SDG can be found in many food prod- report of SDG occurring in flaxseed was done by Bakke and
ucts, flaxseed appears to be the richest source, with values more Klostermann [2].
than 100 times higher than those of the other foods. The content Many extraction solvents have been utilized to extract
of SDG in flaxseed is 0.2–0.3%, and about 1–4% in defatted flaxseed lignans from flaxseed. In 1956, Bakke and Kloster-
flaxseed powder [2,18,19]. mann described a laboratory process for extracting SDG from
defatted flaxseed powder (DFP) using equal parts of 95% ethanol
∗ Corresponding author at: College of Engineering, China Agricultural Uni-
and 1,4-dioxane [18]. Degenhardt and Charlet obtained flaxseed
versity, P.O. Box 50, 17 Qinghua Donglu, Beijing 100083, PR China.
lignans using methanol–water in their experiments [3,25]. And
Fax: +86 10 62737351. a solvent comprising acetone and water was adopted by Dob-
E-mail address: wlj@cau.edu.cn (D. Li). 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 (LG10-2.4A, Beijing Jing Li Centrifuge Co., Ltd., China) at
by Westcott, but he did not investigate the optimal extraction 2000 rpm for 10 min to precipitate and remove water-soluble
conditions. Complex solvent of ethanol and water is a prefer- polysaccharides and proteins after filtering the salt with a sand
able solvent from considerations of safety to health and handling core funnel [29]. After freeze-drying process, the weight of lig-
[26]. nans (M) was determined and the acquired ratio of lignans was
The acquired ratio of flaxseed lignans is affected by various calculated using the following equation:
factors, such as extraction time, extraction temperature, solvent
concentration, solid to liquid ratio, and particle size of flaxseed. acquired ratio of lignans (%)
The traditional one factor at a time approach to process optimiza- weight of freeze-dried lignans M
= × 100 = × 100
tion is time consuming and neglects the interaction of the factors. weight of defatted flaxseed powder M0
Response surface methodology (RSM), which takes interaction (1)
into consideration, can reduce the times of experiments [27,28].
So it is an effective method to optimize the conditions of lignans 2.4. Experimental design
extraction from flaxseed.
The purpose of present work was to investigate the effect 2.4.1. Single factor experiments
of ethanol concentration, extraction temperature, and extraction
time on the acquired ratio of lignans by single factor experiment, (1) Lignans were extracted using complex solvent of ethanol
respectively. A second-order polynomial model was set up to and water at room temperature for 24 h. The proportion of
predict the acquired ratio of lignans using RSM. The interaction ethanol in the solvent varied in the range of 50–100% (v/v).
among ethanol concentration, extraction time, and extraction The best solvent concentration was selected according to
temperature was investigated by the response surface analysis. the acquired ratio of lignans.
Finally, the optimal condition was obtained through solving the (2) Lignans were extracted using the best solvent selected in the
predicted equation. previous step. The extraction temperature varied from 20 to
60 ◦ C while fixing the extraction time constant at 24 h.
2. Materials and methods (3) Using the best ethanol concentration selected in first step,
lignans were extracted during various extraction time course
2.1. Materials ranges from 4 to 28 h at the optimum temperature deter-
mined in the second step.
The flaxseed was planted in Lanxi County of Heilongjiang
province, China. Its species is Neiya 3. Analytical grade ethanol, The ranges of three factors were determined for RSM accord-
sodium hydroxide (NaOH) and hydrochloric acid (HCL) were ing to the results of experiment.
purchased from Beijing Beihua Chemical Company.
2.4.2. Experiment of RSM
2.2. Preparation of defatted flaxseed powder Optimization of extracting lignans from flaxseed was car-
ried out using RSM, which is used as a generic means for
Flaxseeds were ground in a coffee grinder to obtain a fine optimization. A three-factor and five-level second-order regres-
powder. The DFP samples used in this study were defatted by sion for rotation-orthogonal composite designs consisting of 23
blending the ground material with hexane (1:6, w/v, 12 h) at experimental runs was employed including 9 replicates at the
ambient temperature (about 23 ◦ C). The DFP was air dried for center point. Nine replicate runs at the central of the design
12 h and stored in a sealed container for later use. were performed to allow the estimate of pure error [30]. Three
independent variables of the design were ethanol concentra-
2.3. Process of extracting lignans tion (X1 , % (v/v, ethanol/water)), extraction time (X2 , h) and
extraction temperature (X3 , ◦ C) while the response variable was
Lignans was extracted using a method, which was slightly the acquired ratio of lignans. In the optimization process the
modified adaptation of a previously reported technique [26]. Two response could be related to the selected variables by a second-
hundred grams of DFP (M0 ) was blended with 1.2 L complex sol- order polynomial model. The generalized model proposed for
vent of ethanol and water. The values of solvent concentration, the response was given in the following equation:
extraction temperature, and extraction time were set accord-
ing to the requirement of experiment. The extract was filtered
3
3
2
3
Y = β0 + βi xi + βii xi2 + βij xi xj (2)
using a sand core funnel, and then it was concentrated at 50 ◦ C
i=1 i=1 i=1 j=i+1
with a rotary evaporator (RE-2S, Beijing Jing Zhi Jie laboratory
apparatus Co., Ltd., China) at 90 rpm. Light yellow syrup was where Y is the response, β0 the constant coefficient, βI ’s are the
obtained. The syrup was hydrolyzed with 1 M NaOH at room linear coefficients, βii ’s are the quadratic coefficients, βij ’s are
temperature for 12 h. The hydrolyzed syrup was acidified with the interaction coefficients, and xi and xj are the coded value
0.5 M HCL to pH 6, which was measured using a pH meter (PB- of independent variables. MATLAB® software (Version 6.5.1)
20, Sartorius AG, Germany). The solution was cooled down to was used to generate the response surfaces and contour plots
15 ◦ C, and then it was centrifuged with a high-speed centrifuge while holding one variable constant in the second-order poly-
Z.-S. Zhang et al. / Separation and Purification Technology 57 (2007) 17–24 19
Fig. 1. Influence of ethanol concentration on the acquired ratio of lignans. 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
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
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 Fig. 5 denoted the effects of extraction time and temperature
natural variables and the following equation was obtained. on the acquired ratio of lignans at fixed ethanol concentration of
70%. The acquired ratio of lignans was found to be a function of
Y = 6.3644 + 0.5545X1 − 0.6225X2 − 0.925X3 the linear and quadratic effects of extraction temperature. The
+ 0.0261X2 X3 − 0.0040X12 + 0.005521 (6) linear and quadratic effects were both positive, which resulted
in a curvilinear increase with the extraction temperature for
This predicted polynomial model was used to obtain the response all the extraction times employed (Fig. 5). Other factors that
surfaces and contour plots for all the interaction among the contributed to the acquired ratio of lignans included the linear
ethanol concentration of the extraction solvent, the extraction effect of the extraction time and its interaction effect with the
time, and the extraction temperature.
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 23
Table 4
The quantity and frequency of the acquired ratio of lignans beyond 8%
Factors
Coding
−1.682 0 0 0
−1 2 0 0
0 3 0 0
1 2 3 1
1.682 0 4 6
Average value (x̄) 0 1.390 1.585
95% confidence interval (xi ) −1.12–1.12 1.166–1.613 1.473–1.696
Parameter interval (Xi ) 56.6–83.4 26.7–27.9 38.7–40.1
The higher acquired ratio of lignans might be obtained at extraction temperature. The F-test and p value indicated that
certain point or area beyond limitative ranges, such as under the the quadratic term of ethanol concentration (x12 ) had the largest
condition that extraction temperature exceeded 40 ◦ C, extraction effect on the acquired ratio of lignans, and followed by the
time exceeded 28 h, and both extraction temperature and time extraction time (x2 ) and the interaction of the extraction time
exceeded them maximum values (>40 ◦ C, >28 h). However, In and the extraction temperature (x2 x3 ). Frequency analysis indi-
the light of practical factors, such as the acquired ratio of lignans, cated that the acquired ratio of lignans might be beyond 8%
economic costs, safety and credibility, product quality and so at a probability of 95% in the range of ethanol concentration:
on, it was unadvisable to increase extraction temperature and 56.6–83.4%, extraction time: 26.7–27.9 h, extraction tempera-
prolong extraction time excessively. ture: 38.7–40.1 ◦ C. And the optimal extraction conditions were
obtained at the ethanol concentration of 70%, the extraction time
3.3. Frequency analysis of 28 h, and extraction temperature of 40 ◦ C using the RSM. In
this condition, the experimental acquired ratio of lignans was
A three-factor and five-level experiment design can require 8.975%, which was close to the predicted value (9.316%).
53 = 125 arrangements. According to the method presented in
literature [31], frequency analysis could be carried out in the Acknowledgements
arrangements, which the acquired ratio of lignans were beyond
8%. Seven arrangements were up to the demand by statistics, Research support was provided by the Funding System
which occupied 5.6% in all arrangements. The results of fre- for Scientific Research Projects of Doctor Subject of Chi-
quency analysis were showed in Table 4. nese Advanced University (No. 20050019029), the Key Project
Lignans were extracted under the conditions listed in the last of Chinese Ministry of Education (No. 105014) and Funding
row of Table 4. The conditions were the ethanol concentration System for Scientific Research Projects of China Agricultural
ranging from 56.6 to 83.4%, the extraction times ranging from University (No. 2004010).
26.7 to 27.9 h, and the extraction temperatures ranging from 38.7
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