Study on Separation and Purification of Genistein in the Soybean

ARTICLE pubs.acs.org/IECR

Study on Separation and Purification of Genistein in the Soybean Residue Using Macroporous Resin Adsorption Hua Li, Juan Liu,* Dan Li, and Hongkai Wang School of Chemical and Energy Engineering, Zhengzhou University, No. 100 Zhengzhou Science Road, Zhengzhou, Henan, China 450001 ABSTRACT: Soybean residue is a rich byproduct that is discarded in soybean processing factories. In this research, soybean residue was used to prepare highly valued genistein by separation and purification through the combination of isoelectric point precipitation, centrifugation, solvent extraction, and macroporous resin adsorption. The purification process had been studied in detail. The results showed that AB-8 resin, feathering high adsorption and desorption recovery, is a suitable resin for purifying genistein, and a optimized process for gradient elution was obtained: the extracts was sequentially eluted with deionized water, 20% ethanol, and 70% ethanol in the first process, and with 40% ethanol and 70% ethanol in the second process; the purity of genistein was about 90% by high-performance liquid chromatography (HPLC) analysis.

1. INTRODUCTION Genistein, which is one of the major isoflavones in soybeans, is associated with a broad variety of beneficial properties on human health and are found in soybean.1,2 Genistein has been extensively studied as a chemopreventive or therapeutic agent in several types of cancer.2 The soybean is mainly used in processing bean product and extracting oil or protein, but the active compounds in residue or byproducts are often abandoned in large amounts. There are 12 main isoflavones in soybeans: genistein, daidzein, and glycitein, and their respective acetyl, malonyl and aglycone forms. Genistein in the waste residue is one of them that can be extracted. If the active compounds in the residue are utilized, the value of soybeans will be greatly raised.3 However, the soybean has complicated components, and the content of target product of genistein is very low; the obtained extract usually contains some impurities, such as fat, protein, salt and sugar, etc.,4,5 To obtain a high content of genistein, it is necessary to confirm a suitable purification process. There are several methods, such as liquid
liquid extraction and ion exchange, that have been employed for the separation of bioactive compounds from natural resources.6 However, these methods are inefficient and they require longer times and more solvent consumption. Recently, there has been a growing interest in employing sorbents to enrichment bioactive compounds from traditional herbal medicines, because of their moderate purification effect. In all sorbents, macroporous resins, because of their relative low cost and easy regeneration, have been employed in the enrichment of many secondary metabolites, including hesperidin, anthocyanins, licorice flavonoids and polyphenols, genistein, and apigenin.7 However, there is no report on using macroporous resin to separate and purify genistein in the soybean residue yet. In the present study, the separation and purification of genistein in the soybean residue was studied, based on AB-8 macroporous resin,8 and the specific aim was to develop an efficient method for the enrichment of genistein from the soybean residue with the AB-8 resin and optimal conditions. The information of this study is significant for resin selection and process optimization in purifying genistein, as well as other natural resources. r 2011 American Chemical Society

2. EXPERIMENTAL SECTION 2.1. Reagents and Materials. Analytical reagent (AR)-grade reagents (such as ethanol, acetone, ethyl acetate ester, cyclohexane, and sulfuric acid) were used. The standard sample for genistein was purchased from Sigma
Aldrich Group. AB-8, ADS-8 macroporous adsorption resins were purchased from Nankai Chemical Factory, China. LSA-10, LSA-20 macroporous adsorption resin was purchased from Shandong Dongda Chemical Industry Group Corp Resin Institute, China. The soybean residue was purchased from Beijing Heyuan Natural Product Co., Ltd., China. Four macroporous resins were dried to constant weight and pulverized, and then stored in an airtight black bag before the experiment. In addition, 5% α-naphthol ethanol solution, 5% hydrochloric acid, 5% sodium hydroxide, and 6 mol L
1 hydrochloric acid were also prepared. 2.2. Instruments and Equipment. The instrumentation used in this experiment included the following: Model WFZUV-2102 UV spectrophotometer (UNICO Instrument Co., Ltd.), Model P200 high-performance liquid chromatography system (Dalian Elite Analytical Instruments Co., Ltd.), and a chromatography column (Shim-Pack CLC ODS 150  4.6 mm). The mobile phase was acetonitrile (denoted as “A”) or a 0.5% aqueous solution of acetic acid (denoted as “B”); there was a linear variation of 83% A to 55% A within 50 min. The flow rate was 0.4 mL/min, the column temperature was 38 °C, and the detection wavelength was 260 nm. 2.3. Sample Preparation and Analytical Methods. 2.3.1. Preparation of Crude Extract of Soybean Isoflavone. The wet soybean residuals were first dried, and then soaked with cyclohexane at room temperature for 24 h to remove lipid, then dried. A 5.00 g sample was added into a 250-mL flask. The extraction was carried out using 100 mL of 70% aqueous ethanol at 80 °C for 2 h. When the extraction was completed, the cooled extract was filtered by vacuum and collected in a volumetric flask. Received: January 10, 2011 Accepted: December 4, 2011 Revised: November 13, 2011 Published: December 05, 2011 44

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at 25 °C for 24 h, then 1 mL of supernatant was taken out to measure the absorbance, and static adsorption capacity and adsorption recovery and desorption recovery were obtained. Dynamic adsorption experiments were carried out using a glass column wet-packed with the resin particles. First, 58 mL of wet resin is placed into the column. Then the solution of soy isoflavone extracts are introduce downward into the column, the feed rate was 4 BV h
1; After finishing the adsorption equilibrium, desorption of genistein from the sorbents was performed in turn with deionized water and 70% ethanol, at a flow rate of 1 BV h
1. Samples were withdrawn from the effluent and analyzed using the UV spectrophotometer at 260 nm. During the above procedures, according to different concentration of the soy isoflavone extracts and different flow rate of the soy isoflavone extracts introdued the column, curves of dynamic adsorption and dissolution, as well as the adsorption and desorption characteristics, were studied. All the dynamic experiments were performed at room temperature. 2.6. Gradient Elution of AB-8 Macroporous Resin. Soluble sugar, salt, and strong polar impurities were removed by deionized water at a rate of 1 BV h
1, until the Molish reaction was negative. [The Molish reaction is described as follows. Furfural and its derivatives were formed by dehydration of sugar under concentrated sulfuric acid or concentrated hydrochloric acid, then the effect of furfural and its derivatives and α-naphthol would form purple and red conjugates. If the elution is not complete, the purple and red would exist, and the result would be considered to be positive; otherwise, the result is negative.] Then, it was eluted with different concentrations of ethanol; when the concentration of eluent was constant, the first elution was finished. After the eluents were combined and concentrated, the second elution began. The above procedure was repeated until the concentration of elution became constant. When elution was performed, 10 mL eluent was collected every time, and the genistein concentration of each 10 mL of eluent was analyzed.

The residue was taken back and re-extracted one more time using fresh solvent each time with the same conditions as noted above. The combined extracts were collected and concentrated to a certain volume; thus, the crude extract of soybean isoflavone was obtained. 2.3.2. Analytical Methods. The standard curve of isoflavone content was analyzed using genistein as the standard at 260 nm, using an ultraviolet (UV) spectrophotometer, and the standard curve was obtained, C ¼ 9:738A
0:2539 with (r2 = 0.9984). Here, C is the concentration (in units of mg L
1) of genistein in the solution and A is the absorbance of the solution. 2.4. Preliminary Purification of Soybean Isoflavone. The crude extraction solution was placed into a separating funnel and was extracted three more times using cyclohexane as the solvent. The color of upper layer was yellow (the upper layer mainly consists of oil), while the color of the lower layer was bright yellow; the lower layer solution was collected and concentrated, then its pH was adjusted to 4.0
4.5 with 6 mol L
1 hydrochloric acid, to make the pH the same as the isoelectric point (IEP) of protein. The solubility of the protein is dependent on, among other things, the pH of the solution. Protein is an ampholyte, and protein itself can be either positively or negatively charged overall, because of the terminal amine
NH2 and carboxyl (
COOH) groups and the groups on the side chain. It is positively charged at low pH and negatively charged at high pH. The intermediate pH at which a protein molecule has a net charge of zero is called the IEP of that protein. Protein is the least soluble when the pH of the solution is at its IEP; therefore, protein can be removed with isoelectric precipitation. Furthermore, the mixture was centrifugally frozen under the conditions of 0 °C and 3000 rpm for 30 min, and the filtrate was collected after the upper sediments (sediments mainly consist of protein) was removed. The filtrates were extracted three times with ethyl acetate, and the upper layer solution was collected and the extraction solution was obtained. 2.5. Adsorption Capacities of AB-8 Macroporous Resin. 2.5.1. The Pretreatment of Resin. AB-8 macroporous resin is a weak polar polymer of styrene, with an average diameter of 13
14 nm; a particle size range of 0.315
1.25 mm g90% (60
16 meshes); a porosity of 42%
46%; a moisture content of 60
70%; a pore volume of 0.73
0.77 mL/g; a specific surface area of 480
520 m2/g; a wet real density of 1.05
1.09 g/mL; and a skeleton density of 1.13
1.17 g/mL. Before use, the macroporous resin was leached with 95% ethanol for 24 h and washed several times using the deionized water. The resins were placed into a 2.6-cm inner diameter (id) glass column, and 2BV (where BV represents the column bed volume) of 5% HCl and the same quantity of NaOH (5%) were sequentially passed through the column at a flow rate of 5 BV/h to remove impurities. Finally, the column was washed with deionized water at the same flow rate until the effluent was chemically neutral.9 2.5.2. Dynamic Adsorption and Desorption of AB-8 Macroporous Resin. Two grams (2 g) of wet resin and 50 mL of an aqueous crude flavonoids solution was added into a 100-mL flask, which was then shaken in a water-bath shaker at 25 °C for 24 h. One milliliter (1 mL) aliqouts of the upper solutions were taken out to measure the absorbance, then followed by filtration. The solid resin was washed by deionized water, after filtration, 50 mL of 70% ethanol was added into the flask, and shaken in the shaker

3. RESULTS AND DISCUSSION 3.1. Static Adsorption Capacity, Adsorption Recovery, and Desorption Recovery of AB-8 Macroporous Resin. The

equilibrium adsorption capacity is given as10 q ¼ ðC0
CV Þ 

V0 G

ð1Þ

where q is the equilibrium adsorption capacity (expressed in terms of milligrams per gram of resin); C0 and CV are the initial and equilibrium concentrations of solute in the solution, respectively (expressed in units of mg mL
1); V0 is the volume of the initial feed solution (mL); and G is the mass of the dry adsorbent (g). Adsorption recovery: E1 ð%Þ ¼

C0
C1  100 C0

ð2Þ

Desorption recovery: E2 ð%Þ ¼

C2  100 C0
C1

ð3Þ

where E1 is the adsorption recovery, E2 the desorption recovery, C2 the concentration of the solute in the desorption solution (mg mL
1), and C1 the concentration of the solute in the supernatant after 24 h (mg mL
1). 45

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Table 1. Adsorption and Desorption Properties of Reins on Genistein reins

adsorption recovery (%)

desorption recovery (%)

AB-8

90.8

86.5

LSA-10 LSA-20

70 68

60 55

ADS-8

45.8

85.7

The performance of adsorption and desorption with four macroporous resins (AB-8, LSA-10, LSA-20, and ADS-8) were investigated as the procedures in section 2.5. It had been reported that the four macroporous resins all had good adsorptive effects, with large saturated adsorption capacity. In addition, the four resins are weak polar polymers, which are fit for the purification of genistein.11 The adsorption and desorption properties of resins on genistein are listed in Table 1. From Table 1, we can find that the adsorption recovery of these four macroporous resins decreased in the order of AB-8 > LSA-10 > LSA-20 > ADS-8, the adsorption recovery of 90.8% for AB-8 is higher than other resins. On the other hand, the desorption recovery of 88% for AB-8 is also higher than that for other resins. The AB-8 macroporous adsorption resin is a weak-polarity adsorbent resin, on the basis of the theory of similarity and intermiscibility; the resin had small adsorption capacity for the strong polarity inorganic salt, sugar, and impurities in feed solution. Moreover, soy isoflavone was weak in polarity; thus, the resin had high adsorption capacity for soy genistein. In soy genistein, the polarity of glucoside isoflavone is higher than aglycone isoflavone, so glucoside isoflavone was easily soluble in low concentrations of ethanol solution while the isoflavone of daidzein was easily soluble in higher concentrations of ethanol solution. Therefore, in the gradient incremental elution, using different concentrations of ethanol solution, glucoside isoflavone was eluted before aglycone isoflavone. Therefore, a higher-purity genistein could be obtained. From the above result, it could be seen that AB-8 macroporous resin had high adsorption and desorption recovery and was a suitable resin for purifying genistein. 3.2. Dynamic Adsorption and Desorption on AB-8 Macroporous Resin12. 3.2.1. Dynamic Adsorption Curves. The dynamic adsorption curve on AB-8 was obtained by contacting 20 mL of aqueous solution of crude flavoniods (0.51 mg mL
1, 0.23 mg mL
1, and 0.11 mg mL
1, respectively) with l g of resin at a rate of 3.6 mL min
1 at 25 °C.11 The flavonoids concentration in the liquid phase was monitored at different time intervals until equilibration.13 The dynamic adsorption curve on AB-8 resin at different concentration is shown in Figure 1. As can be seen from Figure 1, with the increase of concentration of crude flavoniods, the breakthrough point of genistein on AB-8 resin was advanced (i.e., the adsorption time of reached saturated adsorption capacity was decreased, and the breakthrough volume of resin had no obvious difference from three different concentrations of crude flavoniods). The adsorption rate increased as the solution concentration increased; however, if the concentration is too big, it will cause the improvement of mass-transfer resistance, difficulty with feed-in, and big energy consumption. For economical reasons, the feeding concentration of 0.23 mg mL
1 was finally selected. The 0.23 mg mL
1 crude flavoniod was flowed through the resin layer at flow rates of 1.7 mL min
1, 3.6 mL min
1, and 5.9 mL min
1. The concentration and adsorption volume were measured, and the dynamic adsorption curve is shown in Figure 2.

Figure 1. Dynamic adsorption curve on AB-8 resin, caused by contacting 20 mL of aqueous solution of crude flavoniods ((b) 0.51 mg mL
1, (9) 0.23 mg mL
1, and (2) 0.11 mg mL
1) with l g of resin at a rate of 3.6 mL min
1 at 25 °C.

Figure 2. Dynamic adsorption curve of AB-8 resin contacting 0.23 mg mL
1 crude flavoniods at a rate of (b) 1.7 mL min
1, (9) 3.6 mL min
1, and (2) 5.9 mL min
1 at 25 °C.

As seen from Figure 2, the breakthrough point of genistein on AB-8 resin was advanced and the adsorption capacity was decreased as the flow rates of aqueous solution of crude flavoniods increased. When the adsorption volume (V) is the same, the flow rate of 1.7 mL min
1 has larger adsorption capacity than flow rates of 3.6 mL min
1 and 5.9 mL min
1. The slower the flow rate, the greater the adsorption capacity of resin. The reason for this is likely that the low flow rate was favorable to the diffusion in particle and liquid film of soybean genistein molecules in the resin layer, but productivity decreased. For economical reasons, the 3.6 mL min
1 was finally chosen. 3.2.2. Dynamic Desorption Curves. The dynamic desorption curves were determinated with the 0.23 mg mL
1 of the crude 46

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Figure 4. The first elution on the column with different solvents in Process I. The first elution on the column of different solvents was B f 30% A f 60% A f 70% A. (“A” is ethanol and “B” is distilled water.)

Figure 3. Dynamic desorption curve of the AB-8 resin. [Experimental conditions: temperature, 25 °C; flow rate, 3.6 mL min
1, and concentration of soy isoflavone extracts, 0.23 mg mL
1.]

flavoniods at a flow rate of 3.6 mL min
1; the result was shown in Figure 3. It was found that the concentration of genistein increased with the increase in the volume of elute, reaching a maximum value at v ≈ 25 mL and then decreased; the genistein content of outflow liquid decreased gradually after v = 50 mL, and it was eluted completely until v = 90 mL. 3.3. The Technological Process of Gradient Elution on AB-8 Macroporous Adsorptive Resin. 3.3.1. Gradient Elution on AB-8 Macroporous Resin. The gradient elution curves were determinated with the 0.23 mg mL
1 of the crude flavoniods at a flow rate of 3.6 mL min
1. Soluble sugar, salt, and strong polar impurities were removed by deionized water at a rate of 1 BV h
1, until the Molish reaction was negative. Then, it was eluted with different concentrations of ethanol solution; when the concentration of eluent was constant, the first elution was finished. After eluent were combined and concentrated, the second elution was begun. The procedure above was repeated until the concentration of elution became constant. In the above operations, when elution was performed, 10 mL of eluent was collected every time, and the genistein concentration of each 10 mL of eluent was analyzed. Process I: The first elution, in turn, was

Figure 5. The first elution on the column with different solvents in process II. The first elution of different solvents on the column was B f 20% A f 30% A f 40% A f 70% A.

the elution solution is a white cloudy liquid, which perhaps contains some soluble protein, and the color of the elution solution changes from yellow to light yellow gradually with the increase of elution time (the yellow was pigment in an aqueous solution of crude flavoniods), and the Molish reaction was positive. The first elution curves are shown in Figures 4 and 5. As shown in Figure 4, the concentration of soy isoflavones decreased as the ethanol concentration increased and reached their peak value at a concentration of 30%, then decreased as the ethanol concentration increased. When the concentration exceeded 30%, the impurities desorbed were increased, so the purities of genistein were decreased; therefore, 30% ethanol is suitable to elute glycosides. As seen in Figure 5, when eluted with deionized water, 20% ethanol, and 30% ethanol solution, respectively, the soy isoflavones concentration in all increased first and then decreased later; when eluted with 40% ethanol and 70% ethanol solution, the flavoniod concentration were both low, and concentration trend changed insignificantly. Figures 4 and 5 show that the purity and recovery of concentration in the first elution was lower and the first elution is completed when Molish reaction is negative. The Second Elution: The elution solution is colorless in the elution process using deionized water, and the Molish reaction is negative; the second elution curves are shown in Figures 6 and 7. These figures show that the contentration of soy isoflavon of eluent decrease greatly, compared with the first elution when eluted with deionized water. Therefore, the elution process using deionized water could be omitted in the second elution on the column. 3.3.3. Comparison of the First Elution with Different Concentrations of Ethanol Elution Solution. Process I: When the elution solution is 30% ethanol, the elution rate is the fastest and the concentration of soy isoflavones is the highest (50.3 μg mL
1),

B f 30% A f 60% A f 70% A After the first elution, the eluent was collected and applied to the column again. Then, the second elution was carried out sequentially with B f 30% A f 70% A where “A” is ethanol and “B” represents deionized water. Process II: The first elution order sequentially was B f 20% A f 30% A f 40% A f 70% A The second elution order was B f 40% A f 70% A 3.3.2. Comparison of Elution Process with Deionized Water. The First Elution: In the elution process with deionized water, 47

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which indicates that 30% ethanol is suitable to elute glycosides, because both have similar polarities. With increasing ethanol concentration, the concentration of soy isoflavones of the elution solution decreases greatly; the elution solution has little or no isoflavone when eluted with a 50% ethanol solution. Process II: When eluted with 30% ethanol, the elution solution had a lower soy isoflavones content, compared with the 20% ethanol elution; therefore, in order to simplify the production process, 30% ethanol elution could be saved during production. 3.3.4. Comparison of the Second Elution with Different Concentrations of Ethanol Elution Solution. Compared with Figures 6 and 7, it can be found that the separating efficiency when eluted by the 30% ethanol solution was inferior to that of the

40% ethanol solution, which illustrated that glycosides removal was incomplete by the 30% ethanol solution. However, the eluent content by 70% ethanol was higher than that 40% ethanol, which indicated that 70% ethanol was a better elution solution. From the above analysis, the suitable elution process for the first elution was deionized water f 20% ethanol f 70% ethanol The suitable elution process for the second elution was 40% ethanol f 70% ethanol There was a report that a certain volume water was used to wash the column, then the concentration gradient of ethanol solution was 50%, 80%, 95%, but the eluted effects are not better than those determined from our method.6 The above process had been tested to be optimum by many repeated experiments. 3.4. Determination of Elution Product by HPLC. The elution product and standards were determinated by highperformance liquid chromatography (HPLC). The results are shown in Figure 8. Figure 8 shows that the retention time of the elution product and standards is within the range of 18.0
24.0 min, and by comparing the areas of peaks as well as the locations of peaks, the product obtained is obviously high in purity, being 90.1%; a high purity of genistein could be obtained by gradient elution with AB-8 resin as the stationary phase.

Figure 6. The second elution on the column with different solvents in process I. The second on the column elution of different solvents was B f 30% A f 70% A. (“A” is ethanol and “B” is distilled water.)

4. CONCLUSIONS Most of the proteins and fats can be removed by freeze centrifugation, as well as cyclohexane and ethyl acetate extraction, which can be used for further purification of soy isoflavones. The adsorption and desorption properties of AB-8 for genistein were demonstrated in detail. The results showed that AB-8 microporous resin had higher adsorption and desorption recovery for soybean genistein than other resins, it was a suitable resin for the purification of flavonoids. The results of the adsorption experiments demonstrated that the breakthrough times of adsorbent decreased and breakthrough point increased as the concentration of extracted solution increased; as the concentration of extracted solution decreased, the contrary trend was observed. Because of the high flow rate, the breakthrough point

Figure 7. The second elution on the column with different solvents in process II. The second elution of different solvents on the column was B f 40% A f 70% A.

Figure 8. High-performance liquid chromatography (HPLC) chromatograph of soybean residue extract after purification with AB-8 resin and genistein standards. [Note that the ordinate is the voltage (mV), whereas the absicissa is retention time (min).] 48

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would be large at the fixed concentration: the slower the flow rates, the longer the breakthrough times. Therefore, in the purification of high concentrations of crude flavoniods, lower flow rates must be chosen. In addition, the desorption rate was faster with 70% ethanol solution and the resin was easy to recycle. The results of gradient elution on AB-8 macroporous resin showed that the optimized process was as follows. The extracted solution was sequentially eluted with deionized water, 20% ethanol, and 70% ethanol in the first process, and with 40% ethanol and 70% ethanol in the second process; the purity of genistein was ∼90%, and the recovery was 93.3%. AB-8 resin feathering high adsorption and desorption recovery is a suitable resin for purifying genistein.

’ AUTHOR INFORMATION Corresponding Author

*Tel.: 0086-371-67781712. Fax: 0086-371-63886154. E-mail: [email protected].

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