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Soybean isoflavones: Efficacy of extraction conditions and effect of food type on extractability
Food Research International 38 (2005) 1199–1204 www.elsevier.com/locate/foodres
Soybean isoflavones: Efficacy of extraction conditions and effect of food type on extractability Allaoua Achouri, Joyce Irene Boye *, Denis Belanger Agriculture and Agri-Food Canada, Food Research and Development Centre, 3600 Casavant Boulevard West, St. Hyacinthe, Que., Canada J2S 8E3 Received 26 October 2004; accepted 10 May 2005
Abstract Soy isoflavones are phytochemicals of intense interest due to their association with a variety of health protective effects. Analytical techniques to identify and quantify these compounds vary in accuracy, reproducibility and sensitivity. In this study, solvent extraction efficiencies, and the effect of replicate extractions, and sonication on the isoflavone content of two soy products (high and low protein content) were evaluated. The later study was conducted to determine the effect of protein content on isoflavone extractability. Repeated extractions (up to five sequential extractions) of soy meal sample increased the total concentration of isoflavones by 74%, 69% and 65%, using acetonitrile–HCl, methanol and ethanol. The increment for soy protein isolate was 147%, 103% and 105%, respectively. Sonication of both samples in the three solvents for 15 min extracted as much isoflavones as the total of the five sequential extractions. These results strongly indicates that, a one step extraction without sonication can markedly under-estimate the concentration of isoflavones in foods, and that protein content of foods may have a marked impact on isoflavone extractability. Crown Copyright 2005 Published by Elsevier Ltd. All rights reserved. Keywords: Soy meal; Soy protein isolate; Quantitative analysis; Isoflavones; HPLC
1. Introduction Interest in soybeans and soy based products has grown significantly in the last decade due to their reported nutritional and health-promoting benefits. Researchers have credited phytochemicals in soybeans, especially isoflavones, for some of these beneficial health effects. Soy isoflavones are reported to play a role in the prevention of osteoporosis, and several hormonallyinfluenced cancers (Coward, Barnes, Setchell, & Barnes, 1993; Jenkins et al., 2003; Messina & Barnes, 1991) and to act as phytoestrogens in humans (Adlercreutz & Mazur, 1997; Brzezinski et al., 1997; Jenkins et al., 2002; Murkies, Wilcox, & Davis, 1998). Their ability to act as antioxidants may also serve to prevent oxidative damage in living *
Corresponding author. Tel.: + 1 450 773 1105; fax: + 1 450 773 8461. E-mail address: [email protected] (J.I. Boye).
tissue (Jha, Von Recklinghausen, & Zilliken, 1985; Pratt & Birac, 1979; Ruiz Larrea et al., 1997; Wei, Bowen, Cai, Barnes, & Wang, 1995). There continues to be considerable variation in measurement techniques used by researchers, testing centers and ingredient manufacturers to determine the isoflavone content of food product (Schryver, 2002). A standardized analytical technique to quantify these compounds in foods has, therefore, become essential. High performance liquid chromatography (HPLC) using reversed-phase C18 stationary matrices, mostly with mixtures of methanol or acetonitrile, has proved to be the method of choice for the analysis of isoflavones (Murphy, 1981; Song, Barua, Buseman, & Murphy, 1998). However, no information has so far been reported on the effects of replicate extractions and the food matrix on the yield of isoflavones. Isoflavones are polyphenolic compounds, and several reports indicate that they can interact differentially with components in
0963-9969/$ - see front matter. Crown Copyright 2005 Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.foodres.2005.05.005
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the food matrix (e.g., proteins) (Boye, 1999; Franke, Custer, Cerna, & Narala, 1994) which can directly influence their extractability. The present investigation was, therefore, aimed at evaluating the extraction yield of isoflavones using three different solvents, namely methanol, ethanol and acetonitrile–HCl, with repeated extractions, with and without sonication, and compare their efficiency in extracting isoflavones from a low protein (defatted soy meal) and a high protein (soy protein isolate) soy product. The objective of the later study was to determine if the concentration of protein in a food matrix would influence the extractability of the isoflavones.
2. Materials and methods 2.1. Materials Defatted soybean meal (SM) (53% protein, 30% carbohydrates, 1% fat) and soy protein isolate-ProFam 873 (SPI) (90% protein, 1% fat, 6% ash) was supplied by ADM Co. (4666 East Faries Paekway, Decatur, Illinois). Genistin, genistein, daidzein and flavone (internal standard), were purchased from Sigma–Aldrich (St. Louis, MO). Daidzin, glycitein and glycitin were from LC Laboratories (Woburn, MA 01801, USA). All other reagents were of HPLC grade.
80, 100 lg/mL). A mixture of the standards was also analyzed and used for identification purposes. 2.4. Isoflavone extraction Isoflavones were extracted from the soybean samples using three different solvents (i.e., 80% acetonitrile–HCl 0.1 N, 80% methanol and 80% ethanol). Samples (2 g) were dispersed in 10 mL of each solvent and vigorously mixed at room temperature for 2 h using a VWR-Rocking Platform Shaker (Model 200). The dispersions were then centrifuged for 30 min at 3000 rpm, using a Backman CS-6 centrifuge. The extracts were taken to dryness under a flow of nitrogen, re-dissolved in 80% methanol and filtered through a 0.45 lm filter unit prior to HPLC analysis. The precipitates from the first extractions were subjected to repeated extractions (total of 5 extractions), with each extraction lasting 2 h. A second set of samples, dispersed in the same solvents were subjected to sonication in a VWR-Aquasonic (Model 750D) at ultrasonic frequency of 50 to 60 Hz. The samples were maintained at 22 C (with a thermostatic controller) and sonicated for three different time periods (15, 30 and 60 min). All extracts were then centrifuged for 30 min at 3000 rpm, dried with nitrogen, re-dissolved in 80% methanol and filtered prior to HPLC analysis. 2.5. Statistical analysis
2.2. Chromatographic conditions Analyses were carried out by HPLC. A Waters system with a Model 717 autosampler, a Model 600 dual pump, a Model 996 photodiode array detector, and a digital PC 3000 using Millennium HPLC data processing software (version 3.2) was used. A C18-YMC-Pack ODS AM (5 lm, 25 cm · 4.6 mm) was used for chromatographic separations. Column temperature was held constant at 22 C in a Waters thermal chamber controller. Elution was carried out at a flow rate of 1 mL/min with the following solvent system: A: acetonitrile, B: 0.2% trifluoroacetic acid (TFA) in deionized water. After injection of 10 lL of sample, the system was maintained at 15% A for 5 min, then increased to 30% in 10 min and held for 10 min, and then increased to 50% within 10 min and held for another 10 min. At the end of the 45 min, the system was recycled back to 15% A and held at that level for 10 min for a total run of 60 min per sample. Eluted isoflavones were monitored with UV at 254 nm. 2.3. Isoflavone standards Isoflavone stock solutions were prepared by dissolving the standards in methanol to give a 1 mg/mL concentration. Calibration curves were made for each standard with six different concentrations (Daidzin at 15, 30, 60, 90, 120, 150 lg/mL, and the other five standards at 10, 20, 40, 60,
Data are the means of three determinations, and error bars indicate the standard deviation. ANOVA was carried out to determine differences that were significant at the 5% level of probability, using the data analysis ToolPak of EXCEL-Microsoft Corporation program (2002).
3. Results and discussion The HPLC chromatogram of the isoflavone standards is represented in Fig. 1(a). The use of a C18YMC-Pack ODS AM column with acetonitrile–0.2% TFA/water as a binary mobile phase, using the solvent gradient applied, resulted in a good resolution of the standards within 50 min. The chromatograms of the isoflavones extracted from SM and SPI (first extraction) using ACN–HCl showed similar pattern of isoflavones (Fig. 1(b) and (c)), with noticeable variations in their concentrations. Nine different isoflavones were identified including daidzein, glycitein, genistein, daidzin, glycitin, genistin, malonyl-daidzin, malonyl-glycitin and malonyl-genistin. The first three are the aglycone forms and the last six are the glycoside forms. Malonyl derivatives were identified based on information in the literature (Murphy, Song, Buseman, & Barua, 1997) due to the unavailability of standards.
Total isoflavone concentration (µg/g sample)
A. Achouri et al. / Food Research International 38 (2005) 1199–1204
5000 4500 4000 3500 3000 2500 2000 1500 1000 500 0
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+74 %
+69 %
+103 %
+147 %
SM
+65 %
SPI
SM
ACNM
MeOH
SPI
+107 %
SM
SPI EtOH
Extraction solvent Total isoflavone (E1)
Total isoflavone (E5)
Fig. 2. Total isoflavones of soy meal (SM) and soy protein isolate (SPI) extracted with one single step extraction (E1), and with five P sequential extractions ( 5E), using three different solvents.
Fig. 1. RP-HPLC chromatograms of isoflavone standards mixture (a), isoflavones extracted from soy meal (b) and from soy protein isolate (c). (a): 1, daidzin; 2, glycitin; 3, genistin; 4, daidzein; 5, glycitein; 6, genistein; 7, flavone (internal standard). (b) and (c): X1, X2 and X3 are unidentified compounds, possibly malonyl-daidzin, malonyl-glycitin and malonyl genistin, as reported in the literature.
3.1. Extraction efficiency 3.1.1. Effect of sequential extractions The total amount of isoflavones extracted after the first extraction (E1) for each solvent is given in Fig. 2. Repeating the extraction five times increased the total
yield of individual isoflavones by 56%, 61%, 56%, 53%, 42%, 54%, 100%, 60% and 83% in SM for daidzin, glycitin, genistin, daidzein, glycitein, genistein, malonyldaidzin, malonyl-glycitin and malonyl-genistin, respectively (Table 1). For SPI a similar trend was observed. The second extraction (E2) yielded almost as much isoflavones as the first extraction, and a total increase of approximately 123%, 119%, 120%, 128%, 89%, 101%, 101%, 153% and 142% for daidzin, glycitin, genistin, daidzein, glycitein, genistein, malonyl-daidzin, malonylglycitin and malonyl-genistin respectively, was obtained after the fifth extraction. For the different solvents used, the yield of total isoflavones after five extractions (compared to only one extraction, E1) increased by 74%, 69% and 65% for SM, and 147%, 103% and 107% for SPI, using acetonitrile, methanol and ethanol, respectively (Fig. 2). These results strongly suggest that a one time extraction of isoflavones using the above-mentioned method markedly under-estimates the concentration of isoflavones in these products. The variation in the extraction yield of isoflavones between SM and SPI is very interesting. For the high protein sample (SPI), a one time extraction extracted only 41% of the total isoflavone compared to 58% for the lower protein product (SM), using ACN– HCl solvent. This difference might be due to the stronger protein–polyphenol interaction in the SPI sample. In a review paper (Boye, 1999) reported that a variety of interactions including hydrogen bonding, ionic and covalent binding, and mainly hydrophobic interactions are involved in the formation of the protein–polyphenol complex. These interactions are strongly influenced by factors such as, temperature, pH, and salt which occur during acidic precipitation of soy proteins. 3.1.2. Effect of sonication The effect of sonication on the extractability of the isoflavones content is represented in Fig. 3. The results
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Table 1 Effect of sequential extractions on the recovery of individual isoflavones from soy meal (SM) and soy protein isolate (SPI) using three different solvents Total isoflavone (lg/g sample)
ACN SM
Dein
24 ± 0.3 22.8 ± 2.7 8 ± 0.3 3.8 ± 0.8 1.1 ± 0.2 59.6
48 ± 3 19 ± 2 6 ± 1.1 1.5 ± 0.03 0.6 ± 0.03 75.1
34 ± 1.3 22.8 ± 0.3 6.6 ± 0.13 3.2 ± 0.1 1.1 ± 0.5 67.5
E1 E2 E3 E4 E5
40 ± 1.2 6.6 ± 0.4 1.9 ± 0.1 1.2 ± 0.08 1 ± 0.14 50.7
33.5 ± 4.2 20 ± 1.1 7.3 ± 0.3 2.7 ± 0.2 1.3 ± 0.04 64.7
23 ± 1 13.3 ± 2.2 3.4 ± 0.2 1.9 ± 0.01 1.2 ± 0.05 42.6
31.4 ± 1.1 21.4 ± 0.4 7.8 ± 0.4 5.5 ± 0.2 2.5 ± 0.2 68.6
28.2 ± 1.6 8.9 ± 0.5 2.9 ± 0.5 1.3 ± 0.01 0.9 ± 0.01 42.2
35.4 ± 0.05 19.7 ± 0.1 6.4 ± 0.1 3.7 ± 0.1 2 ± 0.1 67.3
E1 E2 E3 E4 E5
7 ± 0.25 1.3 ± 0.7 0.3 ± 0.07 0 0 8.3
5.2 ± 0.6 3.6 ± 0.8 1.4 ± 0.3 1 ± 0.1 0.3 ± 0.01 11.4
4.9 ± 0.9 2.1 ± 0.2 0.4 ± 0.01 0.3 ± 0.02 0 7.7
12 ± 0.2 4.5 ± 0.1 1.3 ± 0.3 0.5 ± 0.08 0.4 ± 0.02 18.8
6 ± 0.5 2 ± 0.16 0.5 ± 0.01 0.3 ± 0.07 0 8.5
7.5 ± 0.6 4.1 ± 0.2 1.8 ± 0.6 0.5 ± 0.03 0.6 ± 0.07 14.5
E1 E2 E3 E4 E5
365 ± 11.5 130 ± 8 43 ± 1.6 13 ± 0.7 7.2 ± 0.4 557.7
138 ± 2.1 141 ± 11 68 ± 5 30 ± 5.5 11.4 ± 0.9 388.1
462 ± 7.4 237 ± 19.5 48.4 ± 1.8 16.7 ± 0.5 6 ± 0.2 770.3
226 ± 17 131 ± 10 44.5 ± 3.5 24.6 ± 2.8 8.6 ± 0.86 434
514 ± 18 169 ± 15 54.5 ± 3.5 12.5 ± 0.6 5 ± 0.3 755.3
E1 E2 E3 E4 E5
606 ± 18 206 ± 14 55 ± 2 17 ± 0.8 8.8 ± 0.5
285 ± 43 248.5 ± 20 108 ± 10 44 ± 1 14 ± 1.5 700
669 ± 7.8 339 ± 25 84 ± 3 32 ± 1 12 ± 0.5
377 ± 35 247 ± 22 89 ± 0.8 56 ± 0.8 21 ± 2.4 789
750 ± 20 256 ± 22 92 ± 5.7 21 ± 1 8 ± 0.5
358 ± 14 236 ± 22 77 ± 1.5 44 ± 7 19 ± 3 734
E1 E2 E3 E4 E5
81 ± 2.6 29 ± 1.6 11 ± 2.7 4.7 ± 0.2 3.6 ± 0.1 128.8
33 ± 4.5 31.5 ± 2 14 ± 0.4 6 ± 0.7 2.8 ± 0.3 87.3
102 ± 5.5 48 ± 3.5 11 ± 0.5 4.5 ± 0.2 2.3 ± 0.07 168.2
54 ± 3 30.5 ± 1.8 10 ± 0.5 6 ± 0.5 2.6 ± 0.1 103.3
107 ± 8.8 40 ± 3.7 14.6 ± 1 4.7 ± 0.3 2.7 ± 0.8 169
48 ± 1.4 29.3 ± 1.5 9.5 ± 0.9 5.5 ± 1 3.1 ± 0.5 95.3
E1 E2 E3 E4 E5
354 ± 11 172 ± 6 176 ± 2.5 60 ± 6.5 48 ± 0.8
69 ± 2.3 20.4 ± 0.3 11 ± 0.6 4.5 ± 0.4 5.5 ± 0.1 111
342 ± 27 154 ± 9 47 ± 1.2 15 ± 0.5 4.4 ± 0.6
42 ± 3.3 24 ± 0.3 7.8 ± 0.01 11.3 ± 1.1 0.8 ± 0.2 86
336 ± 32 273 ± 21 63 ± 7 19 ± 5.6 4 ± 0.4
28 ± 3.8 23.7 ± 2.8 8.3 ± 1 4.3 ± 0.1 2.4 ± 0.5 66.4
E1 E2 E3 E4 E5
662 ± 20 279 ± 11 224 ± 33 65 ± 8 46 ± 2.6 1276
69 ± 5.3 62 ± 1.3 32 ± 1.8 15.3 ± 0.8 13.2 ± 0.2 191.6
490 ± 32 242 ± 15 80 ± 2 27 ± 1 8 ± 0.2 846.8
103 ± 1.7 60 ± 1.3 20.1 ± 1 20.4 ± 1.9 2 ± 0.3 206.2
490 ± 41 268 ± 12 99.5 ± 7.3 28 ± 0.8 8 ± 0.2 894
70.3 ± 1.4 64 ± 1.1 22 ± 0.5 13 ± 0.2 6.5 ± 0.7 175.1
E1 E2 E3 E4 E5
59 ± 1.8 15.5 ± 0.7 6 ± 0.3 4 ± 0.4 3.4 ± 0.1 87.2
8.8 ± 0.3 12 ± 1 4.6 ± 0.1 2.4 ± 0.1 1.6 ± 0.1 29.3
57.4 ± 0.6 27 ± 1.8 7 ± 0.3 3.2 ± 0.3 1.6 ± 0.2 96
55 ± 0.4 22 ± 1.3 7.3 ± 0.4 4 ± 0.6 2.3 ± 0.5 90.7
20 ± 0.2 14 ± 0.4 5.4 ± 0.2 3.6 ± 0.6 2.2 ± 0.3 44.3
Total genistin Glin
Total glycitin MDin
Total malonyl-Daidzin MGin
Total malonyl-Genistin MGly
Total malonyl-Glycitin
SPI
41.5 ± 2.8 24 ± 2.6 4 .2 ± 0.3 2.3 ± 0.04 0.9 ± 0.05 72.6
Total daidzin Gin
SM
21 ± 0.4 18.7 ± 0.8 6.6 ± 1.8 1.9 ± 0.06 0.7 ± 0.1 48.5
Total glycitein Din
SPI
62 ± 2.2 13 ± 1.4 3 ± 0.2 0.7 ± 0.2 0.4 ± 0.04 78.8
Total genistein Glein
EtOH
SM
E1 E2 E3 E4 E5
Total daidzein Gein
MeOH SPI
23 ± 3 14 ± 0.6 5.2 ± 0.03 2.4 ± 0.2 1.4 ± 0.08 45.6
206 ± 9 125 ± 9 39 ± 6 21 ± 3 9±1 399.9
Total isoflavone (E1) P Total isoflavone ( 5E)
2235 ± 125 662 ± 12 2191 ± 110 892 ± 11 2334 ± 152 806 ± 9 3890 ± 275 1631 ± 28 3702 ± 311 1811 ± 27 3893 ± 374 1664 ± 24 P E1 to E5: Refers to the number of extractions; 5E: Sum of five extractions; ACN: Acetonitrile; MeOH: Methanol; EtOH: Ethanol; Dein: Daidzein; Gein: Genistein; Glein: Glycitein; Din: Daizin; Gin: Genistin; Glin: Glycitin; MDin: Malonyl daidzin; MGin: Malonyl Genistin; MGly: Malonyl glycitin.
clearly show that sonication for 15 min extracted as much isoflavones as the total of the five sequential extractions (with ordinary shaking for a total of
10 h). Prolonging the time of sonication to 30 and 60 min, however, did not increase the total amount of isoflavone extracted. Sonication may, therefore, provide a
A. Achouri et al. / Food Research International 38 (2005) 1199–1204
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solvent systems, 80% ACN–HCl–H2O, 80% methanol– HCl–H2O and 80% ethanol–HCl–H2O, Wang, Kuan, Octave, Ware, and Carman (1990) and Wang, Ma, Pagadala, Sherrard, and Krishnan (1998), reported no differences in extraction efficiencies. Extraction with acetonitrile–HCl was, however, preferred because this system resulted in fast settling of suspending sample particles and less interfering impurities in the extract. 3.2. Quantitation of aglycones and glycosides extractability
Fig. 3. Effects of sonication and shaking (five sequential extractions) on the total isoflavone content from soy meal (SM) and soy protein P isolate (SPI) using three different solvents. ( 5E: represent the sum of five extractions.)
means of speeding the extraction and analysis of isoflavones. 3.1.3. Effect of solvent The analysis of variance showed no significant (p 6 0.05) differences between the three extraction solvents used. The total concentration of isoflavones after the fifth extraction, yielded 3890, 3702 and 3856 lg/g for SM, and 1631, 1811 and 1664 lg/g for SPI, using acetonitrile–HCl, methanol and ethanol, respectively. Sonication for 15 min yielded 3273, 3678 and 3770 lg/g sample in SM, and 1130, 1817 and 1755 lg/g sample in SPI, using the same solvents, respectively. Closer examination of the results, however, showed that the extraction efficiency of ACN–HCl was significantly (p 6 0.05) higher than the other two solvents in extracting the malonyl-glycosides from SM sample. The presence of hydrochloric acid in acetonitrile may have enhanced the extraction of this particular isoflavone form. In her early work, Murphy (1981) reported that addition of water and/or hydrochloric acid to organic solvents greatly improved the extraction efficiency; and that 80% ACN was superior to other extraction solvents (Murphy, Barua, & Hauck, 2002). Using the same three
SPI contained much lower concentrations of isoflavones (both aglycones and glycosides isomers) compared to SM, irrespective of the number of extractions and the type of solvent used (Fig. 4). This significant (p 6 0.05) difference in the content of isoflavones is likely due to losses of isoflavones during processing of soy protein isolate. This processing effect has been reported by several authors (Eldridge, 1982a, 1982b; Murphy, 1982; Wang & Murphy, 1994a, 1994b, 1996). Differences in the distribution of the different isoflavone forms were also observed in the two samples (Table 2). SM contained the same amount of b-glycosides and malonyl-glycosides equalling about 50% of total isoflavones. SPI contained predominantly the b-glycosides
Fig. 4. Soy meal (SM) and soy protein isolate (SPI) total concentration of aglycone and glucoside isoflavones from extraction 1 (E1), and P sum of five extractions ( 5E), using three different solvents.
Table 2 Proportion of different isoflavone forms from soy meal (SM) and soy protein isolate (SPI) samples Solvent
Aglycones (%/total isoflavonesa) SM
SPI
SM
SPI
SM
SPI
ACN–HCl MeOH EtOH
3.54 ± 0.1 3.32 ± 0.02 3.26 ± 0.05
7.64 ± 0.13 8.12 ± 0.23 8.97 ± 0.53
40.6 ± 0.9 56.0 ± 0.2 53.0 ± 0.6
72.00 ± 0.66 73.25 ± 0.38 73.85 ± 0.56
55.86 ± 0.37 40.67 ± 0.22 44.1 ± 0.49
20.33 ± 0.21 18.64 ± 0.13 17.17 ± 0.43
b-Glycosides (%/total isoflavonesa)
ACN–HCl: Acetonitrile–hydrochloric acid; MeOH: Methanol; EtOH: Ethanol. a Sum of five extractions.
Malonyl-glycosides (%/total isoflavones a)
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equalling 75% of total isoflavones. The higher percentages of the aglycone and b-glycoside isoflavones in the SPI sample may be attributable to hydrolysis of the glycosides during processing (Murphy, 1982). For the two samples studied, the proportion of the different isoflavone forms remained the same irrespective of the extraction technique (sonication or shaking).
4. Conclusion A single step extraction of isoflavones markedly under-estimates the true concentration of isoflavones in a food product. The food matrix, specifically the concentration of protein, may have an impact on the extractability of the isoflavones. For quantitation purposes, care must be taken to use the best extraction system for the food matrix under examination. The poor extractability of isoflavones with one extraction compared to the total yield of five sequential extractions in high protein samples, such as SPI, may be due to stronger protein–polyphenol interactions. Although, the evidence in the literature suggests that the biological effects of soy isoflavones depend upon the aglycone form, the recent trend has been to quantify all type of derivative consumed, as these may have differing biological activities. The results presented in this paper could prove helpful in selecting analytical conditions for the extraction of isoflavones from different food matrices.
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Soybean isoflavones: Efficacy of extraction conditions and effect of food type on extractability Allaoua Achouri, Joyce Irene Boye *, Denis Belanger Agriculture and Agri-Food Canada, Food Research and Development Centre, 3600 Casavant Boulevard West, St. Hyacinthe, Que., Canada J2S 8E3 Received 26 October 2004; accepted 10 May 2005
Abstract Soy isoflavones are phytochemicals of intense interest due to their association with a variety of health protective effects. Analytical techniques to identify and quantify these compounds vary in accuracy, reproducibility and sensitivity. In this study, solvent extraction efficiencies, and the effect of replicate extractions, and sonication on the isoflavone content of two soy products (high and low protein content) were evaluated. The later study was conducted to determine the effect of protein content on isoflavone extractability. Repeated extractions (up to five sequential extractions) of soy meal sample increased the total concentration of isoflavones by 74%, 69% and 65%, using acetonitrile–HCl, methanol and ethanol. The increment for soy protein isolate was 147%, 103% and 105%, respectively. Sonication of both samples in the three solvents for 15 min extracted as much isoflavones as the total of the five sequential extractions. These results strongly indicates that, a one step extraction without sonication can markedly under-estimate the concentration of isoflavones in foods, and that protein content of foods may have a marked impact on isoflavone extractability. Crown Copyright 2005 Published by Elsevier Ltd. All rights reserved. Keywords: Soy meal; Soy protein isolate; Quantitative analysis; Isoflavones; HPLC
1. Introduction Interest in soybeans and soy based products has grown significantly in the last decade due to their reported nutritional and health-promoting benefits. Researchers have credited phytochemicals in soybeans, especially isoflavones, for some of these beneficial health effects. Soy isoflavones are reported to play a role in the prevention of osteoporosis, and several hormonallyinfluenced cancers (Coward, Barnes, Setchell, & Barnes, 1993; Jenkins et al., 2003; Messina & Barnes, 1991) and to act as phytoestrogens in humans (Adlercreutz & Mazur, 1997; Brzezinski et al., 1997; Jenkins et al., 2002; Murkies, Wilcox, & Davis, 1998). Their ability to act as antioxidants may also serve to prevent oxidative damage in living *
Corresponding author. Tel.: + 1 450 773 1105; fax: + 1 450 773 8461. E-mail address: [email protected] (J.I. Boye).
tissue (Jha, Von Recklinghausen, & Zilliken, 1985; Pratt & Birac, 1979; Ruiz Larrea et al., 1997; Wei, Bowen, Cai, Barnes, & Wang, 1995). There continues to be considerable variation in measurement techniques used by researchers, testing centers and ingredient manufacturers to determine the isoflavone content of food product (Schryver, 2002). A standardized analytical technique to quantify these compounds in foods has, therefore, become essential. High performance liquid chromatography (HPLC) using reversed-phase C18 stationary matrices, mostly with mixtures of methanol or acetonitrile, has proved to be the method of choice for the analysis of isoflavones (Murphy, 1981; Song, Barua, Buseman, & Murphy, 1998). However, no information has so far been reported on the effects of replicate extractions and the food matrix on the yield of isoflavones. Isoflavones are polyphenolic compounds, and several reports indicate that they can interact differentially with components in
0963-9969/$ - see front matter. Crown Copyright 2005 Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.foodres.2005.05.005
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the food matrix (e.g., proteins) (Boye, 1999; Franke, Custer, Cerna, & Narala, 1994) which can directly influence their extractability. The present investigation was, therefore, aimed at evaluating the extraction yield of isoflavones using three different solvents, namely methanol, ethanol and acetonitrile–HCl, with repeated extractions, with and without sonication, and compare their efficiency in extracting isoflavones from a low protein (defatted soy meal) and a high protein (soy protein isolate) soy product. The objective of the later study was to determine if the concentration of protein in a food matrix would influence the extractability of the isoflavones.
2. Materials and methods 2.1. Materials Defatted soybean meal (SM) (53% protein, 30% carbohydrates, 1% fat) and soy protein isolate-ProFam 873 (SPI) (90% protein, 1% fat, 6% ash) was supplied by ADM Co. (4666 East Faries Paekway, Decatur, Illinois). Genistin, genistein, daidzein and flavone (internal standard), were purchased from Sigma–Aldrich (St. Louis, MO). Daidzin, glycitein and glycitin were from LC Laboratories (Woburn, MA 01801, USA). All other reagents were of HPLC grade.
80, 100 lg/mL). A mixture of the standards was also analyzed and used for identification purposes. 2.4. Isoflavone extraction Isoflavones were extracted from the soybean samples using three different solvents (i.e., 80% acetonitrile–HCl 0.1 N, 80% methanol and 80% ethanol). Samples (2 g) were dispersed in 10 mL of each solvent and vigorously mixed at room temperature for 2 h using a VWR-Rocking Platform Shaker (Model 200). The dispersions were then centrifuged for 30 min at 3000 rpm, using a Backman CS-6 centrifuge. The extracts were taken to dryness under a flow of nitrogen, re-dissolved in 80% methanol and filtered through a 0.45 lm filter unit prior to HPLC analysis. The precipitates from the first extractions were subjected to repeated extractions (total of 5 extractions), with each extraction lasting 2 h. A second set of samples, dispersed in the same solvents were subjected to sonication in a VWR-Aquasonic (Model 750D) at ultrasonic frequency of 50 to 60 Hz. The samples were maintained at 22 C (with a thermostatic controller) and sonicated for three different time periods (15, 30 and 60 min). All extracts were then centrifuged for 30 min at 3000 rpm, dried with nitrogen, re-dissolved in 80% methanol and filtered prior to HPLC analysis. 2.5. Statistical analysis
2.2. Chromatographic conditions Analyses were carried out by HPLC. A Waters system with a Model 717 autosampler, a Model 600 dual pump, a Model 996 photodiode array detector, and a digital PC 3000 using Millennium HPLC data processing software (version 3.2) was used. A C18-YMC-Pack ODS AM (5 lm, 25 cm · 4.6 mm) was used for chromatographic separations. Column temperature was held constant at 22 C in a Waters thermal chamber controller. Elution was carried out at a flow rate of 1 mL/min with the following solvent system: A: acetonitrile, B: 0.2% trifluoroacetic acid (TFA) in deionized water. After injection of 10 lL of sample, the system was maintained at 15% A for 5 min, then increased to 30% in 10 min and held for 10 min, and then increased to 50% within 10 min and held for another 10 min. At the end of the 45 min, the system was recycled back to 15% A and held at that level for 10 min for a total run of 60 min per sample. Eluted isoflavones were monitored with UV at 254 nm. 2.3. Isoflavone standards Isoflavone stock solutions were prepared by dissolving the standards in methanol to give a 1 mg/mL concentration. Calibration curves were made for each standard with six different concentrations (Daidzin at 15, 30, 60, 90, 120, 150 lg/mL, and the other five standards at 10, 20, 40, 60,
Data are the means of three determinations, and error bars indicate the standard deviation. ANOVA was carried out to determine differences that were significant at the 5% level of probability, using the data analysis ToolPak of EXCEL-Microsoft Corporation program (2002).
3. Results and discussion The HPLC chromatogram of the isoflavone standards is represented in Fig. 1(a). The use of a C18YMC-Pack ODS AM column with acetonitrile–0.2% TFA/water as a binary mobile phase, using the solvent gradient applied, resulted in a good resolution of the standards within 50 min. The chromatograms of the isoflavones extracted from SM and SPI (first extraction) using ACN–HCl showed similar pattern of isoflavones (Fig. 1(b) and (c)), with noticeable variations in their concentrations. Nine different isoflavones were identified including daidzein, glycitein, genistein, daidzin, glycitin, genistin, malonyl-daidzin, malonyl-glycitin and malonyl-genistin. The first three are the aglycone forms and the last six are the glycoside forms. Malonyl derivatives were identified based on information in the literature (Murphy, Song, Buseman, & Barua, 1997) due to the unavailability of standards.
Total isoflavone concentration (µg/g sample)
A. Achouri et al. / Food Research International 38 (2005) 1199–1204
5000 4500 4000 3500 3000 2500 2000 1500 1000 500 0
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+74 %
+69 %
+103 %
+147 %
SM
+65 %
SPI
SM
ACNM
MeOH
SPI
+107 %
SM
SPI EtOH
Extraction solvent Total isoflavone (E1)
Total isoflavone (E5)
Fig. 2. Total isoflavones of soy meal (SM) and soy protein isolate (SPI) extracted with one single step extraction (E1), and with five P sequential extractions ( 5E), using three different solvents.
Fig. 1. RP-HPLC chromatograms of isoflavone standards mixture (a), isoflavones extracted from soy meal (b) and from soy protein isolate (c). (a): 1, daidzin; 2, glycitin; 3, genistin; 4, daidzein; 5, glycitein; 6, genistein; 7, flavone (internal standard). (b) and (c): X1, X2 and X3 are unidentified compounds, possibly malonyl-daidzin, malonyl-glycitin and malonyl genistin, as reported in the literature.
3.1. Extraction efficiency 3.1.1. Effect of sequential extractions The total amount of isoflavones extracted after the first extraction (E1) for each solvent is given in Fig. 2. Repeating the extraction five times increased the total
yield of individual isoflavones by 56%, 61%, 56%, 53%, 42%, 54%, 100%, 60% and 83% in SM for daidzin, glycitin, genistin, daidzein, glycitein, genistein, malonyldaidzin, malonyl-glycitin and malonyl-genistin, respectively (Table 1). For SPI a similar trend was observed. The second extraction (E2) yielded almost as much isoflavones as the first extraction, and a total increase of approximately 123%, 119%, 120%, 128%, 89%, 101%, 101%, 153% and 142% for daidzin, glycitin, genistin, daidzein, glycitein, genistein, malonyl-daidzin, malonylglycitin and malonyl-genistin respectively, was obtained after the fifth extraction. For the different solvents used, the yield of total isoflavones after five extractions (compared to only one extraction, E1) increased by 74%, 69% and 65% for SM, and 147%, 103% and 107% for SPI, using acetonitrile, methanol and ethanol, respectively (Fig. 2). These results strongly suggest that a one time extraction of isoflavones using the above-mentioned method markedly under-estimates the concentration of isoflavones in these products. The variation in the extraction yield of isoflavones between SM and SPI is very interesting. For the high protein sample (SPI), a one time extraction extracted only 41% of the total isoflavone compared to 58% for the lower protein product (SM), using ACN– HCl solvent. This difference might be due to the stronger protein–polyphenol interaction in the SPI sample. In a review paper (Boye, 1999) reported that a variety of interactions including hydrogen bonding, ionic and covalent binding, and mainly hydrophobic interactions are involved in the formation of the protein–polyphenol complex. These interactions are strongly influenced by factors such as, temperature, pH, and salt which occur during acidic precipitation of soy proteins. 3.1.2. Effect of sonication The effect of sonication on the extractability of the isoflavones content is represented in Fig. 3. The results
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Table 1 Effect of sequential extractions on the recovery of individual isoflavones from soy meal (SM) and soy protein isolate (SPI) using three different solvents Total isoflavone (lg/g sample)
ACN SM
Dein
24 ± 0.3 22.8 ± 2.7 8 ± 0.3 3.8 ± 0.8 1.1 ± 0.2 59.6
48 ± 3 19 ± 2 6 ± 1.1 1.5 ± 0.03 0.6 ± 0.03 75.1
34 ± 1.3 22.8 ± 0.3 6.6 ± 0.13 3.2 ± 0.1 1.1 ± 0.5 67.5
E1 E2 E3 E4 E5
40 ± 1.2 6.6 ± 0.4 1.9 ± 0.1 1.2 ± 0.08 1 ± 0.14 50.7
33.5 ± 4.2 20 ± 1.1 7.3 ± 0.3 2.7 ± 0.2 1.3 ± 0.04 64.7
23 ± 1 13.3 ± 2.2 3.4 ± 0.2 1.9 ± 0.01 1.2 ± 0.05 42.6
31.4 ± 1.1 21.4 ± 0.4 7.8 ± 0.4 5.5 ± 0.2 2.5 ± 0.2 68.6
28.2 ± 1.6 8.9 ± 0.5 2.9 ± 0.5 1.3 ± 0.01 0.9 ± 0.01 42.2
35.4 ± 0.05 19.7 ± 0.1 6.4 ± 0.1 3.7 ± 0.1 2 ± 0.1 67.3
E1 E2 E3 E4 E5
7 ± 0.25 1.3 ± 0.7 0.3 ± 0.07 0 0 8.3
5.2 ± 0.6 3.6 ± 0.8 1.4 ± 0.3 1 ± 0.1 0.3 ± 0.01 11.4
4.9 ± 0.9 2.1 ± 0.2 0.4 ± 0.01 0.3 ± 0.02 0 7.7
12 ± 0.2 4.5 ± 0.1 1.3 ± 0.3 0.5 ± 0.08 0.4 ± 0.02 18.8
6 ± 0.5 2 ± 0.16 0.5 ± 0.01 0.3 ± 0.07 0 8.5
7.5 ± 0.6 4.1 ± 0.2 1.8 ± 0.6 0.5 ± 0.03 0.6 ± 0.07 14.5
E1 E2 E3 E4 E5
365 ± 11.5 130 ± 8 43 ± 1.6 13 ± 0.7 7.2 ± 0.4 557.7
138 ± 2.1 141 ± 11 68 ± 5 30 ± 5.5 11.4 ± 0.9 388.1
462 ± 7.4 237 ± 19.5 48.4 ± 1.8 16.7 ± 0.5 6 ± 0.2 770.3
226 ± 17 131 ± 10 44.5 ± 3.5 24.6 ± 2.8 8.6 ± 0.86 434
514 ± 18 169 ± 15 54.5 ± 3.5 12.5 ± 0.6 5 ± 0.3 755.3
E1 E2 E3 E4 E5
606 ± 18 206 ± 14 55 ± 2 17 ± 0.8 8.8 ± 0.5
285 ± 43 248.5 ± 20 108 ± 10 44 ± 1 14 ± 1.5 700
669 ± 7.8 339 ± 25 84 ± 3 32 ± 1 12 ± 0.5
377 ± 35 247 ± 22 89 ± 0.8 56 ± 0.8 21 ± 2.4 789
750 ± 20 256 ± 22 92 ± 5.7 21 ± 1 8 ± 0.5
358 ± 14 236 ± 22 77 ± 1.5 44 ± 7 19 ± 3 734
E1 E2 E3 E4 E5
81 ± 2.6 29 ± 1.6 11 ± 2.7 4.7 ± 0.2 3.6 ± 0.1 128.8
33 ± 4.5 31.5 ± 2 14 ± 0.4 6 ± 0.7 2.8 ± 0.3 87.3
102 ± 5.5 48 ± 3.5 11 ± 0.5 4.5 ± 0.2 2.3 ± 0.07 168.2
54 ± 3 30.5 ± 1.8 10 ± 0.5 6 ± 0.5 2.6 ± 0.1 103.3
107 ± 8.8 40 ± 3.7 14.6 ± 1 4.7 ± 0.3 2.7 ± 0.8 169
48 ± 1.4 29.3 ± 1.5 9.5 ± 0.9 5.5 ± 1 3.1 ± 0.5 95.3
E1 E2 E3 E4 E5
354 ± 11 172 ± 6 176 ± 2.5 60 ± 6.5 48 ± 0.8
69 ± 2.3 20.4 ± 0.3 11 ± 0.6 4.5 ± 0.4 5.5 ± 0.1 111
342 ± 27 154 ± 9 47 ± 1.2 15 ± 0.5 4.4 ± 0.6
42 ± 3.3 24 ± 0.3 7.8 ± 0.01 11.3 ± 1.1 0.8 ± 0.2 86
336 ± 32 273 ± 21 63 ± 7 19 ± 5.6 4 ± 0.4
28 ± 3.8 23.7 ± 2.8 8.3 ± 1 4.3 ± 0.1 2.4 ± 0.5 66.4
E1 E2 E3 E4 E5
662 ± 20 279 ± 11 224 ± 33 65 ± 8 46 ± 2.6 1276
69 ± 5.3 62 ± 1.3 32 ± 1.8 15.3 ± 0.8 13.2 ± 0.2 191.6
490 ± 32 242 ± 15 80 ± 2 27 ± 1 8 ± 0.2 846.8
103 ± 1.7 60 ± 1.3 20.1 ± 1 20.4 ± 1.9 2 ± 0.3 206.2
490 ± 41 268 ± 12 99.5 ± 7.3 28 ± 0.8 8 ± 0.2 894
70.3 ± 1.4 64 ± 1.1 22 ± 0.5 13 ± 0.2 6.5 ± 0.7 175.1
E1 E2 E3 E4 E5
59 ± 1.8 15.5 ± 0.7 6 ± 0.3 4 ± 0.4 3.4 ± 0.1 87.2
8.8 ± 0.3 12 ± 1 4.6 ± 0.1 2.4 ± 0.1 1.6 ± 0.1 29.3
57.4 ± 0.6 27 ± 1.8 7 ± 0.3 3.2 ± 0.3 1.6 ± 0.2 96
55 ± 0.4 22 ± 1.3 7.3 ± 0.4 4 ± 0.6 2.3 ± 0.5 90.7
20 ± 0.2 14 ± 0.4 5.4 ± 0.2 3.6 ± 0.6 2.2 ± 0.3 44.3
Total genistin Glin
Total glycitin MDin
Total malonyl-Daidzin MGin
Total malonyl-Genistin MGly
Total malonyl-Glycitin
SPI
41.5 ± 2.8 24 ± 2.6 4 .2 ± 0.3 2.3 ± 0.04 0.9 ± 0.05 72.6
Total daidzin Gin
SM
21 ± 0.4 18.7 ± 0.8 6.6 ± 1.8 1.9 ± 0.06 0.7 ± 0.1 48.5
Total glycitein Din
SPI
62 ± 2.2 13 ± 1.4 3 ± 0.2 0.7 ± 0.2 0.4 ± 0.04 78.8
Total genistein Glein
EtOH
SM
E1 E2 E3 E4 E5
Total daidzein Gein
MeOH SPI
23 ± 3 14 ± 0.6 5.2 ± 0.03 2.4 ± 0.2 1.4 ± 0.08 45.6
206 ± 9 125 ± 9 39 ± 6 21 ± 3 9±1 399.9
Total isoflavone (E1) P Total isoflavone ( 5E)
2235 ± 125 662 ± 12 2191 ± 110 892 ± 11 2334 ± 152 806 ± 9 3890 ± 275 1631 ± 28 3702 ± 311 1811 ± 27 3893 ± 374 1664 ± 24 P E1 to E5: Refers to the number of extractions; 5E: Sum of five extractions; ACN: Acetonitrile; MeOH: Methanol; EtOH: Ethanol; Dein: Daidzein; Gein: Genistein; Glein: Glycitein; Din: Daizin; Gin: Genistin; Glin: Glycitin; MDin: Malonyl daidzin; MGin: Malonyl Genistin; MGly: Malonyl glycitin.
clearly show that sonication for 15 min extracted as much isoflavones as the total of the five sequential extractions (with ordinary shaking for a total of
10 h). Prolonging the time of sonication to 30 and 60 min, however, did not increase the total amount of isoflavone extracted. Sonication may, therefore, provide a
A. Achouri et al. / Food Research International 38 (2005) 1199–1204
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solvent systems, 80% ACN–HCl–H2O, 80% methanol– HCl–H2O and 80% ethanol–HCl–H2O, Wang, Kuan, Octave, Ware, and Carman (1990) and Wang, Ma, Pagadala, Sherrard, and Krishnan (1998), reported no differences in extraction efficiencies. Extraction with acetonitrile–HCl was, however, preferred because this system resulted in fast settling of suspending sample particles and less interfering impurities in the extract. 3.2. Quantitation of aglycones and glycosides extractability
Fig. 3. Effects of sonication and shaking (five sequential extractions) on the total isoflavone content from soy meal (SM) and soy protein P isolate (SPI) using three different solvents. ( 5E: represent the sum of five extractions.)
means of speeding the extraction and analysis of isoflavones. 3.1.3. Effect of solvent The analysis of variance showed no significant (p 6 0.05) differences between the three extraction solvents used. The total concentration of isoflavones after the fifth extraction, yielded 3890, 3702 and 3856 lg/g for SM, and 1631, 1811 and 1664 lg/g for SPI, using acetonitrile–HCl, methanol and ethanol, respectively. Sonication for 15 min yielded 3273, 3678 and 3770 lg/g sample in SM, and 1130, 1817 and 1755 lg/g sample in SPI, using the same solvents, respectively. Closer examination of the results, however, showed that the extraction efficiency of ACN–HCl was significantly (p 6 0.05) higher than the other two solvents in extracting the malonyl-glycosides from SM sample. The presence of hydrochloric acid in acetonitrile may have enhanced the extraction of this particular isoflavone form. In her early work, Murphy (1981) reported that addition of water and/or hydrochloric acid to organic solvents greatly improved the extraction efficiency; and that 80% ACN was superior to other extraction solvents (Murphy, Barua, & Hauck, 2002). Using the same three
SPI contained much lower concentrations of isoflavones (both aglycones and glycosides isomers) compared to SM, irrespective of the number of extractions and the type of solvent used (Fig. 4). This significant (p 6 0.05) difference in the content of isoflavones is likely due to losses of isoflavones during processing of soy protein isolate. This processing effect has been reported by several authors (Eldridge, 1982a, 1982b; Murphy, 1982; Wang & Murphy, 1994a, 1994b, 1996). Differences in the distribution of the different isoflavone forms were also observed in the two samples (Table 2). SM contained the same amount of b-glycosides and malonyl-glycosides equalling about 50% of total isoflavones. SPI contained predominantly the b-glycosides
Fig. 4. Soy meal (SM) and soy protein isolate (SPI) total concentration of aglycone and glucoside isoflavones from extraction 1 (E1), and P sum of five extractions ( 5E), using three different solvents.
Table 2 Proportion of different isoflavone forms from soy meal (SM) and soy protein isolate (SPI) samples Solvent
Aglycones (%/total isoflavonesa) SM
SPI
SM
SPI
SM
SPI
ACN–HCl MeOH EtOH
3.54 ± 0.1 3.32 ± 0.02 3.26 ± 0.05
7.64 ± 0.13 8.12 ± 0.23 8.97 ± 0.53
40.6 ± 0.9 56.0 ± 0.2 53.0 ± 0.6
72.00 ± 0.66 73.25 ± 0.38 73.85 ± 0.56
55.86 ± 0.37 40.67 ± 0.22 44.1 ± 0.49
20.33 ± 0.21 18.64 ± 0.13 17.17 ± 0.43
b-Glycosides (%/total isoflavonesa)
ACN–HCl: Acetonitrile–hydrochloric acid; MeOH: Methanol; EtOH: Ethanol. a Sum of five extractions.
Malonyl-glycosides (%/total isoflavones a)
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equalling 75% of total isoflavones. The higher percentages of the aglycone and b-glycoside isoflavones in the SPI sample may be attributable to hydrolysis of the glycosides during processing (Murphy, 1982). For the two samples studied, the proportion of the different isoflavone forms remained the same irrespective of the extraction technique (sonication or shaking).
4. Conclusion A single step extraction of isoflavones markedly under-estimates the true concentration of isoflavones in a food product. The food matrix, specifically the concentration of protein, may have an impact on the extractability of the isoflavones. For quantitation purposes, care must be taken to use the best extraction system for the food matrix under examination. The poor extractability of isoflavones with one extraction compared to the total yield of five sequential extractions in high protein samples, such as SPI, may be due to stronger protein–polyphenol interactions. Although, the evidence in the literature suggests that the biological effects of soy isoflavones depend upon the aglycone form, the recent trend has been to quantify all type of derivative consumed, as these may have differing biological activities. The results presented in this paper could prove helpful in selecting analytical conditions for the extraction of isoflavones from different food matrices.
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