Extraction isoflavones

Table 11.6.2 Solvent Mixture for Extracting Isoflavones from Soy Foods"...

Soybeans and soybean products contain high levels of protein, carbohydrates, and lipids (Table 11.6.1). As minor components of complex mixtures, isoflavones must first be separated from the bulk of the matrix constituents prior to analysis. Efficient extraction methods for isoflavones should account for their diverse structures, chemical properties, and the food matrix of which they are constituents. This unit describes a practical way of extracting isoflavones from soybean products in their natural forms using readily available solvents and laboratory equipment. 

The solvents used for extracting isoflavones from soy foods were chosen according to the solubility of isoflavones and the food matrix involved. The diversity of soy isoflavones in polarity requires the use of a combination of organic solvent and water for extraction. The organic-to-water ratio (10 5) was established based on Murphy s study on solvent selection (Murphy et al., 2002). Water content in the solvent needs to be adjusted according to the moisture content in soy foods. Freeze-drying of samples before extraction simplifies the extraction process, but is not a prerequisite. 

Acetonitrile, acetone, ethanol, and methanol have been used to extract isoflavones from soy foods. Among them, acetonitrile proved to be the most efficient (Griffith et al., 2001 Murphy et al., 2002). The solvent is supplemented with 0.1 M HC1 to completely un ionize the isoflavones and to release them from protein complexes by denaturing and precipitating the proteins. Room temperature is recommended for extraction to avoid alteration of the natural forms of the isoflavones. The time for extraction, 2 hr, was chosen for maximum recovery and shortest processing time. Ultrasound is used to aid the extraction process by degrading and weakening the cellular matrix. 

Besides solvent extraction, supercritical carbon dioxide has been applied to extract isoflavones (Rostagno et al., 2002). Due to the hydrophobicity of carbon dioxide, supercritical fluid extraction is more suitable for extracting nonpolar aglycones than polar glycosides of isoflavones, and may not be quantitative. 

When resuspending isoflavones in pure organic solvent, extra care should be taken to completely dissolve extracted isoflavones in the solvent. Solublization can be achieved by ultrasonification and providing sufficient time for the isoflavones to dissolve. Vortexing helps to release and recover isoflavones from precipitated proteins that may stick to the wall of glass vials and containers. All solvents must be of a high degree of purity. All extracted samples should be filtered to remove small particles. 

Isoflavones are responsible for the slightly bitter flavor of soy isolate. The flavor of soy isolate can be improved by extracting soy flakes with an azeotropic mixture of hexane and alcohol (Eldridge et al., 1971). After extraction, the isolate has a bland taste. Acetonitrile containing HC1 and water is more effective than methanol for extracting isoflavones from soybean products (Murphy, 1981). Isoflavones can be hydrolyzed by acid hydrolysis and followed by thin-layer chromatography (TLC) and GC analysis. GC analysis is performed on silylated isoflavones (Naim et al., 1974). 

Heat treatment involved in the chemical analysis of isoflavones may affect their structure. Kudou et al. (1991) separated the soybean into three fractions (seed coat, hypocotyl and the cotyledons) and extracted isoflavones using 70% aqueous ethanol at room temperature for 24 h or at 80°C for 15 h. Total isoflavone concentration in hypocotyl was 5.5 to 6 times higher than that in the cotyledon. Glycitin and its derivatives occurred only in the hypocotyl fraction. Most of the malonyl isoflavones were converted to respective glucosides after extraction at 80°C for 15 h. 

Extracted isoflavones may be consumed as dietary supplements, and are marketed for perimenopausal women and postmenopausal women (PMW) looking for an alternative to hormone replacement therapy. Many of these supplements contain isoflavones from red elover rather than soybeans, and are supplied in daily doses of 50-200 mg/d (Setch-ell et al 2001 Howes and Howes 2002 Nurmi et al 2002 Tice et al 2003 Chua et al 2004 Delmonte and Rader 2006), although actual content upon analysis often differs to that stated on the label (Setehell et al 2001 Howes and Howes 2002 Nurmi et al. 2002 Tice et al. 2003 Chua et al 2004 Krenn and Potsch 2006). 

KNIGHT D c, HOWES J B, EDEN J A (1999) The effect of Promensil , an isoflavone extract, on menopausal symptoms. Climacteric. 2 79-84. 

UPMALIS D H, LOBO R, BRADLEY L, WARREN M, CONE F L, LAMIA C A (2000) VaSOmotor symptom relief by soy isoflavone extract tablets in postmenopausal women a multicenter, double-blind, randomized, placebo-controlled study. Menopause. 1 236-42. 

Table 6.3 Effect of soy protein/extracts containing isoflavones on OVX-induced bone...

Picherit et al., 2001 7 month old OVX adult rats 3 doses of isoflavone extract (20, 40, 80 mg/kg body wt/d) for 91 d Isoflavones reversed femoral failure load, total femoral, diphyseal or metaphyseal BMDs and normalized bone turnover markers induced by OVX. Optimal dose without uterotrophic effect was 40 mg/kg body wt... 

Similar to the human studies, the animal studies are not entirely consistent, due to the different study designs (source and dose of soy protein/isoflavones time, method and length of administration age of rats, etc.). Nevertheless, a certain number of conclusions may be drawn. Overall, soy extracts or pure isoflavones show an osteoprotective effect in the ovariectomized rat model of menopausal bone loss. The time of administration is important and they must be given at the time of ovariectomy which allows prevention but not reversal of bone loss. Although the OVX-induced bone loss in the rat is a... 

In order to produce soy protein, soybeans are first dehulled, flaked, and defatted to make white flakes . Soy protein concentrates are obtained by removing a portion of the carbohydrates from defatted and dehulled soybeans. Alcohol extraction is the method most commonly used to manufacture soy protein concentrates even though it results in the loss of isoflavones. Soy protein concentrates retain most of the fiber in the original soybean and must contain at least 65% protein on a moisture-free basis to meet quality standards. The most concentrated source of soy protein is soy protein isolates (or isolated soy protein, ISP), which is required to be at least 90% protein on a moisture-free basis. It is heat-treated during processing to insure inactivation of trypsin inhibitors. Most isolated soy protein is manufactured by water extraction from defatted and dehulled soybeans and it retains the natural isoflavones. 

Isoflavones have been implicated in goiter induction. Soybean extracts inhibit reactions catalyzed by thyroid peroxidase (TPO), essential to the synthesis of thyroid hormones (Divi et al., 1997). Genistein and daidzein (at about 1-10 p,M of IC50) may act as alternative substrates for tyrosine iodination (Divi et al., 1997). Furthermore, genistein and daidzein have also been shown to cause the irreversible inactivation of TPO in the presence of hydrogen peroxide. Genistein also inhibits thyroxine synthesis in the presence of iodinated... 

Klejdus B, Vitamvasova D and Kuban V. 2001. Identification of isoflavone conjugates in red clover (Trifolium pratense) by liquid chromatography-mass spectrometry after two-dimensional solid-phase extraction. Anal Chim Acta 450 81-97. 

A similar RP-HPLC method has been developed for the determination of isoflavones in soybean powder. Ground soybean powder (100 g) was extracted four times with 300 ml of hexane, then the defatted powder was extracted with six solvents or solvent mixtures such as acetone, methanol-water (4 1, v/v), methanol-ethyl acetate-acetone (1 1 1, v/v), acetone-0.1 M HC1 (5 1, v/v), methanol-water (4 1, v/v) plus 0.1 M HC1 (5 1, v/v), and methanol-ethyl acetate-acetone (1 1 1, v/v) plus 0.1 M HC1 (5 1, v/v). An aliquot of 0.5 g defatted or non-defatted soybean powder was shaken for 2h or sonificated for lh with 5 ml... 

Z. Kuklenyik, X. Ye, J.A. Reich, L.L. Needham and A.M. Calafat, Automated online and off-line solid-phase extraction methods for measuring isoflavones and lignans in urine. J. Chromatogr. Sci. 42 (2004) 495-500. 

One could argue that efficacy may be improved by extracting and concentrating in tablets the compound considered to be the active principal of soy. This should also, at least theoretically, improve compliance. Western women, in general, do not find the traditional Japanese diet easy to follow, hence, isolated isoflavones in tablet form have been used. 

Numerous questions concerning the effects of phytoestrogen supplements for women remain unanswered. Further studies should be performed to clarify the effects of isoflavone extracts, their optimal doses, the significance of individual variation in phytoestrogen metabolism and long-term effects, particularly on men. Risk, benefits, and interactions between phytoestrogens and drugs also need to be carefully evaluated. 

Clifton-Bligh, P.B., Barber, R.J., Fulcher, G.R., Nery, M.L. and Moreton, T. (2001). The effect of isoflavones extracted from red clover (rimostril) on lipid and bone metabolism. Menopause, 8, 259-265. 

Greaves, K.A., Parks, J.S., Williams, J.K. and Wagner, J.D. (1999). Intact dietary soy protein, but not adding an isoflavone-rich soy extract to casein, improves plasma lipids in ovariectomized cunomologus monkeys, J. Nutr., 129, 1585-1592. 

Tice, J.A., Ettinger, B., Ensrud, K., Wallace, E.R., Blackwell, T. and Cummings, R.S. (2003). Phytoestrogen supplements for the treatment of hot flushes The Isoflavone Clover Extract (ICE) Study A randomized controlled trial, JAMA, 290, 207-214. 

Four pure isoflavones were obtained from a crude soybean extract after CPC with the solvent system CHCl3-Me0H-H20 (4 3 2) (Figure 1.2). The isoflavones were isolated in amounts of 5 to 10 mg after the introduction of 150 mg of sample. 

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