Elsevier

Food Chemistry

Volume 105, Issue 1, 2007, Pages 266-272
Food Chemistry

Analytical, Nutritional and Clinical Methods
Supercritical fluid extraction of daidzein and genistein isoflavones from soybean hypocotyl after hydrolysis with endogenous β-glucosidases

https://doi.org/10.1016/j.foodchem.2006.11.019 Get rights and content

Abstract

An optimal condition of supercritical fluid extraction (SFE) for isoflavone aglycones (daidzein and genistein) in soybean hypocotyls previously subjected to thermohydration at pH 5.0 and a temperature of 50 °C for 6, 12 and 18 h was developed. Different temperatures, pressures and cosolvents (methanol, ethanol, and acetonitrile) was tested and compared with solid–liquid extraction using aqueous methanol solution (80% v v−1) conducted in parallel for comparison. The extraction conditions were 50–70 °C, 176–380 bar, adding 0, 5, 10 mol% of cosolvents 80% in water as a modifier. The results from SC–CO2 showed that the cosolvent and pressure have significant effects in the extraction efficiency. It was found that the extraction conditions promoting the highest extraction of daidzein and genistein were at the temperature of 60 °C, pressure of 380 bar and both static and dynamic extraction of 15 min with the addition of 10% acetonitrile (80% v v−1). The maximum amounts of daidzein and genistein extracted by each method were solid–liquid extraction (70.07 mg 100 g−1) and carbon dioxide–acetonitrile (17.97 mg 100 g−1). The yield of daidzein and genistein achieved by a 30 min SC–CO2 extraction on soybean hypocotyls after 12 h soaking time was markedly improved by the addition of a modifier (acetonitrile) to the CO2 fluid. HPLC analysis of the obtained extracts revealed that extraction of isoflavone aglycones by SC–CO2 was 4.78 and 13.19 mg 100 g−1 for daidzein and genistein, respectively. The contents of daidzein and genistein obtained in the solid–liquid extraction were superior to 86% and 63%, respectively, compared to supercritical extraction.

Introduction

Daidzein and genistein, the major isoflavones in soybeans, are associated with a broad variety of beneficial properties on human health and are found in high concentrations in soybean hypocotyl (10 times the concentration in soybeans) (Liu, 1999, Zhu et al., 2005). After consumption, primary isoflavonoids are metabolized in the gut and transformed into active aglycones, some of which are absorbed as free isoflavones (Arjmandi and Smith, 2002, Fritz et al., 2003, Morel et al., 2003, Wei et al., 2002, Wuttke et al., 2003, Yamaguchi, 2002).

From the structure of the isoflavone glucosides and their aglycones we presume that the isoflavone glucoside is hydrolyzed by the β-glucosidase. Matsuura et al., 1989, Matsuura and Obata, 1993, Pandjaitan et al., 2000 reported that β-glucosidase was related to the production of daidzein and genistein during the soaking of soybeans.

The traditional method for the extraction of plant materials include steam distillation and organic solvent extraction using percolation, maceration or Soxhlet techniques (Liggins et al., 2000, Simonne et al., 2000, Tura and Robards, 2002). These procedures however they are have distinct drawbacks such as time-consuming and labour-intensive operations, handling of large volumes of hazardous solvents and extended concentration steps which can result in the loss or degradation of target analytes. Moreover, there is an increasing interest for alternative extraction technologies consuming less organic solvents, because of the rising solvent acquisition and disposal cost and regulatory restriction.

Supercritical fluid extraction (SFE) offers several advantages over conventional solvent extraction methods. SFE can penetrate into the pores of solid materials more effectively than techniques based upon liquid solvents, so it enables a much faster mass transfer, resulting in faster extractions. For instance, the extraction time can be reduced from hours or days for a liquid–solid extraction to a few minutes for SFE, with comparable or better recoveries. Also, in SFE, fresh fluid is continuously pumped through the samples, so it can provide quantitative or complete extraction, and the solvation power of the fluid can be manipulated by changing pressure and/or temperature, facilitating a remarkable high selectivity (Lang and Wai, 2001, Smith, 1999, Tura and Robards, 2002). The most important advantage of utilizing SC–CO2 is the easy separation of the solvent from the extracted material and it operates at an ambient temperature that does not affect the heat sensitive compounds. Further, SC–CO2 provides lower mass transfer resistance than do those in conventional separation process. These advantages have attracted an increasing interest from researchers, especially from the food, pharmacy and environmental-engineering industries.

However, due to the limited solubility of polar organic compounds in SC–CO2 or to their interaction with the matrix, quantitative extraction of these compounds with pure SC–CO2 is not possible. The addition of a polar modifier (e.g. methanol) to SC–CO2 is the simplest and the most effective way to obtain the desired polarity of CO2-based fluids. Modifiers can also overcome interactions between the analyte and the matrix, increasing the extraction efficiency of polar organic compounds (Lang & Wai, 2001).

Several researchers successfully applied SFE to extract similar compounds from different matrices, like phenolic compounds from olive leaves (Le Floch, Tena, Rios, & Valcarel, 1998), flavonoids from Scutellaria radix (Lin, Tsai, & Wen, 1999), from Ginkgo biloba (Liu, Zhao, Wang, & Yang, 1999) and from Chamomile flowers (Scalia, Guiffreda, & Pallado, 1999), polyphenols from grape seeds (Palma, Taylor, Zoecklein, & Douglas, 2000), isoflavones from soybean flour (Rostagno, Araújo, & Sandi, 2002) and isoflavone from soybean products (Chandra & Nair, 1996).

In the present study, SFE was used to extract isoflavone aglycones (daidzein and genistein) from soybean hypocotyls after enzymatic hydrolysis of the glucosidic isoflavones by endogenous β-glucosidases, and the extracts were analyzed by HPLC. Extraction conditions were adjusted in order to obtain the highest yield of daidzein and genistein, and the influence of the extraction conditions of the method was examined. The results obtained was compared with the results obtained using solid–liquid extraction.

Section snippets

Materials

SFE grade carbon dioxide (99.99% pure) supplied in a cylinder with a dip tube was purchased from White Martins (Brazil); genistin, genistein, daidzin, daidzein, flavone and p-nitrophenyl-β-d-glucoside (p-NPG) were obtained from Sigma Chemical Co. (USA). Acetronitrile, methanol, dimethyl sulfoxide (DMSO), trifluoroacetic (TFA) (Vetec Química-Brazil) were of analytical grade (99%). The solvents used were filtered through a 0.45 μm nylon membrane filter prior to utilization.

Sample preparation

The hypocotyls of the

Hydrolysis of isoflavone in soybean hypocotyl by endogenous β-glucosidases

The effect of soaking time on enzyme activity was investigated by using p-NPG at 50 °C and pH 5.0, incubated for 18 h (Fig. 2). The quantities of daidzein and genistein increased with soaking time up to 12 h. Thus, isoflavone glucosides hydrolysis was time dependent. At 12 h incubation time, the percent hydrolysis was 60% for daidzin and 56% for genistin. Matsuura and Obata (1993) found 50% hydrolysis for daidzin and 51% for genistin. In this experiment, daidzin was more easily hydrolyzed than

Conclusions

These results indicate that extraction efficiencies achieved by solid–liquid extraction are higher than those attained by SFE. However, the higher complexity of the chromatographic patterns produced by solid–liquid extraction indicates that SFE affords enhanced extraction selectivity compared to the classical techniques. The SFE extraction with the addition of 10% acetonitrile after 12 h soaking time of the soybean hypocotyls resulted in 13.87% and 37% recoveries of active aglycones daidzein and

Acknowledgements

This work was supported by CAPES (Coordenação de Aperfeiçoamento de Pessoal de Nível Superior) and FAPEMIG (Fundação de Amparo à Pesquisa do Estado de Minas Gerais.

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