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Re-evaluation of analysis procedures for phytoestrogen compounds in soy products

T. Wong, F. Sherkat and D.M. Small

School of Applied Sciences, RMIT University, Melbourne VIC 3000, Australia

Introduction

Phtoestrogens are present at low levels in various foods, most notably in soy based foods. Analysis is an important issue and is of particular significance to studies of the physiological roles of these compounds. Sample extraction procedures typically involve sonication of a freeze-dried sample in a mixture of strong acid and ethanol prior to reflux for 1 hours. However there is a lack of the uniformity of extraction methods in the current literature (Barnes et al, 1994; Griffith et al, 2001). For subsequent quantitation, HPLC has been widely used conferring advantages of both speed and simplicity of analysis (Penalvo et al, 2004). The main objective of this study has been to investigate and optimize the extraction and analysis conditions as a basis for further research on various soy products.

Materials and methods

Materials

The extraction solvents hydrochloric acid and 96% ethanol solution were supplied by Ajax Chemicals Co. The analyte enzymatic soy flour was provided by the Oppenheimer company.

HPLC equipment

Reversed-phase HPLC procedures used a Waters 600i pump coupled with Shimadzu UV-vis spectrophotometer with a Shimadzu 3395 integrator. A Nova-Pak C18 column (150 3.9mm I.D.; 4μm) was selected for isoflavone analysis due to its reported efficiency, and optimum recovery of the analytes (Pettersson and Kiessling, 1984).

Chromatographic conditions

The mobile system was 33% of 1% acetic acid in water (solvent A) and 67% of acetonitrile (solvent B). The flow rate was set on 0.8mL/min with 20μL injection into the HPLC system with isocratic elution and monitored at 280nm (Hutabarat, 2000).

Results and discussion

The two isoflavones which represent a large proportion of the total in soy flour are daidzein and genistein. For this work a sample of soy flour was selected for investigation. When differing extraction periods for measurement of isoflavone contents were used the results showed varying recoveries (Figure 1). The preliminary results here indicated that the longer period of reflux showed decreasing amounts both with and without sonication. This indicated the need to study a wider range of reflux times.

The results of further investigations into the reflux period are presented in Figures 2 and 3. The apparent isoflavone content reached a maximum value at 1 hours (Figure 2) for both daidzein and genistein. The results also showed solid evidence that sonication successfully enhanced the apparent isoflavone content to a certain extent. The results suggested the contribution of 10 minutes sonication facilitated the extraction process by showing the optimum level of isoflavone content at 1 hours. By taking the results for both daidzein and genistein into account, it was it was apparent that the sum value for the two isoflavones was optimal at 2 hours refluxing time (Figures 2 and 3). The results indicate that sonication did not significantly impact on the measurements at this point of refluxing. The longer sonication period (20 minutes) did not enhance the extraction process or optimize quantitation of isoflavones.

Therefore the methodology selected for further use involved 10 minutes sonication and 1 hours refluxing time for the quantitation of isoflavones in the extraction process.

Figure 1. Extraction of soy flour with application of 10 minutes sonicaton
Note: mean and standard deviation are from six separate analyses.
1-daidzein 10min sonication; 2-daidzein no sonication; 3-genistein 10min sonication; 4-genistein no sonication

Figure 2. Extraction of soy flour by varying time of reflux (hrs)
Note: mean and standard deviation are from six separate analyses.
1-daidzein 10min sonication; 2-daidzein no sonication; 3-genistein 10min sonication; 4-genistein no sonication

Figure 3. Extraction of soy flour by varying time of reflux and period of sonication
Note: mean and standard deviation are from six separate analyses.
1-daidzein 10min sonication; 2-daidzein no sonication; 3-genistein 10min sonication; 4-genistein no sonication; 5-daidzein 20min sonication; 6-genistein 20min sonication

Conclusion

For effective extraction of isoflavone compounds from soy flour, sonication for 10 minutes together with one and a half hours reflux achieved optimumal extraction when the extracts contained both daidzein and genistein. These procedures in conjunction with reversed phase HPLC were found to be appropriate and have been successfully applied to studies of a wider range of products and the changes occurring during processing of soy grains into beverage and soy bean curd foods.

Acknowledgements

We are grateful to Renuka Mayadunne for her support with the setting up of the HPLC instrumentation.

References

Barnes, S., Kirk, M., and Coward, L. (1994) J. Agric. Food Chem., 42:2466-2474.

Griffith, P. & Collison, W. (2001) J. Chromatogr. A, 913:397-413.

Hutabarat, S., Greenfield, H. and Mullholland, M. (2000) J. Chromatogr. A, 886:55-63.

Penalvo, L., Nurmi, T. and Adlercreutz, H. (2004) Food Chem., 87:297-305.

Pettersson, H. & Kiessling, K-H. (1984) J. Assoc. Off. Anal. Chem., 67:503-506.

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