An Effective and Green Method for the Extraction and Purification of Aglycone Isoflavones From Soybean

Isoflavones, belonging to phytoestrogen and with significant amounts in soybean [Elizabeth JZ 2007], have obvious effects in the prevention and therapy of chronic diseases such as heart diseases, osteoporosis and cancers (prostrate, breast, thyroid and colon) [Cohen LA 2000]. Especially the aglycone isoflavones (genistein and daidzein) exhibit higher biological activity, so the production of aglycone isoflavones has been attracting more and more attention in recent years. However, the high energy consumption, poor efficiency, the use of toxic solvents are the problems in the extraction and purification of aglycone isoflavones.

In this paper, an effective and green strategy was developed to obtain aglycone isoflavones  from soybean through the extraction, enzymatic hydrolysis and purification steps (Fig A). In the extraction step, a novel ethanol-alkaline extraction method was designed to promote the conversion of conjugated forms (acetyl glycoside isoflavones and malonyl glycoside isoflavones) into their non-conjugate forms (glycoside isoflavones) [Ismail B 2005]. A high extraction yield (94.34%) of isoflavones was achieved at the optimum conditions (pH 9.0, 65% ethanol, 70^oC, solid to liquid ratio (m:v) 1:15, 1h and 400rpm stirring). Then, in the enzymatic hydrolysis step, cellulase (GC-220) instead of traditional b-glucosidase was used to convert the glycoside isoflavones into aglycone isoflavones with an excellent conversion of 94.80%. Finally, the crude aglycone isoflavones were purified by an environmental-friendly procedure comprised of ethanol precipitation, ethyl acetate extraction and water rinse. The final amount of aglycone isoflavones was 1.279mg from every gram of defatted soybean, and the final recovery and purity of aglycone isoflavones calculated based on HPLC (Fig B), could achieve 80.38% and 87.21%, respectively. Additionally, the organic solvent of ethanol and acetic ether used in the whole process could be recycled with the recoveries of 87.60% and 90.00%. The whole extraction, enzymatic hydrolysis and purification process is an environment-friendly, economical and simple process, which will increase the yield of aglycone isoflavones and make the production safe and green.

(1) Elizabeth JZ, Ka MN and Kathy QL. Extraction and purification of isoflavones from soybeans and characterization of their estrogenic activities. J. Agr. Food Chem 55:6940-6950 (2007).

(2) Cohen LA, Zhao Z, Pittman B and Scimeca JA. Effect of intact and isoflavone-depleted soyprotein on NMU-induced rat mammary tumorigenesis. Carcinogenesis 21:929-935 (2000).

(3) Ismail B and Hayes K. b-Glycosidase activity toward different glycosidic forms of isoflavones. J. Agr. Food Chem 53:4918-4924 (2005).

This work was supported by the Program for New Century Excellent Talents in Chinese University (NCET-08-0386; NCET-11-0372), the 863 Program of China (2008AA10Z318), the Natural Science Foundation of China (20976125; 31071509; 51173128) and Tianjin (10JCYBJC05100), and the Program of Introducing Talents of Discipline to Universities of China (No. B06006).

Figure (A) Extraction, hydorlysis and purification of aglycone isoflavones. (B) The HPLC chromatogram of final products