(594b) 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