Title: Comment on extraction and purification of isoflavones from soybeans and characterization of their estrogenic activities Author
Submitted to: Journal of Agricultural and Food Chemistry
Publication Type: Other
Publication Acceptance Date: February 22, 2008
Publication Date: March 20, 2008
Citation: Shappell, N.W. 2008. Comment on extraction and purification of isoflavones from soybeans and characterization of their estrogenic activities. Journal of Agricultural and Food Chemistry 56:3427. Interpretive Summary: Letter to Editor - no interpretive summary
Technical Abstract: The recently published paper by Zhang et al. (1) describes a method for extraction and quantitation of isoflavones from soybean flour, in addition to comparing estrogenic activity using the E-Screen (3). While the paper was well written in many respects including literature review and method development, certain omissions leave the reader with more questions than should be reasonably expected. A critical omission is found under Method Validation of HPLC Analysis (p 6943). The reader is referred to the Supporting Information, Figure 2, but at the time of this writing (10/5/07, original placed on Web 7/21/07) there is no Supporting Information for this article on line. Because the whole premise of this paper is method validation for quantitation, it is critical to have access to calibration curves. While Table 1 does report regression equations and R2 for these curves, AS, LODs, and LOQs for each aglycone isoflavone, the concentration of standards included is unknown. The reference to “Supporting Information Fig.2” implies at least a Figure 1, and potentially more than two figures. In Figure 5, chromatograms are presented of isoflavone product before optimization (A) and after (B). The extraction process “before optimization” is not defined. Does this refer to product extracted with 100% ethanol, stirring for 2 h at 37 degrees followed by centrifugation? If so, then it could be assumed that increased absorbance units obtained “after optimization”, is a reflection predominantly to the hydrolytic processing of glycones to aglycones, rather than an enhanced extraction efficiency. In the discussion of optimization an improvement in yield of isoflavones is cited, without “change in the product composition”. While the authors go to great length in the introduction to discuss differences in concentrations, bioavailability, and relative estrogenic activity of isoflavones in the glycosylated or aglycosylated state, they fail to quantitate just what % change in yield is due to hydrolysis (with conversion to an aglycone) versus extraction efficiency. This could have been addressed by subjecting one set of samples to hydrolysis and one not. In the discussion the authors state “In this study, >85% of genistin and daidzin was degraded by hydrolysis, whereas the concentrations of genistein and daidzein were increased about 40-fold (details not shown).” The lack of data makes it impossible to assess what % of isoflavones in the unextracted sample was glycosylated. In the evaluation of “Our Extracts” the authors seem to draw make implications that are not supported by the data presented. For instance, “according to the HPLC chromatogram (Figure 5b), the composition of our product was relatively simple.” Compositional complexity of a sample would not be evaluated from such a limited HPLC gradient (30 to 50% methanol, monitoring absorbance at 255nm) to claim “only minor impurities detected in this product.” Taken together, the implication is that there are no major “impurities” or other compounds in this product. Such implications are worrisome, as these products are intended for human consumption. In the discussion of “Our product” to 4 other commercial products tested, all data presented in Figures 5 and 6 are apparently from a single extraction of their product. The authors base their claims of superior estrogenic activity over 3 of the four other supplements tested, to the “MTT assay (results not shown)”. One large paragraph goes on to generally describe the results - with no data given - yet P values are included. In conclusion, while the paper provided a nice description of a method, it falls short in providing much needed information about the composition of isoflavones found in commercial products and the relative merits of the new extraction procedure.