dual
pump and System 32 Karat HPLC processing software (Beckman, Fullerton,
CA). Isoflavone forms were separated and quantified by reversed-phase HPLC
on a C18 column (Hypersil 5 mm ODS 200 ¥ 2.1 mm i.d., Agilent Technologies,
Palo Alto, CA). The extraction supernatant (20 mL) was injected, and
elution was achieved by a gradient from 95% of buffer A (88:10:2 ratio of
H2O : methanol : 10% [v/v]; acetic acid) to 100% of buffer B (98:2 ratio of
methanol : 10% [v/v]; acetic acid) in 40 min. The solvent flow rate was
0.4 mL/min. Isoflavone stock solutions were prepared by dissolving known
concentrations of pure standards in HPLC-grade methanol. Standard curves
were obtained by plotting the standard concentration as a function of peak
areas integrated by the Beckman System Gold software. The samples were run
with two replications. The results were adjusted for molecular weight of the
correspondent aglycon (Song et al. 1998) and were expressed as microgram of
aglycon isoflavone equivalent per gram of protein (dry basis).