Interconversions of isoflavones in soybean..

Isoflavones as affected by storage of soybeans . . .
Food Chemistry and Toxicology
tin, glycitin, daidzein, genistein, and glycitein were purchased
from INDOFINE Chemical Company (Somerville, N.J., U.S.A.).
Three acetylglucosides of isoflavones, acetyldaidzin, acetylgenistin,
and acetylglycitin, were purchased from LC Laboratories
(Woburn, Mass., U.S.A.).
Storage of soybeans
Soybeans were stored under 4 conditions, including adverse
conditions (84% relative humidity, 30 °C), mild conditions (57%
relative humidity, 20 °C), cold conditions (4 °C in walk-in cooler),
and uncontrolled ambient conditions (in a garage). Soybeans in
the conditions of uncontrolled ambient garage, cold condition,
and 57% RH, 20 °C were stored for up to 18 mo and sampled at 3
mo intervals. Soybeans in 84% RH, 30 °C were stored for up to 9
mo and sampled at 1 mo intervals. For the uncontrolled ambient
condition, soybeans were packed in a paper bag and placed in a
garage without temperature control. For cold conditions, soybeans
were packed in a paper bag and put into a plastic bag (air
and water impermeable type), then stored in a refrigerator (3 to
4 °C). For temperature and humidity controlled conditions, soybeans
(120 g) were sealed in a can (size 303 mm ´ 406 mm) which
contained 40 mL of saturated salt solution in a cup with a lid on
which many holes were punched, and stored in a temperaturecontrolled
incubator. The saturated sodium bromide solution
was used for 57% RH and 20 °C, and the saturated potassium
chloride solution was used for 84% RH and 30 °C (Rockland
1960). Soybeans sealed in a plastic bag and stored in –20 °C
freezer was served used as the control. At the end of the storage
interval, soybeans were placed in a –20 °C freezer until analysis.
Preparation of internal standard
The internal standard, THB, was synthesized according to procedures
described in Murphy and others (1997). In a flask containing
a mixture of 4-hydroxyphenylacetic acid (2 g) and boron trifluoride
etherate (4.5 mL), resorcinol (2.9 g) was added. This mixture
was stirred and heated in boiling water bath on a hot plate to refluxed
for 10 min. After cooling to room temperature, 30 mL of saturated
sodium acetate was slowly added to the flask, then 15 mL
of saturated sodium bicarbonate were added drop by drop. The
orange-yellow precipitate was filtered and washed with distilled
water and then chloroform, and dried at room temperature under
hood. The orange crystals were redissolved in 100% ethanol and
purified through a Sephadex LH 20 chromatography column with
50% ethanol as the eluent. A single peak as determined by UV detection
at 262 nm was collected and freeze-dried. The THB purity
was more than 99% as checked by HPLC and there was no contamination
peak at the retention time of genistein.
Table 1—Moisture content of soybeans stored in 3 conditions
for up to 18 mo 1
57% RH Cold Ambient
Month 20 °C 4 °C Condition Condition
0 5.40 ± 0.01 b 5.40 ± 0.01 e 5.40 ± 0.01 c
3 9.37 ± 0.03 a 8.18 ± 0.05 d 8.82 ± 0.01 b
6 9.65 ± 0.44 a 8.99 ± 0.05 c 8.94 ± 0.05 b
9 9.36 ± 0.06 a 9.38 ± 0.02 b 9.00 ± 0.06 b
12 9.74 ± 0.06 a 9.81 ± 0.00 a 9.40 ± 0.06 a
18 9.79 ± 0.02 a 9.41 ± 0.11 b 8.87 ± 0.17 b
1 Data were expressed as means ± sd of triplicate and in the same column
with different superscripts are statistically different at P £ 0.05.
sample solution was filtered again through a 0.20 mm PTFE (polytetrafluoroethylene)
membrane filter (Nalge Nunc International
Corp., Rochester, N.Y., U.S.A.) and analyzed by HPLC within 10 h
after extraction to minimize the conversion of malonylglucosides
to glucosides (Murphy and others 1999). Each sample was extracted
in duplicate.
Isoflavone analysis
The quantitative analysis of soybean isoflavones was performed
by HPLC according to Murphy and others (1997) with a slight modification.
A Waters Associates (Milford, Mass., U.S.A.) chromatography
system equipped with a Model 720 system controller, Model
6000A solvent delivery system, Model 7125 loading sample injector,
and Model 418 LC spectrophotometer set at 262 nm was used. A
YMC-pack ODS-AM-303 C 18 reversed phase column (5 mm, 250
mm ´ 4.6 mm internal dia) was obtained from Waters and employed
for chromatographic separations at 34 °C, which was maintained
with a column heater. A linear gradient mobile phase consisted of
solvent A (0.1% acetic acid in water) and solvent B (0.1% acetic acid
in acetonitrile). After injection of 10 mL of sample, the system was
eluted with 15% of solvent B for 5 min at the flow rate of 1.0 mL/min,
then increased to 29% in 31 min at the flow rate up to 1.5 mL/min,
and then to 35% in 8 min at the same flow rate of 1.5 mL/min. Then
the gradient increased to 50% of solvent B for 10 min at the flow rate
of 1.5 mL/min prior to recycling back to 15% B at the flow rate of 1.0
mL/min at zero time. Each sample containing standards was separated
and analyzed at least in duplicate.
Quantification of isoflavones
Isoflavone quantification was performed by calculating the
peak area obtained from HPLC analyses. Aglycones, b-gluco-
Extraction of isoflavones
The soybeans were ground with a Tekmar (A-10) Analytic Mill
to pass through a 60-mesh sieve. Moisture content was measured
by a vacuum oven method (AOAC 925.09 1990). Isoflavones
were extracted by modifying the method of Murphy and
others (1997). One mL of internal standard (THB, 1.25 mg/mL)
was added to 1 g of soybean flour in a screw-top centrifuge tube,
and mixed thoroughly for 20 min, then adding 5 mL of acetonitrile,
1 mL of 0.1N HCl, and 3.5 mL of distilled water. The mixture
was stirred for 2 h at room temperature. Then the slurry was centrifuged
at 6000 g for 20 min. The supernatant was filtered
through Whatman No. 42 filter paper into a 125 mL flask and taken
to dryness on a rotary evaporator at < 35 °C. The residues in
the flask were dissolved in 10 mL of 80% methanol and kept in a
freezer (–20 °C) for less than 10 h before analysis. An aliquot of
Figure 1—Moisture of soybeans stored in 84% RH and 30 °C
for 9 mo. Column with different letter is significantly (p <
0.05) different.