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Research and Development Division, Kikkoman Corporation, 399 Noda, Noda City, Chiba, 278-0037, Japan and * Noda Institute for Scientific Research, 399 Noda, Noda City, Chiba, 278-0037, Japan
1To whom correspondence should be addressed.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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KEY WORDS: • isoflavone aglycone • fermented soy • antioxidant • atherosclerosis • rabbits
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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, Cassidy and Griffin 1999
). Monkeys fed an atherogenic diet containing isolated soy
protein with the isoflavone glucosides [isoflavone glucosides intake,
9.41 mg/(kg body · d)] had significantly lower plasma LDL and VLDL
cholesterol levels, and higher HDL cholesterol levels compared with
those fed an atherogenic diet containing isolated soy protein with the
isoflavones removed [isoflavone glucosides intake, 0.97 mg/(kg body
· d)], and the coronary artery atherosclerotic lesions were smaller
in the soy protein–containing isoflavone group (Anthony et al. 1997
). Feeding high fat, soy protein–based diets containing
isoflavones to C57BL/6 mice resulted in lower plasma cholesterol
concentrations and smaller atherosclerotic lesion areas compared with
mice fed the same diets without the isoflavones (Kirk et al. 1998
). Therefore, these reports have suggested that isoflavones
could be responsible in part for the hypolipidemic and
antiatherosclerotic activities of soy. However, when isoflavone
aglycone–rich soy extract was added to casein, the serum lipid profile
in ovariectomized monkeys was not altered (Greaves et al. 1999
), nor did dietary supplementations with isoflavone
aglycones from soy or subterranean clover (Trifolium
subterraneum) affect serum lipids in normolipidemic humans
(Hodgson et al. 1998
, Nestel et al. 1997
).
Soy isoflavones have been shown in vitro to exert effects that may
prevent the development of atherosclerosis and subsequent coronary
heart disease. The isoflavone aglycones, genistein and daidzein, have
scavenging activity (Ruiz-Larrea et al. 1997
) and
antioxidant properties (Arora et al. 1998
,
Lehtonen et al. 1996
, Ruiz-Larrea et al. 1997
). Isoflavones display antioxidative activities in both
aqueous (Ruiz-Larrea et al. 1997
) and lipophilic phases
(Arora et al. 1998
, Lehtonen et al. 1996
)
and can inhibit lipoprotein oxidation in serum (Hodgson et al. 1996
) and LDL oxidation in vitro (de Whalley et al. 1990
, Kanazawa et al. 1995
, Kapiotis et al. 1997
). The daidzein metabolites, equol and
O-desmethylangolesin
(O-DMA),3
were more potent inhibitors of lipoprotein oxidation than daidzein or
genistein in serum (Hodgson et al. 1996
). Therefore,
dietary isoflavones may reduce atherosclerosis through their
antioxidant activity. However, the antiatherogenic effects of
isoflavones have not been demonstrated in vivo.
In this study, we investigated the effect of an isoflavone aglycone–rich extract free of any soy protein, prepared from fermented soy, on experimental atherogenesis in rabbits.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Daidzein (>99%) and genistein (>99%) were purchased from Sigma Chemical (St. Louis, MO). Glycitein (>95%) and equol (98%) were purchased from Fujicco (Kobe, Japan) and Funakoshi (Tokyo, Japan), respectively. Other chemicals were of analytical or HPLC grade.
Isoflavone aglycone–rich extract and soy saponin–rich extract.
The isoflavone aglycone–rich extract (SoyActTM) was provided by
Kikkoman Corporation (Noda City, Chiba, Japan). The extract was
produced by the fermentation of soy, followed by ethanol/water
extraction and purification. Soy saponin-rich extract was prepared
from the isoflavone aglycone–rich extract, followed by ethanol/ethyl
acetate extraction. For analysis of isoflavones in these extracts, 1 mg
of the extract was mixed with 2.5 mL dimethyl sulfoxide, and the solution was sonicated with an ultrasonicator (Branson, Model
B1210J-DTH, Tokyo, Japan). Concentrations of isoflavones (genistein,
daidzein and glycitein) in the solution were analyzed by HPLC with a
CAPCELLPAK C18 AG 120 column (4.6 x 150 mm, Shiseido, Tokyo,
Japan). A mixture of acetonitrile/water/trifluoroacetic acid
(25:75:0.1, v/v/v) was used as the mobile phase at a flow rate of 1.0
mL/min. Isoflavones in the eluate were detected by their absorbance at
254 nm. The concentration was calculated from the standard curve of the
authentic compound. For determination of main soy saponins, those
extracts were heated at 90°C for 1 h in 2 mol/L NaOH and
analyzed on a HPLC system with ODSAQ AQ-312 column (6.0 x 150 mm,
YMC, Kyoto, Japan) (Ireland and Dziedzic 1985
). Protein
content was determined by the Kjeldahl method. Sugar content was
determined by the method of Somogyi (1952)
. Fat was
extracted using chloroform:methanol (2:1, v/v) and weighed. Moisture
was determined as the loss in weight after drying at 105°C for
24 h. Ash content was determined by the direct ignition method
(550°C overnight). Fiber was determined by the method of
Prosky et al. (1984)
. The isoflavone aglycone–rich
extract was subjected to SDS-PAGE on a 12.5% gel by the method of
Laemmli (1970)
; the gel was stained with Coomassie
Brilliant Blue R-250.
Animals and diets.
Cholesterol-fed New Zealand white rabbits were used because of their
extensive utilization in research on antiatherogenic agents. New
Zealand white male rabbits (n = 31; 12-wk-old;
Nippon Bio-Supply, Tokyo, Japan) were divided into five groups.
They were individually housed in metal cages in an air-conditioned
room (23 ± 1°C, 55 ± 5%, humidity). Powdered
diets were prepared every 8 wk and stored at 4°C. Throughout the
experiment period, the rabbits were given restricted amounts (90 g/d)
of each diet, because the extent of atherosclerosis depended on
cholesterol intake (Bocan et al. 1993
). Rabbits had free
access to tap water. A control group of seven rabbits was fed a
standard nonpurified diet (RM-4, Funabashi Farm, Chiba, Japan), which
was composed of 142 g/kg protein as alfalfa meal flour, wheat bran,
barley, casein, corn grain and meat meal flour, 508 g/kg carbohydrate,
120 g/kg fiber, 119 g/kg moisture, 79 g/kg ash and 32 g/kg fat. The
seven rabbits in the cholesterol group were fed the standard diet
containing 1 g/100 g cholesterol. The seven rabbits in the low
isoflavone group were fed the standard diet containing a 0.78 g/100 g
isoflavone aglycone–rich extract plus 1 g/100 g cholesterol, and the
seven rabbits in the high isoflavone group were fed the standard diet
containing a 2.33 g/100 g isoflavone aglycone–rich extract plus 1
g/100 g cholesterol. Three rabbits in the soy saponin group were fed
the standard diet containing a 1.09 g/100 g saponin-rich extract
plus 1 g/100 g cholesterol. Food intake was monitored by measuring the
food remaining each day, and body weight was determined every 2 wk.
Isoflavone aglycone–rich extract intakes, isoflavone aglycone
(genistein plus daidzein plus glycitein) intakes and saponin intake
were calculated from food intakes and body weights. Blood samples were
drawn from the marginal ear veins at 6 and 8 wk and centrifuged at 1000
x g for 10 min. at 4°C to obtain serum or
plasma. The blood was collected at 6 wk into tubes coated with EDTA or
sodium heparin, and centrifuged to obtain plasma. At the end of 8 wk,
the rabbits were killed with an overdose of sodium phenobarbital.
During the experiment, the rabbits received humane care consistent with
institutional guidelines.
Biochemical analyses.
Serum total cholesterol and triglyceride were measured enzymatically
(Richmond 1973
, Tamaoku et al. 1982
). The
density of serum was adjusted with KBr solution, and lipoproteins
(VLDL, LDL, HDL and HDL3) were fractionated by
ultracentrifugation at 223,000 x g for 4 h
(Havel et al. 1955
). HDL2 cholesterol was
calculated by the equation (total HDL cholesterol - HDL3 cholesterol). Serum lipoperoxide were measured as
thiobarbituric acid-reactive substances (TBARS) by fluorometric
assay (Yagi 1976
).
LDL were isolated from fresh plasma in the density range of
1.019–1.063 kg/L by short-run ultracentrifugation
(Kleinveld et al. 1992
). LDL were dialyzed for 20 h
at 4°C against PBS (pH 7.4). The protein concentration of LDL was
determined by the method of Bradford (1976)
.
CuSO4 was added to both plasma and the LDL suspension. The
final concentrations of CuSO4 in reaction mixtures were 200
µmol/L in plasma and 5 µmol/L in the
LDL suspension. The reaction mixtures were incubated at 37°C for
4 h and cholesteryl ester hydroperoxide (ChE-OOH) was measured by
HPLC using a TSK gel Octyl-80 Ts column (4.6 x 100 mm; TOSOH,
Tokyo, Japan). The ChE-OOH concentrations were calculated from the
standard curve of the hydroperoxy derivative of cholesteryl linoleate
(Arai et al. 1996
).
Serum concentrations of isoflavones (genistein and daidzein) and their
metabolites (p-ethyl phenol and equol) at 6 wk were
measured by a method described in detail elsewhere (Piskula and Terao 1998
, Piskula et al. 1999
). Briefly,
nonconjugated (free) isoflavones were extracted from serum with
methanol/acetic acid (95:5, v/v), centrifuged at 5000 x g for 10 min at 4°C and after dilution, the
supernatant was analyzed by HPLC with a TSK gel ODS-80TS column (4.6
x 150 mm, TOSOH, Japan). The flow of the mobile phase composed of
water/methanol/acetic acid (58:40:2, v/v/v) containing 50 mmol/L
lithium acetate was set at 0.9 mL/min. The elute was monitored with an
amperometric electrochemical detector (ICA-3060, TOA, Tokyo, Japan)
with the working potential set at +950 mA. For determination of the
serum total concentration of isoflavones (conjugated and free), serum
was subjected to enzymatic hydrolysis with H-5 sulfatase containing 25
units of sulfatase and 500 units of ß-glucuronidase (Sigma Chemical).
Conjugated isoflavones (glucuronide and sulfate conjugates) were
calculated by the equation (total isoflavone - free isoflavone).
The authentic standards of genistein, daidzein, p-ethyl
phenol and equol methanolic solution were used as references.
Portions of the aortic arch and thoracic aorta were homogenized in
chloroform/methanol (2:1, v/v) and analyzed for total cholesterol. The
remaining aorta samples were added to 10 volumes (wt/v) of cold 0.1
mol/L PBS (pH 7.0) and homogenized. The homogenates were analyzed for
malondialdehyde (MDA) (Esterbauer and Cheeseman 1990
).
Aorta preparation and staining.
The aortic arch and thoracic aorta were fixed in 10% buffered formalin
solution and stained with Sudan IV to reveal sudanophilic plaques
(Holman et al. 1958
). The surface area of the
atherosclerotic lesions was measured with an Olympus SP500F image
analyzer (Olympus Optical, Tokyo, Japan) and expressed as a percentage
of the total surface area of the aortic intima.
Immunohistochemistry.
The aortic arch was fixed in 4% buffered paraformaldehyde solution.
Serial sections of the aortic arch were stained with monoclonal
antibody RAM11 (a rabbit macrophage-specific antibody: DAKO,
Copenhagen, Denmark), HHF35 (a muscle actin-specific antibody:
DAKO), and FOH1a/DLH3 (oxidized LDL antibody) (Itabe et al. 1994
) by the streptavidin-peroxidase conjugate method
(Shi et al. 1988
). The stained sections of the aortic
arch of all rabbits were examined by microscope.
Statistical analysis.
Statistical analysis was evaluated by one-way ANOVA followed by Tukey’s significant difference test to identify significantly different means; SPSS for Windows software, Release 8.0 (SPSS, Chicago, IL) was used. Log transformations of raw data were done in cases of unequal variances. Values in the text are means ± SD.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The isoflavone aglycone–rich extract was composed of 429.4 mg/g isoflavones as the aglycone form (192.0 mg/g genistein, 211.2 mg/g daidzein and 26.2 mg/g glycitein), 112.3 mg/g saponin, 132.4 mg/g protein, 126.0 mg/g sugar, 50.0 mg/g fat, 29.1 mg/g moisture, 20.4 mg/g ash and 1 mg/g fiber. The protein component of the extract was composed of amino acids and peptides, which had molecular weights < 10,000 because no clear bands were detected by SDS-PAGE of the extract. The extract did not contain vitamin E. The saponin-rich extract was composed of 239.8 mg/g soy saponin, 24.2 mg/g isoflavones, 265.3 mg/g protein, 282.1 mg/g sugar, 10.9 mg/g fat, 32.8 mg/g moisture, 53.5 mg/g ash.
Food intake, growth, isoflavone and soy saponin intakes.
There were no significant differences in food intake among the groups (data not shown). Body weight at end of the 8-wk feeding period was 2.6 ± 0.3 kg. The isoflavone aglycone–rich extract intakes were 272 ± 14 mg/(kg body · d) in the low isoflavone group and 754 ± 66 mg/(kg body · d) in the high isoflavone group. Isoflavone aglycone and soy saponin intakes were 116 ± 6 and 31 ± 2 mg/(kg body · d) in the low isoflavone group, and 324 ± 29 and 85 ± 7 mg/(kg body · d) in the high isoflavone group, respectively. In the soy saponin group, isoflavone aglycone and saponin intakes were 10 ± 2 and 121 ± 23 mg/(kg body · d), respectively.
Serum lipid and TBARS concentrations.
The serum lipid profile (total cholesterol, LDL-cholesterol, VLDL
cholesterol, HDL-cholesterol, etc.) and TBARS levels in the
isoflavone groups did not differ from the cholesterol group
(Table 1
). In the saponin group, the serum lipid profile and TBARS level also
did not differ from the cholesterol group (Table 1)
.
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In the sera of rabbits fed the isoflavone aglycone–rich extract, most of the isoflavones and their metabolites were present in conjugated forms, and a small amount in the free form. The serum concentration of daidzein in the conjugated form was significantly higher in the high isoflavone group (10.15 ± 7.75 µmol/L) than in the low isoflavone group (1.75 ± 0.96 µmol/L) (P < 0.05, statistical analysis was performed on log-transformed data.). Serum concentration of genistein in the conjugated form tended to be higher in the high isoflavone group than in the low isoflavone group (P = 0.085). Serum concentrations of p-ethyl phenol and equol in the conjugated forms in the high isoflavone group did not differ from those in the low isoflavone group. Neither isoflavones nor their metabolites were detectable in sera from control and cholesterol group rabbits.
Copper-mediated plasma or LDL oxidation.
The plasma ChE-OOH level in the high isoflavone group was
significantly lower than in the cholesterol group (Table 2
). ChE-OOH levels of LDL were significantly lower in the low and
high isoflavone groups than in the cholesterol group (Table 2)
.
ChE-OOH levels of plasma and LDL in the saponin group did not
differ from the cholesterol group (Table 2)
.
|
Cholesterol concentration in the aortic arch was significantly lower in
the high isoflavone group than in the cholesterol group (Table 3
). The MDA concentration in the aortic arch was significantly lower in
the low and high isoflavone groups than in the cholesterol group (Table 3)
. Cholesterol and MDA concentrations in the aortic arch of the soy
saponin–fed group did not differ from the cholesterol group (Table 3)
.
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The extent of atherosclerosis in the aortic arch was
significantly reduced in the low and high isoflavone groups by 26.3
(P < 0.05) and 36.9% (P < 0.01),
respectively, and also in the thoracic aorta by 41.6 and 57.3%
(P < 0.01), respectively, compared with those of the
cholesterol group (Table 4
). The lesion area of the aortic arch in the saponin group did not
differ from the cholesterol group (Table 4)
.
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Oxidized LDL were detected in the cytoplasm of the
macrophage-derived foam cells (RAM 11-positive cells) in the
atherosclerotic lesions by immunohistochemical analysis (Fig. 1
). The number of oxidized LDL-positive macrophage-derived foam cells
in atherosclerotic lesions in the aortic arch was lower in both
isoflavone groups compared with the cholesterol group (Fig. 1)
. No
smooth muscle cells (HHF 35-positive cells) were observed in the
atherosclerotic lesions in the cholesterol-fed groups.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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), estrogenic (Shutt and Cox 1972
),
antiosteoporotic (Anderson and Garner 1997
) and
anticarcinogenic activities (Herman et al. 1995
). Soy
isoflavone aglycones reduced the development of mammary carcinoma
(Lamartiniere et al. 1995
), carcinoma in prostate and
seminal vesicles (Onozawa et al. 1999
) and skin tumors
(Wei et al. 1998
), but had no effect on colon
carcinogenesis (Davies et al. 1999
) or intestinal
neoplasia in ApcMin mouse (Sorensen et al. 1998
). On the other hand, soy isoflavone aglycones might
enhance colon carcinogenesis in azoxymethane-treated rats
(Rao et al. 1997
). In vitro, soy isoflavones aglycones
inhibited the oxidative modification of LDL by macrophages
(Kapiotis et al. 1997
), enhanced the resistance of LDL
to oxidation (de Whalley et al. 1990
, Kanazawa et al. 1995
) and exhibited antioxidative activities in both the
aqueous (Ruiz-Larrea et al. 1997
) and lipophilic phases
(Arora et al. 1998
, Lehtonen et al. 1996
), which may prevent the development of atherosclerosis and
subsequent coronary heart disease. However, the antiatherogenic effect
of isoflavones without soy protein has not been demonstrated in vivo.
In this study, we showed that an isoflavone aglycone–rich extract from
fermented soy inhibited progression of atherosclerosis in
cholesterol-fed rabbits, without affecting serum cholesterol
levels. A reduction in atherosclerotic lesions in the aorta was
associated with increasing doses of the isoflavone aglycone–rich
extract in the diet (Table 4)
. The isoflavone aglycone–rich extract
contained small amounts of soy saponin, amino acids and peptides. We
examined the antiatherosclerotic effect of the soy saponins and found
that atherosclerotic lesion areas were not different in the saponin and
cholesterol groups. These results suggest the antiatherosclerotic
activity of the isoflavone aglycone–rich extract is due mainly to
isoflavones.
In our study, the standard diet contained 6.44 g/100 g alfalfa meal.
Hanson et al. (1973)
reported that the saponin
concentration in alfalfa was 1.78–3.59 g/100 g; thus our standard diet
contained a maximum of 0.231 g/100 g saponin from alfalfa.
Malinow et al. (1980)
reported that plasma cholesterol
and atherosclerotic lesions were lower in rabbits fed a diet containing
1.0–1.2 g/100 g alfalfa saponin plus 0.1 g/100 g cholesterol than in
rabbits fed a diet containing 0.1 g/100 g cholesterol alone. However,
the rabbits in our study fed the standard diet containing 1 g/100 g
cholesterol developed severe atherosclerosis. These results suggested
that the small amount of saponin from alfalfa in the diet did not
influence atherosclerosis development in the cholesterol-fed
rabbits in our study.
Feeding an isoflavone aglycone–rich extract to rabbits decreased
ChE-OOH levels induced by CuSO4 in both
plasma and LDL (Table 2)
. The concentrations of serum isoflavones
increased after the extract feeding. The isoflavone aglycone–rich
extract also decreased the aortic MDA concentration in rabbits (Table 3)
. In our immunohistochemical study, the number of foam cells
originating from macrophages that had contained oxidized LDL decreased
more in the atherosclerotic lesions of rabbits fed the isoflavone
aglycone–rich extract plus cholesterol than in the lesions of rabbits
fed cholesterol alone. These results suggest that isoflavones and/or
their antioxidative metabolites inhibit oxidation of LDL, thereby
exerting an antiatherosclerotic effect.
Subendothelial accumulation of foam cells, derived primarily from
monocytes and macrophages by uptake of oxidized LDL, plays a key role
in the initiation of atherosclerosis (Holvoet and Collen 1998
). In this and other recent studies, we showed that aortic
MDA concentration and the number of oxidized LDL-positive
macrophage-derived foam cells in atherosclerotic lesions increased in
the aortae of rabbits fed cholesterol (Yamakoshi et al. 1999
). These results suggest that lipid peroxidation is an
important step in atherogenesis in cholesterol-fed rabbits.
Isoflavone aglycone–rich extract feeding decreased ChE-OOH levels
in both plasma and LDL (Table 2)
. In vitro, soy isoflavones and their
metabolic products inhibited lipoprotein oxidation in serum
(Hodgson et al. 1996
). In vivo, most of the main
isoflavoids of soy, genistein and daidzein, and the genistein
metabolite, p-ethyl phenol, and the daidzein metabolites,
equol and O-DMA, are conjugated in the liver and released
into the circulation. Glucuronide or sulfate conjugates may associate
with the LDL surface layer. LDL isolated from subjects after the intake
of soy-containing isoflavones were less susceptible to oxidation
than LDL isolated from individuals consuming a soy-free diet.
However, only minute amounts of unmodified isoflavone, corresponding to
approximately one isoflavone molecule/500 LDL molecules, were found in
LDL (Tikkanen et al. 1998
). Some reports have indicated
that endogenous human estrogens can be converted to lipid-soluble
esters in human tissues and become incorporated into LDL
(Hochberg et al. 1991
, Larner et al. 1993
, Shwaery et al. 1997
). Meng et al. (1999)
reported that some isoflavone oleic acid esters
increased the oxidation resistance of LDL after their incorporation in
vitro. In our study, some lipid-soluble isoflavones such as
esterified isoflavones might also have been incorporated into LDL,
thereby inhibiting LDL oxidation. However, there is currently no
evidence that isoflavones are converted to such lipid metabolites in
vivo.
In this study, the isoflavone aglycone–rich extract did not affect the
serum lipid profile of cholesterol-fed rabbits. The isoflavone
aglycone–rich soy extract added to casein did not alter the serum
lipid profile in ovariectomized monkeys (Greaves et al. 1999
), nor did dietary supplementation with isoflavone
aglycones from soy or subterranean clover change serum lipid
concentrations in normolipidemic humans (Hodgson et al. 1998
, Nestel et al. 1997
). Taken together, these
results suggest that isoflavones without soy protein did not alter the
serum lipid profile. It has been suggested that isoflavones could be
responsible in part for the hypocholesterolemic effect of soy protein
(Anderson et al. 1995
). Greaves et al. (1999)
reported that other components of soy protein, either
alone or in combination with isoflavones, might be involved in this
effect.
Genistein inhibited the migration and proliferation of smooth muscle
cells in vitro (Fujio et al. 1993
, Shimokado et al. 1994
and 1995
), and might also suppress thrombus formation
by inhibiting platelet activation (Kuruvilla et al. 1993
, Murphy et al.1993
) and aggregation
(Asahi et al. 1992
, McNicol 1993
), and
reducing platelet serotonin uptake in vitro (Helmeste and Tang 1995
). The migration and proliferation of smooth muscle cells
and thrombus formation are important steps in the atherosclerotic
process. In this study, neither smooth muscle cells nor thrombus was
observed in the atherosclerotic lesions of cholesterol-fed groups.
In this study, we showed that the serum concentration of daidzein in
the conjugated form differed significantly according to isoflavone
level, but serum concentration of genistein in the conjugated form did
not differed significantly, although it tended (P = 0.085) to increase in serum with increasing isoflavone level in the
diet. The plasma concentration of daidzein in the conjugated form
tended to be slightly higher than that of genistein in the
conjugated form when daidzein and genistein were administered orally to
rats at a dose of 7.9 µmol/kg body weight (Piskula et al. 1999
). Daidzein may be absorbed more than genistein in
rabbits as well. Further studies are required to elucidate the
absorption and excretion of isoflavones in rabbits.
We did not measure the concentration of O-DMA, which is one
of the daidzein metabolites, in serum. Although O-DMA, along
with equol, was a more potent inhibitor of in vitro lipoprotein
oxidation in serum than the two major dietary isoflavones, genistein
and daidzein (Hodgson et al. 1996
), O-DMA
production from daidzein was minor compared with equol in humans
(Setchell and Aldercreutz 1988
).
In conclusion, an isoflavone aglycone–rich extract inhibited progression of atherosclerosis in cholesterol-fed rabbits. These results suggest that the antioxidative action of isoflavones and their antioxidative metabolites inhibit the oxidation of LDL, thereby exerting an antiatherosclerotic effect.
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ACKNOWLEDGMENTS |
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|
FOOTNOTES |
|---|
3 Abbreviations used: ChE-OOH, cholesteryl ester hydroperoxide; MDA, malondialdehyde; O-DMA, O-desmethylangolesin; TBARS, thiobarbituric acid-reactive substances.
Manuscript received September 17, 1999. Initial review completed October 29, 1999. Revision accepted February 14, 2000.
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