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Prolonged Intake of Isoflavone- and Saponin- Containing Soybean Extract (Nijiru) Supplement Enhances Circulating - Carboxylated Osteocalcin Concentrations in Healthy Individuals

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The effect of nijiru, which is a by-product of the processing of soybeans to make the fermented soybeans called natto, on circulating blood chemistry levels re- lated to calcium and bone metabolism in healthy indi- viduals was investigated. Twelve volunteers (six men and six women) were received nijiru twice a day for 60 days at a dose of 1500 mg (6 tablets) per day. The serum ?-carboxylated osteocalcin concentration was significantly increased by the intake of nijiru in both men and women to about 2-fold that in the control group. The serum calcium concentration was signifi- cantly decreased by nijiru supplementation in women, and the serum inorganic phosphorus concentration was significantly reduced in both men and women. However, the intake of nijiru did not have a significant effect on serum glucose, nitrogen urea, albumin, free cholesterol, triglyceride, high-density lipoprotein cho- lesterol, and ?-glutamyltranspeptidase concentrations in men or women, indicating that liver and renal func- tion is not affected by nijiru supplementation. The re- sults of the present study suggest that the intake of isoflavone- and saponin-containing nijiru can stimu- late the ?-carboxylation of osteocalsin, which plays an important role in bone formation and mineralization, in healthy individuals.
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Journal of Health Science, 47(6) 579–582 (2001)
579
Prolonged Intake of
Key words —–— genistein, saponin, ?-carboxylated
osteocalcin, bone metabolism, osteoporosis
Isoflavone- and Saponin-
Containing Soybean Extract
(Nijiru) Supplement

INTRODUCTION
Enhances Circulating ?-
It is known that bone mass decreases in both men
Carboxylated Osteocalcin
and women with increasing age. The decrease in
Concentrations in Healthy
bone mass is due to increased bone resorption and
to decreased bone formation. Osteoporosis with a
Individuals
decrease in bone mass is widely recognized as a
major public health problem. The most dramatic
expression of this disease is represented by fractures
Masayoshi Yamaguchi,*, a Rie Ono,a, b
of the proximal femur, of which the incidence in-
and Zhong Jie Maa
creases as the population ages.1,2) A deficiency in both
aLaboratory of Endocrinology and Molecular Metabolism,
micronutrients and macronutrients appears to be
Graduate School of Nutritional Sciences, University of
strongly implicated in the pathogenesis and conse-
Shizuoka, 52–1 Yada, Shizuoka 422–8526, Japan and bDepart-
quences of hip fracture in the osteoporotic elderly.3)
ment of Research and Development, Marumiya K.K., 2211
Nutritional factors may prevent bone loss with in-
Uchida, Kikusui-machi, Tamana-gun, Kumamoto 865–0104,
creasing age, although this has not been fully clari-
Japan
fied.
(Received July 24, 2001; Accepted August 9, 2001)
Daidzein and genistein, natural isoflavonoid
phytoestrogens found in Leguminosae, have an ana-
The effect of nijiru, which is a by-product of the
bolic effect on bone metabolism in rats.4–6)
processing of soybeans to make the fermented soybeans
Isoflavones including daidzin, daidzein, genistin, and
called natto, on circulating blood chemistry levels re-
genistein are present in soybeans high concentra-
lated to calcium and bone metabolism in healthy indi-
viduals was investigated. Twelve volunteers (six men

tions. Daidzin and genistin are hydrolyzed to
and six women) were received nijiru twice a day for
daidzein and genistein, respectively, by ?-glucosi-
60 days at a dose of 1500 mg (6 tablets) per day. The
dase in the gastrointestinal tract. Genistein and
serum ?-carboxylated osteocalcin concentration was
daidzein have been demonstrated to stimulate bone
significantly increased by the intake of nijiru in both
formation and to inhibit bone resorption, thereby
men and women to about 2-fold that in the control
increasing bone mass.7–13)
group. The serum calcium concentration was signifi-
Nijiru is produced in the processing of the fer-
cantly decreased by nijiru supplementation in women,
mented soybean preparation natto, and contains sig-
and the serum inorganic phosphorus concentration
nificant quantities of isoflavone and saponin. The
was significantly reduced in both men and women.
intake of isoflavone- and saponin-containing nijiru
However, the intake of nijiru did not have a significant
has been found to have an anabolic effect on bone
effect on serum glucose, nitrogen urea, albumin, free
components in rats, suggesting its role in the pre-
cholesterol, triglyceride, high-density lipoprotein cho-
vention of osteoporosis.14) Soybean saponin has been
lesterol, and ?-glutamyltranspeptidase concentrations
demonstrated to have an anabolic effect on bone
in men or women, indicating that liver and renal func-
component in vitro and in vivo.15) Moreover, the pro-
tion is not affected by nijiru supplementation. The re-
longed intake of natto supplemented with nijiru con-
sults of the present study suggest that the intake of
taining isoflavone and saponin has a preventive ef-
isoflavone- and saponin-containing nijiru can stimu-
late the
?-carboxylation of osteocalsin, which plays an
fect on ovariectomy-induced bone loss, suggesting
important role in bone formation and mineralization,
that it may have a role in the prevention of osteoporo-
in healthy individuals.
sis.16) Thus soybean-based foods may be useful for
the prevention of osteoporosis with increasing age.
The present study was undertaken to determine
*To whom correspondence should be addressed: Laboratory of
the effect of supplemental nijiru intake on bone
Endocrinology and Molecular Metabolism, Graduate School of
Nutritional Sciences, University of Shizuoka, 52–1 Yada,
metabolic marker in healthy individuals over the long
Shizuoka 422–8526, Japan. Tel. & Fax: +81-54-264-5580; E-
term.
mail: yamaguch@u-shizuoka-ken.ac.jp

580
Vol. 47 (2001)
MATERIALS AND METHODS
Table 1. Change in Serum Calcium and Inorganic Phosphorus
Concentrations Following Intake of Iso?avone- and
Materials —–— Soybeans were boiled under 1.5 at-
Saponin-Containing Soybean Extract in Healthy In-
dividuals
mospheric pressure for 40 min at 160°C, and the
nijiru produced was then freeze-dried. The
Serum level
Control
At day 60
isoflavone content was measured after removal from
(baseline)
the nijiru powder by extraction with 80% hot etha-
Calcium (mg/dl)
nol solution. This ethanol solution was filtered, and
All
9.00±0.09
8.61±0.21
the filtrate was subjected to reverse-phase high-per-
Men
9.18±0.12
9.02±0.29
Women
8.81±0.08
8.28±0.25*
formance liquid chromato-graphy. The isoflavone
Inorganic phosphorus (mg/dl)
concentration was expressed as micrograms per gram
All
3.36±0.12
4.02±0.16***
of nijiru powder. The calcium content in the nijiru
Men
3.12±0.15
3.89±0.27**
powder was measured by atomic absorption spec-
Women
3.60±0.15
4.22±0.07**
trophotometry after digestion with HNO . The
3
Each value is the mean ± S.E.M. of 12 (both men and women)
composistion of saponin, daidzin, daidzein, genistin,
or 6 (men or women) individuals. *p < 0.05, **p < 0.025, and
genistein, and calcium in nijiru prepared from soy-
***p < 0.01 compared with the baseline value.
beans was 320, 1800, 12, 1600, 15, and 820 µg/g
dry powder, respectively. One tablet contained
250 mg of nijiru dry powder.
Table 2.
Serum Metabolic Findings Following Intake of
Iso?avone- and Saponin-Containing Soybean Extract
Experimental Procedures —–— Twelve adults aged
in Healthy Individuals
17–58 years (six men and six women), who were
judged to be healthy with no abnormal liver or kid-
Serum level
Control
At day 60
(baseline)
ney function as assayed by standard biochemical
Glucose (mg/dl)
100.3±4.00
91.7±4.67
data, were enrolled as volunteers in this study. In-
Nitrogen urea (mg/dl)
14.1±0.96
13.4±1.08
formed consent was obtained from all. The intake
Albumin (g/dl)
5.1±0.11
5.1±0.08
of other foods with an abundance of vitamin K,
Free cholesterol (mg/dl)
49.0±4.61
44.8±2.23
which stimulates production of ?-carboxylated
Triglyceride (mg/dl)
56.9±5.68
67.2±8.06
osteocalcin, was prohibited during the study. All
HDL cholesterol (mg/dl)
55.3±3.36
59.1±3.30
volunteers were 1500 mg (6 tablets) of nijiru pow-
?-GTP (IU/l)
32.9±4.81
29.7±5.06
der twice a day for 60 days. Blood samples were
Each value is the mean ± S.E.M. of 12 healthy volunteers.
collected at 10:00 on the day prior to the beginning
of administration and 60 days thereafter. Serum ?-
carboxylated osteocalcin, calcium, and inorganic
RESULTS
phosphorus levels and other biochemical levels were
measured using routine methods.
The changes in serum calcium and inorganic
Analytical Procedures —–— Serum samples were
phosphorus concentrations in healthy individuals
obtained by centrifugation (2500 rpm for 5 min)
before and after administration of nijiru are shown
between 20 and 40 min after blood collection, and
in Table 1. The serum calcium concentration was
then stored at –20°C until assayed. The serum ?-car-
significantly decreased with the intake of nijiru tab-
boxylated osteocalcin concentration was assayed
lets in women, although not seen in men. The serum
using a Gla-type Osteocalcin (Gla-OC) EIA Kit
inorganic phosphorus concentration was signifi-
(Takara Shuzou, Shiga, Japan).17) Serum calcium and
cantly reduced in both men and women at 60 days
inorganic phosphorus concentrations were deter-
compared with baseline.
mined using KIT (Wako Junyaku, Osaka, Japan).
The effect of nijiru tablet intake on serum meta-
Serum glucose, nitrogen urea, albumin, free-choles-
bolic findings is shown in Table 2. There was no sig-
terol, triglyceride, high-density lipoprotein (HDL)
nificant alteration in glucose, nitrogen urea, albu-
cholesterol, and ?-GTP were assayed using KIT.
min, free-cholesterol, triglyceride, HDL cholesterol,
Statistical Analysis —–— Differences in values be-
and ?-GTP levels in either men or women.
fore and after the intake of nijiru tablets were esti-
The changes in serum ?-carboxylated osteocalcin
mated using Student’s t-test. p values of less than
concentration after 60 days of nijiru intake are shown
0.05 were considered statistically significant.
in Fig. 1. Serum ?-carboxylated osteocalcin in-
creased significantly in individuals of both sexes.

No. 6
581
mineralization in healthy individuals.
The intake of nijiru supplement caused a sig-
nificant decrease in serum calcium and inorganic
phosphorus concentrations in healthy volunteers.
This decrease may be partly involved in bone min-
eralization , which is related to an increase in ?-car-
boxylated osteocalcin following the intake of nijiru.
Serum findings associated with liver and kid-
ney functions were not significantly altered by the
intake of nijiru supplement in healthy individuals.
This indicates that prolonged intake of nijiru does
Fig. 1. Changes in Serum ?-Carboxylated Osteocalcin
not influence metabolic functions of the liver and
Concentrations Following Intake of Isoflavone- and
kidney in such individuals. In addition, the body
Saponin-Containing Soybean Extract
weight of the healthy volunteers did not change sig-
Each value is the mean ± S.E.M. of 12 (men and women) or 6 (men
nificantly with prolonged intake of nijiru (data not
or women) individuals. *p < 0.01 compared with the baseline value.
White bars, control; black bars, after nijiru intake.
shown). From these observations, it is assumed that
nijiru is a safety and healthy dietary supplement.
Nutrition may have a role in the prevention of
DISCUSSION
bone loss with increasing age. Numerous elements
in food have been shown to have an anabolic effect
Nutritional factors may be significant in prevent-
on bone metabolism.21,22) The isoflavone and sapo-
ing bone loss with increasing age. Soybean
nin in nijiru may be related to an anabolic effect an
isoflavone has been shown to have an anabolic ef-
bone mineralization in healthy individuals. The in-
fect on bone metabolism in rats, suggesting its role
take of nijiru as a supplement may thus have a role
in prevention of osteoporosis.4–13) Nijiru has been
in the prevention of age-related bone loss.
demonstrated to have a stimulatory effect on bone
formation and mineralization in normal rats and an
inhibitory effect on bone loss in ovariectomized
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