123

Journal of Health Science, 49(2) 123–128 (2003)

Anabolic Effect of Wasabi Leafstalk (Wasabia
japonica MATSUM.) Extract on Bone Components
in the Femoral-Diaphyseal and -Metaphyseal
Tissues of Aged Female Rats in Vitro and in Vivo
Masayoshi Yamaguchi,*, a Zhong Jie Ma,a and Toshihiro SuzukibaLaboratory of Endocrinology and Molecular Metabolism, Graduate School of Nutritional Sciences, University of Shizuoka, 52–1 Yada,
Shizuoka 422–8526, Japan and bFunctional Food Project Team, Shizuoka Industrial Research Institute of Shizuoka Prefecture, 2078
Makigaya, Shizuoka 421–1298, Japan(Received November 20, 2002; Accepted December 2, 2002)
The effect of wasabi leafstalk (Wasabia japonica MATTUM.) extract on bone components in the femoraldiaphyseal and -metaphyseal tissues of aged female rats in vitro and in vivo was investigated. Femoral-diaphyseal
and -metaphyseal tissues were cultured for 48 hr in Dulbecco’s modified Eagle’s medium (serum free) containing
either vehicle or wasabi leafstalk extract (10, 25 or 50 µg/ml of medium). The presence of wasabi leafstalk extract(50 µg/ml) caused a significant increase in calcium content, alkaline phosphatase activity and deoxyribonucleic acid
(DNA) content in the diaphyseal and metaphyseal tissues in vitro. However, the effect of wasabi leafstalk extract(50 µg/ml) in increasing bone components was completely abolished in the presence of cycloheximide (10–6 M), an
inhibitor of protein synthesis. Moreover, rats were orally administered wasabi leafstalk extract (10 or 20 mg/100 gbody weight) once daily for 7 days. The calcium content, alkaline phosphatase activity and DNA content in thefemoral-diaphyseal and -metaphyseal tissues of aged rats was significantly increased by the administration of wasabi
leafstalk extract (10 or 20 mg/100 g) for 7 days in vivo. Meanwhile, body weight, serum calcium and inorganic
phosphorus concentrations of female aged rats were not significantly altered by the administration of wasabi leafstalk extract (10 or 20 mg/100 g) for 7 days. The present study demonstrates that wasabi leafstalk extract has ananabolic effect on bone components in vitro and in vivo. The intake of wasabi leafstalk extract may have a preventive
effect on bone loss with increasing age.
Key words —–— bone metabolism, Wasabia japonica, wasabi leafstalk, osteoporosis, aging, rat femur

INTRODUCTION
Bone loss with increasing age induces osteoporosis. Osteoporosis is widely recognized as a major
public health problem.4) Bone loss may be due toincreased bone resorption and decreased bone formation. A decrease in bone mass leads to bone fracture. The most dramatic expression of osteoporosis
can be countered by factors which help to preventbone loss with increasing age.5)
Recent studies have shown that isoflavones (including genistein and daidzein), which are containedin large quantities in soybean, have a stimulatory
effect on osteoblastic bone formation6–8) and an in1–3)*To whom correspondence should be addressed: Laboratory of
Endocrinology and Molecular Metabolism, Graduate School of
Nutritional Sciences, University of Shizuoka, 52–1 Yada,
Shizuoka 422–8526, Japan. Tel. & Fax: +81-54-264-5580; Email: yamaguch@u-shizuoka-ken.ac.jp

hibitory effect on osteoclastic bone resorption,9–11)
thereby increasing bone mass. Also, menaquinone7, an analogue of vitamin K2 which is essential forthe γ-carboxylation of the osteocalcin of a bone matrix protein, is abundant in fermented soybean. It
stimulates osteoblastic bone formation12) and inhibits osteoclastic bone resorption13) in vitro. Its prolonged dietary preventive effect on bone loss inducedby ovariectomy in rats, which is an animal modelfor osteoporosis, has been demonstrated.14) Thus,
nutritional factors play a role in bone health and may
be important in the prevention of bone loss with increasing age in humans.15–17)
Other food factors have been shown to have ananabolic effect on bone metabolism. More recently,
it has been demonstrated that among various marine algae, Sargassum horneri (S. horneri) has aunique anabolic effect on bone calcification in vitro
and in vivo.18,19) The anabolic effect of S. horneri

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extract on bone components is due to increased boneformation, caused by a newly synthesized protein
component, and also to decreased bone resorption.20,21)
Of various plant extracts, wasabi leafstalk(Wasabia japonica MATSUM.) extract has a uniquestimulatory effect on bone calcification in vitro.22,23)
Whether an anabolic effect of wasabi leafstalk extract on bone components is demonstrated in aged
female rats, in which bone mass is decreased by aging, has remained unknown. The present study was
undertaken to determine the anabolic effect of wasabi
leafstalk extract on bone components in aged female
rats. We found that wasabi leafstalk extract has ananabolic effect on bone components in the femoraldiaphyseal (cortical bone) and -metaphyseal (trabecular bone) tissues of aged female rats in vitro andin vivo.

MATERIALS AND METHODS
Chemicals —–— Dulbecco’s modified Eagle’s medium (MEM) (high glucose, 4.5 g/dl) and a penicillin-streptomycin solution (penicillin 5000 U/mg;
streptomycin 5000 µg/ml) were purchased from
Gibco Laboratories (Grand Islasnd, NY, U.S.A.).
Bovine serum albumin (fraction V) and cycloheximide were purchased from Sigma Chemical Co. (St.
Louis, MO, U.S.A.). Other chemicals, of reagent
grade, were from Wako Pure Chemical Industries(Osaka, Japan).
Wasabi Leafstalk Extracts —–— Fresh wasabi leafstalk (Wasabia japonica MATSUM.; about 50 g) was
homogenized for 3 min in distilled water or 20%
ethanol solution (150 ml), and the homogenate wascentrifuged at 8000 rpm for 20 min.22) The resulting
supernatant was filtered through filtration paper, thenthe filtration solution was extracted 3 times with diethyl ether (about 150 ml). The ether phase was removed, and the resulting aqueous phase was lyophilized. About 0.9 g of the frozen powder was obtained. The powder was dissolved in distilled water.
Before its use in bone culture experiments, wasabi
leafstalk extract was aseptically filtered through amembrane.
Animals —–— Female Wistar rats (conventional)
weighing 200–250 g (50 weeks old) were obtained
from Japan SLC (Hamamatsu, Japan). The animals
were fed commercial laboratory chow (solid) containing 1.1% calcium and 1.1% phosphorus at a room

temperature of 25°C, with free access to distilledwater.
Bone Culture —–— The femurs were removed aseptically after bleeding, and were then soaked in icecold 0.25 M sucrose solution. The femur was cleanedof soft tissue and marrow, and the diaphysis andmetaphysis (not containing epiphyseal tissue) wereseparated. The femoral-diaphyaseal and -metaphyseal tissues were cut into small pieces. Diaphyseal
or metaphyseal fragments were cultured for 48 hr in
a 35 mm dish in 2.0 ml of medium consisting ofDulbecco’s MEM (high glucose, 4.5 g/dl) supplemented with 0.25% bovine serum albumin plus antibiotics (penicillin 100 units and streptomycin
100 µg/ml of medium).24) In our experiments, bone
tissues were cultured in a medium containing either
vehicle or Wasabi leafstalk extracts (10, 25 or 50 µg/
ml of medium). Cultures were maintained at 37°C
in a water-saturated atmosphere containing 5% CO2
and 95% air.
Administration Procedures —–— The water suspension (10 and 20 mg/ml/100 g body weight) of
the powder of an ethanol-solubilized extract ofwasabi leafstalk was orally administered to rats
through a stomach tube once daily for 7 days. Control rats received distilled water (1.0 ml/ 100 g bodyweight) orally. The animals were killed 24 hr after
the last administration by cardiac puncture underlight ether anesthesia, and the blood and femur were
removed immediately.
Analytical Procedures —–— Blood samples were
centrifuged for 30 min after collection, and the serum was separated and analyzed immediately. Serum calcium was determined by the method ofWillis.25) Serum inorganic phosphorus was measured
by the method of Taussky and Shon.26)
The diaphyseal and metaphyseal tissues weredried for 16 hr at 110°C, weighed, and then dissolvedin nitric acid solution. Calcium was determined byatomic absorption spectrophotometry.24) The calcium
content in bone tissues was expressed as milligrams
per gram of dry bone.
To assay alkaline phosphatase activity, the metaphyseal tissues were immersed in 3.0 ml of ice-coldbarbital buffer 6.6 mM (pH 7.4), cut into small
pieces, and disrupted for 60 sec with an ultrasonicdevice. The supernatant, centrifuged at 600 × g for
5 min, was used to measure enzymes The assay was
carried out under optimal conditions. Alkaline phosphatase activity was determined by the method ofWalter and Schutt.27) Enzyme activity was expressed

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No. 2

Fig. 1. Effect of Wasabi Leafstalk Extract on Calcium Content
in Rat Femoral-diaphyseal and -metaphyseal Tissues in
Vitro

Fig. 2. Effect of Wasabi Leafstalk Extract on Alkaline
Phosphatase Activity in Rat Femoral-diaphyseal and-metaphyseal Tissues in Vitro

Femoral-diaphyseal and -metaphyseal tissues obtained from normal
aged rats were cultured for 48 hr in a medium containing either vehicle
or wasabi leafstalk extract (0, 10, 25 or 50 µg/ml of medium) in theabsence or presence of cycloheximide (10–6 M). Each value is the mean
± standard error of the means (S.E.M.) of six rats. *p < 0.01 comparedwith the control (none) value. White bars, cycloheximide; black bars,
wasabi leafstalk extract (50 µg/ml) plus cycloheximide.

Femoral-diaphyseal and -metaphyseal tissues obtained from normal
aged rats were cultured for 48 hr in a medium containing either vehicle
or wasabi leafstalk extract (0, 10, 25 or 50 µg/ml of medium) in theabsence or presence of cycloheximide (10–6 M). Each value is the mean
± S.E.M. of six rats. *p < 0.01 compared with the control (none) value.
White bars, cycloheximide; black bars, wasabi leafstalk extract (50 µg/ml) plus cycloheximide.

as micromol of p-nitrophenol liberated per minuteper milligram of protein. Protein concentration wasdetermined by the method of Lowry et al.28)
To measure bone DNA content, the diaphyseal
and metaphyseal tissues were shaken with 4.0 ml
ice-cold 0.1 N NaOH solution for 24 hr after homogenization of the bone tissues.29) After alkali extraction, the samples were centrifuged at 1000 × g for
5 min, and the supernatant was collected. DNA content in the supernatant was determined by the methodof Ceriotti30) and expressed as the amount of DNA(mg)/g wet weight of bone tissue.
Statistical Analysis —–— The significance of difference between values was estimated by Student’s
t-test. p-Values of less than 0.05 were considered toindicate statistically significant differences.

and metaphyseal calcium content was completely
prevented in the presence of cycloheximide (10–6 M).
The presence of wasabi leafstalk extract (25 or
50 µg/ml) caused a significant increase in alkaline
phosphatase activity in the diaphyseal and metaphyseal tissues in vitro (Fig. 2). The stimulatory effectof wasabi leafstalk (50 µg/ml) on diaphyseal andmetaphyseal enzyme activity was not seen in thepresence of cycloheximide (10–6 M).
Diaphyseal and metaphyseal DNA content wassignificantly raised in the presence of wasabi leafstalk extract (50 µg/ml) (Fig. 3). This effect was completely prevented in the presence of cycloheximide(10–6 M).

RESULTS
Effect of Wasabi Leafstalk Extract on Bone Components in Vitro
The effect of wasabi leafstalk extract on calcium
content in the femoral-diaphyseal and -metaphyseal
tissues obtained from female aged rats in vitro isshown in Fig. 1. Bone tissues were cultured for 48 hr
in the presence of wasabi leafstalk extract. Diaphyseal and metaphyseal calcium content was significantly increased in the presence of wasabi leafstalkextract (25 or 50 µg/ml), but increasing diaphyseal

Effect of Administration of Wasabi Extract on
Bone Components in Aged Female Rats in Vivo
Body weight, serum calcium and inorganic phosphorus concentrations of aged (50-week-old) rats
were not significantly altered by the oral administration of wasabi leafstalk extract (10 or 20 mg/100 gbody weight) for 7 days (Table 1).
The oral administration of wasabi leafstalk extract (10 or 20 mg./100 g) to aged female rats caused
a significant increase in calcium content (Fig. 4),
alkaline phosphatase activity (Fig. 5) and DNA content (Fig. 6) in the femoral-diaphyseal and -metaphyseal tissues in vivo.

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Vol. 49 (2003)

Fig. 3. Effect of Wasabi Leafstalk Extract on DNA Content in
Rat Femoral-diaphyseal and -metaphyseal Tissues in
Vitro
Femoral-diaphyseal and -metaphyseal tissues obtained from normal
aged rats were cultured for 48 hr in a medium containing either vehicle
or wasabi leafstalk extract (0, 10, 25 or 50 µg/ml of medium) in theabsence or presence of cycloheximide (10–6 M). Each value is the mean
± S.E.M. of six rats. *p < 0.01 compared with the control (none) value.
White bars, cycloheximide; black bars, wasabi leafstalk extract (50 µg/ml) plus cycloheximide.

DISCUSION
Of various plant extracts, wasabi leafstalk(Wasabia japonica MATSUM.) extract has been
demonstrated to have a unique stimulatory effect onbone calcification in vitro.22) Characterization of theactive component of wasabi leafstalk extract instimulating bone calcification in vitro has been examined.23) The components of wasabi leafstalk extract which increase bone calcium content are stable
when treated by heating or with acidity or alkalinization.23) Thus, the active component of wasabi
leaftsalk extract thus may not be a protein.
The extract of wasabi leafstalk has been subjected to gel filtration chromatography with a highpressure liquid chromatography system.23) From thisstudy, it is assumed that the active component of
wasabi leafstalk extract in stimulating bone calcifi-

cation has a longer retention time than that of cytidine (molecular weight; MW. 243) that is used as astandard.23) The molecular weight of genistein, which
can stimulate bone calcification in vitro,31) is 270.
The active component of wasabi leafstalk may differ from cytidine or genistein. In addition, sinigrin
has been shown to have no effect on bone calcification.23) Identification of the active component inwasabi leafstalk extract remains to be elucidated.
The present study was undertaken to determine
whether wasabi leafstalk extract has a preventive
effect on bone loss with increasing age, which induces osteoporosis. Bone metabolism in the femoral-diaphyseal and -metaphyseal tissues is deteriorated with increasing age (50 weeks old), as compared with that of young rats (4 weeks old).19,32) The
presence of wasabi leafstalk extract in culture medium caused a significant increase in calcium content, alkaline phosphatase activity and DNA content
in the femoral-diaphyseal and -metaphyseal tissuesof aged female rats in vitro. The effect of wasabi
leafstalk extract in increasing bone components wascompletely prevented in the presence of cycloheximide, an inhibitor of protein synthesis. This result
suggests that an anabolic effect of wasabi leafstalkextract on bone metabolism results from newly synthesized protein components.
Moreover, the oral administration of wasabi leafstalk extract was found to induce a significant increase in calcium content, alkaline phosphatase activity and DNA content in the femoral-diaphyseal
and -metaphyseal tissues of aged female rats in vitro.
Administration of wasabi leafstalk extract did notcause a significant alteration in body weight, serum
calcium or inorganic phosphorus concentrations inaged female rats, indicating that the extract may nothave a toxic effect. Thus, the intake of wasabi leafstalk extract may have a preventive effect on the deterioration of bone metabolism with increasing age.
Presumably, dietary wasabi leafstalk has a role in

Table 1. Body Weight, Serum Calcium and Inorganic Phosphorus Concentrations in Female Aged Rats Orally
Administered Wasabi Leafstalk Extract
Treatment
Control
Wasabi leafstalk extract
10 mg /100 g
20 mg /100 g

Body Weight(g)
238.3 ± 5.23

Serum concentration (mg/dl)
Calcium
Inorganic phosphorus
9.00 ± 0.11
5.28 ± 0.18

235.3 ± 4.48
225.3 ± 4.25

9.01 ± 0.14
9.02 ± 0.15

5.24 ± 0.17
5.30 ± 0.13

Rats (50 weeks old) were orally administered wasabi leafstalk extract (10 or 20 mg/100 g body weight) for 7 days. Eachvalue is the mean ± S.E.M. of six rats. Data were not significant.

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Fig. 4. Change in Calcium Content in the Femoral-diaphyseal
and -metaphyseal Tissues of Aged Rats Orally
Administered Wasabi Leafstalk Extract in Vivo

Fig. 6. Change in DNA Content in the Femoral-diaphyseal and-metaphyseal Tissues of Aged rats Orally Administered
Wasabi Leafstalk Extract in Vivo

Rats were orally administered wasabi leafstalk extract (10 or 20 mg/
ml/100 g body weight) once daily for 7 days, and the animals were killed
24 hr after the last administration. Each value is the mean ± S.E.M. of
six rats. *p < 0.01 compared with the control (none) value.

The procedure of administration was described in the legend of
Fig. 4. Each value is the mean ± S.E.M. of six rats. *p < 0.01 comparedwith the control (none) value.

Fig. 5. Change in Alkaline Phosphatase Activity in the Femoraldiaphyseal and -metaphyseal Tissues of Aged rats Orally
Administered Wasabi Leafstalk Extract in Vivo
The procedure of administration was described in the legend of
Fig. 4. Each value is the mean ± S.E.M. of six rats. *p < 0.01 comparedwith the control (none) value.

the prevention of osteoporosis with aging.
In conclusion, it has been demonstrated thatwasabi leafstalk extract has an anabolic effect onbone components in the femoral-diaphyseal (cortical bone) and -metaphyseal (trabecular bone) tissuesof aged female rats in vitro and in vivo.

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