Chinese Medicine, 2011, 2, 130-137
doi:10.4236/cm.2011.24022 Published Online December 2011 (http://www.SciRP.org/journal/cm)
Effects of Bofutsushosan and Gardeniae Fructus on
Diabetic Serum Parameters in Stre ptozotocin-Induced
Diabetic Mice
Qing Yu1, Mai Yasuda1, Tatsuo Taka h a sh i 1, Masaaki Nomura2, Nobuyoshi Hagino3,
Shinjiro Kobayashi1*
1Department of Clinical Pharmacy, Faculty of Pharmaceutical Sciences, Hokuriku University,
Kanazawa, Japan
2Department of Education Center of Clinical Pharmacy, Faculty of Pharmaceutical Sciences, Hokuriku University,
Kanazawa, Japan
3Laboratory of Integrative Medicine, US-Japan Cooperative Biomedical Research Laboratories, Tulane
University Herbert Research Center and Department of Medicine, Tulane University Health Science Center, School of
Medicine, Belle Chasse, USA
Received July 9, 2011; revised August 14, 2011; accepted August 30, 2011
Abstract
Streptozotocin (STZ)-induced diabetic mice increased levels of serum glucose, triglyceride and cholesterol, and
decreased level of serum insulin. Effects of Bofutsushosan (BOF: Pulvis ledebouriellae compositae: 防風通聖
散) and its composed crude drug, gardeniae fructus (GF: 山梔子) were investigated on levels of these diabetic
parameters (serum glucose, insulin, triglyceride and cholesterol) in STZ-diabetic mice. BOF and GF were ex-
tracted in 10 volumes of distilled water with an automatic extractor “Torobi”. STZ-induced diabetic mice with
serum glucose level of over 600 mg/dl at 3 - 4 weeks after intravenous injection of 150 mg/kg STZ were used
for experiments. BOF extract, GF extract, geniposide (a main constituent of GF), and glibenclamide were ad-
ministered intraperitoneally into 3-hour-fasted STZ-diabetic mice. At 6 hours after administration, BOF extract
(100 - 300 mg/kg) decreased high levels of serum glucose, triglyceride and cholesterol, and also increased low
level of serum insulin in STZ-diabetic mice in a dose-dependent manner, respectively. Anti-diabetic drug
glibenclamide (0.3 - 1 mg/kg) as positive control significantly decreased serum glucose and cholesterol levels,
and increased serum insulin level in the diabetic mice. GF extract (30 - 300 mg/kg) decreased serum glucose,
triglyceride and cholesterol levels but did not affect serum insulin level in the diabetic mice. Geniposide (10 -
100 mg/kg), decreased serum glucose level but did not affect serum insulin and triglyceride levels in the dia-
betic mice. These results demonstrated that intraperitoneally administrated BOF extract improved abnormal
levels of serum glucose, insulin, triglyceride and cholesterol in the STZ-diabetic mice as being similar to
glibenclamide. GF extract has an important role in a part of improving actions of BOF in the diabetic mice. The
action of GF extract on serum glucose was parallel with the action of geniposide in the diabetic mice, support-
ing roles of geniposide in anti-hyperglycemic action of GF.
Keywords: Bofutsushosan (Pulvis Ledebouriellae Compositae: 防風通聖散), Gardeniae Fructus (山梔子),
Geniposide, Streptozotocin-Induced Diabetic Mice, Anti-Hyperglycemic Action,
Anti-Hyperlipidemic Action
1. Introduction
Diabetes mellitus is a chronic heterogeneous disease cha-
racterized by hyperglycemia resulting from both insulin
resistance and insulin deficiency secondary to pancreatic
cell failure. Diabetes mellitus through hyperglycemia is
widely known to be a major factor that leads to micro-
vascular and neural complication [1]. Hyperglycemia-

131 Q. YU ET AL.
induced end organ damage in diabetes mellitus is associ-
ated with 1) accumulation of advanced glycation end
products [2,3]; 2) increased oxidative stress [4,5]; 3) pol-
yol metabolic pathway [6,7]; and 4) activation of protein
kinase C pathway [8,9]. Insulin deficiency in diabetes
mellitus stimulates lipolysis in the adipose tissue, and
gives rise to hyperlipidemia and fatty liver [10]. Althou-
gh diabetes mellitus is characterized as a disease of car-
bohydrate metabolism, abnormalities of lipid and lipo-
protein metabolism are commonly observed. Hyperlipi-
demia [11] and atherosclerosis [12] are frequently asso-
ciated with diabetes mellitus, indicating alterations of
cholesterol metabolism in this diabetes [13]. In addition,
there is triglyceride enrichment of both high-density
lipoprotein (HDL) and low-density lipoprotein (LDL).
Therefore, the lipid profile in type 2 diabetic subjects
generally consists of elevated triglycerides and LDL cho-
lesterol, and reduced HDL cholesterol [14,15]. Diabetes-
related dyslipidaemia has also been shown to be an im-
portant contributor to vascular dysfunction. Atheroscle-
rosis is, however, associated with macrovascular disease
and thus the role of dyslipidaemia versus hyperglycemia
in the pathogenesis of microvascular disease remains
unclear and continues to be debated [16].
Streptozotocin (STZ) is N-nitroso derivative of gluco-
samine and has been commonly used to induce not only
animal models of insulin-dependent diabetes mellitus
(IDDM, type 1) but also non-insulin-dependent diabetes
mellitus (NIDDM, type 2) with hypoinsulinemia by STZ
administration to neonates (1 or 2 day old mice) [17-19].
It has been reported that STZ is capable of producing
mild to severe types of diabetes according to the dosages
used when it is given to animals by either single intrave-
nous or intraperitoneal (i.p.) injection [20].
Using this experimental model, we sought to investi-
gate the efficacy of traditional Chinese medicine, bofu-
tsushosan (BOF: Pulvis ledebouriellae compositae) on
the prevention of abnormal levels of diabetic parameters,
serum glucose, insulin, triglyceride and cholesterol. BOF
is consisted of 18 crude drugs as described in Table 1
[21]. BOF is indicated for the relief of the following sy-
mptoms of those patients with hypertension, insulin re-
sistance and thick subcutaneous fat in the abdomen and
tendency to constipation [21]. BOF has been successfully
used in patients with hyper functional constitution who
exhibit risk factors for cerebrovascular events. Gardeniae
fructus (GF) has traditionally classified as an antipyretic
agent in BOF. Chemical isolated from GF has been sug-
gested to increase glucose-stimulated insulin release fr-
om pancreatic cells of type 2 diabetes mellitus model
[22]. In the present study, effects of extracts of BOF and
GF were compared on levels of the diabetic parameters
in STZ-diabetic mice to study a role of GF in action of
BOF in the STZ-diabetic mice.
2. Materials and Methods
2.1. Preparation of Streptozotocin-Diabetic Mice
Fed male mice (ddY strain; 4 weeks of age; 16 - 20 g;
Japan SLC, Shizuoka, Japan) were injected with a single
dose (150 mg/kg) of STZ (Sigma, St. Louis, MO, U.S.A.)
in saline into the tail vein. STZ-induced diabetic mice (7-
8 weeks of age; blood glucose over 600 mg/dl) were
used for experiments at 3 - 4 weeks after the injection of
STZ. Age-matched normal male mice (ddY strain; 7 - 8
weeks of age) were used in the control experiments.
These mice were given CRF-1 (Oriental Yeast Co., To-
kyo, Japan) and water ad libitum and kept at 25˚C - 26˚C
with lights on from 7 a.m. to 7 p.m. Drugs were adminis-
tered intraperitoneally to mice that had been fasted for 3
hours. The Ethics Review Committee for Animal Expe-
rimentation of Hokuriku University approved the expe-
rimental protocol.
2.2. Preparation and Administration of Drugs
BOF consists of Ephedrae Herba, Saposhnikoviae Radix,
Zingiberis Rhizoma, Schizonepetae Spica, Rhei Rhizo-
ma, Natrium Sulfuricum, Glycyrrhizae Radix, Forsythiae
Table 1. Crude drugs composed in bofutsushosan.
Crude drugs content (g)a
1 Ephedrae Herba 1.2
2 Saposhnikoviae Radix 1.2
3 Zingiberis Rhizoma 0.4
4 Schizonepetae Spica 1.2
5 Rhei Rhizoma 1.5
6 Natrium Sulfuricum 0.6
7 Glycyrrhizae Radix 2.0
8 Forsythiae Fructus 1.2
9 Platycodi Radix 2.0
10 Cnidii Rhizoma 1.2
11 Scutellariae Radix 2.0
12 Gardeniae Fructus 1.2
13 Gypsum Fibrosum 2.0
14 Talcum 3.0
15 Angelicae Radix 1.2
16 Paeoniae Radix 1.2
17 Atractylodis Lanceae Rhizoma 2.0
18 Menthae Herba 1.2
aEach value (g) was represented as dry weight.

Q. YU ET AL.
132
Fructus, Platycodi Radix, Cnidii Rhizoma, Scutellariae
Radix, GF, Gypsum Fibrosum, Talcum, Angelicae Radix,
Paeoniae Radix, Atractylodis Lanceae Rhizoma, and Men-
thae Herba. BOF and GF were purchased from Tsumura
Co. (Tokyo) and extracted in 10 volumes of distilled wa-
ter with an automatic extractor “Torobi” (Tochimoto,
Osaka, Japan) for 60 min. A water extract of the drug
was filtered through a mesh (No. 42, Sanpo, Tokyo), ly-
ophilized with a freeze-drier (DF-03G, ULVAC, Tokyo),
and stored at 4˚C [23,24]. The dry weight yields of BOF
and GF extracts were 27.5% and 67.7% (w/w), respecti-
vely. BOF extract, GF extract, geniposide (Wako, Osaka),
a main constituent of GF, and glibenclamide (Wako) we-
re administered intraperitoneally (0.1 ml/10 g body wei-
ght) into 3-hour-fasted STZ-diabetic mice.
2.3. Measurement of Glucose, Insulin,
Triglyceride and Cholesterol Levels in
Serum
Blood was collected from the neck vein plexus of diabe-
tic mice at 6 hours after the administration of drugs, and
centrifuged at 8000 rpm at 25˚C for 5 min. Serum gluco-
se level of the supernatant was measured by the glucose
oxidase method with a serum glucose monitor set (MED-
ISAFE MINI, Terumo, Tokyo). Serum glucose levels
were measured in fasted mice before, and 6 hours after
the administration of drugs or saline, respectively. The
fall % of serum glucose (SG) was calculated as [SG (be-
fore drug treatment) – SG (after drug treatment)]/[SG
(before drug treatment) – 85] × 100. The average of SG
of 3-hour-fasted normal mice is 85 mg/dl [23,24]. Serum
insulin levels of 3-hour-fasted mice were measured with
a mouse ELISA kit for insulin (Morinaga, Yokohama,
Japan) 6 hours after the administration of drugs or saline.
Serum total triglyceride and cholesterol levels were
measured with ELISA kits for triglyceride and choles-
terol (Wako), respectively.
2.4. Statistical Analyses
All values were expressed as means ± S.E.M.. Differen-
ces between group data were evaluated by unpaired t-test
at p = 0.05 or 0.01. A value of p < 0.05 was considered
statistically significant.
3. Results
3.1. STZ-Induced Changes of Levels of Body
Weight, Serum Glucose, Insulin,
Triglyceride and Cholesterol in ddY Mice
Levels of body weight, serum glucose, insulin, triglyc-
eride and cholesterol in ddY mice during 3 weeks after
injection of STZ were compared with those in age-
matched normal mice in Table 2. The body weights of
STZ-treated mice were significantly decreased to 93% of
those of age-matched normal mice. Levels of diabetic
parameters in sera of 3-hour-fasted ddY mice with STZ
treatment were compared with those of 3-hour-fasted
normal mice. Serum glucose levels of STZ-treated mice
were 996.8 mg/dl and significantly 5.4-fold greater than
those of normal mice. Serum insulin levels of STZ-
treated mice were 183.7 pg/ml and lowered by 82% of
normal insulin level. Levels of serum triglyceride and
serum cholesterol of STZ-treated mice were 187.3 and
183.2 mg/dl, respectively and increased to 87% and 35%
of normal levels. Therefore, we used STZ-treated mice
as the diabetic model.
3.2. Effects of Bofutsushosan (BOF) and
Glibenclamide on Levels of Serum Glucose
and Insulin in STZ-Diabetic Mice
Effects of BOF extract (30 - 300 mg/kg) on level of
blood glucose were compared with that of anti-diabetic
drug glibenclamide as positive control in i.p. treatment
during 6 hours. After the drug injection, BOF extract (30
- 300 mg/kg) decreased high level of serum glucose of
STZ-diabetic mice in a dose-dependent manner (Figure
1). Glibenclamide (Glc; 0.3 - 1 mg/kg) also significantly
decreased high serum glucose level of STZ-diabetic mice.
Glibenclamide showed anti-hyperglycemic action by its
i.p. administration as well as BOF extract did. BOF ex-
tract (30 - 100 mg/kg) was significantly elevated serum
insulin level in a dose-dependent manner. Glibenclamide
(0.3 - 1 mg/kg) also significantly increased serum insulin
level (Figure 2 ) in the STZ-diabetic mice. Effects of BOF
extract and glibenclamide on elevation of serum insulin
levels were parallel with those anti-hyperglycemic ef-
fects in STZ-diabetic mice.
Table 2. Body weight, serum glucose, insulin, triglyceride (TG) and cholesterol (CH) of STZ-diabetic mice.
Body weight (g) Glucose (mg/dl) Insulin (pg/ml) TG (mg/dl) CH (mg/dl)
Normal mice 37.5 ± 0.3 183.3 ± 3.7 1024 ± 64.2 100.2 ± 5.2 135.4 ± 3.7
STZ mice 34.9 ± 0.3** 996.8 ± 43.3** 183.7 ± 25.0** 187.3 ± 10.3** 183.2 ± 4.8**
Values represent means ± S.E.M. **p < 0.01: Significantly different from normal mice.

133 Q. YU ET AL.
Figure 1. Effects of bofutsushosan (BOF) extract and gli-
benclamide (Glc) on serum glucose level in STZ-diabetic mi-
ce. Serum glucose levels were measured before and 6 hours
after i.p. administration of BOF extract or Glc into 3-hour-
fasted diabetic mice. The values are expressed as means ±
S.E.M. of 5 - 25 data. *p < 0.05, **p < 0.01: Significantly
different from contr ol water group.
Figure 2. Effects of bofutsushosan (BOF) extract and gli-
benclamide (Glc) on serum insulin level in STZ-diabetic
mice. Serum insulin level was measured at 6 hours after i.p.
administration of BOF extract or Glc into 3-hour-fasted
diabetic mice. The values are expressed as means ± S.E.M.
of 5 - 25 data. *p < 0.05, **p < 0.01: Significantly different
from control water group.
3.3. Effects of Bofutsushosan (BOF) and
Glibenclamide on Levels of Serum
Triglyceride and Serum Cholesterol in
STZ-Diabetic Mice
BOF extract (30 - 300 mg/kg) lowered serum triglyceride
level of STZ-diabetic mice in a dose-dependent manner.
However, glibenclamide (0.3 - 1 mg/kg) did not affect
the serum triglyceride level (Figure 3). BOF extract (30
- 300 mg/kg) also significantly lowered serum choles-
terol level of STZ-diabetic mice. Glibenclamide had a
weak action for lowering serum cholesterol level when
administered only a high dose of 1 mg/kg (Figure 4 ).
Figure 3. Effects of bofutsushosan (BOF) extract and gli-
benclamide (Glc) on serum triglyceride level in STZ-dia-
betic mice. Serum triglyceride level was measured at 6
hours after i.p. administration with BOF extract or Glc into
3-hour-fasted diabetic mice. The values are expressed as
means ± S.E.M. of 5-25 data. *p < 0.05, **p < 0.01: Sig-
nificantly different from the control water group.
Figure 4. Effect of bofutsushosan (BOF) extract and gli-
benclamide (Glc) on serum cholesterol in STZ-diabetic mice.
Serum cholesterol level was measured at 6 hours after i.p.
administration with BOF extract or Glc into 3-hour-fasted
diabetic mice. The values are expressed as means ± S.E.M.
of 5 - 25 data. *p < 0.05, **p < 0.01: Significantly different
from the control water group.

Q. YU ET AL.
134
3.4. Effects of Gardeniae Fructus (GF) and
Geniposide on Serum Diabetic Parameters
in STZ-Diabetic Mice
Effects of extract of GF composed in BOF were investi-
gated on levels of the diabetic parameters in STZ-dia-
betic mice. GF extract (30 - 300 mg/kg) in an i.p. ad-
ministration lowered high serum glucose level in a
dose-dependent manner (Figure 5). Geniposide (Gep; 10
- 100 mg/kg), a main compound of GF, also lowered
high blood glucose level in a dose-dependent manner
(Figure 5). Anti-hyperglycemic action of GF extract was
parallel with that of geniposide. GF extract (30 - 300
mg/kg) in an i.p. administration did not affect serum in-
sulin level of STZ-diabetic mice. Geniposide (10 - 100
mg/kg) also did not affect serum insulin level of the dia-
betic mice (Figure 6). GF extract (30 - 300 mg/kg) low-
ered serum triglyceride level (Figure 7) and serum cho-
lesterol level (Figure 8) in a dose-dependent manner.
However, geniposide (10 - 100 mg/kg) did not affect
serum triglyceride level (Figure 7) and increased serum
cholesterol level conversely (Figure 8).
4. Discussion
Diabetes mellitus is characterized as a disease of carbo-
hydrate metabolism and abnormalities of lipid and lipo-
protein metabolism. Insulin deficiency stimulates lipoly-
sis in the adipose tissue, and gives rise to hyperlipidemia
and fatty liver in diabetes mellitus [10]. Hyperlipidemia
Figure 5. Effects of gardeniae fructus (GF) extract and
geniposide (Gep) on the serum glucose level in STZ-diabetic
mice. Serum glucose levels were measured before and 6
hours after i.p. administration with GF extract or Gep into
3-hour-fasted diabetic mice. The values are expressed as
means ± S.E.M. of 5 - 15 data. *p < 0.05, **p < 0.01:
Significan t ly different from the control water group.
Figure 6. Effects of gardeniae fructus (GF) extract and ge-
niposide (Gep) on serum insulin level in STZ-diabetic mice.
The immunoreactive insulin level was measured at 6 hours
after i.p. administration with GF extract or Gep into
3-hour-fasted diabetic mice. The values are expressed as
means ± S.E.M. of 5 - 15 data.
Figure 7. Effects of gardeniae fructus (GF) extract and ge-
niposide (Gep) on serum triglyceride level in STZ-diabetic
mice. Serum triglyceride level was measured at 6 hours
after i.p. administration with GF extract or Gep into
3-hour-fasted diabetic mice. The values are expressed as
means ± S.E.M. of 5 - 15 data. *p < 0.05, **p < 0.01: Sig-
nificantly different from the control water group.
and atherosclerosis are frequently associated with diabe-
tes mellitus, indicating alterations of cholesterol metabo-
lism [11-13]. The lipid profile in type 2 diabetic subjects
generally consists of elevated triglycerides and LDL
cholesterol, and reduced HDL cholesterol. In the present
study, STZ-diabetic mice were used as diabetic model
and showed significant increase of serum glucose, total
triglyceride and total cholesterol levels, and decrease of
serum insulin level (Table 2).

135 Q. YU ET AL.
Figure 8. Effects of gardeniae fructus (GF) extract and
geniposide (Gep) on the serum cholesterol level in
STZ-diabetic mice. Serum cholesterol level was measured
at 6 hours after i.p. administration with GF extract or Gep
into 3-hour-fasted diabetic mice. The values are expressed
as means ± S.E.M. of 5 - 15 data. *p < 0.05, **p < 0.01: Sig-
nificantly different from the control water group.
BOF is a traditional Chinese medicine consisted of 18
crude drugs as described in Table 1 [21]. BOF extract
and glibenclamide, an oral anti-diabetic agent, were
given a single i.p. administration to the STZ-diabetic mi-
ce. BOF extract lowered high serum glucose level in a
dose-dependent manner as parallel to elevate low serum
insulin level. The improving actions of BOF extract on
serum levels of glucose and insulin were similar to those
of glibenclamide in STZ-diabetic mice (Figures 1 and 2).
These results indicate that our STZ-diabetic mice can be
released insulin from pancreatic β cells by both gliben-
clamide and BOF extract. STZ has been reported to pro-
duce mild to severe types of diabetes mellitus according
to the dosages used and experimental conditions [20].
Type 2 diabetes mellitus is associated with a combina-
tion of resistance to insulin action and impaired insulin
secretion [25]. We have unpublished data that both BOF
extract and glibenclamide in their oral administration
decreased serum glucose level and increased insulin level
in serum of alloxan-induced diabetic mice, which show
type 2 model of diabetes mellitus [26]. It is also reported
that low dose STZ combined with high-energy intake can
effectively induce type 2 diabetes through altering the
related gene expression [27]. These results demonstrate
that BOF improves hyperglycemia through releasing
insulin from pancreatic β cell of STZ-diabetic mice.
BOF extract also lowered high levels of serum trigly-
ceride and serum cholesterol of STZ-diabetic mice in a
dose-dependent manner. However, glibenclamide decr-
eased only high level of serum cholesterol at a high dose
but not affect serum triglyceride even at the high dose
(Figures 3 and 4). These results indicated that actions of
BOF extract on levels of serum triglyceride and choles-
terol did not depend on action of released insulin in the
STZ-diabetic mice.
GF extract is traditionally classified as antipyretic ag-
ent in BOF and decreased high level of serum glucose in
STZ-diabetic mice in a dose-dependent manner. How-
ever, GF extract did not affect level of insulin in the
STZ-diabetic mice (Figures 5 and 6). These results de-
monstrate that anti-hyperglycemic action of GF differed
from those of BOF and glibenclamide. The anti-hyper-
glycemic action of GF is supported by action of genipo-
side, a main compound of GF on serum glucose level.
Yield of geniposide in GF extract is estimated to be 37%
and the effect of geniposide is almost 3-time greater than
that of GF extract. Geniposide also did not affect serum
insulin level of STZ-diabetic mice. These results indica-
ted that anti-hyperglycemic action of GF extract de-
pended on the action of geniposide in the STZ-diabetic
mice. The anti-hyperglycemic action of GF extract was
independent on release of serum insulin. GF extract also
lowered levels of serum triglyceride and serum choles-
terol of STZ-diabetic mice in a dose-dependent manner.
However, geniposide did not affect level of serum trigl-
yceride and increased level of serum cholesterol (Fig-
ures 7 and 8), being different from the actions of GF
extract for serum levels of triglyceride and cholesterol.
The results suggest that some compounds different from
geniposide in GF may have a role for the actions of GF
extract on serum triglyceride and cholesterol levels.
Mitochondrial uncoupling protein 2 (UCP2) has re-
ported to alter the yield of ATP synthesis from glucose,
and is proposed as a negative regulator of glucose-sti-
mulated insulin secretion in pancreatic β cells of type 2
diabetes mellitus model [28]. The absence of mitochon-
drial UCP2 renders animals more sensitive to the onset
of type 1 autoimmune diabetes in mice [29]. UCP2 and
UCP3 gene expressions were increased in skeletal mus-
cle of STZ-diabetic rat, while UCP1, UCP2 and UCP3
gene expressions were reduced in brown adipose tissue
of these rats [30]. UCP2 also modulates myocardial ex-
citation-contraction coupling [31]. Genipin has been re-
ported to be the bioactive compound of geniposide, one
of major effective compounds of GF and a natural
cross-linking agent. Genipin is inhibitor of UCP2. UCP2
deficiency improves obesity- and high glucose-induced β
cell dysfunction and consequently improves type 2 dia-
betes [22]. The inhibition of UCP2 with genipin also
sensitizes multidrug-resistant cancer cells to cytotoxic
agents [32]. The present study demonstrate that adminis-
tered GF extract and geniposide decreased high serum
glucose level but not affect serum insulin level in the

Q. YU ET AL.
136
STZ-diabetic mice. Genipin may involve the anti-hyper-
glycemic and anti-hyperlipidemic actions of GF extract.
We need further experiments for high doses of geni-
poside and genipin, and duration of their treatment.
5. Conclusions
BOF extract improved abnormal levels of serum glucose,
insulin, triglyceride and cholesterol in the STZ-diabetic
mice. Extract of GF in BOF also improved levels of se-
rum glucose, triglyceride and cholesterol but not level of
serum insulin in the diabetic mice. These results indicate
that GF extract has an important role in a part of im-
proving actions of BOF in the diabetic mice. Anti-hy-
perglycemic action of BOF extract may have two, insulin
release-dependent and non-insulin release-dependent me-
chanisms in the STZ-diabetic mice.
6. Acknowledgements
This work was supported in part by a grant (to SK) for
the “Academic Frontier” project for Private Universities
(2005-2009) from the Ministry of Education, Culture,
Sports, Science and Technology of Japan, Japan.
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