Vol.2, No.5, 493-498 (2010)
doi:10.4236/health.2010.25073
Copyright © 2010 SciRes. Openly accessible at http://www.scirp.org/journal/HEALTH/
Health
Long-term administration of traditional kampo medicine
shimotsuto, juzentaihoto and unseiin inhibits
experimental thrombosis in mice
Yoshinobu Ijiri1,2, Hiroko Anzai3, Weifua Gao4, Kunio Takahashi5, Naemi Kajiwara3,
Masahiro Murakami2,4, Junichiro Yamamoto2,6*
1Department of Food Nutritional Science, Faculty of Liberal Arts, Osaka Shoin Women’s University, Osaka, Japan
2Antithrombotic Diet Discussion Group, Laboratory of Physiology, Faculty of Nutrition, Kobe Gakuin University, Kobe, Japan
3Laboratory of Nutrition Physiology, Faculty of Health and Welfare, Kobe Women’s University, Kobe, Japan
4Laboratory of Pharmaceutics, Faculty of Pharmacy, Osaka-ohtani University, Osaka, Japan
5Zaiseido Pharmaceutical Co Ltd, Wakayama, Japan
6Laboratory of Physiology, Faculty of Nutrition and Cooperative Research Center of Life Sciences, Kobe Gakuin University, Kobe,
Japan; *Corresponding Author:
Received 13 January 2010; revised 27 January 2010; accepted 30 January 2010.
ABSTRACT
Traditional Chinese herbal medicines (Kampo)
are used to improve flow characteristics of
blood (Oketsu). We assumed that by preventing
stagnation of blood, these medicines may be
beneficial not only in venous but in arterial thro-
mbotic conditions. The present study aimed to
assess the antithrombotic effect of three Kampo,
using well-established in vitro and animal mod-
els of thrombosis. Western-style highfat diet
containing 1% Kampo (Shimotsuto, Juzentaih-
oto or Unseiin) was administered to C57BL/6
mice for 12 weeks. The effect on thrombus for-
mation by laser irradiation of the carotid artery
of mice was assessed. In addition the ex-vivo
technique of shear-induced platelet reactivity
measurement (haemostatometry) and the in vivo
test of endothelial function (flowmediated vaso-
dilation) were also used to assess the mecha-
nism of antithrombotic effect. All three medici-
nes have significantly inhibited arterial throm-
bus formation in mice. According to our studies,
the mechanism of antithrombotic effect is based
on the inhibition of shear-induced platelet reac-
tivity and stimulation of endothelial function
(Unseiin). It is assumed that the common ingre-
dients Japanese Angelica Root, Cnidium Rhiz-
ome, Peony Root and Rehmannia Root could be
responsible for the observed antithrombotic
effect.
Keywords: Kampo; Chinese Medicine; Thrombosis;
Platelet; Endothelial Function; Stroke;
Cardiovascular Disease
1. INTRODUCTION
Prevention of lifestyle-related atherothrombotic disea-
ses such as myocardial infarction and stroke is an im-
portant and urgent social task in many developed coun-
tries. Epidemiological studies have provided evidence
for the causative role of inappropriate diet in the dev-
elopment of atherothrombotic diseases and the regular
life style and exercise in the prevention of such dis-
eases.
Chinese herbal medicine (Kampo) has a long history
of treating various diseases. Currently used Kampo is
believed to prevent stagnation thus improve the flow
characteristics of blood. In our earlier studies, we used
sensitive animal models of arterial thrombosis to assess
the actual thrombotic status. We succeeded overcoming
the main difficulty of the relative resistance of rodents to
the prothrombotic effect of high fat diet. We showed that
in apolipoprotein E and low-density lipoprotein receptor
double deficient C57BL/6J mice, high fat diet induced a
prothrombotic state, similar to humans [1]. By inducing
and measuring the rate of arterial thrombus formation in
response to laser irradiation in such spontaneously ather-
ogenic mice, we could test the antithrombotic effect of
various substances and diets. In the past, the use of this
in vivo test together with other global in vitro thrombo-
sis tests enabled us to find various fruits and vegetables
with significant experimental antithrombotic activity
[2,3].

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2. MATERIALS AND METHODS
2.1. Chinese Herbal Medicines (Kampo) for
Preventing Stagnation of Blood (Oketsu)
Shimotsuto, Juzentaihoto and Unseiin were used (Table
1). These were supplied by Zaiseido Pharmaceutical Co
Ltd, Wakayama, Japan.
2.2. Diets Containing Kampo
Experimental diets containing the three Kampos in 1%
(w/w) were prepared by adding Kampo to the Western-
style high fat model diet (Table 2). Diets were stored at
–30℃ until use.
2.3. Animals and Administration of Diet
Five week old male C57BL/6J mice were purchased
from SLC Co Ltd (Hamamatsu, Japan) one week before
the experiments and raised for one week on standard
solid chow (CE-2, Clea Japan Inc., Tokyo, Japan) and
drinking water ad libitum. Subsequently the chow was
changed to the experimental diet, starting at age of 6
week, and lasted for 12 weeks. Mice were kept in the
Animal Unit of Kobe Women’s University, had free
access to both diets and water, the room was air-
conditioned (22.5 ± 2℃ and humidity 40-60%) having
12-h light and dark cycle. Animals were fasted overnight
before the test and kept in compliance with the “Guiding
Principles for the Care and Use of Animals in the field of
Physiological Sciences,” published by Physiological
Table 1. Composition of the three Kampo for blood stagnancy
(Oketsu).
Content (g)
Ingredient
ShimotsutoJuzentaihoto Unseiin
Japanese Angelica 4.0 3.0 4.0
Cnidium Rhizome 4.0 3.0 4.0
Peony Root 4.0 3.0 4.0
Rehmannia Root 4.0 3.0 4.0
Atractylodes Rhizome 0 3.0 0
Poria Sclerotium 0 3.0 0
Cinnamon Bark 0 3.0 0
Ginseng 0 3.0 0
Glycyrrhiza 0 1.0 0
AStragalus Root 0 3.0 0
Coptis Rhizome 0 0 1.5
Scutellaria Root 0 0 3.0
Phellodendron Bark 0 0 1.5
Gardenia Fruit 0 0 2.0
Society of Japan. The experiments were approved by the
Animal Experiment Committee of Kobe Women’s Uni-
versity.
2.4. He-Ne Laser-Induced Carotid Artery
Thrombosis Test
This technique has been described in detail [1-4]. Briefly,
the left femoral artery and the carotid artery (450-500 μM
in diameter) of Nembutal anaesthetized mouse were ex-
posed. The mouse was placed on a special microscope
stage and through the femoral artery, a heat-absorbing
dye Evans blue was injected. Subsequently the centre of
the exposed carotid artery was irradiated with He-Ne
laser. Thrombus formation at the site of irradiation was
monitored under epi-illumination and recorded on vid-
eotape using CCD camera.
2.5. Calculation of Thrombus Size
From the start of laser irradiation, the computer-image of
the forming thrombus was recorded in every 10 seconds
for 10 minutes. The thrombus mass was delineated and
its size was calculated by a software (Image Processing
and Analysis Java version 1.30, National Institutes of
Health, Bethesda, Maryland, USA). Due to frequent
embolisation, the size of thrombus increased (building
up the thrombus) or decreased (partial embolisation).
Thrombotic status was defined by the total sum of
thrombus sizes. An increase of such total sum indicated a
prothrombotic state (enhanced thrombus formation),
while decrease of such sum indicated an antithrombotic
effect.
2.6. Endothelial Function Test
The endothelium-dependent flow-mediated vasodilation
(FMV) technique originally described for rat was ada-
pted to mice [5,6]. The anaesthetized mouse was kept on
a heated pad to maintain body temperature; the left fe-
moral artery was exposed, isolated and covered with
gauze saturated with 37℃ saline. Blood flow in the ar-
tery was stopped by clamping for 3 minutes and then the
flow was restored by releasing the clamp. Diameter of
the artery was monitored 2-4 mm distal from the site of
clamping by a CCD camera (Model CS900, Takenaka
System Co. Ltd., Kyoto, Japan). Baseline images were
taken before clamping and then in every 10 seconds over
60 seconds and further in 30 seconds intervals over 450
seconds after restoration of blood flow. Nitroglycerin-
mediated vasodilation was induced by placing 70 micro-
liters of 2.2 mM nitroglycerin/saline solution on the ar-
tery. The recorded images of the artery were transferred
to a computer and the diameter changes were calculated
with a software (Image Processing and Analysis; Java
version 1.30). Changes in vessel diameter after restora-
tion of flow were expressed as percentage of the baseline
alues (before clamping or nitroglycerin). v

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Table 2. Experimental diets containing Chinese herbal prescriptions (Kampo) for blood stagnancy (Oketsu).
Ingredient Control diet
(High fat diet) Shimotsuto Juzentaihoto Unseiin
g/kg
Casein 232 232 232 232
Cystine 3 3 3 3
Corn starch 362 362 362 362
Sucrose 99.5 99.5 99.5 99.5
Soy oil 30 30 30 30
Butter 75 75 75 75
Beef tallow 100 100 100 100
Cellulose 50 50 50 50
Mineral mix 35 35 35 35
Vitamin mix 10 10 10 10
Choline bitartrate 2.5 2.5 2.5 2.5
tert-Butylhydroquinon 0.041 0.041 0.041 0.041
Cholesterol 0.5 0.5 0.5 0.5
Shimotsuto - 10 - -
Juzentaihoto - - 10 -
Unseiin - - - 10
Energy, KJ/100g 1956
2.7. Platelet Function Test
The shear-induced platelet reactivity test (haemosta-
tometry) was performed with a three-channel purpose-
built Haemostatometer constructed in the Physiology
Laboratory of the Faculty of Nutrition at Kobe Gakuin
University. Details of haemostatometry have been desc-
ribed in detail elsewhere [7,8]. In brief, non-anticoagu-
lated blood withdrawn from the abdominal aorta was
perfused through polyethylene tubing by oil displace-
ment technique and the perfusion pressure was continu-
ously monitored. When the pressure stabilized at 60
mmHg, through-holes were pierced in the tubing by a
fine needle. Escape of blood (“bleeding”) through the
pierced holes into the surrounding saline resulted in a
sharp drop and then a gradual return of the perfusion
pressure to the baseline level. The recovery of pressure
after the initial drop reflected platelet-rich haemostatic
plug formation in the pierced holes. The area of pressure
changes (mmHg.s) were calculated (H1 and H2) and
used as an index of platelet reactivity. An increase or
decrease of H1 and H2 over controls indicated sup-
pressed or enhanced platelet reactivity, respectively. The
time from the start of the test until the first decrease of
perfusion pressure of at least 10 mmHg (CT1) and to a
level not higher than 10 mmHg (CT2) reflected the ini-
tial and completed coagulations, respectively. Prolonga-
tion of CT1 and CT2 indicated inhibition of dynamic
coagulation, whereas shortening of CT1 and CT2 sug-
gested hypercoagulation.
2.8. Statistical Analysis
The effect of Shimotsuto, Juzentaihoto and Unseiin in
the applied tests groups was analysed by factorial
ANOVA, followed by the post hoc test of Fisher’s PLSD
using commercially available statistical package Stat
View (v. 5.0; SAS Institute Inc., North Carolina, USA).
Logarithmic H1, H2 data and FMV data were analyzed
by Student’s unpaired t-test; CT1 and CT2 values were
analyzed without logarithmic conversion. Results were
expressed as mean ± SEM. P < 0.05 was considered to
be statistically significant.
3. RESULTS AND DISCUSSIONS
3.1. Antithrombotic Effect of Diets
Containing Kampos
Results are shown in Figure 1. Long-term administration

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496
Openly accessible at
0
2
4
6
8
10
12
14
16
Control
Thrombogenicity (Mean±SEM)
Shimotsuto JyuzentaihotoUnseiin
(X10
7
)
****
0
2
4
6
8
10
12
14
16
Control
Thrombogenicity (Mean±SEM)
Shimotsuto JyuzentaihotoUnseiin
(X10
7
)
******
N = 5-6, *P < 0.05, **P < 0.01.
Figure 1. Antithrombotic effect of diets containing various
Kampo.
of all Kampo significantly decreased experimental thro-
mbogenicity.
Shimotsuto contains four components common in
three antithrombotic Kampos (Table 2): Japanese angel-
ica, Cnidium rhizome, Peony root and Rehmannia root.
In contrast, Juzentaihoto and Unseiin contain other addi-
tional components than the above common four. These
components are added to adjust the efficacy of medicine
to individual personality considering the physical and
mental characters. The present results show that anti-
thrombotic activity of the tested three Kampos derives
from Japanese angelica, Cnidium rhizome, Peony root
and Rehmannia root. As Unseiin showed the strongest
antithrombotic activity, its effect was further investi-
gated.
3.2. Effect of Unseiin on Endothelial
Function
The effect of diet containing Unseiin on endothelial
function was investigated after 12 weeks feeding by the
flow-mediated or endothelium-mediated vasodilation
(FMV) and nitroglycerin-mediated vasodilation (NMV)
techniques. Results are shown in Figure 2. Endothe-
lium-mediated vasodilation was significantly higher in
mice with Unseiin feeding than in the controls but Un-
seiin did not affect nitroglycerin-induced vasodilation.
Thus our finding suggests that Unseiin enhances endo-
thelial function but not medial or muscular layer of the
blood vessel.
Keishi-bukuryo-gan (Gui-zhi-fu-ling-wan) has been
used for the improvement of blood circulation and re-
cently it is often used to prevent arteriosclerosis. One of
the mechanisms involved is thought to be the improve-
ment of endothelial dysfunction [9]. Choto-san has also
been shown to improve blood circulation by protecting
endothelium [10].
Flow-mediated vasodilation test was developed for di-
agnosing arteriosclerosis in humans [11-15] and we have
employed this test to animal experiments [5,6]. Publica-
tions using FMV test are tremendously increasing in hu-
mans and demonstrating that nitric oxide and prostacyclin
are involved in FMV [16]. Unseiin enhances endothelial
function by nitric oxide and prostacyclin generation, and
this could be a mechanism of the inhibition of arterial
thrombosis.
3.3. Effect of Unseiin on Shear-Induced
Platelet Reactivity
The diet containing Unseiin was given to mice for 12
weeks and the effect of Unseiin on platelet function was
assessed by shear-induced platelet function test (haemo-
statometry) using non-anticoagulated blood. Results are
shown in Figure 3. Unseiin inhibited shear-induced plate-
let reactivity, but no effect on blood coagulation [7,8].
Kangen-karyu (KGK) is a traditional Chinese herbal
medicine to invigorate circulation. It contains 6 herbs,
peony root, cnidium rhizome, safflower, cyperus rhizome,
saussurea root (JP XIV), and Salvia miltiorrhiza root.
KGK significantly extended tail-bleeding time and sup-
pressed exvivo platelet aggregation in mice three days
after oral administration, while it did not extend proth-
rombin time. These findings suggest that the anti-
thrombotic effect of KGK may be due to the inhibition
Relative vessel diameter (%)
FMV NMV
100
120
140
160
control Unseiin
*
N.S.
100
120
140
160
control Unseiin
Relative vessel diameter (%) Relative vessel diameter (%)
FMV NMV
100
120
140
160
control Unseiin
*
N.S.
100
120
140
160
control Unseiin
Relative vessel diameter (%)
FMV: n = 8, *P = 0.023, NMV: n = 7, N.S.: not significant (P = 0.199).
Figure 2. Effect of diets containing Unseiin on flow-mediated
vasodilation (FMV) and nitroglycerin-mediated vasodilation
(NMV).
0
2
4
6
8
(x10
3
)
(mmHg・sec)
5
8
9
10
7
6
(min)
H2 CT1
*
N.S.
control Unseiin
control Unseiin
0
2
4
6
8
(x10
3
)
(mmHg・sec)
5
8
9
10
7
6
(min)
H2 CT1
*
N.S.
control Unseiin
control Unseiin
H2: n = 4, *P = 0.013; CT1: n = 4, N.S.: not significant (P = 0.643).
Figure 3. Effect of diets containing Unseiin on shear-induced
platelet reactivity and dynamic coagulation.

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Openly accessible at
of platelet aggregation but not to anticoagulation [17].
The components, peony root and cnidium rhizome are
common in KGK and in the currently investigated three
Kampos. Peony root and cnidium rhizome might play a
major role in antithrombotic activity of Kampo.
Agonist-induced platelet aggregation tests were used
to assess of antithrombotic effect of Gosha-jinki-gan
[18]. However, there are differences in the effects ob-
tained by agonist-induced and shear-induced platelet
function test [19]. We have compared agonist-induced
platelet function test using anticoagulated whole blood
and shear-induced platelet function test using non-anticoa-
gulated whole blood, and concluded that shear-induced
platelet function test using non-anticoagulated whole
blood is physiologically more relevant to platelet func-
tion existing in vivo.
In conclusion, long-term intake of Shimotsuto, Juzen-
taihoto and Unseiin inhibited arterial thrombogenesis by
their anti-platelet activities and by stimulating the vas-
cular endothelium.
4. ACKNOWLEDGEMENTS
We thank Mrs Kinuyo Matsumoto, Ms Miho Hayashi and Ms Ikue Ku-
suda, Faculty of Health and Welfare, Kobe Women’s University for their
help of animal care.
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