Chinese Medicine, 2011, 2, 62-70
doi:10.4236/cm.2011.22012 Published Online June 2011 (htt p://www.SciRP.org/journa l/cm)
Pharmacological Modulation by Shakuyakukanzoto
(Shao-Yao-Gan-Cao-Tang) and the Ingredients
in Rat Intestinal Smooth Muscle
Hiroyasu Satoh, Kiminori Tsuro
Departm e nt of P harmacol ogy, Traditional Herbal Medicine, School of Medi c ine,
Nara Medical University, Kashihara, Japan
Received January 21, 2011; revised March 10, 2011; accepted May 24, 2011
Abstract
Shakuyakukanzoto (Shao-Yao-Gan-Cao-Tang), a formulation of Japanese herbal (Kampo) medicines, is
composed of Paeoniae Radix and Glycyrrhizae Radix. Effects of Shakuyakukanzoto and the ingredients on
rat intestinal tract were ex amined. Shakuyakuk anzoto (0.01 - 0.3 mg/ml) relaxed a carbachol (CCh, 0.3 μM)
-induced contraction in a concentration-dependent manner. Both components (Paeoniae Radix and Glycyr-
rhizae Radix) also relaxed the CCh-induced contraction. At 0.1 to 1 mM, their constituents (paeoniflorin and
glycyrrhetic acid) and the metabolic products (18-α- and 18-β-glycyrrhetinic acids) exerted almost the same
actions. The relaxations induced by Shakuyakukanzoto were not modified by 1 μM nicardipine, 10 μM su-
ramin (ATP receptor inhibitor) and several K+ channel inhibitors, but was attenuated by 20 μM IBMX (a
phosphodiesterase inhibitor). Also, IBMX inhibited the relaxations induced by paeoniflorin and glycyrrhetic
acid, but not by other ingredients. Nicardipine decreased the relaxation of just 18-α-glycyrrhetinic acid. Even
in non-treatment with CCh, Shakuyakukanzoto relaxed the intestinal tract. CCh (0.3 μM) elicited spontane-
ous contractions in 23% specimens, depressed by application of Shakuyakukanzoto. These results indicate
that Shakuyakukanzoto causes a remarkable relaxation by the anti-cholinergic and the PDE inhibitory actions,
but by minor contribution of Ca2+ channel inhibition. Thus, Shakuyakukanzoto exerts an anti-spasmodic ac-
tion due to the interaction with pharmacological effects of its ingredients.
Keywords: Shakuyakukanzoto, Paeoniae Radix, Paeoniflorin, Glycyrrhetic Acid, PDE Inhibition,
Anti-Cholinergic Action, Ca2+ Channel Inhibition, Intestinal Tract
1. Introduction
Since traditional Japanese herbal (Kampo) medicines are
composed of a mixture with lots of herbs, they produce
multiple pharmacological and physiological functions.
Shakuyakukanzoto (Shao-Yao-Gan-Cao-Tang), a kind of
Kampo formulations, is composed of just two compo-
nents; Paeoniae Radix and Glycyrrhizae Radix. The main
ingredient of Paeoniae Radix is paeoniflorin, and that of
Glycyrrhizae Radix is glycyrrhetic acid.
Shakuyakukanzoto has been mostly used for the re-
laxant effect of skeletal muscle [1]. Nicotinic ACh re-
ceptors on neuromuscular junction play an important role
for the contraction. Paeoniflorin produced the relaxation
by means of a depolarized blockade like succinylcholine
[2]. Paeoniflorin regulates Ca2+ movement near around
neuromuscular junction, and glycyrrhetic acid inhibits
Ca2+-activated K+ (IKCa) channel to repolarize or hyper-
polarize the membrane [3]. The combination with Paeo-
niae radix and Glycyrrhizae radix enforces the relaxant
action of skeletal muscle.
Also, Shakuyakukanzoto may be useful to relieve a
pain, and exhibit an anti-spasmodic action in gastrointes-
tinal smooth muscle [4]. The relaxations of smooth mus-
cles induced by Kampo medicines depend on mainly a
phosphodiesterase (PDE) inhibition [5]. Most recent re-
ports have also demonstrated to play a key role for regu-
lation of the gap junction on gastrointestinal smooth
muscle [6-8].
Until now, there is less information of more detailed
pharmacological mechanisms for the gastrointestinal ac-
tions of Shakuyakukanzoto, the components (Paeoniae

H. SATOH ET AL.
63
Radix and Glycyrrhizae Radix), especially the ingredients
(paeoniflorin and glycyrrhetic acid) and the metabolic
products (18-β- and 18-α-glycyrrhetinic acids). In the
present experiments, therefore, the pharmacological ac-
tions of Shakuyakukanzoto and the ingredients on the re-
laxation were investigated using rat intestinal smooth
muscle.
2. Material and Methods
All experiments were carried out, according to the guide-
lines laid down by the Nara Medical University Animal
Welfare Committee, and also under the terms of the
Declaration of Helsinki.
2.1. Experimental Procedures
Wistar rats (8 to 15 weeks-old), weighing approximately
300 g, were anesthetized with ether, and euthanized by
exsanguination. The intestinal tract was quickly removed,
and the isolated intestinal tract was cut into rings of 1.5 cm
in length. The strips were suspended in a jacketed organ
chamber fill ed wit h 20 m l m odifi ed Ty rode s olution.
The strips were suspended between both sides with
stainless st e el st irrups. The lower s tirrup was anchore d a nd
the upper stirrup was attached to a force-displacement
transducer (Nihon Kohden TB-652T, Tokyo, Japan) to
record the isometric force. All strips were stretched to
generate a resting tension of 1.0 g, which was optimal fo r
contractions with muscarin ic ACh receptor agon ist. After
40 min of resting, carbachol (CCh, 0.3 μM) was added to
the tissue bath. After the contractile response became
steady, the drugs were cumulatively administrated into
the bath solution. The effects of each con centration of th e
drugs were measured 5 - 7 min after the responses beca me
steady. To examine the involvement with Ca2+ channel,
PDE or other mechanisms, the pretreatment with 1 μM
nicardipine, 20 μM IBMX or oth er inhibitors was carried
out. Each of the experiments was examined at least qua-
druplicates. The responses were analyzed as a percentage
change from the value before an application of drugs.
2.2. Experiments of Spontaneous Contractions
Pretreatment with CCh (0.3 μM) was usually carried out.
Under the conditions, the spontaneous contractions were
exhibited occasionally in some specimens. The effects of
Shakuyakukanzoto on the spontaneous contractions were
investigated.
2.3. Experiments in the Absence of CCh
Using rat ileum in non-pretreatment with CCh, the ef-
fects of Shakuyakukanzoto and the ingredients on ga-
strointestinal smooth muscle were examined using the
same experimental techniques.
2.4. Solution and Drugs
The modified Tyrode solution was comprised of (in mM);
136.8 mM NaCl, 2.7 mM KCl, 1.8 mM CaCl2, 1.1 mM
MgCl2, 0.4 mM NaH2PO4, 11.9 mM NaHCO3, and 5.6
mM glucose. The chamber solution was kept at 36.5˚C
and oxygenated with 95% O2 and 5% CO2.
The drugs used were Shakuyakukanzoto, Paeoniae
Radix and Glycyrrhizae Radix (Tsumura Co., Tokyo,
Japan), as a spray-dried powder extracted with boiling
water of a ground raw materials. Each drug was dis-
solved with DMSO. Other drugs used were CCh,
18-α-glycyrrhetinic acid and 18-β-glycyrrhetinic acid
(Sigma Chemical, MO. U.S.A.), and paeoniflorin and
glycyrrhtic acid (Wako Chemical, Kyoto, Japan). Nicar-
dipine (Ca2+ channel inhibitor), Bay K 8644 (Ca2+ chan-
nel stimulator), suramin (ATP receptor inhibitor), apa-
min (Ca2+-activated K+ channel inhibitor), glibencl amide
(ATP-sensitive K+ channel inhibitor), tetraethyammo-
nium (TEA, voltage-dependent K+ channel inhibitor) and
3-isobutyl-1-methylxanthine (IBMX, phosphodiesterase
inhibitor) (Sigma) were also used.
2.5. Statistical Analyses
To compare the pair values, we are used statistical me-
thods of the Student’s t-test and ANOVA followed by
post-hoc tests (Dunn-Bonferonii test) using Excel (Mi-
crosoft Inc., Washington, U.S.A.) and S-PLUS (Mathe-
matical System Inc., Washington, U.S.A.). All values are
represented as means ± SEM. A p value of less than 0.05
was considered significant.
3. Results
3.1. Effects of Shakuya kukan zoto on CCh-Induced
Contraction
Pretreatment with 0.3 μM CCh produced a strong con-
traction of isolated ileum; by 1.4 ± 0.2 g (n = 157). Then,
Shakuyakukanzoto (0.01 to 0.3 mg/ml) was admini-
strated cumulatively into the bath, and at over 0.1 mg/ml
significantly relaxed the CCh-indu ced contraction; at 0.3
mg/ml by 27.7 ± 3.3% (n = 12, P < 0.001). The res-
ponses were concentration-dependent. These results are
summarized in Table 1.
The relaxation induced by Shakuyakukanzoto (0.3
mg/ml) increased (by 37.7 ± 2.0%, n = 8, P < 0.001) at 1
μM nicardipine, but decreased to 15.1 ± 3.2% (n = 5, P <
0.05) at 20 μM IBMX. Interestingly nicardipine rather

H. SATOH ET AL.
64
Table 1. Relaxant effects of shakuyakukanzoto and the components on CCh-induced con t raction.
n 0.01 0.03 0.1 0.3 mg/ml
Shaku yakukanzoto
Control 12 0.2 ± 0.1 1.8 ± 1.1 11.1 ± 2.7*** 27.7 ± 3.3***
Nicardipine 1 μM 8 1.0 ± 0.5 11.5 ± 2.7*,## 21.8 ± 2.3**,# 37.7 ± 2.0***,#
IBMX 20 μM 5 0 ± 0 0 ± 0 6.0 ± 2.6# # 15.1 ± 3 .2*,#
Paeoniae Radix
Control 8 2.3 ± 1.3 4.3 ± 2.8 10.5 ± 4.5** 27.3 ± 6.3**
Nicardipine 1 μM 6 1.1 ± 0.4 9 .6 ± 2.4*,# 13.6 ± 3. 2 * 25.4 ± 2.6* *
IBMX 20 μM 6 0 ± 0 0 ± 0 10.4 ± 2.3* 24.7 ± 3.3**
Glycyrrhiziae Radix
Control 8 0 ± 0 0 ± 0 5.6 ± 1.8* 19.2 ± 2.2***
Nicardipine 1 μM 6 1.3 ± 0.5 6.4 ± 1.7*,# 11.3 ± 1 .5*,# 20.7 ± 1.8**
IBMX 20 μM 9 1.6 ± 1.0 8. 3 ± 2.5**,# 21.2 ± 2.8**,# 34.7 ± 3.0**,##
Values (%) are represent ed as mean ± S.E.M. *,#: P < 0.05, **,##: P < 0.01, ***: P < 0.001, * means a significant difference be-
tween the value at each concentration and control value. # means a significant difference of the values in the presence, as com-
pared with the values in the absence of inhibitors at each concentration.
enhanced the relaxation concentration-dependently. Sha-
kuyakukanzoto also did not affect on Bay K 8644 (3
nM)-induced contraction significantly.
In addition, the relaxation induced by Shakuyakukan-
zoto (0.3 mg/ml) was not modified by 100 μM suramin,
1 μM glibenclamide, and 100 mM tetraethyammonium
(TEA). Apamin at 0.1 μM attenuated the Shakuyaku-
kanzoton-induced relaxation, but did not cause it to sig-
nificant extent.
3.2. Modulation by Paeoniae Radix and
Glycyrrhizae Radix
At 0.01 - 0.3 mg/ml, the components of Paeoniae radix
and Glycyrrhizae Radix relaxed the CCh-induced con-
traction concentration-dependently (Table 1). Their re-
laxant effects at 0.1 mg/ml was 10.5 ± 4.5% (n = 8, P <
0.01) and 5.6 ± 1.8% (n = 8, P < 0.05), respectively.
Glycyrrhizae Radix had the weaker relaxation at all
ranges of c oncentrat i ons.
The relaxation against the CCh-induced contraction
was enhanced by nicardipine. The CCh-induced contrac-
tion was relaxed by 25.4 ± 2.6% (n = 6, P < 0.01) at 0.3
mg/ml Paeoniae Radix, but even low concentrations
(0.03 mg/ml) of Paeoniae radix enhanced the relaxation,
as compared with the value in the absence of nicardipine.
Glycyrrhizae Radix (0.01 to 0.3 mg/ml) also enhanced
the relaxation concentration-dependently; at 0.3 mg/ml
by 20.7 ± 1.8% (n = 6, P < 0.01).
On the other hand, in the presence of 20 μM IBMX,
Paeoniae Radix at 0.1 mg/ml relaxed the CCh-induced
contraction by 10.4 ± 2.3% (n = 6, P < 0.05). And 0.1
mg/ml Glycyrrhizae Radix potentiated the relaxation by
21.2 ± 2.8% (n = 9, P < 0.01). Glycyrrhizae Radix pro-
duced the stronger relaxation in the presence of IBMX.
These results indicate that both components make minor
contribution to the PDE- and Ca2+ channel-dependent
relaxations.
3.3. Effects of Paeoniflorin and Glycyrrhetic Acid
Paeoniflorin, a constituen t of Paeoniae Radix, a t 0.1 to 1
mM relaxed the CCh-induced contraction (Table 2). At
0.3 mM, the relaxing effect was 16.3 ± 6.3% (n = 4, P <
0.05). Glycyrrhetic acid, a constituent of Glycyrrhizae
radix, at 0.3 mM also relaxed by 6.4 ± 2.0% (n = 4, P <
0.05). These responses behaved concentration-depen-
dently. Paeoniflorin exhibited a stronger relaxation than
glycyrrhetic acid.
Paeoniflorin markedly attenuated the CCh-induced
contraction, but not in the presence of nicardipine. The
relaxation induced by paeoniflorin (1 mM) was 20.5 ±
2.9% (n = 8, P < 0.01) at 1 μM nicardipine and 4.7 ±
0.4% (n = 6, P > 0.05) at 20 μM IBMX. Glycyrrhetic
acid (1 mM) also relaxed the contraction by 16.4 ± 2.1%
(n = 6, P < 0.01) at 1 μM nicardipine, and by 7.1 ± 2.7%
(n = 6, P > 0.05) at 20 μM IBMX. Both glycyrrhetic acid
and paeoniflorin had no Ca2+ channel inhibitory action,
but possessed the PDE inhibitory action (P < 0.05 -
0.01).
3.4. Effects of 18-β- and 18-α-Glycyrrhetic Acids
Metabolic products (bioactive components) of glycyr-
rhetic acid are 18-β-glycyrrhetinic acid (a main product)
and 18-α-glycyrrhetinic acids. Both products (0.1 to 1

H. SATOH ET AL.
65
Table 2. Relaxant effects of the constituents and the products from Shakuyakukanzoto on CCh-in-
duced contr action.
n
0.1
0.3
1 mM
Paeoniflorin
Control
4
4.1 ± 1.6*
16.3 ± 6.3*
19.8 ± 6.9*
Nicardipine 1 μM
8
6.2 ± 0.4
14.2 ± 2.0*
20.5 ± 2.9**
IBMX 20 μM
6
0 ± 0
1.7 ± 0.8#
4.7 ± 0.4# #
Glycyrrhetinic acid
Control
4
0.9 ± 0.6
6.4 ± 2.0*
12.1 ± 3.5*
Nicardipine 1 μM
6
5.1 ± 2.1*, #
9.0 ± 1.7
16.4 ± 2.1* *,#
IBMX 20 μM 6 0.8 ± 0.8 1.7 ± 1.1
##
7.1 ± 2.7
#
18-β-glycyrrhetinic acid
Control
8
1.4 ± 1.4
2.5 ± 1.6
15.1 ± 6.1*
Nicardipine 1 μM 11 6.7 ± 1. 4 15.8 ± 4.5**
,##
25.2 ± 4.4**
IBMX 20 μM
8
5.3 ± 3.6
10.5 ± 4.3*
18.8 ± 3.3**
18-α-glycyrrhetinic acid
Control
5
0 ± 0
7.0 ± 2.6
18.6 ± 3.8* *
Nicardipine 1 μM
6
0 ± 0
-3.1 ± 1.9**,# #
-23.2 ± 5.0**,###
IBMX 20 μM
6
7.7 ± 2.1*,#
12.4 ± 2.9*
20.5 ± 3.3* *
Values (%) are represented as mean ± S.E.M. *,#: P < 0.05, **,##: P < 0.01, ###: P < 0.001, * means a significant difference between
the valu e at each con centr ation and co ntro l valu e. # means a sig nifi cant d ifferen ce of t he val ues i n the p resen ce, as co mpared with
the values in the absence of inhibitors at each concentration.
mM) also relaxed the CCh-induced contraction concen-
tration-dependently (Table 2). At 1 mM, the relaxing
effects of 18-α- and 18-β-glycyrrhetinic acids were 18.6
± 3.8% (n = 5, P < 0.01) and 15.1 ± 6.1% (n = 8, P <
0.05), respectively.
In the presence of 1 μM nicardipine, 18-β-glycyrrhe-
tinic acid (1 mM) relaxed the CCh-induced contraction
by 25.2 ± 4.4% (n = 11, P < 0.01), stronger relaxation
than control value. On the other hand, 18-α-glycyrrhe-
tinic acid (1 mM) caused rather a contraction by 23.2 ±
5.0% (n = 6, P < 0.01). In the presence of 20 μM IBMX,
18-β- and 18-α-glycyrrhetinic acids at 1 mM did not af-
fect it; by 18.8 ± 3.3% (n = 8, P < 0.01) and by 20.5 ±
3.3% (n = 6, P < 0.01), respectively.
3.5. Effects on Spontaneous Contractions
Prior administration of CCh (0.3 μM) elicited occasio-
nally spontaneous contraction in some specimens (Fig-
ure 1(a)). The incidence was in 36 out of 157 specimens
(approximately 23%). Under the condition, application
of Shakuyakukanzoto (0.01 to 0.3 mg/ml) depressed the
amplitude and prolonged the cycle length of spontaneous
contractions (Figures 1(b) and (c)). The depression be-
haved in a concentration-dependent manner.
3.6. Effects in th e Absence of CCh
Using rat ileum in non-pretreatment with CCh, the ef-
fects of Shakuyakukanzoto and the ingredients on ga-
strointestinal smooth muscle were examined. Under the
condition, Shakuyakukanzoto (0.01 - 0.3 mg/ml) by itself
similarly caused the relaxations; at 0.3 mg/ml by 12.6 ±
3.1% (n = 13, P < 0.05) (Table 3(A)). The components
of Paeoniae radix and Glycyrrhizae radix also had simi-
lar relaxant actions; at 0.3 mg/ml by 16.3 ± 3.6% (n = 12,
P < 0.001) and by 17.5 ± 3.3% (n = 16, P < 0.001), re-
spectively. The response was weaker, and was 19.2 ±
2.2% (n = 8, P < 0.001) at 0.3 mg/ml in CCh-treated
ileum, although the effects of Glycyrrhizae radix were
not affected.
At 0.1 - 1 mM, the constituents (paeoniflorin and gly-
cyrrhetic acid) and metabolic products (18-β- and 18-α-
glycyrrhetinic acids) similarly relaxed the intestinal
smooth muscle concentration-dependently (Table 3(B)).
The relaxations induced by paeoniflorin and glycyrrhetic
acid at 1 mM were 2.7 ± 0.9% (n = 4, P < 0.05) and 5.4 ±
1.0% (n = 4, P < 0.01), r espectively. And 18 -β- and 18-α
-glycyrrhetinic acids at 1 mM also decreased by 16.1 ±
4.8% (n = 6, P < 0.01) and by 14.3 ± 2.1% (n = 4, P <
0.01), respectively. The relaxations were observed more
markedly in the pre s e nce of CCh.
4. Discussions
The present experiments showed that Shakuyakukanzoto
and its ingredients caused the potent relaxant action on
the CCh-induced contraction. They possess an-
ti-cholinergic action. Also the relaxations were due to
PDE inhibition or Ca2+ channel inhibition. Shakuyaku-

H. SATOH ET AL.
66
(a)
(b) (c)
Figure 1. Spontaneous contractions induced by CCh. (a) Concentration-dependent suppression by Shakuya-
kukanzoto of the spontaneous contractions. (b) Changes in amplitude of the contractions. (c) Changes in cycle
length of the contractions. Values (%) represent mean ± S.E.M. *: P < 0.05, **: P < 0.01, ***: P < 0.001, with
respect to control value.
Table 3. Comparative relaxing actions of Shakuyakukanzoto, its constituents and the products on the ileum in
the absence of CCh.
A n 0.01 0.03 0.1 0.3 mg/ml
Shakuyakukanzoto 13 1. 6 ± 0.3* 3.0 ± 0. 4 * 6.3 ± 1.1** 12.6 ± 3.1*
Paeoniae radix 12 3.3 ± 0.6* 4.8 ± 1.8 * 8.4 ± 2.3* 16.3 ± 3.6***
Glycyrrhizae radix 16 1.6 ± 0.4 4.8 ± 0.3** 11.5 ± 2.4*** 17.5 ± 3.3***
B n 0.1 0.3 1 mM
Paeoniflorin 4 0.2 ± 0.1 2.8 ± 0.3* 2.7 ± 0.9*
Glycyrrhetinic acid 4 0.1 ± 0.2 2.9 ± 1.0* 5.4 ± 1.0**
18-β-glycyrrhetinic acid
6
2.0 ± 1.1
2.4 ± 1.2
16.1 ± 4.8**
18-α-glycyrrhetinic acid 4 0.3 ± 0.2 1.8 ± 2.4 14.3 ± 2.1**
Values (%) represent mean ± S.E.M. *: P < 0.05, **: P < 0.01, ***: P < 0.001, with respect to control value.
kanzoto relaxed the intestinal tract even in
non-pretreatment with CCh. Occasionally CCh elicited
the spontaneous contractions in some specimens, and the
application of Shakuyakukanzoto depresse d t he m.
4.1. Anti-Cholinergic Action
Prior administration of CCh produced a marked contrac-
tion of isolated ileu m, consistent with previous report [9].
Shakuyakukanzoto relaxed the CCh-induced contra ction.
Paeoniae Radix and Glycyrrhizae Rad ix also relaxed it.
At the same concentrations, Shakuyakukanzoto and
Paeoniae Radix had stronger relaxing effect than Gly-
cyrrhizae Radix. Even in the absence of CCh, Shakuya-
kukanzoto itself caused the relaxation and the related
compounds also did it, but had much weaker relaxations
0.01
0.03
0.1
0.3 mg/ml
Shakuyakukanzoto

H. SATOH ET AL.
67
as compared with those in presence of CCh. Glycyrrhi-
zae Radix makes minor contribution to the CCh-induced
relaxation, because it also relaxed the ileum without
treatment with CCh to almost the same extent.
Paeoniae Radix has been reported to inhibit the con-
tractions induced by nicotin e and electric stimulations [3].
The contractions are produced by ACh release from
nerve ending mediated through neuroganglionic ACh
receptors. Paeoniflorin prevents the damages mediated
through muscarinic (M1) receptor in rat hipp ocampal ( CA1)
neurons and ameliorates the dysfunctions [10,11]. In the
present experiments, both paeoniflorin and glycyrrhetic
acid relaxed the CCh-induced contraction. Paeoniflorin
had stronger relaxation. 18-α- and 18-β-glycyrrhetinic
acids also had the concentration-dependent relaxation.
These findings indicate that Shakuyakukanzoto and its
containing compounds exhibit the anti-cholinergic action,
presumably mediated through M1 (or M3) receptor. Espe-
cially Paeoniae Radix and paeoniflorin in Shakuyakukan-
zoto possess the s tronge r ant i-cholinergic action.
4.2. Ca2+ Channel Inhibition
The inhibition of Ca2+ channel has previously been
shown. Paeoniflorin inhibits Ca2+ current in NG108-15
neuronal ce lls, and Na+ current in hippocampus neurons
[12], presumably leading to the relaxation of intestinal
tract. The inhibition of Na+ current results in a decline
of cellular Ca2+ concentration ([Ca2+]i) via Na/Ca ex-
change. Paeoniflorin also relaxes the isolated rat aorta
due to the [Ca2+]i decline and the increases in NO and
cGMP [13].
In the present experiments, however, Shakuyakukan-
zoto and the containing compounds had no Ca2+ channel
inhibitory action. This is supported by the minor action of
Shakuyakukanzoto on Bay K 8644 (3 nM)-induced con-
traction. Interestingly, they d ecreased the relaxant actions
by addition of nicardipine. Only 18-α-glycyrrhetinic acid
had the inhibitory action of Ca2+ channel, and produced
rather the contraction. The mechanisms are now unclear
yet, but cellular signaling pathways such as Rho kinase,
MLCK and P K-C might be involved [14-17].
Therefore, Shakuyakukanzoto and the related com-
pounds have less or no effect on Ca2+ channel, but might
exert the relaxation by the inhibitions of Na+ and KCa
channels and the activation of cGMP signaling pathway.
4.3. PDE Inhibition
Shakuyakukanzoto and the ingredients caused the strong
PDE inhibitory action. Especially, Glycyrrhizae Radix,
paeoniflorin and glycyrrhetic acid were marked. The
PDE inhibition (as a result cAMP accumulation) can
produce the potent relaxation of smooth muscle. Most
Kampo formulations have been reported to exert the
PDE inhibitory action [5]. The PDE inhibition was ob-
served in paeoniflorin but not in Paeoniae Radix. So, the
resultant effects are responsible for an interaction among
the containi ng compounds.
4.4. Depression of the Spontaneous Contractions
and Spasms
The smooth muscle cells may exhibit the spontaneous
contractions. The foundation of pacemaker mechanisms
is very similar to that of sino-atrial (SA) nodal cells of
heart [18]. It is a pendulum movement with a repetitive
depolarization and repolarization. In rat pregnant uterus
smooth muscle cells with spontaneous contractions, a
hyperpolarization-activated inward (or pacemaker) cur-
rent (If), is similarly identified [19]. In general, the spon-
taneous activity of smooth muscle cells may be largely
dependent on transient Ca2+ sparks. Interstitial cells of
Cajal, gastrointestinal pacemakers, exhibit Ca2+ release
from IP3-dependent Ca2+ stores by activating a Ca2+-
dependent cationic current that drives pacemaker depola-
rization [20]. The [Ca2+]i elevation activates the KCa
channels to produce the repolarization of spontaneous
action potentials [21], as well as the Ca2+-activated Cl-
channels to produce the depolarization during pacemaker
potential [22]. Most recently in guinea pig SA nodal cells,
however, we have been found minor contribution of
transient Ca2+ sparks [23]. It is not yet clear now, but
might be closely related with the connection of Ca2+
channels on the plasma membrane and the sarcoplasmic
reticulum (SR) in interstitial smooth muscle.
In this study, after administration of CCh, spontaneous
contractions occurred with approximately 23% incidence.
The KCa channel in aortic smooth muscle cells is inhi-
bited by β-adrenoceptor and muscarinic receptor stimula-
tions and also by PK-C stimulation [24]. Glycyrrhetic
acid also inhibits KCa channel [3]. In this study, applica-
tion of Shakuyakukanzoto ceased the spontaneous con-
tractions. Thus, the depression would be partly due to
inhibition of KCa channel and regulation of Ca2+ move-
ment near around neuromuscular junction, because of
minor contribution of Ca2+ channel.
In skeletal muscles, nicotinic ACh receptor on neuro-
muscular junction plays an important role for the con-
traction. Paeoniflorin produces the relaxation by means
of a depolarized blockade like succinylcholine [2]. Paeo-
niflorin regulates Ca2+ movement near around neuro-
muscular junction, and glycyrrhetic acid inhibits KCa
channel [3]. In rat hippocampal slice, furthermore, the
dependence of anti-cholinergic action has been found
[10]. The combination with Paeoniae Radix and Glycyr-
rhizae Radix would enforce the relaxant action of skelet-
al muscle. Most recent reports have demonstrated that

H. SATOH ET AL.
68
Table 4. Pharmacological actions of Shakuyakukanzoto and the ingredients.
Anti-cholinergic action Ca2+ channel inhibition PDE inhibition
Shakuyakukanzoto + – +
Paeoniae radix + – –
Glycyrrhiziae radix – – –
Paeoniflorin + – +
Glycyrrhetinic acid + – +
18-β-glycyrrhetinic acid + – –
18-α-glycyrrhetinic acid + + –
Shakuyakukanzoto may be sufficiently effective for
spasmodic diseases of gastrointestinal tract, as well as
for cramp and twitch of skeletal muscle. The anti-
spasmodic and muscle relaxing effects are exerted not
only by the anti-cholinergic action (mediated through
nicotinic and muscarinic ACh receptors) but also by
PDE inhibitory actions. Glycyrrhetic acid and the me-
tabolic products can slow myocardial conduction via
modulation of gap junction, but no t via that o f the ion ic
channels [25]. Also 18-β- and 18-α-glycyrrhetinic acids
exert a blocking action of gap junction [6,8]. The
blockade of gap junction prevents spasmodic diseases
in gastrointestinal smooth muscle, and reduces epilep-
togenicity and arrhythmogenesis [7]. Thus, the phar-
macokinetic properties may be well suitable for the
transient clamp in leg skeletal muscle and the gastroin-
testinal spasms.
5. Conclusions
Shakuyakukanzoto possesses the higher bioactivities for
rat ileum, and is so effective for many diseases in clinical
uses. Shakuyakukanzoto produced a remarkable relaxa-
tion by 1) the anti-cholinergic and 2) the PDE inhibitory
actions, but by 3) minor contribution of Ca2+ channel
inhibition. Also, Shakuyakukanzoto exerted an anti-
spasmodic action due to the interaction with pharmaco-
logical effects of its ingredients.
The absorption is rapid from intestine, and the plasma
concentration-time curves are fitted with a mean terminal
half-life (T1/2) of 116.2 min [26]. Glycyrrhetic and 18-β-
glycyrrhetinic acids possess a free radical scaven ging
property [27,28], and anti-allergic activi ti es su ch as p ass iv e
cutaneous anaphylaxis and skin contact inflammation
[29,30]. However, the pathological findings of the meta-
bolic products from glycyrrhetic acid have also been well
known [31].
Finally, the pharmacological characteristics of Sha-
kuyakukanzoto, the ingredients and the related com-
pounds are summarized on Table 4. Further extensive
studies are needed to elucidate in more detail mechan-
is ms.
6. Acknowledgements
The authors wish to express thanks for the supply of
Shakuyakukanzoto extract (Tsumura Co.).
7. References
[1] M. Kimura, I. Kimura, K. Takahashi, M. Muroi, M. Yo-
shizaki, M. Kanaoka and I. Kitagawa, “Blocking Effects
of Blended Paeoniflorin or Its Related Compounds with
Glycyrrhizin on Neuromuscular Junctions in Frog and
Mouse,” Japanese Journal of Pharmacology, Vol. 36, No.
3, 1984, pp. 275-282. doi:10.1254/jjp.36.275
[2] M. Kimura, I. Kimura and H. Nojima, “Depolarizing
Neuromuscular Blocking Action Induced by Electro-
pharmacological Coupling in the Combined Effect of
Paeoniflorin and Glycyrrhizin,” Japanese Journal of
Pharmacology, Vol. 37, No. 4, April 1985, pp. 395-399.
doi:10.1254/jjp.37.395
[3] M. Kimura, I. Ki mura and M. Kimura, “Dec reasing Effects
by Glycyrrhizin and Paeoniflorin on Intracellular Ca2+- Ae-
quorin Luminescence Transients with or without Caffeine in
Directly Stimulated-Diaphragm Muscle of Mouse,” Japa-
nese Journal of Pharmacology, Vol. 39, No. 3, November
1985, pp. 387-390. doi:10.1254/jjp.39.387
[4] J. X. He, E. Goto, T. Akao and T. Tani, “Interaction be-
tween Shaoyao-Gancao-Tang and a Laxative with Respect
to Alteration of Paeoniflorin Metabolism by Intestinal Bac-
teria in Rats,” Phytomedicine, Vol. 14, No. 7-8, August
2007, pp. 454-459. doi:10.1016/j.phymed.2006.09.014
[5] Y. Saegusa, A. Sugiyama, A. Takahara, Y. Nagasawa and
K. Hashimoto, “Relationship between Phoshpodiesterase
Inhibition Induced by Several Kampo Medicines and
Smooth Muscle Relaxation of Gastrointestinal Tract Tis-
sues of Rats,” Journal of Pharmacology, Vol. 93, No. 1,
2003, pp. 62-68.
[6] Y. Takeda, S. M. Ward, K. M. Sanders and S. D. Koh,
“Effects of the Gap Junction Blocker Glycyrrhetinic Acid
on Gastrointestinal Smooth Muscle Cells,” American
Journal of Physiology, Gastrointestinal Liver Physiology,
Vol. 289, No.4, April 2005, pp. G832-G841.
doi:10.1152/ajpgi.00389.2004
[7] Z. Gajda, E. Hermesz, E. Gyengesi, Z. Szupera and M.
Szente, “The Functional Significance of Gap Junction
Channels in the Epileptogenicity and Seizure Susceptibil-
ity of Juvenile Rats,” Epilepsia, Vol. 47, No. 6, 2006, pp.
1009-1022. doi:10.1111/j.1528-1167.2006.00573.x

H. SATOH ET AL.
69
[8] E. E. Daniel, A. F. EI-Yazbi, M. Mannarino, G. Galante,
G. Boddy, J. Livergant and T. E. Oskouei, “Do Gap Junc-
tions Play a Role in Nerve Transmissions as well a s Pac-
ing in Mouse Intestine,” American Journal of Physiology,
Gastrointestinal Liver Physiology, Vol. 292, No. 3, 2007,
pp. G734-G745. doi:10.1152/ajpgi.00428.2006
[9] A. Sugaya, H. Okita, Y. Takazu and E. Sugaya, “Neru-
pharmacological Study of Saiko-keisi-to,” Shoyaku, Vol.
29, 1975, pp. 160-165.
[10] K. Tabata, K. Matsumoto and H. Watanabe, “Paeoniflorin,
a Major Constituent of Peony Root, Reverses Muscarinic
M1-Receptor Antagonist-Induced Suppression of Long-
Term Potentiation in the Rat Hipp ocampal Slice ,” Japanese
Journal of Pharmacology, Vol. 83, 2000, pp. 25-30.
doi:10.1254/jjp.83.25
[11] J. Liu, D. Z. Jin, L. Xiao and X. Z. Zhu, “Paeoniflorin
Attenuates Chronic Cerebral Hypoperfusion-Induced
Learning Dysfunction and Brain Damage in Rat,” Brain
Research, Vol. 1089, No. 1, May 2006, pp. 162-170.
doi:10.1016/j.brainres.2006.02.115
[12] G. Q. Zhang, X. M. Hao, S. Z. Chen, P. A. Zhou, H. P.
Cheng and C. H. Wu, “Blockade of Paeoniflorin on So-
dium Current in Mouse Hippocampal CA1 Neurons,” Ac-
ta Pharmacologica Sinica, Vol. 24, No. 12, December
2003, pp. 1248-1252.
[13] T. Y. Tsai, S. N. Wu, Y. C. Liu, A. Z. Wu and Y. C. Tsai,
“Inhibitory Action of L -type C a2+ Current by Paeoniflorin,
a Major Constituent of Peony Root, in NG108-15 Neu-
ronal Cells,” European. Journal of Pharmacology, Vol.
523, No. 1-3, October 2005, pp. 16-24.
doi:10.1016/j.ejphar.2005.08.042
[14] K. S. Murth y, Y. S. Yee, J. R. Grider and G. M. Ma khlouf,
“Phorbol-Stimulated Ca2+ Mobilization and Contraction in
Dispersed Intestinal Smooth Muscle Cells,” Journal of
Pharmacology and Experimental Therapeutics, Vol. 294,
No. 3, 2000, pp. 991-996.
[15] K. S. Murthy, J. R. Grider, J. F. Kuemmerle and G. M.
Makhlouf, “Sustained Muscle Contraction Induced by
Agonists, Growth Factors, and Ca2+ Mediated by Distinct
PKC Isozymes,” American Journal of Physiology, Ga-
strointestinal Liver Physiology, Vol. 279, No. 1, July 2000,
pp. G201-G210.
[16] F. Nakao, S. Kobayashi, K. Mogami, Y. Mizukami, S. Shi-
rao, S. Miwa, N. Todoroki-Ikeda, M. Ito and M. Mats uzaki,
“Involvement of Src Family Protein Tyrosine Kinase in
Ca2+ Sensitization of Coronary Artery Contraction Mediated
by a Sphingosylphosphorylcholin-Rho-Kinase Pathway,”
Circulation Research, Vol. 91, 2002, pp. 952-960.
doi:10.1161/01.RES.0000042702.04920.BF
[17] S-K. Ryu, D. S. Ahn, Y-E. Cho, S-K. Choi, Y-H. Kim, K,
G. Morgan and Y-H. Lee, “Augmented-Induced Ca2+-
Sensitization of Mesenteric Artery Contraction in Spon-
taneous Hypertensive Rat,” Naunyn-Schmiedeberg Arc-
hive of Pharmacology, Vol. 373, No. 1, 2006, pp. 30-36.
doi:10.1007/s00210-006-0036-7
[18] H. Satoh, “Sino-Atrial Nodal Cells of Mammalian Hearts:
Ionic Currents and Gene Expression of Pacemaker Ionic
Channels,” J ournal of Smooth Muscle Research, Vol. 39,
No. 5, 2003, pp.175-193. doi:10.1540/jsmr.39.175
[19] H. Satoh, “Identification of Hyperpolarization-Activated
Inward Current in Uterine Smooth Muscle Cell during
Pregnancy,” General Pharmacology, Vol. 26, No. 6, Oc-
tober 1995, pp. 1335-1338.
doi:10.1016/0306-3623(95)00006-M
[20] L. Thomsen, T. L. Robinson, J. C. F. Lee, L. A. Farraway,
M. J. G. Hughes, S. W. Andrews and J. D. Huizinga, “In-
terstitial Cells of Cajal Generate a Rhythmic Pacemaker
Current,” Nature Medicine, Vol. 4, No. 7, July 1998, pp.
848-851. doi:10.1038/nm0798-848
[21] M. T. Nelson, H. Cheng, M. Rubart, L. F. Santana, A. D.
Bonev, H. J. Knot and W. J. Lederer, “Relaxation of Ar-
terial Smooth Muscle by Calcium Sparks,” Science, Vol.
270, No. 5236, October 1995, pp. 633-637.
doi:10.1126/science.270.5236.633
[22] R. ZhuGe, S. M. Sims, R. A. Tuft, K. E. Fogarty and J. V.
Jr. Walsh, “Ca2+ Sparks Activate K+ and Cl- Channels
Resulting in Spontaneous Transient Currents in Guinea-
Pig Tracheal Myocytes,” Journal of Physiology (Lon-
don) , Vol. 513, No. 3, December 1998, pp. 711-718.
doi:10.1111/j.1469-7793.1998.711ba.x
[23] Y. Himeno, F. Toyoda, H. Satoh, A. Amano, C. Y. Cha,
H. Matsumura and A. Noma, ”Minor Contribution to
Pacemaking in Guinea Pig SA Node,” American Journal
of Physiology, Heart Circulation Physiology, Vol. 300,
2011, pp. H251-H261.
doi:10.1152/ajpheart.00764.2010
[24] H. Satoh, “Modulation of Ca2+-Activated K+ Current by
Isoprenaline, Carbachol, and Phorbol Ester in Cultured
(and Fresh) Rat Aortic Vascular Smooth Muscle Cells,”
General Pharmacology, Vol. 27, No. 2, March 1996, pp.
319-324. doi:10.1016/0306-3623(95)02005-5
[25] P. Kojodjojo, P. Kanagaratnam, O. R. Segal, W. Hussain
and N. S. Peters, “The Effects of Carbenoxolone on Hu-
man Myocardial Conduction: A Tool to Investigate the
Role of Gap Junction Uncoupling in Human Arrhythmo-
genesis,” Journal of the American College of Cardiology,
Vol. 48, No. 6, September 2006, pp. 1242-1249.
doi:10.1016/j.jacc.2006.04.093
[26] L. C. Chen, M. H. Chou, M. F. Lin and L. L. Yang,
“Pharmacokinetics of Paeoniflorin after Oral Administr a-
tion of Dshao-yao Gan-chao Tang in Mice,” Japanese
Journal of Pharmacology, Vol. 88, No. 3, 2002, pp. 250-
255. doi:10.1254/jjp.88.250
[27] H. Hosseinzadeh, A. M. Nassiri and S. Parvardeh, “The
Effects of Carbenoxolone, a Semisynthetic Derivative of
Glycyrrhizinic Acid, on Peripheral and Central Ischemia-
Reperfusion Injuries in the Skeletal Muscle and Hippo-
campus of Rats,” Phytomedicine, Vol. 12, No. 9, Sep-
tember 2005, pp. 632-637.
doi:10.1016/j.phymed.2004.07.007
[28] I. G. Sava, V. Battaglia, C. A. Rossi, M. Salvi and A. Toni-
nello, “Free Radical Scavenging Action of the Natual Po-
lyamine Spermine in Rat Liver Mitochondria,” Free Radi-
cal Biology and Medicine, Vol. 41, No. 8, October 2006, pp.
1272-1281. doi:10.1016/j.freeradbiomed.2006.07.008

H. SATOH ET AL.
70
[29] H. Y. Park, S. H. Pa rk, H. K. Yoon, M. J. Han and D. H.
Kim, “Anti-Allergic Activity of 18-β-Glycyrrhetinic Ac-
id-3-O-β-D-Glucuronide,” Archives of Pharmacal Re-
search, Vol. 27, 2004, pp. 57-60.
doi:10.1007/BF02980047
[30] O. H. Kang, J. A. Kim, Y. A. Choi, H. J. Park, D. K. Kim,
Y. H. An, S. C. Choi, K. J. Yun, Y. H. Nah, X. F. Cai, Y.
H. Kim, K. H. Bae and Y. M. Lee, “Inhibition OH Inter-
leukin-8 Production in the Human Col onic Epit helia l Cell
Line HT-29 by 18-β-Glycyrrhetinic Acid,” International
Journal of Molecular Medicine, Vol. 15, 2005, pp. 981-
985.
[31] H. Kato, M. Kanaoka, S. Yano and M. Kobayashi, “3-
Monoglucuronyl-Glycyrrehetic Acid is a Major Metabo-
lite That Causes Licorice-Induced Pseudoaldosteronism,”
Journal of Clinical Endocrinology and Metabolism, Vol.
80, No. 6, June 1995, pp. 1929-1933.
Abbreviations
ACh: acetylcholine
ATP: adenosine triphosphate
ANOVA: analysis of varience
CA: hippocampal neuron
CCh: carbachol
DMSO: dimethyl sulfoxide
T1/2: half-life
IBMX: 3-isobutyl-1-methylxanthine
If: hyperpolarization-activated inward (or pacemaker)
current
IL: interleukin
IP3: inositol triphosphate
KCa: Ca2+-activated K+ channel
M1 receptor: muscarinic receptor
MLCK: myosin light chain kinase
PDE: phosphodiesterase
PK-C: protein kinase C
SA node: sino-atrial node
SR: sarcoplasmic reticulum
TEA: tetraethyammonium