Pharmacological Modulation by Shakuyakukanzoto (shao-yao-gan-cao-tang) and the Ingredients in Rat Intestinal Smooth Muscle

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 examined. Shakuyakukanzoto (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 spontaneous 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 Ca 2+ channel inhibition. Thus, Shakuyakukanzoto exerts an anti-spasmodic action due to the interaction with pharmacological effects of its ingredients.


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 components; 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 relaxant effect of skeletal muscle [1].Nicotinic ACh receptors 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 Ca 2+ movement near around neuromuscular junction, and glycyrrhetic acid inhibits Ca 2+ -activated K + (I KCa ) channel to repolarize or hyperpolarize the membrane [3].The combination with Paeoniae 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 gastrointestinal smooth muscle [4].The relaxations of smooth muscles induced by Kampo medicines depend on mainly a phosphodiesterase (PDE) inhibition [5].Most recent reports have also demonstrated to play a key role for regulation of the gap junction on gastrointestinal smooth muscle [6][7][8].
Until now, there is less information of more detailed pharmacological mechanisms for the gastrointestinal actions of Shakuyakukanzoto, the components (Paeoniae 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 actions of Shakuyakukanzoto and the ingredients on the relaxation were investigated using rat intestinal smooth muscle.

Material and Methods
All experiments were carried out, according to the guidelines laid down by the Nara Medical University Animal Welfare Committee, and also under the terms of the Declaration of Helsinki.

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 filled with 20 ml modified Tyrode solution.
The strips were suspended between both sides with stainless steel stirrups.The lower stirrup was anchored and 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 for contractions with muscarinic ACh receptor agonist.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 concentration of the drugs were measured 5 -7 min after the responses became steady.To examine the involvement with Ca 2+ channel, PDE or other mechanisms, the pretreatment with 1 μM nicardipine, 20 μM IBMX or other inhibitors was carried out.Each of the experiments was examined at least quadruplicates.The responses were analyzed as a percentage change from the value before an application of drugs.

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.

Experiments in the Absence of CCh
Using rat ileum in non-pretreatment with CCh, the ef-fects of Shakuyakukanzoto and the ingredients on gastrointestinal smooth muscle were examined using the same experimental techniques.

Statistical Analyses
To compare the pair values, we are used statistical methods of the Student's t-test and ANOVA followed by post-hoc tests (Dunn-Bonferonii test) using Excel (Microsoft Inc., Washington, U.S.A.) and S-PLUS (Mathematical System Inc., Washington, U.S.A.).All values are represented as means ± SEM.A p value of less than 0.05 was considered significant.
enhanced the relaxation concentration-dependently. Shakuyakukanzoto also did not affect on Bay K 8644 (3 nM)-induced contraction significantly.
In addition, the relaxation induced by Shakuyakukanzoto (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 Shakuyakukanzoton-induced relaxation, but did not cause it to significant extent.
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 produced the stronger relaxation in the presence of IBMX.These results indicate that both components make minor contribution to the PDE-and Ca 2+ channel-dependent relaxations.

Effects on Spontaneous Contractions
Prior administration of CCh (0.3 μM) elicited occasionally spontaneous contraction in some specimens (Figure 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 behaved in a concentration-dependent manner.

Discussions
The present experiments showed that Shakuyakukanzoto and its ingredients caused the potent relaxant action on the CCh-induced contraction.They possess anti-cholinergic action.Also the relaxations were due to PDE inhibition or Ca 2+ channel inhibition.Shakuyaku-  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 depressed them.

Anti-Cholinergic Action
Prior administration of CCh produced a marked contrac-tion of isolated ileum, consistent with previous report [9].Shakuyakukanzoto relaxed the CCh-induced contraction.
Paeoniae Radix and Glycyrrhizae Radix also relaxed it.At the same concentrations, Shakuyakukanzoto and Paeoniae Radix had stronger relaxing effect than Glycyrrhizae Radix.Even in the absence of CCh, Shakuyakukanzoto 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 as compared with those in presence of CCh.Glycyrrhizae 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 contractions induced by nicotine 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 (M 1 ) receptor in rat hippocampal (CA 1 ) 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 M 1 (or M 3 ) receptor.Especially Paeoniae Radix and paeoniflorin in Shakuyakukanzoto possess the stronger anti-cholinergic action.

Ca 2+ Channel Inhibition
The inhibition of Ca 2+ channel has previously been shown.Paeoniflorin inhibits Ca 2+ current in NG108-15 neuronal cells, 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 Ca 2+ concentration ([Ca 2+ ] i ) via Na/Ca exchange.Paeoniflorin also relaxes the isolated rat aorta due to the [Ca 2+ ] i decline and the increases in NO and cGMP [13].
In the present experiments, however, Shakuyakukanzoto and the containing compounds had no Ca 2+ channel inhibitory action.This is supported by the minor action of Shakuyakukanzoto on Bay K 8644 (3 nM)-induced contraction.Interestingly, they decreased the relaxant actions by addition of nicardipine.Only 18-α-glycyrrhetinic acid had the inhibitory action of Ca 2+ channel, and produced rather the contraction.The mechanisms are now unclear yet, but cellular signaling pathways such as Rho kinase, MLCK and PK-C might be involved [14][15][16][17].
Therefore, Shakuyakukanzoto and the related compounds have less or no effect on Ca 2+ channel, but might exert the relaxation by the inhibitions of Na + and K Ca channels and the activation of cGMP signaling pathway.

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 observed in paeoniflorin but not in Paeoniae Radix.So, the resultant effects are responsible for an interaction among the containing compounds.

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) current (I f ), is similarly identified [19].In general, the spontaneous activity of smooth muscle cells may be largely dependent on transient Ca 2+ sparks.Interstitial cells of Cajal, gastrointestinal pacemakers, exhibit Ca 2+ release from IP 3 -dependent Ca 2+ stores by activating a Ca 2+dependent cationic current that drives pacemaker depolarization [20].The [Ca 2+ ] i elevation activates the K Ca channels to produce the repolarization of spontaneous action potentials [21], as well as the Ca 2+ -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 Ca 2+ sparks [23].It is not yet clear now, but might be closely related with the connection of Ca 2+ 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 K Ca channel in aortic smooth muscle cells is inhibited by β-adrenoceptor and muscarinic receptor stimulations and also by PK-C stimulation [24].Glycyrrhetic acid also inhibits K Ca channel [3].In this study, application of Shakuyakukanzoto ceased the spontaneous contractions.Thus, the depression would be partly due to inhibition of K Ca channel and regulation of Ca 2+ movement near around neuromuscular junction, because of minor contribution of Ca 2+ channel.
In skeletal muscles, nicotinic ACh receptor on neuromuscular junction plays an important role for the contraction.Paeoniflorin produces the relaxation by means of a depolarized blockade like succinylcholine [2].Paeoniflorin regulates Ca 2+ movement near around neuromuscular junction, and glycyrrhetic acid inhibits K Ca channel [3].In rat hippocampal slice, furthermore, the dependence of anti-cholinergic action has been found [10].The combination with Paeoniae Radix and Glycyrrhizae Radix would enforce the relaxant action of skeletal muscle.Most recent reports have demonstrated that Shakuyakukanzoto may be sufficiently effective for spasmodic diseases of gastrointestinal tract, as well as for cramp and twitch of skeletal muscle.The antispasmodic 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 metabolic products can slow myocardial conduction via modulation of gap junction, but not via that of the ionic 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 epileptogenicity and arrhythmogenesis [7].Thus, the pharmacokinetic properties may be well suitable for the transient clamp in leg skeletal muscle and the gastrointestinal spasms.

Conclusions
Shakuyakukanzoto possesses the higher bioactivities for rat ileum, and is so effective for many diseases in clinical uses.Shakuyakukanzoto produced a remarkable relaxation by 1) the anti-cholinergic and 2) the PDE inhibitory actions, but by 3) minor contribution of Ca 2+ channel inhibition.Also, Shakuyakukanzoto exerted an antispasmodic action due to the interaction with pharmacological effects of its ingredients.The absorption is rapid from intestine, and the plasma concentration-time curves are fitted with a mean terminal half-life (T 1/2 ) of 116.2 min [26].Glycyrrhetic and 18-βglycyrrhetinic acids possess a free radical scavenging property [27,28], and anti-allergic activities such as passive cutaneous anaphylaxis and skin contact inflammation [29,30].However, the pathological findings of the metabolic products from glycyrrhetic acid have also been well known [31].
Finally, the pharmacological characteristics of Shakuyakukanzoto, the ingredients and the related compounds are summarized on Table 4.Further extensive studies are needed to elucidate in more detail mechanisms.

Table 2 . Relaxant effects of the constituents and the products from Shakuyakukanzoto on CCh-in- duced contraction.
Values (%) are represented as mean ± S.E.M. * ,# : P < 0.05, ** ,## : P < 0.01, ### : P < 0.001, * means a significant difference between the value at each concentration and control value.# means a significant difference of the values in the presence, as compared with the values in the absence of inhibitors at each concentration.