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The Anxiolytic Effect of Sho-ju-sen, a Japanese Herbal Medicine, Assessed by an Elevated Plus-maze Test in Mice Hisashi Kuribara,* Hisato Iwata, Hideo Tomioka, Reiko Takahashi, Kazumi Goto, Naomi Murohashi and Sakuji Koya Laboratory of Development, Wakanyaku Medical Institute Ltd, 1193 Akagiyama, Fujimi-mura, Seta-gun, Gunma 371-0101, Japan. Sho-ju-sen (SK), a Japanese herbal medicine with a nourishing tonic action, is composed of a water extract of Kumazasa leaves (Sasa kurinensis Makino et Sibata) (SS), and ethanol extracts of Japanese red pine needles (Pinus densiflora Sieb. et Zucc) (PN) and Ginseng roots (Panax ginseng C. A. Meyer) (PX) in the ratio 8:1:1. In this study, an elevated plus-maze test in mice was carried out to assess whether SK had an anxiolytic effect. No significant change was observed in either the plus-maze or activity test following a single administration of SK (10 and 20 mL/kg p.o.). However, mice allowed a free intake of SK (10% solution) for 5 days and longer showed a significant prolongation of the time spent in the open arms (an anxiolytic effect), as long as that caused by the benzodiazepine anxiolytic diazepam (1 mg/kg p.o.). SK (1%, 3% and 30% solutions for 7 days) tended to develop the anxiolytic effect. Of the constitu- ents of SK, SS (8% solution), but not PN (1% solution) or PX (1% solution), resulted in the anxiolytic effect. Except for a slight acceleration in the motor activity by PN (1% solution), no significant change in the motor activity was produced by any treatment with SK, SS or PX. The combined treatment of SK (10% solution) or SS (8% solution) with 1 mg/kg diazepam enhanced the anxiolytic effect. Flumazenil (0.1 mg/kg s.c.), a benzodiazepine receptor antagonist, alone did not change the time spent in the open arms. However, it completely reversed the anxiolytic effect of SK, SS and diazepam. The present results suggest that: (1) long-term treatment with SK develops an anxiolytic effect, (2) SS is the main constituent for the anxiolytic effect of SK, and (3) benzodiazepine receptors are involved in the anxiolytic effect of SK and SS. Copyright # 2001 John Wiley & Sons, Ltd. Keywords: Sho-ju-sen; herbal medicine; elevated plus-maze test; anxiolytic effect; benzodiazepine receptors; mice. INTRODUCTION Sho-ju-sen (SK), a Japanese herbal medicine with a nourishing tonic action, is composed of a water extract of Kumazasa leaves (Sasa kurinensis Makino et Sibata) (SS), and ethanol extracts of Japanese red pine needles (Pinus densiflora Sieb. et Zucc) (PN) and Ginseng roots (Panax ginseng C. A. Meyer) (PX) in the ratio 8:1:1. Clinical case reports of the long-term consumption of SK (Wakanyaku Medical Institute, 1996a,b) suggested that SK was effective for the amelioration of various symptoms including fatigue, vegetative dystonia, depres- sion, anxiety, sleeping disturbance etc. A non-placebo controlled clinical study demonstrated that consumption of SK for 12 weeks (12 mL/day during weeks 1–4, 24 mL/day during weeks 5–8, and 36 mL/day during weeks 9–12) resulted in an improvement of unidentified clinical syndrome in 99/110 cases (approx. 90%) (Ichikawa et al., 1998). These reports indicate that SK produces an anxiolytic and/or antidepressant effect after consumption for several days. The aims of this study were to assess preclinically whether SK showed an anxiolytic effect, and the characteristics of this action by using an elevated plus- maze test in mice. This was used because the validity of the plus-maze test originally established by Pellow et al. (1985) for evaluation of an anxiolytic effect of drugs has been well discussed (Dawson and Tricklebank, 1995; Kulkarni and Shaema, 1991; Kulkarni and Reddy, 1996). To estimate a possible change in the plus-maze performance caused by motor activity (Dawson and Tricklebank, 1995), an ambulatory activity test was also carried out. MATERIALS AND METHODS Animals. Male mice of the ddY strain (SLC Japan, Hamamatsu) were purchased at 6 weeks of age. Groups of ten mice were housed in aluminium cages (20 w 30 l 10 h cm) with paper bedding (SLC Japan) and allowed free access to a solid diet (MF: Oriental Yeast, Tokyo) and tap water. The conditions of the breeding room were controlled (temperature; 23 Æ 1 °C. relative humidity; 55 Æ 3%, and a 12:12 h light–dark cycle with lights on 0700–1900 h). PHYTOTHERAPY RESEARCH Phytother. Res. 15, 142–147 (2001) DOI: 10.1002/ptr.698 Copyright # 2001 John Wiley & Sons, Ltd. * Correspondence to: H. Kuribara, Laboratory of Development, Wakanyaku Medical Institute Ltd, 1193 Akagiyama, Fujimi-mura, Seta-gun, Gunma 371- 0101, Japan. Received 22 March 1999 Accepted 20 December 1999

The anxiolytic effect of Sho-ju-sen, a Japanese herbal medicine, assessed by an elevated plus-maze test in mice

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The Anxiolytic Effect of Sho-ju-sen, a JapaneseHerbal Medicine, Assessed by an ElevatedPlus-maze Test in Mice

Hisashi Kuribara,* Hisato Iwata, Hideo Tomioka, Reiko Takahashi, Kazumi Goto, NaomiMurohashi and Sakuji KoyaLaboratory of Development, Wakanyaku Medical Institute Ltd, 1193 Akagiyama, Fujimi-mura, Seta-gun, Gunma 371-0101, Japan.

Sho-ju-sen (SK), a Japanese herbal medicine with a nourishing tonic action, is composed of a waterextract of Kumazasa leaves (Sasa kurinensis Makino et Sibata) (SS), and ethanol extracts of Japanesered pine needles (Pinus densiflora Sieb. et Zucc) (PN) and Ginseng roots (Panax ginseng C. A. Meyer)(PX) in the ratio 8:1:1. In this study, an elevated plus-maze test in mice was carried out to assess whetherSK had an anxiolytic effect. No significant change was observed in either the plus-maze or activity testfollowing a single administration of SK (10 and 20 mL/kg p.o.). However, mice allowed a free intake ofSK (10% solution) for 5 days and longer showed a significant prolongation of the time spent in the openarms (an anxiolytic effect), as long as that caused by the benzodiazepine anxiolytic diazepam (1 mg/kgp.o.). SK (1%, 3% and 30% solutions for 7 days) tended to develop the anxiolytic effect. Of the constitu-ents of SK, SS (8% solution), but not PN (1% solution) or PX (1% solution), resulted in the anxiolyticeffect. Except for a slight acceleration in the motor activity by PN (1% solution), no significant change inthe motor activity was produced by any treatment with SK, SS or PX. The combined treatment of SK(10% solution) or SS (8% solution) with 1 mg/kg diazepam enhanced the anxiolytic effect. Flumazenil(0.1 mg/kg s.c.), a benzodiazepine receptor antagonist, alone did not change the time spent in the openarms. However, it completely reversed the anxiolytic effect of SK, SS and diazepam. The present resultssuggest that: (1) long-term treatment with SK develops an anxiolytic effect, (2) SS is the main constituentfor the anxiolytic effect of SK, and (3) benzodiazepine receptors are involved in the anxiolytic effect ofSK and SS. Copyright � 2001 John Wiley & Sons, Ltd.

Keywords: Sho-ju-sen; herbal medicine; elevated plus-maze test; anxiolytic effect; benzodiazepine receptors; mice.

INTRODUCTION

Sho-ju-sen (SK), a Japanese herbal medicine with anourishing tonic action, is composed of a water extract ofKumazasa leaves (Sasa kurinensis Makino et Sibata)(SS), and ethanol extracts of Japanese red pine needles(Pinus densiflora Sieb. et Zucc) (PN) and Ginseng roots(Panax ginseng C. A. Meyer) (PX) in the ratio 8:1:1.

Clinical case reports of the long-term consumption ofSK (Wakanyaku Medical Institute, 1996a,b) suggestedthat SK was effective for the amelioration of varioussymptoms including fatigue, vegetative dystonia, depres-sion, anxiety, sleeping disturbance etc. A non-placebocontrolled clinical study demonstrated that consumptionof SK for 12 weeks (12 mL/day during weeks 1–4,24 mL/day during weeks 5–8, and 36 mL/day duringweeks 9–12) resulted in an improvement of unidentifiedclinical syndrome in 99/110 cases (approx. 90%)(Ichikawa et al., 1998). These reports indicate that SKproduces an anxiolytic and/or antidepressant effect afterconsumption for several days.

The aims of this study were to assess preclinicallywhether SK showed an anxiolytic effect, and thecharacteristics of this action by using an elevated plus-maze test in mice. This was used because the validity ofthe plus-maze test originally established by Pellow et al.(1985) for evaluation of an anxiolytic effect of drugs hasbeen well discussed (Dawson and Tricklebank, 1995;Kulkarni and Shaema, 1991; Kulkarni and Reddy, 1996).To estimate a possible change in the plus-mazeperformance caused by motor activity (Dawson andTricklebank, 1995), an ambulatory activity test was alsocarried out.

MATERIALS AND METHODS

Animals. Male mice of the ddY strain (SLC Japan,Hamamatsu) were purchased at 6 weeks of age. Groupsof ten mice were housed in aluminium cages(20 w � 30 l � 10 h cm) with paper bedding (SLC Japan)and allowed free access to a solid diet (MF: OrientalYeast, Tokyo) and tap water. The conditions of thebreeding room were controlled (temperature; 23 � 1°C.relative humidity; 55 � 3%, and a 12:12 h light–darkcycle with lights on 0700–1900 h).

PHYTOTHERAPY RESEARCHPhytother. Res. 15, 142–147 (2001)DOI: 10.1002/ptr.698

Copyright � 2001 John Wiley & Sons, Ltd.

* Correspondence to: H. Kuribara, Laboratory of Development, WakanyakuMedical Institute Ltd, 1193 Akagiyama, Fujimi-mura, Seta-gun, Gunma 371-0101, Japan.

Received 22 March 1999Accepted 20 December 1999

Apparatus and measurement procedures

The elevated plus-maze test. The elevated plus-mazeused in this study was the same as that used in ourprevious studies (Kuribara and Maruyama, 1996; Kuri-bara et al., 1996, 1998), and was an improvement of theoriginal apparatus for rats (Pellow et al., 1985) and mice(Lister, 1987). Briefly, the floor and side wall of theclosed arms (6w � 30 l � 10 h cm) and the floor of thecentre platform (8 � 8 cm) were made of grey non-transparent polyvinylchloride fibre. In contrast, the openarms (6w � 30 l cm) had no side walls, and the floor wasmade of transparent polyvinylchloride fibre. The plus-maze was placed 40 cm above the base.

Each mouse was placed on the centre platform facingone of the closed arms, and the cumulative time spent inthe open arms was recorded for 5 min. When all fourpaws of the mouse crossed the borderline space betweenthe centre platform and the open arm, it was considered tohave entered the open arm.

Activity test. Immediately after the end of the 5 minplus-maze test horizontal movements of the mouse weremeasured for 5 min with a tilting-type ambulometerwhich had a bucket-like Plexiglas activity cage of 20 cmdiameter (SMA-1: O’Hara & Co., Tokyo).

Drugs

Sho-ju-sen (SK) and its constituents. A water extract ofKumazasa leaves (SS), and ethanol extracts of Japanesered pine needles (PN) and Ginseng roots (PX) wereobtained from Wakanyaku Medical Institute (Tokyo).Commercial preparations of diazepam (Cercine Inj.,Takeda Chemical, Osaka) and flumazenil (Anexate Inj.,Yamanouchi, Tokyo) were purchased.

For the single administration of SK, the originalpreparation was used. For the free intake, the originalpreparations of SK, SS, PN and PX were diluted with tapwater, and the solutions were presented to mice as asubstitute for tap water. The control mice were given tapwater throughout the experimental period.

Injectable preparations of diazepam and flumazenilwere diluted with physiological saline. Diazepam wasadministered orally (p.o.), and flumazenil subcutaneously(s.c.). The concentrations of diazepam and flumazenilwere adjusted so that each volume administered wasconstant at 0.1 mL/10 g body weight of the mouse.

Experimental schedules

All experimental treatments mentioned below werecarried out between 0900–1500 h. Mice were used onlyonce in either experiment mentioned below.

Single administration of SK and diazepam. Groups of10 mice each were given either SK (10 or 20 mL/kg) ortap water (control for SK) 1 h before, or diazepam (1 or2 mg/kg p.o.) or physiological saline (control fordiazepam) 10 min before the behavioural tests.

Free intake of SK and its constituents for 7 days.Groups of 10–20 mice each were given either SK (1%,3%, 10% or 30%), SS (8%), PN (1%) or PX (1%) for 7days. On day 7, 1 h after the withdrawal of the free drugintake, the plus-maze test was carried out and wassubsequently followed by the activity test.

During the free intake of drug solution, the bodyweight of mice and the fluid intake (group of 10 mice)were measured once a day.

Evaluation of the time course of changes in the effect.To evaluate the time-course of changes in the develop-ment of effects, groups of 20 mice each were given eitherSK (10%) or SS (8%) for 1, 2, 3, 4, 5, 7 or 14 days prior tothe behavioural tests. The control groups of 20 mice eachwere given tap water for 1, 3, 5, 7 or 14 days prior to thebehavioural tests.

To evaluate the time-course of changes in thedisappearance of the effects, groups of 20 mice eachwere first given either SK (10%) or SS (8%) for 7 days.One h (0 day), 1, 2, 3, 4, 5 or 7 days after the withdrawal,the behavioural tests were carried out. The control groupsreceived the behavioural tests on either day 0, 2, 4, 5 or 7.

Combined drug treatments. Groups of 10 mice eachwere first given SK (10%) or SS (8%), and the controlgroups tap water for 7 days. Fifty min after the with-drawal on day 7, diazepam (1 mg/kg p.o.) or flumazenil(0.1 mg/kg s.c.) was challenge administered. The beha-vioural tests were carried out 10 min after the challengeadministration. Single administration of diazepam andflumazenil, and combined administration of diazepamand flumazenil were also carried out.

Table 1. Effects of single p.o. administration of Sho-ju-sen and diazepam on the elevated plus-maze performance and motoractivity in mice

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ANXIOLYTIC EFFECT OF SHO-JU-SEN 143

Copyright � 2001 John Wiley & Sons, Ltd. Phytother. Res. 15, 142–147 (2001)

Statistical analysis

The time spent in the open arms in the plus-maze test andthe activity count in the activity test were analysed byStudents’s t-test. Values of p less than 0.05 wereconsidered significant.

RESULTS

Single administration

As shown in Table 1, the single administration of SK (10and 20 mL/kg p.o.) produced no significant change ineither plus-maze or activity tests. However, diazepam (1and 2 mg/kg p.o.) prolonged the time spent in the openarms (anxiolytic effect) in a dose-dependent manner witha significant increase in the motor activity at 2 mg/kg.

Free intake of SK and its constituents for 7 days

The average daily fluid intakes on days 1 and 7 were 1.5–2.0 mL/10 g and 1.2–1.8 mL/10 g body weight, respec-tively, and mice gained 3.0–4.5 g body weight during thefree intake of drug solutions or tap water (control) for 7days. There were no marked differences in these valuesamong the groups.

As shown in Fig. 1, SK (10%) showed a significantanxiolytic effect as potent as that of the singleadministration of 1 mg/kg diazepam (Table 1). Theanxiolytic potential of SK (1%, 3% and 30%) was lessthan that of SK (10%). Among the constituents of SK, SS(8%) but not PN (1%) or PX (1%) showed a significantanxiolytic effect.

As shown in Fig. 2, PN (1%) slightly but significantlyincreased the motor activity. However, no significant

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Table 2. Process of the development of anxiolytic effect following free intake of Sho-ju-sen (SK) and Kumazasa (SS)

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144 H. KURIBARA ET AL.

Copyright � 2001 John Wiley & Sons, Ltd. Phytother. Res. 15, 142–147 (2001)

change in the motor activity was produced by SK (1%,3%, 10% or 30%), SS (8%) or PX (1%).

Time course of changes in the anxiolytic effect

Process of development of the anxiolytic effect. Asshown in Table 2, in both cases of SK (10%) and SS(8%), the anxiolytic effect attained a significant level onday 4, and a peak effect was maintained on day 7 andlater. No significant change in the motor activity wasproduced by either drug treatment.

Process of disappearance of the anxiolytic effect. Asshown in Table 3, the anxiolytic effect produced by thefree intake of SK (10%) and SS (8%) for 7 daysweakened progressively after withdrawal. However, asignificant anxiolytic effect remained for up to 4 and 3days in the cases of SK and SS, respectively. Nosignificant change in the motor activity was manifestedthroughout the observation period of 7 days.

Combined drug treatments

As shown in Fig. 3, the combined treatment with SK(10%) and 1 mg/kg diazepam significantly enhanced theanxiolytic effect. The combination of SS (8%) anddiazepam tended to enhance the anxiolytic effect.Although 0.1 mg/kg flumazenil alone did not changethe time spent in the open arms, it completely reversedthe anxiolytic effect of SK, SS and diazepam.

None of the combined treatments resulted in a markedchange in the motor activity (data not shown).

DISCUSSION

The present study first revealed that free intake of SK(10%) for 7 days produced a significant prolongation inthe time spent in the open arms without a marked changein the motor activity. Such results indicate that the changein the plus-maze performance reflects an anxiolytic effectof SK, supporting the clinical efficacy of SK on variouspsychic and psychosomatic symptoms such as vegetativedystonia, unidentified clinical syndrome, depression,

anxiety, sleeping disturbance, etc (Wakanyaku MedicalInstitute, 1996a,b; Ichikawa et al., 1998).

The daily SK dose consumed was estimated to be 12–18 mL/kg/day during the free intake of SK (10%), and theanxiolytic potential of SK was almost the same as that of1 mg/kg diazepam. However, acute administration of 10and 20 mL/kg SK did not produce an anxiolytic effect.Such results suggest that, rather than a direct effect ofcertain chemical (s) in SK, an increase in metabolite(s) ofunidentified chemical(s) and/or a change in someneuronal functions are involved in the anxiolytic effectof SK. This consideration may be supported by the timecourse of change in the induction of the anxiolytic effect.Thus, a significant anxiolytic effect was produced by thefree intake of SK (10%) for 4 days and longer. A similarslow onset of the anxiolytic effect was demonstratedfollowing the repeated administration of Saiboku-to, aChinese medicine (Kuribara and Maruyama, 1996), andits active chemical honokiol (Kuribara et al., 1998).

The free intake of SK (30%) resulted in less anxiolyticeffect than that produced by the free intake of SK (10%).Comparatively higher doses of benzodiazepine anxioly-tics produce motor dysfunction or sedation. Such effectssometimes mask the appearance of the anxiolytic effectin the plus-maze test, revealing a bell shaped dose-

Table 3. Process of the disappearance of anxiolytic effect following the withdrawal of free intake of Sho-ju-sen (SK) andKumazasa (SS) for 7 days

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ANXIOLYTIC EFFECT OF SHO-JU-SEN 145

Copyright � 2001 John Wiley & Sons, Ltd. Phytother. Res. 15, 142–147 (2001)

response curve. However, the treatment with SK (30%)caused neither a marked change in motor activity nor inbodily condition, indicating a possibility that there areboth anxiolytic and anxiogenic chemicals in SK, and theyinteract to reduce the anxiolytic effect at a high dose ofSK. As seen in Fig. 2, PX tended to shorten the time spentin the open arms. However, further studies, includingidentification of the active chemicals and assessment ofthe dose-response relationships for individual chemicals,are required.

In addition, the present study revealed that, of theconstituents of SK, SS (8%), but not PN (1%) or PX(1%), resulted in almost the same potential and the timecourse of change in the development of the anxiolyticeffect as those of SK. These results clearly indicate thatchemical(s) in SS are responsible for the anxiolytic effectof SK.

After the free intake the anxiolytic effect of both SKand SS attained the maximum level on day 7 and almostthe same anxiolytic potentials were maintained up to day14. Non-placebo controlled clinical trials of SK for 12weeks also demonstrated a progressive improvement invarious conditions related to an unidentified clinicalsyndrome (Ichikawa et al., 1998). The present results andthe clinical evidence suggest that, unlike benzodiazepineanxiolytics (Schweizer et al., 1995; Woods and Winger,1995; Woods et al., 1995), a long-term intake of SK (orSS) scarcely induced tolerance to the anxiolytic effect.Furthermore, the anxiolytic effect once induced by thefree intake of SK (10%) and SS (8%) for 7 days persistedup to 3 to 4 days after withdrawal. Taken together thetime-course of changes in the anxiolytic effect of SK andSS, the delay onset and the persistence for 4 days, lead tothe conclusion that the anxiolytic effect of SK and SS iscaused by an accumulation of metabolite(s) havingcomparatively longer half-lives and/or a change inneuronal functions.

Similar to many reports (Pellow and File, 1986;Kulkarni and Shaema, 1991; Dawson and Tricklebank,1995; Kulkarni and Reddy, 1996), diazepam showed adose-dependent anxiolytic effect. The benzodiazepinereceptor antagonist flumazenil reveals an anxiogeniceffect at high doses in animal studies (Haefely, 1988; Leeand Rodgers, 1991) and in clinical trials (Nutt et al.,1990; Price et al., 1995). However, flumazenil generally

has no marked effect at comparatively low doses, butreverses the effects of benzodiazepine anxiolytics (Boastet al., 1983). The present experiment not only confirmedthe antagonistic interaction between diazepam andflumazenil, but also demonstrated that flumazenil com-pletely reversed the anxiolytic effect of SK and SS.Furthermore, a significant enhancement of the anxiolyticeffect was produced by the combined treatment with SKand diazepam. The combination of SS and diazepam alsotended to enhance the anxiolytic effect. These resultsindicate that benzodiazepine receptors are involved in theanxiolytic effect of SK and SS.

Diazepam increased the motor activity and weakenedthe grip strength, induction of disinhibition and muscle-relaxation, respectively. Furthermore, at doses higherthan 5 mg/kg, diazepam caused sedation (data notshown). However, gross observation revealed that theacute administration of SK, or free intake of SK and SSnever caused changes in either motor activity or muscletone (data not shown). In these respects, SK and SS arelikely to possess a selective anxiolytic effect withouteliciting behavioural disorders such as sedation, disin-hibition, muscle relaxation and ataxia which are gen-erally induced as side effects of benzodiazepineanxiolytics (Schweizer et al., 1995; Woods and Winger,1995; Woods et al., 1995). It has been reported thatbenzodiazepine partial agonists, which have selectiveaction on benzodiazepine receptor subtypes correlated tothe anxiolytic effect, show an anxiolytic effect with lesslikelihood of producing motor dysfunction, tolerance andphysical dependence (Haefely et al., 1990). Thus, it isspeculated that SK and SS may stimulate the benzodia-zepine receptor subtype(s) similar to benzodiazepinepartial agonists. However, neurochemical and pharma-cokinetic evaluations are required to elucidate themechanisms and characteristics of the anxiolytic actionof SK and SS.

Acknowledgements

We thank Mr Kazuo Tanaka and Mr Hisanori Yokote (The Chairmanand President, respectively, of Wakanyaku Medical Institute) forsupport of this study.

REFERENCES

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ANXIOLYTIC EFFECT OF SHO-JU-SEN 147

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