ANTIDEPRESSANT EFFECT OF SHO-JU-SEN 173

Copyright © 2004 John Wiley & Sons, Ltd. Phytother. Res. 18, 173–176 (2004)

Copyright © 2004 John Wiley & Sons, Ltd.

Received 13 January 2003Accepted 3 July 2003

PHYTOTHERAPY RESEARCHPhytother. Res. 18, 173–176 (2004)Published online in Wiley InterScience (www.interscience.wiley.com). DOI: 10.1002/ptr.1412

SHORT COMMUNICATIONAn Antidepressant Effect of Sho-ju-sen,a Japanese Herbal Medicine, Assessed byLearned Helplessness Model in Mice

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

The antidepressant effect of Sho-ju-sen, a Japanese herbal medicine composed of extracts of three herbs;kumazasa leaf (Sasa Kurinensis Makino et Sibata), Japanese red pine leaf (Pinus densiflora Sieb. et Zucc)and ginseng radix (Panax ginseng C.A. Meyer), was assessed using a learned helplessness model in mice. Thelearned helplessness was produced by presenting 120 unavoidable/inescapable shocks for 3 days to the mousein a shuttle box, and the avoidance training was carried out on day 4. Compared with the control group giventap water, free consumption of Sho-ju-sen (1%, 3% and 10%) for 21 days resulted in a significant ameliorationof the response rate at 1% and 3%, and both the response rate and % avoidance at 10%. Although Sho-ju-sen (10%) caused no significant effect following the 7-day intake, it ameliorated the response rate followingthe 14-day intake. The extract of Japanese red pine leaf, but not kumazasa leaf or ginseng radix, mildlyimproved the response rate. Learned helplessness was significantly and dose-dependently reduced by imipramine(10 and 30 mg/kg i.p.), while only mildly by diazepam (1 mg/kg p.o.). These results suggest that a long-termconsumption of Sho-ju-sen is effective for the amelioration of depression, and the effectiveness is derivedmainly from the extract of Japanese red pine leaf. Copyright © 2004 John Wiley & Sons, Ltd.

Keywords: Sho-ju-sen; Japanese herbal medicine; discrete shuttle avoidance; learned helplessness; antidepressant effect.

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

proved many symptoms related to vegetative dystoniaand unidentified clinical syndrome such as fatigue,vegetative dystonia, depression, anxiety, sleeping dis-turbance, etc. (Wakanyaku Medical Institute, 1996;Ichikawa et al., 1998). A preclinical evaluation usingan elevated plus-maze test in mice indicated that ananxiolytic effect was produced following the free con-sumption of Sho-ju-sen for longer than 4 days (Kuribaraet al., 2001).

The aim of this study was to assess, using learnedhelplessness model in mice, whether Sho-ju-sen showedan antidepressant effect. To emphasize the utility ofthe learned helplessness model as well as the effective-ness of this Japanese herbal medicine thereon, theeffects of imipramine and diazepam, a tricyclic anti-depressant and a benzodiazepine anxiolytic, respectively,were also evaluated.

MATERIALS AND METHODS

Animals. The experimental animals used were male ddYmice (SLC Japan, Hamamatsu). They had been housedin standard breeding cages (10 mice/cage) with a roll-paper bedding (Paper Clean, SLC Japan). A solid diet(MF: Oriental Yeast, Tokyo) and tap water (or drugsolutions mentioned below) were freely availableexcept during the period of behavioural treatment. Theconditions of the breeding room were controlled (12:12 hlight–dark schedule with lights on between 7:00 and

INTRODUCTION

When animals have been repeatedly exposed to a nox-ious situation, in which neither avoidance nor escape iseffective, they may show a learning deficit in the samesituation, i.e. a learned helplessness characterized by asignificant impairment and/or retardation of acquisitionof the avoidance/escape response (Seligman and Beagley,1975). Learned helplessness has been applied for evalu-ation of the antidepressant effect of drugs. Many reportshave consistently demonstrated that typical (tricyclic)and atypical antidepressants as well as MAO-inhibitorsameliorate the learned helplessness, and result in sig-nificant improvement of the acquisition of avoidance/escape response (Seligman and Beagley, 1975; Shermanet al., 1982; Petty and Sherman, 1980; Willner, 1984).The recovery of avoidance/escape acquisition is alsoconsidered to reflect a reversal of stress-related beha-vioural disorder by antidepressants (Weiss et al., 1989).

Sho-ju-sen, a liquid-type Japanese herbal medicinemade of a water extract of kumazasa leaves (Sasakurinensis Makino et Sibata; Sasa), and ethanol extractsof Japanese red pine leaves (Pinus densiflora Sieb. etZucc; Pine) and ginseng radixes (Panax ginseng C.A.Meyer; Ginseng), has been used as a nourishingtonic.It has been empirically reported that Sho-ju-sen im-

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Copyright © 2004 John Wiley & Sons, Ltd. Phytother. Res. 18, 173–176 (2004)

19:00 h, temperature; 23 ± 1 °C, and relative humidity;60 ± 2%). The mice were used in the experiment from7 weeks old (weight 28–32 g).

Apparatus and experimental schedules. The apparatusused was a shuttle box for mice (9 × 30 × 10 cm), and acontrol unit for the discrete avoidance test (GT-8450,De CARES GT-M5 and TIDP-10, respectively, O’Hara& Co., Tokyo).

The experiment was carried out following the pro-cedure mentioned previously (Kuribara, 1994). Micewere first given 120 unavoidable/inescapable electricshocks (100 V, 0.3 mA, 50 Hz AC, 3 s) for 3 days (induc-tion sessions). Each shock was preceded by a tonesignal (800 Hz, 5 s). Such pairs of tone signal and shockwere repeated at 30 s intervals. On the next day of thelast induction session, these mice were trained to avoidthe shock under the discrete avoidance task of anintertrial interval of 22 s, a warning duration of 5 s, anda shock duration of 3 s. Thus, in this training session,the mice were allowed to avoid and/or escape from theelectric shock in the shuttle boxes that were the sameas those used in the induction sessions. A movementfrom one side to the opposite side (shuttle) of the boxduring the warning period (presenting tone signal) waseffective for the avoidance of the shock. Shuttles dur-ing shock duration could stop the shock and tone signal(namely escape). In the training session for 1 h, 120trials were held. The indices of the avoidance behaviourwere the response rate (number of shuttles) and % ofavoidance (number of avoidance responses/trials).

Drugs and administration schedules. The drugs usedwere Sho-ju-sen and its constituents; Sasa, Pine andGinseng (Wakanyaku Medical Institute, Tokyo), imipra-mine HCl (Tofranil Inj., Novartis Pharm., Takarazuka)and diazepam (Cercine Inj., Takeda Chem., Osaka).

The original preparation of Sho-ju-sen and its con-stituents were diluted by tap water. Prior to startingthe induction session, groups of 10–20 mice were givenfreely either Sho-ju-sen (1%, 3% or 10%), Sasa (8%),Pine (1%) or Ginseng (1%) for 3 weeks as a substitu-tion for the drinking water. The concentrations of Sasa,Pine and Ginseng were equivalent to those in Sho-ju-sen (10%). Furthermore, to evaluate the criticalperiod of free intake of Sho-ju-sen for the develop-ment of a significant antidepressant effect, other groupsof mice were given Sho-ju-sen (10%) for 1 or 2 weeks.Such drug treatments were continued by starting thethird induction session. The control mice were giventap water throughout the corresponding experimentalperiods.

Table 2. Average body weights of mice (±±±±±SEM) during free intake of Sho-ju-sen and its constituents

Average body weight (g)

Solution n Before Day 7 Day 12 Day 21

Tap water (control) 20 32.7 ± 0.3 35.9 ± 0.6 37.1 ± 0.6 38.1 ± 0.7

Sho-ju-sen 1% 10 32.4 ± 0.3 35.4 ± 0.5 36.4 ± 0.7 37.6 ± 0.63% 10 31.7 ± 0.4 35.2 ± 0.8 36.4 ± 0.8 37.7 ± 0.9

10% 20 32.1 ± 0.3 35.1 ± 0.5 37.8 ± 0.6 37.9 ± 0.7Kumazasa 8% 10 31.5 ± 0.2 35.3 ± 0.3 38.6 ± 0.5 39.9 ± 0.6Japanese red pine 1% 10 32.2 ± 0.2 35.3 ± 0.5 37.0 ± 0.6 38.2 ± 0.7Ginseng 1% 10 31.8 ± 0.2 35.3 ± 0.4 38.2 ± 0.5 39.7 ± 0.5

Imipramine (10 and 30 mg/kg i.p.) was administeredonce a day for 7 days; 4 days before starting the induc-tion session, and 1 h before each induction session.Diazepam (0.3, 1 and 3 mg/kg p.o.) was administered10 min before each induction session. Physiologicalsaline was administered as the control injection. Priorto the training session no drug administration wascarried out.

Statistical analysis. The mean response rates (frequencyof shuttles) and % avoidance in the training sessionwere first analysed by one-way analysis of variance,followed by two-tailed Student’s t-test. p values lessthan 0.05 were defined as a significant difference.

RESULTS

Fluid intakes and body weights

As shown in Table 1, the mice gradually decreased thefluid intake during the free-consumption period for3 weeks. Although the amounts of fluid intake wereslightly smaller in the groups receiving Sho-ju-sen andits constituents than that in the water-control group,the tendency to a decrease in fluid intake was almostthe same among the groups.

As shown in Table 2, there was no marked change inthe body weight gain among groups of mice that hadbeen given Sho-ju-sen (1%, 3% and 10%), Sasa (8%),Pine (1%), Ginseng (1%) and tap water.

Drug effects

As shown in Table 3, the mice given unavoidable/inescapable shocks with no drug treatment (control

Table 1. Average fluid intake during 3 weeks

Fluid intake (mL/kg/day)

Solution n Week 1 Week 2 Week 3

Tap water (control) 20 201 171 132

Sho-ju-sen 1% 10 154 116 1013% 10 152 122 108

10% 20 150 137 104Kumazasa 8% 10 159 152 117Japanese red pine 1% 10 161 132 118Ginseng 1% 10 166 120 121

ANTIDEPRESSANT EFFECT OF SHO-JU-SEN 175

Copyright © 2004 John Wiley & Sons, Ltd. Phytother. Res. 18, 173–176 (2004)

Table 4. Effects of free intake of Sho-ju-sen (10% solution) on learned helplessness in mice

Period of freeSolution intake (week) n Response/min % avoidance

Tap water (control) 1 20 0.95 ± 0.17 35.7 ± 6.62 20 1.10 ± 0.20 40.5 ± 7.83 20 1.05 ± 0.21 37.8 ± 8.2

Sho-ju-sen 1 10 1.10 ± 0.24 37.2 ± 10.22 10 1.84 ± 0.20a 55.0 ± 11.33 20 1.65 ± 0.16a 59.7 ± 7.0a

Data presented are mean ± SEM. a p < 0.05 vs the corresponding control.

Table 3. Effects of 3-week free intake of Sho-ju-sen and its con-stituents on learned helplessness in mice

Treatment n Response/min % avoidance

Tap water/no shock 20 2.19 ± 0.03 68.9 ± 5.9

Tap water/shock (control) 20 1.05 ± 0.21 37.8 ± 8.2Sho-ju-sen 1% 10 2.07 ± 0.01a 48.3 ± 8.8

3% 10 2.25 ± 0.06a 46.3 ± 9.310% 20 1.65 ± 0.16a 59.7 ± 7.0a

Kumazasa 8% 10 1.20 ± 0.30 38.2 ± 11.0Japanese red pine 1% 10 1.60 ± 0.25a 48.5 ± 10.6Ginseng 1% 10 1.11 ± 0.28 34.1 ± 10.3

Data presented are mean ± SEM. a p < 0.05 vs the control.

Table 5. Effects of imipramine (i.p.) and diazepam (p.o.) onlearned helplessness in mice

Drug n Response/min % avoidance

No drug/no shock 10 2.14 ± 0.03 63.3 ± 7.7Saline/shock (control) 10 0.65 ± 0.28 25.7 ± 11.9Imipramine 10 mg/kg 10 1.43 ± 0.24a 54.8 ± 11.2a

30 mg/kg 10 2.07 ± 0.03a 73.8 ± 7.1a

No drug/no shock 10 2.22 ± 0.05 69.4 ± 5.8Saline/shock (control) 20 0.91 ± 0.18 33.8 ± 6.8Diazepam 0.3 mg/kg 10 1.36 ± 0.26 35.2 ± 9.8

1 mg/kg 10 1.89 ± 0.08a 51.8 ± 11.43 mg/kg 10 1.37 ± 0.24 30.3 ± 12.1

Imipramine was i.p. administered for 4 days prior to startingthe induction session, and 1 h before each induction session.Diazepam was p.o. administered 10 min before each inductionsession. Data presented are mean ± SEM. a p < 0.05 vs thecontrol.

group) demonstrated a significantly lower response rateand % avoidance than the mice given no shocks, i.e.induction of learned helplessness.

Compared with the control group, the 3-week treat-ment with Sho-ju-sen (1%, 3% and 10%) resulted inamelioration of the learned helplessness. Thus, a sig-nificant increase in the response rate was demonstratedin the groups given Sho-ju-sen (1% and 3%), and boththe response rate and % avoidance in the group givenSho-ju-sen (10%). Pine, but not Sasa or Ginseng, mildlyincreased the response rate.

As shown in Table 4, the treatment with Sho-ju-senfor 2 weeks, but not for 1 week, caused a significantincrease in the response rate.

As shown in Table 5, seven daily administrations ofimipramine (10 and 30 mg/kg) resulted in a significantincrease in both the response rate and % avoidance.While diazepam, only at 1 mg/kg, mildly increased theresponse rate.

DISCUSSION

Antidepressants can specifically ameliorate learnedhelplessness, and improve avoidance acquisition depres-sed by the pretreatment with unavoidable/inescapableshocks (Seligman and Beagley, 1975; Sherman et al.,1982; Petty and Sherman, 1980; Willner, 1984). Con-sistent with such reports, the present experiment alsodemonstrated that seven daily treatments with thetricyclic antidepressant, imipramine, ameliorated thelearned helplessness, i.e. resulting in a significant im-provement of both the response rate and % avoidancein the training session. Whereas, the benzodiazepineanxiolytic, diazepam, had less potential than imipra-mine in the amelioration of the learned helplessness.

These basic data indicate that the present experimentalcondition was appropriate for the assessment of theantidepressant effect of test drugs.

The effects of Sho-ju-sen on the learned helplesnesswere dose- and time-dependent. Thus, the free con-sumption of Sho-ju-sen for 3 weeks resulted in a signific-ant increase in the response rate at 1% and 3%, andboth the response rate and % avoidance at 10%. Fur-thermore, a significant recovery of response rate wasproduced by a 2-week, but not a 1-week, free consump-tion of Sho-ju-sen (10%). The daily doses of Sho-ju-senconsumed were 1.54–1.01, 4.56–3.24 and 15.0–10.4 mL/kg/day for the groups of mice given 1%, 3% and10% Sho-ju-sen, respectively. However, the establishedavoidance response was never accelerated by the freeconsumption of Sho-ju-sen (1%–10%) or the single p.o.administration of 10–20 mL/kg Sho-ju-sen, rather thesingle administration of a high dose (40 mL/kg) ofSho-ju-sen mildly decreased the response rate (Kuribara,unpublished data). These results therefore suggest that,similar to the tricyclic antidepressant, imipramine, long-term consumption of Sho-ju-sen can reverse the learnedhelplessness, and may be effective for ameliorationof depression. The present results are also consistentwith the clinical efficacy of Sho-ju-sen on unidentifiedclinical syndrome, vegetative dystonia, etc. (WakanyakuMedical Institute, 1996; Ichikawa et al., 1998).

In the present study, the effects of constituents ofSho-ju-sen were also evaluated. The extract of Pine,but not Sasa or Ginseng, mildly ameliorated the learnedhelplessness; a significant increase in the response rate.However, the potential of Pine was less than that of

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Sho-ju-sen. These results suggest that the ameliorationof learned helplessness by Sho-ju-sen is mainly causedby Pine, and Sasa and/or Ginseng acting to enhancethe effect.

During the 3-week consumption of Sho-ju-sen, Sasa,Pine and Ginseng, the mice revealed slight decreases inthe volume consumed compared with the control micegiven tap water. Such a difference may be caused bynon-specific factor(s) including the taste of the fluids asreported elsewhere (Kuribara et al., 2000). Furthermore,all groups of mice showed a progressive decrease influid consumption. A similar change in the fluid andfood intake dependent on the maturation of rodentshas been observed (e.g. Tadokoro et al., 1981). How-ever, Sho-ju-sen, Sasa, Pine or Ginseng neither caused

a marked change in the body weight gain nor causedthe development of any abnormal symptoms, indicat-ing that Sho-ju-sen as well as its constituents may havea lower risk for induction of side-effects.

Our previous study using the elevated plus-maze testin mice revealed that free consumption of Sho-ju-senfor 4 days and longer caused an anxiolytic effect, andthat the effect was mainly caused by Sasa (Kuribaraet al., 2000). Taken together the present and previousresults, it is expected that long-term use of Sho-ju-sendevelops protective effects on stress-related psychic andpsychosomatic symptoms with a low risk of unwantedside-effects. It is also considered that such effects ofSho-ju-sen are produced by interactions of three con-stituents; extracts of Sasa, Pine and Ginseng.

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