Journal of Ethnopharmacology 143 (2012) 533–539

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Journal of Ethnopharmacology

0378-87

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ceutical

Sasebo,

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journal homepage: www.elsevier.com/locate/jep

Anxiolytic effects of yokukansan, a traditional Japanese medicine, viaserotonin 5-HT1A receptors on anxiety-related behaviors in ratsexperienced aversive stress

Taku Yamaguchi a,b,n, Aki Tsujimatsu a, Haruko Kumamoto a, Takeshi Izumi a, Yu Ohmura a,Takayuki Yoshida a, Mitsuhiro Yoshioka a

a Department of Neuropharmacology, Hokkaido University Graduate School of Medicine, Sapporo 060-8638, Japanb Department of Pharmacology, Faculty of Pharmaceutical Sciences, Nagasaki International University, Sasebo 859-3298, Japan

a r t i c l e i n f o

Article history:

Received 16 February 2012

Received in revised form

31 May 2012

Accepted 10 July 2012Available online 20 July 2012

Keywords:

Anxiolytic effects

Yokukansan

Serotonin 5-HT1A receptors

Anxiety-related behaviors

Aversive stress.

41/$ - see front matter & 2012 Elsevier Irelan

x.doi.org/10.1016/j.jep.2012.07.007

esponding author at: Department of Pharm

Sciences, Nagasaki International Universit

Nagasaki 859-3298, Japan. Tel.: þ81 956 20

ail address: taku@kyudai.jp (T. Yamaguchi).

a b s t r a c t

Ethnopharmacological relevance: Yokukansan, a traditional Japanese medicine (Kampo), has been reported

in the treatment of behavioral and psychological symptoms of dementia (BPSD) such as aggression, anxiety

and depression in patients with Alzheimer’s disease and other forms of senile dementia.

Aims of the study: In the present study, we investigated the anxiolytic effects of yokukansan on anxiety-

related behaviors in rats that have experienced aversive stress.

Materials and methods: We used male Wistar/ST rats which received an electrical footshock as aversive

stress. Yokukansan at a dose of 1.0 g/kg was administered orally once a day for 14 or 16 day before

behavioral tests. To evaluate the anxiolytic effects, we used the contextual fear conditioning (CFC) test and

elevated plus-maze (EPM) test. And we also investigated effects of yokukansan on locomotor activity in the

Open-field (OF) test and on the change in plasma corticosterone after CFC stress, in rats that had

experienced footshock stress.

Results: In the CFC test, rats that had experienced footshock showed significant freezing behavior on

re-exposure to the box 14 day after footshock stress. Yokukansan significantly suppressed freezing behavior

in the CFC test. In the EPM test on the 16th day after the CFC test, yokukansan significantly increased the

time spent in open arms after footshock stress compared to control rats. However, repeated administration

of yokukansan on the 14th day did not affect the decrease in locomotor activity and the increase in plasma

corticosterone by re-exposure to the box 14 day after footshock stress in the OF test and determination of

serum corticosterone, respectively. These anxiolytic effects by yokukansan were antagonized by

WAY-100635, a selective 5-HT1A receptor antagonist, in the CFC test, but not the EPM test. Furthermore,

5-HT1A receptor agonist buspirone significantly suppressed freezing behavior in the CFC test; however,

buspirone induced no change in the time spent in open arms in the EPM test.

Conclusion: These findings suggested that yokukansan has anxiolytic effects on anxiety-like behaviors

induced by both innate fear and memory-dependent fear. In particular, yokukansan produced anxiolytic

effects via 5-HT1A receptors in memory-dependent fear induced by aversive stress. Furthermore,

yokukansan could be useful as one of the therapeutic drugs for the treatment of anxiety disorders and

various mental disorders that have comorbid anxiety.

& 2012 Elsevier Ireland Ltd. All rights reserved.

1. Introduction

Yokukansan (Yi-gan san in Chinese) is a traditional Japanesemedicine (Kampo), which was developed in 1555 by Xue Kai as acure for agitation and restlessness in infants. In the traditional use ofyokukansan, it is useful for curing screaming attacks, sleep terrorsand hypnic myoclonia, especially in children. Moreover, it has been

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used as a cure for neurosis, insomnia and irritability in adultsubjects, which has been approved by the Ministry of Health Labourand Welfare of Japan. Thus, yokukansan is assumed to have sometherapeutic effects on the excitability of nerves. However, themechanisms underlying the therapeutic effects of yokukansan arestill unclear.

Behavioral and psychological symptoms of dementia (BPSD),including agitation, aggression, screaming, hallucinations and delu-sions, occur in 20–80% of patients with dementia (Lawlor, 2004;Cerejeira et al., 2012). These symptoms impair the activities of dailyliving (ADL) in patients with dementia, impose great burdens oncaregivers, hasten hospital admission (Steele et al., 1990), and

T. Yamaguchi et al. / Journal of Ethnopharmacology 143 (2012) 533–539534

increase care costs (Beeri et al., 2002). To date, antipsychotics havebeen used as pharmacotherapy for BPSD; however, antipsychoticsinduce extrapyramidal symptoms and other adverse events and, inconsequence, they decrease the quality of life (QOL) or increase thedifficulty of maintaining the ADL (Kales et al., 2012; Huybrechts et al.,2012). In addition, in 2008 and 2012, the U.S. Food and DrugAdministration (FDA) warned that mortality was increased in elderlypatients with dementia who used atypical antipsychotics (http://www.fda.gov/Safety/MedWatch/SafetyInformation/SafetyAlertsforHumanMedicalProducts/ucm110212.htm; 2008, www.fda.gov/downloads/Drugs/DrugSafety/UCM300946.pdf; 2012). Wang et al. (2005)reported that conventional antipsychotics also involve the same risksas atypical antipsychotics. Recently, yokukansan was clinical reportedto improve the symptoms of BPSD without severe adverse events(Iwasaki et al., 2005a; 2005b; Mizukami et al., 2009; Hayashi et al.,2010; Okahara et al., 2010).

In studies using experimental animals, yokukansan was effectiveon aggressiveness induced by intracerebroventricular injection ofamyloid b protein in mice (Sekiguchi et al., 2009). Recently,yokukansan was found to ameliorate abnormal interaction beha-viors, such as enhanced aggressive behavior and decreased socialbehavior induced by para (p)-chloroamphetamine (PCA), which iswell-known to be a potent serotonergic neurotoxin (Kanno et al.,2009). In addition, an in vitro binding assay showed that yokukansanpossesses a partial agonistic effect on 5-HT1A receptors (Terawakiet al., 2010). These reports suggest that the effectiveness of yoku-kansan on impaired social interaction behaviors in PCA-injected ratsmay be mediated through the serotonergic neuronal systems via5-HT1A receptors.

Taken together with these previous reports, yokukansan mayhave therapeutic potential for problematic mental symptoms,including anxiety, induced by the excitability of nerves; however,anxiolytic effects and the mechanism of yokukansan are stillunclear. Therefore, in the present study, we investigated thepharmacological effects of yokukansan on anxiety-related beha-vior in rats that had exposed the footshock stress as an aversivestimulus in the contextual fear conditioning test. Following thetest, using the same rat that had experienced the contextual fearconditioning test, we further evaluated effects of yokukansan onanxiety-related behavior in the elevated plus-maze test. Wefurther examined the involvement of 5-HT1A receptors in thepharmacological effects of yokukansan using WAY-100635, aselective 5-HT1A receptor antagonist.

Fig. 1. Experimental schedule. Experimental schedule, described in Materials

and methods, involved the contextual fear conditioning (CFC) test and elevated

plus-maze (EPM) test. FS: footshock.

2. Materials and methods

2.1. Subjects

The subjects were male Wistar/ST rats (10–13 weeks old)supplied by Nippon SLC Co. (Hamamatsu, Japan) and housed ingroups of three to four rats under an alternating light-dark cycle(light from 7 p.m. to 7 a.m.) at approximately 22 1C. Food and waterwere available ad libitum. The treatment of animals complied withthe guidelines for the care and use of laboratory animals of theAnimal Research Committee of the Hokkaido University, Japan.

2.2. Drugs

Yokukansan (Lot No. 2060054010) was donated and manufac-tured by Tsumura & Co. (Tokyo, Japan) according to the formulationpreviously reported by Terawaki et al. (2010), which was used in thepresent studies. In brief, yokukansan is a dried extract of thefollowing raw materials:4.0 parts Atractylodis Lanceae Rhizoma(rhizome of Atractylodes lancea De Candolle, Compositae), 4.0 partsPoria (sclerotium of Poria cocos Wolf, Polyporaceae), 3.0 parts Cnidii

Rhizoma (rhizome of Cnidium officinale Makino, Umbelliferae),3.0 parts Angelicae Radix (root of Angelica acutiloba Kitagawa,Umbelliferae), 2.0 parts Bupleuri Radix (root of Bupleurum falcatum

Linne, Umbelliferae), 1.5 parts Glycyrrhizae Radix (root and stolon ofGlycyrrhiza uralensis Fisher, Leguminosae), and 3.0 parts UncariaUncis Cum Ramulus (hook of Uncaria rhynchophilla Miquel, Rubia-ceae). The seven medical herbs were extracted with purified waterat 95 1C for 1 h, and the extraction solution was separated from theinsoluble waste and concentrated by removing water under reducedpressure. Spray drying was used to produce a dried extract powder.The yield of the extract was about 15.9%.

The dry powdered extract of yokukansan was dissolved indistilled water (0.01 or 0.1 g/ml). The dosage was decided basedon the results of a previous study (Kanno et al., 2009). Yokukansanwas orally administered at a volume of 1 ml/100 g for rats once aday, according to the experimental schedule in Fig. 1. For controls,distilled water was administered at the same volume. All behavi-oral tests were performed 60 min after the final administration ofyokukansan or distilled water. N-[2-[4-(2-methoxyphenyl)-1-piperazinyl]ethyl]-N-(2-pyridinyl) cyclo-hexanecarboxamide trihy-drochloride (WAY-100635), a selective 5-HT1A receptor antagonist.and buspirone, a 5-HT1A receptor agonist, were purchased fromSigma-Aldrich (St. Louis, MO) and dissolved in saline.

2.3. Contextual fear conditioning (CFC) test

Each rat was acclimated in a footshock box (30.5�24.1�21.0 cm; Med Associates Inc.) for 5 min. Subsequently, ten 2 selectrical footshocks (shock intensity, 0.5 mA) were administeredat 30 s intervals, except for no-footshock controls. No-footshockcontrols did not receive footshocks. After the last footshock, ratswere immediately returned to their home cage (Footshocked [FS]group, see the experimental schedule in Fig. 1). Two weeks later,each rat was placed into the footshock box again without beingshocked. Freezing behavior was defined as a lack of movementexcept for respiration, accompanied by an arched back andretraction of the ears (Fanselow, 1980), and was used as ameasure of memory-dependent fear. In the 15 min testing period,the presence or absence of freezing was estimated by an auto-matic system (FreezeFrame; Actimetrics, Wilmette, IL) using apixel difference method.

Yokukansan (0.1 and 1.0 g/kg, p.o.) or distilled water (10 ml/kg, p.o.) was administered once a day to rats for 14 day afterexposure to electrical footshocks. For assessment of the acuteeffect of yokukansan, a single injection of yokukansan or distilledwater was given to rats on the 14th day after exposure toelectrical footshocks.

Yokukansan (1.0 g/kg, p.o.) was administered to rats injectedonce with WAY-100635 (0.03 and 0.3 ml/kg, i.p.) for 14 day as acontrol. For assessment of the acute effect of WAY-100635, asingle injection of WAY-100635 or saline was given to rats on the

Fig. 3. Effects of yokukansan on locomotor activity in rats that have experienced

footshock stress in the open-field test. (A) Line crossing for every 5 min over

15 min. (B) Total line crossing for 15 min. DW: distilled water, FS: footshocked

group, Non-FS: non-footshocked group. Values are the mean7SEM. npo0.05,nnpo0.01 compared with distilled water-injected rats without footshock stress.

Fig. 2. Effects of yokukansan on freezing behavior in rats that have experienced

footshock stress in the contextual fear conditioning test. DW: distilled water, FS:

footshocked group, Non-FS: non-footshocked group. Values are the mean7SEM.

**po0.01 compared with distilled water-injected rats without footshock stress.#po0.05 compared with distilled water-injected rats that have experienced

footshock stress.

T. Yamaguchi et al. / Journal of Ethnopharmacology 143 (2012) 533–539 535

14th day after repeated yokukansan. As a control, yokukansan(1.0 g/kg, p.o.) was administered for 14 day to rats injected withsaline.

Buspirone (1.0 mg/kg. i.p.) or saline (10 ml/kg, i.p.) was admi-nistered once a day to rats for 14 day after exposure to electricalfootshocks.

2.4. Elevated plus-maze (EPM) test

After the CFC test, rats were again administered drug(s) orvehicle for an additional 2 day, and the EPM test was performed60 min after the final injection of yokukansan or distilled water(see the experimental schedule in Fig. 1). Also, effects of pre-treatments of WAY-100635 or buspirone in the EPM test are dueto the result that animals were re-tested 2 day later after CFC test.

Other anxiety-related behavior was evaluated using the EPMtest (Pellow et al., 1986, 1985), and the results were assessed asdescribed in our previous report (Ohmura et al., 2008). Thebehavior of each rat was monitored using a CCD camera, andthe data were recorded/analyzed using the LimeLight softwarepackage (Actimetrics Inc., Wilmette, IL). All measures of rat move-ments were automatically generated by the software. Briefly, theapparatus consisted of a central platform (10�10 cm) and twoopposite open arms (50 cm length�10 cm width) and closed arms(50 cm length�10 cm width�40 cm height) that were elevated50 cm above the floor. The luminosity measured at the center ofthe maze (central platform) was 400 lx. The rats were placed on thecentral platform and allowed to enter the arms freely for 10 min. Armentry was defined as entry of the center of the body into the arm. Thetime spent in open and closed arms and the number of entries intoopen and closed arms was recorded. The time spent in open arms wascalculated as the percentage of time that the center of the rat’s bodyspent in the arms. Locomotor activity was quantified by counting thenumber of times that the rat crossed the central platform as thenumber of total arm entries judged using the center of the body.

2.5. Open-field (OF) test

An open-field apparatus (90 cm length�90 cm width�40 cmheight) illuminated by a white fluorescent light (200 lx) placed160 cm above the floor was used. Each rat was first placed in thecenter of the open field and left in the apparatus for 15 min. Thelocomotor activity of each rat was monitored using a CCD camera,and the data were recorded/analyzed using the Limelight soft-ware package (Actimetrics Inc., Wilmette, IL). All measurementsof rat movement were quantified by counting the number oftimes the rat crossed a line dividing the floor into 81 (9�9)squares of equal size. These counts were automatically generatedby the software. This open-field test was performed 1 h afterinjection of yokukansan or distilled water on the 14th day afterexposure to electrical footshocks.

2.6. Determination of serum corticosterone

Approximately 50 ml blood collected from the tail vein wassampled into two hematocrit tubes (VITREX Medical A/S, Herlev,Denmark) 30 min after exposure to the contextual fear condition-ing test. Samples were separated using a centrifugal separator(10,000� g for 10 min) (KH-120M; Kubota Co., Ltd., Tokyo, Japan),and serum was stored at �80 1C until analysis. Sampling occurredat a consistent time of day (12:00–16:00 h). Serum corticosteronelevels were measured in duplicate by enzyme immunoassay usinga Corticosterone EIA kit (Cayman Chemical Company, Ann Arbor,MI). The absorbance of each sample was measured at a wave-length of 405 nm.

2.7. Statistical analysis

The resultant values are expressed as the mean7SEM. Thesignificance of differences between groups was determined usingone-way analysis of variance (ANOVA) followed by a two-tailedDunnett’s test (Figs. 2, 4, 6). Repeated ANOVA followed by Fisher’s

Fig. 4. Effects of yokukansan on serum corticosterone in rats that have experi-

enced footshock stress. DW: distilled water, FS: footshocked group, Non-FS: non-

footshocked group. Values are the mean7SEM. npo0.05, compared with distilled

water-injected rats without footshock stress.

T. Yamaguchi et al. / Journal of Ethnopharmacology 143 (2012) 533–539536

PLSD test (Figs. 3 and 5) was used as a post-hoc test. The significanceof differences between sham and buspirone-treated groups wasdetermined using a two-tailed Student’s t-test (Fig. 7). A differencewas considered significant at po0.05.

Fig. 5. Effects of yokukansan on anxiety-like behaviors during the elevated plus-

maze test in rats that have experienced footshock stress. Upper graph shows the

time spent in open arms and bottom graph shows the total arm entry in the

elevated plus-maze test. DW: distilled water, FS: footshocked group, Non-FS: non-

footshocked group. Values are the mean7SEM. npo0.05 compared with distilled

water-injected rats without footshock stress. #po0.05 compared with distilled

water-injected rats that have experienced footshock stress.

Fig. 6. Effects of 5-HT1A receptor antagonist WAY-100635 on the anxiolytic effects

by yokukansan in the Contextual fear conditioning test and elevated plus-maze

test. (A) Contextual fear conditioning test. (B) Elevated plus-maze test. Values are

the mean7SEM. npo0.05 compared with saline and yokukansan-injected rats.

3. Results

3.1. Effects of yokukansan on freezing behavior in rats that had

experienced footshock stress

As shown in Fig. 2, rats that had experienced footshocksignificantly developed freezing behavior by re-exposure to thebox 14 day after footshock stress in the CFC test. Repeated admin-istration of yokukansan at a dose of 1.0 g/kg for 14 day, but not at adose of 0.1 g/kg, significantly suppressed freezing behavior in theCFC test (Fig. 2); however, single administration of yokukansan at adose of 1.0 g/kg on the 14th day did not affect freezing behavior byre-exposure to the box 14 day after footshock stress (Fig. 2).

3.2. Effects of yokukansan on locomotor activity and serum

corticosterone in rats that had experienced footshock stress

Fig. 3 shows the effects of yokukansan on locomotor activity inrats that had experienced footshock stress in the OF test. Rats thathad experienced footshock stress had significantly reduced loco-motor activity in the OF test; however, repeated administration ofyokukansan at a dose of 1.0 g/kg on the 14th day did not affect thedecrease in locomotor activity by re-exposure to the box 14 daysafter footshock stress compared with the footshock-stressedgroup administered distilled water (Fig. 3).

Fig. 4 shows the effects of yokukansan on the change in plasmacorticosterone after contextual fear-conditioning stress. Ratsre-exposed to the footshock box 14 days after footshock stress hadsignificantly elevated plasma corticosterone; however, repeatedadministration of yokukansan at a dose of 1.0 g/kg on the 14thday also did not also affect the increase in plasma corticosterone byre-exposure to the box 14 days after footshock stress, comparedwith the footshock-stressed group administered distilled water(Fig. 4).

3.3. Effects of yokukansan on anxiety-like behaviors during the

elevated plus-maze test in rats that had experienced footshock stress

As shown in Fig. 5, in the EPM test after the CFC test, rats thathad experienced footshock showed a significantly decreased timespent in open arms by re-exposure to the box 14 day after footshockstress. Repeated administration of yokukansan at a dose of both0.1 and 1.0 g/kg for 14 days significantly inhibited the decrease of

T. Yamaguchi et al. / Journal of Ethnopharmacology 143 (2012) 533–539 537

time spent in open arms in rats that had experienced footshockstress in the EPM test, compared to control rats (Fig. 5, upper graph).Moreover, single administration of yokukansan at a dose of 1.0 g/kgon the 14th day also inhibited the decrease of time spent in openarms by re-exposure to the box 14 days after footshock stress in theEPM test (Fig. 5, upper graph). Total arm entry was not affected inany-treated groups (Fig. 5, bottom graph).

3.4. Effects of 5-HT1A receptor antagonist WAY-100635 on the

anxiolytic effects of yokukansan

Fig. 6 shows the effects of WAY-100635, a 5-HT1A receptorantagonist, on the anxiolytic effects of yokukansan in rats thathad experienced footshock stress. Yokukansan (1.0 g/kg, p.o.) wasadministered to rats injected once with WAY-100635 (0.03 and0.3 ml/kg, i.p.) for 14 days as a control. The anxiolytic effects byrepeated administration of yokukansan were significantly antag-onized by co-administration of WAY-100635 (0.3 mg/kg, i.p.) withyokukansan in the CFC test (Fig. 6A), but not the EPM testassessed 2 day later after CFC test (Fig. 6B). Single administrationof WAY-100635 at a dose of 0.3 mg/kg on the 14th day did notaffect the anxiolytic effects by repeated administration of yoku-kansan in both CFC and EPM tests (Fig. 6A and B).

3.5. Effects of 5-HT1A partial agonist buspirone on anxiety-like

behaviors in rats that had experienced footshock stress

As shown in Fig. 7, repeated administration of buspirone, a5-HT1A partial agonist, at a dose of 1.0 mg/kg for 14 day significantly

Fig. 7. Effects of 5-HT1A partial agonist buspirone on anxiety-like behaviors in rats

that have experienced footshock stress in the open-field test and elevated plus-

maze test. (A) Contextual fear conditioning test. (B) Elevated plus-maze test. FS:

footshocked group. Values are the mean7SEM. npo0.05 compared with saline-

injected rats that have experienced footshock stress.

suppressed freezing behavior in rats that had experienced footshockin the CFC test (Fig. 7A); however, repeated administration ofbuspirone did not change the time in spent open arms in the EPMtest assessed 2 day later after CFC test (Fig. 7B).

4. Discussion

Yokukansan (0.1–1.0 g/kg) which we used for the animalexperiments is equivalent to approximately 10–15 times of theclinical dose. It is difficult to extrapolate the dose for animals fromthe clinical dose for humans. Therefore, the dosage (0.1 and 1.0 g/kg)of yokukansan in the present study was decided based on the resultsof previous study demonstrating that yokukansan amelioratedimpairment of social interaction behaviors in PCA-injected rats(Kanno et al., 2009).

The CFC test is used to evaluate anxiety-related behaviors thatdepend on fear memory. Rats that have experienced aversivestresses (electrical footshock) showed emotional responses suchas freezing behavior, defecation and urination in the experimentalbox where the rats had received the footshock stress, in spite ofno exposure to aversive stress. In particular, it is thought thatfreezing behavior was expressed more frequently when rats feltstrong anxiety and fear. In the CFC test, repeated administrationof yokukansan significantly inhibited the expression of freezingbehavior in rats that had experienced footshock stress. Thisinhibitory effect of freezing behavior by yokukansan is not dueto the increase of locomotor activity since yokukansan did notaffect locomotor activity in the present study (see Fig. 3). Further-more, there are no reports that yokukansan induced memoryimpairments in experimental animals and humans, although it isreported that yokukansan ameliorated memory deficits inAPP-Tg(þ) mice (Tabuchi et al., 2009), in mice intracerebroven-tricular-injected amyloid b protein (Sekiguchi et al., 2009), inolfactory bulbectomized mice (Yamada et al., 2011), in thiamine-deficient rats (Ikarashi et al., 2009) and in aged rats (Mizoguchiet al., 2011). On the other hands, conditioned fear paradigm (CFCtest) involves both emotional and cognitive factors different frominnate fear paradigm (EPM test). However, Mizoguchi et al. (2011)show that yokukansan did not change the ability of initiallearning of aged rats in which the initial learning function wasnormal, in the reversal learning test. Thus, since yokukannsan donot enhance the general cognitive function, anxiolytic effects byyokukansan in CFC test is not likely due to memory enhancingeffects. Taken together with these findings, it is suggested thatrepeated administration of yokukansan has anxiolytic effects onanxiety-related behaviors induced by conditioned fear.

In the EPM test after the CFC test, rats that had experiencedfootshock stress showed a significantly decreased time spent inopen arms, which indicated the anxiety-related behavior, espe-cially stress-induced anxiety. Such an experimental paradigmusing not only the footshock but also the forced-swimming andrestraint stress is often used to evaluate effects of anxiolytic onstress-induced anxiety in the EPM test (Smriga and Torii, 2003;Chaki et al., 2004). We also confirmed a footshock stress-inducedanxiety in the EPM test. In the EPM test with this footshockstress-induced anxiety as an index, yokukansan significantlyincreased the time spent in open arms compared with controlsin the EPM test. This result of the EPM test indicates thatyokukansan also had anxiolytic effect as in the CFC test in thisstudy. Anxiolytic effects of yokukansan have been reported usingthe hole-board test and EPM test in mice, which produceanxiolytic effects through the benzodiazepine active site of GABAA

receptors in mice (Kamei et al., 2009). In the present study,yokukansan was also confirmed to have anxiolytic effects in theEPM test in rats. From these results, it is suggested that

T. Yamaguchi et al. / Journal of Ethnopharmacology 143 (2012) 533–539538

yokukansan can improve anxiety-related behaviors induced bynot only innate fear but also memory-dependent fear.

It is known that the hypothalamus-pituitary-adrenal axis isactivated by stress, and corticosterone is released from the adrenalgland. The plasma corticosterone levels were significantly increasedin both distilled water and yokukansan-treated groups that hadexperienced footshock stress compared to control rats. Moreover,there was no difference between distilled water and yokukansan-treated groups in the plasma corticosterone levels. It is reported thatchlordiazepoxide, a benzodiazepine receptor agonist, inhibitedincreased the plasma corticosterone levels by stress (Degroot et al.,2004). These observations and our findings in the present study ofincreased plasma corticosterone levels by stress indicated thatbenzodiazepine receptors were not involved in the anxiolytic-effectof yokukansan in the contextual fear conditioning test. Otherwise, a5-HT1A receptor agonist tandospirone, and a selective serotoninreuptake inhibitor fluoxetine themselves elevated plasma corticoster-one levels under and without stress conditions (Lopez et al., 1998;Sullivan et al., 2005). These reports indicated that serotonergicmechanisms were not involved in elevated plasma corticosteronelevels by stress reduced by the anxiolytic effects of yokukansan.Further studies are required to clarify this issue.

The anxiolytic effects of yokukansan were antagonized by WAY-100635, a selective 5-HT1A receptor antagonist, in the CFC test, butnot the EPM test (Fig. 6). Furthermore, 5-HT1A receptor agonistbuspirone significantly induced the anxiolytic-like effect in the CFCtest, but not the EPM test (Fig. 7). Anxiolytic effect of yokukansanwas induced by serotonin release in the prefrontal cortex of agedrats (Mizoguchi et al., 2010). Yokukansan ameliorated aggressivebehavior induced by a potent serotonergic neurotoxin via 5-HT1A

receptors (Kanno et al., 2009). Furthermore, yokukansan had apartial agonistic effect on 5-HT1A receptors in an in vitro bindingassay (Terawaki et al., 2010). It is reported that 5-HT1A receptoragonists, including buspirone, did not induce anxiolytic effects in theEPM test (Klint 1991; Cao and Rodgers 1997). On the other hand, itwas reported that anxiolytic effects by acute and repeated admin-istration of yokukansan in the EPM test were mediated throughactivation of the benzodiazepine active site of GABAA receptors(Kurihara and Maruyama 1996; Kamei et al., 2009). Indeed, ourfindings also revealed the anxiolytic effects of yokukansan in theEPM test (Fig. 5). Therefore, it is suggested that yokukansan mayproduce anxiolytic effects due to the dual activation of 5-HT1A andGABAA receptors. In particular, our findings suggested that theanxiolytic effect by repeated administration of yokukansan wasmediated through the serotonergic neuronal system(s) via 5-HT1A

receptors in rats that had experienced aversive stress.Uncaria hook is one of the components of Yokukansan. Uncaria

hook contains many alkaloids, corynoxeine, hirsuteine, geissoschizinemethyl ether, rhynchophylline and so on, which may play pivotalroles in the effects of yokukansan in the central nervous system(Heitzman et al., 2005). In particular, geissoschizine methyl ether,which is an active ingredient of Uncaria hook, is an indole alkaloidand has pharmacological effects similar to a 5-HT1A receptor agonist(Pengsuparp et al., 2001). Moreover, it is reported that geissoschizinemethyl ether has a partial agonistic binding profile to 5-HT1A

receptors in binding assays (Pengsuparp et al., 2001). In the presentstudy, we show that yokukansan produces anxiolytic-like effectsmediated through the activation of 5-HT1A receptors in the CFC test;therefore indicating that Uncaria hook, especially geissoschizinemethyl ether, might contribute to the effects of yokukansan.

5. Conclusion

In conclusion, these findings suggested that yokukansan hasanxiolytic properties in both innate fear and conditioned fear.

In particular, yokukansan produced anxiolytic effects mediatedthrough 5-HT1A receptors on the learned fear. Furthermore,yokukansan could be useful as one of the therapeutic drugs forthe treatment of anxiety disorders and various mental disordersthat have comorbid anxiety.

Acknowledgments

The authors thank Tsumura Corporation (Tokyo, Japan) for thegift of the dry powdered extract of yokukansan. The authors alsothank Ms. Yukino Futami and Mr. Reona Bandoh for technicalassistance.

References

Beeri, M.S, Werner., P, Davidson., M, Noy, S., 2002. The cost of behavioral andpsychological symptoms of dementia (BPSD) in community dwelling Alzhei-mer’s disease patients. International Journal of Geriatric Psychiatry 17,403–408.

Cao, B.J., Rodgers, R.J., 1997. Comparative behavioural profiles of buspirone and itsmetabolite 1-(2-pyrimidinyl)-piperazine (1-PP) in the murine elevated plus-maze. Neuropharmacology 36, 1089–1097.

Cerejeira, J., Lagarto, L., Mukaetova-Ladinska, E.B., 2012. Behavioral and psycho-logical symptoms of dementia. Frontiers in Neurology 3, 73.

Chaki, S., Nakazato, A., Kennis, L., Nakamura, M., Mackie, C., Sugiura, M., Vinken, P.,Ashton, D., Langlois, X., Steckler, T, 2004. Anxiolytic- and antidepressant-likeprofile of a new CRF1 receptor antagonist, R278995/CRA0450. EuropeanJournal of Pharmacology 485, 145–158.

Degroot, A., Wade, M., Salhoff, C., Davis, R.J., Tzavara, E.T., Nomikos, G.G., 2004.Exposure to an elevated platform increases plasma corticosterone and hippo-campal acetylcholine in the rat: reversal by chlordiazepoxide. EuropeanJournal of Pharmacology 493, 103–109.

Fanselow, M.S., 1980. Conditioned and unconditional components of postshockfreezing. Pavlovian Journal of Biological Science 15, 177–182.

Hayashi, Y., Ishida, Y., Inoue, T., Udagawa, M., Takeuchi, K., Yoshimuta, H., Kiue, K.,Ninomiya, Y., Kawano, J., Sameshima, T., Kawahara, T., Goto, I., Shudo, K.,Kurayama, S., Nakamura, J., Okahara, K., Mitsuyama, Y., 2010. Treatment ofbehavioral and psychological symptoms of Alzheimer-type dementia withyokukansan in clinical practice. Progress in Neuro-Psychopharmacology andBiological Psychiatry 34, 541–545.

Heitzman, M.E., Neto, C.C., Winiarz, E., Vaisberg, A.J., Hammond, G.B., 2005.Ethnobotany, phytochemistry and pharmacology of Uncaria (Rubiaceae).Phytochemistry 66, 5–29.

Huybrechts, K.F., Gerhard, T., Crystal, S., Olfson, M., Avorn, J., Levin, R., Lucas, J.A.,Schneeweiss, S., 2012. Differential risk of death in older residents in nursinghomes prescribed specific antipsychotic drugs: population based cohort study.British Medical Journal 344, e977.

Ikarashi, Y., Iizuka, S., Imamura, S., Yamaguchi, T., Sekiguchi, K., Kanno, H.,Kawakami, Z., Yuzurihara, M., Kase, Y., Takeda, S., 2009. Effects of yokukansan,a traditional Japanese medicine, on memory disturbance and behavioral andpsychological symptoms of dementia in thiamine-deficient rats. Biological andPharmaceutical Bulletin 32, 1701–1719.

Iwasaki, K., Satoh-Nakagawa, T., Maruyama, M., Monma, Y, Nemoto, M., Tomita, N.,Tanji, H., Fujiwara, H., Seki, T., Fujii, M., Arai, H, Sasaki, H., 2005a. Arandomized, observer-blind, controlled trial of the traditional Chinese medi-cine Yi-gan san for improvement of behavioral and psychological symptomsand activities of daily living in dementia patients. Journal of Clinical Psychiatry66, 248–252.

Iwasaki, K., Maruyama, M., Tomita, N., Furukawa, K., Nemoto, M., Fujiwara, H.,Seki, T., Fujii, M., Kodama, M., Arai, H., 2005b. Effects of the traditional Chineseherbal medicine Yi-gan san for cholinesterase inhibitor-resistant visual hallu-cinations and neuropsychiatric symptoms in patients with dementia withLewy bodies. Journal of Clinical Psychiatry 66, 1612–1613.

Kales, H.C., Kim, H.M., Zivin, K., Valenstein, M., Seyfried, L.S., Chiang, C.,Cunningham, F., Schneider, L.S., Blow, F.C., 2012. Risk of mortality amongindividual antipsychotics in patients with dementia. The American Journal ofPsychiatry 169, 71–79.

Kamei, J., Miyata, S., Ohsawa, M., 2009. Involvement of the benzodiazepine systemin the anxiolytic-like effect of Yokukansan (Yi-gan san). Progress in Neuro-Psychopharmacology and Biological Psychiatry 33, 1431–1437.

Kanno, H., Sekiguchi, K., Yamaguchi, T., Terawaki, K., Yuzurihara, M., Kase, Y.,Ikarashi, Y., 2009. Effects of yokukansan, a traditional Japanese medicine, onsocial and aggressive of para-chloroamphetamine-injected rats. Journal ofPharmacy and Pharmacology 61, 1249–1256.

Klint, T., 1991. Effects of 8-OH-DPAT and buspirone in a passive avoidance test andin the elevated plus-maze test in rats. Behavioural Pharmacology 2, 481–489.

Kurihara, H., Maruyama, Y., 1996. The anxiolytic effect of oriental herbal medi-cines by an improved plus-maze test in mice—involvement of benzodiazepinereceptors. Japanese Journal of Neuropsychopharmacology 18, 179–190.

T. Yamaguchi et al. / Journal of Ethnopharmacology 143 (2012) 533–539 539

Lawlor, B.A., 2004. Behavioral and psychological symptoms in dementia: the roleof atypical antipsychotics. Journal of Clinical Psychiatry 65, 5–10.

Lopez, J.F., Chalmers, D.T., Little, K.Y., Watson, S.J., 1998. A.E. Bennett ResearchAward. Regulation of serotonin1A, glucocorticoid, and mineralocorticoidreceptor in rat and human hippocampus: implications for the neurobiologyof depression. Biological Psychiatry 43, 547–573.

Mizoguchi, K., Shoji, H., Tanaka, Y., Tabira, T., 2011. Ameliorative effect oftraditional Japanese medicine yokukansan on age-related impairments ofworking memory and reversal learning in rats. Neuroscience 177, 127–137.

Mizoguchi, K., Tanaka, Y., Tabira, T., 2010. Anxiolytic effect of a herbal medicine,yokukansan, in aged rats: involvement of serotonergic and dopaminergictransmissions in the prefrontal cortex. Journal of Ethnopharmacology 127,70–76.

Mizukami, K., Asada, T., Kinoshita, T., Tanaka, K., Sonohara, K., Nakai, R.,Yamaguchi, K., Hanyu, H., Kanaya, K., Takao, T., Okada, M., Kudo, S., Kotoku,H., Iwakiri, M., Kurita, H., Miyamura, T., Kawasaki, Y., Omori, K., Shiozaki, K.,Odawara, T., Suzuki, T., Yamada, S., Nakamura, Y., Toba, K., 2009. A randomizedcross-over study of a traditional Japanese medicine (kampo), yokukansan, inthe treatment of the behavioural and psychological symptoms of dementia.International Journal of Neuropsychopharmacology 12, 191–199.

Ohmura, Y., Yamaguchi, T., Izumi, T., Matsumoto, M., Yoshioka, M., 2008.Corticotropin releasing factor in the median raphe nucleus is involved in theretrieval of fear memory in rats. European Journal of Pharmacology 584,357–360.

Okahara, K., Ishida, Y., Hayashi, Y., Inoue, T., Tsuruta, K., Takeuchi, K., Yoshimuta, H.,Kiue, K., Ninomiya, Y., Kawano, J., Yoshida, K., Noda, S., Tomita, S., Fujimoto, M.,Hosomi, J., Mitsuyama, Y., 2010. Effects of yokukansan on behavioral andpsychological symptoms of dementia in regular treatment for Alzheimer’s disease.Progress in Neuro-Psychopharmacology and Biological Psychiatry 34, 532–536.

Pellow, S., File, S.E., 1986. Anxiolytic and anxiogenic drug effects on exploratoryactivity in an elevated plus-maze: a novel test of anxiety in the rat.Pharmacology Biochemistry and Behavior 24, 525–529.

Pengsuparp, T., Indra, B., Nakagawasai, O., Tadano, T., Mimaki, Y., Sashida, Y.,Ohizumi, Y, Kisara, K., 2001. Pharmacological studies of geissoschizine methyl

ether, isolated from Uncaria sinensis Oliv., in the central nervous system.European Journal of Pharmacology 425, 211–218.

Sekiguchi, K., Yamaguchi, T., Tabuchi, M., Ikarash,i, Y., Kase, Y., 2009. Effects of

yokukansan, a traditional Japanese medicine, on aggressiveness induced byintracerebroventricular injection of amyloid beta protein into mice. Phytother-

apy Research 23, 1175–1181.Smriga, M., Torii, K., 2003. Prolonged treatment with L-lysine and L-arginine

reduces stress-induced anxiety in an elevated plus maze. Nutritional Neu-roscience 6, 125–128.

Steele, C., Rovner, B., Chase, G.A., Folstein, M., 1990. Psychiatric symptoms and

nursing home placement of patients with Alzheimer’s disease. AmericanJournal of Psychiatry. 147, 1049–1051.

Sullivan, N.R., Crane, J.W., Damjanoska, K.J., Carrasco, G.A., D’Souza, D.N., Garcia, F.,Van de Kar, L.D., 2005. Tandospirone activates neuroendocrine and ERK (MAP

kinase) signaling pathways specifically through 5-HT1A receptor mechanismsin vivo. Naunyn-Schmiedeberg’s Archives of Pharmacology 371, 18–26.

Tabuchi, M., Yamaguchi, T., Iizuka, S., Imamura, S., Ikarashi, Y., Kase, Y., 2009.Ameliorative effects of yokukansan, a traditional Japanese medicine, onlearning and non-cognitive disturbances in the Tg2576 mouse model of

Alzheimer’s disease. Journal of Ethnopharmacology 122, 157–162.Terawaki, K., Ikarashi, Y., Sekiguchi, K., Nakai, Y., Kase, Y., 2010. Partial agonistic

effect of yokukansan on human recombinant serotonin 1A receptors expressedin the membranes of Chinese hamster ovary cells. Journal of Ethnopharmacol-ogy 127, 306–312.

Wang, P.S., Schneeweiss, S., Avorn, J., Fischer, M.A., Mogun, H., Solomon, D.H.,Brookhart, M.A., 2005. Risk of death in elderly users of conventional vs.

atypical antipsychotic medications. New England Journal of Medicine. 353,2335–2341.

Yamada, M., Hayashida, M., Zhao, Q., Shibahara, N., Tanaka, K., Miyata, T.,Matsumoto, K., 2011. Ameliorative effects of yokukansan on learning andmemory deficits in olfactory bulbectomized mice. Journal of Ethnopharmacol-

ogy 135, 737–746.