Evaluation of behavioral and pharmacological effects of Hedyosmum brasiliense and isolated...

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Journal of Ethnopharmacology 128 (2010) 63–70

Contents lists available at ScienceDirect

Journal of Ethnopharmacology

journa l homepage: www.e lsev ier .com/ locate / je thpharm

valuation of behavioral and pharmacological effects of Hedyosmum brasiliensend isolated sesquiterpene lactones in rodents

ogério Tolardoa, Lucas Zettermanb, Daniel Rilo Bitencourttb, Ticiana Camila Morab,ábio Lazzarotto de Oliveirab, Maique Weber Biavatti c, Solomon Kweku Sagoe Amoahc,ristiani Bürgera,b, Márcia Maria de Souzaa,b,∗

Centro de Ciências da Saúde, CCS, Mestrado em Ciências Farmacêuticas/UNIVALI, Itajaí, SC, BrazilNúcleo de Investigacões Químico-Farmacêuticas, NIQFAR, UNIVALI, Itajaí, SC, BrazilDepartamento de Ciências Farmacêuticas, CCS, Universidade Federal de Santa Catarina, UFSC, Campus Universitário/Trindade 88040-900, Florianópolis, SC, Brazil

r t i c l e i n f o

rticle history:eceived 4 May 2009eceived in revised form7 December 2009ccepted 21 December 2009vailable online 28 December 2009

eywords:edyosmum brasilienseypnoticnxiolyticntidepressant

a b s t r a c t

Ethnopharmacological relevance: Hedyosmum brasiliense Miq. (Chloranthaceae) is an essential Brazilianspecies largely found in the Atlantic Forest. It is popularly known as “cidrão” and in folk medicine, thisaromatic species is widely used as a calmative/tranquilizer and to treat sleep disorders.Aim of the study: To examine the neurochemical properties of ethanol extract (EEHb), fractions andcompounds of fresh leaves of Hedyosmum brasiliense and the antidepressant effect of the isolatedsesquiterpene lactones podoandin and 13-hydroxy-8,9-dehydroshizukanolide.Materials, methods and results: The effects of EEHb were demonstrated by the open field, elevated-plus-maze, forced swimming, pentobarbital-induced sleeping time, PTZ-induced seizure, and inhibitoryavoidance tests. EEHb did not show a protective effect against PTZ-induced convulsions. In the plus-maze test, EEHb (100 mg/kg, i.p.) exhibited an anxiolytic effect through the effective enhancement ofthe frequency and time spent in the open arms of the maze. Conversely, the time spent and the num-ber of entrances to the closed arms were decreased. All these effects were also completely reversed bypre-treatment with flumazenil (2.5 mg/kg, i.p./a benzodiazepine receptor agonist), similar to the resultsobserved with diazepam used as a positive standard. In this test, the anxiolytic effect of EEHb was alsototally blocked by pre-treatment with reserpine (2.0 mg/kg, i.p.), a drug known to induce depletion ofbiogenic amines. In the forced swimming test, the treatment of EEHb (100 mg/kg, i.p. or 100 mg/kg, p.o.)given in acute and chronic form (10, 50 and 100 mg/kg), produced a decrease in immobility time, similarto that of imipramine (10 mg/kg, i.p.), the positive control. The dichloromethane and hexane fractions

(100 mg/kg, p.o.) also produced a decrease in immobility time. In addition, the two isolated compoundstested in a single dose (10 mg/kg, i.p.), the antidepressant effect was observed only with the compoundpodoandin, which also caused a decrease in immobility time. EEHb (10–100 mg/kg) a dose-dependentmanner also caused a decrease in barbiturate sleeping time in mice, and in high doses (100 mg/kg), didnot interfere in memory consolidation.

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Conclusions: The resultsolytic, antidepressant, an

. Introduction

Hedyosmum brasiliense is the largest genus of the small fam-

ly Chloranthaceae, and occurs only in Americas. It comprises6 species, which are mainly tropical and subtropical (Souzand Lorenzi, 2005). The name derives from the Greek “one whomells sweet,” alluding to the pleasant aroma of the leaves

∗ Corresponding author at: NIQFAR/CCS/UNIVALI, R. Uruguai, 458, CP 360, 88302-02, Itajaí, SC, Brazil. Tel.: +55 47 3341 7932x8066; fax: +55 47 3341 7600.

E-mail address: msouza@univali.br (M.M. de Souza).

378-8741/$ – see front matter © 2009 Elsevier Ireland Ltd. All rights reserved.oi:10.1016/j.jep.2009.12.026

st that EEHb presents psychopharmacological activities, including anxi-notic effects.

© 2009 Elsevier Ireland Ltd. All rights reserved.

(Reitz, 1965). Recently, some studies focusing on the isola-tion of substances and biological activity have been published(Bercion et al., 2005; Acebey et al., 2007; Su et al., 2008), andonly one study has been conducted with Hedyosmum brasiliense(Trentin et al., 1999) which verified its antinociceptive activ-ity. In folk medicine, this aromatic species is widely used asa sedative, and its leaves produce essential oil reminiscent of

Lemon Balm aroma. It is used as a substitute for green tea andis also recommended for the treatment of migraines and dis-eases of the ovary, as a diuretic, tonic and aphrodisiac. The freshleaves are also macerated in alcoholic beverages such as whitewine.

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4 R. Tolardo et al. / Journal of Eth

The objective of the present work is to evaluate the neuro-hemical properties of ethanol extract, fractions of fresh leaves ofedyosmum brasiliense Miq. (Chloranthaceae), and the antidepres-

ant effect of the isolated sesquiterpene lactones podoandin and3-hydroxy-8,9-dehydroshizukanolide assessed by the open field,levated-plus-maze, pentobarbital induces sleeping time, forcedwimming, strychnine and pentilenotetrazole-induced seizure andnhibitory avoidance tests.

. Material and methods

.1. Animals

Male Swiss albino mice (25–30 g) and Wistar rats (250–300 g)ere used. The animals were housed 4 to a cage and maintained at

2–23 ◦C under a 12 h light/dark cycle (lights on at 7:00 AM) withree access to food and water. All the experiments were conductedlind in terms of the treatment condition of the animals. The USAational Institute of Health Guidelines for Animal Care and Useere followed, and the experiments were approved by the Animalare and Ethical Committees of the University do Vale do Itajaínder number 436/07. Each animal was used only once.

.2. Plant material

Aerial parts of Hedyosmum brasiliense were collected on March006 in Luis Alves (Morro do Baú) at Santa Catarina, Brazil. Voucherpecimens were analysed by the botanist Dr. Ana Claudia Araújo,nd deposited at the Herbarium Lyman Bradford Smith (UNIVALI,tajaí, SC) (number 2031).

.3. Extract and fraction preparation

Fresh leaves (5 kg) were comminuted in a domestic mixer andacerated in bidistilled ethanol for 4 weeks, then filtered. The

xtract obtained was then concentrated in rotary evaporator. Theecovered ethanol was used to re-macerate the plant, and theombined concentrated extracts were kept in a desiccator underacuum to remove residual solvent, yielding 80 g of crude extract.n aliquot was separated to the biological assays, and the exce-ent (20 g) was rediluted in water and partitioned with solvents of

ncreasing polarity yielding the following fractions: hexane (5.5 g),ichloromethane (4 g), ethyl acetate (6.3 g) and the residual aque-us fraction, which was kept in the freezer. The above fractionsere subjected to chromatographic procedures to isolate the tested

ompounds, as described below.

.4. Isolation of compounds

The hexane fraction was successively chromatographed usingilica gel (230–400 mesh) and gradient of hexane–acetone (from:1 to 3:7) as eluent to yield �-sitosterol (24�-ethyl-cholestan--ene-3�-ol, 20 mg) and the sesquiterpene lactone podoandin (1,0 mg), identified in comparison with previously published dataKubo et al., 1992). From the dichloromethane fraction, which waslso successively chromatographed using silica gel (230–400 mesh)nd a gradient of hexane–acetone (from 9:1 to 3:7) as eluent,he sesquiterpene lactone 13-hydroxy-8,9-dehydroshizukanolide2, 100 mg) and the phenolic ethyl caffeate (40 mg) were obtainednd identified in comparison with previously published spectralata (Guedes, 1997; Trentin et al., 1999).

.5. Drugs and treatments

Imipramine, pentylenetetrazole, reserpine sulphate, haloperi-ol and sodium pentobarbital were obtained from Sigma Chem.

rmacology 128 (2010) 63–70

Co. (St. Louis, USA). Flumazenil and diazepam from Cristália Prod.Química Farm. Ltda, São Paulo, Brazil. Imipramine and pentylenete-trazole were dissolved in distilled water, while diazepam wasprepared in 20% propylene glycol. Pentobarbital was dissolved in20% (v/v) Tween-80. EEHb, fractions and compounds were dilutedin distilled water with 1 drop/ml of DMSO. The drugs were preparedbefore use and all the other drugs were of analytical grade. TheEEHb and compounds 1 and 2 were administered by intraperitonealroute (i.p.) 30 min before the tasks. Due to difficulties in obtain-ing new plants for preparation of extracts, fractions and isolationof compounds, the intraperitoneal route was used in most of thepharmacological tests. Only in the forced swimming test was theextract and the fractions administered orally. To address some ofthe mechanisms by which the extract EEHb causes anxiolytic-likeaction in the plus-maze test, animals were treated with differentdrugs. The doses of drugs used were selected on the basis of liter-ature data and on previous results from our laboratory (De-Souzaet al., 2003; Aragão et al., 2006; Machado et al., 2007).

2.6. Open-field test

The open-field arena was made of acrylic (transparent walls andblack floor, 30 cm × 30 cm × 15 cm), divided into 9 squares of equalareas. The open-field test was used to evaluate the exploratoryactivity of the animals (Archer, 1973). The animals received theEEHb (10–100 mg/kg, i.p.) and haloperidol (2.5 mg/kg, i.p.) 30 minprior test. The mouse was placed individually in the center of thearena, and allowed to explore it freely. The parameters observedwere: ambulation or crossing (the number of squares crossed withall 4 paws) and number of rearings, both recorded for the last 5 minof the 6-min testing period.

2.7. Barbiturate-induced sleeping time

In this test, which was performed according to the method ofFerrini et al. (1974). Mice were divided into 5 groups of 8–10 ani-mals each. The animals received extract of Hb (10–100 mg/kg, i.p.)and diazepam (1.0 mg/kg, i.p.) 30 min before sodium pentobarbi-tal (50 mg/kg, i.p.). The control group was treated with a 0.9% NaClsolution in the same conditions. The latency times for the start ofsleep and, the duration of sleep (min) of each animal was observed.The sleeping time was recorded as the time taken to recover therighting reflex.

2.8. Elevated-plus-maze

This test has been widely validated for measuring anxiolyticand anxiogenic-like activities in rodents (Lister, 1987). The appara-tus consisted of two opposite open arms (30 cm × 5 cm), crossedby two closed arms of the same dimensions, with 25 cm highwalls. The arms were connected to a 5 cm × 5 cm central square.The apparatus was raised 45 cm above the floor, in a dimly illu-minated room. The mice were placed individually in the centerof the maze, facing a closed arm, and the number of entries andtime spent on the open arms were recorded for the next 5 min.Entry into an arm was defined as the animal placing all four pawsinside the arm. After each test, the maze was carefully cleaned withwet tissue paper (10% ethanol solution). The animals received theextract of EEHb (10–100 mg/kg, i.p.) and diazepam (0.75 mg/kg, i.p.)30 min prior test. The control group was treated with a 0.9% NaClsolution, under the same conditions. In order to elucidate the mech-

anisms possibly involved with the anxiolytic effect of EEHb, we useddiazepam and flumazenil, administered alone or in association withEEHb. Reserpine (2 mg/kg, i.p.). A drug known to cause depletionof biogenic amines (noradrenaline, dopamine and serotonin) fromstorage granules was also used to evaluate the role of those amines

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assessed by the elevated-plus-maze test. The results (Fig. 3A and B)showed that the number of entrances into the open arms (q = 3.483,p < 0.05) and the time the animals remained in the open arms,(q = 6.622, p < 0.05) were significantly increased with the dose of100 mg/kg, in 52.63% and 35.00%, respectively, compared with

R. Tolardo et al. / Journal of Eth

n the anxiolytic effect of the extract. In the combination protocol,eserpine was administered 10 min before EEHb, and the test waserformed 30 min later (Aragão et al., 2006).

.9. Strychnine and PTZ-induced seizures

The potential anticonvulsant action of the EEHb was analyzedsing the pentylenetetrazole (PTZ) and strychnine methods. Briefly,he mice were immobilized using a transparent plastic restrainernd given vehicle, different doses of extract of EEHb (10–100 mg/kg,.p.) or phenobarbital sodium (50 mg/kg, i.p.), 30 min before receiv-ng PTZ (80 mg/kg, i.p.) or strychnine (4.0 mg/kg, i.p.); the animals

ere observed for 60 min thereafter. The latency of the first clonicr tonic episode was recorded for each animal (Aragão et al., 2006).

.10. Forced swimming test

This test is the most widely used and recognized as pharmaco-ogical model for assessing antidepressant activities. The proceduresed was essentially similar to that previously described by Porsoltt al. (1977), with minor modifications. This method is based on thebservation of animals exposed to a situation of forced swimming:fter a period of vigorous activity (struggling), the animals becameassive and immobile and produced just enough movements toeep their heads above the water (immobility). Swimming sessionsere conducted by placing Swiss albino mice in individual pexi-

lass cylinders (46 cm high × 20 cm diameter) containing 20 cm ofater at 24 ± 1 ◦C. Different doses of extract (10–100 mg/kg, i.p.; or

00 mg/kg, v.o.), vehicle, podoandin (1) (10 mg/kg), 13-hydroxy-,9-dehydroshizukanolide (2) (10 mg/kg) imipramine (50 mg/kg,

.p. – positive control) or fractions were administered 30 or 60 mindepending on the route of administration used) before the test.he observation started immediately thereafter and lasted 6 min.n the other experiment, the animals received the treatments for 15ays and 24 h after the last treatment with the extract and controlshey were submitted to the test.

.11. Inhibitory avoidance

Rats were trained in a one-trial, step-down inhibitory avoidancearadigm (IA), a highly validated learning task in which stepping-own from a platform in a given context is associated with aootshock resulting in an increase in step-down latency (Izquierdot al., 2004). The IA training apparatus was a 50 cm × 25 cm × 25 cmlexiglas box with a 5 cm high, 8 cm wide, and 25 cm long platformn the left end of a series of bronze bars that constitutes the floorf the box. During training, the animals were gently placed on thelatform facing the left rear corner of the training box. When theytepped down and placed their four paws on the grid, they received2 s, 0.4 mA scrambled footshock. They were then immediatelyithdrawn from the training box. Memory retention was evaluated

n a test session carried out 24 h after training. In the test, trainednimals were placed back in the training box platform until theyventually stepped down to the grid. The latency to step-down dur-ng the test session was taken as an indicator of memory retention.

ceiling of 180 s was imposed for the step-down latencies duringhe retention test. To evaluate the effect of the extract on acqui-ition consolidation or evocation of memory in the animals, thereatment with EEHb (10–100 mg/kg) or vehicle were given 30 minefore IA training, 30 min after the IA training and 30 min beforehe IA test.

.12. Statistical analysis

The results are presented as mean ± SEM. For the paramet-ic data, the variables were analyzed by one-way analysis of

rmacology 128 (2010) 63–70 65

variance (ANOVA), followed by Student–Newmann–Keuls analy-sis. P values lower than 0.05 were considered as indicative ofsignificance. For the statistical analysis of non-parametric datafrom the passive avoidance test, the Mann–Whitney U-test wasused.

3. Results

EEHb at doses of 50 and 100 mg/kg, i.p., showed sedative effectsin mice, as assessed by the open-field test (Fig. 1). Significant effectswere detected with both doses, which produced similar percent-ages of inhibition (28.57 and 39.80%, respectively) in the numberof crossings (p < 0.05, p < 0.05) (Fig. 1A) compared with the con-trols. The number of rearings was also significantly lower withboth doses, which produced percentages of inhibition of 48.75%and 56.25% (p < 0.5, p < 0.01) (Fig. 1B). Haloperidol, used as a pos-itive control, produced a statistically significant decrease in bothparameters.

In the barbiturate-induced sleeping time test, intraperitonealadministration of EEHb 10, 50 and 100 mg/kg respectively signifi-cantly decreased (p < 0.05, p < 0.01, and p < 0.01) sleep latency timein 11.41%, 43.17% and 38.38%, respectively (Fig. 2A). Conversely,the treatment with EEHb produced an increased total sleep time(31.67%, 48.15% and 56.07% respectively) which was both statisti-cally significant and dose-dependent with all the doses (p < 0.05,p < 0.01, p < 0.01), suggesting a potentiation of the pentobarbitaleffect (Fig. 2B).

A possible anxiolytic activity of EEHb f (100 mg/kg, i.p.) was

Fig. 1. Effects of acute administration of EEHb (10, 50 and 100 mg/kg, i.p.) andhaloperidol (2.5 mg/kg, i.p.) in the open-field test in mice. A = number of cross-ings and B = number of rearings. Each bar represents the mean ± SEM, from 6 to10 animals per group. *p < 0.05, **p < 0.01 and ***p < 0.001, compared with the corre-sponding control value. The data were analyzed by the analysis of variance (ANOVA)or t-test and complemented by the Dunnett’s or Newman–Keuls post hoc tests.

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Fig. 2. Effect of EEHb (10, 50 and 100 mg/kg) and diazepam (1 mg/kg) on (A) sleeplatency and (B) total time of sleep induced by pentobarbital sodium. Data arereported as means ± SEM. N = 8–10 mice. *p < 0.05,**p < 0.01 compared to the controlgroup (ANOVA followed by Student–Newmann–Keuls analysis).

Fig. 3. Effects of EEHb (10, 50 and 100 mg/kg, i.p.) and diazepam (0.75 mg/kg, i.p.) in the espent in the open arms; C = number of entrances to the closed arms and D = time spent igroup*p < 0.05, **p < 0.01, compared with the corresponding control value. Data were anaNewman–Keuls post hoc tests.

rmacology 128 (2010) 63–70

the controls. Conversely, EEHb (100 mg/kg, i.p.) only significantlyreduced the time spent in the closed arms (Fig. 3D) (q = 3.832,p < 0.05) by 42.85%. These effects were similar to those obtainedfor diazepam. Flumazenil (2.5 mg/kg, i.p.) significantly reversed theeffects of diazepam and EEHb (Fig. 4 A–D), although it showedno effect when used alone. These data suggest the participationof GABAergic system in the anxiolytic effect of EEHb. In this samemodel we also observed that reserpine (2.0 mg/kg, i.p.) significantlyreversed the effects of EEHb (Fig. 5 A–D) in all the parametersobserved.

The comparison between the treatment of animals with EEHb(10–100 mg/kg), phenobarbital (40 mg/kg) and the control groupshowed that only phenobarbital significantly suppressed strych-nine and pentilenetetrazol-induced colon seizures, with completesuppression being observed (results not shown).

As shown in Fig. 6A, using the i.p. route, it was observedthat acute treatment with EEHb (100 mg/kg) exhibited a signifi-cant reduction in the length of immobility (F3.16 = 2.47, p < 0.05)(51.27%). This effect was observed in all the doses used (10, 50and 100 mg/kg, i.p.) when given chronically (F3.16 = 2.47, p < 0.01,F3.16 = 2.47, p < 0.05, F3.16 = 2.47, p < 0.05 specifically) (Fig. 6C).As shown in Fig. 6B, using the v.o. route it was observedthat acute treatment with EEHb extract (100 mg/kg), hexanicor dichloromethane fractions respectively exhibited a significantreduction in the length of immobility (F27.58 = 12.81, p < 0.001)(71.34%), F27.58 = 10.45, p < 0.001) (58.12%), F27.58 = 10.96, p < 0.00160.99%).The antidepressant effect of imipramine was observedin all experiments. In this test, an antidepressant effect wasonly observed for the compound podoandin (1) (Fig. 7). Thiscompound exhibited a significant reduction in immobility time(F3.16 = 2.47, p < 0.01) decreasing to 51.67%, compared with thecontrols.

Finally, EEHb extract did not produce any effects on memoryretention in the animals in the inhibitory avoidance test (resultsnot shown).

levated-plus-maze test, in mice. A = number of entrances to the open arms, B = timen the closed arms. Each bar represents the mean ± SEM, from 8 to 10 animals perlyzed by analysis of variance (ANOVA) or t-test and complemented by Dunnett’s or

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Fig. 4. Effects of pre-treatment of flumazenil (FLU, 2 mg, i.v.) on anxiolytic effect of EEHb (100 mg/kg, i.p.) and diazepam (0.75 mg/kg, i.p.) in the elevated-plus-maze test inmice. A = number of entrances to the open arms; B = time spent in the open arms; C = number of entrances to the closed arms and D = time spent in the closed arms. Eachbar represents the mean ± SEM, from 8 to 10 animals per group*p < 0.05, **p < 0.01, compared with the corresponding control value. #p < 0.05 compared with animals treatedonly with EEHb. Data were analyzed by analysis of variance (ANOVA) or t-test and complemented by the Dunnett’s or Newman–Keuls post hoc tests.

Fig. 5. Effects of pre-treatment of reserpine (2 mg/kg, i.p.) on anxiolytic effect of EEHb (100 mg/kg, i.p.) in the elevated-plus-maze test in mice. A = number of entrances tothe open arms; B = time spent in the open arms; C = number of entrances to the closed arms and D = time spent in the closed arms. Each bar represents the mean ± SEM, from8 to 10 animals per group. *p < 0.05, **p < 0.01, compared with the corresponding control value. #p < 0.05 compared with animals treated only with EEHb. Data were analyzedby analysis of variance (ANOVA) or t-test and complemented by the Dunnett’s or Newmann–Keuls post hoc tests.

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Fig. 6. Effect of EEHb (10, 50 and 100 mg/kg, i.p. or 100 mg/kg, p.o.), fractions(100 mg/kg, p.o.) or imipramine (10 mg/kg, i.p. or p.o.) administered acute (A and B)aN(

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Fig. 7. Effect of EEHb (100 mg/kg, i.p.), podoandina (10 mg/kg, i.p.) and 13-

physiology of states of depression and anxiety. Therefore, a great

nd chronically (C) on immobility time in mice. Data are reported as means ± SEM.= 8–10 mice. *p < 0.05, **p < 0.01, ***p < 0.001 compared to the control group

ANOVA followed by the Student–Newmann–Keuls analysis).

. Discussion

The central effects of Hedyosmum brasiliense were studied, andhe results for the preliminary neuropharmacological screeninghowed that this extract exhibited: (I) sedative effect; (II) hypnoticffect; (III) anxiolytic effect; and (IV) antidepressant effect.

The general depressant activity of EEHb was confirmed by theecrease in sleep latency and by the increase in the pentobarbital-

nduced sleeping time, which may be attributed to an inhibition ofentobarbital metabolism or a sleep regulating action (Morais etl., 1998). The hypnotic action of pentobarbital was demonstrated

y Petty (1995) to be mediated by the GABA-A receptor complex.he decrease in numbers of rearings and crossings in the open-eld test confirms the depressant activity of HB, since it is agreedhat rearing is a result of the level of excitability of the central ner-

hydroxy-8,9-dihydroshizukanoide (10 mg/kg, i.p.) and imipramine (10 mg/kg, i.p.)on immobility time in mice. Data are reported as means ± SEM. N = 8–10 mice.**p < 0.01, ***p < 0.01compared to the control group (ANOVA followed by theStudent–Newmann–Keuls analysis).

vous system (Masur et al., 1971). In addition, this test provides agood indication of the animal’s emotional state. The results showedthat EEHb was able to significantly decrease not only the numberof crossings, indicative of a possible sedative effect, but also thenumber of rearings.

To study the possible anxiolytic effect of EEHb, the elevated-plus-maze test was used. Anxiety, a symptom which accompaniesvarious disorders of the central nervous, and is in itself a disor-der, is characterized in humans by a tense and exhaustive physicalalertness (Jackson and Turkington, 2005). Other animal species dis-play a variety of defensive reactions in response to predators, someof which are understood as correlated states of anxiety (Rodgersand Johnson, 1995). Rodents demonstrate anxiety, fear and curios-ity when placed in a new environment, and an overall assessmentof behavior can be determined through the observation of freezing,grooming (fear) or rearing, head-dips (curiosity) and the number offecal boluses (Nic Dhonnchadha et al., 2003; De-Souza et al., 2003;Treit and Pinel, 2005).

The elevated-plus-maze has been frequently used to detect andevaluate the anxiolytic/anxiogenic properties of drugs (Pellow andFile, 1987). The frequency and time spent in the open arms is themain indicator of anxiety in the plus-maze model, given that anopen area is extremely aversive to rodents (Pellow and File, 1987;File et al., 1993). In addition, in the open arms there is no thig-motaxis (Treit and Fundytus, 1988), which enhances the anxiousstate in the animals. Our results showed that EEHb was also ableto significantly increase the time spent in, as well as the numberof entrances to the open arms, indicating a positive response inthis test. Numerous neural pathways are involved in the patho-

number of neurotransmitters are involved in the underlying mech-anisms of anxiolytic and antidepressant drugs (Aragão et al., 2006).It is widely accepted that benzodiazepinic (BZ) anxiolytic drugs actclinically by enhancing the effect of GABA, in the GABA-A receptor.

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lthough classical BZ agonists enhance the function of the GABA-receptor and are effective anxiolytics, they present unwanted

ide-effects, including sedation, dependence and abuse labialityWhiting, 2006). BZ are widely used as sedative and anxiolyticrugs, agreeing with studies on animal models such as the elevated-lus-maze test, where the effects of DZP are highly reproducible.ased on these findings, it is thought that substances which caneduce the animal’s anxiety exposed to these paradigms could exertheir effects through an action similar to that of benzodiazepinesHerrera-Ruiz et al., 2005). In this study, flumazenil reversed notnly the diazepam effect, but also the EEHb effect, indicating thatoth substances might present a similar mechanism of action. Thusur results indicate that the anxiolytic effects of EEHb may involvehe GABA-A receptor complex, a hypothesis which is re-enforcedy the results obtained in the pentobarbital-induced sleeping timeest.

The relation between anxiety and catecholamines is clinicallynd experimentally important. There is also preliminary evidenceor the use of centrally acting beta-adrenergic antagonists, likeropranolol, to inhibit consolidation of traumatic memories inost-traumatic stress disorder (Garakani et al., 2006). This drug islso used to treat acute performance anxiety, and other types of dis-rder (Brasha et al., 2005). It is concluded that dynamic changes inonoaminergic activity of the brain accompany the development

f anxious depression in animals. In addition, various parametersf monoaminergic systems are differently changed, depending onrain area, mediator system and stage of disorder (Avgustinovich etl., 2004). In this study, anxiolytic EEHb effects were totally blockedy pre-treatment with reserpine, a known inhibitor of the vesicu-

ar catecholamine transporter (that facilitates vesicular storage).similar process occurs at the storage sites for 5-HT, what can

ltimately result in a depletion of biogenic amines. Furthermore,his finding suggests that the anxiolytic effect of EEHb is probablyelated, at least in part, to the increased catecholamine activity inhe CNS.

The forced swimming test has been validated as a suitable toolor predicting the antidepressant properties of drugs (Porsolt et al.,977; Anisman and Matheson, 2005). The forced swimming test is aehavior test in rodents that gives an indication of the clinical effec-iveness of various types of antidepressant drugs. When rodents areorced to swim in a confined space, they tend to become immobilefter vigorous activity (struggling). This inescapable stressful situ-tion can be evaluated by assessing different behavioral strategiesPorsolt et al., 1977). Several authors have proposed that immo-ility during the test could be an efficient adaptative response tohis stress (Porsolt et al., 1977; Galea et al., 2001). In this study,cute and chronic administration of HB prior to the test reducedotal immobility time behavior. EEHb affects the normal pattern ofehavior during the test, suggesting an antidepressive behavior inesponse to an inescapable stress.

The effect of EEHb in memory consolidation and strychninend PTZ-induced seizures was also examined in this study. In thenhibitory avoidance test and PTZ-induced seizures test in particu-ar, no effects were observed on memory and convulsions in thenimals when treated with EEHb, as was expected. Our resultsupport the idea that EEHb interacts with the GABA-A receptor,robably at the level of the receptor subtypes that mediate theffects of BDZ, to produce the sedative and hypnotic activitiesbserved. Sedative and anxiolytic drugs such as BDs also have aropensity to cause cognitive deficits (Barton et al., 2008). Also,hese drugs exhibit anticonvulsant effects (Wheless and Treiman,

008). EEHb could express these unobserved effects at higher doseshan those tested here, but further studies are needed to clarify thisssumption.

Concerning the chemical constituents of EEHb, classical phyto-hemical methods were used to isolate five crystalline substances

rmacology 128 (2010) 63–70 69

identified by nuclear magnetic resonance (NMR) data. The hex-ane fraction of EEHb furnished the ubiquitous steroid �-sitosteroland the sesquiterpene lactone of guaianolide type podoandin (1).From the dichloromethane fraction of EEHb, the sesquiterpene lac-tone of lindenanolide type 13-hydroxy-8,9-dehydroshizukanolide(2) was obtained, previously isolated from Hedyosmum brasiliense,and the phenolic ethyl caffeate, and from ethyl acetate fractionof HB, the phenolic protocatechuic aldehyde was obtained. Thephenolic compounds and compound 1 are first described for thisspecies (Kubo et al., 1992; Oliveira, 2009). In this study both frac-tions were evaluated in the model of depression showing similarefficacy. Due to the low yield obtained for these compounds, theeffect of the two lactones in a single dose (10 mg/kg) was eval-uated only in the depression model. The antidepressant effectwas observed only with the guaianolide compound 1. This showsthat besides the compound podoandin (1), other constituents notyet identified may contribute to the antidepressant effects of theplant.

The sesquiterpene lactones are recognized as characteristicchemotaxonomic compounds, due to the metabolic specificity ofthis class (Cao et al., 2008). Works by Trentin et al. (1999) report thatthe lindenanolide 13-hydroxy-8,9-dehydroshizukanolide (com-pound 2) exhibited graded antinociception against acetic-acidwrithing and capsaicin-induced licking when given by differentroutes in mice. Recently, the glutamatergic system was indicatedas the mechanism of action of the antinociceptive effect of thiscompound (Martini et al., 2007).

From some Helichrysum species (Asteraceae) was also isolatedsome guaianolide sesquiterpene lactones, and this species also pro-duces essential oil. Helichrysum species are used in the treatment ofinsanity, possession, as a sedative to treat insomnia and as a protec-tive cleanser. Their traditional uses indicate that these plants mayexhibit psychotropic effects, and some pharmacological studieshave determined the GABA-receptor binding effect of their extracts(Lourens et al., 2008).

Bilobalide, a sesquiterpene trilactone from Ginkgo biloba hasshown neuroprotective properties. Its mechanism of action isunknown but it was recently found to block GABA-A receptors(Kiewert et al., 2007).

In conclusion the results together suggest that ethanol extractof HB exhibited important effects on the nervous system of mice,which confirms, in part, the popular use of this plant. Furthermore,the results show that the extract and fractions are absorbed orallyhighlighting further the effects of the plant when used in this way.

Acknowledgements

The authors are grateful to Cristália laboratories for theFlumazenil used in experiments. Lucas Zetterman and Daniel RiloBitencourtt, research students in pharmacology, thank UNIVALI forthe fellowship support. The work received financial support fromthe Brazilian National Research Council (CNPq).

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