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Copyright © 2008 John Wiley & Sons, Ltd.

PHYTOTHERAPY RESEARCHPhytother. Res. 22, 000–000 (2008)Published online in Wiley InterScience(www.interscience.wiley.com) DOI: 10.1002/ptr.2664

Antidepressant Profile of Ptychopetalumolacoides Bentham (Marapuama) in Mice

Ângelo L. Piato1,2, Lucas P. Rizon2, Bárbara S. Martins2, Domingos S. Nunes3 and ElaineElisabetsky1,2,*1Programa de Pós-Graduação em Ciências Farmacêuticas, Faculdade de Farmácia, Universidade Federal do Rio Grande do Sul,Porto Alegre, RS, 90610-000, Brazil2Laboratório de Etnofarmacologia, Instituto de Ciências Básicas da Saúde, Universidade Federal do Rio Grande do Sul, PortoAlegre, RS, 90050-170, Brazil3Departamento de Química, Universidade Estadual de Ponta Grossa, Ponta Grossa, PR, 84030-310, Brazil

Depression has become of universal major importance, and it is therefore vital to expand the armamentariumfor treating the condition. Lack of motivation and lassitude are major symptoms treated with the use ofMarapuama (Ptychopetalum olacoides, PO) remedies by communities in the Brazilian Amazon. Consideringthe prominence of such symptoms in depression, the present study was designed to verify the effects of astandardized PO ethanol extract (POEE) on the forced swimming (FST) and tail suspension tests (TST).POEE i.p. (15–100 mg/kg) and oral (300 mg/kg) resulted in a significant and dose-related anti-immobilityeffect. We further examined the involvement of dopamine, noradrenaline and serotonin in these antidepressant-like effects. POEE effects were prevented when catecholamine synthesis was inhibited by -ααααα-methyl-ρρρρρ-tyrosine(AMPT) (100 mg/kg, i.p.), while inhibition of serotonin synthesis with ρρρρρ-chlorophenylalanine methyl esterhydrochloride (PCPA) (100 mg/kg, i.p.) was devoid of effect. The blockade of βββββ-adrenergic (propranolol 2 mg/kg, i.p.) and D1 dopamine (SCH 23390 0.5 mg/kg, i.p.) receptors prevented POEE anti-immobility effects;by contrast, blockade of D2 dopamine (sulpiride 2 and 50 mg/kg, i.p.) receptors was ineffective. Consistentwith traditional use, the results indicate that POEE possesses antidepressant-like effects, possibly mediated byβββββ-adrenergic and D1 dopamine receptors. Copyright © 2008 John Wiley & Sons, Ltd.

Keywords: Ptychopetalum olacoides; Marapuama; Muirapuama; antidepressant; catecholamine; serotonin.

Received 22 April 2007Revised 24 June 2008

Accepted 25 June 2008

* Correspondence to: Elaine Elisabetsky, Laboratório de Etnofarm-acologia, ICBS, UFRGS, Av. Sarmento Leite 500/202, 90050-170, PortoAlegre, RS, Brazil.E-mail: elaine.elisabetsky@gmail.com

INTRODUCTION

It is recognized that depression is associated with con-siderable morbidity and mortality, unfortunately witha consistently high prevalence worldwide. The lifetimeprevalence of depression has been estimated to be ashigh as 21% of the general population in some devel-oped countries (Wong and Licinio, 2001). Despite theintroduction of various classes of antidepressants, in-cluding tricyclics (TCA), selective reversible inhibitorsof monoamine oxidase, selective serotonin reuptakeinhibitors (SSRI) and specific serotonin-noradrenalinereuptake inhibitors, the treatment of depression is notentirely satisfactory. All clinically used antidepressantshave a slow onset of action, present significant unde-sirable side effects, and lack the desired efficacy in asignificant proportion of patients (Healy, 2006). Theexcellent patient acceptance (Bilia et al., 2002) of SaintJohn’s Wort (Hypericum perforatum L., Hypericaceae)has drawn attention to plant extracts as potential sourcesof highly desirable new and innovative antidepressantagents.

Although traditional concepts of health are not alwayseasily translated into biomedical nosological categories,several traditionally used plant species have shown anti-

depressant activity in animal models, including Bacopamonniera Wettst (Sairam et al., 2002), and Clitoria ternateaL. (Jain et al., 2003). Ptychopetalum olacoides Bentham(PO) (Olacaceae), known as Marapuama or Muirapuama,is used by Amazonian peoples for treating ‘nervousweakness’, a syndrome that includes lassitude, a gen-eral lack of desire or motivation (‘a man loses interesteven for soccer and women’), tremors and sexualimpotence as prominent symptoms (Elisabetsky andSiqueira, 1998). The species is also indicated to facili-tate recovery from stroke, and for coping with highlystressful (physically and/or psychologically) circum-stances (Elisabetsky and Siqueira, 1998). Coherent withsuch traditional claims, relevant neuroprotective andmemory facilitating effects were identified with a spe-cific PO root ethanol extract (POEE) (Siqueira et al.,2004; da Silva et al., 2007).

Despite major limitations (Willner, 1990), animalmodels have been useful not only in detecting anti-depressant properties of new drugs, but also in identi-fying the neurotransmitter systems that underlie theirmechanisms of action. We have previously establishedthat POEE potentiates yohimbine-induced lethality, andreverses reserpine-induced ptosis (Siqueira et al., 1998),indicating potentiation of catecholaminergic activity.The aim of the present study was to verify the effectsof POEE in the forced swimming (FST) and tail suspen-sion tests (TST), two widely used screening tests for anti-depressants. In addition, the involvement of dopamine,noradrenaline, and serotonin receptors in POEE effectswas investigated.

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METHODS

Animals. Experiments were performed with two-month-oldmale (CF1, 40-50 g) mice, obtained from the FundaçãoEstadual de Produção e Pesquisa em Saúde (FEPPS).Mice were housed eight per cage (40 × 33 × 16 cm) andmaintained in our own animal facility, under controlledenvironmental conditions (22 ± 1 °C, 12-hr light/darkcycle, free access to food [Nuvilab CR1] and water) forat least two weeks before experiments. All procedureswere carried out in accordance with institutionalpolicies on the handling of experimental animals(approved by the ethics committee, process 2006543)which follow the NIH guidelines (NIH Guide for Careand Use of Laboratory Animals, NIH publicationNo. 85-23, 1985).

Extract. Roots of Ptychopetalum olacoides Bentham(Olacaceae) were collected in Pará (Brazil) and identi-fied by Nelson Rosa (MPEG 108.036, Goeldi Museum).Collections were carried out according to national guide-lines associated with the United Nations Conventionon Biodiversity. P. olacoides ethanol extract (POEE)was prepared as detailed elsewhere (Elisabetsky andSiqueira, 1998): briefly, dried ground roots (2.5 kg) wereextracted with ethanol (12 L) in Soxhlet (40 h), and thiswas evaporated under reduced pressure (6% yield).Analytical HPLC was carried out on an HP 1100 systemequipped with a photodiode array detector (AgilentTechnologies) and HPLC fingerprinting was performedas detailed elsewhere (Siqueira et al., 2007).

Drugs. Imipramine HCl, DL-propranolol HCl, R (+)-SCH 23390 HCl, (±)-Sulpiride, D,L-α-methyl-ρ-tyrosine(AMPT), ρ-chlorophenylalanine methyl ester hydro-chloride (PCPA) were acquired from Sigma (St Louis,MO, USA). All drugs were dissolved in saline (NaCl0.9%), with the exception of POEE dissolved in DMSO20%. Drugs and vehicles were injected intraperitoneally(i.p.) or orally (p.o.) in a constant volume of 0.1 ml/10 gbody weight.

Locomotion. Locomotion was measured in activitycages (45 × 25 × 20 cm, Albarsch Electronic Equip-ment, Brazil), equipped with three parallel photocells,automatically recording the number of crossings. Groups(n = 10) of mice were treated with saline (i.p. or p.o.),DMSO 20% (i.p. or p.o.) or POEE (10, 15, 25, 50 and100 mg/kg i.p., or 100 and 300 mg/kg p.o.). Mice wereindividually placed in the activity cages 30 (i.p.) or90 (p.o.) min after treatments, and locomotion wasrecorded for 6 min. The effects of antagonists in loco-motion were likewise evaluated, with the drug sched-ules as indicated.

Tail Suspension Test (TST). The TST was used asdescribed by Steru et al. (1985): mice were suspended50 cm above the bench by means of a piece of adhesivetape placed approximately 1 cm from the tip of the tail.Mice were observed for 6 min, and the immobility timewas recorded on a stopwatch; mice were considered tobe immobile when hanging passively and motionless.Mice were submitted to the TST 30 or 90 min (for i.p.and p.o. treated groups, respectively) after treatments.Groups (n = 10–12) of mice were treated as above

(2.4), with an additional group treated with imipramine(20 mg/kg, i.p.).

Forced Swimming Test (FST). The FST followed Sunalet al. (1994). Briefly, mice were gently dropped in-dividually into plastic cylinders (height: 30 cm, diam-eter: 24 cm) containing 18 cm of water (25 ± 1 °C), andobserved for 6 min. The duration of immobility wasrecorded on a stopwatch during the last 4 min, afterwhich mice were immediately removed from the appara-tus and towel dried. A mouse was judged to be immo-bile when it floated in an upright position, making onlysmall movements to keep its head above the water.Groups (n = 9–12) of mice were treated as above (2.4)(except POEE 10 and 15 mg/kg), and with an additionalgroup treated with imipramine (15 mg/kg, i.p.). Micewere submitted to the FST 30 or 90 min (for i.p. andp.o. treated groups, respectively) after treatments.

Inhibition of monoamine synthesis. AMPT was used toinhibit the synthesis of dopamine and noradrenaline,while PCPA inhibited that of serotonin. Doses of theinhibitors were selected from literature (Rojas-Corralesand Micó, 1998; Rodrigues et al., 2002) and tested bypilot experiments; selected doses do not modify immob-ility time in the models here used. Groups of mice re-ceived saline or AMPT (100 mg/kg, i.p.) 24 and 2 h beforesaline, imipramine (20 mg/kg, i.p.) or POEE (25 mg/kg,i.p.); 30 min later mice were subjected to the TST.Additional groups were treated with saline or PCPA(100 mg/kg, i.p.), once a day for 4 consecutive days; 15 minafter the last PCPA treatment groups received saline,fluoxetine (32 mg/kg, i.p.) or POEE (25 mg/kg, i.p.) and30 min later were subjected to the TST.

Effects of antagonists on POEE actions in the FST andTST. Appropriate doses for antagonists were selectedfrom literature (Renard et al., 2001; Rodrigues et al.,2002; Zomkowski et al., 2004) as well as pilot experi-ments, and were used doses that do not modify immob-ility. Groups of mice received saline or antagonists(propranolol 2 mg/kg, SCH 23390 0.5 mg/kg, or sulpiride2 and 50 mg/kg) 15 min before vehicle (DMSO 20%,i.p.), imipramine (15 or 20 mg/kg, i.p.) or POEE (25 mg/kg, i.p.), and were submitted 30 min later to the FST orTST.

Statistical analysis. Results (cumulative counts for locomo-tion or time in seconds for immobility) are expressedas mean ± SEM. Comparisons between groups weremade by ANOVA followed by Student-Newman-Keuls(SNK), using SPSS 10.0. Other results were analyzedby two-way ANOVA followed by Tukey’s test. A valueof p < 0.05 was considered significant.

RESULTS

Locomotion of groups treated with POEE i.p. or orallydid not differ from controls (F(8,87) = 1.2, data notshown). There was no significant effect of antagonistsor synthesis inhibitors in locomotion (F(7,41) = 0.67,Table 1). As can be seen in Table 2, i.p. and p.o. POEEsignificantly reduced immobility in the TST (i.p.:F(7,114) = 37.9; p.o.: F(4,52) = 48.9, p < 0.05) and FST

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Table 1. The effects of saline and antagonists on locomotion

LocomotionTreatment Dose (mg/kg) (means ± S.E.M)

Saline 139.6 ± 7.6DMSO 20% 135.5 ± 4.8AMPT 100 131.2 ± 13.1PCPA 100 120.8 ± 9.5SCH 23390 0.5 137.0 ± 6.4Sulpiride 2 126.4 ± 10.8

50 129.0 ± 5.2Propranolol 2 120.4 ± 11.7

(i.p.: F(5,67) = 31.6; p.o.: F(4,52) = 25.7, p < 0.05).Imipramine was more active than POEE in both tests.

Figure 1A shows the influence of AMPT on the effectsof imipramine and POEE in the TST. A two-way ANOVArevealed a main effect of pretreatment (F(1,65) = 151.6,p < 0.01), treatment (F(3,65) = 59.0, p < 0.01), and ofpretreatment × treatment interaction (F(2,65) = 40.2,p < 0.01). Post hoc analyses indicated that pretreat-ment with AMPT prevented the effects of POEE andimipramine in the TST. Figure 1B shows the influenceof PCPA on the effects of fluoxetine and POEE in theTST. A two-way ANOVA likewise revealed a maineffect of pretreatment (F(1,70) = 23.5, p < 0.01), treat-ment (F(3,70) = 51.4, p < 0.01), and of pretreatmentx treatment interaction (F(2,70) = 35.2, p < 0.01). Posthoc analyses indicated that the pretreatment with PCPAprevented the effect of fluoxetine, but not that of POEE,in the TST.

Figure 2A shows the influence of SCH23390 on theeffects of imipramine and POEE in the FST. A two-way ANOVA revealed a main effect of pretreatment(F(1,54) = 44.9, p < 0.01), treatment (F(2,54) = 40.5,p < 0.01), and of pretreatment x treatment interaction(F(2,54) = 16.5, p < 0.01). Post hoc analyses indicatedthat the pretreatment with SCH23390 prevented theeffects of POEE and imipramine in the FST. Figure 2Bshows the influence of sulpiride on the effects of imipramineand POEE. A two-way ANOVA also revealed a maineffect of pretreatment (F(2,127) = 32.4, p < 0.01), treat-ment (F(3,127) = 68.5, p < 0.01), and of pretreatmentx treatment interaction (F(4,127) = 28.7, p < 0.01). Posthoc analyses indicated that the pretreatment with

Table 2. Effects of imipramine and POEE in tail suspension and forced swimming tests (immobility time, sec)

Tail Suspension Test Forced Swimming Test

Treatment Dose (mg/kg) i.p. p.o. i.p. p.o.

Saline 228.0 ± 4.6 230.9 ± 7.5 164.5 ± 3.6 156.6 ± 4.6DMSO 221.0 ± 4.2 234.5 ± 7.1 173.5 ± 4.6 164.8 ± 4.7Imipramine 15 64.6 ± 4.0*# 2

20 112.0 ± 6.4*# 1

POEE 10 221.5 ± 7.015 191.0 ± 13.3*25 139.0 ± 8.7*† 91.1 ± 10.4*†

50 156.5 ± 8.7* 122.2 ± 13.8*100 178.0 ± 13.2* 232.6 ± 6.1 136.2 ± 10.6* 156.6 ± 9.3300 200.2 ± 7.5* 125.8 ± 9.3*

* p < 0.05 × controls (saline and DMSO); # p < 0.05 × POEE; † p < 0.05 × all other POEE doses, ANOVA/SNK. 1 Imipramine, i.p.: 120.1± 5.8*#; 2 Imipramine, i.p.: 75.0 ± 4.6*#, ANOVA/SNK.

Figure 1. xxx

sulpiride blocked the effect of imipramine, but not thatof POEE, in the FST. Figure 2C shows the influenceof propranolol on imipramine and POEE effects inthe FST. A two-way ANOVA revealed a main effectof pretreatment (F(1,66) = 78.6, p < 0.01), treatment(F(3,66) = 28.4, p < 0.01), and of pretreatment × treat-ment interaction (F(2,66) = 16.8, p < 0.01). Post hocanalyses indicated that the pretreatment with propranololblocked the effects of both POEE and imipramine inthe FST.

Figure 3A shows the influence of SCH23390 on theeffects of imipramine and POEE in the TST. A two-way ANOVA revealed a main effect of pretreatment(F(1,65) = 55.5, p < 0.01), treatment (F(3,65) = 40.4,p < 0.01), and of pretreatment × treatment interaction

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Figure 2. xxx

Figure 3. xxx

(F(2,65) = 9.5, p < 0.01). Post hoc analyses indicatedthat the pretreatment with SCH23390 blocked theeffects of POEE and imipramine in the TST. Figure 3Bshows the influence of propranolol on the effects ofimipramine and POEE in the TST. A two-way ANOVArevealed a main effect of pretreatment (F(1,66) = 47.2,p < 0.01), treatment (F(3,66) = 83.7, p < 0.01), and ofpretreatment × treatment interaction (F(2,66) = 11.4,p < 0.01). Post hoc analyses indicated that the pretreat-ment with propranolol attenuated the effect of imipramineand blocked that of POEE in the TST.

DISCUSSION

Despite eventually leading to false-positive results, theFST and TST are animal models that have been usefulin predicting clinical efficacy for several categories ofantidepressants (Steru et al., 1985). The characteristicbehavior scored in these tests is immobility (Willner,1990); however, in analyzing the present findings, changesin motor activity caused by stimulatory or sedativeeffects of POEE or antagonists can be ruled out, sincenone of the doses used in this study had significanteffects on locomotion. POEE significantly and dose-

dependently decreased immobility in the two mousemodels, when given i.p. or orally. In the FST the effectwas dose-related (r = −0.31, p < 0.05, Pearson correla-tion analysis), while in the TST the effect presentedan inverted U shape with the intermediate dose beingmore active. A similar U shaped dose-effect curve wasalso observed with Hypericum perforatum L. (Butterwecket al., 2002) in animal models, as well as with nortrip-tyline, both in animal models and in the clinic (Reis deOliveria et al., 1990). It is likely that isolated activecompound(s) and/or purified formulations would revealsmaller active dosages and differ in their dose-effectprofile.

In agreement with previously reported data, AMPTinhibited the effect of imipramine (De Montis et al.,1993), while PCPA inhibited the effect of fluoxetinein the TST (Rodrigues et al., 2002). The effect of thesesynthesis inhibitors in the TST indicates that POEEantidepressant-like activity may be mediated by dopamineand/or noradrenaline, but not by serotonin. Addition-ally, SCH 23390, propranolol and sulpiride significantlyinhibited the effect of imipramine in the FST (Mancinelliet al., 1991; D’Aquila et al., 1994). POEE activity wasaffected (reversed or attenuated) by SCH 23390 (butnot by sulpiride in doses known to inhibit TCAs andSSRIs), and by propranolol in both models, indicatingthat POEE antidepressant-like activity may be mediatedby dopaminergic D1 and β-noradrenergic receptors.

Despite major attention lately given to the involvementof serotonin in depression, early studies of depressedpatients pointed to a significant role for noradrenaline(NA) and dopamine (D’Aquila et al., 2000). Several stud-ies have established that most antidepressants, includ-ing those with minimal direct biochemical effects onNA, can influence noradrenergic systems (Miller et al.,2001). Thus, a decreased 24-hour excretion of NA andits major metabolites has been observed with lithium,

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electroconvulsive therapy, the dopamine reuptake in-hibitor bupropion, and the serotonin (5-HT) reuptakeinhibitor zimelidine (Golden et al., 1988). Consequently,it has been suggested that increased noradrenergic func-tion is an integral component of antidepressant treatments,or represents one specific way by which an antidepres-sant effect can be achieved through pharmacologicaltreatment (Miller et al., 2001). Accordingly, the datajustify the noradrenergic system as a major researchfocus in this field (Miller et al., 2001).

β-adrenergic receptors are down regulated in severalbrain areas after long-term treatment with most anti-depressants, and this receptor has been repeatedlyimplicated in the pathogenesis of depression and theactions of antidepressants. The relevance of β-adrenergicreceptors for POEE anti-immobility effects is thereforesignificant and a β-adrenergic modulation is consistentwith the increase in yohimbine-induced lethality ob-served with POEE treatment (Siqueira et al., 1998).

Relevant for social functioning, the role of noradrenalinein improving patients’ energy, interest and motivationis listed as one of the advantages of selective noradrenalinereuptake inhibitors (Brunello et al., 2002). As mentionedearlier, lack of energy, motivation and interest are symp-toms usually taken into account by healers when sug-gesting treatments with P. olacoides-based remedies.

Dopamine has recently been associated with depres-sion and antidepressant treatments by several lines ofevidence (Dunlop and Nemeroff, 2007). Because humandepressive syndromes are characterized by anhedoniaand loss of motivation, it makes sense that antidepres-sants that preferentially facilitate dopamine transmis-sion in mesolimbic areas (crucially involved in rewardmechanisms and motivation) would be beneficial in thereversal of depressive states (D’Aquila et al., 2000). Adecrease in the density of dopamine D1 receptors (as-sessed by PET scan) was reported in depressed humans(Suhara et al., 1992). Consistent with the hypothesisthat an enhanced neurotransmission at the dopamineD1 receptor might result in antidepressant effects, acute

administration of selective dopamine D1 agonists hasantidepressant effects similar to that of chronic imipraminein both the FST and the learned helplessness procedures(D’Aquila et al., 2000). Moreover, acute treatment withSCH 23390 (but not with sulpiride) suppresses the effectof chronic imipramine in these models (Gambaranaet al., 1995), and antagonizes the anti-immobility effectsof bupropion and nomifensine in the FST (Yamada et al.,2004). Comparable with this study, the acute effects ofseveral SSRIs (Renard et al., 2001,) and a semi-syntheticester of hyperforin (Cervo et al., 2005) in the FST werereversed by D1 and D2 antagonists. Conversely, thedopamine D1 agonist SKF 38393 enhances the anti-immobility effects of selective serotonin re-uptake in-hibitors, including paroxetine and fluvoxamine (Renardet al., 2001). The involvement of the D1 dopaminereceptor in the effects of POEE in the FST and TST isconsistent with these hypotheses linking dopamine anddepression, as well as with the reported ability of theextract to reverse reserpine-induced ptosis (Siqueiraet al., 1998).

In conclusion, we here provide further evidence thata standardized ethanol extract from P. olacoides possessesantidepressant properties in validated mouse models.The effects of POEE appear to require the D1

dopamineand the β-noradrenergic receptors, both shown to berelevant for antidepressant effects of other antidepres-sant drugs. Clinical studies and the identification of activeantidepressant components of the extract are necessaryto further assess the therapeutic potential of this speciesin the treatment of depression. Nevertheless, its tradi-tional medical use is a strong indication of bioavailabilityand consistent with the experimental profile here reported.

Acknowledgements

This study is associated with patents PI0205432-9/PI0307647-4 (INPI/Br). The authors are grateful to CAPES, CNPq and PROPESQ(UFRGS) for fellowships and support.

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