Journal of Ethnopharmacology 100 (2005) 306–309

Passiflora actinia Hooker extracts and fractions induce catalepsy in mice

K.C. Santosa, C.A.M. Santosa, R.M.W. de Oliveirab,∗a Laboratory of Pharmacognosy, Department of Pharmacy, Federal University of Parana, Rua Prefeito Lothario Meissner 3400,

Jardim Botanico, 80210-170 Curitiba, Parana, Brazilb Laboratory of Psychopharmacology, Department of Pharmacy and Pharmacology, University of Maringa,

Avenida Colombo 5790, 87020-900 Maringa, Parana, Brazil

Received 16 December 2003; received in revised form 19 October 2004; accepted 14 March 2005Available online 10 May 2005

Abstract

Leaves from severalPassiflora species are largely employed in the Brazilian folk medicine for its anxiolytic and sedative properties. Inbehavioral studies, to analyze the tranquillizer action ofPassiflora actinia Hooker, it was noteworthy that animals treated with the hydroethanol(HE) and methanol (ME) extracts presented an abnormal postural immobility compared to control animals. That observation led to aninvestigation of the effects of HE and its fractions on evaluation of catalepsy in mice. The results showed that HE extract, methanol extract,t inm©

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he sequent aqueous crude fractions (AF), and fa, fb and fc chromatographic fractions obtained fromPassiflora actinia induced catalepsyice. Apparently, the active principles responsible for catalepsy are present in all of the fractions of the extract.2005 Elsevier Ireland Ltd. All rights reserved.

eywords: Passifloraceae;Passiflora actinia; Catalepsy; Flavonoids

. Introduction

Passifloraceae family has about 20 genera and 600 speciesf plants.Passiflora genus is the largest one with 80 speciesistributed mostly in American and African continents (Joly,998). The aerial parts ofPassiflora species have been tra-itionally used to treat anxiety, insomnia and nervousnessDi Stasi et al., 1885; Wolfman et al., 1994; Dhawan et al.,004). The principal bioactive compounds described in thisenus areC-glycosil flavonoids and�-carbolinic alkaloidsBlumenthal et al., 2000). Some reports have pointed outhe harman alkaloids (Alonso, 1998), the flavonoid chrysinWolfman et al., 1994; Zanoli et al., 2000) or maltol (Speronind Minghetti, 1988) as the mainly bioactive constituents ofassiflora incarnata Linneau, one of the species ofPassi-ora that have been extensively studied chemically and bi-logically (Soulimani et al., 1997; Dhawan et al., 2001a,b).ecently,Dhawan et al. (2001a,b)have characterized a ben-

∗ Corresponding author. Tel.: +55 44 2614313; fax: +55 44 2636231.

zoflavone moiety fromPassiflora incarnata as the active substance responsible for several central nervous system e

Passiflora actinia Hooker (vernacular name: ‘maracua-do-mato’) is a native species widely distributed throSouthern Brazil. High-performance liquid chromatogra(HPLC) comparative analysis of the active fractions ofPassi-flora actinia Hooker has suggested a closer chromatograprofile with Passiflora incarnata (Santos et al., 2003).Fractionation of the methanol–water extract showedflavonoid fraction was composed mainly of isovitexin whthe alkaloid fraction presented very low flavonoid contenpharmacological studies aimed to analyze the psychotactions of Passiflora actinia, Santos et al. (2003)havedemonstrated that intraperitoneal (i.p.) injections ofPassi-flora actinia crude hydroalcoholic (HE), methanolic (ME)methanolic–aqueous fractions (AF) cause sedative effemice. These effects were observed as a pronounced dein the number of entries in enclosed and open arms oelevated plus maze (EPM) and a decrease in the in the gmotor activity in the open field. Only the aqueous fracof the ME (30 mg/kg) showed selective anxiolytic activity

E-mail address: rmmwoliveira@uem.br (R.M.W. de Oliveira). the EPM. Interestingly, during the behavior test, it was noted

378-8741/$ – see front matter © 2005 Elsevier Ireland Ltd. All rights reserved.oi:10.1016/j.jep.2005.03.010

K.C. Santos et al. / Journal of Ethnopharmacology 100 (2005) 306–309 307

that animals treated with thePassiflora actinia extracts pre-sented an abnormal postural immobility compared to controlanimals. This is possibly due to a cataleptic effect of thetreatments.

Catalepsy is a term used to define a state of abnormalpostural immobility. It has been used to predict tranquillizeractivity (Carlini, 1973) as well as to evaluate motor effects ofdrugs, particularly those related to extra-pyramidal system.Therapeutic doses of haloperidol, a neuroleptic drug, inducecatalepsy in rodents and Parkinson-like symptoms in humans(Sanberg et al., 1988). In the same way, high doses of mor-phine and other opioids produce a state of catalepsy, whichis characterized by muscular rigidity and marked akinesia(Turski et al., 1992).

The aim of this work was to evaluate the plausible catalep-tic effect ofPassiflora actinia extracts and its fractions, usingthe hang bar test.

2. Materials and methods

2.1. Plant material

The leaves ofPassiflora actinia were collected in Can-guiri’s Farm, Pinhais, Parana, Brazil (November 2001), anda voucher specimen was deposited at the Department ofB ed-e

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(( ctedw( andt bse-q dis-t andr ex-t dC c-t xide9 di-e df –16( rudeM pre-p

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light:12-h dark cycle, were used. The experiments were car-ried out according to the National Institute of Health Guidefor Care and Use of Laboratory Animals.

2.4. Drugs

Haloperidol (Haldol®, Janssen, 1 mg/kg), hydroethanolextract (30, 100 and 300 mg/kg), methanol extract (100and 300 mg/kg), the sequent aqueous crude fraction (100and 300 mg/kg), and a and c chromatographic fractions,fa (100 and 300 mg/kg) and fc (100 and 300 mg/kg), re-spectively, ofPassiflora actinia were dissolved in saline.The chromatographic fraction b, fb (100 and 300 mg/kg),was dissolved in vehicle (saline containing DMSO 2%).All the treatments were i.p. administered at a volumeof 10 ml/kg.

2.5. Catalepsy test

Catalepsy was evaluated according to the standard bar-hanging procedure by placing the animals with both forelegsover a horizontal glass bar (0.5 cm o.d.), elevated 4.5 cmfrom the floor (Sanberg et al., 1988). The time the mousemaintained this position was recorded for up to 300 s; afterthree attempts the animal was replaced in cataleptic position.Catalepsy was considered finished when the forepaw touchedt mentw tracta

2

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3

atd (EH:F n(T eri-d forc f-td fa,f g at3f6F oiw after6

otany Herbarium (UPCB no. 30.831), Universidade Fral do Parana, PR, Brazil.

.2. Preparation of Passiflora actinia extracts andractions

The leaves (23.85 kg) were oven-dried at 45◦C and ground7.98 kg). The dried and powderedPassiflora actinia leaves750 g) were exhaustively and successively Soxhlet extraith petroleum ether (37.05 g), CHCl3 (24.67 g) and MeOH

103.86 g). The solvents were removed under vacuumhe residues and water extract were lyophilized. Suuently, the MeOH extract (100 g) was suspended in

illed water (1000 ml), shaken in water-ice bath for 8 hefrigerated for 12 h. An aliquot (800 ml) of the filteredract was partitioned sequentially withn-hexane (6.78 g) anHCl3 (3.77 g). The remaining H2O extract, aqueous fra

ion (70.3 g), was subjected to CC using aluminum o0 Merck (activity I–II seg. Brokmann) eluted with a grant of CHCl3 MeOH H2O (pH 9.5). The resulting joine

ractions: fa = 3–6 (1.8 g), fb = 7–13 (2.7 g) and fc = 142.3 g), were subjected to biological tests as well as the cE and its fractions. The hydroethanol extract (HE) wasared with ethanol (45◦ GL).

.3. Animals

Male albino-Swiss mice (30–45 g), housed in groof five per cage) with free access to food and wn a temperature-controlled room (23± 1◦C), with a 12-h

he floor or when the mouse climbed the bar. Measureas performed 30 and 60 min after saline, vehicle or exdministrations.

.6. Statistical analysis

Data represent mean± S.E.M. and were analyzed by Sent’st-test or one-way analysis of variance (ANOVA). M

iple comparisons were performed by the Tukey’s test.

. Results

Passiflora actinia HE and ME extracts administration,oses of 300 mg/kg, induced catalepsy in mice at 30 min2,29= 9.67,p < 0.001; ME:F2,25= 3.91,p < 0.05) and 60 mi

EH: F2,29= 10.59, p < 0.001; ME: F2,25= 6.73, p < 0.05).he effects of HE extract were comparable with halopol (Fig. 1), a drug that works as a positive controlatalepsy (Sanberg et al., 1988). Haloperidol was effective aer 30 min (t = 7.5, d.f. = 16,p < 0.0001) and 60 min (t = 10.1,.f. = 16,p < 0.0001). In addition, AF and all subsequent

b and fc fractions administered at dose of 300 mg/k0 min (AF: F2,28= 7.26,p < 0.05; fa:F2,25= 5.2, p < 0.05;

b: F2,23= 16.42,p < 0.001; fc:F2,25= 9.78,p < 0.001) and0 min (F2,28= 4.47,p < 0.05; fa:F2,25= 5.89,p < 0.05; fb:2,23= 5.02,p < 0.05; fc:F2,25= 9.31,p < 0.001) were able t

nduce catalepsy in the animals (Figs. 1 and 2). AF 100 mg/kgas at the lower dose able to induce catalepsy in mice0 min (F2,28= 4.47,p < 0.05).

308 K.C. Santos et al. / Journal of Ethnopharmacology 100 (2005) 306–309

Fig. 1. Effects ofPassiflora actinia hydroethanol extract (HE, 30, 100 and300 mg/kg), methanol extract (ME, 100 and 300 mg/kg), aqueous crude frac-tion (AF, 100 and 300 mg/kg), and a and c chromatographic fractions (fa,100 and 300 mg/kg; fc, 100 and 300 mg/kg) on catalepsy in mice. The testwas performed 30 and 60 min after the i.p. administration of the treatments.Haloperidol (1 mg/kg) was used as a positive control. The time during whichthe mouse maintained the cataleptic position was recorded for up 300 s, withthree attempts allowed to replace the animal over the glass bar. Columnsrepresent the means and vertical bars the S.E.M.s of the groups (n = 8–10animals).+p < 0.001 compared to saline by Student’st-test; ∗p < 0.05 and** p < 0.001 compared to saline by ANOVA followed by Tukey’s test.

Fig. 2. Effects ofPassiflora actinia b chromatographic fraction (fb, 100 and300 mg/kg) on catalepsy in mice.∗p < 0.05 and** p < 0.001 compared tovehicle (V) by ANOVA followed by Tukey’s test. Further details inFig. 1.

4. Discussion

The present results show that extracts obtained ofPassi-flora actinia leaves produce catalepsy in mice. HE and ME ex-tracts or AF, fa, fb and fc fractions were equally potent in pro-ducing catalepsy when injected in mice at doses of 300 mg/kgwhile the AF was effective at a lower dose (100 mg/kg).These results are in agreement with others showing thatPas-siflora edulis aqueous (Vale and Leite, 1983) or hydroethanolextracts (Bruschi et al., 2002) induce catalepsy in mice wheni.p. administered at doses of 40 and 80 mg/kg, respectively.

The main chemical constituents ofPassiflora areflavonoids, such as vitexin, isovitexin, orientin, isoorientin,apigenin and kampferol (Dhawan et al., 2004). Recently,De-Paris et al. (2002)have shown a correlation between the con-

tent in flavonoids found in aqueous extracts fromPassiflora’sspecies and their psychotropic actions. Anxiolytic effectswere detected inPassiflora edulis at half the dose (50 mg/kg)of Passiflora alata (100 mg/kg). Concerning the flavonoidcontent, the hydroethanol extract ofPassiflora edulis pre-sented almost twice (4.60%, w/w) the flavonoid content ofPassiflora alata (2.90%, w/w), which was composed by vi-texin, isovitexin, orientin and isoorientin (Petry et al., 2001).Other studies have shown the involvement of the flavonoidsvitexin, isovitexin, orientin, isoorientin (De-Paris et al., 2002)or chrysin (Wolfman et al., 1994; Zanoli et al., 2000), in pro-moting depressor effects in the central nervous system. In ad-dition, Rocha et al. (2002)have suggested that orientin andisoorientin could be responsible for the observed anxiolytic-like effect of theCecropia glazioui butanol fraction. How-ever, other studies reported that pure vitexin and isovitexin donot have psychotropic activity (Speroni and Minghetti, 1988)while orientin has very mild anxiolytic effects (Okuyamaet al., 1996).

The HPLC analysis of the fractions ofPassiflora actiniahas shown that the flavonoid isovitexin was present in theME, AF, fb and fc, while standard alkaloids such as harman,harmin, harmaline, harmol or harmalol, were not found in anyfraction (HE, ME, AF, fa, fb or fc;Santos, 2003). Possibly,the cataleptic activity described herein was not dependent onthe flavonoid or alkaloid content of the extracts or fractionso cec laceiw oninsh .,2 insf 5s tic ac-te

ingc(e rans-m motora bya de ofp .,1 thebe dRa toi hosec eo therm lepsyb dK ,2 lated

f Passiflora actinia, since all of them were able to induatalepsy in mice. However, formation of froth takes pn the active AF fraction when shaken (Santos et al., 2003)hich suggests the presence of saponins. In fact, five sapave been isolated fromPassiflora alata (Reginatto et al001). This is in line with the sedative-like effect of sapon

rom Panax notoginseng (Hosttetmann and Marston, 199)uggesting a correlation between the sedative–catalepivity and the presence of saponins inPassiflora actiniaxtracts.

Little is known about the mechanisms underlyatalepsy induced byPassiflora’s extracts.Vale and Leite1983) have attributed the cataleptic effect ofPassifloradulis aqueous extract to an antagonism of dopamine tission, since this extract decreased spontaneous lococtivity and licking behavior, both of them inducedmphetamines. Catalepsy has been linked to blockaostsynaptic dopamine D1 and D2 receptors (Sanberg et al988). Haloperidol is thought to induce catalepsy throughlockade of dopamine receptors in the striatum (Ellenbroekt al., 1985) and nucleus accumbens (Hartgraves anandall, 1986). Likewise, the administration ofl-NOARG,n inhibitor of nitric oxide (NO) production, is believed

nduce catalepsy by inhibiting dopamine release in terebral areas (Marras et al., 1995). Along with the blockadf postsynaptic striatal dopamine receptors, multiple oechanisms have been proposed to explain the cataehavior such as antagonism of�-opioid (Havemman anuschinsky, 1981) or acetylholine receptors (Moo-Puc et al.003). Furthermore, the catalepsy state may be modu

K.C. Santos et al. / Journal of Ethnopharmacology 100 (2005) 306–309 309

by drugs that modify the serotonergic (Pires et al., 1993)or adenosinergic (Zarrindast et al., 1997) neurotransmittersystems. Thus, the results obtained in the present studyrequire further investigation to elucidate the catalepsyinduced byPassiflora actinia extracts.

In summary, the present results show that acute treatmentwith Passiflora actinia extracts and fractions induce catalepsyin mice. This could potentially causes an adverse effect suchas an increase in extra-pyramidal side effects of neurolep-tic drugs or beneficial such as improving the therapeutic re-sponse. Furthermore, in clinical populations the possibility ofpharmacological interactions between drugs andPassiflora’sextracts must be considered.

Acknowledgements

The authors thank Prof. Dr. A. Cervi (Universidade Fed-eral do Parana), for the identification of the plant material,and S.C.F. de Oliveira for technical support.

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