Journal of Ethnopharmacology 146 (2013) 311–317

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Neuropharmacological in vivo effects and phytochemical profileof the extract from the aerial parts of Heteropterys brachiata (L.)DC. (Malpighiaceae)

Maira Huerta-Reyes a,b,n, Maribel Herrera-Ruiz a, Manases Gonzalez-Cortazar a, Alejandro Zamilpa a,Esther Leon c, Ricardo Reyes-Chilpa d, Arturo Aguilar-Rojas a, Jaime Tortoriello a

a Centro de Investigacion Biomedica del Sur, Instituto Mexicano del Seguro Social, Argentina No. 1, Col. Centro, Xochitepec, Morelos C.P. 62790, Mexicob Unidad de Investigacion Medica en Farmacologıa, Hospital de Especialidades, Centro Medico Nacional Siglo XXI, Instituto Mexicano del Seguro Social,

Av. Cuauhtemoc No. 330, Col. Doctores, Del. Cuauhtemoc, C.P. 06720 Mexico D.F., Mexicoc Herbario Nacional de Mexico (MEXU), Instituto de Biologıa, Universidad Nacional Autonoma de Mexico, Apartado Postal 70-367, Del. Coyoacan, C.P. 04510 Mexico D.F., Mexicod Instituto de Quımica, Universidad Nacional Autonoma de Mexico, Del. Coyoacan, C.P. 04510 Mexico D.F., Mexico

a r t i c l e i n f o

Article history:

Received 28 September 2012

Received in revised form

28 November 2012

Accepted 29 December 2012Available online 10 January 2013

Keywords:

Heteropterys brachiata

Antidepressant

Anxiolytic

Anticonvulsant

Sedative

41/$ - see front matter & 2013 Elsevier Irelan

x.doi.org/10.1016/j.jep.2012.12.049

esponding author at: Centro de Investigacion

o del Seguro Social, Argentina No. 1, Col. Cen

Mexico. Tel.: þ52 777 361 2155; fax: þ52

acion Medica en Farmacologıa, Hospital de E

l Siglo XXI, Instituto Mexicano del Seguro Soc

tores, Del. Cuauhtemoc, C.P. 06720 Mexico D

2 55 5627 6900x21368.

ail address: chilanguisima@yahoo.com (M. Hu

a b s t r a c t

Ethnopharmacological relevance: Heteropterys brachiata is a plant species that has been used in

traditional Mexican medicine for the treatment of nervous disorders.

Aim of the study: To evaluate the anxiolytic, anticonvulsant, antidepressant and sedative effects

produced by the methanolic extract of Heteropterys brachiata (HbMeOH) in ICR mice. Additionally,

we determine the acute toxicity profiles of the extract and the presence of its main constituents.

Material and methods: The neuropharmacological effects of the extract were evaluated using a variety

of models, such as the elevated plus maze (EPM), the forced swimming test (FST), the pentobarbital

potentiation test (PTBt), pentylenetetrazole-induced seizures test (PTZt), and the open field test (OFT).

HPLC was employed for obtention of phytochemical profile.

Results: HbMeOH produced a significant antidepressant effect in FST at 500 and 750 mg/kg doses, while

doses from 500 to 1500 mg/kg exhibited a clear dose-dependent anxiolytic activity in EPM. A dose of

500 mg/kg showed a significant anticonvulsant activity in PTZt and an absence of sedation effects in

PTBt. The main compounds of HbMeOH were chlorogenic acid and chlorogenic acid methyl ester, as

well as less abundant terpene-type compounds. Furthermore, the extract was either safe with no

deaths in mice treated orally with 2000 mg/kg.

Conclusions: HbMeOH extract which contains mainly hydroxycinnamic acids and triterpene-type

compounds, possesses antidepressant, anxiolytic and anticonvulsive properties and can be considered

safe or of low toxicity when orally administrated. These findings lend pharmacological justification to

the traditional use of Heteropterys brachiata in the treatment of nervous disorders.

& 2013 Elsevier Ireland Ltd. All rights reserved.

1. Introduction

Mental disorders affect 450 million people worldwide, with 121million suffering from depression, and approximately 50 millionpeople have epilepsy (World Health Organization (WHO), 2011). InMexico, approximately 15 million individuals experience some type

d Ltd. All rights reserved.

Biomedica del Sur, Instituto

tro, Xochitepec, Morelos C.P.

777 361 2194; Unidad de

specialidades, Centro Medico

ial. Av. Cuauhtemoc No. 330,

.F., Mexico.

erta-Reyes).

of mental health problem, with depression and anxiety being themost common (Valencia, 2007); however, the use of medicalnational health services by Mexican population is poor (o25%)(Medina-Mora et al., 2003). In developed countries, 70% to 80% ofthe population resort to traditional medicine to treat illnesses, andnearly 53% of patients with depression and anxiety utilize comple-mentary and alternative plant-related drugs (World HealthOrganization (WHO), 2008). This fact may be due to several factors,including the desire to avoid adverse effects as well as to takeadvantage of the safety, effectiveness and better quality control ofthe phytomedicines available on the market today (De Souza et al.,2009). Medicinal plants have been an important resource in thedevelopment of drugs. Indeed, nearly 25% of today’s conventionaldrugs originated directly or indirectly from plants (Zhang, 2004). Aconsiderable number of herbal medicines are recognized as active in

M. Huerta-Reyes et al. / Journal of Ethnopharmacology 146 (2013) 311–317312

the central nervous system (CNS), and they have at least ahypothetical therapeutic potential to affect chronic conditions suchas anxiety, depression, headaches or epilepsy in cases wherepatients do not respond well to conventional treatments(Quintans-Junior et al., 2008).

Heteropterys brachiata (L.) DC. is a woody vine belonging to theMalpighiaceae family and is widely distributed throughout thetropics and sub-tropics from Mexico to South America (Anderson,1993). In Mexico, it is popularly known as ‘‘Bejuco de Margarita’’,‘‘tsak ts aah’’ or ‘‘tipite-ek’’ and has been used in traditionalmedicine mainly for the treatment of nervous disorders (Arguetaet al., 1994). Some others reports indicate that Heteropterys

brachiata has also been employed as contraceptive as well as inthe treatment of fever, scabies and rabies (Monroy-Ortiz andCastillo-Espana, 2007). Despite the widespread use of this plant inMexican folk medicine, there is no scientifically investigationreported about the plant and its biological properties. Our studyfocuses on the neuropharmacological activities of Heteropterys

brachiata with respect to understanding its traditional medicinalapplications and potential uses in drug development. The presentstudy was conducted to evaluate the anxiolytic, antidepressant,sedative, anticonvulsant and influence on the motor activityeffects produced by the extract of Heteropterys brachiata in ICRmice using a variety of models, such as the elevated plus maze,the forced swimming test, the pentobarbital potentiation test,pentylenetetrazole-induced seizures test, and the open field test.Furthermore, the extract of Heteropterys brachiata was evaluatedto determine its acute toxicity profile and analyzed to quantify itsmain constituents.

2. Materials and methods

2.1. Plant collection and identification

The aerial parts of Heteropterys brachiata (L.) DC. were collectedfrom the state of Morelos, Mexico (at latitude 18147024.50 0 northand longitude 99110012.10 0 west) in September 2007. The identifica-tion of the plant was authenticated by an expert in the field of planttaxonomy, who is also one of the authors (E. Leon). A voucher wasdeposited as reference at the Mexican Institute of Social SecurityMedicinal Herbarium (IMSSM) under the number 15442.

2.2. Preparation of extracts

The plant material was dried in a dark at room temperatureand powered (1.64 kg). After dewaxing with n-hexane, the plantmaterial was successively extracted (3� ) overnight with metha-nol (100%). The extraction volume used was 7.5 L of solvent pereach kg of plant material. The liquid extract was dried by removalof the solvent under vacuum. The HbMeOH (175.7 g) was thenused in the pharmacological experiments.

2.3. Drugs and chemicals

Imipramine hydrochloride (IMI) and pentylenetetrazole (PTZ)were purchased from Sigma-Aldrich Chemical Co. (St. Louis, MO,USA). Diazepam (DZP) and carboxymethyl cellulose (CMC) wereobtained from Cryopharma S.A. de C. V. (Guadalajara, Jal, Mexico).Sodium pentobarbital (PEN) was purchased from Pfizer Inc. (NewYork, NY, USA).

2.4. Animals

The animal experiments were performed in strict adherence to theofficial requirements of the Mexican Regulations of Experimental

Animal Care (NOM-062-ZOO-1999). The experimental protocol wasapproved by the institutional research and ethics committee (Registrynumber 2007-1701-8). For each neuropharmacological assay, groupsof eight ICR albino mice weighing 30–36 g each were utilized (HarlanMexico S.A. de C. V., Mexico City, Mexico). The animals were housedin community cages and maintained under regular laboratory condi-tions (2572 1C, 12-h light-dark cycle, free access to water andstandard rodent chow: 2018S, Harlan Tekland). All animals wereacclimatized for 3 weeks prior to initiation of the test. The experi-ments were conducted in a special adjacent noise-free room withcontrolled illumination. Each animal was used only once in theexperiment.

2.5. Neuropharmacological assays

HbMeOH was administrated p.o. at a dose of 500 mg/kg.In those assays that exhibited activity at the initial dose of500 mg/kg, the dose-dependent effects were determined using750, 1000 and 1500 mg/kg doses.

2.5.1. FST

The FST is the most widely used pharmacological in vivo modelfor assessing antidepressant activity. The development of immo-bility when mice are placed in an inescapable cylinder filled withwater reflects the cessation of persistent escape-directed behavior(Porsolt et al., 1977). The apparatus utilized to perform the FSTconsisted of a clear glass cylinder (20 cm high�12 cm diameter)with water filled to a depth of 15 cm (2471 1C). The mice weretreated with HbMeOH (500 mg/kg, experimental treatment, n¼8)and CMC 1% (vehicle, p.o., control group, n¼8) at 48, 36, 24, 18,and 1 h prior to the test. IMI (15 mg/kg, i.p., positive control, n¼8)was administered 24 h, 18 h, and 30 min before the test. Prior tothe administration schedule, the mice were subject to a pre-testsession in which each animal was individually placed in thecylinder for 15 min. During the test session, a trained observerrecorded the immobility time.

2.5.2. EPM

The EPM test is the most frequently employed model for theassessment of the anxiolytic activity of novel substances (Lister,1987). The maze was constructed of Plexiglas and consisted of acentral platform (5�5 cm) with two open (30�5 cm) and twoclosed arms (30�5 cm) and 25 cm high walls. The maze waselevated 38.5 cm from the room’s floor. The mice were treated30 min prior to the test with DZP (1 mg/kg, i.p., positive control,n¼8), while CMC 1% (vehicle, p.o., control group, n¼8) andHbMeOH (500 mg/kg, experimental treatment, n¼8) were admi-nistrated 1 h prior to the test. Each animal was placed at thecenter of the maze facing one of the open arms. The number ofentries and the time spent in the enclosed and open arms wererecorded during the 5 min test. All of the test sessions wererecorded by video camera. After each test, the maze was carefullycleaned with wet tissue paper (10% ethanol solution).

2.5.3. OFT

The open field area was comprised of transparent acrylic wallsand a black floor (30�30�15 cm) divided into nine squares ofequal size. One hour before the test, the mice were treated withHbMeOH (500 mg/kg, experimental treatment, n¼8) and CMC 1%(vehicle, p.o., control group, n¼8). The open field test was used toevaluate the locomotor activity of mice that had previously beensubjected to the FST and EPM tests. The observed parametersincluded the number of squares crossed (with four paws) (cross-ings) and the number of rearings (Archer, 1973).

Fig. 1. Effect of oral administration of Heteropterys brachiata methanol extract on

immobility time of ICR mice exposed to FST. npo0.05 with ANOVA followed by a

post hoc Dunnett’s test (mean7S.D.). Veh, vehicle; IMI, imipramine hydrochloride;

HbMeOH, methanolic extract of Heteropterys brachiata.

M. Huerta-Reyes et al. / Journal of Ethnopharmacology 146 (2013) 311–317 313

2.5.4. PTZt

HbMeOH (500 mg/kg, experimental treatment, n¼8) and CMC1% (vehicle, p.o., control group, n¼8) were administered 1 hbefore the PTZ (75 mg/kg, i.p.), while DZP (1 mg/kg, i.p., positivecontrol, n¼8) was administered only 30 min prior to the PTZ.Following the administration of PTZ, mice were placed in separatetransparent Plexiglas cages (25�15�10 cm) and were observedfor the occurrence of seizures over a 30 min time period. Latencyof convulsions (the time prior the onset of clonic convulsions),convulsions number (tonic or clonic convulsions of 5 s duration atleast), and mortality protection (percentage of deaths in 24 h)were recorded (Williamson et al., 1996).

2.5.5. PTBt

HbMeOH (500 mg/kg, experimental treatment, n¼8) and CMC1% (vehicle, p.o., control group, n¼8) were administrated 1 h priorto the test, and DZP (1 mg/kg, i.p., positive control, n¼8) wasadministered 30 min before the test. In this experiment, after theadministration of PEN (30 mg/kg, i.p.), the mice were placedseparately in transparent Plexiglas cages (25�15�10 cm) toobserve the hypnotic effect, which was considered to be the timeinterval between the disappearance (latency) and reappearance(duration) of the righting reflex (Williamson et al., 1996).

2.6. Toxicity assay

The acute toxicity test was performed according to the OECDguidelines for the testing of chemicals (OECD, 2001). Nine ICRfemale mice (26–28 g, 8 weeks old) were acclimatized under regularlaboratory conditions (the same as those describe above). Food waswithheld for 1 h before administration. The animals were caged ingroups of three, and doses of 500 and 2000 mg/kg of HbMeOH and100 ml/10 g of CMC 1% (vehicle, p.o., control group) were adminis-tered orally by gavage. Thirty minutes after administration, theanimals were subjected to an initial period of observation of severalCNS activity-associated behavioral parameters: locomotor activity,tremors, grip strength, bizarre behavior, convulsions, abdominalcontortions, gait incapacity, piloerection, palpebral closure, andconstipation. This procedure was performed three times weeklyfor 2 weeks during which animal deaths, animal weights, and foodconsumption were also recorded.

2.7. HPLC analysis

We performed an HPLC analysis of HbMeOH for detection andquantification of its major constituents. HPLC analysis was con-ducted on a Waters 2695 liquid chromatographer equipped witha Waters 2996 photodiode array detector. Separation was carriedout using a RP C-18 Superspher (Merck) column (120�4 mm;5 mm) with the following solvent ratios for the mobile phasewhere solvent A is water and solvent B is acetonitrile: A:B¼100:0(0–1 min); 90:10 (2–4 min); 80:20 (5–9 min); 70:30 (10–15 min);60:40 (16–18 min); 40:60 (19–20 min); 0:100 (21–30 min); and100:0 (31–32 min). The sample injection volume was 10 ml with a1.0 ml/min flow rate. The detection wavelength was scanned at190–400 nm. Quantification of the main compounds wasachieved using calibration curves that were separately con-structed with pure standards (Herrera-Ruiz et al., 2006).

Purification of the main compounds detected in the extractwas carried out by successive extraction (3� ) of 20 g of HbMeOHwith n-hexane and ethyl acetate. The remaining solid wasdissolved in methanol (Hb-bip-MeOH, 13.1 g) and subjected toSi gel chromatography, eluting with CH2Cl2:(CH3)2CO¼100:0 to50:50; 7 fractions were collected. Fraction 5 (1.2 g) was subjectedto Si gel chromatography, eluting with CH2Cl2:MeOH¼100:0 to

0:100, and 10 fractions were collected. Chlorogenic acid methylester precipitated in Fraction 3 (33.3 mg), while chlorogenic acidwas detected in Fraction 4 (31.7 mg).

2.8. Statistical analysis

The data were analyzed using a one-way ANOVA followed by apost hoc Dunnett test using the SPSS 11.0 program. Differencesbetween the experimental groups were considered statisticallysignificant when po0.05.

3. Results

3.1. FST

HbMeOH induced a significant antidepressant effect in the FSTbecause it significantly diminished the immobility time comparedwith the control (po0.05) (Fig. 1). In addition to the effectsobserved for the 500 mg/kg dose, the 750 mg/kg dose caused asignificant increase in immobility time (po0.0) in the FST withrespect to control group (Veh). However, the 1000 and 1500 mg/kg doses did not induce a similar behavior (Fig. 1).

3.2. EPM

The anxiolytic DZP induced a significantly increased of thepercentage of entries to open arms (EOA) and the percentage oftime spent in the arms (TOA) in the EPM, when comparing tocontrol group (po0.05). Mice treated with HbMeOH in the doserange from 500 to 1500 mg/kg, provoked significantly changes ofEOA and TOA, with respect to the control group (Veh) (po0.05)(Fig. 2).

3.3. OFT

Mice treated with HbMeOH (500 mg/kg), did not showed anychange in the parameters of total crossing and rears in the OFT,comparing with the control group (Veh) (p40.05). In the case ofanimals treated with doses of 750, 1000 and 1500 mg/kg ofHbMeOH exhibited a decrease in the number of the total crossing(po0.05), but not in the number of rears (p40.05) (Fig. 3).

3.4. PTZt

The dose of 500 mg/kg of HbMeOH showed a total protectionagainst death in mice as well as significant diminution of thenumber of convulsions (3.271.3, po0.05) (Table 1) while

Fig. 2. Effect of oral administration of Heteropterys brachiata methanolic extract

on the percentage of time spent in open arms (TOA) and entries into open arms

(EOA) by mice exposed to EPM. npo0.05 in ANOVA followed by a post hoc

Dunnett’s test (mean7S.D.). Veh, vehicle; DZP, diazepam; HbMeOH, methanolic

extract of Heteropterys brachiata.

Fig. 3. Effect of oral administration of Heteropterys brachiata methanolic extract

on the number of total crossings and rearings of ICR mice exposed to the open field

paradigm. npo0.05 with ANOVA followed by a post hoc Dunnett’s test

(mean7S.D.). Veh, vehicle; DZP, diazepam; HbMeOH, methanolic extract of

Heteropterys brachiata.

Table 1Anticonvulsant effect of methanolic extract from Heteropterys brachiata on PTZ-

induced seizures in ICR mice.

Treatment

(mg/kg)

Latency of

convulsions (s)

Convulsions

number

Mortality

protection

(%)

Veh (100 ml/10 g) 91.0718.6 7.373.7 0.0

DZP (1) 0.070.0n 0.070.0n 100.0

HbMeOH (500) 61.8718.9n 3.271.3n 100.0

HbMeOH (750) 95.07 47.8 3.372.5n 57.1

HbMeOH (1000) 79.6736.1 4.873.1 14.2

HbMeOH (1500) 61.1713.0 4.371.5 28.5

Data presented as the mean7S.D. with n¼8.n po0.05 compared to vehicle using ANOVA and a post hoc Dunnett’s test.

Veh, vehicle; DZP, diazepam; HbMeOH, methanolic extract of Heteropterys

brachiata.

Table 2Effect of the methanolic extract of Heteropterys brachiata in the PTB test on ICR

mice.

Treatment (mg/kg) Latency Duration

Veh (100 ml/10 g) 0.070.0 0.070.0

DZP (1) 246.3752.5n 2690.07644.0n

HbMeOH (500) 0.070.0 0.070.0

Data presented as the mean7S.D. with n¼8.n po0.05 compared to vehicle using ANOVA and a post hoc Dunnett’s test.

Veh, vehicle; DZP, diazepam; HbMeOH, methanolic extract of Heteropterys

brachiata.

M. Huerta-Reyes et al. / Journal of Ethnopharmacology 146 (2013) 311–317314

750 mg/kg of HbMeOH diminished the convulsions number(po0.05) with a protection only of the 57.1%. Since treatmentswith 1000 and 1500 mg/kg of HbMeOH offered no anticonvulsantprotection and did not change the onset of seizures, these effectsare not different as compared with those observed for the controlgroup (Table 1).

3.5. PTBt

The treatment of 500 mg/kg of HbMeOH exhibited no changesin mice behavior in the parameters of latency and duration ascompared with the control group (p40.05) (Table 2).

3.6. Toxicity

Normal behavior was observed in CMC 1%-treated micebecause animals did not exhibit any alterations in the parametersanalyzed. No changes in the weight of the animals and in the foodconsumption were detected. There were no deaths during the 14days of observation after the acute i.p. treatment with 2000 mg/kgof HbMeOH.

3.7. HPLC analysis

We analyzed HbMeOH by HPLC for detection and quantificationof major constituents of active extract. Fig. 4 shows the HPLC profileof HbMeOH recorded at 220 nm, in which four peaks were detected.The main compounds of HbMeOH, peak 1 (tR¼3.9 min) and peak 2(tR¼8.1 min), were isolated, identified and quantified as chlorogenicacid (3.2 mg/kg) and chlorogenic acid methyl ester (60 mg/kg),respectively (Fig. 5), since the spectroscopic data were found to bein good agreement with values reported in the literature (Jang et al.,2010; Sari, 2010). Quantification was established with calibrationcurves (linear regression where r240.9850).

4. Discussion

Despite its use in traditional Mexican medicine to treat somemental diseases, Heteropterys brachiata has not yet been evalu-ated for its activity on the CNS. The present study investigated forthe first time the CNS effects of a methanolic extract of the aerialparts of Heteropterys brachiata (HbMeOH) in mice. Our resultsshowed that HbMeOH standardized in its content of hydroxycin-namic acids with doses of chlorogenic acid (3.2 mg/kg) andchlorogenic acid methyl ester (60 mg/kg) induced a significantantidepressant effect in the FST. In this test, an experimentalmodel for testing the efficacy of antidepressant drugs, the animaldevelops a learned helplessness syndrome characterized by alowered motivation for escaping as evidenced by increasedperiods of immobility (Porsolt et al., 1977). Several authors haveproposed that immobility during the test could be an efficientadaptive response to this stress (Porsolt et al., 1977; Tolardo et al.,2010). Interestingly, the higher doses of HbMeOH (1000 and1500 mg/kg) were not effective in the FST. It is possible that thiseffect showed on FST may be influenced by the action ofneurotransmitters or receptors because similar saturated dose-response curves have been already reported after oral administrations

Fig. 4. HPLC chromatogram of the antidepressant, anxiolytic and anticonvulsive HbMeOH extract. The peaks were identified as (1) chlorogenic acid, (2) chlorogenic acid

methyl ester, (3) unknown triterpene, and (4) unknown triterpene.

Fig. 5. Chemical structure of the main compounds, chlorogenic acid and chloro-

genic acid methyl ester isolated from the antidepressant, anxiolytic and antic-

onvulsive HbMeOH extract.

M. Huerta-Reyes et al. / Journal of Ethnopharmacology 146 (2013) 311–317 315

of different crude plant extracts, such as the extract of Allium

macrostemon (Alliaceae) (Lee et al., 2010). Other possible mechanismfor this effect showed on FST may not involve receptor occupation. Innon-competitive antagonism, the threshold dose of agonist is notmarkedly increased, but the maximum response is depressed in thepresence of the antagonist. Crude plant extracts often produce thiskind of effect. It arises when the blocking action takes place, as itwere, beyond the receptor, such as effects on intracellular secondmessengers (Williamson et al., 1996). Indeed, our results are inagreement with other studies where the best effect was shown onthe middle dosage (750 mg/kg) of the extract, while the high dosage(1500 mg/kg) did not shown the antidepressant activity, as the caseof the herbal formulae Kai Sin San used in the traditional Chinesemedicine (Zhou et al., 2012)

On the other hand, in the present work, the oral administrationof HbMeOH exhibited a clear anxiolytic activity in a dose-dependent manner. Because numerous neural pathways areinvolved in the pathophysiology of depression and anxiety states,false positive results can be obtained for agents that stimulatelocomotor activity (Bourin et al., 2001). Therefore, the observationthat HbMeOH did not increase the number of crossings andrearings in the OFT at the 500 mg/kg dose confirms the assump-tion that the anxiolytic effect of the extract does not influence themotor system (Sanchez-Mateo et al., 2005). However, althoughhigher doses of HbMeOH do not exhibit changes in the number ofrears, the registered decrease of the total crossing suggests thatthere is a general depressor effect on the nervous system (dosSantos et al., 2006). This observation is in agreement with thosereports in which higher doses 4500 mg/kg from a crude plantextract used widely in folk medicine not only exhibited anxiolyticproperties but also elicited sedation and myorelaxant effects(Galani and Patel, 2011). Moreover, in this study, our resultsobtained in the PTBt assay also confirm the absence of sedation

effects at the lower dose of HbMeOH because the treatment of500 mg/kg exhibited no changes in the behavior of the mice.

The effect of HbMeOH as an anticonvulsant by the PTZt modelwas also examined in this study because this model is widely usedto investigate anticonvulsive drugs with a high predictive value forthe detection of clinically effective drugs (Galani and Patel, 2011).GABA is the major inhibitory neurotransmitter implicated in epi-lepsy. The enhancement and inhibition GABA neurotransmissionwill attenuate and enhance convulsion, respectively (de Oliveiraet al., 2011). Because there is evidence that PTZ may be exerting itsproconvulsant effect by inhibiting the activity of GABA at GABAA

receptors, it is probable that the delayed occurrence of PTZ convul-sion observed by the administration of 500 mg/kg of HbMeOH maybe interfering with the GABAergic mechanism to exert its antic-onvulsant effect (Quintans-Junior et al., 2008).

Our findings suggest a CNS-depressant action of HbMeOH atdoses 4500 mg/kg. Because the effective dose of such as Valeri-

ana officinalis, Crataegus monogyna and several Hypericum species(Sanchez-Mateo et al., 2002), is between 500 and 1000 mg/kg ofplant extracts, Heteropterys brachiata appears to be not onlywithin a similar range of potency but also interesting to explorefor its potential neuropharmacological properties at doseso500 mg/kg, the lowest dose employed in the present study.

Although some other species belonging to the Heteropterys genushave been studied in some neuropharmacological aspects, theliterature is lacking on pharmacological studies of the plant Het-

eropterys brachiata, the species subject of our study. The extract ofthe Brazilian species Heteropterys aphrodisiaca improved learningand memory deficits in aged rats in the passive avoidance test andin the T maze, respectively (Galv~ao et al., 2002). Heteropterys

aphrodisiaca also showed a strong antioxidative activity in vitro

and induced an increase in superoxide dismutase activities in oldrats (Mattei et al., 2001). In the case of Heteropterys glabra, theethanolic extract induced a reduction in motor activity and altera-tions in EEG parameters (Galietta et al., 2005). However, there isscarce information about the chemical constitution of these extracts.The only report on this topic is a partial phytochemical screeningthat revealed the presence of glycosides, polyphenols, tannins,alkaloids, saponins and anthracene derivatives as major componentsin the species Heteropterys aphrodisiaca (Galv~ao et al., 2002). Then, tothe best of our knowledge, the present study is the first thatcomprises pharmacological and quantitative phytochemical data ofthe genus Heteropterys and ever more, of the plant species Heterop-

terys brachiata. Thus, the main compounds identified in HbMeOHare the hydroxycinnamic acids chlorogenic acid and chlorogenicacid methyl ester. Although hydroxycinnamic acids are present in alarge variety of fruits and vegetables, chlorogenic acid is the mostabundant hydroxycinnamic acid in food and has been reported toprevent different cancers, cardiovascular diseases and type 2 dia-betes mellitus (Suzuki et al., 2006). Like other dietary polyphenols,chlorogenic acid and chlorogenic acid methyl ester are also

M. Huerta-Reyes et al. / Journal of Ethnopharmacology 146 (2013) 311–317316

antioxidants with antiviral, antibacterial and antifungal properties(Lafay et al., 2006; Chen et al., 2010). Additionally, chlorogenic acidhas been considered to be a neuroactive substance because itimproved the impairment of short-term or working memoryinduced by scopolamine in the Y-maze and significantly reversedcognitive impairments in mice as measured by the passive avoid-ance test (Kwon et al., 2010). Moreover, chlorogenic acid has beenshown to have an anxiolytic effect produced by activation of thebenzodiazepine receptor (Bouayed et al., 2007). In this sense, thereis a possibility that the chlorogenic acid due largely to its contentmay be involved at least in part in the anxiolytic effect shown byHbMeOH, since the presence of this hydroxycinnamic acid had beenresponsible to exercise the anxiolytic effect in the EPM model(Bouayed et al., 2007).

In the case of peak 3 (tR¼22.9 min; UV absorption: 192.3 nm)and peak 4 (tR¼23.5 min; UV absorption: 192.3 nm) also detected inHbMeOH, although their identities remain unknown, their tR and UVabsorption values suggest the presence of a complex mixture ofterpene-type compounds (Harborne, 1984), which appear to be thesecond most abundant chemical family in HbMeOH after thehydroxycinnamic acids. Previously, terpenes have been reported tobe partly responsible for the sedative, anxiolytic, anticonvulsant andanalgesic activities in several species of medicinal plants (Arag~aoet al., 2006; Kubacka et al., 2006; Wijeweera et al., 2006; Tavianoet al., 2007). Furthermore, the galphimines, a group of triterpenesdescribed for first time in 1993, have been recognized as thecompounds responsible for the potent anxiolytic properties showedby Galphimia glauca extracts in behavioral models and in clinicalstudies on patients with generalized anxiety disorder (Herrera-Arellano et al., 2007). It is interesting to note that Galphimia glauca

is a species that also belongs to the same plant family as Heterop-

terys: Malpighiaceae. Nonetheless, through the chromatographicanalyses performed using HPLC in the present study, the galphi-mines A, B and E were not detected in HbMeOH. The lack ofgalphimines does not preclude the possibility of the active involve-ment of the terpene-type compounds on the neuropharmacologicalproperties exhibited by HbMeOH, since in one sense, this extract notonly exerted anxiolytic activities, and on the other hand, aswe mentioned before, terpene-type compounds had been detectedas responsible of CNS effects in other medicinal plants extracts.Nevertheless, further studies are needed to confirm this hypothesis.

In the present acute toxicity study, our results showed thatHbMeOH could be consider as safe, or of low toxicity, when orallyadministrated (Adeneye et al., 2006). This lack of signs of acutetoxicity supports the use of Heteropterys brachiata in folkloricmedicine by an oral route and encourages further studies for thedevelopment of HbMeOH as a therapeutic agent.

5. Conclusions

The methanolic extract of Heteropterys brachiata possesses anti-depressant, anxiolytic and anticonvulsive properties and can beconsidered safe or of low toxicity when orally administrated. To thebest of our knowledge, this study presents the first report on thequantitative phytochemical data of this extract which containsmainly hydroxycinnamic acids and terpene-type compounds. Thesefindings lend pharmacological justification to the traditional use ofHeteropterys brachiata in the treatment of nervous disorders.

Acknowledgments

This work was supported by grant 82588 from SEP-CONACyT-Ciencia Basica 2007, Mexico (to M. Huerta-Reyes) and grant FIS/IMSS/PROT/C2007/040 from the FIS, Instituto Mexicano del

Seguro Social, Mexico (to M. Huerta-Reyes). The authors wish tothank Jonathan Orduno and Arturo Perez for technical assistance.

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