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Effects of methyl-eugenol administration on behavioral models related to depression and anxiety, in rats.

Abstract

Croton zehntneri (Cz) is a popular plant in Brazilian folk medicine. Recently, the use of its essential oil showed depressive activity response in the central nervous system (CNS). Chemical studies show that the main compound of this oil is the methyl-eugenol (ME). This work seeks to evaluate the ME activity in behavioral models of depression and anxiety, in the rat. Male rats (60 days old) were divided into four groups (n = 10) and treated with doses of 1.0, 3.0 and 10.0 ml/100 g body wt., v.o., of ME (experimental) and saline (control). One hour after treatment, they were observed in the forced swimming test and 15 min later in the open-field test. A decrease was observed in the immobility time during the forced swimming test for all experimental groups, in comparison with control group (C = 168.8 [+ or -] 27.3; 1.0 [micro]l = 139.1 [+ or -] 23.5; 3.0 [micro]l = 137.2 [+ or -] 18.7 and 10.0 [micro]l = 139.8 [+ or -] 23.6). The open-field results showed no differences in comparison to the control group. The same was observed for social interaction, plus-maze and holeboard tests, suggesting no alterations in anxiety behavior. These data suggest that ME administration induced antidepressive CNS alterations, expressed by the smallest immobility in the swimming model, and not of a level able to alter motor and exploratory activity in the open-field. The absence of effects observed in the open-field can be a result of the experimental contingency, taking low anxiety levels. These data are in contradiction to observations with Cz essential oil in these models.

[c] 2004 Elsevier GmbH. All rights reserved.

Keywords: Methyl-eugenol; Croton zehntneri; Behavior; Depression; Anxiety; Rat

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Introduction

Croton zehntneri Pax et Hoffm. (Cz) (Euphorbiaceae), popular name "canela de cunha", is used in Brasilian folk medicine for "nervous" disturbances such as irritability, anxiety and convulsions, as well as to relieve gastric disturbances. An earlier communication (Bernardi et al., 1991) reported that the tea prepared by pouring water over dried leaves or branches showed stimulant properties in rats (decreased pentobarbital sodium-induced hypnosis time and reduced minimal convulsant dose of strychnine). The same authors showed, however, that the lyophilized branch extract seems to contain one or more active principles with central nervous system (CNS) depressant characteristics, not only because treatment with the extract increases the duration of pentobarbital loss of some reflex, but also because it increases the minimal convulsant doses of picrotoxin and sthychnine. This mixed picture of activity may show that simple manipulation, such as the lyophilizing process, is able to alter the pharmacological effects of Cz.

Behavior models related to the dopaminergic-cholinergic system showed that the aqueous extract of branches and leaves modify the cholinergic-dopaminergic equilibrium in the CNS. Finally, the most important data of these studies were that the results with the tea "in nature" were not compatible with those reported for popular medicinal use. The authors suggested then that the medical properties of Cz are probably related to the presence of the essential oil, reduced in the employed preparations, because this oil is volatile and the leaves and branches were dry (Giorgi et al., 1991).

A decrease in general activity and an elevation of the pentylenetetrazole minimal convulsant dose for rats were observed after Cz essential oil exposure. These results suggest an effect of the oil on central mechanisms related to neural modulation of seizures (Batatinha et al., 1995).

Lazarini et al. (2000) showed that the administration of Cz essential oil increased the immobility duration during the forced swimming test, as well as a reduction in locomotion frequency observed in the open-field test. No effects were observed in social interaction, plus-maze or holeboard test. These data suggest that Cz oil produced central depressor effects in rats, without any anxiety alterations. Those results corroborate the use of that plant in the Brazilian popular medicine to treat nervous disturbances.

Chemical investigations of this plant, in particular of the species from Oieras, State of Piaui, showed the presence of several compounds, such as methyl-eugenol (ME) (24.6%) and estragol (10.3%) (Alencar and Craveiro, 1978) in the Cz essential oil. The present study sought to study the main component of Cz oil (ME) to explain the central mechanisms involved with the oil's CNS activity. Rat behavior anxiety and depression models were used.

Materials and methods

Animals

Male Wistar rats weighted 180-250 g each were used throughout this study. The animals were each housed randomly in appropriate cages at 21-23 [degrees]C on a 12 h light cycle (lights on at 7:00 h) with free access to food (Purina) and tap water. The animals and the experiments followed the guidelines of the Committee on Care and Use of Laboratory Animal Resources, National Research Council, USA.

Drug

ME (from Petite Marie Quimica Fina Industria e Comercio de Produtos Quimicos LTDA) was added (2.5 [micro]l) to water (100 ml) and shaken vigorously for 5 min. Two hours later, the suspension was filtered through filter paper and the filtrate was used at concentrations of 1.0, 3.0 and 10.0 [micro]l/ml. The solutions were given orally (by gavage) to the animals, which were deprived of food for 12 h. Rats received no more than 1 ml/kg body wt. of the ME suspension or the tap water.

Swimming test

The swimming test procedure described by Porsolt et al., (1978) was followed closely. The animals were trained 24 h before the test (15 min) and the test was performed for 5 min. Individual rats were forced to swim inside an upright cylindrical aquarium containing 19 cm of water at 25 [degrees]C. The absence of hind leg movement was record as immobility by a constant observer using a stopwatch during the test.

Open-field

The open-field apparatus was similar to that described by Broadhurst (1960). The number of crossings (locomotion), number of rearings and total immobility time were recorded for 5 min. The open-field apparatus was then cleaned using 5% ethanol before introducing the next animal, to preclude the possible cueing effects of odors left by previous subjects. To minimize possible influences of circadian changes on rat open-field behavior, control and experimental animals were intermixed.

Social interaction

This test was performed following File (1980). Rats were housed singly for 5 days prior to testing and social interaction was carried out in the open-field. At 90 days of age, rats from both groups were submitted individually to a 15-min familiarization session in the test arena, 24 h before the experiment. The pairs of rats were then tested for 5 min and social interaction was evaluated by the total time in seconds spent by the pairs of rats in active social interaction (sniffing, following, grooming, kicking, boxing, biting, wrestling and crawling under or over the partner). The apparatus was washed with a 5% ethanol solution before each behavioral test. Control and experimental rats were intermixed and the observations were made between 14:00 and 17:00 h.

Elevated plus-maze

The elevated plus-maze was made of wood and had two open arms (50 cm X 10 cm) and two enclosed arms of the same size with 40 cm high walls, and was elevated 50 cm above the ground. Each rat was placed in the center square (10cm X 10cm) and observed for the number of entries into the type of arm (all four paws defining an entry) and the time in the open and closed arms. These parameters were recorded for 5 min. The apparatus was washed with a 5% ethanol solution before each behavioral test; control and experimental rats were intermixed, and the observations were made between 14:00 and 17:00 h.

Holeboard

The holeboard apparatus was the same as that used in the open-field test, with one difference: the presence of four holes spaced equally in the floor, each 3.8 cm in diameter. Head dipping was measured with the same hand-operator counter as used to score locomotion, rearing frequency, and immobility. The apparatus was then cleaned using 5% ethanol before introducing the next animal to preclude the possible cueing effects of odors left by previous subjects.

Experiment 1

Swim and open-field tests: Sixty-day-old rats (n = 40) were randomly distributed into four groups (10/group). Animals of the experimental groups received 1.0, 3.0 and 10.0 [micro]l/100 g body wt. (p.o) of the ME suspension, while tap water (1 ml/kg body wt.) was administered (p.o) to the control group. Sixty minutes after the treatments, rats were placed individually in the water cylinder to assess the swim and immobility time over a period of 5 min. Fifteen minutes after the swimming test, these animals were observed in the open-field apparatus for 5 min.

Experiment 2

Social interaction, elevated plus-maze, and holeboard tests: Ninety-day-old rats (n = 80) were distributed randomly into four groups (20/group). Animals of the experimental groups were treated with 1.0, 3.0 and 10.0 [micro]l/100 g body wt. (p.o) of the ME suspension, while tap water (1 ml/kg body wt.) was administered (p.o) to a control group. Sixty minutes after the treatments, ten pairs of rats from each group and from the same treatment were observed in the social interaction test. After this, one animal from each pair, observed previously, was tested in the elevated plus-maze and the other in the holeboard, as described previously.

Statistics

Analysis of variance (ANOVA) followed by the Tukey-Kramer test was used to analyze data. In all experiments p < 0.05 was the criterion for statistical significance.

Results

Experiment 1

Swim and open-field tasks: administration of ME suspension decreased immobility, but not in a dose-dependent manner, in the swimming test, as compared to control animals [F(3.36) = 4.12, p = 0.01)] (Fig. 1A). In addition, locomotion frequency, rearing frequency, and immobility duration in the open-field were not altered after any of the doses employed {locomotion [F(3.36) = 1.91, p = 0.14], rearing [F(3.36) = 0.45, p = 0.72], immobility [F(3.36) = 0.15, p = 0.99]} (Figs. 1B-D, respectively).

Experiment 2

Social interaction, elevated plus-maze, and holeboard tasks: no significant differences were observed between control and experimental groups in terms of the social interaction [F(3.32) = 0.30, p = 0.82], plus-maze {%entries into open arms [F(3.32) = 0.36, p = 0.78], %time on open arms [F(3.32) = 0.21, p = 0.89], and total entries [F(3.32) = 1.27, p = 0.23]}, and holeboard {locomotion [F(3.32) = 0.12, p = 0.95], rearing [F(3.32) = 0.96, p = 0.43], immobility [F(3.32) = 0.41, p = 0.75], number of head-dips [F(3.32) = 1.11, p = 0.36]} tests. These data are summarized in Table 1.

Discussion

Rats forced to swim in a restricted area assume an immobile posture after initial attempts to escape. In a subsequent immersion, the beginning of the immobility is faster and marked. Porsolt et al. (1978) named this phenomenon "behavior despair" and attributed the animals' response to the development of a depression process. Treatment with anti-depressive medicines reduces the immobility time during the swimming test. The authors presented it as a model of animal depression.

In the forced swimming test, which evaluates the depressive behavior noted by Porsolt, a significant decrease of the immobility time was observed after the administration of different doses of ME, to rats. These results suggest that ME induces an anti-depressive, or stimulant activity on the CNS, but not in a dose-dependent manner. The activity of Cz essential oil in this model showed CNS-depressive activity (Lazarini et al., 2000). This essential oil contained 24.6% ME, 10.3% estragol, and other compounds, as determined by gas chromatography. These results suggest that this compound (ME), although it is the principal compound of the Cz essential oil, would not be directly responsible for the depressive activity of the Cz oil in this behavioral model. This suggestion may be supported by the fact that the ME demonstrates a separate CNS-stimulant activity.

[FIGURE 1 OMITTED]

The results observed in the open-field test showed no significant differences among all groups tested. This behavior model is used to study exploratory and motor activity. The purpose of including this test was to analyze the general activity of the animals after the forced swimming test. After the administration of the ME, at the doses used, and after the animals' results in the forced swimming test, no alterations were observed in this behavior model. It is therefore suggested that those substances do not alter the normal CNS paths for that test; in other words, locomotion activity and emotionality.

The administration of the ME showed no effects on anxiety behavior of the rats, according to the tests used to analyze animal anxiety, the social interaction (File et al., 1993) and elevated plus-maze tests. In fact, in the social interaction test, anxiolytic drugs increased (Corbertt et al., 1991), while anxiogenic drugs decreased (Baldwin et al., 1989), the time spent in active social interaction, independently of any alterations in motor activity (File, 1980). Another animal model to test anxiety behavior is the elevated plus-maze. It has been observed that anxyolitic drogs, diazepam-like, increase the number of arm entries and percentage of time spend in the open arms of an elevated plus-maze, and anxiogenic drugs have the opposite effect (Zangrossi and File, 1992). The present study showed no significant differences between control and experimental groups in the models used to study anxiety alterations. The social interaction and plus-maze results indicate that this component of the essential oil of Cz does not modify the anxiety state in these tests. These results are similar to those observed for Cz essential oil administration in these models, showing neither anxiolytic nor anxiogenic responses (Lazarini et al., 2000).

No significant differences was observed among rats of the control and ME groups in the holeboard test This experimental model is used mainly to test exploratory behavior, and was used in these experiments (social interaction and elevated pluz-maze), because it is known that an increase in anxiety reduces exploratory behavior in rats. The lack of observed alterations in the anxiety models could explain these results in the "holeboard".

These data suggest that the administration of the ME, the principal compound of the essential oil of Cz, produces stimulating or "anti-depressive" effects in rats, probably by modifying the CNS mechanisms involved with this behavior, without alterations in structures related to anxiety. These results also suggest that ME, isolated, is not responsible for the Cz essential oil responses observed in these behavior models.
Table 1. Effects of ME administration on social interaction, elevated
plus-maze and holeboard parameters of rats treated orally with different
doses of ME

 Groups
 Control 1 [micro]l/ml

Social interaction (s) 158.6[+ or -]10.0 148.4[+ or -]9.7
Elevated plus-maze
 % Entries into open arms 27.5[+ or -]7.0 33.0[+ or -]7.9
 % Time on open arms 25.9[+ or -]9.5 25.7[+ or -]9.6
 Total entries (freq.) 8.2[+ or -]0.8 7.9[+ or -]1.0
Holeboard
 Locomotion (freq.) 45.8[+ or -]10.0 49.5[+ or -]8.1
 Rearing (freq.) 12.4[+ or -]3.0 8.1[+ or -]2.3
 Immobility (s) 46.1[+ or -]22.1 24.9[+ or -]9.4
 Head-dips (freq.) 2.3[+ or -]0.7 1.3[+ or -]0.6

 Groups
 3 [micro]l/ml 10 [micro]l/ml

Social interaction (s) 153.1[+ or -]15.1 144.2[+ or -]9.0
Elevated plus-maze
 % Entries into open arms 26.4[+ or -]8.2 35.7[+ or -]6.4
 % Time on open arms 22.8[+ or -]8.9 33.0[+ or -]10.0
 Total entries (freq.) 7.6[+ or -]1.0 10.2[+ or -]1.3
Holeboard
 Locomotion (freq.) 53.4[+ or -]8.7 52.2[+ or -]12.3
 Rearing (freq.) 14.8[+ or -]4.4 9.0[+ or -]2.4
 Immobility (s) 29.4[+ or -]12.2 38.0[+ or -]24.0
 Head-dips (freq.) 3.5[+ or -]1.1 2.7[+ or -]1.0


Acknowledgements

This work was supported by the Fundacao de Amparo a Pesquisa do Estado de Sao Paulo (FAPESP, proc. 01/09516-2 and 01/09517-9).

Received 22 September 2003; accepted 3 December 2003

References

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Batatinha, M.J.M., Souza-Spinosa, H., Bernardi, M.M., 1995. Croton zehntneri: possible central nervous system effects of the essential oil in rodents. J. Ethnopharmacol. 45, 53-57.

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Broadhurst, P.L., 1960. Experiments in psychogenetics. In: Eisenk, H.J. (Ed.), Experiments in Personality. Routledge & Kegan Paul, London, pp. 31-71.

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File, S.E., 1980. The use of social interaction as a method for detecting anxiolytic activity of chlordiazepoxide-like drugs. J. Neurosci. Meth. 2, 219-238.

File, S.E., Zangrossi, J.R.H., Andrews, N., 1993. Social interaction and elevated plus maze tests: changes in release and uptake of 5-HT and GABA. Neuropharmacology 32, 217-221.

Giorgi, R., Batatinha, M.J., Bernardi, M.M., DeSouza-Spinosa, H., Spinosa, F.R., Palermo-Neto, J., 1991. Effects of Croton zehntneri aqueous extracts on some cholinergic- and dopaminergic-related behaviors of laboratory rodents. J. Ethnopharmacol. 34, 189-193.

Lazarini, C.A., Uema, A.H., Brandao, G.M.S., Guimaraes, A.P.C., Bernardi, M.M., 2000. Croton zehntneri essential oil: effects on behavioral models related to depression and anxiety. Phytomedicine 7, 477-481.

Porsolt, R.D., Anton, G., Blavet, N., Jalfre, M., 1978. Behavioral despair in rats: a new model sensitive to antidepressant treatments. Eur. J. Pharmacol. 47, 379-384.

Zangrossi, H., File, S.E., 1992. Behavioral consequences in animal tests of anxiety and exploration of exposure to cat odor. Brain Res. Bull. 29, 381-388.

M.C.B. Norte, R.M. Cosentino, C.A. Lazarini*

Laboratorio de Farmacologia, Faculdade de Medicina de Marilia, Famema, Av. Monte Carmelo. 800, Marilia 17-519030, Brasil

*Corresponding author. Tel./fax: +55 14 423 4344.

E-mail address: lazarini@famema.br (C.A. Lazarini).
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Author:Norte, M.C.B.; Cosentino, R.M.; Lazarini, C.A.
Publication:Phytomedicine: International Journal of Phytotherapy & Phytopharmacology
Geographic Code:3BRAZ
Date:Apr 1, 2005
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