Anxiolytic-like and sedative actions of Rollinia mucosa: Possible involvement of the GABA/benzodiazepine receptor complex

Abstract

This study evaluated possible CNS effects of a hexane extract of leaves from Rollinia mucosa (Jacq.) Baill. (Annonaceae). This plant extract induced anxiolytic-like actions similar to those induced by diazepam in the avoidance exploratory behavior paradigm. Its significant activity was shown at doses from 1.62 to 6.25 mg/kg. It also enhanced pentobarbital-induced hypnosis time, and at high doses produced motor coordination impairment. The benzodiazepine (BDZ) receptor binding, evaluated by in vitro autoradiography following a single administration of R. mucosa, revealed that this plant extract reduced BDZ binding in the hippocampus (29%), amygdala (26%), and temporal cortex of mice (36%). In conclusion, the present findings support the proposal that R. mucosa may induce central nervous system (CNS) depressant effects, presumably through an interaction with the GABA/benzodiazepine receptor complex.

Keywords::

• Benzodiazepine receptors
• Rollinia mucosa (Jacq.) Baill.
• sedative

Introduction

Rollinia mucosa (Jacq.) Baill. (Annonaceae), a tropical fruit tree known as “biribia”, is the most widespread species of this genus (Figueiredo et al., 1999). This species is limited to warm lowlands, from 20° north to 30° south latitudes in tropical America and it is distributed in the West Indies, from Mexico to Bolivia, and Brazil. The fruit is regarded as a refrigerant, analeptic, and anti-scorbutic, while the powdered seeds are said to be a remedy for enterocolitis (Morton, 1987). Chemical investigations of this species reported the presence of acetogenins with antineoplastic and cytotoxic activities in seeds and leaves (Pettit et al., 1987; Shi et al., 1997). In addition, we reported that the furafuranic lignans, (+)-epieudesmine and (+)-epimagnolin, and the linear C₃₁-ketone palmitone (16-hentriacontanone), were the principal constituents of the hexane extract of leaves of R. mucosa (Estrada-Reyes et al., 2002). It has been reported that palmitone, isolated from Annona diversifolia Saff. (Annonaceae) induces an anxiolytic-like effect in mice tested in the elevated plus-maze paradigm (González-Trujano et al., 2006). In addition, a lignan mixture present in a methanol extract of the sedative herbal Valeriana officinalis L. (Valerianaceae), produced an increase in the affinity to the BDZ binding site of GABA_A receptors and possessed partial agonistic activity to A₁ adenosine receptors (Schumacher et al., 2002). Therefore, the present study evaluated the effects of R. mucosa in mice tested in an anxiety paradigm, the avoidance exploratory behavior test, and the ability of this plant extract to facilitate pentobarbital-induced hypnosis time. General activity and motor coordination were evaluated in the open field and rota-rod tests, respectively. Finally, possible changes on BDZ binding following a single administration of R. mucosa were evaluated and compared to those obtained after an acute administration of diazepam, a well-known agonist to BDZ receptors.

Materials and methods

Plant material and preparation of the extracts

Leaves of R. mucosa were collected in Ocosingo, Chiapas, Mexico in August 2006. The plant was identified by A. Esquinca, Escuela de Biología Herbarium, UNICACH, Chiapas, where a voucher specimen (R. mucosa No. 16204) was deposited. The hexane extract of R. mucosa was obtained as previously reported (Estrada-Reyes et al., 2002).

Animals

Swiss-Webster male mice weighing 25-30 g were used in this study. Mice were housed in groups of 10 animals in plastic cages (44 × 21 × 21 cm) and kept under 12:12 h inverted light/dark cycle conditions (lights on at 22:00 h). All behavioral evaluations were done between 10:00 and 14:00 h to avoid the variability induced by circadian rhythms. Animals had free access to Purina mouse chow and water throughout the experiment. Principles of laboratory animal care were followed; all procedures were conducted in accordance with established guidelines (ILAR, 1996). The local Committee of Ethics on Animal Experimentation approved all experimental procedures, which followed the regulations established in the Mexican Official Norm for the use and care of laboratory animals “NOM-062-ZOO-1999”. All the experimental sessions were videotaped and analyzed by an observer unaware of treatment conditions.

Drugs

All drugs in this study were intra-peritoneal (i.p.) injected in a total volume of 10 mL/kg body weight. The fresh hexane extract was suspended in 1% Tween 80 and isotonic solution (0.9% of NaCl), sodium pentobarbital (Sigma, St. Louis, MO) was dissolved in saline solution (0.9%), while diazepam (Hoffmann-La Roche, México City) was dissolved in propylene glycol 1%. For the behavioral studies an initial comparison between R. mucosa control group (treated with 1% Tween 80 and isotonic solution, 0.9% of NaCl) and diazepam control group (treated with propylene glycol 1%) was performed. Since no significant differences were found between control groups, only R. mucosa control group was used for comparison purposes.

Behavioral tests

Avoidance Exploratory Behavior Test (AEBT)

Mice were divided into six groups (n = 10) receiving a dose of R. mucosa (1.62, 3.12, 6.25, or 12.5 mg/kg), vehicle (control group) or diazepam (1.0 mg/kg, positive control) one hour before the anxiety test. The AEBT was initially described by Crawley and Goodwin (1980) and it is used to screen new anxiolytic drugs. The apparatus consisted of an acrylic chamber (44 × 21 × 21 cm) divided into a small, darkened compartment and a large and highly illuminated compartment. An opening of 13 × 15 cm separated the dark from the bright area. At the beginning of the test each mouse was introduced into the bright compartment and the number of transitions from one side to the other was recorded for 10 min. An increase in this parameter was interpreted as an anxiolytic-like effect. The test chamber was carefully cleaned after each recording. In order to prevent behavioral changes of animals after the first experience, all mice were tested only once in this paradigm.

Open field test

In order to discard a possible influence of drug-treatments on locomotors activity, all treatments studied in the anxiety paradigm were analyzed in an open field test immediately after the anxiety test. Each animal was placed into an acrylic cage (60 × 40 × 40 cm) with a checkerboard pattern (20 × 20 cm) on the floor. The total number of square crossings by the animal was registered for 10 min.

Sodium pentobarbital-induced sleeping time

The effect of R. mucosa on pentobarbital-induced sleeping time was studied in mice as described previously (Bastidas-Ramírez et al., 1998; Gilani et al., 2000). Fifty-six animals were divided into seven groups (n = 8). One hour before the administration of sodium pentobarbital (42 mg/kg) mice were treated with a dose of R. mucosa (3.12, 6.25, 12.5, 25, or 50 mg/kg). The control group received vehicle, while the last group which served as a positive control, received diazepam (2 mg/kg). The time in minutes elapsing from the loss to the regaining of the righting reflex after sodium pentobarbital administration was recorded and referred to as sleeping time.

Motor coordination

The effect on motor coordination was assessed using a rota-rod apparatus. In brief, mice were trained to remain for 5 min on the rod rotating at speed of 22 rpm. On the next day, the R. mucosa (0.0, 1.62, 3.12, 6.25, 12.5, 25, and 50 mg/kg), or DZ (2 mg/kg) were administered and the ability of mice to remain on the rotating rod was assessed before and 60 min after R. mucosa administration. For each animal, the fall-off time from the rod was registered (De Andrés et al., 1999).

Statistics

Differences between treated and control groups in the anxiety, motor activity, hypnosis, and motor coordination tests were analyzed using a Kruskal-Wallis Analysis of Variance on Ranks (p * < 0.05, p** <  0.01 and p*** <  0.001), followed by the Mann-Whitney Rank Sum Test. All statistical and laboratory analyses were carried out using the Sigma Stat Program (version 3.1, Jandel Scientific).

Autoradiography studies

Mouse manipulation

For habituation to manipulation, animals received a daily i.p. injection of saline solution (0.1 mL/10 g) for 5 days. The freshly hexane extract was i.p. administered at 3.12 and 6.25 mg/kg (6 mice per dose). These doses were used because they produce behavioral depression without inducing toxic effects. A control group (n = 6), was manipulated as the experimental animals, but was injected with vehicle. Mice were sacrificed by decapitation 24 h after treatment; their brains were quickly removed, frozen in pulverized dry ice and stored at -70°C. Frozen coronal sections of 20 μm were cut in a cryostat, thaw-mounted on gelatin-coated slides, and stored at -70°C until the day of incubation.

Autoradiography

The in vitro autoradiography technique was used to determine BDZ binding in specific brain areas of mice following treatment with R. mucosa or vehicle. We used brain slices pre-washed in Tris-HCl buffer in order to avoid binding alterations resulting from GABA levels, or other ligands (Rocha et al., 1993). Initially, the brain sections were pre-washed in Tris HCl buffer (170 mM; pH 7.4) for 30 min at 25°C. The sections were subsequently incubated for 45 min at 4°C in a solution containing 2 nM ³H-flunitrazepam (88 Ci/mM) (Amersham, Arlington Heights, IL), which labels BDZ sites; and 170 mM Tris buffer either in the presence or absence of 1 μM clonazepam, a BDZ agonist. Binding obtained in the presence of clonazepam was considered to be non-specific. Finally, incubation was terminated with two consecutive washes (1 min each) in 170 mM Tris buffer and a distilled water rinse (2 s) at 4°C. The sections were then quickly dried under a gentle stream of cold air. The slides were arrayed in X-ray cassettes together with tritium standards (Amersham), and apposed to tritium-sensitive film (Amersham Hyperfilm) for three weeks at room temperature. The films were developed using Kodak D11 developer and fixer at room temperature. Optical densities of selected structures appearing on the autoradiograms were determined by using a video-computer enhancement program (JAVA Jandel Video Analysis Software). For each structure, 10 optical density readings were taken from at least five sections, and were averaged. The optical density readings of the standards were used to determine tissue radioactivity values for the accompanying tissue sections and to convert them to fmol/mg protein.

BDZ receptor binding was analyzed in the following structures: frontal, temporal and entorhinal cortices, amygdala, hippocampus and thalamus. These particular structures were chosen for analysis because they are known to be involved in anxiety (Belzung, 1992; Davis, 1992).

Data from R. mucosa experiments were examined statistically by analysis of variance (ANOVA). Whenever a significant difference was found across groups within a region, a post hoc Dunnet’s test was used to determine significant differences (p < 0.05).

Results

In the AEBT, the hexane extract of leaves of R. mucosa at doses from 1.62 to 6.25 mg/kg significantly increased the number of transitions between the light and the dark areas, an effect similar to that induced by diazepam. However, at the dose of 12.5 mg/kg, R. mucosa did not show any effect on the anxiety paradigm (Figure 1A). This dose decreased general activity of mice in the open field test (shown by a decrease on the number of counts Figure 1B); none of the other doses of R. mucosa (1.62-6.25 mg/kg range) had an effect on general mouse activity.

Figure 1.  Effect of the hexane extract of Rollinia mucosa (Rm) on the AEBT (A) and on locomotor activity (B).

The effect of both diazepam and R. mucosa on pentobarbital-induced hypnosis is shown in Figure 2. The hexane extract significantly increased in a dose-dependent fashion pentobarbital-induced sleeping time, an effect similar to that obtained with diazepam. However, pentobarbital-induced hypnosis latency was not significantly modified with any doses of R. mucosa or diazepam tested alone.

Figure 2.  Effect of the Rollinia mucosa Rm on pentobarbital-induced hypnosis.

In the rota-rod test, the fall-off time of animals treated with R. mucosa 25 or 50 mg/kg and 2 mg/kg of BDZ, was reduced to 88.2 ± 13.58, 47.46 ± 8.57, and 148.8 ± 16.44 s, respectively, whereas animals treated with lower quantities remained for 300 s on the rotating rod.

In relation to the autoradiographic studies, in those areas of relatively high binding density examined under control situation, specific [³H]flunitrazepam binding ranged from 504 ± 16 fmol/mg of protein in hippocampus to 129 ± 11 fmol/mg of protein in caudate putamen. Non-specific binding ranged from 1% to 5% of the total binding. These values are in the same range as those previously reported (Rocha et al., 1993). In comparison to their control group, mice treated with R. mucosa showed decreased BDZ binding in specific brain areas, depending on the dose injected. Animals receiving 3.12 mg/kg decreased BDZ binding in amygdala (46%) and hippocampus (27%) and the dose of 6.25 mg/kg produced reduction of binding in amygdala (26%), hippocampus (29%) and temporal cortex (36%). Although the thalamus presented reduced BDZ binding following R. mucosa treatment, values were not significantly different when compared with the control group (Table 1).

Table 1.  Effect of a single administration of Rm on [³H]flunitrazepam binding (fmol/mg proteispecific regions of mouse brain.

Structure	Treatment (mg/kg)
	vehicle	3.12	6.25
Frontal cortex	407 ± 22	378 ± 64	471 ± 47
Temporal cortex	463 ± 56	395 ± 33	296 ± 30*
Enthorinal cortex	402 ± 50	405 ± 3	370 ± 74
Caudate putamen	129 ± 11	142 ± 43	140 ± 14
Amygdala	441 ± 36	234 ± 38 *	326 ± 35 *
Hippocampus	504 ± 16	365 ± 35 *	358 ± 40 *
Thalamus	168 ± 28	91 ± 20	80 ± 21

Each value is the mean ± standard error in six animals.

*p < 0.05 as compared to control group.

Discussion

Our results showed that the hexane extract of Rollinia mucosa (1.62, 3.12 and 6.25 mg/kg) produced a dose-dependent increase in the number of transitions between the highly illuminated and darkened compartments of the AEBT. This effect is considered as an anxiolytic-like action (Crawley & Goodwin, 1980) and is similar to that observed with diazepam (López-Rubalcava et al., 1992; Fernández-Guasti et al., 1992) and serotonergic anxiolytics such as ipsapirone (Fernández-Guasti & López-Rubalcava, 1990; López-Rubalcava et al., 1992). It is important to mention that at the highest dose (12.5 mg/kg) no anxiolytic-like effects were observed, however, at this same dose a significant decrease in general activity was found in the open field test. Therefore, it is possible that unspecific collateral motor effects masked R. mucosa anxiolytic-like actions.

The analysis of the in vitro autoradiography experiment demonstrated that R. mucosa was able to modify BDZ binding in specific brain areas of mice such as the amygdala, the hippocampus and the temporal cortex. The decrease in BDZ binding was observed 24 h after R. mucosa administration and could be explained through different mechanisms (Tunnicliff & Ngo, 1986). It is important to note that the amygdala and the hippocampus are structures associated with anxiety (Belzung, 1992; Davis, 1992) and that the reduced BDZ binding observed in these areas following the R. mucosa administration could be associated with the anxiolytic-like effects induced by this species.

On the other hand, the administration of R. mucosa, at the dose range of 12.5-50 mg/kg did not modify the hypnosis latency; therefore, R. mucosa alone (up to 50 mg/kg) has no hypnotic actions. However, it was able to increase pentobarbital-induced sleeping time.

The doses of 25 and 50 mg/kg, R. mucosa produced a severe impairment of motor coordination, similar to those observed with a sedative dose of diazepam. Since pentobarbital hypnotic actions are mediated through the GABA/benzodiazepine receptor complex (Petty, 1995) and since R. mucosa is also able to modify BDZ binding, it could be presumed that the GABAergic system participates in R. mucosa-induced potentiation on pentobarbital-induced hypnotic actions. It is important to mention that the lack of hypnotic actions of R. mucosa alone could indicate that the effects of this plant extract on pentobarbital-induced hypnosis and on BDZ binding are due to an indirect rather than a direct interaction with the GABA/benzodiazepine receptor complex. Future studies should specifically address this issue in order to analyze the exact mechanism of action of this plant extract.

In previous work, we reported that palmitone was the most abundant compound in the hexane extract of R. mucosa, although also furafuranic lignans were present (Estrada-Reyes et al., 2002). We also reported that the administration of the hexane extract of Annona cherimolia (rich in palmitone) produced anxiolytic-like actions similar to those of diazepam in two animal models of anxiety, the AEBT and in the burying behavior paradigm in mice (López-Rubalcava et al., 2006). So it is reasonable to assume that palmitone present in R. mucosa could account for the anxiolytic-like effects of this plant extract. Other behavioral studies reported that palmitone produces anti-anxiety actions, without sedative effects, even at 30 mg/kg (González-Trujano et al., 2006). Since R. mucosa showed sedative effects similar to diazepam at doses higher than those that exert anxiolytic-like actions it is possible that components other than palmitone could account for the impairment of motor activity.

In conclusion, R. mucosa-induced anxiolytic-like actions and was able to facilitate pentobarbital-induced hypnotic actions. Although its exact mechanism of action remains to be elucidated, results obtained with the autoradiography study suggested that R. mucosa could interact with the GABA/benzodiazepine receptor complex and mediate these central depressant actions.

Acknowledgements

The authors wish to thank Isabel Beltrán Villalobos for technical assistance, and Rubén Luviano Jaramillo for animal handing.

Declaration of interest: This study was partially supported by CONACyT. Grant numbers: 32702-M (for RL), 34992-N (for MVM) and 50636-M (for L-RC). The authors alone are responsible for the content and writing of the paper.