Central nervous system activity of the methanol extract of Ficus platyphylla stem bark

Journal of Ethnopharmacology 85 (2003) 131–137

Central nervous system activity of the methanol extract of Ficus platyphylla stem bark B.A. Chindo a , S. Amos a,∗ , A.A. Odutola b , H.O. Vongtau a , J. Abbah a , C. Wambebe a , K.S. Gamaniel a a

Department of Pharmacology and Toxicology, National Institute for Pharmaceutical Research and Development (NIPRD), P.M. B. 21, Abuja, FCT, Nigeria b Department of Pharmacology and Clinical Pharmacy, Ahmadu Bello University, Zaria, Nigeria Received 25 March 2002; received in revised form 27 November 2002; accepted 27 November 2002

Abstract The central nervous system (CNS) activity of the methanolic extract of Ficus platyphylla stem bark was studied on locomotor activity, pentobarbital sleeping time, exploratory behaviour, amphetamine-induced hyperactivity, apomorphine-induced stereotypy, active-avoidance and performance on tread mills (rota-rod), using mice and rats. The results revealed that the extract significantly reduced the locomotor and exploratory activities in mice, prolonged pentobarbital sleeping time in rats, attenuated amphetamine-induced hyperactivity and apomorphine-induced stereotypy in mice, dose-dependently. The extract significantly suppressed the active-avoidance response in rats, with no significant effect on motor co-ordination as determined by the performance on rota-rod. The results suggest that the extract may possess sedative principles with potential neuroleptic properties. © 2002 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Ficus platyphylla; Locomotor activity; Pentobarbital sleeping time; Exploratory behaviour; Motor co-ordination; Active-avoidance

1. Introduction

2. Materials and methods

Ficus platyphylla Del. Holl. (Moraceae) is a deciduous plant locally known as “gamji” and widely distributed throughout the savannah region of West African coast. In Northern Nigeria, the plant is used in traditional medicine for treating several diseases including insomnia, psychosis, depression, and also useful as an analgesic (Audu, 1989). Previous studies revealed that the plant possesses antinociceptive, anti-inflammatory, and gastrointestinal activities (Amos et al., 2002, 2001a) in rodents. The intraperitoneal and oral LD50 in mice were found to be greater than 2000 mg/kg and above 5000 mg/kg, respectively, and the preliminary phytochemical analysis revealed the presence of flavonoids, tannins and saponins (Amos et al., 2001a). In the present study, we evaluated the central nervous system (CNS) activity of F. platyphylla to see if there is any scientific basis for the use of the plant in traditional medicine for the treatment of CNS disorders.

2.1. Plant material

∗ Corresponding

author. Tel.: +234-9-5232459; fax: +234-9-5231043. E-mail address: [email protected] (S. Amos).

The plant material was collected from Kala’a in Hong Local Government Area of Adamawa State, Nigeria. The plant was identified and authenticated by Late (Mr.) A. Ohaeri and Mallam I. Muazzam of the Department of Medicinal Plant Research and Traditional Medicine, National Institute for Pharmaceutical Research and Development (NIPRD), Abuja. A voucher specimen (no. 4035) was deposited at NIPRD Herbarium for future reference. 2.2. Preparation of extract The bark was chopped, cleaned, air dried for 10 days and milled into coarse powder using pestle and mortar. Eighty grams of the coarse powder was extracted to exhaustion with 1 l of methanol using a soxhlet extractor for 12 h. The solvent was removed under reduced pressure using a rotary evaporator and it gave a yield of 35% (w/w).

0378-8741/02/$ – see front matter © 2002 Elsevier Science Ireland Ltd. All rights reserved. doi:10.1016/S0378-8741(02)00376-8

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2.3. Animals

2.7. Test for motor co-ordination (rota-rod test) in mice

Swiss albino mice (18–25 g) and adult Wistar rats (200–250 g) of either sex, obtained from animal facility centre of NIPRD were used for the studies. The animals were kept in plastic cages and housed under standard conditions of temperature, relative humidity and light/dark cycles (12/12 h). They were fed with Ladokun feeds and water ad libitum.

The method used for the assessment of locomotor (Forced Motor) activity in mice was as described previously (Ozturk et al., 1996; Perez et al., 1998). A rota-rod treadmill device (Ugo Basile no. 7600, Varese, Italy) was used for this purpose. The mice were placed on a horizontal rotating rod set at a rate of 16 revolutions per min. Mice that were able to remain on the rod longer than 180 s were selected and divided into four groups of five mice per group. Three groups of the selected animals received graded doses of the extract (25–100 mg/kg, p.o.), while the remaining group received normal saline to serve as control. Thirty minutes later, each mouse was placed on the rod for 180 s, at intervals of 30 min, for 3 h.

2.4. Locomotor activity in mice Adult mice of either sex were randomly divided into four groups of six mice per group. Three groups were given three doses of the extract (25–100 mg/kg, p.o.). Animals in the remaining group received normal saline (10 ml/kg) as control. Thirty minutes later, the animals were transferred individually to Letica activity cages (LE 886) connected to AM1051 (Benwick electronics) data logger. The AM1051 data logger is provided with two layers of infrared sensor placed horizontally to monitor the rearing, mobile and static activities, as well as the active and mobile times. After 1 min of latency period, activity counts were recorded for 6 min, at 30 min intervals for 90 min (Wambebe et al., 1997; Amos et al., 2001b). 2.5. Pentobarbital sleeping time in rats The test was performed in four groups of five rats each. Three groups received the extracts at the doses of 25, 50 and 100 mg/kg, p.o., while the control group received normal saline. The animals in the fifth group received diazepam (1 mg/kg, i.p.). Thirty minutes later, pentobarbital sodium (40 mg/kg, i.p.) was administered to each rat to induce sleep. Each rat was observed for the onset and duration of sleep, with the criterion for sleep being loss of righting reflex (Wambebe, 1985; Rolland et al., 1991). The interval between loss and recovery of righting reflex was used as the index of hypnotic effect (Ramirez et al., 1998).

2.8. Amphetamine-induced hyperactivity in mice Adult mice of either sex were randomly selected and divided into four groups of six mice each. Three groups were pre-treated with graded doses of the extract (25–100 mg/kg, p.o.) while the other group received normal saline as control. Thirty minutes later, d-amphetamine (2 mg/kg, i.p.) was administered to all the mice. The animals were transferred individually to LETICA activity cages (LE 886) each connected to a multicount (LE 3806) and after 1 min latency period, activity counts were recorded for 6 min at 15, 30, 60, 90 and 120 min (Wambebe et al., 1997; Amos et al., 2001b). 2.9. Apomorphine-induced stereotype studies in mice The test was performed in five groups of six mice each. One group received saline as control, while three groups received graded doses of the extract (25–100 mg/kg, p.o.). The animals in the fifth group received chlorpromazine (2 mg/kg, i.p.). Thirty minutes after drug and saline administration, all the animals were injected with apomorphine (2 mg/kg, i.p.). The signs of stereotypic behaviour, which include sniffing, jumping/climbing, limb licking and circling were observed and counted using hand tally counters for 2 h (Nemeroff, 1980; Amos et al., 2001b; Chindo, 1999). 2.10. Active-avoidance test in rats

2.6. Hole-board test for exploratory behaviour in mice The hole-board test procedure for exploratory behaviour in mice was used as described previously by Ozturk et al. (1996), with some modifications. The apparatus used for this test was an Ugo Basile of 60 cm × 30 cm with 16 evenly spaced holes with in build infra red sensors. In brief, adult mice of either sex were randomly divided into five groups of five mice per group. Three groups received graded doses of the extract (25–100 mg/kg, p.o.). One group received clonazepam (0.2 mg/kg, i.p.) and the remaining group received normal saline to serve as control. Thirty minutes later, the number of head dips into the holes was counted for each animal for 5 min (Wolfman et al., 1994).

The animals were trained in an active-avoidance paradigm in a 25 cm×25 cm×40 cm shuttle-box with a buzzer located at the midline on the lid of the box and a floor consisting of stainless steel grid bars. The box was divided into two equal compartments by a wood panel with an 8 cm × 7 cm hole in the middle. The animals which had shown at least 80% avoidance responses during trials were selected for the experiment (Leite, 1978). The selected rats were randomly divided into four groups of six rats per group. Three groups received graded doses of the extract (25–100 mg/kg, p.o.), while the remaining group received normal saline to serve as control. The rats were assessed individually 30 min after pre-treatment with the extract. A sound (conditioned

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stimulus) was presented during 20 s. The unconditioned stimulus was a 0.4 mA scrambled shock for a maximum of 40 s or until the rat escaped to the other compartment of the cage (Leite, 1978; Morais et al., 1998). 2.11. Statistical analysis All the results were expressed as mean ± S.E.M. and differences in means were estimated using ANOVA followed by Dunnet’s post hoc test. Results were considered significant at P < 0.05. 3. Results 3.1. Effect on locomotor activity The extract (25–100 mg/kg, p.o.) produced a significant [F (4, 17) = 6.1; P < 0.05] decrease in the total mobile, and rearing activities, and shortened the mobile time in mice. These effects were dose- and time-dependent (Figs. 1–3). 3.2. Effect on pentobarbital sleeping time The extract (25–100 mg/kg, p.o.) significantly [F (4, 24) = 5.3; P < 0.05] prolonged the duration of pentobarbi-

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Table 1 Effect of Ficus platyphylla on pentobarbital sleeping time in mice Treatment

Dose (mg/kg)

Mean duration of sleep (min)

Normal saline Ficus platyphylla

10 ml/kg 25 50 100 1

70.0 80.0 94.4 125.0 90.0

Diazepam

± ± ± ± ±

1.0 1.4∗ 1.7∗ 2.5∗ 1.5∗

Values are expressed as mean±S.E.M., N = 5. Asterisks denote statistical significance between control and treated group [F (4, 24) = 5.3; P < 0.05].

tal sleeping time in rats, with no effect on the onset of sleep. The increase in duration of sleep was dose-dependent. Diazepam also prolonged the duration of pentobarbital sleeping time in rats (Table 1). 3.3. Effect on exploratory behaviour The extract (25–100 mg/kg, p.o.) exhibited a significant [F (4, 24) = 6.8; P < 0.05] and dose-dependent decrease in exploratory activity as indicated in the hole-board experiment. Similarly, clonazepam caused a decrease in the number of head dips in the hole-board experiment (Fig. 4).

Fig. 1. Effect of the methanol extract of Ficus platyphylla on mobile counts in mice administered orally at doses of 25 mg/kg (䊏), 50 mg/kg (䉱) and 100 mg/kg (䊉). Control group received normal saline at a dose of 10 ml/kg (䉬). ∗ P < 0.05, N = 6.

Fig. 2. Effect of the methanol extract of Ficus platyphylla on rearing counts in mice administered orally at doses of 25 mg/kg (䊏), 50 mg/kg (䉱) and 100 mg/kg (䊉). Control group received normal saline at a dose of 10 ml/kg (䉬). ∗ P < 0.05, N = 6.

Fig. 3. Effect of the methanol extract of Ficus platyphylla on mobile time in mice administered orally at doses of 25 mg/kg (䊏), 50 mg/kg (䉱) and 100 mg/kg (䊉). Control group received normal saline at a dose of 10 ml/kg (䉬). ∗ P < 0.05, N = 6.

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Fig. 4. Effect of the methanol extract of Ficus platyphylla on exploratory behaviour in mice administered orally at doses of 25 mg/kg ( ), 50 mg/kg ( ) and 100 mg/kg ( ). Control group received normal saline at a dose of 10 ml/kg ( ) and clonazepam (0.2 mg/kg, ). ∗ P < 0.05, N = 5.

Fig. 5. Effect of the methanol extract of Ficus platyphylla on apomorphine-induced stereotype behaviour in mice administered orally at doses of 25 mg/kg ( ), 50 mg/kg ( ) and 100 mg/kg ( ). Control group received normal saline at a dose of 10 ml/kg ( ), chlopromazine (2 mg/kg, ). ∗ P < 0.05, N = 6.

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Table 2 Effect of Ficus platyphylla on amphetamine-induced hyperactivity in mice Treatment Mean activity counts Amphetamine Ficus platyphylla

Dose (mg/kg)

0 360 ± 12.2

2

350.2 ± 19.4 345.8 ± 18.2 355.4 ± 18.7

25 50 100

15

30

60

90

574.5 ± 25.3

624.1 ± 26.4

721.0 ± 31

695.4 ± 27.9

298.2 ± 21.0 85.3 ± 15.6∗ 67.3 ± 12.5∗

248.7 ± 23.5 68.4 ± 16.7∗ 50.4 ± 11.2∗

135.2 ± 20.1 47.5 ± 12.4∗ 41.2 ± 13.0∗

120

19∗

102.3 ± 40.6 ± 14.0∗ 30.1 ± 9.8∗

705.4 ± 29.3 95.6 ± 17.6∗ 31.2 ± 10∗ 24.8 ± 8.5∗

Values are expressed as mean ± S.E.M., N = 6. Asterisks denote statistical difference between control and treated groups [F (3, 23) = 4.6; P < 0.05].

Table 3 Effect of Ficus platyphylla on active-avoidance test Drug Normal saline Ficus platyphylla

Dose (mg/kg) 10 ml/kg 25 50 100

Number of escape

Avoidance counts

12.75 ± 2.0

14.75 ± 4.3

11.25 ± 4.0 11.5 ± 4.2 10.0 ± 3.3

2.7∗

8.75 ± 3.25 ± 0.5∗ 0.75 ± 0.25∗

Percent active-avoidance – 59.3 22.0 5.1

Values are expressed as mean ± S.E.M., N = 6. Asterisks denote statistical difference between control and treated groups [F (3, 23) = 4.75; P < 0.05].

3.4. Effect on motor co-ordination The extract (25–100 mg/kg, p.o.) did not produce any significant effect on the motor co-ordination as determined by the treadmill performance. All the mice stayed on the rota-rod for longer than 180 s (data not shown). 3.5. Effect on amphetamine-induced hyperactivity The extract (25–100 mg/kg, p.o.) significantly [F (3, 23) = 4.6; P < 0.05] attenuated amphetamine-induced hyperactivity in mice. The effect was dose- and time-dependent (Table 2). 3.6. Effect on apomorphine-induced stereotype behaviour The extract (25–100 mg/kg, p.o.) significantly [F (4, 29) = 7.3; P < 0.05] attenuated apomorphine-induced stereotyped behaviour in mice dose-dependently. This effect was similar to that produced by chlorpromazine (Fig. 5). 3.7. Effect on active-avoidance The extract (25–100 mg/kg, p.o.) significantly [F (3, 23) = 4.74; P < 0.05] decreased the conditioned avoidance response in rats when compared to control. The extract had no significant effect on the escape response (Table 3).

4. Discussion and conclusion This study provided evidence that the methanolic extract of F. platyphylla (F.P.) stem bark may contain psychoactive substances that are sedative in nature. The extract significantly decreased the mobile and rearing activities, and

the mobile time in mice, indicating a central depressant effect. According to Masur et al. (1971) and Morais et al. (1998), mobile and rearing activities are functions of CNS excitability, and decrease in these parameters is suggestive of sedative property (Ozturk et al., 1996). The central depressant activity of the extract was confirmed by its ability to potentiate the pentobarbital-induced sleep, which may be attributed to an action on the central mechanisms involved in the regulation of sleep (N’Gouemo et al., 1994; Amos et al., 2001c) or an inhibition of pentobarbital metabolism (Kaul and Kulkarni, 1978). The hole-board test is a measure of exploratory behaviour (Crawley, 1985). An agent that decreases this parameter reveals a sedative behaviour (File and Pellow, 1985). Anxiolytics have been shown to increase the number of head dips in the hole-board test (Takeda et al., 1998). The extract remarkably diminished the exploratory behaviour in mice, dose-dependently, thereby suggesting that the extract possess sedative activity rather than anxiolytic potentials. Amphetamine induces hyperactivity by the release of dopamine from the dopaminergic nerve terminals particularly at the striatal pathways in the mesolimbic system (Hoffman and Lefkowitz, 1996). This behavioural effect of amphetamine could be masked by neuroleptics, most of which are believed to be dopamine (D2 ) receptor antagonists (Anca et al., 1993; Chindo, 1999). The effect of the extract against amphetamine-induced hyperactivity suggests a possible interference with the central dopaminergic neurotransmission. The antidopaminergic property of the extract was confirmed by its ability to antagonise apomorphine-induced stereotypy. Agents, which inhibit apomorphine-induced stereotypy, can antagonise dopamine receptors in the nigrostriatal system (Tarsy and Baldessarini, 1986). The effect of the extract against apomorphine may be correlated with its neuroleptic potentials. The neuroleptic

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potential of the extract was confirmed by the results in which it inhibited active-avoidance responses without affecting the escape responses to electric shock. This behaviour differentiates neutoleptics from non-selective depressants, which inhibit both responses (Morais et al., 1998; Baldessarini, 1996). The extract had no effect on the motor co-ordination. All the mice stayed on the rota-rod for longer than 180 s, suggesting that the inhibitory effect of the extract might be elicited via central mechanisms, not by peripheral neuromuscular blockade (Perez et al., 1998; Amos et al., 2001b). The therapeutic benefits of traditional remedies are often attributed to a combination of active constituents (Amos et al., 2001c). For instance, saponins are known to have antagonistic activity against amphetamine, sedative property, and decrease spontaneous motor activity in experimental animals (Wagner et al., 1983; Dubois et al., 1986). It is therefore, probable that the saponins component of the extract might contribute in part for the observed pharmacological activities. We may therefore, conclude that the methanol extract of F. platyphylla contains psychoactive principles that are sedative in nature with possible neuroleptic properties. Further studies are in progress in our laboratory to isolate the useful active components.

Acknowledgements This work was supported by a grant from NIPRD, Abuja, Nigeria. The authors acknowledge the secretariat assistance of Charles Balogun.

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