Journal of Ethnopharmacology 97 (2005) 5357

Neuropharmacological effects of the aqueous extract of Nauclea latifolia root bark in rats and mice
S. Amosa, , J. Abbaha , B. Chindoa , I. Edmonda , L. Bindaa , B. Adzua , S. Buharib , A.A. Odutolab , C. Wambebea , K. Gamaniela
a

Department of Pharmacology and Toxicology, National Institute for Pharmaceutical Research and Development (NIPRD), Idu Industrial Area, P. M. B. 21, Garki, Abuja, Nigeria b Department of Pharmacology and Clinical Pharmacy, Ahmadu Bello University, Zaria, Nigeria Received 9 October 2002; received in revised form 19 January 2004; accepted 6 October 2004 Available online 13 December 2004

Abstract The present study evaluated the neuropharmacological effects of the aqueous extract of Nauclea latifolia root bark in rodents. Effects on the spontaneous motor activity (SMA), exploratory behaviour, pentobarbital sleeping time, apomorphine-induced stereotypic behaviour and motor coordination (rota-rod performance) were investigated. The extract (50200 mg/kg p.o.) signicantly (P < 0.05) decreased the SMA and exploratory behaviour in mice and prolonged pentobarbital sleeping time in rats dose-dependently. The extract also remarkably attenuated the intensity of apomorphine-induced stereotypy dose-dependently in mice, but had no effect on motor coordination as determined by the performance on rota-rod. These results indicate the presence of psychoactive substances in the aqueous extract of the root bark of Nauclea latifolia. 2004 Elsevier Ireland Ltd. All rights reserved.
Keywords: Nauclea latifolia; Exploratory behaviour; Spontaneous motor activity; Pentobarbital sleep; Apomorphine-induced stereotypic behaviour; Motor co-ordination

1. Introduction Nauclea laltifolia Smith (family: Rubiaceae) occur wildly in Savanna forests of continental Africa where it is known as African peach or African g. The plant has been used by the natives of East and West Africa in traditional medicine for treatment of various ailments (Dalziel, 1957). For instance, the pulverized root and bark have been used to treat sores (as topical application by washing of the affected parts) and gonorrhoea in Sudan, Ghana, Ivory Coast and Nigeria. Similarly, a decoction of the root bark is commonly employed in the treatment of stomach disorders, cough and, in particular, malaria fever (Irvine, 1961). In malaria ethnopharmacy, the root bark is the preferred part of the plant used. This part is

Corresponding author. Tel.: +234 9 5232459; fax: ++234 9 5231043. E-mail address: samsonamos@yahoo.co.uk (S. Amos).

usually harvested, sun dried and pulverized to obtain a powder. About 250 g of the powdered material is macerated in gin (local alcoholic beverage) or hot water and administered twice daily for 7 days. Clinically, on a pilot scale, the freeze-dried cold-water extract is formulated into capsules and administered orally at a dose range of 1625 mg/kg. Previous scientic studies of the plant revealed that both the methanol and ethanol extracts of the dried fruit, stem and root bark possess spasmolytic and anti-bacterial activity (Ogunlana and Ramstad, 1975). The hot water extract of the root and stem bark of the plant was found to be active against Plasmodium falciparum in vitro (Gbeassor et al., 1989). Further investigations in our laboratory revealed that the cold water extracts of the leaves of Nauclea latifolia as well as some of its fractions were active against Plasmodium berghei in vivo in addition to their spasmolytic effects (Gamaniel et al., 1997). Udoh et al. (1998) reported the cardiovascular effect of the root and leaf extract of Nauclea latifolia. Recent

0378-8741/$ see front matter 2004 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.jep.2004.10.003

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investigations corroborate the spasmolytic, anti-plasmodial and anti-parasitic effects of the plant (Benoit-Vical et al., 1998; Traore-Keita et al., 2000; Fakae et al., 2000; Onyeyili et al., 2000; Tona et al., 2000). These activities support the traditional notion of the use of this plant. As a step towards development and eventual introduction into clinical use as anti-malarial, we set out to investigate possible central nervous system (CNS) effect of high acute doses of Nauclea latifolia using mice and rats. The results will provide additional safety pharmacology data on the aqueous extract of Nauclea latifolia.

placed on LETICA (Spain) instrument with 16 evenly spaced holes and a counter (LE 3333) before and at 30 and 60 min post-treatment. The number of times mice dipped their heads into the holes during the 5 min trial was automatically counted both for control and treated groups. 2.4.2. Studies on spontaneous motor activity (SMA) Adult mice of either sex were randomly divided into four groups of six mice per group. The base line activity counts of all the animals were taken twice. Three groups were given graded doses of the extract (50, 100 and 200 mg/kg p.o.). Animals in the remaining group received normal saline (20 ml/kg p.o.). Thirty minutes post-treatment, the animals were transferred individually to Letica activity cages (LE 886) connected to a multicounter (LE 3806) and after 1 min latency, activity counts were recorded for 6 min at 30, 60, 90 and 120 min (Amos et al., 2001b). 2.4.3. Studies on pentobarbital-induced sleep in rat The effect of the extract on pentobarbital sleeping time was performed in ve groups of rats (n = 5). Three groups received graded doses of the extract (50, 100 and 20 mg/kg p.o.). One group received diazepam (1 mg/kg i.p.), while animals in the control group were administered normal saline (20 ml/kg p.o.). Thirty minutes post-treatment, pentobarbital sodium (35 mg/kg) was administered i.p. to each rat. The onset and duration of sleep for each rat was recorded, the criterion for sleep being loss of righting reex (Wambebe, 1985; Ramirez et al., 1998). 2.4.4. Apomorphine-induced stereotypic behaviour The effect of the extract on apomorphine-induced stereotypic behaviour was investigated as described by Kenneth and Kenneth (1984). Briey, three groups of mice of either sex (n = 6) were administered graded doses of the extract (50, 100 and 200 mg/kg p.o.), while animals in the control group received normal saline (20 ml/kg p.o.). Thirty minutes later, apomorphine (2 mg/kg i.p.) was administered to each mouse. Signs of stereotypic behaviour, which include mainly sniffing and gnawing, were observed and rated. The stereotypic episodes were scored as follows: absence of stereotypy (0); occasional snifng (1); occasional snifng with occasional gnawing (2); frequent gnawing (3); intense continuous gnawing (4); intense gnawing and staying on the same spot (5). The stereotypic behaviour was measured and scored after every minute and mean of 5 min period was calculated and recorded. 2.4.5. Studies on motor coordination (rota-rod performance) This test was performed using a horizontal rotating rod (Ugo Basile 7560, Milano, Italy) set at a rate of 16 revolutions per minute. Mice that were able to remain on the rod longer than 3 min were selected and grouped into four (n = 6). Groups IIII received the extract at doses of 50, 100 and 200 mg/kg p.o., respectively. Animals in the remaining

2. Materials and methods 2.1. Plant material and preparation of extract The roots of Nauclea latifolia were collected from Idu, in the Federal Capital Territory, Abuja, Nigeria for this study. Taxonomic identication was established by Mallam Muazzam Ibrahim, of the National Institute for Pharmaceutical Research and Development Herbarium, Abuja where voucher (Number 4251) specimens were preserved for reference. The root bark was separated and cleaned, then sun-dried and pulverized using a mechanical grinder. The powdered material was extracted with distilled water by cold maceration for 24 h, then ltered through Whatman no. 1 lter paper and freezedried using LYOVAC, GT2 (Germany). This gave a yield of 9.50% (w/w). The freeze-dried extract was then subsequently reconstituted in distilled water at appropriate concentrations for the various experiments. 2.2. Drugs Apomorphine hydrochloride, pentobarbital sodium, diazepam (Sigma Chemical Co., USA), nitrazepam (Roche, Nigeria), sodium chloride (Fisher Scientic Co. USA). 2.3. Animals Swiss albino mice (1825 g) and adult Wistar rats (200250 g) of either sex were maintained at the Animal Facility Centre (AFC), NIPRD, under standard environmental condition of temperature (22 3), relative humidity (14 1) and light/dark cycles (12/12 h). The animals were fed with Ladokun Feeds, Ibadan, Nigeria and water ad libitum. 2.4. Pharmacological evaluation 2.4.1. Studies on exploratory behaviour The head dip test procedure of Perez et al. (1998) was used for this study. Mice were divided into ve (5) groups (n = 6). Three Groups received graded doses of the extract (50, 100 and 200 mg/kg p.o). One group received nitrazepam (2 mg/kg i.p.) and the remaining group received normal saline (20 ml/kg p.o.) to serve as control. The animals were singly

S. Amos et al. / Journal of Ethnopharmacology 97 (2005) 5357 Table 1 Effects of the aqueous extract of Nauclea latifolia root bark on exploratory behaviour in mice Treatment (p.o.) Mean head-dips S.E.M. 0 min Normal saline (20 ml/kg) Extract (mg/kg) 50 100 200 Nitrazepam (2 mg/kg) 69.60 5.80 63.40 9.30 63.60 6.70 82.00 9.90 75.20 5.40 30 min post-treatment 68.40 5.00 36.80 2.40a 26.00 3.50a 20.10 4.50a 25.30 2.20a

55

1 h post-treatment 67.90 5.10 13.30 4.70a 10.00 4.90a 8.20 3.50a 9.40 4.20a

Values are expressed as mean S.E.M. (n = 6). a Statistical difference between treated and control groups[F(4, 25) = 7.81; P < 0.05)]. Table 2 Effects of the aqueous extract of Nauclea latifolia root bark on spontaneous motor activity in mice Time (post-treatment; minute) Locomotor count S.E.M. Control Pre-dose 0 30 60 90 120 512.50 354.00 139.00 145.00 219.50 9.42 5.94 4.28 3.40 3.91 50a 510.50 68.30 41.30 49.70 50.00 7.55 8.24b 8.87b 5.52b 5.31b 100a 518.50 45.50 24.00 27.60 42.30 6.48 2.80b 5.81b 2.93b 3.12b 200a 521.60 23.50 15.00 10.50 8.50 7.58 1.77b 1.07b 0.35b 0.43b

Values are expressed as mean S.E.M. (n = 6). a Extract in mg/kg. b Statistical difference between treated and control groups[F(4, 25) = 7.62; P < 0.05)].

group were administered normal saline (20 ml/kg i.p.). Mice were placed on the rota-rod 30 min post-treatment and at intervals of 30 min for a total of 120 min, The length of time taken before the animal fell from the rod during the 3 min trial was recorded in seconds (Adzu et al., 2002). 2.4.6. Data analysis Results were expressed as mean S.E.M. Differences were estimated by means of both one and two-ways analysis of variance (ANOVA) followed by Dunnets test for multiple comparisons. Effects were considered signicant at P < 0.05.

tion in SMA was dose and time related. Peak effects occurred between 60 and 90 min for all the treated groups. (Table 2) 3.3. Studies on pentobarbital-induced sleeping time The extract (50200 mg/kg p.o.) prolonged the duration of pentobarbital-induced sleep in rats dose-dependently. The onset of sleep was also shortened in animals treated with the extract. The effect of the extract on duration of sleep was signicantly [F(4, 20) = 4.76; P < 0.05)] different from that of control (Table 3). 3.4. Apomorphine-induced stereotypic behaviour

3. Results 3.1. Studies on exploratory behaviour The extract (50200 mg/kg p.o.) caused a dose-dependent reduction in head-dip response in the mice. The reduction in head-dip was also time dependent, with the effect at 60 min being more pronounced. Similarly nitrazepam caused a decrease in the frequency of head-dips. The observed effects in the treated groups were signicantly [F(4, 25) = 7.81; P < 0.05)] different from that of the control (Table 1). 3.2. Studies on spontaneous motor activity The extract (50200 mg/kg p.o.) signicantly [F(3, 20) = 7.62; P < 0.05)] reduced the SMA in mice. The reduc-

The extract (50200 mg/kg p.o.), in a dose-dependent manner, reduced the intensity of apomorphine-induced
Table 3 Effects of the aqueous extract of Nauclea latifolia root bark on pentobarbitalinduced sleeping time in rats Treatment Control (20 ml/kg) Extract (mg/kg) 50 100 200 Diazepam (1 mg/kg) Onset of sleep (min) 4.70 0.35 2.00 0.00 2.00 0.00 2.00 0.00 2.00 0.00 Duration of sleep (min) 67.00 7.07 113.00 22.63a 150.50 13.08a 180.50 10.20a 98.60 7.50a

Values are expressed as mean S.E.M. (n = 5). a Statistical difference between treated and control groups [F(4, 25) = 4.76; P < 0.05)].

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Table 4 Effects of the aqueous extract of Nauclea latifolia root bark on apomorphineinduced stereotypic behaviour in mice Treatment (p.o.) Control Extract (mg/kg) 50 100 200 Means score per 5 min S.E.M. 4.25 0.40 2.75 0.20a 1.67 0.30a 1.00 0.25a

Values are expressed as mean S.E.M. (n = 6). a Statistical difference between treated and control groups [F(4, 25) = 6.75; P < 0.05)].

stereotypic behaviour, P < 0.05)] (Table 4).

signicantly

[F(3,

19) = 6.75;

3.5. Studies on motor coordination (rota-rod) The extract (50200 mg/kg p.o.) did not exhibit signicant effect on the rota-rod performance of the mice as all the animals stayed on the rod for 180 s (3 min) without falling (data not shown).

4. Discussion The results of this study generally indicate that high acute doses of the aqueous extract of Nauclea latifolia possess sedative properties. This is shown by the marked reduction in locomotor activity in mice in the SMA test. This model has been used in laboratory animals to evaluate the gross behavioural effects of drugs (Hsieh et al., 1991; File and Fernandes, 1994). The model measures the level of the excitability of the CNS (Masur et al., 1971), which correlates well with drug effects in humans. Agents that suppress this behaviour were known to do so through central inhibition (Adzu et al., 2002). Similarly, the extract signicantly diminished the exploratory behaviour in mice as demonstrated by the reduction of the head-dip test. The test is a measure of exploratory behaviour (File and Wardill, 1975; Crawley, 1985) that reveals sedative activity of agents (File and Pellow, 1985; Amos et al., 2001b). It has been established that anxiolytics increase the number of head-dips (Takeda et al., 1998). The effect of the extract is therefore suggestive of a sedative activity rather than anxiolytic potentials. Further evidence of the central depressant activity of the extract is provided by the extract ability to potentiate pentobarbital-induced hypnosis, an effect that may be attributed to an action on the central mechanisms involved in the regulation of sleep (NGouemo et al., 1994; Chindo et al., 2003) or an inhibition of pentobarbital metabolism (Kaul and Kulkarni, 1978). It is generally accepted that the sedative effects of drugs can be evaluated by measurement of pentobarbital sleeping time in laboratory animals (Lu, 1998; Carpendo et al., 1994; Gamaniel et al., 1998). The extract prolongation of pentobarbital sleep corroborate the observa-

tion of Fujimori, 1965 who proposed that prolongation of barbital hypnosis is a good index of central nervous system depressant activity. Apomorphine, according to Stolk and Rech (1970), acts directly on the post-synaptic dopamine D-2 receptors to induce hyperactivity and stereotypic behaviour. The effect of the extract against apomorphine is therefore suggestive of possible interference with central dopaminergic neurotransmission. Furthermore, the inability of the extract to affect motor coordination is additional evidence of centrally mediated actions and not blockade of neuromuscular system (Perez et al., 1998; Amos et al., 2001a). The efcacy of most herbal remedies is attributed to various active principles in combination. For instance, saponins have been shown to have antagonistic activity against amphetamine, sedative property, and decrease spontaneous activity in experimental animals (Wagner et al., 1983; Dubois et al., 1986). It is therefore probable that the saponins that are present in abundance in the extract might contribute in part for the observed CNS effects. This nding could be of immense clinical benet especially in the management of some malaria patients that may present with agitation and acute toxicity resulting from over dose administration of the plant extract by traditional healers. Based on the above studies we concluded that the aqueous extract of Nauclea latifolia might contain some psychoactive principles, which are sedative in nature. Further studies to identify and isolate the active components are in progress.

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