Journal of Ethnopharmacology 143 (2012) 213–220 Contents lists available at SciVerse ScienceDirect Journal of Ethnopharmacology journal homepage: www.elsevier.com/locate/jep Antipsychotic and sedative effects of the leaf extract of Crassocephalum bauchiense (Hutch.) Milne-Redh (Asteraceae) in rodents Germain Sotoing Taı̈we a,b,c,n, Elisabeth Ngo Bum d, Emmanuel Talla e, Amadou Dawe f, Fleur Clarisse Okomolo Moto d,g, Gwladys Temkou Ngoupaye d,h, Neteydji Sidiki d, Bernard Dabole e,i, Paul Désiré Djomeni Dzeufiet b, Théophile Dimo b, Michel De Waard c,j a Department of Zoology and Animal Physiology, Faculty of Science, University of Buea, P.O. Box 63 Buea, Cameroon Department of Animal Biology and Physiology, Faculty of Science, University of Yaoundé I, P.O. Box 812, Yaoundé, Cameroon c Institut National de la Santé et de la Recherche Médicale, Unité 836, Laboratoire Canaux Calciques, Fonctions et Pathologies, Grenoble Institut de Neurosciences, Université Joseph Fourier, Chemin Fortuné Ferrini, Site santé de la Tronche, P.O. Box 170, 38042 Cedex 9, Grenoble, France d Department of Biological Sciences, Faculty of Science, University of Ngaoundéré, P.O. Box 454, Ngaoundéré, Cameroon e Department of Chemistry, Faculty of Science, University of Ngaoundéré, P.O. Box 454, Ngaoundéré, Cameroon f Higher Teachers’ Training College, University of Maroua, P.O. Box 55, Maroua, Cameroon g Higher Teachers’ Training College, University of Yaoundé I, P.O. Box 47, Yaoundé, Cameroon h Department of Animal Biology, Faculty of Science, University of Dschang, P.O. Box 67, Dschang, Cameroon i Department of Organic Chemistry, Faculty of Science, University of Yaoundé I, P.O. Box 812, Yaoundé, Cameroon j Smartox Biotechnologies, Floralis, Biopolis, 5 Avenue du Grand Sablon, 38700 La Tronche, France b a r t i c l e i n f o abstract Article history: Received 19 April 2012 Received in revised form 15 June 2012 Accepted 16 June 2012 Available online 27 June 2012 Ethnopharmacological relevance: Crassocephalum bauchiense (Hutch.) Milne-Redh (Asteraceae) has been used as a medicine for the treatment of epilepsy, insomnia, dementia and psychotic disorders in Cameroonian traditional medicine. Aim of the study: This study was designed to examine whether the aqueous extract and the alkaloid fraction prepared from the leaves of Crassocephalum bauchiense possess antipsychotic and sedative properties in rodents. Materials and methods: The rectal temperature of mice was recorded with a probe thermometer at a constant depth. Novelty-induced rearing behavior is used to evaluate a central excitatory locomotor behavior in mice. The antipsychotic effects of the extracts were assessed using the apomorphine animal model of psychosis. The catalepsy test was tested based on the ability of the leaves extracts of Crassocephalum bauchiense to alter the duration of akinesia by placing the naive mice with both forelegs over a horizontal bar. The extracts of Crassocephalum bauchiense effects were evaluated on sodium pentobarbital-induced sleeping time. In addition, gamma-aminobutyric acid concentrations in the brain treated mice were also estimated. Results: The aqueous extract and the alkaloid fraction from Crassocephalum bauchiense caused dosedependent inhibition of novelty-induced rearing behavior, decreased the apomorphine-induced stereotypy and fighting, and had significant fall of the body temperature. The aqueous extract prolonged the sodium pentobarbital sleeping time. This prolongation was not reversed by bicuculline, a light-sensitive competitive antagonist of GABAA receptors complex. However, the effect of the aqueous extract on sodium pentobarbital-induced sleeping time was blocked by N-methyl-b-carboline3-carboxamide, a partial inverse agonist of the benzodiazepine site in the GABAA receptor complex and flumazenil, a specific antagonist of the benzodiazepine site in the GABAA receptor complex. In biochemical experiments, the concentration of the inhibitory amino acid, gamma-aminobutyric acid, was significantly increased in the brain of animals treated with the aqueous extract of Crassocephalum bauchiense and sodium valproate. Conclusions: The results show that the antipsychotic and sedative properties of Crassocephalum bauchiense are possibly mediated via the blockade of dopamine D-2 receptors and GABAergic activation, Keywords: Crassocephalum bauchiense Antipsychotic Sedative Brain Traditional medicine Abbreviations: ANOVA, analysis of variance; AE, aqueous extract; AF, alkaloid fraction; BIC, bicuculine; CPZ, chlorpromazine; DZP, diazepam; FG 7142, N-methyl- b-carboline-3-carboxamide; GABA, gamma-aminobutyric acid; ID50, dose of extract necessary to reduce the response by 50% relative to the control value; NIH, National Institutes of Health; RO 151788, flumazenil; S.E.M, standard error of the means; SV, sodium valproate n Corresponding author at: Department of Zoology and Animal Physiology, Faculty of Science, University of Buea, P.O. Box 63, Buea, Cameroon. Tel.: þ237 77 71 86 70; fax: þ 237 22 15 73 70. E-mail address: taiwe_sotoing@yahoo.fr (G. Sotoing Taı̈we). 0378-8741/$ - see front matter & 2012 Elsevier Ireland Ltd. All rights reserved. http://dx.doi.org/10.1016/j.jep.2012.06.026 214 G. Sotoing Taı̈we et al. / Journal of Ethnopharmacology 143 (2012) 213–220 respectively. However, pharmacological and chemical studies are continuing in order to characterize the mechanism(s) responsible for these neuropharmacological actions and also to identify the active substances present in the extracts of Crassocephalum bauchiense. & 2012 Elsevier Ireland Ltd. All rights reserved. 1. Introduction Psychosis is a chronic recurrent neuropsychiatric disorder that affects the quality of life of the sufferers and is a major public health concerns (Ehmann et al., 2004). Although the etiology of the disease is unknown, hyperdopaminergic activity is closely linked with the pathogenesis of psychosis (Harrison, 1999). Individuals with psychoses are more prone to suicide, depression, anxiety, aggression, substance abuse, cognitive impairment, victimization, poverty and increased medical problems (Mullen, 2006). Current drugs used for the disorder have failed to alter the course of the disease but only provide symptomatic relief (Baldessarini, 2001; Davis et al., 1991). Adverse events can affect every system of the body and range from the annoying photosensitivity and jaundice, for example to the disabling seizures and blindness, among others to the potentially fatal agranulocytosis and neuroleptic malignant syndrome (Arana, 2000). Moreover, the overall functional and quality of life outcomes of patients still remain poor after treatment (Mullen, 2006) and the clinical efficacy of these drugs is largely limited by adverse effects associated with their use (Ray et al., 2009). Thus, there is a critical need to search for more effective and less toxic agents for the treatment of the disease. An increasing number of herbal products have been introduced into psychiatric practice, as alternative or complementary medicines, and also there is a large number of herbal medicines whose therapeutic potential has been assessed in a variety of animal models (Zhang, 2004). Crassocephalum bauchiense (Hutch.) Milne-Redh (Asteraceae) is a medicinal herb reputed to be of beneficial effect in the Cameroonian traditional system of medicine. It is common in the savanna woodland from Nigeria to northern and southern Cameroon, and is generally widespread in tropical Africa (Biholong; 1986; Burkill, 1985). In ‘‘Fulfuldé’’ language, in northern Cameroon it is known as ‘‘Hako kahdam’’ (Arbonnier, 2000). The leaves decoction of Crassocephalum bauchiense is effective in the cases of cerebral deficit, anxiety, epilepsy, cerebral malaria, behavioral disturbances in mentally retarded children (Adjanohoun et al., 1996; Biholong, 1986) and neuropathic pain (Arbonnier, 2000). Similarly, an aqueous extract of the whole plant is commonly employed in the treatment of insomnia, psychosis and other central nervous system disorders (Adjanohoun et al., 1996; Arbonnier, 2000; Biholong, 1986; Dalziel, 1937). Local people of northern and western Cameroon use the Crassocephalum bauchiense extract to relieve toothache and nervousness. It is also used to treat infantile convulsion, cerebral malaria, gastrointestinal infections as well as liver disorders (Arbonnier, 2000; Biholong, 1986; Mouokeu et al., 2011; Taı̈we et al., 2012). According to Cameroonian traditional healers, the leaves of Crassocephalum bauchiense are the preferred part of the plant used for treating epilepsy, insomnia and dementia (Adjanohoun et al., 1996; Arbonnier, 2000; Biholong, 1986). This part is usually harvested, sun dried, and pulverized to obtain powder. About 100 g of the powdered material is macerated in 500 ml of water. Previous works have shown that the ethyl acetate extract from Crassocephalum bauchiense has antibacterial activities against all the tested microorganisms (Mouokeu et al., 2011). A single dermal dose of this extract up to 32 g/kg body weight did not produce any visible sign of toxicity (Mouokeu et al., 2011). Also, daily dermal application of the Crassocephalum bauchiense extract gel formulation for 28 day did not show any negative effect, instead some biochemical parameters such as alanine aminotransferase, aspartate aminotransferase, low density lipoprotein, high density lipoprotein and triglycerides were significantly affected positively (Mouokeu et al., 2011). In the course of pharmacological studies, antinociceptive property of the aqueous extract and the alkaloid fraction prepared from the leaves of Crassocephalum bauchiense have already been reported from our laboratory (Taı̈we et al., 2012). An extensive search of the literature reveals no reports on the psychopharmacological activity of Crassocephalum bauchiense. Therefore, the present work was undertaken in order to investigate whether the aqueous extract of Crassocephalum bauchiense and the alkaloid fraction from the leaves of Crassocephalum bauchiense have antipsychotic and sedative potentials and if it is able to induce behavioral modifications in rodents. 2. Materials and methods 2.1. Plant material Fresh leaves of Crassocephalum bauchiense used in this study were harvested in the Mawi area of Ngaoundéré, Cameroon in July 2007. The species was authenticated and a voucher was deposited at the National Herbarium, Yaoundé (No. 7954/SRF/Cam). Authenticated leaves of Crassocephalum bauchiense were extracted as described elsewhere (Taı̈we et al., 2012). The aqueous extract of Crassocephalum bauchiense (AE, 20, 40, 80 and 160 mg/kg) and the alkaloid fraction no. Fr. XVI, isolated from Crassocephamum bauchiense (AF, 5, 10, 20 and 40 mg/kg), were dissolved in saline 0.9% containing dimethyl sulfoxyde 2% (vehicle) and administered orally in a volume of 10 ml/kg (Taı̈we et al., 2012). 2.2. Preliminary qualitative phytochemical analysis Preliminary phytochemical properties of the aqueous extract and the alkaloid fraction of Crassocephalum bauchiense were tested using the following chemicals and reagents: flavonoids (NaCl and HCl), alkaloids with Mayer and Dragendoff’s reagents, saponins (frothingtest), tannins (FeCl3), glycosides (NaCl3 and Fehling’s solutions A and B), cardiac glycosides (Salkowski test), anthraquinones (Borntrager’s reaction), phenols (FeCl3 and K3Fe(CN)) and lipids (filter paper) (Evans, 2002). 2.3. Drugs Apomorphine, bicuculine (BIC), chlorpromazine (CPZ), flumazenil (RO 151788), N-methyl-b-carboline-3-carboxamide (FG 7142), sodium pentobarbital are from Sigma Chemical, USA. Diazepam (DZP) and sodium valproate (SV) are from Sanofi-Synthelabo. All other chemicals and reagents used in the brain gamma-aminobutyric acid (GABA) content estimation are from Sigma Chemical, USA. These substances were prepared in saline 0.9% containing dimethyl sulfoxyde 2% (vehicle) and administered in a volume of 10 ml/kg. 2.4. Animals The experiments were conducted using male and female Swiss mice (24–26 g). All animals were housed in a controlled environment, with free access to food, water and were maintained on a 12 h lightdark cycle. Each animal was used only once. All experiments were G. Sotoing Taı̈we et al. / Journal of Ethnopharmacology 143 (2012) 213–220 performed according to the Guide for the Care and Use of Laboratory Animal published by the United States National Institutes of Health (NIH publication no. 85–23, revised 1996). Additionally, the study protocol was approved by the Cameroon National Ethical Committee (Ref. no. FW-IRB00001954) for animal handling and experimental procedure. 215 performed 30 and 60 min after administration of the aqueous extract of Crassocephalum bauchiense (20, 40, 80 and 160 mg/kg, p.o.), the alkaloid fraction from Crassocephalum bauchiense (5, 10, 20 and 40 mg/kg, p.o.), chlorpromazine (1 mg/kg; i.p.) or vehicle (10 ml/kg, p.o.). The time during which the mouse maintained the cataleptic position was recorded for up to 300 s, with three attempts allowed to replace the animal over the bar. 2.5. Behavioral testing Tests were performed every day in the light cycle between 7:30 and 12:30 AM with experimentally naive mice. 2.5.1. Primary observation test The behavioral and eventual neurotoxic effects of Crassocephalum bauchiense were first evaluated according to a standardized observation grid similar to that described by Irwin (1968). The animals received orally various doses of the aqueous extract of Crassocephalum bauchiense (20, 40, 80 and 160 mg/kg, p.o.), the alkaloid fraction from Crassocephalum bauchiense (5, 10, 20 and 40 mg/kg, p.o.) or vehicle (10 ml/kg, p.o.). Rectal temperature was recorded with an electronic thermometer at predetermined times in groups of mice before and after (0.5, 1, 2, 3 and 24 h) the administration of either vehicle or the extracts of Crassocephalum bauchiense. Pre-drug recording served as the reference point for the determination of the temperature change (Taı̈we et al., 2011). 2.5.2. Assessment of novelty-induced rearing behavior Novelty-induced rearing was evaluated by placing the animals directly from home cages to a transparent plexiglas cage (45 cm  25 cm  25 cm) containing sawdust. All mice were observed and assessed singly in the plexiglas cage, after administration of the aqueous extract of Crassocephalum bauchiense (20, 40, 80 and 160 mg/kg, p.o.), the alkaloid fraction from Crassocephalum bauchiense (5, 10, 20 and 40 mg/kg, p.o.), chlorpromazine (1 mg/kg; i.p.) or vehicle (10 ml/kg, p.o.). Novelty-induced rearing considered as a central excitatory locomotor behavior (Ajayi and Ukponmwan, 1994; Labella et al., 1979) was counted as the number of times the mouse was standing on its hind limb with its forelimbs against the wall of the observation cage or in the free air. The number of rears was counted for 30 min. 2.5.3. Apomorphine-induced stereotypy and fighting The effect of the extracts on apomorphine-induced stereotypic behavior was investigated as described by Kenneth and Kenneth (1984). Briefly, 10 groups of mice were administered graded doses of the aqueous extract of Crassocephalum bauchiense (20, 40, 80 and 160 mg/kg, p.o.), the alkaloid fraction from Crassocephalum bauchiense (5, 10, 20 and 40 mg/kg, p.o.), chlorpromazine (1 mg/ kg, i.p.) or vehicle (10 ml/kg p.o.). One hour later, apomorphine (2 mg/kg s.c.) was administered to each mouse. Signs of stereotypic behavior, which include mainly sniffing and gnawing, were observed and rated. The stereotypic episodes were scored as follows: absence of stereotypy (0), occasional sniffing (1), occasional sniffing with occasional gnawing (2), frequent gnawing (3), intense continuous gnawing (4), intense gnawing and staying on the same spot (5). The stereotypic behavior was measured and scored after every minute and mean of 5 min period was calculated and recorded (Amos et al., 2005). 2.5.4. Catalepsy test Catalepsy was evaluated according to the standard bar hanging procedure by placing the naive mice with both forelegs over a horizontal bar, elevated 4.5 cm from the floor (Sanberg et al., 1988). Catalepsy was considered finished when the forepaw touched the floor or when the mouse climbed the bar. Measurement was 2.5.5. Sodium pentobarbital-induced sleep The test was performed in ten groups of six mice each. They received the aqueous extract of Crassocephalum bauchiense (20, 40, 80 and 160 mg/kg, p.o.), the alkaloid fraction from Crassocephalum bauchiense (5, 10, 20 and 40 mg/kg, p.o.) while the control group received vehicle (10 ml/kg, p.o.). The animals in the 10th group received diazepam (3 mg/kg, i.p.). One hour later, sodium pentobarbital (42 mg/kg, i.p.) was administered to each mouse to induce sleep. Each mouse was observed for the onset and duration of sleep, with the criterion for sleep being loss of righting reflex. The interval between loss and recovery of righting reflex was used as the index of hypnotic effect (Gonzalez-Trujano et al., 1998). In the antagonistic experiments, N-methyl-b-carboline-3carboxamide (FG 7142, 10 mg/kg, i.p.), a partial inverse agonist of the benzodiazepine site in the GABAA receptor complex, flumazenil (RO151788, 10 mg/kg, i.p.), a specific antagonist of the benzodiazepine site in the GABAA receptor complex and bicuculline (BIC, 5 mg/kg, i.p.), a light-sensitive competitive antagonist of GABAA receptors, were injected 15 min prior to the aqueous extract of Crassocephalum bauchiense (160 mg/kg) or the alkaloid fraction from Crassocephalum bauchiense (40 mg/kg) treatments. 2.5.6. Estimation of brain GABA content The brain GABA level was estimated in groups of mice. The measurement of GABA, based on the method of Lowe et al. (1958) was carried out as follows. Animals were killed by decapitation at predetermined intervals after the administration of the aqueous extract of Crassocephalum bauchiense (20, 40, 80 and 160 mg/kg, p.o.), the alkaloid fraction from Crassocephalum bauchiense (5, 10, 20 and 40 mg/kg, p.o.), vehicle (10 ml/kg, p.o.) or sodium valproate (300 mg/kg, i.p.). The brains were rapidly removed, blotted, weighed and taken in ice cold 5 ml trichloroacetic acid (10% w/v), homogenized and centrifuged at 10,000  g for 10 min at 0 1C. A sample (0.1 ml) of tissue extract was taken in 0.2 ml of 0.14 M ninhydrin solution in 0.5 M carbonate–bicarbonate buffer (pH 9.9) was kept in a water bath at 60 1C for 30 min then cooled and treated with 5 ml of copper tartrate reagent (0.16% disodium carbonate and 0.03% copper sulphate and 0.0329% tartaric acid). After 10 min, the fluorescence reading was taken at 377/451 nm in a spectrofluorimeter. For GABA standards, different amounts (20, 40, 60, 80, 100 mg) mixed with 1.5 mM glutamic acid were dissolved in 0.1 ml 10% trichloroacetic acid (w/v). GABA was determined by the measurement of the formed fluorescent product resulting from the reaction of GABA with ninhydrin in an alkaline medium, in the presence of glutamate (Sutton and Simmonds, 1974). The GABA content in brain was expressed in mg/g of wet brain tissue. 2.6. Data analysis and statistics Data were expressed as mean7standard error of the means (S.E.M.) per group. Statistical differences between control and treated groups were tested by two-way repeated measures analysis of variance (ANOVA), followed by Newman–Keuls post hoc test. The differences were considered significant at Po0.05. The ID50 (dose of extract necessary to reduce the response by 50% relative to the control value) and 95% confidence intervals values were determined by using linear regression. The statistical 216 G. Sotoing Taı̈we et al. / Journal of Ethnopharmacology 143 (2012) 213–220 package used for the analysis was Graphpad Prism 5.01 for Window (Graphpad Prism Software, San Diego, CA, USA). 3. Results 3.1. Preliminary qualitative phytochemical analysis The aqueous extract of Crassocephalum bauchiense contained flavonoids, alkaloids, saponins, tannins, glycosides, cardiac glycosides, anthraquinones and phenols, but not lipids. The alkaloid fraction from Crassocephalum bauchiense, a colorless liquid, showed a positive reaction with Dragendorff’s reagent, indicating the presence of alkaloids; all other phytochemical tests were negative. 3.2. Primary observation test The experiments reported in this paper show that the aqueous extract and the alkaloid fraction from Crassocephalum bauchiense did not acted as a stimulant in the spontaneous motor activity. Pretreatment with the aqueous extract of Crassocephalum bauchiense at the doses of 80 and 160 mg/kg produced a significant fall of body temperature at 0.5 h [F(4, 32) ¼9.57, P o0.01], 1 h [F(4, 28) ¼14.28, Po0.001], and 2 h [F(4, 29) ¼11.43, Po0.01], after treatment of animals. In control animals, no significant variations of rectal temperature were found. The alkaloid fraction from Crassocephalum bauchiense exhibited significant alteration [F(4, 26)¼6.52, Po0.001] of body temperature compared to the control group. At 1 h and 3 h time interval, the hypothermic effect was observed with all doses of the alkaloids fraction from Crassocephalum bauchiense (5–40 mg/kg). The body temperature returned toward basal values after 24 h (Fig. 1). 3.3. Assessment of novelty-induced rearing behavior The data revealed that the aqueous extract of Crassocephalum bauchiense induced a dose-dependent and significant reduction [F(5, 19)¼ 7.43, Po0.001] in novelty-induced rearing activity in mice 1 h after administration, compared to the control group (Fig. 2). The calculated mean ID50 values for oral administration of the aqueous extract of Crassocephalum bauchiense was 87.15 (54.19–102.29) mg/kg. The maximal inhibition is 67.63%. Likewise, as shown in Fig. 2, the alkaloid fraction from Crassocephalum bauchiense given systematically produced equipotent inhibition in novelty-induced rearing behavior. The calculated mean ID50 values and the maximal inhibition were 7.43 (4.81–9.27) mg/kg and 86.50% [F(5, 16)¼26.42, Po0.001], respectively. Chlorpromazine used as positive control, also reduced the rearing behavior in mice, producing significant inhibition of 64.63% [F(5, 24)¼ 18.51, Po0.001]. 3.4. Apomorphine-induced stereotypy and fighting Apomorphine (2 mg/kg, s.c.) produced characteristic stereotyped behavior, which consisted of persistent sniffing, gnawing, intense licking and chewing in control animals. Interestingly in the apomorphine-induced stereotypic behavior in mice, the aqueous extract and the alkaloid fraction prepared from the leaves of Crassocephalum bauchiense reduced the intensity of apomorphine-induced stereotypic behavior, significantly (Fig. 3). The calculated mean ID50 values and the maximal inhibition of the aqueous extract of Crassocephalum bauchiense were 24.13 (17.45–31.14) mg/kg and 72.46% [F(5, 25)¼46.12, Po0.001], respectively. Besides, the best result was obtained with the higher doses (20 and 40 mg/kg) of the alkaloid fraction from Crassocephalum Fig. 1. Influence of the oral treatment with the leaves extracts of Crassocephalum bauchiense on the body temperature in mice. 0: average rectal temperature recorded just before plant extracts or vehicle injection. Results are expressed as mean 7S.E.M. for six animals. Data were analysis by two-way ANOVA, followed by Newman–Keuls post hoc test: aPo 0.05, bP o0.01, cP o 0.001, significantly different compared to the vehicle. bauchiense produced significant reduction in stereotyped. The calculated mean ID50 values for oral administration of the alkaloid fraction from Crassocephalum bauchiense was 17.24 (14.72–22.17) mg/kg. The maximal inhibition is 65.63% [F(5, 36)¼89.21, Po0.001]. The reference drug chlorpromazine (1 mg/kg, i.p.) significantly [F(5, 28)¼43.75, Po0.001] suppressed the stereotyped behavior induced by apomorphine (Fig. 3). 3.5. Catalepsy test The aqueous extract of Crassocephalum bauchiense administration, at the doses of 80 and 160 mg/kg, induced catalepsy at 30 min [F(5, 28) ¼61.43, Po0.05] and 60 min [F(5, 19) ¼49.13, Po0.01], in animals (Fig. 4). The effects of the aqueous extract were comparable with chlorpromazine, a drug that works as a positive control for catalepsy. As can be seen in Fig. 4, intraperitoneal administration of chlorpromazine (1 mg/kg, i.p.) induced catalepsy at 30 min [F(5, 42)¼ 71.28, Po0.001] and 60 min [F(5, 27)¼86.12, Po0.001]. The alkaloid fraction from Crassocephalum bauchiense administered orally did not induce any cataleptic effect either at low doses or at the higher dose at 30 min [F(5, 36)¼64.21, P40.05] and 60 min [F(5, 28) ¼78.50, P40.05], in mice. 3.6. Sodium pentobarbital-induced sleep Animals given sodium pentobarbital (42 mg/kg i.p.) showed loss of writhing reflex within 5 min of administration (Table 1). The administration of the aqueous extract of Crassocephalum bauchiense (80 and 160 mg/kg, p.o.) decreased the latency of G. Sotoing Taı̈we et al. / Journal of Ethnopharmacology 143 (2012) 213–220 Fig. 2. Influence of the oral treatment with the leaves extracts of Crassocephalum bauchiense or chlorpromazine on novelty-induced rearing behavior in mice. Novelty-induced rearing considered as a central excitatory locomotor behavior was counted as the number of times the mouse was standing on its hind limb with its forelimbs against the wall of the observation cage or in the free air. Results are expressed as mean 7 S.E.M. for six animals. Data were analysis by two-way ANOVA, followed by Newman–Keuls post hoc test: aP o0.05, bP o0.01, c P o0.001, significantly different compared to the vehicle. sleep significantly [F(5, 24) ¼59.17, Po0.001]. The aqueous extract (20–160 mg/kg) prolonged the duration of pentobarbital-induced sleep in mice dose dependently. The effect of the aqueous extract on duration of sleep was significantly [F(5, 36) ¼108.43, Po0.001] different from that of control. While the central depressant drug, diazepam (1 mg/kg, i.p.) and the aqueous extract of Crassocephalum bauchiense (20–160 mg/kg), produced an extension of sodium pentobarbital induced sleep, none of the alkaloid fractions from Crassocephalum bauchiense influenced sleeping time significantly [F(5, 32) ¼8.15, P40.05] 60 min after oral administration at the doses of 5, 10, 20 and 40 mg/kg. The potentiating effect of the aqueous extract of Crassocephalum bauchiense on sodium pentobarbital induced sleep and reduced sleep latency effect was abolished by N-methyl-b-carboline-3carboxamide (FG 7142, 10 mg/kg, i.p.), a partial inverse agonist of the benzodiazepine site in the GABAA receptor complex and flumazenil (RO151788, 10 mg/kg, i.p.), a specific antagonist of 217 Fig. 3. Influence of the oral treatment with the leaves extracts of Crassocephalum bauchiense or chlorpromazine on apomorphine-induced stereotypic behavior in mice. The stereotypic episodes were scored as follows: the absence of stereotypy (0); occasional sniffing (1); occasional sniffing with occasional gnawing (2); frequent gnawing (3); intense continuous gnawing (4); intense gnawing and staying on the same spot (5). Results are expressed as mean 7 S.E.M. for six animals. Data were analysis by two-way ANOVA, followed by Newman–Keuls post hoc test: aPo 0.05, bPo 0.01, cPo 0.001, significantly different compared to the vehicle. the benzodiazepine site in the GABAA receptor complex, pretreating 15 min before the extract was given. Pre-treatment with bicuculline (5 mg/kg, i.p.), a light-sensitive competitive antagonist of GABAA receptors, did not prevent the aqueous extract induced sedation but increased the latency of sleep (Table 1). 3.7. Estimation of brain GABA content A significant increase in the level of brain GABA concentration in animals treated with the aqueous extract of Crassocephalum bauchiense (160 mg/kg, p.o.) [F(6, 33) ¼42.14, Po0.01] and sodium valproate (300 mg/kg, i.p.) [F(6, 26) ¼92.45, Po0.001], a positive control, was observed 1 h after oral administration (Table 2). The systemic administration of the alkaloid fraction from Crassocephalum bauchiense (5–40 mg/kg, p.o.) did not produce any significant effect [F(6, 30)¼15.19, P40.05] in the level of brain GABA concentration in animals (Table 2). 218 G. Sotoing Taı̈we et al. / Journal of Ethnopharmacology 143 (2012) 213–220 Fig. 4. Influence of the oral treatment with the leaves extracts of Crassocephalum bauchiense or chlorpromazine on catalepsy in mice. The time during which the mouse maintained the cataleptic position was recorded for up 300 s, with three attempts allowed to replace the animal over the bar. Results are expressed as mean 7 S.E.M. for six animals. Data were analysis by two-way ANOVA, followed by Newman–Keuls post hoc test. cP o 0.001, significantly different compared to the vehicle. 4. Discussions The primary observation test permits a preliminary orientation concerning neurobehavior and eventual psychotropic activity. The experiments reported in this paper show that the aqueous extract and the alkaloid fraction prepared from the leaves of Crassocephalum bauchiense exhibited significant alteration of body temperature in animals treated. The hypothermia observed in the present studies after oral administration of the extracts of Crassocephalum bauchiense suggests an implication of both central and peripheral mechanisms. This is not surprising since it is well known that certain psychoactive central nervous system depressant drugs (e.g. antipsychotics) reduce temperature both in normal and pyretic conditions (Baldessarini, 1985; Bradley, 1989), while other psychoactive central nervous system depressant drugs (e.g. diazepam) are devoid of any such actions (Bartholini et al., 1973; Taı̈we et al., 2011). The extracts of Crassocephalum bauchiense did not modify the performance of animals in the rotarod test which measures the capacity of animals to maintain their balance (Taı̈we et al., 2012). If preceded by a training period, the test is particularly effective for detecting ataxic or neuroleptic effects (Duncan et al., 1985). Positive or negative findings in these tests are worthy of interest and merit confirmation using other behavioral procedures, because of their lack of specificity. The aqueous extract and the alkaloid fraction prepared from the leaves of Crassocephalum bauchiense decreased the novelty induced rearing behavior in mice. On exposure to a new environment, mice displayed novelty-induced behavior syndrome consisting of rearing, grooming and wet-dog-sakes. In this study, novelty-induced rearing which is a measure of a central nervous system excitation (Labella et al., 1979a,b) was used to test the sedative properties of the extracts. This inhibition in noveltyinduced rearing behavior suggests that the extracts of Crassocephalum bauchiense possess a central nervous action (Lu, 1998; Hellion-lbarrola et al., 1999). The novelty-induced rearing behavior response is regulated by multiple neurotransmitter systems; such transmitters include GABAA, opioid and dopamine D-2 receptors (Walting, 1998). Psychosis has been linked to increased dopaminergic and serotonergic neurotransmissions, and both preclinical and clinical investigations have confirmed their role in the development and treatment of the disease (Baldessarini, 2001; Szechtman, 1986). Stereotyped behavior is one of the prominent features of psychotic symptoms, and in humans it manifests itself as repetitive performance of strange gestures or asking the same questions or making the same kind of comments (Davis et al., 1991). Apomorphine activates post-synaptic dopamine D-2 receptors in the brain directly, and by this mechanism low doses of apomorphine (2 mg/kg, s.c.) increase locomotor activity and produce stereotyped behavior, resulting in a restricted and persevering behavioral pattern (Stolk and Rech, 1970). Antagonism of all three classic effects of a low dose of apomorphine (stereotypes, verticalization and hypothermia) indicates neuroleptic activity. The effect of the aqueous extract and the alkaloid fraction prepared from the leaves of Crassocephalum bauchiense against apomorphine is therefore suggestive of possible interference with central dopaminergic neurotransmission. Furthermore, the inability of the extracts to affect motor coordination is additional evidence of centrally mediated actions and not blockade of neuromuscular system (Perez et al., 1998; Taı̈we et al., 2012). The catalepsy test has been used to predict major tranquillizer activity (Sanberg et al., 1988) as well as to evaluate motor effects of drugs, particularly those related to the extra-pyramidal system. Catalepsy is one of the major adverse effects associated with the use of conventional antipsychotic drugs (Shopsin et al., 1979). Thus, the test for cataleptic behavior forms an integral component involved in the discovery and development of antipsychotic drugs (Zetler, 1981). In this test, the alkaloid fraction from Crassocephalum bauchiense did not produce a cataleptic effect, as measured by the duration of akinesia in mice. In contrast, chlorpromazine, a typical antipsychotic drug and the aqueous extract of Crassocephalum bauchiense, produced a marked cataleptic effect, making the animals incapable of initiating voluntary movement. The blockade of striatal dopamine has been shown to mediate the catalepsies induced by antipsychotics (Baldessarini, 2001). The finding that the alkaloid fraction from Crassocephalum bauchiense did not produce catalepsies may further encourage its use in psychosis. The results obtained in our study suggest that inhibition of apomorphine-induced stereotypic behavior by the alkaloid fraction from Crassocephalum bauchiense is not related to the reduction of spontaneous locomotor activity of animals and it is not induced catalepsy. The failure of the alkaloid fraction from Crassocephalum bauchiense to prolong the duration of sleep produced by sodium pentobarbital in mice suggests a lack of sedative activity (Fujimori, 1995). The prolongation of sodium pentobarbitalinduced sleeping time is an indication of central nervous system depression, as previous studies established a positive correlation between the potentiation of sodium pentobarbital sleeping time and central nervous system depression (Amos et al., 2005). Drugs G. Sotoing Taı̈we et al. / Journal of Ethnopharmacology 143 (2012) 213–220 Table 1 Influence of the oral treatment with the leaves extracts of Crassocephalum bauchiense or diazepam on sodium pentobarbital-induced sleep in mice. Treatments Dose (mg/kg) Sedative action Onset time to the sleep (min) Total sleep time (min) Vehicle Vehicle þFG 7142 Vehicle þRO 151788 Vehicle þBIC – –þ 10 –þ 10 –þ 5 5.57 70.92 5.49 70.28 5.53 70.34 5.82 70.27 14.62 73.16 13.21 71.39 14.35 71.25 13.08 71.34 AE AE AE AE AE þ FG 7142 AE þ RO 151788 AE þ BIC 20 40 80 160 160þ 10 160þ 10 160þ 5 3.87 70.19 4.23 70.52 2.77 70.53** 2.13 70.27** 5.38 70.61 5.41 70.49 5.21 70.52 41.53 75.08* 50.67 74.96** 85.42 75.78*** 88.18 75.72*** 25.32 73.52 20.81 73.41 45.23 75.03* AF AF AF AF AF þ FG 7142 AF þ RO 151788 AF þ BIC 5 10 20 40 40þ 10 40þ 10 40þ 5 5.087 0.62 4.68 70.78 4.27 70.71 4.28 70.62 6.81 70.47 5.75 70.72 8.37 70.72 20.83 73.36 21.02 71.78 21.38 74.42 22.05 73.38 22.65 74.15 20.31 74.05 20.33 75.61 DZP 3 1.42 70.18*** 96.47 76.61*** Results are expressed as mean 7 S.E.M. for six animals. FG 7142, RO151788 or BIC were administered 15 min before the extracts or vehicle. The extracts or vehicle were administered 1 h before pentobarbital, the sleeping time activity were determined. Data were analysis by two-way ANOVA, followed by Newman–Keuls post hoc test. *P o 0.05, **Po 0.01, ***Po 0.001, significantly different compared to the vehicle. AE, aqueous extract; AF, alkaloid fraction. Table 2 Influence of the oral treatment with the leaves extracts of Crassocephalum bauchiense or sodium valproate on brain GABA content in mice. Treatments Dose (mg/kg) GABA level in brain tissue (lg/g 7 S.E.M.) Percentage increase (%) Vehicle – 395.487 10.52 – AE AE AE AE 20 40 80 160 404.65 7 10.13 424.137 10.67 447.177 11.31* 496.157 10.48** 2.26 6.75 11.56 20.29 AF AF AF AF 5 10 20 40 402.98 7 11.92 401.33 7 10.21 399.657 12.91 400.08 710.17 1.86 1.46 4.37 1.15 SV 300 501.92 7 25.08*** 21.20 Results are expressed as mean 7 S.E.M. for seven animals, and units are in mg/g, GABA level in brain tissue. *Po 0.05, **Po 0.01, ***P o 0.001, significantly different compared to the vehicle. Data were analysis by two-way ANOVA, followed by Newman–Keuls post hoc test. AE, aqueous extract; AF, alkaloid fraction. with sedative properties are known to prolong the duration of sleep produced by barbiturates (Fujimori, 1995; Amos et al., 2005). Therefore, it may be concluded that the alkaloid fraction from Crassocephalum bauchiense did not demonstrate sedative properties, as it failed to prolong the duration of sleep induced by sodium pentobarbital in mice. The aqueous extract of Crassocephalum bauchiense (80–160 mg/kg) produced a significant decrease in the time of onset of sleep as well as prolongation of sleep induced 219 by sodium pentobarbital in a manner comparable to diazepam (3 mg/kg). The prolongation of sodium pentobarbital induced sleep indicates a sedative and central nervous system depressant activity of the aqueous extract (File and Wardill, 1975). The reduction in sleep latency time and sleep prolongation effect of the aqueous extract of Crassocephalum bauchiense was antagonized by N-methylb-carboline-3-carboxamide, a partial inverse agonist at the benzodiazepine site in the GABAA receptor complex and flumazenil, a specific antagonist of the benzodiazepine site in the GABAA receptor complex, pre-treating 15 min before the extract was given. These results indicate that the effects of the extracts are mainly mediated via the GABAergic system (Evans and Lowry, 2007; Taı̈we et al., 2010; Walting, 1998). Anticonvulsant, anxiolytic and sedative (e.g. sodium valproate) are known to exert their pharmacological action by causing an increase in GABA acid content in mice cerebral hemisphere (Chapman et al., 1983; Saad, 1972; Taı̈we et al., 2010). It was found that the aqueous extract of Crassocephalum bauchiense and sodium valproate significantly enhanced the brain GABA concentration which again is suggestive of a sedative action of the extract and the reference drug. Taken together, we suggest that the behavioral action of the aqueous extract is correlated to an increase in GABA concentrations in the brain. The efficacy 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 (Amos et al., 2005; Dubois et al., 1986; Wagner et al., 1983). It is therefore probable that the saponins that are present in abundance in the extract might contribute in part for the observed central nervous system effects. Based on the above studies we concluded that the aqueous extract and the alkaloid fraction prepared from the leaves of Crassocephalum bauchiense might contain some psychoactive principles, which are sedative or not in nature. These neuropharmacological properties are possibly mediated via the GABAergic neurotransmission as well as blockade of dopamine D-2 receptors. Further studies to identify and isolate the active components are in progress. This justifies its use in traditional medicine in the management of insomnia and psychosis. Acknowledgments The authors are very thankful to Smartox Biotechnologies, Floralis, Biopolis, 5 Avenue du Grand Sablon, 38700 La Tronche, France, the University of Ngaoundéré, P.O. Box 455 Ngaoundéré, Cameroon and the University of Buea, P.O. Box 63 Buea, Cameroon, for supporting us by providing apparatus and drugs. References Adjanohoun, J.E., Aboukakar, N., Dramane, K., Ebot, M.E., Ekpere, J.A., Enow-Orock, E.G., Focho, D., Gbile, Z.O., Kamanyi, A., Kamsu, K.J., Keita, A., Mbenkum, T., Mbi, C.N., Mbiele, A.L., Mbome, I.L., Mubiru, N.K., Nancy, W.L., Nkongmeneck, B., Satabu, B., Sofowora, A., Tamze, V., Wirmum, C.K., 1996. Traditional medicine and pharmacopoeia. Contribution to Ethnobotanical and Floristic Studies in Cameroon. Centre de Production de Manuels Scolaires, Porto-Novo (Rep. Du Benin), 133. Ajayi, A.A., Ukponmwan, O.E., 1994. Evidence of angiotensin II and endogenous opioid modulation of novelty induced rearing in the rat. African Journal of Medicine and Medical Sciences 23, 287–290. Amos, S., Abbah, J., Chindo, B., Edmond, I., Binda, L., Adzu, B., Buhari, S., Odutola, A.A., Wambebe, C., Gamaniel, K., 2005. Neuropharmacological effects of the aqueous extract of Nauclea latifolia root bark in rats and mice. Journal of Ethnopharmacology 97, 53–57. Arana, G.W., 2000. An overview of side effects caused by typical antipsychotics. Journal of Clinical Psychiatry 61, 5–11. Arbonnier, M., 2000. Arbres, arbustes et lianes des zones se ches d’Afrique de l’Ouest Trees, shrubs and lianas of West Africa dry zones, Mali, Ouagadougou: 220 G. Sotoing Taı̈we et al. / Journal of Ethnopharmacology 143 (2012) 213–220 Centre de Coopération Internationale en Recherche Agronomique pour le développement/Muséum national d’histoire naturelle/Union mondiale pour la nature. First edition, (CIRAD/MNHN/UICN). Baldessarini, R.J., 2001. Drugs and the treatment of psychiatry and mania. In: Hardman, J.G., Limbird, I.E. (Eds.), Goodman and Gilman, the pharmacological basis of therapeutics, 10th edn. McGraw-Hill, New York, pp. 175–191. Bartholini, G., Keller, H., Pieri, L., Pletscher, A., 1973. The effect of diazepam on the turnover of cerebral dopamine. In: Garattini, S., Mueseini, E., Randall, L.O. (Eds.), The Benzodiazepines. Raven Press, New York, pp. 235–240. Biholong, M., 1986. Contribution a l’étude de la flore du Cameroun: les Astéracées. The se de doctorat. Université de Bordeaux III, Bordeaux. France, 10–50. Bradley, B.P., 1989. Neuroleptic drugs. In: Introduction to Neuropharmacology. Wright, London, pp. 201–219. Burkill, H.M., 1985. The Useful Plants of West Tropical Africa, second ed., vol. 1. Families A– D. Royal Botanic Gardens, Kew, London. Chapman, A.G., Meldrum, B.S., Mendes, E., 1983. Acute anticonvulsant activity f structural analogue of valproic acid and change in brain GABA and aspartate content. Life Science 32, 2023–2031. Dalziel, J.M., 1937. The useful plants of West Tropical Africa. Ed. The Crown Agency for the Colonies, London. Davis, K.L., Kahn, R.S., Grant, K.O., Davidson, M., 1991. Dopamine in schizophrenia: a review and reconceptualization. American Journal of Psychiatry 148, 1474–1486. Dubois, M.A., Ilyas, M., Wagner, H., 1986. Cussonosides A and B, two triterpenessaponins from Cussonia barteri. Planta Medica 56, 80–83. Duncan, G.E., Paul, I.A., Harden, T.K., Mueller, R.A., Stumpf, W.E., Breese, G.R., 1985. Rapid down regulation of P-adrenergic receptors by combining antidepressant drugs with forced swim: a model of antidepressant-induced neural adaptation. The Journal of Pharmacology and Experimental Therapeutics 234, 402–408. Ehmann, T., Yager, J., Hanson, L., 2004. Early psychosis: a review of the treatment literature. Children’s Mental Health Policy Research Program 7, 1–52. Evans, W.C., 2002. Trease and Evans’ Pharmacognosy, 15th ed. Saunders Ltd, Edinburgh. Evans, A.K., Lowry, C.A., 2007. Pharmacology of the b-Carboline FG 7142, a partial inverse agonist at the benzodiazepine allosteric site of the GABAA receptor: neurochemical. neurophysiological, and behavioral effects. CNS Drug Reviews 13, 475–501. File, S.E., Wardill, A.G., 1975. Validity of head dipping as a measure of exploration in a modified hole board. Psychopharmacologia 44, 53–59. Fujimori, H., 1995. Potentiation of barbital hypnosis as an evaluation method for CNS depressant. Psychopharmacology 7, 74–77. Gonzalez-Trujano, M.E., Navarrete, C.A., Reyes, B., Hong, E., 1998. Some pharmacological effects of the ethanol extract of leaves of Annona diversifolia on the central nervous system in mice. Phytotherapy Research 2, 600–602. Harrison, P., 1999. The neuropathology of schizophrenia: A critical review of the data and their interpretation. Brain 122, 593–624. Hellion-lbarrola, M.C., Ibarrola, D.A., Montalbetti, I., Villalba, D., Heinichen, O., Ferro, E.A., 1999. Acute toxicity and general pharmacological effect on central nervous system of the crude rhizome extract of Kyllinga brevifolia Rottb. Journal of Ethnopharmacology 66, 271–276. Irwin, S., 1968. Comprehensive observational assessment. A systematic, quantitative procedure for assessing the behavioural and physiologic state of the mouse. Psychoparmacologia 13, 222–256. Kenneth, S.K., Kenneth, I.D., 1984. Genetic control of apomorphineinduced climbing behaviour in two inbred mouse strains. Brain Research 293, 343–351. Labella, F.S., Punsky, C., Havlicek, V., 1979. Morphine derivatives with diminished opiate receptor potency show enhanced control excitatory activity. Brain Research 174, 263–271. Lowe, I.P., Robins, E., Eyermen, G.S., 1958. The fluorimetric measurement of glutamic decarboxylase and its distribution in brain. Journal of Neurochemistry 3, 8–16. Lu, Ming-chin, 1998. Studies on the sedative effects of cistanche deserticola. Journal of Ethnopharmacology 59, 1661–1665. Mouokeu, R.S., Ngono, R.A.N., Lunga, P.K., Koanga, M.M., Tiabou, A.T., Njateng, G.S.S., Tamokou, J.D.D., Kuiate, J.R., 2011. Antibacterial and dermal toxicological profiles of ethyl acetate extract from Crassocephalum bauchiense (Hutch.) Milne-Redh (Asteraceae). BioMed Central, Complementary and Alternative Medicine 11 (43), 1–7. Mullen, P.E., 2006. Schizophrenia and violence: from correlations to preventive strategies. Advances in Psychiatric Treatment 12, 239–248. Perez, R.M.G., Perez, J.A.L., Garcia, L.M.D., Sossa, H.M., 1998. Neuropharmacological activity of Solanum nigrum fruit. Journal of Ethnopharmacology 62, 43–48. Ray, W.A., Chung, C.P., Murray, K.T., Hall, K., Stein, C.M., 2009. Atypical antipsychotic drugs and the risk of sudden cardiac death. The New England Journal of Medicine 360, 225–235. Saad, S.P., 1972. Administration of CNS depressant drugs like barbiturates, hydantoin and diazepam etc. can restore the isoniazid induced fall in brain GABA levels. Journal of Pharmacy and Pharmacology 24, 839–840. Sanberg, P.R., Bunsey, M.D., Giordano, M., Norman, A.B., 1988. The catalepsy test: its ups and downs. Behavioural Neuroscience 102, 748–759. Shopsin, M., Klein, H., Aaronson, M., Collora, M., 1979. Clozapine, chlorpromazine and placebo in newly hospitalized, acutely schizophrenic patients. Archives of General Psychiatry 36, 657–664. Stolk, J.M., Rech, R.H., 1970. Antagonism of O-amphetamine by methylp-tyrosin. Behavioural evidence for participation of cathelcolamine stores and synthesis in the amphetamine stimulant response. Neuropharmacology 9, 249–263. Sutton, I., Simmonds, M., 1974. Effects of acute and chronic pentobarbitone on the g-aminobutyric acid system in rat brain. Biochemical Pharmacology 23, 1801–1808. Szechtman, H., 1986. Behavior performed at onset of drug action and apomorphine stereotypy. European Journal of Pharmacology 121, 49–56. Taı̈we, G.S., Ngo Bum, E., Dimo, T., Talla, E., Weiss, N., Amadou, D., Moto, O.F.C., Sidiki, N., Dzeufiet, P.D., De Waard, M., 2010. Antidepressant, myorelaxant and anti-anxiety-like effects of Nauclea latifolia Smith (Rubiaceae) roots extract in murine models. International Journal of Pharmacology 6 (4), 326–333. Taı̈we, G.S., Ngo Bum, E., Dimo, T., Talla, E., Weiss, N., Sidiki, N., Amadou, D., Moto, O.F.C., Dzeufiet, P.D., De Waard, M., 2011. Antipyretic and antinociceptive effects of Nauclea latifolia and possible mechanisms of action. Pharmaceutical Biology 49, 15–25. Taı̈we, S.G., Ngo Bum, E., Talla, E., Dimo, T., Sidiki, N., Dawe, A., Nguimbou, R.M., Dzeufiet, P.D.D., De Waard, M., 2012. Evaluation of antinociceptive effects of Crassocephalum bauchiense Hutch (Asteraceae) leaf extract in rodents. Journal of Ethnopharmacology 141, 234–241. Wagner, H., Ott, S., Jurcic, K., Morton, J., Neszmelyi, A., 1983. Chemistry 12C NMR study and Pharmacology of two saponins from Colubrina astiatica. Planta Medica 48, 136–141. Walting, Keith, J., 1998. Overview of central nervous system receptors. In: Keith, J., Walting (Ed.), The RBI Handbook of Receptor Clarification and signal Transduction, third ed. RBI, Natick, MA, pp. 2–45. Zetler, G., 1981. Central depressant effects of caerulein and cholecystokinin octapeptide (CCK8) differ from those of diazepam and haloperidol. Neuropharmacology 20, 277–283. Zhang, Z.J., 2004. Therapeutic effects of herbal extracts and constituents in animal models of psychiatric disorders. Life Sciences 75, 1659–1699.