Epilepsy is a disease that affects about 40 million people worldwide (Njamshi et al., 2010). In 1968, the prevalence of epilepsy in Africa was about 4.8 to 40 ‰. In 1996, Diop and collaborators reported in Senegal a prevalence of epilepsy of 21 ‰ (Diop et al., 1996). In 2006, Ngoungou and collaborators estimated the prevalence in sub-Saharan Africa to be two or three time highest than the rate in developed world (Ngounou et al., 2007). In Cameroon, some epidemiological studies on epilepsy have shown that, the prevalence of epilepsy is estimated to vary from 5-136/1000. The highest ones are reported in some villages of the Cameroon Central Province located in the Sanaga and Mbam River Valley (Nchoji Nkwi & Tioko Ndonko, 1989; Dongmo et al., 2000; Preux et al., 2000; Boussinesq et al., 2002; Kamgno et al., 2003; Dongmo et al., 2004; Prischich et al., 2008). Cameroon is one of the countries most affected by epilepsy in Africa and in the world. Thus, epilepsy is among the major public health problems in Cameroon. In Africa and in Cameroon particularly, phytotherapy in traditional medicine still plays an important role in the management of diseases, mainly among populations with very low income (Geoffrey & Kirby, 1996). And phytotherapy relies on the use of a wide variety of plant species. Annona muricata Linn (Annonaceae), Annona senegalensis Pers (Annonaceae), Bidens pilosa Linn (Asteraceae), Bryophyllum pinnatum (Lam) Oken (Crassulaceae), Citrus sinenis (Linn) Osbeck (Rutaceae), Clerodendron thomsoniae Balf (Verbenaceae), Daniellia oliveri (Rolfe) Hutch and Dalz (Caesalpiniaceae), Datura stramonium Linn (Solanaceae), Detarium microcarpum Guil et Perr (Caesalpiniaceae), Euphorbia hirta Linn (Euphorbiaceae), Flacourtia indica Willd (Flacourtiaceae), Hymenocardia acida Tul (Hymenocardiaceae), Jatropha gossypiifolia Linn (Euphorbiaceae), Khaya senegalensis A Juss (Desrousseaux) (Meliaceae), Mentha cordifolia Auct (Lamiaceae), Prosopis Africana Guill and Perr (Taub) (Mimosaceae), Ricinus communis Linn (Euphorbiaceae), Securidaca longepedunculata Fres (Polygalaceae), Senna singueana (Delile) Lock 1988 (Caesalpiniaceae), Terminalia glaucescens Planch. ex Benth (Combretaceae), Terminalia mollis Laws (Combretaceae), Tetrapleura tétraptera Taub (Schum Thonn) (Mimosaceae), Trichilia emetica Vahl (Meliaceae) and Vitellaria paradoxa C F Gaertn (Sapotaceae) are plants that are being used empirically in traditional medicine in Cameroon to treat epilepsy and diseases related to the brain like agitations, anxiety, convulsions, dizziness, headaches, insomnia, migraines, pains and schizophrenia according to our traditional Healers and the literature (Abbiw, 1990; Adjanohoun et al., 1984, 1996; Arbonnier, 2000; Berhaut, 1975; Biholong, 1986; Bouquet, 1969; Brenan, 1959; Dalziel, 1937; Hutchinson & Dalziel, 1958; Iwu, 1993; Joyner, 2004; Malgras, 1992; Mutasa et al., 1990; Nwaiwu & Akah, 1986; Pousset, 1989; Raponda-Walker & Silans, 1961; Saulnier, 1998) (Table 1). Though the literature showed a lot of pharmacological studies done
with these plants, very few were done to study their sedative and anticonvulsant properties. This study was undertaken to evaluate the anticonvulsant and sedative properties of these plants used in the treatment of insomnia and epilepsy in traditional medicine in Africa, particularly in Cameroon.
2. Materials and methods
Adult male mice (Mus musculus Swiss; 22 2 g; 6 or 8 per group) were used for this study. The animals were housed in standard cages at 25°C, on a 12/12 h light-dark cycle. They were supplied with food and water ad libitum.
Drugs were administered in a volume of 10 ml/kg of mice body weight. The study was conducted in accordance with the nationally (N°.FWA-IRB00001954) and internationally accepted principles for laboratory animal use and care. In diazepam or sodium thiopental- induced sleep tests, mice were divided into negative control group that received distilled water and four test groups that received different doses of the plant extracts. In anticonvulsant tests, there was one more group that received a known anticonvulsant compound and served as a positive control.
2.2. Plant material
A voucher specimen of each plant was authenticated by a botanist, Professor Mapongmetsem Pierre Marie, Department of Biological Sciences, University of Ngaoundéré and deposited at the National Herbarium of Cameroon in Yaoundé.
2.3. Preparation of the extracts
10 g of each plant material were macerated for 1 h in an amount of distilled water (25, 50, 75, 100 or 150 ml) according to the plant. The mixture was boiled for 20 min. After cooling, the supernatant (decoction) was collected and filtered. The decoction of each plant was diluted in distilled water to obtain less concentrated solutions. In another experiment, the decoction was dried and the w/w yield of the extract was calculated (table 2). The decoctions were prepared according to the methods close to the ones used in traditional medicine.
10 g of dried fruits of Datura stramonium were macerated in 50 ml of distilled water. After 1 h the supernatant was collected, filtered and used in mice. The w/w yield of the extract was obtained (table 2).
2.4. Anticonvulsant tests
2.4.1. N-methyl-D-aspartate (NMDA) test
Six groups of 6 or 8 mice received different treatments. Group I (negative control) was treated with distilled water. Groups II to V (test groups) were treated with 4 doses of the plant extracts. Group VI (positive control) was treated with 3 mg/kg of CGP 37849 i.p. or 33 ηmol/kg of D-AP7 i.p. Mice were injected subcutaneously with NMDA, 75 mg/kg 1 h after administration of the different treatments. They were observed for 30 min. Animals that did not exhibit turning behaviour within the 30 min of observation were declared protected. Turning behaviour was characterised by two consecutive 360° cycles fulfilled by the same animal (Croucher et al., 1982; Ngo Bum et al., 2001; 2009a; 2009b; Schmutz et al., 1990).
2.4.2. Strychnine (STR) test
Six groups of 6 or 8 mice received different treatments as above, except that group VI (positive control) was treated with clonazepam (3 mg/kg, i.p.). Convulsions followed by death were induced in mice by the i.p. injection of 2.5 mg/kg STR nitrate 1 h after administration of the different treatments. The animals which survived more than 10 min after strychnine injection were qualified protected (Ngo Bum et al., 2001, 2009a).
2.4.3. Picrotoxine (PIC) test
Six groups of 6 or 8 mice received different treatments as above, except that group VI (positive control) was treated with clonazepam (0.4 mg/kg, i.p.). Clonic seizures were induced in mice by the i.p. injection of 7.5 mg/kg PIC 1 h after administration of the different treatments. The animals which did not convulse within the 15 min of observation after PIC injection were qualified protected (Lehmann et al., 1988; Ngo Bum et al., 2001).
2.4.4. Pentylenetetrazol (PTZ) test
Six groups of 6 or 8 mice received different treatments as above, except that group VI (positive control) was treated with clonazepam (0.1 mg/kg, i.p.). Clonic seizures were induced in mice by the i.p. injection of 70 mg/kg PTZ 1 h after administration of the different treatments. The animals that did not convulse within the 10 min from the injection of PTZ were qualified protected (Ngo Bum et al., 2001, 2009a, 2009b).
2.4.5. Isonicotinic hydrazide acid (INH) test
Six groups of 6 or 8 mice received different treatments as above, except that group VI (positive control) was treated with diazepam, 10 mg/kg (per os). Animals were injected i.p. with INH 250 mg/kg 1 h after the administration of the different treatments. The time to the onset of clonic or tonic seizures was recorded. (Bernasconi et al., 1988; Ngo Bum et al., 2001).
2.5. Diazepam or sodium thiopental-induced sleep in mice
Five groups of 6 or 8 mice received different treatments. Group I (negative control) was treated with distilled water and groups II to V (test groups) were treated with 4 doses of the plant extracts.The methods described by Beretz et al., (1978) and modified by Rakotonirina et al., (2001) were used. Sleep potentiating effects of the plant were studied in mice that received sodium thiopental or diazepam at a dose of 50 mg/kg (i.p.) 1 hour after the administration of the different treatments. The time between the loss of the straightening reflex and the regain of this reflex measured the sleeping time. The loss or the regain of the straightening reflex was measured by stimulating the external ear. When the mouse anterior paw does not move after stimulation with horsehair, the animal is sleeping. When the mouse is awakened, it moves and shakes its paw.
2.6. Statistical analysis
Three parameters were measured: the protection against chemically-induced seizures, the latency to the onset of seizures (min) in INH test, the latency to the onset of sleep and the sleeping time (min) in the sleep potentiation test. Data of the control groups were compared to data of groups treated with the plants extracts and to data of the positive control groups. The statistical analysis were done using Fisher exact test and Anova followed by Dunnett (REGWQ). P<0.05 was considered significant.
D-2-amino-7-phosphonoheptanoate, Clonazepam, Isonicotinic hydrazide acid, N-methyl-D-aspartate, penthylenetetrazol, picrotoxine, sodium thiopental and strychnine are from Sigma Chemical, USA. Diazepam is from Roche, France.
3.1. Sedative properties
The extracts of twenty one plants increased in a dose-dependent manner the sleeping time induced by sodium thiopental or diazepam. The most potent was Datura stramonium. it multiplied by a factor of 5 the sleeping time of the control group (from 16 ± 7 to 94 ± 25 min at a dose of 70 mg/kg), but this extract was very toxic for animals. The decoctions of eight plants multiplied by a factor of 4 the sleeping time of their control group: Annona senegalensis (from 19 ± 4 to 89 ± 29 min at a dose of 67 mg/kg), Clerodendron thomsoniae (from 19 ± 3 to 94 ± 30 min at a dose of 134 mg/kg), Daniellia oliveri (from 20 ± 8 to 81 ± 13 min at a dose of 198 mg/kg), Hymenocardia acida (from 20 ± 11 to 85 ± 21 min at a dose of 87.6 mg/kg), Securidaca longepedunculata (from 18 ± 3 to 78 ± 14 min at a dose of 66.7 mg/kg), Terminalia mollis (from 17 ± 1 to 84 ± 15 min at a dose of 70 mg/kg), Tetrapleura tetraptera (from 19 ± 3 to 91 ± 15 min at a dose of 84 mg/kg) and Trichilia emetica (from 17 ± 1 to 84 ± 10 min at a dose of 126 mg/kg). The sleeping time of the control groups were multiplied by a factor of 3 by six plants: Flacourtia indica (from 16 ± 12 to 49 ± 3 min at a dose of 100 mg/kg), Jatropha gossypiifolia (from 11 ± 5 to 43 ± 15 min at a dose of 140 mg/kg), Prosopis Africana (from 19 ± 3 to 61 ± 26 min at a dose of 112 mg/kg), Senna singueana (from 24 ± 2 to 86 ± 5 min at a dose of 20 mg/kg),Terminalia glaucescens (from 37 ± 13 to 120 ± 21 min at a dose of 76 mg/kg), and Vitellaria paradoxa (from 25 ± 4 to 84 ± 20 min at a dose of 84 mg/kg). The decoctions of five plants multiplied by a factor of 2 the sleeping time of their control group: Annona muricata (from 31 ± 11 to 71 ± 15 min at a dose of 120 mg/kg), Bidens pilosa (from 31 ± 2 to 80 ± 2 min at a dose of 140 mg/kg), Detarium microcarpum (from 20 ± 6 to 52 ± 12 min at a dose of 111.45 mg/kg), Euphorbia hirta (from 56 ± 16 to 145 ± 10 min at a dose of 140 mg/kg) and Mentha cordifolia (from 10 ± 2 to 24 ± 3 min at a dose of 140 mg/kg). Bryophyllum pinnatum induced a slight increase of the sleeping time. Only Citrus sinenis and Kaya senegalensis could not increase the total sleep time of mice (table 3). Some of those plants also reduced the onset time of sleep (Table 4).
3.2. Anticonvulsant properties
3.2.1. On PTZ- induced convulsions
78.3% of plants extract were effective against PTZ-induced convulsions. Annona muricata, Annona senegalensis, Bidens pilosa, Clerodendron thomsoniae, Daniellia oliveri, Datura stramonium, Detarium microcarpum, Euphorbia hirta, Flacourtia indica, Hymenocardia acida, Mentha cordifolia, Ricinus communis, Securidaca longepedunculata, Senna singueana, Terminalia glaucescens, Terminalia mollis, Tetrapleura tétraptera, Trichilia emetica and Vitellaria paradoxa protected mice against convulsions induced by PTZ (table 5).
3.2.2. On STR- induced convulsions
The percentage of plants extracts that protected mice against STR-induced convulsions was 77.8%. Annona muricata, Bidens pilosa, Daniellia oliveri, Detarium microcarpum, Flacourtia indica, Hymenocardia acida, Jatropha gossypiifolia, Khaya senegalensis, Mentha cordifolia, Prosopis Africana, Securidaca longepedunculata, Senna singueana, Terminalia mollis, Trichilia emetica protected mice against STR- induced convulsions (table 5).
3.2.3. On PIC- induced convulsions
The percentage of plants extracts that protected mice against PIC-induced convulsions was 87.5%. Clerodendron thomsoniae, Flacourtia indica, Mentha cordifolia, Securidaca longepedunculata, Senna singueana, Terminalia glaucescens and Vitellaria paradoxa protected mice against convulsions induced by PIC (table 5).
3.2.4. On NMDA- induced turning behaviour
The percentage of plants extracts that protected mice against NMDA-induced turning behaviour was 100%. Annona muricata, Bidens pilosa, Bryophyllum pinnatum, Citrus sinenis, Euphorbia hirta, Khaya senegalensis protected mice against turning behaviour induced by NMDA (table 5).
3.2.5. On MES- induced convulsions
The percentage of plants extracts that protected mice against MES-induced convulsions was 25%. Securidaca longepedunculata protected mice against convulsions induced by MES (table 5).
3.2.6. On INH- induced convulsions
The percentage of plants extracts that were effective against INH-induced convulsions in mice was 60%. Ricinus communis, Securidaca longepedunculata, Senna singueana delayed the onset of seizures in INH test (table 5).
3.2.7. Plants efficacy
Flacourtia indica, Ricinus communis, Securidaca longepedunculata, Senna singueana, Terminalia glaucescens showed very good anticonvulsant activities (80 to 100% of protection against PTZ, PIC or INH induced seizures). The other eighteen plants tested protected 50 to 75% of
mice against the induced convulsions. 78% of plants protected both PTZ and STR-induced convulsions. 80.6% of plants protected both PTZ and PIC-induced convulsions. 80.8% of plants protected both STR and PIC-induced convulsions. Finally, 66.7% of plants at the same time protected PTZ, STR and PIC-induced convulsions.
3.2.8. Plants toxicity
Datura stramonium, Ricinus communis and Securidaca longepedunculata were also showed to be toxic. Their extract killed animal in 24h after their administration to mice.
4. Discussion and conclusions
The extracts of twenty one plants (91.3% of plants) increased the sleeping time induced by sodium thiopental or diazepam. The potentiation of the sleep time suggests the presence of sedative properties in the extracts of these plants (Rakotonirina et al., 2001; Ngo Bum et al., 2009a; 2009b). These sedative properties could be related to the presence of some components in the extracts activating the benzodiazepine, barbiturate and/or GABA receptors in the GABAA receptor complex (Rang et al., 1999; Bonin & Orser, 2008; Olkkola & Ahonen, 2008). Diazepam (benzodiazepine) and sodium thiopental (barbiturate) all bind to the GABAA receptor complex. Diazepam potentiates GABA-mediated inhibition via the increase in the affinity of this inhibitory neurotransmitter to its recognition sites within the GABAA receptor complex, by increasing the opening frequency of the chloride ion channel which leads to the enhancement of influx of chloride anions into the neuron and subsequent hyperpolarisation (Czapinsky et al., 2005). While sodium thiopental that act on the barbiturate binding site directly gate the chloride ion channel of the GABAA receptor complex. The sedative properties found here could explain the use of the twenty one plants in traditional medicine in Africa, particularly in Cameroon in the treatment of insomnia. The first eight more potent plants to induced sedation were: Datura stramonium > Clerodendron thomsoniae > Terminalia mollis > Trichilia emetica > Tetrapleura tétraptera > Annona senegalensis > Securidaca longepedunculata > Hymenocardia acida > Daniellia oliveri. Two plants, Citrus sinenis and Kaya senegalensis did not show sedative properties. The results also showed that 95.6% of the tested plants possess anticonvulsant properties by inhibiting convulsions induced chemically or electrically. Five plants (Flacourtia indica, Ricinus communis, Securidaca longepedunculata, Senna singueana, Terminalia glaucescens) showed very good anticonvulsant activities against PTZ, PIC or INH induced seizures.
The effect was moderate for the rest of plants. Tetrapleura tetraptera one of the plants studied showed also anticonvulsant properties in fruits (Nwaiwu, 1986; Ojewole, 2005). The antagonism of INH, PTZ- and PIC-induced seizures suggests the interaction of these plants with the GABA-ergic neurotransmission (De Deyn et al., 1992; Doctor et al., 1982; Löscher & Schmidt, 1988; Salih & Mustafa, 2008; Perez-Saad & Buznego, 2008). GABA is the main inhibitory neurotransmitter substance in the brain and is widely implicated in epilepsy. Inhibition of GABA-ergic neurotransmission or activity has been shown to promote and facilitate seizures, while enhancement of GABA-ergic neurotransmission is known to inhibit or attenuate seizures (Gale, 1992; Li-Ping et al., 2008). Moreover, some studies indicated that PTZ diminishes the GABAergic tone (Mcdonald & Baker. 1977; Ahmadiani, 2003), probably by a competitive antagonist action on the BZD receptors (Rehavi et al., 1982). Correspondingly, drugs that enhance GABAA-receptor neurotransmission, such as BZDs (White, 1997; Ahmadiani et al., 2003) can block seizures induced by PTZ. PIC is known to be a non competitive GABA antagonist exerting his effect by blocking the chloride channel in the GABAA receptor complex. Isoniazide can precipitate convulsions in patients with seizure disorders, and it is regarded as a GABA-synthesis inhibitor (Kale Shubhangi et al., 2010). The antagonism of STR -induced convulsions suggests the presence of anticonvulsant effect through glycine-STR-sensitive receptors (Findlay et al., 2002). Few plants extract antatagonized MES induced convulsions, by probably prolonging neurons sodium channels inactivation (Holmes, 2007). The results show no difference in plants inhibiting convulsions induced by PTZ, PIC and STR. GABA and glycine-STR-sensitive neurotransmission are equally involved. But very few plants produced their anticonvulsant activities by prolonging neurons sodium channels inactivation. Datura stramonium, Ricinus communis and Securidaca longepedunculata were found toxic and therefore they are not suitable to be used to treat people. The toxicity of Ricinus communis could be related to the presence of a very toxic component named ricin (Iwu, 1993). The toxicity of Datura stramonium could be related to its delirants or anticholinergics compounds.
The purported anticonvulsant and sedative properties of the medicinal plants are scientifically shown. The ethnopharmacological study on Cameroon anticonvulsant and sedative medicinal plants is accurate in 90% of cases. A great amount of plants extract interacted through GABA and glycine-STR-sensitive neurotransmissions to inhibit convulsions. Many anticonvulsant plants also possess sedative properties. Twenty one plants possess sedative properties, but only eighteen plants could be used in traditional medicine in Africa in the treatment of insomnia. Eighteen plants possess at least moderate anticonvulsant effects, while five plants possess very good anticonvulsant properties. However only twenty medicinal plants could be used in the treatment of epilepsy. Three plants were found very toxic.