Extracts of Feretia apodanthera Del. demonstrated anticonvulsant activities against seizures induced by chemicals and maximal electroshock

Epilepsy Research 127 (2016) 30–39

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Extracts of Feretia apodanthera Del. demonstrated anticonvulsant activities against seizures induced by chemicals and maximal electroshock G.S. Taiwe a , F.C.O. Moto b , S. Pale a , A.K. Kandeda c , Amadou Dawe d , N. Kouemou a , E.R.M. Ayissi b , G.T. Ngoupaye e , J.S.K. Njapdounke f , G.C.N. Nkantchoua f , J.P.O. Omam b , D. Pahaye f , E. Ngo Bum f,∗ a

Department of Zoology and Animal Physiology, Faculty of Sciences, University of Buea, P. O. Box 63, Buea, Cameroon Department of Biological Sciences, Higher Teachers’ Training College, University of Yaoundé 1, P. O. Box 47, Yaoundé, Cameroon c Department of Animal Biology and Physiology, Faculty of Science, University of Yaoundé I, P.O. Box 812, Yaoundé, Cameroon d Department of Chemistry, Higher Teachers’ Training College, University of Maroua, P. O. Box 55, Maroua, Cameroon e Department of Animal Biology, Faculty of Sciences, University of Dschang, P. O. Box 67, Dschang, Cameroon f Department of Biological Sciences, Faculty of Science, University of Ngaoundéré, P.O. Box 454, Ngaoundéré, Cameroon b

a r t i c l e

i n f o

Article history: Received 22 November 2014 Received in revised form 2 August 2016 Accepted 11 August 2016 Available online 12 August 2016 Keywords: Feretia apodanthera Aqueous extract Alkaloid fraction Anticonvulsant Traditional medicine

a b s t r a c t Extracts of Feretia apodanthera Del. (Rubiaceae) have been extensively used in traditional Cameroonian medicine to treat a variety of diseases, including some neurological disorders. The present study was aimed to tests the anticonvulsant properties of the aqueous extract and the alkaloid fraction of the stem barks of Feretia apodanthera. The anticonvulsant investigation was carried out against bicuculline-, picrotoxin-, pentylenetetrazol-, Methyl-␤-carboline-3-carboxylate-, N-Methyl-Daspartate-, 4-aminopyridine-, and maximal electroshock-induced seizures or turning behavior in mice. The aqueous extract protected mice against bicuculline-, picrotoxin-, pentylenetetrazol-, Methyl␤-carboline-3-carboxylate-, N-methyl-D-aspartate −, 4-aminopyridine- and maximal electroshockinduced seizures or turning behavior. Also, N-Methyl-D-aspartate-, 4-aminopyridine- and maximal electroshock- induced seizures or turning behavior, were significantly antagonized by the alkaloid fraction (80 mg/kg) from Feretia apodanthera. The total protection of mice provided by the aqueous extract against convulsions induced by pentylenetetrazol or picrotoxin was anagonized by flumazenil, a specific antagonist of the benzodiazepine site in the GABAA receptor complex. The aqueous extract of Feretia apodanthera (but not the alkaloid fraction) increased the brain GABA content and inhibited the GABA transaminase activity. In conclusion, Feretia apodanthera was revealed possessing anticonvulsant effects in mice, likely via the GABAergic neurotransmission. © 2016 Elsevier B.V. All rights reserved.

1. Introduction Epilepsy is the second most common neurological disorder after stroke, affecting at least 50 million persons worldwide (Scheuer and Pedley, 1990). It shows a prevalence rate in 1–2% of the world population (Kamboj et al., 2009). Various anticonvulsant drugs are available to grapple with this neurological disorder. Dose-related neurotoxicity, cognitive impairment, and systemic side effects are

∗ Corresponding author at: Department of Biological Sciences, Faculty of Science, University of Ngaoundere, Cameroon, P.O. Box 565, Ngaoundere, Cameroon. E-mail address: eli [email protected] (E. Ngo Bum). http://dx.doi.org/10.1016/j.eplepsyres.2016.08.009 0920-1211/© 2016 Elsevier B.V. All rights reserved.

the major problems caused by antiepileptic drugs (Reynolds and Trimble, 1985). Despite treatment improvement has occurred with the panel of available antiepileptic drugs, epilepsy remains refractory in one third of patients. Furthermore, it should be mentioned that adverse effects associated with antiepileptic drugs and recurrent seizures limit their use (Maertns et al., 1995). Therefore, the search for new therapeutic agents continues, and medicinal plants have emerged as a crucial source for the development of drugs to treat neurological disorders and play an important role for patients who respond poorly to conventional treatments (HerreraRuiz et al., 2006; Carlini, 2003). Feretia apodanthera Del. (Rubiaceae) is a medium-sized flowering tree distributed in some countries of Western Africa. The stem barks of Feretia apodanthera is being used

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empirically in Cameroonian traditional medicine to treat infantile convulsions, epilepsy, agitations, anxiety, schizophrenia, various forms of pain and headaches (Adjanohoun et al., 1996; Kerharo and Adams, 1974; Dalziel, 1937). In Senegal, the leaves of Feretia apodanthera are used to treat different urinary and renal infections. The leaves are also used to treat stomach aches as well as nausea and syphilis (Adjanohoun et al., 1996; Kerharo and Adams, 1974). Phytochemical studies of Feretia apodanthera allowed the isolation and identification of seven iridoid glucosides: feretoside, gardenoside, geniposide, desacetyl asperulosidic acid, 11-methyl ixoside, apodanthoside and 10-ethyl apodanthoside (Bailleul et al., 1980, 1977). The first three occur in the flowers. Feretoside and gardenoside were isolated from the root barks of Feretia apodanthera (Bailleul et al., 1980). Only few studies pointed to the possible neuropharmacological effects of Feretia apodanthera. Stem and root barks extracts of Feretia apodanthera decreased the exploratory and spontaneous motor activities in mice, increased hexobarbital sleeping time in mice and protected rabbits against pentylenetetrazolinduced seizures (Bailleul et al., 1981, 1980). The present study was undertaken to explore the antiepileptic effects of both the aqueous extract and the alkaloid fraction from the stem barks of Feretia apodanthera, by using chemicals and maximal electroshock induced seizures. Part of the results was published in abstract form (Taiwe et al., 2014). 2. Material and methods 2.1. Plant material The stem barks of Feretia apodanthera used in this study was harvested from the north region of Cameroon in April 2009. The species was authenticated and a voucher was deposited at the National Herbarium, Yaoundé (Num. 31225/HNC). 2.2. Preparation of the aqueous extract and alkaloid extraction The stem barks were separated and cleaned, then sun-dried and crushed using a mechanical grinder. The powdered material was extracted with distilled water (50 g of powder per 375 ml water) by cold maceration for 24 h, then filtered through Whatman n◦ . 1 filter paper and freeze-dried (FreeZone® Dry 4.5, USA). This procedure resulted in a yield of 8.62% (w/w). For fractionation, the dried and powdered stem barks of Feretia apodanthera (1000 g) were extracted with acetone/H2 O (7/3; 5 l) at room temperature. The extract was evaporated in vacuo to afford a dark residue (649.17 g). The residue was suspended in warm water (1 l) and then extracted successively with ethyl acetate (0.5 l × 3) and n-butanol (0.5 l × 3), and concentrated to give residue A (153.71 g) and B (392.54 g), respectively. The latter was resolved in warm water (1 l), acidified with 1 mol/l HCl to pH between 4 and 5, and extracted with CHCl3 (0.5 l × 3). The aqueous layer was neutralized with 1 mol/l NaOH to pH 9–10 and extracted with CHCl3 (0.5 l × 3) once again and concentrated in vacuo to obtain the crude base (alkaloid fraction; 195.18 g) (Taïwe et al., 2012a,b; Taiwe et al., 2014). The freeze-dried extract (aqueous extract) and the alkaloid fraction from the stem barks of Feretia apodanthera were dissolved in saline 0.90% containing dimethyl sulfoxyde 2% (vehicle) at the appropriate concentrations as indicated in the various experiments and administered orally in a volume of 10 ml/kg. 2.3. Chemicals Bicuculline, picrotoxin, pentylenetetrazol, clonazepam, methyl-␤-carboline-3-carboxylate, diazepam, flumazenil, glacial acetic acid, N-Methyl-D-aspartate, D-2-amino-7phosphonoheptanoate, 4-aminopyridine and phenobarbital

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were obtained from Sigma Chemical, USA. Diazepam was obtained from Roche. All other chemicals and reagents used in the brain gamma-aminobutyric acid (GABA) content estimation and in the determination of brain GABA-T activity are from Sigma Chemical, USA. 2.4. Animals Swiss albino mice (25–30 g) of either sex were used in this study. The animals were randomly housed in appropriate cages at 22 ± 2 ◦ C on a 12 h light/dark cycle with free access to food and water. In all the experimental studies, each group consisted of six to ten animals. Each animal was used only once. The investigation conforms to the Guide for the Care and Use of Laboratory Animal, according to the ethical guidelines of Cameroon Bioethics Committee (Ref n◦ FW-IRB00001954) and the US National Institutes of Health (NIH No. 85-23, revised 1996). 2.5. Pharmacological tests 2.5.1. Bicuculline-induced seizures Briefly, ten groups of six mice were administered graded doses of the aqueous extract of Feretia apodanthera (50, 100, 150 and 200 mg/kg; p.o.), the alkaloid fraction from Feretia apodanthera (10, 20, 40 and 80 mg/kg; p.o.), diazepam (positive control; 3 mg/kg, i.p.) or vehicle (10 ml/kg p.o.). One hour later, all animals were injected intraperitonealy with bicuculline (4 mg/kg) and placed in isolated cages. The time to onset of clonic or tonic seizures was recorded. A threshold convulsion lasting for at least 5 s has been considered as an episode of clonic spasms. Absence of this threshold convulsion over 30 min indicated that the animal was protected from the convulsant-induced seizures (Masereel et al., 1998). 2.5.2. Picrotoxin-induced seizures Mice were divided into twelve groups of six mice each, and received the aqueous extract of Feretia apodanthera (50, 100, 150 and 200 mg/kg; p.o.), the aqueous extract + flumazenil (200 mg/kg, p.o. + 10 mg/kg, i.p.), the alkaloid fraction from Feretia apodanthera (10, 20, 40 and 80 mg/kg; p.o.), the alkaloid fraction + flumazenil (80 mg/kg, p.o. + 10 mg/kg, i.p.), clonazepam (1 mg/kg, i.p.) or vehicle (10 ml/kg p.o.). One hour later, clonic seizures were induced in mice by intraperitoneal injection of 7.5 mg/kg picrotoxin. Mice were observed for 15 min and the protective effect of the different treatments was recorded. Animals that did not convulse within the 15 min of observation were qualified as protected (Ngo Bum et al., 2001). The time to onset of clonic or tonic seizures was recorded. In the antagonistic experiment, flumazenil was administered 30 min before the test. 2.5.3. Pentylenetetrazol-induced seizures Twelve groups of six mice were treated as discussed previously. However, the positive control group received 0.1 mg/kg clonazepam i.p. Clonic seizures were induced in mice by the i.p. injection of 70 mg/kg pentylenetetrazol. The protective effect of the different treatments given 1 h before pentylenetetrazol injection was recorded. Animals that did not convulse within the 10 min of observation were qualified as protected (Schmutz et al., 1990; Ngo Bum et al., 2009a). The time to onset of clonic seizures was recorded. 2.5.4. Methyl-ˇ-carboline-3-carboxylate-induced seizures Ten groups of six mice were administered graded doses of the aqueous extract of Feretia apodanthera (50, 100, 150 and 200 mg/kg; p.o.), the alkaloid fraction from Feretia apodanthera (10, 20, 40 and 80 mg/kg; p.o.), diazepam (positive control; 4 mg/kg, i.p.) or vehicle (10 ml/kg p.o.). Seizures were induced in mice by the i.p. injection of

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methyl-␤-carboline-3-carboxylate (12 mg/kg) dissolved in a minimal amount (<5% of final volume) of glacial acetic acid, and brought to volume with saline. The final solution showed a pH value of 5–5.5. The mice were observed 30 min for the incidence of clonic seizures (De Sarro and De Sarro, 1993). 2.5.5. N-Methyl-D-aspartate-induced turning behavior Ten groups of six mice were treated as discussed previously. However, the positive control group received D-2-amino-7phosphonoheptanoate (D-AP7; 33 nmol/kg, i.p.). Turning behavior was induced in mice by subcutaneous injection of N-Methyl-Daspartate 75 mg/kg, 1 h after treatment. Mice were observed for 30 min. The latency time to turning behavior was recorded. Animals that did not exhibit turning behavior within the 30 min were declared protected. Turning behavior was characterized by two consecutive 360◦ cycles completed by the same animal (Schmutz et al., 1990; Ngo Bum et al., 2009a,b). 2.5.6. 4-Aminopyridine-induced seizures Briefly, Twelve groups of six mice were administered graded doses of the aqueous extract of Feretia apodanthera (50, 100, 150 and 200 mg/kg; p.o.), the alkaloid fraction from Feretia apodanthera (10, 20, 40 and 80 mg/kg; p.o.), phenobarbital (30 mg/kg, i.p.) or vehicle (10 ml/kg p.o.). One hour later, the K+ channel blocker 4aminopyridine was administered subcutaneously to all animals at the dose 15 mg/kg, then placed singly in a box and observed for 60 min. The occurrence of clonic and tonic seizures was recorded. Animals showing tonic extension or death were scored as nonprotected according to Yamaguchi and Rogawski (1992). 2.5.7. Maximal electroshock seizures test Mice divided into ten groups of six mice were administered graded doses of the aqueous extract of Feretia apodanthera (50, 100, 150 and 200 mg/kg; p.o.), the alkaloid fraction from Feretia apodanthera (10, 20, 40 and 80 mg/kg; p.o.), diazepam (5 mg/kg, i.p.) or vehicle (10 ml/kg p.o.). Tonic extension of the hind extremities of mice were induced by passing alternating electrical current (50 Hz, 30 mA, 0.2 s) through eye electrodes. The time to onset of tonic seizures, the duration of tonic hind limb extension and the number of animals protected from tonic hind limb extension were determined in each dose group (Ngo Bum et al., 2009a,b). 2.6. Estimation of brain GABA content The measurement of brain GABA level, based on the method of Lowe et al. (1958) was carried out as follows. Animals were killed by decapitation 1 h after administration of the aqueous extract of Feretia apodanthera (50, 100, 150 and 200 mg/kg; p.o.), the alkaloid fraction from Feretia apodanthera (10, 20, 40 and 80 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 10000g for 10 min at 0 ◦ C. 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 ◦ C 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 ␮g) mixed with 1.5 ␮M 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 ␮g/g of wet brain tissue.

2.7. Determination of GABA transaminase (GABA-T) activity in the brain Male mice were sacrificed by decapitation. The brains were removed and immediately submerged in ice-cold artificial cerebrospinal fluid. The brain tissues were then washed to remove blood, blotted to dry and submerged in 5 ml of methanol, homogenized using a glass Teflon homogenizer for 2 min and centrifuged at 10,000 rpm at −10 ◦ C for 15 min (Nayak and Chatterjee, 2001a,b). GABA-T activity in the brain homogenates was measured spectrophotometrically as described by Sytinsky et al. (1975) and with few modifications (Nayak and Chatterjee, 2001a,b). To a 10 ml volumetric flask, 15 ␮mol from each of ␣-oxoglutarate and GABA, 10 ␮g of pyridoxal phosphate and 1 ml of supernatant of the brain tissue homogenate (10% in sucrose, 0.32 mol/l) were added and the final volume was made up to 3 ml with buffer containing 0.2 M Tris-HCl (pH 8.6). The final mixture were incubated at 37 ◦ C for 30 min for reaction in 96-well plates with the various concentrations of the aqueous extract of Feretia apodanthera (25, 50, 75, 100, 150, 175 and 200 ␮g/ml), the alkaloid fraction from Feretia apodanthera (10, 20, 40, 60, 80, 100 and 120 ␮g/ml), sodium valproate (positive control; 100 ␮g/ml) or vehicle (saline 0.90% containing dimethyl sulfoxyde 2%). The reaction was terminated by adding 0.5 ml ice-cold 20% trichloroacetic acid. The blank was prepared by replacing the homogenate with methanol from the mixture. The succinic semialdehyde (SSA) produced in the incubation mixture was estimated at 610 nm. The complex color of SSA and 3methyl 2-benzothia-zolone-2-hydrazone in the presence of 12% FeCl3 was measured against the blank. GABA-T activity was measured in units/mg of protein.

2.8. Acute toxicity test The acute toxicity test for the aqueous extract and the alkaloid fraction from the stem barks of Feretia apodanthera was carried out to evaluate any possible toxicity. Mice of either sex were divided into control and test groups. The first group of mice served as a normal control treated with vehicle. The aqueous extract of Feretia apodanthera (50, 100, 150, 200, 400, 800, 1600, 3600 and 7200 mg/kg) and the alkaloid fraction from Feretia apodanthera (10, 20, 40, 80, 160, 320, 640, 1280, 2560 and 5120 mg/kg) were administered orally to different groups of mice. After administration of these extracts, mice were allowed access to food and water ad libitum. Behaviour parameters including convulsion, hyperactivity, sedation, grooming, loss of righting reflex, increased or decreased respiration, food and water intake and mortality were recorded for a period of 14 days (Taïwe et al., 2011). The median lethal dose (LD50 ) was estimated according to the method described by Litchfield and Wilcoxon (1949).

2.9. Data analysis Fisher’s exact test (two-tailed) was used to compare percentages of protection against chemicals and maximal electroshock-induced seizures or turning behavior. For the latency to the onset of seizures and the duration of tonic hind limb extension in the maximal electroshock test, the control groups were compared to the other groups by the analysis of variance (ANOVA), two-way, followed by Newman-Keuls post hoc test. The differences were considered significant at p < 0.05. The ED50 (dose of extract necessary to reduce the response by 50% relative to the control value) and 95% confidence intervals values were determined according to the method of Litchfield and Wilcoxon (1949).

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Table 1 Effects of the aqueous extract and the alkaloid fraction from Feretia apodanthera on the latency to tonic-clonic seizures or turning behavior induced by bicuculline, picrotoxin, pentylenetetrazole, Methyl-␤-carboline-3-carboxylate, N-Methyl-D-aspartate and 4-aminopyridine. Treatments

Vehicle FA FA FA FA FA + Flu AF AF AF AF AF + Flu CZP DZP D-AP7 PHO

Dose

The latency to tonic-clonic seizures (min)

(mg/kg)

BIC test (D/T)

– 50 100 150 200 200 + 10 10 20 40 80 80 + 10 – – – –

10.17 ± 0.86 22.69 ± 0.54a 23.59 ± 0.27a 24.82 ± 0.31b 25.19 ± 0.00b Not tested 10.53 ± 0.66 10.55 ± 0.78 10.67 ± 0.91 10.15 ± 0.81 Not tested Not tested – Not tested Not tested

PIC test (D/T) (6/6) (2/6) (1/6) (1/6) (0/6) (3/6) (2/6) (2/6) (1/6)

(0/6)

04.15 ± 0.91 09.44 ± 1.19a 13.24 ± 0.71b 14.38 ± 0.11b – 03.72 ± 0.37 04.46 ± 0.55 04.85 ± 0.33 04.61 ± 0.33 04.65 ± 0.25 04.25 ± 0.11 – Not tested Not tested Not tested

PTZ test (D/T) (6/6) (2/6) (2/6) (1/6) (0/6) (0/6) (3/6) (3/6) (2/6) (2/6) (1/6) (0/6)

04.03 ± 0.49 06.81 ± 0.54 08.17 ± 0.45a 08.25 ± 0.00b – 05.48 ± 0.65 04.77 ± 1.51 04.94 ± 0.91 04.11 ± 0.77 04.41 ± 0.44 04.17 ± 0.32 – Not tested Not tested Not tested

(6/6) (1/6) (1/6) (0/6) (0/6) (0/6) (4/6) (3/6) (3/6) (2/6) (2/6) (0/6) –

␤-CCM test (D/T)

NMDA test (D/T)

4-AP test (D/T)

16.77 ± 1.35 18.33 ± 0.12 24.76 ± 0.61a 29.12 ± 0.00a – Not tested 16.69 ± 0.59 18.19 ± 0.26 19.53 ± 2.67 18.70 ± 1.02 Not tested Not tested (0/6) Not tested Not tested

8.96 ± 0.78 16.14 ± 1.07a 18.28 ± 0.54a 19.73 ± 0.26a 20.26 ± 0.35b Not tested 25.45 ± 0.72b 27.74 ± 0.43b 29.52 ± 0.00c – Not tested Not tested Not tested (0/6) Not tested

22.32 ± 1.71 34.52 ± 1.57a 32.29 ± 0.13a 31.78 ± 0.53a 36.77 ± 1.34a Not tested 45.52 ± 2.42a 54.29 ± 2.13b – – Not tested Not tested Not tested Not tested (0/6)

(6/6) (1/6) (0/6) (0/6) (0/6) (3/6) (2/6) (2/6) (2/6)

–

(6/6) (0/6) (0/6) (0/6) (0/6) (0/6) (0/6) (0/6) (0/6)

–

(6/6) (1/6) (1/6) (0/6) (0/6) (0/6) (0/6) (0/6) (0/6)

Results are expressed as mean ± S.E.M., for 6 animals. Data were analyzed by two-way ANOVA, followed by Newman-Keuls post hoc test. a P < 0.05, b P < 0.01, c P < 0.001, significantly different compared to the vehicle. FA, aqueous extract; AF, alkaloid fraction; BIC, bicuculline; PIC, picrotoxin, PTZ, pentylenetetrazol; ␤-CCM, methyl-␤-carboline3-carboxylate; NMDA, N-Methyl-D-aspartate; 4-AP, 4-aminopyridine; CZP, clonazepam (1 mg/kg for Pic test and 0.1 mg/kg for PTZ test); DZP, diazepam (3 mg/kg for Bic test, 4 mg/kg for ␤-Carb test and 5 mg/kg for MES test); D-AP7, D-2-amino-7-phosphonoheptanoate (33 nmol/kg for NMDA test); PHO, phenobarbital (30 mg/kg for 4-AP test); Flu, flumazenil (10 mg/kg); (D/T), Dead/Treated mice are in parentheses.

Table 2 The ED50 of the aqueous extract and the alkaloid fraction in protecting mice against seizures induced by chemical convulsant or maximal electroshock and their confidence intervals values. Convulsant tests

Aqueous extract (mg/kg)

Alkaloid fraction (mg/kg)

BIC PIC PTZ ␤-CCM NMDA 4-AP MES

136.81 (91.42–142.54) 124.92 (94.56–135.29) 117.57 (84.31–121.8) 61.53 (41.96–72.49) –-– –-– 125.35 (48.37–141.54)

–-– –-– –-– –-– 9.84 (7.91–12.42) 8.77 (7.34–11.98) –-–

Results are ED50 and their confidence intervals values for 6 animals. Data are. BIC, bicuculline; PIC, picrotoxin, PTZ, pentylenetetrazol; ␤-CCM, methyl-␤-carboline-3carboxylate; NMDA, N-Methyl-D-aspartate; 4-AP, 4-aminopyridine.

3. Results 3.1. Effects of Feretia apodanthera on bicuculline-induced seizures The aqueous extract of Feretia apodanthera antagonized bicuculline-induced seizures. The doses of 150 and 200 mg/kg of the aqueous extract protected 66.7% (P < 0.01) and 83.3% (P < 0.01) of mice, respectively. Diazepam, a known anticonvulsant, completely protected the mice against bicuculline-induced seizures (Fig. 1A). The aqueous extract also increased [F(5, 42) = 108.25; P < 0.001] the time to onset of seizures (Table 1). The ED50 of the aqueous extract of Feretia apodanthera was 136.81 (91.42–142.54) mg/kg (Table 2). Contrarily, the alkaloid fraction from Feretia apodanthera did not protect animals against clonic seizures induced by bicuculline (Fig. 1B). 3.2. Effects of Feretia apodanthera on picrotoxin-induced seizures Clonazepam, a known anticonvulsant, completely protected the mice against picrotoxin-induced seizures and exitus. In the same way, the aqueous extract of Feretia apodanthera dose-dependently increased the number of mice protected. The 200 mg/kg dose protected 100% (P < 0.001) of mice (Fig. 2A). Additionally, the aqueous extract showed a significant increase [F(6, 39) = 215.72; P < 0.001] in the delay of the onset to seizures induced by picrotoxin (Table 1).

The ED50 of the aqueous extract was 124.92 (94.56–135.29) mg/kg (Table 2). Flumazenil antagonized the protection provided by the aqueous extract against seizures. The alkaloid fraction from Feretia apodanthera could not protect mice against seizures induced by picrotoxin (Fig. 2B). 3.3. Effects of Feretia apodanthera on pentylenetetrazol-induced seizures The aqueous extract of Feretia apodanthera dose-dependently protected animals against clonic seizures induced by pentylenetetrazol. At the dose of 100 mg/kg, the aqueous extract protected 50% (P < 0.05) of mice against seizures. The dose of 200 mg/kg provided protection to 100% (P < 0.001) of mice (Fig. 3A). The aqueous extract also increased the time to the onset of seizures (Table 1). This effect was comparable to that of clonazepam, a standard antiepileptic drug (Fig. 3A). The ED50 of the aqueous extract was 117.57 (84.31–121.80) mg/kg (Table 2).The total protection of mice provided by the aqueous extract against seizures induced by pentylenetetrazol was strongly antagonized by flumazenil. However, the alkaloid fraction from Feretia apodanthera did not affect pentylenetetrazol-induced seizures (Fig. 3B). 3.4. Effects of Feretia apodanthera on methyl-ˇ-carboline-3-carboxylate-induced seizures The aqueous extract of Feretia apodanthera at the doses of 100, 150 and 200 mg/kg exhibited a 66.7% (P < 0.01), 83.3% (P < 0.01) and 100% (P < 0.001) protection respectively against threshold seizures induced by methyl-␤-carboline-3-carboxylate (Fig. 4A). It also significantly increased the time to the onset of seizures [F(5, 39) = 206,18; P < 0.001]. The ED50 of the aqueous extract was 61.53 (41.96–72.49) mg/kg (Table 2). The alkaloid fraction from Feretia apodanthera had no effect on methyl-␤-carboline-3-carboxylateinduced seizures (Fig. 4B). 3.5. Effects of Feretia apodanthera on N-Methyl-D-aspartate-induced turning behavior The aqueous extract of Feretia apodanthera had a moderate effect on N-Methyl-D-aspartate-induced turning behavior (Fig. 5A). 50% (P < 0.05) of mice were protected at the doses of 150 and

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Fig. 1. Effects of the aqueous extract and the alkaloid fraction of Feretia apodanthera on bicuculline-induced seizures in mice. (A) Aqueous extract, (B) Alkaloid fraction. Illustrated are the percentages of protected animals. N = 6 animals per dose. Data were analysed by Fisher Exact Test (two-tailed), a P < 0.05, b P < 0.01, c P < 0.001, significantly different compared to the vehicle (CON); FA50 = aqueous extract 50 mg/kg; 10AF = alkaloid fraction 10 mg/kg; DZP = diazepam 3 mg/kg.

Fig. 2. Effects of the aqueous extract and the alkaloid fraction of Feretia apodanthera on picrotoxin-induced seizures in mice. (A) Aqueous extract, (B) Alkaloid fraction. Illustrated are the percentages of protected animals. N = 6 animals per dose. Data were analysed by Fisher Exact Test (two-tailed), a P < 0.05, c P < 0.001, significantly different compared to the vehicle (CON); FA50 = aqueous extract 50 mg/kg; 10AF = alkaloid fraction 10 mg/kg; CZP = clonazepam 1 mg/kg; Flu10 = flumazenil 10 mg/kg.

Fig. 3. Effects of the aqueous extract and the alkaloid fraction of Feretia apodanthera on pentylenetetrazol-induced seizures in mice. (A) Aqueous extract, (B) Alkaloid fraction. Illustrated are the percentages of protected animals. N = 6 animals per dose. Data were analysed by Fisher Exact Test (two-tailed), a P < 0.05, b P < 0.01, c P < 0.001, significantly different compared to the vehicle (CON); FA50 = aqueous extract 50 mg/kg; 10AF = alkaloid fraction 10 mg/kg; CZP = clonazepam 1 mg/kg; Flu10 = flumazenil 10 mg/kg.

200 mg/kg aqueous extract. Contrary, the turning behavior induced by N-Methyl-D-aspartate (75 mg/kg) was significantly antagonized by the alkaloid fraction from Feretia apodanthera. Animals were

completely protected both by the alkaloid fraction (80 mg/kg) and by D-AP7 (Fig. 5B). The ED50 of the alkaloid fraction in protecting animals against turning behavior was 9.84 (7.91–12.42)

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Fig. 4. Effects of the aqueous extract and the alkaloid fraction of Feretia apodanthera on methyl-␤-carboline-3-carboxylate-induced seizures in mice. (A) Aqueous extract, (B) Alkaloid fraction. Illustrated are the percentages of protected animals. N = 6 animals per dose. Data were analysed by Fisher Exact Test (two-tailed), a P < 0.05, b P < 0.01, c P < 0.001, significantly different compared to the vehicle (CON); FA50 = aqueous extract 50 mg/kg; 10AF = alkaloid fraction 10 mg/kg; DZP = diazepam 3 mg/kg.

Fig. 5. Effects of the aqueous extract and the alkaloid fraction of Feretia apodanthera on N-Methyl-D-aspartate-induced turning behavior in mice. (A) Aqueous extract, (B) Alkaloid fraction. Illustrated are the percentages of protected animals. N = 6 animals per dose. Data were analysed by Fisher Exact Test (two-tailed), a P < 0.05, b P < 0.01, c P < 0.001, significantly different compared to the vehicle (CON); FA50 = aqueous extract 50 mg/kg; 10AF = alkaloid fraction 10 mg/kg; D-AP7 = D-2-amino7-phosphonoheptanoate 33 nmol/kg.

mg/kg, respectively (Table 2). Also, the time to the onset of turning behavior was significantly increased by the aqueous extract [F(5, 41) = 162,53; P < 0.001] and the alkaloid fraction [F(5, 75) = 241,31; P < 0.001] from Feretia apodanthera (Table 1). 3.6. Effects of Feretia apodanthera on 4-aminopyridine-induced seizures The aqueous extract protected 66.7% of mice against 4aminopyridine-induced seizures at the doses of 100, 150 and 200 mg/kg (P < 0.01) (Fig. 6A). The doses of 10 and 20 mg/kg of the alkaloid fraction protected 50% (P < 0.05) and 66.66% (P < 0.01) of mice, respectively. The dose 80 mg/kg alkaloid fraction, similar to phenobarbital, totally protected mice (p < 0.001) (Fig. 6B). Its ED50 was 8.77 (7.34–11.98) mg/kg (Table 2). 3.7. Effects of Feretia apodanthera on maximal electroshock-induced seizures The aqueous extract significantly increased the number of mice protected in this test. The doses of 100 and 150 mg/kg of the aque-

ous extract protected 66.7% (P < 0.01) and 83.3% (P < 0.01) of mice, respectively. The highest dose 200 mg/kg of the extract, similar to phenobarbital, completely protected the mice (Fig. 7A). The ED50 was 125.35 (48.37–141.54) mg/kg (Table 2). The aqueous extract also significantly decreased the duration of tonic hind limb extension induced by maximal electroshock from 6.83 ± 0.56 min in the control group to 2.54 ± 0.00 min in the group treated with the dose 150 mg/kg of the extract (Table 3). The alkaloid fraction from Feretia apodanthera demonstrated a moderate effect in this test.

3.8. Estimation of brain GABA content A significant increase in the level of brain GABA concentration was observed in animals 1 h after oral administration of the aqueous extract of Feretia apodanthera (150 and 200 mg/kg, p.o.) [F(5, 28) = 61,42; p < 0.05]. Contrarily the administration of the alkaloid fraction from Feretia apodanthera (10–80 mg/kg, p.o.) did not produce any significant effect [F(5, 51) = 74,21; p > 0.05] in the level of brain GABA concentration of animals (Table 4).

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Fig. 6. Effects of the aqueous extract and the alkaloid fraction of Feretia apodanthera on 4-aminopyridine-induced seizures in mice. (A) Aqueous extract, (B) Alkaloid fraction. Illustrated are the percentages of protected animals. N = 6 animals per dose. Data were analysed by Fisher Exact Test (two-tailed), a P < 0.05, c P < 0.001, significantly different compared to the vehicle (CON); FA50 = aqueous extract 50 mg/kg; 10AF = alkaloid fraction 10 mg/kg; PHO = Phenobarbital 30 mg/kg.

Fig. 7. Effects of the aqueous extract and the alkaloid fraction of Feretia apodanthera on maximal electroshock-induced seizures in mice. (A) Aqueous extract, (A) Alkaloid fraction. Illustrated are the percentages of protected animals. N = 6 animals per dose. Data were analysed by Fisher Exact Test (two-tailed), a P < 0.05, b P < 0.01, c P < 0.001, significantly different compared to the vehicle (CON); FA50 = aqueous extract 50 mg/kg; 10AF = alkaloid fraction 10 mg/kg; DZP = diazepam 5 mg/kg.

Table 3 Effects of the aqueous extract and the alkaloid fraction from Feretia apodanthera on the duration of tonic hind limb extension induced by maximal electroshock. Treatments

Dose (mg/kg)

Time of tonic hind limb extension seizure (s)

Vehicle FA FA FA FA AF AF AF AF DZP

– 50 100 150 200 10 20 40 80 5

06.83 ± 0.56 05.83 ± 0.07 03.36 ± 0.06a 02.54 ± 0.00b – 04.42 ± 0.07 04.55 ± 0.44 04.86 ± 0.48 05.08 ± 0.57 –

Results are expressed as mean ± S.E.M., for 6 animals. Data were analysed by two-way ANOVA, followed by Newman-Keuls post hoc test. a P < 0.05, b P < 0.01, significantly different compared to the vehicle, FA, aqueous extract; AF, alkaloid fraction. DZP, diazepam.

3.9. Effect of Feretia apodanthera on GABA-T activity The brain homogenate incubated with the aqueous extract of Feretia apodanthera, showed a significant (F(7, 42) = 76.2, P < 0.001)

reduction in the brain GABA-T activity (Table 5). The inhibition induced by the aqueous extracts was dose-dependent with 24.02% reduction (P < 0.05) observed at the concentration 75 ␮g/ml and 68.72% reduction (P < 00.001) seen for 175 ␮g/ml. The effect provided by 200 ␮g/ml, 70.03% (P < 0.001) was comparable to that of sodium valproate 80.43% (P < 0.001). The IC50 of the inhibition induced by Feretia apodanthera aqueous extract was 97.29 ␮g/ml. Contrarily the brain homogenate incubation with the alkaloid fraction from Feretia apodanthera (10–120 ␮g/ml) did not produce any significant effect [F(5, 51) = 74,21; p > 0.05] on GABA-T activity (Table 5). 3.10. Acute toxicity No deaths were observed during 14 days after oral administration of single doses of the aqueous extract and the alkaloid fraction from Feretia apodanthera up to the highest doses tested 7200 and 5120 mg/kg, respectively. Doses that were tested for anticonvulsant activity did not produce any behavioral changes. The behavioural changes observed at high doses of the aqueous extract (from dose

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Table 4 Effects of the aqueous extract and the alkaloid fraction from Feretia apodanthera or sodium valproate on brain GABA content in mice. Treatments

Dose (mg/kg)

GABA level in brain tissue (mmol/L)

Percentage of Increase (%)

Vehicle FA FA FA FA AF AF AF AF SVA

– 50 100 150 200 10 20 40 80 300

3.79 ± 0.10 3.92 ± 0.11 4.05 ± 0.12 4.37 ± 0.11a 4.68 ± 0.12a 3.89 ± 0.11 3.89 ± 0.10 3.89 ± 0.11 3.90 ± 0.10 4.83 ± 0.1b

– 3.30 6.82 15.06 23.31 2.46 2.47 2.56 2.63 27.16

Results are expressed as mean ± S.E.M., for 6 animals, a P < 0.05, b P < 0.01 significantly different compared to the vehicle, data were analysed by two-way ANOVA, followed by Newman-Keuls post hoc test, FA, aqueous extract; AF, alkaloid fraction; SVA, sodium valproate.

Table 5 Effects of the aqueous extract and the alkaloid fraction from Feretia apodenthera or sodium valproate on brain GABA-T activity in mice. Treatments

Dose (␮g/kg)

GABA-T activity in brain tissue (units/mg protein ± S.E.M.)

Percentage of Inhibition (%)

Vehicle FA FA FA FA FA FA FA

– 25 50 75 100 150 175 200

3.92 ± 0.05 3.15 ± 0.03 2.98 ± 0.32a 2.91 ± 0.35a 1.45 ± 0.36b 1.35 ± 0.23c 1.22 ± 0.04c 1.17 ± 0.06c

– 19.73 24.02a 25.68a 63.07b 65.45c 68.72c 70.03c

FA FA FA FA FA FA FA

10 20 40 60 80 100 120

3.64 ± 0.09 3.71 ± 0.05 3.71 ± 0.08 3.74 ± 0.09 3.73 ± 0.13 3.66 ± 0.14 3.72 ± 0.09

05.81 03.92 03.92 03.32 03.53 05.25 03.70

0.76 ± 0.04c

80.43c

300

SVA

Results are expressed as mean ± S.E.M., P < 0.05, P < 0.01, P < 0.01 significantly different compared to the vehicle, data were analysed by two-way ANOVA, followed by Newman-Keuls post hoc test, FA, aqueous extract; AF, alkaloid fraction; SVA, sodium valproate. Units are referred to the number of GABA transaminase activity inhibited per mg protein (GABA-T) in the brain homogenates by the studied extracts. a

b

c

of 800 mg/kg) were sedation, hypoactivity, hyperventilation and motor dysfunction. However, alkaloid fraction from Feretia apodanthera did not produce any sedation or alteration in locomotor activity of animals. 4. Discussion The aqueous extract of Feretia apodanthera strongly protected mice against the convulsions induced by bicuculline. Bicuculline, a selective competitive GABAA receptor antagonist acts directly on the postsynaptic GABAA receptor complex to induce hyperactivity behaviour and seizures (Sperber et al., 1989). GABA is the major known inhibitory neurotransmitter of the mammalian nervous system (Vicini et al., 1986; Meldrum and Nilsson, 1976). The dosedependent increase in the anticonvulsant effect of the aqueous extract of Feretia apodanthera in the bicuculline-induced seizures test suggested the presence of compounds capable of interacting with GABA neurotransmission at the GABAA receptor site in central nervous system. Picrotoxin also blocks the neuronal inhibitory effect of GABA to induce generalized seizures in animal (Nicoll, 2007). The dose-dependent effect and the 100% of mice protection by the aqueous extract against the convulsions induced by picrotoxin suggested that the anticonvulsant activity of the aqueous extract of Feretia apodanthera could be related to the picrotoxin

site of the GABAA receptor complex (Ngo Bum et al., 2012). The aqueous extract of Feretia apodanthera strongly protected mice against pentylenetetrazol-induced convulsions. The antagonism of pentylenetetrazol-induced seizures also suggested the existence of anticonvulsant activity and the interaction of the aqueous extract with GABAergic neurotransmission (Ngo Bum et al., 2012; PérezSaad and Buznego, 2008). In addition, the inhibition by flumazenil of the anticonvulsant effects of the aqueous extract of Feretia apodanthera in PIC and PTZ tests indicated the interaction with the GABAergic system (Taïwe et al., 2010; Rang et al., 1999; Matsumoto et al., 1997). Methyl-␤-carboline-3-carboxylate has affinity for benzodiazepine receptors and produces anxiogenic and proconvulsant effects, which can be completely reversed by benzodiazepine agonists (Thiebot et al., 1988). The aqueous extract of Feretia apodanthera exhibited a dose-dependent protection against threshold seizures induced by methyl-␤-carboline-3-carboxylate, supporting that the anticonvulsant properties of this plant could be related to the presence of some components in the aqueous extract activating the benzodiazepine site of the GABAA receptor complex (Rang et al., 1999). This result supported the first one where flumazenil antagonized the anticonvulsant effect of aqueous extract of Feretia apodanthera. Both the aqueous extract and the alkaloid fraction from Feretia apodanthera completely antagonized NMDA-induced turning behavior in mice. But the effect of the alkaloid fraction

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was better. Given the involvement of the NMDA receptor complex in epileptic and epileptiform activity in vivo and since excitatory amino acid antagonists acting at the NMDA or non-NMDA receptor have been shown to possess anticonvulsant and antiepileptic properties in several animal models of epilepsy (Rogawski, 1992; Löscher and Hönack, 1991; Davies et al., 1986), it could be suggested that the alkaloid fraction from Feretia apodanthera also possess anticonvulsant properties. 4-aminopyridine is a K+ channel blocker and Ca2+ channel stimulator, both voltage dependent gated (Rogawski and Barker, 1983; Thesleff, 1980), which shows convulsant action when administered systemically to a variety of species. Furthermore, the convulsant effects of 4-aminopyridine is due to the release of excitatory neurotransmitters. The glutamate release results in over activation of excitatory amino-acid receptors, mainly the NMDA-type (Thesleff, 1980). And an enhancement in the glutamatergic neurotransmission has been linked to the 4-aminopyridine convulsant action (Morales-Villagran et al., 1996), since the administration of NMDA receptor antagonists protects against 4-aminopyridine induced seizures (Fragoso-Veloz and Tapia, 1992). The aqueous extract and the alkaloid fraction from Feretia apodanthera exhibiting anticonvulsant effects here might be link to the glutamate signal pathway. Finally, only the aqueous extracts of Feretia apodanthera inhibited maximal electroshockinduced convulsions, probably by prolonging the inactivation of sodium channels (Holmes, 2007). The maximal electroshock and PTZ tests are of predictive relevance considering the clinical spectrum of activity of experimental compounds (Kupferberg and Schmutz, 1997). They are assumed to identify anticonvulsant drugs effective against generalized tonic-clonic/partial seizures in man (Ngo Bum et al., 2009b; Holmes, 2007; Kupferberg and Schmutz, 1997). Some anticonvulsant, anxiolytic and sedative compounds (e.g. sodium valproate) are known to exert their pharmacological action also by causing an increase in GABA content in mice cerebral hemisphere (Taïwe et al., 2010; Chapman et al., 1983; Saad, 1972). It was found that the aqueous extract of Feretia apodanthera significantly enhanced the brain GABA concentration which again was suggestive of the interaction of the plant aqueous extract with the GABA neurotransmission. The involvement of GABA neurotransmission is supported by the inhibition of the activity of GABA-T by the aqueous extract of Feretia apodanthera that also explained the increase of brain GABA concentration in pretreated mice with the aqueous extract. GABA-T is the primary catabolic enzyme in the mammalian brain that catalyzes the transfer of amino group from GABA to ␣- ketoglutarate leading to the depletion in the level of GABA (Sherif and Saleem Ahmed, 1995). Taken together, it could be suggested that the behavioural action (sedation, hypo-activity) of the very high doses of aqueous extract was correlated to GABAergic neurotransmission in the brain (Taïwe et al., 2012b; Chapman et al., 1983). The efficacy of most herbal remedies could be attributed to various active principles the plant (Dubois et al., 1986; Wagner et al., 1983).

5. Conclusions Feretia apodanthera possess anticonvulsant activity and provides scientific rationale for the use of the aqueous extract of Feretia apodanthera for the amelioration of epilepsy in traditional medicine in Cameroon. More studies are necessary to clarify the antiepileptic components and the mechanisms underlying the plant activiyies.

Conflict of interest The authors declare that they have no conflict of interests.

Acknowledgments The authors are very thankful to the University of Ngaoundere, Cameroon and the University of Buea, Cameroon, for supporting us by providing apparatus and chemicals.

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