Anti-convulsivant and sedative like-effect of Abyssinone V-4 ’ methyl ether Isolated from Erythrina droogmansiana ( Leguminosae )

1 Department of chemistry, Faculty of Sciences, University of Ngaoundere, POBOX 454, Ngaoundere, Cameroon. 2 Département of biological Sciences, Faculty of Sciences, University of Ngaoundere, POBOX 454, Ngaoundere, Cameroon. 3 Institute of Medical Research and Medicinal plants Studies (IMPM), POBOX 6163, Yaounde, Cameroon. 4 Department of Organic Chemistry, University of Yaounde 1, POBOX 812, Yaounde, Cameroon.


INTRODUCTION
Epilepsy is a disease that affects about 40 millions 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, Ngounou and collaborators estimated the prevalence in sub-Saharian Africa to be two or three times higher 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.Thus, epilepsy is one of 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, mostly amongst .populations with very low income (Geoffrey and Kirby, 1996).Phytotherapy relies on the use of a wide variety of plant species including Erythrina droogmansiana.Abyssinone V-4' methyl ether is a prenylated flavonoid isolated from plant Erythrina droogmansiana.50 flavonoids have been obtained during the last three decades from about 15 species of Erythrina genus (Kamat et al., 1981;Njamen et al., 2003;Kebenei et al., 2011;Mvondo et al., 2012), with prenylated flavonone, isoflavones and pterocarpans being the major non alkaloid secondary metabolites isolates so far (Mvondo et al., 2012;Wandji et al., 1994).Among these metabolites, there are abyssinones which are prenylated flavonoids isolated from Erythrina abyssinica (Kebenei et al., 2011).
These molecules have gained attention since abyssinone II was reported to show aromatase inhibitory activity (Maiti et al., 2007).The antioxidant and cytotoxic activities of abyssinone I, abyssinone II and related compounds have been reported (Rao GV et al., 2009).
The anti-inflammatory and antioxidant activities of prenylflavanones isolated from Erythrina sigmoidea have been reported (Njamen et al., 2004).Some abyssionones especially abyssinone V have recently been reported to inhibit the activity of the protein tyrosine phosphatase-1B (PTP 1B), which is directly linked to type-2 diabetes and obesity therapy (Na MK et al., 2006;Kone et al., 2011) and to exhibit estrogenic properties (Mvondo et al., 2012).The anticonvulsivant and sedative property of abyssinone V-4-methyl ether has not yet been evaluated.Therefore, the present study was undertaken to investigate the anticonvulsivant and sedative effect of abyssinone V-4-methyl ether, a prenylated flavonoid isolated from Erythrina droogmansiana.

Plant material
The root bark of E. droogmansiana T. Durand was collected from Nkomekoui, Yaounde-Cameroon in August 2010.Identification and authentication of the plant material was done at the National Herbarium Yaoundé, Cameroon, where a voucher specimen N°4261/SRFK has been deposited.

Extraction and isolation of the compound
Air-dried and pulverized root bark of E. droogmansiana (1.2 Kg) was extracted successively with ethyl acetate and methanol.The extract was filtered and the solvent evaporated under reduced pressure, 150 g of residue was obtained.This extract was subjected to chromatography over silica gel packed in n-hexane.Gradient elution was done using n-hexane, ethyl acetate and methanol in increasing polarity to give 7 series of fractions mixed on the basis of TLC.Repeated column chromatography with hexane-EtAOc (90:10) yielded YG4 and other compounds.The structures have been elucidated using spectral methods (MS, NMR, and element analysis).
The compound YD4 was obtained as a white powder (500 mg) and showed a [M]+at m/z 422.2094 corresponding to molecular formula (C 26 H 30 O 5 ).This compound was identified as abyssinone V-4'-methyl ether (Figure 1).The presence of a flavonone skeleton was evident from the 1 HNMR spectra at 5.33 (1H, dd) and 2.76 (1H, 2q) corresponding to the H-2 and to the H-3 proton of the C-ring of flavonones respectively.From the 13 CNMR spectra, the presence of signal at 79.3 and 42.5 respectively indicated the C-2 and C-3 of the C-ring of flavonone.The 1 H and 13 CNMR spectra data of this compound were compared to those previously published (Yenesew et al., 1998).Other compounds obtained were droogmascarpine, phaseollidin, stigmasterol and β-sitosterol.ccAll extract were prepared according to traditional healers and chemists, and were administered 1 hour before the tests at the following doses 12.5, 25, 50 and 100 mg/kg.

Animals and drug administration
Adult male mice: Musmusculus Swiss 22 ± 3 g, 2 months old and obtained from the animal laboratory of our University were used for this study.Abyssinone V-4'-methyl ether and distilled water were given orally to experimental animals after suspending in a mixture of distilled water and 0.5% dimethylsulfoxide (DMSO).The positive control groups received the same experimental handling as those of the test groups.

Diazepam-induced sleep test
Mice were divided into five groups of 5 mice and received different treatments.Group I (negative control) was treated with distilled water.Groups II to V (test groups) were treated with 4 doses of Abyssinone.The sleep potentiating effects of the plant extracts were studied in mice that had received diazepam (ip) at a dose of 50 mg/kg 1 hour after treatment.The time taken from the loss of the straightening reflex to its regain gave the sleeping time (Ngo Bum et al., 2009a,b;Rakotonirina et al., 2001).

Pentylenetetrazol (PTZ) test
Mice were divided into six groups of 5 mice each and 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 abyssinone.Group VI clonazepam, 0.1 mg/kg ip, was used as positive control.Clonic seizures were induced in mice by the ip injection of 70 mg/kg PTZ.The protective effect of the different treatments given 1 h before PTZ injection was recorded.Animals that did not convulse within the 10 min of observation were qualified protected (Ngo Bum et al., 2010;Ngo Bum et al., 2001;Wamil et al., 1994.).

Picrotoxine (PIC) test
Six groups of 5 mice each were treated as above.However the positive control group received 0.4 mg/kg clonazepam ip.Clonic seizures were induced in mice by the ip injection of 7.5 mg/kg PIC.Mice were observed for 15 min.A protective effect of the different treatments given 1 h before PICinduced clonic seizures was recorded.Animals that did not convulse within 15 min of observation were qualified protected (Ngo Bum et al., 2005;Ngo Bum et al., 2001;Bernasconi et al., 1988;Lehmann et al., 1988).

Pilocarpine (PILO) test
Mice were divided into six groups of 7 mice each and 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 abyssinone.Group VI Diazepam, 0.3 mg/kg ip, was used as positive control.Generalized convulsions were induced in mice by the ip injection of 375 mg/kg PILO.The scopolamine administered ip (1 mg/kg) 15 min after different treatments and 45 min before PILO injection was recorded.Animals that did not convulse and did not die within the following one to 24hours of observation were qualified protected (Ethel et al 2010;Henrik et al., 2003).

Statistical analysis
Three parameters were measured: the protection against convulsion induced by PTZ, PIC and PILO, the latency and the sleeping time.The percentages of protected animals were analyzed using the Fisher Exact Test (two-tail).The Fischer Exact test was used to compare the percentage of protected mice.Data were p < 0.05 were qualified significant.The ED50 were determined with Statgraphics Plus (confident limits at 95%).

Effect of Abyssinone on diazepam-induced sleep in mice.
Abyssinone V-4' methyl ether protected mice for convulsions induced by chemically substances.In diazepaminduced sleep test, the abyssinone not increased sleeping time of the control group (fig.2).

Effect of Abyssinone V-4' methyl etherPIC-induced convulsions in mice.
The doses of 25 and 100 mg/kg provided all protection against PIC-induced seizures (p < 0.001) and at a doses 12.5 and 50 mg/kg the extract protected 80% of mice (p < 0.001) (fig.4)

DISCUSSION
Abyssinone did not potentialise the sleep time.The sedative properties are absent in this extracts (Ngo Bum et al., 2009a;2009b;Rakotonirina et al., 2001).This result could be related to the absence of some components in the extracts activating the benzodiazepine, barbiturate and/or GABA receptors in the GABA A receptor complex (Bonin and Orser, 2008;Olkkola and Ahonen, 2008;Rang et al., 1999).While sodium thiopental that act on the barbiturate binding site directly gate the chloride ion channel of the GABA A receptor complex.
Abyssinone presents anticonvulsivant properties.This extract antagonizes the PTZ, PIC-and PILO-induced seizures, this suggests the interaction of these extract with the GABA-ergic neurotransmission (Salihand Mustafa, 2008;Perez-Saad and 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 (Li-Ping et al., 2008).Moreover, some studies showed that PTZ diminishes the GABA-ergic tone (Ahmadiani, 2003), probably by a competitive antagonist action on the BZD receptors (Rehavi et al., 1982).Drugs that enhance GABA A -receptor neurotransmission, such as BZDs (Ahmadiani et al., 2003;White, 1997) 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 GABA A receptor complex.Isoniazide can enhance convulsions in patients with seizure disorders, and it is regarded as a GABA-synthesis inhibitor (Kale Shubhangi et al., 2010).
The antagonism of PILO -induced convulsions suggests the presence of anticonvulsant effect through in the limbic system, particularly in the hippocampus, entorhinal cortex and amygdala (Bartolomei et al., 2005).The subject of this review, the pilocarpine model, belongs to status Epileticus models.This model appears to be highly isomorphic with the human disease, so it has been used in many laboratories since its first description a quarter of a century ago (Turski et al., 1983a, b).

CONCLUSION
Abyssinone V-4' methyl ether did not show sedative properties but instead showed very good anticonvulsant activities against PTZ, PIC or PILO-induced seizures.However, Erythrina, medicinal plants could be used in the treatment of epilepsy.

Fig. 2 :
Fig. 2: Effect of Abyssinone on diazepam-induced on sleep time in mice.

Figure 6
Figure 6 presents the different stages reached by mice after administration of the pilocarpine.At doses of 25 and 50