the plant antiaris toxicaria ( family moraceae ) is an indigenous plant common in ghanaian forests .
foto  or  kyenkyen  in akan and the bark cloth tree in english . despite considerable advancements in the treatment of neurological disorders
currently available antiepileptic drugs ( aeds ) have debilitating adverse effects on cognition and behaviour .
these adverse effects are commonly and consistently observed with barbiturates , benzodiazepines , and topiramate [ 4 , 5 ] .
these problems are known to prevail more in developing countries due to lack of facilities for proper diagnosis and treatment along with monitoring of aed serum levels [ 6 , 7 ] .
natural products and plants already used in traditional medicine can be a good place to start in the search for safer and more effective options .
numerous plants used for the treatment of epilepsy traditionally have been shown to be potent in models of epilepsy and several such plants remain to be scientifically validated .
leonotis leonurus , delphinium denudatum , mimosa pudica , and synedrella nodiflora are but a few examples [ 810 ] .
preliminary screening of the aqueous extract of antiaris toxicaria revealed significant anticonvulsant effect in pentylenetetrazole - induced seizure test .
hence , this study seeks to further explore the extract 's potential as an anticonvulsant .
stem bark of antiaris toxicaria was harvested from the knust campus , kumasi , and authenticated at the pharmacognosy department of the faculty of pharmacy , knust , kumasi , ghana . a voucher specimen ( knust / hm1/011/s007 )
dried stem bark was milled into powder using a commercial grinder . the coarse powder ( 431  g )
was extracted by cold maceration with distilled water as solvent at room temperature for five days .
filtrate was oven - dried to obtain a yield of 23.40%  w / w of antiaris toxicaria aqueous extract ( aae ) .
male icr mice and sprague - dawley rats weighing between 20 and 25  g and 120 and 145  g , respectively , were obtained from noguchi memorial institute for medical research , accra , ghana , and kept in the departmental animal house .
animals were maintained under laboratory conditions of temperature , humidity , and light , housed in stainless steel cages ( 34  47  18  cm ) , and allowed access to water and food ad libitum .
all animals were handled in accordance to the guide for the care and use of laboratory animals   and experiments were approved by the faculty ethics committee .
diazepam ( dzp ) , pentylenetetrazole ( ptz ) , picrotoxin ( pct ) , 4-aminopyridine ( 4-ap ) , and strychnine ( str ) were purchased from sigma - aldrich inc . , st .
flumazenil was obtained from app pharmaceuticals , llc , schaumburg , il , usa . the method as described by vellucci and webster , 1984 , and moezi et al . , 2011 ,
the plant extract was administered at doses of 200 , 400 , and 800  mg  kg body weight orally .
intraperitoneal ( i.p ) injection of diazepam ( 0.1 , 0.3 , and 1  mg  kg ) was used as reference anticonvulsant drug .
animals were pretreated with the plant extract thirty minutes and diazepam fifteen minutes before administration of pentylenetetrazole ( ptz ) 85  mg  kg subcutaneously .
control animals were pretreated with distilled water ( 10  ml  kg , p.o . ) .
the onset of , total duration as well as frequency of clonic seizures were measured within a thirty minute period .
animals received aae at doses of 200 , 400 , and 800  mg  kg ( p.o . ) body weight .
picrotoxin ( pct ) was injected intraperitoneally at a dose of 3  mg  kg fifteen minutes after pretreatment with diazepam and thirty minutes after aae .
diazepam ( 0.1 , 0.3 , and 1  mg  kg ) served as positive control .
control animals received distilled water ( 10  ml  kg , p.o . ) .
anticonvulsant activity was scored similarly to that stated in the ptz test [ 13 , 15 ] .
tonic convulsions of hind limb extremities of mice were induced using electrical current ( 50  ma , 60  hz , and 0.2 seconds ) via ear clip electrodes .
control group animals received distilled water orally ( 10  ml  kg , p.o . ) .
carbamazepine at doses of 3 , 10 , and 30  mg  kg orally served as reference anticonvulsant .
aae was tested at doses of 200 , 400 , and 800  mg  kg orally .
aae was administered orally at doses of 200 , 400 , and 800  mg  kg body weight .
strychnine was injected subcutaneously at a dose of 2  mg  kg thirty minutes after aae administration .
standard anticonvulsant employed was diazepam ( 0.1 , 0.3 , and 1  mg  kg , i.p ) .
the onset of , decrease in the total duration plus frequency of tonic convulsions were taken as indication of anticonvulsant activity .
animals received aae orally at doses of 200 , 400 , and 800  mg  kg body weight .
4-aminopyridine was dissolved in normal saline and injected subcutaneously at a dose of 10  mg  kg body weight thirty minutes after drug treatments .
control animals were pretreated with normal saline ( 10  ml  kg ) and sodium valproate at 100 , 200 , and 400  mg  kg served as positive control .
hind limb tonic extensions and decrease in the total duration of convulsions were recorded . method as described by morales - villagrn and tapia , 1996 ,   was used with slight modifications .   in order to investigate the mechanism of action of aae as an anticonvulsant
, mice were treated with flumazenil , a benzodiazepine antagonist ( 1  mg  kg , i.p ) fifteen minutes before the administration of aae ( 400  mg  kg , p.o . ) .
thirty ( 30 ) minutes later , convulsions were induced with pentylenetetrazole at 85  mg  kg , intraperitoneally .
other groups of animals received aae or flumazenil or diazepam ( 0.3  mg  kg , i.p ) only .
animals were observed for thirty minutes after treatment via video recording for latency and duration of convulsions .  
analysis of variance ( anova ) followed by newman - keuls ' post hoc test was used to determine significant differences between means .
two - way anova followed by bonferroni test was used in the flumazenil experiment . in the 4-aminopyridine seizure test
, the kaplan - meier method was used in estimating survival relative to time and survival differences were analyzed with the log - rank test .
statistical analyses were carried out with graph pad prism version 5.0 ( graphpad software , san diego , ca , usa ) and sigmaplot version 11.0 ( systat software , inc . ,
pentylenetetrazole ( 85  mg  kg , s.c . ) produced myoclonic jerks in all mice pretreated with the distilled water .
aae ( 200800  mg  kg ) produced a significant ( p = 0.0021 ;  f3,16 = 7.671 ; figure 1(a ) ) dose - dependent increase in time taken to the onset of clonic seizures . in the extract - treated animals ,
frequency of seizures was also decreased nondose - dependently , and only the 800  mg  kg dose was significant ( p = 0.0511 ; f3,16 = 3.213 ; figure 1(a ) ) . aae produced significant ( p = 0.0018 ; f3,16 = 8.005 ; figure 1(c ) ) dose - dependent decrease in the total duration of convulsions in all animals pretreated with the various doses of the extract . the reference anticonvulsant diazepam ( 0.11.0  mg  kg , i.p ) also profoundly delayed the onset of myoclonic jerks , decreased frequency of jerks and significantly ( p < 0.0001 ; f3,16 = 125.8 ; figures 1(b ) and 1(d ) ) antagonized ptz - induced seizures .
picrotoxin ( 3  mg  kg , i.p ) produced clonic seizures in all mice pretreated with the distilled water .
aae ( 200800  mg  kg ) produced a dose - dependent increase in latency to seizures which was significant ( p = 0.0009 ; f3,16 = 9.108 ; figure 2(a ) ) at all doses .
the frequency of seizures was also significantly ( p = 0.0081 ; f3,16 = 5.591 ; figure 2(a ) ) decreased dose - dependently .
aae produced significant ( p = 0.0002 ; f3,16 = 12.53 ; figure 2(c ) ) dose - dependent decrease in the total duration of seizures in all animals .
the reference anticonvulsant diazepam ( 0.11.0  mg  kg , i.p . ) also profoundly delayed the onset of myoclonic jerks , decreased frequency of jerks and significantly antagonised ( p < 0.0001 ; f3,16 = 17.72 ; figures 2(b ) and 2(d ) ) pct - induced seizures .  
the extract did not affect the latency to onset of hind limb tonic extensions and duration of convulsions significantly ( figure 3 ) .
the reference anticonvulsant carbamazepine ( 3 , 10 , and 30  mg  kg , p.o . ) , however , delayed the onset of hles significantly ( p = 0.0054 ; f3,16 = 6.193 ; figure 3 ) and decreased the total duration of electroshock - induced convulsions .  
aae did not affect the latency to onset of hind limb tonic extensions and duration of convulsions significantly ( figure 4 ) .
the reference anticonvulsant diazepam ( 1  mg  kg , i.p ) , on the other hand , delayed the latency to convulsions significantly ( p < 0.0001 ; f4,20 = 36.60 ; figure 4 ) .
the duration of convulsions was also significantly ( p < 0.0001 ; f4,20 = 14.31 ;
4-aminopyridine ( 10  mg  kg , i.p . ) produced hind limb tonic extensions in all animals .
the extract produced a significant ( p < 0.0001 ;  f3,28 = 22.02 ; figure 5(a ) ) increase in time taken to the onset of convulsions .
sodium valproate ( 100400  mg  kg ) also significantly ( p < 0.0001 ; f3,28 = 17.61 ; figure 5(b ) ) delayed the onset of convulsions .
the extract significantly ( p = 0.0004 ,  (df = 3 ) = 18.11 ; figure 6(a ) ) improved survival of the animals after induction of convulsions .
the test for trend presented significant ( p < 0.0001 , (df = 1 ) = 15.79 ) effect of the treatment groups on median survival indicating a linear trend .
sodium valproate also produced similar effects on survival ( p < 0.0001 , (df = 3 ) = 21.23 ; figure 6(b ) ) and linear trends ( p < 0.0001 ,  (df = 1 ) = 19.17 ) .
survival curves show a decrease in probability of survival with time . however , probability of survival increased with increasing dose .
flumazenil significantly ( p = 0.0009 ; f1,8 = 26.09 ; figure 7(c ) ) reversed the reduction in duration of seizures produced by the extract . a significant reversal ( p = 0.0003 ; f1,8 = 37.51 ; figure 7(a ) ) was also obtained for the latency to seizures .
effects of diazepam were significantly ( p < 0.0001 ; figures 7(a ) , 7(b ) , and 7(c ) ) reversed by flumazenil .
the outcome of this study provides evidence that the aqueous extract of the stem bark of antiaris toxicaria possesses anticonvulsant activity .
the ability of aae to delay the onset of convulsions and/or shorten the duration of convulsions was considered an indication of anticonvulsant activity .
judging from the data obtained , the plant extract exhibited anticonvulsant activity in the ptz test just like diazepam which can be due to action on gaba system [ 1921 ] .
it , however , has been shown to be due to inhibition and/or attenuation of gabaergic neurotransmission [ 22 , 23 ] .
it is therefore likely that aae produces its anticonvulsant effect directly or indirectly by enhancing gabaergic neurotransmission in the brain .
- synaptic inhibition mediated by gaba often leads to inhibition of neurotransmitter release from the excitatory arm .
pct - induced seizures which are due to the decreased gabaa - receptor - mediated inhibition in turn promote the excitatory arm of the cns mainly mediated by glutamate [ 20 , 22 ] .
the extract being effective in the pct - induced seizure test points to a more specific action on gaba - mediated neurotransmission .
this has been further confirmed by the use of flumazenil to reverse the anticonvulsant properties of aae in the ptz - induced seizure model .
flumazenil is a benzodiazepine antagonist at the gabaa receptor possessing the ability to reverse anticonvulsant effects and the accompanying alterations in extracellular glutamate concentration .
the specificity of flumazenil to this receptor has been proven as several authors have demonstrated that the antagonist has no effect on diazepam 's action on voltage - dependent na channels [ 2931 ] .
retigabine is currently the only approved drug for the treatment of epilepsy which functions through activation of potassium currents [ 3234 ] and has promise in the treatment of pharmacoresistant epilepsy .
it is also known that 4-aminopyridine stimulates the release of neurotransmitters , including glutamate , in numerous preparations such as brain slices [ 3538 ] and the neuromuscular junction .
furthermore , it has been recently found by a microdialysis procedure in vivo that glutamate is by far the amino acid predominantly released by 4-aminopyridine in the striatum .
therefore , due to the well - recognized function of excitatory amino acid receptors in convulsive and excitotoxic mechanisms , it seems feasible that the excess glutamate released may be involved in the excitatory effects .
this indicates that the extract may be acting , in addition to the above , by modulating the release of glutamate . from the pattern of survival curves
, it can be inferred that the dose of the extract does affect survival rate offering significant protection against death from 4-aminopyridine - induced convulsions .
this was evident from the log - rank analysis which indicated significant differences between survival curves for each dose level .
median survival time ranged from 30  min and death may be prevented totally at the highest dose of aae .
the exact mechanism by which the extract offers this protection may be linked to its ability to reduce excitotoxic effects mediated by glutamate release .
this indicates that it is not effective in partial and generalized tonic seizures   and is unable to prevent seizure spread .
the fact that aae produced no protective effects against strychnine - induced convulsions suggests it does not interact with the glycine - mediate inhibitory pathway .
the results presented here indicate that the aqueous extract of antiaris toxicaria exhibits anticonvulsant activity , possibly through gaba - mediated inhibition and inhibition of glutamate mediated excitation via activation of potassium ion channels .
 
 antiaris toxicaria ( moraceae ) was evaluated for anticonvulsant activity in rodents . 
 animal models used include maximal electroshock test ( mest ) ; pentylenetetrazole - induced ( ptz ) convulsions ; picrotoxin - induced ( pct ) convulsions ; strychnine- ( str- ) and 4-aminopyridine - induced convulsions . 
 increase in latency to seizures as well as reduction in duration and frequency of seizures indicated anticonvulsant activity . 
 the extract was more effective in all models used except the maximal electroshock test and strychnine - induced convulsions . 
 antiaris toxicaria aqueous extract ( 200 , 400 , and 800  mg  kg1 ) significantly ( p < 0.05  0.01 ) shortened the duration of convulsions in ptz- and pct - induced seizures . 
 delay in the onset of convulsions in the two tests was significant ( p < 0.001 ) . 
 reduction in the frequency of seizures was also significant ( p < 0.05  0.001 ) in both tests . 
 antiaris further delayed the onset of seizures in 4-aminopyridine model while producing 75% protection against death in mice . 
 diazepam ( 0.1 , 0.3 , and 1  mg  kg1 ) , carbamazepine ( 3 , 10 , and 30  mg  kg1 ) , and sodium valproate ( 100400  mg  kg1 ) were used as reference anticonvulsant drugs for various models . 
 flumazenil blocked the effect of the extract in the ptz test significantly suggesting that antiaris toxicaria may be acting by enhancing the effects of the gabaergic system . 
 antiaris toxicaria aqueous extract therefore possesses anticonvulsant activity . 
1. Introduction
2. Materials and Methods
3. Results 
4. Discussion 
5. Conclusion