current available anticonvulsant drugs are able to efficiently control epileptic seizures in about 50% of the patients ; another 25% may show improvement whereas the remainder does not benefit significantly ( 1 ) . furthermore , undesirable side effects of the drugs used clinically often render treatment difficult so that a demand for new types of anticonvulsants exists . one of the approaches to search for new antiepileptic drugs is the investigation of naturally - occurring compounds , which may belong to new structural classes . glycyrrhiza glabra l. , fabaceae , is a tall , erect perennial herb with branched stalks which grow to 1.5 m. g. glabra is a native of south - east europe and south - west asia , which includes iran . it is among the world s most ancient herbal remedies with a wide range of pharmacological activities including expectorant , antitussive , emollient , anti - inflammatory , antipyretic , antiviral , antibacterial , antiprotozoal , hepatoprotective , antitumor , vasorelaxant , antiplatelet aggregation , immunomodulatory , endocrinological , antidepressant , memory enhancing , sedative , muscle relaxant ( 2 ) and antifungal effects ( 3 ) . the root of g. glabra has also been studied for anticonvulsant effect and was found to be effective ( 4 , 5 ) . there is no report regarding the possible anticonvulsant effects of the different varieties of g. glabra . on the other hand , some compounds believed to be responsible for anticonvulsant effects of the root of g. glabra , such as flavonoids which are also found in g. glabra leaves ( 6 - 8 ) . if the leaves are also proven to be effective , considering the perennial nature of this plant together with the easy harvest of the leaves , which provide a high yield of readily accessible target tissue , they could be a renewable source of bioactive anticonvulsant material . therefore , in this study the possible anticonvulsant and toxic effects of the leaves of g. glabra var . glandulifera were collected from borujen ( chahar mahall and bakhtiari province , iran ) in may 2007 glandulifera was authenticated by soroush sardari and a voucher specimen ( no . 74 - 86 ) was deposited in the herbarium of pasteur institute of iran . pentylenetetrazole ( ptz ) , phenytoin sodium and ethosuximide were purchased from sigma - aldrich ( pool , uk ) . n - hexane , tween 80 , dimethyl sulfoxide ( dmso ) , methanol , ethanol , dichloromethane , ethyl acetate , toluene , antimony trichloride , dragendorrf s reagent , potassium hydroxide , glacial acetic acid , vanillin , sulphuric acid , ferric chloride , hydrochloric acid and sodium hydroxide were all from merck ( darmstadt , germany ) . the extract and the fractions were dissolved in tween 80 ( 25% ) : dmso ( 2 : 1 v / v ) mixture and used freshly . extract preparation 
  the air - dried leaves ( 100 g ) of the plant were ground and extracted at the room temperature for 48 h by percolation method using 80% ethanol ( 900 ml ) . the extract was then concentrated with a rotary evaporator apparatus at temperature not exceeding 40c . the crude extract was suspended in 200 ml distilled water and extracted with dichloromethane for three times ( each time 150 ml ) . the dichloromethane ( f1 ) and the aqueous ( f2 ) parts were collected separately and dried by rotary evaporator at 40c . f1 was further partitioned by methanol ( 110 ml , f1a ) and n - hexane ( 44 ml , f1b ) ( figure 1 ) . flow diagram of fractionation of the crude extract obtained from the leaves of g. glabra var . glandulifera 
 preliminary phytochemical screening 
  the crude extract and the fractions were screened for the presence of triterpenes / sterols , alkaloids , flavonoids , anthraquinones , anthrones , coumarines , valepotriates , essential oil and tannins by thin layer chromatography using silica gel g ( merck , germany ) plates of 0.25 mm thickness ( 9 ) . the extract and fractions were dissolved in tween 80 ( 25% ) : dmso ( 2 : 1v / v ) . development was carried out with ethyl acetate : methanol : water ( 100 : 13.5 : 10 v / v / v ) and ethyl acetate : toluene ( 93 : 7 ) . after development , the plates were sprayed with the following reagents for detecting the respective classes of compounds : antimony trichloride ( triterpenes / sterols ) , dragendorrf s reagent ( alkaloids ) , potassium hydroxide ( anthraquinones , anthrones and coumarins ) , hydrochloric acid - glacial acetic acid ( valpotriates ) , vanillin - sulphuric acid ( essential oil ) and ferric chloride ( tannins ) . detection was carried out visually in visible light and under the uv light (  = 365 nm ) . male nmri mice ( 20 - 28 g , pasteur institute of iran ) were used . the animals were housed in standard cages with free access to food ( standard laboratory rodent s chow ) and water . the animals house temperature was maintained at 23  1c with a 1 h light/12 h dark cycle ( light on from 06:00 to 18:00 ) . the study was approved by the ethics committee of pasteur institute of iran and conforms to the european communities council directive of 24 november 1986 ( 86/609/eec ) . all animals experiments were carried out in such a way that minimized the number of animals and their suffering . all the injections were intraperitoneal ( ip ) in volume of 0.1 ml/10 g of mice body weight . ptz - induced seizures 
  the minimal ip dose of ptz at which 99% of the animals showed general clonus was determined by a dose - percent effect curve . general clonus was considered as the criteria of clonic seizure which characterized by clonus of four limbs with transient loss of righting reflex ( 11 ) . the dose ( 60 mg / kg ) was then injected to 18 groups of 10 mice each were pretreated ip 30 min before that , with the crude extract ( 1 , 1.5 , 2 , 3 and 4 g / kg ) , f1 ( 1 , 1.5 and 2 g / kg ) , f2 ( 2 and 4 g / kg ) , f1a ( 1 , 2 and 3 g / kg ) , f1b ( 1 and 1.5 g / kg ) , ethosuximide ( 150 mg / kg , as positive control ) , saline ( 10 ml / kg , as control ) and the solvent of the extract and the fractions ( 10 ml / kg , as control ) . if no general clonus occurred during a 30 min period of observation , the animals were considered protected . mes - induced seizure 
  electro - convulsive shock , inducing hind limb tonic extension ( hlte ) in 99% of the animals ( 11 ) was previously determined ( 12 ) . the electrical stimulus ( 50 ma ; 50 hz ; 1 sec duration ) was applied through ear - clip electrodes using a stimulator apparatus ( mgh-777 , development of electronic industry , iran ) . five groups of 10 mice each were pretreated ip with the crude extract ( 3 and 4 g / kg ) , phenytoin ( 25 mg / kg , as positive control ) , saline ( 10 ml / kg , as control ) and the solvent of the extract and fractions ( 10 ml / kg , as control ) . after 30 min the animals received transauricular electroshock . abolition of hlte within 10 sec after delivery of the electroshock was the criterion for anticonvulsant effect . seven groups of 10 mice each were treated ip with the solvent of the extract and the fractions ( 10 ml / kg , as control ) , the crude extract ( 1.5 , 2 , 3 and 4 g / kg ) , f1 ( 2 g / kg ) and f1a ( 3g / kg ) . the mortality rate was recorded after 24 h. the dose of the extract required to produce an anticonvulsant effect ( ed50 ) or death ( ld50 ) in 50% of the animals and its associated 95% confidence limit was calculated by the method of litchfield and wilcoxon ( 13 ) using a commercial computer program ( graphpad instat 3 , version 2003 ) . the therapeutic index ( ti ) of the extract was calculated via dividing the ld50 by the ed50 . data obtained from the convulsive tests were expressed as the percentage of the animals showing convulsions and fisher s exact test was used to analyze the data . anticonvulsant activity 
  the crude extract and f1 fraction showed anticonvulsant activity ( table 1 ) . the ed50 value of 2.11 g / kg and 1.30 g / kg were obtained for the crude extract and f1 fraction , respectively . however , f2 , f1a and f1b fractions had no protective effect against clonic seizures induced by ptz ( table 1 ) . glandulifera ethanol extract and fractions on clonic seizures induced by pentylenetetrazole in mice n = 10 ,  : p < 0.05 ,  : p < 0.01 and  : control 1 : saline , solvent of ethosuximide ; control 2 : tween 80 ( 25% ) : dmso ( 2 : 1 , v / v ) , solvent of the extract and fractions . f1 : ch2cl2 fraction , f2 : aqueous fraction , f1a : n - hexane fraction , f1b : methanol fraction . the crude extract up to the dose of 4 g / kg did not show any anticonvulsant effect against tonic seizures induced by mes ( table 2 ) . glandulifera ethanol extract on tonic seizures induced by maximal electroshock in mice n = 10 ,  : p < 0.001 compared to control value . control 1 : saline , solvent of phenytoin ; control 2 : tween 80 ( 25% ) : dmso ( 2 : 1 , v / v ) , solvent of the extract . at the anticonvulsant doses , the crude extract and fractions had lethal effects on the animals ( table 3 ) glandulifera ethanol extract and the fractions in mice n = 10 ; control : tween 80 ( 25% ) : dmso ( 2 : 1 , v / v ) , solvent of the extract and the fractions ; f1 : ch2cl2 fraction ; f1a : n - hexane fraction 
 preliminary phytochemical analysis 
  the constituents of the ethanol extract and the fractions are demonstrated in table 4 . the extract and f1 contain triterpenes / sterols , flavonoids , alkaloids , anthraquinones and tannin . anthrones , coumarines , valepotriates and essential oil were not found in any of the extract or fractions . glandulifera + : positive ; - : negative ; f1 : ch2cl2 fraction ; f2 : aqueous fraction ; f1a : n - hexane fraction ; f1b : methanol fraction the results of the present study indicate that the crude extract of g. glabra var . glandulifera blocks clonic seizures induced by ptz and the ed50 value of 2.11 g / kg was obtained for the extract . in order to pick out the anticonvulsant components of the leaves , bioactivity - guided fractionation showed that the active anticonvulsant principle ( s ) were non - polar as the activity was observed in dichloromethane fraction ( f1 ) and not in the aqueous fraction ( f2 ) . therefore , it seems that in f1 fraction , both toxic and anticonvulsant principles are present . by further fractionation neither n - hexane ( f1a ) nor methanol ( f1b ) sub - fractions had anticonvulsant activity . thus , anticonvulsant mixtures or combination of principles that are responsible for the activity are absent in sub - fractions ( f1a and f1b ) . the phytochemical tests performed in this study revealed the presence of triterpenes / sterols , alkaloids , flavonoids , anthraquinones and tannins in the leaf extract and f1 fraction . however , in f2 , triterpenes / sterols , alkaloids and anthraquinones , in f1a , triterpenes / sterols , anthraquinones and tannins , and in f1b , alkaloids , were not found . triterpenoids , flavonoids and alkaloids are detected in the root of g. glabra ( 2 , 14 ) and their anticonvulsant activity has been demonstrated previously ( 15 - 18 ) . therefore , the anticonvulsant activity of the leaf extract and f1 could be attributed to the combined activity of triterpenoids , flavonoids and alkaloids present in the plant . although f1a fraction is mainly composed of lipophilic substances , the phytochemical investigation showed only traces of certain secondary metabolites such as flavonoids , which are most likely inactive for anticonvulsant property . the rest of this fraction could be made from primary metabolites of lipid nature that are not expected to show such bioactivity . on the other hand , the highest concentrations of triterpenes together with four other categories of phytochemicals present in the crude extract and f1 , corresponded to anticonvulsant activity . thus , it seems that the absence of such pattern of phytochemicals in n - hexane fingerprint of chemicals could be the reason of non - effectiveness of f1a fraction against seizures . we obtained the ld50 value of 3.03 g / kg for the extract , which is close to the ed50 value and the calculated ti value of 1.43 g / kg for the leaf extract was narrow . it seems that the toxicity of the extract and the fractions are the result of the combined toxicity of the constituents such as alkaloids , anthraquinones and tannins whose toxicity has been reported by many researchers ( 19 - 31 ) . the anticonvulsant effect of an ethanol extract obtained from the roots of g. glabra has been previously reported ( 4 ) . in that study , ed50 value of about 20 mg / kg obtained for the extract against clonic seizures induced by ptz in mice . the root ethanol extract of g. glabra has wide ti and low toxicity where even at the doses 50 times higher than ed50 , no death or toxicity has been observed in mice ( 4 ) . in conclusion , therefore , compared to the roots , the leaf has weak potential to be considered as a source for anticonvulsant compounds . investigation on the possible anticonvulsant activity of the leaves of the other varieties of g. glabra grown in iran ( e.g. , var .
a review of the publications in traditional medicine indicates that the root of glycyrrhiza glabra l. , fabaceae , is recommended for treatment of epilepsy . as a renewable source , the leaves of g. glabra var . 
 glandulifera growing in iran were examined for possible anticonvulsant activity.the anticonvulsant activity of the leaves ethanol extract and dichloromethane , f1 , n - hexane , f1a , and methanol , f1b , fractions were evaluated intraperitoneally in mice using maximal electroshock ( mes ) and pentylenetetrazol ( ptz ) seizure tests . 
 acute toxicity of the extract and the fractions were also assessed . 
 phytochemical screening of the extract and the fractions for their active constituents was also carried out by thin layer chromatography and various chemical reagents . 
 the extract and the fractions showed anticonvulsant effect in ptz test . 
 the ed50 value of 2.11 g / kg and 1.30 g / kg was obtained for the crude extract and f1 fraction , respectively . the ld50 value of 3.0 g / kg was found for the extract . 
 triterpenes / sterols , alkaloids , flavonoids , anthraquinones and tannins were present in the extract and fractions . 
 triterpenes and anthraquinones were the highest in the extract , while triterpenes and tannins were prevailing in f1 fraction . 
 the anticonvulsant activity of the extract and f1 fraction could be mainly attributed to the compounds of triterpenes / sterols class present in the leaves of the plant . 
 the therapeutic index of the leaves extract was narrow and in this regard it has low anticonvulsant potential . 
 evaluation of the possible anticonvulsant activity of the leaves of the other varieties of g. glabra grown in iran ( e.g. , var . 
 violacea ) is suggested .