Neurochemical Research

, Volume 37, Issue 12, pp 2725–2730

Anticonvulsant Activity and Toxicity of Essential Oil and Methanolic Extract of Zhumeria majdae Rech, a Unique Iranian Plant in Mice

Authors

  • Ali Mandegary
    • Department of Toxicology and Pharmacology, School of Pharmacy, Neuroscience Research CenterKerman University of Medical Sciences
    • Department of Pharmacognosy, School of Pharmacy, Herbal and Traditional Medicines Research CenterKerman University of Medical Sciences
  • Maryam Abdar
    • Committee of Student Research, School of PharmacyKerman University of Medical Sciences
  • Milad Arab-Nozari
    • Committee of Student Research, School of PharmacyKerman University of Medical Sciences
Original Paper

DOI: 10.1007/s11064-012-0863-5

Cite this article as:
Mandegary, A., Sharififar, F., Abdar, M. et al. Neurochem Res (2012) 37: 2725. doi:10.1007/s11064-012-0863-5

Abstract

Drug-resistance and adverse effects of current drugs are the most obstacles in the treatment of epilepsy. In a plan for finding new natural anticonvulsant agents, we studied the anticonvulsant effects of essential oil (ZMEO) and methanolic extract (ZMME) of Zhumeria majdae in pentylene tetrazol (PTZ) and maximal electro-shock (MES) models in mice. Mice received different doses of ZMEO and ZMME, 30 min before induction of chemical and electrical convulsions. Neurotoxicity (movement toxicity and sedation) was evaluated using rota-rod test. The mortality was determined after 24 h following injection of different doses of the ZMEO and ZMME. The obtained results show that ZMEO dose-dependently protected mice from tonic convulsions induced by PTZ and MES with effective doses (ED50) of 0.26 (0.13–0.39) and 0.27 (0.17–0.37) ml/kg respectively. Toxic doses (TD50) in rota-rod test for ZMEO was 0.55 (0.42–0.70) ml/kg. ZMME at dose of 2 g/kg decreased tonic convulsions as much as 40 %. For ZMEO, TD50 of 0.55 (0.45–0.69) ml/kg was obtained. ZMME significantly decreased the walking time in rota-rod test at dose of 2 g/kg. Lethal dose (LD50) of ZMEO was determined as 2.35 (1.98–2.65) ml/kg. ZMME showed about 34 % death of the animals at dose 5 g/kg. The essential oil of Z. majdae could be a good candidate for further anticonvulsive studies.

Keywords

Zhumeria majdaeEssential oilMethanolic extractAnticonvulsantRota-rodPTZMES

Introduction

Epilepsy is a general term for conditions with recurrent unprovoked seizures that causes substantial mortality and morbidity. Seizures are found in many kinds, but with one thing in common: uncontrolled firing of the neurons in the brain. Epilepsy affects 50 million people worldwide with incidence ranged from 16 to 51 cases per 100,000 people every year [2, 9]. Drug-resistance and adverse effects of current drugs are the most obstacles in the treatment of epilepsy [9]. The rationale behind the use of traditional medicinal plants is exploring the traditional herbal medicines for newer agents with potential different mechanisms of actions and lower toxicity in epilepsy.

Zhumeria majdae Rech (Labiatae) is a perennial fragrant shrub, native to the southern parts of Iran. This unique plant found in Hormozgan province in south of Iran and is used potentially in folk medicine as anticonvulsant, antispasmodic and for dysmenorrhoea. There are reports about the antileishmanial and antiplasmodial [13], antibacterial [15], anti inflammatory [19] and antioxidant activities of the essential oil and extract of Z. majdae [14, 20]. The antioxidant activity of the plant has been attributed to the presence of phenolic compounds in the alcoholic extract [14, 20]. The GC/MS analysis of the essential oil of the aerial parts of the plant indicated the presence of terpenoids like linalool and camphor as major components [11, 20]. There are also some reports in the literature about the anticonvulsant effects of terpenoids [35, 22]. With these observations in mind, in the present work we have examined the possible protective effect of both essential oil and methanolic extract of Z. majdae Rech (ZMEO and ZMME respectively) against seizures induced by maximal electroshock (MES) and pentylenetetrazol (PTZ) as models of grand-mal and petit-mal seizures respectively in human. Meanwhile, neurotoxicity (sedation and motor impairment) and lethality of ZMEO and ZMME were also investigated.

Materials and Methods

Chemicals and Equipments

Pentylenetetrazol, ethosuximide, methanol and tween 80 were purchased from Sigma Co. (Deisenhofen, Germany). Phenytoin sodium was prepared from Loghman Co., Iran and dissolved in saline that was alkalinized slightly with potassium hydrochloride 0.1 mM. PTZ and ethosuximide were dissolved in saline solution (0.9 %). The extract and essential oil were dissolved in tween 80 (5 % v/v) in distilled water and sesame oil respectively. All drugs and the extracts were administrated intra peritoneally (i.p.) in volume of 0.1 ml/10 g of mice body weight.

Electro-stimulant (SATADIS-8810, Iran) and rota-rod apparatus (TSE3375-4R, Iran) were the equipments used in the present work.

Extraction and Isolation of the Essential Oil

Zhumeria majdae was gathered from Bandar-Abbas in Hormozgan province in June 2011 and authenticated by botanist. A voucher specimen was deposited in Herbarium center of Faculty of Pharmacy (KF1387). After air-drying of the plant, 100 g of aerial parts were extracted three times with methanol 80 % using percolation method for 72 h. The filtrated extract was concentrated under vacuum and was stored in −20 °C until the time of test.

The essential oil of the plant (100 g) was prepared by a cleavenger apparatus using hydro-distillation method for 4 h. The oil was separated and dehydrated with suitable adsorbent and was stored in −20 °C until the time of experiment.

Animals

NMRI male mice (18–28 g) were prepared from the animal house of Neuroscience Research Center, Kerman University of Medical Sciences, Kerman, Iran. They were maintained on normal diet in individual metabolic cages with access to food and water in standard conditions (in a room temperature 25 ± 2 °C with 12-h light, 12-h darkness). The ethical guidelines for the investigation of experimental seizures in conscious animals were followed in all tests according to the rules of our school (EC/KNRC/88-13, 2010) which were approved by Ethic committee of Research Chancellor of Kerman University of Medical Sciences. All efforts were made to minimize animal suffering and to reduce the number of animals used.

Acute Toxicity

Groups of 6 mice were treated with doses of 1.5, 1.75 and 2 ml/kg of essential oil and doses of 4 and 5 g/kg of the extract (5 g/kg was the maximum injectable dose for the extract) and the number of deaths was determined after 24 h.

PTZ Induced Convulsion in Mice

The minimal i.p. dose of PTZ at which caused hind limp tonic extension (HLTE) in 99.9 % of animals was calculated using time-course curve and was determined as 110 mg/kg [1, 12, 18]. The time of maximum effect for the essential oil and extract was determined 30 min. Twelve groups each 6 mice were treated for 30 min with the solvents (sesame oil and tween 80 (5 % v/v) preparation, 0.1 ml/10 g, as controls), ethosuximide (150 mg/kg, as positive control), extract (0.5, 1, 1.5, 2, 3 and 4 g/kg) and essential oil (0.125, 0.25 and 0.5 ml/kg) 30 min before receiving PTZ (110 mg/kg). The time between PTZ injection and onset of clonic jerks and HLTE and the percentage of HLTE were recorded for each animal. The cut-off point for HLTE was considered 30 min.

Maximal Electroshock (MES) Test in Mice

The groups of animals each 6 mice were treated with the solvents (sesame oil and tween 80 (5 % v/v), 0.1 ml/10 g, as controls), phenytoin sodium (25 mg/kg, as positive control), extract (1 and 3 g/kg) and essential oil (0. 25, 0.35, 0.5 and 0.65 ml/kg). Tonic convulsions were induced in 99.99 % of the untreated animals by alternative electrical current (50 mA, 50 Hz, 1 s duration) through ear-clip electrodes using a stimulator apparatus [1, 12, 18]. The HTLE (%) was determined in 10 s. Phenytoin (25 mg/kg) was used as positive control.

Neurotoxicity Experiment

Neurotoxicity was evaluated according to the Dunham and Miya method [6]. Briefly, the trained animals received different doses of extract (doses of 3 and 4 g/kg), essential oil (doses of 1.5, 1.75 and 2 ml/kg) and the solvents of essential oil and extract (sesame oil and tween 80 (5 % v/v), 0.1 ml/10 g) and tested on the rota-rod as described before [1, 12].

Data Analysis

The dose of the extract required to produce an anticonvulsant effect (ED50) or motor impairment (TD50) or death (LD50) in 50 % of animals and its associated 95 % confidence limit was calculated using probit analysis. The percentage of HLTE in animals in the PTZ and MES methods and the percentage of animals that fell off the bar in rota-rod within 1 min was considered and analyzed using Fisher’s exact test. The time of remaining on the rod and the onset time of clonic and tonic convulsions after injection of PTZ were expressed as mean ± SEM and were analyzed by one-way analysis of variance (ANOVA) followed by the Tukey–Kramer multiple comparisons test. The criteria for significance were p-values of less than 0.05. All the statistical analysis were performed by SPSS software.

Results

The Yield of Essential Oil Isolation and Extraction

The hydro-distillation of the aerial parts of Z. majdae gave a pale colored essential oil (8.3 % v/w). The yield of extraction was about 10 %.

Effect of Essential Oil of Z. majdae Against PTZ and MES Induced Convulsions

ZMEO significantly increased the onset of tonic convulsions at dose of 0.25 ml/kg in PTZ induced seizures. The onset of clonic seizures was not significantly increased by the essential oil (Fig. 1).
https://static-content.springer.com/image/art%3A10.1007%2Fs11064-012-0863-5/MediaObjects/11064_2012_863_Fig1_HTML.gif
Fig. 1

The effect of Z. majdae essential oil on the onset of clonus (square) and tonic (question mark) seizures 30 min after i.p. administration of PTZ to mice. Histograms represent mean ± SEM (n = 6–8); *p < 0.05 and **p < 0.01 compared with control values (sesame oil). One-way ANOVA test followed by the Tukey–Kramer multiple comparisons test

Effect of ZMEO on the percentage of HLTE is shown in Table 1. The essential oil significantly prevented tonic convulsions induced by PTZ and MES at doses of 0.25 and 0.35 ml/kg respectively.
Table 1

Effect of Z. majdae essential oil on tonic seizures induced by PTZ and MES in mice

Treatment

Dose (i.p.)

HLTE (%)

PTZ

MES

Sesame oil

0.1 ml/10 g

100

100

Ethosuximide

150 mg/kg

0.0***

Phenytoin

25 mg/kg

0.0***

Z. majdae

0.125 ml/kg

100

100

Z. majdae

0.25 ml/kg

16.6**

66.6

Z. majdae

0.35 ml/kg

33.3*

Z. majdae

0.5 ml/kg

16.6**

16.6**

Z. majdae

0.65 ml/kg

0.0***

Data are presented as percentage of tonic seizure (n = 6–8)

p < 0.05; ** p < 0.01 and *** p < 0.001 compared to sesame oil

Effect of Methanolic Extract of Z. majdae Against PTZ and MES Induced Convulsions

As shown in Fig. 2, ZMME increased the onset times of tonic convulsions in a dose-dependent manner. This effect was significantly different from control at doses of 2 g/kg. The extract was not effective to prevent HLTE in MES and PTZ models (Table 2). Administration of higher doses was not possible for the extract.
https://static-content.springer.com/image/art%3A10.1007%2Fs11064-012-0863-5/MediaObjects/11064_2012_863_Fig2_HTML.gif
Fig. 2

The effect of Z. majdae methanolic extract on the onset of clonus (square) and tonic (question mark) seizure 30 min after i.p. administration of PTZ to mice. Histograms represent mean ± SEM (n = 6–8); *p < 0.05 compared with control values (normal saline, N.S, and tween 80 (5 % v/v)). One-way AXOVA test followed by the Tukey–Kramer multiple comparisons test

Table 2

Effect of Z. majdae methanolic extract on tonic seizures induced by PTZ and MES in mice

Treatment

Dose (i.p.)

HLTE (%)

PTZ

MES

Tween 80 (5 % v/v)

0.1 ml/10 g

100

100

Ethosuximide

150 mg/kg

0.0***

Phenytoin

25 mg/kg

0.0***

Z. majdae

0.5 g/kg

100

Z. majdae

0.75 g/kg

100

Z. majdae

1 g/kg

100

100

Z. majdae

1.5 g/kg

100

Z. majdae

2 g/kg

60

Z. majdae

3 g/kg

100

100

Z. majdae

4 g/kg

100

Data are presented as percentage of tonic seizure (n = 6–8)

*** p < 0.001 compared to tween 80 (5 % v/v)

Neurotoxicity and Lethality of Essential Oil and Methanolic Extract of Z. majdae

The ZMEO and ZMME significantly reduced the time spent on rota-rod at doses of 0.65 ml/kg and 2 g/kg respectively (Fig. 3). These sedation and movement toxicities were dose dependent with TD50 of 0.55 ml/kg for the ZMEO. LD50 value of the ZMEO was determined as 2.35 (1.98–2.65) ml/kg. Mortality was 34 % for the methanolic extract at the dose of 5 g/kg. Therapeutic index (TI) values were 8.7 and 9.1 against seizures induced by MES and PTZ, respectively.
https://static-content.springer.com/image/art%3A10.1007%2Fs11064-012-0863-5/MediaObjects/11064_2012_863_Fig3_HTML.gif
Fig. 3

Neurotoxicity of the methanolic extract (a) and essential oil (b) of Z. majdae rota-rod performance 30 min after i.p. administration to mice. Histograms represent mean ± SEM for 6 animals *p < 0.05 and **p < 0.01 versus control (normal saline, N.S, and tween 80 (5 % v/v)) by analysis of variance with Tukey’s post hoc test

Discussion

Drug-resistance and adverse effects of current drugs are the most obstacles in the treatment of epilepsy [9]. Hence, exploring new types of anticonvulsants with broad spectrum and lower toxicity is needed. One of the approaches to search for new antiepileptic drugs is the investigation of naturally occurring compounds; including those occur in the traditional medicinal plants. In the present study we have evaluated essential oil and methanolic extract of the aerial parts of Z. majdae, a unique plant from Iran on the seizures induced by PTZ and MES.

The results indicate that essential oil dose-dependently protected mice from PTZ and MES induced convulsions (ED50s of 0.26 and 0.27 ml/kg respectively). It also increased the onset time of tonic seizures which reached to the peak at dose of 0.25 ml/kg. Even though the essential oil produced neurotoxicity too close to the anticonvulsant dose (TD50 = 0.55 ml/kg), its lethality dose was much (9 time) higher than effective dose (LD50 = 2.35 vs ED50 = 0.27 ml/kg).

We previously reported that linalool and camphor from terpenoids were the major constituents of the plant essential oil. Borneol is also another component of the essential oil (1.32 % v/v) [20] which might induce anticonvulsant activity [16].

PTZ and MES are among the standard tests proposed to screen the anticonvulsive compounds [8, 21]. MES produce the spread of seizure similar to grandmal epilepsy and it is stated that MES-induced tonic extension can be prevented either by drugs that inhibit voltage-dependent Na+-channels, such as phenytoin, valproate, felbamate and lamotrigine; or by drugs that block glutamatergic excitation mediated by the N-methyl-D-aspartate (NMDA) receptor, such as felbamate [17]. This type of seizures can be prevented by drugs that enhance gamma amino butyric acid type A (GABAA) receptor mediated inhibitory neurotransmission, such as benzodiazepines, phenobarbital and perhaps valproate and felbamate [18].

The results also revealed that ZMEO produces sedation and motor deficits at concentration close to anticonvulsant doses (TD50 = 0.55 ml/kg). Some terpene compounds such as linalool exhibited sedation in mice [10].

The lethal dose of the essential oil was much higher than therapeutic concentrations for MES and PTZ-induced seizures (TI of 8.7 and 9.1, respectively). This suggests acceptable therapeutic effect for the essential oil. However, chronic toxicity studies must be performed in order to assess the real toxicological profile of that. Farhat et al. [7] has linked the higher lethality of essential oil extracted from plants collected in the winter season to the higher level of camphor, α,β-thujone, and camphene. Camphor has been reported as one of the main constituents (26.15 %) of ZMEO [20].

The methanolic extract of Z. majdae exhibited anticonvulsant effects in neither PTZ nor MES models. This extract only dose-dependently increased the time of tonic convulsion after injection of PTZ. The extract significantly impaired motor coordination at the dose of 2 g/kg. Terpenoids are from those plant metabolites which exhibited anticonvulsant activity [35]. We have reported the chemical composition of the Z. majdae essential oil. As this report, linalool (53.28 %), camphor (26.15 %), D-limonene (4.17 %), camphene (2.16 %) and borneol (1.32 %) were determined as the major components of the oil [21]. Terpenoids especially monoterpenes constitute more than 90 % of the oil composition and therefore might be responsible for anticonvulsant activity of ZMEO. Due to nonpolar nature of terpenoids, methanol extract of the plant has low capacity to extract terpenoids and so ZMME exhibited non significant anticonvulsant activity in both PTZ and electroshock method.

Conclusions

The results of the present study indicated that the essential oil of Z. majdae would be effective as an anticonvulsant agent in both absence and generalized tonic–clonic convulsions. Even though this oil produced neurotoxicity too close to the anticonvulsant dose, its lethality dose was much (9 time) higher than effective dose (LD50 = 2.35 vs ED50 = 0.27 ml/kg). The lethal dose of the essential oil was also much higher than therapeutic concentrations for MES and PTZ-induced seizures. Further studies for understanding the accurate mechanism(s) and active components involved in this effect need to be investigated.

Acknowledgments

This work was supported by the Neurosciences Research Center and Vice Chancellor for research, Kerman University of Medical Scieces, Kerman (Grant number 89/882). This article also has been derived from the thesis of Pharm.D students in Kerman University of medical Sciences, Faculty of Pharmacy, Kerman, Iran.

Conflict of interest

There are no conflict in design, financial support, report, and submission of article to the journal.

Copyright information

© Springer Science+Business Media, LLC 2012