Effect of Hydroalcoholic Extract of Stachys lavandulifolia on Pentylenetetrazole-induced Seizures in Male Mice: The Role of GABAergic and Opioidergic Systems

Abstract

Introduction: Epilepsy is one of the most common neurological disorders. Though there are several effective drugs for treating epilepsy, most drugs are associated with side effects and drug interactions. Stachys lavandulifolia used in Iranian traditional medicine has proven anti-anxiety and sedative properties. The current study aimed to evaluate the anticonvulsant effect of hydroalcoholic extract of S. lavandulifoliaon the Pentylenetetrazole (PTZ)-induced seizure in male mice and the role of benzodiazepine and opioid receptors.

Methods: This study was conducted on 100 male mice, randomly categorized into 10 groups: Normal Saline (NS), two diazepam groups (0.025 and 0.1 mg/kg), three S. lavandulifolia extract groups (50, 100, and 200 mg/kg), diazepam 0.025 mg/kg+S. lavandulifolia extract 50 mg/kg, and three groups that pretreated with NS, flumazenil, or naloxone, 5 min before injection of 200 mg/kg S. lavandulifolia extract. After 30 min, PTZ (80 mg/kg) was injected into animals, and seizure indices were evaluated.

Results: The S. lavandulifoliaextract attenuated the PTZ-induced seizures in a dose-dependent manner, and pretreatment with flumazenil reversed this effect. However, pretreatment with naloxone could not reverse this effect because seizure indices in the naloxone pretreated group were lower than that in the normal saline group. The combination of an ineffective dose of diazepam and S. lavandulifoliaextract decreased PTZ-induced seizures.

Conclusion: The results of our study showed the anticonvulsant properties of hydroalcoholic extract of S. lavandulifolia. These effects might be due to the impact of the components of this extract on the central benzodiazepine system.

Highlights: Hydroalcoholic extract of S. lavandulifolia attenuated the PTZ-induced seizures in a dose dependent manner.Pretreatment with flumazenil (blocker of benzodiazepines receptor) reversed anti-seizure effect of S. lavandulifolia extract.Combination of an ineffective dose of diazepam and S. lavandulifolia extract decreased PTZ-induced seizures.

Plain language summary: Epilepsy is one of the most common neurological disorders after stroke and is characterized by recurrent seizures due to abnormal excessive neural activity in the brain. Although there are many anticonvulsant drugs on the market, not all patients with epilepsy can be treated and one-third of patients suffer from recurring epilepsy despite using different antiepileptic drugs and more than 50% of them show side effects drugs during treatment. So, it is necessary to conduct further studies to develop more effective anti-epilepsy drugs with the minimum side effects. In recent years, plenty of studies have been conducted on medical plants, and S. lavandulifolia reported among the Iranian traditional medicine with antianxiety and sedative features. Some studies have mentioned the sedative and anti-inflammatory function of S. lavandulifolia, and its significant effects on anxiety have been approved comparable to diazepam. Overall, considering the anti-anxiety, analgesic, and sedative effects of the hydroalcoholic extract of S. lavandulifolia, it might possess anti-convulsive effects, too. The purpose of the current study was designed to investigate whether the effect of intra peritoneal injection of hydroalcoholic extract of S. lavandulifolia on the PTZ-induced convulsion in male mice and assessed the role of benzodiazepine and opioid receptors. Results of this study demonstrated that S. lavandulifolia extract attenuated the PTZ-induced seizures in a dose dependent manner, and pretreatment with flumazenil (blocker of benzodiazepines receptor) reversed this effect. However, pretreatment with naloxone (Non-selective blocker of opioids receptor) could not reverse this effect but the combination of an ineffective dose of diazepam and S. lavandulifolia extract decreased PTZ-induced seizures, thus anti-epileptic effect of S. lavandulifolia mediated by benzodiazepine receptors.

Keywords: Flumazenil; Naloxone; Pentylenetetrazole; Seizure; Stachys lavandulifolia.

Figure 1.

Flowchart for different groups and drug administration Phase 1, dose-response of hydroalcoholic extract of S. lavandulifolia (SL) compared with Normal Saline (NS) and diazepam (positive control groups) on Pentylenetetrazole (PTZ)-induced seizure; Phase 2, exploring benzodiazepine and opioids receptor roles on the antiepileptic effect of SL extract by pretreatment with flumazenil and naloxone; Phase 3, comparing the effects of alone and simultaneous administration of low doses of SL extract and diazepam on PTZ-induced seizure. Drugs and saline were injected intraperitoneally.

Figure 2.

The effects of hydroalcoholic extract of S. Lavandulifolia on Pentylenetetrazole (PTZ)-induced seizure parameters and comparing them with the normal saline and diazepam groups A) The latency period of clonic seizure initiation, B) the delay time of tonic-clonic seizure initiation, and C) the mortality rate after 24 hours. ^*P<0.05, ^**P<0.01 and ^***P<0.001 compared to the normal saline group.

Figure 3.

The effects of flumazenil and naloxone pretreatment on anticonvulsant properties of hydroalcoholic extract of S. lavandulifolia (SL) A) The latency period of clonic seizure initiation, B) the delay time of tonic-clonic seizure initiation, and C) the mortality rate after 24 hours. ^*P<0.05, ^**P<0.01 and ^***P<0.001 compared to the normal saline group, and ††P<0.01 and †††P<0.001 compared to 200 mg/kg SL extract group.

Figure 4.

The effect of simultaneous administration of hydroalcoholic extract of S. lavandulifolia and diazepam on Pentylenetetrazole (PTZ)-induced seizure parameters A) The latency period of clonic seizure initiation, B) the delay time of tonic-clonic seizure initiation, and C) the mortality rate after 24 hours. ^*P<0.05, ^**P<0.01, and ^***P<0.001 compared to the normal saline group.