Magnolol Exerts Anti-epileptic Effects Via Gaba-benzodiazepine Receptor Complex In Mice

The bark of Magnolia officinalis has been used in traditional Chinese medicine to treat epilepsy, but the exact mechanism and major constituent involved in antiepileptic effects are still unknown. Magnolol is one of major bioactive constituents and the content of magnolol is in the range of 2-11 % in magnolia bark. Magnolol has been demonstrated to exhibit depressant effects on the central nervous system. Thus, we suggest that magnolol may have the effects of ant-epilepsy. GABAA receptor is an important drug target for anticonvulsive effect. The benzodiazepine (BZ) binding site is one of the potent modulating sites. The actions of benzodiazepines, are due to interactions with a benzodiazepines binding site on the GABAA receptor complex, and these interactions subsequently increase the probability of GABA to open the chloride channel associated with the receptor inducing inhibition. Therefore, we hypothesized that magnolol exerts anti-epileptic effect via GABA-benzodiazepine receptor complex.We used kungming mice by using animal models of behavior and EEG seizure, in vitro electrophysiology and immunohistochemisty to examine the anti-seizure potential of magnolol. In vivo, we divided mice to three groups, group of pentylenetetrazole(PTZ), group of protective effects of magnolol, group of reversal effects by flumazenil, a benzodiazepine receptors antagonist. The behavioral tests began immediately after intraperitoneal injection of PTZ for 60 min according to seizure stage evaluation. EEG recording began before 10 min of IP. injection of PTZ for 60 min. Brains were removed for immunohistochemisty after injection of PTZ immediately. For electrophysiological studies, recording lasted for 20 min before, during bath applied magnolol in the presence or absence of flumazenil.The results showed that magnolol at a dose of 40 mg/kg delayed the onset of myoclonic jerks (MJ) from 54.6±3.4 s to 84.4±5.6 s. Magnolol at 80 mg/kg significantly delayed the onset of myoclonic jerks to 135.2±8.9 s. There was no GCS or GTCS observed in magnolol-treated mice. Magnolol also decreased seizure stage and mortality of PTZ-treated mice compared to vehicle-treated animals. In vehicle treated mice, the mean seizure stage was 4.2±0.1. Magnolol at 40 or 80 mg/kg dose-dependently decreased the mean seizure stage to 2.5±0.2 or 2.1±0.1 respectively. In the recording of EEG signals, magnolol at doses of 40 and 80 mg/kg prolonged the latency of seizure onset from 43±3.7 s to 72.3±6.3, and 116.2±15.4 s, respectively. Furthermore, magnolol decreased the number of the spike-wave discharges. In Mg2+-free model of epileptiform activity via extracellular recordings in hippocampal slices, magnolol attetuated epileptiform power in CA3 of the hippocampus. The average power of seizure-like burst decreased 59±3% within 20 min after 8 min applied with magnolol. Magnolol also significantly decreased seizure-induced Fos immunoreactivity in the piriform cortex, dentate gyrus(DG), and CA1.CA2 and CA3 of hippocampal areas. In PTZ treated mice, Fos-positive cells were significantly increased to 9020±300 /mm2 in DG. Pretreatment with magnolol 40,80 mg/kg, Fos-positive cell in the DG dramatically decreased to 920±60 and 430±30/mm2, respectively. These findings indicated the hippocampus and piriform cortex may be the brain structures selectively involved in the anti-epileptic effect of magnolol. On the other hand, the anti-epileptic effects of magnolol were reversed by flumazenil. Flumazenil 10 mg/kg blocked the prolonged latency of seizure behaviors and EEG onset, decreased seizure stages and numbers of EEG spikes by magnolol. In vitro, flumazenil 1μM reversed the antiepileptic effects of magnolol, whereas flumazenil was unable to reverse the protective effect of magnolol on mortality in the behavioral test.Taken together, these findings indicated that inhibitory epileptiform activity of magnolol may be mainly mediated by GABA-benzodiazepine receptors complex.