There is a growing demand for natural sleep aids due to various side effects of long-term administration of pharmacological treatments for insomnia. Honey has been reported to exhibit numerous potential health benefits, and it is hypothesized that honey may favorably affect insomnia treatment. Therefore, this study was performed to investigate the possible hypnotic effect of clover honey (CH) and to determine its in vivo mechanism. The total flavonoid content (TFC) of CH and fractions extracted with ethylacetate (EtOAc) and H2O was measured. The pentobarbital-induced sleep test using GABAA-benzodiazepine (BZD) agonists and antagonists was conducted to evaluate the potential mechanism of action behind the sedative-hypnotic activity of CH in mice. The results showed that administration of 500 and 1,000 mg/kg of CH significantly (p<0.01) reduced the sleep latency to a level similar to that of diazepam (DZP, 2 mg/kg), and 1,000 mg/kg of CH significantly (p<0.01) prolonged the sleep duration, which was comparable to that of DZP (2 mg/kg). Administration of the EtOAc fraction with a higher TFC significantly reduced the sleep latency at 50 to 200 mg/kg and prolonged the sleep duration at 100 to 200 mg/kg, which were comparable to those after administration of DZP (2 mg/kg). However, co-administration of CH and EtOAc with flumazenil, a specific GABAA-BZD receptor antagonist, blocked the hypnotic effect. Our findings suggest that the hypnotic activity of CH may be attributed to allosteric modulation of GABAA-BZD receptors. The TFC of CH is expected to be a key factor that contributes to its hypnotic effect.

Bicuculline is one of the most commonly used GABAд eceptor antagonists in electrophysiological research. Because of its poor water solubility, bicuculline quaternary ammonium salts such as bicuculline methiodide (BMI) and bicuculline methbromide are preferred. However, a number of studies have shown that BMI has non-GABAд eceptor-mediated effects. The substantia gelatinosa (SG) of the trigeminal subnucleus caudalis (Vc) is implicated in the processing of nociceptive signaling. In this study, we investigated whether BMI has non-GABA receptor-mediated activity in Vc SG neurons using a whole cell patch clamp technique. SG neurons were depolarized by application of BMI (20M) using a high Cℓ⁻ipette solution. GABA ( 30-100μM) also induced membrane depolarization of SG neuron. Although BMI is known to be a GABAд receptor antagonist, GABA-induced membrane depolarization was enhanced by co-application with BMI. However, free base bicuculline (fBIC) and picrotoxin (PTX), a GABAд and GABAс receptor antagonist, blocked the GABA-induced response. Furthermore, BMI-induced membrane depolarization persisted in the presence of PTX or an antagonist cocktail consisting of tetrodotoxin (Nα+ nnel blocker),AP-5 (NMDA receptor antagonist), CNQX (non-NMDA receptor antagonist), and strychnine (glycine receptor antagonist). Thus BMI induces membrane depolarization by directly acting on postsynaptic Vc SG neurons in a manner which is independent of GABAд receptors. These results suggest that other unknown mechanisms may be involved in BMI-induced membrane depolarization.