Dieckol, a Major Marine Polyphenol, Enhances Non-Rapid Eye Movement Sleep in Mice via the GABAA-Benzodiazepine Receptor

Abstract

We had previously demonstrated that phlorotannins, which are marine polyphenols, enhance sleep in mice via the GABA_A-benzodiazepine (BZD) receptor. Among the constituents of phlorotannin, dieckol is a major marine polyphenol from the brown alga Ecklonia cava. Although phlorotannins are known to exert hypnotic effects, the sleep-enhancing effect of dieckol has not yet been determined. We evaluated the effect of dieckol on sleep-wake state of mice by analyzing electroencephalograms (EEGs) and electromyograms. Flumazenil, a GABA_A-BZD antagonist, was used to investigate the molecular mechanism underlying the effects of dieckol on sleep. The polygraphic recordings and corresponding hypnograms revealed that dieckol accelerated the initiation of non-rapid eye movement sleep (NREMS); it shortened sleep latency and increased NREMS duration. According to the change in time-course, dieckol showed sleep-enhancing effects by increasing the amount of NREMS and decreasing wakefulness during the same hours. Additionally, sleep quality was evaluated by analyzing the EEG power density, and dieckol was found to not affect sleep intensity while zolpidem was found to reduce it. Finally, we treated mice with zolpidem or dieckol in combination with flumazenil and found the latter to inhibit the sleep-enhancing effect of dieckol and zolpidem, thereby indicating that dieckol exerts sleep-enhancing effects by activating the GABA_A-BZD receptor, similar to zolpidem. These results implied that dieckol can be used as a promising herbal sleep aid with minimal side effects, unlike the existing hypnotics.

Keywords: dieckol; electroencephalogram; hypnotic; marine polyphenols; phlorotannins; sleep.

Figure 1

Chemical structures and molecular weights (MW) of zolpidem (A) and dieckol (B).

Figure 2

Experimental procedure (A), typical EEG, EMG, and FFT spectra (B), and definition of sleep-wake episodes (C) in C57BL/6N mice. EEG, electroencephalogram; EMG, electromyogram; FFT, fast Fourier transform; NREMS, non-rapid eye movement sleep; REMS, rapid eye movement sleep; Wake, wakefulness.

Figure 3

Effect of dieckol and zolpidem on sleep-wake profiles in C57BL/6N mice. (A) Representative EEG and EMG signals, and corresponding hypnograms in a mouse treated with dieckol or zolpidem. (B) Effects of dieckol and zolpidem on sleep latency. (C) Amounts of NREMS and REMS during the 2 h period after administration of dieckol or zolpidem. Gray bars indicate the baseline day (vehicle administration). Each value represents the mean ± SEM of each group (n = 6–7) with data points. *p < 0.05 and **p < 0.01, significant difference compared to the vehicle (paired Student’s t-test). EEG, electroencephalogram; EMG, electromyogram; NREMS, non-rapid eye movement sleep; NS, no significance; REMS, rapid eye movement sleep; Wake, wakefulness.

Figure 4

Effects of dieckol (A) and zolpidem (B) on time-course changes in NREMS, REMS, and Wake for 24 h in C57BL/6N mice. Light and dark circles indicate the baseline day (vehicle) and experimental day (dieckol or zolpidem), respectively. Each circle represents the hourly mean ± SEM (n = 6–7) of NREMS, REMS, and Wake. *p < 0.05 and **p < 0.01, significant difference compared to the vehicle (two-way ANOVA with Bonferroni post-test). NREMS, non-rapid eye movement sleep; REMS, rapid eye movement sleep; Wake, wakefulness.

Figure 5

Characteristics of sleep-wake episodes in C57BL/6N mice after oral administration of dieckol and zolpidem. (A) Mean duration and (B) total number of NREMS, REMS, and Wake bouts in a 2 h period after the administration of dieckol and zolpidem. (C) Sleep-wake state transitions during the 2 h following the administration of dieckol and zolpidem. Light and dark bars indicate the baseline day (vehicle administration) and experimental day (dieckol or zolpidem administration), respectively. Each column represents the mean ± SEM (n = 6–7) with data points. *p < 0.05 and **p < 0.01, significant difference compared to the vehicle (paired Student’s t-test). EEG, electroencephalogram; EMG, electromyogram; NREMS (or N), non-rapid eye movement sleep; REMS (or R), rapid eye movement sleep; Wake (or W), wakefulness.

Figure 6

(A) Changes in the number of NREMS and Wake bouts of different durations in C57BL/6N mice after oral administration of dieckol or zolpidem. Light and dark bars indicate the baseline day (vehicle administration) and experimental day (dieckol or zolpidem administration), respectively. Each column represents the mean ± SEM (n = 6–7) with data points. *p < 0.05 and **p < 0.01, significant difference compared to the vehicle (paired Student’s t-test). (B) Electroencephalogram (EEG) power density curves of NREMS caused by dieckol and zolpidem. Delta activity, an index of sleep intensity, is shown in the inset histogram. The dash (▬) represents the range of the delta wave (0.5‒4 Hz). *p < 0.05, significant difference compared to the vehicle (two-way ANOVA with Bonferroni post-test). NREMS, non-rapid eye movement sleep.

Figure 7

Effect of flumazenil treatment on dieckol- and zolpidem-induced sleep in C57BL/6N mice. (A) Amount of NREMS, REMS, and Wake for 2 h after pretreatment with flumazenil (1 mg/kg, i.p. at 16:45 h) and oral administration of dieckol (150 mg/kg, at 17:00 h) or zolpidem (10 mg/kg, at 17:00 h) and each vehicle in mice. Each column represents the mean ± SEM (n = 6-7) with data points. **p < 0.01, significant difference compared to the vehicle (paired Student’s t-test). (B) Time-course changes in NREMS, REMS, and Wake after administration of vehicle, flumazenil, and dieckol. The horizontal filled and open bars on the X-axis (time) indicate the 12 h dark and 12 h light periods, respectively.