Sleep-Promoting Effect of Ecklonia cava: Ethanol Extract Promotes Non-rapid Eye Movement Sleep in C57BL/6N Mice
Sleep-Promoting Effect of Ecklonia cava: Ethanol Extract Promotes Non-rapid Eye Movement Sleep in C57BL/6N Mice
Fisheries and aquatic sciences. 2014. Mar, 17(1): 19-25
Copyright © 2014, The Korean Society of Fisheries and Aquatic Science
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial Licens ( which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
  • Received : July 06, 2013
  • Accepted : August 26, 2013
  • Published : March 31, 2014
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About the Authors
Minseok, Yoon
Department of Seafood Science and Technology, Institute of Marine Industry, Gyeongsang National University, Tongyeong 650-160, Korea
Jin Soo, Kim
Department of Seafood Science and Technology, Institute of Marine Industry, Gyeongsang National University, Tongyeong 650-160, Korea
Jinho, Jo
Korea Food Research Institute, Seongnam 463-746, Korea
Daeseok, Han
Korea Food Research Institute, Seongnam 463-746, Korea
Suengmok, Cho
Korea Food Research Institute, Seongnam 463-746, Korea

We investigated the effects of Ecklonia cava ethanol extract (ECE) on sleep architecture and sleep profiles. ECE was orally administered at a dose of 100, 250, or 500 mg/kg to C57BL/6N mice and its effects were measured by recording electroencephalogram (EEG) and electromyogram. Administration of ECE (250 and 500 mg/kg) significantly induced non-rapid eye movement sleep (NREMS) without affecting rapid eye movement sleep. The increase in NREMS by ECE (500 mg/kg) was significant ( P < 0.05) during the first 2 h after administration. In addition, ECE had no effect on EEG power density (an indicator of sleep quality) in NREMS. These results suggest that ECE induces NREMS in a manner similar to physiological sleep.
Ecklonia cava is a brown alga distributed in the coastal areas of Jeju Island, Korea. It has been used as an ingredient in functional foods and traditional medicines ( Cho et al., 2012b ). Phlorotannins, fucoidans, fucoxanthins, and carotenoids have been identified as its major bioactive compounds ( Heo et al., 2005 ; Kim and Bae, 2010 ). Extracts of this alga show various biological properties, including antioxidative ( Li et al., 2009 ; Kim and Kim, 2010 ), immune-enhancing ( Ahn et al., 2008 , 2011 ), anti-allergic ( Le et al., 2009 ; Shim et al., 2009 ), anticancer ( Kong et al., 2009 ; Lee et al., 2011 ), and anti-inflammatory ( Kim and Bae, 2010 ) effects.
Recently, the sedative-hypnotic effect of E. cava ethanol extract (ECE) was demonstrated using pentobarbital-induced sleep tests in mice ( Cho et al., 2012a , 2012b8 ). In addition, it has been reported that ECE induces sleep via the benzodiazepine (BZD) site of the gamma-aminobutyric acid (GABA A ) receptor. Eckol, eckstolonol, dieckol, and triphlorethol A have been characterized as ligands of the GABA A -BZD receptor ( Cho et al., 2012b ).
In humans, sleep is essential to maintain health due to its primary function of providing rest and restoring the body’s energy levels ( Krueger et al. 2008 , ). Sleep is important for physical and cognitive performance, the immune system, mood stability, productivity, and quality of life ( Krueger et al., 2008 ; Imeri and Opp, 2009 ). Disorders and deprivation of sleep impair cognitive and psychological functioning and worsen physical health ( Brand and Kirov, 2011 ). Although sleep is fundamental to human health, insomnia is currently a widespread health complaint and has become a prevalent and disruptive problem in modern society ( Borja and Daniel, 2006 ; Doghramji, 2006 ; Erman, 2008 ). Sleep aids that contain constituents or extracts from foods and plants have become popular as alternatives to prescription sleep drugs to improve sleep quality and avoid adverse side effects ( Meletis and Zabriskie, 2008 ). Therefore, there is a growing demand for a novel class of hypnotic food constituents.
In the present study, to better understand the hypnotic effects of ECE, changes in the sleep-wake profiles of animals after oral administration of ECE were studied by recording electroencephalogram (EEG) and electromyogram (EMG). C57BL/6N mice were used because their sleep-wake cycle and delta activity are more stable than other strains ( Huber et al., 2000 ).
Materials and Methods
- Materials
To prepare ECE, dried E. cava from Jeju Island, Korea, was washed with water to remove salt, sand, and epiphytes attached to the surface. Then the samples were extracted with 70% (v/v) ethanol at 75℃ for 16 h. The extract solution was filtered and lyophilized. Diazepam (DZP; Myungin Pharm. Co. Ltd., Seoul, Korea), a GABA A -BZD agonist, was used as a reference hypnotic drug. All other chemicals and reagents were of the highest grade available.
- Animals
All procedures involving animals were conducted in accordance with the animal care and use guidelines of the Korea Food Research Institutional Animal Care and Use Committee (permission number: KFRI-M-12027). C57BL/6N mice (male; 27-30 g; 12 weeks old) were purchased from Koatech Animal Inc. (Pyeongtaek, Korea). Animals were housed in an insulated, sound-proof recording room maintained at an ambient temperatures of 23 ± 0.5℃, with a constant relative humidity (55 ± 2%) on an automatically controlled 12 h light/12 h dark cycle (lights on at 09:00). Mice had free access to food and water. All efforts were made to minimize animal suffering and to use only the number of animals required for the production of reliable scientific data.
- Pharmacological treatments
ECE was dissolved in sterile saline containing 0.5% carboxymethyl cellulose immediately before use, and was administered orally (p.o.) to mice ( n = 8) at 09:00 on the experimental day at doses of 100, 250, or 500 mg/kg. The positive control DZP (2 mg/kg) was administered in the same manner as ECE.
- Polygraphic recordings and vigilance state analysis
Under pentobarbital anesthesia (50 mg/kg, i.p.), mice were chronically implanted with a head-mount (#8201; Pinnacle Technology Inc., Lawrence, KS, USA) installed with EEG and EMG electrodes for polysomnographic recordings. The front edge of the head-mount was placed 3.0 mm anterior to the bregma, and four electrode screws for EEG recordings were positioned in holes perforating the skull ( Fig. 1A ). Two EMG wire electrodes were inserted into the nuchal muscles. The head-mount was fixed to the skull with dental cement. After surgery, mice were allowed to recover in individual cages for 1 week and to acclimate to the recording conditions for 3-4 days before the experiment.
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Surgery for EEG and EMG recordings (A). An inside view of the recording chamber (B). Typical EEG, EMG and FFT spectra in mice (C). The EEG and EMG signals and FFT spectra were collected from a vehicle control mouse in this study. EEG, electroencephalogram; EMG, electromyogram; FFT, fast Fourier transform; NREMS, non-rapid eye movement sleep; REMS, rapid eye movement sleep; Wake, wakefulness.
The EEG and EMG recordings were performed using a slip ring designed to allow the mice to move freely ( Fig. 1B ). Two EEG channels and one EMG channel were recorded using the PAL-8200 data acquisition system (Pinnacle Technology Inc.). The signals were amplified (100×), filtered (low-pass filter: 25 Hz EEG and 100 Hz EMG), and stored at a sampling rate of 200 Hz. Recording started at 09:00 and continued for 12 h. To evaluate sleep-promoting effects, recording was performed for 2 days. Data collected during the first day served as a baseline comparison (vehicle) for the second experimental day (test article).
The vigilance states were automatically scored by a 10 s epochs as wakefulness (Wake), rapid eye movement sleep (REMS), or non-REM sleep (NREMS) based on fast Fourier transform (FFT) analyses by SleepSign ver. 3.0 (Kissei Comtec, Nagano, Japan), according to the standard criteria ( Qu et al., 2010 ). The FFT is a computational tool that facilitates signal analysis, such as power spectrum analysis and filter simulation using digital computers ( Cochran et al., 1967 ). FFT analyses of EEG could be classified into two frequency bands; namely, δ (0.75-4 Hz) and θ (6-10 Hz) ( Kohtoh et al., 2008 ). As a final step, defined sleep–wake stages were examined visually and corrected if necessary.
- Data analysis
All data are expressed as the mean ± SEM ( n = 6-8). Statistical analysis was performed using Prism 5.0 (GraphPad Software Inc., San Diego, CA, USA). The time-course of hourly amounts of sleep and Wake at each stage, histograms of the amounts of sleep and Wake, sleep latency, and mean duration of sleep and Wake were analyzed using the paired t -test, with each animal serving as its own control. For sleep latency and the total amount of each vigilance stage during the 3 h period immediately following drug treatment, one-way repeated measures analysis of variance (ANOVA) was performed, followed by the Dunnett’s test to determine whether the differences among groups were statistically significant. The significance level was set at P < 0.05 for all statistical tests.
Results and Discussion
- Effects of ECE on sleep latency and the amounts of NREMS and REMS
EEG and EMG signals in mice were recorded for 12 h after oral administration of ECE (100, 250, or 500 mg/kg) at 09:00, and its effects were compared to the positive control DZP (2 mg/kg). Fig. 2A shows representative EEG and EMG signals and the corresponding hypnograms for vehicle, ECE, and DZP. As shown in Fig. 2B , ECE (250 and 500 mg/kg) significantly ( P < 0.01) decreased sleep latency compared to the vehicle. DZP also significantly ( P < 0.01) decreased sleep latency, and was not significantly different from ECE (500 mg/kg). The short sleep latency in mice administered ECE indicates that ECE accelerated the initiation of NREMS.
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(A) Representative examples of EEG and EMG signals and corresponding hypnograms in a mouse treated with vehicle, ECE, and DZP. (B) Effects of ECE and DZP on sleep latency. (C) Total time spent in NREMS and REMS for 3 h after administration. Each column represents the mean ± SEM (n = 8). **P < 0.01, compared with vehicle (unpaired Student’s t-test). ##P < 0.01, significant as compared with ECE (100 mg/kg; Dunnett’s test). DZP, diazepam; ECE, Ecklonia cava ethanol extract; EEG, electroencephalogram; EMG, electromyogram; NREMS (or NR), non-rapid eye movement sleep; NS, not significant; REMS (or R), rapid eye movement sleep; Wake (or W), wakefulness.
The sleep structure of animals is composed of NREMS and REMS. The sleep-wake states are generally characterized as follows: Wake, low-amplitude EEG and high-voltage EMG activity; NREMS, high-amplitude slow or spindle EEG and low-voltage EMG activity; and REMS, low-voltage EEG and EMG activity ( Fig. 1C ) ( Bastien et al., 2003 ; Kohotoh et al., 2008 ). The total time spent in NREMS and REMS for the first 3 h after ECE or DZP administration was calculated ( Fig. 2C ). ECE (250 and 500 mg/kg) significantly ( P < 0.05) increased the total amount of NREMS by 47.8% and 71.4%, respectively. The rate of increase in NREMS after administering DZP (2 mg/kg) was 103.8% ( P < 0.01). However, ECE and DZP did not produce significant changes in the total amount of REMS. BZD agents, such as DZP, are known to increase NREMS without changing REMS ( Tobler et al., 2001 ; Qiu et al., 2009 )
The sedative-hypnotic effects of ECE are caused by the phlorotannins found in brown seaweed. The phlorotannin fraction of ECE significantly promotes pentobarbital-induced sleep in mice; however, the residual fraction does not show any significant hypnotic effects ( Cho et al., 2012a ). The phlorotannin fraction induces sleep via the positive allosteric modulation of GABA A -BZD receptors ( Cho et al., 2012a ). In addition, major phlorotannin constituents dieckol, eckol, eckstolonol, and triphlorethol A have been found to have binding affinity to the GABA A -BZD receptors ( Cho et al., 2012b ) and potentiate the pentobarbital-induced sleep in mice (data not shown).
- Effects of ECE on the time spent in each sleep stage
Fig. 3 shows the time course changes in NREMS, REMS, and Wake for 12 h after the administration of ECE or DZP. After ECE administration, the amount of NREMS immediately increased, while the amount of Wake decreased. These effects were significant ( P < 0.05) compared to the vehicle for the first 2 h. There was no further significant disruption of sleep architecture during subsequent periods. These results indicate that ECE induces NREMS without having adverse effects after sleep induction ( Masaki et al., 2012 ). DZP also showed a significant difference ( P < 0.01) for the first 2 h; however, during the subsequent period, the hourly amount of NREMS was higher than for ECE.
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Time courses of NREMS, REMS, and Wake after the administration of ECE (A) and DZP (B). Each circle represents the hourly mean ± SEM (n = 8) of NREMS, REMS, and Wake. *P < 0.05, **P < 0.01, compared with vehicle (unpaired Student’s t-test). DZP, diazepam; ECE, Ecklonia cava ethanol extract; NREMS, non-rapid eye movement sleep; REMS, rapid eye movement sleep; Wake, wakefulness.
- Effects of ECE on the mean duration of each sleep stage and EEG power density in NREMS
To better understand the sleep profile caused by ECE, the mean duration of each sleep stage and EEG power density in NREMS were calculated. ECE and DZP significantly ( P < 0.05) decreased the mean duration of Wake by 54.0% and 58.8%, respectively; however, they did not affect the mean duration of NREMS or REMS ( Fig. 4A and 4B ). This suggests that ECE decreased the maintenance of Wake ( Masaki et al., 2012 ). ECE did not affect EEG power density (0-20 Hz) in NREMS compared to the vehicle ( Fig. 4C ), whereas DZP produced a significant ( P < 0.05) decrease in delta (0.5-4 Hz) activity, as shown in the inset histogram of Fig. 4D . Delta activity is an indicator of the depth or intensity of NREMS ( Bastien et al., 2003 ; Winsky-Sommerer, 2009 ). A decrease in delta activity caused by DZP has been reported in humans and rodents ( Tobler et al., 2001 ; van Lier et al., 2004 ). BZD agents increase sleep duration but decrease sleep quality ( Bastien et al., 2003 ; Ishida et al., 2009 ). Generally, DZP increase beta activity (13–30 Hz), which is the highest EEG frequency associated with attention and arousal. Although BZD sleep drugs induce sleep, they also induce beta activity ( Coenen and van Luijtelaar, 1991 ; van Lier et al., 2004 ). In the present study, DZP also increased beta activity; however, ECE did not. In summary, ECE decreased sleep latency and increased the amount of NREMS, similar to DZP; however, it did not affect EEG power density, unlike DZP. These results suggest that ECE induces NREMS in a similar manner as physiological sleep.
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Effects of ECE and DZP on changes in the mean duration of each sleep stage (A, B) and EEG power density in NREMS (C, D). Delta activity in NREMS, an index of sleep intensity, is shown in the inset histogram of (C) and (D). The bar (▬) represents the range of the delta wave (0.5‒4 Hz). *P < 0.05, compared with vehicle (unpaired Student’s t-test). DZP, diazepam; ECE, Ecklonia cava ethanol extract; EEG, electroencephalogram; NREMS, non-rapid eye movement sleep; REMS, rapid eye movement sleep; Wake, wakefulness.
This study was supported by grants from the Korea Food Research Institute (E0131402) and Korea Small and Medium Business Administration (S2060252).
Ahn G , Hwang I , Park E , Kim J , Jeon YJ , Lee J , Park JW , Jee Y 2008 Immunomodulatory effects of an enzymatic extract from Ecklonia cava on murine splenocytes Mar Biotechnol 10 278 - 289
Ahn G , Park E , Lee WW , Hyun JW , Lee KW , Shin T , Jeon YJ , Jee Y 2011 Enzymatic extract from Ecklonia cava induces the activation of lymphocytes by IL-2 production through the classical NF-κB pathway Mar Biotechnol 13 66 - 73
Bastien CH , LeBlanc M , Carrier J , Morin CM 2003 Sleep EEG power spectra, insomnia, and chronic use of benzodiazepines Sleep 26 313 - 317
Borja NL , Daniel KL 2006 Ramelteon for the treatment of insomnia Clin Ther 28 1540 - 1555
Brand S , Kirov R 2011 Sleep and its importance in adolescence and in common adolescent somatic and psychiatric conditions Int J Gen Med 4 425 - 442
Cho S , Han D , Kim SB , Yoon M , Yang H , Jin YH , Jo J , Yong H , Lee SH , Jeon YJ , Shimizu M 2012 Depressive effects on the central nervous system and underlying mechanism of the enzymatic extract and its phlorotannin-rich fraction from Ecklonia cava edible brown seaweed Biosci Biotechnol Biochem 76 163 - 168
Cho S , Yang H , Jeon YJ , Lee CJ , Jin YH , Baek NI , Kim D , Kang SM , Yoon M , Yong H , Shimizu M , Han D 2012 Phlorotannins of the edible brown seaweed Ecklonia cava Kjellman induce sleep via positive allosteric modulation of gamma-aminobutyric acid type A-benzodiazepine receptor: a novel neurological activity of seaweed polyphenols Food Chem 132 1133 - 1142
Cochran WT , Cooley JW , Favin DL , Helms HD , Kaenel RA , Lang WW , Maling GC Jr , Nelson DE , Rader CM , Welch PD 1967 What is the fast Fourier transform? Proc IEEE Inst Electr Electron Eng 55 1664 - 1674
Coenen AM , van Luijtelaar EL 1991 Pharmacological dissociation of EEG and behavior: a basic problem in sleep-wake classification Sleep 14 464 - 465
Doghramji K 2006 The epidemiology and diagnosis of insomnia Am J Manag Care 12 ((8 Suppl 12)) S214 - S220
Erman MK 2008 New perspectives in the diagnosis and management of insomnia CNS Spectr 13 ((12 Suppl 17)) 3 -
Heo SJ , Park EJ , Lee KW , Jeon YJ 2005 Antioxidant activities of enzymatic extracts from brown seaweeds Bioresource Technol 96 1613 - 1623
Huber R , Deboer T , Tobler I 2000 Effects of sleep deprivation on sleep and sleep EEG in three mouse strains: empirical data and simulations Brain Res 857 8 - 19
Imeri L , Opp MR 2009 How (and why) the immune system makes us sleep Nat Rev Neurosci 10 199 - 210
Ishida T , Obara Y , Kamei C 2009 Effects of some antipsychotics and a benzodiazepine hypnotic on the sleep-wake pattern in an animal model of schizophrenia J Pharmacol Sci 111 44 - 52
Kim MM , Kim SK 2010 Effect of phloroglucinol on oxidative stress and inflammation Food Chem Toxicol 48 2925 - 2933
Kim TH , Bae JS 2010 Ecklonia cava extracts inhibit lipopolysaccharide induced inflammatory responses in human endothelial cells Food Chem Toxicol 48 1682 - 1687
Kohtoh S , Taguchi Y , Matsumoto N , Wada M , Huang ZL , Urade Y 2008 Algorithm for sleep scoring in experimental animals based on fast Fourier transform power spectrum analysis of the electroencephalogram Sleep Biol Rhythms 6 163 - 171
Kong CS , Kim JA , Yoon NY , Kim SK 2009 Induction of apoptosis by phloroglucinol derivative from Ecklonia cava in MCF-7 human breast cancer cells Food Chem Toxicol 47 1653 - 1658
Krueger JM , Rector DM , Roy S , Van Dongen HPA , Belenky G , Panksepp J 2008 Sleep as a fundamental property of neuronal assemblies Nat Rev Neurosci 9 910 - 919
Le QT , Li Y , Qian ZJ , Kim MM , Kim SK 2009 Inhibitory effects of polyphenols isolated from marine alga Ecklonia cava on histamine release Process Biochem 44 168 - 176
Lee H , Kang C , Jung ES , Kim JS , Kim E 2011 Antimetastatic activity of polyphenol-rich extract of Ecklonia cava through the inhibition of the Akt pathway in A549 human lung cancer cells Food Chem 127 1229 - 1236
Li Y , Qian ZJ , Ryu BM , Lee SH , Kim MM , Kim SK 2009 Chemical components and its antioxidant properties in vitro: an edible marine brown alga, Ecklonia cava Bioorg Med Chem 17 1963 - 1973
Masaki M , Aritake K , Tanaka H , Shoyama Y , Huang ZL , Urade Y 2012 Crocin promotes non-rapid eye movement sleep in mice Mol Nutr Food Res 56 304 - 308
Meletis CD , Zabriskie N 2008 Natural approaches for optimal sleep Alternat Complement Ther 14 181 - 188
Qiu MH , Qu WM , Xu XH , Yan MM , Urade Y , Huang ZL 2009 D1/D2receptor-targeting L-stepholidine, an active ingredient of the Chinese herb Stephonia, induces non-rapid eye movement sleep in mice Pharmacol Biochem Be 94 16 - 23
Qu WM , Xu XH , Yan MM , Wang YQ , Urade Y , Huang ZL 2010 Essential role of dopamine D2receptor in the maintenance of wakefulness, but not in homeostatic regulation of sleep, in mice J Neurosci 30 4382 - 4389
Shim SY , Quang-To L , Lee SH , Kim SK 2009 Ecklonia cava extract suppresses the high-affinity IgE receptor, FcεRI expression Food Chem Toxicol 47 555 - 560
Tobler I , Kopp C , Deboer T , Rudolph U 2001 Diazepam-induced changes in sleep: Role of the α1GABAAreceptor subtype Proc Natl Acad Sci USA 98 6464 - 6469
van Lier H , Drinkenburg WH , van Eeten YJ , Coenen AM 2004 Effects of diazepam and zolpidem on EEG beta frequencies are behavior-specific in rats Neuropharmacology 47 163 - 174
Winsky-Sommerer R 2009 Role of GABAAreceptors in the physiology and pharmacology of sleep Eur J Neurosci 29 1779 - 1794