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Ecklonia cava Ethanol Extract promotes Non-Rapid Eye 1

Movement Sleep in Mice 2

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Running title: Sleep-Promoting Effects of Ecklonia cava Ethanol Extract 4

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* Corresponding Author: 7

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Abstract 1

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Our primary objective was to investigate the effects of the Ecklonia cava 3

ethanol extract (ECE) on sleep architecture and profile. ECE was orally administered at 4

a dose of 100, 250, or 500 mg/kg to C57BL/6N mice. The effects of ECE were 5

measured by recording electroencephalograms (EEG) and electromyograms. 6

Administration of ECE (250 and 500 mg/kg) significantly induced the amount of non-7

rapid eye movement sleep (NREMS) without changes in rapid eye movement sleep. The 8

increase in NREMS by ECE (500 mg/kg) was significant (p < 0.05) during the first 2 h 9

after administration. In addition, ECE had no effect on either EEG power density (an 10

indicator of sleep quality) in NREMS. These results suggest that ECE induces NREMS 11

that is similar to physiological sleep. 12

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Key words: 14

Ecklonia cava, Electroencephalogram, Electromyogram, Non-rapid eye movement sleep, 15

Delta power 16

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Introduction 1

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Ecklonia cava is the brown alga that is distributed in the coastal areas of Jeju 3

Island, Korea. E. cava has been used as an ingredient of functional foods and traditional 4

medicines (Cho et al., 2012a). Phlorotannins, fucoidans, fucoxanthins, and carotenoids 5

were identified as major bioactive compounds of E. cava (Kim and Bae, 2010; Heo et 6

al., 2005). It has been reported that extracts and constituents from E. cava have various 7

biological properties, e.g., antioxidative (Li et al., 2009; Kim and Kim, 2010), immune-8

enhancing (Ahn et al., 2008; Ahn et al., 2011), anti-allergic (Le et al., 2009; Shim et al., 9

2009), anticancer (Kong et al., 2009; Lee et al., 2011), and anti-inflammatory (Kim and 10

Bae, 2010 ) effects. 11

In our previous studies (Cho et al., 2012a; Cho et al., 2012b), the sedative-12

hypnotic effect of the E. cava ethanol extract (ECE) was demonstrated by using the 13

pentobarbital-induced sleep test in mice. In addition, we also reported that ECE induces 14

sleep via the benzodiazepine site of the GABAA receptor. Eckol, eckstolonol, dieckol, 15

and triphlorethol-A were characterized as a ligand to the benzodiazepine site of the 16

GABAA receptor (Cho et al., 2012a). However, the effects of ECE on sleep architecture 17

and profile have not yet been evaluated. 18

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In this study, the effects of ECE on changes in the sleep-wake profiles in 1

C57BL/6N mice were studied by recording electroencephalograms (EEG) and 2

electromyograms (EMG). 3

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Materials and Methods 1

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Materials 3

For preparation of ECE, the dried E. cava from Jeju Island, Korea was 4

adequately washed with water to remove the salt, sand, and epiphytes attached to the 5

surface. Then the samples were extracted with 70% (v/v) ethanol at 75°C for 16 h. The 6

extract solution was filtered and lyophilized. Diazepam (DZP; Myungin Pharm. Co. 7

Ltd., Seoul, Korea), a GABAA-BZD agonist, was used as a reference hypnotic drug. All 8

other chemicals and reagents were of the highest grade available. 9

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Animals 11

All procedures involving animals were conducted in accordance with the 12

animal care and use guidelines of the Korea Food Research Institutional Animal Care 13

and Use Committee (permission number: KFRI-M-12027). C57BL/6N mice (male; 27–14

30 g; 12 weeks) were purchased from Koatech Animal Inc. (Pyeongtaek, Korea). The 15

animals were housed in an insulated, sound-proof recording room maintained at an 16

ambient temperature of 23 ± 0.5°C, with a constant relative humidity (55 ± 2%) on an 17

automatically controlled 12 h light/12 h dark cycle (lights on at 09:00). They had free 18

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access to food and water. All efforts were made to minimize animal suffering and to use 1

only the number of animals required for the production of reliable scientific data. 2

3

Pharmacological treatments 4

ECE was dissolved in sterile saline containing 0.5% carboxymethyl cellulose 5

(CMC) immediately before use, and administered orally (p.o.) at 09:00 on the 6

experimental day at a dose of 100, 250, or 500 mg/kg. The positive control DZP (2 7

mg/kg) was administered in the same manner as ECE. 8

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Polygraphic recordings and vigilance state analysis 10

Under pentobarbital anesthesia (50 mg/kg, i.p.), the mice were chronically 11

implanted with a headmount (#8201, Pinnacle Technology Inc., Lawrence, KS, USA) 12

installed with EEG and EMG electrodes for polysomnographic recordings. The front 13

edge of the headmount was placed 3.0 mm anterior to bregma, and four electrode 14

screws for EEG recording were positioned in holes perforated into the skull (Fig. 1A). 15

Two EMG wire electrodes were inserted into the nuchal muscles. The headmount was 16

fixed to the skull with dental cement. After surgery, mice were allowed to recover in 17

individual cages for 1 week, and acclimated to the recording conditions for 3–4 days 18

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before the experiment. 1

The EEG and EMG recordings were carried out by means of a slip ring 2

designed so that the movement of the mice was not restricted (Fig. 1B). Two channels 3

of EEG and one channel of EMG were recorded using the PAL-8200 data acquisition 4

system (Pinnacle Technology Inc., Lawrence, KS, USA). The EEG and EMG signals 5

were amplified (100×), filtered (low-pass filter: 25 Hz EEG and 100 Hz EMG), and 6

stored at a sampling rate of 200 Hz. Recording was started at 09:00 AM, and was 7

continued for 12 h. For evaluation of sleep-promoting effects, the recording was 8

performed for 2 days. The data collected during the first day was served as baseline 9

comparison data (vehicle) for the second experimental day (test article). The vigilance 10

states were automatically classified by a 10-s epoch as wakefulness (Wake), rapid eye 11

movement sleep (REMS), or non-REM sleep (NREMS) by SleepSign ver. 3.0 (Kissei 12

Comtec, Nagano, Japan), according to the standard criteria (Qu et al., 2010). As a final 13

step, defined sleep-wake stages were examined visually and corrected if necessary. 14

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Data analysis 16

All data were expressed as the mean ± SEM (n = 6–8). Statistical analysis was 17

performed with the Prism 5.0 (GraphPad Software Inc., San Diego, CA, USA). The 18

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time-course of the hourly amounts of sleep and Wake at each stage, histograms of the 1

amounts of sleep and Wake, sleep latency, and mean duration of sleep and Wake were 2

analyzed using the paired t-test, with each animal serving as its own control. For sleep 3

latency and the total number of each vigilance stage during the 3 h immediately 4

following drug treatment, one-way repeated measures analysis of variance (ANOVA) 5

was performed, followed by the Dunnett’s test to determine whether the differences 6

among groups were statistically significant. The significance level was set at P < 0.05 7

for all statistical tests. 8

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Results and Discussion 1

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Effects of ECE on sleep latency and the amounts of NREMS and REMS 3

The EEG and EMG signals in mice were recorded for 12 h after the oral 4

administration of ECE (100, 250, or 500 mg/kg) at 09:00, and its effects were compared 5

with the positive control DZP (2 mg/kg). Fig. 2A shows the representative EEG and 6

EMG signals and corresponding hypnograms for vehicle, ECE, and DZP. As shown in 7

Fig. 2B, ECE (250 and 500 mg/kg) significantly (p < 0.01) decreased sleep latency 8

compared to vehicle. The administration of DZP also significantly (p < 0.01) decreased 9

sleep latency, and was not significantly different to ECE (500 mg/kg). The short sleep 10

latency in mice administered ECE indicates that ECE accelerated the initiation of 11

NREMS. 12

Sleep structure of animals is composed of NREMS and REMS. The sleep-wake 13

states are generally characterized as follows: Wake, low-amplitude EEG and high-14

voltage EMG activity; NREMS, high-amplitude slow or spindle EEG and low-voltage 15

EMG activity; and REMS, low-voltage EEG and EMG activity (Fig. 1C) (Kohotoh et 16

al., 2008; Bastien et al., 2003). The total time spent in NREMS and REMS for the first 3 17

h after ECE or DZP administration was calculated (Fig. 2C). ECE (250 and 500 mg/kg) 18

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significantly (p < 0.05) increased the total amount of NREMS by 47.8% and 71.4%, 1

respectively. The rate of increase in the amount of NREMS of DZP (2 mg/kg) was 2

103.8% (p < 0.01). However, ECE and DZP did not produce significant changes in the 3

total amount of REMS. BZD agents, e.g., DZP, are known to increase NREMS without 4

changing REMS (Qiu et al., 2009; Tobler et al., 2001) 5

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Effects of ECE on the time spent in each sleep stage 7

Fig. 3 shows the time course changes in NREMS, REMS, and Wake for 12 h 8

after the administration of ECE or DZP. After the injection of ECE, the amount of 9

NREMS was immediately increased, and the amount of Wake was decreased. These 10

effects of ECE were significant (p < 0.05) compared with vehicle for the first 2 h. There 11

was no further significant disruption of sleep architecture during the subsequent period. 12

These results indicate that ECE induce NREMS without causing adverse effects after 13

sleep induction (Masaki et al., 2012). DZP also showed a significant difference (p < 14

0.01) for the first 2 h; however, during the subsequent period, its hourly amount of 15

NREMS was higher than for ECE. 16

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Effects of ECE on the mean duration of each sleep stage and EEG power density in 18

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NREMS 1

To better understand the sleep profile caused by ECE, the mean duration of 2

each sleep stage and EEG power density in NREMS were calculated. ECE and DZP 3

significantly (p < 0.05) decreased the mean duration of Wake by 54.0% and 58.8%, 4

respectively; however, they did not affect the mean duration of NREMS and REMS 5

(Fig. 4A and B). A decrease in the mean duration of Wake by ECE without affecting 6

NREMS and REMS means that ECE decreased the maintenance of Wake (Masaki et al., 7

2012). ECE did not affect the EEG power density (0‒20 Hz) in NREMS compared with 8

vehicle (Fig. 4C), whereas DZP produced a significant (p < 0.05) decrease in delta (0.5–9

4 Hz) activity, as shown in the inset histogram of Fig. 4D. Delta activity is an indicator 10

of the depth or intensity of NREMS (Bastien et al., 2003; Winsky-Sommerer, 2009). 11

The decrease in delta activity caused by DZP in humans and rodents has been reported 12

previously (Tobler et al., 2001; van Lier et al., 2004). BZD agents produce an increase 13

in the quantity of sleep (sleep duration), but a decrease in the sleep quality (delta 14

activity) (Bastien et al., 2003; Ishida et al., 2009). DZP exerts an increase in beta 15

activity (13–30 Hz), which is the highest EEG frequency generally associated with 16

attention and arousal. Although BZD sleep drugs induce sleep, this increase in beta 17

activity is their own property (van Lier et al., 2004; Coenen and van Luijtelaar, 1991). 18

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In this study, DZP also showed this typical increase in beta activity; however, ECE did 1

not. In summary, ECE decreased sleep latency and increased the amount of NREMS, 2

similar to DZP; however, it did not change the EEG power density, unlike DZP. These 3

results suggest that ECE induces NREMS that is very similar to physiological sleep. 4

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Acknowledgements 1

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References 1

2

Cho S, Yang H, Jeon YJ, Lee CJ, Jin YH, Baek NI, Kim D, Kang SM, Yoon M, Yong 3

H, Shimizu M and Han D. 2012a. Phlorotannins of the edible brown seaweed 4

Ecklonia cava Kjellman induce sleep via positive allosteric modulation of 5

gamma-aminobutyric acid type A–benzodiazepine receptor: A novel 6

neurological activity of seaweed polyphenols. Food Chem 132, 1133-1142. 7

Ahn GN, Hwang IS, Park EJ, Kim JH, Jeon YJ, Lee JH, Park JW and Jee YH. 2008. 8

Immunomodulatory effects of an enzymatic extract from Ecklonia cava on 9

murine splenocytes. Mar Biotechnol (NY) 10, 278–289. 10

Ahn GN, Park EJ, Lee WW, Hyun JW, Lee KW, Shin TH, Jeon YJ and Jee YH. 2011. 11

Enzymatic extract from Ecklonia cava induces the activation of lymphocytes 12

by IL-2 production through the classical NF-κB pathway. Mar Biotechnol 13, 13

66–73. 14

Bastien CH, LeBlanc M, Carrier J and Morin CM. 2003. Sleep EEG power spectra, 15

insomnia, and chronic use of benzodiazepines. Sleep 26, 313–317. 16

Cho S, Han D, Kim SB, Yoon M, Yang H, Jin YH, Jo J, Yong H, Lee SH, Jeon YJ and 17

Shimizu M. 2012b. Depressive effects on the central nervous system and 18

underlying mechanism of the enzymatic extract and its phlorotannin-rich 19

fraction from Ecklonia cava edible brown seaweed. Biosci Biotechnol 20

Biochem 76, 163-168. 21

Cho S, Yang H, Jeon YJ, Lee CJ, Jin YH, Baek NI, Kim D, Kang SM, Yoon M, Yong 22

H, Shimizu M and Han D. 2012a. Phlorotannins of the edible brown seaweed 23

Ecklonia cava Kjellman induce sleep via positive allosteric modulation of 24

- 15 -

gamma-aminobutyric acid type A–benzodiazepine receptor: A novel 1

neurological activity of seaweed polyphenols. Food Chem 132, 1133-1142. 2

Coenen AM and van Luijtelaar EL. 1991. Pharmacological dissociation of EEG and 3

behavior: a basic problem in sleep-wake classification. Sleep 14, 464–465. 4

Heo SJ, Park EJ, Lee KW and Jeon YJ. 2005. Antioxidant activities of enzymatic 5

extracts from brown seaweeds. Bioresource Technol 96, 1613–1623. 6

Ishida T, Obara Y and Kamei C. 2009. Effects of some antipsychotics and a 7

benzodiazepine hypnotic on the sleep-wake pattern in an animal model of 8

schizophrenia. J Pharmacol Sci 111, 44–52. 9

Kim MM and Kim SK. 2010. Effect of phloroglucinol on oxidative stress and 10

inflammation. Food Chem Toxicol 48, 2925–2933. 11

Kim TH and Bae JS. 2010. Ecklonia cava extracts inhibit lipopolysaccharide induced 12

inflammatory responses in human endothelial cells. Food Chem Toxicol 48, 13

1682–1687. 14

Kohtoh S, Taguchi Y, Matsumoto N and Wada M. 2008. Algorithm for sleep scoring in 15

experimental animals based on fast Fourier transform power spectrum 16

analysis of the electroencephalogram. Sleep Biol Rhythms 6, 163–171. 17

Kong CS, Kim JA, Yoon NY and Kim SK. 2009. Induction of apoptosis by 18

phloroglucinol derivative from Ecklonia cava in MCF-7 human breast cancer 19

cells. Food Chem Toxicol 47, 1653–1658. 20

Le QT, Li Y, Qian ZJ, Kim MM and Kim SK. 2009. Inhibitory effects of polyphenols 21

isolated from marine alga Ecklonia cava on histamine release. Process 22

Biochem 44, 168–176. 23

- 16 -

Lee HK, Kang CK, Jung ES, Kim JS and Kim EK. 2011. Antimetastatic activity of 1

polyphenol-rich extract of Ecklonia cava through the inhibition of the Akt 2

pathway in A549 human lung cancer cells. Food Chem 127, 1229–1236. 3

Li Y, Qian ZJ, Ryu BM, Lee SH, Kim MM and Kim SK. 2009. Chemical components 4

and its antioxidant properties in vitro: an edible marine brown alga, Ecklonia 5

cava. Bioorg Med Chem 17, 1963–1973. 6

Masaki M, Aritake K, Tanaka H, Shoyama Y, Huang ZL and Urade Y. 2012. Crocin 7

promotes non-rapid eye movement sleep in mice. Mol Nutr Food Res 56, 8

304-308. 9

Qiu MH, Qu WM, Xu XH, Yan MM, Urade Y and Huang ZL. 2009. D1/D2 receptor-10

targeting L-stepholidine, an active ingredient of the Chinese herb Stephonia, 11

induces non-rapid eye movement sleep in mice. Pharmacol Biochem Behav 12

94, 16–23. 13

Qu WM, Xu XH, Yan MM, Wang YQ, Urade Y and Huang ZL. 2010. Essential role of 14

dopamine D2 receptor in the maintenance of wakefulness, but not in 15

homeostatic regulation of sleep in mice. J Neurosci 30, 4382–4389. 16

Shim SY, Quang-To L, Lee SH and Kim SK. 2009. Ecklonia cava extract suppresses 17

the high-affinity IgE receptor, FcεRI expression. Food Chem Toxicol 47, 18

555–560. 19

Tobler I, Kopp C, Deboer T and Rudolph U. 2001. Diazepam-induced changes in sleep: 20

role of the alpha 1 GABAA receptor subtype. Proc Natl Acad Sci USA 98, 21

6464-6469. 22

- 17 -

van Lier H, Drinkenburg WH, van Eeten YJ and Coenen AM. 2004. Effects of 1

diazepam and zolpidem on EEG beta frequencies are behavior-specific in rats. 2

Neuropharmacology 47, 163–174. 3

Winsky-Sommerer R. 2009. Role of GABAA receptors in the physiology and 4

pharmacology of sleep. Eur J Neurosci 29, 1779–1794. 5

6

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Figure legends (4 figures) 1

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Fig. 1. Surgery for EEG and EMG recordings (A). An inside view of the recording 3

chamber (B). Typical EEG, EMG and FFT spectra in mice (C). The EEG and EMG 4

signals and FFT spectra were collected from a vehicle control mouse in this study. 5

Abbreviations: EEG, electroencephalogram; EMG, electromyogram; FFT, fast Fourier 6

transform; NREMS, non-rapid eye movement sleep; REMS, rapid eye movement sleep; 7

Wake, wakefulness. 8

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Fig. 2. (A) Representative examples of EEG and EMG signals and corresponding 10

hypnograms in a mouse treated with vehicle, ECE, and DZP. (B) Effects of ECE 11

and DZP on sleep latency. (C) Total time spent in NREMS and REMS for 3 h after 12

administration. Each column represents the mean ± SEM (n = 8). **p < 0.01, 13

compared with vehicle (unpaired Student’s t-test). ##

p < 0.01, significant as compared 14

with ECE (100 mg/kg; Dunnett’s test). Abbreviations: DZP, diazepam; ECE, Ecklonia 15

cava ethanol extract; EEG, electroencephalogram; EMG, electromyogram; NREMS (or 16

NR), non-rapid eye movement sleep; NS, not significant; REMS (or R), rapid eye 17

movement sleep; Wake (or W), wakefulness. 18

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Fig. 3. Time courses of NREMS, REMS, and Wake after the administration of 20

ECE (A) and DZP (B). Each circle represents the hourly mean ± SEM (n = 8) of 21

NREMS, REMS, and Wake. *p < 0.05, **p < 0.01, compared with vehicle (unpaired 22

Student’s t-test). Abbreviations: DZP, diazepam; ECE, Ecklonia cava ethanol extract; 23

NREMS, non-rapid eye movement sleep; REMS, rapid eye movement sleep; Wake, 24

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wakefulness. 1

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Fig. 4. Effects of ECE and DZP on changes in the mean duration of each sleep 3

stage (A and B) and EEG power density in NREMS (C and D). Delta activity in 4

NREMS, an index of sleep intensity, is shown in the inset histogram of (C) and (D). The 5

bar (▬) represents the range of the delta wave (0.5‒4 Hz). *p < 0.05, compared with 6

vehicle (unpaired Student’s t-test). Abbreviations: DZP, diazepam; ECE, Ecklonia cava 7

ethanol extract; EEG, electroencephalogram; NREMS, non-rapid eye movement sleep; 8

REMS, rapid eye movement sleep; Wake, wakefulness. 9