Arousal Inhibitory Effect of Phlorotannins on Caffeine in Pentobarbital-Induced Mice
Arousal Inhibitory Effect of Phlorotannins on Caffeine in Pentobarbital-Induced Mice
Fisheries and aquatic sciences. 2014. Mar, 17(1): 13-18
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 : September 30, 2013
  • Published : March 31, 2014
Export by style
Cited by
About the Authors
Suengmok, Cho
Korea Food Research Institute, Seongnam 463-746, Korea
Hyejin, Yang
Korea Food Research Institute, Seongnam 463-746, Korea
Minseok, Yoon
Korea Food Research Institute, Seongnam 463-746, Korea
Jiyoung, Kim
Korea Food Research Institute, Seongnam 463-746, Korea
Dongsoo, Kim
Korea Food Research Institute, Seongnam 463-746, Korea
Jinkyung, Kim
KGC Lifengin, Seoul 135-549, Korea
Seon-Bong, Kim
Department of Food Science and Technology, Pukyong National University, Busan 608-737, Korea

Sleep is vital to maintain health and well-being; however, insomnia is currently a widespread health complaint worldwide. In particular, caffeine, a psychoactive component of coffee, tea, and caffeine beverages may lead to sleep disorders such as insomnia. In this study, our primary objective was to investigate the inhibitory effect of high-purity phlorotannin preparation (HP-PRT) on caffeine-induced wakefulness. The sleep test of pentobarbital-induced mice was used as an in vivo animal model. Caffeine (50 and 100 mg/kg) showed significant arousal effects (an increase in sleep latency and a decrease in sleep duration). Co-administration of caffeine (50 mg/kg) and the sedative-hypnotic diazepam (DZP, 1 mg/kg) did not result in similar arousal activity. HP-PRT (500 mg/kg) also inhibited caffeine-induced wakefulness. Our results suggest that HP-PRT would be a useful additive for developing coffee products without the arousal effect.
Sleep is vital to maintain health and well-being because of its primary function of providing rest and restoring the body’s energy levels ( Krueger et al., 2008 ). Sleep disorders impair not only cognitive and psychological functioning but also physical health ( Brand and Kirov, 2011 ). In addition, obesity and cardiovascular disease are closely related to sleep disorders ( Gangwisch et al., 2005 ; Wolk et al., 2005 ; Miller and Cappuccio, 2007 ).
Coffee is one of the most consumed beverages in the world ( Tuomilehto et al., 2004 ), and has an arousal effect on the central nervous system ( Davis et al., 2003 ). Caffeine, the major psychoactive constituent in coffee ( Brown et al., 2001 ), induces wakefulness by inhibiting adenosine A 2A receptors ( Huang et al., 2005 ). According to the “Caffeine Intake by the U.S. Population” report, the average amount of caffeine consumed is approximately 300 mg/person/day ( Somogyi, 2010 ). A large amount of caffeine may lead to sleep disorders, such as insomnia ( Sanchez-Ortuno et al., 2005 ; Strassnig et al., 2006 ). Therefore, to identify foods, and their constituents, that can inhibit the arousal effect of caffeine would be promising from the perspective of the food industry.
In our previous studies ( Cho et al., 2012a ; Cho et al., 2012b ), we have demonstrated that phlorotannin extracts from the brown alga Ecklonia cava induce sleep via modulation of the benzodiazepine site of the GABA A receptor. In the present study, the effect of phlorotannins on caffeine-induced wakefulness was evaluated using a pentobarbital-induced sleep test in mice.
Materials and Methods
- Materials
The high-purity phlorotannin preparation (HP-PRT, lot no. SD-GT-E-004) was obtained from S&D Co., Ltd. (Yeongigun, Chungcheongnam-do, Korea). HP-PRT was purified from the ethanol extract of the brown alga Ecklonia cava , and was standardized to 67 mg/g dieckol. The total phlorotannin content of HP-PRT is 90% (900 mg phloroglucinol equivalents/g, w/w). Diazepam (DZP; Myungin Pharm. Co. Ltd., Seoul, Korea), a GABA A -benzodiazepine 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). Every effort was made to minimize the number of animals used and any pain and discomfort that they might experience. ICR mice (male; 23–28 g; 4 weeks) were purchased from Koatech Animal Inc. (Pyeongtaek, Korea). The animals were housed in an insulated, soundproof recording room maintained at an ambient temperature of 23 ± 0.5℃, with a relative humidity (55 ± 2%) on an automatically controlled 12-h light/12-h dark cycle (lights on at 5:00). They had free access to food and water.
Pentobarbital-induced sleep test
For better understanding the pentobarbital-induced sleep test in mice, the experimental procedure is shown in Fig. 1 . All samples were dissolved in 0.5% (w/v) carboxymethyl cellulose (CMC)-physiological saline before use. Control mice (0.5% CMC-saline, 10 ml/kg) were tested in parallel with the animals receiving test sample treatment. All experiments were performed between 13:00 and 17:00, and mice were fasted for 24 h prior to the experiment. Test solutions were administered (post-oral injection, p.o.) to mice by using a sonde needle 30 min prior to the intraperitoneal injection (i.p.) of pentobarbital (42 mg/kg). Following the pentobarbital injection, the mice were placed in individual cages and observed for measurements of sleep latency and sleep duration. Observers were blinded to the individual treatments. The sleep latency was recorded from the time of pentobarbital injection to the time of sleep onset, and sleeping duration was defined as the difference in time between the loss and recovery of the righting reflex.
PPT Slide
Lager Image
The experimental procedure of the pentobarbital-induced sleep test in mice.
Statistical analysis
For multiple comparisons in the pentobarbital-induced sleep test, data were analyzed using one-way ANOVA followed by Dunnett’s test. Comparisons between group data were analyzed using an unpaired Student’s t -test. Differences with p <0.05 were considered statistically significant. The significance analysis was performed using Prism 5.0 (GraphPad Software Inc., San Diego, CA, USA).
Results and Discussion
Although a large amount of caffeine intake is known to lead to sleep disorders such as insomnia, few studies have been conducted on foods and their constituents with inhibitory effects on caffeine-induced wakefulness. In this study, we evaluated the effect of HP-PRT with sedative-hypnotic activity on caffeine-induced wakefulness by using the pentobarbitalinduced sleep test in mice. This method is useful to evaluate in vivo hypnotic and arousal activities ( Ma et al., 2009 ). It is important that the active compounds are able to pass the blood–brain barrier to produce hypnotic or arousal activities ( Risa et al., 2004 ). Therefore, it is necessary to confirm activities through in vivo animal model assays ( Fang et al., 2010 ).
To determine a statistically significant dosage of caffeine, its effects on sleep latency and sleep duration in mice were evaluated. With a hypnotic dose of pentobarbital (42 mg/kg), caffeine (12.5, 25, 50, and 100 mg/kg, p.o.) produced a dosedependent increase in sleep latency and a decrease in sleep duration ( Fig. 2 ). It showed a significant ( p < 0.01) arousal activity from a concentration of 50 mg/kg. According to the results of Huang et al. (2005) , caffeine significantly produced arousal activity in analysis of sleep-wake profile based on electroencephalography and electromyography. In this study, 50 mg/kg was selected as a dosage of caffeine for the experiment to test the inhibitory effects of phlorotannins. A dosage of HP-PRT was determined by testing four concentrations of 62.5, 125, 250, and 500 mg/kg. Diazepam (DZP), one of the most wellknown sedative-hypnotics, was tested as a positive control in parallel with HP-PRT. One mg/kg of DZP has been tested as the positive control in the pentobarbital-induced sleep test ( Amos et al., 2003 ; de Sousa et al., 2005 ). As expected, it was found that DZP (1 mg/kg, p.o.) significantly potentiated the pentobarbital-induced sleep in mice (p < 0.01) relative to the control group ( Fig. 3 ). HP-PRT also caused a dose-dependent decrease in sleep latency ( Fig. 3A ) and an increase in sleep duration ( Fig. 3B ). Effect of HP-PRT on caffeine-induced wakefulness was evaluated at 500 mg/kg with the significant hypnotic activity similar to DZP at 1 mg/kg.
PPT Slide
Lager Image
Effects of caffeine on sleep latency (A) and sleep duration (B) in mice administered a hypnotic dose (42 mg/kg, i.p.) of pentobarbital. Mice received pentobarbital 30 min after oral administration (p.o.) of the control group (CON) and caffeine. Each column represents mean ± SEM (n = 10). * p < 0.05, ** p < 0.01, significant difference as compared to the control group (Dunnett’s test).
PPT Slide
Lager Image
Effects of high-purity phlorotannin preparation (HP-PRT) and diazepam (DZP) on sleep latency (A) and sleep duration (B) in mice administered a hypnotic dose (42 mg/kg, i.p.) of pentobarbital. Mice received pentobarbital 30 min after oral administration (p.o.) of samples. Each column represents mean ± SEM (n = 10). ** p < 0.01, significant difference as compared to the control group (Dunnett’s test).
The inhibitory effect of DZP, a positive control on caffeineinduced wakefulness, is shown in Fig. 4 . DZP induces sleep via positive allosteric modulation of the benzodiazepine site of the GABA A receptor ( Kopp et al., 2003 ; Tobler et al., 2001 ). As expected, administration of caffeine (50 mg/kg) alone showed significant arousal effects; however, sleep latency and sleep duration of mice treated with both DZP (1 mg/kg) and caffeine were similar to those of the control group. HPPRT (500 mg/kg) also reversed the arousal effect of caffeine ( Fig. 5 ). The pentobarbital-induced sleep test using rodents is a useful in vivo assay for evaluating hypnotic or arousal effect. Pentobarbital, an anesthetic, produces hypnosis and anesthesia by modulating the barbiturate site of GABA A receptor ( Gerak et al., 2004 ). Regardless of molecular mechanisms of sleep-wake regulation, hypnotic or arousal compounds can accelerate or decelerate the pentobarbital-induced sleep. In this study, arousal effect of caffeine via the adenosine A 2A receptor was offset by hypnotic effect of DZP and HP-PRT via the benzodiazepine site of the GABA A receptor. Therefore, this offset did not alter duration and latency of the pentobarbital-induced sleep. In addition, according to the report by El Yacoubi et al. (2003) , caffeine reduced the hypnotic effect of alcohol by modulating the GABA A receptor ( Koob, 2004 ; Lobo and Harris, 2008 ; Kumar et al., 2009 ).
PPT Slide
Lager Image
Effects of co-administration of diazepam (DZP, 1 mg/kg) and caffeine (CF, 50 mg/kg) on sleep latency (A) and sleep duration (B) in mice administered a hypnotic dose (42 mg/kg, i.p.) of pentobarbital. Each column represents mean ± SEM (n = 10). # p < 0.05, ## p < 0.01, significant difference between each group (unpaired Student’s t-test). NS, not significant.
PPT Slide
Lager Image
Effects of co-administration of high-purity phlorotannin preparation (HP-PRT, 500 mg/kg) and caffeine (CF, 50 mg/kg) on sleep latency (A) and sleep duration (B) in mice administered a hypnotic dose (42 mg/kg, i.p.) of pentobarbital. Each column represents mean ± SEM (n = 10). # p < 0.05, ## p < 0.01, significant difference between each group (unpaired Student’s t-test). NS, not significant.
In this experiment, no adverse effects were observed following HP-PRT administration. It is known that benzodiazepine agents, such as DZP increase non-rapid eye movement sleep without altering rapid eye movement sleep ( Qiu et al., 2009 ) but decrease delta activity, an indicator of sleep quality ( Bastien et al., 2003 ). HP-PRT does not alter delta power (data not shown) and would thus be a better agent for inhibiting caffeine-induced wakefulness. Phlorotannins, which are oligomers and polymers of phloroglucinol (1,3,5-tri-hydroxybenzene), have a brown color and a bitter taste ( Shibata et al., 2002 ). Their sensory characteristics would be suitable as an additive to coffee. In addition, phlorotannins have various biological activities, such as antioxidative ( Zou et al., 2008 ), anti-inflammatory ( Kim et al., 2009 ), antibacterial ( Nagayama et al., 2002 ), and antiallergic ( Sugiura et al., 2006 ) effects. Therefore, HP-PRT may be a good additive for the preparation of coffee products without an arousal effect. Further studies are needed to evaluate the effects of co-administration of HP-PRT and caffeine on sleep-wake architecture and profile.
This study was supported by grants from the Korea Food Research Institute (E0131402) and Korea Small and Medium Business Administration (S2060252).
Amos S , Binda L , Chindo BA , Tseja A , Odutola AA , Wambebe C , Gamaniel K 2003 Neuropharmacological effects of Hibiscus sabdariffa aqueous extract Pharm Biol 41 325 - 329
Bastien CH , LeBlanc M , Carrier J , Morin CM 2003 Sleep EEG power spectra, insomnia, and chronic use of benzodiazepines Sleep 26 313 - 317
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
Brown J , Kreiger N , Darlington GA , Sloan M 2001 Misclassification of exposure: coffee as a surrogate for caffeine intake Am J Epidemiol 153 815 - 820
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
Davis JM , Zhao Z , Stock HS , Mehl KA , Buggy J , Hand GA 2003 Central nervous system effects of caffeine and adenosine on fatigue Am J Physiol Regul Integr Comp Physiol 284 399 - 404
De Sousa FCF , Pereira BA , Lima VTM , Lacerda CDG , Melo CTV , Barbosa-Filho JM , Vasconcelos SMM , Viana GSB 2005 Central nervous system activity of yangambin from Ocotea duckei Vattimo (Lauraceae) in mice Phytother Res 19 282 - 286
El Yacoubi M , Ledent C , Parmentier M , Costentin J , Vaugeois JM 2003 Caffeine reduces hypnotic effects of alcohol through adenosine A2Areceptor blockade Neuropharmacology 45 977 - 985
Fang XS , Hao JF , Zhou HY , Zhu LX , Wang JH , Song FQ 2010 Pharmacological studies on the sedative-hypnotic effect of Semen Ziziphi spinosae (Suanzaoren) and Radix et Rhizoma Salviae miltiorrhizae (Danshen) extracts and the synergistic effect of their combinations Phytomedicine 17 75 - 80
Gangwisch JE , Malaspina D , Boden-Albala B , Heymsfield SB 2005 Inadequate sleep as a risk factor for obesity: analyses of the NHANES I Sleep 28 1289 - 1296
Gerak LR , Stevenson MW , Winsauer PJ , Moerschbaecher JM 2004 Effects of pregnanolone alone and in combination with other positive GABAAmodulators on complex behavior in rats Psychopharmacology 173 195 - 202
Huang ZL , Qu WM , Eguchi N , Chen JF , Schwarzschild MA , Fredholm BB , Urade Y , Hayaishi O 2005 Adenosine A2A, but not A1, receptors mediate the arousal effect of caffeine Nat Neurosci 8 858 - 859
Kim AR , Shin TS , Lee MS , Park JY , Park KE , Yoon NY , Kim JS , Choi JS , Jang BC , Byun DS , Park NK , Kim HR 2009 Isolation and identification of phlorotannins from Ecklonia stolonifera with antioxidant and anti-inflammatory properties J Agric Food Chem 57 3483 - 3489
Koob GF 2004 A role for GABA mechanisms in the motivational effects of alcohol Biochem Pharmacol 68 1515 - 1525
Kopp C , Rudolph U , Keist R , Tobler I 2003 Diazepam-induced changes on sleep and the EEG spectrum in mice: role of the α3-GABAAreceptor subtype Eur J Neurosci 17 2226 - 2230
Krueger JM , Rector DM , Roy S , Van Dongen HP , Belenky G , Panksepp J 2008 Sleep as a fundamental property of neuronal assemblies Nat Rev Neurosci 9 910 - 919
Kumar S , Porcu P , Werner DF , Matthews DB , Diaz-Granados JL , Helfand RS , Morrow AL 2009 The role of GABAAreceptors in the acute and chronic effects of ethanol: a decade of progress Psychopharmacology 205 529 - 564
Lobo IA , Harris RA 2008 GABAAreceptors and alcohol Pharmacol Biochem Behav 90 90 - 94
Ma Y , Ma H , Eun JS , Nam SY , Kim YB , Hong JT , Lee MK , Oh KW 2009 Methanol extract of Longanae Arillus augments pentobarbital-induced sleep behaviors through the modification of GABAergic systems J Ethnopharmacol 122 245 - 250
Miller MA , Cappuccio FP 2007 Inflammation, sleep, obesity and cardiovascular disease Curr Vasc Pharmacol 5 93 - 102
Nagayama K , Iwamura Y , Shibata T , Hirayama I , Nakamura T 2002 Bactericidal activity of phlorotannins from the brown alga Ecklonia kurome. J Antimicrob Chemother 50 889 - 893
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 Behav 94 16 - 23
Risa J , Risa A , Adsersen A , Gauguin B , Stafford GI , van Staden J , Jager AK 2004 Screening of plants used in southern Africa for epilepsy and convulsions in the GABAA-benzodiazepine receptor assay J Ethnopharmacol 93 177 - 182
Sanchez-Ortuno M , Moore N , Taillard J , Valtat C , Leger D , Bioulac B , Philip P 2005 Sleep duration and caffeine consumption in a French middle-aged working population Sleep Med 6 247 - 251
Shibata T , Fujimoto K , Nagayama K , Yamaguchi K , Nakayama T 2002 Inhibitory activity of brown algal phlorotannins against hyaluronidase Int J Food Sci Tech 37 703 - 709
Somogyi LP 2010 Caffeine intake by the U.S. population, FDA comments. Subcontract No. 70000073494 FDA Oakridge, USA 1 - 85
Strassnig M , Brar JS , Ganguli R 2006 Increased caffeine and nicotine consumption in community-dwelling patients with schizophrenia Schizophr Res 86 269 - 275
Sugiura Y , Takeuchi Y , Kakinuma M , Amano H 2006 Inhibitory effects of seaweeds on histamine release from rat basophile leukemia cells (RBL-2H3) Fisheries Sci 72 1286 - 1291
Tobler I , Kopp C , Deboer T , Rudolph U 2001 Diazepam-induced changes in sleep: role of the alpha 1 GABAAreceptor subtype Proc Natl Acad Sci USA 98 6464 - 6469
Tuomilehto J , Hu G , Bidel S , Lindström J , Jousilahti P 2004 Coffee consumption and risk of type 2 diabetes mellitus among middle-aged Finnish men and women JAMA 291 1213 - 1219
Wolk R , Gami AS , Garcia-Touchard A , Somers VK 2005 Sleep and cardiovascular disease Curr Probl Cardiol 30 625 - 662
Zou Y , Qian ZJ , Li Y , Kim MM , Lee SH , Kim SK 2008 Antioxidant effects of phlorotannins isolated from Ishige okamurae in free radical mediated oxidative systems J Agric Food Chem 56 7001 - 7009