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Behavioural Brain Research 313 (2016) 1–7

Contents lists available at ScienceDirect

Behavioural Brain Research

journa l homepage: www.e lsev ier .com/ locate /bbr

esearch report

tryptic hydrolysate from bovine milk as1-casein enhancespentobarbital-induced sleep in mice via the GABAA receptor

rene Joy I. dela Pena a, Hee Jin Kim a, June Bryan de la Pena a, Mikyung Kim a,hrislean Jun Botanas a, Kyung Yi You a, Taeseon Woo a, Yong Soo Leeb, Jae-Chul Jung c,yung-Mi Kim c, Jae Hoon Cheong a,∗

Uimyung Research Institute for Neuroscience, Department of Pharmacy, Sahmyook University, 815 Hwarang-ro, Nowon-gu, Seoul 139-742, Republic of

orea

Department of Pharmacy, Duksung Women’s University, 33 Samyang-ro 144-gil, Dobong-gu, Seoul, 01369, Republic of Korea

Life Science Research Institute, NOVAREX Co., Ltd., Ochang, Cheongwon, Chungbuk 28126, Republic of Korea

i g h l i g h t s

Bovine aS1-casein tryptic hydrolysate did not alter the locomotor activity and motor function of mice.

Bovine aS1-casein tryptic hydrolysate potentiated the sleep induced by pentobarbital sodium in mice.

Bovine aS1-casein tryptic hydrolysate increased the slow (delta) EEG wave in rats.

Bovine aS1-casein tryptic hydrolysate increased Cl− influx, in vitro, which was blocked by bicuculline.

Bovine aS1-casein tryptic hydrolysate has sleep-promoting properties with no or minimal sedative effects.

r t i c l e i n f o

rticle history:

eceived 6 May 2016

eceived in revised form 6 July 2016

ccepted 8 July 2016

vailable online 9 July 2016

eywords:

ovine aS1-casein tryptic hydrolysate (CH)

lpha-casozepine

asein

ilk

leep

enzodiazepine

entobarbital

a b s t r a c t

Studies have shown that enzymatic hydrolysis of casein, the primary protein component of cow’s

milk, produces peptides with various biological activities, and some of these peptides may have sleep-

promoting effects. In the present study, we evaluated the sedative and sleep-promoting effects of bovine

aS1-casein tryptic hydrolysate (CH), containing a decapeptide aS1-casein known as alpha-casozepine.

CH was orally administered to ICR mice at various concentrations (75, 150, 300, or 500 mg/kg). An hour

after administration, assessment of its sedative (open-field and rota-rod tests) and sleep-potentiating

effects (pentobarbital-induced sleeping test and EEG monitoring) were conducted. Although a trend can

be observed, CH treatment did not significantly alter the spontaneous locomotor activity and motor

function of mice in the open-field and rota-rod tests. On the other hand, CH (150 mg/kg, respectively)

enhanced the sleep induced by pentobarbital sodium in mice. It also promoted slow-wave (delta) EEG

activity in rats; a pattern indicative of sleep or relaxation. These behavioral results indicate that CH

has sleep-promoting effects, but no or has minimal sedative effects. To elucidate the probable mecha-

EGnism behind the effects of CH, we examined its action on intracellular chloride ion influx in cultured

human neuroblastoma cells. CH dose-dependently increased chloride ion influx, which was blocked by

co-administration of bicuculline, a competitive GABAA receptor antagonist. Taken together, the results of

the present study suggest that CH has sleep-promoting properties which are probably mediated through

ride i

the GABAA receptor–chlo

. Introduction

Sleep plays a vital role in our health and well-being. A “good

ight’s sleep” can soothe and restore a person’s physical and psy-

∗ Corresponding author.

E-mail address: cheongjh@syu.ac.kr (J.H. Cheong).

o

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ttp://dx.doi.org/10.1016/j.bbr.2016.07.013

166-4328/© 2016 Elsevier B.V. All rights reserved.

on channel complex.

© 2016 Elsevier B.V. All rights reserved.

chological state after a long and tiring day. Adequate and quality

sleep can also protect an individual from some diseases and acci-

dents, and can generally promote a better quality of life [1,2].

However, millions of people in the world do not get enough amount

of sleep or have difficulty falling or staying asleep (i.e. sleep dis-

rders). These conditions can lead to impairments in well-being

nd the development of various health problems such as obe-

ity, diabetes, and hypertension [1,3–5]. To manage sleep disorders

ral Br

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2 I.J.I. dela Pena et al. / Behaviou

nd avoid or mitigate sleep-related problems, sedative-hypnotics

rugs (e.g. benzodiazepines, diazepam) are commonly prescribed

y physicians. However, long-term use of these synthetic sub-

tances may lead to tolerance, dependence, and other unwanted

ide-effects [6,7]. Thus, there is a growing interest in the develop-

ment of new sleep-aids that don’t have or have lesser side effects

than sedative-hypnotic drugs, and most preferably from natural

sources.

Milk has long been known and used as natural sleep-aid.

Recently, we reported that milk collected during night time (Night

milk) produces sedative and sleep-promoting effects in mice, an

effect comparable to that of the benzodiazepine drug, diazepam

[8]. This effect of Night milk is attributed to its rich stores of

sleep-promoting components such as tryptophan and melatonin.

Another milk component that is thought to contribute to its sleep-

promoting properties is casein, the primary protein constituent

of cow’s milk. Studies have shown that enzymatic hydrolysis of

casein produces peptides with various biological activities [9,10].

Of these, bovine aS1-casein tryptic hydrolysate (CH), containing a

decapeptide aS1-casein known as alpha-casozepine, was shown to

have anxiolytic, anticonvulsant, and anti-stress effects [1,11–14].

tudies have also shown that CH can alleviate sleep disturbances in

oth animals and humans [1,12]. These effects of CH are thought to

e mediated through its affinity to the gamma amino butyric acid

GABA)A receptors. This receptor is also the main binding site of

enzodiazepine-type drugs. Despite the putative sleep-promoting

roperties of CH, no study has directly assessed the sedative and

leep-promoting effects of CH.

Thus, in the present study we evaluated the sedative and sleep-

romoting effects of CH in mice. CH was orally administered to

ice in dosages of 75, 150, 300, or 500 mg/kg of body weight.

hen, behavioral changes consequential to CH treatment (acute

r repeated) were observed in various experimental procedures

hat would characterize the sedative (open-field and rota-rod test)

nd sleep-promoting (pentobarbital-induced sleeping test and EEG

onitoring) activities of a substance. The most commonly used

nd prescribed benzodiazepine, diazepam, was used as a reference

rug in these experiments. Moreover, to further characterize the

ffects of CH and its possible mechanism, changes in chloride ion

Cl−) influx (GABAergic neurotransmission) was assessed in cul-

ured human neuroblastoma cells (SH- SY5Y), in vitro.

. Materials and methods

.1. Animals

Male ICR mice (25–30 g) and Sprague-Dawley rats (250–300 g)

ere obtained from Hanlim Laboratory Animals Co. (Hwasung,

outh Korea). They were housed in groups in a temperature-

ontrolled (22 ± 2 ◦C) and humidity-controlled (55 ± 5%) animal

oom on a 12/12 h light/dark (07:00–19:00 h light) schedule. They

ere acclimatized to the facility for seven days before the com-

encement of any experiments. Food and water were freely

vailable, except the night before and during the experiments.

ll experiments were performed between 10:00 to 16:00. Animal

reatment and maintenance were carried out in accordance with

he Principles of Laboratory Animal Care (NIH publication No. 85-23

evised 1985) and the Animal Care and Use Guidelines of Sahmyook

niversity, Korea (SYUIACUC 2015-009).

.2. Drugs and materials

Bovine aS1-casein tryptic hydrolysate (CH, also known as

actium®) was obtained from Ingredia (Arras, France). N-(6-

ethoxyquinolyl) acetoethylester (MQAE) was purchased from

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nvitrogen Co. (Carlsbad, CA, USA). Bicuculline was dissolved in

imethyl sulfoxide (DMSO), with a maximum concentration of

.1%. Pentobarbital sodium was obtained from Hanlim Pharm. Co.,

td (Seoul, South Korea). Diazepam and other materials were pur-

hased from Sigma-Aldrich Co. (St. Louis, Mo, USA). CH was freshly

repared in sterile distilled water and was orally (p.o.) given to mice

n dosages 75, 150, 300, or 500 mg/kg (CH-treated group). Diazepam

as diluted in physiological saline and was intraperitoneally (i.p.)

njected at 1 mg/kg (positive control group). Animals in the control

roup received the vehicle (distilled water, p.o.).

.3. Psychopharmacological evaluation

All experiments were conducted 60 min after the 1st (acute) and

th (repeated) day of treatment. Experiments were performed as

escribed in our previous studies [8,15], but with minor modifica-

tions.

2.3.1. Open-field test (OFT)

The open-field apparatus was a square arena measuring

42 × 42 × 42 cm. CH, diazepam, or the vehicle was given to mice

0 min before the start of each session. To start an experiment,

ice were gently placed in the center of the open-field and were

llowed to spontaneously explore the whole arena. Mice were

llowed to habituate to the open-field arena for 2 min to eliminate

he bias of novelty. Then, an automated system (Ethovision, Noldus,

etherlands) recorded the distance moved (cm) and movement

uration (seconds) of each subject for 10 min.

.3.2. Rota-rod test

The rotating rod or rota-rod apparatus used was purchased from

go Basile Corporation (Model 7650; Varese, Italy). It was set at a

xed speed of 36 rotations per minute (rpm). A day before the actual

est, mice were habituated and trained to run on the rotating rod for

min. Mice with poor performance were excluded. On the actual

ay of the experiment, mice were given CH, diazepam, or vehicle

nd placed on the rotating rod. Each session lasts for 10 min. Latency

ime before the first fall (seconds) and the number of falls (falling

requency) were recorded.

.3.3. Pentobarbital-induced sleeping test

Experiments were performed in a standard mice cage, mea-

uring 50 × 30 × 50 cm, with aspen beddings. Mice were given CH,

iazepam, or vehicle before the start of the test. To start a test and to

nduce sleep, pentobarbital sodium (42 mg/kg, i.p.) was injected to

ach mouse. Immediately after pentobarbital injection, mice were

ndividually placed in cages for observation. The disappearance

onset of sleep) and reappearance (duration of sleep) of the righting

eflex were monitored and recorded, for a maximum of 2 h. Animals

hat did not sleep within 15 min after pentobarbital administration

ere excluded from the experiment.

.3.4. Electrophysiological (EEG) recording

To assess the changes in brain electrical wave activities, rats

ere surgically implanted with wireless telemetry and were

ecorded by LabChart 7.3 software (AD Instruments, USA). Animals

ere anesthetized with pentobarbital sodium (50 mg/kg, i.p.) and

xed in a stereotaxic apparatus (51600 Stoelting Co., Wood Dale,

llinois). A two-channel digital transmitter (Telemetry Research,

uckland, NZ) was intraperitoneally (abdomen) implanted to each

at then electrodes were shunted towards the skull. Electrodes were

ositioned into the left frontal and right occipital cortical regions

hen fixed with a screw and stabilized with dental cement. Seven-

ay post-surgery (recovery period), rats were given vehicle, CH, or

iazepam, and then 60 min after, electroencephalographic (EEG)

ecording were started. Two frequency bands, delta (0.5–4 Hz; slow

ral Br

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I.J.I. dela Pena et al. / Behaviou

ave) and alpha (8–13 Hz; fast wave), were quantified using Fast

ourier-transformed (FFT).

.3.5. Intracellular Cl− measurement assay

The effects of the CH, or CH + bicuculline, on intracellular Cl−

concentration ([Cl−]i) in SH-SY5Y human neuroblastoma cells

were evaluated. Relative changes were measured using the Cl−-

ensitive indicator, N-(6-methoxyquinolyl) acetoetylester (MQAE).

ells were washed twice and re-suspended at a concentration of

× 105 cells/ml in Hank’s solution. For loading MQAE into the cells,

ells were incubated overnight with the dye at a final concentration

f 5 mM at room temperature. Fluorescence (excitation wavelength

et at 365 nm, emission wavelength at 450 nm) was monitored in

well-stirred cuvette. Experiments were executed at room tem-

erature to minimize fluorescent dye loss. Data are presented as

elative fluorescence F/F0, where F0 is the fluorescence without Cl−

ions and F is the fluorescence as a function of time. The F/F0values

are directly proportional to [Cl−]i. Fluorescence values were modi-

fied for background fluorescence which was separately determined

using an HEPES-buffered KSCN solution containing five mM valino-

ycin to maximally quench the MQAE ion-selective signal. In a

eparate experiment the F0 value was determined by bathing the

ells with Cl−-free (KNO3) solution containing 10 mM tributyltin

nd 10 mM nigericin.

.4. Statistical analysis

All data were expressed as mean ± S.E.M. Data were analyzed

sing one-way analysis of variance (ANOVA). When statistically sig-

ificant differences were found, Dunnett’s test was used as a post

oc test to determine the statistical differences between groups.

ll statistical analyses were conducted using GraphPad Prism Ver-

ion 5.02 software (California, USA). Differences were considered

tatistically significant when p < 0.05.

. Results

.1. Open-field test

Fig. 1 shows the distance moved (A, C) and the movement dura-

ion (B, D) of animals treated with CH, diazepam, or vehicle (control

roup). One-way ANOVA showed significant differences in distance

oved (D1 [F (5, 92) = 2.37, p < 0.05], D5 [F (5, 74) = 2.15, p < 0.05)

nd movement duration (D1 [F (5, 92) = 4.49, p < 0.01]) between

he experimental groups. Acute (single) treatment of diazepam

emonstrated a significant decrease in distance moved ([q = 3.204];

ig. 1A) and movement duration ([q = 4.197]; Fig. 1B) compared to

the vehicle-treated (control) group, exhibiting diazepam’s sedative

effect. However, repeated treatment showed a significant increase

in distance moved ([q = 3.114]; Fig. 1C). CH-treated mice showed

a decreasing trend in distance moved and movement duration;

however, it did not show statistical significance compared with the

control group.

3.2. Rotarod test

Fig. 2 shows the latency to first fall (A, C) and falling frequency

(B, D) of mice treated with CH, diazepam, or vehicle (control group).

One-way ANOVA showed significant differences in latency to first

fall (D1 [F (5, 92) = 2.83, p < 0.05]) and falling frequency (D1 [F (5,

92) = 4.24, p < 0.01]) between the experimental groups. Diazepam-

treated mice significantly showed decrease in endurance/latency

time (D1 [q = 3.194]; Fig. 2A) and increased falling frequency (D1

[q = 3.477]; Fig. 2B) on the rotating rod, as compared to the control.

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n support to the open-field test, CH-treated mice did not show a

ignificant difference in latency time and falling frequency.

.3. Pentobarbital-induced sleeping test

Fig. 3 shows the onset/loss of righting reflex (A, C) and total

duration (B, D) of sleep induced by pentobarbital sodium in

mice pretreated with CH, diazepam, or vehicle (control). One-

way ANOVA showed significant differences in sleep onset time

(D1 [F (5, 92) = 11.57, p < 0.001], D5 [F (5, 73) = 2.85, p < 0.01])

and sleeping duration (D1 [F (5, 92) = 34.47, p < 0.001], D5 [F (5,

73) = 5.00, p < 0.001]) between the experimental groups. Diazepam

treatment potentiated pentobarbital-induced sleeping behaviors

as shown by a decreased sleep onset time (D1 [q = 7.148], D5

[q = 3.382]; Fig. 3A) and increased total sleep duration time (D1

[q = 7.148], D5 [q = 4.141]; Fig. 3B & D). CH treatment also poten-

tiated pentobarbital-induced sleep duration at 150 mg/kg (D1

[q = 3.725], D5 [q = 2.616]; Fig. 3B & D).

3.4. Electroencephalogram (EEG) recording

Fig. 4 shows the total power or delta (A, C) and alpha (B, D)

aves in the EEG of rats treated with CH, diazepam, or vehicle.

ne-way ANOVA showed significant differences in slow (delta total

ower) [D1 F (3, 44) = 5.812, p < 0.01] and fast (alpha total power)

[F (3, 44) = 2.992, p < 0.05] waves. Diazepam-treated rats showed a

significant increase in delta waves (D1 [q = 3.939]; Fig. 4A) and a sig-

nificant decrease in alpha waves (D1 [q = 2.942]; Fig. 4B). CH-treated

ats also showed a significant increase (D1 [q = 2.673]; Fig. 4A) in

delta total power and a downward trend in alpha waves; however,

it did not reach statistical significance compared with the vehicle-

treated (control) rats.

3.5. Intracellular Cl− measurement assay

Fig. 5 shows the effects of CH on Cl− influx in human neurob-

lastoma cells in vitro. Treatment of CH increased Cl− influx in a

dose-dependent manner (Fig. 5A). Contrariwise, bicuculline, a com-

petitive GABAA receptor antagonist, inhibited the increased in Cl

influx induced by CH (Fig. 5B).

. Discussion

In the present study, our goal was to evaluate the sedative and

leep-promoting effects of bovine aS1-casein tryptic hydrolysate

CH). We have found that CH has sleep-promoting properties as evi-

enced by an augmented pentobarbital-induced sleep in mice and

n increased slow (delta) EEG wave in rats. This sleep-promoting

ffect is probably mediated through the GABAergic neurotransmit-

er system.

The probable sedative effect of CH was evaluated through the

pen-field and rota-rod test. Consistent with previous studies, the

enzodiazepine drug, diazepam, decreased (after single treatment)

he locomotor activity of mice in the open-field test (Fig. 1). A

decreased locomotor activity in this test is considered to reflect

the sedative effects of a substance [16]. However, mice repeatedly

treated with diazepam showed a significant increase in the open-

field test (Fig. 1). Similar results were observed by Pinna et al. [17],

suggesting that locomotor increase induced by diazepam in repeat-

edly treated subjects might be due to the reduction of neophobia or

drug tolerance [18,19]. Although a trend can be observed, CH did

not significantly alter the locomotor activity of mice. This result

indicates that this substance has no strong sedative effects. Similar

results were observed in the rota-rod test (Fig. 2). The rota- rod test

s another test used to evaluate the sedative and/or muscle-relaxant

roperties of a substance [20]. We have found that CH treatment did

4 I.J.I. dela Pena et al. / Behavioural Brain Research 313 (2016) 1–7

Fig. 1. Effects of bovine aS1-casein tryptic hydrolysate on locomotor activity in mice (n = 14–20). Each bar represents the mean ± SEM of the distance moved (A, C) and

movement duration (B, D), for 10 min. ** p < 0.01 significantly different from the control group. Abbreviations: Con—control; DZP—diazepam.

Fig. 2. Effects of bovine aS1-casein tryptic hydrolysate on the rotating rod performance of mice (n = 14–20). Each bar represents the mean ± SEM of the latency time (A, C) and

falling frequency (B, D) during the 10 min rota-rod test. * p < 0.05, and ** p < 0.01 significantly different from the control group. Abbreviations: Con—control; DZP—diazepam.

I.J.I. dela Pena et al. / Behavioural Brain Research 313 (2016) 1–7 5

Fig. 3. Effects of bovine aS1-casein tryptic hydrolysate on pentobarbital-induced sleeping test in mice (n = 14–20). Each bar represents the mean ± SEM of the onset of sleep

(A, C) and total duration of sleep (B, D) during the 120 min sleeping test. * p < 0.05, ** p < 0.01, and *** p < 0.001 significantly different from the control group. Abbreviations:

Con—control; DZP—diazepam.

Fig. 4. Effects of bovine aS1-casein tryptic hydrolysate on EEG activity in rats (n = 8-12). Each bar represents the mean ± SEM of the total power of delta wave (A, C) and alpha

wave (B, D) for 1 h which was 1 h after administration. * p < 0.05 and ** p < 0.01 significantly different from the control group. Abbreviations: Con—control; DZP—diazepam.

6 I.J.I. dela Pena et al. / Behavioural Brain Research 313 (2016) 1–7

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Fig. 5. Effects of a bovine aS1-casein tryptic hydrolysate on [Cl−]i in neuroblastom

nd the emission wavelength at 450 nm using the Cl−-sensitive indicator, N-(6me

eak (F/F0). Abbreviations: BIC—bicuculline.

ot cause any significant motor alteration in this test. Diazepam still

emonstrated sedative effects as evidenced by decreased falling

atency and increased falling frequency. Together, the results of the

FT and rota-rod tests suggest that CH does not have or have very

inimal sedative effects.

In the pentobarbital-induced sleeping test, diazepam reduced

he sleep onset and increased the sleep duration of mice. CH also

otentiated the duration of sleep but not the onset of sleep induced

y pentobarbital sodium (Fig. 3). This result suggests that CH pos-

sesses sleep-enhancing effect, but it might lack sleep-inducing

effect. Substances that potentiate the pentobarbital-induced sleep

possess centrally acting sleep-promoting properties [21,22]. The

result of the pentobarbital-induced sleeping test is further sup-

ported by the results of the EEG monitoring in rats. EEG is

considered to be a vital and sensitive parameter during sleep

examination and is used to evaluate and diagnose sleep-related

problems/sleep disorders [23]. Treatment with CH (150 mg/kg) sig-

nificantly increased slow (delta) wave activity (Fig. 4), similar to

that of diazepam. This result is considered to be an indication of

sleep. Generally, high frequency (alpha) EEG waves are decreased

during sleep and, as the stages of sleep progress, delta waves gov-

ern the EEG tracing [23–25]. Together these results indicate that

CH has sleep-enhancing effects. This finding is in concordance with

previous studies showing that CH improves sleep in stressed sub-

jects [1,11,12] and consistent with the notion that this substance

ontributes to the sleep-promoting effects of milk.

It may seem confounding that in our experiments CH did not

nduce sedative effects but has sleep-promoting effects. However,

everal studies have suggested that sedative and sleep-promoting

ffects are related but dissociable processes [26,27]. Studies have

suggested that different brain regions mediate these effects. For

instance, a recent study has shown that slow-wave sleep, similar

to the one observed in the present study, is mediated by GABAergic

neurons in the medullary parafacial zone [28], while the seda-

tive effects induced by GABAergic agents are probably mediated

through the tuberomammillary nucleus [29]. Taking these things

into consideration, it is quite rational to conclude that CH pos-

sesses sleep-enhancing effects but is devoid of or has minimal

sedative effects. Further studies are needed to validate this claim

and to dissect the mechanisms by which these effects are medi-

ated. Regardless, our behavioral experiments have demonstrated

that CH has sleep-promoting effects in rodents.

We also investigated the probable mechanism behind the sleep-

enhancing effect of CH. It was reported that CH have a similar

structure with GABA and have an affinity for benzodiazepine

receptors, specifically GABAA receptor subtype [13]. Therefore,

we studied its action on Cl− influx neuroblastoma cells. Indeed,

CH significantly increased Cl− influx in a dose-dependent man-

ner (Fig. 5). Moreover, this CH-induced Cl− influx was blocked

by co-administration of bicuculline, a competitive GABAA recep-

tor antagonist. Altogether, these results strongly suggest that the[

s (SH-SY5Y). Fluorescence was monitored in the excitation wavelength at 365 nm

quinoyl) acetoetylester (MQAE). Contents of influxed Cl− ion were expressed as a

sleep-promoting effect of CH is mediated through GABAergic neu-

rotransmission, notably through the GABAA receptor-Cl− channel

complex.

In conclusion, the present study suggests that CH is capable of

facilitating and promoting sleep, with no or minimal sedative prop-

erties. The sleep-enhancing effects of CH are probably mediated

through the GABAA receptor-Cl− channel complex. CH might be

used as a natural sleep aid for managing sleep-related problems.

Acknowledgement

This work was supported by the Chungbuk Bio International

R&D Project 2014-1-01. We also would like to thank Ingredia SA

(Arras, France) for their supporting knowledge on Lactium®.

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