Pharmaceutical Society of Japan

NII-Electronic Library Service

PharmaceuticalSociety of Japan
80S BioL
Pharm.BulL
28(5)
808
-81O

{2005)
Vbl,28,
No.5
Hypnotic Activities of Chamomile and
Sleep-Disturbed Rats
PassifioraExtracts in
Kazuaki SHiNoMiii,,a [IbshioINouE,b
lk)shiaki UTsu,aShin
ToKuNAGA," fakayoshiMAsuoKA,"
Asae OHMoRi,"and ChiakiKAMEi,*"
"Depantment

of
PharmacolQgy,FLiculty
qf'
PharmaceuticatSciences,Okql'ama Uhiversity;i-1-I tsushima-naka,
Okayama 700-8530,Jltpan:and
bMdteriat
R&D Laberato,?l
Qgawa
& Co..Ltd.;15-7 anidori,Uluyasu,Chiba
279-O032,.ku)an.ReceivedNovember 19,2004;accepted February 1,200S

In the
present
studM we inyestigated hypnoticactiyities of chamemile and
passiflora
extracts using sleep-dis-
turbed model rats. A significant decrease in sleep latency was observed with chamomile extract at a dose of
300 mgtkg, whi}e
passiflora
extract showed no etfects on sleep latencyeyen at a doseof30eO mg/kg. No significant
etfects were observed with both herbal extracts on total times of wakefulness, non-rapid eye movement
(non-
REM)
sleep and REM sleep. Flumazenil,a benzodiazepine
reeeptor
antagon;st, at a dose of 3mgtkg
showed

a
significant antagonistic effect on the shortening insleep latencyinducedby ehamomile extract. No significant eF
fectswere observed with chamomile and
passiflora
extracts on deltaactivity during non-REM sleep. In conclu-
sion, chamomile extract isa herb havingbenzodiazepine-like hypnoticactivity

Key words chamomile;
passiflora;
insomnia;herbaldn]g;sleep-disturbed model; rat

Benzodiazepines and benzodiazepine analogues are the
most widely used hypnotic drugssince the 1960s.i)However,
itiswell known that benzodiazepines
have
many untoward
reactions,
such as drug dependence,tolerance,rebound
insomnia,amnesia

and

muscle

relaxation.2'3}

Therefore. it
seems

likely

that

these

hypnotics
need to be used cautiously.

On the other hand herbaldrugs,such as chamomile and
passiflora,
are extensively used as traditional medicine for
treatmentof insomniaand anxiety. Therehavebeensome re-
ports
on the hypnoticeffects of these herbalextracts in hu-
mans.4'5)

lnanimal

experirnents,

Soulimaniet

aL6)

also

re-
ported
that the aqueous extract ofpasstt7bra incarnata L
caused a decreaseofrearing and locomotioninthe staircase
apparatus or exploratory test.The aqueous extract of
passi-
floraalso indueed sleep after treatment with a subpharmaco-
logical doseof

pentobarbital

inmice.6)

DellaLoggiaet

aL7)
demonstrated

that

the

extracts

frornimtricaria chamomilla
and
Aissij7ora
incarnata showed a significant increase inbar-
biturates-induced sleep time inmice. However,the literature
dealingwith the hypnoticactivities ofchamomile and
passi-
flora

extracts

by

measuTing
electroencephalogram
(EEG)
and
electromyogram
(EMG)
inanimals isscanty. The normal an-
imals have high-baseline
sleep
time, and the hypnotic
poten-
cies of charnomile and
passifiora
may be mild compared with
those of benzodiazepines and benzodiazepine analogues.
Therefbre,itmay be diMeult to evaluate the hypnoticactivi-
tiesofthe herbaldrugs innormal animals.

In the
previous
study, we developeda sleep-disturbed
model that is usefu1 for evaluating hypnotic activities by
placing
rats on a
grid
ina cage fi11ed with water to 1cm
belowthe
grid
surface.S) The hypnoticetllects of drugsinrats
placed
on the
grid
suspended over water were rnore
potent
than

thatinrats

placed

on

sawdust.S'9) Inthe

present

studM
we studied the hypnotic activities of chamomile and
passi-
fiora

extracts

using

the
sleep-disturbed
model

rats.
MMERIALS

AND

METHODS
Animals Thirty-two rnale Wistar rats wcighing 220-
*
To whorn cerrespondence shou]d be addressed.
300
g

(Japan
SLC, Shizuoka, Japan)were used. Allanimals
were

rnaintained

inan

air-conditioned

room

with

controlled
temperature
(24
2OC) and humidiry
(55
15%). They were
housedinaluminium cages with sawdust and keptunder a
]ight-darkcycle
(lights
on from 7:OO to 19:OO).The animals
were
allowed
free
access tofoodand
water

except

during

the
experiments. All
procedures
involvinganimals were con-
dueted inaccordance with the
guidelines
of the Animal Care
and Use Committee,Facultyof Pharmaceutieal Sciences,
Okayama University.

Surgery The animals were anesthetized with
pentobarbi-
talsodium
(Nernbutal'S,
35mg/kg, i.p,,Abbott Laboratories,
NorthChicago,IL,U.S.A.), then fixedina stereotaxic appa-
ratus
(SR-5,
Narishige, Tokyo, Japan). Forelectroencephalo-
gram

(EEG)
recording, a stainless steel screw electrode was
chronically implanted intothe right fi;ontal cortex
(A:
O.S,
L:
3.0) according to the atlas of Paxinos and Watson.iO)Tb
record

the
electromyogram
(EMG),
stainless steel
wire
elec-
trodes
(200 pm)
were implantedintothe dorsalneck muscle.
A

stainless

steel

screw

fixed

inthe

left

frontal boneserved

as
a

reference

electrode.

The

electrodes

were

connected

to

a
miniature receptaele and the whole assembly was fixedto the
skull

with

dentalcement,

At

least7

d

were

allowed

forrecov-
ery fromthe surgery.

EEG and EMG Recordings EEG and EMG were
recorded with an electroencephalograph
(Model
EEG 5113,
Nihon Kohden,
'[bkyo,
Japan) from 9:OO to 15:OO. The
recording was carried out according to the method
described
previously.9,ii,i2)
The signals were amplified and filtered
(EEG,

O,5-30Hz;

EMG,

16-128Hz),
then digitizedat a
sampling rate of 128Hz and
recorded
using
the
data
acquisi-
tion
program
SLeepSign ver. 2.0
(Kissei
Comtec,Nagano,
Japan).EEG and EMG of the
rat
were
measured

in

a

eylin-
drical
plastic
cage. A
grid
floorwas
placed
inside the
plastic
cage. The cage was filledwith watcr up to 1 cm below the
grid
surface. The stainless steel rods of the
grid(3
mm

wide)
were set 2cm apart from each other, The observation cage
was
placed
ina sound-proof and electrically-shielded
box.

Sleep-WakeStateAnalysis The sleep-wake states
were
e-mait/kamei@pheasant.pharm,okayama-u,ac.jp
C/2005 PharmaceuticalSocietyofJapan
Pharmaceutical Society of Japan
NII-Electronic Library Service
PharmaceuticalSociety of Japan
Mav

2005automatically
classified in 10-s epochs as wake, non-rapid
eye

movement

(non-REM)
or REM sleep
by
SleepSign
ver.
2.0according

to the

criteria
previous]y

described.S'T]) As

a
final step, definedslcep-wake stages were exarnined visuallM
and corrected, ifnecessary. Each state
was
characterized as
fo11ows: wake, low-amplitude EEG and high-voltage EMG
activities; non-REM sleep, high-amplitude slow or spindle
EEG and low-EMG
activities;
REM
sleep,
low-voltageEEG
and

EMG

activities.

Calculation for Detta Activityduring Non-REM SIeep
The deltaactivity within non-REM sleep was determined
using a
program
of SleepSignver. 2.0.The
power
spectrum
densities, integratedand averagect courd be dividedintothe
4 frequencyareas: deltawave
(O.5--4
Hz),theta
wave

(4-
8Hz),alpha wave
(8-i3
Hz) and betawave
(l3-30Hz),
The

data
of delta
power
innon-REM sleep were expressed as
a
percentage
of the average deltaactivity during
non-REM
sleep during the entire recording
period
of each control

14,[S)
group.

Drugs The fo11owing
drugs
were used: chamomile
ex-
tract
{Ogawa
& Co., Ltd.,Chiba, Japan),
passifiora
cxtract
(Ogawa
&

Co..Ltd.)and

fiumazenil
(Sigma,
St.Louis,MO,
U,S.A,),The chamomile and
passiflora
were extracted by re-
fiuxingin water for 1h from the flower
part
of imtricaria
chamomilla or the
aerial
part

of
llassij7ora incarnata, respec-
tively. The charnomile and
passiflora
extract were suspended
inO.S% carboxyrnethyl eellulose
(CMC)
solution and admin-
istered orally

at

9:OO,and

EEG

and

EMG

were

measured

for
6h after drug administration. Inthe antagonism experiments,
flumazenilwas
dissolved
in 109r6dimethyl sulfoxide and in-
jected
intraperitoneally beforethe drugtests.Eightrats werc
used ineach
group,
and counterbalanced design fordrug
dosage was used. Drugs were administered at intervals of 7d
when the same rats were used forrepeated experiments. Each
rat was subjected to
experirnent
for drug study three or four
tlmes.

Data Analysis
and

Statistics
Values shown are
means
S.E,M.One-wayanalysis of
variance

(AN
OVA)
with
the Dunnett'stest was
used
for estimating the drugefTects.
Sleep latencywas definedas the time from drug administra-
tionup tothe first 12consecutive 1O-sepochs of sleep.
RESULTS

Effectsof Herbal Extracts en Sleep Parameters A sig-
nificant shortening of sleep latency was observed with
chamomile extract at a doseof3OO mg/kg. On the other hand
passiflora
extract had no effects on sleep latencyeven at a
dose

of
3000mglkg
(Fig.
1).No significant effects
were

ob-
served

with

both

herbal

extracts

on

total

times

of

wakefu1-
ness, non-REM slecp or REM sleep
(Fig.
2).

Effect
of
Flumazenil
on

the Shortening
in
Sleep
La-
tency Inducedby Chamomile Extract Flumazenilalone
showed no significant etTt)cts on sleep latencyat doses of 1
and 3mgtkg. The shortening of sleep
latency
caused by
chamomile extract
(300
mg/kg) was significantly antagonized
byflumazenil at a dose of3 mgfkg
(Fig.
3),

Effects of Herbal Extracts on Delta Actiyity during
Non-REM S)eep No significant effects were observed with
chamomile
and
passiflora
extracts
on

delta

posN'er
during
non-REM sleep
(Table
1
).
809
ri20
1oo
s:.-E:6e-es
42
oo
o
o
,F1
o

Centre1
3o

loo

3oe

Centro1

3oo

looo

3voO

(mglkg)
Chemomiletxtraet

Passitlersextract
Fig, 1. Effkrets of Herba] Extractson Slcep Latency in Sleep-Disturbed
Rats

C/oluninsandi'orticalbarsrepvesenlnieansS.E,M.Cn'=8).DrugswcreHdministcrcd
eralty.*Significantl)'differcntt'romcontTelgroupatp,/{),O)'..
/
!-
*
z
'
360
rt24oge.l:eR
i2o
11'
/t'J
vaHEMsleep
[]
Nen-REM
sleep
ew
wske
o
Contre1

3o

loe

3oo

CentroL300

MOo

3000(mglkg)
Chamemileextract

Passitlersextract
Fig.
2,
Effects
ef Herbal lixtracts on 1intalTime ot' Each Sleep Statein
Sleep-Djsturbcd Rats

CelLanns r:present the means of cuch slccp stute
Cn=S).
Drugs "'erc administered
oraIly. and EFG and EMG vL'ere mensurcd for6h,
rtgtrgtt"
1201oo
sooo
4020
o
I
!
aa
*
%
Fig,3.by
Chamomile ExtractinSteep-DisturbcdRats

C"lumnsnnd verticul hursruprcsent mc/ms S.F.M.
(n=S).
('
administered erall)', and fiumazcnil -as injected intraperiteneally.
entiremcontrolgroupatp<O.CIS.

Centrel l 3 Centrel l 3
Cmgrkg}
Flumazenil

Flvmalenie
CMCselution
Chamemlleextnlct
Efit]cts ef Flumazenilon the Shorteningin SieepLalency Induced
hHnitiiniTecxtrattwHs
*SignificantlydM'er-
Pharmaceutical Society of Japan
NII-Electronic Library Service
PharmaceuticalSociety of Japan
810 Nlo1,28,No. 5
Table 1.Effects

ot'

Herbal

Extracts

on

Delta

Activity
DuringNon-REM SIeepinSleep-Disturbed Rats
EEG activity
(%)
Dose(mg/kg,p.o.)
or.1 1-2 2-3 3-4 4-5 5-6
(h)
Chamomileextract
Control
30
100
300
Passifloraextract
Control
300
IOOO
3000
67,25.473.8
11.487.0
8,984,O
6,969.3
5.467,6

5,583.9
16.1
72.77,O
86.8 7.4IO12
15.8952
8.097,8
7.894.S
S.283.4
6.480.8
9.188.6
6.6
96,4 9.4I06.9

12.0
107,5 tL]
II2,2 8.1105.S

4.497,2

5.192.0

9,8r07.1
10.6
I04.9 8.6116.8
13.6
111,8l5.4
108.3 8.610S.6
5.1t03,1
4,8108.7
7,894.S
6.8
108.9 7.S1132
13.3
ltO,8 10.1
r13,7 19.l
100.6 32109,4

9,11042
5,O97.6
5.S
104.9 8.293.2

10.599.8
10.3
102.7 16,39g.2
3,9100.4

5.097.2
6.587.9
7,1
Datarepresentrmeaps S.E.M.(n=8).Drugswereadministeredorally.
DISCUSSION

The
present
study was undertaken to investigatethe hyp-
notic
activities of

chamomile
and
passifiora

extracts

using
the sleep-disturbed model rats. As shown in the text,
chamomile extract at a doseof 300mglkg caused a signifi-
cant
shortening
in sleep latency
Gould
et al.
i6)
reported that
hospitalized
patients
were
given
a strong chamornile tea,and
ten of the twelve
patients
immediately fe11intoa deep sleep
lasting
90min,DellaLoggiaet

aL')

also

demonstrated
that
chamomile extract caused a significant
prolongation
ofs]eep-
ingtime induced by barbiturates inmice. In addition, Aval-
loneet aLi7) showed that apigenin, a fiavonoidisolatedfrom
Mbtricariachamomilla, significantly reduced the locomotor
activity inthe open field testofrats, Thesefindings strongly
suggest that charnomile isa herbal
product
havinghypnotic
activity inanirnals.

To investigate
the
detailed mechanisms invotved
in

the
hypnotic
potencies
caused by chamomile extract, the efllect
of fiumazenil
(benzodiazepine
antagonist) on chamomile ex-
tract,thatitinducedthe shortening of sleep latencM
was
studied,

Flumazenil at

a

dosethat

caused

no

obvious

effect
when used alone showed a significant antagonistic effect
on the decreasein sleep latencyinduced by chamomile ex-
tract. Violaet

aLiS)

reported

thatapigenin

extracted

from
chamomile flowersinhibited
[iH]-fiunitrazepam
bindingin
the

bovinecerebral

cortex.

Avallone et

al.i9)

also

demon-
strated

that

the

fractionsof

extracts

from Mdtricaria
chamomiUa

selectively
bound to central benzodiazepine re-
ceptors

using

a

[3H]-flunitrazepam

bindingassay
in
rats.On
the other hana itiswell known that benzodiazepine receptor
agonists cause hypnotie effects on the sleep-wake cycle in
humans

and

animals.B'Y'20'2i)
We have also reported that ben-
zodiazepines, such as triazolamand fiunitrazepam caused
the
decreaseinsleep latency,total waking time and deltaactivity
using

the

present
model.S'Y'i4) From these findings,itseems
likelythat the shortening of sleep latencyinduced by
chamomile
extract may be caused by apigenin, a component
of Matricariaehamomilla,
through
benzodiazepine
recep-
tors,

In the
previous
study using the sleep-disturbed model rats,
drugs
havingthe
powerfuI
anxiolytic
potencies
caused more
potent
hypnotic

potencies
than

those

with

weak

anxiolytic
activities.S)Viola et al.
iS)
reported that apigenin induced anx-
iolyticaction in an elevated
plus-maze
test
of

mice.
From
theseresults,we

assume

thatthe effect of

chamomile

extract
may bedue tonot only hypnoticactivities but also anxiolytic
activities

ofthe

herb,

Inconclusion, chamomile extract isa herbhavingbenzo-
diazepine-like hypnoticactivity.
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