Indi an Jo urnal of Experimental Biology
Vol. 42, June 2004, pp. 632-635
Role of 5-hydroxytryptamine in Moringa
oleifera induced potentiation of
pentobarbitone hypnosis in albino rats
Kausik Ra/, Rimi Ham}l , Pratip Kr Debnath 2 &
Debjani Guha l •
IN e urophy siology Laboratory, Depa rtme nt o f Ph ysio logy,
Uni versity of Calcutta, 92, A.P.C. Road. Ko lkat a 700 009 , Indi a
2 J B Roy State Ayurvedi c Medica l Co ll ege and Hospital s,
170-172. Raja Dine ndra Strcet, Ko lkata 700004. India
·s N Pradhan Centre fo r Ncurosc ie nces, Unive rsity of Calcutta,
244B A.J.C. Bose Road. Kolkata 700 020,India
Received 4 Septelllber 2003; re l'ised 16 March 2004
The role of 5-hydroxytryptaminc (5- HT) in pe ntobarbi lO ne
(PB ) sleeping time, g ross behaviour, e lec tri cal activity o f th e brain
and scrum 5-HT leve l was studi ed in Ho ltz man strain adult albino
rats foll ow in g treatmc nt w ith M. oleijera (MO). MO (350mg/kg)
ca used inhibition of awareness. to uc h response, mo tor acti vity,
ri g htin g re ne x, and g rip stre ngth. It sig nifi can tl y increased the PB
sleeping time. serum 5-HT level (P<O.OOI) and a-wave activity.
These o bse rvation s indicate that the aqu eo us ex trac t o f MO
potentiat ed PB induced sleep in g tim e a nd increased th e a-wave
ac tiv ity throu gh 5-HT.
Key words: Moril/ga ole(fera. PB sleepi ng tim e, Scrotonin , EEG.
5-hydroxytryptamine (5-HT) IS a monoam1l1e
neurotransmitter of the central nervous system. It is
present primarily in the raphae nucleus , hippocampus,
amygdala, blood platelets, and mast cells. Besides
neurotran smission , 5-HT plays an impoltant role in a
variety of physiological responses like mood, appetite,
sl eep, depression and cognitive dysfunction. That central
5-HT has a role in the process of sleep is strongly
supported by ev idence that p-chlorophenylalanine, a
selective 5-HT sy nthesis inhibitor regul arly precipitates
nearly total insomnia in cats u . Similarly, reserpine a
central monoamine depletor, produces insomnia in cats.
5-hydroxytryptophan restores sleep temporarily in p-
chlorophenylalanine pretreated cats t and on chronic
administration, preve nts p-chlorophenylalanine
in somnia' . Conclusive ev idence for serotonergic
med iation of sleep in cats, has been provided by
destructive lesions of raphae areas, which lead to marked
*Correspo nde nt author
Pho ne: 091-033-2223-2084
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Email: debjaniguha @rediffmail. co m
reduction of 5-HT synthesis, release and metabolism,
where total insomnia lasting for several days is seen 2 .
Several studies reported that 5-HT plays an essential role
for the production of sleep4.5.
Moringa oleifera (Ver Sajnae; MO) a perennia l
plant is found and cultivated in West Bengal. Flowers
and leaves of the plant possess depressant propertl· 7
whereas root of the pl ant is used by tribal groups of
India as antiepileptic agentS .Majumdar el a/ 9 reported
that crude methanolic extract of MO possess less
toxic action (LD 50 : 2.8g/kg, i p) in mice. Ray et 011 0
reported that MO decreased the locomoto r activity
and pe nicillin induced convulsion by alte ring brain 5-
HT, dopamine and norepinephrine. Thus it seemed
pertinent to explore its effect on pe ntobarbitone (PB)
induced hypnosis a nd electrical acti vi ty. An attempt
has been done to relate the effect with 5-HT.
Preparation of extractlO-The root pieces (I kg) of
MO were purchased from the locally. The plant
mate rial was ide ntifi ed, auth e nticated and kept in
De partme nt of Physi o logy , Calcutta University. The
root bark was di scarded and the root was sundried,
grinded a nd spread over tray w ith shifting o f materials
every day to avoid growth of fungu s. The powder was
soa ked in water overnight and th e so luti on was
filtered with Whatm a n No. I filte r paper and
subjected to lyophilization. The final y ield was 13 %.
AHilllals and treatment-Adult Holtz man strain
albino rats of either sex(l08) weighing I 50± 109 were
used . The rats were ho used in groups in cages at an
ambient te mpe rature of 25 °±1 °C and 45 .5% RH , with
a 12: 12 hr LD cycle. T he animals had fre e access to
standard laboratory di et and tap water ad IibilulIl . All
animal studies were performed in accordance wit h
institutional ethical committee and all procedures
were followed as per rules and reg ulat io ns.
. In the first set of ex periment, be havioural effect
and PB s leeping time were noted . Fifty four (54) rats
were divided into 9 groups-control (group I) and 8
experimental groups (group II-IX) . Each group
consisted of 6 animals. Group I rats were treated with
saline (5mllkg, po) . Groups II-IX rats were treated
with MO orally usin g orogastric cannula in the doses
of SO, 100, I SO, 200, 250, 300, 350 and 400mg/kg
respectively between 0900-1 IOOhrs and 30 min later
 NOTES
the gross behavioural respo nses were noted. For PB
sleeping time, MO was given orally 4hr prior to PB
admini stration in experimental rats.
In the second set of experiment, electrical activity
and serum 5-HT were noted. Another fifty four (54)
rats were divided same as the first set of experiment i.e;
control and experimental group.The control rats were
treated with saline (5ml/kg,po) and experimental ' rats
were treated with MO (50-400mg/kg , po) and 30 min
later electrical activity was recorded (8 Channel EEG
Medicare & Recorder, Chandigarh) for 5-6 hr without
interruption. After EEG studi es, rats were sacrificed
and blood was collected from jugular vein for serum
separation and 5-HT was estimated.
Behavioural effects"-The effects of MO on
awareness, grip strength , touch response, righting
refl ex and spontaneous motor acti vity were observed
by conventional methods.
PB induced sleeping time'2·IJ-The PB (40mg/k g,
ip, Abbott India Ltd) induced sleeping time was
measured as the time interv al between the loss and the
regain of the ri ghting reflex. The ri ghting re flex was
considered to be lost when the animal placed on its
back fail ed to regain its no rmal posture within 10 sec.
The control rats received o nly PB. The experimental
rats were treated with aqueous ex tract of MO and after
4hr PB was given intraperi toneally .
Surgical procedures for electroencephalographic
studies-Prior to surgery all the animals were fasted
overnight but had free access to water. Under
pentobarb ital anaesthes ia (40 mg/k g, ip; Abbott India
Ltd) rats were mounted o n the stereotaxic apparatus
(lNCO, Indi a Ltd). Care was taken to prevent the
damage of the tympanic membrane. Head was fixed in
such a positi on that lambda and bregma were in the
same hori zontal plane. The scalp was incisio ned in the
midline and the pericranial mu scles were retracted
laterally. After retracting th e nuchal musculature the
overlying bone was drilled at the specific loci (surface
cortex). Bipolar electrodes were impl anted on the
sUlface of the cortex through trephined holes and fixed
with dental cement''' . A reference electrode was
implanted over the frontal bo ne and all electrodes were
then so ldered to a multipl e plug which was fastened to
the calvarium with dental cement. Penicillin (10,000
IU) was inj ected intramuscularly o n the day of
operation and for the next two consecutive day s as
antibiotic meas ure. The electrical activiti es from
cerebral cortex were monitored through an 8 channel
EEG machine (Recorder & Medicare, Chandigarh).
633
Recordings were taken approximately every 5 mm
throughout the session for a period of 5-6 hr. ' 5
Biochemical estimatioll of 5-HT' o, '6 -Blood from
jugular vein was collected for serum separation . Serum
(2 m1) was mixed with 5m1 of 10% heptane and 2.5 m1
of O.003N HC!. It was then shaken for 5 min and
centrifuged at 2000 rpm for 10 min. Acid layer
(2.25ml) was eluted and mixed with 100 mg alumin a
and 0.5ml of 2M sodium acetate. The mi xture was
shaken for 5 min and centrifuged at 2000 rpm for
IOmin .The supernatant was used for the estimation of
5-HT. Supernatant was mixed with 1.5 ml of 10%
isobutanol , shaken twice with I ml of salt saturated
buffer.Then I ml of 10% heptane was added to the
butanol phase and 2 .5 ml of 0.1 N HCI was added and
shaken well and 0.5 ml of 0.3N HC I was added with
repeated shaking This was taken for estimation of 5-
HT spectroflurometrically.
Statistical analysis-The results were analysed
stati stically using Student' s ' t' test. Difference below
the probability level 0 .05 was considered stati sti call y
significant.
Studies of gross behavioural changes after treatment
with MO in different concentrations (50-400mg/kg) are
summarized in Table l. MO in graded doses prod uced
gradual increase in the depressive effect as indi cated by
a reduction in behavioural response. With doses 50-
150mg/k g there was no appreciable change in
behavioural response. But 200-400mg/kg doses
produced behavioural changes in awareness , touch
response', ri ghting refl ex, grip strength and spontaneo us
moto r acti vity. Howeve r MO in the dose of 350mg/kg
produced significant depress ive effect. There was
reduction in the spontaneous motor acti vity, grip
strength and touch response with changes in the
awareness and the ri ghting refl ex.
MO potentiated PB s leepi ng time in a dose
dependent (50-400mg/kg) manner. The onset of acti on
was after 1-2 min and the durati on of action varied
from 4-9 hr depending upon th e dose o f the extract. At
50-150 mg/k g doses there was no appreciable change
in sleeping time , However at 200 and 300mg/kg it
produced a mild potentiation of sleeping time but at
350 mg/k g dose there was marked potentiation of the
s leeping time. However at 400mg/k g dose there was no
further increase of sleeping time (Table 2),
The normal EEG pattern showed predom inance of
low voltage fast waves or f3-waves in normal saline
treated control rats. MO was administered orally, 30
min before EEG studies. In 50-150mg/k g dose there
634 INDI AN J EX P BI OL, JUN E 2004

was occasional occ urrence o f a -acti vity (high voltage
slow waves). At 200-300mg/kg doses the a -wave
acti vity increased predo mina ntly, but at 350 mg/kg
dose the frequency o f a -wave activity increased and
persisted for nearly more than 5 hI'. At 400mg/kg dose
the a -waves decreased w ith gradual increase in the /3-
wav or the low vo ltage fast waves (Fig 1).
Serum 5-HT level was signifi cantly increased at
300-400mg/kg doses as compared to control groups.
T he most effecti ve changes in 5-HT level were found
at the 350 mg/kg dose (Tabl e 2).
The results o f the present study indicate that MO
decreases touch response, ri ghting refl ex of rat in
co mpari son with respecti ve control groups probably
due to its depressant acti on II . Reducti on of awareness,
spontaneous motor acti vity and depressant acti on may
be due to the action of MO on central nervous system
(CNS )17. Takah ashi et a / 18 reported th at 5-HT plays an
important role in animal beha vior such as locomotor
depression. In the present study MO prolonged the
sleeping time with an increase in serum 5-HT level.
Thi s corroborates earli er studi es showing th at PB
induced sleeping time is a 5-HT medi ated response and
prolongation of sleeping time may be due to elevati on
of 5-HT level 19. 20.
5-HT is mainl y found in pl atelets, enterochromaffin
cell s (EC cell s), throughout the GI tract and in specific
21
region of the CNS . Thus increase in serum 5-HT leve l
may be due to the tri ggering o f the secreti on of 5-HT
from platel ets and EC cell s by MO and may be
responsibl e for prolongatio n of sleeping time. There is
considerabl e ev idence which links brain 5-HT w ith
sleep mechani sm 19. 5-HT has direct excitatory and
inhibitory acti on and the 5-HT released from
di encephalon and cerebral cortex plays an essenti al
inhibitory role to cause normal sleepS. From the present
results the exact mechani sm by which MO causes
depress ion of loco moto r acti vity and potenti ati on of the
PB induced hypnos is is not clear. However it is we ll
known that Reticular-activating system (RAS) pl ays an
important role in sleep mechanisms. It has also been
reported earli er that 5-HT is mainly associated with
production and prolo ngation of sleep mechani sm

A.

B.

5D1lvL-
Sec

Fig I-EEG study showing (A) low vo ltage waves in norma l co ntro l rats and (B) after trea tme nt with MO (350 mg/kg), there was hi gh
vo ltage slo w waves (occ urrence o f a-waves) .

T able I-Effect of aqueous ex tract of MO on behav ioura l profil e in rat

Behaviour Control MO (mglkg)
50 100 150 200 250 300 350 400
Awareness 0 0 0 0 + + 3+ 4+ 2+
Touch Respo nse 0 0 0 0 + + 3+ 4+ 2+
Ri ghtin g Reflex 0 0 0 0 + + 3+ 4+ 2+
Gri p Strength 0 0 0 0 + + 3+ 4+ 2+
Spontaneous
Motor acti vity 0 0 0 0 + + 3+ 4+ 2+
0= no effect: + = slight depressio n: 2+ = moderate depression; 3+ = strong depression: 4+ = very stro ng depression.
MO (50- 150mg/kg) had no e ffec t on the awareness. touch response, ri ghting reflex. gri p strength and spontaneolls motor ac ti vit y.200
and 250 mg/kg indicate slight depression effect. At 300mg/kg showed strong depression effec t whereas at 350mg/kg the depressaI1l
effec t was very strong and effecti ve. At 400lllgikg there was a moderate de pression effect.
 NOTES 635

Table 2- Effec t of M. o /eif era (MO) root ext rac t on PB ind uced 1996.327 .
sleeping time and se rum sero tonin level 7 Dasputra P G, Pharmacology of M. o/eijem. hulial/ J
Phanll{[Co/. 9 ( 1977) 82.
[Values are mea n ± SE. from 6 an imals in eac h group]
8 Boddin g P 0 , 501111/(// M edicille (Laxmi Jan ardan press.
Dose Sleep ing time Serum serotonin level Ca lcutta, India) 1983,2 11.
(mg/kg MO ) (min ) ( ~ g/l OO ml se rum ) 9 Mazumdar U K, Gupta M, Chakraborty S & Pal D K.
Contro l (5 ml /k g saline) 175.33±2.42 0.08 1±0.003 Evaluation of hemato logical and hepatorenal functi ons of
50 178 .50± 1.94 0.086±0.004 methano lic ex tract of Moril/ ga o/eilem Lam. roo t treated mi ce.
100 18 1.67±2.43 0.092±0.005 II/diall J Elp Bioi , 37( 1999) 612.
150 189. 17±6.49 0.099±0.008 10 Ray K, Hazra R & Guh a D. Ce nt ra l inhibitory effect of
200 I 93.50±7 .30" O. 105±0.005
h Mor il/ga o/eijera root ex tract: Possible role of
neurotransmitters. II/dioll J Erp Bio/. 4 1 (2003 ) 1279.
250 264. 17±9.98" 0. 11 4±0.008 h
300 II Gupta M, Mazumdar U K & Das S, Effect of leaf ex tract fro m
496.0±8.29'· 0.233±0. 1 Ie
C/erodel/drol/ cole/;rookimllllll on CNS functi on in mice.
350 562 .83± 14.58 c 0.319±0.006"
II/dial/ J Exp Bioi. 36 ( 1998) 17 1.
400 49 1. 67±7.85" 0.22 1±0.008"
12 Vogel H G & Voge l W H. Psyc hotropic and neurotropic
P va lues : " < 0.05: h < 0.0 I: " < 0.00 I. acti vity. in D m g discOI 'e n ' al/d em/l/ariol/: Plwl'/lw('%giml
assuvs (S pringer-Verl ag Berl in He ide lberg) 1997 .267.
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