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7/30/2019 Pharmacological Evaluation of Pachyrrhizus Erosus for Cns
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PHARMACOLOGICAL EVALUATION OF PACHYRRHIZUS
EROSUS (L) SEEDS FOR CENTRAL NERVOUS SYSTEM
DEPRESSANT ACTIVITY
MOHD ABID, H. J. HRISHIKESHAVAN AND MOHAMMED ASAD*
Krupanidhi College of Pharmacy,
#5, Sarjapur Road, Koramangala,
Bangalore 560 034
( R e c ei v e d on J u l y 1 , 2 00 5 )
Abstract : The research work deals with the screening of ethanol and
chloroform ex t rac t s o f Pachyrrhi zus erosus seeds fo r cen t ra l nervous
system (CNS) depressant activity. The Pa ch yrr hi zu s er os us seed is known
to contain rot inoids, f lavonoids and phenylfuranocoumarin derivat ives as
chemical components and i s repor ted to have an t i fungal , an t i secre tory ,
insect i c ides , an t ibacter i a l and spasmoly t i c ac t iv i ty . Since seeds of
Pac hyrrhizus erosus is used as folk medicine in t reatment of insomnia,
we made an a t t empt to s tudy i t s CNS depressan t ef fec t . The d i f feren t
act ivi t ies s tudied were potent iat ion of pentobarbi tone-induced s leep, test
for locomotor activity, effect on muscle co-ordination, antiaggressive and
antianxiety activities. The result of the study reflected that ethanol extract
of the seeds (150 mg/kg, p.o) decreased locomotor activity, produced muscle
re laxat ion and showed an t i anx ie ty and an t i aggress ive ac t iv i ty .
Key words : Pachyrrh izus erosus s ed a t i ve muscle re laxan t
a n t i a n x i e t y an t i agg ress i ve
Indian J Physio l Pharmacol 2006; 50 (2) : 143151
*Corresponding Author : e-mail : mohammedasad@redif fmail .com
INTRODUCTION
Advance in science and technology has
contributed to an enormous improvement in
the qual i ty of l i fe of humankind. However,modern l i fe s t ress , associa ted t r ia l s and
t r ibula t ion are responsible for the surge
in inc idence of var ie ty of psychia t r ic
disorders . Path breaking research in
psychopharmacology has flooded the market
p lace wi th d rugs for speci f ica t ion. For
ins tance , benzodiazepines (diazepam,
ni t raz ipam lorazepam and a lprazolam etc)
are the most frequently prescribed synthetic
drugs for var ie ty of condi t ion par t icular ly
anxiety, depression, epi lepsy and insomnia.
But these psychoneural drugs have very
ser ious s ide ef fec t s l ike chronic use of
benzodiazepines causes deter io ra t ion ofcognitive function, physical dependence and
tolerance . Bes ides addic t ion l iabi l i t i es ,
benzodiazepines adverse ly af fec t the
respi ra tory, d iges t ive and immune sys tem
of body and the chronic t rea tment wi th
benzodiazepines of ten prove more harmful
in the longer run (1).
In this context, a resurgence of interest
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144 Abid et al Indian J Physiol Pharmacol 2006; 50(2)
in medic ine f rom natura l sources (mainly
plant products ) is seen and there i s
t remendous hope that drugs of plant origin
wi l l have s igni f icant ly lesser s ide ef fec t s
than that observed wi th synthet ic drugs
while having comparable eff icacy.
A var ie ty of natura l ly occurr ingdrugs such as Thymus l inear is , Lactuca
seroi la , Papaver somni ferum (opium) and
Atropa bel ladonna were tes ted for
psychopharmacological ef fec ts and were
found to be ef fec t ive in the t rea tment of
psychia t r ic d i sorde rs (2) . The p lant
Pachyrrhizus erosus (L) is a tuberous root
(Leguminasae), cultivated in South East Asia
including India . Seeds of Pachyrrhizus
erosus contain rot inoids, isoflavonoids and
phenylcoumar ine , which are repor ted to
possess potent ac t iv i ty agains t ant i -Herps
Simplex Virus type 1 and type II (3). Seeds
of th i s p lant were found to possess
ant ibacter ia l ac t iv i ty agains t Hel icobac ter
py lor i (4) . Tuberous root of Yam bean
(Pachyrrhizns erosus) conta ins two major
prote ins named as YBG1 and YBG2 that
were found to exhibi t cys te ine proteolyt ic
act iv i ty (5) . Fur ther , seed ext rac t of th i s
pl an t is us ed as pe st ic id e, ant i- ox id an t, ant i-
inf lammatory, ant i - fungal , ant i - secre tory,
insecticide and spasmolytic (6).
Pa chyr rhi zu ss eros us (L) is used as folk
medicine in treatment of insomnia, although
it is not reported in literature. Some of the
plants l i ke Montonoa tomentosa, Kunth,
Cast i l l ega tenui f lora and Penstemen
barbatus and Byrsonima containing chemical
cons t i tuents l ike coumar in , f l avonoids ,
monoterpines , ro t inoids , proanthocyanidine
and glycolipids were reported to possess CNS
depressant activity (6). Based on the above
information, we thought it is worthwhile to
evaluate the Pa chyirrhizus erosus (L) seeds
for CNS depressant act ivi ty.
ME T HODS
E xper imenta l an ima ls
Swiss albino mice of either sex weighing
between 1235 g were used in the present
s tudy. The exper imenta l protocol was
approved by the Institutional Animal Ethics
Commit tee . The animals were mainta ined
under standard conditions in Committee for
the Purpose of Control and Supervision on
Exper iments on Animals (CPCSEA)
approved Ins t i tu t ional Animal House .
C h e m i c a l s
Pentobarbi tone sodium was obta ined
from Sigma-Aldr ich, USA, Diazepam was
p rocured from Cipla , Ahmedabad ( India) ,
Chloroform was purchased f rom Nice
chemicals , Cochin ( India) , Chlorpromazine
from Sun Pharma, Mumbai (India) .
Die thyl e ther f rom Merck Limi ted,
Ahmedabad (India) and petroleum ether was
obtained from S.D. Fine Chemicals, Mumbai( India) .
E xtract ion o f Pachyrrh izus erosus seeds
The Pachyrrhizus erosus seeds were
collected from Kolkata (West Bengal, India),
in winter season. They were dried, powdered
and subjected to ext rac t ion by Soxhle t
method using chloroform and ethyl alcohol
as solvents . Ext rac t ions of dr ied coarse
powder of Pachyrrhizus erosus seeds were
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Indian J Physiol Pharmacol 2006; 50(2) Pharmacological Evaluation of Pac hyrrh izus Erosus 14 5
done in a manner, ini t ial ly with petroleum
ether , chloroform, and then wi th e thyl
a lcohol . Evaporat ion of solvents f rom the
ext rac t was done us ing rotary vacuum
evaporator. A sticky mass was obtained after
evaporat ion of solvent . The ext rac t s were
subjected to qual i ta t ive phytochemical
analysis . A suspension of the extracts wasprepared us ing 2% w/v Tween 80 in di st il led
w a t e r .
Acute ora l tox ic i ty s tudy
The acute ora l toxic i ty s tudy was
performed according to the OPPTS (Office
of Prevent ion, Pes t ic ides and Toxic
Substance) guidel ines fol lowing Up and
Down procedure (7).
Select ion o f the ex tract
The chloroform and e thanol ext rac t of
Pachyrrhizus erosus seeds were evaluated
for sedat ive-hypnot ic ac t iv i ty in
pen tobarbi tone induced s leep tes t . The
ext rac t , which potent ia ted the sedat ive-
hypnot ic ac t iv i ty of pentobarbi tone was
chosen for further study (8) .
T est for locomotor act iv i ty
The spontaneous locomotor ac t iv i ty of
each mouse was recorded individually for 10
min us ing ac tophotometer . The e thanol ic
ext rac t of Pachyrrhizus erosus seeds
(EEPES) was administered at two doses of
EEPES (75 and 150 mg/kg, p .o) 60 min
before the test and ch lorp romazine (3 mg/k g,
i .p) , used as s tandard was given 30 min
before the tes t . The cont rol group was
treated with 2% w/v Tween 80 orally, 60 min
before test (9).
Muscle co -ord ina t ion t es t
This test was carried out using inclined
plane and rotarod appara tus .
1 . I ncl i ned p lan e :
The plane cons is t s of two rec tangular
plywood boards connected at one end by a
hinge. One board is the base; the other is
the movable inc l ined plane . Two plywood
side panels wi th degrees marked on thei r
surface are fixed on the base. A rubber mat
with ridges 0.2 cm in height is fixed to the
inclined plane, which was set at 65 degrees.
Swiss albino mice were taken and divided
into four groups, each group comprised of 6
animals. The two doses of EEPES (75 and
150 mg/kg) were adminis tered ora l ly , thes tandard group was t rea ted wi th diazepam
(4 mg/kg) intraperitonially and control group
received Tween 80 (2% w/v) orally. The test
was car r ied out 30, 60 and 90 min af ter
adminis t ra t ion of drugs and vehic le . The
animals were placed a t the upper par t of
the inclined plane and were given 30 sec to
hang on or to fall off (10).
2 . R o tarod
The rotarod apparatus consists of a metal
rod (3 cm diameter ) coated wi th rubber
attached to a motor with the speed adjusted
to 2 rotations per minute. The rod is 75 cm
in length and is divided into 6 sections by
meta l l ic d i scs , a l lowing the s imul taneous
testing of 6 mice. The rod is in a height of
about 50 cm above the tabletop in order to
discourage the animals from jumping off the
rol ler . Cages below the sec t ion serve to
restrict the movements of the animals when
they fall from the roller.
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146 Abid et al Indian J Physiol Pharmacol 2006; 50(2)
Swiss a lbino mice underwent a pre tes t on
the appara tus . Only those animals , which
had demonstrated their ability to remain on
the revolving rod (20 rpm) for 5 min, were
used for the test. The animals were treated
in the same way as mentioned under inclined
plane test (11).
Ant i -anx ie ty act iv i ty
The ant i-anxiety act ivi ty was evaluated
using staircase test and elevated plus maize
t e s t .
1 . S ta ir ca se t es t
Staircase consists of five identical steps
2.5 cm high, 10 cm wide and 7.5 cm deep.
The internal height of the walls is constanta long whole length of the s ta i rcase . The
drugs and t rea tments were same as
mentioned under inclined plane. The animals
were placed on the floor of the box with its
back to the staircase. The number of steps
cl imbed and the number of rears were
counted over a 3 min per iod. A s tep was
considered to be climbed only if the mouse
had placed a l l four paws on the s tep. In
order to s impl i fy the observat ion, the
numbers of steps descended were not takeninto account . After each step the box was
cleaned in order to el iminate any olfactory
cues , which might modi fy the behavior of
the next animal (12).
2 . E le va te d p lu s m az e
The apparatus consist of two open arms
(5 10 cm) and two closed arms (5 10 l5
cm) radiat ing from a platform (5 5 cm) to
form a plus-sign f igure. The apparatus was
si tuated 40 cm above the f loor. The open-
arms edges were 0.5 cm in height to keep
the mice f rom fa l l ing and the c losed-arms
edges were 15 cm in height. The drugs and
t rea tments were same as ment ioned under
inc l ined plane .
The animal was placed at the center of
the maze, facing one of the closed arms.
During 5 min test period the fol lowing
measures a r e t aken :
T he nu mb er of en tr ie s i nt o o p en ar ms
T he n um be r of e nt ri es i nt o cl os ed a rm s
T ime sp en t i n t he op en ar ms
Arm entry was counted when the animal
had placed al l of i ts four paws on i t . The
procedure was conducted in a sound
at tenuated room, wi th observat ions made
from an adjacent room (14).
Ant i -aggress ive act iv i ty
This was car r ied out us ing i sola t ion
induced aggress ion tes t . Male a lbino mice
weighing about 1220 gm are kept isolatedin small cages for a period of 6 weeks. Prior
to the adminis t ra t ion of the drag, the
aggress ive behavior of the animals was
tes ted. The male mouse being accus tomed
to l ive together wi th other animals was
placed into the cage of an isolated mouse.
The aggress ive behavior of the i sola ted
mouse was recorded. The fol lowing
parameters were used to assess aggress ive
behav io r
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Indian J Physiol Pharmacol 2006; 50(2) Pharmacological Evaluation of Pac hyrrh izus Erosus 14 7
Hit ting the tai l on the bot tom of the cage
S cr ea mi ng ( pi er ci ng n oi se ) an d
Bi t ing.
After these initial tests, the animals were
subjected to di f ferent drug t rea tments as
ment ioned under inc l ined plane . Theaggress iveness was s tudied again af ter
60 and 120 min af ter drug or vehic le
adminis t ra t ion (15) .
Sta t i s t ica l ana ly s i s
Resul t s were expressed as MeanSEM
The di f ferences between exper imenta l
groups were compared us ing one-way
Analysis of Variance (ANOVA) followed by
Dunnet t s t es t and were cons idered
stat ist ical ly signif icant when P
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Indian J Physiol Pharmacol 2006; 50(2) Pharmacological Evaluation of Pac hyrrh izus Erosus 14 9
as indica ted by a s igni f icant decrease in
screaming, h i t t ing the ta i l on the bot tom,
and bi t ing. Low dose did not show any
significant effect (Table IV).
DIS CUS S ION
The study showed that EEPES (150 mg/
kg, p.o) possess sedative, antianxiety, muscle
re laxant and ant iaggress ive ac t iv i ty .
EEPES potent ia ted the s leep induced by
pentobarb i tone sugges t ing that i t possess
some sleep inducing property. The study on
the spontaneous motor act ivi ty showed that
EEPES (150 mg/kg, p .o) decreased the
frequency and the amplitude of movements.
The reduct ion of the spontaneous motor
act ivi ty could be at t r ibuted to the sedat ive
effect of the extract (12).
Incl ined plane method was or iginal ly
developed for tes t ing curare- l ike agents .
T A B LE I I I : E f f ec t o f EE P E S an d d ia z e pa m i n s ta i r c as e t e st a n d e le v a te d p l us - m az e t e s t.
Elevate d plus maz e testStair case test
Groups No. of entry intoNo. of cl imb ing No. of rearing Time spen t in
in 3 min in 3 min Closed arms Open arms open arms
Control 20.16 1.38 9.83 0.60 13.33 1.11 9.66 1.3 91.5 7.50(Vehicle 6 ml/kg, p.o)
Ethanolic extract 13.66 0.80 8.16 0.47 9.83 1.35 6.66 0.80 107.16 10.89(75 mg/kg, p.o)
Ethanolic extract 8.16 0.60** 6.000.57** 7.66 1.20** 6.83 1.66 147.33 9.97*(150 mg/kg, p.o)
Diazepam 6.00 0.68** 5.10.60** 7.16 0.95** 4.83 1.35* 199.51 15.84**(4 mg/kg, p.o)
All values are MeanSEM, n = 6, *P
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150 Abid et al Indian J Physiol Pharmacol 2006; 50(2)
Later on, it has been used by many authors
for tes t ing compounds for muscle re laxing
act ivi ty of both cent ra l ly ac t ing and
per iphe ra l ac t ing muscle re laxants (11) .
EEPES (150 mg/kg, p.o) made the animals
unable to s tay on incl ined plane dur ing
30 sec per iod. EEPES (150 mg/kg, p .o)
also reduced the time spent on the revolvingrod by mice in the rotarod tes t , a t es t
mainly used to screen cent ra l ly ac t ing
muscle relaxants (12). This represented that
EEPES may have muscle relaxant act ivi ty,
which could be due to CNS depressant
act iv i ty .
The mouse s ta i rcase was used for the
assessment of anxie ty (number of rear ing)
and sedat ion (number of s teps ascended) .
Greater number of rear indica tes anxie tyl ike behavior and lesser number of s teps
ascended indicated increased sedat ion (13).
The present inves t igat ion successful ly
detected the anxiolytic-like effects of EEPES
and diazepam; both signif icant ly decreased
the number of rearing and number of steps
ascended compared to control . This showed
that EEPES has both anxiolytic and sedative
p roper t ie s .
The sedat ive , muscle re laxant and
anxiolytic effects of EEPES could be due to
the in terac t ion of i sof lavonoids (chemical
cons t i tuent of the plant ) wi th the GABA/
benzodiazepine receptor complex in bra in
(16).
E levated plus-maze tes t i s used to
evaluate psychomotor per formance and
emot ional aspects of rodents (17) . The
resul t s showed that EEPES s igni f icant ly
increased the t ime spent on the open arms
and decreased the number of ent r ies in to
open and closed arms. This type of effect is
observed with the drugs that act on GABA/
benzodiazep ine receptor complex as well
wi th drugs tha t s t imula te glucocor t icoid
product ion and re lease in the adrena l
cortex (14), af ter administrat ion of 5-HT1B
receptor antagonis t s and 5-HT1 A
agonis t s
(18) . Therefore , wi th the present da ta ,i t i s d i f f icul t to predic t the precise
mechanism for the anxiolytic activity of the
Pachyrrhizus erosus seeds.
EEPES showed inhibition of aggressiveness
in isolated mice. The serotoninergic system
is implicated in aggressive states and it has
been hypothes ized that decreas ing
serotoninergic ac t iv i ty may encourage
aggress ive behavior (19) . S ince , both
ant ianxie ty and ant iaggress ive ef fec t s areseen with 5HT1A
antagonists , i t is assumed
that EEPES may a l so in terac t wi th the 5-
HT1A
receptors .
To conclude, the e thanol ic ext rac t of
seeds of Pachyrrhizus erosus possess
sedat ive, ant i -anxiety, muscle relaxant and
anti-aggressive properties. The result of the
present s tudy subs tant ia tes the t rad i t ional
use of seeds ofPa chyrr hizus erosus for the
t rea tment of insomnia .
ACKNOWLEDGEMENTS
The authors are thankful to Prof. Suresh
Nagpal , Chai rman, Krupanidhi Educat ional
Trus t , Bangalore , India , P rof . Suni l
Dhamanigi , Secre tary , Krupanidhi
Educat ional Trus t , Bangalore , India and
Dr . Ami t Kumar Das , Professor and
Principal , Krupanidhi College of Pharmacy,
Bangalore, India, for providing facilities to
carry out this work.
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