pharmacological evaluation of pachyrrhizus erosus for cns

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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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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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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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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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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pharmacological evaluation of pachyrrhizus erosus for cns

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