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Phytomedicine 15 (2008) 959970

Neuropharmacological studies on Wedelia calendulacea Less stem extract

T. Prakasha,, N. Rama Raob, A.H.M. Viswanatha Swamyc

aDepartment of Pharmacology and Toxicology, Acharya & B.M. Reddy College of Pharmacy, Bangalore 560 090,

Karnataka, IndiabDepartment of Pharmaceutical Chemistry, Chalapathi Institute of Pharmaceutical Science, Guntur 522 034, Andhra Pradesh, IndiacDepartment of Pharmacology, K.L.E. College of Pharmacy, Hubli 580 031, Karnataka, India

Abstract

The neuropharmacological activities of the methanolic and aqueous extract of Wedelia calendulacea stem were

screened in rats and mice. The extracts effect on pentobarbital-induced sleeping time, pentylenetetrazole- and

styrychnine-induced seizure, spontaneous motor activity, exploratory behaviour, and rota-rod performance (motor

coordination) were evaluated. The methanolic extract (20 and 50 mg/kg, i.p.) and aqueous extract (200 and 500 mg/kg,

i.p.) produced a significant (po0.001) prolongation of pentobarbital-induced sleeping time, and reduced the SMA and

exploratory behaviour. The extract prolonged onset of the phases of seizure activity but did not protect mice against

lethality induced by pentylenetetrazole and strychnine. It also failed to affect the motor coordination test. These results

suggest that the extract contained an agent with neuropharmacological activity that may be sedative in nature. In

addition, from the crude methanolic extract of Wedelia calendulacea stem a HPLC fingerprint profile and liquid

chromatography/sequential mass spectrometry (LC/MS) were performed.

r 2008 Elsevier GmbH. All rights reserved.

Keywords: Wedelia calendulacea; Sedative-hypnotic; Pentobarbitone sleeping time; Behavioral despair. HPLC fingerprint; LC/MS;

Wedelolactone

Introduction

Wedelia calendulacea Less (Syn., W. chinensis Merrill)

of the family Asteracea, known as pitabringi (San-

skrit), pila bhangra (Hindi), kalsarji, Gargari

(Kannada) is a procumbent, perennial herb found in

wet and marshy places in Assam, Arunachal Pradesh,Utter Pradesh and costal areas of the Indian Union. The

herb is said to possess properties and main active

constituents (coumestans i.e., wedelolactone and de-

methylwedelolactone) similar to Eclipta alba Hassk,

(Wagner et al., 1986; Thakur and Mengi, 2005) a plant

for which the leaves are regarded as a tonic and

alternative. The plant has been extensively studied for

its hepatoprotective activity and a number of herbal

preparations comprising of Wedelia calendulacea are

available for treatment of jaundice and viral hepatitis

(Wagner et al., 1986). Ayurvedic Medicinal System for

its hepatoprotective efficiency and the herbal extract waseffective in curing induced liver injury in vivo (Sharma

et al., 1989). The leaves are regarded as tonic and

alternative, useful in cough, cephalagia and disease of

the skin. An ethanolic extract of plant inhibits the

growth of Ehrlichs ascites carcinoma. In East and

South Asia W. calendulacea is used to treat hepatitis,

swelling, distended stomach, headaches and baldness. It

also found to affect the central nervous system

(Nadkarni, 1976) and used for its immuno-stimulatory

ARTICLE IN PRESS

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0944-7113/$ - see front matterr 2008 Elsevier GmbH. All rights reserved.

doi:10.1016/j.phymed.2008.05.005

Corresponding author. Tel.: +91 9886967959;

fax: +9180 28393541.

E-mail address: [email protected] (T. Prakash).
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activity (Govindachari and Premila, 1985). Accordingly,

an attempt has been made towards the study of the

pharmacological action of the stem extract of W.

calendulacea and the neuropharmacologiacl profile of

the stem extract thus revealed is reported here.

Recently, commercially available LC/MS bench top

instruments, some with tandem MS capability, have

reduced the time scale taken to characterise natural

products (Baldwin, 1995; Careri et al., 1998). LC/MS

would appear to be an ideal alternative for wedelolac-

tone detection and has the potential to combine

isolation with analysis.

Materials and methods

Plant material

Wedelia calendulacea fresh stem was collected in the

month of September at S.C.S College of Pharmacy,

Botanical garden, Harapanahalli, Karnataka and wereauthenticated by Dr K. P. Shreenath., Reader in

Botany, Bangalore University, Bangalore. A voucher

specimen was deposited in the Department.

Preparation of extract

The stem was separated and cleaned, then shed-dried

and pulverized using a mechanical grinder. Air-dried,

powdered stem of W. calendulacea was extract in a

Soxhelt extractor with petroleum ether (b. p. 6080 1C).

The petroleum ether extract was discarded. The residue

was subsequently extracted with chloroform and the

chloroform extract was also discarded. Subsequently,

the residue was extracted with methanol (yield: 8.3 g)

and the methanolic extract was taken. W. calendulacea

stem powder was extract with water separately. Thus the

obtained extract was concentrated under reduced

pressure in a rotary vacuum evaporator (yield: 16.4 g).

Phytochemical screening of the methanolic extract gave

positive reaction for alkaloids, glycosides, tannins,

resins, phytosterols, reducing sugars and aqueous

extract gave positive reaction for saponins, alkaloids,

glycosides, phytosterols and reducing sugars.

Chemicals and drugs

For following chemicals and drugs were used. All

chemicals were of analytical grade and chemicals

required for estimation of GABA were obtained from

Sigma Chemicals. Sodium pentobarbital, pentylenete-

trazole, strychnin, and diazepam (Sigma Chemical Co.,USA) were used. HPLC grade solvents, normal saline

(5 ml/kg, i.p.) was used as control in all the experiments.

Animals

Swiss mice (1825 g) and rats (150200 g) of either sex

were obtained from National Institute of Mental Health

and Neuro Science, Bangalore. Animals were housed in

group of 68 per cage at a temperature of 2571 1C and

relative humidity of 41.55% and provided food and

water ad libitum. After 1 week of acclimatization the

animals were used for further experiment. The Institu-tional Animal Ethical Committee approved the protocol

of the study was obtained as per the Indian CPCSEA

guidelines.

HPLC analysis

Preparation of extract samples

Transfer sample about 100 mg (methanolic extract of

Wedelia calendulacea stem) to a 100ml calibrated

volumetric flask. Add about 60 ml of methanol (HPLC

grade, 99.9%) to dissolve and sonicate for about 5 min,

dilute to volume with methanol and mix.

Standard sample

Transfer about 10 mg of wedelolactone standard,

accurately weighed, to a 100 ml calibrated volumetric

flask. Add about 60 ml of methanol to dissolve and

sonicate for about 5 min, dilute to volume with

methanol and mix.

Qualitative analysis

The chromatographic analysis of Wedelia calendula-

cea stem, methanolic extract was performed using a

high-performance liquid chromatograph (HPLC) with

an AllianceTM Waters 2690 separations module coupled

to a Waters 996 photodiode array, a binary gradient

liquid chromatograph system equipped with a UV/PDA

detector set at 254 nm and utilizing a Waters stainless-

steel 4.6 mm 250 mm C18 column (Particle size 5m,

Merck) at a flow rate of 1.0 ml/min (Software, LC

solution). The mobile phase consisted of a 0.1 N

orthophosphoric acid (AR grade) in double distilled

water (2.751000 ml); filter and sonicate in pump A and

0.1 N orthophosphoric acid in acetonitrile (HPLC

grade, 99.8%, 0.2 m filtered, Merck) (2.751000 ml) filter

and sonicate in pump B.

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O

O

O

OH

OH

O

CH3

OH

Fig. 1. Structure of wedelolactone.

T. Prakash et al. / Phytomedicine 15 (2008) 959970960


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Inject 20 ml of methanol into the chromatograph as

blank run and discard any peak due to the solvent in

sample and standard chromatogram. Separately inject

20ml each of standard and sample preparation into the

chromatograph, record the chromatograms, and mea-

sure the responses for the wedelolactone. The relative

standard deviation for replicate injections of standardpreparation should not be more than 2.0%.

LC/MS analysis

The crude methanolic extract of Wedelia calendulacea

stem was concentrated and applied to LC/MS (ES-

QUIRE 3000 PLUS) equipped with a HPLC (Agilent,

1100 series) and an 1100 series of PHENOMENEX

column (4.6 50 mm) maintained at room temperature,

eluted with 90% CH3CNH2O (HPLC grade) gradient

at 0.2 ml/min, and monitored by UV 254 nm absorbance

and 10% ultrapure water in a linear gradient elution to

85:15 over 30 min. Data were acquired for the duration

of the separation using the data-dependent scan mode.

Behavioural changes and toxicity studies

Groups of mice (n 10), after intraperitoneal admin-

istration of different doses of W. calendulacea stem

extract of methanolic (10, 20, 50 mg/kg) and aqueous

(100, 200, 500 mg/kg), were observed at 30 min intervals

for up to 2 h for probable behavioural changes (Irwin,

1962).

For the toxicity study, group of mice (n 10) wereadministered orally different doses of the stem extracts

and mortality were recorded after 24 h.

Spontaneous motility

Spontaneous motility in a control vehicle-treated

(0.1 ml/10 g) group of mice (10 in each) was recorded

in a photoactometer for 15 min initially, then on the next

day the same animals received the test substances (in

different doses of methanolic and aqueous extracts) and

photoactometer noted again.

Effect on pentobarbitone sleeping time

Groups of 10 mice each of both sexes were

administered intraperitoneally W. calendulacea stem

extract of methanolic and aqueous (in different dose)

or control group received 10 ml/kg of solution 30 min

after treatment, all animals received 40 mg/kg of sodium

pentobarbitone. The sleeping time was recorded as the

time interval between the loss and the recovery of the

righting reflex (Dandiya and Collumbine, 1959).

Analgesic activity

Analgesic activity of the methanolic and aqueous

extract was tested as anti-nociceptive effect against

chemical and thermal noxious stimuli in mice.

Chemical method (acetic acid-induced writhing)

This was carried out in groups of mice (n 10) by

noting the writhing responses produced by intraperito-

neal administration of 1% acetic acid (0.1 ml/10 g)

15 min after intraperitoneal injection of either control

vehicle or W. calendulacea stem extract of methanolic

and aqueous (in different dose) were compared against

the standard analgesic aspirin (200 mg/kg). The number

of writhes produced in these animals was counted for

30min (Whittle, 1964).

Thermal method (tail flick latent period)

Analgesic activity was recorded by using a Techno

analgesiometer. The rats were placed in a rat holder,

with its tail coming out through a slot in the lid. The tail

was kept on the bride of the analgesiometer called jacket

with an electrically heated nichrome wire underneath.

The tail received radiant heat from the wire, heated by

passing current of 6 mA. The time taken for the

withdrawal of the tail after switching on the current,

was taken as the latent period, in see of tail flicking

response and was considered as the index of nociception.The cut off time for determination of latent period was

taken as 30 s to avoid injury to the skin (Battacharya et

al., 1971). Three tail flick latencies were measured (Basal

reaction time) per rat at each time interval and the

means of tail-flick latencies were used for statistical

analysis. After recording the basal reaction time in

group of rats (n 6) at least 3 consecutive trials were

selected for further experimentation and were adminis-

tered intraperitoneally either control vehicle, W. calen-

dulacea stem extract of methanolic and aqueous (in

different dose) or pentozocine (10 mg/kg) was used as

the reference standard and were tested 30 min later.

Anticonvulsant activity

Maximum-electroshock-induced convulsion (MES)

Control vehicle or W. calendulacea stem extract of

methanolic and aqueous (in different dose) was admi-

nistered to a group of rats (n 6), 30min before

application of electrical shock (150 mA, 0.2 s) using

corneal electrodes. The duration of hindleg extension

was noted (Swinayard et al., 1952). Phenytoin (25 mg/

kg, i.p.) was used for reference standard drug.

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T. Prakash et al. / Phytomedicine 15 (2008) 959970 961


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Pentylenetetrazole-induced convulsion

Pentylenetetrazole (80 mg/kg, i.p.) was injected into

the groups of rats (n 6) pretreated 30 min earlier with

control vehicle, W. calendulacea stem extract of metha-

nolic and aqueous (in different dose) or standard drug

(phenobarbitone sodium, 20 mg/kg, i.p.) intraperitone-

ally, and onset to tonic convulsion and number of rats

showing tonic convulsion as well as mortality was

recorded in each group (Soaje-Echaque and Lim, 1962).

Strychnine-induced convulsion

Control vehicle, W. calendulacea stem extract of

methanolic and aqueous (in different dose) or

reference drug (phenobarbitone sodium, 20 mg/kg, i.p.)

were injected into groups of rats (n 6), 30 min

before administration of strychnine (4 mg/kg, i.p.)

number of rats showing tonic convulsion as well as

mortality was recorded in each group (Rudzik et al.,

1973).

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Fig. 2. HPLC profile of crude methanolic extract of Wedelia calendulacea stem (A) recorded at 254nm. Profile of authentic

wedelolactone is shown in (B).



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Body temperature

Rectal temperature was recorded with an electronic

telethermometer at pre-determined times in groups

of mice (n 10) before and after the administration of

either control vehicle or W. calendulacea stem extract

of methanolic and aqueous (in different dose) for 4 h.

Conditioned avoidance response

This was performed by the method of Maffi (1959).

Male rats were trained to climb a pole on hearing a

sound of a buzzer in order to avoid an electrical shock

passed through the grid floor 15 s later. On further

training of these rats, which had been trained for

conditioned avoidance response (CSR), the animal

claimed the pole immediately after being placed in the

pole climbing apparatus. This phenomenon was termed

a secondary conditioned response (SCR). The rats,

which showed a correct SCR in at least 10 consecutive

trials, were selected for further experimentation. Differ-ent groups of selected animals were injected with control


and aqueous (in different dose) or standard reference

drug (Chlorpromazine, 3 mg/kg, i.p.) and were tested

30 min later and thereafter at end of each hour for 3 h.

Effect of exploratory behaviour pattern

Head dip test

Female mice (n 10), 30 min after injection with

control vehicle or W. calendulacea stem extract of

methanolic and aqueous (in different doses) or

standard reference drug (Diazepam 10 mg/kg, i.p.),

were placed singly on a wooden board with 16 evenly

spaced holes and the of time the head was dipped into

the holes during 3 min interval was counted (Dorr et al.,

1971).

Y-maze test

Female rats (n 6), pretreated with either control


and aqueous (in different doses) or standard reference

drug (Diazepam 10mg/kg, i.p.), 35min before the

experiment, were placed singly in a Y-shaped runway

(33 cm 38cm 13cm) for 5 min and the number of

times that the rat entered the arm of the maze with all

four feet (classed as an entry) were counted (Rushton

et al., 1961).

Evasion test

Those mice which escaped within 5 min form a

rectangular box within an inclined plane by which the

mice could escape from the box were selected for further

testing. Fifteen minutes after administration of control


and aqueous (in different doses) or standard reference

drug (Diazepam 10 mg/kg, i.p.), the mice in each group

(n 10) were placed in the box again. The number of

mice remaining in the box after 5 min in each group was

noted (Turnar, 1965).

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277.

2

287.

0

295.

2

313.

1

314.

2

323.

0335.

0

+MS, 46.6min (#6038), 100%=121663, Background Subtracted, Background Subtracted

0

1

2

3

4

5x104

Intens.

260 270 280 290 300 310 320 330 340 m/zm/z

Fig. 3. LC/MS analysis of the wedelolactone product in crude methanolic extract of Wedelia calendulacea stem. LC/MS

chromatogram of peaks detected in Wedelia calendulacea stem, indicating the same molecular weight as wedelolactone (m/

z 313.1).



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Muscle relaxant activity

Rota rod test

Mice were place 15 min after intraperitoneal admin-

istration saline on a horizontal rotating road (diameter

32 mm, rotating at 5 r.p.m.). Animals reaming on the

road 3 min or more in two successive trials were selectedand placed in group of 10 animals each. The animals in

each group then received control vehicle or W.

calendulacea stem extract of methanolic and aqueous

(in different doses), diazepam 10 mg/kg and 30 min later

were placed on the road at interval of 30 min up to 2.5 h.

If animal failed more than once to remain on the road

for 3 min, the test was considered to be positive i.e.

motor incoordination was present (Kulkarni and

Joseph, 1997).

Chimney test

In a Pyrex glass tube (30 cm long and 28 mm

diameter) marked at point 20 cm from its base, a mouse

was introduced at the end nearest to the mark. When the

animals reached the other end of the tube, the tube was

moved to the vertical position and immediately the

mouse tried to climb backwards. Only those mice that

reached the mark within 30 s were selected for further

testing. The test was repeated with screened mice

(n 10), 30 min after treatment with either control


and aqueous (in different doses), diazepam (10 mg/kg,

i.p.) intraperitoneally (Boissier et al., 1961).

Traction test

The method of Rudzik et al. (1973) was used. The

force paws of a mouse were placed on a small twisted

wire rigidly supported above a laboratory bench top.

Normal mice grasped the wire with the force paws and

when allowed to hang free, placed at least hind foot on

the wire within 5 s. Inability to place at least one hind

foot marked failure in the traction test. Previously

screened male mice (n 10) were used for the test

30 min after the administration ofW. calendulacea stem

extract of methanolic and aqueous (in different dose),

diazepam (10 mg/kg, i.p.) or control vehicle.

Inclined screen test

This test was performed according to Randall et al.

(1960) with minor modification. Groups of mice

(n 10) were left on a glass plane. Inclined at 301 and

the time taken for each mouse to slide of the screen was

recorded 30min after treatment with this test was

carried out 30 min after treatment with control vehicle,

W. calendulacea stem extract of methanolic and aqueous

(in different doses), or diazepam (10 mg/kg, i.p.).

Aggressive behaviour

This was performed by the electroshock-induced

fighting test (Tedeschi et al., 1959). Fighting was

produced in pairs of male albino mice confined in a 2 l

inverted beaker (15 cm diameter and 20 cm high) by

subjecting them to electrical foot shock (interrupted

direct current of 3 mA, 400 V stimulus intensity of 0.2 sduration at a frequency of 5 shocks/s) using a apparatus

consisting of grid floor composed of parallel stainless-

steel rods. Only those mice were selected for further

experimentation which showed at least one fighting

episode in 3 min. Testing was performed on 8 pairs of

previously screened mice after administration of either

control vehicle or W. calendulacea stem extract of

methanolic and aqueous (in different doses), or Diaze-

pam (10 mg/kg, i.p.).

Effect on brain GABA content

The brain aminobutyric acid (GABA) content in mice(n 6) was estimated according to the method of Lowe

et al. (1958). Animals were sacrificed by decapitation at

predetermined time intervals after the intraperitoneal

administration of control vehicle or W. calendulacea

stem extract of methanolic and aqueous (in different

doses). Brain were rapidly removed, blotted, weighed

and placed in 5 ml of ice-cold trichloroacetic acid (10%

w/v), then homogenized and centrifuged at 10,000 rpm

for 10min at 0 1C. A sample (0.1 ml) of tissue extract

was placed in 0.2ml of 0.14 M ninhydrin solution in

0.5 M corbonate-bicorbonate buffer (pH 9.95), kept in a

water bath at 60 1C for 30 min, then cooled and treated

with 5 ml of copper tartrate reagent (0.16% disodiumcarbonate, 0.03% copper sulphate and 0.0329% tartaric

acid). After 10min fluorescence at 377/455 nm in a

spectofluorimeter was recorded.

Statistical analysis

All values are expressed as mean7S.E.M. The results

were analysed by TukeyKramer test for multiple

comparison using Graph Pad Instate Software (GPIS,

Verssion1.13). Differences were considered significant at

po0.05.

Results

Product identification by HPLC and LC/MS

Using the conditions stated in the experimental

section, the wedelolactone in the Wedelia calendulacea

crude extract were well resolved by this column with

excellent peak shapes (Figs. 2 and 3). A peak at

23.093 min, the same retention time with authentic

wedelolactone (Fig. 2) and showed the same LC/MS

fragmentation pattern (Fig. 3), indicating the same

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molecular weight as wedelolactone (m/z 313.1), accu-

mulated product in the crude extract to be wedelolac-

tone. The structure of wedelolactone is shown in Fig. 1.

General behavior and toxicity

The toxicity study was conducted as per the guidelines

of CPCSEA; article no 420. The methanolic extract ofW. calendulacea stem in doses of 300 mg/kg (i.p.)

showed mortality in female albino mice within 24 h

after administration. Aqueous extract of W. calendula-

cea stem in doses up to 2000 mg/kg (i.p.) did not cause

any mortality in mice during the 24h period after

administration. However animals treated with metha-

nolic extract (10 mg/kg and above) and aqueous extract

(100 mg/kg and above) altered some of the behavioral

responses in mice was observed. The animals became

remarkably quiet and there was a considerable decrease

in locomotor activity which lasted nearly for 1.52 h.

Spontaneous motility

The methanolic and aqueous extract in different doses

caused significant reduction in spontaneous locomotor

activity in mice.

The average photoactometers reading in the control

vehicle treated groups were 521.11710.15, but following a

treatment with the methanolic extract (10, 20, 50 mg/kg,

i.p.) and aqueous extract (100, 200, 500 mg/kg, i.p.) there

was a 23.33%, 34.21%, 62.91% and 21.14%, 36.21% and

54.22% inhibition of spontaneous motility, respectively, as

compared to control group (data not showed).

Pentobarbitone sleeping timePrior administration of methanolic and aqueous

extract of stem significantly potentiated pentobarbi-

tone-induced sleeping time in mice in a dose-dependent

fashion (Fig. 4).

Analgesic activity

The methanolic extract in dose of 20, 50 mg/kg and

aqueous extract of 200, 500 mg/kg showed 54.30%,

61.95% and 48.09%, 54.55% inhibition of analgesic

activity against acetic acid-induced writhing test (Fig. 5).

W. calendulaces of stem both the extract showed

significant analgesic activity by tail flick method in dose

dependently (Table 1).

Anticonvulsant activity

The vehicle-treated rats showed tonic hindleg exten-

sion was 16.1671.30 s. Methanolic extract did not

protect the animals from seizures and duration of

hindleg extension was not reduced. The animals treated

with aqueous extract at a dose of 500 mg/kg were

reduced the duration of hindleg extension 9.5070.76 s

(data not showed). Moreover, treatment with stem

extracts is also a dose-related delay of the onset to tonic

convulsion caused by pentylenetetrazole, and strychnine

and even if it was not be found to offer any protection

either against pentalenetetrazole and strychnine-induced

convulsion, an inhibition of mortality was also observed

(Table 2).

Body temperature

The administration of stem extract caused a signifi-

cant decrease in body temperature of mice (Fig. 6).

ARTICLE IN PRESS

I II III IV V VI VII

0

10

20

30

40

50

60

70 *****

**

***

MEANSLEEPINGT

IMEINMINUTES

Fig. 4. Effect ofW. calendulacea stem extracts on pentobarbi-

tone-induced sleeping time in mice. (Sleeping time plotted as

mean7SEM, from 10 animals in each group.) (I) Vehicle

control; (II) methanolic extract 10 mg/kg; (III) methanolic

extract 20 mg/kg; (IV) methanolic extract 50mg/kg; (V)aqueous extract 100mg/kg; (VI) aqueous extract 200 mg/kg;

(VII) aqueous extract 500mg/kg. *P(versus vehicle contro-

l):o0.05; **o0.01; ***o0.001.

I II III IV V VI VII VIII --0

10

20

30

40

50

60

70

80

* **

***

MEANNOO

FWRITHING

Fig. 5. Effect of W. calendulacea stem extracts on acetic acid-

induced writhing in mice. (Writhing plotted as mean7SEM,

from 10 animals in each group). (I) Vehicle control; (II) aspirin

200 mg/kg; (III) methanolic extract 10 mg/kg; (IV) methanolic

extract 20 mg/kg; (V) methanolic extract 50mg/kg; (VI)aqueous extract 100 mg/kg; (VII) aqueous extract 200 mg/kg;

(VIII) aqueous extract 500mg/kg. *P(versus vehicle contro-

l):o0.05; **o0.01; ***o0.001.



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Conditioned avoidance response

Methanolic and aqueous extracts of stem were not

effective in blocking SCR of trained rats without any

effect on CAR. Chloropromazine (as a standard drug)

was found to blocking booth SCR and CAR in all the

animals.

Exploratory behaviour pattern

On the head dip test in mice treated with different

doses of methanolic and aqueous extracts, there was

significant reduction in head dip responses as compared

to control (Table 3).

On the Y-maze test, after being treated with stem

extract, there was a remarkable decrease in exploratory

behaviour of rats as compared to control and also

extract caused a significant inhibition of residual

curiosity in mice as observed in the evasion test

(Table 4).

Muscle relaxant activity

In the rotarod test, no motor in coordination was

found to occur in mice up to a dose of 50mg/kg of

methanolic and 500 mg/kg of aqueous extract. No

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Table 1. Effect of W. calendulacea stem extract on tail flick latent period in rats

Treatment Dose (mg/kg) Mean latent period of tail flick response (s)

Initial After 30 min After 60 min

Control vehicle 0.1 ml/10 g 9.3270.31 9.3370.55 9.26.1670.54

Methanolic extract 10 8.277

0.22 12.667

0.76

**

14.337

1.05

***

Methanolic extract 20 8.5570.54 12.8370.79** 15.3370.80***

Methanolic extract 50 8.7770.77 13.8370.47*** 16.3370.55***

Aqueous extract 100 9.2270.40 10.8370.30 12.5070.67*

Aqueous extract 200 8.7270.35 12.0070.57* 13.5070.67**

Aqueous extract 500 9.1670.32 12.0070.77* 15.6670.66***

Pentozocine 10 9.8370.36 16.1670.54*** 18.3370.30***

Results are expressed as mean7S.E.M. *P(by TukeyKramer multiple comparison v/s respective control);o0.05, **o0.01, and ***o0.001 (n 6).

Table 2. Effect of W. calendulacea stem extract on pentylenetetrazole and strychnine-induced convulsion in rats

Treatment (dose mg/kg) Onset to tonic convulsion (s)

(mean7S.E.M.)

Rat showing convulsion Percentage of mortality (within

1 h)

Pentylenetetrazole Strychnine Pentylenetetrazole Strychnine Pentylenetetrazole Strychnine

Control vehicle (0.1ml/10 g) 32.170.05 5.7570.47 6/6 6/6 50.00 100

Methanolic extract (10) 78.670.4 5.2170.92 6/6 6/6 33.33 100

Methanolic extract (20) 174.671.12 6.5371.20 6/6 6/6 16.16 100

Methanolic extract (50) 219.670.8*** 7.2372.0 5/6 6/6 00.00 83.33

Aqueous extract (100) 83.470.37 5.9471.65 6/6 6/6 33.33 100

Aqueous extract (200) 95.470.36 6.1571.85 6/6 6/6 33.33 100

Aqueous extract (500) 129.070.48* 6.1172.1 6/6 6/6 00.00 100

Phenobarbitone (20) NS NS 0/6 0/6 00.00 00.00

Results are expressed as mean7S.E.M. *P(by TukeyKramer multiple comparison v/s respective control);o0.05, **o0.01, and ***o0.001 (n 6).

NS not showed.

0 1 2 3 435.0

35.5

36.0

36.5

37.0

**

**

* **

*

*

*

*

* *

MEANBODYTEMPERATUREC

TIME IN HOURS

Fig. 6. Effect of W. calendulacea stem extracts on normal body

temperature of mice (temperature values expressed as mean7-

SEM, from 10 animals in each group). () Vehicle control; (K)

methanolic extract 10 mg/kg; (m) methanolic extract 20 mg/kg;

(.) methanolic extract 50 mg/kg; (E) aqueous extract 100 mg/

kg; (b) aqueous extract 200 mg/kg; (c) aqueous extract 500 mg/

kg. *P(versus vehicle control):o0.001; **o0.01.



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motor incoordination was found to occur either in

traction or in inclined screen with higher doses of stem

extracts. However, in the chimney test at higher doses of

stem extract showed that there was an occurrence of

significant loss of coordination and tone of muscle in

mice (Table 5).

Aggressive behavior

On foot shock-induced fighting behavior, a significant

inhibition of aggressive behaviour in mice treated with

the stem extract (50 mg/kg of methanolic and 500 mg/kg

of aqueous extract) could be observed (Table 6).

Estimation of brain GABA content

The results showed that W. calendulacea stem extract

at a dose of 50 and 500 mg/kg of methanolic and

aqueous extract caused a significant increase (19.41%

and 12.58%) in brain GABA concentration in mice

(Table 7).

Discussion

The field of behavioural pharmacology uses concepts

and techniques derived from pharmacology and psy-

chology for the study of interaction between drugs and

behaviour. The discovery of new compounds which act

on CNS processes (for example, drugs that relieve in a

relatively selective way certain symptoms of schizophre-

nia, anxiety or depression) will stimulate not only their

clinical use but will also contribute useful information

for the validation of animal models. This, in turn, will

permit the investigation of new compounds and a better

understanding of physio-pathological and neuro chemi-

cal processes that are involved.

In this work, methodology for the validation of

medicinal plants with action on the CNS has been

applied, specifically methodology which is used to

investigate depressive activity since the search for new

compounds of natural origin with depressivesedative

activity is important in the western world.

As evidenced from the initial part of the present

investigation, the methanolic and aqueous extracts of

W. calendulacea stem produced alteration in general

ARTICLE IN PRESS

Table 3. Effect of W. calendulacea stem extract on exploratory behaviour (Head dip) in mice

Treatment Dose (mg/kg) Mean no of head dips in 3 min after

30 min treatment

Mean no of head dips in 3 min after

1 h treatment

Control vehicle 0.1 ml/10 g 31.8071.02 31.4072.16

Methanolic extract 10 28.8071.82 29.2071.28

Methanolic extract 20 26.4071.52 30.6071.60Methanolic extract 50 9.671.00*** 22.6071.50**

Aqueous extract 100 28.2071.74 29.8071.54

Aqueous extract 200 24.2072.08* 26.6071.89

Aqueous extract 500 13.0071.30*** 14.5071.05***

Diazeam 4 4.1071.17*** 6.2271.10***

Results are expressed as mean7S.E.M.*P (by TukeyKramer multiple comparison v/s respective control);o0.05.**o0.01, and ***o0.001 (n 10).

Table 4. Effect of W. calendulacea stem extract on exploratory behaviour (Y-maze and Evasion test) in mice

Treatment Dose (mg/

kg)

Y-maze test .Men

entry7S.E.M. in 5 min

Evasion test. No of mice

remaining in the boxafter 5 min

% Mice showing

curiosity in evasion test

Control vehicle 0.1 ml/10 g 14.3070.46 0 100

Methanolic extract 10 11.2070.57 1 90

Methanolic extract 20 10.7070.95 3 70

Methanolic extract 50 9.1170.89** 7** 30

Aqueous extract 100 13.6971.20 1 90

Aqueous extract 200 11.4571.05 4 60

Aqueous extract 500 9.9870.75* 6* 40

Diazeam 4 3.870.53*** 10*** 0

Results are expressed as Mean7S.E.M. *P (by TukeyKramer multiple comparison v/s respective control); o0.05, **o0.01, and ***o0.001.



10/12

behaviour patterns, significant reduction of spontaneous

motility, potentiation of pentobarbitone-sleeping time in

a dose-dependent fashion, and reduction in normal body

temperature; all of the above findings are suggestive of a

CNS-depressant action of the both extract.

The effect of the stem extracts was further investi-

gated on certain other characteristic action of the

psychopharmacological agents, e.g., on exploratory

behaviour pattern, aggressive behaviour pattern, and

muscle relaxant activity. The validation of the anxiety

was carried out by measuring external signs, through the

Hole-Board, Y-maze, and evasion test. The exploration

capacity might be considered to be an index of anxiety

although it is difficult to separate it from motor activity.

The results show a significant decrease in exploratory

conduct in the mice caused by the methanolic and

aqueous extracts. The stem extract also significantly

antagonized the electroshock-induced fighting beha-

viour in mice, thereby demonstrating the suppression

of the aggressive behaviour pattern. In tests concerning

the muscle relaxant activity, the stem extract was found

to produce motor in-coordination and loss of muscle

tone in the chimney test, while the stem extract did not

show any such action in the rotarod, 30o inclined screen,

and traction test in the dose employed. Thus, the results

of the test on the muscle relaxant activity seem a little

paradoxical at the moment and indicate the requirement

for further detailed investigations on the muscle relaxant

action of the extract.

Further evidence of the central depressant activity of

the extract is provided by the extract ability to

potentiate pentobarbital-induced hypnosis, an effect

ARTICLE IN PRESS

Table 5. Effect of W. calendulacea stem extract on muscle tone (chimney test) in mice

Treatment Dose (mg/kg) No of mice failed in the test % Failure in the test

Control vehicle 0.1 ml/10 g 0 0

Methanolic extract 10 0 0

Methanolic extract 20 1 10

Methanolic extract 50 5**

50Aqueous extract 100 0 0

Aqueous extract 200 2 20

Aqueous extract 500 4* 40

Diazepam 10 10*** 100

Results are expressed as mean7S.E.M.*P (by TukeyKramer multiple comparison v/s respective control);o0.05.**o0.01, and ***o0.001 (n 10).

Table 6. Effect of W. calendulacea stem extract on electro-shock-induced fighting in mice

Treatment Dose (mg/kg) Fighting (present in Pairs) % Fighting % Inhibition

Control vehicle 0.1 ml/10 g 8 100 0

Methanolic extract 10 8 100 0

Methanolic extract 20 6 75 25

Methanolic extract 50 4 50 50*

Aqueous extract 100 8 100 0

Aqueous extract 200 6 75 25

Aqueous extract 500 5 62.3 37.5**

Diazepam 10 10 0 100***

Results are expressed as mean7S.E.M.*P (by TukeyKramer multiple comparison v/s respective control);o0.05, **o0.01, and ***o0.001.

Table 7. Effect of W. calendulacea stem extract on brain GABA content in mice

Treatment Dose (mg/kg) GABA level in brain tissue (mg/g7S.E.M.) % Increase

Control vehicle 0.1 ml/10 g 394.2376.74

Methanolic extract 50 470.7678.27* 19.41

Aqueous extract 500 443.8477.63* 12.58

Results are expressed as mean7S.E.M.*P (by TukeyKramer multiple comparison v/s respective control);o0.001 (n 6).



11/12

that may be attributed to an action on the central

mechanisms involved in the regulation of sleep (Goue-

mo et al., 1994) or an inhibition of pentobarbital

metabolism (Kaul and Kulkarni, 1978). It is generally

accepted that the sedative effects of drugs can be

evaluated by measurement of pentobarbital sleeping

time in laboratory animals (Carpendo et al., 1994;Gamaniel et al., 1998). The extracts prolongation of

pentobarbital hypnosis is a good index of central

nervous system depressant activity (Fujimori, 1965).

GABA is known as an inhibitory neurotransmitter in

a number of CNS pathways. The widespread distribu-

tion of GABA, coupled with the fact that virtually all

neurons are sensitive to its inhibitory effect, indicates

that GABA function is ubiquitous in the brain. Studies

have also shown that GABA serves as a transmitter at

about 30% of all the synapses in the CNS (Rang et al.,

2005). Our studies with W. calendulacea indicate that the

stem extract significantly increased brain GABA content

in mice. According to a study conducted by Saad (1972),

CNS-depressant drugs increased brain GABA content

in mice, and these findings are in agreement with our

studies with W. calendulacea stem extract.

Finally, body temperature can be interpreted as an

index of alteration of various central neuro-transmitters,

but it also serves to distinguish between total and partial

benzodiazepine receptor agonists (Jackson and Nutt,

1990).

However, on the basis of the above findings of the

present investigation, it can be concluded that the stem

extract of W. calendulacea possess a potent CNS-

depressant action, mostly similar to that of psycho-pharmacological agents. However, it is difficult at the

moment to indicate the exact nature more investigations

before and definite conclusion can be drawn in this

aspects. Further investigations in this regard are in

progress in our laboratory.

Acknowledgement

Authors are grateful to Sha, Bhra, Shri Chandra-

mouleswara Swamiji, President and Shri T.M. Chan-

drashekaraiah, Manager, T.M.A.E. Society, for theirencouragement in carrying out this work through

Principal, S.C.S. College of Pharmacy, Harapanahalli,

Karnataka. The authors extend a special thanks to

Dr Kotresha, Dr Shivaprasad, Dr Roopa Karki and

Dr Vinod Mathew of discussion on analysis of the

compounds.

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