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Phytomedicine 15 (2008) 959970 www.elsevier.de/phymed

Neuropharmacological studies on Wedelia calendulacea Less stem extract


T. Prakasha,, N. Rama Raob, A.H.M. Viswanatha Swamyc
Department of Pharmacology and Toxicology, Acharya & B.M. Reddy College of Pharmacy, Bangalore 560 090, Karnataka, India b Department of Pharmaceutical Chemistry, Chalapathi Institute of Pharmaceutical Science, Guntur 522 034, Andhra Pradesh, India c Department of Pharmacology, K.L.E. College of Pharmacy, Hubli 580 031, Karnataka, India
a

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 signicant (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 ngerprint prole 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 ngerprint; LC/MS; Wedelolactone

Introduction
Wedelia calendulacea Less (Syn., W. chinensis Merrill) of the family Asteracea, known as pitabringi (Sanskrit), 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 demethylwedelolactone) similar to Eclipta alba Hassk, (Wagner et al., 1986; Thakur and Mengi, 2005) a plant
Corresponding author. Tel.: +91 9886967959; fax: +91 80 28393541. E-mail address: prakash_tigari@yahoo.com (T. Prakash).

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 efciency and the herbal extract was effective 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

0944-7113/$ - see front matter r 2008 Elsevier GmbH. All rights reserved. doi:10.1016/j.phymed.2008.05.005

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CH3 O

Chemicals and drugs


OH O O O OH OH

Fig. 1. Structure of wedelolactone.

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, pentylenetetrazole, 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.

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 prole 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 wedelolactone detection and has the potential to combine isolation with analysis.

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 Institutional Animal Ethical Committee approved the protocol of the study was obtained as per the Indian CPCSEA guidelines.

HPLC 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 were authenticated by Dr K. P. Shreenath., Reader in Botany, Bangalore University, Bangalore. A voucher specimen was deposited in the Department.

Preparation of extract samples Transfer sample about 100 mg (methanolic extract of Wedelia calendulacea stem) to a 100 ml calibrated volumetric ask. 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 ask. 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 calendulacea 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 stainlesssteel 4.6 mm 250 mm C18 column (Particle size 5 m, Merck) at a ow 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); lter and sonicate in pump A and 0.1 N orthophosphoric acid in acetonitrile (HPLC grade, 99.8%, 0.2 m ltered, Merck) (2.751000 ml) lter and sonicate in pump B.

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.

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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 20 ml each of standard and sample preparation into the chromatograph, record the chromatograms, and measure the responses for the wedelolactone. The relative standard deviation for replicate injections of standard preparation should not be more than 2.0%.

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 intraperitoneal 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 30 min (Whittle, 1964).

LC/MS analysis The crude methanolic extract of Wedelia calendulacea stem was concentrated and applied to LC/MS (ESQUIRE 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.

Thermal method (tail ick 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 icking 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 ick latencies were measured (Basal reaction time) per rat at each time interval and the means of tail-ick 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 administered intraperitoneally either control vehicle, W. calendulacea 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.

Behavioural changes and toxicity studies


Groups of mice (n 10), after intraperitoneal administration 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) were administered 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 reex (Dandiya and Collumbine, 1959).

Anticonvulsant activity
Maximum-electroshock-induced convulsion (MES) Control vehicle or W. calendulacea stem extract of methanolic and aqueous (in different dose) was administered to a group of rats (n 6), 30 min 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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Fig. 2. HPLC prole of crude methanolic extract of Wedelia calendulacea stem (A) recorded at 254 nm. Prole of authentic wedelolactone is shown in (B).

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 methanolic and aqueous (in different dose) or standard drug (phenobarbitone sodium, 20 mg/kg, i.p.) intraperitoneally, 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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T. Prakash et al. / Phytomedicine 15 (2008) 959970
Intens. x104 5 +MS, 46.6min (#6038), 100%=121663, Background Subtracted, Background Subtracted 295.2

963

3 277.2

287.0

314.2

0 260 270 280 290 300 m/z 310 320 330 340 m/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).

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 Maf (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 oor 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. Different groups of selected animals were injected with control vehicle or W. calendulacea stem extract of methanolic 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.

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 vehicle or W. calendulacea stem extract of methanolic and aqueous (in different doses) or standard reference drug (Diazepam 10 mg/kg, i.p.), 35 min before the experiment, were placed singly in a Y-shaped runway (33 cm 38 cm 13 cm) 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).

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

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 vehicle or W. calendulacea stem extract of methanolic 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).

323.0

335.0

313.1

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Muscle relaxant activity


Rota rod test Mice were place 15 min after intraperitoneal administration 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 selected and 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).

Aggressive behaviour This was performed by the electroshock-induced ghting test (Tedeschi et al., 1959). Fighting was produced in pairs of male albino mice conned 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 s duration at a frequency of 5 shocks/s) using a apparatus consisting of grid oor composed of parallel stainlesssteel rods. Only those mice were selected for further experimentation which showed at least one ghting 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 Diazepam (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 sacriced 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 10 min at 0 1C. A sample (0.1 ml) of tissue extract was placed in 0.2 ml 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% disodium carbonate, 0.03% copper sulphate and 0.0329% tartaric acid). After 10 min uorescence at 377/455 nm in a spectouorimeter 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 signicant at po0.05.

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 vehicle or W. calendulacea stem extract of methanolic 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 of W. calendulacea stem extract of methanolic and aqueous (in different dose), diazepam (10 mg/kg, i.p.) or control vehicle.

Results
Inclined screen test This test was performed according to Randall et al. (1960) with minor modication. 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 30 min 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.).

Product identication 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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***

MEAN SLEEPING TIME IN MINUTES

molecular weight as wedelolactone (m/z 313.1), accumulated product in the crude extract to be wedelolactone. 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 of W. 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. calendulacea stem in doses up to 2000 mg/kg (i.p.) did not cause any mortality in mice during the 24 h period after administration. However animals treated with methanolic 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 signicant 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 time Prior administration of methanolic and aqueous extract of stem signicantly potentiated pentobarbitone-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 signicant analgesic activity by tail ick method in dose dependently (Table 1). Anticonvulsant activity The vehicle-treated rats showed tonic hindleg extension 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

70 60 50 40 30 20 10 0 I II III **

***

**

IV

VI

VII

Fig. 4. Effect of W. calendulacea stem extracts on pentobarbitone-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 50 mg/kg; (V) aqueous extract 100 mg/kg; (VI) aqueous extract 200 mg/kg; (VII) aqueous extract 500 mg/kg. *P(versus vehicle control):o0.05; **o0.01; ***o0.001.

80 70
MEAN NO OF WRITHING

60 50 40 * 30 20 10 0 I II III IV V VI VII VIII -*** **

Fig. 5. Effect of W. calendulacea stem extracts on acetic acidinduced 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 50 mg/kg; (VI) aqueous extract 100 mg/kg; (VII) aqueous extract 200 mg/kg; (VIII) aqueous extract 500 mg/kg. *P(versus vehicle control):o0.05; **o0.01; ***o0.001.

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

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Table 1. Treatment

Effect of W. calendulacea stem extract on tail ick latent period in rats Dose (mg/kg) Mean latent period of tail ick response (s) Initial After 30 min 9.3370.55 12.6670.76** 12.8370.79** 13.8370.47*** 10.8370.30 12.0070.57* 12.0070.77* 16.1670.54*** After 60 min 9.26.1670.54 14.3371.05*** 15.3370.80*** 16.3370.55*** 12.5070.67* 13.5070.67** 15.6670.66*** 18.3370.30***

Control vehicle Methanolic extract Methanolic extract Methanolic extract Aqueous extract Aqueous extract Aqueous extract Pentozocine

0.1 ml/10 g 10 20 50 100 200 500 10

9.3270.31 8.2770.22 8.5570.54 8.7770.77 9.2270.40 8.7270.35 9.1670.32 9.8370.36

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


Onset to tonic convulsion (s) (mean7S.E.M.) Pentylenetetrazole Strychnine 5.7570.47 5.2170.92 6.5371.20 7.2372.0 5.9471.65 6.1571.85 6.1172.1 NS Rat showing convulsion Percentage of mortality (within 1 h) Pentylenetetrazole 50.00 33.33 16.16 00.00 33.33 33.33 00.00 00.00 Strychnine 100 100 100 83.33 100 100 100 00.00

Treatment (dose mg/kg)

Pentylenetetrazole 6/6 6/6 6/6 5/6 6/6 6/6 6/6 0/6

Strychnine 6/6 6/6 6/6 6/6 6/6 6/6 6/6 0/6

Control vehicle (0.1 ml/10 g) Methanolic extract (10) Methanolic extract (20) Methanolic extract (50) Aqueous extract (100) Aqueous extract (200) Aqueous extract (500) Phenobarbitone (20)

32.170.05 78.670.4 174.671.12 219.670.8*** 83.470.37 95.470.36 129.070.48* NS

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.

MEAN BODY TEMPERATURE C

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 signicant 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 signicant 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 50 mg/kg of methanolic and 500 mg/kg of aqueous extract. No

37.0

36.5 * * 36.0 *

** ** ** * * * *

35.5

35.0 0 1 2 3 TIME IN HOURS 4

Fig. 6. Effect of W. calendulacea stem extracts on normal body temperature of mice (temperature values expressed as mean7SEM, 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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Table 3. Treatment

Effect of W. calendulacea stem extract on exploratory behaviour (Head dip) in mice Dose (mg/kg) Mean no of head dips in 3 min after 30 min treatment 31.8071.02 28.8071.82 26.4071.52 9.671.00*** 28.2071.74 24.2072.08* 13.0071.30*** 4.1071.17*** Mean no of head dips in 3 min after 1 h treatment 31.4072.16 29.2071.28 30.6071.60 22.6071.50** 29.8071.54 26.6071.89 14.5071.05*** 6.2271.10***
***

Control vehicle Methanolic extract Methanolic extract Methanolic extract Aqueous extract Aqueous extract Aqueous extract Diazeam

0.1 ml/10 g 10 20 50 100 200 500 4

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. Treatment

Effect of W. calendulacea stem extract on exploratory behaviour (Y-maze and Evasion test) in mice Dose (mg/ kg) Y-maze test .Men entry7S.E.M. in 5 min 14.3070.46 11.2070.57 10.7070.95 9.1170.89** 13.6971.20 11.4571.05 9.9870.75* 3.870.53*** Evasion test. No of mice remaining in the box after 5 min 0 1 3 7** 1 4 6* 10*** % Mice showing curiosity in evasion test 100 90 70 30 90 60 40 0
**

Control vehicle Methanolic extract Methanolic extract Methanolic extract Aqueous extract Aqueous extract Aqueous extract Diazeam

0.1 ml/10 g 10 20 50 100 200 500 4

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

o0.01, and

***

o0.001.

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 signicant loss of coordination and tone of muscle in mice (Table 5).

Discussion
The eld of behavioural pharmacology uses concepts and techniques derived from pharmacology and psychology 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 schizophrenia, 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 chemical processes that are involved. In this work, methodology for the validation of medicinal plants with action on the CNS has been applied, specically 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

Aggressive behavior On foot shock-induced ghting behavior, a signicant 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 signicant increase (19.41% and 12.58%) in brain GABA concentration in mice (Table 7).

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Table 5. Treatment

Effect of W. calendulacea stem extract on muscle tone (chimney test) in mice Dose (mg/kg) 0.1 ml/10 g 10 20 50 100 200 500 10 No of mice failed in the test 0 0 1 5** 0 2 4* 10*** % Failure in the test 0 0 10 50 0 20 40 100
***

Control vehicle Methanolic extract Methanolic extract Methanolic extract Aqueous extract Aqueous extract Aqueous extract Diazepam

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. Treatment

Effect of W. calendulacea stem extract on electro-shock-induced ghting in mice Dose (mg/kg) 0.1 ml/10 g 10 20 50 100 200 500 10 Fighting (present in Pairs) 8 8 6 4 8 6 5 10 % Fighting 100 100 75 50 100 75 62.3 0
**

% Inhibition 0 0 25 50* 0 25 37.5** 100***

Control vehicle Methanolic extract Methanolic extract Methanolic extract Aqueous extract Aqueous extract Aqueous extract Diazepam

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. Treatment

Effect of W. calendulacea stem extract on brain GABA content in mice Dose (mg/kg) 0.1 ml/10 g 50 500 GABA level in brain tissue (mg/g7S.E.M.) 394.2376.74 470.7678.27* 443.8477.63* % Increase 19.41 12.58

Control vehicle Methanolic extract Aqueous extract

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

behaviour patterns, signicant reduction of spontaneous motility, potentiation of pentobarbitone-sleeping time in a dose-dependent fashion, and reduction in normal body temperature; all of the above ndings are suggestive of a CNS-depressant action of the both extract. The effect of the stem extracts was further investigated 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 difcult to separate it from motor activity. The results show a signicant decrease in exploratory conduct in the mice caused by the methanolic and

aqueous extracts. The stem extract also signicantly antagonized the electroshock-induced ghting behaviour 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

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that may be attributed to an action on the central mechanisms involved in the regulation of sleep (Gouemo 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 distribution 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 signicantly 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 ndings 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 ndings of the present investigation, it can be concluded that the stem extract of W. calendulacea possess a potent CNSdepressant action, mostly similar to that of psychopharmacological agents. However, it is difcult at the moment to indicate the exact nature more investigations before and denite 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 Chandramouleswara Swamiji, President and Shri T.M. Chandrashekaraiah, Manager, T.M.A.E. Society, for their encouragement 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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