PHYTOTHERAPY RESEARCH, VOL. 6 , 175-179 (1992)

Studies on the Neuropharmacological Aspects of Pluchea indica Root Extract

T. Sen and A. K. Nag Chaudhuri* Division of Pharmacology, Department of Pharmaceutical Technology, Jadavpur University, Calcutta 700032, India

Neuropharmacological studies were conducted in rodents with Pluchea indica Less root extract. On investigation with difterent experimental models it was found that the extract produced alteration of behaviour pattern, reduction in spontaneous motility, prolongation of pentobarbitone-induced sleep, suppression of aggressive behaviour pattern and of the conditioned avoidance response. The observations suggest that the root extract of P. indica possesses a potent central nervous system depressant action.

Keywords: Pluchea indica; neuropharmacology in rodents.

INTRODUCTION

Pluchea indica is a shrub widely found in the Indian subcontinent. In traditional medicine systems, different parts of the plant have been used in the treatment of a variety of diseases; the leaves and roots have been reported to possess astringent and antipyretic actions while decoctions of roots and leaves are mentioned for use in the treatment of lumbago, leucorrhoea and dysentery, as diaphoretics, nerve tonics and in poultices against atonic and gangrenous ulcer (Kirtikar and Basu, 1975; Nadkarni, 1976). On a survey of the literature it was revealed that practically nothing was known concerning the pharmacological actions of P. indica. Accordingly, an investigation concerning the pharmacological actions of the roots of P. indica has been carried out in our laboratory. We have already reported the antiinflammatory (Sen and Nag Chaudhuri, 1991) and antiulcer (Pal and Nag Chaudhuri, 1989) actions of the root extract. Here we report the neuropharmacological actions of the root extract revealed during the course of further pharmaco- logical investigation.

MATERIALS AND METHODS

Plant material. Air dried roots of P. indica (Compositae) were supplied by United Chemicals and Allied Products (Calcutta, India).

Preparation of the root extract. Air-dried powdered roots of P. indica were extracted in a Soxhlet extractor with petroleum-ether (60-80 "C). The petroleum-ether frac- tion was separated and the defatted residue was again subjected to extraction with chloroform. The chloro- form fraction was rejected and the residue was further extracted with methanol. The methanol was removed by drying in uacuo, and a blackish-brown residue was obtained which was kept at 4 "C until used. Just prior to

Author to whom correspondence should be addressed.

use, the dried extract was dissolved in physiological saline, which was also used as control in pharmacologi- cal experiments.

Experimental animals. Charles-Foster rats (120-180 g) and Swiss albino mice (18-22g) maintained under uniform laboratory conditions for at least 10 days were used for pharmacological experiments. Injections were normally made intraperitoneally unless otherwise specified.

Behavioural change and toxicity studies. Different doses of the test material were administered to groups of mice (n=10) which were observed every 30min up to a period of 2 h for studying behavioural changes (Irwin, 1962). For the toxicity study, groups of male mice (n = 10) were injected with the test substance in differ- ent doses and the mortality was recorded after 24 h.

Spontaneous motility. Spontaneous motility of the control animals treated with saline (0.1 mL/10 g) was recorded in a photoactometer for 15 min. The same animals were injected with the test drug on the following day at the same time of day and the spontaneous motility was again recorded.

Pentobarbitone sleeping time. Groups of animals received pentobarbitone (40 mg/kg) after 15 min of test drug or control vehicle (0.1 mL/10 g) administration. The time interval between the loss and regaining of the righting reflex was measured as sleeping time (Dandiya and Collumbine, 1959).

Analgesic activity. Analgesic activity was measured against chemical and mechanical noxious stimuli.

Acetic acid-induced writhing. Groups of mice (n = 10) received 3% acetic acid 15 min after the administration of control or test material and the writhing response was measured for the subsequent 20min (Turner, 1965).

Caudal compression (tail clip method). The mice which tried to remove the clip applied to the base of the tail within 15s were used in this test. The test was per-

0951-418X/92/040175-05 $07.50 0 1992 by John Wiley & Sons, Ltd. Accepted (revised) 21 October I991

116 T. SEN AND A. K. NAG CHAUDHURI

Table 1. Effect of P. i&u on pentobarbitone induced sleep- ing time in mice (mean & SE; II = 10)

Sleeping time Dose in minutes P

Treatment (mglkg) (mean? SE) (vs control) Vehicle 0.2 rnL 42 f 4.7 - P. indica 25 47 k 2.1 -

50 54 k 4.2 < 0.05 100 68k3.1 < 0.001 200 81 f 5.2 < 0.001

formed in groups of mice (n = 10) at 15 and 30 min after the administration of the extract (Bianchi and Franceschini, 1954).

Pentylenetetrazole-induced convulsion. Pentylenetetrazole (100 mg/kg) was administered to groups of mice (n = 10) 30 rnin after the administration of the extract or control vehicle (0.1 mL/lOg) and the mortality was recorded (Soaje-Echaque and Lim, 1962).

Strychnine-induced convulsion. Test material (in predeter- mined doses) or control vehicle (0.1 mL/10 g) was injected to groups of mice (n = 10) 30 min prior to the administration of strychnine (3 mg/kg) and the number of deaths were recorded at the end of 4 h (Rudzik et al., 1973).

Body temperature. Rectal temperatures were recorded at predetermined time intervals in groups of mice (n = 10) before and after the administration of control vehicle or test material.

Conditioned avoidance response. Male rats were trained to climb a pole on hearing the sound of a buzzer in order to avoid an electric shock applied subsequently on the metal grid floor 15 s later (conditioned avoidance response-CAR) . Different groups of screened ani- mals were injected with either control vehicle (0.1 mL/ log) or test material and obervations were recorded after 30 rnin and then hourly for 3 h (Maffi, 1959).

30 r

U

v) U (L 1 5 t

(L

5 5 'i 0 L

* T

A 0 C O Figure 1. Effect of P. indica extract (i.p.) on acetic acid-induced writhing in mice. 0 Vehicle control; El 50 rnglkg; E4 100 rnglkg; P3 300 rnglkg. Significance relative to respective control: 'p< 0.01; *~p<0.001.

Exploratory behaviour pattern

Head dip test. 30 minutes after injection of the test material or control vehicle, female mice were placed on top of a wooden box with 16 evenly placed holes; the number of times the mice dipped its head inside the holes was counted for a period of 3 min (Dorr et al., 1971). The same experiment was repeated using a mixture of amphetamine and the root extract (1:lO) (Rushton et al., 1961).

Y-Maze test. Female rats were placed in a Y-shaped runway (33 x 38 x 13 cm) for 5 rnin and the number of times the rat entered the arm of the maze with all four feet (an 'entry') were counted. The experiment was carried out on groups of 10 rats, 35 min after the administration of control vehicle or test material.

Evasion test. Groups of mice (n=10) were kept in a rectangular wooden box having an inclined plane. Those mice which escaped from the box within 5 rnin were used for the test. The screened animals were injected with the test material or control vehicle (0.1 mL/lOg); 15 rnin later the test was repeated and the number of mice remaining in the box at the end of 5 rnin was recorded (Turner, 1965).

Muscle relaxant activity

The effect on muscle relaxant activity was tested by (a) Rotarod test (b) Chimney test (c) Traction test and (d) Inclined screen test.

Rotarod test. Mice were placed on a rotating rod (32 mm diameter, rotating at 5 rpm). Animals remaining on the rod for 3min or more in two successive trials were selected for testing. 30min after the injection of test material or control vehicle the same test was repeated (with the screened mice) at intervals of 30 rnin for 2.5 h. If a mouse failed to remain on the rod more than once in 3 min, the test was considered as positive (Dunham and Miya, 1957).

Chimney test. Into a pyrex glass tube (30 cm long, 2.5 cm diameter) marked at 20 cm from the base, a mouse was introduced at one end and was allowed to move to the other end of the tube; as soon as it reached the other end the tube was moved to a vertical position and the mouse was allowed to climb backwards to the 20 cm mark. Those mice which successfully reached the mark within 30 s were selected for further testing after admi- nistration of the test material or control vehicle. The same test was conducted with the screened animals 15min after injection of the drug or control vehicle (Boissier et al., 1961).

Traction test. The forepaws of a mouse were placed in a small twisted wire rigidly supported above a bench top. Normal mice grasped the wire with forepaws and when allowed to hang free, placed at least one hind foot on the wire within 5 s. Inability to place at least one hind foot marked failure in traction. Previously screened male mice in groups of 10 were used for the test 30 rnin after the injection of the drug or control vehicle (Rud- zik et al., 1973).

NEUROPHARMACOLOGICAL ASPECTS OF P. INDICA

36.0

U 0

W LL 3 I- 4 LL W n r W t

+ 0 0 m

a z W I

35.0

177

-

+- I I I I I I

T

Figure 2. Effect of P. indica extract (i.p.1 on body temperature in mice. 0 Vehicle control; A 25 mg/kg; x 50 mglkg; 0 100 mglkg. Significance relative to respective control: *p<0.05; **p<0.02; ***p<O.Ol; ****p<O.OOl.

Inclined screen. Muscular competence was also tested by a 30" inclined screen. Groups of 10 male mice, 15 min after injection of test material or control vehicle, were left on the screen for 4 h to observe a paralysant effect servere enough to cause the mouse to slide off the screen (Randall et al., 1960).

Aggressive behaviour. Fighting was produced in pairs of male abino mice by subjecting them to electrical foot shock (interrupted direct current of 3 mA, 400 V, stimulus intensity for 0.2s duration at frequency of 5 shocksh) placed in an apparatus with grid floor com- posed of parallel stainless steel rods. The pairs of mice which fought at least once in 3min were used for further tests. The same test was repeated with 10 pairs of screened mice 30 min after the administration of test material or control vehicle (Tedeshi et al., 1959).

Estimation of brain GABA content. The brain GABA content in mice was estimated ( h w e et al., 1958), in groups of mice (n = 5). Animals were killed by decapi- tation at predetermined time intervals after the admi- nistration of vehicle or test substance. Brains were rapidly removed, blotted, weighed and taken in ice cold 5 mL trichloroacetic acid (lo%, wh) , homogenized and centrifuged at 10 OOO x g for 10 min at 0 "C; 0.1 mL of tissue extract was taken in 0.2 mL of 0.14 M ninhyd- rin solution in 0.5 M carbonate-bicarbonate buffer (pH 9.95), was kept in a water bath at 60 "C for 30 min then cooled and treated with 5 mL of copper tartrate reagent (0.16% disodium carbonate and 0.03% copper sulphate and 0.0329% tartaric acid). After 10 min the fluores- cence reading was taken at 377/451 nm in a Shimadzu RF-5000 spectrofluorimeter (Japan).

Table 2. Effect of P. indica root extract on conditioned avoi- dance response (CAR)

Number % of animals not responding

Treatment (mglkg) tested 0.5 h 1 h 2 h 3 h Dose of animals to CAR after

Control vehicle 10 10 0 0 0 0 P. indica 100 10 0 0 10 10

300 10 10 20 30 20 600 10 20 50b 40" 60b

1000 10 20 70b 60b 50b Chlorpromazine 20 10 1OOb loob l O O b loob p compared with control ~ ~ 0 . 0 2 ; p<O.OOl.

~ ~ ~~~

Table3. Effect of P. indica on exploratory behaviour (head dip test) in mice (mean k SE; n = 10)

Dose Treatment (mglkgl Vehicle 0.2 mL P. indica 50

100 200 300

Diazepam 10 Amphetamine 5.0 Mixture (amphetamine 5.0

+ root extract 50)

Mean number of head dips

in 3 min

23.5 + 0.82 12.16f1.21

9.0 f 1.74 7.4 f 1.03 5.0 f 0.76 5.0 f 0.76

31.8f 4.4 41.1 f4.16

P - < 0.001 < 0.001 < 0.001 < 0.001 < 0.001 - -

178 T. SEN AND A. K. NAG CHAUDHURI

For GABA standards different amounts (20,40, 60, 80, 100 pg) mixed with 1.5 p~ glutamic acid were dis- solved in 0.1 mL 10% trichloro acetic acid (w/v). The GABA content in brain was expressed in pg/g of wet brain tissue.

Statistical analysis. Statistical analysis of data was done by Student’s f-test or chi-squared test (as applicable).

RESULTS

The root extract of P. indicu in doses up to 1200 mg/kg did not produce any mortality in groups of male albino mice up to a period of 24 h after injection. However the extract in doses of 25 mg/kg and above, caused certain behavioural alterations in mice. The animals became remarkably quiet, remained in groups at the corner of the cage and the spontaneous locomotor activity was also remarkably reduced for 3 to 4 h.

Spontaneous motility. The root extract in a dose of 100 and 200 mg/kg (i.p.) caused significant reduction of spontaneous motility in mice. The average photoact- ometer reading in the control vehicle treated groups were 543.33 f 96.96 and 506 f 40.9 while following treatment with the extract (100 and 200mg/kg) the readings were only 257 k 49.99 and 215.33 f 21.58 res- pectively, which was highly significant (p vs control, c0.05 and 0.001 respectively).

Pentobarbitone sleeping time. The root extract signifi- cantly potentiated pentobarbitone-induced sleeping time (Table 1).

Analgesic activity. The root extract at doses of 50 mg/kg and above significantly reduced acetic acid-induced writhing response in mice (Figure 1). However, no analgesic response could be observed with the extract in the caudal compression test.

Anticonvulsant action. The root extract up to a dose of 400mg/kg did not offer any protection either against pentylenetetrazol or strychnine-induced seizures.

Body temperature. The root extract at doses of 25 mg/kg and above caused a significant decrease of normal body temperature (Figure 2).

Conditioned avoidance response. The root extract at doses of 600 mg/kg and above showed significant inhibition of conditioned avoidance response (CAR) (Table 2).

Table4. Effect of P. indicu exploratory behaviour test) in rats (mean & SE; n = 10)

Dose Mean entry 2 SE Treatment (mglkg) in 5 min

Vehicle - 12.42 + 0.782 P. indica 25 10.83 + 0.60

50 8.5 f 1.62 100 7.00 + 0.36 200 5.33 + 0.61

Diazepam 10 2.16+0.47 NS, not significant.

(Y-maze

P (vs control)

NS NS

< 0.00 1 (0.001 (0.001

-

Table 5. Effect of P. i&u on residual curiosity (evasion test) Number of mice

Number remaining in Dose of mice the box after P

Treatment (mglkg) tested 5 min (VS control) - Vehicle 0.2 mL/20 g 10 0

P. indica 25 10 60 < 0.005 50 10 70 < 0.002

100 10 100 < 0.001 Diazepam 10 10 100 < 0.001

Exploratory behaviour pattern. The root extract at the doses employed showed a definite reduction in head dip reactions compared with the control. However, with the extract-amphetamine mixture (10 : 1) there was an increase in the number of head dip responses compared with the individual drug effect (Table 3). The root extract significantly inhibited the exploratory behaviour in rats tested in the Y-maze (Table 4). A significant reduction of residual curiosity was observed with the root extract evasion test (Table 5).

Muscle relaxant activity. No muscle relaxant activity could be observed with the root extract (up to a dose of 600 kg/mg) when tested with rotarod, inclined screen or traction tests. However, a significant loss of muscle coordination was observed to occur with the root extract in the chimney test (Table 6).

Aggressive behaviour. The root extract was found to inhibit significantly the electroshock-induced fighting in mice (Table 7).

GABA content estimation. The results show that the root extract at a dose of 300 mg/kg caused a significant increase in the level of brain GABA concentration in mice (Table 8).

DISCUSSION

The initial experimental data indicated that the P. indicu root extract produced definite alterations of general behavioural profile, considerably reduced spontaneous motility, showed analgesic action against acetic acid induced writhing, caused potentiation of pentobarbitone-induced sleeping time in a dose- dependent manner and produced hypothermia. These findings are altogether suggestive of a potent CNS-depressant action of the extract. The root extract was also found to antagonize the conditioned avoidance

Table 6. Effect of P. indica on muscle tone (chimney test)

Dose Treatment (mg/kgl Vehicle - P. indica 25

50 100 200

Diazepam 10

Number 96 of animals of mice showing tested negative test

10 0 10 10 10 20 10 60 10 80 10 100

P (vs control)

NS NS

< 0.005 < 0.002 < 0.001

-

NS, not significant.

NEUROPHARMACOLOGICAL ASPECTS OF P . INDICA 179

Table 7. Effect of P. indica on electroshock-induced aggress- ive behaviour in mice

Number Number Dose of pairs of pairs %

Treatment (mglkg) used fighting inhibition - Vehicle 0.02 mL 10 10

f . indica 50 10 10 100 10 9 10 200 10 8 20 400 10 5' 50 600 10 2' 80

Diazepam 10 10 0' 100 ' Compared with control p<O.OOl.

response. Accordingly other psychopharmacological properties of the root extract were further explored. In exploratory behaviour pattern studies it was found that the root extract had inhibitory influence on the head dip test, Y-maze test and evasion test but on treatment with mixtures of amphetamine and root extract the exploratory behaviour of the animals in the head dip test was potentiated. This potentiation with the root extract is in conformity with and similar to certain tranquilizers (Dorr et al., 1971). Electroshock-induced aggressive behaviour was also inhibited by the root extract. In studies related to muscle relaxant activity it was found that the root extract had a significant inhibi- tory action on muscle coordination and muscle tone as

Table 8. Effect of P. indica extract on brain GABA content in mice (n = 5; mean f SE)

GABA level Dose (PB4 of %

Treatment (mg/kg) brain tissue) Increase

Vehicle 0.2 mL 414.2 k 6.256 f. indica 100 430 f 7.844 3.86

300 472f7.190' 13.95 ' Compared with control p<O.OOl.

evident from the chimney test. All of the above actions are similar to the psychoactive drugs. Such drugs (e.g. Diazepam) are known to exert their pharmacological action by virtue of causing an increase in the GABA content in the cerebral hemisphere in mice, which indicates that the activity of the drugs under investi- gation in brain disorders is related to decreased GABA content (Saad et al., 1972). The observation of an enhancement of the brain GABA concentration by the root extract in the present experiments is further suggestive of a tranquillizing action of the root extract. Thus on the basis of the above finding it may be inferred that the root extract has a potent CNS-depressant action. However, the precise nature and category of such an action is difficult to predict at the moment and further investigations are necessary before any definite conclusions can be drawn in this respect.

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