1. INTRODUCTION:

Anamirta cocculus, belonging to family Menispermaceae, is an Southest Asian and Indian climbing plant.^[1] Anamirta cocculus holds a reputed position as medicinal herb. Its leaves and fruits are used in medicine the fruit is slightly bitter , good expectorant , removes gases from intestine, good for rheumatism and as applications for inflammation Various alkaloids are isolated from the plants. This is popular folk remedy in Asia and adjacent regions in the Philippiens, and infusion of roots of Anamita cocculus is used to treat fever, dyspepsia and menstrual problem. Extract of the stem is added to native wine and is drunk to make blood strong.

One of the important pharmacological action of minor tranquillizers or anti anxiety agents is the skeletal muscle relaxation together with taming or calming effect and reduction in anxiety and tension. The site of this activity is the CNS. Disturbance in the maintenance of tone and posture is the first sign of centrally mediated skeletal muscle relaxation. A drug increasing or decreasing CNS activity will also produce increase or decrease in spontaneous motor activity (SMA) in the animal. Locomotor activity refers to an increase in alertness and decrease in locomotor activity considered as sedative. The locomotor activity can be an index of wakefulness (alertness) of mental activity. Pyrexia is a condition, where the body temperature is elevated than normal value. Antipyretics reduce the elevated body temperature which occurs in pyrexia.

Pharmacological studies of effect of plants and their extracts on central nervous system could be beneficial in identification of new lead molecules. Thus the aim of the present study was to evaluate ethanolic extract of stem bark of Anamita cocculus for CNS activities. Studies on muscle relaxant, locomotor, analgesic and antipyretic activities could be studied for screening CNS activity.

2. MATERIALS AND METHODS:

2.1. Materials

An alcoholic extract prepared from the stem bark of Anamirta cocculus prepared as reported previously was used for the study.^[2] Since the extract was partially soluble in distilled water a suspension was prepared with 0.1% carboxymethyl cellulose (CMC) and this suspension was used.

2.2. Ethical clearance, animal maintenance and care

Experiments on animals were carried out after getting approval from Institutional Animal Ethics Committee, Govt. Medical College, Thiruvananthapuram (No. 39/IAEC/MCT/06; dated 19.09.2006).

Albino rats and mice were collected from the animal house, Govt. Medical College, Thiruvananthapuram, India. The mice were maintained on pellet diet and water ad libitum. Animals were housed in propylene cages with dust free rice husk as bedding material under laboratory conditions with control environment of temperature 30±2^oC. They were fed ad libitum with rodents chow and free access to drinking water.

2.3. Muscle relaxant activity

Rotarod apparatus was used to study muscle relaxant activity. Healthy albino mice of both sexes weighing between 20 and 25 g, fed on water alone for 18 hours were used. The mice were trained to walk on a rotating rod, rotated at 25 rpm and selected only those animals which stayed for a period of 2-5 minutes in the successive trials. After selection, they were divided in to 5 groups of 6 each. Each animal was placed on the rotating rod, and noted down the ‘fall off time’ when the mouse falls from the rotating rod. The extract as a suspension in 0.1% CMC was given orally at a dose of 400 and 800mg/kg body weight of animal. The third group served as the control and was given vehicle (10 mL/kg body weight). Diazepam (4 mg/kg) was given orally to the standard group of animals. Forty minutes later, each animal was re-tested for the ‘fall off time’ and difference was noted.^[3-7]

2.4. Effect on locomotor activity

Photoactometer was used to study locomotor activity. Healthy albino mice of both sexes weighing between 20 and 25 g were used for the study. They were fed on water alone for 18 hours. Animals were individually placed in activity cage for 10 minutes and checked basal activity score of all the animals. Selected animals were divided into 4 groups each containing six animals. The first group served as the control and was given the vehicle (10 mL/kg body weight). Diazepam in a dose of 4 mg/kg body weight was given orally to the second group. The extract as suspension in 0.1% CMC was given orally in a dose of 400 mg/kg to third group and 800mg/kg body weight to group IV.After 60 minutes, re-checked each mouse for activity scores for 10 minutes and change in the activity was noted. The change in activity of the test group was compared with that of the standard and control.^[8-11]

2.5. Analgesic activity

Analgesic activity was studied by hot plate method where heat is used as source of pain. Albino mice of both sexes, weighing between 20 and 25 g, starved overnight, being allowed free access of water, were used. Weight of the entire animal was taken and each one was marked for identification. They were divided in to 4 groups of 6 mice each. The initial reaction time of all the animals of control and test groups were recorded by putting them on the Hot plate maintained at 55^oC. Licking of paw or jumping was taken as the index of reaction to heat. A cut off time of 15 s was used to prevent injury to the paws. To the control group, vehicle (0.1% CMC) of the extract solution in a dose of 10 ml/kg body weight was given. The standard drug morphine sulphate, 5 mg/kg was given to the second group. The other three groups received extract solution in a dose of 400 and 800mg/kg body weight respectively. All the above drugs are administered orally. The reaction time of each of animal at 30, 60 and 120 min after administration were noted.^[12-15]

2.6. Antipyretic activity

Study to evaluate antipyretic activity was carried out as per previously reported method. ^[16-20] Albino rats weighing 150-200 g were fed till 24 h prior administering drugs. The room temperature was kept at 30-32^oC. Animals were divided in to four groups of 6 animals each. Each of them was marked for proper identification using marking ink. Then food was withdrawn prior to measurement of rectal temperature. One and half centimetres of a digital thermometer was introduced in rectum of the rats and the rectal temperature was recorded. Pyrexia was induced by subcutaneous injection in the back below the nape of the neck of a 20% suspension of dried yeast in 0.1% CMC at a dose of 20 mL/kg of body weight. The site of injection was massaged in order to spread the suspension beneath the skin. After 18 h of yeast injection, rats which showed a rise in temperature of at least 1^o F (0.6^°C) were taken for the study. Animals in the various groups were treated as follows:

Group I : 0.1% CMC (10 ml/kg) as vehicle, orally.

Group II : Aqueous suspension of alcoholic extract of Anamirta cocculus 400 mg/kg in 0.1% CMC suspension, orally.

Group III: Aqueous suspension of alcoholic extract of Anamirta cocculus 800 mg/kg in 0.1% CMC suspension, orally.

Group IV: Paracetamol, 100 mg/kg in 0.1% CMC suspension, orally.

Rectal temperature was recorded every hour for 2 hours after administration of drugs.

3. RESULTS AND DISCUSSION:

3.1. Muscle relaxant activity

In the rota-rod test, the extract in doses of 400 and 800 mg/kg body weight, produced a decrease in ‘fall off time’ of 52.3% and 78.2% respectively and diazepam (4 mg/kg) showed 88.4% decrease in ‘fall off time’. The two doses of the extract at 60 minutes after administration of the extract showed significant effects (p<0.05) when compared with control group. Results are shown in Figure 1.

Fig 1: Muscle relaxant activity of Anamirta cocculus extract

3.2. Effect on locomotor activity

Diazepam (4 mg/kg) reduced the locomotor activity by 83.6% when compared to the initial value. The group treated with extract of 400 mg/kg showed a reduction of activity to the extent of 72.42% from initial value and the group received extract at a dose of 800mg/kg showed a reduction of activity by 87.83% from the initial value. These effects were found to be dose dependent and statistically significant (p<0.05) when compared to the control groups. Results are shown in Figure 2. The reduction in locomotor activity may be due to the CNS depressant effect. It may be due to its tranquilizing, centrally acting muscle relaxant or analgesic effect.

Fig 2: Locomotor activity of Anamirta cocculus extract

3.3. Analgesic activity

The group treated with morphine sulphate 5 mg/kg body weight showed marked increase in reaction time and peak effect was attained at 30 minute, after drug administration the analgesic effects sustained 1 h. The extract treated groups also showed increase in the reaction time and it was higher than that of standard. The effect of the two dose of extract at 30, 60 min after injection were found to be statistically significant (p<0.05) when compared with control group. Results are shown in Table 1.

Table 1: Analgesic activity of Anamirta cocculus extract

Treatments	Reaction time in seconds (Mean ± SEM)
Treatments	0 min	30 min	60 min	120 min
Control (Vehicle 1%CMC)	4.0±0.44	3.5±0.42	3.3±0.49	3.3±0.42
Morphine sulphate 5 mg/kg	3.83±0.30	7.8±0.44	5.0±0.44	3.7±0.42
Extract 400 mg/kg	4.2±0.47	7.7±0.66	5.6±0.89	5.3±0.75
Extract 800 mg/kg	3.83±0.30	11.5±0.76	7.16±0.40	3.3±0.33

3.4. Antipyretic activity

The administration of yeast suspension caused an elevation in body temperature up to 1-1.6 ^oC in rats. The groups treated with extract 400 and 800 mg/kg showed inhibition of pyrexia by a respectively when compared with the rise of body temperature among the same group. The group treated with paracetamol showed a reduction of pyrexia by 27.7%. The results are statistically significant for p =0.000. Results are shown in Figure 3. The antipyretic effect of the test drug may be due to the presence of flavonoid compounds which could cause predominant inhibition of cyclooxygenase or lipooxygenase.

Fig 3: Antipyretic activity of Anamirta cocculus extract

4. CONCLUSION:

Based on the results of the present study we could conclude that the extract of Anamirta cocculus possess significant antidepressant like effect, muscle relaxant activity, analgesic activity and antipyretic effects. However, further studies are required to find out the exact mechanism behind these activities. Pharmacological studies of effect of plants and their extracts on central nervous system could be beneficial in identification of new lead molecules.

5. CONFLICT OF INTEREST:

None

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Received on 15.06.2016 Modified on 28.06.2016

Res. J. Pharmacognosy and Phytochem. 2016; 8(3): 141-144.

DOI: 10.5958/0975-4385.2016.00025.X