Studies on antidiabetic potential of aqueous and alcoholic leaf extract, free radical-scavenging properties and antimycobacterial activity of afzelin isolated from the alcoholic extracts of leaves of P. dulce were recently reported^3,4. Also the CNS depressant and anti-inflammatory activities of this plant have been demonstrated^{6, 8}.

In spite of considerable literature available on P. dulce Benth, there is no known data regarding CNS activities of this plant. Therefore the present study was undertaken to investigate in experimental mouse models, the neuropharmacological effects of the Pithecellobium dulce leaves extracts on spontaneous motor activity, motor coordination, sodium pentobarbital-induced hypnosis and pentylenetetrazole (PTZ)-induced convulsions.

MATERIALS AND METHODS:

Animals:

Swiss albino mice of either sex of 7-9 weeks of age; weighing between 20–35 g were obtained from our animal house and maintained under the standard conditions: room temperature (25±3) °C, humidity 45% - 55%, 12 /12 hours-light/dark cycle. Animals had ad libitum access to food and water. The experimental protocol was approved by the Institutional Animal Ethics Committee of the college and was in accordance with the guidelines of the Committee for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA), Govt. of India.

Plant material and preparation of the extracts:

Pithecellobium dulce Benth leaves were collected from local area of Warananagar. The plant was authenticated by Botanist, Dr. Mrs. U. S. Yadav at Willingdon College, Sangli, MS, India and the voucher specimen deposited in same college. The leaves were shade dried for a week then they were mechanically powered and sieved.

The powder was subjected for aqueous and ethanolic extraction. Leaves powder was extracted with chloroform water by maceration for six h at room temperature with occasional shaking on orbital shaker and then stand for the next 18 hrs. Then the extract was filtered by using Whatman filter paper and concentrated over heating on water bath, and then the concentrated extract was air dried. The ethanolic extract was prepared by Soxhlet apparatus using ethyl alcohol as solvent. This extract was oven-dried at 40^oC giving a dried extract.

Preliminary phytochemical screening:

The preliminary phytochemical screening of aqueous and ethanolic extracts was carried out to determine the nature of phytoconstituents present in it. The results of preliminary phytochemical investigation are shown in table 1.

Acute oral toxicity study:

Acute toxicity study was performed in mice. The extracts were administered orally at doses of 175, 550, 2000 mg/kg. Animals were observed for signs of toxicity, continuously for two h, and for mortality up to 48 h, after oral administration of different doses of extract⁹.

Table 1: Preliminary phytochemical study of aqueous and ethanolic extract of P. dulce

Chemical constituents	Aqueous Extract	Ethanolic Extract
Proteins	+	+
Steroids	-	-
Cardiac glycosides	-	-
Saponin glycosides	+	+
Flavonoids	+	+
Tannins and phenolic compounds	+	+
Sugars	+	+
Alkaloids	-	-

Spontaneous motor activity:

The spontaneous motor activity was measured using an actophotometer. Actophotometer operated on photoelectric cells which were connected in circuit with a counter. When the beam of light falling on the photocell was cut off by the animal, a count was recorded. These cutoffs were counted for a period of 10 min and the figure was taken as a measure of the locomotor activity of the animal.

Swiss albino mice of either sex were divided into four groups of five animals each. Each mouse was placed individually in the actophotometer for ten min and basal activity score was obtained. First group was treated with control vehicle distilled water and group two with standard drug Diazepam (4 mg/ kg i.p.); group three and four received aqueous and ethanolic extracts at 200 mg/kg p.o. respectively. One h after treatment, mice were placed again in the actophotometer for recording the activity score¹⁰.

Effect on motor coordination:

The effect of extract on motor coordination (muscle relaxant property) was studied by the rotarod test. The untreated fresh mice were placed on a horizontal rod rotating at a speed of 25 rpm. The animals were divided into four groups of five animals each. The first and second group received control vehicle distilled water and Diazepam (4 mg/ kg i.p.) respectively, while the third and fourth groups received aqueous and ethanolic extract (200 mg/kg). Each group of the mice was then placed on the rod and time taken for the mice to fall from the rotating rod was noted ¹¹.

Pentobarbital induced hypnosis:

This test evaluates the depressive action of a given drug in CNS that possess sedative activity and characteristics of a hypnotic drug¹². Swiss albino mice were divided into four group’s of five animals each. Groups received vehicle, Diazepam (4 mg/ kg i.p.); aqueous and ethanolic extract (200 mg/kg) p.o respectively. After 30 min of diazepam injection and 60 min of extract administration, sodium pentobarbital (SP) (42 mg/kg, i.p.) was administered to all animals. The interval between the administration of SP and the loss of the righting reflex was recorded as onset of sleep, while the time between loss and recovery of the righting reflex was taken as sleeping time¹³.

Effect on pentylenetetrazole (PTZ)-induced convulsions in mice:

Four groups containing five mice each were made and administered 80 mg/kg dose of PTZ i.p. after 60 min of administration of extracts and 30 min after injection of reference drug diazepam (4 mg/ kg i.p.). Mice were observed for the onset of action (indicated by Straub’s tail, jerky movements of whole body and convulsions) and severity of convulsions due to the PTZ¹⁰. The percentage of animals that died was also recorded. Mice that did not convulse within 40 min after PTZ administration were considered protected.

Statistical analysis:

Results are expressed as mean±SEM. The statistical analysis of data was done by analysis of variance (ANOVA) followed by Dunnett’s test. A value of P < 0.05 was considered significant.

RESULTS:

Acute oral toxicity study:

Under the presence of the experimental conditions the absence of toxic symptoms and mortality in animals indicates that the extract might be having the LD₅₀ value above 2000 mg/kg p. o. body weight. Thus the extracts were considered to be safe for further pharmacological screening and dose of 200 mg/kg p. o. of each extract (aqueous and ethanolic) was selected for the further study.

Spontaneous motor activity:

The results of the spontaneous locomotor activity of aqueous and ethanolic extracts are shown in Fig 1. The mice treated with aqueous and ethanolic extracts showed the significant reduction in locomotion when compared with control group (P<0.01).

Fig 1: Effect of aqueous (AE) and ethanolic extracts (EE) of P. dulce on spontaneous motor activity

Each bar represents mean response from 5 mice ±SEM (**p<0.01)

Effect on motor coordination:

Both the aqueous and ethanolic extract caused failure of motor coordination at 200 mg/kg p.o. dose. The aqueous extract being more potent showed the significant failure of motor coordination (P<0.01) as compared to control but less than diazepam. (Fig. 2)

Fig 2: Effect of aqueous (AE) and ethanolic extracts (EE) of P. dulce on motor coordination

Each bar represents mean response from 5 mice ± SEM (* P < 0.05; ** P < 0.01)

Pentobarbital induced hypnosis:

Significant potentiation of sodium pentobarbital sleeping time in mice was observed by aqueous and ethanolic extracts with respect to the control drug diazepam. (Fig. 3)

Fig 3: Effect of aqueous (AE) and ethanolic extracts (EE) of P. dulce on Pentobarbital induced hypnosis

Each bar represents mean response from 5 mice ± SEM (**p<0.01)

Effect on pentylenetetrazole-induced convulsions in mice:

In this model diazepam treated mice did not produce any convulsions. (Table-2) Aqueous and ethanolic extracts increased onset of convulsions and reduced percentage of mortality as compared to control group. But increase in onset of convulsions was found to be statistically non-significant when compared with control group (p>0.05)

Table 2: Effect of aqueous and ethanolic extracts of P. dulce on PTZ induced convulsions in mice

Treatment + PTZ (80 mg/kg, i. p.)	Onset of convulsions (Sec)	% Mortality
Control (D.W.)	68.2±9.53	100
Diazepam (10 mg/ kg i.p.)	-	0
AE (200 mg/kg p.o.)	74.4±6.43	80
EE (200 mg/kg p.o.)	83.2±9.47	80

Values are mean±S.E.M

DISCUSSION:

Effects of aqueous and ethanolic extracts of Pithecellobium dulce leaves were tested for spontaneous motor activity, motor coordination, sodium pentobarbital-induced hypnosis and pentylenetetrazole induced convulsions in mice. The outcome of the present research demonstrates that Pithecellobium dulce produces significant pharmacological effects in the CNS.

The results showed that both extracts exerts depressant effects on the CNS. They significantly decreased the locomotor activity. Decrease in locomotion reveals depression effect on CNS¹⁴. The CNS depressant activity may be due to the increase in the concentration of GABA in brain ¹⁵.

The effect of extract on motor coordination reveals that both extracts produced significant lack in motor co-ordination and exhibited muscle relaxant activity.

It is generally accepted that the sedative effects of drugs can be evaluated by measurement of the sleep time induced by pentobarbital in laboratory animals¹⁶. At the dose tested, both extracts potentiated sodium pentobarbital induced sedation and hypnosis. The ability of Pithecellobium dulce to potentiate pentobarbital induced hypnosis could be attributed to effects on the central mechanisms involved in the regulation of sleep or to an inhibition of pentobarbital metabolism^{17, 18}.

Prolongation of pentobarbital hypnosis is an indicator of central nervous system depressant activity¹⁹. Furthermore, it is well known that these depressant effects are mediated through the GABA/benzodiazepine receptor complex²⁰. Therefore Pithecellobium dulce induced enhancement of the effects of SP could be attributed to the participation of GABAnergic system.

PTZ is the most frequently used substance, as well as an acute experimental model in a preliminary screening to test potential anticonvulsant drugs²¹. The mechanism by which PTZ is believed to exert its action is by acting as an antagonist at the GABA-A receptor complex²². Pithecellobium dulce extracts does not exhibit a protection against convulsions induced by PTZ.

The above studies indicate that the aqueous and ethanolic extracts of the Pithecellobium dulce leaves possesses CNS depressant, hypnotic and skeletal muscle relaxation activity. Further studies are in progress to isolate the active constituents responsible for these activities.

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Received on 03.11.2010

Accepted on 03.12.2010

Research J. Pharmacology and Pharmacodynamics. 3(1): Jan. –Feb. 2011, 27-30