Some Central Nervous System Activities of Nerium Oleander Linn (Kaner) Flower Extract

Purpose: The purpose of the study was to evaluate the activity of 50 % hydroalcohol flower extract of Nerium oleander Linn. on the central nervous system (CNS) of mice. Methods: The effect of the 50 % hydroalcohol extract of N. oleander flowers at dosage levels of 100 and 200 mg/kg p.o. on the locomotor activity of mice was measured with an actophotometer while muscle relaxant activity was evaluated on rotarod apparatus. Also, the anticonvulsant activity of the extract in mice (following maximal electroshock and pentyleneterazol-induced convulsion) as well as the potentiation of pentobarbital-induced sleep were determined using standard procedures. Diazepam was used as the reference standard. Result: The extract (at doses of 100 and 200 mg/kg) significantly reduced ( p < 0.01) spontaneous locomotor activity and also potentiated pentobarbital-induced sleep. At the higher dose (200 mg/kg) the extract showed 66 % protection against electroshock-induced convulsions while the lower dose (100 mg/kg) produced a significant reduction ( p < 0.01) in pentylenetetrazol (PTZ)-induced convulsions. Conclusion: The ethanol extract of Nerium oleander flowers has anticonvulsant activity in an experimental animal model. Anticonvulsant activity.


INTRODUCTION
Nerium oleander Linn. (Kaner) belongs to the family Apocynaceae. It is a large glabrous evergreen shrub that produces milky juice. It is native to Iran, the Mediterranean region, as well as India. The leaves are in pairs of three, shortly stalked, coriaceous, 10 -15 cm long, linear lanceolate with dark green colour. The flowers are salver-shaped pink or white without any fragrance [1].
In the traditional medicine system, parts of this plant are used for the treatment of various human ailments. The leaf is used as a cardiotonic, diuretic, anti-bacterial in cutaneous eruptions, and is also effective against snake-bites; the root is used for curing different types of cancers, ulcers and leprosy. The root-bark is used specifically against ring worm and the aqueous extracts of the leaves, branches, roots and flowers are toxic to certain insects [2]. Several phytochemicals have been identified in various parts of the plant and they include mainly cardiotonic glycosides, terpenoids and steroids [1].
A recent study has established that the hot aqueous extract of N. oleander leaves (Anvirzel) inhibits fibroblast growth factor-2 and also has anti-tumor activity [3]. Another study demonstrated that the leaf extract has antibacterial and sedative-hypnotic activities and also affects behavioral pattern in mice [4]. The dried and fresh flowers of the plant have been reported to exhibit potent antiinflammatory activity against carrageenan hind paw edema model in mice without inducing any gastric damage [5].
Although the leaves and the root have been the subject of several studies, there has been little investigation of the flowers. The objective of this study, therefore, was to investigate the anticonvulsant activities of the 50 % hydroalcohol flower extract.

Preparation of extract
The flowers (2 kg) were shade-dried and coarsely powdered with a grinding mill. The powder was extracted with petroleum ether (40 -60 ºC) to defat it and then macerated with ethanol:water (1:1) with constant stirring (1200 rpm, 3 h). The solvent incorporating the extractives was filtered and the marc pressed to squeeze out residual extractives. This process was repeated thrice to achieve complete extraction. The extracts obtained during the three cycles were combined and reduced to 1/8 th of its original volume in a rotary evaporator at 45 °C and then lyophilized in a freeze dryer to obtain a yield of 11 %w/w.

Acute toxicity study
Determination of maximum tolerable dose was performed according to OECD (Organization for Economic Corporation and Development) guideline 423 (11) [6]. The study was performed at graded doses of 5, 50, 300 and 2000 mg/kg p.o. using 3 female mice (18 -22 g) at each dose level. The mice were deprived of food 3 -4 h prior to the experiment and thereafter individually administered the extract. Each animal was continuously monitored during the first 30 min, then on hourly basis for the next 4 h, and subsequently, at four hourly interval. Finally, they were placed under observation for 14 days to monitor any abnormal signs and symptoms depicting toxicity. The animals were then humanely killed by a high inhalation dose of diethyl ether and observed for any changes in skin and fur, eyes, mucous membrane (ear), respiratory, circulatory, autonomic and central nervous systems, a swell as somatosensory activity and behavioral pattern. Attention was given to phenomena such as tremors, convulsions, salivation, diarrhoea, lethargy, sedation, hypnosis and coma [6].

Assessment of spontaneous motor activity
The animals were divided into four groups of 6 mice each. Group I served as control and received normal saline (1 ml/100 g body weight); Group II received the reference standard (diazepam, 2 mg/kg i.p.) while Groups III and IV received 100 and 200 mg/kg p.o. of the extract, respectively. To evaluate spontaneous motor activity, each mouse was introduced into an actophotometer and its score of locomotor was measured after a period of 10 min [7].

Potentiation of pentobarbital-induced hypnosis
The animals (excluding those used in the preceding experiment) were divided into three groups (n = 6). Group I was administered pentobarbital (50 mg/kg, i.p.); Groups II and III received 100 and 200 mg/kg of the extract orally, respectively, and then were injected with pentobarbital (50 mg/kg, i.p.) 2 h later. The time elapsed between loss and recovery of the righting reflex was noted and taken as sleeping time [8].

Evaluation of muscle relaxant activity
Before performing this experiment, the fresh mice were trained to remain on rotarod apparatus (with the rod rotating at a speed of 25 rpm) for 3 min. After training, the mice were divided into three groups (n = 6). Group I served as control, Group II as reference standard (diazepam, 4 mg/kg i.p.) while Groups III and IV were administered the extract (100 and 200 mg/kg p.o., respectively).
The effect on motor coordination was assessed with the rotarod apparatus as the fall-off time from the rotarod [7].

Determination of anticonvulsant activity Pentylenetetrazol (PTZ)-induced convulsion test
Four groups of mice (n = 10) were used. Group I was administered the vehicle, i.e., normal saline (1 ml/100g body weight) and served as control, Group II received reference standard (diazepam, 2 mg/kg, i.p.) while Groups III and IV were administered 100 and 200 mg/kg, p.o., respectively, of the extract. Two hours later, PTZ was administered (60 mg/kg, i.p.) to all four groups. The animals were observed for 30 min and the onset and duration of convulsion noted [8].

Maximum electroshock convulsion test
Four groups of mice were used in this test. Group I ingested normal saline (1 ml/100 g) and served as control; Group II received the reference drug (diazepam, 2mg/kg, i.p.) while Groups III and IV were administered the extract (100 and 200 mg/kg, p.o., respectively. Two hours later, they were given a current of 12 mA 50 Hz for 0.2 sec in an electroconvulsiometer via an ear electrode. The onset of convulsion and duration of tonus were noted. Complete protection (100 %) against convulsion is achieved only when hind limb tonic extension is completely abolished [8].

Statistical analysis
All the data obtained were analyzed using one way analysis of variance (ANOVA) followed by Dunnett's test or Student 't' test (Graph-pad Prism statistical software). P < 0.05 was considered statistically significant.

Acute toxicity of extract
The maximum tolerable dose of the extract via oral administration was 2000 mg/kg body weight. Table 1 shows the effect of the extract on spontaneous locomotor activity. Compared with the control group, a small but significant reduction (p < 0.05) in locomotor activity was observed after 1 h. However, much higher reduction in locomotor activity by the reference standard, diazepam, was found. Both the extract and standard effected very drastic reduction (p < 0.001) in locomotor activity after 2 and 3 h, respectively.

Potentiation of pentobarbital-induced hypnosis
Pentobarbital did not modify the latency to induce sleep after extract administration. However, as Fig 1 shows, the former increased the duration of extract-induced hypnosis significantly (p < 0.05 and p < 0.001) at doses of 100 and 200 mg/kg, respectively.

Muscle relaxant activity of extract
The animals did not show any motor uncoordination after administration of the extract. Table 2 shows that the extract increased the onset of pentylenetetrazol-induced seizures but reduced the duration of the convulsions. The increase in the onset of seizure was statistically significant, p < 0.01) and p < 0.05) for extract doses of 100 and 200 mg/kg, respectively, and highly significant (p < 0.001) for the standard drug, diazepam; with regard to their duration reduction effect, the difference was statistically significant at p < 0.01 for both doses. However, the clonus phase was completely abolished only at the lower dose of the extract and also with diazepam.

Effect of extract on maximal electroshock convulsions (MEC)
As Table 3 indicates, the higher dose of the extract (200 mg/kg) was more effective than the lower dose in lowering MEC. However, while the lower dose failed to provide protection against convulsion, the higher dose provided 60 % protection, compared with 100 % for the reference standard, diazepam.

DISCUSSION
Acute toxicity data indicate that the maximum tolerable dose of N. oleander flower extract in mice is 2000 mg/kg body weight, suggesting that the extract is not highly toxic.
The reduction in locomotor activity following the administration of the extract implies that it exerted a depressive effect on the CNS [9]. It has been established that increase in the concentration of gamma-amino butyric acid (GABA) may lead to CNS depressant effect [10]. This led to further exploration of the effect of the extract on activities responsible for increase in GABA concentration, such as potentiation of hexobarbital-induced hypnosis, motor coordination, anticonvulsant activity. The fact that extract activity was potentiated by pentobarbitone in induced hypnosis suggest a GABA-mediated effect on the CNS since CNS depressants extend barbiturate sleeping time [11].
It is known that sedative-hypnotic drugs induce their effect on the Gabaergic system in the brain and inhibition of neuronal output could be facilitated by GABA (an inhibitory neurotransmitter) release [12]. Loss of righting reflex induced by phenobarbital is potentiated by GABA agonist (muscimol and 4,5,6,7-tetrahydroisoxazolo [5,4-c]pyridin-3-ol (THIP) and inhibited by GABA antagonist (bicucullin); the activation of GABA receptor partially mediates the sleep response [13]. It is thus plausible to assert that the sedative effect of the extract is due to the facilitation of GABAergic transmission. The findings in respect of the extract of N. oleander flower are in agreement with those reported for some other plants (Rauwolfia canescens [14] and Carissa edulis [15]). The fact that the extract had no effect on motor coordination also implies that it exerts significant CNS depressant effect through GABA receptor stimulation.
The test on the anticonvulsant activity of the extract is in effect a measure of its stimulatory effect on GABA receptor. Two different anticonvulsant animal models were used: MES-and PTZ-induced convulsion models. The extract showed anticonvulsant activity in PTZ-induced convulsion model and the higher dose of the extract (200 mg/kg) was more effective than the lower dose in lowering MEC. PTZ-induced activation of Ttype Ca 2+ channels of the thalamic region produces petit mal seizures [16]. Compounds that are effective in PTZ-induced seizure models are effective against petit mal type of epilepsy [17]. Petit mal seizures can also be prevented by drugs that enhance GABAA-BZD receptor mediated neurotransmission such as benzodiazepines and phenobarbitone [18]. Protection against hind limb tonic extension also shows the capability of the extract to prevent seizure discharge within the brain stem seizure substrate [19], suggesting that the extract may be useful in the treatment of generalized tonic-clonic seizures.

CONCLUSION
On the basis of results obtained, the hydroalcohol extract of Nerium oleander flowers exhibited anticonvulsant activity against PTZ and MES in experimental animal models. Further fractionation of this extract, may yield antiepileptic drug(s). Also, the results further buttresses the pharmacological basis for the ethnomedicinal use of the plant as antiepileptic agent. .