Anti-Epileptic Activity of the Hydro-Alcoholic Extract of Erythrina fusca Lour. Bark through Behavioral Studies against the Animal Models of Epilepsy.

 

M. Kannadasan1, Subal Debnath*2, Nilesh P. Babre2, P. Parameshwar2, V.V. Rajesham2 and Habibur rehman3

1College of Pharmacy, SRIPMS, Coimbatore, Tamilnadu. 2Srikrupa Institute of Pharmaceutical Sciences, Vil. Velkatta, Kondapak (mdl), Dist. Medak, Siddipet. Andra Pradesh – 502 277. 3PSG College of Pharmacy, Department of Pharmaceutics, Peelamedu, Coimbatore, T.N.

*Corresponding Author E-mail: subal_2007@yahoo.co.in

 

ABSTRACT:

The objective of the present study was to determine the anti-epileptic and behavioural studies of hydroalcoholic extract of the bark of Erythrina fusca Lour. (HAEEF). In the in vivo methods, whole animals are used for the demonstration of an injury by exogenous agents of epileptic seizure on the brain with its physiological significance. The anti-epileptic and behaviuoral studies of Erythrina fusca Lour. bark extract was studied by using  different experimental models. Experimental models of epileptiform seizures (EMES) have constituted the nearest physiological approach to the development of new antiepileptic drugs for seizure control in epileptic patients. Epileptic seizure challenged animals treated with HAEEF at doses of 250 mg/kg and 500 mg/kg showed reduction MES-induced tonic phase latency and also inhibited the clonic seizure. Seizure duration was found to be lower in the extract treated animals than that shown by control group. Thus, it can be inferred that the hydro-alcoholic extract of Erythrina fusca Lour. bark possess antiepileptic effect against the animal models of epilepsy.

 

KEYWORDS: Erythrina fusca Lour. bark, Phenobarbitone - induced sleep, Ketamine- induced anesthesia, Locomotor activity, Motor co-ordination, Haloperidol - induced catalepsy

 


INTRODUCTION:

Epilepsy is more than convulsion; a broad variety of clinical phenomena may reflect epileptic seizures activity for an example, behavioral (or) gastrointestinal signs. The typical behavioral manifestation common to all forms of epilepsy is the aberrant synchronized discharge of neuronal population termed the “seizures”. Epileptic seizure can be induced in any normal human (or) vertebrate brain with a variety of different electrical (or) chemical stimuli. The classification of epilepsy is based upon the underlying etiology. Classification of epilepsy deals with three categories. They are, 1) Idiopathic epilepsy, 2) Symptomatic epilepsy, 3) Cryptogenic epilepsy1.

 

Epileptic seizures are classified by according to “International Classification of Epileptic Seizures” [ICES] introduced in the year 1981. The epileptic seizures are mainly divided into two categories. They are i) Partial seizures and ii)

 

Generalized seizures, where partial seizures are subdivided into i) simple partial seizures, ii) complex partial seizures (psychomotor or limbic seizures), iii) partial seizures evolving to secondary seizures. The generalized seizures also further divided into the followings, i) absence seizures, known as ‘petit mal epilepsy’, ii) myoclonic seizures, iii) tonic seizures, iv) clonic seizures, v) Tonic – clonic  seizures known as ‘grand mal epilepsy’. In the past, convulsion and absence have been refered to as ‘grandmal’ and ‘petitmal’ respectively2.

 

E. fusca is a deciduous tree with spiny bark and light orange flowers. Its legume pods reach 20 centimetres (7.9 in) in length and contain dark brown seeds. The seeds are buoyant, allowing them disperse across oceans. The tree is highly adapted to coastal conditions, tolerant of both flooding and salinity. Like many other species in the genus Erythrina, E. fusca Lour. contains toxic alkaloids which have been utilized for medicinal value but are poisonous in larger amounts. The most common alkaloid is erythraline, which is named for the genus. The new buds and leaves are eaten as a vegetable. The easy-to-grow and attractive flowering tree is cultivated as an ornamental shade and hedge plant. It is a common shade tree in cacao plantations. It attracts hummingbirds, which pollinate its flowers3.

In the assessment of behavioral studies, it was shown that CNS inhibitory in nature as evident by the behavioral assessment of HAEEF. The HAEEF appeared to significantly potentiate phenobarbitone–induced sleeping time in a dose–dependent manner. Phenobarbitone is an intermediate acting barbiturate and is known to produce sleep up to 50 min. the extract may have acted synergistically with phenobarbitone to increase the duration of sleep.

 

MATERIALS AND METHODS:

Collection and authentication of plant material:

The plant material consists of dried powdered stem bark of Erythrina fusca Lour. belonging to the family Fabaceae. The fresh bark of Erythrina fusca Lour. were collected from the Connur, Nilgris district, Tamil Nadu. The bark was authenticated by a taxonomist from Botanical Survey of India (BSI), Tamil Nadu Agricultural University (TNAU), Coimbatore.

 

Preparation of the extract:

Fresh stem bark of Erythrina fusca Lour. were collected and air dried in shade under the room temperature. The dried stem bark material was powdered mechanically and sieved through No. 20 mesh sieve. The fine powder was kept separately in an airtight container until the time of use. Around 100 g of finely powdered bark material was evenly packed in a soxhlet apparatus and the extraction was done with water: ethanol in the ratio of 30 : 70 for 48 hours. The solvent was then evaporated under reduced pressure. The percentage (%) yield of the extract was calculated.

 

Drugs and chemicals:

Standard drugs like diazepam, ketamine, haloperidol, chlorpromazine, phenobarbitone, and phenytoin were purchased from local pharmacy, Coimbatore. All other drugs and chemicals used in the study were obtained commercially and were of analytical grade.

 

Instruments:

Electro convulsivo meter, Actophotometer, Rotarod, UV-Visible spectrophotometer, Shimadzu Jasco V-530.

 

Experimental animals

Wistar rats of either sex weighing between 100-150 g were used for the study. They were housed individually in polypropylene cases in well ventilated room. The animals were fed with commercial rat feed pellets and given drinking water ad libitum.

 

Acute toxicity studies:

It was found that the animals were safe up to a maximum dose of HAEEF at 2000 mg/kg of body weight. There were no changes in normal behavioral pattern and no signs and symptoms of toxicity and mortality were observed.

 

Evaluation of behavioural studies

Phenobarbitone - induced sleep:4, 5

Wistar rats weighing between 100 – 125 g were randomly divided into four groups consisting of six animals each. Group I received phenobarbitone 20 mg/kg, i.p. and Group II animals received 10 mg/kg of diazepam orally before 60 min of phenobarbitone sodium injection. Group III animals received 250 mg/kg of HAEEF orally, before 60 min of phenobarbitone injection, and animals of the group IV received 500 mg/kg of HAEEF orally, before 60 min of phenobarbitone injection.  Latency in the onset of action and duration of sleeping time was then recorded. The duration of sleep was defined as the time between loss and regain of righting reflex.

 

Ketamine - induced anesthesia:6, 7

Wistar rats of either sex weighing between 100 – 125 g were randomly divided into three groups consisting of six animals each. The animals were treated with ketamine HCl (60 mg/kg, i.p.) one hour before test drug treatment. Group I served as control and rats in this group received ketamine HCl (60 mg/kg i.p.) only. Animals of groups II and III rats were treated with (250 mg/kg and 500 mg/kg,  p.o.) HAEEF (one hour prior to ketamine HCl, 60 mg/kg, i.p.). Onset of action and duration of anaesthesia was then recorded.

 

Locomotor activity:8, 9

Rats were assigned into three groups consisting of six animals each. Animals were placed individually inside an Actophotometer for 10 min and their locomotor activity was noted. They were selected according to the normal basal score. Group I animals received standard drug chlorpromazine 3 mg/kg orally. HAEEF was given in two divided doses of 250 mg/kg and 500 mg/kg orally to groups II and III respectively. Animals were placed for 10 min in the Actophotometer and the score was noted down. After 60 min of drug administration the total count was recorded and the values of HAEEF are compared with that of standard. The percentage (%) increase or decrease in locomotor activity was calculated as follows

% of change in activity =   1  -  After treatment  ´ 100  

                                                         Before treatment

 

Motor co-ordination:10- 12

Wistar rats weighing about 100 – 125 g were selected for their muscle grip strength (motor co-ordination). The Rotarod apparatus consist of a metal rod placed in the centre and is divided into four chambers. An appropriate speed of 25 rpm is selected. Only rats remaining on the rod for 3 min or more in successive trial before drug treatment (diazepam and HAEEF) and there by animals are selected for this muscle co-ordination test. Animals were divided into three groups consisting of six animals each. Group-I served as a reference group treated with diazepam (10 mg/kg, p.o) and the test group of animals administered with HAEEF in two doses of 250 mg/kg and 500 mg/kg by orally. The fall of time after treatment was noted down and the mean value was compared with the reference value.

 

Haloperidol - induced catalepsy:13- 15

Wistar rats of either sex weighing between 100-125 g were randomly divided into three groups consisting of six animals of each. Group I served as control and received haloperidol (1.5 mg/kg p.o.) only. Animals of groups II and III were received HAEEF orally at doses of 250 mg/kg and 500 mg/kg respectively. Catalepsy was measured by bar test method after haloperidol treatment with considerable intervals at 5, 15, 30, 45, 60, 90 and 120 mins. The score was noted down as follows,

Stage I -Rat moves normally when placed on the table, score = 0.

Stage II -Rat moves only when touched or pushed, score = 0.

Stage III - Rat placed  on the table with front paw set alternately on a 3 cm high block fails to correct posture in 10 seconds, score = 0.5 for each paw with total of one for each of this stage.

Stage IV -Rat fails to remove when the front paw are placed alternately on a 9 cm block, score = 1 for each paw with a total score of two for this stage.

 

Thus for a single rat, the maximum possible score would be 3.5 revealing total catatonia. The onset and severity of cataleptic response in each group of animals was compared.

 

RESULTS AND DISCUSSION:

Effect of HAEEF on phenobarbitone – induced sleep

In vehicle treated rat loss of righting reflex was observed, 18.55 ± 0.726 min after phenobarbitone administration. The loss and regain of righting reflex was observed till 44.09 ± 0.831 min. The effect of HAEEF at doses 250 mg/kg and 500 mg/kg significantly increased the duration of sleeping time  to 54.76 ± 4.096 and 68.00 ± 0.734 min (P<0.01) respectively (Table 1 and Fig. 1).

 

Fig. 1: Effect of HAEEF on phenobarbitone-induced sleep

 

Effect of HAEEF on ketamine – induced anaesthesia:

HAEEF increased the duration of anaesthesia induced by ketamine. The extract caused a significant increase in the duration of anaesthesia 18.10 ± 0.354 and 25.67 ± 0.867 (P<0.01) at dose of 250 mg/kg and 500 mg/kg respectively. Administration of the extract to ketamine -injected rats resulted in the abolition of palpebral, pedal and inner ear reflexes. Animals had their eyes closed while anaesthetized and during recovery they demonstrated ataxia (Table 2 and Fig. 2).

 

Effect of HAEEF on locomotor activity:

HAEEF at doses of 250 mg/kg and 500 mg/kg significantly decreased the the locomotor activity to 61.55 ± 1.163 and 84.27 ± 0.772 respectively (P<0.01) when compared with control rats (Table 3 and Fig. 3).

 

Fig. 2: Effect of HAEEF on ketamine–induced anaesthesia

 

Fig. 3:  Effect of HAEEF on locomotor activity

 

Effect of HAEEF on motor co-ordination:

Animals treated with HAEEF at doses of 250 mg/kg and 500 mg/kg showed early fall from the Rota rod significantly 49.15 ± 0.913 and 74.66 ± 0 0.685 (P<0.01) respectively (Table 4 and Fig. 4).

 

Fig. 4: Effect of HAEEF of motor co-ordination activity

 

 


Table 1: Effect of HAEEF on phenobarbitone-induced sleep

Groups

Treatment

Dose

Onset of action

Duration  of sleep

I

Control PHB

30 mg/kg

18.55 ± 0.726

44.09 ± 0.831

II

HAEEF + PHB

250 mg/kg

15.36 ± 0.542

54.76 ± 4.096

III

HAEEF + PHB

500 mg/kg

13.53 ± 0.814

68.00 ± 0.734

IV

DZP + PHB

10 mg/kg

11.20 ± 0.672

75.93 ± 0.381

Values are mean ± SEM; n= 6, P<0.01 when compared to control.

 

Table 2: Effect of HAEEF on ketamine - induced anaesthesia

Groups

Treatment

Dose

Onset of action

Duration  of sleep

I

Control Ketamine

60 mg/kg

15.50 ± 0.920

13.91 ± 0.622

II

HAEEF + Ketamine

250 mg/kg

12.01 ± 0.602

18.10 ± 0.354

III

HAEEF + Ketamine

500 mg/kg

6.722 ± 0.556

25.67 ± 0.867

Values are mean ± SEM; n= 6, aP<0.01 when compared to control.

 

Table 3:  Effect of HAEEF on locomotor activity

Groups

Treatment

Dose

Locomotor activity

 

 

 

Before treatment

After treatment

I

HAEEF

250 mg/kg

543.4 ± 0.586

61.55 ± 1.163

II

HAEEF

500 mg/kg

590.2 ± 1.012

84.27 ± 0.772

III

Chlorpromazine

3 mg/kg

542.9 ± 0.7621

90.50 ± 0.897

Values are mean ± SEM; n= 6, P<0.01 when compared to control.

 

Table 4: Effect of HAEEF of motor co-ordination activity

Groups

Treatment groups

Dose

Locomotor activity

I

HAEEF

250 mg/kg

49.15 ± 0.913

II             

HAEEF

500 mg/kg

74.66 ± 0.685

III

Chlorpromazine

3 mg/kg

94.51 ± 0.945

Values are mean ± SEM; n= 6, P<0.01 when compared to control.

 

Table 5: Effect of HAEEF on haloperidol - induced catalepsy.

Groups

Treatment

Dose

Degree of catalepsy response after min

 

I

Control

Haloperidol

1.5 mg/kg

3 ± 0.00

3 ± 0.00

3 ± 0.00

3 ±  0.00

 

3 ±  0.00

 

1.86 ± 0.15

0.00 ± 0.00

II

HAEEF

250 mg/kg

2.92 ±  0.14

2.05 ±0.10

1.47 ± 0.20

0.97 ±0.19

0.26 ±0.20

0.00  ± 0.00

0.00 ± 0.00

III

HAEEF

500 mg/kg

3.77 ±  0.30

2.43 ± 0.26

0.92 ± 0.31

0.00  ± 0.00

0.00  ± 0.00

0.00  ± 0.00

0.00 ± 0.00

 

 


Effect of HAEEF on haloperidol – induced catalepsy:

HAEEF treated animals at doses 250 mg/kg and 500 mg/kg reduced the increased severity of haloperidol induced catalepsy. HAEEF treated groups significantly reduced the scores of catalepsy (P<0.01). The values of observations are given in Table 5 and Fig. 5.

 

Fig. 5: Effect of HAEEF on haloperidol –induced catalepsy.

 

In the assessment of behavioral studies, it was shown that CNS inhibitory in nature as evident by the behavioral assessment of HAEEF. The HAEEF appeared to significantly potentiate phenobarbitone–induced sleeping time in a dose–dependent manner. Phenobarbitone is an intermediate acting barbiturate and is known to produce sleep upto 50 min. The extract may have acted synergistically with phenobarbitone to increase the duration of sleep.

 

In addition to increased the duration of anaesthesia of ketamine significantly. It also abolished palpebral, pedal and inner ear reflex. The HAEEF treated animals also shown that significant reduction in spontaneous locomotor activity and also motor co–ordination test. The results of behavioural studies indicated depressive effect on CNS may due to interaction with GABA/ benzodiazepine receptor complex in the brain. The reduced locomotor activity was compared with the standard drug chlorpromazine. An agent with antiaggressive effect often has useful anxiolytic effects. Therefore, the sedative effect of HAEEF may indicate potentially useful anxiolytic effect.

 

CONCLUSION:

In this studies showed the results that HAEEF significantly decrease the score of neuroleptic-induced catalepsy in rats. The catalepsy effect produced by haloperidol, a dopaminergic blockers of neuroleptics. The mechanism of action of HAEEF may be inhibiting the blockade of striatal D1 and D2 recptors. In conclusion the results of the present studies demonstrated that hydroalcoholic extract of the E. stricta L. bark has shown the antiepileptic. Further studies are needed to be ascertaining its clinical effectiveness and mechanism of action.

 

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Received on 29.07.2010          Modified on 07.08.2010

Accepted on 11.08.2010         © RJPT All right reserved

Research J. Pharm. and Tech. 4(2): February 2011; Page 315-319