Effect of Curcumin on Sub-Therapeutic Doses of AED’S And Long Term Memory In MES Induced GTC Type of Seizures in Rats
Jithendra Chimakurthy *, Talasila EGK Murthy1 and Lokesh Upadhyay2
1Department of Pharmacology and Pharmaceutics, Bapatla College of Pharmacy, Bapatla, AP, India-522101
2 CARISM, SASTRA Univerisity, Thanjavur, TN, India-613402
*Corresponding Author E-mail: jithu_indra@rediffmail.com
ABSTRACT:
Treatment of epilepsy includes the use of multiple dose regimen for prolonged periods, making the side and adverse effects inevitable to the patients. This can be decreased by reducing the dose of antiepileptic drugs administered or prescribing drugs that can potentiate their antiepileptic effect and decrease the unwanted effects. The potentiating effect of curcumin (10 mg/kg p.o.) on the maximal electro shock [MES] induced Generalized tonic clonic [GTC] seizures was studied against sub therapeutic doses of phenytoin [PHT] (13 mg/kg i.p.) and sodium valproate [VPT] (252 mg/kg p.o.) for 14 days, along with its effect on memory retention in seizure induced rats. Rats were trained for conditioned avoidance response before the initiation of treatment and the effect on memory was studied after the induction of epilepsy using conditioned avoidance response task. A significant increase in the percentage protection was observed with PHT (83.3) and VPT (100), when curcumin was administered with sub therapeutic doses of PHT and VPT. Memory retention was also preserved on combined administration of curcumin with PHT and VPT when compared to PHT and VPT alone. Hence a potentiating effect on sub therapeutic doses of PHT and VPT against GTC seizures, along with significant memory retentive effects can be attributed to curcumin.
KEY WORDS : Curcumin, Phenytoin, Sodium Valproate, Epilepsy, Memory.
INTRODUCTION:
Epilepsy is characterized as a group of convulsive seizures, recurring in episodes and is associated with sensory disturbances, abnormal behavior, loss of consciousness or all. Uncontrolled electric discharge from the nerve cells of brain is the common mechanism involved in the occurrence of different types of seizures, although the cause of occurrence of most of the seizures is unknown, it may some times be associated with cerebral trauma, intra cranial infection, brain tumor, vascular disturbances, intoxication or chemical imbalance. According to several publications about 70% of the people with epilepsy are lacking proper treatment and increased incidence of recurrence with a high prevalence of about 0.8 % in children below the age of 7 years1 was reported.
Since the existing antiepileptic drugs [AED’s] are necessitating prolonged treatments including questionable efficacy, studies on seizure induction2, plethora of side
effects and impairment of memory3 by some of the existing AED’s, a need for improved treatment methods which will counteract the side effects and drawbacks of the existing AED’s is crucial.
Combination of AED’s is being used, as a means to increase the efficacy of AED therapy. The purpose of the present study was to evaluate the effect of curcumin on the sub therapeutic dose of standard AED’s, Phenytoin and sodium valproate, we have chosen curcumin in view of the reports such as, the rhizomes of curcuma longa being cited in the ‘Database for Indian medicinal plants’ in the treatment of epilepsy4, the protective effect of norepinephrine [NE] against electroshock induced GTC seizures5, the MAO A and B inhibitory activity of curcuma longa6, studies on the learning and memory retentive effects of curcuma longa7, its potential to increase the brain monoamine levels in discrete regions of brain8 and the efficacy of curcumin in the treatment of Generalized Tonic Clonic [GTC] seizures being observed in our previous studies. Reports on the sub therapeutic doses of phenytoin and sodium valproate9 are taken as an index for the estimation of the extent of potentiating effect of curcumin on seizure incidence and memory, when administered along with ED 50 of these AED’s. Since most of the existing AED’s are reported to be involved in memory disruption and many other side effects. This unwanted effects can be decreased by decreasing the dose of AED or substituting with a safer herbal remedy, which are free of side effects or
administration of herbal AED’S along with sub therapeutic dose of the standard AED’S, which on prolonged use will render them free of unwanted effects. Hence, the objective of the present study was to evaluate the 1.Potentiating effect of curcumin (10 mg/kg) on sub therapeutic doses of Phenytoin (13 mg/kg i.p.) and Sodium valproate (252 mg/kg p.o.) 2. Effect on memory retention after exposure to seizures.
Inbred Adult male wistar rats (200 – 250 g) were procured from the animal house of Bapatla College of pharmacy, Bapatla, India and were housed at a constant room temperature (22 ± 1ºC), 40-50% relative humidity and 12 – 12 h light/dark cycles were maintained. Standard pellet feed (Rayan’s Biotech, Hyderabad) and water was provided ad libitum through out the experimentation period. Animals were acclimatized to laboratory conditions one week prior to initiation of experiments. The experimental design was approved by Institutional animal ethics committee (IAEC/I-6/BCOP/2007-2008) and all the experiments involved in this work were performed in accordance with CPCSEA guidelines for the use and care of experimental animals.
Drugs and drug administration:
Curcumin was obtained from Lailla Impex, Vijayawada, India and was characterized by H+ NMR studies. For oral administration, Curcumin (10 mg/kg p.o.) was dissolved in peanut oil and diluted to the desired concentration with the same, on the day of administration. PHT (13 mg/kg i.p.) was diluted with sterile saline for i.p. administration. VPT (252 mg/kg p.o.) was suspended in 1% w/v CMC. The peanut oil, CMC and sterile saline were used as control treatments and the behavioral data did not differ between rats that received these vehicles, hence the results were compared with peanut oil treated control group.
Conditioned avoidance response: Training procedure10:
Rats were subjected to conditioned avoidance response before the induction of MES seizures. Each rat was placed individually in a pole climbing apparatus (M.K.M Industries, Chennai, India) provided with a grid floor that can be electrified and conditioned with a buzzer. Rats were trained 3 times a day initially to avoid the electroshock (80 V, 5 pulses/sec), shock was applied to the grid floor intermittently preceded by the beep (conditioned stimulus) for two weeks. Rats learned to avoid the shock by climbing the pole after the conditioned stimulus, were selected for studying the effect on MES induced seizures and later on the memory retention effect after induction of seizure.
Curcumin, PHT and VPT were administered for 14 days and 60 min prior to the induction of MES on the last day of treatment. Group I animals served as epileptic control, group II, III and IV rats were treated with curcumin 10 mg/kg p.o., PHT 13 mg/kg i.p. and VPT 252 mg/kg p.o. respectively. Group V rats were treated with PHT + curcumin 10 mg/kg and group VI rats were treated with VPT + curcumin 10 mg/kg. In the determination of memory retention an additional control group which was not exposed to MES was used.
Induction of seizures using MES:
GTC seizures were induced, using an Electro convulsiometer (Inco Co., Ambala, India). Maximal seizures were elicited by 60 Hz alternating current of 150 mA intensity for 0.2 sec using corneal electrodes. A drop of electrolyte solution 0.9% sodium chloride with lignocaine was applied to the corneal electrodes, which ensures better contact and the mortality rate to zero. Percentage protection of drugs from seizures was estimated by considering inhibition of complete hind limb tonic extension (HLTE) or extension not greater than 90º angle with the plane of the body11. Various other parameters measured were, duration of tonic flexion, tonic extension, clonic convulsions and righting reflex.
After ensuring recovery from seizures, rats were placed individually in the pole climbing apparatus for determining the effect on memory retention. A fixed number (i.e. ten) of shocks were applied to the electric grid floor and the number of shocks avoided by each animal of a group were determined and tabulated.
All the data obtained from the observations were statistically analyzed using one way ANOVA, when the F test ratio was significant, the inter group differences were evaluated using Dunnet’s ‘t’ test and P< 0.05 was considered to be significant.
Results from the study have suggested that, curcumin had a high synergistic activity in the treatment of GTC seizures with sub therapeutic doses of PHT and VPT. Treatment with curcumin at a dose of 10 mg/kg exhibited a percentage protection of 66. Whereas, PHT and VPT treated rats have shown a percentage protection of 50 and 66, when administered alone, and this increased to 83.3 and 100, when administered along with curcumin, against the MES induced seizures. Further more, a significant decrease in the duration of tonic flexion and clonic convulsions was also observed on combined administration of PHT & VPT with curcumin. Neither PHT nor VPT alone had shown any significant changes in the duration of tonic flexion and clonic convulsions, but a
Table 1. Effect of curcumin on sub therapeutic doses of standard AED’s against GTC seizures
|
Groups Treatment |
Duration in different phases (sec) |
|||||
|
Percentage protection |
Tonic Flexion |
Tonic Extension |
Clonic Convulsions |
Righting Reflex |
||
|
I |
Control |
0% |
5 ± 0.85 |
13.3 ± 0.86 |
13.4 ± 1.62 |
5.83 ± 1.014 |
|
II |
Curcumin10 mg/kg p.o. |
66% |
3.42 ± 0.66ns |
3.74 ± 1.21# |
8.25 ± 1.62ns |
3.86 ± 0.82* |
|
III |
PHT13.5 mg/kg i.p. |
50% |
3.16 ± 0.73 ns |
6.3 ± 2.06 ** |
14.8 ± 2.35 ns |
5.5 ± 1.05 ns |
|
IV |
Sod. Val252 mg/kg p.o. |
66% |
3.33 ± 0.61 ns |
6.5 ± 2.27 ** |
5.5 ± 1.74** |
6.16 ± 1.24ns |
|
V |
curcumin + PHT |
83.3% |
1.3 ± 0.49 ** |
0.83 ± 0.54# |
2.33 ± 1.66# |
2.33 ± 1.74* |
|
VI |
curcumin + VPT |
100% |
2.33 ± 0.76* |
1 ± 0.68# |
5.66 ± 1.72** |
2.83 ± 1.93* |
|
d.f. 5, 30 F= |
- |
3.63 |
10.2 |
7.74 |
0.35 |
|
Values are mean ± SEM of six observations, Data of all the groups were compared with Group I *p<0.05; **p<0.01; #p<0.001 ns-non significant. PHT-Phenytoin; VPT-Valproate; d.f.- degrees of freedom.
Data are expressed as mean ± SEM of six observations, representing the number of shocks avoided out of 10 applied All the groups were compared with Epileptic control, **p<0.01; #p<0.001; ns-non significant; d.f.-degrees of freedom. PHT-Phenytoin, Control-Vehicle treated, MES was administered to all the groups except Control (Vehicle treated).
significant reduction was observed in the time taken for the righting reflex in group V and VI rats (P<0.05) and the results were tabulated in Table 1.
Effect on memory retention:
Conditioned avoidance response is an experimental model to study procedural type of long term memory, a significant decrease in the retention of the learned task was observed in epileptic control rats, curcumin alone and in combination with sub therapeutic doses of PHT and VPT had shown a significant, P<0.01 and P<0.001 respectively, increase in the number of shocks avoided, an index of memory retention. Whereas, no significant increase in memory retention was observed when PHT and VPT were administered alone. Fig. 1.
DISCUSSION:
In most instances antiepileptic drug therapy includes the use of more than one drug if the therapy with single drug fails, this multiple regimen is increasing the existence of frequency and severity of the toxic effects which are further aggravated with an increase in the dose. Hence, for optimal management of epilepsy, drugs administered at a low dose can be of rescue from the adverse effects of
multiple regimens, moreover it should provide adequate seizure control, curcumin being found to have considerable protective effect over GTC seizures in our previous studies, was studied for its effect on low dose (sub therapeutic dose) of standard AED’s. This potentiating effect of curcumin on the sub therapeutic doses of PHT and VPT, will ensure to decrease the side effects and adverse effects that exists with AED’s at normal dose and when used in combination. The probable mechanism involved in the antiepileptic effect of curcumin can be attributed to the influencing effect on the monoamine levels8, as NE is considered to produce adequate protection from GTC type of seizures5 in addition to the sodium channel blockade effect of PHT and sodium channel blockade, blockade of ca2+ influx and facilitation of GABA mediated cl- ion channel by VPT.
A good memory retentive effect in seizure induced animals was observed, when treated with curcumin alone and in combination with sub therapeutic doses of PHT and VPT, when compared with epileptic control. This increase in memory retention can be interpreted to the increased NE and DA concentrations, as drugs like L-Dopa have shown an improvement in cognitive test of patients of Alzheimer’s disease by increasing DA levels. Other possible mechanism for the memory retentive effect of curcumin can be attributed to the MAO-B inhibitory activity of curcumin8, as MAO-B inhibitory action of selegiline is useful in the treatment of Alzheimer’s disease12. In the forebrain of Alzheimer’s patients, pathological abnormalities in Serotonergic and Noradrenergic innervations are known to exist in addition to cholinergic innervations abnormality. This also indicates the rationality of a combination therapy of cholinergic and monoaminergic drugs in Alzheimer’s disease13.
These studies, and the data obtained allowed us to substantiate the use of curcumin in protection from GTC seizures and enhancing memory. The effects of curcumin on the brain monoaminergic system are of particular interest, since it has been suggested that monoamines are of great clinical significance in the treatment of seizures and memory disorders5,14. The exact mechanism involved in the potentiation of sub anticonvulsant doses has to be substantiated with further studies.
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Received on 05.08.2008 Modified on 15.10.2008
Accepted on 16.11.2008 © RJPT All right reserved
Research J. Pharm. and Tech. 1(4): Oct.-Dec. 2008;Page 401-404