|Year : 2009 | Volume
| Issue : 19 | Page : 46-50
Synergistic effect of Withania somnifera dunal and L-dopa in the inhiition of haloperidol-induced catalepsy in mice
Lal Gupta Girdhari, Chand Rana Avtar
Department of Pharmacology, B. N. College of Pharmacy, Udaipur-313 002 (Rajasthan), India
|Date of Submission||10-Oct-2008|
|Date of Decision||05-Apr-2009|
|Date of Acceptance||06-Jun-2009|
|Date of Web Publication||16-Feb-2010|
Chand Rana Avtar
Department of Pharmacology, B. N. College of Pharmacy, Udaipur-313 002 (Rajasthan)
Source of Support: None, Conflict of Interest: None
| Abstract|| |
The possible synergism between WS and dopamine precursor L-dopa to inhibit haloperidol-induced catalepsy was investigated by using standard bar test in mice. The effect of WS (20-200 mg/kg, oral), L-dopa (20-200 mg/kg, oral) plus carbidopa and combination of subeffective doses of WS (20 or 50 mg/kg, oral) prior to L-dopa (20, 50 or 100 mg/kg, oral) plus carbidopa was assessed in haloperidol (1 mg/kg, i.p.) induced catalepsy. L-dopa and carbidopa combination was always administered in 10:1 ratio. WS (100 or 200 mg/kg, oral) and L-dopa (50, 100 or 200 mg/kg, oral) plus carbidopa treated groups showed a dose dependent reduction in cataleptic scores. Subeffective doses of WS (20 or 50 mg/kg, oral) prior to L-dopa (20, 50 or 100 mg/kg, oral) also potentiated the anticataleptic effect of L-dopa. These results indicate that subeffective doses of WS enhance the anticataleptic actions of L-dopa and the possibility of using WS as adjunctive therapy to reduce the doses and the adverse effects of dopamine precursor in Parkinson's disease.
Keywords: Withania somnifera , Parkinsonism, Catalepsy, L-dopa, Carbidopa
|How to cite this article:|
Girdhari LG, Avtar CR. Synergistic effect of Withania somnifera dunal and L-dopa in the inhiition of haloperidol-induced catalepsy in mice. Phcog Mag 2009;5, Suppl S2:46-50
|How to cite this URL:|
Girdhari LG, Avtar CR. Synergistic effect of Withania somnifera dunal and L-dopa in the inhiition of haloperidol-induced catalepsy in mice. Phcog Mag [serial online] 2009 [cited 2019 Nov 20];5, Suppl S2:46-50. Available from: http://www.phcog.com/text.asp?2009/5/19/46/59782
| Introduction|| |
Parkinson's disease is characterized by a motor impairment caused by the degeneration of dopaminergic neurons located in the substantia nigra pars compacta and by the reduction of dopamine levels in the striatum. Replacement therapy with the dopamine precursor L-dihydroxy-phenylalanine (L-dopa) is successful in most parkinsonian patients. In modern practice, L-dopa is always administered with peripheral decarboxylase inhibitor carbidopa or benserazide which prevents peripheral decarboxylation of L-dopa and allows maximum amount of L-dopa to reach the CNS  . This will not only reduce the dose but also the peripheral side effects of L-dopa per se treatment. A principal limitation of long-term use of L-dopa therapy is that, with time there is a development of motor fluctuations such as "wearing off" and "on-off" phenomena. Increasing the dose of medication may help but this leads to development of dyskinesias , . To avoid such problems, there has been an increasing thrust worldwide to opt for safer and effective herbal drugs mentioned in the traditional medical systems.
Withania somnifera Dunal (WS) (family, Solanaceae), known as ashwagandha in Ayurveda, the ancient Hindu system of medicine, has been in use for more than 2500 years. Historically, WS, or its major active principles, has been used as an antioxidant, adaptogen, anxiolytic, antidepressant, memory enhancer, antiinflammatory, antiulcerogenic, antiparkinsonian and anticarcinogenic agents. The active principles of WS, consisting of sitoindosides VII-X and withaferin-A, have been shown to exhibit significant antistress and antioxidant effect in rat brain frontal cortex and striatum  .
A decoction containing cow's milk powdered Mucuna pruriens seeds and WS has been reported to be effective in 18 clinically diagnosed parkinsonian patients  . Moreover, haloperidol induced catalepsy has been blocked by WS and a polyherbal preparation, BR-16A (Mentat), which has WS as a one of its ingredient , . WS is also reported to be safe in long term use with no adverse effects  . Therefore, the present study was undertaken to investigate the effects of WS as an adjunct to L-dopa against haloperidol-induced catalepsy in mice.
| Materials and Methods|| |
Male, Swiss albino mice weighing 25 to 30 g were housed in group of 4 per cage under controlled light (12:12 light: dark cycle, light on at 0700 h) and temperature (25 ± 20C) environment and behavioral assessment was conducted during the light cycle. Food (Rat chow, Lipton, India) and water was provided ad libitum. All procedures were carried out under strict compliance with ethical principles and guidelines of the Institutional Animal Ethical Committee constituted as per the direction of the Committee for the Purpose of Control and Supervision of Experimental Animals; Madras (Reg. No. 870/ac/05/CPCSEA).
Commercial ethanolic WS root extract (Dabur, New Delhi, India), haloperidol (RPG Life Sciences Ltd, Ankleshwar, India), L-dopa plus carbidopa in 10:1 ratio (Sun Pharmaceuticals, Mumbai, India) were obtained from respective sources. WS root extract, l-dopa plus carbidopa was suspended in 0.5% w/v carboxy methyl cellulose (CMC) in distilled water and administered via oral route. The stock solution contained 100 mg/ml of WS. Haloperidol was obtained in an injectable form and diluted with water for injection I.P. Haloperidol was injected via i.p. route.
In this test, front paw of the mice was gently placed on a horizontal metal bar with 2 mm diameter and placed 5 cm above ground level. The length of time the mouse maintain this abnormal posture with atleast one paw was measured. The test was stopped when the paw of animal touch the ground or 180 sec pass. If the animals do not hold on to the bar after three attempts, it receive the score of 0 seconds  .
All subjects were experimentally naοve at the beginning of each study. The behavioral observations were made during the light cycle between 0900 and 1600 h to minimize variations due to diurnal fluctuations. Overnight-fasted mice were used in order to increase the drug absorption of orally administered drugs. The degree of catalepsy was assessed at different time intervals (30, 60, 120, 180 or 240 min) after haloperidol injection by subjecting individual mouse to the bar test.
(a) Effect of haloperidol on catalepsy
Mice were randomly divided in different groups (n=6 in each) and vehicle (0.2 ml of water for injection, I.P., n=6) or haloperidol (0.3, 1, 5 or 10 mg/kg, n=6 per group) was injected via i.p. route. The degree of catalepsy was assessed at different time intervals (30, 60, 120, 180 or 240 min) after haloperidol injection by subjecting individual mouse to the bar test.
(b) Effect of WS on haloperidol-induced catalepsy
Vehicle or WS (20-200 mg/kg, n=6 per group) was administered through oral route, 60 minutes before haloperidol (1 mg/kg, i.p., n=6 per group) and 30 min thereafter individual mouse was subjected to the bar test at different time intervals (30, 60, 120, 180 or 240 min after haloperidol injection).
(c) Effect of L-dopa plus carbidopa on haloperidol-induced catalepsy
To assess the influence of L-dopa plus carbidopa on haloperidol-induced catalepsy, separate groups of mice administered with L-dopa at different doses 20, 50, 100 or 200 mg/kg, oral, n=6 per group. Haloperidol (1 mg/kg, i.p., n=6 per group) was administered 30 min after L-dopa plus carbidopa combination and 30 min thereafter individual mouse was subjected to the bar test for different time intervals (30, 60, 120, 180 or 240 min after haloperidol injection). L-dopa and carbidopa combination was always administered in 10:1 ratio.
(d) Effect of combined treatment of WS and L-dopa plus carbidopa on haloperidol-induced catalepsy
To assess the influence of WS in the effect of L-dopa plus carbidopa on haloperidol-induced catalepsy, separate groups of mice administered with subeffective doses of WS (20 or 50 mg/kg, n=6 per group) 30 min prior to L-dopa (20, 50 or 100 mg/kg, oral, n=6 per group). Haloperidol (1 mg/kg, i.p., n=6 per group) was administered 30 min after L-dopa plus carbidopa combination and 30 min thereafter individual mouse was subjected to the bar test for different time intervals (or 30, 60, 120, 180 or 240 min after haloperidol injection).
TLC study of WS root extract
Thin-layer chromatography (TLC) was used to identify the steroidal lactones (withanolides) present in WS. The solvent system used was chloroform:methanol:water (64:50:10, v/v) and spots were finally identified with vanillin-phosphoric acid  .
The data are presented as Mean ± SEM. Statistical significance was determined by one-way analysis of variance (ANOVA) followed by post hoc Dunnett's multiple comparison test. Differences were considered to be significant at P<0.05.
| Results|| |
Effect of haloperidol on cataleptic behavior on bar test
In the present study, haloperidol (0.3, 1, 5 or 10 mg/kg, i.p.) produced significant and dose dependent increase in cataleptic score on bar test as compared to vehicle treated group during whole assessment time, except 30 and 60 min (P>0.05) after haloperidol (0.3 mg/kg, i.p.) injection [Table 1].
Effect of WS on haloperidol-induced catalepsy
WS (20 or 50 mg/kg) showed insignificant protection against haloperidol induced catalepsy during whole assessment time, as compared to respective control group in haloperidol-induced catatonia (P>0.05). On the other hand, WS (100 or 200 mg/kg) significantly and dose dependently decreased cataleptic score on the bar test as compared to respective control group during whole assessment time, except 30 min (P>0.05) after haloperidol injection [Table 2].
Effect of L-dopa plus carbidopa on haloperidol-induced catalepsy
L-dopa (20 mg/kg) plus carbidopa showed insignificant protection against haloperidol-induced catatonia during whole assessment time (P>0.05) while higher doses of L-dopa (50, 100 or 200 mg/kg) plus carbidopa significantly and dose dependently decreased cataleptic score on the bar test as compared to respective control group [Table 2].
Effect of combined treatment of WS and L-dopa plus carbidopa on haloperidol-induced catalepsy
Prior administration of subeffective doses of WS (20 or 50 mg/kg) potentiated the effect of subeffective dose of L-dopa (20 mg/kg) plus carbidopa by significantly decreasing cataleptic score on the bar test as compared to subeffective dose of L-dopa treated control group. The enhancement of anticataleptic effect was significant during whole assessment time except at dose WS (20 mg/kg) with L-dopa (20 mg/kg) at 240 min [Table 3]. On the other hand, prior administration of subeffective doses of WS (20 or 50 mg/kg) also potentiated anticataleptic effect of L-dopa (50 or 100 mg/kg) plus carbidopa by significantly decreasing descent time on the bar test as compared to respective dose of L-dopa treated group during whole observation period [Table 3]. WS (20 mg/kg) prior to L-dopa (100 mg/kg) plus carbidopa showed complete reversal of catatonia in the bar test upto 180 min after haloperidol injection and also showed significant decrease in descent time on bar test at 240 min after haloperidol injection [Table 3].
TLC study of WS root extract
TLC showed the presence of four blueish violet spots (Rf value: 0.8-0.9).
| Discussion|| |
In the present study, WS and L-dopa showed synergistic effect in the inhibition of haloperidol-induced catalepsy in mice. Haloperidol is a well known neuroleptic for the treatment of schizophrenia and other affective disorders  , primarily acting as a D2 receptor antagonist in the mesolimbic-mesocortical pathway. Due to its non-selective action, it also produces blockade of post-synaptic D2 receptors in the nigrostriatal pathway leading to the development of extrapyramidal side effects in humans  and catalepsy in animals  . Catalepsy has been defined as "an inability to correct an imposed abnormal posture while maintaining the righting reflex". Haloperidol induced catalepsy is also associated with an increase in oxidative stress in the brain  . In this study, haloperidol (0.3, 1, 5 or 10 mg/kg, i.p.) produced dose dependent increase in cataleptic state, this was significant as compared to vehicle treated group. For further studies, haloperidol (1 mg/kg, i.p.) was chosen to produce a moderate degree of catalepsy, so that attenuation or potentiation of the phenomenon could be detected.
The anticataleptic effect of WS has already been reported , . The anticataleptic effect of WS could be attributed to the direct scavenging of free radicals by polyphenols present in it and also by inhibition of lipid peroxidation in the central nervous system. In our study also, WS (100 or 200 mg/kg, oral) significantly and dose dependently reduced cataleptic score. The standard drug L-dopa (50, 100 or 200 mg/kg, oral) plus carbidopa showed significant protection against haloperidol-induced catatonia. Prior administration of subeffective dose of WS potentiated the effect of sub-effective dose of L-dopa plus carbidopa as indicated by decreased cataleptic score.
When compared with the standard drug L-dopa (100 or 200 mg/kg, oral), WS at the dose of 100 and 200 mg/kg was found to be less effective but significant in reversing haloperidol-induced catalepsy. WS (20 mg/kg) prior to L-dopa (100 mg/kg) plus carbidopa showed complete reversal of catatonia in the bar test after haloperidol injection.
The anticataleptic effect of WS and potentiation of the effect to the different doses of L-dopa may be due to the antioxidant or neuroprotective action, in stress induced dopamine neuron degeneration  . A polyherbal preparation, EuMil, which has WS as a one of its ingredient, significantly increased the stress-induced decrease in dopamine level in striatal, hypothalamal region of the rat brain  . However, further investigations are needed to determine the active compound(s) and correlation between dopamine levels with the effect of WS.
These results indicate that subeffective doses of WS enhance the anticataleptic action of L-dopa and leave open the possibility of using WS as adjunctive therapy to reduce the doses and the adverse effects on long term use of dopamine precursor in Parkinson's disease.
| Acknowledgements|| |
The authors wish to thanks the management members of B. N. Sansthan, Udaipur, for providing the necessary facilities to carry out the research work. The supply of WS root extract by M/s Dabur, New Delhi is also acknowledged.
| References|| |
|1.||Aminoff M.J. Parkinson's disease. Neurol. Clin. 19 : 119-28 (2001). |
|2.||Kopin I.J. The pharmacology of Parkinson's disease therapy: an update. Annu. Rev. Pharmacol. Toxicol. 33 : 467-95 (1993). |
|3.||Lang A.E. and Lozano A.M. Parkinson's disease (review). Second of two parts. N. Engl. J. Med. 339 : 1130-43 (1998). |
|4.||Gupta G.L. and Rana A.C. Withania somnifera (Ashwagandha): A Review. Phcog. Rev. 1 : 129-36 (2007). |
|5.||Nagashayana N., Sankarankutty P., Nampoothiri M.R., Mohan P.K. and Mohanakumar K.P. Association of L-DOPA with recovery following Ayurveda medication in Parkinson's disease. J. Neurol. Sci.. 176 : 124-7 (2000). |
|6.||Kumar A. and Kulkarni S.K. Effect of BR-16A (Mentat;), a polyherbal formulation on drug-induced catalepsy in mice. Ind. J. Exp. Biol.. 44 : 45-8 (2006). |
|7.||Nair V., Arjuman A., Gopalakrishna H.N. and Nandini M. Effect of Withania somnifera root extract on haloperidol-induced catalepsy in albino mice. Phytother. Res.. 22 : 243-6 (2008). |
|8.||Costall B. and Naylor R.J. On catalepsy and catatonia and the predictability of the catalepsy test for the neuroleptic activity. Psychopharmacology. 34 : 233-41 (1974). |
|9.||Gupta G.L. and Rana A.C. Protective effect of Withania somnifera Dunal root extract against protracted social isolation induced behavior in rats. Ind. J. Physiol. Pharmacol. 51 : 345-53 (2007). |
|10.||Seeman P., Corbett R., Nam D. and Van Tol H.H. Dopamine and serotonin receptors: amino acid sequences, and clinical role in neuroleptic Parkinsonism. Jpn. J. Pharmacol. 71 : 187-204 (1996). |
|11.||Farde L., Nordstrom A.L., Wiesel F.A., Pauli S., Halldin C. and Sedvall G. Positron emission tomographic analysis of central D1 and D2 dopamine receptor occupancy in patients treated with classical neuroleptics and clozapine: Relation to extrapyramidal side effects. Arch. Gen. Psychiatry 49 : 538-44 (1992). |
|12.||Sanberg P.R. Haloperidol-induced catalepsy is mediated by postsynaptic dopamine receptors. Nature 284 : 472-3 (1980). |
|13.||Sagara Y. Induction of reactive oxygen species in neurons by haloperidol. J. Neurochem. 71 : 1002-12 (1998). |
|14.||Shukla S.D., Jain S., Sharma K. and Bhatnagar M. Stress induced neuron degeneration and protective effects of Semecarpus anacardium Linn. and Withania somnifera Dunn. in hippocampus of albino rats: An ultra structural study. Ind. J. Exp. Biol. 38 : 1007-13 (2000). |
|15.||Bhattacharya A., Muruganandam A.V., Kumar V. and Bhattacharya S.K. Effect of poly herbal formulation, EuMil, on neurochemical perturbations induced by chronic stress. Ind. J. Exp. Biol. 40 : 1161-3 (2002). |
[Table 1], [Table 2], [Table 3]