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Effect of Withania somnifera glycowithanolides on a rat model of tardive dyskinesia. (Letter to the Editor).


Withania somnifera glycowithanolides (WSG) were investigated for their preventive effect on the animal model of tardive dyskinesia (TD), induced by once daily administration of the neuroleptic, haloperidol (1.5 mg/kg, i.p.), for 28 days. Involuntary orofacial movements (chewing movements, tongue protusion and buccal tremors) were assessed as TD parameters. WSG (100 and 200 mg, p.o.), administered concomitantly with haloperidol for 28 days, inhibited the induction of the neuroleptic TD. Haloperidol-induced TD was also attenuated by the antioxidant, vitamin E (400 and 800 mg/kg, p.o.), but remained unaffected by the GABA-mimetic antiepileptic agent, sodium valproate (200 and 400 mg/kg, p.o.), both agents being administered for 28 days like WSG. The results indicate that the reported antioxidant effect of WSG, rather than its GABA-mimetic action, may be responsible for the prevention of haloperidol-induced TD.

Key words: Withania somnifera, glycowithanolides, haloperidol, tardive dyskinesia, antioxidant action


Tardive dyskinesia (TD) is a delayed extrapyramidal neurological syndrome associated with clinical use of neuroleptics. The pathophysiological basis of TD remains obscure. It has been postulated that compensatory increase in dopamine (DA) function in the basal ganglia, following the blockade of DA receptors by neuroleptics, is involved (Penis et al., 1979). The other theories of TD implicate central cholinergic dysfunction (Alphs and Davis, 1983), GABA deficiency (Ogawa et al., 1984) and oxidative stress (Egan et al., 1992). Animal models of TD have used the induction of abnormal involuntary orofacial movements following long-term administration of neuroleptics (Takeuchi et al., 1998; Bhattacharya et al., 2000). Haloperidol-induced TD has been utilized to investigate the prophylactic and therapeutic effects of anti-TD agents.

Withania somnifera Dunal (WS) (family: Solanaceae), is categorized as a rasayana in Ayurveda, the classical Indian system of medicine. The rasayanas, apart from their use for promoting physical and mental health, providing defence against disease and noxious external factors, and for arresting the aging process, have been used to improve cognitive functions in the elderly. WS has also been used in convulsive disorders, tremors and in diseases which closely resemble the modern concept of neurodegenerative disorders (Weiner and Weiner, 1994). The major bioactive chemical principles of WS appear to be the glycowithanolides (WSG) (Bhattacharya et al., 1987, 1995, 1997; Ghosal et al., 1989). WSG has been shown to inhibit ibotenic acid-induced cognitive deficits in rats by reversing rat brain frontal cortical and hippocampal decreases in acetylcholine, choline acetylase and cholinergic muscarinic receptors produced by the neurotoxin (Bhattacharya et al., 1995). WSG exerts significant antioxidant effect in various r at brain areas, including striatum (Bhattacharya et al., 1997). Autoradiographic studies on rat brain indicate that WSG affects preferentially events in the cortical and basal forebrain cholinergic signal transduction cascade (Schliebs et al., 1997). WS appears to have an in vitro and in vivo GABA-mimetic action (Kulkarni and George, 1996).


WS has several chemotypes as ascertained by systematic chemical analysis (Ghosal, 1999). The Indian chemotype-I, rich in withanolide glycosides (= sitoindosides) (Ghosal, 1999), was used for the isolation of the test compounds. A herbarium specimen of the plant has been preserved at the R&D Centre, Indian Herbs Limited, Saharanpur, India.

Freshly harvested 2-year old thin roots of WS were dried, coarsely powdered and then exhaustively extracted with aqueous-alcohol (1:1) at 55 [+ or -] 5 [degrees]C. The extract was concentrated under reduced pressure to remove ethylalcohol and the aqueous concentrate was exhaustively extracted with chloroform to remove fatty material and free withanolides. The chloroform-insoluble (water-soluble) fraction was spray-dried to give a free-flowing colourless powder (yield, 12-15%, w/w). It contained sitoindosides VII-X and withaferin-A as the major bioactive entities, the relative abundance of these compounds (collectively referred to as glycowithanolides, WSG) in the extract powder being 28-30%. Besides these, oligosaccharides (= polyglucans, molecular wt. <2000 D) with relative abundance of 12-15%) constituted the carriers of the bioactive compounds, in the form of inclusion complex (Ghosal, 1999). The structures of the sitoindosides were established by comprehensive spectroscopic analysis and crucial chemical t ransformation, as described earlier (Bhattacharya et al., 1987; Ghosal et al., 1989). The composition of the sitoindosides and withaferin-A in the standardized extract powder was determined by HPTLC (Bhattacharya et al., 1995). Briefly, a CAMAG assembly [software (C) 1990, scanner II.V, 3.14/PC/CATS version, 3.05 1 amp deuterium, wavelength 254 nm, quenching mode] was used. The detection and quantification of the bioactive ingredients was carried out in two solvent systems, chloroformmethanol (90:10) and n-butylalcohol-acetic acid-water (4:12), using authentic markers. The equimolar composition of the five components was made by adding appropriate amounts of the deficient compounds. The combined formulation was freely soluble in normal saline.

Adult male Wistar rats (160-180 g) were used. The rats were housed in colony cages (4-5 rats/cage) at an ambient temperature of 25 [+ or -] 2 [degrees]C and 45-55% relative humidity, with a 12 h light/12 h dark cycle. The animals had free access to standard pellet chow and drinking water. Experiments were conducted between 9.00 and 14.00 hours.

An injectable preparation of haloperidol (Searle, India) diluted in normal saline, was administered (1.5 mg/kg, i.p.) to the rats, once daily at 10.00 h for 28 days, including Sundays. The rats were shifted to the observation room 48 h after the final dose. After a 2 h habituation period, the rats were placed in a transparent observation cage (25 x 15 x 10 cm). After a further 5 min period of habituation, the rats were observed for involuntary orofacial movements (chewing moments, tongue protrusions and buccal tremors) for the next 15 min by an unbiased observer. Since these involuntary moments do not appear while the rat is walking, grooming, rearing or sleeping, the time period for these activities was deducted from the observation period of 15 mm and the remaining period (approx. 50%) was assessed as the actual observation period. The total number of each abnormal orofacial movement was divided by this observation period (mm) to calculation the frequency of chewing movements, tongue protrusions and buccal tremors (Takeuchi et al., 1998). Earlier studies have indicated that an increase of the observation period to 30 min, does not significantly after the data (Bhattacharya et al., 2000).

WSG (100 and 200 mg/kg), sodium valproate (Sun Pharma, India, 200 and 500 mg/kg) and vitamin E (Torrent Laboratories, India, 400 and 800 mg/kg) were administered orally (p.o.), suspended in 0.3% carboxymethylcellulose in distilled water, once daily for 28 days. Control animals received equivalent volume (2.5 ml/kg, p.o.) for the same period. The drug or vehicle administrations were done one hour before haloperidol injections.

Results and Discussion

The results of the study indicate that WSG can prevent neuroleptic-induced TD in rats. Sodium valproate proved ineffective whereas vitamin E was able to prevent induction of TD symptoms. These results are in conformity with earlier studies (Takeuchi et al., 1998; Bhattacharya et al., 2000) in which GABA-mimetic agents like sodium valproate and clonazepam were unable to prevent haloperidol-induced TD, unlike vitamin E, which had a prophylactic preventive effect. It appears that the reported antioxidant effect of WSG (Bhattacharya et al., 1997; Bhattacharya et al., 2001) rather than its GABA-mimetic action (Kulkarni and George, 1996) may be responsible for the observed prevention of neuroleptic-induced TD. However, facilitation of striatal cholinergic function by WSG (Bhattacharya et al., 1995; Schliebs et al., 1997) may be an additional mechanism. Ibotenic acid-induced rat model of Alzheimer's disease is associated with perturbed striatal cholinergic function. WSG not only inhibits cognitive deficits in this m odel but also reverses the dysfunction in presynaptic and postsynaptic cholinergic markers induced by the neurotoxin (Bhattacharya et al., 1995). The results indicate that the clinical use of W. ashwagandha in some undefined neurodegenerative disorders in Ayurveda, merits investigation, using modern paradigms and investigative methods.

Table 1

Effects of Withania somnifera glycowithanolides (WSG), sodium valproate
and vitamin E on haloperidol (HP, 1.5 mg/kg, i.p. X 28 days) -- induced
rat model of tardive dyskinesia (data are means [+ or -] SEM).

Treatments (mg/kg, p.o.) n Chewing *

Vehicle 8 6.4 [+ or -] 0.8
Haloperiodl (HP) 12 13.6 [+ or -] 1.2 (a)
WSG (100) + HP 8 9.6 [+ or -] 0.9 (b)
WSG (200) + HP 8 8.1 [+ or -] 0.8 (b)
Sodium valproate (200) + HP 8 10.2 [+ or -] 1.8
Sodium valproate (400) + HP 8 9.6 [+ or -] 1.9
Vitamin E (400) + HP 8 8.8 [+ or -] 0.8 (b)
Vitamin E (800) + HP 6 7.9 [+ or -] 0.6 (b)

Treatments (mg/kg, p.o.) Tongue *

Vehicle 1.6 [+ or -] 0.6
Haloperiodl (HP) 4.2 [+ or -] 0.9 (a)
WSG (100) + HP 2.2 [+ or -] 0.6 (b)
WSG (200) + HP 1.9 [+ or -] 0.4 (b)
Sodium valproate (200) + HP 3.0 [+ or -] 0.7
Sodium valproate (400) + HP 2.8 [+ or -] 0.9
Vitamin E (400) + HP 2.3 [+ or -] 0.8 (b)
Vitamin E (800) + HP 2.0 [+ or -] 0.5 (b)

Treatments (mg/kg, p.o.) Buccal *

Vehicle 0.6 [+ or -] 0.2
Haloperiodl (HP) 2.0 [+ or -] 0.6 (a)
WSG (100) + HP 0.9 [+ or -] 0.3 (b)
WSG (200) + HP 0.8 [+ or -] 0.4 (b)
Sodium valproate (200) + HP 1.4 [+ or -] 0.6
Sodium valproate (400) + HP 1.2 [+ or -] 0.8
Vitamin E (400) + HP 0.9 [+ or -] 0.4 (b)
Vitamin E (800) + HP 0.7 [+ or -] 0.2 (b)

* Data represents frequency of the test parameters.

(a)P < 0.05 different from vehicle-treated group

(b)P < 0.05 different from haloperidol treated group (Tukey test).


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Bhattacharya, S.K., Goel, R.K., Kaur, R., Ghosal, S.: Antistress activity of sitoindosides VII and VIII, new acylsteryglucosides from Withania somnifera. Phytother. Res. 1: 32-37, 1987.

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Ogawa, T., Nagao, T., Kashiwabara, K., Fukiwara, Y., Harada, T., Otsuki, S.: Tardive dyskinesia and neurotransmitters effects of sodium valproate, cyproheptadine, oxypertine, hydroxyzine pamoate and Ca-hopatenate on monoamine metabolites, cyclic nucleotides and gammaaminobutyric acid in human cerebrospinal fluid. Clin. Ther. 7S: 1-17, 1984.

Perris, C., Dimitrijevic, P., Jacobsson, L., Paulsson, P., Rapp, W., Froberg, H.: Tardive dyskinesia in psychiatric patients treated with neuroleptics. Br. J. Psychint. 135: 509-514, 1979.

Schliebs, R., Liebmann, A., Bhattacharya, S.K., Kumar, A., Ghosal, S., Bigl, V.: Systemic administration of defined extracts from Withania somnifera (Indian Ginseng) and shilajit differentially affects cholinergic but not glutamatergic and gabaergic markers in rat brain. Neurochem. Int. 30: 181-190, 1997.

Takeuchi, H., Ishigooka, J., Kobayashi, K., Watanabe, S., Miura, S.: Study on the suitability of a rat model for tardive dyskinesia and the preventive effects of various drugs. Prog. Neuropsychol. Biol. Psychiat. 22: 679-691, 1998.

Weiner, M.A., Weiner, J.: Ashwagandha (Indian Ginseng). In: Herbs that Heal, Quantum Books, Mill Valley, California, 1994, pp. 70-72.

Salil K. Bhattacharya (1), D. Bhattacharya (2), K. Sairam (1) and S. Ghosal (3)

(1) Department of Pharmacology, Institute of Medical Sciences, Banaras Hindu University, Varanasi, India

(2) Department of Pharmacology, Postgraduate Institute of Basic Medical Sciences, Calcutta University, Calcutta, India

(3) Drug Research and Development Centre, Calcutta, India


S. K. Bhattacharya, Department of Pharmacology, Institute of Medical Sciences, Banaras Hindu University, Varanasi - 221 005, India

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Author:Bhattacharya, Salil K.; Bhattacharya, D.; Sairam, K.; Ghosal, S.
Publication:Phytomedicine: International Journal of Phytotherapy & Phytopharmacology
Geographic Code:9INDI
Date:Mar 1, 2002
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