INTRODUCTION:

Tardive dyskinesia (TD) is a movement disorder (hyperkinetic) influenced by repetitive and involuntary movements, mostly affecting the orofacial part of the body, including trunk and limbs¹. The extrapyramidal system regulates movement via "direct" and "indirect" pathways and dopamine is a key regulator of these two pathways that restrict and facilitate motor movements^2,3.

In the schizophrenia condition, excessive release of dopamine is a major concern. To overcome this problem, dopamine receptor blocking agents (DRBAs) such as typical antipsychotics are used to treat schizophrenia^4,5. Their prolonged treatment (months or years) increases the super sensitivity of D2 receptors, which results from aberrant hyperkinetic/involuntary movements^6,7. The antipsychotics-induced pathogenic mechanisms are linked to the degradation of striatal neurones, a critical factor in the course of TD progression⁸. The earlier study found that prolonged antipsychotic treatment enhances oxidative stress, neuroinflammation, and neuronal excitotoxicity results from hyperkinetic motor activity^9–11. Furthermore, anti-psychotic altered antioxidant enzyme activity leads to excessive oxidative insults that contribute to the pathophysiology of TD; several antipsychotic-induced preclinical animal models have been developed to investigate the neuroprotective effect of drugs against TD^10,12,13.

Haloperidol-induced neurotoxicity is a well-established model used to investigate the therapeutic effects of compounds against TD. Acute haloperidol administration enhances the neurotoxicity in rats and causes orofacial dyskinesia (OD), which is marked by increased vacuous chewing movement (VCM), facial jerking, and tongue protrusion (TP)^13–15. An accumulative study indicates that haloperidol has the potential to induce TD-like pathogenic conditions in animals via modifying oxidative stress, mitochondrial functioning, and the apoptosis process^9,16,17.

Arbutin is a bioactive hydroquinone glucoside that is produced mostly by the Ericaceae family plant Arctostaphyllos uva-ursi, it is naturally present in many other plants, animals, and microbes¹⁸. According to the report, arbutin was found to possess antioxidant and anti-inflammatory properties, traditionally utilised for the management of type 2 diabetes and hypertension¹⁹. In contrast to that, it has been utilized as an anti-microbial, anti-tussive, and anti-infective agent in the treatment of several disease conditions^20,21. Additionally, arbutin shows neuroprotective effects on various diseases such as ischemia-reperfusion²¹, Alzheimer's diseas²², Parkinson's disease²³, and epilepsy²⁴. Moreover, arbutin can improve cognitive function²⁵.

Thus, the purpose of this study was to evaluate arbutin's neuroprotective effect against the behavioural and biochemical changes caused by haloperidol in TD rats.

MATERIALS AND METHODS:

Experimental Animals:

This study utilised Wistar rats weighing between 180-250g obtained from the Central Animal House of Panjab University in Chandigarh, India. Four rats were kept collectively under typical conditions: a room temperature of 22±2°C, 60% relative humidity, and a 12hour light/dark cycle. The rats had unrestricted access to purified fresh water and food ad libitum. All behavioural activity was performed between 9:00 and 17:00hours, the current study includes similar-age rats to reduce variation across experiments, and this protocol was approved by Institutional Animal Ethics Committee (IAEC) with protocol no. CUPB/IAEC/2022/005.

Drug and chemicals:

Haloperidol (Serenace® Inj.) dissolved in distilled water before administration; Arbutin (Sigma-Aldrich® Pvt. Ltd. India), and other high-quality analytical grade chemicals were used in the present study.

Experimental Protocol:

For 21 days, Haloperidol (1mg/kg) was injected intraperitoneally. Arbutin at 50 and 100mg/kg was given intraperitoneally for 21 days after giving the haloperidol treatment. All experimental rats were divided into four groups (n=6 animals in each group) namely, Group I (vehicle control); Group-II (Haloperidol at 1mg/kg, i.p.; Group-III (Haloperidol at 1mg/kg, i.p. + Arbutin at 50mg/kg, i.p.; Group-IV (Haloperidol at 1mg/kg, i.p. + Arbutin at 100mg/kg, i.p. for 21 days.

On the 21st day, various behavioural parameters were performed to examine the impact of arbutin on motor abnormalities. At the end of the protocol (22nd day), all experimental rats were sacrificed, and the striatum was removed, the homogenates was prepared for further biochemical analysis.

Behavioral Assessments:

For behavioral assessment, orofacial movements for oral dyskinesia^10,26, rotarod activity for motor coordination and balance abilities¹³, locomotor activty²⁷, narrow beam walking activity for gait abnormalities²⁸ was employed.

Dissection and Homogenization:

At the end of protocol day 22, each experimental animal was euthanised using thiopentone at a dosage of 100 mg/kg, administered intraperitoneally. Subsequently, the brain was extracted, the striatum was isolated, and a homogenate was prepared utilising 0.1 M phosphate buffer solution (pH 7.4), and centrifuged for 15 minutes at 10,000 g. After that, the supernatant was collected and stored for further biochemical examination²⁹.

Biochemical Estimations:

The biochemical estimations of MDA²⁶, Nitrite²⁷, Catalase³⁰, superoxide dismutase (SOD)³¹, reduced glutathione (GSH)²⁸, total proteins³² in the striatum supernatant were done. Additionally, TNF-α and IL-1β were quantified in the stratum rat brain via ELISA kit (Krishgen Biosciences USA)^33,34.

Statistical Analysis:

GraphPad Prism (8.0.1 V) was applied for data analysis. 2-way ANOVA and Bonferroni's post hoc test for multiple comparisons were used to assess the behavioural data. Tukey's post hoc test was used after a one-way ANOVA to assess all biochemical data. Every observed result was calculated as Mean±S.D. all the results are reliable and statistically significant.

RESULTS:

Arbutin's effect on rats' facial jerks, tongue protrusions, and VCMs induced by haloperidol

Acute haloperidol (1mg/kg) injection for duration of 21 days showed a significant (p<0.0001) increase in VCMs (Fig 1a), tongue protrusion (Fig 1b), and facial jerking (Fig 1c) on the 21st day than the control group. Long-term treatment with arbutin (50 and 100mg/kg) substantial declines the VCMs (p<0.001), facial jerking (p<0.001), and tongue protrusion(p<0.05) on the 21st day in comparison with haloperidol group.

Fig. 1: Effect of Arbutin on a) VCMs, b) tongue protrusions, c) facial jerking in haloperidol-induced neurotoxicity in rats. Mean ± SD is used to express the data. ^ap <0.0001 Halo vs control, ^bp <0.0001 Arb vs haloperidol, ^cp<0.01 Arb (100) vs Arb (50). Halo: Haloperidol, Arb: Arbutin

Effect of arbutin on motor coordination in haloperidol-induced neurotoxicity in rats:

Acute haloperidol injection for duration of 21 days (p<0.0001) dramatically reduces motor coordination on the 21st day compare with the control group. Prolonged injection of arbutin (50 and 100mg/kg) considerably (P<0.0001) improved motor coordination compare to the disease group (Fig. 2a).

Fig. 5: Effect of Arbutin on a) motor coordination, b) locomotor activity, c) narrow beam walking activity in haloperidol-induced neurotoxicity in rats. Mean±SD is used to express the data. ^ap <0.0001 Halo vs control, ^bp<0.0001 Arb vs haloperidol, ^cp<0.001 Arb (100) vs Arb (50). Halo: Haloperidol, Arb: Arbutin

Effect of arbutin on locomotor activity in haloperidol-induced neurotoxicity in rats:

Acute treatment with haloperidol results in a reduction in locomotor activity significantly (P<0.0001) on the 21st day in comparison to the vehicle control group. Arbutin (50 and 100mg/kg), substantially (P<0.0001) improved locomotor activity in comparison to haloperidol-injected rats (Fig. 2b)

Effect of arbutin on narrow beam walking activity in haloperidol-induced neurotoxicity in rats:

Chronic treatment with haloperidol for 21 days shows a substantial (P<0.0001) reduction in the transfer latency on the 21st day in contrast with a control group. Continuously administration of arbutin (50 and 100 mg/kg) showed significantly (P<0.0001) improvement in transfer latency than the haloperidol injected group (Fig. 2c).

Effect of arbutin on MDA and nitrite in haloperidol-induced neurotoxicity in rats:

Acute haloperidol injection significantly (P<0.0001) increased in MDA and nitrite levels in the striatum then the control group. Arbutin (50 and 100mg/kg) treatment decreased MDA significantly (P<0.001) (Fig. 3a), and nitrite (Fig. 3b) levels in comparison with haloperidol-treated rats.

Fig. 3: Effect of Arbutin on a) MDA, b) nitrite, c) SOD, d) GSH and e) Cat in haloperidol-induced neurotoxicity in rats. Mean ± SD is used to express the data. ^ap <0.0001 Halo vs control, ^bp <0.001 Arb vs haloperidol, ^cp<0.05 Arb (100) vs Arb (50). Halo: Haloperidol, Arb: Arbutin

Effect of arbutin on antioxidants in haloperidol-induced neurotoxicity in rats:

Haloperidol injection reduced in the SOD, GSH, and catalase levels significantly (P<0.0001) in contrast with control group. Co-administration of arbutin (50 and 100 mg/kg) dramatically (P<0.001) enhanced SOD (Fig. 3a), GSH (Fig. 3b), and catalase (Fig. 3c) levels as compared to the haloperidol-treated group.

Effect of arbutin on TNF-α and IL-1β levels in haloperidol-induced neurotoxicity in rats:

Acute treatment with haloperidol showed a considerable (P<0.0001) increase in the TNF-α and IL-1β levels in comparison to the control group. Long-term administration of arbutin at the dose of 50 mg/kg as well as the dose of 100 mg/kg substantially (p<0.001) decreased the TNF-α and IL-1β in the striatum in comparison to the haloperidol group (Fig. 4).

Fig. 4: Effect of Arbutin on TNF-α and IL-1β levels in haloperidol-induced neurotoxicity in rats. Mean±SD is used to express the data. ap <0.0001 Halo vs control, bp <0.0001 Arb vs haloperidol, cp<0.0001 Arb (100) vs Arb (50). Halo: Haloperidol, Arb: Arbutin

DISCUSSION:

Arbutin is a bioactive hydroquinone glucoside obtained from Arctostaphyllos uva-ursi, it is known for its antioxidant and anti-inflammatory properties, which provide neuroprotective action against brain disorders ^22,23. The present study shows arbutin has the ability safeguard neurones from neurotoxicity induced by haloperidol-induced experimental rat model to investigate behavioral and biochemical changes in rats presenting TD-like symptoms¹². The earlier study revealed that haloperidol treatment increases the reactive oxygen species that induce mitochondrial dysfunction, leading to neurodegeneration^9,35,36. The haloperidol (1 mg/kg) treated group caused a significant increase in VCMs, tongue protrusion, and facial jerking in TD rats, similar to that observed in patients taking antipsychotic medication³⁷. While arbutin at low dose 50 and high dose 100mg/kg has potential to reverse the orofacial dysfunctions in haloperidol-treated rats. Evidence from prior research suggests that antipsychotic treatment like haloperidol enhances the generation of free radicals, which is associated with hyperkinetic movement³⁸. Furthermore, another study revealed that arbutin administration exerted a neuroprotective effect by improving motor function in MPTP-treated rats. Their results revealed that arbutin has the potential to reverse motor abnormalities³⁹. In this experiment, arbutin showed a significant increase in muscular coordination observed by using rotarod, locomotor activity by using an actophotometer, and gait abnormality by using the narrow-beam walk test in comparison with haloperidol-treated group.

Furthermore, several studies have found that haloperidol treatment causes dopamine receptor hypersensitivity by inhibiting the dopamine D2 receptors specifically located in the striatum. As a result, there is an increase in oxidative stress that leads to neurodegeneration^7,40,41. Another study reported higher levels of free radical formation in TD patients compared to those who do not have the illness⁴². According to the report, arbutin has antioxidant and anti-inflammatory properties which provide a neuroprotective effect against various neurodegenerative disorders^23,24. The present study found that arbutin treatment dramatically reduced TBARS and nitrite concentrations in rats treated with haloperidol. The increase in MDA showed the formation of lipid peroxidation, which is associated with TD pathogenesis. Previous study outcomes suggested that arbutin treatment has the capacity to reduce MDA and nitrite levels in rat brains subjected to streptozotocin treatment²².

Additionally, prolonged treatment with antipsychotic drugs altered the antioxidant enzyme activity and imbalanced the oxidative stress that is responsible for the progression of TD^7,43–45. Furthermore, another study found that arbutin has the potential to enhance the antioxidant enzyme activity by stimulating the Nrf2/HO-1 signaling pathway, which is responsible for the transcription of several antioxidant enzymes that provide neuroprotection against isolectin-induced multiple sclerosis in rats⁴⁶. In the current study, the arbutin treatment showed a considerable increase in antioxidant enzymes such as SOD, GSH, as well as catalase amounts in the brain of rats in comparison with disease group. This result indicates that arbutin can halt the pathogenesis of TD. Moreover, the prior researcher observed that the long-term arbutin treatment inhibit the generation of pro-inflammatory cytokines (such as TNF-α and IL-1β) showing neuroprotection against MPTP-treated PD rats²³. Furthermore, other research evidence revealed that prolonged antipsychotic treatment impaired the production of inflammatory markers that lead to the degeneration of striatal neurons, which is linked to the pathogenesis of TD⁴⁷. In this research, arbutin treatment significantly reduced the TNF-α as well as IL-1β levels which was found to be increased in haloperidol-treated rats. The study outcomes indicate that arbutin has a neuroprotective potential that reduces the behavioural and biochemical abnormalities in haloperidol induced TD rats. The present study indicates that arbutin has a neuroprotective effect; arbutin ameliorates the behaviour deficit in haloperidol-induced TD in rats. Besides that, arbutin administrations restored the antioxidant enzyme levels, alleviating oxidative stress and decreasing the pro-inflammatory cytokine levels in the rat’s brain and thereby providing a reason for arbutin could be used in the treatment of TD. The outcomes indicate that arbutin could be a better option to treat TD-like symptoms. Nevertheless, cellular and molecular studies are needed to explore and verify the potential neuroprotective mechanism of arbutin.

ACKNOWLEDGMENT:

Author would like to acknowledge the facility provided by the Central University of Punjab (CUPB), Bathinda, India. The authors would like to acknowledge Indian Council of Medical Research, New Delhi, India for providing a Senior Research Fellowship to Mr. Shubham Upadhayay to pursue his research at the Department of Pharmacology, CUPB. And also, Chitkara College of Pharmacy, Chitkara University, Rajpura, Patiala, Punjab, India for providing the necessary facilities to carry out this work.

AUTHOR CONTRIBUTION STATEMENT:

Reviewed the literature, and contribute to behavioural experiments and data acquisition: Gursewak Singh, Thakur Gurjeet Singh. Manuscript drafting, data collection, and statistical analysis: Shubham Upadhayay, Ashi Mannan. Manuscript editing, data interpretation: Uma Shanker Navik, Ashi Mannan. Concept design: Puneet Kumar. Critically reviewed the manuscript: Thakur Gurjeet Singh, Puneet Kumar. Supervision: Thakur Gurjeet Singh, Puneet Kumar.

All authors read the final version of the manuscript and approved the authorship list.

DATA AVAILABILITY STATEMENT:

The authors confirm that the data supporting the findings of this study are available in the article.

ETHICS APPROVAL:

Ethical clearance for conducting this study was obtained from Institutional Animal Ethics Committee (IAEC) at the Central University of Punjab, Bhatinda, India with protocol no CUPB/IAEC/2022/005.

CONFLICT OF INTEREST:

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

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Received on 02.03.2024 Revised on 11.11.2024

Accepted on 12.05.2025 Published on 08.11.2025

Available online from November 13, 2025

Research J. Pharmacy and Technology. 2025;18(11):5121-5127.

DOI: 10.52711/0974-360X.2025.00739

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