NEUROPROTECTIVE EFFECT OF METHANOLIC EXTRACT OF SARGASSUM WIGHTII ON HALOPERIDOL INDUCED CATALEPSY AND TARDIVE DYSKINESIA IN ALBINO RATS

Objective: The present study was designed to evaluate the neuroprotective effect of methanolic extract of Sargassum wightii on haloperidol- induced catalepsy and tardive dyskinesia in Wistar albino rats. Methods: In this study, thirty Wistar albino rats were randomly divided into six groups. Gr-I served as control. Haloperidol (1 mg/kg intraperitoneally) was administered to rats of Gr-II to Gr-V for twenty-one consecutive days to induce catalepsy and tardive dyskinesia. Animals of Gr-II to Gr-V were orally administered with vehicle, levodopa carbidopa combination (30 mg/kg), Sargassum extract 200 and 400 mg/kg respectively. All the drugs and vehicles were given orally one hour before haloperidol injection for twenty one consecutive days. The cataleptic scores were recorded using standard bar test. Tardive dyskinesia was assessed in terms of vacuous chewing movement (VCM) and tongue protrusion (TP) scores. After behavioural testing, all animals were sacrificed on twenty-second day and various biochemical parameters like MDA, SOD and GSH were estimated in brain tissue. Results: Chronic administration of haloperidol significantly increased cataleptic scores, VCM and TP scores. (p<0.001) Sargassum wightii extract (400 mg/kg) significantly inhibited haloperidol-induced catalepsy, VCM and TP (p<0.001) Haloperidol increased MDA and decreased SOD and GSH in brain tissue to a highly significant extent (p<0.001) Sargassum extract at 400 mg/kg also significantly reversed the haloperidol-induced alteration in brain oxidative stress markers. Conclusion: Sargassum wightii inhibits haloperidol-induced catalepsy and tardive dyskinesia. Thus it may be used as a unique therapeutic adjunct for the prevention of neuroleptic-induced extrapyramidal symptoms, however, it has to be explored more. to its promising antioxidant activity. Further study is required in other experimental and clinical models of PD to elucidate the exact mechanism of neuroprotective activity of S wightii . In the present investigation, the isolation of individual phytochemicals and their effects on biogenic amines in the brain could not be done which directs future research.


INTRODUCTION
Neuroleptics used in the treatment of schizophrenia and other psychiatric illness are very often associated with extrapyramidal side effects like catatonia and tardive dyskinesia [1,2]. Haloperidol, a typical antipsychotic produces extrapyramidal syndrome (EPS) like Parkinsonism, due to a blockade of D2 receptors within the striatum and reduced dopaminergic transmission [3,4]. It produces a behavioural state of catalepsy and movement disorders like akathisia, dystonia, and at last, chronic tardive dyskinesia [5]. Hence haloperidol-induced catalepsy is considered as a robust model in rodents to evaluate nigrostriatal functions [6]. Evidence from many experimental and clinical studies also suggests that neuroleptics induce oxidative stress and cell death. Even lipid peroxidation byproducts in blood and cerebrospinal fluid are increased in patients with tardive dyskinesia [7,8].
Marine algae or seaweeds have created a significant place in pharmaceutical and biomedical fields because they are recognized as the rich source of bioactive compounds and secondary metabolites like peptides, carotenoids, phenols, terpenoids, phlorotannins, flavonoids, fucoidans, phytosterols and glycolipids [9,10]. Emerging researches have scientifically proved the anti-inflammatory, anticoagulant, antimicrobial, anticancer, antiviral, antioxidant, hypoglycaemic, antiulcer, neuroprotective and hepatoprotective effects of some of the seaweeds [11][12][13][14]. Several study reports have shown that the sulphated polysaccharide of some algae possesses free radical scavenging activity which might normalize lipid peroxidation [15,16]. The chemical composition of Sargassum has been extensively studied however literature on its bioactivity is scarce. One earlier study report has shown that some of the constituents of Hypnea musciformis (one of the brown algae) raise the level of dopamine and its metabolites that alleviate the symptoms in Parkinsonism [17]. Hence it may be hypothesized that Sargassum wightii, another species of brown algae containing similar phytoconstituents could have a beneficial effect in Parkinsonism (PD). Interestingly, over the past decade, there is an amazing rise in researches on marine algae. But to our knowledge, scientific data on the neuroprotective activity of Sargassum wightii against PD are lacking. To bridge this knowledge gap, the current study was designed to evaluate the potential benefits of methanolic extract of brown seaweeds Sargassum wightii on haloperidol-induced catalepsy and tardive dyskinesia in wistar albino rats.

Animal
Wistar albino rats of either sex (100-150g) were procured from Saha Enterprise, Kolkata and maintained in the animal house of Roland Institute of Pharmaceutical Sciences, Berhampur. Animals were housed and fed with standard pellet diet and water ad libitum and maintained under standard conditions temperature (24±1 °C), relative humidity (45-55%) and 12:12 light: dark cycle. The animals were kept under observation in the laboratory and allowed to acclimatize for one week before experimentation. With prior permission from the Institutional Animal Ethics Committee (IAEC), (Regd. No: 926/PO/Re/S/06/CPCSEA), Approval No-86/IAEC/ RIPS/07.04.2017, handling and care of animals during experimentation were followed as per CPCSEA guidelines. The experiments were conducted between 10.00 to 16.00h.

Plant material
The plant material was a gift sample from Microbiotech limited, Gujrat India. The dried samples were coarsely ground to a fine powder using electrical blender before extraction. To prepare the methanolic extract of Sargassum, 40 gm of powder sample was extracted with 400 ml of methanol by using soxhlet's apparatus for 72h. The extract obtained was dried in an evaporator and stored at 4 °C for further use [18]. Different concentration of drug solution was freshly prepared on the day of the experiment using 1% CMC suspension.

Chemicals
Haloperidol was purchased from Sigma, Aldrich USA; Levodopa and carbidopa combination: Glaxo Smithkline Pharmaceuticals, India; Carboxymethyl Cellulose(CMC), Thiobarbituric Acid (TBA), Trichloroacetic Acid (TCA), Dithiobis Nitro benzoic Acid (DTNB), phosphate buffer and all other chemicals used were of analytical grade and obtained from Himedia Laboratories Pvt Ltd, Mumbai, India.

Phytochemical screening of plant extract
The methanolic extract of S wightii was subjected to phytochemical screening test by the method described earlier [19] and it revealed the presence of polyphenols, terpenoids, tannins, flavonoids, polysaccharides like glycolipids, etc.

Acute toxicity test
Acute toxicity study was done as per OECD Guidelines 423 [Limit test]. The extract at doses 5, 50, 1000, 2000 mg/kg were given orally in stepwise form. The rats were observed for 24 h. There was no mortality or behavioral changes observed during the study period [20].

Experimental design
Haloperidol (1 mg/kg) was injected intraperitoneally for 21 d to induce catalepsy and tardive dyskinesia [21]. The test drugs and vehicles were given orally for 21 d, one hour before haloperidol injection. Thirty albino rats were randomly divided into five groups (n=6 in each group) to receive treatments as follows.
Group-II (disease control) received 1% CMC.+Haloperidol Group-III (standard) was treated with L-Dopa, carbidopa combination (30 mg/kg)+Haloperidol [21] Group IV and V animals (test groups) received the methanolic extract of Sargassum wightii at the doses of 200 and 400 mg/kg respectively and were co-administered with Haloperidol (ip) Then all the rats were subjected to behavioral and biochemical assessments. On the 22 nd day, following behavioral assessment, the animals were sacrificed by cervical dislocation. The brains were removed, the forebrain was dissected and a 10% (w/v) of brain tissue homogenate was prepared in 0.1M phosphate buffer (PH-7.4)

Haloperidol-induced catalepsy
In this model, haloperidol 1mg/kg/ip was administered daily for 21 successive days to elicit a moderate to the high degree of catalepsy and it was scored as a measure of reduced ability to move and failure to maintain the correct posture using standard bar test. [22] Catalepsy was assessed in terms of time for which the animal maintained an imposed posture with both the front limb raised and resting on the 9 cm height wooden bar and scored accordingly. The endpoint of the catalepsy was considered when both the front paws were removed from the bar. If the animal maintained this position for 20 s or more it was said to cataleptic, given one point and every further 20 s was given more extra points (numbers). A cut-off time 300 sec was applied for scoring. The severity of catatonia was observed on the 7 th , 14 th and 21 st days of haloperidol and test drug administration [23].

Haloperidol-induced tardive dyskinesia
For this experiment, 24 h after the last test dose (on the 22 nd day), the animals were placed individually in small cages (30 x 20 x 30 cm). Then each animal was allowed to acclimatize to the observation cage for 10 min. The vacuous chewing movement (VCM) and tongue protrusion (TP) were observed for 5 min and scored. VCM was referred to as single mouth openings in the vertical plane not directed toward physical material. Individual tongue protrusions during oral dyskinesia were preceded by visible retraction of the tongue. If tongue protrusions or vacuous chewing movements occurred during a period of grooming, they were not taken into account [24].

Lipid peroxidation assay (TBARS)
Lipid peroxidation was assayed by measuring the level of malondialdehyde (MDA) in the brain. Malondialdehyde was determined by measuring thiobarbituric reactive species using the method of Okhawa et al. 1996. In which the thiobarbituric acid reactive substances react with thiobarbituric acid to produce a red colour complex having peak absorbance at 532 nm [25].

Reduced glutathione assay
Reduced glutathione (GSH) was determined by the method of Sedlak J et al. 1968. The procedure is based on the reduction of Ellman´s reagent by-SH groups of GSH to form 2-nitro-s-mercaptobenzoic acid, the nitromercaptobenzoic acid anion has an intense yellow color which can be determined spectrophotometrically. (Shimadzu UV-1800) The amount of glutathione in tissue is expressed as μg/mg tissue [26].

Superoxide dismutase assay
The enzyme activity of superoxide dismutase in brain tissue was assessed using the method of Marklund and was expressed in terms of units of SOD activity/mg of tissue [27].

Statistical analysis
The observations were shown as mean±SEM. The data were analyzed using One-Way ANOVA followed by Tukey's post hoc test. Data analyses were performed using statistical software, Graph pad Prism 7. A probability value of less than 0.05 was considered as a minimum level of significance.

Phytochemical screening
The phytochemical screening of Sargassum extract revealed the presence of flavonoids, phenolic compounds, sterols, tannins, saponins, and glycolipids.

Acute toxicity test
On acute toxicity test, up to 2000 mg/kg body weight, no behavioral changes or mortality were observed within the study period.

Effect of methanolic extract of S wightii (SWE) on haloperidolinduced catalepsy
In this experiment, Haloperidol induced cataleptic score was significantly increased on 7 th , 14 th and 21 st days of treatment in comparison to that of the control group (p<0.001). Standard drug Ldopa-carbidopa (30 mg/kg) decreased cataleptic score significantly on the 7 th , 14 th and 21 st days of treatment as compared to that of haloperidol treated group on respective days. (p<0.001) However, pre-treatment with the test drug (SWE) at 200 mg/kg and 400 mg/kg reduced the cataleptic score to a highly significant extent only on the 14 th and 21 st days of observation when compared with the haloperidol control group on respective days. (p<0.001) table 1

Effect of administration S wightii on haloperidol-induced tardive dyskinesia in rats
On chronic administration, haloperidol increased the VCM and TP on the14 th and 22 nd days of treatment to a highly significant extent in comparison to that of the normal control group (p<0.001). With L dopa-carbidopa 30 mg/kg, the reference standard drug treatment, there was a highly significant decrease in the Haloperidol induced VCM and TP throughout the study period. (p<0.001) Sargassum wightii 200 mg/kg reduced the haloperidol-induced VCM and TP significantly only on the 22 nd day of treatment as compared to that of Haloperidol control. (p<0.01) However, pre-treatment of S wightii 400 mg/kg reduced VCM and TP to a highly significant extent both on the 14 th and 22 nd days of treatment when compared with the haloperidol treated control group (table 2).   Values are expressed as mean±SEM, n=6. One way-ANOVA followed by Tukey's multiple comparison tests was applied for analysis. # P<0.001 when compared with the vehicle group. *: p<0.05 **: P<0.01; ***: P<0.001 test group vs haloperidol control group.

Effect of methanolic extract of S wightii (SWE) on haloperidolinduced changes in various biochemical parameters
On chronic haloperidol administration, the TBAR level in the brain was increased to a highly significant extent with a concomitant decrease in GSH and SOD in comparison to that of the control group (p<0.001).
When compared with the haloperidol treated control group, L dopacarbidopa (30 mg/kg-Gr III), as well as S wightii (400 mg/kg-GR V), significantly reduced the haloperidol-induced increase in TBAR with a concurrent significant rise in GSH and SOD. fig. 1, 2, 3.

DISCUSSION
Haloperidol induced catalepsy (HIC) is a widely accepted animal model to test the effect of drugs modulating the extrapyramidal side effects of antipsychotic drugs [22,28]. Haloperidol, a non-selective dopamine antagonist provides an experimental model in rodents simulating PD in human beings. It blocks dopamine receptors and interferes with dopamine transmission in the striatum. Thus the drugs which increase the dopamine transmission in the nigrostriatal pathway and inhibit HIC. Moreover, evidence in support of the "free radical hypothesis" proposes that the free radical byproducts of dopamine metabolism impart neurotoxic effects at basal ganglia and substantia nigra which explains one of the mechanisms involved in the process of neurodegeneration in neuroleptic-induced tardive dyskinesia and PD [29,30].
In the present study, chronic administration of haloperidol ( [24]. Oxidative stress generated as a result of mitochondrial dysfunction plays an important role in the pathogenesis of PD. Lipid peroxidation, a sensitive marker of oxidative stress impairs membrane function, distorts structural integrity and inactivates several membrane-bound enzymes present in all biological membranes. Superoxide dismutase (SOD) helps in neutralizing the free radical-induced toxic effects. Even depletion of glutathione is a positive correlate of the extent of neuronal loss. Evidences show that a rise in MDA, decrease SOD and depletion of GSH in the brain result in neuronal loss (substantia nigra) and are important factors of the etiology of PD [34].
In the present study, haloperidol-treated animals exhibited an increase in the level of lipid peroxidation and decreased level of protective antioxidants such as GSH and SOD, suggesting a possible free radical generation. Previous studies have also reported that haloperidol-induced catalepsy is associated with oxidative stress [23,24,30] Treatment with methanolic extract of S wightii reversed the haloperidol-induced changes in oxidative stress markers like MDA, GSH and SOD significantly ( fig. 1,2,3). Our observations are in accordance with that of Yuvraj et al., 2014 who have shown the antioxidant activity of S wightti in an in vitro model [17]. Since the neuroleptic-induced extrapyramidal syndrome is correlated with an increase in free radical production it can be assumed that the antioxidant property of SWE could be another protective factor in the prevention of neurodegeneration in PD. Such correlation was also made by Perwez et al. who have shown the neuroprotective effect of Mentha arvensis against haloperidol-induced catalepsy in mice [23].
Studies on neuroprotective and antioxidant activities of Sargassum species have substantiated the role of various phytoconstituents. Presence of phenols, phytosterols, flavonoids, carotenoids, fucoxanthin, polysaccharides (fucoidan) and tannins in Sargassum (seaweeds) contribute to their promising antioxidant, antiinflammatory and neuroprotective properties [35][36][37]. The plant of our research interest, Sargassum wightii contains the same phytoconstituents. Hence its neuroprotective effect observed in our research could also be attributed to its promising antioxidant activity. Further study is required in other experimental and clinical models of PD to elucidate the exact mechanism of neuroprotective activity of S wightii. In the present investigation, the isolation of individual phytochemicals and their effects on biogenic amines in the brain could not be done which directs future research.

CONCLUSION
Our study suggests that the Sargassum wightii inhibited the extrapyramidal features and reversed the altered oxidative stress parameters induced by haloperidol. Hence it can be a novel approach for the prevention of extrapyramidal side effects of neuroleptics. The naturally occurring phytochemicals present in Sargasssum wighitii possessing neuroprotective and antioxidant properties with little toxicities pose a new insight for further research in this field.

ACKNOWLEDGMENT
The author is thankful to Roland Institute of Pharmaceutical Sciences and Department of Pharmacology, M K C G Medical College, Berhampur, Odisha for providing necessary facilities for this research work.

AUTHORS CONTRIBUTIONS
The corresponding author, Dr. B Rath designed the work and prepared the manuscript. Mrs. Sradhsini Rout performed the experiment and collected data, Dr. S K Bhattamisra supervised, Dr. A Kumar supported in the extraction and phytochemical analysis part of this work. Dr. S Rath performed the statistical analyses, reviewed the manuscript and edited.