Neuroprotective effect of methanolic extract of Sargassum wightii on rotenone-induced parkinsonism-like symptoms in Wistar albino rats

Introduction Parkinson’s disease (PD) is the second most common neurodegenerative disease that mostly affects people 60 years and above. The cardinal features of PD are akinesia, bradykinesia, resting tremor, rigidity, postural instabilities, and is often associated with cognitive dysfunction with disease progression (1). The neuropathological hallmark of PD is the selective and progressive degeneration of dopaminergic neurons in Substantia Nigra pars compacta, resulting in dopamine deficiency in the striatum (2,3). http://www.herbmedpharmacol.com doi: 10.34172/jhp.2021.54


Introduction
Parkinson's disease (PD) is the second most common neurodegenerative disease that mostly affects people 60 years and above. The cardinal features of PD are akinesia, bradykinesia, resting tremor, rigidity, postural instabilities, and is often associated with cognitive dysfunction with disease progression (1). The neuropathological hallmark of PD is the selective and progressive degeneration of dopaminergic neurons in Substantia Nigra pars compacta, resulting in dopamine deficiency in the striatum (2,3).
Though the genetic and environmental factors pose the leading causes, other factors such as reactive oxygen species induced damage, excitotoxicity, mitochondrial dysfunctions, and inflammation-mediated cell injury have also been implicated in the etiology of PD. Over the decades, experimental evidence has focused on mitochondrial dysfunction as an important pathogenic component of PD (3,5). Dopaminergic neurons are more sensitive to oxidative stress. An increase in the metabolism of dopamine generates high levels of Reactive Oxygen free radicals that cause cellular damage (6,7). Rotenone, a natural pesticide, is a highly lipophilic and specific mitochondrial complex-I inhibitor. Therefore, it has been widely used as a mitochondrial poison model (7,8). On chronic administration, it also induces oxidative stress. Both mitochondrial inhibition and oxidative stress contribute to dopaminergic neuronal degeneration and the development of PD-like symptoms (9,10).
Levodopa has been considered as the gold standard drug therapy for PD, but its side effects on long term use are alarming. Recently, neuroprotective approaches with the use of antioxidants, antiapoptotic and anti-inflammatory compounds of natural sources have taken the upper hand in preventing disease progression in PD.
Over the past decade, considerable attention has been focused on researches on marine macroalgae as they offer enormous resources for novel compounds. The macroalgae (seaweeds) have been identified as the largest reservoir of structurally diverse, bioactive compounds and secondary metabolites like peptides, carotenoids, phenols, terpenoids, phlorotannins, flavonoids, complex polysaccharides, phytosterols, glycolipids, vitamins, and minerals. Hence the pharmaceutical and agri-food industries have shown the demand for seaweeds as a functional food. Moreover, scientifically seaweeds have been validated for their antibacterial, antifungal, antiinflammatory, anticoagulant, antioxidant, hypolipidemic, anti-diabatic, hepatoprotective and neuroprotective activities (11,14). Sargassum wightii, brown algae that shares similar phytoconstituents such as alkaloids, phenols, flavonoids, phlorotannins, has been proved as a powerful free radical scavenger (15). In our previous study, we observed the neuroprotective effect of S. wightii against haloperidol induced catalepsy (16). Thus, it is hypothesized that it has potential protective effect on other neurodegenerative diseases such as PD. This study aimed to evaluate the possible neuroprotective effect of methanolic extract of S. wightii on rotenone induced PD model in Wistar albino rats.

Animals
Male Wistar albino rats of 150-200 g body weight were maintained in the animal house of Roland Institute of Pharmaceutical Sciences, Berhampur, under ambient conditions of temperature (24 ± 1°C), relative humidity (45-55%) and 12:12 light: dark cycle. The animals were fed with standard pellet diet and water ad libitum and were allowed to acclimatize to laboratory conditions for 7 days before the day of the experiment. With prior permission from the Institutional Animal Ethics Committee (IAEC), (Regd No: 926/PO/Re/S/06/ CPCSEA), the experimental procedures were conducted in accordance with the Guidelines of Committee for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA) formulated by the Ministry of Fisheries, Animal Husbandry and Dairying, Government of India. All the experiments were conducted between 10.00 to 16.00 hours.

Plant material
The plant materials were collected as a gift sample from Micobiotech limited (Manufacturers of natural agricultural inputs) New Gundlav, Valsad, Gujarat, India. To prepare a methanolic extract of Sargassum, 40 g of powder sample was extracted with 400 mL of methanol by using Soxhlet's apparatus for 72 hours. The extract obtained was dried in the evaporator and stored at 4°C for further use. Different concentrations of drug solution were freshly prepared on the day of the experiment using 1% CMC suspension (17).

Drugs and chemicals
Rotenone, dopamine HCl, and glycyl glycine buffer were purchased from Sigma-Aldrich, USA. Nitroblue-tetrazolium chloride, dithionitrobenzoic acid, thiobarbituric acid, and trichloro-acetic acid were purchased from HiMedia Laboratories Pvt. Ltd. Mumbai, India. All other chemicals were of analytical grade and purchased from standard manufacturers.
Phytochemical screening of plant extract The methanolic extract of S. wightii was subjected to a phytochemical screening test in our earlier study and was found to contain polyphenols, terpenoids, tannins, flavonoids, and polysaccharides (16).

Acute toxicity test
The acute toxicity study was done in our previous study as per OECD Guidelines 423 [Limit test]. The extract at doses 5, 50, 1000, and 2000 mg/kg were given orally in a stepwise manner. The animals were observed for 24 hours. No mortality or behavioral changes were observed during the study period (16).
Experimental design Thirty Wistar albino rats were randomly divided into five groups (n=6) to receive treatments as follows: Group-I (Normal control) was administered with vehicle olive oil (1 mL/kg p.o). Group-II was treated with Rotenone (10 mg/kg p.o.) daily. Group-III rats (standard group) received levodopa and carbidopa combination (10 mg/kg orally) and rotenone (10 mg/kg p.o) (1,6). Group-IV (S. wightii 200 mg/kg + rotenone 10 mg/kg) and group-V (S. wightii 400 mg/kg + rotenone 10 mg/kg) were considered as the test groups. All the treatments were given orally for a period of 28 days. The drugs and vehicle were given one hour before rotenone administration. Behavioral tests were performed on days 0, 14, and 28 of the study. After the last behavioral assessments on day 28, rats were sacrificed by cervical dislocation and the brains were harvested quickly for the biochemical estimations.

Behavioral assessments
Anti-Parkinson's activity On chronic administration of rotenone, the animals developed Parkinson's-like symptoms such as tremor and akinesia. The severity of tremor was measured by giving different scores like no tremors (score 0), occasional tremors (score 1), moderate tremors (score 2), and continuous tremors (score 3). To measure akinesia, the tail of the animal was held in hand and the animal was allowed to move forward on his forelimbs. The number of steps taken by the fore-limbs of the animal was counted for three minutes (18,19).

Motor coordination
The motor coordination was evaluated by using the rotarod apparatus (Inco, Ambala, India). The performance was measured by the duration of animal stay on the rod in relation to the rod speed. The rats were allowed to adjust their postures and balance them on the rotating rods at different speeds. On the day of the experiment, the rats were placed individually on the rotating rod with a speed of 25 rpm, the fall-off time was recorded and the cut-off time was fixed at 180 seconds (20).

Spontaneous locomotor activity
The locomotor activity (horizontal activity) was measured using a digital photoactometer (Inco, Ambala, India). The animals were placed individually in the activity cage and acclimatized for 5 minutes. Interruption in the photo beams inside the cage by the movement of the animals represents activity count, which was recorded for five minutes (20). The chamber was swabbed with 10% ethanol each time to avoid animal odor.

Biochemical estimations Preparation of solubilised brain mitochondrial sample
To obtain a solubilized mitochondrial sample, the method of Berman et al, was followed (21). The rats were decapitated, and the whole brains were removed quickly, washed, minced, and homogenized at 4°C in 10 mL of isolation medium (225 mM mannitol, 75 mM sucrose, 5 mM HEPES, 1 mM EGTA, 1 mg/mL bovine serum albumin, pH 7.4), then centrifuged at 2000 g for 5 minutes. The pellets were resuspended with 10 mL isolation buffer and recentrifuged as above. The resulting supernatants were collected in separate tubes and centrifuged at 12 000 g for 8 minutes. The pellets, including the fluffy synaptosomal layer, were resuspended with an isolation medium and centrifuged at 12 000 g for 10 minutes to get brown-colored pellets. These pellets containing mitochondria without the synaptosomal layer were resuspended in 10 mL of isolated medium and recentrifuged as above. Finally, the mitochondrial pellets were resuspended with 50 μL of medium and used as the sample.
Mitochondrial nicotinamide adenine dinucleotide dehydrogenase (NADH) (Complex-I) enzyme activity The method of King and Howard was followed for the estimation of NADH dehydrogenase enzyme activity. It involves catalytic oxidation (releasing H + ) of NADH to NAD + with subsequent reduction of cytochrome C. The reaction mixture contained 0.2 M glycylglycine buffer (pH 8.5), 6 mM NADH in 2 mM glycylglycine buffer, and 10.5 mM cytochrome C. The reaction was initiated after the addition of the prepared solubilised mitochondrial sample and the absorbance was measured spectrophotometrically at 550 nm for 2 minutes band. The values were expressed as nM/mg protein (22,23).
Dopamine assay Determination of dopamine was done using highperformance liquid chromatography (reverse phase method), and the total brain dopamine content was expressed in micrograms per gram of tissue by using formula (24).

Monoamine (µg/g tissue) =AT/AS× CS× dilution factor
AT= Area under the curve for the sample, AS= Area under the curve for the standard, CS= concentration of the standard µg/mL and dilution factor=10.
Lipid peroxidation assay The quantitative analysis of lipid peroxidation was assayed by measuring the level of malondialdehyde (MDA) in the brain tissue following the method of Okhawa. The MDA activity was determined by measuring the absorbance of thiobarbituric acid reactive species spectrophotometrically (Shimadzu UV 1800) at 532 nm (25).

Superoxide dismutase assay
The superoxide dismutase (SOD) activity in brain tissue homogenates was assayed by measuring the inhibition of reduction of nitro-blue-tetrazolium following the method of Marklund et al and was expressed in terms of units/mg tissue (26).

Reduced glutathione assay
The reduced glutathione (GSH) of brain tissue homogenates was determined by the method of Sedlak et al. The principle was based on the reduction of Ellman's reagent by -SH groups of GSH to form 2-nitro-smercaptobenzoic acid. The absorbance of yellow colour imparted by nitromercaptobenzoic acid anion was recorded spectrophotometrically at 412 nm (Shimadzu UV-1800). The amount of GSH in tissue was expressed as μg/mg tissue (27).

Statistical analysis
The values were expressed as mean ± SEM. The data were analyzed for statistical significance applying ANOVA followed by Tukey's multiple comparison tests using GraphPad Prism 7.0. A probability values less than 0.05 were considered significance.

Results
Effect of methanolic extract of Sargassum wightii on rotenone induced changes on body weight Rotenone reduced the bodyweight of rats significantly at the end of the fourth week of treatment. Treatment with levodopa + carbidopa (10 mg/kg) body weight significantly prevented the rotenone-induced loss in body weight. Further treatment with S. wightii at 200 mg/kg and 400 mg/kg body weight also inhibited the rotenone induced loss in body weight after 28 days of treatment (P < 0.01) (Figure 1).

Behavioral indices Effect of methanolic extract of Sargassum wightii on rotenone induced akinesia and tremor
On the 14 th and 28 th days of rotenone administration, the rats showed a significant increase in akinesia score to 25.83 ± 3.38 and 17.83 ± 3.44, respectively compared with normal control rats (P < 0.001) (Figure 2A). In comparison to the rotenone control group, levodopa + carbidopa (10 mg/kg) body weight significantly prevented rotenone-induced increase in akinesia to 48.33 ± 4.40 and 60.33 ± 4.28 on the 14 th and 28 th days, respectively (P < 0.001). S. wightii 200 mg/kg treatment for fourteen days did not reveal any significant change in rotenone-induced akinesia, but on the 28 th day, it showed a significant decrease in akinesia (38.67 ± 3.92) (P < 0.01). S. wightii with 400 mg/kg body weight significantly reduced the akinesia both on 14 th (46.67 ± 3.27) (P < 0.01) and 28 th days (50.67 ± 4.28) of treatment (P < 0.001) (Figure 2A). However, on the 28 th day, only the higher dose of S. wightii (400 mg/kg) reduced the tremor score in rotenone-induced PD rats (P < 0.05) ( Figure 2B).

Effect of methanolic extract of Sargassum wightii on motor coordination
The motor coordination of the animals was tested using rotarod apparatus. In comparison to the control rats, rotenone-treated rats showed a significant decrease in motor coordination on the 14 th and 28 th days of treatment (71 ± 9.93 seconds and 26.8 ± 5.17 seconds, respectively). Levodopa + carbidopa (10 mg/kg) treated rats significantly prevented rotenone-induced changes in motor coordination activity significantly on 14 th day 124 ± 7.97 seconds (P < 0.001) and to a highly significant extent on 28 th day 159.2 ± 9.60 seconds (P < 0.001).
Sargassum wightii 200 mg/kg and 400 mg/kg body weight after 14 days of treatment did not show any significant change in motor activity as compared to the rotenone-treated control group. Nevertheless, S. wightii 200 mg/kg and 400 mg/kg increased the motor coordination activity after 28 days of administration to a highly significant level 94.80 ± 11.94 seconds and 122.6 ± 8.52 seconds, respectively compared to rotenonetreated group (P < 0.001) ( Figure 3A).

Effect of methanolic extract of Sargassum wightii on locomotor activity
The locomotor activity of the rats in rotenone-treated group was significantly reduced on the 14 th and 28 th Figure 1. Effect of Sargassum wightii on body weight. One-way ANOVA followed by Tukey's multiple comparison tests was applied for analysis. #P < 0.001 compared to the vehicle control group. **P < 0.01, ***P < 0.001 compared to the rotenone control group. days of treatment by 64.5 ± 7.54 seconds and 40.5±4.05 seconds compared to the vehicle control. Administration of levodopa + carbidopa (10 mg/kg) showed a significant increase in the locomotor activity both on the 14 th day (140±16.33) and 28 th day (182 ± 32.57) (P < 0.001) compared to the rotenone-treated group. S. wightii 200 mg/ kg treatment for fourteen days did not reveal a significant change in rotenone-induced locomotor activity, but on the 28 th day, it showed a significant increase in activity (114 ± 7.12) (P < 0.01). S. wightii with 400 mg/kg body weight significantly increased the locomotor activity both on 14 th (140.7 ± 14.9) (P < 0.01) and 28 th day (152.2 ± 12.19) (P < 0.001) ( Figure 3B).

Discussion
Rotenone, a naturally derived pesticide, acts as a specific mitochondrial complex1 inhibitor and induces oxidative stress by increasing the formation of reactive oxygen species by its prominent mitochondrial complex inhibition and impaired ATP production. Evidence suggests that rotenone-induced PD is a suitable model as it simulates the behavioral and neuropathological features of disease due to selective degeneration of dopaminergic neurons (28)(29)(30). Oxidative stress may play a greater role in the demise of dopaminergic neurons indirectly by activating intracellular, cell death-related molecular pathways (30,31). During the pathogenesis of PD, the production of reactive oxygen species damages the substantia nigra through lipid peroxidation, protein oxidation, and DNA oxidation. This phenomenon seems to be induced mainly by mitochondrial dysfunction, monoamine oxidase activation, or even by changes in the antioxidant defence system. Earlier studies postulated that the neurotoxicity induced by rotenone is a consequence of dopamine oxidation that produces free radicals and damages the dopaminergic neurons, which is the key point in the neuropathology of PD (32,34). Currently seaweeds Figure 2. Effects of Sargassum wightii on akinesia (A) and tremor (B). Statistical analysis was carried out using one-way ANOVA followed by Tukey's multiple comparison tests. # P < 0.001 compared to the vehicle control group. *P < 0.05, **P < 0.01, ***P < 0.001 compared to the rotenone control group.
have attracted food industries as nutraceuticals for their numerous bioactive components. S. Wightii, brown seaweed has been proved to possess antioxidant and anti-inflammatory activities in our earlier study. In this context, this study was an attempt to explore the potential neuroprotective effect of methanolic extract of S. wightii against rotenone-induced Parkinson's-like symptoms in rats. We used levodopa and carbidopa combination as the reference standard drug for comparison.
Rotenone administration (10 mg/kg) for 28 days, exhibited Parkinson's-like symptoms as evidenced from an increase in akinesia, tremor, and lack of motor coordination on rotarod test and deficit in locomotor activity in Photoactometer test. Our observations were in accordance with earlier studies (1,6). The reduced body weight of rotenone-treated group animal was seen in this study also corroborate with other reports. The reporters explained that this might be due to changes in liver enzymes levels, gastrointestinal dysfunction, and changes in neurotransmitter level (35,36). Mitochondria are a double membrane-bounded organelle residing in the cell cytoplasm involved in cellular energy metabolism, which may play a central role in survival and apoptosis. The bio-energetic dysfunction of it is an important pathogenic component in PD (37,38). In our study, rotenone depleted mitochondrial NADH dehydrogenase that explains rotenone-induced mitochondrial damage. Moreover, dopamine depletion reduced SOD and GSH levels and simultaneously enhanced lipid peroxidation in the rotenone-treated control rats. These may suggest that oxidative stress has been induced by rotenone (Table 1). In our observation, treatment with methanolic extract S. wightii both at 200 and 400 mg/kg reversed the rotenoneinduced changes in the body weight, motor coordination, Values are expressed as mean ± SEM, n=6. Statistical analysis was carried out using One-Way ANOVA followed by Tukey's multiple comparison tests. # P < 0.001 compared to the vehicle control group. **P < 0.01; ***P < 0.001 compared to the rotenone control group. Statistical analysis was carried out using one-way ANOVA followed by Tukey's multiple comparison tests. # P < 0.001 when compared to the vehicle control group. **P < 0.01, ***P < 0.001, test group as compared to rotenone control group.
locomotor activity, and akinesia; however, they were not comparable to levodopa and carbidopa combination. We also observed S. wightii, with both doses restored rotenone-induced dopamine and mitochondrial NADH dehydrogenase depletion (Table 1). Evidence supporting the oxidative stress hypothesis in the pathogenesis of PD suggests that increased lipid peroxidation and reduced antioxidants such as SOD and GSH are associated with neuronal cell death in Substantia nigra (39). In this work, methanol extract of S. wightii 400 mg/kg restored the oxidative stress biomarkers like MDA, SOD, and GSH levels, revealing its antioxidant property (Table 1). Thus, we can explain that the neuroprotective effect shown by S. wightii is possibly due to restoration of dopamine as well as mitochondrial function. Additionally, the prevention of neurodegeneration from oxidative stress in PD could be correlated to its potent antioxidant property. However, this research is in dearth of providing the evidence correlating the neuroprotective effect of S. wightii with other neurochemical markers specific to PD, hence addressing further research. Also, the phytoconstituents responsible for this beneficial effect of S. wightii need to be isolated to provide a clue in the direction of the drug development process.

Conclusion
Results of this study conclude that S. wightii possesses neuroprotective activity in the animal model of PDs indicating its potential role as a novel therapeutic candidate to manage neurodegeneration and prevent PD progression. However, it needs further evaluation in different experimental and clinical models to establish its efficacy and safety in long-term use.

Acknowledgments
Authors are thankful to Professor CS Maharana, HOD Pharmacology, MKCG Medical College, Berhampur, Odisha, India for providing laboratory facility to accomplish this work.

Authors' contributions
BR designed and guided the study. SR conceived the research idea and performed the experiment following all experimental guidelines, provided the data and wrote the first draft of the manuscript. AK carried out the literature search, IR and PKP performed statistical analysis and helped in the interpretation of results, SKB supervised the study. All authors reviewed and approve the final manuscript.

Conflicts of interest
Authors declare that there are no conflicts of interest.

Ethical considerations
The experiment was conducted upon receiving approval

Funding/Support
This research received no specific grant from any funding agency and was carried out on the expense of the authors.