INTRODUCTION:

Parkinson’s disease (PD) is the second most common age-related human neurodegenerative disorder next to Alzheimer’s disease. The incidence rate varies from 1 to 4% in individuals over 60 years¹.

The frequency of Parkinson’s disease increases with ageing ². PD is a complex neurodegenerative disease with motor and non-motor symptoms.

According to the United Kingdom Parkinson Disease Society Brain Bank, the clinical findings are based on the prevalence of two or three motor symptoms; bradykinesia and rigidity or muscle tremor at rest. Despite intense research, an effective therapy is not available to prevent the onset and the progression of the disease. The basic pathophysiology of PD results from the progressive and selective loss of dopaminergic neurons in the substantia nigra pars compacta (SNpc) with consequent depletion of dopamine in striatal projections, and other brainstem regions. Even though the mechanism of neurodegeneration is not fully understood, oxidative stress, free-radical generation, environmental toxins, and endogenous neurotoxins may be involved in the augmented loss of nigrostriatal dopamine (DA) neurons. This leads to disruption of the cerebral neuronal systems that are responsible for motor function³. The pathogenesis of Parkinson's disease includes abnormalities in cellular protein transport, interaction between proteins and protein aggregation. The neurochemistry has shown involvement of excitotoxicity and oxidative stress in cell death³. The oxidation hypothesis suggests that dopamine oxidative metabolism produces free radicals that could play role in the development or progression of PD⁴. The progressive striatal dorsoventral dopamine depletion leads to the cardinal motor signs of PD, bradykinesia, resting tremor, rigidity, and postural instability⁵. The rest tremor is a sign that distinguishes PD from other diseases although medical treatment is initially effective but may become ineffective later. Experimental animal models of tremor have primarily been applied to examine drugs with probable therapeutic value for PD tremor⁶. Often PD is idiopathic, whereas secondary causes include severe brain injury, followed by stroke, viral infections, environmental toxins like MPTP (1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine), and drug induced. Biochemical measurements and imaging modalities suggest that at least 70% decrease in striatal dopamine occurs before the onset of clinical Parkinsonism and it progresses over time.

The extent of these signs in PD has been revealed by some large studies. Moreover most of the PD patients may experience psychiatric symptoms at some point, in the disease. Depression and hallucinations are the most commonly described psychiatric symptoms, but many others like cognitive defects and dementia may occur. Studies have shown that psychiatric symptoms are often unrecognized in PD patients by the physicians and when they are recognized go undertreated. In early PD cognitive deficits have been described, and at least a third of PD patients develop condition like dementia. Along with reactive mood changes, changes in the neurochemicals also contribute to the development of mood symptoms in PD. Decrease as well as loss of dopamine neurons in the ventral tegmental area is the most possible neuropathological cause of mood symptoms in PD, since changes in the 5-hydroxytryptophan and noradrenaline systems occur. The current treatments of PD aim to increase the dopamine level in striatum to ameliorate the associated motor deficits. These include the precursor of dopamine (L-Dopa), agonists of dopamine, MAO-B inhibitors, catechol O methyl transferase inhibitors and NMDA receptor antagonists⁷. L-Dopa is a neurotransmitter; content of dopa in brain tissues is reduced due to blockade/conversion of tyrosine to L-Dopa. The L- isomer of dopa [3-(3,4-dihydroxyphenyl) alanine] is being used for symptomatic relief of PD⁸.

Rotenone is an insecticide and is found naturally in plants that belongs to the family of Leguminosae. It is also used in organic farming. Due to high lipophilicity, rotenone can readily cross the blood–brain barrier and enter all cells without depending on specific transporter. Rotenone mainly has affinity for mitochondrial complex I and inhibits it. Rotenone produces cytotoxicity by causing oxidative stress and by producing reactive oxygen species⁹. Rotenone selectively ablates the dopaminergic neurons in the nigrostriatal pathway in mice and develop Parkinsonism. The effects are similar to the behavioural manifestation and neurochemical mechanisms of PD, including mitochondrial dysfunction, apoptosis, and oxidative stress¹⁰. Findings from earlier studies have shown that the substantia nigra region is more vulnerable to damage by rotenone¹¹. Histological findings in the midbrain are correlated with impaired motor coordination response along with biochemical evidences¹². This might specify that the midbrain part responds highly to oxidative stress caused by rotenone. This vulnerability of the midbrain to rotenone-induced oxidative stress has been demonstrated in other studies¹³.

Embelin, chemically 2,5-dihydroxy-3-undecyl-1,4- benzoquinone is an active constituent of the fruits of Embelia ribes Burm (Family: Myrsinaceae). The vernacular names of this plant are Krimighna in Sanskrit, Vidang in Assamese, Marathi, Oriya, Bengali, Vavding in Gujarati, Vayavidanga in Hindi, Kannada, Babading in Kashmiri: Vizhalari in Malayalam, Babrung in Punjabi, Vayuvidangam in Tamil, Vayuvidangalu in Telugu and Baobarang in Urdu language.¹⁴

Embelin can cross the blood-brain barrier (BBB) and produce an effect on the CNS¹⁵. The structure of embelin has similarity with that of natural coenzyme Q10 i.e., ubiquinones and the role of the latter is well defined in various biochemical defensive mechanisms¹⁶. Embelin has many proved pharmacological effects like anxiolytic¹⁷, analgesic, anti-inflammatory¹², anti-depressant¹³, antidiabetic¹⁴, anti-bacterial¹⁵, anti-Huntington’s ¹⁶, anti-convulsant¹⁷, anti-oxidant¹⁸. A recent study also revealed that embelin ameliorates the effect of MPTP in a mouse model of PD¹⁹. In our earlier investigation the therapeutic potential of embelin and combination with L-Dopa revealed beneficial effects by reducing α-synuclein and various oxidative stress parameters in PD mice²⁰. The aim of the present investigation is to study the therapeutic potential of embelin monotherapy and embelin plus L-Dopa combination in rotenone induced PD in Swiss mice on neurobehavioral changes.

MATERIALS AND METHODS:

Materials:

Chemicals:

Rotenone was procured from TCI (Plau, USA) and olive oil from Thuruthel Drug Lines, (ERUMBA Pharmacy Kerala, India). Embelin was extracted in the phytochemistry facility of Department of Research and Development, Saveetha Institute of Medical and Technical Sciences, Chennai, India. Levodopa (L-Dopa) was obtained from SR Laboratories (Maharashtra, India) dimethyl sulfoxide (DMSO) from Pure Chemicals, and hydroxypropyl cellulose (HPC) from HIMEDIA, Mumbai, (India)

Experimental animals:

Adult male Swiss albino mice (25 to 30g; 8 to 12 weeks) were procured from Biogen animal facility (Bengaluru, India). Animals were housed under standard laboratory conditions and maintained on a natural light/dark cycle with free access to food and water ad libitum. Before initiating the experiments, the animals were acclimatized for seven days to laboratory conditions.

Ethical approval:

The animal experimental protocol was approved by the Institutional Animal Ethics Committee (Ref no. SU/CLAR/RD/001/2017), and the animal procedures were performed as per the guidelines of the Committee for the of Control and Supervision of Experiments on Animals (CCSEA, Government of India)

Experimental protocols:

The mice were randomly divided into seven groups of six animals each. The grouping was done one day prior to the start of the experiments. The duration of the experiment (injection and oral dosing) was 21 days. The tail of each mouse was marked with non-toxic permanent marker pen for identification. Animals were weighed separately and the body weights were noted. Group-1(Control) received olive oil (2mL/kg) by oral administration (p.o.) each day, Group-2 received 2.5 mg/kg rotenone by intraperitoneal route (i.p.) each day. Group-3 received L-Dopa 7.5mg/kg p.o. along with 2.5 mg/kg rotenone everyday by i.p. Group-4 received embelin 20mg/kg p. o along with 2.5mg/kg rotenone everyday by i.p. Group-5 received embelin 40mg/kg p. o along with 2.5mg/kg rotenone everyday by i.p. Group-6 received embelin 20mg/kg and L-Dopa 7.5mg/kg p. o along with 2.5mg/kg rotenone everyday by i.p. and group-7 received embelin 40mg/kg and L-Dopa 7.5 mg/kg p. o along with 2.5mg/kg rotenone everyday by i.p.

Preparation of rotenone stock solution:

50mg rotenone was weighed and dissolved in 1mL of DMSO. From this, an aliquot 0.2mL was taken and diluted with 19.8 mL of olive oil²¹.

Preparation of embelin and L-Dopa treatment drugs:

Embelin drug was prepared as described earlier in our published protocol²⁰. L-Dopa was weighed 15mg and dissolved in 20mL of 10% of hydroxypropyl cellulose. (HPC).

Rotarod apparatus:

Rotarod, an apparatus used to evaluate motor coordination and balance in rodents was employed in the study. Rotarod test is the measurement of duration of time spent by mice to maintain their balance on a moving rod which will assess the motor coordination. Mice were allowed to adjust their posture so that they could maintain the balance on a rotating rod at speeds of 5, 10 and 15 rotations per minute (RPM) three times per day at 30 minutes interval. The retention time (average) on the rotarod was calculated as explained in the previous studies²². The first test was performed 2.5 hour after the previous rotenone dose. The mice were placed separately on rotating cylinder suspended above a cage floor. By doing this, mice were trained to walk against the motion of a rotating drum at a constant speed of 12 RPM for a maximum of 3 min. Overall we performed four training trials per day at an interval time of one hour. Mice falling off throughout a training trial were put back on the rotating drum of rotarod apparatus. Rotarod apparatus was cleaned with ethanol and made dry before each trial. The mean latency to fall off the rotarod apparatus was noted and mice that stayed on the drum for more than 180 seconds were scored as 180 seconds.

Actophotometer Test:

Digital actophotometer is designed to study the locomotor activity in small animals like mice or rats. Optical sensors and emitters are used in digital actophotometer apparatus to record the horizontal movements. The animal locomotor activity was examined using this apparatus. The mouse was placed on actophotometer apparatus and the basal activity score of each animal was recorded over a period of 5 min. The control mice, PD mice and drug treated PD mice groups were explored, and activity score was recorded after 30 min and 60 minutes. Reduced locomotor activity score in the animals was taken as index of CNS depression.

Elevated Plus Maze (EPM):

The elevated plus maze apparatus is employed to evaluate the behaviour of rodents and has been accepted to measure anti-anxiety properties^23,24. The time spent in the closed and open arms by the rodents are assessed by this test which is measure of anxiety. Behaviour of the rodents in this task reflects a conflict between their preference for safe areas and innate motivation to explore novel environments. EPM relies upon mice inclination toward dark areas, natural fear of heights and open spaces and enclosed spaces.

The apparatus comprised of two open arms (51 × 12cm) and two closed arms (51 × 12 × 40.6cm) that extended form a common central platform (10 × 10cm), raised to 72cm above the floor. During the procedure each animal from various groups were separately placed in the central platform making them to face the open arm and permitted to explore the maze at will for five minutes. After completion of each session the apparatus was wiped and cleaned with ethanol. The test was recorded in a video camera and the behavioural analysis was carried out based on the number of entries into the open and closed arms. The percentage of time spent in the closed arms was used as an index of anxiety-like behaviour.

Open Field Test (OFT):

OFT measures and assesses general activity and exploratory behaviour in both rats and mice. OFT measures both quantity and quality activity in animals. The shape of open field apparatus is, usually circular, square or rectangular, which is enclosed with surrounding walls that prevent escape. The OFT is often used to evaluate the toxic, stimulant or sedative properties of different compounds. Thus, the OFT measures a number of facets of rodent behaviour rather than simple locomotion assessment. The mice were placed in the centre of a square (43 cm × 43 cm) arena and allowed to explore the arena for 5 min. The central square is used as some mouse strains have high locomotive activity and cross the lines of the test chamber several times in a test session. Also, the central square has adequate space surrounding it to give meaning to the central location as being distinct from the outer locations ²⁵ Time in rest, number of rearing’s and ambulatory episodes were recorded. The arena was cleaned with ethanol between each animal.

Statistical analysis:

The results are expressed as mean ± SEM. One Way ANOVA followed by Bonferroni ‘t’ test was used for comparison between the groups. P <0.05 was taken as statistically significant. Sigma Plot 14.5 (Systat, USA) was used for statistical analysis.

RESULT:

Administration of rotenone (2.5 mg/kg) for seven days to mice consecutively produced motor problems in rotarod test, decreased locomotor activity in actophotometer test, anxiety-like behaviours in the elevated plus maze, bradykinesia and postural instability seen in the open field test.

Evaluation of motor coordination using rotarod apparatus:

The mean time stays on rotarod in PD mice were significantly decreased in comparison to the control animals. PD mice treated with combination of embelin and L-Dopa had significantly increased the duration of stay on the accelerating rotarod in comparison with the other treated groups. (Table 1 and Figure1)

Group 2 (PD) mice showed 4.82-fold decrease in motor coordination compared to Group 1 (control group), whereas group 7 subjected to treatment with a combination of embelin (40 mg/kg) and L-Dopa along with rotenone (Group 7) showed only 1.1-fold decrease in motor coordination compared to control group in rotarod fall 1. PD mice showed 5.81-fold decrease in motor coordination compared to control group, whereas group 7 showed only 1.16-fold decrease in motor coordination compared to control group in rotarod fall 2. PD mice showed 5.77-fold decrease in motor coordination compared to control group, whereas group 7 showed only 1.11-fold decrease in motor coordination compared to control group in rotarod fall 3.

Table 1: Protective effect of embelin and embelin L-DOPA combination therapy on PD induced changes on motor coordination in mice. Values are mean + SE (n = 6 each).

Group	Rotarod fall 1 Time in Seconds	Rotarod fall 2 Time in Seconds	Rotarod fall 3 Time in Seconds
Control	163.1 ±1.7	160.8 ±0.9	156.5 ±1.4
Rotenone	33.8± 0.9	27.6± 0.4	27.1 ±0.4
Rotenone +Levodopa	94.5 ±1.5	88.6± 1.3	86.8 ±0.8
Rotenone+Embelin 20mg/kg	57± 0.5	54.8 ± 0.7	55.5± 0.7
Rotenone+Embelin 40mg/kg	83.5± 1.1	79.6 ±1.9	76 ±0.8
Rotenone+ Embelin20mg/kg+ Levodopa	118.1 ±0.8	116.5 ±1.0	117± 0.5
Rotenone+ Embelin40mg/kg+ Levodopa	145.8± 1.5	138.3± 1.4	140± 1.6

Evaluation of locomotor activity using actophotometer:

In locomotor activity it was found that there is significantly decreased (p<0.001) locomotor activity in PD mice group compared to control. The mean and SE of movement activity in groups 1 to 7 were 354.3± 4.8, 51.1± 2.4, 222.3± 2.3, 108.1 ±1.7, 176.3 ±2.2, 255.3± 2.6 and 288.5 ±2.7 respectively. (Table 2 and Figure 2) PD mice showed 6.86-fold decrease in locomotor activity as compared to control group, whereas group 7 showed only 1.2-fold decrease in locomotor activity compared to control group. The increase in the locomotor activity with group 7 is statistically significant (p<0.001) compared to PD mice group. The ‘F’ value (1346.979) and ‘P’ values (p<0.001) are by one-way ANOVA with Bonferroni multiple comparison test.

Figure 2. Protective effect of embelin and embelin L-Dopa combination therapy on PD induced changes in locomotor activity in mice

^aSignificantly different from control group.

^bSignificantly different from rotenone group.

^cSignificantly different from rotenone+L-Dopagroup.

Table 2: Protective effect of embelin and embelin L-DOPA combination therapy on PD induced changes in locomotor activity in mice. Values are mean + SE (n = 6 each).

Group	Movement activity
Control	354.3± 4.8
Rotenone	51.1± 2.4
Rotenone +Levodopa	222.3± 2.3
Rotenone+Embelin 20mg/kg	108.1 ±1.7
Rotenone+Embelin 40mg/kg	176.3±2.2
Rotenone+ Embelin20mg/kg+ Levodopa	255.3± 2.6
Rotenone+ Embelin40mg/kg+ Levodopa	288.5 ±2.7

Assessment of anxiety behaviour using EPM:

There is significantly increase (p<0.001) in anxiety behaviour in PD mice group compared to control group. The decrease in the anxiety behaviour following treatment showed that combination of embelin 40 mg/kg and L-Dopa is statistically significant (p<0.001) compared to PD mice group. (Table 3 and Figure 3). PD mice showed 3.25 -fold increase in time spent in closed arm to control group whereas group 7 showed 2-fold increase time spent in closed arm compared to control group. PD mice showed 4.4 -fold decrease in time spent in closed arm to control group whereas group 7 showed 1.19-fold decrease time spent in open arm compared to control group.

DISCUSSION:

Several epidemiological studies have suggested that pesticides and environmental toxins are inhibitors of mitochondrial complex I and are involved in the pathogenesis of PD^25-28_.

Being lipophilic in nature rotenone easily crosses biological membranes. Rotenone gets evenly distributed following systemic administration, throughout the body and gains access to all cells and tissues of the body. Earlier works have revealed that major inhibition of mitochondrial complex I and peripheral toxicity impair locomotion rather than nigrostriatal DA neurodegeneration²⁹. Rotenone selectively decreases the dopaminergic neurons in the nigrostriatal pathway in mice to develop PD. The effects are similar to the behavioral changes manifested and neurochemical abnormalities in PD in humans includes mitochondrial dysfunction, apoptosis and oxidative stress³⁰. Exposure of mice to rotenone develops PD symptoms and triggers nigrostriatal dopaminergic degeneration apart from α-synuclein-positive cytoplasmic inclusions. Earlier research studies have shown that substantia nigra is one of the most affected brain regions following rotenone toxicity³¹_.Daily i.p. injection of rotenone produces a more consistent lesion in the nigrostriatal pathway, and causes accumulation of α-synuclein and ubiquitin-positive inclusions and motor deficits. Even though neurodegenerative events and cascade in PD are not clear yet. Increased generation of free-radicals and increased oxidative stress may be related to decrease and loss of dopaminergic neurons. Pathophysiology of PD suggested that the mitochondrial complex I inhibition in substantia nigra plays consistent role in the development of PD. Previous study indicates that rotenone causes decrease as well as loss of dopaminergic neurons in the substantia nigral region and reduces striatal dopamine levels thereby accounting for motor deficits in mice³²_.

To evaluate the beneficial effects of embelin on PD induced motor dysfunction rotarod test was performed. In the current work, embelin either alone or in combination with L-Dopa treatment increased the latency. These results demonstrated that embelin (40mg/kg) along with L-Dopa (7.5mg/kg) rendered better neuroprotective effects that manifests

improvements of motor dysfunction in PD mice. These results suggested the neuroprotective effects of embelin in PD condition and hence could be due to the anti-oxidative properties of embelin with increased GST and GSH levels and decreased lipid peroxidation and malondialdehyde (MDA) content¹⁸_.

The locomotor activity of the mice was assessed using an actophotometer test. Decreased locomotor activity in PD groups shows the depression of central nervous system which was induced by rotenone. The drug rotenone induced immobility was a measure of depressant property. We found that embelin (20mg/ kg and 40mg/kg) therapy along with L-Dopa (7.5mg/kg) increased the locomotor activity by producing anti-depressant like property. Previous study explained the possible anti-depressant property of embelin and is due to inhibiting the reuptake of nor adrenaline and 5 hydroxytryptophan¹³_.

Anxiety and hypnosedation are mainly mediated in the CNS involving GABA receptor complex. It is well known that GABA is involved in other physiological functions related to behavior and hence implicated in various psychological and neurological disorders such as amyotrophic lateral sclerosis (ALS), schizophrenia, bipolar disorder, Parkinson and Alzheimer’s disease³³_. Anxiety-like behavior was expressed in elevated plus maze test. Rotenone administered mice spent more time in the closed arms rather than the open arms when compared with control and embelin treated groups. Decrease in anxiety could be confirmed from EPM test if there is an increase in the amount of time spent or entries in the open arms (time or entries in openarms/ total entries or entire time in open or closed arms). Rodents have an urge to explore a perceived threatening stimulus and also exhibit a natural aversion to brightly light areas. The outcomes based on two conflicting drives is anxiety. Increase in exploratory behavior in animals leads to reduction in anxiety. Increased anxiety suggests less locomotor motion and the animal willhave a preference to remain in the edges of the field³⁴_.Our EPM test result shows that anxiolytic effect of embelin is exhibited in a dose-dependent manner. The anxiolytic effect of embelin is same as of benzodiazepines by acting on benzodiazepine receptors enhancing the GABA action¹⁴_.

CONCLUSION:

The present work explored the neuroprotective efficacy of orally administered drug viz., embelin drug tested alone and in combination with Levodopa in PD model mice. The findings suggest that embelin when administered either alone and in combination with levodopa therapy significantly prevented the damage caused by rotenone on neuro behavioral changes. However, better effect was seen with embelin and L-Dopa (7.5 mg/kg) combination treatment rather than embelin alone treatments.

CONFLICT OF INTEREST:

The authors declare that they have no conflict of interest.

ACKNOWLEDGMENTS:

We thank the Department of Pharmacology, Saveetha Medical College, Chennai for providing the facilities to conduct behavioral studies. We also thank S. Madhankumar and S. Praveenkumar, for providing their assistance during animal experiments.

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Received on 04.06.2021 Modified on 09.04.2022

Research J. Pharm. and Tech 2023; 16(5):2107-2114.

DOI: 10.52711/0974-360X.2023.00346

Group	Closed arm Time in Seconds	Open arm Time in Seconds	Center Time in Seconds	Standing Time in Seconds
Control	31.3 ± 5.4	55.5 ± 5.2	33.1±8.2	2.5± 0.2
Rotenone	101± 3.1	12.6 ± 2.6	4.6±1.2	0.3 ±0.2
Rotenone +Levodopa	84.6 ± 1.9	27.3± 1.7	8±1.4	1±0.2
Rotenone+Embelin 20mg/kg	91 ± 2.2	19.3 ± 2	9.6±1.4	0.6 ±0.2
Rotenone+Embelin 40mg/kg	88.3 ± 2.028	19.3 ± 1.5	12.1±1.7	0.8 ± 0.3
Rotenone+Embelin20mg/kg+ Levodopa	83.6 ± 1.8	31± 0.8	15.3±2.4	1± 0.2
Rotenone+ Embelin40mg/kg+ Levodopa	49.6± 2.9	46.3± 1.6	24 ± 4	1.6±0.4

Group	Closed arm Time in Seconds	Open arm Time in Seconds	Center Time in Seconds	Standing Time in Seconds
Control	31.3 ± 5.4	55.5 ± 5.2	33.1±8.2	2.5± 0.2
Rotenone	101± 3.1	12.6 ± 2.6	4.6±1.2	0.3 ±0.2
Rotenone +Levodopa	84.6 ± 1.9	27.3± 1.7	8±1.4	1±0.2
Rotenone+Embelin 20mg/kg	91 ± 2.2	19.3 ± 2	9.6±1.4	0.6 ±0.2
Rotenone+Embelin 40mg/kg	88.3 ± 2.028	19.3 ± 1.5	12.1±1.7	0.8 ± 0.3
Rotenone+Embelin20mg/kg+ Levodopa	83.6 ± 1.8	31± 0.8	15.3±2.4	1± 0.2
Rotenone+ Embelin40mg/kg+ Levodopa	49.6± 2.9	46.3± 1.6	24 ± 4	1.6±0.4