Vidhyabharti college of Pharmacy, camp road, Amravati 444601, Maharashtra India
The present study investigated the neuroprotective effect of ethanolic extract of Pterocarpus marsupium (EEPM) on haloperidol-induced catalepsy in experimental animals. Phytochemical screening of EEPM revealed the presence of carbohydrates, glycosides, phenols, phytosterols, saponins, tannins, and flavonoids. Male albino mice were divided into six groups (n=6) and subjected to various drug treatments for 21 days. Catalepsy was induced by administering haloperidol (1 mg/kg, i.p.) and assessed using the bar test and actophotometer. EEPM was administered orally at doses of 100, 200, and 400 mg/kg, 30 minutes prior to haloperidol. The standard group received levodopa (20 mg/kg) with haloperidol. Behavioral tests were performed on the 7th, 14th, and 21st day of dosing. EEPM significantly attenuated haloperidol-induced catalepsy in a dose-dependent manner, with the highest dose (400 mg/kg) producing a statistically significant improvement in activity counts by day 21. The neuroprotective effect of EEPM may be attributed to the presence of bioactive compounds, including flavonoids and phenolic compounds, which are potent antioxidants. The results suggest that EEPM possesses neuroprotective and anti-cataleptic properties, potentially due to its antioxidant and anti-inflammatory effects. Further research is needed to elucidate the molecular mechanism of EEPM in neuroprotection and to isolate the individual constituents responsible for its therapeutic potential in the treatment of Parkinson's disease.
Parkinson's disease (PD) is the second most common aging-related neurodegenerative progressive condition after Alzheimer's disease (AD) With a frequency of 1-2% in those over 65. It affects the basal ganglia circuitry. Clinical recognition of this illness is based on four cardinal symptoms: bradykinesia, postural instability, stiffness, and resting tremor It is defined by a large-scale degeneration of dopamine neurons in the substantia nigra pars compacta, which affects target cortical and limbic locations in addition to the striatum Parkinson as paralysis agitans (1817): also referred to as "shaking palsy" Numerous factors can lead to neurodegeneration, such as the inhibition of mitochondrial complex-1, the interplay between genetic and environmental factors, exposure to metal-containing environmental contaminants, problems with proteasomal function, and the activation of microglia Catalepsy is a condition characterized by inactivity, decreased responsiveness to stimuli and a tendency to maintain an immobile posture. Catalepsy constitutes a neurological illness of persistent rigidity of the muscle and a lack of motion where one's limps sustain and in appropriate, rigid state [5] It may be associated with the nervous system drug toxicity, psychotic disorders and other conditions[6] Catalepsy is the neurodegenerative disease of unknown etiology and characterized by motor symptoms of tremor, rigidity, bradykinesia, and postural instability[7] Catalepsy is characterized by an abnormal basal ganglia activity. Non motor comorbidities, such as cognitive impairments (the comorbidity of anxiety and depression like Parkinson’s disease) are likely the result of an intricate interplay of multi-system degenerations neurotransmitter deficiencies extending beyond the loss of dopaminergic nigral neurons [8] The disorder promoted a diminished sensitivity for discomfort and a deceased reaction to stimuli from the environment. The typical symptoms of catalepsy comprise a somnolent State with minimal voluntary motion and responsiveness as well as remarkable muscular flexibility. Because of the muscle flexibility, it is possible to mold the entire body and its parts into distinctive postures that can be preserved for a prolonged amount of time[9]A state of slippery mobility and tonic paralysis in any number of areas of the body has been referred to as catalepsy. It is commonly referred to as paralysis or somatoform separation and is frequently seen to take place as a reaction of defense to stress in individuals as well as animals. The plant Pterocarpus marsupium Roxb. belonging to the family Fabaceae is popularly known as Indian Kino tree or Malabar Tree in English , Vijayasar or Bija in Hindi and Asana in Sanskrit. In the Ayurvedic medical system, P. marsupium is a plant medication that is a member of the "Rasayana" class. It is primarily found in the states of Orissa, Bihar, Madhya Pradesh, and Gujarat. It is a tree of moderate to great size. It is between 15 and 30 meters high. Pterosupin, pterostilbene, liquirtigenin, isoliquiritigenin, epicatechin, kinoin, kinotannic acid, kino-red beta-eudesmol, carsupin, marsupol, marsupinol, and other phytoconstituents are among the many that are significantly concentrated in it. it has antipyretic, anthelmintic, and liver-tonic properties, and is beneficial for all body ailments and styptic vulnerants. It is also effective for biliousness, griping, opthalmiya, boils, and urine discharges. The heartwood and root of the plant shows presence of chemical constituents[16] like carbohydrates, proteins, alkaloids, flavonoids, polyphenols, pentosans, pseudobaptogenin, pterosupin, pterostilbene, liquiritigenin, isoliquiritigenin, garbazol, 5 deoxy kaempferol, p- hydroxy benzaldehyde, b- eudesmol, erythrodirol- 3- mono acetate, marsupol, irisolodinone -7 rhamnopyranose. Due to the Presence of flavonoids, Polyphenols and Tannins. The present study was planned to Evaluate Neuroprotective effect of Pterocarpus Marsupium on Haloperidol induced catalepsy in Experimental Animal.
MATERIALS AND METHODS
Animal and Housing condition
The experiment is performed on albino mice (weighing 25-30gm), which are obtained from the animal house of Department of Pharmacology. Vidyabharati college of pharmacy, Amravati.( 1504/PO/RE/S/11/CPCSEA). All the animals are acclimatized to the animal house prior to use. They are kept in cages in animal house with n 12 h light: 12 h dark cycle. Animals are fed on pellets and top water ad libitum. The care and handling of mice were in accordance with the internationally accepted standard guidelines for use of animals (CPCSEA).Permission und approval animal studies were obtained from the Institutional Animal Ethics Committee (IAEC) of Vidyabharati college of Pharmacy, Amravati. SG) Amravati University.
PLANT COLLECTION IDENTIFICATION AND EXTRACT PREPERATION
Collection and Authentication of plant
The heartwood of Pterocarpus Marsupium was collected from Chandrapur district, Maharashtra, India. The plant were identified and authenticated by prof L.P Khalid , Department of Botany, Vidyabharati Mahavidyalaya, Amravati. Heartwood were cleaned, dried in shade.
Extraction Process
The heartwood of Pterocarpus Marsupium was processed by washing with clean water, air-drying, pulverizing, and sieving through a 0.3 mm sieve. tool consists of several parts including a heat round bottom flask, Soxhlet extractor, and condenser. The solid coarsely powdered were placed in thimble and placed in an extractor. The bottom end of the extractor was connected to a round bottom flask containing a solvent (Ethanol 1000ml was chosen as the solvent), and was connected to a reflux Condenser. Process was repeated until the all the material extracted from the solid leaves powder.
The percentage yield of the extract was calculated and the extract was then subjected to different phytochemical tests.
DRUGS AND CHEMICALS
Haloperidol injection IP 5mg/ml (RPG Life sciences limited) purchased from local pharmacy Chandrapur, Maharashtra 442401. Levodopa and carbidopa tablet (syndopa Plus) from sun pharma laboratory.
PHYTOCHEMICAL SCREENING
Qualitative Phytochemical investigation
Qualitative chemical tests were conducted in order to identify the various phytoconstituents Present in Pterocarpus Marsupium
PREPERATION OF DOSES
Haloperidol was diluted to 1 mg/10 ml with distilled water. Two different concentrations ( 100mg/kg, 200 mg/kg, and 400 mg/kg) of the EEPM were prepared by dissolving the extracts in distilled water. All solutions were freshly prepared at the time of administration to the animals. Extract solution and vehicle (0.9% NaCl) were given orally and inducing drug (Haloperidol) intraperitoneally & standard drug (Levodopa and carbidopa) orally.
EXPERIMENTAL PROCEDURE
Male albino mice (Swiss strain) weighing 25-30 g will be house under standard laboratory conditions of light and dark cycle (12:12), in groups of six each. The animal had free access to food and water. Following one-week acclimatization period, mice were randomly divided into six groups, each group containing six animals (n =6), and subjected to various drug treatments for 21 days. Ist group is control group administered with saline 1ml i.e. animal kept as it is without any drug treatment. The IInd group is administered with inducing agent i.e. Haloperidol 1mg/kg intraperitonially for 21 days. catalepsy was measured by performing bar test. This procedure was performed 30 min after the administration of haloperidol at 30 min intervals until 120 minutes The IIIrd group i.e. standard group receive Levodopa 20mg/kg with haloperidol 1mg/kg. The group IVth, Vth and VIth received drug extract orally at dose 100mg/kg 200mg/kg and 400mg/kg dose, respectively for 21 days test drug extract were given 30 min prior to the haloperidol administration. All the Behavioural test was Performed at 7th, 14th and 21th day of dosing.
BEHAVIOURAL STUDIES
BAR TEST: Catalepsy was assessed as the time during which the mouse maintained an imposed with both forelimbs extended and rested on a 4 cm high wooden bar (1 cm in diameter). The endpoint of catalepsy was considered to occur when both forepaws Were removed from the bar or if the animal moved its head in an exploratory manner. A cut-off time of 180s was applied. Between the 2 determinations, the animals were returned their home cages. All the observations were made in a quiet room at 23-25°C As for the scoring method, if the animal maintained the imposed posture for at least 20s it was considered to be cataleptic and the time was recorded in seconds. The test was performed on the 7th, 14th and 21th day of dosing.
Actophotometer Test : An actophotometer was used to assess the horizontal & vertical spontaneous moments of the animal. Set up comprises of an electronic unit and infrared beam chamber complete with two sets of sensor arrays for horizontal & vertical activity the animal was placed into metal chamber for 5 min. the moment makes inside the chamber interrupt one or more infrared beams. The beam interruptions are counted and recorded by the electric devices. The test was performed on the 7th, 14th and 21th day of dosing for testing, each mouse was individually placed in an activity cage for 5 min after 30, 60, and 120 min of dosing, and total locomotor activity was recorded.
STATASTICAL ANALYSIS
All results were represented as mean ± SEM (standard error of mean). The statistical significance between groups was evaluated by one- and two-way analysis of variance (ANOVA) using Turkeys post-tests & Dunnetts test via Graph pad prism version 10.4.2
RESULT
1.Phytochemical Screening
Table No 1: Phytochemical screening
|
Sr no |
Chemical Constituents |
Results |
|
1 |
Alkaloids |
- |
|
2 |
Flavonoids |
+ |
|
3 |
Carbohydrate |
+ |
|
4 |
Tannins |
+ |
|
5 |
Glycosides |
+ |
|
6 |
Phenols |
+ |
|
7 |
Saponins |
+ |
|
8 |
Steroids |
- |
Where, indicates (+) Present, (-) Absent.
Phytochemical testing carried out to find out the secondary metabolite because secondary metabolic possess biological activity. Phytochemical studies of Pterocarpus Marsupium performed for the presence. of Carbohydrate, Glycosides, Phenols, Phytosterols, Saponins, Tannins, Flavonoids.
2 Pharmacological Screening:
Table No 2: Effect of Extract of EEPM On Catalepsy Bar Test on Haloperidol Induced Catalepsy in Mice 7 Day Cataleptic score
|
Test
|
Time
|
Groups |
||||||
|
Controls |
Haloperidol |
Levodopa +Carbidopa |
EEPM (100mg/kg) |
EEPM (200mg/kg)
|
EEPM (400mg/kg)
|
|||
|
Catalepsy Test |
5 |
4.1± 0.3 |
167.8 ± 0.7 |
15.5± 0.4 |
92.1 ± 0.7 |
72.1 ± 0.7 |
52.1 ± 0.7 |
|
|
60 |
3.1± 0.3 |
172 ± 0.5 |
17.5±0.4 |
95.1 ± 0.7 |
75.1± 0.7 |
55.1 ± 0.7 |
||
|
120 |
2.5 ±0.2 |
170 ± 0.5 |
12.5± 0.4 |
85.1±0.7 |
65.1 ± 0.7 |
45.1 ± 0.7 |
||
|
180 |
1.5 ±0.2 |
160 ± 0.5 |
10.5 ±0.4* |
80.1 ± 0.7* |
60.1 ± 0.7* |
40.1 ± 0.7* |
||
All Values are expressed as a Mean ± SEM (n = 6 ) statistical analysis was performed using two way ANOVA followed by Turkey’s post hoc test for multiple comparison When compared with control(*p?0.0001) & Haloperidol(* p?0.0001)
The results indicate that EEPM may have promising neuroprotective potential, especially at higher doses, but levodopa remains the superior agent in managing haloperidol-induced extrapyramidal symptoms.
Table No 3: Effect of Extract of EEPM On Catalepsy Bar Test on Haloperidol Induced Catalepsy in Mice
14 Day Cataleptic score
|
Test
|
Time
|
Groups |
||||||
|
Controls |
Haloperidol |
Levodopa+ Carbidopa |
EEPM (100mg/kg) |
EEPM (200mg/kg) |
EEPM (400mg/kg) |
|||
|
Catalepsy Test |
5 |
5.16±0.3
|
167 ± 0.7
|
12.5 ± 0.4 |
90 ±0.5
|
70 ± 05
|
50 ± 0.5
|
|
|
60 |
4.16±0.3 |
174 ± 0.7 |
16.5 ±0.4
|
95 ±0.5
|
75 ± 0.5 |
55 ± 0.5 |
||
|
120 |
3.16±0.3 |
170 ±0.5 |
11.5 ± 0.4
|
85 ± 0.5
|
65 ±0.5
|
45± 0.5
|
||
|
180 |
2.5 ± 0.2 |
162 ± 0.5 |
9.5 ± 0.4*
|
80 ± 0.5 *
|
60 ± 0.5*
|
40 ± 0.5*
|
||
All Values are expressed as a Mean ± SEM (n = 6 ) statistical analysis was performed using two way ANOVA followed by Turkey’s post hoc test for multiple comparison When compared with control(*p?0.0001) & Haloperidol(* p?0.0001)
The results indicate that EEPM may have promising neuroprotective potential, especially at higher doses.
Table No 4 : Effect of Extract of EEPM On Catalepsy Bar Test on Haloperidol Induced Catalepsy in Mice 21 Day Cataleptic score
|
Test
|
Time
|
Groups |
||||||
|
Controls |
Haloperidol |
Levodopa +Carbidopa |
EEPM (100mg/kg) |
EEPM (200mg/kg)
|
EEPM (400mg/kg)
|
|||
|
Catalepsy Test |
5 |
4.3± 0.2
|
165 ± 0.5 |
12.5 ± 0.4 |
90 ± 0.7 |
70 ± 0.7 |
50 ± 0.7 |
|
|
60 |
3.3 ±0.2 |
170 ± 0.5 |
14.5±0.4 |
93 ± 0.7 |
73 ± 0.7
|
53 ± 0.7
|
||
|
120 |
2.5 ±0.2
|
168 ± 0.5 |
11.5± 0.4 |
82 ±0.7 |
62 ± 0.7 |
42 ± 0.7 |
||
|
180 |
1.5± 0.2 |
158 ± 0.5 |
9.5 ± 0.4* |
77 ± 0.7* |
57 ± 0.7* |
37 ± 0.7* |
||
All Values are expressed as a Mean ± SEM (n = 6 ) statistical analysis was performed using two way ANOVA followed by Turkey’s post hoc test for multiple comparison When compared with control(*p?0.0001) & Haloperidol(* p?0.0001).
The results indicate that EEPM may have promising neuroprotective potential, especially at higher doses, but levodopa remains the superior agent in managing haloperidol-induced extrapyramidal symptoms.
Table No 5: Effect of Extract of EEPM On Actophotometer test On Haloperidol Induced Catalepsy in Mice
|
Groups
|
Treatment and Doses |
7th day No. of counts /5min |
14th day No. of counts /5min |
21st day No. of counts /5min |
|||
|
I |
Control
|
149.1 ± 0.6
|
150 ± 0.5
|
150.8 ± 0.6 |
|||
|
II |
Haloperidol
|
60.1 ± 0.5
|
60 ± 0.5
|
58 ± 0.5
|
|||
|
III |
Levodopa + Carbidopa
|
136 ± 0.5 |
140 ± 0.5 |
142 ± 0.5*
|
|||
|
IV |
EEPM (100mg/kg)
|
68.8 ± 0.6 |
70 ± 0.5 |
72 ± 0.5* |
|||
|
V |
EEPM (200mg/kg)
|
73.8 ± 0.6 |
75 ± 0.5
|
78 ± 0.5*
|
|||
|
VI |
EEPM (400mg/kg)
|
76.8 ± 0.6
|
78 ± 0.5 |
82 ± 0.5*
|
All Values are expressed as a Mean ± SEM (n = 6 ) statistical analysis was performed using one way ANOVA followed by Dunnett’s test for multiple comparison When compared with control(*p?0.0001) & Haloperidol(* p?0.0001)
The highest dose (400 mg/kg) produced a statistically significant improvement in activity counts by 21 day.
DISCUSSION
In the present study, the animals which were treated for 21 days haloperidol showed severe cataleptic responses alone with decreased locomotory and coordination However the exact mechanism by which haloperidol increases free radical production was not clear. The enzymatic degradation by MAOs was associated with the production of hydrogen peroxide, which was readily converted to the hydroxyl radical in the presence of iron. Thus, it could initiate a destructive LPO cascade, but an increased dopamine (DA) turnover, leading to hydrogen peroxide production which might not be exclusively involved in the degeneration of oxidative stress. The auto oxidation of DA which resulted in the production of superoxide radicals might have contributed to the unbalanced production of the free radicals however, other mechanisms may also be involved Haloperidol was reported to suppress the activity of certain detoxifying enzymes, thus leaving the cell unprotected, especially if the basal enzyme activity was low or if the free radical scavenging mechanisms were less effective. Haloperidol (HP) is converted to potentially toxic (HHP+) metabolites which may play a role in the extrapyramidal side effects which are observed in the patients treated with haloperidol. Another possible mechanism could be the effect of neuroleptics on the mitochondrial respiration, loss of dopamine neurons of the SNpc. The pathogenesis of PD includes oxidative stress, protein accumulation like a-synuclein, mitochondrial dysfunction, apoptosis, and neuronal excitotoxicity. Among all, oxidative stress is a crucial pathological mechanism for PD. SNpc is more vulnerable to reactive oxygen species as it contains more amount of dopamine.
In the present study, we evaluated the effect of EEPM in haloperidol induced Parkinson disease in experimental animals. Haloperidol induced catalepsy is a widely accepted animal model of PD. Haloperidol (nonselective D2 dopaminergic antagonists) provides a pharmacological model of parkinsonism by interfering with the storage of catecholamine's intracellularly, resulting in dopamine depletion in nerve
In the Presesnt investigation of Preliminary Phytochemical analysis of ethanolic extract of Pterocarpus Marsupium revelase the Presence of Carbohydrate, Glycosides, Phenols, Phytosterols, Saponins, Tannins, Flavonoids. Neuroprotective effect of ethanolic extract of Pterocarpus Marsupium is due to Presence of bioactive compounds in EEPM, including flavonoids and phenolic compounds, which are potent antioxidants. Hence, the observed reduction in cataleptic behavior may be attributed to the antioxidant potential of PM, which neutralizes reactive oxygen species and reduces lipid peroxidation. Haloperidol, a typical antipsychotic, induces catalepsy by blocking dopamine D2 receptors, particularly in the nigrostriatal pathway, leading to motor dysfunction. In the current study, administration of haloperidol significantly increased catalepsy scores in all behavioral parameters, such as the bar test, actophotometer test, pole test, wire hanging test, water maze test, and passive avoidance test.
The bar test showed significant reduction in immobility time, indicating improved motor coordination. The actophotometer and pole tests demonstrated increased locomotor activity and improved descent time, respectively. These findings suggest that EEPM may improve motor function and delay disease progression in PD models.Furthermore, results from the passive avoidance and water maze tests suggest that PM also offers cognitive benefits. These findings are significant, considering that non-motor symptoms such as memory impairment are also prevalent in PD.
However, treatment with ethanolic extract of Pterocarpus marsupium (EEPM) significantly attenuated haloperidol-induced catalepsy in a dose-dependent manner. This suggests that Pterocarpus Marsupium possesses potent neuroprotective and anti-cataleptic properties, likely due to its rich phytoconstituents such as flavonoids and tannins, which have known antioxidant and anti-inflammatory effects.
CONCLUSION:
EEPM significantly reduced the symptoms of PD may be due to antioxidant and neuroprotective activities or increase in the level of brain dopamine similar to L-dopa and carbidopa. Thus, EEPM may have therapeutic potential in the treatment of PD. Further, it is necessary to estimate the brain dopamine level and isolate the individual constituents responsible for neuroprotective potential, and also characterization of active constituents responsible for neuroprotective effect. Future work needs to be done in the direction to elucidate the molecular mechanism of EEPM leaves in neuroprotection.. It could be the next better, safer & cheaper herbal alternate in management of Parkinsonism and also modulation of herbal drug with dopaminergic agonist to potentiate the activity.
REFERENCES
Dr. Sagar Magar, Sachin Bhartal, Sushmita Bhendekar*, Pradnya Bhosale, Snehal Bodhak, Subhadra Bramharakshas, Green Synthesis of Metal Nanoparticles Using Plant Extracts, Int. J. of Pharm. Sci., 2025, Vol 3, Issue 6, 1384-1393. https://doi.org/10.5281/zenodo.15613766
10.5281/zenodo.15613766
