A Study to Evaluate the Neuroprotective property of Aqueous Extract of Mentha piperita Leaves on Haloperidol Induced Parkinsonism in Experimental rats

 

Naveen K L*, Ananya Bhattacharjee, Karunakar Hegde

Department of Pharmacology, Srinivas College of Pharmacy, Valachil, Mangalore - 574143. Karnataka, India.

*Corresponding Author E-mail: t.naveen.nulenur@gmail.com

 

ABSTRACT:

The main objective of the proposed study was designed to investigate the neuroprotective property of aqueous extract of the Mentha piperita leaves (AEMPL) against haloperidol induced Parkinsonism in experimental animals. Rats were divided into 5 groups (n=6 animals). Group 1 received vehicle control, Group 2 received Haloperidol (1mg/kg, i.p.), Group 3receivedstandard drug Sinemet (125mg/kg p.o.), Group4andGroup5treated with lower (100mg/kg, p.o.) and higher (250mg/kg, p.o.) doses of AEMPL respectively for 14 days. Later motor co-ordination (rota-rod), locomotor activity (actophotometer) and catalepsy bar test was performed and also brain histopathological examination was also done. Both the lower and higher doses of aqueous extract of Mentha piperita leaves showed dose dependent, extremely significant increases in locomotor activity by increasing number of cuts, decreases fall off time and also decreases in latency period in metal bar test, histopathological examination shows reduction in neuronal loss and normal brain architecture was observed when compared haloperidol treated group. Hence Mentha piperita shows neuroprotective activity because of potential anti-oxidant property. The obtained result was comparable with that of the standard drug levodopa+carbidopa (Sinemet). The outcome of the present study provides the evidence that the aqueous extract of Mentha piperita leaves reported that it has dose dependent beneficial neuroprotective effect against haloperidol induced Parkinson’s disease in experimental rats.

 

KEYWORDS: Mentha piperita, Actophotometer, Anti-Parkinson, Haloperidol, Neuroprotective.

 

 


INTRODUCTION:

Neurodegenerative diseases illustrate a major threat or risk to human health and they are age-dependent disorders increasing rapidly in the recent years, mainly these disorders affects elderly population. Such illnesses, impacting people of all ages, are one of the most important medical and socio-economic issues of our time. Neurodegenerative disorders has significant impact on patient's education, social and family level and they can contribute to a total inability to perform any kind of daily activities.

 

The main problem is that these conditions are typically diagnosed late and the effectiveness of treatment options are limited1,2. Parkinson’s disease (PD) is progressive, complex neurodegenerative disorder that affects the overall quality of life and it’s also called as Shaking Palsy3. PD is one of most common clinical syndrome characterized by motor impairment such as bradykinesia, resting tremor, rigidity, freezing phenomenon and postural instability. Most of the current treatment approaches are based on restoration of dopaminergic tone in the brain striatum4. The exact cause for Parkinson’s disease is unknown, a neurological disorder resulting with the loss of dopaminergic neurons in the substantia nigra pars compacta (SNc) in brain5. The hallmark of PD patients are; loss of neuromelanin containing dopamine (DA) neurons in substantia nigra, presence of lewy bodies in cytoplasm, and abnormal accumulation of α-synuclein protein in different brain regions and peripheral tissues6. Upon exposure to some toxic chemicals such as 6-OHDA, paraquat, rotenone, MPTP, methamphetamine and isoquinoline derivatives are may increases risk of PD7.

 

Mitochondrial dysfunction, oxidative stress, nerve inflammation and insufficient Autophagic Proteasomal degeneration leads to death of dopamine neuron and mutation of some important genes such as LLRK, PARK-2 (encoded as parkin), PARK-7, PINK-1 and SNC-A (coding α-synuclein) have been attributed only to 10% of PD cases8.

 

Drugs which blocks dopamine action may results in the Parkinsonism. Neuroleptic drug like Haloperidol causes drug induced Parkinson’s in the world wide. Mainly haloperidol blocks the Dopamine D2 receptor and which induces state of catalepsy in human or experimental animals by inhibiting the dopaminergic neuronal transmission at basal ganglion.9

 

Among the various pharmacological treatment, L-dopa remains as the most efficacious, mainstay of therapy. However, prolong use of L-dopa leads to disabling the motor complications, mainly dyskinesia and motor fluctuations. Because of these side effects of conventional medicine, researchers look back into use of natural herbs and their product as an alternative medicine10.

 

Mentha piperita popularly known as peppermint belongs to the family Lamiaceae. It is one of the oldest medicinal and traditional known species found at eastern and western countries. The main aim to cultivate this plant is to produce peppermint oil around the world10. The major active constituents like flavonoids, tannins and some essential oils are present in this herb so that those components are responsible for different medicinal properties12.

 

Some of the herbal drugs proved their neuroprotective property against drug induced Parkinson. Earlier research study has been reported that mentha species having neuroprotective and significant anti-oxidant property on CNS, so it can be a good alternative in improving the treatment of Parkinson’s disease (PD). Existing literature is lacking in studies showing Anti-Parkinson’s effect of M. piperita. Hence, the present study to explore the effects of aqueous extract of M. piperita on the animal model of PD by investigating its effect on behavioral models and oxidative stress changes induced by haloperidol in rats13.

 

MATERIALS AND METHODS:

Animals:

Swiss albino rats of either sex weighing between 150 and 250g were used for the study. The animals were housed under standard laboratory conditions and fed with pellet diet and water ad libitum. On the day of the experiment, animals were fasted overnight to avoid the food on drug absorption, though water was given. Animals were maintained on natural light-dark cycle, 22±2°C temperature and 50±5% humidity. The animals were acclimatized to laboratory conditions prior to experimentation. Permission was taken from the Institutional Animal Ethics Committee and animal care as per the Committee for the Purpose of Control and Supervision of Experiments on Animals, India.

 

Collection and authentication of plant material:

The leaves of M. piperita belongs to the family Lamiaceae were collected from local market Mangalore, Karnataka. It was authenticated by botanist Dr. Siddaraju M N. M.Sc. PhD Assistant professor, Department of Botany, University College, Mangalore.

 

Extraction:

Fresh leaves was collected, cleaned and washed them by using tap water, dried in the open away from direct sunlight. The dried leaves was subjected for grinding to get fine powder. The dried powdered leaves 100-150g was taken in 250ml of beaker and extracted with by adding 1.5L of boiling water (Decoction) for a period of 10min. the obtained liquid extract was filtered, combined and concentrated to get almost to dryness using water bath at 60˚C for 3h. Thereafter, the concentrated aqueous extract was stored in a refrigerator for subsequent evaluation14.

 

Dose selection:

Doses were selected according to the earlier research work carried on. Two doses were selected and was administered post orally at a constant volume of 100 mg/kg (low dose) and 250mg/kg (high dose) for each animals15.

 

Experimental design:

Rats weighing about 150–250g were selected and randomly divided into five groups (n = 6 in each groups).

Group I   - Control (distilled water)

Group II  - Haloperidol (1mg/kg i.p.)

Group III - received L-dopa and carbidopa (Sinemet 125 mg/kg   p.o.)

Group IV - Received M. piperita lower dose (100mg/kg, p.o.)

Group V - Received M. piperita higher dose (250mg/kg, p.o.)

 

Behavirol assessment:

Acute study;

Haloperidol induced catalepsy: Metal bar test:

Catalepsy was measured by means of a standard bar test mainly to evaluate the effect of drugs on the extrapyramidal system.  Catalepsy is the state of reduced ability to initiate the body movement and failures correct abnormal body posture. After 30 min administration of haloperidol (1mg/kg, i.p.), the time duration of catalepsy score was measured for a period of 5 min at an interval of every 30, 60, 90 and 120min. Duration of a catalepsy score was determined by placing an animal on the horizontal metal bar at an height of 9cm in such a way that the fore-limbs of the animal should be on the horizontal bar while the hind-limb touches the surface. The animal are considered as  cataleptic, if they shows reduced ability to initiate body movement and also failure to correct the abnormal body posture within cut off time16.

 

Chronic study:

As like acute study, to induce motor in coordination dosing was continue with all groups except group 1 and all other groups were treated with Haloperidol through intra peritoneal route with the dose of 1mg/kg. All other treatments as per the protocol were continued for a period of 14 days. After 30min of last treatment motor co-ordination, locomotor activity and other behavioral assessment was carried out.

 

Locomotor Activity:(Actophotometer):

This test measures the time of exploration, locomotor activity and the voluntary locomotion within an enclosed area. Here locomotor activity was determined by using an Actophotometer (activity cage). The animals were placed individually into a 30 cm × 30 cm black metal chamber with a screen floor and a light-tight lid. Six beams of red light were focused 2 cm above the floor into photocells on the opposite side. Each beam interruption i.e. number of rays cut by the animal was registered as an event on the external counter. The number of light rays cutter by the animal was counted for a period of 5 min17.

 

Motor co-ordination: (Rota rod):

The speed was select in such a way that the roller rod would make 30 rpm. Before the test, each animal were trained for a period of 1min on the moving rod. The animals were placed on the roller for 3 min. Latency to fall from rolling rod was noted. A normal animal could maintain its equilibrium for an indefinite period. Movement impairment was indicated by the inability of the animal to remain on the roller for a 3 min test period18.

 

Histopathological analysis:

The brains from control and experimental groups were fixed with 10% formalin and embedded in paraffin wax and cut into longitudinal section of 5 μm thickness. The sections were stained with hematoxylin and eosin dye for histopathological observation19.

 

Statisticalanalysis:

All the data were expressed in terms of mean ± SEM using Graphad Prism. Statistical significance was assessed using One-way Analysis of variance (ANOVA) and column statistics followed by Tukey-Kramer multiple comparisons tests. p<0.05 was considered significant.

 

RESULTS:

Preliminary phytochemical screening of M. piperita leaves:

Preliminary phytochemical analysis of aqueous extract of M. piperita leaves was performed and the results are shown in as below.

 

Table no 1: Phytochemical Study of aqueous extract of M. piperita leaves

Sl no

Phytochemical Tests

Names of Conducted Tests

Inference

1

Alkaloids

Mayer’s Test, Wagner’s Test, Hager’s Test

Presence of alkaloids

2

Flavonoids

Ferric Chloride Test, Alkaline Reagent Test, Lead Acetate Test

Presence of flavonoids

4

Tannins

Ferric Chloride Test, Lead Acetate Test

Presence of tannins

5

Glycosides

Keller-Killiani Test

Presence of glycosides

6

Anthocyanin

2 mL of Plant Extract + 2 mL of 2N HCL

Absence of anthocyanin

7

Proteins

Biuret Test, Ninhydrin Test, Millon’s Test

Absence of proteins s

 

Acute Study:

Behavioral Assessment:

The Effects of AEMPL on Haloperidol Induced Catalepsy:

Haloperidol control group showed extremely significant (p<0.001) results by increase in all time dependent in the cataleptic behaviour when compared to vehicle control group. The duration of cataleptic score was extremely significant decrease with time (p<0.001) in standard group when compared with haloperidol control. Pre-treatment with AEMPL 100 mg/kg showed moderately significant (p<0.01) whereas 250 mg/kg showed extremely significant (p<0.001) reduction in the duration of cataleptic score as compared to haloperidol control group. (Table no:2).


 

Table no 2: Acute study- Effect of AEMPL in haloperidol induced catalepsy

Treatment (min)

Duration of catalepsy in seconds

0 min

30 min

60 min

90 min

120 min

Vehicle control

3.29±0.04

3.58±0.09

3.58±0.04

3.69±0.06

3.78±0.05

Haloperidol control

3.3±0.035

124.3±0.41***

130.42±0.45***

125.32±0.54***

132.25±0.74 ***

Standard (Sinemet 125 mg/kg   p.o)

3.4±0.42

86.32±0.47###

54.32±0.35###

38.23±0.39###

26.34±0.41###

Low dose

3.29±0.37

101.3±0.51##

84.24±0.65##

74.23±0.74###

65.39±0.65##

High dose

3.42±0.24

94.32±0.54###

75.32±0.67###

65.39±0.77###

52.32±0.99###

 


All the values are expressed in terms of Mean±SEM, n=6 ***P<0.001, when haloperidol   group compared with vehicle control group. ###P<0.001, ##P<0.01. When Standard, Low dose and High dose of AEMPL compared with Haloperidol treated group.

 

Chronic Study:

Behavioral Assessment:

Locomotor activity test:

Haloperidol treated group showed extremely significant (p<0.001) reduction in locomotor activity when compared to the vehicle treated group. Standard and high dose of AEMPL showed extremely significant (p<0.001) whereas low dose group showed moderately significant (p<0.01) increase in the locomotor activity in dose dependent manner as compared to haloperidol treated group. (Table no 3).

 

Motor co-ordination activity test:

Haloperidol treated group showed extremely significant (p<0.001) reduction in motor coordination activity when compared to vehicle treated group. Standard and high dose of AEMPL showed extremely significant (p<0.001), whereas in case of low dose AEMPL also showed extremely significant (p<0.001) increase in the number of fall of time (Sec) when compared with haloperidol treated group. (Table no 3).

 

Table 3:Chronicstudy of Effect of AEMPL in motor co-ordination (Rota rod),  Locomotor activity (Actophotometer) on haloperidol induced Parkinsonian Rats.

Groups

Motor Coordination (Mean fall of time in s)

Locomotor Activity

(No. of counts/ 5 min)

Vehicle control

72.66±0.666

128.16±0.945

Haloperidol control

23.16±0.542***

25.16±0.307***

Standard(Sinemet 125 mg/kg   p.o)

56.83±0.792 ^^^

112.51±0.670 ^^^

Low dose

35.23±0.703 ^^^

69.66±0.494 ^^

High dose

42.32±0.477 ^^^

81.33±0.614 ^^^

All the values are in Mean±SEM, n=6 ***P<0.001 when haloperidol group compared with vehicle control group.

^^^P<0.001, ^^P<0.01 when Standard, Low dose and High dose of AEMPL compared with Haloperidol treated group.

 

Effect of various drugs on histological changes induced by haloperidol:

In the vehicle control group, histological analysis showed normal architecture of the striatal region of rat brain with darkly stained neurons (Figure A), while the haloperidol-treated group showed moderate decrease in neurons, with moderate cellular hypertrophy, alteration in brain architecture when compared to the vehicle control group (Figure B). The Sinemet (125mg/kg)-treated group showed significant protection from neuronal damage or neuronal alterations observed in standard. When compared to the haloperidol-treated group (Figure C). Further, Rats treated with Haloperidol control and AEMPL 100mg/kg and 250mg/kg showed minimal changes in neuronal cell populations and shows significant neuronal protection when compared with haloperidol treated group. (Figure D and E).

 

DISCUSSION:

Herbal drugs which derived from plant origin plays vital role in both modern and traditional medicine. Hence plant and its derivatives had undoubtable medicinal properties in alleviating human health issues. Parkinson’s disease is the most common type of progressive neurodegenerative disorder characterized by the presence of bradykinesia, resting tremors, muscular rigidity, shuffling gait and flexed posture. PD is most commonly diagnosed as a neurodegenerative disorder, due to degeneration of dopamine producing neurons in the substantia nigra pars compacta. Still, the exact cause of the degeneration of neurons is not well defined. Oxidative stress may plays a major role in the disease progression20.Oxidative stress may arises from the rapid metabolism of dopamine with the harmful free radicals generation in our barin21. In case of PD, the major part of brain region such as substantia nigra pars compacta (SNpc) is highly exposed to a higher rate of free radical formation when compared with the other part of the brain, resulting in the increased level of oxidative stress. This may be related to the energy metabolism of these cells or to their more content of dopamine in brain. Various studies have revealed oxidative stress changes evident in the brain of PD patients22.

 

Haloperidol is a neuroleptic drug, which blocks post synaptic striatal, central DA receptor (D2) in the striatum, produces a behavioral state in animals such as mice and rats in which they fail to correct their externally imposed postures. This is referred to as state of catalepsy. Catalepsy is one of the animal models to test the haloperidol-induced extrapyramidal side effects of neuroleptic drugs. Haloperidol is a non-selective D2 DA antagonist, which induces catalepsy due to the blockade of DA receptors in the brain striatum23. Drugs which attenuate haloperidol-induced motor disorders might reduce the extrapyramidal signs of PD. In our study, three behavioral parameters - Rota rod performance, Actophotometer and a catatonic i.e. catalepsy response were measured as retention time (sec), number of counts and the latency period (sec), respectively.

 

The important parameter for the selection of aqueous extract of M. piperita leaves(AEMPL)as the test drug is because of presence of flavonoid sand alkaloids as a major phytoconstituents, due to their significant antioxidant potential, which are responsible for anti-parkinsonian activities i.e. Neuroprotective action in PD. Most of the research studies on extract of M. piperita are based on its richness in anti-oxidants properties and other important nutrients had medicinal values in different studies24.

 

The present study investigates neuroprotective potential of M. piperita leaves extract were determined by using three different pharmacological models such as metal bar test, locomotor activity and motor co-ordination. Acute study was carried out for a time period of 24hrs and chronic study continued for 14 days as per the previous protocol. In acute study i.e. day of starting dose, all standard and extract dose are administered to animals, after the 60 min haloperidol was given via I.P. route except group-1. After 30 min of administration of haloperidol (1mg/kg, i.p.), the duration of catalepsy (catatonia) was measured for a period of 5 min at a time interval of every 30, 60, 90 and 120min. Duration of catalepsy score was measured or determined by placing an animal on the horizontal metal bar at a height of 9cm respectively. The rats are considered cataleptic if it shows reduced ability to initiate movement and a failure to correct their abnormal posture. Pre-treatment with AEMPL100 and 250mg/kg shows significant reduction in the time duration of catalepsy induced by the haloperidol when compared with haloperidol treated group in dose dependent manner. Duration of catalepsy was also significantly decreased by standard drug Sinemet 125mg/kg treated group.

 

In chronic study, after 14 days of treatment with haloperidol into experimental rats causes a significant decrease in locomotor and muscle co-ordination activity. The test drug is compared with haloperidol control and haloperidol group compared with vehicle control. In case of Rotarod test, the group which received only haloperidol shows significant, decreases fall off time as compared to the normal group. In case of standard treated group, a significant increases in fall off time as compared to the haloperidol treated group. In extract treated group a significant increase in fall off time as compared to the halo peridol treated group. In locomotor activity the group which received only haloperidol, shows significant, decreases locomotor activity as compared to the normal group. In standard treated group an increase in locomotor activity was seen as compared to the Haloperidol treated group. In extract treated group a significant dose dependent increase in locomotor activity was observed as compared to the haloperidol treated group in experimental animals.

 

Histopathological findings showed haloperidol treated group showed the congestion of degenerative changes due to decrease the number of neural cells in SNpc in brain tissue and also changes in brain architecture. Pre-treatment with AEMPL showed regenerative changes in SNpc, decreased infiltration of neutrophils, reduced intracellular space, increased density of cells, and regained normal architecture and moderate necrosis in striatum region of brain. It further substantiates the neuroprotective activity against haloperidol induced disease model.

 

By observing all above pharmacological and histopathological results suggest that M. piperita leaves has the ability to improve symptoms of Parkinsonism and also has Neuroprotective activity, in part, by the restoring the level of dopamine and by the regulation of the anti-oxidant system. Thus, anti-oxidant and neuroprotective activities may be responsible.

 

 

Whether, M. piperita has anti-oxidant and neuroprotective activity in other experimentally induced Parkinsonism models like reserpine, 6-hydroxy dopamine, rotenone, MPTP needs further evaluation. Further studies may also be undertaken to identify and investigate the mechanism of action of active anti-parkinsonian compounds in M. piperita.

 

CONCLUSION:

Parkinson disease is a progressive, second most common neurodegenerative disorder accompanied by loss of dopaminergic neurons in the substantia nigra pars compacta (SNpc). Haloperidol is most commonly used neuroleptic drug to create experimental model of PD. Haloperidol toxicity leads to formation of free radicals leading to generation oxidative stress. The results of the present study conclusively showed that M. piperita has neuroprotective property in haloperidol experimental model of PD. M. piperita was also found to be effective in increasing rota rod, actophotometer performance and also decreasing catatonic response. Histopathological study also shows neuroprotective property against haloperidol treated rats. Hence, the neuroprotective effect of M. piperita on behavioral and histopathology study is due to its anti-oxidant properties. In this regard, future clinical studies can be undertaken which may provide a ray of hope to use M. piperita in the treatment of PD.

 

ACKNOWLEGMENT:

Authors are thankful to Srinivas College of Pharmacy for the help and facilities provided to conduct this study.

 

CONFLICT OF INTEREST:

None.

 

ETHICAL APPROVAL:

The study was approved by the Institutional Animal Ethics Committee.

 

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Received on 05.02.2022         Modified on 11.07.2022

Accepted on 14.12.2022   ©Asian Pharma Press All Right Reserved

Asian J. Pharm. Res. 2023; 13(3):139-144.

DOI: 10.52711/2231-5691.2023.00027