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Kartika B, Muralidharan P, Habibur Rahman

Pharmacology department , C.L.Baid Metha College of Pharmacy , Chennai-97, Tamilnadu



Parkinsonism is a progressively increasing neurodegenerative disorder, characterized by the

clinical signs involved in motor co-ordination and body movements. The present study is carried
out to determine the neuroprotective effect of m
 
 against Parkinsonism. In the
present study animals were pretreated with the hydroalcoholic extract of m
 

leaves for 3weeks (200 and 400mg/kg). On 21st day animals were anaesthetized and
Parkinsonism was induced by 6-Hydroxydopamine (8 µg 6-hydroxydopamine in 4 µl saline
containing 0.02% ascorbic acid) into left substantia nigra and treatment continued for another
week. After 1 week behavioural tests were evaluated using Rota rod test, Catalepsy test, Beam
walking test, Grid test, Pole test and also antioxidant, neurotransmitter levels
estimated.Pretreated groups significantly reduced the neurological deficits, increase in
antioxidant enzymes and neurotransmitter glutamate levels was significantly reduced and
increased Dopamine level in dose dependent compared to negative control group.
Hydroalcoholicleavesextract of m

 showed significant antiparkinsonism activity
in rats.
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Parkinsonism describes a syndrome of which Parkinson¶s disease is the main cause. Parkinson¶s
disease (PD) is a chronic neurodegenerative disorder characterized by the progressive loss of
dopaminergic neurons of substantia nigra pars compacta in the ventral midbrain. The loss of
dopaminergic neurons, leads to the reduction of dopamine being released into the striatum. These
processes are then responsible for the clinical features of PD including bradykinesia, resting
tremor, rigidity, and difficulty in initiating movements4 Mutations in the Į-synuclein or Parkin
gene have been associated with familial PD.The prevalence of Parkinson¶s disease in
industrialized countries is estimated at 0·3% of the general population and about 1% of the
population older than age 60 years. The causes of parkinsonism are: oxidative stress,free radical
formation,mitochondria dysfunction, apoptosis, neuroinflammation ,genetic susceptibility,
toxins such as 6-hydroxydopamine and 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine , rotenone,
Paraquat, Maneb, manganese,toluene, N-Hexane, carbonmonoxide ,Mercury, Cyanide, Copper,
Lead and Trichloroethylene,certain medications, viral infection, Alzheimer's disease,
amyotrophic lateral sclerosis (ALS), Creutzfeldt-Jakob disease, Wilson's disease and
Huntington's disease, Administration of dopamine directly into brain and cell loss in the
dopaminergic nigrostriatal tract of the brain, ageing causes the parkinsonism.

Pharmacologic management of parkinsonism involves administering „




   
m 
   
 
    
 
 .All the drugs suffer from side effects
and there is a need for alternative medicine in the management of parkinsonism. Today, herbal
therapy has made a comeback as an alternative to prescription medication. Annona squamosa
contains flavanoids, polyphenols, dopamine, Cardiotonic and several alkaloids, quinoline, and
bullatacinone, terpene derivatives, and a novel diazepine, squamolone as major active
constituents which have role in treatment of parkinsonism. As there was no scientific data
available on evaluation of the antiparkinsonism activity of hydroalcoholic extract of Annona
squamosa leaves against parkinsonism.

Plant introduction

m
 
 a small well-branched tree or shrub that bears edible fruits called !#
$$%&, species of the genus m
 and member of the family Annonaceae more willing to
grow at lower altitudes 0 metres (0 ft) to 2,000 metres (6,600 ft)

Uses: Analgesic, anti-inflmmatory, anthelmentic, anti-oxidant, anti-bacterial, anti-head lice, anti

diabetic, hepatoprotectivity,anti-emetic,expectorant, anti-cancer, insecticidal, antispasmodic
agents and rheumatism and painful spleen. antipyretic, antiulcer, and antiseptic, anti-ovulatory
and abortifacient activities to treat hysteria,wounds to clarify the urine.

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The leaves of m
 
 were dried under shade and powered. A weighed quantity of
Powered was subjected to continuous hot extraction with water and ethanol (4:10) in soxhlet
apparatus. The extract was evaporated under reduced pressure using rotatory vaccum evaporator
until all solvent was removed to give a molten extract of 16.4% w/w and the extract is used for
the study.

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Preliminary phytochemial screening was carried out for Hydroalcoholic extract of Annona
squamosa, it shows positive results for the presence of alkaloids, flavonoids, polyphenols,gums
mucilage, vitamins, minerals and flavonones.

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The procedure was followed by using OECD guide lines (Organization of Economic
Cooperation and Development) 423. The acute toxic class method is a step wise procedure with
3 male animals per step. Depending upon the mortality and/or morbidity status of animals, on the
average 2-3 steps may be necessary to allow judgment on the acute toxicity of the test substance.
This procedure results in the use of minimal number of animals while allowing for acceptable
data based scientific conclusion. The method uses defined doses (2000mg/kg body weight) and
the results allow a substance to be ranked and classified according to the Globally Harmonized
System (GHS) for the classification of chemicals which cause acute toxicity. Behavioral profile ,
Neurological profile , Autonomic profile. After a period of 24 and 72h they were observed for
any lethality or death

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Colony inbreed strains of Male Sprague-Dawlely rat (200-250 g) were used for pharmacological
studies, 4 different groups with 6 animals in each were housed at controlled temperature
(25±2°C) and light dark cycle (12/12 hr); on a standard rodent diet(Hindustan lever pvt ltd.,
banglore) and water . Animals were handled and acclimatized to laboratory conditions
at least 1 week prior to experimentation. All protocols were approved by IAEC, and studies
were carried out in accordance of CPCSEA, guidelines.

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GROUP I : control (0.9 % saline, p.o)

GROUP II : 6-OHDA (8 µg 6-hydroxydopamine in 4 µl saline containing 0.02% ascorbic

acid) into left substantia nigr

GROUP III : 6-OHDA + low dose of Hydroalcoholic extract of Annona squamosa

GROUP IV : 6-OHDA + high dose of Hydroalcoholic extract of Annona squamosa

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1. Open field spontaneous activity

Animals were placed in the corner of an open field (33×33×30cm) chamber with floor
grated into squares under weak light. Number of square crossed (in 5 mins) was counted
manually in triplicates as forepaw criterion (horizontal activity). The vertical activity was
measured by number of observations including grooming and rearing.

2. Catalepsy test

Catalepsy was measured by placing the animal on a flat horizontal surface with both hind limbs
on square wooden block of 3cm in height and the latency in seconds required to move the limbs
from the block to the ground was measured.

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Neurotransmittors such as Dopamine, Acetylcholine esterase and anti-oxidant enzymes such as

Lipid Peroxidation, Superoxide Dismutase activity were evaluated.




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Group 1= Control, Group 2=Negative control, Group 3=Low dose and Group 4= High dose

The statistical analysis result showed that application of 6-OHDA led to a pronounced reduction
in the movement (p < 0.001, p < 0.01, respectively) in group 2 as compared to group 1, treatment
with HAEASwere markedly increased in crossing number, rearing number and grooming
activities in group 3 and 4 as compared to the corresponding group 2. It also showed that
cataleptic rigor occurs in group 2, which was reduced significantly (p<0.01 and p<0.001) in
group 3 and group 4. The significant (p<0.01 and p<0.001) decrease in the levels of
Acetylcholine esterase and dopamine in group 2 as compared to group 1 was observed which
was then restored significantly in group 3 and group 4.
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% 10.81± 0.5070 2.343± 0.07192 2.813±0.1226 32.84±0.3824 33.12±0.4908 0.8908±0.0142
&#",& 6.668± 0.2244a*** 3.935± 0.1028a*** 1.698±0.04990a** 22.12±0.6462a** 24.49±0.3512a*** 0.4508±0.0105a**

%

/ & 7.890± 0.09656b* 3.310± 0.1646b** 2.293±0.1558b** 25.16±0.6525b** 27.24±0.4923b** 0.5345±0.02699b*
"#( & 8.740± 0.2074b** 2.915±0.7735b*** 2.415±0.07240b** 29.34±0.3424b*** 28.98±0.6632b*** 0.6610±0.0238b2***

"#"0"&1 <0.05 **P<0.01 and *** P<0.001, Values are expressed as mean ±SEM of n=6
a
animals.Comparisions were made between Control Vs Negative control; b Negative control Vs
Treatment groups. Symbol represents the statistical significance done by ANOVA, followed by
Dunnet¶s multiple comparision test.

  


 

The Hydroalcoholic extract of leaves of Annona squamosa were subjected to preliminary

phytochemical screening and also acute toxicity studies. It showed the presence of alkaloids,
flavonoids, polyphenols,gums mucilage, vitamins, minerals and flavonones and found that
HAEAS at dose of 2000mg/kg orally was free of toxicity.

The HAEAS was given to animal models at two dose levels ie low
dose(200mg/kg) and high dose(400mg/kg) by oral route. It showed the increase in locomotion,
motor co-ordination and balance, muscular strength ,neuromuscular function, catching reflex and
cataleptic rigor in treated group as compared to negative control.

The elevated levels of antioxidant enzymes, Ascorbic acid , neurotransmitter

Glutamate and Monoaminooxidase B were decreased and diminished levels of Dopamine,
Acetylcholine esterase were increased significantly in dose dependent manner.

The present study proved that Hydroalcoholic extract of Annona squamosa possess
antiparkinsonsm activity and By the statistical analysis it was found that the higher dose of
extract (400mg/kg) produced significant antiparkinsonism activity than the lower dose
(200mg/kg).



 
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I am much grateful to My guide and Head of the department of pahrmaclogy Dr. P.Murlidharan,
C.L.Baid Metha College of Pharmacy Research Foundation Chennai for his support.