Evaluating the effect of α-pinene on motor activity, avoidance memory and lipid peroxidation in animal model of Parkinson disease in adult male rats

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*Corresponding author: rafieirad.m@gmail.com, Tel/Fax: +9861-43643374
Research Journal of Pharmacognosy (RJP) 4(2), 2017: 53-63
Received: Nov 2016
Accepted: Feb 2017
Original article
Evaluating the effect of α-pinene on motor activity, avoidance memory and
lipid peroxidation in animal model of Parkinson disease in adult male rats
S. Goudarzi, M. Rafieirad*
Department of Biology, Faculty of Sciences, Izeh Branch, Islamic Azad University, Izeh, Iran.
Abstract
Background and objectives: Parkinson's disease (PD) is a common neuropathologic disorder that is
caused by degeneration of dopaminergic neurons of dense part of nigra. Oxidative stress has been
found in the pathophysiology of PD. Since α-pinene has strong anti-oxidant effects, the purpose of this
research was to study its effects on movement disorders and memory and lipid peroxidation in PD.
Methods: Thirty five male rats were divided in 5 groups: control, vehicle, PD (received injection of 6-
hydroxydopamine (6-OHDA)) and Parkinson's groups receiving doses of 100 and 200 mg/kg via
gavage for two weeks. Generating animal models for Parkinson was done by intracerebral injection of
6-OHDA in the left side of the brain in medial forebrain bundle (MFB). After the injection, the
movement balance of the rats was measured by Rotarod. Memory test was done by shuttle box; their
brain was extracted to analyze malondialdehyde (MDA) in striatum, hippocampus and blood. Results:
The results showed that Parkinson caused, movement disorder (p<0.01), avoidance memory reduction
(p<0.001) and malondialdehyde accumulation in hippocampus (p<0.05) and striatum (p<0.001)
tissues and in blood (p<0.001). Administration of 200 and 100 mg/kg α-pinene improved the
movement disorder (p<0.05). Administration of both doses of 200 and 100mg/kg showed
improvement in avoidance memory (p<0.001) and (p<0.01), respectively. Malondialdehyde showed
reduction in striatum (p<0.001) and hippocampus (p<0.05, p<0.001), respectively in the treatment
groups after administration of both doses. In the blood, the dose of 200 α -pinene significantly reduced
MDA in the tretment groups. Conclosion: The results of this research show that α-pinene could
reduce the symptoms of PD in rats.
Keywords: lipid peroxidation, memory, movement, Parkinson`s disease, α-pinene
Introduction
Parkinson disease (PD) was first described by
James Parkinson in 1817. This disease
demonstrates a progressive neurodegeneration,
which has already affected almost 1% of the
population over 50 years old [1]. Disturbances in
consciousness, memory, perception ability, and
visual-spatial function decline are observed in
these patients [2]. Parkinson is a degenerative
disease of the central nervous system in which
the substantia nigra cells in the midbrain
gradually vanish and dopamine production
gradually reduces [3]. The middle brain damage
can cause disruptions in the dopamine content of
striated objects, decrease in the density of

Goudarzi S. and Rafieirad M.
54 RJP 4(2), 2017: 53-63
dopamine receptors, decrease in neuronal
activity, and increase in the activity of brain free
radicals [4]. Degeneration of dopaminergic
neurons in the midbrain and the sharp decline of
dopamine at the back-middle of the dense part of
the substantia nigra [3,5]cause debilitating and
motor disorders such as bradykinesia, resting
tremor, rigidity, and postural instability [6].
Several hypotheses have been suggested about
the pathology and the death cause of pars
compacta dopaminergic neurons in substantia
nigra including oxidative stress, lipid
peroxidation, reducing glutathione level,
destruction of DNA, iron accumulation and
increasing free radical formation [7]. Oxidative
stress not only destroys dopaminergic neurons
but also leads to cell death by impairing the
oxidative phosphorylation process and reducing
energy production [8]. Endogenous sources of
oxidative stress include free radicals resulting
from the metabolism of dopamine and melanin.
Reactive radicals of oxygen are produced
constantly in the midbrain dopaminergic neurons
by dopamine metabolism and monoamine
oxidase B auto-oxidation enzyme [9]. The
pharmacologic treatment of PD can be further
divided into neuroprotective and symptomatic
therapy. In practice, nearly all of the available
treatments are symptomatic in nature and do not
appear to slow or reverse the natural course of the
disease. However, several potential
neuroprotective agents for PD have shown some
promise in animals and/or humans and are
undergoing further investigations. Antioxidants
with low molecular weight such as vitamins and
protein molecules such as superoxide dismutase,
glutathione peroxidase and glutathione can
protect the body from creating oxidative stress
induced by free radicals in the central nervous
system dopaminergic neurons [10]. Studies have
shown that plant phenols, such as flavonoids,
phenolic acids and flavonolignan acids can act as
effective antioxidants [11]. Dopamine has been
introduced as a potential substrate in synaptic
plasticity and memory mechanisms [12]. There is
pharmacological evidence for the role of
dopamine in learning and memory [13]. Both
dopamine receptors (D1, D2) are involved in
learning and memory processes [14]. It has been
reported that dopamine receptors enhance the
passive recognition [15] and improve cognitive
performance in rats while they do not affect
learning [16]. In recent years, a number of studies
have described cognitive dysfunction in
Parkinson's disease, which is significantly
associated with their disability status. Cognitive
dysfunction may include memory loss, difficulty
in concentrating, slowed information processing,
and cognitive problems in different tasks [17,18].
Memory disorder is one of the most common
cognitive dysfunctions in Parkinson's disease. α-
Pinene is an organic compound of
the terpene class, one of two isomers of pinene. It
is an alkene and contains a reactive four-
membered ring. It is found in the oils of many
species of coniferous trees. α-Pinene is naturally
found in plants such as Prangos ferulacea [19],
Hypericum richeri [20], Ferula gummosa.[21],
Teucrium stocksianum [22] Salvia officinalis
[23], Ferulago angulata [24], Origanum
majorana [25], and Salvia lachnocalyx [26]. α-
Pinene is an isomer used in multiple reactions
such as isomerization, oxidation, hydration,
acetylation, etc. It is also used in the preparation
of many terpenoids such as ocimene, terpinolene,
terpinene hydrate, and camphor [26]. α-Pinene is
the most important ingredient of turpentine that is
used as a flavoring. It is an important interface of
the aromatic compounds which is used as
flavoring in salts, household sprays, disinfectants,
and pesticides [27]. α-Pinene is the main
ingredient of essential oils from various plants
and has shown inhibitory effect of
acetylcholinesterase activity [28]. It also has
shown anti-depressant [29], anticonvulsants [30],
antioxidant [31], antispasmodic [19],
antibacterial [32], anti-inflammatory [33], anti-
tumor properties [34]. In the present study, due to
the antioxidant effects of α-pinene, the effect of
its chronic administration on behavioral disorders
and malondialdehyde (MDA) caused by the toxin
6-hydroxydopamine (6-OHDA) has been
investigated for the first time.
Experimental
Animals
Thirty five adult male rats were used with the
weight range of 200-250g, which were randomly

Effect of α-pinene on Parkinson disease in rats
55
divided into five groups of seven rats. All rats
were maintained under the same conditions,
including 21±2 °C, 12 h of light and 12 h of
darkness. All tests were done based on the ethical
protocols and standards of laboratory animal
protection (No. 1394.2002).
Experimental Group
The control group: no surgery was performed
(intact); vehicle group: stereotoxic injection
surgery was performed on this group without
neurotoxin 6-hydroxy-dopamine and they
recieved 3% Tween 80 [35] for 14 days by
gavage; Parkinson Group (PD): this group
received 2 µL containing 8 micrograms of
neurotoxin 6-hydroxy dopamine at MFB area by
stereotoxic surgery; Parkinson group received a
daily dose of 100 µg/Kg of α-pinene for 14 days
by gavage (α-pinene 100 mg/kg + PD) [35];
Parkinson group received a daily dose of 200
µg/Kg of α-pinene for 14 days by gavage
administration. (α-pinene 200 mg/kg + PD) [35].
Stereotoxic injection surgery procedure
Initially, the rats were weighed and then
anesthetized by intraperitoneal injection of 90
mg/kg ketamine hydrochloride (Alusan Co.,
Netherlands) and 10 mg\kg xylazine (Alusan Co.,
Netherlands). The rats were placed in stereo tax
device and fixed by the mouthpiece and bars on
the device. The skull dorsal hair was shaved and
the animal’s scalp was disinfected by alcohol and
a longitudinal cutting was created through the
back of the head between the eyes to the dorsal
surface between the ears. The crossbred tissues
were removed from the external of the cranium in
a way that bregma part was visible. Lambda and
bregma points were placed equal on a level and
the device index was set on. Then, (MFB)
peculiarities were determined according to the
extracted coordinates from the Atlas of brain
surgery AP;-3/8 DV;-8/2, ML±1/6. In this study,
the unilateral injection of 6-hydroxy-dopamine
was used anterior-middle category (MFB) to
create Parkinson's disease [3].
6-Hydroxy-dopamine solution preparation
6-Hydroxy-dopamine (Sigma, USA) was
prepared at a concentration of 8 µg per 2 µl of
normal saline dissolved in 0.01% ascorbic acid.
α-Pinene: α-pinene (Betagen, Iran) was
administered by gavage method after dissolving
in 3% tween 80.
Behavioral tests
Apomorphine-induced rotational behavior
Contralateral rotations of each animal were
recorded after subcutaneous injection of
apomorphine (0.5 mg/kg in normal saline
containing 0.01% ascorbic acid) to confirm the
dopamine depletion. Full spin were measured in a
cylindrical proper place for 60 min in 10-min
intervals [36].
Rotarod (motor coordination test)
This test aims to measure the motor balance and
harmony in movement (motor efficiency and
coordination). It measures the time (latency) it
takes for the mouse to fall off the rod rotating at
different speeds or under continuous acceleration.
Briefly, the animals were placed on a rotarod
device bar whose speed varied. The primery
speed of the bar was 5 rpm. Then, the speed of
rotation bars gradually increased within 300
seconds. The main standard for balance in all
groups was 25 rpm. Rats became familiar with
this device. Then, each animal was assessed 3
times and 45 minutes intervals between the
sessions and the average time was calculated
[37].
Passive avoidance memory test
This test was conducted using a shuttle box
(5500-ST, Borj Sanaat Co., Iran), which
contained two compartments, one was dark and
the other light. Their bottom was covered by
stainless steel wires with a diameter of 1 to 2 mm
and at a distance of one centimeter. A slight
shock was applied to animals’ paw by an electric
current generator (75 volt, 0.3 mA for 3 seconds)
in the dark compartment only once. Initially, the
animals were placed in the shuttle box with
guillotine doors for 10 min in order to become
familiar with the device (training) to move freely
between the inside and outside of their enclosure.

Goudarzi S. and Rafieirad M.
56 RJP 4(2), 2017: 53-63
Then, the animals were placed in the light box
and their delay time to go to the dark box was
recorded (learning). The guillotine door was
closed as soon as the animal entered the dark
compartment and the electric shock was applied
to their feet. After 24 h, their delay time for
entering the dark compartment was measured in
seconds (no electric shock) as the passive
avoidance memory. This operation was
conducted for all rats in all groups [38].
Malondialdehyde (MDA) measurement
For (MDA) measurement striatum and
hippocampus tissues of the brain were removed
and blood samples were also taken from the tail.
The MDA level of tissues was measured by
spectrophotometric method, using the thio-
barbituric acid (TBA) reagent, based on the
response of a chromogenic reagent, (TBA) with
MDA at 100 °C. Molecules of MDA would react
with TBA to yield a complex dye. MDA
concentration was measured at 532 nm [39].
Standard curve
Three mL of the 1% phosphoric acid solution
was added to 0.5 mL of the standard solution
with concentrations of 8, 10, 6, 4, 2, 1, 5.0 mM
and the rest of the steps were performed as
beofore and the absorption was recorded at 532
nm [39].
Statistical analysis
Data have been reported as mean ± SEM. The
results were analyzed using SPSS software.
ANOVA analysis was used to check the results in
different groups and discrepancy between groups
were considered significant if p<0.05.
Results and Discussion
All animals tolerated the stereotaxic surgical
procedures and no deaths were observed during
the study.
The results showed that rotation test of the
Parkinson's groups significantly increased in
contrast to the control group after the creation of
MFB lesion due to infusion of 6-OHDA in the
rats (p<0.001). In the Parkinson's groups which
were treated with α-pinene 100, 200 mg/kg for 14
days, it was found that the treatment groups had
less rotation [100, 200 mg/kg (p<0.001)].
Motor coordination in Parkinson group showed a
significant decrease compared to the control
group (p<0.01) and treatment with doses of 100
and 200 mg/kg of α-pinene could increase the
motor coordination significantly compared with
the Parkinson group (p <0.05) (figure 1).
The passive avoidance memory significantly
decreased in the group with Parkinson compared
to the control group (p<0.001) )figure 2(
furthermore, no perceptible difference was
observed between the control group and the
vehicle group. In addition, in all Parkinson's
groups doses of 100 and 200 of α-pinene
increased memory compared to the Parkinson
group (p<0.01) and (p<0.001), respectively.
Lipid peroxidation levels of experimental groups
have been shown in figures 4 and 5 and 6. MDA
level of hippocampal tissue homogenates
increased significantly compared to the control
group (p<0.05). In addition, no significant
difference was observed between the control and
the vehicle group. The results of fourteen-day
prescribing α-pinene with two doses of 100 and
200 mg/kg redused MDA in the hippocampus
tissue compared to Parkinson group (p<0.05) and
(p <0.001), respectively. On the other hand,
MDA levels increased in Parkinson's group
compared to the control group in the striatum
tissue (p<0.001). Similarly, there was no
significant difference between the control group
and vehicle group. By comparing MDA
measurement among Parkinson group that
recieved α-pinene 100, 200mg/kg for 14 days, it
was found that MDA had a significant reduction
in the group receiving α-pinene in comparison
with Parkinson group (p<0.001). For MDA
measurement in the blood, only the dose of 200
mg/kgof α-pinene significantly decreased lipid
peroxidation (p<0.001)
In the present study, the minimum amount of 6-
OHDA was used to induce Parkinson's disease.
The advantage of this model is that it is very
similar to the early stages of Parkinson's disease
in humans as well as minimizing or eliminating

Effect of α-pinene on Parkinson disease in rats
57
Figure 1. Effect of 14-day gastric gavage of of 100 and 200 mg/kg α-pinene on circling behavior in Parkinson's disease (PD).
(mean ± SEM; One-way ANOVA and Tukey's test (n=7) ); * shows significance between the control group and PD . # shows
significant difference between PD and treated groups.
Figure 2. Effect of 14-day gastric gavage of of 100 and 200 mg/kg of α-pinene a on rotarod in Parkinson's disease (PD). (mean ±
SEM; One-way ANOVA and Tukey's test (n=7) ). * shows significance between the control group and PD. # shows significant
difference between PD and treated groups.
Figure 3. Effect of 14-day gastric gavage of of 100 and 200 mg/kg of α-pinene on memory in Parkinson's disease (PD). (mean ±
SEM; One-way ANOVA and Tukey's test (n=7) ). * shows significant between the control group and PD . # shows significant
difference between PD and treated groups.

Goudarzi S. and Rafieirad M.
58 RJP 4(2), 2017: 53-63
Figure 4. Effect of α-pinene on MDA levels in hippocampus tissue between control group, PD and PD groups orally receiving
100 and 200 mg/kg of α-pinene for 14 days. (mean ± SEM; One-way ANOVA and Tukey's test (n=7) ). * shows significant
between the control group and PD . # shows significant difference between PD and treated groups.
Figure 5. Effect of α-pinene on MDA levels in striatum tissue between control group, PD and PD groups orally receiving 100
and 200 mg/kg of α-pinene for 14 days. (mean ± SEM One-way ANOVA and Tukey's test (n=7) ). * shows significant
difference between the control group and PD . # shows significant difference between PD and treated groups).
Figure 6. Effect of α-pinene on MDA levels in blood between control group, PD and PD groups orally receiving 100 and 200
mg/kg of α-pinene for 14 days. (mean ± SEM; One-way ANOVA and Tukey's test (n=7) ). * shows significant difference
between the control group and PD . # shows significant difference between PD and treated groups

Effect of α-pinene on Parkinson disease in rats
59
non-specific effects of neurotoxin on other
systems. The antioxidative treatment in the early
stages of Parkinson's disease has been examined
in the clinic. One treatment is to use antioxidants
to reduce the oxidative stress and protect
dopaminergic neurons. Biological antioxidants
play a vital role in protecting cells against
oxidative stress caused by free radicalsThe
results of the present study showed that the
chronic consumption of α-pinene improved
movement disorders in rats with Parkinson's
disease. The results of rotation testing in
Parkinson's group compared with the control
group demonstrated stricture and devastation of
dopaminergic neurons, while rotation in the
groups receiving α-pinene were much lower than
that in the lesion group, which can display
prevention from the demolition of dopaminergic
cells and reduction of motor coordination
following this destruction with the gavage of α-
pinene. Learning and passive memory avoidance
tests were performed under the same conditions
in the shuttle box for all groups. Increasing the
delay time for the first entry to the dark room and
the total time for spending in the light room and
decreasing the total time for spending in the dark
room indicated the improvement of passive
avoidance memory by α-pinene. In addition, our
results showed that the level of malondialdehyde
(MDA) in the hippocampus and striatum of
Parkinson group receiving α-pinene significantly
reduced and it possibly prevented the progression
of the disease. Some previous researches have
also investigated the effects of plant extracts on
animal models of PD. For example, oral
prescribtion of ginseng extract has led to stopping
cell damage of the substantia nigra and
decreasing the dysfunction in Parkinson rats [40].
In another study which was carried out on
Ginkgo biloba, it was found that the leaf extract
of this plant reduced the behavioral disorders
resulting from injuries caused by 6-
hydroxydopamine [41]. Studies have shown that
the unilateral damage of nigrostriatum
dopaminergic system by injecting striatal 6-
hydroxydopamine decreases the level of
dopamine and up-regulation of postsynaptic
dopaminergic receptors located on the affected
side of striatal neurons [42]. Salar et al. have
reported that the aqueous extract of barberry
could reduce the behavioral symptoms of
Parkinson by inhibiting the acetylcholinesterase
enzyme in the brain. In previous studies, barberry
extract had an antioxidant property and prevented
the production of lipid peroxidation and MAO-A
activity. As a result, the amount of dopamine and
monoamines increased in brain. MAO inhibitors
increase the amount of dopamine and
norepinephrine in the nerve synapse and they
have antidepressant effects [43]. It has been
reported that oral administration of rosemary leaf
extract induced neuroprotective effects on the
hippocampus and prevented memory impairment
caused by neurotoxin 6-hydroxydopamine.
Therefore, it might be considered to be used in
improving memory disorders in Parkinson
patients [4]. There are several reports showing
that oxidative stress is involved in the
pathogenesis of Parkinson disease by producing
free radicals and weakening the brain antioxidant
system [10,44]. Oxidative stresses cause
apoptosis and loss of dopamine cells [44,45].
Findings show that the use of plant extracts with
antioxidant substances can improve the cognitive
and motor symptoms of Parkinson disease [46].
α-Pinene is a single ring monoterpenoid [47].
Some terpenoids act as serotonin reuptake
inhibitors and increase the norepinephrine and
dopamine activity (like monoamine oxidase
inhibitors) [48-50]. It was also reported that
taking α-pinene at a dose of 100 mg/kg prevents
damage to gastric mucosa walls and protects the
gastric mucosa against acidification,
accumulation of bacteria, and mechanical forces
resulting from proteolytic digestion [51]. Various
pharmacological effects have been reported for α-
pinene, including anti-microbial, anti-
inflammatory, analgesic, antioxidant, memory-
enhancing, anti-anxiety and neuronal protection
properties [52]. Previous studies have shown that
α-pinene and many monotrepenoids have anti
acetyl-cholinesterase activity [53,54]. In addition,
such plants contain monoterpenes that are useful
for the treatment of memory disorders, including
Alzheimer's disease [55]. Findings have shown
that some plants possess terpenoids such as

Goudarzi S. and Rafieirad M.
60 RJP 4(2), 2017: 53-63
carvacrol and pinene and it has also been
reported that carvacrol, α-pinene, β-pinene, and
β-caryophyllene are able to increase GABA
evoked current responses. [56]. It seems that the
antioxidant properties of α-pinene plays an
important role in its protective effects which
suggest it to be used as an adjuvant treatment in
patients with Parkinson's disease. Antioxidant
effect of α-pinene was confirmed in the present
study by measuring oxidative stress parameters
such as malondialdehyde (lipid peroxidation) in
brain tissues and blood. α-Pinene enhances the
memory probably due to its anti-
acetylcholinesterase activity. On the other hand,
monoterpenes increase dopamine by interfering
with monoamine systems and MAO inhibition.
Terpenoids might adjust GABAergic system
modulation, which cause extra movements in the
weakened Parkinson patients. In order to treat
Parkinson, dopamine must be increased and
acetylcholine must be decreased. The dual
function of α-pinene might improve Parkinson's
symptoms and impaired memory.
Based on the results of this study, consumption of
α-pinene might protect dopaminergic neurons
against 6-OHDA-induced lesions, and possibly
could have a protective effect against Parkinson's
disease.
Acknowledgements
The authors would like to thank the Research
Deputy of Islamic Azad University, Izeh Branch
for its support to carry out this study. This article
was extracted from Mrs. Samira Goudarzi MSc
thesis. The research protocol was adopted by
Islamic Azad University, Izeh Branch based on
research ethics charter laws. This project was
financially supported by the student herself.
Declaration of interest
The authors declare that there is no conflict of
interest. The authors alone are responsible for the
content of the paper.
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