1. The study aims to evaluate the protective effects of Zataria multiflora Boiss essential oil in a rat model of Aβ-induced Alzheimer's disease. 2. Forty rats will be divided into four groups - control, sham, Alzheimer's disease, and Zataria multiflora oil treatment. Biomarkers like MDA, acetylcholinesterase activity, and BDNF levels will be measured and histopathological changes examined. 3. Results will provide insight into how Zataria multiflora oil impacts oxidative stress, inflammation, and cholinergic function that are impaired in Alzheimer's disease.

2. Evaluation of Protective Mechanism of
Zataria multiflora Boiss. Essential Oil in
Rats With Aβ-induced Alzheimer’s disease
Shiraz University
International Division
Department of Pharmacology
By:
Narges Eskandari Roozbahani
Supervised By:
Dr. Shomali

3. Alzheimer's disease (AD), is the most common form of dementia.
Alois Alzheimer in 1906
Relation between AD and increasing in Aβ
Neuro-inflammatory
Oxidative stress
Zataria multiflora Boiss (antiseptic, anti microbial, Carminative,
anti-oxidative, anti cholinesterase, anti-inflammation and
analgesic)
 AchE, MDA, BDNF
Problem statement

4. Literature review

5. Glenner & Wong 1984; Masters et al 1985; Glenner 1988
The senile plaque in its mature form consist of a central core of an extracellular
thioflavin S and Congo-red –positive fibrous protein (Aβ). Surrounding the
amyloid core are degenerating nerve endings.
Grundke-Iqbal et al, 1986; Nukina & Ihara 1986; Wood et al., 1986
NFT consist of pair helical filaments and the related straight filaments, which are
made of the hyper phosphorylated microtubule-associated protein tau.
McGeer 1995
Although classically defined inflammation which typically includes edema and
neutrophil invasion, is not a characteristic of the AD brain, numerous acute-
phase reactant and immune related markers are present with Aβ deposition.
Microglial activation is central to the inflammatory response in AD.
Blessed et al, 1968
The number of senile plaques averaged over several neocortical association areas
correlated well with functional dementia scores and mental status score, but
abundant amyloid deposits can be present in cognitively normal individuals.

6.  Arriagada et al, 1992; Neve & Robakis 1998
 The interacellular neurofibrillary lesions correlate better
with the presence of dementia
 Coyle et al, 1983
 Cholinergic neurons in the nucleus basalis of Meynert
are reduced early in the course of AD, and the
dysfunction of cholinergic neurons is belived to be
primarily responsible for cognitive deficits in the
disease.

7.  Amyloid cascade hypothesis
 Tau hypothesis
 Cholinergic hypothesis
 Oxidative degeneration
 Slow inflammation process
AD Hypothesis

8. 1.The amyloid precursor protein (APP) is broken down by a
series of secretases.
2.During this process, a non soluble fragment of the APP protein
(called Ab-42) accumulates and is deposited outside the cell.
3.The non soluble or “sticky” nature of Ab-42 helps other
protein fragments (including ApoE) to gather into plaques.
Somehow the plaques (or possible the migration of Ab-42
outside the cell) cause neuronal death.
The plaque formation cause inflammation in brain tissue,
release AchE of plaque and has toxic effect on neurons.

10. b-secretase Pathway:

13. 1. The t (tau) protein is a microtubule-associated protein that
acts as a three-dimensional “railroad tie” for the microtubule. The
microtubule is responsible for axonal transport.
2.Accumulation of phosphate on the tau proteins cause “paired helical
filaments” or PHFs (like two ropes twisted around each other) that
accumulate and lead to the neurofibrillary tangles (NFT). PHFs are the
main component in NFTs.
3.Impaired axonal transport is the probable cause of cell death.

14. Microtubules are like railroad tracks that transport nutrition and other molecules. Tau-
proteins act as “ties” that stabilize the structure of the microtubules. In AD, tau proteins
become tangled, unstabilizing the structure of the microtubule. Loss of axonal transport
results in cell death.

15. Oxidative Stress Hypothesis
 Oxidative stress results from either a decrease of natural
cell antioxidant capacity or an increased amount of ROS
in organisms (Finkel & Holbrook, 2008).
 The brain and nervous system are vulnerable to
oxidative stress due to limited antioxidant capacity,
consumes 20% of the metabolic oxygen, neuron can not
synthesize glutathione and contain more of
polyunsaturated fatty acids. (Oboh &Rocha, 2007).

16.  Ach is found in the synapses of the cerebral cortex is
one of the major features seen in suffer of Alzheimer’s
disease (Bierer et al, 1995) and due to the high activity
of AchE, an enzyme that converts Ach into inactive
choline and acetate.
 AchE inhibitors such as tacrine, donepezil and
rivastagmine are commonly used synthetic drugs for the
treatment of AD.
Cholinergic Hypothesis

17. Brain-derived neurotrophic factor (BDNF) belongs to neurotrophin
family and has a critical role during brain development, maintains
neuronal function and structural integrity in adulthood
In AD patients, significant decrease in the level of BDNF has been
documented in hippocampal and cortical regions.

18. Transgenic mice
Destructive:
 STZ, TMT,
Non-destructive:
Aβ 1-42, Aβ 1-40, Aβ 25-35
Animal models of AD

19. Two classes of medications have been approved by the US FDA
for the treatment of AD:
 AChEIs: tacrine, donepezil, rivastigmine, galantamine. mostly
for mild-to-moderate AD,
 The noncompetitive NMDA receptor antagonist memantine for
the moderate-to-severe stages of AD
Disadvantages of current medicines:
 duration of their efficacy is limited
 undesired effects such as gastrointestinal side effects of
AChEIs, (nausea, diarrhea, vomiting, and weight loss).
And about Memantine are constipation, dizziness, headache, and
confusion

20. It is a thyme-like plant belonging to Lamiaceae family that grows wild in Iran,
Pakistan and Afghanistan.
This plant with the vernacular name of Avishan-Shirazi (Shirazi thyme) in Iran is a
valuable medicinal and condimental plant.
It has several traditional uses as an antiseptic, carminative, stimulant, diaphoretic,
diuretic, anesthetic, antispasmodic and analgesic.

21.  Jukic et al, 2007
 The anti-cholinesterase effect of thymol and carvacrol
 Hosseinzadeh et al, 2000
 Anti-inflammatory effect of the ethanoic extract
 Jaffary et al, 2000
 Anti-inflammatory effect of the essential
 Babaie et al, 2007
 Anti-oxidative effect

22.  Sharififar et al, 2007; Shahsavari et al, 2008; Saei Dehkordi et al, 2010
 Anti-oxidative effect, Scavenging the free radical, β- caroten bleaching
 Hosseinimehr et al, 2010
 Reduce the oxidative stress and genotoxicity induced cyclophosmaid
 Karamian et al, 2011
 Scavenging the nitric oxide and MDA, preventing of nitrosative stress and
lipid peroxidation
 Majlesi et al, 2011
 Amelioration of cognitive deficits in MWM test

23. Main
purpose
Evaluation of protective mechanism of
Zataria multiflora Boiss. essential oil in
rats with Aβ –induced Alzheimer's disease

24. 1. Presence of amyloid plaques in hippocampal
region
2. Level of BDNF in brain
3. MDA in brain tissue
4. Activity of the enzyme acetylcholinesterase
(AchE) in brain tissue

25. Animal
Evaluation of
Biomarkers and
Histopathological
Changes
Experimental
Design

26. In this study, 40 Wistar male rats with about 200 gr
weight will be use. Animals will be keep under standard
laboratory conditions (23 ± 2°C, 12 light and 12 dark
cycles, and standard humidity) and had free access to
water and food.
Procedures are in accordance with institutional ethical
guidelines for care and use of laboratory animals in
experiments
Animals

27. Authenticated pure essential oils of Zattaria multiflora
Boiss will purchase from Barij Essence Company and
the phytochemical analysis will be done by GC/MS.
In this study amyloid beta 1-42 (Sigma Aldrich) will
dissolve in sterile deionized water (vehicle) at the
concentration of 5 μg/ μL and incubated at 37ͦ C for 7
days to obtain the aggregated form
Essential oil

28. Experimental Design
# Group Number treatment AD induction
1 Control 10 _________ -
2 Sham 10 Vehicle (5% tween 80) orally
100 μL/Kg
20 days after induction the
AD
2μL D.W bilaterally
infused into lateral
ventricle
3 Alzheim
er ‘s
10 __________ 2μL of aggregated Aβ1-42
(10μg) bilaterally infused
into lateral ventricle
4 ZMEO 10 Z.M essential oil orally
100 μL kg-1 day-1
20 days after induction the
AD
1μL beta-amyloid 1-42
bilaterally in to CA1reagion
of the hippocampus
28

29. A-P: - 0.5 mm
DV: 4 mm
ML: ±1.5 mm
Paxinos Atlas

30. 20 days after treatment all animals will be sacrificed under deep
anesthesia and brain will be removed immediately.

31.  MDA
 Spectrophotometry
 AchE activity
 Colorimetery
 BDNF
 ELISA

32. Data analysis
Data will be expressed as Mean±SEM
The statistical significance will be
assessed by one-way ANOVA and
followed by suitable test as post hoc.
Threshold of significance p< 0.05