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CHAPTER 1
THE PROBLEM AND ITS BACKGROUND

Introduction
The memory is the most important function of the brain. It is the process
by which organisms are able to record their experiences and use this information
to adapt their responses to the environment.

Hence it is vital for survival

(Dunning J, et al, 2003). A large number of the elderly population experience

neurodegenerative conditions which includes amnesia or memory loss. Amnesia
may be due to organic or functional causes.

Organic causes include brain

damage from injury or taking CNS depressants. A psychological factor such as

defense mechanism is a type of the functional cause of amnesia (Nordqvist C,
2015).
The hippocampus plays a crucial role in memory, damage to it and related
systems is central to amnesic syndrome (Scoville & Milner, 1957). To lose
memories, a person must experience widespread brain deterioration such as
Alzheimers disease (AD) or other forms of dementia in which amnesia is the
main symptom.
In AD, the hippocampus is one of the first regions of the brain to suffer
damage. High carbohydrate meal has been shown to worsen cognitive
performance and behavior in Alzheimers patients. When somebody gets
Alzheimers, their brain has trouble metabolizing glucose, and while it is
sometimes claimed that the brain can only run on glucose, this isnt true.

Research shows that ketones from the medium chain fatty acids of coconut oil
cross the blood-brain barrier and act as neuroprotective agent and are actually a
more efficient fuel than glucose. It might help explain the cognitive benefits some
Alzheimers patients report (Kashiwaya et al,1999).
There have been some testimonies that the use of coconut oil (Cocos
nucifera L., Arecaceae) on patients with Alzheimers disease has made quite an
improvement on them. One popular case is Steve Newport who was diagnosed
with AD at the age of 51. His wife, Dr. Mary Newport who is a neonatologist at a
hospital in Tampa, Florida fed him 2 tablespoons of virgin coconut oil with every
meal and in 18 months, Steves severe dementia became mild.
Until now, the use of coconut oil as treatment for AD is not yet proven but
since the major symptom of Alzheimers disease is amnesia, the researchers
became interested to prove if the coconut oil, which is now becoming a popular
trend to cure AD, has the anti-amnesic properties.
Background of the Study
Amnesia, also called memory loss, happens when a person loses the
ability to remember information and events they would normally be able to recall.
It could be something that happened seconds or minutes ago, or a memorable
event that occurred in the past. It's normal to become a bit forgetful as a person
gets older but memory loss could be a symptom of something more serious, like
Alzheimers disease (National Health Service, UK).

Amnesia is the major

symptom of AD which is characterized by loss of memories, such as facts,

information and experiences.

Until now, though there are studies concerning the certain cause of
Alzheimers disease, it is still unclear unto how and why it affects only some
people while others are immune to it. The continuous failures of drug companies
to develop an effective Alzheimers drug led to many researches and reports.
News about how effective coconut oil was in treating Alzheimers disease is
spreading fast and is seen to be a new hope after the continuous failure of the
attempts of developing new drug spreads to the people. There was convincing
anecdotal evidence that coconut oil can halt and even reverse the progression of
symptoms of degenerative brain diseases (Alban D, 2012). It was found out that
coconut oil contains medium-chain fatty acids (MCFAs), which when converted to
ketone bodies, are responsible for enhancing the memory. Ketone bodies from
MCFAs in coconut oil bypass glucose metabolism to directly feed the brain which
alleviates the symptoms of AD and enhance memory skills (Dr. Mercola, 2013).
With that, it can be used as an alternative fuel for the brain that the body makes
when digested might offer profound benefits against AD. At this time, only few
studies are made on the prevention and treatment of Alzheimers while some are
still ongoing and people are hopefully waiting for its positive results.
The coconut is an important fruit tree in the world, especially in the tropical
and subtropical regions and with its many uses is often called the tree of life
(Chan E, Elevitch CR, 2006). Coconut oil has been consumed for thousands of
years in tropical cultures, for example; Philippines which is one of the largest
coconut-producing countries in the world and coconut has been an indelible part
of Philippine food and culture. With that, the research on the health benefits of

coconut oil has existed for a very long time, from anti-oxidant, antibacterial,
antiviral, anti-cancer activity etc. using almost all of its parts.
The researchers thought that with the abundance of coconut in the
Philippines, the researchers will have adequate resources to use to in the study
of the anti-amnesic activity of coconut oil. If managed to be proven then it will
possibly affect the economy of the Philippines. There would be an increase in
the economy due to the increase in profit of industries involved in the production
of coconut oil. The researchers would also like to venture on its property to fight
Alzheimers disease and pursue this study to contribute to the growth of new
studies for this degenerative disease and hopefully will produce good result.
Statement of the Problem
This study aims to answer the following problems:
1. What are the physical and chemical properties of prepared oil from
coconut endosperm (Cocos nucifera L., Arecaceae)?
2. What is the dose of coconut oil (Cocos nucifera L., Arecaceae) that is toxic
to 50% of the Diazepam-induced amnesic animal model population?
3. What dose of coconut oil (Cocos nucifera L., Arecaceae)
combats
Diazepam-induced amnesia in Swiss Albino mice measured by:
a) Elevated plus maze
b) Novel object recognition
4. Is there a significant difference in the treatment of amnesia in prepared oil
from coconut endosperm when compared with Donepezil (Aricept)?
Scope and delimitation of the study
The study will be conducted to determine if the coconut oil (Cocos
nucifera L., Arecaceae) is effective against amnesia. The coconut endosperm

will be used in preparation of the oil. The researchers will determine physical
properties of coconut oil to confirm its identity. Researchers will make use of
Swiss albino mice as the animal model that will be induced with Diazepam to
effectuate the amnesia.

The Swiss albino mice will undergo Marble test to

identify if Diazepam-induced amnesia effectively worked on them. Elevated plus

maze and Novel object recognition tests will be used to detect behavior of the
mice.

Donepezil will be used as reference standard and its activity will be

compared with coconut oil (Cocos nucifera L., Arecaceae) by measuring their
acetylcholinesterase inhibitory effect against amnesia. The limitation of the study
is the isolation and identification tests of the constituent that causes the antiamnesic effect.
Significance of the Study
The study of the effectiveness of Coconut endosperms oil as anti-amnesic
will be beneficial to the following:

People who have amnesia-related diseases.

The researchers are trying to find a new possible treatment for the
AD which is an amnesia-related disease. Until today, there is no existing
drug that can stop Alzheimers which is why this study will serve as a

hopeful step in finding it.

The family and/or loved ones of people with Amnesia.
It can help them financially and emotionally.

It can help them

financially because they can save a lot of money from going in and out of
the hospital, considering the expensive laboratory tests and different
medicines.

Emotionally because they would not go through the

experience where their loved one has completely forgotten them and

doesnt even recognize them.

Coconut growers/producers
They will also profit from the study because the findings will make
people use the new benefit of coconut as a big business opportunity.
Producing new medicinal products from coconut allow researchers to

conduct other related studies.

Healthcare professionals
They experience high level of stress in taking care of patients with
amnesia especially Alzheimer patients. This study can help them because
hopefully, in the future, there will be lower cases of AD, thereby lessening

their workload at the same time.

The future researchers
Researchers who will focus on amnesia or the coconut fruit can
have the opportunity to gain additional knowledge about the effects and
the treatment of Swiss albino mice induced with Diazepam which prompts
the amnesia.

Definition of Terms
Alzheimers disease (AD). A slow progressing neurodegenerative disease of the
brain and a type of dementia that causes problems with memory, thinking and
behavior (Bradbury, 2007).
Amnesia. A major symptom of AD and is defined as loss of memory, or the
inability to remember facts or events (Mastin, 2010).
Acetylcholine (ACh). Neurotransmitter which nerves use to signal muscles to
initiate or cease movement (Myers, 2005).

Acetylcholinesterase (AChE). An enzyme that degrades acetylcholine to

acetate and choline (Colovic et al., 2011).
Gamma-amino butyric acid (GABA). An amino acid which acts as a major
inhibitory neurotransmitter in the central nervous system (Deans, 2011).
Medium-chain Fatty Acids (MCFA). A type of fatty acid that by passes the liver
and is easily converted to ketone bodies in which are responsible for enhancing
memory (Henderson, 2008).
Ketone bodies. An alternative to glucose and also served as a fuel of the brain
and body when MCFA from coconut oil were digested (Henderson, 2008).
Diazepam. A CNS depressant used to induce amnesia to Swiss Albino mice
(Wolkowits, 1987).
Marble Test. Behavioral model in which Swiss Albino mice will undergo; test
used to identify if Diazepam-induced amnesia effectively worked on them
(Gulinello, 2003).
Elevated Plus Maze (EPM). Behavioral model that will be used to evaluate if the
memory of Swiss Albino mice enhanced after receiving the MCFA extracted from
coconut oil (Komada, 2008).
Novel Object Recognition (NOR). Test used to evaluate the behavior of
Diazepam-induced Swiss Albino mice, in which they will try recognizing objects
inserted in their cage during given time intervals (Antunes, 2012).

CHAPTER 2
REVIEW OF RELATED LITERATURE
This chapter presents the available literature and studies conducted that
are crucial to the investigation of the anti-amnesic activity of prepared oil from
Coconut endosperm (Cocos nucifera L., Arecaceae) against Diazepam-induced
amnesia in Swiss Albino mice.

Plant Material

Plate 1. Coconut endosperm (Cocos nucifera L., Arecaceae)

(Retrieved from: http://www.essentialcoconut.com)
The coconut (Cocos nucifera L.) is a member of the family Arecaceae of
the genus Cocos.

The coconut tree (Cocos nucifera L.) can be found in

rainforests and countries with tropical climate such as in Africa, Asia, Latin
America and the Pacific and is extensively cultivated in the Philippines, especially
in regions where the dry season is not too prolonged (Mercola, 2013). It is locally
known as Niyog and is abundant in the Southern Tagalog and Bicol regions of
Luzon and the Eastern Visayas which were the centers of coconut production
(Quisumbing, 1978).
The fruit, commonly called coconut, is often obscurely 3-angled, 15 to 25
centimeters long. The coconut has a smooth hard protective outer layer, usually
green, but others may be pale yellow, silver-gold or brown depending on variety
and age. Coconut fruit has a husk of about 1 to 2 inches thick with an inner
hollow round seed of about 5 to 7 inches in diameter. The coconut seed has a
hard shell that when cracked open will reveal a layer of white edible meat, called
endosperm, and about a glassful of watery coconut milk. Endosperm forms a

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thick white layer of fleshy fibrous substance adherent to the membranous testa
which is adherent to the stony-black shell (Yong et al., 2009).
Folkloric Use
The oil from the coconut endosperm is highly nutritious and rich in fiber,
vitamins, and minerals. Coconut is classified as a "functional food" because it
provides many health benefits beyond its nutritional content (Fife, 2015).
Coconut oil is used in traditional medicine for baldness, bruises, burns,
wounds, colds, constipation, cough, dysentery, earache, fever, irregular or painful
menstruation, lice, dandruff, malnutrition, nausea, rash, scabies, scurvy, skin
infections, sore throat, swelling, toothache, and upset stomach (Hahn, 1997).
In Amboinia, coconut oil is used as vermifuge. Lauric acid, the dominant fatty
acid in coconut oil, finds application in cooking, detergents, soaps and cosmetics.
Medicinal Use
Coconut oil contains antimicrobial lipids, capric acid, caprylic acid and
lauric acid that are known to possess antibacterial property. It can also be used
as to treat fungi and yeast infections such as ringworm, athlete's foot, thrush,
diaper rash and candidiasis.

Coconut oil stabilizes blood sugar and insulin

production (Bardot, 2013).

A study of warm water crude extract of coconut endosperm showed that it
has protective effects on ulcerated gastric mucosa (Quisumbing, 1978). Another
study showed the crude methanol extract of oil from coconut endosperm contains

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phytochemical constituents that significantly reduced the parasitemia in all 3 in

vivo assessment assays in the treatment of malaria (Jenson, 2011).
Coconut oil helps promote weight loss, protects against heart disease,
cancer, diabetes, arthritis, stops premature aging of the skin, strengthens the
immune system and improves digestion (Fife, 2015).
Studies involving medium chain fatty acids (MCFAs) from coconut oil were
reported to alleviate the symptoms of AD and enhance memory skills. Studies
suggest further research in other neurodegenerative disorders (Mercola, 2013).
Plant Constituent
The composition of crude coconut oil includes triacylglycerols, free fatty
acids, partial glycerides, phospholipids, sterol, tocopherols, pigments, volatiles,
trace metals and oxidized products (Rahman, 2000). Coconut oil is made up of a
mixture it is composed with roughly 92% saturated fatty acids, 6%
monounsaturated fatty acids, and 2% polyunsaturated fatty acids (Creasy, 2015).
It is one of the richest sources of saturated fat you can find.
It contains predominantly medium chain triglycerides.

Medium chain

triglycerides (MCTs) are a class of lipids in which three saturated fats are bound
to a glycerol backbone. What distinguishes MCTs from other triglycerides is the
fact that each fat molecule is between six and twelve carbons in length. MCTs
are a component of many foods, with coconut one of being the dietary sources
with the highest concentration of MCTs.

The oil is highly stable towards

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atmospheric oxidation.

The oil is characterized by a low iodine value, high

saponification value, and high saturated fatty acids content and is a liquid at
room temperatures of 27C (Prakash et.al, 2007). It contains a high proportion of
glycerides of lower chain fatty acids known as medium chain fatty acids.
The saturated fat in coconut oil is made up of seven different types of
fatty acids, including caproic, caprylic, capric, lauric, myristic, palmitic, and stearic
acid.

The most abundant medium-chain fatty acid in coconut oil is the 12-

carbon Lauric acid, which is broken down into a compound called monolaurin in
the body. It is primarily 44.6% lauric acid, 16.8% myristic acid, 8.2% palmitic acid
and 8% caprylic acid. Although it contains seven different saturated fatty acids in
total, its only monounsaturated fatty acid is oleic acid while its only
polyunsaturated fatty acid is linoleic acid. Most of the fatty acids in the diet are
long-chain fatty acids, but the medium-chain fatty acids in coconut oil are
metabolized differently (Gunnars, 2012).
The Acetylcholinesterase Inhibitor Activity of Donepezil
The treatment of memory loss or amnesia is by modifying the
neurotransmitters involved in the regions of the brain that affect the memory
function with the use of acetylcholinesterase (AChE) inhibitors. These regions
include the Hippocampus, Cerebellum, Amygdala, Basal ganglia and cortical
structures (Westmoreland, B., 1994). The members of AChE inhibitors which are
widely used as pharmacotherapy for amnesia, dementia, and Alzheimers
disease include Donepezil, Tacrine, Galantamine, and Rivastigmine. In the

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1970s, three laboratories independently found that the activity of choline

acetyltransferase (ChAT), the enzyme synthesizing acetylcholine (ACh), was
markedly reduced in the cerebral cortex of AD brain which causes amnesia
(Bowen D., Davies P., Perry E., 1976). This shows that the central cholinergic
system is involved in cognitive dysfunctions so studies about AChE inhibitors
were made to alleviate the cognitive deficits. The mechanism of action of AChE
inhibitors is to stop the activity of AChE which is the enzyme that breaksdown
ACh.
Donepezil hydrochloride is the second drug approved by the U.S. FDA for
the treatment of mild to moderate AD. It is a new class of AChE inhibitor having
an N-benzylpiperidine and an indanone moiety that shows longer and more
selective action. Donepezil was associated with better performance in memory
and thinking tests in patients who were on the active medication compared with
patients taking a placebo but it should be stressed that the degree of
improvement was modest, and more than half of the patients showed no
improvement at all (Morris, J., 2003).
The Anti-Amnesic Activity of Coconut Oil
The therapeutic effects of the FDA approved AchE inhibitors to alleviate
symptoms of amnesia, dementia, and Alzheimers disease are still not significant
because no permanent improvement on the patients have been achieved
(Youdim, K., et al, 2004). This is why medicinal plants have been considered in

14

searching for new drugs. The antioxidant property of most medicinal plants that
show efficacy against amnesia makes them more preferred over synthetic drugs.
According to an article of Philippine Coconut Authority, the coconut oil was
first tried by Dr. Mary Newport to her husband Steve as treatment for his
Alzheimers disease. Mary was able to dig up the results for the new drug called
AC-1202 (later known as Axona).

She learned from the journal, BMC

Neuroscience, that the active ingredient in the drug was MCT (medium-chain
triglycerides), an oil that is used to treat epilepsy and also placed in hospital
feeding programs for newborns. She knew as a pediatrician that babies needed
to develop their brains with the right brain foods and MCT was found in
great quantities in virgin coconut oil (VCO). Axona works by targeting brain cells
to nutritionally support cognition. According to Accera Inc, developer of the said
medicine, adding Axona to Alzheimer's disease management may address a
specific nutritional deficiency not addressed by FDA-approved Alzheimer's
treatments.
Coconut oil works the same way as Axona because of the medium chain
fatty acids (MCFAs) that is then converted into ketones which is effective for
slowing cognitive decline. In Alzheimers disease, the engines inside neurons
become defective. Even if there is enough glucose in the body, neurons cannot
use it properly. This is called diminished cerebral glucose metabolism (DCGM),
or glucose hypometabolism, and it most often occurs in the areas of the brain
involved in memory and cognition (Cunnane S, et al. 2011). Clinical studies have
shown that raised ketone body levels can enhance the memory and cognition.

15

Ketones are a special type of high-energy fuel produced in the liver specifically to
nourish the brain.

Ketones are produced from fat stored in the liver. Under

normal conditions, only a small amount of ketones circulate in our blood, but as
blood glucose levels go down, ketone production steps up. This way the brain
has a continual supply of either glucose or ketones to rely on (Reger, M.A., et
al. 2004)
Mechanism of Action of Coconut Oil
Medium-chain fatty acid metabolized straight to the liver from the digestive
tract, where they are used as a quick source energy or turned into so-called
ketone bodies, which can have therapeutic effects on brain disorders like
epilepsy and Alzheimers disease (Gunnars, 2012).
In Alzheimers patients, there appears to be a reduced ability to use
glucose for energy in certain parts of the brain. Ketone bodies can supply energy
for the brain and researchers have speculated that ketones can provide an
alternative energy source for these malfunctioning cells and reduce symptoms of
Alzheimers such as amnesia.
Carbohydrates, when digested, are turned into glucose for energy. When
the bodys energy requirements have been satisfied, and there is still glucose left
over, the body restructures the glucose into fat molecules for storage. In order for
the body to use the glucose as energy, insulin is required, like a key in the lock,
to open the door for the glucose. Again, its a regulating mechanism to ensure the
furnace doesnt keep burning when no energy is required.

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Like glucose, ketones can provide the body with energy. Unlike glucose,
however, no insulin is required for the cells to accept the ketones. This makes
ketones necessary for people whose cells have become insulin resistant. One
area this has had an incredible impact on is in neurodegenerative diseases like
dementia, Alzheimers, and Parkinsons. Brain cells that are no longer able to
accept glucose for energy can thrive on the ketones supplied by the ingestion of
medium chain triglycerides (Gursche, 2013).
Ketone bodies are a key physiological replacement fuel preserving brain
function during periods of low glucose availability, and the brain has a transport
system for ketones independent of glucose transport. If brain ketone metabolism
is not lower in AD or is less affected than glucose, one potential strategy to
improve brain fuel availability and reduce the risk of AD that has already been
targeted in clinical studies would be to develop a way to safely and reliably
provide the brain with ketones as an alternative fuel to glucose (Cunnane et.al.,
2011).
Diazepam Induce In Swiss Albino Mice
Diazepam is a benzodiazepine derivative with anti-anxiety, sedative,
hypnotic and anticonvulsant properties. Diazepam potentiates the inhibitory
activities of gamma-aminobutyric acid (GABA) by binding to the GABA receptor,
located in the limbic system and the hypothalamus thereby producing a calming
effect. This increases the frequency of chloride channel opening, allowing the
flow of chloride ions into the neuron and ultimately leading to membrane

17

hyperpolarization and a decrease in neuronal excitability (National Cancer

Institute, US). It should only be used for the shortest period of time possible.
Otherwise Diazepam can cause tolerance, dependence and even withdrawal
symptoms in some people. The effects of Diazepam may last for a few hours or
even days after you stop having it (National Health Institute, UK).
Diazepam is given to Swiss albino mice to induce amnesia. Diazepaminduced amnesia serves as the interoceptive behavioral model. Central
cholinergic system plays a major role in regulation of cognitive function. Drugs
that reduce cholinergic function produce amnesia in laboratory animals. In the
present study, scopolamine and diazepam significantly impaired memory of mice
(Dhingra D., Kumar V., 2012). Benzodiazepine receptor agonists such as
diazepam and alprazolam have been shown to produce anterograde amnesia in
rodents (Preston et al., 1989; Singh et al., 1998) and human beings (Lister et al.,
1985)
Pathophysiology of Amnesia
Amnesia refers to a specific deficit in new learning and memory (Erickson
KR, 1990).
experiences.

It involves the loss of memories, such as facts, information and

Real-life amnesia generally doesn't cause a loss of self-identity.

Instead, people with amnesia also called amnestic syndrome are usually
lucid and know who they are, but may have trouble learning new information and
forming new memories (MayoClinic, 2014).
Processing of memories involves registration which is taking in new
information, encoding which is forming associations, time stamps, and retrieval.

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Deficits in any of these steps can cause amnesia. Amnesia, by definition, results
from impairment of memory functions, not impairment of other functions. For
example: attention, motivation, reasoning and language, which may cause similar
symptoms. (Huang J., 2013)
Amnesia can result from diffuse cerebral impairment, bilateral lesions, or
multifocal injuries that impair memory-storage areas in the cerebral hemispheres.
Predominant pathways for declarative memory are located along the medial
parahippocampal region and hippocampus as well as in the inferomedial
temporal lobes, orbital surface of the frontal lobes or basal forebrain, and
diencephalon which contains the thalamus and hypothalamus (Huang J., 1013).
There are two causes of amnesia, Organic and Functional. Organic
causes of amnesia may include brain damage through injury, or the use of
specific drugs - usually sedative drugs. Amnesia is one of the symptoms of some
degenerative brain diseases, such as Alzheimer's disease wherein cholinergic
neurons deficit is involved.

Functional causes of amnesia are psychological

factors, such as defense mechanisms (Nordqvist, C., 2015).

Acetylcholine is a neurotransmitter, one of the chemicals that neurons use
to communicate with each other.

In the body, acetylcholine is the primary

neurotransmitter which nerves use to signal muscles to initiate or cease

movement. In the brain, acetylcholine is produced in several locations including
the basal forebrain. It may promote learning. Acetylcholine-producing cells in the
basal forebrain are damaged in the early stages of Alzheimer's disease, which

19

may contribute to the memory impairments which are an early symptom of the
disease. Currently marketed as Alzheimer's drugs, are cholinesterase inhibitors,
meaning that they increase the effectiveness of acetylcholine in the brain (Myers
C.E., 2006).
On the other hand, Acetylcholinesterase is involved in the termination of
impulse transmission by rapid hydrolysis of the neurotransmitter acetylcholine in
numerous cholinergic pathways (Colovic M., Bondzic A., et. al., 2013). It is found
in the synapse between nerve cells and muscle cells. It waits patiently and
springs into action soon after signals are passed, breaking down the
acetylcholine into its two component parts, acetic acid and choline. This
effectively stops the signal, allowing the pieces to be recycled and rebuilt into
new neurotransmitters for the next message (Goodsell D., 2004).
Marble Test
Marble burying test is used as a quick, simple, high throughput test with
predictive validity for pharmacologically active CNS compounds such as
anxiolytics and selective serotonin reuptake inhibitors (SSRIs). This burying task
can be used both as an indicator of obsessive compulsive-like (OCD) behavior
and/or anxiety-like behavior. When mice are placed in a novel cage, a common
response is to show increased digging behavior. Mice with OCD-like symptoms
tend to engage in a high degree of repetitive behaviors (including digging). Mice
with a high degree of anxiety tend to engage in a high degree of digging in novel
contexts (potentially as a means to attempt to escape the new cage).

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It is also to record the number of marbles buried by mice placed in a novel

environment. Mice, which are placed individually in a cage, bury glass marbles
that are present in the cage. This behavior belongs is probably a type of
defensive burying typical of rodents. It does not seem that marbles are
specifically aversive, although making them so by coating them or pairing them
with aversive substances does increase burying.

It has been shown to be

sensitive to benzodiazepines as these compounds reduce burying behavior when

compared to vehicle-treated control mice, without inducing changes in locomotor
activity. Results have shown that in addition to benzodiazepines, this test system
is sensitive to the effects of selective serotonin reuptake inhibitors (SSRIs),
serotonin noradrenaline reuptake inhibitors (SNRIs) and tricyclic antidepressants
(TCAs).
Elevated Plus Maze
The Elevated Plus Maze (EPM) is an animal model test which is used to
assess anxiety-related behavior in rodent models of CNS disorders. The elevated
plus maze test is one of the most popular tests of all currently available animal
models of anxiety (Rodgers and Dalvi, 1997; Crawley, 2007). EPM was modified
from Y-shaped apparatus described by Montgomery into four arms (two open and
two enclosed) that are arranged to form a plus sign as described by Handley and
Mithan (Walf, A., et al., 2007).

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Plate 2. Elevated Plus Maze (EPM)

(Retrieved from: www.cidd.unc.edu)
It was described as the number of entries onto the open arms versus
number of total arm entries, and time spent on the open arms versus closed
arms; provide the measures of anxiety-like behavior (Munekazu, K., 2008).
Unlike other behavioral assays used to assess anxiety responses that rely upon
the presentation of harmful stimuli (i.e., electric shock, food/water deprivation,
loud noises, exposure to predator odor, etc.) that typically produce a conditioned
response, this test relies upon rodents' natural tendency to go toward dark,
enclosed spaces (approach/comfortable places) and an unconditioned fear of
heights/open spaces (avoidance/ risky spaces) (Frye, C., et al., 2007).
As subjects freely explore the maze, their behavior responses are easily
assessed and quantified by an observer. Other ethological measures that can be
observed in rodents in the maze are the number of rears, head dips, defecation,
and freezing or stretched-attend postures. Furthermore, beyond its utility as a
model to detect anxiolytic effects of benzodiazepine-related compounds, the
elevated plus maze can be used as a behavioral assay to study the brain sites
(e.g., limbic regions, hippocampus, amygdala, dorsal raphe nucleus, etc.) and
mechanisms (e.g., GABA, glutamate, serotonin, hypothalamicpituitaryadrenal

22

axis neuromodulators, etc.) underlying anxiety behavior, because of the ease of

employment and the vast number of studies already in the literature (Walf, A., et
al., 2007).
Novel Object Recognition
Since the beginning of the twentieth century, Animal models of memory
have been considered as the subject of many scientific publications. In humans,
memory is often accessed through spoken or written language, while in animals,
cognitive functions must be accessed through different kind of behaviors in many
specific, experimental models of memory and learning (Antunes, M., 2011). One
of them is the Novel Object Recognition (NOR) test that can be evaluated by the
differences in the exploration time of novel and familiar objects. The NOR test is
particularly attractive because it does not require external motivation, reward, or
punishment but a little training or habituation is required, and it can be completed
in a relatively short time (Silvers et al. 2007). A mouse is presented with two
similar objects during the first session, and then one of the two objects is
replaced by a new object during a second session. The amount of time taken to
explore the new object provides an index of recognition memory (Leger, M.,
2013).
The test consists of three phases: habituation, familiarization, and test
phase. In the habituation phase, each animal is allowed freely exploring the
open-field arena in the absence of objects. Familiarization phase, the improvised
box contains two identical sample objects in which the mouse will be placed for a
few minutes. In the test phase, the animal is returned to the open-field arena with

23

two objects, one is identical to the sample and the other is novel (Ennaceur,
2010). The NOR task is very useful to study short-term memory, intermediateterm memory, and long-term memory, through manipulation of the retention
interval, i.e., the amount of time animals must retain memory of the sample
objects presented during the familiarization phase before to the test phase, when
one of the familiar objects is replaced by a novel one (Taglialatela et al., 2009).
When animals are exposed to a familiar and a novel object, they approach
frequently and spend more time exploring the novel than the familiar one
(Ennaceur, 2010). Despite animals spent more time exploring the novel object,
the recognition performance varies according to the delay between the
familiarization and the test phase, as well as the time of exploration of the sample
during the familiarization phase (Ennaceur and Delacour 1988).
Brain structure (parahippocampal regions of the temporal lobe) plays an
important role in recognition memory formation, and when some damage exists,
the performance in recognition memory tasks is impaired (Albasser et al. 2009).

CHAPTER 3
METHODOLOGY

24

This chapter presents the methods and procedures that will be used for
the determination of the anti-amnesic activity of prepared oil from coconut
endosperm (Cocos nucifera L., Arecaceae) against Diazepam-induced amnesia
in Swiss Albino mice.
Preparation of Virgin Coconut Oil
Researchers will use a mature, brown coconut, rather than a young green
one and split with a sharp cleaver. Then scrape the meat of the coconut from the
shell. Cut the coconut meat into small pieces or shred the coconut flesh with the
scraper. Place the pieces into a food processor. Turn on the food processor to a
medium speed and blend until well shredded. Add a little water to help it blend if
necessary. Filter the coconut milk. Put a coffee filter or cheesecloth over a widemouth jar. Pour or spoon a small amount of the coconut mixture onto the cloth.
Wrap the cloth around the coconut mixture and squeeze the milk into the jar.
Squeeze hard, to make sure you get every last drop. Repeat this process until all
of the coconut mixture has been used. Leave the jar unattended for at least 24
hours. As it sets, the coconut milk and oil will separate and a layer of curd will
appear at the top of the jar. Refrigerate the jar so the curd hardens more quickly.
Scoop out the curd with a spoon and discard it. The pure virgin coconut oil is left
in the jar.
Physical determination of coconut oil

25

After the preparation of the coconut oil, researchers will determine its
physical properties. Researcher will be using organoleptic evaluation, solubility
test and stain test and pH determination.
Diazepam-Induced Amnesia in Swiss Albino mice
Diazepam injection is dissolved separately in normal saline and injected
IP, volume of IP injection is 1 ml/100 g of mouse. Amnesia is induced in separate
groups (interoceptive model) of mice by diazepam (1 mg/kg, IP) on 15th day after
90 minutes of the last dose of Coconut oil.
Marble Test
The test is carried out in a cage (26 x 16 x 14mm) filled approximately 10
cm deep with wood chip bedding, slightly tamped down to make a flat, even
surface. Place a regular pattern of glass marbles on the surface, evenly spaced,
each about 4 cm apart. The room should be quiet enough so that the mice would
be undisturbed. A digital video camera is placed overhead on the ceiling which
detects and records the behavior of the Swiss Albino mice. Place one mouse in a
cage and leave for 30 minutes. The mouse is then carefully removed from the
cage after 30 minutes. The number of marbles buried (to 2/3 their depth) with
bedding will be counted. The lesser number of marbles covered is used as an
indicator of amnesia, as a major symptom of Alzheimers disease.
Coconut oil induced in Swiss Albino mice

26

One group of Swiss Albino mice is fed of very low carbohydrate, high
saturated fat food and water for 15 days. After 15 days, each mouse will be
injected 0.1ml/kg of the prepared coconut oil intraperitoneally. The coconut oil will
be injected to the mice every day.
Elevated Plus Maze (EPM)
The Elevated Plus Maze for Swiss Albino mice is made of illustration
board, and consist of two open arms (15.25 cm 5 cm) and two covered arms
(15.25cm 5cm 14cm) extended from a central platform (5 cm 5 cm). Each
arm of the maze is attached to sturdy wood legs such that it is elevated 30 cm off
from the floor. The maze is placed in a separate brightly lit room that is
illuminated with fluorescent overhead lights which produce consistent illumination
within the room. A digital video camera is mounted overhead on the ceiling which
detects and records when the mice enter the open or closed arms of the maze
and the time spent in each.
On the first day, each mouse was placed at the end of an open arm, facing
away from the central platform. Transfer latency (TL) was defined as the time
taken by the animal to move from the open arm into one of the covered arms with
all its four legs. TL was recorded on the first day for each mouse. If the animal did
not enter into one of the covered arm within 90 seconds, it was gently pushed
into one of the two covered arms and TL was assigned as 90 seconds. The
mouse was allowed to explore the maze for another 2 minutes and then returned
to its home cage. Retention of this learned-task was examined 24 hours (11th

27

day) after the first day trial. The distance moved, mean velocity, time and
frequency in each zone, total and percent time in open arms, and the number of
entries into open arms are collected for analysis.
Novel Object Recognition (NOR)
The improvised testing arena is made of an illustration board which is 40
cm long, 40cm wide and 50cm tall. Testing is conducted under dim white-light
illumination.

A digital video camera is placed overhead on the ceiling which

detects and records the behavior of the mice.

On the first day, mouse is placed into the center of the arena. After five
minutes, return the mouse to its cage. Test the remaining mouse. Two novel
objects are placed into the arena. Place the mouse into the center of the arena.
After five minutes, return the mouse to its cage.
On the second day, four identical novel objects are placed in the testing
arena. The mouse is then placed into the center of the arena, after five minutes,
return the mouse to its cage. Four identical novel objects are placed in each
corner of the testing arena. Fifty minutes after, place the mouse into the center of
the arena. After five minutes, return the mouse to its cage.
If memory is functioning normally, the mouse will spent more time
exploring the novel object than it does exploring the familiar objects. If
exploration of all objects is the same, this can be interpreted as a memory
deficit. The time spent with only nose point in object zone, time spent with nose

28

point in proximity of the object, percentage of body elongation and the orientation
of the animal relative to an object is measured to determine if the prepared
coconut oil exhibits an anti-amnesic activity in the Diazepam-induced amnesia in
Swiss Albino mice.
Brain Isolation
The hippocampus is one of the most widely studied areas in the brain
because of its important functional role in memory processing and learning, its
remarkable

neuronal

cell

plasticity,

and

its

involvement

in

epilepsy,

neurodegenerative diseases, and psychiatric disorders. (Hagihara, H., 2009). In

isolation of the mouse brain, use cervical dislocation to prevent pre- and
postsynaptic effects of anesthesia and a surgical scissor to remove the head with
a cut posterior from the ears. Make a small incision (Iris scissors) on the top of
the skull starting from the caudal part at the point of the (inter) parietal bone, be
careful not to cut through the brain. Make a firm cut through the most anterior
part of the skull, between the eyes. This enables to remove the brain more easily.
Take care of the meninges as these could rupture the brain while taking it out.
Carefully cut the cranial nerves and gently lift the brain out of the skull. Wipe off
excess blood and transfer the brain on ice to cool down immediately.
Nitric oxide (NO) is an important signaling molecule that is widely used in
the nervous system. Abnormal NO signaling could therefore contribute to a
variety

of

neurodegenerative

pathologies

such

as

stroke/excitotoxicity,

Alzheimer's disease, multiple sclerosis, and Parkinson's disease. NO level in

29

hippocampal tissue might increase significantly by Diazepam injection;

pretreatment with coconut oil will reverse the NO induced amnesia in mice.
Measurement of Acetylcholinesterase activity in isolated brain of Swiss
Albino mice
After the mice were killed by cervical dislocation their brains were
removed and rapidly dissected at 00C to obtain total cortex, hippocampus,
midbrain and striatum. After weighing the dissected areas were stored at - 20OC
until analysis which was within four weeks. Brain regions were homogenized in
20 volumes per wet weight of ice-cold 0.03 M sucrose, and homogenates were
frozen and thawed once to liberate, as far as possible, the soluble AChE. Aliquots
of this total homogenate were then centrifuged at 50,000g for 120 min to
sediment the membranes and their bound AChE. Acetylcholinesterase activities
of the total homogenate and supernatant were measured by a stopped assay
version (Chubb & Smith, 1976) of the Ellman assay (Ellman et al., 1961) using
1.0mM acetylthiocholine as substrate. Protein was measured in the soluble and
total fractions by the method of Lowry et al. (1951) after precipitation by 6%
trichloroacetic acid.
Using Donepezil as control
Donepezil hydrochloride will be dissolve freshly each day in physiological
saline. After the Swiss Albino mice received diazepam to model amnesia for
hours, mice will be receiving one of 3 doses of donepezil (0.03 mg/kg, 0.1 mg/kg,
0.3 mg/kg). Doses of donepezil were chosen according to our previous
validations (Bartko et al., 2010) and other published findings with transgenic

30

mouse models (Yuede et al., 2007). The order of doses was counterbalanced in
a Latin square design for each group and stimulus duration. Each dosing day
was followed by three drug-free washout days where mice were tested with a
stimulus duration of 2 s.