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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

Alzheimer’s 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 Alzheimer’s patients. When somebody gets

Alzheimer’s, their brain has trouble metabolizing glucose, and while it is

sometimes claimed that the brain can only run on glucose, this isn’t true.

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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

Alzheimer’s patients report (Kashiwaya et al,1999).

There have been some testimonies that the use of coconut oil (Cocos

nucifera L., Arecaceae) on patients with Alzheimer’s 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, Steve’s 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 Alzheimer’s 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

Alzheimer’s 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.

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Until now, though there are studies concerning the certain cause of

Alzheimer’s 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 Alzheimer’s drug led to many researches and reports.

News about how effective coconut oil was in treating Alzheimer’s 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 Alzheimer’s 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

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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

Alzheimer’s 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

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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 anti-amnesic effect.

Significance of the Study

The study of the effectiveness of Coconut endosperm’s 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 Alzheimer’s which is why this study will serve as a

hopeful step in finding it.

The family and/or loved ones of people with Amnesia.

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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

doesn’t 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

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Alzheimer’s 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).

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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).

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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).

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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

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

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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

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.

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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

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 27°C (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

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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 Alzheimer’s

disease include Donepezil, Tacrine, Galantamine, and Rivastigmine. In the

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).

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The Anti-Amnesic Activity of Coconut Oil

The therapeutic effects of the FDA approved AchE inhibitors to alleviate

symptoms of amnesia, dementia, and Alzheimer’s 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

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

Alzheimer’s 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

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slowing cognitive decline. In Alzheimer’s 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.

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 Alzheimer’s disease (Gunnars, 2012).

In Alzheimer’s 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

Alzheimer’s such as amnesia.

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Carbohydrates, when digested, are turned into glucose for energy. When

the body’s 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, it’s a regulating mechanism to ensure

the furnace doesn’t keep burning when no energy is required.

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, Alzheimer’s, and Parkinson’s. 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

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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

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. Diazepam-

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

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experiences. 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.

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).

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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

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

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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).

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

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and two enclosed) that are arranged to form a plus sign as described by Handley

and Mithan (Walf, A., et al., 2007).

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

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(e.g., limbic regions, hippocampus, amygdala, dorsal raphe nucleus, etc.) and

mechanisms (e.g., GABA, glutamate, serotonin, hypothalamic–pituitary–adrenal

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

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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

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, intermediate-

term 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).

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CHAPTER 3

METHODOLOGY

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 wide-

mouth 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

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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 Alzheimer’s disease.

Coconut oil induced in Swiss Albino mice

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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

(15.25 cm × 5 cm × 14 cm) 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

27

(11th 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

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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

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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.