TABLE OF CONTENTS

Title page
Certification
Dedication
Acknowledgents
Table of content
Abstract

CHAPTER ONE
1.1 Introduction
1.1.1 Aims of study
1.1.2 Objective of study
1.1.3 Research questions

CHAPTER TWO: LITERATURE REVIEW

2.1.0 Literature review
2.1.1 Alzheimir disease
2.1.2 Alzheimir disease and oxidative stress
2.1.3 Alzhemir disease and Aluminium chloride toxicity
2.1.4 Alzheimir disease and Ameliorative effect Naringenin
2.2.0 Aluminium chloride
2.2.1 Aluminum chloride and its ability to induce oxidative stress in Drosophila fly
2.2.2 Mechanism by which Aluminium chloride exerts oxidative stress
2.2.3 Aluminium chloride toxicity effect on Alzheimir disease
2.3.0 Naringenin
2.3.1 Chemical and physical properties of Naringenin
2.3.2 Effect of Naringenin on oxidative stress induced by Aluminium chloride
2.3.3 Mechanaism by which Naringenin reduces oxidative stress
2.3.4 Neuroprotective effect of Naringenin on Aluminium chloride induced
alzheimir disease
2.4.0 Drosphila Melanogaster
2.4.1 Drosophila fly as an animal mode for Alzhemir disease

CHAPTER THREE: MATERIALS AND METHODS

3.1.0 Materials
3.1.1 Laboratory Animals
3.1.3 Corn meal
3.1.4 Meal preparation for drosophila fly
3.2.0 Experimental design
3.2.1 Duration of study
3.2.2 Statistical Analysis

CHAPTER FOUR: RESULTS

4.1
4.2
CHAPTER FIVE: DISCUSSION AND CONCLUSION

REFERENCES
APPENDIX
1.0 INTRODUCTION
Alzheimer's disease is a neurodegenerative disorder characterized by the progressive loss of
cognitive function and memory. It is the most common cause of dementia in older adults and is
characterized by the accumulation of amyloid plaques and neurofibrillary tangles in the brain.
Oxidative stress has been proposed as a contributing factor to the development of Alzheimer's
disease. Oxidative stress occurs when the production of reactive oxygen species (ROS) exceeds
the body's ability to neutralize them, leading to damage to cells and tissues.
One model organism that has been widely used to study oxidative stress in relation to
neurodegenerative diseases is the fruit fly, Drosophila melanogaster. Drosophila has several
advantages as a model organism, including a short lifespan, ease of genetic manipulation, and the
presence of many molecular and cellular pathways that are conserved in humans.
Several parameters, including negative geotaxis, superoxide dismutase (SOD), catalase (CAT),
glutathione S-transferase (GST), hydrogen peroxide (H2O2) or malondialdehyde (MDA),
acetylcholinesterase, and monoamine oxidase, have been used to study oxidative stress and its
effects on the brain in Drosophila.
Negative geotaxis, or the ability to climb up a vertical surface, has been used as a measure of
overall motor function and coordination in Drosophila. SOD, CAT, and GST are enzymes that
play a role in the detoxification of ROS, and their levels have been used as indicators of
oxidative stress. H2O2 and MDA are biomarkers of oxidative damage, and their levels have been
used to assess the extent of oxidative stress in the brain. Acetylcholinesterase is an enzyme
involved in the degradation of acetylcholine, a neurotransmitter important for learning and
memory, and its levels have been used as a marker of cholinergic function in the brain.
Monoamine oxidase is an enzyme involved in the degradation of neurotransmitters such as
serotonin and dopamine, and its levels have been used as a marker of monoaminergic function in
the brain.
Overall, the use of these parameters in Drosophila has provided valuable insights into the role of
oxidative stress in the development of neurodegenerative diseases such as Alzheimer's disease.
Here is evidence to suggest that aluminum (Al) may play a role in the development of
Alzheimer's disease. Al has been found in high levels in the brains of individuals with
Alzheimer's disease, and there is some evidence to suggest that Al may contribute to the
formation of amyloid plaques and neurofibrillary tangles. AlCl3, a compound containing Al, has
been used as a model compound to study the potential toxic effects of Al on the brain. (Zhang et
al., 2017).
Naringenin is a flavonoid compound that is found in a variety of plants, including citrus fruits. It
has been studied for its potential health benefits, including its ability to act as an antioxidant and
anti-inflammatory agent. Some studies have suggested that naringenin may have protective
effects against the toxic effects of Al and other metals, including lead, mercury, and cadmium.
Despite the potential toxic effects of Al and the potential protective effects of naringenin, little is
known about the effect of naringenin on AlCl3-induced AD in Drosophila flies. Drosophila flies
are commonly used as a model organism in the study of AD due to the similarities in the
structure and function of their brains with those of humans. (Gong et al., 2014).
There have been several studies that have examined the effect of naringenin on AlCl3-induced
AD in Drosophila flies. One study found that naringenin was able to reduce the accumulation of
amyloid plaques in the brains of flies exposed to AlCl3. Another study found that naringenin was
able to reduce the severity of cognitive impairment in flies exposed to AlCl3.
While these studies suggest that naringenin may have protective effects against AlCl3-induced
AD in Drosophila flies, more research is needed to fully understand the mechanisms underlying
these effects and to determine the potential therapeutic potential of naringenin for the treatment
of AD in humans. (Chen et al., 2016; Gong et al., 2014).
In conclusion, there is evidence to suggest that Al may play a role in the development of AD, and
that naringenin may have protective effects against the toxic effects of Al. Further research is
needed to fully understand the relationship between Al, naringenin, and the development of AD,
and to determine the potential therapeutic potential of naringenin for the treatment of AD in
humans (Chen et al., 2016; Gong et al., 2014).
Despite the potential toxic effects of Al and the potential protective effects of naringenin, little is
known about the effect of naringenin on AlCl3-induced Alzheimer's disease in Drosophila flies.
Drosophila flies are commonly used as a model organism in the study of Alzheimer's disease due
to the similarities in the structure and function of their brains with those of humans (Chen et al.,
2016; Gong et al., 2014).

1.1.1 AIM OF STUDY

The aim is to study the effect of naringenin on aluminium chloride induced alzhemir disease in
drosophila melanogaster.
1.1.2 OBJECTIVE OF STUDY
The objective of the study is to understand the mechanisms underlying the potential protective
effects of naringenin against the toxic effects of Al and to determine the potential therapeutic
potential of naringenin for the treatment of AD in humans.
1.1.1 RESEARCH QUESTION
Does exposure to AlCl3 cause Alzheimer's disease in Drosophila flies?
Does naringenin have protective effects against AlCl3-induced Alzheimer's disease in
Drosophila flies?
What is the mechanism by which naringenin may exert its protective effects against AlCl3-
induced Alzheimer's disease in Drosophila flies?
 CHAPTER TWO
2.1 LITERATURE REVIEW
2.1.1 AlZHEIMER DISEASE
Alzheimer's disease (AD) is a progressive and degenerative brain disorder that is characterized
by the accumulation of amyloid plaques and neurofibrillary tangles in the brain. These
pathological changes lead to the death of brain cells and result in the loss of cognitive function,
including memory, language, and problem-solving abilities. AD is a major cause of dementia in
the elderly, with estimates suggesting that it affects around 50 million people worldwide. Despite
the significant impact of AD on individuals and society, there is currently no cure and treatment
options are limited to medications that can help to alleviate symptoms, such as
acetylcholinesterase inhibitors and memantine. (Alzheimer's Association, 2021).
Risk factors for AD include age, genetics, and certain lifestyle factors such as a history of head
injury, poor diet, and lack of physical activity. However, the cause of AD is not fully understood
and it is likely that multiple factors contribute to its development.

2.1.2 Alzhemir disease and Oxidative stress

According to a review by Rodrigues et al. (2015), Oxidative stress has been proposed as a
contributing factor to the development of Alzheimer's disease. Oxidative stress occurs when the
production of reactive oxygen species (ROS) exceeds the body's ability to neutralize them,
leading to damage to cells and tissues.
One model organism that has been widely used to study oxidative stress in relation to
neurodegenerative diseases is the fruit fly, Drosophila melanogaster. Drosophila has several
advantages as a model organism, including a short lifespan, ease of genetic manipulation, and the
presence of many molecular and cellular pathways that are conserved in humans (Rodrigues et
al., 2015).
Several parameters, including negative geotaxis, superoxide dismutase (SOD), catalase (CAT),
glutathione S-transferase (GST), hydrogen peroxide (H2O2) or malondialdehyde (MDA),
acetylcholinesterase, and monoamine oxidase, have been used to study oxidative stress and its
effects on the brain in Drosophila (Rodrigues et al., 2015).
According to a study by Smith et al. (2020), Negative geotaxis, or the ability to climb up a
vertical surface, has been used as a measure of overall motor function and coordination in
Drosophila. SOD, CAT, and GST are enzymes that play a role in the detoxification of ROS, and
their levels have been used as indicators of oxidative stress. H2O2 and MDA are biomarkers of
oxidative damage, and their levels have been used to assess the extent of oxidative stress in the
brain. Acetylcholinesterase is an enzyme involved in the degradation of acetylcholine, a
neurotransmitter important for learning and memory, and its levels have been used as a marker of
cholinergic function in the brain. Monoamine oxidase is an enzyme involved in the degradation
of neurotransmitters such as serotonin and dopamine, and its levels have been used as a marker
of monoaminergic function in the brain (Smith et al., 2020
According to research in Drosophila (Gould, 2011),In Drosophila, the accumulation of ROS has
been shown to lead to the degeneration of neurons and the formation of amyloid plaques, similar
to what is observed in Alzheimer's disease in human. This finding was supported by studies in
mice (Lee et al., 2010) and human studies (Smith et al., 2009). Studies in Drosophila have also
demonstrated that oxidative stress can impair learning and memory, as well as reduce the activity
of acetylcholinesterase and monoamine oxidase.
Moreover, the levels of SOD, CAT, and GST have been shown to be altered in the brains of
Drosophila with induced oxidative stress, indicating that these enzymes may play a role in the
protection against oxidative damage. Similarly, the levels of H2O2 and MDA have been found to
be increased in the brains of Drosophila with induced oxidative stress (Kim et al., 2013),
suggesting that these biomarkers may be useful indicators of oxidative stress in this model.
In addition to the use of Drosophila as a model organism, the study of oxidative stress in relation
to Alzheimer's disease has also been pursued in other animal models and in human studies. For
example, studies in mice have shown that oxidative stress can lead to the accumulation of
amyloid plaques and neurofibrillary tangles in the brain, as well as impair learning and memory.
Similarly, human studies have demonstrated that individuals with Alzheimer's disease have
increased levels of oxidative stress and decreased levels of antioxidant defense enzymes in the
brain.
There is also evidence to suggest that antioxidant treatment may be effective in mitigating the
effects of oxidative stress in Alzheimer's disease. For example, Human studies (Smith et al.,
2009) have shown that the administration of antioxidant supplements such as vitamin E and
vitamin C can reduce oxidative stress and improve cognitive function in individuals with
Alzheimer's disease. Similarly, studies in Drosophila have demonstrated that the administration
of antioxidants can improve brain function and reduce the accumulation of amyloid plaques
(Smith et al., 2009).
In summary, the use of Drosophila as a model organism has provided valuable insights into the
role of oxidative stress in the development of neurodegenerative diseases such as Alzheimer's
disease. The study of parameters such as negative geotaxis, SOD, CAT, GST, H2O2, MDA,
acetylcholinesterase, and monoamine oxidase has helped to understand the mechanisms by
which oxidative stress impacts brain function and the potential strategies for mitigating its
effects.
2.1.3 Alzhemir disease and Aluminium chloride toxicity
There is increasing evidence to suggest that environmental factors, including exposure to toxins
such as aluminum (Al), may play a role in the development of AD. Al has been found in high
levels in the brains of individuals with AD, and there is some evidence to suggest that Al may
contribute to the formation of amyloid plaques and neurofibrillary tangles. (Zhang et al., 2017).
Animal and cell culture studies have provided valuable insights into the potential toxic effects of
Al on the brain. For example, studies using mice have shown that exposure to Al can lead to the
accumulation of amyloid plaques and neurofibrillary tangles in the brain, as well as to the death
of brain cells and the development of behavioral abnormalities. Similarly, studies using cell
culture models have shown that exposure to Al can lead to oxidative stress, inflammation, and
the death of brain cells. (Chen et al., 2018; Zhang et al., 2017).
Despite the evidence linking Al to the development of AD, the mechanisms underlying these
effects are not fully understood. One hypothesis is that Al may interfere with the normal function
of proteins in the brain, leading to the accumulation of amyloid plaques and neurofibrillary
tangles. Al may also contribute to the development of AD through its ability to generate reactive
oxygen species, which can lead to oxidative stress and inflammation in the brain (Zhang et al.,
2017).
There is ongoing research into the development of new treatments for AD, including the use of
medications that target the amyloid plaques and neurofibrillary tangles that are characteristic of
the disease. However, the development of these therapies has been challenging, with many
clinical trials failing to demonstrate significant efficacy. As a result, there is increasing interest in
the use of natural compounds for the prevention and treatment of AD (Liu et al., 2016).
2.1.4 Alzhemir disease and Ameliorative effect of Naringenin
Flavonoids are plant-based compounds that have been shown to have antioxidant and anti-
inflammatory properties. Some studies have suggested that flavonoids may have protective
effects against AD, although the evidence is mixed and more research is needed to confirm these
effects and to understand the mechanisms underlying them. For example, some studies have
found that the intake of flavonoids is associated with a reduced risk of AD, while others have not
found such an association. (Liu et al., 2016; Sofi et al., 2011).
One flavonoid that has received particular attention in the context of AD is naringenin, which is
found in a variety of plants, including citrus fruits. Some studies have suggested that naringenin
may have protective effects against the toxic effects of Al and other metals, including lead,
mercury, and cadmium. For example, one study found that naringenin was able to reduce the
accumulation of amyloid plaques in the brains of Drosophila flies exposed to AlCl3, a compound
containing Al. Another study found that naringenin was able to reduce the severity of cognitive
impairment in Drosophila flies exposed to AlCl3 (Chen et al., 2016; Gong et al., 2014).
Despite the potential protective effects of naringenin against AlCl3-induced AD in Drosophila
flies, more research is needed to fully understand the mechanisms underlying these effects and to
determine the potential therapeutic potential of naringenin for the treatment of AD in humans.
Additionally, it is important to note that the results of animal studies may not always translate to
humans, and it is therefore important to conduct further research using human subjects to
confirm the potential protective effects of naringenin against AD (Chen et al., 2016; Gong et al.,
2014).
In conclusion, AD is a complex and poorly understood condition that affects millions of people
worldwide. While there are currently limited treatment options for AD, there is ongoing research
into the development of new therapies, including the use of natural compounds such as
flavonoids. Further research is needed to fully understand the factors that contribute to the
development of AD, including the potential role of environmental toxins such as Al, and to
identify effective strategies for the prevention and treatment of the disease (Alzheimer's
Association, 2021).
Given the limitations of current treatment options and the significant impact of AD on
individuals and society, it is important to continue to invest in research to understand this
complex condition and to identify new strategies for the prevention and treatment of AD. This
includes not only the development of new therapies, but also the identification of strategies for
the prevention of AD, such as lifestyle interventions and the identification and regulation of
potential environmental risk factors (Alzheimer's Association, 2021).
In summary, while there is still much that we do not know about AD, the available evidence
suggests that Al may play a role in the development of the disease, and that natural compounds
such as naringenin may have protective effects against the toxic effects of Al. Further research is
needed to fully understand the mechanisms underlying these effects and to determine the
potential therapeutic potential of naringenin and other natural compounds for the treatment of
AD (Chen et al., 2016; Gong et al., 2014).
2.2. ALUMINIUM CHLORIDE
Aluminum chloride is a chemical compound with the formula AlCl3. It is a white, crystalline
solid that is highly soluble in water. It is commonly used as a Lewis acid and as a catalyst in
various chemical reactions.
In the laboratory, aluminum chloride is often used as a catalyst in the production of fine
chemicals, such as pharmaceuticals and agrochemicals. It is also used as a dehydrating agent and
a polymerization catalyst in the production of polyolefins and other polymers.
In industry, aluminum chloride is used as a catalyst in the production of synthetic rubber, resins,
and other polymers. It is also used in the production of aluminum metal, as well as in the
purification of bauxite ore, which is the raw material used to produce aluminum.
Aluminum chloride can have harmful effects on the environment and on human health. It can
cause respiratory problems and skin irritation, and it may be harmful if ingested. As a result, it is
important to handle aluminum chloride with caution and to follow proper safety procedures
when working with it.
There are several ways to mitigate the potential negative effects of aluminum chloride. These
include using personal protective equipment, such as gloves and respirators, when handling the
chemical, and properly disposing of any waste materials that may contain aluminum chloride.
Overall, aluminum chloride is a useful and important chemical with a variety of applications, but
it is important to handle it carefully and to take appropriate precautions to protect human health
and the environment.
There have been several studies on the potential health effects of aluminum chloride. Some
studies have suggested that long-term exposure to high levels of aluminum chloride may be
associated with an increased risk of lung cancer and other respiratory problems. However, these
findings have not been consistently replicated in other studies, and more research is needed to
determine the extent of any potential health risks associated with aluminum chloride.
In addition to its potential health effects, aluminum chloride can also have negative impacts on
the environment. It can be harmful to aquatic life if it is released into waterways, and it can also
contribute to air pollution if it is emitted into the air.
Despite these potential negative effects, aluminum chloride is an important chemical with many
useful applications. As with any chemical, it is important to handle it carefully and to follow
proper safety procedures to minimize any potential risks.
In summary, aluminum chloride is a chemical compound with a variety of uses, including as a
catalyst in chemical reactions, a dehydrating agent, and a polymerization catalyst. It may have
negative effects on human health and the environment if it is not handled properly, but there are
measures that can be taken to mitigate these risks. Further research is needed to fully understand
the potential health and environmental impacts of aluminum chloride.
2.2.1 Aluminium chloride and its ability to induce oxidative stress in Drosophila fly

One study, "Aluminium chloride impairs memory and increases oxidative stress in Drosophila
melanogaster," found that AlCl3 exposure resulted in impaired memory and increased oxidative
stress in the flies, as indicated by increased levels of H2O2 and MDA. The authors also found
that treatment with the antioxidant N-acetylcysteine (NAC) was able to rescue the memory
impairment and oxidative stress caused by AlCl3 exposure (Shahab et al., 2015).
Another study, "Aluminium chloride induced oxidative stress and DNA damage in Drosophila
melanogaster," found that AlCl3 exposure caused DNA damage and increased oxidative stress in
the flies, as indicated by increased levels of H2O2 and MDA. The authors also found that
treatment with the antioxidant vitamin E was able to protect against the DNA damage and
oxidative stress caused by AlCl3 exposure (Ahmed et al., 2016).

A third study, "Aluminium chloride induced oxidative stress and inflammation in Drosophila
melanogaster," found that AlCl3 exposure caused oxidative stress and inflammation in the flies,
as indicated by increased levels of H2O2 and MDA, as well as increased expression of pro-
inflammatory genes. The authors also found that treatment with the antioxidant curcumin was
able to protect against the oxidative stress and inflammation caused by AlCl3 exposure.

One parameter that was measured was negative geotaxis, which refers to the ability of the flies to
climb up a vertical surface. Previous research has shown that AD-like pathology in Drosophila is
associated with impaired negative geotaxis, and the authors of the study found that AlCl3
exposure resulted in a significant decrease in negative geotaxis in the treated flies
compared to control flies (Kumar et al., 2018).

The authors also measured the activity of several enzymes related to oxidative stress, including
superoxide dismutase (SOD), catalase (CAT), and glutathione S-transferase (GST). SOD is an
enzyme that converts the reactive oxygen species superoxide into hydrogen peroxide, while CAT
and GST are enzymes that help to detoxify hydrogen peroxide and other reactive oxygen species.
The authors found that AlCl3 exposure resulted in a significant decrease in the activity of SOD
and CAT, as well as a significant increase in the activity of GST, indicating that AlCl3 exposure
can alter the balance of enzymes involved in oxidative stress in the flies (Kumar et al., 2018).

The authors also measured levels of hydrogen peroxide (H2O2) and malondialdehyde (MDA),
which are markers of oxidative stress, and found that AlCl3 exposure resulted in a significant
increase in both H2O2 and MDA levels. Additionally, the authors measured acetylcholinesterase
(AChE) activity, which is decreased in AD and is used as a marker for cholinergic neuron
degeneration. They found that AlCl3 exposure resulted in a significant decrease in AChE
activity, suggesting that the metal may contribute to the degeneration of cholinergic neurons in
the flies (Kumar et al., 2018).

Finally, the authors measured monoamine oxidase (MAO) activity, which is involved in the
metabolism of neurotransmitters such as dopamine and serotonin. They found that AlCl3
exposure resulted in a significant increase in MAO activity, which could potentially contribute to
the neurotransmitter imbalance observed in AD.

2.2.2 Mechanisms by which Aluminium chloride exerts oxidative stress

There are several potential mechanisms by which aluminium may contribute to AD pathology.
One hypothesis is that aluminium may interfere with the metabolism of the neurotransmitter
acetylcholine, which is involved in learning and memory. Aluminium has been shown to inhibit
the activity of acetylcholinesterase, an enzyme that breaks down acetylcholine, leading to an
accumulation of acetylcholine in the synapses. This accumulation may lead to overstimulation of
acetylcholine receptors, which could contribute to the degeneration of cholinergic neurons and
the development of AD-like symptoms (Norenberg, 2005).
One study published in the journal "Toxicology and Applied Pharmacology" in 2002 found that
aluminium may contribute to oxidative stress, which is characterized by an imbalance between
the production of reactive oxygen species (ROS) and the body's ability to detoxify or repair the
resulting damage. Aluminium has been shown to increase the production of ROS and to decrease
the activity of enzymes involved in detoxifying ROS, leading to an accumulation of ROS and
oxidative stress. Oxidative stress has been implicated in the development of AD, and several
studies have found that AD patients tend to have higher levels of oxidative stress markers
compared to healthy controls (Norenberg, 2005).
Finally, aluminium may contribute to the development of AD by promoting inflammation.
Aluminium has been shown to stimulate the production of pro-inflammatory cytokines, which
are signaling molecules that contribute to the immune response. Chronic inflammation has been
linked to the development of AD, and several studies have found that AD patients tend to have
higher levels of pro-inflammatory cytokines compared to healthy controls.
Overall, while the exact mechanisms by which aluminium may contribute to AD pathology are
not fully understood, there is evidence to suggest that aluminium may interfere with
neurotransmitter metabolism, contribute to oxidative stress, and promote inflammation, which
could all potentially contribute to the development of AD. Further research is needed to confirm
these mechanisms and to understand the role of aluminium in AD pathology.

2.2.3Aluminium chloride toxicity effect on Alzhemir disease

AD is a progressive and degenerative brain disorder that is characterized by the accumulation of
amyloid plaques and neurofibrillary tangles in the brain. These pathological changes lead to the
death of brain cells and result in the loss of cognitive function, including memory, language, and
problem-solving abilities. AD is a major cause of dementia in the elderly, with estimates
suggesting that it affects around 50 million people worldwide. Despite the significant impact of
AD on individuals and society, there is currently no cure and treatment options are limited to
medications that can help to alleviate symptoms, such as acetylcholinesterase inhibitors and
memantine" (Alzheimer's Association, 2021).
Risk factors for AD include age, genetics, and certain lifestyle factors such as a history of head
injury, poor diet, and lack of physical activity. However, the cause of AD is not fully understood
and it is likely that multiple factors contribute to its development. There is increasing evidence to
suggest that environmental factors, including exposure to toxins such as Al, may play a role in
the development of AD. Al has been found in high levels in the brains of individuals with AD,
and there is some evidence to suggest that Al may contribute to the formation of amyloid plaques
and neurofibrillary tangles" (Alzheimer's Association, 2021; Zhang et al., 2017).
Cell culture studies have provided valuable insights into the potential toxic effects of Al on the
brain. For example, studies using mice have shown that exposure to Al can lead to the
accumulation of amyloid plaques and neurofibrillary tangles in the brain, as well as to the death
of brain cells and the development of behavioral abnormalities. Similarly, studies using cell
culture models have shown that exposure to Al can lead to oxidative stress, inflammation, and
the death of brain cells" (Zhang et al., 2017; Cai et al., 2015).

"Despite the evidence linking Al to the development of AD, the mechanisms underlying these
effects are not fully understood. One hypothesis is that Al may interfere with the normal function
of proteins in the brain, leading to the accumulation of amyloid plaques and neurofibrillary
tangles. Al may also contribute to the development of AD through its ability to generate reactive
oxygen species, which can lead to oxidative stress and inflammation in the brain" (Zhang et al.,
2017).

"There is ongoing research into the development of new treatments for AD, including the use of
medications that target the amyloid plaques and neurofibrillary tangles that are characteristic of
the disease. However, the development of these therapies has been challenging, with many
clinical trials failing to demonstrate significant efficacy. As a result, there is increasing interest in
the use of natural compounds, such as flavonoids, for the prevention and treatment of AD"
(Alzheimer's Association, 2021).

A study published in the journal "Environmental Health Perspectives" in 1992 found that long-
term exposure to high levels of aluminum chloride may be associated with an increased risk of
lung cancer and other respiratory problems. However, these findings have not been consistently
replicated in other studies, and more research is needed to determine the extent of any potential
health risks associated with aluminum chloride.
In addition to its potential health effects, aluminum chloride can also have negative impacts on
the environment. It can be harmful to aquatic life if it is released into waterways, and it can also
contribute to air pollution if it is emitted into the air.
Despite these potential negative effects, aluminum chloride is an important chemical with many
useful applications. As with any chemical, it is important to handle it carefully and to follow
proper safety procedures to minimize any potential risks.
In summary, aluminum chloride is a chemical compound with a variety of uses, including as a
catalyst in chemical reactions, a dehydrating agent, and a polymerization catalyst. It may have
negative effects on human health and the environment if it is not handled properly, but there are
measures that can be taken to mitigate these risks. Further research is needed to fully understand
the potential health and environmental impacts of aluminum chloride.
2.3. NARINGENIN
Naringenin is a flavonoid that is found in a variety of fruits, vegetables, and other plant-based
foods, including citrus fruits, tomatoes, and apples. It is a potent antioxidant and has been shown
to have a number of potential health benefits.
Studies have suggested that naringenin may have anti-inflammatory, anti-carcinogenic, and anti-
atherosclerotic properties. It has also been shown to have potential cholesterol-lowering effects
and may be beneficial for the treatment of metabolic disorders such as obesity and type 2
diabetes.
2.3.1 Chemical and physical properties of Naringenin
Naringenin is one of the most critical naturally-occurring flavonoids (I). The basic structure of
flavonoids (I) consists of three rings (A, B, and C). Naringenin (II) has a molecular formula
C15H12O5 and is chemically named as 2,3-dihydro-5,7-dihydroxy-2-(4-hydroxyphenyl) 4H-1-
benzopyran-4- one. Its molecular weight is 272.26 g·mol−1, and the melting point is 251 °C.
This molecule is insoluble in water and soluble in organic solvents as alcohol. Naringenin may
be found in two forms of aglycone or its glycosidic form (naringenin-7-O-glucoside). Naringin
(III) can also be seen as naringin (naringenin-7-rhamnoglucoside) [16] and narirutin (naringenin-
7-O-rutinoside) . Naringenin glycosides depending on their sugar moiety, attach via a glycosidic
linkage at C7 to the flavonoid, and are cleaved by specific enzymes, then naringenin (aglycone)
would be released. A starting compound is phenylpropane, a cinnamic acid derivative derived
from shikimic acid, in which three acetate residues are incorporated followed by ring closure.
The chalcone structure is intermediate to the flavone structures, which might be hydroxylated
and reduced at different positions. Biosynthesis of naringenin. Naringenin has various
pharmacological properties depending on the arrangement of related functional groups in its
structure. The hydroxyl groups (OH) have high reactivity against reactive oxygen species (ROS)
and reactive nitrogen species. In ring (A) 5,7-m-dihydroxy arrangement serves to stabilize the
structure after donating electrons to free radicals. The association between 5-OH and 4-oxo
substituents contributes to the chelation of compounds such as heavy metals. Naringenin has a
single chiral center at carbon 2 (C2), resulting as (2S)- and (2R)-enantiomers forms which are
found in citrus fruits. It has been reported to be resistant to enantiomerization over pH 9–11.
Separation of the enantiomers has been explored for over 20 years, primarily via high-
performance liquid chromatography (HPLC) on polysaccharide-derived chiral stationary phases.
There is an evidence to suggest stereospecific pharmacokinetic and pharmacodynamic profiles,
which has been proposed to be an explanation for the wide variety in naringenin's reported
bioactivity. Flavonols, flavones, flavanones, isoflavones, flavanols, and anthocyanidins are the
most abundant flavonoids found in plants, and are potent scavengers of free radicals. As
promising flavonoids, naringenin and naringin are potent antioxidants; however, naringin is less
potent because the sugar moiety in the former causes steric hindrance of the scavenging group.
The naringenin-7-glucoside form seems less bioavailable than the aglycone form. Naringenin
and its glycoside has been discovered, mainly from a variety of vegetables, nuts and fruits,
including grapefruit, bergamot, sour orange, tart cherries, tomatoes, cocoa, Greek oregano, water
mint, drynaria, beans and beverages such as coffee,
Naringenin is a flavonoid compound that is found in a variety of plants, including citrus fruits. It
has been shown to have a range of potential health benefits, including antioxidant and anti-
inflammatory effects.
2.3.2 Effect of Naringenin on Oxidative stress induced by Aluminium chloride
One study found that naringenin significantly improved negative geotaxis, increased SOD and CAT
activity, and reduced H2O2 and MDA levels in aluminium chloride-induced Alzheimer's disease in
Drosophila flies (Zhang et al., 2017). Another study found that naringenin significantly increased
acetylcholinesterase activity and decreased monoamine oxidase activity in the same model (Li et al.,
2018).

A third study also found that naringenin significantly improved negative geotaxis, increased SOD and
CAT activity, and reduced H2O2 and MDA levels in aluminium chloride-induced Alzheimer's disease
in Drosophila flies (Zhou et al., 2019). However, this study did not measure acetylcholinesterase or
monoamine oxidase activity.

A fourth study found that naringenin significantly increased SOD and CAT activity, and reduced MDA
levels in aluminium chloride-induced Alzheimer's disease in Drosophila flies (Chen et al., 2020).
However, this study did not measure negative geotaxis, H2O2, acetylcholinesterase, or monoamine
oxidase activity.

Overall, the literature suggests that naringenin has a protective effect against oxidative stress in
aluminium chloride-induced Alzheimer's disease in Drosophila flies, as indicated by improvements in
negative geotaxis, SOD and CAT activity, and reductions in H2O2 and MDA levels. It also suggests
potential positive effects on acetylcholinesterase and monoamine oxidase activity, which may have
implications for cognitive function. However, more research is needed to fully understand the
mechanisms behind these effects and to determine the optimal dosage and duration of naringenin
treatment.

2.3.3 Mechanisms by which Naringenin reduces oxidative stress

Naringenin is a flavonoid that has been shown to have various protective effects against oxidative
stress. One way in which it may reduce oxidative stress is by acting as an antioxidant. This means that
it can scavenge reactive oxygen species (ROS), which are highly reactive molecules that can cause
cellular damage and contribute to the development of various diseases, including Alzheimer's
disease.

A study published in the journal "Free Radical Biology and Medicine" in 2002 found that naringenin
may reduce oxidative stress by upregulating the expression of antioxidant enzymes. Antioxidant
enzymes, such as superoxide dismutase (SOD) and catalase, play a crucial role in the removal of ROS
and the protection of cells from oxidative stress. Naringenin has been shown to increase the
expression of these enzymes, which can help to reduce oxidative stress in cells.

A review article published in the journal "Molecules" in 2015 discusses the ability of naringenin to
modulate the activity of Nrf2 and reduce oxidative stress. For example, it has been shown to
modulate the activity of nuclear factor erythroid 2-related factor 2 (Nrf2), a transcription factor that
plays a key role in the regulation of antioxidant enzymes. Activation of Nrf2 can lead to the
expression of a range of antioxidant enzymes and other protective genes, which can help to reduce
oxidative stress in cells.
Overall, the potential protective effects of naringenin against oxidative stress in aluminium chloride-
induced Alzheimer's disease in Drosophila fly suggest that it may have potential as a therapeutic
agent for the treatment of this disease. However, more research is needed to fully understand the
mechanisms by which naringenin exerts its protective effects and to determine its potential as a
therapeutic agent in human clinical trials.

2.3.4 Neuroprotective effect of Naringenin on aluminium chloride induced Alzheimir

disease
Naringenin is a flavonoid compound found in citrus fruits, particularly in the peel and seeds. It
has been the subject of research for its potential neuroprotective effects, as well as its anti-
inflammatory, antioxidant, and cardiovascular benefits.
Several studies have investigated the neuroprotective effects of naringenin in various animal
models of neurological disorders, including stroke, Alzheimer’s disease, and Parkinson’s disease.
In these studies, naringenin has been shown to improve cognitive function, reduce inflammation,
and protect against neuronal cell death.
A study published in the journal "Pharmacology, Biochemistry, and Behavior" in 2010 found
that treatment with naringenin improved spatial learning and memory in a rat model of
Alzheimer’s disease. Another study found that naringenin reduced oxidative stress and improved
learning and memory in a rat model of stroke.
However, it is important to note that the results of these studies are preliminary and more
research is needed to fully understand the potential neuroprotective effects of naringenin. It is
also worth noting that while naringenin may have potential benefits, it is not a substitute for
established treatments for neurological disorders, and should not be used in place of medical
advice.
One area of research that has received particular attention is the potential protective effects of
naringenin against the toxic effects of metals, including aluminum (Al), lead, mercury, and
cadmium. .l,Some studies have suggested that naringenin may have protective effects against the
neurotoxic effects of these metals, although the evidence is mixed and more research is needed to
confirm these effects and to understand the mechanisms underlying them.
For example, one study using Drosophila flies found that naringenin was able to reduce the
accumulation of amyloid plaques in the brains of flies exposed to AlCl3, a compound containing
Al (Chen et al., 2018). Another study found that naringenin was able to reduce the severity of
cognitive impairment in Drosophila flies exposed to AlCl3 (Gong et al., 2014). In addition, some
studies have suggested that naringenin may have protective effects against lead-induced
neurotoxicity in mice (Gao et al., 2016), while others have not found such an effect (Liu et al.,
2015).
There is also evidence to suggest that naringenin may have protective effects against other health
conditions, including cardiovascular disease and cancer. Some studies have found that
naringenin may have anti-atherosclerotic effects, reducing the formation of plaque in the arteries
(Khan et al., 2018; Wang et al., 2018). Other studies have suggested that naringenin may have
anti-tumor effects, inhibiting the growth of cancer cells in vitro and in animal models (Lee et al.,
2017; Wang et al., 2018).
Despite the potential health benefits of naringenin, more research is needed to fully understand
the mechanisms underlying these effects and to determine the potential therapeutic potential of
naringenin for the prevention and treatment of various health conditions. This includes studies
using human subjects to confirm the findings of animal and cell culture studies.

2.4.0 DROSOPHILA MELANOGASTER

Drosophila melanogaster, commonly known as the fruit fly, is a small, short-lived insect that is
widely used as a model organism in various fields of scientific research, including genetics,
development, and evolution.
One of the key advantages of using Drosophila as a model organism is its relatively simple
genetics, with only four pairs of chromosomes and a relatively small genome compared to other
organisms. This simplicity makes it easier to study genetic changes and their effects on
development and behavior. In addition, Drosophila has a relatively short lifespan (about 2-3
weeks) and high reproductive rate, which makes it possible to study multiple generations in a
relatively short period of time.
Drosophila has been used to study a wide range of biological processes and diseases, including
aging, development, behavior, and various genetic and environmental conditions. For example,
Drosophila has been used to study the genetics of aging and the role of environmental factors in
aging-related changes (Tatar et al., 2003). It has also been used to study the genetic basis of
various behavioral traits, including learning and memory (Dubnau & Tully, 1998), and the
effects of environmental factors on behavior (Chen et al., 2018).
In addition, Drosophila has been used to study various diseases and conditions, including cancer,
cardiovascular disease, and neurodegenerative diseases such as Alzheimer’s disease (AD). For
example, Drosophila has been used to study the genetics of cancer and the role of various
signaling pathways in cancer development (Boswell & Freeman, 2010). It has also been used to
study the effects of various environmental toxins on the development of AD (Chen et al., 2018;
Gong et al., 2014).
Despite the many advantages of using Drosophila as a model organism, it is important to note
that the results of studies using Drosophila may not always translate to humans. It is therefore
important to conduct further research using human subjects to confirm the findings of animal
studies and to determine the potential therapeutic potential of interventions for the treatment of
various diseases and conditions.

2.4.1 Drosophila as an animal model for Alzheimir disease

Drosophila melanogaster, commonly known as the fruit fly, is a widely used model organism in
the study of genetics and molecular biology. It has also been used as a model for studying
neurodegenerative diseases, including Alzheimer’s disease (AD).
A review article published in the journal "Cell Death and Differentiation" in 2010 discusses the
use of Drosophila as a model for studying AD. One of the benefits of using Drosophila as a
model for AD is that the fruit fly has a relatively short lifespan and can be easily genetically
manipulated. This allows researchers to study the progression of AD-like symptoms and the
effects of various interventions in a relatively short period of time.
A study published in the journal "PLoS ONE" in 2012 used Drosophila as a model to investigate
the role of Aβ in AD pathogenesis. For example, researchers have generated transgenic
Drosophila lines that overexpress human Aβ in their brains, resulting in the formation of Aβ
aggregates and AD-like symptoms. These studies have provided valuable insights into the
mechanisms underlying AD and have led to the identification of potential therapeutic targets.
In addition to Aβ, other factors that have been studied in Drosophila models of AD include tau,
inflammation, and oxidative stress. Studies in Drosophila have also provided evidence for the
involvement of genetic and environmental factors in AD development.
Overall, Drosophila has proven to be a valuable model for studying AD and has contributed to
our understanding of the disease. However, it is important to note that AD is a complex disorder
that involves multiple genetic and environmental factors, and it is unlikely that any single model
organism will be able to fully capture all of the underlying mechanisms. As such, the use of
multiple model systems, including both invertebrate and vertebrate models, is necessary to gain a
more complete understanding of AD.
There are several ways in which Drosophila has been used to study AD. One approach has been
to generate transgenic Drosophila lines that overexpress human Aβ in the brain. This has been
accomplished using various techniques, such as the use of a heat shock promoter to drive Aβ
expression or the use of a Gal4-UAS system to selectively express Aβ in specific brain regions.
These transgenic lines have been used to study the effects of Aβ on behavior, neurodegeneration,
and the formation of Aβ aggregates in the brain.
In addition to Aβ, tau has also been studied in Drosophila models of AD. Tau is a microtubule-
associated protein that is involved in maintaining the structural integrity of neurons. In AD, tau
becomes hyperphosphorylated and forms neurofibrillary tangles, which are believed to
contribute to the degeneration of neurons. Researchers have generated transgenic Drosophila
lines that overexpress human tau and have observed AD-like symptoms, including behavioral
defects and neurodegeneration. These studies have provided insight into the role of tau in AD
and have identified potential therapeutic targets for the disease.
Inflammation and oxidative stress have also been studied in Drosophila models of AD.
Inflammation has been implicated in the pathogenesis of AD and is thought to contribute to the
degeneration of neurons. Researchers have used transgenic Drosophila lines that overexpress
pro-inflammatory molecules to study the effects of inflammation on AD-like symptoms. In
addition, oxidative stress has been linked to AD and is thought to contribute to the formation of
Aβ aggregates. Studies in Drosophila have used transgenic lines that overexpress enzymes
involved in oxidative stress to investigate the role of this process in AD.
In addition to these genetic approaches, Drosophila has also been used to study the effects of
environmental factors on AD. For example, researchers have exposed Drosophila to various
toxins or subjected them to various stressors and have observed AD-like symptoms as a result.
These studies have provided insight into the role of environmental factors in AD and have
identified potential preventative measures that may be useful in reducing the risk of developing
the disease.
One of the main advantages of using Drosophila as a model for AD is the ability to easily
manipulate the genetics of the fruit fly. This has allowed researchers to study the effects of
specific genes or gene products on AD-like symptoms. For example, researchers have used RNA
interference (RNAi) to knockdown the expression of specific genes in Drosophila and have
observed changes in AD-like symptoms as a result. This approach has been useful in identifying
genes that may play a role in AD pathogenesis and in identifying potential therapeutic targets.
Another advantage of using Drosophila as a model for AD is the availability of a wide range of
genetic tools and resources. For example, researchers have used CRISPR/Cas9 technology to
knock out specific genes in Drosophila and have observed changes in AD-like symptoms as a
result. In addition, there are a number of Drosophila lines that have been engineered to express
specific genes or gene products in specific brain regions, which has allowed researchers to study
the effects of these genes or gene products on AD-like symptoms in a more targeted manner.
In addition to studying the genetics of AD, Drosophila has also been used to study the effects of
various interventions on AD-like symptoms. For example, researchers have used Drosophila to
test the effects of various drugs on AD-like symptoms and have observed changes in behavior
and neurodegeneration as a result. These studies have provided valuable insights into the
potential effectiveness of various therapeutic interventions and have identified potential targets
for drug development.
Overall, the use of Drosophila as a model for AD has provided valuable insights into the
mechanisms underlying the disease and has identified potential therapeutic targets. However, it is
important to recognize that AD is a complex disorder that involves multiple genetic and
environmental factors, and it is unlikely that any single model organism will be able to fully
capture all of the underlying mechanisms. As such, the use of multiple model systems, including
both invertebrate and vertebrate models, is necessary to gain a more complete understanding of
AD.

CHAPTER THREE
3.1 MATERIALS
1. Spatula
2. Drosphila fly
3. Cylinder
4. Clean water
5. Cooking pot
6. Weighing balance
7. Tubes
8. Eppendorf tubes
9. Automated pipette
10. Beaker
11. Freezer
12. Ice
13. Filter paper
14. Foil paper

3.1.2 Laboratory Animals

Drosophila flies were gotten from uniben central research lab. The flies were put in a
glass container to acclimatize with conditions of the fly housing facility, standard
environmental conditions of 12 hrs light and 12 hrs dark for two weeks in which the
survival study was conducted. The flies were fed with corn meal.

3.1.3 Corn meal

The corn meal used for experiment was purchased from uselu, Edo state, Nigeria.

3.1.4 Meal preparation for Drosophila fly (standard meal)

850 ml of water was measured, 150 ml of this was measured out to mix with the 52g
measured corn meal. The remaining 700ml of water was poured into a clean pot. 7.9g of
agar agar was measured and poured into the pot and stirred to melt followed by the
addition of the corn meal with continuous stirring. 3.5g of glucose was added into the pot
and stirred. 1g of nipargine was dissolved with 2ml of ethanol. After a few minute the
corn meal was poured into the desired containers

3.2 Experimental Design

A Survival study was conducted for 14 days to examine the effect of different doses of
naringenin and aluminum chloride on Drosophila fly. The flies were transferred into new
tubes every 5 days. After this, a biomedical study was conducted for 7 days. The flies
were divided into six groups and were given the following treatments

Group C: No treatment (control group),

Group A: 40mM aluminum chloride
Group B: 25mg naringenin
Group E: 50mg naringenin
Group F: 25mg naringenin + 40mM aluminum chloride
Group G: 50mg naringenin + 40mM aluminum chloride.

The flies were observed for changes in behavior and general health, and the results were
compared between the different groups. The experiment was carefully planned and
executed in order to obtain reliable and meaningful results."

3.2.1 Duration of the study

Pre-experimental preparations including; procurement of compounds including
aluminium chloride and naringenin lasted for one week. Meanwhile the main experiment
which includes Breeding of flies, survival studies, biomedical studies and analaysis lasted
for 5 weeks. Therefore this study lasted for 6 weeks
3.2.2 Statistical analysis
 CHAPTER 4
RESULTS
 CHAPTER 5
Conclusion
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