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An Undergraduate Research
Presented to
by
Ranielle D. Samson
March 2018
TABLE OF CONTENTS
Page
Abstract………….…………………………………………………………………………i
Acknowledgment…………………………………………………………………………ii
List of Tables
List of Figures
Chapter
1. The Problem and Its Background
Introduction…………………………………………………………………………..…1
Background of the Study……………………………………………………………...3
Conceptual Framework……………………………………………………………….5
Research Objectives……………………………………………………………….…5
Research Hypothesis…………………………………………………......................6
Significance of the Study……………………………………………………………..6
Scope and Delimitation of the Study………………………………..….................. 7
Definition of Terms………………………………………………….……………...…8
The researcher would like to express their sincere gratitude to those who
inspired, guided, helped, supported and contributed to their thesis throughout the
To their parents and family members who gave physical, spiritual, financial
regarding research;
To their classmates and friends that contributed for the cause of their
research;
And most importantly to the Sovereign Lord who made all these things
possible, for giving them heavenly wisdom, knowledge, and strength to finish this
study.
C.J.E.A
C.J.C.C
S.R.D
S.K.M.G
THE NEUROPROTECTIVE PROPERTY OF THE SEMI-PURIFIED FLAVONOIDS FROM
THE INDIAN MANGO (Mangifera indica Linn,. Family Anachardiacaea) LEAVES
AGAINST ALUMINUM INDUCED NEUROTOXICITY IN FEMALE SPRAGUE
DAWLEY RATS.
ABSTRACT
This study was undertaken to evaluate the neuroprotective property of the semi-
purified flavonoids from the leaves of Indian Mango (Mangifera alitissima Linn. Family
Anachardiaceae) leaves against aluminum induced neurotoxocity in female Sprague
dawley rats.
Fifteen (15) female Sprague dawley rats, weighing 100-200 grams were randomly
divided into five groups: The positive control, pre-treated with ascorbic acid 250mg/kg. The
negative control, using plain NSS only. The experimental groups, pre-treated with mango
flavonoid extract 100, 200, and 400mg/kg. All were pre-treated orally on the first to ten (1st-
10th) day of experimentation. On the 11th – 14th day, the researcher induced the aluminum
chloride. After seven days, brains were collected and submitted through histopathological
analysis to determine the neuroprotective effect. Histopathological analysis graded the CA3
neuronal cell necrosis on the rat brains using the established neurological score.
Groups pre-treated with mango flavonoid extract 100 and 200 mg/kg showed no
significant difference on neurological score compared with the positive control group. While
group pretreated with mango flavonoid extract 400mg/kg showed significant difference on
neurological score compared to the positive control group.
Conclusion: As the results of histopathological analysis of the rat brains, from the flavonoid
extract from the leaves of Indian Mango (Mangifera alitissima linn. Fam. Anachardiaceae)
specifically, 400mg/kg inhibited the neuronal damage induced by aluminum chloride, and
therefore have a neuroprotective potential against neurotoxicity.
Introduction
Group 3
100mg/kg Semi-
purified flavonoids
extract
Severe degeneration Moderate to Severe Moderate to Severe
degeneration degeneration
deformed the arc the more damaged the
brain is. The degree of severity was
interpreted through the table given at
table 1. Group 5 having a dose of 400
mg/ml of the semi purified flavonoid
extract showed a significant
neuroprotective property wherein it
Severe degeneration Severe degeneration resulted to a mild degeneration of the
hippocampus comparable to the
standard drug, ascorbic acid.
Group 4 CONCLUSION
200mg/kg Semi-
Based on the results presented, the
purified flavonoids
semi- As the result of the
extract histopathological test in the rats’
brain, the researchers concluded that
Moderate to Severe flavonoids present in the leaves of the
degeneration Indian Mango exhibits
neuroprotective activity against
Aluminum induced neurotoxicity in
the brains for the rats. The dose
400mg/kg/day of the semi-purified
flavonoid extract showed comparable
neuroprotective activity with that of
the positive control, Ascorbic Acid.
Recommendations
The recommendations based on
Mild to Moderate Mild to Moderate
the study conducted are hereby
degeneration degeneration
presented:
1. Development of a better
extraction method of flavonoids
Group 5 from the leaves of the Indian
400mg/kg Semi- Mango.
purified flavonoids 2. Determination of the safe and
effective dose of the flavonoids
extract
present in the leaves of Indian
Mango.
3. Toxicity study regarding the
Moderate to severe
dosing of the extract.
degeneration
4. Phamacokinetic and
Pharmadynamic evaluation of
Histopathological report shows the flavonoids.
different result within the five groups 5. Formulate a dosage form of the
based on its cellular degeneration in the flavonoid extracts from Indian
CA3 region of the hippocampus of the rat Mango.
brain. Interpretations were based on the
severity in the arc shape of the CA3
region which means that the more
Reference: lipidium-sativum-water-extract-on-
hippocampus-rats-brain-tissue
Alberto, Jhoemar P., et al. The
Neuroprotective Actions of the Barrita, J.L., et al (2013). Antioxidant
Flavonoids from the Fuji Apple Fruits Role of Ascorbic Acid and His Protective
(Malus domestica Family Rosaseae) on Effects on Chronic Diseases.
Induced Brain Ischemia using Middle https://www.intechopen.com/books/oxid
Carotid Artery Occlusion Stroke Model in ative-stress-and-chronic-degenerative-
Male Sprague Dawley Rats (Centro diseases-a-role-for-
Escolar University, 2014) antioxidants/antioxidant-role-of-ascorbic-
acid-and-his-protective-effects-on-
Balgoon, M.J. et al (2015). ATR-IR Study chronic-diseases
of the Mechanism of Aluminum Chloride
Induced Alzheimer’s Disease; Curative Ceccatelli S. (2013). Mechanisms of
and Protective Effect of Lipidium sativum neurotoxicity and implications for
Water Extract on Hippocampus Rats neurological disorders. Journal of
Brain Tissue. World Academy of Internal Medicine Volume 273, Issue 5,
Science, Engineering and Technology Version of
International Journal of Pharmacological
and Pharmaceutical Sciences Vol:9,
No:11, 2015. Record online: 22 APR 2013
http://waset.org/publications/10002963/a http://onlinelibrary.wiley.com/doi/10.111
tr-ir-study-of-the-mechanism-of- 1/joim.12053/pdf
aluminum-chloride-induced-alzheimer-s-
disease-curative-and-protective-effectof- Colak, S., et al (2011). The
neuroprotective role of boric acid on
aluminum chloride-induced neurotoxicity.
CHAPTER 1
Introduction
drugs, heavy metals, certain food preservatives pesticides, cosmetics and for the
cancer patients who uses chemotherapy drugs and radiation treatment frequently,
can affect the normal activity and function of the nervous system which may cause
damage to the nervous tissues (Robert C. Repetto et al., 1996). This can kill the
neurons which are responsible for the transmittance and processing of the
information in the form of electrical and chemical signal in the brain. (S. Robertson.,
2014). Some may immediately produce effects that may occur for an hour or
several hours. These includes the effect of alcoholic beverages, fumes from a can
permanent brain damage, while some neurotoxins may only produce visible sign
and symptoms after repeated exposures over weeks or even years. Examples are
eating food and drinking water contaminated with lead and frequent exposure to
neurotoxin, the dose a person has been exposed to, the ability of the person to
metabolise and excrete the toxin, the ability of the structure and mechanism to
recover and how vulnerable the cells are. The symptoms may include weakness
Robertson., 2014).
used for the prevention and management of neurotoxicity which is associated with
brain ischemic stroke, Parkinson’s disease and Alzheirmer’s disease. The goal of
a neuroprotectant is to lessen the risk of brain damage in patients, slow the disease
inflammation and free radical production leading to oxidative stress. (Ikezu et al.,
2008)
Aluminum (Al) is the third most abundant element in the earth’s crust next
to oxygen and silicon. It enters the body in various ways. Aluminum Chloride (AlCl3)
was once present in the formulation of antiperspirant and some drugs (Exley,
1998). Due to studies proving AlCl3 can cause neurotoxins, some companies
reformulated their antiperspirant products although at this time there are still some
Aluminum compounds can contribute to serious toxic effect to both animals and
humans (AD; Yokel, 2000; Rondeau et al., 2009; Miu et al., 2004). Aluminum is
formation in the brain of the mice (Practico D. et al., FASEB J 2002). Aluminum
and Aluminum compounds are known to be potent neurotoxin that can cause
sclerosis, Parkinson’s and Alzheimer’s disease (AD; Yokel, 2000; Rondeau et al.,
2009; Miu et al., 2004). Recent studies have shown that the possible mechanism
blood-brain barrier (BBB; Ku¨c¸u¨k et al., 2001). Both amyloid beta and aluminium
are capable of potentiating reactive oxygen species (ROS) formation that will lead
study aims to study the basic principles and potential therapeutic interventions
its significance in the medical field specifically for the prevention and management
shown that mangoes have been associated with a decreased risk of chronic
disease such as diabetes and cancer. In some studies mangoes were proven to
shown that mangoes contains approximately 76% of vitamin C and 25% of vitamin
2000).
blue and purple anthocyanin pigment of plant tissues. They are group of plant
health benefits through cell signalling pathways and antioxidant effects, Ingestion
diabetes type II. In the study of these disease it is assumed that neurotoxicity is
associated, thus flavonoids have been suggested to exert health benefits through
Conceptual Framework
flavonoid from the leaves of the mangoes, the identification of flavonoid extract
leaves of mangoes through the evaluation of the dissected brains of the pre-treated
Research Objectives
Specifically, it aimed:
activity.
of the semi purified flavonoid from the Mangifera indica and the possible
property of the semi purified flavonoid with the standard drug which is
ascorbic acid.
Aluminum, being one of the most widely used metal that is an integral part
in the daily lives of the people, is currently known to be one of the most toxic metal
that people are exposed to. Hence, the need for an effective, safe and cheap
effects brought by synthetic agents. Thus, the researchers was able to adopt to
study the use brought about by the Mango leaves, Mangifera indica, to improve
to frequent exposure to these toxic metals, which includes all the children, adults
and elderly. This study would also like to prove the efficacy of flavonoids present
in the leaves of the Mango and to be able to appreciate Mango, Mangifera indica,
study is able to provide such information and evidence, then this could be useful
to the future development of new drugs that could be obtained in a cheaper way,
thus helping those who suffer from the complications who can’t afford the support
they need and those who aren’t able to buy the prophylaxis they ought to use for
preventive purposes.
indica) leaves.
2. The extraction of the crude constituents, macerated for 5 days using 50%
3. The isolation of the flavonoids by defatting the crude extract, with petroleum
6. The use of Ascorbic Acid as the standard drug for the biological test.
Definition of Terms
The following terms were defined for the convenience and optimal
Aluminum. It is the third most abundant element in the earth’s crust next to
2014).
regeneration of the nervous system, its cells, structure and function. (Vajda FJ,.
2009).
(Robertson, 2014)
CHAPTER 2
This chapter synthesizes the comprehensive review of the local and foreign
studies and literature about the study. This summary of applicable information
provides as the groundwork of knowledge and basis for the ideas, methodology,
results and discussion conferred in the whole study of the determination of the
Manga from the species Mangifera indica has been considered as the
national fruit of the Philippines. Mangoes are eaten as raw, cooked, frozen,
preserved or dried. Ripe mangoes are used for confectioneries, ice cream,
sherbet, and bakery products while unripe mangoes (usually the Indian variety)
are a good source of juice. The demand for processed mango is increasing, as
Mangoes are a sub-tropical fruit, requiring a long hot period to properly set
the fruit. More mangoes are eaten fresh than any other fruit in the world. There are
over thousand varieties of mango. The enzyme in mangoes are tenderizing as are
the enzyme in papaya. Mangoes help lower cholesterol and they are rich in the
vitamins A and C.
into many classes depending on their chemical structure. They are responsible
mainly of the flavor and color of the fruits and vegetables. The flavonols and
flavones give yellow or orange color, the anthocyanins red, violet or blue colors.
The aurones are golden yellow pigments while flavonones and flavonoids are ither
substances alters the normal activity of the nervous system. This can eventually
disrupt or even kill neurons, key cells that transmit and process signals in the brain
and on other parts of the nervous system. Neurotoxicity can result from exposure
organ transplants, as well as exposure to heavy metals such as lead and mercury,
certain food and food additives, pesticides, industrial and/or cleaning solvents,
due to the direct action of the substance, with the impairment and neurocognitive
deficits being temporary, and resolving when the substance is metabolised from
the body. In some cases the level or exposure-time may be critical, with some
the most common naturally occurring brain toxins that lead to neurotoxicity as a
result of excessive dosage are beta amyloid (Aβ), glutamate and oxygen radicals.
When present in high concentrations they can lead to neurotoxicity and death
(apoptosis). Some of the symptoms that result from cell death include loss of motor
et al., 2007)
chlorine. It is white, but samples are often contaminated with iron (III) chloride,
giving it a yellow color. The solid has a low melting and boiling point. It is mainly
produced and consumed in the production of aluminium metal, but large amounts
are also used in other areas of chemical industry. The compound is often cited as
bases, so suitable precautions are required. It can cause irritation to the eyes,
common form of dementia, a general term for memory loss and other cognitive
abilities serious enough to interfere with daily life. Alzheimer's disease accounts
aging. The greatest known risk factor is increasing age, and the majority of people
with Alzheimer's are 65 and older. But Alzheimer's is not just a disease of old age.
its early stages, memory loss is mild, but with late-stage Alzheimer's, individuals
Alzheimer's is the sixth leading cause of death in the United States. Those with
Alzheimer's live an average of eight years after their symptoms become noticeable
to others, but survival can range from four to 20 years, depending on age and other
health conditions. .
in the human body, although its function at the cellular level is not yet clear. It is
necessary for the synthesis of collagen, a protein that has many connective
functions in the body. Among the substances and structures that contain collagen
are bone, cartilage and the surrounding material, as well as carrier substances and
materials of union muscle, skin and other tissues. It also requires (AA) for the
acids and vitamins. Participate in the liver for detoxification of toxic substances and
blood level for immunity. As an antioxidant reacts with histamine and peroxide for
reduced incidence of cancer. The requirement for vitamin C for adults is well
defined but they have not been uniform across different cultures, so their need has
been defined as culture-specific. They have also defined other roles in cellular
sometimes ingest an overdose of it. In most reports mention that discrete increases
in blood levels of this vitamin reduces the risk of death in all conditions. Although
there are many functions of vitamin C, his role in health is discussed mostly in
relation to its role as an antioxidant and its effects on cancer, blood pressure,
play important roles in cellular function and have been implicated in processes
associated with aging, including vascular, inflammatory damage and cancer. In the
case of
antioxidant role, the AA has actions at the molecular level because it acts as a
monosodium glutamate.
Alzheimer's has no current cure, but treatments for symptoms are available
dementia symptoms and improve quality of life for those with Alzheimer's and their
caregivers. Today, there is a worldwide effort under way to find better ways to treat
nervous system that mainly affects the motor system. The symptoms generally
come on slowly over time. Early in the disease, the most obvious
Thinking and behavioral problems may also occur. Dementia becomes common in
the advanced stages of the disease. Depression and anxiety are also common
occurring in more than a third of people with PD. Other symptoms include
sensory, sleep, and emotional problems. The main motor symptoms are
involve both genetic and environmental factors. Those with a family member
affected are more likely to get the disease themselves. There is also an increased
risk in people exposed to certain pesticides and among those who have had
prior head injuries, while there is a reduced risk in tobacco smokers and those who
drink coffee or tea. The motor symptoms of the disease result from the death of
cells in the substantia nigra, a region of the midbrain. This results in not
enough dopamine in these areas. The reason for this cell death is poorly
the neurons. Diagnosis of typical cases is mainly based on symptoms, with tests
levodopa becomes less effective. As the disease progresses and neurons continue
to be lost, these medications become less effective while at the same time they
symptoms. Surgery to place microelectrodes for deep brain stimulation has been
PD, such as sleep disturbances and emotional problems, is less strong. (Elkouzi,
2014).
excitotoxicity, cells were stimulated with 1-glutamic acid having the dose at 50
protection was obtained with 2.5 ml of Mangifera indica extract. Then, the
production of reactive oxygen species, which can kill neurons and glial cells
(Matute et al., 2002). It has been advised that excitotoxicity is the major
chemicals that have neuroprotective properties. They prevent or decrease the free
radical production, iron chelation and it has anti-inflammatory effect both in in vitro
et al, 2002)
mangiferin and morin, another antioxidant polyphenols has been identified to have
the Center Pharmaceutical Chemistry. The data showed that the extract contains
the following composition: moisture 50%, total phenol in anhydrous base 30%, and
culture of neurons from the cerebral cortex of 18-day rat embryos. The cell was
Mangifera indica extract ranging from 0.5 to 5 microgram/ml and mangiferin from
exposure.
The result showed that the highest protection from glutamate toxicity by Mangifera
indica extract was obtained at 2.5 microgram/ml. Mangifera indica extract at 5 and
lowest and the highest mangiferin concentration had a lower neuroprotective effect
Studies have shown that mangiferin saves neurons from cell death in acute
injury and lessen neurological deficit caused by ischemic damage to the rat brain.
However, this is not excluded the facts that other components present in the
extract may also contribute to those properties. Indeed, other constituents of the
extract are also neuroprotective. This include quercetin, a natural flavonoids, also
The researcher observed that Mangifera indica extract and mangiferin have
cortical neurons and both have similar antioxidant activity. This advice that these
stress occurs.
CHAPTER 3
This chapter includes the discussion of the research methodology that was
from the extract of the leaves of Indian mango against induced neurotoxicity using
Research Methodology
The semi-purified flavonoids were extracted from the Indian mango leaves
using 80% ethanol, distilled water, 2M HCl and ethyl acetate. The extracts
underwent physical and chemical tests, and confirmatory test for the presence of
flavonoids.
histopathological analysis.
Settings of the Study
Research Procedure
The leaves of plant sample (Manigifera indica) used in the study were
collected at Laguna, Philippines throughout the month of June to August 2017. The
leaves were air-dried for 7 days. Then, it was cut into smaller pieces and then
100 grams of the dried leaves were weighed and placed in a 500 mL glass
jar and macerated using 80% ethanol for 48 hours, the leaves were then filtered
The crude extract obtained is defatted using petroleum ether and the
aqueous layer is collected, it was the evaporated into incipient dryness using water
bath. The sample is dissolved using 2M HCl, filtered and ethyl acetate is added
until the extract is colorless. Using separatory funnel, ethyl acetate layer is
separated from the aqueous layer. The ethyl acetate layer obtained is then
subjected to incipient dryness. The residue obtained is the semi-purified
flavonoids.
The odor, color, and appearance of the extract were studied and physically
examined.
adding about 0.1g of the extract in four (4) separate test tubes containing 5ml of
the following: distilled water, 80% ethanol, acetone, chloroform, and petroleum
ether. It was evaluated as soluble, very soluble, freely soluble, sparingly soluble,
defatted sample and warmed using water bath for 15 minutes. Positive result
In a tets tube with the sample, 10 mL of ethyl acetate was added and
ammonia solution was added. Positive result would be the appearance of yellow
coloration.
A few drops of 10% ferric chloride solution were added in the test
tube containing the sample. The positive result would include a green-blue or violet
coloration.
About 0.5 mL of concentrated HCl was added together with three (3)
pieces of magnesium turnings. Observe any color change within 10 minutes, if the
color changes, the solution will be diluted with an equal volume of water and octyl
alcohol. The solution was shaken and allowed to stand, note the positive result:
observe colors ranging from orange to red, crimson, and magenta occasionally to
green or blue.
extracts
Fifteen (15) male Spague-Dawley rats were used as the test subject
for this study. These were provided by the Department of Science and Technology
The rats were grouped into five (5) groups, which consist of three (3)
rats each. They were housed in per group in a 12”x16”x7” plastic cage and were
12h:12h dark/light photo period and relative humidity of NMT 65%. They were
acclimatized for one (1) week prior to the onset of the experiment. All animals
were fed pellets (rabbit pellets/ dog food/ corn pellets) twice a day (morning and
afternoon) and were given 60-80 mL of potable water daily. To identify each rat,
each rat was marked on their tail with one, two, three, four, or five lines according
to their group.
All experiments were performed prior to the approval from the
Institutional Animal Care and Use Committee (IACUC) of Centro Escolar University
were used as the experimental variable in this study. It was dissolved in distilled
prior to administration.
chloride was administered in each rat until the fourteenth (14th) day. After one (1)
week, the rats were euthanized and each brain was extracted by an IACUC
purified flavonoids and ascorbic acid that were both dissolved in distilled water via
oral gavage for a period of 10 days. Rats were weighed twice a week for the basis
of the computation of the volume to be given. The volume of the extract given was
computed as follows:
𝑚𝑔
𝑊𝑒𝑖𝑔ℎ𝑡 𝑜𝑓 𝑡ℎ𝑒 𝑟𝑎𝑡 (𝑘𝑔) 𝑥 𝐷𝑜𝑠𝑒 𝑡𝑜 𝑏𝑒 𝑎𝑑𝑚𝑖𝑛𝑖𝑠𝑡𝑒𝑟𝑒𝑑 ( )
𝑘𝑔
𝑉𝑜𝑙𝑢𝑚𝑒 (𝑚𝑙) = 𝑚𝑔
𝐶𝑜𝑛𝑐𝑒𝑛𝑡𝑟𝑎𝑡𝑖𝑜𝑛 𝑜𝑓 𝑡ℎ𝑒 𝑠𝑜𝑙𝑢𝑡𝑖𝑜𝑛 ( 𝑚𝐿 )
Group 1 served as the negative control group. The rats in this group
were fed with normal diet and no treatment received from 1st to 14th day.
Group 2 served as the positive control group. The rats in this group
were fed with normal diet and received ascorbic acid through oral gavage, 250
these groups were fed with normal diet and received with an increasing dose of
semi-purified flavonoids from Indian mango, 100 mg/kg, 200 mg/kg, and 400
On the 11th day, all rats received Aluminum Chloride until the 14th
One (1) week after inducing neurotoxicity, the rats will be euthanized
and a professional IAUC laboratory veterinarian will extract the rat’s brain. In 10%
formalin, the obtained brain tissues from the dissection were fixed before being
micrometer and will be stained with Haematoxylin and Eosin (H&E) stain using
routine protocol. Digital micrographs of the slides were taken after the observation
testing and the results were interpreted using the legends below:
Indication Score
The table shows the established neurological score to be used in indicating the degree of CA3
neuronal cell necrosis upon the histopathological analysis of individual rat brains.
5. Statistical Treatment of Data
The validity of the test drug samples used in the study as compared with
the positive control was verified by using the following statistical calculations:
The mean was used to compare the severity of CA3 neuronal cell necrosis
The standard deviation was used to determine he spread out of the group
items.
This chapter included the discussion, analysis and interpretation of data and
results of the study conducted. The data and results were obtained based on the
Table 1
Table 1 shows that 100 grams of Mango leaves macerated in 80% ethanol
Table 2
Color Yellowish-brown
Odor Pungent
Table 2 shows the extract from the leaves of Mango was yellowish-
Table 3
Solvent Results
Acetone Soluble
Benzene Insoluble
Chloroform Insoluble
Ether Insoluble
Lead Acetate test, Ferric chloride test, Alkaline Reagent test, and
Table 4
Table 5
Table 5. Shows that the negative control has moderate to severe CA3
neuronal cell necrosis whereas the positive control has moderate CA3
concentration.
Table 6
Table 7 shows that there is no significant difference between the positive control
and the flavonoid extract at 400 mg/ml concentration. This means that the
Summary Findings
Based on the procedure and methods performed by the researchers, findings are
hereby presented:
1. The percentage yield per 100 grams of shade dried leaves was 0.998% of
semi-purified flavonoids.
3. The solubility test showed that the semi-purified flavonoid extract from
Indian Mango leaves was soluble in Acetone, Distilled water and 80%
Conclusion
As the result of the histopathological test in the rats’ brain, the researchers
concluded that flavonoids present in the leaves of the Indian Mango exhibits
neuroprotective activity against Aluminum induced neurotoxicity in the brains
Recommendations
presented:
10. Formulate a dosage form of the flavonoid extracts from Indian Mango.
References
Afifi, O. et al., (2016). Histological Study on the Protective Role of Ascorbic Acid
Rats. https://doi.org/10.1016/j.jmau.2015.10.001
Alberto, Jhoemar P., et al,. The Neuroprotective Actions of the Flavonoids from
http://waset.org/publications/10002963/atr-ir-study-of-the-mechanism-of-
aluminum-chloride-induced-alzheimer-s-disease-curative-and-protective-
effect-of-lipidium-sativum-water-extract-on-hippocampus-rats-brain-tissue
Barrita, J.L., et al., (2013). Antioxidant Role of Ascorbic Acid and His Protective
diseases-a-role-for-antioxidants/antioxidant-role-of-ascorbic-acid-and-his-
protective-effects-on-chronic-diseases
http://onlinelibrary.wiley.com/doi/10.1111/joim.12053/pdf
Colak, S., et al., (2011). The neuroprotective role of boric acid on aluminum
chloride-induced neurotoxicity.
Jetti, R., et al., (2014) Neuroprotective effect of Ascorbic acid and Ginkgo biloba
http://fluoridealert.org/wp-content/uploads/jetti-20141.pdf
Brain Injury
http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0051324
Nijveldt, R., et al., (2001) Flavonoids: a review of probable mechanisms of action
http://ajcn.nutrition.org/content/74/4/418.full
Toting, J.P. et al., (2016). Formulation of Hand Sanitizer Gel using the
= 0.998g x100
100 g
= 0.998 = 1.0 %
APPENDIX E
Beaker- A beaker is a simple container for stirring, mixing and heating liquids
with a flat bottom. Most also have a small spout to aid pouring.
moisture-sensitive items such as cobalt chloride paper for another use. A common
use for desiccators is to protect chemicals which are hygroscopic or which react
with water from humidity. It also requires some time to achieve a low humidity.
Erlenmeyer flask- a flask having a wide base, narrow neck, and conical form,
Funnel- is a pipe with a wide (often conical) mouth and a narrow stem. It is used
Without a funnel, spillage may occur. Funnels are usually made of stainless
compared to other tools, such as volumetric flasks. Volumetric flasks are used
immiscible solvent phases of different densities. Typically, one of the phases will
be aqueous, and the other a lipophilic organic solvent such as ether, MTBE,
delineation, between the two liquids. The more dense liquid, typically the aqueous
phase unless the organic phase is halogenated, sinks and can be drained out
through a valve away from the less dense liquid, which remains in the separatory
funnel.
liquids for laboratory purposes. They are usually made of solid glass, about the
thickness and slightly longer than a drinking straw, with rounded ends.
Test tube- widely used by chemists to handle chemicals, especially for qualitative
experiments and assays. Their spherical bottom and vertical sides reduce mass
loss when pouring, make them easier to wash out, and allow convenient monitoring
of the contents.
APPENDIX F
Extraction of Flavonoids
Evaporating the extract through water bath Final product of the sample
APPENDIX G
Animal handling during Biological Testing
Collecting the brain of the rat with the help of the veterinarian in-charge.
APPENDIX H
Pre-treatment Dosage Computation in Group 2 Rats
Formula:
Week 1 Week 2
Rat 1
Rat 2
Rat 3
Formula:
Week 1 Week 2
Rat 1
Rat 2
Rat 3
Formula:
Week 1 Week 2
Rat 1
Rat 2
Rat 3
= 0.144 kg x200mg/kg = 0.183kg x200mg/kg
100mg/ml 100mg/ml
= 029 ml = 0.37 ml
APPENDIX K
Pre-treatment Dosage Computation in Group 5 Rats
Formula:
Week 1 Week 2
Rat 1
= 0.145kg x400mg/kg = 0.155kg x400mg/kg
100mg/ml 100mg/ml
= 0.58 ml = 0.62 ml
Rat 2
Rat 3
= 0.150kg x400mg/kg = 0.172kg x400mg/kg
100mg/ml 100mg/ml
= 0.60 ml = 0.69 ml
APPENDIX L
Statistical Analysis
GROUP
I II III IV V
N 3 3 3 3 3
Min 1.00 1.00 1.00 1.00 1.50
DESCRIPTIVE STATISTICS
PERSONAL INFORMATION
EDUCATIONAL BACKGROUND
PERSONAL INFORMATION
EDUCATIONAL BACKGROUND
PERSONAL INFORMATION
EDUCATIONAL BACKGROUND
PERSONAL INFORMATION
EDUCATIONAL BACKGROUND