THE NEUROPROTECTIVE PROPERTY OF THE SEMI-PURIFIED FLAVONOIDS

FROM THE INDIAN MANGO (Mangifera indica Linn. Fam. Anacardiaceae)

LEAVES AGAINST ALUMINUM INDUCED NEUROTOXICITY IN

FEMALE SPRAGUE DAWLEY RATS.

An Undergraduate Research

Presented to

The School of Pharmacy

Centro Escolar University Manila

In Partial Fulfillment of the Requirements for the Degree

Bachelor of Science in Pharmacy

by

Joshua Elijah A. Castroverde

Chris Jason C. Concepcion

Ranielle D. Samson

Keane Mikkolaine G. Silva

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

2. Review of Related Literature and Studies……………...……………….……….. 9

3. Methods and Procedures

Research Methodology……………………….…………………………...19
Research Procedure…………………………….…………………………20
Collection and Preparation of Plant Sample………………………….…20
Extraction and Isolation of the Semi-purified flavonoids…………….…20
Identification Test for Flavonoids…………………………………. …….21
Biological Testing…………………………………………………........... 23

4. Presentation, Analysis and Interpretation of Data…………….....………… 28

Collection and Preparation of Plant Sample…………………………… 28
Extraction and Purification of Flavonoids………………………………..28
 Physical Evaluation of the Flavonoid Extract……………………………29
Chemical Evaluation of the Flavonoid Extract…………………………..30

5. Summary, Conclusions and Recommendations…………………….…………33

Summary of findings……………………………………………………….33
Conclusion…………………………………………………………………..33
Recommendation…………………………………………………………..34
Reference List
Appendices
Curriculum vitae
 ACKNOWLEDGEMENT

The researcher would like to express their sincere gratitude to those who

inspired, guided, helped, supported and contributed to their thesis throughout the

entire course of the study.

To their parents and family members who gave physical, spiritual, financial

and moral support to make this study successful;

To their advisers, Susan M. Montemayor and Mylene S. Andal, for their

patience, motivation, enthusiasm, immense knowledge, constructive criticism,

encouragement and valuable suggestions in answering some of their questions

regarding research;

To the faculty of School of Pharmacy, for their opinions and suggestions;

To the laboratory technicians, for providing them the necessary instruments,

equipment, and chemical reagents needed in their 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.

Castroverde, Joshua Elijah A. Concepcion, Chris Jason C.

Samson, Ranielle D Silva, Keane Mikkolaine G.

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.

KEYWORDS: Neuroprotective, Mango leaves, histopathology, flavonoids

Introduction

Neurotoxicity is the damage to the chemotherapy drugs and radiation

brain or peripheral nervous system
caused by exposure to natural or
man-made toxic substances (S.
Robertson., 2014). Regular exposure
to these natural or man-made toxic
substances which can be part of our
daily lives such as pharmaceutical
and non-pharmaceutical drugs, heavy
metals, certain food preservatives
pesticides, cosmetics and for the
cancer patients who uses
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 of paint and inhalation of
organophosphates which may last
long or cause 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 chemotherapy drugs
(Peter S. Spencer et al., 1990).
The effect of neurotoxicity may
vary depending on the characteristic
of the 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 or
numbness of the limbs, loss of
memory, vision, compulsive
behaviours, imbalance, loss of
circulation, chronic fatigue,
autoimmune conditions such as
irritable bowel syndrome or
rheumatoid arthritis and sexual
dysfunction.
Neuroprotection is defined as the
“protection of neurons” and is now
widely used for the prevention and
management of neurotoxicity which is
associated with brain ischemic stroke,
Parkinson’s disease and Alzheimer’s
disease. The goal of a sclerosis,
neuroprotectant is to lessen the risk of
brain damage in patients, slow the
disease progression by halting or at
least slowing the loss of neurons.
Neuroprotective treatment often
target oxidative stress, excitotoxicity,
mitochondrial dysfunction, apoptosis,
glutamate receptor activation,
excessive dosage of amyloid beta,
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 who uses this as it is the
best antiperspirant component of
each deodorants. 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 now well known as a
neurotoxin that enhances
neuroinflammatory events in the brain
by different mechanism. Some of this
mechanism incudes the ability of
Aluminum to exacerbates oxidative
stress, amyloid beta desposition and
plaque 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
neurodegenerative disease such as
multiple sclerosis, amyotrophic lateral
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 of
aluminium-induced neurotoxicity is
related to distorted cythoarchitecture
of 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 to genotoxicity
and DNA damage. (Izumi T., et al.,
2003).
METHODS AND PROCEDURE
The experimental method of research
was used in the determination of the
neuroprotective property of the Semi-
purified flavonoid extract from the
leaves of Indian Mango (Mangifera
alitissima linn. Fam.
Anachardiaceae). Mnago leaves
were gathered from Calamba,
Laguna. The leaves of mango were
cut into small pieces and air dried for
7 days. A specimen was submitted to
the Bureau of Plant Industry for
authentication of the plant. About 100
grams of the ground dried plant were
weighed and treated with 80% ethyl
alcohol and keep macerated for 7
days. The mixture was then filtered
and the filtrate was then evaporated
through water bath until it became
syrupy. It was deffated using
petroleum ether and was re-extracted
using ethyl acetate and 2M HCl and
was separated using separatory
funnel. This was repeated until the
solvent is almost colorless. The
concentrated flavonoid extract was
weighed and stored in a tightly
stoppered container in 2-8°C ready
for physical and chemical tests,
instrumental analysis and biological
testing.
Fifteen female Sprague dawley rats
were purchase from the Department
of Science and Techonology. They
were acclimatized at Centro Escolar
University, Manila Animal house for 7
days. The test animals were divided
into five groups of 3 Sprague dawley
rats each. Group I Negative control
(Plain NSS), Group II Positive control
(Ascorbic acid 250mg/kg/ml), Group
III Experimental group (Semi-purified
flavonoids 100mg/kg/ml), Group IV
Experimental group (Semi-purified
flavonoids 200mg/kg/ml), and Group
V Experimental group (Semi-purified
flavonoids 400mg/kg/ml)
Histopathological Analysis
Photomicrograph of the
histopathological analysis of the
individual rat brains were evaluated
by an IACUC laboratory veterinarian
technologist. Histopathological
criterion, CA3 neuronal cells necrosis
on each rat brain was graded semi-
quantitatively using the established
neurological score indicating the
degree of CA3 neuronal cell necrosis.
Statistical Analysis
All data were statistically treated by
one-way analysis of variance
(ANOVA), followed by LSD (least
significant difference) multiple
comparison test assess significant
differences of mean between group.
All results were expresses as mean
+/- S.E.M (standard error of mean) at
5% significant level. Difference were
considered significant at p <0.05.
Indication Score
Normal CA3 neuronal cell necrosis 4.0
Mild CA3 neuronal cell necrosis 3.0
Mild to Moderate CA3 neuronal cell 2.5
necrosis
Moderate CA3 neuronal cell necrosis 2.0
Moderate to severe CA3 neuronal cell 1.5
necrosis
Severe CA3 neuronal cell necrosis 1.0
 Table 1 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

RESULTS AND DISCUSSION Group 1

Negative Control
Percentage Yield of Flavonoids (Plain NSS)
After being extracted from the leaves
of Mango leaves, the flavonoids Moderate to Severe
degeneration
obtained were weighed. The
percentage yield showed that every
100g of the cut mango leaves there is
0.998% of flavonoids.

Identification Tests for Flavonoids

For the physical tests, organoleptic

test shows that the flavonoid extract
was yellowish brown in color, sweet Severe degeneration Moderate degeneration
odor and semi-solid. While solubility
tests shows that the flavonoids
extract from Indian Mango leaves was
soluble in 80% ethyl alcohol and
acetone, spraringly soluble in distilled Group 2
water and insoluble in chloroform and Positive Control
petroleum ether. (Ascorbic Acid)
For the Phytochemical screening.
The Results of the chemical
evaluation revealed that the crude Mild degeneration
extract was positive in Bate-smith
Metcalf test, Wilstatter Cyanidin test,
Lead Acetate test, Shinoda test, Ethyl
Acetate test, Alkaline reagent test and
Ferric chloride test.
Histopathological Analysis

Figure 1 Histopathological Analysis

of Cellular Degeneration of Rats Brain
N Severe degeneration Severe degeneration

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:
Alberto, Jhoemar P., et al. The
Neuroprotective Actions of the
Flavonoids from the Fuji Apple Fruits
(Malus domestica Family Rosaseae) on
Induced Brain Ischemia using Middle
Carotid Artery Occlusion Stroke Model in
Male Sprague Dawley Rats (Centro
Escolar University, 2014)
Balgoon, M.J. et al (2015). 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. World Academy of
Science, Engineering and Technology
International Journal of Pharmacological
and Pharmaceutical Sciences Vol:9,
No:11, 2015.
http://waset.org/publications/10002963/a
tr-ir-study-of-the-mechanism-of-
aluminum-chloride-induced-alzheimer-s-
disease-curative-and-protective-effectof-
lipidium-sativum-water-extract-on-
hippocampus-rats-brain-tissue
Barrita, J.L., et al (2013). Antioxidant
Role of Ascorbic Acid and His Protective
Effects on Chronic Diseases.
https://www.intechopen.com/books/oxid
ative-stress-and-chronic-degenerative-
diseases-a-role-for-
antioxidants/antioxidant-role-of-ascorbic-
acid-and-his-protective-effects-on-
chronic-diseases
Ceccatelli S. (2013). Mechanisms of
neurotoxicity and implications for
neurological disorders. Journal of
Internal Medicine Volume 273, Issue 5,
Version of
Record online: 22 APR 2013
http://onlinelibrary.wiley.com/doi/10.111
1/joim.12053/pdf
Colak, S., et al (2011). The
neuroprotective role of boric acid on
aluminum chloride-induced neurotoxicity.
 CHAPTER 1

The Problem and Its Background

Introduction

Neurotoxicity is the damage to the brain or peripheral nervous system

caused by exposure to natural or man-made toxic substances (S. Robertson.,

2014). Regular exposure to these natural or man-made toxic substances which

can be part of our daily lives such as pharmaceutical and non-pharmaceutical

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

of paint and inhalation of organophosphates which may last long or cause

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

chemotherapy drugs (Peter S. Spencer et al., 1990).

 The effect of neurotoxicity may vary depending on the characteristic of the

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

or numbness of the limbs, loss of memory, vision, compulsive behaviours,

imbalance, loss of circulation, chronic fatigue, autoimmune conditions such as

irritable bowel syndrome or rheumatoid arthritis and sexual dysfunction. (S.

Robertson., 2014).

Neuroprotection is defined as the “protection of neurons” and is now widely

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

progression by halting or at least slowing the loss of neurons. Neuroprotective

treatment often target oxidative stress, excitotoxicity, mitochondrial dysfunction,

apoptosis, glutamate receptor activation, excessive dosage of amyloid beta,

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

who uses this as it is the best antiperspirant component of each deodorants.

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

now well known as a neurotoxin that enhances neuroinflammatory events in the

brain by different mechanism. Some of this mechanism incudes the ability of

Aluminum to exacerbates oxidative stress, amyloid beta desposition and plaque

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

neurodegenerative disease such as multiple sclerosis, amyotrophic lateral

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

of aluminium-induced neurotoxicity is related to distorted cythoarchitecture of

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

to genotoxicity and DNA damage. (Izumi T., et al., 2003).

Background of the Study

With a minimal yet dangerous risk of patients developing neurotoxicity, this

study aims to study the basic principles and potential therapeutic interventions

associated with neurotoxicity and to explore further regarding the

pathophysiological and improvement of the medical treatment for this disease.

 The researchers have found a potential plant of interest to further study for

its significance in the medical field specifically for the prevention and management

of neurotoxicity. Hence, the researchers have chosen Mango (Mangifera indica,

Family Anacardiaceae) as the subject for potential substance to be used in the

management and prevention of neurotoxicity. Many epidemiological studies have

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

have an anti-oxidant, antiviral, anthelmintic, anti-allergy, antiparasitic,

antispasmodic, antipyretic, immunomodulatory, anti-diarrhoeal, anti-inflammatory,

antibacterial, antifungal and hepatoprotective (J. Szalay., 2015) Studies have

shown that mangoes contains approximately 76% of vitamin C and 25% of vitamin

A, which is a primary contributor to its anti-oxidant activity (R. Fritz., 2015).

Mangoes contain a wide range of various phytochemicals such as polyphenols

flavonoids and triterpenoids. Mangiferin a xanthone glycoside a major bio-active

constituent, isomangiferin, tannins and gallic acid derivatives. (Scartezzini P, et al.,

2000).

Flavonoids are phenolic compounds known for its characteristics as red,

blue and purple anthocyanin pigment of plant tissues. They are group of plant

metabolites commonly found in variety of fruits and vegetables thought to provide

health benefits through cell signalling pathways and antioxidant effects, Ingestion

of flavonoid-rich food is associated with lesser risk of having cardiovascular

disease, cancer, neurodegenerative diseases and having a better management of

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

its neuroprotective property (Sandhar et al., 2011).

Conceptual Framework

The study was composed of the following procedures: extraction of the

flavonoid from the leaves of the mangoes, the identification of flavonoid extract

though physical test, phytochemical screening and instrumental analysis, the

induction of neurotoxicity on rats induced by Aluminum Chloride, and the

determination of the neuroprotective activity of the flavonoid extract from the

leaves of mangoes through the evaluation of the dissected brains of the pre-treated

rats by histopathological analysis. The following figure shows these steps.

Research Objectives

This research topic generally aimed to determine the neuroprotective

property of the flavonoids present in Mango in induced brain neurotoxicity in rats.

Specifically, it aimed:

1. To extract and isolate the flavonoids present from Mangifera indica.

2. To determine the concentration of flavonoids that exerts neuroprotective

activity.

3. To compare the neuroprotective property of the semi purified flavonoid

extract from mango leaves with the standard or comparative drug.

Research Hypothesis

The study aims to prove the effectiveness of the neuroprotective property

of the semi purified flavonoid from the Mangifera indica and the possible

outcome is that there is no significant difference in the neuroprotective

property of the semi purified flavonoid with the standard drug which is

ascorbic acid.

Significance of the Study

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

neuroprotective is rapidly increasing. Even with the current marketed drugs

associated with its neuroprotective property, a remarkable number of patients still

continue to suffer from the complications that correspond to the neurotoxicity.

Natural neuroprotective agents can prevent or supress many

pathophysiological mechanism of known brain disorders without undesirable

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

quality of life of people exposed to these toxic metals, specifically aluminium, in

their daily lives.

 This study will provide a prophylactic therapy to people who are subjected

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,

as a natural source of flavonoids exhibiting potential neuroprotective activity. If this

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.

Scope and Delimitations of the Study

The study focused on the:

1. The collection and preparation of the plant sample, Mango (Mangifera

indica) leaves.

2. The extraction of the crude constituents, macerated for 5 days using 50%

ethyl alcohol as a solvent.

3. The isolation of the flavonoids by defatting the crude extract, with petroleum

ether and purification by ethyl acetate.

4. The confirmatory test for flavonoids by physical, phytochemical screening,

instrumental analysis and biological test.

5. The use of Aluminum Chloride (AlCl3) as the neurotoxin agent in male

Sprague Dawley Rats.

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

understanding of the readers:

Aluminum. It is the third most abundant element in the earth’s crust next to

oxygen and silicon. (Haney, et al., 2011)

Extraction. It is a way of pulling or separating a component from one

another. (Edison, 2011)

Flavonoid. It is a group of plant metabolites thought to provide health

benefits through cell signalling pathways and antioxidant effects. (Robertson,

2014).

Neuroprotection. It is an effect that may result in salvage, recovery or

regeneration of the nervous system, its cells, structure and function. (Vajda FJ,.

2009).

Neurotoxicity. It refers to damage to the brain or peripheral nervous

system caused by exposure to natural or man-made toxic substances.

(Robertson, 2014)
 CHAPTER 2

Review of Related Literature and Studies

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

neuroprotective property if the semi-purified flavonoids from Indian mango

(mangifera alitissima linn. Fam. Anacardiaceae) leaves against Aluminum chloride

induced neurotoxicity in male sprague dawley rats

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

seen in the proliferation of mango products in supermarkets and groceries.

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.

Flavonoids include many of the common plant pigments. It can be divided

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

colorless or only slightly yellow. (Villasenor, 2011)

Natural food-derived flavonoids may help reduce the incidence of

atherosclerosis, cancer, cardiovascular diseases, diabetes, thrombosis,

inflammation in arthritis, asthma, encephalomyelitis, and atherosclerosis,

neurodegenerative diseases such as Alzheimer’s and Parkinson’s diseases,

obesity, hyperlipidemia, nerve injury, and hypertension. (Cassidy et al., 2011)

Neurotoxicity happens when the exposure to natural or manmade toxic

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

to substances used in chemotherapy, radiation treatment, drug therapies, and

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,

cosmetics, and some naturally occurring substances. Symptoms may appear

immediately after exposure or be delayed. They may include limb weakness or

numbness, loss of memory, vision, and/or intellect, uncontrollable obsessive

and/or compulsive behaviors, delusions, headache, cognitive and behavioral

problems and sexual dysfunction. Individuals with certain disorders may be

especially vulnerable to neurotoxins.

 The presence of neurocognitive deficits alone is not usually considered

sufficient evidence of neurotoxicity, as many substances may impair

neurocognitive performance without resulting in the death of neurons. This may be

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

substances only becoming neurotoxic in certain doses or time periods. Some of

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

control, cognitive deterioration and autonomic nervous system dysfunction.

Additionally, neurotoxicity has been found to be a major cause of

neurodegenerative diseases such as Alzheimer's disease (AD). (Vulih-Shultzman

et al., 2007)

Aluminium chloride (AlCl3) is the main compound of aluminium and

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

a Lewis acid. It is an example of an inorganic compound that "cracks" at mild

temperature, reversibly changing from a polymer to a monomer.

Aluminum chloride is a neurotoxin. Anhydrous AlCl3 reacts vigorously with

bases, so suitable precautions are required. It can cause irritation to the eyes,

skin, and the respiratory system if inhaled or on contact.

According to Alzheimer's Association in USA, Alzheimer's is the most

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

for 60 to 80 percent of dementia cases. Alzheimer's is not a normal part of

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.

Approximately 200,000 Americans under the age of 65 have younger-onset

Alzheimer’s disease (also known as early-onset Alzheimer’s). It is a progressive

disease, where dementia symptoms gradually worsen over a number of years. In

its early stages, memory loss is mild, but with late-stage Alzheimer's, individuals

lose the ability to carry on a conversation and respond to their environment.

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. .

Ascorbic acid (AA), commonly known as vitamin C, plays an important role

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

synthesis of hormones, neurotransmitters and in the metabolism of certain amino

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

reducing inflammatory symptoms. Its antioxidant capacity is associated with

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

processes and reactions. Some epidemiological data mentioned its usefulness in

reducing cold with increasing consumption of foods rich in vitamin, so people

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,

immunity, drug metabolism and urinary excretion of hydroxyproline. Antioxidants

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

AA its antioxidant role is useful since it contributes to the maintenance of

the vascular system and the reduction of atherogenesis through regulation in

collagen synthesis, production of prostacyclin and nitric oxide. In addition to this

antioxidant role, the AA has actions at the molecular level because it acts as a

cofactor of enzymes such as dopamine hydroxylase, influencing neurotransmitter

concentration, improves lysosomal protein degradation and mediates consumer

monosodium glutamate.

Alzheimer's has no current cure, but treatments for symptoms are available

and research continues. Although current Alzheimer's treatments cannot stop

Alzheimer's from progressing, they can temporarily slow the worsening of

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

the disease, delay its onset, and prevent it from developing.

Parkinson's disease (PD) is a long-term degenerative disorder of the central

nervous system that mainly affects the motor system. The symptoms generally

come on slowly over time. Early in the disease, the most obvious

are shaking, rigidity, slowness of movement, and difficulty with walking.

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

collectively called "parkinsonism", or a "parkinsonian syndrome".

The cause of Parkinson's disease is generally unknown, but believed to

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

understood, but involves the build-up of proteins into Lewy bodies in

the neurons. Diagnosis of typical cases is mainly based on symptoms, with tests

such as neuroimaging being used to rule out other diseases.

There is no cure for Parkinson's disease, with treatment directed at

improving symptoms. Initial treatment is typically with the antiparkinson

medication levodopa (L-DOPA), with dopamine agonists being used once

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

produce a complication marked by involuntary writhing movements. Diet and

some forms of rehabilitation have shown some effectiveness at improving

symptoms. Surgery to place microelectrodes for deep brain stimulation has been

used to reduce motor symptoms in severe cases where drugs are

ineffective. Evidence for treatments for the non-movement-related symptoms of

PD, such as sleep disturbances and emotional problems, is less strong. (Elkouzi,

2014).

The researcher evaluates the effect of Mangifera indica extract on

excitotoxicity, cells were stimulated with 1-glutamic acid having the dose at 50

micrometer alone or in the presence of Mangifera indica extract. 56% maximal

protection was obtained with 2.5 ml of Mangifera indica extract. Then, the

researchers measured the effects of Mangifera indica extracts on excitotoxic-

induced oxidative stress and the mitochondrial depolarization by fluorimeter using

5,6-chloromethyl-2,7-dichlororodihydrofluorescein diacetate and

tetramethylrhodamine, respectivel. Both parameters were effectively reduced by

the extract at concentrations which showed neuroprotection. Mangiferin, an

antioxidant polyphenol which is a major component of Mangifera indica, was also

effective in preventing neuronal death, oxidative stress, and mitochondrial

depolarization. Maximal protection at 64% was obtained at 12.5 microgram/ml of

mangiferin which also attenuated oxidative stress and mitochondrial depolarization

at the neuroprotective concentrations.

Glutamate is the major excitatory neurotransmitter in the central nervous

system however excessive amount of glutamate cause over activation of post-

synaptic neurons, which is known as excitotoxicity and it will increase 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

mechanism that contributes to the neurodegeneration leading to stroke and

trauma, and other progressive neurological diseases such as Parkinson’s disease

and Alzheimer’s disease (Michaelis et al., 2003).

I flavonoids and other antioxidant polyphenols have recently defines as the

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

and in vivo (Gilgun-sherki et al, 2004).

An aqueous stem bark extract from Mangifera indica which contains a

defined and standardized mixture of components such as polyphenols, terpenoids,

steroids, fatty acids, microelements and xanthone mangiferin which is the

predominant components has been used as nutritional supplement. (Nuez-Selles

et al, 2002)

Mangiferin is the major component of Mangifera indica and it has been

advised that it is primarily responsible for the activity of manga. Recently,

mangiferin and morin, another antioxidant polyphenols has been identified to have

neuroprotective properties in both in vivo and in vitro (Gootlieb et al., 2006)

The Mangifera indica extract was analyzed by the Quality Department of

the Center Pharmaceutical Chemistry. The data showed that the extract contains

the following composition: moisture 50%, total phenol in anhydrous base 30%, and

mangiferin 20% according to the regulatory quality specification.

The researcher made a dose-response curve of glutamate excitotoxicity in

culture of neurons from the cerebral cortex of 18-day rat embryos. The cell was

exposed to glutamate ranging to 1-2 micrometers for 10 minutes. To test the

efficacy of Mangifera indica extract and mangiferin against excitotoxicity, the

Mangifera indica extract ranging from 0.5 to 5 microgram/ml and mangiferin from

6.25-25 microgram/ml was injected in the culture medium during glutamate

exposure.

The neuroprotective effect of Mangifera indica are note dose-independent.

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

0.5 microgram/ml also showed excitotoxicity induced by glutamate however with a

lower efficacy. Also, neuronal viability after exposure to glutamate in conjuction

with mangiferin was highest at 12.5 microgram/ml. same with the extract, the

lowest and the highest mangiferin concentration had a lower neuroprotective effect

against excitotoxicity than the average concentration showed by the result.

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

other polyphenolic compounds present in Mangifera indica including gallic acid,

and epicatequin galate have neuroprotector effects (Bastianetto et al., 2006)

The researcher observed that Mangifera indica extract and mangiferin have

both similar neuroprotective property against glutamate-excitotoxic damage in

cortical neurons and both have similar antioxidant activity. This advice that these

extracts can be used as dietary supplement to attenuate neuronal death during

nirmal aging and neurodegenerative disorders in which excitotoxicity and oxidative

stress occurs.
 CHAPTER 3

Methods and Procedures

This chapter includes the discussion of the research methodology that was

utilized in the determination of neuroprotective property of semi-purified flavonoids

from the extract of the leaves of Indian mango against induced neurotoxicity using

aluminum chloride. It covers the procedure used in the physical, chemical,

biological testing, histopathological evaluation, and the statistical analysis.

Research Methodology

To determine the neuroprotective property of semi-purified flavonoids from

Indian mango (Mangifera indica Lin. Fam. Anacardiacaea) leaves, experimental

method was used to conduct this study.

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.

The researchers used 15 laboratory animals to induced neurotoxicity and

aluminum chloride was used as neurotoxicity inducer in male Sprague-dawley rats.

The neuroprotective property of semi-purified flavonoids was assessed using

histopathological analysis.
Settings of the Study

The study was conducted at Centro Escolar University, Manila at

Concepcion Aguila Hall room 305.

Research Procedure

1. Collection and Preparation of Plant Sample

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

pulverized using blender, and stored in a well-closed container.

2. Extraction and Isolation of the Semi-purified Flavonoids

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

using muslin cloth.

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 semi-purified flavonoids were collected in a tared evaporating dish and

weighed. Percentage yield was computed based on this formula:

𝑊𝑒𝑖𝑔ℎ𝑡 𝑜𝑓 𝑡ℎ𝑒 𝑆𝑒𝑚𝑖 − 𝑝𝑢𝑟𝑖𝑓𝑖𝑒𝑑 𝐹𝑙𝑎𝑣𝑜𝑛𝑜𝑖𝑑𝑠

% 𝑌𝑖𝑒𝑙𝑑 = 𝑥 100
𝑊𝑒𝑖𝑔ℎ𝑡 𝑜𝑓 𝑡ℎ𝑒 𝑑𝑟𝑖𝑒𝑑 𝑠𝑎𝑚𝑝𝑙𝑒

3. Identification Tests for Flavonoids

3.1 Physical Test

3.1.1 Organoleptic Test

The odor, color, and appearance of the extract were studied and physically

examined.

3.1.2 Solubility Test

The solubility of the extracted semi-purified flavonoids was determined by

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,

slightly soluble, very slightly soluble, or insoluble.

 3.2 Chemical Test

3.2.1 Bate-Smith and Metcalf test

0.5 mL of concentrated HCl was added to a test tube containing the

defatted sample and warmed using water bath for 15 minutes. Positive result

would be the appearance of strong red or violet color.

3.2.2 Ethyl Acetate Test

In a tets tube with the sample, 10 mL of ethyl acetate was added and

subjected in a steam bath for 3 minutes. Using 4 mL of the filtrate, about 1 mL of

ammonia solution was added. Positive result would be the appearance of yellow

coloration.

3.2.3 Ferric Chloride Test

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.

3.2.4 Lead Acetate Test

A few mL of lead acetate solution was added to drop by drop.

Formation of flesh-brown colored precipitate indicates the presence of flavonoids.

3.2.5 Shinoda’s Test

In a test tube, three (3) pieces of magnesium ribbon were added

followed by a few drops of concentrated HCl. The appearance of pink, orange, or

red to purple coloration indicates the presence of flavonoids.

 3.2.6 Wilstatter “Cyanidin Test”

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.

4. Biological Testing in Neuroprotective property of semi-purified flavonoid

extracts

4.1 Experimental Animals

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

(DOST) and kept at an animal facility of Centro Escolar University, Manila.

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

stored in an air conditioned room at a constant temperature of 25 ±2℃ and

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

Mendiola, Manila, Philippines.

4.2 Experimental Variable

The extracted semi-purified flavonoids from Indian mango leaves

were used as the experimental variable in this study. It was dissolved in distilled

water at 100mg/mL prior to administration.

4.3 Positive Control

Ascorbic acid was dissolved having a concentration of 100mg/mL

prior to administration.

4.4 Neurotoxicity Induction

On the eleventh (11th) day of the experiment proper, aluminum

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

laboratory veterinarian for the histoptahological assay.

4.5 Experimental Proper

4.5.1 Dose Preparation and Administration

The rats received a dose of 100mg/mL concentration of semi-

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 )

4.5.2 Experimental Protocol

All rats were treated according to the group they belong.

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

mg/kg on the 1st to 10th day.

Groups 3, 4 and 5 served as the experimental groups. The rats in

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

mg/kg, respectively, on the 1st to 10th day.

On the 11th day, all rats received Aluminum Chloride until the 14th

day for the induction of neurotoxicity.

 4.5.3 Preparation of tissue sample

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

processed in an automatic tissue processor. The processed tissues were

embedded in a paraffin wax, sectioned with microtome at a thickness of 4

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

of the stained slides under light microscope.

4.5.4 Histopathological Analysis

Histopathological analysis of the rat’s brain was observed and

evaluated by a professional laboratory veterinarian and a medical technologist.

The neuronal cell degeneration was measured using histopathoogical

testing and the results were interpreted using the legends below:

Indication Score

Normal CA3 neuronal cell necrosis 4.0

Mild CA3 neuronal cell necrosis 3.0

Mild to moderate CA3 neuronal cell necrosis 2.5

Moderate CA3 neuronal cell necrosis 2.0

Moderate to severe CA3 neuronal cell necrosis 1.5

Severe CA3 neuronal cell necrosis 1.0

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

of the test animal with the positive control group.

The standard deviation was used to determine he spread out of the group

items.

One-way Analysis of Variance (ANOVA) was used to test the non-

significance of difference among several groups.

The test was applied to determine if the is no significant differences among

the groups of test animals as regards to the pharmacologic effects of semi-purified

flavonoid extracts. The level of significance was set at p<0.05.

 CHAPTER 4

Presentation, Analysis, and Interpretation of Data

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

method and procedures done in the study.

1. Collection and Preparation of Plant Sample

The plant samples used in the study were collected in Laguna,

Philippines from June to August 2017.

2. Extraction and Purification of Flavonoids

The extraction and purification of flavonoids from the Mango leaves

obtained a percentage yield of 0.998%.

Table 1

Percentage Yield of Flavonoid Extract

Weight of the sample = 100g

Weight of the residue = 0.998g

Percentage Yield = 0.998%

Table 1 shows that 100 grams of Mango leaves macerated in 80% ethanol

yields 0.998g equivalent to 0.998% percentage yield.

2. Physical Test of Flavonoid Extracts

Table 2

Results for Organoleptic Evaluation

Test Performed Results

Appearance Semi solid

Color Yellowish-brown

Odor Pungent

Table 2 shows the extract from the leaves of Mango was yellowish-

brown in color with pungent odor, and semisolid consistency.

Table 3

Results for Solubility Test

Solvent Results

Acetone Soluble

Benzene Insoluble

Chloroform Insoluble

Ether Insoluble

Distilled Water Soluble

80% Ethanol Soluble

Table 3 shows that the semi-purified flavonoid extract is soluble

in acetone, distilled water and 80% ethanol.

3. Chemical Test

Lead Acetate test, Ferric chloride test, Alkaline Reagent test, and

Bate-Smith and Metcalf test were used to determine the presence of

flavonoids in the leaves of Mango.

Table 4

Results for Chemical Evaluation

Test Expected Result Actual Result

Lead Acetate Formation of yellow Formation of yellow
Test precipitate precipitate

Ferric Chloride Formation of intense Formation of intense

Test green color dark green color

Alkaline Reagent Formation of intense Formation of intense

Test yellow color which dark yellow color
becomes colorless on
further addition of dilute
acid
Bate-Smith and Appearance of strong No change in color
Metcalf Test red or violet coloration

Table 4 shows that flavonoids were present in the leaves of Mango.

 4. Evaluation of degradation of CA3 Neuronal cells

Table 5

Results of degradation of CA3 Neuronal cells

Treatment group Interpretation

Mean = 1.33 Moderate to severe CA3
Negative N=3 neuronal cell necrosis.
SD = 0.29
Mean = 2
Positive N=3 Moderate CA3 neuronal
SD = 1 cell necrosis
Flavonoid Extract
Mean = 1.17 Severe CA3 neuronal cell
100 mg/mL N=3 necrosis.
SD = 0.29

200 mg/mL Mean = 1.17

N=3 Severe CA3 neuronal cell
SD = 0.29 necrosis.

Mean = 2.17 Moderate CA3 neuronal

400 mg/mL N= 3 cell necrosis
SD = 0.58

Table 5. Shows that the negative control has moderate to severe CA3

neuronal cell necrosis whereas the positive control has moderate CA3

neuronal cell necrosis as well as the flavonoid extract at 400 mg/ml

concentration.
 Table 6

Comparison of Degradation of CA3 Cells between the

semi-purified flavonoid extract and Ascorbic Acid.

Groups Mean SD F- Comparison P- Remarks

value value value
Negative 1.33 0.29
(Normal
Saline p>0.05
Solution) Means
Positive 2.00 1.00 are not
(Ascorbi signific
c Acid) antly
100mg/ 1.17 0.29 differe POSITIVE 3.98E- Means are
mL nt 01 not
(Extract) significantly
different
200mg/ 1.17 0.29 POSITIVE 3.98E- Means are
mL 01 not
(Extract) significantly
different
400mg/ 2.17 0.58 POSITIVE 9.86E- Means are
mL 01 not
(Extract) significantly
different

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

flavonoid extract is comparable with the positive control in terms of degradation of

CA3 neuronal cells.

 CHAPTER 5

Summary, Conclusions and Recommendations

This chapter includes the summary findings, conclusions and

recommendations of the study of the Neuroprotective Property of Semi-purified

Flavonoids from Mango (Mangifera indica) leaves.

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.

2. The semi-purified flavonoid extract from Indian Mango leaves was

yellowish-brown in color, pungent in odor and semi-solid in physical state.

3. The solubility test showed that the semi-purified flavonoid extract from

Indian Mango leaves was soluble in Acetone, Distilled water and 80%

Ethanol. While insoluble with Benzene, Chloroform and Ether.

4. The 400mg/kg/day semi-purified flavonoid extract showed comparable

neuroprotective activity with the standard drug, Ascorbic Acid.

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

for the rats at the dose 400mg/kg/day.

Recommendations

The recommendations based on the study conducted are hereby

presented:

6. Development of a better extraction method of flavonoids from the leaves

of the Indian Mango.

7. Determination of the safe and effective dose of the flavonoids present in

the leaves of Indian Mango.

8. Toxicity study regarding the dosing of the extract.

9. Phamacokinetic and Pharmadynamic evaluation of flavonoids.

10. Formulate a dosage form of the flavonoid extracts from Indian Mango.
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2402,p- ISSN: 2319-2399.Volume 8, Issue 1 Ver. V (Feb. 2014), PP 30-36

http://fluoridealert.org/wp-content/uploads/jetti-20141.pdf

Keddy, P., et al., (2012). Neuroprotective and Anti-Inflammatory Effects of the

Flavonoid-Enriched Fraction AF4 in a Mouse Model of Hypoxic-Ischemic

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

and potential applications 1, 2, 3.

http://ajcn.nutrition.org/content/74/4/418.full

Sandhar, H., et al., (2011). A review of Phytochemistry and Pharmacology of

flavonoids. InternationalPharmaceuticaSciencia 1(1), 25-41

Toting, J.P. et al., (2016). Formulation of Hand Sanitizer Gel using the

SemiPurified Flavonoids from the outer coverings of the Red Creole

variety of Allium cepa Linn.: A preliminary investigation. John Paul

Tolentino Toting et al /JAASP 2016;1:346-50

APPENDICES
APPENDIX A
Mango tree
APPENDIX B
Mango leaves
APPENDIX C
 APPENDIX D

% yield = weight of semi-purified flavonoid x100

Weight of plant sample

= 0.998g x100
100 g

= 0.998 = 1.0 %
 APPENDIX E

Beaker- A beaker is a simple container for stirring, mixing and heating liquids

commonly used in many laboratories. Beakers are generally cylindrical in shape,

with a flat bottom. Most also have a small spout to aid pouring.

Dessicator-are sealable enclosures containing desiccants used for preserving

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,

convenient in laboratory experimentation for swirling liquids by hand.

Evaporating dish- is used to evaporate excess solvents - most commonly water

- to produce a concentrated solution or a solid precipitate of the dissolved

substance. Most are made of porcelain or borosilicate glass.

Funnel- is a pipe with a wide (often conical) mouth and a narrow stem. It is used

to channel liquid or fine-grained substances into containers with a small opening.

Without a funnel, spillage may occur. Funnels are usually made of stainless

steel, aluminium, glass, or plastic.

Graduated Cylinder- is used for measuring volumes (amounts) of liquids. This

piece of equipment is used routinely, although it is only moderately accurate

compared to other tools, such as volumetric flasks. Volumetric flasks are used

when absolute precision (accuracy) is needed.

Hot plate- is a portable self-contained tabletop small appliance that features one,

two or more electric heating elements or gas burners.

Separatory Funnel - also known as separation funnel, separating funnel, or

colloquially separatory funnel, is a piece of laboratory glassware used in liquid-

liquid extractions to separate (partition) the components of a mixture into two

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,

dichloromethane, chloroform, or ethyl acetate. All of these solvents form a clear

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.

Stirring rod- is a piece of laboratory equipment used to mix chemicals and

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

Indian Mango leaves Cutting of plant sample into small pieces

Grounding of plant sample Maceration with 80% ethanol

Evaporating the extract through water bath Final product of the sample
 APPENDIX G
Animal handling during Biological Testing

Administration of the dose Died rats using cervical dislocation

Collecting the brain of the rat with the help of the veterinarian in-charge.
 APPENDIX H
Pre-treatment Dosage Computation in Group 2 Rats

GROUP 2 POSITIVE CO CONTROL (Ascorbic Acid 250mg/kg)

RAT Weight 1st week (g) Dose (ml) Weight 2nd week (g) Dose (ml)
Rat 1 153 g 0.38ml 155g 0.39ml
Rat 2 162 g 0.41 ml 162g 0.41 ml
Rat3 161 g 0.40 ml 162g 0.41ml

Formula:

Weight of the rat (kg) x Dose to be administered (mg/kg)

Volume (ml) =
Concentration of the solution (mg/ml)

Week 1 Week 2
Rat 1

= 0.153kg x 250mg/kg = 0.155kg x 250mg/kg

100mg/ml 100mg/ml
= 0.38 ml = 0.39 ml

Rat 2

= 0.162kg x 250mg/kg = 0.162kg x 250mg/kg

100mg/ml 100mg/ml
=0.41ml =0.41 ml

Rat 3

= 0.161kg x 250mg/kg = 0.162kg x 250mg/kg

100mg/ml 100mg/ml
=0.40ml = 0.41 ml
 APPENDIX I
Pre-treatment Dosage Computation in Group 3 Rats

Group 3- Flavonoid 100 mg

st
RAT Weight 1 week (g) Dose (ml) Weight 2nd week (g) Dose (ml)
Rat 1 148 g 0.14 ml 153g 0.15 ml
Rat 2 139 g 0.14ml 151 g 0.15 ml
Rat3 145 g 0. 15 ml 140g 0.14 ml

Formula:

Weight of the rat (kg) x Dose to be administered (mg/kg)

Volume (ml) =
Concentration of the solution (mg/ml)

Week 1 Week 2
Rat 1

= 0.148kg x100mg/kg = 0.153kg x100mg/kg

100mg/ml 100mg/ml
= 0.14 ml = 0.15 ml

Rat 2

= 0.139kg x100mg/kg = 0.151kg x100mg/kg

100mg/ml 100mg/ml
= 0.14 ml = 0.15 ml

Rat 3

= 0.145kg x100mg/kg = 0.140kg x100mg/kg

100mg/ml 100mg/ml
= 0.15 ml = 0.14 ml
 APPENDIX J
Pre-treatment Dosage Computation in Group 4 Rats

Group 4- Flavonoid 200 mg

st
RAT Weight 1 week (g) Dose (ml) Weight 2nd week (g) Dose (ml)
Rat 1 143 g 0.29 ml 167 g 0.33 ml
Rat 2 148 g 0.30ml 181 g 0.36 ml
Rat3 144 g 0. 29 ml 183 g 0.37 ml

Formula:

Weight of the rat (kg) x Dose to be administered (mg/kg)

Volume (ml) =
Concentration of the solution (mg/ml)

Week 1 Week 2
Rat 1

= 0.143kg x200mg/kg = 0.167kg x200mg/kg

100mg/ml 100mg/ml
= 0.29ml = 0.33 ml

Rat 2

= 0.148kg x200mg/kg = 0.181kg x200mg/kg

100mg/ml 100mg/ml
= 0.30 ml = 0.36 ml

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

Group 5- Flavonoid 400 mg

st
RAT Weight 1 week (g) Dose (ml) Weight 2nd week (g) Dose (ml)
Rat 1 145 g 0.58 ml 155g 0.62 ml
Rat 2 168 g 0.67 ml 192g 0.79 ml
Rat3 150 g 0. 60 ml 172g 0.69 ml

Formula:

Weight of the rat (kg) x Dose to be administered (mg/kg)

Volume (ml) =
Concentration of the solution (mg/ml)

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

= 0.168kg x400mg/kg = 0.192kg x400mg/kg

100mg/ml 100mg/ml
= 0.67 ml = 0.79 ml

Rat 3
= 0.150kg x400mg/kg = 0.172kg x400mg/kg
100mg/ml 100mg/ml
= 0.60 ml = 0.69 ml
 APPENDIX L
Statistical Analysis

Severity of Degeneration of C3 cells

GROUP
I II III IV V

NEGATIVE POSITIVE 100mg/mL 200mg/mL 400mg/mL

1 1.00 3.00 1.00 1.00 2.50

2 1.50 1.00 1.00 1.00 2.50
TRIAL

3 1.50 2.00 1.50 1.50 1.50

4
5
6

N 3 3 3 3 3
Min 1.00 1.00 1.00 1.00 1.50
DESCRIPTIVE STATISTICS

Max 1.50 3.00 1.50 1.50 2.50

Sum 4.00 6.00 3.50 3.50 6.50

Mean 1.33 2.00 1.17 1.17 2.17

Std. error 0.17 0.58 0.17 0.17 0.33

Variance 0.08 1.00 0.08 0.08 0.33

Std. dev 0.29 1.00 0.29 0.29 0.58
Median 1.50 2.00 1.00 1.00 2.50
APPENDIX M
Castroverde, Joshua Elijah A.
6F Kaginhawaan St. Countryside Subdivision
Concepcion 1, Marikina City
Email add: elijahcastroverde@gmail.com
Mobile number: 09061832906

PERSONAL INFORMATION

Date of Birth: April 23, 1998

Place of Birth: Marikina City
Civil Status: Single
Citizenship: Filipino
Religion: Born Again Christian
Sex: Male
Language: Filipino and English

Father: Jun Castroverde

Mother: Editha CastroverdE

EDUCATIONAL BACKGROUND

TERTIARY Centro Escolar University

9 Mendiola St, San Miguel, Manila
2014-2018

SECONDARY National Christian Life College

Marikina City
2013-2014

PRIMARY National Christian Life College

Marikina
2009-2010
Concepcion, Chris Jason C.
11 JP Rizal Street Subay, Cardona, Rizal
Email add: Jason_concepcion03@yahoo.com
Mobile number: 09772068102

PERSONAL INFORMATION

Date of Birth: November 03, 1997

Place of Birth: Cardona, Rizal
Civil Status: Single
Citizenship: Filipino
Religion: Roman Catholic
Sex: Male
Language: Filipino and English

Father: Jun J. Concepcion

Mother: Criselda C. Concepcion

EDUCATIONAL BACKGROUND

TERTIARY Centro Escolar University

9 Mendiola St, San Miguel, Manila
2014-2018

SECONDARY Catalino D. Salazar National High School

Subay, Cardona, Rizal
2013-2014

PRIMARY Subay Elementary School

Subay, Cardona, Rizal
2009-2010
Samson, Ranielle D.
467 DNE Subdivision Brgy 3. Calamba City,
Laguna
Email add: raniellesamsond@gmail.com
Mobile number: 09155228085

PERSONAL INFORMATION

Date of Birth: September 20, 1997

Place of Birth: Los Baños, Laguna
Civil Status: Single
Citizenship: Filipino
Religion: Roman Catholic
Sex: Female
Language: Filipino and English

Father: Nathaniel V. Samson

Mother: Romina A. Delos Santos

EDUCATIONAL BACKGROUND

TERTIARY Centro Escolar University

9 Mendiola St, San Miguel, Manila
2014-2018

SECONDARY Colegio De San Juan De Letran-Calamba

Ipil-Ipil Street, Barrio Bucal, Calamba City, Laguna
2010-2014

PRIMARY Colegio De San Juan De Letran-Calamba

Ipil-Ipil Street, Barrio Bucal, Calamba City, Laguna
2004-2010
Silva, Keane Mikkolaine G.
15th St. Claire St. Santiago Subd. Brgy. Sta.
Monica, Novaliches, Quezon City
Email add: slvmikkolaine@gmail.com
Mobile number: 09277375368

PERSONAL INFORMATION

Date of Birth: November 07, 1997

Place of Birth: Quezon City
Civil Status: Single
Citizenship: Filipino
Religion: Iglesia ni Cristo
Sex: Female
Language: Filipino and English

Father: Michael P. Silva

Mother: Lalaine G. Silva

EDUCATIONAL BACKGROUND

TERTIARY Centro Escolar University

9 Mendiola St, San Miguel, Manila
2014-2018

SECONDARY School of Saint Anthony

Block 89 lot 43C Largro QC, Greater Lagro
2013-2014

PRIMARY Hold Child Academy

London, Drive Novaliches, Quezon City, Metro Manila
2009-2010