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EXTRACTING BIOACTIVE COMPOUNDS FROM MORINGA OLEIFERA LEAVES FOR ANTICANCER PRODUCT CHARLENE CASSANDRA CHATTERTON BACHELOR OF CHEMICAL ENGINEERING UNIVERSITI MALAYSIA PAHANG

CHARLENE CASSANDRA CHATTERTON

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Page 1: CHARLENE CASSANDRA CHATTERTON

EXTRACTING BIOACTIVE COMPOUNDS FROM MORINGA

OLEIFERA LEAVES FOR ANTICANCER PRODUCT

CHARLENE CASSANDRA CHATTERTON

BACHELOR OF CHEMICAL ENGINEERING

UNIVERSITI MALAYSIA PAHANG

Page 2: CHARLENE CASSANDRA CHATTERTON

EXTRACTING BIOACTIVE COMPOUNDS FROM MORINGA

OLEIFERA LEAVES FOR ANTICANCER PRODUCT

CHARLENE CASSANDRA CHATTERTON

Thesis submitted in partial fulfilment of the requirements

for the award of the degree of

Bachelor of Chemical Engineering

Faculty of Chemical & Natural Resources Engineering

UNIVERSITI MALAYSIA PAHANG

DECEMBER 2016

Page 3: CHARLENE CASSANDRA CHATTERTON

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SUPERVISOR’S DECLARATION

We hereby declare that we have checked this thesis and in our opinion, this thesis is

adequate in terms of scope and quality for the award of the degree of Bachelor of

Chemical Engineering.

Signature :

Name of main supervisor :

Position : SENIOR LECTURER

Date :

Page 4: CHARLENE CASSANDRA CHATTERTON

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STUDENT’S DECLARATION

I hereby declare that the work in this thesis is my own except for quotations and

summaries which have been duly acknowledged. The thesis has not been accepted for

any degree and is not concurrently submitted for award of other degree.

Signature :

Name : CHARLENE CASSANDRA CHATTERTON

ID Number : KE 13061

Date :

Page 5: CHARLENE CASSANDRA CHATTERTON

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Dedicated to my family

Page 6: CHARLENE CASSANDRA CHATTERTON

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ACKNOWLEDGEMENT

I would like to express my deepest gratitude and appreciation to my beloved

supervisor, Dr. Eman N. Ali for the endless motivation and guidance throughout my

undergraduate research project. I am thankful for the suggestions, honest comments and

advice on my research project as they served as a continuous encouragement in order

for me to work harder and improve my research work. You have been a wonderful

supervisor as well as a friend to me, always responsive to any question that I bring

forward to you regardless on weekends or even public holidays. Without your support,

my journey would have been a difficult one.

A special thanks to my family for showering me with love and support

throughout my entire research project as well as keeping me in your prayers. Apart from

that, I thank my friends and course mates who have helped me in one way or another in

my research project in terms of knowledge sharing, inspiration and support towards

achieving my goal.

I am also indebted to the Ministry of Higher Education and Universiti Malaysia

Pahang for funding my study.

Besides that, I am sincerely grateful to the staffs of Chemical Engineering and

Natural Resources Faculty who have been kind and resourceful in assisting me

whenever I had any enquiries.

Page 7: CHARLENE CASSANDRA CHATTERTON

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TABLE OF CONTENTS

Page

SUPERVISOR’S DECLARATION ii

STUDENT’S DECLARATION iii

ACKNOWLEDGEMENT v

ABSTRACT vi

ABSTRAK vii

TABLE OF CONTENTS viii

LIST OF TABLES x

LIST OF FIGURES xi

LIST OF ABBREVIATIONS 1

CHAPTER 1 INTRODUCTION 2

1.1 Background of the Study 2

1.1.1 Botanical Description of Moringa oleifera 5

1.1.2 Nutritional value of Moringa oleifera 7

1.1.3 Chemical composition of Moringa oleifera leaves 8

1.2 Motivation 1

1.3 Problem Statement 1

1.4 Objectives 1

1.5 Scopes of Study 1

CHAPTER 2 LITERATURE REVIEW 2

2.1 Introduction 2

2.2 Anticancer properties of Moringa oleifera leaves against various cancer cells 2

2.3 Bioactive compounds extracted from Moringa oleifera leaves 8

2.4 Summary of literature review 13

CHAPTER 3 METHODOLOGY 15

3.1 Introduction 15

3.2 Extraction 16

3.3 Total phenolic content and total flavonoid content 21

3.4 DPPH radical scavenging activity 23

3.5 Cytotoxic activity 25

CHAPTER 4 RESULTS AND DISCUSSION 27

4.1 Introduction 27

4.2 Total phenolic content and total flavonoid content 27

4.3 The DPPH radical scavenging activity 30

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4.4 Cytotoxic activity of Moringa oleifera leaves extracts 33

CHAPTER 5 CONCLUSION AND RECOMMENDATION 34

5.1 Conclusion 34

5.2 Recommendation 34

REFERENCES 35

Appendix 41

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LIST OF TABLES

Table No. Title Page

Table 1: Chemical composition of dried Moringa oleifera leaves. 8

Table 2: Amino acids composition of dried Moringa oleifera leaves. 9

Table 3: Mineral contents of dried Moringa oleifera leaves. 9

Table 4: Fatty acids composition of dried Moringa oleifera leaves. 10

Table 5: The nutritional value of Moringa oleifera. 11

Table 6: The A549 cell viability after exposure to Moringa oleifera leaves extract. 7

Table 7: Yields of crude extracts, contents of total phenolics and flavonoids. 8

Table 8: Phenolic acids content in Moringa oleifera leaves. 9

Table 9: Flavonoids content in Moringa oleifera leaves. 10

Table 10: Test conducted to identify the phytochemicals. 11

Table 11: The phytochemicals present from extraction using different solvents. 12

Table 12: Disadvantages of various extraction methods. 20

Table 13: The solvents and method used in the extraction of the samples. 26

Table 14: TPC and TFC values of the S1, S2, S3, S4 and S5 extracts. 28

Table 15: TPC and TFC values of the water extracts. 28

Table 16: DPPH IC50 values of the samples. 30

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LIST OF FIGURES

Figure No. Title Page

Figure 1: Moringa oleifera flowers. 6

Figure 2: Moringa oleifera leaves. 6

Figure 3: Moringa oleifera pods. 7

Figure 4: Moringa oleifera seeds. 7

Figure 5(A): Microscopic view of the cytotoxic effect, (B): Number of viable colonies. 5

Figure 6: The percentage survival of cancer cells and normal cells after treatment. 5

Figure 7: The set-up of Soxhlet extraction apparatus used in the actual experiment. 17

Figure 8: Rotary Evaporator. 19

Figure 9: Layout of 96-well plates for cytotoxicity study. 25

Figure 10: TPC test for S1, S2, S3, S4 and S5 extracts. 27

Figure 11: TFC test for S1, S2, S3, S4 and S5 extracts. 27

Figure 12: TPC test for water extracts by Soxhlet (left) and boiling (right). 27

Figure 13: TFC test for water extracts by Soxhlet (left) and boiling (right). 27

Figure 14: DPPH radical scavenging activity of S1-S5. 29

Figure 15: The DPPH test for water extracts by Soxhlet (left) and boiling (right). 30

Figure 16: The % inhibition of DPPH radicals of S1-S5. 31

Figure 17: The % inhibition of DPPH radicals of the water extracts. 31

Figure 18: The effect of the extracts on MCF-7 cells after 72 hours of treatment. 32

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1

LIST OF ABBREVIATIONS

EBV Epstein-Barr virus

HCT-8 ileocecal adenocarcinoma cancer cells of a 67 year old male

MDA-MB-231 breast mammary gland cancer cells

DPPH 2,2’diphenylpicrylhydrazyl assay

ALL acute lymphoblastic leukemia

AML acute myeloid leukemia

Panc-1 human pancreatic cancer cell lines

COLO-357 human pancreatic cancer cell lines

p34 human pancreatic cancer cell lines

A549 human lung cancer cell

MCF 7 epithelial breast cancer cell line

MDA MB 231 mesenchymal breast cancer cell line

CAE chlorogenic acid equivalents

1QE isoquercetin equivalents

SZ Squeezing

DF Decoction of fresh leaves

DD Decoction of dried leaves

MF70 Maceration of fresh leaves with 70% ethanol

MD50 Maceration of dried leaves with 50 % ethanol

MD70 Maceration of dried leaves with 70% ethanol

PD50 Percolation of dried leaves with 50 ethanol

PD70 Percolation of dried leaves with 70% ethanol

SD50 Soxhlet extraction of dried leaves with 50 % ethanol

SD70 Soxhlet extraction of dried leaves with 70% ethanol

ND Not detected

N/A Not available

LE Leucocyanidin equivalent

DE Diosgenin equivalent.

IC50 50% inhibitory concentration

HepG2 human hepatocellular carcinoma

Caco-2 colorectal adenocarcinoma

MCF-7 breast adenocarcinoma

COS-7 African green monkey normal cells

TPC Total phenolic content

TFC Total flavonoid content

GAE gallic acid equivalence

CE catechin equivalence

QE quercetin equivalence