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SCREENING OF THE POTENTIAL ANTIOXIDANT,
ANTIMICROBIAL AND ANTIPROLIFERATIVE
ACTIVITIES OF DONAX GRANDIS
BY
HUSNA HAWA MOHD HASSAN
A thesis submitted in fulfilment of the requirements for
the degree of Master of Biotechnology Science
Kulliyyah of Science
International Islamic University
Malaysia
MAY 2013
ii
ABSTRACT
In this study, crude extracts of different parts (leaves, fruits, roots and stems) of
Donax grandis using different types of organic solvents (hexane, dichloromethane and
methanol) and water extracts were evaluated by performing phytochemicals screening
and proximate analysis of saponins. The methanolic crude extracts of D. grandis were
further evaluated for antioxidant, antimicrobial and antiproliferative activities.
Phytochemical screening of the crude extracts (leaves, fruits, stems and roots)
revealed the presence of phenolic compounds, alkaloids, tannins, phytosterols, cardiac
glycosides, terpenoids, steroids, saponins and flavonoids. The results showed that the
fruits contained the highest amount of saponins (2.39 wt. %). Total phenolic content
(TPC) which was determined according to the Folin-Ciocalteu methods showed that
all parts of the plant samples had low total phenolic content ranged from 0.18 to 0.65
mg GAE/g extract, with fruit having the highest phenolic content. Free radical
scavenging activity of the methonolic crude extracts were measured by using 2,2-
diphenyl-1-picrylhydrazyl (DPPH) free radical, and the IC50 value of the leaf, fruit,
stem and root extracts ranged from 14.86 to 21.85 mg/mL. Antimicrobial activity
using disc diffusion method on methanolic crude extracts at concentration of 40
µg/disc were evaluated against two Gram-positive bacteria, two Gram-negative
bacteria and three fungi strains. The extracts showed significant antimicrobial
activities against bacteria and fungi strains. The inhibition zones ranged from 7.00 ±
0.57 mm – 18.00 ± 0.5 mm, whereas MIC values ranged from 6.25 µg/mL – 100
µg/mL. 3-[4,5-Dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide (MTT) assay
was used to screen for anticancer activity of the respective D. grandis extracts, at
various concentrations, against human lung carcinoma (H1299) and human oral
tumor (HSC-3) cell lines. The methanolic extracts of fruits and roots proved to have
medium inhibition with the IC50 values of 28.3 ± 2.9 µg/mL and 273.3 ± 15.3 µg/mL
for H1299, and 520.0 ± 36.1 µg/mL and 583.3 ± 12.3 µg/mL for HSC-3 respectively.
The isolation and structural elucidation of a pure compound from methanolic extract
of D. grandis’ fruits revealed one potent antiproliferative compound, i.e. (25R)-
Spirost-5-en-3beta-ol; 3b-hydroxy-5-spirostene, also known as diosgenin which
collectively responsible for unleashing the antiproliferative activity against both
H1299 and HSC-3 cell lines. The finding, vividly support its traditional claims and
form a strong basis to explore D. grandis anticancer properties.
iii
ملخص البحث
الأراق انثار، اندذر )أخزاء يخرهفح ي انضرخهظاخ اصرخزاج ذىف ذ انذراصح،
انكض )خزاذز لد تاصرخذاو أاع يخرهفح ي انذثاخ انعضح ثاخ انذاكزل( انضما
انا ع طزك إخزاء فسض اناد انكائح انثاذح ذسهم يضرخهظاخ( انثا انثال
كضاداخ انشاطاخخزاذز لد انذاكز ي لانثا يضرخهظاخ يذة. طاتيثاشز نم
انثاذ انكائ ذمى انفسض .كثثظ ن انخلاا انضزطا نكزتاخ، لنلأكضذج، يضاداخ
كشفد ع خد انزكثاخ انفنح ، ( ، انضما اندذرنثارالأراق ا)يضرخهظاخ ل
،انفارصرزل ،ذاشالخهكصذاخ انمهة، ذزتذس، لهااخ، (wt. % انظات
2.39) تضث أظزخ انرائح أ انثار رض أكثز لذر ي انظات. فلافذاخ ذسذد
نذ إخان يسر انفنح فما نهطزق صكانر فه أظز أ خع أخزاء ي عاخ انثاخ
، يع خد أعه غزاو/يهغى 0.65إن 0.18ي خيدع انفل ذزاذ .يسر يخفض
2,2نهاد يسر -diphenyl-1-picrylhydrazyl (DPPH) لضد .ف انثار انفنح
.يذة انثال نضرخهض انسزجنهدذرشاط انكضر IC50 الاراق ،اندذر،انثار)لح
يم/يغ 21.85 14.86ذزازد (انضما نضرخهض يذة لد شاط يضاداخ انكزتاخ
انمزص ضذ انثكرزا إداتح /يكزغزاو 40ذزكز بتاصرخذاو أصهب شز انمزص انثال
انضرخهظاخ أظزخ. انفطزاخي اث اث ي انثكرزا صهثح انغزاو ثلاز صلالاخ
7.00± 0.57ذزازد ياطك ذثثظ . نهكزتاخ ضذ صلالاخ انثكرزا انفطزاخ ايأشطح
100 –6.25ي ذزكز ذثثظ انسذ الاد و، تا ذزازد لىلو 0.5± 18.00–يهى
تفسضنشاط انضزطاا ذى لاس. (يهى/يكزغزاو 3-[4,5-Dimethylthiazol-2-yl]-2,5-
diphenyl tetrazolium bromide (MTT) نضرخهظاخ (HSC-3) انفىصزطا
(H1299) ضذ صزطا انزئح يضرخهظاخ يذة ،ترزاكز يخرهفح خزاذز ثاخ انذاكز
± 273.3يم /خذر ثثد نذا إعالح يرصطح يع انمى يكزغزاوالثار الي انثال
يم /يكزغزاو 15.3 IC50 28.3 ± 2.9 ل H1299 يم /يكزغزاو ±36.1 520.0،
ي ركة انمنىلنكه اانرشخض عزل .عه انران يم/يكزغزاو ±12.3 583.3
ا نذ خظائض لح ذثثظ لاراو كشف يضرخهض يذة انثال ي انثارانذ
،(صثزصرل) خزاذز انضزطاح نثاخ انذاكز (25R)-Spirost-5-en-3beta-ol;
3b-hydroxy-5-spirostene انر نذا انضؤنح لاعالح أضا انعزف دصد انزكة
. انشاط انضزطا انثشز نهخلاا انشفز H1299 HSC-3 ذ انرائح ذذعى تشكم اضر
انضزطاح انمح لاصركشاف ثاخ انذاكز خزادز نهخظائض انطانة انرمهذح
iv
Zarina Zainuddin
Head Department of
Biotechnology Science
Kamaruzzaman Yunus
Dean, Kulliyyah of
Science
Nazlina Ibrahim
External Examiner
Ridzwan Hashim
Co-Supervisor
Jamaluddin Mohd Daud
Main Supervisor
Shafida Abd Hamid
Inetrnal Examiner
APPROVAL PAGE
I certify that I have supervised and read this study and that in my opinion it conforms
to acceptable standards of scholarly presentation and is fully adequate, in scope and
quality, as a dissertation for the degree of
Master of Science (Biotechnology Sciences).
_____________________
_____________________
_____________________
I certify that I have read this study and that in my opinion it conforms to acceptable
standards of scholarly presentation and is fully adequate, in scope and quality as a
dissertation for the degree of Master of
Biotechnology.
_____________________
_____________________
This thesis was submitted to the Department of Biotechnology and is accepted as a
fulfillment of the requirements for the degree
of Master of Science (Biotechnology Science).
_____________________
This thesis was submitted to the Kulliyah of Science and is accepted as a fulfillment
of the requirements for the degree of Master of
Biotechnology.
_____________________
M. Taher Bakhtiar
Co-Supervisor
v
DECLARATION
I hereby declare that this dissertation is the results of my own investigations, except
where otherwise stated. I also declare that it has not been previously or concurrently
submitted as a whole for any other degrees at IIUM or other institutions.
Husna Hawa Mohd Hassan
Signature ……………………………………… Date …...….….……………
vi
INTERNATIONAL ISLAMIC UNIVERSITY MALAYSIA
DECLARATION OF COPYRIGHT AND
AFFIRMATION OF FAIR USE OF UNPUBLISHED
RESEARCH
Copyright © 2013 by Husna Hawa Mohd Hassan. All right reserved.
SCREENING OF THE POTENTIAL ANTIOXIDANT,
ANTIMICROBIAL AND ANTIPROLIFERATIVE ACTIVITIES
OF DONAX GRANDIS
No part of this unpublished research may be reproduced, stored in a retrieval
system, or transmitted, in any form or by any means, electronic, mechanical,
photocopying, recording or otherwise without prior written permission of the
copyright holder except as provided below.
1. Any material contained in or derived from this unpublished research may
only be used by others in their writing with due acknowledgment.
2. IIUM or its library will have the right to make and transmit copies (print or
electronic) for institutional and academic purposes.
3. The IIUM library will have the right to make, store in a retrieval system
and supply copies of this unpublished research if requested by other
universities and research libraries.
Affirmed by Husna Hawa Binti Mohd Hassan
……………………………….. ..……………….…..
Signature Date
vii
Dedication
I dedicate my master thesis to ALLAH (s.w.t.) the almighty, to my beloved mother, late
Kamariah Mohd Yatib, my father (Mohd Hassan Alil), my husband (Ahmad Kamal)
and my loving children (Umar, Akif, Qaisara and Kamilia).
viii
ACKNOWLEDGEMENTS
First and foremost, I would like to express sincere gratitude to my advisor Assc. Prof.
Dr Jamaluddin Mohamad Daud, Prof. Ridzwan Hashim and Assc. Prof. M.Taher for
their continuous support and for their constructive criticism and valuable comments of
my research. During this time, they shown me patience and enthusiasm, as well as
being a great source of acknowledgement. Under their guidance, I was able to
complete my research and thesis with greater efficiency.
A very special thanks to Assc. Prof. Dr Muhammad Nor, Dr. Deny Susanti and
Dr. Norziana, for sharing many helpful interesting discussions with me an for taking
me on board at the start of my project.
I would like to thank to Assc. Prof Zurina Mahmud and sister Raudhatul
Jannah Mohd Zuki from Faculty of Applied Science, University Teknologi MARA,
Perlis for helping me in confirming my compound.
I also wish to thank everyone at Kulliyah of Sience and Kulliyah of Pharmacy
especially, Dr Mohammed Nor, Dr Deny Susanti, Br. Muzzammil, Br. Amir, Br.
Zaffar, Sis. Putri and Sis. Tuti for great working atmosphere and being so helpful at
any time.
I would like to thank all my friends especially Sis Maryana and Sis Azizah for
their great support and understanding. Finally, I want to thank my loving family, my
dear husband Ahmad Kamal and our wonderful children Umar, Akif, Qaisara and
Kamilia. I thank you for being so patient when I have been busy with my lab works
and thesis.
Husna Hawa Mohd Hassan
ix
TABLES OF CONTENTS
Abstract ..…………………………………………………………………………….. i
Abstract in Arabic ..………………………………………………………………...... ii
Approval Page ......………………………………………………………………….. iii
Declaration Page ......………………………………………………………………... iv
Copyright Page …..…………………………………………………………......….....v
Dedication …..…………………………………………………………………….....vi
Acknowledgements .…………………………………………………………….…..vii
List of Tables …...…..………………………………………………………….....…xii
List of Figures …..……...………………………………………………………......xiii
List of Appendices ...……………...……………………….……………………...... xv
List of Abbreviations ….....…………………………….…………………....…..… xvi
.
CHAPTER ONE: INTRODUCTION AND OBJECTIVE …………….....…...…1
1.1 General Introduction .................................................................................... 1
1.2 Medicinal Plants Potential............................................................................ 1
1.3 Significance of Study ................................................................................... 3
1.4 General Objectives ....................................................................................... 4
1.5 Research Objectives ..................................................................................... 4
1.6 Reseach Hyphotheses ................................................................................... 5
CHAPTER TWO: LITERATURE REVIEW ..........................……………...……6
2.1 Marantaceae ................................................................................................. 6
2.1.1 General Information .................................................................... 6
2.1.2 Medicinal Uses of Marantaceae .................................................. 7
2.2 Donax Grandis ............................................................................................. 7
2.2.1 Botanical Description ................................................................ 11
2.2.2 Traditional and Medicinal Uses of Donax grandis ................... 13
2.2.3 Ethnobotanical Uses of Donax Grandis ................................... 14
2.3 Benefits of phytochemicals ........................................................................ 14
2.3.1 Phenolic or Polyphenols ........................................................... 15
2.3.2 Flavonoids ................................................................................. 16
2.3.3 Tannins ...................................................................................... 17
2.3.3.1 Hydrolyzable Tannins ............................................... 18
2.3.3.2 Condensed Tannins ................................................... 19
2.3.4 Alkaloids ................................................................................... 19
2.3.5 Saponins .................................................................................... 20
2.4 Antioxidant Activity .................................................................................. 22
2.4.1 Benefit of Antioxidant .............................................................. 24
2.4.2 Natural Antioxidants ................................................................. 24
2.4.3 Antioxidant Activity Estimation ............................................... 25
2.5 Antimicrobial Activities in Plants ............................................................. 26
2.5.1 Plants as Antimicrobial Agents ................................................. 26
2.5.2 Microbial Strains used in the Antimicrobial Bioassay.............. 27
2.5.2.1 Escherichia coli ........................................................ 27
2.5.2.2 Pseudomonas aeruginosa ......................................... 28
x
2.5.2.3 Bacillus cereus .......................................................... 28
2.5.2.4 Staphylococcus aureus .............................................. 28
2.5.2.5 Candida spp. ............................................................. 29
2.6 Cancer ........................................................................................................ 30
2.6.1 Types of cancer ......................................................................... 31
2.6.1.1 Lung Cancer .............................................................. 33
2.6.1.2 Oral / Mouth Cancer ................................................. 33
2.6.2 Antiproliferative activity, Apoptosis and Cytotoxicity ............. 34
2.6.3 Determination of Cytotoxicity by MTT assay .......................... 34
2.6.4 Saponins in Treating Cancer ..................................................... 35
CHAPTER THREE: MATERIALS AND METHODS .………………….………37
3.1 Sample Collection and Preparation ............................................................ 37
3.2 Reagents and Chemicals ............................................................................ 39
3.3 Equipment and Apparatus .......................................................................... 39
3.4 Disposable and Consumable Items ............................................................ 40
3.5 Cancer Cell Lines ....................................................................................... 40
3.6 Extraction of Plant Materials ..................................................................... 40
3.7 Phytochemicals Screening Assays ............................................................. 41
3.7.1 Evaluation of Phytochemicals Screening Test .......................... 41
3.7.1.1 Tests for Phenolic compounds .................................. 41
3.7.1.1.1 Ferric Chloride Test ........................................ 41
3.7.1.1.2 Gelatin Test .................................................... 41
3.7.1.2 Test for Flavonoids ................................................... 42
3.7.1.3 Test for Tannins ........................................................ 42
3.7.1.4 Test for Phytosterols (Libermann-
Burchard‟s Test) ........................................................ 42
3.7.1.5 Test for Terpenoids and Steroids .............................. 42
3.7.1.6 Test for Terpenoids (Salkowski test) ........................ 42
3.7.1.7 Test for Alkaloids ..................................................... 43
3.7.1.8 Test for Saponins ...................................................... 43
3.7.1.9 Test for cardiac glycosides (Keller-Killiani
test) ............................................................................ 43
3.7.1.10 Determination of Saponins Content ........................... 44
3.7.1.11 Determination of Total Phenolic Contents
(TPC) ......................................................................... 44
3.8 Determination of Antioxidant Activity ...................................................... 45
3.9 Isolation and Purification of Steroidal Saponin from Methanolic
Extract of D. grandis fruits ........................................................................ 47
3.10 Disc Diffusion Assays ................................................................................ 48
3.10.1 Tested Microorganisms ............................................................. 48
3.10.2 Media Preparation ..................................................................... 49
3.10.3 Preparation of Antibiotics ......................................................... 50
3.10.4 Preparation Mc Farland Standard ............................................. 50
3.10.5 Preparation of Inoculums .......................................................... 50
3.10.6 Preparation of Disc ................................................................... 51
3.10.7 Antimicrobial Disc Diffusion Test ............................................ 52
3.10.8 Minimum Inhibitory Concentrations (MIC) Test ..................... 53
3.11 Antiproliferative Activity Assay ............................................................... 54
xi
3.11.1 Preparation of Stock Solution ................................................... 54
3.11.2 Cell Revival .............................................................................. 54
3.11.3 Cell Subculture and Cell Maintenance...................................... 55
3.11.4 Cell Counting ............................................................................ 55
3.11.5 MTT Assay ................................................................................ 57
3.12 Statistical Analysis .................................................................................... 59
3.13 Compound Structural Elucidation ............................................................. 59
3.13.1 Nuclear Magnetic Resonance (NMR) ....................................... 59
3.13.2 NMR Sample Preparation ......................................................... 59
CHAPTER FOUR: RESULTS AND DISCUSSION …….……………...……….60
4.1 Percentage of Dried Samples and Extraction Yield of D.grandis. ............ 60
4.2 Phytochemical Screening Assays of D. grandis. ....................................... 62
4.3 Free Radical Scavenging Activity of Methanolic Crude Extract
from Different Parts of D. grandis ............................................................. 68
4.4 Relationship Bbetween Total Phenolic Contents and DPPH Free
Radical Scavenging .................................................................................... 70
4.5 Evaluation of Antimicrobial Activity of methanolic crude
extract of D. grandis and Crude Saponins From Fruits ............................. 72
4.5.1 Minimum Inhibitory Concentration (MIC) of D. grandis ............ 78
4.6 Antiproliferative Activities ........................................................................ 82
4.6.1 Evaluation of Antiproliferative Activities of
Methanolic Crude Extracts From Different Parts of D.
grandis ...................................................................................... 82
4.6.2 Evaluation of Antiproliferative Activity of Crude
Saponins and Isolated Compound from D. grandis
Fruits ......................................................................................... 88
4.7 Identification of Pure Compound ............................................................... 93
CHAPTER FIVE: CONCLUSION AND RECOMMENDATION ….…………..96
5.1 Conclusion ................................................................................................. 96
5.2 Recommendation........................................................................................ 97
BIBLIOGRAPHY.......................................................................................................99
APPENDICES...........................................................................................................112
xii
LIST OF TABLE
Table No. Page No.
2.1 Common names of D. grandis by different cultures 10
2.2 Medicinal uses of D. grandis in Malaysia 13
2.3 Major group of Phenolic Compound 16
2.4 In vitro and in vivo Anticancer Activities of Saponins 37
4.1 The percentage weight of various parts of D. grandis plant
material after drying
61
4.2 The percentage yield of crude extracts after extraction from
various plant parts of D. grandis
61
4.3 Phytochemical screening of D. grandis using different solvent
for extraction
63
4.4 The percentage of saponins content in different parts of D.
grandis
65
4.5 Free radical scavenging activity (IC50) of methanolic extract
from different parts of D. grandis and BHT
68
4.6 Antimicrobial activity from methanolic crude extract and crude
saponins from fruits of D. grandis
73
4.7 Minimum Inhibitory Concentration (MIC) values of methanolic
crude extract of D. grandis parts and crude saponins from fruits
of D. grandis
80
4.8 Antiproliferative activity of methanolic extract from different
parts of D. grandis
83
4.9 Antiproliferative activity of crude saponins and isolated saponins
compound from D. grandis
88
4.10 1H and
13C-NMR Chemical Shifts of Pure Saponin Compound
from D. grandis
94
xiii
LIST OF FIGURES
Figure No. Page No.
2.1 Malaysian Donax grandis 9
2.2 Taxonomy classification of D. grandis 10
2.3 Different parts of D. grandis 12
2.4 Structure of the six main classes of flavanoids 17
2.5 Basic structure of tannins 18
2.6 A molecular structure of Paclitaxel 19
2.7 Molecular Structure of Saponins 20
2.8 Classification of Saponins 21
2.9 Basic Structure of Sapogenin 22
3.1 Dried powdered of leaves, fruits, stems and roots of D. grandis 38
3.2 Zone of inhibition of antimicrobial test by disc diffusion method 53
3.3 Cell counting preparation using hemacytometer 56
3.4 Cell counting on hemacytometer 56
3.5 96 well plates for determination of MTT assay 58
4.1 Standard curve for total phenolic content as gallic acid
equivalents
66
4.2 Total phenolic content of methanolic extract from different parts
of D. grandis
67
4.3 Free radical scavenging activity of methanolic extract from
different parts of D. grandis
69
4.4 Correlation between Total Phenol Contents and Free Radical
Scavenging Activity (IC50) of D. grandis methanolic crude
extracts.
71
4.5 Plates showing zone inhibition of bacteria 75
xiv
4.6 Plates showing zone inhibition of fungi 75
4.7 Dose-dependent antiproliferative activity of methanolic extract
from different parts of D. grandis against H1299 cell line
84
4.8 Dose-dependent antiproliferative activity of methanolic extract
from different parts of D. grandis against HSC-3 cell line
85
4.9 Dose-dependent antiproliferative effects of crude and Pure
Saponin Compound from D. grandis fruits against H1299 cell
line
90
4.10 Dose-dependent antiproliferative effects of crude and Pure
Saponin Compound from D. grandis fruits against HSC-3 cell
line
91
4.11 Structure of diosgenin 95
xv
LIST OF APPENDICES
Appendix No Page No.
1 Classification for Zone Diameter Interpretive 112
2 Interpretation of Liquid Chromatography Mass
Spectrophotometry (LCMC)
113
3 Compund 9 Mass Spectrum 114
4 13C - NMR 115
5 1H - NMR 116
6 NMR - COSY 117
7 NMR 13C HMBC 118
8 NMR 13C HMQC 119
9 NMR 13C HSQC 120
10 NMR NOESY 121
xvi
LIST OF ABBREVIATIONS
WHO World Health Organization
NCI American National Cancer Institute
DNA Dinucleotide Disease
HIV Human Immunodeficiency Virus
ATP Adenosine Triphosphate
ABTS 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulphonic
acid)
ROS Reduction Oxygen Species
DPPH 1,1-diphenyl-2-picrylhydrazyl radicals
MTT 3-(4,5-dimethylthiozolyl-2)-2,5 diphenyltetrazolium
Bromide
DMEM Dulbecco's Modified Eagles Medium
PBS Phosphate Buffer saline Solution
et al. (et alia): and others
etc (et cetra): and so forth
e.g example
IMR Institute For Medical Reaserch
ATCC American Type Culture Collection
ºC Degree Celcius
% Percent
g Grams
mg Miligram
mL Mililiter
µg Micrograms
µL Microliter
mm Milimeter
xvii
nm Nanometer
µg/disc Microgram per disc
µg/mL Microgram per mililiter
UV Ultra-violet
CFU/ml Colony forming unit/mlililiter
R2 Correlation
Rf Retention factor
Rt Retention time
ppm Part per milion
DCM Dichloromethane
DF Dilution factor
MeOH Methanol
DMSO Dimethyl sulfoxide
CO2 Carbon dioxide
1H Proton NMR
13C Carbon 13
MIC Minimum Inhibitory Concentration
LCMS Liquid Chromatography Mass Spectrometry
IR Infrared Spectroscopy
BaCl2•2H2O Barium chloride dihydrate
HMBC Heteronuclear Multiple Bond Correlation
HMQC Heteronuclear Multiple-Quantum Correlation
HSQC Heteronuclear Single-Quantum Correlation
NOESY Nuclear Overhauser Enhancement Spectroscopy
1
CHAPTER ONE
CHAPTER 1 INTRODUCTION AND OBJECTIVES
1.1 GENERAL INTRODUCTION
In Malaysia, traditional medicines are widely used and practiced in the community for
maintaining health and also for treatment of many diseases. Plants and herbal extracts
are considered as important materials in modern medicine, due to their phytochemical
and medicinal contents in their natural form. Their secondary metabolites may act as
an agent for the structural moieties that can work together for delaying a wide range of
biological activities (Gulfraz, 2011). The medicinal values of plants have been
claimed to lie in their phytochemical components including alkaloids, tannins,
flavonoids, saponins and other phenolic compounds, which can give strong
physiological action on the human body (Phan et al., 2001). Many herbal plants
products have attracted the attention of chemists in exploring new phytochemical
compounds for their antioxidant, antimicrobial the antiproliferatives activities.
1.2 MEDICINAL PLANTS POTENTIAL
Plants have been used as medicines throughout history. In Asia, the practice of herbal
medicine is extremely well established and documented. For this reason, many of the
medicinal plants from Asian region were internationally recognized. Nowadays,
people used herbal medicinal plants for treating illness and maintaining health because
of the establishment in research and development of medicinal plants. (CTA, 2007).
2
The medicinal value of plants lies in some chemical substances or group of
compounds that produce a definite physiological action in the human body. These
chemical substances are called secondary metabolites. The most important of these
bioactive groups of plants are alkaloids, terpenoids, steroids, flavonoids, tannins,
saponins and phenolic compounds (Edeoga et al., 2005).
The presence of phytochemicals compound in many plants is important for
normal growth and defense against infection and injury. Intake of phytochemicals by
human can be better through the dietary sources rather than supplement. Phenolic
compounds found in vegetables, fruits or medicinal plants are known for their
potential of antioxidant activities in preventing diseases. A number of researches have
indicated that a positive correlation between the antioxidant activity and the total
phenolic content. (Cai et al., 2004; Tawaha et al., 2007; Andre et al., 2010). Nowadays,
there is a growing interest toward natural antioxidants from herbal sources.
Antioxidants, both exogenous and endogenous, whether synthetic or natural, can be
effectively useful in preventing the free radical formation by scavenging or promotion
of their decomposition and suppression of such disorders (Souri et al., 2008).
Many medicinal plants have been screened for their antimicrobial potential
worldwide. Scientists have discovered a wide range of antimicrobial agents to
overcome variety of diseases. The antimicrobial compounds produced by plants are
active against plant and human phatogenic microorganisms. Thus, rapid emergence of
multiple drug resistance strains of pathogenic to current antimicrobial agents has
generated urgent intensive studies for new antibiotics from medicinal plants (Sarojini,
2011; Govindappa et al., 2011). Many plants used as antimicrobial agent originated
from compounds synthesized by secondary metabolites of the plants. For example,
3
saponins which have been observed to kill protozoans and mollusks can also act as
antifungal and antiviral agents (EFSA, 2009).
Cancer burden worldwide is increasing even though in 2002 Malaysian
National Cancer reported there are decreasing number of cancer incidence. It may be
resulting from successful eliminating prevalence cases. However, the latest National
Register Report in 2003 estimated that the number of cancer burden suddenly
increased in parallel with increasing Malaysian population. However, the actual
incidence and outcome of treatment for these patients is still largely unknown. At
present, the cancer treatment by chemotherapeutic agents, surgery and radiation have
not been fully effective because of the few reported incidence of the disease (Lim &
Azura, 2008).
The development of new active anticancer drugs remains one of the most
challenging areas of researches. It is considered to be important for the evaluation of
plant extract to be used for alternative medicine and also in food industry. Natural
products and other related substances or extracts of folk medicine accounted for 30%
of the top 35 worldwide natural product-based drugs sold in recent years
(Venkateshwar et al., 2008). Thus, the use of traditional plant products as chemo
preventative or chemotherapeutic treatments requires further study to find the most
efficient, cheapest and less painful treatment.
1.3 SIGNIFICANCE OF STUDY
Natural products can be consumed as a daily food or as an alternative medicine that
may not have given any side effects to the individuals. Nowadays, much interest on
maintaining health care and efforts are made to accomplish this through the extraction
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of plants compound and evolution in medicinal uses. Today, traditional knowledge
and practices have contributed to our modern medicine which is attested by more than
40% of commonly prescribed medicines throughout the world found their origins
directly or indirectly in plants or animals (Fasihuddin & Ghazally, 2003).
There is a great potential of D. grandis may be useful in food industry and also
pharmacology. In this study, different parts of D. grandis which are traditionally used
for herbal medicine are not been studied scientifically for antioxidant, antimicrobial
and antiproliferative activities properties.
1.4 GENERAL OBJECTIVES
The aim of this research was to evaluate the different parts of Donax grandis (leaves,
fruits, stems and roots) extracts for their quantitative phytochemicals constituents and
to study their potential antioxidant, antimicrobial and also the antiproliferatives
activities.
1.5 RESEARCH OBJECTIVES
Objectives:
1. To carry out screening test for the presence of major secondary
metabolites in crude extracts of different parts of D. grandis.
2. To determine the antimicrobial activity in methanolic crude extracts of
different parts of D. grandis and isolated crude saponin from fruits.
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3. To determine the total phenolic content and free radical DPPH
scavenging activities in methanolic crude extracts of different parts of
D. grandis.
4. To determine the antiproliferative activity in methanolic crude extracts
of isolated crude saponins and pure saponins compounds from fruits of
D. grandis
5. To elucidate the chemical structure of the pure isolated compounds
from D. grandis fruits.
1.6 RESEARCH HYPOTHESES
Donax grandis contains active compounds that could be exploited as antioxidant,
antimicrobial and antiproliferative agents.
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CHAPTER TWO
2 CHAPTER 2 LITERATURE REVIEW
2.1 MARANTACEAE
2.1.1 General Information
Donax grandis is a member of plant Marantaceae. The Marantaceae is classified as
perennial and rhizomatous herbs. It includes about 23 genera and 550 species
worldwide with eight genera and an estimated 55 species reported are found in
tropical Asia. There are several publications on Marantaceae made over the last few
years in Malaysia. However, research on medicinal properties taxanomy and
distribution of Marantaceae were poorly documented. Futhermore, Marantaceae is
classified among the most poorly understood members of the family (Andersson, 1998;
Zhu & Helen, 2000).
Most of Marantaceae species found in scattered or lumps of the forest and do
not covering the large scale areas. Marantaceae have many of the clumps which
represent single individual and it usually found in soggy soil. However, most species
of Marantaceae, can be found in moderate shade area in a tropical forest. Due to that,
it becomes the important factor for their local distribution (Helen, 1978).
They often exhibit a wide range of economic uses from ornamental plants mainly
through species from Asia and America, consumer goods and nutrition sweetener.
Furthermore, some African species of the large leaves of Marantaceae are used to wrap up
various dishes such as fish or mash made of manioc flour (Alexandra, 2008).
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2.1.2 Medicinal Uses of Marantaceae
Members of the family Marantaceae possess great medicinal values. The leaves, stems
and rhizomes were mostly used for medicinal purposes. In India, Phryrnium
pubinerve of Marantaceae has been used to heal leprosy and also in paralytic patient.
In addition, it is effectively used in treating skin diseases (Hynniewta & Yogendra,
2008).
Schumannianthus virgatus of Marantaceae has been used as medicinal herbs
for curing dysentery, vomiting and also skin diseases (Rajith & Ramachandran, 2010;
Raji & Raveendran, 2011). In a report of the medicinal plant in Vanuatu, Gesine (2008)
found that Marattia smithii from a family of Marantaceae had been used to treat skin
disease such as neurodermatitis and infantile eczema.
2.2 DONAX GRANDIS
Common Donax (Donax grandis) is from a family of Marantaceae. In also known
scientificily as Donax canniformis or Clinogyne grandis. It can be found widely
distributed in South East Asia region such as Malaysia, Thailand, Brunei, Philippine
and, Papua New Guinea. D. grandis can usually be found distributed in jungle along
the river and in wet places in the secondary forest and bamboo bush. It can be easily
propagated by rhizomes and also through the seed (Ong, 2008). Traditionally, D.
grandis is useful in making mats. The split stems can also be used for weaving baskets,
making a fish traps and hats.
Fatan (1990), reported that D. grandis rhizomes are used traditionally in Malay
culture to cure shingles. Medicinally, the leaves and roots decoctions are taken in bath to
cool body during fever and the juice from stem is effectively used against snake bite.
