THE EFFECTS OF ACTIVATED VIRGIN COCONUT OIL (AVCO) AND VIRGIN COCONUT OIL (VCO) ON CELL MEMBRANE OF Candida albicans BY NOR IZZAH BINTI MUKHTAR A thesis submitted in fulfillment of the requirement for the Master of Science (Biosciences) Kulliyyah of Science International Islamic University Malaysia MAY 2019
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THE EFFECTS OF ACTIVATED VIRGIN COCONUT
OIL (AVCO) AND VIRGIN COCONUT OIL (VCO) ON
CELL MEMBRANE OF Candida albicans
BY
NOR IZZAH BINTI MUKHTAR
A thesis submitted in fulfillment of the requirement for the
Master of Science (Biosciences)
Kulliyyah of Science
International Islamic University Malaysia
MAY 2019
ii
ABSTRACT
Virgin coconut oil (VCO) has been used in treating infections for ages. VCO can be
used in preventing and protecting human body from microbial and fungal infections
which can cause disease such as oral candidiasis. Oral candidiasis is an infection which
is caused by the overgrowth of Candida albicans. However, the knowledge on the
mechanism of action of AVCO and VCO on the cell membrane of C. albicans is limited.
The aim of this study is to investigate the effects of activated virgin coconut oil (AVCO)
and the crude extract of VCO on the cell membrane lysis and the components of
cytoplasmic contents of Candida albicans. The differences of fatty acids composition
in AVCO and VCO was analysed by using gas chromatography-mass spectrometry
(GC-MS) method. To elucidate the effects of AVCO and VCO, live/dead bacterial
viability kit was used to investigate the cell viability of Candida albicans in vitro
cultures and was observed under fluorescence microscopy. Transmission electron
microscopy was used to examine the cellular morphology of Candida albicans after
being treated with AVCO and VCO. The cellular leakage of Candida albicans was
detected by using UV/Vis spectrophotometer. Cytoplasmic leakage confirms creation
of holes in the membrane by AVCO. Disturbance of cell membrane causes pore through
which cellular materials (protein and DNA) leakage takes place. AVCO has been
proven to have an antifungal effect on Candida albicans by altering the cell walls,
disintegrating fungal cell membranes. The results of this study suggested that AVCO
may disrupt the structure of the cell membrane and causes the release of cytoplasmic
materials of Candida albicans due to the destruction of membrane network.
Nevertheless, VCO has shown limited or no antifungal activity on the cell membrane
of C. albicans. The study of cellular leakage of cell membrane components of Candida
albicans may help to understand the mechanism of action of AVCO which has the
potential to be a new antifungal treatment in treating oral candidiasis that can be an
alternative to combat this pathogenic fungus which increases its resistance against
current antifungal agents. In conclusion, this study proposes that AVCO has higher
antifungal activity against Candida albicans compared to the crude extract of VCO.
iii
خلاصة البحث
من قديم علاج الالتهاباتفي ( virgin coconut oil, VCO) البكرستخدم زيت جوز الهند يض امر لأل ةسببالفطرية المو جسم الإنسان وحمايته من العدوى الميكروبية كما يستخدم أيضا لوقاية الزمن،كروبات المبيضات مي المبيضات الفموي. داء المبيضات الفموي هو عدوى ناتجة عن فرط نمو داءمثل
activatedط )المنشالبكر دراسة تأثير زيت جوز الهند ا البحث هو الهدف من هذكان . البيضاءvirgin coconut oil, AVCOـلل ةالخام ات( والمستخلصVCO لوي على تحلل الغشاء الخ
الأحماض محتوىفي تلافات. تم تحليل الاخالبيضاء بيضاتلفطريات الم لازميةبتويات السيتو مكونات المحو ،(GC-MSز )باستخدام طريقة التحليل الطيفي الكتلي للغا VCOالــو AVCOالــ الدهنية في
لميتة والحيةاطقم اختبار حيوية البكتيريا تم استخدام ،VCOو AVCO تأثير كلا منلتوضيح و تحت مجهر اوتم معاينته مستنبتات خارج الجسم الحي،في بيضاءلمبيضات اللية و الخل من الحيويةللتحقق بعد معالجتها بيضاءلامبيضات للفحص التشكل الخلوي ل النافذ . تم استخدام المجهر الإلكترونيفسفوري
طيافية الأشعة باستخدام م ءبيضاالبيضات في الم. تم الكشف عن تسرب خلوي VCOالــو AVCOبالــ الخلوي الغشاءفيمي على حدوث ثقوب التسرب السيتوبلاز أكد. (UV/Vis) المرئية وفوق البنفسجية
لمواد الخلوية اتسرب امسام يحدث من خلاله نشوءي إلى و غشاء الخلالاضطراب أدى. AVCOبواسطة ى المبيضات البيضاء للفطريات عل امضاد اتأثير AVCO لدى أن تم إثبات)البروتين والحمض النووي(.
ه بإمكان هذه الدراسة أن لايا الفطرية. اقترحت نتائججدران الخلايا، وتفكيك أغشية الخ تشويهعن طريق AVCO ات البيضاء بسبب من المبيض يةواد السيتوبلازمالمل بنية الغشاء الخلوي ويسبب إطلاق يعطت
على د الفطرياتضمعدوما نشاطاً محدودًا أو فقد أظهر VCOأما بالنسبة للــشبكة الغشاء. تعطيللمبيضات ل ويالخل غشاءالدراسة التسرب الخلوي لمكونات . تساعدلمبيضات البيضاءوي لغشاء الخلال
ديدًا مضادًا للفطريات جالتي لديها القدرة على أن تكون علاجًا و AVCOالــ في فهم آلية عمل اءالبيضسببة للأمراض والتي الذي يمكن أن يكون بديلًا لمكافحة هذه الفطريات المو ،داء المبيضات الفمويل
لدى ح هذه الدراسة أنتقتر ا ضد العوامل الحالية المضادة للفطريات. ختام يوية في تزايدها الحمقاومت.VCOللــلخام امقارنة مع المستخلص أقوى المبيضات البيضضد فطريات لل مضادا نشاط AVCOالــ
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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 thesis for the degree of Master of Science (Biosciences)
…………………………………..
Zurainie Abllah
Supervisor
…………………………………..
Mohd. Azrul Naim Mohamad
Co-Supervisor
…………………………………..
Intan Azura Shahdan
Co-supervisor
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 thesis
for the degree of Master of Science (Biosciences)
…………………………………..
Hanani Ahmad Yusof @ Hanafi
Internal examiner
…………………………………..
Wan Himratul Aznita Wan Harun
External examiner
This thesis was submitted to the Department of Biotechnology and is accepted as a
fulfilment of the requirement for the degree of Master of Science (Biosciences)
………..……………………….......
Mardiana Mohd Ashaari
Head, Department of Biotechnology
This thesis was submitted to the Kulliyyah of Science and is accepted as a fulfilment of
the requirement for the degree of Master of Science (Biosciences)
…………………………
Shahbudin Saad
Dean, Kulliyyah of Science
v
DECLARATION
I hereby declare that this thesis is the result of my own investigations, except where
otherwise stated. I also declare that it has not been previously or concurrently submitted
for any other degrees at IIUM or other institutions.
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
be used by others in their writing with due acknowledgement.
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 retrieved system
and supply copies of this unpublished research if requested by other
universities and research libraries.
By signing this form, I acknowledged that I have read and understand the IIUM
Intellectual Property Right and Commercialization policy.
Affirmed by Nor Izzah binti Mukhtar
……..……..…………… …………………..
Signature Date
vii
This thesis is dedicated to my beloved parents for laying the
foundation of what I turned out to be in life.
viii
ACKNOWLEDGEMENT
In the name of Allah, The Most Gracious and The Merciful. Alhamdulillah, all praise
to Allah for the strength and His blessing on me throughout the sweat and tears in
preparing this thesis. It will never be possible without all the sincere prayers from the
amazing servants of Allah around me.
I would like to express my deepest gratitude to my beloved parents; Mukhtar
bin Ahmad and Rohani binti Ahmad and to my siblings for their endless love and
support throughout my study. They have been with me through thick and thin till the
end of this journey.
Special appreciation goes to my supervisor and co-supervisor, Asst. Prof. Dr.
Zurainie Abllah, Asst. Prof. Dr. Intan Azura Shahadan and Dr. Mohd. Azrul Naim for
their supervision and constant support. Their invaluable help through their constructive
comments and suggestions throughout the experimental and thesis-writing works have
led to the success of this research.
Sincere thanks to all my friends Ummi Aqilah, Amilin, Fatin, Umi Kalthum,
Ainatul, Fatimah, Fatihah, Faiqah, Kasmadiana, Maisarah, Syazila and Ruhan for their
kindness during my study. Thanks for the friendship and memories. Not to forget, all
Science Officers and the laboratory staffs who have been assisting me with the
laboratory works and acquiring the most valuable knowledge and experiences.
To all the above specifically mentioned, and to others not mentioned in this
piece of writing, I would like to say jazakumullahu khairan kathira and thank you so
much.
ix
TABLE OF CONTENTS
Abstract ....................................................................................................................... ii Abstract in Arabic ....................................................................................................... iii Approval Page ............................................................................................................. iv Declaration .................................................................................................................. v
Copyright Page ............................................................................................................ vi Acknowledgement ...................................................................................................... viii Table of Contents ........................................................................................................ ix List of Tables .............................................................................................................. xi List of Figures ............................................................................................................. xiii
List of Symbols ........................................................................................................... xv List of Abbreviations .................................................................................................. xvi
CHAPTER ONE INTRODUCTION ...................................................................... 1 1.1 Research Background ................................................................................ 1
1.2 Problem Statement ..................................................................................... 3 1.3 General Research Objective ....................................................................... 4 1.4 Research Objectives ................................................................................... 4
1.5 Research Questions .................................................................................... 4 1.6 Research Hypothesis .................................................................................. 5
1.7 Significance of Study ................................................................................. 5
CHAPTER TWO LITERATURE REVIEW ......................................................... 6 2.1 Oral Candidiasis ......................................................................................... 6
2.2 Candida albicans ....................................................................................... 9 2.2.1 Structure of C. albicans................................................................... 10
2.2.2 Pathogenicity of C. albicans ........................................................... 14
2.3 Current Treatment of Oral Candidiasis ...................................................... 18 2.3.1 Mechanism of Action of Antifungal Agents ................................... 20
2.3.2 Resistance against Antifungal Agents ............................................. 21 2.4 Virgin Coconut Oil (Cocos nucifera L.) .................................................... 23
2.4.1 Hydrolysis of Coconut Oil .............................................................. 24
2.4.2 Medium Chain Fatty Acids (MCFA) .............................................. 25 2.4.3 VCO as A Potential Treatment against Oral Candidiasis ............... 26 2.4.4 AVCO with Broad Antimicrobial/Antifungal Spectrum ................ 28
2.5 Mechanism of Action of Plant Oils against C. albicans ............................ 28 2.5.1 Antifungal Action of Plant Oils on C. albicans .............................. 28
CHAPTER THREE MATERIALS AND METHOD ............................................ 30 3.1 Preparation of Virgin Coconut Oil ....................................................................... 30
3.1.1 AVCO and VCO Stock Preparations .............................................. 31 3.2 Preparation of Fatty Acids Methyl Esters (Fame) ..................................... 31
3.2.1 Gas Chromatography Mass Spectrometry ...................................... 31 3.3 Growth and Maintenance of Candida albicans ......................................... 32 3.4 Cell Viability .............................................................................................. 32
x
3.4.1 Culture Conditions and Preparation of Fungal Suspensions ........... 32
3.4.2 Staining Fungal Suspension with Kit L7012 .................................. 33 3.4.3 Microscopic Observations Using Fluorescence Microscopy .......... 34
3.5 Release of Cytoplasmic Material ............................................................... 34
3.5.1 Release of Cytoplasmic Material Absorbing at 260 Nm and
280 Nm Assay .......................................................................................... 35 3.5.2 Concentration of Protein Content Measurement ............................. 35
CHAPTER FOUR RESULTS AND DISCUSSION .............................................. 40 4.1 Analysis of the Composition of Fatty Acids In AVCO and VCO ............. 40 4.2 Determining the Viability of C. albicans Using Fluorescence
4.3 Quantification of Cytoplasmic Leakage of C. albicans ............................. 49 4.4 Transmission Electron Microscopy ........................................................... 58
CHAPTER FIVE CONCLUSION AND OUTLOOK ........................................... 62
CHAPTER SIX REFERENCES ............................................................................. 63
APPENDIX A ............................................................................................................ 76
APPENDIX B ............................................................................................................ 79
APPENDIX C ............................................................................................................ 80
xi
LIST OF TABLES
Table 2.1 Clinical Classification of Oral Candidiasis 6
Table 2.2 Types of Secreted Proteins of C. albicans 15
Table 2.3 Virulence factors of C. albicans 16
Table 2.4 Summary of Drugs for the Treatment of Oral
Candidiasis
19
Table 2.5 Summary of Previous Studies on Virgin Coconut Oil 27
Table 3.1 Category of Sample Used for Cell Viability Test 33
Table 3.2 Category of Sample Used for Release of Cytoplasmic
Material
34
Table 4.1 Fatty Acid Composition (%) of VCO and AVCO
Compared with APCC Standard Range
40
Table 4.2 Percentage Distribution of Dead Fungi after Staining 43
Table 4.3 Significance Value for Means Comparison among
AVCO, VCO, PC and NC throughout 3 Hours.
43
Table 4.4 Cytoplasmic Release of DNA at 260 Nm from C.
albicans after Treatment with AVCO, VCO, Nystatin
and Tween.
52
Table 4.5 Significance Value for Means Comparison among
AVCO, VCO, Nystatin and Tween throughout 4
Hours of Cytoplasmic Release of DNA.
52
Table 4.6 Cytoplasmic Release of Protein at 280 Nm from C.
albicans after Treatment with AVCO, VCO, Nystatin
and Tween.
53
Table 4.7 Significance Value for Means Comparison among
AVCO, VCO, Nystatin and Tween throughout 4
Hours of Cytoplasmic Release of Protein.
53
Table 4.8 Concentration of Protein Leakage of C. albicans after
Treatment with AVCO, VCO, Nystatin and Tween.
57
xii
Table 4.9 Significance Value for Means Comparison among
AVCO, VCO, Nystatin and Tween throughout 4
Hours of Total Protein Concentration.
57
xiii
LIST OF FIGURES
Figure 2.1 The Image of Different Lesions of Oral Candidiasis. 9
Figure 2.2 Different Growth Morphologies of C. albicans. 11
Figure 2.3 Cellular Organization of Candida albicans 11