abstract - studentsrepo.um.edu.mystudentsrepo.um.edu.my/3756/2/front_part.pdf · ii abstract...

ii

ABSTRACT

Candida species is an opportunistic microorganism residing in the human oral cavity.

There is increasing prevalence of candidal infections in the oral cavity largely because

of the increasing size of the population at risk. The rise in diagnosed cases of oral

candidiasis is also due to the immunosuppressive effect of prescribed antifungal agents

on resistant hosts. The ability of Candida species to adhere on mucosal tissues and/or

denture surfaces, and the production of hydrolytic enzymes along with defective host

immunity are among the known key factors of invasions and pathogenesis of oral

candida. Although Candida albicans remains as the most pathogenic organism, the

emergence of non-Candida albicans Candida (NCAC) species with reduced

susceptibility to prescribed antifungal agents has prompted efforts to study antifungal

agents from natural sources. Plants have long been known to possess medicinal values

and are rich in chemical constituents that can be used in the development of antifungal

products. Initially, seven local plants were systematically screened for their antifungal

activity and two of them - Brucea javanica L. and Piper betle L. were selected based on

their positive antifungal activities on seven Candida species tested. The specific

objectives of this study were; (i) to determine the minimum inhibition concentration

(MIC) and minimum fungicidal concentration (MFC) values of the extracts, (ii) to

investigate the growth inhibitory effect of the extracts based on changes in the pattern of

growth profile of each Candida species, (iii) to investigate the influence of extracts on

adherence mechanisms which include the non-specific and specific bindings, (iv) to

investigate any morphological changes of the cells following treatment with the extracts,

and (v) to explore the differential expressions of multigene family of secreted aspartyl

proteinases (SAPs) and hyphal cell wall protein 1 (HWP1).

iii

Candida species purchased from the American Types Culture Collection (ATCC) were

Candida albicans ATCC 14053, Candida dubliniensis ATCC MYA-2975, Candida

glabrata ATCC 90030, Candida krusei ATCC 14243, Candida lusitaniae ATCC 64125,

Candida parapsilosis ATCC 22019 and Candida tropicalis ATCC 13803. Growth

inhibitory responses of the Candida species to the extracts include determination of the

MIC and MFC, while effect on the growth curve was determined based on

spectrophotometric assay. Deviations in the doubling time (g) and specific growth rates

(µ) were computed as percentage to extract-treated cells relative to that of the total cells

in the absence of extracts. In addition, the anti-adherence effect of the B. javanica and P.

betle extracts which included study on the cell-surface hydrophobicity (CSH) and

specific bindings on pellicles, ultrastructure and the regulations of SAP1-10 and HWP1

were also analysed. 0.12% w/v chlorhexidine gluconate-containing mouthrinse was

used as a reference.

In preliminary screening, the diameter of inhibition zone (DIZ) values showed that B.

javanica and P. betle aqueous extracts exhibited a wide range of antifungal activities

over the seven Candida species with C. dubliniensis identified as the most sensitive. Of

the seven Candida species, C. tropicalis showed the highest growth rates (0.319 ± 0.002

h-1

) while the others were in the range of 0.141 ± 0.001 to 0.265 ± 0.005 h-1

. This

indicated that different species of candida reproduce at different rate. In the presence of

extracts, the lag and log phases were extended and shifted to the right. This resulted in

the deviations of the g- and µ-values, indicating that the extracts may have exerted

fungistatic activity towards the candidal cells. Growth kinetics of the candidal species

was also elucidated based on colony forming unit (CFU) enumeration. Different

Candida species have shown different CSH values and adhering capacity to the pellicle.

iv

In view of the CSH, C. krusei, C. dubliniensis and C. tropicalis showed the highest

adsorption to hexadecane at 30.23%, 26.19% and 19.70%, respectively, while the others

were much lower within the range of 7% to 10%. The CSH of all Candida species were

significantly affected by these two extracts (P < 0.05), with B. javanica exhibiting more

than 60% reduction of CSH than P. betle. Specific bindings of the candidal cells on the

pellicles were also shown to be affected by the treatment of extracts. Exposure to P.

betle-treated pellicle drastically reduced the adhering capacity of three out of seven

candidal species by more than 50% (C. tropicalis 86.02%, C. albicans 61.41% and C.

krusei 56.34%). Pellicles treated with B. javanica exhibited similar effect on C.

tropicalis (89.86%), C. lusitaniae (89.66%), C. albicans (79.74%), C. glabrata (76.85%)

and C. krusei (67.61%). Comparatively, the adherence interference effect of B. javanica

towards the candidal cells was slightly higher than P. betle. In addition to the growth

inhibitory and anti-adherence effects, physical changes in the cell walls of Candida

species were also demonstrated following treatment of the candidal cells with the

extracts. The expressions of SAP1-10 and HWP1 were affected by the extracts treatment,

suggesting that the extracts have successfully penetrated and disrupted the intrinsic

environment of the cells. The genes seemed to be suppressed and this may then revoke

the pathogenesis of oral Candida.

As a conclusion, B. javanica and P. betle exhibited antifungal activities towards the

seven oral Candida species tested. Data from this study strongly suggest the fungistatic

and growth inhibitory effects of the extracts. Thus, B. javanica and P. betle extracts may

be considered as promising adjuncts in oral health products.

v

ABSTRAK

Spesies Candida merupakan mikroorganisma oportunis yang menetap di dalam mulut

manusia. Jangkitan Candida dalam mulut (oral kandidiasis) biasanya berlaku kepada

golongan yang berisiko masalah kesihatan. Peningkatan kes kandidiasis juga berpunca

daripada kesan imunosupresif agen antikulat yang diberikan kepada hos secara

konsistan. Keupayaan spesies Candida untuk melekat ke atas tisu mukosa dan/atau

permukaan dentur, dan penghasilan enzim-enzim hidrolitik ketika sistem imunisasi

tidak sempurna antara faktor utama kolonisasi dan patogenesis bagi spesies Candida.

Walaupun Candida albicans masih kekal sebagai organisma patogen, tetapi kemunculan

spesies Candida selain C. albicans yang rintang terhadap agen antikulat telah

mendorong usaha untuk mengkaji agen antikulat yang baru daripada sumber semulajadi.

Tumbuh-tumbuhan telah lama diketahui mempunyai nilai perubatan dan kaya dengan

juzuk kimia yang boleh digunakan dalam pembangunan produk-produk antikulat.

Dalam penyelidikan ini, tujuh spesies tumbuhan tempatan telah disaring secara

sistematik bagi mengetahui aktiviti antikulatnya, dan dua daripadanya iaitu Brucea

javanica L. dan Piper betle L. telah dipilih untuk kajian seterusnya kerana menunjukkan

potensi aktiviti antikulat ke atas tujuh spesies Candida yang diuji. Objektif khusus

penyelidikan ini adalah untuk (i) menentukan kepekatan perencatan minimum (MIC)

dan kepekatan fungisidal minimum (MFC) bagi ekstrak yang diuji, (ii) mengkaji kesan

perencatan ekstrak terhadap pertumbuhan sel Candida, (iii) mengkaji pengaruh ekstrak

ke atas mekanisma perlekatan spesies Candida meliputi pengikatan khusus dan bukan

khusus, (iv) mengkaji sebarang perubahan struktur permukaan sel Candida selepas

rawatan ekstrak diberikan, dan (v) meneroka perbezaan ekspresi gen SAP (SAP1-10)

dan HWP1 di bawah pengaruh ekstrak.

vi

Tujuh spesies Candida yang telah dibeli daripada American Types Culture Collection

(ATCC) adalah Candida albicans ATCC 14053, Candida dubliniensis ATCC MYA-

2975, Candida glabrata ATCC 90030, Candida krusei ATCC 14243, Candida

lusitaniae ATCC 64125, Candida parapsilosis ATCC 22019 dan Candida tropicalis

ATCC 13803. Tindak balas perencatan pertumbuhan tujuh spesies Candida terhadap

ekstrak merangkumi penentuan MIC and MFC, manakala kesan ke atas lengkok

pertumbuhan ditentukan dengan kaedah asai spektrofotometri. Perubahan bagi

peratusan masa ganda dua (g) dan kadar pertumbuhan spesifik (µ) selepas rawatan

ekstrak berbanding jumlah sel sebelum rawatan ekstrak ditentukan. Kesan ekstrak B.

javanica dan P. betle terhadap mekanisma perlekatan, struktur morfologi sel, dan aturan

expresi gen SAP1-10 dan HWP1 turut dikaji. 0.12% w/v chlorhexidine digluconate

diguna sebagai kawalan positif.

Dalam ujian penyaringan awal, diameter zon perencatan menunjukkan ekstrak B.

javanica dan P. betle mempunyai kepelbagaian aktiviti antikulat ke atas tujuh spesies

Candida yang diuji, di mana C. dubliniensis merupakan spesies yang paling sensitif

berbanding yang lain. C. tropicalis merupakan spesies yang mempunyai kadar

pertumbuhan yang tinggi (0.319 ± 0.002 h-1

), manakala spesies Candida lain sekitar

julat 0.141 ± 0.001 to 0.265 ± 0.005 h-1

. Ini menunjukkan bahawa spesies yang berbeza

dalam genus Candida mempunyai kebolehulangan yang berbeza. Dengan kehadiran

ekstrak, fasa-fasa lag dan log telah diperluas dan beralih ke kanan. Ini menunjukkan

ekstrak mempunyai aktiviti fungistatik. Kesan ekstrak ke atas kinetik pertumbuhan

spesies Candida juga dikaji berdasarkan jumlah pembentukan koloni unit (CFU). C.

krusei, C. dubliniensis dan C. tropicalis mempunyai kapasiti penyerapan hexadecane

yang tinggi masing-masing pada 30.23%, 26.19% dan 19.70%, berbanding yang lain

vii

dalam julat 7% hingga 10%. Kehidrofobian permukaan sel bagi semua spesies Candida

telah terjejas berikutan rawatan ekstrak yang diberi (P < 0.05), di mana ekstrak B.

javanica lebih berkesan dengan 60% pengurangan kehidrofobian berbanding ekstrak P.

betle. Kapasiti pengikatan spesifik setiap spesies Candida ke atas pelikel turut terjejas

dengan rawatan kedua-dua ekstrak yang diberikan. Pendedahan sel ke atas pelikel yang

dirawat ektrak P. betle telah mengurangkan kapasiti pelekatan tiga daripada tujuh

spesies Candida lebih daripada 50% (C. tropicalis 86.02%, C. albicans 61.41% dan C.

krusei 56.34%). Pelikel yang dirawat dengan ekstrak B. javanica menunjukkan kesan

yang sama ke atas C. tropicalis (89.86%), C. lusitaniae (89.66%), C. albicans (79.74%),

C. glabrata (76.85%) dan C. krusei (67.61%). Secara perbandingan, kesan gangguan

pelekatan yang dikenakan ekstrak B. javanica terhadap sel Candida sedikit tinggi

daripada ekstrak P. betle. Di samping kesan perencatan pertumbuhan dan anti-pelekatan,

perubahan fizikal pada dinding sel spesies Candida juga kelihatan selepas rawatan

ekstrak diberi. Ekpresi gen SAP1-10 dan HWP1 juga terjejas menunjukkan bahawa

ekstrak berjaya menembusi membran sel dan membeku persekitaran intrinsik sel-sel.

Gen-gen ini kelihatannya ditindas dan menafikan proses patogenesis spesies Candida.

Kesimpulannya, ekstrak B. javanica dan P. betle mempunyai kesan anti-kulat terhadap

tujuh spesies Candida yang diuji. Data yang diperoleh daripada kajian ini juga

membuktikan bahawa ekstrak B. javanica dan P. betle mempunyai aktiviti fungistatik

dan kesan perencatan pada kepekatan yang lebih tinggi. Justeru, kedua-dua tumbuhan

tempatan ini berpotensi untuk pembangunan sebagai agen antikulat khususnya dalam

penghasilan produk-produk kesihatan mulut.

viii

ACKNOWLEDGEMENT

This project has worked out smoothly through the tireless dedication of talented

workaholics:

I could not have done this work without the help of several people who contributed to it

directly or indirectly. First and foremost, I would like to express my gratitude to my

supervisors, Dr. Wan Himratul Aznita Wan Harun and Assoc. Prof. Dr. Fathilah

Abdul Razak for giving me the opportunity to work on this interesting project. Their

continuous support and guidance, the unlimited enthusiasm and the numerous helpful

discussions were the cornerstones of this project.

I would like to thank the staff of the Department of Oral Biology, Faculty of Dentistry,

University of Malaya, the staff of Electron Microscopy Unit, Faculty of Medicine,

University of Malaya, and the staff of Microscopy Unit, Institute of Bioscience,

Universiti Putra Malaysia for their assistance throughout the project. I want to thank all

former and current members of the lab for all the lively discussions that have created an

atmosphere, which made the whole work fun!

I am also thankful for the financial supports from the University of Malaya Research

Grant (RG095/09HTM), Postgraduate Research Fund (PS160/2010B), and the

Malaysian Government Scholarship (MyBrain15).

Special thanks to my parents who tempered each other’s advice to give me strength and

endless support throughout this life.

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LIST OF CONTENTS PAGE

ABSTRACT i

ABSTRAK v

ACKNOWLEDGEMENT viii

LIST OF CONTENTS ix

LIST OF FIGURES xvii

LIST OF TABLES xx

LIST OF ABBREVIATIONS xxi

CHAPTER ONE: INTRODUCTION AND OBJECTIVES

1.1 Introduction 1

1.2 Objectives 4

CHAPTER TWO: LITERATURE REVIEW

2.1 The Oral Cavity and Its Indigenous Microbial 5

2.2 The Genus Candida 5

2.2.1 Biology and Taxonomy 5

2.2.2 General Morphology 6

2.2.3 Structures and Functions of Cell Wall 7

2.3 Normal Microbial Flora 13

2.4 Transition of the Normal Flora to Pathogenic Flora 13

2.5 Types of Candida Species 14

2.5.1 Candida albicans 14

2.5.2 Candida dubliniensis 15

2.5.3 Candida glabrata 16

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2.5.4 Candida krusei 17

2.5.5 Candida lusitaniae 18

2.5.6 Candida parapsilosis 19

2.5.7 Candida tropicalis 20

2.6 Emergence of the Non-Candida albicans Candida Species 21

2.7 Oral Candidiasis 21

2.7.1 Classification of Oral Candidiasis 22

2.7.2 Predisposing Factors to Oral Candidiasis 22

2.7.2.1 Local Host Factors 22

2.7.2.2 Systemic Factors 23

2.7.2.3 Iatrogenic Factors 24

2.7.3 Denture Wearing Associated with Oral Candidiasis 26

2.8 Virulence Factors 27

2.8.1 Adherence Mechanisms 27

2.8.1.1 Specific Adhesion: Acquired Pellicles 27

2.8.1.2 Non-Specific Adhesion: Cell Surface Hydrophobicity 29

2.8.2 Virulence-Associated Genes in Candida Species 30

2.8.2.1 Adhesin-like Hyphal Cell Wall Protein 1 (Hwp1) 31

2.8.2.2 Secreted Aspartyl Proteinases (Saps) 32

2.9 Chemical-based Antimicrobial: Chlorhexidine gluconate 34

2.10 Plant-based Antimicrobial: An Alternative to Antifungal Agents 34

2.11 Types of Plant Species 35

2.11.1 Brucea javanica Linn. 35

2.11.1.1 General Information 35

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2.11.1.2 The Uses of Brucea javanica (L.) Merr. 36

2.11.1.3 The Chemical Constituents of Brucea javanica (L.) Merr. 36

2.11.2 Centella asiatica Linn. 37


2.11.2.2 The Uses of Centella asiatica L. 38

2.11.2.3 The Chemical Constituents of Centella asiatica L. 38

2.11.3 Garcinia mangostana Linn. 39


2.11.3.2 The Uses of Garcinia mangostana L. 39

2.11.3.3 The Chemical Constituents of Garcinia mangostana L. 40

2.11.4 Mangifera indica Linn. 41


2.11.4.2 The Uses of Mangifera indica L. 41

2.11.4.3 The Chemical Constituents of Mangifera indica L. 42

2.11.5 Piper betle Linn. 43


2.11.5.2 The Uses of Piper betle L. 43

2.11.5.3 The Chemical Constituents of Piper betle L. 44

2.11.6 Piper sarmentosum Roxb. 44


2.11.6.2 The Uses of Piper sarmentosum Roxb. 45

2.11.6.3 The Chemical Constituents of Piper sarmentosum Roxb. 46

2.11.7 Psidium guajava Linn. 46


xii


2.11.7.2 The Uses of Psidium guajava L. 47

2.11.7.3 The Chemical Constituents of Psidium guajava L. 48

CHAPTER THREE: MATERIALS AND METHODS

3.1 Materials 49

3.1.1 Plant Samples 49

3.1.2 Candida Species 49

3.1.3 Culture Media 50

3.1.4 Commercial Kits 50

3.1.5 Consumables 50

3.1.6 Chemical Reagents 50

3.1.7 Equipments 51

3.2 Methods 53

3.2.1 Collection of Plant Samples 53

3.2.2 Preparation of Crude Aqueous Extracts 53

3.2.3 Preparation of Culture Media 55

3.2.4 Stock Cultures Preparation and Condition 55

3.2.5 Preparation of Standard Candida Cell Suspension 56

3.2.6 Screening of the Plant Extracts for Antifungal Response 56

3.2.6.1 The Kirby-Bauer Susceptibility Test 56

3.2.7 Screening for Antifungal Activity 57

3.2.7.1 Preparation of Candidal Suspension 57

3.2.7.2 Preparation of Extract’s Stock Solution 59

3.2.7.3 Determination of Minimum Inhibition Concentration 59

xiii


3.2.7.4 Determination of Minimum Fungicidal Concentration 61

3.2.8 The Effect of Brucea javanica L. and Piper betle L. Extracts 61

on the Growth Profiles of Candida Species

3.2.8.1 Experimental Design 61

3.2.8.2 Growth Profiles of Untreated Candida Species 62

3.2.8.3 Growth Profiles of Extract-treated Candida Species 62

3.2.9 The Effects of Brucea javanica L. and Piper betle L. Extracts 65

on the Cell Surface Hydrophobicity of Candida Species


3.2.9.2 Preparation of Standard Candidal Cell Suspension 65

3.2.9.3 Determination of Cell Surface Hydrophobicity without 67

Treatment

3.2.9.4 Cell Surface Hydrophobicity of Candida Species 67

Following Treatment of the Extracts


on the Adherence of Candida Species


3.2.10.2 Collection of Saliva 69

3.2.10.3 Preparation of Acquired-pellicles 69

3.2.10.4 Adherence Affinities of Candida Species to Salivary 69

Pellicles

3.2.10.5 Anti-adherence Activity of Brucea javanica L. and 70

Piper betle L. Extracts

xiv



on the Morphology of Candida Species


3.2.11.2 Preparation of Samples for Scanning Electron Microscopy 72


on the Expression of Secretory Aspartyl Proteinases (SAP1-10)

and Hyphal Wall Protein (HWP1) Genes


3.2.12.2 Total RNA Extraction 75

3.2.12.3 RNA Quantification 76

3.2.12.4 Specific Primers 77

3.2.12.5 Reverse Transcription-Polymerase Chain Reaction 78

3.2.13 Statistical Analysis 79

CHAPTER FOUR: RESULTS

4.1 Screening of Plant Extracts for Antifungal Activity 80

4.1.1 The Kirby-Bauer Susceptibility Test 80

4.2 Determination of Antifungal Activity 84

4.3 The Effect of Brucea javanica L. and Piper betle L. Extracts on the 86

Growth Profiles of Candida Species

4.3.1 Growth Profiles of Candida Species 86

4.3.2 Growth Profiles of Candida Species Following Treatment of 92

Brucea javanica and Piper betle Extracts

xv



Cell Surface Hydrophobicity of Candida Species

4.4.1 Cell Surface Hydrophobicity of Selected Oral Candida Species 102

4.4.2 Cell Surface Hydrophobicity of Selected Oral Candida Species 104

Following Treatment of Brucea javanica and Piper betle Extracts


Adherence Affinity of Candida Species to Acquired Pellicles

4.5.1 The Adherence Affinity of Candida Species 108

4.5.2 Anti-adherence Activity of Brucea javanica and Piper betle 110

Extracts


Morphology of Candida Species

4.6.1 Scanning Electron Microscope Examination 113


Expressions of Secretory Aspartyl Proteinases (SAP1-10) and Hyphal

Wall Protein (HWP1) Genes

4.7.1 Differential Expression of Secretory Aspartyl Proteinases 129

(SAP1-10)

4.7.2 Differential Expression of Hyphal Wall Protein (HWP1) 130

CHAPTER FIVE: DISCUSSION

5.1 The Susceptibility of Oral Candida to Piper betle and Brucea javanica 134

Extracts

xvi


5.2 Growh Inhibitory Responses of Candida Species to Piper betle and 136

Brucea javanica Extracts

5.3 Adherence Capacity of Oral Candida Species: The Cell Surface 139

Hydrophobicity and Specific Adhesion Mechanism of Candidal Cells

under Influence of Brucea javanica and Piper betle Extracts

5.4 Ultrastructure of Candida Species Following Treatment with the 143

Piper betle and Brucea javanica Extracts

5.5 Differential Expression of SAP1-10 and HWP1 Genes Following 145

Treatment with the Piper betle and Brucea javanica Extracts

CHAPTER SIX: SUMMARY AND CONCLUSIONS

6.1 Summary and Conclusions 152

CHAPTER SEVEN: CLINICAL SIGNIFICANCE AND

RECOMMENDATIONS

7.1 Significance of the Study in Clinical Application 155

7.2 Recommendations for Future Research 156

REFERENCES 158

APPENDIX 193

xvii

LIST OF FIGURES PAGE

Figure 2.1 Chitin structure – a simple polysaccharide in the cell walls of all 11

fungi

Figure 2.2 Schematic representation of cell wall components 12

Figure 2.3 Schematic diagram summarizing the possible predisposing 25

factors influenced the interaction between Candida and the host

Figure 2.4 Brucea javanica Linn. 35

Figure 2.5 Centella asiatica Linn. 37

Figure 2.6 Garcinia mangostana Linn. 39

Figure 2.7 Mangifera indica Linn. 41

Figure 2.8 Piper betle Linn. 43

Figure 2.9 Piper sarmentosum Roxb. 45

Figure 2.10 Psidium guajava Linn. 47

Figure 3.1 An outline of the research procedures 54

Figure 3.2 An illustration of the screening method using the Kirby-Bauer 58

susceptibility test

Figure 3.3 An illustration of procedure to determine the MIC and MFC of 60

extracts against candidal species

Figure 3.4 An illustration of the growth kinetic assays 64

Figure 3.5 An illustration of determination of cell surface hydrophobicity of 66

candidal species

Figure 3.6 An illustration of the Nordini Artificial Mouth (NAM) model 71

Figure 3.7 An outline of the molecular study 74

Figure 4.1 The antifungal responses of Candida species using the 82

Kirby-Bauer susceptibility test

xviii


Figure 4.2 Growth curve of Candida species cultured in YPD broth 87

Figure 4.3 A composite figure illustrating the normal growth of Candida 88

species cultured in YPD broth

Figure 4.4 Composite figures illustrating the population of Candida species 94

following treatment with P. betle extract

Figure 4.5 Composite figures illustrating the population of Candida species 97

following treatment with B. javanica extract

Figure 4.6 The cell surface hydrophobicity of Candida species measured by 103

their binding affinities to hexadecane

Figure 4.7 Comparison of the effect of P. betle and B. javanica extracts 106

on the CSH of oral candidal species

Figure 4.8 The adherence of candida to saliva-coated glass surface 109

Figure 4.9 The adhering capacity of oral candida to salivary-pellicle 111

(untreated) and extract-treated pellicle

Figure 4.10 The anti-adherence effect of P. betle and B. javanica extracts 112

on the binding capacity of oral Candida species to the acquired

pellicles

Figure 4.11 SEM micrographs of C. albicans 122

Figure 4.12 SEM micrographs of C. dubliniensis 123

Figure 4.13 SEM micrographs of C. glabrata 124

Figure 4.14 SEM micrographs of C. krusei 125

Figure 4.15 SEM micrographs of C. lusitaniae 126

Figure 4.16 SEM micrographs of C. parapsilosis 127

Figure 4.17 SEM micrographs of C. tropicalis 128

xix


Figure 4.18 Differential expression of SAP1-10 genes in C. albicans 132

cultured in different conditions

Figure 4.19 Differential expression of HWP1 gene in C. albicans 133

cultured in different conditions

Figure 6.1 Summary of the effect of crude extract of B. javanica and 154

P. betle on oral candida

xx

LIST OF TABLES PAGE

Table 2.1 Virulence attributes of C. albicans 31

Table 3.1 Oligonucleotide sequences of SAP1-10 and ACT1 gene 77

Table 3.2 Oligonucleotide sequences of HWP1 gene 78

Table 4.1 The diameter of growth inhibition zones produced by the seven 83

plant extracts on the seven different Candida species

Table 4.2 MIC and MFC values of B. javanica and P. betle extracts 85

towards seven species of candida

Table 4.3 Changes in the doubling time (g) and specific growth rates (µ) 100

of the candidal species cultured in the absence (untreated) and

presence of P. betle extract (1, 3 and 6 mg/mL)

Table 4.4 Changes in the doubling time (g) and specific growth rates (µ) 101

of the candidal species cultured in the absence (untreated) and

presence of B. javanica extract (1, 3 and 6 mg/mL)

Table 4.5 The means percentage of adsorption of seven Candida species 107

to hexadecane following treatment with B. javanica and P. betle

extracts at different concentrations of 1, 3 and 6 mg/mL

Table 4.6 Deviations in the sizes of candidal cells following treatment of 114

P. betle and B. javanica extracts.

Table 4.7 The changes in the ultrastructural morphological features of 115

seven Candida species cells observed following treatment of

P. betle and B. javanica extracts (6 mg/mL)

xxi

LIST OF ABBREVIATIONS

Abbreviation Description

bp Base pair

cm Centimetre

CSH Cell surface hydrophobicity

CWP Cell wall protein

CHX Chlorhexidine gluconate

CFU/mL Colony forming units per millilitre

° C Degree Celsius

DNA Deoxyribonucleic acid

dNTP Deoxyribonucleoside triphosphates

dH2O Distilled water

EtBr Ethidium bromide

GPI Glycophophatidylinositol

g Gravity

g Gram

g/mL Gram per millilitre

g/L Gram per litre

h Hour(s)

L Litre

µg Microgram

μg/mL Microgram per millilitre

µL Microlitre

mL Millilitre

mL/min Millilitre per minute

xxii



µL Microlitre

μm Micromolar

µmole/ml Micromoles per millilitre

mg Milligram

mg/mL Milligram per millilitre

mm Millimetre

mM Millimolar

min Minute

M Molar

MW Molecular weight

nm Nanometer

OD Optical density

pp. Pages

% Percentage

RT-PCR Reverse transcription-polymerase chain reaction

rDNA Ribosomal deoxynucleic acid

RNA Ribonucleic acid

RNase Ribonuclease

sec Seconds

NaCl Sodium chloride

sp. Species

i.e. That is

TBE Tris-Borate-EDTA

xxiii



U/mL Unit per millilitre

V Voltage

v/v Volume per volume

w/v Weight per volume

YPD Yeast peptone dextrose

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