UNIVERSITI PUTRA MALAYSIA

FATEMEH EHTESHAM FAR

FPSK(m) 2012 39

ESTABLISHING STANDARD WORKING PROCEDURES TO INVESTIGATE EFFECTS OF CHANNA STRIATUS BLOCH CRUDE EXTRACT ON MALASSEZIA FURFUR GROWTH

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ESTABLISHING STANDARD WORKING PROCEDURES TO

INVESTIGATE EFFECTS OF CHANNA STRIATUS BLOCH CRUDE EXTRACT ON MALASSEZIA FURFUR GROWTH

By

FATEMEH EHTESHAM FAR

Thesis Submitted to the School of Graduate Studies, Universiti Putra Malaysia, in Fulfillment of the Requirements for the Degree of Master of Sciences.

August 2012

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DEDICATION

This thesis is dedicated to all of the following persons who have accompanied me in this way:

My husband, Sadegh A., without Whose Caring Support it would not have been possible

,and

To my Grandmother & Grandfather who support and encouraged me

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Abstract of thesis presented to the Senate of Universiti Putra Malaysia in fulfillment of the requirement for the degree of Master of Science

ESTABLISHING STANDARD WORKING PROCEDURES TO INVESTIGATE EFFECTS OF CHANNA STRIATUS BLOCH CRUDE

EXTRACT ON MALASSEZIA FURFUR GROWTH

By

FATEMEH EHTESHAM FAR

August 2012

Chairman: Professor Abdul Manan Mat Jais, PhD

Faculty: Medicine and Health Sciences

Skin fungal infections are a common type of skin diseases that has become a global

problem. Fungal infections commonly constitute a health problem in developing

countries particularly in tropical and subtropical regions. Malassezia and Candida

species are the most prevalent pathogens in human and animals. Yeast-like fungus

Malassezia furfur is associated with several dermatological diseases such as

Pityriasis versicolor, Malassezia folliculitis, Atopic dermatitis and Seborrheic

dermatitis (Dandruff). The available drugs known as antimycotic act against

extensive range of molecular targets of fungi but the numbers of antifungal resistance

pathogens have increased. Furthermore, majority of these antifungal drugs are

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synthetic that lead to some health-threatening side-effects. Consequently, increasing

demand for seeking the natural therapeutic medicines has made much attention to

pelagic organisms, especially, fish that can be found throughout the ocean. There are

no standard parameters for minimum fungicidal concentration (MFC), as well as

definitive guideline for evaluating the antifungal drugs against Malassezia species.

So, this study attempted to apply some modified methods which are more adjustable

for M. furfur in order to perform antifungal susceptibility tests. Therefore, this

research was planned to evaluate effects of Channa striatus crude extract on yeast-

like fungi M. furfur ATCC 14521 through bioassays. Bioassay studies demonstrated

that Channa striatus crude extract in various concentrations did not show inhibitory

activity against M. furfur which was evidenced in terms of minimum inhibitory

concentration (MIC), minimum fungicidal concentration (MFC) tests and percentage

of cell viability using colorimetric assay. Besides, colorimetric resazurin microtitre

assay (REMA) Plate Method expressed Channa striatus crude extract to have

proliferative effect on M. furfur in comparison with growth control. As a conclusion,

it was previously hypothesized that possibly Channa striatus crude extract has

sufficient antifungal properties to inhibit M. furfur growth. But, based on the in vitro

data of this study, Channa striatus crude extract exacerbate the diseases associated

with M. furfur. Therefore, it cannot be an effective natural antimycotic medicine to

treat skin diseases related to M. furfur.

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Abstrak tesis yang dikemukakan kepada Senat Universiti Putra Malaysia sebagai memenuhi keperluan untuk ijazah Master Sains

PEMBANGUNAN STANDARD PROSEDUR KERJA DALAM MENYIASAT KESAN EKSTRAK MENTAH CHANNA STRIATUS PADA

PERTUMBUHAN MALASSEZIA FURFUR

Oleh

FATEMEH EHTESHAM FAR

Ogos 2012

Pengerusi: Profesor Abdul Manan Mat Jais, PhD

Fakulti: Perubatan dan Sains Kesihatan

Jangkitan kulit oleh kulat amat mudah terjadi dan menjadi satu masalah global

terutamanya di negara-negara membangun di kawasan-kawasan tropika dan

subtropika. Spesies Malassezia dan Candida adalah agen jangkitan yang paling lazim

ke atas manusia dan haiwan. Malassezia furfur adalah sejenis yis yang dikaitkan

dengan beberapa penyakit dermatologi seperti versicolor Pityriasis, Malassezia

folliculitis, dermatitis atopik dan dermatitis Seborrheic (Kelemumur). Ubatan yang

sedia ada dikenali sebagai ubat ‘antimycotic’ bertindak terhadap pelbagai sasaran

molekul di dalam kulat tetapi bilangan patogen yang resistan kepada dadah tersebut

telah meningkat. Tambahan pula, kebanyakan ubat antikulat sintetik membawa

kepada beberapa kesan sampingan yang boleh mengancam kesihatan. Oleh yang

demikian, permintaan yang semakin meningkat untuk menemui ubat-ubatan semula

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jadi terapeutik telah membawa perhatian dan kajian ke atas organisma pelagik

terutamanya ikan yang boleh didapati di seluruh lautan. Permasalahan timbul apabila

tiada garis panduan yang jelas bagi menentukan Kepekatan Fungisidal Minima

(MFC) ke atas spesies Malassezia agar keberkesanan ubat-ubatan semula jadi

terapeutik ini dapat dinilai secara muktamad. Jadi, kajian ini cuba untuk

menggunakan beberapa kaedah yang diubahsuai untuk dijalankan ke atas spesies

Malassezia. Oleh itu, penyelidikan ini telah dirancang untuk menilai kesan ekstrak

mentah Channa striatus ke atas yis penyebab kelumumur iaitu M. furfur ATCC

14521 untuk menilai sifat ‘antimycotic’ ekstrak mentah Channa striatus melalui

beberapa kaedah bioassei. Kajian yang dijalankan menunjukkan bahawa ekstrak

mentah Channa striatus pada kepekatan berlainan tidak menunjukkan aktiviti prencat

kulat terhadap M. furfur yang telah dibuktikan dari segi kaedah Kepekatan

perencatan Minima (MIC), Kepekatan Fungisidal Minima (MFC) dan peratusan sel

viabiliti menggunakan kolorimetrik asai. Selain itu, kaedah kalorimetrik resazurin

mikrotitre asai (REMA) menunjukkan ekstrak mentah Channa striatus mempunyai

kesan proliferatif ke atas M. furfur berbanding dengan experimen kawalan. Sebagai

kesimpulan, hipotesis awal adalah berkemungkinan ekstrak mentah Channa striatus

mempunyai ciri-ciri anti-kulat yang mencukupi untuk menghalang pertumbuhan M.

furfur. Tetapi, berdasarkan data in vitro dalam kajian ini, didapati ekstrak mentah

Channa striatus berpotensi untuk menggalakkan lagi pertumbuhan M. furfur di

dalam sesuatu jangkitan kulit yang berkaitan. Jadi, ia tidak boleh menjadi ubat

semulajadi yang berkesan untuk merawat penyakit kulit yang berkaitan dengan M.

furfur.

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ACKNOWLEDGEMENTS

First and foremost I offer my sincerest to the chairman of my supervisory committee;

Professor Dr. Abdul Manan B. Mat Jais, who has supported me throughout my

research project with his patience and knowledge whilst allowing me to complete

this work. I attribute the level of my master degree to his encouragement and effort

and without him this thesis, too, would not have been done. One simply could not

wish for a better or friendlier supervisor.

I am also very grateful to my co-supervisor; Dr. Mohd Nasir Mohd Desa, who with

his kindness, affection, and support gave me courage and ability to overcome all the

problems I have faced during course of my study.

Special thanks to the Ministry of Science of Malaysia and Faculty of Medicine and

Health Sciences for providing the financial means and laboratory facilities.

Special appreciations to the Institute of Bioscience (IBS) of UPM for providing the

laboratory facilities.

The author would also like to convey thanks to great Malaysia; for its hospitality and

smiles of its people.

Lastly, I also want to take this opportunity to dedicate my appreciation to my beloved

husband, Grandmother, Grandfather and friends for their understanding and endless

love, throughout the duration of my study.

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I certify that a Thesis Examination Committee has met on 27 August 2012 to conduct the final examination of Fatemeh Ehtesham Far on her thesis entitled " Establishing Standard Working Procedures to Investigate Effects of Channa striatus BLOCH Crude Extract on Malassezia furfur Growth" in accordance with the Universities and University Colleges Act 1971 and the Constitution of the Universiti Putra Malaysia [P.U.(A) 106] 15 March 1998. The Committee recommends that the student be awarded the Master of Science. Members of the Thesis Examination Committee were as follows: Sabrina binti Sukardi, PhD Associate Professor (Medical) Faculty of Medicine and Health Sciences Universiti Putra Malaysia (Chairman) Malina binti Osman, PhD Associate Professor Faculty of Medicine and Health Sciences Universiti Putra Malaysia (Internal Examiner) Roslida binti Hamid @ Abdul Razak, PhD Faculty of Medicine and Health Sciences Universiti Putra Malaysia (Internal Examiner) Ridzwan Hashim, PhD Professor Kulliyyah of Scince International Islamic University Malaysia (External Examiner)

SEOW HENG FONG, PhD Professor and Deputy Dean School of Graduate Studies Universiti Putra Malaysia Date:

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This thesis was submitted to the Senate of Universiti Putra Malaysia and has been accepted as fulfillment of the requirement for the degree of Master Science. The members of the Supervisory Committee were as follows:

Abdul Manan Mat Jais, PhD Professor Faculty of Medicine and Health Sciences Universiti Putra Malaysia (Chairman) Mohd Nasir Mohd Desa, PhD Senior Lecturer Faculty of Medicine and Health Sciences Universiti Putra Malaysia (Member)

BUJANG BIN KIM HUAT, PhD Professor and Dean School of Graduate Studies Universiti Putra Malaysia Date:

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DECLARATION

I declare that the thesis is my original work expect for quotations and citations which have been duly acknowledged. I also, declare that it is has not been previously, and is not concurrently, submitted for any other degree at Universiti Putra Malaysia or at other institution.

FATEMEH EHTESHAM FAR

Date: 27 August 2012

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TABLE OF CONTENTS

Page

DEDICATION ii ABSTRACT iii ABSTRAK v ACKNOWLEDGMENT vii APPROVAL viii DECLARATION x LIST OF TABLES xiv LIST OF FIGURES xv LIST OF ABBREVIATIONS xvii CHAPTER

1 INTRODUCTION 1 1.1 Introduction 1 1.2 Objectives 7

1.2.1 General Objective 7 1.2.2 Specific Objectives 7 1.3 Research Hypothesis 8 2 LITERATURE REVIEW 9

2.1 Haruan (Channa striatus) 9 2.1.1 Some Biomedical properties of Channa striatus 10

2.2 Human yeast–like Fungi Pathogen 12 2.2.1 Malassezia 12 2.2.2 Some Skin Diseases Associated with Malassezia furfur

13

2.3 Review of Some Synthetic and Natural Compounds Investigated for Antimycotic Activity Against the Malassezia furfur

21

2.4 Limitations of the Study 23 2.5 Conventional Antifungal Assays 25 3 MATERIALS AND METHODS

30

3.1 Channa striatus Source 30 3.2 Yeast–like fungi Source and Its Maintenance 30 3.3 Identification of Malassezia furfur 31 3.3.1 Selective Culture Media 31 3.3.2 Growth at 37ºC and 40ºC 34 3.3.3 Microscopic Examination 34 3.3.4 Biochemical Tests 35 3.4 Preparation of the Channa striatus fillet and Extracts 35 3.4.1 Preparation of the Fresh Channa striatus Fillet 35

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3.4.2 Preparation of the Channa striatus Aqueous Extract

36

3.4.3 Preparation of the Channa striatus Crude Extract

37

3.5 Bioassays 37 3.5.1 Minimal Inhibitory and Fungicidal Concentration Test

37

3.5.2 Colorimetric Resazurin Microtiter Assay (REMA) Plate Method

39

3.5.3 Agar Well Diffusion Assay 41 3.6 Statistical Analysis 42 4 RESULTS 44 4.1 Identification of Malassezia furfur 44 4.1.1 Selective Culture Media 44 4.1.2 Growth at 37ºC and 40ºC 46 4.1.3 Microscopic Examination 46 4.1.4 Biochemical Tests 48 4.2 Bioassays 48 4.2.1 Minimal Inhibition Concentration and Minimal Fungicidal Concentration Tests

48

4.2.2 Colorimetric Resazurin Microtiter Assay (REMA) Plate Method

50

4.2.3 Agar-Well Diffusion Assay 62 5 DISSCUSION 68 5.1 Identification of Malassezia furfur 68 5.2 The Effect of Channa striatus Crude extract on Malassezia furfur by Bioassays

73

5.2.1 Assessment of the Effects of Channa striatus Crude Extract on Malassezia furfur by MIC and MFC Tests

74

5.2.2 Screening of the Effect of Channa striatus Crude Extract on Malassezia furfur by Qualitative Colorimetric Resazurin Microtiter Assay (REMA) Plate Method

76

5.2.3 Screening of the Effect of Channa striatus Crude Extract on Growth Percentage of the Malassezia furfur by Quantitative Colorimetric Resazurin Microtiter Assay (REMA) Plate Method

78

5.2.4 Screening the Effect of Channa striatus Crude Extract by Agar- Well Diffusion Assay

86

6 CONCLUSIONS 88

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REFERENCES 91 APPENDICES 107 BIODATA OF STUDENT 110