UNIVERSITI PUTRA MALAYSIA ESTABLISHING STANDARD WORKING PROCEDURES TO INVESTIGATE EFFECTS OF CHANNA STRIATUS BLOCH CRUDE EXTRACT ON MALASSEZIA FURFUR GROWTH FATEMEH EHTESHAM FAR FPSK(m) 2012 39
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
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 ii
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 iii
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 yeastlike 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. iv
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 v
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. vi
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. vii
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: viii
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: ix
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 x
TABLE OF CONTENTS Page DEDICATION ABSTRACT ABSTRAK ACKNOWLEDGMENT APPROVAL DECLARATION LIST OF TABLES LIST OF FIGURES LIST OF ABBREVIATIONS 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 ii iii v vii viii x xiv xv xvii 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 2.3 Review of Some Synthetic and Natural 21 Compounds Investigated for Antimycotic Activity Against the Malassezia furfur 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 13 xi
3.4.2 Preparation of the Channa striatus 36 Aqueous Extract 3.4.3 Preparation of the Channa striatus Crude Extract 37 3.5 Bioassays 37 3.5.1 Minimal Inhibitory and Fungicidal 37 Concentration Test 3.5.2 Colorimetric Resazurin Microtiter Assay 39 (REMA) Plate Method 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 48 Minimal Fungicidal Concentration Tests 4.2.2 Colorimetric Resazurin Microtiter Assay 50 (REMA) Plate Method 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 73 Malassezia furfur by Bioassays 5.2.1 Assessment of the Effects of Channa 74 striatus Crude Extract on Malassezia furfur by MIC and MFC Tests 5.2.2 Screening of the Effect of Channa 76 striatus Crude Extract on Malassezia furfur by Qualitative Colorimetric Resazurin Microtiter Assay (REMA) Plate Method 5.2.3 Screening of the Effect of Channa 78 striatus Crude Extract on Growth Percentage of the Malassezia furfur by Quantitative Colorimetric Resazurin Microtiter Assay (REMA) Plate Method 5.2.4 Screening the Effect of Channa striatus Crude Extract by Agar- Well Diffusion Assay 86 6 CONCLUSIONS 88 xii
REFERENCES 91 APPENDICES 107 BIODATA OF STUDENT 110 xiii