The Efficacy of Herbal Plants Extract Against Infection of Aeromonas Hydrophila, Staphylococcus Xylosus and Streptococcus Agalac
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THE EFFICACY OF HERBAL PLANTS EXTRACT AGAINST INFECTION OF AEROMONAS HYDROPHILA, STAPHYLOCOCCUS XYLOSUS AND STREPTOCOCCUS AGALACTIAE IN SNAKEHEAD (CHANNA STRIATA) FISH AZIRAH BINTI AKBAR ALI UNIVERSITI SAINS MALAYSIA 2014 THE EFFICACY OF HERBAL PLANTS EXTRACT AGAINST INFECTION OF AEROMONAS HYDROPHILA, STAPHYLOCOCCUS XYLOSUS AND STREPTOCOCCUS AGALACTIAE IN SNAKEHEAD (CHANNA STRIATA) FISH by AZIRAH BINTI AKBAR ALI Thesis submitted in fulfilment of the requirements for the degree of Master of Sciences February 2014 KEBERKESANAN EKSTRAK TUMBUH-TUMBUHAN HERBA TERHADAP PENYAKIT AEROMONAS HYDROPHILA, STAPHYLOCOCCUS XYLOSUS DAN STREPTOCOCCUS AGALACTIAE PADA IKAN HARUAN (CHANNA STRIATA) oleh AZIRAH BINTI AKBAR ALI Tesis yang diserahkan untuk memenuhi keperluan bagi Ijazah Sarjana Sains Februari 2014 ACKNOWLEDGEMENT In the name of Allah S.W.T, the Most Gracious, Most Merciful thus this thesis has been possible. With great honour, my deepest appreciation to my supervisor, Dr. Zary Shariman Yahaya, for his excellent guidance, unlimited support and motivation, patience, willingness of time in sharing ideas and knowledge and also his Do’a. Thank you very much Dr. A special thanks goes to Mr. Sinan Al-Hakeem for his assistance, suggestion, and patience. Not forgotten thanks to Ms. Ainul Farhana, in helping me a lot during at laboratory time, collecting the plants samples, being patience and supportive. I would like to convey my heartfelt thanks to all my friends, Kak Amelia and Nahid for their support and help during in fish trials experiment, lab 418 members (Kak Shamim, Kak Khairiah, Firdaus, Rajiv, Keat Lay, Boon Aun, Julian, Gaikling, Sally and others) and also Nik aisyah, and Mimi for their word inspiration and encourage. Finally, I express my sincere gratitude to my beloved parents, sister and brothers for their prayer, patience, trust, untiring support in every way, valuable motivation, constant love, inspiration and made my dream come true. Last but not least, I would like to thank Universiti Sains Malaysia (USM), Penang for providing the necessary facilities especially Staffs in Biology School. Not forgotten thanks to government which provide me the financial support during my study and also project grant which able the project to carry on. Azirah Akbar Ali, February 2014 ii TABLE OF CONTENTS PAGES ACKNOWLEDGEMENT…………………………………………………. ii TABLE OF CONTENTS………………………………………………...... iii LIST OF TABLES…………………………………………………………. xi LIST OF FIGURES……………………………………………………….. xiv LIST OF PLATES…………………………………………………………. xv LIST OF ABBREVIATIONS……………………………………………… xvii ABSTRAK…………………………………………………………………. xix ABTRACT……………………………………………….…………………. xxi CHAPTER ONE: INTRODUCTION 1.1 Problem Statement……..……………………………………………...... 2 1.2 Research objectives…………………………………………………… 3 CHAPTER TWO: LITERATURE REVIEW 2.1 Aquaculture…………………………………………………………….. 4 2.1.1 Aquaculture in Malaysia…………………………………………. 5 2.2 Snakehead fish………………………………………………………….. 7 2.2.1 Taxonomy and Synonymy of Snakehead fish…………………… 7 2.2.2 Snakehead fish (Channa straita)………………………………... 8 2.2.2. (a) Taxonomy and distribution………………………………… 9 2.2.2. (b) Biology of C. striata…………………………………………… 9 2.2.2. (c) Morphology of C. striata……………………………………… 10 2.2.2. (d) Importance medicinal properties of C. striata……………. 11 A. Amino acids……………………………………………… 12 iii B. Fatty acids………………………………………………... 12 2.2.2. (e) Traditional health treatment………………………………. 13 2.2.2. (f) Pharmacological properties of Channa striata…………….. 13 A. Post-partum and energy booster meal……………………. 14 B. Wound healing…………………………………………… 14 C. Anti-pain…………………………………………………. 14 D. Anti-inflammatory and anti-pyretic……………………… 15 E. Anti-oxidants…………………………………………….. 15 F. Anti-fungal and anti-bacterial……………………………. 15 G. Cardiological effect………………………………………. 16 H. Haematological treatments………………………………. 16 I. Neurology and neurophysiology…………………………. 16 J. Skin disease………………………………………………. 16 K. Platelet-aggregation……………………………………… 17 L. Antinociceptive properties……………………………….. 17 M. Other uses………………………………………………… 17 2.3 Fish diseases……………………………………………………………. 18 2.3.1 Types of fish diseases……………………………………………… 19 2.3.1(a) Microbial diseases……………………………………………. 19 2.3.1. (a).1Viral….………………………………………………. 19 2.3.1. (a).2 Bacteria………………………………………………... 20 2.3.1. (a).3 Fungi………………………………………………….. 22 2.3.1. (a).4 Protozoa…………………………………………………. 23 2.4 Bath treatments for sick fish……………………………………………. 24 2.4.1 Types of bath treatment…………………………………………… 24 iv 2.4.1(a) Dip Bath………………………………………………….... 24 2.4.1(b) Short Bath…………………………………………………. 24 2.4.1(c) Prolonged Bath……………………………………………… 24 2.4.2 Types of Chemotherapeutants used in Malaysia and Singapore….. 25 2.5 Phytotherapeutics in Aquaculture……………………………………… 27 2.5.1 Role of phytotherapeutic in aquaculture.………………………… 29 2.5.1. (a) Growth promoter………………………………………… 29 2.5.1. (b) Immunostimulants………………………………………... 29 2.5.1. (c) Antibacterial, Antiviral and Antifungal agents…………… 30 2.5.1. (d) Anti-stress agent………………………………………….. 31 2.5.1. (e) Appetite stimulators………………………………………. 31 2.6 Plants used in present study……………………………………………. 32 2.6.1 Senna spectabilis………………………………………………………... 32 2.6.2 Jacaranda filicifolia……………………………………………………. 33 2.6.3 Samanae saman……………………………………………………........ 36 2.6.4 Tamarindus indica…………………………………………………….... 37 2.6.5 Carica papaya…………………………………………………….......... 39 2.6.6 Andira inermis…………………………………………………….......... 40 2.6.7 Morinda elliptica……………………………………………………..... 41 2.6.8 Coleus aromaticus benth………………………………………… 42 2.6.9 Citrus hystrix……………………………………………………............ 44 2.6.10 Milletia pinnata……………………………………………………..... 45 2.6.11 Cymbopogon nardus…………………………………………………. 47 2.6.12 Cymbopogon citratus………………………………………………… 48 2.6.13 Polyalthia longifolia………………………………………………… 49 v CHAPTER THREE: MATERIALS AND METHODS 3.1 Flow chart……………………………………………………………… 51 3.2 Plant materials…………………………………………………….......... 52 3.3 Preparation of water plants extract…………………………………….. 54 3.4 Antimicrobial activity screening of the crude plants extracts………….. 54 3.4.1 Microorganisms…………………………………………………… 54 3.4.2 Antimicrobial activity test (Well Diffusion Method)……………... 55 3.4.2. (a) 1. Nutrient Agar preparation………………………………. 55 3.4.2. (a) 2 Mueller-Hinton Agar preparation……………………….. 55 3.4.2. (b) Water plants extract preparation…………………………... 55 3.4.2. (c) Bacteria suspension preparation…………………………... 56 3.4.2. (d) Well Diffusion Method…………………………………… 56 3.5 Minimum Inhibitory Concentration (MIC)…………………………….. 56 3.5.1 Justification of using three plants extract…………………………. 56 3.5.2 Mueller-Hinton Broth Preparation………………………………… 57 3.5.3 Water plants extract preparation…………………………………... 57 3.5.4 Bacteria suspension preparation…………………………………... 57 3.5.5 Microtitre Plate Assay…………………………………………….. 57 3.6 Brine Shrimp toxicity test……………………………………………… 58 3.6.1 Artificial seawater preparation…………………………………… 58 3.6.2 Artermia salina hatchings and collection………………………… 58 3.6.3 Water plants extract preparation………………………………….. 59 3.6.4 Toxicity test Assay……………………………………………….. 59 3.6.5 Toxicity test determination……………………………………….. 59 vi 3.7 Scanning Electron Microscope (SEM)………………………………… 60 3.7.1 Justification of using S. saman water extract……………………… 60 3.7.2 Bacteria preparation……………………………………………….. 60 3.7.3 Water extracts preparation………………………………………… 60 3.7.4 Sample preparation for SEM……………………………………… 61 3.8 Phytochemical test of water plants extract of S. saman………………….. 62 3.8.1 Alkaloids test…………………………………………………….... 62 3.8.2 Saponins test……………………………………………………..... 62 3.8.3 Flavonoids test…………………………………………………….. 62 3.8.4 Tannins and Polyphenolic test……………………………………. 63 3.8.5 Triterpenes or Steroids test………………………………………... 63 3.9 Gas chromatography and Mass-spectrum (GC-MS) of S. saman……….. 63 3.9.1 Samanae saman extract preparation………………………………. 63 3.9.2 Gas chromatography and Mass-spectrum for S. saman…………….. 63 3.10 Pathogenicity test…………………………………………………….. 64 3.10.1 Justification of using gram positive bacteria…………………… 64 3.10.2 Bacteria suspension preparation for injection………………….. 64 3.10.3 Snakehead fish………………………………………………….. 65 3.10.3. (a) Collection and storage experimental animal……………… 65 3.10.3. (b) Challenging fish pathogens……………………………….. 65 3.11 Therapeutic investigation of S. saman extracts in-vivo……………….. 66 3.11.1 Preparation of pathogenic bacteria and injection………………… 66 3.11.2 Collection and storage of experimental animals…………………. 67 3.11.3 Treatment protocol………………………………………………. 67 3.11.4 Preparation of S. saman water extract…………………………… 67 vii 3.11.5 Bath treatment……………………………………………………. 67 3.11.6 Exposure design experimental…………………………………… 68 3.12 Molecular identification and confirmation of S. xylosus……………… 68 3.12.1 Bacteria preparation……………………………………………… 68 3.12.2 DNA extraction from bacteria…………………………………… 68 3.12.3 Polymerase Chain Reaction (PCR)………………………………. 69 3.12.4 Agarose gel electrophoresis……………………………………… 71 3.12.4. (a) Agarose gel preparation…………………………………. 71 3.12.4. (b) Agarose gel running…………………………………….. 71 3.12.4(c) Agarose gel visualizing…………………………………….. 71 3.12.5 Purification of DNA……………………………………………... 71 3.12.6 Sequencing and Analysis………………………………………… 72 3.13 Confirmation of S. xylosus using API STAPH test kit……………… 72 3.13.1 Bacteria suspension preparation ……………………………… 72 3.13.2 API STAPH method…………………………………………….. 73 3.13.3 API STAPH analysis……………………………………………. 73 CHAPTER FOUR: RESULTS 4.1 Antimicrobial activity test (Well Diffusion Method)…………………... 75 4.2 Minimum Inhibitory Concentration (MIC)…………………………….. 78 4.3 Brine Shrimp Toxicity Test…………………………………………….