BIOLOGICAL ACTIVITIVIES OF ACANTHOPHORA SPICIFERA (VAHL) BORGESEN FROM PULAU GEDUNG, PENANG by NURUL AILI BINTI ZAKARIA Thesis submitted in fulfillment of the requirements for the degree of Master of Science June 2011 ACKNOWLEDGEMENTS (In the name of Allah, The Most Merciful and The most Gracious) Upon completion of this research, I would like to express my gratitude to many parties. My heartfelt thanks go to my supervisor, Prof Darah Ibrahim and my co-supervisor, Assoc. Prof. Dr. Shaida Fariza Sulaiman for their unending guidance, supports and encouragements. Without their precious critics and scientific advises, I may not be able to complete this thesis on time. I would also like to address my thankful to the University Sains Malaysia for giving me the USM fellowship as financial support throughout my study. I am also grateful to En. Hamid from School of Chemistry, En. Razak and En. Hilman from Drug Center and all staffs of Electron Microscopy (EM) Unit, USM for their great guidance and assistances. Not to forget all the lab members of Industrial Biotechnology Research Laboratory (IBRL) and Phytochemistry Laboratory, USM for their endless support in any respect during the completion of this research. Lastly, I am heartily thankful to Him for giving me such a wonderful family. I am indebted to my lovely parents, Ayahanda Zakaria Endut, Ibunda Nooriah Idris and my siblings for their loves, prayers and trust. Thank you for always being there to support me through hard and joyous times. Nurul Aili Binti Zakaria ii TABLE OF CONTENTS ACKNOWLEDGEMENTS ii TABLE OF CONTENTS iii LIST OF TABLES viii LIST OF FIGURES x LIST OF ABBREVIATIONS xii ABSTRAK xiii ABSTRACT xv CHAPTER 1 INTRODUCTION 1 1.1 Acanthophora spicifera (Vahl) Borgesen 1 1.1.1 Morphological description 1 1.1.2 Utilization and therapeutic values 2 1.1.3 Phytochemical constituents and bioactivity 3 1.2 The needs of drugs from natural products 4 1.3 Potential drugs from natural products 6 1.3.1 Seaweeds as source of drugs 7 1.4 Problem statement 9 1.5 Objectives of study 10 CHAPTER 2 LITERATURE REVIEW 11 2.1 Fading miracle: The needs for new antibiotics 11 2.1.1 Declining of antibiotics in pipeline 11 2.1.2 Antibiotic resistance 12 2.1.3 The emergence of diseases and infections 13 2.2 History of antibiotic discovery 15 2.3 Types of antibiotics and mechanisms of action 16 2.3.1 Antibacterial drugs 17 2.4 Antioxidant activity 22 2.4.1 Oxidative damage and diseases 22 2.4.2 Natural antioxidant from algae 25 iii 2.5 Bioactive compounds from nature 27 2.5.1 Bioassay-guided technique for natural products 27 2.5.2 Secondary metabolites from algae 28 2.6 Previous studies on A. spicifera 37 CHAPTER 3 MATERIALS AND METHODS 42 3.1 Algal material 42 3.2 Preparation of algal material 43 3.3 Proximate chemical composition analysis 43 3.4 Extraction procedures 47 3.4.1 Soxhlet extraction 48 3.4.2 Maceration 49 3.5 Antioxidant study 51 3.5.1 DPPH free radical scavenging activity 51 3.5.1.1 Preparation of DPPH solution 51 3.5.1.2 Preparation of sample 51 3.5.1.3 DPPH free radical scavenging activity assay 51 3.5.2 Follin- Ciocalteau phenol reagent assay 53 3.5.3 Correlation between total phenolic contents and free radical 54 scavenging activity 3.6 Antimicrobial study 54 3.6.1 Test microorganism and culture condition 54 3.6.2 Preservation of stock cultures using agar slants 55 3.6.3 Test for antimicrobial activity 57 3.6.3.1 Preparation of microbial suspension 57 3.6.3.1.1 Bacterial suspension 57 3.6.3.1.2 Yeast suspension 57 3.6.3.1.3 Fungal suspension 58 3.6.3.2 Preparation of microbial seeded agar plates 58 3.6.3.3 Preparation of sample extracts 59 3.6.3.4 Preparation of disc extracts 59 3.6.3.5 Disc diffusion test 59 3.6.4 Minimum Inhibitory Concentration (MIC) assay 60 iv 3.6.5 Minimum Bactericidal Concentration (MBC) assay 63 3.6.6 Determination of mechanisms of antibiosis (bacteriostatic or 63 bactericidal) 3.6.7 Effect of crude methanol extract on the morphology of 64 bacterial cells 3.6.7.1 Determination of Time-kill growth curve 64 3.6.7.2 Structural degeneration and morphological changes of 65 bacterial cell 3.6.7.2.1 Scanning Electron Microscopic (SEM) study 67 3.6.7.2.2 Transmission Electron Microscopic (TEM) study 68 3.6.8 Fractionation of crude methanol extract 68 3.6.8.1 Thin-Layer Chromatography (TLC) 68 3.6.8.1.1 Detection of monoterpenoids 70 3.6.8.1.2 Detection of diterpenoids 70 3.6.8.1.3 Detection of triterpenoids 71 3.6.8.2 Antibacterial activity of crude methanol extract on TLC 71 plate 3.6.8.3 Column Chromatography (CC) technique 72 3.6.8.3.1 Column packing 72 3.6.8.3.2 Column development 73 3.6.8.4 Purification of fractions 74 3.6.8.5 Identification of active compounds 74 3.6.8.5.1 LC-MS analysis 74 3.6.8.5.2 GC-MS analysis 75 3.7 In-vitro Toxicity study 76 3.7.1 Preparation of growth medium 76 3.7.2 Preparation of extract 76 3.7.3 Hatching of brine shrimp (Artemia salina) 77 3.7.4 Brine Shrimp Lethality Test (BSLT) 78 3.7.5 Determination of 50 % Lethality concentration (LC50) 79 3.8 Statistical Analysis 79 v CHAPTER 4 RESULTS 81 4.1 Proximate Chemical Composition Analysis 81 4.2 Yield of extracts 82 4.3 Antioxidant activity 83 4.3.1 Total phenolic content of various A. spicifera extracts 83 4.3.2 DPPH Free radical scavenging activity of various A. 85 spicifera extracts 4.3.3 Correlation between total phenolic content and antioxidant 90 Activity 4.4 Antimicrobial activity of crude extract and fraction extracts of 92 A. spicifera 4.4.1 Determination of Minimum Inhibitory Concentration (MIC) 98 and Minimum Bactericidal Concentration (MBC) 4.5 Effect of crude methanol extract on the morphology of bacterial 105 cells 4.5.1 Time-kill study of bacterial cells treated with A. spicifera 105 extract 4.5.2 Structural and morphological study on P. aeruginosa ATCC 108 27853 treated with crude methanol extract of A. spicifera 4.5.2.1 Scanning Electron Microscopic (SEM) studies 108 4.5.2.2 Transmission Electron Microscopic (TEM) studies 111 4.6 Bioassay-guided fractionation of crude methanol extract of A. 113 spicifera 4.6.1 Fractionation of crude methanol extract 113 4.6.1.1 Thin-Layer Chromatography (TLC) 113 4.6.1.2 Antibacterial activity of TLC chromatograms of 119 crude methanol extract 4.6.2 Column chromatography technique 121 4.6.3 Minimum Inhibitory Concentration (MIC) of fractions of 124 crude methanol extract 4.6.4 Re-fractionation of active fractions 126 4.6.5 Identification of active compounds 127 4.6.5.1 LC-MS analysis 127 vi 4.6.5.2 GC-MS analysis 130 4.7 In-vitro toxicity test 136 CHAPTER 5 DISCUSSION 140 5.1 Proximate Chemical Composition Analysis of A. spicifera 140 5.2 Extraction and Yield of extracts 143 5.3 Antioxidant activity 148 5.3.1 Total phenolic content of various A. spicifera extracts 148 5.3.2 DPPH radical scavenging activity of A. spicifera extracts 151 5.3.3 Correlation between Total phenolic content and Antioxidant 156 activity of A. spicifera 5.4 Antimicrobial activity of A. spicifera extracts 159 5.4.1 Disc susceptibility test of A. spicifera extracts 159 5.4.2 Minimum Inhibitory Concentration (MIC) and Minimum 165 Bactericidal Concentration (MBC) 5.5 Morphological and structural effect of P. aeruginosa ATCC 27853 168 cells treated with A. spicifera crude methanol extract 5.5.1 Time-Kill Curve study 168 5.5.2 Scanning Electron Microscope (SEM) and Transmission 170 Electron Microscope (TEM) study 5.6 Bioassay-guided fractionation of crude methanol extract of 173 A. spicifera 5.7 GC-MS analysis of the active compounds 176 5.8 In-vitro toxicity of crude methanol extract of A. spicifera 178 CHAPTER 6 CONCLUSION AND RECOMMENDATION FOR 181 FUTURE RESEARCH REFERENCES 184 APPENDICES 210 PUBLICATION FROM THIS RESEARCH 216 vii LIST OF TABLES Page Table 1.1 Taxonomy of A. spicifera 2 Table 1.2 Phytochemical constituents and bioactivity of A. spicifera 4 Table 1.3 Drugs derived from algae 8 Table 2.1 Discovery of antibiotics 15 Table 2.2 Summary of antibiotics type, action and classes 18 Table 2.3 Important physiological functions that involve free radicals or 24 their derivatives Table 2.4 Antioxidant evaluation of A. spicifera 27 Table 2.5 Secondary metabolites from algae 31 Table 2.6 Secondary metabolites of red algae from family, 34 Rhodomelaceae Table 2.7 Previous studies on A. spicifera 37 Table 3.1 List of microorganisms used in the present study 56 Table 3.2 Summary of serial dilutions for MIC determination of crude 61 extract Table 3.3 Medium and extract preparation for MIC test for fraction 62 using micro-well broth dilution test Table 3.4 Parameter for GC-MS and oven for GC-MS analysis 76 Table 3.5 Summary of preparation of extract for toxicity test 77 Table 4.1 Total yield of extracts of A. spicifera from Soxhlet extraction 82 Table 4.2 Total yield of extracts of A. spicifera from maceration and 82 solvent partition extraction Table 4.3 Total phenolic content of various A.