View metadata, citation and similar papers at core.ac.uk brought to you by CORE provided by Repository@USM COMPARATIVE ANALYSIS OF IN VITRO BIOACTIVITIES AND PHENOLIC CONTENT OF LEAF EXTRACTS FROM SIX SPECIES OF Aquilaria AZLINDA BINTI IBRAHIM UNIVERSITI SAINS MALAYSIA 2016 COMPARATIVE ANALYSIS OF IN VITRO BIOACTIVITIES AND PHENOLIC CONTENT OF LEAF EXTRACTS FROM SIX SPECIES OF Aquilaria by AZLINDA BINTI IBRAHIM Thesis submitted in fulfillment of the requirements for the degree of Master of Science (Biotechnology) September 2016 ACKNOWLEDGEMENT First of all, I would like to express my heartfelt appreciation to my supervisor, Professor Dr. Shaida Fariza Sulaiman for trusting me and giving me the great opportunity to learn under her supervision. I deeply appreciate her kindness, invaluable advices, unceasing support, guidance and knowledge, which inspired me throughout my study. Without her support, I would never have succeeded in achieving this milestone. Also, I would like to offer my gratitude to my co- supervisor, Associate Professor Dr. Hideyuki Nagao for his precious support and guidance. Special thanks to my labmates in phytochemical laboratories, Dr. Ooi Kheng Leong, Ms. Nurul Shafiqah Hashim and Mr. Joshua Jeyentheren Anantham, who had supported, encouraged and assisted me tremendously during my experimental work. I am overwhelmed with their kindness and guidance. Next, I would like to express my gratitude to Professor Baharuddin Salleh and Mrs. Siti Nurdijati for providing the plant materials in this study. I profusely thank all the staff of School of Biological Sciences, Universiti Sains Malaysia especially Mr. Kamarudin and Mrs. Shafawati for their kind help and co-operation throughout my research project. I am also highly indebted to Ministry of Higher Education (MyBrain15) for the funding of my study. ii I would also like to acknowledge my greatest appreciation and thanks to my beloved parents, Ayahanda Haji Ibrahim Salleh and Bonda Hajjah Zaleha Daiman for their trust, love, prayer, patience and moral support. I dedicate my warm thanks to my dearest sister, Ms. Azreen Ibrahim, who is always there for me through thick and thin in making this study a success, and also not forgetting my beloved friends, Ms. Nur Amalina Kamarudin, Mr. Nazirul Mubin Mohamed, Ms. Khairun Dalila Salihin, Ms. Nurul Hamizah Hamidon, Ms. Nurul Ashiqin Zawawi, and Ms. Nor Farah Anis Kamaruzaman who always motivated me during this project. Most of all, thank you to Almighty Allah s.w.t, for giving me His endless love, blessing, strength and knowledge to complete this study. Thank you. Azlinda Ibrahim iii TABLE OF CONTENTS ACKNOWLEDGEMENT ii TABLE OF CONTENTS iv LIST OF TABLES viii LIST OF FIGURES ix LIST OF SYMBOLS AND ABBREVIATIONS xi ABSTRAK xiii ABSTRACT xv CHAPTER 1: INTRODUCTION 1 1.1 Background of study 1 1.2 Problem statements 4 1.3 Objectives of the study 5 CHAPTER 2: LITERATURE REVIEW 6 2.1 Genus Aquilaria 6 2.1.1 Morphological description of Aquilaria used in this study 7 2.1.2 Uses of Aquilaria leaves 14 2.2 Bioactivities of Aquilaria leaves 15 2.2.1 Antioxidant activity 16 2.2.2 Anti-diabetic activity 17 2.2.3 Anti-inflammatory activity 19 2.2.4 Antibacterial activity 21 iv 2.2.4(a) Antibacterial activity of herbal teas from Aquilaria 23 species 2.2.4(b) Gastroenteritis bacteria 25 2.3 Phytochemicals and phenolic compounds in leaves of Aquilaria 32 CHAPTER 3: MATERIALS AND METHODS 38 3.1 Plant materials 38 3.2 Preparation of samples 39 3.3 Extraction 39 3.3.1 Extraction of dried samples into crude extracts 39 3.3.2 Hydrolysis of the crude extracts 40 3.4 Antioxidant tests 41 3.4.1 Preparation of extracts 41 3.4.2 Preparation of DPPH (2,2-diphenyl-1-picrylhydrazyl) solution 42 3.4.2(a) DPPH radical scavenging assay 43 3.4.3 Preparation of ferric reducing antioxidant power (FRAP) reagent 44 3.4.3(a) FRAP assay 44 3.4.4 Preparation of metal chelating solution 45 3.4.4(a) Metal chelating test 45 3.5 Determination of total phenolic content 46 3.5.1 Preparation of extracts and standard 46 3.5.2 Preparation of Folin-Ciocalteu’s (FC) reagent 47 3.5.2(a) Determination of total phenolic content 47 3.6 Enzyme inhibition assays 48 3.6.1 Preparation of extracts 48 v 3.6.2 Determination of α-glucosidase inhibitory activity 49 3.6.3 Determination of α-amylase inhibitory activity 50 3.6.4 Determination of COX-2 inhibitory activity 51 3.7 Antibacterial activity 52 3.7.1 Bacterial strains 52 3.7.1(a) Preparation and maintenance of stock cultures 53 3.7.2 Culture media 53 3.7.2(a) Nutrient agar (NA) preparation 53 3.7.2(b) Nutrient broth (NB) preparation 54 3.7.2(c) Saline solution preparation 54 3.7.3 Antibacterial test 55 3.7.3(a) Preparation of extracts 55 3.7.3(b) Preparation of inoculums 55 3.7.3(c) Determination of the minimum inhibitory 56 concentration (MIC) 3.8 Ultra Performance Liquid Chromatography (UPLC) 57 CHAPTER 4: RESULTS 61 4.1 Selection of solvent for extraction based on the antioxidant 61 activities 4.1.1 DPPH radical scavenging activity of Aquilaria leaf extracts 61 using five polar solvents 4.1.2 FRAP activity of Aquilaria leaf extracts using five solvents 64 4.1.3 Comparison between the antioxidant activities derived from 67 the two assays 4.2 Yield of Aquilaria leaf extracts 68 4.3 Antioxidant properties of Aquilaria leaf extracts 69 vi 4.3.1 DPPH radical scavenging activity 69 4.3.2 FRAP activity 72 4.3.3 Metal chelating activity 75 4.4 Total phenolic contents of Aquilaria leaf extracts 77 4.5 Enzyme inhibition assays 81 4.5.1 α-Glucosidase inhibitory potentials of Aquilaria leaf extracts 81 4.5.2 α-Amylase inhibitory potentials of Aquilaria leaf extracts 83 4.5.3 Anti-inflammatory properties of Aquilaria leaf extracts 85 4.6 Antibacterial potentials of Aquilaria leaf extracts 87 4.7 UPLC analysis 91 CHAPTER 5: DISCUSSION 113 CHAPTER 6: CONCLUSION 131 REFERENCES 133 vii LIST OF TABLES Page Table 1.1 Aquilaria conservation status in Peninsular Malaysia and 3 the IUCN Red List of Threatened Species. Table 2.1 Morphological similarities and differences of Aquilaria 9 species. Table 2.2 Lists of the gastroenteritis bacteria with their diseases 26 caused Table 2.3 Food poisoning caused by Bacillus cereus. 27 Table 2.4 Gastroenteritis caused by Escherichia coli. 30 Table 2.5 Phenolic compounds and other compounds and their 34 contents in leaves of Aquilaria species. Table 3.1 Leaf samples of Aquilaria 38 Table 3.2(a) UPLC gradient mode. 59 Table 3.2(b) UPLC gradient mode. 59 Table 4.1 Percentage yields of methanolic and hydrolyzed extracts of 68 Aquilaria species. Table 4.2 Total phenolic contents of Aquilaria extracts. 78 Table 4.3 α-Glucosidase inhibitory potentials of Aquilaria extracts. 82 Table 4.4 α-Amylase inhibitory potentials of Aquilaria extracts. 84 Table 4.5 Minimum inhibitory concentrations of methanolic and 89 hydrolysed leaf extracts of six species of Aquilaria against selected gastroenteritis pathogens. Table 4.6 Contents of phenolic constituents in extracts of six 109 Aquilaria species. viii LIST OF FIGURES Page Figure 4.1 Graph of free radical scavenging percentage versus five 62 extracts of each Aquilaria. Figure 4.2 Graph of ferric reducing potentials (FRAP) percentage 65 versus extracts of each Aquilaria. Figure 4.3 Graph of DPPH radical scavenging percentage versus 70 methanolic and hydrolyzed leaf extracts of six Aquilaria species. Figure 4.4 Graph of ferric reducing potentials (FRAP) percentage 73 versus methanolic and hydrolyzed leaf extracts of six Aquilaria species. Figure 4.5 Graph of MC assay percentage versus methanolic and 76 hydrolyzed leaf extracts of six Aquilaria species. Figure 4.6 Linear calibration curve of gallic acid for determination of 79 total phenolic content. Figure 4.7 Cyclooxygenase inhibiting potentials percentage versus 86 methanolic and hydrolyzed leaf extracts of six Aquilaria species. Figure 4.8 UPLC chromatogram and UV spectra of (a) methanolic 92 extract of Aquilaria hirta at 290 nm (b) methanolic extract of Aquilaria hirta at 330 nm. Figure 4.9 UPLC chromatogram and UV spectra of (a) hydrolyzed 93 extract of Aquilaria hirta at 290 nm (b) hydrolyzed extract of Aquilaria hirta at 330 nm. Figure 4.10 UPLC chromatogram and UV spectra of (a) methanolic 95 extract of Aquilaria beccariana at 290 nm (b) methanolic extract of Aquilaria beccariana at 330 nm. ix Figure 4.11 UPLC chromatogram and UV spectra of (a) hydrolyzed 96 extract of Aquilaria beccariana at 330 nm. Figure 4.12 UPLC chromatogram and UV spectra of (a) methanolic 98 extract of Aquilaria malaccensis at 290 nm (b) methanolic extract of Aquilaria malaccensis at 330 nm. Figure 4.13 UPLC chromatogram and UV spectra of (a) hydrolyzed 99 extract of Aquilaria malaccensis at 290 nm (b) hydrolyzed extract of Aquilaria malaccensis at 330 nm. Figure 4.14 UPLC chromatogram and UV spectra of (a) methanolic 101 extract of Aquilaria rostrata at 290 nm (b) methanolic extract of Aquilaria rostrata at 330 nm. Figure 4.15 UPLC chromatogram and UV spectra of (a) hydrolyzed 102 extract of Aquilaria rostrata at 290 nm (b) hydrolyzed extract of Aquilaria rostrata at 330 nm. Figure 4.16 UPLC chromatogram and UV spectra of (a) methanolic 104 extract of Aquilaria sinensis at 290 nm (b) methanolic extract of Aquilaria sinensis at 330 nm. Figure 4.17 UPLC chromatogram and UV spectra of (a) hydrolyzed 105 extract of Aquilaria sinensis at 290 nm (b) hydrolyzed extract of Aquilaria sinensis at 330 nm.