QUALITY CONTROL ANALYSIS, STANDARDIZATION AND STABILITY STUDIES OF EURYCOMA LONGIFOLIA AND EFFECT OF ITS SAPONIN CONTENT ON SOLUBILITY AND TOXICITY OF EURYCOMANONE NURSYAZURA BINTI KHARI UNIVERSITI SAINS MALAYSIA 2014 QUALITY CONTROL ANALYSIS, STANDARDIZATION AND STABILITY STUDIES OF EURYCOMA LONGIFOLIA AND EFFECT OF ITS SAPONIN CONTENT ON SOLUBILITY AND TOXICITY OF EURYCOMANONE By NURSYAZURA BINTI KHARI Thesis submitted in fulfilment of the requirements for the degree of Master of Science AUGUST 2014 1 ACKNOWLEDGEMENTS Alhamdulillah, all praises to Allah the almighty who gave me the knowledge, strength and the determination to complete this study and finish my thesis successfully. I would like to express my sincere gratitude to my supervisor, Prof. Zhari Ismail for his support, guidance, encouragement and supervision throughout my research. This study would not have been completed without his help. I would like to convey my sincere words of appreciation to my colleague Dr. Abdalrahim F.A. Aisha for his invaluable assistance, support and guidance which made the work possible. Special thanks to Dr. Amin Malik Shah Bin Abdul Majid, for allowed me to work in EMAN lab and to all the students at the EMAN lab for their support and assistance, particularly Miss. Suzana Hashim helped in MTT assay. My sincere thanks to both Ms. Nor Dyana Binti Zakaria from INFORMM, USM and Mr. Masrul Mansor from School of Biological Sciences, USM whom has helped me in performing particle size and Transmission Electron Microscope (TEM) analysis. I would like to thank Universiti Sains Malaysia for giving me the opportunity and providing me with all the necessary facilities that made my study possible and to the Malaysian Ministry of Higher Education for awarding me the Budget Mini scholarship for two years, which has supported me to pay the fees. Special thanks to my colleagues and my entire friend at Pharmaceutical Chemistry research laboratory especially Dr. Beh Hooi Kheng, Nurul Najwa, Noor Hafizoh, Gheniya, Munira, Shahrul and Amirul for their encouragement and support. Last but not least, I wish to express my deepest love and gratitude to my parents, Khari Bin Yaakob and Sabehah Binti Shuib, my sister Nurfarina Binti Khari and beloved husband Mohd Baijuri Bin Azmi for their support, understanding and endless love, through the duration of my studies. ii 2 TABLE OF CONTENTS 1 ACKNOWLEDGEMENTS .................................................................................. ii 2 TABLE OF CONTENTS ..................................................................................... iii 3 LIST OF TABLES..............................................................................................viii 4 LIST OF FIGURES...............................................................................................x 5 LIST OF ABBREVIATION...............................................................................xvi 6 ABSTRAK...........................................................................................................xix 7 ABSTRACT ........................................................................................................ xix 1 CHAPTER ‎1 INTRODUCTION ......................................................................... 1 1.1 Significance of Medicinal Plants ................................................................. 1 1.2 Efficacy, Safety and Quality Control in the Standardization of Herbal Medicine ................................................................................................................... 2 1.3 Problem Statements and Justification of the Research ................................ 5 1.4 Objectives of the Study .............................................................................. .6 2 CHAPTER 2‎ LITERATURE REVIEW .......................................................... .7 2.1 Eurycoma longifolia Jack ........................................................................... .7 2.1.1 Taxonomy ....................................................................................... .7 2.1.2 Vernacular Names .......................................................................... .7 2.1.3 Plant Description ............................................................................ 8 2.1.4 Review of Chemical Constituents of Eurycoma longifolia ........... 10 2.1.5 Review of the Pharmacological Activities of Eurycoma longifolia....................................................................................................14 2.2 Stability Studies ......................................................................................... 18 2.2.1 Physical Factors Effecting Stability ............................................... 19 2.2.1.1 Temperature ..................................................................... 19 2.2.1.2 Moisture ........................................................................... 19 2.2.1.3 Light................................................................................. 19 2.2.2 Chemicals Factors Effecting Stability ........................................... 20 iii 2.2.2.1 Hydrolysis ........................................................................ 20 2.2.2.2 Oxidation ......................................................................... 20 2.2.2.3 Isomerisation and Polymerization ................................... 20 2.2.3 Accelerated Stability ...................................................................... 21 2.3 Saponins .................................................................................................... 22 2.3.1 Definition and Chemical Compositions ......................................... 22 2.3.2 A Review of the Pharmacological Activities of Saponins ............. 23 3 CHAPTER ‎3 MATERIALS AND METHODS ................................................ 28 3.1 Quality Control Analysis of Eurycoma longifolia Raw Material .............. 28 3.1.1 Preparation of Plant Materials .......................................................... 28 3.1.2 Moisture and Ash Content ................................................................ 29 3.1.3 Acid Insoluble Ash Content ............................................................. 29 3.1.4 Alcohol Soluble Extractives ............................................................. 29 3.1.5 Water Soluble Extractives ................................................................ 30 3.1.6 Determination of Heavy Metals ........................................................ 31 3.1.7 Optimization of Extraction Time and Solvent Ratio ........................ 31 3.1.7.1 Plant Material and Small Scale Extraction ........................ 31 3.1.7.2 Chemical Profiling of Extracts from Different Extraction Time and Solvent Ratio ................................................................. 32 3.2 Analytical Studies of Eurycoma longifolia Extracts and Commercial Products Using Spectroscopic and Chromatographic Techniques ......................... 33 3.2.1 Material and Methods........................................................................33 3.2.1.1 Chemicals and Solvents......................................................33 3.2.1.2 Instrumentations..................................................................33 3.2.1.3 Marker.................................................................................34 3.2.2 Preparation of Extracts from Raw Materials....................................34 3.2.3 Preparation of Commercial Products................................................35 3.2.4 Fourier Transform Infrared (FTIR) Spectroscopy and PCA ............ 36 3.2.5 High Performance Thin-Layer Chromatography (HPTLC)..............36 3.2.6 High Performance Liquid Chromatography (HPLC) ...................... 37 3.2.6.1 Preparation of Marker Compound ..................................... 37 3.2.6.2 Preparation of Samples ...................................................... 38 3.2.6.3 Optimization and Validation of HPLC Method ................. 38 iv 3.2.6.4 Quantification of Eurycomanone in Eurycoma longifolia Extracts and Commercial Products ................................................ 40 3.2.7 Analysis of Primary and Secondary Metabolites in Eurycoma longifolia Extracts and Commercial Products.................................... 40 3.2.7.1 Material and Methods ........................................................ 40 3.2.7.2 Instruments.........................................................................40 3.2.7.3 Determination of Total Phenols ......................................... 41 3.2.7.4 Determination of Glycosaponins ....................................... 42 3.2.7.5 Determination of Total Polysaccharides ............................ 42 3.2.7.6 Determination of Total Protein Using Lowry’s Method ........................................................................................................43 3.3 Stability Studies on Eurycoma longifolia Root, Stem and Leaf Water Extracts ................................................................................................................... 44 3.3.1 Stability Study Protocol .................................................................... 44 3.3.2 Quantification of Eurycomanone by HPLC ..................................... 45 3.3.3 Calculation of Chemical Kinetics ..................................................... 45 3.3.3.1 Determination of the Order of Degradation Reaction ....... 45 3.3.3.2 Determination of the Rate Constant (K) ............................ 46 3.3.3.3 Determination