Torch Ginger) Inflorescence
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CHEMICAL COMPOSITION AND BIOLOGICAL PROPERTIES OF Etlingera elatior Jack. (TORCH GINGER) INFLORESCENCE By MARASINGHE MUDIYANSELAGE JEEVANI OSADEE WIJEKOON Thesis submitted in fulfillment of the requirements for the degree of Master of Science July 2011 ACKNOWLEDGEMENT First and for most, I offer my sincere gratitude to my main supervisor Prof. A. A. Karim for his invaluable guidance and inspiration which was the great power for me to complete my research. Also I am very much grateful to my co-supervisor Dr. Rajeev Bhat for the precious guidance and encouragement that he provided throughout this study. Appreciation is also extended to all the academic staff members in School of Industrial Technology for providing all resources necessary to conduct this research. Thanks are extended to all my friends and lab assistants from School of Industrial Technology who helped me in completion of this research. Greatest thanks to the Institute of Post Graduate Studies, University Sains Malaysia for their financial assistance provided under USM fellowship during my studies in Malaysia. I extend my deepest appreciation to my loving parents for encouraging and guiding me to study throughout my life. I am indebted to my husband, Champika for his love, care and support all the way, without him this work will not be completed. Studying as a foreign student in Malaysia gave me a lot of unforgettable experiences during my studies. Feeling sometimes depressed and sometimes happy….. It was the most remarkable time I spent, and now, after two years, I am harvesting the fruits of my effort. Lastly, I am very much grateful to all of those who supported me in any respect during the completion of this research. M. M. Jeevani Osadee Wijekoon July 2011 ii TABLE OF CONTENTS Page ACKNOWLEDGEMENT ii TABLE OF CONTENTS iii LIST OF TABLES ix LIST OF FIGURES xi LIST OF ABBREVIATIONS/SYMBOLS xii ABSTRAK xiv ABSTRACT xvi CHAPTER 1 - INTRODUCTION 1.1 Background……………………………………………………………….. 1 1.2 Rationale of study………………………………………………………… 2 1.3 Objectives ………………………………………………………………… 3 1.3.1 Main objective…………………………………………………… 5 1.3.2 Specific objectives……………………………………………….. 5 iii CHAPTER 2 - LITERATURE REVIEW 2.1 Overview of the recent research on medicinal plants………………….... 6 2.2 Functional foods and nutraceuticals……………………………………... 8 2.3 Zingiberaceae family…………………………………………………….. 9 2.3.1 Etlingera elatior…………………………………………………. 19 2.3.1.1 Common notes and morphology of the plant Etlingera elatior……………………………………...................... 19 2.3.1.2 Taxonomic classification of Etlingera elatior (Jack.) R.M. Smith…………………………………………….. 20 2.3.1.3 Overview on nutraceutical value of Etlingera elatior…. 21 2.4 Plant phenolic compounds………………………………………………. 24 2.5 Flavonoids……………………………………………………………….. 27 2.6 Tannins…………………………………………………………………... 30 2.7 Anthocyanins……………………………………………………………. 31 2.8 Plant essential oils……………………………………………………….. 33 2.9 Nutritional quality of foods……………………………………………… 39 2.9.1 Proximate composition of foods………………………………… 39 2.9.2 Amino acid profile………………………………………………. 39 2.9.3 Fatty acid profile………………………………………………… 43 iv 2.9.4 Minerals in nutrition……………………………………………... 44 2.10 Anti nutrients…………………………………………………………….. 44 2.10.1 Saponins………………………………………………………... 45 2.10.2 Phytic acids…………………………………………………...... 46 CHAPTER 3 - MATERIALS AND METHODS 3.1 Chemicals and reagents…………………………………………………… 48 3.2 Sample collection and preparation………………………………………... 49 3.3 Assessment of antioxidant activity of E. elatior………………………….. 50 3.3.1 Preparation of extracts…………………………………………. 50 3.3.2 Determination of total phenols………………………………… 51 3.3.4 Determination of total flavonoid content…………………….... 51 3.3.5 Determination of anthocyanins……………………………....... 52 3.3.6 Determination of condensed tannins ………………………….. 52 3.3.7 Determination of antioxidant activity using DPPH radical- scavenging activity…………………………………………….. 53 3.3.8 Ferric reducing/ antioxidant power assay (FRAP assay)……… 53 3.4 Analysis of essential oils…………………………………………………. 54 v 3.4.1 Hydrodistillation of essential oil………………………………. 54 3.4.2 Relative quantification of volatile compounds by GC-MS……. 56 3.5 Antimicrobial activity…………………………………………………....... 56 3.5.1 Microorganisms and growth conditions………………………….. 56 3.5.2 Media preparation and growth conditions for bacterial stock cultures……………………………………………………………. 56 3.5.3 Media preparation and growth conditions for fungi stock cultures……………………………………………………………. 57 3.5.4 Activation of cultures and preparation of bacterial and fungal suspensions……………………………………………………...... 57 3.5.5 Antimicrobial susceptibility testing (Agar disc diffusion method). 58 3.5.5.1 Preparation of agar plates for disc diffusion assay……... 58 3.5.5.2 Extract preparation……………………………………… 58 3.5.5.3 Preparation of culture inoculated plates for disc diffusion assay………………………………………….. 58 3.5.5.4 Disc diffusion assay……………………………….......... 59 3.6 Determination of minimum inhibitory concentration (MIC)……………... 60 3.7 Nutritional quality analysis of the inflorescence………………………….. 61 3.7.1 Proximate composition analysis………………………………....... 61 vi 3.7.2 Fatty acid analysis………………………………………………… 61 3.7.3 Amino acid analysis………………………………………………. 62 3.7.4 Determination of mineral and heavy metals content by Inductively Coupled Plasma Optical Emission Spectrophotometer 63 (ICP-OES)………………………………………………………… 3.8 Analysis of antinutritional compounds…………………………… 64 3.8.1 Determination of saponin ……………………………... 64 3.8.2 Determination of phytic acid ………………………....... 65 3.9 Statistical analysis………………………………………………………… 65 CHAPTER 4 - RESULTS AND DISCUSSION 4.1 Assessment antioxidant activity of the E. elatior inflorescence………...... 66 4.1.1 Selection of plant material and extraction conditions……………. 66 4.1.2 Total phenols……………………………………………………... 67 4.1.3 Total flavonoid content…………………………………………... 68 4.1.4 Anthocyanins…………………………………………….............. 70 4.1.5 Tannins…………………………………………………………… 70 4.1.6 Antioxidant activity (percent inhibition of DPPH and FRAP assay)……………………………………………………………... 71 vii 4.2 Chemical composition of essential oil……………………………………. 72 4.3 Antimicrobial activity E. elatior inflorescence ………………………... 87 4.4 Assessment of nutritional quality and antinutrients of E. elatior………… 92 4.4.1 Proximate composition of E. elatior inflorescence……………… 92 4.4.2 Analysis of individual profiles of macronutrients……………… 93 4.4.2.1 Fatty acid composition………………………………… 93 4.4.2.2 Amino acid profile of proteins in E. elatior inflorescence…………………………………………... 96 4.4.2.3 Mineral composition of the E. elatior inflorescence…... 97 4.4.3 Analysis of antinutrients and heavy metals……………………..... 100 CHAPTER 5 - CONCLUSION 102 CHAPTER 6 - RECOMMENDATIONS 104 REFERENCES 106 APPENDICES 123 LIST OF PUBLICATIONS 132 viii LIST OF TABLES Page Table 2.1 Drugs discovered from medicinal plants 8 Table 2.2 Zingiberaceae species classification in different genera 11 Table 2.3 Chemical compounds and their bioactivity of different 17 Zingiberaceae plants Table 2.4 Main classes of phenolic compounds 26 Table 2.5 Different classes of flavonoids, their substitution patterns and 28 dietary sources Table 2.6 Bioactivity of some plant essential oils 37 Table 2.7 Structure, discovery, occurrence and importance of amino 41 acids Table 3.1 Combination of the solvents used for extraction 50 Table 3.2 Operating conditions used in ICP-OES 63 Table 4.1 Extractability of bioactive antioxidative compounds in 69 different solvent systems Table 4. 2 DPPH and FRAP values of E. elatior inflorescence extracted 72 with various solvents Table 4.3 Chemical composition of essential oils of E. elatior 74 inflorescence ix Table 4.4 Potential application of major chemical compounds of the 78 essential oil of E. elatior Table 4.5 Potential application of minor chemical compounds of the 81 essential oil of E. elatior Table 4.6 Zones of inhibition representing the antimicrobial activity of 88 essential oils, ethanolic and water extracts of E. elatior inflorescence Table 4.7 Minimum inhibitory concentration (MIC) levels of the 90 essential oil and the extracts against Gram-positive bacteria and Gram- negative bacteria Table 4.8 Proximate composition and energy value of E. elatior 93 inflorescence Table 4.9 Fatty acid profiles of the oil of E. elatior inflorescence 95 Table 4.10 Amino acid profile of proteins in E. elatior inflorescence 97 Table 4.11 Mineral composition of E. elatior inflorescence 99 Table 4.12 Antinutrients and heavy metal contents in E. elatior 101 inflorescence x LIST OF FIGURES Page Figure 1.1 Conceptual framework of the research 4 Figure 2.1 Natural habitat of Etlingera elatior plant in Penang 20 Figure 2.2 Antioxidative compounds isolated from E. elatior 23 Figure 2.3 Structure of Chalcones (a) and Aurones (b) 27 Figure 2.4 Nuclear structure of flavonoid 27 Figure 2.5 Chemical structures of condensed tannins (a) and hydrolyzable 30 tannins (b) Figure 2.6 Structure of major anthocyanin-3-O-glucoside present in fruit and 32 respective wavelength at the maximum of absorption in the visible region Figure 2.7 Examples for monoterpenes (a. menthol, b. limonene) and 35 sesquiterpenoids (c. β-caryophyllene, d. artemisinin) structures Figure 2.8 Basic structures of saponin (a) triterpenoid and (b) steroid 45 Figure 2.9 Structure of phytic acid 46 Figure 3.1 Selected inflorescences of Etlingera elatior 49 Figure 3.2 Schematic diagrams of Likens-Nickerson apparatus for 55 distillation of essential oil Figure 4.2 Chromatogram for the analysis of essential oils by GC-MS. 73 xi LIST OF ABBREVIATIONS / SYMBOLS