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IDENTIFICATION and CHARACTERIZATION of Stevia Rebaudiana EXTRACTION for TOTAL PHENOLIC COMPOUND USING SOXHLET EXTRACTION and MICROWAVE-ASSISTED EXTRACTION TECHNIQUES

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IDENTIFICATION and CHARACTERIZATION of Stevia Rebaudiana EXTRACTION for TOTAL PHENOLIC COMPOUND USING SOXHLET EXTRACTION and MICROWAVE-ASSISTED EXTRACTION TECHNIQUES View metadata, citation and similar papers at core.ac.uk brought to you by CORE provided by UMP Institutional Repository IDENTIFICATION AND CHARACTERIZATION OF Stevia rebaudiana EXTRACTION FOR TOTAL PHENOLIC COMPOUND USING SOXHLET EXTRACTION AND MICROWAVE-ASSISTED EXTRACTION TECHNIQUES ERNA WATI IBNU HAJAR Thesis submitted in fulfilment of the requirements For the award of the degree of Master of Engineering in Chemical Faculty of Chemical and Natural Resources Engineering UNIVERSITI MALAYSIA PAHANG December 2013 ABSTRACT This study focused on identifying extraction condition (e.g. sample concentration, extraction time) of total phenolic compound (TPC) from leaves, stems and flowers of Stevia rebaudiana. Two method processes were used: 1) using Soxhlet Extraction (SE) and 2) Microwave-Assisted Extraction (MAE). Possible mechanisms of productivity enhancement by both methods were investigated. The extract were analyzed with an appropriate Gas Chromatography-Mass Spectrometry (GC-MS) and Fourier Transform Infrared Spectroscopy (FTIR). The reproducibility of extraction and of chromatographic analysis was proved. The identification of extraction in both extraction technique has been intensively studied. In comparison of these two techniques to extraction of TPC in leaves, stems and flowers of Stevia rebaudiana, MAE had a higher extraction efficiency with reduces extraction time. The yield of TPC in leaves of Stevia rebaudiana was 100.385 mg GAE/g dw and in stems was 18.702 mg GAE/g dw at concentration 0.1 g/ml. Meanwhile, the yield TPC in flowers was 64.461 mg GAE/g dw at the concentration 0.06 g/ml after 12 min running using MAE. The qualitative GC-MS analysis of the extract from leaves, stems and flowers of Stevia rebaudiana showed that total ion chromatogram (TIC) of Soxhlet and MAE shows some valuable constituents of extracts such as Octadecanoic acid, n- Hexadecanoic acid, 1-Octadecene, Taraxasterol, Tridecanoic acid, D-allose, Morpinan, Benzene rings, Stigmasterol, β-Sitosterol, γ-Sitosterol, α-Amyrin, Longifolenaldehyde, Allo-inisitol, Neo-inositol, Azulene, and Phenol. The results suggest that Stevia rebaudiana can also be consider as a potential source of essential oil for the use of cosmeceutical, medicinal and pharmacological application. FTIR analysis of raw and extracted from leaves, stems, and flowers of Stevia rebaudiana showed a great abundance of alcohols, phenols, aromatic rings, carboxylic acids, ketones, aldehydes, alkanes, ethers, ester, acetylenic as well as olefinic. Heavy metals accumulation in the Stevia rebaudiana from leaves, stems, and flowers is reported. The analysis of sample was carried out by using an Inductively Coupled Plasma Mass-Spectrophotometer (ICP-MS) to analyze the Arsenic (As), Cadmium (Cd), Chromium (Cr), Copper (Cu), Iron (Fe), Magnesium (Mg), plumbum (Pb), Selenium (Se), Zinc (Zn), aluminium (Al), silver (Ag), Cobalt (Co), calcium (Ca), manganese (Mn) and nickel (Ni). The accumulation of heavy metals in leaves, stems, and flowers of Stevia rebaudiana contain fifteen heavy metals below tolerance limit in plant and can be used as food products. Keywords: Soxhlet Extraction, Microwave-assisted extraction, Stevia rebaudiana, GC-MS, FTIR, ICP-MS, Heavy metals. iv TABLE OF CONTENTS Page SUPERVISOR’S DECLARATION i STUDENT’S DECLARATION ii ACKNOWLEDGEMENTS iii ABSTRACT iv ABSTRAK v TABLE OF CONTENTS vi LIST OF TABLES x LIST OF FIGURES xii LIST OF SYMBOLS xiv CHAPTER 1 INRTODUCTION 1.1 Background 1 1.2 Problem Statement 5 1.3 Objectives of the Research 6 1.4 Research Scope 6 1.5 Rationale and Significant of Research 6 CHAPTER 2 LITERATURE REVIEW 2.1 Introduction 8 2.2 Medicinal Plants 8 2.3 Stevia rebaudiana 10 2.4 Antioxidant 12 2.5 Phenolic Compound 13 2.6 Heavy Metals 15 2.7 Extraction Techniques 18 2.8 Soxhlet Extraction 20 vi 2.9 Microwave-assisted Extraction 22 2.10 Microwave Theory 23 2.11 Factors Effecting Microwave-assisted Extraction 25 2.11.1 Solvent Nature 26 2.11.2 Extraction Time 27 2.11.3 Microwave Power 28 2.11.4 Matrix Characterization 29 2.11.5 Temperature 30 2.12 Optimization 31 CHAPTER 3 MATERIALS AND METHODS 3.1 Introduction 33 3.2 Materials 33 3.3 Preparation of Sample 34 3.4 Soxhlet Extraction 34 3.5 Microwave-assisted Extraction 35 3.6 Gallic acid standard Calculation 36 3.6.1 Reagents 36 3.6.2 Procedure 36 3.7 Total Phenolic Compound (TPC) Analysis by Folin-Ciocaltue Method 37 3.8 FTIR analysis 38 3.8.1 Solid Sampling 38 3.8 2 Liquid Sampling 38 3.9 GC-MS Analysis 39 3.10 ICP-MS Analysis 40 3.10.1 Preparation Sample 40 3.10.2 Analysis of Heavy Metals 41 3.11 Data Collection and Analysis by Statistical 43 CHAPTER 4 RESULTS AND DISCUSSION 4.1 Introduction 44 4.2 Optimization of TPC using Soxhlet extraction and Microwave-assisted 44 vii extraction 4.3 Characterization of Stevia rebaudiana using SE and MAE analyzed by 60 GC-MS and FTIR 4.3.1 GC-MS Analysis 60 4.3.2 FTIR Analysis 71 4.4 Assessment of heavy metals using microwave digestion system analyzed 78 by ICP-MS CHAPTER 5 CONCLUSION AND RECOMMENDATIONS 5.1 Introduction 85 5.2 Conclusions 85 5.3 Recommendations for the Future Research 87 REFERENCES 88 APPENDICES A.1 Experimental Flowchart 105 A.2 TPC in Stevia rebaudiana by Folin - Ciocaltue Method 106 A.3 Output of TPC in Leaves of Stevia rebaudiana using SE by Statistics 121 A.4 Output of TPC in Stems of Stevia rebaudiana using SE by Statistics 127 A.5 Output of TPC in Flowers of Stevia rebaudiana using SE by Statistics 134 A.6 Output of TPC in Leaves of Stevia rebaudiana using MAE by Statistics 137 A.7 Output of TPC in Stems of Stevia rebaudiana using MAE by Statistics 143 A.8 Output of TPC in Flowers of Stevia rebaudiana using MAE by Statistics 150 A.9 GC-MS Profile Releated Results of Stevia rebaudiana using SE 153 A.10 GC-MS Profile Releated Results of Stevia rebaudiana using MAE 159 B.1 FTIR Spectra of Raw Material Stevia rebaudiana 167 B.2 FTIR Spectra of Stevia rebaudiana Extract using SE 168 B.3 FTIR Spectra of Stevia rebaudiana Extract using MAE 169 B.4 Principle IR Absorbance for Certain Fungtional 170 B.5 Accumulation Heavy Metals in Stevia rebaudiana Extract by ICPMS 172 Agilent 7500a viii B.6 Accumulation Heavy Metals in Stevia rebaudiana Extract by ICP-MS 176 Elan 9000 C Published 185 ix LIST OF TABLES Table No. Title Page 2.1 Chemical and physical properties of phenol 14 2.2 Total concentration of heavy metals normal range in plants, normal range 17 In plants, normal range in soils and toxic soils for plants 2.3 Dissipation factor and dielectric constants for same solvents commonly 25 used in microwave-assisted extraction 2.4 Boiling temperatures and temperatures under microwave at 175 psig of 31 different solvents 3.1 Details of UV-Vis Spectrophotometer equipment 37 3.2 Details of FTIR Spectrometer equipment 38 3.3 Details of GC-MS equipment and column 39 3.4 Details of ICPMS Elan 9000 equipment 42 3.5 Operating conditions for ICP-MS Agilent 7500a 42 4.1 TPC yield in leaves of Stevia rebaudiana using SE 48 4.2 TPC yield in stems of Stevia rebaudiana using SE 48 4.3 TPC yield in flowers of Stevia rebaudiana using SE 48 4.4 TPC yield in leaves of Stevia rebaudiana using MAE 49 4.5 TPC yield in stems of Stevia rebaudiana using MAE 49 4.6 TPC yield in flowers of Stevia rebaudiana using MAE 49 4.7 The volume rate of heat generation at 450 Watt for 2-12 min 58 4.8 The identified components present in leaves extract using SE 66 4.9 The identified components present in stems extract using SE 63 4.10 The identified components present in flowers extract using SE 63 4.11 The identified components present in leaves extract using MAE 70 4.12 The identified components present in stems extract using MAE 70 4.13 The identified components present in flowers extract using MAE 71 4.14 Stuctural features of raw material from Stevia rebaudiana (leaves, stems 73 x and flowers) by FTIR 4.15 Stuctural features of leaves, stems and flowers extracts from Stevia 76 rebaudiana using SE by FTIR 4.16 Stuctural features of leaves, stems and flowers extracts from Stevia 78 rebaudiana using MAE by FTIR 4.17 The accumulation of heavy metals in Stevia rebaudiana (leaves, stems 79 Flowers) by using ICP-MS Elan 9000 and Agilent 7500a xi LIST OF FIGURES Figure No. Title Page 2.1 Stevia rebaudiana Bertoni 11 2.2 Phenol chemical structure 14 3.1 Sample of Stevia rebaudiana purchase from Malacca Malaysia 33 3.2 Soxhlet Extraction (SE) 34 3.3 Microwave-assisted extraction (MAE) 35 3.4 The Spectrum 100 FTIR Spectrometer 38 3.5 GC-MS an Agilent 5973N Mass Selective Detector 39 3.6 (a) The vessels (b) Microwave digestion System 40 3.7 Inductively Coupled Plasma Mass – Spectrophotometer Elan 9000 41 3.8 Inductively Coupled Plasma Mass – Spectrophotomete Agilent 7500a 42 4.1 Gallic acid standard curve 45 4.2 TPC yield in leaves and stems of Stevia rebaudiana at concentration 50 0.01 g/ml using SE 4.3 TPC yield in leaves and stems of Stevia rebaudiana at concentration 50 0.02 g/ml using SE 4.4 TPC yield in leaves and stems of Stevia rebaudiana at concentration 51 0.04 g/ml using SE 4.5 TPC yield in leaves, stems and flowers of Stevia rebaudiana at concen- 51 tration 0.06 g/ml using SE 4.6 TPC yield in leaves and stems of Stevia rebaudiana at concentration 52 0.08 g/ml using SE 4.7 TPC yield in leaves and stems of Stevia rebaudiana at concentration 52 0.1 g/ml using SE 4.8 TPC yield in leaves and stems of Stevia rebaudiana at concentration 53 0.01 g/ml using MAE 4.9 TPC yield in leaves and stems of Stevia rebaudiana at concentration 53 xii 0.02 g/ml using MAE 4.10 TPC yield in leaves and stems of Stevia rebaudiana at concentration 54 0.04 g/ml using MAE 4.11 TPC yield in leaves, stems and flowers of Stevia rebaudiana at concen- 54 0.06 g/ml using MAE 4.12 TPC yield in leaves and stems of Stevia rebaudiana at concentration 55 0.08 g/ml using MAE 4.13 TPC yield in leaves and stems of Stevia rebaudiana at concentration 55 0.1 g/ml using MAE 4.14 The volume of heat generation vs.
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