Antioxidant activities of selected vietnamese sea cucumber species

MINISTRY OF EDUCATION AND TRAINING NHA TRANG UNIVERSITY OLUWAFEMI SEGUN AJIBOYE ANTI-OXIDANT ACTIVITIES OF SELECTED VIETNAMESE SEA CUCUMBER SPECIES MASTER THESIS KHANH HOA - 2020 MINISTRY OF EDUCATION AND TRAINING NHA TRANG UNIVERSITY OLUWAFEMI SEGUN AJIBOYE ANTI-OXIDANT ACTIVITIES OF SELECTED VIETNAMESE SEA CUCUMBER SPECIES MASTER THESIS Major: Food Technology Topic allocation Decision 8540101 Decision on establishing the Committee: 192/QD-DHNT dated 03/03/2020 Defense date: 19/09/2020 Supervisors: Dr NGUYEN THE HAN MSc DO ANH DUY Chairman: TS KHONG TRUNG THANG Faculty of Graduate Studies: (Full name) (signature) KHANH HOA – 2020 UNDERTAKING I undertake that the thesis entitled: “Antioxidant activities of selected Vietnamese Sea Cucumber Species” is my own work The work has not been presented elsewhere for assessment until the time this thesis is submitted 31st August, 2020 Oluwafemi Segun Ajiboye iii ACKNOWLEDGEMENT I would like to express my deepest appreciation to the Faculty of Graduate Studies, Nha Trang University and VLIR project for giving best conditions for me to carry out and finish my Master’s study My heartiest gratitude goes to both of my supervisors, Dr The Han Nguyen, MSc Anh Duy Do and Dr Hong Ngoc Thuy Pham for their continuous support of my M.Sc study and research, for their patience, motivation, enthusiasm, and immense knowledge Their guidance helped me in all the time of research and writing of this thesis Without them this would never be a success Furthermore, I would like to extend my sincere thanks to all my teachers and colleagues of the M.Sc programme for hearing me out and having to tolerate me over the last two years I cannot begin to express my gratitude and appreciation for their friendship They have been a wonderful support for me and mentioning a few names would not be enough Nobody has been more important to me in the pursuit of this project than my loving and supportive wife, Gbemisola Ebunoluwa I want to thank her for her care and unending inspiration I would also like to appreciate my parents, Chief & Mrs J.O Ajiboye and my wife’s parents, Engr & Mrs J.A Adewole whose love and guidance are with me in whatever I pursue They are the ultimate role models I would also like to extend my gratitude to my amazing uncle, Gbenga Alao and aunt, Bola Adeyemi for their unrelentless encouragement throughout the course of my program My appreciation will be incomplete without recognizing my wonderful siblings, Shola, Opeyemi, Ibukun, Oreofe and Tolu for their love and unending support My greatest appreciation goes to God, the Almighty, for his grace and blessings throughout my study and the successful completion of my research work May his name be praised forevermore 31st August, 2020 Oluwafemi Segun Ajiboye iv TABLE OF CONTENTS UNDERTAKING iii ACKNOWLEDGEMENT iv LIST OF SYMBOLS viii LIST OF ABBREVIATIONS ix LIST OF TABLES x LIST OF FIGURES xi ABSTRACT xii Chapter 1: INTRODUCTION 1.1 Problem statement 1.2 Research questions 1.3 Objectives of the study 1.3.1 General objective 1.3.2 Specific objectives Chapter 2: LITERATURE REVIEW .5 2.1 General overview of sea cucumber species production 2.2 Properties of sea cucumber 2.3 Potential bioactive compounds from sea cucumbers 2.3.1 Saponin 2.3.2 Mycosporine-like amino acids 2.3.3 Polysaccharides 10 2.3.4 Collagen .12 2.3.5 Vitamins and minerals 13 2.4 Biological activities of sea cucumber ingredients .14 2.4.1 Anti-aging activities 14 2.4.2 Skin whitening activities 15 v 2.4.3 Anti-microbial activities .16 2.4.4 Wound healing activities 16 2.5 Tyrosinase role in the biosynthesis of melanin 17 2.6 Extraction of Bioactive Compounds 19 Chapter 3: MATERIALS AND METHODS 23 3.1 Materials 23 3.1.1 Sample collection and preparation 23 3.1.2 Analytical chemicals 23 3.2 Methods 24 3.2.1 Experimental design 24 3.2.2 Screening test .24 3.3 Determination of the extraction parameter ranges for optimization 25 3.4 Response Surface Methodology 26 3.5 Determination of Total Phenolic Content and Antioxidant Activity of Holothuria sp2 27 3.5.1 Total Phenolic Content (TPC) 27 3.5.2 DPPH Radical Scavenging Capacity 27 3.5.3 Ferric Reducing Antioxidant Power (FRAP) 27 3.6 Fractionation of Holothuria sp2 extract .27 3.7 Recovery yield 28 3.8 Determination of tyrosinase inhibitory activity of Holothuria sp2 extract and fractions 28 3.9 Statistical analyses 28 Chapter 4: RESULTS AND DISCUSSION 29 4.1 Total phenolic content and antioxidant of different sea cucumber extracts .29 4.2 Determination of extraction parameters 30 4.2.1 Extraction solvent .30 4.2.2 Ultrasonic temperature .31 vi 4.2.3 Ultrasonic time 32 4.2.4 The sample to solvent ratio 32 4.3 Fitting the models 33 4.4 The effect of factors on total phenolic content of Holothuria sp2 .35 4.5 The effect of factors on the antioxidant capacity of Holothuria sp2 37 4.6 Optimization and verification of the predictive models 39 4.7 Total phenolic content, antioxidant activities and yield of Holothuria sp2 fractions 40 4.8 Evaluation of tyrosinase inhibitory activity of Holothuria sp2 fractions 41 Chapter 5: CONCLUSION AND RECOMMENDATION 43 5.1 Conclusion 43 5.2 Recommendations .43 APPENDICES I Appendix Standard curve of gallic acid for TPC assay I Appendix Standard curve of Trolox for DPPH radical scavenging capacity I Appendix Standard curve of Trolox for ferric reducing antioxidant power II Appendix Standard curve of ascorbic acid for tyrosinase inhibitory assay III Appendix Box-Behnken design and observed responses III vii LIST OF SYMBOLS βo : Regression coefficients for intercept βi : Regression coefficients for linear equation βii : Quadratic coefficients for quadratic equation βij : Regression coefficient for interaction terms viii LIST OF ABBREVIATIONS UAE : Ultrasound-Assisted Extraction RSM : Response surface methodology BBD : Box-Behnken Design MAAs : Mycosporine-like amino acids FucCS : Fucosylated chondroitin sulfate TPTZ : 2,4,6-Tris(2-pyridyl)-s-triazine DPPH : 2,2-diphenyl-1-picrylhydrazyl radical TPC : Total phenolic content mg : Milligram g : Gram GAE : Gallic Acid Equivalent TE : Trolox Equivalent FRAP : Ferric Reducing Antioxidant Power ACE : Ascorbic Acid Equivalent 2D : Dimensions 3D : Dimensions ANOVA : Analysis of Variance PRESS : Predicted Residual Sum of Square ix LIST OF TABLES Table 3.1 Independent variables and their levels used for Box-Behnken design (BBD) 26 Table 4.2 The effect of extraction solvent on TPC and antioxidant capacity of Holothuria sp2 .30 Table 4.3 The effect of extraction temperatures on TPC and antioxidant capacity .31 Table 4.4 The effect of extraction time on TPC and antioxidant capacity 32 Table 4.5 The effect of solid to solvent ratio on TPC and antioxidant capacity 33 Table 4.6 Analysis of variance for determination of model fitting .34 Table 4.7 Regression coefficients (RC) and analysis of variance for experimental results 35 Table 4.8 Validation of the predicted values for TPC and antioxidant capacities 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60 30 0.375 182.49 298.81 77.523 60 50 0.375 211.72 277.76 91.90 50 30 0.25 196.33 403.52 55.50 50 30 0.5 163.64 203.70 58.62 50 50 0.25 262.47 433.13 86.69 50 50 0.5 200.11 175.48 66.19 40 40 0.25 239.83 435.68 77.47 10 60 40 0.25 258.32 471.40 84.65 11 40 40 0.5 168.04 208.74 51.29 12 60 40 0.5 245.62 196.31 77.60 13 50 40 0.375 194.97 337.36 76.64 14 50 40 0.375 183.12 361.57 64.02 15 50 40 0.375 188.65 368.27 62.56 X1: ultrasonic temperature; X2: ultrasonic time; X3: sample-to-solvent ratio; TPC: total phenolic content; GAE: gallic acid equivalents; TE: trolox equivalents FRAP: ferric reducing antioxidant power III ... the selected Vietnamese sea cucumber species?  What is the optimum extraction condition for antioxidant activity and tyrosinase inhibitory activity of the selected Vietnamese sea cucumber species? ... .5 2.1 General overview of sea cucumber species production 2.2 Properties of sea cucumber 2.3 Potential bioactive compounds from sea cucumbers 2.3.1 Saponin ... extraction of novel bioactive compounds, especially soft bodied animals like Sea cucumbers (Dhinakaran, 2014) Sea cucumber is a group of animals that lives in the sea, and are classified as soft-bodied