AN INVESTIGATION OF THE VOLTAMMETRIC BEHAVIOUR OF ANTIOXIDANTS IN FLAVONOIDS By Lee-Ann Ramsarup Student Number: 20427580 Submitted in fulfilment of the requirements for the degree of Master of Applied Science in Chemistry in the Faculty of Applied Sciences at the Durban University of Technology April 2020 DECLARATION I, Lee-Ann Ramsarup, hereby declare that the work presented in this thesis entitle “An Investigation of the Voltammetric Behaviour of Antioxidants in Flavonoids” has not been submitted to any other University or Institution for any degree or examination. This is my own work and where the work of others was used, it was to the best of my knowledge accurately reported and duly acknowledged. Signature of Lee-Ann Ramsarup (Student) Date Signature of Prof Krishna Bisetty (Supervisor) Date Signature of Dr Suvardhan Kanchi (Co- Supervisor) Date i DEDICATION This work is dedicated to my late uncle, Gayson Samuel. I am thankful for his presence and support throughout my life. His memory lives on forever. ii ACKNOWLEDGMENTS All that I have and all that I am is because of my Lord and Saviour Jesus Christ. All honour and glory to Him for giving me the strength and faith to see this project to completion. I wish to express my sincere appreciation to my Supervisor, Prof Krishna Bisetty for his assistance and constant motivation throughout my research. I extend my heartfelt appreciation to my Co-Supervisor, Dr Suvardhan Kanchi for his time, support and direction during this journey. Special thanks to Dr Myalo I Sabela (Lecturer) and Nokukhanya Xhakaza (Technician) for assisting me with their time and experience during the experimental phase of the research work. I would also like to thank the management and fellow colleagues at Metrohm South Africa for their unwavering support and understanding. To my parents, Pat and Ashok Ramsarup, my brother Karl and Narintha, I am thankful for your prayers and support. I have learned to be strong and courageous because of you. I am eternally thankful to my husband, Dr Viren Chunilall and my son Anvir. Thank you both for being supportive, encouraging and understanding throughout this process. Your dedication and love for me is priceless. Thank you for the inspiration to make each day memorable. iii ABSTRACT The two case studies in this work involve the development and fabrication of an electrochemical biosensor using various enzymes for the evaluation of the electrochemical responses, relating to the total phenolic (TP) content and the antioxidant activities in wine and tea samples respectively. The modification of the glassy carbon electrode (GCE) was carried out using green apple as an enzymatic source of polyphenol oxidase and laccase enzyme. The experimental variables were optimized using the Box-Behnken experimental design as a predictive model, for a better understanding of the parameters and their interaction responses with each other during an electrochemical analysis. This multivariate optimization method is based on a factorial design, where the three most influential factors include the electrolyte pH, the deposition time (td) and the scan rate (sr). The design was run in a single block fashion while random order of experiment was selected to provide greater protection against the effects of outlying variables. The optimized results obtained yielded the most suitable conditions for the determination of the TP content in wine samples. They were selected as follows: phosphate buffer of pH 7.65 as supporting electrolyte, td 29.8 s and sr 25.0 mV/s respectively. The method was optimized for the current signal at a deposition potential of 0.2 V and within an oxidation potential of -0.2 V to 0.6 V. Good analytical responses were obtained with apple sensors for the detection of TP content in wine samples, with a higher concentration in red wines than in white wines. Differential pulse voltammetry (DPV) and cyclic voltammetry (CV) were used to establish and interpret the redox mechanisms of flavonoids present in alcoholic and non-alcoholic beverages. The sensor responses were evaluated by first, investigating the changes in the total phenolic (TP) content in wine samples using catechin as a standard. Thereafter, the electrochemical behaviour of rutin and ascorbic acid, antioxidant capacities (trolox reagents) were established in tea samples, yielding a positive linear correlation between the trolox equivalent antioxidant capacities (TEAC) and TP content (R2 = 0.9812 ± 0.012). DPV was applied to the laccase modified GCE, and the experimental results indicate that this sensor shows good reducing properties. The scavenging ability of 2,2’-Azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) a diammonium salt, was assessed in the sample extracts, which yielded half-maximal effective concentration (EC50) values of 10.80 µg/ml and 11.62 µg/ml for ascorbic acid and rutin respectively. These findings indicated that the experimental design was a convenient method to evaluate the statistical significance of the optimised parameters, and the positive linearity for the TEAC and the TP content confirms the robustness of this methodology. iv TABLE OF CONTENTS Declaration ............................................................................................................................................... i Dedication ............................................................................................................................................... ii ACKNOWLEDGMENTS ............................................................................................................................ iii Abstract .................................................................................................................................................. iv TABLE OF CONTENTS ............................................................................................................................... v List of Tables ........................................................................................................................................ viii List of Figures ......................................................................................................................................... ix Acronyms ................................................................................................................................................ x Research OutputS ................................................................................................................................. xii CHAPTER 1 .............................................................................................................................................. 1 INTRODUCTION ....................................................................................................................................... 1 1.1 Flavonoids ..................................................................................................................................... 1 1.2 Antioxidants .................................................................................................................................. 2 1.3 Problem Statement ....................................................................................................................... 4 1.4 Aim and Objectives ....................................................................................................................... 4 1.5 Thesis Outline ................................................................................................................................ 5 CHAPTER 2 .............................................................................................................................................. 6 LITERATURE REVIEW ............................................................................................................................... 6 2.1 Catechin ........................................................................................................................................ 6 2.1.1 Antioxidant properties of catechins....................................................................................... 7 2.2 Antioxidant Evaluation Models ..................................................................................................... 9 2.2.1 DPPH method (1, 1-diphenyl 2, picryl hydrazyl) .................................................................... 9 2.2.2 Superoxide radical scavenging activity ................................................................................ 10 2.2.3 Conjugated diene assay ....................................................................................................... 10 2.2.4 ABTS (2,2-azinobis(3-ethylbenzothiazoline-6-sulfonate) Method ....................................... 10 2.2.5 Hydroxyl radical scavenging activity .................................................................................... 10 2.2.6 Xanthine oxidase method .................................................................................................... 11 2.2.7 Cytochrome C test ................................................................................................................ 11 2.2.8 Oxygen Radical Absorbance Capacity (ORAC)...................................................................... 11 2.3 Enzymes ...................................................................................................................................... 11 2.3.1 Cytochrome C ......................................................................................................................