Extraction of grape seed to produce a proanthocyanidin rich extract By Havanakwavo Chikoto Submitted in fulfilment of the requirements for the degree of Master of Science Department of Paraclinical Sciences Faculty of Veterinary Science University of Pretoria 2004 Promoter: Prof J.N. Eloff © University of Pretoria Preface The experimental work described in this thesis was conducted in the Programme for Phytomedicine, Department of Paraclinical Sciences, Faculty of Veterinary Science, University of Pretoria, under the supervision of Prof J.N Eloff. These studies have not been submitted in any form to any other University and the results represent work done by Havanakwavo Chikoto, except where the work of others is acknowledged. Havanakwavo Chikoto ii © University of Pretoria Acknowledgements I am deeply indebted to my supervisor, my wife, my supervisor’s wife, my colleagues and my family back at home. My supervisor has expertly guided me through this work, and together with his wife, Christna, made my two years comfortable, imparting lessons, implied and expressed, which I shall always cherish and use. Thank you. My wife, Pelagia, my friend and confidante, who has been (and still is!), very loving, patient, and supportive through out the duration of this endeavour. My colleagues who have been a great family away from home. My family back in Zimbabwe who have been very supportive through this period I have been away from home I use “my” with such great pride! iii © University of Pretoria Table of contents Preface……………………………………………………………………………………….ii Acknowledgements………………………………………………………….…………..…iii Table of contents……………………………………………………………………..….…iv List of Figures……………………………………………………………………..…….….ix List of Tables………………………………………………………………………….…...xiii Abbreviations used…………………………………………………………………...…...xiv Summary………………………………………………………………..………………….xv Opsomming…………………………………………….…………………………………xvii 1 Introduction ........................................................................................................ 19 1.1 Oxidative stress........................................................................................... 19 1.1.1 Occurrence and nature of free radicals .................................................. 2 1.1.2 Causes of oxidative stress ..................................................................... 4 1.1.2.1 Pollution ............................................................................................. 4 1.1.2.2 Ultraviolet radiation ............................................................................. 5 1.1.2.3 Drugs .................................................................................................. 6 1.1.3 Effects of oxidative stress ...................................................................... 7 1.1.3.1 DNA damage ...................................................................................... 7 1.1.3.2 Lipid peroxidation ............................................................................... 7 1.1.3.3 Protein modification ............................................................................ 9 1.1.4 Combating oxidative stress .................................................................... 9 1.1.4.1 Enzymatic Defence ............................................................................ 9 1.1.4.2 Non enzymatic Defense ..................................................................... 9 1.1.4.2.1 Metal Binding Proteins .................................................................. 9 1.1.4.2.2 Small Molecular antioxidants ...................................................... 10 1.2 Phytochemical antioxidants ......................................................................... 14 1.2.1 Phenolics ............................................................................................. 14 1.2.1.1 Flavonoids ........................................................................................ 15 1.2.1.1.1 Proanthocyanidins ...................................................................... 18 1.2.1.2 Coumarins ........................................................................................ 21 1.2.1.3 Phenylpropanoids ............................................................................. 22 1.2.2 Non-phenolic antioxidants .................................................................... 22 iv © University of Pretoria 1.2.2.1 Terpenoids ....................................................................................... 22 1.3 Botanical sources of antioxidants ................................................................ 24 1.3.1 Pine Bark ............................................................................................. 24 1.3.1 Green Tea ............................................................................................ 24 1.3.2 Ginkgo biloba ....................................................................................... 24 1.3.3 Grape seed .......................................................................................... 25 1.4 Methods that have been used for extraction of grape seed ........................ 25 1.5 Aim of study ................................................................................................ 26 2 General Materials and Methods ......................................................................... 27 2.1 Materials ..................................................................................................... 27 2.2 Preparation of grape seed ........................................................................... 28 2.3 Initial extraction procedures ........................................................................ 29 2.3.1 Serial extraction ................................................................................... 29 2.3.2 Direct extraction ................................................................................... 29 2.4 TLC analysis of extracts .............................................................................. 30 3 Selection of the best extractant .......................................................................... 31 3.1 Use of individual solvents ............................................................................ 31 3.1.1 Introduction .......................................................................................... 31 3.1.2 Results and Discussion ........................................................................ 31 3.1.2.1 Serial extraction ................................................................................ 31 3.1.2.2 Direct extraction ............................................................................... 32 3.1.3 Conclusions ......................................................................................... 35 3.2 Extraction with ethanolic solutions .............................................................. 36 3.2.1 Introduction .......................................................................................... 36 3.2.2 Materials and methods ......................................................................... 36 3.2.3 Results and Discussion ........................................................................ 36 3.2.4 Conclusions ......................................................................................... 38 4 Investigation of factors affecting extraction yield ................................................ 39 4.1 Investigations on red seed .......................................................................... 39 4.1.1 Introduction .......................................................................................... 39 4.1.2 Materials and Methods ......................................................................... 39 4.1.2.1 Influence of varying temperature ...................................................... 39 v © University of Pretoria 4.1.2.2 Influence of duration of heating ........................................................ 40 4.1.2.3 Sample mass to solvent volume proportions .................................... 40 4.1.3 Results and discussion ........................................................................ 40 4.1.3.1 Influence of varying temperature ...................................................... 40 4.1.3.2 Influence of duration of heating ........................................................ 41 4.1.3.3 Sample mass to solvent volume proportions .................................... 41 4.1.4 Conclusions ......................................................................................... 42 4.2 Investigations on oil expressed grape seed (OEGS) .................................. 42 4.2.1 Introduction .......................................................................................... 42 4.2.2 Materials and Methods ......................................................................... 43 4.2.3 Results and Discussion ........................................................................ 43 4.2.4 Conclusion ........................................................................................... 44 5 Quantitative and qualitative analysis .................................................................. 45 5.1 Free radical scavenging activity .................................................................. 45 5.1.1 Introduction .......................................................................................... 45 5.1.2 Materials and methods ........................................................................