View metadata, citation and similar papers at core.ac.uk brought to you by CORE provided by Wits Institutional Repository on DSPACE ISOLATION OF PURE CASSAVA LINAMARIN AS AN ANTI CANCER AGENT CHRISTOPHER AVWOGHOKOGHENE, IDIBIE A Dissertation Submitted to the Faculty of Engineering and the Built Environment, University of the Witwatersrand, in Fulfillment of the requirement for the Degree of Master of Science in Engineering. Johannesburg, 2006. DECLARATION I declare that this dissertation is my own, unaided work. It is being submitted for the degree of Master of Science in the University of Witwatersrand, Johannesburg. It has not been submitted before for any degree or examination in any other University. (Signature of candidature) Day of ii ABSTRACT Cassava is a known source of linamarin, but difficulties associated with its isolation have prevented it from being exploited as a source. A batch adsorption process using activated carbon at the appropriate contact time proved successful in its isolation with ultrafiltration playing a pivotal role in the purification process. Result revealed that optimum purification was obtained with increasing amount of crude cassava extract (CCE) purified. 60g of CCE took 32 mins, 80 g, 34 mins while 100 g took 36 mins of contact time, where 1.7 g, 2.0 g and 2.5 g of purified product were obtained, respectively. The purification process in batch mode was also carried out at different temperatures ranging from 25 to 65oC. Results showed that purification increases with increase in temperature. In a bid to ascertain the moles of linamarin adsorbed per pore volume of activated carbon used, the composite isotherm was found to represent the measured adsorption data quite well. The adsorption of linamarin was used to study the goodness of fit criteria (R2) for the entire process. Results showed that R2 value was best with decreasing amount of CCE purified (R2=1 for 60 g) at the temperature of 45oC. Compound elucidation of purified product by Picrate paper test, IR and 1HNMR confirmed the structure of linamarin. Cytotoxic effects of linamarin on MCF-7, HT-29, and HL-60 cells were determined using the 3 - (4, 5 – dimethylthiazol-2-yl) – 2, 5 – diphenyltetrazolium bromide (MTT) assay. Cytotoxic effects were significantly increased in the presence of linamarase, which catalysed the hydrolysis of linamarin to hydrogen cyanide. A 10–fold decrease in the IC50 values obtained for linamarin or crude extract in the presence of linamarase was determined for HL-60 cells. This study thus describes a method for the isolation and purification of linamarin from cassava, as well as the potential of this compound as an anticancer agent. iii DEDICATION This dissertation is exclusively dedicated to our Lord Jesus Christ who is, who was and who is to come in the glory of the Father Almighty God forever and ever. Amen. iv ACKNOWLEDGEMENTS I ascribe all praise, glory and thanks to the Almighty God, the wonder, miracle way maker who gave all enablement against all odds for making this study a befitting success. His grace is truly ‘all sufficient’. I wish to first of all express my appreciation to my supervisor, Prof. S. E. Iyuke and Dr H. Davids for their guidance, support, suggestions, perseverance and encouragement throughout the course of this research. I also wish to express my sincere appreciation to Griffin Cancer Trust Foundation, University of the Witwatersrand, Johannesburg, South Africa, for the grant they provided for this research. I also wish to express my heart-felt appreciation to Professor Emeritus, W. Neuse, who granted me free assess to his equipment to perform some of my laboratory work in polymer chemistry laboratory at the School of Chemistry of this university. God bless you. I also stand to appreciate the post-graduate students under Prof. Neusa in the likes of sister Blessing, Zenaib (a post doctoral student), Jacob, Allen, Eli, Bavon, Jack, January and Tumi for the love, concern and high understanding they demonstrated during the short period of experimental work in polymer chemistry laboratory. May the good Lord bless and grant you all your heart desires. Special thanks to my loving parents Mr. and Mrs. Sunday Ufuoma Idibie and the entire family for all the prayer, support, love and encouragement. May the good Lord, our God cause His face to shine on you and be gracious to everyone in Jesus’ name. Amen. Special thanks are extended also to different individuals most especially to Eric Efurhievwe and family, Engr. Igbafe and family, Olubambi Peter and his family, Toyin Kolawole, Dr Ilemobade and family, Adeyemi Sola, Engr. Saka Abdulkareem, Samuel Afolabi, Maria Modiba, Mala Raghununan, Gladys Mabasa, all West Campus and International House Fellowship Members, students and staff of School of Chemical and Metallurgical Engineering, WITS University and to those whose names are not mentioned here that have in v one way or the other been vitally helpful to me in the course of this research. God bless you all richly in Jesus’ name. My best regards and thanks also go to Willie and Diamond Akele for their strong support on every side. May the Almighty God bless you. This dissertation will not be complete if I fail to acknowledge the laudable work of physical, moral, and financial support of Chief Ama Johnson, the Adjerese 1 of Agbarho Kingdom. Your good works must surely grant you a glorious place in God’s kingdom. God bless you and your entire family richly in Jesus’ name. Amen. vi CONFERENCE ATTENDED Idibie, C.A., Davids, H and Iyuke, S.E. (2006). Anticancer candidature of purified cassava (Manihot esculenta Crantz) linamarin. Proceedings of the 1st symposium on Biomaterials Science and Application in South Africa. Wits School of Health, Johannesburg. 4th September, 2006. JOURNAL PAPER SUBMITTED .Idibie, C.A., Davids, H and Iyuke, S.E. (2006). Cytotoxicity of purified cassava (Manihot esculenta Crantz) linamarin to a selected cancer cell lines. Bioprocess and biosystems Engineering Journal. (Submitted) vii TABLE OF CONTENTS Page DECLARATION ii ABSTRACT iii DEDICATION iv ACKNOWLEDGEMENTS v CONFERENCE ATTENDED/JOURNAL PAPER SUBMITTED vii TABLE OF CONTENTS viii LIST OF FIGURES xii LIST OF TABLES xiv LIST OF ABBREVIATIONS AND SYMBOLS xv CHAPTER 1: INTRODUCTION 1 1.1 BACKGROUND AND MOTIVATION 1 1.2 RESEARCH PROBLEM 3 1.3 HYPOTHESIS 4 1.4 JUSTIFICATION OF STUDY 5 1.5 SCOPE OF PROJECT 5 1.6 RESEARCH QUESTION 6 1.7 PURPOSE AND AIM 6 1.8 EXPECTED CONTRIBUTION TO KNOWLEGDE 6 1.9 DISSERTATION OUTLINE 7 CHAPTER 2: LITERATURE REVIEW 8 2.1 CASSAVA 8 2.1.1 Cassava as a cyanogenic plant 9 viii 2.1.2 Cyanogenocity of cassava (hydrolysis of linamarin) 10 2.1.3 Linamarin as nitriloside 11 2.1.4 Biosynthesis of linamarin 13 2.1.5 Analysis of linamarin 14 2.1.6 Anti cancer potential of nitriloside (linamarin) 15 2.1.7 Physicochemical parameters of linamarin 18 2.2 Overview of ulrafiltration in membrane technology 20 2.3 Activated carbon in liquid-phase adsorption 28 2.3.1 Theory of adsorption 28 2.3.2 Activated carbon in adsorption 30 2.3.3 Batch adsorption 33 2.3.4 Composite isotherm 34 2.3.5 Types of composite isotherm 36 2.4 Invitro evaluation of antitumoral agent 38 CHAPTER 3: EXPERIMENTAL 40 3.1 Reagents and chemicals 40 3.2 Preparation of alkaline picric paper 40 3.3 Preparation of hydrolyzing enzyme solution from cassava root peel 41 3.4 Preparation of crude cassava extracts 41 3.5 Optimum amount of linamarase and linamarin determination 42 3.6 Determination of purification/adsorption point 43 3.7 Purification of linamarin by batch adsorption with activated carbon 43 3.8 Preparation of calibration curve 43 3.9 Determining the effect of temperature on the rate of purification/adsorption 44 3.10 Analysis of results 44 3.10.1 Infra-red spectra 44 3.10.2 Nuclear magnetic resonance test 44 3.10.3 Melting point determination 44 ix 3.11 Cell culture 45 3.12 In vitro anticancer effect 45 3.13 Analysis of in vitro anticancer effect results 45 CHAPTER 4: RESULTS AND DISCUSSION 46 4.1 Separation of linamarin (cyanogenic component) from cassava 46 4.2 Optimum amount of linamarase and linamarin determination 47 4.3 Calibration curve 48 4.4 Determination of linamarin purification-adsorption point 49 4.5 Effect of temperature on purification/adsorption of linamarin 53 4.6 Adsorption isotherm 55 4.7 Effect of temperature on linamarin removal from solution 57 4.8 Composite isotherm correlation 58 4.9 Effect of temperature on pore volume efficiency 60 4.10 Compound characterisation 61 4.10.1 Nuclear magnetic resonance test 61 4.10.2 Infra-red spectrometer (IR) test 63 4.10.3 Melting point determination 64 4.11 Efficacy of purified cassava linamarin and crude cassava extract on cancer cells 64 CHAPTER 5: CONCLUSION AND RECOMMENDATION 70 5.1 Conclusion 70 5.2 Recommendations 71 REFERENCES 72 APPENDIX 1 87 APPENDIX 2 89 x APPENDIX 3 90 APPENDIX 4 91 APPENDIX 5 101 APPENDIX 6 104 xi LIST OF FIGURES Figure 2.1: Cassava tubers (roots) 9 Figure 2.2 a: Cassava planted at two months old (Wits garden) 10 Figure 2.2 b: Cassava planted at eight months old (Wits garden) 11 Figure 2.3: Linamarin hydrolysis 12 Figure 2.4: Detoxification reaction of hydrogen cyanide by rhodenase 15 Figure 2.5: Structure of some related -glucosidase compounds 19 Figure 2.6: Simple membrane concept 21 Figure 2.7 Schematic diagrams of the principal types of membrane 22 Figure 2.8: Schematic diagram of ultrafiltration crossflow System 24 Figure 2.9: Mechanism of adsorption and desorption process 29 Figure 2.10: Mechanism of adsorption isotherms 37 Figure 3.1: Laboratory