In vitro propagation, phytochemistry and pharmacology of the blood lily, Scadoxus puniceus Devashan Naidoo Research Centre for Plant Growth and Development School of Life Sciences University of KwaZulu-Natal, Pietermaritzburg 1 In vitro propagation, phytochemistry and pharmacology of the blood lily, Scadoxus puniceus By Devashan Naidoo Submitted in fulfilment of the requirements for the degree of Doctor of Philosophy Research Centre for Plant Growth and Development School of Life Sciences University of KwaZulu-Natal Pietermaritzburg November, 2016 I STUDENT DECLARATION In vitro propagation, phytochemistry and pharmacology of the blood lily, Scadoxus puniceus I, Devashan Naidoo, student number: 209506613 declare that: 1. The research reported in this thesis, except where otherwise indicated is the result of my own endeavours in the Research Centre for Plant Growth and Development, School of Life Sciences, University of KwaZulu-Natal, Pietermaritzburg; 2. This thesis has not been submitted for any degrees or examination at any other University; 3. This thesis does not contain data, figures or writing, unless specifically acknowledged, copied from other researchers; and 4. Where I have reproduced a publication of which I am an author or co-author, I have indicated which part of the publication was contributed by me. Signed at …………………………….....on the ………day of…………………….2016 Signature II DECLARATION BY SUPERVISORS We hereby declare that we acted as Supervisors for this PhD student: Students Full Name: Devashan Naidoo Student Number: 209506613 Thesis Title: In vitro propagation, phytochemistry and pharmacology of the blood lily, Scadoxus puniceus Regular consultation took place between the student and ourselves throughout the investigation. We advised the student to the best of our ability and approved the final document for submission to the College of Agriculture, Engineering and Science, Higher Degrees Office for examination by the University appointed Examiners. SUPERVISOR PROFESSOR J.F. FINNIE CO-SUPERVISOR PROFESSOR J. VAN STADEN III COLLEGE OF AGRICULTURE, ENGINEERING AND SCIENCE DECLARATION 1 - PLAGIARISM I, Devashan Naidoo, student number: 209506613, declare that 1. The research reported in this thesis, except where otherwise indicated, is my original research. 2. This thesis has not been submitted for any degree or examination at any other University. 3. This thesis does not contain other persons‟ data, pictures, graphs or other information unless specifically acknowledged as being sourced from other persons. 4. This thesis does not contain other persons‟ writing, unless specifically acknowledged as being sourced from other researchers. Where other written sources have been quoted, then: a. Their words have been re-written but the general information attributed to them has been referenced. b. Where their exact words have been used, then their writing has been placed in italics and inside quotation marks, and referenced. 5. This thesis does not contain text, graphics or tables copied and pasted from the internet, unless specifically acknowledged, and the source being detailed in the thesis and in the reference section. Signed at ……………..………..…….....on the ………day of…………………….2016 Signature IV PUBLICATIONS FROM THIS THESIS 1. D. Naidoo, A.O. Aremu, J. Grúz, B. Ondřej, K. Doležal, J. Van Staden, J.F. Finnie, (manuscript under review - Phytochemistry). UHPLC-MS/MS quantification of phenolic compounds and pharmacology of Scadoxus puniceus, a highly traded South African medicinal plant. 2. D. Naidoo, A.O. Aremu, J. Van Staden, J.F. Finnie, (manuscript Accepted – South African Journal of Botany). In vitro plant regeneration and alleviation of physiological disorders in Scadoxus puniceus. 3. D. Naidoo, L.P. Slavětínská, J. Van Staden, J.F. Finnie, (Submitted – Journal of Natural Products). Inhibition of the biotransformation of acetylcholine by alkaloids and a naturally occurring chlorinated amide isolated from Scadoxus puniceus (Amaryllidaceae). V CONFERENCE CONTRIBUTIONS D. Naidoo, A.O. Aremu, J.F. Finnie, J. Van Staden., 2014. Phytochemical and pharmacological evaluations of different organs of Scadoxus puniceus: motivation for plant part substitution. 41st Annual Conference of the South African Association of Botanists (SAAB), University of Venda, Tshipise Forever Resort, South Africa (11-15 January 2015). Oral Presentation. D. Naidoo, A.O. Aremu, J. Grúz, B. Ondřej, K. Doležal, J. Van Staden, J.F. Finnie. 2015. Phenylpropanoid metabolism and pharmacology of the blood lily, Scadoxus puniceus, a highly traded South African medicinal plant. 63rd International Congress and Annual Meeting of the Society for Medicinal Plant and Natural Product Research. Budapest, Hungary, 23-27 August 2015. Abstract published in Planta Medica, 81. VI COLLEGE OF AGRICULTURE, ENGINEERING AND SCIENCE DECLARATION 2 - PUBLICATIONS DETAILS OF CONTRIBUTION TO PUBLICATIONS that form part and/or include research presented in this thesis (include publications in preparation, submitted, in press and published and give details of the contributions of each author to the experimental work and writing of each publication). Publication 1 Contributions: AOA, JG, BO and KD performed the ultra high performance liquid chromatography. DN designed and performed pharmacological assays, and prepared the manuscript under the supervision of JFF and JVS. Publication 2 Contributions: DN designed and performed all experimental work and prepared the manuscript under the supervision of JFF and JVS. AOA assisted with experimental design. Publication 3 Contributions: DN designed and performed all experimental work and prepared the manuscript under the supervision of JFF and JVS. LPS interpreted NMR spectra and provided spectroscopic data. Author’s abbreviation DN Devashan Naidoo AOA Adeyemi O. Aremu JG Jiřĺ Grúz BO Bíba Ondřej KD Karel Doležal JFF Jeffrey F. Finnie JVS Johannes Van Staden LPS Lenka Poštová Slavětínská VII ACKNOWLEDGEMENTS I would like to thank: My supervisor Prof. J. F. Finnie. Thank you for all the advice, ideas and knowledge that you have imparted onto me through the course of my studies. You are responsible for planting the seed that eventually developed into this thesis. Most importantly though, thank you for the encouragement and your calming influence. When I used to walk into your office worried and stressed, I always walked out feeling like I was on top of things. My co-supervisor Prof. J. Van Staden. Thank you for always keeping me on my toes and for the push when it was needed. Most of all thank you for the opportunity of working with you; it‟s only a privilege to work beside a legend such as yourself. My advisor or un-official co-supervisor Dr. A. O. Aremu. Thank you for teaching me everything I know, all the help with experimental design and for the countless times you‟ve read and edited this document. Your work ethic will always be something I admire. The Department of Chemical Biology and Genetics, Centre of the Region Haná for Biotechnological and Agricultural Research, Faculty of Science, Palacký University & Institute of Experimental Botany is thanked for the use of research facilities, reagents and instrumentation. Dr Lenka Poštová Slavětínská from the Institute of Organic Chemistry and Biochemistry, Academy of Sciences of the Czech Republic, who interpreted 1D and 2D NMR data and provided the spectroscopic information of the isolated compounds. The National Research Foundation, Pretoria, for awarding me a scholarship to pursue my Masters and Doctoral degrees. All the students and staff at the Research Centre for Plant Growth and Development. Most importantly my family. To my mother and father, thank you for everything you have done for me, from teaching me how to write once upon a time, to allowing the opportunity to achieve a Doctoral degree. Your sacrifices will not go in vain for without your guidance, encouragement, love and care, I wouldn‟t be half the person I am today. My brothers Kobershan and Keeran thank you for the coffee breaks and conversations during those endless nights during the process of writing up. VIII TABLE OF CONTENTS STUDENT DECLARATION ................................................................................................. II DECLARATION BY SUPERVISORS ............................................................................... III COLLEGE OF AGRICULTURE, ENGINEERING AND SCIENCE DECLARATION 1 - PLAGIARISM ................................................................................................... IV PUBLICATIONS FROM THIS THESIS ............................................................................. V CONFERENCE CONTRIBUTIONS.................................................................................. VI COLLEGE OF AGRICULTURE, ENGINEERING AND SCIENCE DECLARATION 2 - PUBLICATIONS.............................................................................................. VII ACKNOWLEDGEMENTS .............................................................................................. VIII TABLE OF CONTENTS ..................................................................................................... IX LIST OF FIGURES ........................................................................................................... XIV LIST OF TABLES ............................................................................................................ XVII LIST OF ABBREVIATIONS ........................................................................................... XIX