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Micropropagation of Three Brachystelma Species and Investigations of Their Phytochemical Content and Antioxidant Activity

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Micropropagation of Three Brachystelma Species and Investigations of Their Phytochemical Content and Antioxidant Activity Micropropagation of three Brachystelma species and investigations of their phytochemical content and antioxidant activity By Nqobile P. Hlophe (BSc. Hons. University of KwaZulu-Natal, South Africa) Submitted in fulfilment of the requirements for the degree of Master of Science Research Centre for Plant Growth and Development School of Life Sciences College of Agriculture, Engineering and Sciences University of KwaZulu-Natal Pietermaritzburg, South Africa March 2017 Table of Contents College of Agriculture, Engineering and Science declaration 1 – Plagiarism ........................ iv Student declaration ................................................................................................................ v Supervisor declaration .......................................................................................................... vi Conference contribution from this thesis ............................................................................. vii Potential publications from this thesis ................................................................................. vii Acknowledgements ............................................................................................................ viii List of Figures ...................................................................................................................... ix List of Tables ...................................................................................................................... xii List of Acronyms ............................................................................................................... xiii Abstract .............................................................................................................................. xv Chapter 1: Introduction ......................................................................................................... 1 1.1 Plant utilization in African Traditional Medicine (ATM) ............................................. 1 1.2 Micropropagation in plant conservation ....................................................................... 2 1.3 Value of plant secondary metabolites in medicinal properties....................................... 4 1.4 Brachystelma species ................................................................................................... 6 1.4.1 Introduction ........................................................................................................... 6 1.4.2 Distribution ........................................................................................................... 7 1.4.3 Botanical description ............................................................................................. 8 1.4.4 Ethnobotanical uses ............................................................................................. 10 1.4.5 Conservation status.............................................................................................. 11 1.5 Study aims and objectives .......................................................................................... 12 1.6 General thesis overview ............................................................................................. 12 Chapter 2: Literature review ................................................................................................ 14 2.1 Micropropagation ...................................................................................................... 14 2.1.1 Introduction ......................................................................................................... 14 2.1.2 Nutrient media..................................................................................................... 16 2.1.3 Effect of auxins and cytokinins on plant regeneration .......................................... 17 2.1.4 The explant material ............................................................................................ 18 2.1.5 Effect of in vitro ecological factors ...................................................................... 18 2.1.6 Challenges of contamination in micropropagtion ................................................. 19 2.1.7 Micropropagation in the family Apocynaceae ...................................................... 20 i 2.2 Phytochemicals in plants and their pharmacological potential .................................... 21 2.2.1 Phytochemicals in plants ..................................................................................... 21 2.2.1.1 Phytochemicals of the Apocynaceae ............................................................. 22 2.2.1.2 Phytochemicals in Brachystelma ................................................................... 23 2.2.2 Pharmacological activity ..................................................................................... 24 2.2.2.1 Pharmacological activity of the Apocynaceae................................................ 25 2.2.2.2 Antioxidant activity ...................................................................................... 26 2.3 Conclusions ............................................................................................................... 27 Chapter 3: Micropropagation of three Brachystelma species ................................................ 28 3.1 Introduction ............................................................................................................... 28 3.2 Materials and methods ............................................................................................... 30 3.2.1 Explant decontamination and bulking up of experimental material ...................... 30 3.2.2 In vitro shoot proliferation ................................................................................... 31 3.2.3 In vitro rooting .................................................................................................... 32 3.2.4 Ex vitro rooting and acclimatization .................................................................... 32 3.2.5 Effect of type of culture vessel on Brachystelma pygmaeum growth .................... 33 3.2.6 Effect of plant density ......................................................................................... 33 3.2.7 Data analysis ....................................................................................................... 33 3.3 Results and discussion ............................................................................................... 34 3.3.1 Explant decontamination and bulking up of plant material ................................... 34 3.3.2 Effect of cytokinins on shoot proliferation and other shoot growth parameters..... 36 3.3.3 In vitro rooting of Brachystelma pygmaeum ........................................................ 45 3.3.4 Ex vitro rooting and acclimatization of B. ngomense, B. pulchellum and B. pygmaeum .................................................................................................................... 47 3.3.5 Effect of type of culture vessel on Brachystelma pygmaeum growth .................... 50 3.3.6 Effect of plant density ......................................................................................... 51 3.4. Conclusions .............................................................................................................. 53 Chapter 4: Phytochemical profiling and antioxidant evaluation of three Brachystelma species ........................................................................................................................................... 54 4.1 Introduction ............................................................................................................... 54 4.2 Materials and methods ............................................................................................... 55 4.2.1 Collection and preparation of plant material ........................................................ 55 ii 4.2.2 Phytochemical investigations............................................................................... 56 4.2.2.1 Extract preparation ........................................................................................ 56 4.2.2.2 Flavonoid determination ............................................................................... 56 4.2.2.3 Phenolic acid determination .......................................................................... 57 4.2.2.4 Phenolic acid profile ..................................................................................... 57 4.2.2.4.1 Extract preparation ................................................................................. 57 4.2.2.4.2 Specific phenolic acid quantification ...................................................... 58 4.2.3 Antioxidant activity ............................................................................................. 58 4.2.3.1 Extract preparation ........................................................................................ 58 4.2.3.2 2,2 Diphenyl-1-picryl hydrazyl (DPPH) radical scavenging activity ............. 59 4.2.3.3 Oxygen radical absorbance capacity (ORAC) ............................................... 59 4.2.4 Data analysis ....................................................................................................... 60 4.3 Results and discussion ............................................................................................... 60 4.3.1 Phytochemical investigations..............................................................................
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