Heavy metals in South African medicinal plants with reference to safety, efficacy and quality AMBROSE OKEM Submitted in fulfilment of the academic requirements for the degree of Doctor of Philosophy In the Research Centre for Plant Growth and Development School of Life Sciences University of KwaZulu-Natal, Pietermaritzburg November 2014 Contents Contents ................................................................................................................................... i Abstract .................................................................................................................................vii College of Agriculture, Engineering and Science Declaration 1 - Plagiarism .....xi Publications from this Thesis ........................................................................................ xiv Conference Contributions from this Thesis ............................................................... xv College of Agriculture, Engineering and Science Declaration 2 - Publications ................................................................................................................................................ xvi Acknowledgements ........................................................................................................ xviii List of Figures .................................................................................................................... xix List of Tables ..................................................................................................................... xxv List of Abbreviations ..................................................................................................... xxvii Chapter 1 .................................................................................................................................1 Introduction and literature review ....................................................................................1 1.1 Introduction ....................................................................................................................1 1.2 South African traditional medicine: an overview ......................................................1 1.2.1 Trade of medicinal plant products in South Africa ............................................2 1.2.1.1 Trade of medicinal plant products in outdoor-street markets .............3 1.2.1.2 Trade of medicinal plant products in indoor muthi shops ...................5 1.3 Medicinal plant product contamination ......................................................................6 1.3.1 Cases of heavy metal contamination in South African traditional medicine .7 1.4 Heavy metal uptake in plants ......................................................................................9 1.4.1 Mechanisms of heavy metal toxicity and tolerance strategies in plants..... 10 1.4.2 Physiological and biochemical responses of plants to heavy metal toxicity ................................................................................................................................ 11 1.5 Safety assessment of medicinal plant .................................................................... 12 1.6 Adverse event reactions associated with medicinal plant products................... 14 1.7 Quality assurance of traditional medicinal plant products ................................... 15 1.7.1 Approaches to ensure quality of medicinal plant products ........................... 16 1.7.1.1 Safe cultivation of medicinal plants ..................................................... 16 1.7.1.2 Good agricultural and collection practices.......................................... 17 i 1.7.1.3 Analytical techniques for quality assurance of medicinal plant products................................................................................................... 18 1.7.1.4 Macroscopic and microscopic analysis of medicinal plant products .................................................................................................................. 19 1.8 Pharmacovigilance in traditional medicine............................................................. 19 1.8.1 Pharmacovigilance of South African traditional medicine ............................ 21 1.9 South African medicinal plants investigated in this study.................................... 22 1.9.1 Adenia gummifera (Harv.) Harms..................................................................... 22 1.9.2 Alepidea amatymbica Eckl. & Zeyh. ................................................................ 23 1.9.3 Bulbine natalensis A. Rich ................................................................................. 23 1.9.4 Cassine transvaalensis (Burtt Davy) Codd ..................................................... 24 1.9.5 Drimia elata (Jacq.)............................................................................................. 24 1.9.6 Hypoxis hemerocallidea Fisch. & C.A. Mey.................................................... 25 1.9.7 Lycopodium clavatum L. .................................................................................... 26 1.9.8 Momordica foetida Schumach. ......................................................................... 26 1.9.9 Ocotea bullata (Burch.) E. Mey......................................................................... 27 1.9.10 Rapanea melanophloeos (L.) Mez ................................................................. 28 1.9.11 Schizocarphus nervosus (Burch.) van der Merwe....................................... 29 1.10 Aims and objectives ................................................................................................ 29 Chapter 2 .............................................................................................................................. 32 Heavy metals in medicinal plants obtained from outdoor-street markets and muthi shops ......................................................................................................................... 32 2.1 Introduction ................................................................................................................. 32 2.1.1 Techniques for assessing heavy metals in plant material ............................ 32 2.1.2 Multivariate analysis – hierarchical cluster analysis ...................................... 33 2.2 Materials and Methods.............................................................................................. 34 2.2.1 Sample collection and preparation ................................................................... 34 2.2.2 Elemental analysis .............................................................................................. 34 2.2.2.1 Reagents and standard solutions ........................................................ 34 2.2.2.2 Microwave acid-assisted digestion ...................................................... 35 2.2.2.3 Elemental analysis using ICP-OES ..................................................... 35 2.2.3 Antibacterial and phytochemical screening .................................................... 35 2.2.3.1 Sample preparation ................................................................................ 35 ii 2.2.3.2 Determining total phenolics................................................................... 36 2.2.3.2 Screening for flavonoids ........................................................................ 36 2.2.3.3 Antibacterial microdilution assay.......................................................... 37 2.2.4 Statistical analysis............................................................................................... 37 2.2.5 Hierarchical cluster analysis (HCA).................................................................. 38 2.3 Results ......................................................................................................................... 38 2.3.1 Elemental analysis .............................................................................................. 38 2.3.2 Phytochemical compositions ............................................................................. 43 2.3.3 Antibacterial activity ............................................................................................ 44 2.3.4 Hierarchical Cluster analysis ............................................................................. 45 2.4 Discussion ................................................................................................................... 47 2.5 Conclusions ................................................................................................................ 51 Chapter 3 .............................................................................................................................. 53 Effects of Cd and Al uptake and distribution on physiochemical parameters in Drimia elata and Bulbine natalensis ............................................................................. 53 3.1 Introduction ................................................................................................................. 53 3.2 Materials and Methods.............................................................................................. 55 3.2.1 Pot trials ................................................................................................................ 55 3.2.2 Sample preparation and data collection .......................................................... 55 3.2.3 Elemental analysis .............................................................................................