Herbal formulations claimed to cure several medical conditions including skin eruptions, chest pains, wounds, gout, menstrual pains, stress, nervous disorders, microbial and viral infections as well as stomach ailments have recently appeared as part of South African traditional medicine. The formulations consist of mixtures of extracts of different plant parts from several different plant species packaged in labelled bottles or boxes. The mixtures are available for sale in herbal shops and public places. While there has been widespread use of these herbal mixtures, there has been no scientific evidence to support their use. This project was aimed at documenting, validating claimed efficacy and testing the safety of fourteen unregistered commercial herbal preparations commonly sold in Pietermaritzburg. A detailed investigation of the pharmacological effects and safety of the plant components of one of the mixtures, Imbiza ephuzwato was also carried out. / Thesis (Ph.D.) - University of KwaZulu-Natal, Pietermaritzburg, 2009.

Plants are able to take up and accumulate certain environmental contaminants such as heavy metals. When the plants are ingested by man, these contaminants are transferred along the food chain. Due to the poorly regulated medicinal plant trade in South Africa, many opportunities exist for heavy metal contamination of medicinal plants namely contaminated harvest sites as well as poor drying, processing, storage, transport and manufacturing conditions. The concentrations of five heavy metals (As, Cd, Co, Ni, Pb) and six microelements (B, Cu, Fe, Mn, Mo, Zn) were determined in some commonly used South African medicinal plants obtained from street markets. Elemental content was determined using inductively coupled plasma optical emission spectrophotometry (ICP-OES). Some of the medicinal plant samples investigated contained As and Cd at levels exceeding the World Health Organization limits of 1 and 0.3 mg kg-1 respectively. Lead and Ni were detected in all the samples. Elevated Fe and Mn levels were recorded in certain plant species. The results revealed multiple metal contamination in some medicinal plant parts sold in local markets and is thus grounds for concern. The effects of Cd application on growth parameters of some medicinal plant species belonging to the Hyacinthaceae (Albuca setosa, Eucomis autumnalis, Eucomis humilis, Merwilla plumbea) gave insight into heavy metal accumulation and distribution in these species. Application of Cd at 5 mg l-1 over a 12 week period reduced growth in A. setosa. The medicinally used A. setosa bulbs accumulated 37 mg kg-1 Cd after 12 weeks. Cadmium application at 2 mg l-1 over a six week period had no effect on growth parameters of E. autumnalis or E. humilis. However, a substantial difference in total Cd accumulation was detected in the plants (40.2 and 15.3 mg kg-1 respectively). Cadmium application at 2 mg l-1 significantly reduced the fresh weight of leaves, bulbs and roots of M. plumbea. Although most of the Cd was stored in the roots, the medicinally used bulbs accumulated up to 11.6 mg kg-1 when applied at 10 mg l-1. The antagonistic effect between Cd and Zn treatments and their effect on micronutrient distribution in M. plumbea were investigated. Five treatments were evaluated: (1) Hoagland’s nutrient solution (HS) (control) (2) HS + Cd 2 mg l-1 (single) (3) HS + Cd 2 mg l-1 + Zn 50 mg l-1 (combination) (4) HS + Cd 2 mg l-1 + Zn 100 mg l-1 (combination) (5) HS + Cd 2 mg l-1 + Zn 150 mg l-1 (combination). Cadmium readily accumulated in leaves, bulbs and roots of M. plumbea when supplied at 2 mg l-1. Zinc at 50 mg l-1 led to increased Cd accumulation. However, further increases in Zn concentration showed an antagonistic effect of Zn on Cd uptake and accumulation. Thus, increasing Zn levels in soils may be favourable for reducing toxic Cd accumulation in M. plumbea plants. Boron was not significantly affected by the addition of Cd to the media. However, with an increase in Zn, leaf B content increased while the B content in the bulbs and roots decreased. Copper and Mo levels were not significantly affected by treatments with Cd or Cd/Zn combinations. Compared to the control, Cd and Cd/Zn applications caused an increase in Mn content in leaves, bulbs and roots. Iron levels of M. plumbea were not significantly affected by Cd in the media. However, with an increase of Zn in the Cd-containing media, Fe content in the leaves, bulbs and roots increased. Tulbaghia violacea is one of the few medicinal plants that is also frequently used as a leafy vegetable. Application of Cd at 2 and 5 mg l-1 to T. violacea of varying sizes (small 8 - 10 g, medium 16 - 20 g, large 80 – 95 g) elicited a difference in growth response, Cd accumulation and micronutrient distribution. Leaf length and fresh weight of leaves of the medium-size plants decreased with application of Cd at 2 mg l-1 whilst 5 mg l-1 Cd significantly decreased the number of leaves in small-sized plants. Small plants accumulated more Cd in the leaves than medium- or large-sized plants. Application of Cd at 2 mg l-1 and 5 mg l-1 lowered the leaf Cu, Fe, Mo and Zn contents in small- and medium-size plants. This study indicated that T. violacea has the ability to accumulate Cd. In addition, plant size plays an important role with regards to Cd accumulation and elemental distribution. The effect of various nutrient applications (10%, 50% and 100% Hoagland’s nutrient solutions (HS); and HS deficient in N, P or K) on growth parameters and micronutrient distribution in Dioscorea dregeana were investigated. Irrigating plants with 50% HS resulted in better growth performance, whereas a deficiency of either N, P or K negatively affected seedling growth. Plants grown in 10% HS contained higher total B, Fe and Mo levels compared to seedlings grown in 50% and 100% HS. Compared to the control, P deficiency resulted in a Fe increase in the leaves, tuber and roots while a lack of P and K significantly increased total Mn content in D. dregeana. The effect of excess Zn (100, 200 and 300 mg l-1) on growth performance, chlorophyll content and microelemental distribution on Dioscorea sylvatica was investigated. Growth parameters showed a significant decrease when supplied with Zn at 100 mg l-1. Zinc phytotoxicity was evident by the reduction in chlorophyll content. Highest Zn concentrations were detected in the roots. Certain micronutrients appear to be redistributed due to Zn toxicity. The effect of microelements (Cu, Zn) and heavy metals (Cd, Pb, Hg) on germination and seedling development of Bowiea volubilis, Eucomis autumnalis and Merwilla plumbea was investigated. Copper and Zn applied at 1 mg l.1 significantly reduced the percentage germination of E. autumnalis. Low concentrations (. 1 mg l.1) of Cu and Zn negatively affected the root growth of all three species. Mercury concentrations of 0.5 and 1 mg l.1 significantly decreased the percentage germination of B. volubilis and E. autumnalis respectively. Cadmium and Hg at 2 mg l.1 showed a negative effect on the root growth of B. volubilis. Concentrations of 0.5 mg l.1 of all heavy metals tested significantly decreased shoot length of M. plumbea. The effect of Cd on biological activity (anti-inflammatory, antibacterial and antifungal) of medicinal plants with previously confirmed activity was evaluated. When supplied with Cd at 2 mg l-1, Eucomis humilis bulbous extracts showed lower anti-inflammatory activity than the control for both COX-1 and COX-2 activity. Eucomis autumnalis bulbous extracts had greater COX-1 activity compared to the control. However, Cd suppressed the activity of COX-2. Compared with non-Cd-treated Merwilla plumbea plants (control), those supplied with Cd at 10 mg l-1 showed increased antibacterial activity against Bacillus subtilis, Klebsiella pneumoniae and Staphylococcus aureus. However, no change in activity against Escherichia coli was observed. Cadmium accumulation in the bulbs had no effect on antifungal activity of Tulbaghia violacea. Thus, optimized agricultural practices are essential for quality control of cultivated medicinal plants. The studies presented in this thesis collectively answer several questions related to heavy metal involvement in South African medicinal plants. The findings substantiate the need to regulate and monitor the South African medicinal plant trade against heavy metal contamination which will in turn provide a product of safety and quality to the consumer. / Thesis (Ph.D.)-University of KwaZulu-Natal, Pietermaritzburg, 2008.

Greyia radlkoferi is a South African indigenous tree, which has recently been discovered to be a source of extracts that have a potential in the development of cosmeceutical herbal products having the ability to treat hyperpigmentation disorders. For product development however, G. radlkoferi would need to be available in a commercial scale. Greyia radlkoferi grows naturally in the wild and is often available for cultivation as an ornamental plant. In order to establish this plant into cultivation, suitable propagation techniques must be established for rapid multiplication of trees and thus a sustainable leaf production. For consistent and improved leaf supply to the market, agronomic practices that will enhance leaf production were investigated in the current study. Furthermore, in order to meet market demand in terms of good quality extracts with guaranteed therapeutic efficiency, pre-harvest and post-harvest factors that affect the chemical composition of the extracts were investigated. Recently developed biotechnology techniques such as metabolomics using 1H-NMR and multivariate data analysis offered a platform to study the chemical variation of extracts. Therefore, the current study was aimed at understanding the requirements for propagation and optimum leaf production as well as conditions that favour optimum production of secondary metabolite of G. radlkoferi plant material (at pre and post-harvest) and thus assess its commercial viability. To understand the effects of temperature on seed germination of G. radlkoferi, seeds were exposed to five temperatures (10°C, 15°C, 20°C, 25°C and 30°C) in the incubators in the laboratory. Germination of G. radlkoferi by seeds was discovered to be temperature dependent. The optimum germination temperature of 81% was obtained at 25°C. In the case of vegetative propagation by stem cuttings, the effect of cutting position (basal or apical), exogenous rooting hormone (Seradix1, Seradix 2, 0.1% IBA, 0.3% IBA and 0.8% IBA) and cutting position were investigated in the glasshouse. The cutting position had a significant effect on rooting of G. radlkoferi cuttings with basal cuttings exhibiting 35% rooting as compared to 6% rooting attained for the apical cuttings. A clear trend in rooting response to application of rooting hormones was observed, with 0.1% Indole butyric acid (IBA) showing the highest rooting percentage of 63%. Considering the outcomes of the propagation studies as well as the limited material for vegetative propagation, seed propagation appears to be the most suitable technique for large-scale multiplication of G. radlkoferi. The effect of different pruning techniques as well as harvesting frequencies on fresh and dry weights of G. radlkoferi leaves were evaluated. Factors considered were four pruning treatments (‘pruned but not tipped’, ‘tipped but not pruned’, ‘not pruned nor tipped’ as well as ‘pruned and tipped’) and three harvesting periods (monthly, bimonthly and once–off). Bimonthly harvests highly increased leaf production compared to trees that were harvested monthly and once-off with higher leaf fresh weight yield of 238 g per tree or 2.38 tons/per hectare as well as dry weight yield of 83 g per tree or 0.830 tons/hectare. This outcomes of this study further suggested that a suitable pruning practice for G. radlkoferi would be to either ‘prune only’ or ‘cut back the main stem’ rather than a combination of the two treatments. The influence of seasons (summer, autumn, winter and spring) on the anti-tyrosinase activity and metabolomics profile of G. radlkoferi leaf extracts were investigated. Seasons significantly influenced the chemical composition and the efficacy of the plant extracts. Tyrosinase enzyme inhibition was investigated against monophenolase (tyrosine) with kojic acid as positive control. The highest tyrosinase inhibition concentration with IC50 (50% tyrosinase inhibition concentration) value of 30.3±1.8 μg/ml were obtained in winter harvested leaves compared to the other seasons. The lowest IC50 values were obtained in spring. Metabolomics analysis using orthogonal partial least square discriminant analysis (OPLS-DA) provided a clear class separation according to the harvest season. Extracts from winter harvested leaves contained sucrose, acetamide, alanine and a compound of the catechin group (gallocatechin-(4 alpha->8)-epigallocatechin) as revealed by 1H-NMR metabolomics with assistance of LC-MS. Since compounds of the catechin group are well-known tyrosinase inhibitors, the high tyrosinase activity exhibited in extracts of winter harvested G. radlkoferi leaves could be ascribed to the presence of gallocatechin-(4 alpha->8)-epigallocatechin. Based on the outcomes of the seasonal study, we suggest that in order to obtain extracts with high bioactivity, the best suitable time for harvesting leaf samples is in late autumn-early winter. Processing leaf material using three different drying methods (sun, oven and air drying) significantly influenced chemical composition and the efficacy of the plant extracts. Extracts prepared from air-dried leaf material showed the highest tyrosinase inhibition with IC50 value of 17.80 μg/ml compared to extracts of the other drying methods. Extracts of leaves processed with air drying preserved most metabolites during processing while extracts of sun-dried and oven-dried leaves clearly depleted some metabolites especially amino acids and some aromatic compounds. 1H-NMR metabolomics approach with the assistance of LC-MS data successfully determined a positive association of alanine, acetamide, sucrose and gallocatechin-(4 alpha->8)-epigallocatechin as the chemical constituents contributing to the variation in the air-dried leaves compared to the oven-dried leaves. A positive association of valine, alanine, leucine, isoleucine, gallocatechin-(4 alpha->8)-epigallocatechin and glucose contributed to the variation in air-dried group, compared to the sun-dried group. The highest tyrosinase inhibitory activity exhibited in air-dried samples compared to the other drying methods was associated with the presence of gallocatechin-(4 alpha->8)-epigallocatechin. Because air drying preserved most leaf metabolites compared to sun and oven drying, it was regarded as the most suitable method for processing G. radlkoferi leaf material. This study is the first scientific account that provides guidelines and recommendations to (1) establish G. radlkoferi as a cultivated plant for commercialization, (2) optimize leaf production for sustainable supply to the commercial markets and (3) optimize medicinal content of G. radlkoferi related to harvesting time and post-harvest processing (drying), for enhanced quality of extracts and its products / Agriculture, Animal Health and Human Ecology / Ph. D. (Agriculture)