Dissertations / Theses on the topic 'Medicinal plants Botany'

The methanolic crude extract of aerial parts of the plant Scrophularia nodosa was shown to have potent DPPH radical scavenging activity (IC50 = 48.75 ± 7.00 μg/ml). Activity-guided fractionation resulted in the isolation of three principal antioxidant compounds; acteoside, angoroside C and angoroside A. Acteoside (yield = 1.21%, IC50 = 15.2 μM) appeared to be the most abundant and most antioxidant-active. The potent antioxidant activity is in support of the traditional use of the plant for wound healing and anti-inflammatory conditions. The methanolic extract of aerial parts of Tanacetum vulgare has potent DPPH radical scavenging activity (IC50 = 37.00 ± 1.20 μg/ml). Activity-guided fractionation on the methanolic extract of T. vulgare resulted in the isolation of 3,5-di-caffeoylquinic acid (3,5-DCQA), axillarin and luteolin. 3,5-DCQA appeared to be the most abundant and most antioxidant-active compound (yield = 7.28%, IC50 = 9.7 μM). The potent antioxidant activity is in support of the traditional use of the herb for fever, rheumatic conditions and anti-inflammatory conditions. The methanolic crude extract of Cassia auriculata and its fractions were shown to have potent scavenging activity on DPPH, hydroxyl and hydroperoxide radicals, moderate superoxide radical scavenging activity and potent ion(III) reducing power. The activity-directed studies resulted in the isolation of kaempferol-3-0-β-D-rutinoside, kaempferol, luteolin, quercetin and unknown antioxidant inactive compound. The previously reported pharmacological aspects of the above flavonoids and flavonoid glycosides (anti-carcinogenic, anti-inflammatory, hyperglycaemic, antidiabetic) along with the shown antioxidant behaviour explain the traditional medicinal values of the plant. Cassia alata L crude extract and its fractions showed potent radical scavenging activity against formation of lipid peroxide radicals. The activity directed isolations resulted in the isolation of kaempferol along with p-hydroxybenzoic acid. These may contribute towards the traditional medicinal values of the plant as an antidiabetic, anti-microbial and for skin diseases.

Four endemic medicinal plants from the Yucatan peninsula belonging to genera with little pharmacological and phytochemical reported information and used for medicinal purposes by local communities were selected. The species selected included Jacquinia flammea Millsp. ex Mez, Sideroxylon foetidissimum Jacq. subsp. gaumeri, Serjania yucatanensis Standl., and Serjania adiantoides Radlk. The root, stem/bank and leaves of each plant species were extracted using ethanol and the resulting crude extracts were tested for their cytotoxic effect using the modified MTT (3-[4,5-Dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide) assay followed by a bioassay-guided fractionation of the most active extracts in order to identify the active metabolites. The initial cytotoxic evaluation against HeLa cells at two fixed concentrations (100 and 33.3 µg/mL) identified the root extracts f J. flammea, S. foetidissimum subsp. gaumeri and S. yucatanensis, and the stem/bank extract of S. adiantoides as the most active extracts. The crude extract of roots of J. flammea was subjected to solvent partition using solvents of ascending polarity (pet. ether, CHCI3, EtOAc, BuOH and water). The resulting fractions were tested for their cytotoxic activity. The water fraction of the solvent partition showed the strongest activity against HeLa cells (IC50 = 28.61 ± 2.27 µg/mL). When tested against RAW 264.7 cells, the water fraction also showed significant activity (IC50 = 10.60 ± 1.83 µg/mL). The water fraction was subjected to chromatographic fractionation using open silica gel columns resulting in the isolation of a saponin as the most active metabolite against RAW 264.7 cells (IC50 = 4.76 ± 0.32 µg/mL). The isolated compound was identified using 1D (1H and 13C and DEPT-135) and 2D (COSY, HMBC, HSQC and NOESY and ROESY) NMR and mass spectrometry analysis as sakurasosaponin. The molluscicidal and antifungal activities of sakurasosaponin have been reported but no studies on its cytotoxic activity have been previously reported. The crude extract of roots of S. foetidissimum subsp. gaumeri was subjected to solvent partition using solvents of ascending polarity (pet. ether, CHC13, EtOAc, and BuOH). The resulting fractions were tested for their cytotoxic activity. The BuOH extract of S. foetidissimum subsp. gaumeri showed the strongest activity against RAW 264.7 cells (IC50 = 35.12 ± 4.32 µg/mL) and it was subjected to further chromatographic fractionation using open silica gel columns yielding mixtures of saponin-containing fractions. The crude extract of roots of S. yucatanensis was subjected to solvent partition using solvents of ascending polarity (pet. ether, CHCI3, EtOAc, and BuOH). The resulting fractions were tested for their cytotoxic activity. The crude extract of S. adiantoiodes did not show cytotoxic activity when tested against RAW 264.7 cells.

Thesis (Ph. D.)--Harvard University, 1975.
"Annex: 'Renaissance humanism and botany, ' Annals of science 33 (1976), 519-542 [and] 'Publishing scholarly books in the sixteenth century, ' Scholarly publishing, April 1983, 259-274." Includes bibliographical references (p. 261-283) and index.

Up until the early 1990s conservation practices in South Africa were culturally biased, focusing largely on the value systems of the affluent. However, with the release in 1997 of the White Paper on the Conservation and Sustainable Use of South Africa's Biological diversity, the role that biological resources play in providing for the needs of all South Africans, is now emphasized. According to this policy, human needs must be considered if conservation is to be successfully implemented. Using this document as the framework for this study I chose to investigate various aspects of medicinal plant use in a rural community in Paulshoek, Namaqualand. The main aims were as follows: to evaluate the local knowledge regarding medicinal plants; to document the plants used and collected in Paulshoek; and to determine potential threats to the biological resource. This was achieved by employing a variety of social and ecological methods. It became apparent from the interactions and interviews with the residents that medicinal plants are an important resource to the Paulshoek community since more than 70% of the population regularly use herbal remedies. While there is some evidence to suggest that the local knowledge of medicinal plants is dying out, I would speculate that most of the knowledge has already been lost. Of the 15 plants used and collected in Paulshoek, most appear to be highly sustainable in the landscape. This conclusion was based on people's perceptions regarding the change in abundance of each of these species over time and by further comparing plant size between Paulshoek and adjacent commercial farms. As most medicinal species seem unaffected by either: harvesting or land use practices this indicates that it is possible to achieve a sustainable harvest. Certain species do, however, show evidence of decline. Fuelwood harvesting most probably accounts for the change in abundance of Rhus burchelli over time, while Mentha longifolia may be facing some reduction in plant fitness due to harvesting for medicinal purposes. Sceletium emarcidum is on the verge of local extinction due to a combination of intensive harvesting and high grazing pressures. In contrast, high stocking densities appear to account for the increased abundance in both Galenia africana and Ballota africana. These findings clearly show that while the resource as a whole may be fairly resilient to harvesting and land use practices, certain species are in need of urgent conservation. This study further highlights the need to look beyond the direct impacts of harvesting and consider all possible threats, if the resource is to be sustainably managed. While this case study is atypical of the state of the medicinal plant resource in most of South Africa, this survey serves as a novel protocol for evaluating the sustainability of any resource which is regularly utilized.

Diabetes mellitus (DM) is a group of non-infectious diseases that cause hyperglycemia. DM symptoms were first clinically described by ancient Greek physicians whose prescriptions included plant-based remedies. Today, DM affects >400 million people globally and prevalence rates are rapidly increasing in developing countries where basic healthcare relies on local knowledge of botanical remedies. Many developing countries are home to diverse peoples and plants—providing fodder for varied plant-selection strategies and unique botanical pharmacopoeias. I addressed the plant-selection strategies used in a multi-ethnic, developing country, Trinidad and Tobago (T&T), to ascertain their role in shaping the local antidiabetic pharmacopoeia and to assess their benefits and risks in identifying safe and useful remedies. Using literature reviews, field surveys, and laboratory bioassays, I completed three categories of analysis. Ethnobotanical analyses showed that T&T’s antidiabetic pharmacopoeia is primarily of recent origin as >50% of the 48 historical DM remedies were Neotropical natives, including congenerics of well-known medicinal Paleotropical genera. Nevertheless, conservative knowledge transmission was also evident as several Paleotropical species of T&T’s pharmacopoeia, including Momordica charantia and Catharanthus roseus were also used in Africa, India and across the Caribbean. Paleotropical natives with a long history of use are likely to be safer remedies. Ethno-medicinal analyses of the pre- and post-2000 DM remedies of T&T, totaling 99 species, suggest that the centuries-old hot/cold folk disease-model was the model predominantly used in plant-selection. Parallels found between T&T folk concepts and biomedical mechanisms of DM provide probable bases for efficacy but the chronic use of purgatives and bitter-tasting plants is likely to be risky. Phytochemical analyses revealed that 69% of the tested plant extracts contained phenolic compounds, with more than half producing >80% alpha-glucosidase inhibition. Phenolic content and alpha-glucosidase inhibition were strongly correlated among food plants used as medicines, suggesting higher probability of selection as a result of non-target effects. The medicinal use of food plants may provide the best margins of safety and efficacy in identifying antidiabetic remedies. Together, these analyses showed how culture-specific plant-selection strategies can identify safe, useful remedies for developing countries to address their increasing DM prevalence in a cost-effective and sustainable manner.

Hermannia incana Cav. (Sterculiaceae), known as sweet yellow bells, is a medicinal plant used by the people of the Eastern Cape for the treatment of stomach-ache and diarrhoea. It has purgative and diaphoretic effects. It is a prostrate herb with yellow flowers and sparsely hairy and slightly glandular leaves, occurring in grassland and marshes in the Eastern Cape Province of South Africa. Based on the ethnomedical uses of this plant, the research project was designed to evaluate its antidiarrhoeal and toxicological properties. An ethnobotanical study of plants used for the treatment of diarrhoea in the Eastern Cape Province was carried out, using a questionnaire which was administered to herbalists, traditional healers and rural dwellers. This survey indicated a total of 17 plant species from 14 families. Elephantorrhiza elephantine (Burch.) Skeels, Hermannia incana Cav., Pelargonium reniforme Curt., Alepidea amatymbica Eckl. & Zeyh. and Bulbine latifolia (L.f.) Roem. et Schult. were the most frequently mentioned and highly recommended plants for the treatment of diarrhoea by both the traditional healers and rural dwellers. The root, bark and leaves are the common parts of plants used, while decoctions and infusions are the main methods of preparation. The agar dilution method was used to study the antimicrobial activity. The methanol extracts of the plant showed appreciable activity against Gram-positive and Gram-negative bacteria at concentrations ranging from 0.5 to 7.0 mg/ml. The acetone and water extracts of both the leaves and the roots showed moderate activity against Gram positive bacteria and less activity against Gram negative bacteria. All the extracts inhibited the growth of the fungi Aspergillus flavus, Aspergillus niger, and Mucor hiemalis with growth inhibition ranging from 54.31 percent to 96.67 percent at 0.1-10 mg/ml. None of the extracts suppressed the growth of Candida albicans at the maximum concentration (10 mg/ml) tested. iii In the in vivo antidiarrhoeal evaluation using Wistar rats, the aqueous extract at all the doses tested, significantly prolonged the time of induction of diarrhoea and also reduced the frequency of diarrhoeal episodes and fecal parameters (total number, number of wet, fresh and dry weight and water content of the faeces). The percentage inhibition of defecation and intestinal content (enteropooling) were increased in dose dependent manner. The doses also reduced the intestinal transit time of charcoal, masses and volumes of intestinal fluid (gastrointestinal motility). These results are indications of antidiarrhoeal property of H. incana leaf extract with the 600 mg/kg body weight of the extract being the most effective. In the toxicological evaluation using Wistar rats, the oral administration of the extract did not produce any significant effect on the liver and kidney body weight ratios, RBC, HB, PCV, MCV MCH, MCHC, RCDW, WBC, neutrophils, monocytes and basophils cholesterol, triacylglycerol, high-density lipoprotein cholesterol, low-density lipoprotein cholesterol and atherogenic index. The extract also did not affect the levels of sodium, potassium, chloride, inorganic phosphorus, urea, creatinine, total protein, globulin, albumin, total and conjugated bilirubin. The activities of alkaline phosphatase, gamma glutamyl transferase and alanine aminotransaminase in the serum were increased by the extract whereas aspartate aminotransaminase was decreased. The levels of LUC, platelets, lymphocytes and eosinophils were significantly affected at 600 mg/kg body weight. The available evidence in this study suggests that the extract of H. incana leaf is mild, parameter and dose specific. The structure and distribution of foliar appendages on the leaves of this plant were investigated with the JEOL (JSM-6390LV) scanning electron microscope (SEM). Both glandular and non-glandular trichomes were observed. Long stalked glandular trichomes were present on both the abaxial and adaxial surfaces while short stalked glandular trichomes were present only on the adaxial surface. Glandular trichomes were capitate while nonglandular trichomes were stellate with many arms. Energy dispersive X-ray spectroscopyiv SEM showed that Al, Ca, K, Na, Ti and Si were the major constituents of the crystals analyzed from the leaf surfaces. The phytochemical screening of H. incana revealed the presence of bioactive antidiarrhoeal agents such as alkaloids, tannins, saponins, phenolics, triterpenes, cardiac glycosides, flavonoids, cardenolides and dienolides. Two flavonoids, epicatechin and 3, 5, 7, 2’ tetra-hydroxy flavone-3- O--D-glucopyranoside were isolated from the leaves of the plant through bio-active guided fractionation. Both these compounds were screened against diarrhoea causative organisms (Echerichia coli, Shigella flexneri, Bacillus cereus and Staphylococcus aureus) and exhibiting minimum inhibitory concentrations ranging from 12.5 to 100 μg/ml. The findings from this research have generally justified the traditional use of this plant for the treatment of diarrhoea in this province.

Morindae Officinalis Radix (MOR), Bajitian in Chinese, is the dried root of Morinda officinalis F.C.How. (Rubiaceae). It is one of the most popular herbal medicines used in the southeast region of China. Various types of chemical constituents have been experimentally shown to be bioactive components of MOR, among which secondary metabolites and saccharides predominate. Pharmacological studies revealed MOR shows kidney tonifying, anti- osteoporosis, antidepressant, anti-inflammatory and antioxidant effect. Since 2002, MOR has been approved as a food supplement for daily healthcare, hence increasing consumption and demand for better quality of MOR. However, selection of MOR with superior quality is largely based on traditional experience which lacks scientific basis. For example, 3-4-year-old MOR is usually used without xylem; and processed MOR are believed to show different bioactivities. Therefore, to promote the rational utilization and ensure efficacy of MOR, overall qualitative and quantitative characterization of MOR in different traditional usage is needed. Anthraquinones, iridoid glycosides and oligosaccharides are the common reference compounds for chemical characterization of MOR. However, they are usually selectively characterized, which is not comprehensive enough in herbal quality evaluation. To deal with this, metabolomics targeting secondary metabolome and glycomics targeting glycome can be applied. And the integration of metabolomics and glycomics could be a promising approach to investigate overall chemical variations in MOR according to its traditional usage. Therefore, in this study, chromatographic methods for metabololmics and glycomics were firstly developed to study the traditional usage of MOR. In Chapter 2, they were applied for studying chemical variation and differences in growth year and plant tissue of MOR. In Chapter 3, chemical differences in processed products of MOR were also studied using the established metabololmics and glycomics methods. Further bioactivity differences of them were studied by cell metabolomics with HEK 293 cells under high glucose microenvironment. Besides that, in Chapter 4, consumption method of not only MOR, but other herbal medicines were studied. Conventional boiling water extraction (BWE) and ultrasound-assisted extraction (UAE) were compared to understand their effects on polysaccharides. For the study of growth year and tissues of MOR, the results showed that various types of bioactive components reached a maximum between 3-4 years of growth; and that xylem contained more potentially toxic constituents, but less bioactive components, than cortex. For the study of processing products, the results showed that secondary metabolome and glycome of raw MOR and other processing products was found qualitatively and quantitatively different. Contents of secondary metabolites were generally increased in processed products, while saccharides were decreased instead. Also, steamed MOR (F) seemed to show preventive effect of diabetic nephropathy and different MOR processing products had induced different metabolic changes on high glucose induced HEK 293 cells. In the study of extraction methods, the results showed that the polysaccharides from the herbal medicines by UAE were quantitatively and qualitatively different with those by BWE. The powerful extraction ability and polysaccharide degradation caused by ultrasound collectively contributed to these differences. It was revealed that not only the UAE conditions but also the polysaccharide structures could affect the extraction ability and polysaccharide degradation To conclude, metabolomics and glycomics were integrated in this study to investigate the variations in secondary metabolome and glycome in MOR. We had successfully applied these methods to study and provide scientific basis for traditional practice of MOR. We had proved that 3rd to 4th years of growth are the key period for the development of the biochemical signature of MOR. Xylem and cortex of MOR were qualitatively and quantitatively different and removing xylem could help to remove potentially toxic components. This study also provided scientific evidences for the justification of MOR and its processed products, as well as their metabolic effects on high glucose induced DN in HEK 293 cells. Besides, this study revealed both UAE parameters and structural properties of polysaccharides affects extraction recovery of polysaccharides in herbal medicines. Hence, we suggest UAE should be carefully considered before employing it in relevant chemical and pharmacological analysis.

This quantitative study seeks to determine the impacts of harvesting three plant species traditionally used for wound healing during circumcision. Three localities where these plant species occur have been identified. The population size for each species was determined and an assessment of the extent of harvesting was determined through repeated assessment of marked plants. A significant harvest of these species resulted in the unsustainable use of our natural resources. Out of 25 Boophone disticha plants marked, only one plant was remaining after two circumcision seasons. The Brunsvigia grandiflora and Scadoxus multiflorus populations monitored disappeared completely, with no single marked plant found after two circumcision seasons. Growth rates of wild populations of Boophone disticha and cultivated Brunsvigia grandiflora and Scadoxus multiflorus plants were determined. The seedling bulbs of Brunsvigia grandiflora grew significantly more slowly at less than 0.6 cm per year, while Scadoxus multiflorus grew faster at over 1 cm per year. A model for population dynamics of the three plant species was designed which showed that the plants are being harvested before they even flower. It is therefore recommended in this study that a conservation plan for these plant species must be done to save the little that is left in the wild before they become critically endangered.

Pelargonium sidoides DC. (Geraniaceae) root extracts are used in the Eastern Cape Province of South Africa as a traditional medicine for the treatment of respiratory tract and gastro-intestinal infections. Ethanolic extracts are used globally as herbal treatments for bronchitis, asthma and as an immune system booster. Despite documented exploitation of wild populations by illegal harvesters, this species has not been awarded a protected status. The high level of harvest in the years preceding this study prompted this investigation of the prospects for sustainable root harvest through wild harvest and greenhouse cultivation. A novel method was developed for the purification of umckalin, a bioactive constituent in root extracts, such that the root umckalin concentrations of wild and cultivated plants could be quantified by HPLC. As part of the cultivation experiments, the concentration of umckalin in roots was measured for plants across part of the species’ distribution range in the Eastern Cape Province. This survey revealed that root umckalin concentrations were inversely related to the average annual rainfall of the collection site (r² = 0.94, p = 0.007) and directly related to soil pH (r² = 0.97, p = 0.002). Thus, the possibility of inducing high umckalin concentrations in greenhouse-cultivated plants was investigated by subjecting plants to rapid and prolonged water stress treatments. Two leaf applied hormone treatments (cytokinin and gibberellin) and a root competition treatment with a fast growing annual (Conyza albida) were also investigated based on the potential function of umckalin in P. sidoides plants. These five treatments did not significantly affect root umckalin concentrations compared to well-watered controls. The results of further experiments suggested that umckalin production may have been influenced by the geographical origin and genetics of plants rather than environmental variation. Following wild harvest experiments, the regrowth of replanted shoots from which a standard proportion of the root was harvested showed that water availability affected shoot survival but not root regrowth rate. Regrowth rates were low, questioning the viability of wild harvest. In contrast, greenhouse cultivated plants showed ca. six times greater growth rates, supporting the cultivation of roots to supply future market demand.

Este estudio se realizó en la región naua de la Huasteca (México), que se encuentra en la parte noreste del país y en que confluyen los estados de Veracruz, Hidalgo y San Luís Potosí. El objetivo de esta tesis fue Investigar los recursos herbolarios y las prácticas terapéuticas utilizados por el pueblo naua de la Huasteca. Para ello se realizaron entrevistas a ocho médicos indígenas de esta región, y además se realizó una búsqueda bibliográfica de la misma y de los saberes médicos indígenas y demás relacionados con el tema. De esta investigación se colectó información sobre 136 especies de plantas medicinales utilizadas en la región naua de la Huasteca, cuyas familias botánicas mayormente representadas son Asteraceae, Solanaceae, Fabaceae, Lamiaceae, Euphorbiaceae, Rutaceae y Myrtaceae, lo que muestra la diversidad biológica de esta región, del total de las especies el 53% son hierbas, 33% son arbustos y 14% son árboles, lo que está acorde con el tipo de vegetación de bosque tropical y con el mayor uso antropogénico: el estrato herbáceo; el 76% son nativas y el 24% son introducidas, lo que demuestra un amplio uso y conocimiento de la flora medicinal local; el 40% de la totalidad colectada son también comestibles. La mayoría de las plantas nativas son colectadas en la vegetación secundaria, es decir, en donde la actividad antropogénica tiene una mayor incidencia. La tisana es forma de preparación mayormente usada con un 51% de las plantas, un 21% se aplica directamente, sin ningún tipo de preparación, existes otros tipos de preparación como cataplasmas, baño, limpia, tallada o frotada en el cuerpo. Todo lo anterior es usado dentro de la cosmovisión médica de los nauas de la Huasteca, mayoritariamente por los médicos indígenas para sanar lo corpóreo y lo espiritual o tonali; en concordancia con su pensamiento médico, en donde el equilibrio entre estos dos elementos es lo que integra el concepto de salud, en esto último se encuentran padecimientos como el mal aire, susto, mal de ojo, entre otras, para fines académicos estas enfermedades se engloban en lo que la antropología médica ha denominado como los síndromes de filiación cultural. De tal forma que se pude hablar de un conocimiento médico estructurado por parte de los pobladores de esta región, que responde a las necesidades de curación enmarcado dentro de su contexto sociocultural. Todo este conjunto de conocimientos es lo que conforma la cosmovisión médica de los nauas de la Huasteca. Sin embargo, existe una contraposisión entre este modelo médico y la medicina académica, que es el otro modelo médico existente en la región, en ella existe una clara subordinación de la medicina indígena a la académica que además de ser la medicina oficial, le representa un modelo empírico, caduco o rebasado. Para explicar este fenómeno hemos propuesto la categoría y el término de diglosia médica; que hace visible esta relación y que al adjetivarla se pretende facilitar el estudio y abordaje de este fenómeno. Finalmente, se proponen algunas directrices de acción basados en los resultados obtenidos en esta investigación de hacia donde se debería de caminar para que en un futuro se pueda hacer realidad el derecho que tienen los pueblos indígenas a contar con una medicina con pertinencia cultural, mediante el acercamiento de ambos modelos en una ambiente de respeto y de igualdad es decir, desde una relación dialógica en el ámbito médico.
This research was performed in the Huastec region, in the territory of the naua people in Mexico, located in the northeast of this country, in a land where the Veracruz, Hidalgo and San Luís Potosí states converge. The objective of this PhD thesis was to investigate the use of medicinal plants and the therapeutic practices used by the indigenous nauas of the Huastec region. Participant observation and ethnobotanical interviews were conducted to eigth indigenous medical practitioners of this region, and also archive and literature searches were conducted about this region, wisdom of the indigenous medical practitioners and other related topics. Information was collected about 136 medicinal plants, whose most represented botanical families are Asteraceae, Solanaceae, Fabaceae, Lamiaceae, Euphorbiaceae, Rutaceae and Myrtaceae, of which 53% are grasses, 33 % shrubs and 14 % trees, 76% are native and 24% introduced, and 40% of the reported plants are also edible. The infusion is the most used method of preparation, for 51% of plants, whereas 21% are applied directly, without any preparation. The above-mentioned plants are used in the frame of the medical worldview of the naua people of the Huasteca, especially by the indigenous doctors to heal both the body and the spirit. For academic purposes, many diseases of nauas of the Huastec region are included into syndromes of cultural affiliation. This set of knowledge and skills constitutes the medical worldview of the nauas of the Huastec region. There is a contrast between this medical model and academic medicine, which is the other existing medical model in the region, in which there is a clear subordination of indigenous medicine to academic one. To explain this phenomenon, we have proposed the concept and term of medical diglossia, because of the analogy with this linguistic concept and, indeed, because both medical patterns are practiced in different languages (Nauatl for the indigenous one, Spanish for the official one); this is intended to make this phenomenon visible and to enable in the future indigenous peoples to have a culturally congruent medical model by bringing both medical kinds of practices together with no subordination between each other.

This thesis analyses how Andean people’s knowledge of medicinal plants and the relationship between environment and health is represented, transmitted and commoditised in the marketplaces of the department of Oruro, Bolivia. Considering the increase in urban population and their dependence on marketplaces for medicinal plant remedies, this thesis examines the role of marketplaces and the importance of specialist stallholders in the transmission of knowledge. The central research site of Oruro is a multi-cultural city located on the Andean plateau in southwest Bolivia, a population of Spanish, Quechua and Aymara speakers with a pluralistic medical system. Fieldwork was carried out over 18 months with market stallholders in Oruro combining quantitative and qualitative methods with ethnographic documentation of knowledge transmission events. This thesis found that medicinal plant marketplaces in Oruro are highly regulated social systems that incorporate Andean socio-economic mechanisms, including ritual performance for the transmission of cultural knowledge, and the regulation of resource distribution and use. The development of a ‘chemical landscape’ model demonstrated that social exchange and trade between ecosystems and altitudinal zones broadens the spectrum of medicinal compounds available, contributes to the complexity of herbal mixtures and can limit exploitation of local plant populations. The market stallholders use specialist classifications that identify chemical properties, toxicity and variations between plant species and ecological regions. Plant classifications varied with the context and location in which they were used, and humoral classification enabled the selection and combination of plants in mixtures and justified remedy efficacy for specialists and non-specialists. Andean cultural beliefs including complementary opposites enabled transmission of knowledge on the medicinal properties of plants between highland consumers and lowland producers, and defined traditional Andean mixture efficacy. These findings demonstrate that, although state intervention and identity politics are redefining perceptions of medicinal knowledge, the market exchange system centred in Oruro city creates localised specialist knowledge and continuity of cultural knowledge transmission.

A Review of manuscripts written by European explorers and colonists affords the opportunity to develop a clearer understanding both of types of plants employed and their significance in religion and medicine during the 16th to 19th centuries. This paper is a distillation of accounts by thirteen European explorers, written between 1545 until 1861, about Laos and the Lao people in Siam. All of the references to plants and plant use have been extracted for an analysis of which plants European explorers viewed being used traditionally in Laos during this time period and information on how these plants were used and collected. Many of the plants described in the texts were medicinal in nature and some have been examined for modern pharmaceutical use. These pharmaceutical studies have substantiated the effectiveness of historical medicinal plant use. The texts also describe plants that were used in religious ceremonies and that continue to play an important role in Lao culture. Future comparative analysis of these early records with modern day observations of plant use should prove productive in formulating assessments of Traditional Environmental Knowledge loss and the impact of this loss on daily life. Understanding the plants that are important to native Lao in the past can lead to better methods of conservation in the future.

Cancer and malaria are among the most life-threatening diseases globally. Cancer is responsible for about 125,000 annual deaths globally. In 2015, the World Health Organization report estimated that 236000-635000 people died of malaria. These diseases are complicated by the development of resistance to available chemotherapeutic agents. Natural products have been recognized for their major applications in the identification of drug leads in drug discovery. Viola philippica Car, Viola yedoensis Makino (Violaceae), Triclisia subcordata Oliv (Menispermeaceae) and Cyclicodiscus gabunensis Harms (Fabaceae) are medicinal plants traditionally used for the treatment of various diseases including malaria or cancer in China and West Africa. However, the bioactive compounds are unknown. Therefore, this study evaluated the in vitro anticancer and antimalarial activities of the four medicinal plants and searched their bioactive compounds. The in vitro anti-ovarian cancer and antimalarial assays were demonstrated respectively using sulforhodamine B dye and Syber green 1 fluorescence assay methods. Bioassay-guided fractionation and purification were performed. Structural elucidation was performed by nuclear magnetic resonance spectroscopy and mass spectrometry analysis. Results revealed the anticancer and antimalarial activities of T. subcordata; V. philippica, and V. yedoensis to be bisbenzylisoquinoline alkaloids (cycleanine, isochondodendrine and 2′-norcocsuline) and/or cyclotides. The cycleanine analogues were synthesized and found to be more potent than cycleanine. Induction of apoptosis by these alkaloids has also been determined. This study could serve as basis for the support of use of these plants in cancer and/or malaria treatment. The BBIQ alkaloids and analogues could serve as lead compounds in drug discovery. Future in vivo studies need to be carried out on these alkaloids to get drug approval.

Siphonochilus aethiopicus (Schweinf.) B.L. Burtt (Zingiberaceae), commonly known as wild ginger, is a highly sought after plant for use in traditional medicine in South Africa. Over-exploitation of this medicinal plant has resulted in regional extinction in the wild. As a result, there is great interest in the medicinal properties of S. aethiopicus, and as a plant for small scale cultivation to increase the supply for use in traditional medicine. Water, ethanol and ethyl acetate extracts were prepared from the leaves, rhizomes and roots of S. aethiopicus. These extracts were tested for in vitro anti-inflammatory activity in the cyclooxygenase-1 (COX-1) and cyclooxygenase-2 (COX-2) assays, and in the microdilution antibacterial assay. The aqueous extracts showed no significant prostaglandin synthesis inhibition in the COX-1 and COX-2 assays. The ethanol and ethyl acetate extracts of the leaves showed the highest levels of activity at a concentration of 250 µg ml¯¹ per test solution, in both the COX-1 and COX-2 assays. The ethanol and ethyl acetate extracts of the rhizomes and roots also had moderate levels of activity in the COX-1 assay. These results provide some evidence for the rational use of S. aethiopicus in traditional medicine for anti-inflammatory purposes. In the microdilution antibacterial assay, no inhibitory activity against the test bacteria was detected with the aqueous extracts. The ethanol and ethyl acetate extracts tested showed greater antibacterial activity at minimal inhibitory concentrations ranging from 0.78 to 3.13 mg ml¯¹ against the gram-positive bacteria (Bacillus subtilis, Staphylococcus aureus) than the Gram-negative bacteria (Escherichia coli, Klebsiella pneumoniae). No distinct differences were observed between the ethanol and ethyl acetate extracts, or between the different plant parts. A serial extraction of S. aethiopicus rhizome material was conducted and the extracts were tested in the COX-1 assay and the microdilution assay as a preliminary investigation for a bulk extraction. The hexane and ethyl acetate extracts gave slightly higher COX-1 inhibition than the ethanol extract. No distinct differences were observed in the microdilution assay. A bulk ethyl acetate extract of S. aethiopicus rhizome material was prepared, yielding 6.3 g of a thin orange oil. Vacuum liquid chromatography (VLC) was used to fractionate ≈4 g of the extract. The VLC fractions were evaluated using thin layer chromatography (TLC) and a bioautographic assay, using S. aureus as a test organism. The fractions were also tested in the COX-1 assay. The bioautography revealed a number of compounds which exhibited antibacterial activity. Fraction C was purified further using preparative TLC, and 24.9 mg of a pure compound from R,0.54 (toluene:ethyl acetate 93:7) was isolated. The structure of the compound was elucidated from nuclear magnetic resonance (NMR) spectra, and mass spectroscopy of the compound was also recorded. The compound was identified as the sesquiterpenoid furanoeremophil-2-en-1-one, which is structurally identical to the recently reported compound 4aαH-3,5α,8aβ-trimethyl-4,4a,9-tetrahydro-naphtho[2,3-b]-furan-8-one. The compound showed only a very minimal bacteriostatic effect in the microdilution assay. S. aethiopicus plants were harvested before and after seasonal senescence. Ethanol extracts were prepared from fresh or dried material of the leaves, rhizomes and roots, and tested in the COX-1 assay and the microdilution assay TLC fingerprints of the various extracts were also prepared. No noteworthy changes in COX-1 inhibition, due to senescence, were observed with extracts prepared from fresh material, although there did appear to be a slight decrease in activity in the α-roots and an increase in the β-roots after senescence (fresh and dry). A decrease in the antibacterial activity of the leaves and an increase in the antibacterial activity of the α-roots was observed after senescence. These results suggest that the time of harvest may only have a minimal influence on the degree of anti-inflammatory and antibacterial activity.
Thesis (M.Sc.)-University of Natal, Pietermaritzburg, 2002.

Schumanniophyton magnificum and Glypheae brevis are important medicinal plants growing wild in the West African rain forest. They are used in folkloric medicine in the treatment of epilepsy and convulsion as well as for some other diseases. The purpose of this work was to investigate the aspect of folkloric use in order to support folkloric claims and document the findings. The extracts were prepared from ground plant material by a continuous extraction method. Five hundred grams of ground plant material were continuously de-fatted with 2 L petroleum ether (60°- 80°) in a Soxhlet apparatus for about 5 h. The resulting marc was dried and the chemical constituents extracted hot in a Soxhlet apparatus for about 8 to 10 h with 2 L aqueous ethanol (70%). The efficacy of the extraction method was confirmed using standard bioassays and phytochemical analyses. The anti-convulsant activity of the crude extracts was evaluated in vivo against chemically induced convulsions using three different animal models, namely the strychnine, the picrotoxin and the pentylenetetrazole tests. The acute and delayed toxicity test results showed that in all the animal models investigated very high doses, about four times higher than the protective doses of the extracts, were required to kill 50% of the population of animal used. Phytochemical assays of the extracts indicated the presence of alkaloids only in S. magnificum root extract and glycosides in extracts from both species. The glycosides were positive to Baljet, Xanthydrol and Keller-Kiliani tests for cardiac glycosides. S. magnificum and G. brevis chemical constituents were initially isolated with a sequential fractionation method starting with a highly non-polar solvent and gradually increasing to a more polar solvent. The fractions were pooled on the basis of TLC similarity profiles when viewed under the UV light at 254 and 366 nm and were found to have two and four major UV absorbing fractions for S. magnificum and G. brevis respectively. Radio-receptor binding tests were used to assess the anti-convulsant activities of the hydro-alcoholic crude extracts, the organic and aqueous fractions of the crude extracts, partially purified components and pure components in in vitro tests against some standard GABA[A] receptor antagonists, muscimol and isoguvacine respectively. The anti-convulsant activities resided in the aqueous fractions of the hydro-alcoholic crude extracts of both plants. The purely organic fractions of G. brevis demonstrated no activity while all the fractions of the aqueous component demonstrated some degree of activity. The anti-convulsant activity of S. magnificum was found only in one fraction-Fraction 1. This Fraction was further investigated and one of the components appear to be responsible for the activity. The structure of the active constituent was 5,7dihdroxy-2 methylbenzopyran-4-one, a noreugenin. A second bioactive compound, schumanniofoside, was identified from Fraction M[5.2] from S. magnificum.
Thesis (Ph.D.)-University of Natal, Pietermaritzburg, 2002.

Petroleum ether, dichloromethane, and 80% ethanol extracts of 15 plant species collected in Nigeria were screened for in vitro antibacterial, anti-inflammatory and antimalarial activities. Antibacterial activity was tested using the agar diffusion method, while the minimum inhibitory concentrations (MIC) of the active extracts were determined using the microtitre serial dilution method. Most antibacterial activity detected was against Gram-positive bacteria with Staphylococcus aureus being the most susceptible. The highest activity was found in petroleum ether and dichloromethane leaf extracts of Mallotus oppositifolius; petroleum ether, dichloromethane and ethanolic root extracts of Newbouldia laevis; and ethanolic root extracts of Morinda lucida and Canthium subcordatum. Against the Gram-negative bacterium Escherichia coli, the highest activity was found in dichloromethane leaf extracts of Newbouldia laevis, ethanolic root extracts of Phyllanthus amarus, Mallotus oppositifolius, and Canthium subcordatum. A total of 60 plant extracts were screened for antiplasmodial activity. A chloroquine sensitive strain of Plasmodium falciparum (D10) was used. In the assay, the parasite lactate dehydrogenase (pLDH) activity was used to measure parasite viability. About 11 extracts showed promising activity with an IC₅₀ ranging from 2.5 to 13.4 µg/ml. The petroleum ether leaf extract of Hyptis suaveolens had the highest activity (IC₅₀ = 2.5 µg/ml). The cyclooxygenase (COX-1 and COX-2) assays were used to test for anti-inflammatory activity. All the plant species, with the exception of Hedranthera barteri and Picralima nitida showed anti-inflammatory activity. Apart for a few ethanolic extracts, all the activities were recorded with petroleum ether and dichloromethane extracts. Employing bioassay-guided activity fractionation, an antibacterial anthraquinone identified as emodin was isolated from ethanolic root extract of Senna occidentalis. Although this compound had been isolated from other sources, this was the first report of isolation from Senna occidentalis. Using a similar approach a novel antimalarial diterpenoid was isolated from the petroleum ether leaves extract of Hyptis suaveolens. It had IC₅₀ of 0.1 µg/ml. This new compound is worthy of further investigation and may act as an important lead compound for future antimalarial drugs.
Thesis (Ph.D.)-University of KwaZulu-Natal, 2005.

In this ethnopharmacological study to isolate, purify, identify and test crude and isolated compounds from organic and aqueous extracts from stem and leaves of Protorhus longifolia and Sclerocarya birrea, stem bark of Hibiscus cannabinus and Heteropyxis natalensis, leaves of Acokanthera venenata, Carissa marcrocarpa and Syzygium cordatum, seeds of Chiononthus foveolatus and calyces of Hibiscus sabdariffa were tested against seven pathogenic microorganisms which included six bacterial species [Klebsiella pneumoniae (ATCC 12265), Bacillus cereus (ATCC 11778), Salmonella typhimurium (ATCC 13311), Pseudomonas aeruginosa (ATCC 27853), Escherichia coli (ATCC 35219), Staphylococcus aureus (ATCC 29213)) and fungal yeast Candida albicans (ATCC 10231)]. Both organic and aqueous extracts from these medicinal plants exhibited antimicrobial properties against one or more mlcroorgamsms. The extracts of stem bark and leaves were tested for antimicrobial properties. Crude extracts that showed the highest activity were analysed through chromatographic and spectroscopic techniques to isolate, purify and characterise their active components. The highly active antimicrobial extracts were further tested for antimicrobial activity. Chromatographic (TLC and CC) spectroscopic (IR, 13C, 1HNMR) analyses of Syzygium cordatum leaf extract in ethyl acetate revealed the presence of C-30 triterpenes, ursolic (3B-hydroxyurs-12-en-28-oic acid) (UA) and oleanolic (3-hydroxylean-12-en-28-oic acid) (OA) acids; a mixture of methyl maslinate (2a, 3B-dihydroxyolean-12-en-28-oic acid methyl ester) (MM) and methyl corosolate (MC). Analyses of Protorhus longifolia leaf extract in hexane and ethylacetate revealed the presence of the alkaloid lupeol (lup-20(29)-en-3pB-ol), lupenone [lup - 20 (29) - en - 3 - one or lup - 20 ( 30 ) - en - 3 - one], lupinine (octahydro-2H-quinolizine-lmethanol), lupulon (3 ,5-dihydroxy-2,6,6-tris(3-methyl-2-butenyl)-4-(3-methy1-1-oxobutyl)-2,4-yclohexadien-1-one) or (3,5-dihdroxy-4-isovaleryl-2,6,6-tris(3-methyl-2-butenyl)-2, 4-cyclohexadien-1-one) and luteolin [(2-(3, 4-dihydroxyphenyl)-5, 7-dihydroxy-4H -1-benzopyran-4-one), 3',4', 5,7 -tetrahydroxyflavone or 5,7,3' 4' - tetrahydroxyflavone] and other compounds to be characterised in future studies. Sclerocarya birrea bark extract in methanol was found to contain mixtures of compounds that could not be separated due to solvent complications. Heteropyxis natalensis stem bark in ethyl acetate gave betunilic acid (3B-hydroxy-20(29)-lupaene- 28-oic acid) as a major compound.
Thesis (M.Sc.)-University of KwaZulu-Natal, 2006.

The post-harvest physiology of nine frequently used indigenous southern African medicinal plants was investigated, in particular the effects of storage time and accelerated ageing on the biological activity and chemical constituents of these plants. Water, ethanol and hexane extracts of fresh plant material as well as material that had been stored in dry form in paper bags at room temperature for 90 days (short-term) were tested. Three bioassays, the COX-1 anti-inflammatory assay, nematode anthelmintic assay and minimum inhibitory concentration anti-bacterial assay, were used to determine biological activity. Thin layer chromatography of all the plant extracts were used to determine changes in chemical composition. The plants tested were Alepidea amatymbica Eckl. & Zeyh., Leonotis leonurus (L.) R. Br., Drimia robusta Bak., Vernonia colorata (Willd.) Drake, Scilla natalensis Planch., Eucomis autumnalis (Mill.) Chitt. subsp. autumnalis, Bowiea volubilis Harv. ex Hook. f., Helichrysum cymosum (L.) D. Don and Siphonochilus aethiopicus (Schweinf.) B. L. Burtt. Only those plants, which are known to exhibit a particular biological activity either traditionally or scientifically, were tested in the relevant bioassays. Of the plant extracts tested for anthelmintic activity only the water extracts showed activity and very little change in activity was observed after storage. Of the plant extracts tested for anti-inflammatory activity the ethanol extracts generally yielded highest activity. S. natalensis and B. volubilis both showed an increase in cyclooxygenase inhibition (anti-inflammatory) activity after storage whereas S. aethiopicus, H. cymosum, D. robusta and V. colorata showed a loss in activity after storage. The anti-inflammatory activity of E. autumnalis did not change. The water extracts of plants tested for antibacterial activity showed no activity, whereas the ethanol extracts generally showed an increase in activity. The TLC fingerprints indicated that there was chemical break-down during storage in certain species. These corresponded to the changes in biological activity. Alepidea amatymbica, Eucomis autumnalis, Helichrysum cymosum, Leonotis leonurus, Siphonochilus aethiopicus and Vernonia colorata were investigated further as to the effect of one year's storage (long-term storage) on their chemical composition and biological activity. Similar trends to that of the 90-day storage were observed. Activity gained in plants that were stored for 90 days was retained after a year of storage. Elevated temperature and humidity (55 C and 100% relative humidity) were used to accelerate the ageing process of Alepidea amatymbica, Leonotis leonurus and Vernonia colorata plant material. Again changes in the chemical composition and biological activity were observed, and the extent of these changes was greater than those in the stored material. The compounds responsible for the cyclooxygenase inhibition in the ethanolic extracts of Alepidea amatymbica leaf material appear to be stable and were not affected by the conditions of the accelerated ageing procedure (55 C and 100% humidity for seven days), but the root material lost activity, as did the leaf material of Leonotis leonurus. The leaf material of Vernonia colorata showed a slight (8%) increase in cyclooxygenase inhibition activity. The response of the plant material to accelerated ageing with respect to antibacterial activity varied with plant species. Alepidea amatymbica root material and Vernonia colorata leaf material appear to be stable whereas the other plant materials lost activity after prolonged (25 days) ageing.
Thesis (M.Sc.)-University of Natal, Pietermaritzburg, 2003.

Due to over-exploitation of commonly-used medicinal plants, mainly from KwaZuluNatal, because of ever-increasing human population growth, many of the useful medicinal plants are becoming depleted in their natural habitats. Some species like Warburgia salutaris, which is currently declared very rare in the KwaZulu-Natal province, appear to be on the verge of extinction. In order to counteract this overexploitation, this study sought to provide information that could help resource users to grow these threatened species through ex situ conservation methods. A short list of heavily utilised medicinal tree specles was selected from the approximately 700 tree species indigenous to KwaZulu/Natal. The criteria considered for short listing were; life form, species scarcity, past population status and part used. A total of 23 species were short listed, but a subset of 12 species was selected based on the availability of fruits and seeds. The aim of short-listing was to work on a manageable number of commonly utilised medicinal tree species. The seed physiology and growth of these species were studied. With the exception of Erythrophleum lasianthum and Curtisia dentata, all of them had a moisture content of 2': 20 % (on a dry mass basis), which is indicative of a recalcitrant behaviour. However, it could not be concluded that these seeds were truly recalcitrant because desiccation sensitivity was not directly assessed. Using the triphenyl tetrazolium chloride (TTC) viability test, most of the seeds of the 12 species seemed to be of good quality. Results of the TTC test for seed viability were similar to results obtained v using direct germination for most species. Results of flotation test for seed viability were different from the results obtained using direct germination for most spcies. The pre-treatment which achieved the highest germination percentage in almost all the seed types was cracking the outer coverings. Cracking pre-treatment appeared to be efficient in enhancing the removal of some substances which might inhibit germination of seeds. Hot water and acid pre-treatments frequently reduced germination. Growth of young seedlings was assessed in terms of stem diameter, height, and leaf area under sun and shade. Seedling growth in terms of stem diameter and height of most species did not show any significant difference. One of the few species which showed statistically significant differences in stem diameter growth was Ekebergia capensis. It was found that 3 out of lO of the species showed statistically significant differences in height growth. Two of the statistically significant differences in height occured on seedlings in the sun while one had statistically significant difference in the 40% shadecloth while 7 did not. Significant differences in leaf area occured on 7 out of lO species. Of these, 4 species had higher growth in the shade than in the sun while 3 had higher growth in the sun than in the shade. Generally, it appears that young developing seedlings establish themselves well under shade environment; this could be because most of the species used in this study are forest species.
Thesis (M.Sc.)-University of Natal, 1996.

The large geophytic monocotyledon, Hypoxis colchicifolia Baker, has been identified for the importance of its corm extracts in the development of a potential non-toxic prodrug for the treatment of inflammation, certain malignancies and HIV-infection. The underground corms of this plant are also commonly used for therapeutic applications in traditional medicine in Kwazulu-Natal where it primarily occurs. A review of published literature revealed, however, that H. colchicifolia plants are currently harvested in an unsustainable manner from traditional collecting sites due largely to population growth, increased land use for urban development and agriculture, and the popularisation of Hypoxis plants for herbal remedies. A further search of historical records established that H. colchicifolia plants were dominant in grassland vegetation prior to 1950, but had rapidly declined since then. Quantitative data subsequently gathered in this study from comparative surveys of both H. colchicifolia and H. hemerocallidea populations from sites with near-pristine, disturbed, burnt and mown grassland vegetation showed for the first time that exposure to human activity and the grassland management practices of mowing and burning incurred not only a 75% reduction in plant density of both these Hypoxis species, but also the total destruction of mature plants of H. colchicifolia in frequently mown and burnt areas. Flowering data recorded in these surveys, and confirmed by monitoring field performance of cultivated H. colchicifolia plants, showed that a contributing factor to the plant's inability to withstand these pressures was that juvenile forms only reached flowering maturity after three to four years growth, thus adversely affecting seedling recruitment. It was concluded therefore that, since Hypoxis species responded differently to mowing and burning, geophytic plants should be considered individually and not as "forbs" during the planning of grassland management programmes for natural conservation areas. The need to cultivate H. colchicifolia to ensure its survival was also established using the new field data gathered in this study. Methods to propagate this species have, however, not been established. Data gathered on all the plants comprising a single population confirmed that mature plants survive to an estimated 20 years and longer in natural areas. Greatest hypoxoside yields were also obtained from corms with a fresh mass of 350g to 400g. Since these corms were estimated to be 10-years-old and older, propagation and cultivation methods that could sustain plant production and survival for long periods, and therefore increased hypoxoside yields, would have to be developed. Several micropropagation systems suitable for the mass production of H. colchicifolia and from which phenotypically normal plantlets were recovered, were therefore established via organogenesis, embryo culture and somatic embryogenesis. The latter cultures have not been reported previously for Hypoxis. In the former culture the toxic effects of phenolic leachates and browning were controlled, and improved plantlet regeneration achieved, by adding polyvinyl pyrrolidone to the medium and introducing distinct sequential aseptic steps into the micropropagation procedure developed. Defined protocols for the different phases of in vitro somatic embryogenesis are not readily available for monocotyledons, however, neither are the factors controlling embryogenesis and organ regeneration known. In this study the process of somatic embryogenesis from excised zygotic embryos of H. colchicifolia was shown to be complex and the resultant cultures very heterogeneous. Although the stage of development of the zygotic embryo explants was important at the time of inoculation, data showed that the induction and regulation of the processes of embryo culture and somatic embryogenesis were ultimately determined by the exogenously applied plant growth regulators. By comparing the different pathways leading to plantlet regeneration, and the morphological stages of development of the structures produced both on solid and in liquid media, not only photographically, but also quantitatively and schematically, the repeated formation of pseudoembryonic structures and neomorphs confirmed that they form an integral part in the in vitro somatic embryogenic pathway of H. colchicifolia. Evidence suggested not only that two types of somatic embryos are produced in the embryogenic cultures of H. colchicifolia, but that the pseudoembryonic structures produced resemble the pseudobulbils produced in polyembryonic cultures of Citrus. The success of the somatic embryogenic cultures was confirmed by the estimation that 28 112 somatic embryos and embryo clusters of H. colchicifolia could be obtained from 16 ml of somatic embryogenic liquid culture. Furthermore phenotypically normal plantlets regenerated from all of the micropropagation procedures developed were successfully transplanted from the laboratory, acclimatized under greenhouse conditions and their horticultural and field performances evaluated.
Thesis (Ph.D.)-University of KwaZulu- Natal, Pietermaritzburg, 2004.

A significant potion of the population in Lesotho relies on traditional medicine to meet its health care requirements. Traditional healers and herbalists were interviewed from Qacha's Nek (Highlands) and Mohale's Hoek (Lowlands) districts in Lesotho on plants used by the Basotho in traditional remedies. Fifteen plants were reported to be used for bacterial infections while thirteen plants were used for diseases associated with inflammation . Plant roots were most often used to make water extracts. Mainly high altitude plants are used with lowland healers obtaining most of their plant material from the highlands, either by collecting them or buying them from highland gatherers. Leaves and roots of plants used to treat bacterial infections were extracted with hexane, methanol and water and the respective extracts screened at 100 mg ml¯¹ for anti-bacterial activity using the disc diffusion bioassay. Seven species displayed very high anti-bacterial activity against both Gram-positive and Gram-negative bacteria. A number of plant extracts had medium inhibitory activity, mostly against Gram-positive bacteria. This activity was mainly found in the root extracts. Six of the thirteen plants screened for anti-inflammatory activity using the cyclooxygenase-1 (COX-1) bioassay had activity above 90%. Hexane and methanol extracts were the most active while water extracts usually had lower activity. Malva parviflora, Eriocephalus punctulatus and Asparagus microraphis exhibited high anti-inflammatory activity from hexane, methanol and water extracts made from leaf and root material. High anti-bacterial activity was also recorded from M. parviflora and E. punctulatus hexane, methanol and water extracts. An investigation on seasonal variation and plant part substitution in medicinal activities for these plants was carried out. Extracts of M. parviflora collected between June 1999 and July 2001 showed variation in anti-bacterial activity. Extracts made from leaves and roots inhibited the growth of both Gram-positive and Gram-negative bacteria. More bacterial strains were inhibited by extracts made from roots collected in cooler months. However, a trend in seasonal activity was not evident for either the roots or leaves because there was no detection of activity in some of the extracts made within the same months or seasons of the adjacent years. Variation in anti-inflammatory was detected for M. parviflora extracts. E. punctulatus leaf extracts did not exhibit any seasonal variation in anti-bacterial activity. Anti-inflammatory activity of E. punctulatus showed seasonal variation with the highest activity noted when material was collected during the cooler months and a decline in activity when collections were made during the warmer months. Hexane, methanol and water extracts made from leaves and roots of A. microraphis did not show any seasonal variation in anti-inflammatory activity. Thus, M. parviflora and E. punctulatus should be collected during the cooler months while A. microraphis can be collected throughout the year. Traditional healers, herbalists and vendors need to be encouraged to use aerial parts in substitution of ground parts which are reported to be highly utilized. Effect of storage on anti-bacterial and anti-inflammatory activities of M. parviflora, E. punctulatus and A. microraphis were monitored. Dried, ground leaf and root material of the three plants was stored in a cold room, at room temperature and in the Botanical Garden where the material was exposed to high and large changes in temperature. Dried hexane and methanol extracts made from leaves and roots of these plants were stored in a cold room and at room temperature. Initially, storage of the plant material under the three storage conditions caused an increase in antibacterial activity of the hexane, methanol and water extracts made from leaf and root material of M. parviflora and E. punctulatus. Storage for a longer period resulted in a decrease in inhibitory activity. TLC fingerprints developed from hexane and methanol extracts made from M. parviflora and E. punctulatus stored in a cold room and at room temperature showed a consistent number and colour of spots during the initial storage period. Prolonged storage resulted in a decline in the number and colour of detected spots. The stored hexane and methanol extracts made from leaves and roots showed a similar trend of increases and decreases in anti-bacterial activity as well as changes in spots with the storage of the extracts. Testing of the effect on anti-inflammatory activity of hexane, methanol and water extracts made from leaves and roots of M. parviflora, E. punctulatus and A. microraphis showed no change in inhibitory activity of hexane extracts obtained from the material and the extracts stored at the three storage conditions. Methanol and water extracts made from leaves exhibited an increase in activity with prolonged storage. Generally, the stability of the inhibitory activity was longer for the stored dried material than the plant extracts. Isolation of biological active compounds from M. parviflora was not successful due to loss in anti-bacterial activity as a result of collection of plant material from a different locality. Anti-inflammatory compounds could not be isolated due to insufficient amount and the synergistic effect of the active compounds . The purified compounds exhibited loss of activity following HPLC purification which then re-appeared upon recombining the fractions. A number of compounds were detected from essential oils of E. punctulatus using GC. Fractions containing these compounds gave positive anti-bacterial activity in the disc-diffusion , bioautographic and MIC bioassays as well as high anti-inflammatory activity with COX-1 and COX-2 anti-inflammatory bioassays. No anti-inflammatory compounds were isolated from A. microraphis.
Thesis (Ph.D.)-University of Natal, Pietermaritzburg, 2003.

Traditional medicine in southern Africa draws upon a vast selection of plants to treat gastrointestinal disorders such as diarrhoea and intestinal parasites. The evaluation of these plants for biological activity is necessary, both to substantiate the use of these plants by healers, and also a possible lead for new drugs or herbal preparations. After a survey of the existing ethnobotanical literature, plants used to treat stomach ailments such as diarrhoea, dysentery or intestinal worm infestations were selected and submitted to bioassays according to their traditional uses. Extracts of the chosen plants were made using the solvents hexane, ethanol and water, to ensure the extraction of compounds with a wide range of polarity. In total, 138 extracts were tested for antibacterial activity, 72 for anthelmintic activity, and 42 for antiamoebic activity. Antibacterial activity was evaluated using the disc-diffusion assay, and Minimal Inhibitory Concentration (MIC) values were determined using a microdilution assay. The extracts were tested against the Gram-positive bacteria Bacillus subtilis and Staphylococcus aureus, and the Gram-negative bacteria Escherichia coli and Klebsiella pneumoniae. Ethanolic extracts showed the greatest activity and Gram-positive bacteria were the most susceptible microorganisms. The free-living nematode Caenorhabditis elegans, which is morphologically similar to parasitic nematodes, was used in two different assays to evaluate anthelmintic activity. A microdilution technique was employed to investigate antiamoebic activity against the enteropathogenic Entamoeba histolytica, the causal organism of amoebic dysentery. These assays were suitable for the screening of a large number of extracts at one time. Several plants exhibited significant activity against these test organisms. Many species of plants belonging to the family Combretaceae are used in southern African traditional medicine against a variety of ailments, including abdominal complaints, bilharzia and diarrhoea. Extracts of powdered leaf material of 24 species belonging to the Combretaceae were prepared using the solvents ethyl acetate, acetone, methanol and water. These extracts were screened for anthelmintic activity. Significant activity was exhibited by C. apiculatum, C. hereroense and C. mossambicense. The most anthelmintic activity was shown by acetone extracts, followed by ethyl acetate, water and then methanol extracts. The aromatic rhizomes of Acarus calamus L. are used extensively in traditional medicine worldwide. They reportedly relieve stomach cramps and dysentery, and are used as anthelmintics. Rhizome extracts of A. calamus growing in KwaZulu-Natal, South Africa, exhibited anthelmintic and antibacterial activity in the initial general screening. Using bioassay-guided fractionation, the phenylpropanoid β-asarone was isolated from the rhizome. This compound possessed both anthelmintic and antibacterial activity. It has previously been isolated from A. calamus, and a related species, A. gramineus. Different varieties of A. calamus exhibit different levels of β-asarone, with the diploid variety containing none of the compound. Mammalian toxicity and carcinogenicity of asarones has been demonstrated by other researchers, supporting the discouragement of the medicinal use of Acarus calamus by traditional healers in South Africa. Schotia brachypetala was another plant to show good antibacterial activity in the initial screening. The roots and bark of S. brachypetala are used in South African traditional medicine as a remedy for dysentery and diarrhoea. The lack of pharmacological and chemical data on this plant prompted a further investigation into its antibacterial activity. The differences in activity of ethanol and water extracts with respect to plant part, season and geographical position were analysed. No extreme fluctuations in activity were noted. Two other Schotia species, S. afra and S. capitata, were included in the study, and both displayed good antibacterial activity. The storage of the plant, either as dried, ground plant material at room temperature, or as an extract residue at -15°C, had little effect on the antibacterial activity. Preparing the extracts from fresh or dry material also did not notably affect the activity. In general, the ethanolic extracts were more active than the aqueous extracts. The chemical profiles on TLC chromatograms were compared and found to be very similar in the case of ethanol extracts prepared in different months of the year, and from different trees. The extracts of the three species, and of the leaves stored under various conditions, as well as extracts prepared from fresh or dry material, also showed similar TLC fingerprints. However, various plant parts of S. brachypetala showed distinctly different chemical compositions. The leaves of S. brachypetala showed slightly higher antibacterial activity than the roots. Fractionation of the ethanol extract of the dried leaves using liquid-liquid partitioning and chromatographic techniques yielded 9,12,15-octadecatrienoic (linolenic) acid and methyl-5, 11,14,17-eicosatetraenoate. These fatty acids displayed antibacterial activity against the Gram-positive bacteria Bacillus subtilis and Staphylococcus aureus, and activity to a lesser extent against the Gram-negative Escherichia coli and Klebsiella pneumoniae. Linolenic acid is known to have antibacterial activity. The screening of plants for biological activity yielded valuable preliminary information about the plants used by traditional healers to treat gastrointestinal illnesses. The isolation of biologically active compounds from two highly active plants was achieved.
Thesis (Ph.D.)-University of Natal, Pietermaritzburg, 2001.

Ocimum obovatum E. Mey ex Benth. var. obovatum is a traditionally used medicinal plant that grows along the KwaZulu-Natal coast and the western Cape of South Africa. The plant is noted for its hair restoration properties, remedy for infantile abdominal pain and cramps and its use as an enema to treat epigastric conditions in children. The aims of this study were to document the micromorphology and ultrastructure of the foliar secretory structures responsible for the production and secretion of the essential oils and chemical composition of the secretion, which gives the plant a distinct aroma. It is believed that these oils contain the active ingredients that contribute to the medicinal properties of the plant. A variety of microscopic methods and histochemical and phytochemical tests were used to achieve this. Leaves in all stages of development were pubescent and gland dots, characteristic of plants in the genus, were found on both adaxial and abaxial surfaces. Three types of trichomes were found on both leaf surfaces across all stages of development; non-glandular trichomes and two types of glandular trichomes. Non-glandular trichomes are single, multicellular and uniseriate with microornamentation and a supportive cellular pedestal. The glandular trichomes consisted of peltate and capitate trichomes. Peltate trichomes are made up of four head cells and a very small basal cell that gives the glands the appearance of being sessile. The capitate trichomes were further divided into two types based on the morphology of the glands. Type I capitate trichomes are smaller than the larger peltate trichomes and are composed of one basal cell and a head consisting of two broad head cells. Type II capitate trichomes consisted of one basal cell, one stalk cell and a single oval head cell. Histochemical tests showed that peltate and Type I capitate trichomes have cutinized or suberized walls in the stalk cell to prevent the apoplastic flow of secretory material into neighbouring mesophyll tissue. The histochemical stains also showed that the secretory material present in the glandular trichomes are lipid in nature and essential oils are present. Ultrastructural studies showed polymorphic leucoplasts, few Golgi bodies, numerous vesicles and mini vacuoles, mitochondria and short profiles of endoplasmic reticulum cisternae. Phytochemical tests revealed the presence of essential oils that are terpene-rich. Flavonoids, tannins, saponins, terpenoids, fixed oils and fat, phenolics and cardiac glycosides were also detected in a crude ethanolic extract of the leaves. These chemical compounds appear to be responsible for the medicinal properties for which the plant is traditionally exploited.
Thesis (M.Sc.)-University of KwaZulu-Natal, Westville, 2011.

The use of traditional medicine is a popular practice in South Africa especially among rural dwellers due to several reasons such as availability of natural products, cultural beliefs, preference of natural products to synthetically derived drugs and the high cost of modern drugs. Traditional healers in South Africa play key roles in administering treatment for all sorts of ailments using plants. The aim of this study was to evaluate the efficacy of seven selected medicinal plants that are used in traditional medicine to treat stomach-related ailments for their pharmacological and phytochemical properties. Plant material was extracted sequentially with ethyl acetate (EtOAc), ethanol (EtOH) and water. The extracts were evaluated for their antimicrobial activities using the microdilution technique against two Gram-positive (Enterococcus faecalis ATCC 19433 and Staphylococcus aureus ATCC 12600) bacteria and a Gram-negative (Escherichia coli ATCC 11775) bacterium. A modified microdilution technique was used to screen for antifungal activity against a yeast-like fungus (Candida albicans ATCC 10231). Only the EtOAc extract of Tetradenia riparia demonstrated good antibacterial activity against the Gram-negative E. coli, all the other extracts that were active only showed good antibacterial activity against the two Gram-positive (E. faecalis and S. aureus) bacteria with MIC values <1 mg/ml. None of the extracts that exhibited good inhibitory activity showed corresponding bactericidal activity against the bacterial test strains, suggesting that the observed activity were all inhibitory. Good antifungal activity with an MIC value <1 mg/ml was observed in only 5 extracts, and none of the extracts exhibited corresponding fungicidal activity. The in vitro colorimetric assay for anthelmintic activity against Caenorhabditis elegans revealed that almost all the extracts possessed moderate to high anthelmintic properties. The EtOAc extract of T. riparia had the best activity at MLC value of 0.004 mg/ml. The anti-inflammatory activity of the plant extracts was tested using the cyclooxygenase assays to determine their inhibitory potential against COX-1 and COX-2 enzymes. All the EtOAc extracts demonstrated both COX-1 and COX-2 inhibitory activity in the range of 50.7 ± 2.4 to 99.5 ± 0.5%. Apart from the EtOH extracts of C. multicava that showed high inhibitory activity against both COX-1 and COX-2, all the other EtOH extracts were COX-2 selective. Aqueous extracts exhibited poor inhibitory activity against both COX-1 and COX-2 enzymes with the exception of T. riparia and Coddia rudis that showed good inhibitory activity (69.1 ± 0.9 and 92.65 ± 0.7%) against COX-1 and COX-2 respectively. The standard plate incorporation assay for the Ames test was carried out to determine the potential genotoxic effects of the plant extracts and this revealed that all the extracts were non-mutagenic towards Salmonella typhimurium tester strains TA98, TA100 and TA1537 without metabolic activation. However, further studies incorporating metabolizing enzymes are needed to confirm the safe use of the studied plants. Phytochemical analysis revealed relatively high amounts of total phenolics, gallotannins and flavonoids in all the evaluated plants. Total and steroidal saponins were detected in only two plant samples, Canthium spinosum and Cassinopsis ilicifolia (bark). These findings present useful information on the types of bioactive compounds that could be responsible for the pharmacological activities observed among some of the plant extracts. The results obtained in this study showed different levels of pharmacological activities among all the evaluated medicinal plants which provide scientific validation for their use in traditional medicine as antimicrobial agents. Phytochemical analysis provides valuable information for further study that will be aimed at isolation and identification of the bioactive principles in the evaluated plant species.
Thesis (M.Sc.)-University of KwaZulu-Natal, Pietermaritzburg, 2011.

Infectious diarrhoea is the second largest single cause of mortality in children under the age of five globally. Bacteria are responsible for most diarrhoeal episodes especially in developing countries, and progressive increase in antimicrobial resistance has given rise to the need to investigate other sources of therapy such as medicinal plants. Ten plant extracts were analysed for their antimicrobial activities using the agar well diffusion and broth microdilution method. Their phytochemical contents were screened, and their effect on 1, 1-diphenyl-2-picrylhydrazyl (DPPH) was used to assess their antioxidant activities. Their toxicity profiles were evaluated using the XTT Cytotoxicity Assay. Water and methanol extracts of Adansonia digitata v ABSTRACT Infectious diarrhoea is the second largest single cause of mortality in children under the age of five globally. Bacteria are responsible for most diarrhoeal episodes especially in developing countries, and progressive increase in antimicrobial resistance has given rise to the need to investigate other sources of therapy such as medicinal plants. Ten plant extracts were analysed for their antimicrobial activities using the agar well diffusion and broth microdilution method. Their phytochemical contents were screened, and their effect on 1, 1-diphenyl-2-picrylhydrazyl (DPPH) was used to assess their antioxidant activities. Their toxicity profiles were evaluated using the XTT Cytotoxicity Assay. Water and methanol extracts of Adansonia digitata seeds and pulp showed no inhibition against all the test organisms, while water and methanol extracts of A. digitata leaves showed inhibition, with minimum inhibitory concentration (MIC) ranging from 0.39 to 6.25mg/ml. Water and methanol extracts of Garcinia livingstonei and Sclerocarya birrea barks showed good activity against all the test organisms, with MICs between 0.39 and 1.56 mg/ml. Alkaloids, phenols, flavonoids, saponins, tannins, and terpenoids were found in one or more of the plant extracts, and all the plant extracts demonstrated scavenging power against DPPH.The cytotoxicity of extracts of Garcinia livingstonei, and Sclerocarya birrea barks ranged between 105.9 μg/ml and 769.9 μg/ml. The results obtained in this study validate the traditional use of A. digitata leaves, G. livingstonei and S. birrea bark in treating bacteria causing diarrhoea.
Life Sciences
M. Sc. (Life Sciences)

Healthcare in South Africa is polarised between western and traditional African systems of therapy. The latter is consulted by the majority of the population and therefore plays an integral role in the delivery of healthcare to South Africans. Traditional medicines are primarily plant products with long storage lives, among which the dominance of bark is typical of southern African traditional healthcare systems. Expansion of the traditional healthcare sector during the twentieth century, in response to rising consumer demands, stimulated a lucrative trade in medicinal plants that is centred in KwaZulu-Natal. Since herbal medicines are sourced almost exclusively from indigenous vegetation, harvesting pressures exerted on the indigenous flora to meet demands for traditional medicines have rendered such resources non-sustainable. Although trees comprise a small fraction of South African medicinal plant species, bark from them constitutes a substantial proportion of the plant products used medicinally. Trees are among the most threatened medicinal plants in South African due to their limited abundance, the ecological sensitivity of the vegetation in which they occur, and destructive methods of commercial bark harvesting that frequently take place within protected areas. In KwaZulu-Natal, bark is harvested primarily from forests that occupy an extent of only 0.1 % in the province. Conservation of economically valuable tree species is particularly problematic since data necessary for the establishment of sustainable usage systems are absent or inaccessible. Alternatives to in situ conservation for renewable bark resources include propagation, multi-use timber systems and reintroduction of locally extinct species. To facilitate appropriate management of bark resources, there is a need for specialist publications and consolidated data with which sustainable usage levels may be determined. The importance of bark in South African traditional healthcare is poorly reflected by the ethno botanical literature. In this study, 180 bark species used in traditional healthcare in KwaZulu-Natal were inventoried from thorough literature surveys, but this number is anticipated to be a conservative reflection of actual statistics. Where trade data were recorded in the literature, they indicated intensive exploitation of bark resources in KwaZulu-Natal and throughout South Africa, but conservation and management data were lacking for 72 % of the species inventoried. A number of problems were encountered in the literature, of which vague information and the documentation of local vernacular nomenclature were the most troublesome. Despite the importance of traditional medicine, the country's political history led to the prevailing situation, where the traditional healthcare sector is largely unregulated. Coupled with increasingly limited availability of medicinal plants, the quality and appropriate use of traditional medicines is negatively affected by growing numbers of inadequately trained practitioners, herbalist retailers and plant gatherers. Possibilities of misidentification or purposeful adulteration of medicinal bark products therefore lead to concerns for patient safety, since dried bark is difficult or impossible to identify. Whilst bark characters are useful for field identifications, many useful diagnostic characters are lost through desiccation, and anatomy and morphology of bark are variable. Additionally, medicinal bark products used in KwaZulu-Natal, and their identification, are largely undocumented. This study focussed on eight bark species used medicinally in the province, elected by an esteemed traditional medical practitioner as having problematic identity. Monograph-type characterisation profiles were drawn up for reference specimens collected from various localities, and their medicinal bark products traded under vernacular names recorded in the literature. In the absence of standardised traditional medicines, there is a need for reliable and affordable methods for their authentication. Phytochemical bark characters identified by Thin Layer Chromatography (TLC) have proved useful in chemotaxonomic studies, and the technique is widely used for herbal drug authentication. TLC was tested here for authentication of medicinal bark products from the aforementioned study species. Three reference samples of each species were collected, and TLC-generated fingerprints compared. At the intraspecific level, TLC was useful in confirming the relationship of ethanol and hexane bark extracts, but was less meaningful in distinguishing between fingerprints of different species. Three medicinal bark products of each study species were purchased and fingerprints compared to a reference. The technique proved useful in confirming the identity of several medicinal bark products. Authentication of medicinal bark products may be useful in toxicology cases and in the accurate documentation of their trade. This research identified a complexity of issues surrounding the use of bark in traditional healthcare in KwaZulu-Natal, and indeed South Africa. A multi-faceted approach is required to secure their sustainability. Critical, however, to factors such as effective conservation and regulation of the traditional healthcare sector, is recognition of the importance, and documentation, of traditional bark medicines. The integrity of traditional healthcare, and the future of the South African flora, hinge upon the sustainable use of medicinal products such as bark.
Thesis (M.Sc.)-University of Natal, Pietermaritzburg, 2002.

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.

Candida species were discovered more than a century ago as a causative organism of oral thrush. In HIV patients, the presence of oral candidiasis has been shown to be the earliest opportunistic infection. Candidiasis lesions associated with HIV infections are primarily a reflection of the specific change of the host's immune response caused by the virus. Studies of AIDS all over the world show that 58-81% of all patients contract a fungal infection at some time during the primordial stage or after developing AIDS and 10-20% have died as a direct consequence of fungal infections. Twenty four South African medicinal plants were screened using a modification of the NCCSL broth microdilution antifungal test against Candida albicans standard strain ATCC 10231 and two clinical isolates from a 5-month- old baby and an adult. This assay was performed in order to find a traditional remedy to treat oral candidiasis. Of all the screened plants Allium sativum L., Glycyrrhiza glabra L., Polygala myrtifolia L. and Tulbaghia violacea L. aqueous extracts were found to have the best activity. Allium sativum and Tulbaghia violacea aqueous bulb extracts had MIC values of 0.56 mgml-1 and 3.25 mgml-1 respectively, whilst Polygala myrtifolia leaf extracts and Glycyrrhiza glabra rhizome extracts had MIC values of 1.56 mgml-1 and 3.25 mgml-1 respectively when tested against the isolate from a 5-month-old baby, which was the most susceptible of the isolates used. All the extracts had higher MIC values against the standard strain (ATTC 10231), which was the least susceptible to the extracts used. Stability testing was performed on fresh aqueous extracts of A. sativum, G. glabra, T. violacea and P. myrtifolia stored at 4°C, 23°C and 33°C over a period of one week, to determine the stability of the extracts in solution. All A. sativum extracts maintained stability for three days in solution, whilst T. violacea extracts remained stable for only two days in solution. TLC fingerprinting of A. sativum and T. violacea extracts indicated the presence of the known antibacterial and antifungal compound allicin. The activity of allicin and other active compounds was observed by using the bioautographic assay, which was performed on these extracts. P. myrtifolia and G. glabra extracts lost stability 24 hours after preparation at all tested temperatures. However, it was clear with the four plant extracts tested that storage of solutions at higher temperatures reduced their activity and stability. The unpleasant taste and smell of A. sativum and G. glabra could however not be masked, since the intake of these two extracts would result in HIV patients being recognised. These two plants where therefore not considered for further investigation. G. glabra and P. myrtifolia are both saponin containing plants. These could be the active constituents responsible for the anticandidal action. G. glabra is known for its biological activity as an antibacterial agent, whilst other Polygala species have been reported to possess antifungal saponins. Although P. myrtifolia and G. glabra are not stable for more than 24 hours, they do not have an unpleasant smell or taste. These plants are therefore further investigated for use as oral mouthwash in clinics and homes.
Thesis (M.Sc.)-University of Natal, Pietermaritzburg, 2003.

Throughout the history of mankind, many infectious diseases have been treated with plant extracts. Venereal infections are one such group and are regarded as conditions that are highly responsive to traditional treatment. Aqueous, ethanol and ethyl acetate extracts of 13 plants used in South Africa for the treatment of venereal diseases were screened for in vitro antibacterial, antifungal, mutagenic and antimutagenic activities. Antibacterial activity was evaluated using the disc-diffusion and microdilution assays to determine the minimal inhibitory concentration (MIC) values of the extracts. The extracts were tested against the Gram-positive bacteria Bacillus subtilis and Staphylococcus aureus, and the Gram-negative bacteria Escherichia coli and Klebsiella pneumoniae. Among the plants tested, Gunnera perpensa, Harpephyllum caffrum, Hypoxis latifolia and Ledebouria ovatifolia showed the best antibacterial activity. The aqueous rhizome extract of Gunnera perpensa displayed good activity against Gram-negative bacteria with an MIC value of 0.78 mg/ml, and against S. aureus (0.78 mg/ml). Aqueous and ethanol extracts of H. caffrum bark were active against both Gram-positive and Gram-negative bacteria. Hypoxis latifolia aqueous corm extracts exhibited very good MIC values against K. pneumoniae (0.78 mg/ml), E. coli and S. aureus (1.56 mg/ml). Ethanol and ethyl acetate bulb extracts of Ledebouria ovatifolia displayed good activity against Bacillus subtilis bacteria with MIC values of 0.78 mg/ml and 0.39 mg/ml respectively. Antifungal activity was evaluated using the microdilution bioassay. Good activity was shown by the ethanolic bark extracts of Bersama lucens and Harpephyllum caffrum against Candida albicans. Only in the case of Harpephyllum caffrum did aqueous extracts have activity against Candida albicans. In the Ames test, all plant extracts showed a negative genotoxic response except for ethanol and ethyl acetate bulb extracts of Cyrtanthus obliquus which induced mutations in TA98. Moderate antimutagenic activity was observed with the ethyl acetate extract of G. perpensa and the ethanolic extract of H. latifolia. High antibacterial and antifungal activity detected with Harpephyllum caffrum bark extracts resulted in an investigation on seasonal and geographical variation of this inhibitory activity. Seasonal variation in antibacterial and antifungal activities was investigated in order to determine the best collection time to ensure potential high medicinal activity in plant preparations. The highest inhibitory activity was detected with plant material collected in June and December 2003, with a decline in activity when collections were made in September 2004. The chemical profiles of TLC chromatograms were compared and little variation was found, particularly in the case of plant material obtained from the Botanic Garden of the University of KwaZulu-Natal and a 'Muthi' Shop in Pietermaritzburg. Identification of active compounds from G. perpensa and H. caffrum was not successful due to insufficient amounts of isolated fractions.
Thesis (Ph.D.)-University of KwaZulu-Natal, 2006.

The herbal medicine trade is thriving in KwaZulu Natal with an ever-increasing number of people harvesting and trading in indigenous plants, especially those species with medicinal and/or magical properties. The number of plants harvested has increased whereas the size of the plants collected has decreased, resulting in low recruitment into wild populations. As a result of these two factors, species diversity has decreased. To this end, the aim of these investigations was to establish micropropagation protocols for the selected species i.e. Bowiea volubilis, Haworthia_ limifolia and Cryptocarya latifolia. In addition, hardening-off protocols were also developed. The bulbous plant, Bowiea volubilis, was propagated via organogenesis using the inflorescence stem. Bulblet formation occurred directly without an intervening callus phase. Bulblets were produced on explants on Linsmaier and Skoog (1965) (LS) medium containing 30 g.r' sucrose and either I mg.r' BAP and I mg.r' 2,4-D or 1 mg.r' BAP and 1 mg.r' NAA. Shoots and roots were induced upon transfer to the basal medium devoid of plant growth regulators. Regenerated plantlets were successfully hardened-off. Haworthia limifolia, a succulent, was propagated via direct somatic embryogenesis using leaf material. Embryo formation was induced on a modified Murashige and Skoog (1962) (MS) medium containing 20 g.r' sucrose and 1 - 5 mg.r' 2,4-D. secondary embryogenesis occurred when the explants were transferred to the basal medium supplemented with activated charcoal and devoid of growth hormones. Healthy plantlets, produced from secondary embryos, were transferred to pots and acclimatised to greenhouse conditions. A large proportion of the plantlets regenerated were vitrified and as a result, this problem was addressed by changing the medium composition or culture environment. Silica gel, when placed in the culture vessel, was the best treatment for reversal of the vitrified condition. The establishment of leaf and nodal segment cultures of Cryptocarya latifolia required extensive investigation of sterilants to reduce fungal contamination. Several fungicides were tested and a successful sterilisation protocol was established. A number of media were tested for the induction of dormant axillary buds and multiplication of shoots. The best medium for both bud induction and proliferation was MS medium containing 30 g.r1 sucrose and 1 mg.r1 BAP and 0.01 mg.r1 NAA. Callus cultures were established on MS medium containing 30 g.r1 sucrose and 3 mg.rl 2,4-D. These calli, however, were non-embryogenic. Application of the established protocols and future research strategies are discussed.
Thesis (M.Sc.)-University of Natal, 1995.

Cussonia species (commonly known as Cabbage trees) are indigenous to South Africa and are used in traditional medicine to treat an assortment of diseases. Due to their attractive growth form, they are assets in gardens. However, there are no developed methods for propagating these species. The use of three selected species, Cussonia paniculata (Eckl. & Zeyh.), C. spicata (Thunb.) and Schefflera umbellifera (Sond.) Baill, = C. umbellifera), in traditional medicine was validated. Rapid propagation protocols for C. paniculata and C. spicata were investigated and ultimately developed for the former species. Cussonia paniculata, C. spicata and C. umbellifera were screened for their medicinal properties, mainly focussing on anti-bacterial, anti-inflammatory and anti-malarial activities. In the anti-bacterial screening, C. spicata bark and root extracts showed activity against selected Gram-positive and Gram-negative bacterial strains at a concentration of 50 mg ml ¯¹ . The highest inhibition was observed with ethanol and ethyl acetate root extracts against Staphylococcus aureus. The other two species did not show anti-bacterial activity. Ethanol and ethyl acetate extracts of all species showed anti-inflammatory activity in the cyclooxygenase assay (COX-1) at a concentration of 8 μg ml ¯¹, These active extracts showed an inhibition percentage that was greater than 50 % against cyclooxygenase. In the anti-malarial screening , bark extracts were screened. C. umbellifera bark extracts exhibited the best inhibition against P. falciparum, a malaria-causing agent in humans. The percentage inhibition of these extracts was up to 100% at a concentration of 200 μg ml ¯¹ . While C. spicata is known to be used to treat malaria, the screening results showed much less activity (less than or equal to 35 %) as compared to C. umbellifera, which is preferably used to treat malaria. The results obtained from screening these three species validated their use in traditional medicine. This means that the people or traditional healers use these species for different treatments by possibly relying on past knowledge about the effects after administering the medicine. Fingerprinting using Thin Layer Chromatography (TLC) was used in an attempt to determine whether there are any chemical differences or similarities between the three species. There were similarities between the plant parts across the species as well as some differences. However, this method cannot be used as an unequivocal test to deduce that compounds that are present in a certain species and not in others are the ones responsible for bringing about a certain biological activity. That can only be achieved by a bioassay-guided isolation of possible compounds. A tissue culture protocol was developed to produce a large -number of plants of C. paniculata. Explants were derived from nodal explants of in vitro germinated seeds and cultured on Murashige and Skoog (MS) (1962) medium supplemented with 3% sucrose, 2.5 mg l ¯¹ BA and solidified with 3 g l ¯¹ Gelrite. These explants produced multiple shoots. The average number of shoots per explant ranged between 1 to 3.5. Multishoots were subcultured on to rooting media and roots were produced on MS with 0.75 mg l ¯¹ IBA and 1 mg l ¯¹ NAA. Callus from zygotic embryos also produced plantlets on MS supplemented with 1.5 mg l ¯¹ 2,4-D and 0.5 mg l ¯¹ BA. Hyperhydricity was encountered in this study. This problem was reversed successfully by transferring the shoots from medium solidified with 3 g l ¯¹ Gelrite to medium solidified with 8 g l ¯¹ agar. Plantlets were successfully acclimatized for planting ex vitro. The percentage of healthy plants after a 35-day acclimatization period was 63 %. C. spicata was not successfully micropropagated from shoot-tip explants. However, a protocol was developed for decontaminating shoot-tips from the mother plants. The plant material was successfully decontaminated with 0.01% HgCl₂ for 15 min. The decontamination percentage was up to 80 %. Browning of the explants was observed and it was successfully treated with soaking the explants in a 15 mg l ¯¹ ascorbic acid solution for 15 min. A high percentage of shoot-tip regeneration (80 %) was observed when they were cultured on MS medium supplemented with 2 mg l ¯¹ BA, 1 mg l ¯¹ IAA and 1 mg l ¯¹ GA₃. However, multishoots were not observed as in C. panicualata. Shoot elongation in vitro was similar to shoot elongation as it occurs in nature. The shoots elongated and a flush of palmitately arranged leaves were produced. Further research is required to investigate a commercially viable protocol for rapid propagation and conservation of the germplasm of Cussonia species.
Thesis (M.Sc.)-University of Natal, Pietermaritzburg, 2000.

Merwilla natalensis (Planchon) Speta is ranked as one of the most commonly sold medicinal plants at most of the informal medicinal plant markets found throughout South Africa. The increasing demand for medicinal plants has resulted in over-exploitation of many of the wild populations. Overharvesting has resulted in M. natalensis being declared vulnerable. Although this species is so popular, and reports state that the bulbs are used for a variety of ailments, very little is known about its pharmacological activity or phytochemical composition. Extracts were made from mature M. natalensis bulbs using hexane, dichloromethane, methanol and water. These extracts were screened for antibacterial, anticancer, anti-inflammatory, antischistosomal and anthelmintic activity. Antibacterial activity was evaluated using the minimal inhibitory concentration (MIC) assay. Methanol extracts displayed good antibacterial activity against both Gram-positive (Bacillus subtilis and Staphylococcus aureus) and Gram-negative (Escherichia coli and Klebsiella pneumoniae) bacteria. Anti-inflammatory activity was evaluated using the COX-1 and COX-2 bioassays. Dichloromethane extracts displayed the highest inhibitory activity against both COX-1 and -2 enzymes. (80% and 91% inhibition respectively) Very good activity was displayed against the free-living nematode Caenorhabditis elegans and the schistosomula worms of Schistosoma haematobium using microdilution techniques. Anticancer activity was evaluated using the biochemical induction assay (BIA) in which DNA-damaging properties are tested for. No activity was found using this assay, however, these results do not prove that M. natalensis does not have other anticancer properties. The phytochemical investigation of mature M. natalensis plants showed the bulbs to contain both saponins and bufadienolides. One of the bufadienolides had the same Rf value as proscillaridin A. Cytotoxicity tests reveal M. natalensis to be extremely cytotoxic, yet the bulbs are commonly sold at traditional medicine markets around South Africa. This cytotoxicity may be accredited to the presence of saponins within the bulbs. No alkaloids or tannins were detected in the bulbs. With the growing population in South Africa, there is an increasing demand for traditional medicines. This increasing demand is placing tremendous strain on natural populations growing in the wild. However, as the demand cannot continue to be met other sources are needed. Tissue cultured plants have been grown at two different regions of South Africa. These plants have been grown under different conditions to determine the optimal ones needed to grow M. natalensis as a commercial crop on small-scale farms. Plantlets taken directly from tissue culture were acclimatized successfully for cultivation by means of simple and cost effective methods. Cultivated plants were harvested on a six-monthly basis for a period of two years. Field cultivation produced bulbs of almost marketable size (±300g fresh weight) after 24 months. Bulb size was not dependent on additional fertilizer or irrigation. No significant differences (p<_0.05) were shown in the average dry weights of bulbs grown under different treatments (control, fertilizer without irrigation, fertilizer with irrigation). Leaf senescence and dormancy of young plants were prevented with irrigation. Flowering occurred after 24 months, with the irrigation and fertilizer plot having the most flowering plants. TLC fingerprinting revealed differences in the chemical composition of the bulbs harvested at different stages of growth. Noticeable differences were found between bulbs cultivated at the different growing sites. Pharmacological screenings were done of the harvested bulbs to investigate the effect of age (time of harvest) and growing conditions on antibacterial, anti-inflammatory and anthelmintic activity. Methanol extracts were screened against Gram-positive (Bacillus subtilis and Staphylococcus aureus) and Gram-negative (Escherichia coli and Klebsiella pneumoniae) bacteria. Variations in activity were found. The time of harvest had a significant effect (p<_0.05) on biological activity, with the younger plants being more active. Antibacterial activity decreased with an increase in plants age. Methanol extracts were also screened for anthelmintic activity against Caenorhabditis elegans. Activity was found to increase with plant maturity. Irrigation was found to increase activity at the low rainfall (Fort Hare) site. Bulbs harvested from the irrigation treatment had significantly higher anthelmintic activity (p<_0.05) than bulbs harvested from treatments without irrigation. Dichloromethane extracts from bulbs grown at both sites had high anti-inflammatory activity. There were no significant differences (p<_0.05) in the activity of bulbs harvested from the different treatment plots. The time of harvest had an effect on the inhibition of prostaglandin synthesis by COX-1 enzymes. This study provides not only scientific verification for the use of M. natalensis to some extent as a medicinal plant, but also important data needed to successfully cultivate this species as a crop for small-scale farming.
Thesis (Ph.D.)-University of Natal, Pietermaritzburg, 2003.

Medicinal bulbs form part of the diversified flora in South Africa. The plants are used extensively in South African traditional medicine in the treatment of various ailments. Due to the ever-increasing demand and the unrestricted collection of medicinal plants from the wild, many of these slow growing bulbous plant species are driven into over-exploitation and extinction. The main parts collected for use are the underground bulbs, leading to the destructive harvesting of the whole plant. This form of plant harvesting poses threats to the long term sustainability of these plant resources from their natural habitats. Sustainable harvesting of these plants should be within the limits of their capacity for self-renewal. However, this seldom occurs with the often inconsiderate medicinal plant gatherers. Conservation of these plants is therefore necessary. A strategy that would take into consideration the sustainable harvesting and perhaps simultaneously provide similar medicinal benefits, would be the substitution of bulbs with leaves of the same plant. This study was aimed at evaluating the seasonal pharmacological and phytochemical properties in bulbs/corms and leaves of medicinal bulbs with a view of promoting the substitution of bulbs with leaves in traditional medicinal use. Four medicinal bulbous plants, Tulbaghia violacea, Hypoxis hemerocallidea, Drimia robusta and Merwilla plumbea were evaluated for the pharmacological and phytochemical properties in their bulbs/corms and leaves in spring, summer, autumn and winter seasons, with a view of promoting the use of leaves as a conservation strategy. Dried plant materials were sequentially extracted with petroleum ether (PE), dichloromethane (DCM), 80% ethanol (EtOH) and water in each season. The extracts were tested for activities against Gram-positive (Bacillus subtilis and Staphylococcus aureus), Gram-negative (Escherichia coli and Klebsiella pneumoniae) bacteria and the fungus Candida albicans using the in vitro microdilution assays to obtain minimum inhibitory concentrations (MIC) and minimum fungicidal concentrations (MFC). The four plant species were also evaluated for their ability to inhibit cyclooxygenase (COX-1 and COX-2) enzymes. Spectrophotometric methods were used to evaluate saponin and phenolic contents of samples from the four plant species in each season. Antibacterial activity was fairly comparable between bulbs/corms and leaves of H. hemerocallidea, T. violacea, and M. plumbea, with at least one extract showing some good activity (MIC < 1 mg/ml) in most of the seasons. Bulb extracts of D. robusta did not show good antibacterial activity while the leaf extracts showed good activity (0.78 mg/ml) against B. subtilis in spring, summer, and autumn and S. aureus (0.78 mg/ml) in autumn. The best antibacterial activity was recorded in winter, with MIC values as low as 0.195 mg/ml from the DCM bulb extracts of T. violacea against K. pneumoniae and S. aureus and PE corm extracts of H. hemerocallidea (0.195 mg/ml) against B. subtilis. Good antibacterial activity from water extracts were only recorded from corm extracts of H. hemerocallidea in summer, autumn and winter, H. hemerocallidea leaf extracts in autumn and winter, and M. plumbea bulb extracts in autumn. The leaf extracts of all the screened plant species demonstrated good fungicidal activity in autumn, with H. hemerocallidea corm water extracts recording an MFC value as low as 0.39 mg/ml. The leaf extracts of H. hemerocallidea (water), D. robusta (DCM) and M. plumbea (DCM) had good MFC values of 0.78 mg/ml each, in spring. The DCM leaf extracts of T. violacea also showed good fungicidal activity (0.78 mg/ml) in summer, while corm water extracts of H. hemerocallidea had an MFC value of 0.39 mg/ml in winter. There were no fungicidal activities recorded from all the bulb extracts in all the seasons. All the PE and DCM extracts in all the tested plant samples recorded between moderate (40-70%) and high (> 70%) COX-1 and COX-2 inhibition levels across all seasons. The EtOH corm extracts of H. hemerocallidea also demonstrated moderate to high inhibitory activity against COX-1 enzyme across all seasons. Bulb and leaf extracts of T. violacea showed selective inhibitory activity for COX-2 enzyme in all the seasons. The highest COX inhibitory levels were recorded in COX-2 from the PE leaf (spring) and bulb (autumn) extracts of T. violacea, with both recording 100% inhibitory activity. Phytochemical analysis revealed higher total phenolic compounds in bulbs/corms and leaves of all the analysed plant species, to be either higher in spring or winter. Plant material collected in autumn had the least levels of total phenolics. An almost similar trend to that of total phenolics was observed for flavonoids, gallotannins and condensed tannins in most plant samples, with higher levels either in spring or winter. Total saponins were consistently higher in winter than in the other seasons in all the screened plant species. There were in some cases, relationships between the peaks in the levels of some phytochemical compounds and the observed levels of bioactivity in different assays. The results obtained from this study demonstrate that the leaves of the screened plant species may substitute or complement bulbs in the treatment of certain ailments in traditional medicine. Thus, plant part substitution can be sustainably utilised in the conservation of these plant species while retaining the same medicinal benefits.
Thesis (M.Sc.)-University of KwaZulu-Natal, Pietermaritzburg, 2010.

The crisis of newly emerging diseases and the resistance of many pathogens to currently used drugs, coupled with the adverse side-effects of many of these drugs have necessitated the continuous search for new drugs that are potent and efficacious with minimal or no adverse side-effects. The plant kingdom is known to contain many novel biologically active compounds, many of which could potentially have a higher medicinal value when compared to some of the current medications. Indeed, the use of plants in traditional medicine, especially in African communities, is gaining more importance due to their affordability and accessibility as well as their effectiveness. Exponential population growth rates in many developing countries has resulted in heavy exploitation of our plant resources for their medicinal values. In addition, plant habitat destruction arising from human developmental activities has contributed to the fragmentation or loss of many plant populations. Owing to these factors, many plant species with horticultural and/or medicinal potential have become either extinct or are threatened with extinction. These threatened species cut across different taxonomic categories including shrubs, trees and succulents. Without the application of effective conservation strategies, the medicinal and/or horticultural potential of such threatened species may be totally lost with time. The extinction of such species could lead to the loss of potential therapeutic compounds and/or genes capable of being exploited in the biosynthesis of new potent pharmaceutical compounds.
Thesis (Ph.D.)-University of KwaZulu-Natal, Pietermaritzburg, 2009.

Eucomis (Family Hyacinthaceae) are deciduous geophytes with long, narrow leaves and erect, densely packed flower spikes. The bulbs are greatly valued in traditional medicine for the treatment a variety of ailments, and are thus heavily harvested for trade in South Africa's "muthi" markets. Eucomis species propagate relatively slowly from offsets and seed, and this, together with their over-utilization ethnopharmacologically, has led to their threatened status. This investigation focussed mainly on the study of the anti-inflammatory activity of plant extracts prepared from the leaves, bulbs and roots, and the development of suitable tissue culture protocols for the bulk propagation of the species under study. Common underlying symptoms in the majority of ailments treated with traditional remedies prepared from Eucomis species are pain and inflammation. Prostaglandins are the primary mediators of the body's response to pain and inflammation, and are formed from essential fatty acids found in cell membranes. This reaction is catalysed by cyclooxygenase, a membrane-associated enzyme occurring in two isoforms, COX-1 and COX-2. Non-steroidal anti-inflammatory drugs (NSAIDs) act by inhibiting the activity of COX. The use of commercially available COX-1 inhibitors is associated with side-effects, including gastric and renal damage. Selective COX-2 inhibitors do not have these undesired effects, and are thus potentially very valuable to the pharmaceutical industry. The relative inhibitory effects of different extracts of Eucomis species on the activities of purified cyclooxygenase enzyme preparations (COX-1 in sheep seminal vesicles, COX-2 in sheep placenta) were assessed. The COX-1 assay was used to screen extracts from 10 species of Eucomis and one hybrid species at a concentration of 250 μg mℓ ¯¹ in the assay. High levels of anti-inflammatory activity were exhibited by the ethanolic extracts prepared from the dried leaves, bulbs and roots. Aqueous extracts (screened at 500 μgmℓ ¯¹) showed much lower levels of activity. In general, the highest levels of anti-inflammatory activity were observed for the ethanol bulb and root extracts. Comparison of the activity of the bulb extracts from bulbs harvested in summer and winter revealed very little difference in COX-1 inhibitory activity. Eucomis extracts were separated using thin layer chromatography. The plates were developed in a solvent system of benzene : 1,4-dioxan : acetic acid, 90:25:4 and stained with anisaldehyde-sulphuric acid. The TLC fingerprints prepared from these extracts showed different chemical profiles for the leaf, bulb and root extracts, but many similarities between the different species. The position of the active R[f] fractions was determined and correlated with the TLC-fingerprints. The most widely utilized species medicinally, E. autumnalis subspecies autumnalis, was chosen for further investigation. The fluctuation of anti-inflammatory activity with season and physiological age was determined. Young plants were found to have high levels of COX-1 inhibitory activity, particularly in the leaves. As the plant matured, higher levels of activity were associated with the bulb and root extracts. The antiinflammatory activity of the leaf, bulb and root extracts varied slightly throughout the year, with the highest levels detected towards the end of the growing season, shortly before the onset of dormancy. This study of E. autumnalis autumnalis was extended to investigate the effects of environmental conditions on the levels of COX-1 inhibitory activity. The extent to which high temperature and light intensity, fertilization of the plants in summer with Kelpak preparations, and cold storage of the dry bulbs during winter, affected the levels of active compounds accumulated, was determined. Kelpak application decreased the anti-inflammatory activity of the leaf, bulb and root extracts, while high temperature / high light intensity had no significant effect on the COX-1 inhibitory activity of the leaf or bulb extracts. The root extract did show a significant increase in anti-inflammatory activity. Bulbs that were removed from the soil and stored at 10°C exhibited significantly higher COX-1 inhibitory activity than the control bulbs maintained in the soil. Higher COX-1 inhibition was observed in the leaf extracts from these plants when harvested half-way through the growing season. No significant difference was observed at this stage between the bulb and root extracts from the different treatments. Bioassay-guided fractionation (using the COX-1 assay) was used to isolate the active principle(s) in the bulb extract. The bulb material was subjected to serial extraction using a Soxhlet apparatus. The ethyl acetate fraction showed the highest levels of COX-1 inhibition, and this was further fractionated using a Sephadex LH-20 column and a solvent system of cyclohexane : dichloromethane : methanol (7:4:1). The most active fraction from this separation was then purified using semi-preparative TLC and HPLC. The primary compound eluting in this fraction had an IC₅₀ value of 14.4 μgmℓ ¯¹ in the COX-1 assay, and 30.5 μgmℓ ¯¹ in the COX-2 assay. This compound was tentatively characterized as a phenol ring attached to a conjugated hydrocarbon chain (with a molecular weight of 390), and was a potent COX-1 inhibitor. The COX-2 / COX-1 inhibitory ratio was calculated to be 2.1. A second, highly active compound, with IC₅₀ values of 25.7 μgmℓ ¯¹ and 21.8 μgmℓ ¯¹ in the COX-1 and COX-2 assays respectively, crystalized from one of the Sephadex LH-20 column fractions. This compound was identified as a spirostane-type triterpenoid, eucosterol, previously isolated from Eucomis species but not specifically linked to the pharmacological activity of the extracts. This compound showed COX-2 / COX-1 inhibitory ratio of 0.8, indicating that it was a selective COX-2 inhibitor. Two further compounds were identified from this extract, after crystallization from different fractions obtained from Sephadex LH-20 chromatography. These were both homoisoflavanones, 5,7-dihydroxy-6-methoxy-3-(4-methoxy benzyl)-chroman-4-one, and 5,7-dihydroxy-3-(4-methoxy benzyl)-chroman-4-one [eucomin], the latter having been isolated previously. The first compound exhibited very low levels of both COX-1 and COX-2 inhibition, and the second compound (eucomin) exhibited high COX-1, but low COX-2 inhibitory activity. The in vitro propagation of the genus Eucomis was undertaken primarily to provide a source of material for experimentation, and also to optimize this technique for the bulk production of plants for commercial and conservation purposes. Multiple shoot production was initiated from leaf explants, in all species studied. A Murashige and Skoog (MS) medium, supplemented with 100 mg ℓ ¯¹ myo-inositol, 20 g ℓ ¯¹ sucrose, and solidified with 2 g ℓ ¯¹ Gelrite® was used. The optimal hormone combination for shoot initiation in the majority of species was determined to be 1 mg ℓ ¯¹ NAA and 1 mg ℓ ¯¹ BA. Optimal root initiation was demonstrated on media supplemented with 1 mg ℓ ¯¹ IAA, IBA or NAA, depending on species. A continuous culture system using this protocol produced 25-30 plantlets per culture bottle, with 10-25 specimens per bottle available for acclimatization. To maximize plantlet survival, different support media used during the acclimatization process were necessary. Certain species responded best on a vermiculite medium, while perlite (which holds less water) was necessary for the optimal survival rate of other species. Acclimatized plantlets were repotted in a sand : soil mix (1:1). Further experimental work aimed to determine the factors affecting the accumulation of anti-inflammatory compounds in in vitro plantlets. Extracts prepared from in vitro plantlets showed high levels of COX-1 and COX-2 inhibitory activity, with a C0X-2/C0X-1 ratio of 1.1. High levels of sucrose (40 g ℓ ¯¹) significantly increased the number of shoots initiated, but had no effect on the anti-inflammatory activity. Low levels of sucrose (10 g ℓ ¯¹) led to a significant decrease in COX-1 inhibition. Changing the levels of nitrogen in the medium (but not the ratio of nitrate to ammonium ions) had no significant effect on the COX-1 inhibitory activity of the extracts. Callus was initiated from leaf explants and experiments were conducted to maximize callus proliferation. Optimal callus growth occurred on an MS medium supplemented with 100 mg ℓ ¯¹ myo-inositol, 30 g ℓ ¯¹ sucrose, 2 g ℓ ¯¹ Gelrite® , and a hormone combination of 10 mg ℓ ¯¹ 2,4-D and 2 mg ℓ ¯¹ kinetin. Callus cultures maintained in the dark grew best. Callus extracts tested in the COX assays (250 μgmℓ ¯¹) showed a higher level of COX-2 inhibition (69%) than COX-1 inhibition (46%). Lastly, the conclusive identification of the species under study was attempted, using DNA fingerprinting. Protocols were developed for the extraction of DNA from the leaves of Eucomis plants, and the optimization of the AP-PCR technique. Random sequence (10-base) oligonucleotide primers were screened, each primer used singly. Primers were selected on the basis that more than five distinct bands were detected. Differences were detected in the amplification products visualized using nondenaturing agarose gel electrophoresis stained with ethidium bromide. This work provides the basis for further studies into the phylogenetic relationships between the various species (and hybrids) of Eucomis.
Thesis (Ph.D.)-University of Natal, Pietermaritzburg, 1999.

The cancer bush (Lessertia frutescens L.) is an important leguminous perennial native to southern Africa and has been used for centuries in traditional medicine by the continent’s diverse cultural groups. Like many other legumes, the seeds of this species exhibit dormancy. Moreover, woody plants are typically difficult to propagate in in vitro culture systems. But in vitro shoot cultures are valuable in providing an alternative means of deriving desired secondary metabolites or phytocompounds, under controlled conditions. This study describes novel protocols for breaking seed dormancy, rapid and efficient in vitro propagation, bioreactor culture, and comprehensive phytochemical data following screening and analysis of in vitro and field extracts of L. frutescens. Experiments using physical, mechanical and chemical pre-sowing treatments were conducted to determine the germination response of this species. The results indicated that seeds of L. frutescens exhibited exogenous dormancy due to the inhibitory effect of the hard coat on germination. Seed dormancy was released by mechanical scarification in which 100 % germination was achieved. In vitro propagation studies using single node explants in Murashige and Skoog (MS) medium supplemented with combinations of different concentrations of benzyladenine and naphthaleneacetic acid revealed a maximum number of 10 shoots per explant in solid medium, and 12.9 shoots per explant in liquid medium inside a temporary immersion bioreactor. Indirect shoot organogenesis and plant regeneration using rachis and stem segments was achieved with the highest percentage of explants forming shoots (88.8 %) from rachis explants cultured onto MS medium supplemented with thidiazuron. Direct shoot organogenesis from hypocotyl and cotyledon segments was also achieved in L. frutescens. The highest shoot regeneration using hypocotyls (83 %) was obtained in MS medium supplemented with kinetin whilst the highest shoot regeneration using cotyledons (46 %) was obtained in MS medium supplemented with kinetin in combination with benzyladenine. Successful rooting (up to 80 %) and acclimatization (up to 90 % survival rate) was attained. Spectrophotometric and gravimetric methods indicated that saponins were the most abundant, followed by phenolics, flavonoids and then alkaloids in in vitro leaf extracts then in field leaf extracts and seed extracts, respectively. After qualitative analysis these extracts were also found to contain tannins, phlobatannins and cardiac glycosides of medicinal interest. By using gas and liquid chromatography the presence of the medicinally important L-canavanine, gamma amino-butyric acid and D-pinitol was verified in in vitro leaf, field leaf and seed extracts. In vitro leaves had higher quantities of all compounds, except for D-pinitol. Phytocompound analysis of shoots derived from several of the cytokinin-enhanced media showed that these organs contained higher quantities of L-canavanine compared to the control. This study, therefore, highlights the potential techno-economic production of medicinal phytocompounds from in vitro leaves of L. frutescens following large scale production using the protocols described in this study.
Thesis (Ph.D.)-University of KwaZulu-Natal, Westville, 2011.