Submitted in fulfillment for the Degree of Master of Technology (Biotechnology) in the Department of Biotechnology and Food Technology, Durban University of Technology, Durban, 2009. / Information regarding the nutritional value of wild food plants in Africa and current information varies from source to source. Prior to commercialization of wild foods the nutritional, ethnobotanical, medical, chemical, anthropological and toxicity requires investigation. Plants from the Amaranthaceae family were chosen because the family is characterized by several species which are used by indigenous communities as a source of nutrition in different plants of the world. The focus of this study was to investigate the nutritional and biological activities of three plants from the Amaranthaceae family viz. Achyranthes aspera, Alternanthera sessilis and Guilleminea densa that are considered famine plants. This study aimed to determine the nutritional value (proximate, minerals and vitamins), biological activity, toxicity and potential of a tissue culture system for three species from the family Amaranthaceae. Nutritional analysis comprised of determining moisture, ash, protein, fat, carbohydrate, dietary fibre and energy. Mineral analysis of calcium, copper, iron, magnesium, manganese, phosphorus, sodium and zinc was performed by microwave digestion and then analyzed by ICP Spectrophotometry. Vitamin A, Vitamin B1, Vitamin B2, Vitamin B3 and Vitamin C were also analyzed. For biological and safety analyses aqueous and methanolic extracts were prepared. Anti-oxidative and anti-inflammatory properties of the extracts were tested; antimicrobial activity was tested by evaluating the bactericidal, fungal effect and minimum inhibitory concentration on selected bacteria and fungi using the agar disk diffusion method. Anti mosquito potential was determined by setting up repellency, larvacidal assay and insecticidal assay. The safety and toxicity analysis was carried out by measuring cytotoxicity, toxicity and mutagenicity. The potential of an in vitro tissue culture system of A. aspera, A. sessilis and G. densa was determined using micropropagation. A. aspera indicated significant amounts moisture, ash, dietary fibre, protein, vitamin B1, vitamin B2, magnesium and manganese. Plant extracts of A. aspera had antibacterial activity against the Gram negative bacteria Esherichia coli, Pseudomas aeroginosa and Salmonella typhi; Gram positive bacteria Staphylococcus epidermis and Staphylococcus aureus. The methanolic extract had antifungal activity against Sacchromyces cerevisiae and exhibited significant free radical scavenging activity as well as 85% repellency against Anopheles arabiensis. The aqueous extract stimulated the growth of the K562 (Chronic Myclogenous Leukaemia) cell line and the plant extracts showed no mutagenicity or toxicity. A. sessilis indicated significant levels of ash, dietary fibre, protein, energy, vitamin A, vitamin B1, vitamin B2, vitamin B3, iron, magnesium and manganese present. Plant extracts of A. sessilis had antibacterial activity against Gram negative bacteria P. aeroginosa and Gram positive bacteria S. epidermis. The plant also showed antifungal activity against the yeasts S. cerevisiae and Candida albicans. The methanolic plant extract showed excellent antioxidant activity. The aqueous plant extract stimulated the growth of the K562 cell line and the plant extracts possessed no mutagenicity or toxicity. This plant grew well in a tissue culture system where it was propagated from callus to a fully grown plant able to survive in environmental conditions. G. densa has ash and dietary fibre, vitamin B2, vitamin B3 and iron. The plant extracts had antibacterial activity against Gram negative bacteria E. coli, P. aeroginosa and Klebsiella. oxytoca; Gram positive bacteria Baccilus stereathermophilus and S. aureus. The plant also has antifungal activity against C. albicans and significant repellency activity against A. arabiensis where it showed 100% repellency. This plant was not found to be mutagenic or toxic. The results obtained from this study show promising potential for the plants to be exploited as famine food plants. The nutritional value, biological activity and ability to micropropagate A. aspera, A. sessilis and G. densa indicates a good potential for purposes of harnessing biotechnological products.

Submitted in fulfillment of the requirements for the Degree of Master of Technology: Biotechnology, Durban University of Technology, 2009. / Dichrostachys cinerea, Carpobrotus dimidiatus, Capparis tomentosa and Leonotis leonurus are all plants that are indigenous to South Africa. These plants are used in traditional medicine to treat various ailments. However, there is little or no scientific data to justify these traditional uses. Furthermore, it is difficult to reconcile traditional knowledge with scientific evidence because of the overwhelming targeting of signal-responsive systems by plant defensive compounds, multiple sites of action and the connectedness of the signaling pathways, which provide many cures and have pleiotropic effects. In order to evaluate the action spectrum of these plants, and validate its widespread use, this research evaluated the antibacterial, antioxidant, anti-inflammatory, anti-mosquito and immunomodulatory properties of these plants. Antimicrobial activity of the extract was determined by evaluating the bactericidal and fungicidal action using the agar disc diffusion assay. Anti-oxidative properties of the extracts were tested using the DPPH photometric assay. Anti-inflammatory properties were carried out using the 5-lipoxygenase assay. The larvicidal, repellency and insecticidal assay was determined against A.arabiensis. The safe use of these plant extracts was determined by evaluating toxicity, a brine shrimp lethality assay and an in vitro cell culture system using human myelogenous leukemia cell line. Potential carcinogenic activity was evaluated using the Ames Salmonella Mutagenecity assay. The immunomodulatory activity of the extracts on human peripheral blood mononuclear cells 6 was evaluated on freshly harvested lymphocytes using the MTT assay. Cytokine response was evaluated by measuring the secretion of interferon-gamma and interleukin-10. Elucidation of the B cells, T cells, activated T cells, CD 4+, CD 8+ and NK cells was performed by flow cytometry. The extracts showed anti-microbial activity against Escherichia coli, Pseudomonas aeruginosa, Klebsiella oxytoca, Salmonella typhimurium, Serratia marcescens, Bacillus cereus and Tricoderm sp. The highest activity was shown by methanolic and aqueous extracts of L. leonurus leaves followed by methanolic and aqueous extracts of D. cinerea. Extracts of C. tomentosa and D.cinerea demonstrated a higher degree of free radical scavenging than rutin, which was used as a standard indicating that these plants have strong antioxidant properties. None of the plants showed significant anti-inflammatory activity when compared to NDGA. In the anti-mosquito assays, the extracts showed strong repellency and insecticidal activity. L. leonurus extracts demonstrated the highest insecticidal and repellency activity against the mosquito, and was also found to cause ‗knockdown‘ and mortality. The extracts display no toxicity, cytotoxicity and mutagenicity. The immunological studies for immune modulation showed that the methanol extracts of these plants induce a Th1- predominant immune response because they significantly suppressed the secretion of IL-10 and augment IFN-γ production, which are hallmarks used to indicate a stimulation of the innate immune response. This study also provides new information, with respect to the potential use of these plants in producing a mosquito repellent and an immunostimulant.

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 genus Helichrysum (Asteraceae) consists of approximately 500 species worldwide, with 245 indigenous to South Africa. As a result of the large number of species, the chemistry and biological activity of several species have not yet been investigated. The aim of this project was to investigate the phytochemistry of three species and propose a synthetic route to one of the antibacterial compounds isolated. An extensive literature review regarding the widespread traditional uses, biological activity and phytochemistry of the South African Helichrysum species is provided. From Helichrysum splendidum, a plant used traditionally to treat rheumatism, two monomeric guaianolides and a dimeric guaianolide, helisplendidilactone, were isolated. The stereochemistry of these known compounds was confirmed and the NMR assignments for certain peaks of helisplendidilactone were corrected. An X-ray structure for helisplendidilactone was obtained for the first time. The phytochemistry of Helichrysum montanum was investigated for the first time and new diastereoisomers of known guaianolides were isolated. The phytochemistry of H. splendidum and H. montanum is remarkably similar and supports their morphological classification in the same taxonomic group. The chloroform:methanol extract of H. montanum yielded a new dimeric guaianolide, 13’-epihelisplendidilactone, which is related to helisplendidilactone, as well as three monomeric guaianolides (of which one is a new diastereomer of a known compound). The extract also yielded spathulenol (a sesquiterpene), umbelliferone (a coumarin) and 4’,5,7-trihydroxy-3,3’,8-trimethoxyflavone (a flavonoid). Thirty-five Helichrysum species were screened for antimicrobial activity against six microorganisms and a preliminary cytotoxic assay, which included the use of “normal” and cancer cell lines, was performed. H. excisum was selected for further study based on the fact that it exhibited promising antimicrobial activity and relative low toxicity. Furthermore, with the exception of the essential oil, the phytochemistry of this species has not been investigated. From the aerial parts of H. excisum, five flavonoids, identified as pinocembrin, gnaphaliin, lepidissipyrone, 5-hydroxy-7,8-dimethoxyflavone and isoscutellarein 7-O-b-glucoside were isolated. Four of these flavonoids have an unsubstituted B-ring, a phenomenon often observed in flavonoids isolated from Helichrysum species. The active antimicrobial component of H. excisum has been identified as lepidissipyrone. Owing to the interesting biological activities reported for phloroglucinol a-pyrones and the synthetic challenges associated with these molecules, lepidissipyrone was selected for a synthetic study. Both the flavanone and pyrone moieties present in lepidissipyrone have been successfully synthesised. A successful strategy towards the CH2 linker between the two units has been illustrated. The strategy could be used to synthesise similar phloroglucinol-derived pyrones. / Thesis (Ph.D.) - University of KwaZulu-Natal, Pietermaritzburg, 2008.

This work is an account of the investigations into the chemistry of two Cedrelopsis species from the Ptaeroxylaceae, Cedrelopsis grevei and Cedrelopsis microfoliata and a species from the Mesembryanthemaceae, Khadia alticola, as well as investigations into the synthesis of hydroxylated and prenylated chalcones. Cedrelopsis grevei, commonly called Katrafay, is amongst the many medicinal plants of Madagascar, being used to relieve muscle fatigue when the bark is soaked in hot water. Previous investigations of the wood and stem bark of this plant, have yielded chromones and coumarins and a recent investigation of the stem bark of a specimen collected in the north of Madagascar has yielded two novel limonoids of unusual structure, cedmilinol and cedmiline. The fruit and seed of Cedrelopsis grevei have not been studied previously and a phytochemical investigation of these plant parts was undertaken in this work. The dichloromethane extract of the fruit and seeds yielded, after column chromatography, a dihydrochalcone, uvangoletin, a flavanone, 5,7dimethoxypinocembrin, two simple chalcones, cardamonin and flavokawin Band three prenylated chalcones, 2’-methoxyhelikrausichalcone, cedreprenone and cedrediprenone. Three of these compounds, 2’-methoxyhelikrausichalcone, cedreprenone and cedrediprenone have not been isolated previously. Cedrelopsis microfoliata is another medicinal plant used in Madagascar. The leaves of this plant are used to prepare a decoction for woman to drink after childbirth. This is the first phytochemical investigation of Cedrelopsis microfoliata. The hexane extract of the dried stem bark yielded three compounds after column chromatography, a chalcone, microfolian and two flavanones, microfolione and (+)-agrandol. The dicholoromethane extract of this compound yielded four compounds after column chromatography, three coumarins, cedrecoumarin A, obliquin and microfolicoumarin and a sesquiterpenoid, sesquichamaenol. Four of the compounds isolated from Cedrelopsis microfoliata, microfolidione, microfolione, (+)-agrandol and microfolicoumarin have not been isolated previously. Khadia alticola is one of the species added to "Khadi", a Tswana/South Sotho name for beer brewed traditionally using the fleshy roots of a variety of taxa. Khadia is also reported to be used medicinally by the Manyika people of the Umtali district of Zimbabwe. The phytochemical investigation of the roots of Khadia alticola, which have not been studied previously, was undertaken to determine whether mesembrine type alkaloids were present in this species and thus contributing to the "potency" of the beer brewed traditionally. No mesembrine alkaloids were isolated in this work, however, a common sterol, sitosterol was isolated from the acidic chloroform fraction of the roots of this species and a flavonoid, 3,4',5,7-tetrahydroxyflavan was isolated from the basic chloroform fraction. Two chalcones, 3’,5’-dihydroxychalcone and 2’-hydroxychalcone were synthesised using the Claisen condensation. An isoprenylated acetophenone intermediate and an isoprenylated chalcone were also synthesised. / Thesis (Ph.D.)-University of Natal, Durban, 2001.

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.

The present study investigated pharmacological properties of Capsicum frutescens-derived capsaicin, including its analgesic, anti-inflammatory and coagulatory properties. The effects of capsaicin on gastrointestinal and myocardial muscles, as well as on myocardial ischaemic-reperfusion, were also investigated. Capsaicin pre-treatment in neonatal rats has been found to abolish the development of thermal hyperalgesia produced in a model of neuropathic pain in rats (Toth-Kasa et al., 1986). In addition, capsaicin sensitivity has been found to be dependent on continued presence of nerve growth factor (NGF), whose concentration increases in inflamed tissues (Bevan and Winter, 1995). By stimulating the release of excitatory amino acids (EAA); such as glutamate and neuropeptides [(CGRP, neurokinin A (NKA) and Substance P (SP)] from both the peripheral and central terminals of sensory neurones by two mechanisms (Kroll et al., 1990; Del Bianco et al., 1991; Lou et al., 1992; 1994; Woolf et al., 1994); capsaicin has been shown to produce a longer-term inhibitory effect. This is one likely mechanism for capsaicin analgesic and anti-inflammatory actions (Bleakman et al., 1990). Within the gastro-intestinal tract, SP and NKA are involved in the physiological control of several digestive functions, such as motility, fluid and electrolyte secretion, blood flow, and tissue homeostasis (Otsuka, 1993; Holzer et al., 1997). Consistent with this finding, upsurge of SP in irritable bowel syndrome (IBD) was confirmed by Mantyh et al, (1988). Pre-treatment of rats with either capsaicin or NK-1R antagonists dramatically reduced fluid secretion, mucosal permeability, and intestinal inflammation in animal models of acute and chronic inflammation (McCafferty et al, 1994; Pothoulakis et al., 1994). Capsaicin can modulate endocrine and paracrine activities, immune responses, as well as gastro-intestinal and cardiovascular functions. Moreover, up-regulation of Substance P receptors was found to be associated with chronic inflammatory conditions (De et al., 1990). Stimulation of transient receptor potential vanilloid 1 also results in the activation of nociceptive and neurogenic inflammatory responses (Rigoni et al., 2003). vi The pharmacodynamic effects of capsaicin on the cardiovascular system remain elusive. Some actions of capsaicin on the heart were attributed to an interaction at K+ channels (Castle, 1992), or liberation of neuropeptides, most notably calcitonin-gene-related-peptide (CGRP) from the vanilloid-sensitive innervation of the heart (Franco-Cereceda et al., 1988; 1991). The possibility of a direct effect of capsaicin on the heart via a cardiac vanilloid receptor (VR), or through interaction of vanilloid receptors with purinergic receptors, and subsequent release of nitric oxide (NO), leading to vasodilatation were considered. Evidence abound in the literature that Ca2+ ions are released through 1, 4, 5 inositol phosphatase by the release of phospholipase C, or through interaction of the vanilloid receptors with cannabinoids. In an earlier study, Jaiarj et al. (1998) found that capsaicin acting on the heat-sensitive vanilloid receptors, had thrombolytic effects. Though weak evidence, Jaiarj et al. (1998) observed that individuals who consume large amounts of Capsicum have lower incidence of thromboembolism. Following ethical approval, the study reported in this thesis was conducted in phases. Identification of Capsicum frutescens (facilitated by a botanist in the Department of Botany, Westville campus of the University of KwaZulu Natal). Chromatographic extraction of capsaicin from Capsicum frutescens was followed by Nuclear Magnetic Resonance (NMR) analysis of the extract. Animal studies were conducted using capsaicin extract (CFE) and/or a reference capsaicin (CPF), using „hot plate. and „acetic acid. test methods to investigate the role of capsaicin on analgesia. Fresh egg albumin-induced inflammation was used to investigate the role of capsaicin in inflammation, following pre-treatment with CFE and CPF. Concentraton-response curves of increasing concentrations of capsaicin, acetylcholine and other agonist drugs with specific antagonists on strips of chick oesophagus, guinea-pig ileum, and rabbit duodenum were constructed following investigations on gastrointestinal (GIT) smooth muscles. The effect of capsaicin on coagulation was assessed by measuring international normalized ratio (INR) of animals that were exposed to different concentrations of capsaicin (CFE and CPF). Furthermore, parallel control studies were conducted in each of these investigations using distilled water or saline as placebo-control or specific-prototype agonists. negative-control. Cardiovascular investigations included studies on the effects of capsaicin on the heart rate, inotropy, vii coronary perfusion pressure, and ischaemic-reperfusion injury, using Langendorf.s rat heart models. Collated data were triangulated by manual hand-written and PowerLab data acquisition, or computerised capture. Statistical analysis were performed by either one or two of the following: Student.s t-test, ANOVA (repeated or single–use modes), facilitated and confirmed by Graph Pad Prism, Microsoft Excel or CPSS software(s). Reproducibility and relevance to the stated objectives of the various studies were confirmed by assessing which of the Null or Alternative hypothesis is validated by the results from the test. Treatment with CFE or CPF at all doses significantly (p<0.01) increased MRT. By comparison with control, writhing responses to acetic acid were significantly reduced following pre-treatment with various doses of CFE or CPF. The results in both parallel groups of CFE and CPF in the hot plate and acetic acid tests had Pearson correlation of one (1). Compared to the diclofenac (DIC) group, the degree of inhibition of paw oedema by CFE and CPF was statistically significant (P<0.05-0.001), best in the first 4 hours of treatment. The results of the in vitro laboratory animal study indicate that relatively low concentration of CPF (20 or 40 .g) produced significant (p.0.05), concentration-related inhibitions of acetylcholine (0.1-5 .g)-induced contractions of the chick isolated oesophagus, guinea-pig isolated ileum and rabbit isolated duodenum. Biphasic effects, which were noticed at low concentrations, consisted of initial brief contractions, followed by longer-lasting relaxations and reductions of the contractile amplitudes of the muscle preparations. Percentage inhibitions of the smooth muscle contractions by CFE or CPF were concentration-dependent, ranging from 20-70% (p<0.02). / Thesis (Ph.D.)-University of KwaZulu-Natal, Westville, 2012.

Boophone disticha (L.f.) Herb is one of the most widely distributed bulbous species in southern Africa. Of Africa’s many bulbous plants, it is widely known for its poisonous and medicinal properties. It is of considerable ethnobotanical interest in traditional medicine because of its hallucinogenic alkaloids and it has great potential as an ornamental due to its fan-shaped foliage and large umbel of bright pink to deep red flowers. In South Africa, many bulbous plants are used in traditional medicine which are collected from wild populations. The high demand for trade and use of such plants, that are destructively harvested, places an enormous pressure on natural populations. According to the Red List of South African Plants, the conservation status of B. disticha has been listed as ‘declining’. It is, therefore, important to develop conservation strategies for these medicinal plants, such as the development of alternative propagation methods. Micropropagation is a useful technique for rapid clonal multiplication of plant material which could alleviate the pressure on the wild plant populations, as well as potentially producing useful secondary metabolites. The in vitro induction of storage organs is especially beneficial as it can limit the loss of plants during acclimatization since bulblets are generally hardier than shoots or plantlets. Thus, the main aim of this research was to establish a micropropagation protocol which could be a valuable tool for conservation of this plant species. In addition, B. disticha plants were assessed in various ethnopharmacological assays to evaluate their medicinal properties, and a preliminary study on the population genetics was also conducted. As part of the development of a suitable micropropagation protocol, the effect of environmental and physiological factors on the initiation and growth of bulblets were investigated. These factors included the effect of various plant growth regulators, carbohydrates, temperature, photoperiod and liquid culture. Different explants (i.e. ovaries, anthers, filaments, pedicels, embryos, seeds and bulb twin-scales) were tested to determine which explants were the most suitable for subsequent experiments. Although success was limited, twin-scales proved to be the most suitable explant and it was demonstrated that activated charcoal, ascorbic acid and N6- benzyladenine were required as media supplements. Antimicrobial activity was tested between different plant parts and seasons. The plant parts (roots, leaves, outer and inner bulb scales) were extracted with a range of differing polarity solvents. These were screened for antibacterial activity against Bacillus subtilis, Staphylococcus aureus, Escherichia coli and Klebsiella pneumoniae, and for antifungal activity against Candida albicans. Extracts from roots of plants collected in spring and summer showed the best antimicrobial activity against B. subtilis, E. coli and K. pneumoniae, indicating that plant part and collection time do affect activity. In vitro grown bulblets also showed antimicrobial activity, demonstrating that antibacterial properties were maintained in cultured plantlets. Extracts from plants collected in summer were tested for mutagenicity using the Ames test (Salmonella/microsome assay; plate incorporation method, with or without metabolic activation). None of the extracts tested were found to induce mutations and also did not modify the effect of the mutagenic compounds (2AA with S9 and 4NQO without S9). Although the results do not indicate a mutagenic response, this does not necessarily confirm that it is not mutagenic nor carcinogenic to other bacterial strains, however, B. disticha must be used with caution, especially considering the levels of alkaloids in the plant. The two major constituent alkaloids of B. disticha were identified as buphanidrine and distichamine. In the antibacterial assay, both compounds exhibited broad-spectrum micromolarlevel activity against the two Gram-positive and two Gram-negative bacteria tested. The best MIC value, of 0.063 mg/ml, was found for bupanidrine/distichamine against S. aureus, E. coli and K. pneumonia. The isolated compounds were tested and found to be neither mutagenic nor toxic at the concentrations tested. Thus, buphanidrine and distichamine are thought to be the constituents likely responsible for the medicinal properties of the plant. To determine the level of genetic variation between different populations of B. disticha, plants were collected from six wild populations in KwaZulu-Natal, South Africa. DNA was isolated and tested for genetic variation using ten Inter Simple Sequence Repeat (ISSR) primers. The level of inter-population polymorphism ranged between 23% and 39%, showing that the populations had low genetic polymorphism. From the genetic distance results, it was found that the Midmar and Umgeni Valley populations are closely related, and these populations are similar to two sister populations. The Amatikulu and Lions River populations were similar but slightly different to the other populations. Antimicrobial assays showed minor difference in activity from the six wild populations. Although the micropropagation of B. disticha had limited success, this study did develop a successful decontamination protocol as well as determine the most useful explant and supplements. This information provides an important starting point for the development of a successful micropropagation protocol for the conservation of B. disticha. Since, B. disticha is an important medicinal plant in South Africa, this study has also deepened our understanding of the constituents that could be responsible for the medicinal properties of B. disticha and, in so doing, confirmed the value of this plant for use in traditional medicine in South Africa. / Thesis (Ph.D.)-University of KwaZulu-Natal, Pietermaritzburg, 2013.

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.

Studies have shown that the traditional healers have used Tulbaghia violacea (TV) (also known as ‘wild garlic’) for the treatment of a number of ailments including fever, tuberculosis, stomach problems, and oesophageal cancer. However, little is known with regards to the anticancer and antiproliferative properties of this plant. Therefore, this study investigated the effects of TV and domesticated garlic extracts on Jurkat cells, in order to determine whether or not these extracts possess anti-proliferative properties. Cultured Jurkat cells were treated with IC50 concentrations of garlic (14μg/ml), TV leaf (256μg/ml), TV bulb (225μg/ml) and TV stalk (216μg/ml) extracts as determined by the methylthiazol tetrazolium assay. Free radical production was measured using the thiobarbituric acid reactive substance (TBARS) and nitric oxide (NO) assays, while glutathione (GSH) concentration was measured using the GSH-Glo™ assay. The apoptosis inducing properties of each extract were measured using flow cytometry (Annexin V- Fluos and JC-1 assays) and luminometry (caspases 3/7, 8, 9 and ATP). Western blots were run to determine protein expression, while comet and DNA fragmentation assays were used to determine the level of DNA damage induced. Wild and domesticated garlic extracts induced a significant increase in malondialdehyde concentration ([MDA]), with TV bulb extract inducing the highest concentration (p<0.0001). A significant increase in NO concentration was observed in the bulb (p<0.0001) and stalk (p<0.001) extracts, and leaf (p<0.05) and stalk (p<0.05) TV extracts significantly increasing GSH concentration. The longest comet tails were observed in TV bulb extracts (p<0.0001) and comprised mainly of single strand breaks, while the comets induced following garlic exposure contained double strand breaks. All extracts, except TV leaf, increased the percentage of cells undergoing apoptosis. Tulbaghia violacea leaf induced a significant (p<0.0001) increase in percentage of cells undergoing necrosis, whereas TV bulb resulted in a significant (p<0.0001) decrease. All TV extracts induced caspase 3/7 and 9 activity, with the most significant increase in caspase 9 activity observed for TV leaf and bulb. No significant change in caspase 3/7 activity was evident for domesticated garlic. Cleavage of PARP and expression of NF B and HSP 70 occured for all extracts. However, HSP 70 was not differentially expressed. Exposure to wild and domesticated garlic extracts induced peroxidative lipid and DNA damage within the cells, indicating oxidative stress. This damage occurred in conjunction with increased percentage of cells undergoing apoptosis and expression of caspase 3/7. Therefore, these findings suggest that TV is inducing cell death through apoptosis in Jurkat cells using a number of mechanisms, including the induction of oxidative stress. This is of clinical significance, as cell death through apoptosis is the preferred method of action for anti-cancer drugs. / Thesis (M.Med.)-University of KwaZulu-Natal, Durban, 2012.