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.

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.

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.

Treatment of diabetes mellitus is mainly focused on glycaemic control regulated by insulin and takes place in insulin sensitive tissues like skeletal muscle which accounts for 75% of glucose metabolism. Plant derived compounds that have anti-diabetic potential are currently being investigated for diabetes treatment as they are cheap and non-toxic. Oleanolic acid (OA), a triterpene found in a wide variety of plants has been shown to have anti-diabetic effects but its mechanism of action, especially on the insulin signalling cascade has not been fully elucidated. The aim of the present study was to investigate the effects of OA on the PI3K/Akt insulin signalling cascade in skeletal muscle and skin of streptozotocin induced diabetic male Sprague-Dawley rats. Male Sprague-Dawley rats (non-diabetic and diabetic) were treated with insulin (4IU/ kg bw), OA (80 mg/kg bw) and a combination of OA + insulin in an acute and sub-chronic study. The study showed that OA does not reduce blood glucose levels in type 1 diabetic rats but enhances insulin stimulated hypoglycaemic effects. In the acute study OA was shown to activate Akt and dephosphorylate GS in skeletal muscle of streptozotocin induced diabetic rats. In the sub-chronic study OA and OA + insulin increased expression of GS in skeletal muscle of diabetic rats. GP expression was decreased by OA and OA + insulin treatments in skeletal muscle whilst in skin it was increased by both treatments. OA increased both GS and GP in skeletal muscle whilst in skin they were decreased. OA + insulin treatment increased GS and decreased GP activities in skeletal muscle and increased activity of both enzymes in skin of diabetic rats. OA increased the amount of glycogen in both muscle and skin whilst OA + insulin reduced the amount of glycogen. OA and OA + insulin treatment showed some protective effects against liver and muscle damage as there were reductions in serum LDH, ALT and AST levels. In conclusion, oleanolic acid in synergy with insulin can enhance activation of the insulin signalling pathway and there was evidence of OA activation of insulin signaling enzymes independent of insulin. / Thesis (M.Sc.)-University of KwaZulu-Natal, Durban, 2013.

Diabetes mellitus (DM) is a metabolic disease characterized by chronic hyperglycaemia; this condition is caused by lack of insulin secretion (Type 1) and/or insulin resistance (Type 2). In diabetic patients; carbohydrate, protein and lipid metabolism is disturbed due to the lack of the body’s ability to utilise glucose efficiently. Management of type 1 diabetes involves insulin therapy which may be inconvenient for patients. Therefore alternative methods for management of type 1 diabetes involving medicinal products are being investigated. This study is aimed at investigating the effect of OA on markers of lipid metabolism and on proteins of the insulin signalling pathway in Type 1 diabetic rats as this plant product has anti-hyperglycaemic effects. Male Sprague-Dawley rats were divided into two groups (diabetic and normal). In both groups the rats were further divided into four groups and assigned to treatment as follows: vehicle, insulin, OA and OA plus insulin. Oral glucose tolerance test was performed in fasted and non-fasted diabetic rats for 2 hours. In acute studies the effect OA following treatment of rats was evaluated at 15, 30 and 60 minutes. In sub-chronic studies rats were treated daily for 14 days. OA did not improve glucose tolerance in diabetic rats after 2 hours of administration. However, it enhanced blood glucose lowering effect of insulin and this was statistically significant in fasted rats. In acute studies OA enhanced the effect of insulin in normal and diabetic animals as AKT phosphorylation was increased when insulin was used in combination with OA. OA reduced the expression and activity of HSL in liver tissue after 14 days of treatment in both normal and diabetic rats. In adipose tissue, OA reduced the expression of HSL in diabetic rats. However, OA alone did not reduce the activity of HSL but when it was combined with insulin, a reduction of HSL activity was observed. OA administration had no significant effect on TGA and HDL-c levels but significantly (p < 0.05) reduced total cholesterol and LDL-c in diabetic rats. It had no significant effect on total cholesterol, and increased LDL-c levels in normal rats. Serum AST and ALT levels in diabetic rats were reduced by OA administration but this reduction was not statistically significant. The results of this study suggest that OA enhances the hypoglycaemic effect of insulin, improves lipid profile and possesses hepatoprotective effects. Lastly, OA independently increases AKT phosphorylation and decreases HSL expression and activity. / Thesis (M.Sc.)-University of KwaZulu-Natal, Durban, 2013.

Thesis (MSc (Plant Biotechnology))--University of Stellenbosch, 2006. / The development of a reliable and reproducible method for the genetic characterisation and identification of the commercially important Agathosma species was investigated. Previous research attempts aimed at developing a reliable and reproducible method of identifying these Agathosma species failed, mostly because these studies were based on phenotypic traits and these methods were therefore influenced by environmental factors. In this study amplified fragment length polymorphisms (AFLPs) were successfully used to quantify the genetic variation between the Agathosma species and as a result three distinct groups could be identified. The data obtained were elaborated with the Dice genetic similarity coefficient, and analysed using different clustering methods and Principle Coordinate Analysis (PCoA). Cluster analysis of the genotypes revealed an overall genetic similarity between the populations of between 0.85 and 0.99. The AFLP-based dendrogram divided the populations into three major groups: (1) the A. serratifolia and A. crenulata populations, (2) the putative hybrid, A. betulina X A crenulata populations, and (3) the A. betulina populations, confirming that this technique can be used to identify species. The question of hybridisation was also clarified by the results of the PCoA, confirming that the putative hybrid is not genetically intermediately spread between the A. crenulata and A. betulina populations, and that it is genetically very similar to A. betulina. The putative hybrid can therefore rather be viewed as a genetically distinct ecological variant of A. betulina. As the AFLP technique cannot be directly applied in large-scale, routine investigations due to its high cost and complicated technology, the development of polymerase chain reaction (PCR)-based molecular markers, able to accurately identify the species, was undertaken. Due to the superior quality of A. betulina oil, the development of such markers is especially critical for this species. Several species-specific AFLP markers were identified, converted to sequence characterised amplified regions (SCARs) and ultimately single nucleotide polymorphisms (SNPs) were characterised. The developed SCARs were unable to distinguish between the species. The conversion of AFLP fragments to SCARs is problematic due to multiple fragments being amplified with the AFLP fragment of interest. The diagnostic feature of the SNP-based markers was not sensitive enough, since this technique could not distinguish between the A. betulina and A. crenulata and/or the putative hybrid populations. The SNPs that were characterised were found not to be species-specific; they were only specific to the particular clone. Although a quick and robust marker specific for A. betulina has not yet been developed, this study sets the stage for future genetic studies on Agathosma species. Such a marker, or set of markers, would be an invaluable contribution to a blooming buchu oil industry.

Diabetes mellitus is a chronic physiological carbohydrate metabolic disorder with significant impact on the economy, quality of life and life expectancy in South Africa. Herbal medicine has become the alternative therapy in the management of this disease. However, their safety and effectiveness have not been investigated. To address this, one of the plants used in Eastern Cape Province, South Africa, Bulbine abyssinica A. Rich (Asphodelaceae), was evaluated. Bulbine abyssinica is one of the species used in the management of diabetes mellitus. This plant was mentioned during an ethnobotanical survey conducted in Nkonkobe municipality of the Eastern Cape Province. Though a decoction prepared from the whole plant is used in the treatment of diabetes mellitus, the mechanism of action and its safety has not been elucidated. Thus, this research work was designed to contribute to the understanding of the possible mechanism of action of B. abyssinica as an antidiabetic medicinal plant and its toxic potentials to the users. The aqueous extract exhibited remarkable inhibitory activity onα-amylase (estimated inhibitory concentration (IC)50 value of 3.28 μg/ml), while the acetone extract exhibited weak inhibitory activity. The acetone extract exhibited notable α-glucosidase inhibitory activity (IC50 value of 4.27 μg/ml) while aqueous extract had significantly weak activity. The Lineweaver-Burk double reciprocal plots revealed that the aqueous extract exerts noncompetitive inhibition on the α-amylase activity while the acetone extract exerts a near competitive inhibitory pattern on the α-glucosidase activity. The extracts from the plant possessed high free radical scavenging activities, with acetone extract exhibiting the highest activities in all assay models used except with ferric reducing power and nitric oxide (NO) scavenging ability. The aqueous extract exhibited the highest ferric reducing power and nitric oxide radical mopping strength while the essential oil exhibited the highest scavenging activities with 1,1-diphenyl-2-picrylhydrazyl (DPPH) and relatively high ferric reducing power and nitric oxide scavenging ability. The acetone extract and the essential oil of this species exhibited higher albumin denaturation inhibition than the aqueous extract while the latter showed the greatest membrane lysis protection. The essential oil, acetone and aqueous extracts from this plant significantly inhibited the growth of Shigelle flexneri, Pseudomonas aeruginosa, Staphylococcus aureus and Enterococcus faecalis. Klebsiella pneumonia, Proteus vulgaris and Streptococcus pyogens growth were inhibited by acetone and aqueous extracts. The essential oil also showed inhibitory activity against Proteus vulgaris. However, the extracts were active against the growth of only three fungi species. The essential oil showed significant inhibitory activity against Trichophyton rubrum. The aqueous extract inhibited the growth of Microsporum gypseum while the acetone extract was active against Microsporum canis, and Microsporum gypseum. The carbohydrate, crude fibre, moisture, ash, crude protein and crude fat of approximately 74.8 percent, 8.9 percent, 8.8 percent, 8 percent, 7.7 percent and 0.6 percent, respectively, were detected in this plant. The species is characterized by moderate levels of oxalates, phytic acids, Vitamin A, Vitamin C and Vitamin E. Potassium and calcium were present in highest levels, while magnesium, iron, sodium, aluminium and phosphorus were moderately present. Manganese, zinc and copper where in low amounts. These vitamins and mineral elements were within their recommended daily allowance (RDA) in humans. The investigation also revealed appreciable amounts of total phenols, flavonoids, flavanols, proanthocyanidins and alkaloids in both acetone and aqueous extracts while saponins and tannins were in trace amounts. The essential oil was characterized by large quantities of terpenes (91.9 percent) and small fraction of esters (8.01 percent).

Currently, the approved anti-HIV drugs on the market only target the three HIV enzymes: reverse transcriptase, protease and more recently, integrase. Due to the limited nature of the current therapy, it is possible that a multi-drug resistant virus can emerge. The main concerns in developing countries however, are the expense and availability of the drugs and because of this, it is essential to investigate all alternatives. Traditional medicine offers many advantages as compared to allopathic treatment in so far as being relatively cheaper, accessible and it is broadly accepted in the population groups of the developing countries. Little is known though, of the exact efficacy and toxicity of these remedies so it is vital that these possible leads be investigated thoroughly. For the purpose of this study, two plants, Sutherlandia frutescens and Lobostemon trigonum were studied to ascertain their potential anti-HIV activity. Sutherlandia has received international attention as a possible cheap herbal remedy to improve the health of AIDS sufferers. Anecdotal evidence from health workers claim that HIV- infected patients on Sutherlandia treatment have shown improved CD4 counts, decreased viral loads and a general improvement in well-being. Extracts were prepared from dried leaves and flowers in methanol, ethanol, acetone, methylene dichloride or distilled water. Sulphated polysaccharides have been described extensively in literature with regards to their anti-HIV activity, so as a form of dereplication; an ethanol precipitation was performed on the aqueous extracts to remove sulphated polysaccharides. A toxicity study was performed on all crude extracts using uninfected peripheral mononuclear blood cells (PBMCs) isolated from whole blood. To measure anti-HIV activity, HIV-infected PBMCs were cultured with each of the crude extracts and cell viability measured using the tetrazolium salt, XTT. HIV-infected CEM-NKR-CCR5 cells were also used and supernatant from the viral studies was tested for the HIV antigen p24. xii Results varied greatly between assays but with the inclusion of a point-scale system to evaluate the extracts it was clear that overall the organic extracts of the Sutherlandia flowers, especially the acetone extract (SFA), showed great anti-HIV potential. SFA in every case decreased p24 levels and in the toxicity study did not decrease cell proliferation. With the HIV-infected PBMCs SFA actually helped improve cell proliferation despite the infection. To determine the specific anti- HIV activity, all crude extracts were tested for inhibition of HIV-I reverse transcriptase, the glycohydrolase enzymes: a-glucosidase, ß-glucosidase, ßglucuronidase, HIV-I integrase and HIV-II protease. No significant inhibition was seen with these experiments except for the HIV-I RT assay. The aqueous extract of the Lobostemon leaves produced an inhibitor of HIV-RT with a very low IC50 value of 0.049mg/ml. Some inhibitory effect was lost with the removal of the sulphated polysaccharides and the addition of BSA to the assay, but still 64% inhibition of the HIVRT remained, which confirmed that the inhibitor could be something novel, and not of the polysaccharide or tannin compounds.

This study explores knowledge of some medicinal plants amongst the sub-urban community of and around a township in the Eastern Cape province. This qualitative interpretivist case study presents the prior knowledge of medicinal plants possessed by Grade 9 learners, which is used as a springboard toward interviewing traditional healers, herbal practitioners and lecturers at a university in the departments of Botany and Pharmacy. The data obtained from the informants reveals the factors that can influence integration of indigenous knowledge of medicinal plants in the learning programme for grade 9 General Science. These factors include: prior knowledge and enthusiasm of Grade 9 learners and teachers, support of the community which include parents, traditional healers, herbal practitioners and professionals who could introduce indigenous knowledge of medicinal plants into formal education, availability of resource materials and complexity of identifying pharmacologically tested plants from other indigenous medicinal plants. The analysis and discussion of the findings, have led me to conclude that the enthusiasm of learners who have a rich background of indigenous knowledge on medicinal plants is likely to be hampered by the unenthusiastic teachers as well as the reluctance of herbal practitioners in their communities to part with this knowledge. Hence I recommend that teachers be motivated through workshops and in-service training, conducted by goverr\ment paid herbal practitioners using the prior knowledge of learners as a stepping-stone.

Human immunodeficiency virus (HIV) and its gradual weakening of the immune system is an ever growing threat. Acquired immune deficiency syndrome (AIDS), the final stage of HIV, renders a person vulnerable to various opportunistic infections, which in the end lead to death. Apart from intensive vaccine studies, treatment research mainly focuses on preventing the individual HIV enzymes (reverse transcriptase, integrase and protease) from performing their functions. Entry inhibitors, however, block viral entry into the cell, while antisense drugs lock onto the viral genome to keep it from functioning. In this study production of active recombinant HIV-1 protease and integrase was attempted for future drug screening programs. HIV-1 protease was cloned into a pET28b(+) vector and expressed in ROSETTA(DE3)pLysS cells. The protein was purified using a nickel-affinity column utilizing the hexa-histidine tag encoded by the vector. Gel filtration chromatography was attempted after refolding of the protease, but protease yield seemed to decrease with the additional purification step. Partially purified protease was characterized with kinetic studies. Kinetic parameters of HIV-1 protease were determined to be Km = 592 μM, Vmax = 0.59 μM/min and kcat = 31 s-1. HIV-1 integrase, which was cloned into a pET15b vector, was expressed in E. coli BL21(DE3) cells. The coding sequence had been mutated to introduce the amino acid substitutions F185K and C280S, increasing solubility of the protein. The first step in purification of this protein was nickel-affinity chromatography, after which cation exchange chromatography was attempted. HIV-1 integrase concentration was low throughout experiments and no clear elution from the cation exchange column could be observed. A non-radioactive enzyme linked HIV-1 integrase assay failed to detect integrase activity. Modifications to future studies of the integrase are suggested in the chapter involved.