M.Sc. / Cephalanthus natalensis of the Rubiaceae family of plants is used in traditional medical systems. Different parts of this shrubby plant are used to treat different ailments. In this study, the use of C. natalensis for medicinal purposes was investigated. Plant materials (leaves, bark and fruits) were collected from different localities in Swaziland. Extraction of phytochemicals in C. natalensis was done by maceration in four solvents of increasing polarities: hexane, dichloromethane, ethyl acetate and methanol. Amino acids and alkaloids were also extracted in boiling water and mild acid, respectively. Phytochemical screening of compounds was done by means of TLC, HPLC and GC-MS. Previously reported TLC screening of C. natalensis indicated the presence of alkaloids. This study also confirmed the presence of alkaloids, although these were not isolated. No other chemical compounds have been reported from this plant in literature. Phenolic compounds and terpenes were extracted and detected by the chromatographic methods employed. GC-MS analysis was also used to identify and quantify amino acids in the extracts. A phytochemical assessment of compounds was done by investigating the leaves, bark and fruits of C. natalensis. A further phytochemical variation study was conducted by comparing these phytochemicals and their relative concentrations in C. natalensis plants collected from five different localities. While the same compounds were present in all the plants sampled, the concentrations of the different compounds seemed to vary irrespective of the geographic location of the plants sampled. In the different plant parts collected, some common compounds were present in the leaves, bark and fruits and other compounds varied between the different parts studied. The traditional usage of C. natalensis as fever and malaria remedy was investigated in vitro. Antibacterial and antimalarial tests of crude extracts were done. The antibacterial test on the extracts against five bacteria: Bacillus cereus, Staphylococcus aureus, Staphylococcus epidermidis, Escherichia coli and Pseudomonas aeruginosa showed an average activity in all the plant parts tested. The average MIC values ranged from 0.917 mg/mL to >100 mg/mL. In the antimalarial test the extracts inhibited 50% of Plasmodium falciparum growth at concentrations ranging from 27 μg/mL to >100 μg/mL. Hexane extracts of the bark had notable antimalarial activity with the highest inhibition at the lowest concentration of 27 μg/mL. The fruits were not as active as expected since they are particularly used as malaria remedy in traditional medicine. Two compounds were also isolated from methanol extracts of C. natalensis for the first time. A volatile isoprenoid ketone, (5E,9E)-6,10,14-trimethylpentadeca-5,9,13-trien-2-one, was isolated and characterised. The structure of the second compound could not be completely characterised due to complex data obtained from the NMR experiments.

D.Phil. (Biochemistry) / Plants are able to recognise and respond to signals from the environment through a complex array of biochemical pathways, which enables them to deter pathogenic micro-organisms and herbivores. Thousands of different structures of low-molecular weight organic compounds / natural products can be produced through an inducible chemical defence system; that can be manipulated for biotechnological purposes. The importance of natural products in medicine, agriculture and industry has led to numerous studies such as this, to understand the biosynthesis and biological activity of these substances...

TB is still one of the world's biggest killers. Immunosuppresion induced by AIDS caused a rise in the incidence of TB during the past decade. The search for new drugs to effectively treat TB remains one of the big challenges facing the scientific community. Drugs from plants have been used for centuries to treat various human diseases with varying degrees of success. South Africa with its big resource of plants and ethnobotanical knowledge is an ideal place to screen for anti- TB compounds. The Zulu tribe of South Africa used the root bark of Euclea nata/ensis A.DC. to treat TB related symptoms. Naphthoquinones isolated from E. nata/ensis proved to have good activity against TB. Nine compounds were isolated from the chloroform extract of E. nata/ensis root material. Three of these compounds were newly isolated from this species (mamegakinone, neodiospyrin and 5-hydroxy-4-methoxy-2-napthaldehyde). The structures of the isolated compounds were confirmed using NMR methods and where possible the HPLC and TLC results were compared to authentic standards. Most of the compounds were tested for anti- TB activity with only mamegakinone, lupeol and betulin not showing any activity (5-hydroxy-4-methoxy-2-napthaldehyde still needs to be tested). The activity of the naphthoquinones, especially 7-methyljuglone, diospyrin, isodiospyrin and neodiospyrin, show promise that these compounds could develop into an affordable medicine to treat TB. The activity of the crude extract against the resistant DP48 110 1 TB strain showed that there are probably unknown active compounds remaining in the extract. The most active compound, 7 -methyljuglone, was synthesised and an improved synthetic pathway was developed. The synthesis of naphthoquinones remains important in order to produce the compounds on a larger scale. This will make further studies into the mode of action, biosynthesis, bioactivity etc. of these compounds possible. Attempts were made to synthesise diospyrin with 7 -methyljuglone as the starting material. These experiments failed up to now. By altering the reaction parameters such as pH and temperature it should be possible to synthesise diospyrin in future attempts. Neodiospyrin were synthesised from reduced 7 -methyljuglone. This synthesis will yield information on the naphthoquinone chemistry and on how to synthesise diospyrin and isodiospyrin. The enzymatic synthesis of naphthoquinones was also investigated with the use of a cell-free extract. These experiments indicated that it might be possible to enzymatically synthesise diospyrin and the other dimers. / Dissertation (MSc (Plant Physiology))--University of Pretoria, 2005. / Plant Science / unrestricted

Submitted in partial compliance with the requirements of the Master’s Degree in Technology: Homoeopathy, Durban University of Technology, Durban, South Africa, 2017. / The C4 trituration method is a unique method for homoeopathically proving a substance and determining its therapeutic properties. The process requires the use of a mortar and pestle and involves many rounds of grinding (triturating) the raw substance. During the trituration process symptoms are experienced by the triturator. Withania somnifera, commonly known as Ashwagandha in Ayurvedic medicine is highly regarded for its clinical efficacy as an adaptogenic herb. Due to the herb’s therapeutic properties and extensive use in Ayurvedic medicine a comprehensive homoeopathic drug proving was conducted at The Durban University of Technology by Laidlaw in 2015. Aim The aim of this study was to conduct a homoeopathic C4 trituration proving of Withania somnifera with a subsequent comparison to the conventional 30CH proving of Withania somnifera which was conducted by Laidlaw in 2015. The study was of a qualitative and exploratory nature hence employed a qualitative methodology with purposive sampling. Participants were required to triturate up to the fourth potency [C4] and all the symptoms experienced were recorded in the journals provided. The study incorporated thematic analyses of data. The data was transliterated by the researcher, thereafter those symptoms were classified systematically and converted into rubrics using Schroyens (2012) repertory. Recorded symptoms were collated to create a C4 trituration representation. Subsequently the symptoms obtained from the C4 trituration proving of Withania somnifera was comparatively analysed against the symptoms of the conventional 30CH proving of Withania somnifera, which was conducted by Laidlaw (2015). Method This research study followed a qualitative methodology which comprised a single blind study, and a proving sample. This was a qualitative study; the requirement for sample size is usually up to 10 participants until saturation of data, but this study utilised 18 participants to ensure the generation of rich data. Participants triturated 91: 100 lactose to the fourth potency [C4] using a mortar and pestle. All symptoms experienced were recorded in journals. The analysis of the data followed a qualitative phenomenological methodology. Results The C4 trituration proving yielded 145 rubrics. Two of these rubrics were newly formulated. The majority of the rubrics were produced in the MIND and HEAD section. There were 100 Grade 1 rubrics, 44 Grade 2 rubrics and 1 Grade 3 rubrics. Conlcusion The C4 trituration produced a symptom picture of Withania somnifera, which was comparable to the 30CH conventional proving. The C4 trituration proving produced observable changes in the triturators’ state of health but predominantly MIND symptoms were evoked. The symptom picture created with the C4 trituration had many similarities to the 30CH conventional proving. The polarity of symptoms that existed in Withania somnifera was observed in both provings and included symptoms such as: anxiety vs tranquillity, increased concentration vs decrease in concentration. Differences between the C4 trituration proving and the 30CH conventional proving were found in the description of physical symptoms such as headaches, in the types of delusions that occurred in each proving, and in some mental symptoms such as anger, sadness, clarity, and introspection. / M

Traditional medicine has a long history of being used for treating various ailments ranging in severity. Although traditional medicine has typically been the health care for the poorest levels of society, there is a worldwide growth in popularity. The growing popularity of traditional medicine, termed the green boom, may be ascribed to people taking a more holistic approach to maintain their health. Traditional medicine is widely used on a regular basis by 70% of South Africans. Various indigenous medicinal plants are used for the preparation of traditional herbal medicine. These plants are mostly indigenous to the regions were it is used. In this study four medicinal plants (Bulbine frutescens, Leonotis leounurus, Melianthus major & Zantedecshia aethiopica) that are traditionally used in the Eastern Cape region for treating burn wound infections, were collected for investigation. The in vitro antibacterial activity of these plants was tested against different bacterial strains of eight different bacteria. The bacteria used in this investigation included bacterial strains of four Gram-positive bacteria, S. aureus, methicillin-resistant S. aureus (MRSA), E. feacalis, S. pyogenes and four Gramnegative bacteria, P. aeruginosa, A. baumanii, K. pneumoniae and P. mirabilis. Traditional preparations as well as three different extracts (methanol, aqueous & acetone) of the plants were used for in vitro antibacterial activity testing. The microtitre plate assay and agar dilution assay were used for determining the antibacterial activity of the traditional preparations and plant extracts against the different bacterial strains. In the microtitre plate assay the antibacterial activity was tested using the bacterial growth indicator, INT and a microtitre plate spectrophotometer to determine the minimal inhibitory concentrations of the plant extracts and traditional preparations. The microtitre plate assay was used for testing the antibacterial activity of the plants against the bacterial strains of five bacteria, S. aureus, MRSA, P. aeruginosa, A. baumanii and K. pneumoniae. The bacterial strains of the three bacteria, S. pyogenes, E. feacalis and P. mirabilis were not compatible with the microtitre plate assay using INT and spectrophotometric readings to determine bacterial inhibition. Therefore the agar dilution assay were used as an alternative method for determining the MIC’s of the plant extracts against the bacterial strains of these bacteria. The initial plant extract concentration in the microtitre plate assay differed with the different plant extracts in the microtitre plate assay. Acetone followed by methanol extracted the highest plant extract concentrations with the different medicinal plants. M. major followed by L. leonurus produced the highest plant extract concentrations following extraction with the different extraction solvents. Consequently the acetone extract of M. major had the highest plant extract concentration before serial dilution in the microtitre plate assay. Uniform plant extract concentrations were tested in the agar dilution assay. The methanol extract followed by the acetone extract of the plants gave the highest antibacterial activity against the different bacterial strains. The extracts of M. major followed by L. leonurus inhibited the highest number of bacterial strains in the microtitre plate assay and the extracts of B. frutescens inhibited the lowest number of bacterial strains. The acetone and methanol extracts of M. major were the only extracts that displayed antibacterial activity in the agar dilution assay. The bacterial strains of P. mirabilis were the only bacteria that were inhibited using this method. The bacterial strains of S. pyogenes and E. feacalis were not inhibited at any of the plant extract concentrations in the agar dilution assay.

Bacterial resistance to antibiotics has been a great problem for many years. The degree of resistance and the speed with which resistance develops varies with different organisms and different drugs. Enzymes called β-lactamases are produced by bacteria and are one mechanism in which bacteria develop antimicrobial resistance. Gram-negative bacteria producing enzymes called ESBLs because of their wide substrate range are of a particular concern in nosocomial infections. In many countries people still use traditional medicine derived from plants as an alternative to the Western medicine due to increased cost of Western medicine and microbial resistance of antibiotic treatments. Biologically active compounds isolated from plants species are used in herbal medicine. Because of the high prevalence of the ESBLs and their increasing resistance to the antibiotics, this research study was done to test the antimicrobial activities of selected medicinal plants of the Eastern Cape; G. incanum, D. angustifolia and E. autumnalis which were traditionally used to treat various infections. The in vitro antimicrobial activity of three different extracts (acetone, methanol & distilled water) and the traditional preparations of the three plants were tested against the selected strains of ESBL-producing bacteria, non β-lactamase producers and the different fungal species. The extracts were screened against 26 Gram-positive bacterial strains, 53 Gram-negative bacterial strains and 15 fungal strains. The Gram-positive bacteria included strains from S. aureus, B. cereus and E. faecalis. The Gram-negative bacteria included strains from E. ii coli, E. cloacae, K. pneumoniae, P. aeruginosa and Acinetobacter spp. The fungal strains included 9 strains of Candida albicans and a single strain of each of the following opportunistic fungi, Mucor sp, Geotrichium sp, Penicillium sp, Fusarium sp and Rhizopus sp. The agar dilution assay was used for the antimicrobial screening of the plants extracts and for the determination of the MICs. The Ames test was performed for the determination of probable carcinogenicity of the extracts of G. incanum and D. angustifolia. The distilled water extracts followed by acetone extracts of the plants revealed the highest antimicrobial activity against the different microbial strains. The extracts of G. incanum followed by the extracts of D. angustifolia inhibited the highest number of microbial strains. The extracts of E. autumnalis did not show any antimicrobial activity against all the pathogens in this study. More of the Gram-positive bacteria were inhibited by the plant extracts. The lowest MIC was obtained with Gram-positive bacteria. The bacterial strains of E. faecalis and P. aeruginosa were not inhibited by any of the plants extracts in the agar dilution assay yet Acinetobacter species which are MDR were inhibited by the distilled water and methanol extracts of G. incanum. A single strain of Mucor sp was the only spore forming fungi that was inhibited by the distilled water extracts of G. incanum. None of the plants extracts showed any mutagenic effects on the TA100 S. typhimurium strains incorporated on the Ames test. Apart from revealing of new antimicrobial agents that may be used against resistant organisms, the proper use of antimicrobial agents should be recommended. The study has highlighted a need for further investigations on the properties of the medicinal plants used in this study.

The need for alternative strategies for the prevention and treatment of diabetes is growing rapidly as type II diabetes is reaching epidemic status in our society. This need was the basis for the creation of this study, as it was necessary to start looking towards medicinal plants as potential antidiabetic treatment and no comprehensive in vitro model existed. In creating a model for determining the effects of alternative traditional medicines as antidiabetic potentiates, it was necessary that two metabolic pathways, namely glucose uptake and insulin secretion, which play a significant role in glucose homeostasis, be at the centre of our investigations. The objective of this project was to optimize the methodology required to screen and ultimately determine the effectiveness of the plant extracts Kankerbos and MRC2003, as antidiabetic potentiates, through observing their effects on glucose utilisation and insulin secretion. If these medicinal plants are going to make a positive contribution to the health of type II diabetic South Africans, then the determination of their efficacy is essential. The cell lines used in this study included 3T3-L1 preadipocytes, Chang liver, C2C12 muscle and INS-1 rat pancreatic cells. Each cell line represents a different in vivo organ that is known to have an influence on glucose homeostasis in our bodies, each with its own unique metabolic pathways and mechanisms of activity, thereby making each one a vital component in the study. The positive controls for the two models were insulin and metformin (glucose utilisation) and glibenclamide (insulin secretion). Insulin was shown to provide a significant increase in the amount of glucose taken up in C2C12 muscle and Chang liver cells for acute conditions. Chronic treatments with metformin provided a significant increase in glucose utilised by Chang liver cells. Glibenclamide was an effective positive control for stimulating insulin secretion by INS-1 cells under acute conditions as there was a significant increase in the amount of insulin secreted. MRC2003 did not show any significant antidiabetic activity. Sutherlandia frutescens (Kankerbos) showed biological activities comparable to some of the more recognized antidiabetic compounds throughout the study. With regards to the glucose utilisation model, Kankerbos was seen to have both acute and chronic effects in different cell lines. In the C2C12 muscle cell line, Kankerbos significantly increased glucose uptake when they were exposed to acute conditions. Kankerbos also had a significant effect on the Chang liver cells as it was observed that under both acute and chronic conditions, this plant extract induced the uptake of glucose into these cells. With respect to the insulin secretion model involving INS-1 cells, no significant effect was seen during acute exposure with Kankerbos treatment. However during chronic exposure, an increase in insulin secretion was initiated by this plant extract. Overall, the results of this study suggest that Kankerbos has a twofold mechanism of action for its glucose-lowering effects. Given that Kankerbos is widely available in South Africa, this study was valuable as it provided an indication that Kankerbos has antidiabetic activities and could possibly be used as an alternative antidiabetic medication.

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.

The rising costs of prescription drugs in the maintenance of personal health and wellbeing have increased the interest in medicinal plants. The World Health Organization estimates that 65 percent-80 percent of the world’s population use traditional medicine as their primary form of health care. In this project the focus has been on the use of Leonotis leonurus extracts as a traditional medicine. The major chemical constituent of this plant is marrubiin, which is a diterpenoid labdane lactone formed from a precursor called premarrubiin. Aqueous and acetone extract (AL and OL extract, respectively) of this plant has been found to have an antithrombotic effect, with IC50 values of 3mg/ml and 6mg/ml, respectively. The extracts also have an effect on fibrinolysis, where the lysis time was decreased by more than 50 percent by the organic extract and standard marrubiin. In whole blood ADP-induced platelet aggregation, the organic extract inhibited aggregation by 68 percent at a final concentration of 138μg/ml (equivalent to 7.2μg/ml marrubiin). Marrubiin has also been screened for antithrombotic/anticoagulant activity; no antithrombotic activity has been observed but it increased the rate of fibrinolysis, by decreasing lysis time by 64 percent and also decreasing fibrin formation. From these findings it can be concluded that marrubiin has a fibrinolytic effect and antiplatelet aggregation effect. In the diabetic studies, in hyperglycemic condition, the OL (10μg/ml) extract and standard marrubiin significantly increased insulin secretion by 200 percent (2-fold) and 400 percent (4-fold), respectively, with respect to the control. The OL extract and standard marrubiin stimulated the release of insulin, the stimulatory index was significantly increased by 450 percent (4.5-fold) and 500 percent (5-fold), respectively, with respect to the control. In the apoptotic studies, in the normoglycemic and hyperglycemic conditions, the OL extract decreased the occurrence of apoptosis, in a dose-dependent manner, with the lower concentrations inducing apoptosis significantly higher than the relevant controls. Standard marrubiin did not have an effect on apoptosis in hyperglycemic condition, but it decreased the occurrence of apoptosis by 200 percent (2-fold) under normoglycemic conditions. The OL extract increased proliferation by 148 percent (1.48- fold) and 155 percent (1.55-fold) in normoglycemic and hyperglycemic conditions, respectively. The same effect was observed for standard marrubiin, where, proliferation was increased by 180 percent (1.8-fold) and 200 percent (2.0-fold) in normoglycemic and hyperglycemic conditions, respectively. RT-PCR displayed that standard marrubiin inhibited the expression of insulin by 50 percent under normoglycemic conditions.

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