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 ecology of blue duikers Philantomba monticola and red duikers Cephalophus natalensis was studied in three lowland coastal evergreen forests in Natal. Variation in population densities, estimated with numerous census techniques and between and within study areas permitted comparisons of habitat parameters. Both species are strictly diurnal, and are active between 18 and 40% of the nycthemeral cycle. Usually a pair of blue duikers occupy a permanent territory of about 0,7ha while red duiker home ranges, between 2 and 15ha, overlap extensively with each other and those of blue duikers. Habitat quality of both species is determined by canopy cover (stem densities, spatial patterning, and, diversity of the tree and shrub layers), tree to shrub ratios, and, bedsites. The main dietary item, freshly fallen leaves, is not that of a highconcentrate feeder as hypothesised. Food appears to be abundant while condensed tannins have no influence on duiker densities. Management recommendations include land acquisition, forest ecology research, monitoring interaction with other species, genetic aspects, translocations and captive propagation. / Thesis (Ph.D.)-University of Natal, Pietermaritzburg, 1990.

Thesis (M.Sc.) - University of KwaZulu-Natal, Pietermaritzburg, 2009. / Full text also available online. Scroll down for electronic link.

>Magister Scientiae - MSc / Extracts from the root bark, stem bark, and leaves of R. natalensis were screened for antibacterial activity against standard bacterial strains; Staphylococcus aureas, Escherichia coli and Pseudomonas aureginosa, and fungi; Candida albicans, Trichophyton mentagrophytes or Microsporum gyseum. Chromatographic techniques were utilized to isolate pure compounds. This study validates and documents, in a systematic way, the antimicrobial properties of the R.natalensis used for many years by many people of the world. It also provides valuable information for further phytochemical isolation and characterization studies of active compounds, necessary for the development of new drugs. The extractions were carried out using broad spectrum of solvents (hexane, dichloromethane, ethyl acetate, and methanol).Fractionation was done using standard chromatographic techniques. A total of seven (7)compounds were isolated from R. natalensis. Three of the isolates were characterized and their structures were unambiguously established by detailed spectroscopic analysis that involved high resolution mass spectrometry, 1D and 2D-NMR spectral data experiments 1H, 13C, DEPT,COSY, HMBC, and NOESY. These compounds are: 3-(1-(2,4-dihydroxyphenyl)-3,3-bis(4- hydroxyphenyl)-1-oxopropan-2-yl)-7-methoxy-4H-chromone-4-one (39), Rhuschromone, a novel compound isolated for the first time, 2’,4’ dihydroxychalcone-(4-O-5’’’)-4’’,2’’’,4’’’-trihydroxychalcone (40) and 3-((Z)-heptadec-13-enyl) benzene-1,2-diol (41), an urushiol.Compound 39 recorded the highest activity zone of inhibition (21mm) against S. aureas, which was found to be 50% as active the chloramphenicol standard used. The traditional use of the extracts in infections and inflammatory conditions is rationalized based on the content of the isolated compounds, and it has been proposed that the total crude extract, with its contents of so many bioactive compounds, could be formulated for use in many infections, microbial or fungal.Furthermore, not all of the species studied to date have been fully characterized for potential bioactivities. Thus, there remains a significant research gap spanning the range from lead chemical discovery through process development and optimization in order to better understand the full bioactive potential of many of these plants.

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.

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

Euclea natalensis A.DC. belongs to the Ebenaceae family, and is extensively distributed along the eastern coast of southern Africa. Many Euclea species are widely gathered by indigenous people because of their medicinal properties. Roots of these plant species are frequently used to treat respiratory complications such as chest pains, bronchitis, pleurisy and asthma. Ground root powder is topically applied in cases of leprosy and is used by some ethnic groups to treat toothache and headache. The bioactivity encountered is attributable to naphthoquinones, which are common phenolic compounds in the Ebenaceae family. Naphthoquinones isolated from E. natalensis (shinanolone, 7-methyljuglone, diospyrin, isodiospyrin and neodiospyrin) have exhibited a broad spectrum of antimicrobial activity. The demand for these products will escalate due the amount of plant material required to further research. We need to explore techniques that can maximize their productivity. The present study was conducted on E. natalensis, in an attempt to establish if there exists any correlation between the accumulation of naphthoquinones and stages of seedling growth, seasonal fluctuations and application of fertilizers. A possible correlation between seedling growth stages and the accumulation of naphthoquinones (shinanolone, 7-methyljuglone and diospyrin) was investigated in seeds and seedlings of Euclea natalensis. In this study, the seeds represented the first stage, whereas the second seedling stage was defined as the stage when the radicles were about 6 cm long. The lengths of the seedlings at the third, fourth and fifth seedling stages were 9 cm, 12 cm and 16 cm respectively. Plant materials collected from the five seedling stages were separately extracted using chloroform and the naphthoquinones were then quantified by means of High Performance Liquid Chromatography (HPLC). Mobile phase of MeCN: H2O: AcOH (62.5: 32.5: 5) was used as an eluent in an isocratic mode and at a flow rate of 0.8 ml/min. Standard curves of each of the four compounds were obtained by making a series of dilutions in the concentration range of 22.5 µg/ml to 2.25 µg/ml. Ten microlitres of each dilution was injected three times into the HPLC, and the run time for each injection was 20 minutes. Calibration curves were then generated and used for the quantification of each compound. Shinanolone, which was the only naphthoquinone detectible in seeds, accumulated at variable rates (P<0.01) and no trend could be established between its synthesis and seedling growth. The content of shinanolone ranged from 87.5 mg/kg dry weight (dw) in seeds to a high mean value of 1047 mg/kg (dw) during the fourth seedling stage. A significant correlation (P<0.01) was found between the mean concentrations of 7-methyljuglone and seedling growth. 7-Methyljuglone was quantified at a high mean level of 5003 mg/kg during the third seedling stage and was not detected in the seed samples. A positive correlation (P<0.01) was established between the concentration of diospyrin and seedling stages. Diospyrin was detected at an elevated mean concentration of 6182 mg/kg during the fifth seedling stage, which was higher than the other quantified naphthoquinones. Seasonal variation of naphthoquinones (shinanolone, 7-methyljuglone, diospyrin, isodiospyrin and neodiospyrin) was investigated from eleven plants of E. natalensis subsp. natalensis growing in natural populations, over a period of four seasons. The roots were harvested, dried, extracted and analysed as in the previous study. The mean levels of shinanolone and 7-methyljuglone were found to be uniform in all the seasons and no statistically significant variation could be found between seasonal changes and their mean concentrations. Accumulation of isodiospyrin and neodiospyrin varied significantly with seasonal changes (P<0.05). These two bioactive naphthoquinones were detected only in summer and autumn respectively, and not in winter. A statistically significant variation (P<0.05) was established between the levels of diospyrin and seasonal fluctuations. Diospyrin was detected at a mean concentration of 3190 mg/kg (dw) during spring, which was higher than the other naphthoquinones quantified in all four seasons. The effect of NPK fertilizers on growth performance and accumulation of naphthoquinones (shinanolone, 7-methyljuglone, diospyrin, isodiospyrin and neodiospyrin) in seedlings of E. natalensis grown in shade and under field conditions was investigated. Each group was subdivided into four subgroups, which were then subjected to four respective treatments of water-soluble foliar feed (2:1:2 (44) NPK) at three different concentrations. Treatments tested were as follows: Treatment 1 at 40 g/l, Treatment 2 and Treatment 3 at 20 g/l and 10 g/l respectively. The control group received only supplemental water. The first harvest was conducted after 6 months of application of fertilizers and the second one was done after 12 months of treatment. Roots and shoots were harvested and analysed separately. The naphthoquinones were quantified as previously described. The bioactivity of root extracts from seedlings was tested against Mycobacterium smegmatis and extracts with lower MIC were further tested on M. tuberculosis. Growth parameters differed between the two groups, with seedlings from the shadehouse showing more plant vigour than the field grown plants. No significant interaction could be established between the measured growth factors and treatment. A significant interaction (P<0.001) was found between Treatment 2 and shadehouse seedlings. Treatment 2 enhanced vegetative performance with the mean values of fresh weight of shoots and roots being twice as much as their respective control mean values. A significantly positive correlation was established between the concentration of shinanolone (P<0.01), isodiospyrin (P<0.05) and neodiospyrin (P<0.05) with fertilization from field-grown seedlings. Application of NPK fertilizers significantly (P<0.05) increased the accumulation of neodiospyrin in seedlings subjected to shadehouse conditions. The most potent naphthoquinone, 7-methyljuglone, was found to be abundant in all the extracts and was quantified at a high mean concentration of 10200 mg/kg from shadehouse seedlings. Root extracts of E. natalensis seedlings grown under field conditions were generally more active against the bacterial strain of M. smegmatis as compared to extracts acquired from roots of seedlings maintained under a shadehouse setting. A lowest minimum inhibitory concentration (MIC) of 0.78mg/ml against M. smegmatis was observed from the second harvest of field-cultivated seedlings of the control and Treatment 1 subgroups. The MIC values for shadehouse seedlings ranged from 1.6 to 6.3 mg/ml. Minimum bactericidal concentration (MBC) values from all the extracts tested were relatively higher than their respective MIC’s. Root extracts of E. natalensis were more active against M. tuberculosis and their MIC values were lower than the tested concentrations. Extracts acquired from field-grown seedlings were more active against M. smegmatis with a lowest MIC value of 0.78 mg/ml. Extracts from the control group and Treatment 1, which had less application of fertilizers were more active against strains of M. tuberculosis with MIC value of 10 µg/ml. This shows the selectivity of E. natalensis against the mycobacterial strain of M. tuberculosis. Based on the findings, synthesis and accumulation of naphthoquinones in E. natalensis is highly variable within individuals of the species investigated. Naphthoquinones accumulate in relatively higher amounts in roots of E. natalensis than in the aboveground structures, which validate their harvest by indigenous people. The concentration of shinanolone varied slightly and its production increased with seedling growth. The synthesis of 7-methyljuglone is independent of fertilisation as its accumulation was enhanced in seedlings subjected to control treatment. Neodiospyrin and isodiospyrin were always present in every sample obtained from the seedlings but they were not detectible in every profile of samples from mature plants. Diospyrin is the only naphthoquinone that was detected in every sample analysed and also quantified in high concentrations from mature plants harvested in spring. The study showed that depending on the requirement of a particular naphthoquinone for research, one could target the seasons and seedling stages recommended from this study. This study also showed that field-cultivated seedlings produced more potent naphthoquinones than the ones subjected to controlled environments. / Dissertation (MSc (Plant Science))--University of Pretoria, 2008. / Plant Production and Soil Science / unrestricted

Background: In order to optimize net transmission success, parasites are hypothesized to evolve towards causing minimal damage to their reservoir host while obtaining high shedding rates. For many parasite species however this paradigm has not been tested, and conflicting results have been found regarding the effect of arenaviruses on their rodent host species. The rodent Mastomys natalensis is the natural reservoir host of several arenaviruses, including Lassa virus that is known to cause Lassa haemorrhagic fever in humans. Here, we examined the effect of three arenaviruses (Gairo, Morogoro and Lassa virus) on four parameters of wild-caught Mastomys natalensis: body mass, head-body length, sexual maturity and fertility. After correcting for the effect of age, we compared these parameters between arenavirus-positive (arenavirus RNA or antibody) and negative animals using data from different field studies in Guinea (Lassa virus) and Tanzania (Morogoro and Gairo viruses). Results: Although the sample sizes of our studies (1297, 749 and 259 animals respectively) were large enough to statistically detect small differences in body conditions, we did not observe any adverse effects of these viruses on Mastomys natalensis. We did find that sexual maturity was significantly positively related with Lassa virus antibody presence until a certain age, and with Gairo virus antibody presence in general. Gairo virus antibody-positive animals were also significantly heavier and larger than antibody-free animals. Conclusion: Together, these results suggest that the pathogenicity of arenaviruses is not severe in M. natalensis, which is likely to be an adaptation of these viruses to optimize transmission success. They also suggest that sexual behaviour might increase the probability of M. natalensis to become infected with arenaviruses.

The primary aim of this study was to investigate the role of food supply and ground cover on the community structure, population dynamics and demography of terrestrial small mammals in a subtropical grassland. This aim was achieved through a series of food supplementation and cover manipulation experiments conducted at eKundizeni Farm near Matsapha, Swaziland, over a 28 month period. The effects of five different diets on the rodent Mastomys natalensis were investigated in the laboratory, and the results showed that rolled oats and rabbit pellets were suitable for growth and reproduction in this species. The effects of supplementary food were investigated on two supplemented grids and one control over a twelve month period. Small mammal biomass increased significantly on the supplemented grids in relation to the control. This increase in biomass was the result of a twofold increase in the numbers of M natalensis. Food supplementation further affected M. natalensis by: extending the breeding season of females; increasing body weight; increasing survival; and decreasing home range area. Food supplementation had a weak positive effect on the density of another rodent Lemniscomys rosalia, but did not affect any other demographic feature of this species. Food supplementation did not have a demographic effect on any other species of small mammal captured. The effects of vegetative cover were investigated, over a twelve month period, on two control grids and four manipulated grids on which the vegetative cover was mechanically reduced. Supplementary food was added to two of the latter four manipulated grids. The biomass of small mammals, including M. natalensis, was lower on grids with reduced vegetative cover than on the controls. However, food supplementation resulted in a significant increase in the biomass of M. natalensis on one of the manipulated grids. Hence, M. natalensis was induced to shift to a habitat with reduced cover by the provision of supplementary food. Additional information on the population dynamics, age structure, reproduction and diet of M natalensis, L. rosalia, Mus minutoides and Steatomys pratensis is also presented. / Thesis (Ph.D.)-University of Natal, Pietermaritzburg, 1997.

During the past two decades, panicle-feeding Hemiptera have become pests of sorghum in West and Central Africa, and particularly in Mali, where this is a staple food crop. Of the more than 100 sorghum insect pests reported in Africa, 42 species were found to be panicle-feeding pests. Prior to this study, no research had been done on the paniclefeeding Hemiptera in South Africa. The objectives of the study were to determine the abundance and diversity of panicle-feeding Hemiptera on sorghum. A check list was compiled and the temporal distribution of different Hemiptera species determined during the different panicle stages of development. In addition, the effect of insecticide application on Hemiptera numbers was evaluated and the correlation between grain mould severity and Hemiptera feeding damage was investigated. To determine the abundance and diversity of Hemiptera on sorghum panicles, surveys were conducted between November 2004 and June 2006 at 26 sites in four provinces of South Africa. Two methods of collection were used viz. the plastic bag and D-Vac methods. The total number of the adults and nymphs collected during this study was 23 798. Forty-three different herbivorous Hemiptera species were collected. The most abundant family was the Miridae (41 %), followed by the Lygaeidae (17 %). Eurystylus spp., Calidea dregii, Campylomma sp., Creontiades pallidus, Nysius natalensis and Nezara viridula were the most abundant species and also occurred widely in the sorghum production area. Infestation levels of these species were low compared to that in other parts of Africa and it cannot be concluded that they have pest status in South Africa. There was no clear distinction between the stages during which panicles were infested by different species. The general tendency was that nearly all species were present from the flowering stage onwards and that numbers declined when grain hardened. In general, Campylomma sp. and C. pallidus numbers peaked during the flowering stage and Eurystylus spp. and N. natalensis during the milk stage. Hemiptera feeding damage resulted in an increase in incidence of seeds with discoloured germ, therefore contributing significantly to reduction in grain quality. / Thesis (M. Environmental Science (Plant Protection))--North-West University, Potchefstroom Campus, 2007.