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Item Characterisation of bacterial symbionts in selected drought-tolerant legumes for biofertilisers development for use in Namibia(University of Namibia, 2023) Mataranyika, Paidamoyo NatashaNamibia is a semi-arid country with approximately 1% of arable land. Crop cultivation is profoundly challenged by nutrient-poor sandy soils combined with low water retention. To meet the increasing food demand, farmers resort to applying synthetic fertilisers and pesticides despite their environmental consequences. However, there is increasing evidence that arid or semi-arid plant microbiomes offer an unexploited reservoir that is pivotal to plant health, growth, and development. Plant growth promoting bacteria (PGPB) are of increased interest as they offer sustainable alternatives to environmentally unfriendly and unsustainable chemical fertilisers. The present study aimed to isolate, identify, and characterise plant-associated bacteria from five drought-tolerant legumes grown in Namibia. Identification was done using 16S rRNA sequencing and bioinformatics. Plant growth-promoting (PGP) abilities were characterised based on exopolysaccharide production, antifungal activity, indole acetic acid production, phosphate solubilization, siderophore production, and bacterial nitrogen fixation. Using 16S Illumina metagenomic sequencing, this study characterised the plant microbiomes of the nodules, roots, rhizosphere, and seeds. Isolates identified from the roots and rhizosphere were from the Proteobacteria (72%), Actinomycetota (15%), Bacteroidetes (5%) and Firmicutes (8%) phyla and included known plant growth-promoting species such as Stenotrophomonas pavanii, Streptomyces murinus, and Enterobacter cloacae. Nodule endophytes were mostly from the phylum Firmicutes (88%). The identified genera include Bacillus, Priestia, Paenibacillus, Gottfriedia, Neobacillus, Lysinibacillus, Fictibacillus, and Brevibacillus. Characterisation found that rhizobacteria expressed more plant growth promoting traits compared to root endophytes. Siderophore production was observed iii in most root endophytes and rhizobacteria. The following isolates, CRhi10, CRhi15, MBRhi17, HR5, RMBRhi4, RMBRhi1 and IPCRhi7 from the legume root endospheres and rhizospheres showed the most potential as plant growth promoters. A total of 34 nodule endophytes tested positive for at least one plant growth-promoting trait. Isolates MB1, MB3.1, H14, M25-11, M8-16.1 and M8-16.2 showed the most potential as plant-growth promoters. CRhi15 (S. maltophilia), HR5 (E. mori), H14 (P. aryabhattai), M25-11 (L. boronitolerans), and M8-16.1 (Bacillus sp.) were selected and assessed for their ability to induce drought tolerance on Vigna unguiculata seeds in potted trials. The inoculants were also combined and assessed in a consortium. Drought tolerance was observed to be highest with S. maltophilia (CRhi15), Bacillus sp. (M8-16.1) and E. mori (HR5). The average root length under drought stress was 37.5 cm, 51.8 cm, and 33.7 cm respectively while the average shoot length was 63.1 cm, 80.6 cm, and 75.3 cm. Microbiome analysis of the root, rhizosphere and seed microbiomes found important plant growth-promoting genera. These include Bacillus, Mesorhizobium, Pseudomonas, and Bradyrhizobium. The nodule microbiome was predominantly Bradyrhizobium. The relative abundance of the nitrogen-fixing Bradyrhizobium genus was determined in four drought-tolerant legume species- Vigna aconitifolia (mothbean), V. unguiculata (cowpea), Lablab purpureus (dolichos), and Macrotyloma uniflorum (horsegram). Both culture-dependent and independent methods revealed that these PGP bacteria can promote plant growth under drought, nutrient and biotic stress conditions. Therefore, S. maltophilia (CRhi15), Bacillus sp. (M8-16.1) and E. mori (HR5) may be further explored in field trials in efforts to develop commercial biofertilisersItem Population genetics and transmission dynamics of Plasmodium falciparum in the Kavango East and Zambezi regions of Namibia(University of Namibia, 2019) Tambo, MunyaradziIn 2010, Namibia, declared a goal to eliminate malaria within its borders, this was revised in 2017 to 2022 due to new challenges in achieving elimination. Some of the key challenges associated with malaria control and elimination are; 1) a lack of comprehensive data including malaria case distribution and resources especially in Africa where the burden is highest 2) there is no accurate classification of imported and local malaria cases or quantification of the level of importation 3) a lack of validated tools to supplement transmission estimates. Malaria positive samples tested with rapid diagnostic tests (RDTs) and dried blood spots (DBS) were collected with corresponding epidemiological data from health districts in the Kavango East and Zambezi regions of Namibia. DNA was extracted using the chelex DNA extraction method, the parasite DNA was genotyped with capillary electrophoresis using a 26 microsatellite marker set to determine P. falciparum genetic structure in the Kavango East and Zambezi regions of Namibia The study through population genetics analysis showed that the genetic structure of P. falciparum in Namibia follows the pattern of a high transmission setting, there are high levels of genetic diversity, low genetic relatedness, random mating and population admixture. Secondly, there are high levels of importation contributing to local transmission. Lastly, population genetic matrices are good surrogate markers for measuring malaria transmission intensity and importation.Item Optimation of Ontaku/ Oshikundu: Pearl millet and sorghum malts quality and conveniet premix development(University of Namibia, 2020) Embashu, WernerOshikundu/Ontaku is a nonalcoholic, acidic, opaque fermented beverage. It is comprised of pearl millet (Pennisetum glaucum (L.) R. Br) meal/flour, commonly known as mahangu, malts of pearl millet or sorghum (Sorghum bicolour (L.) Moench) and/or brans (pearl millet). Brewing of oshikundu remains an art in households with no empirical improvement of a controlled fermentation flow process to give a consistent product. Some of the major limitations to the formal commercialisation of this brew are the absence of standardised malting process that gives a consistent malt quality and microbial safety. The lack of standard ingredient ratios (flour/meal: malt: water) of brewing. Also, the sedimentation of adjunct particles at the bottom (dreg), thus creating the difference in viscosity of oshikundu. Welldefined fermenting microorganisms are not used, and the preparation method still relays on a laborious time-consuming process. Therefore, this study investigated conditions for malting, reduction of dreg, identification of fermenting microorganisms and formulation of ingredient ratio for an improved preparation process. Grains used in the study were collected from Omahenene Agricultural Research Station (20 15 harvest), of the Ministry of Agriculture, Water and Forestry. Pearl millet varieties Okashana 2 (SDMV 93032), Kantana (landrace) and Kangara (SDMV 92040), while sorghum varieties are Macia (SDS3220) and landrace commonly referred to as red sorghum. Malts of the two cereals were prepared by steeping in static water at 20-22°C for 2 hours wet and 2 hours air-rest for a total of 8 hours and germinated at 30°C. Malts were dried between 50-55°C for 24 hours. Cereals germinative energy was above 90% as recommended for sorghum by the European Brewery Convention. Malting loss was high up to 30% in pearl millet varieties and Macia. Crude protein and fibre were found . to increase following malting. Reducing sugars were not detected in nongerminated pearl millet graiJ.'ls. Malts reducing sugars were statistically significant (p ~0.05) between cereals. The malt reducing sugars was as follows Macia> Red sorghum> time reduce dregs. Also, ingredient ratios were formulated by the use of dry ingredients premix for making oshikundu that was easy to prepare.Item In vitro and in vivo anticancer activity of Gomphocarpus Fruticosus extracts and development of LIPID-based nanoparticles(University of Namibia, 2019) Dushimemariya, FlorenceCancer is on the rise globally, with incidences and mortalities increasing in developing countries including in Namibia. The ongoing search for efficacious and safe medicines to treat cancer is often cumbered by poor solubility and toxicity of potential treatments. Gomphocarpus fruticosus is a medicinal plant used to treat tumors in amibia through topical application and it has been shown to have anticancer action against several cell lines. I lowcver, due to its cardiotoxic properties, it has not been investigated further. This study was conducted to develop a lipid-based drug delivery system based on Gomphocarpusfruticosus extracts, to alleviate the plants toxicity profile while harnessing its anticancer properties. The phytochemistry of G. fruticosus harvested in Namibia was investigated using thin layer chromatography and Gas Chromatography-Mass Spectroscopy. The anticancer activity was evaluated against three cells lines (PC3, I leLa and HcpG2) and cytotoxicity against primary cell lines (Hck293 and KMST6). Apoptosis induction was investigated using the APOPercentageru assay, while general oxidative stress and caspase induction was investigated using the Reactive oxygen species (ROS) assay in HcLa and Hek293 cel ls. The toxicity of the G. fruticosus extracts was also assessed using in vivo toxicity assays in Balb/C mice. Finally, to improve solubility of phytochemicals and reduce non-specific toxicity, a lipid based delivery system was fonnulated through thin-layer hydration of phosphatidylcholine, cholesterol and plant extract (250: J: I 0) in phosphate buffered sal inc The nanoparticlcs were characterized for physically for zeta potential and polydispcrsity using Dynamic Light Scattering and Scanning Electron Microscopy. The encapsulation efficiency of the drug delivery system and its stabi lity in media was also investigated before its biological activity was evaluated against the lleLa cervical cancer cell line. Phytochemical analysis showed G. fruticosus had alkaloids, coumarins, steroids, anthraquinones, Oavonoids and triterpenoids. Furthermore, anti-inflammatory, anticancer and antioxidant compounds such as betasitosterol, alpha-amyrin, lupeol and their esters and acetates were detected by GCMS. The plant extract showed selective antiproliferativc activity against HeLa cel ls (ICso 8.35"7 ± 0.13 g/L) although the mechanism of cell dcalh was most likely necrosis not apoptosis as expected. This conclusion was supported by detected ROS levels and lack of caspase cleavage. In vivo toxicity in Balb C mice was observed (LDso 1.968"1 ± 59.16 glkg) and classified as category 3 under GHS. Lipid based nanoparticles showed high encapsulation efficiency of 96.5 I % with a particle size range between 1 30-150 nm and narrow particle size spread. The zeta potential was low at -1.6 ± 0.6 mV, contributing to agglomeration and low stabi lity in biological media. Fluorescent microscopy and flow cytometry analysis showed uptake of G. fruticosus phytosomes based on the rhodamine fluorescence, treatment of HeLa cells with phytosomes (3.976"4 g/L), increased apoptosis. Cells also had reduced viability in a short time space after exposure, alluding to accelerated cell death in comparison to free extract. This study raises the possibility that the toxicity of G. fruticosus can be managed using a lipid based delivery system especially for cervical cancer following optimization in in vivo cancer disease models. It validates the ethnomedicinal use of G. fruticosus and adds value to a plant whose development was previously abandonded due to toxicity. Furthermore, it elucidates the mechanism of action of G. fruticosus extracts which was not previously known, while describing the phytochemistry of Namibian G. fruticosus. This study can lead to the development of commercially viable, safe and locally sourced treatment alternatives, especially for cervical cancer.Item Antitubercular and antimalarial activity of metabolites isolated from crude and lead-like enhanced (LLE) extracts from selected Namibian medicinal plants(University of Namibia, 2020) Raidron, Celestine VidaMedicinal plants remain an important source of new lead compounds and drugs, but the re-isolation of known compounds and the loss of bioactivity during purification impedes the discovery of novel bioactive compounds. Of interest to this study, is a protocol developed by Camp and co-workers, which involves enhancing the quality of plant extracts by frontloading them with metabolites with drug-like properties, that is, generating lead like enhanced (LLE) extracts. This approach was successfully applied and yielded novel antiplasmodial and antitrypanosomal compounds but has not been explored to search for antitubercular compounds or drug leads. The aim of this study was (i) to prepare crude organic and aqueous extracts of 25 plant parts obtained from eight indigenous Namibian medicinal plants, (ii) to evaluate their antiplasmodial and antimycobacterial activity, and (iii) to correlate their ethnomedicinal use with the biological results obtained in this study. It was further aimed at evaluating the antiplasmodial and antimycobacterial activity of the LLE extracts and MeOH fractions – obtained from the active crude extracts – as well as to isolate and characterize the bioactive compounds. Eight plant species which are used traditionally for the treatment of tuberculosis, malaria and associated symptoms, were collected at Uis in the Erongo region and Tsumkwe in the Otjozondjupa region. Plants included were: Terminalia sericea, Adansonia digitata, Ozoroa paniculosa, Diospyros lycioides, Albizia anthelmintica, Combretum imberbe, Aloe dichotoma and Sarcocaulon marlothii Engl. The antiplasmodial activity was tested in vitro using the parasite lactate dehydrogenase assay against Plasmodium falciparum (CQS) NF54 and the in vitro antimycobacterial activity testing was done using the standard broth microdilution method against Mycobacterium tuberculosis H37Rv-GFP strains. The preliminary biological activity results showed that 10 crude extracts (8 organic and 2 aqueous) displayed antimycobacterial activity with an MIC90 < 90.0 μg/mL, whereas 4 crude extracts (1 organic and 3 aqueous) displayed antiplasmodial activity with IC50 ≤ 18.0 μg/mL. The antimycobacterial and antiplasmodial activities for the fourteen crude extracts ranged from MIC90 9.94 – 86.8 μg/mL and IC50 5.20 - 17.8 μg/mL, respectively. The African baobab tree, A. digitata, displayed the best antimycobacterial (bark, aq.: MIC90 9.94 μg/mL) and antiplasmodial (stems, org.: IC50 5.20 μg/mL) activity. The stems of S. marlothii, an endemic and phytochemically unexplored medicinal plant, are traditionally used to treat tuberculosis. However, both the organic and aqueous extracts displayed poor antimycobacterial activity with MIC90s of 103 μg/mL and >125 μg/mL, respectively. Instead, both the organic and aqueous extracts displayed in vitro antiplasmodial activity with IC50s of 8.80 μg/mL and 17.8 μg/mL, respectively. The antiplasmodial and antimycobacterial bioactive crudes were then subjected to solid phase extraction using Strata-X reversed phase cartridges prepacked with N-vinylpyrrolidone (NVP). The solvent systems used for elution was 70% MeOH:H2O containing 1% trifluoroacetic acid followed by 100% MeOH, to yield the LLE and MeOH fractions, respectively. Compared to the bioactive antimycobacterial crudes, none of the LLE extracts displayed antimycobacterial activity. The MeOH fraction of the bark of A. digitata however, displayed a more than threefold increase in activity (MIC90 19.5 μg/mL) compared to the organic crude (MIC90 70.7 μg/mL). With regard to antiplasmodial activity, the MeOH fraction of the organic extract of the stems of S.mmarlothii displayed a twofold increase in antiplasmodial activity (IC50 4.30 μg/mL) compared to the crude (IC50 8.80 μg/mL). In accordance with the objective of the study, the crude organic extract of the stems of S. marlothii was subjected to flash chromatography, that is, to isolate and characterize potentially novel antiplasmodial compounds. Subfraction on E9 (IC50 6.464 ± 1.767 μg/mL), originating from the organic extract of the stems of S. marlothii, was obtained in small quantities (29.4 mg) and was impure as revealed by LC-MS data. The aromatic region of the 1H-NMR of subfraction E9 revealed that the major bioactive compound, contain a hydroxylated, trisubstituted aromatic ring with an unsaturated side chain, most likely a caffeic acid derivative. The results obtained in this study supports the traditional use of T. sericea and A. digitata for the treatment of malaria and tuberculosis and their associated symptoms. It also supports the enrichment of extracts to expedite antimalarial drug research and recommends further purification of the crude or LLE extracts of S. marlothii for the unambiguously identification of the active compound/s which could serve as leads in antimalarial drug discoveryItem Optimisation of Ontaku/ Oshikundu: Pearl millet and sorghum malts quality and convinient premix development(University of Namibia, 2020) Embashu, WernerOshikundu/Ontaku is a nonalcoholic, acidic, opaque fermented beverage. It is comprised of pearl millet (Pennisetum glaucum (L.) R. Br) meal/flour, commonly known as mahangu, malts of pearl millet or sorghum (Sorghum bicolour (L.) Moench) and/or brans (pearl millet). Brewing of oshikundu remains an art in households with no empirical improvement of a controlled fermentation flow process to give a consistent product. Some of the major limitations to the formal commercialisation of this brew are the absence of standardised malting process that gives a consistent malt quality and microbial safety. The lack of standard ingredient ratios (flour/meal: malt: water) of brewing. Also, the sedimentation of adjunct particles at the bottom (dreg), thus creating the difference in viscosity of oshikundu. Well-defined fermenting microorganisms are not used, and the preparation method still relays on a laborious time-consuming process. Therefore, this study investigated conditions for malting, reduction of dreg, identification of fermenting microorganisms and formulation of ingredient ratio for an improved preparation process. Grains used in the study were collected from Omahenene Agricultural Research Station (2015 harvest), of the Ministry of Agriculture, Water and Forestry. Pearl millet varieties Okashana 2 (SDMV 93032), Kantana (landrace) and Kangara (SDMV 92040), while sorghum varieties are Macia (SDS3220) and landrace commonly referred to as red sorghum. Malts of the two cereals were prepared by steeping in static water at 20-22oC for 2 hours wet and 2 hours air-rest for a total of 8 hours and germinated at 30oC. Malts were dried between 50-55oC for 24 hours. Cereals germinative energy was above 90% as recommended for sorghum by the European Brewery Convention. Malting loss was high up to 30% in pearl millet varieties and Macia. Crude protein and fibre were found to increase following malting. Reducing sugars were not detected in nongerminated pearl millet grains. Malts reducing sugars were statistically significant (p ≤ 0.05) between cereals. The malt reducing sugars was as follows Macia> Red sorghum> Kantana>Okashana 2 = Kangara. Malting resulted in significantly increased free amino nitrogen (FAN) content. Kantana had the highest FAN followed by Macia malt. No amylolytic activity was detected in nongerminated grains irrespective of the cereal. Pearl millet was found not to contain condensed tannins. Malts had an unacceptable high aerobic plate count load above 6.3 Log cfu/g or (2 × 107 cfu/g) as specified for Southern African sorghum malts. However, results show that the malts were not contaminated by Salmonella spp., Shigella and coliforms. Regulated mycotoxins in malts were found to be below the legal limits. Cereal malts are not of safety concern from coliforms and mycotoxins under these malting conditions. Oshikundu is likely fermented by lactic acid bacteria (Lactobacillus. plantarum, L. pentosus, L. acidifarinae, L. paraplantarum, L. spicheri, L. namurensis, L. zymae, L. fermentum, L. brevis, L. delbrueckii subsp bulgaricus, L. buncheri, Leuconostoc gurlium and Pediococcus acidilactici) and yeast (Saccharomyces cerevisiae and S. paradoxus). However, the dynamics of LAB and yeast during fermentation are not known. The use of smaller amounts of dry ingredients and pre-gelatinisation of pearl millet meal in the process of making oshikundu significantly decreases total solids. This suggests that the amount of suspended particles in oshikundu that tend to settle during storage can also be reduced through this route, in the absence of consumer acceptability test. The study demonstrated a creative formulation of a dry powder premix for brewing oshikundu. Preliminary sensory evaluation showed that panellists extremely liked the ease of preparation method, where only water was required to be added to the premix. The study demonstrated that malting pearl millet and sorghum grains under set conditions gave malts of acceptable quality (reducing sugars, free amino nitrogen, alpha and beta amylase activity, phenolic content, radical scavenging activity, mycotoxins and microbial load). Also, a lower amount of pre-gelatinised adjunct can be used to achieve the same yield of oshikundu and at the same time reduce dregs. Also, ingredient ratios were formulated by the use of dry ingredients premix for making oshikundu that was easy to prepare.Item Dynamics of the floodplain fisheries of the Zambezi and Chobe floodplain, Zambezi region, Namibia(University of Namibia, 2019) Simasiku, Evans KamwiFloodplain fisheries are typically multi-species and in most cases harvested with a variety of gears. In the upper- Zambezi, artisanal gillnet fishers mainly target three cichlids: Oreochromis andersonii, Oreochromis macrochir and Coptodon rendalli. However, their abundance is declining because of increased fishing pressure and their stocks are becoming vulnerable to overfishing. The principal aim of this PhD thesis was to assess the fish population dynamics in a highly variable system and the implications of these dynamics to fisheries management by investigating the response of floodplain fisheries to water flow and to determine fish harvesting patterns and catch rates on the Zambezi/Chobe floodplain. The specific objectives of this study were: (1) to link littoral fish colonization rates with water quality and water level in floodplain littoral habitats; (2) to determine the feeding ecology of Hydrocynus vittatus (Tigerfish) in the Zambezi/Chobe floodplain (3) to assess the fishing patterns and harvesting rates of the gillnet fishery on the Zambezi/Chobe floodplain; (4) determine the volume and turnover of fish exports from the Zambezi/Chobe floodplain and finally (5) to assess the significance of fish protected areas in protecting the fish stocks of the Zambezi/Chobe floodplains. Seine net surveys showed that the marginal zone of the Zambezi/Chobe floodplain were dominated by fishes of the fish families Cichlidae, Cyprinidae and Characidae. Individual fish densities of small fishes showed spatiotemporal variations among the different stages of flooding with a marked increase in juvenile cichlids during the peak flooding phase while cyprinids were most abundant during the receding (fall) phase. Among other environmental filters, dissolved oxygen and water level had a marked influence on the distribution and abundance of the littoral fish species. The feeding ecology of Hydrocynus vittatus (Tigerfish) (Characidae), in the Zambezi River was investigated between February - December 2016. The findings indicated that large size classes of H. vittatus (>176mm) were largely piscivorous, and showed a diet shift with change in size. The small size classes (<140mm) consumed mainly aquatic insects (21.1%), Synodontis spp. (17.8%), and Micralestes acutidens (12.1%). They later shifted to a diet in which Synodontis spp. (26.1%), Brycinus lateralis (15.2%) and M. acutidens (13.0%) dominated. The study showed that H. vittatus plays an ecological role with the ability of converting un-exploitable non-commercial species such as B. lateralis (15.2%) M. acutidens and Synodontis spp. into exploitable protein. Hence, the population of Hydrocynus vittatus must be of conservation priority to sustain a balanced fishery in the Zambezi River. The fishery on the Zambezi/Chobe floodplain was investigated by means of Catch Assessment Surveys (CAS) from 2010 to 2017. The fishery is dominated by gillnets in the form of monofilament net types. The principal species in gillnet catches were three cichlids: O. andersonii, O. macrochir and C. rendalli. Length at maximum selectivity for the three cichlids showed that they were caught at a length close to their reported length at 50% maturity, raising the fear, risk of growth overfishing. Analysis of annual catch rates of the three commercially important species; O. andesonii, O. macrochir and C. rendalli showed a significant decline from 2010 to 2017. Annual production from the 1700 km2 of the Zambezi/Chobe floodplain was estimated at 2248 t/yr in 2010 dropping to 939 t/yr in 2017. A survey on the floodplain fish processing and exports was conducted at Wenela Border Post, north of Katima Mulilo. Daily fish loads destined for exports were weighed and recorded using a hanging scale at Wenela Border. The study showed that women are the key players in the fish processing and preservation on the Zambezi/Chobe floodplain. The major processing techniques employed by the fish vendors were sun drying and smoking. Approximately 1575.8 t/yr of fish products worth N$22 million were exported to foreign markets in Zambia between June 2015 and December 2016. The active involvement of women in the fish processing and export on the Zambezi/Chobe floodplain as well as the large markets available (both local and foreign) suggest that this sector has the potential to contribute immensely to improving the economic and livelihood of the Zambezi Region. However, the fish vendors faced challenges of inadequate cold storage facilities, poor weather and packaging. Thus, the study recommended private and government’s intervention in the provision of storage facilities and training programs for the fish processors in hygienic handling of fish products, quality control and packaging of processed fish products to conform to human health standards. The fisheries of the Zambezi/Chobe River and its associated floodplains are currently threatened by an increase in fishing pressure. With increasing fishing pressure, fish populations may undergo a series of changes in size, species composition and abundance. As a result, two Fish Protected Areas (FPA) on the Zambezi/Chobe River (Kalimbeza Channel and Kasaya Channel) were recently established to protect and conserve the fish stocks of the Zambezi/Chobe River. However, accrued benefits from these FPAs have not been assessed. Comparative experiments using gillnets of a graded set of mesh sizes (12 mm -150 mm) were conducted between FPA (Kalimbeza channel) and non-FPA (Hippo channel) between March and December 2016 to test the hypothesis that FPA yield high fish abundance (CPUE) than non- FPA. Experimental fishing trials showed higher CPUE by weight and number (p < 0.05) of the five dominant species (H. vittatus, Schilbe intermedius, Pharyngochromis acuticeps, M. acutidens and B. lateralis) in the FPA than non-FPA. The study confirmed the importance of the protected areas in conserving fish resources in the Zambezi/Chobe River. Recommendations were made to manage the fish stocks of the Zambezi/Chobe Rivers by imposing restrictions on gear type, establish more fish reserves and advocate for community engagement in managing their own resources under strict guidance from government.Item Characterisation, bioactivity and qsar studies of natural products from selected Namibian red marine algae(University of Namibia, 2020) Ishola, AnthonyThe objective of this research was to discover new drug leads from Namibian marine algae. Plocamium extracts were screened and their phytochemical contents were quantified. Both the antioxidant activity and the antimicrobial activity of the crude extracts were determined, as well as the dose-response relationship for Plocamium extracts in BALB/c mice. This was done by using acute and sub-acute toxicity parameters. In addition, the structural elucidation of the major metabolite found in the crude extract was determined and the Quantitative Structure Activity Relationships (QSARs) of the compound were determined. Methods: Frozen Plocamium leaves were soaked in dichloromethane (DCM) and methanol (MeOH) in a ratio 1:1 (v/v) for 48 hours. Concentrated Plocamium extracts were screened for phytochemical constituents. Total phenolic and flavonoid contents as well as antioxidant activity were quantified. Dried algal extracts were also reconstituted with different solvents and tested in vitro for antimicrobial activity against 12 pathogens using the Kirby Bauer disc diffusion method. Mice of known weights were infected with Escherichia coli and Pseudomonas aeruginosa by intravenous injection and sub-cutaneous methods respectively. The mice were later treated with gentamycin and ampicillin injections. Other groups of mice were treated with different concentrations of Plocamium extract over a period of five days. E. coli and P. aeruginosa loads in the faeces of the test mice were quantified daily. Plocamium extracts were purified using HPLC to fractionate the extracts. Major fractions were collected and identified by means of one and two dimensional NMR spectroscopy data and MS analysis. In terms of QSAR, the structures of the metabolites were theoretically optimized using the Merck Molecular Force Field. In addition, several physicochemical properties were computed by using the B3LYP variant of Density Functional Theory in conjunction with the 6-31G (d) basis set. Results: Plocamium cornutum and Plocamium rigidum extracted using DCM had total phenolic content of 132.85 ± 0.82 mg and 188.65 ± 0.45 mg Gallic acid equivalents per gram respectively. The IC50 values for Plocamium rigidum and Plocamium cornutum were 28.87 ± 0.82 μM and 40.11 ± 0.38 μM respectively. Ethanolic extracts of Plocamium rigidum showed a zone of inhibition of 6.35 ± 0.25 mm against Listeria monocytogenes while the standard ampicillin had no activity. From the probit plot, the LD50 was calculated to be 3556 mg/kg. A therapeutic dosage of P. rigidum of 355 mg/kg in BALB/c mice reduced E. coli load to pre-evaluation levels on the fifth day. The chemical structure of Plocamium sample I and II (Plocamium rigidum and Plocamium cornutum respectively) yielded two known compounds namely, 3,4-erythro-7-dichloromethyl-3-methyl-3,4,8-trichloro-1,5E,7E-octatriene (from sample I) and 1E,3R,4S,5E,7Z-1-bromo-3,4,8-trichloro-7-(dichloromethyl)-3-methylocta-1,5,7-triene (from sample II) respectively. CPKOvality and HLgap are physicochemical properties that best describe the variation in biological activity of the metabolites. The equation of the best fit was determined as: pIC50 = 9.91CPKovality + 0.270HL-gap-17.149 (R2 = 0.71, Adj. R2 = 0.56, R = 0.84, Std error = 0.31, and q2 = 0.55) Conclusion: DCM is a better solvent than methanol for the extraction of natural products from Plocamium species. P. rigidum showed inhibition against E. coli and L. monocytogenes in vitro. Although P. rigid inhibited the growth of E. coli, the possible development of liver lesions (in vivo) after chronic exposure is an indication of liver injury which is a sign of the chronic toxicity of P. rigidum, even at low concentrations. The probable compound responsible for this action is sample (II) above. The variables in the equation above are the parameters that best describe the variation in biological activity of the halogenated monoterpenes extracted and identified. The equation also shows that CPKovality (moderate size and shape of ligands) and a small H-L gap with high reactivity are the parameters that best optimised structure to predict improved biological activity of Plocamium metabolites.Item Evaluation of indigenous Namibian mushrooms and plants for antimalarial properties(University of Namibia, 2019) Kadhila, Nailoke PaulineMalaria is a parasitic disease caused by Plasmodium species and transmitted by Anopheles mosquitoes. The disease is currently ranked high among the most problematic infectious diseases around the world. Despite the significant progress that has been made toward reducing the global burden of malaria, it remains one of the most significant public health threats in sub-Saharan Africa and many other parts of the developing world. Current malaria control measures have adverse environmental and human effects. The synthetic repellents used for controlling vectors are causing irreversible damage to the ecosystem since the chemicals are non-degradable in nature. The current antimalarial drugs are also facing the specter of parasite resistance, hence the need to discover novel drugs from natural products. The overall objective of the study was to perform bioassay-guided fractionation and determine the antiplasmodial activities, phytochemical profiles, active compounds and cytotoxicities of mushroom and plant extracts. The study involved an ethnobotanical survey of putative antimalarial mushrooms and plants. Preparation of mushroom and plant organic extracts; evaluation of antiplasmodial activity of the extracts using the parasite lactate dehydrogenase (pLDH) assay; verification of phytochemical profiles (saponins, terpenoids, anthraquinones, coumarins, alkaloids, and flavonoids) using standard procedures; elucidation of active antiplasmodial compounds using Gas chromatography-Mass Spectrometry (GC-MS), Fourier Transform Infrared (FTIR) and Nuclear Magnetic Resonance spectroscopy (NMR) and determination of cytotoxicity using the (3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide) (MTT) method. Four mushroom and thirteen plant species used in this study were collected from north-central Namibian regions namely Kavango East, Kavango West, Ohangwena, and Oshikoto. Of these, only two plants were active against 3D7 strains of the malaria parasite, Plasmodium falciparum. None of the evaluated mushrooms showed activity. The phytochemical screening of the plants revealed presence of anthraquinones, saponins, terpenoids, coumarins, alkaloids, and flavonoids. Two antiplasmodial compounds namely Npk1 F70 (IC50 = 2.63 ± 0.48 μg/mL) and NPk1 F78 (IC50 2.64 ± 0.32 μg/mL) were isolated from Npk1 (Pechuel-loeschea leubnitiziae) dichloromethane (DCM) extracts. The compounds were screened for cytotoxicity on the Chinese hamster ovary (CHO) cell line using the MTT assay and were found to be toxic to the mammalian cells. GraphPad Prism was used to calculate the compounds’ IC50 values of 2.7 μg/mL and 2.22 μg/mL for NPk1 F78, and Npk1 F70, respectively. The molecular weight of the two anti-3D7 compounds was determined by GC-MS to be 246 g/mol. When FTIR and NMR were performed, the structure of the active compound was identified as xerantholide. This is the first study to elucidate an active antiplasmodial compound from this indigenous Namibian plant P. leubnitiziae which is used by local people to manage malaria. This study, to the best of my knowledge is the first to report the antimalarial activity of xerantholide, a known guaianolide extracted from aerial parts of P. leubnitiziae. It is recommended that pharmacomodulation study be carried out on the compound identified in this study in order to decrease the compound’s toxicity, while maintaining or improving its activity. Further studies also need to be done on xerantholide to determine the diastereomers (stereoisomers that are not mirror images of one another). Modification on xerantholide chemical structure also needs to be carried out so that the toxic compounds can be used for possible anti-cancer therapy.Item Factors influencing the establishment of translocated eland (Taurotragus oryx) and springbok (Antidorcas marsupialis) in the Nyae Nyae conservancy, Namibia(University of Namibia, 2018) Lendelvo, Selma M.The emergence of the Community-Based Natural Resources Management (CBNRM) approach in Namibia contributed to the establishment of communal conservancies that aim to conserve wildlife outside protected areas, as well as benefit local communities. Recent translocations in Namibia involved the movement of wildlife from protected areas to communal conservancies in order to expand the range of wildlife species in the areas they once occupied, as part of effective community-based conservation efforts. Little research has been done to understand the outcome of the translocation of ungulates for the purpose of restocking wildlife populations in communal conservancies, and to determine the factors affecting the establishment of these ungulates, as well as the contribution of translocation to sustainable wildlife management in Namibia. The aim of this study was to establish the factors that contributed to outcomes of translocated eland (Taurotragus oryx) and springbok (Antidorcas marsupialis) in the Nyae Nyae conservancy. The study utilised a mixed-method design that involved the employment of both qualitative and quantitative data collection methodologies in obtaining primary and secondary data. Field observations were carried out to collect data on the current population structure of the eland and springbok. A total of 56 questionnaires, 19 key-informants and 6 focussed-group discussions (FGDs) with community members and stakeholders, were administered during the period of July 2013 to March 2015. Stakeholders comprised the relevant local, regional and national institutions that worked closely with the Nyae Nyae conservancy in different aspects of wildlife and conservancy management. Secondary data, consisting of long-term wildlife count data, were obtained from the conservancy and the office of the Ministry of Environment and Tourism both in Tsumkwe and Windhoek. Following translocations, a stable trend of the overall wildlife population sizes in the Nyae Nyae conservancy (r=0.477; t10=1.574; p=0.145) was found. The springbok (r= 0.181; t10=0.580; p=0.575) showed a positive population trend while the eland (r= -0.429; t10= -1.502; p=0.164) showed a declining trend, according to the long-term monitoring data collected between 2001 and 2013. The outcome of the field assessments on the habitat utilised by the springbok was consistent with the analysis of the long-term data, indicating that the free-roaming and growing springbok population was found along the Nyae Nyae Pans in the conservancy. However, a free-roaming eland population was not detected but a population confined in the Buffalo Camp was found. The composition of the social groupings of the observed eland and springbok populations comprised solitary, bachelor, mixed, nursery and/or female groups. Perceptions gained from the surveys concluded that the factors such as availability and distribution water resources, range condition, veld fires frequency and level predation had limited negative influence on the translocated populations of the two species, while human-related activities were attributed to the translocation failure of the eland. There was little evidence of hunting of the eland for both subsistence and trophy purposes, although evidence existed that the eland had been allocated a hunting quota. Although springbok was not commonly utilised for trophy hunting, this species had been hunted sustainably for subsistence and the purposes of meat distribution since 2004. The selection of a suitable, natural habitat, coupled with limited human disturbance, contributed to the establishment of the translocated springbok population. However, the eland population not increasing after translocation in 2001 remains complex because the confined eland population that was moved into the Buffalo Camp of the conservancy since 1994 was able to survive in a similar habitat for over two decades. Suitability to natural habitat factors alone is not sufficient to contribute to the establishment of translocated wildlife species. Therefore, the anthropogenic elements of a habitat should form part of the suitability assessments during the translocation process of wildlife into conservancies because of the nature of community-based conservation areas.Item Prevalence, antibiotic resistance trends, virulence and effect of some medicinal plants on Staphylococcus froms school children in the Mariental district, Namibia(University of Namibia, 2018) Walter, SunetteThe main aim of this study was to investigate potentially pathogenic community-associated staphylococci in school children. Objectives were: To determine the prevalence of nasal Staphylococcus aureus and coagulase-negative staphylococci (CoNS) in school children from the Mariental District; to characterize the bacteria in terms of their antibiograms and drug resistance patterns; to screen bacterial isolates for their ability to produce enterotoxins and produce biofilms as potential virulence factors; and to assess the antimicrobial and anti-biofilm activity of crude methanolic extracts of Aptosimum albomarginatum (Marloth and Engl.) roots, Albizia anthelmintica (A. Rich Brongn.) twigs and Dicoma schinzii (O. Hoffm.) against staphylococci (including multi-drug resistant strains) isolated from the learners. To our knowledge, this is the first study undertaken to report on the prevalence, antibiotic resistance trends and virulence characteristics of potentially pathogenic staphylococci among school children in the Mariental District. This was a cross-sectional study involving five schools. With informed consent from parents/guardians, nasal specimens (swabs) were obtained from 272 randomly selected learners aged 6-14 years. Specimens from swabs were enriched for Staphylococcus in brain heart infusion broth prior to isolation on Staphylococcus medium no. 110 and tryptone soy agar. Pure cultures were obtained from mixed cultures, Gram-stained and biochemically tested for identification of S. aureus and CoNS. Methicillin-resistant Staphylococcus aureus (MRSA) and methicillin-resistant coagulase-negative staphylococci (MRCoNS) were identified by their resistance towards cefoxitin using Kirby-Bauer disk diffusion assay. By disk diffusion assay, 352 S. aureus and 81 coagulase-negative staphylococcal isolates underwent susceptibility testing against the antibiotics ampicillin, cefoxitin, ciprofloxacin, erythromycin, gentamicin, rifampicin and tetracycline. The American Type Culture Collection reference strains S. aureus ATCC 25923 and S. aureus ATCC 33591 were used for quality control. Isolates were classified as multi-drug resistant when they displayed resistance towards three or more classes of antibiotics. Twenty-two multi-drug resistant MRSA nasal isolates and the multi-drug resistant MRSA reference strain S. aureus ATCC 33591 were screened for production of enterotoxins A-D, using a SET-RPLA toxin detection kit. The microtiter plate assay was employed to determine biofilm production in 10 nasal S. aureus isolates (including one MRSA isolate), as well as S. aureus ATCC 25923 and S. aureus ATCC 33591. Crude methanolic plant extracts were prepared by maceration, filtration, rotary evaporation and freeze-drying. Qualitative chemical assays and thin layer chromatography (TLC) were used to screen for flavonoids, saponins and anthraquinones in the plant material. To test for antimicrobial activity of the crude methanolic extracts, disk diffusion assays were used. The antibiotics gentamicin and chloramphenicol were used as positive controls, and discs flooded with dimethyl sulfoxide (DMSO) as negative control. A microtiter plate assay determined if extracts could inhibit and/or eradicate S. aureus and MRSA biofilms. Four hundred and thirty-three isolates from 272 swabs were morphologically and biochemically identified as Staphylococcus bacteria. Of these isolates, 352 (81.3%) were S. aureus, while 81 (18.7%) were CoNS. Furthermore, 51/433 (11.8%) isolates were MRSA and 7/433 (1.6%) MRCoNS. The overall prevalence of S. aureus in the study population of 272 learners was 80.5%, and that of CoNS was 25.0%. Methicillin-resistant S. aureus was isolated from 48/272 (17.6%) learners and MRCoNS in only 7/272 (2.6%) learners. From the study participants who reported getting nosebleeds, 75.4% were colonized with S. aureus, whereas 81.4% who were exposed to cigarette smoke from a household member carried this bacterium. Out of 433 staphylococcal isolates, 96.0% S. aureus and 66.7% CoNS were resistant to ampicillin, with this resistance being significantly higher in S. aureus (P < 0.0001). Ciprofloxacin and gentamicin were most effective against S. aureus isolates, with 99.7% and 93.2% of isolates that were susceptible to these drugs, respectively. Ciprofloxacin was also the most effective drug against isolates of CoNS, with 100.0% susceptibility. Of 352 S. aureus isolates, 51 (14.5%) were cefoxitin/methicillin-resistant. Only seven (8.6%) of 81 CoNS isolates were cefoxitin/methicillin-resistant. In total 31 antibiotic resistance patterns were observed, 27 for S. aureus and 14 for CoNS. Overall, the three most frequently observed patterns were AP, AP-E, and AP-T. Of all isolates, 12.5% were multi-drug resistant. These include 50 isolates of S. aureus and four CoNS. From the 51 MRSA isolates, 43.1% were multi-drug resistant. One of these MRSA isolates showed resistance towards 6/7 antibiotics tested with only ciprofloxacin that was effective against it. Methicillin-resistant CoNS was not multi-drug resistant, with the most common resistance pattern being AP-RP-FOX. Twenty-three multi-drug resistant MRSA isolates were screened for enterotoxins A-D. Of these, seven were enterotoxigenic. Enterotoxin A was the most prevalent, produced by five isolates. Enterotoxin B was found in one isolate, while enterotoxin C was produced by two isolates. One isolate tested positive for both enterotoxins B and C. Enterotoxin D was not detected in the isolates screened. All 12 S. aureus isolates, including two MRSA strains, which were evaluated for biofilm formation were strong biofilm formers in microtiter plates. Aptosimum albomarginatum root extract was a moderately active antimicrobial agent against 7/12 S. aureus isolates, including two MRSA isolates. Moderate antimicrobial activity was also observed with this extract in 9/54 multi-drug resistant isolates, of which two were MRSA with the same antibiotic resistance pattern of AP-T-RP-FOX. These two isolates may therefore be the same S. aureus strain. The root extract was the best biofilm inhibition agent, with highly active inhibition (86.0%) observed in S. aureus ATCC 33591 (MRSA), and moderate activity in four other isolates. This extract eradicated the biofilm of S. aureus isolate S110 S73 Pure by 40.0% (moderate activity). Flavonoids and saponins/triterpenes may contribute to the root’s antimicrobial and anti-biofilm properties. Root extract from D. schinzii displayed moderate antimicrobial activity against 8/12 S. aureus isolates, while the plant’s leaf extract was moderately active against 2/12 S. aureus isolates. The leaf extract also moderately inhibited biofilms in three isolates. Flavonoids, coumarins, saponins or triterpenes in the leaves may contribute to its antimicrobial and anti-biofilm properties. In conclusion, the high prevalence of S. aureus and presence of MRSA (including multi-drug resistant bacteria) among study participants calls for improvement in current hygiene practices at schools in the Mariental District to prevent staphylococcal disease. Nosebleeds and exposure to cigarette smoke were identified as possible risk factors for colonization with S. aureus in the children. Overall, multi-drug resistance in our study was relatively low. However, almost half of the MRSA isolates were multi-drug resistant, which is of concern. Results from toxin screening indicated that multi-drug resistant MRSA may produce enterotoxins, whereas S. aureus and MRSA can produce strong biofilms. Self-infection by these bacteria poses various health risks for the children. Nevertheless, learners should be encouraged to frequently wash their hands to prevent spread of antibiotic-resistant bacteria within the community. Ciprofloxacin and gentamicin may effectively be used to treat staphylococcal infections in this study population. Antimicrobial activity was observed with A. albomarginatum roots and D. schinzii roots and leaves, indicating potential use against staphylococcal infections. Noteworthy is this activity of A. albomarginatum root extract against multi-drug resistant strains, including MRSA. The root extract was the best anti-biofilm agent against S. aureus. It was highly active in inhibiting biofilm formation in one MRSA isolate, and moderately active in inhibiting formation in four isolates. The extract moderately eradicated the biofilm in one isolate. This activity may to some extent be attributed to the presence of the secondary metabolites flavonoids, saponins or triterpenes in the roots. The D. schinzii leaf extract moderately inhibited biofilms in three isolates. Flavonoids, coumarins, saponins or triterpenes may play a role its anti-biofilm activity. The present work thus supports the traditional medicinal use of A. albomarginatum roots and D. schinzii roots and leaves as natural antimicrobial agents, and A. albomarginatum roots and D. schinzii leaves as anti-biofilm agents, in infections involving the bacteria under study.Item Profiling studies of five Namibian indigenous seed oils obtained using three different extraction methods(University of Namibia, 2018) Cheikhyoussef, NataschaNamibia has a rich biodiversity of plant species of which a great variety is used for food, cosmetics and medicinal applications. Seed oil production from indigenous sources has economic importance for Namibia and is an asset for improving the livelihoods of local communities. Knowledge of the quality and composition of these oils can be used towards value-added product development strategies and improved marketing of indigenous resources. A comprehensive profile of indigenous seed oils currently produced in Namibia at traditional and commercial level was developed. This profile included a presentation of the physico-chemical properties of the oils and their quality characteristics among three different extraction techniques, namely the traditional extraction method, machine-operated cold pressing and Soxhlet extraction. The oils studied were Citrullus lanatus (Kalahari melon) oil, Schinziophyton rautanenii (Schinz) Radcl.-Sm. (Manketti) nut oil, Sclerocarya birrea (A. Rich) Hochst. (Marula) nut oil, Ximenia americana (Blue sour plum) nut oil and Acanthosicyos horridus Welw. ex Hook.f. (!Nara) oil. Significant differences (p<0.05) among the three extraction methods for Manketti nut oil were found for the characteristics of AV, p-AV, IV and RI. Cold pressed Manketti nut oil was significantly different (p<0.05) in terms of its PV from the traditionally and the Soxhlet extracted Manketti nut oil, with a higher PV. Significant differences (p<0.05) were found for the characteristics of AV, PV and IV for Marula nut oil. No significant differences (p≥0.05) were found for the p-AV among the three extraction techniques. Significant differences (p<0.05) were found for the characteristic of the RI for Ximenia nut oil. The traditionally extracted Ximenia nut oil was significantly different (p<0.05) from the cold pressed and the Soxhlet extracted Ximenia nut oil for the characteristics of PV and p-AV, with higher values. Significant differences (p<0.05) among the two extraction techniques for the !Nara seed oil were found for the characteristics of SV, AMW, EV, IV, SG and RI and non-significiant differences (p≥0.05) for the characteristics of AV, PV and p-AV. Major fatty acids found were linoleic (31.2-32.2%) acid, α-eleostearic (24.2-35.7%) acid (Manketti), oleic (66-6-67.6%) acid (Marula), oleic (46.3-44.1%) acid, ximenynic (6.5-12.0%) acid (Ximenia), linoleic (52.6-57.0%) acid, oleic (10.5-17.7%) acid (Melon) and linoleic (53.1-54.5%) acid, oleic (12.8-13.9%) acid (!Nara). Highest total tocopherol content found was 205.64 mg/100g (Manketti; Soxhlet extracted), 29.72 mg/100g (Marula; traditionally extracted), 10.75 mg/100g (Ximenia; traditionally extracted), 46.10 mg/100g (!Nara, Soxhlet extracted) and 74.39 mg/100g (Melon; cold pressed). The highest stigmasterol (53.11 mg/100g) was found in traditionally extracted Marula nut oil and the highest β-sitosterol content (682.43 mg/100g) was found in Soxhlet extracted Manketti nut oil. The hydrolysis of Marula and Manketti nut oils for fatty acid production with Candida rugosa lipase was studied using a 23 full factorial design was applied to study the interactions of experimental factors such as pH, temperature, oil concentration and C. rugosa lipase concentration. The degree of hydrolysis ranged from 49.5% to 77.0% and 56.3% to 75.7% for Manketti oil and Marula oil, respectively. The effect of initial pH, temperature, oil and enzyme concentration and their interaction had significant effect on the degree of hydrolysis of Manketti oil. For the Marula oil hydrolysis, oil concentration, enzyme conentration and the interaction between temperature and oil concentration were observed to be significant. The optimal conditions for achieving a %H of 76.5% for Manketti oil was initial pH of 6.0, a temperature of 30 °C, an oil concentration of 30% and an enzyme concentration of 10 mg/g of oil. The optimal condition for achieving %H of 62.5% for Marula oil hydrolysis, was temperature of 36.8 °C, oil concentration of 11.3% and enzyme concentration of 23.6 mg/g of oil. The present study revealed that the indigenous seed and nut oils obtained from three different extraction techniques have the potential for promotion in the food, in particular as functional foods, cosmetics and the pharmaceutical industry.Item Ethnopharmacological assessement of Guibourtia Coleosperma and Diospyros Chamaethamnus extracts as elaternative treatment options for Malaria(University of Namibia, 2016) Du Preez, Iwanette C.Malaria, a parasitic infectious disease, remains one of the world’s foremost health concerns, even more so in developing countries. Much progress has been made in fighting the disease, particularly in Southern Africa where four countries (Namibia included) have targeted malaria elimination by 2020. Challenges such as the absence of a vaccine, resistance to insecticides, and particularly the emergence of resistance to current antimalarial treatment regimens threaten to undermine the current successes in malaria control efforts. Secondly, the lack of access and acceptance of conventional antimalarial treatment by populations in malaria endemic areas reduces the feasibility of eliminating and consequently eradicating malaria. Local communities in Namibia use plant-based medicines to treat malaria and malaria associated symptoms based on traditional observations or beliefs over decades. As to whether these plants are efficacious for the indication and cause toxicity is yet to be validated scientifically. The aims of this study were, therefore, to evaluate the biological activities of the extracts of two Namibian plant species to provide a scientific rationale for their traditional uses. Guibourtia coleosperma and Diospyros chamaethamnus, which are used to alleviate symptoms of malaria in Namibia, were investigated using phytochemical analyses, and in vitro and in vivo bioassays. Extracts were prepared by using solvent extraction of varying polarities to obtain a wide range of metabolites. Ground plant material was macerated in distilled water (aqueous extracts) and dichloromethane-methanol (1:1v/v) (organic extracts) respectively. The extracts were dried in vacuo, and examined for six classes of compounds known to have antiplasmodial activity using TLC. GC-MS was used to identify compounds in the plant extracts related to biological activity, using a BP5MS column. Radical scavenging abilities of the plant extracts were ascertained by the 2,2-Diphenyl-1-picrylhydrazyl (DPPH) method. In vitro antimalarial activity was determined by reconstituting the plant extracts in water and DMSO at varying concentrations, and incubating extracts with Plasmodium falciparum D10 infected RBCs for 48 hours. Subsequently, growth inhibition of the P. falciparum parasites was determined using parasitaemia. In vitro assays to determine cytotoxic effects were conducted with the plant extracts using a fibroblast cell line (W138). In vivo inhibition of the growth of P. berghei in Swiss albino mice (20±4 g) was evaluated using optical microscopy on blood smears. Survival curves post-infection were also used to determine suppressive and prophylactic activities of extracts. The plant extracts were also evaluated for their toxicity in healthy mice using a dose escalation method with a starting dose of 300 mgkg-1. The crude extracts contained alkaloids, anthraquinones, flavonoids, steroids and terpenoids. Secondary metabolites with antiplasmodial, antibacterial and antioxidant properties were tentatively identified as 3,5-dihydroxy-6-methyl-2,3-dihydro-4H-pyran-4- one, phloroglucinol, stigmasterol, glycerol 2-hexadecanoate, α-amyrin, 9,12-octadecdenoic acid (Z,Z)-, hexadecanoic acid, oleic acid, lanosterol, spiculesporic acid, squalene, campesterol and vitamin E. Antioxidant activity results showed that the extracts of D. chamaethamnus yielded the highest antioxidant activities with IC50 values of ranging from 7.63 to 10.74 μg/mL, whilst the extracts G. coleosperma yielded antioxidant activities with IC50 values of ranging from 22.03 to 36.05 μg/mL. Moderate in vitro antiplasmodial activity (IC50 < 50 μg/ml) against P. falciparum D10 was observed for the two plants ranging from 18.30 to 31.61 μg/mL. All plant extracts showed no cytotoxicity with IC50 values above 100 μg/mL, except for the organic extract of D. chamaethamnus (CC50=29.73). The organic extracts for both D. chamaethamnus and G. coleosperma (800 mgkg-1) exhibited significant (P< 0.05) blood schizonticidal activity in the 4-day early infection test with parasite growth inhibition of 44.66 and 29.59 %, respectively. This dose also prolonged the survival of the mice by 50 and 58 % (i.e. with 6 and 7 days), respectively. The plant extracts also exhibited prophylactic activity in the mice inhibiting parasitaemia with 56.13 (D. chamaethamnus) and 55.48 % (G. coleosperma) at the highest dose (800 mgkg-1) and increasing survival by 155.6 and 22.2 % (i.e. with 14 and 3 days), respectively. Oral administration of crude extracts at the highest dose of 2000 mgkg-1 resulted in no mortalities or evidence of adverse effects, indicating that D. chamaethamnus and G. coleosperma extracts were non-toxic. The study showed promising antimalarial activities of D. chamaethamnus and G. coleosperma. The results show that the ethnomedicinal use of these plants to treat symptoms of malaria is rational and safe. This is a step in the right direction towards incorporating their use in mainstream health care policies as alternative treatment options for malaria. Identification of bioactive compounds to standardize extracts should the next step. Further studies should also include the optimization of doses to improve efficacy, and studies to assess the antiplasmodial activities of the two plants in combination treatments as used in an ethnomedicinal setting. Lastly, this study has shown that the plant extracts can also be used as a prophylactic. This is new knowledge should be shared with indigenous communities to maximize the medicinal use of these plants.Item Diversity and distribution of Tylosema esculentum (Marama bean) endophytic bacteria communities in Omitara, Harnas and Otjinene, eastern Namibia(University of Namibia, 2016) Uzabakiriho, Jean D.Tylosema esculentum is a nutritious drought avoiding plant endemic to the Kalahari Desert. Our study assessed the density, diversity and distribution of endophytic microbial community structures associated with leaves, stems and tuberous roots of T. esculentum in Eastern Namibia. Culture-dependent and PCR-based 454 pyrosequencing methods were used. ANOVA with pairwise comparison revealed a significant difference in bacterial density between below and above ground. Endophytic bacterial isolates (605) were identified, grouped into 24 genera and three phyla. Proteobacteria was the most represented (67.4%) followed by Firmicutes (23.7%) and Actinobacteria (4.3%). Shannon diversity index, revealed a significant difference between the tuberous roots and leaves (p = 0.005) and stems (p = 0.006) microbial communities. The cluster analysis revealed a separation between the above and below ground microbial communities. The PCA and the Jaccard diversity indices confirmed these findings. Our results suggested that the microbial community composition was mainly governed by the plant parts rather than the location or sampling time. The phylogenetic analysis showed that all these microbial communities fell into two clades distinct from known cultivated bacteria from NCBI. All isolates associated with T. esculentum were positive for the nifH gene amplification. Only 42% nested with known strain in the NCBI GeneBank Database. This finding showed the presence of putative novel nitrogen-fixing bacteria associated with T. esculentum. Ten samples (leaves, stems and tuberous roots) from Omitara were examined using 16S rRNA gene pyrosequencing method. The presence of the three phyla Firmicutes (50.3%), Proteobacteria (38.32%) and Actinobacteria (4.46%) was confirmed. Two more phyla Fusobacteria (5.7%) and Bacteriodetes (1.368%) were revealed. Strikingly, 2 phyla (Firmicutes and Proteobacteria) of the five phyla represented 89 % of the total sequences. Similarly, four families (Enterobacteriaceae, Bacillaceae, Pasteurellaceae and Fusobacteriaceae) of the 18 recorded, represented nearly 91% of the total T. esculentum bacterial community assemblage. The genus Bacillus was predominantly found in the tuberous roots though shared across all samples. The class Clostridiale was exclusively found in leaves and stems. Within the Gammaproteobacteria class, the sequence grouping showed the Enterobacteriaceae family and the yet to be identified Enterobacteriaceae dominated. Unlike the Enterobactericeae that are mostly found in the tuberous roots, the Pasteurellaceae family was preferably found in the leaves and stems. Actinobacteria have shown a ubiquitous colonization compared with the Bacteriodetes that colonized the above ground part of the plant. T. esculentum organs exerted selective pressures on their associated bacterial communities. Only 68% of all reads could be classified at the phylum level. Firmicutes are the most dominant phylum in the current study with 46.9% sequences that have not yet been classified to any existing family, order or genus. Also, the rarefaction curves predicted that additional sampling will lead to significantly increased estimates of diversity. Sequences in this study, have shown similarities with sequences occurring in water-stressed environments with plant growth promoting traits. In conclusion, T. esculentum bean lives in community with a large diversity of potentially plant growth promoting bacteriaItem Domestication in Marama bean (Tylosema Esculentum): Agronomy, phenotypic and molecular characterization for its improvement(University of Namibia, 2014) Takundwa, Mutsa M.Marama bean [Tylosema esculentum (Burchell) Schreiber] belongs to the family Fabaceae and is a candidate for domestication in arid zones. It is indigenous to the Kalahari regions of Southern Africa thriving in low nutrient and low moisture soils. Marama bean seeds exhibit high oil (up to 48%) and protein (up to 42%) content comparable to peanut and soybean respectively. The main purpose of this study was to determine the usefulness of previously developed microsatellite markers in distinguishing phenotypically characterized marama plants and plants treated with gamma irradiation for improvement. The chromosome number was determined to lay a foundation for molecular mapping. Further to this, the study sought to establish the effect of improved nutrients, moisture and hormone treatments on vegetative growth of marama bean. Grafting was explored as a propagation method to side step juvenility and molecular identification of potential fungal pathogens of leaf tissue was achieved. SSR primers were screened using DNA isolated from phenotypically characterized individuals representing 13 marama bean ecotypes. Two microsatellite markers MARA 039 and MARA077 were found to be candidates for use in detecting differences in internode length as well as distinguishing seeds treated with gamma radiation from untreated seeds. The chromosome number in T. esculentum was found to be n=22 (2n=44) and this will be useful in future mapping efforts. A completely randomized block design was used in assessing the possibilities of enhancing vegetative growth with fertilizer, hormones and water: Lucky plant fertilizer (LS004990-00-00) (1g/L), 100μg/L of hormone Gibberellin (GA3), 200mL water for the control, 400mL water for the high water treatments were applied. The results obtained were analysed by one way ANOVA and showed no significant difference in internode length (p=0.362>0.05), stem length (p=0.256>0.05) and number of leaves (p=0.466>0.05) suggesting the treatments had no effect on the vegetative growth of T. esculentum. Marama bean was observed to be non-responsive to grafting in the trials carried out prompting the exploration of tissue culture methods for future studies. The overall inoculations of PDA and PDB with leaf tissue showing signs of necrosis and DNA isolation together with the internal transcribed spacer (ITS) region amplification of the total 8 single spore cultures plus sequencing followed by a comparison of the DNA sequences with GenBank revealed the presence of a complex with 8 known species: Penicillium brevicompactum, Epicoccum sorghi, Rhizopus stolonifer, Alternari solani, Fusarium equiseti, Penicillium olsonii, Fusarium chlamydosporum and Fusarium incarnatum. This study has made several contributions to knowledge and current understanding of plant sciences. To our knowledge, this is the first report describing the presence of these fungi on marama bean seedling leaf tissue. This study has made a major contribution to mapping efforts as it identified regions of the genome that can be used in these studies. This study has also confirmed the marama bean chromosome number. Molecular markers linked to internode length or germination rate or any other trait that may be of agronomic importance had not been reported previously. The results from the work on grafting and plant growth regulators will prevent farmers from using high cost agricultural practices in cultivation of marama bean. Based on the findings of this study, the recommended next steps would be to explore Next Generation Sequence data with genome walking to identify genes in marama, particularly genes controlling flower formation, as well as establishing F1 populations for mapping studies.Item Evaluation of selected Namibian ethno-medicinal plants for anti-HIV properties(University of Namibia, 2015) Hedimbi, MariusNamibian ethno-medicinal plants have not yet been evaluated for their efficacy in inhibiting the activities of Human Immunodeficiency Virus (HIV) reverse transcriptase (RT) and their toxicological effects to mammalian cells have not yet been reported. Hence the aims of this study were: (1) to evaluate selected Namibian plants for their biological activities against RT; (2) to evaluate their toxicity to mammalian cells, and (3) to isolate and characterize the active compounds from selected plant extracts. Colorimetric assays were used to evaluate RT inhibition and cell viability.Item Microbial ecology of anaerobic carbon mineralization in Namibian shelf sediments(2007) Julies, Elsabe M.This PhD thesis is an essential component of the 'Namibia Gas' (NAMIBGAS) project, which aims to improve the understanding of the rate and fluxes of hydrogen sulfide and methane to the sediment surface and into the water column on the Namibian shelf, which is one of the most productive upwelling systems on Earth. Organic matter degradation in the sediment drives hydrogen sulfide production, the maintenance of anoxia, and methane formation. Therefore, the primary aims of the thesis are to study carbon transformation processes within the sediment, determine the reactivity of organic carbon in the sediment and, investigate the control of microbial community structure and activity by the amount and accessibility of carbon sources. The emphasis is on linking microbial community structure (identity) to their function (activity) to provide new insights into the microbial ecology that controls carbon turnover in these upwelling sediments. By studying the stepwise degradation of organic carbon a complete process overview of organic carbon mineralization could be obtained.Firstly, the diagenetic transformation of dissolved organic carbon (DOC), in particular dissolved carbohydrates, was studied by using both biogeochemical methods and molecular techniques. The bulk sediment composition, pore water chemistry, polysaccharide hydrolysis rates, 35S-sulfate reduction rates, and the abundance of active bacteria involved in the initial and terminal processes of organic carbon degradation within the top 15 cm of the sediment from two sampling stations were determined (Chapter 2). Secondly, the diversity of bacteria from the same two sampling stations within the top 12 cm of the sediment, using the 16S rRNA library approach were investigated (Chapter 3). The central question was whether shifts of the community structure of bacteria involved in the major carbon transformation steps of hydrolysis, fermentation, and terminal oxidation are reflected in changes of biogeochemical rates. Finally, the effect of a sudden high input of DOC into the sediment in the form of high molecular weight substances (the polysaccharide laminarin) and low molecular weight substances (lactate and acetate) on the metabolic activity and community structure of bacteria involved in initial hydrolytic and fermentation steps and terminal oxidation was investigated during two separate experiments (Chapter 4). Substrate addition simulates the high input of organic matter into the sediment after a phytoplankton bloom, following upwelling events.Item The reactions of ozone with compounds relevant to drinking-water processing: Phenol and its derivatives(University of Namibia, 2002) Mvula, Eino N.Kinetic and mechanistic investigations on the reactions of ozone with phenol and some of its derivatives (hydroquinone, catechol, phloroglucinol, pentachlorophenol, pentabromophenol, 1,2-dimethoxybenzene, 1,4-dimethoxybenzene and 1,3,5-trimethoxybenzene) have been carried out. These compounds were chosen, as they can serve as models for compounds which are abundant in surface waters and wastewater. The scope of the investigation was widened by including dihydrogen sulfide, often a contaminant of ground waters in arid areas, and its organic analogues. By employing various analytical techniques, such as high performance liquid chromatography (HPLC); ion chromatography (IC); nuclear magnetic resonance spectroscopy (NMR); gas chromatography (GC) and gas chromatography coupled to mass spectroscopy (GC-MS), it was possible to identify and quantify reaction products. The stopped-flow technique was employed to measure rate constants of ozone reactions. When strong substrate absorptions at 240–280 nm prevented the use of this technique, rate constants were measured by competition kinetics. Pulse radiolysis was used for the study of OH-induced reactions. Methods were developed for the detection and quantification of reactive intermediates in ozone reactions such as singlet dioxygen, OH, O2 and hydroperoxides. A germanium diode detector was used for the quantification of O2(1g) formation. For the detection of OH formation, 2-methyl-2-propanol and DMSO were used and tetranitromethane was applied for the detection of O2. Based on the products in the reactions of ozone with phenol and its derivatives, various reaction pathways have been identified. Ozone reacts with phenol and its derivatives by ozone addition (Criegee mechanism), electron transfer and O-atom transfer reactions. The Criegee mechanism, practically the only reaction with olefins, often occurs to only a small extent here. Instead, OH (up to 26%), O2 and O2(1g) are major intermediates. The occurrence of the Criegee mechanism was confirmed by the yields of hydrogen peroxide and their corresponding carbonyl compounds. The Criegee mechanism is shown to be more pronounced in methoxybenzenes as compared to phenols. For example, in the reaction of ozone with 1,4-dimethoxybenzene, the hydrogen peroxide yield (56%) and that of methyl(2Z,4E)-4-methoxy-6-oxo-hexa-2,4-dienoate (52%), which are the products of the Criegee type reaction, are much higher than that of hydrogen peroxide (5.6%) in the reaction of ozone with hydroquinone. A possible reason for this is that, i.e. the zwitterion formed in the reaction of ozone with hydroquinone may undergo a deprotonation reaction [reaction (1)] which competes with the 1,3- dipolar cyloaddition [reaction (2)] whereas in the case of the zwitterion formed in the reaction of ozone with 1,4-dimethoxybenzene, the 1,3-dipolar cycloaddition may occur without hindrance.Item A taxonomic, ecological and nutritional study of Porphyra Capensis Kutzing population from the Namibian coast(University of Namibia, 2015) Kavishe, DevotaThe Namibian Porphyra capensis Kützing was studied as very little was known of its taxonomy, ecology, phylogeny or nutritional status. It was hypothesized that it was phlogenetically related to the South African P. capensis as they share common ancestry. DNA was extracted from thalli and partial sequences obtained from their 18SrDNA and ITS regions. GenBank sequences of interest were incorporated, alignments made and phylogenetic trees were generated using MEGA 5.1. The results showed the existence of a Namibian P. capensis clade, implying preliminarily that it is a subspecies. It was also hypothesized that the abundance (standing crop biomass and cover) of P.capensis along Lüderitz and Swakopmund shores would be similar because of similar environmental conditions created by the cold Benguela Current System. This was tested in tandem with another hypothesis that the frequeny of harvesting thalli from the field would not significantly affect annual yield as nutrients and propagules would constantly be replenished from the system. A best fit model describing the relationship between standing crop and percentage foliar cover of Namibian Porphyra was developed. Cover and standing crop biomass were not significantly different between Lüderitz and Swakopmund but standing crop biomass varied significantly with seasons (p=0.001). Harvesting frequency increased the mean annual yield (p=0.008). The regression power equation Y = with R2 = 0.649 was proposed as the best model for estimating biomass (Y, g) from cover (X, %) in the field while being cognizant of the relatively low coefficient of determination which resulted from its patchy distribution and multilayering of thalli. The hypothesis that the nutritional content of P. capensis was comparable to that of lettuce was tested and laboratory cultures on the Namibian P. capensis life cycle were also initiated. Nutritional analyses were carried out according to standard procedures and it was established that fibre and calcium were significantly higher in P. capensis (p=0.016 and p=0. 036 respectively) while fat was significantly higher in lettuce (p=0. 0026). Magnesium, phosphorus, crude protein, iron, carbon and β–carotene were not significantly different. The life cycle of P. capensis in laboratory cultures was completed. The study proposed a subspecies status to the Namibian P. capensis; assessed its nutritional content while opening up possibilities for its mariculture to conserve wild populations. The study contributes to the promotion of P. capensis as a ‘health food’. Further taxonomic exploration of local Porphyra populations was recommended and a prudent use of the abundance model encouraged.Item A study of the immunity to malaria among the San people in the Tsumeb area of Oshikoto and Kavango regions of Namibia(University of Namibia, 2014) Amoo, Chipo C.The San people are indigenous minorities in Namibia that are known for their hunting and gathering lifestyle. From personal observations, discussions, interviews, raw data from Tsintsabis clinic and results obtained from the research, one had to assume that the San people are immune to malaria. It is unclear how the disease does not affect them although they live in endemic areas. The objective of this study was to investigate how the San have survived without a major outbreak of malaria as well as the mechanism underlying this immunity. The sample consisted of two hundred participants that included the San and the other ethnic groups (“the other ethnic groups” in this study refers to tribes other than the San that lived in the same regions studied). Questionnaires were administered and focus group discussions were conducted to both groups in Oshikoto and Kavango Regions to determine their knowledge of malaria. The presence of malaria parasites and the structure characteristics of the red blood cells in the blood samples of the San and other ethnic groups were examined microscopically by using thin and thick blood smears. Full blood cell count was measured and the role of nutrition played in the immunity boosting against malaria was investigated. The results were analyzed through descriptive and inferential statistics that included t-test and for indicator variables for the San and the other ethnic groups at 5% level of significance. Chi-square and t-test were used to evaluate differences between the shapes of red blood cell and presence of malaria parasites in the blood of the San and the other ethnic groups. The results showed that the control groups had better knowledge of malaria (56%) compared to the San people who showed no knowledge of malaria. All San people (100%) took traditional medicinal herbs but not specifically against malaria disease compared to 12% in the other ethnic groups. The chi square test indicated that the shapes of the red blood cells of the San (80%), displayed spikes and 2% showed mixed shape on the surface of the erythrocytes compared to the other ethnic groups of which 20% showed spikes and 1% showed a mixed shape. The t-test showed significant differences in the mean numbers of RBC, Hb, MCV, WBC, MON, BAS, LIC between the San and other ethnic groups. A t-test of the haemoglobin indicated that LAIcCHBbI (p=0.003) was significantly higher than the other Hb variants. The mean of the WBC of the San was significantly higher than that of the ethnic groups which suggested that the San are more likely immune to the malaria parasite. There were phenotypic variations in the San red blood cells which most likely as a result of genetic influences. Food samples of the San analyzed showed the means of the following; 73.88% moisture; 0.83mg/100g Iron; 0.39mg/100g Zinc; 12.37mg/100g Vitamin C; 0.48mg/100g Antioxidants and 0.83mg/100g Flavonoids were all slightly higher than in the other ethnic groups of Sorghum biocolor and Pennisetum glaucum. Pennisetum glaucum had the highest content in Zinc 3.2mg/100g compared to that of the San food. A study on the presence of fungi on the food of the San people was carried out and the following species were identified R. stolonifer, S. cerevisiae, P. notatum, A. pezia, A. niger. The fungi were possibly producing secondary metabolites that boost immunity against bacterial and some protozoan infections like malaria parasites. The statistics above explain the immunity of the San, which have not been studied before and for which there is dearth of data in this regard. This knowledge could be useful in determining and developing interventions against transmission of malaria. Among others, the significant contribution to the field of malariology is that the San people have a distinct morphology in haemoglobin C whereby 63.1% of the San people showed the presence of HbC and 36.9% in the other ethnic groups. Spiculated RBC, HbC and nutritional elements helped build immunity against malaria parasites in the San people. It is recommended that IgG of the San can be used to test for acquired immunity and development of vaccine in animal studies and therefore can be used to form basis for antimalarial vaccines and drugs.