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

Diabetes mellitus is a growing threat to human health. Current pharmacological agents cause undesirable side-effects. Herbal remedies offer the potential for alternative treatment strategies that may prove more cost-effective and devoid of the undesirable side-effects. The purpose of this study was to evaluate the in vitro anti-diabetic activity of aqueous and methanol extracts of Sclerocarya birrea (A. Rich.) Hochst. (Anacardiaceae) and Ziziphus mucronata Willd. (Rhamnaceae), which are traditionally used for the treatment of diabetes mellitus in southern Africa. Polyphenolic contents of extracts were quantified using the aluminium trichloride and Folin-Ciocalteau methods. The capacity of individual extracts to scavenge both the 2,2’-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) and 2,2-diphenyl-1-picrylhydrazyl radicals was used as a measure of antioxidant activity. The inhibitory activities of the crude extracts of both plants on the enzymes, α-amylase and α-glucosidase, were determined using colorimetric assays. The effects of the crude extracts on cell viability was assessed in C2C12 myotubes, HepG2 hepatocarcinoma cells, 3T3-L1 adipocytes and RIN-m5F pancreatic β-islet cells, using the Sulforhodamine B assay. Fluorescence detection was used to investigate the effects of the crude extracts on glucose uptake in C2C12, HepG2 and 3T3-L1 cells. Insulin secretion was assessed in RIN-m5F cells, using ELISA. Crude extracts of both plants contained flavonoids and phenols, but flavonoid content was predominantly higher. All the extracts displayed antioxidant activity, with the methanol extract of S. birrea possessing the most potent free radical scavenging ability (IC50 = 2.16 μg/ml). Aqueous and methanol extracts of S. birrea displayed significantly (p < 0.05) greater inhibition of α-amylase, than the positive control, acarbose. Only the methanol extract of Z. mucronata inhibited α-amylase activity. Furthermore, crude extracts of both plants also displayed potent α-glucosidase inhibitory activity. Most of the crude extracts had low toxicity, where concentrations of 100 μg/ml of crude extract of the plants did not induce 50% cell death. iv Although no significant increase in insulin secretion from cultured RIN-m5F cells was noted, the crude extracts of both plants significantly (p < 0.05) increased glucose uptake in C2C12, HepG2 and 3T3-L1 cells, with efficacy significantly (p < 0.05) higher than the positive control, insulin. From the results, the plant extracts appear to exert their hypoglycaemic effects independently of insulin, via an extra-pancreatic mechanism, possibly involving interactions with the different receptors. An additive hypoglycaemic effect originates from the inhibition of both α-amylase and α-glucosidase. The findings of the present study provide evidence that S. birrea and Z. mucronata possess in vitro anti-diabetic activity. Further investigations are required to elucidate the mechanism(s) of action of the crude extracts using more targeted in vitro assays. / Dissertation (MSc)--University of Pretoria, 2013. / gm2014 / Pharmacology / unrestricted

MSc (Chemistry) / Department of Chemistry / Malaria, an infectious disease affecting both human beings and other animals, is transmitted by parasitic protozoans belonging to the Plasmodium genus. Malaria is commonly treated with drugs such as quinine, chloroquine, and artesunate. However, the incidence of treatment failure due to drug-drug interactions and parasite resistance is increasing. Therefore, the rich medicinal potential of plants found in nature in Africa is increasingly being explored. The traditional use of Lippia javanica, Sclerocarya birrea, Melia azedarach and Capparis tomentosa for the treatment of malaria is well-known, but the phytochemistry of these four plants is not fully known. Parts of these plants were extracted and column chromatography was used to fractionate the extracts. The antioxidant activities of the fractions were determined using free radical scavenging and reducing power assays, while the cytotoxic, antiplasmodial and antitrypanosomal activities were determined using cell toxicity assay, parasite lactate dehydrogenase (pLDH) and trypanosome assay. The methanol stem bark extract of Melia azedarach (Fraction 2) had the highest phenolic content (59.39 mg GAE/g), while the methanol leaf extract of Melia azedarach had the highest flavonoid content of 188.65 mg QE/g. In the reducing power tests and DPPH free radical scavenging activity, the methanol stem bark extract of Melia azedarach had the lowest IC50 value of 0.1074 μg/mL and an IC0.5 value of 0.5296 μg/mL, respectively. Furthermore, the methanol stem bark extract of Melia azedarach at a concentration of 50 μg/mL showed significant cytotoxicity against HeLa cells (-1.22±0.07 %). The methanol stem bark extract of Melia azedarach at the tested concentration (250 μg/mL) decreased the viability of Plasmodium falciparum to 36.38±11.96 % with an IC50 value of 6.5 μg/mL. Concerning the antitrypanosomal activity, the methanol stem bark extract of Melia azedarach affected the viability of the trypanosomes at the tested concentration (250 μg/mL), giving a viability of 14.05 ± 0.59 %, with an IC50 value of 0.4 μg/mL. The presence of epicatechin (29) and catechin (31) in this extract was confirmed using several spectroscopic techniques (IR, NMR, UPLC-MS and HRMS). / NRF

Thesis ( PhD. (Biotechnology )) --University of Limpopo, 2005 / The marula tree (Sclerocarya birrea subsp. caffra), an indigenous, multipurpose, drought tolerant tree of Africa harbors great economic potential. Acceptance of marula-derived products internationally will directly increase the demand for marula resource. Rapid multiplication of marula trees of superior quality forms the basis of sustainable export growth. In vitro propagation and genetic improvement offer the opportunity for accelerated multiplication of selected tree material as well as to dramatically increase production, quality and efficiencies. The objectives of the study were therefore to develop a protocol for in vitro multiplication of marula and to determine the feasibility of Agrobacterium-mediated transformation of the marula tree. Nodal sections with axillary bud (s) were cultured on Murashige and Skoog (MS) medium supplemented with 4.8μM BA and 2.4μM KN and 0.1% polyvinylpyrrolidone (PVP) to obtain on average 2.5 microshoots per responding explant. The proliferated microshoots were elongated on MS medium supplemented with 1.2μM BA and 1.0μM KN. Elongated microshoots were rooted in MS medium at half salts strength supplemented with 10μM IBA and 0.3% activated charcoal (AC). On average 82% of the shoots rooted. Survival of acclimatized plantlets was 90%. RAPD analysis confirmed intraclonal genetic stability between parent plants and their clones within the limits of the technique.Nodal sections cocultivated with Agrobacterium tumefaciens for 3 days on MS multiplication medium supplemented with 100μM acetosyringone resulted on average in transient expression of 52.5% of the explants with 1.6 blue stained zones per explant. Cocultivated explants on MS selection medium containing 300mgl-1 kanamycin resulted in 1.5% chimeric putative transgenic shoots. This is the first report on the micropropagation and genetic transformation of marula, Sclerocarya birrea subsp caffra. / South Africa’s National Research Foundation Institutional Research Development Program (NRF-IRDP)

MSc (Botany) / Department of Botany / See the attached abstract below

Thesis (Ph.D. (Microbiology)) -- University of Limpopo, 2022 / NRF and TIA

MaxEnt modelling uses only the known locations of a species to predict the overall distribution of a species. Large trees are important for the functioning of savanna ecosystems, bringing nutrients to the surface, providing shelter to animals and providing a number of ecological functions. Large trees have been identified as declining in density in many southern African reserves, making the conservation of large trees within reserves an issue in park management, such as in Kruger National Park (KNP) and Hluhluwe iMfolzi Parks (HiP). Two species of primary concern are Acacia nigrescens and Sclerocarya birrea, which have similar distributions in Southern Africa. Effective management of large trees requires understanding their distribution within reserves and any potential distribution changes. By determining the current locations of a species, and using GIS layers of environmental variables to predict the extent of habitats that could support the species, niche models can predict species distribution. Maximum Entropy techniques evaluate the probability of finding the species in raster squares, with values for environmental factors controlling distribution. For this study, the locations of A. nigrescens and S. birrea trees higher than 5 m were recorded in KNP and HiP, and were used in conjunction with MaxEnt to produce distribution probability maps for both species in each reserve. In HiP, the distribution map was compared with an independent existing data set to determine if the predicted distributions were accurate. The factors effecting their distribution were compared between HiP and KNP to determine why the species were found together in KNP but not in HiP. MaxEnt could predict the locations of the species within HiP, but predictions were better for A. nigrescens than S. birrea. In both Reserves, rainfall was the best predictor of tree location, along with elevation. The niche overlap was higher in KNP, where both species are well within their total species range, than in HiP where A. nigrescens was at the edge of its distribution. These variables that are limiting distribution at a reserve scale will have an influence on the overall distribution of the species. Niche models can be used to inform the establishment of botanical reserves or other management strategies that can help preserve large trees within reserves. / Thesis (M.Sc.)-University of KwaZulu-Natal, Westville, 2011.

Sclerocarya birrea (marula, Anacardiaceae) is a highly-valued indigenous tree in most parts of sub-Saharan Africa because of its medicinal and nutritional properties. The marula tree is adapted to the semi-arid conditions that characterise most parts of sub-Saharan Africa and renders them unsuitable for conventional crop agriculture. The unique nutritional properties of marula and its high tolerance to dry conditions provide opportunities for its development into a plantation crop. On the other hand, the demand for marula plant parts, mainly the bark and roots as medicinal remedies, poses a great threat to wild populations. In the long term, the growing demand of marula products in the food, pharmaceutical and cosmetic industries will not be sustainable from wild populations alone. Plant tissue culture technologies can be useful for in vitro manipulation and mass propagation of the plant in the process of domestication and conservation. The aims of the project were to determine the optimum conditions for seed germination, in vitro propagation and plant regeneration, and to evaluate the potential bioactivity of secondary metabolites from its renewable plant parts as an alternative option in the conservation of S. birrea. An ex vitro seed germination study indicated that after-ripening and cold stratification are critical factors. Cold stratification (5 °C) of marula nuts for 14 days improved germination (65%) as compared to non-stratified nuts (32%). Direct shoot organogenesis was achieved from leaf explants through the induction of nodular meristemoids on Murashige and Skoog (MS) (1962) medium and woody plant medium (WPM) supplemented with 6-benzyladenine (BA) in combination with naphthalene acetic acid (NAA), indole-3-butryric acid (IBA) and indole-3-acetic acid (IAA). Induction of nodular meristemoids from 86% of the leaf cultures was achieved on a MS medium with 4.0 ìM BA and 1.0 ìM NAA. High levels (78–100%) of induction were also achieved on WPM with different concentrations of BA (1.0–4.0 ìM) and IBA (1.0–4.0 ìM). The highest conversion of nodular meristemoids into shoots on MS initiation medium was only 22% for 4.0 ìM BA and 1.0 ìM NAA. This was improved to 62% when nodular clusters were cultured in MS liquid medium. Histological studies revealed high numbers of unipolar meristematic buds developing from globular nodules. These embryo-like structures have in the past been mistaken for true somatic embryos. The initiation of high numbers of nodular meristemoids per explant provides potential for automated large-scale clonal propagation in bioreactors, in vitro phytochemical production and the development of synthetic seed technology, similar to somatic embryogenesis. Plant regeneration through nodule culture has potential for application in mass micropropagation and plant breeding of S. birrea. Adventitious shoot and root induction are important phases in micropropagation. Plant growth regulators play an important role in these developmental processes, and the type and concentration used have major influences on the eventual organogenic pathway. Three auxins (IAA, IBA and NAA) and four aromatic cytokinins (6-benzyladenine, meta-topolin, meta-topolin riboside, and meta-methoxytopolin riboside) were evaluated for their potential to induce adventitious shoot and root formation in S. birrea shoots, hypocotyls and epicotyls. Among the evaluated cytokinins, the highest adventitious shoot induction (62%) was achieved on MS medium supplemented with meta-topolin (8.0 ìM). The lowest adventitious shoot induction (2.5%) was obtained on MS basal medium containing 2.0 ìM meta-methoxytopolin riboside. The highest adventitious shoot induction for hypocotyls was 55% on MS medium supplemented with 8.0 ìM meta-topolin. For the tested auxins, IBA induced adventitious rooting in 91% of shoots at a concentration of 4.0 ìM after 8 weeks in culture. However, the in vitro rooted plants only survived for two weeks when transferred ex vitro. A temperature of 25 °C and 16-h photoperiod were optimum for adventitious root induction. Stomatal density (number per mm2) on the abaxial leaf surfaces was higher for the 16-h photoperiod treatment (206.6 ± 15.28) compared to that for a 24-h photoperiod (134.6 ± 12.98). Normal mature stomata with kidney-shaped guard cells and an outer ledge over the stomatal pore were observed for in vitro plants growing under a 16-h photoperiod. Total phenolic content, proanthocyanidins, gallotannins, flavonoids, and antioxidant activities of S. birrea methanolic extracts were evaluated using in vitro bioassays. Methanolic extracts of the young stem bark and leaves contained high levels of these phytochemicals. Sclerocarya birrea young stem extracts contained the highest levels of total phenolics (14.15 ± 0.03 mg GAE g-1), flavonoids (1219.39 ± 16.62 ìg CE g-1) and gallotannins (246.12 ± 3.76 ìg GAE g-1). Sclerocarya birrea leaf extracts had the highest concentration of proanthocyanidins (1.25%). The EC50 values of the extracts in the DPPH free radical scavenging assay ranged from 5.028 to 6.921 ìg ml-1, compared to ascorbic acid (6.868 ìg ml-1). A dose-dependent linear curve was obtained for all extracts in the ferric-reducing power assay. All the extracts exhibited high antioxidant activity comparable to butylated hydroxytoluene based on the rate of â-carotene bleaching (89.6 to 93.9%). Sclerocarya birrea provides a source of secondary metabolites which have potent antioxidant properties and may be beneficial to the health of consumers. Sclerocarya birrea young stem and leaf ethanolic extracts exhibited high bioactivity (MIC < 1.0 mg ml-1) against both Gram-positive (Bacillus subtilis and Staphylococcus aureus) and Gram-negative (Escherichia coli and Klebsiella pneumoniae) bacteria. The highest activity (MIC = 0.098 mg ml-1 and total activity = 1609.1 ml g-1) was recorded for young stem extracts against B. subtilis. The highest activity (MIC = 1.56 mg ml-1 and MFC = 1.56 mg ml-1) in the antifungal assay against Candida albicans was observed for young stem ethanolic extracts. Sclerocarya birrea extracts had moderate acetylcholinesterase (AChE) inhibition activity. The dichloromethane (DCM) and methanol (MeOH) fractions exhibited dose-dependent acetylcholinesterase inhibitory activity. The highest AChE inhibitory activities were from leaves (DCM fraction, IC50 = 0.1053 mg ml-1) and young stems (MeOH fraction, IC50 = 0.478 mg ml-1). High inhibitory activity against cyclooxygenase (COX-1 and COX-2) enzymes was observed. All extracts and fractions showed high COX-1 enzyme inhibition (90.7-100%). Petroleum ether (PE) and dichloromethane fractions also exhibited high inhibition against COX-2 enzyme (77.7-92.6%). The pharmacological activities observed suggest that S. birrea renewable plant parts (leaves and young stems) provide a substantial source of medicinal secondary metabolites. Based on these results, plant part substitution can be a practical conservation strategy for this species. / Thesis (Ph.D.)-University of KwaZulu-Natal, Pietermaritzburg, 2009.

The current research was done to evaluate the nutritive values and the ruminal degradation of dry matter (DM) and crude protein (CP) from three non-conventional oil cakes, viz: amarula (Sclerocarya birrea) (AOC), macadamia (Integrifolia) (MOC) and baobab (Adansonia digitate L.) (BOC). The oil cakes were collected from biodiesel producers in Limpopo Province, transported to the ARC-Animal Production campus, where proximate and ruminal nutrient degradation analysis were conducted. Triplicates samples from each oil cake were analyzed for the nutritive values, mineral and amino acids contents. Three rumen cannulated mid-lactating (days in milk; DIM: 180±5) Holstein cows weighing 667±43 kg body weight were allocated to determine the in situ ruminal dry matter (DM) and crude protein (CP) degradation. The cows were offered a totally mixed ration (TMR) (60 concentrate: 40 forage ratio) that was compounded according to their daily nutrient requirements, and were milking was done twice per day at 12 hrs intervals. The three oil cake samples were ground using a 2-mm screen after which sub-samples (6.5 g) were put in 10 x 20 cm; 50 μm pore size polyester bags to achieve 15 mg/cm² (ratio of the sample size to surface area). The bags were then fistulated in each cow’s rumen in triplicate for a period of 2, 4, 8, 16, 24, or 48 hrs. After being incubated, the bags were removed from the rumen and washed with cold (4°C) water in 20-L buckets. Following immersing in cold water, the bags were machine washed until clean water was obtained. The bags were then dried at 60 °C in an oven for 48 hrs. The dried bags were individually weighed, and the content of each bag were removed and stored into glass vial until analysis. The remaining two duplicate sets of each sample were rinsed using cold water in order to determine solubility at 0 hrs. The AOC had higher (P<0.05) ether extract (EE) and CP content than both BOC and MOC. Macadamia oilcake (MOC) and BOC had higher (P<0.05) fractions of fibre (NDF, ADF and ADL) compared to the AOC. The AOC had greater (P<0.05) content of essential amino acids than in the BOC and MOC. Additionally, AOC had a high (P<0.05) phosphorus, but low calcium and potassium concentration. While AOC had high effective degradability of DM, it also had high water soluble as well as DM and CP rapidly degradable fractions. Effective degradation of CP was higher in AOC and BOC than in MOC. However, BOC had a high insoluble but degradable fraction of CP. Further work to determine the toxicology of these non-conventional oil cakes and animal feeding experiments is needed / Agriculture and  Animal Health / MSc. Agriculture

MSCAGR (Plant Production) / Department of Plant Production / See the attached abstract below