Doctoral Degrees (Physiology)

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Alzheimer’s disease dementia risk in post-traumatic stress disorder: identification of common underlying mechanisms using rat models.
(2021) Faborode, Oluwaseun Samuel.; Mabandla, Musa Vuyisile.
Post-traumatic stress disorder (PTSD) is a neuropsychiatric disorder that can develop from exposure to a trauma. Studies have shown that people who have PTSD are susceptible to developing dementia, mostly Alzheimer’s disease (AD), suggesting common underlying risk factors in the comorbidity. Although several molecular pathways have been implicated in AD and PTSD, including oxidative stress, cellular apoptosis, synaptic dysfunction and stress dysregulation, the underlying neurobiological mechanisms linking AD and PTSD are less understood. This study, therefore, investigated the effect of trauma-like exposure in an amyloid-beta (Aβ) rat model of AD. Seventy-two adult male Sprague-Dawley rats were used throughout the study. The animals were randomly divided into four groups where they received either footshocks or Aβ(1-42) injection or were exposed to footshocks and Aβ(1-42) injection or remained naive. Following inductions, the animals were tested for cognitive, locomotor and anxiety-like behaviours. Thereafter, brain samples were collected for further neurochemical analyses. Our results show that footshocks increased anxiety-like behaviour and impaired fear memory extinction in Aβ(1-42) lesioned rats. A combination of footshocks and Aβ(1-42) also reduced the expression of nuclear factor erythroid 2-related factor 2 (Nrf2), NAD (P) H: quinone oxidoreductase 1 (NQO1), heme oxygenase-1 (HO-1), and increased the expression of Kelch-like ECH-associated protein 1 (Keap1) in the amygdala and hippocampus. Prior exposure to footshocks before Aβ(1-42) lesion caused a decrease in the number of crossing in the target quadrant of the Morris water maze test and reduced percentage alternation in the Y-maze test, indicating memory deficits. There was an interactive effect of footshocks and Aβ(1-42) lesion on the downregulation of BIN1 and the upregulation of NR2B in the hippocampus. There was also an interactive effect of footshocks and Aβ(1-42) lesion on the upregulation of FKBP5 in the hippocampus, amygdala, and PFC. Our finding suggests that footshock stress can exacerbate AD-like pathology via dysregulated redox balance, BIN1 downregulation, FKBP5 and NR2B upregulation, and increased apoptosis in the brain of Aβ(1-42)-lesioned rats. These molecular changes were associated with increased anxiety, impaired fear extinction and memory deficits. These findings, therefore, suggest common molecular mechanisms in PTSD and AD. Isifo sengcindezi eba semva kwesehlakalo esibi i-post-traumatic stress disorder (i-PTSD) yisifo sokusebenzelana kwezinzwa nensebenzamqondo esidala wukwehlelwa yisehlakalo esibi. Ucwaningo selukhombise ukuthi abantu abane-PTSD basengcupheni yokuba nesifo sokukhohlwa, ikakhulukazi i- Alzheimer’s (i-AD), okuchaza ukuthi izimo eziyingcuphe enkulu ekubeni nezifo eziyizimbelambela. Nakuba izindlelamigudu eziningi zamamolekhyuli zisoleka kwi-AD ne-PTSD, okufaka nengcindezi ye-oxidative, i-cellular apoptosis, i-synaptic dysfunction kanye nokungalawuleki kwengcindezi, izindlelamigudu zempiliswanozinzwa nomzimba ezixhumanisa i-AD ne-PTSD akuqondwa ngokuphelele. Ngakho-ke lolu cwaningo, luphenye umthelela wokubekeka endaweni ecisho ibe yisehlakalo esinzima kwimodeli yamagundane i-amyloid-beta (Aβ) ye-rat model of AD. Amagundane amadala esilisa angamashumi ayisikhombisa nambili asetshenziswa kulolu cwaningo. Izilwane zehlukaniswa ngokungahleliwe zaba amaqembu amane lapho zanikwa khona amafootshocks noma umjovo we-Aβ(1-42) noma ayengathola i-footshocks nomjovo we-Aβ(1-42) noma ahlale engazi. Ukulandela ukwethulwa, izilwane zahlolelwa ukuziphatha kwazo ngokomcabango, ukuhamba nokuba nexhala. Emva kwalokho, amasampula obuchopho aqoqwa ukuze aphinde acutshungulelwe amanyurokhemikhali. Imiphumela yethu iveze ukuthi ama-footshocks enyuse ukuziphatha sakuba nexhala kanye nokuqeda ukukhumbula ukwesaba okugwegwile kuma-Aβ(1-42) onikwe amalesioned rats. Ingxubevange yefootshocks ne-Aβ(1-42) iphinde yehlisa ukuvela kwe-nuclear factor erythroid 2-related factor 2 (Nrf2), i-NAD (P) H: i-quinone oxidoreductase 1 (NQO1), i-heme oxygenase-1 (HO-1), kanye nokuvela okusezingeni eliphezulu kwe-Kelch-like ECH-associated protein 1 (Keap1) kwi-amygdala ne-hippocampus. Ukusondelana nama-footshocks kwangaphambi kwe-Aβ(1-42) kwdala ukwehla esibalweni sokuhlanganayo kwi-target quadrant yokuhlolwa kwe-Morris water maze test nenguqunguquko yephesenti kwi-Y-maze test, okuveza ukushoda kokukhumbula. Kwaba nomthelela onokungenelana wama-footshocks kanye ne-Aβ(1-42) lesion ekulawulenikwehlisa i-BIN1 nasekulawulenikukhuphula i-NR2B kwi-hippocampus. Kwaphinde kwaba nomthelela ongenelanayo we-footshocks ne- Aβ(1-42) lesion ekulawulenikukhuphula i- FKBP5 kwi-hippocampus, i-amygdala, ne- PFC. Esikutholile kuphakamise ukuthi ingcindezi ye-footshock ingabhebhezela isakhiwosifo esifuze i-AD nge-dysregulated redox balance, i-BIN1 downregulation, i-FKBP5 ne-NR2B upreguation, i-apoptosis ephezulu ebuchosheni be-Aβ(1-42)-ekuma-lesioned rats. Lezi zinguquko kumamolekhyuli zazihlotshaniswe nokwenyuka kwexhala, inqedakwesaba egwegwile nokulahlekelwa wukukhumbula. Ngakho-ke lokhu okutholakele, kuphakamisa izindlela ezejwayelekile zomumo wamamolekhyuli kwi-PTSD ne-AD.
An investigation on the effects of a rhenium (V) compound with uracil-derived ligands on markers associated with hepatic, cardiovascular and renal complications in diet-induced prediabetic rats.
(2023) Siboto, Angezwa.; Khathi, Andile.; Ngubane, Phikelelani Siphosethu.; Sibiya, Ntethelelo Hopewell.
Prediabetes is a metabolic disorder that often precedes the onset of type 2 diabetes mellitus. This development of this asymptomatic condition is associated with chronic consumption of high calorie diets and sedentary lifestyles. Prediabetes results in decreased insulin sensitivity in the peripheral tissues resulting in elevated blood glucose levels that are not high enough for a diagnosis of type 2 diabetes. This moderate hyperglycaemia has been shown to lead to trigger complications such as non-alcoholic fatty liver disease, renal dysfunction and cardiovascular disease which are generally only diagnosed during type 2 diabetes. The current management strategy for prediabetes consists of a combination of pharmacological and lifestyle intervention. The pharmacological agents such as metformin while lifestyle intervention involves dietary modification to lower calorie diets. Studies show that prediabetic patients tend to be more dependent pharmacological intervention and struggle with changing diets thus lowering the efficacy of drugs such as metformin. This often leads to the eventual development of prediabetes. Therefore, there is a need for new pharmacological agents that can remain effective in both the presence and absence of dietary intervention. In our laboratory we have synthesised a novel rhenium (v) compound with uracil-derived ligands that has shown promising biological activities that include anti-hyperglycaemic effects in diet-induced prediabetic rats. This compound was shown to improve insulin sensitivity in peripheral tissues in prediabetic rats. To advance from this knowledge, this study sought to investigate the effects of the rhenium (V) compound with uracil-derived ligands on markers associated with hepatic, cardiovascular and renal complications in diet induced prediabetic rats model.
The case for critical thought: an investigation into contemporary determinist knowledge, its social effects, and the alternative offered by a 'mode 2' approach to teaching, learning and research.
(2002) Skinner, Jane.
This thesis is centrally concerned with the current nee-liberal world order and its effects upon society. It is concerned to expose the contradictions and weaknesses within the knowledge systems that underpin our political reality. It considers economics as the determining discourse of neo-liberal politics, analytic biology as its determining discourse of individual persons, and analytic and neo-pragmatist philosophy as its leading systems of thought. In each case it finds a linear rationalism compatible with the determinist materialism of noo-Darwinism, and indeed explicitly invoking Darwin. This seems to vindicate Manuel Castells's fmding of this 'Knowledge Society' as driven by 'an abstract, universal instrumentalism'. The thought systems of this economic liberalism have seen politics subsumed within economics, de-humanising most of the institutions of the earlier Liberal tradition, to the detriment of both freedom and democracy. But it disputes Castells's assumption that this is a necessary reality and finds in neo-liberal education the exception to this dehumanising trend. Revitalised as 'Mode 2' knowledge production, this form of teaching, learning and research is found to be ideally suited to challenge the underpinnings of the very social order which initially produced it. The thesis as a whole is designed to employ Mode 2 methods in order to support this contention. Using this approach it seeks to demonstrate that in place of neo-Darwinism the ideas of the South African natural scientist Eugene Marais, concerning the significance of conscious thought itself within evolution, can provide a more convincing epistemoloy than the behaviourism and materialism of analytic biology. It finds John Maynard Keynes's acceptance of economics as a moral and not a natural science, more logically convincing and more inherently useful for social reconstruction than the current mathematicisation of economic theory. Prevalent philosophical approaches appear to serve only to reinforce the systems of thought already found (and found wanting) in politics, biology and economics. But again these philosophies are shown to be vulnerable to a Mode 2 critique, particularly employing the ontological understanding of the contemporary pragmatist philosopher Joseph Margolis, whose strong version of relativism allows for both bivalent and multivalent truth values more appropriate to understanding the complex realities of ethical and democratic societies.
Characterising epigenetic alterations following cocaine consumption.
(2017) Ajonijebu, Duyilemi Chris.; Mabandla, Musa Vuyisile.
Complementary data from clinical and animal research have converged on the hypothesis that persistent use of psychostimulant drugs such as cocaine may not only involve pathological alterations in neural processes that subserve reward–related learning, but also complex interactions between genes and the environment through epigenetic modifications. Fosb and Crem (cAMP response element modulator) are among the central trans-factors suspected to mediate these long–term neurobiological changes due to their potential roles in drug reward. However, the critical question that concerns inheritance of epigenetic marks associated with parental cocaine experience in social settings, offspring vulnerability and modifying maternal–foetal environment by fostering, remains poorly understood. The present study therefore aimed to investigate possible associations between cocaine–induced behavioural changes in social contexts and DNA methylation patterns of inducible transcription factors in the prefrontal cortex (PFC) and hippocampus (HPC) of the exposed parent mice. We further examined whether the induced epigenetic changes were inheritable and then determined the impact of early postnatal (PN) fostering on associated neurobiological changes in the offspring of drug-exposed parents. In doing so, we were able to investigate the interaction between epigenetic and environmental factors in relation to drug consumption. Behavioural response of C57BL/6 mice to cocaine treatments were examined using conditioned place preference (CPP) and IntelliCage (IC) phenotyping techniques. In the CPP experiment, male mice received 6 cocaine injections (10mg/kg, i.p.) on alternate days followed by 6 days of extinction learning. A subthreshold dose of cocaine (5mg/kg, i.p) was later injected to reinstate CPP behaviour. In the IC, female mice were group–housed and initially had free access to drugs (300mg/L cocaine and 12% v/v ethanol in their drinking bottles) and water for 30 days to investigate consumption preference. Subsequently, withdrawal effect and alternate nose-poke learning tasks (ANT) were examined in the following 28 days with concurrent access to cocaine and water. In both experiments, locomotor activity and novelty exploration/recognition memory of mice were examined post cocaine treatments. DNA methylation status of Crem and Fosb gene promoters, within the HPC and PFC, were also assessed using quantitative real–time polymerase chain reaction. Thereafter, cocaine exposed or unexposed male and female mice were matched for mating to produce offspring with lineal phenotypes. At birth, some of the offspring were cross-fostered to further examine the impact of PN fostering on drug-induced epigenetic changes. Locomotion, memory competence and DNA methylation status were also evaluated in the offspring similar to the parent mice. In male mice, cocaine treatment resulted in significant changes in CPP during conditioning. After extinction learning, the subthreshold dose of cocaine did not reinstate the conditioned behaviour. The treatment increased locomotor activity in the open field but decreased novelty exploration in the object recognition task. These changes were characterized by significant hypomethylation in Crem and Fosb gene promoters only in the PFC. During the first 30 days of free access to cocaine, ethanol or water in the IC, the female mice spent significantly more time licking and consuming cocaine than ethanol, whereas consumption of either drugs was significantly less than water. Overall, the mice exhibited motivational deficits as manifested by their inability to learn the ANT. Our data also showed that prolonged access to cocaine in the IC decreased locomotor activity while recognition memory remained intact in the cocaine–experienced mice compared to their controls. These changes were accompanied by hypomethylation or hypermethylation in the promoters of Fosb and Crem genes in the PFC and HPC of the cocaine–experienced mice, respectively. In the offspring, memory performance and locomotor activity were not affected by parental cocaine exposure, except that recognition memory was impaired by early PN fostering in offspring lineally inclined to either paternal and/or combined parental cocaine experience. Crem was hypomethylated only in the PFC of offspring of cocaine–exposed parent mice, while fostering the offspring reversed the expression. Significant change in Fosb methylation was only observed in the HPC of fostered offspring. Together, these findings suggest differential responses of the substrate brain regions to the converging environmental stimuli and dynamic regulation of induced neurobiological changes via DNA methylation. Overall, the data also provide some evidence that cocaine–induced epigenetic marks can be inherited by the non-drug exposed offspring while early PN fostering may enhance molecular switching that may render the individual vulnerable to drug consumption. Key words: Cocaine; social environment; IntelliCage, conditioned place preference; Crem; Fosb; DNA methylation; hippocampus; prefrontal cortex; epigenetic inheritance; postnatal fostering.
Chemical investigation of isihlambezo or traditional pregnancy-related medicines.
(2004) Brookes, Kathleen Bridget.; Dutton, Michael Francis.
This study was undertaken to redress the scant knowledge regarding the chemistry and mode of action of pregnancy-related traditional medicines, or isihlambezo (Zulu), which are used by 60 to 80% of women in South Africa. The three selected plants are among the six most frequently cited species from the approximately 90 used by traditional healers. The purpose of the study was to identify components which could cause uterine contractions, those with nutritional value for the foetus and mother, and those with any toxic effects. Plant root extracts were purified via silica gel column chromatography and bioassays were carried out on the fractions, using isolated rat uterine tissue. Purified compounds were identified via spectral techniques, and some were characterised by comparison to authentic standards using HPLC, and others by matching their GC-MS spectra to library standards. Thirty-eight compounds were identified in total, the majority of these being novel to the species concerned. Those isolated from Combretum kraussii were 1 sitosterol, 2 combretastatin, 3 3',4-tri-O-methylellagic acid, 4 combretastatin B-1, 5 combretastatin A-1, 6 3,3'-di-O-ellagic acid lactone, 7a ellagic acid lactone, 7b ellagic acid, 8 and 9 a mixture of combretastatin B-1 and A-1 glucosides, 10 and 11 partly characterised glucosides of ellagic acid. Those isolated from Gunnera perpensa were 12 3',4-tri-methylellagic acid, 13 ellagic acid lactone, 14 1,1'-biphenyl-4,4'-diacetic acid, 15 p-hydroxybenzaldehyde, 16 Z-methyl lespedezate, 17 and 18 partly characterized higher glucosides of Z-methyllespedezate. Those isolated rom Rhoicissus tridentata were 19 (-)-epigallocatechin, 20 (+)-gallocatechin, 21 procyanidin B3, 22 procyanidin B4, 23 (+)-catechin hydrate, 24 (+)-mollisacacidin, 25 (+)-epicatechin, 26 fisetinidol-(4a-8) catechin, 27 (-)-fisetinidol, 28 fisetinidol-(4b-8)catechin, 29 gallic acid, 30 epicatechin-3-0-gallate, 31 partly characterized hydrogel of glucose, 32 sitosterol, 33 sitosterolin, 34 y-sitosterol, 35 oleanolic acid, 36 lupen-3-one, 37 20-epi-y-taraxastananol and 38 triacontanol. The compounds with the greatest in vitro uteroactivity were predominantly proanthocyanidins or phenolic glucosides. It is proposed that effects of phenolic glucosides could be due to the interaction of the sugar moiety as well as the phenolic moiety with the receptor site in muscle tissue. The corresponding phenolic aglycones isolated were only moderately uterotonic, or unreactive by comparison. Non-polar compounds such as sitosterol and sitosterolin showed minimal enhancement of the uterine response at low concentrations, and inhibition at higher concentrations. Aqueous root extracts of the plants were all found to be non-toxic according to cell-viability tests using monkey vero cells and human fibroblasts. Extracts are therefore considered safe for human consumption, although it is recommended that Rhoicissus tridentata be used with caution because it showed the lowest cell viability of the three species, and uterine hyperstimulation has been attributed to this species, as well as CNS depression and respiratory arrest. Ions which could be nutritionally beneficial in pregnancy, calcium, iron, and phospate, were present in low in aqueous extracts. Levels of calcium and potassium ions were considered to be too low to directly stimulate uterine muscle. Proanthocyanidins, combretastatins, ellagic acid derivatives and phytosterols, with health-promoting properties, were also identified.
The cytotoxic effects, anti-iflammatory, antioxidant, antibacterial, and antidiabetic properties of eight selected South African plants for medicinal purposes.
(2020) Nkala, Bongani Alphouse.; Mbongwa, Hlengiwe Prosperity.; Qwebani-Ogunleye, Tozama.
People from the Southern African region have been using the fauna and flora of the region in their homes for millennia to treat all sorts of ailments and complaints with great success. This knowledge transfer was done through ’apprenticeships’ and oral communication. Certain communities consider medicinal plants to be safer than drugs and that they can treat more than one ailment. This study investigated cytotoxic effect, antimicrobial, anti-inflammatory, antioxidant, and antidiabetic properties of eight selected South African plants for medicinal purposes. Plant species were collected from the Walter Sisulu National Botanical-Gardens and were extracted with 90% methanol (1 g/10 ml) and concentrate to 10 mg/ml. Antimicrobial activities were determined by the microplate dilution method to establish the ability of the plant extracts to inhibit or kill pathogenic organisms with minimal inhibitory concentrations and minimum bactericidal concentration. Cytotoxicity effects were determined with Alamar blue and crystal violet cell viability assays, against C2C12 and RAW 264.7 cells. Anti-inflammatory effects were identified with stimulated lipopolysaccharide RAW 264.7 cells, and nitric oxide inhibition was measured with Griess reagent assay. The estimation of preliminary phytochemical, antioxidant (DPPH and ABTS radical scavenging), and alpha-amylase inhibition were determined with standard methods. The plant extracts inhibition and bactericidal effects were observed against all bacteria, namely: Lippia javanica (0.25±0.00 to 1.13±0.29 mg/ml); Ziziphus mucronata leaf (0.44±0.00 to 1.00±0.00 mg/ml); Erythrina lysistemon (0.44±0.00 to 1.08±0.00 mg/ml) and Schkuhria pinnata (0.5±0.00 to 1.34±0.00 mg/ml). All plant extracts exhibited flavonoids, phenols, terpenoids, and coumarins. The antioxidant inhibition was observed above 80% for Schkuhria pinnata, Lippia javanica, Clerodendrum myricoides, and Erythrina lysistemon. Also, these plant species exhibited an alpha-amylase inhibitory effect of 80%. The IC50 values were > 1000 μg/ml. All plant extracts demonstrated some degree of an antiinflammatory effect. However, Clerondendrum myricoides (35% - 89%), Lippia javanica (26% - 77%), Erythrina lysistemon (23% - 76%), Schkuhria pinnata (27% - 65%), and Vernonia oligocephala (16% - 58%) with IC50 value >1000 μg/ml, exhibited a marked antiinflammatory effect. Therefore, the presence of phenolic, flavonoids, anti-inflammatory, antioxidants, and α-amylase properties are potential solutions towards the management of diabetes and other chronic inflammatory diseases. Keywords: medicinal plants, antimicrobial, cytotoxicity, anti-inflammatory, antidiabetic, antioxidant.
Developmental methylmercury toxicity in a 6- hydroxydopamine Parkinsonian rat model: evaluating Searsia chirindensis as a potential neuroprotectant.
(2017) Moosa, Zulfiah Mohamed.; Mabandla, Musa Vuyisile.
Methylmercury (MeHg) pollution in South Africa has escalated due to increased demand from industrial sources such as coal-fired power stations. This had led to a growing interest in the effects of this metal toxin on human health. Prenatal MeHg exposure has been suggested to be a silent neurotoxicant, which may display its effects when triggered by a further neurotoxic insult. MeHg exposure during the perinatal period leads to neurodevelopmental deficits resulting in motor and cognitive dysfunction. This suggests that developmental MeHg exposure may predispose to the development of neurodegenerative diseases such as Parkinson’s disease (PD). In this study, we investigate the effects of prenatal MeHg exposure at adolescence and furthermore when subjected to an additional neurotoxic insult in a parkinsonian rat model. Behavioural tests were conducted to assess motor deficits with neurochemical assessment of trace element levels, total antioxidant capacity, dopamine and cytokine concentrations as well as gene expression profiling. We also investigated a novel plant extract Searsia chirindensis (SC) as a potential neuroprotectant by alleviating neurotoxicity. Overall the results of our study show that prenatal MeHg exposure disrupts trace element homeostasis at adolescence asymptomatically however, these imbalances are exaggerated following a further neurotoxic insult leading to motor deficits. Treatment with SC reduced motor deficits in MeHg-exposed offspring as reflected by higher dopamine levels. Contrastingly, treatment in the absence of MeHg exacerbated motor deficits with higher copper levels and upregulation of antioxidant genes fth1 and nqo1 in response to the neurotoxic effect. Therefore the overall total antioxidant capacity was not affected by SC. We also investigated the effect of SC on normal body parameters to assess for toxicity. Our findings showed that SC did not affect either liver or renal function and therefore does not affect the homeostasis of other body systems. Therefore conclusively our study showed that developmental MeHg exposure results in altered trace element homeostasis which may predispose to the development of neurodegenerative diseases such as Parkinson’s. We also showed that SC stem-bark extract reduced motor deficits caused by 6-hydroxydopamine in MeHg-exposed offspring but exacerbated neurotoxicity in its absence. SC also did not have any adverse effect on the homeostasis of other body systems. Overall, this suggests that SC has potential as a neuroprotectant however further studies must be conducted to fully elucidate the mechanisms involved in its effect.
The effect of isolated and nanoencapsulated flavonoids from Eriocephalus africanus on apoptotic factors and microRNA expression in cancer.
(2020) Magura, Judie.; Mackraj, Irene.; Moodley, Roshila.
Cancer continues to be a major health burden worldwide, with millions of new cases being diagnosed each year. Among women breast cancer remains a leading cause of cancer-related morbidity and mortality globally, despite the significant advances in detection and individualised treatments. The ideal non-surgical approach for the treatment of breast cancer would be anticancer therapeutics that are delivered directly to the tumour site for complete elimination of cancerous cells without being toxic to surrounding healthy cells. However, current chemotherapeutics encounter numerous challenges due to adverse side effects and progressive drug resistance albeit effective. In light of this, identifying new effective therapies with minimal toxic and chemosensitizing effects as well as target specificity is crucial in combating cancer. Emerging evidence has supported the use of plant-derived chemicals as novel alternative treatment options, owing to their minimal side effects and toxicity. Plant-derived polyphenols have gained considerable research interest due to their ability to inhibit proliferation, initiate apoptosis and arrest the cell cycle of cancerous cells by modulating related pathways. Furthermore, incorporation of active plant-derived polyphenols into novel technologies such as nanosystems, offers more optimal therapies through improved bioavailability and target specificity. In this regard, this study demonstrates, for the first time, the potential of phytochemicals isolated from the methanolic extract of the medicinal plant, Eriocephalus africanus, as an alternative therapeutic strategy in breast cancer treatment using ER-positive human adenocarcinoma (MCF-7) cell lines. Spectroscopic techniques including nuclear magnetic resonance spectroscopy (NMR), infrared spectroscopy (IR) and mass spectrometry (MS) were used to identify the isolated compounds as hesperidin (flavanone), luteolin (flavone) and apigenin (flavone). Preliminary anticancer screening using the 3-(4,5dimethylthiazolyl)-2,5-diphenyl-tetrazolium bromide (MTT) assay revealed hesperidin and luteolin to be potent against MCF-7. Dysregulated cellular apoptotic death is a hallmark of cancer and chemotherapy resistance; thus, the development of anticancer drugs targeting apoptosis is a widely used, effective anticancer treatment strategy. In this study, the efficacy of hesperidin and luteolin in targeting the apoptotic pathway was evaluated. Treatment of breast cancer cells with hesperidin and luteolin resulted in the downregulated expression of key anti-apoptotic Bcl-2; upregulated expression of pro-apoptotic Bax and caspases -8, -9 and -3. In addition, hesperidin and luteolin demonstrated the ability to effect epigenetic control through altering the expression of apoptotic microRNAs (-16, -21 and -34a). Moreover, treatment with hesperidin and luteolin resulted in significant accumulation of MCF-7 apoptotic cells into the G0/G1 and sub-G1 cell cycle phases, respectively. Encapsulation of hesperidin into nanoemulsions improved the cytotoxic and apoptotic effects in MCF-7 without being cytotoxic to non-cancerous human cell lines (HEK 293), halted the progression of the MCF-7 cells in the G2/M phase, and exhibited potential therapeutic activity through inhibiting the expression of oncomirs miR-21 and -155 overexpressed in breast cancer. Encapsulation of luteolin into solid nanoparticles generated from cleaved stearylamine exhibited non-selective cytotoxicity and decreased cell viability (< 10%) in both MCF-7 and HEK 293 cells, thus no further investigations were conducted using luteolin-loaded solid nanoparticles. Collectively, findings from this study provide new evidence on the effects of flavonoids isolated from E. africanus on apoptotic and epigenetic control in breast cancer, increasing our knowledge of the molecular basis of their anticancer activity.
The effect of prenatal Mycobacterium tuberculosis infection on offspring neurodevelopment and autistic-like behaviours in a valproic acid mouse model of autism.
(2021) Manjeese, Wadzanai.; Mpofana, Thabisile.; Mvubu, Nontobeko Eunice.; Steyn, Adrie J.C.
Autism spectrum disorder (ASD) is a neurodevelopmental disorder characterized by restricted repetitive patterns, communication challenges and lack of social skills. ASD has no distinct biomarkers, with symptoms overlapping with related developmental disorders like Schizophrenia. Maternal immune activation (MIA) is when the maternal immune system is invaded by a pathogen causing an immune response that interferes with the normal fetal brain development process. Mycobacterium tuberculosis (Mtb) infections are common during pregnancy and are known to affect fetal health, often causing spontaneous abortions and low birth weights. Valproic acid (VPA) is an anticonvulsant and mood stabilizer associated with ASD when administered during pregnancy. Gestational VPA exposure of mice on Embryonic day 12.5 (E12.5) induces ASD-traits in offspring, as such, this study employed VPA as a positive control. This study investigated the effects of prenatal exposure to Mycobacterium tuberculosis (Mtb) (singularly and in combination with VPA) on developmental delays and offspring behaviour. Pregnant mice were divided into saline, VPA, Mtb, and VPA+Mtb; treatments were administered on E12.5. Developmental milestones were measured between post-natal day 7 (PND 7) and 28. Offspring were subjected to neurobehavioural studies to test for social interaction and repetitive behaviours on PND 35. Ionised calcium binding molecule 1 (IBA-1) and Glial Fibrillary acid protein (GFAP) expression in the prefrontal cortex (PFC) and cerebellum regions were analysed using immunohistochemistry (IHC). The effect on the BBB’s function was determined using Evans blue dye-albumin extravasation method on PND 35. Additionally, cerebellar tissues were homogenized and processed for molecular analyses of NRXN1, NRXN2, NLGN1, NLGN2 and SHANK3 expression. Changes in expression patterns of NRXNs and NLGNs causes an imbalance in the excitation and inhibition of neurons, a feature associated with ASD. The Mtb treated group had significantly low litter count and high fetal resorption compared to saline treated group. Neuroinflammation was evident in the Mtb offspring at PND 35 as shown by a significant increase in GFAP and IBA-1 expressing astrocytes and microglia in the PFC and cerebellum compared to saline group. The BBB’s integrity was compromised as shown by the increased permeability to EB-dye in the PFC and cerebellum of Mtb, VPA and VPA+Mtb offspring. The Mtb offspring also displayed systemic inflammation and altered ASD-linked behaviours. NRXN1 and NLGN1 were overexpressed in the cerebellum of Mtb-induced MIA offspring compared to saline offspring. Dual exposure to VPA and Mtb restored NRXN1 expression levels, reduced astrocyte and microglia injury in the PFC, rescued social behaviours and restored normal eye-opening patterns in offspring. The study demonstrates impaired fetal development which persists into the post-natal period. The impaired development was accompanied by neuroanatomical changes and behavioural patterns consistent with ASD pathophysiology. These findings might be attributed to Mtb-induced maternal system inflammation in pregnancy that induces fetal inflammation via the placenta and BBB of a developing fetus causing insult in the brain. Immune dysregulation and synaptic defects are hallmarks of ASD. We therefore conclude that prenatal Mtb infection predisposes offspring to a higher risk of neurodevelopmental challenges later in life and dual exposure to VPA and Mtb rescues some of these challenges.
The effect of sildenafil citrate and kraussianone-2 on pre-eclampsia-like manifestations in Sprague-Dawley rats.
(2011) Ramesar, Shamal Vinesh.; Mackraj, Irene.; Moodley, Jagidesa.; Gathiram, Premjith.
Pre-eclampsia, often described as toxaemia of pregnancy, historically represents one of the most widely investigated conditions relating to human reproduction. To date no firm cure has been found and a clear, well defined mechanism has not been ascribed to the pathogenesis of the disease. Researchers seem to focus on single pathways in isolation of others. The disease rather represents a multitude of possible underlying pathologies nvolving genetics, immune dysregulation, vascular maladaptation, and sociobiological factors thus complicating the approach to treatment. However, a central theme is the presence of reduced placental perfusion resulting in a hypoxic and/or ischaemic placenta and the subsequent secretion of various factors that initiate the maternal syndrome. It is within this context that we examine how an intervention such as increasing placental perfusion may represent a promising treatment strategy for this disease. We sought to manipulate the vasodilatory mechanisms of the uterine vasculature using sildenafil citrate and a flavonoid extracted from Eriosema kraussianum (Kr2), in Sprague-Dawley rats that exhibited preeclampsia-like manifestations. Both treatment regimens improved fetal outcomes and reduced blood pressure amplification and proteinuria. They also reduced the plasma concentrations of the two anti-angiogenic factors; sFlt1 and sEng. Only sildenafil citrate improved nitric oxide levels which was expected, suggesting that Kr2 causes vasodilation by some other mechanism. Nevertheless, both compounds improved both pup and placental weights, suggesting that they also improve utero-placental perfusion. These findings that selective uterine vascular dilation improves placental perfusion may be promising in averting possible death to mothers and their babies from pre-eclampsia especially in low resource environments.
The effects of insulin and Syzygium aromaticum-derived oleanolic acid containing dermal patches on kidney function and renal expression of glucose transporters in streptozotocin-induced diabetic rats.
(2014) Ngubane, Phikelelani Siphosethu.; Musabayane, Cephas Tagumirwa.
Introduction The tight glycaemic control required to attenuate chronic complications in type 1 diabetes mellitus requires multiple daily injections of bolus insulin which have been reported to be associated with Na+ retention resulting in hyperinsulinaemic oedema and hypertension. Current research on insulin delivery methods include buccal, oral, nasal, and transdermal delivery systems. Transdermal delivery system is of great interest as this offers sustained controlled release of insulin into the systemic circulation. We have previously reported that transdermal application of pectin hydrogel insulin (PI) matrix patches sustain controlled insulin delivery into the bloodstream of STZ-induced diabetic rats to perhaps ameliorate diabetic complications. Since we have previously reported that STZ-induced diabetic rats retain Na+ following hypotonic saline challenge, this study investigated whether insulin-containing dermal patches can avert and improve the impaired renal fluid and electrolyte handling of STZ-induced diabetic rats. We have also shown that oral administration of OA in addition to possessing hypoglycaemic effects, improves kidney function STZ-induced diabetic rats. The study therefore also investigated whether OA-containing dermal patches can improve kidney function STZ-induced diabetic rats. Materials and methods Pectin insulin (PI)-containing dermal patches of various doses (3.99, 9.57, 16.80 μg/kg) and pectin oleanolic acid (P-OA) containing dermal patches of various doses (21, 42, 84 mg/kg) were prepared by dissolving pectin/insulin or pectin/OA in deionized water and solidified with CaCl2. Short-term (5 weeks) effects on renal function of thrice daily treatments with PI and P-OA patches 8 hours apart were assessed in diabetic animals. Rats sham treated with the pectin drug free patch and insulin (175 μg/kg sc) acted as negative and positive controls, respectively. Daily urine volume, urinary glucose, Na+, K+ and creatinine excretion rates were monitored over 5-weeks. Blood was collected 6 h following treatments for insulin determination. Blood and kidney samples were also collected after 5 weeks for hormonal analysis and measurement of selected biochemical parameters. Results Untreated STZ-induced diabetic rats exhibited elevated weekly urinary glucose, K+ outputs and depressed urinary Na+ outputs throughout the 5-week compared to non-diabetic control animals. Application of PI-containing dermal patches significantly increased urinary Na+ output and reduced urine volume and urinary outputs of glucose and K+ in weeks 4 and 5. Plasma AVP concentrations of untreated STZ-induced diabetic rats were significantly low at end of the 5-week experimental period by comparison with control non-diabetic animals while plasma aldosterone levels were significantly elevated. The highest dose of the insulin-containing dermal patch (16.80 μg/kg) significantly (p < 0.05) elevated plasma AVP concentrations while decreasing plasma aldosterone concentrations of STZ-induced rats by comparison to untreated STZ-diabetic rats. GFR of untreated STZ-induced diabetic rats was significantly decreased while plasma creatinine concentrations were significantly elevated by comparison to non-diabetic control animals. PI containing dermal patches increased GFR of STZ-induced diabetic rats with a concomitant reduction of plasma creatinine concentrations by comparison to untreated STZ-induced diabetic rats. Interestingly, P-OA dermal patches also increased GFR of STZ-induced diabetic rats while reducing plasma creatinine concentrations. The effects of both PI and P-OA containing dermal patch compared with subcutaneous insulin. Significant increase of MDA and decreases of SOD and GPx were found in the skin, kidney and heart tissues of STZ-diabetic animals as compared to non-diabetic control animals. PI (16.80 μg/kg) -treated STZ-induced diabetic animals however showed low concentrations of MDA and increased the activities of SOD and GPx in the skin, kidney and heart tissues compared to untreated STZ-induced diabetic animals. P-OA-treated STZ-induced diabetic animals similarly and significantly showed decreased MDA, and increased activity of antioxidant enzymes; SOD and GPx in skin, kidney and heart tissues. H and E kidney stained sections of untreated non-diabetic control, untreated STZ-induced diabetic rats and diabetic animals topically applied with insulin and OA-containing dermal patches were observed under light microscope. However, STZ-induced diabetic rats showed thickened basement membrane of the Bowmans capsule, thickened glomerular basement membrane and hypercellularity of the proximal tubules by comparison to the non-diabetic animals after 5 weeks of the study. Treatment with insulin containing dermal patches and subcutaneous insulin for 5 weeks however attenuated these features when compared with the untreated STZ-diabetic rats. Like PI dermal patches, OA containing dermal patches also ameliorated structural changes of kidney of STZ-induced diabetic rats. The increased urinary glucose concentrations of the untreated STZ-induced diabetic rats were associated with increased expression of GLUT 1 and SGLT 1 to normalcy by comparison to nondiabetic rats. The highest dose of PI containing dermal patch however, like subcutaneous insulin, significantly decreased the expressions of GLUT 1 and SGLT 1 by comparison to STZ-induced diabetic controls. Plasma insulin concentrations of untreated STZ-induced diabetic rats were significantly low in comparison with control non-diabetic animals. Acute (6 h) and short-term (5 weeks) daily application of PI containing dermal patches to STZ induced diabetic rats significantly elevated plasma insulin concentrations by comparison with untreated diabetic animals. However, the plasma insulin concentrations in animals treated with the high insulin doses (9.57, 16.80 μg/kg) were significantly higher than those found in diabetic groups treated with the low insulin dose (3.99 μg/kg). There were no differences in the plasma insulin concentrations in STZ-induced diabetic animals treated with P-OA containing dermal patches by comparison to STZ- diabetic untreated controls both acutely and chronically. To determine whether insulin was transported across skin of STZ-induced diabetic rats following topical application of PI and P-OA containing dermal patches, we also monitored the density of phosphorylated insulin receptor substrates (IRS) in the skin by immunohistochemical staining with specific insulin receptor antibodies. Non-diabetic treated skin sections showed slight immunostaining of insulin receptors in comparison STZ-induced diabetic rats which stained negative. Immunohistochemical staining for phosphorylated IRS in the skin of animals following application of insulin and sc insulin treatment for 5 weeks clearly demonstrated widespread localization of IRS in cell bodies of the dermis, collagen and subcutaneous layer. Interestingly, OA-containing dermal patches also showed widespread localization of IRS in cell bodies of the dermis, collagen and subcutaneous layer. H and E skin sections of untreated non-diabetic control, untreated STZ-induced diabetic rats and diabetic animals topically applied insulin and OA-containing dermal patches showed no significant histological differences in dermis compared to the untreated non diabetic control skin sections. Discussion Previous studies indicate compromised renal function in experimental diabetes and diabetic patients. The results herein however indicate that insulin containing dermal patches increase Na+ excretion probably by decreasing plasma aldosterone and increasing plasma AVP concentrations of STZ-induced diabetic rats. PI containing dermal patches also improve kidney function by increasing GFR with concomitant reduction of plasma creatinine concentrations. Like PI containing dermal patches, P-OA containing dermal patches increased Na+ excretion by decreasing plasma aldosterone and increasing plasma AVP concentrations of STZ-induced diabetic rats. P-OA containing dermal patches also increased GFR and reduced plasma creatinine concentrations of STZ-induced diabetic rats. Conclusion From these results, we conclude that PI and P-OA dermal patches deliver physiological amounts that can improve kidney function in diabetes.
Effects of maslimic acid and related triterpene derivatives on kidney function of male Sprague-Dawley rats.
(2014) Mkhwanazi, Blessing Nkazimulo.; Musabayane, Cephas Tagumirwa.; Van Heerden, Fanie Retief.
Reports indicate that hyperglycaemia leads to development of kidney complications which result in sodium retention, decrease in glomerular filtration rate (GFR) and high blood pressure. Various biochemical processes such as polyol pathway, AGEs formation are thought to gives rise to a development and progression of these complications. Clinical trials show that there is currently no commercially available compound that lowers blood glucose concentration while alleviating diabetic nephropathy (DN). Current methods involve the use of ACE blockers which are associated with side effects. Previous reports in our laboratories indicate that triterpene constituents of Syzygium spp. such as oleanolic acid (OA) possess hypoglycaemic and renoprotetive effects in STZ-induced diabetic rats. The important question is whether MA, a related triterpene also possesses the same properties. MA is a hydrophobic triterpene and we therefore synthesised derivatives to improve solubility, bioavailability and efficacy. Accordingly this study was designed to investigate the effects of MA and related triterpene derivatives on renal function of STZ-induced diabetic rats.
An EM investigation of skeletal muscle regeneration in cultured explants.
(1995) Naidoo, Poovalingam Ramsamy.; Gathiram, Premjith.
Abstract available in PDF file.
Endotoxaemia in intestinal dysfunction in experimental animals : intestinal ischaemia and hyperthermia.
(1988) Gathiram, Premjith.; Gaffin, Stephen L.
Endotoxins or lipopolysaccharides (LPS), highly toxic component of the outer membrane of gram-negative bacteria, are normally present in the mammalian gut lumen.In this thesis, I investigated, in laboratory animals, whether these gut-derived endotoxins play a role in pathophysiology resulting from intestinal dysfunctions caused by intestinal ischaemia and heat-stress.In primates, reperfusion of the splanchnic region after a temporary ischaemia was followed by a rapid increase in LPS concentration, first in the hepatic portal plasma and, ten minutes later, in the systemic arterial plasma. Rises in plasma LPS concentrations during or following the temporary intestinal ischaemia was prevented by prophylactic administrations of corticosteroids, anti-LPS IgG antibodies and oral, non-absorpable, antibiotics agents which appear to stabilize cellular membranes, aid the reticuloendothelial system in removal of LPS from the circulation and destroy the intestinal aerobic gramnegative bacteria respectively. In addition, administration of therapeutic anti-LPS antibodies also rapidly reduced the plasma LPS concentrations to baseline during an endotoxaemia. In a control heat-stress model, elevations in plasma LPS concentration commenced at rectal temperatures greater than 41,SoC. Like the intestinal ischaemia model, this occurred first in the hepatic portal plasma, and 10-15 minutes later, in the systemic arterial plasma. Peak plasma LPS levels of about 0,3 ng/ml, measured in heat-stressed primates, have proved in previous studies, to be toxic. A rapid decline in mean arterial pressure was followed by increases in plasma LPS concentrations and heart rates. Reductions in splanchnic blood flow and consequent local ischaemia coupled with thermal injury to the intestinal wall and the liver, may have permitted rises in plasma LPS concentration. Furthermore, as in the ischaemia model, prophylactic administrations of corticosteroids, anti-LPS IgG antibodies, and oral, nonabsorbable antibiotics prevented a rise in plasma LPS concentration. Of importance, prophylaxis with intravenous corticosteroids and 'anti-LPS IgG antibodies increased the survival rates significantly in heat stroke in primates. In addition, monkeys having high titres of "natural" antiLPS IgG antibodies had lower plasma LPS concentrations and survived the induced-heat stroke. It is suggested that other pathophysiologic conditions which compromise the integrity of the gut wall would also lead to the development of an endotoxaemia, and that gutderived endotoxins contribute to the athogenesis of heat stroke and treatments with corticosteroids and anti-LPS IgG antibodies may prove beneficial in other endotoxinrelated disorders.
Evaluating immune activation and cellular determinants of thrombosis in pre-diabetes.
(2018) Mkandla, Zibusiso.; Nkambule, Bongani Brian.; Dludla, Philani.
Introduction Platelet dysregulation in pre-diabetes plays a major role in the progression of prothrombotic and pro-inflammatory conditions. Cardiovascular disease (CVD), which may occur at the pre-diabetic stage, are a common cause of morbidity in diabetic individuals. Despite treatment, people living with type 2 diabetes are at an increased risk of developing CVD, which is attributed to increased platelet activation and platelet mediated cellular cross-talks. The aim of the study was to investigate platelet activation and function in prediabetes and to further evaluate the effects of oral glucose lowering and anti-inflammatory therapy on platelet function a pre-diabetic state. Methodology Male mice were fed experimental diets, control low-fat diet (D12450J 10 kcal% fat) and a high-fat diet (HFD) (D12492, 60 kcal% fat) (Research Diets, NJ, USA). Platelet activation was determined by measuring the formation of spontaneous platelet-monocyte aggregate (PMA). The high-fat diet (HFD) fed mice were then randomized into 3 treatment groups; metformin (150mg/kg) and low-dose aspirin (3mg/kg) dual therapy; low-dose aspirin (3mg/kg); and clopidogrel (0.25mg/kg). The drugs were administered orally, once a day, every day for 3-weeks. We determined the pre-diabetic status of the mice by measuring their oral glucose tolerance and insulin levels. We further measured the haematological parameters. Platelet function and reactivity were determined by stimulating them with endogenous agonists, adenosine diphosphate (ADP), collagen and arachidonic acid, before and after treatment. Results Overall, there were no significant differences in the baseline characteristics such as body weights and insulin levels. However, after three weeks on the experimental diets, the high-fat diet (HFD) fed group exhibited delayed glucose clearance (p=0.0362). Baseline levels of platelet-monocyte aggregates were increased in the HFD group, p=0.0156. Post-stimulation with 4μM ADP and 20μM ADP, the HFD group expressed elevated levels of activated platelets, p<0.05. Metformin and low-dose aspirin-treatment inhibited reversible platelet aggregation, p=0.0220. While, residual platelet activation was observed at a concentration of 20μM ADP, p=0.0535. In the low-dose aspirin group, there were no significant variations in platelet reactivity following stimulation with ADP, p>0.05. Clopidogrel treatment inhibited the platelet response to 20μM ADP (p=0.0313). Conclusion Despite anti-platelet treatment in pre-diabetes, platelets exhibit a varied response to endogenous agonists. Therapeutic targeting of these pathways may reduce the risk of thrombotic complications in pre-diabetes. We further highlight the potential synergistic benefit of using dual oral glucose lowering and antiplatelet treatment to minimize the high on-treatment platelet responses observed in pre-diabetes and T2DM.
Evaluating plasmodium berghei infection influence and asiaticacid administration efficacy in sprague dawley male rats: effects on parasitamia, glucose homeostasis and renal electrolyte handling.
(2016) Mavondo, Greanious Alfred.; Mabandla, Musa Vuyisele.
Abstract Introduction: Five human-infecting Plasmodium species orchestrate varied pathophysiology culminating in malaria. Despite being treatable, malaria has high mortality and morbidity in pregnant women and children under five years. Current treatment is hampered by drug resistance, toxicity and failure to address malaria induced pathology directly. Asiatic acid has antioxidant, pro-oxidant, antihyperglycaemiac renoprotective qualities which may be anti-disease properties in malaria. Very little is currently known or reported about these asiatic acid anti-disease perspectives in malaria and therefore require further investigations. The aim of this study was to investigate the influence of asiatic acid administration and malaria infection on parasitaemia suppression, glucose homeostasis, renal function and electrolyte handling in Plasmodium berghei-infected Sprague Dawley male rats. Methods: Three sub-chronic studies and one acute study were conducted. The sub-chronic protocol involved per-oral pre- and post-infection administration of asiatic acid (5, 10, 20 mg/kg) and post-infection transdermal drug delivery system application of asiatic acid (5, 10, 20 mg/kgamidated hydrogel matrix pectin patch). Acute studies included post-infection oral glucose tolerance response to asiatic acid. Influence of asiatic acid on %parasitaemia changes, physicochemical changes, immunological effects of malaria, haematological results of malaria, antioxidant capacity, glucose homeostasis, renal function and renal electrolyte handling were investigated. Results: Asiatic acid suppressed parasitaemia to varying extents with asiatic acid 10mg/kg and 5mg/kg emerging as the most efficacious amongst the three doses by per oral administration and transdermal delivery drug delivery system, respectively. Malaria suppression occurred in both pre-infection and post-infection administration of asiatic acid. Asiatic acid preserved physicochemical parameters, ameliorated haematological effects of malaria, influenced immunological effects of malaria, modulated glucose homeostasis in malaria, protected renal function and electrolyte handling in malaria. Asiatic acid improved glucose tolerance response in acute malarial states. Antioxidant status was also improved in malaria. Conclusions: Asiatic acid displayed chemoprophylactic and chemotherapeutic effects in malaria. Asiatic acid has both glucose homeostatic and renoprotective properties in malaria. The antioxidant characteristics of the amphiphilic asiatic acid seem to exert anti-disease and antiparasitic effects in malaria. Asiatic acid may be used as an antimalarial compound with ability to ameliorate malaria associated pathophysiology.
An evaluation of the chemical composition and the in vitro and in vivo antihypertensive activity of extracts of Tulbaghia acutiloba Harv. in an L-NAME induced hypertensive model.
(2019) Arhin, Isaiah.; Mackraj, Irene.
The increasing prevalence of hypertension over the years has been identified as a major contributor to high morbidity and mortality, globally and locally, in Africa, posing a serious global health threat. The demand for novel therapeutic strategies has become increasingly important, given that conventional options may be inaccessible and costly, and are often associated with side effects, hampering patient compliance. The scientific validation of alternative strategies, such as phytotherapy has, therefore, become a major focus in the treatment and management of hypertension, as it is perceived as being cheap, accessible, and possessing minimal side effects. Hence the investigation of medicinal plants, within the field of novel drug discovery, is of interest as plants possess various phytochemicals displaying biological activities, which may be beneficial in hypertension, and it’s associated complications. Since there is no existing scientific data available to validate its medicinal usage, this study, therefore, evaluated the in vitro and in vivo antihypertensive effects of Tulbaghia acutiloba. The hydro-methanolic extracts of the plant parts (i.e. leaves, flowers, rhizomes and roots) were initially evaluated in vitro for their phytochemistry, antioxidant potential, angiotensin-converting enzyme (ACEI) inhibition activities, and heavy metal content. The phytochemical investigation of the various parts of the plants showed the presence of phenols, amino acids and alkaloids in all parts, with the leaves exhibiting a higher total phenolic content, in comparison to the other parts. Further analysis, using gas chromatography–mass spectrometry (GC-MS), revealed the presence of bioactive compounds, such as α-linolenic acid, which was found only in the leaves. Other compounds such as oleic acid and palmitic acid were found in all the parts of the plants. All parts of the plant showed antioxidant activity in vitro. Heavy metal toxicity analysis revealed the safety profile for all parts of the plants. All parts also showed a potential ACE inhibitory effect of greater than 50%, with the leaves showing the most significant effects, comparable to the conventional drug, Ramipril. We further investigated the effect of the hydro-methanolic leaf extract on oxidative stress, endothelial function, cardiovascular, renal and haematological parameters, associated with hypertension, in an L-NAME induced hypertensive rat model. The administration of the hydro-methanolic leaf extract of Tulbaghia acutiloba at different concentrations of 40, 60 and 80mg/kg b.w., reduced systolic, diastolic and mean arterial pressure in the model, with a pronounced effect at the dosage of 80mg/kg b.w. Additionally, the leaves of T. acutiloba significantly enhanced bradykinin receptor levels (B1 and B2), nitric oxide (NO) availability, promoted antioxidant activities and significantly reduced ACE activity in serum and cardiac tissues in hypertensive rats. Cardioprotection was significantly enhanced at 80mg/kg b.w. of T.acutiloba, as depicted by the cardiac function and morphology, and cardiac gene expression in experimental rats. There was no evidence of toxicity as depicted in the liver enzymatic activity after the administration of T.acutiloba in the hypertensive rats. Administration of T. acutiloba improved renal function as evidenced by the increased creatinine clearance (Ccr), improved fractional excretion of sodium and decreased urine protein-creatinine ratio (UPr/UCr). Additionally, decreased levels of leucocyte infiltration, decrease in both, neutrophil to lymphocyte ratio (NLR) and lymphocyte to monocyte ratio (LMR), was found after administration of T.acutiloba, with a maximal effect occurring at a dose of 80mg/kg b.w. Together, these findings provide scientific validation for T.acutiloba as a medicinal plant that has cardioprotective and antihypertensive properties, and is able to improve renal function and haematological parameters in an L-NAME induced hypertensive rat model. Overall, the data also provides substantive evidence for the possible usage of T.acutiloba as an alternate antihypertensive agent, in resource limited areas where conventional drugs are inaccessible. Key Words: Tulbaghia acutiloba, phytotherapy, cardioprotection, L-NAME, renal function, haematology.
Evaluation of the potential benefits of L-ergothioneine on selected complications associated with type-2 diabetes in a rat model.
(2021) Dare, Ayobami.; Nadar, Anand.; Channa, Mahendra Lala.
Several pathogenic factors promote type-2 diabetic complications in patients, including cardiomyopathy, nephropathy, and non-alcoholic fatty liver disease (NAFLD). Specific nutraceuticals from food may act as a medicinal adjuvant in managing diabetic complications. L-ergothioneine (L-egt), a bioactive compound obtained from medicinal mushrooms, beans and some meat products, has been shown to reduce lipid accumulation, provide cytoprotection in tissue injury and enhances therapeutic efficacy when used as adjuvant. This study investigated the effect of L-ergothioneine with or without metformin on pathogenic metabolic pathways and biomarkers associated with selected diabetic complications in a type-2 diabetic rat model. Ninety (90) adult male Sprague-Dawley (175±20)g rats were divided into three study groups [study 1 (36), study 2 (30) and study 3 (24)]. A 10% fructose solution was provided ad libitum to adult male Sprague- Dawley (175±20)g rats for 14 days followed by a single intraperitoneal injection of low dose streptozotocin (STZ 40mg/kg bwt, i.p) to induce type-2 diabetes after which the animals were randomly divided into six, five, and four groups (n=6) in studies 1 (liver), 2 (kidney), and 3 (heart), respectively. The control groups were administered 1ml/100g distilled water, while L-egt (35mg/kg bwt), metformin (500mg/kg bwt), and losartan (20mg/kg bwt) were administered in the other groups. At the end of each study, animals were euthanized via decapitation, blood samples were collected, while the heart, kidney, and liver tissue were excised and used for biochemical, RT-qPCR, ELISA, western blotting, and histopathological analysis. An in-silico study was done to evaluate the molecular antioxidant mechanism of L-egt. Administration of L-egt, with or without metformin, to diabetic animals positively altered selected biomarkers of hepatic, renal, and cardiac dysfunction and prevented structural damage in these tissues. This treatment regimen mitigated oxidative stress, inflammation, and fibrosis by downregulating (p<0.05) SREBP1c, FAS, NF-kB, fibronectin, TGFβ1, and Keap1 expression and upregulating (p<0.05) Nrf2, Sirt1, NQO1, and HO1 expression compared with the diabetic control animals. Interestingly, co-administration of L-egt and metformin improved glucose homeostasis and reduced HOMA-IR. The in-silico study showed that L-egt binds to the active site of Nrf2 and may serve as a ligand to activate this potent antioxidant molecule. The overall result from this study showed the potential benefits of L-ergothioneine in the management of selected complications associated with type-2 diabetes. This bioactive compound may be an effective adjuvant to attenuate hypertriglyceridemia, oxidative stress, and inflammation, thereby protecting vital organs associated with diabetic complications against injury and improving glycemic control when coadministered with metformin to delay the onset of diabetic complications.
Evaluation of the therapeutic properties of a Ruthenium(ll) uracil-derived diimine complex on selected complications associated with diet-induced pre-diabetes.
(2019) Mabuza, Lindokuhle Patience.; Khathi, Andile.; Ngubane, Phikelelani Siphosethu.
Pre-diabetes is a chronic metabolic condition where blood glucose levels are above the upper threshold considered normal but below the threshold for a diagnosis of diabetes. Pre-diabetes predisposes individuals to a high probability of future progression to overt T2DM. Pre-diabetic patients are at increased risk of developing other pathologies such as NAFLD, diabetic nephropathy (DN) and immune dysregulation complications. As a chronic disease, the long-term implications of diabetes contribute to poor quality of life and significantly increase costs associated with healthcare. Pre-diabetes may however be reversible, through the implementation of lifestyle modification programmes based around dietary modification and increased physical activity. Where lifestyle modifications are ineffective, both pharmacotherapy and lifestyle modification are recommended. However, there is reported poor patient compliance in terms of dietary intervention as patients tend to heavily rely on the pharmacological treatment, thus reducing the efficacy of the drug and increasing the possibility to develop T2DM. Hence, there is a need for novel drugs that will remain therapeutic even in the absence of dietary modification. In our laboratory, we have synthesized a novel ruthenium(II) uracil-derived diimine complex that has been shown to improve insulin sensitivity and restore glucose homeostasis in diet-induced prediabetes. In this study, we further sought to evaluate the effects of this compound on selected complications associated with diet-induced pre-diabetes. Estimating whether ruthenium(II) uracil-derived diimine complex in both the presence and/or absence of dietary intervention will show to be effective in the management of prediabetic-related complications. A high fat high carbohydrate (HFHC) diet was used to induce pre-diabetes for 20 weeks. After the induction, pre-diabetic rats were randomly allocated to the following treatment groups: non-prediabetes (NPD); pre-diabetic (PD); metformin plus HFHC; metformin plus normal diet (ND); Ruthenium plus HFHC and ruthenium plus ND. The animals were treated with subcutaneous injection of ruthenium complex (15 mg/ kg) and oral dose of metformin (500 mg/ kg). The rats were treated once a day every third day at 09:00 am for 12 weeks. Every 4 weeks, parameters such as body weight, food intake, fasting blood glucose, fluid intake and urinary output were monitored for 12 weeks treatment period. In study 1, the administration of ruthenium(II) complex resulted in the restoration of liver and body weights in the pre-diabetic treated rats when compared to the PD group. This treatment also reduced liver damage enzyme biomarkers and plasma total bilirubin levels in the pre-diabetic treated rats when compared to the PD group whilst administration of ruthenium(II) with dietary intervention reduced plasma sterol regulatory element binding protein 1c (SREBP-1c) concentration in the pre-diabetic treated rats when compared to the PD group. These findings were further supported by the histological analysis of the liver, showing reduced hepatic lipid droplet accumulation, hepatocyte ballooning and locular disarray in the ruthenium(II)-treated rats when compared to the PD group as seen in chapter 2 of the study. In study 2, the administration of ruthenium(II) complex resulted in reduced blood glucose, aldosterone, fluid intake and urinary output in the pre-diabetic treated rats when compared to the PD group which positively correlated with a restoration in plasma and urinary electrolytes along with plasma antioxidants concentrations in the ruthenium(II)-treated rats. Furthermore, there was a decrease in kidney injury molecular-1 (KIM-1) concentration, albumin excretion rate (AER) albumin creatinine ratio (ACR) and mRNA expression of podocin in urine in the ruthenium(II)-treaded rats. These observetions were further demonstrated by the histological analysis of the kidney, displaying improved histology of renal glomerulus in ruthenium-treated rats when compared to the PD group as seen in chapter 3 of the study. In study 3, treatment with ruthenium(II) complex resulted in reduction of platelet activation markers mean platelet volume (MPV) and CD40 Ligand (CD40 L) concentrations, which positively correlated with decreased plasma triglycerides (TG) and very low-density lipoproteins (VLDL) levels in the pre-diabetic treated rats when compared to the PD group. Whilst administration of ruthenium(II) with dietary intervention reduced plasma fibrinogen concentration in the pre-diabetic treated rats when compared to the PD group. These was further evidenced by normalization of immune cell counts in the ruthenium(II)-treated rats. Furthermore, there was a decrease in pro-inflammatory cytokines, tumor necrosis factor α (TNF-α) concentration in the ruthenium(II)-treated rats and decreased interleukin-1β (IL-1β) concentration in the ruthenium(II) with dietary interventiontreated rats when compared to the PD group as seen in chapter 4 of the study. Taken together, the results observed suggest that ruthenium(II) complex exhibited hepato and renoprotective effects while ameliorating immune dysregulation underlying pre-diabetes in diet-induced pre-diabetic rats. However, further studies are still required to find out the exact mechanism behind potential effect of this metal-based compound.
Fungal endophytes: isolation, identification and assessment of bioactive potential of their natural products.
(2017) Sibanda, Edson Panganayi.; Mduluza, Takafira.; Mabandla, Musa Vuyisile.
Fungal endophytes produce a broad variety of bioactive compounds with potential to address some of the unmet human needs. Medicinal plants have an important role to play in the search for new strains of endophytes fungi, as it is possible that their beneficial characteristics are as a result of the metabolites produced by their endophytic community. However, inspite of this potential as repositories of bioactive compounds, the fungal endophytes of African medicinal plants remain largely underexplored. This thesis reports on studies that were conducted to bioprospect for endophytic fungi with antioxidant and antimicrobial activity hosted by the plants Warburgia salutaris, Annona senegalensis, Kigelia africana and Vitex payos used in Zimbabwean traditional medicine. The surface sterilization technique was used to isolate the endophytic fungi that were identified by ribosomal DNA sequencing of the nuclear ribosomal internal transcribed spacer region. Crude extracts obtained from the fermentation of the isolated endophytic fungi were screened for antimicrobial activity using the agar diffusion method and evaluated for total antioxidant activity using a commercial kit that used the single electron transfer mechanism. Fourier-transform infrared spectroscopy (FT-IR) and Gas Chromatography – Mass Spectrometry (GC-MS) were used to provide a snapshot of the metabolites present in the endophyte fungi extracts. A total of 33 endophytic fungi were isolated from the medicinal plants and the fungal endophyte colonisation rates varied by plant species and plant tissue. The isolated fungi across the different plant species and tissue types were found to be dominated by members of the phylum Ascomycota. The endophytic fungi Penicillium chloroleucon was isolated from all the plant species except for Cladosporium uredinicola and Myrothecium gramineum (both isolated from Kigelia africana) which had an inhibitory effect against Escherichia coli (ATCC1056). Whilst Epicoccum sorghinum isolated from Annona senegalensis exhibited the most potent antioxidant activity, a significant number of the screened endophytic fungi from the different plant species were also found to have some antioxidant activity. The total phenolic content was found to have a positive correlational relationship with total antioxidant activity of the screened endophytic fungi crude extracts. The endophytic fungi were shown to produce a diverse range of metabolites including phenolic and polyphenolic compounds through FT-IR and GC-MS analysis. The isolate Cladosporium uredinicola has potential as a source of antimicrobial compounds whilst the isolate Epicoccum sorghinum has potential as a source of natural antioxidant. Antioxidant activity is a common phenomenon in the studied endophytic fungi and the fungal endophytes of the medicinal plants of Zimbabwe have potential as sources of bioactive compounds. Keywords: Bioprospecting, antioxidant, antimicrobial, endophytic fungi, medicinal plants, Zimbabwe.

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