Biochemistry

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Antioxidative and antidiabetic activity and phytochemicals analysis of some selected Sudanese traditional medicinal plants.
(2021) Idris, Almahi Idris Mohamed.; Islam, Shahidul.
This study was conducted to evaluate the antioxidant and anti-diabetic properties of selected traditional Sudanese medicinal plants (Cyperus rotundus, Nauclea latifolia, and Hibiscus sabdariffa) using in vitro, ex vivo, and in silico experimental models. The crude extracts (ethyl acetate, ethanol, and aqueous) were screened in vitro for their antioxidant activities using ferricreducing antioxidant power (FRAP), 1,1-diphenyl-2-picrylhydrazyl (DPPH), and nitric oxide radical (NO) scavenging activities, as well as their carbohydrate digesting enzyme inhibitory activities for antidiabetic evaluation. Subsequently, the extracts were subjected to Gas Chromatography-Mass Spectrometry (GC-MS) analysis to elucidate their possible bioactive compounds. Additionally, ex vivo studies was conducted to investigate their capability to promote muscle glucose uptake and suppress glucose absorption in the intestine as well as to analyze antioxidative effects in iron–induced oxidative stress in hepatic tissue. Molecular docking was carried out to determine the probable enzymes' inhibitory mode of action by ligands identified through GC-MS. This study indicates that these traditional Sudanese medicinal plants have remarkable antioxidant and antidiabetic activities, which may help to ameliorate oxidative stress and diabetes. Therefore, these plants may be considered a natural source of bioactive compounds beneficial for human health, particularly for managing diabetes and oxidative stress-related metabolic disorders.
Antioxidative and antidiabetic activity and phytochemicals, analysis of some selected Sudanese traditional medicinal plants.
(2021) Idris, Almahi Mohamed.; Islam, Shahidul.
This study was conducted to evaluate the antioxidant and anti-diabetic properties of selected traditional Sudanese medicinal plants (Cyperus rotundus, Nauclea latifolia, and Hibiscus sabdariffa) using in vitro, ex vivo, and in silico experimental models. The crude extracts (ethyl acetate, ethanol, and aqueous) were screened in vitro for their antioxidant activities using ferricreducing antioxidant power (FRAP), 1,1-diphenyl-2-picrylhydrazyl (DPPH), and nitric oxide radical (NO) scavenging activities, as well as their carbohydrate digesting enzyme inhibitory activities for antidiabetic evaluation. Subsequently, the extracts were subjected to Gas Chromatography-Mass Spectrometry (GC-MS) analysis to elucidate their possible bioactive compounds. Additionally, ex vivo studies was conducted to investigate their capability to promote muscle glucose uptake and suppress glucose absorption in the intestine as well as to analyze antioxidative effects in iron–induced oxidative stress in hepatic tissue. Molecular docking was carried out to determine the probable enzymes' inhibitory mode of action by ligands identified through GC-MS. This study indicates that these traditional Sudanese medicinal plants have remarkable antioxidant and antidiabetic activities, which may help to ameliorate oxidative stress and diabetes. Therefore, these plants may be considered a natural source of bioactive compounds beneficial for human health, particularly for managing diabetes and oxidative stress-related metabolic disorders.
Comparative antidiabetic effects and mechanisms of actions of five Chinese and South African indigenous teas.
(2020) Xiao, Xin.; Islam, Shahidul.
The present thesis assessed the in vitro, ex vivo and in vivo anti-oxidative and antidiabetic activities of five teas which are widely consumed in China or South Africa. Three of the selected five teas are from South Africa, namely red rooibos (Aspalathus linearis), green rooibos (Aspalathus linearis) and red honeybush (Cyclopia genistoides) tea. The remaining two from China are jasmine green (Camellia sinensis) and zhengshanxiaozhong (ZSXZ) black tea (Camellia sinensis). The different sequential solvent extracts following increasing polarity index (dichloromethane, ethyl acetate, ethanol, and water) and hot water extract of different teas were evaluated at in vitro and ex vivo conditions for their antioxidant properties, inhibitory potentials on α-glucosidase, α-amylase and pancreatic lipase, effects on ameliorating Fe2+- induced oxidative pancreatic or hepatic injury, as well as the glucose absorption inhibition in small intestine and the glucose uptake stimulation in isolated psoas muscle of rats. Possible bioactive components responsible for the activities of the extracts were identified by using Gas Chromatography-Mass Spectrometry (GC-MS) analysis or liquid chromatograph-mass spectrometry (LC-MS) analysis. In vitro and ex vivo tests presented promising antioxidant and antidiabetic activities of these five teas. The red honeybush, jasmine green and green rooibos teas, were further subjected to an in vivo intervention trial in a fructose-streptozotocin (STZ) induced T2D model of Sprague-Dawley rats. Assays were carried out to reveal the effects of these teas on lowering blood glucose level, improving oral glucose tolerance ability, stimulating insulin secretion and hepatic glycogen synthesis and ameliorating some diabetes related parameters such as serum lipid profile, hepatic and renal function tests and calculated insulin resistance (HOMA-IR), β-cell function (HOMA-β) from the blood glucose and serum insulin data. Furthermore, in vivo oxidative stress markers such as reduced glutathione, superoxide dismutase and catalase activity and lipid peroxidation were analysed in harvested organs (liver, kidney, heart and pancreas). The results of in vivo tests demonstrated that high dose of jasmine green tea showing the best activity followed by the high dose of red honeybush tea, low dose of jasmine green tea, high dose of green rooibos tea, low dose of red honeybush tea, when lowest activity was observed for the low dose green rooibos tea. The results of this study indicated promising anti-T2D properties of the above-mentioned teas. However, further clinical trials are needed to ascertain the results of these in vitro, ex vivo and in vivo studies.
Recombinant expression and enzymatic characterisation of Trypanosoma vivax cathepsin L-like protease (TviCATL) for single chain variable fragment antibody production.
(2022) Ramjeawon, Bhavana Roshenlal.; Coetzer, Theresa Helen Taillefer.
Humans and animals in sub-Saharan Africa are at risk of African trypanosomiasis (AT), caused by tsetse fly-transmitted protozoan blood parasites of the Trypanosoma genus. Animal African trypanosomiasis (AAT), or nagana, is caused by T. brucei, T. congolense and T. vivax and negatively impacts livestock farming and consequently the economy of the continent. Since AAT occurs in rural areas, affordable rapid diagnostic tests (RDTs) and drugs are required. Diagnostic tests focus on antibody detection; however, antigen detection is more favorable since anti-trypanosome antibodies persist in blood for years following recovery. Due to the parasite’s defense by antigenic variation, development of a vaccine is unlikely. Molecules that are essential for parasite survival, such as peptidases, are currently being targeted for diagnosis and chemotherapy. A cathepsin-L-like cysteine protease from T. vivax, TviCATL, is released by dying parasites in the host bloodstream and was shown to be a diagnostic target for detecting host antibodies. To achieve diagnosis of current infections, detection of TviCATL is being explored. The overall aim of this study was to enzymatically characterise TviCATL; and to study the interaction of antibodies against the TviCATL antigen which could be used as a chemotherapeutic drug for the diagnosis of T. vivax infections. The protease, TviCATL, was recombinantly expressed in E. coli using the pET-28a expression vector and purified using a nickel chelate affinity column. The resulting 47 kDa protein was identified using western blot and was shown to hydrolyse H-D-Ala-Leu-Lys-AMC and was inhibited by bestatin and E-64 and had optimal activity between pH 6.5 and 7.5. The crossreactivity between TviCATL and antibodies produced against other Trypanosoma spp cysteine proteases was evaluated in western blots, and results confirmed cross-reactivity. In addition, chicken anti-TviCATL antibodies were able to detect TviCATL in TviCATL-spiked bovine serum. The production of antibodies using the Nkuku® phage library was employed as an alternative to the animal-based antibody production and single-chain variable fragment (scFvs) antibodies were selected by panning against the TviCATL antigen. After four rounds of panning, TviCATL-scFvs binders were enriched and four clones gave the highest signal when evaluated using a monospecific ELISA. Due to the low values obtained, optimisation of panning is necessary for improved results. Optimisation of recombinant expression and purification of the identified scFvs for use in a sandwich ELISA were explored to this end. This study showed that TviCATL is a promising chemotherapeutic and diagnostic target for African animal trypanosomiasis.
The role of MMP-14 and MMP-2 in mediating myoblast fusion.
(2016) Nkosi, Mthokozisi Siphesihle.; Niesler, Carola Ulrike.
Satellite cells are muscle precursor cells that have the ability to self-renew, proliferate and differentiate into myoblasts that eventually elongate and fuse to form myotubes which are vital for regeneration and repair of muscle. Satellite cells reside in a niche, between the sarcolemma of the muscle fiber and the basal lamina, which consists of mostly collagen IV, proteoglycans and laminin. Matrigel is a gelatinous protein mixture that consists primarily of collagen IV and laminin and therefore resembles the basal lamina. Matrix Metalloproteinases (MMPs) are zinc endopeptidases, proteolytic peptidases which break peptide bonds within their substrates. MMP-14 (membrane bound) also known as membrane-type 1 matrix metalloproteinase (MT1-MMP) is one of the major matrix metalloproteinases (MMPs) involved in muscle repair and regeneration, together with MMP-2 (secreted). MMP-2 is a secreted gelatinase A, which is activated by MMP-14. MMP-2 is also known to be activated by nitric oxide (NO), therefore allowing active MMP-2 to release growth factors such as Hepatocyte Growth Factor (HGF) from the extracellular matrix (ECM). There are two forms of MMP-2, intracellular MMP-2 and extracellular (secreted) MMP-2. Secreted MMP-2 contains a peptide signal that helps direct it outside the cell, while intracellular MMP-2 lacks this feature and is therefore retained within the cell. Intracellular MMP-2 activity is known to be a major cause of muscular atrophy. Secreted MMP-2 is known to degrade ECM components, facilitating satellite cell mobility and release of growth factors such as HGF, aiding in muscle regeneration. MMP-2 can cleave collagen IV due to the presence of a fibronectin-like domain within its catalytic domain; this is not the case with MMP-14. MMP-14 and MMP-2 together degrade collagens, fibronectin, laminin-2/4 and other adhesion molecules. This clears the path for the myoblast to align and fuse to form myotubes which then finally align to form mature muscle fibers. The levels of MMP-14 and MMP-2 must be regulated; low levels can cause muscular dystrophy. The current study analysed expression levels, activity and role of MMP-14 and MMP-2 in C2C12 myoblast differentiation. C2C12 myoblasts first proliferated (Day 0), then aligned and elongated (Days 1-2) and then finally fused into myotubes (Days 3-5) during differentiation. MMP-14 and MMP-2 protein levels were high during the elongation period and also during fusion of C2C12 myoblasts. MMP-14 was localised at the focal adhesions, where actin filaments terminate during myoblast proliferation and fusion. ii Inhibition of MMPs using BB94 (10 µM) was observed to significantly reduce C2C12 myoblasts fusion. Secreted MMP-2 seems to play a vital role in the C2C12 differentiation, as activity was seen during myogenesis; when neutralised with an antibody, an 18% decrease in fusion was observed. Matrigel promoted an increase of MMP-2 expression within the cell during fusion (day 5 of differentiation), while no intracellular MMP-2 protein was observed at day 2 of differentiation. Levels of secreted MMP-2 increased significantly from day 2 to day 5 of differentiation; however, the presence of Matrigel significantly reduced levels of secreted MMP-2 detected in conditioned media at day 5 compared to uncoated conditions. The decrease is, in part, due to the fact that MMP-2 was found to bind to Matrigel. In conclusion, MMP-14 and MMP-2 play an important role in C2C12 myoblast elongation and fusion. This study provides further insight into the role of MMPs in myogenesis and lays the foundation for future work.

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