Department of Biochemistry and Microbiology

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Browsing Department of Biochemistry and Microbiology by Author "Bradley, G."

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    A comparative study of the in vitro antidiabetic properties, cytotoxicity and mechanism of action of Albuca bracteata and Albuca setosa bulb extracts
    (University of Fort Hare, 2015) Odeyemi, Samuel Wale; Bradley, G.
    The search for cheap, non toxic and readily available antidiabetic drugs has been a challenge for researchers and the pharmaceutical industries. Diabetes mellitus is a metabolic disease characterized by defects in the synthesis of insulin and/or insensitivity to the action of insulin at the target cells. The disease has been on the increase mostly in developing countries where large proportions of the population have little access to good medical care due to either accessibility or non availability of synthetic drugs. This has led to the use of medicinal plants to treat diabetes because it is safe, cheap and with few side effects. There is little scientific evidence on the dosages, active compounds, mechanisms of action and toxicity of these traditionally used plants. Two of the most frequently used plants; Albuca setosa and Albuca bracteata were investigated in this study. The qualitative analysis of different extractions of these plants revealed the presence of phenolics, alkaloids, tannins and saponins. The antioxidant properties of aqueous, acetone and methanollic extracts of Albuca setosa and Albuca bracteata were investigated using models such as Diphenyl-1-Picrylhydrazyl (DPPH), 2, 2’-azino-bis (3-ethylbenzothiazoline-6-sulphonic acid) (ABTS), Ferric ion reducing antioxidant potential (FRAP), Nitric Oxide and Hydrogen Peroxide (H2O2). Both plants revealed inhibitions against DPPH in a concentration - dependent manner with Albuca setosa (0.330 mg/ml) showing higher activity than Albuca bracteata (0.647 mg/ml) determined from the IC50. The aqueous extract of Albuca setosa showed a higher inhibition against DPPH radical compared to the Albuca bracteata aqueous extract at all concentrations investigated. The isolated saponins from Albuca bracteata had a higher DPPH scavenging activity than the crude methanolic extract of the plant in a concentration - dependent manner but are significantly different from each other at 0.4, 0.6 and 1.0 mg/ml only. The IC50 of the saponins was also observed to be higher than the crude extracts and standards. The Albuca setosa aqueous extract showed a higher percentage inhibition of ABTS radicals than Albuca bracteata at all the concentrations investigated. Overall, the Albuca setosa aqueous extract (0.0809 mg/ml) showed maximum activity against ABTS radicals. The iron reducing power was significantly higher (P < 0.05) in the methanolic extract of both plants compared to the aqueous counterpart. Overall, the Albuca bracteata aqueous extract (0.344 mg/ml) showed maximum activity as indicated by the IC50. The aqueous extracts of both plants also revealed percentage inhibitions in a concentration - dependent manner against NO2. The aqueous extract of Albuca bracteata bulb was more active against nitric oxide and hydrogen peroxide inhibition. In this study, the cytotoxicity of the extracts was evaluated at a high dose of 100 μg/ml on Chang liver cells and determined using MTT, crystal violet, glucose consumption, lactate production and lactate dehydrogenase release and FRAP. The aqueous extracts of both Albuca setosa and Albuca bracteata were non-toxic on Chang liver cells at the concentrations investigated. The MTT revealed that the aqueous extract of Albuca setosa bulb had the optimum cell viability of 108.09% while the acetonic extract of Albuca bracteata showed the least cell viability (37.72%) compared with the control. The crystal violet test also revealed the acetone extract of Albuca bracteata to have the least percentage of cell viability at 31.47%, while the aqueous extract of Albuca setosa showed the maximum cell viability at 112.5%.The aqueous extracts of both plants showed higher percentage cell density on the second day of incubation from the proliferation assay. All the tested samples were observed to consume more glucose than the blank except for the methanollic and acetone extracts of Albuca bracteata bulb. The aqueous and methanolic extracts of Albuca setosa bulbs produced the highest lactate with 120.2 μg/ml and 113.7 μg/ml respectively. The acetone extracts of both Albuca setosa and Albuca bracteata revealed toxicity with a higher lactate dehydrogenase release compared to the control. The aqueous extracts of both Albuca setosa and Albuca bracteata bulbs showed reducing power of 18.7 and 22.1 μmol/l of FeSO4 respectively. The in vitro antidiabetic potentials investigated revealed that the aqueous extracts of both plants inhibited the activity of α- glucosidase but have weak inhibition against α- amylase. The acetone extract of Albuca bracteata showed the highest inhibition against the alpha amylase followed by the acetone extract of Albuca setosa. Both plant extracts also showed weak inhibition against the dipeptidylpeptidase-IV (DPP-IV) at the concentration investigated. The glucose uptake in HepG2 for both aqueous and methanol extracts of Albuca setosa were not significantly different from the control but the MTT result of the treatments in HepG2 for both Albuca setosa and Albuca bracteata aqueous extracts revealed low cell density compared to the control. The aqueous extract of Albuca setosa demonstrated glucose utilization in L6 cells with a response of 188.93% of the control at 25 μg/ml while the aqueous extract of Albuca bracteata demonstrated glucose utilisation in L6 with a response of 89.17% of the control. The MTT of the treatments in L6 revealed no sign of cytotoxicity, as the cell density of all the treatments were not significantly different from the control. The aqueous extracts of both Albuca setosa and Albuca bracteata also showed high glucose utilization in 3T3-L1 cells with 123.21% and 131.56% respectively. In conclusion, Albuca setosa possesses better in vitro strong antidiabetic activity compared to Albuca bracteata. The mechanism of glucose utilization in L6 and 3T3-L1 of Albuca setosa and Albuca bracteata may be due to the activation of some molecules in the insulin signaling pathway, while the in vitro antidiabetic activity of Albuca bracteata may also involve its inhibitory effect on α-glucosidase. The antioxidant properties of both plants may also play an important role in ameliorating the complications of diabetes. These findings therefore support the folkloric usage of these plants for the management of diabetes mellitus.
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    Identification of agricultural and industrial pollutants in the Kat river, Eastern Cape and their effect on agricultural products found along the river banks.
    (University of Fort Hare, 2015) Mutingwende, Nhamo; Bradley, G.
    There is growing concern that commonly used Pharmaceuticals and Personal Care Products (PPCPs) and pesticides are entering and contaminating drinking water supplies. The use of targeted quantitation of PPCP has been well established but there is an emerging trend to also screen for and identify unexpected environmental pollutants. Chemicals like pesticides hormones and antibiotics are especially of interest because of proven endocrine disrupting effects and a possible development of bacterial resistance. Powerful screening methods are required to detect and quantify the presence of these compounds in our environment. PPCP encompass a wide range of pollutants, including Endocrine Disrupting Compounds (EDC), pesticides, hormones, antibiotics, drugs of abuse, x-ray contrast agents and drinking water disinfection by-products to name a few. In order to properly assess the effects of these compounds on our environment, it is necessary to accurately monitor their presence. The diversity of chemical properties of these compounds makes method development challenging. LC/MS/MS is able to analyse polar, semi-volatile, and thermally labile compounds covering a wide molecular weight range. The new AB SCIEX TripleTOF™5600 LC/MS/MS was used to profile environmental samples for unexpected pollutants, to identify and characterise the chemical composition and structure of the pollutants, and to quantify (based on intensity) the concentration in collected water samples. Liquid Chromatography coupled to tandem Mass Spectrometry (LCMS/ MS) is able to analyse polar, semi-volatile, and thermally labile compounds covering a wide molecular weight range, such as pesticides, antibiotics, drugs of abuse, x-ray contrast agents, drinking water disinfection by-products etc. More recently there is a growing interest from environmental researchers to also screen for and identifies non-targeted compounds in environmental samples, including metabolites and degradates, but also completely unexpected pollutants. The new AB SCIEX TripleTOF™5600 LC/MS/MS system is capable of performing highly sensitive and fast MS scanning experiments to search for unknown molecular ions while also performing selective and characteristic MS/MS scanning for further compound identification and, therefore, is the instrument of choice for this challenging task. General unknown screening workflows do not use a target analyte list and compound detection is not based on any prior knowledge, including retention times and information on possible molecular and fragment ions. Therefore, acquired chromatograms are very rich in information and can easily contain thousands of ions from both any compounds present in the sample as well as from the sample matrix itself. Thus, powerful software tools are needed to explore such data to identify the unexpected compound. Water samples were collected both upstream and downstream of two WWTPs (Seymour and Fort Beaufort) and were directly injected on the AB SCIEX TripleTOF™5600 LC/MS/MS after being filtered. 15 sample points along the Kat River, ranging from a point as close to the source as possible to a point just before it joins the Great Fish River were used. The samples collected from the source were used as the control in each of the experiments, the assumption being the closer you get to the source, the less contaminated the water would be for the analysis of pesticides. Points were selected where the Kat River crosses the R67 or on farms where the river was accessible using farm roads. Samples were collected from October 2013 to November 2014.The Peak view software and Analyst software were used in the analysis of PPCPs. The XIC Manager allows you to manage large lists of compounds and perform automatic extracted ion chromatogram (XIC) calculations and review results operations. The results were displayed in the chromatogram pane and the XIC table (see results). The results reported here in this thesis indicate that there is contamination in the Kat River water due to both pesticides and PPCPs. The results also indicate that the food products are also contaminated and hence both the Kat River agricultural produce and its water need to be closely monitored for both pesticide and PPCPs contaminants. Further studies to investigate the quantitative levels of pesticides and PPCPs in the Kat river water to determine if the concentration levels of the detected pesticides are below the reported Maximum Residues Limits will be explored in the future.
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    Mechanism and synchronicity of wheat (Triticum aestivum) resistance to leaf rust (Puccinia triticina) and Russian wheat aphid (Duiraphis noxia) SA1.
    (2016) Njom, Henry Akum; Bradley, G.
    Wheat (Triticum aestivum and T. Durum) is an extremely important agronomic crop produced worldwide. Wheat consumption has doubled in the last 30 years with approximately 600 million tons consumed per annum. According to the International Maize and Wheat Improvement Center, worldwide wheat demand will increase over 40% by 2020, while land as well as resources available for the production will decrease significantly if the current trend prevails. The wheat industry is challenged with abiotic and biotic stressors that lead to reduction in crop yields. Increase knowledge of wheat’s biochemical constitution and functional biology is of paramount importance to improve wheat so as to meet with this demand. Pesticides and fungicides are being used to control biotic stress imposed by insect pest and fungi pathogens but these chemicals pose a risk to the environment and human health. To this effect, there is re-evaluation of pesticides currently in use by the Environmental Protection Agency, via mandates of the 1996 Food Quality Protection Act and those with higher perceived risks are banned. Genetic resistance is now a more environmental friendly and effective method of controlling insect pest and rust diseases of wheat than the costly spraying with pesticides and fungicides. Although, resistant cultivars effectively prevent current prevailing pathotypes of leaf rust and biotypes of Russian wheat aphid from attacking wheat, new pathotypes and biotypes of the pathogen/pest may develop and infect resistant cultivars. Therefore, breeders are continually searching for new sources of resistance. Proteomic approaches can be utilised to ascertain target enzymes and proteins from resistant lines that could be utilised to augment the natural tolerance of agronomically favourable varieties of wheat. With this ultimate goal in mind, the aim of this study was to elucidate the mechanism and synchronicity of wheat resistance to leaf rust (Puccinia triticina) and Russian wheat aphid (Duiraphis noxia) SA1. To determine the resistance mechanism of the wheat cultivars to leaf rust infection and Russian wheat aphid infestation, a proteomics approach using two-dimensional gel electrophoresis was used in order to determine the effect of RWA SA1 on the wheat cultivars proteome. Differentially expressed proteins that were up or down regulated (appearing or disappearing) were identified using PDQuestTM Basic 2-DE Gel analysis software. Proteins bands of interest were in-gel trypsin digested as per the protocol described in Schevchenko et al. (2007) and analysed using a Dionex Ultimate 3000 RSLC system coupled to an AB Sciex 6600 TripleTOF mass spectrometer. Protein pilot v5 using Paragon search engine (AB Sciex) was used for comparison of the obtained MS/MS spectra with a custom database containing sequences of Puccinia triticina (Uniprot Swissprot), Triticum aestivum (Uniprot TrEMBL) and Russian wheat aphid (Uniprot TrEMBL) as well as a list of sequences from common contaminating proteins. Proteins with a threshold of ≥99.9% confidence were reported. A total of 72 proteins were putatively identified from the 37 protein spots excised originating from either leaf rust or Russian wheat aphid experiments. Sixty-three of these proteins were associated with wheat response to stress imposed by RWA SA1 feeding while 39 were associated with infection by Puccinia triticina. Several enzymes involved in the Calvin cycle, electron transport and ATP synthesis were observed to be differentially regulated suggesting greater metabolic requirements in the wheat plants following aphid infestation and leaf rust infection. Proteins directly associated with photosynthesis were also differentially regulated following RWA SA1 infestation and P. triticina race 3SA145 infection including a key enzyme of the Calvin cycle, ribulose bisphosphate carboxylase/oxygenase (RubisCO. These changes observed may reflect the reallocation of metabolites from normal growth processes to defensive functions after induction of plant responses by aphid feeding as well as pathogen infection. Some of the differentially accumulated proteins were found to be involved in ATP synthesis. The increased energy demand by the infected or infested plants was to cope with the impeding stress. Also, a few of the differentially regulated proteins were found to be related to stress. Most of these proteins were expressed early in the resistant cultivar but only occurred much later in the moderately resistant cultivar. These results suggest that RWA SA1 infestation and leaf rust infection of wheat may result in an increase photosynthesis, photorespiration, ATP synthesis and production of stress related proteins to cope with the stress. The effect of leaf rust infection on wheat plant colonization with RWA was evaluated. Two wheat cultivars, SST 347 (resistant to both leaf rust race 3SA145 and Russian wheat aphid biotype SA1) and SST 356 (susceptible to both 3SA145 and RWA SA1) were grown to the 2-3 leaf stage. Treatments consisted of untreated control (wheat seedling infested with aphids only) and test (wheat seedlings inoculated with urediniospores of Puccinia triticina race 3SA145 suspended in Soltrol-170® and later infested with aphids at day 3, 5, 7 and 9 post infection). Aphid population on each plant was determined by counting the total number of aphids on each leaf with the aid of a hand lens for 21 days post infestation. Disease response in inoculated seedlings was scored using the 0 to 4 infection type scale. Results obtained showed infection types; 1+ and 3++ for SST 347 and SST 356 respectively. Few aphids chose to colonise previously infected plants at the beginning as aphid numbers on the preinfected SST 347 only reached >10 after day 16 whereas on preinfected SST 356 reached >10 after day 13-15. We therefore concluded that preinfection with leaf rust delayed aphid colonization of both the resistant SST 347 and susceptible SST 356 cultivars to the same extent. Therefore prior infection of wheat plants under laboratory conditions appears to induce resistance to Russian wheat aphids (antixenosis) in both resistant and susceptible cultivars. However, this resistance was later overcome with doubling of aphid populations occurring at a higher rate on the susceptible cultivar as compared to the resistant cultivar. This was the first study to show that infection of plants with rust primed resistance to insect herbivores.