Browsing by Author "White, Charlize"

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    Inhibitory effect of selected herbal supplements on CYP450-mediated metabolism : an in vitro approach
    (Stellenbosch : Stellenbosch University, 2016-03) White, Charlize; Rosenkranz, Bernd; Bouic, Patrick J. D.; Stellenbosch University. Faculty of Medicine and Health Sciences. Dept. of Medicine. Clinical Pharmacology.
    ENGLISH ABSTRACT: INTRODUCTION Herbal products are popularly used as complementary and alternative medicines to treat a variety of conditions. Often patients use them in conjunction with conventional medicines. Herbal products contain many pharmacologically active phytochemicals that may interfere with the absorption, distribution, metabolism, and elimination of medicines. This interaction can lead to an increase of the plasma concentrations of other medicines to toxic levels, or to their decrease below therapeutic levels, resulting in lack of efficacy. The liver cytochrome P450 (CYP) enzymes are responsible for the metabolism of a large majority of medicines. In order to provide more information on the potential interaction between African herbal medicines and conventional medicines, the present study has investigated the inhibition of selected CYP enzymes by three popular South African medicinal plants, Buchu, African ginger, and Warburgia salutaris. METHODS Buchu capsules, African ginger, and Warburgia salutaris tablets were obtained in a local pharmacy. 60% methanol/water extracts were prepared and analysed by GC-MS to reveal the composition of the volatile components of each product. Fluorogenic inhibition assays were conducted using Vivid® recombinant CYP screening kits according to the manufacturer’s protocol. This protocol included the pre-incubation of herbal extracts, recombinant CYP isoform and cofactor solution. The metabolic reaction was initiated by the addition of CYP-specific substrate and NADP+; the solution was incubated for 30 minutes at 37°C, after which fluorescence was measured using a microplate reader. The percentage remaining activity was calculated and used to determine the IC50 values of each herbal product. Time - dependent inhibition (TDI) was evaluated using the normalized ratio, NADP+-, concentration -, and time - dependent approaches. RESULTS The GC-MS analysis revealed monoterpenes, sesquiterpenes, and alkane hydrocarbons in the volatile component. Warburgia salutaris, African ginger, and Buchu inhibited CYP2C19 with IC50 values of 5.88 μg/ml, 32.38 μg/ml, and 53.52 μg/ml, respectively. Likewise, the IC50 values of 5.64 μg/ml, 1.09 μg/ml, and > 100 μg/ml were obtained for inhibition of CYP3A4 by Warburgia salutaris, African ginger, and Buchu, respectively. Using the normalized ratio, Warburgia salutaris and African ginger showed time- and concentration - dependent inhibition of CYP1A2, and Buchu showed intermediate TDI effects that were not concentration dependent. All three extracts showed TDI of CYP3A4; the inhibition displayed by Buchu and Warburgia salutaris was NADP+ dependent. African ginger was the only extract to show NADP+ dependent inhibition of CYP1A2. A kinetic TDI assay Stellenbosch University https://scholar.sun.ac.za iii showed that the IC50 value of African ginger decreased over time, indicating TDI. Warburgia salutaris was not a time-dependent inhibitor of CYP3A4, and Buchu may have a limited time-dependent inhibitory effect. CONCLUSION Warburgia salutaris, African ginger, and Buchu have the potential to cause clinically relevant herb-drug interaction, if sufficient concentrations are achieved in vivo. Further studies are needed to confirm this finding.
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    Protein phosphorylation in Ischaemia and reperfusion of the heart : a focus on protein phosphatase 2A
    (Stellenbosch : Stellenbosch University, 2020-03) White, Charlize; Van Vuuren, Derick; Marais, Erna; Lochner, Amanda; Stellenbosch University. Faculty of Health and Medical Sciences. Dept. of Biomedical Sciences: Medical Physiology.
    ENGLISH ABSTRACT: Understanding the mechanism and signalling pathways involved in myocardial ischaemia/reperfusion (I/R) injury is a prerequisite for elucidating any novel therapeutic intervention to reduce I/R injury. Historically, protein kinases have been the focus of research on I/R injury while information regarding phosphatases in I/R injury is lacking. Clarifying the role of phosphatases in the cellular response to I/R injury is critical to enhance existing cardioprotective interventions or identify new therapeutic targets. This study aimed to investigate the effects of protein phosphatase 2A (PP2A, a major phosphatase in the heart) inhibition on the outcomes of I/R and to characterize the phosphoproteome of myocardial tissue exposed to these interventions. For this study, tissue was generated using the isolated working mouse heart model. Administration of a PP2A inhibitor, okadaic acid (OA; 50 nM), prior to 20 minutes global ischaemia (GI) reduced infarct size (28 ± 19%) when compared to control (56 ± 23%; n=9; p < 0.0106), but did not exert a major effect on functional recovery. Phosphoproteomic analysis conducted on hearts exposed to OA prior to 20 minutes GI followed by 10 minutes reperfusion revealed that 898 proteins and 184 phosphorylated proteins were influenced by PP2A inhibition. Pathway analysis indicated that the major pathways affected by PP2A inhibition were mitochondrial function and oxidative phosphorylation, epithelial adherens signalling and remodelling, and cytoskeletal signalling. Two proteins from the data set, striatin and heat shock protein (HSP) 90α, were selected for confirmation and further investigation. Mitochondrial respiration was also investigated. In line with the phosphoproteomic results, OA administration increased the amount of phosphorylated HSP90α during reperfusion (1.83 ± 0.83 Arbitrary Units (AU) vs 0.69 ± 0.09 AU; n = 2-4; p = 0.0480) and increased the amount of striatin during reperfusion (1.93 ± 0.84 AU vs 0.80 ± 0.06 AU; n = 2-4; p = 0.0436). Striatin, but not HSP90α coimmunoprecipitated with PP2A, indicating that the increased phosphorylation of HSP90α was an indirect effect of PP2A inhibition. As striatin recruits regulatory proteins into large signalling complexes that mostly include PP2A, co-immunoprecipitation may indicate the presence of these complexes in the heart. OA was also administered directly to isolated mitochondria, where oxidative phosphorylation analysis was performed polarographically (using a Clark-type electrode). No differences in oxidative phosphorylation or respiratory control ratio were observed. Mitochondria were separated into the outer mitochondrial membrane (OMM) and mitoplast (inner mitochondrial membrane and matrix) using digitonin. Western blot analysis indicated that that catalytic and scaffolding subunits of PP2A are probably localized to the OMM, while striatin is located within the mitoplast, a novel finding. Finally, following an incidental observation, the inhibitory effects of sodium orthovanadate (SOV) on PP2A was investigated. This study is the first to demonstrate that SOV is a weak, competitive inhibitor of PP2A (IC50 ≈ 2.2 mM). This study is, to our knowledge, the first to use a phosphoproteomic approach to investigate the effects of phosphatase inhibition during myocardial I/R. Our data indicate that PP2A is associated with numerous proteins and processes during I/R, including oxidative phosphorylation and cytoskeletal dynamics.