Steenkamp, Vanessa2025-02-262025-02-262025-042024-12*A2025http://hdl.handle.net/2263/101245Dissertation (MSc (Pharmacology))--University of Pretoria, 2024.Parkinson’s disease is an incurable, progressive disorder characterised by the loss of dopaminergic neurons in the brain's substantia nigra, primarily due to oxidative stress and mitochondrial dysfunction. Current treatments focus on symptomatic relief, by increasing endogenous dopamine levels, mimicking dopamine's function, and slowing oxidative metabolism, but do not prevent disease progression. Medicinal plants are widely used to treat neurological disorders. The aim of this study was to evaluate the effects of crude and fractionated extracts of Catha edulis and Datura stramonium, plants known for their psychoactive properties and traditional use in treating neurological disorders, on 6-hydroxydopamine (6-OHDA)-induced cytotoxicity in the SH-SY5Y human neuroblastoma cell line, a model for Parkinson's disease. Catha edulis (leaves) and Datura stramonium (leaf/root mixture) crude extracts were prepared using dichloromethane/methanol (50/50). Fractionation of the extract was conducted using semi-automated C8 solid phase extraction. Ultra performance liquid chromatography mass spectrometry analysis was employed to determine the presence of chemotaxonomic markers of the plants. Inherent cytotoxicity, of the crude extract and fractions (1, 3.1, and 10 μg/mL per fraction) and the cytoprotective ability of the crude extract and fractions against SH-SY5Y cells after 6-OHDA-induced cytotoxicity (72.83 µM), was assessed using the sulforhodamine B (SRB) assay after 48 h exposure. The half maximal inhibitory concentration (IC50) values of commercially purchased pure compounds (atropine and scopolamine) and their cytoprotective effects against SH-SY5Y cells were assessed using the SRB assay after 48 h exposure. Cell morphology was visualised using phase contrast microscopy. Intracellular reactive oxygen species (ROS) levels were determined using the diacetyl dichlorofluorescein assay (H2DCFDA). Mechanistic assays were performed using a single concentration (3.1 μg/mL) due to the inconsistent dose-dependent responses and unclear results in the cytoprotection and intracellular ROS assays at lower and higher concentrations. Caspase 3/7 activity was determined by the Acetyl-Asp-Glu-Val-Asp-7-amido-4-methylcoumarin Ac-DEVD-AMC) cleavage assay and mitochondrial membrane integrity using the mitochondrial staining assay. In silico analysis was performed to assess the binding affinities and interactions of chemotaxonomic markers identified in the plant crude extracts with dopamine 1 and 2 (D1 and D2) receptors. Chemotaxonomic markers detected in C. edulis were cathine and norephedrine, while atropine, hyoscyamine, and noradrenaline, were identified in D. stramonium. After 48 h treatment, both plant extracts and fractions exhibited minimal cytotoxicity. The crude extract and all fractions of C. edulis provided cytoprotection of between 5.8 and 10.07%, with fraction 1 displaying the highest cytoprotection. All fractions and the crude extract of D. stramonium provided cytoprotection ranging from 22.56 to 34.28%, with fraction 4 exhibiting the highest cytoprotection. The IC50 of the pure compounds, atropine and scopolamine, was determined as 49.48 µM and 48.26 µM, respectively. Atropine increased cell density by 30.69%, and scopolamine by 27.83%, indicating cytoprotective activity for both compounds. Microscopic analysis confirmed these results. 6-Hydroxydopamine reduced the cell density by approximately 18.62%, increased intracellular ROS generation by 2.82-fold, increased caspase 3/7 activation by 4.17-fold, and increased the ratio of healthy mitochondria to cell density by 0.5 relative to the negative control. All fractions of C. edulis and D. stramonium reduced intracellular ROS levels between 1.21 to 2.66-fold and 1.12 to 1.82-fold, respectively. Additionally, all fractions significantly (p < 0.0001) reduced caspase 3/7 activation, with C. edulis and D. stramonium reducing this parameter by approximately 1.35-fold and 3.24-fold, respectively, compared to 6-OHDA. All fractions significantly (p < 0.05) decreased the mitochondrial membrane integrity of healthy mitochondria to cell density by 0.59 and 0.88 for C. edulis and D. stramonium, respectively, compared to the 6-OHDA. In silico analysis revealed that norephedrine and noradrenaline exhibited high binding affinities for both D1 and D2 receptors, with the binding free energy of -4.34 and -6.73 kJ/mol for D1, and -5.91 and -7.43 kJ/mol for D2, respectively. Both plant extracts and their fractions maintained cell viability, provided cytoprotection, reduced ROS and caspase 3/7 activity, and preserved mitochondrial integrity. These findings suggest that C. edulis and D. stramonium extracts could provide protection against cellular damage caused by Parkinson's disease, indicating potential for future research into their therapeutic applications. Further studies should explore the mechanisms and efficacy of these extracts in vivo.en© 2023 University of Pretoria. All rights reserved. The copyright in this work vests in the University of Pretoria. No part of this work may be reproduced or transmitted in any form or by any means, without the prior written permission of the University of Pretoria.UCTDSustainable Development Goals (SDGs)Oxidative stressParkinson's diseasePlant extractsCytotoxicityCytoprotectionDissertationu19236612https://doi.org/10.25403/UPresearchdata.28498004