Anti-ferroptotic properties of isolates from traditional South African medicinal plants in a 6-hydroxydopamine-induced SH-SY5Y cytotoxicity model

Abstract

Introduction: Parkinson’s disease (PD), a progressive neurodegenerative disorder, lacks disease-modifying treatments, and thus relies on symptomatic relief only. A potential untapped disease-modifying pathway for PD is the iron-dependent cell death process ferroptosis, which promotes oxidative stress within cells. Medicinal plants in Sub-Saharan Africa are a potential source of new chemical entities or scaffolds for anti-ferroptotic pharmacotherapy. During the study, a series of semi-automated fractions from African herbal remedies with ethnomedicinal claims of central nervous system activities were investigated to protect against 6-hydroxydopamine (6-OHDA)-induced cytotoxicity in the SH-SY5Y neuroblastoma cell line as an in vitro model of PD. Methods: Twenty-four plants were extracted and fractionated to produce a crude extract and seven associated fractions. All samples were screened for both inherent cytotoxicity in the SH-SY5Y cell line, as well as cytoprotection against 6-OHDA-induced cytotoxicity. Acellular ferroptotic-associated assays were conducted, including the iron chelation and ferric reducing antioxidant power (FRAP) assays. Cytoprotective fractions of five plants were selected for further analyses to assess their ability to reduce reactive oxygen species (ROS; 2′,7′-dichlorodihydrofluorescein diacetate assay), modulate glutathione (GSH) levels (monochlorobimane fluorescence), inhibit lipid peroxidation (thiobarbituric acid reactive substances assay), and alter mitochondrial integrity (CytoPainter Red) in the SH-SY5Y cell line. For the fractions subjected to further analyses, compound identification was done using high-performance liquid chromatography mass spectrometry and the Dictionary of Natural Products, and the identified compounds’ physicochemical properties were determined. These compounds were also investigated using in silico molecular docking against ferroptosis-relevant targets. Results and discussion: A total of 24 plants were screened, with only 15 selected fractions that were elucidated to assess their effects ferroptosis role-players. No correlation between FRAP or iron chelation and cytoprotection was present. Of the screened fractions, one fraction of Acokanthera oppositifolia, two fractions of Bulbine latifolia and Aptosimum procumbens, four fractions of Schotia brachypetala and five fractions of Polygala virgata increased cell density by 30% after 6-OHDA exposure and had little inherent cytotoxicity (≤ 10% reduction in cell density). The fractions of P. virgata exhibited significant (p < 0.05) reductions in intracellular ROS, with fraction 4 displaying the largest reduction (2.09-fold). Additionally, P. virgata fractions 3, 5, 6, and 7 demonstrated similar ROS reductions (1.28- to 1.47-fold). A. procumbens fraction 3 increased GSH levels by 25.79%, followed by A. procumbens fraction 4 (22.98%, p < 0.05), B. latifolia fraction 3 (22.96%), and P. virgata fraction 6 (19.25%). These findings suggest that plant fractions from P. virgata and A. procumbens offer promising antioxidant and cytoprotective effects, with fraction 4 of P. virgata being particularly effective in reducing lipid peroxidation (0.284-fold reduction). In addition, several fractions enhanced mitochondrial integrity, with B. latifolia (fraction 5) and S. brachypetala (fraction 3) normalising the active mitochondrial ratio. Physicochemical elucidation via SwissADME shows that there are some compounds with theoretical blood-brain barrier penetration. Molecular docking of identified compounds also provided a base for the investigation of the compounds as potential leads for drug design, with xanthones (P. virgata fractions 5 to 7) and butanedioate (A. procumbens fraction 4) being identified as promising scaffolds. Conclusion: The semi-automated fractions of the selected plants offer a promising approach to mitigating the complex cellular processes involved in oxidative stress patterns and ferroptotic role-players (GSH, lipid peroxides and mitochondrial integrity), but whether the ferroptosis pathway is the underlying pathway modulated for cytoprotection remains unclear. While the results indicate that these compounds may protect cells and maintain mitochondrial function, further research is needed to optimise their pharmacological properties, including bioavailability and blood-brain barrier penetration. Fractions 5 to 7 from P. virgata, due to high ROS scrubbing, and fraction 4 from A. procumbens, due to GSH increases, are currently primed for further research. The research also shows the need to advocate for the systematic investigation and cataloguing of traditional plant medicines, aiming to provide a foundation for the development of novel therapeutic strategies for PD and other neurodegenerative diseases. These results highlight the therapeutic potential of multi-target plant-based therapies in managing PD, supporting the integration of traditional healing practices into modern medicine. Further studies are needed to optimise bioavailability and target specific neuroprotective pathways.