Parkinson’s disease, characterised by loss of dopaminergic neurons in the substantia nigra of the brain, is attributed to oxidative stress and mitochondrial dysfunction. As no cure is available, and dopamine-replacement therapy only offers symptomatic relief, other avenues of treatment are sought. Acokanthera oppositifolia, Boophone disticha and Xysmalobium undulatum are used ethnomedicinally for the treatment of neurological disorders, however, these plants have not been assessed in vitro for cytoprotective activity. The aim of the study was to assess the cytoprotective activity of these three plants in an in vitro SH-SY5Y cellular model of Parkinson’s disease induced by 6-hydroxydopamine (6-OHDA).
Plant material was extracted using acetone and methanol ultrasonic maceration. Cytotoxicity was induced by exposing cells to 33.3 μM 6-OHDA for 2 h, followed by 24 h incubation with the crude extracts. Ultra-performance liquid chromatography high definition mass spectrometry was used for tentative identification of phytochemicals. Cytoprotection was initially assessed using the sulforhodamine B staining assay to determine concentration ranges. Mitochondrial membrane potential (MMP), reactive oxygen species (ROS) levels, reduced glutathione (GSH) content, intracellular Ca2+ flux and ATP levels were assessed using the JC-1 ratiometric, dihydrodichlorofluorescein cleavage, monochlorobamine adduct formation, Fura-2AM and bioluminescence assays, respectively. Cell morphology was visualized using polarisation-optical transmitted light differential interference contrast microscopy.
Several phytochemicals were tentatively identified that are known markers in the plant species, however, little difference was noted between the acetone and methanol extracts qualitatively. Extracts reduced cell density by 91%, increased ROS (217.7%) and GSH (102.1%) levels. Mitochondrial depolarisation (54.2%) was evident. Crude extracts attenuated cytotoxicity by reducing ROS and sustaining ATP production, however, no alteration to MMP was observed. Furthermore, Ca2+ effects were maintained by B. disticha and X. undulatum, but reduced by A. oppositifolia. Morphological changes, characteristic of cytotoxicity, was observed when exposed to 6-OHDA-in the micrographs. Intermediate-polarity extracts reduced the detrimental effects associated with 6-OHDA-induced cytotoxicity. Xysmalobium undulatum (5 μg/mL) displayed the greatest level of cytoprotection, however, the inherent cytotoxicity may limit the usefulness of extracts during treatment of disease.
Although none of the plant extracts showed potential to reverse in vitro characteristics of Parkinson’s disease completely, concomitant use with dopamine replacement therapy should be investigated as a possible treatment modality. Adjunct use of the crude extracts with traditional dopamine replacement therapy may offer an alternative approach, however, future studies are required to elucidate the feasibility of such a combination.