Neuroprotective and antioxidant properties of Polygala virgata fractions in a 6-hydroxydopamine-induced neurotoxicity model

Abstract

INTRODUCTION : Ferroptosis contributes to Parkinson's disease progression given dysregulation of iron homeostasis and redox status. Polygala virgata is used ethnomedicinally for memory enhancement. This study assessed the cytoprotective and antioxidant properties of crude extracts and fractions of P. virgata using a 6-hydroxydopamine-induced (6-OHDA) SH-SY5Y neuroblastoma cytotoxicity model. METHOD : Dried roots of P. virgata (14% w/v) were sequentially extracted using dichloromethane/methanol (1:1) and methanol, which was combined to give a crude extract. The crude extract was separated into seven fractions using different ratios of water, acetonitrile and methanol on solid phase extraction (SPE). Inherent cytotoxicity of the samples (10 μg/mL), as well as their ability to reduce 6-OHDA-induced cytotoxicity (35 μM), was determined using the sulforhodamine B (SRB) assay after 48-h (h) exposure. The active fractions' cytoprotective effect in relation to reactive oxygen species (ROS), glutathione levels (GSH), lipid peroxidation, and mitochondrial integrity was determined fluorometrically. Cytoprotective fractions’ phytochemical constituency was elucidated using liquid chromatography high resolution mass-spectrometry (UPLC-HRMS). RESULTS : Fractions 3 to 7 increased cell density after exposure to 6-OHDA by 31.14%, 28.08%, 30.72%, 40.58% (p < 0.01) and 28.86%, respectively, with no inherent cytotoxicity observed. Fraction 4 reduced 6-OHDA-induced ROS generation (2.09-fold) and lipid peroxidation (0.28-fold). Non-significant increases in GSH were noted (1.34 to 19.25%), while all fractions hyperpolarised the mitochondrial membrane. Multi-hydroxylated xanthones, flavones and flavans were tentatively identified using UPLC-HRMS. CONCLUSION : P. virgata fractions reduced 6-OHDA-induced cytotoxicity via decreased oxidative stress and hyperpolarisation of the mitochondrial membrane, most likely ascribed to the identified xanthones, flavones and flavans. Isolation and purification of these compounds are warranted as potential antioxidant scaffolds.

HIGHLIGHTS • First in vitro neuroprotective activity of Polygala virgata identified. • Little to no cytotoxicity present in fractions. • Neuroprotection linked to reactive oxygen species reduction (F4) and glutathione modulation (F6). • Possible synergism with identified compounds due to ratiometric differences.