Redox State in Moringa Oleifera Treated SH-SY5Y Neuroblastoma Cell Line

Abstract

Moringa Oleifera (MO), the “Miracle Tree”, is a dietary staple that originates from India. All edible components of the tree (pods, flowers, seeds, bark, leaves) have been extensively studied and showed a richness in an array of essential amino acids, vitamins, minerals, and antioxidants. Evidence-based studies highlight the significance of the administrative treatment of MO extracts as a preventative measure against cancer, heart disease, diabetes, hepatitis, and infectious diseases. However, the majority of studies are conducted with ethanolic extracts. This study depicts the in vitro effect of aqueous MO leaf extract on human neuronal SH-SY5Y. MO extract was prepared by soaking leaves powder in hot water to mimic consumers' daily consumption as a hot tea. The viability of MO-treated cells at conc. 0.01%-0.08% was tested using MTT and trypan blue exclusion assays at 24 and 48 hours. Changes in reactive oxygen species (ROS), NADPH, and ATP levels in treated cells were measured using NBT, NADPH, and ATP luminescence assays respectively. The possible protective effect of pre-/ or co-treatment of antioxidants NAC, MitoQ, and catalase with MO was determined. Molecular docking was conducted on phytochemicals found within MO (quercetin, gallic acid, p-coumaric acid, and 4-hydroxy 3-methoxy cinnamic acid) and their interaction with antioxidant enzymes (SOD, NOX, CAT, & GR). Treatment of SH-SY5Y cells with MO extract (24 and 48 hours) induced a dose- dependent, time-independent a) decrease in viability by 85% to 25%, b) increase in ROS by ~5% to ~60% at conc. of 0.02%-0.03% respectively, with c) a preliminary decrease in ATP levels. In addition, at MO % ≤ 0.02, an increase in NADPH levels was obtained in a time-dependent manner that maintained ROS levels. However, pre-/ co- treatment with antioxidants NAC, MitoQ, and catalase exhibited no protective effect in restoring viability. Following this, the docking of the phytochemicals with antioxidant enzymes had negative binding affinities, with quercetin demonstrating the highest affinity. Three of the antioxidants (NOX, SOD & GR) displayed binding affinity with all phytochemicals except catalase, which only had affinity with quercetin. In essence, MO treatment exerted a cytotoxic effect on SH-SY5Y cells. While glial cells (N9) were more sensitive, the neuronal cells illustrated a higher sensitivity compared to previously tested cell lines (A549, CaCo2, HepG2). The dose-dependent decrease in viability may be attributed to the increase in ROS levels, which were in line with our previous findings on other cell lines. The increase in NADPH levels may have resulted from a) the inhibition of NADPH oxidases (NOX) by one or more MO

2 components, b) the activation of antioxidant enzymes, and c) the differential inhibitory or stimulatory effect (Km, Vmax) of various MO components on pro/ anti-oxidant metabolic enzymes. Our findings in molecular docking using the computational analysis tool PyRx indicate favorable interactions between phytochemicals and pro/ anti-oxidant enzymes. Future metabolomics will further elucidate the metabolite profiles of different pathways in MO-treated compared to non-treated neuronal cells.