DOI: 10.25381/cput.25429264.v2
title: The neuroprotective effects of Rooibos extracts against oxidative stress-induced cytotoxicity in human neuroblastoma SH-SY5Y cells

description: Oxidative stress, a key player in neurodegenerative disorders such as Alzheimer's and Parkinson's disease, is a critical target for therapeutic interventions. Plant polyphenols demonstrate substantial neuroprotective effects in numerous neurodegenerative disorders, underpinning future preclinical and clinical studies. South African Rooibos (Aspalathus linearis) herbal tea, which is widely consumed for its health benefits and caffeine-free nature, has been shown to positively affect various processes observed in the pathogenesis of neurodegenerative disorders. In this study, we investigate the neuroprotective potential of fermented and unfermented aqueous and ethanolic Rooibos extracts on neuroblastoma cells, addressing a critical gap in current research. Aqueous and ethanolic extracts [15-500 µg/mL] were evaluated in an SH-SY5Y cellular model subjected to oxidative stress induced by hydrogen peroxide (H2O2). The antioxidant properties of the extracts were measured using cell-free systems. The total antioxidant content and capacity of the Rooibos extracts were determined by measuring using total polyphenol content (TPC), trolox equivalent antioxidant capacity (TEAC) and ferric ion reducing antioxidant power (FRAP). HPLC was employed to quantify major phenolic compounds, revealing aspalathin as the predominant polyphenol. The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay was used to assess the maximum non-toxic dosage (MNTD) of Rooibos extracts against SH-SY5Y cells. Subsequently, the cells were pretreated with the MNTDs of Rooibos extracts, and thereafter exposed for 3 hours to 150 µM H2O2. The effect of Rooibos extracts on cell viability and metabolic activity was determined using MTT and intracellular adenosine triphosphate (ATP) assays; oxidative status was assessed using thiobarbituric acid reactive substance (TBARS), conjugated diene (CDs), protein carboxylation, and reduced glutathione (GSH) assays; and endogenous antioxidant enzyme activity in SH-SY5Y cells was assessed using superoxide dismutase (SOD) and catalase (CAT) colorimetric assays. In addition, the activities of lactate dehydrogenase (LDH) and caspase -3/7, -8, and -9 were used to measure cellular death. Rooibos extracts exhibited a dose-dependent increase in antioxidant capacity in cell-free systems. The results showed that treatment with Rooibos extracts did not reduce cell viability of SH-SY5Y cells. Furthermore, when SH-SY5Y cells were exposed to H2O2, Rooibos extracts exhibited dose-dependent cytoprotective effects. Rooibos fermented aqueous extract at 60 µg/mL increased (P≤0.05) intracellular ATP levels, while the fermented ethanolic extract at 125 and 250 µg/mL increased (P≤0.001) intracellular ATP levels and (P≤0.001), respectively. Both unfermented and fermented aqueous and ethanolic extracts reduced CDs, an early marker of lipid peroxidation (P≤0.01, and P≤0.001, when compared to cells treated only with H2O2), except for fermented aqueous extract at 60 µg/mL, where CDS formation was comparable to cells treated only with H2O2. Furthermore, a significant decrease in protein carbonyls was observed in cells pre-treated with 250 µg/mL of unfermented aqueous Rooibos extract (P≤0.01), 125 and 250 µg/mL of unfermented ethanolic Rooibos extract (P≤0.001) and (P≤0.05), respectively and 250 µg/mL of fermented ethanolic Rooibos extract (P≤0.001) compared to cells treated only with H2O2. Pre-treatment with 60 µg/mL of unfermented ethanolic Rooibos extract prevented H2O2 depletion of reduced GSH (P&lt;0.05). Both Rooibos extracts significantly (P≤0.01, P≤0.001) restored endogenous antioxidant enzyme, CAT activity, with varying effects on SOD enzyme activity in the cells. The current study demonstrates that Rooibos, a renowned South African herbal tea at certain concentrations, may enhance the endogenous antioxidant defence system in oxidatively challenged H2O2 human neuroblastoma SH-SY5Y cells. However, this was not the case for all concentrations tested in this model, implying that Rooibos may possess pro-oxidant properties at certain concentrations. The interactions between Rooibos and H2O2 must be further elucidated. Nevertheless, these findings suggest a compelling avenue for the use of Rooibos in preventative strategies against neurodegenerative disorders, including Alzheimer's disease and Parkinson’s disease.