# _Applied_
## 4 Discussion  
Most neurodegenerative diseases are caused by the death of brain nerve cells due to oxidative stress [29]. An antioxidant can donate an electron or a hydrogen atom, transfer unpaired electrons, and can chelate metals [30]. Since DPPH and ABTS assays can be applied to both water-soluble and fat-soluble antioxidant measurements, they are widely used to measure the antioxidant activity of foods [31]. [PERSON] et al. [32] showed that 70% ethanol extract showed a significantly higher electron donating ability than water extraction, and our analysis showed that SBE had higher radical scavenging activity than SBW (Table 1). Thus, SBE can scavenge free radicals by delivering hydrogen (DPPH) or single electrons (ABTS assay). Cellular antioxidants quench free radicals by transferring a hydrogen or electron, thereby inducing the synthesis of detoxifying and antioxidant proteins in phase II [33]. Substances such as friedelin and glutinol (triterpene-based compounds) and isoorientin (flavonoid-based compounds) have been reported as component studies for SB [33]. Isoorientin, a family of rooibos flavonoids, has antioxidant activity and has been reported to protect cells from 6 OHDA-induced neurotoxicity in PC12 cells [34]. In addition, it protects cells from oxidative damage caused by t-BOOH in HepG2 cells, and has effects on DPPH free radical scavenging activity, iron (III) reducing activity, linoleic acid peroxidation, and MDA formation [35]. Isoorientin reportedly has anti-inflammatory activity and the ability to diminish mitochondrial ROS production [36,37]. Isoorientin and orientn, rooibos flavonoids, enhance mitochondrial function by regulating mitochondrial respiration, leading to a reduced production of ROS in C2C12 skeletal muscle cells [38]. In addition, isoorientin attenuates the oxidative damage induced by oleic acid in buffalo rat liver 3A (BRL-3A) cells [39], and reduces ROS generation in UV-stimulated human dermal fibroblasts [36]. In this study, the isoorientin content of SBE was higher than that of SBW. This is consistent with our finding that the antioxidant and neuroprotective activities of SBE are superior to those of SBW. Therefore, the antioxidant and neuroprotective effects of SBE are likely mediated by isoorientin. However, the bioactive activities of various phytochemicals present in trace amounts cannot be excluded. Further studies on unidentified phenolic acids and flavonoids in SB extracts that mediate its antioxidant effect in vitro and in vivo are warranted. SBE with a high radical scavenging activity reduced the intracellular ROS accumulation induced by H\({}_{2}\)O\({}_{2}\) (Figure 2). This implies that the neuroprotective effect of SBE is mediated by quenching or neutralizing free radicals. The expression of genes encoding antioxidant proteins is induced by molecular mechanisms (Figure 5). Antioxidant response elements (ARE), Nrf2, and Keap1 regulate the expression of genes encoding SOD2, CAT, and GPx [40]. Under normal conditions, Nrf2 is bound to Keap1 in the cytoplasm. Upon exposure to oxidative stress, Nrf2 is released from Keap1, is translocated into the nucleus,and activates phase II antioxidant enzymes, such as heme oxygenase (HO-1) [41; 42]. HO-1 catalyzes the conversion of heme to CO, free iron, and biliverdin, which is converted into bilirubin. Bilirubin has potent antioxidant and cytoprotective activities [43]. Thus, an elevated expression of Nrf2 and HO-1 can protect cells from oxidative damage. SOD catalyzes the conversion of superoxide anion (O2\({}^{-}\)) into a molecule of H2O2. In peroxisomes, CAT converts H2O2 to water (H2O) [44]. Glutathione peroxidase (GPx) contains selenium and catalyzes the degradation of H2O2 to H2O. These enzymes act collectively in the metabolic pathway of free radicals [45; 46]. We found that SBE increased the expression levels of Nrf2 and HO-1 in PC12 cells.