# Cytoprotective-Antioxidant Effect of _Brunfelsia grandiflora_ Extract on Neuron-like Cells
## 4 Discussion  
A very recent chemical characterization of _Brunfelsia grandiflora_ has revealed that its main phenolic components are found in high amounts and that all these bioactive compounds support the traditional use of this solanacea as a medicinal therapy in the Peruvian Amazon [9]. In the same study, the in vitro antioxidant capacity of _B. grandiflora_ extracts was reported [9], and data from our laboratory suggest the antioxidative stress capacity of the extracts in cultured endothelial cells [27]. In the present study, _Brunfelsia grandiflora_ extract was prepared and its cytoprotective and antioxidative stress potential was evaluated in a culture of neuron-like cells. Realistic concentrations of the extract showed significant cytoprotection of neuron-like cells in culture, including the restriction of ROS generation and apoptotic activity as well as a remarkable antioxidant effect and molecular regulation of oxidative stress/cell death-related genes. The selection of the range of extract doses of _B. grandiflora_ to assay the antioxidant capacity was based on data from preceding studies testing the neuroprotective activity of plant extracts and other plant-derived products. In the last few years, a range of doses of different plant extracts like that used in this study has proved effective in neuronal  
Figure 5: Effects of _B. grandiflora_ extract (50–200 \(\upmu\)M) on the mRNA levels of oxidative stress-related genes (**A**) NFxB, (**B**) SOD, and (**C**) NRF2 in SH-SYSY cells after co- (green bars) and pretreatment (blue bars). mRNA expression was normalized with respect to control (mean \(\pm\) SEM) of three replicates. Different letters (a, b, c, d) show significance (\(p<0.05\)) between groups. \(\uparrow\) represents increase (%) and \(\downarrow\) represents decrease (%) compared with control and compared with t-BOOH, respectively. and glial protection against induced challenges. Thus, 10-20 \(\upmu\)g/mL of extracts from _Tinospora cordifolia_ showed neuroprotective and neurodegenerative potential against glutamate-mediated excitotoxicity [28]. Likewise, 10-100 \(\upmu\)g/mL concentrations of _Danggui Buxue Tang_, an ancient Chinese herbal decoction, protected against \(\upbeta\)-amyloid-induced cell death in cultured cortical neurons [29]. Similarly, 1-50 \(\upmu\)g/mL extracts from _Lithospermum erythrorhizon_ showed significant cytoprotection of rat embryonic precursor neuroretinal cells in culture [30]. Concentrations of _Gynostemma pentaphyllum_ extract from 10 to 100 \(\upmu\)g/mL showed positive effects on injured neuroblastoma-spinal cord hybrids NSC-34 and activated BV-2 microglial cells [31]. In the same year, 5-50 \(\upmu\)g/mL extract from _Graptopetalum parasquayense_ inhibited \(\upbeta\)-amyloid-induced damage in SH-SYSY cells [32], and similar extract concentrations from two other medicinal plants were found to be neuroprotective in primary cultures of retinal ganglion cells [33]. Finally, last year, 10 \(\upmu\)g/mL of a _Melissa officinalis_ extract showed significant cytoprotection and reduced neuroinflammation in microglial cells [34]. We have recently standardized an oxidative stress assay in neuron-like cells by challenging them with the potent oxidative inducer t-BOOH [5,6]. In a reaction assisted by metal ions, organic peroxide t-BOOH decomposes to other peroxyl and alkoxyl groups that may produce ROS [35]. In a situation of long-lasting overproduction of ROS, namely oxidative stress, there might be excessive damage to cellular macromolecules that can irrevocably compromise cell viability, such as when t-BOOH induces cellular stress [36]. In this model of oxidative stress in SH-SYSY cells, we report that concentrations of 25-200 \(\upmu\)g/mL of phenolic compounds of cocoa [5], and of _Desmodium tortuosum_ extract (25-200 \(\upmu\)g/mL) [6], protect neuron-like cells from oxidative stress. Therefore, the _B.