## 3 Results
### In Vitro Results of Sage in Combination with Rutin  
Based on the results observed in the individual botanical extracts, sage (_Salvia officinalis_) standardized in 4% rosmarinic acid was revealed to present the most noticeable effects on reducing oxidative stress and cell death, while increasing the gene expression of trophic factors implicated in neuronal survival.  
Next, in vitro studies were performed related to the potential effect of sage in promoting neuronal function. Specifically, brain-derived neurotrophic factor (BDNF) protein expression was analyzed in human neural stem cells, and acetylcholine esterase (AChE) activity was analyzed in mouse hippocampal cells.  
BDNF presents numerous effects on the nervous system and is especially relevant for learning and memory, particularly long-term memory [15]. Based on this premise, the expression of this trophic factor in neural stem cells exposed to sage extract was analyzed (Figure 6). Furthermore, the combination of sage with rutin, at a 65:35 ratio, was also assessed and compared. Rutin is a phenolic compound with multiple health benefits, including potential neuroprotective effects [14; 16]. The reason for this was to evaluate if the combination of these two extracts could have possible complementary effects.  
\begin{table}
\begin{tabular}{l l l l l l} \hline \hline
**Botanical Extract** & **ROS** & **RNS** & **NRF2** & **NGF** & **VEGF** \\ \hline Bilberry & NS & 0.0001\% (*) & NS & 0.001\% (*) & NS \\  & & & & 0.0001\% (**) & \\ \hline Blueberry & 0.001\% (*) & 0.001\% (***) & NS & 0.001\% (*) & NS \\  & & 0.0001\% (**) & & & \\ \hline Bacopa & NS & NS & NS & 0.0001\% (*) & NS \\ \hline Sage & 0.01\% (***) & 0.01\% (***) & 0.001\% (***) & 0.0001\% (***) & 0.01\% (*) \\  & & 0.0001\% (*) & 0.01\% (***) & & 0.001\% (*) \\ \hline Safron & P-ox: & NS & NS & 0.00005\% (*) & NS \\  & 0.0001\% (*) & & & & \\  & 0.00005\% (**) & & & & \\ \hline Arctic Root & 0.01\% (***) & 0.01\% (*) & NS & NS & NS \\  & & 0.001\% (*) & & & \\ \hline Ginkgo & P-ox: & 0.00005\% (***) & NS & 0.0001\% (**) & NS \\  & 0.0001\% (***) & & & & \\  & A-ox: & & & & \\  & 0.00005\% (**) & & & & \\ \hline Gins & 0.01\% (**) & NS & NS & 0.0001\% (**) & NS \\  & & 0.0001\% (**) & & & 0.001\% (*) & \\  & & & & 0.01\% (***) & \\ \hline Gotu kola & NS & NS & NS & 0.0001\% (*) & NS \\ \hline \hline \end{tabular}  
* NS: not significant; P-ox: pro-oxidant effect; A-ox: antioxidant effect.  
\end{table}
Table 2: Summary of results observed of neural stem cells submitted to oxidative stress and exposed to the various botanical extracts. Values indicate the concentrations where significant values were detected. ROS: reactive oxygen species, RNS: reactive nitrogen species, Nrf2: nuclear factor erythroid 2-related factor 2, NGF: nerve growth factor, VEGF: vascular endothelial growth factor. * \(p<0.05\), ** \(p<0.01\), *** \(p<0.005\), *** \(p<0.001\).  
As a result, it was observed that sage extract presented a tendency, which was not quite significant (\(p\) = 0.11), to increase BDNF protein levels. However, when combined with ruin, BDNF expression was significantly increased, confirming a potential combinatory effect.  
Finally, AChE activity was assessed in mouse hippocampus cells with the combination of sage and ruin (Figure 7). The results here showed that AChE activity was significantly reduced by approximately 63.4% at the highest dose and 35.2% at the lowest, confirming the inhibitory effect of the combination in hippocampal cell cultures.