# Enhancement of the Neuroprotective Effect of
## 4 Discussion  
Among the many biologically active substances in _S. maxima_, \(\beta\)-carotene was selected and analyzed for content determination in the extracts of _S. maxima_ from various extraction processes because it is known to be effective for improving cognitive ability and memory [29]. Although a fat-soluble \(\beta\)-carotene is normally extracted using an organic solvent such as methanol, ethanol, or acetone, water should be used in this case, as organic solvents are not suitable for use in fermentation [30]. Therefore, the concentrations of \(\beta\)-carotene in the extracts processed with only hot water and from ultrasonic extraction with water solvent should also be measured as controls, to compare with those from a combined process of fermentation and ultrasonic extraction [17]. The result of this work proved this combined process is effective in eluting \(\beta\)-carotene with water solvent. These are considered to be the first convincing results where an increase of \(\beta\)-carotene content from _S. maxima_ was observed by using a combination of lactic acid fermentation and ultrasonic extraction with only water solvents, which is critically important in industrial applications, although previous reports have indicated that the \(\beta\)-carotene content and the content of the indicator components in natural products could be increased through fermentation [31]. It was also clearly shown that the highest antioxidant activity exhibited in the fermentation extracts can be attributed to the highest \(\beta\)-carotene content in the extracts from a combined process compared to the other extracts, because \(\beta\)-carotene has mainly worked on strong antioxidant effects in the extracts \(\beta\)-carotene from various processes [29; 32; 33]. Also, from previous reports of the function of \(\beta\)-carotene, such as a high neuroprotective activity [29; 34], the above results strongly indicate that the extract with high amounts of \(\beta\)-carotene could be expected to improve cognitive function and memory. More interestingly, the neuroprotective activities of the extracts that contained lower amounts of \(\beta\)-carotene performed better than when treating with pure \(\beta\)-carotene, possibly due to the synergistic effects between the \(\beta\)-carotene and other biologically active substances, along with the high extraction yield. Similar results were also shown in other works [35]. However, more detailed experiments investigating these effects are required. \(\beta\)-carotene is known to be a potent antioxidant, and its strong antioxidant activity is known to be effective for ischemia treatment and neuroprotective activity [27; 29; 30], which can result in neuroprotective effects of _S. maxima_. These features are considered to be the reasons for the high neuroprotective activity observed above [29; 34]. Since the fermentation extracts and \(\beta\)-carotene showed high neuroprotective activity in the above results, it is possible to expect the correlation between the activities against oxidative stress and the neuroprotective activity, which can derive a neuroprotective mechanism that is associated with antioxidant activities, whose mechanism was also proposed in  
Figure 6: (**a**) Brain-derived neurotrophic factor (BDNF) and (**b**) p-CREB expression levels from fermented _Spirulina maxima_ after glutamate-induced ROS production in HT22 cells. other works [25; 35; 36]. To confirm this hypothesis, the reduction of ROS production was also observed in this work, because this treatment induces oxidative damage, Ca\({}^{2+}\) ions induce ROS activity, and Glutathione (GSH) inhibits ROS formation [36]. Therefore, this clearly proved that that such high ROS activity and Ca\({}^{2+}\) concentration still led to a reduced oxidative stress in the fermentation extract is considered to be attributable to the high \(\beta\)-carotene content. Based on such a strong antioxidant effect, _S.