## 3 Results and Discussion
### Inhibition of mRNA Expression and Secretion of Pro-Inflammatory Cytokines by the Extracts  
Because the _Spirulina_ extracts proved to be capable of suppressing the production of mediators that directly affect inflammation, Figures 4-7 show the effects of the two extracts on the mRNA expression of TNF-\(\alpha\), IL-6 and IL-1\(\beta\), which are pro-inflammatory cytokines that directly affect the inflammation of brain nerve cells. These experiments also assess the degree to which the extracts suppress the actual production of these cytokines in mouse nerve cells. Figure 4 shows RT-PCR products that reflect the expression of mRNA in the cells treated with only LPS, cells treated with both LPS and extracts, and the control with no not treatment. As shown in Figure 4a, the amount of TNF-\(\alpha\) increased sharply in the mouse microglial cells treated with LPS alone compared to the control without any treatment. In contrast, when LPS and _Spirulina_ extracts were administered together, TNF-\(\alpha\) expression decreased in a concentration-dependent manner. Similar to the effects on NO and PGE2 production, shown in Figures 2 and 3, the ultrasonic extract had greater inhibitory effects on expression than the extract obtained through the general extraction process. To quantitatively compare the electrophoretic bands, shown in Figure 4a, the sizes of individual bands were normalized to the beta-actin band, which is a house-keeping gene, using a program that quantitatively compares the band sizes, as shown in Figure 4b. Similar to the pattern in Figure 4a, mRNA expression relative to beta-actin decreased as the concentration of the extracts administered increased. Additionally, the ultrasonic extract had much greater inhibitory activity than the general extract, as shown in Figure 4a. In particular, the difference in TNF-\(\alpha\) expression inhibition between the two extracts was larger higher extract concentrations of 0.1 mg/mL than at lower concentrations,  
Figure 3: Comparison of PGE2 secretion from BV-2 cells by the treatment of various concentrations of the _S.maxima_ extracts. EE, 70% ethanol extraction at 80 \({}^{\circ}\)C for 12 h, UE, ultrasonic pretreatment with 70% ethanol at 40 kHz and room temperature for 8 h, and further extraction at 65 \({}^{\circ}\)C for 4 h. Values are presented as means \(\pm\)SD; \({}^{*}\)\(p<0.05\) and \({}^{**}\)\(p<0.01\) compared with the non-treatment group. possibly, due to higher amounts of chlorophylls in the extracts. Therefore, this result indicates once again that extraction of bioactive substances sensitive to heat requires a low-temperature extraction process and that the UE is the most efficient extraction process in such cases. As shown in Figure 5, the secretion of TNF-\(\alpha\) from BV-2 cells was observed to confirm whether down-regulation of mRNA expression actually reflects the suppression of TNF-\(\alpha\) secretion in mouse central nervous system (CNS) nerve cells. Similar to the pattern of the transcription of mRNA in Figure 4, the secretion of TNF-\(\alpha\) was also decreased as a concentration-dependent manner with the greatest reductions at high concentrations. Unusually, although the number of general extracts produced concentration-dependent reductions, the reductions were relatively smaller compared to the ultrasonic extract. Although mRNA expression suppression, as shown in Figure 4, appeared to be concentration-dependent for both extractions, the degree of TNF-\(\alpha\) suppression was lower in the general extract treatment. This means that mRNA expression is more sensitive to the useful components in the extract, but dramatic effects occur only when the concentration of useful substances is high enough in the following transcription stage.