*3.2. Inhibition of Nitric Oxide (NO) and Prostaglandin E2 (PGE2) Production by the Extracts*

Prior to carrying out the anti-inflammatory experiments, in Figure 1 the cell cytotoxicity of the samples against mouse microglial cells was compared by treating the UE or EE (0.01 to 0.1 mg/mL) with (black bars) or without 1 μg/mL of LPS (white bars).

**Figure 1.** Cytotoxicity of the *S. maxima* extracts with (black bars) and without (white bars) adding 1 μg/mL of lipopolysaccharide (LPS) against BV-2 cells. EE, 70% ethanol extraction at 80 ◦C for 12 h, UE, ultrasonic pretreatment with 70 % ethanol at 40 kHz and room temperature for 8 h, and further extraction at 65 ◦C for 4 h. Values are presented as means ±SD; \* *p* < 0.05 and \*\* *p* < 0.01 compared with the non-treatment group.

Interestingly, the EE from the hot 70% ethanol extraction process generally had higher cytotoxicity than the UE both with and without LPS, which was presumably attributable to the high heat-mediated degeneration of chlorophyll and resultant increases in toxic substances, such as pheophorbide. The effects of the samples on cell death also showed concentration dependency. The highest cell cytotoxicity (approximately 14%) was observed upon treatment of 0.1 mg/mL EE with LPS, compared to approximately 12% upon treatment of the UE at the same concentration; approximately 9% and 5% of the cells died upon treatment of 0.01 mg/mL UE and EE with LPS, respectively. In general, less cell cytotoxicity was observed in both samples without LPS, but the difference in the cytotoxicities of extracts treated with and without LPS was not significant. Therefore, the extracts from both processes could be used to investigate their anti-inflammatory effects because ca. 90% and 86% of the lowest survival rates were observed at the maximum dosage of 0.1 mg/mL without and with LPS, respectively. Figures 2 and 3 demonstrate the NO and PGE2 measurements after treatment with the two extracts in BV-2 cells. In Figure 2, the amount of NO produced in the untreated group not treated with LPS was extremely small at 5.21 μM, but the amount of NO produced in the LPS-treated group sharply increased to 40.31 μM. In contrast, when BV-2 cells were treated with 70% ethanol extract together with LPS, 35.6 μM NO was generated at the 0.01 mg/mL EE concentration, while 20.3 μM of NO was generated at the maximum concentration of 0.1 mg/mL. Additionally, when BV-2 cells were treated with UE extracts, 31.4 μM NO was generated at the low concentration, and 19.6 μM NO was generated

at the high concentration, indicating that the extracts obtained from the UE had greater ability to suppress NO generation than extracts from conventional extraction processes. Additionally, both extracts suppressed the generation of NO in a concentration-dependent manner, demonstrating that UEs exert remarkable anti-inflammatory effects on brain nerve cells and that 70% EEs also exhibit anti-inflammatory activity in brain nerve cells. These results are consistent with the findings of other studies [49], indicating that chlorophyll has anti-inflammatory effects and strongly suggesting that the anti-inflammatory effect of *Spirulina* extract is attributable to chlorophyll; similar results regarding the antioxidant effect of chlorophyll were also reported in other studies [37,50]. Additionally, when the extracts were administered alone at high concentrations, NO was minimally produced to levels similar to the control group not treated with LPS (data not shown).

**Figure 2.** Secretion of nitric oxide from BV-2 cells by the treatment of various concentrations of the *S. maxima* extracts. EE, 70% ethanol extraction at 80 ◦C for 12 h, UE, ultrasonic pretreatment with 70% ethanol at 40 kHz and room temperature for 8 h, and further extraction at 65 ◦C for 4 h. Values are presented as means ±SD; \* *p* < 0.05 and \*\* *p* < 0.01 compared with the LPS group.

Figure 3 demonstrates the production of PGE2, a major product of phlogogenic mechanisms.

As shown in Figure 3, 88.4 pg/mL PGE2 was produced in the control without any treatment, while 1580.9 pg/mL PGE2 was produced when BV2 cells were treated with LPS, indicating that inflammation was induced. When the extracts were administered at concentrations ranging from 0.01 to 0.1 mg/mL, PGE2 production decreased in a concentration-dependent manner in response to both extracts. Approximately 826.5 pg/mL PGE2 was produced when the UE was administered at a maximum concentration of 0.1 mg/mL. In general, the amount of PGE2 produced decreased according to concentration in the same pattern as that of NO production. The ultrasonic extract had a more dramatic reduction compared to the extract obtained by the general extraction process, reconfirming that ultrasound extracts have stronger anti-inflammatory effects. When compared to *Coridalyis bungeana* [51], *Coptids rhizome* [52], and Royal jelly [53], the *Spirulina* extracts from this UE showed anti-inflammatory effects selectively on brain nerve cells similar to or higher than those of the other substances. The extract containing large amounts of the useful bioactive substance in intact forms without the destruction of activity obtained through ultrasonic extraction has better anti-inflammatory properties, therefore confirming the excellence of ultrasonic low-temperature extraction once again. In particular, NO is known to be excessively produced by macrophages in response to stimulation with substances, such as LPS and amyloid-beta which are toxic intracellular substances to cause cytotoxicity and inflammation [54]. PEG2 is synthesized by COX-2 to mediate the pain and fever

on damaged tissues or cell regions and is known to be involved in the induction of Parkinson's and Alzheimer's disease at high levels [55]. Therefore, effective inhibition of these substances may reduce factors that cause inflammation in brain nerve cells, thereby improving cognitive functions through anti-inflammatory effects. This information can be understood in the same context as previously reported findings, indicating that *Spirulina* can protect brain nerve cells and improve cognitive function and memory [31,32,35].

**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 ◦C for 12 h, UE, ultrasonic pretreatment with 70% ethanol at 40 kHz and room temperature for 8 h, and further extraction at 65 ◦C for 4 h. Values are presented as means ±SD; \* *p* < 0.05 and \*\* *p* < 0.01 compared with the non-treatment group.
