## 2 Materials and Methods
### Measurement of the Chlorophyll Contents in the Extracts  
The amounts of total chlorophylls in the extracts were estimated by the following method [41]: First, 1.4 g of sodium oxide was dissolved in 40 mL of distilled water and 16.6 g of pyridine was added to make 100 mL of the final volume. Then, 10 mg of the extract was placed in a foil-covered test tube and added with 10 mL of distilled water, and further incubated for 30 min. Thereafter, 2 mL of the suspension was mixed with 5 mL of an alkaline pyridine solution and incubated at 60 \({}^{\circ}\)C with aluminum foiled cover, and then allowed to stand for 15 min. After that, the solution was centrifuged at 3000\(\times\) g for 3 min. and the supernatant was transferred to a 10 mL brown flask and stored in a cool place. Then, the absorbance of the sample solution was compared with that of alkaline pyridine solution by a UV spectrophotometer (UTX-20M, Biostep co., Burkhardsdorf, Germany) at 419 nm and 454 nm of wavelengths. The actual concentrations in the extracts were estimated by the following Equation (1):  
\[\text{Total chlorophyll (mg/100 g)}=\text{C/S}\times 100. \tag{1}\]  
\[\text{C: Chlorophyll (mg/L)}=8.970\times(7.19\times\text{A419 nm}+3.33\times\text{A454 nm)}\]  
\[\text{S: Weight (mg)}\text{ of the extracted sample in 2 mL of alkaline pyridine solution}\]  
\[\text{A419 nm: Absorbance at a wavelength of 419 nm}\]  
\[\text{A454 nm: Absorbance at a wavelength of 454 nm}\]  
Measurement of Cell Cytotoxicity and the Production of Nitric Oxide (NO) and Prostaglandin E2 (PGE2)  
The cytotoxicity of the _Spirulina_ extracts to mouse microglial cells (BV-2, ATCC, Manassas, VA, USA) was observed with the following method [42]. First, BV-2 cells were seeded in a 24-well plate at a concentration of \(1\times 10^{5}\) viable cells/mL with Dulbecco's modified Eagle's Medium (DMEM, Gibco, Carlsbad, CA, USA) enriched with 10% fetal bovine serum (FBS, Gibco, Carlsbad, CA, USA), and 100 U/mL penicillin and 100 ug/mL streptomycin at 37 \({}^{\circ}\)C with 5% CO\({}_{2}\). Then, the cells were treated with various concentrations of two samples (UE and EE) with or without 1 ug/mLof lipopolysaccharideacharide (LPS, L2630, Sigma, St. Louis, MO, USA), as well as with no treatment as a control, and cultured for one more day [43]. Next, the culture medium was removed and 1 mg/mL 3-(4,5-methylthiazol-2-yl)-2,5-diphenyltetrazolzolium bromide (MTT, Sigma) solution was added. The cells were cultured again for 4 h at 37 \({}^{\circ}\)C with 5% CO\({}_{2}\) with minimal light. The MTT solution was then removed, 200 \(\upmu\)l of dimethyl sulfoxide (DMSO, Sigma) was added to the wells, and the wells were incubated for 30 min in the dark. After that, the absorbance was measured at a wavelength of 570 nm by a microplate ELISA reader (Thermo Fisher Scientific, Waltham, MA, USA). The cytotoxicity was estimated as the ratio of the absorbance of the untreated group (Au) to that of the sample group (As), as shown in the following Equation (2) [44]:  
\[\text{Cytotoxicity (\%)}=(1-\text{Au/As})\times 100. \tag{2}\]  
The concentrations of NO and PGE2 secreted from BV-2 cells were measured with the following method. To measure NO production from BV-2 cells, the BV-2 cells were seeded into 96-well plates at a concentration of \(1.0\times 10^{5}\) cells/mL, certain concentrations of each of the samples were added to the wells, and the cells were incubated for 4 h. Then 1 \(\upmu\)g/mL of LPS was added, and the cells were cultured for 24 h at 37 \({}^{\circ}\)C in a CO\({}_{2}\) incubator (CB150, Binder, Bohemia, NY, USA) with 5% CO\({}_{2}\). Next, 50 \(\upmu\)L of Griess reagent and 50 \(\upmu\)L of cell culture supernatant were mixed, added to the cells, and allowed to react for 5 min in 96-well plates. The amount of NO in the medium was measured at 540 nm using an ELISA Reader (Thermo Fisher Scientific, Waltham, MA, USA) with NaNO\({}_{2}\) as a standard [45].