# Microalgae as Potential Sources of Bioactive Compounds for Functional Foods and Pharmaceuticals
## 3 Results and Discussion
### Evaluation of Bioactivities of Microalgal Extracts
#### 3.1.4 Anti-Inflammatory Activity  
Cell viability of the POC W, NANNO W, NANNO ET, TCTP4 ET and TCH ET extracts at 10, 50 and 100 ug/mL was tested in Mac THP-1 cells. All ethanol extracts revealed not to have a cytotoxic effect on Mac THP-1 cells, as cell viability remained over 80%. The NANNO and POC water extracts showed a reduction in cell viability to 60% at concentrations over 50 ug/mL (Figure 1A). Considering these results, the anti-inflammatory assays were performed with 10, 20 and 50 ug/mL for all extracts. DMSO was shown not to affect cell viability (data not shown). A clear downregulation of the TNF-\(\alpha\) production occurred in the LPS-stimulated Mac THP-1 cells pretreated with TCTP4 ET and TCH ET extracts at all concentrations tested (\(p\) < 0.001) (Figure 1B). Pretreatments with NANNO ET and POC W extracts showed significant TNF-\(\alpha\) downregulation at concentrations of 50 ug/mL (\(p\) < 0.01) and 10 ug/mL (\(p\) < 0.001), and 50 ug/mL (\(p\) < 0.01), respectively (Figure 1B), whereas NANNO W extract showed no significant decrease in TNF-\(\alpha\) production (Figure 1B). Since the TCTP4 ET extract showed a dose-dependent decrease in TNF-\(\alpha\) production, presenting the highest percentage of TNF- \(\alpha\) inhibition of 83.0 \(\pm\) 9.4% at 50 ug/mL, an effect even higher than the positive anti-inflammation control dexamethasone (DXM) (57.0 \(\pm\) 8.6%) (\(p\) < 0.001), this extract was selected for further fractionation. The resulting four fractions (hexane, dichloromethane, ethyl acetate and water/ethanol) of the TCTP4 ET extract were evaluated for cell viability (Figure 2A) and anti-inflammatory activity (Figure 2B) in Mac THP-1 cells. Cell viability was shown to be above 90% for all fractions at all concentrations tested (10, 20, and 50 ug/mL) (Figure 2A). The hexane (H) and dichloromethane (DCM) fractions displayed high TNF-\(\alpha\) inhibition in Mac THP-1 cells, with both fractions presenting similar TNF-\(\alpha\) levels as the control non-stimulated cells and showing even higher TNF-\(\alpha\) inhibition than dexamethasone (DXM), at all concentrations tested (Figure 2B). These results clearly indicate a strong and promising anti-inflammatory potential of hexane and dichloromethane fractions of the TCTP4 ET extract in LPS-stimulated Mac THP-1 cells. Water extracts presented no significant anti-inflammatory activity, possibly due to the extracted compounds being mainly polar and ionic and therefore presenting low anti-inflammatory effect (besides polysaccharides) [70]. The high TNF \(\alpha\) inhibitory effect obtained for the hexane and dichloromethane fractions of the TCTP4 ET extract may be related to the presence of compounds with anti-inflammatory activity, such as phenolic compounds, pigments, and non-polar lipids, which are commonly extracted with hexane and DCM and have reported anti-inflammatory activity [13]. Figure 1: **(A)** Cell Viability (%) of macrophage-differentiated THP-1 (Mac THP-1) cells after incubation with _Porphyridium_ sp. water (POC W), _Nannochloropsis_ sp. water (NANNO W) and ethanol (NANANO ET), _Tetraselmis striata_ CTP4 ethanol (TCTP4 ET) and _Tetraselmis chui_ ethanol (TCH ET) microalgal crude extracts at 10, 50 and 100 \(\mu\)g/mL for 48 h, assessed through the MTS assay. **(B)** Anti-inflammatory activity of the microalgal crude extracts defined in panel **(A)**, in LPS-stimulated Mac THP-1 cells. Evaluation of the inflammatory marker TNF-\(\alpha\) was performed by ELISA in the cell culture media of Mac THP-1 cells treated for 24 h with the microalgae crude extracts at 10, 20 and 50 \(\mu\)g/mL, and then stimulated with LPS (100 ng/mL) for a further 24 h. Dexamethasone (DXM) (2 \(\mu\)M) was used as a positive anti-inflammatory control and non-stimulated cells (CTR) as controls to LPS stimulation (LPS). Data are representative of three independent experiments, and presented as mean \(\pm\) SD.