## 4 Discussion  
Purified R-PE was obtained from _C. formosaum_, and similar to the R-PE extracted from the Mediterranean red algae _Corallina elongata_[41], characteristic peaks were observed at 280, 498, and 566 nm, but not 620 nm, indicating that the purified R-PE exhibits high purity and a low PC content (\(\lambda\): 610-620 nm) [42]. In addition, R-PE purified from _C. formosaum_ was a classical \"three-peak\" phycoerythrin with maximum absorption observed at 498, 539, and 565 nm, respectively (Figure 2). This is a typical absorption spectrum of R-PE, as described in a previous study [43]. The maximum fluorescence emission of R-PE obtained from _C. formosaum_ was observed at 580 nm, which was consistent with a previous study related to the fluorescence spectrum of PE [44]. Therefore, using the above-described spectral parameters, the purified PE in this study was confirmed to be typical R-PE. Generally, the purity of R-PE is estimated using three indexes: A\({}_{565}\)/A\({}_{280}\) > 5.3, A\({}_{565}\)/A\({}_{495}\) < 1.5, and A\({}_{620}\)/A\({}_{566}\) < 0.005 [16; 41; 43]. A\({}_{565}\)/A\({}_{280}\) represents the purity of the product as a result of the preparation protocol with respect to contaminating proteins, and A\({}_{565}\)/A\({}_{495}\) is an indicator of the property of the purified pigment. A pigment is considered to not be overly contaminated with B-phycoerythrin (B-PE) when A\({}_{565}\)/A\({}_{495}\) < 1.5. The high ratio of A\({}_{620}\)/A\({}_{566}\) indicates the contamination of the product with PC [41]. As a reference, purified PE with purity ratios of 0.7, 3.9, and \(>\)4.0 is classified as food-grade, reactive-grade, and analytical-grade PE, respectively [45]. The purity ratio (A\({}_{566}\)/A\({}_{280}\)) of purified R-PE in this study reached 5.79 (Figure 2), far exceeding the standard for food-grade PE. The absorbance ratio of A\({}_{620}\)/A\({}_{566}\) was 0.003, and A\({}_{566}\)/A\({}_{498}\) was 1.40, which demonstrated the absence of PC and B-PE contaminants, respectively. In this study, the potential bioactivities and functions of R-PE purified from _C. formosaum_ were analyzed by testing the pigment in several cell lines and bionic epidermal tissues by using multiple functional models. The morphological grading and cell viability assay results indicated that purified R-PE does not exert significant toxic effects on NIH-3T3 cells and that morphological changes are not observed, indicating that R-PE does not exhibit significant cytotoxic effects on the examined cell lines. Previous studies reported that PE extract from the red alga _Porphyna vietnamensis_ had the potential anti-inflammatory effect on paw edema inhibition in the in vivo system using Wistar rats [46], this effect above be regarded as the term'soothing effect' in folk medicine [47]. In addition, PE extract from cyanobacterium _Pseudanabanea tenuis_ showed the potential anti-oxidant effects on mercury chloride-caused oxidative stress and renal damage inhibition in the in vivo system using male mice [48]. The release of proinflammatory cytokines leads to the inflammation of the skin, and these cytokines include IL-6 and tumor necrosis factor \(\alpha\) (TNF-\(\alpha\)). IL-6 exhibits a pro-inflammatory effect when it comes into contact with outer and inner stimulants, while TNF-\(\alpha\) regulates multiple critical functions including apoptosis, proliferation, and inflammatory cytokine production. In this study, the anti-inflammatory effects of R-PE were determined by assessing its ability to inhibit IL-6 and TNF-\(\alpha\). Compared to the vehicle control cells, the production rate of these two pro-inflammatory cytokines did not differ between RAW264.7 cell samples that were treated with or without R-PE in the presence of LPS, suggesting that R-PE purified from _C. formosaum_ per se may not exhibit inhibitory effects on IL-6 and TNF-\(\alpha\) production. This result was supported by a past research using macrophages [49].