## 3 Results and Discussion  
### SG1-SG4 Exhibit Cytotoxicity to Colon Cancer Cells  
Four crude extracts of fucoidan (SG1-SG4) were produced according to the procedures reported in our previous investigation [10]. The purpose of high temperature puffing pretreatment (up to 220 \({}^{\circ}\)C) is to disrupt the cellular structure of the algal sample and facilitate the extraction of fucoidan by warm water [10]. The extracted fucoidans SG1-SG4 exhibited characteristics of fucoidan as illustrated by TLC, monosaccharide composition, and FTIR analyses [10]. Moreover, SG1-SG4 had similar molecular weight distributions, monosaccharide compositions, and FTIR spectra. In addition, the total sugar contents of SG1, SG2, SG3, and SG4 were 53.72% \(\pm\) 0.97%, 66.42% \(\pm\) 2.76%, 80.37% \(\pm\) 3.00%, and 78.38% \(\pm\) 1.72%, respectively, indicating they had distinct total sugar contents and carbohydrates comprised more than 50% of the composition of SG1-SG4 [10]. Furthermore, the amounts of fucose, sulfate, and uronic acids in SG1-SG4 varied [10]. Of note, the extract with the most impurities (alginate, protein, and polyphenols) was SG1 (6.26 + 3.78 + 2.70 = 12.74, g/100 g, dry basis), followed by SG2 (4.08 + 3.75 + 2.56 = 10.39, g/100 g, dry basis), and SG3 (1.80 + 2.76 + 2.02 = 6.58, g/100 g, dry basis), and the lowest impurity content was found in SG4 (0.64 + 2.97 + 1.07 = 4.68 g/100 g) [10]. These results are consistent with previous findings that demonstrated certain impurities such as polyuronic acids, proteins, and phenolic compounds are usually coextracted with fucoidan [23]. Since SG1-SG4 have different compositions, their biological activities such as anti-cancer activity warrant further elucidation. In our preliminary experiments, we treated HT-29 cells with SG1-SG4 at a concentration of 500 ug/mL and different time courses (24, 48, and 72 h) and found that treatment duration of 48 h was optimal for inducing cytotoxicity of HT-29 cells. In order to ensure consistency across all cellular experiments, the duration of treatment of cells was thus set at 48 h. The results of HT-29 cells treated with different concentrations of SG1-SG4 for 48 h and the cytotoxicity of SG1-SG4 against HT-29 cells are shown in Figure 1a. All of the fucoidans (SG1-SG4) showed cytotoxicity to HT-29 cells with IC\({}_{50}\) of 93.3 \(\pm\) 8.5 ug/mL for SG1, 297.3 \(\pm\) 5.2 ug/mL for SG2, 331.7 \(\pm\) 51.4 ug/mL for SG3, and 272.0 \(\pm\) 13.4 ug/mL for SG4, respectively (Figure 1b). Among SG1-SG4, SG1 showed the highest cytotoxicity to HT-29 cells. In addition, among SG2-SG4, their cytotoxicities to HT-29 cells were not significantly different (\(p>0.05\)) (Figure 1b). Interestingly, SG1 also possessed a higher antioxidant activity as compared to other fucoidans extracted [10]. The high cytotoxicity to cells and high antioxidant activity exhibited by SG1 may be attributed to the higher polyphenols content present in SG1 in the form of impurities or other structural factors. However, further research is needed to elucidate this issue. Our previous studies suggested that SG4 had the highest extraction yield of fucoidan (9.83%\(\pm\)0.11%), followed by SG3 (8.27%\(\pm\)0.01%) and SG2 (6.57%\(\pm\)0.02%), and then SG1 (2.02%\(\pm\)0.02%) [10]. Although SG1 showed slightly more potent cytotoxicity to HT-29 cells as compared to the other SGs, the extraction yield for SG1 was very low. From a cost perspective, SG4 is thus a better choice for further development as an anti-colon cancer agent. Additionally, we performed a similar experiment utilizing three commonly used human epithelial normal cells from different tissues (e.g., SV-HUC-1 cells derived from ureter, HEK-293 cells derived from embryonic kidney, and BEAS-2B cells derived from lung) to examine the toxic effects exerted by SG1 and SG4 on normal cells. As shown in Figure 1c, SG1 and SG4 showed cytotoxicities to three normal cells with an IC\({}_{50}\) value more than 500 \(\mathrm{\SIUnitSymbolMicro g}\)/mL.