*2.2. E*ff*ects on Cell Viability*

Cell viability of the uveal melanoma cell line OMM-1 was determined after treatment with the di fferent fucoidans for 24 h using the commercially available 3-(4,5-dimethylthiazol-2-yl)-5-(3- carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) assay (Figure 1). None of the tested fucoidans significantly lowered cell viability, but some significantly increased the viability, for instance 100 μg/mL LD\_SiAT2 increased viability up to 113% ± 6% (*p* < 0.001) and the SL fucoidan SL\_SiAT2 increased viability to 115% ± 4% (*p* < 0.001). This might be related to the SiAT2 extraction method (in case of SL and LD fucoidan), which perhaps leads to fucoidans with beneficial e ffects for the cell viability of OMM-1 cells.

**Figure 1.** The cell viability of the uveal melanoma cell line OMM-1 was assessed after treatment for 24 h with *Laminaria digitata* (LD) fucoidan (**a**), *Saccharina latissima* (SL) fucoidans (**b**) and *Fucus distichus* subsp. *evanescens* (FE) fucoidans (**c**) extracted with SiAT2/3 or SAT2/3 (SiAT2/3 = Cellic ®CTec2 or 3 enzyme mix + Sigma-Aldrich alginate lyase (SigmALy), SAT2/3 = Cellic ®CTec2 or 3 enzyme mix + alginate lyase expressed from *Sphingomonas* sp. (SALy), ad = acid treatment and dialysis). Also, three SL ion-exchange chromatography (IEX) fractions (SL\_F1, SL\_F2 and SL\_F3) were invastigated. Cell viability was analyzed with a MTS (3-(4,5-Dimethylthiazol-2-yl)-5-(3- carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium) assay and is shown as the mean and standard deviation in relation to the 100% control. Significance was determined with ANOVA; +*p* < 0.05, ++ *p* < 0.01, +++ *p* < 0.001 compared to control (*n* ≥ 4; number of independent experiments). No fucoidan exhibited antiproliferative e ffects.

The same procedure was performed for the human RPE cell line ARPE-19 (Figure 2). Cell viability was determined after 24 h with the MTS assay. Again, none of the fucoidan displayed significant antiproliferative e ffects. SiAT2 extracts from all three seaweed species, increased cell viability slightly at 100 μg/mL, again, although under 10% di fference compared to control and therefore not biological relevant.

**Figure 2.** The cell viability of the human RPE cell line ARPE-19 was assessed after treatment for 24 h with LD fucoidan (**a**), SL fucoidans (**b**) and FE fucoidans (**c**). Cell viability was analyzed with a MTS assay and is shown as the mean and standard deviation in relation to the 100% control. Significance was determined with ANOVA; + *p* < 0.05, ++ *p* < 0.01 compared to control (*n* ≥ 4, number of independent experiments). No fucoidan showed antiproliferative e ffects.

### *2.3. E*ff*ects on Oxidative Stress Protection*

The LD fucoidan showed no significant protective e ffects against any tested concentration of H2O2, in the melanoma cell line OMM-1 (Figure 3). From SL, only the SL\_F2 increased cell viability significantly at all concentrations tested with 10 μg/mL and 50 μg/mL showing the best protection (both 51% ± 1, *p* < 0.001 against 39% ± 1 stress control). FE fucoidans showed heterogeneous results depending on the tested concentration and used extraction method. FE\_SAT2ad showed significant protective e ffects (49% ± 4%, *p* < 0.01; 49% ± 5%, *p* < 0.01; 47% ± 3%, *p* < 0.05 against 37% ± 2% stress control) at di fferent concentrations 10 μg/mL, 50 μg/mL and 100 μg/mL, respectively In addition, 10 μg/mL FE\_SAT3ad and FE\_SiAT2 increased cell viability up to 61% ± 2%, *p* < 0.01 and 60% ± 8%, *p* < 0.01. It seems that the protective e ffects are more dependent on the tested FE fucoidan concentration then on the extraction method.

**Figure 3.** OMM-1 cell survival after 30 min treatment with LD fucoidan (**a**), SL fucoidans (**b**) and FE fucoidans (**c**) and 24 h stress insult with 1 mM H2O2, which reduced cell viability to at least 60% in all cases. Viability was determined with MTS assay. Values are pictured as the mean and standard deviation in relation to an untreated control (100%). Significance was evaluated via ANOVA; + *p* < 0.05, ++ *p* < 0.01, +++ *p* < 0.001 versus 0 μg/mL fucoidan + 1 mM H2O2 (*n* ≥ 4, number of independent experiments).

One role of RPE cells is to limit the oxidative stress in the human retina [4]. ARPE-19 cells as an RPE cell line are very resistant against hydrogen peroxide [26]. Therefore we used 0.5 mM *tert*-butyl hydroperoxide (TBHP) to lower the cell viability of ARPE-19 significantly after 24 h, as previously shown [19]. Again, the LD fucoidan showed no significant e ffect (Figure 4). Some of the FE fucoidans also had a slight additional toxic e ffect at 50 and 100 μg/mL, while 10 μg/mL FE\_SAT2ad, FE\_SiAT2ad and FE\_SiAT3ad had a minimal protective e ffect. FE fucoidan at 10 μg/mL seems to be the best concentration concerning oxidative stress protection, but the e ffects are small and not relevant, corresponding to the fact that ARPE-19 are rather resistant against oxidative stress on their own and are hardly a ffected by fucoidan extracts [17,19]. SL\_F2 and SL\_F3 at concentrations of 50 μg/mL slightly decreased cell viability significantly down to 47% ± 3, *p* < 0.05 and 49% ± 2%, *p* < 0.05, respectively compared to 54% ± 3% stress control, but this not likely to be biological relevant. Otherwise, there were no significant e ffects for the SL fucoidans.

**Figure 4.** ARPE-19 cell survival after 30 min treatment with LD fucoidan (**a**), SL fucoidans (**b**) and FE fucoidans (**c**) and 24 h stress insult with 0.5 mM TBHP *(tert*-butyl hydroperoxide), which reduced cell viability below 60% in all cases. Viability was determined with MTS assay. Values are pictured as the mean and standard deviation in relation to an untreated control (100%). Significance was evaluated via ANOVA; + / \* *p* < 0.05, \*\* *p* < 0.01, \*\*\* *p* < 0.001 versus 0 μg/mL fucoidan + 0.5 mM TBHP (*n* ≥ 4, number of independent experiments).
