**3. Discussion**

## *3.1. Integration in Previous Studies*

This study is the continuation of fucoidan research within the EU FucoSan project in regards to finding a possible treatment for AMD [16–19]. One main goal of this international project is to characterize di fferent fucoidans to choose the best fucoidan for a potential medical application. Factors like algae species, extraction method, purity, chemical composition, harvest place and time are important to define fucoidans with the best beneficial e ffects for further treatment development in AMD. In previous studies, factors like algae species and molecular weight were in focus. We could show that fucoidans from SL and LH (extracted with hot water extraction followed by CaCl2 precipitation and ultrafiltration or dialysis) showed the most promising e ffects of the species tested so far. In addition, high-molecular weight correlates with beneficial activities relevant for AMD [17,19]. Another study, testing fucoidan of FE, gave first indication that purity is an important factor for the relevant biological activities [18]. It has been suspected that in addition to the algae species the extraction method is a huge influencing factor concerning the biological activity, because it influences the structure and overall composition of the extract, leading to di fferential e ffects dependent on the method of extraction. Therefore, we investigated fucoidans purified by a novel technique using di fferent enzymes. Additionally, we compared the e ffects in the three species LD, SL and FE, with di fferent molecular weights and di fferent monosaccharide compositions, to elucidate the best suited extraction method and algal species.

#### *3.2. Slightly Increased Cell Viability in OMM-1 and ARPE-19 Relation to Uronic Acids, Molecular Weight and Concentration*

Toxicity was not found after treatment with any extracts, which corresponds to previously published studies [16–19]. Crude LD\_SiAT2 and SL\_SiAT2 improved the cell viability of the OMM-1 cell line slightly. Our data indicate that further fractionation could attenuate this e ffect, as can be seen for the SL fractions in this work, suggesting that this e ffect may be due to contaminating agents. Glucose, mannitol or guluronic acid were reduced by fractionation. In addition, other not investigated agents like phenols, could have been diminished by purification. Mak et al., 2014 described that crude fucoidans from *Undaria pinnatifida* have a higher toxicity on tumor cells because of the higher yield of uronic acids [27]. However, while the crude fucoidans of this work also have higher amounts of uronic acids compared to the fractions, there were protective for OMM-1. Our fractions have although high molecular weights, which could influence the protective properties [27] and in addition di fferent species may exert di fferent e ffects.

We also gathered some data indicating that fucoidans can increase the cell viability of ARPE-19 in higher concentrations. It is unknown whether this is due to an actual protective e ffect of the fucoidan (which is not the case for the oxidative stress protection in ARPE-19, here only 10 μg/mL FE fucoidans had a small protective e ffect). It could be speculated that fucoidans in higher concentration starting with 100 μg/mL increase the cell metabolism and leads to an increased proliferation rate. It was also reported that 100 μg/mL of commercially available fucoidan from Sigma Aldrich can reduce the apoptosis of ARPE-19 cells via di fferent cellular pathways [28].

#### *3.3. E*ff*ects on Oxidative Stress Protection-Heterogeneous Results, Dependency on Alginates and Galactose*

We also tested the influence of oxidative stress protection of H2O2 treated OMM-1 and TBHP treated ARPE-19. FE\_SAT2ad, FE\_SiAT2ad and FE\_SiAT3ad at 10 μg/mL showed a small protective effect in ARPE-19, which was also seen in OMM-1 cells. However, the e ffects of FE was highly heterogeneous as for the FE\_SiAT2 extract (no acid precipitation) and also FE\_SAT3ad displaying no effect at all. Furthermore, the measured protective e ffects were small and their biological relevance questionable. This corresponds to early studies, which showed no protective e ffects of FE fucoidans on oxidative stress toxicity in ARPE-19 [17,19]. As described above, FE\_SiAT2 has the highest yield of guluronic and mannonic acid the main component of alginates and lowest mol% of fucose. FE\_SAT3ad has the second highest content of guluronic acid. Mannuronic acid yield of FE\_SiAT2 is rather high in these samples due to lack of alginate precipitation. It could be speculated that alginates are cumbersome for the protective e ffects. FE-SAT3ad has the lowest glucose content, an important nutrient for the growth of tumor cells, which could lower the metabolism of cancer cells and interferes with the protective e ffects on OMM-1 cells. The SL\_F2 extract showed the best protective e ffects in OMM-1 and this is in contrast to the other three SL extracts. The SL\_F2 extract had the highest amount of fucose, the main component of fucoidans, with 64.7 mol%. However, SL\_F3 had nearly the same content (63.3 mol%), so the loss of the protective e ffect and overall biological activity against H2O2 is not due to the fucose content. Also the size and sulfation content between these two SL extracts is similar [15]. It could be suspected that the overall antioxidative e ffects are due to accompanying substances like phenols [21,29]. The molecular weight and the monosaccharide composition are similar, with the exception that SL\_F3 had a higher amount of galactose. It could be speculated that the amount of galactose plays an important role regarding the antioxidative e ffects, because it is much higher in SL\_F3. In relation to heart aging, galactose is described as antioxidants reducing as well as oxidative stress and inflammation inducing [30], so it could interfere with the protective e ffects. SL\_F1 has nearly no fucose but consists of high amounts of alginates. This extract showed no biological activity in any of the tests in this study, which leads us to the conclusion, that algae extracts with lower contents of alginates are recommended for obtaining protective e ffects.

#### *3.4. E*ff*ects on VEGF-Acid Precipitation Lowers and Higher Molecular Weight Improves VEGF Inhibition*

Several extracts reduced the VEGF secretion after three days of stimulation. FE\_SiAT2 was the only FE extract, which inhibited VEGF significantly. This extract has the highest amount of fucose, arabinose/rhamnose and the lowest amount of glucuronic acid, compared to LD\_SiAT2 and SL\_SiAT2. All other FE extracts were treated with acid and did not lower VEGF significantly. This strongly indicates that acid may alter the structure of FE fucoidan, interfering with VEGF interaction. Indeed, in this study, no fucoidan treated with acid displayed any VEGF reduction e ffects. The LD extract showed a VEGF inhibiting e ffect. It had the lowest fucose content out of all extracts, but the highest mannitol content. VEGF inhibition could be caused by mannitol, although this has not ye<sup>t</sup> been investigated no literature regarding the e ffect of mannitol and VEGF secretion can currently be found. In contrast to the crude SL\_SiAT2 and the first fraction SL\_F1, the extracts SL\_F2 and SL\_F3 lowered VEGF very efficiently in comparison to all other extracts. They had both a much higher fucose content and were the purest extracts. The fucose content alone seems not to be the only biological factor contributing to VEGF inhibition, since the FE extracts also had higher fucose content and was not causing VEGF reduction. In addition to fucose content, molecular weight could be of high importance. The two SL fractions SL\_F2 and SL\_F3 had a significant higher molecular weight (>800 kDa (Table 3)) than all other tested extracts, which supports our findings that bigger fucoidans from *Laminaria hyperborea* were more e ffective regarding VEGF inhibition [17]. This could be due to a steric interference with the VEGF molecules. This corresponds with previous findings, which suggested that fucoidans with higher molecular weight are generally more anti-angiogenic, while fucoidans with low molecular weight are considered more pro-angiogenic [31].

#### *3.5. Comparison of Cellic* ®*CTec2 and 3, Alginate Lyases as Well as Precipitation Technique*

The Cellic ®CTec2 vs. Cellic ®CTec3 relates to di fferent enzyme-mixes added in the first step of the purification procedure. They are both enzyme mixes made for degradation of land plant cell walls. They were applied to degrade the brown algal cell wall cellulose and hemicelluloses as well as the storage compound laminarin. In addition, two di fferent alginate lyases (SigmALy and SALy) were used to degrade the cell wall alginate and together these enzymes release the fucoidans gently from the cell wall matrix. FE extracts of this study can be utilized to compare the biological e ffects after application of these four enzyme purification techniques. Regarding VEGF inhibition they showed

no significant effects with the exception of FE\_SiAT2 which was VEGF inhibiting. The use of an additional acid precipitation step seems to be more important for this function than the enzyme mix applied, because only the fraction not treated with acid, FE\_SiAT2, exhibited VEGF inhibition. The effects on oxidative stress protection were rather heterogeneous and seem to be more related to the concentration applied. The influence on cell viability of ARPE-19 and OMM-1 were rather similar. Overall, we cannot determine a significant biological difference between the extracts treated with the four enzymatic techniques, suggesting that all methods can be used to purify fucoidans resulting in a slightly different amount of monosaccharide and uronic acid compositions only. Overall, acid precipitation is not recommended, because beneficial biological effects important to AMD could be lost due to a possible removal of sidechains or sulfates from the fucoidans. or perhaps more importantly, reduce the size of the fucoidans by partial hydrolysis of the fucoidan backbone. Since, high molecular weight fucoidans are considered antiangiogenic and of grea<sup>t</sup> importance when using fucoidans against AMD [17,31]. CaCl2 precipitation of alginate is preferred. Indeed, fucoidans with very high molecular weight of over 800 kDa were obtained in the SL\_F2 and F3 fractions treated with CaCl2, and these extracts showed the most promising biological effects.

#### *3.6. Di*ff*erent Fucoidan Structures between Algal Species Lead to the Described Biological E*ff*ects*

It is well known that the biological properties of the fuciodans are highly dependent on the fucoidan structure and composition. While no structural data of fucoidan from LD can currently be found in literature, the structure of SL and FE fucoidans is different. FE fucoidan consists of alternating α-(1-3)- and α-(1-4)-linked L-fucopyranose unit with sulfate group primarily at C2 [10,32,33] while sulfate groups have been found at C2 and C4 of some fucose residues [34]. Fucoidans from SL are very diverse. Their structure is depending on the overall harvest time, extraction method and further fractionation, which could lead to mixture of different low-sulfated heteropolysaccharides with proteins and uronic acids [29]. Four partial structures of fucoidans from SL have been reported: fucan sulfate, fucogalactan, fucoglucuronomannan, and fucoglucuronan [29,35]. It can be speculated that the different algal origins with very heterogeneous structural compositions, leads to the different biological effects that we described.
