**1. Introduction**

AMD (age-related macular degeneration) as main cause of central vision loss in the elderly is an irreversible disease with the number of patients annually increasing [1]. In the late phase of the disease, two forms exist which both lead to a degeneration of retinal components in the macula lutea. In the early form of AMD oxidized lipid protein molecules are deposited, terminating in accumulated drusen, which may interfere with retinal pigment epithelium (RPE) function. RPE cells are important for the maintenance of the photoreceptors. In the late stages of the disease geographic atrophies can occur, with large areas of RPE and photoreceptor degeneration [2,3]. In the exudative ("wet") late form, an excessive production of the vascular endothelial growth factor (VEGF) leads to the formation of new blood vessels growing under and into the retina, causing edema and bleeding which ruptures the retina [2]. The pathology and causes of AMD development are not completely understood, but factors like complement activation, oxidative stress, inflammatory milieu and the excess VEGF are correlated with the development of AMD [2,4–6]. Up until now, the only treatment available are VEGF inhibiting agents, which need to be regularly injected in the human eye and slow down the deterioration but cannot cure the disease [7].

Brown seaweeds produce numerous very promising chemical compounds that are interesting for medical research, because of their beneficial e ffects for human health. An example are fucoidans, also designated as sulfated fucans, which are marine polysaccharides mainly composed of sulfated fucosyl moieties and sulfate ester groups. Minor constituents in fucoidans include other sugar moieties like galactose, mannose, xylose and glucuronic acids. Fucoidans are cell wall components and serve mainly as protective agen<sup>t</sup> against pathogens and other environmental influences in the ocean [8]. In addition, they are also important as structural component and protect against dehydration [9]. Fucoidans exert many additional biological e ffects. These biological activities depend on the structure and this in turn depends on factors like algal species [10], harvest place and harvest time [11]. Among the di fferent biological e ffects are the capability to lower inflammatory cytokines, to reduce oxidative burden and to inhibit VEGF as well as blood lipids [12]. These e ffects pave the way for a possible treatment option for AMD and other diseases in the human eye [13]. However, the activities of the fucoidans are highly dependent on the biological systems they are applied to. Therefore, appropriate testing systems are vital for investigating its potential and furthermore, the extraction method, as this e ffects the structure and the purity of the tested extracts are of high importance for reproducible beneficial e ffects.

In order to properly elucidate structure-function relationships of fucoidans for prevention of AMD or other degenerative diseases, it is essential to focus on the extraction technology. In particular, to obtain pure fucoidans, while maintaining the relevant structural features required for specific biological activities. Early work on fucoidans relied on several steps of acidic extraction at elevated temperatures (70–100 ◦C), but such extractions may a ffect the chemical composition and size of the extracted fucoidans [14]. Instead, we have developed several targeted enzymatic assisted extraction procedures that gently and precisely loosens up the cell wall matrix, releasing fucoidans in a gentle way, obtaining crude fucoidan extracts, containing also low molecular weight alginates In previous work one of these new enzymatic treatments were used and followed by ion-exchange chromatographic (IEX) purification obtaining well-defined, pure fucoidans from *Saccharina latissima* (SL) [15].

We showed already promising e ffects of di fferent fucoidans on ocular cells. In brief, fucoidans from past studies were extracted with hot water, followed by precipitation with CaCl2 and ultrafiltration or dialysis [16–19]. *Fucus vesiculosus* fucoidan from Sigma-Aldrich can reduce angiogenesis and VEGF of RPE [13]. Fucoidans from *Fucus serratus*, *Laminaria digitata* (LD) and *Fucus distichus* subsp. *evanescens* (FE) were protective against oxidative stress in the uveal melanoma cell line OMM-1 and could inhibit ARPE-19 VEGF production [19] exactly like a other *Saccharina latissima* fucoidan, which was protective in ARPE-19 and lowered VEGF of primary RPE [19]. Di fferent sized fucoidans from *Laminaria hyperborea* showed that the large, non-degraded fucoidan is most suitable for oxidative stress protection and VEGF inhibition [17]. Moreover, the tested fucoidans are not antiproliferative for ocular cells in general [16], which is necessary for use in medical treatments. However, the biological e ffects of fucoidan di ffer strongly in relation to their chemical characteristics, which are influenced by the extraction method, and activities may be confounded by contaminants in the extracts [20,21].

The objective of this work was to examine fucoidans from three di fferent algal species (LD, SL, FE), which were extracted with four di fferent enzymatic treatments, followed by alginate precipitation with either HCl or CaCl2. Additionally, crude fucoidan from SL was further purified and separated by ion-exchange chromatographic (IEX). In our previous studies related to fucoidans and their e ffect on ocular cells, we focused on the comparison of di fferent algal species, fucoidans with di fferent molecular weights or the e ffects on cell viability in tumor and non-tumor cell lines. In this study we tested fucoidans from di fferent species, extracted from the seaweeds by di fferent enzymatic

methods. In addition, SL extracts were further purified and fractionated by IEX thereby removing contaminating compounds like alginate and polyphenols and achieving a higher fucose content. This study focuses on investigating whether the biological activity of fucoidans can be improved by different enzyme assisted purification methods. We compare fucoidans from different brown algal species and different enzymatically assisted treatments as well as IEX fractionation, to choose the most promising combination for further AMD research. Performed tests include detection of toxic effects, the ability to protect ocular cells against oxidative stress and to inhibit VEGF secretion. Furthermore, molecular weights and monosaccharide composition was determined to make a connection to the biological effects. Taken together, this study is well equipped to compare the bioactivity of fucoidans in relation to enzymatic extraction methods (different FE enzymatic purified extracts), further isolating steps (fractionated SL extracts), and also different species of origin (LD, SL, FE) in relation to the molecular weight and monosaccharide composition.
