*Article* **E** ff**ects of a Newly Developed Enzyme-Assisted Extraction Method on the Biological Activities of Fucoidans in Ocular Cells**

#### **Philipp Dörschmann 1,\*, Maria Dalgaard Mikkelsen 2, Thuan Nguyen Thi 2, Johann Roider 1, Anne S. Meyer 2 and Alexa Klettner 1**


 **\***Correspondence:philipp.doerschmann@uksh.de;Tel.:+49-431-500-13712

Received: 30 April 2020; Accepted: 25 May 2020; Published: 26 May 2020

**Abstract:** Fucoidans from brown seaweeds are promising substances as potential drugs against age-related macular degeneration (AMD). The heterogeneity of fucoidans requires intensive research in order to find suitable species and extraction methods. Ten di fferent fucoidan samples extracted enzymatically from *Laminaria digitata* (LD), *Saccharina latissima* (SL) and *Fucus distichus* subsp. *evanescens* (FE) were tested for toxicity, oxidative stress protection and VEGF (vascular endothelial growth factor) inhibition. For this study crude fucoidans were extracted from seaweeds using di fferent enzymes and SL fucoidans were further separated into three fractions (SL\_F1-F3) by ion-exchange chromatography (IEX). Fucoidan composition was analyzed by high performance anion exchange chromatography (HPAEC) after acid hydrolysis. The crude extracts contained alginate, while two of the fractionated SL fucoidans SL\_F2 and SL\_F3 were highly pure. Cell viability was assessed with an 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) assay in OMM-1 and ARPE-19. Protective e ffects were investigated after 24 h of stress insult in OMM-1 and ARPE-19. Secreted VEGF was analyzed via ELISA (enzyme-linked immunosorbent assay) in ARPE-19 cells. Fucoidans showed no toxic e ffects. In OMM-1 SL\_F2 and several FE fucoidans were protective. LD\_SiAT2 (Cellic ®CTec2 + Sigma-Aldrich alginate lyase), FE\_SiAT3 (Cellic ® CTec3 + Sigma-Aldrich alginate lyase), SL\_F2 and SL\_F3 inhibited VEGF with the latter two as the most effective. We could show that enzyme treated fucoidans in general and the fractionated SL fucoidans SL\_F2 and SL\_F3 are very promising for beneficial AMD relevant biological activities.

**Keywords:** fucoidan; fucose; enzymatic purification; age-related macular degeneration; VEGF; oxidative stress; *Laminaria digitata*; *Fucus distichus* subsp. *evanescens*; *Saccharina latissima*; retinal pigment epithelium
