Purification of fucoidan from Bulgarian Black sea algae Cystoseira crinita (Desf.) Bory.

Department of Pharmacognosy and Pharmaceutical Chemistry, Faculty of Pharmacy, Medical University Plovdiv, 15-A Vassil Aprilov Blvd., 4002 Plovdiv, Bulgaria. Department of Pharmaceutical sciences, Faculty of Pharmacy, Medical University – Plovdiv, 15-A Vassil Aprilov Blvd., 4002 Plovdiv, Bulgaria. Research Institute at Medical University-Plovdiv (RIMU), 15-A Vassil Aprilov Blvd., 4002 Plovdiv, Bulgaria. Department of Pharmacology and Drug Toxicology, Faculty of Pharmacy, Medical University Plovdiv, 15-A Vassil Aprilov Blvd., 4002 Plovdiv, Bulgaria. Department of Biochemistry and Microbiology, Faculty of Biology, Plovdiv University Paisii Hilendarski, 24 Tsar Asen Str., 4000 Plovdiv, Bulgaria. Université Clermont Auvergne, Clermont Auvergne INP, CNRS, Institut Pascal, F-63000 Clermont-Ferrand, France Institut Universitaire de France (IUF), 1 rue Descartes 75005 Paris, France


Introduction
Fucoidans are highly complex and heterogeneous polysaccharides composed of Lfucose and sulfate groups, but may also contain uronic acids, galactose, xylose, mannose, arabinose and glucose. They are mainly found in brown seaweeds and marine invertebrates. In recent years, fucoidans are attracting great interest for their various activities such as anti-inflammatory, immunomodulatory, antioxidant, anticoagulant, antiangiogenic, antihyperlipidemic, antibacterial, antitumor, prebiotic and wound healing activity (Hahn et al., 2012;Hentati et al., 2018).
Brown algae fucoidans are usually in complexes with different molecules as polyphenols, proteins, lipids and other polysaccharides (alginates). The coextraction of other algal compounds could influence the purity of the isolated fucoidan and respectively its biological activity (Hahn et al., 2012). Therefore, a pretreatment of the algae mass before the fucoidan isolation is required. The aim of this study was to optimize the extraction process of fucoidan from the Bulgarian Black sea algae Cystoseira crinita in order to achieve high polysaccharide yield and purity.
The purity of the isolated fucoidan was estimated by quantifying the content of total polyphenols and proteins. Total phenolic compounds were determined by the colorimetric Folin-Ciocalteu assay using gallic acid as a standard (Singleton et al., 1999). Protein content was estimated according to the Bradford method (1976) calibrated against a standard of bovine serum albumin.

Results and discussion
The extraction yield of fucoidan from Cystoseira crinita was approximately 5% of the dry biomass.
Other algal compounds such as phenols, terpenes, lipids and some pigments have a high affinity for fucoidan, adsorbing tightly to it during the process of extraction (Hahn et al., 2012). Therefore, an appropriate pretreatment procedure was performed to achieve high purity of the target product. Polysaccharide stability was also taken into consideration when choosing the purification agent. The results from the five different purification protocols used were as follows: The highest yield of fucoidan was estimated using methanol:chloroform:water mixture (method 2) and a consecutive acetone:chlorophorm:ethanol extraction (method 3). On the other hand, these models showed highest phenols and proteins content. Acetone and chloroform were used to remove fatty acids, terpenes, chlorophyll and some phenols. Alcohols as methanol and ethanol, and their aqueous solutions, were used to remove chlorophyll and the major reserve carbohydrate mannitol. Method 1, method 2 and method 3 are not expected to alter fucoidan structure but they usually result in high traces of coextracted compounds, which was also confirmed by our results (Hahn et al., 2012).
Method 4 and method 5 using formaldehyde showed the lowest content of impurities (<0.1% of phenols and <0.6% of proteins). This could be attributed to the polymerization of polyphenols by formaldehyde which linked and fixed phenols, making them insoluble (Hahn et al., 2012). The lowest yield of fucoidan when aqueous solution of formaldehyde was used, could be due to the watersolubility of the polysaccharide. Hence, in method 5 an ethanol:water solution (80:15) was applied to prevent the extraction of fucoidan during the purification procedure.

Conclusion
Water-ethanol solution of formaldehyde was determined as the optimal purification agent for the pretreatment procedure of fucoidan extraction, due to the obtained high yield and purity of the isolated polysaccharide.