Methods for quantification and extraction of fucoidan, and quantification of the release of total carbohydrate and fucoidan from the brown algae Laminaria hyperborea
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Fucoidan is a sulphated heteropolysaccharide with muglicious nature. It has recently gained much attention because of its bioactive properties. The first objective of this research was to evaluate methods for qualitative determination of fucoidan. As laminaran is co extracted with fucoidan when using acid for extraction, main focus was to determine, to what extent glucose would interfere with the overall quantification for fucose. Two methods were chosen, the Dische method and an enzymatic method. The Dische method was cheap and rapid but it showed a limitation for quantitatively detecting fucose. The method worked precisely if the glucose concentration is 5 times or less, than the concentration of fucose within the samples. The enzymatic method was more sensitive, specific and reliable. The method worked efficiently even if glucose concentration was 40 times higher than fucose. The second objective was to optimize a method for extraction of fucoidan from L. hyperborean to obtain a quantitative yield of fucoidan. Different acid concentrations and extraction times were tested and the conditions were optimized for extraction with 0.1 molar HCl at 80oC for 2 hours. The third objective was to determine the release of fucoidan from whole live algae with damaged parts of the same algae i.e. old leaf, new leaf and stem. L. hyperborea were harvested form Storsteinan (Trondheim) at the end of February 2015 and analyzed for the content of fucoidan. Whole algae of L.hyperborea submerged in distilled water and seawater in darkness and at 4oC were found to release very low amounts. The release of total carbohydrate from plants in seawater was determined to increase with time, with a maximum of about 100 mg/kg wet weight of the algae. The release of fucoidan from the same whole algae was very low, less than what could be reliably detected with the method (< 2 mg fucose /kg wet weight of algae). For the corresponding experiment with whole plants in distilled water, the total carbohydrate content could not be reliably determined. The release of fucose from whole plants in distilled water was similar to in seawater. For the release of total carbohydrate from old leaf in distilled water could not be determined reliably, while the release of fucose from old leaf was found to increase up to about 400 mg/kg wet weight of algae. For the corresponding experiment with old leaf in seawater, both the total carbohydrate and fucose was found to increase about 400 mg/kg wet weight, i.e. almost all carbohydrate released is fucoidan. For the release of total carbohydrate from new leaf in both distilled water and seawater were found to increase to up to around 2500 mg/kg wet weight. The release of fucose from new leaf both in fresh water and seawater were found to increase up to around 120-140 mg/kg wet weight of algal i.e. very much lower that the total carbohydrate content.For the release of total carbohydrate and fucose from the stem in distilled water, the total carbohydrate was found to increase up to around 200 mg/kg wet weight of algae, while fucose increased up to 50-100 mg/kg wet weight of algae, i.e. about half of the carbohydrate released from the stem is fucoidan. Release of total carbohydrate from stem in seawater could not be reliable determined. The release of fucose from stem in seawater was found to increase to about 50 mg/kg wet weight of algae, was found to be not very different from the release of fucose from stem in distilled water. Overall, the release of carbohydrate and fucoidan from whole plants both in distilled water and seawater was very much lower (on the level of few percent), than the release from damaged parts of the plants. No large differences between the release from plants / plants in distilled water as compared to seawater could be found. Interestingly, the release of fucoidan from the individual parts of the plant was quite different, where old leaf released almost exclusively fucoidan, where new leaf released quite low amounts (around 10 %) and stem releasing about half of the total carbohydrates as fucoidan.Whole plant in distilled water, Old leaf in distilled water and stem is sea water could not be determined reliable for total carbohydrate content and gave a higher absorbance at 269 nm which is in the rang for the poly phenols.The hypothesis if fucoidan is produced by the plant in respond to damage could neither be proved nor disproved. Several dried algae samples were showing an increased content of fucoidan after the extraction but this also could have been caused by a faster release of other compounds e.g. laminaran, proteins, salts and phenols, resulting in a counterfeit increase of the fucoidan amount. The determined change in content was too small to answer that question precisely. So it will remain a topic of further research.