Isolation and Purification of Anticoagulant Polysaccharide Compound from Fermented Edible Brown Seaweed, Laminaria ochotensis

다국어 초록 (Multilingual Abstract)

Anticoagulant activities of a fermented edible brown alga, Laminaria ochotensis was investigated. L. ochotensis was fermented with 15% sugar (w/v) at $25^{\circ}C$ for 10 weeks. Anticoagulant activity was measured from the supernatant of algal mixture at biweekly intervals up to $10^{th}$ week by activated partial thromboplastin (APTT), prothrombin time (PT) and thrombin time (TT) assay using citrated human plasma. Sample having high APTT activity $(6^{th}\;week)$ was filtered, ethanol precipitated and freeze-dried. The polysaccharide compound having anticoagulant activity was purified by DEAE ion exchange chromatography followed by Sepharose-4B gel filtration chromatography. Anticoagulant activity, polysaccharide concentration, and heparin like activity were determined for the collected fractions by APTT, $phenol-H_2SO_4$, and glycosaminoglycan assay, respectively. The anticoagulant activity assay showed that the activity was increased up to $6^{th}$ week, and decreased thereafter. The concentration of our purified compound was $31.0{\mu}g/ml$ and showed higher APTT activity than commercial heparin. At the same concentration of $31.0{\mu}g/ml$, the heparin showed 186.5 sec activity while our purified compound showed an activity of 386 sec. Single spot on agarose gel electrophoresis showed that the compound was purified and polyacrylamide gel electrophoresis (PAGE) results revealed that the molecular mass of the purified polysaccharide compound was between 60 and 500 kDa. Therapeutic interest of the algal polysaccharide as an anticoagulant has recently been in highlighted. This purified anticoagulant compound from fermented L. ochotensis can be used as a model for anticoagulant agent or could be developed as an anticoagulant agent. This study can be extended to identify the structure and chemical composition of the purified polysaccharide, and to establish a relationship between structure and the function of the identified anticoagulant compounds.

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