Optimization of chemical sulfation, structural characterization and anticoagulant activity of Agaricus bisporus fucogalactan

• Agaricus bisporus fucogalactan was chemically characterized.
• The fucogalactan was chemically sulfated in different conditions.
• The sulfated fucogalactans were evaluated on APTT and PT assays.
• The sulfated fucogalactan with the best anticoagulant activity (E100) was characterized.
• E100 acted mainly by inhibiting the intrinsic pathway of blood coagulation.

A fucogalactan (E) was isolated from aqueous extract of Agaricus bisporus. The monosaccharide composition, methylation, and NMR analyses showed it is constituted by a (1→6)-linked α-d-Galp main-chain, partially methylated at O-3, and partially substituted at O-2 by non-reducing end-units of α-l-Fucp or α-d-Galp. HPSEC analysis showed it had Mw of 1.28 × 104 g mol−1. The polysaccharide was sulfated modifying reaction time, molar ratio of sulfation agent to hydroxyl group on the polysaccharide (ηClSO3H/OH ratio), and ratio of total reaction volume to weight of sample (VT/w ratio; μL mg−1). The degree of substitution (DS) was evaluated for all sulfated derivatives. The sulfated fucogalactan with the highest DS value (2.83) had the best anticoagulant activity on Activated Partial Thromboplastin Time (APTT) and Protrombin Time (PT) assays. This sulfated fucogalactan, named E100, was obtained with the optimal conditions of ηClSO3H/OH ratio of 18, VT/w ratio of 100, in 6 h of reaction. The results showed that E100 produces a linear increment of APTT for concentrations of 15–45 μg mL−1, whereas PT was almost constant between 20 and 400 μg mL−1, suggesting an anticoagulant activity via inhibition of the intrinsic pathway of blood coagulation. NMR and methylation analyses showed that α-d-Galp units of the main chain were greatly sulfated on 2-O-, 3-O-, and 4-O-positions.