Anticoagulant Properties of a Green Algal Rhamnan-type Sulfated Polysaccharide and Its Low-molecular-weight Fragments Prepared by Mild Acid Degradation

Abstract

The active sulfated polysaccharide from seaweed possesses important pharmaceutical and biomedical potential. In the study, Monostroma sulfated polysaccharide (MSP) was obtained from Monostroma angicava, and the low-molecular-weight fragments of MSP (MSP-Fs: MSP-F1⁻MSP-F6) were prepared by controlled acid degradation. The molecular weights of MSP and MSP-F1⁻MSP-F6 were 335 kDa, 240 kDa, 90 kDa, 40 kDa, 24 kDa, 12 kDa, and 6.8 kDa, respectively. The polysaccharides were sulfated rhamnans that consisted of →3)-α-l-Rhap-(1→ and →2)-α-l-Rhap-(1→ units with partial sulfation at C-2 of →3)-α-l-Rhap-(1→ and C-3 of →2)-α-l-Rhap-(1→. Anticoagulant properties in vitro of MSP and MSP-F1⁻MSP-F6 were evaluated by studying the activated partial thromboplastin time, thrombin time, and prothrombin time. Anticoagulant activities in vivo of MSP and MSP-F4 were further evaluated; their fibrin(ogen)olytic activities in vivo and thrombolytic properties in vitro were also assessed by D-dimer, fibrin degradation products, plasminogen activator inhibitior-1, and clot lytic rate assays. The results showed that MSP and MSP-F1⁻MSP-F4 with molecular weights of 24⁻240 kDa had strong anticoagulant activities. A decrease in the molecular weight of MSP-Fs was accompanied by a decrease in the anticoagulant activity, and higher anticoagulant activity requires a molecular weight of over 12 kDa. MSP and MSP-F4 possessed strong anticoagulant activities in vivo, as well as high fibrin(ogen)olytic and thrombolytic activities. MSP and MSP-F4 have potential as drug or helpful food supplements for human health.

Keywords: anticoagulant activity; depolymerization; fibrin(ogen)olytic activity; low-molecular-weight fractions; sulfated polysaccharide; thrombolytic activity.

Figure 1

High-performance gel permeation chromatography (HPGPC) chromatograms of Monostroma sulfated polysaccharide (MSP) and low-molecular-weight MSP fractions (MSP-Fs). (a) HPGPC chromatogram of MSP (A) and the standard curve of molecular weight (B); (b) HPGPC chromatograms of MSP-Fs.

Figure 2

NMR spectra of MSP-Ds. (a) ¹H NMR; (b) ¹³C NMR; (c) ¹H–¹H COSY; (d) ¹H–¹³C HSQC; (e) ¹H–¹³C HMBC. A: →3)-α-l-Rhap-(1→; B: →3)-α-l-Rhap-(1→; C: →2)-α-l-Rhap-(1→; D: →2)-α-l-Rhap(3SO₄)-(1→. Rhap: rhamnopyranose.

Figure 2

NMR spectra of MSP-Ds. (a) ¹H NMR; (b) ¹³C NMR; (c) ¹H–¹H COSY; (d) ¹H–¹³C HSQC; (e) ¹H–¹³C HMBC. A: →3)-α-l-Rhap-(1→; B: →3)-α-l-Rhap-(1→; C: →2)-α-l-Rhap-(1→; D: →2)-α-l-Rhap(3SO₄)-(1→. Rhap: rhamnopyranose.

Figure 3

¹H NMR spectra of MSP and MSP-Fs.

Figure 4

Anticoagulant activities in vitro by APTT, TT, PT assays on MSP and MSP-Fs. (A) APTT; (B) TT; (C) PT.

Figure 4

Anticoagulant activities in vitro by APTT, TT, PT assays on MSP and MSP-Fs. (A) APTT; (B) TT; (C) PT.

Figure 5

Anticoagulant and fibrin(ogen)olytic activities in vivo of MSP and MSP-F4. (A) APTT; (B) TT; (C) D-dimer, the D-dimer levels of control, urokinase, MSP at 5 mg/kg and MSP-F4 at 5 mg/kg as well as 10 mg/kg groups were below detection limit in this assay; (D) Plasminogen activator inhibitior-1 (PAI-1), the PAI-1 value of MSP at 10/20 mg/kg and MSP-F4 at 20 mg/kg was up to 0; (E) Fibrin(ogen) degradation products (FDP). Statistical significance: for the anticoagulant activity assay, where ** and ^## represented p < 0.01; for the fibrin(ogen)olytic and thrombolytic activity assays, where ^□ represented p < 0.05, ^△△ and ^□□ represented p < 0.01.

Figure 5

Anticoagulant and fibrin(ogen)olytic activities in vivo of MSP and MSP-F4. (A) APTT; (B) TT; (C) D-dimer, the D-dimer levels of control, urokinase, MSP at 5 mg/kg and MSP-F4 at 5 mg/kg as well as 10 mg/kg groups were below detection limit in this assay; (D) Plasminogen activator inhibitior-1 (PAI-1), the PAI-1 value of MSP at 10/20 mg/kg and MSP-F4 at 20 mg/kg was up to 0; (E) Fibrin(ogen) degradation products (FDP). Statistical significance: for the anticoagulant activity assay, where ** and ^## represented p < 0.01; for the fibrin(ogen)olytic and thrombolytic activity assays, where ^□ represented p < 0.05, ^△△ and ^□□ represented p < 0.01.