Sulfonation of papain-treated chitosan and its mechanism for anticoagulant activity

Abstract

The novel low molecular weight chitosan polysulfate (MW 5,120–26,200 Da) was prepared using the depolymerization of chitosan with papain (EC. 3.4.22.2). The sulfonation of depolymerized products was performed using chlorosulfonic acid in N, N-dimethylformamide under semi-heterogeneous conditions. The structures of the products were characterized by FTIR, 13C NMR, and 1H NMR (1D, 2D NMR) spectroscopy. The present study sheds light on the mechanism of anticoagulant activity of chitosan polysulfate. Anticoagulant activity was investigated by an activated partial thromboplastin assay, a thrombin time assay, a prothrombin time assay and thrombelastography. Surface plasmon resonance also provided valuable data for understanding the relationship between the molecular binding of sulfated chitosan to two important blood clotting regulators, antithrombin III and heparin cofactor II. These results show that the principal mechanism by which this chitosan polysulfate exhibits anticoagulant activity is mediated through heparin cofactor II and is dependent on polysaccharide molecular weight.

Physicochemical characterization of chitosan derivatives

“…This behaviour indicates that the non-existence of free amino groups is (Gamzazade et al, 1997) that have used the same synthesis procedure to produce the substitution of the sulphate group in hydroxide and amino groups, but not with a 100% of substitution. On the other hand, other studies (Suwan et al, 2009) also show the inclusion of the sulphate group in both kinds of centres with a 100% of inclusion, which is in agreement with the results obtained in this work. The behaviours commented in relation to the titration curves shown in Figure 1 allows us the calculation of the polymers deacetilation degree using Equation (1) (see material and methods section), which are shown in Table 2.…”

“…On the other hand, the opposite behaviour is observed for chitosan sulphate: a decrease in the molecular weight when compared to the chitosan. This different behaviour is due to the fact that the reaction between chlorosulphonic acid produces an aggressive chemical reaction with chitosan that causes the rupture of polymer chains, in agreement with previous studies (Suwan et al, 2009) that had observed a decrease of the molecular weight close to 99% when the chemical reaction between these compounds was performed for 5 h. The chemical reaction was performed for only 1 h in this work, and for this reason, the reduction in the molecular weight was low. The present work also studies the flow behaviour of different aqueous solutions corresponding to the polymers previously analysed, taking into account the exceptional importance of this physical property upon different industrial operations (Á lvarez, Sanjutjo, Cancela, & Navaza, 2000;Jiao, Xueqiong, & Juntag, 1998;Sovilj & Petrovi, 2007).…”

“…The amide band corresponding to the CÀ ÀO stretch of the acetyl group appears at 1640 cm 71 , and at 1567 cm 71 , we can observe the amide band corresponding to the NÀ ÀH stretch. The FTIR spectrum corresponding to CS showed the appearance of characteristic S¼O and SÀ ÀO bond-stretching absorptions at 1235, 1068 and 807 cm 71 , respectively, being in accordance with the sulphonation of chitosan (Suwan et al, 2009). The spectrum of CC shows the band at 1629 cm 71 , which can be attributed to the carboxylation.…”

“…Sulphonation of chitosan was performed using the methodology developed in previous studies (Gamzazade, Skyyar, Nasibov, Suskov, & Knirel, 1997), although with several modifications to improve the synthesis of this kind of chitosan (Suwan et al, 2009). The preparation of the sulphating reagent to produce the chitosan modification consisted of the drop addition of 4.5 mL chlorosulphonic acid (Sigma-Aldrich, 99%) with stirring to 30 mL of dimethylformamide (Panreac), which had been previously cooled to a temperature in the range of 0-48C.…”

RETRACTED ARTICLE: Biological and Biochemical Potential of Sea Snake Venom and Characterization of Phospholipase A2 and Anticoagulation Activity

“…Present investigation, in invitro anticoagulant potential and the levels of coagulation factors were assayed in normal human plasma with APTT, PT and TT reagents. In humans, normal hemostasis depends on the complex interaction of clotting of blood plasma and aggregation of platelets through the release of clotting factors [21]. The APTT assay measures the activity of all coagulation factors in the intrinsic pathway.…”