Purification and structural elucidation of chondroitin sulfate/dermatan sulfate from Atlantic bluefin tuna (Thunnus thynnus) skins and their anticoagulant and ACE inhibitory activities

Abstract

Chondroitin sulfate/dermatan sulfate (CS/DS) was extracted from Atlantic bluefin tuna (Thunnus thynnus) skin (SGAT) and was purified and characterized. SGAT was characterized by acetate cellulose electrophoresis, FTIR spectroscopy, ¹³C NMR spectroscopy and SAX-HPLC. According to the results obtained for specific chondroitinases (ABC and AC) and the SAX-HPLC separation of generated unsaturated repeating disaccharides, the polymer was found to contain a disaccharide monosulfated in positions 6 and 4 of GalNAc and disulfated disaccharides in different percentages. These results were confirmed by ¹³C NMR experiments. The average molecular mass was 24.07 kDa, as determined by PAGE analysis. SGAT was evaluated for its in vitro anticoagulant activity via activated partial thromboplastin time, thrombin time and prothrombin time tests. The polymer showed strong inhibitory activity against angiotensin I-converting enzyme (IC₅₀ = 0.25 mg mL^-1). Overall, the results suggest that this newly extracted CS/DS can be useful for pharmacological applications.

Fig. 1. (A) UV spectrum of the sulfated glycosaminoglycans purified from Atlantic bluefin tuna skin (SGAT) in the wavelength range from 190 to 700 nm obtained using a UV-vis spectrophotometer. (B) Acetate cellulose electrophoresis of sulfated glycosaminoglycans from Atlantic bluefin tuna skin. Standard GAGs (dermatan sulfate (DS), chondroitin sulfate (CS) and heparan sulfate (HS)) (Track 1), purified GAGs from Atlantic bluefin tuna skin (Track 2). (C) PAGE analysis of CS/DS purified from Atlantic bluefin tuna skin (SGAT). The calibration curve was constructed using standards prepared from chondroitin sulfate with known molecular masses of 4.63 kDa, 16.75 kDa and 28.34 kDa.

Fig. 2. Structural characterization of SGAT. (A) FT-IR spectrometry of purified CS/DS from Atlantic bluefin tuna skin (SGAT). (B) ¹³C NMR spectrum of the purified CS/DS from Atlantic bluefin tuna skin. (C) SAX-HPLC separation of the unsaturated disaccharides produced by CS/DS purified from Atlantic bluefin tuna skin and treated with chondroitinase ABC or AC. ΔDi0S (ΔUA-GalNAc), ΔDi6S (ΔUA-GalNAc6S), ΔDi4S (ΔUA-GalNAc4S), ΔDi2,6diS (ΔUA2S-GalNAc6S) ΔDi4,6diS (ΔUA GalNAc4, 6diS), ΔDi2,4diS (ΔUA2S-GalNAc4S), ΔDi2,4,6triS (ΔUA2S-GalNAc4,6 diS). The identities of the disaccharide species were determined by co-elution with purified standards (Seikagaku Co./Sigma-Aldrich). All measurements were performed in triplicate.

Fig. 3. (A) Anticoagulant activity of the purified CS/DS from Atlantic bluefin tuna skin (SGAT) at different concentrations evaluated by measurement of the activated partial thromboplastin time (aPTT). (B) Anticoagulant activity of SGAT at different concentrations evaluated by measurement of the thrombin time (TT). (C) Anticoagulant activity of SGAT at different concentrations evaluated by measurement of the prothrombin time (PT). All results are expressed as mean ± SD (n = 3) and all measurements were performed in triplicate.

Fig. 4. Angiotensin I-converting enzyme inhibitory activity of SGAT. The values are the means of three replications ± SD.