Hyaluronic acid from bluefin tuna by-product: Structural analysis and pharmacological activities.

The fishing and aquaculture industries generate a huge amount of waste during processing and preservation operations, especially those of tuna. Recovering these by-products is a major economic and environmental challenge for manufacturers seeking to produce new active biomolecules of interest. A new hyaluronic acid was extracted from bluefin tuna's vitreous humour to assess its antioxidant and pharmacological activities. The characterization by infrared spectroscopy (FT-IR), nuclear magnetic resonance ((1D1H) and 2D (1H COSY, 1H/13C HSQC)) and size exclusion chromatography (SEC/MALS/DRI/VD) revealed that the extracted polysaccharide was a hyaluronic acid with high uronic acid content (55.8 %) and a weight average molecular weight of 888 kDa. This polymer possesses significant anti-radical activity and ferrous chelating capacity. In addition, pharmacological evaluation of its anti-inflammatory and analgesic potential, using preclinical models, in comparison with reference drugs (Dexamethasone, diclofenac, and acetylsalicylate of lysine), revealed promising anti-inflammatory activity as well as interesting peripheral and central antinociceptive activity. Therefore, our new hyaluronic acid compound may therefore serve as a potential drug candidate for the treatment of pain sensation and inflammation of various pathological origins.

Characterization and anticoagulant activity of a fucosylated chondroitin sulfate with unusually procoagulant effect from sea cucumber.

The fucosylated chondroitin sulfate (FuCS) was isolated from the sea cucumber Holothuria polii body wall and then purified by anion exchange chromatography and the structure was characterized by FT-IR and NMR spectroscopy. Anticoagulant activity was measured in plasma by classical anticoagulation tests and the thrombin generation was assessed by calibrated automated thrombography. The results showed that the FuCS obtained at a yield of 4.66% possesses high sulfate content 43% and an average molecular mass of 45.8kDa. Structural analysis revealed the presence of alternating units of chondroitine sulfate branched at position 3 of the ß-d-glucuronic acid by two types of fucose 2,4-O-di and 3,4-O-disulfated residues. The FuCS exhibited a high anticoagulant effect mediated essentially by heparin cofactor II and to lesser extent by antithrombin. Moreover, the CAT assay depicted a dual effect of FuCS with a procoagulant tendency for low doses and an anticoagulant effect at higher doses.

Characterization of compressed earth blocks using low frequency guided acoustic waves.

The objective of this work was to analyze the influence of compaction pressure on the intrinsic acoustic parameters (porosity, tortuosity, air-flow resistivity, viscous, and thermal characteristic lengths) of compressed earth blocks through their identification by solving an inverse acoustic wave transmission problem. A low frequency acoustic pipe (60-6000 Hz of length 22 m, internal diameter 3.4 cm) was used for the experimental characterization of the samples. The parameters were identified by the minimization of the difference between the transmissions coefficients data obtained in the pipe with that from an analytical interaction model in which the compressed earth blocks were considered as having rigid frames. The viscous and thermal effects in the pores were accounted for by employing the Johnson-Champoux-Allard-Lafarge model. The results obtained by inversion for high-density compressed earth blocks showed some discordance between the model and experiment especially for the high frequency limit of the acoustic characteristics studied. This was as a consequence of applying high compaction pressure rendering them very highly resistive therefore degrading the signal-to-noise ratios of the transmitted waves. The results showed that the airflow resistivity was very sensitive to the degree of the applied compaction pressure used to form the blocks.

Anticoagulant properties and cytotoxic effect against HCT116 human colon cell line of sulfated glycosaminoglycans isolated from the Norway lobster (Nephrops norvegicus) shell.

Sulfated glycosaminoglycans (SGNL) were extracted for the first time from Norway lobster (Nephrops norvegicus) shell. The monosaccharide composition analysed by GC/MS revealed the presence of galacturonic acid, glucuronic acid, N-acetylgalactosamine and N-acetylglucosamine. The analysis of SGNL with acetate cellulose electrophoresis in Zn-acetate revealed the presence of heparan sulfate (HS) and dermatan sulfate (DS). SGNL were evaluated for their anticoagulant activities using activated partial thromboplastin time (aPTT), thrombin time (TT) and prothrombine time (PT) tests. After 21h incubation, HCT116 cell proliferation was inhibited (p<0.05) between 39.7 and 54.8% at 1.5-7.5mg/mL of SGNL. SGNL don't show hemolytic activity towards bovine erythrocytes and no cytotoxicity against the normal lymphocytes. The antiproliferative efficacy of these lobster glycosaminoglycans were probably related with the higher sulfate content. SGNL demonstrated promising antiproliferative and anticoagulant potential, which may be used as a novel, effective and promising antithrombotic agent.

Design and synthesis of antimicrobial, anticoagulant, and anticholinesterase hybrid molecules from 4-methylumbelliferone.

We designed and synthesized new series of diverse triazoles, isoxazoles, isoxazolines, and aziridines linked 4-methylumbelliferone 1 using intermolecular 1,3-dipolar cycloaddition reactions. Structures of these compounds were established on the basis of (1)H NMR, (13)C NMR, and ESI-HRMS. All prepared compounds were evaluated for their antimicrobial, anticoagulant, and anticholinesterase activities. Interestingly, among the tested molecules, some of the analogs displayed better activities than the parent 4-methylumbelliferone 1 such as 6a and 6d for their antifungal properties. Moreover, compounds 4, 5, 6, and 7 showed the importance of the added fragments to 4-methylumbelliferone 1 via the linker methylene to have good activity.

Triacylglycerols composition and volatile compounds of virgin olive oil from Chemlali cultivar: comparison among different planting densities.

The present study focused on the comparison the chemical composition of virgin olive oil samples obtained from fruits of the main Tunisian olive cultivar (Chemlali) grown in four planting densities (156, 100, 69, and 51 trees ha(-1)). Despite the variability in the triacylglycerols and volatile compounds composition, the quality indices (free fatty acids, peroxide value, and spectrophotometric indices K(232) and K(270)) all of the virgin olive oils samples studied met the commercial standards. Decanal was the major constituent, accounting for about 30% of the whole volatiles. Moreover, the chemical composition of the volatile fraction of the oil from fruits of trees grown at the planting density of 156, 100, and 51 trees ha(-1) was also characterised by the preeminence of 1-hexanol, while oils from fruits of trees grown at the planting density of 69 trees ha(-1) had higher content of (E)-2-hexenal (20.3%). Our results confirm that planting density is a crucial parameter that may influence the quality of olive oils.

Highly sulfated dermatan sulfate from the skin of the ray Raja montagui: anticoagulant activity and mechanism of action.

The dermatan sulfate (DS) isolated from the ray skin Raja montagui was identified and characterized. Its average molecular weight (Mw) and sulfate content were 39 kDa and 25% w/w, respectively. This DS prolonged thrombin time and activated partial thromboplastin time and inhibited the thrombin generation in a concentration-dependent manner whereas it had no effect on the anti-Xa assay and on platelet function. Data from the anti-IIa assay allowed the assessment of the specific anticoagulant activity which was 40 units/mg. The kinetics of the thrombin inhibition by heparin cofactor II (HCII) has been studied as a function of DS concentration according to a kinetic model in which the polysaccharide binds quickly to the inhibitor and forms a complex more reactive than the free inhibitor towards thrombin. This DS accelerated thrombin inhibition exclusively by HCII. The dissociation constant of the DS-HCII complex, K(DSHCII), and the rate constant of the thrombin inhibition by this complex, k, were (2.93+/-0.25)x10(-6)M and (2.2+/-0.35)x10(9)M(-1)min(-1), respectively. Our findings indicated that the major polysaccharide in the skin of the ray Raja montagui was a DS endowed with a high anticoagulant effect mediated by HCII and which may constitute an anticoagulant drug of interest in anticoagulant therapy.

Anticoagulant activity of a sulfated polysaccharide from the green alga Arthrospira platensis.

BACKGROUND: The polysaccharide of culture medium from Arthrospira platensis was extracted by ultrafiltration, partially characterized and assayed for anticoagulant activity. METHODS: The crude polysaccharidic fraction was fractionated by anion exchange chromatography on DEAE-cellulose, subjected to acetate cellulose electrophoresis and characterized by physicochemical procedures. The anticoagulant effect of the ultrafiltrated polysaccharide was checked by several coagulation tests. RESULTS: Anion exchange chromatography revealed in the whole ultrafiltrated polysaccharidic fraction the occurrence of a sulfated spirulan-like component designated PUF2. The average molecular weight of PUF2 was determined by size exclusion chromatography combined with multi-angle light scattering (SEC-MALS) and viscosimetry and was 199 kDa and the sulfate content was 20% weight/dry weight. The physicochemical characterization indicated the occurrence of rhamnose (49.7%), galacturonic and glucuronic acid (32% of total sugar). The anticoagulant effect of this sulfated polysaccharide was mainly due to the potentiation of thrombin inhibition by heparin cofactor II and was 4-times higher than that of the porcine dermatan sulfate whereas it had no effect on anti-Xa activity. CONCLUSIONS: An ultrafiltrated sulfated polysaccharide, likely a calcium spirulan was obtained from the culture medium of A. platensis and showed an anticoagulant activity mediated by heparin cofactor II. GENERAL SIGNIFICANCE: Old culture medium of A. platensis may represent an important source for the spirulan-like PUF2 which was endowed with potentially useful anticoagulant properties whereas its obtention by ultrafiltration may represent an extraction procedure of interest.

Polysaccharides from the skin of the ray Raja radula. Partial characterization and anticoagulant activity.

INTRODUCTION: The polysaccharide fraction from the skin of the ray Raja radula was extracted, characterized and assayed for anticoagulant activity. MATERIALS AND METHODS: A whole polysaccharidic fraction was extracted from the skin of the ray Raja radula by papain digestion followed by cetylpyridinium chloride and ethanol precipitation and was subjected to gel chromatography and anion exchange chromatography, acetate cellulose electrophoresis and characterized by physicochemical procedures. APTT and anti Xa assays were performed to assess the anticoagulant activity of the polysaccharidic fractions in comparison with unfractionated heparin. RESULTS: Gel and anion-exchange chromatography revealed two negatively charged polysaccharidic populations different in both molecular weight and charge. Infrared spectra suggested the occurrence of uronic acids and acetylated hexosamines. The second polysaccharide was highly sulfated, with a sulfate content of approximately 29%. These data suggested that dermatan sulfate (DS) is the sulfate rich polysaccharide whereas hyaluronic acid (HA) is the polysaccharide devoid of sulfate groups. Molecular mass characterization indicated that their average molecular masses were 22 kDa and 85 kDa, respectively. The sulfated polysaccharide, i.e. presumably DS, accounted alone for the observed concentration-dependent anticoagulant activity which was, as measured by APTT, 2 to 3-fold lower than that of heparin. In addition, it had a significant anti-Xa activity. CONCLUSION: A major-sulfated polysaccharide, likely a dermatan sulfate, was extracted from the ray Raja radula skin. The results indicated that it exhibited a high anticoagulant activity and suggested that it was mediated by both heparin cofactor II and antithrombin.

Mechanism of thrombin inhibition by heparin cofactor II and antithrombin in the presence of the ray (Raja radula) skin dermatan sulfate.

INTRODUCTION: The kinetics of the thrombin inhibition by heparin cofactor II (HCII) and antithrombin (AT) have been studied as a function of the concentration of a dermatan sulfate (DS) from the skin of the ray Raja radula. MATERIALS AND METHODS: The initial concentrations of inhibitor (I), HCII or AT, and thrombin (E) were set at equimolecular levels (3.10(-9) M). Analysis of the experimental data obtained for DS concentrations ranging from 10(-8) to 10(-4) M was performed according to a previously described model in which DS binds quickly to the inhibitor and forms a complex more reactive than the free inhibitor towards thrombin. RESULTS: The apparent rate constant of the thrombin inhibition, k(app), by either HCII or AT, increased in a concentration-dependent manner for DS concentrations up to 10(-5) M or 10(-6) M, respectively. At higher DS concentrations, k(app) remained unchanged for thrombin inhibition by HCII whereas a decrease in k(app) was observed for the thrombin-AT reaction. The dissociation constant of the polysaccharide-inhibitor complex, K(DSI), and the rate constant of the thrombin inhibition by this complex, k, were (7.81+/-0.75).10(-7) M and (2.84+/-0.42).10(9) M(-1).min(-1), whereas they were (4.93+/-0.31).10(-7) M and (2.47+/-0.28).10(8) M(-1).min(-1), when the inhibitor was either HCII or AT, respectively. CONCLUSION: DS from ray skin catalyzes the thrombin inhibition by HCII or AT primarily by forming a DS-inhibitor complex more reactive than the free inhibitor towards the protease. The affinity of DS for HCII was approximately 2-fold higher whereas the catalyzed reaction rate constant was approximately 20-fold higher when compared to AT.

Characterization of a novel dermatan sulfate with high antithrombin activity from ray skin (Raja radula).

INTRODUCTION: A novel dermatan sulfate (DS) from the skin of the ray Raja radula with high anticoagulant activity was identified and its monosaccharide composition and anticoagulant mode of action and potency were determined. MATERIALS AND METHODS: The DS isolated from the ray skin was identified by chondroitinase treatment and characterized by FT-IR and (1)H NMR spectroscopy. Its anticoagulant activity was checked by activated partial thromboplastin time (aPTT), thrombin time (TT), thrombin generation (TG), heparin cofactor II (HCII) and antithrombin (AT)-mediated inhibition of thrombin. The effects on platelet activation and aggregation were investigated using flow cytometry and aggregometry, respectively. RESULTS: Chemical backbone structures of DS from Raja radula were close to that of DS from porcine intestinal mucosa. However, (1)H NMR indicated that iduronic acid was the major hexuronic acid moiety in the ray skin DS and also suggested that the amount of 2-O-sulfonated iduronic acid was higher in comparison with mammalian DS along with the occurrence of 4-O-sulfonated N-acetylgalactosamine residues. The anticoagulant effect of the ray skin DS was mainly due to the potentiation of thrombin inhibition by HCII but also, although to a lesser extent, by AT and was higher than that of the DS standard. Moreover, it had no effect on platelet activation and aggregation induced by various agonists. CONCLUSION: Altogether, these results indicated that DS from raja radula skin is an anticoagulant drug of interest potentially useful in anticoagulant therapy.