Isolation, Purification and Structural Characterestics of Chondroitin Sulfate from Smooth hound Cartilage: In vitro Anticoagulant and Antiproliferative Properties.

Chondroitin sulfate was extracted from the cartilage of smooth hound (CSSH) and then purified by anion exchange chromatography. The structual characteristic of CSSH was evaluated by acetate cellulose electrophoresis, FTIR, 13C NMR and SAX-HPLC. Molecular weight of CSSH was average 68.78 KDa. Disaccharide analysis indicated that CSSH was predominately composed of monosulfated disaccharides in position 6 and 4 of the N-acetylgalactosamine (45.34% and 32.49%, respectively). CSSH was tested for in vitro anticoagulant activity using the three classical coagulation assays (activated partial thromboplastin time (aPTT), prothrombine time (TT) and thrombin time (PT) tests). The finding showed that CSSH prolonged significatively (p < 0.05), aPTT, TT and PT about 1.4, 3.44 and 1.21 fold, respectively, greater than that of the negative control at a concentration of 100 µg/ml. The CSSH caused a significant antiproliferative activity against HCT116 cell, which was 79% of cell proliferation inhibition at the concentration of 1000 µg/ml. Further, CSSH presented no toxicity against the normal cells and no hemolysis towards bovine erythrocytes for all concentrations tested. CSSH demonstrated hopeful antiproliferative and anticoagulant potential, which may be used as a novel and effective drug.

Development and characterization of bioactive edible films from spider crab (Maja crispata) chitosan incorporated with Spirulina extract.

Active food packaging films based on crab chitosan and Spirulina extract (SE) were developed. The effects of the SE incorporation at different levels on physical (color, opacity water vapor and oxygen permeability) and mechanical (tensile strength and elongation at break) properties of chitosan films were investigated. FTIR was carried out to observe the potential modifications of the chitosan films when incorporated with SE. The obtained results suggested that incorporation of SE into chitosan films improved mechanical and barrier properties. The antioxidant activity of the chitosan/SE films was characterized by means of three different analytical assays (DPPH, FRAP and FIC). Crab chitosan edible films containing SE showed higher antioxidant activity, regardless concentrations and methods assayed. Furthermore, the antioxidant activity occurred in a concentration-dependent manner. The agar disc diffusion method was used to determine the antibacterial activities of chitosan edible films against Escherichia coli, Staphylococcus aureus, Pseudomonas aeruginosa, Listeria monocytogenes, Salmonella typhimurium, Bacillus subtilis and Bacillus cereus. The chitosan/SE films were more effective (p<0.05) against five of the seven tested bacteria. The obtained crab chitosan edible films incorporated with SE showed great potential to be used for active food packaging due to its excellent antioxidant and antibacterial activities.

In vitro and in vivo anti-coagulant activity and toxicological studies of marine sulfated glycosaminoglycans.

The present study aimed to characterize and evaluate the in vitro and in vivo anticoagulant activity of sulfated glycosaminoglycans from the skins of smooth hound (SHSG) and grey triggerfish (GTSG). The analysis of SHSG and GTSG with acetate cellulose electrophoresis in Zn-acetate revealed the presence of hyaluronic acid (HA), chondroitin sulfate (CS) and dermatan sulfate (DS). Both glycosaminoglycans were evaluated for their in vitro anticoagulant activities using activated partial thromboplastin time (aPTT), thrombin time (TT) and prothrombine time (PT) tests. SHSG and GTSG and calciparin were tested as in vivo anticoagulants by subcutaneous (s.c) injection to adult female Wistar rats in a concentration of 75mg/kg of body weight. The administration of SHSG, GTSG and calciparin to rats induced a significant decrease of platelet rates compared to the control. The aPTT assay of SHSG and GTSG was prolonged 1.3 and 1.23-fold respectively compared with the control. Toxicity studies were performed to investigate whether or not SHSG and GTSG can cause pathological changes in the liver, proteins and DNA. The concentration and catalytic activity of liver oxidative stress markers and enzymes, respectively, as well as the observed hepatic morphological changes indicated that calciparin induced hepatic toxicity and oxidative damage in the liver. The higher activity and lower toxicity of SHSG and GTSG recommended these compounds as a better drug candidate than calciparin.

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.

Chitin and chitosan from the Norway lobster by-products: Antimicrobial and anti-proliferative activities.

Chitin was recovered through enzymatic deproteinization of the Norway lobster (Nephrops norvegicus) processing by-products. The obtained chitin was characterized and converted into chitosan by N-deacetylation, the acid-soluble form of chitin. Chitosan samples were then characterized by Fourier transform infrared spectroscopy (FTIR) and 13 Cross polarization magic angle spinning nuclear magnetic resonance (CP/MAS)-NMR spectroscopy. The antimicrobial activity and anti-proliferative capacity of chitosan were evaluated. Antimicrobial activity assays indicated that prepared chitosan exhibited marked inhibitory activity against the bacterial and fungal strains tested. Further, cytotoxic effects of chitosan samples on human colon carcinoma cells HCT116 was evaluated using the MTT assay. Chitosan showed the antiproliferative capacity against the colon-cancer-cell HCT116 in a dose dependent manner with IC50 of 4.6mg/ml. Indeed, HCT116 cell proliferation was significantly inhibited (p<0.05) between 13.5 and 67.5% at 0.5-6mg/mL of chitosan after 24h of cell treatment. The chitosan showed high antitumor activity which seemed to be dependent on its characteristics such as acetylation degree.

Valorisation of smooth hound (Mustelus mustelus) waste biomass through recovery of functional, antioxidative and antihypertensive bioactive peptides.

Concerns over the environmental and waste disposal problems created by the large amounts of by-products generated from fish processing industries are increasing worldwide. The bioconversion of those marine waste by-products through the enzymatic hydrolysis of their protein content offers the possibility for the development of bioactive peptides for use in various biotechnological applications. The present study aimed to investigate and evaluate the biological and functional properties of smooth hound (Mustelus mustelus) protein hydrolysates (SHPHs) obtained by treatment with intestinal and gastric enzyme preparations from M. mustelus viscera and porcine pancreatin. The results revealed that the SHPHs exhibited different degrees of hydrolysis and antioxidant activity. The hydrolysate produced by the intestinal crude extract presented the highest rate of antioxidative activity, showing an IC50 value of 1.47 ± 0.07 mg/mL in 1,1-diphenyl-2-picrylhydrazyl (DPPH) scavenging assays. The alkaline protease extract from the intestine of M. mustelus produced hydrolysate with the highest angiotensin I-converting enzyme (ACE) inhibitory activity (82 ± 1.52% at 2 mg/mL). All the protein hydrolysates showed excellent solubility and interfacial properties that were governed by pH. The major amino acids detected in SHPHs were glutamic acid/glutamine, aspartic acid/asparagine, histidine and arginine, followed by methionine, phenylalanine, serine, valine and leucine. Overall, the results indicated that smooth hound by-products can be used to generate high value-added products, thus offering a valuable source of bioactive peptides for application in wide range of biotechnological and functional food applications.

Assessment of pectinase production by Bacillus mojavensis I4 using an economical substrate and its potential application in oil sesame extraction.

Carrot (Daucus carota) peels, local agricultural waste product, is rich in lignocellulolytic material, including pectin which can act as an inducer of pectinase production. Pectinolytic enzymes production by Bacillus mojavensis I4 was studied in liquid state fermentation using carrot peel as a substrate. Medium composition and culture conditions for the pectinase production by I4 were optimized using two statistical methods: Taguchi design was applied to find the key ingredients and conditions for the best yield of enzyme production and The Box-Behnken design was used to optimize the value of the four significant variables: carrot peels powder, NH4Cl, inoculum size and incubation time. The optimal conditions for higher production of pectinase were carrot peels powder 6.5 %, NH4Cl 0.3 %, inoculum level 3 % and cultivation time 32 h. Under these conditions, the pectinase experimental yield (64.8 U/ml) closely matched the yield predicted by the statistical model (63.55 U/ml) with R (2) = 0.963. The best pectinase activity was observed at the temperature of 60 °C and at pH 8.0. The enzyme retained more than 90 % of its activity after 24 h at pH ranging from 6.0 to 10.0. The enzyme preserved more than 85 % of its initial activity after 60 min of pre-incubation at 30-40 °C and more than 67 % at 50 °C. The extracellular juice of I4 was applied in the process of sesame seeds oil extraction. An improvement of 3 % on the oil yield was obtained. The findings demonstrated that the B. mojavensis I4 has a promising potential for future use in a wide range of industrial and biotechnological applications.

Biochemical and antioxidant properties of peptidic fraction of carotenoproteins generated from shrimp by-products by enzymatic hydrolysis.

The composition, functional properties and in vitro antioxidative activity of the peptidic fraction of carotenoproteins from shrimp (Parapenaeus longirostris) by-products generated by enzymatic treatment with Alcalase was evaluated. The peptidic fraction of carotenoproteins (PFCP) contained 80.8 ± 0.21% protein, 2.74 ± 0.3% lipid, 14.4 ± 0.14% ash, 1.13 ± 0.08% chitin and 1.08 ± 0.02 µg total carotenoid/g of sample. The amino acid profile of PFCP showed a high percentage of essential amino acids, such as arginine, lysine, histidine and leucine. Therefore, PFCP had a high nutritional value and could be used as a supplement to poorly balanced dietary proteins. PFCP showed an excellent solubility and possessed interfacial properties, which were governed by their concentrations. The antioxidant activities of PFCP at different concentrations were evaluated using various in vitro antioxidant assays, including the 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical method, reducing power, chelating effects assay and ß-carotene bleaching. The antioxidant activity of PFCP, based on their protection of supercoiled DNA strand from scission by peroxyl and hydroxyl radicals into the nicked circular form was also investigated. Results from this study suggest that the peptidic fraction of carotenoproteins is a good source of natural antioxidants and peptides with interesting functionalities.