Isolation and partial characterization of collagen from outer skin of Sepia pharaonis (Ehrenberg, 1831) from Puducherry coast.

Type I collagen from outer skin of Sepia pharaonis was extracted and partially characterized. Yield of Acid Soluble Collagen (ASC) and Pepsin Soluble Collagen (PSC) were calculated as 1.66% and 3.93% and the total protein content of ASC and PSC were found as 18.4% and 48.6%. FT-IR spectrum of ASC and PSC recorded 12 and 14 peaks, respectively. 1H NMR spectrum of ASC showed singlets at 1.23 ppm, 3.1 ppm, 3.55 ppm and 3.7 ppm and PSC at 1.23 ppm and 2.08 ppm. The molecular weight for ASC was calculated as 102 kDa and for PSC as 110, 108 and 102 kDa through SDS-PAGE. Differential Scanning Calorimetry (DSC) results supported that PSC withstand high thermal stability (82.85 °C) than ASC (73.13 °C). Higher denaturation temperature with high molecular weight well support the property of type I collagen from skin of S. pharaonis and it could be used as another potent source for the extraction of collagen.

Chitosan nanoparticles from marine squid protect liver cells against N-diethylnitrosoamine-induced hepatocellular carcinoma.

Rationale of this study was framed to investigate the protective effect and anti-cancer property of nanoparticles based on chitosan isolated from squid, Sepioteuthis lessoniana, on hepatic cells in N-Nitrosodiethylamine-induced hepatocellular carcinoma in rats. The results conferred that the chitosan nanoparticle supplementation had a protective effect on liver cells by reducing the levels of marker enzymes and bilirubin and thus increasing the albumin levels. The level of reduced glutathione, ascorbic acid and α-tocopherol significantly increased in both post- and pre-treatment with chitosan nanoparticles. The levels of antioxidant enzymes were enhanced and lipid peroxidation products were diminished while treating nitrosodiethylamine-induced hepatocellular carcinoma with chitosan nanoparticles. Supplementation of chitosan nanoparticles had potent anti-hyperlipidemic property that was evidenced by monitoring the serum lipid levels and its components. Animals pre-treated with chitosan nanoparticles along with nitrosodiethylamine showed a significant reduction in the total cholesterol and triglycerides levels with increase in the levels of phospholipids and free fatty acids. Chitosan nanoparticles treated rats showed significant increment in high-density lipoprotein cholesterol and reduction in low-density lipoprotein and very low-density lipoprotein cholesterol when compared with levels in nitrosodiethylamine-induced hepatocellular carcinoma. Nitrosodiethylamine-induced carcinoma changes on circulation and hepatic antioxidant defense mechanism were regulated by chitosan nanoparticles, concluding that the chitosan nanoparticles have a potent protective effect on liver cells which might be due to its robust antioxidant and anti-lipidemic property.

Characterization of bioactive chitosan and sulfated chitosan from Doryteuthis singhalensis (Ortmann, 1891).

Chitosan was extracted from the pen of squid Doryteuthis singhalensis and characterized using FT-IR, NMR, CHN, SEM and DSC analysis. Purified chitosan was sulfated with chlorosulfonic acid in N,N-dimethylformamide and the added sulfate group was confirmed with FT-IR analysis. The molecular weight and degree of deacetylation (DDA) of chitosan was found 226.6kDa and 83.76% respectively. Chitosan exhibited potent antioxidant activity evidenced by reducing power, chelating ability on ferrous ions and scavenging activity on DPPH, superoxide and hydroxyl radicals. The anticoagulant assay using activated partial thromboplastin time (APTT) and prothrombin time (PT) showed chitosan as a strong anticoagulant. The results of this study showed possibility of using D. singhalensis pen as a non-conventional source of natural antioxidants and anticoagulant which can be incorporated in functional food formulations.

Antioxidant and anticoagulant activity of sulfated polysaccharide from Gracilaria debilis (Forsskal).

Sulfated polysaccharide was isolated from Gracilaria debilis and purified through gel chromatography and their molecular weight was determined through AGE and PAGE. The total sugars in the crude, fractionated and purified polysaccharide were estimated as 52.65%, 59.70% and 67.60%, respectively. The ash and moisture content of crude and purified polysaccharide was found to be 14.2% and 23.5% and the polysaccharide was free from protein contamination. The sulfate and uronic acid contents in the crude, fractionated and purified were estimated as 14.08%, 15.33% and 16.01% and 10.12%, 13.56%, 16.70%. The elemental composition including carbon (crude - 23.12%, purified - 21.05%), hydrogen (crude - 3.4%, purified - 4.13%) and nitrogen (crude - 1.22%, purified - 0.56%) were also analyzed. The anticoagulant activity of the sulfated polysaccharide through APTT and PT was estimated at 14.11 and 8.23IU/mg. The purified polysaccharide with the molecular mass of 20kDa showed highest antioxidant activity (38.57%, 43.48% and 38.88%) in all the assays tested such as DPPH hydroxyl radical, superoxide radical, hydroxyl radical scavenging activities and the structural property was analyzed through FT-IR and (1)H NMR spectrum. The results together suggest that the isolated low molecular weight sulfated polysaccharide will demonstrate as a enormously available alternative natural source of antioxidant for industrial uses.

Protective effect of chitosan from Sepia kobiensis (Hoyle 1885) cuttlebone against CCl4 induced hepatic injury.

Carbon tetrachloride (CCl4) is a potent hepatotoxic agent causing hepatic necrosis and it is widely used in animal models for induction of acute and chronic liver damage. The antioxidative and hepatoprotective effects of chitosan from Sepia kobiensis against CCl4 induced liver toxicity in Wistar rats was studied by measuring the activity of lipid peroxidation (TBARS, lipid hydroperoxides), non enzymatic antioxidant (GSH), antioxidant enzyme activities (SOD, CAT and GPx), liver marker enzymes (ALT and AST), lipid profile (FFA, TG, cholesterol and HDL cholesterol) and histopathological changes. Rats treated with chitosan against CCl4 toxicity showed significantly decreased levels of ALT and AST activities, total cholesterol, triglyceride and free fatty acid in plasma and tissue. Whereas the treatment with chitosan along with CCl4 showed markedly increased level of hepatic and circulatory in SOD, CAT, GPx and reduced glutathione and decreased the malondialdehyde level. Histopathological observations proved the marked hepatoprotective effect of chitosan. The CCl4 induced alterations in circulatory and hepatic antioxidant defense system were normalized by chitosan and it could be concluded that the hepatoprotective effect of chitosan may be due to its antioxidant and antilipidemic properties.

Isolation and structural characterisation of acid- and pepsin-soluble collagen from the skin of squid Sepioteuthis lessoniana (Lesson, 1830).

Acid-solubilised collagen (ASC) and pepsin-solubilised collagen (PSC) were effectively isolated from squid skin with good yield and total protein content. ASC and PSC consist of two α-chains with an imino acid content of 182.6 and 184 imino acid residues/1000 residues. The molecular weight was determined to be between 73 and 107 kDa by using SDS-PAGE. For peptide mapping, collagens were digested with achromo endopeptidase, and all components, including α, ß-chains, were markedly hydrolysed. Degradation peptides with molecular weights between 106.9 and 15.47 kDa were obtained. UV-vis absorption spectrum revealed distinct absorption at 220-240 nm. FT-IR spectra of collagens were almost similar when compared with standard. In differential scanning calorimetry profile, ASC and PSC exhibited a To of 59.10, 62.18°C and TP of 104.91, 98.10 °C, respectively. This investigation indicates that the collagen isolated from the squid skin, which is thrown as waste in the seafood-processing plant, might supplement the vertebrate collagen in industrial applications.

Hepatoprotective effect of ß-chitosan from gladius of Sepioteuthis lessoniana against carbon tetrachloride-induced oxidative stress in Wistar rats.

Chitosan has attracted much attention as a biomedical material, owing to its unique biological activities. In this study, hepatoprotective effect of ß-chitosan obtained from the gladius of squid Sepioteuthis lessoniana was studied against carbon tetrachloride (CCl4)-induced oxidative stress and liver injury in rats. The rats that received ß-chitosan along with the administration of CCl4 showed significantly decreased plasma and tissue alanine aminotransferase (ALT) and aspartate aminotransferase (AST) activities and total cholesterol, triglyceride (TG) and free fatty acid (FFA) contents, whereas the treatment with ß-chitosan alone markedly increased rat hepatic and circulatory superoxide dismutase (SOD), catalase and glutathione peroxidase (GPx) and reduced glutathione (GSH) levels and decreased the malondialdehyde level. Histopathological observations recommended the marked hepatoprotective effect of ß-chitosan. The CCl4-induced alterations on circulatory and hepatic antioxidant defence system were normalised by ß-chitosan, and it could be concluded that the hepatoprotective effect of chitosan may be due to its antioxidant and antilipidemic property. Therefore, ß-chitosan could be considered as antihepatotoxic agent.

Extraction, characterization and antioxidant property of chitosan from cuttlebone Sepia kobiensis (Hoyle 1885).

Chitin was extracted from the cuttlebone of Sepia kobiensis and chitosan was prepared through deacetylation. The chitosan was characterized for its structural, physical and thermal (CHN, DDA, FT-IR, NMR, XRD, Viscometric analysis, SEM and DSC) properties. Further, the chitosan exhibited the antioxidant activity of 50.68-74.36% at 1-10 mg ml(-1) and it also showed the reducing power of 0.28% at 1 mg ml(-1). At 10 mg ml(-1), the chitosan exhibited the scavenging ability of 46.17%, on 1,1-diphenyl-2-picrylhydrazyl radicals, 23.38-73.70% on superoxide radicals at 0.05-1.6 mg ml(-1) and 18.34% to 62.39% (0.1-3.2 mg ml(-1)) on hydroxyl radicals; whereas at 1-10 mg ml(-1) the chelating ability on ferrous ions was calculated as 49.74-73.59%. Based on the potential antioxidant activity, scavenging ability on hydroxyl radicals and chelating abilities on ferrous ions, the chitosan from the cuttlebone of S. kobiensis may not only be used as a potent natural antioxidant but also as a possible food quality enhancer ingredient in the pharmaceutical industry.

Sulfation of ß-chitosan and evaluation of biological activity from gladius of Sepioteuthis lessoniana.

ß-Chitosan extracted from the gladius of Sepioteuthis lessoniana was sulfated with chlorosufonic acid. The structural features of sulfated chitosan were analyzed by NMR spectroscopy. The elemental composition was analyzed using elemental analyzer. The molecular weight of sulfated chitosan estimated through viscometry was calculated as 12.47 × 10(4) Da. The water solubility of sulfated chitosan was found to be 81%. The sulfated chitosan was able to scavenge the DPPH, superoxide and hydroxyl radicals and the inhibitory concentration (IC50) of those free radicals was found to be 4, 0.428 and 0.473 mg/ml respectively. Sulfated chitosan also showed reducing and chelating property. The IC50 value of reducing and chelating ability was found to be 0.316 and 5.64 mg/ml respectively. The anticoagulant activity was determined for human plasma with respect to activated partial thromboplastin time and prothrombin time and it was 7.13 IU and 1.26 IU, respectively.

Physicochemical characterisation of ß-chitosan from Sepioteuthis lessoniana gladius.

ß-Chitin and its chitosan from the gladius of Sepioteuthis lessoniana have been isolated, purified, characterised and compared with the commercial chitosan. Ash, moisture, mineral, metal and elemental content were analyzed using standard techniques. The optical activity of chitin was found to be levorotatory. The degree of deacetylation was calculated by potentiometric titration and (1)H NMR. Viscosity average molecular weight of ß-chitosan was calculated by viscometry and size average molecular weight by GPC. The structure of ß-chitosan was elucidated with FT-IR and NMR. Thermal nature, crystalline structure and morphology of ß-chitosan were characterised through DSC, XRD and SEM, respectively. The water and fat binding capacity of ß-chitosan presently studied was significantly higher than that of the commercial chitosan. The result of the present study adds that S. lessoniana gladius is also an additional source of ß-chitin and chitosan of higher yield, lower molecular weight and higher degree of deacetylation.