Accelerated identification of serine racemase inhibitor from Centella asiatica.

Serine racemase (SR) converts the free form of L-serine into D-serine (DS) in the mammalian brain. The DS functions as a co-agonist of N-methyl D-aspartate (NMDA) receptor. The over- activation of NMDA receptor leads to many neurological disorders like stroke, amyotrophic lateral sclerosis, Alzheimer's disease and an effective inhibitor of SR could be a corrective method for the receptor over-activation. We report for the first time here a rapid way of purifying and identifying an inhibitor from medicinal plants known to have the neuro-protective effect. We have purified SR inhibitor from the methanolic extract of Centella asiatica by affinity method. High resolution mass spectrometry and infrared spectroscopy were used to identify the ligand to be madecassoside. We have shown the madecassoside binding in silico and its inhibition of recombinant human serine racemase in vitro and ex vivo.

Anti-diabetic activity of crude polysaccharide and rhamnose-enriched polysaccharide from G. lithophila on Streptozotocin (STZ)-induced in Wistar rats.

The aim of the present study was to elucidate the anti-diabetic effects of the crude polysaccharide and rhamnose-enriched polysaccharide derived from G. lithophila on streptozotocin (STZ)-induced diabetic Wistar rats. Treatment with crude polysaccharide and rhamnose-enriched polysaccharide showed increases in body weight and pancreatic insulin levels and a decrease in blood glucose levels compared with control diabetic rats. The blood concentrations of total cholesterol (TC), triglycerides (TGs), low-density lipoprotein (LDL) and very-low-density lipoprotein (VLDL) decreased, and high-density lipoprotein (HDL) increased both in the crude polysaccharide- and rhamnose-enriched polysaccharide-treated rats. Superoxide dismutase (SOD) and glutathione peroxidase (GPx) levels increased, and malondialdehyde (MDA) levels decreased in the livers, kidneys and pancreases of crude polysaccharide- and rhamnose-enriched polysaccharide-treated rats. Immunohistological examination further confirmed that restoration of the normal cellular size of the islets of Langerhans and the rebirth of ß-cells were found to be greater in the body region than in the head and tail regions of the pancreas. The crude polysaccharide- and rhamnose-enriched polysaccharide-treated diabetic rats showed normal blood glucose levels and insulin production, and reversed cholesterol levels and enzymatic actions. Therefore, rhamnose-enriched polysaccharide from G. lithophila acts as a potent anti-diabetic agent to treat diabetes and can lead to the development of an alternative medicine for diabetes in the future.

Multi-elemental concentration in different body parts of Sepiella inermis by inductively coupled plasma mass spectrometry.

The present study examined the accumulation of metal on Sepiella inermis from the Mudasalodai Landing Center, from southeast coastal region of India. Inductively coupled plasma mass spectrometry (ICP-MS) was used to determine metals including aluminium, boron, cadmium, cobalt, chromium, copper, iron, manganese, magnesium, nickel, lead and zinc. The results showed that metal concentration in S. inermis detected in the head, arm, mantle, eye, ink, liver and nidamental gland with higher concentration of magnesium up to 992.78 mg/kg, and tentacle showed maximum concentration of aluminium 306.72 mg/kg. Further, copper found in low concentration ranges from 0.04 to 0.55 mg/kg in different parts of S. inermis. Heavy metal like cadmium detected high in tentacle with 0.24 mg/kg, and the manganese present in eye was 0.55 mg/kg. However, no accumulation of nickel was found in the tentacle part.

Chemical structure and biological properties of a polysaccharide isolated from Pleurotus sajor-caju.

Herein, a polysaccharide obtained from Pleurotus sajor-caju was fractionated via anion-exchange column chromatography and purified using gel permeation column chromatography. The chemical characterization of the polysaccharide indicated that it contained 90.16% total carbohydrate, 0% protein, 12.7% ash and 5.2% moisture; on the other hand, the carbon, hydrogen and nitrogen contents were found to be 31.53, 4.28 and 3.01%, respectively. The polysaccharide has the molecular weight of 79 kDa; the chemical structure of the polysaccharide is →6)α-d-Glciv(1→6)α-d-Glciii(1→6)ß-d-Glcii(1→6)α-d-Glci(1→units. The polysaccharide exhibited the DPPH radical scavenging activity of 21.67-68.35% at 10-160 µg ml-1, ABTS radical scavenging activity of 16.01-70.09% at 25-125 µg ml-1, superoxide radical scavenging activity of 24.31-73.64% at 50-250 µg ml-1, hydroxyl radical scavenging activity of 16.64-63.51% at 25-125 µg ml-1 and reducing power of 0.366-1.678% at 10-120 µg ml; further evaluation of the polysaccharide revealed its anticancer activity of 18.61-63.21% at 100-500 µg ml-1 concentration against the AGS human gastric carcinoma cell line. The active principle of the polysaccharide may be used in the food and pharmacological industry in the future.

Isolation and chemical characteristics of rhamnose enriched polysaccharide from Grateloupia lithophila.

The crude polysaccharide was extracted from Grateloupia lithophila through hot-water extraction and deproteinization. Further, fractionated by anion-exchange column using Q-Sepharose and purified by gel-permeation chromatography using Sepharose 4-LB column. The crude and purified polysaccharide contains high carbohydrate (75.7 and 89.7%), ash (18.2 and 3.2%) and moisture (14.8 and 1.3%); the protein and uronic acid were absent. The molecular weight of crude, fractionated and purified polysaccharide was found to be 37 kDa, 29 kDa and 24 kDa. The monosaccharide composition of the crude polysaccharide was found to be having rhamnose (79.82%), fructose (8.38%), galactose (3.95%), xylose (3.31%) and glucose (1.48%); whereas the purified polysaccharide reported higher amount of rhamnose (95.88%), 1.13% of xylose and 2.21% of fructose. The structural elucidation of the purified polysaccharide was conformed as α-l-rhamnose through polarimetry, FT-IR and 1H NMR spectroscopy.

Isolation, characterization and bioactive potential of sulfated galactans from Spyridia hypnoides (Bory) Papenfuss.

The sulfated galactans (SG) of mass 16 kDa was purified from S.hypnoides through anion exchange and gel permeation chromatography. The biochemical properties of SG including carbohydrate, 3,6 anhydrogalactose, sulfate, uronic acid, moisture, ash, carbon, hydrogen, nitrogen contents were estimated. In the purified SG, the presence of major sugars such as galactose and glucose were identified through HPLC and it was further structurally characterised through FT-IR and NMR spectroscopy. Anticoagulant activity of SG was estimated as 25.36 & 2.46 IU at 25 µg/ml (aPTT & PT). SG also showed potential dose dependent antioxidant activity against free radicals such as DPPH (56.41% at 2 mg/ml), hydroxyl radicals (65.58% at 3 mg/ml) and superoxide radicals (73.12% at 0.6 mg/ml). The maximum metal chelating and total antioxidant property (76.42%, 66.81%) was exhibited at 1 mg/ml. The results indicate that the SG from red seaweed represents a good source of polysaccharide with significant anticoagulant and antioxidant properties.

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.

Mucopolysaccharide from cuttlefish: Purification, chemical characterization and bioactive potential.

The sulfated mucopolysaccharide (GAG) was isolated from S. pharonis and the carbohydrate and protein content was found to be 62.4% and 3.9%. The disaccharide profile of sulfated GAG composed glucuronic acid, N-acetyl glucosamine and sulfate content by contributing 50.11%, 38.00% and 27.69% respectively. The carbon, hydrogen and nitrogen content of the sulfated GAG showed 14.80%, 1.68% and 2.99% respectively. The molecular weight of sulfated GAG was calculated as 27kDa and the structural characterization was done by Fourier Transform Infrared (FT-IR) and NMR Spectroscopy. The Activated Partial Thromboplastin Time (APTT) and Prothrombin Time (PT) of sulfated GAG were determined as 91 IU and 39.55 IU at 25µg/ml respectively. Further the sulfated GAG reported the cytotoxic effect (CC50) of 1100µg/ml concentration on Vero cell line. The sulfated GAG reported the anticancer activity against HeLa cell line with an inhibition rate of 18.65%-66.13% at 50-250µg/ml concentration. The sulfated GAG can be considered as a potent anticoagulant and anticancer drug in future.

Evaluation of antioxidant activities and chemical analysis of sulfated chitosan from Sepia prashadi.

The chitin and chitosan of S. prashadi was prepared through demineralization, deproteinzation, deacetylation process and sulfation were carried by chlorosulfonic acid in N,N-dimethylformamide. The sulfate content in chitosan was found to be 18.9%. The carbon, hydrogen and nitrogen composition of the sulfated chitosan were recorded 39.09%, 6.95% and 6.58% respectively. The structural analysis was done by using FT-IR and NMR spectroscopy technique. The DSC curves of sulfated chitosan showed a large endothermic peak resolved with To value of 54.57°C and TP value of 97.46°C. The morphology of sulfated chitin and sulfated chitosan were studied by SEM. The Further in vitro antioxidant activity of sulfated chitosan was screened by scavenging activity of superoxide radical assay, hydroxyl radical scavenging assay, metal-ion chelating effect and reducing power. Its anticoagulant activity was tested for human plasma with respect to Activated Partial Thromboplastin Time (APTT) and Prothrombin Time (PT). Results prove that sulfated chitosan has potent antioxidant and anticoagulant activity.

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.

Fabrication of ß-chitosan nanoparticles and its anticancer potential against human hepatoma cells.

ß-Chitosan from the gladius was enzymatically depolymerized and utilized for the synthesis of ß-chitosan nanoparticles using sodium tripolyphosphate by ionotropic gelation. The size and zeta potential of ß-Chitosan nanoparticles (ß-CNP) were determined. The structural features were evaluated by FT-IR and NMR spectral analysis. The morphological characterization, composition and surface topography of ß-CNP were explored by SEM, EDAX and AFM techniques. The thermal and crystallographic nature of ß-CNP was also studied. The cell viability of HepG2 cells inhibited by ß-CNP was detected in a dose-dependent manner. The inhibitory concentration of ß-CNP was 30µg/ml. Various biochemical parameters such as TBARS and lipid hydroperoxides, enzymatic and non-enzymatic antioxidant (SOD, CAT, GPx and GSH) studies proved the anticancer property of ß-CNP in HepG2 cells. This study suggests that ß-CNP should be a promising drug for treating hepatocellular carcinoma in future.

Preparation, characterization and antibacterial activity of chitosan and phosphorylated chitosan from cuttlebone of Sepia kobiensis (Hoyle, 1885).

Chitosan is a commercially available derivative of chitin that has been extensively studied for its antimicrobial properties. In order to improve the water solubility and its biological activity, the chemical modification or derivatisation is attempted. In the present investigation, the chitosan prepared from the cuttlebone of Sepia kobiensis was being chemically modified by reacting it with orthophosphoric acid so as to obtain phosphorylated chitosan. Then the chitosan and phosphorylated chitosan were structurally characterized through FT-IR spectroscopy. Further the antibacterial activity of chitosan and phosphorylated chitosan was tested against clinically isolated human pathogens (Gram-positive: Streptococcus sp., Streptococcus pneumoniae and Staphylococcus aureus and Gram-negative: Escherichia coli, Vibrio cholerae, V. alginolyticus, Vibrio parahaemolyticus, Pseudomonas aeruginosa, Klebsiella pneumoniae, Salmonella sp. and Proteus vulgaris) by well diffusion method and the Minimum Inhibitory Concentration (MIC) was also calculated. The results of the present study suggests that the chitosan and phosphorylated chitosan has concentration dependent antibacterial activity with variation against several pathogenic human pathogenic bacterial strains which indicates their possible use as antibacterial agents.

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.

Structural characterization and bioactivities of sulfated polysaccharide from Monostroma oxyspermum.

Sulfated polysaccharide was isolated from Monostroma oxyspermum through hot water extraction, anion-exchange and gel permeation column chromatography. The sulfated polysaccharide contained 92% of carbohydrate, 0% of protein, 7.8% of uronic acid, 22% of ash and 33% of moisture respectively. The elemental composition was analyzed using CHNS/O analyzer. The molecular weight of sulfated polysaccharide determined through PAGE was found to be as 55 kDa. Monosaccharides analysis revealed that sulfated polysaccharide was composed of rhamnose, fructose, galactose, xylose, and glucose. The structural features of sulfated polysaccharide were analyzed by NMR spectroscopy. Further the sulfated polysaccharide showed total antioxidant and DPPH free radical scavenging activity were as 66.29% at 250 µg/ml and 66.83% at 160 µg/ml respectively. The sulfated polysaccharide also showed ABTS scavenging ability and reducing power were as 83.88% at 125 µg/ml and 15.81% at 400 µg/ml respectively. The anticoagulant activity was determined for human plasma with respect to Activated Partial Thromboplastin Time (APTT) and Prothrombin Time (PT) was 20.09 IU and 1.79 IU at 25 µg/ml respectively. These results indicated that the sulfated polysaccharide from M. oxyspermum had potent antioxidant and anticoagulant activities.

Characterization and wound healing property of collagen-chitosan film from Sepia kobiensis (Hoyle, 1885).

The present findings were aimed to develop an effective form of collagen-chitosan film to investigate the wound healing property in Wistar rats. Primarily, the acid-soluble collagen (ASC) isolated, structurally and physicochemically characterized. FT-IR and 1H NMR spectra were confirmed the collagen peptide crosslinks. The molecular weight of ASC subunits was about 86 kDa and 79 kDa, the proline and hydroxyproline content was 13.15% and 7.55%, respectively. In addition, collagen-chitosan films were successfully prepared in a controlled environment and characterized by FT-IR and DSC. In wound healing, the experimental animal model showed the increased wound size reduction in the CChF treated rats (96.25%) when compared to control rats (33.75%) followed by CF treated rats (65%) and ChF (55%). The histological results well supported these findings. It can be concluded that a better and faster epithelization was observed in the CChF group compared to the other groups.

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.