Carbohydrate-Binding Properties and Antimicrobial and Anticancer Potential of a New Lectin from the Phloem Sap of Cucurbita pepo.

A Cucurbita phloem exudate lectin (CPL) from summer squash (Cucurbita pepo) fruits was isolated and its sugar-binding properties and biological activities were studied. The lectin was purified by affinity chromatography and the hemagglutination assay method was used to determine its pH, heat stability, metal-dependency and sugar specificity. Antimicrobial and anticancer activities were also studied by disc diffusion assays and in vivo and in vitro methods. The molecular weight of CPL was 30 ± 1 KDa and it was stable at different pH (5.0 to 9.0) and temperatures (30 to 60 °C). CPL recovered its hemagglutination activity in the presence of Ca2+. 4-nitrophenyl-α-D-glucopyranoside, lactose, rhamnose and N-acetyl-D-glucosamine strongly inhibited the activity. With an LC50 value of 265 µg/mL, CPL was moderately toxic and exhibited bacteriostatic, bactericidal and antibiofilm activities against different pathogenic bacteria. It also exhibited marked antifungal activity against Aspergillus niger and agglutinated A. flavus spores. In vivo antiproliferative activity against Ehrlich ascites carcinoma (EAC) cells in Swiss albino mice was observed when CPL exerted 36.44% and 66.66% growth inhibition at doses of 3.0 mg/kg/day and 6.0 mg/kg/day, respectively. A 12-day treatment by CPL could reverse their RBC and WBC counts as well as restore the hemoglobin percentage to normal levels. The MTT assay of CPL performed against human breast (MCF-7) and lung (A-549) cancer cell lines showed 29.53% and 18.30% of inhibitory activity at concentrations of 128 and 256 µg/mL, respectively.

Antimicrobial, antioxidant and antiproliferative activities of a galactose-binding seed lectin from Manilkara zapota.

A seed lectin from Manilkara zapota (MZSL) was purified using ammonium sulphate precipitation and affinity chromatography. Hemagglutination activity, neutral sugar content and physicochemical properties of the lectin were determined and toxicity was checked by brine shrimp toxicity assay. Antimicrobial, antioxidant as well as in vitro anticancer activities of MZSL were also evaluated. Our findings showed the molecular weight of MZSL to be 33.0 ± 1 kDa. Minimum hemagglutination concentration of the lectin was 15.625 µg/ml. With a neutral sugar content of 6.32 %, the lectin was fully active at a temperature range of 30-50 °C and pH 7.0-8.0 and it was mildly toxic with an LC50 value of 107.93 µg/ml. The lectin demonstrated bacteriostatic activity against gram-positive bacteria in contrast to gram-negative bacteria at a concentration of 31.25 µg/ml, agglutinated Staphylococcus aureus and Shigella dysenteriae and exerted fungistatic activity against Aspergillus niger. MZSL dose-dependently reduced the formation of biofilm by E. coli. DPPH assay confirmed its antioxidant activity with an IC50 value of 96.42 µg/ml. MZSL showed 21.64 % growth inhibition against Ehrlich ascites carcinoma (EAC) cells at 80 µg/ml whereas its antiproliferative potential against MCF-7 and A-549 cancer cell lines became evident with IC50 values of 70.66 µg/ml and 107.64 µg/ml, respectively.

Antitumor Activity of a Lectin Purified from Punica granatum Pulps against Ehrlich Ascites Carcinoma (EAC) Cells.

BACKGROUND: Lectins are carbohydrate-binding proteins with various pharmacological activities, such as antimicrobial, antidiabetic, antioxidant, and anticancer. Punica granatum fruit extract has traditional uses, however, the anti-cancer activity of purified lectin isolated from P. granatum pulp is yet to be reported. OBJECTIVE: The goals of this study are purification, characterization of the lectin from P. granatum, and examination of the purified lectin's anticancer potential. METHODS: Diethylaminoethyl (DEAE) ion-exchange chromatography was used to purify the lectin, and SDSPAGE was used to check the purity and homogeneity of the lectin. Spectrometric and chemical analysis were used to characterize the lectin. The anticancer activity of the lectin was examined using in vivo and in vitro functional assays. RESULTS: A lectin, designated as PgL of 28.0 ± 1.0 kDa molecular mass, was isolated and purified from the pulps of P. granatum and the lectin contains 40% sugar. Also, it is a bivalent ion-dependent lectin and lost its 75% activity in the presence of urea (8M). The lectin agglutinated blood cells of humans and rats, and sugar molecules such as 4-nitrophenyl-α-D-manopyranoside and 2- nitrophenyl -ß- D-glucopyranoside inhibited PgL's hemagglutination activity. At pH ranges of 6.0-8.0 and temperature ranges of 30°C -80°C, PgL exhibited the highest agglutination activity. In vitro MTT assay showed that PgL inhibited Ehrlich ascites carcinoma (EAC) cell growth in a dose-dependent manner. PgL exhibited 39 % and 58.52 % growth inhibition of EAC cells in the mice model at 1.5 and 3.0 mg/kg/day (i.p.), respectively. In addition, PgL significantly increased the survival time (32.0 % and 49.3 %) of EAC-bearing mice at 1.5 and 3.0 mg/kg/day doses (i.p.), respectively, in comparison to untreated EAC-bearing animals (p < 0.01). Also, PgL reduced the tumor weight of EAC-bearing mice (66.6 versus 39.13%; p < 0.01) at the dose of 3.0 mg/kg/day treatment. Furthermore, supplementation of PgL restored the haematological parameters toward normal levels deteriorated in EAC-bearing animals by the toxicity of EAC cells. CONCLUSION: The results indicated that the purified lectin has anticancer activity and has the potential to be developed as an effective chemotherapy agent.

Biogenic silver/silver chloride nanoparticles inhibit human cancer cells proliferation in vitro and Ehrlich ascites carcinoma cells growth in vivo.

Silver/silver chloride nanoparticles (Ag/AgCl-NPs) were synthesized for the first time from the herbal Geodorum densiflorum rhizome extracts and characterized by different techniques. The surface plasmon resonance peak at 455 nm was observed in the UV-Visible spectrum, the average particle size of 25 nm was determined by SEM, XRD reflection peaks (28.00°, 32.42°, 38.28°, 46.38°, 54.94°, 57.60°, 64.64°, and 67.48°) indicated the presence of Ag-NPs and AgCl-NPs, heat stability was confirmed by TGA and FTIR analysis indicated the presence of alcohol/phenol, alkanes, primary amines, nitro compounds, alkyl chloride functional groups. The synthesized Ag/AgCl-NPs, previously synthesized Kaempferia rotunda and Zizyphus mauritiana mediated Ag/AgCl-NPs separately inhibited the proliferation of BxPC-3 cells with the IC50 values of 7.8, 17.1, and 20.1 µg/ml, respectively. In the case of MCF-7 cells, the IC50 values of G. densiflorum- Ag/AgCl-NPs and K. rotunda-Ag/AgCl-NPs were 21.5 and 23.5 µg/ml, respectively. Whereas the IC50 of G. densiflorum-Ag/AgCl-NPs was 28.0 µg/ml against glioblastoma stem cells (GSCs). Induction of apoptosis in GSCs, BxPC-3 and MCF-7 cells was noted followed by NPs treatment. In GSCs, the expression level of NFκB, TNFα, p21, and TLR9 genes were upregulated after treatment with G. densiflorum-Ag/AgCl-NPs while in the MCF-7 cells, the expression of p53, FAS, Caspase-8 and -9, NFκB, MAPK, JNK and p21 genes were increased. G. densiflorum-Ag/AgCl-NPs inhibited 60% and 95% of EAC cells growth at the doses of 2 and 4 mg/Kg/day after intraperitoneal treatment with five consequent days, respectively. A remarkable improvement of hematological parameters with the decreased average tumor weight and increase of 75% life span of G. densiflorum-Ag/AgCl-NPs treated mice were observed. Altogether, this study reported for the first time in vitro anticancer activity of biogenic G. densiflorum-Ag/AgCl-NPs against GSC cells along with MCF-7 and BxPC-3 cells and in vivo anticancer properties against EAC cells.

Hypnea musciformis-mediated Ag/AgCl-NPs inhibit pathogenic bacteria, HCT-116 and MCF-7 cells' growth in vitro and Ehrlich ascites carcinoma cells in vivo in mice.

In the present study, Ag/AgCl-NPs were biosynthesised using Hypnea musciformis seaweed extract; NPs synthesis was confirmed by a change of colour and observation of a razor-sharp peak at 424 nm by UV-visible spectroscopy. Synthesised nanoparticles were characterised by transmission electron microscopy, energy-dispersive X-ray spectroscopy, X-ray powder diffraction and Fourier transform infrared spectroscopy. Bacterial cell growth inhibition proves that the Ag/AgCl-NPs have strong antibacterial activity and cell morphological alteration was observed in treated bacterial cells using propidium iodide (PI). Ag/AgCl-NPs inhibited Ehrlich ascites carcinoma (EAC) cells, colorectal cancer (HCT-116) and breast cancer (MCF-7) cell line in vitro with the IC50 values of 40.45, 24.08 and 36.95 µg/ml, respectively. Initiation of apoptosis in HCT-116 and MCF-7 cells was confirmed using PI, FITC-annexin V and Hoechst 33342 dye. No reaction oxygen species generation was observed in both treated and untreated cell lines. A significant increase of ATG-5 gene expression indicates the possibility of autophagy cell death besides apoptosis in MCF-7 cells. The initiation of apoptosis in EAC cells was confirmed by observing caspase-3 protein expression. Ag/AgCl-NPs inhibited 22.83% and 51% of the EAC cell growth in vivo in mice when administered 1.5 and 3.0 mg/kg/day (i.p.), respectively, for 5 consequent days.

Antiproliferative and Antimicrobial Potentials of a Lectin from Aplysia kurodai (Sea Hare) Eggs.

In recent years, there has been considerable interest in lectins from marine invertebrates. In this study, the biological activities of a lectin protein isolated from the eggs of Sea hare (Aplysia kurodai) were evaluated. The 40 kDa Aplysia kurodai egg lectin (or AKL-40) binds to D-galacturonic acid and D-galactose sugars similar to previously purified isotypes with various molecular weights (32/30 and 16 kDa). The N-terminal sequence of AKL-40 was similar to other sea hare egg lectins. The lectin was shown to be moderately toxic to brine shrimp nauplii, with an LC50 value of 63.63 µg/mL. It agglutinated Ehrlich ascites carcinoma cells and reduced their growth, up to 58.3% in vivo when injected into Swiss albino mice at a rate of 2 mg/kg/day. The morphology of these cells apparently changed due to AKL-40, while the expression of apoptosis-related genes (p53, Bax, and Bcl-XL) suggested a possible apoptotic pathway of cell death. AKL-40 also inhibited the growth of human erythroleukemia cells, probably via activating the MAPK/ERK pathway, but did not affect human B-lymphoma cells (Raji) or rat basophilic leukemia cells (RBL-1). In vitro, lectin suppressed the growth of Ehrlich ascites carcinoma and U937 cells by 37.9% and 31.8%, respectively. Along with strong antifungal activity against Talaromyces verruculosus, AKL showed antibacterial activity against Staphylococcus aureus, Shigella sonnei, and Bacillus cereus whereas the growth of Escherichia coli was not affected by the lectin. This study explores the antiproliferative and antimicrobial potentials of AKL as well as its involvement in embryo defense of sea hare.

Trichosanthes dioica seed lectin inhibits Ehrlich ascites carcinoma cells growth in vivo in mice by inducing G0 /G1 cell cycle arrest.

Trichosanthes dioica seed lectin (TDSL), having a molecular mass of 57 ± 2 kDa was purified in an alternative way. For the purification process, the galactose-sepharose-4B affinity column was used. The purified TDSL agglutinated human and mouse erythrocytes at the minimum concentration of 8  µg/ml. d-lactose and d-galactose were the most potent inhibitory sugars as observed. The purified lectin was a glycoprotein having 3.0% of a neutral sugar. The lectin exhibited maximum activity up to 60°C and pH range from 7.0 to 10.0 and stable up to 4.0 M urea as tested. The lectin demonstrated mild toxicity when administered against brine shrimp nauplii, and the LC50 value was calculated to be 84.0 µg/ml. Minimum agglutination of Ehrlich ascites carcinoma (EAC) cells caused by the lectin was found at the protein concentration of 1.56 µg/ml. TDSL inhibited 7, 50.2%, and 60.3% of the EAC cells growth in vivo in mice when administered with 0.75, 1.5, and 3.0 mg kg-1  day-1 (i.p.), respectively, for five consecutive days. After lectin treatment, red blood cell (RBC) and hemoglobin levels were increased significantly toward the normal compared with EAC cells-bearing control and normal mice. The tumor burden reduced to 29.5% and 67% after treatment with 1.5 and 3.0 mg kg-1  day-1 of the lectin. TDSL triggered the cell cycle arrest at the G0 /G1 phase, which was observed using flow cytometry. In conclusion, TDSL can be a candidate for the potent anticancer agents that exerts low toxicity toward brine shrimp nauplii. PRACTICAL APPLICATIONS: A 57 ± 2 kDa lectin (designated TDSL) was purified from Trichosanthes dioica seeds using a galactose-sepharose-4B affinity column. The lectin demonstrated mild toxicity and agglutinated Ehrlich ascites carcinoma (EAC) cells. The lectin inhibited 50.2% and 60.3% of the EAC cell growth in vivo in mice when administered with 1.5 and 3.0 mg kg-1  day-1 (i.p.), respectively, for five consecutive days. The lectin increased RBC and hemoglobin level toward the normal compared with lectin-treated EAC cells-bearing, EAC cells-bearing control and normal mice. The tumor burden reduced to 29.5% and 67% after treatment with 1.5 and 3.0 mg kg-1  day-1 lectin. TDSL triggered the cell cycle arrest at the G0 /G1 phase. The lectin can be a candidate for potent anticancer agents.

Biogenic silver/silver chloride nanoparticles inhibit human glioblastoma stem cells growth in vitro and Ehrlich ascites carcinoma cell growth in vivo.

The importance of biogenic silver/silver chloride nanoparticles has become increasing day by day. In the present study, silver/silver chloride nanoparticles (Ag/AgCl-NPs) were synthesized from Kaempferia rotunda tuberous rhizome extract to evaluate the antiproliferative activity against human glioblastoma stem cells (GSCs) in vitro and Ehrlich ascites carcinoma (EAC) cells in vivo in mice. Synthesis of nanoparticles was confirmed by colour change and UV-visible spectrum and characterized by TEM, XRD, TGA, AFM and FTIR. K rotunda and recently synthesized Zizyphus mauritiana fruit extract-mediated Ag/AgCl-NPs inhibited 77.2% and 71% of GSCs growth at 32 µg/mL concentration with the IC50 values of 6.8 and 10.4 µg/mL, respectively. Cell morphological studies and caspase-3 immunofluorescence assay revealed that both biogenic nanoparticles induced apoptosis in GSCs. Expression levels of several genes were checked by real-time PCR after treatment with K rotunda tuberous rhizome-mediated Ag/AgCl-NPs. PARP, EGFR, NOTCH2 and STAT3 gene expression were decreased with the increase of NFκB, TLR9, IL1, TNFα, IKK and p21 gene that would be the cause of induction of apoptosis in GSCs. The cell cycle arrest at G2 /M phase was confirmed by flow cytometric assay. Both nanoparticles were injected intraperitoneally to rapidly growing EAC cells for 5 consecutive days. Approximately, 32.3% and 55% EAC cells growth were inhibited by K rotunda tuberous rhizome-mediated Ag/AgCl-NPs at 6 and 12 mg/kg/day doses, respectively while only 20% cell growth inhibition was monitored at 12 mg/kg/day dose of Z mauritiana-mediated Ag/AgCl-NPs. From the above results, it can be concluded that presently synthesized nanoparticles would be a potent anticancer agent.

Characterization of pepsin-solubilised collagen recovered from mackerel (Scomber japonicus) bone and skin using subcritical water hydrolysis.

This study was aimed at isolation of pepsin-solubilised collagen (PSC) from Mackerel (Scomber japonicus) bone and skin in order to effectively valorise these abundant wastes. The yield of PSC (8.10%) from skin was considerably higher than that from bone (1.75%). Based on the protein patterns, both PSCs were type Ι, and consisted of two α-chains. Fourier-transform infrared spectra demonstrated that PSCs from the bone and skin exhibited a triple-helical structure. The denaturation temperatures (Td) of the PSCs from bone and skin were 27 and 30 °C, respectively. Low-molecular-weight peptides (<1650 Da) were generated from both PSCs after subcritical water hydrolysis treatment. Glycine accounted for 30% of the total amino acids identified in both PSC hydrolysates. The antioxidant activities of both PSC hydrolysates were significantly higher than those of the isolated PSCs. Therefore, PSC hydrolysates can be used as a functional ingredient in the food, cosmetic and pharmaceutical industries.

MytiLec-1 Shows Glycan-Dependent Toxicity against Brine Shrimp Artemia and Induces Apoptotic Death of Ehrlich Ascites Carcinoma Cells In Vivo.

MytiLec-1, a 17 kDa lectin with ß-trefoil folding that was isolated from the Mediterranean mussel (Mytilus galloprovincialis) bound to the disaccharide melibiose, Galα(1,6) Glc, and the trisaccharide globotriose, Galα(1,4) Galß(1,4) Glc. Toxicity of the lectin was found to be low with an LC50 value of 384.53 µg/mL, determined using the Artemia nauplii lethality assay. A fluorescence assay was carried out to evaluate the glycan-dependent binding of MytiLec-1 to Artemia nauplii. The lectin strongly agglutinated Ehrlich ascites carcinoma (EAC) cells cultured in vivo in Swiss albino mice. When injected intraperitoneally to the mice at doses of 1.0 mg/kg/day and 2.0 mg/kg/day for five consecutive days, MytiLec-1 inhibited 27.62% and 48.57% of cancer cell growth, respectively. Antiproliferative activity of the lectin against U937 and HeLa cells was studied by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay in vitro in RPMI-1640 medium. MytiLec-1 internalized into U937 cells and 50 µg/mL of the lectin inhibited their growth of to 62.70% whereas 53.59% cell growth inhibition was observed against EAC cells when incubated for 24 h. Cell morphological study and expression of apoptosis-related genes (p53, Bax, Bcl-X, and NF-κB) showed that the lectin possibly triggered apoptosis in these cells.

Geodorum densiflorum rhizome lectin inhibits Ehrlich ascites carcinoma cell growth by inducing apoptosis through the regulation of BAX, p53 and NF-κB genes expression.

A lectin with a molecular mass of 12 ±â€¯1 kDa was isolated for the first time from Geodorum densiflorum (Lam.) rhizome (GDL). The lectin exhibited hemagglutination activity both in mice and human erythrocytes which was inhibited by 4-nitrophenyl-ß-D-glucopyranoside among the tested 26 sugars. The lectin was heat stable and showed its full activity in the pH range from 5.0 to 9.0. The lectin did not lose its activity in the presence of urea but the activity lost significantly when treated with EDTA. Divalent cation Ca2+ and Mg2+ also partially inhibited the activity of the lectin. The lectin strongly agglutinated Ehrlich ascites carcinoma (EAC) and inhibited the cells growth by 60% at 160 µg/ml protein concentration but unable to inhibit the growth of HeLa cells in vitro. The growth inhibition was due to the induction of apoptosis in the EAC cells which was confirmed by annexin-V and caspase-3 substrate and finally by apoptosis-related genes expression. An intensive expression of anti-apoptotic Bcl-X gene was observed only in untreated EAC cells while pro-apoptotic Bak and Bax genes expressed only in GDL treated EAC cells with the remarkable increase of the p53 gene expression. In the treated EAC cells NFκB expression was down regulated.

Moringa oleifera seed lectin inhibits Ehrlich ascites carcinoma cell growth by inducing apoptosis through the regulation of Bak and NF-κB gene expression.

A Moringa oleifera seed lectin (MOSL) was purified by using chitin column with the molecular mass of 17±1kDa. The lectin agglutinated mouse, cow and human erythrocytes and the hemagglutination activity was inhibited by methyl-α-d-mannopyranoside, methyl-ß-d-galactopyranoside, lactose and glucose. The lectin exhibited 100% hemagglutination activity at the pH range from 8.0 to 9.0 and temperature range from 30 to 60°C. Additionally, the lectin gradually lost its activity in the presence of urea but the activity abolish completely when treated with EDTA. MOSL showed mild toxicity against brine shrimp nauplii with a LC50 value of 131.0µg/ml. Antiproliferative activity was studied against Ehrlich ascites carcinoma (EAC) cells and 71.08% cell growth inhibition was observed in vitro at 200µg/ml. The lectin was injected (i.p.) into EAC mice at the doses of 2.0 and 4.0mg/kg/day for five consecutive days and 25.38% and 55% of cell growth inhibition was observed, respectively. MOSL caused the cell cycle arrest at G2/M phase as determined by FACS flow cytometry. The cell growth inhibition was due to the induction of apoptosis in the EAC cells which was confirmed by cell morphological study, caspase-3 inhibitor and activation of Bak and suppression of Bcl-2 and NF-κB genes expression.

Antitumor properties of a methyl-ß-d-galactopyranoside specific lectin from Kaempferia rotunda against Ehrlich ascites carcinoma cells.

A lectin was isolated from the tuberous rhizome of Keampferia rotunda by using different chromatographic methods with the molecular weight of 21±1kDa. The lectin contained highest percentage of leucine and lowest percentage of tryptophan residues as determined by LC-MS. The lectin agglutinated mice and human erythrocytes and the hemagglutination activity was inhibited by Methyl-ß-d-galactopyranoside. The lectin did not lose its activity in the presence of urea but the activity abolished completely when treated with EDTA. The lectin exhibited its activity at the pH ranging from 6.0 to 9.0 and in a temperature range of 30-80°C. Antiproliferative activity was studied against Ehrlich ascites carcinoma (EAC) and U87 cell lines. No inhibitory effect was observed against U87 cell line whereas 43.7% cell growth inhibition was observed in vitro against EAC cells at 160µg/ml. The lectin was injected (i.p.) in EAC bearing Swiss albino mice at the doses of 3.0 and 6.0mg/kg/day for five consecutive days and 41 and 59% of EAC cell growth inhibition was observed, respectively. The cell growth inhibition was due to the induction of apoptosis in the EAC cells which was confirmed by cell morphological study, caspase-3 inhibitor and apoptosis-related gene expression.

Purification and characterization of a novel chitinase from Trichosanthes dioica seed with antifungal activity.

Chitinases are a group of enzymes that show differences in their molecular structure, substrate specificity, and catalytic mechanism and widely found in organisms like bacteria, yeasts, fungi, arthropods actinomycetes, plants and humans. A novel chitinase enzyme (designated as TDSC) was purified from Trichosanthes dioica seed with a molecular mass of 39±1 kDa in the presence and absence of ß-mercaptoethanol. The enzyme was a glycoprotein in nature containing 8% neutral sugar. The N-terminal sequence was determined to be EINGGGA which did not match with other proteins. Amino acid analysis performed by LC-MS revealed that the protein was rich in leucine. The enzyme was stable at a wide range of pH (5.0-11.0) and temperature (30-90 °C). Chitinase activity was little bit inhibited in the presence of chelating agent EDTA (ethylenediaminetetraaceticacid), urea and Ca(2+). A strong fluorescence quenching effect was found when dithiothreitol and sodium dodecyl sulfate were added to the enzyme. TDSC showed antifungal activity against Aspergillus niger and Trichoderma sp. as tested by MTT assay and disc diffusion method.

Characterization of digestive enzymes from de-oiled mackerel (Scomber japonicus) muscle obtained by supercritical carbon dioxide and n-hexane extraction as a comparative study.

The oil in mackerel muscle was extracted using an environmental friendly solvent, supercritical carbon dioxide (SC-CO2) at a semi-batch flow extraction process and an n-hexane. The SC-CO2 was carried out at temperature 45 °C and pressures ranging from 15 to 25 MPa. The flow rate of CO2 (27 g/min) was constant at the entire extraction period of 2 h. The highest oil extracted residues after SC-CO2 extraction was used for activity measurement of digestive enzymes. Four digestive enzymes were found in water soluble extracts after n-hexane and SC-CO2 treated samples. Amylase, lipase and trypsin activities were higher in water soluble extracts after SC-CO2 treated samples except protease. Among the four digestive enzymes, the activity of amylase was highest and the value was 44.57 uM/min/mg of protein. The water soluble extracts of SC-CO2 and n-hexane treated mackerel samples showed same alkaline optimum pH and pH stability for each of the digestive enzymes. Optimum temperature of amylase, lipase, protease and trypsin was 40, 50, 60 and 30 °C, respectively of both extracts. More than 80 % temperature stability of amylase, lipase, protease and trypsin were retained at mentioned optimum temperature in water soluble extracts of both treated samples. Based on protein patterns, prominent protein band showed in water soluble extracts after SC-CO2 treated samples indicates no denaturation of protein than untreated and n-hexane.

Recovery of functional materials with thermally stable antioxidative properties in squid muscle hydrolyzates by subcritical water.

Subcritical water hydrolysis was carried out to produce functional materials from squid muscle using a batch reactor. The reaction temperatures and pressures for hydrolysis of thermal dried squid muscle were maintained from 160 to 280 °C and 6 to 66 bar for 3 min. The ratio of material to water for hydrolysis was 1:25 (w/v) and it was stirred at 140 rpm. Hydrolysis yield was increased after increasing the temperature and pressure while the protein in hydrolyzate decreased with the rise of temperature. The reducing sugar yield was high at temperature 220 °C in subcritical water hydrolysis of squid muscle. Low molecular weight peptides were found in all hydrolyzates by SDS-PAGE. The highest yield of free and structural amino acid in hydrolyzate was 421.53 ± 1.24 and 380.58 ± 2.25 mg/100 g, respectively at 250 °C. All essential amino acids were identified in muscle hydrolyzates and it was high at 220 °C. Among the essential amino acids, leucine was the most abundant. Antioxidative properties were found in all hydrolyzates and it was high at 220 °C. More than 98 ± 0.26 % ABTS antioxidant activity was retained in hydrolyzates after long time heat treatment.

Characterization of lecithin isolated from anchovy (Engraulis japonica) residues deoiled by supercritical carbon dioxide and organic solvent extraction.

UNLABELLED: Lecithin was isolated and characterized from anchovy (Engraulis japonica) deoiled residues using supercritical carbon dioxide (SC-CO(2)) at a semibatch flow extraction process and an organic solvent (hexane) extraction. SC-CO(2) extraction was carried out to extract oil from anchovy at different temperatures (35 to 45 °C) and pressures (15 to 25 MPa). Extraction yield of oil was influenced by physical properties of SC-CO(2) with temperature and pressure changes. The major phospholipids of anchovy lecithin were quantitatively analyzed by high-performance liquid chromatography. Phosphatidylcholine (PC) (68%± 1.00%) and phosphatidylethanolamine (PE) (29%± 0.50%) were the main phospholipids. Thin layer chromatography was performed to purify the individual phospholipids. The fatty acid compositions of lecithin, PC, and PE were analyzed by gas chromatography. A significant amount of eicosapentaenoic acid and docosahexaenoic acid were present in both phospholipids of PC and PE. Emulsions of lecithin in water were prepared through the use of a homogenizer. Oxidative stability of anchovy lecithin was high in spite of its high concentration of long-chain polyunsaturated fatty acids. PRACTICAL APPLICATION: Lecithin can be totally metabolized by humans, so is well tolerated by humans and nontoxic when ingested. Lecithin from anchovy contain higher amounts of ω-3 fatty acids especially EPA and DHA, it may have positive outcome to use in food and pharmaceutical industries.