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Angiotensin II (Ang II)-induced vascular endothelial cell injury and dysfunction are important pathophysiological factors in the occurrence and development of hypertension. In this study, the amelioration effects of two peptides KA-8 (KLHDEEVA) and PG-7 (PSRILYG) from Harpadon nehereus bone on Ang II-induced damage and dysfunction in human umbilical vein endothelial cells (HUVECs) were investigated. The results showed that they could significantly decrease the reactive oxygen species (ROS) level and increase the activity of antioxidant enzymes in Ang II-induced HUVEC. Two peptides, especially PG-7, significantly upregulated the expression of nuclear factor erythroid 2-related factor 2 (Nrf2). In addition, PG-7 significantly reduced the level of expression of endothelin-1(ET-1) and increased the phosphorylation level of phosphoinositide 3-kinase (PI3K), serine/threonine kinase (AKT), and nitric oxide synthase (eNOS). These results indicated that the two peptides, especially PG-7, can ameliorate angiotensin II-induced HUVEC injury and dysfunction through activation of the AKT/eNOS and Nrf2 pathway. Furthermore, PG-7 showed a stronger affinity with angiotensin-converting enzyme (ACE) and ACE inhibitory than KA-8. In conclusion, peptide PG-7 reveals potential in the prevention and treatment of hypertension.
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Oxidative stress and abnormal glucose metabolism are the important physiological mechanisms in the occurrence and development of diabetes. Antioxidant peptides have been reported to attenuate diabetes complications by regulating levels of oxidative stress, but few studies have focused on peptides from marine bone collagen. In this study, we prepared the peptides with a molecular weight of less than 1 kD (HNCP) by enzymolysis and ultrafiltration derived from Harpadon nehereus bone collagen. Furthermore, the effects of HNCP on blood glucose, blood lipid, liver structure and function, oxidative stress, and glucose metabolism were studied using HE staining, kit detection, and Western blotting experiment in streptozocin-induced type 1 diabetes mice. After the 240 mg/kg HNCP treatment, the levels of blood glucose, triglyceride (TG), and low-density lipoprotein cholesterol (LDL-C) in streptozotocin-induced diabetes mice decreased by 32.8%, 42.2%, and 43.2%, respectively, while the levels of serum insulin and hepatic glycogen increased by 142.0% and 96.4%, respectively. The antioxidant enzymes levels and liver function in the diabetic mice were markedly improved after HNCP intervention. In addition, the levels of nuclear factor E2-related factor 2 (Nrf2), glucokinase (GK), and phosphorylation of glycogen synthase kinase-3 (p-GSK3ß) in the liver were markedly up-regulated after HNCP treatment, but the glucose-6-phosphatase (G6Pase) and phosphoenolpyruvate carboxykinase1 (PEPCK1) were down-regulated. In conclusion, HNCP could attenuate oxidative stress, reduce blood glucose, and improve glycolipid metabolism in streptozocin-induced type 1 diabetes mice.
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Diabetes Mellitus Experimental , Diabetes Mellitus Tipo 1 , Animales , Ratones , Hipoglucemiantes/farmacología , Hipoglucemiantes/uso terapéutico , Hipoglucemiantes/química , Estreptozocina , Glucemia , Antioxidantes/química , Diabetes Mellitus Tipo 1/inducido químicamente , Diabetes Mellitus Tipo 1/tratamiento farmacológico , Diabetes Mellitus Tipo 1/metabolismo , Diabetes Mellitus Experimental/metabolismo , Estrés Oxidativo , Hígado , Glucosa/metabolismo , Péptidos/farmacología , Péptidos/uso terapéutico , Péptidos/metabolismoRESUMEN
Acute liver injury (ALI) and acute kidney injury (AKI) are significantly affected by the antioxidant status. In the present study, the protective effect and mechanism of the collagen peptide Phe-Leu-Ala-Pro (FLAP) in mice with ALI and AKI induced by carbon tetrachloride (CCl4) were examined. The results showed that FLAP effectively improved the liver mass index, the renal mass index, and the histopathological morphology. FLAP treatment significantly decreased the levels of serum aspartate aminotransferase (AST), alanine aminotransferase (ALT), urea nitrogen (BUN), and creatinine (CRE) but increased the activity of catalase (CAT), superoxide dismutase (SOD), and glutathione peroxidase (GSH-Px). The protein expression levels of nuclear factor E2-related factor 2 (Nrf2), heme oxygenase-1 (HO-1), p-protein kinase B (p-AKT), and p-phosphatidylinositol-3-kinase (p-PI3K) in the liver and kidneys were significantly up-regulated after FLAP treatment. FLAP down-regulated the levels of interleukin-6 (IL-6), IL-1ß, tumor necrosis factor-α (TNF-α), and nuclear factor-κ B (NF-κB) in liver and kidney tissues. Thus, FLAP may play a protective role in ALI and AKI by attenuating oxidative stress and inflammation mediated by the Nrf2/anti-response element (ARE) and PI3K/AKT/NF-κB pathways.
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The physicochemical characterization of a new collagen from the bone of Harpadon nehereus (HNBC) and its protective effects against Ang II-induced injury of human umbilical vein endothelial cells (HUVEC) were investigated. The triple helix of HNBC analyzed by SDS-PAGE was formed as (α1)2α2, belonging to type I collagen. UV spectra showed that HNBC had a maximum absorbance at 230 nm. FTIR spectra indicated the triple helical structure and activity of HNBC. The high solubility of HNBC was observed in the low pH ranges (≤4) and NaCl concentrations (≤2%, w/v). The maximum transition (T m) of HNBC was determined to be 48.5 °C. The amino acid composition analysis showed that glycine, glutamic acid, proline, and alanine were the abundant amino acids available in HNBC. HNBC showed free radical (DPPH and OH·) scavenging activities in the tested concentrations (0.5-6 mg/mL). In addition, HNBC could effectively protect against Ang II-induced injury of HUVEC by increasing the activities of antioxidant enzymes, such as CAT, SOD, and GSH-Px. Overall, collagen from the bone of H. nehereus has promising prospects in functional food and biomedical industries.
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The protective effect of collagen peptide from Acaudina molpadioides (Amp) on acute kidney injury (AKI) in mice and its mechanism were explored. The results showed that Amp-fed could effectively improve the renal mass index and histopathological morphology. The levels of serum creatinine and urea nitrogen decreased significantly, while the antioxidant enzyme catalase (CAT) and superoxide dismutase (SOD) increased significantly in Amp-fed groups. Western blot results disclosed that Amp significantly upregulates the levels of heme oxygenase-1 (HO-1), Nrf2, p-PI3K, and p-AKT in the kidney. In addition, Amp could significantly downregulate the levels of nuclear factor NF-kappa-B (NF-κB), tumor necrosis factor α (TNF-α), inflammatory cytokines interleukin 6 (IL-6) and interleukin 1ß (IL-1ß). These findings provide evidence that Amp plays a protective role in AKI via attenuation of oxidative stress and inflammation mediated by PI3K/AKT/Nrf2 and PI3K/AKT/NF-κB pathways. This study laid a foundation for the application of Amp in the prevention of AKI.
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Lesión Renal Aguda , Pepinos de Mar , Animales , Ratones , Lesión Renal Aguda/tratamiento farmacológico , Lesión Renal Aguda/prevención & control , Colágeno/metabolismo , Inflamación , Factor 2 Relacionado con NF-E2/metabolismo , FN-kappa B/metabolismo , Estrés Oxidativo , Péptidos/metabolismo , Fosfatidilinositol 3-Quinasas/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismo , Pepinos de Mar/metabolismoRESUMEN
Collagen peptide from Acaudina molpadioides (AMP) showed antioxidative activity in H2 O2 -induced RAW264.7 cells in our pervious study. In this study, it was observed that AMP could effectively improve the morphology and function of liver in CCl4 -induced mice. After 200 mg/kg AMP treatment, the content of MDA in liver decreased by 62.3%, and the level of antioxidant enzymes (SOD, GSH-Px, and CAT) increased by more than 65%. Western blot results disclosed that AMP (200 mg/kg) upregulated the Nrf2 level by 73.8% and downregulated Keap1 by 41.0% in CCl4 -induced mice liver. The levels of p-ERK, p-JNK, and p-p38 in 200 mg/kg AMP treatment groups decreased by 57.3%, 40.9%, and 40.6%, but the levels of p-PI3K and p-AKT increased by 162.6% and 60.3%, respectively. Furthermore, the trends of Nrf2, Keap1, p-ERK, p-JNK, p-p38, p-PI3K, and p-AKT levels in H2 O2 -induced RAW264.7 cells after AMP treatment were similar to the results in CCl4 -induced mice liver. These findings provided evidence that AMP exerted antioxidant activity via Keap1/Nrf2-ARE, PI3K/AKT, and MAPKs pathways in vivo and in vitro. Therefore, the collagen peptide from A. molpadioides might represent a novel functional food to prevent acute liver injury via attenuation of oxidative stress.
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Enfermedad Hepática Crónica Inducida por Sustancias y Drogas , Pepinos de Mar , Adenosina Monofosfato/metabolismo , Animales , Antioxidantes/metabolismo , Antioxidantes/farmacología , Enfermedad Hepática Crónica Inducida por Sustancias y Drogas/metabolismo , Colágeno/metabolismo , Proteína 1 Asociada A ECH Tipo Kelch/genética , Proteína 1 Asociada A ECH Tipo Kelch/metabolismo , Hígado/metabolismo , Ratones , Factor 2 Relacionado con NF-E2/genética , Factor 2 Relacionado con NF-E2/metabolismo , Estrés Oxidativo , Péptidos/metabolismo , Péptidos/farmacología , Fosfatidilinositol 3-Quinasas/genética , Fosfatidilinositol 3-Quinasas/metabolismo , Proteínas Proto-Oncogénicas c-akt/genética , Proteínas Proto-Oncogénicas c-akt/metabolismo , Pepinos de Mar/química , Transducción de SeñalRESUMEN
A novel peptide (Cya-Phe-Leu-Ala-Pro, SCP) was formulated through non-protein amino acid-cysteic acid (Cya) modification of collagen peptide (Phe-Leu-Ala-Pro, CP) from Acaudina molpadioides. Introduction of this Cya showed remarkable improvement in the scavenging activities of OH·. SCP exhibited stronger effects than CP in preventing H2O2-induced oxidative damage due to lower levels of ROS and MDA, and higher activities of antioxidant enzymes, such as SOD, GSH-Px, HO-1, and NQO1. It was speculated that SCP could significantly increase the expression level of Nrf2 compared to CP, thereby activating the expression of downstream ARE genes. The expression levels of p38 in the upstream pathway to regulate Nrf2 content were significantly higher in both the CP and SCP-treated groups, while a higher level of JNK was observed only in the SCP-treated groups. The present study provided insights towards the application of cysteic acid modified peptide in protecting cell from oxidative damage through the JNK/Nrf2 pathway.
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Ácido Cisteico/farmacología , Peróxido de Hidrógeno/metabolismo , Factor 2 Relacionado con NF-E2/metabolismo , Péptidos/farmacología , Animales , Ácido Cisteico/química , Relación Dosis-Respuesta a Droga , Estructura Molecular , Factor 2 Relacionado con NF-E2/genética , Péptidos/química , Pepinos de Mar/química , Transducción de Señal/efectos de los fármacos , Relación Estructura-ActividadRESUMEN
Collagen is a promising biomaterial used in the beauty and biomedical industries. In this study, the physicochemical characterization, antioxidant activities, and protective effects against H2O2-induced injury of collagen isolated from Acaudina molpadioides were investigated. The amino acid composition analysis showed that the collagen was rich in glycine (Gly), alanine (Ala), and glutamic acid (Glu), but poor in tyrosine (Tyr) and phenylalanine (Phe). Zeta potential analysis revealed that the isoelectric point (pI) of collagen from Acaudina molpadioides was about 4.25. It possessed moderate scavenging activities of 2,2-diphenyl-1-picrylhydrazyl (DPPH) and 2,2'-azino-bis-3-ethylbenzothiazoline-6-sulfonic acid (ABTS) radicals in a dose-dependent manner. In addition, the collagen was able to effectively improve cell viability and morphology, inhibit the production of Malondialdehyde (MDA), and increase the activities of Superoxide Dismutase (SOD) and Glutathione Peroxidase (GSH-Px) in cultured RAW264.7 cells, resulting in a protective effect against H2O2-induced injury. Overall, the results showed that collagen extracted from A. molpadioides has promising prospects in the beauty and cosmetics industries.
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Antioxidantes/farmacología , Colágeno/farmacología , Peróxido de Hidrógeno/toxicidad , Macrófagos/efectos de los fármacos , Estrés Oxidativo/efectos de los fármacos , Pepinos de Mar/química , Secuencia de Aminoácidos , Animales , Antioxidantes/química , Antioxidantes/aislamiento & purificación , Colágeno/química , Colágeno/aislamiento & purificación , Glutatión Peroxidasa/metabolismo , Macrófagos/metabolismo , Macrófagos/patología , Malondialdehído/metabolismo , Ratones , Células RAW 264.7 , Superóxido Dismutasa/metabolismoRESUMEN
Antioxidant peptides have elicited interest for the versatility of their use in the food and pharmaceutical industry. In the current study, antioxidant peptides were prepared by microwave-assisted alkaline protease hydrolysis of collagen from sea cucumber (Acaudina molpadioides). The results showed that microwave irradiation significantly improved the degree of hydrolysis of collagen and the hydroxyl radical (OHâ ) scavenging activity of hydrolysate. The content and OHâ scavenging activity of collagen peptides with molecular weight ≤ 1 kDa (CPS) in the hydrolysate obtained at 250 W increased significantly compared with the non-microwave-assisted control. CPS could scavenge OHâ and 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical in a dose-dependent manner. The scavenging activity of OHâ and DPPH radical was 93.1% and 41.2%, respectively, at CPS concentration of 1 mg/mL. CPS could significantly promote RAW264.7 cell proliferation and reduce the Reactive Oxygen Species (ROS) level of H2O2-induced damage in RAW264.7 cells in a dose-dependent manner. Furthermore, all CPS-treated groups exhibited an increase in superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px) and a decrease in malondialdehyde (MDA) level compared with the control. These results showed that CPS could effectively protect RAW264.7 cells from H2O2-induced damage, implying the potential utilization of CPS as a natural antioxidant for food and pharmaceutical applications.
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Antioxidantes/farmacología , Colágeno/metabolismo , Peróxido de Hidrógeno/farmacología , Péptidos/farmacología , Sustancias Protectoras/farmacología , Animales , Compuestos de Bifenilo/metabolismo , Línea Celular , Proliferación Celular/efectos de los fármacos , Glutatión Peroxidasa/metabolismo , Hidrólisis , Radical Hidroxilo/metabolismo , Malondialdehído/metabolismo , Ratones , Microondas , Estrés Oxidativo/efectos de los fármacos , Células RAW 264.7 , Especies Reactivas de Oxígeno/metabolismo , Superóxido Dismutasa/metabolismoRESUMEN
Acid-solubilized collagen (ASC) and pepsin-solubilized collagen (PSC) were obtained from Nibea japonica swim bladders. The denaturation temperature (Td) of ASC and PSC was approximately 33.8⯰C. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Fourier transform infrared spectroscopy (FTIR) analyses indicated that ASC and PSC contained triple-helical type I collagen when compared to rat tail collagen type I. Moreover, the microstructure of collagen sponges was uniform and porous. In addition, ASC and PSC exhibited antioxidant properties and in vitro scratch assays showed that PSC at various concentrations (0, 12.5, 25, and 50⯵g/mL) had significant effects on the scratch closure rate. Furthermore, collagen sponge from Nibea japonica swim bladders exhibited an increased efficacy of wound healing when compared to the control mice. The levels of interleukin (IL)-1ß, IL-6 and tumor necrosis factor (TNF)-α in the collagen sponge treated mice were significantly decreased when compared to the control group. Thus, our results suggested that collagen sponge from Nibea japonica swim bladders has potential wound healing applications.
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Antioxidantes/química , Antioxidantes/farmacología , Productos Biológicos/química , Productos Biológicos/farmacología , Fenómenos Químicos , Colágeno/química , Perciformes , Cicatrización de Heridas/efectos de los fármacos , Secuencia de Aminoácidos , Animales , Proteínas de Peces/química , Ratones , Células 3T3 NIH , Solubilidad , Análisis Espectral , TemperaturaRESUMEN
Enantiopure epoxides are versatile synthetic intermediates for producing optically active pharmaceuticals. In an effort to provide more options for the preparation of enantiopure epoxides, a variant of the epoxide hydrolase (vEH-Am) gene from a marine microorganism Agromyces mediolanus was synthesized and expressed in Escherichia coli. Recombiant vEH-Am displayed a molecular weight of 43 kDa and showed high stability with a half-life of 51.1 h at 30 °C. The purified vEH-Am exhibited high enantioselectivity towards styrene oxide (SO) and benzyl glycidyl ether (BGE). The vEH-Am preferentially converted (S)-SO, leaving (R)-SO with the enantiomeric excess (ee) >99%. However, (R)-BGE was preferentially hydrolyzed by vEH-Am, resulting in (S)-BGE with >99% ee. To investigate the origin of regioselectivity, the interactions between vEH-Am and enantiomers of SO and BGE were analyzed by molecular docking simulation. In addition, it was observed that the yields of (R)-SO and (S)-BGE decreased with the increase of substrate concentrations. The yield of (R)-SO was significantly increased by adding 2% (v/v) Tween-20 or intermittent supplementation of the substrate. To our knowledge, vEH-Am displayed the highest enantioselectivity for the kinetic resolution of racemic BGE among the known EHs, suggesting promising applications of vEH-Am in the preparation of optically active BGE.
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Organismos Acuáticos/metabolismo , Epóxido Hidrolasas/metabolismo , Compuestos Epoxi/metabolismo , Escherichia coli/metabolismo , Hidrólisis , Cinética , Simulación del Acoplamiento Molecular/métodos , EstereoisomerismoRESUMEN
Collagen was extracted from bigeye tuna (Thunnus obesus) skins by salting-out (PSC-SO) and isoelectric precipitation (PSC-IP) methods. The yield of the PSC-IP product was approximately 17.17% (dry weight), which was greater than the yield obtained from PSC-SO (14.14% dry weight). Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis indicated that collagen from bigeye tuna skin belongs to collagen type I. Inductively coupled plasma mass spectrometry results indicate that the heavy metal abundance in PSC-IP was lower than the maximum acceptable amounts according to Chinese regulatory standards. In addition, results from a methylthiazolyldiphenyl-tetrazolium bromide assay and an in vitro scratch assay demonstrated that PSC-IP could promote the proliferation and migration of NIH-3T3 fibroblasts. Overall, results suggest PSC-IP could be used to rapidly extract collagen from marine by-products instead of traditional salting-out methods. Collagen from bigeye tuna skin may also have strong potential for cosmetic and biomedical applications.
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Colágeno/análisis , Colágeno/aislamiento & purificación , Proteínas de Peces/análisis , Proteínas de Peces/aislamiento & purificación , Animales , Ensayos de Migración Celular , Proliferación Celular , Colágeno/química , Colágeno Tipo I , Proteínas de Peces/química , Ratones , Células 3T3 NIH/efectos de los fármacos , Piel/química , Piel/metabolismo , Solubilidad , AtúnRESUMEN
The present study was focused on the preparation and characterization of the antioxidant peptides by microwave-assisted enzymatic hydrolysis of collagen from sea cucumber Acaudina molpadioides (ASC-Am) obtained from Zhejiang Province in China. The results exhibited the effects of microwave irradiation on hydrolysis of ASC-Am with different protease. Neutrase was selected from the four common proteases (papain, pepsin, trypsin, and neutrase) based on the highest content and DPPH scavenging activity of hydrolysate Fa (Molecular weight < 1 kDa). The content and 2,2-diphenyl-1-picrylhydrazyl (DPPH) scavenging activity of Fa obtained by hydrolysis of neutrase increased by 100% and 109% respectively at a microwave power of 300 W compared with no microwave irradiation. Five subfractions were obtained after performing the gel filtration chromatography, and the Fa.2 exhibited the highest DPPH scavenging activity. The amino acid analysis showed that the contents of Glutamic acid, Alanine, Tyrosine, and Phenylalanine in fraction Fa.2 increased significantly, but an obvious decrease in the content of Glycine was observed compared to Fa. Four peptides (Fa.2-A, Fa.2-B, Fa.2-C, and Fa.2-D) were purified from Fa.2 by high performance liquid chromatography, and Fa.2-C showed the highest DPPH scavenging activity. The sequence of Fa.2-C was identified as Phenylalanine-Leucine- Alanine-Proline with a half elimination ratio (EC50) of 0.385 mg/mL. The antioxidant activity of Fa.2-C was probably attributed to the small molecular sizes and the presence of hydrophobic amino acid residues in its sequence. This report provided a promising method for the preparation of antioxidant peptides from collagen for food and medicinal purposes.
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Colágeno/química , Péptidos/aislamiento & purificación , Péptidos/farmacología , Aminoácidos/química , Animales , Antioxidantes/química , Antioxidantes/aislamiento & purificación , Antioxidantes/farmacología , Compuestos de Bifenilo/química , Colágeno/aislamiento & purificación , Hidrólisis , Microondas , Péptido Hidrolasas/química , Péptidos/química , Pepinos de Mar/químicaRESUMEN
The ability to remove toxic heavy metals, such as Pb(II), from the environment is an important objective from both human-health and ecological perspectives. Herein, we describe the fabrication of a novel carboxymethylcellulose-coated metal organic material (MOF-5â»CMC) adsorbent that removed lead ions from aqueous solutions. The adsorption material was characterized by Fourier-transform infrared spectroscopy, X-ray diffractometry, scanning electron microscopy, and X-ray photoelectron spectroscopy. We studied the functions of the contact time, pH, the original concentration of the Pb(II) solution, and adsorption temperature on adsorption capacity. MOF-5â»CMC beads exhibit good adsorption performance; the maximum adsorption capacity obtained from the Langmuir isotherm-model is 322.58 mg/g, and the adsorption equilibrium was reached in 120 min at a concentration of 300 mg/L. The adsorption kinetics is well described by pseudo-second-order kinetics, and the adsorption equilibrium data are well fitted to the Langmuir isotherm model (R² = 0.988). Thermodynamics experiments indicate that the adsorption process is both spontaneous and endothermic. In addition, the adsorbent is reusable. We conclude that MOF-5â»CMC is a good adsorbent that can be used to remove Pb(II) from aqueous solutions.
RESUMEN
In the present study, peptide fractions of Cyclina sinensis hydrolysates, with molecular weight (MW) < 3 kDa and highest relative proliferation rate of murine macrophage cell line RAW 264.7, were purified by a series of chromatographic purification methods, to obtain peptide fractions with immunomodulatory activity. The amino acid sequence of the peptide was identified to be Arg-Val-Ala-Pro-Glu-Glu-His-Pro-Val-Glu-Gly-Arg-Tyr-Leu-Val (RVAPEEHPVEGRYLV) with MW of 1750.81 Da, and the novel pentadecapeptide (named SCSP) was synthesized for subsequent immunomodulatory activity experiments. Results showed the SCSP enhanced macrophage phagocytosis, increased productions of nitric oxide (NO), tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), and interleukin-1ß (IL-1ß), and up-regulated the protein level of inducible nitric oxide synthase (iNOS), nuclear factor κB (NF-κB), and NOD-like receptor protein 3 (NLRP3) in RAW 264.7 cells. Furthermore, the expression of inhibitor of nuclear factor κB-α (IκB-α) was down-regulated. These findings suggest that SCSP might stimulate macrophage activities by activating the NF-κB signaling pathway and can be used as a potential immunomodulatory agent in functional food or medicine.
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Bivalvos/química , Factores Inmunológicos/química , Factores Inmunológicos/farmacología , Péptidos/química , Péptidos/farmacología , Hidrolisados de Proteína/química , Hidrolisados de Proteína/farmacología , Animales , Bivalvos/metabolismo , Línea Celular , Regulación hacia Abajo/efectos de los fármacos , Factores Inmunológicos/metabolismo , Interleucina-1beta/metabolismo , Interleucina-6/metabolismo , Macrófagos/efectos de los fármacos , Macrófagos/metabolismo , Ratones , Inhibidor NF-kappaB alfa/metabolismo , FN-kappa B/metabolismo , Proteína con Dominio Pirina 3 de la Familia NLR/metabolismo , Óxido Nítrico/metabolismo , Óxido Nítrico Sintasa de Tipo II/metabolismo , Péptidos/metabolismo , Fagocitosis/efectos de los fármacos , Hidrolisados de Proteína/metabolismo , Células RAW 264.7 , Transducción de Señal/efectos de los fármacos , Factor de Necrosis Tumoral alfa/metabolismo , Regulación hacia Arriba/efectos de los fármacosRESUMEN
The structure of pepsin-solubilized collagen (PSC) obtained from the skin of Lophius litulon was analyzed using the sodium dodecylsulphate polyacrylamide gel electrophoresis (SDS-PAGE), Fourier transform infrared spectroscopy (FTIR), and scanning electron microscopy (SEM). SDS-PAGE results showed that PSC from Lophius litulon skin was collagen type I and had collagen-specific α1, α2, ß, and γ chains. FTIR results indicated that the infrared spectrum of PSC ranged from 400 to 4000 cm-1, with five main amide bands. SEM revealed the microstructure of PSC, which consisted of clear fibrous and porous structures. In vitro antioxidant studies demonstrated that PSC revealed the scavenging ability for 2,2-diphenyl-1-picrylhydrazyl (DPPH), HO·, O2-·, and ABTS·. Moreover, animal experiments were conducted to evaluate the biocompatibility of PSC. The collagen sponge group showed a good biocompatibility in the skin wound model and may play a positive role in the progression of the healing process. The cumulative results suggest that collagen from the skin of Lophius litulon has potential applications in wound healing due to its good biocompatibility.
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Colágeno/química , Colágeno/farmacología , Peces/metabolismo , Piel/química , Piel/metabolismo , Aminoácidos/química , Animales , Antioxidantes/química , Antioxidantes/farmacología , Colágeno/metabolismo , Electroforesis en Gel de Poliacrilamida , Masculino , Ratones , Ratones Endogámicos ICR , Pepsina A/química , Solubilidad , Cicatrización de Heridas/efectos de los fármacosRESUMEN
An effective epoxide hydrolase (EH) production strain was mutagenized using 60 Co gamma and UV irradiation. Among positive mutant strains, the EH activity of C2-44 reached 33.7 U/g, which was 267% as much as that of the original Aspergillus niger ZJB-09103. Compared with the wild type, there were significant changes in morphology for C2-44, including the color of mycelia on the slants and the shape of conidial head. In addition, glucose and soybean cake were the optimal carbon and nitrogen source in terms of EH activity for the mutant C2-44 instead of soluble starch and peptone for the wild-type strain. The reaction time required to reach 99% enantiomeric excesses of (S)-epichlorohydrin from racemic substrate was shortened significantly by the mutant C2-44. This phenomenon was probably explained by the higher Vmax for hydrolysis of racemic epichlorohydrin by C2-44 compared with Aspergillus niger ZJB-09103.
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Aspergillus niger , Epóxido Hidrolasas , Proteínas Fúngicas , Rayos gamma , Mutagénesis/efectos de la radiación , Rayos Ultravioleta , Aspergillus niger/enzimología , Aspergillus niger/genética , Epóxido Hidrolasas/biosíntesis , Epóxido Hidrolasas/genética , Proteínas Fúngicas/biosíntesis , Proteínas Fúngicas/genéticaRESUMEN
In this study the analysis and confirmation of flumequine enantiomers in rat plasma by ultra-fast liquid chromatography coupled with electron spray ionization mass spectrometry (using propranolol as an internal standard [IS]) was developed and validated. Plasma samples were prepared by liquid-liquid extraction using methyl tert-butyl ether as the extraction solvent. Direct resolution of the R- and S-isomers was performed on a CHIRALCEL OJ-RH column (4.6 × 150 mm, 5 µm) using acetonitrile / 0.1% formic acid / 1 mM ammonium acetate as the mobile phase. Detection was operated by electron spray ionization in the selected ion monitoring and positive ion mode. The target ions at m/z 262.1 and m/z 260.1 were selected for the quantification of the enantiomers and IS, respectively. The linear range was 0.5-500 ng/mL. The precisions (coefficient of variation, CV%) and recoveries were 1.43-8.68 and 94.24-106.76%, respectively. The lowest quantitation limit for both enantiomers is 0.5 ng/mL, which is sensitive enough to be applied to sample analysis in other related studies.
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Cromatografía Liquida/métodos , Fluoroquinolonas/sangre , Fluoroquinolonas/química , Espectrometría de Masa por Ionización de Electrospray/métodos , Espectrometría de Masas en Tándem/métodos , Animales , Extracción Líquido-Líquido , Éteres Metílicos/química , Propranolol/sangre , Ratas , Reproducibilidad de los Resultados , EstereoisomerismoRESUMEN
In this work, flumequine (FLU) enantiomers were separated using a Chiralpak OD-H column, with n-hexane-ethanol (20:80, v/v) as the mobile phase at a flow rate of 0.6 mL/min. Solid phase extraction (SPE) was used for cleanup and enrichment. The limit of detection, limit of quantitation, linearity, precision, and intra/interday variation of the chiral high-performance liquid chromatography (HPLC) method were determined. The developed method was then applied to investigate the degradation behavior of FLU enantiomers in mariculture pond water samples. The results showed that the degradation of FLU enantiomers under natural, sterile, or dark conditions was not enantioselective. Chirality 28:649-655, 2016. © 2016 Wiley Periodicals, Inc.
Asunto(s)
Cromatografía Líquida de Alta Presión/métodos , Fluoroquinolonas/análisis , Fluoroquinolonas/química , Contaminantes Químicos del Agua/análisis , Acuicultura , Biodegradación Ambiental , Cromatografía Líquida de Alta Presión/instrumentación , Estanques , Extracción en Fase Sólida , Estereoisomerismo , Contaminantes Químicos del Agua/químicaRESUMEN
A new method for the isolation and enrichment of ofloxacin enantiomers from fish samples was developed using magnetic molecularly imprinted polymers (MMIPs). These polymers can be easily collected and rapidly separated using an external magnetic field, and also exhibit a high specific recognition for ofloxacin enantiomers. The preparation of amino-functionalized MMIPs was carried out via suspension polymerization and a ring-opening reaction using rac-ofloxacin as a template, ethylenediamine as an active group, glycidyl methacrylate and methyl methacrylate as functional monomers, divinylbenzene as a cross-linker, and Fe3O4 nanoparticles as magnetic cores. The characteristics of the MMIPs were assessed using transmission electron microscopy (TEM), X-ray powder diffraction (XRD), Fourier-transform infrared spectroscopy (FT-IR), and vibrating sample magnetometer (VSM) measurements. Furthermore, the adsorption properties were determined using Langmuir and Freundlich isotherm models. The conditions for use of these MMIPs as magnetic solid-phase extraction (MSPE) sorbents, including pH, adsorption time, desorption time, and eluent, were investigated in detail. An extraction method using MMIPs coupled with high performance liquid chromatography (HPLC) was developed for the determination of ofloxacin enantiomers in fish samples. The limits of quantitation (LOQ) for the developed method were 0.059 and 0.067 µgâmL(-1) for levofloxacin and dextrofloxacin, respectively. The recovery of ofloxacin enantiomers ranged from 79.2% ± 5.6% to 84.4% ± 4.6% and ofloxacin enantiomers had good linear relationships within the concentration range of 0.25-5.0 µgâmL(-1) (R² > 0.999). The obtained results demonstrate that MSPE-HPLC is a promising approach for preconcentration, purification, and simultaneous separation of ofloxacin enantiomers in biomatrix samples.