鱿鱼皮胶原蛋白医用止血材料的研究

目的 从鱿鱼皮中提取胶原蛋白并研究经化学和物理方法改性的鱿鱼皮胶原海绵的止血效果。方法 从新鲜鱿鱼皮中提取得到酸溶性胶原蛋白 (ASC)和酶溶性胶原蛋白 (PSC),冷冻干燥制备胶原海绵,分别采用EDC交联、干热交联 (DHT)、EDC/DHT结合交联这3种方法对其进行改性处理。采用兔耳创伤止血和肝脏止血试验来评价改性后胶原海绵的止血性能。结果 鱿鱼皮胶原蛋白ASC和PSC为典型的I型胶原蛋白。动物止血实验结果表明ASC-E、PSC-E、ASC-E/D和PSC-E/D等4种改性鱿鱼皮胶原海绵组均能对创面起到一定止血作用,其中尤以PSC-E组的止血效果最好,优于市售明胶海绵。 结论ASC-E、PSC-E、ASC-E/D和PSC-E/D等4种改性鱿鱼皮胶原海绵组均能有效的缩短出血时间,减少出血量,达到快速止血的效果,其中PSC-E止血效果最佳。     

Extracted pepsin of trout waste and ultrasound-promoted method for green recovery of fish collagen

Collagen type I is a prominent protein in animal tissues. This structural protein can be commercially derived from fish processing waste without any health risk. The current study aimed to investigate the efficacy of rainbow trout pepsin and the ultrasound-promoted method on collagen recovery. Pepsin extract was purified from rainbow trout stomach in order to use in collagen extraction from wasted yellowfin skin through ultrasound technique in five durations (0, 5, 10, 15, and 20 min). The 15 min ultrasound treated pepsin soluble collagen indicated the highest recovery yield (23.8%) as well as the highest imino acid content (18.2%). The structure of type I collagen was observed in the SDS-PAGE pattern of collagens and no structural change occurred during the extraction process. The presence of the helical structure in collagen samples was confirmed by UV and FTIR spectra. The ultrasound cavitation advanced the viscosity of collagens and resulted in excellent solubility in acidic environments and lower salt concentrations. Ultrasound-treated samples suggested superior water holding capacity along with emulsifying, and foam attributes. The improved gel strength of collagens was parallel with increasing the time of ultrasound up to 15 min. Free radicals scavenging ability and ferric reducing power of collagens were positively stimulated by increasing the time of the ultrasound up to 15 min. Therefore, this study contributes to the development of green technology and the promotion of by-product utilization for collagen recovery as a potential practical protein in biomaterial, wound dressing, drug delivery, food, and cosmetics products.     

In-vitro interaction of mitomycin C and streptomycin A with collagen.

Mitomycin C and streptomycin A were studied for their interaction with soluble collagens from sepia, fish and rat skins using UV absorption spectroscopy at pH 7.4 and 3.0 and equilibrium dialysis. Both the drugs bound to collagen as shown from association constants and could be quantitatively recovered by prolonged dialysis against water showing formation of dissociable bonds between drug and collagen. At pH 7.4, mitomycin C showed greater binding affinity than streptomycin A with collagen from all three species. The reverse trend was seen at pH 3.0. Binding capacity of sepia and fish skin collagens for streptomycin A at pH 3.0 was found to be significantly greater than for mitomycin C.     

TEMPERATURE-MODULATED CARCINOGENICITY OF 7,12-DIMETHYLBENZ[a]ANTHRACENE IN RAINBOW TROUT

Temperature-modulated hepatic disposition, covalent binding of radiolabeled genotoxin to hepatic DNA, and cancer incidence in rainbow trout ( Oncorhyncus mykiss ) were assessed after a single exposure to 7,12-dimethylbenz[a]anthracene (DMBA). Fish (2 g) were acclimated at 10, 14, or 18°C for 1 mo and then exposed to 1 ppm DMBA in their water for 20 h. Exposures were at respective acclimation temperatures, or 10 and 18°C acclimated fish were shifted to 14°C for DMBA exposures. After 4 but not 20 h of exposure, hepatic [ 3 H]DMBA equivalents increased with temperature for fish exposed at their respective acclimation temperatures (10 or 18°C). Covalent binding of [ 3 H]DMBA to hepatic DNA was similar after 3 d in fish exposed at their respective acclimation temperatures. However, in fish exposed at 14°C, after 3 d the concentration of [ 3 H]DMBA covalently bound to hepatic DNA was higher in 10°C than 18°C acclimated fish. After 21 d, covalent binding of [ 3 H]DMBA to hepatic DNA was less persistent in 18°C than 10°C acclimated, exposed, and reared fish. There were no differences between temperature-shifted groups at that time. Temperature effects on tumor incidence were assessed 9 mo after DMBA waterborne exposures in fish that were reared at (1) their respective acclimation and exposure temperatures, (2) 14°C after exposure at their respective acclimation temperature, and (3) 14°C after 14°C exposures. Incidence of stomach, liver, and swimbladder cancer increased dramatically with rearing temperature. Differences in tumor incidence were less marked in fish reared at the same temperature (14°C). A strong negative correlation between liver tumor incidence and persistence of [ 3 H]DMBA equivalents covalently bound to hepatic DNA suggested increased error-prone DNA repair at warmer temperature played an important role in increased tumor incidence.     

Extraction,optimization and characterization of collagen from sole fish skin

In this study, collagen was successfully extracted from marine waste i.e. Sole fish skin, which is available in the coastal area of Mangalore, India. The extraction process was optimized using One Variable at a Time (OVAT) and Response Surface Methodology (RSM) with Box-Behnken Design (BBD) was to achieve maximum yield and the extracted collagen was characterized. The optimal conditions to obtain highest collagen yield was determined to be, an acetic acid concentration of 0.54 M, NaCl concentration of 1.90 M, solvent/solid ratio of 8.97 ml/g and time of 32.32 h. The maximum collagen yield of 19.27 ± 0.05 mg/g of fish skin was achieved under the optimal conditions. The analysis of variance and contour plots exhibited a significant interaction of all the selected variables over collagen extraction process. The SDS-PAGE (Sodium dodecyl sulfate - polyacrylamide gel electrophoresis) analysis suggested that the extracted collagen contained three α-chains i.e. (α1)₂, α2 (M.W. 118, 116 kDa) and one β chain (M.W. 200 kDa) which was similar to commercially available calfskin Type I collagen. FT-IR (Fourier Transform Infrared Spectroscopy) analysis confirmed the existence of helical arrangements of collagen. SEM (Scanning electron microscopy) observation revealed that the extracted collagen was in the form of fibrils with irregular linkages.     

Study of HMG-CoA Reductase Inhibition Activity of the Hydrolyzed Product of Snakehead Fish (Channa striata) Skin Collagen with 50 kDa Collagenase from Bacillus licheniformis F11.4

Bioactive peptides produced from enzymatic hydrolysis fibrous protein have been proven to have several biological activities. Previous study showed that the hydrolysis product of snakehead fish skin collagen with 26 kDa collagenase from Bacillus licheniformis F11.4 showed HMG-CoA (HMGR) inhibition activity. The aim of this research was to determine the ability of the hydrolysis product produced from snakehead fish skin collagen hydrolysed by 50 kDa collagenase from B. licheniformis F11.4 in inhibiting HMGR activity. Snakehead fish skin collagen was extracted using an acid method and collagenase was produced from B. licheniformis F11.4 using half-strength Luria Bertani (LB) medium containing 5% collagen. Crude collagenase was concentrated and fractionated using the DEAE Sephadex A-25 column eluted with increasing gradient concentrations of NaCl. Collagen, collagenase, and fractions were analyzed using SDS-PAGE and collagenolytic activity was analyzed by the zymography method. Collagenase with 50 kDa molecular weight presented in fraction one was used to hydrolyze the collagen. The reaction was done in 18 hours at 50°C. The hydrolysis product using 3.51 μg collagen and 9 ng collagenase showed 25.8% inhibition activity against pravastatin. This work shows for the first time that the hydrolysis product of snakehead fish skin collagen and 50 kDa collagenase from B. licheniformis F11.4 has potential as an anticholesterol agent.     

Pinealectomy modifies the thermoregulatory effects of bombesin in goldfish

Intraventrieular administration of the neuropeptide, bombesin, to intact and sham-pinealectomized fish significantly decreased their behaviorally selected internal body temperatures in horizontal thermal gradients. A behavioral hypothermia of 4–7°C was evident regardless of the previous acclimatization temperature (10 or 25°C) and preferred body temperatures selected by individual fish. Pinealectomy increased the body temperatures of all control fish by 2–4°C while markedly reducing the hypo-thermic effects of administration of bombesin. The hypothermic effects of bombesin were most evident in the pinealectomized fish acclimatized to 10°C. The possible relations between neuropeptides and the pineal gland in the regulation of body temperature are considered.     

Physiological disturbances in rainbow trout, Salmo gairdneri (R.), exposed at two temperatures to effluents from a titanium dioxide industry

Rainbow trout (Salmo gairdneri R.) were exposed to the supernatant waste water (260 μl/l) from a TiO₂ producing plant in a continuous flow test (Diluent: brackish water 7‰) for 2 wk at two temperatures (7–8 and 13–15°C). Presedimentation chambers were used to separate the solid, precipitated fraction of the waste water from the soluble part.After 2 wk of exposure the fish were killed and blood samples for hematological and biochemical analyses were taken via caudal vessels. The effect pattern of the fish exposed at low temperature (7–8°C) differed from that of the fish exposed at 13–15°C. The exposure at 13–15°C significantly increased the blood hematocrit level and increased the plasma sodium and chloride concentrations. Moreover, increased plasma glucose and lactate contents were found in these fish. These parameters were unaffected in the fish exposed at 7–8°C. Instead this group suffered from a plasma hypocalcemia and a sex-dependent inhibition of erythrocytic delta-aminolevulinic dehydrates (ALA-D) enzyme activity.The effects obtained at 13–15°C were characterized as general stress symptoms. However, the effects obtained at 7–8°C indicated a more serious toxic response of the exposed fish. The reason behind this more pronounced effect at low temperature is assumed to be related to a higher concentration of metals present in the waste water at low temperature.     

Stability of paclitaxel-loaded solid lipid nanoparticles in the presence of 2-hydoxypropyl-β-cyclodextrin

Paclitaxel (PTX)-loaded solid lipid nanoparticles without hydroxyl-β-cyclodextrin (PS) or with hydroxypropyl-β-cyclodextrin (PSC) were prepared by hot-melted sonication. Biocompatible and biodegradable stearic acid was used to produce the solid matrix. The stability of PS and PSC was assessed at different temperatures. Drug stability, as assessed by encapsulation efficiency (EE; %), particle size, and the polydispersity index (PDI), was examined and in vitro release of PTX from PS or PSC for up to 180 days was assessed. After 180 days of storage at 25 °C, no significant change in particle size, PDI, or EE of PS or PSC was observed. PS and PSC displayed similar sustained PTX release patterns. The particle size, PDI, EE, PTX release profile, and cytotoxicity of PS changed significantly with increasing incubation time, whereas those of PSC showed no significant change, when samples were stored at 40 ± 2 °C. PSC was more stable than PS in plasma with regard to particle size and PDI. These results demonstrate that PSC could be a promising formulation to increase drug stability.     

Collagen from Turkey (Meleagris gallopavo) tendon: A promising sustainable biomaterial for pharmaceutical use

Collagen is the major fibrillar component and protein in both human and animal connective tissue. It is applied in medical preparations, e.g. wound dressings and tissue engineering. Meat and poultry production industries result in large amounts of organic waste, rich in collagen. Our aim was to isolate and characterize pepsin soluble collagen from turkey tendon. Structural analysis indicated the presence of α-chains from both collagen type I and III, β-dimers and γ-trimers, consistent with the estimated molecular weight of 477.3 kDa. Circular dichroism spectroscopy confirmed an intact triple helix. The collagen demonstrated excellent thermal stability, with denaturation temperatures (T_max) at 38.5 °C and 44.5 °C and partial refolding after extensive heating. Biocompatibility was confirmed through cell viability tests. The collagen was investigated for its potential drug carrier ability. Freeze dried collagen scaffolds containing prilocaine hydrochloride and riboflavin were prepared in the presence or absence of photo-crosslinking. Photochemical crosslinking was confirmed by SEM and enhanced mechanical properties were observed. Scaffolds had a significant slower in vitro release of the active ingredient than a reference solution. Altogether, our study suggests collagen from turkey tendon as a promising sustainable biomaterial for pharmaceutical use.     

Biochemical and ultrastructural changes of the liver and kidney of the phytoplanktivorous silver carp feeding naturally on toxic Microcystis blooms in Taihu Lake, China.

Many experimental studies have documented the impact of microcystins (MC) on fish based on either intraperitoneal injection, or oral gavaging via the diet, but few experiments were conducted by MC exposure through natural food uptake in lakes. In this study, the phytoplanktivorous silver carp were stocked in a large pen set in Meiliang Bay of Taihu Lake where toxic Microcystis blooms occurred in the warm seasons. Fish samples were collected monthly and MC concentrations in liver and kidney of the fish were determined by LC-MS. The maximum MC concentrations in liver and kidney were present in July when damages in ultrastructures of the liver and kidney were revealed by electron microscope. In comparison with previous studies on common carp, silver carp showed less damage and presence of lysosome proliferation in liver and kidney. Silver carp might eliminate or lessen cell damage caused by MC through lysosome activation. Recovery in the ultrastructures of liver and kidney after Microcystis blooms was companied with a significant decrease or even disappearance of MC. Catalase and glutathione S-transferase in liver and kidney of silver carp during Microcystis blooms were significantly higher than before and after Microcystis blooms. The high glutathione pool in liver and kidney of silver carp suggests their high resistance to MC exposure. The efficient antioxidant defence may be an important mechanism of phytoplanktivorous fish like silver carp to counteract toxic Microcystis blooms.     

Resveratrol ameliorates carbon tetrachloride-induced acute liver injury in mice

The planktivorous filter-feeding silver carp (Hypophthalmichthys molitrix) and bighead carp (Aristichthys nobilis) are the attractive candidates for bio-control of plankton communities to eliminate odorous populations of cyanobacteria. However, few studies focused on the health of such fishes in natural water body with vigorous toxic blooms. Blood parameters are useful and sensitive for diagnosis of diseases and monitoring of the physiological status of fish exposed to toxicants. To evaluate the impact of toxic cyanobacterial blooms on the planktivorous fish, 12 serum chemistry variables were investigated in silver carp and bighead carp for 9 months, in a large net cage in Meiliang Bay, a hypereutrophic region of Lake Taihu. The results confirmed adverse effects of cyanobacterial blooms on two phytoplanktivorous fish, which mainly characterized with potential toxicogenomic effects and metabolism disorders in liver, and kidney dysfunction. In addition, cholestasis was intensively implied by distinct elevation of all four related biomarkers (ALP, GGT, DBIL, TBIL) in bighead carp. The combination of LDH, AST activities and DBIL, URIC contents for silver carp, and the combination of ALT, ALP activities and TBIL, DBIL, URIC concentrations for bighead carps were found to most strongly indicate toxic effects from cyanobacterial blooms in such fishes by a multivariate discriminant analysis.     

In vitro effects of Se-allylselenocysteine and Se-propylselenocysteine on cell growth, DNA integrity, and apoptosis

Two previously unevaluated selenium compounds, Se-allylselenocysteine (ASC) and Se-propylselenocysteine (PSC), have been shown recently to be active in the chemoprevention of experimentally induced mammary carcinogenesis. Other than their potential as chemopreventive agents, little is known about the pharmacological properties of these compounds. In this article, we report on the in vitro effects of ASC and PSC on cell growth inhibition, apoptosis, and the induction of DNA damage. The effects of ASC and PSC were examined in two mouse mammary epithelial cell lines derived from mammary hyperplasias. These cell lines, designated TM2H and TM12, have mutant or wild-type p53, respectively. It was observed that ASC but not PSC reduced, in a concentration- and time-dependent manner, the number of adherent cells in culture, and this suppressive effect was more prominent in TM12 than in TM2H cells. ASC was also found to induce alkaline-labile DNA damage and the oxidation of pyrimidines, and it also increased the rate of apoptosis. These changes were not seen by exposure to PSC or the sulfur analog of ASC. However, additional data obtained from the intact rat mammary gland suggest that the loss of DNA integrity induced by ASC might not be manifest in vivo at doses of ASC that inhibit carcinogenesis.     

Fatty acid and amino acid composition in haruan as a potential role in wound healing

1. Two species of snakehead fish are available in Sabah, i.e. Channa striatus and Channa melanosoma, and are commonly known as haruan. Haruan is consumed by many Malaysians to induce healing after a clinical operations. However, there is no scientific evidence as yet to substantiate the claim, and so it was decided to analyse the biochemical composition in haruan to determine which compounds may have a possible role or potential in wound healing.2. Samples (midline fillet) of both species were extracted separately in hexane for the qualitative analysis of fatty acids by a gas chromatography, Hewlett-Packard 5890A, using a 10 meter superox 11 column (Alltech) at temperature between 190 and 245°C. Peak areas were calculated automatically using Hewlett-Packard 3393A computing integrator. Subsequently, the amino acid composition was analysed using a precolumn derivatization reverse phase HPLC waters PICO-TAG system.3. Haruan is found to contain unusually high arachidonic acid (AA) but almost no eicosapentaenoic acid (EPA). AA which is a precursor of prostaglandin may initiate blood clotting and be responsible for growth. Haruan also contains all the essential amino acids for wound healing, particularly glycine which is the most important component of human skin collagen. Therefore, haruan contained all the basic biochemical requirements for wound healing.     

Spatial and interspecific patterns in persistent contaminant loads in bighead and silver carp from the Illinois River

We measured concentrations of selected organohalogens, fluorinated compounds and mercury in whole, ground silver (Hypophthalmichthys molitrix) and bighead (H. nobilis) carp from the Illinois River, Illinois, in 2010 and 2011 to characterize spatial and interspecific patterns of contaminant burdens. Silver carp, which had greater lipid content, tended to have greater concentrations of lipophilic compounds. Concentrations of organohalogens were generally greater in carp from the upper reaches of the river. The halogenated compounds were associated with length and lipid content in silver carp. Bighead carp had greater mercury concentrations than did silver carp; total mercury concentrations were negatively associated with lipid content of bighead carp. Perfluorinated compounds, comprised predominantly of perfluorooctane sulfonic acid, did not vary by species or river reach. Chlordanes and polychlorinated biphenyls were of potential concern with regard to the use of these carp as animal feed additives. Our results indicated that, even though they occupy a lower trophic level than many similarly-sized fish, these carp may accumulate measureable concentrations of organic contaminants.     

Reversibility of d-penicillamine induced collagen alterations in rat skin and granulation tissue

Granulation tissue was produced in rats by subcutaneous implantation of Visella^® sponges. d-penicillamine (d-pen) 100 or 500 mg/kg was administered daily for 42 days by gastric tubing. Pairfed, placebo treated animals were included as controls. Half of the groups were kept for additionally 28 days without medication. The inhibitory effect of d-pen on cross-link formation in newly synthesized collagen was readily reversible. By contrast, cross-link deficiency lasting beyond the observation period was observed in the higher polymeric collagen variants released by dilute acid, heat exposure or limited pepsin proteolysis as estimated by solubility, α/β chain ratio and/or aldehyde content. By SDS-polyacrylamide gel electrophoresis on gels containing 3.6 M urea it was shown that purified dermal acid soluble collagen from treated animals consisted of a mixture of type I and III collagen, whereas only type I collagen was detected in controls. The band pattern was identical in reduced and unreduced collagen samples. Four weeks after d-pen discontinuance type III collagen had disappeared from the acid extract. Moreover, the ratio of type III to type I collagen in the pepsin digest from both granulation tissue and skin showed a persistent rise with d-pen. These observations indicate that d-pen destabilized type III collagen in particular by interference with its disulfide linkages. The amount of granulation tissue remained unaffected throughout the experiment, whereas the skin collagen content decreased at the higher dose level. The regeneration was not completed by the end of the observation period. Modulation of the molecular stability of granuloma collagens may be of relevance for the antirheumatoid effect of d-pen, but the sustained effect on normal tissues may imply a long standing impairment of their supportive capacity.     

Antibacterial,mechanical and physical properties of collagen - chitosan sponges from aquatic source

Collagen and chitosan, used for the preparation of sponges, were isolated from the residues of aquatic origin, and co-adjuvant material alginate was added. All sponges are light-colored, but those formulated with chitosan have a yellow hue. The chemical interaction between the components was confirmed by Fourier-transform infrared spectroscopy. The porosity of the sponges is in the range from 88 to 95%. The sponges based on collagen/chitosan/alginate exhibited the highest capacity for absorption in water at 25 °C, with values of up to 2838%. Collagen-chitosan sponges have less rigidity and mechanical strength than collagen-alginate sponges. Collagen-alginate sponges contained the highest maximum stress and Young's modulus, with values of 0.5463 MPa and 0.0086 MPa, respectively. Collagen sponges with chitosan have a greater elongation at break (6.1566%). The sponges with the highest antimicrobial activity were collagen-chitosan mixtures, with inhibition zones of up to 46.62% against Staphylococcus aureus. We anticipate that the sponges based on fish collagen and shrimp chitosan can serve as an alternative of biomaterial for the healing of cutaneous wounds due to their mechanical and antibacterial properties.     

Temperature affects the toxicity of lead-contaminated food in Geophagus brasiliensis (QUOY & GAIMARD, 1824)

Lead is toxic to fish, and its toxicity can be aggravated by the water temperature. Geophagus brasiliensis populations are geographically widespread and thus live in areas with different temperatures. The objective of this work was to evaluate the effects of lead-contaminated feed in fish (Geophagus brasiliensis) exposed to different temperatures. A factorial experiment was performed with two temperatures (25 and 28°C), and two feeds (control and lead contaminated - 60 mg/kg) for a sum of four treatments (25°C, Control = 25/C; 25°C, 60 mg/kg = 25/60; 28°C, Control = 28/C and 28°C, 60 mg/kg = 28/60). Analyses of the lead accumulation, oxidative stress and genotoxic damage were performed. The gills and liver showed increased lead concentrations in fish receiving lead-contaminated food at both temperatures. The lead concentrations in the intestines and muscles of fish exposed to the 25/60 treatment was greater than it was in fish exposed to the 25/C treatment. The enzyme response in the gills and the micronuclei count increased in fish exposed to the 25/60 treatment. Higher temperatures can be a beneficial factor for Geophagus brasiliensis because they can hinder the absorption of lead, thereby reducing the damage caused to the organism.     

Recombinant collagen and gelatin for drug delivery

The tools of recombinant protein expression are now being used to provide recombinant sources of both collagen and gelatin. The primary focus of this review is to discuss alternatives to bovine collagen for biomedical applications. Several recombinant systems have been developed for production of human sequence collagens. Mammalian and insect cells were initially used, but were thought to be too costly for commercial production. Yeast have been engineered to express high levels of type I homotrimer and heterotrimer and type II and type III collagen. Co-expression of collagen genes and cDNAs encoding the subunits of prolyl hydroxylase has lead to the synthesis of completely hydroxylated, thermostable collagens. Human types I and III collagen homotrimers have been expressed in transgenic tobacco plants, while transgenic mice have been engineered to produce full-length type I procollagen homotrimer as well as a alpha2 (I) homotrimeric mini-collagen. Most recently, a transgenic silkworm system was used to produce a fusion protein containing a collagenous sequence. Each of these transgenic systems holds great promise for the cost-effective large-scale production of recombinant human collagens. As seen in other recombinant expression systems, transgenic silkworms, tobacco, and mice lack sufficient endogenous prolyl hydroxylase activity to produce fully hydroxylated collagen. In mice and tobacco, this was overcome by over-expression of prolyl hydroxylase, analogous to what has been done in yeast and insect cell culture. In addition to recombinant alternatives to bovine collagen, other sources such as fish and sponge collagen are discussed briefly. Recombinant gelatin has been expressed in Pichia pastoris and Hansenula polymorpha in both non-hydroxylated and hydroxylated forms. Pichia was shown to be a highly productive system for gelatin production. The recombinant gelatins produced in yeast are of defined molecular weight and physio-chemical properties and represent a new biomaterial not previously available from animal sources. Genetic engineering has made great progress in the areas of recombinant collagen and gelatin expression, and there are now several alternatives to bovine material that offer an enhanced safety profile, greater reproducibility and quality, and the ability of these materials to be tailored to enhance product performance.