OBJECTIVETo study collagen structure of the traditional Chinese medicine elephant skin and the proposed alternatives such as pig skin, fish scale, and antioxidant activity.

METHODOrthogonal experimental design method was employed to determine the optimal extraction condition of collagen from the elephant skin, and the structure and content of collagen of proposed alternatives were compared, their scavenging ability were determined by salicylic acid.

RESULTCollagen extracted from elephant skin with the optimal conditions was the structural integrity and good quality first time, and collagen structure of the elephant skin was similar to the proposed alternatives. Free radical scavenging capacity of collagen, values of IC50, were 0.51 g x L(-1) of elephant skin, 0.60 g x L(-1) of pig skin and 0.42 g x L(-1) of fish scale.

CONCLUSIONBy comparing and identification of proteins that the collagen of elephant skin is type I collagen, with a strong antioxidant capacity, is the active ingredients of elephant skin. It provides a further study of alternatives as an important reference.

Animals ; Antioxidants ; isolation & purification ; pharmacology ; Collagen ; isolation & purification ; pharmacology ; Elephants ; Fishes ; Skin ; chemistry ; Swine

OBJECTIVETo describe the methods which were used to develop collagen-based materials for wound dressing.

METHODSFresh frozen bovine tendon was treated with 0.05 mol/L acetic acid at pH 3.2 for 48-72 hours, homogenized, filtered, mixed with 8% chondroitin sulphate, for creating a deaerated 1.5%-2.5% collagen solution. The solution was lyophilized in either a pre-frozen or non-pre-frozen mould. The collagen sponge was then cross-linked with 0.25% glutaraldehyde for 24 hours. Three other types of wound dressings were developed using a similar method: collagen membrane with a polyurethane membrane onlay, polyurethane-coated collagen membrane and collagen membrane on gauze.

RESULTSIt was demonstrated that the use of frozen bovine tendon was stable, and that the prepared collagen sponge contained pores of 50-400 microm in diameter.

CONCLUSIONSCollagen could be used as wound dressing.

Amino Acids ; analysis ; Animals ; Biological Dressings ; Cattle ; Collagen ; analysis ; chemistry ; isolation & purification ; Freeze Drying ; Polyurethanes

OBJECTIVETo investigate the selective removal of tannins from Polygonum cuspidatum extracts by using collagen fiber adsorbent, and to evaluate the adsorption and desorption performances of collagen fiber adsorbent to tannins.

METHODThe adsorbent was prepared from bovine skin collagen fiber through crosslinking reaction of glutaraldehyde, and then used for the selective removal of tannins from P. cuspidatum extracts. Gelatin-turbidity method, gelatin-ultraviolet spectrometry method and HPLC were used for detection of tannins in the solutions. Ethanol-water solutions with varying concentration were used to test their desorption ability of tannins in order to choose proper desorption solution. On the basis of batch experimental results, the column adsorption and desorption tests were carried out, by using gelatin-turbidity method for detection of tannins.

RESULTThe collagen fiber adsorbent exhibited excellent adsorption selectivity to tannins. It was found that tannins of P. cuspidatum were completely removed, while nearly no adsorption of active components (resveratrol as representative) was found. Moreover, the collagen fiber adsorbent could be regenerated by using 30% ethanol-water solution and then reused.

CONCLUSIONThe collagen fiber adsorbent can be considered as a promising material for selective removal of tannins from P. cuspidatum extracts.

Adsorption ; Collagen ; chemistry ; Fallopia japonica ; chemistry ; Plant Extracts ; analysis ; Tannins ; isolation & purification

Acid-soluble collagen (ASC) and pepsin-soluble collagen (PSC) from the spine (ASC-SP and PSC-SP) and skull (ASC-SK and PSC-SK) of the skipjack tuna, Katsuwonus pelamis, were successfully isolated and characterized. The yields of ASC-SP, PSC-SP, ASC-SK and PSC-SK were (2.47 ± 0.39)%, (5.62 ± 0.82)%, (3.57 ± 0.40)%, and (6.71 ± 0.81)%, respectively, on the basis of dry weight. The four collagens contained Gly (330.2-339.1 residues/1 000 residues) as the major amino acid, and their imino acid contents were between 168.8 and 178.2 residues/1 000 residues. Amino acid composition, SDS-PAGE, and FTIR investigations confirmed that ASC-SP and ASC-SK were mainly composed of type I collagen, and had higher contents of high-molecular weight cross-links than those of PSC-SK and PSC-SP. The FTIR investigation also certified all the collagens had triple helical structure. The denaturation temperatures of ASC-SK, PSC-SK, ASC-SP, and PSC-SP were 17.8, 16.6, 17.6, and 16.5 °C, respectively. All isolated collagens were soluble at acidic pH (1-5) and lost their solubilities when the NaCl concentration was above 2% (W/V). The isolated collagens from the spines and skulls of skipjack tuna could serve as an alternative source of collagens for further application in food, cosmetic, biomedical, and pharmaceutical industries.
Acids ; chemistry ; Amino Acids ; analysis ; Animals ; Collagen ; chemistry ; isolation & purification ; Collagen Type I ; chemistry ; isolation & purification ; Hydrogen-Ion Concentration ; Molecular Structure ; Molecular Weight ; Pepsin A ; chemistry ; Skull ; chemistry ; Sodium Chloride ; Solubility ; Spine ; chemistry ; Temperature ; Tuna

We have prepared a wound dressing made from chitosan and collagen. Its clinical curative effect was detected. Chitosan solution was put into purified collagen solution. Then, the solution became sponge by means of freeze drying, and it was subjected to a series of toxicology tests, including acute toxicity, stimulation test, allergic and hemolysis tests, as well as the clinical test of openning trauma in orthopedics. All of the results of toxicology tests were negative. The chitosan-collagen sponge could not only accelerate the speed of curing but also restrain the extravasate. Therefore, the chitosan-collagen sponge has good biocompatibility and clinical curative effect. It is a prospective security-biomaterial for medical use.
Biocompatible Materials ; administration & dosage ; Biological Dressings ; Chitin ; analogs & derivatives ; isolation & purification ; therapeutic use ; Chitosan ; Collagen ; isolation & purification ; therapeutic use ; Humans ; Membranes, Artificial ; Wound Healing ; drug effects

The object of the research was to extract, purify and identify the type II collagen of Agkistrodon acutus. Type II collagen of A. acutus was extracted by enzyme decomposition method, and purified by ion exchange column chromatography. It was characterized by SDS-PAGE gel electrophoresis, ultraviolet spectrophotometry, infrared absorption spectroscopy and mass spectroscopy. The results showed that the size of C II was about 130 kDa. It absorbed at 223 nm. IR spectrum obtained showed that the triple helical domains of amino-acid sequences were characterized by the repetition of triplets Gly-X-Y. The MS spectrum graphically stated that C II extracted from cow and A. acutus have the similar peptides. The C II of A. acutus was obtained by extraction and purification. Appraisal analysis by SDS-PAGE, UV, IR and MS, C II of A. acutus was consistent with the standard C II of cow. It was proved that the extracted protein was C II.
Agkistrodon ; metabolism ; Animals ; Collagen Type II ; chemistry ; isolation & purification ; metabolism ; Electrophoresis, Polyacrylamide Gel ; Mass Spectrometry ; Reptilian Proteins ; chemistry ; isolation & purification ; metabolism

OBJECTIVETo prepare anti-von Willebrand factor A3 (vWF-A3) domain monoclonal antibodies(mAbs) which block vWF-A3 binding to collagen, and characterize their biochemical properties and functions.

METHODSBALB/c mice were immunized with purified recombinant vWF-A3 protein (rvWF-A3). Murine anti-human vWF-A3 mAbs were developed by standard hybridoma technology and identified with ELISA. The recognition of the mAbs with rvWF -A3 and reduced human vWF was identified by Western-blot. The effect of mAbs on binding of purified human vWF to human placenta or calf skin collagen III was studied with collagen binding inhibition test.

RESULTSA group of 30 murine anti-human vWF-A3 mAbs was obtained, from which 2 clones were identified as inhibitory ones and designated as SZ-123 and SZ-125. SZ-123 and SZ-125 could react specifically with human vWF and rvWF-A3 respectively, while neither of them reacted with rvWF-A1 and rvWF-A2. Western-blot showed that SZ-123 and SZ-125 could recognize a 27 x 10(3) band of rvWF-A3 and 2 reduced human vWF bands at 250 x 10(3) and 170 x 10(3). SZ-123 and SZ-125 not only inhibited the binding of purified human vWF (1.5 and 3.0 microg/ml) to human type III collagen and to calf skin collagen III in a dose dependent manner, but also inhibited the binding of plasma vWF from human, rhesus monkeys or Beagle dogs to the two collagens.

CONCLUSIONSZ-123 and SZ-125 are neutralizing mAbs against vWF-A3 domain and may have therapeutic potential as an antithrombotic agent.

Animals ; Antibodies, Monoclonal ; immunology ; isolation & purification ; Collagen ; immunology ; Mice ; Mice, Inbred BALB C ; von Willebrand Factor ; immunology

Endostatin, a carboxyl-terminal fragment of collagen XVIII is known as an anti-angiogenic agent, that specifically inhibits the proliferation of endothelial cell and the growth of several primary tumor. We report here the purification and characterization of the recombinant murine endostatin (rmEndostatin) which was expressed in a prokaryotic expression system. This rmEndostatin has similar physiochemical properties of yeast-produced recombinant endostatin, and it also specifically inhibits the proliferation and migration of bovine capillary endothelial cells stimulated by basic fibroblast growth factor. The biological activity of rmEndostatin was also shown by its anti-angiogenic ability on the chorioallantoic membrane of chick embryo in vivo. In this article, we demonstrate the refolding and purification of rmEndostatin, expressed using E. coli system, to a biologically active and soluble form. In addition, these results confirm the activity of endostatin as a potent anti-angiogenic agent. Copyright 2000 Academic Press.
Angiogenesis Inhibitors/pharmacology* ; Angiogenesis Inhibitors/isolation & purification ; Angiogenesis Inhibitors/genetics* ; Animal ; Blotting, Western ; Cattle ; Cell Movement/drug effects ; Chick Embryo ; Chorion/pathology ; Chorion/drug effects ; Circular Dichroism ; Collagen/pharmacology* ; Collagen/isolation & purification ; Collagen/genetics* ; Electrophoresis, Polyacrylamide Gel ; Endothelium, Vascular/drug effects ; Endothelium, Vascular/cytology ; Escherichia coli/genetics* ; Fibroblast Growth Factor, Basic/pharmacology ; Mice ; Neovascularization, Physiologic/drug effects ; Peptide Fragments/pharmacology* ; Peptide Fragments/isolation & purification ; Peptide Fragments/genetics* ; Protein Folding ; Recombinant Proteins/pharmacology ; Recombinant Proteins/isolation & purification ; Recombinant Proteins/genetics ; Solubility ; Yeasts/genetics

This study was aimed to further investigate the function of platelet collagen receptor-glycoprotein VI and to screen its specific inhibitor. The extracellular domain of platelet glycoprotein VI (GPVI) in E. coli was expressed by recombinant technology, the extracellular domain cDNA of GPVI was amplified from pBluescript KS(-)-GPVI plasmid by PCR. Proved by sequencing, the expression vector pET-20b(+)-GPVI was constructed, which was then transformed into E. coli (BL21(DE3)pLysS) and induced by IPTG. The recombinant GPVI was purified on Ni-NTA resin column and renatured in PBS containing GSH and GSSG. The anti-penta His McAb and anti-GPVI polyclonal antibody were used to identify the recombinant GPVI in Western blotting. Collagen binding test was conducted to investigate the biological activity of recombinant GPVI. The results showed that the recombinant GPVI was expressed in E. coli and successfully purified, which was confirmed to be similar to the native GPVI in Western blotting. The recombinant GPVI can bind the type I collagen in dose-dependent manner. In conclusion, the recombinant GPVI can be achieved in E. coli and restore its native characteristics after renaturation.
Blood Platelets ; metabolism ; Blotting, Western ; Escherichia coli ; genetics ; Humans ; Integrin alpha2beta1 ; Platelet Membrane Glycoproteins ; biosynthesis ; genetics ; Protein Binding ; Receptors, Collagen ; biosynthesis ; genetics ; Recombinant Proteins ; biosynthesis ; isolation & purification

Recently, research on collagen attracts more interests due to its good biological compatibility. The present study attempted to establish a fast and efficient method to purify collagen from soft-shelled turtle and to explore its application in biological materials. The structure and type of collagen fiber in calipash were determined by van Gieson staining and Picrosirius red staining, which could contribute to the isolation of collagen from soft-shelled turtle Calipash (STCC). Collagen fibers were in high content and the main collagen fiber was type I in STCC. The crude STCC solution was purified by dialysis with different cut-off molecular weight. SDS-PAGE demonstrated that the best purification was in applying 100 kDa dialysis bags after 48 h. The water absorbing capacity and holding capacity of STCC were up to 12.06 g/g and 98.21%, respectively. STCC can be degraded by collagenase in vitro entirely after 72 h. The hemolysis, skin sensitization, hemostatic and wound healing of STCC were determined by using SD rat model, and the collagen cross-linked by glutaric dialdehyde was set as a comparison. STCC and STCC cross-linked did not result in destructed red blood cell, inflamed and sensitized skin. Both materials exhibited good hemostatic effect. Thus, STCC improved the wound healing efficiently. This study implies a potential of STCC in the field of biomaterial.
Animals ; Biocompatible Materials ; Biotechnology ; Collagen ; isolation & purification ; Electrophoresis, Polyacrylamide Gel ; Molecular Weight ; Rats ; Rats, Sprague-Dawley ; Skin ; drug effects ; Turtles ; Wound Healing