Method for isolating collagen from bone tissue of stock-farm animals
FIELD: chemical engineering.
SUBSTANCE: method involves pretreating bone tissue of stock-farm animals, comminuting it, precipitating the end product and drying it by lyophilizing. The bone tissue is degreased with alcohol-ether mixture. Demineralization 0.5 and HCl is carried out after grinding during 20 h. Non-resolvable matrix is subjected to proteolysis in HCl of pH=2.0 in pepsin presence at 37°C during 18 h. Then it is centrifuged at 40000g. The end product is precipitated from supernatant with ammonium sulfate to 25% saturation and centrifuged at 6000g. The precipitate is lyophilized against distilled water and chromatographically purified using CM-Sephadex at pH=4.8 and dried with lyophilization.
EFFECT: low cost collagen production; high purity cosmetic preparation production.
The invention relates to chemical technology, in particular to pharmaceutical production, namely the production of drugs, deustche aimed at improving the state of the connective tissue and its regeneration, and is intended for use in cosmetics, it is also possible its use in medicine.
It is now known that the collagen fibers of the matrix of connective tissue perform structural, supporting and stabilizing role in respect of proteoglycans and water. In addition, the collagen framework has great elasticity under the action of forces in tension and compression, playing a major role in limiting the degree of hydration of the matrix of connective tissue (Miller E.J. Biochemical characteristics and biological significance of the genetically distinct collagens // Molec. Cell. Biochem. - 1976. - Vol.13. - N.2. - P.165-192).
A method of obtaining collagen from demineralizing connective tissue of the dermis, which is subjected to alkali-salt treatment, neutralization with acetic acid, followed by freezing, freeze drying and grinding, without further sterilization (RF Patent No. 2076718, publ. 10.04.1997).
However, the known method involves the secretion of collagen from demineralizing connective tissue, in the case of the mineralized materials selection collagen and according to this scheme difficult.
adaca of the present invention is to develop a consumption of time and reagents method of producing collagen from bone tissue of farm animals.
The problem is solved in that in the method of extraction of collagen from bone tissue of farm animals, including pre-treatment tissue, grinding, precipitation of the desired product and its freeze-dried bone degreasing alcohol-ether mixture after grinding spend the demineralization of 0.5 N. HCl for 20 hours, nerastvorim matrix is subjected to proteolysis in the presence of pepsin in HCl with a pH of 2.0 for 18 hours at 37°C, then centrifuged at 40000g, from the supernatant precipitated target product ammonium sulfate up to 25% saturation, centrifuged at 6000g, sediment cialiswhat against distilled water and clean chromatographies on CM-Sephadex at pH of 4.8, freeze-dried.
The present invention explaining a detailed description of the sample lab execution and illustrations, in which:
Figure 1 - illustrates the allocation of mineralized collagen from connective tissue;
Figure 2 shows the chromatographic profile of the collagen of the bone tissue on the cation-exchanger.
The method is as follows.
After pre-processing, which consists in removing soft tissue, mineralized connective tissue were crushed to pieces of a size about 5×5 mm Then the fabric was subjected to demineralization of 0.5 N. HCl for 20 h, after that the procedure demineralized tissue was liberated from a solution of bone mineral Nikolayevich proteins and spent basenowy proteolysis in the presence of enzyme in 0.1 M HCl to pH 2. Pepsin was taken from a rate of 1 mg per 100 mg tissue. For 18 h incubation at 37 ° °With magnetic stirrer fabric was completely destroyed with the formation of a colloidal solution from which laid siege to the collagen by adding ammonium sulfate up to 25%saturation. The precipitate was formed within 1 h, after which it was separated by centrifugation (10 min, 40.000 gx). To obtain highly purified collagen using a chromatographic separation on CM-Sephadex. After that, the collagen freeze-dried.
Examples of laboratory execution.
Cattle bone is cleaned from soft tissue, degrease the alcohol-ether mixture 1:1, is crushed to pieces size 5×5 mm
100 g of degreased crushed bone tissue is placed in a round bottom flask, into which pour 200 ml of 0.5 N. HCl and left for 20 h at room temperature. Obtained after removal nadeshiko nerastvorim bone matrix filled with 0.1 M HCl and make 1 mg of pepsin. The reaction mass is placed on the magnetic stirrer and carry out the enzymatic hydrolysis of bone tissue for 18 h until complete dissolution of bones and formation of a colloidal solution. The precipitate was separated by centrifugation (40.000 g × 30 minutes)
Then to the solution FR is olidata was added a saturated solution of ammonium sulfate to a concentration of 25%. The precipitate of collagen, formed for 24 hours, was separated by centrifugation (6.000 g × 60 minutes), suspended and were dialyzed against distilled water for 2 days, then freeze-dried. The obtained collagen was dissolved in 0.02 M solution of CH3COONa (pH of 4.8) in 1.0 M urea, centrifuged and applied to the column with the dimensions of 2.5×20 cm with CM-Sephadex (ion-exchange capacity of 4.5 to+0.5 meg/g, grain size 40-120 μm). For elution used a linear gradient of NaCl (0,00-1.5 M) in acetate buffer (pH 4.8). Chromatography was performed under denaturing conditions. Fractions were densitometrically at a wavelength of 226 nm, the fraction of collagen within a single chromatographic peak were collected, were dialyzed against water and freeze-dried.
The proposed method is easy to use, allows to reproduce it in the laboratory of the medical institution, for its implementation does not require the use of expensive reagents and it allows to obtain a highly purified product from cheapest available raw materials.
In addition, obtaining vysokochistogo collagen may be conducted in conjunction with the secretion of other biologically active substances (GAG is a glycosaminoglycan, NCU - not collagenous proteins of bone, thereby reducing the cost of the target product.
Method of extraction of collagen from the bone weste the animals, involving pre-treatment tissue, grinding, precipitation of the desired product and its freeze drying, characterized in that the bone degreasing alcohol-ether mixture after grinding spend the demineralization of 0.5 n HCl for 20 h, nerastvorim matrix is subjected to proteolysis in the presence of pepsin in HCl with a pH of 2.0 for 18 h at 37°C, then centrifuged at 40000g, from the supernatant precipitated target product ammonium sulfate up to 25% saturation, centrifuged at 6,000g, sediment deleteroute against distilled water and purified by chromatography on CM-Sephadex at pH 4,8, freeze-dried.
FIELD: chemical-pharmaceutical industry, pharmacy.
SUBSTANCE: invention relates to a pantohematogen-base medicinal formulation for external using that comprises the following components, wt.-%: dry pantohematogen, 0.01-6; polymeric gel-forming composition, 0.1-20, and water, the balance. Proposed medicinal formulation provides using pantohematogen an aqueous solution without addition of alcohol and therefore it can be used in cases wherein alcohol-containing formulations are contraindicative - in burns of different degree, small skin damages and others and can be used in treatment of patients showing intolerance to alcohol-containing preparations. Method for preparing the pantohematogen medicinal formulation for external using involves preparing "dry" pantohematogen aqueous solution in the ratio limits pantohematogen : water = (1:50)-(1:200) by weight and solution is kept under normal conditions for 1 h and filtered. Additionally an aqueous solution of rare-linked polymer is prepared in the ratio = (1:10)-(1:200) and an aqueous solution of neutralizing agent in the amount necessary for providing slightly alkaline medium of final solution. In the volume ratio of pantohematogen aqueous solution and rare-linked polymer in the ranges = (3:1)-(1:10) components are mixed and stirred to obtain the homogenous solution. Then neutralizing agent aqueous solution is added to a mixture and stirred to obtain homogenous solution with slightly alkaline reaction (pH = 6-8). The advantage of proposed technology in preparing the medicinal formulation involves its simplicity and providing prolonged fitness period.
EFFECT: improved preparing method.
6 cl, 1 ex
FIELD: medicine, stomatology, pharmacy.
SUBSTANCE: invention proposes a composition used in treatment of alveolitis that comprises anesthetic, antiseptic and a hemostatic component wherein the composition comprises anaesthesinum as an anesthetic, crystalline iodine and potassium iodide an aqueous solution as an antiseptic and pectin as a hemostatic component. The composition comprises lanolin and vaseline as ointment base wherein components are taken in the definite ratio. Invention provides high effectiveness in treatment of alveolitis and its capacity to activate regenerative healing processes. Invention can be used in treatment of complications after teeth removal, in particular, alveolitis.
EFFECT: valuable medicinal properties of composition.
SUBSTANCE: the present innovation deals with obtaining compositions being of bactericidal, antiphlogistic, regenerating, antioxidant and antimicrobial properties at no allergic action. Wound-healing preparation (variant 1) contains: stearic acid 4.5-6.0; purified lanolin and its derivatives 3.0-6.0; vegetable oil 3.0-5.0; Vaseline 1.5-2.5; glycerol 4.0-8.0; castor oil 8.0-12.0; zinc stearate 1.0-2.5; 70%- alcoholic extract of phytospecies 2.5-3.5; extract of common John's wort grass 3.5-8.5; dog rose oil or sea buckthorn oil 2.6-6.0; Na-salts of fatty acids of wool fat 0.3-0.6; triethanolamine 0.7-1.1; anesthesin 2.0-3.0; 10%-butyric solution of propolis 0.3-1.0; boric acid 0.4-0.6; purified water - the rest. Wound-healing preparation (variant 2) contains: stearic acid 4.5-6.0; purified lanolin and its derivatives 3.0-6.0; vegetable oil 3.0-5.0; Vaseline 1.5-2.5; glycerol 4.0-8.0; castor oil 8.0-12.0; zinc stearate 1.0-2.5; 70%-alcoholic extract of phytospecies 2.5-3.5; extract of common John's wort 3.5-8.5; dog rose oil or sea buckthorn oil 2.6-6.0; Na-salts of fatty acids of wool fat 0.3-0.6; triethanolamine 0.7-1.1; anesthesin 2.0-3.0; levomycetin or gentamycine 2.0-3.0; 10%-butyric solution of propolis 0.3-1.0; boric acid 0.4-0.6; purified water - the rest. Additional introduction of the extract of common John's wort has increased antioxidant properties; 10%-butyric propolis - antioxidant and bactericidal properties, and levomycetin and gentamycine - has widened the range of antimicrobial action. Application of the preparation suggested in patients with burns and wounds revealed positive therapeutic effect without any cicatricial neoplasms and allergic action. No contraindications had been established.
EFFECT: higher efficiency of application.
4 cl, 8 ex
SUBSTANCE: the present innovation deals with new compositions in the form of a single-stage gel that contains minoxidyl, a thickening agent and a pharmaceutically acceptable solvent. The composition contains 3-8 weight% minoxidyl. Noncarbometric thickening agent is not a heterobiopolysaccharide or cellulose derivative. Carbometric thickening agent being tolerant to a solvent is not a carbometer of Carbopol 934P type. Minoxidyl is practically solubilized in the composition. It is, also, described the method for preparing minoxidyl-containing composition. Also, the present innovation deals with the ways of treating or preventing alopecia in patients, moreover, these methods deal with local application of these compositions at the site of alopecia according to the innovation suggested. Thus, it is possible to obtain a single-phase composition by its consistence of moderate viscosity that could be obtained upon conventionally applied equipment.
EFFECT: higher efficiency of application.
83 cl, 8 ex
SUBSTANCE: the present innovation deals with compositions of ingredients that have curative indication due to external application at patient's body. The suggested ointment contains vegetable oil, wax as fatty product, rivanol, tannin, glycerol as antiseptic at the following ratio of components, weight%: wax 13-15; rivanol solution 1.2-1.3; tannin 3-5; glycerol 15-20; vegetable oil - the rest. The innovation provides increased efficiency of healing action at no side complications.
EFFECT: higher efficiency of therapy.
4 ex, 1 tbl
FIELD: pharmaceutical industry, in particular analgesic, vasodilatation, anti-inflammation ointment for topical application.
SUBSTANCE: claimed ointment is prepared in form of fine dispersed formulation containing bee venom extract melittin, lavender oil, camphor, arnica tincture, rosemary oil, eucalyptus oil, revitalin, carbopol, 30 % solution of sodium hydroxide, hydrophilic anionic ointment, soy lecithin, glycerol, conserving solution, ethanol, distilled water.
EFFECT: reduced toxicity of bee venom, decreased side effects and improved therapeutic effect.
2 tbl, 3 ex
SUBSTANCE: claimed ointment contains (mass %): fir oil 5.0-10; xeroform 3.0-6.0; and balance: ointment base.
EFFECT: nontoxic and hypoallergic ointment of with high effectiveness and prolonged storage time.
FIELD: pharmaceutical industry, in particular anesthetic and vasodilating ointment.
SUBSTANCE: claimed ointment is prepared on the base of fine dispersed formulation contains apitoxin extract melittin, camphor, lavender, rosemary and eucalyptus oils and revitalin in specific component ratio. Ointment of present invention is useful in treatment of reumathysm, muscle and joint diseases, myodynia, radiculitis, ischias, neurodynia, polyneuritis, etc.
EFFECT: ointment with reduced apiotoxin toxicity, decreased side effects and improved therapeutic effect.
FIELD: pharmaceutical industry, in particular pharmaceutical preparation in ointment form for treatment of foul wounds.
SUBSTANCE: claimed agent contains as active ingredient combination of antibacterial agent dioxidine; methyluracil as agent accelerating cell regeneration; lidoxaine hydrochloride as topical anesthetic agent and polyethylene oxide 400 and 1500 as carrier in ratio of 1:4:2:(3.5-3.6):1, respectively. Claimed agent may be used in the first step of wound process.
EFFECT: agent having minimal collateral effect due to application of low toxic components.
3 tbl, 1 ex
FIELD: pharmaceutical industry, in particular production of ointment having antiinflammation and antimicrobial activity.
SUBSTANCE: method for ointment production includes extraction of ground dogwood stones with chloroform during certain time followed by solvent removing and isolation of lipophilic fraction. Then dried solvent cake from dogwood stones is extracted with boiled purified water, solvent is removed and hydrophilic fraction is isolated. Further emulsifier, petrolatum and lipophilic fraction taken in specific ratio are fused at certain temperature; mixture is cooled and blended with water purified and heated up to certain temperature; obtained mixture is blended with solution comprising purified hydrophilic fraction, glycerol and water taken in specific ratio.
EFFECT: ointment of increased antiinflammation and antimicrobial activity.
2 tbl, 6 ex
SUBSTANCE: the present innovation deals with the method to accelerate mucosal healing due to the following technique: one should apply a membrane consisted of purified collagenic material obtained out of natural collagen-containing tissue onto the part of affected mucosa to provide the chance for mucosal reconstruction in this part and, also, it deals with mucosa-regenerating preparation and application of purified collagenic material obtained out of collagen-containing natural tissue for preparing mucosa-regenerating preparation. The innovation provides more modified method that accelerates mucosal regeneration, as a whole, and, particularly, after surgical operations associated with the plasty of oral fornix.
EFFECT: higher efficiency.
12 cl, 3 dwg, 5 ex
FIELD: medicine, dermatology, in particular treatment of trophic ulcer (TU) and long-term open septic wound (LTOSW).
SUBSTANCE: claimed method includes application of wound-healthing composition onto TU and LTOSW in the first step of wound process. Said composition contains (mass %): methyluracil 0.9-1.1; pepsin 3.4-4.4; bentaine hydrochloride 3.6-4.4; sodium chloride 9.0-11.0; and balance up to 100,0: distilled water. In the second and third phases fine cut collagen preparations in form of 3-4 mm thickness layer are applied on purified TU and LTOSW surface. Then cotton carrier freely moistened with wound-healthing composition diluted with water in ratio of 1:2 is layered onto collagen layer. Multihole microirrigator and further sterile cotton carrier are sequentially applied over abovementioned cotton carrier.
EFFECT: effective treatment due to accelerated abruption of necrotic tissues, inhibition of microbial growth, decreased risk of infective, toxic and allergic affects, and improved tissue regeneration.
2 ex, 3 cl
FIELD: medicine, experimental medicine.
SUBSTANCE: one should introduce tripeptide Pro-Gly-Pro for laboratory animals as injections at the quantity of 0.09-1.0 mg/kg body weight, and, also, gelatin as fodder additive. The method suggested enables to suppress appetite, decrease the quantity of fodder intake that leads to decreased body weight as a result.
EFFECT: higher efficiency.
2 cl, 5 dwg, 5 ex
FIELD: peptides, pharmacy.
SUBSTANCE: invention relates to low-molecular derivatives of peptides that are able to act as inhibitors in interaction between laminine and nidogen (interactions laminine/nidogen). Also, invention relates to a method for their preparing, pharmaceutical composition prepared on thereof and their using for preparing pharmaceutical agents, and for identification of inhibitors in interaction laminine/nidogen.
EFFECT: valuable properties of peptides.
5 cl, 12 dwg
FIELD: medicine, traumatology.
SUBSTANCE: osseous defect after filling with collapan and usage of metallofixative should be covered with absorbable collagen film that provides orientation of osseous regenerate's organization.
EFFECT: higher efficiency of therapy.
SUBSTANCE: the suggested multi-purpose heterogeneous collagen matrix for implantation is being flexible-elastic mass obtained out of two collagen sources, moreover, one source is a tissue of vertebrates of one and the same class, and another source - that of an animal of another class, moreover, collagen sources are animal tissues of, for example, mammalian class and avian class, matrix consists of two phases: solid phase - as microspheres out of mammalian tissue collagen, and liquid phase - out of denaturated avian tissue collagen at the ratio of phases being (1-10) : (1-10), at microspheres size being 100-300 mcm, as for final products of biodegradation they are represented by CO2 and H2O. Matrix, additionally, contains components of physiological culture media, and, also, additives that favor the growth and differentiation of cells and tissues, antibacterial and/or antiviral components, and, also, antiaggregation preparations in their efficient quantity, for example, additionally, it contains embryonic cells of nervous tissue. Another aspect of the present innovation deals with the method to obtain the matrix due to preparing mammalian collagen solution (MCS) and denaturated avian collagen solution (ACS), moreover, it is necessary to apply 0.3 M acetic acid, at final concentration for MCS being approximately 0.5-1.5%, and for ACS - approximately 3.0-5.0%, then MCS should be treated with γ -irradiation at the dosage of 1.0 Mrad to be further homogenized to obtain microspheres. Then both MCS and ACS should be washed off with distilled water up to pH of not less than 6.0 and with phosphate buffer solution to mix washed off mammalian collagen and avian collagen at 1:1 ratio at obtaining matrix. The matrix obtained should be additionally supplemented with antibacterial and/or antiviral components, and, also, stimulating agents for tissue regeneration and antiaggregation preparations. The matrix obtained should be sterilized due to γ -irradiation at the dosage of 0.5 Mrad/1 ml. The present innovation enables to obtain new heterogeneous collagen matrix which is considered to be a multi-purpose one applied for transplantology and substitution surgery of different tissues and organs in alive body in case of tissue lesions. Moreover, it is distinguished by controlled terms of biodegradation.
EFFECT: higher efficiency of application.
13 cl, 16 ex, 4 tbl
SUBSTANCE: method involves applying surgical intervention and antitumor therapy. After having removed a part or the whole lung, mediastinum is opened by cutting out horseshoe-shaped or rectangular flap of mediastinal pleura and carrying out lymphodissection. Then, hemostatic sponge is placed in mediastinum. The sponge is impregnated with antitumor chemotherapeutical preparations in intraoperative mode. Pleura is sutured above the sponge. Another hemostatic sponge impregnated with cytostatic preparations in intraoperative mode is attached to visceral pleura of interlobular sulcus.
EFFECT: prolonged chemotherapeutical preparations delivery to malignant neoplasm foci or subclinical metastases.
FIELD: medicine, oncology.
SUBSTANCE: the present innovation deals with local chemotherapy of malignant cerebral tumors. Thus, after removing the tumor one should apply a collagen hemostatic sponge with a cavity inside it with a powder-like chemopreparation into tumor's bed, then the cavity should be covered with another fragment of collagen sponge. The innovation enables to minimize toxic manifestations of chemotherapy at applying maximal concentration of anti-tumor preparation.
EFFECT: higher efficiency.
6 dwg, 2 ex