DE2507223A1 - Wound-healing partially degraded collagen prod - contg 25-50 pts monomeric collagen, 25-50 pts polymeric collagen and 0-40 wt pts highly polymeric collagen - Google Patents

Abstract

New product obtainable from collagen contains 25-50 pts. monomeric, 25-50 pts. polymeric and 0-40 pts. highly polymeric collagen. At advantageously additionally contains proline so that the total proline content is up to 40%. The product is suitable for the treatment of open wounds, since it strongly stimulates wound granulation. It is also suitable for cosmetic purposes, on account of its moisturizing and reparative action.

Description

Product obtainable from collagen and process for its production The present invention relates to a product obtainable from collagen, a process for its manufacture as well as pharmaceuticals and cosmetic products that use this product contain.

Collagen is a protein whose properties for the most part are known (TRAUB, W. and PIEZ, K.A. in Advanc. Prot. Dem., 25, 243, Academic Press, New York (1971); VIIDIK, A., Intern. Rev. Conn. Tissue Res., 6, 127, Academic Press, New York (1973); RAMACHANDRAN, G.N., and HODGE, AG., In "Treatise on Collagen, Vol. 1, pp. 103 and 185, Academic Press, London (1967)). It's already tried been using collagen to heal wounds.

However, the results of these tests were very changeable and not reproducible.

In the native state, the collagen consists of three chains 2 2 1 chains, 1 42 chain), which are linked in a helix-like manner and which are connected by non-covalent Ties are held together. This collagen is hereinafter referred to as monomeric Collagen ". Some or a whole series of" molecules "of the monomer Collagens can bond with each other through covalent bonds, creating what is known as "polymeric collagen" (containing 2 to about 4 collagen Units) and "high polymer collagen" (containing more than 4 collagen units) is formed.

It has now been found that it can be used to achieve a good effect Collagen crucially depends on that in the collagen-containing preparations a certain ratio of monomeric: polymeric: high polymer collagen is observed will.

The invention relates to a product which contains 25-50 parts of monomeric, 25-50 parts polymeric and 0-40 parts high polymer collagen in a buffer contains.

The invention also relates to a method of production of the product, which consists in a) collagen in an alkali salt solution, which contains a hexose or glycerine, stir at low temperature and the insoluble Separating constituents, a solution I containing monomeric collagen, and a residue A is obtained, b) the residue A in 0.01-1.0 molar acid under Stirring addition of 0.05 -3%, -Ketoglutaric acid at room temperature and then the insoluble constituents are separated off, a supernatant II, the polymer Contains collagen, and a residue B is obtained, c) the residue B in an aqueous Solution containing 0.2 to 8 moles of urethane and optionally 0.3 to 8 moles of urea per Liters, or in Tris buffer (pH4'0 bits8'0), which contains 0.05 to 0.5 mol / l calcium chloride contains, stirs and centrifuges, with the high polymer collagen in the supernatant III receives, and d) the supernatants I, II and III separated by dialysis against water or with sodium chloride precipitates, the precipitates individually in one dissolves physiologically compatible buffer (pH 2 - 7) and mixes the solutions in such a way that that the ratio of components I, II and III to one another (25 to 50): (25 to 50 (O to 40).

In a preferred embodiment, the solution obtained in this way is used Proline added so that the total proline content up to 40%, preferably 30%, based on the total content of amino acids after hydrolysis.

Finally, the invention relates to pharmaceuticals and cosmetic products Means containing the new collagen product.

Commercially available collagen can be used as the starting material. This is always a mixture of monomeric, polymeric and high-polymer collagen. A collagen with as high a proportion of monomer as possible is best suited which can be obtained from young animals.

In the case of collagen from old animals, the proportion of monomers can be so low be that working up by the process according to the invention is uneconomical will.

It can be useful to use the collagen used as the starting material to subject to a cleaning step before working up according to the invention. Purification can be done by removing impurities from the collagens e.g. extracted with a saline solution containing a complexing agent.

The first reaction step a) is expediently carried out at 2-8 ° C. The alkali salt concentration should not be below 0.01 mol / l, upwards no limit set.

In particular, glucose, fructose and galactose are used as hexoses Consideration, which are preferably used in an amount of 0.05 to 0.5 mol / l. Suitable acids for step b) are e.g. sulfuric acid, phosphoric acid, formic acid, Succinic acid, oxalic acid and the like.

Acetic acid and lactic acid are particularly suitable. For the step c) Urethanes are those with lower alkyl groups, especially ethyl urethane, in an amount of normally 0.2-5.0, preferably 1.5-2.5, mol / l. It has proven to be beneficial to add urea in an amount of 0.3 - 8.0, preferably 0.5-1.5, mol / l to be added to the urethane solution.

The buffer used for d) should have a pH of 2-7 and 0.01 to 1.0 molar. All suitable salts can be used for its preparation and acids are used which are physiologically acceptable. Particularly suitable are acetate and citrate buffers with a pH of 3.5 to 6.0.

It is advisable to add an antibiotic to the solution obtained, to increase the shelf life. Other substances such as vitamins can also be used (Ascorbic acid), amino acids (glycine, DL-threonine, L-cysteine) or Cu, Zn and iron salts can be added.

The new product available from collagen is very suitable for Treatment of open wounds because it strongly stimulates wound granulation. Also for cosmetic Purposes it is very useful because of its moisturizing and reparative effects.

Example 1 1. Pre-purification of the collagen 6 kg of collagen are used in 20 1 deionized water in which 4% EDTA (800 g) and 1% NaCl (200 g) are dissolved, stirred at 4 ° C. for 24 hours. Then it is centrifuged. The supernatant is discarded.

II. Production of the end product a) The pre-purified collagen (5.5 kg) is in 100 l of water in which 1 kg of glucose and 1.5 kg of NaCl are dissolved, 24 hours stirred at 4 ° C. and then centrifuged. The supernatant (I) is saved (80 1).

b) The residue (2.5 kg) is with 40 1 0.05 M acetic acid, in the 0.5% (200 g), ketoglutaric acid are included, stirred for 24 hours at room temperature and then centrifuged again. The supernatant (II) is saved (35 1).

c) The centrifugation residue (1.5 kg) is in 25 l of water in which 1.5 kg of urea and 1.125 kg of ethyl urethane have been dissolved beforehand, 24 hours at 4 ° C touched. It is then centrifuged again and the supernatant (III) (20 l) is stored. The residue (400 g) is discarded.

d) The supernatants I, II and III are dialyzed separately against water (40 hours at 15 ° C). The precipitated collagen fibers are separated, in 0.1 M citrate buffer (pH 4.0) added at 40C (I in 45 1, II in 15 1, III in 16.5 1) and centrifuged.

40 parts I, 40 parts II and 20 parts III are mixed together and then diluted 1:10 with the citrate buffer. The total concentration I, II and III is in the final product; at about 0.6%. The proline content is after hydrolysis 15.7% based on the total protein content.

Example 2 Example 1 was repeated, but step c) the centrifugation residue in 25 1 of 0.2 M Tris buffer (pH 7.0) containing 0.1 mol / l CaCl2 contained, stirred.

The yield was the same as in Example 1.

Example 3 a) 5 kg of collagen are in 40 1 of water, in which 1.136 kg Disodium hydrogen phosphate and 4 kg of glycerol are dissolved, stirred at 80C for 24 hours and centrifuged.

The supernatant I is saved. (30 1) b) The residue (3.5 kg) is with 30 1 0.5 M lactic acid and 300 g -ketoglutaric acid for 20 hours at room temperature stirred and then centrifuged.

The supernatant II (20 1) is saved.

c) The centrifugation residue (2.2 kg) is in 25 1 of water in which 9 kg of ethyl urethane were previously dissolved, stirred at 4 ° C. for 24 hours. After that, will centrifuged and the supernatant III saved. (20 1) d) The protrusions I (3Ö 1), II (20 1) and III (20 1) are saturated separately with NaCl and over at 4 0C Left at night. The precipitated collagens are individually filtered, with Washed small amounts of water, weighed and in each 5 1 0.1 M citrate buffer (pH 4.5) solved. Solutions I, II and III are mixed so that the mixture is monomeric (I), polymer (II) and high polymer (III) collagen in a ratio of 45: 45: 10 contains.

The proline content after hydrolysis is 16.4% based on the total protein content.

Leave the collagen products obtained according to Examples 1, 2 and 3 can be used directly for medical and cosmetic purposes. Further examples for medicinal and cosmetic compositions are the following: A. 100 ml Solution according to Example 1 0.19 g L-proline 1.0 g neomycin sulfate B. 100 ml solution according to Example 2 0.22 g L-proline 0.5 g glycine 0.5 g D, L-threonine 0.5 g L-cysteine 1.0 g Neomycin sulfate C. 100 ml solution according to Example 3 0.23 g L-proline 0.5 g glycine 0.5 g D, L-threonine 0.5 g L-cysteine 0.1 g ascorbic acid 1.0 g Zn salt 0.5 g neomycin sulfate 0.5 g bacitracin

Claims (9)

Claims t. Product available from collagen, characterized that there are 25-50 parts monomeric, 25-50 parts polymeric and 0-40 parts high polymer Contains collagen in a buffer. 2. Product according to claim 1, characterized in that it is additionally Contains proline, so that the total proline content is up to 40%. 3. A method for producing the product according to claim 1, characterized characterized in that a) collagen in an alkali salt solution containing a hexose or Contains glycerin, stirred at low temperature and the insoluble components separated off, a solution I containing monomeric collagen and a residue A receives, b) the residue A in 0.01-1.0 molar acid with the addition of 0.5 to 3% qt-ketoglutaric acid is stirred at room temperature and then the insoluble Separating constituents, a supernatant II containing polymeric collagen, and a residue B is obtained, c) the residue B in an aqueous solution which is 0.2 contains up to 8 moles of urethane and optionally 0.3 to 8 moles of urea per liter, or in Tris buffer pH 4.0 to 8.0), which contains 0.05 to 0.5 mol / l calcium chloride, stirred and centrifuged to obtain the high polymer collagen in supernatant III, and d) the supernatants I, II and III separated by dialysis against water or with Sodium chloride precipitates, the precipitates individually in a physiological way tolerable Buffer (pH 2-7) suspended and the solutions mixed so that the ratio of Components I, II and III to each other (25 to 50): (25 to 50): t 0 to 40) amounts to. 4. The method according to claim 3, characterized in that one to adding proline to the solution obtained, so that the proline content is up to 40%, preferably 30%, based on the total content of amino acids after hydrolysis. 5. The method according to claim 3, characterized in that one to of the solution obtained 0.1-5%, preferably 1%, glycine and / or cysteine and / or Threonine adds. 6. The method according to claim 3, characterized in that one to 0.01 to 2% zinc and / or copper and / or iron salts are added to the resulting solution. 7. The method according to claim 3, characterized in that to the solution obtained 0.5-2%, preferably 1%, antibiotics such as neomycin sulfate or adding bacitracin. 8. Medicaments containing products according to claim 1 or 2. 9. Cosmetic agents containing products according to claim 1 or 2.