DE2507223B2 - COLLAGEN PRODUCT AND METHOD FOR MANUFACTURING THE COLLAGEN PRODUCT - Google Patents

Description

The invention relates to a collagen product, the process for its production and medicaments and cosmetic products containing this product.

Collagen is a protein, the properties of which are for the most part known (T raub, W. and Pie, K A. in Avanc. ProtChem., 25, 243, Academic Press, New York [1971]; Viidik, A. Intern Rev. Conn. Tissue Res, 6, 127, Academic Press, New York (1973); Ramachandran, G.N _M and Hodge, AG in "Treatise on Collagen", Vol. 1, pp. 103 and 185, Academic Press , London [1967]). Attempts have been made to use 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 ap chains, 1 a ₂ chain), which are linked in a helix-like manner and which are held together by non-covalent bonds. This collagen is referred to below as "monomeric collagen". Some or a whole series of "molecules" of the monomeric collagen can bond with one another through covalent bonds, whereby so-called "polymeric collagen" (containing 2 to about 4 collagen units) and "high polymer") collagen "(containing more than 4 collagen units) Units).

It has now been found that in order to get a good effect with collagen it is crucial What matters is that in the collagen-containing preparations a certain ratio of monomeric to polymeric to high polymer collagen is adhered to.

The invention relates to a method for producing a collagen product which consists in that he

a) Collagen in an alkali salt solution containing a hexose or glycerine, stirred at low temperature and the insoluble constituents separated off, a solution I containing monomeric collagen and a residue A being obtained,

b) the residue A is stirred in 0.01 to 1 molar acid with the addition of 0.5 to 3% a-ketoglutaric acid at room temperature and then the insoluble constituents are separated off, a supernatant II containing polymeric collagen and a Residue B receives,

c) the residue B in an aqueous solution containing 0.2 to 8 mol urethane and optionally 0.3 to 8 mol urea pno liter, or in Tris buffer (pH 4.0 to 8.0) which is 0.05 to Contains 0.5 mol / l calcium chloride, stirred and centrifuged to obtain the high polymer collagen in supernatant III, and

d) the solution I and the supernatants II and III separated by dialysis against water or by saturation with sodium chloride precipitates, the precipitates individually in a physiologically compatible buffer (pH 2 to 7) suspended, and the solutions so

mixes that the ratio of the ingredients 1.11 and IH to each other (25 to 50): (25 to 50): (0 to 40) contributes, and then adds proline so that the Proline content up to 40% based on the total content of amino acids after hydrolysis amounts to

The invention also relates to the product obtained by the process formed and to medicaments and cosmetic agents consisting of the new collagen product as well as usual known additives exist.

Commercially available collagen can be used as the starting material for the manufacture of the product will. This is always a mixture of monomeric, polymeric and high-polymer collagen. Preferably a collagen with the highest possible proportion of monomers, that from young animals, is suitable can be won. In the case of collagen from old animals, the proportion of monomer can be so low that one Working up by the process according to the invention becomes uneconomical.

It can be useful to purify the collagen used as the starting material subject before working it up according to the invention. The cleaning can be done in that one from the collagens impurities z. B. with a saline solution that contains a complexing agent, extracted.

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, there is no upper limit.

Particularly suitable hexoses are glucose, fructose and galactose, which are preferably in one Amount of 0.05 to 0.5 mol / l can be used. Suitable acids for step b) are, for. B. Sulfuric acid, phosphoric acid, formic acid, succinic acid and oxalic acid. Are particularly suitable Acetic acid and lactic acid. For step c) urethanes come 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 additionally use urea in an amount of 0.3-8.0, preferably 0.5-1.5 mol / l to add to the urethane solution. The buffer used for d) should have a pH of 2 to 7 and 0.01 to l omolar. All suitable salts and acids can be used for its preparation are physiologically compatible. Acetate and citrate buffers with a pH of 3.5 are particularly suitable to 6.0.

It is advisable to add an antibiotic to the solution obtained in order to increase the shelf life raise. There can also be other substances such. B. Vitamins (ascorbic acid), amino acids (glycine, D, L-threonine, L-cysteine) or Cu, Zn and iron salts can be added.

The new product made from collagen is very suitable for the treatment of open wounds because it has the Strongly stimulates wound granulation. It is also used for cosmetic purposes because of its moisturizing properties and reparative effect well usable.

example 1
I. Pre-purification of the collagen

6 kg of collagen are dissolved in 201 deionized water, in the 4% EDTA (800 g) and 1% NaCl (200 g) are dissolved,

stirred at 4 ° C. for 24 hours, followed by centrifugation The supernatant is discarded.

II. Manufacture of the end product

a) The pre-purified collagen (5.5 kg) is in 1001 Water in which 1 kg of glucose and 1.5 kg of NaCl are dissolved, stirred for 24 hours at 4 ° C and then centrifuged The supernatant (I) is saved (801).

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 (H) is saved (351).

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 ethyluretcan are dissolved, 24 hours at 4 ° C touched. Then it is centrifuged again and the supernatant (HI) (20 1) is saved. The residue (400 g) is discarded.

d) The solution I and the supernatants 1, II and III are dialyzed separately against water (40 Hours at 15 ° C). The failed collagen fibers are separated off, taken up in 0.1 M citrate buffer (pH 4.0) at 4 ° C. (I in 45 1, II in 15 1, III in 16.51) and centrifuged.

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

Example 2

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

The yield was the same as in Example 1.

Example 3

a) 5 kg of collagen are in 40 l of water, in which 1.136 kg of disodium hydrogen phosphate and 4 kg of glycerol are dissolved, stirred at 8 ° C for 24 hours and centrifuged. The supernatant I is saved (30 1).

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

c) The centrifugation residue (2.2 kg) is dissolved in 25 l of water in which 9 kg of ethyl urethane were previously dissolved were stirred at 4 ° C for 24 hours. It is then centrifuged and the supernatant III stored (20 1).

d) The supernatants I (301), II (201) and III (201) become separately saturated with NaCl and left to stand at 4 ° C overnight. The failed collagens are individually filtered, washed with small amounts of water, weighed and each 51 0.1 M Dissolved citrate buffer (pH 4.5). The solutions 1, II and III are mixed so that the mixture contains monomeric (I), polymeric (II) and high polymer (111) collagen in a ratio of 45: 45: 10.

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

The collagen products obtained according to Examples 1, 2 and 3 can be used directly for medical purposes

and use cosmetic purposes. Further examples of medical and cosmetic compositions are the following:

A. 100 ml of solution according to Example 1

0.19 g L-proline 1.0 g neomycin sulfate

B. 100 ml of solution according to Example 2

0.22 g L-proline 0.5 g glycine C5g D, L-threonine 0.5 g L-cysteine 1.0 g neomytine sulfate

C. 100 ml solution according to example 3

o, 23 g L-proline 0.5 g glycine 0.5 g D, L-threonine

0.1 g of ascorbic acid

1.0 g Zn salt

0.5 g neomycin sulfate

0.5g bacitracin

Claims (4)

Claims: 25 223 1. A method for producing a collagen product, characterized in that a) collagen into one. Alkali salt solution, which contains a hexose or glycerin, is stirred at low temperature and the insoluble constituents are separated off, whereby a solution 1, which mainly contains monomeric solvent, and a residue A are obtained, b) the residue A is stirred in 0.01 to 1 molar acid, in particular acetic or lactic acid, with the addition of 0.5 to 3% a-ketoglutaric acid at room temperature and then the insoluble constituents are separated off, a supernatant II, which contains polymeric collagen, and contains a residue B , 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 liter, or in Tris buffer (pH 4.0 to 8.0), which is 0.05 to Contains 0.5 mol / l calcium chloride, stirred and centrifuged to obtain the high polymer collagen in supernatant III, and d) the solution I and the supernatants II and III separated by dialysis against water or by saturation with sodium chloride precipitates, the precipitates individually in a physiological way compatible buffer (pH 2 to 7) and the solutions are mixed so that the ratio of components I, II and III to one another (25 to 50): (25 to 50): (0 to 40), and then proline is added, so that the proline content is up to 40%, based on the total content of amino acids after hydrolysis. 2. Collagen product obtained by a process in which one a) Collagen in an alkali salt solution containing a hexose or glycerine, stirred at low temperature and the insoluble constituents separated off, a solution I, which mainly contains monomeric collagen, and a residue A being obtained, b) the residue A is stirred in 0.01 to 1 molar acid, in particular acetic or lactic acid, with the addition of 0.5 to 3% -ketoglutaric acid at room temperature and then the insoluble constituents are separated off, a supernatant II, which contains polymeric collagen, and contains a residue B , 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 liter, or in Tris buffer (pH 4.0 to 8.0), which is 0.05 to Contains 0.5 mol / l calcium chloride, stirred and centrifuged to obtain the high polymer collagen in supernatant III, and d) the solution I and the supernatants II and III separated by dialysis against water or by saturation with sodium chloride precipitates, the precipitates individually in a physiological way compatible buffer (pH 2 to 7) and the solutions are mixed so that the ratio of components I, II and IU to one another (25 to 50): (25 to 50) :( 0 to 40), and then proline is added, so that the proline content is up to 40%, based on the total amount of amino acids after hydrolysis 3. Medicament, consisting of the collagen product according to claim 2 and customary known ones Additives. 4. Cosmetic agent consisting of the collagen product according to claim 2 and the usual known additives.