BE1004915A3 - COLLAGEN, PROCESS FOR PREPARING THE SAME, AND COSMETIC AGENT CONTAINING THE SAME. - Google Patents

Abstract

The invention relates to a collagen comprising sulfoxidized methionine residues, a process for preparing such collagen and a cosmetic agent containing such collagen. To prepare this collagen, a material containing collagen is subjected to an enzymatic or chemical treatment in the presence of an oxidant, in particular in the presence of hydrogen peroxide. The collagens of the invention are remarkably compatible with the skin and do not cause allergic reactions. They can therefore be used in cosmetic agents, and in particular in cleaning products.

Description

COLLAGEN. PROCESS FOR THE PREPARATIONS. AND COSMETIC AGENT

CONTAINING

The invention relates to a collagen in which the methionine residues are at least partially sulfoxidized, a process for preparing it and a cosmetic agent containing it.

Collagen plays a very important role in the connective tissue of mammals. It represents 25 to 30% of all 11 proteins in the body. It is found in particular in the skin, tendons, ligaments, blood vessels, cartilage and bones.

Studies have shown that collagen is not a single compound. So far, at least twelve different types have been characterized. These various types of collagen, which have different properties and which have biological functions which are also different in the organism, are characterized in particular by their amino acid compositions, their amino acid sequences, the disulfide bridges between the chains , the pitch of the triple helix, and their behavior in an electric field. By immunolocation using monoclonal antibodies, it was also possible to study their distribution in the various tissues. This is how, in the skin, we mainly find types I and III of collagen. These two types of collagen have very similar amino acid compositions.

Due to its amino acid composition, collagen occupies a special place among proteins. It is particularly rich in glycocolle. In addition, it is the only protein that contains the amino acid hydroxyproline. Its sulfur content is relatively low since, for example, type I collagen does not contain cysteine, and methionine is the only other sulfur amino acid.

It is in the form of procollagen that the collagen molecules are synthesized in fibroblasts and brought into intercellular voids. This is where the transformation into collagen takes place, by degradation of the procollagen. By crosslinking between the various collagen molecules, insoluble collagen is finally formed, such as is found mainly in aged connective tissue. As a general rule, collagen consists of three polypeptide chains (molecular mass of approximately 100,000 for each chain), which, taken in isolation, each form a helix with left pitch, and wind together in a triple helix with right pitch. The two ends of the molecule are made up of globular telopeptides. These telopeptides play an important role in the natural crosslinking of collagen molecules. They can easily be eliminated enzymatically (for example using pepsin or trypsin). The biochemistry, biology, biosynthesis and metabolism of collagen are described in many scientific works and journal articles, for example in the following:

Berg, A.; Einsatz von Proteinen in Kosmetika (Use of proteins in cosmetics); Parfümerie und Kosmetik 65 (7), 391 - 401 (1984)

Hein, R., Nehrlich, A., Müller, P., Krieg, T.; Angeborene Erkrankungen des Kollagens. Klinische Heterogenität und molekulare Defekte (Congenital collagen disorders) (Clinical heterogeneity and molecular defects); Internist 26, 420-428 (1985)

Krieg, T., Hein, R., Hatamochi, H., Aumailley, M.; Molecular and clinical aspects of connective tissue; Eur. J. Clin. Invest. 18 (2), 105-123 (1988)

Lindner, H.; Kollagen in der Kosmetik (Collagen in cosmetics); Parfümerie und Kosmetik 65 (6), 340-346 (1984)

Mayne, B.; Preparation and applications of monoclonal antibodies to different collagen types (Preparation and applications of monoclonal antibodies for various types of collagen); Clin. Biochera. 21 (2), 111-115 (1988)

Mayne, R., Burgeson, R.E. (Eds.); Biology of extracellular matrix: Structure and function of collagen types (Biology of extracellular matrix: Structure and function of types of collagen); Academie Press (1987) Nimni, M.E.; Collagen: Structure, function and biomaterial properties (Collagen: Structure, function and properties as a biomaterial); NATO ASI, Ser. E 116, 365-383 (1986)

Parkany M. ƒ Polymers natural origin as biomaterials. 2. Collagen and gelatin (Natural polymers as biomaterials. 2. Collagen and gelatin); in: Macromol. Biomater. ; G.W. Hastings, P. Ducheyne (Eds.); CRC, Boca Raton, Florida; 111-117 (1984)

Piez, K. A.; Collagen (Collagen); Encycl. Polym. Sei. Eng. 3, 699-727 (1985)

Prockop, D.J., Kivirikko, K.I. ; Heritable diseases of collagen (Hereditary diseases of collagen); New Engl. J. Med. 311 (6), 376-386 (1984)

Reference may also be made to the publications which are cited as references in these articles.

Collagen has many advantageous properties. Due to its high water binding capacity, it plays an important role in regulating the water content of the skin. It also exerts softening and calming actions. It stimulates the growth of fibroblasts and accelerates healing. In addition, it also has hemostatic properties.

Due to its advantageous properties and its very good possibilities of adaptation, collagen is currently used in medicine in a large number of indications (for example suture material, implant material, wound dressing, haemorrhage stop).

Collagen can be extracted from different sources of collagen, for use in cosmetics. It is mainly extracted from the connective tissue of young animals or animal embryos. Calfskin is preferably used, the collagen of which is hardly distinguished from a physiological point of view from human collagen. Collagen from calf skin is made up of at least 90% collagen type I, and it also contains a small amount of collagen type III. It is used either without having modified it, in the form of native soluble collagen, or after having modified it by enzymatic way, in the form of atelocollagen or hydrolyzate of collagen, or else after having modified it by way chemical, in the form of deamidocollagen, collagen methyl ester, succinyl-collagen or guanidino-collagen.

The processes for extracting collagen and for enzymatic or chemical modification of collagen are known and described in a large number of publications and patents. But it is important, during these operations, to avoid denaturation of the helical structure of collagen. It is also necessary to operate under the most sterile conditions possible. In this regard, reference may be made to the review article by Light, N.D.,; Collagen in skin:

Preaparation and analysis (Collagen in the skin: preparation and analysis), in "Methods in skin research"; (D. Skerrow, CJ Skerrow Eds.), John Wiley and Sons Ltd., 559-586 (1985), as well as in patents DE - 20 64 604 and 26 16 939, in European Patent Applications N ° 0 052 288 , 0 083 868, 0 124 659, 0 132 979, 0 214 035, 0 224 453, 0 233 770 and 0 284 789, and to US Patents Nos. 4 404 033, 4 582 640 and 4 687 518; reference may also be made to the publications indicated in this article and in these patents.

Application DE-20 64 604, as well as the corresponding US patent No. 3,991,184, describes skin care products which contain native soluble collagen, having an unmodified collagen structure and very little crosslinked. Native soluble collagen is obtained from the skin of young animals or animal embryos, by extraction in a weakly acidic aqueous medium, at low temperature. The molecular mass of the collagen thus obtained is from 5000 to 50,000.

The object of the invention is to prepare cosmetic active substances, based on collagen, the modes of action of which are well defined and which have no undesirable side effects. In addition, their preparation must be simple and economically feasible.

It has now been found that collagen comprising methionine sulfoxide residues has advantageous cosmetic properties.

The object of the invention consists of collagens which are characterized in that at least part of the methionine residues contained in the collagen is in the form of methionine sulfoxide. In addition, the collagen derivatives according to the invention are not altered with respect to the respective collagens used as starting materials. The collagen derivatives according to the invention are practically no longer sensitive to oxygen, since their sulfur atoms are already oxidized. In addition, compared to the initial collagens, they exhibit better solubility in water, in particular in acidic aqueous media, so that the collagen derivatives according to the invention can be incorporated in a higher concentration in the products of application.

The collagen derivatives according to the invention are remarkably compatible with the skin: no allergic reactions have been observed. They form a protective layer on the skin and hair.

The preparation of the collagen derivatives according to the invention can be carried out from all the usual sources of collagen. Calf skin is preferably used, the collagen of which is hardly distinguished from human collagen, from a physiological point of view, as well as native soluble collagen, collagen consisting, for at least 90%, of type I collagen, collagen type I or III, 1 · atelocollagen or deamidocollagen. Intermediate products from the collagen processing industry can also be used.

The collagen preparation according to the invention is carried out by treating a material containing collagen (for example calfskin or intermediate products from the collagen processing industry) in the presence of an oxidant, in order to obtain therefrom collagen. If one wishes to carry out the preparation of a collagen derivative, the formation of this derivative is carried out at the same time. This is how, to prepare an atelocollagen from calf skins, the enzymatic degradation is carried out by proteases, for example trypsin or pepsin, in the presence of an oxidant, so that an atelocollagen is obtained sulfoxide. Similarly, to prepare a deamidocollagen, deamidation is carried out in the presence of an oxidant, so that a deamidocollagen sulfoxide is obtained.

It is also possible to prepare the collagen according to the invention from isolated collagen, or from collagens, from an isolated derivative, by treating this derivative with an appropriate oxidant.

Suitable oxidants are those which cause sulfoxidation of the sulfur atoms of the methionine residues contained in the collagen, without otherwise modifying this collagen. Examples of such oxidants are hydrogen peroxide, peroxides of alkali metals such as sodium peroxide, sodium perborate, peracids and peresters; hypohalites, such as alkaline hypohalites, in particular an alkaline hypochlorite, for example sodium hypochlorite, and compounds which release hypohalites in situ; finally chloramines, for example chloramine-T. This oxidant is used in an amount of 1.5 to 2.0 equivalents, in particular 1.6 to 1.9 equivalents, per equivalent of methionine. If necessary, the excess oxidant is destroyed at the end of the reaction in the usual way, by addition of a reducing agent, or else eliminated by salting out of the collagen with NaCl and redissolution using a citrate buffer.

The reaction is preferably carried out in an acid medium, in particular at pH 2-5, better still at pH 3-4. It is advantageous to add a buffer, for example a citrate buffer, to keep the pH value within the desired range.

The reaction temperature is preferably in the range from 0 to 20 ° C, in particular from 10 to 20 ° C, and better still from 15 to 17 ° C. In general, the oxidant is left to act on the collagen for at least 1 day, in particular for 2 to 6 days, and better still for 3 to 4 days.

Preferably, the material containing collagen is treated beforehand with the oxidant, for example by spraying a solution of the oxidant or immersion in this solution. Advantageously, from 0.05 to 0.5 equivalent of oxidant is used per kilo of material containing collagen (collagen content of approximately 30%), in aqueous solution at 0.05-0.5% by weight. Preferably, this preliminary treatment lasts from 1 to 10 hours, and in particular from 2 to 5 hours. Preferably, one operates at a temperature situated in the range from 20 to 50 ° C., in particular from 30 to 50 ° C., or else the material containing the collagen is sprayed, in the frozen state, with the oxidant and it is let thaw.

Under the specified conditions indicated, the sulfoxidation of the methionine residues in the collagen is carried out, without the corresponding sulfone functions then being formed. The yield is from 91 to 96%, relative to the collagen used. This sulfoxidation is reversible: for example, the derivatives according to the invention can be transformed again into the starting materials, by reaction with mercaptoethanol.

Collagens generally contain 5 to 6 methionine residues per 1000 amino acid residues. In the collagens in accordance with the invention, from 2 to 5, and preferably from 2 to 3 of these methionine residues are sulfoxides (see the table below).

The collagens according to the invention have a molecular mass of approximately 270,000 to approximately 300,000 (determined by electrophoresis on SDS-polyacrylamide gel, under the conditions indicated by U. K. Laemmli, Nature 227, 680-685 (1970)).

For further characterization and estimation of the purity of collagens, the usual biochemical and physicochemical methods can be applied, for example HPLC, SDS-polyacrylamide gel electrophoresis, amino acid analysis, nitrogen analysis, mapping techniques peptides, cleavage by cyanogen bromide, optical rotary dispersion. In particular, one can determine, among other things, the content of hydroxyproline, the amino acid characteristic of collagen. The proportion of this is 14% in collagen from the skin (Nimni, MEf (2986), Nato Asi, Ser. E. 116. 365-383).

In the process according to the invention, the products are in the form of an aqueous solution. This solution can be transformed as it is, optionally after concentration, into a cosmetic agent. The solutions are practically sterile, due to the use of the oxidant, so that it may be possible to dispense with incorporating a preservative therein.

From the solutions indicated, the collagen derivatives according to the invention can be obtained in the usual way, for example by spray drying, lyophilization, etc. The solid products obtained in this way can also be transformed into cosmetic agents.

According to a particularly preferred method, one starts from slaughtered calf skins. The skins used are preferably frozen and disinfected, for example using chloramine T.

The skins are then reduced to pieces, in the thawed state or preferably in the frozen state. It is during this grinding or following it that the skins undergo the preliminary treatment with the oxidant used according to the invention, under the conditions indicated above.

The skin pieces that have been pretreated are then minced in a meat grinder. The mince obtained is then treated with the oxidant under the conditions indicated above. Preferably, the mince is subjected to an extraction with a citrate buffer, at 15-17 ° C., with stirring. It is then particularly advantageous to keep the concentration of the oxidant, in particular hydrogen peroxide, constant during the extraction step, and for example at a SO value at 120 ppm, in particular from 90 to 110 ppm.

The porridge of skins thus obtained is then subjected to filtration through a cotton filter cloth filter bag folded in double. If desired, the excess oxidant is destroyed in the usual manner. The product thus obtained has a molecular mass of approximately 295,000 and gives the gel electrophoresis chromatogram shown in FIG. 1. The amino acid analysis of this product gives the results indicated in the table given below.

The invention also relates to cosmetic products, which contain collagen derivatives according to the invention. These cosmetic products are in particular maintenance products, which can be in the form of cream, mask, grouped products, lotion, gel, milk, etc. To this end, the collagen derivatives in accordance with the invention are incorporated into usual cosmetic supports and auxiliaries.

Suitable carriers are, for example, oils, such as petrolatum oil, avocado oil, paraffin oil and the like. The auxiliaries which can be used are, for example, emulsifiers, such as mixtures of oils and / or fatty alcohols or polyethoxylated alcohols, soaps and the like; thickeners, such as sodium alginate, gum arabic, xanthan gum, cellulose derivatives and the like; propellants for aerosol formulations, such as carbon dioxide and nitrogen; solvents, such as alcohols and the like.

The cosmetic products in accordance with the invention may also contain constituents usually incorporated in products of this type. Among these constituents, mention may be made, for example, of perfumes, dyes, preservatives, antioxidants, sequestrants, plasticizers, emulsifiers and the like.

The cosmetic products in accordance with the invention may also contain cosmetically active adjuvants. Among these, mention may be made, for example, of hydrating agents, carotenoids, stabilizers, humidity regulating agents, pH regulating agents, agents screening out UV-A and UV-B rays, and the like.

The cosmetic products in accordance with the invention generally contain from approximately 0.01 to 5%, preferably from approximately 0.1 to 2% by weight of collagen derivative, relative to the total weight of the cosmetic product.

The following examples illustrate the preparation of the active products in accordance with the invention.

Example 1 a) 250 kg of frozen calf skins (masks, disinfected with chloramine T) are coarsely ground, in the frozen state, and simultaneously sprayed with a solution of 2500 ml of hydrogen peroxide (30%) in 500 1 of water. The pieces are then left to thaw, for about 2 to 3 hours. The water which has collected is removed, and the pieces are possibly washed once again with 500 l of water. After having separated them from the water, the pieces of skin are passed through a chopper.

In a 400 L container, 100 L of a 0.128 M citrate buffer solution (pH 3.6), at 15-17 ° C, and 400 ml of hydrogen peroxide (30%) are available. 50 kg of skins passed through the mincer are then added. After 30 minutes of stirring at 15-17 ° c, the concentration of hydrogen peroxide is measured. By adding an additional amount of hydrogen peroxide, the concentration of hydrogen peroxide is maintained at about 100 ppm. Thanks to this procedure, solutions are obtained which contain from 0.4 to 0.8% of dissolved collagen, that is to say from 600 g to 1200 g per charge. The consistency of the hydrogen peroxide concentration, here 100 ppm, corresponding to 40 ml per 150 l of charge, is of crucial importance for the collagen which goes into solution, and which has not yet reacted in the heterogeneous phase, can transform into a collagen comprising oxidized tallow methionine residues. In addition, this concentration of hydrogen peroxide provides an optimal bacteriostatic medium. The suspension is stirred for 3 days at 15-17 ° C., then it is diluted with 100 l of buffer and stirred for 24 h.

By centrifugation at 10,000 rpm, a solution of native, slightly opalescent soluble collagen is obtained, which is combined with other extracts, so that the solution contains 0.225% of soluble collagen (300 Mg of hydroxyproline / ml ). The excess hydrogen peroxide is destroyed according to known methods.

Alternatively, to remove the hydrogen peroxide, sodium chloride is added to the centrifuge, until the sodium chloride concentration is 6% by weight, the precipitated fibrils are separated by centrifugation, and finally puts them in solution in 0.128 M citrate buffer.

Storage is carried out with usual preservatives, for example Phenonip (mixture of p-hydroxybenzoate esters with phenoxyethanol).

The molecular mass of the sulfurized collagen, determined by electrophoresis on polyacrylamide-SDS gel, is approximately 295,000.

The product is characterized by the fact that, during the analysis of amino acids, methionine sulfoxide appears in the elution diagram before the aspartic acid, and that all the less methionine is detectable further on.

For the amino acid analysis, an aliquot of the solution is removed and sodium chloride is added thereto, until the solution contains 6% by weight of sodium chloride. The precipitated collagen is separated by centrifugation, dialyzed three times against 0.1 M acetic acid, then lyophilized.

For the amino acid analysis, 5 mg of lyophilized collagen is hydrolyzed in 10 ml of 6N HCl, under a nitrogen atmosphere, for 2 hours, at 110 ± 1 ° C. Excess hydrochloric acid is removed in vacuo, the residue is taken up in an elution buffer, and the amino acids are assayed by ion exchange chromatography.

TABLE 1

Amino acid composition, number of residues per 1000

b) Under the reaction conditions indicated above, it is possible to transform native soluble collagen into a corresponding collagen, comprising sulphoxidized residues of methionine (1000 g of native soluble collagen in 100 1 of 0.128 M citrate buffer (pH 3.6 ) and 400 ml of hydrogen peroxide; the treatment conditions and the temperature are those indicated in Example 1).

Example 2 a) The skin residues obtained according to Example 1, or alternatively fresh chopped skin, can be reacted, in the presence of hydrogen peroxide, as described in Example 1, with enzymes such as trypine or pepsin (see the references indicated above), to obtain an atelocollagen containing sulphoxidized methionine residues. The molecular mass of the product obtained, determined by electrophoresis on SDS polyacrylamide gel, is approximately 270,000, and its amino acid composition is shown in Table 1.

b) In an analogous manner and under the reaction conditions mentioned in Example 1b, a pure atelocollagen can be transformed into an atelocollagen comprising residues of methionine sulfoxides.

Example 3 a) Skin residues obtained in Example 1, or alternatively fresh chopped skin, can be reacted in the presence of hydrogen peroxide, as described in Example 1, with alkalis or acids (see the references indicated above) to obtain a deamidocollagen comprising residues of methionine sulfoxides. The molecular mass of the product obtained, determined by electrophoresis on polyacrylamide-SDS gel, is approximately 295,000, and its amino acid composition is given in Table 1.

b) in a similar manner and under the reaction conditions mentioned in Example 1b, it is possible to convert pure deamidocollagen into deamidocollagen comprising sulphoxidized methionine residues.

FIG. 1 represents the electrophoresis chromatograms on polyacrylamide-SDS gel, for the products obtained according to examples 1 to 3. This electrophoresis was carried out on a 7% gel, under the conditions indicated by UK Laemmli in Nature, 227 , 680-685 (1970). Under these conditions, a partial degradation of the collagen triple helix occurs, so that the chromatogram reveals chains there having a molecular mass of approximately 300 kD, chains B having a molecular mass of approximately 200 kD and a chains having a molecular weight of about 100 kD. Proteolytic degradation products are not detectable.

In the following examples, the collagen derivatives of Examples 1 to 3 can be used in the form of the solution obtained or in the form of solid products.

Example 4

Skin cream: collagen derivative 0.1 g polyoxyethylene cetyl ether 1.5 g cetyl alcohol 2 g petroleum jelly oil 6 g avocado oil 4 g lanolin 4 g perfume, as much as necessary sterile water, supplement to 100 g

Example 5

Body milk: collagen derivative 1 g paraffin oil 5 g petroleum jelly oil 7 g preservative 0.15 g triethanolamine stearate 5 g stearic acid 3 g sterile water, supplement to 100 g

Example 6

Skin lotion: collagen derivative 0.5 g preservative 0.15 g polyvinylpyrrolidone 3 g ethanol 20 g sterile water, supplement to 100 g

Example 7 skin gel: collagen derivative 0.5 g preservative 0.15 g ethanol 40 g propylene glycol 42 g polymerized acrylic acid (Carbopol 240, from the firm Goodrich Chemical Co.) 1 g sterile water, complement to 100 g

Claims (12)

1. Collagen, characterized in that at least part of the methionine residues contained in this collagen is in the form of methionine sulfoxide. 2. Collagen according to claim 1, characterized in that it is derived from a collagen chosen from a native soluble collagen, an atelocollagen, a deamidocollagen and a collagen consisting, for at least 90%, of type I collagen. 3. Collagen according to claim 1 or 2, characterized in that, for 1000 amino acid residues in this collagen, from 2 to 5, in particular 2 or 3 methionine residues are sulfoxidized. 4. Method for preparing a collagen according to claim 1, characterized in that a material containing collagen is transformed, in the presence of an appropriate oxidant, into a collagen or a derivative of collagen, or else in that isolated collagen or an isolated collagen derivative is treated with an appropriate oxidant. 5. Method according to claim 4, characterized in that one uses, as oxidant, hydrogen peroxide or an alkali metal peroxide. 6. Method according to claim 4 or 5, characterized in that the oxidant is used in an amount of 1.5 to 2.0 equivalents, in particular 1.6 to 1.9 equivalents, per equivalent of methionine . 7. Method according to one of claims 4 to 6, characterized in that one uses, as starting material, calf skin, a native soluble collagen, an atelocollagen or a deamidocollagen. 8. Method according to one of claims 4 to 7, characterized in that one operates in an acid medium, in particular at a pH of 2 to 5, in particular from 3 to 4. 9. Method according to one of claims 4 to 8, characterized in that one operates at a temperature in the range from 0 to 20eC, and in particular from 10 to 20eC. 10. Method according to one of claims 4 to 9, characterized in that the material containing collagen is treated beforehand with the oxidant. 11. Method according to claim 10, characterized in that, before the preliminary treatment, the material containing collagen is reduced into small pieces, in the frozen state. 12. Cosmetic agent, containing at least one collagen according to one of claims 1 to 3 or a collagen obtained according to one of claims 4 to 11.