JPS6128645B2 - - Google Patents

Description

[Detailed description of the invention]

TECHNICAL FIELD The present invention relates to a skin protective agent having a reactivating effect. As is well known, the main aging phenomenon of the skin occurs in the form of tissue remodeling in the connective tissue (dermis). The main component of protective and connective tissue is the so-called collagen, which is used as an umbrella name for the fibrous scleroprotein group of proteins. During the aging process of the skin, collagen-containing fibers change their structure in such a way that the initially uncrosslinked fibers undergo inter- or intramolecular lateral cross-linking, which increases with age. Due to significant cross-linking of the fibers, the water binding power of the skin decreases, so the skin loses its swelling properties and
Becomes inelastic. Skin ages and has a tendency to form increased wrinkles. It is known from German Patent No. 1 194 098 that certain properties of gelatin derived from collagen can be used as cosmetic preparations to improve the swelling of the skin. With this preparation the water binding capacity of the skin is improved. Gelatin is obtained by acidic hydrolysis of animal skin. Finally, a sufficient increase in the elasticity of the skin is the aim of German Published Patent Application No. 2064604, and for this purpose skin protective agents are described therein.
It contains soluble natural collagen with a fully uncrosslinked and unaltered collagen structure. Soluble natural collagen is obtained by direct extraction of connective tissue from the skin of young and/or fetal animals. The extraction is carried out in a mild manner in a weakly acidic aqueous medium with the addition of organic acids and organic salts at low temperatures, so that the structure of the collagen remains unchanged. The use of this substance as a mixed component of a skin protectant can reverse the depletion of soluble collagen in the skin or compensate for the loss of soluble collagen. However, tests have shown that neither gelatin obtained from collagen nor collagen extracted by acid treatment are unsuitable for producing the desired effect. This is because acid-soluble collagen and collagen gelatin already have a certain degree of aging. In addition, acid-soluble collagen is generally only a very small component in natural collagen tissue.
It has therefore been established that, at least with this method, it is not possible to compensate for the loss of soluble collagen in the skin. Now, it has been found that the neutral salt-soluble precursors of natural collagen are suitable in an excellent manner for cosmetic and skin protection purposes both in pure form and as a mixed component. As is well known, collagen formation takes place in fibroblasts. The basic constituent is the so-called tropocollagen, which polymerizes over time to form natural collagen. Newly formed tropocollagen is considered to be the earliest form of extracellular collagen, and therefore by simultaneously supplementing it with the chemical building blocks for collagen formation, it is no longer possible to form collagen. It is particularly suitable for activating senescent cells that are unable to function. For the experiments carried out, it was established that the connective tissue cells activated within this method are able to give rise to collagen with new functions. The favorable adjustment of the structure of the connective tissue can be further increased if the precursors of natural collagen are subsequently stabilized by the addition of acids. For this purpose, ascorbic acid can preferably be used. This is important in collagen biosynthesis. Finally, mixtures of neutral salt-soluble precursors of natural collagen and water-soluble placental extracts have proven to be particularly preferred. As will be explained in more detail in the examples given below, a 1:1 mixing ratio of extracts already soluble in neutral salts and placental extracts shows an additive positive effect. The placenta extract used is prepared from 3-7 month old placentas. By isotopic methods, the neutral salt soluble extract was demonstrated to be a direct precursor to natural collagen. Furthermore, the solubility of neutral salts decreases with increasing age.The precursors soluble in neutral salts are composed of 90% alpha chains and 10% beta chains, which are later converted in tissues. It has been proven that this is nothing more than natural collagen. Therefore, since natural collagen originates in large units from the constituents of tropocollagen, the newly formed natural collagen already has a somewhat cross-linked fibrous structure. Unlike the precursors, natural collagen is acid soluble to some extent, at least in young connective tissue. Acid solubility decreases significantly as crosslinking increases. Tests have shown that collagen obtained by acid treatment has a very small proportion of alpha chains, and for this reason the acid-soluble fraction is
It can be used to activate connective tissue.
It turned out that good results could not be obtained. Said precursor to natural collagen, then still only the so-called procollagen, is obtained from the connective tissue (skin, tendon, cartilage, bone) of an unborn, newborn or dead mammalian fetus by the treatment of neutral salts. It can be extracted by Examples of such animals include: calves, piglets, camels, elephants, and others. The treatment of neutral salts is the action of NaCl, in which
The concentration of NaCl is preferably 0.01-10 in the buffer solution
It is a mole. Subsequent precipitation is carried out with concentrated NaCl solution or by saturation with ammonium sulphate. Salt-soluble procollagen can be freeze-dried and/or treated with organic acids such as ascorbic acid, citric acid or CH ₃ (CH ₂ ) _o ·COOH [n=0, 1, 2,
3] Maintain the system in a 0.1-0.3N solution of acid. It is desirable to add preservatives and penetrants as soon as possible to begin extraction of the ground animal material. As preservatives, for example toluol or Nipagine are useful. The resulting solution is evaluated based on its protein, hydroxyproline and hydroxylysine content. Kjeldahl measurements give a protein content which is generally present at 0.3-0.6%. The hydroxyproline content of pure procollagen is 13-14%. Next, the production of procollagen extract will be explained with reference to Examples. Example 1 50 kg of ground calf skin was extracted twice for 12-48 hours with 6-11 times the volume of 0.1-0.5M NaCl solution buffered with 0.02M phosphate. The buffer used was
It consisted of KH ₂ PO ₄ −NaOH. After centrifugation - at an initial and final temperature of 0°C - solid NaCl was added at a concentration of 15-22%.
Added up to. After standing and stirring at 0°C,
Centrifuge at 3000-6000 rpm, stir the precipitate in 5-10 times the volume of 0.05M acetic acid containing 0.1% Nipagin preparation, and after further centrifugation at 0°C for a long time, add phosphate buffer. Dialysis was performed against a 0.5M NaCl solution adjusted to pH 7.4. Solid NaCl concentration 15-20%
After addition, the precipitated procollagen was removed by centrifugation, dissolved in 0.1-0.3M citric acid, acetic acid, ascorbic acid, optionally dialyzed several times against the corresponding acid solution, and centrifuged. The appropriate Nipagin formulation, Tween-80 and propylene glycol were added to produce a shelf-stable final product. Analysis yielded 0.6% N and theoretical hydroxyproline. Example 2 After removing blood, meat, and fat, grind 20 kg of fetal pig skin in a meat grinder and grind 10% of the 5 times the amount.
Extracted with NaCl solution for 4 times at 3°C. Triol or Nipagin was added to prevent bacterial action. After centrifugation, the solution was saturated with 55-75% ammonium sulfate depending on temperature, and the precipitate was removed by centrifugation. The precipitate, which can be removed at the bottom or top of the centrifuge depending on the density of the solution, was washed with water, further centrifuged and dissolved in 0.5M acetic acid, citric acid. After centrifugation, it was dialyzed against 0.5M-NaCl. All operations are carried out at as low a temperature as possible.
Procollagen is precipitated by adding 10-13% NaCl and subsequently dissolved in a 0.1-0.4M organic acid solution containing the following components ascorbic acid, Nipagin preparation, Tween-80 and propylene glycol;
Centrifuge. Analysis value: Protein in collagen
0.4% and hydroxyproline 14%. Example 3 In utero mammalian connective tissue is extracted with 0.5M NaCl solution at PH7.1 and 1° C. for 5 times for 15-42 hours and centrifuged. After addition of NaCl, further centrifugation
Dissolve the residue in 0.05M organic acid. Acid solution is 0.1M
- Prepared by adding the calculated amount of acid to the NaCl solution, which is used for the second extraction of connective tissue at PH 7.1. This extract contains large amounts of mucopolysaccharides,
This is important for the water binding capacity of the skin. The product according to the invention is incorporated (oil/water emulsion) into, for example, the following preparations (creams): Example: Lanette N 150 g Cetiol V 100 g Tonsil oil 100 g Glycerin 50 g Water 500 g Procollagen extract 100 g Preservative + aromatic oil example: Lanette N 120g Cetiol V 80g White wax 60g Vaseline 10g Paraffin oil 100g Isolinolate ester 5g Vitamin E 2g Holagiinol oil solution 2g Preservative 2g Water 390g Preservative 2g Glycerin 120g procollagen extract 100g aromatic oil 10g Example: Ceramol 130g tonsil oil (fragrant) 60g spermaceti 10g cetyl/steryl alcohol 40g lanolin 20g preservative 2g glycerin 50g water 635g procollagen extract 50g aromatic oil 3g Example: Lanette N 125g Grape seed oil 100g Oleyl alcohol 10g Preservative 2g Cetyl alcohol 5g Propylene glycol 50g Citric acid 5g Water 605g Placenta extract 50g Procollagen extract 50g As is clear from the examples, samples and procollagen extracts Each sample contained 100 g of sample and 50 g of procollagen extract, with 50 g of placenta extract also being added to the sample. To test the action of the products according to the invention,
A comparative test was conducted according to the table below. At that time, 1 in the table
The sample designated 2 contains water in place of the amount of procollagen extract or the amount of placental extract, and the amount of procollagen extract in the sample designated 2 is greater than the amount of procollagen extract in the acid soluble collagen preparation. I operated it by replacing it. Finally, in the test series designated by 3, the amount of the relevant procollagen extract was replaced by the corresponding amount of collagen gelatin.

【table】

Table: The general function of the skin's capillaries can be determined by measuring the temperature using a medical electronic skin thermometer. In the dry, poorly circulatory skin of test subjects between the ages of 40 and 60, a markedly fluctuating skin temperature is found, corresponding to the blocked capillary action (see column 1 of the table). After 3 weeks of use of the oil/water emulsion ~ and the corresponding comparison sample, the sample with procollagen extract (,,,) only showed a stabilizing effect on the skin, which was due to the change in skin temperature. This was confirmed by stability over several days (see column 3 of the table). On the contrary, the corresponding comparison samples _1-3 , _1
-3 , ₁ and ₁ showed almost no stabilizing effect. After use, significant fluctuations in the temperature of the skin are still observed, indicating that no improvement in the action of the skin capillaries occurs. All samples were tested on 40 subjects each.

【table】

[Table] N (1)−(3)
(one two three) (
Four)

Claims (1)

[Claims] 1. In skin protection agents with reactivating action and containing soluble collagen as active substance, the active substance is extracted from the connective tissue with sodium chloride in a neutral buffer solution and subsequently precipitated with concentrated sodium chloride solution. Or prepare by saturating ammonium sulfate, centrifuging, freeze-drying the precipitate and/or
A skin protectant consisting of a neutral salt-soluble precursor of natural collagen by stabilizing it by dissolving it in a 0.3N ascorbic acid solution and dialysis before or after dissolution.