Rich hydroxyprolin proteins containing cosmetic formulations of

 

(57) Abstract:

The invention relates to a rich hydroxyprolin the glycoproteins, which have an average molecular weight of 20,000 daltons, and high solubility in water. Glycoproteins get acid-alcohol extraction from cell cultures Taxus spp., Gingko biloba, Lycopersicum esculentum and Daucus carota. The glycoproteins of this invention can be used in cosmetic formulation, in which previously it was assumed the use of animal collagen. 2 c. and 1 C.p. f-crystals.

The invention relates to enriched hydroxyprolin the glycoproteins, which can be obtained from plant sources, and to their pharmaceutical and cosmetic use.

More specifically, the invention relates to a rich hydroxypropane the glycoproteins, which can be obtained by using the acid-alcohol extraction from cell cultures Taxus spp., Gingko biloba, Lycopersicum esculentum and Daucus carota, with the following characteristics:

- average molecular weight of 20,000 daltons interval variability from 12,000 to 38000 determined by means of gel filtration and electrophoresis;

- solubility in acidic aqueous solutions.

It is known that some glycoproteins of animal p the Institute as such, or being included in a suitable formulation.

Collagen, which is a glycoprotein rich in Proline and hydroxyprolin, used especially as such or in combination with other polypeptide substrates in the treatment of wrinkles and other unsightly stains associated with poor hydration and skin elasticity. However, the collagen of animal origin has limitations in its use due to risk of contamination by viruses and toxins. Although compounds of plant origin are not associated with a similar risk, so far their use in cosmetics was extremely limited: for example, a well-known cosmetic formulation, which contains the crude extracts of plants such as Aloe or even pulverized plant foods such as avocado.

Known plant glycoproteins, called estensioni, which are obtained from plant cells under proliferation and which have a structure similar to the collagen of animals. In EP-A-05334078 describes cosmetic use of extension having an average molecular weight greater than 100,000 daltons. However, the methods of extraction of extension described to date, which include the extraction of vegetable materials of different origin with the PTA, do not allow to obtain products suitable for cosmetics, due to problems related to solubility, reproducibility and durability of their chemical-physical characteristics.

Presently discovered that you can get hydroxyprolin-rich glycoproteins, which are structurally similar to the above extensively, but with lesser mol. mass and higher solubility in acidic aqueous solutions, using the method comprising culturing in vitro cells of the selected plants and extraction of the acid-alcohol solutions of cells growing in a suitable environment.

Glycoproteins produced according to this invention, are hydrating, film-forming, semi-permanent and promotes scarring properties, a higher level than the properties of collagen. The glycoproteins of this invention can, therefore, be used in the cosmetic or dermatological formulation for the treatment of dry skin, psoriasis, ichthyosis, dandruff, keratoses, wrinkles, acne, eczema, inflammatory dermatosis, skin aging and in all other areas of application for which it was supposed the use of animal collagen.

Aqueous solutions of glycoproteins invented this is the FL. In addition, the viscosity of these solutions is particularly high and does not depend on concentrations; 0.1% concentration suddenly have the same film-forming and moisturizing effect, what are the solutions of 1% collagen or 5% of vegetable albumin.

Subject extraction the plant material is obtained from cultivated in a fermenter cultures of cells of Taxus spp., Gingko biloba, Lycopersicum esculentum and Daucus carota. Particularly preferably, the cell line of the species of Taxus spp., Gingko biloba and Lycopersicum esculentum. Methods of culturing cells are conventional and include the use of suspension cultures derived on the basis of calli cultures of different parts of these plants, such as leaves, bark, roots, stem or seeds, as described by the authors Dobbs and Roberts, Experiments in Plant Tissue Culture, 2nd ed. Cambridge University Press, New York, 1985.

Plant tissue callus after sterilization and optional add antibacterial agents usually used for inoculum suitable liquid culture media, as described in the aforementioned guide Dobbs and Roberts. Particularly suitable environment for this invention is the environment Skoog and Murashige. Adding specific additives, such as Proline, reducing agents, ethylene or junction sleep to increase productivity or generate the desired glycoproteins.

Preferred use naphthylacetic acid, such as auxin, 6-(,-dimethylamino)-purine as cytokinins, vitamins and 3% sucrose as carbon source.

Depending on the selected material may be useful to add vitamin C to prevent purchasing the end product of a brown color.

The fermentation time can vary from 3 to 12 days and is preferably between 5-6 days. Immediately upon completion of the fermentation culture medium and centrifuged cell mass is extracted with alcohols, preferably ethanol, in the presence of dilute mineral acids, preferably hydrochloric or sulfuric acid. This procedure inactivates several enzymes, which can endanger the stability of glycoproteins of the present invention, in particular, polyphenoloxidase and tyrosinekinase that promote polymerization of glycoproteins followed by the formation of insoluble products.

Alcohol extraction in the presence of mineral acids makes possible complete extraction of the major glycoproteins, and was ultimately extremely selective. In addition to ethanol, you can use the others who form and then concentrate and heated to a temperature of 70-100oC, preferably about 80oC, before completion of the deposition denaturirovannykh proteins. Then the suspension lighten the concentration and the liquid fractional ultrafiltration to remove high molecular weight and low molecular weight substances. The ultrafiltration is carried out with the use of polysulfone membranes having a cut-off molecular masses capabilities from 10,000 to 40,000 daltons, such as MethodRor RomiconRfibers which can be hollow or alternative twisted. The obtained filtered product is subjected to electrodialysis to remove unwanted substances, such as salts and low molecular weight sugar. After filtration and dialysis the solution obtained can be used as such in the cosmetics or pharmaceutical products, or it can be concentrated to a smaller volume and then liofilizirovanny or fine grind.

Analytical characterization of the products of this invention was performed using gel filtration using a liquid chromatograph, high pressure, consisting of a pump unit (Waters pump unit and is equipped with a battery column (Ultrahydrogel Linear WatersR) H,5 cm and UV-detector absorption Waters UV absorbtion detektor, model 484). As an eluent of apollinopolis was dissolved in the same solution as eluent (3 mg/10 ml) and graded quantities of the substance, as well as standard substances, selected as standards with molecular masses between cytochrome C (12400 daltons) and dextran blue (2000000 daltons), products or intermediate products can be defined alternative or simultaneously by means of electrophoresis on a 12.5% pohjakallio gel and 4% concentrating gel. The sample is dissolved in a buffer containing LTOs and 0.1% mercaptoethanol, choosing a number between 100 mg and 300, the Migration is carried out at constant current at 20 mA for 4 hours a Calibration curve is built using 5 standards of molecular weights (7, 14, 24, 54, and 66 KD). Molecular weight of 22.5 and 25 KD calculated on the basis of this calibration curve for two main bands, and molecular masses of 31 and 34 KD calculated for the less intense bands. The procedures described here give a mixture of products with comparable molecular masses and comparable amino acid compositions of different cell explants originating from different plants. The results of amino acid analysis of glycoproteins extracted from the cells, Gingko biloba, is presented below as an example.

Amino acid is the peak Area, %

Asp 4,399

Glu - 4,328

Hyp - 17,505

Ser - 7,065

Gly - 6,056

Hys - 1,78 eu - 5,525

Phe - UAH 1,862

Lys - 14,254

The above data refer to the percentage of the total number of amino acids present in a mixture of glycoproteins. Sugar in this mixture are the arabinose and galactose. The ratio of amino acids to sugars is about 2:1 for different products.

As mentioned above, the products of this invention can be used in pharmaceuticals and cosmetics. For a pharmaceutical product may be included in the gels or ointments or be applied to the saturable drug Marley for special treatment of burns or wounds. In this case, the product is usually subjected to sterilization or sterile filtration and lyophilization.

Cosmetic and dermatological preparations of the present invention can be prepared according to conventional methods. Examples of forms of introduction are water sprays, lotions, solutions, emulsions, gels, ointments and creams.

Cosmetic and dermatological preparations of the present invention may contain hydroxyprolin-rich glycoproteins in the amount of from about 0.01 to about 50 wt.%, preferably from 0.05 to 5 wt.%, as well as conventional fillers. With this high Sarata, containing more than 50% soluble hydroxyprolin-rich glycoproteins.

The glycoproteins of this invention can be added to the pharmaceutical and cosmetic preparations as such, or they can microencapsulates thus, in order to ensure a long lasting hydrating effect. Microcapsules can be either hydrophilic or lipophilic. The preparations of this invention may include other active ingredients with complementary or useful activity for the desired goals.

Further, the invention is illustrated by the following examples.

Example 1

Obtaining callus and liquid culture Gingko biloba for the production of glycoproteins

Prepare the Explant young leaves of Gingko biloba washing leaves in a solution of 0.1% Tween 80 (Tween 80R). The lamina is cut into sections of approximately 0.5 cm and pre-sterilized for 1 min 75% ethanol. Then complete sterilization with 2% sodium hypochlorite solution and three times with washing Explant in sterile water. The resulting explants are transferred into a Petri dish environment Murashige and Skoog medium containing 3% sucrose, supplemented with vitamins of Linsmeier and Skoog and hormones, the/SUP>C for 20 days. At the end of this period, given the friable calli that are easy to grow and develop in a continuous series under subcultivation in the same conditions in which they can be used for reproduction in a liquid medium. The calli used for inokulirovanny Erlenmeyer flasks containing 200 ml of medium Murashige and Skoog, with the addition of nattukottai acid and 6-(,-dimethylamino)-purine, vitamins of Linsmeier and Skoog and 3% sucrose as carbon source. The flask is incubated with stirring in continuous light for 4 days, after which the biomass of cells harvested for the extraction of glycoproteins.

Example 2

Receiving glycoproteins from cells Gingko biloba

5 l of the culture obtained according to example 1, centrifuged at low speed and the collected cells (1.5 kg wet weight) extracted with 1.5 l of 70% ethanol containing 1% sulfuric acid. The extraction was repeated twice, thereby removing quantitatively the major glycoproteins. After neutralization of the extracts was filtered to remove turbidity and concentrated in vacuo at 50oC to remove the ethanol. The aqueous concentrate is heated at 85oC for 30 min and centrifuged again to remove the sediment, which she cut 40000 daltons to exclude substances with high molecular masses.

Then the filtered product Ultrafiltered using a membrane with hollow fibers with a cut-off of 10,000 daltons to remove not-glycoprotein, low molecular weight substances. Then the filtrate is subjected to dialysis and concentrated to 1% of the solid residue. Get 1.5 liters of slightly viscous product, which is used as such in the cosmetic formulation. In the analysis using electrophoresis, the product contained 6 pages, 4 of which had a molecular weight of 16,000, 22000, of 33,000 and 36,000 daltons.

Example 3

Receiving glycoproteins from Lycopersicum esculentum

Following the procedure of example 1, prepared cell mass of the sterile kidney Lycopersicum esculentum in 14-liter fermenter containing 10 l of medium Murashige and Skoog medium with the addition of naphthylacetic acid and 6-(,-dimethylamino)-purine, vitamins of Linsmeier and Skoog and 3% sucrose as carbon source. The fermentation is conducted for 5 days at 23oC under stirring at 150 rpm in the presence of yeast extract with a concentration of 0.05% and a concentration of dissolved oxygen of approximately 70%. At the end of the fermentation broth is collected and Microfiltered through the ceramic membrane of 0.2 μm to concentrate cells. To the obtained is about, described in example 2. Get 3.5 l of a solution with 0.5% of dry residue. Analysis of lyophilised solutions gave, respectively, the content of Proline 10% and the content of hydroxyproline 31%.

Example 4

Cosmetic preparative form

100 g of the emulsion oil-in-water contains, g:

The solution of example 2 or 3 - 10,0

Acetylated lanolin alcohol PEG-10 - 2,0

Cetyl-stearyl alcohol - 1,5

Cetylpalmitate - 2,0

Stearic acid - 7,0

Attractant - 7,5

Cetilistat potassium - 0,5

Preservatives, if necessary

Flavor - if necessary

Purified water - as needed to 100

Example 5

Cosmetic preparative form

100 g of the emulsion oil-in-water contains, g:

The solution of example 2 or 3 - 10,0

Cetylstearyl - 5,0

Jojoba oil - 10,0

Isopropylmyristate - 8,0

Dimethicone - 0,5

Antioxidant as needed

Preservatives, if necessary

Flavor - if necessary

Purified water In the amount necessary to 100

Additional experimental data.

Example 6

Cosmetic preparative form

The solution in water, g:

The solution of the mixture of example 2 or 3 - >/BR>Cosmetic preparative form

Gel, g:

The solution of the mixture of example 2 or 3 - 1,00

Propylene glycol, diazolidinylurea, methylparaben, propylparaben - 1,00

Purified water as needed to 100 IFL

1. The mixture enriched hydroxyprolin glycoprotein (HPRG), we obtain the acid-alcohol extraction of Gingko biloba, Lycopersicum esculentum, or from cultures of cells according to a method that includes (a) the cultivation of Gingko biloba, Lycopersicum esculentum and cells in liquid medium for 3 to 12 days, b) extraction of the resulting mass of cells is mixed with water to alcohols in the presence of dilute mineral acids, (C) neutralization, concentration and heating the extracts at a temperature of 70 - 100oC, d) centrifugation with a drop of sediment, fractional ultrafiltration and dialysis of the supernatant, and these enriched hydroxyprolin glycoprotein (HPRG) have an average molecular weight of 20,000 daltons interval variability 12000 - 38000 daltons determined using gel filtration and electrophoresis.

2. Cosmetic preparation for skin hydration comprising as active agent a mixture of glycoproteins under item 1.

3. A cosmetic product under item 2 in fo the

 

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