RU2807113C1 - Cosmetic composition that promotes activation of restorative and regenerative processes in various types of skin and its derivatives, hair and nails - Google Patents

Abstract

FIELD: cosmetology.

SUBSTANCE: cosmetic composition that promotes the activation of restorative and regenerative processes in the skin of various types and in its derivatives - hair and nails, contains solutions in a pharmaceutically acceptable carrier and/or diluent of a protein-peptide complex isolated from mammalian blood serum (10 wt.%), chromatographically purified fraction isolated from the fruit extract of Bertholletiaexcelsa (10 wt.%), chromatographically purified fraction of the mineral Brakshun (10 wt.%) in concentrations ranging from 10^-12-10^-8 mg protein/ml, fractions of hyaluronic acid (HA) with different molecular masses: ultra-low molecular weight HA - from 2 kDa to 10 kDa; low molecular weight HA - from 40 kDa to 100 kDa; medium molecular weight HA - from 200 kDa to 500 kDa; high molecular weight HA - from 1000 kDa to 2000 kDa, with a ratio between these fractions of 1:1:1:1 - total (0.2-1.0 wt.%), calcium chloride (0.01 wt.%), citrate silver (1.00 wt.%) and demineralized water (up to 100%).

EFFECT: ability to activate regeneration processes in the skin.

2 cl, 4 dwg, 12 ex

Description

The invention relates to cosmetology and can be used to develop a line of medical and cosmetic products of a new generation that promote the activation of regenerative processes in the skin of various types, as well as in its derivatives - hair and nails, which include protein-peptide complexes (BPC), which have the property of regulating physiological regeneration processes and activating reparative regeneration processes in the skin and its derivatives, helping to restore the morphological structure and function of the organ, which do not cause the development of negative reactions in the skin and the body as a whole.

One of the main biological processes that determine the condition of the skin and its derivatives - nails and hair - is physiological regeneration, which occurs constantly during the normal functioning of the body. During physiological regeneration, which is a process opposite to apoptosis - “programmed” cell death, natural renewal of skin tissue and its derivatives occurs. This equilibrium occurs in young, normally developing organisms. It can be disrupted as the consequences of negative effects on the skin from environmental factors, diseases, and stress increase. In this case, the processes of reparative regeneration in the skin are disrupted and cannot lead to a complete restoration of the structure and function of the organ: changes occur, for example, in the dermis, causing dryness, sagging skin, the formation of wrinkles, and in case of infection and inflammation - connective tissue scars. Hair and nails become thin and brittle, and alopecia begins. The development of these processes begins and intensifies with age as an inevitable attribute of the aging of the body.

Currently, the search and study of substances that can activate the processes of physiological and reparative regeneration of the skin, without causing the development of negative side processes, are becoming relevant in the problem associated with maintaining healthy, unfading skin.

In this aspect, interest may be generated by membranotropic homeostatic tissue-specific bioregulators (MHTBs) - a group of previously unstudied endogenous substances, the basis of which are complexes of interacting proteins and peptides, which are present in the form of a nano-sized structure in the extracellular space of animal tissues [1].

MGTB were found in all tissues and biological fluids of vertebrates, including mammals [1, 2]. They influence the basic biological processes in tissues: cell adhesion, their migration, proliferation, differentiation, apoptosis. The biological effect of MGTB is characterized by the presence of tissue and lack of species specificity and is carried out at very low concentrations of solutions corresponding to the range of 10 ^-8 -10 ^-17 mg of protein/ml. The most important property of MGTB is their ability to stimulate recovery and repair processes in pathologically altered tissues due to additional activation of cellular sources of regeneration in the body [1, 3].

In particular, it has been shown that MGTB, isolated from cattle blood serum, stimulates the proliferation of mammalian fibroblasts, increases their viability and clonogenicity in vitro [2, 3]. This MGTB in a mouse skin wound model in vivo causes complete restoration of the morphological structure of the skin, the formation of hair follicles, sebaceous and sweat glands, and damaged muscle fibers [4].

In the invention RU 2372922 “Method for treating deep skin burns” dated November 20, 2009, it was shown that when applied to the burn surface of the skin of rats with a depth of IIIB degree of a biodegradable material containing allogeneic cultured fibroblasts of animal fetuses, in the form of a sponge composition containing collagen, chitosan, and also ascorbic acid, chondroitinsulfuric acid, D-glucuronic acid, heparin and protein obtained from cattle blood serum using a previously developed method for isolating bioregulators of the MGTB group. As a result, a significant acceleration of the healing process, complete restoration of the dermal layer of the skin, as well as the epidermis, was observed.

In the invention RU 2468814 “Wound healing gel” dated December 10, 2012, it was shown that when a composition containing chitosan, salicylic acid and a bioregulator of the MGTB group, isolated from cattle blood serum, was exposed to a purulent wound in an experiment in rats, complete epithelization of the wound occurred , peeling of the scab, the structure of the dermis was restored, there were no foci of inflammation in the dermis, the formation of hair follicles occurred in the subcutaneous adipose tissue. The formation of gland ducts could be observed, and in some areas under the wound, partial restoration of muscle elements was noted. The wound healing gel promoted the formation of morphologically completely restored tissue in the area of injury, stimulating regeneration in an infected skin wound. In this invention, it was shown that the wound-healing gel suppresses the pathogenic flora of a purulent wound and causes activation of regenerative processes.

In the invention RU 2481121 “Anti-burn gel” dated May 10, 2013, it was shown that the gel composition, which includes chitosan, salicylic acid and a bioregulator of the MGTB group, isolated from cattle blood serum, has a pronounced anti-burn effect. On the 12th and, especially, on the 18th day, complete and complete epithelization of the planar burn wound occurs, complete maturation of the granulation tissue, and the absence of necrotic changes in the striated muscles. This composition has a pronounced reparative effect and promotes the restoration of morphologically and biochemically normal skin tissue. Such a pronounced effect on skin restoration processes in case of damage is facilitated by the protective effect of this MGTB, isolated from mammalian blood serum, on vascular endothelial cells, which ensures the normal function of the body’s circulatory system, including skin capillaries and its derivatives [5].

The ability of this MGTB to restore blood circulation is shown in the invention RU No. 2787479 “A set of pharmacological agents intended to improve blood flow in the cerebral cortex and restore the motor and cognitive functions of the body” dated December 15, 2021.

The present invention presents a new cosmetic composition, which includes a bioregulator of the MGTB group, isolated from the blood serum of mammals, as well as chromatographically purified fractions isolated, respectively, from the extract of the seeds of the Bertholletia excelsa tree, and an aqueous solution of the Brakshun mineral. In addition, the cosmetic composition includes hyaluronic acid (HA), which is known to be the most important component of the extracellular matrix, expressed by all skin cells and ensures constant hydration of the tissue of this organ [6]. HA molecules interact with water, ensuring the maintenance of a high moisture concentration in all layers of the skin. It is precisely in this state of the intercellular space of the skin that physiological regeneration processes, which are necessary to maintain the homeostasis of this organ in contact with the external environment, are constantly occurring. HA in a cosmetic substance is used in the form of a mixture of fractions with different molecular weights, which differ in permeability into the skin layers [7]. For example, super low molecular weight fractions of HA (up to 10 kDa) are able to penetrate into the dermis, and medium molecular weight fractions of HA (from 200 kDa to 500 kDa) - only into the epidermis; the high molecular weight fraction (1000 kDa - 2000 kDa) does not penetrate the layers of the skin, forms a film on its surface.

The claimed cosmetic composition uses four fractions of HA, each of which, by retaining water molecules, provides hydration to all layers of the skin and its surface, necessary for the biological effect of MGTB isolated from mammalian blood serum [4].

The cosmetic composition also includes silver citrate, which is a bactericidal component; calcium chloride, necessary to maintain a stable conformation of the protein-peptide complex (PPC), which is the biologically active basis of MGTB isolated from animal blood serum [1].

The technical objective of the claimed invention is to expand the range and create highly effective cosmetics for the skin and its derivatives - hair and nails, which ensure the maintenance of smooth, elastic skin, its elastic turgor, prevent the formation of wrinkles, prevent and eliminate hair loss, brittleness and unevenness of nails.

The technical result of the claimed invention in accordance with the stated technical task is to expand the range and create a cosmetic composition that exhibits the property of activating restoration and reparation processes in flabby and aging skin, as well as helping to restore the structure and function of its derivatives - nails and hair.

This activation helps maintain the biosynthesis of collagen and elastin and restore the function of the skin’s vascular system.

The use of a new cosmetic substance is characterized by the absence of any negative effects on individual tissues, organs and the body as a whole.

The specified technical task and technical result are achieved by a cosmetic composition that promotes the activation of regenerative processes in various types of skin, as well as in its derivatives - hair and nails, including solutions in a pharmaceutically acceptable carrier and/or diluent of a protein-peptide complex isolated from the blood serum of mammals, chromatographically purified fraction isolated from the fruit extract of Bertholletia excelsa and the chromatographically purified fraction of the Brakshun mineral, in the concentration range of 10 ^-12 -10 ^- 8 mg of protein / ml, and also contains a mixture of hyaluronic acid fractions with different molecular weights, including ultra-low molecular weight hyaluronic acid with they say weighing from 2 kDa to 10 kDa, low molecular weight hyaluronic acid with a mol. weighing from 40 kDa to 100 kDa, medium molecular weight hyaluronic acid with a mol. weighing from 200 kDa to 500 kDa, high molecular weight hyaluronic acid with a mol. weighing from 1000 kDa to 2000 kDa, with a ratio between these fractions of 1:1:1:1, as well as calcium chloride, silver citrate and demineralized water with the following ratio of components in wt.%:

the above solution in pharmaceutical acceptable carrier and/or diluent protein-peptide complex from whey mammalian blood 10.0-20.0 the above solution in pharmaceutical acceptable carrier and/or diluent Bertholletia excelsa fruit extract fractions 10.0-20.0 the above solution in pharmaceutical acceptable carrier and/or diluent fractions of the Brakshun mineral 10.0-20.0 the above fraction mixture hyaluronic acid - 0.2-1.0 calcium chloride - 0.01 silver citrate - 1.0 demineralized water - the rest up to 100

Moreover, the cosmetic composition according to the invention contains demineralized water or saline solution as a pharmaceutically acceptable carrier and/or diluent.

The following examples and figures 1-4 illustrate the invention (preparation of the claimed cosmetic composition and its properties), but do not limit it.

Fig. 1. Membranotropic activity of the supernatant fraction isolated from cattle blood serum. The abscissa indicates the degree of tenfold sequential dilution of the fraction under study; along the ordinate - the magnitude of the membranotropic effect, Ma (%). Asterisks indicate the concentrations of the studied fraction at which the Ma values differed from the control ones (p<0.05), i.e. were biologically active.

Fig. 2. Membranotropic activity of the supernatant fraction isolated from horse blood serum. The abscissa indicates the degree of tenfold sequential dilution of the fraction under study; along the ordinate - the magnitude of the membranotropic effect, Ma (%). Asterisks indicate the concentrations of the studied fraction at which the Ma values differed from the control ones (p<0.05), i.e. were biologically active.

Fig. 3. Membranotropic activity of the supernatant fraction isolated from the Bertholletia excelsa seed extract. The abscissa is the degree of tenfold sequential dilution of the fraction under study; along the ordinate - the magnitude of the membranotropic effect, Ma (%). Concentrations of the studied fraction at which Ma values differed from the control (p <0.05) are marked in black, i.e. were biologically active.

Fig. 4. Membranotropic activity of a chromatographically purified fraction isolated from a solution of the Brakshun mineral by gel filtration. The abscissa indicates the degree of tenfold sequential dilution of the fraction under study; along the ordinate - the magnitude of the membranotropic effect, Ma (%). Asterisks indicate the concentrations of the studied fraction at which the Ma values differed from the control ones (p<0.05), i.e. were biologically active.

Example. 1. Method for obtaining VPA from blood serum of cattle (for example, bull) or horse

The raw materials used are commercial preparations “Liquid bovine serum, for cell cultures”, sterile, reg. udost. - FSR 2008/03104, 200 ml, as well as “Horse serum liquid”, test for the absence of mycoplasma, sterile, glass, reg. udost. - FSR 2008/03322, 100 ml, “Biolot”.

Ammonium sulfate is gradually added to the cooled blood serum solution with constant stirring until a saturated salt solution is formed (78 g of salt was added per 100 ml of serum) in the presence of 10 ^-2 M EDTA. The protein suspension is left for 7 days at a temperature of +4°C, after which it is centrifuged at 15000 g for 60 minutes. The supernatant and sediment fractions are collected. Both fractions are dialyzed against water until ammonium ions are completely removed. The biologically active fraction of the supernatant is concentrated 5 times at 40°C using a vacuum rotary evaporator, and then separated on a Superdex™ 75 (10×300 mm) GL “GE Healthcare” (Sweden) column, equilibrated with water, using gel filtration on a Bio-chromatograph. Rad NGC Chromatography System Quest Plus (USA). Elution rate - 0.5 ml/min. Detection is carried out spectrophotometrically at 210 and 280 nm (Fig. 1, 2). The fraction is collected at 280 nm, concentrated and tested for the presence of membranotropic activity characteristic of MGTB [2] (Fig. 3, 4).

To assess the homogeneity of this fraction, electrophoresis is carried out according to the Laemmli method [9] using PAGE plates 1 mm thick; 12.5% and 4.0% constitute the concentrating and separating gels, respectively. To determine the molecular weight, a set of marker proteins 2000-250000 Da (BioRad, USA) is used. Electrophoresis is carried out at a constant current of 20-30 mA for 1 hour. Then a strip 1 cm wide is cut out, fixed with isopropanol/acetic acid solution and stained with Coomassie R250, and then washed in 5% acetic acid; the rest of the gel is stored on a glass plate at 4°C. Based on the electrophoretic separation pattern obtained after staining the 1-cm control strip of the gel with Coomassie R250, a horizontal strip corresponding to a protein with a molecular weight of 65-66 kDa is cut out from the rest of the gel, and this part of the gel is repeatedly extracted with demineralized water for 24 hours. The extracts are collected. , combined, dialyzed against demineralized water for 24 hours with its repeated replacement, then concentrated using a vacuum rotary evaporator at 40°C.

The analysis of this fraction is carried out using a time-of-flight MALDI-TOF mass spectrometer equipped with a nitrogen laser (337 nm) with a pulse frequency of up to 20 Hz. All measurements are carried out in linear and reflex mode, determining positive ions. To accumulate mass spectra, the laser radiation power is set at the minimum threshold value sufficient for desorption-ionization of the sample. External calibration is carried out using precise molecular weights of known proteins. The sample is applied to three wells of the plate, for each of which the spectrum obtained by summing 10 series of spectra of 50 laser pulses for each is recorded. To record, process and analyze mass spectra, flexControl 2.4 (Build 38) and flexAnalysis 2.4 (Build 11) software (Bruker Daltonics, Germany) is used. The mass measurement accuracy is ±2 Da. A saturated solution of a-cyano-4-hydroxycinnamic acid in a mixture of 70% acetonitrile and 0.1% trifluoroacetic acid is used as a matrix.

At all stages of the study, the quantitative protein content in all studied fractions is determined spectrophotometrically using the method of Warburg and Christian [6]. The results obtained show that the fraction isolated from cattle serum contains peptides with molecular weights from 1131 Da to 3121 Da and protein with a molecular mass of 66367±2 Da; the fraction isolated from horse blood serum contains peptides with a molecular mass of 1060-6931 Da and a protein with a molecular mass of 75866±2 Da [1].

The resulting fractions exhibit membranotropic activity characteristic of MGTB, represent BOD and are used for the preparation of cosmetic compositions.

Example. 2. Determination of membranotropic activity of fractions used to obtain a cosmetic composition

To study fractions for biological activity, a previously developed adhesiometric method was used, designed to identify MGTB of animal origin [2]. This method is based on determining a parameter that characterizes the viscoelastic properties of liver tissue under conditions of multiple short-term organ cultivation in vitro using standard deformation.

The method is used for biotesting of fractions obtained in the process of isolation and purification of fractions isolated from various sources, and always correlates with its specific activity.

The study was carried out on F1 CBA/C57BL hybrid mice weighing about 20 g. Fragments weighing about 1 mg were taken from a large portion of the mouse liver (areas of the liver with large blood vessels were not used). In cultivation bottles containing a nutrient medium - 0.9 ml of 199 medium and 0.1 ml of a solution of the test fraction in a certain concentration, prepared by tenfold serial dilution in nutrient medium 199; instead of the test solution, 1 ml of nutrient medium was added to the control bottles. To study the dose dependence of the membranotropic effect of the studied fraction, the protein concentration in it was determined [6] and then its solutions with different concentrations were prepared in separate bottles (see example 3). Five fragments of liver tissue were placed in all bottles of the experimental and control series. Organ cultures were then incubated at 37°C for 2 hours. Next, each tissue fragment was dried and dispersed in a special tissue disintegrator with a gap of 50 μm in 0.1 ml of a 0.1% trypan blue solution prepared in a nutrient medium under standard deformation conditions. The resulting suspension of cells and cell nuclei was examined microscopically in a Goryaev chamber (0.02 mm ³ ). Determination of the range of active concentrations of the studied fractions was carried out by studying their biological effects with serial dilutions of 10, 100, 1000 and further times.

The biological effect was calculated using the formula:

Ma = 200% - (Noп/Nk) × 100%,

where: Ma - biological effect caused by the substance;

Nop and Nk are the number of cell nuclei released from 1 mg of tissue, respectively, in the experiment (organ cultures in the presence of the drug being studied) and in the control (organ cultures without the addition of the drug).

In each individual experiment, at least 5 tissue fragments were counted for each experimental point (corresponding solution concentration).

To determine the biological activity of each studied fraction, at least 3-4 experiments were carried out. Statistical processing of the data was carried out using the method of variation statistics using the Student's test (Origin 6.0 Profession program).

Example 3. Preparation of solutions of various concentrations of fractions isolated from various raw materials for the claimed cosmetic composition

Solutions of various concentrations of fractions isolated from the corresponding raw materials were prepared on the basis of the base drug.

The concentration of the substance in the fractions is determined by the amount of total protein using the spectrophotometric method [6], in some cases the colorimetric method was used [10].

To a bottle containing 0.1 ml of an aqueous solution of the fraction being studied (protein concentration, for example, 100 ± 10 μg/ml), add 0.9 ml of a solvent, for example, demineralized water, and shake vigorously 25 times. Take 0.1 ml of the resulting solution into the next bottle and add 0.9 ml of the same solvent, shake 25 times. This procedure is repeated 15 times, obtaining solutions of the studied fraction with 10-fold serial dilution. In this way, fraction solutions with concentrations corresponding to, for example, 10 ^-1 to 10 ^-15 mg protein/ml can be obtained.

All solutions can be sterilized by passing through sterile GV membrane filters with a pore diameter of 0.22 μm (Millipore, USA).

Example 4. Method for obtaining chromatographically purified fraction from Bertholletia excelsa fruits

The source of isolation of the biologically active fraction, which is used to prepare a cosmetic composition, is the extract of seeds of a South American plant of the lecithis family, Bertholletia excelsa. The fruits contain many microelements: calcium, zinc, copper, selenium, magnesium, potassium, phosphorus, sodium; vitamins C, D, E, group B, pantothenic and folic acids, thiamine, riboflavin, choline and niacin, betaine, etc. Fruits contain a lot of selenium, which is present in them in a biogenic form - non-toxic and easily absorbed by the body. Selenium is extremely important for the absorption of fat-soluble vitamins; it regulates the functioning of the endocrine and reproductive systems in both women and men. Biogenic selenium is of fundamental importance in the prevention and suppression of tumor growth and metastasis, and it also prevents premature aging: the development of early menopause, atherosclerosis, and cataracts. Bertholletia excelsa seeds contain large amounts of unsaturated fatty acids, which are extremely important for the functioning of the cardiovascular system. Consumption of nuts improves intestinal function, prevents intoxication of the body, and regulates carbohydrate metabolism, including glucose.

For extraction, use 1 kg of fresh Bertholletia excelsa seeds without a shell and without a brown surface film: crushed into fragments 2-3 mm in size in a blender and placed in an extracting physiological solution (0.15 M sodium chloride solution; 10 ^-3 M solution -p calcium chloride). Extraction is carried out for 24-30 hours at 4°C. Then the plant mass is discarded, the extract is filtered, and centrifuged at 15000g for 30 minutes at 4°C. The supernatant is collected and ammonium sulfate is added with constant stirring until a saturated salt solution (780 g/l) is formed. The resulting protein suspension is left for 7 days at 4°C, then centrifuged at 15,000 g for 45 minutes. The supernatant and sediment are collected separately, and after dialysis and removal of ammonium ions, the membranotropic activity of each fraction is determined. 1 ml of biologically active supernatant is separated on a medium-pressure Superdex™ 75 (10×300 mm) GL “GE Healthcare” (Sweden) column, equilibrated with water, by gel filtration on a Bio-Rad NGC Chromatography System Quest Plus chromatograph (USA). Elution rate - 0.5 ml/min. Detection is carried out spectrophotometrically at 210 and 280 nm. The major fraction is collected at 280 nm, dialyzed against demineralized water for 24 hours with repeated replacements, then concentrated using a vacuum rotary evaporator at 40°C, and tested for the presence of membranotropic activity.

The fraction is examined by PAGE electrophoresis under denaturing conditions. The fraction is then used to prepare a cosmetic composition. At each stage of the study, the quantitative protein content in all studied fractions was determined spectrophotometrically using the method of Warburg and Christian [6].

Example 5. Method for obtaining a chromatographically purified fraction of a Brakshun mineral solution

A mineral substance that forms on the surface of rocks in hard-to-reach grottoes and rock crevices, mainly located in Altai. It is believed that the basis of the mineral is aluminum-magnesium alum, which contains many elements, including rare metals. In addition, C, N, O, H, and S were found in the mineral in small quantities, which suggests the presence of organic compounds in the mineral that can determine its inherent biological activity. Brakshun has been used in folk medicine for centuries as an active stimulator of regenerative processes and an effective wound healing agent. It has a general strengthening effect on the body as a whole, at the same time, it helps to normalize the function of each individual system of the body. Taking the mineral is effective in the treatment of cancer. It is taken to rejuvenate facial skin, against hair loss, and strengthen nails.

5 mg of the Brakshun mineral (Doctor Altai phyto-market) is dissolved in 5 ml of demineralized water with stirring at 35-37°C for 24 hours, filtered through 6 layers of gauze, centrifuged at 15000g, 30 minutes. The opalescent, slightly colored solution is separated on a medium-pressure Superdex™ 75 (10×300 mm) GL “GE Healthcare” (Sweden) column, equilibrated with water, by gel filtration on a Bio-Rad NGC Chromatography System Quest Plus chromatograph (USA). Elution rate - 0.5 ml/min. Detection is carried out spectrophotometrically at 210 and 280. The major fraction, absorbing at 280 nm, is collected and its membranotropic activity is determined. The fraction eluting at 32-34 minutes was biologically active. The UV spectrum of the fraction showed the presence of aromatic compounds and possibly a peptide bond. The protein concentration, according to data obtained by the colorimetric method [10], corresponded to 1-3 μg of protein/ml. A study of the membranotropic activity of this fraction showed that it exhibits membranotropic activity characteristic of MGTB. The study of this fraction by MALDI TOF mass spectrometry showed the presence of peptides with molecular masses of 1104, 1476, 2486, 3875 Da, determined at the sensitivity limit of the device. The resulting fraction is used to prepare the claimed cosmetic composition.

Example 6. Preparation of a cosmetic composition

In demineralized water (60.0 wt.%), calcium chloride (0.01 wt.%), silver citrate (1.00 wt.%), a mixture of hyaluronic acid fractions with different molecular weights, for example, 5 “Yes, are dissolved with stirring. , 42 kDa, 500 kDa and 2000 kDa, taken in a ratio of 1.0: 1.0: 1.0: 1.0 (total wt.%) for several hours. Next, prepared solutions of the following fractions are added to this solution in the concentration range of 10 ^-12 -10 ^-8 mg of protein / ml: BOD isolated from mammalian blood serum (10.0 wt.%), chromatographically purified fractions isolated, respectively, from the extract seeds of Bertholletia excelsa (10.0 wt.%) and Brakshun mineral (10.0 wt.%). The resulting gel is intensively stirred until all components are completely dissolved and demineralized water is added to 100 wt. %. This cosmetic composition is stored in a closed container for 1 year at room temperature, protected from light.

Example 7. Determination of membranotropic activity of a cosmetic composition

To a small volume of the cosmetic composition (3-5 ml), placed in a penicillin bottle, a few milliliters of nutrient medium 199 are carefully layered with a pipette and left for 16-18 hours at +4-+8°C. Carefully pipette the extract obtained by treating the cosmetic composition with medium 199. This extract is diluted with fresh nutrient medium 199 10, 100 and 1000 times, as indicated in example 3, thus preparing the medium for culturing liver tissue (see example 3). Mouse liver fragments Fl C57BL/CBA (male, 20-22g) are placed 5 pieces in each penicillin bottle containing an extract of the cosmetic composition in nutrient medium 199, in different concentrations. Liver organ cultures are incubated at 37°C for 2 hours and a parameter is determined that reflects the viscoelastic properties of the tissue in each test bottle relative to the control, which was a mouse liver organ culture incubated in nutrient medium 199 (see example 2). When the nutrient medium 199 comes into contact with the claimed cosmetic composition, substances are transferred into it, the activity of which is determined by the method of assessing membranotropic activity characteristic of MGTB. Using this method, the quality of each batch of cosmetic products prepared on the basis of the claimed cosmetic composition is checked.

Example 8. Effect of a cosmetic composition on skin wound healing in mice in vivo

The claimed cosmetic composition for the skin of the face and neck was studied on an experimental model of a skin wound in mice in vivo. Previously, using this model, it was shown that MGTB, isolated from the blood serum of mammals (cattle, horses, rats), promoted the regeneration of an experimental wound with restoration of the structure of all morphological elements of the skin [1, 3].

Experiments were carried out on male Fl (CBA/C57BI) mice, weighing 20-22 g, kept under standard conditions in the vivarium of the Institute of Gene Biology, Russian Academy of Sciences.

Under anesthesia, a piece of skin measuring about 1 cm ² was cut out from each animal's back on its back.

The following variants of experimental points were used in the experiment:

1. Control group of mice that were injured and not further exposed to any influence;

2. An experimental group of mice, to which, after an experimental injury, 100 μl of the studied cosmetic composition was applied daily to the wound area.

On the 12th day after the wound was inflicted, the animals were removed from the experiment, skin fragments were cut out, including the wound area, and fixed in Bouin’s solution. Then, for 12 hours, they were washed three times with 70% ethanol. Then, according to standard methods, tissue fragments were embedded in paraffin and histological sections 7 μm thick were prepared. Sections were stained with hematoxylin-eosin according to standard techniques. Histological preparations were viewed and photographed using a Keyence BZ 9000 microscope (Japan).

In the control group of animals (untreated wound), almost complete epithelization and slight inflammation were observed 12 days after the wound was applied. In the dermis, many small capillaries were observed, collagen fibers arranged in dense strands parallel to the epidermis, that is, a fibrous scar was formed. The subcutaneous tissue contained fat cells that were irregularly shaped and separated by patches of connective tissue. Restoration of a small number of gland ducts was observed. Scar formation also occurred in the subcutaneous tissue; collagen fibers were looser than in the dermis.

In the animals of the experimental group, in the area of the wound, a strong tightening of the edges of the wound and almost complete epithelialization were observed, more pronounced than in the animals of the control series - restoration of all layers of multilayered epithelium occurred. There was no foci of inflammation. The structure of the dermis was almost completely restored. In the dermis (especially under the wound) and in the subcutaneous tissue, restoration of numerous gland ducts and hair follicles was noted. Adipose tissue was developed. In some areas under the wound, partial restoration of muscle elements was noted.

The data obtained indicate an extremely effective wound-healing effect of the studied cosmetic composition, which not only stimulated the regeneration process in the skin wound, but, most importantly, contributed to the formation of morphologically completely restored tissue in the area of injury.

Example 9. Study of the effect of the claimed composition on the condition of the skin of the face and neck

On 33 volunteers - 24 women, 9 men, aged from 38 to 68 years, a study was carried out of the influence of the claimed composition containing a mixture of fractions isolated from cattle blood serum, isolated from the seeds of Bertholletia excelsa, fractions from the Brakshun mineral, as well as a mixture of hyaluronic acid fractions. acids with different molecular weights, on the condition of the skin of the face and neck. The experiment was carried out for 30 days.

All volunteers during the experiment were healthy, did not take pharmacological drugs or dietary supplements, and had no history of allergic reactions or atopy. Experienced group - 18 people in total: 13 women and 5 men, age 43-68 years; control group of volunteers - a total of 15 people: 11 women, 4 men, aged 38-54 years.

Several volunteers in both the experimental and control groups had oily skin; All male participants in the experiment had sensitive skin, on which, after shaving, redness formed in the neck area, it could become inflamed.

Composition of the cosmetic composition (wt.% per 100 ml of cosmetic composition)

The composition contains calcium chloride (0.01), silver citrate (1.00); a mixture of hyaluronic acid fractions with different molecular weights of 5 kDa, 42 kDa, 500 kDa and 2000 kDa at a mixture ratio of 1:1:1:1 (0.25 wt.% of each fraction, total 1.0 wt.% ) and the following solutions:

- BOD isolated from mammalian blood serum, containing 10 ^-10 mg of protein/ml (protein concentration) (10.0 wt.%);

- chromatographically purified fraction isolated from the seeds of Bertholletia excelsa, containing 10 ^-10 mg of protein / ml (protein concentration (10.0 wt.%));

- chromatographically purified fraction isolated from the Brakshun mineral, containing 10 ^-10 mg of protein/ml (protein concentration) (10.0 wt.%);

the rest: demineralized water - up to 100 wt. %.

As a comparison drug, we studied “Children’s Cream”, produced by AOA “Svoboda”, 68 g tube, active ingredients: chamomile extract, lanolin, vitamin A, menthol. The cream nourishes, softens and protects the skin from irritation, diaper rash, has a slight cooling effect, and does not contain a perfume composition.

During the experiment, the studied gel composition according to the invention or baby cream was placed in standard plastic bottles with a pipette and distributed to the participants of the experiment.

A cosmetic composition or baby cream was applied as a standard, at approximately the same time of day, to cleansed facial skin after wiping with a cotton swab moistened with boiled water at room temperature and drying as follows: on the center of the forehead, chin and nose - 1 drop each; on the central area of the cheeks, chin and neck - 2 drops each. The applied composition or baby cream was carefully distributed with the pads of the index and middle fingers over the surface of the skin. Using light movements, distribute the composition over the skin in the eyelid area.

Parameters reflecting the degree of skin hydration and a number of its properties were analyzed.

The skin hydration index was determined for all participants before the start of the experiment using a Corneometer ^® CM 825 sensor (Courage & Khazaka), examining the center of the forehead and chin. Three people in the experimental group were identified (women, ages 47, 54, 68 years) who had a humidity parameter below 50. These data indicated dry skin in three volunteers. The remaining participants in the experiment had values of this parameter above 50. By the end of the experiment, in these three volunteers the parameter reflecting skin moisture acquired a value above 50 (55-58), which indicated the elimination of dry skin when exposed to the composition under study. Men noted a significant improvement in skin condition after daily shaving: after using the gel composition, its moisture content increased, the feeling of tightness, redness and irritation disappeared, while when using baby cream, these unpleasant sensations after shaving lasted longer.

Using the VISIOSCAN VC 98 dermatoscan, a number of characteristics of the facial skin of the experiment participants were studied: on the 3rd, 8th, 15th, 22nd and 30th days of the study. It should be noted that in the control group (using baby cream) there was no fundamental change in the parameters reflecting the condition of the skin, while in the experimental group significant changes were observed in such characteristics as smoothness, roughness, rejection of the stratum corneum (flakyness), dynamics were noted reducing the parameters of major wrinkles. Volunteers in the experimental group observed the disappearance of small wrinkles (“mesh” on the temples and cheeks), a decrease in the parameters of large nasolabial folds, wrinkles on the forehead and “crow’s feet” near the eyes, scaly skin and roughness of the skin almost disappeared, along with parameters reflecting skin moisture , reached the normal value. There was also a decrease in facial swelling and restoration of the oval. The results obtained were not subject to statistical analysis for both groups, but for each participant in the experiment it was possible to note a completely reliable change in individual parameters. For example, for a volunteer, female. 68 years old, there was an increase in skin smoothness by the end of the experiment by 25-30%, humidity by 15%, and a decrease in the parameters of nasolabial folds by 23-27%. It is important that all volunteers in the experimental group did not detect any negative skin reactions during or after the experiment. It was noted that the composition under study was quickly absorbed into the skin (10-12 minutes), without leaving a film on its surface. The results of the study indicate the effective effect of this composition on facial skin of various types in people of different ages.

Example 10. The influence of the claimed composition on the condition of facial skin with rosacea

A study was conducted on 12 volunteers - all women, aged from 45 to 62 years - to study the effect of the claimed composition on the condition of facial skin with rosacea.

Composition of the cosmetic composition (wt.% per 100 ml of cosmetic composition)

The composition contains calcium chloride (0.01), silver citrate (1.00); a mixture of hyaluronic acid fractions with different molecular weights of 5 kDa, 42 kDa, 500 kDa and 2000 kDa at a mixture ratio of 1:1:1:1 (0.25 wt.% of each fraction, total 1.0 wt.% ) and the following solutions:

- BOD isolated from horse blood serum, containing ^10-12 mg of protein/ml (protein concentration) (10.0 wt.%);

- chromatographically purified fraction isolated from the seeds of Bertholletia excelsa, containing 10 ^-8 mg of protein/ml (protein concentration) (10.0 wt.%);

- chromatographically purified fraction isolated from the Brakshun mineral, containing 10 ^-8 mg of protein/ml (protein concentration) (10.0 wt.%);

demineralized water - up to 100 wt. %.

All volunteers did not take pharmacological drugs or dietary supplements during the experiment, and had no history of allergic reactions or atopy. All volunteers showed changes on their facial skin that were characteristic of rosacea: in the area of the nose and on the cheeks, networks of capillaries protruding like colored islands were found. The skin of all participants was thin, dry, prone to inflammation, redness, and also had increased sensitivity to changes in ambient temperature. All participants strictly avoided direct sunlight. After examining all the participants, the cosmetologist confirmed that they had symptoms of rosacea.

During the experiment, the composition under study according to the invention was placed in standard plastic bottles with a pipette and distributed to the participants.

The composition under study was applied twice (morning and evening) to cleansed facial skin at approximately the same time of day after carefully wiping with a cotton swab moistened with boiled water at room temperature and drying as follows: on the center of the forehead, chin and nose; on the central area of the cheeks, on the chin area, 1 drop. The applied composition was carefully distributed using a cotton swab over the surface of the facial skin. All participants in the experiment did not use any more cosmetic or medicinal products or wear makeup.

On the 28th day of the study, all participants were examined by a cosmetologist to assess the condition of their facial skin. All participants noted a significant improvement in skin condition, which was reflected in the disappearance of increased sensitivity and dryness. The thermal sensitivity of the facial skin has significantly decreased, it has become more elastic and moisturized. The color intensity and the area occupied by the capillary networks sharply decreased. An examination by a cosmetologist showed a decrease in the symptoms of rosacea and characterized the skin condition as close to normal.

Example 11. The effect of the claimed composition on the condition of the scalp and its derivatives - hair and nails

A study was conducted on the effect of the claimed composition on the condition of the scalp and its derivatives - hair and nails - on 14 volunteers, women aged from 25 to 47 years. The experiment was carried out for 45 days. All participants in the experiment suffered a viral infection, after which they developed dryness and severe itching of the scalp, and the formation of areas of inflammation due to regular scratching of the skin. A sharp thinning of hair and intense hair loss began. The area behind the ear and the area above the forehead were especially susceptible to alopecia. Many experienced a significant deterioration in the condition of their nails - their fragility and dullness increased, and surface irregularities (grooves) appeared. All volunteers' nails grew slowly and split. The cuticle was in a damaged state: it had either peeled off from the surface of the nail, or, on the contrary, had grown greatly.

All participants in the experiment were healthy during the experiment, did not take pharmacological drugs or blood pressure, and did not have allergic reactions or atopy.

The composition contains: calcium chloride (0.01 wt.%), silver citrate (1.00 wt.%); a mixture of hyaluronic acid fractions with different molecular weights of 2.5 kDa, 50 kDa, 200 kDa and 1000 kDa at a mixture ratio of 1:1:1:1 (0.05 wt.% of each fraction, total 0.2 wt. . %), and:

- BOD solution isolated from cattle blood serum, containing 10 ^-10 mg of protein/ml (protein concentration) (10.0 wt.%);

- a solution of a chromatographically purified fraction isolated from the seeds of Bertholletia excelsa, containing 10 ^-10 mg of protein/ml (protein concentration (10.0 wt.%));

- a solution of a chromatographically purified fraction isolated from the Brakshun mineral, containing 10 ^-10 mg of protein / ml (protein concentration (10.0 wt.%));

demineralized water - up to 100 wt. %.

Participants in the experiment used baby shampoo to wash their hair once for 5 days. The composition under study was applied 1 drop at a time to different areas of the scalp, 10 drops in total, using a standard plastic bottle with a pipette. Next, the product was rubbed into the scalp, massaging it with your fingers until the composition was completely absorbed. The procedure was carried out once a day, in the morning. One drop was applied to each individual nail of both hands with the composition distributed over the surface of the nails and the skin of the phalanx. The experiment lasted 45 days. At the end, all participants in the experiment noted an increase in the number of hairs on their heads - an increase in their density and the appearance of new hairs, their thickening, the complete disappearance of itching of the scalp and hair loss. Several participants in the experiment began to recover curly hair, which completely disappeared before the experiment began, and a decrease in gray hair was also noted. In addition, the experiment participants noted an improvement in the condition of their nails: they lost their brittleness, layering, and their unevenness and dullness disappeared. Nails gained shine and began to grow faster. The cuticle was restored: it became soft and elastic, its normal thickness was restored.

Example 12. Effect of a cosmetic composition in the form of a cleansing lotion for oily and problem skin

A study was conducted on the effect of a composition in the form of a cleansing lotion for oily and problematic skin on 9 volunteers - all women, aged from 19 to 35 years.

All volunteers did not take pharmacological drugs or dietary supplements during the experiment, and had no history of allergic reactions or atopy. All volunteers showed changes on their facial skin characteristic of problematic, combination skin, prone to inflammation, and with wide pores. In 3 women (26-35 years old), the skin was described by a cosmetologist as oily, aged, with wide pores. In 6 volunteers (age 19-24 g), facial skin was characterized as problematic, prone to inflammation with wide pores; All volunteers had acne.

Composition of the cosmetic composition (wt.% per 100 ml of cosmetic composition)

The composition contains calcium chloride (0.01), silver citrate (1.00); a mixture of hyaluronic acid fractions with different molecular weights of 5 kDa, 42 kDa, 200 kDa and 1000 kDa at a mixture ratio of 1:1:1:1 (0.1 wt.% of each fraction, total 0.4 wt.% ) and the following solutions:

- BOD isolated from horse blood serum, containing ^10-12 mg of protein/ml (protein concentration) (20.0 wt.%);

- chromatographically purified fraction isolated from the seeds of Bertholletia excelsa, containing 10 ^-10 mg of protein/ml (protein concentration) (20.0 wt.%);

- chromatographically purified fraction isolated from the Brakshun mineral, containing 10 ^-8 mg of protein/ml (protein concentration) (20.0 wt.%);

demineralized water - up to 100 wt. %.

During the experiment, the composition under study according to the invention was placed in standard plastic bottles with a pipette and distributed to the participants.

The composition was applied twice (morning and evening) at approximately the same time of day to cleansed facial skin after carefully wiping with a cotton swab moistened with boiled water at room temperature and drying as follows: on the center of the forehead, chin and nose; 1 drop on the central area of the cheeks, on the chin, neck and décolleté line. The applied composition was carefully distributed over the surface of the facial skin using the pads of the index and middle fingers. All participants in the experiment did not use any more cosmetic or medicinal products or wear makeup.

On the 29th day of the experiment, all participants were examined by a cosmetologist to assess the condition of their facial skin. All participants showed a significant improvement in their skin condition: their complexion improved significantly and was closer to healthy. The oily sheen of the skin completely disappeared, it acquired a matte tint and became more hydrated and elastic. Areas of inflammation have completely disappeared; acne disappeared in 5 volunteers; in the rest, acne decreased significantly (by more than 50%).

conclusions

A new cosmetic composition has been developed for the care of skin and its derivatives (hair and nails). The composition contains a protein-peptide complex isolated from the blood serum of mammals, as well as chromatographically purified fractions obtained, respectively, from the extract of Bertholletia excelsa seeds of the Brakshun mineral. As shown by the results of studying these fractions using PAGE electrophoresis and mass spectrometry, they contain proteins and peptides. Since these fractions exhibit membranotropic activity in low concentrations, characteristic of natural bioregulators of the MGTB group [1, 2], it can be assumed that they also represent protein-peptide complexes. It is possible that the Brakshun mineral, which is a mixture of aluminum-magnesium alum, also contains organic substances that can stimulate regenerative processes in the tissues of higher organisms.

The cosmetic composition contains fractions of hyaluronic acid with molecular weights of four intervals: 2-10 kDa; 40 - 100 “Yes; 200-500 kDa and 1000 - 2000 kDa in the ratio between fractions as 1:1:1:1, which have different penetrating abilities into skin tissue. Hyaluronic acid ensures the preservation and deposition of water in tissues, which is necessary for the biological action of protein-peptide fractions, for example, BOD, isolated from mammalian blood serum.

Silver and calcium chlorides, components that provide the bactericidal properties of cosmetics and maintain the conformation of protein and peptide molecules. It has been shown that cosmetics developed on the basis of the claimed cosmetic composition have no contraindications for long-term use and do not cause the development of negative processes in the skin, its derivatives and in the body as a whole. The developed cosmetics can be recommended as a base for makeup, day or night cosmetics, and after shaving. These products exhibit the ability to activate regeneration processes in the skin and its derivatives, which is expressed in the restoration of tissue turgor, its hydration, the disappearance of fine wrinkles, the reduction of the area of large wrinkles in people of different ages, in maintaining the elasticity of thin sensitive skin, and the rapid repair of minor skin defects. The use of the claimed cosmetic composition promotes the disappearance of acne, normalization of the function of the sebaceous and sweat glands of the skin, as well as restoration of the structure and function of capillaries and small vessels of the skin, and restores microcirculation processes in it. In men, after shaving, the use of a cosmetic composition prevents the development of skin irritation and redness, and promotes the healing of small cuts, scratches and abrasions.

The use of the claimed cosmetic composition for the scalp and hair growth contributed to the restoration of hair in women after an infectious viral disease, the reduction or disappearance of itching of the scalp, stopped hair loss, and caused the restoration of its structure. The cosmetic composition had a positive effect on the condition of the nails - their structure and appearance.

The claimed cosmetic composition did not have a negative effect on the skin during long-term use, and did not cause adverse reactions from other tissues and the body as a whole.

Literature

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2. Yamskova V.P., Reznikova M.M. Low-molecular-weight polypeptide in the blood serum of warm-blooded animals: influence on cell adhesion and proliferation // Journal of General Biology. 1991. T. 52. No. 2. pp. 181-191.

3. Bueverova E.I., Bragina E.V., Reznikova M.M., Yamskova V.P., Khrushchov N.G. The effect of the adhesion factor of blood serum on the proliferation of mammalian cells in vitro // DAN USSR. 1985. T. 281. No. 1. pp. 158-160.

4. Yamskova V.P., Krasnov M.S., Rybakova E.Yu., Vecherkin V.V., Borisenko A.V., Yamskov I.A. "Analysis of regulatory proteins from bovine blood serum that display biological activity at ultra low doses: 2. Tissue localization and role in wound healing", pp. 71-78 // In the book "Biochemical Physics Frontal Research", Ed. by S.D. Varfolomeev, E.B. Burlakova, A.A. Popov and G.E. Zaikov, Hauppauge NY, Nova Science Publishers Inc. 2007. 127 P.

5. Krasnov M.S., Yamskova V.P. The influence of bioregulators isolated from blood serum and bull cornea on the condition of tissue and cells of the rabbit cornea during cultivation and storage // Ophthalmology. 2021. T. 18(3). pp. 488-494.

6. Snetkov P. ¹ , Zakharova K. ¹ , Morozkina S. ¹ , Olekhnovich R. ¹ , Uspenskaya M. ¹ Hyaluronic Acid: The Influence of Molecular Weight on Structural, Physical, Physico-Chemical, and Degradable Properties of Biopolymer // Polymers (Basel). 2020 Aug 11. T. 12(8). P. 1800.

7. Essendoubi M., Gobinet C, Reynaud R., Angiboust J. F., Manfait M., Piot O. Human skin penetration of hyaluronic acid of different molecular weights as probed by Raman spectroscopy // Skin Research and Technology. 2018. V. 24, Issuel. February. P. 129-134.

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Claims (4)

1. A cosmetic composition that promotes the activation of restorative and regenerative processes in the skin of various types and in its derivatives - hair and nails, containing solutions in a pharmaceutically acceptable carrier and/or diluent of a protein-peptide complex isolated from mammalian blood serum, a chromatographically purified fraction isolated from the fruit extract of Bertholletia excelsa and the chromatographically purified fraction of the mineral Brakshun, in the concentration range of 10 ^-12 -10 ^-8 mg of protein / ml, and also contains a mixture of hyaluronic acid fractions with different molecular weights, including ultra-low molecular weight hyaluronic acid with a mol. weighing from 2 kDa to 10 kDa, low molecular weight hyaluronic acid with a mol. weighing from 40 kDa to 100 kDa, medium molecular weight hyaluronic acid with a mol. weighing from 200 kDa to 500 kDa, high molecular weight hyaluronic acid with a mol. weighing from 1000 kDa to 2000 kDa, with a ratio between these fractions of 1:1:1:1, as well as calcium chloride, silver citrate and demineralized water with the following ratio of components in mass%: the above solution in a pharmaceutically acceptable carrier and/or diluent of a protein-peptide complex from mammalian blood serum 10.0-20.0 the above solution in a pharmaceutically acceptable carrier and/or diluent of the protein-peptide complex from Bertholletia excelsa fruit extract 10.0-20.0 the above solution in a pharmaceutically acceptable carrier and/or diluent of the Brakshun mineral fraction 10.0-20.0 the above mixture of hyaluronic acid fractions 0.2 - 1.0 calcium chloride 0.01 silver citrate 1.0 demineralized water the rest up to 100 2. A cosmetic composition that promotes the activation of restorative and regenerative processes in the skin of various types and in its derivatives - hair and nails, according to claim 1, characterized in that it contains demineralized water or saline solution as a pharmaceutically acceptable carrier and/or diluent.