KR20060094704A - Cosmetic composition containing collagenase inhibition and collagen synthesis promoting effect containing milk extract - Google Patents

Abstract

The present invention relates to a cosmetic composition, and more particularly, to a cosmetic composition containing the milky skin extract having the effect of inhibiting the activity of the collagenase (Collagenase) enzyme that breaks down the collagen protein and promotes collagen protein synthesis.

Milky skin extract, collagen, laminin-10, cosmetics

Description

Scalp care cosmetic composition containing collagenase inhibition and collagen synthesis promoting effect containing milk extract

The present invention relates to a cosmetic composition containing the milk extract. More specifically, the present invention is characterized in that it contains a milk extract containing the effect of inhibiting the activity of collagenase (Collagenase) enzyme that degrades collagen protein as an active ingredient and at the same time promotes collagen protein synthesis. In addition, the milk extract of the present invention promotes the expression of laminin-10 protein, which is essential for hair follicle formation and development, and is effective in maintaining strong hair roots.

The skin consists of the epidermis and the dermis. The dermis has an extracellular matrix (ECM), which is elastic. It consists of amorphous substrates (glycosaminoglycans) and fibrous proteins such as collagen and elastic fibers embedded in them. They function to fix the vascular system such as the vascular system, nerves and lymphatic vessels, and support the epidermal organs such as sweat glands and sebaceous glands. Especially in the case of the scalp, since the hair is present, the role of the extracellular matrix of the scalp is to additionally support the hair support.

As the skin ages, structural modification of the extracellular matrix is induced. Representative examples include changes in structural proteins of extracellular matrix such as collagen, elastin, and fibrin. Among them, collagen is reduced or denatured due to endogenous aging. This causes skin wrinkles to be promoted. In addition, the number of elastic fibers that give elasticity is reduced, and there is also a form deformation in which the vertical structure is arranged horizontally. These morphological changes are known to have an important effect on reducing the elasticity of the skin. Therefore, the development of substances capable of promoting the production of scarce extracellular matrix molecules or inhibit enzymes that degrade the extracellular matrix molecules produced. More specifically in the scalp, the distribution of proteins that make up the extracellular matrix is directly related to the development of hair follicles. Hair follicles are characterized by the movement of tissue up and down from the epidermis to the dermis, depending on the hair cycle. If hair follicle development fails for any reason, no hair is produced, eventually leading to hair loss. For example, laminin-10, an extracellular matrix component, is known to be a factor that promotes the development of hair follicles. Laminin-10 expression is suppressed at the lesion site of hair loss patients, and laminin is overexpressed.

According to Korean Patent No. 0163118, cosmetic cleansing composition containing the milk extract is effective in improving the skin and scalp by the amount of blood, antibacterial and astringent effects.

There is no description of the specific mechanism by which the effect of the milk extract is exhibited. In the present invention, the milk extract has the effect of inhibiting collagenase activity and promoting collagen synthesis and stimulating the expression of laminin-10, which is an important component for hair follicle development, inducing normal hair follicle development, thereby aging the skin by strengthening the elasticity and strengthening the hair root. It has been shown at the molecular level and in the clinical evaluation that there is an efficacy in prevention.

The present inventors have selected dozens of plant extracts that have a safety for the human body to screen the herbal extracts having the effect of enhancing the elasticity of the scalp and strengthening the hair root, and evaluated the inhibition of collagenase enzymes and the promotion of collagen synthesis. An extract was found. In addition, it was confirmed through tissue staining that the extracellular matrix portion of the mouse's back tissue became thick even when the milky skin extract was applied to the animal and evaluated.

The cosmetic composition according to the present invention has a clear mechanism of action that the milk extract extracts collagenase and promotes collagen synthesis, thereby enhancing scalp elasticity. This increase in scalp elasticity was recognized as an excellent effect even when applied to humans. In addition, the milk extract shows excellent results in the evaluation of the protein expression of laminin-10, which is known to promote the development of hair follicle tissue, and confirmed the hair root strengthening effect of the actual human hair pulling test to complete the present invention.

The present invention relates to a cosmetic composition having collagenase inhibition and collagen synthesis promotion, hair root strengthening effect, characterized in that it contains 0.01 to 10% by weight of the milky skin extract as an active ingredient.

Yubaekpi used in the cosmetic composition of the present invention is the stem part of the elm family Ulm macrocarpa, Ulmus pumila, Ulmus davidiana, which are distributed in North Korea, China and Japan, respectively. The root part of the root is collected and dried in the sun, and pharmacological action is known to be diuretic, small seedling, canned, species, and independent action. The main ingredients are beta-sitosterol, phytosterol, and tannin. It consists of.

Milky skin extract used in the present invention is a dry herbal medicine is dried and finely pulverized and powdered to 50g of powder to extract the extraction solvent 500ml after extracting the extract for 3 days, the extract is filtered and then concentrated under reduced pressure and used in the present invention It was. Although the extraction solvent used at this time is not specifically limited in the selection, Polyhydric alcohols, such as lower alcohols, such as purified water, methanol, ethanol, isopropyl alcohol, n-butanol, glycerol, propylene glycol, 1, 3- butylene glycol, And hydrocarbon solvents such as methyl acetate, ethyl acetate, benzene, n-hexane, diethyl ether and dichloromethane. However, lower alcohols such as methanol, ethanol, and n-butanol are preferably used as extraction solvents, and one or two or more of them may be mixed and used for extraction.

Preparation Example: Method of Preparing Milky Skin Extract

In the method of preparing the milk extract, the root of the milk extract was first chopped, and 500 ml of the medicinal product ethanol was added to 50 g of the powder passed through the sieve of No. 10, followed by cooling at room temperature for 3 days. The extracted stock solution was filtered using Whatman # 1 filter paper, and the filtered extract was evaporated completely at 45 ° C. using a vacuum condenser to obtain 2.5 g of the dried extract.

The content of the milky skin extract used in the cosmetic composition of the present invention is from 0.01 to 10% by weight as a solid content relative to the total weight of the composition, and supplements such as sebum control agents, anti-dandruff agents, keratin softeners, softening agents, moisturizing agents, anti-inflammatory agents, etc. for comprehensive scalp care. You can add ingredients.

In addition, the milky skin extract used in the cell experiments exemplified in the present invention was prepared as follows. First, the concentration of the solid content obtained by completely evaporating the solvent after cooling was defined as 100% by weight, and diluted with 1% by weight stock solution using 70% ethanol to apply it to the cells. In evaluating the efficacy of this extract, a cytotoxicity assessment was performed in a concentration range of 0.000001% by weight to 0.01% by weight to determine the concentration without toxicity to the cells. As a result, the cytotoxicity was shown at a concentration of 0.001% by weight or more, and the cell titration treatment concentration was determined by setting it in the range of 0.000001% by weight to 0.0001% by weight.

<Evaluation of Collagen Aid Inhibition of Milky Skin Extracts>

Human scalp tissue obtained from plastic surgery scalp surgery is digested with a homogenizer, and only the supernatant is taken by centrifugation. The collagen is purely separated from the supernatant through Sephacryl S-200 chromatography. 100 ml of 2% red collagen substrate Azocoll solution was added to 1.5 ml Eppendorf, and one eppendorf tube was used as a blank and the other tube was obtained from Sigma. Add AIDS type I standard enzyme solution (collagen breakdown activity: 315 units / mg) to 10, 100 or 200 ppm.

After adding 100 µl of the collagenase enzyme isolated before each tube, one tube was used as a control, and each of the remaining tubes was diluted with distilled water and concentrated at a concentration of 0.000001% to 0.0001% by weight. Mix at a ratio of 2: 2 to fully homogenize and centrifuge at 5000 g for 10 minutes. After centrifugation, 10 µl of the supernatant was taken, buffer solution (0.05 M Tris-HCl, 1 nM CaCl _2, 7.8) was added to 500 µl of the total reaction solution, and reacted for 18 hours at 37 ° C. incubator at 10000 g. Undigested collagen was precipitated by centrifugation for 5 minutes, and the supernatant containing the degraded collagen was taken. The absorbance was measured at 540 nm to prepare a standard activity curve, and the enzyme concentration was converted from the standard and control groups. Comparative evaluation yielded the following results. TABLE 1

      Evaluation Results of Collagen Aid Inhibition of Milky Peel Extract Milk Extract Extract Concentration Control 0.000001% 0.00001% 0.0001% Enzyme Activity (%) 100 61 34 24

In the above test results, the inhibitory effect on collagenase activity was increased with increasing the concentration of milky skin regardless of the formulation.

<Efficacy Evaluation Experiments on Collagen Protein Synthesis of Milky Skin Extracts>

After scalp surgery from plastic surgery, human scalp tissues were obtained and skin fibroblasts were isolated and cultured therefrom.

Dispense 10 ⁶ cells / well in a 24-well plate and incubate in DMEM medium containing 10% FBS for one day. The next day, replace the medium with fresh medium, incubate for 24 hours, and attach to the bottom with HBSS buffer solution. After washing the cell layer, 0.8ml of MEM medium without serum and proline was added, and then the experimental group (milky skin extract 0.000001% to 0.0001% treated group) was mixed with distilled water at a ratio of 1: 2 and completely homogenized. The supernatant was treated by centrifugation at 5000 g for 10 minutes, and then the cells were incubated with a culture solution containing 100 μl of ¹⁴ C-Proline (10 μCi). After 24 hours, total protein and collagen protein were measured.

First, in order to measure the amount of extracellular total protein, the culture medium of each well was placed in a sealed dialysis tube at one end and sealed at the other end of the Cold Buffer (Tris-Hcl 0.05mol / L, NaCl 0.2mol / L, CaCl). ₂ 0.05mol / L, Phenylmethylsulfonyl fluoride 0.3mN) was completed for 24 hours, and then 100 μl each was added to the Counting Vial and 10 ml of Scintillation cocktail was added to the Liquid Scintillation Counter (LSC) for 1 minute.

To measure the total amount of protein in the cell, 0.1N NaOH and 0.3mN of Phenylmethylsulfonyl fluoride were added to each well from which the cell culture medium was removed, and then maintained at 60 ° C. for 30 minutes to destroy the cell membrane. Place in a sealed dialysis tube and seal the other end to complete dialysis with Cold Buffer (Tris-Hcl 0.05mol / L, NaCl 0.2mol / L, CaCl ₂ 0.05mol / L, Phenylmethylsulfonyl fluoride 0.3mN) for 24 hours. 100 μl was taken and placed in Counting Vial. 10 ml of Scintillation cocktail was added and the radioactivity was measured by LSC for 1 minute.

To measure the amount of intracellular / extracellular collagen protein synthesis, 100 μl of dialysis-produced cell culture solution and cell homogenate were placed in 1.5 ml microtubes, and the collagen buffer solution (0.05 M Tris-Hcl, 1 mM CaCl _2, 0.3). After adding 100 μl of mM Phenylmethylsulfonyl fluoride) and collagenase enzyme (100 ppm), it was maintained at 37 ° C. for 3 hours to completely decompose Collagen, and then remove 50% of Trichloroacetic acid and 1% Tannic to remove undigested protein. After adding 500µl of acid-containing solution, settling at 4 ℃ for 30 minutes, centrifuging at 1000xg for 5 minutes, and then taking 100µl of supernatant into a counting vial, and adding 10ml Scintillation cocktail to LSC for 1 minute. Radioactivity was measured. From the radioactivity measurement, the collagen synthesis amount of the untreated control group was 100%, and the relative collagen synthesis rate was expressed as a percentage. TABLE 2

    Evaluation of Collagen Synthesis Rate by Milk Extract Milk Extract Extract Concentration Control 0.000001% 0.00001% 0.0001% Collagen synthesis rate (%) 100 168 207 249

In order to study the efficacy and effect on the collagen synthesis of human fibroblasts, the milk culture extract, which is the active ingredient of the present invention, was treated with the cell culture medium containing ¹⁴ C-Proline as described above. It appeared to increase with the concentration of. In particular, when treated with 0.0001% of baekpipi extract collagen synthesis rate increased more than two times.

Histopathological experiments on the promotion of extracellular matrix protein synthesis of milk extract

To perform histopathological experiments, mice aged 42 to 56 days old (C57BL / 6, female) were used. Hairless mice are generally used for histopathological evaluation of skin. However, since the object of the present invention is cosmetic for scalp, it is determined that the condition of normal hair and hair follicles is similar to the actual human scalp environment. Hairless mice were not used.

First, the hair on the back of the mouse was removed using an electric razor, and each mouse was weighed and divided into 5 pieces so that the weight was evenly distributed. After the adaptation period for one day, the control drug and the milky skin extract were applied to the hair removal site from the next day. At this time, 40% ethanol was used as the control drug and the concentrated solid of the milky skin extract according to the preparation was 40% ethanol. Diluted to use.

100 microliters were applied to the dorsal area per mouse subject for 4 weeks. At the end of the test, all animals were euthanized and necropsied. The dorsal skin was detached 2 per head (2 x 2 cm), attached flat to filter paper, fixed in 10% neutral formalin and then normal tissue. After treatment, paraffin embedding, 4 micrometer sections were cut and subjected to H & E (hematoxylin & eosin) staining, Verhoeff staining (elastine fiber and collagen differentiation). After all the staining process, the samples were measured by using an image analyzer to measure the thickness of the dermal layer. The relative amounts of these proteins were determined based on the degree of staining of the extracellular matrix constituent proteins elastin and collagen. It showed by the ratio and the result similar to <Table 3> was obtained.

    Changes in Dermal Thickness and Extracellular Protein Contents by Milky Skin Extracts Milk Extract Extract Concentration Control 0.1% 0.5% One% Dermis thickness (mm) 0.97 0.99 1.11 1.25 Elastin, collagen protein amount (ratio) One 1.1 1.7 2.1

<Evaluation of Laminin-10 Protein Promoting Effect of Milky Skin Extracts>

Human skin fibroblasts were cultured in the same manner as the cell isolation / culture method. Using DMEM medium containing 10% FBS, put the cells in a 100mm culture dish at a concentration of 1.8 x 10 ⁶ cells / ml, and incubate overnight. The next day, after changing to a medium without FBS, after 6 hours, the milk extract is treated with 0.000001% to 0.0001% and incubated for 48 hours. After 48 hours, rinse twice with phosphate buffer solution. The cells are scraped by applying 500 ul of cytolysis buffer prepared beforehand. After standing on ice for 20 minutes, only the supernatant is taken by centrifugation, and the absorbance is measured using the Bradford method to quantify the total amount of protein. To two 4-20% Tris-Glycine SDS-PAGE gels, add a dye containing beta-mercaptoethanol (beta-mercaptoethaol) and load 100 ug of each boiled protein for 5 minutes and electrophoresis. When the dye is placed in the 2/3 position of the gel, the electrophoresis is stopped, the gel is separated and transferred at 4 ° C. at 100V voltage using two Nitrocellulose membranes and 3M paper. After applying the voltage for 1 hour, the nitro cellulose membrane is carefully separated and blocked with 1% BSA (Bovine Serum Albumin) for 1 hour at room temperature. Rinse the membrane twice with TTBS (Tween-20 TBS) buffer solution. 10ml each of the solution containing laminin-10 and the beta-actin antibody to be used as a control is added, and the antigen-antibody reaction is carried out for 3 hours while moving the membrane. Rinse quickly with TTBS buffer and add a second antibody with fluorescent material to react for 30 minutes. After the reaction is rinsed twice with Tris buffer (TBS), and fluorescence reaction using ECL solution, a solution that induces fluorescence reaction. While adjusting the exposure time, the fluorescence reaction is detected using an X-ray film. The film is scanned and the density of each band is quantified using the image master software. When the value is corrected based on the expression pattern of beta-actin protein, which is known to be constantly expressed in cells, based on the basic condition, and the expression level of laminin-10 is 100% in the control group (no milk extract). , Relative values for the expression level of the milk extract treatment group was recorded as shown in Table 4.

     Measurement of Laminin-10 Expression by Milky Skin Extract Milk Extract Extract Concentration Control 0.000001% 0.00001% 0.0001% Laminin-10 protein expression rate (%) 100 119 129 153

As shown in Table 4, when the milk extract was treated with 0.000001% to 0.0001%, it was confirmed that the laminin-10 protein expression rate increased in proportion to the concentration. It was proved that the milky skin extract increased the expression of laminin-10 and promoted the development of hair follicle tissues, thereby being an effective ingredient for maintaining healthy hair roots.

The cosmetic composition of the present invention can be prepared in all formulations that can be applied to the scalp, such as liquid, cream, paste, or solid phase, and emulsion type scalp lotion and liquid skin for scalp care by adding conventional additives. And ampule types and the like, and also include aerosol types of these formulations.

It was confirmed in the previous evaluation that the milk extract of the present invention promotes the synthesis of collagen and elastin fibers in human fibroblasts and animals. In addition, the milk extract extract also promoted the expression of laminin-10, an important factor for hair follicle formation and development. Actually, the milky skin extract was applied to the cosmetic composition for scalp to confirm whether the composition is effective in enhancing the elasticity of the human scalp and strengthening the hair root, and clinical evaluation was performed based on the following comparative examples and examples. The following examples are merely illustrative of the present invention, and the scope of the present invention is not limited to the following examples.

   Comparative Examples and Examples (Unit: weight%) Ingredient Comparative Example 1 Comparative Example 2 Example 1 Example 2 ethanol 5.0 5.0 5.0 5.0 Milky Skin Extract - - 1.0 1.0 Red ginseng extract - 0.5 - 0.5 Fruit Extract - 0.5 - 0.5 glycerin - 5.0 - 5.0 Hyaluronic acid - 0.5 - 0.5 cellulose 0.5 0.5 0.5 0.5 Pyrotonolamine 0.1 0.1 0.1 0.1 L-menthol 0.1 0.1 0.1 0.1 antiseptic 0.15 0.15 0.15 0.15 Purified water Add until 100% by weight

Clinical Evaluation of Increased Scalp Elasticity

20 healthy 25-35 year old male and female subjects were divided into 4 groups of 5 people each, and the composition of the Comparative Examples and Examples of <Table 5> was used for 2 months, and skin elasticity was measured. The elasticity measurement was carried out using a skin elasticity measuring instrument (Cutometer SM575, C + K Electronic Co., Germany) in a constant temperature and humidity room maintained at a temperature of 25 ℃, humidity 75%, the skin elasticity is expressed as ΔR2 value, The elasticity of the skin (elasticity) is indicated, which means that the higher the skin elasticity is increased. TABLE 6

        Scalp Elasticity Clinical Evaluation Results division ΔR2 Comparative Example 1 0.019 Comparative Example 2 0.021 Example 1 0.087 Example 2 0.094

As shown in Table 6, in the comparative example, the elasticity did not increase significantly, whereas in the example containing the milky skin extract, the elasticity increased.

In particular, when it contains an auxiliary component such as a moisturizer, sebum control agent, anti-inflammatory agent in addition to the milk extract, it was confirmed that the elasticity is further increased to become an excellent composition.

<Clinical Evaluation of Hair Root Strengthening Effect>

A hair pull test was conducted to verify the hair root strengthening effect of the milk extract. Pulling the hair is an evaluation method introduced as a method for diagnosing hair cycle abnormality in dermatology (Rev. 4), Chapter 25 Hair Disease, p.476-477. . The subjects who participated in the scalp resilience test were to fix the subject's head and hang a strand of hair in the load cell of the tensile strength tester (Zwicki 1120) to obtain a force of 21.5 gmf (average value of the force of the growing scalp's scalp). Pulled the hair down. 50 hairs per person (10 strands each from five sites including the frontal, head and two temporal and posterior heads) were pulled and the number of hairs pulled out was counted to obtain the average number of hairs lost per person. In the case of the comparative example, there was no significant difference before and after use, but in the case of the example, the number of hairs falling out after use was significantly reduced, thereby confirming that the hair root was strengthened. TABLE 7

       Results of hair root strengthening clinical evaluation (number: number of hairs falling out per person) division Hair Loss Before Use Hair loss after use Comparative Example 1 7.4 8.2 Comparative Example 2 6.4 6.0 Example 1 6.8 4.4 Example 2 7.0 4.2

In summary, the milk extract of the present invention was very effective in inhibiting collagenase enzyme and promoting collagen synthesis in human skin cells, and also confirmed that the dermal layer was thickened in actual animal evaluation. In addition, it promoted the protein expression of laminin-10, a factor that promotes the development of hair follicle tissue, and showed an excellent effect in clinical evaluation of scalp elasticity enhancement and hair root strengthening efficacy.

The present invention relates to a cosmetic composition containing the milk extract. More specifically, the present invention provides a scalp cosmetic composition comprising a milky skin extract having the effect of inhibiting the activity of the collagenase (Collagenase) enzyme that breaks down the collagen protein as an active ingredient and promoting collagen protein synthesis. Related to the present invention, the milk extract of the milk extract has almost no side effects, and has the effect of preventing aging of the scalp by strengthening the hair root due to the enhancement of scalp elasticity and the promotion of laminin-10 expression according to the inhibition of collagenase and the promotion of collagen synthesis.

Claims (4)

Cosmetic composition having the effect of inhibiting collagen and promoting the synthesis of collagen containing milky skin extract. The cosmetic composition according to claim 1, wherein the content of the milky skin extract is 0.01 to 10% by weight of the total weight of the composition as a solid content. The cosmetic composition according to claim 1 or 2, wherein the extraction solvent of the milky skin extract is used by mixing one or more kinds of lower alcohols selected from methanol, ethanol and n-butanol. The cosmetic composition according to claim 1 or 2, wherein the cosmetic composition is a transdermal topical formulation that can be applied to the scalp, and is selected from a liquid, a cream, a paste, and an aerosol.