KR20120003749A - Cosmetic composition containing silicate - Google Patents

Abstract

PURPOSE: A cosmetic composition containing silicate is provided to promote collagen type I biosynthesis and to ensure anti-oxidation. CONSTITUTION: A cosmetic composition for anti-wrinkling contains silicate as an active ingredient. The cosmetic composition for anti-wrinkling is manufactured in the form of a lotion, gel, aqueous liquid, cream, essence, oil-in-water type or water-in-oil type makeup base, foundation, lip stick, face powder, eye shadow, or eyebrow pencil. A cosmetic composition for anti-aging contains silicate as an active ingredient.

Description

Cosmetic composition containing silicate

The present invention relates to a cosmetic composition, and more particularly to a cosmetic composition containing a silicate.

Aging can result from UV, stress, disease conditions, environmental factors, wounds, and the activation of free radicals with age, which intensifies the destruction of antioxidant defenses in vivo and damages cells and tissues. Adult disease and aging are promoted. More specifically, the activation of free radicals causes oxidation of lipids, proteins, polysaccharides, and nucleic acids, which are the major constituents of the skin, to destroy skin cells and tissues, resulting in skin aging. Among them, the oxidation of protein is caused by severe cuts in connective tissues such as collagen, hyaluronic acid, elastin, proteoglycan, and fibronectin, which severely inhibit inflammatory reactions and elasticity of skin. Immune function is caused. Therefore, it protects the cell membrane by eliminating free radicals generated by metabolic processes of the body, irradiation by UV irradiation or inflammatory reactions, and the already damaged cells are regenerated by active metabolism to proliferate the cells. Can restore and maintain healthy skin.

On the other hand, aging involves not only free radicals but also enzymes called substrate metalloproteinases (MMPs). When the expression of MMPs is promoted, elasticity of the skin is reduced and wrinkles are formed. Therefore, there is a need for development of a substance capable of regulating or inhibiting MMP expression in which activation is induced in cells.

On the other hand, alopecia in which hair falls from the scalp is caused by a number of factors.The causes of alopecia include internal factors such as genetic constitution and the action of male hormones, and mental stress or lipid peroxide accumulation in daily life. There are external factors, and these factors are known to be complicated and show signs of hair loss. In recent years, hair loss of women as well as male hair loss occurs a lot, especially in the young people are a trend that is gradually spreading, many kinds of hair growth or hair growth agents are commercially available to improve the hair loss phenomenon.

Commercially available hair regrowths or hair growth agents include vasodilators such as capronium chloride, minoxidil and various extracts, and hormones such as estrogens and estradiol for inhibiting male hormone action. Among these, minoxidil and capronium chloride are widely used for the treatment and prevention of alopecia, but the clinical effects are not excellent and there are side effects that inhibit male function.

Accordingly, an object of the present invention is to develop and provide a novel cosmetic composition having wrinkle improvement, anti-aging and hair growth promoting ability.

In order to achieve the above object, the present invention provides a cosmetic composition for improving wrinkles, comprising a silicate containing an active ingredient.

The present invention also provides an anti-aging cosmetic composition characterized in that the silicate contains an active ingredient.

The present invention also provides a cosmetic composition for promoting hair growth, comprising silicate as an active ingredient.

Hereinafter, the means for solving the problems of the present invention will be described in detail.

In the present invention, to investigate the effect of silicate on the skin, cell cytotoxicity, cell activity, degree of inhibition of MMP-1 that degrades collagen, collagen type I biosynthesis, degree of antioxidant effect Was investigated.

In the cytotoxicity experiment to determine the irritation of the raw materials used in cosmetics, the minimum concentration of the raw materials causing the irritation is set to 1.0%. That is, when 1.0% treatment of the cell survival rate of 90% or more evaluated as a safe raw material, the silicate as an active ingredient of the present invention was evaluated as a safe raw material without causing cell death at 1.0%.

In the field of cosmetics, the evaluation of cellular activity has been used as an important indicator for evaluating the overall efficacy of skin aging, wrinkle improvement and wound healing effects. This is because fibroblasts synthesize collagen involved in the prevention of wrinkles depending on the activity of the cells constituting the dermal layer. As a result of the cell activity test, silicate was weaker than TGF-β (148%), but showed excellent cell activity effect at 142% and 138% in 1.0% ~ 0.5%, respectively.

On the other hand, among the dozens of MMPs produced in the body, MMP-1 is a proteinase that acts specifically on collagen. By inhibiting the activity of MMP-1, it protects collagen and maintains elasticity of skin tissues. It is known to prevent production (Mosmann et al; 1983). The expression of MMP-1, which plays an important role in skin photoaging, increases MMP-1 expression through a signal transduction pathway that increases JNK / p38 activity in cells by UVA and increases the activity of the transcription factor AP-1. Is known to cause collagen deficiency in skin (Chung et al; 2000).

In the evaluation of inhibition of collagen-degrading MMP-1, the silicate was not different from the control group that was not irradiated with UV light, but the inhibition of MMP-1 expressed after UV induction was about 33% at 1.0% of treatment concentration. It was shown to inhibit about%.

Collagen is a major constituent protein of about 90% of the dry weight of skin among the components constituting the connective tissue of skin cells. Therefore, the degradation of collagen can be evaluated to directly affect the elasticity of connective tissue and wrinkles and sagging of the skin. So far, about 19 kinds of collagen are known, and a difference in elasticity and tensile strength appears depending on the type of collagen. In the skin, collagen is mainly synthesized in the fibroblasts constituting the dermal layer, and collagen type I and collagen type III account for about 85%, and collagen type II, IV, and IX. This is about 15%. In infancy, collagen type III accounts for about 85%, and as adulthood, collagen type III gradually decreases and collagen type I increases. Thus, in adulthood around 20 years old, collagen type I accounts for 85%. For this reason, collagen type I is quantified in evaluating the degree of collagen synthesis.

In the collagen type I biosynthesis promotion evaluation, silicates showed lower biosynthesis promotion results than positive control, but collagen is effective in cosmetics given that positive control is a growth factor. It was evaluated to be usable as a production promoting factor.

On the other hand, in order to measure the antioxidant effect of the silicate was measured the superoxide (superoxide) anion inhibitory action by DPPH and xanthine / xanthine oxidase (Xanthine / xanthine oxidase).

From the reduction of DPPH (1,1-Diphenyl-2-picrylhydrazyl) radical, it can be seen that the scavenging reaction of free radical proceeds, and the degree of inhibition of the initial reaction of lipid peroxidation can be evaluated. Inhibition of superoxide anion by xanthine / xanthine oxidase is shown by superoxide anion scavenging and xanthine oxidase inhibition.

Silicate was lower than the positive control group for the two radical scavenging functions, but it was significant compared to the negative control group.

On the other hand, the present invention is to measure the hair growth effect of silicate gross observation, histological observation, ALP (Alkaline phosphatase) and γ-GT (γ-glutamyl transpeptidase) activity, EGF (Epidermal growth factor) and VEGF (Vesicular) epidermal growth factor) activity and TGF-β1 expression levels were measured. Silicate aqueous solution showed a hair growth effect similar to minoxidil used as a hair regrowth through the animal test, and histological observation confirmed the effect of promoting hair growth by affecting the hair follicle and hair root growth of the skin. .

 Alkaline phosphatase (ALP) is known as an indicator of angiogenesis in the hair growth cycle, and γ-glutamyl transpeptidase (γ-GT) is known as an indicator of angiogenesis in the hair growth cycle. At the beginning of the cycle (1-2 weeks), it showed an effect of activating ALP and γ-GT enzymes that promote angiogenesis.

 EGF (Epidermal growth factor) is a substance that induces epithelial production. It is a regenerative factor that affects hair follicles and dermis. It directly affects skin regeneration and hair growth. Vesicular epidermal growth factor (VEGF) forms blood vessels in the epithelium. It is a substance that induces. Silicate aqueous solution was found to promote EGF production and slightly increase the production of VEGF.

As a result of confirming the expression of TGF-β1 growth factor, a negative regulator in skin tissue, it was confirmed that silicate inhibits the expression of TGF-β1 growth factor in skin tissue.

On the other hand, the silicate used in the present invention is preferably used in an aqueous solution, and in addition to the silicate, other components that can give a synergistic effect to the main effect, preferably within the range of not impairing the main effect of the present invention. It may also contain such.

In any one selected from the cosmetic composition for anti-wrinkle and anti-aging cosmetic composition containing the silicate of the present invention as an active ingredient, the formulation is not limited to a specific kind, for example, lotion, gel, water-soluble liquid, cream, essence Foundation cosmetic formulation consisting of oil-in-water (O / W) and water-in-oil (W / O) type, oil-in-water and water-in-oil makeup base, foundation, skin cover, lipstick, lip gloss, two-way cake, face powder, eye bird Also, it may have any one appearance selected from tincture cosmetic formulations consisting of cheek color and eyebrow pencils. It may optionally be applied to the skin in the form of an aerosol or may be in the form of a solid, such as a stick. It may be used as a skin care product and / or makeup product.

In addition, the hair growth cosmetic composition of the present invention may be any one selected from hair tonic, hair lotion, hair cream, shampoo, rinse.

The composition according to the invention may also contain conventional auxiliaries such as hydrophilic or lipophilic gelling agents, hydrophilic or lipophilic active agents, preservatives, antioxidants, solvents, fragrances, fillers, blockers, pigments, odorants and dyes in the cosmetic field. have. The amounts of these various adjuvants are amounts commonly used in the art, such as 0.0001 to 30% by weight relative to the total weight of the composition. In any case, the adjuvant and its proportions will be chosen so as not to adversely affect the desirable properties of the cosmetic composition according to the invention.

As described above, the present invention has no cytotoxicity, excellent cell activity, inhibits the activity of collagen degrading enzyme MMP-1, and promotes collagen type I biosynthesis to exert an excellent wrinkle improvement effect. .

In addition, the present invention is excellent in the antioxidant effect exhibits an anti-aging effect.

In addition, the present invention is the activity of ALP (Alkaline phosphatase) known as an indicator of angiogenesis in the hair growth cycle and γ-glutamyl transpeptidase (γ-GT) known as an indicator of angiogenesis in the hair growth cycle. Substantial hair growth due to its excellent effect of promoting the production of epidermal growth factor (EGF), which induces epithelial production, and vein epidermal growth factor (VEGF), which induces blood vessel formation in the epithelium, and suppressing the expression of TGF-β1 growth factor. It is effective.

1 is a diagram showing the cytotoxicity of silicates.
2 is a diagram showing the cell activity of silicate.
3 is a diagram showing the MMP-1 inhibitory effect of silicate.
4 is a diagram showing the collagen type I biosynthesis effect of silicate.
5 is a diagram showing the scavenging ability of the silicate DPPH radicals.
6 is a diagram showing the superoxide radical scavenging ability of the silicate.
Figure 7 is a diagram showing the weight change of the experimental animals coated with silicate.
Figure 8 is a diagram showing the feed intake of experimental animals coated with silicate.
9 is a diagram showing the drinking water of the experimental animals coated with silicate.
10 is a diagram of visual observation of wool of experimental animals coated with silicate.
11 is a view showing the histological changes of wool of the experimental animals coated with silicate.
12 is a view showing the change in ALP (Alkaline phosphatase) activity of the experimental animals coated with silicate.
Figure 13 is a diagram showing the change of γ-GT (γ-glutamyl transpeptidase) activity of the experimental animals coated with silicate.
14 is a diagram showing changes in EGF (Epidermal growth factor) activity of the experimental animals coated with silicate.
15 is a view showing the change of VEGF (Vesicular epidermal growth factor) activity of the experimental animals coated with silicate.
Figure 16 is a diagram showing the expression level of TGF-β1 of experimental animals coated with silicate.

Hereinafter, the content of the present invention will be described in more detail in the following examples, but the scope of the present invention is not limited only to the following examples, and includes modifications of equivalent technical ideas.

Preparation Example 1 Preparation of Silicate Standard Aqueous Solution

After adding 1000 mL of distilled water to 14 g of silicate finely ground to 0.01 to 0.05 mm, the silicate was dissolved while mixing at 60 ° C. to 70 ° C. for 24 hours. Filtered with 2 filter paper 'to prepare a saturated solution of 100% silicate.

Experimental Example 1: Measurement of Cytotoxicity

The most important thing in cosmetics should not cause skin irritation due to cosmetic base. By evaluating the cytotoxicity of the cosmetic raw material can be predicted the presence or absence of skin irritation, the cytotoxicity measurement on the standard silicate aqueous solution obtained in Preparation Example 1 was performed to set the concentration for various evaluation.

Experimental cells were human normal fibroblast and melanoma cells (Mouse melanoma, B16F1).

As a test material, 100% silicate saturated aqueous solution obtained in Preparation Example 1 was diluted to 0.125, 0.25, 0.5, 1.0, 2.0%, and PBS Buffer was used as a control.

Cytotoxicity was modified by Mosmann's method of measuring cell viability using MTT (3- (4,5-dimethylthiazol-2-yl) -2-5-diphenyltetrazolium bromide) reagent. Thawed cells were inoculated into 96-well plates at 2 × 10 ⁴ cells / well concentration using 10% FBS DMEM, and then incubated in a CO ₂ incubator for 24 hours. After 24 hours, the fresh culture medium was replaced and the experimental sample was processed. After 24 hours, MTT solution (5 µg / ml) was added and after 4 hours, the supernatant was removed by centrifugation, and 100 µl acid-isopropanol (0.04 N HCl in isopropanol) was added. Absorbance was measured at 565 nm with a micro plate reader (BIO-TEK Instruments, USA) to allow color formazan to elute. Three replicates were performed to evaluate the significance of the experiment.

All experimental results were expressed as mean ± standard deviation, and statistical significance test was conducted by Students t-test, and statistically significant when p value was less than 0.05. The same applies to the analysis of the experimental results.

Measurement result (Fig. 1), judging the presence or absence of irritation by the cosmetic substrate is evaluated as a safe raw material when the survival rate of the cell more than 90% at 1.0% treatment, the silicate added as an active ingredient of the present invention constitutes the human dermal layer Both the fibroblasts and the melanoma in the basal layer of the epidermis showed over 90% survival even with 2.0% treatment, making it a safe material for skin.

In addition, the optimum concentration for the efficacy and efficacy evaluation using the cell culture technology was set to 1.0% by the experimental results.

Experimental Example  2: cell activity measurement

In Experimental Example 2, the effect of silicate on the fibroblasts constituting the dermal layer was evaluated to determine whether it can be used as a material for wrinkle improvement and wound healing.

Experimental cells were human normal fibroblast (HDF).

As a test material, 100% silicate saturated aqueous solution obtained in Preparation Example 1 was diluted to 0.125, 0.25, 0.5, and 1.0%, respectively, and 10 ng / TGF-β (Transforming growth factor-β) was used as a positive control. ml treated.

Cell activity was measured by the following method.

The thawed HDF was inoculated into a 24-well plate at a concentration of 1 × 10 ⁵ cells / well using DMEM containing 10% FBS, followed by CO ₂ The incubator was incubated for 24 hours. After 24 hours, the cells were exchanged with DMEM containing 0.5% FBS (growth 0%), and the experimental samples were treated with 1.00%, 0.50%, 0.25%, and 0.13%, respectively, and cultured continuously for 48 hours. After 48 hours, Mosmann's method of measuring cell activity using MTT (3- (4,5-dimethylthiazol-2-yl) -2-5-diphenyltetrazolium bromide) reagent was modified. After 48 hours, MTT solution (5 μg / ml) was added and after 4 hours, the supernatant was removed by centrifugation, and 100 μl acid-isopropanol (0.04 N HCl in isopropanol) was added. The blue formazan was eluted and the absorbance was measured at 565 nm with a micro plate reader (BIO-TEK Instruments, USA). As a positive control group, 10 ng / ml of TGF-β was used, and three replicates were performed to evaluate the significance of the experiment.

As a result of the measurement (Fig. 2), the silicate was weaker than the positive control TGF-β (148%), but showed a relatively good cell activity effect of 142% and 138% in 1.0% to 0.5%, respectively. This generally belongs to the category of excellent efficacy in the development of cosmetic materials aimed at cellular activity. From these results, silicate promotes the activity of fibroblasts to prevent wrinkle formation and to be used as a material for wound healing. Inferred.

Experimental Example  3: ELISA Through the MMP -1 activity inhibition mechanism evaluation

To investigate the effect of silicate on the expression of MMP-1, which is increased by UV light, human skin fibroblasts were irradiated with UV light, cultured for 48 hours with various concentrations of silicate, and the amount of MMP-1 synthesis was measured. .

Experimental cells were human normal fibroblasts.

As a test material, 100% silicate saturated aqueous solution obtained in Preparation Example 1 was diluted, and a positive control was treated with 10 ng / ml of TGF-β (Transforming growth factor-β).

As a result of the experiment of cell survival rate in Experimental Example 1, the silicate affects the survival rate of cells at 1.0% or more. In Experimental Example 3, MMP-1 expression inhibition mechanism was evaluated at 1.0%, 0.5%, and 0.25% of silicate. .

Human skin fibroblasts were incubated at a concentration of 1.5 × 10 ⁵ cells / ml until they were grown to about 80% in 35 mm diameter dishes. After removing the original medium before the ultraviolet irradiation, washed with PBS to remove the serum components in the medium, and then irradiated with ultraviolet A (6.3 J / ㎠) with an ultraviolet irradiation device (Sankyo Denki, Japan). After irradiation with UV A, the silicate was diluted to 0.50, 1.00, 2.00% in DMEM / F12 (3: 1) medium without FBS, and then incubated for 48 hours. TGF-b (10 ng / ml) was used as a positive control.

The amount of MMP-1 synthesis induced by UVA irradiation was measured by an enzyme linked immunosorbent assay (ELISA) method using the MMP-1 Biotrok ™ ELISA kit (UK). First, the human skin fibroblasts were irradiated with ultraviolet A (6.3 J / cm 2), and then treated with a sample and cultured for 48 hours, the micro-coated medium coated with a monoclonal antibody (monoclonal anti-MMP-1) against MMP-1. 100 μl was dispensed into each well of the plate, and the lid was kept warm at room temperature (20-25 ° C.) for 2 hours. After removing the contents of each well, the washing process was repeated three times with washing buffer. After the washing solution was completely removed, 100 μl of a polyclonal anti-MMP-1 antibody to MMP-1 was dispensed into each well, and the lid was kept warm at room temperature for 2 hours. After the contents of each well were removed, the washing process of the washing buffer was repeated three times. After the washing solution was completely removed, 100 μl of a oxidase conjugate was dispensed into each well. Warm for 1 hour at room temperature. After the warming, the washing process of the washing buffer was repeated three times to remove the unreacted antibody, and 100 μl of the diluted substrate solution was dispensed into each well, followed by warming at room temperature for 30 minutes. . 100 μl of the stop solution was added to each well, and the absorbance was measured at 450 nm using a micro plate reader (USA) within 30 minutes. The concentration of the sample against the measured standard was calculated.

As a result of the measurement (FIG. 3), the silicate was not different from the control group not irradiated with ultraviolet rays. However, inhibition of MMP-1 expressed after induction of ultraviolet rays was shown to inhibit from 1.0% to about 33%. From this, it could be deduced that silicate has an effect of inhibiting the synthesis of MMP-1, which is increased in expression by ultraviolet rays.

Experimental Example  4: ELISA To accelerate collagen type I synthesis

In Experimental Example 4, the degree of promoting synthesis of collagen type I to silicate was evaluated using fibroblasts (HDF, Human Dermal Fibroblast, Normal, passage 11).

Human cells used human normal fibroblasts.

As a test substance, 100% silicate saturated aqueous solution obtained in Preparation Example 1 was diluted, and a positive control was treated with 10 ng / ml of TGF-β (Transforming growth factor-β). The negative control was a medium containing 10% FBS without any treatment.

As a result of evaluating the cell viability in Experimental Example 1, it was confirmed that the silicate affects the survival rate of the cell at 1.0% or more, this Experimental Example 4 collagen at 1.0%, 0.5%, 0.25%, 0.125%, 0.064% silicate The degree of synthesis of type I was evaluated.

The thawed cells were incubated with DMEM (high glucose) containing 10% FBS in a T75 culture flask, and the cells were suspended with Trypsin-EDTA to form a 48-well plate at a concentration of 5.0 × 10 ⁴ cells / ml. Seeding was performed by 500 ul. After 24 hours, the cells were replaced with fresh culture medium, and then treated with the culture medium, diluted with 1.0%, 0.5%, 0.25%, 0.125%, 0.064%, and incubated for 48 hours.

The determination of collagen type I in fibroblasts was carried out by an immunosorbent assay (ELISA) using the 'Procollagen Type I c-peptide EIA kit (TaKaRa, Japan)'. The cell culture supernatants incubated for hours were carefully collected. First, 100 ul of culture supernatant cultured for 48 hours was added to an antibody-coated strip that binds to 'Procollagen Type I c-peptide' and reacted at 37 ° C. for 2 hours. After the reaction, washing three times using a washing buffer (washing buffer) to remove the unreacted material, and then put a blocking buffer (blocking buffer) 100 ul each, and reacted for 2 hours at 37 ℃ again. After the reaction, washed three times using a washing buffer (washing buffer) to remove the unreacted material, a solution conjugated to the POD (Antibody-conjugated) to another antibody binding to 'Procollagen Type I c-peptide' (Antiobody- POD conjugate solution) 100 ul each was reacted for 2 hours at 37 ℃. After the reaction, washing three times with a washing buffer (washing buffer) to remove the unreacted material, the same amount of the substrate (substrate) solution, reacted for 30 minutes and then stop solution (1N H ₂ SO ₄ ) The experiment was terminated. The strips at which the experiment was completed were measured for absorbance at 450 nm to evaluate the degree of collagen synthesis in comparison with the collagen standard.

As a result (Fig. 4), 0.5% silicate treatment showed a collagen type I synthesis promoting effect significant for the negative control (negative control), especially when treated with silicate 1.0% (211.4 ng / ml) It was evaluated to promote 57.4% collagen type I biosynthesis than negative control.

From these results, it was inferred that silicate could be used as an effective collagen production promoting factor in cosmetics.

Experimental Example  5: measure antioxidant effect

In Experimental Example 5, the effectiveness of the silicate was evaluated to verify the effectiveness on the skin.

As a test substance, 100% silicate saturated aqueous solution obtained in Preparation Example 1 was diluted to 0.25, 0.5, 1.0, 2.0, and 4.0%, respectively. As a positive control, vitamin C (10 ug / ml) and BHA (30 ug) were used. / ml) was used.

Anti-oxidative effects were measured by measuring DPPH radical scavenging activity and Superoxide radical scavenging activity.

Free radical scavenging activity using DPPH (2,2-Di (4-tert-octylphenyl) -1-picrylhydrazyl) was measured by measuring the radical scavenging effect using 0.1 mM DPPH (Blois). et al., 1958).

The same amount of sample was added to a 0.1 mM DPPH-containing methanol solution, mixed well with a vortex mixer, and then reacted at 37 ° C. for 10 minutes, using a spectrophotometer at 565 nm. Absorbance was measured.

Superoxide radical scavenging ability formed in the xanthine / xanthine oxidase reaction was measured by the 'SOD test' method (Furuno et al., 2002).

3 mM xanthine, 3 mM EDTA, 0.72 mM NBT and DFO (3,9-diferuloyl-6-oxopterocarpen) were added to 0.05 M Na ₂ CO ₃ buffer (pH 10.2), followed by reaction at 25 ° C for 10 minutes. . 0.25 U / ml xanthine oxidase was added to the reaction solution and reacted at 25 ° C. for 25 minutes, and then the absorbance was measured at 565 nm for the superoxide radical scavenging effect.

First, as a result of measuring the antioxidant effect of silicate using DPPH (Fig. 5, when the control is 100%, expressed as% for the control), the silicate is 4.00% to 27.44%, 2.00% to 18.26% DPPH radical (radical) was erased.

As a result of measuring the inhibition of the production of superoxide anions formed by Xanthine oxidase (FIG. 6, when the control is 100%, it is expressed in% of the control), the silicate is 4.00% to 36.56 %, 2.00% showed a superoxide radical scavenging effect of 18.20%.

From the above results, it could be inferred that silicate showed significance in the scavenging function of the two radicals.

Experimental Example  6: measurement of hair growth effect

Saturated aqueous silicate solution (100%), 50% diluent was used as a test substance to measure the hair growth effect of silicate. Distilled water as a negative control, 3% or 5% MXD (minoxidil) as a positive control was purchased from Hyundai Pharmaceutical (Cheonan, Chungcheongnam-do) was used.

Six weeks old male C57BL / 6 was purchased and adapted to the laboratory for one week, then seven weeks old was used for the experiment. Animal cages were kept at a constant humidity (50 ± 10%) and at a constant temperature (23 ± 3 ° C), and were housed in 5 mice in an isolated mouse cage. Feed and water were freely fed. A total of 72 animals were used for the experiment, 18 animals per group. The mouse cage used the cage of Samkwang Co., Ltd.

Experimental Example  6-1: drinking water, feed intake and weight

  Body weight was measured once a week from the day of the first start until the end of the experiment. Negative and feed intakes were also measured immediately before application of the hair growth test material and measured at a constant time (2 pm) until the end of the experiment. Clinical symptoms were also observed daily.

As a result of the measurement (Figs. 7 to 9), the body weight of 57BL / 6 mice gradually increased with the duration of the test, and there was no significant difference between the test substance administration group and the control group, and the change in feed intake and drinking volume was also tested. No difference between groups was observed.

Experimental Example  6-2: Hair Loss Treatment and Application of Hair Loss Agent

 In order to apply the resting period of the hair circulator, the hair of the back was removed using an electric razor. No damage was done to the skin during removal, and there was no evidence of inflammatory symptoms. After 1 day of depilation, each sample was applied to the depilated site. Sample processing was performed once every 21 days at 2 pm. After spraying a certain amount (0.2 ml) of test substance on the shaved part, it was evenly distributed by rubbing with a brush. Distilled water as a negative control, 5% minoxidil (MXD) as a positive control, and test substance were applied.

The observation of hair growth was performed by visual observation and histological analysis.

  On the 0, 4, 7, 10, 17, 14, and 21 days after the start of the experiment, anesthesia was performed with ether to visually confirm the hair growth condition. Then, photographs were taken. Judged by visual comparison. The camera used Nikon's D70 model (Tokyo, Japan). The hair growth effect of each group was observed by visual observation of each animal, depending on the degree of hair growth, 0-19% (-), 20-39% (±), 40-59% (+), 60-79% ( ++), 80-100% (+++).

Samples were removed on day 21 of the test for histological analysis of hair growth and fixed using 10% formalin buffer. After stepwise dehydration with alcohol and xylene and embedded in paraffin, 4μm sections were prepared using a microtom (HM-315 model, Microm Co., Germany) and paraffin was removed again with alcohol and xylene. Staining with hematoxylin and eosin was used to observe the histological changes of the hair follicle tissue under an optical microscope (Olympus, Tokyo, Japan).

Weeks Groups Distilled Water (D.W.) MXD 50% Silicate (Na ₂ SiO ₃ ) 100% Silicate (Na ₂ SiO ₃ ) One - - - - 2 - ++ ± + 3 + +++ +++ +++

As a result of visual observation of hair growth (Table 1, Fig. 10), almost no hair grew up to about 7 days in all test groups. The MXD treated group, which was a positive control group, showed an increase in hairs after 10 days, compared to the negative control group, and the silicate (Na ₂ SiO ₃ ) treated group also started to grow hair on the back 10 days after the treatment. . After 14 days of experiment, the MXD treated group was able to observe almost 80% hair growth on the back of the mouse. The silicate (Na ₂ SiO ₃ ) treated group grew hair faster than the negative control on day 14 but was slower than the positive control. Hair growth effect.

As a result of histological analysis of hair growth (FIG. 11), the positive control group and the test substance applying group increased the number and size of hair follicles more than distilled water, which was a negative control group, and there were no inflammatory or other pathological findings. The shape of was formed long.

Experimental Example 6-3: Enzyme Activity Measurement

  In Experimental Example 6-3, the activities of ALP (Alkaline phosphatase) and γ-GT (γ-glutamyl transpeptidase) were measured.

On the 21st day of the test, the dorsal skin was dissected and rapidly frozen in liquid nitrogen. A constant weight (1 g) of skin was excised and placed in 4-fold 0.1M phosphate buffer to homogenize tissue and buffer. After centrifugation at 12,000 rpm for 20 minutes, the supernatant was collected and used for enzyme activity measurement. ALP (Alkaline phosphatase) activity was measured by enzyme ratio measurement (GSCC) method, γ-GT (γ-glutamyl transpeptidase) activity was measured using the 'Þ-nitrophenolphosphate (PNPP) -DEAV' method.

Hitachi-747, an automatic biochemical analyzer, was used for the measurement. All reagents were used by Daichi Corporation. Others were based on the clinical pathology standard.

First, as a result of measuring the activity of ALP (Alkaline phosphatase) (FIG. 12), the silicate treated group increased slightly after 1 to 2 compared with the control group, but no significant difference was observed, and the MXD treated group, which was a positive control group, also showed ALP after 1-2. Enzyme activity increased but no significant change.

As a result of measuring the activity of γ-GT (γ-glutamyl transpeptidase) (FIG. 13), the MXD treated group increased significantly compared to the control group after 2 weeks, and the silicate treated group was not significantly different but slightly after 2 weeks. There was a tendency to increase.

Experimental Example 6-4: Growth Factor Analysis

In Experimental Example 6-4, VEGF (vesicular epidermal growth factor) and EGF (epidermal growth factor) were measured.

After binding a specific antibody to the vesicular epidermal growth factor (VEGF) and epidermal growth factor (EGF) of the mouse, it was measured.

The experimental group and the control group were put in each well and allowed to react, followed by washing after 2 hours, and the enzyme-linked polyantibody was added to each well. After the reaction for 2 hours, washed again, when the blue color of the reaction stop material was added to each well (well) was measured. VEGF and EGF measurements were purchased from R & D SYSTEMS kit (Mineapolis, MN, USA). Growth factor analysis was carried out by the test method listed in the kit.

First, as a result of measuring EGF (epidermal growth factor) (Fig. 14), it showed a significant increase in the MXD treated group and the silicate treated group compared to the control group (p <0.05), from which the silicate was the same as minoxidil (minoxidil) It could be inferred from the mechanism that it directly affects hair growth.

As a result of measuring VEGF (Vesicular epidermal growth factor) (FIG. 15), the MXD and silicate treatment groups showed higher VEGF activity than the negative control group, but no statistically significant level was observed.

Experimental Example 6-5: Western blot analysis of TGF-β1

Skin tissue was homogenized in protein prep (iNtRON Biotechnology, Seongnam, Korea). Proteins were quantified by Bradford protein assay (Bradford MM, 1976). After loading 30 μg of protein, electrophoresis was performed on 12% SDS PAGE, and the protein was transferred to PVDF membranr (Hoefer Inc., San Francisco, USA). Blocking on 5% BSA, TGF-β1 antibody (1: 500, Santa Cruz Biotechnology, Inc., California, USA) or β-Actin (1: 2000, Cell Signaling Technology Inc., Boston, USA) primary antibody diluted in 3% BSA Overnight at ° C. Then, the secondary antibody 'anti-rabbit horseradish peroxidase-conjugated antibody (1: 4000, Santa Cruz Biotechnology, Inc., California, USA)' or 'anti-mouse horseradish peroxidase-conjugated antibody (1: 4000, Santa Cruz Biotechnology , Inc., California, USA) 'was reacted for 1 hour at room temperature, and then developed to measure protein expression rate.

As a result of the measurement (FIG. 16), it was confirmed that the expression level of the silicate treated group as a test substance was weak compared to the 5% MXD treated group as the positive control group. However, the expression level was confirmed to show a similar pattern through the hair cycle.

In addition, the expression level of the silicate treatment group was similar to that of the positive control group in the period from catagen to talogen phase where the growth factor of TGF-β1 showed the highest activity. When compared, the difference was obvious. By confirming the expression of the negative regulator TGF-β1 in the skin tissue of the silicate treatment group, it could be inferred that the use of the silicate compound can help hair regrowth.

Claims (3)

Wrinkle improvement cosmetic composition comprising a silicate as an active ingredient.
Anti-aging cosmetic composition comprising a silicate containing an active ingredient.
Hair growth promoting cosmetic composition comprising a silicate containing an active ingredient.

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