KR20050059026A - N-butyric acid with preventing or improving activity of skin wrinkle and a cosmetics composition containing histone deacetylase inhibitor - Google Patents

Abstract

The present invention is a histone deacetylase containing n-Butyric Acid (n-Butyric Acid) excellent in preventing and improving skin wrinkles by promoting the expression of enzymes that promote the expression of dermal constitutive proteins and inhibit the dermal constituent proteins The present invention relates to a cosmetic composition comprising an inhibitor, and may be used in a functional cosmetic form by using a diluent or excipient such as a filler, an extender, a binder, a humectant, a disintegrant, a surfactant, or by adding to a variety of cosmetic bases. Suspensions, solvents, emulsions and the like can be used as various cosmetic base materials using simple diluents such as water, liquid, paraffin, and excipients such as wetting agents, sweeteners, fragrances, and preservatives as liquid preparations for skin administration.

Description

Cosmetic composition comprising a histone deacetylase inhibitor including en-butyric acid having excellent skin anti-wrinkle activity {N-BUTYRIC ACID WITH PREVENTING OR IMPROVING ACTIVITY OF Skin Wrinking

The present invention relates to a cosmetic composition comprising a histone deacetylase inhibitor including n-butyric acid having excellent skin wrinkle prevention and improvement activity, and more specifically, collagen and elastin in cells. It relates to a cosmetic composition comprising a histone deacetylase inhibitor including en-butyric acid which increases the expression of and has the effect of preventing or improving skin wrinkles in a hairless mouse.

The skin consists of an upper layer, an epidermis of about 0.1 mm in thickness, a lower layer, a dermis of about 1-4 mm in thickness, and a basal layer. The causes of the appearance of wrinkles on the skin can be largely divided into the generation of wrinkles by natural aging and the generation of wrinkles by exposure to ultraviolet light (photoaging), and the mechanism of wrinkle formation by two causes has not yet been accurately identified. Not in condition.

Common phenomena in both aging are wrinkles and skin loss due to the loss and elasticity of collagen and elastin, the major constituent proteins in the dermis, as well as the reduction of the hyaluronic acid, the substrate of the dermis. According to the results of the previous studies, reduction and modification of these main proteins, collagen and elastin, are known to be directly involved in the generation of wrinkles and decrease in elasticity of the skin.

There are various kinds according to the mechanism of inhibiting aging of skin, but representatively, collagen production promoter, elastin activator, hyaluronic acid production increase agent, free radical scavenging agent, cell proliferative agent, photoaging inhibitor, keratinocyte proliferation inhibitor, It can be divided into moisturizer, harmful free radical generation agent, cell regeneration material and the like.

Currently applied methods to improve skin wrinkles include the prevention of wrinkle formation by antioxidants such as vitamin C, vitamin E, and carotenoids that inhibit free radical formation or reactions, and collagen biosynthesis in the dermis such as retinol. How to increase the amount, collagen is injected into the skin, botulinum toxin injection into the skin, and the like.

However, vitamin C, vitamin E, carotenoids, and retinol all break easily at room temperature. Retinol, in particular, is known to have side effects on the skin at high concentrations. Collagen infusions and toxin injections also provide temporary relief. There is a problem that can cause side effects.

The present invention was developed to solve the above problems, and provides a cosmetic composition comprising a histone deacetylase inhibitor including n-Butyric acid excellent in skin wrinkle prevention and improvement activity. There is a purpose.

In order to achieve the above object, the present invention first provides a Histone Deacetylase inhibitor including n-butyric acid excellent in fms skin wrinkle prevention and improvement activity according to the preferred embodiment 1 described below. Furthermore, the present invention provides a composition comprising a Histone Deacetylase inhibitor including n-butyric acid having excellent skin wrinkle prevention and improvement activities according to Example 2.

In general, n-Butyric Acid (n-BA) is one of enzyme inhibitors of Histone Deacetylase (HDAC) and is known to inhibit cell growth. Enzyme inhibitors of Histone Deacetylase have been attempted to be developed as anticancer drugs, but they are still not developed as anticancer drugs due to the effectiveness of their efficacy. In addition, n-Butyric Acid (n-BA) is known as a substance produced by decomposition of vegetables by the activity of digestive enzymes in the intestines when ingested. To date, it is known that there is no toxicity to the human body.

According to preferred embodiment 1 of the present invention described below, it is possible to provide a Histone Deacetylase inhibitor including n-Butyric Acid excellent in the prevention and improvement of skin wrinkles. That is, the Histone Deacetylase inhibitor including n-Butyric Acid of the present invention promotes the expression of dermal constituent proteins such as collagen or elastin to prevent the formation of skin wrinkles or to improve skin wrinkles that have already been formed.

The compounds of the present invention may be used in the form of general external medicines or functional cosmetic preparations, and when formulated, a diluent or excipient such as fillers, extenders, binders, wetting agents, disintegrating agents, surfactants, etc., which are commonly used, may be used, or various types may be used. It is prepared by adding to a cosmetic base.

In addition, liquid preparations for skin administration include suspensions, solutions, emulsions, and the like, and various excipients such as wetting agents, sweeteners, fragrances, and preservatives, in addition to commonly used simple diluents such as water, liquid, and paraffin, are included. And various kinds of cosmetic bases may be included.

That is, the composition including the Histone Deacetylase inhibitor including n-Butyric Acid of the present invention can be added to the functional cosmetics or drugs, and the kind of the functional cosmetics or drugs is not particularly limited.

Hereinafter, the present invention is intended to help more accurate understanding of the present invention. However, the following preferred embodiments are only presented to aid the understanding of the present invention, and the present invention is not limited to the following examples.

Until now, methods for measuring the prevention and improvement of skin wrinkles include dermal composition through changes in expression of dermal constituent proteins, that is, expression level of collagen or elastin that supports skin and collagenase protein that breaks down collagen. And a method for measuring the expression level of the protein. In Vivo models using a hairless mouse have also been accepted as the main method for conducting research on skin wrinkles.

In the following examples, the method for measuring the expression changes of the dermal constituent proteins through the expression analysis of proteins and the anti-wrinkle effect and improvement of the anti-wrinkle effect of the Histone Deacetylase inhibitor including n-Butyric Acid through In Vivo experiment using a hairless mouse It was.

Example 1 Preparation of Histone Deacetylase Inhibitor Composition Including n-Butyric Acid.

After dissolving n-Butyric acid in dimethyl sulfoxide (DMSO) at 100 mM, an aqueous solution was prepared at 5 mM and used for the experiment.

Example 2 Expression Changes of RNA and Protein of Dermal Constituent Protein Through Cell Culture.

end. Cell Culture of Human Fibroblasts

Dermal constitutive protein RNA expression and protein expression were investigated through cell culture of human fetal fibroblast cells established at Hallym University in Korea. As a cell culture method of human fibroblasts, a new petri with a dilution of 1: 4 (corresponding to division number 2) by culturing a human fibroblast cell line with Ethylen Diamine Tetra Acetic acid (EDTA) Substituting a large number of dishes with a continuous increase in number, the culture medium is DMEM (Dulbe's Modified Eagle's Medium) -10% fetal bovine serum (including penicillin-streptomycin) as a medium. And medium was changed every 3 days at 37 ° C., 5% CO ₂ .

I. Analysis of RNA Expression of Dermal Constituent Proteins through Cell Culture

By using the passaged human fibroblasts, each herbal medicine was treated to young cells with high expression of dermal constitutive RNA and aged cells with low expression of dermal constitutive protein RNA, and then expressed in cells three days later. Expression of dermal constitutive protein RNA was measured using Reverse Transciptase PCR (RT-PCR) method.

After washing the Petri dishes cells (young cells: fission 30, old cells: fission 60) treated with PBS (Phosphate Buffered Saline) twice to prepare RNA, RNA Zol B (Biotecx Co) Total RNA was prepared (0.2ml / 10 ⁶ Cells) and quantified by RT-PCR.

In other words, total RNA was first made of single-stranded DNA using 3′-side primer and reverse transcriptase, and the product was amplified using two primers and Taq Polymerase. The following known amplification products were separated by electrophoresis on Agarose Gel, stained with ethidium bromide and photographed. RNA expression of collagen and elastin is measured and compared for untreated and treated groups.

Therefore, as shown in FIG. 1, which shows the expression level of collagen RNA in Histone Deacetylase inhibitor treatment group including n-Butyric Acid and other herbal medicine treatment groups, n-Butyric Acid and herbal medicine extracts, etc., were shown to increase collagen RNA expression. As shown in the graph of FIG. 2, which is a result of measuring the RNA expression level of elastin by the same experimental method, n-Butyric Acid and herbal medicine extracts, etc. have been shown to increase elastin RNA expression.

All. Changes in Dermal Constituent Protein Expression through Cell Culture

Using the passaged human fibroblasts, n-Butyric Acid and each herbal medicine were treated to young cells with high expression of dermal constitutive proteins and aged cells with low expression of dermal constituent proteins, and then intracellularly 3 days later. The expression of dermal constitutive protein expressed in SDS PAGE (Polyacryamide Gel Electrophoresis) gel was electrophoretically separated and measured by Western blot method.

For measurement, the Petri dish cells treated with n-Butyric Acid and medicinal herb extracts (young cells: split water 30, old cells: split water 60) were washed twice with PBS (Phosphate Buffered Saline), and n-Butyric Acid and In order to investigate the expression of dermal constituent proteins after Chinese herbal medicine treatment, the cells were recovered using NET Buffer (150mM NaCl, 50mM Tris-HCl pH 7.4, 5mM EDTA) and the cells were eluted. After 20 μg of protein was electrophoresed at 150 V for 1 hour on an SDS PAGE gel, 70 V was transferred to a transfer membrane for 30 minutes, and the membrane was immersed in a milk solution and left overnight.

After washing with TBST washing solution (buffer solution) three times for 10 minutes, and reacted for 1 hour and 30 minutes using the primary antibody, washed three times with the washing solution and then for 1 hour with the secondary antibody and three times with the washing solution. Afterwards, the expression of the protein was examined using a method of photosensitive to the film by using the Enhanced Chemiluminsdcence (ECL) kit. As shown in FIG. 3, n-Butyric Acid, herbal medicines, and the like have been shown to increase collagen protein expression.

The same experimental method was used to investigate the expression change of elastin. As a result, n-Butyric Acid and herbal extract oak, etc. were shown to increase elastin protein expression.

As a result of investigating the expression changes and protein expression of the dermal genes using human fibroblasts, it can be seen that n-butyric acid and herbal medicine extracts increase the expression of dermal protein collagen RNA and elastin RNA at RT-PCR results. . In addition, n-butyric acid was found to increase the expression of collagen and elastin protein in protein expression analysis.

Example 3 Determination of wrinkle removal efficacy using a hairless mouse.

The anti-wrinkle effect of n-Butyric Acid, which was determined to have an anti-wrinkle effect in RNA expression analysis and protein expression analysis, was confirmed to be effective in preventing and improving wrinkles using hairless mice. Hairless mice used in the experiment were purchased from Charles River. The age of the mice used in the experiment was experimented for 8 weeks using the 6-week-old mice, which used mice that have not yet been wrinkled considering the degree of wrinkles of the hairless mouse.

During the experiment, the environment was maintained at 23 ± 2 ℃ and relative humidity of 55 ㅁ 10%. The animals were reared in four cages for 12 hours under artificial lighting. Feed and drinking water for experimental animals were supplied freely. In order to verify the anti-wrinkle effect of n-Butyric Acid on the bred mice, n-Butyric Acid was diluted with 1/50 of 5mM aqueous solution and herbal medicine extract oak to the concentration of 1/50 of the stock solution. I applied it. The reared mice were exposed to UV lamps three times a week, each time irradiated with UVA at 14 J / cm 2 and UVB at 20 mJ / cm 2. The extent of wrinkles was measured using the Donald Methods (Photochemistry And Photobiology 1987) method.

On the other hand, Figure 5a is a photograph of a mouse not exposed to UV and Figure 5b is a graph showing the result of measuring the wrinkles of the mouse not exposed to UV. In addition, Figure 6a is a picture of a mouse exposed to UV, Figure 6b is a graph showing the result of measuring the wrinkles of the mouse exposed to UV. In addition, Figure 7a is a photograph of a mouse exposed to UV after administration of herbal medicines, pill, Figure 7b is a graph showing the result of measuring the wrinkles of the mice exposed to UV after administering herbal medicines.

In addition, Figure 8a is a picture of a mouse exposed to UV after administration of n-Butyric Acid, Figure 8b is a graph showing the result of measuring the wrinkles of mice exposed to UV after administration of n-Butyric Acid. The results showed that the anti-wrinkle effect of n-Butyric acid was significantly higher than that of other extracts.

Reference numeral 1 is a control (untreated young fibroblasts), 2 is Bupyeongcho, 3 is Gangjinhyang, 4 is Plywood, 5 is Cheonhwangyeon, 6 is Geumchocho, 7 is Samchoroot, 8 is 5 oak, 9 is Sangbaekri , 10 is ginseng extract, 11 is ganoderma lucidum, 12 is phosphorus, 13 is motherwort, 14 is male, 15 is sujijihwang, 16 is geonjihwang, 17 is roots of bark, 18 is mold opening, 19 is n-Butyric Acid, 20 is DMSO, 21 represents a trade transcription reaction (non-response control) and 22 represents DNA (a DNA addition control instead of RNA). In addition, the same reference numerals shown in FIGS. 2 to 4 will be omitted the description overlapping with the same configuration.

In conclusion, Histone Deacetylase inhibitor including n-Butyric Acid induced not only increase expression of constituent protein of dermal cells but also increase RNA expression in experiments using human fibroblasts. Wrinkle prevention and improvement in wrinkle production experiment using hairless mouse It has been shown to have an effect. In addition, the treatment of n-Butyric Acid did not cause cancer or other side effects on the skin of hairless mice.

As described above, the present invention has an effect of developing and supplying a functional cosmetic for inhibiting wrinkles and a material derived therefrom using a Histone Deacetylase inhibitor including n-Butyric Acid which can remove and delay wrinkles.

In addition, the compounds of the present invention may be prepared by adding a diluent or excipient such as fillers, extenders, binders, humectants, disintegrating agents, surfactants, etc., which are commonly used, or by adding them to various cosmetic bases to form external pharmaceuticals or functional cosmetic preparations. It can be used as an effect.

In addition, the Histone Deacetylase inhibitor including n-Butyric Acid of the present invention has the effect of preventing the formation of skin wrinkles by improving the expression of dermal constituent proteins such as collagen or elastin or to improve the wrinkles already formed.

1 is a diagram showing a change in RNA expression of collagen in human fibroblasts treated with n-Butyric Acid and various herbal extracts of the present invention.

Figure 2 is a graph of the change in elastin RNA expression in human fibroblasts treated with n-butyric acid and herbal extract of the present invention.

Figure 3 shows the collagen protein expression changes in human fetal fibroblasts treated with n-butyric acid and various herbal extracts of the present invention.

4 is a diagram showing changes in elastin protein expression in human fibroblasts treated with the en-butyric acid and various herbal extracts of the present invention.

5A is a photograph of a mouse not exposed to UV.

Figure 5b is a graph of the results of measuring wrinkles of mice not exposed to UV.

Figure 6a is a photograph of the mouse exposed to UV.

Figure 6b is a graph of the results of measuring the wrinkles of mice exposed to UV.

Figure 7a is a photograph of the mouse exposed to UV after administration of the oak extract.

Figure 7b is a graph of the results of measuring the wrinkles of mice exposed to UV after administration of the oak extract.

8A is a photograph of mice exposed to UV after administration of en-butyric acid.

Figure 8b is a graph of the results of measuring the wrinkles of mice exposed to UV after administration of en-butyric acid.

Claims (1)

Histone Deacetylase Inhibitor with n-Butyric Acid with Excellent Activity to Prevent and Improve Skin Wrinkles by Promoting Expression of Dermal Protein and Inhibiting Dermal Protein Degrading Cosmetic composition comprising a.