KR20100104703A - Functional cosmetic composition for renewing skin and method for using the same - Google Patents

Abstract

PURPOSE: A functional cosmetic composition for skin regeneration is provided to easily facilitate transdermal absorption and to prevent skin aging. CONSTITUTION: A functional cosmetic composition for skin regeneration contains 5-aminolevulinic acid ester or cosmetically acceptable salt thereof as an active ingredient. The cosmetically acceptable salt of 5-aminolevulinic acid ester is a compound of chemical formula II (R_2·NH_2-CH_2-CO-CH_2-CH_2-CO-O-R_1). In chemical formula II, R1 is 2-prophenyl group(CH_2-CH=CH_2), 3-butenyl group(CH_2-CH_2-CH=CH_2), 4-pentenyl group (CH_2-CH_2-CH_2-CH=CH_2), 5-hexenyl group(CH_2-CH_2-CH_2-CH_2-CH=CH_2), methyl group(CH_3), or alkyl group; and R2 is HCl, H_2SO_4, H_3PO_4, acetic acid, benzoic acid, benzene sulfonic acid.

Description

Functional cosmetic composition for renewing skin and method for using the same

The present invention relates to a functional cosmetic composition, and more particularly, to a functional cosmetic composition for skin regeneration comprising a 5-aminolevulinic acid ester or a salt thereof as an active ingredient and a functional cosmetic composition for skin rejuvenation.

In general, 5-aminolevulinic acid (abbreviated as "ALA") is a major precursor of tetrapyrrole compounds such as heme, bacterio-chlorophyll and corinoid in all living systems. Animals and crops can be used as environmentally friendly herbicides that selectively weed without damaging them (Rebeiz CA, Montazer-Zouhoor A., Hopen H., and Wu SM, Enzyme Microb.Technol., 6). : 390, 1984).

In addition, ALA has been reported to be used as a bioactive material in medical fields such as skin cancer treatment and microbial resistance drugs. Photodynamics treatment using ALA is effective for actinic keratoses, psoriasis, and warts (verrucae). And skin cancers, and recently, in the workshops of the British Photodermatology Group, ALA-photodynamic therapy is used for UV keratosis, Bowen's disease and superficial basal cells. Good evidence has been shown to be effective against superficial basal cell carcinoma.

As such, 5-aminolevulinic acid, which is used as a topical photosensitizer for photodynamic therapy (PDT), is currently used in the treatment of acne and skin cancer as a topical photosensitizer certified by the FDA (food and drug administration). . 5-aminolevulinic acid is a precursor of photosensitizers used in photodynamic therapy. The application of ALA to the skin causes it to migrate into the cell, transforming the ALA into a practical photosensitive agent used for photodynamic therapy called Protoporphyrin IX (PpIX) through the biosynthesis process of Heme.

 PpIX is a very effective photoresist that is excited in triplet by light energy and reacts with surrounding oxygen to produce generator oxygen. The generated generator oxygen can cause damage to cell membranes and cell death, thereby treating skin diseases such as the above. On the other hand, ALA is relatively selectively absorbed in skin cancer or other pathological conditions and thus causes little harm to the surrounding normal tissues.

That is, when a light (light) of visible light (400nm-650nm) wavelength is irradiated to cells or tissues (skin) where PpIX is accumulated at a high concentration, PpIX selectively absorbs light and generates free active oxygen such as singlet oxygen. This is because the cells are selectively destroyed.

However, although the use of ALA appears to be quite advanced in the art, it is not always entirely satisfactory in photochemical treatment using ALA. This is because ALA cannot penetrate all tumors and other tissues with sufficient efficiency to treat a wide range of tumors or other conditions, and ALA also has the problem of becoming unstable in pharmaceutical formulations.

In order to solve this problem, 5-aminolevulinic acid ester has been proposed and used as a more stable photochemical therapeutic agent, but 5-aminolevulinic acid ester is similar to that of 5-aminolevulinic acid for photodynamic therapy (PDT). It is only used as a topical photosensitizer.

Cosmetics is a general term for all kinds of things used for make-up, such as creams and lotions. The definition of cosmetics in the Pharmacist Act is "a product used by painting, spraying and other similar methods to clean or beautify the human body. This little thing. " At the same time, they are regulated in all aspects, including manufacturing and sales, and advertising.

Cosmetics are primarily a means to keep the human body in a healthy state, but gradually protects the body from aging, ultraviolet rays, various contaminants and dryness, and makes it more beautiful and attractive to make you feel psychological satisfaction. Its meaning is expanding. Accordingly, cosmetics having various functionalities, such as UV protection, whitening, wrinkle improvement, and skin moisturizing ability, have been developed. Such cosmetics have their effects as ingredients containing respective functions.

In particular, cosmetics and medicines to improve skin wrinkles have been continuously developed so far, and recently, popular cosmetics are widely used cosmetics such as sunscreens, moisturizers and antioxidants to prevent wrinkles caused by external factors. The effect that the product can actually have on wrinkles is minimal. Therefore, there is a need to develop a novel functional cosmetic composition that can prevent or improve aging through skin wrinkle improvement.

The present inventors researched to develop a novel functional cosmetic composition that can prevent aging through skin wrinkle improvement, and as a result, the new use of ALA ester to accelerate the skin regeneration cycle when PpIX synthesized by ALA ester is activated The present invention has been completed.

Accordingly, an object of the present invention is to provide a functional cosmetic composition for skin regeneration comprising 5-aminolevulinic acid ester or a salt thereof as an active ingredient for skin regeneration, and to provide a novel use of 5-aminolevulinic acid ester or a salt thereof. To provide.

Another object of the present invention is to provide a functional cosmetic composition for skin regeneration useful for preventing and improving aging and skin care as well as having easy transdermal absorption and low cytotoxicity when applied to the skin.

Another object of the present invention is to regenerate the skin to obtain a skin regeneration effect by activating PpIX synthesized by 5-aminolevulinic acid ester only by exposure to sunlight or indoor light for a specific time without performing a separate phototherapy To provide functional cosmetic compositions.

Still another object of the present invention is to apply a functional cosmetic composition for skin regeneration comprising 5-aminolevulinic acid ester or a salt thereof as an active ingredient to the skin to speed up the skin regeneration cycle, thereby effectively preventing or improving aging. It is to provide a method of using a functional cosmetic composition for regeneration.

The objects of the present invention are not limited to the above-mentioned objects, and other objects that are not mentioned will be clearly understood by those skilled in the art from the following description.

In order to achieve the above object, the present invention provides a functional cosmetic composition for skin regeneration comprising 5-aminolevulinic acid ester or a cosmetically acceptable salt thereof as an active ingredient.

In a preferred embodiment, the cosmetically acceptable salt of the 5-aminolevulinic acid ester is a compound having the formula

Formula (II) R ₂ ㆍ NH ₂ -CH ₂ -CO-CH ₂ -CH ₂ -CO-OR ₁

Wherein R ₁ is 2-propenyl group (CH ₂ -CH = CH ₂ ), 3-butenyl group (CH ₂ -CH ₂ -CH = CH ₂ ), 4-pentenyl group (CH ₂ -CH ₂ -CH _2- CH = CH ₂ ), 5-hexenyl group (CH ₂ -CH ₂ -CH ₂ -CH ₂ -CH = CH ₂ ), methyl group (CH ₃ ), and a general alkyl group, and R ₂ is It is selected from the group consisting of inorganic acids or organic acids such as HCl, H ₂ SO ₄ , H ₃ PO _4, acetic acid, benzoic acid, benzene sulfonic acid.

In a preferred embodiment, the 5-aminolevulinic acid ester or cosmetically acceptable salt thereof is included in an amount of 5 to 15 parts by weight per 100 parts by weight of the cosmetic base.

In a preferred embodiment, the cosmetic base comprises at least one selected from the group consisting of stearyl alcohol, polyethylene glycol or purified water.

In a preferred embodiment, the cosmetically acceptable salt of the 5-aminolevulinic acid ester is hydrochloride.

The present invention is a skin regeneration, characterized in that the 5-aminolevulinic acid ester or a salt thereof contained in a single use amount of the functional cosmetic composition for skin regeneration according to any one of claims 1 to 5 is 50 to 75mg. It provides a method of using functional cosmetic composition.

In a preferred embodiment, the functional cosmetic composition for skin regeneration is applied to the skin and exposed to sunlight for 1 hour to 2 hours outdoors or indoors for 2 hours or more after a predetermined time.

The present invention has the following excellent effects.

First, the functional cosmetic composition for skin regeneration of the present invention includes 5-amino levulinic acid ester or a salt thereof as an active ingredient of skin regeneration, thereby providing a new use of 5-amino levulinic acid ester or a salt thereof.

The functional cosmetic composition for skin regeneration of the present invention is not only easy to transdermal absorption and low cytotoxicity when applied to the skin, but also has excellent skin regeneration effect and is useful for preventing and improving aging and skin care.

The functional cosmetic composition for skin regeneration of the present invention can obtain a skin regeneration effect by activating PpIX synthesized by 5-aminolevulinic acid ester only by being exposed to sunlight or indoor light for a specific time without performing a separate phototherapy. Therefore, it has ease of use.

By using the functional cosmetic composition for skin regeneration of the present invention by applying the functional cosmetic composition for skin regeneration comprising 5-amino levulinic acid ester or a salt thereof as an active ingredient to the skin to accelerate the skin regeneration cycle effectively aging Can be prevented or improved.

The terms used in the present invention were selected as general terms as widely used as possible, but in some cases, the terms arbitrarily selected by the applicant are included. In this case, the meanings described or used in the detailed description of the present invention are considered, rather than simply the names of the terms. The meaning should be grasped.

Hereinafter, the technical structure of the present invention will be described in detail with reference to the accompanying drawings and preferred embodiments.

However, the present invention is not limited to the embodiments described herein but may be embodied in other forms. Like reference numerals designate like elements throughout the specification.

First, the present invention is a PpIX synthesized by the ALA ester is activated only by being exposed to sunlight or a room for a specific time without performing a separate phototherapy after the ALA ester is applied to the skin to accelerate the skin regeneration cycle A new use of ALA esters and / or salts thereof is said to have an effective effect on regeneration. That is, after applying the functional cosmetic composition for skin regeneration of the present invention to the skin and after a predetermined time, preferably about 1 hour, it is exposed to sunlight for 1 to 2 hours outdoors or indoors for 2 hours or more. This is because PpIX can also be activated.

Accordingly, the present invention provides a functional cosmetic composition for skin regeneration comprising 5-aminolevulinic acid ester or a salt thereof as an active ingredient of skin regeneration, wherein the functional cosmetic composition provided by the present invention is transdermal absorption when applied to the skin. Not only is it easier and less cytotoxic, it can also speed up the skin regeneration cycle, effectively preventing or improving aging.

In particular, the 5-aminolevulinic acid ester contained in the functional cosmetic composition for skin regeneration of the present invention as an active ingredient of skin regeneration is preferably having the following formula.

Formula (I)

NH ₂ -CH ₂ -CO-CH ₂ -CH ₂ -CO-OR ₁

Wherein R ₁ is 2-propenyl group (CH ₂ -CH = CH ₂ ), 3-butenyl group (CH ₂ -CH ₂ -CH = CH ₂ ), 4-pentenyl group (CH ₂ -CH ₂ -CH _2- CH = CH ₂ ), 5-hexenyl group (CH ₂ -CH ₂ -CH ₂ -CH ₂ -CH = CH ₂ ), methyl group (CH ₃ ), and a general alkyl group.

In addition, it is preferable that the cosmetically acceptable salt of 5-aminolevulinic acid ester has the following formula (II).

Formula (II)

R ₂ ㆍ NH ₂ -CH ₂ -CO-CH ₂ -CH ₂ -CO-OR ₁

Wherein R ₁ is the same as formula (I), and R ₂ is an inorganic acid and organic acids forming a cosmetically acceptable salt, preferably HCl, H ₂ SO ₄ , H ₃ PO _4, acetic acid, benzoic acid, benzene sulfonic acid It is selected from the group which consists of inorganic acids or organic acids, such as these, Especially HCl is more preferable.

In addition, the functional cosmetic composition of the present invention preferably comprises 5 to 15 parts by weight of 5-aminolevulinic acid ester or a salt thereof per 100 parts by weight of the cosmetic base. If the content of the 5-aminolevulinic acid ester or cosmetically acceptable salt thereof is less than 5 parts by weight, it is difficult to obtain a skin regeneration effect through a simple procedure of the user, and if it exceeds 15 parts by weight, safety is achieved due to the high concentration without significant improvement of the effect. Not only does this fall, but the cost rises.

On the other hand, the functional cosmetic composition for skin regeneration of the present invention is prepared according to a conventional method. That is, the functional cosmetic composition containing the ALA ester or cosmetically acceptable salt thereof may be prepared in the form of a cream, essence, etc. by applying a known cosmetic base containing various cosmetically or dermatologically acceptable additives. Because.

Here, the cosmetic base includes water and / or an alcoholic solvent according to the solubility characteristics (described later) of the ALA ester or cosmetically acceptable salt thereof, any one selected from the group consisting of stearyl alcohol, polyethylene glycol or purified water. It is preferable to include the above. The additive may include a cosmetically acceptable skin softener, skin penetration enhancer, colorant, fragrance, emulsifier, thickening agent, and solvent, and the like, and in some cases, may include various functional ingredients effective for skin care such as collagen. There will be.

Example 1

ALA methyl ester hydrochloride was synthesized in the laboratory by known methods, which ALA methyl ester hydrochloride was produced in powder form.

An essence composition 1 was prepared by including 5 parts by weight of ALA methyl ester hydrochloride produced per 100 parts by weight of the cosmetic base configured as shown in Table 1 below.

That is, the thickener is added to purified water with slow stirring to disperse uniformly. The aqueous phase components are mixed to produce an aqueous phase. Sodium hymarunate is added to and dissolved in other cosmetic bases, including emulsifiers (glycerine), skin softeners (witch hazel), fragrances, and preservatives. The alcohol phase was added to the aqueous phase with stirring and an alkali agent was added to complete the essence composition 1.

ingredient content(%) Purified water 73.9 Polyethylene glycol 5.0 Stearyl alcohol 2.0 Vytein 3.0 glycerin 5.0 Medium Hi-Marunate 5.0 Caste oil 0.4 Witch hazel 2.0 Other cosmetic base 3.7 Sum 100

Example 2

An essence composition 2 was prepared in the same manner as in Example 1, except that 7.5 parts by weight of ALA methyl ester hydrochloride produced per 100 parts by weight of the cosmetic base was included.

Example 3

An essence composition 3 was prepared in the same manner as in Example 1, except that 10 parts by weight of the produced ALA methyl ester hydrochloride was included per 100 parts by weight of the cosmetic base.

Example 4

An essence composition 4 was prepared in the same manner as in Example 1, except that 15 parts by weight of ALA methyl ester hydrochloride produced per 100 parts by weight of the cosmetic base was included.

Hereinafter, various experiments were conducted to confirm the human stability and skin regeneration effect of the functional cosmetic composition of the present invention. The ALA and ALA esters used in the experimental examples mean hydrochloride salts of ALA and hydrochloride salts of ALA esters. Therefore, in order to activate the PpIX, a quick experiment was performed by irradiating the LED (25J) for 10 minutes instead of sunlight or indoor light.

Experimental Example 1

Solubility Test of ALA-Ester

Since ALA and ALA-ester prepared by a known method are produced in powder form, solubility experiments were performed to use them as cosmetic compositions, and the results are shown in Table 2.

From Table 2 it can be seen that ALA or ALA-ester is well soluble in alcohol and water. Therefore, in order to use the cosmetic composition, it can be seen that it is preferable to use the compound as a vehicle as a water-soluble solvent.

Experimental Example 2

Skin Cell Toxicity Test of ALA-Ester

The cytotoxicity of ALA and ALA-esters was tested using normal keratinocytes and human fibroblasts, which are normal cells of the skin, and the results are shown in Table 3 and FIG. 1. At this time, ALA (5-aminolevulinic acid) and ALA esters administered to the cells were ALA, ALA methylester (ALA-me), ALA butenylester (ALA-bu), ALA pentenylester (ALA-pe), and ALA hexenylester (ALA-hx ), And various concentrations ranging from 10 mM to 0.01 mM in serum free medium were administered to the cells.

In other words, in order to confirm cytotoxicity by ALA and ALA esters, colorimetric (MTT) kit (CHEMICON International Inc., USA), which can test cell viability, was performed according to the manufacturer's instructions. In more detail, after dispensing the cells in 96 well plates and treated with 100ul each of the medium containing ALA and ALA ester for each concentration was further incubated for 24 hours. 10 μl of each well of MTT (3,-(4,5-dimethylthiazol-2-yl) -2,5-diphenyl tetrasodium bromide) reaction solution was added and reacted for 2 hours to form a progressively colored Formazan crystal on the bottom of the well. To form. The crystals formed were dissolved in 100ul of dimethylsulfoxide (DMSO) to obtain a homogeneous solution. The absorbance was measured at an ELISA reader (Molecular Device, USA) under a wavelength of 570 nm.

As shown in Table 3 and FIG. 1, ALA esters in skin human keratinocytes (FIG. 1A) and fibroblasts (FIG. 1B) maintained cell viability of 80% or more at a concentration of 1 mM or less. In conclusion, the ALA esters of the present invention showed a slight difference in the concentration of each cell compared to the conventional ALA concentration, but generally less than 1mM cytotoxicity was less affected the cell viability.

Experimental Example 3

Since the measurement of PpIX synthesis amount is an important measure of whether the ALA ester of the present invention is an effective photosensitive material, in order to confirm how the actual photosensitive material PpIX is synthesized in the cell, the protoporphyrin IX (PpIX) Experiment was performed to measure the amount of synthesis, and the results are shown in FIGS. 2 and 4 and 5.

That is, in order to measure the amount of PpIX synthesized in the cells, 2 × 10 ⁵ cells were dispensed in 10 cm ² dishes (Falcon) and then ALA-ester was added to the medium when 70-80% confluent was obtained. To 0.001-1 mM and incubate for 4 hours. Next, after washing with PBS containing 1M HClO ₄ 50% methanol solution was added and collected by cell scraper. After standing at room temperature for 5 minutes, the resultant was centrifuged to extract PpIX from the supernatant.

As compared with the amount of PpIX synthesis induced by the ALA ester of the present invention and the conventionally developed ALA from Table 4, Table 5, and FIG. 2, the amount of PpIX synthesized in normal and cancer cell lines was compared with that of the conventional photosensitizer ALA. At low concentrations (0.01-0.1 mM) more effectively, at high concentrations (0.5-1 mM) almost the same amount of PpIX synthesis was found in normal skin cells.

Experimental Example 4

An experiment was conducted to measure the amount of protoporphyrin IX (PpIX) synthesis in skin tissues. It was found that the amount of PpIX produced by ALA and ALA esters in skin tissues was much higher than in cells. Dilute 2 mL of the sample collected according to the same sampling method as Experimental Example 3 according to the fluorescence intensity, and then add 50ul of 8.4 x 10 ^-6 acridine to the slit of excitation and emission of the fluorescence spectrophotometer. The fluorescence spectrum was measured using 408 nm as the excitation wavelength, and the results are shown in Tables 6 to 8 and FIGS. 3A to 3C, respectively.

From Tables 6 to 8 and FIGS. 3A to 3C, when comparing the amounts of PpIX synthesis with time and concentration of ALA and ALA esters applied, the amount of PpIX synthesis over time was determined by the type and concentration of ALA and ALA-ester. Regardless of the amount of PpIX synthesized, the amount of PpIX increased 3 hours after application, but the significant increase in both ALA and 4 types of ALA esters was observed due to the small sample size and large standard deviation. It can be seen that it was not visible.

In addition, each concentration (5%, 7.5%, 10%: concentration is the weight / weight percent, which is calculated by converting the weight of the ALA ester mixed in 1 g of the base in terms of percentage, i.e., 1 g of base) When mixing 75 mg of ALA ester, the concentration becomes 7.5%.) After comparing the amount of PpIX produced after 1 hour, 5% to 10% after applying ALA and ALA-ester As the concentration increased to%, only ALA-pe increased PpIX synthesis, but ALA decreased PpXI synthesis at 10% than 5%, while remaining ALA-ester increased PpXI synthesis at 10%. There was no significant change in both ALA and four ALA esters due to the large sample size and standard deviation. As a result of comparing the amount of PpIX synthesized after 3 hours of application by concentration (5%, 7.5%, 10%), the amount of PpIX was increased at 10% concentration than 5% concentration. As in 1 hour after application, it was not statistically significant (ALA p = 0.491, ALA- me p = 0.252, ALA- bu p = 0.252). However, after 3 hours of application of ALA-pe and ALA-hx, the amount of PpIX synthesis increased significantly (ALA-pe p = 0.045 , ALA-he p = 0.041 ). As a result, when applied at a concentration of 5%, the amount of PpIX synthesized in ALA and ALA esters did not show a big difference, but when applied at a concentration of 7.5% or higher, the amount of PpIX synthesized in ALA esters was increased. The amount was observed to be higher than ALA. At the 10% concentration, the amount of PpIX synthesized from ALA-me and ALA-bu was the highest. There was also no statistical significance because of the small number of samples and large standard deviation (p> 0.05). Based on this, it was found that ALA-me, ALA-bu, ALA-pe, and ALE-hx of the present invention can synthesize more PpIX than ALA.

Experimental Example 5

In order to check whether the functional cosmetic composition comprising the ALA-ester or salt thereof of the present invention has skin regeneration function, the essence composition 2 prepared in Example 2 was applied to the skin of an experimental animal and then exposed to sunlight for 30 minutes. After observation, the results are shown in FIG. In addition, the control mouse was irradiated with LED (25J) only without the application of the essence composition 2.

At this time, a female 8-week-old ICR mouse was used as an experimental animal. Before applying the essence composition 2, the hair on the back of the mouse was removed using a hair removal device and a hair removal agent to remove the hair so as not to damage the skin. Used.

After 1, 2, 4, 8, 12, 16 and 21 days of treatment, skin biopsies were performed on the distribution of ICR mice. Biopsy tissue was partially treated with paraffin embedded tissue and the remaining sites were stored as frozen tissue using liquefied nitrogen. Paraffin-embedded tissue was stained with Hematoxylin-Eosin, and procollagen type expression was confirmed using N- & C-terminus of procollagens type 1 antibody.

As a result of observing the change over time through H & E staining, epidermal necrosis with phobia of the epidermal basal cell layer and slight inflammatory cell infiltration was observed after 1 day of PDT treatment from FIG. 4, and from the epidermal basal layer from day 2 Regeneration was observed. From day 4 the epidermis was fully restored and the dermal matrix layer began to thicken. On the 8th day, the epithelial and dermal matrix changes were remarkable in the PDT group, whereas no significant change was observed in the group irradiated with only red LED light.

Experimental Example 6

RT-PCR was performed to confirm mRNA expression of the essence composition 2 and the control mouse of the present invention and the results are shown in FIG. At this time, the control mouse was irradiated with the essence composition 2 and not exposed to the LED, and irradiated with the LED 25J only without the application of the essence composition 2.

1. Manufacture of mouse skin tissue protein homogenate

The back skin tissues of control mice and mice coated with essence composition 2 were cut out and placed in a membrane bowl, and ground to a small section with liquefied nitrogen. This was again added with an appropriate amount of lysis buffer (1% Triton X-100, 20mM Tris-HCl, pH 7.4, 150mM NaCl, 5% glycerol, 1 mM EDTA, pH 8.0, protease inhibitor cocktail) and a tissue mill (Ultra ^Turrax® , KikaLaboritechnik T25, Malaysia). After centrifugation at 3,000 rpm for 20 minutes, the supernatant was transferred to a new tube and then centrifuged at 14,000 rpm for 20 minutes to use the supernatant as a skin homogenate. All of the above procedures were performed at 4 ° C to prevent protein degeneration.

2. Total RNA Isolation and RT-PCR

Total RNA was isolated from mouse skin tissue using RNeasy mini kit (Qiagen, CA, USA) and quantified using UV-spectrophotometer (Beckman) (OD ₂₆₀ / OD ₂₈₀ ). As a result, total RNA having a ratio between 1.8 and 2.0 was used for the experiment. CDNA was synthesized using Omniscript RT kit (Qiagen, CA, USA) (conditions: 1 μg RNA, 10Xbuffer, dNTP, oligodT, inhibitor, reverse trascriptase, 1 hour at 37 ℃, 5 minutes at 93 ℃). Each synthesized cDNA was diluted and PCR was performed using a PCR-premixture kit (ELPIS, Taejeon, Korea). Perkin Elmer 9600 (Shelton, CT, USA) instrument was used. mcollagen type I alpha1 (336bp) is 5'-gat gtg cca ctc tga ctg gaa-3 ', 5'-ggc tac gct gtt ctt gca gtg-3' (60 ° C), mProcollagen III (443bp) is 5'-cca ccc cga act caa gag tgg -3 ', 5'- cca tcc tct aga act gtg taa gtg-3' (60 ° C), mMMP2 (396bp) is 5'- aga aaa gat tga cgc tgt gt -3 ', 5' ctt cac gct ctt gag act tt-3 '(56 ° C), mMMP3 is 5'- tgt acc cag tct aca agt cct cca -3', 5'-ctg cga aga tcc act gaa gaa -3 (62 ° C), 5'- gca tac ttg tac cgc tat gg -3 ', 5'- tgt gat gtt atg atg gtc cc -3' (57 ° C), mMMP13 (307bp) is 5'- gac ttc tac cca ttt gat gg -3 ', 5'- tta ggg ttg ggg tct tca tc -3' (59 ℃), mTGF-β1 (169bp) is 5'-tga cgt cac tgg agt tgt acg g-3 ', 5'-ggt tca tgt cat gga tgg tgc-3 '(60 ° C.) and mTNF-α were carried out with 5' gga ggt cta ctt tgg agt cat tga 3 ', 5' tcc ctt tgc aga act cag gaa tgg 3 ', (60 ° C.). Amplified PCR fragments were identified by 1.5% agarose gel electrophoresis.

 As a result, as shown in FIG. 5, the inflammatory cytokines TNF-α and TGF-β1 in the mRNA expression of the group irradiated with LED after the essence composition 2 (including ALA-methylester) were applied were initially decreased after the expression was increased. It was. In addition, MMP-2, MMP-3, MMp-9 was strongly expressed from day 1 and decreased after 8 days. MMP-13 decreased immediately after expressing strongly on day 2. The mRNA expression of collagen I and III in the ALA-1 and LED groups was strongly expressed from day 4 and increased to day 12. Only the essence composition 2 (ALA-me) was applied or the control group irradiated only with LED, there was a slight change in the mRNA expression of MMP-2, MMP-9, MMP-3, MMP-13. In conclusion, it was confirmed that cytokines and MMPs involved in inflammation of TNF-a, TGF-β1, MMP-2, 3, 9, and collagen remodeling were initially involved in PDT, and then procollagen type I increased.

Experimental Example 7

Red wavelength LED (25J) was irradiated on the mouse to which the essence composition 2 and the mouse to which the essence composition 4 was applied, and to the control mouse to which the essence composition was not applied, and the tissue homogeneous solution was made over time to perform western blot. The analysis results are shown in FIGS. 6 and 7.

At this time, Western blot analysis for Procollagen I and MMPs was performed as follows.

Protein quantification of mouse skin tissue homogenates was determined using a BCA ^™ protein assay kit (Pierce) using bovine serum albumin (BSA, Pierce, Rockford, IL, USA) as a standard protein, and then -70 ° C until used in experiments. It was stored in and used. Electrophoresis was performed by Laemmli (1970). Cell homogenate was added to 1 × SDS sample buffer (50mM Trix HCl, pH 6.8, 10% glycerol, 2% bromophenol blue, 5% β-mercaptoethanol) and then boiled for 5 minutes and placed on ice for 5 minutes. Each sample was transferred to 0.45 μm PVDF membrane (Millipore, MA, USA) after electrophoresis using 6% and 10% SDS-PAGE. After confirming the transfer to the membrane using Ponceau'S solution (Sigma, MO, USA) and reacted with a blocking solution (5% skim milk, PBS, 0.1% tween-20, PBS-T) at room temperature for 1 hour. Procollagen type I (Y-18, N-terminus, sc-8787, Santacruz, 1: 200 D), Procollagen type I (A-17, C-terminus, sc-) 25973, Santacruz, 1: 200 D), MMP-9 (AF911, R & D, 1: 100 D), MMP-3 (ab53015, abcam, 1: 1,000 D), MMP-2 (MAB3308, Chemicon, 1: 1,000 D ) And β-actin (abcam, 1: 5,000 D) were each diluted and reacted at 4 ° C. for overnigt. After washing 4 times with PBS-T, horseradish peroxidase-conjugated goat anti-goat IgG, anti-mouse IgG and anti-rabbit IgG were diluted 1: 5,000 and reacted for 1 hour, followed by ECL kit (Amersham, UK Was confirmed by color development. A standard marker for sizing was used with a pre-stained protein ladder (10-180 kDa, IBM, USA).

As shown in FIG. 6, after applying the essence composition 2 and the essence composition 4 to a mouse, irradiating the red wavelength LED (25J) and making a tissue homogenate over time, performing western blot and analyzing the resultant, the essence composition was applied. Afterwards, the protein expression of MMP-3, MMP-2 and MMP-9 increased in maximum from day 1 and decreased after 2 days in the group irradiated with LED. The amount of expression was minimal in the group irradiated with LED only. Collagen type I (N-terminus) began to increase from day 4 and collagen expression increased significantly from day 8 and day 12. In addition, in the group irradiated with LED only, the expression increased slightly over time, but the increase was small.

In addition, expression of collagen type I (C-terminus) was increased at 8 and 12 days, but the expression was weak in the control group irradiated only with LED. It was confirmed that when the essence composition was applied to the mouse skin and the LED was irradiated, it secreted inflammatory cytokines and induced the expression of MMPs. Then, as the expression of MMPs decreased, it was confirmed that the expression of collagen required for skin regeneration was increased.

On the other hand, as shown in Figure 7, after applying the essence composition 2 and the essence composition 4 to the mouse, and irradiated with LED (25J) of the same red wavelength and making a tissue homogeneous solution over time to analyze the western blot result essence Compared to the composition 4, the essence composition 2 was less in the amount of collagen production, but it was confirmed that the collagen is produced in almost the same time and aspect. In conclusion, the functional cosmetic composition of the present invention was able to produce collagen in the skin significantly compared to the control group, it could be confirmed that it can help in the prevention and treatment of skin regeneration, that is, skin aging.

Experimental Example 8

Essence composition 1 to 4 to determine whether allergic and primary contact dermatitis for the essence composition 1 to 4 prepared in Examples 1 to 4 in normal subjects A patch test was performed on the back skin. After 48 hours the patch was removed and the result of the skin reaction was observed and the result was read.

As a result of performing a patch test on the essence compositions 1 to 4 containing ALA-methyl ester and measuring the occurrence of allergic contact dermatitis or primary contact dermatitis, it is safe to not cause allergic or primary contact dermatitis to any normal person. It was found to be a cosmetic composition.

Experimental Example 9

The skin regeneration effect of the essence composition 2 was observed by applying the essence composition 2 to a normal subject having skin problems such as wrinkles and blemishes on the face and exposing it to the room for 3 hours. For follow-up observation, photographs were taken before and two weeks after the procedure, and clinical effects were observed. Some of them are shown in FIGS. 8 to 12.

As a result of measuring the skin regeneration effect by performing an essence composition 2 containing ALA-methyl ester once, as a result, more than 90% of the test subjects recognized that wrinkles and skin troubles were reduced, and thus the functional cosmetic for skin regeneration of the present invention. The composition was found to be excellent skin regeneration effect.

In particular, the photos shown in FIGS. 8 to 12 show that the color of the skin becomes brighter after the procedure compared to before the single treatment of the essence composition 2, the pores are reduced, and the color of the skin and the wrinkles of the skin are improved, as well as the effect on the acne. You can see that there was.

More specifically, FIG. 8 shows that the color of the skin and the wrinkles of the skin are improved on the right side after the procedure compared to the left side before the procedure, and FIG. 9 shows the color of the skin is brighter on the right side after the procedure compared to the left side before the procedure. 10 shows that the size of the pores decreased in the right picture after the procedure compared to the left before the procedure.

In addition, FIG. 11 shows that acne is improved and skin color is improved in the right picture after the procedure compared to the left side before the procedure, and FIG. 12 shows that acne is improved as the skin is cleared on the right side after the procedure compared to the left side before the procedure. .

While the present invention has been particularly shown and described with reference to exemplary embodiments thereof, it is clearly understood that the same is by way of illustration and example only and is not to be taken by way of limitation, Various changes and modifications will be possible.

1 is a graph showing the results of toxicity tests of ALA esters on skin human keratinocytes and fibroblasts;

2 is a graph showing the synthesis amount of PpIX in skin human keratinocytes and fibroblasts,

3a to 3c are graphs comparing the concentrations of ALA and ALA esters contained in the functional cosmetic composition applied to the skin and the amount of synthesis of PpIX over time,

Figure 4 is a photograph showing the effect of skin aging treatment of ALA ester after applying the essence composition 2 to the skin of experimental animals,

Figure 5 is a photograph of the RT-PCR experiment results for confirming the mRNA expression,

6 is a result of Western blot analysis and analysis,

7 is a result of Western blot analysis and collagen production graph,

8 to 12 are photographs before and after the procedure of the experimenter who applied the essence composition 2 to the face to determine the skin regeneration effect of the essence composition 2.

Claims (7)

A functional cosmetic composition for skin regeneration comprising 5-aminolevulinic acid ester or a cosmetically acceptable salt thereof as an active ingredient. The method of claim 1, The cosmetically acceptable salt of the 5-aminolevulinic acid ester is a functional cosmetic composition for skin regeneration, characterized in that the compound having the formula: Formula (II) R ₂ ㆍ NH ₂ -CH ₂ -CO-CH ₂ -CH ₂ -CO-OR ₁ Wherein R ₁ is 2-propenyl group (CH ₂ -CH = CH ₂ ), 3-butenyl group (CH ₂ -CH ₂ -CH = CH ₂ ), 4-pentenyl group (CH ₂ -CH ₂ -CH _2- CH = CH ₂ ), 5-hexenyl group (CH ₂ -CH ₂ -CH ₂ -CH ₂ -CH = CH ₂ ), methyl group (CH ₃ ), and a general alkyl group, and R ₂ is It is selected from the group consisting of inorganic acids or organic acids such as HCl, H ₂ SO ₄ , H ₃ PO _4, acetic acid, benzoic acid, benzene sulfonic acid. The method of claim 1, The 5-aminolevulinic acid ester or its cosmetically acceptable salt thereof is a functional cosmetic composition for skin regeneration, characterized in that it comprises 5 to 10 parts by weight per 100 parts by weight of the cosmetic base. The method of claim 3, wherein The cosmetic base is a functional cosmetic composition for skin regeneration comprising any one or more selected from the group consisting of stearyl alcohol, polyethylene glycol or purified water. The method of claim 1, The cosmetically acceptable salt of the 5-aminolevulinic acid ester is a functional cosmetic composition for skin regeneration, characterized in that the hydrochloride. The functional cosmetic composition for skin regeneration, characterized in that 50 to 75 mg of 5-aminolevulinic acid ester or a salt thereof contained in the one-time use amount of the functional cosmetic composition for skin regeneration according to claim 1. How to use. The method of claim 6, The functional cosmetic composition for skin regeneration is applied to the skin and after a certain period of time, it is exposed to sunlight for 1 to 2 hours outdoors or indoors for more than 2 hours to use the functional cosmetic composition for skin regeneration. Way.

Images