TWI675849B - Use of compound or cosmetically acceptable salt thereof for wrinkle reduction - Google Patents

Abstract

The present invention provides a fatty acid-conjugated tripeptide and a cosmetic composition containing the same. The present invention provides excellent effects in preventing skin aging, reducing wrinkles, enhancing skin elasticity, and penetrating the skin.

Description

Use of a compound or a cosmetically acceptable salt thereof to reduce wrinkles Cross-references to related applications

This application claims the benefit of Korean Patent Application No. 10-2016-0127375, filed with the Korean Intellectual Property Office on October 4, 2016, the disclosure of which is incorporated herein by reference.

The present invention relates to a fatty acid-conjugated tripeptide and a cosmetic composition containing the fatty acid-conjugated tripeptide having an excellent effect of reducing skin wrinkles.

The skin is an important organ, which can protect the human body, play a variety of functions such as barrier functions, and excretory functions. However, due to external environmental factors such as pollutants, ultraviolet rays, stress, etc., the skin cells are damaged and the regeneration of the cells is performed incorrectly. As a result, the thickness of the epidermis, dermis, and subcutaneous tissue becomes thinner, and the elasticity of the skin decreases, and wrinkles are formed. Ultraviolet rays induce active oxygen on the skin, which increases the damage to skin cells and pigmentation, which in turn leads to the loss of direct functions of the skin's main components and skin aging. Reactive oxygen is the cause of human disease and aging, and can be evaluated as an index to measure antioxidative activity and anti-aging action. Features of skin aging may include decreased elasticity, wrinkles, freckles, ephelides, and the like. One reason is that collagen and elastin from elastic fibers are made from lipids. Lipid peroxidation-induced chain scission, its abnormal cross-linking, and hyaluronic acid chain scission. Lipid peroxidation is caused by the oxidative pressure of free radicals generated as reactive oxygen species (ROS) during this period. It is reported that this process of free radical generation not only occurs before the harmful metabolic activation to the skin, but also causes damage to skin cells and tissues. Specifically, for wrinkles, active oxygen can induce the expression of matrix metalloproteinase (MMP), and degrade or damage the skin's elastic collagen or elastin, resulting in wrinkles. Collagen is the main component of the dermal matrix, so regulating the biosynthesis and degradation of collagen has become the key to skin aging. It has been known that collagen-promoting substances such as ascorbic acid (U.S. Patent No. 4,983,382), vitamin A (Retinoid) (U.S. Patent No. 4,877,805) and derivatives thereof have been developed, and cosmetic compositions containing the same . However, ascorbic acid is easily oxidized in air and moisture, and therefore has a problem of stability, and vitamin A is reported to be unstable and cause skin irritation problems when applied to the skin.

As a compound that is less irritating to the skin and has the effect of reducing skin wrinkles, peptides are the main component of protein, which has the advantages of reducing wrinkles and having no side effects on the skin. In addition to peptides, the RGD (Arginine-Glycine-Aspartic Acid) (Arg-Gly-Asp) sequence has been known as a cell recognition sequence since the late 1980s, and Synthetic RGD peptides have been used since the early 1990s (Biochem J. (1993) 296, 21-24). In addition, according to the paper, RGD-containing peptides are known to have an effect on integrin-mediated cell attachment, and the above functions affect cell growth (Annu Rev Cell Dev Biol. (1996) 12: 697-715). Now, RGD is used as a component that activates collagen and reduces skin wrinkles, but the peptide is limited in the effect of actually reducing wrinkles due to its hydrophilicity and difficulty in penetrating the skin barrier.

In order to overcome the above-mentioned problems of the prior art, the present invention aims to solve the difficulty of the hydrophilic tripeptide RGD (arginine-glycine-aspartic acid) to penetrate the skin barrier. Conjugation to prepare a variety of derivatives having a hydrophilic-lipophilic structure, and thus identifying the derivatives with high efficacy for reducing skin wrinkles represents the completion of the present invention.

Therefore, an object to be achieved by the present invention is to provide a fatty acid conjugated tripeptide of the following formula I or a cosmetically acceptable salt thereof.

In addition, another object to be achieved by the present invention is to provide a cosmetic composition comprising as an active ingredient a fatty acid conjugated tripeptide of the following formula I or a cosmetically acceptable salt thereof, and a method for reducing wrinkles use.

The present invention relates to a fatty acid conjugated tripeptide and a cosmetic composition containing the fatty acid-conjugated tripeptide having an excellent effect of reducing skin wrinkles.

Hereinafter, the present invention will be described in more detail.

The present invention provides a fatty acid-conjugated tripeptide or a cosmetically acceptable salt thereof that is a fatty acid-conjugated RGD, more preferably palmitoyl-RGD (palmitoyl-RGD) or a cosmetically acceptable salt thereof.

The RGD is a tripeptide linked to three amino acids of arginine, glycine, and aspartic acid, which are known to have a collagen synthesis effect, and is therefore used as a cosmetic ingredient.

The above-mentioned fatty acid-conjugated RGD is represented by a general formula such as Chemical Formula I: [Chemical Formula I]

Where n is an integer from 11 to 16.

Moreover, the above palmito-RGD is a compound represented by the following Chemical Formula II, or Palm-Arg-Gly-Asp, wherein n in Chemical Formula I is 15.

Also, the present invention provides the fatty acid-conjugated tripeptide (RGD) of the above-mentioned chemical formula I or a cosmetic composition containing the same.

The above-mentioned cosmetic composition provides excellent effects in preventing skin aging, reducing wrinkles, enhancing skin elasticity, and penetrating the skin, and has good effects on long-term storage stability and skin safety.

The amount of procollagen synthesis of cells treated with the compound of the above formula I may be at least 20% more than that of cells treated with the compound of the following formula III at the same concentration.

[Chemical Formula III]

Relative to the total weight of the cosmetic composition, the cosmetic composition of the present invention may include 0.0001 to 2% by weight, preferably 0.01 to 0.05% by weight of the compound of Formula I as an active ingredient.

And, the present invention provides a cosmetic composition for reducing wrinkles, which comprises the fatty acid-conjugated tripeptide (RGD) of the above-mentioned chemical formula I or a cosmetically acceptable salt thereof.

Relative to the total weight of the cosmetic composition, the cosmetic composition of the present invention may include 0.0001 to 2% by weight, preferably 0.01 to 0.05% by weight of the compound of Formula I as an active ingredient.

The formulation of the cosmetic composition according to the present invention is not particularly limited, and the cosmetic composition of the present invention may include a conventional cosmetic composition combination known in the art according to the formulation to be prepared. The cosmetic composition of the present invention can be formulated into formulations such as lotion, emulsion, nutrition cream, pack, beauty liquid, essential oil, and the like. According to the formula to be prepared, it is combined by selecting oil, emulsifier, emulsion stabilizer, humectant, thickener, preservative, fragrance, etc. .

The present invention provides a method for preparing the above-mentioned compound of formula I, which comprises the following steps: (a) by using arginine derivative having a protected N-terminus, C-terminus or a reactive residue thereof Synthesis of tripeptide (arginine-glycine-aspartic acid) derivatives in the liquid phase by using compounds, glycine derivatives and aspartic acid derivatives; (b) conjugating the N-terminus of arginine with a fatty acid by reacting the above tripeptide (arginine-glycine-aspartic acid) derivative with a fatty acid salt; and (c) conjugating from the above fatty acid The combined tripeptide (arginine-glycine-aspartic acid) derivative removes the protective group of the tripeptide moiety.

Hereinafter, a method for preparing palmito-RGD of the above-mentioned chemical formula II according to the present invention will be described in detail with reference to the following reaction formula I. The method shown in the following reaction formula I is only a representative of possible preparation methods, and the order of unit operations, reagents, reaction conditions, and the like shown in the following reaction formula I can be appropriately modified if necessary. In addition, RGD prepared by a solid synthesis method which is a general peptide synthesis method can be used.

The palm pupa-RGD of the present invention prepared according to the above method is not only stable, but also has excellent effects in preventing skin aging, reducing wrinkles, enhancing skin elasticity, and penetrating the skin.

Advantages of the invention

The fatty acid-conjugated tripeptide compound according to the present invention provides excellent effects in preventing skin aging, reducing wrinkles, enhancing skin elasticity, and penetrating the skin.

FIG. 1 is a graph showing the results of a cytotoxicity experiment of fatty acid-conjugated RGD.

FIG. 2 is a diagram illustrating a comparison of the effects of procollagen synthesis by fatty acid-conjugated RGD.

Fig. 3 is a diagram illustrating a comparison of the transdermal absorption effect of fatty acid-conjugated RGD.

Figures 4 to 6 are diagrams illustrating the effect of a composition containing palmarium-RGD on reducing periorbital wrinkles.

FIG. 7 is a diagram illustrating a skin elasticity-enhancing effect of a composition containing palmarium-RGD.

FIG. 8 is a diagram illustrating the effect of a composition containing palmarium-RGD on improving skin dermis density.

The present invention provides a fatty acid-conjugated tripeptide and a cosmetic composition containing the same for reducing skin wrinkles.

In a specific embodiment of the present invention, the fatty acid-conjugated tripeptide may be palmarium-RGD.

Hereinafter, the present invention will be described in further detail through examples. However, the descriptions of these examples are merely illustrative descriptions of the present invention, and are not to be construed as limiting the scope of the present invention.

Preparation Example 1: Preparation of palmarium-RGD

Preparation Example 1-1: Synthesis of Boc-Gly-Asp (OBn) ₂

N- (tert-butoxycarbonyl) glycine (N- (tert-butoxycarbonyl) glycine (N-Boc-Gly-OH), 20g, 114.17mmol) and L-aspartic acid dibenzyl ester (L- aspartic acid dibenzyl ester, 55.43 g, 114.17 mmol) was dissolved in tetrahydrofuran (THF), 600 mL, after which the temperature was lowered to 0 ° C, and then 1-hydroxybenzotriazole hydrate (1-hydroxybenzotriazole hydrate) (HOBt), 18.51 g, 137.00 mmol) and 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (1- (3-dimethylaminopropyl) -3-ethylcarbodiimidehydrochloride (EDC HCl), 27.00 g, 137.00 mmol) was added to the resulting solution, and triethylamine (TEA), 55.69 mL, 399.59 mmol) was slowly added thereto. Then, the resulting mixture was stirred at 0 ° C for 30 minutes, and then the resulting solution was removed from the cooling bath and stirred at room temperature overnight. The reaction solution was concentrated, and then water (320 mL) and ethyl acetate (400 mL) were added to the obtained concentrate to extract twice (400 mL × 2), and the organic layer was rinsed with NaCl _(aq) (300 mL), and Dry over sodium sulfate (Na ₂ SO ₄ ), then filter under pressure and concentrate. The concentrate was dried under reduced pressure to obtain (S) -dibenzyl-2- (2-((tert-butoxycarbonyl) amino) acetamido) succinate ((S) -dibenzyl2- (2- ((tert-butoxycarbonyl) amino) acetamido) succinate (Boc-Gly-Asp (OBn) ₂ ), 64.53 g, 120%), as a yellow oil, allowed the following reaction to proceed without its purification process.

Preparation Example 1-2: Synthesis of Gly-Asp (OBn) ₂ HCl

(S) -Dibenzyl-2- (2-((tert-butoxycarbonyl) amino) acetamido) succinate (53.71 g, 114.15 mmol) was dissolved in ethyl acetate (500 mL), and then A solution of 4M hydrochloric acid (HCl) 1,4-di [oxan] ane (1,4-dioxane) (113 mL, 114.15 mmol) was added thereto, and the resulting mixture was stirred at room temperature for 6 hours. The resulting solid was filtered and dried under reduced pressure, thereby obtaining (S) -dibenzyl 2- (2-aminoacetamido) succinate ((S) -dibenzyl2- (2-aminoacetamido) succinate hydrochloride (Gly-Asp (OBn) ₂ HCl), 39.47 g, 85%) as a white solid.

Preparation Example 1-3: Synthesis of Boc-Arg (NO ₂ ) -Gly-Asp (OBn) ₂

(S) -Dibenzyl 2- (2-aminoacetamido) succinate (39.47 g, 97.01 mmol) and 2-((tert-butoxycarbonyl) amino) -5- (3 -Nitroguanidyl) pentanoic acid (2-((tert-butoxycarbonyl) amino) -5- (3-nitroguanidino) pentanoicacid (N-Boc-Arg (NO ₂ ) -OH), 30.98 g, 97.01 mmol) was dissolved in After tetrahydrofuran (600 mL), the temperature was lowered to 0 ° C, followed by 1-hydroxybenzotriazole (15.73 g, 116.41 mmol) and 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide. Hydrochloric acid (22.31 g, 116.41 mmol) was added to the resulting solution, and triethylamine (47.33 mL, 339.54 mmol) was slowly added thereto. Then, the resulting mixture was stirred at 0 ° C for 30 minutes, and then the resulting solution was removed from the cooling bath and stirred at room temperature overnight. The reaction solution was concentrated, and then water (320 mL) and ethyl acetate (400 mL) were added to the resulting concentrate to extract twice (400 mL × 2), and the organic layer was rinsed with NaCl _(aq) (300 mL), and sulfuric acid Sodium is dried, filtered and concentrated under pressure. The concentrate was dried under reduced pressure to obtain (S) -dibenzyl 2- (2-((S) -2 ((tert-butoxycarbonyl) amino) -5- (3-nitroguanidyl) pentanidine Amine) Ethylamino) Succinate ((S) -dibenzyl 2- (2-((S) -2 ((tert-butoxycarbonyl) amino) -5- (3-nitroguanidino) pentanamido) acetamido) succinate ( Boc-Arg (NO ₂ ) -Gly-Asp (OBn) ₂ ), 65.16 g, 102%) as a yellow oil, so that the following reaction was performed without performing its purification process.

Preparation Example 1-4: Synthesis of Arg (NO ₂ ) -Gly-Asp (OBn) ₂ HCl salt

(S) -Dibenzyl 2- (2-((S) -2 ((tert-butoxycarbonyl) amino) -5- (3-nitroguanidyl) pentamidino) acetamido) ) Succinate (65.16g, 97.00mmol) was dissolved in ethyl acetate (500mL), and then a 4M solution of hydrochloric acid (HCl) 1,4-bis [orthofluorene] alkane (94.58mL, 378.30mmol) was added, The resulting mixture was stirred at room temperature for 6 hours. The obtained solid was filtered and dried under reduced pressure, thereby obtaining (S) -dibenzyl-2- (2-((S) -2-amino) -5- (3-nitroguanidino) as a white solid. ) Pentamidine) Acetylamino) Succinate ((S) -dibenzyl 2- (2-((S) -2-amino) -5- (3-nitroguanidino) pentanamido) acetamido) succinate hydrochloride (Arg (NO ₂ ) -Gly-Asp (OBn) ₂ HCl salt), 59.08 g, 100%).

Preparation Example 1-5: Synthesis of Palm-Arg (NO ₂ ) -Gly-Asp (OBn) ₂

(S) -Dibenzyl-2- (2-((S) -2-amino) -5- (3-nitroguanidyl) pentamidine) acetamidine) succinate (59.06 g, 97.04 mmol) was dissolved in dichloromethane (CH ₂ Cl ₂ ) (800 mL), and then the temperature was lowered to 0 ° C., and palmitoyl chloride (30.76 mL, 101.85 mmol), followed by slowly adding triethylamine (27.04 mL, 194.01 mmol). Then, the resulting mixture was stirred at 0 ° C for 30 minutes, and then the resulting solution was removed from the cooling bath and stirred at room temperature overnight. After the reaction was completed, the resulting solution was concentrated under pressure, after which dichloromethane was partially removed and water was added, followed by filtration. The obtained solid was dried under reduced pressure to obtain (S) -dibenzyl 2- (2-((S) -5- (3-nitroguanidyl) -2-palmitinamidopentylamido) acetamidine Amine) succinate ((S) -dibenzyl 2- (2-((S) -5- (3-nitroguanidino) -2-palmitamidopentanamido) acetamido) succinate (Palm-Arg (NO ₂ ) -Gly-Asp ( OBn) ₂ ), 73.01g, 93%).

Preparation Example 1-6: Synthesis of Palm-RGD-HCl salt

(S) -Dibenzyl 2- (2-((S) -5- (3-nitroguanidino) -2-palmitinamidopentylamido) acetamido) succinate (70.00 g, 86.42 mmol) and tetrahydrofuran (1.5 L), and then 3M hydrochloric acid (288.07 mL, 864.21 mmol) was added thereto. Then, under argon (Ar), 10% palladium carbon (Pd / C), 8.64 g was added to the obtained mixture. The solution was stirred for 3 hours at room temperature under hydrogen (H _2). The solution filtered from the reaction mixture by means of diatomaceous earth was decompressed, thereby partially removing its solvent therefrom. To the remaining solution, sodium hydroxide (NaOH) _(aq) was slowly added, and then a solid formed at a pH of 2 or higher was washed with water, filtered, and dried. The solid was refluxed in isopropyl alcohol (IPA), then filtered in a hot state and dried again. When 5M sodium hydroxide and 6M hydrochloric acid were added to the obtained solid, the solid melted as the solution became hot, and the solid was generated as the solution cooled. After that, the solid was filtered and dried in a vacuum oven, thereby obtaining (S) -dibenzyl 2- (2-((S) -5-guanidyl) -2-palmitamidopentylamido) ethyl Amido) succinate ((S) -dibenzyl2- (2-((S) -5-guanidino) -2-palmitamidopentanamido) acetamido) succinate hydrochloride)) RGD-HCl salt, 33.82g, 63%).

Experimental Example 1: Cytotoxicity Experiment

In order to identify the cytotoxicity of fatty acid-conjugated RGD, Lauroyl-Arg-RGD (Lauroyl-Arg-Gly-Asp) and Myristyl-RGD were used as the four materials with fatty acids of different lengths by the following methods. (Myristoyl-Arg-Gly-Asp), Palmito-RGD (Palmitoyl-Arg-Gly-Asp) and Stearoyl-Arg-Gly-Asp.

Newly generated human dermal fbbroblast neonatal (HDFn cells, Cascade Co.) was seeded into 24-well plates (5 × 10 ⁴ cells / well) at 37 ° C and 5% CO ₂ Incubate for 24 hours. Then, the medium was removed, and the microtiter plate was washed with phosphate buffered saline (PBS), and Dulbecco's Modified Eagle's medium (DMEM) to which 1% penicillins / streptomycin (P / S) was added was added. The medium was thus cultured for 24 hours. The medium was removed, the microplate was washed with PBS, then treated according to the final concentration, and thereto was added 900 μ L each of the culture medium and 100 μ L -RGD lauroyl acyl, acyl -RGD myristic, palmitic acyl and stearic acyl -RGD -RGD, Incubate for 48 hours. Then, the medium was removed, the microplate was washed with PBS, and then thereto was added 100 μ LMTT (3- [4,5- dimethylthiazol-2-yl] -2,5-diphenyl tetrazolium bromide ( 3- [4,5-dimethylthiazol-2-yl] -2,5-diphenyltetrazolium bromide)) and 900 μL of culture medium, and cultured for 4 hours. The culture medium was removed, and 300 μL of a dimethyl sulfoxide (DMSO) solution was added thereto, and then the resulting mixture was shaken for 10 minutes, and the optical density at 570 nm was measured using a microplate reader device To identify cell viability.

The results are shown in Tables 1 and 1 below.

As shown in Table 1 above, it was confirmed that Laurel-RGD and Myristica-RGD did not exhibit cytotoxicity at a concentration of 10.0ug / mL or less, and Palmito-RGD did not exhibit cells at a concentration of 4.0ug / mL or less toxicity. On the other hand, it was confirmed that stearylamine-RGD exhibited high cytotoxicity at a concentration of 2.0 u g / mL or more.

Experimental Example 2: Analysis of the effect of procollagen synthesis

The effect of laurel-RGD, myristium-RGD, palmarium-RGD and stearylamine-RGD as fatty acid conjugated tripeptides was analyzed to find fatty acids of appropriate length. Comparative experiments were performed by using RGD and acetamidine-RGD unconjugated with fatty acids as control groups.

The newly generated human dermal fibroblasts were seeded into a 24-well microplate (5 × 10 ⁴ cells / well) by using a medium of Dulbecco's Modified Eagle's Medium (DMEM) to which 10% fetal bovine serum was added. Incubate at 37 ° C and 5% CO ₂ for 24 hours. Then, the medium was changed to a serum-free DMEM medium, cultured for 24 hours, and then an appropriate amount of each test compound was processed. After culturing for 48 hours, the cell culture fluid was collected, and then the amount of procollagen was measured using a collagen measurement kit of Takara Shuzo Co., Ltd. Japan. First, the collected cell culture solution was poured into a 96-well microplate, and the primary collagen antibody was evenly applied thereto, so that the antigen-antibody reaction could occur at 37 ° C for 3 hours. The cell culture was removed from the wells and washed four times with phosphate buffered saline (PBS). A color causing material was injected into each well, and then a reaction was performed at room temperature for 15 minutes, and a 1N sulfuric acid solution was added thereto to stop the reaction. The optical density was measured at 450 nm using a spectrophotometer device. A standard curve was drawn using a standard solution, and the optical density obtained by the method described above was assigned to the standard curve to calculate the amount of procollagen produced by the cell culture solution to which each test compound was added.

The results are shown in Tables 2 and 2 below.

As shown in Table 2 above, it was confirmed that fatty acid-conjugated RGD had a better procollagen synthesis effect than RGD or acetamidine-RGD at the same concentration, and especially at the same concentration, palmito-RGD had the best effect.

As confirmed in the above-mentioned Experimental Examples 1 to 2, it was confirmed that in the fatty acid-conjugated RGD, palm dysprosium-RGD is a substance exhibiting no cytotoxicity at a concentration exhibiting an effective effect, but a high collagen synthesis efficiency . Therefore, in the following experiments, Palmito-RGD is tested in the following experimental examples.

Experimental Example 3: Percutaneous absorption (infiltration into skin cells) experiment

Transdermal absorption experiments were performed by using palmarium-RGD (substance represented by the above Chemical Formula II), and comparative experiments were also performed through RGD not conjugated to fatty acids as a control group (Reference: Lehman PA, Slattery JT, Franz TJ. Percutaneous absorption of vitamin A: effects of vehicle, light, and dose (Percutaneous absorption of retinoids: Influence of vehicle, light exposure, and dose, J. Invest Dermatol., 91; 56-61, 1988).

Specifically, permeability is measured in a Franz diffusion cell. RGD not conjugated to fatty acids was used as the control group. The classified skin stored at -20 ° C was used to identify skin permeability. In order to identify the structural integrity of the skin before testing, it was confirmed that the skin (700 μm thick, stored at -20 ° C) was not damaged by transepidermal water loss (TEWL).

10% RGD and palmito-RGD were dissolved in a solvent of dipropylene glycol / octyldodecanol (1: 1), and then 1 mL of the resulting solution was loaded onto a chamber provided with a sample So that its patch is applied to human skin. Then, after 24 hours, taken out 500 μ L sample port through the lower solution, and analyzed by means of quantitative HPLC. Five skin penetration experiments were performed on each sample.

The results are shown in Tables 3 and 3 below.

As shown in Table 3 above, the detectable amount of RGD is minimal even after 24 hours of application to the skin. This means that the above RGD has hydrophilicity including R (arginine) which is a basic amino acid and D (aspartic acid) which is an acidic amino acid, so that the above RGD cannot penetrate the cuticle of the skin containing a large amount of hydrophobic lipids, but cannot Passed directly to skin cells. In addition, stearylamine-RGD has excellent skin permeability, but has cytotoxicity at concentrations with effective effects as shown in Table 1, making stearylamine-RGD unsuitable as a cosmetic. However, palm ray-RGD is not cytotoxic at effective concentrations. Because RGD is conjugated with palmitoyl, which is a hydrophobic fatty acid, its skin permeability is increased by more than 15 times, and the procollagen synthesis effect is also improved.

Experimental example 4: Skin safety test

In order to measure the skin safety of palmarium-RGD, palmarium-RGD components were dissolved in propylene glycol at various concentrations, and the skin irritation was confirmed.

Specifically, 5 × 20cm contains a defined amount (20 μ L) patches applied palmitic acyl -RGD propylene glycol solutions for 30 adults aged 20 to 50 in the back region for 24 hours. Then, the patch was removed so as to check the change in skin condition with the naked eye after 1 hour and 24 hours, and the results are shown in Table 4. At this time, the degree of change in skin condition is classified and evaluated according to the following skin condition evaluation criteria.

<Evaluation criteria for skin condition>

Researchers performed visual assessments based on the following criteria.

0: No change 1: Very small change

2: slight change 3: slightly change

4: drastic change 5: very drastic change

As shown in Table 4 above, it was confirmed that palmitosalm-RGD content was 0.05 wt% without skin irritation.

Experimental example 5: Humidity and heat stability test

Humidity and heat stability of the active ingredients used in cosmetic compositions are important factors for the composition during storage. Therefore, in order to confirm the suitability of palmarium-RGD as an active ingredient used in a cosmetic composition, its stability to humidity and heat was evaluated.

Specifically, each sample was stored in a sealed state under severe conditions of 4 ° C, 30 ° C, 45 ° C, and a relative humidity of 75% for 12 weeks, and then measured by high performance liquid chromatography (HPLC). Residual rate (%) of the sample of active ingredient value. The results are shown in Table 5 below.

As shown in Table 5 above, it confirms that palmarium-RGD has excellent stability to moisture and heat.

Formula Example 1. Preparation of a solubilization formulation containing palmarium-RGD

In order to confirm the stability of a cosmetic composition containing palmarium-RGD as an active ingredient, a dissolution formulation thereof was prepared as shown in Table 6 below.

<Preparation method>

(1) Each water phase and ethanol phase are uniformly mixed and dissolved.

(2) The ethanol phase is added to the water phase and the resulting solution is mixed and dissolved.

(3) Palmito-RGD of the additive I is dissolved and added to the obtained mixture, and the additive II is mixed therein to complete the dissolution formula.

Formula Example 2. Preparation of essential oil formula containing palmarium-RGD

To confirm the stability of a cosmetic composition containing palmarium-RGD as an active ingredient, an essential oil formulation was prepared as shown in Table 7 below.

<Preparation method>

(1) The water phase and the oil phase are heated, and each of the water phase and the oil phase is uniformly mixed and dissolved.

(2) The oil phase was added to the water phase at 75 ° C to mix and emulsify together.

(3) Palmito-RGD of additive phase I is added to the obtained mixture at 45 ° C. and mixed together, and additive phase II is mixed together.

Formula Example 3. Preparation of a cream formula containing palmarium-RGD

In order to confirm the stability of a cosmetic composition containing new palmarium-RGD as an active ingredient, a cream formula was prepared as shown in Table 8 below.

<Preparation method>

(1) The water phase and the oil phase are heated, and each of the water phase and the oil phase is uniformly mixed and dissolved.

(2) The oil phase was added to the water phase at 75 ° C to mix and emulsify together.

(3) Palmito-RGD of additive phase I is added to the obtained mixture at 45 ° C. and mixed together, and additive phase II is mixed together.

Experimental Example 6: Identification of ingredient stability of a cosmetic composition containing palmarium-RGD

In order to identify the stability of palmarium-RGD in the formula, the dissolving formula, essential oil formula and cream formula of the above Formulation Examples 1 to 3 were stored at 4 ° C, 30 ° C, 45 ° C and sunlight for 12 weeks, and thereafter The same high-performance liquid chromatography (HPLC) as in the above-mentioned Experimental Example 5 was used to observe changes in the content of Palmito-RGD-containing cosmetics over time, and the results are shown in Table 9 below.

As shown in Table 9 above, it was confirmed that the dissolving formula, the essential oil formula and the cream formula had excellent ingredient stability in the formula under high temperature and sunlight conditions.

Experimental Example 7: Identification of Formulation Stability of Cosmetic Compositions Containing Palmarium-RGD

In order to identify the formula stability of the cosmetic composition containing palmarium-RGD, the dissolving formula, essential oil formula, and cream formula of the above Formulation Examples 1 to 3 were set at 4 ° C, 30 ° C (room temperature, RT), 45 ° C and The conditions of sunlight were stored for 12 weeks, and then the changes in color, odor, and formulation were observed by visual and sensory evaluation, and the results are shown in Tables 10 to 12 below. At this time, changes in color, odor, and formulation were classified and evaluated according to the following evaluation criteria.

<Evaluation criteria>

No changes:-Very slight changes: + Minor changes: ++

Observable change: +3 Significant change: +4 Significant change: +5

As shown in Tables 10 to 12 above, it confirms that the dissolving formula, the essential oil formula and the cream formula have excellent stability under high temperature and sunlight conditions.

Experimental Example 8: Identification of skin irritation of a cosmetic composition containing palmarium-RGD

In order to identify the skin irritation of a cosmetic composition containing palmarium-RGD, the skin irritation of the cream formulation of Formulation Example 3 described above was evaluated.

Specifically, a concentration-dependent manner of a 5 × 20 cm patch containing a certain amount (60 uL) of a palm-rhodium-RGD cream formulation prepared in the above Formulation Example 3 was applied to 50 Adults had a total of 9 sessions per back region for 24 hours at a time. During the repeated application of the patch for a total of 9 times, the skin reaction was observed every 30 minutes after the patch was removed, and then the patch was applied again to identify the sensitization induction. After 2 weeks of rest, the patch was applied again, and the degree of sensitization induction was identified at 30 minutes and 24 hours and 48 hours after the patch was removed. The results of the sensitization induction are shown in Table 13 and the results of the sensitization induction test are shown in Table 14. At that time, the skin reaction was judged according to the criteria of the International Contact Dermatitis Research Group (ICDRG).

<Evaluation criteria for skin condition>

0: negative reaction (non-irritating)

1: Suspect reaction-only weak erythema

2: Weak positive reaction-erythema, infiltration, possible pimples

3: Strong positive reaction-erythema, infiltration, pimples, blisters

4: Extremely strong reaction-severe erythema and infiltration and accumulation of blisters

As shown in Tables 13 to 14 above, it was confirmed that the cream formula containing palmarium-RGD has low skin irritation.

Experimental Example 9: Clinical effect of cosmetic composition containing palmarium-RGD in reducing skin wrinkles

The skin wrinkle reduction effect of the above-mentioned cosmetic composition containing palmaridine-RGD was measured.

Specifically, in order to evaluate the effectiveness of a cosmetic composition containing palmarium-RGD, 23 women aged 30 to 60 years were clinically tested. A cosmetic composition containing 0.04 wt% palmarium-RGD was used as the test group product. As a control product, a placebo cream containing distilled water instead of palmarium-RGD used in the test product was used. The products of the above test and control groups were randomly applied to the left and right areas of the subject's face, respectively, and reapplied at intervals of twice a day, and the changes were observed for 12 weeks.

Experimental Example 9-1: Identification test for reducing orbital wrinkles

In order to identify the reduction of peri-orbital wrinkles by the means of using the test group product and the control group product, values were obtained as follows: before and at 4 weeks, 8 weeks, and 12 weeks after use, light was penetrated to the The results were analyzed in a translucent skin wrinkle replica made in the area and analyzed with a Visiometer SV600 (Courage-Khazaka electronic GmbH, Germany). Among these analysis values, R2 and R3 values were evaluated as key parameters for orbital wrinkles. R2 corresponds to five peaks and five valleys for a section in which the fold profile is equally divided into five fifths The maximum difference between. R3 is an arithmetic mean calculated by equally dividing the wrinkle contour into five sections along the X axis and using the difference between the maximum and minimum values measured in each section.

The results of the above R2 and R3 values are shown in the graphs of FIGS. 4 and 5.

As a result, the R2 values at 4 weeks, 8 weeks, and 12 weeks after use were significantly reduced compared to before use (p <0.05). Even when compared with the products of the control group, the R2 values of the products of the test group showed significant differences at 8 and 12 weeks after use (p <0.05). In addition, compared with before use, the R3 value of the product group in the test group was significantly reduced at 4 weeks, 8 weeks, and 12 weeks after use (p <0.05). Even when compared with the control group products, the R3 value of the test group product group showed a significant difference at 8 weeks and 12 weeks after use (p <0.05).

Experimental Example 9-2: Periorbital Wrinkle Measurement Test Using Primos

In order to identify changes in wrinkle measurements around the orbit by using the product, Primos premium (GFMesstechnik GmbH, Germany), a 3D skin photography device, was analyzed before and at 4 weeks, 8 weeks, and 12 weeks after use. Ra value of wrinkles around the orbit. Ra represents the average roughness of the wrinkle profile. The smaller the value, the less wrinkles around the orbit. The results of the measured Ra values are shown in the graph in FIG. 6.

As a result, compared with before use, the Ra value of the test group product group was significantly reduced within 12 weeks after use (p <0.05). Even when compared with the control group products, there were significant differences in Ra values at 4 and 12 weeks after use (p <0.05).

Therefore, the results of Experimental Examples 9-1 and 9-2 above confirmed that the cosmetic composition containing palmarium-RGD had an effect on reducing wrinkles around the orbit.

Experimental Example 9-3: Skin elasticity measurement test

To identify changes in skin elasticity measurements by using products containing palmarium-RGD, skin elasticity was measured before and at 4 weeks, 8 weeks, and 12 weeks after use. Cutometer CM580 (CK Electronics, Koln, Germany) was used to measure skin elasticity once in each of the same areas around both eyes. Among the resulting values, the R2 (Restoration from Skin Deformation) value is used as the evaluation for skin elasticity Sexuality data. The R2 value refers to the overall elasticity. The closer the value is to 1, the more elastic the skin is. The results of the measured R2 values are shown in the graph in FIG. 7.

As a result, the skin elasticity measurement of the product group of the test group was significantly increased at 4 weeks, 8 weeks, and 12 weeks after use compared to before use (p <0.05). Even when compared with the products of the control group, the measured skin elasticity values at 4 weeks, 8 weeks, and 12 weeks after use showed significant differences (p <0.05). Therefore, it has been confirmed that a cosmetic composition containing palmarium-RGD has an effect on enhancing skin elasticity.

Experimental Example 9-4: Skin dermis density measurement test

In order to measure the dermal density of the skin, it was photographed through a skin scanner high resolution ultrasound (taberna pro medicum, Germany) before and after 4 weeks, 8 weeks, and 12 weeks after use. The area around the eyes, and the measured images were used to analyze the dermal density of the skin before and after using the product. The unit of measurement of skin dermis density is%, which indicates that the higher the measured value, the higher the dermis density. The measurement results are shown in Figure 8.

As a result, compared with before use, the dermal density measurements in the product group of the test group increased significantly at 8 and 12 weeks after use (p <0.05). Even when compared with the products of the control group, the dermal density measurements at 8 and 12 weeks after use showed significant differences (p <0.05). Therefore, it was confirmed that the cosmetic composition containing palmarium-RGD has an effect on improving the density of the dermis of the skin.

Claims (1)

Use of a compound of formula I or a cosmetically acceptable salt thereof for reducing wrinkles: Wherein, n is 15, wherein the content of the compound is 0.04 wt% based on the total weight of a cosmetic composition.