WO2018066914A1 - Tripeptide having fatty acids bonded thereto, and anti-wrinkle cosmetic composition comprising same - Google Patents
Tripeptide having fatty acids bonded thereto, and anti-wrinkle cosmetic composition comprising same Download PDFInfo
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- WO2018066914A1 WO2018066914A1 PCT/KR2017/010913 KR2017010913W WO2018066914A1 WO 2018066914 A1 WO2018066914 A1 WO 2018066914A1 KR 2017010913 W KR2017010913 W KR 2017010913W WO 2018066914 A1 WO2018066914 A1 WO 2018066914A1
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- rgd
- cosmetic composition
- skin
- compound
- palmitoyl
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Images
Classifications
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K8/00—Cosmetics or similar toiletry preparations
- A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
- A61K8/30—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds
- A61K8/64—Proteins; Peptides; Derivatives or degradation products thereof
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
- A61Q19/00—Preparations for care of the skin
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
- A61Q19/00—Preparations for care of the skin
- A61Q19/08—Anti-ageing preparations
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
- C07K1/02—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length in solution
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
- C07K1/06—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length using protecting groups or activating agents
- C07K1/061—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length using protecting groups or activating agents using protecting groups
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K19/00—Hybrid peptides, i.e. peptides covalently bound to nucleic acids, or non-covalently bound protein-protein complexes
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
- C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
- C07K5/08—Tripeptides
- C07K5/0815—Tripeptides with the first amino acid being basic
- C07K5/0817—Tripeptides with the first amino acid being basic the first amino acid being Arg
Definitions
- the present invention relates to a tripeptide having a fatty acid bound thereto, and a cosmetic composition having an excellent skin wrinkle improvement effect.
- Skin is an important organ that plays a variety of roles such as protecting the human body, barrier function, and excretory function.
- skin cells are damaged by external environmental factors such as pollutants, ultraviolet rays, and stress, and cell turnover is not done properly, resulting in thinning of the epidermis, dermis and subcutaneous tissue, and deterioration of skin elasticity and wrinkles. Is formed.
- Ultraviolet rays cause free radicals in the skin, resulting in increased damage and pigmentation of the skin cells, leading to a loss of direct function of the skin's major components, leading to skin aging.
- Free radicals can be evaluated as a measure of antioxidant activity and anti-aging activity of free radicals as a cause of disease and aging in the human body.
- the characteristics of skin aging include a decrease in elasticity, wrinkles, blemishes or freckles, and one of the causes is chain breakage of collagen and elastin, which are elastic fibers due to lipid peroxidation, abnormal crosslinking and chain breakage of hyaluronic acid. .
- Lipid peroxidation is caused by oxidative stress, and in the process, free radicals, reactive oxygen species (ROS), are produced. This free radical production process has been reported to not only be a preliminary step of metabolic activation that is harmful to skin, but also cause skin cell and tissue damage.
- proteolytic enzymes are induced by the active oxygen in relation to wrinkles may cause wrinkles by denaturing or destroying collagen or elastin, which gives elasticity to the skin.
- collagen is the most abundant component of the matrix of the dermal layer of the skin, the control of biosynthesis and degradation of collagen is the key during skin aging process.
- Collagen synthesis promoters are known compounds such as ascorbic acid (US Pat. No. 4,983,382), and retinoids (US Pat. No. 4,877,805) and their derivatives, and cosmetic compositions containing the same have been developed.
- ascorbic acid is easily oxidized in the air and moisture, there is a stability problem, retinoids are unstable, and skin irritation problems have been reported in the skin application.
- the peptide As a compound having a small skin irritation and having a skin wrinkle improvement effect, there is a peptide, the peptide is a major component of the protein, there is an advantage that can improve the skin wrinkles without side effects on the skin.
- the RGD (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).
- RGD-containing peptides affect integrin-mediated cell attachment and the function affects cell growth (Annu Rev Cell Dev Biol. (1996) 12). : 697-715.).
- RGD is used as an ingredient for collagen activation and skin wrinkle improvement, but the tripeptide is hydrophilic and difficult to cross the skin barrier.
- the present invention is to overcome the problems of the prior art as described above, in order to solve the difficulty of penetration of the RGD (arginine-glycine-aspartic acid) of the hydrophilic tripeptides into the skin barrier, by combining various hydrophobic fatty acids to the RGD -A number of derivatives having a lipophilic structure were prepared, and the present invention was completed by confirming that their skin wrinkle improvement efficacy is good.
- RGD arginine-glycine-aspartic acid
- the problem to be solved by the present invention is to provide a salt that is acceptable as a tripeptide or a cosmetic thereof to which a fatty acid of the formula [I] is bound.
- Another problem to be solved of the present invention is to provide a cosmetic composition
- a cosmetic composition comprising the tripeptide of the following [Formula I] or a salt acceptable as a cosmetic as an active ingredient and its use for improving wrinkles.
- the present invention relates to a tripeptide combined with a fatty acid and a cosmetic composition having excellent skin anti-wrinkle effect.
- the present invention provides a tripeptide to which a fatty acid is bound, an RGD to which a fatty acid is bound or a cosmetically acceptable salt thereof, more preferably palmitoyl-RGD or a cosmetically acceptable salt thereof.
- the RGD is a tripeptide in which three amino acids of arginine, glycine, and aspartic acid are connected, and is known to have collagen synthesis efficacy and is used as a cosmetic raw material.
- the RGD to which the fatty acid is bound is represented by the general formula of Formula (I).
- n is an integer from 11 to 16.
- palmitoyl-RGD is a compound represented by Formula (II) or Palm-Arg-Gly-Asp, wherein n is 15 in Formula (I).
- the present invention also provides a tripeptide (RGD) to which the fatty acid of formula (I) is bound or a cosmetic composition comprising the same.
- RGD tripeptide
- the cosmetic composition is not only excellent skin anti-aging, wrinkle improvement, skin elasticity enhancement and skin penetration effect, but also provides a high effect in long-term storage stability and skin safety.
- the amount of procollagen synthesis of the cells treated with the compound of Formula I may be 20% or more compared with the case where the compound of Formula III is treated at the same concentration.
- the cosmetic composition of the present invention comprises 0.0001 to 2% by weight, preferably 0.01 to 0.05% by weight of the compound of formula (I) as an active ingredient based on the total weight of the cosmetic composition.
- the present invention also provides a cosmetic composition for improving skin wrinkles comprising a tripeptide (RGD) to which the fatty acid of Formula (I) is bound or a salt thereof that is acceptable as a cosmetic.
- a cosmetic composition for improving skin wrinkles comprising a tripeptide (RGD) to which the fatty acid of Formula (I) is bound or a salt thereof that is acceptable as a cosmetic.
- the cosmetic composition of the present invention comprises 0.0001 to 2% by weight, preferably 0.01 to 0.05% by weight of the compound of formula (I) as an active ingredient based on the total weight of the cosmetic composition.
- the cosmetic composition of the present invention is not particularly limited in its formulation, and may include a cosmetic composition combination commonly used in the art to which the invention pertains, depending on the formulation to be prepared.
- the cosmetic composition of the present invention can be prepared in a formulation such as lotion, milky lotion, nourishing cream, pack, essence, essence, etc., according to the formulation to be prepared, emulsion, emulsifier, emulsion stabilizer, moisturizer, thickener, preservative, fragrance Can be selected and blended.
- the present invention provides a method for preparing a compound of formula (I), comprising the following steps.
- the palmitoyl-RGD of the present invention prepared according to the above method is not only stable but also shows an excellent effect on excellent skin anti-aging, wrinkle improvement, skin elasticity enhancement and skin penetration.
- the tripeptide compound in combination with the fatty acid according to the present invention provides excellent skin anti-aging, wrinkle improvement, skin elasticity enhancement and skin penetration effect.
- Figure 1 is a graph showing the results for the cytotoxicity test of fatty acid-bound RGD.
- Figure 2 is a graph showing a comparison of the collagen synthesis effect of fatty acid-bound RGD.
- Figure 3 is a graph comparing the skin transdermal absorption effect of the fatty acid-bound RGD.
- 4 to 6 is a graph showing the effect of improving the wrinkles around the eyes of the composition containing palmitoyl-RGD.
- the present invention provides a cosmetic composition for improving wrinkles comprising a tripeptide and a fatty acid is bound thereto.
- the tripeptide to which the fatty acid is bound may be Palmitoyl-RGD.
- N- (t-butoxycarbonyl) glycine N- (tert-Butoxycarbonyl) glycine, N-Boc-Gly-OH) (20 g, 114.17 mmol) with L-aspartic acid dibenzyl ester (L-aspartic acid dibenzyl ester) (55.43 g, 114.17 mmol) was dissolved in tetrahydrofuran (THF) (600 mL), then the temperature was lowered to 0 ° C. and 1-hydroxybenzotriazole hydrate (HOBt) (18.51) was added to the solution.
- THF tetrahydrofuran
- Human normal fibroblasts Human Dermal Fibroblast neonatal, Cascade Co., HDFn cells
- Human Dermal Fibroblast neonatal, Cascade Co., HDFn cells were seeded in 24-micro well plates (5 ⁇ 10 4 cells / well) and incubated at 37 ° C., 5% carbon dioxide for 24 hours. . Discard the medium and wash with Phosphate Buffered Saline (PBS) and add 1% Penicillins / Streptomycin (P / S) to Dulbecco's Modified Eagle's Medium (DMEM). One medium was added and incubated for 24 hours.
- PBS Phosphate Buffered Saline
- P / S Penicillins / Streptomycin
- DMEM Dulbecco's Modified Eagle's Medium
- DMSO dimethyl sulfoside
- Human normal fibroblasts were inoculated into 24-well microplates using Dulbo nasal eagle eggs supplemented with 10% fetal calf serum (5 ⁇ 10 4 cells / well), at 37 ° C., 5% CO 2 . Incubated for 24 hours. Serum was replaced with doubling nose fertilized eagle medium and incubated for 24 hours, and then each test compound was treated with an appropriate amount. After 48 hours of incubation, the cell culture solution was collected, and then the amount of procollagen was measured using a collagen measurement kit of Takara Corporation (Takara Shuzo Co., Ltd., Japan).
- the cell culture fluids were collected into 96-well microplates uniformly coated with primary collagen antibodies, and antigen-antibody reactions occurred at 37 ° C. for 3 hours.
- Cell cultures in the wells were removed and washed four times with phosphate buffered saline.
- a color-causing substance was added and reacted at room temperature for 15 minutes, and then 1 N sulfuric acid solution was added to stop the reaction.
- Absorbance at wavelength 450 nm was measured with a spectrophotometer.
- a standard curve was prepared using the standard solution, and the absorbance obtained by the above method was substituted into the standard curve to calculate the amount of procollagen production of the cell culture solution to which each test compound was added.
- the transdermal absorption experiment was conducted using the substance represented by Formula II, palmitoyl-RGD, and as a control, a comparison experiment was performed using RGD to which no fatty acid was bound.
- RGD fatty acid
- the transmittance was measured in a Franz diffusion cell.
- RGD without fatty acid was used.
- Human skin stored at ⁇ 20 ° C. was used to confirm skin permeability.
- Transepidermal Water Loss (TEWL) confirmed that there was no skin damage to the skin (700 ⁇ m in thickness, -20 ° C. storage) to confirm the integrity of the structural skin before the test.
- the detection amount was insignificant. It has a hydrophilic property that the RGD includes a basic amino acid R (arginine) and an acidic amino acid D (aspartic acid), and thus does not penetrate the stratum corneum, which contains a large amount of hydrophobic lipids, and thus is directly delivered to the skin cells. There is no problem.
- stearoyl-RGD has excellent skin permeability, but is not suitable as a cosmetic because it is cytotoxic at a content that shows effective efficacy as shown in Table 1.
- the hydrophobic fatty acid palmitoyl and RGD were conjugated, and skin permeability was improved by 15 times and procollagen synthesis efficiency was also improved.
- palmitoyl-RGD raw material was dissolved in propanediol for each concentration to identify skin irritation.
- palmitoyl-RGD was confirmed that there is no skin irritation up to a concentration of 0.05% by weight.
- palmitoyl-RGD shows excellent stability to moisture and heat.
- solubilized formulations were prepared as shown in Table 6 below.
- Palmitoyl-RGD of addition I was solubilized, added and mixed, followed by mixing addition II.
- Palmitoyl-RGD Concentration in Compositions Number of respondents Responsiveness 0.01 wt% 0 0.0 0.02 wt% 0 0.0 0.05 wt% 0 0.0
- Palmitoyl-RGD Concentration in Compositions Number of respondents Responsiveness 0.01 wt% 0 0.0 0.02 wt% 0 0.0 0.05 wt% 0 0.0
- the skin wrinkle improvement effect on the cosmetic composition including the palmitoyl-RGD was measured.
- R2 and R3 values were evaluated as the main parameters of the wrinkles around the eyes, as measured by Visiometer SV600 (Courage-Khazaka electronic GmbH, Germany).
- R2 corresponds to the largest of the difference between the five highest peaks and the lowest valleys for the five equally divided sections of the wrinkle profile
- R3 defines the five equal segments along the X axis. It is the arithmetic average of the differences between the maximum and minimum values within each segment after dividing.
- the R2 value decreased significantly after 4 weeks, after 8 weeks, and after 12 weeks of use (p ⁇ 0.05), and also after 8 weeks and after 12 weeks of use compared to the control. (p ⁇ 0.05).
- the R3 values of the test product group were significantly decreased (p ⁇ 0.05) after 4 weeks, 8 weeks, and 12 weeks after use, and also significant after 8 weeks and 12 weeks after use. Difference (p ⁇ 0.05).
- Ra means the average roughness (average roughness) of the wrinkle profile, the smaller the value can be seen that the wrinkles around the eyes is improved, the results of the measured Ra value is shown in the graph in FIG.
- the Ra value of the test product group was significantly decreased after 12 weeks of use (p ⁇ 0.05) compared to before use, and also showed a significant difference (p ⁇ 0.05) after 4 weeks of use and 12 weeks after comparison with the control product. Confirmed.
- Skin elasticity was measured before use, 4 weeks after use, 8 weeks after, and 12 weeks after the use of palmitoyl-RGD. Skin elasticity was measured once using the Cutometer CM 580 (CK Electronics, Koln, Germany) at the same site of both eyes, and R2 (skin remodeling force) was used as an evaluation data for skin elasticity. The value of R2 is the total elasticity, and the closer to 1, the more elastic the skin is. The result of the measured R2 value is graphically shown in FIG. 7.
- the measured value of skin elasticity of the test product group was significantly increased after 4 weeks, 8 weeks, and 12 weeks after use (p ⁇ 0.05), and even after 4 weeks and 8 weeks of use compared to the control product. After 12 weeks, there was a significant difference (p ⁇ 0.05). Therefore, it was confirmed that the cosmetic composition including palmitoyl-RGD is effective in promoting skin elasticity.
- the measured value of the dermal density of the test product group was significantly increased after 8 weeks and after 12 weeks of use (p ⁇ 0.05). p ⁇ 0.05). Therefore, it was confirmed that the cosmetic composition including palmitoyl-RGD is effective in improving skin dermal density.
Abstract
The present invention relates to a tripeptide having fatty acids bonded thereto and a cosmetic composition comprising same and provides excellent skin anti-aging, wrinkle reduction, skin elasticity enhancement, and skin permeation effects.
Description
본 발명은 지방산이 결합된 트리펩타이드와, 이를 포함하여 피부 주름개선 효과가 우수한 화장료 조성물에 관한 것이다.The present invention relates to a tripeptide having a fatty acid bound thereto, and a cosmetic composition having an excellent skin wrinkle improvement effect.
피부는 인체를 보호하는 기능, 장벽기능, 배설기능 등 다양한 역할을 하는 중요한 기관이다. 그러나 오염물질, 자외선, 스트레스 등의 외부 환경요인으로 피부 세포들이 손상을 입고, 세포의 턴 오버 등이 제대로 이루어지지 않아 표피, 진피 및 피하조직의 두께가 얇아지고, 피부의 탄력이 저하되어 주름이 형성된다. 자외선은 피부에 활성산소를 유발시키고 그 결과 피부세포의 손상, 색소 침착을 증가시키게 되며 이는 피부 주요성분들의 직접적인 기능의 손실을 초래하며 피부 노화로 이어진다. 활성산소는 인체 내에서 질병과 노화를 일으키는 원인으로서 활성산소의 항산화력 및 노화억제 작용의 척도로 평가될 수 있다. 피부 노화의 특성으로는 탄력성의 감소, 주름살, 기미 또는 주근깨 등을 들 수 있고, 그 원인 중의 하나는 지질과산화에 의한 탄력섬유인 콜라겐과 엘라스틴의 사슬 절단과 비정상적인 교차 결합 및 히알루론산의 사슬 절단이다. 지질과산화는 산화스트레스 (oxidative stress)에 의해서 초래되며, 그 과정 중에서 반응 산소종(Reactive Oxygen Species, ROS)인 프리라디컬이 생성된다. 이러한 프리라디컬 생성과정은 피부에 유해한 대사적 활성화의 전 단계일 뿐만 아니라, 피부 세포 및 조직 손상을 일으킨다고 보고되었다. 특히 주름과 관련하여 활성산소에 의해 단백질 분해효소(Matrix MetalloProteinases, MMPs)의 발현이 유발되어 피부에 탄력을 부여하는 콜라겐 또는 엘라스틴 등을 변성 또는 파괴시켜 주름을 야기시킬 수 있다. 콜라겐은 피부 진피층의 매트릭스를 이루는 성분 중 가장 많은 성분이기 때문에 콜라겐의 생합성과 분해의 조절은 피부노화 과정 중에서 핵심이 되고 있다. 콜라겐 합성 촉진물질들은 아스코르빈산(미국등록특허 제4,983,382호), 및 레티노이드(미국 등록특허 제4,877,805호)와 그들의 유도체 등과 같은 화합물이 알려져 있으며, 이를 함유하고 있는 화장품 조성물이 개발되어 있다. 그러나 아스코르빈산은 공기 및 수분에 쉽게 산화되어 안정성의 문제가 있으며, 레티노이드의 경우 불안정하고 피부 적용 시에 피부 자극 문제들이 보고되고 있다.Skin is an important organ that plays a variety of roles such as protecting the human body, barrier function, and excretory function. However, skin cells are damaged by external environmental factors such as pollutants, ultraviolet rays, and stress, and cell turnover is not done properly, resulting in thinning of the epidermis, dermis and subcutaneous tissue, and deterioration of skin elasticity and wrinkles. Is formed. Ultraviolet rays cause free radicals in the skin, resulting in increased damage and pigmentation of the skin cells, leading to a loss of direct function of the skin's major components, leading to skin aging. Free radicals can be evaluated as a measure of antioxidant activity and anti-aging activity of free radicals as a cause of disease and aging in the human body. The characteristics of skin aging include a decrease in elasticity, wrinkles, blemishes or freckles, and one of the causes is chain breakage of collagen and elastin, which are elastic fibers due to lipid peroxidation, abnormal crosslinking and chain breakage of hyaluronic acid. . Lipid peroxidation is caused by oxidative stress, and in the process, free radicals, reactive oxygen species (ROS), are produced. This free radical production process has been reported to not only be a preliminary step of metabolic activation that is harmful to skin, but also cause skin cell and tissue damage. In particular, the expression of proteolytic enzymes (Matrix MetalloProteinases, MMPs) are induced by the active oxygen in relation to wrinkles may cause wrinkles by denaturing or destroying collagen or elastin, which gives elasticity to the skin. Since collagen is the most abundant component of the matrix of the dermal layer of the skin, the control of biosynthesis and degradation of collagen is the key during skin aging process. Collagen synthesis promoters are known compounds such as ascorbic acid (US Pat. No. 4,983,382), and retinoids (US Pat. No. 4,877,805) and their derivatives, and cosmetic compositions containing the same have been developed. However, ascorbic acid is easily oxidized in the air and moisture, there is a stability problem, retinoids are unstable, and skin irritation problems have been reported in the skin application.
피부자극이 적으면서 피부 주름개선 효과를 갖는 화합물로는 펩타이드가 있는데, 펩타이드는 단백질의 주요 구성성분으로서, 피부에 대한 부작용이 없이 피부 주름개선을 시킬 수 있는 장점이 있다. 이 중에 RGD(Arg-Gly-Asp) 서열은 1980년대 후반부터 세포인식(cell recognition) 서열로 알려져 있으며, 합성 RGD 펩타이드는 이미 1990년대 초반부터 사용되고 있다(Biochem. J. (1993) 296, 21-24). 또한 논문에 따르면 RGD 포함 펩타이드가 인테그린 매개 세포부착(integrin-mediated cell attachment)에 영향을 주고 상기 기능은 세포성장(cell growth)에 영향을 미친다고 알려져 있다(Annu Rev Cell Dev Biol. (1996) 12:697-715.). 현재 RGD는 콜라겐 활성화 및 피부 주름개선을 위한 성분으로서 이용되고 있으나, 이 트리펩타이드는 친수성으로서 피부장벽을 통과하는데 어려움이 있어 실제적으로 주름개선 효능을 기대하기에 한계가 있다.As a compound having a small skin irritation and having a skin wrinkle improvement effect, there is a peptide, the peptide is a major component of the protein, there is an advantage that can improve the skin wrinkles without side effects on the skin. Among them, the RGD (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, it is known that RGD-containing peptides affect integrin-mediated cell attachment and the function affects cell growth (Annu Rev Cell Dev Biol. (1996) 12). : 697-715.). Currently, RGD is used as an ingredient for collagen activation and skin wrinkle improvement, but the tripeptide is hydrophilic and difficult to cross the skin barrier.
본 발명은 상기와 같은 종래 기술의 문제점을 극복하고자 친수성 트리펩타이드인 RGD(아르기닌-글리신-아스파르트산)의 피부장벽에 대한 침투의 어려움을 해결하기 위하여, 상기 RGD에 다양한 소수성의 지방산들을 결합시켜 친수-친유 구조를 가지는 다수의 유도체들을 제조하였으며, 이들의 피부 주름개선 효능이 좋은 것을 확인함으로써 본 발명을 완성하게 되었다.The present invention is to overcome the problems of the prior art as described above, in order to solve the difficulty of penetration of the RGD (arginine-glycine-aspartic acid) of the hydrophilic tripeptides into the skin barrier, by combining various hydrophobic fatty acids to the RGD -A number of derivatives having a lipophilic structure were prepared, and the present invention was completed by confirming that their skin wrinkle improvement efficacy is good.
따라서, 본 발명의 해결하고자 하는 과제는 하기 [화학식 I]의 지방산이 결합된 트리펩타이드 또는 그의 화장료로 허용되는 염을 제공하는 것이다.Therefore, the problem to be solved by the present invention is to provide a salt that is acceptable as a tripeptide or a cosmetic thereof to which a fatty acid of the formula [I] is bound.
또한, 본 발명의 해결하고자 하는 다른 과제는 하기 [화학식 I]의 지방산이 결합된 트리펩타이드 또는 그의 화장료로 허용되는 염을 유효성분으로 포함하는 화장료 조성물 및 그의 주름개선 용도를 제공하는 것이다.In addition, another problem to be solved of the present invention is to provide a cosmetic composition comprising the tripeptide of the following [Formula I] or a salt acceptable as a cosmetic as an active ingredient and its use for improving wrinkles.
본 발명은 지방산이 결합된 트리펩타이드 및 이를 포함하여 피부 주름개선 효과가 우수한 화장료 조성물에 관한 것이다.The present invention relates to a tripeptide combined with a fatty acid and a cosmetic composition having excellent skin anti-wrinkle effect.
이하, 본 발명을 보다 구체적으로 설명한다.Hereinafter, the present invention will be described in more detail.
본 발명은 지방산이 결합된 트리펩타이드로서 지방산이 결합된 RGD 또는 그의 화장료로 허용되는 염, 더욱 바람직하게는 팔미토일-RGD(Palmitoyl-RGD) 또는 그의 화장료로 허용되는 염을 제공한다.The present invention provides a tripeptide to which a fatty acid is bound, an RGD to which a fatty acid is bound or a cosmetically acceptable salt thereof, more preferably palmitoyl-RGD or a cosmetically acceptable salt thereof.
상기 RGD는 아르기닌, 글리신, 아스파르트산의 세가지 아미노산이 연결된 트리펩타이드로서, 콜라겐 합성 효능이 있는 것으로 알려져 화장품 원료로 사용되고 있다.The RGD is a tripeptide in which three amino acids of arginine, glycine, and aspartic acid are connected, and is known to have collagen synthesis efficacy and is used as a cosmetic raw material.
상기 지방산이 결합된 RGD는 화학식 I과 같은 일반식으로 표시된다.The RGD to which the fatty acid is bound is represented by the general formula of Formula (I).
[화학식 I][Formula I]
상기 식에서, n은 11 내지 16의 정수이다.Wherein n is an integer from 11 to 16.
또한, 상기 팔미토일-RGD는 화학식 I에서 n이 15인 것으로서, 하기 화학식 Ⅱ 또는 Palm-Arg-Gly-Asp로 표시되는 화합물이다.In addition, the palmitoyl-RGD is a compound represented by Formula (II) or Palm-Arg-Gly-Asp, wherein n is 15 in Formula (I).
[화학식 Ⅱ][Formula II]
또한, 본 발명은 상기 화학식 I의 지방산이 결합된 트리펩타이드(RGD) 또는 그를 포함하는 화장료 조성물을 제공한다.The present invention also provides a tripeptide (RGD) to which the fatty acid of formula (I) is bound or a cosmetic composition comprising the same.
상기 화장료 조성물은 피부 노화 방지, 주름 개선, 피부 탄력 증진 및 피부 침투 효과가 탁월할 뿐만 아니라 장기 보존 안정성 및 피부 안전성에 있어서도 높은 효과를 제공한다.The cosmetic composition is not only excellent skin anti-aging, wrinkle improvement, skin elasticity enhancement and skin penetration effect, but also provides a high effect in long-term storage stability and skin safety.
상기 화학식 I의 화합물이 처리된 세포의 프로콜라겐 합성양은 하기 화학식 Ⅲ의 화합물을 동일 농도로 처리한 경우에 비하여 20 % 이상 많을 수 있다.The amount of procollagen synthesis of the cells treated with the compound of Formula I may be 20% or more compared with the case where the compound of Formula III is treated at the same concentration.
[화학식 Ⅲ][Formula III]
본 발명의 화장료 조성물은 화장료 조성물 총 중량에 대하여 0.0001 내지 2 중량%, 바람직하게는 0.01 내지 0.05 중량%의 화학식 I의 화합물을 유효성분으로 포함한다.The cosmetic composition of the present invention comprises 0.0001 to 2% by weight, preferably 0.01 to 0.05% by weight of the compound of formula (I) as an active ingredient based on the total weight of the cosmetic composition.
또한, 본 발명은 상기 화학식 Ⅰ의 지방산이 결합된 트리펩타이드(RGD) 또는 그의 화장료로 허용되는 염을 포함하는 피부 주름개선용 화장료 조성물을 제공한다. The present invention also provides a cosmetic composition for improving skin wrinkles comprising a tripeptide (RGD) to which the fatty acid of Formula (I) is bound or a salt thereof that is acceptable as a cosmetic.
본 발명의 화장료 조성물은 화장료 조성물 총 중량에 대하여 0.0001 내지 2 중량%, 바람직하게는 0.01 내지 0.05 중량%의 화학식 I의 화합물을 유효성분으로 포함한다.The cosmetic composition of the present invention comprises 0.0001 to 2% by weight, preferably 0.01 to 0.05% by weight of the compound of formula (I) as an active ingredient based on the total weight of the cosmetic composition.
본 발명의 화장료 조성물은 그 제형이 특별히 한정되는 것은 아니며, 제조하고자 하는 제형에 따라 발명이 속하는 기술 분야에서 통상적으로 사용되는 화장료 조성물 배합을 포함할 수 있다. 본 발명의 화장료 조성물은 화장수, 유액, 영양크림, 팩, 미용액, 에센스 등의 제형으로 제조할 수 있으며, 제조하고자 하는 제형에 따라, 유제, 유화제, 유화안정화제, 보습제, 증점제, 보존제, 향료 등을 선택하여 배합할 수 있다. The cosmetic composition of the present invention is not particularly limited in its formulation, and may include a cosmetic composition combination commonly used in the art to which the invention pertains, depending on the formulation to be prepared. The cosmetic composition of the present invention can be prepared in a formulation such as lotion, milky lotion, nourishing cream, pack, essence, essence, etc., according to the formulation to be prepared, emulsion, emulsifier, emulsion stabilizer, moisturizer, thickener, preservative, fragrance Can be selected and blended.
본 발명은 다음의 단계를 포함하는 상기 화학식 I의 화합물 제조방법을 제공한다.The present invention provides a method for preparing a compound of formula (I), comprising the following steps.
(a) N-말단, C-말단 또는 반응성 잔기가 보호된 아르기닌 유도체, 글리신 유도체 및 아스파르트산 유도체 이용하여 액상에서 트리펩타이드(아르기닌-글리신-아스파르트산) 유도체를 합성하는 단계;(a) synthesizing tripeptides (arginine-glycine-aspartic acid) derivatives in a liquid phase using arginine derivatives, glycine derivatives and aspartic acid derivatives protected with N-terminal, C-terminal or reactive moieties;
(b) 상기 트리펩타이드(아르기닌-글리신-아스파르트산) 유도체와 지방산염을 반응시켜 아르기닌의 N-말단에 지방산을 결합시키는 단계; 및(b) reacting the tripeptide (arginine-glycine-aspartic acid) derivative with a fatty acid salt to bind a fatty acid to the N-terminus of arginine; And
(c) 상기 지방산이 결합된 트리펩타이드(아르기닌-글리신-아스파르트산) 유도체에서 트리펩타이드 부분의 보호기를 제거하는 단계.(c) removing the protecting group of the tripeptide moiety from the tripeptide (arginine-glycine-aspartic acid) derivative to which the fatty acid is bound.
이하, 본 발명에 따른 상기 화학식 Ⅱ의 팔미토일-RGD 제조방법을 하기 반응식 I을 참조하여 상세히 설명한다. 하기 반응식 I에 기재된 방법은 대표적으로 사용된 방법을 예시한 것일 뿐, 단위 조작의 순서, 반응 시약, 반응 조건 등은 경우에 따라 얼마든지 변경될 수 있다. 또한, 일반적인 펩타이드 합성 방법인 고체합성 방법으로 제조된 RGD를 사용할 수 있다.Hereinafter, the method for preparing palmitoyl-RGD of Formula II according to the present invention will be described in detail with reference to Scheme I below. The method described in Scheme I below is merely an example of a method used, and the order of unit operations, reaction reagents, reaction conditions, and the like may be changed as desired. In addition, RGD prepared by the solid synthesis method, which is a general peptide synthesis method can be used.
[반응식 I]Scheme I
상기 방법에 따라 제조된 본 발명의 팔미토일-RGD는 안정할 뿐만 아니라, 우수한 피부 노화 방지, 주름 개선, 피부 탄력 증진 및 피부 침투에 탁월한 효과를 나타낸다.The palmitoyl-RGD of the present invention prepared according to the above method is not only stable but also shows an excellent effect on excellent skin anti-aging, wrinkle improvement, skin elasticity enhancement and skin penetration.
본 발명에 따른 지방산이 결합된 트리펩타이드 화합물은 우수한 피부 노화 방지, 주름 개선, 피부 탄력 증진 및 피부 침투 효과를 제공한다.The tripeptide compound in combination with the fatty acid according to the present invention provides excellent skin anti-aging, wrinkle improvement, skin elasticity enhancement and skin penetration effect.
도 1은 지방산이 결합된 RGD의 세포독성 실험에 대한 결과를 나타낸 그래프이다.Figure 1 is a graph showing the results for the cytotoxicity test of fatty acid-bound RGD.
도 2는 지방산이 결합된 RGD의 프로콜라겐 합성 효과를 비교하여 나타낸 그래프이다.Figure 2 is a graph showing a comparison of the collagen synthesis effect of fatty acid-bound RGD.
도 3은 지방산이 결합된 RGD의 피부 경피 흡수 효과를 비교하여 나타낸 그래프이다. Figure 3 is a graph comparing the skin transdermal absorption effect of the fatty acid-bound RGD.
도 4 내지 6은 팔미토일-RGD를 포함한 조성물의 눈가 주름개선 효과를 나타낸 그래프이다.4 to 6 is a graph showing the effect of improving the wrinkles around the eyes of the composition containing palmitoyl-RGD.
도 7은 팔미토일-RGD를 포함한 조성물의 피부 탄력 개선 효과를 나타낸 그래프이다.7 is a graph showing the skin elasticity improving effect of the composition containing palmitoyl-RGD.
도 8은 팔미토일-RGD를 포함한 조성물의 피부 진피치밀도 개선 효과를 나타낸 그래프이다.8 is a graph showing the skin dermal density improvement effect of the composition containing palmitoyl-RGD.
본 발명은 지방산이 결합된 트리펩타이드 및 이를 포함하는 주름개선용 화장료 조성물을 제공한다.The present invention provides a cosmetic composition for improving wrinkles comprising a tripeptide and a fatty acid is bound thereto.
본 발명의 일 구체예에서, 상기 지방산이 결합된 트리펩타이드는 팔미토일-RGD(Palmitoyl-RGD)일 수 있다.In one embodiment of the present invention, the tripeptide to which the fatty acid is bound may be Palmitoyl-RGD.
이하, 실시예를 통해 본 발명을 보다 구체적으로 설명한다. 그러나, 이들 실시예는 본 발명을 설명하기 위한 것일 뿐이므로 발명의 범위가 이에 한정되는 것은 아니다.Hereinafter, the present invention will be described in more detail with reference to Examples. However, these examples are only for illustrating the present invention, and the scope of the present invention is not limited thereto.
제조예 1: 팔미토일-RGD의 제조Preparation Example 1 Preparation of Palmitoyl-RGD
제조예 1-1: Boc-Gly-Asp(OBn)2 합성Preparation Example 1-1 Synthesis of Boc-Gly-Asp (OBn) 2
N-(t-부톡시카르보닐)글리신(N-(tert-Butoxycarbonyl)glycine, N-Boc-Gly-OH)(20 g, 114.17 mmol)과 L-아스파틱애씨드 디벤질 에스터(L-aspartic acid dibenzyl ester)(55.43 g, 114.17 mmol)를 테트라하이드로퓨란(THF)(600 mL)에 녹인 후 0 ℃로 온도를 낮추고 그 용액에 1-하이드록시벤조트리아졸(1-hydroxybenzotriazole hydrate, HOBt)(18.51 g, 137.00 mmol), 1-(3-디메틸아미노프로필)-3-에틸카보디이미드 하이드로클로라이드(1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, EDC. HCl)(27.00 g, 137.00 mmol)를 첨가하고, 트리에틸아민(TEA)(55.69 mL, 399.59 mmol)을 천천히 첨가하였다. 0 ℃에서 30분 교반 후 냉각 중탕기(cooling bath) 제거 후 상온에서 밤새 교반하였다. 반응용액을 농축하고 물(320 mL)과 에틸아세테이트(400 mL)를 넣고 2번 추출한 뒤(400 mL x 2) 유기층을 소금물(300 mL)로 씻어주고, 소듐 설페이트(Na2SO4)로 건조하여 여과 한 후 압력하에서 농축시켰다. 농축시킨 물질을 감압 하에서 건조하여 (S)-디벤질2-(2-((t-부톡시카르보닐)아미노)아세트아미도)숙시네이트((S)-dibenzyl 2-(2-((tert-butoxycarbonyl)amino)acetamido)succinate, Boc-Gly-Asp(OBn)2)(64.53 g, 120 %)를 노란색 오일로 얻어서 정제과정 없이 다음 반응을 진행하였다.N- (t-butoxycarbonyl) glycine (N- (tert-Butoxycarbonyl) glycine, N-Boc-Gly-OH) (20 g, 114.17 mmol) with L-aspartic acid dibenzyl ester (L-aspartic acid dibenzyl ester) (55.43 g, 114.17 mmol) was dissolved in tetrahydrofuran (THF) (600 mL), then the temperature was lowered to 0 ° C. and 1-hydroxybenzotriazole hydrate (HOBt) (18.51) was added to the solution. g, 137.00 mmol), 1- ( 3- dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (1- (3-dimethylaminopropyl) -3 -ethylcarbodiimide hydrochloride, EDC. HCl) (27.00 g, 137.00 mmol) to Triethylamine (TEA) (55.69 mL, 399.59 mmol) was added slowly. After stirring at 0 ° C. for 30 minutes, a cooling bath was removed, followed by stirring at room temperature overnight. Concentrate the reaction solution, add water (320 mL) and ethyl acetate (400 mL), extract twice (400 mL x 2), and wash the organic layer with brine (300 mL), and dry with sodium sulfate (Na 2 SO 4 ). After filtration and concentration under pressure. The concentrated material was dried under reduced pressure to afford (S) -dibenzyl2- (2-((t-butoxycarbonyl) amino) acetamido) succinate ((S) -dibenzyl 2- (2-((tert -butoxycarbonyl) amino) acetamido) succinate, Boc-Gly-Asp (OBn) 2 ) (64.53 g, 120%) was obtained as a yellow oil, and the following reaction was carried out without purification.
제조예 1-2: Gly-Asp(OBn)2 HCl 합성Preparation Example 1-2 Synthesis of Gly-Asp (OBn) 2 HCl
(S)-디벤질2-(2-((t-부톡시카르보닐)아미노)아세트아미도)숙시네이트(53.71 g, 114.15 mmol)를 에틸아세테이트(500 mL)에 녹인 후 4 M 하이드로클로라이드(HCl) 1,4-디옥산(1,4-dioxane)(113 mL, 114.15 mmol) 용액을 첨가하여 상온에서 6시간 교반하였다. 생성된 고체를 여과하고 감압 하에서 건조하여 (S)-디벤질2-(2-아미노아세트아미도)숙시네이트 하이드로클로라이드((S)-dibenzyl 2-(2-aminoacetamido)succinate hydrochloride, Gly-Asp(OBn)2 HCl)(39.47 g, 85 %)의 흰색고체를 얻었다.(S) -dibenzyl2- (2-((t-butoxycarbonyl) amino) acetamido) succinate (53.71 g, 114.15 mmol) was dissolved in ethyl acetate (500 mL) and 4M hydrochloride ( HCl) 1,4-dioxane (1,4-dioxane) (113 mL, 114.15 mmol) was added thereto, followed by stirring at room temperature for 6 hours. The resulting solid was filtered and dried under reduced pressure to afford (S) -dibenzyl2- (2-aminoacetamido) succinate hydrochloride ((S) -dibenzyl 2- (2-aminoacetamido) succinate hydrochloride, Gly-Asp ( OBn) 2 HCl) (39.47 g, 85%) to obtain a white solid.
제조예 1-3: Boc-Arg(NO2)-Gly-Asp(OBn)2 합성Preparation Example 1-3 Synthesis of Boc-Arg (NO 2 ) -Gly-Asp (OBn) 2
(S)-디벤질2-(2-아미노아세트아미도)숙시네이트 하이드로클로라이드(39.47 g, 97.01 mmol)와 2-((t-부톡시카르보닐)아미노)-5-(3-니트로구아니디노펜타노익산(2-((tert-Butoxycarbonyl)amino)-5-(3-nitroguanidino)pentanoic acid, N-Boc-Arg(NO2)-OH)(30.98 g, 97.01 mmol)을 테트라하이드로퓨란(600 mL)에 녹인 후 0 ℃로 온도를 낮추고 그 용액에 1-하이드록시벤조트리아졸(15.73 g, 116.41 mmol), 1-(3-디메틸아미노프로필)-3-에틸카보디이미드 하이드로클로라이드(22.31 g, 116.41 mmol)를 첨가하고, 트리에틸아민(47.33 mL, 339.54 mmol)을 천천히 첨가하였다. 0 ℃에서 30분 교반 후 냉각 중탕기(cooling bath) 제거 후 상온에서 밤새 교반하였다. 반응용액을 농축하고 물(320 mL)과 에틸아세테이트(400 mL)를 넣고 2번 추출한 뒤(400 mL x 2) 유기층을 소금물(300 mL)로 씻어주고, 소듐 설페이트로 건조하여 여과한 후 압력하에서 농축시켰다. 농축시킨 물질을 감압 하에서 건조하여 (S)-디벤질2-(2-((S)-2((t-부톡시카르보닐)아미노)-5-(3-니트로구아니디노)펜탄아미도)아세타아미도)숙시네이트((S)-dibenzyl 2-(2-((S)-2((tert-Butoxycarbonyl)amino)-5-(3-nitroguanidino)pentanamido)acetamido)succinate, Boc-Arg(NO2)-Gly-Asp(OBn)2)(65.16 g, 102 %)를 노란색 오일로 얻어서 정제과정 없이 다음 반응을 진행하였다.(S) -dibenzyl2- (2-aminoacetamido) succinate hydrochloride (39.47 g, 97.01 mmol) and 2-((t-butoxycarbonyl) amino) -5- (3-nitroguani Dinopentanoic acid (2-((tert-Butoxycarbonyl) amino) -5- (3-nitroguanidino) pentanoic acid, N-Boc-Arg (NO 2 ) -OH) (30.98 g, 97.01 mmol) was dissolved in tetrahydrofuran (600 mL) and the temperature was lowered to 0 ° C. and 1-hydroxybenzotriazole (15.73 g, 116.41 mmol), 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (22.31 g, 116.41 mmol) was added and triethylamine (47.33 mL, 339.54 mmol) was added slowly. After stirring at 0 ° C. for 30 minutes, a cooling bath was removed, followed by stirring at room temperature overnight. Concentrate the reaction solution, add water (320 mL) and ethyl acetate (400 mL), and extract twice (400 mL x 2). The organic layer is washed with brine (300 mL), dried over sodium sulfate and filtered. Concentrated. The concentrated material was dried under reduced pressure to afford (S) -dibenzyl2- (2-((S) -2 ((t-butoxycarbonyl) amino) -5- (3-nitroguanidino) pentaneamido Acetaamido) 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%) was obtained as a yellow oil, and the following reaction was carried out without purification.
제조예 1-4: Arg(NO2)-Gly-Asp(OBn)2 HCl salt 합성Preparation Example 1-4: Synthesis of Arg (NO 2 ) -Gly-Asp (OBn) 2 HCl salt
(S)-디벤질2-(2-((S)-2((t-부톡시카르보닐)아미노)-5-(3-니트로구아니디노)펜탄아미도)아세타아미도)숙시네이트(65.16 g, 97.00 mmol)를 에틸아세이트(500 mL)에 녹여 4 M 하이드로클로라이드 1,4-디옥산(1,4-dioxane)(94.58 mL, 378.30 mmol) 용액을 첨가하여 상온에서 6 시간 교반하였다. 생성된 고체를 여과하여 감압 하에서 건조하여 (S)-디벤질2-(2-((S)-2-아미노)-5-(3-니트로구아니디노)펜탄아미도)아세타아미도)숙시네이트 하이드로클로라이드((S)-dibenzyl 2-(2-((S)-2-amino)-5-(3-nitroguanidino)pentanamido)acetamido)succinate hydrochloride, Arg(NO2)-Gly-Asp(OBn)2 HCl salt)(59.08 g, 100 %)의 흰색고체를 얻었다. (S) -dibenzyl2- (2-((S) -2 ((t-butoxycarbonyl) amino) -5- (3-nitroguanidino) pentaneamido) acetaamido) succinate (65.16 g, 97.00 mmol) was dissolved in ethyl acetate (500 mL), and a solution of 4M hydrochloride 1,4-dioxane (94.58 mL, 378.30 mmol) was added thereto, followed by stirring at room temperature for 6 hours. It was. The resulting solid was filtered and dried under reduced pressure (S) -dibenzyl2- (2-((S) -2-amino) -5- (3-nitroguanidino) pentaneamido) acetaamido) succinate hydrochloride ((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 %) White solid was obtained.
제조예 1-5: Palm-Arg(NO2)-Gly-Asp(OBn)2 합성Preparation Example 1-5 Synthesis of Palm-Arg (NO 2 ) -Gly-Asp (OBn) 2
(S)-디벤질2-(2-((S)-2-아미노)-5-(3-니트로구아니디노)펜탄아미도)아세타아미도)숙시네이트 하이드로클로라이드(59.06 g, 97.04 mmol)를 디클로로메탄(CH2Cl2)(800 mL)에 녹인 후 0 ℃로 온도를 낮춘 후 이 용액에 팔미토일 클로라이드(palmitoyl chloride)(30.76 mL, 101.85 mmol)를 첨가하고, 트리에틸아민(27.04 mL, 194.01 mmol)을 천천히 첨가하였다. 0 ℃에서 30 분 동안 교반 후 냉각 중탕기(cooling bath)를 제거하고, 상온에서 밤새 교반하였다. 반응 후, 압력하에서 농축하여 디클로로메탄을 일부 제거 후 물을 넣어 여과하였다. 그 고체를 감압 하에서 건조하여 (S)-디벤질2-(2-((S)-5-(3-니트로구아니디노)-2팔미트아미도펜탄아미도)아세타아미도)숙시네이트((S)-dibenzyl 2-(2-((S)-5-(3-nitroguanidino)-2-palmitamidopentanamido)acetamido)succinate, Palm-Arg(NO2)-Gly-Asp(OBn)2)(73.01 g, 93 %)를 얻었다.(S) -dibenzyl2- (2-((S) -2-amino) -5- (3-nitroguanidino) pentaneamido) acetaamido) succinate hydrochloride (59.06 g, 97.04 mmol ) Was dissolved in dichloromethane (CH 2 Cl 2 ) (800 mL), the temperature was lowered to 0 ° C., and palmitoyl chloride (30.76 mL, 101.85 mmol) was added to the solution, and triethylamine (27.04) was added. mL, 194.01 mmol) was added slowly. After stirring at 0 ° C. for 30 minutes, the cooling bath was removed and stirred at room temperature overnight. After the reaction, the mixture was concentrated under pressure, and some of the dichloromethane was removed, followed by filtering with water. The solid was dried under reduced pressure to afford (S) -dibenzyl2- (2-((S) -5- (3-nitroguanidino) -2palmitamidopentaneamido) acetaamido) succinate ((S) -dibenzyl 2- (2-((S) -5- (3-nitroguanidino) -2-palmitamidopentanamido) acetamido) succinate, Palm-Arg (NO 2 ) -Gly-Asp (OBn) 2 ) (73.01 g, 93%).
제조예 1-6: Palm-RGD-HCl salt 합성Preparation Example 1-6: Palm-RGD-HCl salt synthesis
(S)-디벤질2-(2-((S)-5-(3-니트로구아니디노)-2팔미트아미도펜탄아미도)아세타아미도)숙시네이트 (70.00 g. 86.42 mmol)와 테트라하이드로퓨란(1.5L)을 넣은 후 3 M 하이드로클로라이드(288.07 mL, 864.21 mmol)를 첨가하였다. 여기에 알곤(Ar) 하에서 10 % 팔라듐카본(Pd/C)(8.64 g)을 넣었다. 그 용액을 수소(H2) 하에서 3시간 동안 상온에서 교반하였다. 반응 혼합물을 셀라이트를 이용해 여과하여 나온 용액을 감압하여 용매를 일부 제거하였다. 남아 있는 용액에 소듐하이드록사이드(NaOH)(aq.)를 천천히 첨가해 pH 2 이상에서 생성된 고체를 물로 세척 하면서 여과하고 건조하였다. 이 고체를 이소프로필알콜(IPA)에서 환류하여 뜨거워진 상태에서 여과하고 다시 건조하였다. 얻어진 고체에 5 M 소듐하이드록사이드와 6 M 하이드로클로라이드 첨가하면 용액이 뜨거워지면서 녹았다가 식으면서 고체가 생성된 후 이 고체를 여과하여 진공 오븐에서 건조한 후 (S)-디벤질2-(2-((S)-5-구아니디노-2팔미트아미도펜탄아미도)아세타아미도)숙시네이트 하이드로클로라이드((S)-dibenzyl 2-(2-((S)-5-guanidino)-2-palmitamidopentanamido)acetamido)succinate hydrochloride), 즉 팔미토일-RGD 염(Palm-RGD-HCl salt)(33.82 g, 63 %)을 얻었다.(S) -dibenzyl2- (2-((S) -5- (3-nitroguanidino) -2palmitamidopentaneamido) acetamido) succinate (70.00 g. 86.42 mmol) Tetrahydrofuran (1.5L) was added, followed by 3 M hydrochloride (288.07 mL, 864.21 mmol). To this was added 10% palladium carbon (Pd / C) (8.64 g) under argon (Ar). The solution was stirred at room temperature for 3 hours under hydrogen (H 2 ). The reaction mixture was filtered through celite, and the solvent was removed by reducing the pressure of the solution. Sodium hydroxide (NaOH) (aq.) Was slowly added to the remaining solution, and the resulting solid was filtered and dried with water washing at pH 2 or above. The solid was refluxed in isopropyl alcohol (IPA), filtered while hot and dried again. When 5 M sodium hydroxide and 6 M hydrochloride were added to the obtained solid, the solution became hot and dissolved, and after cooling, the solid was formed. The solid was filtered and dried in a vacuum oven, followed by (S) -dibenzyl2- (2 -((S) -5-guanidino-2palmitamidopentaneamido) acetaamido) succinate hydrochloride ((S) -dibenzyl 2- (2-((S) -5-guanidino) -2-palmitamidopentanamido) acetamido) succinate hydrochloride), ie Palm-RGD-HCl salt (33.82 g, 63%).
실험예 1: 세포 독성 실험Experimental Example 1: Cytotoxicity Test
지방산이 결합된 RGD의 세포 독성을 확인하기 위하여, 각각 지방산의 길이가 상이한 4가지 물질로서 라우로일-RGD(Lauroyl-Arg-Gly-Asp), 미리스토일-RGD(Myristoyl-Arg-Gly-Asp), 팔미토일-RGD(Palmitoyl-Arg-Gly-Asp), 스테로일-RGD(Stearoyl-Arg-Gly-Asp)에 대하여 다음과 같은 방법으로 세포 독성 실험을 진행하였다.In order to confirm the cytotoxicity of fatty acid-bound RGDs, four substances having different lengths of fatty acids were respectively Lauroyl-Arg-Gly-Asp and Myristoyl-Arg-Gly-. Asp), palmitoyl-Arg-Gly-Asp (Palmitoyl-Arg-Gly-Asp), steroyl-RGD (Stearoyl-Arg-Gly-Asp) was carried out in the following cytotoxicity experiments.
사람의 정상 섬유아세포(Human Dermal Fibroblast neonatal, Cascade Co., HDFn cell)를 24-마이크로 웰 플레이드에 접종하고(5×104 세포/웰), 37 ℃, 5 % 이산화탄소 조건에서 24시간 배양하였다. 배지를 버리고 인산완충식염수(Phosphate Buffered Saline, PBS)로 세척한 다음, 둘배코수정이글배지(Dulbecco's Modified Eagle's Medium, DMEM)에 1 % 페니실린/스트랩토마이신(Penicillins/Streptomycin, P/S)을 첨가한 배지를 넣고 24시간 배양하였다. 배지를 버리고 인산완충식염수로 세척한 다음, 배지 900 uL와 라우로일-RGD, 미리스토일-RGD, 팔미토일-RGD 및 스테로일-RGD를 최종 처리 농도에 맞추어 각각 100 uL씩 넣고 48시간 배양하였다. 배지를 버리고 인산완충식염수로 세척한 다음, MTT(3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) 용액 100 uL 와 배지 900 uL을 넣고, 4시간 동안 배양하였다. 배양액을 제거한 다음 디메틸설포사이드(DMSO) 용액 300 uL씩을 넣고 10분간 흔들어 준 다음 마이크로 플레이트 리더로 570 nm에서 흡광도를 측정함으로써 세포생존율을 확인하였다.Human normal fibroblasts (Human Dermal Fibroblast neonatal, Cascade Co., HDFn cells) were seeded in 24-micro well plates (5 × 10 4 cells / well) and incubated at 37 ° C., 5% carbon dioxide for 24 hours. . Discard the medium and wash with Phosphate Buffered Saline (PBS) and add 1% Penicillins / Streptomycin (P / S) to Dulbecco's Modified Eagle's Medium (DMEM). One medium was added and incubated for 24 hours. Discard the medium and wash with phosphate-buffered saline, then add 900 uL of medium and 100 uL of lauroyl-RGD, myristoyl-RGD, palmitoyl-RGD and steroyl-RGD to the final treatment concentration for 48 hours. Incubated. The medium was discarded and washed with phosphate buffered saline. Then, 100 uL of MTT (3- [4,5-dimethylthiazol-2-yl] -2,5-diphenyltetrazolium bromide) solution and 900 uL of medium were added thereto, and the culture was performed for 4 hours. After removing the culture solution, 300 ul each of dimethyl sulfoside (DMSO) solution was added thereto, shaken for 10 minutes, and the cell viability was confirmed by measuring absorbance at 570 nm using a microplate reader.
그 결과를 하기 표 1 및 도 1에 나타내었다.The results are shown in Table 1 and FIG. 1.
| Sample 명칭 및 농도Sample Name and Concentration | 세포생존율Cell survival rate | |
| 음성 대조군Negative control | 102 %102% | |
| Lauroyl-RGDLauroyl-rgd | 1.0 ug/mL1.0 ug / mL | 101 %101% |
|
2.0 ug/mL2.0 ug / |
100 %100% | |
| 10.0 ug/mL10.0 ug / mL | 101 %101% | |
| Myristoyl-RGDMyristoyl-rgd |
1.0 ug/mL1.0 ug / |
100 %100% |
| 4.0 ug/mL4.0 ug / mL | 99 %99% | |
| 10.0 ug/mL10.0 ug / mL | 102 %102% | |
| Palmitoyl-RGDPalmitoyl-rgd |
1.0 ug/mL1.0 ug / |
100 %100% |
| 2.0 ug/mL2.0 ug / mL | 102 %102% | |
| 4.0 ug/mL4.0 ug / mL | 103 %103% | |
| Stearoyl-RGDStearoyl-rgd | 1.0 ug/mL1.0 ug / mL | 102 %102% |
| 2.0 ug/mL2.0 ug / mL | 54 %54% | |
| 4.0 ug/mL4.0 ug / mL | 24 %24% | |
|
8.0 ug/mL8.0 ug / |
20 %20% | |
상기 표 1에서 보는 바와 같이, 라우로일-RGD 및 미리스토일-RGD는 10.0 ug/mL 이하의 농도에서, 팔미토일-RGD는 4.0 ug/mL 이하의 농도에서는 세포 독성을 나타내지 않는 것을 확인하였다. 반면, 스테로일-RGD의 경우 2.0 ug/mL 이상에서 높은 세포 독성을 나타내는 것을 확인하였다.As shown in Table 1, it was confirmed that lauroyl-RGD and myristoyl-RGD did not show cytotoxicity at concentrations of 10.0 ug / mL or less and palmitoyl-RGD at concentrations of 4.0 ug / mL or less. . On the other hand, it was confirmed that the steroyl-RGD shows high cytotoxicity at 2.0 ug / mL or more.
실험예 2: 프로콜라겐 합성 효능 분석Experimental Example 2: Procollagen Synthesis Efficacy Analysis
지방산 결합된 트리펩타이드로서 라우로일-RGD, 미리스토일-RGD, 팔미토일-RGD 및 스테로일-RGD의 프로콜라겐 합성 효능을 분석하여 적절한 지방산 길이를 찾고자 하였으며, 대조군으로서 지방산이 결합하지 않은 RGD 및 아세틸-RGD를 사용하여 비교 실험하였다.We tried to find the proper fatty acid length by analyzing the procollagen synthesis efficacy of lauroyl-RGD, myristoyl-RGD, palmitoyl-RGD and steroyl-RGD as fatty acid-bound tripeptides. Comparison experiments were performed using RGD and acetyl-RGD.
사람의 정상 섬유아세포를 10 %의 우태아 혈청이 보충된 둘배코수정이글배지를 이용하여 24-웰 마이크로 플레이트에 접종하고(5×104 세포/웰), 37 ℃, 5 % CO2 조건에서 24시간 동안 배양하였다. 혈청이 들어 있지 않은 둘배코수정이글배지로 교체하여 24시간 동안 배양한 후, 각 시험 화합물을 적정량 처리하였다. 48시간 배양 후 세포배양액을 채취하여 후 타카라사(Takara Shuzo Co., Ltd., 일본)의 콜라겐 측정키트를 사용하여 프로콜라겐 양을 측정하였다. 먼저 1차 콜라겐 항체가 균일하게 도포된 96-웰 마이크로 플레이트에 채취한 세포배양액을 넣고 37 ℃에서 3시간 동안 항원-항체 반응이 일어나도록 하였다. 웰 내의 세포배양액을 제거하고 인산완충식염수로 4회 세척하였다. 각 웰에 발색유발물질을 넣고 상온에서 15분 간 반응시킨 후, 1 N 황산용액을 첨가하여 반응을 중지시켰다. 분광광도계로 파장 450 nm에서의 흡광도를 측정하였다. 표준용액을 이용하여 표준곡선을 작성하고, 상기의 방법으로 얻은 흡광도를 표준곡선에 대입하여 각 시험 화합물이 첨가된 세포배양액의 프로콜라겐 생성량을 산출하였다. Human normal fibroblasts were inoculated into 24-well microplates using Dulbo nasal eagle eggs supplemented with 10% fetal calf serum (5 × 10 4 cells / well), at 37 ° C., 5% CO 2 . Incubated for 24 hours. Serum was replaced with doubling nose fertilized eagle medium and incubated for 24 hours, and then each test compound was treated with an appropriate amount. After 48 hours of incubation, the cell culture solution was collected, and then the amount of procollagen was measured using a collagen measurement kit of Takara Corporation (Takara Shuzo Co., Ltd., Japan). First, the cell culture fluids were collected into 96-well microplates uniformly coated with primary collagen antibodies, and antigen-antibody reactions occurred at 37 ° C. for 3 hours. Cell cultures in the wells were removed and washed four times with phosphate buffered saline. In each well, a color-causing substance was added and reacted at room temperature for 15 minutes, and then 1 N sulfuric acid solution was added to stop the reaction. Absorbance at wavelength 450 nm was measured with a spectrophotometer. A standard curve was prepared using the standard solution, and the absorbance obtained by the above method was substituted into the standard curve to calculate the amount of procollagen production of the cell culture solution to which each test compound was added.
그 결과를 하기 표 2 및 도 2에 나타내었다.The results are shown in Table 2 and FIG. 2.
| 성분명Ingredient Name | 농도density | 프로콜라겐 생성량(% control)Procollagen production (% control) |
| RGDRGD | 1.0 ug/mL1.0 ug / mL | 99 %99% |
| 4.0 ug/mL4.0 ug / mL | 101 %101% | |
| Acetyl-RGDAcetyl-RGD | 1.0 ug/mL1.0 ug / mL | 102 %102% |
|
4.0 ug/mL4.0 ug / |
100 %100% | |
| Lauroyl-RGDLauroyl-rgd | 1.0 ug/mL1.0 ug / mL | 98 %98% |
|
4.0 ug/mL4.0 ug / |
105 %105% | |
| Myristoyl-RGDMyristoyl-rgd | 1.0 ug/mL1.0 ug / mL | 97 %97% |
| 4.0 ug/mL4.0 ug / mL | 112 %112% | |
| Palmitoyl-RGDPalmitoyl-rgd |
1.0 ug/mL1.0 ug / |
115 %115% |
| 4.0 ug/mL4.0 ug / mL | 122 %122% | |
| Stearoyl-RGDStearoyl-rgd | 1.0 ug/mL1.0 ug / mL | 117 %117% |
| 4.0 ug/mL4.0 ug / mL | 세포독성 있음Cytotoxic |
상기 표 2에서 보는 바와 같이, RGD나 아세틸-RGD보다 지방산 결합된 RGD의 프로콜라겐 합성 효능이 우수함을 확인하였으며, 특히 팔미토일-RGD의 효능이 동일한 농도에서 가장 우수함을 확인하였다.As shown in Table 2, it was confirmed that the procollagen synthesis efficacy of fatty acid-bound RGD than RGD or acetyl-RGD, in particular, the efficacy of palmitoyl-RGD was the best at the same concentration.
상기 실험예 1 내지 2에서 확인한 바와 같이, 유효한 효능을 내기 위한 농도에서 세포 독성이 없으며 콜라겐 합성 효능이 높은 물질은 지방산 결합 RGD 중 팔미토일-RGD임을 확인하였으므로, 이하 실험예에서는 팔미토일-RGD에 대하여 실험을 진행하였다.As confirmed in Experimental Examples 1 and 2, it was confirmed that the substance having no cytotoxicity and high collagen synthesis efficiency was palmitoyl-RGD among fatty acid-bound RGDs in the concentrations for effective efficacy. The experiment was conducted.
실험예 3: 경피흡수(피부 세포내 침투) 실험Experimental Example 3: Percutaneous Absorption (Intradermal Penetration) Experiment
상기 화학식 II로 표시되는 물질, 팔미토일-RGD를 사용하여 경피흡수 실험을 진행하였으며, 대조군으로서는 지방산이 결합되지 않은 RGD를 사용하여 비교 실험하였다. (참고문헌: LehmanPA, Slattery JT, Franz TJ. Percutaneous absorption of retinoids : Influence of vehicle, light exposure, and dose, J. Invest Dermatol., 91; 56-61, 1988).The transdermal absorption experiment was conducted using the substance represented by Formula II, palmitoyl-RGD, and as a control, a comparison experiment was performed using RGD to which no fatty acid was bound. (RehmanPA, Slattery JT, Franz TJ.Percutaneous absorption of retinoids: Influence of vehicle, light exposure, and dose, J. Invest Dermatol., 91; 56-61, 1988).
구체적으로는, 프란츠 디퓨전 셀(Franz diffusion cell)에서 투과도를 측정하였다. 대조군으로 지방산이 결합되지 않은 RGD를 사용하였다. 피부 투과력을 확인하기 위하여 -20 ℃에 보관된 인체 피부(Human Skin)를 사용하였다. 시험 전에 구조적인 피부의 무결성을 확인하기 위하여 피부(두께 700 μm, -20 ℃ 보관)에 대한 피부 손상이 없음을 경피수분손실량 (Transepidermal Water Loss, TEWL)을 통하여 확인하였다.Specifically, the transmittance was measured in a Franz diffusion cell. As a control, RGD without fatty acid was used. Human skin stored at −20 ° C. was used to confirm skin permeability. Transepidermal Water Loss (TEWL) confirmed that there was no skin damage to the skin (700 μm in thickness, -20 ° C. storage) to confirm the integrity of the structural skin before the test.
10 %의 RGD와 팔미토일-RGD를 디프로필렌 글라이콜(dipropylene Glycol) / 옥틸도데칸올(octyldodecanol) (1:1) 용매에 용해하여 1mL 용액을 시료 적용 챔버에 로딩하여 인체 피부에 첩포하였으며, 24시간이 경과한 뒤 샘플 포트를 통하여 하층 용액을 500 uL 취하여 HPLC로 정량 분석하였다. 피부 투과 실험은 각 샘플 당 5회 실험을 실시하였다. 10% of RGD and palmitoyl-RGD were dissolved in dipropylene glycol / octyldodecanol (1: 1) solvent and 1mL solution was loaded into the sample application chamber and applied to human skin. After 24 hours, 500 uL of the lower layer solution was taken through the sample port and quantitatively analyzed by HPLC. Skin permeation experiments were carried out five times for each sample.
그 결과를 하기 표 3 및 도 3에 나타내었다.The results are shown in Table 3 and FIG. 3.
| 샘플명Sample name | 24시간 후 피부 투과율 (%) Skin penetration rate (%) after 24 hours |
| RGDRGD | 0.1310.131 |
| Acetyl-RGDAcetyl-RGD | 0.1250.125 |
| Lauroyl-RGDLauroyl-rgd | 0.5240.524 |
| Myristoyl-RGDMyristoyl-rgd | 1.0261.026 |
| Palmitoyl-RGDPalmitoyl-rgd | 2.4622.462 |
| Stearoyl-RGDStearoyl-rgd | 2.7252.725 |
상기 표 3에서 보는 바와 같이, RGD의 경우 피부 첩포 후 24시간이 지나도 검출량이 미미하였다. 이는 상기 RGD가 염기성 아미노산인 R(아르기닌)과 산성 아미노산인 D(아스파르트산)을 포함하는 친수성 성질을 가지고 있고, 이로 인해 소수성인 지질이 다량 포함되어 있는 피부 각질층을 투과하지 못하여 피부 세포에 직접 전달되지 못하는 문제가 있다. 또한, 스테아로일-RGD는 피부 투과율이 뛰어나지만, 표 1에서와 같이 유효한 효능이 나타나는 함량에서 세포독성이 있어 화장료로 적합하지 않다. 그러나, 팔미토일-RGD의 경우에는 소수성 지방산인 팔미토일과 RGD가 접합되어 있어서, 피부 투과도가 15배 이상 개선되었으며, 프로콜라겐 합성 효능 또한 개선되었다. As shown in Table 3, in the case of RGD, even after 24 hours after the skin patch, the detection amount was insignificant. It has a hydrophilic property that the RGD includes a basic amino acid R (arginine) and an acidic amino acid D (aspartic acid), and thus does not penetrate the stratum corneum, which contains a large amount of hydrophobic lipids, and thus is directly delivered to the skin cells. There is no problem. In addition, stearoyl-RGD has excellent skin permeability, but is not suitable as a cosmetic because it is cytotoxic at a content that shows effective efficacy as shown in Table 1. However, in the case of palmitoyl-RGD, the hydrophobic fatty acid palmitoyl and RGD were conjugated, and skin permeability was improved by 15 times and procollagen synthesis efficiency was also improved.
실험예 4: 피부 안전성 시험Experimental Example 4: Skin Safety Test
팔미토일-RGD에 대한 피부 안정성을 측정하기 위하여, 팔미토일-RGD 원료를 농도 별로 프로판디올(propanediol)에 용해시켜 피부 자극을 확인하였다.In order to measure skin stability against palmitoyl-RGD, palmitoyl-RGD raw material was dissolved in propanediol for each concentration to identify skin irritation.
구체적으로, 20~50세인 성인 30명을 대상으로 등 부위에 5×20 cm 크기로 팔미토일-RGD 프로판디올 용액의 일정량(20 uL)을 24시간 동안 첩포한 후, 제거하고, 1시간, 24시간 경과 후 육안으로 피부 상태 변화를 판독하였으며, 그 결과를 표 4에 나타내었다. 이때, 피부상태의 변화 정도는 하기의 피부상태 평가 기준에 따라 분류하여 평가하였다. Specifically, for 30 adults aged 20 to 50 years of age 5 × 20 cm in the back portion of the palmitoyl-RGD propanediol solution in a fixed amount (20 uL) of the patch for 24 hours, and then removed, 1 hour, 24 The skin condition change was read visually after the passage of time, and the results are shown in Table 4. At this time, the degree of change of the skin condition was evaluated by classifying according to the following skin condition evaluation criteria.
<피부상태의 평가 기준>Evaluation criteria of skin condition
연구자 육안평가는 다음의 기준에 따라 평가하였다.The researcher's visual evaluation was evaluated according to the following criteria.
0: 변화 없음 1: 극히 조금 변함 2: 조금 변함 0: no change 1: very little change 2: little change
3: 조금 심하게 변함 4: 심하게 변함 5: 극히 심하게 변함 3: slightly worse 4: very badly 5: very severely
| 시험대상자 번호 Subject Number | 팔미토일-RGD0.01 중량%Palmitoyl-RGD0.01 wt% | 팔미토일-RGD0.02 중량%Palmitoyl-RGD0.02 wt% | 팔미토일-RGD0.05 중량%Palmitoyl-RGD0.05 wt% | |||
| 1hr1hr | 24hr24hr | 1hr1hr | 24hr24hr | 1hr1hr | 24hr24hr | |
| 1One | 00 | 00 | 00 | 00 | 00 | 00 |
| 22 | 00 | 00 | 00 | 00 | 00 | 00 |
| 33 | 00 | 00 | 00 | 00 | 00 | 00 |
| 44 | 00 | 00 | 00 | 00 | 00 | 00 |
| 55 | 00 | 00 | 00 | 00 | 00 | 00 |
| 66 | 00 | 00 | 00 | 00 | 00 | 00 |
| 77 | 00 | 00 | 00 | 00 | 00 | 00 |
| 88 | 00 | 00 | 00 | 00 | 00 | 00 |
| 99 | 00 | 00 | 00 | 00 | 00 | 00 |
| 1010 | 00 | 00 | 00 | 00 | 00 | 00 |
| 1111 | 00 | 00 | 00 | 00 | 00 | 00 |
| 1212 | 00 | 00 | 00 | 00 | 00 | 00 |
| 1313 | 00 | 00 | 00 | 00 | 00 | 00 |
| 1414 | 00 | 00 | 00 | 00 | 00 | 00 |
| 1515 | 00 | 00 | 00 | 00 | 00 | 00 |
| 1616 | 00 | 00 | 00 | 00 | 00 | 00 |
| 1717 | 00 | 00 | 00 | 00 | 00 | 00 |
| 1818 | 00 | 00 | 00 | 00 | 00 | 00 |
| 1919 | 00 | 00 | 00 | 00 | 00 | 00 |
| 2020 | 00 | 00 | 00 | 00 | 00 | 00 |
| 2121 | 00 | 00 | 00 | 00 | 00 | 00 |
| 2222 | 00 | 00 | 00 | 00 | 00 | 00 |
| 2323 | 00 | 00 | 00 | 00 | 00 | 00 |
| 2424 | 00 | 00 | 00 | 00 | 00 | 00 |
| 2525 | 00 | 00 | 00 | 00 | 00 | 00 |
| 2626 | 00 | 00 | 00 | 00 | 00 | 00 |
| 2727 | 00 | 00 | 00 | 00 | 00 | 00 |
| 2828 | 00 | 00 | 00 | 00 | 00 | 00 |
| 2929 | 00 | 00 | 00 | 00 | 00 | 00 |
| 3030 | 00 | 00 | 00 | 00 | 00 | 00 |
상기 표 4에서 보는 바와 같이, 팔미토일-RGD는 0.05 중량%의 농도까지 피부자극이 없음을 확인하였다.As shown in Table 4, palmitoyl-RGD was confirmed that there is no skin irritation up to a concentration of 0.05% by weight.
실험예 5: 수분 및 열에 대한 안정성 시험 Experimental Example 5: Stability test against moisture and heat
화장품 조성물에 사용되는 유효성분의 수분 및 열에 대한 안정성은 조성물 보관 기간에 중요한 요소이므로, 팔미토일-RGD의 화장품 조성물에 사용되는 유효성분으로서의 적합성을 확인하기 위하여, 수분 및 열에 대한 안정성을 측정하였다. Since the stability against moisture and heat of the active ingredient used in the cosmetic composition is an important factor in the storage period of the composition, the stability against moisture and heat was measured to confirm the suitability of palmitoyl-RGD as an active ingredient used in the cosmetic composition.
구체적으로 4 ℃, 30 ℃, 45 ℃의 온도 및 75 %의 상대습도인 가혹조건에서 밀봉상태로 보관하면서 각각 12주 동안 시료에 대해서 초기 활성성분 값에 대한 잔사율(%)을 고성능액체크로마토그래피(HPLC)로 분석하였다. 그 결과를 하기 표 5에 나타내었다.Specifically, the residue ratio (%) of the initial active ingredient value for the samples for 12 weeks while stored in a sealed condition at 4 ℃, 30 ℃, 45 ℃ harsh conditions of 75% relative humidity, respectively, high performance liquid chromatography Analysis by (HPLC). The results are shown in Table 5 below.
| 온도Temperature | 초기Early |
1주1 |
2주2 weeks | 4주4 Weeks | 8주8 Weeks | 12주12 Weeks |
| 4 ℃4 ℃ | 100.0 %100.0% | 100.1 %100.1% | 99.7 %99.7% | 98.4 %98.4% | 99.2 %99.2% | 99.0 %99.0% |
| 30 ℃30 ℃ | 100.0 %100.0% | 99.9 %99.9% | 99.5 %99.5% | 98.2 %98.2% | 98.1 %98.1% | 97.8 %97.8% |
| 45 ℃45 ℃ | 100.0 %100.0% | 99.9 %99.9% | 98.8 %98.8% | 98.5 %98.5% | 97.6 %97.6% | 96.3 %96.3% |
상기 표 5에서 보는 바와 같이, 팔미토일-RGD는 수분과 열에 우수한 안정성을 보여주고 있음을 확인할 수 있었다.As shown in Table 5, it was confirmed that palmitoyl-RGD shows excellent stability to moisture and heat.
제형예 1. 팔미토일-RGD를 포함하는 가용화 제형의 제조Formulation Example 1 Preparation of a Solubilized Formulation Containing Palmitoyl-RGD
팔미토일-RGD를 유효성분으로 포함하는 화장료 조성물의 안정성을 확인하기 위하여 하기 표 6과 같이 가용화 제형을 제조하였다.To confirm the stability of the cosmetic composition containing palmitoyl-RGD as an active ingredient, solubilized formulations were prepared as shown in Table 6 below.
| 상Prize | 성분ingredient | 함량 (중량%)Content (% by weight) |
| 수상Awards | 정제수Purified water | To 100To 100 |
|
보습성분 |
10 ~ 2510 to 25 | |
| 점증제Thickener | 적량Quantity | |
| 금속이온 봉쇄제Metal Ion Blocker | 적량Quantity | |
| 가용화상Available image |
피이지-60하이드로제네이티드캐스터오일Fiji-60 |
0 ~ 20 to 2 |
|
피이지-40하이드로제네이티드캐스터오일Fiji-40 |
0 ~ 20 to 2 | |
|
다가알코올 |
0 ~ 100 to 10 | |
|
향료 | 적량Quantity | |
| 에탄올ethanol | 0 ~ 200 to 20 | |
| 첨가 IAddition I | 팔미토일-RGDPalmitoyl-RGD | 0.040.04 |
| 첨가 IIAddition II | 보존제Preservative | 적량Quantity |
| 기타 첨가제Other additives | 적량Quantity |
<제조방법><Production method>
(1) 수상과 가용화상을 각각 균일하게 혼합 및 용해시켰다.(1) The aqueous phase and the solubilized image were mixed and dissolved uniformly, respectively.
(2) 수상에 가용화상을 넣고 혼합 및 가용화 시켰다.(2) The solubilized image was added to the aqueous phase and mixed and solubilized.
(3) 첨가 I의 팔미토일-RGD는 가용화 한 후 투입하여 혼합시킨 후 첨가II를 혼합하여 완료하였다.(3) Palmitoyl-RGD of addition I was solubilized, added and mixed, followed by mixing addition II.
제형예 2. 팔미토일-RGD를 포함하는 에센스 제형의 제조Formulation Example 2 Preparation of Essence Formulations Containing Palmitoyl-RGD
팔미토일-RGD를 유효성분으로 포함하는 화장료 조성물의 안정성을 확인하기 위하여 하기 표 7과 같이 에센스 제형을 제조하였다.In order to confirm the stability of the cosmetic composition containing palmitoyl-RGD as an active ingredient, an essence formulation was prepared as shown in Table 7 below.
| 상Prize | 성분ingredient | 함량 (중량%)Content (% by weight) |
| 수상Awards | 정제수Purified water | To 100To 100 |
|
보습성분 |
10 ~ 2510 to 25 | |
| 점증제Thickener | 적량Quantity | |
| 금속이온 봉쇄제Metal Ion Blocker | 적량Quantity | |
| 유상Paid | 피이지-100스테아레이트Fiji-100 Stearate | 0.1 ~ 10.1 to 1 |
| 글리세릴스테아레이트Glyceryl Stearate | 0.1 ~ 10.1 to 1 | |
| 폴리소르베이트 60Polysorbate 60 | 0.1 ~ 10.1 to 1 | |
| 세틸알코올Cetyl alcohol | 0.1 ~ 10.1 to 1 | |
| 베헤닐알코올Behenyl Alcohol | 0.1 ~ 10.1 to 1 | |
|
스쿠알란 |
5 ~ 205 to 20 | |
| 토코페릴아세테이트Tocopheryl Acetate | 0.1 ~ 0.50.1 to 0.5 | |
| 첨가 IAddition I | 팔미토일-RGDPalmitoyl-RGD | 0.040.04 |
| 첨가 IIAddition II | 향료Spices | 적량Quantity |
| 보존제Preservative | 적량Quantity | |
| 기타 첨가제Other additives | 적량Quantity |
<제조방법><Production method>
(1) 수상과 유상을 가온 하여 각각 균일하게 혼합 및 용해시켰다.(1) The aqueous and oil phases were warmed and mixed and dissolved, respectively.
(2) 75 ℃에서 수상에 유상을 넣고 혼합시켜 유화시켰다(2) The oil phase was added to the aqueous phase at 75 ° C., mixed and emulsified.
(3) 첨가상 I은 팔미토일-RGD로 45 ℃에서 투입하고 혼합시킨 후 첨가상 II를 혼합하였다.(3) Addition phase I was charged with palmitoyl-RGD at 45 ° C. and mixed before addition phase II was mixed.
제형예 3. 팔미토일-RGD를 포함하는 크림 제형의 제조Formulation Example 3 Preparation of a Cream Formulation Containing Palmitoyl-RGD
신규 팔미토일-RGD를 유효성분으로 포함하는 화장료 조성물의 안정성을 확인하기 위하여 하기 표 8과 같이 크림 제형을 제조하였다.To confirm the stability of the cosmetic composition comprising a novel palmitoyl-RGD as an active ingredient, a cream formulation was prepared as shown in Table 8 below.
| 상Prize | 성분ingredient | 함량 (중량%)Content (% by weight) |
| 수상Awards | 정제수Purified water | To 100To 100 |
|
보습성분 |
10 ~ 2510 to 25 | |
| 점증제Thickener | 적량Quantity | |
| 금속이온 봉쇄제Metal Ion Blocker | 적량Quantity | |
| 유상Paid | 피이지-100스테아레이트Fiji-100 Stearate | 0.1 ~ 20.1 to 2 |
| 글리세릴스테아레이트Glyceryl Stearate | 0.1 ~ 20.1 to 2 | |
| 폴리소르베이트 60Polysorbate 60 | 0.1 ~ 20.1 to 2 | |
| 스테아릭애씨드Stearic acid | 0.1 ~ 20.1 to 2 | |
| 세테아릴알코올Cetearyl Alcohol | 0.1 ~ 20.1 to 2 | |
|
카프릭카프릴릭트리글리세라이드 |
10 ~ 3010 to 30 | |
| 토코페릴아세테이트Tocopheryl Acetate | 0.1 ~ 0.50.1 to 0.5 | |
| 첨가 IAddition I | 팔미토일-RGDPalmitoyl-RGD | 0.040.04 |
| 첨가 IIAddition II | 향료Spices | 적량Quantity |
| 보존제Preservative | 적량Quantity | |
| 기타 첨가제Other additives | 적량Quantity |
<제조방법><Production method>
(1) 수상과 유상을 가온 하여 각각 균일하게 혼합 및 용해시켰다.(1) The aqueous and oil phases were warmed and mixed and dissolved, respectively.
(2) 75 ℃에서 수상에 유상을 넣고 혼합시켜 유화시켰다(2) The oil phase was added to the aqueous phase at 75 ° C., mixed and emulsified.
(3) 첨가상 I은 팔미토일-RGD로 45 ℃에서 투입하고 혼합시킨 후 첨가상 II를 혼합하였다.(3) Addition phase I was charged with palmitoyl-RGD at 45 ° C. and mixed before addition phase II was mixed.
실험예 6: 팔미토일-RGD를 포함하는 화장료 조성물의 원료 안정성 확인Experimental Example 6: Confirming the raw material stability of the cosmetic composition containing palmitoyl-RGD
제형 내에서의 팔미토일-RGD의 안정성을 확인하기 위하여, 상기 제형예 1 내지 제형예 3의 가용화 제형, 에센스 제형, 크림 제형을 4 ℃, 30 ℃, 45 ℃, 일광의 조건에 12주 동안 보관한 후, 상기 실험예 5와 동일한 고성능액체크로마토그래피 분석을 통해 팔미토일-RGD를 함유한 화장료 내에서의 함량의 경시 변화를 관찰하였고, 그 결과를 하기 표 9에 나타내었다.In order to confirm the stability of palmitoyl-RGD in the formulation, the solubilized formulation, the essence formulation, and the cream formulation of Formulation Examples 1 to 3 were stored at 4 ° C., 30 ° C., 45 ° C., and sunlight for 12 weeks. Then, the change in the aging content in the cosmetic containing palmitoyl-RGD was observed through the same high performance liquid chromatography analysis as in Experimental Example 5, and the results are shown in Table 9 below.
| 경과시간Elapsed time | 신규 팔미토일트리펩타이드(초기 함량 대비 %)New Palmitoyl Tripeptide (% of Initial Content) | |||||||||||
| 가용화 제형Solubilized Formulations | 에센스 제형Essence Formulation | 크림 제형Cream formulation | ||||||||||
|
4 ℃4 |
30℃30 ℃ | 45℃45 ℃ | 일광daylight |
4 ℃4 |
30 ℃30 ℃ | 45 ℃45 ℃ | 일광daylight |
4 ℃4 |
30 ℃30 ℃ | 45 ℃45 ℃ | 일광daylight | |
| 초기Early | 100100 | 100100 | 100100 | 100100 | 100100 | 100100 | 100100 | 100100 | 100100 | 100100 | 100100 | 100100 |
| 1주1 week | 99.999.9 | 99.399.3 | 98.298.2 | 99.199.1 | 9999 | 99.599.5 | 99.399.3 | 97.497.4 | 97.397.3 | 99.899.8 | 96.096.0 | 97.797.7 |
| 3주3 weeks | 98.698.6 | 96.296.2 | 97.297.2 | 99.299.2 | 97.297.2 | 96.296.2 | 96.296.2 | 97.497.4 | 97.097.0 | 98.198.1 | 98.298.2 | 97.197.1 |
| 5주5 Weeks | 99.199.1 | 97.297.2 | 95.595.5 | 98.198.1 | 97.597.5 | 97.797.7 | 97.797.7 | 96.396.3 | 96.196.1 | 96.296.2 | 98.898.8 | 96.396.3 |
| 8주8 Weeks | 97.697.6 | 96.196.1 | 94.894.8 | 97.597.5 | 96.796.7 | 96.896.8 | 96.896.8 | 96.996.9 | 94.394.3 | 95.595.5 | 96.296.2 | 96.596.5 |
| 12주12 Weeks | 95.995.9 | 9494 | 95.295.2 | 96.296.2 | 95.895.8 | 95.295.2 | 95.295.2 | 94.294.2 | 95.795.7 | 95.795.7 | 98.198.1 | 94.194.1 |
| 결과result | 안정stability | 안정stability | 안정stability | 안정stability | 안정stability | 안정stability | 안정stability | 안정stability | 안정stability | 안정stability | 안정stability | 안정stability |
상기 표 9에서 보는 바와 같이, 가용화, 에센스 및 크림 제형 모두 고온 및 일광 조건에서 제형 내에서의 원료 안정성이 우수함을 확인하였다.As shown in Table 9, it was confirmed that all of the solubilization, essence and cream formulations are excellent raw material stability in the formulation under high temperature and sunlight conditions.
실험예 7: 팔미토일-RGD를 포함하는 화장료 조성물의 제형 안정성 확인Experimental Example 7: Confirming the formulation stability of the cosmetic composition containing palmitoyl-RGD
팔미토일-RGD를 포함하는 화장료 조성물 제형 자체의 안정성을 확인하기 위하여, 상기 제형예 1 내지 제형예 3의 가용화 제형, 에센스, 제형, 크림 제형을 4 ℃, 30 ℃(Room Temperature, RT), 45 ℃ 및 일광의 조건에 12주 동안 보관한 후, 육안평가와 관능평가를 통해 색, 취, 제형 변화를 관찰하였으며, 그 결과를 하기 표 10 내지 표 12에 나타내었다. 이 때, 색, 취, 제형 변화 정도는 하기의 평가 기준에 따라 분류하여 평가하였다.In order to confirm the stability of the cosmetic composition formulation itself including palmitoyl-RGD, solubilization formulation, essence, formulation, cream formulation of Formulation Examples 1 to 3, 4 ℃, 30 ℃ (Room Temperature, RT), 45 After storage for 12 weeks in the conditions of ℃ and daylight, color, odor, and the change in the formulation was observed through visual evaluation and sensory evaluation, the results are shown in Table 10 to Table 12. At this time, the degree of change in color, odor, and dosage form was evaluated according to the following evaluation criteria.
<평가 기준><Evaluation Criteria>
변화 무: - 아주 미세한 변화: + 약간의 변화: ++No change:-Very slight change: + Slight change: ++
변화가 관찰됨: +3 유의적 변화: +4 확연한 변화: +5Change observed: +3 Significant change: +4 Significant change: +5
| 경과시간Elapsed time | 가용화 제형Solubilized Formulations | |||||||||||
| 취 변화Odor change | 색 변화Color change | 제형 변화Formulation change | ||||||||||
|
4 ℃4 |
30 ℃30 ℃ | 45 ℃45 ℃ | 일광daylight |
4 ℃4 |
30 ℃30 ℃ | 45 ℃45 ℃ | 일광daylight |
4 ℃4 |
30 ℃30 ℃ |
45 ℃45 | 일광daylight | |
| 1주1 week | -- | -- | -- | -- | -- | -- | -- | -- | -- | -- | -- | -- |
| 2주2 weeks | -- | -- | -- | -- | -- | -- | -- | -- | -- | -- | -- | -- |
| 4주4 Weeks | -- | -- | -- | -- | -- | -- | -- | -- | -- | -- | -- | -- |
| 8주8 Weeks | -- | -- | -- | -- | -- | -- | -- | -- | -- | -- | -- | -- |
| 12주12 Weeks | -- | -- | -- | -- | -- | -- | ++ | ++ | -- | -- | ++ | -- |
| 경과시간Elapsed time | 에센스 제형Essence Formulation | |||||||||||
| 취 변화Odor change | 색 변화Color change | 제형 변화Formulation change | ||||||||||
|
4 ℃4 |
30 ℃30 ℃ | 45 ℃45 ℃ | 일광daylight |
4 ℃4 |
30 ℃30 ℃ | 45 ℃45 ℃ | 일광daylight |
4 ℃4 |
30 ℃30 ℃ |
45 ℃45 | 일광daylight | |
| 1주1 week | -- | -- | -- | -- | -- | -- | -- | -- | -- | -- | -- | -- |
| 2주2 weeks | -- | -- | -- | -- | -- | -- | -- | -- | -- | -- | -- | -- |
| 4주4 Weeks | -- | -- | -- | -- | -- | -- | -- | -- | -- | -- | -- | -- |
| 8주8 Weeks | -- | -- | -- | -- | -- | -- | -- | -- | -- | -- | -- | -- |
| 12주12 Weeks | -- | -- | -- | -- | -- | -- | ++ | ++ | -- | -- | ++ | -- |
| 경과시간Elapsed time | 에센스 제형Essence Formulation | |||||||||||
| 취 변화Odor change | 색 변화Color change | 제형 변화Formulation change | ||||||||||
|
4 ℃4 |
30 ℃30 ℃ | 45 ℃45 ℃ | 일광daylight |
4 ℃4 |
30 ℃30 ℃ | 45 ℃45 ℃ | 일광daylight |
4 ℃4 |
30 ℃30 ℃ |
45 ℃45 | 일광daylight | |
| 1주1 week | -- | -- | -- | -- | -- | -- | -- | -- | -- | -- | -- | -- |
| 2주2 weeks | -- | -- | -- | -- | -- | -- | -- | -- | -- | -- | -- | -- |
| 4주4 Weeks | -- | -- | -- | -- | -- | -- | -- | -- | -- | -- | -- | -- |
| 8주8 Weeks | -- | -- | -- | -- | -- | -- | -- | -- | -- | -- | -- | -- |
| 12주12 Weeks | -- | -- | -- | -- | -- | -- | ++ | ++ | -- | -- | ++ | -- |
상기 표 10 내지 표 12에서 보는 바와 같이, 가용화, 에센스 및 크림 제형 모두 고온 조건 및 일광 조건에서 안정성이 우수함을 확인하였다. As shown in Table 10 to Table 12, it was confirmed that all of the solubilization, essence and cream formulation is excellent in high temperature and daylight conditions.
실험예 8: 팔미토일-RGD를 포함하는 화장료 조성물의 피부 자극 확인Experimental Example 8: Confirming skin irritation of the cosmetic composition containing palmitoyl-RGD
팔미토일-RGD를 포함하는 화장료 조성물의 피부 자극을 확인하기 위하여, 상기 제형예 3의 크림 제형의 피부 자극에 대한 평가를 실시하였다.In order to confirm the skin irritation of the cosmetic composition containing palmitoyl-RGD, the skin irritation of the cream formulation of Formulation Example 3 was evaluated.
구체적으로, 성인 50명을 대상으로 등 부위에 5×20 cm 크기로 상기 제형예 3에서 제조한 팔미토일-RGD를 농도의존적으로 포함하는 크림 제형의 일정량(60 uL)을 24시간마다 총 9회 첩포하였다. 9회 반복 첩포, 첩포 제거 30분 경과 후 마다 반응을 관찰하고 재첩포하여 감작유도를 확인하였으며, 2주 간의 휴지기를 가진 후에 1회 첩포, 첩포 제거 30분, 24시간 및 48시간 경과 후 감작유발 정도를 확인하였다. 그 감작유도 결과를 표 13에 나타내었으며, 감작유발 시험 결과를 표 14에 나타내었다. 이때, 피부 반응 판정은 국제 접촉피부염연구회(ICDRG)의 기준에 준하여 평가하였다. Specifically, a total of 9 times a predetermined amount (60 uL) of a cream formulation containing palmitoyl-RGD prepared in Formulation Example 3 concentration-dependently 5 x 20 cm in the back region for 50 adults every 24 hours Patched. Nine repeated patches and patches were observed every 30 minutes after the removal of the patch, and the sensation induction was confirmed by re-patching. After having a two-week rest period, one-time patch and patch removal were performed after 30 minutes, 24 hours and 48 hours. The degree was confirmed. The sensitization induction results are shown in Table 13, and the sensitization induction test results are shown in Table 14. At this time, the skin reaction was evaluated according to the criteria of the International Contact Dermatitis Research Association (ICDRG).
<피부 상태 평가 기준><Skin Condition Evaluation Criteria>
0: 변화 없음 (무자극) 0: no change (no irritation)
1: 미자극 - 희미한 또는 가까스로 감지할 수 있는 가벼운 홍반1: non-irritating-faint or barely detectable erythema
2: 경자극 - 경계가 뚜렷하나 약한 홍반, 부종 및 구진2: hard stimulation-erythema, edema and papules with clear but weak boundaries
3: 중자극 - 뚜렷한 홍반, 구진 및 소수포3: medium irritation-pronounced erythema, papules and vesicles
4: 강자극 - 심한 홍반 및 대수포, 가피형성4: strong irritation-severe erythema and alveolar, scalp formation
| 조성물 내 팔미토일-RGD 농도Palmitoyl-RGD Concentration in Compositions | 반응자 수Number of respondents | 반응도Responsiveness |
|
0.01 중량% 0.01 |
00 | 0.00.0 |
|
0.02 중량% 0.02 |
00 | 0.00.0 |
|
0.05 중량% 0.05 |
00 | 0.00.0 |
| 조성물 내 팔미토일-RGD 농도Palmitoyl-RGD Concentration in Compositions | 반응자 수Number of respondents | 반응도Responsiveness |
|
0.01 중량% 0.01 |
00 | 0.00.0 |
|
0.02 중량% 0.02 |
00 | 0.00.0 |
|
0.05 중량% 0.05 |
00 | 0.00.0 |
상기 표 13 내지 표 14에서 보는 바와 같이, 팔미토일-RGD를 포함한 크림 제형의 피부 자극은 적은 것을 확인하였다.As shown in Table 13 to Table 14, it was confirmed that the skin irritation of the cream formulation including palmitoyl-RGD.
실험예 9: 팔미토일-RGD를 포함하는 화장료 조성물의 피부주름개선의 임상 효과Experimental Example 9: Clinical Effect of Skin Wrinkle Improvement of Cosmetic Composition Containing Palmitoyl-RGD
상기 팔미토일-RGD를 포함한 화장료 조성물에 대한 피부주름개선 효과를 측정하였다.The skin wrinkle improvement effect on the cosmetic composition including the palmitoyl-RGD was measured.
구체적으로, 팔미토일-RGD를 함유하는 화장료 조성물의 유효성 평가를 위하여, 만 30~60세의 여성 23명을 대상으로 임상을 수행하였다. 시험제품으로서 팔미토일-RGD 0.04 중량%가 함유된 화장료 조성물을 사용하였으며, 대조제품으로서는 시험제품에서 팔미토일-RGD 대신 정제수를 포함한 플라시보 크림을 사용하였다. 상기 시험제품과 대조제품을 피험자의 얼굴 왼쪽과 오른쪽에 무작위로 각각 도포하였으며, 1일 2회 주기로 재도포하면서 12주 동안 변화를 관찰하였다.Specifically, to evaluate the effectiveness of the cosmetic composition containing palmitoyl-RGD, clinical trials were performed on 23 women aged 30 to 60 years. A cosmetic composition containing 0.04% by weight of palmitoyl-RGD was used as a test product, and a placebo cream containing purified water was used as a control product instead of palmitoyl-RGD. The test and control products were randomly applied to the left and right sides of the subject's face, respectively, and observed for 12 weeks while reapplying twice a day.
실험예 9-1: 눈가 주름개선 확인 시험Experimental Example 9-1: Eye wrinkle improvement test
시험제품 및 대조제품 사용에 의한 눈가 주름개선을 확인하기 위하여 사용 전, 사용 4주 후, 사용 8주 후, 사용 12주 후에 제품을 적용한 부위에 대하여 제작한 반투명의 피부 주름 모사판에 빛을 투과시켜 Visiometer SV600(Courage-Khazaka electronic GmbH, Germany)으로 분석한 값들 중 R2, R3값을 눈가 주름의 주요한 파라미터로서 평가하였다. R2는 주름 프로필을 5개로 균등하게 분할한 구역에 대하여 구한 5개의 가장 높은 꼭대기 값과 가장 낮은 계곡 값의 차이 중 가장 큰 값에 해당되며, R3는 주름 프로필을 X축을 따라 5개의 균등한 분절을 나눈 다음 각 분절 내에서 최대 값과 최소 값의 차이를 측정하여 산술 평균한 값이다.In order to confirm the improvement of the wrinkles around the eyes by using the test product and the control product, light is transmitted to the translucent skin wrinkle miter plate manufactured on the areas where the product is applied before, after 4 weeks, after 8 weeks, and after 12 weeks of use. The R2 and R3 values were evaluated as the main parameters of the wrinkles around the eyes, as measured by Visiometer SV600 (Courage-Khazaka electronic GmbH, Germany). R2 corresponds to the largest of the difference between the five highest peaks and the lowest valleys for the five equally divided sections of the wrinkle profile, and R3 defines the five equal segments along the X axis. It is the arithmetic average of the differences between the maximum and minimum values within each segment after dividing.
상기 R2 값과 R3 값의 결과를 도 4와 도 5에 그래프로 나타내었다.The results of the R2 and R3 values are shown graphically in FIGS. 4 and 5.
그 결과 R2값이 사용전과 비교하여 사용 4주 후, 8주 후, 12주 후 모두 유의하게 감소(p < 0.05)하였으며, 대조제품과 비교하였을 때에도 사용 8주 후, 12주 후에서 유의한 차이(p < 0.05)를 나타내었다. 또한, 시험제품군의 R3 값은 사용 전에 비해 사용 4주 후, 8주 후, 12주 후 모두 유의하게 감소(p < 0.05)하였으며, 대조제품과 비교하였을 때에도 사용 8주 후, 12주 후 유의한 차이(p < 0.05)를 나타내었다. As a result, the R2 value decreased significantly after 4 weeks, after 8 weeks, and after 12 weeks of use (p <0.05), and also after 8 weeks and after 12 weeks of use compared to the control. (p <0.05). In addition, the R3 values of the test product group were significantly decreased (p <0.05) after 4 weeks, 8 weeks, and 12 weeks after use, and also significant after 8 weeks and 12 weeks after use. Difference (p <0.05).
실험예 9-2: Primos를 이용한 눈가 주름 측정 시험Experimental Example 9-2: Eye wrinkle measurement test using Primos
제품 사용에 의한 눈가 주름 측정값의 변화를 확인하기 위하여 3D 피부 촬영 장치 Primos premium (GFMesstechnik GmbH, Germany)을 이용하여 사용 전, 사용 4주 후, 8주 후, 12주 후의 눈가 주름의 Ra값을 분석하였다. Ra는 주름 프로필의 평균 거칠기(average roughness)를 의미하며, 값이 작아질수록 눈가 주름이 개선된다고 볼 수 있으며, 측정한 Ra 값의 결과를 도 6에 그래프로 나타내었다.In order to confirm the change in the wrinkles of the eyes caused by the use of the product, the Ra value of the eye wrinkles before and after 4 weeks, 8 weeks, and 12 weeks after using the 3D skin imaging device Primos premium (GFMesstechnik GmbH, Germany) was measured. Analyzed. Ra means the average roughness (average roughness) of the wrinkle profile, the smaller the value can be seen that the wrinkles around the eyes is improved, the results of the measured Ra value is shown in the graph in FIG.
그 결과 시험제품군의 Ra값이 사용 전에 비해 사용 12주 후 유의하게 감소(p < 0.05)하였으며, 대조제품과 비교하였을 때에도 사용 4주 후, 12주 후 유의한 차이(p < 0.05)를 나타냄을 확인하였다.As a result, the Ra value of the test product group was significantly decreased after 12 weeks of use (p <0.05) compared to before use, and also showed a significant difference (p <0.05) after 4 weeks of use and 12 weeks after comparison with the control product. Confirmed.
따라서, 상기 실험예 9-1 및 9-2의 결과를 통하여 팔미토일-RGD를 포함한 화장료 조성물이 눈가 주름개선에 효과가 있음을 확인하였다.Therefore, the results of Experimental Examples 9-1 and 9-2 confirmed that the cosmetic composition including palmitoyl-RGD is effective in improving wrinkles around the eyes.
실험예 9-3: 피부 탄력 측정 시험Experimental Example 9-3: Skin elasticity test
팔미토일-RGD 포함 제품 사용에 의한 피부 탄력 측정값의 변화를 확인하기 위하여 사용 전, 사용 4주 후, 8주 후, 12주 후의 피부 탄력을 측정하였다. 피부 탄력 측정은 Cutometer CM 580(CK Electronics, Koln, Germany)을 사용하여 1회씩 양쪽 눈가의 동일한 부위에 실시하였으며, 결과값 중 R2(피부의 재변형력)값을 피부탄력도 평가자료로 사용하였다. R2값은 총체적인 탄성으로서 1에 가까운 값일수록 피부가 더욱 탄력적임을 의미하며, 측정한 R2 값의 결과를 도 7에 그래프로 나타내었다.Skin elasticity was measured before use, 4 weeks after use, 8 weeks after, and 12 weeks after the use of palmitoyl-RGD. Skin elasticity was measured once using the Cutometer CM 580 (CK Electronics, Koln, Germany) at the same site of both eyes, and R2 (skin remodeling force) was used as an evaluation data for skin elasticity. The value of R2 is the total elasticity, and the closer to 1, the more elastic the skin is. The result of the measured R2 value is graphically shown in FIG. 7.
그 결과 시험제품군의 피부탄력 측정값은 사용 전에 비해 사용 4주 후, 8주 후, 12주 후 모두 유의하게 증가(p < 0.05)하였으며, 대조제품과 비교하였을 때에도 사용 4주 후, 8주 후, 12주 후 모두 유의한 차이(p < 0.05)를 나타내었다. 따라서, 팔미토일-RGD를 포함한 화장료 조성물이 피부 탄력증진에 효과가 있음을 확인하였다.As a result, the measured value of skin elasticity of the test product group was significantly increased after 4 weeks, 8 weeks, and 12 weeks after use (p <0.05), and even after 4 weeks and 8 weeks of use compared to the control product. After 12 weeks, there was a significant difference (p <0.05). Therefore, it was confirmed that the cosmetic composition including palmitoyl-RGD is effective in promoting skin elasticity.
실험예 9-4: 피부 진피치밀도 측정 시험Experimental Example 9-4: Skin dermal density measurement test
피부 진피치밀도 측정을 위하여 사용 전, 사용 4주 후, 8주 후, 12주 후에 Skin Scanner High Resolution Ultrasound(taberna promedicum, Germany)로 양쪽 눈가 부위를 촬영하고 측정된 화상을 이용하여 제품 사용 전후의 피부 진피치밀도를 분석하였다. 피부 진피치밀도의 측정 단위는 %로, 측정값이 높아질수록 진피치밀도가 개선되었음을 나타내며, 측정한 결과를 도 8에 그래프로 나타내었다.For skin dermal density measurement, use the Skin Scanner High Resolution Ultrasound (taberna promedicum, Germany) before and after 4 weeks, 8 weeks, and 12 weeks of use. Skin dermal density was analyzed. The unit of measurement of the skin dermal density is%, indicating that the dermal density is improved as the measured value is increased, and the measurement results are shown in the graph of FIG. 8.
그 결과 시험제품군의 진피치밀도 측정값이 사용 전에 비해 사용 8주 후, 12주 후 유의하게 증가(p < 0.05)하였으며, 대조제품과 비교하였을 때에도 사용 8주 후, 12주 후 유의한 차이(p < 0.05)를 나타내었다. 따라서, 팔미토일-RGD를 포함한 화장료 조성물이 피부 진피치밀도 개선에 효과가 있음을 확인하였다.As a result, the measured value of the dermal density of the test product group was significantly increased after 8 weeks and after 12 weeks of use (p <0.05). p <0.05). Therefore, it was confirmed that the cosmetic composition including palmitoyl-RGD is effective in improving skin dermal density.
Claims (10)
- 하기 화학식 I의 화합물 또는 그의 화장료로 허용되는 염:Salts acceptable as compounds of formula (I) or cosmetics thereof:[화학식 I][Formula I]
상기 식에서, n은 11 내지 16의 정수이다.Wherein n is an integer from 11 to 16. - 제 1 항에 있어서, The method of claim 1,상기 n은 15인 것을 특징으로 하는 화합물 또는 그의 화장료로 허용되는 염.N is 15, or a compound or a cosmetic acceptable salt thereof.
- 제 1 항에 있어서, The method of claim 1,상기 화학식 I의 화합물이 처리된 세포의 프로콜라겐 합성양이 하기 화학식 Ⅲ의 화합물을 동일 농도로 처리한 경우에 비하여 20 % 이상 많은 것을 특징으로 하는 화합물 또는 그의 화장료로 허용되는 염.A compound or a cosmetic acceptable salt thereof, characterized in that the amount of procollagen synthesis of the cells treated with the compound of the formula (I) is 20% or more compared with the case where the compound of the formula (III) is treated at the same concentration.[화학식 Ⅲ][Formula III]
- 제 1 항 내지 제 3 항 중 어느 한 항의 화합물 또는 그의 화장료로 허용되는 염을 포함하는 화장료 조성물.Cosmetic composition containing the compound of any one of Claims 1-3, or its salt accept | permitted as cosmetics.
- 제 1 항 내지 제 3 항 중 어느 한 항의 화합물 또는 그의 화장료로 허용되는 염을 포함하는 피부 주름개선용 화장료 조성물.A cosmetic composition for improving skin wrinkles, comprising a compound according to any one of claims 1 to 3 or a salt thereof as a cosmetic.
- 제 4 항에 있어서, The method of claim 4, wherein상기 화합물은 화장료 조성물 총 중량에 대하여 0.0001 내지 2 중량%로 포함되는 것을 특징으로 하는 화장료 조성물.The compound is a cosmetic composition, characterized in that it comprises 0.0001 to 2% by weight based on the total weight of the cosmetic composition.
- 제 4 항에 있어서, The method of claim 4, wherein상기 화합물은 화장료 조성물 총 중량에 대하여 0.01 내지 0.05 중량%로 포함되는 것을 특징으로 하는 화장료 조성물.The compound is a cosmetic composition, characterized in that it comprises 0.01 to 0.05% by weight relative to the total weight of the cosmetic composition.
- 제 5 항에 있어서, The method of claim 5, wherein상기 화합물은 화장료 조성물 총 중량에 대하여 0.0001 내지 2 중량%로 포함되는 것을 특징으로 하는 화장료 조성물.The compound is a cosmetic composition, characterized in that it comprises 0.0001 to 2% by weight based on the total weight of the cosmetic composition.
- 제 5 항에 있어서, The method of claim 5, wherein상기 화합물은 화장료 조성물 총 중량에 대하여 0.01 내지 0.05 중량%로 포함되는 것을 특징으로 하는 화장료 조성물.The compound is a cosmetic composition, characterized in that it comprises 0.01 to 0.05% by weight relative to the total weight of the cosmetic composition.
- 다음의 단계를 포함하는 하기 화학식 I의 화합물 제조방법:A process for preparing a compound of formula (I) comprising the following steps:[화학식 I][Formula I]
상기 식에서, n은 11 내지 16의 정수이고, Wherein n is an integer from 11 to 16,(a) N-말단, C-말단 또는 반응성 잔기가 보호된 아르기닌 유도체, 글리신 유도체 및 아스파르트산 유도체를 이용하여 액상에서 트리펩타이드(아르기닌-글리신-아스파르트산) 유도체를 합성하는 단계;(a) synthesizing tripeptides (arginine-glycine-aspartic acid) derivatives in the liquid phase using arginine derivatives, glycine derivatives and aspartic acid derivatives protected with N-terminal, C-terminal or reactive moieties;(b) 상기 트리펩타이드(아르기닌-글리신-아스파르트산) 유도체와 지방산염을 반응시켜 아르기닌의 N-말단에 지방산을 결합시키는 단계; 및(b) reacting the tripeptide (arginine-glycine-aspartic acid) derivative with a fatty acid salt to bind a fatty acid to the N-terminus of arginine; And(c) 상기 지방산이 결합된 트리펩타이드(아르기닌-글리신-아스파르트산) 유도체에서 트리펩타이드 부분의 보호기를 제거하는 단계.(c) removing the protecting group of the tripeptide moiety from the tripeptide (arginine-glycine-aspartic acid) derivative to which the fatty acid is bound.
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| KR1020160127375A KR101715599B1 (en) | 2016-10-04 | 2016-10-04 | Tripeptide combined with fatty acid and anti-wrinkle cosmetic composition comprising the same |
| KR10-2016-0127375 | 2016-10-04 |
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| Publication Number | Publication Date |
|---|---|
| WO2018066914A1 true WO2018066914A1 (en) | 2018-04-12 |
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| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| PCT/KR2017/010913 WO2018066914A1 (en) | 2016-10-04 | 2017-09-29 | Tripeptide having fatty acids bonded thereto, and anti-wrinkle cosmetic composition comprising same |
Country Status (3)
| Country | Link |
|---|---|
| KR (1) | KR101715599B1 (en) |
| TW (1) | TWI675849B (en) |
| WO (1) | WO2018066914A1 (en) |
Families Citing this family (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| KR101715599B1 (en) * | 2016-10-04 | 2017-03-13 | (주)셀트리온 | Tripeptide combined with fatty acid and anti-wrinkle cosmetic composition comprising the same |
| CN112367968B (en) * | 2018-07-06 | 2023-06-16 | 安医健有限公司 | Cosmetic composition for removing or adsorbing fine dust comprising peptide complex as active ingredient |
| CN112321674A (en) * | 2020-10-30 | 2021-02-05 | 润辉生物技术（威海）有限公司 | Snake venom-like tripeptide fatty acid derivative and preparation method and application thereof |
Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| KR20090133069A (en) * | 2009-04-14 | 2009-12-31 | (주)케어젠 | Peptides having activities of transforming growth factor-beta and their uses |
| KR20100031862A (en) * | 2008-09-16 | 2010-03-25 | (주)마임 | Novel palmitoyltripeptide and anti-wrinkle cosmetic composition containing it |
| KR20120107648A (en) * | 2011-03-22 | 2012-10-04 | (주)셀인바이오 | Palmitoyl dipeptide that is glycylproline or hydroxyglycylproline conjugated palmitic acid and method for peparing the same |
| KR20130055505A (en) * | 2011-11-14 | 2013-05-28 | (주)노바셀테크놀로지 | Novel peptide derivative capable of synthesizing collagen and use thereof |
| KR101715599B1 (en) * | 2016-10-04 | 2017-03-13 | (주)셀트리온 | Tripeptide combined with fatty acid and anti-wrinkle cosmetic composition comprising the same |
Family Cites Families (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US4877805A (en) | 1985-07-26 | 1989-10-31 | Kligman Albert M | Methods for treatment of sundamaged human skin with retinoids |
| US4983382A (en) | 1987-01-27 | 1991-01-08 | Avon Products, Inc. | Cosmetic preparation incorporating stabilized ascorbic acid |
| CN102250205A (en) * | 2011-05-18 | 2011-11-23 | 首都医科大学 | Preparation method of fatty acyl-RGD (Arg Gly Asp) induced docetaxel target liposome and antitumor activity thereof |
| US20130164265A1 (en) * | 2011-12-21 | 2013-06-27 | Dana FLAVIN | Skin care compositions |
-
2016
- 2016-10-04 KR KR1020160127375A patent/KR101715599B1/en active IP Right Grant
-
2017
- 2017-09-29 WO PCT/KR2017/010913 patent/WO2018066914A1/en active Application Filing
- 2017-10-03 TW TW106134242A patent/TWI675849B/en not_active IP Right Cessation
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| KR20100031862A (en) * | 2008-09-16 | 2010-03-25 | (주)마임 | Novel palmitoyltripeptide and anti-wrinkle cosmetic composition containing it |
| KR20090133069A (en) * | 2009-04-14 | 2009-12-31 | (주)케어젠 | Peptides having activities of transforming growth factor-beta and their uses |
| KR20120107648A (en) * | 2011-03-22 | 2012-10-04 | (주)셀인바이오 | Palmitoyl dipeptide that is glycylproline or hydroxyglycylproline conjugated palmitic acid and method for peparing the same |
| KR20130055505A (en) * | 2011-11-14 | 2013-05-28 | (주)노바셀테크놀로지 | Novel peptide derivative capable of synthesizing collagen and use thereof |
| KR101715599B1 (en) * | 2016-10-04 | 2017-03-13 | (주)셀트리온 | Tripeptide combined with fatty acid and anti-wrinkle cosmetic composition comprising the same |
Also Published As
| Publication number | Publication date |
|---|---|
| KR101715599B1 (en) | 2017-03-13 |
| TW201817740A (en) | 2018-05-16 |
| TWI675849B (en) | 2019-11-01 |
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