TWI675849B - Use of compound or cosmetically acceptable salt thereof for wrinkle reduction - Google Patents
Use of compound or cosmetically acceptable salt thereof for wrinkle reduction Download PDFInfo
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- TWI675849B TWI675849B TW106134242A TW106134242A TWI675849B TW I675849 B TWI675849 B TW I675849B TW 106134242 A TW106134242 A TW 106134242A TW 106134242 A TW106134242 A TW 106134242A TW I675849 B TWI675849 B TW I675849B
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Classifications
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- 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
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- 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
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- 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
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- 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
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- C07—ORGANIC CHEMISTRY
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- 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
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- 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
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- 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
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- Animal Behavior & Ethology (AREA)
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- Gerontology & Geriatric Medicine (AREA)
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- Cosmetics (AREA)
Abstract
本發明提供一種脂肪酸綴合的三肽和包含其的化妝品組合物,本發明在防止皮膚老化、減少皺紋、增強皮膚彈性和滲透皮膚方面提供優異的效果。 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
本申請主張於2016年10月4日向韓國智財局提交的韓國專利申請第10-2016-0127375號的權益,其公開內容透過引用併入本文。 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.
本發明涉及脂肪酸綴合的三肽(fatty acid-conjugated tripeptide)和包含其的具有優異的減少皮膚皺紋的效果之化妝品組合物。 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.
皮膚是重要的器官,其起到可保護人體,起到屏障功能、以及排泄功能等多種作用。然而,由於諸如污染物、紫外線、壓力等的外部環境因素,皮膚細胞受損並且使細胞的更新不正確地進行。結果,表皮、真皮和皮下組織的厚度變薄,皮膚的彈性降低,繼而形成皺紋。紫外線會對皮膚誘發活性氧(active oxygen),從而增加對皮膚細胞的損壞和色素沉著,繼而導致皮膚主要成分的直接功能的損失以及皮膚老化。活性氧是造成人體疾病和老化的原因,且可作為衡量抗氧化活性(antioxidative activity)和抗衰老作用(anti-aging action)的指標來評估。皮膚老化的特徵可包含彈性下降、皺紋、斑點(freckles)、雀斑(ephelides)及其類似物。原因之一是為彈性纖維(elastic fibers)之膠原蛋白和彈性蛋白由脂質 過氧化引起(lipid peroxidation-induced)的的斷鍊、其異常的交聯以及玻尿酸(hyaluronic acid)的斷鍊。脂質過氧化是由在此期間產生為活性含氧物(reactive oxygen species,ROS)之自由基的氧化壓力所引起的。據載,這種自由基生成的過程不僅在對皮膚有害的代謝活化之前的階段發生,而且還對皮膚細胞和組織造成損害。具體來說,對於皺紋,活性氧可誘導基質金屬蛋白酶(matrix metalloproteinase,MMP)的表達,並且使肌膚有彈性的膠原蛋白或彈性蛋白退化或受到破壞,從而導致皺紋。膠原蛋白為構成真皮層基質的主要成分,因此調節膠原蛋白的生物合成和降解成為皮膚老化過程的關鍵。已知已開發出像是抗壞血酸(ascorbic acid)(美國專利號4,983,382)、維生素A(retinoid)(美國專利號4,877,805)及其衍生物之化合物之促進膠原蛋白產生的物質,以及包含其的化妝品組合物。然而,抗壞血酸在空氣和濕氣中容易被氧化,因此具有穩定性的問題,且據報導,維生素A並不穩定,並且在應用於皮膚時會引起皮膚刺激問題。 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.
作為一種對皮膚刺激性小且具有減少皮膚皺紋作用的化合物,肽係為蛋白質的主要成分,其具有減少皺紋且對皮膚無副作用的優點。除了肽之外,RGD(精胺酸-甘胺酸-天門冬胺酸)(Arg-Gly-Asp)序列從二十世紀八十年代後期就是已知的細胞識別序列(cell recognition sequence),並且從二十世紀九十年代早期就已經使用合成的RGD肽(Biochem J.(1993)296,21-24)。此外,根據論文,已知包含RGD的肽對整合素(intergin)介導的細胞附著具有影響,並且上述功能影響細胞生長(Annu Rev Cell Dev Biol.(1996)12:697-715)。現在,RGD被用作為活化膠原蛋白和減少皮膚皺紋的成分,但是肽由於其親水性並且難以穿透皮膚屏障而在實際減少皺紋的效果上受到限制。 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.
為了克服現有技術的上述問題,本發明致力於解決親水性三肽RGD(精胺酸-甘胺酸-天門冬胺酸)穿透皮膚屏障的困難,其係藉由將各種疏水性脂肪酸與RGD綴合來製備具有親水-親脂結構的多種衍生物,因此透過鑑定衍生物對於減少皮膚皺紋具有高效力代表本發明已完成。 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.
因此,本發明要達成的目的是提供下述化學式I的脂肪酸綴合的三肽或其美容學上可接受的鹽。 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.
此外,本發明要達成的另一個目的是提供一種化妝品組合物,其包含下述化學式I的脂肪酸綴合的三肽或其美容學上可接受的鹽作為活性成分,以及其用於減少皺紋之用途。 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.
本發明涉及脂肪酸綴合的三肽和包含其的具有優異的減少皮膚皺紋作用之化妝品組合物。 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.
在下文中,將更詳細地描述本發明。 Hereinafter, the present invention will be described in more detail.
本發明提供為脂肪酸綴合的RGD的脂肪酸綴合的三肽或其美容學上可接受的鹽,更優選棕櫚醯-RGD(palmitoyl-RGD)或其美容學上可接受的鹽。 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.
上述RGD是與已知具有膠原蛋白合成效果的精胺酸、甘胺酸和天門冬胺酸的三個氨基酸連接的三肽,因此被用作化妝品成分。 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.
上述脂肪酸綴合的RGD係由如化學式I之通式表示:[化學式I]
其中n是11至16的整數。 Where n is an integer from 11 to 16.
而且,上述棕櫚醯-RGD是由下述化學式II,或Palm-Arg-Gly-Asp表示的化合物,其中化學式I中的n是15。 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.
而且,本發明提供了上述化學式I的脂肪酸綴合的三肽(RGD)或包含其的化妝品組合物。 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.
上述化妝品組合物在防止皮膚老化、減少皺紋、增強皮膚彈性和滲透皮膚方面提供了極好的效果,並在長期儲存的穩定性以及皮膚安全性上具有良好效果。 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.
用上述化學式I的化合物處理的細胞的原膠原蛋白(procollagen)合成量可比用相同濃度的下述化學式III化合物處理的細胞多至少20%。 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.
[化學式Ⅲ]
相對於化妝品組合物的總重量,本發明的化妝品組合物可包含0.0001~2wt%,優選地為0.01~0.05wt%的化學式I的化合物作為活性成分。 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.
以及,本發明提供用於減少皺紋的化妝品組合物,其包含上述化學式I的脂肪酸綴合的三肽(RGD)或其美容學上可接受的鹽。 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.
相對於化妝品組合物的總重量,本發明的化妝品組合物可包含0.0001~2wt%,優選地為0.01~0.05wt%的化學式I的化合物作為活性成分。 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.
根據本發明的化妝品組合物的配方無特別限制,並且本發明的化妝品組合物可根據待製備的配方包含本領域已知的常規化妝品組合物組合。本發明的化妝品組合物可製成像是洗劑(lotion)、乳液(emulsion)、營養霜(nourishing cream)、面膜(pack)、美容液(beauty liquid)、精油(essence)等配方,也可以根據待製備的配方,透過選擇油、乳化劑(emulsifier)、乳化穩定劑(emulsion stabilizer)、保濕劑(humectant)、增稠劑(thickener)、防腐劑(preservative)、香料(fragrance)等來組合。 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. .
本發明提供了上述化學式I的化合物的製備方法,其包含以下步驟:(a)藉由使用具有受保護之N-末端,C-末端或其反應性殘基(reactive residue)之精胺酸衍生物、甘胺酸衍生物和天門冬胺酸衍生物在液相中合成三肽(精胺酸-甘胺酸-天門冬胺酸)衍生物; (b)藉由使上述三肽(精胺酸-甘胺酸-天門冬胺酸)衍生物與脂肪酸鹽反應,使精胺酸的N末端與脂肪酸綴合;以及(c)從上述脂肪酸綴合的三肽(精胺酸-甘胺酸-天門冬胺酸)衍生物除去三肽部分的保護基。 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.
在下文中,將參照下面的反應式I詳細描述根據本發明的上述化學式II的棕櫚醯-RGD的製備方法。以下反應式I中所示的方法僅僅是可能的製備方法的代表,並且如果需要,可以適當地修改在以下反應式I中顯示的單元操作的順序、試劑、反應條件等。此外,可以使用藉由為一般肽合成方法的固相合成(solid synthesis)法所製備的RGD。 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.
按照上述方法製備的本發明的棕櫚醯-RGD不僅穩定,而且在防止皮膚老化、減少皺紋、增強皮膚彈性和滲透皮膚方面具有優異的效果。 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.
根據本發明的脂肪酸綴合的三肽化合物在防止皮膚老化、減少皺紋、增強皮膚彈性和滲透皮膚方面提供了優異的效果。 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.
第1圖係繪示說明脂肪酸綴合的RGD的細胞毒性實驗結果的圖。 FIG. 1 is a graph showing the results of a cytotoxicity experiment of fatty acid-conjugated RGD.
第2圖係繪示說明脂肪酸綴合的RGD的原膠原蛋白合成效果比較的圖。 FIG. 2 is a diagram illustrating a comparison of the effects of procollagen synthesis by fatty acid-conjugated RGD.
第3圖係繪示說明脂肪酸綴合的RGD的經皮吸收效果比較的圖。 Fig. 3 is a diagram illustrating a comparison of the transdermal absorption effect of fatty acid-conjugated RGD.
第4圖至第6圖係繪示說明包含棕櫚醯-RGD的組合物減少眼眶周圍(periorbital)皺紋效果的圖。 Figures 4 to 6 are diagrams illustrating the effect of a composition containing palmarium-RGD on reducing periorbital wrinkles.
第7圖係繪示說明包含棕櫚醯-RGD的組合物增強皮膚彈性效果的圖。 FIG. 7 is a diagram illustrating a skin elasticity-enhancing effect of a composition containing palmarium-RGD.
第8圖係繪示說明包含棕櫚醯-RGD的組合物改善皮膚真皮密度效果的圖。 FIG. 8 is a diagram illustrating the effect of a composition containing palmarium-RGD on improving skin dermis density.
本發明提供脂肪酸綴合的三肽和包含其的用於減少皮膚皺紋之化妝品組合物。 The present invention provides a fatty acid-conjugated tripeptide and a cosmetic composition containing the same for reducing skin wrinkles.
在本發明的一個具體實施例中,脂肪酸綴合的三肽可為棕櫚醯-RGD。 In a specific embodiment of the present invention, the fatty acid-conjugated tripeptide may be palmarium-RGD.
下文中,本發明將透過實施例進一步詳細描述。然而,此些實施例的記述僅作為本發明說明性之敘述,而不被解釋為對本發明範圍之限制。 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.
製備實施例1:製備棕櫚醯-RGD Preparation Example 1: Preparation of palmarium-RGD
製備實施例1-1:合成Boc-Gly-Asp(OBn)2 Preparation Example 1-1: Synthesis of Boc-Gly-Asp (OBn) 2
將N-(叔丁氧基羰基)甘胺酸(N-(tert-butoxycarbonyl)glycine(N-Boc-Gly-OH),20g,114.17mmol)和L-天門冬胺酸二芐酯(L-aspartic acid dibenzyl ester,55.43g,114.17mmol)溶於四氫呋喃(tetrahydrofuran(THF),600mL),在這之後,將其溫度降低到0℃,接著將1-羥基苯並三唑水合物(1-hydroxybenzotriazolehydrate(HOBt),18.51g,137.00mmol)和1-(3-二甲基胺基丙基)-3-乙基碳化二亞胺鹽酸鹽(1-(3-dimethylaminopropyl)-3-ethylcarbodiimidehydrochloride(EDC.HCl),27.00g,137.00mmol)加入所得溶液中,再將三乙胺(triethylamine(TEA),55.69mL,399.59mmol)緩慢加入於其中。然後,將所得混合物在0℃下攪拌30分鐘,然後將所得溶液從冷卻浴中移出,並在室溫下攪拌整夜(over night)。將反應溶液濃縮,然後向所得濃縮物中加入水(320mL)和乙酸乙酯(ethyl acetate,400mL)以萃取兩次(400mL×2),有機層用NaCl(aq)(300mL)潤洗,並以硫酸鈉(Na2SO4)乾燥,接著在壓力下過濾及濃縮。將濃縮物減壓乾燥,得到(S)-二芐基-2-(2-((叔丁氧基羰基)胺基)乙醯胺基)琥珀酸酯((S)-dibenzyl2-(2-((tert-butoxycarbonyl)amino)acetamido)succinate(Boc-Gly-Asp(OBn)2),64.53g,120%)的黃色油狀物,使得在沒有其純化過程的情況下進行下列反應。 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.
製備實施例1-2:合成Gly-Asp(OBn)2HCl Preparation Example 1-2: Synthesis of Gly-Asp (OBn) 2 HCl
(S)-二芐基-2-(2-((叔丁氧基羰基)胺基)乙醯胺基)琥珀酸酯(53.71g,114.15mmol)溶於乙酸乙酯(500mL),之後向其中加入4M鹽酸(HCl)1,4-二[口咢]烷(1,4-dioxane)(113mL,114.15mmol)溶液,使得到的混合物在室溫下攪拌6小時。於減壓下過濾並乾燥生成的固體,由此得到(S)-二芐基2-(2-胺基乙醯胺基) 琥珀酸鹽酸鹽((S)-dibenzyl2-(2-aminoacetamido)succinate hydrochloride(Gly-Asp(OBn)2HCl),39.47g,85%)的白色固體。 (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.
製備實施例1-3:合成Boc-Arg(NO2)-Gly-Asp(OBn)2 Preparation Example 1-3: Synthesis of Boc-Arg (NO 2 ) -Gly-Asp (OBn) 2
(S)-二芐基2-(2-胺基乙醯胺基)琥珀酸鹽酸鹽(39.47g,97.01mmol)和2-((叔丁氧基羰基)胺基)-5-(3-硝基胍基)戊酸(2-((tert-butoxycarbonyl)amino)-5-(3-nitroguanidino)pentanoicacid(N-Boc-Arg(NO2)-OH),30.98g,97.01mmol)溶解於四氫呋喃(600mL)後,降溫至0℃,接著將1-羥基苯並三唑(15.73g,116.41mmol)和1-(3-二甲基胺基丙基)-3-乙基碳化二亞胺鹽酸(22.31g,116.41mmol)加入到所得溶液中,並向其中緩慢加入三乙胺(47.33mL,339.54mmol)。然後,將所得混合物在0℃下攪拌30分鐘,然後將所得溶液從冷卻浴中移出,並在室溫下攪拌整夜。將反應物溶液濃縮,然後向所得濃縮物中加入水(320mL)和乙酸乙酯(400mL)以萃取兩次(400mL×2),且有機層用NaCl(aq)(300mL)潤洗,並用硫酸鈉乾燥,並在壓力下過濾及濃縮。將濃縮物減壓乾燥,得到(S)-二芐基2-(2-((S)-2((叔丁氧基羰基)胺基)-5-(3-硝基胍基)戊醯胺基)乙醯胺基)琥珀酸酯((S)-dibenzyl 2-(2-((S)-2((tert-butoxycarbonyl)amino)-5-(3-nitroguanidino)pentanamido)acetamido)succinate(Boc-Arg(NO2)-Gly-Asp(OBn)2),65.16g,102%)的黃色油狀物,使得不進行其純化過程而進行下列反應。 (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.
製備實施例1-4:合成Arg(NO2)-Gly-Asp(OBn)2HCl鹽 Preparation Example 1-4: Synthesis of Arg (NO 2 ) -Gly-Asp (OBn) 2 HCl salt
將(S)-二芐基2-(2-((S)-2((叔丁氧基羰基)胺基)-5-(3-硝基胍基)戊醯胺基)乙醯胺基)琥珀酸酯(65.16g,97.00mmol)溶於乙酸乙酯(500mL)中,然後向其中加入4M鹽酸(HCl)1,4-二[口咢]烷(94.58mL,378.30mmol)溶液,並將得到的混合物在室溫下攪拌6小時。在減壓下過濾並乾燥取得的固體,由此獲得白色固體的(S)-二芐基-2-(2-((S)-2-胺基)-5-(3-硝基胍基)戊醯胺基)乙醯胺基)琥珀酸鹽 酸鹽((S)-dibenzyl 2-(2-((S)-2-amino)-5-(3-nitroguanidino)pentanamido)acetamido)succinate hydrochloride(Arg(NO2)-Gly-Asp(OBn)2 HCl鹽),59.08g,100%)。 (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%).
製備實施例1-5:合成Palm-Arg(NO2)-Gly-Asp(OBn)2 Preparation Example 1-5: Synthesis of Palm-Arg (NO 2 ) -Gly-Asp (OBn) 2
將(S)-二芐基-2-(2-((S)-2-胺基)-5-(3-硝基胍基)戊醯胺基)乙醯胺基)琥珀酸鹽酸鹽(59.06g,97.04mmol)溶於二氯甲烷(dichloromethane(CH2Cl2),800mL)中,然後使其溫度降溫至0℃,向得到的溶液中加入棕櫚醯氯(palmitoyl chloride,30.76mL,101.85mmol),接著緩慢加入三乙胺(27.04mL,194.01mmol)。然後,將所得混合物在0℃下攪拌30分鐘,然後將所得溶液從冷卻浴中移出,並在室溫下攪拌整夜。完成反應後,將得到的溶液在壓力下濃縮,之後,自其部分去除二氯甲烷並加入水,接著過濾。所得固體在減壓下乾燥,得到(S)-二芐基2-(2-((S)-5-(3-硝基胍基)-2-棕櫚醯胺基戊醯胺基)乙醯胺基)琥珀酸酯((S)-dibenzyl 2-(2-((S)-5-(3-nitroguanidino)-2-palmitamidopentanamido)acetamido)succinate(Palm-Arg(NO2)-Gly-Asp(OBn)2),73.01g,93%)。 (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%).
製備實施例1-6:合成Palm-RGD-HCl鹽 Preparation Example 1-6: Synthesis of Palm-RGD-HCl salt
將(S)-二芐基2-(2-((S)-5-(3-硝基胍基)-2-棕櫚醯胺基戊醯胺基)乙醯胺基)琥珀酸酯(70.00g,86.42mmol)和四氫呋喃(1.5L)加在一起,然後向其中加入3M鹽酸(288.07mL,864.21mmol)。然後,在氬氣(Ar)下,將10%鈀碳(palladium carbon(Pd/C),8.64g)加入到所得混合物中。在氫氣(H2)下,將溶液在室溫中攪拌3小時。將藉由矽藻土的手段從反應混合物中過濾的溶液減壓,從而使其溶劑部分地自其除去。向剩餘的溶液中緩慢加入氫氧化鈉(NaOH)(aq),然後將在pH 2或更高的pH下生成的固體用水洗滌、過濾並乾燥。固體在異丙醇(isopropyl alcohol(IPA))中回流,接著在高溫態(hot state)下過濾並再次乾燥。當將5M氫氧化鈉和6M鹽酸加入到所得固體中時,固體隨著溶液變熱而融化,使固 體隨著溶液冷卻而產生。之後,將固體過濾並在真空烘箱中乾燥,由此獲得(S)-二芐基2-(2-((S)-5-胍基)-2-棕櫚醯胺基戊醯胺基)乙醯胺基)琥珀酸鹽酸鹽((S)-dibenzyl2-(2-((S)-5-guanidino)-2-palmitamidopentanamido)acetamido)succinate hydrochloride),即棕櫚醯-RGD-HCl鹽(palmitoyl-RGD-HCl salt,33.82g,63%)。 (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%).
實驗例1:細胞毒性實驗 Experimental Example 1: Cytotoxicity Experiment
為了鑑定脂肪酸綴合的RGD的細胞毒性,藉由以下方法之手段,在分別具有不同長度的脂肪酸之作為四種材料的月桂醯-RGD(Lauroyl-Arg-Gly-Asp)、肉荳蔻醯-RGD(Myristoyl-Arg-Gly-Asp)、棕櫚醯-RGD(Palmitoyl-Arg-Gly-Asp)和硬脂醯-RGD(Stearoyl-Arg-Gly-Asp)上進行細胞毒性實驗。 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.
將新生成的人真皮纖維細胞(human dermal fbbroblast neonatal,HDFn細胞,Cascade Co.)接種到24-微孔板(5×104個細胞/孔)中,於37℃及5%CO2條件下培養24小時。然後,除去培養基,用磷酸緩衝生理鹽水(PBS)清洗微孔板,向其中添加其中添加有1%青黴素/鏈黴素(penicillins/streptomycin(P/S))的Dulbecco's Modified Eagle's培養基(DMEM)的培養基從而培養24小時。除去培養基,用PBS清洗微孔板,然後根據最終處理濃度,向其中加入900μL培養基以及各100μL月桂醯-RGD、肉荳蔻醯-RGD、棕櫚醯-RGD和硬脂醯-RGD,培養48小時。然後,除去培養基,用PBS洗滌微孔板,然後向其中加入100μLMTT(3-[4,5-二甲基噻唑-2-基]-2,5-二苯基四唑鎓溴化物(3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide))和900μL培養基,培養4小時。除去培養液,並向其中分別添加300μL二甲基亞碸(dimethyl sulfoxide(DMSO))溶液,之後將所得混合物振盪10分鐘,並利用微量盤檢測儀(microplate reader)之裝置測量在570nm的光密度,以鑑定細胞活性。 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.
結果顯示在下表1和第1圖中。 The results are shown in Tables 1 and 1 below.
如以上表1所示,證實月桂醯-RGD和肉荳蔻醯-RGD在濃度為10.0ug/mL或以下時不表現細胞毒性,棕櫚醯-RGD在濃度為4.0ug/mL或以下時不表現細胞毒性。另一方面,證實硬脂醯-RGD在濃度為2.0u g/mL或以上時表現出高細胞毒性。 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.
實驗例2:原膠原蛋白合成效果的分析 Experimental Example 2: Analysis of the effect of procollagen synthesis
分析月桂醯-RGD、肉荳蔻醯-RGD、棕櫚醯-RGD和硬脂醯-RGD作為脂肪酸綴合的三肽的原膠原蛋白合成效果,以找到合適長度的脂肪酸。並透過使用未與脂肪酸綴合的RGD和乙醯-RGD作為控制組進行比較實驗。 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.
透過使用其中添加有10%胎牛血清的Dulbecco's Modified Eagle's培養基(DMEM)的培養基,將新生成的人真皮纖維細胞接種到24孔微量培養板(5×104個細胞/孔)中,由此在37℃和5%CO2的條件下培養24小時。然後,將培養基 換成無血清的DMEM培養基,培養24小時,然後處理適量的各測試化合物。培養48小時後,收集細胞培養液,然後使用Takara公司(Takara Shuzo Co.,Ltd.Japan)的膠原蛋白測定試劑盒測定原膠原蛋白量。首先,將收集的細胞培養液注入96孔微量培養板中,初級膠原蛋白抗體均勻地塗佈至其,使抗原抗體反應可在37℃下發生3小時。從孔中取出細胞培養液並用磷酸鹽緩衝鹽水(PBS)洗滌四次。向每個孔中注入染色物質(color causing material),然後在室溫下進行反應15分鐘,並加入1N硫酸溶液於其中以停止反應。用分光光度計之裝置在450nm波長測量光密度(optical density)。使用標準溶液繪製標準曲線,將透過上述方法之手段獲得的光密度歸屬於(assigned to)標準曲線,從而計算向其添加了各測試化合物的細胞培養液所產生的原膠原蛋白量。 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.
結果顯示在下表2和第2圖中。 The results are shown in Tables 2 and 2 below.
如上述表2所示,證實在相同濃度下脂肪酸綴合的RGD的原膠原蛋白合成效果比RGD或乙醯-RGD更優異,特別是在相同濃度下,棕櫚醯-RGD的效果最優異。 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.
如在上述實驗例1至2中證實的那樣,其證實了在脂肪酸綴合的RGD中,棕櫚醯-RGD是在呈現有效效果的濃度中表現無細胞毒性,但是高的膠原蛋白合成效率的物質。因此,下文中在以下的實驗例中對棕櫚醯-RGD進行實驗。 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.
實驗例3:經皮吸收(滲入皮膚細胞)實驗 Experimental Example 3: Percutaneous absorption (infiltration into skin cells) experiment
通過使用棕櫚醯-RGD(由上化學式II表示的物質)進行經皮吸收實驗,並且還透過未與脂肪酸綴合的RGD作為控制組進行比較實驗(參考:Lehman PA,Slattery JT,Franz TJ。類維生素A的經皮吸收:載體、光照和劑量的影響(Percutaneous absorption of retinoids:Influence of vehicle,light exposure,and dose),J.Invest Dermatol.,91;56-61,1988)。 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).
具體而言,在Franz擴散槽(Franz diffusion cell)測量滲透性。以未與脂肪酸綴合的RGD作為控制組。使用儲存在-20℃的入類皮膚以鑑別皮膚滲透性。為了在測試之前鑑別皮膚的結構完整性,通過經皮水分散失(transepidermal water loss(TEWL))證實皮膚(700μm厚,在-20℃保存)沒有損傷。 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).
將10%RGD和棕櫚醯-RGD溶解在二丙烯甘醇/辛基十二烷醇(dipropylene glycol/octyldodecanol,1:1)的溶劑中,然後將1mL所得溶液裝載到提供有樣品的腔室上,使其貼劑應用於人體皮膚。然後,24小時後,通過樣品端口取出500μL下層溶液,並通過HPLC的手段定量分析。每個樣品進行5次皮膚滲透實驗。 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.
結果顯示在下表3和第3圖中。 The results are shown in Tables 3 and 3 below.
如上表3所示,即使在施用於皮膚後24小時,可檢測量的RGD也是極微量的。這意味著上述RGD具有包含為鹼性氨基酸的R(精胺酸)以及為酸性氨基酸的D(天門冬胺酸)的親水性,使得上述RGD不能滲透含有大量疏水脂質的皮膚角質層,而不能直接傳遞到皮膚細胞。此外,硬脂醯-RGD具有優異的皮膚滲透性,但在如表1所示之具有有效效果的濃度時具有細胞毒性,使得硬脂醯-RGD不適合作為化妝品。然而,棕櫚醯-RGD在有效濃度下不具有細胞毒性。由於RGD與為疏水性脂肪酸的棕櫚醯基(palmitoyl)綴合,其皮膚滲透性提高了15倍以上,且原膠原蛋白合成效果也得到改善。 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.
實驗例4:皮膚安全性測試 Experimental example 4: Skin safety test
為了測定棕櫚醯-RGD的皮膚安全性,將棕櫚醯-RGD成分以各濃度溶解於丙二醇中,確認其皮膚刺激性。 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.
具體而言,將5×20cm的含有一定量(20μL)棕櫚醯-RGD丙二醇溶液的貼劑施用於30名20至50歲成年人的背部區域24小時。然後,將貼劑取下,以便在1小時和24小時後用肉眼檢查皮膚狀況的變化,其結果在表4中示出。此時,根據下面的皮膚狀況評估標準對皮膚狀況的變化程度進行分類及評估。 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.
<皮膚狀況的評估標準> <Evaluation criteria for skin condition>
研究人員根據以下標準進行視覺評估。 Researchers performed visual assessments based on the following criteria.
0:沒有變化1:非常微小的變化 0: No change 1: Very small change
2:輕微變化3:略劇烈變化 2: slight change 3: slightly change
4:劇烈變化5:非常劇烈變化 4: drastic change 5: very drastic change
如上表4所示,證實棕櫚醯-RGD的含量達到0.05wt%時不具皮膚刺激性。 As shown in Table 4 above, it was confirmed that palmitosalm-RGD content was 0.05 wt% without skin irritation.
實驗例5:濕度和熱的穩定性測試 Experimental example 5: Humidity and heat stability test
化妝品組合物中使用的活性成分對濕度和熱的穩定性是組合物在儲存期間的重要因素。因此,為了證實棕櫚醯-RGD作為化妝品組合物中使用的活性成分的適合性,評估其對濕度和熱的穩定性。 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.
具體而言,各樣品以密封狀態在4℃、30℃、45℃,相對濕度75%的惡劣條件下保存12週後,用高效液相色譜法(high performance liquid chromatography(HPLC))測定對於初始活性成分值的樣品的殘留率(%)。其結果列於下表5中。 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.
如以上表5所示,其證實棕櫚醯-RGD對水分和熱優異的穩定性。 As shown in Table 5 above, it confirms that palmarium-RGD has excellent stability to moisture and heat.
配方例1。製備包含棕櫚醯-RGD的溶解配方(solubilization formulation) Formula Example 1. Preparation of a solubilization formulation containing palmarium-RGD
為了證實包含棕櫚醯-RGD作為活性成分的化妝品組合物的穩定性,如下表6所示地製備其溶解配方。 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.
<製備方法> <Preparation method>
(1)分別將各水相和乙醇相均勻混合並溶解。 (1) Each water phase and ethanol phase are uniformly mixed and dissolved.
(2)將乙醇相加入到水相中且混合並溶解所得溶液。 (2) The ethanol phase is added to the water phase and the resulting solution is mixed and dissolved.
(3)將添加物I的棕櫚醯-RGD溶解,並將其加到所得的混合物中,並將添加物II混合於其中以完成溶解配方。 (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.
配方例2。製備包含棕櫚醯-RGD的精油配方 Formula Example 2. Preparation of essential oil formula containing palmarium-RGD
為了證實包含棕櫚醯-RGD作為活性成分的化妝品組合物的穩定性,如下表7所示地製備精油配方。 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.
<製備方法> <Preparation method>
(1)加熱水相和油相,並分別均勻混合並溶解各水相和油相。 (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)於75℃將油相加入水相中以混合並乳化在一起。 (2) The oil phase was added to the water phase at 75 ° C to mix and emulsify together.
(3)在45℃加入添加物相I的棕櫚醯-RGD於所得混合物中,並混合在一起,且將添加物相II混合在一起。 (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.
配方例3。製備包含棕櫚醯-RGD的乳霜配方 Formula Example 3. Preparation of a cream formula containing palmarium-RGD
為了證實包含新的棕櫚醯-RGD作為活性成分的化妝品組合物的穩定性,如下表8所示地製備乳霜配方。 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.
<製備方法> <Preparation method>
(1)加熱水相和油相,並分別均勻混合並溶解各水相和油相。 (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)於75℃將油相加入水相中以混合並乳化在一起。 (2) The oil phase was added to the water phase at 75 ° C to mix and emulsify together.
(3)在45℃加入添加物相I的棕櫚醯-RGD於所得混合物中,並混合在一起,且將添加物相II混合在一起。 (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.
實驗例6:鑑定包含棕櫚醯-RGD的化妝品組合物的成分穩定性 Experimental Example 6: Identification of ingredient stability of a cosmetic composition containing palmarium-RGD
為了鑑定配方中棕櫚醯-RGD的穩定性,將上述配方例1至3的溶解配方、精油配方和乳霜配方在4℃、30℃、45℃和日照條件下儲存12週,之後 以與上述實驗例5相同的高效液相色譜(HPLC),觀察包含棕櫚醯-RGD的化妝品中隨時間推移的含量變化,且其結果列於下表9中。 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.
如上表9所示,其證實在高溫和日照條件下溶解配方、精油配方和乳霜配方都具有於配方中優異的成分穩定性。 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.
實驗例7:鑑定包含棕櫚醯-RGD的化妝品組合物的配方穩定性 Experimental Example 7: Identification of Formulation Stability of Cosmetic Compositions Containing Palmarium-RGD
為了鑑定包含棕櫚醯-RGD的化妝品組合物本身的配方穩定性,將上述配方例1至3的溶解配方、精油配方和乳霜配方在4℃、30℃(室溫,RT)、45℃且日照之條件儲存12週,然後通過目視和感官評估觀察顏色、氣味和配方的變化,其結果示於下表10至12中。此時,根據以下評估標準對顏色、氣味和配方的變化進行分類和評估。 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.
<評估標準> <Evaluation criteria>
沒有變化:- 非常些微的變化:+ 輕微的變化:++ No changes:-Very slight changes: + Minor changes: ++
可觀察到的變化:+3 顯著變化:+4 明顯變化:+5 Observable change: +3 Significant change: +4 Significant change: +5
如上表10~12所示,其證實在高溫和日光條件下,溶解配方、精油配方和乳霜配方都具有優異的穩定性。 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.
實驗例8:鑑定包含棕櫚醯-RGD的化妝品組合物的皮膚刺激性 Experimental Example 8: Identification of skin irritation of a cosmetic composition containing palmarium-RGD
為了鑑定包含棕櫚醯-RGD的化妝品組合物的皮膚刺激性,評估上述配方例3的乳霜配方的皮膚刺激性。 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.
具體而言,將濃度依賴性方式(a concentration-dependent manner)之含有於上述配方例3中製備的包含棕櫚醯-RGD的一定量(60uL)之乳霜配方的5×20cm貼劑應用於50名成人的每個背部區域每次24小時,共計9次。在總共9次之貼劑重複應用的過程中,在移除貼劑後每30分鐘觀察一次皮膚反應,之後再次貼上貼劑,從而鑑定致敏誘導(sensitization induction)。休息2週後,再用一次貼劑,並在移除貼劑後30分鐘和24小時和48小時中鑑定致敏誘導的程度。致敏誘導的結果表示在表13和致敏誘導測試的結果表明在表14中。當時,根據國際接觸性皮炎研究組織(International Contact Dermatitis Research Group(ICDRG))的標準判定皮膚反應判斷。 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).
<皮膚狀況的評估標準> <Evaluation criteria for skin condition>
0:負面反應(無刺激性) 0: negative reaction (non-irritating)
1:疑似反應-只有微弱的紅斑 1: Suspect reaction-only weak erythema
2:弱陽性反應-紅斑、浸潤(infiltration)、可能的丘疹 2: Weak positive reaction-erythema, infiltration, possible pimples
3:強烈的陽性反應-紅斑、浸潤、丘疹、水泡 3: Strong positive reaction-erythema, infiltration, pimples, blisters
4:極度強烈的反應-嚴重的紅斑和浸潤和聚集水泡 4: Extremely strong reaction-severe erythema and infiltration and accumulation of blisters
如上表13~14所示,證實包含棕櫚醯-RGD的乳霜配方的具有低皮膚刺激性。 As shown in Tables 13 to 14 above, it was confirmed that the cream formula containing palmarium-RGD has low skin irritation.
實驗例9:包含棕櫚醯-RGD的化妝品組合物在減少皮膚皺紋的臨床效果 Experimental Example 9: Clinical effect of cosmetic composition containing palmarium-RGD in reducing skin wrinkles
測定上述包含棕櫚醯-RGD的化妝品組合物減少皮膚皺紋的效果。 The skin wrinkle reduction effect of the above-mentioned cosmetic composition containing palmaridine-RGD was measured.
具體而言,為了評估包含棕櫚醯-RGD的化妝品組合物的有效性,對23名30至60歲的女性進行臨床測試。使用包含0.04wt%的棕櫚醯-RGD的化妝品組合物作為測試組產品。使用包含蒸餾水代替用於測試組產品中之棕櫚醯-RGD的安慰劑乳霜作為控制組產品。將上述測試和控制組產品隨機地分別施用到對象面部的左及右區域,並以每天兩次的間隔再次施用,並觀察12週的變化。 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.
實驗例9-1:眼眶周圍皺紋減少的鑑定測試 Experimental Example 9-1: Identification test for reducing orbital wrinkles
為了鑑定通過使用測試組產品和控制組產品的手段之眼眶周圍皺紋減少,以如下方式獲得數值:在使用之前和使用後的4週、8週和12週,將光穿透到對於產品施用的區域製造的半透明皮膚皺紋複製品(translucent skin wrinkle replica)中,並用VisiometerSV600(Courage-Khazaka electronic GmbH,Germany)分析其結果。在這些分析值中,R2和R3值作為眼眶周圍皺紋的關鍵參數評估。R2對應於對於其中褶皺輪廓等分為五分之區段之五個峰值和五個谷值 之間的最大差值。R3是通過沿著X軸將皺紋輪廓等分成五個區段並且用每個區段測量的最大值和最小值之間的差值來計算算術平均值。 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.
上述R2和R3值的結果在第4圖和第5圖的圖表中表示。 The results of the above R2 and R3 values are shown in the graphs of FIGS. 4 and 5.
結果,與使用前相比,使用後4週、8週和12週的R2值皆顯著降低(p<0.05)。即使與控制組產品相比時,測試組產品的R2值在使用後8週和12週時顯示出顯著差異(p<0.05)。此外,與使用前相比,測試組產品組的R3值在使用後的4週、8週和12週皆顯著降低(p<0.05)。即使與控制組產品比較時,測試組產品組的R3值在使用後的8週和12週時也顯示出顯著差異(p<0.05)。 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).
實驗例9-2:使用Primos的眼眶周圍皺紋測量測試 Experimental Example 9-2: Periorbital Wrinkle Measurement Test Using Primos
為了鑑定透過使用產品的手段,眼眶周圍皺紋測量值的變化,在使用前和在使用後的4週、8週和12週時使用為一種3D皮膚攝影裝置之Primos premium(GFMesstechnik GmbH,Germany)分析眼眶周圍皺紋的Ra值。Ra代表皺紋輪廓的平均粗糙度。值越小,表示眼眶周圍皺紋就越少。測量的Ra值的結果在第6圖的圖表中表示。 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.
結果,與使用前相比,測試組產品組的Ra值在使用後12週內均顯著降低(p<0.05)。即使與控制組產品比較,使用後4週和12週的Ra值也有顯著差異(p<0.05)。 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).
因此,通過上述實驗例9-1和9-2的結果證實,包含棕櫚醯-RGD的化妝品組合物在減少眼眶周圍皺紋上具有效果。 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.
實驗例9-3:皮膚彈性測量測試 Experimental Example 9-3: Skin elasticity measurement test
為了鑑定通過使用包含棕櫚醯-RGD的產品之手段,皮膚彈性測量值的變化,在使用前和使用後的4週、8週和12週時測量皮膚彈性。使用CutometerCM580(CK Electronics,Koln,Germany)分別在雙眼周圍的每個相同區域測量一次皮膚彈性。在結果值中,將R2(從皮膚變形恢復)值用作為評估皮膚彈 性程度的數據。R2值是指整體的彈性。值越接近1,皮膚越有彈性。測量的R2值的結果在第7圖的圖表中表示。 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.
結果,與使用前相比,測試組產品組的皮膚彈性測量值在使用後的4週、8週和12週中顯著增加(p<0.05)。即使與控制組產品比較,在使用後4週、8週、12週的皮膚彈性測定值皆顯示顯著差異(p<0.05)。因此,證實包含棕櫚醯-RGD的化妝品組合物在增強皮膚彈性上具有效果。 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.
實驗例9-4:皮膚真皮密度測量測試 Experimental Example 9-4: Skin dermis density measurement test
為了測量皮膚真皮密度,在使用之前和在使用後的4週、8週和12週期間,透過皮膚掃描儀高分辨率超音波(Skin Scanner High Resolution Ultrasound(taberna pro medicum,Germany))的手段拍攝雙眼周圍的區域,並使用測量的圖像來分析使用產品前後皮膚的真皮密度。皮膚真皮密度的測量單位為%,表明測量值越高,真皮密度越提升。測量結果如第8圖所示。 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.
結果,與使用前相比,測試組產品組的真皮密度測量值在使用後的8週和12週皆顯著增加(p<0.05)。即使與控制組產品比較,使用後8週和12週的真皮密度測量值也皆顯示出顯著的差異(p<0.05)。因此,證實包含棕櫚醯-RGD的化妝品組合物在改善皮膚真皮密度上具有效果。 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.
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