KR100817191B1 - Ascorbic acid derivatives grafted with tat peptide, its preparation method and costmetic composition for skin whitening comprising the same - Google Patents
Ascorbic acid derivatives grafted with tat peptide, its preparation method and costmetic composition for skin whitening comprising the same Download PDFInfo
- Publication number
- KR100817191B1 KR100817191B1 KR1020010060244A KR20010060244A KR100817191B1 KR 100817191 B1 KR100817191 B1 KR 100817191B1 KR 1020010060244 A KR1020010060244 A KR 1020010060244A KR 20010060244 A KR20010060244 A KR 20010060244A KR 100817191 B1 KR100817191 B1 KR 100817191B1
- Authority
- KR
- South Korea
- Prior art keywords
- ascorbic acid
- tat
- peptide
- tat peptide
- skin
- Prior art date
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- 125000002221 trityl group Chemical group [H]C1=C([H])C([H])=C([H])C([H])=C1C([*])(C1=C(C(=C(C(=C1[H])[H])[H])[H])[H])C1=C([H])C([H])=C([H])C([H])=C1[H] 0.000 description 1
- OUYCCCASQSFEME-UHFFFAOYSA-N tyrosine Natural products OC(=O)C(N)CC1=CC=C(O)C=C1 OUYCCCASQSFEME-UHFFFAOYSA-N 0.000 description 1
- 239000003981 vehicle Substances 0.000 description 1
- 239000011534 wash buffer Substances 0.000 description 1
- 239000001993 wax Substances 0.000 description 1
- 238000001262 western blot Methods 0.000 description 1
- 239000011787 zinc oxide Substances 0.000 description 1
Classifications
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
- C07K7/04—Linear peptides containing only normal peptide links
- C07K7/06—Linear peptides containing only normal peptide links having 5 to 11 amino acids
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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
- A61K8/67—Vitamins
- A61K8/676—Ascorbic acid, i.e. vitamin C
-
- 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
- A61Q19/02—Preparations for care of the skin for chemically bleaching or whitening the skin
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07D—HETEROCYCLIC COMPOUNDS
- C07D307/00—Heterocyclic compounds containing five-membered rings having one oxygen atom as the only ring hetero atom
- C07D307/02—Heterocyclic compounds containing five-membered rings having one oxygen atom as the only ring hetero atom not condensed with other rings
- C07D307/34—Heterocyclic compounds containing five-membered rings having one oxygen atom as the only ring hetero atom not condensed with other rings having two or three double bonds between ring members or between ring members and non-ring members
- C07D307/56—Heterocyclic compounds containing five-membered rings having one oxygen atom as the only ring hetero atom not condensed with other rings having two or three double bonds between ring members or between ring members and non-ring members with hetero atoms or with carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals, directly attached to ring carbon atoms
- C07D307/62—Three oxygen atoms, e.g. ascorbic acid
-
- 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
Abstract
본 발명은 자가침투신호를 갖는 세포 침투성 Tat 펩타이드가 결합된 아스코르브산 유도체, 이의 제조방법 그리고 이를 포함하는 피부 미백용 화장료 조성물에 관한 것이다. 상기 아스코르브산 유도체는 물과 유기용매에 모두 비교적 좋은 용해도를 보이며 높은 안정성과 우수한 피부 침투성을 가지므로, 우수한 미백효과 및 그 효과의 지속성을 향상시킨 피부 미백제를 제공한다.
The present invention relates to an ascorbic acid derivative having a cell penetrating Tat peptide having a self-invasive signal, a method for preparing the same, and a cosmetic composition for skin whitening comprising the same. Since the ascorbic acid derivative shows relatively good solubility in both water and organic solvents and has high stability and excellent skin permeability, it provides a skin whitening agent that has an excellent whitening effect and a sustaining effect thereof.
피부 미백, 아스코르브산, Tat 펩타이드, 자가침투신호Skin whitening, ascorbic acid, Tat peptide, self penetration signal
Description
본 발명은 펩타이드가 결합된 아스코르브산 유도체에 관한 것으로, 더욱 상세하게는 안정성과 우수한 피부침투성을 갖는 아스코르브산 유도체 및 이를 포함하는 피부 미백용 화장료 조성물에 관한 것이다.The present invention relates to an ascorbic acid derivative to which a peptide is bound, and more particularly, to an ascorbic acid derivative having stability and excellent skin permeability and a cosmetic composition for skin whitening comprising the same.
사람의 피부색은 피부내 멜라닌(melanin)의 농도와 분포에 따라 유전적으로 결정되며, 태양 자외선이나 피로, 스트레스와 같은 환경적 또는 생리적 조건에 의해서도 영향을 받는다. 생체 내 멜라닌의 제조는 색소 세포 내에 존재하는 타이로시나제 (Tyrosinase) 라는 효소의 작용에 의해 타이로신(Tyrosine)으로부터 도파(DOPA), 도파퀴논(Dopaquinone)으로 변환된 후, 비효소적인 산화반응을 통해서 이루어진다. 이와 같이, 멜라닌이 합성되는 경로가 알려져 있으나, 아직까지 타이로시나제가 작용하는 단계의 이전 단계인 멜라닌 합성의 유도 메커니즘에 대해서는 자세히 밝혀져 있지 않다. 멜라닌은 피부에 분포함으로써 신체를 보호하는 중요한 기능을 갖고 있으나, 멜라닌의 과잉생산은 피부암(Melanoma)과 밀접한 관계가 있고 피부 흑화를 유발하며 기미, 주근깨 등을 생성하는 것으로 알려져 있다. 최근에는 희고 고운 피부를 갖고자 하는 사람들의 소망에 부응하여, 멜라닌의 과잉생산 예방을 목적으로 하는 화장품과 약제의 개발이 활발히 이루어지고 있다. 이와 같은 멜라닌의 과잉생산 예방용 물질로서 종래에는 하이드로퀴논(Hydroquinone) 이 주로 사용되었다. 그러나, 이 화합물은 색소의 변성 또는 치사를 일으키고 세포의 기능을 손상시키는 등의 부작용을 나타냄에 따라, 현재 한국, 일본 등에서는 화장품으로 사용하는 것이 금지되어 있는 실정이다(참조: J. Soc. Cosmet. Chem., 42, 361(1991)). 또한, 하이드로퀴논의 당 유도체인 알부틴(Arbutin)이 상품화된 바 있으나 효과가 미약하고, 아스코르브산(Ascorbic Acid) 및 코직산 (Kojic Acid) 등은 제품 안정성이 좋지 못하여 사용이 제한되는 단점이 있다. Human skin color is genetically determined by the concentration and distribution of melanin in the skin and is also influenced by environmental or physiological conditions such as solar ultraviolet light, fatigue and stress. The production of melanin in vivo is converted from tyrosine to dopa (DOPA) and dopaquinone by the action of an enzyme called tyrosinase present in pigment cells, and then undergoes a non-enzymatic oxidation reaction. It is done through As such, the pathway by which melanin is synthesized is known, but the mechanism of induction of melanin synthesis, which is a step prior to the action of tyrosinase, has not been elucidated. Melanin has an important function of protecting the body by distributing it to the skin, but the overproduction of melanin is known to be closely related to skin cancer (Melanoma), cause skin blackening, and produce spots and freckles. In recent years, in response to the desire of those who want to have white and fair skin, the development of cosmetics and pharmaceuticals for the purpose of preventing overproduction of melanin has been actively made. As a material for preventing overproduction of melanin, hydroquinone has been mainly used. However, this compound has side effects such as degeneration or lethality of pigment and impaired cell function. Currently, the compound is prohibited from being used in cosmetics in Korea and Japan (see J. Soc. Cosmet). Chem., 42, 361 (1991). In addition, although arbutin, a sugar derivative of hydroquinone, has been commercialized, its effect is weak, and ascorbic acid and kojic acid have disadvantages of limited product stability and limited use.
아스코르브산은 수용성 항 산화제로 미백 효과, 프롤린하이드록실라제(Prolinehydroxylase)의 코펙터(Cofactor)로 콜라겐의 합성 증진효과가 있다[Quaglino, D. Jr., et. al., J. Biol. Chem. 1997, 272, 345]. 그러나 아스코르브산은 불안정하여 장시간의 사용이 요구되는 제품에는 사용하기 어렵기 때문에, 화학적 불안정성의 주요 원인인 아스코르브산의 2번 혹은 3번 위치를 화학적으로 변화시킨 유도체의 개발에 대한 많은 연구가 알려져 있다(한국특허공고공보 94-73 및 94-6115). 현재까지 상용화된 아스코르브산의 유도체로는 아스코르브산 6-팔미테이트, 2,6-디팔미테이트, 6-스테아레이트, 그리고 L-아스코르브산-2-인산에스테르 마그네슘염등의 화합물이 항산화 효과 및 미백 효과를 가진 화 장품의 원료로써 사용되고 있다(한국특허공고공보 제 91-8733호, 미국특허 제 4,179,445). 그러나 이러한 유도체들은 화학적으로 안정화하더라도 생체 내에서 분해 속도가 빨라서 지속적인 항 산화력을 유지 못하는 단점이 있다. Ascorbic acid is a water-soluble antioxidant and has a whitening effect and a cofactor of prolinehydroxylase to enhance collagen synthesis [Quaglino, D. Jr., et. al., J. Biol. Chem. 1997, 272, 345. However, ascorbic acid is unstable and difficult to use in products requiring long-term use, so much research is known on the development of derivatives that chemically change the 2nd or 3rd position of ascorbic acid, which is the main cause of chemical instability. Korean Patent Publication Nos. 94-73 and 94-6115). Commercially available derivatives of ascorbic acid include ascorbic acid 6-palmitate, 2,6-dipalmitate, 6-stearate, and L-ascorbic acid-2-phosphate ester magnesium salts. It is used as a raw material for cosmetics having an effect (Korean Patent Publication No. 91-8733, US Patent No. 4,179,445). However, even though these derivatives are chemically stabilized, they have a disadvantage in that they do not maintain sustained antioxidant capacity due to their rapid degradation rate in vivo.
그러나 상기 언급된 아스코르브산 에스테르는 수용성으로 인해 피부 흡수력이 약하므로, 타이로시나제의 활성억제, 항 산화 효과 및 콜라겐합성효과를 크게 기대하기 어렵다. 또한, 이 화합물은 생체 내에서 빠른 효소분해로 인한 안정성 문제가 대두되고 있다. 그러므로 기존의 아스코르브산 유도체의 단점인 피부 자극성이 다소 극복되었다 하더라도 이것만으로는 부족하며, 외피로부터의 흡수력이 증가되어 생체 내에서의 미백기능이 극대화되고, 안정성이 향상된 새로운 피부 미백제의 개발이 시급하다고 하겠다.However, the above-mentioned ascorbic acid ester is weak in skin absorption due to water solubility, so it is difficult to expect the inhibitory activity, antioxidant effect and collagen synthesis effect of tyrosinase. In addition, this compound has raised stability problems due to rapid enzymatic degradation in vivo. Therefore, even if the skin irritation, which is a disadvantage of the conventional ascorbic acid derivative, has been overcome somewhat, this is not enough, and the absorption from the skin is increased, thereby maximizing the whitening function in the living body, and it is urgent to develop a new skin whitening agent with improved stability. .
또한, 대한민국 출원특허 제98-10752호에서 폴리에틸렌글리콜 유도체의 계면활성능력을 이용하여 아스코르브산에 축합 반응시킨 폴리에톡실화 아스코르브산 유도체도 피부 미백제로서 피부 흡수력 및 안정성을 높이기 위한 하나의 방법으로 제시하고 있으나 아직 만족할 만한 수준은 아니었다. In addition, in Korean Patent Application No. 98-10752, a polyethoxylated ascorbic acid derivative condensed with ascorbic acid using the surface active ability of a polyethylene glycol derivative is also presented as a method for enhancing skin absorption and stability as a skin whitening agent. It was not yet satisfactory.
따라서, 피부에 자극이 없고, 외피로부터의 흡수력이 증가되고, 안정성이 향상되어, 생체 내에서의 미백기능이 극대화된 새로운 피부 미백제의 개발이 시급하다고 하겠다. Therefore, it is urgent to develop a new skin whitening agent that has no irritation to the skin, the absorption from the skin is increased, the stability is improved, and the whitening function is maximized in vivo.
상기 종래 기술의 문제점을 해결하고자, 비자극성이고, 외피와 내피에 쉽고 안전하게 침투하면서도 피부질환의 유발문제 없고, 우수한 타이로시나제 억제능, 자유 라디칼 소거능, 및 항 산화기능을 지속적으로 갖는, 세포 침투성 Tat 펩타이드(Transactivator of transcription peptide)가 결합된 가 결합된 아스코르브산 유도체를 제공하는 것이다. In order to solve the problems of the prior art, it is non-irritating, easily and safely penetrates the outer skin and the endothelium, without causing problems of skin diseases, and has excellent tyrosinase inhibitory activity, free radical scavenging activity, and antioxidant function, and cell permeability. Tat peptide (Transactivator of transcription peptide) is to provide a combined ascorbic acid derivative is bound.
본 발명의 또 다른 목적은 상기 세포 침투성 Tat 펩타이드가 결합된 아스코르브산 유도체의 제조방법을 제공하는 것이다.Still another object of the present invention is to provide a method for preparing an ascorbic acid derivative to which the cell penetrating Tat peptide is bound.
본 발명의 또 다른 목적은 세포 침투성, 안정성, 및 비자극성이고, 미백효과 및 그 지속성이 우수한, 상기 세포 침투성 Tat 펩타이드가 결합된 아스코르브산 유도체를 유효성분으로 포함하는 피부 미백용 화장료 조성물을 제공하는 것이다.Still another object of the present invention is to provide a cosmetic composition for skin whitening, comprising ascorbic acid derivatives having the cell permeability, stability, and non-irritancy, and excellent in whitening effect and its persistence, the ascorbic acid derivatives bound to the cell permeable Tat peptide. will be.
본 발명은 펩타이드가 결합된 아스코르브산 유도체, 더욱 자세하게는 자가침투신호(self penetration)를 갖는 인간 면역 결핍 바이러스 단백질의 일종인 Tat 펩타이드에서 유래된 세포 침투성 Tat 펩타이드가 결합된 아스코르브산 유도체, 및 이를 포함하는 피부 미백용 화장료 조성물에 관한 것이다. The present invention includes an ascorbic acid derivative in which a peptide is bound, more specifically, an ascorbic acid derivative in which a cell penetrating Tat peptide derived from a Tat peptide, which is a kind of human immunodeficiency virus protein having a self penetration signal, and a combination thereof. It relates to a cosmetic composition for skin whitening.
본 발명자는 세포 침투성 Tat 펩타이드를 아스코르브산 또는 그 유도체의 2번 혹은 3번 위치에 도입시켜, 신규한 아스코르브산 유도체를 제공한다. 본 발명의 아스코르브산 유도체는 기존의 아스코르브산 유도체들에 비해, Tat 단백질이 가지는 특징으로 인해 물과 유기 용매에 모두 비교적 좋은 용해도를 보이며, 안정성이 우수하고, 피부 흡수가 좋고, 지속적인 항산화 그리고 타이로시나제의 활성 억제제로 좋은 효과를 나타낸다. 따라서 본 발명의 세포 침투성 Tat 펩타이드는 우수한 피부미백제로 사용될 수 있다. The present inventors introduce a cell-permeable Tat peptide at positions 2 or 3 of ascorbic acid or a derivative thereof to provide a novel ascorbic acid derivative. Ascorbic acid derivatives of the present invention have a relatively good solubility in both water and organic solvents due to the characteristics of the Tat protein, as compared to conventional ascorbic acid derivatives, and have excellent stability, good skin absorption, continuous antioxidant and tyro It has a good effect as an activity inhibitor of cinases. Therefore, the cell penetrating Tat peptide of the present invention can be used as an excellent skin lightening agent.
이하에서 본 발명을 더욱 자세히 설명하고자 한다.Hereinafter, the present invention will be described in more detail.
본 발명은 하기 화학식 1a 또는 화학식 1b을 갖는 세포 침투성 Tat 단백질이 결합된 아스코르브산 유도체를 제공한다. The present invention provides an ascorbic acid derivative to which a cell penetrating Tat protein having the formula 1a or 1b is bound.
상기식에서, Tat는 세포침투성 Tat 펩타이드로서, 상기 R은 글루타민, 라이신, 알지닌, 및 글리신의 곁사슬(side chain)로 이루어진 군에서 1종 이상 선택되며, n은 4 내지 12 범위의 정수이다.Wherein Tat is a cell penetrating Tat peptide, wherein R is at least one selected from the group consisting of side chains of glutamine, lysine, arginine, and glycine, and n is an integer ranging from 4 to 12.
본 발명의 하기 화학식 2a 또는 화학식 2b을 갖는 세포침투성 Tat 단백질이 결합된 아스코르브산 유도체를 제공한다. The present invention provides an ascorbic acid derivative having a cell penetrating Tat protein having the formula (2a) or (2b).
상기식에서, []Tat는 세포침투성 Tat 펩타이드로서, 상기 R은 글루타민, 라이신, 알지닌, 및 글리신의 곁사슬(side chain)로 이루어진 군에서 1종 이상 선택되며, n은 4 내지 12 범위의 정수이고, p는 2 내지 100의 정수이다. Wherein [] Tat is a cell penetrating Tat peptide, wherein R is at least one selected from the group consisting of side chains of glutamine, lysine, arginine, and glycine, and n is an integer ranging from 4 to 12 , p is an integer from 2 to 100.
최근 다양한 인체의 질환이 세포 단백질의 비정상적인 활성에 기인한다는 사실에 따라 이들 단백질의 활성을 조절함으로써 치명적인 인체질환을 치료할 수 있는 물질의 개발이 전 세계적으로 관심의 대상이 되고 있다. 그러나 펩타이드와 단백질은 다른 화합물에 비하여 생리작용에 대한 선택성과 효용성이 매우 우수함에도 불구하고, 세포내부로 직접 전달될 수 없는 단점 때문에 효과적인 약물 전달 수단으로서의 실용화가 현실적으로 어려운 실정이었다. Recently, according to the fact that various human diseases are caused by abnormal activity of cellular proteins, the development of substances capable of treating fatal human diseases by regulating the activity of these proteins has been of global interest. However, although peptides and proteins have excellent selectivity and utility for physiological action compared to other compounds, practical use as effective drug delivery means has been difficult due to the disadvantage that they cannot be delivered directly into cells.
상기와 같은 문제를 해결하기 위하여, 중요한 생리활성을 갖는 다양한 생체 기능 단백질을 세포내로 효율적으로 침투시키는 기술(protein penetration technology)을 이용함으로써 인체 질환 치료에 필요한 물질을 직접적이고 효율적으로 전달 또는 흡수시킬 수 있게 되었다. 상기 단백질 침투기술은 자가침투신호(penetration)를 갖는 인간 면역결핍 바이러스(HIV: Human Immunodeficiency Virus type-1) 단백질의 일종인 Tat 펩타이드가 자발적으로 세포막을 통과하여 쉽게 세포내로 침투 및 이동하는 것을 이용한 것임이 밝혀졌다. 이러한 기능은 Tat 펩타이드 시퀀스의 중간 부위인 단백질 형질도입 부위(protein transduction domain)의 특성 때문에 나타나며, 아직 그 정확한 메카니즘은 알려지지 않은 상태이다(Frankel, A.D. and Pabo, C.O.(1988) Cell 55, 1189-1193. Green, M. and Loewenstein, P.M.(1988) Cell 55, 1179-1188. Ma, M. and Nath, A.(1997) J. Virol. 71, 2495-2499. Vives, E., Brodin, P. and Lebleu, B.(1997) J. Biol. Chem. 272, 16010-16017.). In order to solve the above problems, it is possible to directly or efficiently deliver or absorb the substances necessary for the treatment of human diseases by using a protein penetration technology that efficiently penetrates various biofunctional proteins having important physiological activities into cells. It became. The protein penetration technology uses Tat peptide, a type of Human Immunodeficiency Virus type-1 (HIV) protein, having a self-penetration signal, spontaneously penetrates and migrates through the cell membrane. Turned out. This function is due to the nature of the protein transduction domain, which is the intermediate region of the Tat peptide sequence, and its exact mechanism is unknown (Frankel, AD and Pabo, CO (1988) Cell 55, 1189-1193). Green, M. and Loewenstein, PM (1988) Cell 55, 1179-1188. Ma, M. and Nath, A. (1997) J. Virol. 71, 2495-2499. Vives, E., Brodin, P. and Lebleu, B. (1997) J. Biol. Chem. 272, 16010-16017.).
본 발명자들은 상기와 같은 Tat 펩타이드의 침투 특성에 주시하여 연구한 결과, 아스코르브산 또는 레틴아미드와 같은 원하는 피부 활성 성분을 자가침투신호를 갖는 Tat 펩타이드에 부착하여 이들을 피부내 세포로 직접적이고 효율적으로 침투시키는 것을 완성하여 이를 토대로 본 발명을 완성하게 되었다.The present inventors have studied and studied the penetration characteristics of the Tat peptide as described above. As a result, the desired skin active ingredient such as ascorbic acid or retinamide is attached to the Tat peptide having a self-invasive signal to infiltrate them directly into the cells of the skin. The present invention has been completed based on this.
본 명세서에서 사용된 “세포침투성 Tat 펩타이드”는 그 자체 또는 이와 결합된 물질과 함께 세포침투능을 갖는, Tat 펩타이드 또는 그로부터 유래된 펩타이드를 의미한다. 구체적으로 HIV-1의 Tat 펩타이드의 주요 특징은 단백질의 벡터중 특정 구간의 시퀀스가 세포막(barrier)를 여는 신호를 갖고 있다는 것이며, 서열은 RKKRRQRRR로서 다음 화학식 4에 나타낸다. As used herein, “cell penetrating Tat peptide” refers to a Tat peptide or a peptide derived therefrom that has cell penetrating ability with itself or with a substance bound thereto. Specifically, the main characteristic of the Tat peptide of HIV-1 is that a sequence of a specific section of the vector of the protein has a signal that opens a cell membrane, and the sequence is represented by the following formula (4) as RKKRRQRRR.
세포침투성 Tat 펩타이드의 구성 아미노산은 20 개의 아미노산 중에 라이신(이하, Lys 또는 K이라 함), 알지닌(이하, Arg 또는 R이라 함), 글루타민(이하, Gln 또는 Q이라 함) 등은 모두 구조적으로 아민기(amine group)와 카르복실기(carboxyl group)를 가지고 있기 때문에, 알코올과의 반응을 통하여 카르복시기와 에스테르화반응으로 아스코르브산과 결합시킬 수 있다. 세포침투성9-Tat 펩타이드(n = 9), 예를 들면, Arg-Lys-Lys-Arg-Arg-Gln-Arg-Arg-Arg 또는 RKKRRQRRR로 표시되는 9-Tat 펩타이드, Lys-Lys-Lys-Lys-Lys-Lys-Lys-Lys-Lys 또는 KKKKKKKKK로 표시되는 9-Tat 펩타이드, Arg-Arg-Arg-Arg-Arg-Arg-Arg-Arg-Arg 또는 RRRRRRRRR로 표시되는 9-Tat 펩타이드 등은 아스코르브산과 에스테르화 반응으로 결합될 수 있다. Among the 20 amino acids, the constituent amino acids of the cell penetrating Tat peptide are lysine (hereinafter referred to as Lys or K), arginine (hereinafter referred to as Arg or R), and glutamine (hereinafter referred to as Gln or Q). Since it has an amine group and a carboxyl group, it can be combined with ascorbic acid by esterification with a carboxyl group through reaction with an alcohol. Cell penetrating 9-Tat peptide (n = 9), for example 9-Tat peptide, Lys-Lys-Lys-Lys, represented by Arg-Lys-Lys-Arg-Arg-Gln-Arg-Arg-Arg or RKKRRQRRR 9-Tat peptides represented by -Lys-Lys-Lys-Lys-Lys or KKKKKKKKK, 9-Tat peptides represented by Arg-Arg-Arg-Arg-Arg-Arg-Arg-Arg-Arg or RRRRRRRRR, and the like. Can be combined in an esterification reaction.
상기 세포침투성 Tat 펩타이드는 다음의 화학식 3a를 가지며, The cell penetrating Tat peptide has the following formula 3a,
글루타민(glutamine : Gln), 라이신(lysine : Lys), 알지닌(arginine : Arg) 및 글리신 (glycine : Gly)으로 이루어지는 군으로부터 선택된 1 종 이상의 아미노산을 치환기를 포함하며, 4개 아미노산 내지 12개 아미노산으로 이루어지는 펩타이드이며, 더욱 바람직하게는 라이신 및 알지닌으로 이루어진 군에서 선택된 1종 이상의 아미노산으로 이루어진 펩타이드이다. 또한 상기 화학식 1중에서 n은 4 내지 12인 정수이며, 더욱 바람직하게는 8 내지 10이며, 가장 바람직하게는 n이 9인 것이다. One or more amino acids selected from the group consisting of glutamine (Gln), lysine (lysine: Lys), arginine (arginine: Arg), and glycine (glycine: Gly); It is a peptide consisting of, more preferably a peptide consisting of one or more amino acids selected from the group consisting of lysine and arginine. In Formula 1, n is an integer of 4 to 12, more preferably 8 to 10, most preferably n is 9.
본 명세서에서 사용된 “아스코르브산 유도체”라 함은 피부 미백제로 알려진 아스코르브산 유도체이면 모두 가능하다. 예컨대, 상기 아스코르브산의 유도체로는 아스코르브산 6-팔미테이트, 아스코르브산 2,6-디팔미테이트, 아스코르브산 6-스테아레이트, 그리고 L-아스코브르산-2-인산에스테르 마그네슘염(한국특허공고공보 제 91-8733호, 미국 특허 제 4,179,445호), 폴리에톡실레이티드 아스코르브산 등을 포함하나 이에 한정되지는 않는다. The term "ascorbic acid derivative" as used herein may be any ascorbic acid derivative known as a skin lightening agent. For example, ascorbic acid derivatives include ascorbic acid 6-palmitate, ascorbic acid 2,6-dipalmitate, ascorbic acid 6-stearate, and L-ascorbic acid-2-phosphate ester magnesium salt (Korean Patent Publication). Publication 91-8733, US Pat. No. 4,179,445), polyethoxylated ascorbic acid, and the like.
본 발명은 세포침투성 Tat 펩타이드를 제조 및 정제하고, 유기용매, 촉매 및 축합제의 존재하에 아스코르브산 또는 그 유도체에 상기 Tat 펩타이드를 에스테르반응으로 결합시켜, 세포침투성 Tat 펩타이드가 결합된 아스코르브산 유도체 를 제조하는 방법을 제공한다. 또한, 상기 세포침투성 Tat 펩타이드의 C-말단에 할로겐화물로 활성화시킨 후에 아스코르브산 또는 그 유도체와 에스테르 반응을 수행하는 것이 바람직하다.The present invention provides an ascorbic acid derivative in which a cell penetrating Tat peptide is bound by preparing and purifying a cell penetrating Tat peptide and binding the Tat peptide to an ascorbic acid or a derivative thereof in the presence of an organic solvent, a catalyst and a condensation agent. It provides a method of manufacturing. In addition, it is preferable to perform an ester reaction with ascorbic acid or a derivative thereof after activating a halide at the C-terminus of the cell penetrating Tat peptide.
본 발명의 일례에서, 아스코르브산 세슘염을 출발물질로 하여 Tat 펩타이드-할라이드(예, 염소, 브롬, 또는 요오드 등)을 반응시켜 화학식 1a의 화합물을 제조하는 방법을 제공한다. 또 다른 본 발명의 일례에서, 아스코르브산 세슘염을 이용하여 3-벤질 아스코르브산을 합성하고, 염기하에서 용매중 Tat-펩타이드-할라이드와 반응시켜 3-벤질-2-Tat 펩타이드-아스코르브산을 제조하고, 수소화반응을 수행하여 화학식 1b의 화합물을 제조하는 방법을 제공한다. In one embodiment of the present invention, a method of preparing a compound of Formula 1a is prepared by reacting a Tat peptide-halide (eg, chlorine, bromine, iodine or the like) with cesium ascorbate salt as a starting material. In another example of the present invention, 3-benzyl ascorbic acid is synthesized using cesium ascorbic acid salt, and reacted with Tat-peptide-halide in a solvent under a base to prepare 3-benzyl-2-Tat peptide-ascorbic acid. It provides a method for preparing a compound of formula 1b by performing a hydrogenation reaction.
본 발명의 일례에서, 유기용매, 촉매 및 축합제의 존재하에서 세포 침투성 Tat 펩타이드와 아스코르브산 유도체를 반응시키기 전에, 아스코르브산 유도체의 말단 작용기를 알콜로 바꾼 후, 아스코르브산 유도체를 제조하는 방법을 제공한다. In one example of the present invention, before reacting a cell-permeable Tat peptide and an ascorbic acid derivative in the presence of an organic solvent, a catalyst and a condensing agent, a method of preparing an ascorbic acid derivative after changing the terminal functional group of the ascorbic acid derivative to alcohol do.
더욱 자세하게는, 본 발명의 세포침투성 Tat 펩타이드가 결합된 아스코르브산 유도체의 제조는 Tat 펩타이드를 제조하는 단계와 상기 Tat 펩타이드와 아스코르브산 또는 그 유도체와 결합하는 단계로 구분할 수 있다. More specifically, the preparation of ascorbic acid derivatives bound to the cell penetrating Tat peptide of the present invention may be divided into a step of preparing a Tat peptide and a step of binding the Tat peptide with ascorbic acid or a derivative thereof.
세포침투성 Tat 펩타이드의 제조Preparation of Cell Penetrating Tat Peptides
첫째는 재조합 발현벡터를 이용하는 생물학적 방법으로서, a)pSVC21의 HIV-1 Tat 유전자로부터 자가침투신호 영역 유전자를 6 개의 히스티딘 택(6 His tag)을 갖고 있는 pET 벡터에 PCR 기법을 이용하여 HIV-1의 Tat 유전자를 클로닝(cloning) 하여 융합 단백질의 형태로 발현시킬 수 있는 벡터를 제조하는 단계, 그리고 b) 상기 pET-Tat 발현 벡터를 이용하여 대장균 내에서의 재조합 His-Tat 펩타이드의 형태로 대량 발현시킨 후, 순수 분리 정제하는 단계를 포함하는 Tat 펩타이드의 제조방법이다.First, a biological method using a recombinant expression vector, a) HIV-1 Tp gene from pSVC21 to the pET vector containing 6 histidine tags (6 His tags) from the HIV-1 Tat gene by using the PCR technique Cloning the Tat gene of (c) to prepare a vector that can be expressed in the form of a fusion protein, and b) using the pET-Tat expression vector in mass expression in the form of recombinant His-Tat peptide in E. coli After the purification, the method for preparing a Tat peptide comprising pure separation and purification.
두 번째 방법은, a) pET-Tat 발현 벡터에 목표 단백질(target protein), 예컨대 SOD 또는 GST을 삽입하여 His-Tat-목표단백질의 융합단백질로 대량 발현하고 순수분리 정제하는 단계를 포함하는 Tat 펩타이드의 제조방법이다.The second method comprises the steps of: a) inserting a target protein, such as SOD or GST, into a pET-Tat expression vector, expressing the mass as a fusion protein of His-Tat-target protein and purifying and purifying the purified protein. It is a manufacturing method of.
셋째 방법은, 펩타이드 합성용 유기합성기기를 이용하여 순수한 His-Tat 펩타이드를 합성하는 이 Tat 펩타이드를 제조하는 데는 Merrifield의 고체상(Solid Phase)펩타이드 합성법으로 (J. Am. Chem.Soc. 85, 2149-2154(1963)) ABI사의 펩타이드 합성기를 (Model 431A)를 이용하여 합성하였다. 고체상 합성은 적당한 레진에 α-아미노기가 보호된 아미노산을 카르복실 말단의 아미노산부터 커플링 시킴으로서 시작된다. 이때 α-아미노기가 보호된 아미노산을 히드록시 메틸 레진이나 클로로메틸레이티드 레진에 에스테르 결합으로 붙인다. α-아미노기를 보호하는 그룹으로는 Fmoc(9-fluorenyl methoxycarbonyl)이 Boc(t-butyloxycarbonyl)을 사용하고 Fmoc으로 보호된 아미노산은 ABI나 Pharmacia사 혹은 Calbiochem사를 통해 구입하였다. 아미노산 잔기가 반응성이 있는 아미노산은, 예컨대 Arg잔기는 트리틸(Trt), 4-메톡시-2,3,트리메틸벤젠 술포닐(Mrt), 터르-부틸(t-Bu)과 같은 적당한 그룹으로 보호된 Fmoc-아미노산을 이용하였다. 펩타이드 합성은 고체지지체(Solid Support) 레진에 붙어있는 펩타이드 체인의 아미노 말단에 순차적으로 α-아미노기가 보호된 아미노산을 활성화시킨 후 커플링시키며, 합성이 끝나면 레진으로부터 펩타이드를 절단하고, 보호그룹은 트리플로오로아세트산(TPA)과 같은 시약으로 제거한다. 펩타이드는 TFA 용액으로부터 여과나 원심분리 또는 디에틸에테르로 추출하여 분리하며 고성능 액체크로마토그라피(HPLC)나 다른 방법으로 정제한다. The third method is Merrifield's solid phase peptide synthesis method for synthesizing a pure His-Tat peptide using an organic synthesizer for peptide synthesis (J. Am. Chem. Soc. 85, 2149). -2154 (1963)) ABI's peptide synthesizer was synthesized using (Model 431A). Solid phase synthesis begins by coupling an amino acid having an α-amino group protected to a suitable resin starting from the amino acid at the carboxyl end. At this time, the amino acid in which the α-amino group is protected is attached to hydroxy methyl resin or chloromethylated resin as an ester bond. Fmoc (9-fluorenyl methoxycarbonyl) used Boc (t-butyloxycarbonyl) as a group protecting the α-amino group, and amino acids protected with Fmoc were purchased through ABI, Pharmacia or Calbiochem. Amino acids whose amino acid residues are reactive, for example, Arg residues are protected by suitable groups such as trityl (Trt), 4-methoxy-2,3, trimethylbenzene sulfonyl (Mrt), tert-butyl (t-Bu) Fmoc-amino acid was used. Peptide synthesis activates and couples the amino acid protected by the α-amino group sequentially to the amino terminus of the peptide chain attached to the solid support resin, and after synthesis, the peptide is cleaved from the resin, and the protecting group is triple Remove with a reagent such as uroacetic acid (TPA). Peptides are separated from the TFA solution by filtration, centrifugation or diethyl ether and separated and purified by high performance liquid chromatography (HPLC) or other methods.
세포침투성 Tat 펩타이드와 아스코르브산 또는 그 유도체의 결합Binding of Cell Invasive Tat Peptides to Ascorbic Acid or Derivatives thereof
아스코르브산 또는 그 유도체의 2’또는 3’위치에 치환기를 도입하는 방법은 아스코르브산의 5,6 위치에 아세탈 혹은 케탈을 합성하고 이를 염기의 존재 하에 3번 위치에 치환체를 도입한 뒤 아세탈 혹은 케탈을 제거하여 최종 화합물을 얻는다. 그러나, 본 발명의 제조방법에서는, 5 및 6번 탄소위치에 있는 히드록시기를 보호기, 예컨대 아세탈 또는 켈탈로 변환시키지 않고, 유기용매, 촉매 및 축합제의 존재하에서, 화학식 (5a)의 아스코르브산의 세슘염과 다음의 화학식(3b)의 할라이드기가 결합된 Tat 펩타이드 유도체(Tat-X)를 반응시켜서 얻는다(반응식 1) The method of introducing a substituent at the 2 'or 3' position of ascorbic acid or a derivative thereof synthesizes acetal or ketal at positions 5 and 6 of ascorbic acid and introduces a substituent at position 3 in the presence of a base, followed by acetal or ketal To remove the final compound. However, in the production method of the present invention, cesium of ascorbic acid of formula (5a) in the presence of an organic solvent, a catalyst and a condensing agent, without converting the hydroxy groups at carbon positions 5 and 6 into a protecting group such as acetal or ketal Obtained by reacting a salt with a Tat peptide derivative (Tat-X) having a halide group represented by the following formula (3b) (Scheme 1)
그러므로 본 발명은 아스코르브산의 5번 탄소 및 6번 탄소 위치에 있는 히드록시기를 보호기로 보호하지 않고 최종 화합물을 제조함으로써 높은 수율과 반응 단계를 단축시킨 것을 특징으로 한다. Therefore, the present invention is characterized in that the high yield and reaction step are shortened by preparing the final compound without protecting the hydroxyl groups at the 5th and 6th carbon positions of ascorbic acid with protecting groups.
상기 에스테르 반응은 무수조건에서 진행된다. 아스코르브산 및 그 유도체는 물에 녹지 않으므로 무수 유기 용매, 예를 들어 디메틸술폭시드(DMSO), 디메틸포름아미드(DMF), 헥사메틸포스포르아미드(HMPA),N-메틸피롤리돈, 피롤리돈, 디메틸아세트아미드(DMAC), 1,3-디메틸-3,4,5,6-테트라히드로-2(1H) 리미디논(DMPU)을 단독 혹은 혼합하여 사용할 수 있다. 에스테르화 반응은 무촉매의 경우도 반응이 진행되 지만, 촉매로는 공지의 루이스산을 사용하거나 할로겐화물, 예를 들어 티오닐클로라이드(thionyl chloride)를 이용하여 카르복실산(carbonyl acid)을 활성화시킨 후 알코올과의 반응을 유도하거나, 메탄슬폰산(methanesulfonic acid) 등을 촉매로 사용하여 에스테르화 반응으로 본 발명의 화합물을 제조한다. 또한 에스테르화 반응은 가역 반응이므로 반응을 정방향으로 보내기 위해서는 통상 아스코르브산 및/또는 그 유도체를 10당량 이상을 사용하는 것이 바람직하다. 상기축합제는 N,N-카보닐디이미다졸(CDI), 또는 N,N-디사이클로헥실카보디이미드(DCC)를 사용하는 것이 바람직하고, 축합반응을 촉진시키는 촉매로는 N,N-디메틸아미노피리딘(DMAP), 또는 1-하이드록시벤조트리아졸[1-HOBT]을 사용하는 것이 바람직하다. The ester reaction proceeds under anhydrous conditions. Ascorbic acid and its derivatives are insoluble in water and therefore anhydrous organic solvents such as dimethyl sulfoxide (DMSO), dimethylformamide (DMF), hexamethylphosphoramide (HMPA), N-methylpyrrolidone, pyrrolidone , Dimethylacetamide (DMAC), 1,3-dimethyl-3,4,5,6-tetrahydro-2 (1H) limidinone (DMPU) can be used alone or in combination. The esterification reaction proceeds even in the case of a non-catalyst, but as a catalyst, a known Lewis acid is used or a halide such as thionyl chloride is used to activate the carboxylic acid. After the reaction to induce a reaction with alcohol, or methanesulfonic acid (methanesulfonic acid) and the like using a catalyst to prepare a compound of the present invention. In addition, since esterification reaction is a reversible reaction, it is preferable to use 10 equivalent or more of ascorbic acid and / or its derivative normally, in order to direct reaction. The condensing agent is preferably N, N-carbonyldiimidazole (CDI) or N, N-dicyclohexylcarbodiimide (DCC), and N, N-dimethyl as a catalyst for promoting the condensation reaction. Preference is given to using aminopyridine (DMAP) or 1-hydroxybenzotriazole [1-HOBT].
상기 화학식 3b에서, X는 할로겐족 치환기로서, 더욱 바람직하게는 염소이다. 상기 반응식1에서 사용된 화합물(5a)은 아스코르브산을 물을 용매로 하여 탄산수소세슘으로 처리한 후, 아이소프로필알콜을 부가하여 결정화하여 제조한다. In Formula 3b, X is a halogen substituent, more preferably chlorine. Compound (5a) used in Scheme 1 was prepared by treating ascorbic acid with cesium bicarbonate using water as a solvent, followed by crystallization by addition of isopropyl alcohol.
상기 반응식 2을 참조하면, 화합물 (1b)의 제조법은 아스코르브산 세슘염을 이용하여 3-벤질 아스코르브산을 합성하고 이를 탄산수소세슘으로 처리한 후 Tat-X와 반응하여 3-벤질-2-Tat-아스코르브산을 제조한 뒤, 이를 수소화 반응으로 화합물 (1b)를 제조하였다. Referring to Scheme 2, the method of preparing compound (1b) is synthesized with 3-benzyl ascorbic acid using cesium ascorbate salt and treated with cesium bicarbonate and then reacted with Tat-X to 3-benzyl-2-Tat -Ascorbic acid was prepared, and then this was hydrogenated to prepare compound (1b).
본 발명에서 사용될 수 있는 축합제, 촉매, 및 용매는 반응에 악영향을 끼치지 않는 범위 내에서 당 업계에 통상적으로 공지된 모든 것이 사용될 수 있다. Condensing agents, catalysts, and solvents that can be used in the present invention can be used as is commonly known in the art within the scope that does not adversely affect the reaction.
상기와 같은 방법으로 제조된 상기 화학식 1로 표시되는 Tat 펩타이드가 접합된 아스코르브산 유도체는 통상의 분리, 정제 방법, 예를 들어 재결정이나 칼럼 크로마토그래피법을 사용하여 정제할 수 있다. Ascorbic acid derivatives conjugated to the Tat peptide represented by Chemical Formula 1 prepared by the above method may be purified using conventional separation and purification methods, for example, recrystallization or column chromatography.
또한 아스코르브산 유도체, 예컨대 아스코르브산 6-팔미테이트, 아스코르브 산 2,6-디팔미테이트, 아스코르브산 6-스테아레이트, 그리고 L-아스코브르산-2-인산에스테르마그네슘염, 폴리에톡실레이티드 아스코르브산 등을 사용하여 제조하는 경우에는, 아스코르브산 유도체의 2번 탄소 또는 3번 탄소의 작용기를 알콜기로 바꾼 후 에스테르화 반응을 시키면 다음 화학식 2의 화합물 (2a)와 (2b)와 같은 Tat 펩타이드가 결합된 폴리에톡실레이트화 아스코르브산을 얻을 수 있다. Ascorbic acid derivatives such as ascorbic acid 6-palmitate, ascorbic acid 2,6-dipalmitate, ascorbic acid 6-stearate, and L-ascorbic acid-2-phosphate ester magnesium salt, polyethoxylated ascorbic acid In the case of using an acid or the like, when the functional group of carbon 2 or carbon 3 of the ascorbic acid derivative is changed to an alcohol group and subjected to an esterification reaction, a Tat peptide such as compounds (2a) and (2b) Combined polyethoxylated ascorbic acid can be obtained.
이와 같이 제조한 신규의 2 혹은 3 위치에, Tat 펩타이드가 결합된 아스코르브산 유도체는 화학적인 안정성과 타이로시나제 활성 저해제로 작용하며 지속적인 항산화력을 나타냄으로 새로운 미백제 및 항산화제로써 사용될 수 있다. 이에 대한 자세한 활성 평가는 실시 예에서 기술한다. Ascorbic acid derivatives in which the Tat peptide is bound to the new 2 or 3 positions prepared as described above act as chemical stability and tyrosinase activity inhibitors and exhibit continuous antioxidant power, and thus can be used as new whitening and antioxidant agents. Detailed activity evaluation for this is described in the Examples.
본 발명은 상기 화학식 1 및 2로 표시되는 화합물 중에서 선택된 1 종이상을 함유하는 피부 미백용 화장료 조성물을 제공한다. 본 발명에 따른 조성물은 화학식 1 및 2로 표시되는 화합물 중에서 선택된 1 종 이상의 물질을 0.001 내지 5%(w/w), 바람직하게는 0.01 내지 3%(w/w) 함유한다. 0.001% 미만의 농도에서는 실질적인 미백효과를 기대하기 어렵고, 5% 농도를 초과하는 경우에는 낮은 용해도로 인하여 제형 및 제품의 안정성에 좋지 않은 영향을 미칠 수 있기 때문이다.The present invention provides a cosmetic composition for skin whitening containing one paper selected from the compounds represented by Formulas 1 and 2. The composition according to the present invention contains 0.001 to 5% (w / w), preferably 0.01 to 3% (w / w) of at least one substance selected from the compounds represented by formulas (1) and (2). It is difficult to expect a substantial whitening effect at concentrations less than 0.001%, and if it exceeds 5% concentration, it may adversely affect the stability of the formulation and the product due to low solubility.
본 발명의 피부 미백용 조성물을 제조하는 경우에 용매로서 에탄올, 글리세린, 부틸렌글리콜, 프로필렌글리콜, 글리세레스-26, 메틸글루세스-20, 이소세틸미리스테이트, 이소세틸옥타노에이트, 옥틸도데실미리스테이트, 옥틸도데칸올, 이소스테아릴이소스테아레이트, 세틸옥타노에이트 및 네오펜틸글리콜디카프레이트 중에서 선택된 1 종 이상을 이용한다. 이러한 용매를 사용하여 본 발명의 조성물을 제 조하는 경우 화합물의 종류에 따라, 용매의 혼합비에 따라 용매에 대한 화합물의 용해도가 조금씩 다르나, 본 발명이 속하는 기술분야의 당업자라면 제품의 특성에 따라 용매의 종류 및 사용량을 알맞게 선택하여 적용할 수 있다. Ethanol, Glycerine, Butylene Glycol, Propylene Glycol, Glyceres-26, Methylgluses-20, Isocetyl Myristate, Isocetyl Octanoate, Octyldodecyl as a solvent when preparing the composition for skin whitening of the present invention One or more selected from myristate, octyldodecanol, isostearyl isostearate, cetyloctanoate and neopentyl glycol dicaprate are used. When the composition of the present invention is prepared using such a solvent, the solubility of the compound in the solvent is slightly different depending on the kind of the compound and the mixing ratio of the solvent, but those skilled in the art to which the present invention pertains depend on the characteristics of the product. The type and amount of use can be selected and applied appropriately.
본 발명에 따른 조성물은 피부외용연고나, 유연화장수, 영양화장수, 영양크림, 마사지크림, 에센스, 팩 등의 화장료 형태로 제조될 수 있다. 여기에서 피부외용연고는 화학식 1 또는 2의 화합물의 유효성분 이외에 바셀린 50.0 내지 97.0중량% 및 폴리옥시에틸렌올레일-에테르 포스페이트 0.1 내지 5.0중량%를 함유하도록 제조되며, 유연화장수는 프로필렌글리콜, 글리세린 등의다가알콜류 1.0 내지 10.0중량% 및 폴리에틸렌올레일에테르, 폴리옥시에틸렌 경화피마자유 등의 계면활성제 0.05 내지2.0중량%를 함유하도록 제조된다. 영양화장수 및 영양크림은 화학식 1 또는 2의 유효성분 이외에 스쿠알란, 바셀린, 옥틸도데칸올과 같은 오일류 5.0 내지 20.0중량% 및 세탄올, 스테아릴알콜, 밀납 등의 왁스성분 3.0 내지 15.0중량%을 함유하고,에센스는 글리세린, 프로필렌글리콜 등 다가알콜류 5.0 내지 30.0중량%를 함유한다. 마사지크림은 화학식 1 또는 2의 유효성분 이외에 유동파라핀, 바셀린, 이소노닐이소노나노에이트 등의 오일을 30.0 내지 70.0중량% 함유하여 제조되며, 팩은 폴리비닐알콜을 5.0 내지 20.0중량% 함유하는 필오프 팩 또는 일반 유화형 화장료에 카올린, 탈크, 산화아연, 이산화티탄 등의 안료가 5.0 내지 30.0 중량% 함유된 워시오프 팩으로 제조된다. The composition according to the present invention may be prepared in the form of cosmetics such as skin external ointment, softening longevity, nourishing cosmetics, nutrition cream, massage cream, essence, pack. Here, the external skin ointment is prepared to contain 50.0 to 97.0% by weight of petrolatum and 0.1 to 5.0% by weight of polyoxyethylene oleyl-ether phosphate in addition to the active ingredient of the compound of formula 1 or 2, and the softening lotion is propylene glycol, glycerin, etc. It is prepared to contain 1.0 to 10.0% by weight of polyalcohols and 0.05 to 2.0% by weight of surfactants such as polyethylene oleyl ether and polyoxyethylene hydrogenated castor oil. Nourishing cream and nourishing cream contains 5.0 to 20.0% by weight of oils such as squalane, petrolatum and octyldodecanol and 3.0 to 15.0% by weight of wax components such as cetanol, stearyl alcohol and beeswax in addition to the active ingredients of formula 1 or 2 The essence contains 5.0 to 30.0% by weight of polyhydric alcohols such as glycerin and propylene glycol. Massage cream is prepared by containing 30.0 to 70.0% by weight of oil, such as liquid paraffin, petrolatum, isononylisononanoate, etc. in addition to the active ingredient of Formula 1 or 2, the pack is a peel containing 5.0 to 20.0% by weight of polyvinyl alcohol It is prepared as a wash-off pack containing 5.0 to 30.0% by weight of pigments such as kaolin, talc, zinc oxide and titanium dioxide in the off-pack or general emulsified cosmetics.
한편, 또한 본 발명의 상기 화학식 1 또는 2로 표시되는 화합물을 함유하는 피부 미백 화장료 피부노화 방지용 화장료 조성물은 일반 피부 화장료에 배합되는 보통의 성분, 예를 들면 유분, 물, 계면활성제, 보습제, 저급알콜, 증점제, 킬레이트제, 색소, 방부제, 향료 등을 필요한 만큼 적용 배합하는 것이 가능하다. On the other hand, the skin whitening cosmetics containing the compound represented by the formula (1) or (2) of the present invention is a cosmetic composition for preventing skin aging is common components, such as oil, water, surfactants, moisturizers, lower grades that are formulated in general skin cosmetics It is possible to apply and mix alcohols, thickeners, chelating agents, colorants, preservatives, fragrances and the like as necessary.
본 발명의 화합물의 생리활성을 확인하기 위하여 실시된 경피 흡수 실험, 피부 자극성 실험, 타이로시나제 저해활성 및 라디칼소거 실험을 수행한 결과, 본발명의 화합물은 아스코르브산의 9.7배 및 3-O-에틸아스코르브산의 2.7배의 경피 흡수력을 가지며, 타이로시나제 활성억제능, 자유 라디칼 소거능 등의 실험에서 우수한 효과가 있음이 입증되었다. 따라서, 본 발명의 화합물은 그의 활성도와 비자극성, 피부 흡수력이 뛰어난 미백효과 지속성에 기인하여 화장품의 크림, 로션, 겔 등의 모든 형태로 첨가하여 사용할 수 있다. As a result of performing transdermal absorption experiment, skin irritation experiment, tyrosinase inhibitory activity and radical scavenging experiment conducted to confirm the physiological activity of the compound of the present invention, the compound of the present invention is 9.7 times and 3-O of ascorbic acid. It has a transdermal absorption of 2.7 times that of ethyl ascorbic acid, and proved to be excellent in experiments such as tyrosinase activity inhibitory activity and free radical scavenging activity. Therefore, the compound of the present invention can be added and used in all forms such as creams, lotions, gels, etc. of cosmetics due to its activity, non-irritability, and excellent skin absorption and long lasting effect.
이하, 본 발명의 이해를 돕기 위하여 바람직한 실시예를 제시한다. 그러나 다음의 실시예는 본 발명을 보다 쉽게 이해하기 위하여 제공되는 것일 뿐 본 발명이 다음의 실시예에 한정되는 것은 아니다.Hereinafter, preferred examples are provided to aid in understanding the present invention. However, the following examples are merely provided to more easily understand the present invention, and the present invention is not limited to the following examples.
[실시예]EXAMPLE
실시예 1 : 유전적 재조합 기법에 의한 9-Tat 펩타이드(R = KKKKKKKKK)의 제조Example 1 Preparation of 9-Tat Peptides (R = KKKKKKKKK) by Genetic Recombination Technique
(1) 9-Tat 융합 단백질(KKKKKKKKK : 올리고라이신) 발현 벡터 제조(1) Preparation of 9-Tat fusion protein (KKKKKKKKK: oligolysine) expression vector
먼저, 세포 내로 침투 기능을 갖는 목표 펩타이드를 전달할 수 있는 융합 펩타이드 발현 벡터인 pLys를 제조하였다. 9개의 라이신 펩타이드를 세포 내로 전달할 수 있는 능력을 용이하게 분석할 수 있도록 하기 위하여 녹색형광 단백질(Green Fluorescence Protein; "GFP"라 약칭함)를 목표 펩타이드로 선택하여 GFP의 염기서 열에 해당하는 DNA 절편을 pLys 벡터에 삽입시켜 올리고 Lys-GFP 융합 펩타이드를 발현할 수 있는 벡터인 pLys-GFP 융합 펩타이드를 제조하였다. 올리고Lys-GFP 융합 펩타이드에 대한 대조 펩타이드인 GFP를 과대 발현시키기 위하여 9개의 라이신만 포함되지 않고 나머지 부위는 동일한 pGFP 발현 벡터를 개발하였다. First, pLys, a fusion peptide expression vector capable of delivering a target peptide having a penetrating function into cells, was prepared. To facilitate the analysis of the ability to deliver nine lysine peptides into cells, a DNA fragment corresponding to the base sequence of GFP was selected by selecting Green Fluorescence Protein (abbreviated as "GFP") as the target peptide. Was inserted into the pLys vector to prepare a pLys-GFP fusion peptide, a vector capable of expressing the oligo-Lys-GFP fusion peptide. In order to overexpress GFP, a control peptide to the oligo-Lys-GFP fusion peptide, only 9 lysines were included and the remaining sites developed the same pGFP expression vector.
9개의 라이신을 발현할 수 있는 염기서열을 가진 올리고뉴클레오타이드, 정방향 개시체(forward primer): 5'-TAAGAAGAAAAAGAAAAAGAAAAAGAAGC-3'; 역방향 개시체(reverse primer): 5'-TCGAGC TTCTTTTTCTTTTTCTTTTTCTTCT-3' 두 개를 100℃에서 5분 동안 어닐링(annealing)시켜서 만든 이중가닥의 올리고뉴클레오타이드를 NdeI과 XhoI 제한효소로 절단된 pET-15b 벡터(Invitrogen, Carlsbad, CA)에 삽입시켜 9개의 라이신을 발현시킬 수 있는 벡터인 pLys를 제조하였다. 따라서 신규 제조된 벡터는 아미노 말단부터 6개의 히스티딘, 9개의 라이신 잔기와 목표 펩타이드를 융합 펩타이드 형태로 발현시킬 수 있도록 고안되었다. Oligonucleotide having a nucleotide sequence capable of expressing 9 lysines, forward primer: 5'-TAAGAAGAAAAAGAAAAAGAAAAAGAAGC-3 '; Reverse primer: A double-stranded oligonucleotide made by annealing two 5'-TCGAGC TTCTTTTTCTTTTTCTTTTTCTTCT-3's for 5 minutes at 100 ° C. to a pET-15b vector cleaved with NdeI and XhoI restriction enzymes. PLys, a vector capable of expressing nine lysines, was prepared by insertion into Invitrogen, Carlsbad, CA). Therefore, the newly prepared vector is designed to express 6 histidines, 9 lysine residues and target peptides in the form of fusion peptides from the amino terminus.
다음으로 목표 펩타이드를 녹색형광 단백질(GFP)로 선택하여 GFP의 cDNA의 서열을 기본으로 하여 2종류의 올리고뉴클레오타이드를 합성하였다. 정방향 개시체는 5'-CTCGAGGTGAGCAAGGGCG AGGAGCTG-3'로 Xho I 제한효소 분해자리를 지니고 있으며, 역방향 개시체의 서열은 5'-GGATCCTTACTTGT ACAGCTCGTCCATGCCGAG-3' 로 BamH I 제한효소 분해자리를 갖고 있다. Next, the target peptide was selected as a green fluorescent protein (GFP) to synthesize two kinds of oligonucleotides based on the cDNA sequence of GFP. The forward initiator has an Xho I restriction enzyme site of 5'-CTCGAGGTGAGCAAGGGCG AGGAGCTG-3 ', and the sequence of the reverse initiator has a BamH I restriction site of 5'-GGATCCTTACTTGT ACAGCTCGTCCATGCCGAG-3'.
합성된 올리고뉴클레오타이드를 이용하여 pEGFP-C2 (Clontech)로부터 다음과 같이 중합효소 연쇄반응(PCR)을 수행하여 GFP 염기서열을 가진 DNA 절편을 준비하였다. PCR은 thermal cycler(Perkin-Elmer, model 9600)에서 수행하였으며, 반응 혼합액을 50 ㎕ 실리콘 튜브(siliconized reaction tube)에 넣고 94℃에서 3분간 가열하였다. PCR 반응은 94℃에서 30초간 30회의 연장(extension)반응, 51℃에서 45초간 변성(denaturation), 70℃에서 1분 30초간 어닐링(annealing), 그리고 72℃에서 10분, 20℃에서 5분간 최종 연장반응을 유도하였다. DNA fragments having GFP sequences were prepared by polymerase chain reaction (PCR) from pEGFP-C2 (Clontech) using the synthesized oligonucleotides as follows. PCR was performed in a thermal cycler (Perkin-Elmer, model 9600), and the reaction mixture was placed in a 50 µl silicon tube (siliconized reaction tube) and heated at 94 ° C for 3 minutes. PCR reactions were performed 30 times at 94 ° C for 30 seconds, denaturation at 51 ° C for 45 seconds, annealing at 70 ° C for 1 minute and 30 seconds, and 10 minutes at 72 ° C, and 5 minutes at 20 ° C. The final extension reaction was induced.
PCR 수행 후 아가로즈 젤 전기영동으로 반응물을 분리하고 이것을 pGEM-T 벡터(Promega, USA)에 삽입하였다. 이어서 이 벡터로 적절한 세포(competent cell)를 형질 전환시키고, 형질 전환된 박테리아로부터 플라스미드를 알칼리 용균 방법(alkaline lysis method; Sambrook et al., 1989)으로 분리하였다. GFP cDNA가 포함된 pGEM-T 벡터를 Xho I 과 BamH I로 절단한 다음 pET-15b 및 pLys 발현 벡터에 삽입하였다. 여기서 얻어진 벡터를 pGFP 및 pLys-GFP로 각각 명명하였다. After PCR, the reaction was separated by agarose gel electrophoresis and inserted into the pGEM-T vector (Promega, USA). Appropriate cells were then transformed with this vector, and plasmids from the transformed bacteria were isolated by alkaline lysis method (Sambrook et al., 1989). PGEM-T vector containing GFP cDNA was digested with Xho I and BamH I and inserted into pET-15b and pLys expression vectors. The vectors obtained here were named pGFP and pLys-GFP, respectively.
(2) 융합 펩타이드의 발현 및 확인 (2) Expression and Identification of Fusion Peptides
IPTG로 융합 펩타이드의 과대 발현을 유도한 대장균 세포를 4℃에서 초음파처리로 파쇄한 다음, 원심 분리하여 상등액에 있는 펩타이드를 12% SDS-폴리아크릴아미드 젤 전기영동으로 분리하였다. Escherichia coli cells induced overexpression of the fusion peptide with IPTG were sonicated at 4 ° C, and then centrifuged to separate peptides in the supernatant by 12% SDS-polyacrylamide gel electrophoresis.
pGFP 및 pLys-GFP로 형질전환된 E. coli BL21(DE3)를 선택한 다음, 콜로니를 50 ml LB 배지에 접종하고 IPTG (0.5 mM)를 배지 내에 첨가하여 융합 펩타이드의 과대 발현을 유도시킨 후 세포 추출물을 준비하였다. 재조합된 GFP 및 올리고Lys-GFP 융합 펩타이드의 과대 발현을 유도하였다. 세포 추출물에 존재하는 과대 발현된 GFP 및 올리고 Lys-GFP 융합 펩타이드를 SDS-폴리아크릴아미드 젤 전기영동(polyacrylamide gel electrophoresis)과 웨스턴 블랏 분석으로 확인하였 다. E. coli BL21 (DE3) transformed with pGFP and pLys-GFP were selected, and then colonies were inoculated into 50 ml LB medium and IPTG (0.5 mM) was added into the medium to induce overexpression of the fusion peptide, followed by cell extracts. Was prepared. Overexpression of the recombinant GFP and oligoLys-GFP fusion peptides was induced. Overexpressed GFP and oligo Lys-GFP fusion peptides present in the cell extracts were confirmed by SDS-polyacrylamide gel electrophoresis and Western blot analysis.
(3) 9-Tat 융합 펩타이드의 정제 (3) Purification of 9-Tat Fusion Peptide
pLys-GFP 벡터로 형질전환된 E. coli BL21을 앰피실린이 포함된 LB 배지에 넣고 37℃에서 250rpm으로 교반하며 배양하였다. 배양액내의 박테리아 농도가 O.D600 = 0.6 ~ 1.0을 나타낼 때 IPTG를 배지 내에 첨가하여 최종농도가 0.5mM 되게 한 다음 3시간을 더 배양하였다. 배양한 세포를 원심분리하여 모은 뒤 5ml 결합완충용액(binding buffer; 5mM 이미다졸, 0.5M NaCl, 20mM Tris-HCl, pH 7.9)를 넣고 초음파처리를 하였다. 재조합 올리고 Lys-GFP 융합 펩타이드는 두 가지의 조건하에서 즉, 변성상태(denaturing condition)와 변성시키지 않은 자연상태(native condition)에서 각각 정제하였다. 10g의 박테리아 펠렛을 40ml 완충액 A(buffer A; 6M 우레아, 20mM HEPES, pH 8.0, 100mM NaCl)에 넣고 초음파 처리하여 세포를 파쇄시킨 다음, 원심분리를 2회(16,500rpm 30분, 40,000rpm 30 분, 4℃) 연속 수행하여 불용성의 세포 파편을 제거하였다. E. coli BL21 transformed with pLys-GFP vector was placed in LB medium containing ampicillin and incubated at 37 ° C. and 250 rpm. When the bacterial concentration in the culture medium was O.D600 = 0.6 ~ 1.0, IPTG was added to the medium to bring the final concentration to 0.5mM, followed by further incubation for 3 hours. The cultured cells were collected by centrifugation, followed by sonication with 5 ml binding buffer (5 mM imidazole, 0.5 M NaCl, 20 mM Tris-HCl, pH 7.9). Recombinant oligo Lys-GFP fusion peptides were purified under two conditions, respectively, in denatured condition and native condition. 10 g of bacterial pellet was placed in 40 ml buffer A (6 M urea, 20 mM HEPES, pH 8.0, 100 mM NaCl) and sonicated to disrupt the cells, followed by two centrifugations (16,500 rpm 30 minutes, 40,000 rpm 30 minutes). , 4 ° C.) was performed continuously to remove insoluble cell debris.
완충액 B(buffer B; 6M 우레아, 20mM HEPES, pH 8.0, 100mM NaCl, 20mM 이미다졸)로 평형을 유지시킨 2.5ml Ni2+-니트릴로삼아세트산 세파로즈 컬럼(nitrilotriacetic acid sepharose column; Quagen, USA)에 원심 분리하여 얻은 상등액을 즉시 부하하고, 10배 부피의 결합 완충액과 6배 부피의 세척 완충액(washing buffer; 60mM 이미다졸, 0.5M NaCl, 20mM Tris-HCl, pH 7.9)으로 세척한 다음, 용출 완충액(elution buffer; 1M 이미다졸, 0.5M NaCl, 20mM Tris-HCl, pH 7.9)로 올리고Lys-GFP 융합 펩타이드를 용출하였다. 이어서 올리고Lys-GFP 융합 펩타이드가 포함된 분획들을 모아 세파덱스(Sephadex) G-25M을 함유한 PD-10 컬럼 크로마토그래피(column chromatography)를 수행하여 분획중에 포함된 염분을 제거하였다. 다시 이것을 동결 건조하여 수분이 완전히 제거된 분말 상태의 9개의 라이신으로 이루어진 Tat 펩타이드(KKKKKKKKK)를 얻었다.Centrifuge on a 2.5 ml Ni2 + -nitriloacetic acid sepharose column (Quagen, USA) equilibrated with buffer B (6M urea, 20 mM HEPES, pH 8.0, 100 mM NaCl, 20 mM imidazole). The supernatant obtained is immediately loaded and washed with 10 volumes of binding buffer and 6 volumes of washing buffer (60 mM imidazole, 0.5 M NaCl, 20 mM Tris-HCl, pH 7.9), followed by elution buffer ( elution buffer; 1M imidazole, 0.5M NaCl, 20mM Tris-HCl, pH 7.9) eluted the Olys-GFP fusion peptide. Subsequently, the fractions containing the oligo-Lys-GFP fusion peptide were collected and PD-10 column chromatography containing Sephadex G-25M was performed to remove the salts contained in the fractions. It was freeze-dried again to obtain a Tat peptide (KKKKKKKKK) consisting of nine lysines in powder form, from which moisture was completely removed.
실시예 2: 화학적 합성법에 의한 9-Tat 펩타이드(R = KKKKKKKKK)의 제조Example 2: Preparation of 9-Tat Peptide (R = KKKKKKKKK) by Chemical Synthesis
(1) 9-Tat 펩타이드(KKKKKKKKK : 올리고라이신)의 합성(1) Synthesis of 9-Tat Peptide (KKKKKKKKK: Oligolysine)
본 발명에 사용된 9- Tat 펩타이드(올리고라이신)은 Applied Biosystems사의 431A 자동합성기를 이용하여 고체상 방법으로 합성하였다. 0.25m㏖의 파라히드록시 메틸 페닐옥시메칠 폴리스티(HMP)레진을 표준 반응용기(Standard reaction vellel, 38㎖)에 넣고, Fmoc-아미노산을 합성하려는 펩타이드의 카르복시 말단의 Fmoc-아미노산을 넣고 합성을 시작하였다. 1 m㏖의 Fmoc-아미노산이 들어있는 카트리지를 C-말단 아미노산에서부터 N-말단의 아미노산까지 배열순서대로 가이드웨이에 배열한다. 이때 카트리지의 금속 뚜껑을 제거하고 맨 처음과 맨 끝에는 아무 아미노산도 들어있지 않은 빈카트리지를 놓았다. 펩타이드 합성은 ABI사에서 개발한 표준 스케일 Fmoc 커플링 프로토콜(Protocol)에 따라 시행 전에 파라메타를 편집하고 자동합성 메뉴에 따라 시행하였다(ABI User's Manual. Jan, 1992 참조). 표준 스케일 Fmoc을 사용할 때는 디프로텍션(Deprotection)을 NMP(N-메팅 피롤리딘)으로 희석한 20% 피페리딘을 사용하여 21 분동안 수행하였으며 NMP로 9분 동안 세척하고 커플링을 71분 동안 실시하였다. 커플링에는 HOBT(1-히드록시-벤조트리아졸 사용하였고 NMP로 7분간 세척하는 시스템을 이용하였다. The 9-Tat peptide (oligolysine) used in the present invention was synthesized by a solid phase method using a 431A autosynthesizer of Applied Biosystems. Put 0.25 mmol of parahydroxy methyl phenyloxymethyl polystyrene (HMP) resin into a standard reaction vessel (38 ml), add Fmoc-amino acid at the carboxy terminus of the peptide to synthesize Fmoc-amino acid, and perform synthesis. Started. Cartridges containing 1 mmol of Fmoc-amino acids are arranged in the guideway in the order of C-terminal amino acids to N-terminal amino acids. The metal lid of the cartridge was removed and the empty cartridge containing no amino acids at the beginning and end. Peptide synthesis was performed before editing according to the standard scale Fmoc coupling protocol developed by ABI and following the autosynthesis menu (see ABI User's Manual. Jan, 1992). When using a standard scale Fmoc, deprotection was performed for 21 minutes using 20% piperidine diluted with NMP (N-Metting pyrrolidine), washed for 9 minutes with NMP and coupling for 71 minutes. Was carried out. HOBT (1-hydroxy-benzotriazole was used for the coupling, and the system was washed with NMP for 7 minutes.
(2) 상기 9-Tat 펩타이드(KKKKKKKKK : 올리고라이신)의 분리 및 정제 (2) Isolation and Purification of the 9-Tat Peptide (KKKKKKKKK: Oligolysine)
합성이 완료된 후 9-Tat 펩타이드의 분리는 트리플루오로아세트산(TFA)을 사용하여 고체 지지체(Solid Support)로부터 절단하였다. ABI사 매뉴얼(Introduction to Cleavage Techniques, P6-19(1990))을 참고로 하여 펩타이드를 분리하였다. 즉 합성이 끝나면 펩타이드가 붙은 레진을 둥근 플라스크에 넣고 냉각시킨 후 0.75g 결정 페놀, 0.25㎖ EDT(1,2-에탄디티올), 0.5㎖ 티오아니솔, 0.5㎖ 증류수 및 10㎖ TFA를 넣고 뚜껑을 닫고 실온에서 1-2시간 반응시켰다. 반응이 끝난 후 레진과 반응액을 신털드(Sintered) 유리 깔대기를 통해 저진공으로 여과하여 레진과 펩타이드 용액을 분리한다. 플라스크와 유리깔대기를 5-10㎖ DCM(디클로로메탄)으로 씻어 이 용액이 펩타이드 용액과 섞이게 한다. 50㎖이상의 차가운 디에틸에티르를 첨가하여 펩타이드의 침전물을 얻는다. 이 침전물을 저진공으로 깔대기로 여과하여 깔대기 위에 모아진 침전물을 건조시킨 후 30% 초산에 녹여 냉동 건조시킨다. 이렇게 하여 얻어진 펩타이드는 HPLC (High Performance Liquid Chromatography)로 정제하였다. 이때 칼럼은 C18 analytical column (Pharmacia)을 사용하였으며 완충용액 A는 10% 아세토니트닐 + 0.05% TFA를 사용하여 평형화시키고 B는 80% 아세토니트닐 + 0.05% TFA를 사용하여 펩타이드를 용출하였다. 그결과, 고순도로 정제된 펩타이드를 얻었으며 합성수율은 30 5% 정도였다. After synthesis was complete, separation of 9-Tat peptide was cleaved from solid support using trifluoroacetic acid (TFA). Peptides were isolated by referring to the ABI manual (Introduction to Cleavage Techniques, P6-19 (1990)). After the synthesis, the resin with peptides was placed in a round flask and cooled. Then, 0.75 g crystalline phenol, 0.25 ml EDT (1,2-ethanedithiol), 0.5 ml thioanisole, 0.5 ml distilled water and 10 ml TFA were added to the lid. The reaction mixture was closed and reacted at room temperature for 1-2 hours. After the reaction, the resin and the reaction solution are filtered through low vacuum through a sintered glass funnel to separate the resin and the peptide solution. Wash the flask and glass funnel with 5-10 ml DCM (dichloromethane) to allow the solution to mix with the peptide solution. At least 50 ml of cold diethylether is added to obtain a precipitate of peptides. The precipitate was filtered with a funnel under low vacuum to dry the precipitate collected on the funnel, and then dissolved in 30% acetic acid and freeze-dried. The peptide thus obtained was purified by HPLC (High Performance Liquid Chromatography). The column was a C18 analytical column (Pharmacia), buffer A was equilibrated with 10% acetonitrile + 0.05% TFA and B was eluted with 80% acetonitrile + 0.05% TFA. As a result, a highly purified peptide was obtained and the synthetic yield was about 30 5%.
실시예 3: 아스코르브산 세슘염(4a 화합물)의 합성Example 3: Synthesis of Cesium Ascorbate (Compound 4a)
아스코르브산(5g, 28.4 mmol)을 물 20 mL에 용해한 뒤, 이 용액을 0℃내지 5℃로 유지하면서 고체 탄산수소세슘 (4.63g, 14.2 mmol)을 서서히 부가하였다. 탄 산가스의 발생이 완전히 중단되면 이소프로필알콜 40 mL를 천천히 부가하여 정치시키면 고체가 얻어진다. 생성된 고체를 여과하고 세척한 후 건조하여 8.4 g(95%)의 백색 고체(4a)를 얻었다.Ascorbic acid (5 g, 28.4 mmol) was dissolved in 20 mL of water, and then solid cesium bicarbonate (4.63 g, 14.2 mmol) was slowly added while maintaining the solution at 0 ° C to 5 ° C. When the generation of carbon dioxide ceases completely, 40 mL of isopropyl alcohol is added slowly and left to stand to obtain a solid. The resulting solid was filtered, washed and dried to give 8.4 g (95%) of white solid (4a).
실시예 4: 3-Tat 펩타이드-아스코르브산(화합물 1a)의 제조 Example 4: Preparation of 3-Tat Peptide-Ascorbic Acid (Compound 1a)
상기 실시예 1또는 2에서 제조된 세포침투성 Tat 펩타이드(3.1g, 5.45 mmol)를 10mL의 DMF에 용해하였다. 한다. 무수 SOCl2를 부가한 후 2시간 동안 교반한다. 아스코르브산 세슘염(4a) (560mg, 3.18 mmol)을 첨가한 후 반응물을 60℃에서 24시간 반응하였다. 감압 하에서 용매를 제거한 뒤, 다이에틸에테르 20ml로 결정화시킨 후 건조하여 역상 칼럼 크로마토그라피를 이용하여 정제한 3-Tat-아스코르브산 (1a)을 1.23g(66%) 얻었다. The cell penetrating Tat peptide (3.1 g, 5.45 mmol) prepared in Example 1 or 2 was dissolved in 10 mL of DMF. do. Anhydrous SOCl2 is added and stirred for 2 hours. Cesium ascorbate salt (4a) (560 mg, 3.18 mmol) was added and the reaction was then reacted at 60 ° C. for 24 hours. After removal of the solvent under reduced pressure, crystallization was carried out with 20 ml of diethyl ether, followed by drying to obtain 1.23 g (66%) of 3-Tat-ascorbic acid (1a), which was purified using reversed phase column chromatography.
실시예 5: 2-Tat-아스코르브산(화합물 1b)의 합성 Example 5: Synthesis of 2-Tat-ascorbic acid (Compound 1b)
(1) 3-벤질 아스코르브산(화합물 5b)의 제조(1) Preparation of 3-benzyl ascorbic acid (Compound 5b)
아스코르브산(10g, 56.8mmol)을 디메틸설폭시드 60 mL에 용해한 후에,이 용액에 고체 탄산수소세슘(9.25g, 28.4 mmol)을 부가하였다. 20분 뒤 벤질브로마이드(6.75 mL, 56.8mmol)를 부가한 후 반응물을 50℃에서 18시간 반응하였다. 반응물의 온도를 상온으로 냉각한 디메틸렌글로라이드 (150mL)로 희석하고 생성된 고체를 여과하여 제거하였다. 여액은 감압 하에서 농축한 뒤 실리카젤 칼럼을 이용하여 3-벤질-아스코르브산(화합물 4b)을 7.2g (48%) 얻었다. After ascorbic acid (10 g, 56.8 mmol) was dissolved in 60 mL of dimethylsulfoxide, solid cesium hydrogen carbonate (9.25 g, 28.4 mmol) was added to this solution. After 20 minutes, benzyl bromide (6.75 mL, 56.8 mmol) was added, and the reaction was reacted at 50 ° C. for 18 hours. The temperature of the reaction was diluted with dimethylene glolide (150 mL) cooled to room temperature and the resulting solid was filtered off. The filtrate was concentrated under reduced pressure and 7.2 g (48%) of 3-benzyl-ascorbic acid (Compound 4b) was obtained using a silica gel column.
(2) 3-벤질-2-Tat-아스코르브산(화합물 5c)의 합성 (2) Synthesis of 3-benzyl-2-Tat-ascorbic acid (Compound 5c)
Tat 펩타이드(3.1g, 5.45 mmol)를 10mL의 DMF에 용해한다. 무수 SOCl2를 부 가한 후 2시간 동안 교반하였다. 3-벤질 아스코르브산 (560 mg, 3.18 mmol)을 첨가한 후 반응물을 60℃에서 24시간 반응하였다. 감압 하에서 용매를 제거한 뒤, 디에틸에테르 20ml로 결정화시킨 후 건조하여 역상 칼럼 크로마토그라피를 이용하여 정제한 후 흰색고체 3-벤질-2-Tat-아스코르브산(화합물 5c)을 얻었다.Tat peptide (3.1 g, 5.45 mmol) is dissolved in 10 mL of DMF. Anhydrous SOCl2 was added and stirred for 2 hours. 3-benzyl ascorbic acid (560 mg, 3.18 mmol) was added and the reaction was then reacted at 60 ° C for 24 hours. The solvent was removed under reduced pressure, crystallized with 20 ml of diethyl ether, dried and purified by reverse phase column chromatography to obtain white solid 3-benzyl-2-Tat-ascorbic acid (Compound 5c).
(3) 2-Tat-아스코르브산(화합물 1b)의 합성 (3) Synthesis of 2-Tat-ascorbic acid (Compound 1b)
3-벤질-2-Tat펩타이드-아스코르브산(4.04g, 5 mmol)을 메탄올(40 mL)에 용해한 뒤, 팔라듐-차콜(400 mg, 10%)을 부가하였다. 이 반응 용기를 감압과 수소 주입방법으로 수소 분위기 상태로 변화시키고, 상온에서 2시간 교반하였다. 반응물은 차콜과 셀라이트를 통하여 여과시키고 메탄올로 세척하였다. 여액을 감압하에 농축하여 표제의 화합물, 2-Tat-아스코르브산(화합물 1b) 3g (85%)을 흰색 고체를 얻었다.3-benzyl-2-Tat peptide-ascorbic acid (4.04 g, 5 mmol) was dissolved in methanol (40 mL) followed by the addition of palladium-charcoal (400 mg, 10%). The reaction vessel was changed to a hydrogen atmosphere state under reduced pressure and hydrogen injection, and stirred at room temperature for 2 hours. The reaction was filtered through charcoal and celite and washed with methanol. The filtrate was concentrated under reduced pressure to give 3 g (85%) of the title compound, 2-Tat-ascorbic acid (compound 1b), as a white solid.
실시예 6 : 경피흡수 실험 Example 6 Percutaneous Absorption Experiment
상기 화합물 (1a)의 피부 세포내 침투, 이를 통상적으로 경피흡수라 하므로 본 명세서에서는 경피흡수로 통칭한다. 경피흡수 실험은, 공지의 미백용 화장품원료로써 사용되고 있는 아스코르브산 및 3-0-에틸아스코르브산과 운반매개로 정제수를 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).Intradermal penetration of the compound (1a), commonly referred to as transdermal absorption, is referred to herein as transdermal absorption. Percutaneous absorption experiments were carried out using ascorbic acid and 3-0-ethyl ascorbic acid, which are used as known cosmetic ingredients for whitening, and purified water as carrier media (RehmanPA, Slattery JT, Franz TJ.Percutaneous absorption of retinoids Influence of vehicle, light exposure, and dose, J. Invest Dermatol., 91; 56-61, 1988).
자세하게는, 8주령의 암컷 쥐(헤어리스 마우스)의 등쪽 부위 피부를 1.7 cm2크기로 절취하고, 여기에 35nM의 시료 250㎕을 적용한 후 경피흡수 측정기기(Franz cell)를 이용하여 24시간 후 수용기(receptor)용액 [2% 계면활성제를 포함하는 PBS 완충용액]과 쥐의 등쪽 피부에 흡수된 물질을 추출하여 고압액체 크로마토그래피(HPLC)로 정량 분석하여, 화학식 1a의 본 발명의 화합물과 아스코르브산 및 3-0-에틸 아스코르브산이 경피 흡수량을 정량 비교하였다. 그 결과 화학식 1a) 화합물은 상대적으로 기존의 아스코르브산 및 3-O-에칠아스코르브산에 비해 우수한 경피 흡수 증가율을 나타냈다. Specifically, the skin of the dorsal region of 8-week-old female rat (hairless mouse) was cut to 1.7 cm 2 size, and 250 μl of 35 nM sample was applied thereto, followed by a transdermal absorption device (Franz cell) after 24 hours. (Receptor) The solution [PBS buffer containing 2% surfactant] and the material absorbed in the dorsal skin of the rat was extracted and quantitatively analyzed by high pressure liquid chromatography (HPLC), the compound of the present invention of formula 1a and ascorbic acid And 3-0-ethyl ascorbic acid for quantitative comparison of transdermal absorption. As a result, the compound of Formula 1a) showed a relatively good increase in transdermal absorption compared to conventional ascorbic acid and 3-O-ethylascorbic acid.
실시예 7 : 타이로시나제 저해 활성 Example 7: Tyrosinase Inhibitory Activity
타이로시나제 효소로는 버섯류로 부터 추출한 것으로, 시그마(Sigma)사의 것을 사용하였다. 먼저, 기질인 L-타이로신을 1.5 mM의 농도가 되도록 인산완충액 (0.05M 농도, pH 6.8)에 용해시킨 다음 이 용액 0.01 mL를 0.3 mL 용량의 분광광도계 큐벳(Cuvette)에 가하고 코펙터(Cofactor)인 도파를 0.06 mM 농도로 만들어 0.01mL 첨가하였다. 여기에 본 발명에 따른 화학식 (1a)와 (1b)의 화합물를 첨가하고 0.1 mL가 되도록 상기 인산 완충액을 첨가하였다. 이 반응액에 타이로시나제를 인산 완충액에 60 U/mL로 용해시킨 효소 용액을 0.1 mL 첨가함으로써 반응을 진행시켰다. 이때 대조군(Blank)으로는 타이로시나제대신 완충액 만을 0.1 mL 첨가한 것으로 하였다. 반응은 37℃에서 10분간 진행 시켰으며, 분광광도계(Spectrophotometer, Beckman DU-7500)를 이용하여 475 nm에서의 흡광도를 측정하여 타이로시나제에 대한 저해율을 구하고, IC50값은 효소 활성 저해율이 50%에 달하는 저해제의 농도로 결정하였다. 저해율을 산정하는 식은 아래에 나타낸 바와 같으며 그 결과 화학식 1a) 화합물은 상대적으로 기존의 아스코르브산 및 3- O-에칠아스코르브산에 비해 우수한 타이로시나제 저해 활성을 나타내었다.The tyrosinase enzyme was extracted from mushrooms, and Sigma Corporation was used. First, L-tyrosine, a substrate, is dissolved in phosphate buffer solution (0.05M concentration, pH 6.8) to a concentration of 1.5 mM, and then 0.01 mL of this solution is added to a 0.3 mL volume of spectrophotometer Cuvettes and Cofactor. Phosphorous waveguides were added at a concentration of 0.06 mM and 0.01 mL. To this was added the compounds of formulas (1a) and (1b) according to the invention and the phosphate buffer was added to 0.1 mL. The reaction was advanced by adding 0.1 mL of an enzyme solution in which tyrosinase was dissolved at 60 U / mL in phosphate buffer. In this case, 0.1 mL of a buffer solution instead of tyrosinase was added as a control group. The reaction was carried out at 37 ° C. for 10 minutes, and the absorbance at 475 nm was measured by using a spectrophotometer (Spectrophotometer, Beckman DU-7500), and the inhibition rate against tyrosinase was determined. It was determined by the concentration of inhibitors reaching%. The formula for calculating the inhibition rate is as shown below, and as a result, the compound of Formula 1a) showed relatively superior tyrosinase inhibitory activity compared to conventional ascorbic acid and 3-O-ethylascorbic acid.
저해율(%) = 
A: 저해제가 첨가되지 않은 것의 475 nm 에서의 흡광도 A: absorbance at 475 nm without addition of inhibitor
B: 저해제가 첨가된 것의 475 nm 에서의 흡광도 B: absorbance at 475 nm with inhibitor added
실시예 8 : 라디칼 소거 작용 Example 8 radical scavenging action
화합물 (1a)과 (1b)에 대하여, 자유 라디칼 소거능을 다음과 같이 측정하였다. 1,1-디페닐-2-피크릴-하이드라질 (DPPH) 방법으로 수행하였다 [Blois, M. S. Nature, 1958, 181, 1190]. DPPH는 비교적 안정한 라디칼로 존재시 517nm에서 최대 흡광도를 보이며 라디칼이 소거되면 이 파장에서 흡광성을 잃는다. DPPH는 시그마(Sigma)사의 것을 사용하였으며 0.15 mM농도로 메틸알콜에 녹여 사용하였다. 먼저 화합물 (1a) 혹은 (1b)를 각 농도 별로 제조하여 (0.1-0.000005% (w/v)) 96-웰 플레이트의 각 웰에 100㎕ 씩 넣는다. 여기에 DPPH 용액을 100㎕씩 첨가한 다음 상온에서 방치한 후 마이크로 플레이트 리더 (BioTek EL-340)를 이용하여 517 nm에서의 흡광도를 측정하였다. 이때 대조군으로 상기 화합물 대신 메틸알콜 100㎕를 넣은 것으로 이용하였다. 시료를 처리한 것의 흡광도가 대조군의 흡광도의 절반이 될 때의 상기 화합물의 농도IC50라고 표시하였으며 그 결과 화학식 1a)와 1b) 화합물은 상대적으로 기존의 아스코르브산 및 3-O-에틸아스코르브산에 비해 10배 이상의 우수한 자유 라디칼 소거능을 나타내었다. 상기 결과에서 알 수 있듯이 화학식 1a)와 1b)의 화합물은 매우 강력한 자유 라디칼 소거제이고 그 효과의 지속성이 우수 함을 알 수 있다. For compounds (1a) and (1b), free radical scavenging ability was measured as follows. Performed by 1,1-diphenyl-2-picryl-hydrazyl (DPPH) method [Blois, M. S. Nature, 1958, 181, 1190]. DPPH, as a relatively stable radical, exhibits maximum absorbance at 517 nm and loses absorbance at this wavelength when radicals are scavenged. DPPH was used by Sigma Co., Ltd. and dissolved in methyl alcohol at a concentration of 0.15 mM. First, compound (1a) or (1b) is prepared at each concentration (0.1-0.000005% (w / v)) and put into 100 μl of each well of a 96-well plate. 100 μl of DPPH solution was added thereto, and the mixture was left at room temperature and then absorbed at 517 nm using a microplate reader (BioTek EL-340). In this case, 100 μl of methyl alcohol was used instead of the compound as a control. When the absorbance of the sample treated was half the absorbance of the control group, the concentration of the compound was expressed as IC50. As a result, the compounds of Formulas 1a) and 1b) were relatively compared to conventional ascorbic acid and 3-O-ethyl ascorbic acid. It showed at least 10 times better free radical scavenging ability. As can be seen from the above results, it is understood that the compounds of Formulas 1a) and 1b) are very powerful free radical scavengers and have excellent sustainability.
실시예 9 : 알러지 테스트(LLNA) Example 9 Allergy Test (LLNA)
화장품 원료로서의 화학식 1의 화합물에 대한 안전성 실험으로는 운반 매개물로 에탄올을 사용하는 실험법을 택하였다[참고 문헌 : Kimber Ⅰ(1990) : Identification of contact allergens using the murine local lymph node assay, J. Appl. Toxicol. 10(3); 173~180]. 미백용 화장품 원료로 사용되고 있는 3-0-에틸아스코르브산과 화학식 1의 화합물을 각각 0.3%, 1% 용액으로 제조 시행하였다. 즉 쥐(Balb/c)의 양쪽 귀에 50 ㎕씩 3일간 발라준 후 쥐로부터 이개임파절(Auricular lymph node)을 분리한다. 임파절을 분쇄하여 단일세포 상태로 만든 후 방사성 동위원소(3H-티미딘)를 첨가하여 24시간 배양한 다음, 세포의 증폭도[cpm]를 측정한 결과, 화학식 1의 (1a) 화합물과 3-0-에틸아스코르브산 모두 0.3%와 1% 용액 모두에서 에탄올에 비해 3배 이하의 낮은 유발성을 나타내었다. As a safety test for the compound of Formula 1 as a cosmetic raw material, ethanol was used as a transport medium [Kimber I (1990): Identification of contact allergens using the murine local lymph node assay, J. Appl. Toxicol. 10 (3); 173-180]. 3-0-ethyl ascorbic acid and the compound of formula 1, which are used as cosmetic raw materials for whitening, were prepared as 0.3% and 1% solutions, respectively. In other words, 50 μl was applied to both ears of the mouse (Balb / c) for 3 days, and then the auricular lymph node is separated from the mouse. Lymph nodes were pulverized into a single cell state, and then cultured for 24 hours with the addition of radioisotope (3H-thymidine). Both 0-ethyl ascorbic acid showed less than three times lower induction than ethanol in both 0.3% and 1% solutions.
실시예 10 : Tat 펩타이드 그래프팅 아스코르브산의 피부 자극성 시험 Example 10 Skin Irritation Test of Tat Peptide Grafting Ascorbic Acid
화합물 (1a)의 피부 자극성 시험을 위하여 기니아 피그Guinea Pigs for Skin Irritation Test of Compound (1a)
(Guinea Pig)를 이용한 첩포시험(Patch test)를 수행하였다[참고문헌 : ①Draize, J.H.(1959) : Dermal toxicity. Assoc. Food and Drug officials, US. Appraisal of the safety of chemicals in Food, Drugs and Cosmetics., pp46-59, Texas State Dept. of Health, Austin, Texas. ②Federal Register(1973) : Method of testing primary irritant substances 38(187) : pp1500-1541]. Patch test using (Guinea Pig) was performed [Reference: ①Draize, J.H. (1959): Dermal toxicity. Assoc. Food and Drug officials, US. Appraisal of the safety of chemicals in Food, Drugs and Cosmetics., Pp 46-59, Texas State Dept. of Health, Austin, Texas. ② Federal Register (1973): Method of testing primary irritant substances 38 (187): pp1500-1541].
즉, 정제수를 운반매개로 한 0.3%의 7종류의 비교 시료 용액을 제조 시험하 였다. 먼저 시료 도포 부위(등)의 털을 제거하고 피부 자극을 최소화하기 위해 24시간 동안 환경 적응을 시킨 후 시료 도포 부위를 설정 (1.5cm 1.5cm) 시료와 가제를 적용한 다음 시료의 증발 및 손실을 방지하기 위해 고형 재질의 박지로 밀봉하고 탄력 붕대로 48시간 고정하였다. 폐쇄첩포 제거 후 2시간, 24시간(첩포후 48시간, 72시간)째 자극의 정도를 판정하였다. 그 결과를 다음 표 1에 나타내었다. That is, seven types of comparative sample solutions of 0.3% using purified water as a transport medium were tested. First, remove the hair from the sample application area (back) and adjust the environment for 24 hours to minimize skin irritation, and then set the sample application area (1.5cm 1.5cm) .Apply the sample and gauze, and then prevent the evaporation and loss of the sample. In order to seal it with a solid foil and fixed with an elastic bandage for 48 hours. The degree of stimulation was determined at 2 hours and 24 hours (48 hours and 72 hours after the patch) after removal of the closed patch. The results are shown in Table 1 below.
시료 1. RPLC #1 : 대조군 Sample 1. RPLC # 1: control
2. PALC #2 : 대조군 + 화학식 1의 화합물 0.3% 2. PALC # 2: control + compound of formula 1 0.3%
3. PALC #3 : 대조군 + 액정캡슐(화학식 1의 화합물 없음) 3. PALC # 3: control + liquid crystal capsule (no compound of Formula 1)
4. PALC #4 : 대조군 + 액정캡슐(화학식 1의 화합물 0.3% 함유) 4. PALC # 4: control + liquid crystal capsule (containing 0.3% of the compound of Formula 1)
5. PALC #5 : 대조군 + 지질 액정젤(화학식 1의 화합물 0.3% 함유)5. PALC # 5: control + lipid liquid crystal gel (containing 0.3% of the compound of Formula 1)
6. RPLC : 정제수 중 3-0-에틸아스코르브산 0.3% 6.RPRP: 0.3% of 3-0-ethyl ascorbic acid in purified water
7. PALC : 정제수 중 화학식 1의 화합물 0.3% 7. PALC 0.3% compound of formula 1 in purified water
표 1에서와 같이 3-0-에틸아스코르브산과 화합물 (1a)의 자극 Stimulation of 3-0-ethylascorbic acid with compound (1a) as in Table 1
정도는 큰 유의차가 없음을 알 수 있다. The degree can be seen that there is no significant difference.
실시예 11: 세포독성 실험Example 11: Cytotoxicity Experiment
화장품에 사용되는 원료로서의 1차 안전성을 검증하고자 화학식 1의 화합물(1a)에 대하여 V794 세포(차이니스 햄스터, 폐조직 섬유아세포의 연속세포주)을 배양하여 MTT 시험을 하는 방법 [참고 문헌 : Mossman T.(1983). Rapid colorimetric assay for cellular growth & survival : application to proliferation & cytotoxicity assays. Journal of Immunological Methods 65, 55~63]을 수행하여 세포독성을 시험한 결과, 화학식1의 화합물은 10-3의 농도 이상에서는 점차 아주 약한 독성을 나타내었으나10-4 이하의 농도에서는 거의 독성을 나타내지 않음을 나타내었다. 이 결과는 독성을 나타내지 않는 농도인 10-4 이하에서는 세포증진 효과를 보이기 시작하여 10-7 농도에서 최고의 콜라겐 합성 증진 효과를 보이며, 상대적으로 세포독성을 아주 약하지만 발현되기 시작하는 10-3 이상의 농도에서는 세포 증진 효과가 떨어지는 실험 결과의 일치성을 잘 보여 주었다. Method of MTT test by culturing V794 cells (Chinese hamster, continuous cell line of lung tissue fibroblast) on compound (1a) of formula 1 to verify primary safety as a raw material used in cosmetics [Reference: Mossman T (1983). Rapid colorimetric assay for cellular growth & survival: application to proliferation & cytotoxicity assays. Journal of Immunological Methods 65, 55 ~ 63] showed that the cytotoxicity of the compounds of Formula 1 gradually became very weak at concentrations above 10-3, but at levels below 10-4. Not shown. This result shows the effect of increasing the collagen synthesis at the concentration of 10-7 and below the non-toxic concentration of 10-4 or less, showing the highest collagen synthesis promoting effect, and relatively weak cytotoxic but 10-3 above Concentration showed good agreement with the experimental results, which showed less cellular enhancement.
본 발명은 자가침투신호를 갖는 Tat 펩타이드가 결합된 아스코르브산 및 그 유도체, 그리고 이를 포함하는 피부 미백제를 제공하여, 본 발명의 미백제는 세포 침투성 Tat 펩타이드의 존재로 인해, 기존의 아스코르브산 유도체 미백제에 비해, 피부에 대한 흡수력이 뛰어나고, 비자극성이며, 생체 내에서 안정하여 우수한 미백효과 및 그 지속성을 갖는 장점이 있다. The present invention provides ascorbic acid and derivatives thereof bound to Tat peptides having a self-invasive signal, and a skin whitening agent comprising the same. In comparison, the skin has excellent absorption, non-irritating properties, stable in vivo, and has an excellent whitening effect and its sustainability.
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| KR102607534B1 (en) * | 2015-12-28 | 2023-11-29 | 오비다트 주식회사 | COSMETIC COMPOSITION FOR SKIN WHITENING COMPRISING TATdMt PEPTIDES |
| KR102038697B1 (en) * | 2019-05-08 | 2019-10-30 | 주식회사 앱타이론바이오 | Cosmetic Composition for Anti-oxidation and Whitening of Skin |
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2001
- 2001-09-27 KR KR1020010060244A patent/KR100817191B1/en active IP Right Grant
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPH04149115A (en) * | 1990-10-11 | 1992-05-22 | Kanebo Ltd | Whitening cosmetic |
| JPH07206840A (en) * | 1994-01-14 | 1995-08-08 | Kanto Denka Kogyo Co Ltd | Ascorbic acid-hydroxycarboxylic acid combination product and method for producing the same |
| KR19990076090A (en) * | 1998-03-27 | 1999-10-15 | 성재갑 | Polyethoxylated Vitamin C Derivatives and Methods for Making the Same |
| KR20000060467A (en) * | 1999-03-16 | 2000-10-16 | 성재갑 | process for the preparation antioxidants, polyethoxylated ascorbic acid derivatives |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2022215946A1 (en) * | 2021-04-07 | 2022-10-13 | (주)스템메디텍 | Cell-penetrating peptide variant and use thereof |
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| Publication number | Publication date |
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| KR20030028232A (en) | 2003-04-08 |
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