KR102607534B1 - COSMETIC COMPOSITION FOR SKIN WHITENING COMPRISING TATdMt PEPTIDES - Google Patents
COSMETIC COMPOSITION FOR SKIN WHITENING COMPRISING TATdMt PEPTIDES Download PDFInfo
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- KR102607534B1 KR102607534B1 KR1020150187948A KR20150187948A KR102607534B1 KR 102607534 B1 KR102607534 B1 KR 102607534B1 KR 1020150187948 A KR1020150187948 A KR 1020150187948A KR 20150187948 A KR20150187948 A KR 20150187948A KR 102607534 B1 KR102607534 B1 KR 102607534B1
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Classifications
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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
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K8/00—Cosmetics or similar toiletry preparations
- A61K8/02—Cosmetics or similar toiletry preparations characterised by special physical form
-
- 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
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Animal Behavior & Ethology (AREA)
- General Health & Medical Sciences (AREA)
- Public Health (AREA)
- Veterinary Medicine (AREA)
- Birds (AREA)
- Epidemiology (AREA)
- Dermatology (AREA)
- Cosmetics (AREA)
- Peptides Or Proteins (AREA)
Abstract
본 발명은 원래의 TAT(Trans-Activator of Transcription) 펩타이드의 중간 절편들의 아미노산 서열을 인위적으로 변형시킨 TATdMt(TAT double mutant) 펩타이드의를 포함하는 미백 조성물에 관한 것으로, 구체적으로는 티로시나아제 활성 억제 및 멜라닌 생성을 억제하여 피부 미백 효과가 우수하여, 미백과 관련한 화장품, 의약품, 식품 등 다양한 분야에 적용할 수 있다.The present invention relates to a whitening composition comprising a TATdMt (TAT double mutant) peptide in which the amino acid sequence of the middle fragment of the original TAT (Trans-Activator of Transcription) peptide is artificially modified, and specifically, to inhibit tyrosinase activity. It has an excellent skin whitening effect by suppressing melanin production, and can be applied to various fields related to whitening, such as cosmetics, medicine, and food.
Description
본 발명은 원래의TAT(Trans-Activator of Transcription) 펩타이드의 중간 절편들의 아미노산 서열을 인위적으로 변형시킨 TATdMt(TAT double mutant) 펩타이드를 포함하는 티로시나아제의 활성 억제 및 멜라닌 생성을 억제하는 미백 조성물에 관한 것이다. The present invention relates to a whitening composition that inhibits the activity of tyrosinase and inhibits melanin production, including TATdMt (TAT double mutant) peptide, in which the amino acid sequence of the middle fragment of the original TAT (Trans-Activator of Transcription) peptide is artificially modified. It's about.
피부색을 결정하는 색소로는 멜라닌, 헤모글로빈, 카로티노이드가 있으며, 인간의 피부, 모발, 눈 등의 다양한 색깔은 이들 색소에 의하여 결정된다. 그 중 피부에 있어 색깔을 결정하는 가장 중요한 색소는 멜라닌(melanin)이며, 멜라닌의 성질 및 분포 정도에 따라 피부색이 결정된다.Pigments that determine skin color include melanin, hemoglobin, and carotenoids, and the various colors of human skin, hair, and eyes are determined by these pigments. Among them, the most important pigment that determines skin color is melanin, and skin color is determined by the nature and distribution of melanin.
멜라닌은 피부 기저층에 존재하는 색소 세포(melanocyte)에서 합성되며 주변 각질 세포로 전이되어 사람의 피부색을 나타낸다. 멜라닌이 비정상적으로 적게 되면 백반증과 같은 피부 병변이 유발되며, 반대로 과잉으로 생산될 경우에는 기미, 잡티를 형성하는 것으로 알려져 있다. Melanin is synthesized in pigment cells (melanocytes) present in the basal layer of the skin and is transferred to surrounding keratinocytes, giving a person's skin color. It is known that abnormally low melanin causes skin lesions such as vitiligo, and conversely, when melanin is produced in excess, it is known to form freckles and blemishes.
멜라닌 생성과 소멸 사이클은 다음과 같다. 멜라닌 세포(melanocytes)가 자극을 받으면, 세포내 함유되어 있는 티로신(tyrosine)이라는 아미노산으로부터 자극에 의해 활성화되는 티로시나아제(tyrosinase)가 멜라노좀(melanosome)에서 멜라닌을 생성한다. 이때, 티로신은 산소와 결합하여 산화되고 티로시나아제는 구리와 결합되는데, 만일 피부에 구리가 결핍되면 정상적인 멜라닌 생성에 장애를 주게 된다. 자외선 등의 자극은 멜라닌 생성 세포에 직접적인 영향을 미칠 뿐 아니라, 자극 호르몬계에도 영향을 미쳐 뇌하수체중엽에서 멜라닌 세포 자극호르몬 (melanocyte stimulating hormone, MSH)분비가 많아지면 멜라닌의 생성을 촉진시킨다. 멜라닌 세포 내에 포함되어 있는 티로신이라는 아미노산은 티로시나아제의 작용으로 도파(dopa)로 변하고, 이어 도파옥시다아제(dopaoxidase)의 작용으로 도파키논(dopa-quinone)으로 변한 후, 티로시나아제 관련 단백질-1(tyrosinase-related protein-1, 이하 TRP-1이라고 함)과 티로시나아제 관련 단백질-2(TRP-2)의 작용에 의하여 멜라닌을 생성한다. 이와 같이 생성된 멜라닌은 각질형성세포로 전달되고 표피 박리와 함께 멜라닌이 상실되어 소멸되는 순환 작용을 보인다. The melanin production and extinction cycle is as follows. When melanocytes are stimulated, tyrosinase, which is activated by stimulation from the amino acid tyrosine contained within the cells, produces melanin in melanosomes. At this time, tyrosine is oxidized by combining with oxygen, and tyrosinase is combining with copper. If copper is deficient in the skin, normal melanin production is impaired. Stimuli such as ultraviolet rays not only have a direct effect on melanin-producing cells, but also affect the stimulating hormone system, so that increased secretion of melanocyte stimulating hormone (MSH) from the middle lobe of the pituitary gland promotes the production of melanin. The amino acid tyrosine contained in melanocytes is converted into dopa through the action of tyrosinase, and then into dopa-quinone through the action of dopaoxidase, and then into tyrosinase-related protein-1. Melanin is produced through the action of tyrosinase-related protein-1 (hereinafter referred to as TRP-1) and tyrosinase-related protein-2 (TRP-2). The melanin produced in this way is delivered to keratinocytes and exhibits a cyclical effect in which melanin is lost and disappears with epidermal peeling.
국내 및 일본 등지에서 피부 미백효과 개념의 화장품들이 큰 시장을 형성하고 있는데, 미백 화장품에 있어 멜라닌 생성을 억제하는 방법은 크게 다음과 같이 나눌 수 있다. 첫째로, 자외선을 차단하여 멜라닌 생성의 주원인을 제거하는 방법으로, 이 방법은 화장품 조성물로서 광산란제 또는 광차단제를 함유케 하여 좋은 결과를 기대할 수 있다. 둘째로, 티로시나아제가 활성을 나타내기 위해 필요한 글루코사민(glucosamine)과 같은 코어 탄수화물의 합성을 저해함으로써 멜라닌 생성을 억제시키는 방법이다. 셋째로, 코직산(kojic acid) 또는 알부틴 등을 이용하여 멜라닌 생성에 관여하는 효소인 티로시나아제의 기능을 방해하는 방법이다. 넷째로, 하이드로퀴논과 같이 멜라닌을 생성하는 세포인 멜라닌 세포에 대하여 특이적인 독성을 가지고 있는 물질을 이용하여 멜라닌 세포의 분열을 방해하는 방법이다. 다섯째로, 생성된 멜라닌을 환원시켜 탈색하는 방법이 있다.Cosmetics with a skin whitening effect concept are forming a large market in Korea and Japan. Methods for suppressing melanin production in whitening cosmetics can be broadly divided into the following. First, it is a method of removing the main cause of melanin production by blocking ultraviolet rays. This method can expect good results by containing a light scattering agent or light blocking agent as a cosmetic composition. Second, it is a method of suppressing melanin production by inhibiting the synthesis of core carbohydrates such as glucosamine, which are required for tyrosinase to be active. Third, there is a method of interfering with the function of tyrosinase, an enzyme involved in melanin production, using kojic acid or arbutin. Fourth, it is a method of interfering with the division of melanocytes using a substance that has specific toxicity to melanocytes, which are cells that produce melanin, such as hydroquinone. Fifth, there is a method of decolorizing the produced melanin by reducing it.
한편, 종래에 티로시나아제의 활성억제제로서 미백화장품에 배합되는 원료들에는 아스코르빈산(비타민 C) 및 그 유도체, 상백피 추출물, 녹차 추출물, 알로에 추출물, 황금 추출물 등 식물 추출물뿐만 아니라, 코직산, 알부틴, 유용성 감초추출물 등을 들 수 있다. 그러나 각종 식물 추출물들은 불안정하며 제품에 배합할 경우 효과가 지속적이지 못하며, 티로시나아제 활성 억제력도 미미하다는 문제점이 있다. 또한 코직산(kojic acid)은 티로시나아제 억제제로서 통상적으로 사용되고 있으나, 알레르기 반응을 일으킬 수 있으며(Contact allergy to kojic acid in skin care products, Nakagawa M.et.al., Contact Dermatitis, Jan 95, vol 43(1), PP9-3) 제품 내에서 불안정하여 상기 물질을 함유한 조성물의 제조가 어렵다는 문제점이 있다.Meanwhile, raw materials that are conventionally mixed into whitening cosmetics as tyrosinase activity inhibitors include ascorbic acid (vitamin C) and its derivatives, botanical extracts such as Morus bark extract, green tea extract, aloe extract, and goldenrod extract, as well as kojic acid and arbutin. , oil-soluble licorice extract, etc. However, various plant extracts are unstable, their effects are not continuous when mixed into products, and their ability to inhibit tyrosinase activity is minimal. In addition, kojic acid is commonly used as a tyrosinase inhibitor, but it can cause allergic reactions (Contact allergy to kojic acid in skin care products, Nakagawa M.et.al., Contact Dermatitis, Jan 95, vol 43 (1), PP9-3) There is a problem that it is difficult to manufacture a composition containing the above material because it is unstable in the product.
종래 TAT(Trans-Activator of Transcription) 펩타이드는 서브타입의 유형에 따라 86개 내지 101개의 아미노산으로 구성되는, 후천성 면역 결핍 증후군을 발생시키는 HIV-1의 전사인사로서 N-터미널(a.a. 1-21) , 시스테인 리치(a.a. 22-37), 코어(a.a. 38-48), 베이직(a.a. 49-57), C-터미널(a.a. 73-86/101)의 5개의 기능적 도메인으로 분류되며, 감염된 세포에서 HIV-1의 전사 및 복제를 유도하고, 휴지기에 있는 바이러스를 재활성화 시키는 것으로 알려져 있다. Conventionally, TAT (Trans-Activator of Transcription) peptide is a transcription factor of HIV-1 that causes acquired immune deficiency syndrome, consisting of 86 to 101 amino acids depending on the subtype, and has an N-terminal (a.a. 1-21). , classified into five functional domains: cysteine rich (a.a. 22-37), core (a.a. 38-48), basic (a.a. 49-57), and C-terminal (a.a. 73-86/101), It is known to induce transcription and replication of -1 and reactivate dormant viruses.
TAT 펩타이드는 펩타이드 시퀀스의 중간 부위인 단백질 형질도입 부위(protein transduction domain)의 특성으로 자발적으로 세포막을 통과하여 쉽게 세포 내로 침투 및 이동할 수 있어, TAT의 작은 절편들(아미노산 9-11개)은 주로 CPP(cell penetrating peptides, 세포 투과 펩타이드)로서 연구되고 있으며, 나아가 TAT 펩타이드의 약력학적 효과에 대해서도 연구가 되어, 일부 TAT 펩타이드 절편의 비만억제 효과에 대한 연구도 알려져 있다.TAT peptide can spontaneously pass through the cell membrane and easily penetrate and move into cells due to the characteristics of the protein transduction domain, which is the middle part of the peptide sequence. Small fragments of TAT (amino acids 9-11) are mainly It is being studied as CPP (cell penetrating peptides), and furthermore, the pharmacodynamic effects of TAT peptides have been studied, and studies on the obesity-suppressing effects of some TAT peptide fragments are also known.
그러나 원래의 TAT(Trans-Activator of Transcription) 펩타이드의 중간 절편들의 아미노산 서열을 인위적으로 변형시킨 TATdMt(TAT double mutant) 펩타이드의 미백효과에 대해서는 보고된 바가 없다. However, there has been no report on the whitening effect of TATdMt (TAT double mutant) peptide, which artificially modified the amino acid sequence of the middle fragment of the original TAT (Trans-Activator of Transcription) peptide.
따라서 본 발명은 티로시나아제 활성 억제 및 멜라닌 생성을 억제하여 미백 효과가 우수한, 신규 미백 조성물을 제공한다.Therefore, the present invention provides a novel whitening composition that has excellent whitening effect by inhibiting tyrosinase activity and melanin production.
상기와 같은 과제를 해결하기 위하여, In order to solve the above problems,
본 발명은 서열번호 1, 서열번호 2 또는 서열번호 3의 아미노산 서열로 표현되는 TATdMt 펩타이드를 포함하는 미백 조성물을 제공한다.The present invention provides a whitening composition comprising the TATdMt peptide represented by the amino acid sequence of SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3.
본 발명에 있어서, 서열번호 1의 아미노산 서열은 TAT 펩타이드의 a.a 20-45에 위치하는 아미노산에 상응하며, 하나 이상의 아미노산 서열을 인위적으로 변형시킨 것일 수 있다.In the present invention, the amino acid sequence of SEQ ID NO: 1 corresponds to the amino acid located at a.a 20-45 of the TAT peptide, and one or more amino acid sequences may be artificially modified.
본 발명에 있어서, 상기 서열번호 2의 아미노산 서열은 TAT 펩타이드의 a.a 20-57에 위치하는 아미노산에 상응하며, 하나 이상의 아미노산 서열을 인위적으로 변형시킨 것일 수 있다.In the present invention, the amino acid sequence of SEQ ID NO: 2 corresponds to the amino acid located at a.a 20-57 of the TAT peptide, and one or more amino acid sequences may be artificially modified.
본 발명에 있어서, 상기 서열번호 3의 아미노산 서열은 TAT 펩타이드의 a.a 1-71에 위치하는 아미노산에 상응하며, 하나 이상의 아미노산 서열을 인위적으로 변형시킨 것일 수 있다.In the present invention, the amino acid sequence of SEQ ID NO: 3 corresponds to the amino acid located at a.a 1-71 of the TAT peptide, and one or more amino acid sequences may be artificially modified.
본 발명에 있어서, 상기 펩타이드의 농도는 0.5 내지 50 ㎍/㎖일 수 있다.In the present invention, the concentration of the peptide may be 0.5 to 50 μg/ml.
본 발명에 있어서, 상기 펩타이드의 함량이 미백 조성물 전체 중량에 대하여 0.001 내지 10 중량%일 수 있다.In the present invention, the content of the peptide may be 0.001 to 10% by weight based on the total weight of the whitening composition.
본 발명에 있어서, 상기 미백 조성물은 티로시나아제 활성을 억제시키는 것 일 수 있다.In the present invention, the whitening composition may inhibit tyrosinase activity.
본 발명에 있어서, 상기 미백 조성물은 멜라닌 생성을 억제시키는 것 일 수 있다.In the present invention, the whitening composition may inhibit melanin production.
본 발명에 있어서, 상기 서열번호 1의 아미노산 서열은 서열번호 4의 뉴클레오타이드 서열에 의해 코딩되는 것일 수 있다.In the present invention, the amino acid sequence of SEQ ID NO: 1 may be encoded by the nucleotide sequence of SEQ ID NO: 4.
본 발명에 있어서, 상기 서열번호 2의 아미노산 서열은 서열번호 5의 뉴클레오타이드 서열에 의해 코딩되는 것일 수 있다.In the present invention, the amino acid sequence of SEQ ID NO: 2 may be encoded by the nucleotide sequence of SEQ ID NO: 5.
본 발명에 있어서, 상기 서열번호 3의 아미노산 서열은 서열번호 6의 뉴클레오타이드 서열에 의해 코딩되는 것일 수 있다.In the present invention, the amino acid sequence of SEQ ID NO: 3 may be encoded by the nucleotide sequence of SEQ ID NO: 6.
본 발명에 있어서, 상기 조성물은 화장수, 유액, 크림, 팩, 미용액 및 피부외용 연고로 이루어지는 군으로부터 선택된 것일 수 있다.In the present invention, the composition may be selected from the group consisting of lotion, emulsion, cream, pack, serum, and ointment for external use.
상기에서 살펴본 바와 같이, 본 발명의 서열번호 1, 서열번호 2 또는 서열번호 3의 아미노산 서열로 표현되는 인위적 돌연변이에 의한 TATdMt 펩타이드를 포함하는 미백 조성물은 티로시나아제 활성 억제 및 멜라닌 생성을 억제하여 피부를 미백시키는 효과가 우수하다. 그 결과, 화장품, 의약품, 식품 등 다양한 분야에 적용할 수 있다.As seen above, the whitening composition containing the artificially mutated TATdMt peptide represented by the amino acid sequence of SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3 of the present invention inhibits tyrosinase activity and melanin production to improve skin health. It has excellent whitening effect. As a result, it can be applied to various fields such as cosmetics, medicine, and food.
도 1은 본 발명의 일 실시예에 따른 세포 독성 시험 결과를 나타낸 것이다.
도 2는 본 발명의 일 실시예에 따른 펩타이드 무처리군 및 처리군의 세포 형태를 관찰한 사진을 나타낸 것이다.
도 3은 본 발명의 일 실시예에 따른 멜라닌 생성 억제 결과를 나타낸 것이다.
도 4는 본 발명의 일 실시예에 따른 티로시나아제 활성 억제 결과를 나타낸 것이다.
도 5는 본 발명의 일 실시예에 따른 티로시나아제 mRNA 발현량 측정 결과를 나타낸 것이다.
도 6은 본 발명의 일 실시예에 따른 MITF mRNA 발현량 측정 결과를 나타낸 것이다.Figure 1 shows the results of a cytotoxicity test according to an embodiment of the present invention.
Figure 2 shows a photograph observing the cell morphology of the peptide untreated and treated groups according to an embodiment of the present invention.
Figure 3 shows the results of inhibiting melanin production according to an embodiment of the present invention.
Figure 4 shows the results of tyrosinase activity inhibition according to an embodiment of the present invention.
Figure 5 shows the results of measuring tyrosinase mRNA expression level according to an embodiment of the present invention.
Figure 6 shows the results of measuring MITF mRNA expression level according to an embodiment of the present invention.
이하, 본 발명을 보다 상세하게 설명한다. Hereinafter, the present invention will be described in more detail.
종래 TAT(Trans-Activator of Transcription) 펩타이드는 서브타입의 유형에 따라 86개 내지 101개의 아미노산으로 구성되는, 후천성 면역 결핍 증후군을 발생시키는 HIV-1의 전사인사로서 N-터미널(a.a. 1-21) , 시스테인 리치(a.a. 22-37), 코어(a.a. 38-48), 베이직(a.a. 49-57), C-터미널(a.a. 73-86/101)의 5개의 기능적 도메인으로 분류되며, 감염된 세포에서 HIV-1의 전사 및 복제를 유도하고, 휴지기에 있는 바이러스를 재활성화 시키는 것으로 알려져 있다. Conventionally, TAT (Trans-Activator of Transcription) peptide is a transcription factor of HIV-1 that causes acquired immune deficiency syndrome, consisting of 86 to 101 amino acids depending on the subtype, and has an N-terminal (a.a. 1-21). , classified into five functional domains: cysteine rich (a.a. 22-37), core (a.a. 38-48), basic (a.a. 49-57), and C-terminal (a.a. 73-86/101), It is known to induce transcription and replication of -1 and reactivate dormant viruses.
TAT 펩타이드는 펩타이드 시퀀스의 중간 부위인 단백질 형질도입 부위(protein transduction domain)의 특성으로 자발적으로 세포막을 통과하여 쉽게 세포 내로 침투 및 이동할 수 있어, TAT의 작은 절편들(아미노산 9-11개)은 주로 CPP(cell penetrating peptides, 세포 투과 펩타이드)로서 연구되고 있다. 예를 들어 특허 등록번호 10-0817191호 “TAT 펩타이드가 결합된 아스코르브산 유도체, 그의 제조방법 및 이를 포함하는 피부 미백용 화장료 조성”는 TAT 펩타이드를 세포투과성을 높이는데 사용하고 있다.TAT peptide can spontaneously pass through the cell membrane and easily penetrate and move into cells due to the characteristics of the protein transduction domain, which is the middle part of the peptide sequence. Small fragments of TAT (amino acids 9-11) are mainly It is being studied as CPP (cell penetrating peptides). For example, Patent Registration No. 10-0817191, “Ascorbic acid derivative bound to TAT peptide, manufacturing method thereof, and cosmetic composition for skin whitening comprising the same,” uses TAT peptide to increase cell permeability.
본 명세서에서 용어 “펩타이드”는 펩타이드 결합에 의해 아미노산 잔기들이 서로 결합되어 형성된 분자를 의미한다. As used herein, the term “peptide” refers to a molecule formed by combining amino acid residues with each other through peptide bonds.
본 명세서에서 용어 “TATdMt(TAT double mutant)”는 원래의 TAT 펩타이드의 중간 절편들의 아미노산 서열을 인위적으로 변형시킨 것을 의미한다.As used herein, the term “TATdMt (TAT double mutant)” refers to artificially modifying the amino acid sequence of the middle fragments of the original TAT peptide.
본 발명은, 서열번호 1, 서열번호 2 또는 서열번호 3의 아미노산 서열로 표현되는 TATdMt(TAT double mutant) 펩타이드를 포함하는 미백 조성물을 제공한다. The present invention provides a whitening composition comprising a TATdMt (TAT double mutant) peptide represented by the amino acid sequence of SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3.
상기 서열번호 1의 아미노산 서열은 TAT 펩타이드의 a.a 20-45에 위치하는 아미노산에 상응하며, 하나 이상의 아미노산 서열을 인위적으로 변형시킨 것이 바람직할 수 있으며, 더욱 바람직하게는 TACTNCYC A KCCFHCQVCFITKALGI 일 수 있다.The amino acid sequence of SEQ ID NO: 1 corresponds to the amino acid located at aa 20-45 of the TAT peptide, and it may be preferable to artificially modify one or more amino acid sequences, more preferably TACTNCYC A KCCFHCQVCFITKALGI.
상기 서열번호 2의 아미노산 서열은 TAT 펩타이드의 a.a 20-57에 위치하는 아미노산에 상응하며, 하나 이상의 아미노산 서열을 인위적으로 변형시킨 것이 바람직 할 수 있으며, 더욱 바람직하게는 TACTNCYC A KCCFHCQVCFITKALGISYGR A KRRQRR일 수 있다.The amino acid sequence of SEQ ID NO: 2 corresponds to the amino acid located at aa 20-57 of the TAT peptide, and it may be preferable to artificially modify one or more amino acid sequences, more preferably TACTNCYC A KCCFHCQVCFITKALGISYGR A KRRQRR. .
상기 서열번호 3의 아미노산 서열은 TAT 펩타이드의 a.a 1-71에 위치하는 아미노산에 상응하며, 하나 이상의 아미노산 서열을 인위적으로 변형시킨 것이 바람직 할 수 있으며, 더욱 바람직하게는 MEPVNPSLEPWKHPGSQPKTACTNCYC A KCCFHCQVCFITKA LGISYGR A KRRQRRRPPQGSQTHQVSLSK 일 수 있다.The amino acid sequence of SEQ ID NO: 3 corresponds to the amino acid located at aa 1-71 of the TAT peptide, and it may be preferable to artificially modify one or more amino acid sequences, more preferably MEPVNPSLEPWKHPGSQPKTACTNCYC A KCCFHCQVCFITKA LGISYGR A KRRQRRRPPQGSQTHQVSLSK there is.
상기 인위적 돌연변이는 TAT 펩타이드의 a.a. 28 및/또는 a.a.50의 아미노산을 변이시키는 것이 바람직 할 수 있으며, 더욱 바람직하게는 TAT 펩타이드의 a.a. 28의 라이신(Lys, K)을 알라닌(Ala, A)으로 및/또는 a.a. 50의 라이신(Lys, K) 또는 글라이신(Gly, G)을 알라닌(Ala, A)으로 변이 시키는 것 일 수 있다.The artificial mutation is a.a. of the TAT peptide. It may be desirable to mutate amino acids 28 and/or a.a.50, more preferably a.a.50 of the TAT peptide. 28 lysine (Lys, K) to alanine (Ala, A) and/or a.a. It may be mutating 50 lysine (Lys, K) or glycine (Gly, G) to alanine (Ala, A).
상기 아미노산 서열에 하나 이상의 변이를 유발하여 형성된 본 발명에서 목적하고자 하는 활성을 가진 모든 펩타이드들도 본 발명의 보호범위에 포함된다. All peptides having the desired activity in the present invention formed by causing one or more mutations in the amino acid sequence are also included in the protection scope of the present invention.
본 발명의 상기 TAT의 변이(Mutation)된 절편들(TatdMt)은 그 자체로 우수한 미백효과가 있음을 확인하였다.It was confirmed that the mutated fragments (TatdMt) of TAT of the present invention have an excellent whitening effect in themselves.
본 발명의 목적과 관련된 기능을 가지는 서열번호 1, 서열번호 2 또는 서열번호 3의 절편 및 상기 아미노산 서열에 하나 이상의 변이를 유발하여 본 발명에서 목적하고자 하는 활성을 가진 펩타이드를 사용할 수 있으며, 서열번호 1, 서열번호 2 또는 서열번호 3의 절편만을 사용하는 경우 합성이 용이하고, 효과가 우수하며, 부작용이 없거나 적을 수 있다.A fragment of SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3, which has a function related to the purpose of the present invention, and a peptide having the desired activity in the present invention by causing one or more mutations in the amino acid sequence can be used, and SEQ ID NO: 1, If only the fragment of SEQ ID NO 2 or SEQ ID NO 3 is used, synthesis is easy, the effect is excellent, and there may be no or few side effects.
상기 미백 조성물은 MITF(마이크로프탈미아-연관 전사인자, microphthalmia-associated transcription factor)의 발현을 감소시킬 수 있다. The whitening composition can reduce the expression of MITF (microphthalmia-associated transcription factor).
본 발명의 서열번호 1, 서열번호 2 또는 서열번호 3의 아미노산 서열을 포함하는 펩타이드는, TAT(Trans activator of transcription) 펩타이드의 일부 절편의 시퀀스가 하나 이상 변이(mutant)된 형태일 수 있으며, 종래 알려진 TAT 펩타이드의 세포 투과 기능 외에, 멜라닌 생성 관여 유전자인 MITF의 발현을 감소시킴으로써 티로시나아제의 활성을 억제시키고 멜라닌 합성을 현저히 감소시킬 수 있다.The peptide containing the amino acid sequence of SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3 of the present invention may be in the form of one or more mutant sequences of some fragments of the TAT (Trans activator of transcription) peptide, and may be In addition to the known cell-penetrating function of TAT peptide, it can inhibit the activity of tyrosinase and significantly reduce melanin synthesis by reducing the expression of MITF, a gene involved in melanin production.
피부색은 피부에 존재하는 멜라닌 색소에 의해서 결정된다. 멜라닌 합성 과정에서 티로시나아제(Tyrosinase), 티로시나아제 관련 단백질 1(Tyrosinase related protein 1, TRP1) 및 티로시나아제 관련 단백질 2(Trysoinase related protein 2, TRP2)와 같은 효소들이 중요한 역할을 하며, 상기 효소들은 MITF 전사인자에 의해서 발현이 조절될 수 있다. MITF는 bHLH-Zip( basic-helix-loop-helix-leucine zipper)의 구조를 갖는 전사인자로 E-box(Enhancer box)라는 특정 염기 서열을 인식하고 결합함으로써 여러 유전자들의 발현을 조절한다. 도 5 및 도 6으로부터 MITF와 티로시나아제의 mRNA의 발현양을 확인한 결과, 본 발명의 서열번호 1, 서열번호 2 또는 서열번호 3의 아미노산 서열을 포함하는 펩타이드는 결과적으로 티로시나아제의 활성을 현저히 저하시키며, MITF의 발현을 감소시키는 것을 확인할 수 있었다.Skin color is determined by the melanin pigment present in the skin. In the process of melanin synthesis, enzymes such as tyrosinase, tyrosinase related protein 1 (TRP1), and tyrosinase related protein 2 (TRP2) play an important role. The expression of enzymes can be regulated by the MITF transcription factor. MITF is a transcription factor with the structure of bHLH-Zip (basic-helix-loop-helix-leucine zipper) and regulates the expression of several genes by recognizing and binding to a specific nucleotide sequence called E-box (Enhancer box). As a result of confirming the expression level of MITF and tyrosinase mRNA from Figures 5 and 6, the peptide containing the amino acid sequence of SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3 of the present invention resulted in tyrosinase activity. It was confirmed that it significantly decreased the expression of MITF.
본 발명에 있어서, 상기 서열번호 1, 서열번호 2 또는 서열번호 3의 아미노산 서열로 표현되는 TATdMt펩타이드의 농도는 0.5 내지 50 ㎍/㎖인 것이 바람직하며, 더욱 바람직하게는 0.5 내지 25 ㎍/㎖일 수 있다. 도 1 및 도 2에 나타난 것과 같이, 서열번호 1, 서열번호 2 또는 서열번호 3의 아미노산 서열로 표현되는 TATdMt 펩타이드는 독성을 거의 나타내지 않았으며, 상기 TATdMt 펩타이드를 50㎍/㎖ 이하의 농도로 처리한 경우 70%이상의 세포생존율을 나타내었고, 25㎍/㎖ 이하의 농도로 처리한 경우 80%이상의 우수한 세포생존율을 나타내는 것을 관찰할 수 있었다. 상기의 결과로 상기 TATdMt 펩타이드의 안정성이 우수한 것을 확인할 수 있었다. In the present invention, the concentration of the TATdMt peptide represented by the amino acid sequence of SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3 is preferably 0.5 to 50 μg/ml, more preferably 0.5 to 25 μg/ml day. You can. As shown in Figures 1 and 2, the TATdMt peptide represented by the amino acid sequence of SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3 showed little toxicity, and the TATdMt peptide was treated at a concentration of 50 μg/ml or less. In one case, a cell viability of more than 70% was observed, and when treated at a concentration of 25㎍/㎖ or less, an excellent cell viability of more than 80% was observed. As a result of the above, it was confirmed that the stability of the TATdMt peptide was excellent.
본 발명에 있어서, 상기 서열번호 1, 서열번호 2 또는 서열번호 3의 아미노산 서열로 표현되는 TATdMt펩타이드의 함량은, 미백 조성물 전체 중량에 대하여 0.001 내지 10 중량% 인 것이 바람직하며, 더욱 바람직하게는 0.01 내지 5중량%일 수 있다. 상기 TATdMt 펩타이드의 함량이 0.001 중량% 미만일 경우에는 미백 효과가 미미하며, 10 중량%를 초과할 경우에는 낮은 용해도로 인하여 제형 및 제품의 안정성에 좋지 않은 영향을 미칠 수 있기 때문이다.In the present invention, the content of TATdMt peptide represented by the amino acid sequence of SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3 is preferably 0.001 to 10% by weight, more preferably 0.01%, based on the total weight of the whitening composition. It may be from 5% by weight. If the content of the TATdMt peptide is less than 0.001% by weight, the whitening effect is minimal, and if it exceeds 10% by weight, it may adversely affect the stability of the formulation and product due to low solubility.
본 발명에 있어서, 상기 서열번호 1의 아미노산 서열은 서열번호 4의 뉴클레오타이드 서열에 의해 코딩 될 수 있으며, 상기 서열번호 4의 뉴클레오타이드 서열은 5'-ACTGCTTGTACCAATTGCTATTGTGGAAAGTGTTGCTTTCATTGCCAAGTTTGTTTCATAACAAAAGCCTT AGGCATC-3' 인 것이 바람직할 수 있다.In the present invention, the amino acid sequence of SEQ ID NO: 1 may be encoded by the nucleotide sequence of SEQ ID NO: 4, and the nucleotide sequence of SEQ ID NO: 4 may be preferably 5'-ACTGCTTGTACCAATTGCTATTGTGGAAAGTGTTGCTTTCATTGCCAAGTTTGTTTCATAACAAAAGCCTT AGGCATC-3'.
본 발명에 있어서, 상기 서열번호 2의 아미노산 서열은 서열번호 5의 뉴클레오타이드 서열에 의해 코딩 될 수 있으며, 상기 서열번호 5의 뉴클레오타이드 서열은 5'-ACTGCTTGTACCAATTGCTATTGTGGAAAGTGTTGCTTTCATTGCCAAGTTTGTTTCATAACAAAAGCCTT AGGCATCTCCTATGGCAGGGCAAAGCGGAGACAGCGACGAAGA -3' 인 것이 바람직할 수 있다.In the present invention, the amino acid sequence of SEQ ID NO: 2 may be encoded by the nucleotide sequence of SEQ ID NO: 5, and the nucleotide sequence of SEQ ID NO: 5 may be 5'-ACTGCTTGTACCAATTGCTATTGTGGAAAGTGTTGCTTTCATTGCCAAGTTTGTTTCATAACAAAAGCCTT AGGCATCTCCTATGGCAGGGCAAGCGGAGACAGCGACGAAGA -3'.
본 발명에 있어서, 상기 서열번호 3의 아미노산 서열은 서열번호 6의 뉴클레오타이드 서열에 의해 코딩 될 수 있으며, 상기 서열번호 6의 뉴클레오타이드 서열은 5'-ATGGAGCCAGTAAATCCTAGCCTAGAGCCCTGGAAGCATCCAGGAAGTCAGCCTAAAACTGCTTGTACCA ATTGCTATTGTGCAAAGTGTTGCTTTCATTGCCAAGTTTGTTTCATAACAAAAGCCTTAGGCATCTCCTATGGCAGGGCAAAGCGGAGACAGCGACGAAGACCTCCTCAAGGCAGTCAGACTCATCAAGTTTCTCTATCAAAG-3' 인 것이 바람직할 수 있다.In the present invention, the amino acid sequence of SEQ ID NO: 3 can be encoded by the nucleotide sequence of SEQ ID NO: 6, and the nucleotide sequence of SEQ ID NO: 6 is 5'-ATGGAGCCAGTAAATCCTAGCCTAGAGCCCTGGAAGCATCCAGGAAGTCAGCCTAAAACTGCTTGTACCA ATTGCTATTGTGCAAAGTGTTGCTTTCATTGCCAAGTTTGTTTCATAACAAAAGCCTTAGGCATCTCCTATGGCAGGGCAAAGCGGAGA CAGCGACGAAGACCTCCTCAAGGCAGTCAGACTCATCAAGTTTCTCTATCAAAG-3' may be preferred.
상기 뉴클레오타이드는 첨부한 서열 목록에 기재된 뉴클레오타이드 서열에 한정되지 않는다는 것은 통상의 기술자들에게 자명한 것이며, 뉴클레오타이드에서의 변이는 펩타이드에서 변화를 가져오지 않을 수도 있다. 이러한 뉴클레오타이드는 기능적으로 균등한 코돈 또는 동일한 아미노산을 코딩하는 코돈(예를 들어, 코돈의 축퇴성에 의해, 아르기닌 또는 세린에 대한 코돈은 여섯 개이다), 또는 생물학적으로 균등한 아미노산을 코딩하는 코돈을 포함하는 뉴클레오타이드를 포함한다. It is obvious to those skilled in the art that the nucleotide sequence is not limited to the nucleotide sequence described in the attached sequence list, and variations in the nucleotide may not result in changes in the peptide. These nucleotides include functionally equivalent codons or codons that code for identical amino acids (e.g., due to codon degeneracy, there are six codons for arginine or serine), or codons that code for biologically equivalent amino acids. Contains nucleotides that
상술한 생물학적 균등 활성을 갖는 변이를 고려한다면, 본 발명에서 이용되는 뉴클레오타이드 분자는 서열 목록에 기재된 서열과 실질적으로 동일성(substantial identity)을 나타내는 서열도 포함하는 것으로 해석된다. 상기 실질적인 동일성은, 상기 본 발명의 서열번호 4, 서열번호 5 또는 서열번호 6와 임의의 다른 서열을 최대한 대응되도록 얼라인(align)하고, 본 발명이 속하는 기술 분야에서 통상적으로 이용되는 알고리즘을 이용하여 얼라인된 서열을 분석한 경우에, 최소 60%의 상동성, 보다 바람직하게는 70%의 상동성, 보다 더 바람직하게는 80%의 상동성, 가장 바람직하게는 90%의 상동성을 나타내는 서열을 의미한다. 서열 비교를 위한 얼라인먼트 방법은 본 발명이 속하는 기술 분야에 공지되어 있다. 얼라인먼트에 대한 다양한 방법 및 알고리즘은 Smith and Waterman, Adv. Appi. Math. 2:482 (1981); Needleman and Wunsch, J. Mol. Bio. 48:443 (1970); Pearson and Lipman, Methods in Mol. Biol. 24: 307-31 (1988); Higgins and Sharp, Gene 73:237-44 (1988); Higgins and Sharp, CABIOS 5:151-3 (1989) Corpet et al., Nuc. Acids Res. 16:10881-90 (1988) Huang et al., Comp. Appi. BioSci. 8:155-65 (1992) and Pearson et al., Meth. Mol. Biol. 24:307-31 (1994)에 개시되어 있으며, The NCBI Basic Local Alignment Search Tool (BLAST) [Altschul et al., J. Mol. Biol. 215:403-10 (1990)]는 National Center for Biological Information (NBCI, Bethesda, Md.) 등을 이용하여 접근 가능하며, 인터넷 상에서 blastp, blasm, blastx, tblastn 및 tblastx와 같은 서열 분석 프로그램과 연동되어 이용할 수 있다. Considering the mutations having the above-mentioned biological equivalent activity, the nucleotide molecules used in the present invention are interpreted to also include sequences showing substantial identity with the sequences described in the sequence listing. The substantial identity is achieved by aligning SEQ ID NO: 4, SEQ ID NO: 5, or SEQ ID NO: 6 of the present invention and any other sequence to correspond as much as possible, and using an algorithm commonly used in the technical field to which the present invention pertains. When the aligned sequence is analyzed, it exhibits at least 60% homology, more preferably 70% homology, even more preferably 80% homology, and most preferably 90% homology. It means sequence. Alignment methods for sequence comparison are known in the art to which the present invention pertains. Various methods and algorithms for alignment are discussed in Smith and Waterman, Adv. Appi. Math. 2:482 (1981); Needleman and Wunsch, J. Mol. Bio. 48:443 (1970); Pearson and Lipman, Methods in Mol. Biol. 24: 307-31 (1988); Higgins and Sharp, Gene 73:237-44 (1988); Higgins and Sharp, CABIOS 5:151-3 (1989) Corpet et al., Nuc. Acids Res. 16:10881-90 (1988) Huang et al., Comp. Appi. BioSci. 8:155-65 (1992) and Pearson et al., Meth. Mol. Biol. 24:307-31 (1994), The NCBI Basic Local Alignment Search Tool (BLAST) [Altschul et al., J. Mol. Biol. 215:403-10 (1990)] can be accessed using the National Center for Biological Information (NBCI, Bethesda, Md.), etc., and is linked to sequence analysis programs such as blastp, blasm, blastx, tblastn, and tblastx on the Internet. Available.
서열번호 1, 서열번호 2 또는 서열번호 3의 아미노산 서열을 코딩하는 뉴클레오타이드는 유전자 전달체(gene delivery system)에 포함되어 있다. Nucleotides encoding the amino acid sequence of SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3 are included in the gene delivery system.
본 발명에서 용어“유전자 전달체”는 원하는 타겟 유전자를 대상 세포에 도입하여 발현시키기 위한 매개체를 의미한다. 이상적인 유전자 전달체는 인체에 무해하고 대량 생산이 용이하며 효율적으로 유전자를 전달할 수 있어야 한다.In the present invention, the term “gene carrier” refers to a vehicle for introducing and expressing a desired target gene into a target cell. An ideal gene carrier should be harmless to the human body, easy to mass produce, and efficiently deliver genes.
또한, “유전자 전달”은 유전자가 세포 내로 운반되는 것을 의미하며, 유전자의 세포내 침투(transduction)와 동일한 의미를 가진다. 조직 수준에서, 유전자 전달은 유전자의 확산(spread)과 동일한 의미를 가진다. 따라서 본 발명의 유전자 전달체는 유전자 침투 시스템 및 유전자 확산 시스템으로 기재될 수 있다. Additionally, “gene transfer” means the transfer of genes into cells and has the same meaning as transduction of genes into cells. At the tissue level, gene transfer is synonymous with gene spread. Therefore, the gene delivery system of the present invention can be described as a gene penetration system and a gene diffusion system.
본 발명의 유전자 전달체를 제조하기 위해, 본 발명의 뉴클레오타이드 서열은 적합한 발현 컨스트럭트(expression construct) 내에 존재하는 것 일 수 있다. To prepare the gene delivery vehicle of the present invention, the nucleotide sequence of the present invention may be present in a suitable expression construct.
상기 발현 컨스트럭트에서, 본 발명의 뉴클레오타이드 서열은 프로모터에 작동적으로 연결될 수 있다. In the expression construct, the nucleotide sequence of the invention can be operably linked to a promoter.
용어 “작동적으로 연결”은 핵산 발현 조절 서열(예: 프로모터, 시그널 서열, 또는 전사조절인자 결합 위치의 어레이)과 다른 핵산 서열 사이에 기능적인 결합을 의미하며, 이에 상기 조절 서열은 상기 다른 핵산 서열의 전사 및/또는 해독을 조절하게 된다. The term “operably linked” refers to a functional linkage between a nucleic acid expression control sequence (e.g., a promoter, signal sequence, or array of transcriptional regulator binding sites) and another nucleic acid sequence, wherein the control sequence is connected to the other nucleic acid. It regulates the transcription and/or translation of the sequence.
본 발명의 뉴클레오타이드 서열에 결합된 프로모터는, 동물세포, 포유동물 세포에서 작동하여 유전자의 전사를 조절할 수 있는 것으로서, 포유동물 바이러스로부터 유래된 프로모터 및 포유동물 세포의 지놈으로부터 유래된 프로모터를 포함할 수 있으며, 예를 들어 CMV(포유동물 사이토 메갈로 바이러스)프로모터, 아데노바이러스 프로모터, 백시니아 바이러스 7.5K 프로모터, SV40 프로모터, HSV의 tk 프로모터, RSV 프로모터, EF1 알파 프로모터, 메탈로티오닌 프로모터, 베타-액틴 프로모터, 인간 IL-2 유전자의 프로모터, 인간 IFN 유전자의 프로모터, 인간 IL-4 유전자의 프로모터, 인간 림포톡신 유전자의 프로모터 및 인간 GM-CSF 유전자의 프로모터를 포함할 수 있으며, 이에 한정되는 것은 아니다.The promoter bound to the nucleotide sequence of the present invention is capable of operating in animal cells and mammalian cells to regulate gene transcription, and may include a promoter derived from a mammalian virus and a promoter derived from the genome of a mammalian cell. For example, CMV (mammalian cytomegalovirus) promoter, adenovirus promoter, vaccinia virus 7.5K promoter, SV40 promoter, HSV tk promoter, RSV promoter, EF1 alpha promoter, metallothioneine promoter, beta-actin It may include, but is not limited to, a promoter, the promoter of the human IL-2 gene, the promoter of the human IFN gene, the promoter of the human IL-4 gene, the promoter of the human lymphotoxin gene, and the promoter of the human GM-CSF gene.
유전자 전달체는 다양한 형태로 제작할 수 있는데, 이는 내이키드(naked) 재조합 DNA 분자, 플라스미드, 바이러스 벡터, 내이키드 재조합 DNA 분자 또는 플라스미드를 내포하는 리포좀 또는 니오좀의 형태로 제작할 수 있다. TATdMt 펩타이드-인코딩 뉴클레오타이드 서열은 통상적인 유전자 전달 시스템에 적용될 수 있으며, 플라스미드, 아데노바이러스(Lockett U, et al., Clin. Cancer Res. 3:2075-2080 (1997)), 아데노-관련 바이러스(AAV, Lashford L S., et al., Gene Therapy Technologies, Applications and Regulations Ed. A. Meager, 1999), 레트로바이러스(Gunzberg W H, et al., Retroviral vectors. Gene Therapy Technologies, Applications and Regulations Ed. A. Meager, 1999), 렌티바이러스 (Wang G. et al., J. Clin. Invest. 104(11):R55-62(1999)), 헤르페스 심플렉스 바이러스 (Chamber R., et al., Proc. Natl. Acad. Sci. USA 92:1411-1415 (1995)), 백시니아 바이러스(Puhlmann M. et al., Human Gene Therapy 10:649-657 (1999)), 리포좀(Methods in Molecular Biology, Vol 199, S.C. Basu and M. Basu (Eds.), Human Press 2002) 또는 니오좀에 적용될 수 있고, 바람직하게는 TATdMt를 코딩하는 뉴클레오타이드를 플라스미드에 적용하여 제조될 수 있다.Gene carriers can be manufactured in various forms, such as naked recombinant DNA molecules, plasmids, viral vectors, liposomes or niosomes containing naked recombinant DNA molecules or plasmids. The TATdMt peptide-encoding nucleotide sequence can be applied to conventional gene delivery systems, including plasmids, adenovirus (Lockett U, et al., Clin. Cancer Res. 3:2075-2080 (1997)), and adeno-associated virus (AAV). , Lashford L S., et al., Gene Therapy Technologies, Applications and Regulations Ed. A. Meager, 1999), Retroviruses (Gunzberg W H, et al., Retroviral vectors. Gene Therapy Technologies, Applications and Regulations Ed. A. Meager, 1999), lentivirus (Wang G. et al., J. Clin. Invest. 104(11):R55-62(1999)), herpes simplex virus (Chamber R., et al., Proc. Natl Acad. Sci. USA 92:1411-1415 (1995)), vaccinia virus (Puhlmann M. et al., Human Gene Therapy 10:649-657 (1999)), liposome (Methods in Molecular Biology, Vol 199, S.C. Basu and M. Basu (Eds.), Human Press 2002) or niosomes, and preferably can be prepared by applying the nucleotide encoding TATdMt to a plasmid.
유전자 전달체가 바이러스 벡터에 기초하여 제작된 경우, 상기 접촉시키는 단계에는 본 발명이 속하는 기술 분야에서 공지된 바이러스 감염 방법에 따라 실시된다. 또는 유전자 전달체가 내이키드(naked) 재조합 DNA 분자 또는 플라스미드인 경우, 미세주입법 (Capecchi, M.R., Cell, 22:479 (1980) and Harland and Weintraub, J. Cell Biol. 101:1094-1099 (1985)), 칼슘 포스페이트 침전법(calcium phosphate co-precipitation (Graham, F. L. et al., Virology, 52:456 (1973) and Chen and Okayama, Mol. Cell. Biol. 7:2745-2752 (1987)), 전기천공법 (Neumann, E. et al., EMBO J., 1:841 (1982) and Tur-Kaspa et al., Mol. Cell. Biol., 6:716-718 (1986)), 리포좀 매개 형질감염법(Wong, T. K. et al., Gene, 10:87 (1980) and Nicolau and Sene, Biochim. Biophys. Acta, 721:185-190 (1982); and Nicolau et al., Methods Enzymol., 149:157-176 (1987)), DEAE-덱스트란 처리법 (Gopal, Mol. Cell. Biol., 5:1188-1190 (1985)) 및 유전자 밤바드먼트 방법(Yang et al., Proc. Natl. Acad. Sci., 87:9568-9572 (1990))에 의해 유전자를 세포 내로 이입시킬 수 있다.When the gene carrier is produced based on a viral vector, the contacting step is performed according to a viral infection method known in the art to which the present invention pertains. or when the gene carrier is a naked recombinant DNA molecule or plasmid, microinjection (Capecchi, M.R., Cell, 22:479 (1980) and Harland and Weintraub, J. Cell Biol. 101:1094-1099 (1985) ), calcium phosphate co-precipitation (Graham, F. L. et al., Virology, 52:456 (1973) and Chen and Okayama, Mol. Cell. Biol. 7:2745-2752 (1987)), electric Perforation (Neumann, E. et al., EMBO J., 1:841 (1982) and Tur-Kaspa et al., Mol. Cell. Biol., 6:716-718 (1986)), liposome-mediated transfection Wong, T. K. et al., Gene, 10:87 (1980) and Nicolau and Sene, Biochim. Biophys. Acta, 721:185-190 (1982); and Nicolau et al., Methods Enzymol., 149:157 -176 (1987)), DEAE-dextran treatment method (Gopal, Mol. Cell. Biol., 5:1188-1190 (1985)), and gene bombardment method (Yang et al., Proc. Natl. Acad. Sci ., 87:9568-9572 (1990)), genes can be transferred into cells.
펩타이드를 제조하는 방법으로서, 본 발명이 속하는 기술분야에서 통상의 방법을 사용할 수 있다. 예를 들어, 재조합 발현벡터를 이용하는 생물학적 방법으로서, cDNA절편(서열번호 4, 서열번호 5 또는 서열번호 6)을 pGex4T3 벡터에 클로닝(cloning)하여 융합 단백질의 형태로 발현시킬 수 있는 벡터를 제조하는 단계, 그리고 상기 발현벡터를 이용하여 대장균 내에서 재조합 TATdMt 펩타이드의 형태로 대량 발현시킨 후, 순수 분리 정제하는 단계를 포함하는 TATdMt 펩타이드의 제조 방법이다. As a method for producing peptides, conventional methods in the technical field to which the present invention pertains can be used. For example, as a biological method using a recombinant expression vector, a cDNA fragment (SEQ ID NO: 4, SEQ ID NO: 5, or SEQ ID NO: 6) is cloned into the pGex4T3 vector to prepare a vector that can be expressed in the form of a fusion protein. This is a method of producing a TATdMt peptide, including the step of mass-expressing the recombinant TATdMt peptide in E. coli using the expression vector, followed by separation and purification.
또는 펩타이드 합성용 유기합성기기를 이용하여 순수한 TATdMt 펩타이드(서열번호 1, 서열번호 2 또는 서열번호 3)를 합성하는 방법으로, Merrifield의 고체상(solid phase) 펩타이드 합성법으로서(J. Am. Chem. Soc. 85, 2149-2154(1963)) 펩타이드 합성기 등을 이용할 수 있다. 고체 지지체 레진에 붙어있는 펩타이드 체인의 아미노 말단에 순차적으로 a-아미노기가 보호된 아미노산을 활성화 시킨 후 커플링시키며, 합성이 끝나면 레진으로부터 펩타이드를 절단한다. 이 때, a-아미노기가 보호된 아미노산을 히드록시 메틸 레진이나 클로로메틸레이티드 레진에 에스테르 결합으로 붙인다. 보호그룹은 트리플로오로아세트산(TPA)과 같은 시약으로 제거한다. 펩타이드는 TFA 용액으로부터 여과나 원심분리 또는 디에틸에테르로 추출하여 분리하며 고성능 액체크로마토그래피(HPLC)나 다른 방법으로 정제할 수 있다.Alternatively, a method of synthesizing pure TATdMt peptide (SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3) using an organic synthesis device for peptide synthesis, using Merrifield's solid phase peptide synthesis method (J. Am. Chem. Soc 85, 2149-2154 (1963)), a peptide synthesizer, etc. can be used. Amino acids with protected a-amino groups are sequentially activated and coupled to the amino terminus of the peptide chain attached to the solid support resin, and upon completion of synthesis, the peptide is cleaved from the resin. At this time, the amino acid with the a-amino group protected is attached to hydroxymethyl resin or chloromethylated resin through an ester bond. The protecting group is removed with a reagent such as trifluoroacetic acid (TPA). Peptides are separated from the TFA solution by filtration, centrifugation, or extraction with diethyl ether, and can be purified by high-performance liquid chromatography (HPLC) or other methods.
본 발명에 있어서, 상기 조성물은 화장수, 유액, 크림, 팩, 미용액, 및 피부외용 연고로 이루어지는 군으로부터 선택된 것일 수 있다. 화장료 조성물에 통상적으로 이용되는 성분들을 포함하며, 예컨대 항산화제, 안정화제, 용해화제, 비타민, 안료 및 향료와 같은 통상적인 보조제, 그리고 담체를 포함한다. 또한, 투여 경로에 제한이 없으나 미백 목적상 경피 투여, 피하 투여 등의 국부 투여가 바람직하다.In the present invention, the composition may be selected from the group consisting of lotion, emulsion, cream, pack, serum, and ointment for external use. It contains ingredients commonly used in cosmetic compositions, such as antioxidants, stabilizers, solubilizers, vitamins, conventional auxiliaries such as pigments and fragrances, and carriers. Additionally, there are no restrictions on the route of administration, but for whitening purposes, local administration such as transdermal administration or subcutaneous administration is preferable.
본 발명에 있어서, 통상적으로 제조되는 어떠한 제형으로도 제조될 수 있으며, 예를 들어, 상기 조성물은 용액, 현탁액, 유탁액, 페이스트, 겔, 크림, 로션, 파우더, 비누, 계면활성제-함유 클린싱 오일, 분말파운데이션, 유탁액 파운데이션, 왁스 파운데이션 및 스프레이로 이루어지는 군으로부터 선택될 수 있으나, 이에 한정되는 것은 아니다. 보다 상세하게는, 유연화장수, 수렴화장수, 영양화장수, 영양크림, 마사지크림, 에센스, 아이크림, 클렌징크림, 클렌징폼, 클렌징워터, 팩, 스프레이 또는 파우더의 제형으로 제조될 수 있다.In the present invention, it can be prepared in any conventionally prepared formulation, for example, the composition may be a solution, suspension, emulsion, paste, gel, cream, lotion, powder, soap, surfactant-containing cleansing oil. , may be selected from the group consisting of powder foundation, emulsion foundation, wax foundation, and spray, but is not limited thereto. More specifically, it can be manufactured in the form of softening lotion, astringent lotion, nourishing lotion, nourishing cream, massage cream, essence, eye cream, cleansing cream, cleansing foam, cleansing water, pack, spray, or powder.
본 발명의 화장품 조성물에 함유된 화장품학적으로 유효한 담체는 제형에 따라, 통상적으로 이용되는 담체가 이용될 수 있다.The cosmetically effective carrier contained in the cosmetic composition of the present invention may be a commonly used carrier depending on the formulation.
본 발명의 제형이 연고, 페이스트, 크림 또는 겔인 경우에는 담체성분으로서 동물성유, 식물성유, 왁스, 파라핀, 전분, 트라칸트, 셀룰로오스유도체, 폴리에틸렌글리콜, 실리콘, 벤토나이트, 실리카, 탈크 또는 산화아연 등이 이용될 수 있다.When the formulation of the present invention is an ointment, paste, cream or gel, the carrier ingredients include animal oil, vegetable oil, wax, paraffin, starch, tracant, cellulose derivative, polyethylene glycol, silicone, bentonite, silica, talc or zinc oxide. It can be used.
본 발명의 제형이 파우더 또는 스프레이인 경우에는 담체성분으로서 락토스, 탈크, 실리카, 알루미늄히드록시드, 칼슘실리케이트 또는 폴리아미드파우더가 이용될 수 있고, 특히 스프레이인 경우에는 추가적으로 클로로플루오로히드로카본, 프로판/부탄 또는 디메틸에테르와 같은 추진체를 포함할 수 있다.When the formulation of the present invention is a powder or spray, lactose, talc, silica, aluminum hydroxide, calcium silicate, or polyamide powder can be used as the carrier ingredient. In particular, when the formulation is a spray, chlorofluorohydrocarbon and propane may be used as carrier ingredients. /May contain propellants such as butane or dimethyl ether.
본 발명의 제형이 용액 또는 유탁액인 경우에는 담체성분으로서 용매, 용해화제 또는 유탁화제가 이용되고, 예컨대 물, 에탄올, 이소프로판올, 에틸카보네이트, 에틸아세테이트, 벤질알코올, 벤질벤조에이트, 프로필렌글리콜, 1,3-부틸글리콜오일이 있으며, 특히 목화씨오일, 땅콩오일, 옥수수배종오일, 올리브오일, 피마자오일 및 참깨오일, 글리세롤지방족에스테르, 폴리에틸렌글리콜 또는 소르비탄의 지방산에스테르가 있다.When the formulation of the present invention is a solution or emulsion, a solvent, solubilizing agent, or emulsifying agent is used as a carrier component, such as water, ethanol, isopropanol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1 , 3-butyl glycol oil, especially cottonseed oil, peanut oil, corn germ oil, olive oil, castor oil and sesame oil, glycerol aliphatic ester, polyethylene glycol or fatty acid ester of sorbitan.
본 발명의 제형이 현탁액인 경우에는 담체성분으로서 물, 에탄올 또는 프로필렌글리콜과 같은 액상의 희석제, 에톡실화이소스테아릴알코올, 폴리옥시에틸렌소르비톨에스테르 및 폴리옥시에틸렌소르비탄에스테르와 같은 현탁제, 미소결정성셀룰로오스, 알루미늄메타히드록시드, 벤토나이트, 아가 또는 트라칸트 등이 이용될 수 있다.When the formulation of the present invention is a suspension, the carrier ingredients include water, a liquid diluent such as ethanol or propylene glycol, a suspending agent such as ethoxylated isostearyl alcohol, polyoxyethylene sorbitol ester, and polyoxyethylene sorbitan ester, and microcrystals. Cellulose, aluminum metahydroxide, bentonite, agar, or tracant may be used.
본 발명의 제형이 계면-활성제 함유 클린징인 경우에는 담체성분으로서 지방족알코올설페이트, 지방족알코올에테르설페이트, 설포숙신산모노에스테르, 이세티오네이트, 이미다졸리늄유도체, 메틸타우레이트, 사르코시네이트, 지방산아미드에테르설페이트, 알킬아미도베타인, 지방족알코올, 지방산글리세리드, 지방산디에탄올아미드, 식물성유, 라놀린유도체 또는 에톡실화글리세롤지방산에스테르 등이 이용될 수 있다.When the formulation of the present invention is a cleansing agent containing a surfactant, the carrier ingredients include aliphatic alcohol sulfate, aliphatic alcohol ether sulfate, sulfosuccinic acid monoester, isethionate, imidazolinium derivative, methyl taurate, sarcosinate, and fatty acid amide. Ether sulfate, alkylamidobetaine, aliphatic alcohol, fatty acid glyceride, fatty acid diethanolamide, vegetable oil, lanolin derivative, or ethoxylated glycerol fatty acid ester can be used.
본 발명의 제형이 비누인 경우에는 담체성분으로서 지방산의 알칼리금속염, 지방산헤미에스테르염, 지방산단백질히드롤리제이트, 이세티오네이트, 라놀린유도체, 지방족알코올, 식물성유, 글리세롤, 당 등이 이용될 수 있다.When the formulation of the present invention is soap, alkali metal salts of fatty acids, hemiester salts of fatty acids, fatty acid protein hydrolyzates, isethionates, lanolin derivatives, fatty alcohols, vegetable oils, glycerol, sugars, etc. can be used as carrier ingredients. there is.
이하, 본 발명의 이해를 돕기 위하여 실시예를 제시하나, 하기 실시예는 본 발명을 예시하는 것일 뿐, 본 발명의 범주 및 기술사상 범위 내에서 다양한 변경 및 수정이 가능함은 본 기술분야에서 통상의 지식을 가진 자에게 있어서 명백한 것이며, 이러한 변형 및 수정이 첨부된 특허청구범위에 속하는 것도 당연한 것이다.Hereinafter, examples are presented to aid understanding of the present invention. However, the following examples are merely illustrative of the present invention, and various changes and modifications are possible within the scope and technical idea of the present invention as is known in the art. It is obvious to those with knowledge, and it is natural that such changes and modifications fall within the scope of the appended patent claims.
<< 실시예Example > 인위적 돌연변이에 의한 > Due to artificial mutation TATdMtTATdMt 펩타이드의of peptides 발 foot 현hyeon
서열번호 1 내지 3의 각각의 아미노산 서열로 표현되는 TATdMt 펩타이드를 코딩하는 서열번호 4 내지 6의 각각의 뉴크레오타이드 서열로 표현되는 cDNA 절편을 pGex4T3(Pharmacia, Piscataway, NJ) 벡터에 클로닝하고 E.coli BL21(DE3)에서 과발현시킨 후, Glutathione Agarose 4B(Sigma,St.Louis,MO)를 사용하는 친화 크로마토그래피를 이용하여 정제하여, 각각의 TAT 펩타이드의 a.a. 28번째 및 a.a. 50번째에 인위적으로 돌연변이를 유발시킨 하기 표 1과 같은 아미노산 서열을 가지는 각각의 TATdMt 펩타이드를 발현하였다.The cDNA fragment represented by each nucleotide sequence of SEQ ID NOs: 4 to 6, which encodes the TATdMt peptide represented by each amino acid sequence of SEQ ID NOs: 1 to 3, was cloned into the pGex4T3 (Pharmacia, Piscataway, NJ) vector and incubated with E. After overexpression in coli BL21 (DE3), purification was performed using affinity chromatography using Glutathione Agarose 4B (Sigma, St. Louis, MO), and a.a. of each TAT peptide was obtained. 28th and a.a. At the 50th time, each artificially mutated TATdMt peptide having the amino acid sequence shown in Table 1 below was expressed.
상기 표 1의 TATdMt 펩타이드 아미노산 서열의 밑줄 친 아미노산 시퀀스 부분이 인위적 돌연변이를 유발시킨 부분이다. 앞에 있는 알라닌(A)는 a.a. 28 에 위치하는 아미노산 시퀀스이며, 뒤에 오는 알라닌(A)는 a.a. 50에 위치하는 아미노산 시퀀스이다.The underlined amino acid sequence portion of the TATdMt peptide amino acid sequence in Table 1 is the portion that caused artificial mutation. Alanine (A) in front is a.a. This is the amino acid sequence located at 28, and the alanine (A) that follows is a.a. This is the amino acid sequence located at position 50.
상기 표 2는 상기TATdMt 펩타이드로 발현되는 cDNA의 뉴클레오타이드 서열을 나타낸다.Table 2 shows the nucleotide sequence of cDNA expressed with the TATdMt peptide.
<< 실험예Experiment example 1 내지 4> 1 to 4>
<B16F10 마우스 <B16F10 mouse 흑생종melanoma 세포의 배양> Culture of cells>
B16F10 마우스 흑색종 세포는 페니실린-스트렙토마이신(Penicillin-Streptomycin) 100units/mL과 10% 소태아혈청(FBS, Fetal bovine serum)이 포함된 DMEM(Dulbecco's Modified Eagle's Medium) 배지 (ATCC, USA)를 사용하여 37℃, 5% CO2 분위기의 배양기(Thermo Fisher scientific, USA)에서 배양하였으며, 3일에 한번씩 계대배양 하였다.B16F10 mouse melanoma cells were grown using DMEM (Dulbecco's Modified Eagle's Medium) medium (ATCC, USA) containing 100 units/mL of penicillin-streptomycin and 10% fetal bovine serum (FBS). Cultured in an incubator (Thermo Fisher scientific, USA) in a 5% CO 2 atmosphere at 37°C, and subcultured once every 3 days.
<< 실험예Experiment example 1> B16F10 흑색종 세포에서의 세포 독성 측정 1> Measurement of cytotoxicity in B16F10 melanoma cells
상기 실시예에서 제조된 서열번호 3에 따른 TATdMt 펩타이드가 B16F10 마우스 흑색종(B16F10 mouse melanoma) 세포 생존율에 미치는 영향을 MTT Assay를 통하여 알아보았다.The effect of the TATdMt peptide according to SEQ ID NO: 3 prepared in the above example on B16F10 mouse melanoma cell survival rate was examined through MTT Assay.
상기에서 배양된 B16F10 마우스 흑색종 세포를 24-well 플레이트(plate)에 3×104 cells/well의 농도로 분주한 다음, 18 시간 후 DMEM 배지에 상기 실시예에서 제조된 TATdMt 펩타이드를 0, 0.5, 1, 5, 10, 50 및 100 ug/mL로 처리하고, 37℃, 5% CO2 분위기 배양기에서 48시간 배양하였다.The B16F10 mouse melanoma cells cultured above were dispensed into a 24-well plate at a concentration of 3 × 10 4 cells/well, and then 18 hours later, the TATdMt peptide prepared in the above example was added to DMEM medium at a concentration of 0, 0.5. , 1, 5, 10, 50, and 100 ug/mL, and cultured at 37°C in a 5% CO 2 atmosphere incubator for 48 hours.
이후, 각 well은 PBS(Phosphate Buffer Saline)용액으로 1회 세척하고, 2mg/mL MTT 시약(Sigma, USA) 50 ul과 새 배지 450 ul을 첨가하여 2~3 시간 배양한 후 상등액을 제거하였다. 각 well에 생성된 포르마잔(formazan) 결정에 DMSO(Dimethylsulfoxide)를 배지와 동량(450ul)으로 가한 뒤, 차광 상태에서 30분간 가볍게 흔든 다음 540nm에서 흡광도를 측정하였다.Afterwards, each well was washed once with PBS (Phosphate Buffer Saline) solution, 50 ul of 2 mg/mL MTT reagent (Sigma, USA) and 450 ul of new medium were added and incubated for 2 to 3 hours, and the supernatant was removed. Dimethylsulfoxide (DMSO) was added in the same amount (450ul) as the medium to the formazan crystals formed in each well, shaken lightly for 30 minutes in a light-proof state, and then the absorbance was measured at 540nm.
도 1에 나타낸 바와 같이, 서열번호 3에 따른 TATdMt 펩타이드를 50 ㎍/㎖ 이하의 농도도 처리한 경우 70%이상의 세포생존율을 나타내었으며, 따라서, 사람의 피부에 안전하게 사용할 수 있음을 확인할 수 있었다. 또한, 도 2에 나타낸 바와 같이, 서열번호 3에 따른 TATdMt 펩타이드 무처리군와 처리군의 세포모양을 관찰한 결과 형태학적 차이는 없는 것으로 나타났다. 따라서, 본 발명의 펩타이드가 세포 독성을 나타내지 않는 것을 확인 하였다.As shown in Figure 1, when treated with the TATdMt peptide according to SEQ ID NO: 3 at a concentration of 50 ㎍/㎖ or less, the cell viability was more than 70%, and therefore, it was confirmed that it can be safely used on human skin. In addition, as shown in Figure 2, as a result of observing the cell shapes of the TATdMt peptide untreated group and the treated group according to SEQ ID NO: 3, it was found that there was no morphological difference. Therefore, it was confirmed that the peptide of the present invention does not exhibit cytotoxicity.
<< 실험예Experiment example 2> 멜라닌 생성 억제 측정 2> Measurement of melanin production inhibition
상기에서 배양된 B16F10 마우스 흑색종 세포를 6-well 플레이트에 1 x 105 cells/well의 농도로 분주하여 24시간 배양하였다. 상기 배양된 세포에 하기 표 3과 같이, 서열번호 3에 따른 TATdMt 펩타이드 5, 10 및 25 ug/mL및 멜라닌 생성 자극 호르몬제인 α-MSH(10 nM)를 함께 처리하여 37℃, 5% CO2 분위기 배양기에서 48 시간 배양하였다. 이후 플레이트의 상층액를 제거한 후 세포를 회수하였다. 세포 펠렛(Pellet)에 1N NaOH를 첨가하고 60℃, 10분간 정치시켜 멜라닌을 완전히 용해시킨 후, 490nm에서 흡광도를 측정하였다.The B16F10 mouse melanoma cells cultured above were distributed in a 6-well plate at a concentration of 1 x 10 5 cells/well and cultured for 24 hours. As shown in Table 3 below, the cultured cells were treated with 5, 10 and 25 ug/mL of TATdMt peptide according to SEQ ID NO: 3 and α-MSH (10 nM), a melanin production stimulating hormone, at 37°C and 5% CO 2. Cultured for 48 hours in an atmosphere incubator. Afterwards, the supernatant of the plate was removed and the cells were recovered. 1N NaOH was added to the cell pellet and left at 60°C for 10 minutes to completely dissolve melanin, and then the absorbance was measured at 490 nm.
(대조군)Comparative Example 1
(Control group)
(α- MSH 10nM)Comparative Example 2
(α-MSH 10nM)
(TATdMt 5㎍/ml)Example 1
(TATdMt 5㎍/ml)
(TATdMt 10㎍/ml)Example 2
(TATdMt 10㎍/ml)
(ATdMt 25㎍/ml)Example 3
(ATdMt 25㎍/ml)
상기 실험 결과, 도 3에 나타낸 바와 같이 10nM의 α-MSH를 처리한 비교예 2의 멜라닌 함량은 193% , 10 nM α-MSH와 서열번호 3에 따른 TATdMt 펩타이드 5, 10 및 25 ug/mL을 농도별로 동시에 처리한 실시예 1 내지 3의 멜라닌 함량은 174, 159 및 133% 으로, 본 발명의 펩타이드를 처리한 경우 10 nM α-MSH 단독 처리군보다 멜라닌 함량이 감소하는 것을 확인하였다. 또한 멜라닌 생성은, 10 nM α-MSH의 멜라닌 함량을 기준으로 서열번호 3에 따른 TATdMt 펩타이드 25㎍/㎖의 농도에서 30% 이상 감소된 것으로 나타났다.As a result of the above experiment, as shown in Figure 3, the melanin content of Comparative Example 2 treated with 10 nM α-MSH was 193%, and 10 nM α-MSH and TATdMt peptides according to SEQ ID NO: 3 were 5, 10, and 25 ug/mL. The melanin content of Examples 1 to 3 treated simultaneously at different concentrations was 174, 159, and 133%, and it was confirmed that the melanin content decreased when treated with the peptide of the present invention compared to the group treated with 10 nM α-MSH alone. In addition, melanin production was found to be reduced by more than 30% at a concentration of 25 μg/ml of the TATdMt peptide according to SEQ ID NO: 3, based on the melanin content of 10 nM α-MSH.
<< 실험예Experiment example 3> 티로시나아제( 3> Tyrosinase ( TyrosinaseTyrosinase ) 활성 억제 측정) Measurement of activity inhibition
상기에서 배양된 B16F10 마우스 흑색종 세포를 6-well 플레이트에 1x105 cells/well의 농도로 분주하여 24시간 배양하였다. 배양된 세포에 5, 10 및 25ug/mL농도의 서열번호 3에 따른 TATdMt 펩타이드와 멜라닌 생성 자극 호르몬제인 α-MSH(10 nM)를 함께 처리하여 37℃, 5% CO2 분위기 배양기에서 48 시간 배양하였다. 이후 플레이트의 배지를 제거하고, PBS를 이용하여 세포를 2회 세척한 뒤 1% Tripton X-100을 500ul를 첨가하여 세포를 용해시켰다. 용해된 세포는 세포 스크래퍼(cell scraper)로 회수하여 12,000 rpm에서 10 분간 원심 분리한 뒤 상층액을 취하여 10 nM L-DOPA와 0.1M, pH 6.8 인산완충액(phosphate buffer)을 투입하여 37℃, 1시간 동안 반응시킨 후, 490nm에서 흡광도를 측정하였다.The B16F10 mouse melanoma cells cultured above were distributed in a 6-well plate at a concentration of 1x10 5 cells/well and cultured for 24 hours. Cultured cells were treated with TATdMt peptide according to SEQ ID NO: 3 at concentrations of 5, 10, and 25 ug/mL and α-MSH (10 nM), a melanin production stimulating hormone, and cultured for 48 hours in an incubator at 37°C and 5% CO 2 atmosphere. did. Afterwards, the medium on the plate was removed, the cells were washed twice using PBS, and 500ul of 1% Tripton X-100 was added to lyse the cells. The dissolved cells were recovered with a cell scraper, centrifuged at 12,000 rpm for 10 minutes, the supernatant was taken, and 10 nM L-DOPA and 0.1M, pH 6.8 phosphate buffer were added and incubated at 37°C for 1 minute. After reacting for a while, the absorbance was measured at 490 nm.
상기 실혐 결과, 도 4에 나타낸 바와같이 α-MSH를 처리한 실시예 2에서는 높은 티로시나아제 활성이 관찰되었지만, 서열번호 3에 따른 TATdMt 펩타이드25 ㎍/㎖를 처리한 실험예 3에서는, 약 36%의 티로시나아제 활성 억제 효능을 나타내는 것을 확인할 수 있었다. 따라서 본 발명의 펩타이드는 멜라닌 생성 억제할 뿐 아니라 티로시나아제 활성을 억제하는 것을 확인할 수 있었다.As a result of the above experiments, as shown in Figure 4, high tyrosinase activity was observed in Example 2 treated with α-MSH, but in Experimental Example 3 treated with 25 μg/ml of TATdMt peptide according to SEQ ID NO: 3, about 36 It was confirmed that it exhibited tyrosinase activity inhibition efficacy of %. Therefore, it was confirmed that the peptide of the present invention not only inhibits melanin production but also inhibits tyrosinase activity.
<< 실험예Experiment example 4> 티로시나아제 및 4> Tyrosinase and MITFMITF 의 of mRNAmRNA 발현 측정 Expression measurements
본 발명의 펩타이드의 미백 관련 유전자 발현에 대한 영향을 확인하기 위하여, 멜라닌 형성 세포를 이용하여 실험을 진행하였다. 미백 관련 인자인 티로시나아제(Tyrosinase)와 MITF mRNA의 발현량을 qRT-PCR(quantitative real time PCR)을 통해 확인함으로써 본 발명의 펩타이드의 미백 효능을 확인하였다.In order to confirm the effect of the peptide of the present invention on whitening-related gene expression, an experiment was conducted using melanin-forming cells. The whitening efficacy of the peptide of the present invention was confirmed by confirming the expression levels of tyrosinase and MITF mRNA, which are whitening-related factors, through qRT-PCR (quantitative real time PCR).
RNA추출 및 cDNA합성RNA extraction and cDNA synthesis
실험을 위하여 멜라닌 형성 세포를 6-well 플레이트에 1x105 cells/well로 분주하고, 세포가 안정화 된 후, 멜라닌 생성을 유도하는 호르몬인 α-MSH와 서열번호 3에 따른 펩타이드를 48시간 동안 처리하였다. 5, 10 및 25 ug/mL농도의 서열번호 3에 따른 TATdMt 펩타이드(a.a, 20-45, 20-57, 1-71)와 멜라닌 생성 자극 호르몬제인 α-MSH(10 nM)가 함께 처리된 B16F10 마우스 흑색종 세포에, 페놀(Phenol) 성분이 첨가된 Trizol 시약(Invitrogen, USA)를 이용하여 RNA를 추출하였다. 추출된 RNA는 Nanodrop(Nanodrop, USA)으로 260/280 nm의 비율(ratio) 및 농도를 측정하여 순도 및 농도를 확인한 후 사용하였다. For the experiment, melanin-forming cells were distributed in a 6-well plate at 1x10 5 cells/well, and after the cells were stabilized, they were treated with α-MSH, a hormone that induces melanin production, and the peptide according to SEQ ID NO: 3 for 48 hours. . B16F10 treated with TATdMt peptide (aa, 20-45, 20-57, 1-71) according to SEQ ID NO: 3 at concentrations of 5, 10, and 25 ug/mL and α-MSH (10 nM), a melanin production stimulating hormone. RNA was extracted from mouse melanoma cells using Trizol reagent (Invitrogen, USA) containing phenol. The extracted RNA was used after confirming its purity and concentration by measuring the ratio and concentration of 260/280 nm with Nanodrop (Nanodrop, USA).
cDNA합성은 추출된 1ug의 RNA를 대상으로 M-MLV 역전사 효소(reverse transcriptase) (Enzynomics, Korea)를 사용하여 제조하였다. 1 ug RNA와 0.5 ng oligo dT18에 DEPC-water를 첨가한 10ul의 용액을 70℃에서 10분간 반응시켜, RNA를 변성(denaturation)시킨 후 얼음에서 쿨링 하였다. 여기에 완충액(buffer), 10mM dNTP 및 200 unit/ul 역전사효소를 첨가하여 37℃에서 1시간 반응시켜 cDNA를 합성하여 실험에 사용하였다. cDNA synthesis was prepared using M-MLV reverse transcriptase (Enzynomics, Korea) using 1ug of extracted RNA. 10ul of DEPC-water solution added to 1ug RNA and 0.5ng oligo dT18 was reacted at 70°C for 10 minutes to denature the RNA and then cooled on ice. Buffer, 10mM dNTP, and 200 unit/ul reverse transcriptase were added here, and reacted at 37°C for 1 hour to synthesize cDNA and use it in the experiment.
qRTqRT -- PCRPCR (Quantitative real-time (Quantitative real-time PCRPCR ))
5, 10 및 25 ug/mL농도의 서열번호 3에 따른 TATdMt 펩타이드와 멜라닌 생성 자극 호르몬제인 α-MSH(10 nM)를 함께 처리한 B16F10 마우스 흑색종 세포 내에서 일어나는 유전자 발현 변화를 정량적으로 확인하기 위하여, EvaGreen 염료(dye)를 사용하여 qRT-PCR (quantitative RT-PCR)을 실시하였다. qRT-PCR은 HOT FITEPol EvaGreen PCR Mix Plus(Solis BioDyne, Estonia)와 1 pmole 정방향 프라이머(forword primer), 1 pmole 역방향 프라이머(reverse primer), 10ng cDNA를 혼합하고, 상기 반응액을 Line gene K (BioER, China)를 이용하여 실시하였다. qRT-PCR 결과는 각 유전자의 Ct(threshold cycle) 값을 β-actin의 Ct값으로 표준화한 후 Ct값의 변화량을 비교하여 분석하였다. 실험에 사용된 티로시나아제와 MITF의 mRNA 유전자의 프라이머(primer)는 표 4 및 5와 같다.Quantitatively confirm gene expression changes occurring in B16F10 mouse melanoma cells treated with TATdMt peptide according to SEQ ID NO: 3 at concentrations of 5, 10, and 25 ug/mL and α-MSH (10 nM), a melanin production stimulating hormone. For this purpose, qRT-PCR (quantitative RT-PCR) was performed using EvaGreen dye (dye). qRT-PCR was performed by mixing HOT FITEPol EvaGreen PCR Mix Plus (Solis BioDyne, Estonia) with 1 pmole forward primer, 1 pmole reverse primer, and 10ng cDNA, and the reaction solution was incubated with Line gene K (BioER). , China) was used. qRT-PCR results were analyzed by normalizing the Ct (threshold cycle) value of each gene to the Ct value of β-actin and then comparing the amount of change in Ct value. The primers for the tyrosinase and MITF mRNA genes used in the experiment are shown in Tables 4 and 5.
티로시나아제의 of tyrosinase mRNAmRNA 발현량 확인 Check expression level
상기 cDNA를 이용하여 서열번호 3에 따른 TATdMt 펩타이드의 티로시나아제 mRNA 억제 효능을 확인하였다. 이 때, 사용한 티로시나아제의 염기서열은 표 4 와 같다.Using the above cDNA, the tyrosinase mRNA inhibition effect of the TATdMt peptide according to SEQ ID NO: 3 was confirmed. At this time, the base sequence of the tyrosinase used is shown in Table 4.
PCR 반응 조건은, 94℃에서 30 초간 변성시키고, 60℃에서 20 초간 어닐링 및 72℃에서 60 초간 확장하는 사이클을 40 회 반복하여 수행하였으며, 그 결과를 도 5에 나타내었다.The PCR reaction conditions were denaturation at 94°C for 30 seconds, annealing at 60°C for 20 seconds, and extension at 72°C for 60 seconds, repeated 40 times. The results are shown in Figure 5.
본 발명의 펩타이드가 티로시나아제의 mRNA 발현량에 미치는 영향을 확인해 본 결과, 티로시나아제는 서열번호 3에 따른 TATdMt 펩타이드 25ug/ml 처리한 실시예 3에서, α-MSH만을 처리한 비교예 2 의 경우보다 약 180% 낮은 mRNA의 발현율을 나타내었다. 상기 도 5의 결과로부터 본 발명의 펩타이드가 티로시나아제의 발현을 현저하게 억제하여 최종적으로 멜라닌 생성을 억제한다는 것을 알 수 있다. 즉, 본 발명의 펩타이드는 미백 조성물로 유용할 수 있음을 알 수 있다.As a result of confirming the effect of the peptide of the present invention on the mRNA expression level of tyrosinase, tyrosinase was treated with 25ug/ml of TATdMt peptide according to SEQ ID NO: 3 in Example 3, and in Comparative Example 2 with only α-MSH treated. The mRNA expression rate was approximately 180% lower than that of . From the results shown in FIG. 5, it can be seen that the peptide of the present invention significantly inhibits the expression of tyrosinase and ultimately inhibits melanin production. In other words, it can be seen that the peptide of the present invention can be useful as a whitening composition.
MITFMITF 의 of mRNAmRNA 발현량 확인 Check expression level
상기 cDNA를 이용하여 본 발명의 펩타이드의 MITF mRNA 억제 효능을 확인하였다. 이 때, 사용한 MITF 프라이머의 염기서열은 아래 표 5와 같다.The MITF mRNA inhibition efficacy of the peptide of the present invention was confirmed using the above cDNA. At this time, the base sequence of the MITF primer used is shown in Table 5 below.
PCR 반응 조건은 94℃에서 30 초간 변성, 60℃에서 20초간 어닐링 및 72℃에서 60초간 확장하는 사이클을 40 회 반복하여 수행하였으며, 그 결과를 도 6에 나타내었다.The PCR reaction conditions were 40 cycles of denaturation at 94°C for 30 seconds, annealing at 60°C for 20 seconds, and extension at 72°C for 60 seconds, and the results are shown in Figure 6.
본 발명의 펩타이드가 MITF의 mRNA 발현량에 미치는 영향을 확인해본 결과, MITF는 서열번호 3에 따른 TATdMt 펩타이드 25 ug/ml 처리한 실시예 3에서 α-MSH만을 처리한 비교예 2의 경우보다 약 36% 낮은 mRNA의 발현율을 나타내었다. 따라서 본 발명의 펩타이드는 티로시나아제의 발현을 감소시키는 다양한 메커니즘 가운데 하나로서 티로시나아제의 발현을 조절하는 전사인자인 MITF의 유전자 발현을 억제시키고, α-MSH에 의해 유도된 MITF의 발현을 감소시키면, 티로시나아제의 발현을 감소시켜, 최종적으로 멜라닌 생성을 억제하는 것을 알 수 있었다. 또한, 상기 MITF의 발현 수준 대비 티로시나아제 mRNA 발현 수준으로부터, MITF 발현 억제 외에 티로시나아제 발현을 직접적 또는 간접적으로 조절하는 다른 메커니즘이 작용할 수 있음을 알 수 있다.As a result of confirming the effect of the peptide of the present invention on the mRNA expression level of MITF, MITF was approximately higher in Example 3 treated with 25 ug/ml of TATdMt peptide according to SEQ ID NO: 3 than in Comparative Example 2 treated with only α-MSH. It showed a 36% low mRNA expression rate. Therefore, the peptide of the present invention inhibits the gene expression of MITF, a transcription factor that regulates the expression of tyrosinase, as one of various mechanisms for reducing the expression of tyrosinase, and reduces the expression of MITF induced by α-MSH. It was found that this reduces the expression of tyrosinase and ultimately inhibits melanin production. In addition, from the level of tyrosinase mRNA expression compared to the expression level of MITF, it can be seen that other mechanisms that directly or indirectly regulate tyrosinase expression may act in addition to suppressing MITF expression.
<< 제조예Manufacturing example >>
<제조예 1> 유연화장수 제조<Preparation Example 1> Production of flexible lotion
서열번호 3에 따른 TATdMt 펩타이드 0.1 중량%, 글리세롤 3 중량%, 에탄올 5 중량%, 프로필렌글리콜 2 중량% 적량의 향 및 잔량의 정제수를 혼합하여 유연화장수를 제조하였다.Soft lotion was prepared by mixing 0.1% by weight of TATdMt peptide according to SEQ ID NO: 3, 3% by weight of glycerol, 5% by weight of ethanol, and 2% by weight of propylene glycol with an appropriate amount of fragrance and the remaining amount of purified water.
<제조예 2> 수렴화장수 제조<Preparation Example 2> Preparation of astringent lotion
서열번호 3에 따른 TATdMt 펩타이드 0.1 중량%, 밀랍 4 중량%, 유동파라핀 54 중량%, 스쿠알렌 4 중량%, 글리세롤 3 중량%, 적량의 방부제와 향 및 잔량의 정제수를 혼합하여 수렴화장수를 제조하였다.An astringent lotion was prepared by mixing 0.1% by weight of TATdMt peptide according to SEQ ID NO: 3, 4% by weight of beeswax, 54% by weight of liquid paraffin, 4% by weight of squalene, 3% by weight of glycerol, an appropriate amount of preservative and fragrance, and the remaining amount of purified water.
<제조예 3> 영양크림 제조<Preparation Example 3> Nutritional cream production
서열번호 3에 따른 TATdMt 펩타이드 0.2 중량%, 프로필렌글리콜 6 중량%, 글리세린 4 중량%, 유동파라핀 5 중량%, 스쿠알렌 3 중량%, 폴리솔베이트60 1.5 중량% 및 잔량의 정제수를 혼합하여 영양크림을 제조하였다.Make a nutritional cream by mixing 0.2% by weight of TATdMt peptide according to SEQ ID NO: 3, 6% by weight of propylene glycol, 4% by weight of glycerin, 5% by weight of liquid paraffin, 3% by weight of squalene, 1.5% by weight of polysorbate 60 and the remaining amount of purified water. Manufactured.
<제조예 4> 마사지크림 제조<Production Example 4> Massage cream production
서열번호 3에 따른 TATdMt 펩타이드 0.2 중량%, 바셀린 7 중량%, 유동파라핀 10 중량%, 밀랍 2 중량%, 폴리솔베이트 2.5 중량%, 스쿠알렌 3 중량%, 글리세린 4 중량%, 프로필렌글리콜 6 중량%, 적량의 방부제 및 잔량의 정제수를 혼합하여 마사지크림을 제조하였다.0.2% by weight of TATdMt peptide according to SEQ ID NO: 3, 7% by weight of vaseline, 10% by weight of liquid paraffin, 2% by weight of beeswax, 2.5% by weight of polysorbate, 3% by weight of squalene, 4% by weight of glycerin, 6% by weight of propylene glycol, Massage cream was prepared by mixing an appropriate amount of preservative and the remaining amount of purified water.
<제조예 5> 에센스 제조<Preparation Example 5> Essence preparation
서열번호 3에 따른 TATdMt 펩타이드 0.2 중량%, 프로필렌글리콜 3 중량%, 글리세린 4 중량%, 에탄올 7 중량%, 적량의 방부제와 향 및 잔량의 정제수를 혼합하여 에센스를 제조하였다.Essence was prepared by mixing 0.2% by weight of TATdMt peptide according to SEQ ID NO: 3, 3% by weight of propylene glycol, 4% by weight of glycerin, 7% by weight of ethanol, an appropriate amount of preservative and fragrance, and the remaining amount of purified water.
<제조예 6> 팩 제조<Manufacturing Example 6> Pack manufacturing
서열번호 3에 따른 TATdMt 펩타이드 0.1 중량%, 프로필렌글리콜 6 중량%, 글리세린 4 중량%, 밀랍 2 중량%, 유동파라핀 30 중량%, 적량의 방부제와 향 및 잔량의 정제수를 혼합하여 팩을 제조하였다.A pack was prepared by mixing 0.1% by weight of TATdMt peptide according to SEQ ID NO: 3, 6% by weight of propylene glycol, 4% by weight of glycerin, 2% by weight of beeswax, 30% by weight of liquid paraffin, an appropriate amount of preservative and fragrance, and the remaining amount of purified water.
<비교예 3> 에센스 제조<Comparative Example 3> Essence production
프로필렌글리콜 3 중량%, 글리세린 4 중량%, 에탄올 7 중량%, 적량의 방부제와 향 및 잔량의 정제수를 혼합하여 에센스를 제조하였다.Essence was prepared by mixing 3% by weight of propylene glycol, 4% by weight of glycerin, 7% by weight of ethanol, an appropriate amount of preservative and fragrance, and the remaining amount of purified water.
<실험예 5> 미백도 증가 효과 확인<Experimental Example 5> Confirmation of the effect of increasing whitening degree
건강한 30세 내지 40세, 20명의 성인 여성을 대상으로 미백 효과에 대한 인체 시험을 시행하였다. 각 피험자에게 자외선 조사기로 0.7MED 광량으로 3 회에 걸쳐 자외선을 조사하여 인위적으로 색소침착을 유발한 후, 피험자에게 상기 제조예 5 및 비교예 1의 조성으로 제조된 화장품을 매일 2회(아침, 저녁) 지속적으로 도포하게 하고 8주 경과 후에 기기적 평가(Mexameter MX16)에 의한 Melanin index(MI)를 측정하였다.A human test on the whitening effect was conducted on 20 healthy adult women aged 30 to 40. Pigmentation was artificially induced by irradiating each subject with ultraviolet rays three times at a dose of 0.7 MED using an ultraviolet irradiator. After that, the subjects were given cosmetics prepared with the composition of Preparation Example 5 and Comparative Example 1 twice daily (in the morning, It was applied continuously in the evening) and after 8 weeks, the Melanin index (MI) was measured by instrumental evaluation (Mexameter MX16).
결과는 ΔMI 값으로 나타내었으며, ΔMI는 UV에 의한 피부 흑화 유도 직후의 MI 값에서 샘플 도포 8 주 후에 측정한 MI 값을 뺀 값을 의미한다. 즉 ΔMI 값이 클수록 미백 효과가 큰 것을 알 수 있다. 하기 표 4에 피험자들의 결과를 평균값을 나타내었다.The results were expressed as ΔMI values, and ΔMI means the MI value measured 8 weeks after sample application from the MI value immediately after skin darkening was induced by UV. In other words, it can be seen that the larger the ΔMI value, the greater the whitening effect. Table 4 below shows the average results of the subjects.
표 6을 참고하면, 서열번호 3에 따른 TATdMt 펩타이드를 포함하는 제조 예 5의 경우, 비교예 3에 비하여 ΔMI 값이 60% 이상 큰 것을 알 수 있다. 이는 본 발명의 TATdMt펩타이드를 함유한 미백 조성물의 미백 효과 우수한 것을 의미하는 결과이다.Referring to Table 6, it can be seen that in the case of Preparation Example 5 containing the TATdMt peptide according to SEQ ID NO: 3, the ΔMI value is more than 60% greater than that of Comparative Example 3. This result means that the whitening composition containing the TATdMt peptide of the present invention has an excellent whitening effect.
<110> Obetat Biopharm Inc <120> COSMETIC COMPOSITION FOR SKIN WHITENING COMPRISING TATdMt PEPTIDES <130> DPA-0380 <160> 10 <170> KopatentIn 2.0 <210> 1 <211> 26 <212> PRT <213> Artificial Sequence <220> <223> TATdMt_20-45 <400> 1 Thr Ala Cys Thr Asn Cys Tyr Cys Ala Lys Cys Cys Phe His Cys Gln 1 5 10 15 Val Cys Phe Ile Thr Lys Ala Leu Gly Ile 20 25 <210> 2 <211> 38 <212> PRT <213> Artificial Sequence <220> <223> TATdMt_20-57 <400> 2 Thr Ala Cys Thr Asn Cys Tyr Cys Ala Lys Cys Cys Phe His Cys Gln 1 5 10 15 Val Cys Phe Ile Thr Lys Ala Leu Gly Ile Ser Tyr Gly Arg Ala Lys 20 25 30 Arg Arg Gln Arg Arg Arg 35 <210> 3 <211> 71 <212> PRT <213> Artificial Sequence <220> <223> TATdMt_1-71 <400> 3 Met Glu Pro Val Asn Pro Ser Leu Glu Pro Trp Lys His Pro Gly Ser 1 5 10 15 Gln Pro Lys Thr Ala Cys Thr Asn Cys Tyr Cys Ala Lys Cys Cys Phe 20 25 30 His Cys Gln Val Cys Phe Ile Thr Lys Ala Leu Gly Ile Ser Tyr Gly 35 40 45 Arg Ala Lys Arg Arg Gln Arg Arg Arg Pro Pro Gln Gly Ser Gln Thr 50 55 60 His Gln Val Ser Leu Ser Lys 65 70 <210> 4 <211> 78 <212> DNA <213> Artificial Sequence <220> <223> TATdMt_20-45 <400> 4 actgcttgta ccaattgcta ttgtggaaag tgttgctttc attgccaagt ttgtttcata 60 acaaaagcct taggcatc 78 <210> 5 <211> 114 <212> DNA <213> Artificial Sequence <220> <223> TATdMt_20-57 <400> 5 actgcttgta ccaattgcta ttgtggaaag tgttgctttc attgccaagt ttgtttcata 60 acaaaagcct taggcatctc ctatggcagg gcaaagcgga gacagcgacg aaga 114 <210> 6 <211> 213 <212> DNA <213> Artificial Sequence <220> <223> TATdMt_1-71 <400> 6 atggagccag taaatcctag cctagagccc tggaagcatc caggaagtca gcctaaaact 60 gcttgtacca attgctattg tgcaaagtgt tgctttcatt gccaagtttg tttcataaca 120 aaagccttag gcatctccta tggcagggca aagcggagac agcgacgaag acctcctcaa 180 ggcagtcaga ctcatcaagt ttctctatca aag 213 <210> 7 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Tyrosinase primer-F <400> 7 caagtacagg gatcggccaa c 21 <210> 8 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Tyrosinase primer-R <400> 8 ggtgcattgg cttctgggta a 21 <210> 9 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> MITF primer-F <400> 9 tgatgatccg attcaccaga 20 <210> 10 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> MITF primer-R <400> 10 ggaacagcaa cgagctaagg 20 <110> Obetat Biopharm Inc <120> COSMETIC COMPOSITION FOR SKIN WHITENING COMPRISING TATdMt PEPTIDES <130> DPA-0380 <160> 10 <170>CopatentIn 2.0 <210> 1 <211> 26 <212> PRT <213> Artificial Sequence <220> <223> TATdMt_20-45 <400> 1 Thr Ala Cys Thr Asn Cys Tyr Cys Ala Lys Cys Cys Phe His Cys Gln 1 5 10 15 Val Cys Phe Ile Thr Lys Ala Leu Gly Ile 20 25 <210> 2 <211> 38 <212> PRT <213> Artificial Sequence <220> <223> TATdMt_20-57 <400> 2 Thr Ala Cys Thr Asn Cys Tyr Cys Ala Lys Cys Cys Phe His Cys Gln 1 5 10 15 Val Cys Phe Ile Thr Lys Ala Leu Gly Ile Ser Tyr Gly Arg Ala Lys 20 25 30 Arg Arg Gln Arg Arg Arg 35 <210> 3 <211> 71 <212> PRT <213> Artificial Sequence <220> <223> TATdMt_1-71 <400> 3 Met Glu Pro Val Asn Pro Ser Leu Glu Pro Trp Lys His Pro Gly Ser 1 5 10 15 Gln Pro Lys Thr Ala Cys Thr Asn Cys Tyr Cys Ala Lys Cys Cys Phe 20 25 30 His Cys Gln Val Cys Phe Ile Thr Lys Ala Leu Gly Ile Ser Tyr Gly 35 40 45 Arg Ala Lys Arg Arg Gln Arg Arg Arg Pro Pro Gln Gly Ser Gln Thr 50 55 60 His Gln Val Ser Leu Ser Lys 65 70 <210> 4 <211> 78 <212> DNA <213> Artificial Sequence <220> <223> TATdMt_20-45 <400> 4 actgcttgta ccaattgcta ttgtggaaag tgttgctttc attgccaagt ttgtttcata 60 acaaaagcct taggcatc 78 <210> 5 <211> 114 <212> DNA <213> Artificial Sequence <220> <223> TATdMt_20-57 <400> 5 actgcttgta ccaattgcta ttgtggaaag tgttgctttc attgccaagt ttgtttcata 60 acaaaagcct taggcatctc ctatggcagg gcaaagcgga gacagcgacg aaga 114 <210> 6 <211> 213 <212> DNA <213> Artificial Sequence <220> <223> TATdMt_1-71 <400> 6 atggagccag taaatcctag cctagagccc tggaagcatc caggaagtca gcctaaaact 60 gcttgtacca attgctattg tgcaaagtgt tgctttcatt gccaagtttg tttcataaca 120 aaagccttag gcatctccta tggcagggca aagcggagac agcgacgaag acctcctcaa 180 ggcagtcaga ctcatcaagt ttctctatca aag 213 <210> 7 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Tyrosinase primer-F <400> 7 caagtacagg gatcggccaa c 21 <210> 8 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Tyrosinase primer-R <400> 8 ggtgcattgg cttctgggta a 21 <210> 9 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> MITF primer-F <400> 9 tgatgatccg attcaccaga 20 <210> 10 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> MITF primer-R <400> 10 ggaacagcaa cgagctaagg 20
Claims (13)
상기 서열번호 1의 아미노산 서열은 TAT 펩타이드의 a.a 20-45에 위치하는 아미노산에 상응하며, 하나 이상의 아미노산 서열을 인위적으로 변형시킨 것인 미백 조성물.According to clause 1,
The amino acid sequence of SEQ ID NO: 1 corresponds to the amino acid located at aa 20-45 of the TAT peptide, and is a whitening composition in which one or more amino acid sequences are artificially modified.
상기 서열번호 2의 아미노산 서열은 TAT 펩타이드의 a.a 20-57에 위치하는 아미노산에 상응하며, 하나 이상의 아미노산 서열을 인위적으로 변형시킨 것인 미백 조성물.According to claim 1,
The amino acid sequence of SEQ ID NO: 2 corresponds to the amino acid located at aa 20-57 of the TAT peptide, and is a whitening composition in which one or more amino acid sequences are artificially modified.
상기 서열번호 3의 아미노산 서열은 TAT 펩타이드의 a.a 1-71에 위치하는 아미노산에 상응하며, 하나 이상의 아미노산 서열을 인위적으로 변형시킨 것인 미백 조성물.According to clause 1,
The amino acid sequence of SEQ ID NO: 3 corresponds to the amino acid located at aa 1-71 of the TAT peptide, and is a whitening composition in which one or more amino acid sequences are artificially modified.
상기 아미노산 서열의 인위적 변형은 TAT 펩타이드의 a.a. 28번째 및 a.a.50번째 중 하나 이상에 나타나는 것인 미백 조성물.According to any one of claims 2 to 4,
A whitening composition wherein the artificial modification of the amino acid sequence occurs at one or more of aa 28th and aa50th of the TAT peptide.
상기 펩타이드의 농도는 0.5 내지 50 ㎍/㎖인 미백 조성물.According to paragraph 1,
A whitening composition wherein the concentration of the peptide is 0.5 to 50 μg/ml.
상기 펩타이드의 함량이 미백 조성물 전체 중량에 대하여 0.001 내지 10 중량%인 미백 조성물.According to paragraph 1,
A whitening composition wherein the content of the peptide is 0.001 to 10% by weight based on the total weight of the whitening composition.
상기 미백 조성물은 멜라닌 생성을 억제시키는 미백 조성물.According to clause 1,
The whitening composition is a whitening composition that inhibits melanin production.
상기 서열번호 1의 아미노산 서열은 서열번호 4의 뉴클레오타이드 서열에 의해 코딩되는 것인 미백 조성물.According to clause 1,
A whitening composition in which the amino acid sequence of SEQ ID NO: 1 is encoded by the nucleotide sequence of SEQ ID NO: 4.
상기 서열번호 2의 아미노산 서열은 서열번호 5의 뉴클레오타이드 서열에 의해 코딩되는 것인 미백 조성물.According to clause 1,
A whitening composition wherein the amino acid sequence of SEQ ID NO: 2 is encoded by the nucleotide sequence of SEQ ID NO: 5.
상기 서열번호 3의 아미노산 서열은 서열번호 6의 뉴클레오타이드 서열에 의해 코딩되는 것인 미백 조성물.According to clause 1,
A whitening composition wherein the amino acid sequence of SEQ ID NO: 3 is encoded by the nucleotide sequence of SEQ ID NO: 6.
상기 조성물은 화장수, 유액, 크림, 팩, 미용액 및 피부외용 연고로 이루어지는 군으로부터 선택된 것인 미백 조성물.According to paragraph 1,
The composition is a whitening composition selected from the group consisting of lotions, emulsions, creams, packs, serums, and ointments for external use.
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