KR20140074527A - Anti-aging tetrapeptide and cosmetic composition containing the same - Google Patents
Anti-aging tetrapeptide and cosmetic composition containing the same Download PDFInfo
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- KR20140074527A KR20140074527A KR1020120142573A KR20120142573A KR20140074527A KR 20140074527 A KR20140074527 A KR 20140074527A KR 1020120142573 A KR1020120142573 A KR 1020120142573A KR 20120142573 A KR20120142573 A KR 20120142573A KR 20140074527 A KR20140074527 A KR 20140074527A
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- South Korea
- Prior art keywords
- tetrapeptide
- skin
- group
- aapv
- aging
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- C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
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- C07K5/1005—Tetrapeptides with the first amino acid being neutral and aliphatic
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- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K8/00—Cosmetics or similar toiletry preparations
- A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
- A61K8/30—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds
- A61K8/64—Proteins; Peptides; Derivatives or degradation products thereof
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- A—HUMAN NECESSITIES
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- A—HUMAN NECESSITIES
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- A61Q19/00—Preparations for care of the skin
- A61Q19/08—Anti-ageing preparations
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K19/00—Hybrid peptides, i.e. peptides covalently bound to nucleic acids, or non-covalently bound protein-protein complexes
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Abstract
Description
본 발명은 피부노화 방지용 테트라펩타이드 및 이를 유효성분으로 함유하는 피부노화방지 및 피부주름개선 화장료 조성물에 관한 것으로서, 특히 피부노화 방지 및 피부주름 개선용 화장품에서 가장 중요한 요소인 콜라겐의 합성 및 발현을 촉진시키는 신규의 테트라펩타이드 및 이를 함유하는 화장료 및 의약용 조성물에 관한 것이다.TECHNICAL FIELD The present invention relates to a tetrapeptide for preventing skin aging and an anti-aging and cosmetic composition for improving skin wrinkles containing the same as an active ingredient. More particularly, the present invention relates to a cosmetic composition for promoting the synthesis and expression of collagen, And a cosmetic composition and a pharmaceutical composition containing the novel tetrapeptide.
피부노화는 생활연령과 자외선, 흡연, 공해와 같은 외적 요인 그리고 호르몬적 노화의 조합에 따른 산물이다. 생활수준이 높아지고 인간의 평균수명이 길어짐에 따라 선진국에서는 피부의 기능과 외적인 아름다움과 관련하여 노화가 피부에 미치는 영향에 대한 관심이 크게 증가하고 있다.Skin aging is a product of a combination of life age, extrinsic factors such as ultraviolet rays, smoking, pollution, and hormonal aging. As living standards increase and the average life span of humans increases, there is a growing interest in the effects of aging on skin in developed countries in relation to skin function and external beauty.
피부는 외부 환경과 직접적으로 맞닿아 있어 외상, 감염, 자외선을 포함한 외적 요인으로부터 신체를 보호하는 중요한 장벽 역할을 하는 다양한 세포와 구조로 이루어진 복잡한 기관이다. 피부는 크게 표피, 진피, 피하조직으로 이루어져 있으며 표피(Epidermis)는 주로 케라티노싸이트(Keratinocytes)와 색소를 내는 멜라노싸이트(Melanocytes) 및 랑게르한스(Langerhans) 세포로 이루어진 얇은 층조직이고, 진피(Dermis)는 주로 섬유아세포종(Fibroblasts)에 의해 생성되는 세포 외 조직단백질로 이루어져 있다.Skin is a complex organ composed of various cells and structures that act directly against the external environment and serve as important barriers to protect the body from external factors including trauma, infection, and ultraviolet rays. The skin is mainly composed of epidermis, dermis and subcutaneous tissue. Epidermis is a thin layered structure composed mainly of keratinocytes and pigmented melanocytes and Langerhans cells, and dermis (epidermis) Is composed mainly of extracellular tissue proteins produced by fibroblasts.
지방세포로 구성되어 있는 피하조직은 결합조직의 틀을 담당한다. 가장 많이 존재하는 단백질인 타입 Ⅰ 콜라겐(Type-I collagen)은 다른 타입의 콜라겐과 엘라스틴(Elastin), 프로테오글리칸(Proteoglycan), 파이브로넥틴(Fibronectin) 이외에 다른 세포 외 조직 단백질들과 더불어 결합조직을 구성한다. 새로 생성된 타입 Ⅰ 프로콜라겐은 진피의 세포 외 공간으로 분비된 후 피브릴로제네시스(Fibrilogenesis) 과정을 거쳐 피부에 탄성과 강도에 기여하는 콜라겐을 생성한다.Subcutaneous tissue composed of adipocytes is the framework of connective tissue. Type I collagen, the most abundant protein, forms a connective tissue with other types of collagen and other extracellular tissue proteins besides elastin, proteoglycan, and fibronectin. do. The newly generated type I procollagen is secreted into the extracellular space of the dermis and then undergoes a fibrilogenesis process to produce collagen that contributes elasticity and strength to the skin.
노화된 피부에서는 케라티노사이트 세포 및 결합조직의 구성과 양의 감소에 따라 표피층과 진피층이 얇아지는 현상을 보인다. 피부의 결합조직은 기본적으로 섬유성 콜라겐 다발과 탄력섬유로 구성되어 있는데 피부의 내구성과 탄성을 부여하는 콜라겐과 엘라스틴 같은 단백질들의 변성은 피부가 손상 되기 쉽게 하고 나이 들어 보이게 하는 외모를 야기한다.In aging skin, skin and dermal layers become thinner with decreasing composition and amount of keratinocyte cells and connective tissue. The connective tissue of the skin consists basically of fibrous collagen bundles and elastic fibers. The denaturation of proteins such as collagen and elastin, which gives the durability and elasticity of the skin, causes the skin to be easily damaged and look aged.
조직학적 관점에서 보면 피부노화는 피부의 진피에서 매트릭스를 형성하는 세포외 조직(Extracellular matrix proteins) 조성의 변화로 나타난다. 진피 세포 외 조직 중 콜라겐 단백질들은 피부에 강도와 장력을 부여하며 이로 인해 외부의 자극이나 힘으로부터 피부를 보호하는 역할을 하며 진피층의 90%를 차지하고 있어 콜라겐의 감소는 피부 노화와 주름형성에 밀접한 관련이 있다. 건강한 진피의 주요 가교 구성 성분들은 타입Ⅰ콜라겐과 타입Ⅲ콜라겐이다.From a histological point of view, skin aging is caused by changes in the composition of extracellular matrix proteins that form the matrix in the dermis of the skin. Collagen proteins in the dermal extracellular tissues impart strength and tension to the skin, which protects the skin from external stimuli or forces. It accounts for 90% of the dermal layer. Collagen reduction is closely related to skin aging and wrinkle formation . The major cross-linking components of healthy dermis are type I collagen and type III collagen.
하지만 광노화에 의해 손상된 피부에서는 이 두 콜라겐의 전구체가 확연히 감소하고 이 단백질들의 감소가 임상학적 심각성과 연관되어 있는 것으로 알려져 있다. 콜라겐의 분해는 노화가 진행되면서 분해되기도 하고 자외선 같은 자극에 노출되게 되면 콜라겐 분해효소들(MMPs)의 생합성이 증가하면서 콜라겐 합성이 감소하게 되어 주름이 형성된다.However, in skin damaged by photoaging, it is known that the precursor of these two collagens is markedly reduced and the reduction of these proteins is associated with clinical severity. Decomposition of collagen is decomposed as aging progresses. When exposed to stimuli such as ultraviolet light, collagen synthesis (collagen synthesis) is reduced by increasing biosynthesis of collagenase (MMPs).
콜라겐 분해효소들(MMPs)은 아연-의존적인 세포 내 단백분해 효소로 진피의 세포 외 구조의 성분들을 분해시키거나 재구조화 시킬 수 있는 물질로서 그 중 MMP-1은 진피의 콜라겐에 작용하여 분절시킨다. 따라서 이 효소의 합성 양과 활성도의 조절이 주름에 중요한 요인으로 작용한다.Collagen degrading enzymes (MMPs) are zinc-dependent intracellular proteases that are capable of degrading or restructuring the extracellular components of the dermis, among which MMP-1 acts on collagen of the dermis . Therefore, regulation of the amount and activity of this enzyme is important for wrinkles.
TGF-β는 포유류에서 TGF-β1. TGF-β2 와 TGF-β3의 아이소형이 존재하며 세포 외 기질 축적뿐 만 아니라 세포 분화, 증식, 세포이동, 세포사멸, 세포 외 기질의 재구성 및 발달 등을 광범위한 분야를 조절하는 다양한 기능을 수행하는 단백질이다. TGF-β는 체내에서 분비하는 성장인자로 콜라겐이나 프로테오클리칸과 같은 세포 외 기질의 유전자 발현을 증가 시킴으로써 세포 외 기질축적에 중요한 역할을 하는 것으로 알려져 있다.TGF-β is a member of the TGF-β1 family in mammals. TGF-β2 and TGF-β3 are present in the cells and perform various functions such as cell differentiation, proliferation, cell migration, apoptosis, reconstruction and development of extracellular matrix, as well as extracellular matrix accumulation It is a protein. It is known that TGF-β plays an important role in the accumulation of extracellular matrix by increasing the expression of extracellular matrix, such as collagen or proteoclycan, as a secretory growth factor in the body.
TGF-β의 생물학적 영향은 TGF-β type I receptor(TbRI) 과 type II receptor (TbRII)라 불리는 두 개의 다른 수용체에 의해 개시되며 이 수용체들은 신호전달에 필요하다. 일단 TGF-β가 세포막 단백질인 TGF-β type II 수용체와 결합한 후, 이 리간드를 포함하는 type II 수용체가 type I 수용체와 복합체를 형성하게 된다.The biological effects of TGF-β are initiated by two other receptors called TGF-β type I receptor (TbRI) and type II receptor (TbRII), which are required for signal transduction. Once TGF-β binds to the TGF-β type II receptor, which is a cell membrane protein, the type II receptor containing this ligand forms a complex with type I receptors.
이로 인해 활성화된 TGF-β type I 수용체 복합체는 일시적으로 Smad2/3의 활성화를 포함한 수많은 다음 단계의 시그널을 유도한다. 활성화된 SMAD 2와 SMAD3는 Smad4을 공유하며 결합하여 복합체를 형성한 후 핵 안으로 이동하여 목표유전자의 전사활성에 관여하게 된다.This activated TGF-β type I receptor complex induces a number of next-stage signals including the activation of Smad2 / 3 transiently. Activated SMAD 2 and SMAD 3 share
본 발명의 테트라펩타이드는 TGF-β 유사 펩타이드로써 TGF-β 시그널 경로를 활성화시킴으로써 콜라겐 생합성과 안정성을 향상시킨다.The tetrapeptide of the present invention enhances collagen biosynthesis and stability by activating the TGF-beta signal pathway as a TGF-beta-like peptide.
현재까지 피부의 주름형성을 방지하여 피부의 노화를 개선하는 물질로서 가장 많이 알려진 물질은 레티노이드다. 비타민A의 유도체인 레티노이드의 경우 콜라겐 분해효소인 MMP의 생성을 억제함으로서 콜라겐의 분해를 막고 피부의 주름을 개선하는 것으로 알려져 있다. (Lowe, N. et. al., Pharmacology of retinols in skin, 3, 240,1989)To date, retinoids have been the most commonly known substance to prevent skin wrinkles and improve skin aging. Retinoids, derivatives of vitamin A, are known to inhibit collagen degradation and improve skin wrinkles by inhibiting the production of MMP, a collagenase. (Lowe, N. et al., Pharmacology of retinols in skin, 3, 240, 1989)
Connective Tissue Engineering의 US 6,895,075는 이러한 레티노이드와 펩타이드의 혼합물을 이용하여 콜라겐 및 엘라스틴의 합성을 촉진시키고 피부탄력을 증가시킨다고 기술하고 있으며, US 6,919,072에는 레티노이드와 MMP의 저해제를 이용하여 MMP의 활성을 저해함으로서 자연노화를 방지한다고 기술하고 있다.US 6,895,075 of Connective Tissue Engineering describes the use of such a mixture of retinoids and peptides to promote the synthesis of collagen and elastin and to increase skin elasticity. US 6,919,072 discloses that retinoids and MMP inhibitors inhibit the activity of MMPs Thereby preventing natural aging.
하지만 레티노이드의 경우 피부자극을 유발하고 자외선에 의해 쉽게 반응하는 불안정성으로 인해 사용상의 제약이 있는 것이 사실이다.However, it is true that retinoids cause skin irritation and there are restrictions on their use due to instability that reacts easily with ultraviolet rays.
또한 US 6,906,036에는 MMP의 프로엔자임(proMMP)에서 분리한 펩타이드를 이용하여 콜라겐 분해효소인 MMP를 저해함으로서 건강한 피부를 유지시킨다고 기술하고 있으며, EP 2,510,919에는 올레아노일 펩타펩타이드(Oleanoyl pentapeptide)를 이용하여 이러한 MMP를 저해함으로서 피부의 주름개선 효과를 보인다고 기술하고 있다.US 6,906,036 discloses that peptides isolated from proMMP of MMP are used to maintain healthy skin by inhibiting collagenase, MMP. In EP 2,510,919, Oleanoyl pentapeptide It is described that the inhibition of MMP causes an effect of improving wrinkles of the skin.
이와 같이 MMP의 저해를 통해 콜라겐의 분해를 막음으로 인해 주름이 지속적으로 형성되는 것을 예방할 수 있지만 피부의 매트릭스(Matrix)를 형성하는 단백질들의 추가 생성을 촉진하지 않는다면 완전한 주름개선 효과를 기대하기 어렵다.In this way, it is possible to prevent the formation of wrinkles due to inhibition of collagen degradation through inhibition of MMP, but it is difficult to expect a complete wrinkle-reducing effect unless it promotes the additional production of proteins forming a matrix of the skin.
Sederma(프랑스)사에서 출원한 US 6,620,419는 콜라겐의 카르복실기 말단의 펩타이드를 이용하여 콜라겐의 생성을 촉진시킴으로 노화에 효과적이라고 기술하고 있으며, 같은 Sederma(프랑스)사에서 출원한 US 6,974,799의 경우 트리펩타이드와 테트라펩타이드의 혼합물을 이용하여 콜라겐의 합성을 촉진시킴으로 보다 향상된 항노화 효과를 보이는 것으로 기술하고 있으며, US 5,198,465의 경우 콜라겐 전구체의 펩타이드를 이용하여 콜라겐의 형성을 촉진시킨다고 기술되어 있다.US 6,620,419, filed by Sederma (France), describes effective aging by promoting the production of collagen using peptides at the carboxyl end of collagen, and in US 6,974,799 filed by Sederma (France), tripeptides In addition, US 5,198,465 discloses that the peptide of the collagen precursor is used to promote the formation of collagen.
하지만 위의 특허들의 경우 콜라겐의 분해효소인 MMP의 저해효과를 기대할 수 없고 단순히 매트릭스(Matrix) 단백질의 생성만을 촉진하기 때문에 완전한 주름개선 효과를 기대할 수 없다.However, in the case of the above-mentioned patents, it is impossible to expect the inhibitory effect of collagen degrading enzyme, MMP, and merely promotes the production of matrix protein, so that a complete wrinkle-reducing effect can not be expected.
이러한 배경하에 본 발명자들은 피부의 매트릭스(Matrix) 단백질의 형성을 크게 촉진시키는 동시에 이러한 매트릭스(Matrix) 단백질의 분해를 촉진시키는 MMP를 효과적으로 저해시킬 수 있는 테트라펩타이드를 개발하였으며 본 발명의 테트라펩타이드의 피부노화방지 및 주름 개선 효과를 확인하기 위하여 피부 섬유아세포종 (human skin fibroblst) 세포 주를 이용한 in vitro assay계를 확립하여 콜라겐과 콜라겐 분해효소(MMPs)의 생합성 능력을 TGF-β와 비교 검색하고 화합물 테트라펩타이드의 주름개선 효과를 확인함으로써 본 발명을 완성하였다.
Under these circumstances, the inventors of the present invention have developed a tetrapeptide capable of effectively inhibiting MMP which promotes the formation of Matrix protein of the skin while promoting the degradation of such matrix protein. The tetrapeptide of the present invention in order to confirm the anti-aging and anti-wrinkle effect in the skin using a fiber neuroblastoma (human skin fibroblst) cell lines vitro assay system to compare the biosynthesis ability of collagen and collagenase (MMPs) with that of TGF-β, and confirming the effect of improving the wrinkles of the compound tetrapeptide, thereby completing the present invention.
[문헌 1] Son ED, Lee JY, Lee S, Kim MS, Lee BG, Chang IS, Chung JH, Topical Application of 17b-Estradiol Increases Extracellular Matrix Protein Synthesis by Stimulating TGF-β Signaling in Aged Human Skin In Vivo: J Invest Dermatol, 124: 1149-1161(2005)[1] Son ED, Lee JY, Lee S, Kim MS, Lee BG, Chang IS, Chung JH, Topical Application of 17b-Estradiol Increases Extracellular Matrix Protein Synthesis by Stimulating TGF-β Signaling in Aged Human Skin In Vivo: J Invest Dermatol, 124: 1149-1161 (2005)
[문헌 2] Pilcher BK, Sudbeck BD, Dumin JA, Welgus HG, Parks WC, Collagenase-1 and collagen in epidermal repair: Arch. Dermatol. Res., 290 (Suppl.): S37-S46(1998)[Document 2] Pilcher BK, Sudbeck BD, Dumin JA, Welgus HG, Parks WC, Collagenase-1 and collagen in epidermal repair: Arch. Dermatol. Res., 290 (Suppl.): S37-S46 (1998)
[문헌 3] Griffiths CEM, Russman AN, Majmudar G, Singer RS, Hamilton TA, Voorhees JJ. Restoration of collagen formation in photodamaged human skin by tretinoin (retinoic acid): N Engl J Med, 329: 530-5.8(1993)[Literature 3] Griffiths CEM, Russman AN, Majmudar G, Singer RS, Hamilton TA, Voorhees JJ. Restoration of collagen formation in photodamaged human skin by retinoic acid: N Engl J Med, 329: 530-5.8 (1993)
[문헌 4] Talwar HS, Griffiths CEM, Fisher GJ, Hamilton TA, Voorhees JJ. Reduced type I and type III procollagens in adult human skin: J Invest Dermatol, 105: 285-90(1995)[Document 4] Talwar HS, Griffiths CEM, Fisher GJ, Hamilton TA, Voorhees JJ. Reduced type I and type III procollagens in adult human skin: J Invest Dermatol, 105: 285-90 (1995)
[문헌 5] Bitzer M, Gersdorff GV, Liang D, Rosales AD, Amer AB, Rojkind M, Bottinger EP, A mechanism of suppression of TGF-β/SMAD signaling by NF-kB/RelA: GENES & DEVELOPMENT, 14:187-197(1999)[5] Bitzer M, Gersdorff GV, Liang D, Rosales AD, Amer AB, Rojkind M, Bottinger EP, A mechanism of suppression of TGF-? / SMAD signaling by NF-KB / RelA: GENES & DEVELOPMENT 14: 187 -197 (1999)
[문헌 6] Yamane K, Asano Y, Tamaki K, Ihn H, Epidermal growth factor up-regulates transforming growth factor-b receptor type II in human dermal fibroblasts via p38 mitogen-activated protein kinase pathway: Biochemical and Biophysical Research Communications, 352: 69-77(2007)Biochemical and Biophysical Research Communications, 352 (1986), pp. 263-240, 1998. [6] Yamane K, Asano Y, Tamaki K, Ihn H, Epidermal growth factor up-regulates transforming growth factor-b receptor type II in human dermal fibroblasts via p38 mitogen-activated protein kinase pathway : 69-77 (2007)
[문헌 7] Pujol JP, Chadjichristos C, Legendre F, Bauge C, Beauchef G, Andriamanalijaona R, Galera P, Boumediene K, Interleukin-1 and transforming growth factor β1 as crucial factors in osteoarthritic cartilage metabolism: Connective Tissue Res, 49:293-297(2008)Conjugate Tissue Res., 49: 49-49. [CrossRef], [PubMed], [PubMed], [Web of Science ®] 293-297 (2008)
[문헌 8] Piek E, Heldin CH, Ten Dijke P, Specificity, diversity and regulation in TGF-β superfamily signaling; FASEB J, 13:2105-2124(1999)[Document 8] Piek E, Heldin CH, Ten Dijke P, Specificity, diversity and regulation of TGF-? Superfamily signaling; FASEB J, 13: 2105-2124 (1999)
[문헌 9] Rittie R, Fisher GJ, UV-light-induced signal cascades and skin aging; Age Res Rev, 1: 705-720(2002)[Literature 9] Rittie R, Fisher GJ, UV-light-induced signal cascades and skin aging; Age Res Rev, 1: 705-720 (2002)
[문헌 10] Yaar M, Gilchrest BA, Aging versus photoaging: postulated mechanisms and effectors; J Invest Dermatol Symp Proc, 3: 47-51(1998)[10] Yaar M, Gilchrest BA, Aging versus photoaging: postulated mechanisms and effectors; J Invest Dermatol Symp Proc, 3: 47-51 (1998)
[문헌 11] Fisher GJ, Datta SC, Wang ZQ, Li XY, Quan T, Chung JH, Kang S, Voorhees JJ, c-Jun-dependent inhibition of cutaneous type Ⅰ procollagen transcription following ultraviolet irradiation is reversed by all-trans retinoic acid; J Clin Invest, 106: 663-670(2000)[11] Fisher GJ, Datta SC, Wang ZQ, Li XY, Quan T, Chung JH, Kang S, Voorhees JJ, c-Jun-dependent inhibition of cutaneous type Ⅰ procollagen transcription following ultraviolet irradiation is reversed by all-trans retinoic acid; J Clin Invest, 106: 663-670 (2000)
[문헌 12] US 6,895,075[Literature 12] US 6,895,075
[문헌 13] US 6,919,072[Literature 13] US 6,919,072
[문헌 14] US 6,906,036[Literature 14] US 6,906,036
[문헌 15] EP 2,510,919[Document 15] EP 2,510,919
[문헌 16] US 6,620,419[Document 16] US 6,620,419
[문헌 17] US 6,974,799[Literature 17] US 6,974,799
[문헌 18] US 5,198,465
[Literature 18] US 5,198,465
이에 본 발명의 목적은 인체에 무해하며 피부 침투성이나 안정성이 뛰어난 테트라펩타이드 및 이를 함유하는 피부노화 방지 및 주름개선 효과를 나타내는 약학 조성물 및 화장료 조성물을 제공하는 것이다.Accordingly, an object of the present invention is to provide a tetrapeptide which is harmless to human body and excellent in permeability and stability of the skin, and a pharmaceutical composition and a cosmetic composition containing the same, which exhibit anti-aging and anti-wrinkle effects.
하기 [화학식 1로] 표시되는 테트라펩타이드The tetrapeptide represented by the following formula [1]
[화학식 1][Chemical Formula 1]
(상기 [화학식 1]에서, R은 수소 또는 1~29개의 탄소 원자를 가진 아실기로 포화되거나 포화되지 않은 테트라펩타이드인 아실 유도체를 포함한다.)
(In the above formula (1), R includes hydrogen or an acyl derivative which is a tetrapeptide saturated or unsaturated with an acyl group having 1 to 29 carbon atoms.)
본 발명의 테트라펩타이드는 4개의 연속 아미노산 서열을 포함하는 화합물로, Ala는 알라닌, Pro는 프롤린 그리고 Val는 발린을 의미한다. 본 발명에서 사용한 아미노산은 모두 광학활성을 가진 것으로서 D-form또는 L-form의 아미노산을 포함할 수 있고, C-말단의 경우 일반적으로 카르복실산 형태나 아마이드 형태 또는 알데하이드 형태를 포함할 수 있다.The tetrapeptide of the present invention is a compound containing four consecutive amino acid sequences, Ala means alanine, Pro means proline and Val means valine. The amino acids used in the present invention are all optically active and may include D-form or L-form amino acids, and in the case of C-terminal, they may generally include a carboxylic acid form, an amide form or an aldehyde form.
상기 테트라펩타이드를 유효성분으로 하는 피부노화 방지 및 피부주름 개선용 화장료 또는 약학용 조성물.A cosmetic or pharmaceutical composition comprising the tetrapeptide as an active ingredient for preventing skin aging and improving skin wrinkles.
본 발명에 의하면 피부자극이 없고 피부안정성도 우수한 지방산이 결합한 테트라펩타이드 및 이를 유효성분으로 함유하는, 콜라겐 생합성 증진효과로 인한 주름개선 효과를 나타내는 탁월한 효능을 갖는 피부 약학 조성물 및 화장료 조성물이 제공된다.
According to the present invention, there is provided a tetrapeptide having a fatty acid-bound fatty acid which is free from skin irritation and which has excellent skin stability, and a skin pharmaceutical composition and a cosmetic composition containing the same as an effective ingredient and exhibiting an effect of improving wrinkles due to the effect of promoting collagen biosynthesis.
도 1 은 N-미리스토일 테트라펩타이드(Myristoyl AAPV)의 세포독성을 확인한 결과이다.
도 2는 본 발명의 일실시예로 도 1에서 확인한 안전한 농도에서 미리스토일 테트라펩타이드(Myristoyl AAPV)에 의한 프로콜라겐(Procollagen) 합성 능력을 시험한 결과를 나타낸 도표이다.
도 3은 본 발명의 일실시예로 미리스토일 테트라펩타이드(Myristoyl AAPV)에 의한 파이브로넥틴(Fibronectin) 합성 능력을 시험한 결과를 나타낸 도표이다.
도 4는 본 발명의 일실시예로 미리스토일 테트라펩타이드(Myristoyl AAPV)에 의한 MMP-1 생성 저해능력을 시험한 결과를 나타낸 도표이다.
도 5는 본 발명의 일실시예로 미리스토일 테트라펩타이드(Myristoyl AAPV)에 의한 프로콜라겐 mRNA 발현을 시험한 결과를 나타낸 도표이다.
도 6은 본 발명의 일실시예로 미리스토일 테트라펩타이드(Myristoyl AAPV)에 의한 저해되는 MMP-1과 MMP-3의 mRNA 발현을 시험한 결과를 나타낸 도표이다.
도 7은 본 발명의 일실시예로 미리스토일 테트라펩타이드(Myristoyl AAPV)에 대한 기작으로 TGFβ 수용체 mRNA 발현을 시험한 결과를 나타낸 도표이다.
도 8은 발명의 일실시예로 미리스토일 테트라펩타이드(Myristoyl AAPV) 처리시 발현이 증가하거나 감소하는 유전자들을 색을 달리하여 그림으로 나타낸 도표이다.
도 9은 발명의 일실시예로 미리스토일 테트라펩타이드(Myristoyl AAPV) 처리시 발현하는 유전자들을 TGFβ1처리시와 비교 하여 수치로 나타낸 도표이다.Fig. 1 shows the results of confirming the cytotoxicity of N-myristoyl tetrapeptide (Myristoyl AAPV).
FIG. 2 is a graph showing the results of testing the ability of procollagen synthesis by myristoyl AAPV in a safe concentration as shown in FIG. 1 according to an embodiment of the present invention.
FIG. 3 is a graph showing the results of testing the ability of synthesizing fibronectin by myristoyl AAPV (myristoyl AAPV) as an example of the present invention.
FIG. 4 is a graph showing the results of testing MMP-1 production inhibitory ability by myristoyl AAPV (myristoyl AAPV) as an example of the present invention.
FIG. 5 is a graph showing the results of testing the expression of procollagen mRNA by myristoyl AAPV (myristoyl AAPV) in one embodiment of the present invention.
FIG. 6 is a graph showing the results of testing mRNA expression of MMP-1 and MMP-3 inhibited by myristoyl AAPV (myristoyl AAPV) as an example of the present invention.
FIG. 7 is a graph showing the results of testing the expression of TGFβ receptor mRNA by a mechanism for myristoyl AAPV in an embodiment of the present invention. FIG.
FIG. 8 is a graphical representation of genes whose expression increases or decreases upon treatment with myristoyl AAPV (myristoyl AAPV) according to an embodiment of the present invention.
FIG. 9 is a graph showing numerical values of genes expressed upon treatment with myristoyl AAPV in comparison with treatment with TGFβ1. FIG.
본 발명의 테트라펩타이드는 아래 화학식 1로 표시되는 구조에 기초하며 그 유도체들을 포함한다.The tetrapeptide of the present invention is based on the structure represented by the following formula (1) and includes derivatives thereof.
[화학식 1][Chemical Formula 1]
(상기 [화학식 1]에서, R은 수소 또는 1~29개의 탄소 원자를 가진 아실기로 포화되거나 포화되지 않은 테트라펩타이드인 아실 유도체를 포함한다.)
(In the above formula (1), R includes hydrogen or an acyl derivative which is a tetrapeptide saturated or unsaturated with an acyl group having 1 to 29 carbon atoms.)
본 발명의 테트라펩타이드는 하나 이상의 직쇄 또는 분지쇄, 길거나 짧은 사슬, 포화되거나 포화되지 않은 사슬, 수산기, 아미노기, 아미노아실기로 치환되거나 치환되지 않은 1내지 29개의 탄소원자를 가진 사슬로 치환된 아실유도체를 포함한다.The tetrapeptide of the present invention includes a chain-substituted acyl derivative having 1 to 29 carbon atoms which is substituted or not substituted with one or more straight or branched chain, long or short chain, saturated or unsaturated chain, hydroxyl group, amino group, aminoacyl group .
화학식 1에서 표시되는 R의 구체적인 예로는 수소 또는 아세틸, 에타노일, 프로파노일, 부타노일, 펜타노일, 헥사노일, 옥타노일, 노나노일, 데카노일, 도데카노일, 테트라데카노일, 헥사데카노일, 옥타데카노일, 스테아로일, 이소스테아로일, 라우릴, 언데세노일(Undecenoyl), 올레오일(Oleoyl), 엘라이도일(Elaidoyl), 2-에틸헥세노일 (2-Ethylhexenoyl)등과 같은 아실기가 포함될 수 있다. 특히 R은 수소, 아세틸, 옥타노일, 테트라데카노일 또는 헥사데카노일 중에서 선택되는 것이 바람직하고, 더욱 바람직하게는 R이 테트라데카노일 또는 헥사데카노일인 경우, 항노화 효과가 더욱 우수하다.Specific examples of R shown by the formula (1) include hydrogen or a substituent selected from the group consisting of acetyl, ethanoyl, propanoyl, butanoyl, pentanoyl, hexanoyl, octanoyl, nonanoyl, decanoyl, dodecanoyl, tetradecanoyl, (2-Ethylhexenoyl), and the like can be used in combination with the compound of formula (I) or a pharmaceutically acceptable salt, solvate, prodrug, The same acyl group may be included. Particularly, R is preferably selected from hydrogen, acetyl, octanoyl, tetradecanoyl or hexadecanoyl, and more preferably, when R is tetradecanoyl or hexadecanoyl, the anti-aging effect is more excellent.
본 발명의 테트라펩타이드는 통상적인 펩타이드 합성기를 사용한 고체상 방법에 의하여 합성될 수 있다.The tetrapeptide of the present invention can be synthesized by a solid phase method using a conventional peptide synthesizer.
통상적으로, 사용되는 아미노산은 N-말단이 보호된 형태 그리고 반응성 측쇄가 보호된 형태로 사용된다. N-말단을 보호하는 보호기로는 9-플루오레닐메틸옥시카르보닐기(Fmoc)가 일반적으로 사용된다. 반응성 측쇄를 보호하기 위한 보호기로는 트리페닐메틸, 부틸옥시카르보닐, t-부틸에스테르, 펜타메틸크로만-6-술포닐 등이 사용된다. 본 발명에서 사용되는 세 가지 아미노산의 경우에는 9-플루오레닐메틸옥시카르보닐기(Fmoc)로 N-말단이 보호된 Fmoc-Ala, Fmoc-Pro, Fmoc-Val의 형태로 사용이 될 수 있다. 측쇄고체상 반응을 위하여 사용되는 수지는 2-클로로트리틸 클로라이드(2-Chlorotrityl chloride, CTC), 4-벤질옥시벤질알콜(Wang), 하이드록시메틸페녹시아세틸(HMPA) 또는 트리알콕시벤즈하이드릴아민(Rink Amide) 수지가 사용될 수 있다.Typically, the amino acid used is used in the form in which the N-terminal is protected and the reactive side chain is protected. As the protecting group for protecting the N-terminus, a 9-fluorenylmethyloxycarbonyl group (Fmoc) is generally used. As a protecting group for protecting the reactive side chain, triphenylmethyl, butyloxycarbonyl, t-butyl ester, pentamethylchroman-6-sulfonyl and the like are used. The three amino acids used in the present invention may be used in the form of Fmoc-Ala, Fmoc-Pro, or Fmoc-Val protected with an N-terminal by a 9-fluorenylmethyloxycarbonyl group (Fmoc). The resins used for the side chain solid phase reaction are 2-chlorotrityl chloride (CTC), 4-benzyloxybenzyl alcohol (Wang), hydroxymethylphenoxyacetyl (HMPA) or trialkoxybenzhydrylamine (Rink Amide) resin may be used.
위의 모든 수지를 사용함에 있어서 펩타이드는 C-말단에서부터 N-말단 방향으로 합성된다. 커플링시 아미노산의 카르복실기를 활성제로 활성화시키고, 커플링 후 N-말단의 보호기는 피페리딘(Piperidine)으로 제거되고, 다음 커플링이 수행된다. 카르복실기 활성제로는 HATU(N-[(dimethylamino-)-1H -1,2,3-triazolo[4,5-b]pyridin-1-ylmethylene]-N-methylmethanaminium hexafluorophosphate N-oxide)와 1-히드록시-7-아자벤조트리아졸(1-hydroxy-7-azabenzotriazole, HOAt) 혹은 HBTU(N-[(1H-benzotriazol-1-yl)(dimethylamino)methylene]-N-methylmethanaminium hexafluorophosphate N-oxide)와 1-히드록시벤조트리아졸(1-hydroxybenzotriazole, HOBt)에 DIEA(N,N-diisopropylethylamine)가 함께 이용 될 수 있고, N,N'-디이소프로필카르보디이미드(DIC)와 첨가 보조제로 1-히드록시-7-아자벤조트리아졸(HOAt) 혹은 1-히드록시벤조트리아졸(HOBt)이 함께 사용될 수 있다.In using all of the above resins, peptides are synthesized from the C-terminus to the N-terminus. Upon coupling, the carboxyl group of the amino acid is activated with an activating agent, and after coupling, the protecting group at the N-terminal is removed with piperidine, and the following coupling is carried out. Examples of the carboxyl group activator include HATU ( N - [(dimethylamino) - 1 H -1,2,3-triazolo [4,5-b] pyridin-1-ylmethylene] -N- methylmethanaminium hexafluorophosphate N- (1-hydroxy-7-azabenzotriazole, HOAt) or HBTU ( N - [(1 H- benzotriazol-1-yl) (dimethylamino) methylene] -N- methylmethanaminium hexafluorophosphate N- oxide ( N, N- diisopropylethylamine) may be used together with 1-hydroxybenzotriazole (HOBt), and N, N'-diisopropylcarbodiimide (DIC) Hydroxy-7-azabenzotriazole (HOAt) or 1-hydroxybenzotriazole (HOBt) may be used together.
본 발명에서는 4개의 아미노산이 커플링 된 후에는 N-말단의 아민기는 원하는 알킬기를 가지는 아실 활성체, 예를 들어, 아실안하이드라이드(acylanhydride)와 DMAP(4-(N,N-dimethylamino)pyridine))를 함께 반응하여 N-말단에서 아마이드 결합을 형성하거나 카르복실산(Fatty acid)과 N,N-디이소프로필카르보디이미드(DIC), 1-히드록시-7-아자벤조트리아졸(HOAt) 혹은 1-히드록시벤조트리아졸(HOBt)이 함께 사용되어 아마이드 결합을 형성 할 수 있다. 그런 후 트리플루오로아세트산(TFA)을 포함하는 절단용액에 의하여 폴리펩타이드 유도체는 고체 수지로부터 분리되고 또한 그것의 측쇄에 결합된 보호기가 제거된다.In the present invention, after the four amino acids are coupled, the amine group at the N-terminal is converted to an acyl group having a desired alkyl group, for example, acylanhydride and DMAP (4- ( N, N- dimethylamino) pyridine ) To form an amide bond at the N-terminus, or a carboxylic acid (Fatty acid) and N, N -diisopropylcarbodiimide (DIC), 1 -hydroxy-7-azabenzotriazole ) Or 1-hydroxybenzotriazole (HOBt) may be used together to form an amide bond. The cleavage solution containing trifluoroacetic acid (TFA) then separates the polypeptide derivative from the solid resin and removes the protecting group bound to its side chain.
본 발명은 또한 상기 테트라펩타이드를 포함하는 피부노화 방지 및 피부주름 개선 화장료 조성물을 제공한다.The present invention also provides a cosmetic composition for preventing skin aging and improving skin wrinkles comprising the tetrapeptide.
본 발명의 테트라펩타이드는 피부노화방지 및 피부주름개선 화장료에 0.0001~5 중량%로 함유하는 것이 바람직하며, 0.001~0.5중량%로 함유하는 것이 더욱 바람직하다. 함량이 0.001 중량% 미만일 경우에는 효과가 미미하고, 0.5 중량% 이상인 경우에는 사용량에 따른 경제성이 결여될 경향이 높아진다.The tetrapeptide of the present invention is preferably contained in an amount of 0.0001 to 5% by weight, more preferably 0.001 to 0.5% by weight, in the cosmetic for preventing skin aging and improving skin wrinkles. When the content is less than 0.001% by weight, the effect is insignificant. When the content is more than 0.5% by weight, the economical efficiency due to the amount of use tends to be lacking.
본 발명의 테트라펩타이드를 포함하는 피부노화방지 및 피부주름개선 화장료는 이미 알려진 화장료 제조방법에 의해 제조할 수 있고, 일반적인 피부 화장료에 한정되지 않으며 의약부외품, 외용 의약품에도 적용할 수 있다. 이들의 제형은 통상적으로 제조되는 어떠한 제형으로도 제조될 수 있으며, 예를 들어, 크림, 에멀젼, 액상, 젤, 연고, 팩, 스틱, 파우더 등의 형태로 제조할 수 있으나, 이에 한정되는 것은 아니다. 그리고 각 제형의 화장료 조성물에 있어서, 상기한 테트라펩타이드 이외의 다른 성분들은 기타 화장료의 제형, 사용목적 등에 따라 당업자가 어려움 없이 적의 선정하여 배합할 수 있다.The skin anti-aging and skin wrinkle-improving cosmetic materials containing the tetrapeptide of the present invention can be prepared by a known cosmetic preparation method and are not limited to general skin cosmetic materials, and can be applied to quasi drugs and external medicine. These formulations may be prepared in any conventional formulation and may be in the form of, for example, creams, emulsions, liquids, gels, ointments, packs, sticks, powders, etc., but are not limited thereto . In the cosmetic composition of each formulation, other components than the above-mentioned tetrapeptide can be mixed and selected without difficulty by those skilled in the art depending on the formulation of the other cosmetics, the purpose of use, and the like.
이하, 다음은 상기와 같이 구성된 본 발명에 대해 실시예, 실험예를 통해 보다 구체적으로 설명하고자 한다. 이들 실시예는 오로지 본 발명을 구체적으로 설명하기 위한 것으로, 본 발명의 범위가 이들 실시예에 국한되지 않는다는 것은 당 업계에서 통상의 지식을 가진 자에게 있어서는 자명할 것이다.
Hereinafter, the present invention constructed as described above will be described in more detail through examples and experimental examples. It will be apparent to those skilled in the art that these embodiments are for illustrative purposes only and that the scope of the present invention is not limited to these embodiments.
<실시예 1> 시료준비 (합성 및 분리정제) ≪ Example 1 > Sample preparation (synthesis and separation purification)
본 발명의 테트라펩타이드는 메리필드(Merrifield)의 SPPS(Solid Phase Peptide Synthesis) 방법(J. Am. Chem. Soc., 85(14), 2149-2154)을 이용하여 합성하였다.The tetrapeptides of the present invention were synthesized using Merrifield's SPPS (Solid Phase Peptide Synthesis) method (J. Am. Chem. Soc., 85 (14), 2149-2154).
발명의 펩타이드 합성을 위해 2-클로로트리틸 클로라이드(CTC) 수지를 이용하여 합성을 시작하였으며, 첫 단계의 Fmoc(9-fluorenylmethoxy carbonyl) 아미노산은 N,N-디이소프로필데틸아민(N,N-diisopropylethylamine, DIEA)를 사용하여 디클로로메탄(dichloromethane, DCM) 용매 하에서 커플링한다. 그 다음 단계의 Fmoc 아미노산부터는HATU(N-[(dimethylamino-)-1H -1,2,3-triazolo[4,5-b]pyridin-1-ylmethylene]-N-methylmethanaminium hexafluorophosphate N-oxide)와 1-히드록시-7-아자벤조트리아졸(HOAt) 혹은 HBTU(N-[(1H-benzotriazol-1-yl)(dimethylamino)methylene]-N-methylmethanaminium hexafluorophosphate N-oxide)와 1-히드록시벤조트리아졸(HOBt)에 DIEA(N,N-diisopropylethylamine)를 함께 사용하여 커플링을 해나가는 방법으로 펩타이드의 사슬을 연장시켰다.Synthesis was initiated using 2-chlorotrityl chloride (CTC) resin to synthesize the peptides of the invention. The first step Fmoc (9-fluorenylmethoxy carbonyl) amino acid was N, N -diisopropyldethylamine ( N, N- diisopropylethylamine, DIEA) in dichloromethane (DCM) solvent. In the next step, the Fmoc amino acid was replaced with HATU ( N - [(dimethylamino) - 1 H -1,2,3-triazolo [4,5- b] pyridin- 1-ylmethylene] -N- methylmethanaminium hexafluorophosphate N- oxide 1-Hydroxy-7-azabenzotriazole (HOAt) or HBTU ( N - [(1 H- benzotriazol-1-yl) (dimethylamino) methylene] -N- methylmethanaminium hexafluorophosphate N- oxide) The chain of the peptide was extended by coupling with triazole (HOBt) using DIEA ( N, N- diisopropylethylamine).
이때, C-말단에서부터 N-말단 방향으로 합성해 나가며, 아미노말단에 Fmoc-아미노산을 커플링 시킨 후, 20% piperidine 용액으로 Fmoc기를 제거(Solid-Phase Synthesis: A Practical Guide (1 ed.). CRC Press. 848)하고 DMF(Dimethylforamide)으로 여러번 씻어준 다음 커플링 해나가는 방법으로 진행된다.At this time, the Fmoc-amino acid was synthesized from the C-terminus to the N-terminus, and the Fmoc-amino acid was coupled to the amino terminus. Then, the Fmoc group was removed with 20% piperidine solution (Solid-Phase Synthesis: A Practical Guide (1 ed. CRC Press. 848) and rinsed several times with DMF (Dimethylforamide) followed by coupling.
마지막으로 Fmoc-아미노산을 커플링 시킨 후, 20% piperidine 용액으로 Fmoc기를 제거하고 카르복실산과 N, N'-디이소프로필카르보디이미드(DIC), 1-히드록시-7-아자벤조트리아졸(HOAt)을 함께 사용하여 아실기를 커플링한 다음 DMF로 여러 번 세척하여 건조시켰다. 여기에 TFA(Trifuloroacetic acid)-H2O-Triisopropylsilane(95:2.5:2.5, vol./vol.) 용액을 가하고 2내지 3시간 반응시켜 보호기(Protecting group)의 제거 및 resin으로부터 펩타이드를 분리시킨 후, 디에틸에테르(diethylether)로 펩타이드를 침천시켰다.Finally, the Fmoc-amino acid was coupled and then the Fmoc group was removed with a 20% piperidine solution. The Fmoc group was removed and a solution of the carboxylic acid and N, N' -diisopropylcarbodiimide (DIC), 1-hydroxy-7-azabenzotriazole HOAt) were coupled together and the acyl group was coupled and then washed several times with DMF and dried. After adding a solution of TFA (triflu or o acetic acid) -H 2 O-Triisopropylsilane (95: 2.5: 2.5, vol./vol.) And reacting for 2 to 3 hours, the protecting group was removed and the peptide was separated from the resin , And the peptide was precipitated with diethylether.
상기의 방법으로 얻은 crude 펩타이드는 0.1% TFA가 포함된 아세토니트릴(acetonitrile)과 물의 구배 용매조성으로 RP-HPLC(2996 Detector, 515 Pump, Waters)을 이용하여 순도를 확인하였으며, 순도확인 결과 95% 이하의 펩타이드는 Prep-LC로 분리 정제하여 95% 이상의 펩타이드를 얻었다.The crude peptide thus obtained was purified by RP-HPLC (2996 Detector, 515 Pump, Waters) in the gradient solvent of acetonitrile and water containing 0.1% TFA. The purity was 95% The following peptides were separated and purified with Prep-LC to obtain 95% or more of peptides.
이 펩타이드의 정확한 성분확인을 위하여 Elemental Analyzer(EA1112, CE Instrument, Italy)로 원소분석을 한 결과, 펩타이드의 원소별 함유량이 아미노산 서열로부터 계산하여 얻은 원소별 함유량과 일치하므로 정확한 아미노산 서열을 가짐을 확인하였다. 이렇게 합성된 화합물은 항노화 효과를 측정하는데 이용되었다.
Elemental analysis was performed with an Elemental Analyzer (EA1112, CE Instrument, Italy) to confirm the exact components of the peptide. As a result, the contents of the peptides were consistent with the content of each element calculated from the amino acid sequence, confirming that the peptide had the correct amino acid sequence Respectively. The compound thus synthesized was used to measure the anti-aging effect.
<실시예 2> 피부노화 방지 및 피부주름 개선 효과≪ Example 2 > Effect of preventing skin aging and improving skin wrinkles
인간의 진피 세포(Human Skin Fibroblasts)를 이용하여 본 발명의 테트라펩타이드의 피부노화 방지 효과와 피부주름 개선 효과를 실험하였다.Human skin fibroblasts were used to test the anti-aging effect and the skin wrinkle effect of the tetrapeptides of the present invention.
피부가 주름이 생성되는 것은 피부가 노화가 진행되거나 자외선 같은 자극에 노출되게 되면서 콜라겐 분해효소들(MMPs)의 생합성이 증가함에 따라 콜라겐 합성이 감소하기 때문이다. 따라서 MMP-1과 같은 콜라겐 분해효소의 생합성량을 감소시키고 자외선에 의해 급감하는 Procollagen 1 양을 증진 시키는 것으로 피부 주름을 예방, 개선할 수 있다.Skin wrinkles are caused by reduced collagen synthesis as the skin is aged or exposed to stimuli such as ultraviolet light, resulting in increased biosynthesis of collagenase (MMPs). Therefore, it is possible to prevent and improve the wrinkles of the skin by reducing the amount of biosynthesis of collagenase such as MMP-1 and increasing the amount of
본 실험은 피부 섬유아세포종(human skin fibroblast cells, Hs68) 세포주와 테트라펩타이드를 이용한 in vitro assay계를 확립하여 프로콜라겐 타입1의 생합성 양과 MMP-1 효소의 발현저해를 측정하였고 이들 단백질의mRNA 발현양을 평가함으로써 테트라펩타이드의 피부 노화 방지 효과를 확인하였고, 테트라펩타이드의 기작을 PCR과 CHIP assay를 통해 측정함으로써 피부주름 개선 효과를 측정하였다.
This study was conducted to investigate the effects of human skin fibroblast (Hs68) cell line and tetrapeptide in In vitro assay system was established to measure the amount of biosynthesis of
(1) 세포주 및 세포배양(1) Cell line and cell culture
실험에 사용된 세포주는 human skin fibroblast Hs68세포(ATCC, USA) 를 사용하였다.Human cell skin fibroblast Hs68 cells (ATCC, USA) were used for the experiments.
Hs68세포는 37°C, 5% CO2의 조건에서 10% FBS (Gibco Co), 100 ㎍/㎖ 스트렙토마이신(streptomycin), 100 U/㎖ 페니실린 (penicillin)이 첨가된 DMEM(Dulbecco's modified Eagle medium)을 사용하여 배양하였다(Incubator; Thermo-scientific, USA). Hs68 세포는 100 cm2플라스크 (Corning, USA)에서 충분히 증식시킨 후 배양 3일 간격으로 배양세포 표면을 PBS로 씻어준 후 0.25% 트립신-EDTA 용액을 넣고 배양기에서 3분간 처리한 다음 트립신-EDTA 용액을 버리고 37°C에서5분간 보관하여 세포를 탈착시켰다. 탈착된 세포는 10% FBS가 포함된 DMEM 10 ㎖을 새로운 배양용기에 옮겨 1:5의 분할율 (split ratio)로 37°C, 5% CO2의 조건에서 계대배양하였다.
Hs68 cells is 37 ° C, 5% 10% FBS (Gibco Co) under the conditions of
(2) 세포 (2) Cell 생존률Survival rate 측정 Measure
Hs68 (Human skin fibroblast)를 1*104 cells/well 농도로 96 well plate에 분주하여 37°C, 5% CO2의 조건에서 24 시간 배양하고 시료를 희석한 무혈청배지를 배양배지와 교체한 후 48 시간 동안 추가 배양하였다.Hs68 (human skin fibroblast) was dispensed into 96-well plates at a concentration of 1 × 10 4 cells / well and cultured for 24 hours at 37 ° C and 5% CO 2. The serum-free medium was replaced with culture medium Followed by further incubation for 48 hours.
배양상등액을 제거하고 무혈청배지 100㎕와 MTT용액(3-(4,5-디메틸디아졸-2-일)2,5디페닐테트라졸리움브로마이드(5mg/㎖)을 각각 10㎕씩 넣고 3시간 동안 배양 후 배지를 제거한다. 각 well 에 100㎕ 의 DMSO(Dimethyl superoxide)용액을 첨가하고 포마젠(Formazan)을 용해시킨 후 마이크로 플레이트 판독기(Microplate reader, Tecan)을 이용하여 570nm에서 흡광도를 측정, 아래의 수학식 1과 같이 세포 생존률(%)을 환산하였다.
After removing the culture supernatant, 10 μl of MTT solution (3- (4,5-dimethyldiazol-2-yl) 2,5-diphenyltetrazolium bromide (5 mg / ml) 100 μl of DMSO (Dimethyl Superoxide) solution was added to each well, and the Formazan was dissolved and the absorbance was measured at 570 nm using a microplate reader (Tecan) The cell viability (%) was calculated as shown in the following equation (1).
[수학식 1][Equation 1]
세포 생존률(%) = (Sp)/Sc×100Cell survival rate (%) = (S p ) / S c x 100
Ss: 시료를 처리한 군의 흡광도 S s : absorbance of the group treated with the sample
Sc: 시료를 처리하지 않은 군의 흡광도
S c : absorbance of the untreated group
도1은 미리스토일 테트라펩타이드(Myristoyl AAPV)를 다양한 농도로 처리한 후 세포독성을 실험한 결과이다. 도1에 나타난 바와 같이, MTT 분석결과 1내지 10ppm까지 세포 생존률의 감소를 보이지 않아 미리스토일 테트라펩타이드(Myristoyl AAPV)가 유효 농도 이내에서 안전한 원료임을 확인하였다.
FIG. 1 shows the results of experiments on cytotoxicity after treating myristoyl AAPV at various concentrations. As shown in FIG. 1, MTT analysis showed no reduction in cell viability up to 1 to 10 ppm, confirming that myristoyl AAPV was a safe source within an effective concentration.
(3) (3) 미리스토일Myristico 테트라펩타이드(Myristoyl AAPV)에The tetrapeptide (Myristoyl AAPV) 의한 프로콜라겐 합성 증진 효과 Effect of pro-collagen synthesis
인간 피부 섬유아세포인 Hs68 (human skin fibroblast)를 1*106cells/well 농도로 6 well plate에 분주한 후 70~80% 정도 자랄 때까지 37°C, 5% CO2의 조건에서 24 시간 배양하였다.Human skin fibroblast (Hs68), a human skin fibroblast, was added to 6-well plates at a concentration of 1 * 10 6 cells / well and cultured for 24 hours at 37 ° C and 5% CO 2 until 70-80% Respectively.
Phosphate buffer saline(PBS)로 세포 세척 후 시료를 희석한 무혈청배지를 배양배지와 교체한 후 48 시간 동안 추가 배양한다. 배지를 제거 후 세포 용해 완충액(lysis buffer)(50 mM HEPES, pH 7.5, 150 mM NaCl, 10％ glycerol, 5 mM EDTA, 1 mM sodium orthovanadate, 10 mM NaF, 12 mM bglycerophosphate, 1 mM DTT, 1 mM PMSF, 5 mg/ml aprotinin, and 1％ NP40)으로 세포를 용해시켰다. 용해된 세포를 26,000 g에서 15분간 원심분리하여 세포 추출물을 분리한 Bradford 단백질 측정법으로 단백질 양을 측정하여 동량을 맞추고 95℃에서 5분간 가열하여 단백질을 변성시킨 후 20㎍을 10% SDS-PAGE전기영동을 걸어준다. 전기영동을 걸어준 젤을 나이트로셀룰로스막(nitrocellulose membrane)에 단백질을 이동(transfer)시킨 후 TBST 버퍼에 용해시킨 5% 스킴밀크에서 1시간 동안 배양시킨 후 프로콜라겐 1(Santa Cruze Biotechnology, CA) 항체를 TBST 버퍼에 1:100으로 희석한 용액에 3시간 동안 쉐이킹 하면서 배양시킨다. 항체용액을 제거하고 0.05% Tween20을 첨가한 Phosphate buffer saline(PBS)로 5분씩 3회 세척한 후 HRP-conjugate 된 2차 항체를 TBST 버퍼에 1:2000으로 희석한 용액에1시간 동안 배양한다. 멤브레인을 세척한 후, ECL 용액을 뿌려 imaging densitometer [Labworks ver.4.6. (image acquisition and analysis software), UVP, CA]를 이용하여 측정하였다. 이 때 대조군에는 항-튜불린 항체(Sigma)를 이용하였다.After washing the cells with phosphate buffered saline (PBS), serum-free medium diluted with the sample is replaced with the culture medium, followed by further incubation for 48 hours. After removing the medium, the cells were lysed in a lysis buffer (50 mM HEPES, pH 7.5, 150 mM NaCl, 10% glycerol, 5 mM EDTA, 1 mM sodium orthovanadate, 10 mM NaF, 12 mM bglycerophosphate, 1 mM DTT, PMSF, 5 mg / ml aprotinin, and 1% NP40). The cells were centrifuged at 26,000 g for 15 minutes, and the amount of protein was measured by Bradford protein assay. The proteins were denatured by heating at 95 ° C for 5 minutes, followed by 20 μg of 10% SDS-PAGE electrophoresis It gives you a young man. The electrophoresis gel was transferred to a nitrocellulose membrane and incubated in 5% skim milk dissolved in TBST buffer for 1 hour. Then, the protein was incubated with procollagen 1 (Santa Cruze Biotechnology, CA) The antibody is incubated in a 1: 100 dilution of TBST buffer for 3 hours with shaking. After removing the antibody solution and washing with phosphate-buffered saline (PBS) (0.05% Tween 20) for 5 minutes each, the HRP-conjugated secondary antibody is incubated for 1 hour in 1: 2000 diluted TBST buffer. After washing the membrane, ECL solution was sprayed onto the membrane using an imaging densitometer [Labworks ver.4.6. (image acquisition and analysis software, UVP, CA). An anti-tubulin antibody (Sigma) was used as a control.
아래의 표 1은 본 발명의 일 실시예로서 상기의 방법으로 지방산을 붙이지 않은 화합물과 지방산이 각기 다른 5개의 테트라펩타이드의 프로콜라겐 타입 1 생합성 증진 효과를 비교 실험한 표이다. 각기 다른 지방산이 붙은 테트라펩타이드를 처리한 후 증가한 프로콜라겐 타입1을 비교해 보았을 때 미리스토일 테트라펩타이드(Myristoyl AAPV)가 프로콜라겐 생합성양을 가장 많이 촉진한 것을 확인하였다.
Table 1 below is a comparative experiment of the effect of enhancing the metabolism of
도 2는 본 발명의 일 실시예로서 상기의 방법으로 미리스토일 테트라펩타이드(Myristoyl AAPV)의 procollagen 1 생합성 증진 효과를 실험한 도표이다. 양성대조군(negative control) TGFβ1 처리군과 비교하였을 때, 미리스토일 테트라펩타이드(Myristoyl AAPV)를 처리한 군에서 48%의 프로콜라겐 타입1이 증가한 것을 확인하였다.
FIG. 2 is a graph illustrating the effect of molluscic acid (Myristoyl AAPV) on
(4) 화합물 (4) Compound 미리스토일Myristico 테트라펩타이드(Myristoyl AAPV)의Of the tetrapeptide (Myristoyl AAPV) 파이브로넥틴Fibronectin 합성 증진 효과 Synthetic enhancement effect
인간 피부 섬유아세포인 Hs68 (human skin fibroblast)를 1*106cells/well 농도로 6 well plate에 분주한 후 70~80% 정도 자랄 때까지 37°C, 5% CO2의 조건에서 24 시간 배양하였다.Human skin fibroblast (Hs68), a human skin fibroblast, was added to 6-well plates at a concentration of 1 * 10 6 cells / well and cultured for 24 hours at 37 ° C and 5% CO 2 until 70-80% Respectively.
시료를 희석한 무혈청배지를 배양배지와 교체한 후 48 시간 동안 추가 배양한다. 배양이 끝난 배지를 원심분리기로 죽은 세포와 분리하여 얻은 상등액 100㎕를 파이브로넥틴 항체가 코팅된 96well plate(TAKARA 사, 일본. Cat#: MK115)에 넣고 어두운 곳에서 2시간 동안 37°C에서 배양하였다. 배양액을 제거하고 0.05% Tween20을 첨가한 Phosphate buffer saline(PBS)으로 3회 세척한 후 100 ㎕ HRP-conjugate 된 2차 항체용액를 넣어 1시간 동안 배양한다. 0.05% Tween20을 첨가한 Phosphate buffer saline으로 4회 세척 후 100 ㎕ TMB 용액을 넣어 실온에서 15분간 반응시킨 후 100 ㎕의 1M 황산을 넣어 반응을 종료시킨다. 마이크로 플레이트 판독기(microplate reader, Tecan)을 이용하여 450/595nm에서 흡광도를 측정하였다. 파이브로넥틴 생성 증가율 (%)은 아래의 수학식 2와 같이 환산하였다.
The serum-free medium diluted with the sample is replaced with the culture medium, followed by further incubation for 48 hours. 100 μl of the supernatant obtained by separating the culture medium from the cells dead by centrifugation was added to a 96-well plate (TAKARA, Cat. #: MK115, Japan) coated with fibronectin antibody and incubated in the dark for 2 hours at 37 ° C Lt; / RTI > After removing the culture medium, wash it with phosphate buffer saline (PBS) supplemented with 0.05
[수학식 2]&Quot; (2) "
파이브로넥틴 생성 증가율(%) = [(Fs - Fc)/Fc]×100Nektin generated by five rate (%) = [(F s - F c) / F c] × 100
Fs: 시료를 처리한 군의 흡광도F s : absorbance of the group treated with the sample
Fc: 컨트롤 군의 흡광도F c : absorbance of the control group
도 3은 본 발명의 일 실시예로서 상기의 방법으로 외부 자극을 주지 않은 자연노화상태에서 양성대조군인 TGF-β1과 비교하여 미리스토일 테트라펩타이드의 파이브로넥틴 합성능을 시험한 결과를 나타낸 도표이다. 상기와 같이 실험한 결과 미리스토일 테트라펩타이드(Myristoyl AAPV)를 처리 하였을 때 양성대조군인 TGF-β 이상으로 파이브로넥틴 합성이 증가한 것을 확인하였다.
FIG. 3 is a graph showing the results of testing the fusion activity of the myristoyltetrapide in comparison with the positive control group TGF-β1 in a natural aging state in which no external stimulus is given by the above method as an embodiment of the present invention to be. As a result of the above-mentioned experiment, it was confirmed that the synthesis of fibronectin was more than that of the positive control group TGF-β when the myristoyl AAPV was treated.
(5) (5) 미리스토일Myristico 테트라펩타이드(Myristoyl AAPV)의Of the tetrapeptide (Myristoyl AAPV) MMPMMP -1 생성 억제 효과-1 production inhibitory effect
인간 피부 섬유아세포인 Hs68 (human skin fibroblast)를 1*106cells/well 농도로 6 well plate에 분주한 후 70~80% 정도 자랄 때까지 37°C, 5% CO2의 조건에서 24 시간 배양하였다.Human skin fibroblast (Hs68), a human skin fibroblast, was added to 6-well plates at a concentration of 1 * 10 6 cells / well and cultured for 24 hours at 37 ° C and 5% CO 2 until 70-80% Respectively.
시료를 희석한 무혈청배지를 배양배지와 교체한 후 48 시간 동안 추가 배양한다. 배양이 끝난 배지를 원심분리기로 죽은 세포와 분리하여 얻은 상등액 100㎕를 MMP-1 항체가 코팅된 (anti-MMP-1 antibody coated) 96well plate(머크 사, 미국. Cat#: MK101)에 넣고 어두운 곳에서 2시간 동안 37°C에서 배양하였다. 배양액을 제거하고 0.05% Tween20을 첨가한 Phosphate buffer saline(PBS)으로 4회 세척한 후 100 ㎕ HRP-conjugate 된 2차 항체용액를 넣어 1시간 동안 배양한다. 0.05% Tween20을 첨가한 Phosphate buffer saline으로 4회 세척 후 100 ㎕ TMB 용액을 넣어 실온에서 15분간 반응시킨 후 100 ㎕의 1M 황산을 넣어 반응을 종료시킨다. 마이크로 플레이트 판독기(microplate reader, Tecan)을 이용하여 450/595nm에서 흡광도를 측정하였다. MMP-1 생성 억제율(%)은 아래의 수학식 3와 같이 환산하였다.
The serum-free medium diluted with the sample is replaced with the culture medium, followed by further incubation for 48 hours. 100 μl of the supernatant obtained by separating the culture medium from the cells killed by the centrifugal separator was added to a 96-well plate (Merck, USA, Cat #: MK101) coated with anti-MMP-1 antibody coated Lt; RTI ID = 0.0 > 37 C < / RTI > for 2 hours. After removing the culture medium, wash it with phosphate buffer saline (PBS) supplemented with 0.05
[수학식 3]&Quot; (3) "
MMP-1 생성 억제율(%) = [1 - (Ms)/Mc]×100 MMP-1 production inhibition rate (%) = [1 - (M s ) / M c ] x 100
Ms: 시료를 처리한 군의 흡광도M s : Absorbance of the group treated with the sample
Mc: 컨트롤 군의 흡광도
M c : Absorbance of the control group
도 4는 본 발명의 일 실시예로서 상기의 방법으로 외부 자극을 주지 않은 자연노화상태에서 양성조군인 TGF-β1과 비교하여 미리스토일 테트라펩타이드(Myristoyl AAPV)의 MMP-1 합성 억제능력을 시험한 결과를 나타낸 도표이다. 상기와 같이 실험한 결과 미리스토일 테트라펩타이드(Myristoyl AAPV)를 처리 하였을 때 MMP-1 효소 생성량이 저해되는 것을 확인하였다.
FIG. 4 is a graph showing the ability of Myristoyl AAPV to inhibit MMP-1 synthesis in comparison with TGF-β1 as a positive control in the natural aging state in which no external stimulus is given by the above method Fig. As a result of the above experiment, it was confirmed that the production of MMP-1 enzyme was inhibited when myristoyl AAPV was treated with myristoyl peptide.
(6) (6) 미리스토일Myristico 테트라펩타이드(Myristoyl AAPV)에The tetrapeptide (Myristoyl AAPV) 의한 by 프로콜라젠Procollagen 및 And MMPMMP 유전자 발현 확인 Confirm gene expression
인간 피부 섬유아세포인 Hs68 (human skin fibroblast)를 1*106cells/well 농도로 6 well plate에 분주한 후 70~80% 정도 자랄 때까지 37°C, 5% CO2의 조건에서 24 시간 배양하였다.Human skin fibroblast (Hs68), a human skin fibroblast, was added to 6-well plates at a concentration of 1 * 10 6 cells / well and cultured for 24 hours at 37 ° C and 5% CO 2 until 70-80% Respectively.
각 well plate에 Phosphate buffer saline(PBS) 1㎖씩을 넣어 세포 세척 한 다음 시료를 희석한 무혈청배지를 배양배지와 교체한 후 6시간(수용체), 24시간(프로콜라젠) 또는 48시간(MMP-1, MMP-3)동안 추가 배양하였다.Cells were washed with 1 ml of phosphate buffered saline (PBS) in each well plate, and the serum-free medium diluted with the sample was replaced with the culture medium for 6 hours (receptor), 24 hours (procollagen) 1, MMP-3).
배지를 제거 후 RNA extraction kit(invitrogen, 한국)을 이용하여 RNA 를 분리한 후, cDNA를 합성하였다. Procollaen 1, MMP-1 및 MMP-3의 mRNA 발현량을 측정하기 위하여 합성된 cDNA로 RT-PCR을 실시하였다. 사용된 GAPDH (Glyceraldehyde-3-phosphate dehydrogenase), Procollaen 1, MMP-1 및 MMP-3의 primer sequence는 아래의 표2과 같다. 각 primer를 각각 1 μL (0.5 μg/μL)씩 가하고 합성된 cDNA에 DEPC-Water와 함께 PCR-premix tube ((주)바이오니아, 한국)에 첨가하여 Denaturation은 95℃에서 30초, annealing은60℃에서 30초, extension은 72℃에서 2분 동안 30cycle로 진행하였다. PCR 생성물은 1% agarose gel에서 non-specific PCR product나 primer dimer 등이 없는지를 확인하였고, 생성물의 양을 EC3 UV illuminator (UVP, USA)를 이용하여 확인하였다.
After removing the medium, RNA was isolated using an RNA extraction kit (Invitrogen, Korea), and cDNA was synthesized. RT-PCR was performed on the synthesized cDNA to measure the mRNA expression levels of
도 5는 본 발명의 일 실시예로서 상기의 방법으로 양성대조군인 TGF-β1과 비교하여 미리스토일 테트라펩타이드(Myristoyl AAPV)가 프로콜라겐 타입1 mRNA 발현에 미치는 영향을 확인하였다. FIG. 5 shows the effect of myristoyl AAPV on the expression of
도 6은 본 발명의 일 실시예로서 상기의 방법으로 양성대조군인 TGF-β1과 비교하였을 때 미리스토일 테트라펩타이드(Myristoyl AAPV)가 콜라겐 분해 효소인 MMP-1과 MMP-3의 감소를 PCR 실험을 통해 확인한 결과이다. 도7은 미리스토일 테트라펩타이드(Myristoyl AAPV) 기전의 일환으로 TGFβ 수용체 발현의 증가를 상기와 같이 실험한 결과를 나타낸 것이다.
FIG. 6 is a graph showing the results of a decrease in MMP-1 and MMP-3 collagenase enzymes (Myristoyl AAPV) when compared with the positive control TGF-β1 . FIG. 7 shows the results of the above-mentioned experiment in which the increase of TGFβ receptor expression was observed as a part of the myristoyl AAPV mechanism.
(7) (7) 미리스토일Myristico 테트라펩타이드(Myristoyl AAPV)처리에In the tetrapeptide (Myristoyl AAPV) treatment 따른 피부 관련 유전자들의 발현 확인 Expression of skin-related genes
인간 피부 섬유아세포인 Hs68 (human skin fibroblast)를 1*106cells/well 농도로 6 well plate에 분주한 후 70~80% 정도 자랄 때까지 37°C, 5% CO2의 조건에서 24 시간 배양하였다. Human skin fibroblast (Hs68), a human skin fibroblast, was added to 6-well plates at a concentration of 1 * 10 6 cells / well and cultured for 24 hours at 37 ° C and 5% CO 2 until 70-80% Respectively.
각 well plate에 Phosphate buffer saline(PBS) 1㎖씩을 넣어 세포 세척 한 다음 시료를 희석한 무혈청배지를 배양배지와 교체한 후 24시간 동안 추가 배양하였다. Cells were washed with 1 ml of phosphate buffer saline (PBS) in each well plate, and the serum-free medium diluted with the sample was replaced with the culture medium and further cultured for 24 hours.
배지를 제거 후 RNA extraction kit(Invitrogen, 한국)을 이용하여 RNA 를 분리하였다. 각각의 RNA 샘플 (200ng)들을 표지 시킨 후 Low Input Quick Amp labeling kit (Agilent Technologies, CA.)을 사용하여 증식 시켰다. Cy3로 표지된 aRNA는50 ul의 교잡용액 (Agilent Technologies, CA.)과 섞어준다. 표지된 aRNA는 Agilent SurePrint G3 Human GE 8x60K array (Agilent Technologies, CA.)에 넣어준 후 Gasket 8-plex slide (Agilent Technologies, CA.)로 덮어준다. 이 슬라이드는 17시간 동안 65°C로 세팅된 오븐에서 교잡을 실시한다. 이 슬라이드는 30,000 rpm에서 20초간 분리 후 20초 동안 건조시켰다.After removing the medium, RNA was isolated using RNA extraction kit (Invitrogen, Korea). Each RNA sample (200 ng) was labeled and then propagated using a Low Input Quick Amp labeling kit (Agilent Technologies, CA.). Cy3-labeled aRNA is mixed with 50 μl of hybridization solution (Agilent Technologies, CA.). Labeled aRNA is loaded onto an Agilent SurePrint G3 Human GE 8x60K array (Agilent Technologies, CA.) and covered with a Gasket 8-plex slide (Agilent Technologies, CA.). This slide is carried out in an oven set at 65 ° C for 17 hours. The slides were dried at 30,000 rpm for 20 seconds and then dried for 20 seconds.
DNA chip 결과의 분석 과정은 scanning 을 통하여 얻어진 spot의 signal intensity 수치 데이터를 사용하여 Quantile normalization, RMA(Robust Multi-array Average) normalization을 선별적으로 사용하였으며, 이를 사용하여 scatter plot으로 표시하였다. 실험 결과로 얻어진 모든 유전자들의 분포를 히스토그램으로 표시하였으며 이것은 normalization이 끝난 데이터의 분포도와 비교 검토하여 실험 결과의 정확성을 확인하였다. DNA chip results were analyzed by using quantitative normalization and robust multi-array average (RMA) normalization using spot intensity data obtained from scanning. The distribution of all the genes obtained by the experiment is shown by histogram, and this is compared with the distribution of the normalized data to confirm the accuracy of the experiment results.
도 8로부터 미리스토일 테트라펩타이드(Myristoyl AAPV)를 처리 하였을 때 발현하는 유전자들이 TGFβ1을 처리했을 때 발현하는 유전자들의 양상과 매우 유사함울 확인하였다.From FIG. 8, it was confirmed that genes expressed when myristoyl AAPV was treated with myristoyl were very similar to those expressed when TGFβ1 was treated.
도 9로부터 미리스토일 테트라펩타이드(Myristoyl AAPV)에 의해 세포외 기질 단백질 유전자들이 많이 발현하는 것을 확인하였다.
From FIG. 9, it was confirmed that extracellular matrix protein genes were expressed by myristoyl AAPV.
<실시예 3> 임상에서의 주름 개선 효과 Example 3 Effect of Clinical Wrinkle Reduction
아래 표 3에 나타낸 조성으로 로션을 제조하여 인체에 대한 주름 개선 효과를 실제 사용 테스트를 통하여 평가하였다.
The lotion was prepared by the composition shown in Table 3 below and the wrinkle-improving effect on the human body was evaluated through actual use test.
테트라펩타이드
(Myristoyl AAPV)Myristico
Tetrapeptide
(Myristoyl AAPV)
0.0002
0.0002
(1) (One) 미리스토일Myristico 테트라펩타이드의Tetrapeptide 주름개선 효과 평가 Wrinkle improvement effect evaluation
33명의 45-60세의 건강한 동양 여성들에게 미리스토일 테트라펩타이드(Myristoyl AAPV)를 함유하는 로션을 매일 아침 저녁 2회씩 적당량을 눈가 주름을 포함한 한쪽 얼굴에 3개월간 연속적으로 바르게 하였다. Thirty-three healthy 45-60 year old Oriental women were given a lotion containing Myristoyl AAPV twice daily, morning and evening, each month for 3 months in an appropriate amount, including wrinkles around the eyes.
각 피검자의 주름 개선효과를 Replica analysis(주름 평가법)방법을 통해 전체 주름 수, 주름 길이, 주름 면적을 측정하여 수치화 하였고, 영상분석을 통해 평가하였다. 이 시험은 태국의 임상전문 업체인 Spincontrol Asia를 통해 진행 되었으며, 그 결과를 표 4에 나타내었다.The wrinkle improvement effect of each subject was quantified by measuring the total wrinkle number, wrinkle length and wrinkle area through the replica analysis method and evaluated by image analysis. This test was conducted by Thai clinical specialist company Spincontrol Asia, and the results are shown in Table 4.
표 4에서 나타내듯이 미리스토일 테트라펩타이드(Myristoyl AAPV)를 함유한 임상실험에서 피부주름 파라미터인 전체 주름 수, 주름 면적, 주름 길이를 모두 확연히 감소시킨 것으로 보아 피부 주름 개선 효과가 매우 우수함을 확인하였다. 특히 전체 지원자들 중 45-55세의 여성들에게서 주름 면적과 길이가 전체 평균치에 비해 많이 감소한 것을 확인함으로써 미리스토일 테트라펩타이드(Myristoyl AAPV)이 미세주름에 효과적인 것을 확인하였다.
As shown in Table 4, it was confirmed that the skin wrinkle parameters (total wrinkle number, wrinkle area, and wrinkle length) were remarkably reduced in clinical trials containing myristoyl AAPV, . In particular, it was confirmed that myristoyl AAPV (myristoyl AAPV) is effective for fine wrinkles by confirming that the wrinkle area and length decreased significantly compared with the overall average in women between 45 and 55 years of age among all applicants.
(주)Experiment period
(week)
Claims (6)
[화학식 1]
(상기 [화학식 1]에서, R은 수소 또는 1~29개의 탄소 원자를 가진 아실기로 포화되거나 포화되지 않은 테트라펩타이드인 아실 유도체를 포함한다.)
A tetrapeptide represented by the following formula (1).
[Chemical Formula 1]
(In the above formula (1), R includes hydrogen or an acyl derivative which is a tetrapeptide saturated or unsaturated with an acyl group having 1 to 29 carbon atoms.)
The compound of claim 1, wherein R is selected from the group consisting of hydrogen or a substituent selected from the group consisting of acetyl, ethanoyl, propanoyl, butanoyl, pentanoyl, hexanoyl, octanoyl, nonanoyl, decanoyl, dodecanoyl, tetradecanoyl, hexadecanoyl , Octadecanoyl, stearoyl, isostearoyl, lauryl, undecenoyl, oleoyl, elaidoyl, and 2-ethylhexenoyl. Gt; is selected from the group consisting < RTI ID = 0.0 > of: < / RTI >
The method according to claim 1, wherein Ala in the amino acid of the displayed tetrapeptide is D-Ala or L-Ala, Pro is selected from D-Pro or L-Pro and Val is selected from D-Val or L-Val Lt; / RTI >
The tetrapeptide according to claim 1, wherein the C-terminus of the tetrapeptide to be displayed is selected from carboxylic acid, amide or aldehyde forms.
A cosmetic or pharmaceutical composition for preventing skin aging or improving skin wrinkles, wherein the fatty acid tetrapeptide of any one of claims 1 to 4 is contained in an amount of 0.0001 to 5 wt% based on the total weight of the composition.
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| WO2020144109A1 (en) * | 2019-01-08 | 2020-07-16 | Lipotrue, S.L. | Peptides and compositions for use in cosmetics |
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| ES2330291B1 (en) * | 2008-02-29 | 2010-10-18 | Lipotec Sa | USEFUL PEPTIDES IN THE TREATMENT OF SKIN, MUCOSAS AND / OR LEATHER HAIR AND ITS USE IN COSMETIC OR PHARMACEUTICAL COMPOSITIONS. |
| ES2349972B1 (en) | 2009-02-16 | 2011-11-24 | Lipotec, S.A. | USEFUL PEPTIDES IN THE TREATMENT AND / OR CARE OF SKIN, MUCOUSES AND / OR LEATHER LEATHER AND ITS USE IN COSMETIC OR PHARMACEUTICAL COMPOSITIONS. |
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| CN113271920A (en) * | 2019-01-08 | 2021-08-17 | 利波特鲁有限公司 | Peptides and compositions for use in cosmetics |
| CN113271920B (en) * | 2019-01-08 | 2023-08-01 | 利波特鲁有限公司 | Peptides and compositions for cosmetic use |
| CN111467274A (en) * | 2020-04-30 | 2020-07-31 | 深圳市垂纶远翔科技有限责任公司 | Eye cream for quickly removing wrinkles and bags under eyes |
| KR20210152415A (en) * | 2020-06-08 | 2021-12-15 | 나인바이오팜 주식회사 | Composition for skin protection or wrinkle improvement including Regentide-034 and Regentide-041 |
| KR20210152413A (en) * | 2020-06-08 | 2021-12-15 | 나인바이오팜 주식회사 | Regentide-034 and Composition for improving skin condition comprising the same |
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|---|---|
| KR101437237B1 (en) | 2014-10-30 |
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