KR101342488B1 - Tetrapeptide Having Anti-Aging And Anti-Inflammatory Activities And Cosmetic Composition Containing The Same - Google Patents

Abstract

The present invention relates to a tetrapeptide represented by the following formula (1) and (2) and to skin anti-aging and skin wrinkle improvement, containing the same as an active ingredient, anti-inflammatory cosmetic composition. The tetrapeptide according to the present invention provides a cosmetic having excellent efficacy in improving wrinkles and inflammation by showing an anti-inflammatory effect as well as an anti-wrinkle effect due to the inhibition of the production of collagen degrading enzyme MMPs. In particular, the tetrapeptides for wrinkle improvement and anti-inflammatory are added to various basic cosmetic compositions such as skins, lotions, creams, foundations, essences, gels, packs, forking, body oils, lipsticks, mascaras, makeup bases or soap forms. It can be used and has no skin irritation and excellent skin stability.
[Chemical Formula 1]
RR ¹ -Gly-Leu-Phe
(2)
R-Gly-Leu-Phe-R ²
(In Formula 1 and Formula 2, R is an alkenyl group selected from hydrogen or an acyl group having 1 to 18 carbon atoms or at least one double bond, Leu is D-Leu or L-Leu, Phe is D- can be Phe or L-Phe. in addition may be selected from formula I and the phenylglycine (phenylglycine) portions are each amino acid, especially the 20 natural amino acids and non-associated amino acid represented by the formula (2) in R ¹ and R ^2, and The C-terminus may be in carboxylic acid or amide form.)

Description

Tetrapeptide Having Anti-Aging And Anti-Inflammatory Activities And Cosmetic Composition Containing The Same}

The present invention relates to a novel tetrapeptide (Tetrapeptide) and a cosmetic composition for preventing skin aging, skin wrinkle improvement and anti-inflammatory, containing the same as an active ingredient, in particular, the present invention is collagen decomposition which is the most important factor for the application of cosmetics for skin wrinkle improvement The present invention relates to novel tetrapeptides having an anti-aging effect, improving skin wrinkles, and anti-inflammatory effects by inhibiting synthesis and expression of enzymes and decreasing cytokines increased by ultraviolet rays, and a cosmetic composition containing the same as an active ingredient.

As the industry develops and the average life expectancy of humans increases, interest in the effects of aging on the skin in relation to skin function and external beauty is increasing. Because skin is in direct contact with the external environment, it is a complex organ made up of a variety of cells and structures that serve as an important barrier to protecting the body from external factors, including trauma, infection, and ultraviolet radiation. The skin is largely composed of epidermis, dermis and subcutaneous tissue. The epidermis is a thin layered tissue consisting mainly of keratinocytes and pigmented melanocytes and Langerhans cells, and the dermis is mainly composed of extracellular tissue proteins produced by fibroblasts. Subcutaneous tissue, composed of fat cells (Sebocytes), is responsible for the framework of connective tissue. Type I collagen, the most abundant protein, forms connective tissue along with other extracellular tissue proteins other than collagen, elastin, proteoglycan and fibronectin. Configure. The newly produced type I procollagen is secreted into the extracellular space of the dermis and then undergoes fibrillogenesis to produce collagen, which contributes to the elasticity and strength of the skin.

Skin aging is affected by genetic and environmental exposures (UV irradiation, mechanical shock), hormonal changes, and metabolic processes (reactive compounds such as reactive oxygen species, sugars, and aldehydes). These factors cause cumulative changes in the structure, function, and appearance of the skin. Human skin aging is divided into internal aging that occurs over time according to two different biological processes, and external aging (photoaging) caused by the environment, such as constant light exposure, stress, and smoking. In clinically internally aged skin, the skin is thin, dry, pale, loses its elasticity, and has many fine wrinkles.In the case of aging skin due to external environment such as light and smoking, deep wrinkles, spots, and rough skin are observed. It will have a greater effect on your back skin. Although aging and photo-aged skin are distinctly morphologically histologically distinct, their appearance is clearly distinguished, but recent studies show that both aging promotes the expression of collagen degrading enzymes (MMPs), reduces the synthesis of procollagen, It is reported to share important molecular features, including signaling pathways such as connective tissue damage. Environmental influences cause more apparent variations in aesthetics and tissues than internal factors, the most lethal external factor being UV radiation. The consensus of this molecular mechanism is believed that UV irradiation accelerates important aspects of aging by chronological age of human skin.

From a histological point of view, skin aging is a change in the composition of extracellular matrix proteins that form a 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. However, in skin damaged by photoaging, the precursors of these two collagens are markedly reduced and their decrease is associated with clinical severity. Collagen is degraded as aging progresses, but when exposed to stimuli such as UV rays, collagen breakdown enzymes (MMPs) increase in biosynthesis, which leads to decreased collagen synthesis and wrinkles. Collagen degrading enzymes (MMPs) are zinc-dependent intracellular proteases that can degrade or restructure components of the extracellular structure of the dermis, of which MMP-1 acts to segment collagen in the dermis. . Therefore, regulation of the amount and activity of this enzyme is important for wrinkles.

Ultraviolet rays, the most harmful of the environmental factors that damage the skin, refers to a wavelength band between 200 nm and 400 nm in the electromagnetic spectrum. Depending on the length of the wavelength, UVA (320-400 nm), UVB (280-320 nm) and UVC (200) -280nm). Because UVC is blocked by the ozone layer, the sun's radiation that harms human skin is UVA and UVB. UVA radiation is less intense than UVB radiation, but has high skin permeability, and UVA skin damage is known to include sunburn, cancer, and skin aging. UVB radiation, the most dangerous environmental carcinogen associated with human health, is known to cause gene damage and skin cancer in addition to erythema and sunburn. The biggest changes in skin damaged by photoaging occur in the dermis, the deeper part of the skin, leading to breakdown of connective tissue, a decrease in collagen content and the accumulation of modified elastic fibers.

Retinol and retinoids, the most representative of which are skin wrinkle improving substances, are known to have good effects in various fields such as psoriasis, aging, cancer, and acne. In addition, the retinoids are known to prevent collagen loss by stimulating collagen formation by inhibiting collagenase (MMP-1) when applied to the skin to prevent and restore endogenous and photoaging. Despite these effects, however, it is known to cause local skin irritation reactions such as erythema, itching, psoriasis, and scaling when applied to the skin. Therefore, there is a need for developing a new anti-wrinkle substance which is safer in vivo and suppresses collagenase (MMP-1) than the existing substance and has good collagen synthesis ability.

Damage to the skin by UV irradiation is known to cause inflammation as well as wrinkle formation. In addition to the above mentioned biological mechanisms for skin damage, the skin is known to secrete numerous cytokines, including IL-1, IL-6, IL-8, and TNF-α. Among the most lethal external stimuli, UVB irradiation revealed that keratinocytes secrete extracellular extracellularly of Tumor necrosis factor-α (TNF-α), Interleukin-1β (IL-1β), IL-6, IL-10 and IL-8. To stimulate. UVA irradiation is known to increase the production of these cytokines from fibroblasts in the dermal layer as well as to induce collagen degrading enzymes such as MMP-1. Sun-exposed skin promotes a sustained inflammatory response due to the release of these cytokines. The relationship between chronic inflammation and the production of multiple cancers is not only well known, but has also been shown in many tumor experiments. Skin cells induced to produce cytokines by various external stimuli lose cytokine control and eventually cause various skin diseases including cancer. Therefore, there is also a need for development of a substance that inhibits inflammatory reactions caused by cytokines, along with skin aging damaged by UV stimulation.

1 Rittie R, Fisher GJ. UV-light-induced signal cascades and skin aging. Age Res Rev 2002; 1: 705-720. 2 Yaar M, Gilchrest BA. Aging versus photoaging: postulated mechanisms and effectors. J Invest Dermatol Symp Proc 1998: 3: 47-51. Document 3 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 2000; 106: 663-670. [4] Fisher GJ, Wang ZQ, Datta SC, Varani J, Kang S, Voorhees JJ. Pathophysiology of premature skin aging induced by ultraviolet light. N Engl J Med 1997; 337: 1419-28. [Reference 5] Pilcher, BK, Sudbeck, BD, Dumin, JA, Welgus, HG, Parks, WC. Collagenase-1 and collagen in epidermal repair. Arch Dermatol Res 1998; 290 (Suppl): S37-46. Svobodova, A, Psotova, J, Walterova, D. Natural phenolics in the prevention of UV-induced skin damage. Biomed Papers 2003; 147: 137-145. 7 Glichrest, BA, Yaar, M. Ageing and photoaging of the skin: observations and the cellular molecular level. Br J Dermatol 1992; 127: 25-30. 8, Chen VL, Fleischmajer R, Schwartz E, Palaia M, Timpl R. Immunochemistry of elastotic material in sun-damaged skin. J Invest Dermatol 1986; 87: 334-337. 9 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 1993; 329: 530-538. 10. Talwar HS, Griffiths CEM, Fisher GJ, Hamilton TA, Voorhees JJ. Reduced type I and type III procollagens in adult human skin. J Invest Dermatol 1995; 105: 285-90. [11] Fisher GJ, Datta SC, Talwar HS, Wang ZQ, Varani J, Kang S, Voorhees JJ. Molecular basis of sun induced premature skin ageing and retinoid antagonism. Nature 1996; 379: 335-9. 12. Glichrest BA. A review of skin ageing and its medical therapy. Br J Dermatol 1996; 135: 867-75. Document 13 Alexander JP, Acott TS. Involvement of Erk-MAP kinase pathway in TNFa regulation of trabecular metalloproteinases and TIMPs. Invest Ophthalmol Vis Sci 2001; 42: 2831-2838. [14] Rutter JL, Benbow U, Coon CI, Brinckerhoff CE. Cell-type specific regulation of human interstitial collagenase-1 gene expression by interleukin-1β (IL-1β in human fibroblasts and BC-8701 breast cancer cells. J Cell Biochem 1997; 66: 322-336. Reference 15 Mueller MM. Inflammation in epithelial skin tumours: old stories and new ideas. Eur J Cancer 2006; 42: 735-744. 16. Van Kempen LCLT, Ruiter DJ, van Muijen GNP, Coussens LM. The tumour microenvironment: a critical determinant of neoplastic evolution. Eur J Cell Biol 2003; 82: 539-548. Kupper TS, Chua AO, Flood P, McGuire J, Gubler U. Interleukin 1 gene expression in cultured human keratinocytes is augmented by ultraviolet irradiation. J Clin Invest 1987; 80: 430-436. 18 Kondo S, Kono T, Sauder DN, McKenzie RC. IL-8 gene expression and production in human keratinocytes and their modulation by UVB. J Invest Dermatol 1993; 101: 690-694.  [19] Vamos-vigyazo L. Polyphenol oxidase and peroxidase in fruits and vegetables. Crit Rev Food Sci Nutr 1981; 15: 49-127.

Under this background, the present inventors have found that in using a fiber skin neuroblastoma (human skin fibroblasts) cells with UVA / UVB lamp to identify the anti-aging and anti-wrinkle, anti-inflammatory effect of tetra-peptide vitro by the assay system was established search comparing the biosynthetic ability of collagen and collagen-decomposing enzyme (MMPs) and retinoids, keratinocytes (Keratinocytes), fiber neuroblastoma (human skin fibroblasts) and in using a UVB lamp In vitro assay system was established to confirm the reduction of cytokines increased by UV irradiation and to complete the present invention by confirming the anti-wrinkle effect and anti-inflammatory effect of tetrapeptide (Tetrapeptide).

The present invention is to solve the problems of the prior art as described above is an object of the present invention is harmless to the human body and has excellent skin permeability or stability novel tetrapeptide (Tetrapeptide) and the anti-aging, wrinkle improvement and anti-inflammatory effect containing the same It is providing the external skin pharmaceutical composition and cosmetic composition which are shown.

The tetrapeptide according to the present invention for achieving the above object is represented by the following formula (1) and (2).

[Formula 1]

RR ¹ -Gly-Leu-Phe

(2)

R-Gly-Leu-Phe-R ²

(In Formula 1 and Formula 2, R is an alkenyl group selected from hydrogen or an acyl group having 1 to 18 carbon atoms or at least one double bond, Leu is D-Leu or L-Leu, Phe is D- can be Phe or L-Phe. in addition may be selected from formula I and the phenylglycine (phenylglycine) portions are each amino acid, especially the 20 natural amino acids and non-associated amino acid represented by the formula (2) in R ¹ and R ^2, and The C-terminus may be in carboxylic acid or amide form.)

In the above, R is preferably selected from an acyl group consisting of hydrogen or ethanoyl, octanoyl, tetradecanoyl, hexadecanoyl and octadecanoyl. In particular, R is tetradecanoyl or hexadecanoyl, and among the amino acids of the displayed peptide, Leu is preferably L-Leu, Phe is L-Phe, and the C-terminus is more preferably a carboxylic acid.

R ¹ is preferably selected from amino acids consisting of glycine (leucine), leucine, lysine (lysine) and serine, and R ² is cysteine, glutamic acid, or phenylalanine. (phenylalanine), glycine (glycine), isoleucine (isoleucine), valine, proline, leucine, serine, trionine, tryptopane, tyrosine It is preferably selected from amino acids consisting of. In particular, R ¹ is glycine (glycine) and R ² is preferably selected from phenylalanine (phenylalanine), proline (proline), trionine (thrionine), tryptopane, tyrosine (tyrosine).

The present invention also provides an anti-aging and anti-inflammatory composition comprising the above-described tetrapeptide as an active ingredient. In particular, the tetrapeptide provides an anti-aging and anti-inflammatory composition comprising 0.0001 to 10% by weight based on the total weight of the composition. Such anti-aging agent composition may be a cosmetic composition or a pharmaceutical composition. Such compositions may be formed in the form of skins, lotions, creams, foundations, essences, gels, packs, forking, body oils, lipsticks, mascaras, makeup bases or soaps.

The present invention shows that the composition containing a novel tetrapeptide with no skin irritation and excellent skin stability as an active ingredient has an anti-inflammatory effect as well as an anti-wrinkle effect due to the inhibition of MMP-1 production, a collagen biosynthesis enhancement effect, which is a collagen degrading enzyme. It was confirmed that this is to use it as a skin pharmaceutical composition and cosmetic composition having excellent efficacy in improving wrinkles and inflammation.

1 is a result of confirming the cytotoxicity of tetrapeptide ( A440_19 ).
Figure 2 is a diagram showing the results of testing the MMP-1 inhibitory ability by tetrapeptide ( A440_19 ) at a safe concentration confirmed in Figure 1 as an embodiment of the present invention.
Figure 3 is a diagram showing the results of testing the MMP-1 inhibitory ability of tetrapeptide ( A440_19 ) against MMP-1 increased by UVA irradiation as an embodiment of the present invention.
Figure 4 is a diagram showing the results of testing the MMP-1 inhibitory ability of tetrapeptide ( A440_19 ) against MMP-1 increased by UVB irradiation as an embodiment of the present invention.
5 is a table showing the results of comparing the MMP-1 inhibitory ability of the tetrapeptide ( A440_19 ) with M --1 increased by Retinyl Palmitate by UVA irradiation as an embodiment of the present invention.
6 is a diagram showing the results of comparing the MMP-1 inhibitory ability of the tetrapeptide ( A440_19 ) to MMP-1 increased by UVA irradiation with Retinol as an embodiment of the present invention.
FIG. 7 is a table showing the results of testing the ability of the procollagen type 1 to increase the tetrapeptide ( A440_19 ) to the procollagen type 1 reduced by UVA irradiation as an embodiment of the present invention.
8 is a diagram showing the effect of tetrapeptide ( A440_19 ) on the extracellular protein mRNA expression changes by UVA irradiation as an embodiment of the present invention.
Figure 9 is an embodiment of the present invention as a diagram showing the numerical value of MMP-1 expression in the results shown in Figure 8 by densitometer (Densitometer).
FIG. 10 is a graph showing numerical values of Procollagen Type I expression in a densitometer of the results of FIG. 8 as an embodiment of the present invention.
Figure 11 is an embodiment of the present invention is a graph showing the numerical expression of fibronectin (Fibronectin) of the results shown in Figure 8 by a densitometer.
Figure 12 shows the results obtained by testing the IL-1b and IL- 6 and IL-8 cytokine (Cytokine) inhibitory capacity increase for the UVB irradiation of tetra peptide (A440_19) in dermal cells (Skin Firoblasts) as an embodiment of the present invention It is a chart.
FIG. 13 shows increased IL1-α, IL-1β, IL-6, and IL-8 cytokine (Cytokine) inhibition against UVB irradiation of tetrapeptide ( A440_19 ) in keratinocytes cells in the epidermis as an embodiment of the present invention. This chart shows the results of the ability test.
14 is a graph showing the effect of improving the wrinkles by the tetrapeptide ( A440_19 ) as an embodiment of the present invention in clinical trials.

The tetrapeptide according to the present invention for achieving the above object is represented by the following formula (1) and (2).

[Formula 1]

RR ¹ -Gly-Leu-Phe

(2)

R-Gly-Leu-Phe-R ²

(In Formula 1 and Formula 2, R is an alkenyl group selected from hydrogen or an acyl group having 1 to 18 carbon atoms or at least one double bond, Leu is D-Leu or L-Leu, Phe is D- can be Phe or L-Phe. in addition may be selected from formula I and the phenylglycine (phenylglycine) portions are each amino acid, especially the 20 natural amino acids and non-associated amino acid represented by the formula (2) in R ¹ and R ^2, and The C-terminus may be in carboxylic acid or amide form.)

In the above, R is a residue that binds to the N-terminus of the polypeptide, specific examples of R include hydrogen or acetyl, propanoyl, butanoyl, pentanoyl, hexanoyl, octanoyl, nonanoyl, decanoyl, dodecano Acyl groups such as one, tetradecanoyl, hexadecanoyl, octadecanoyl and the like can be included. In particular, R is preferably selected from hydrogen, acetyl, octanoyl, tetradecanoyl, hexadecanoyl or octadecanoyl, and more preferably, when R is tetradecanoyl or hexadecanoyl, the anti-aging effect is more excellent. Do. R may also be an alkenyl group containing at least one double bond. In particular, it is more preferable that R is tetradecanoyl or hexadecanoyl and the C-terminus is a carboxylic acid.

The amino acid represented by R ¹ in Formula 1 may be selected from 20 natural amino acids and phenylglycine which is an unnatural amino acid. For example, glycine, alanine, serine, aspartic acid, glutamic acid, valine, trionine, arginine , Lysine, proline, leucine, phenylalanine, isoleucine, histidine, tyrosine, metaionine, cysteine, tryptophan (tryptopane), asparagine (asparagine), glutamine (glutamine), phenylglycine and the like, and among these, preferably selected from glycine (glycine), leucine (leucine), lysine (serine), serine (serine) And most preferably in the case of glycine.

The amino acid represented by R ² in Formula 2 may be selected from 20 natural amino acids and phenylglycine which is an unnatural amino acid. For example, glycine, alanine, serine, aspartic acid, glutamic acid, valine, trionine, arginine , Lysine, proline, leucine, phenylalanine, isoleucine, histidine, tyrosine, metaionine, cysteine, tryptophan (tryptopane), asparagine (asparagine), glutamine (glutamine), phenylglycine (phenylglycine), etc. Among these, preferably cysteine (glustein) (glutamic acid), phenylalanine (phenylalanine), glycine (glycine) , Isoleucine (isoleucine), valine (valine), proline (proline), leucine (leucine), serine (serine), trionine (thrionine), tryptopane, tyrosine (tyrosine), the most preferred Preferably, the amino acid represented by R ² is phenylalanine, proline, or trion. onine, tryptopane, tyrosine.

The amino acids of the polypeptides represented by Formula 1 and Formula 2 may include amino acids of L-form and D-form, and more preferably all amino acids have L-form. In addition, the C-terminus of the tetrapeptide may be selected from the carboxylic acid or amide form, and preferably has the carboxylic acid form.

Tetrapeptides according to the invention can be synthesized by solid phase methods using conventional peptide synthesizers. 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 amino acid used in the present invention may be protected at the N-terminus by a 9-fluorenylmethyloxycarbonyl group (Fmoc).

The resin used for the solid phase reaction is applied differently depending on whether the C-terminal form of the polypeptide is carboxylic acid or amide form. When the C-terminus is a carboxylic acid, 2-chlorotrityl chloride (CTC), 4-benzyloxybenzyl alcohol (Wang), hydroxymethylphenoxyacetyl (HMPA) resin may be used. When the C-terminus is in the form of an amide, the terminal of the resin is made of an amine group. For example, trialkoxybenzhydrylamine (Rink amide), methylbenzhydrylamine (MBHA) resin may be used.

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 activator, and after coupling, the N-terminal protecting group is removed with piperidine and the next coupling is performed. 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. In the present invention, after four amino acids are coupled, the N-terminal amine group has an acyl activator having a desired acyl group, for example, acyl anhydride and DMAP (4- ( N, N -dimethylamino) pyridine)) to form an amide bond at the N-terminus or to carboxylic acid (Fatty acid), N, N -diisopropylcarbodiimide (DIC), 1-hydroxy-7-azabenzotriazole ( HOAt) or 1-hydroxybenzotriazole (HOBt) may be used together to form an amide bond. The polypeptide analogue is then separated from the solid resin by the cleavage solution comprising trifluoroacetic acid (TFA) and the protecting group bound to its side chain is removed.

The invention also provides anti-aging or anti-inflammatory compositions comprising such tetrapeptides. The compound of Formula 1 or Formula 2 is preferably contained in an anti-aging and anti-inflammatory cosmetics at 0.0001 to 10% by weight, more preferably at 0.001 to 5% by weight. If the content is less than 0.0001% by weight, the effect is insignificant, and if the content is more than 10% by weight, the economical tendency of lack of use increases.

Cosmetics comprising the tetrapeptides of Formula 1 and Formula 2 may be prepared by a known cosmetic preparation method, and may be applied to quasi-drugs and external medicines without being limited to general skin cosmetics. These formulations may be prepared in any of the formulations commonly prepared, for example in the form of skins, lotions, creams, foundations, essences, gels, packs, pocleaning, body oils, lipsticks, mascaras, makeup bases or soaps. It may be prepared as, but is not limited thereto. And in the cosmetic composition of each formulation, other components in addition to the above-described tetrapeptide can be appropriately selected and blended by those skilled in the art without difficulty according to the formulation, purpose of use, and the like of other cosmetics.

Hereinafter, the present invention configured as described above will be described in more detail through 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.

Example 1 Sample Preparation (Synthesis and Separation Purification)

[Formula 1]

RR ¹ -Gly-Leu-Phe

(2)

R-Gly-Leu-Phe-R ²

Compound synthesized as a sample Compound name Peptide Sequence Compound name Peptide Sequence A440_1 Myr-glfa A440_21 Myr-aglf A440_2 Myr-glfc A440_22 Myr-cglf A440_3 Myr-glfd A440_23 Myr-dglf A440_4 Myr-glfe A440_24 Myr-eglf A440_5 myr-GLFF A440_25 myr-FGLF A440_6 myr-GLFG A440_26 myr-GGLF A440_7 myr-GLFH A440_27 myr-HGLF A440_8 myr-GLFI A440_28 myr-IGLF A440_9 myr-GLFK A440_29 myr-KGLF A440_10 myr-GLFL A440_30 myr-LGLF A440_11 myr-GLFM A440_31 myr-MGLF A440_12 myr-GLFN A440_32 myr-NGLF A440_13 myr-GLFP A440_33 myr-PGLF A440_14 myr-GLFQ A440_34 myr-QGLF A440_15 myr-GLFR A440_35 myr-RGLF A440_16 myr-GLFS A440_36 myr-SGLF A440_17 myr-GLFT A440_37 myr-TGLF A440_18 myr-GLFV A440_38 myr-VGLF A440_19 Myr-glfw A440_39 Myr-wglf A440_20 myr-GLFY A440_40 myr-YGLF

In Table 1, myr refers to a tetradecanoyl group, that is, a myristoyl group.

SPPS (Solid Phase Peptide Synthesis) of Merrifield (Merrifield) to synthesize the compound A440 series of the invention as set forth in Table 1. The above method (J. Am. Chem. Soc. , 85 (14), 2149-2154) the Was used. In order to synthesize the peptide of the present invention, the synthesis was started using 2-chlorotrityl chloride (CTC) resin, and the first step of Fmoc (9-fluorenylmethoxy carbonyl) amino acid was N, N -diisopropylethylamine ( N, N- Coupling under dichloromethane (DCM) solvent using diisopropylethylamine (DIEA). From the next amino acid of Fmoc, 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) and 1-hydroxybenzo Triazole (HOBt) was used together with DIEA ( N, N -diisopropylethylamine) to extend the chain of the peptide by coupling. At this time, the synthesis from the C-terminal to the N-terminal direction, after coupling Fmoc-amino acid to the amino-terminal, remove the Fmoc group with 20% piperidine solution (Solid-Phase Synthesis: A Practical Guide (1 ed.). CRC Press. 848), washing with DMF (Dimethylforamide) several times, and then coupling. 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. To this, trifuloroacetic acid (TFA) -H ₂ O-Triisopropylsilane (95: 2.5: 2.5, vol./vol.) Solution was added and reacted for 2 to 3 hours to remove the protecting group and to separate the peptide from the resin. The peptide was precipitated with diethylether. Crude peptide obtained by the above method was confirmed by using RP-HPLC (2996 Detector, 515 Pump, Waters) as a gradient solvent composition of acetonitrile (acetonitrile) and water containing 0.1% TFA, the purity was confirmed 95% The following peptides were separated and purified by Prep-LC to obtain more than 95% peptide. 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. In this case, when the C-terminus is an amide compound, a trialkoxybenzhydrylamine (Rink amide) resin was used, except for the first coupling method in the synthesis of peptide using 2-chlorotrityl chloride (CTC). Coupling proceeded in the same way. All these compounds were also used to measure anti-aging and anti-inflammatory effects.

<Example 2> skin aging and skin wrinkle improvement

Human dermal cells were used to examine the anti-aging and skin wrinkle improvement effects of the compound tetrapeptide.

Wrinkles are formed in the skin because collagen synthesis decreases as the aging of the skin progresses and exposure to UV-like stimuli increases the biosynthesis of collagen degrading enzymes (MMPs). Therefore, it is possible to prevent and improve skin wrinkles by reducing the amount of biosynthesis of collagen degrading enzymes such as MMP-1 and by increasing the amount of procollagen 1 rapidly reduced by ultraviolet rays.

The experiment in the skin using fiber neuroblastoma (human skin fibroblsts, Hs68) cell lines, and tetra-peptide By establishing an in vitro assay system, the anti-aging effect of tetrapeptides was evaluated by evaluating the amount of MMP-1 biosynthesis and mRNA expression, and the in vitro assay using human skin fibroblst (Hs68) cell line and UVA / UVB lamp. By establishing a system and measuring the activity of tetrapeptide on aging by ultraviolet irradiation, skin wrinkle improvement effect was measured.

(1) Cell line and cell culture

The cell line used in the experiment was human skin fibroblasts Hs68 cells (ATCC, USA). Hs68 cells were Dulbecco's modified Eagle medium (DMEM) supplemented with 10% FBS (Gibco Co), 100 μg / ml streptomycin, and 100 U / ml penicillin at 37 ° C. and 5% CO ₂ . Incubator; Thermo-scientific, USA. Hs68 cells are 100 cm ² After growing sufficiently in a flask (Corning, USA), the surface of the cultured cells was washed with PBS at intervals of 3 days, followed by 0.25% trypsin-EDTA solution, treated in the incubator for 3 minutes, and discarded the trypsin-EDTA solution at 5 ° C. Cells were detached by storage for a minute. The detached cells were subcultured at 37 ° C. and 5% CO ₂ at a split ratio of 1: 5 by transferring 10 ml of DMEM containing 10% FBS to a new culture vessel.

(2) cells Survival rate  Measure

Dispense Hs68 (Human skin fibroblasts) into a 96 well plate at a concentration of 1 * 10 ⁴ cells / well, incubate for 24 hours at 37 ° C and 5% CO ₂ , and replace the serum-free medium with the diluted sample. Further incubation for 48 hours. Remove the culture supernatant and add 100 µl of serum-free medium and 10 µl of MTT solution (3- (4,5-dimethyldiazol-2-yl) 2,5 diphenyltetrazolium bromide (5mg / ml) for 3 hours. After incubation, remove the medium, add 100 μl of DMSO (Dimethyl sulfoxide) solution to each well, dissolve Formazan and measure the absorbance at 570 nm using a Microplate reader (Tecan). Cell survival rate (%) was converted as shown in Equation 1 below.

[ Equation 1 ]

% Cell viability = (S _s ) / S _c × 100

S _s : absorbance of the group treated with the sample

S _c : absorbance of the untreated group

Table 2 below shows the results of the cytotoxicity test after 10 ppm treatment of tetrapeptide. FIG. 1 is a table showing the results of experiments at various concentrations of cytotoxicity of Compound A440_19 , one of the tetrapeptides having no cytotoxicity and the best MMP-1 inhibitory effect, as shown in Table 2 below. As shown in Figure 1, MTT assay showed a cell viability of 95% or more to 1 to 30ppm to confirm that Compound A440_19 is a safe raw material.

Cytotoxicity of Each Compound compound
name order cell
Survival rate (%) compound
name order cell
Survival rate (%) A440_1 myr-GLFA 100 A440_21 myr-AGLF 100 A440_2 myr-GLFC 95 A440_22 myr-CGLF 97 A440_3 myr-GLFD 87 A440_23 myr-DGLF 101 A440_4 myr-GLFE 100 A440_24 myr-EGLF 100 A440_5 myr-GLFF 102 A440_25 myr-FGLF 100 A440_6 myr-GLFG 85 A440_26 myr-GGLF 101 A440_7 myr-GLFH 101 A440_27 myr-HGLF 98 A440_8 myr-GLFI 100 A440_28 myr-IGLF 87 A440_9 myr-GLFK 95 A440_29 myr-KGLF 100 A440_10 myr-GLFL 97 A440_30 myr-LGLF 102 A440_11 myr-GLFM 100 A440_31 myr-MGLF 96 A440_12 myr-GLFN 98 A440_32 myr-NGLF 100 A440_13 myr-GLFP 23 A440_33 myr-PGLF 101 A440_14 myr-GLFQ 97 A440_34 myr-QGLF 102 A440_15 myr-GLFR 103 A440_35 myr-RGLF 99 A440_16 myr-GLFS 75 A440_36 myr-SGLF 100 A440_17 myr-GLFT 101 A440_37 myr-TGLF 97 A440_18 myr-GLFV 98 A440_38 myr-VGLF 98 A440_19 myr-GLFW 102 A440_39 myr-WGLF 101 A440_20 myr-GLFY 103 A440_40 myr-YGLF 20

(3) Compound Tetrapeptide MMP -1 production inhibitory effect

Human skin fibroblasts (Hs68) were dispensed in 6 well plates at a concentration of 1 * 10 ⁶ cells / well and incubated at 37 ° C. and 5% CO ₂ for 24 hours until they grew 70-80%. . 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 dead cells by centrifugation was placed in an anti-MMP-1 antibody coated 96well plate (Merck, USA; Cat #: MK101) and darkened. Incubated at 37 ° C. for 2 hours. After removing the culture medium, wash it with phosphate buffer saline (PBS) supplemented with 0.05% Tween 20 and incubate for 1 hour with 100 μl HRP-conjugated secondary antibody solution. After washing four times with phosphate buffered saline supplemented with 0.05% Tween 20, 100 μl of TMB solution is added and reacted at room temperature for 15 minutes. 100 μl of 1 M sulfuric acid is added to terminate the reaction. Absorbance was measured at 450/595 nm using a microplate reader (Tecan). MMP-1 production inhibition rate (%) was converted as shown in Equation 2 below.

[ Equation 2 ]

MMP-1 production inhibition rate (%) = [1 - (M _s ) / M _c ] x 100

M _s : Absorbance of the group treated with the sample

M _c : Absorbance of the control group

FIG. 2 is a table showing the results of testing the inhibitory ability of MMP-1 synthesis of compound A440_19 compared to retinoic acid, TGF-β1, a positive control group, in a natural aging state without external stimulation by the above method as an embodiment of the present invention. to be. As a result of the experiment as described above, when the concentration of Compound A440_19 was treated with 5, 10, 20, 30ppm, it was shown that the MMP-1 inhibitory effect increased as the concentration was increased, and the effect was constant at the concentration of 10ppm or more. At 10 ppm, which showed the greatest inhibitory effect on MMP-1 synthesis, the inhibitory effect of MMP-1 was 82%, which was comparable to that of 76% retinoic acid and 85% TGF-β1.

(4) Compound Tetrapeptide UVA  or UVB By investigation MMP -1 production inhibitory effect

Human skin fibroblasts (Hs68) were dispensed in 6 well plates at a concentration of 1 * 10 ⁶ cells / well and incubated at 37 ° C. and 5% CO ₂ for 24 hours until they grew 70-80%. . Put 1 ml of PBS into each well plate, and then 30 J / cm ² or 50 mJ / cm ² by UVA (sankyo lamp, Korea) and UVB irradiation device (vilber loumet, France). Ultraviolet rays were irradiated. Remove the phosphate buffer saline (PBS) and replace the serum-free medium with diluted sample and incubate for 48 hours. 100 μl of the supernatant obtained by separating the culture medium from the dead cells by centrifugation was placed in an anti-MMP-1 antibody coated 96well plate (Merck, USA; Cat #: MK101) and darkened. Incubated at 37 ° C. for 2 hours. Remove the culture medium, wash 4 times with Phosphate buffer saline with 0.05% Tween20, and incubate for 1 hour with 100 μl HRP-conjugate secondary antibody. After washing 4 times with Phosphate buffer saline to which 0.05% Tween20 was added, 100 μl TMB solution was added and reacted at room temperature for 15 minutes. Then, 100 μl 1M sulfuric acid was added to terminate the reaction. Absorbance was measured at 450/595 nm using a microplate reader (Tecan), and MMP-1 production inhibition rate (%) was converted as shown in Equation 3 below.

[ Equation 3 ]

% Inhibition of MMP-1 production = [(M _u -M _s ) / (M _u -M _c )] × 100

M _c : absorbance of the control group

M _u : absorbance of group irradiated with UVA or UVB

M _S : absorbance of the group treated with UVA or UVB

Table 3 below shows the results of testing the inhibition of MMP-1 production of tetrapeptide against MMP-1 production by UVA irradiation stimulation by the above method as an embodiment of the present invention. The treatment concentration of each tetrapeptide is 10 ppm. As shown in Table 3, including the compound A440_19 compound A440_4, A440_5 compound, compound A440_6, A440_13 compound, compound A440_16 A440_17 compound, the compound was confirmed to be that inhibit MMP-1 produced by the increased UV stimulation, etc. A440_20.

Comparison of the inhibition rate of MMP-1 production of each compound Compound name order MMP -1 inhibition rate (%) Compound name order MMP -1 inhibition rate (%) A440_1 myr-GLFA (7) A440_21 myr-AGLF 2 A440_2 myr-GLFC 82 A440_22 myr-CGLF 20 A440_3 myr-GLFD 6 A440_23 myr-DGLF 5 A440_4 myr-GLFE 109 A440_24 myr-EGLF 15 A440_5 myr-GLFF 129 A440_25 myr-FGLF (21) A440_6 myr-GLFG 119 A440_26 myr-GGLF 100 A440_7 myr-GLFH (28) A440_27 myr-HGLF 13 A440_8 myr-GLFI 73 A440_28 myr-IGLF 25 A440_9 myr-GLFK (44) A440_29 myr-KGLF 40 A440_10 myr-GLFL 55 A440_30 myr-LGLF 35 A440_11 myr-GLFM 18 A440_31 myr-MGLF 12 A440_12 myr-GLFN (One) A440_32 myr-NGLF One A440_13 myr-GLFP 141 A440_33 myr-PGLF 5 A440_14 myr-GLFQ 27 A440_34 myr-QGLF 0 A440_15 myr-GLFR 21 A440_35 myr-RGLF 25 A440_16 myr-GLFS 102 A440_36 myr-SGLF 33 A440_17 myr-GLFT 132 A440_37 myr-TGLF 15 A440_18 myr-GLFV 98 A440_38 myr-VGLF 19 A440_19 myr-GLFW 163 A440_39 myr-WGLF 21 A440_20 myr-GLFY 145 A440_40 myr-YGLF (120)

3 is an embodiment of the present invention as shown in Table 2 is not cytotoxic and as shown in Table 3 as shown in Table 3 MMP-1 synthesis inhibitory effect of the compound A440_19 that most reduced the increased MMP-1 production most varied Test results at concentration.

4 is a chart showing the results of testing the inhibitory effect of MMP-1 synthesis on UVB irradiation of compound A440_19 . About experiment results surge MMP-1 by MMP-1 production is 128% at 10ppm treatment group of the compound A440_19, UVB surge by the UVA, as Figure 3 and shown in Figure 4 as described above in the tetra-peptide 10ppm treated group 137 It was confirmed that the% decrease.

FIG. 5 is a graph showing the results of comparing the inhibitory effect of MMP-1 synthesis on UVB irradiation of compound A440_19 with Retinyl Palmitate. A retinyl palmitate, 10, 20, 25ppm was alone processing or treatment of the compound A440_ 19 at the same time determine the MMP-1 inhibition produced. As a result, when the tetrapeptide (A440_19) alone or retinyl palmitate at the same time was treated with retinyl palmitate alone it was confirmed that much higher MMP-1 reduction rate.

FIG. 6 is a graph showing the results of a test of the effect of inhibiting MMP-1 synthesis on UVB irradiation of compound A440_19 with Retinol. Retinol was shown to be toxic at 5 ppm or more, so the experiment was conducted at a single concentration of 5 ppm. As compared with the case above, a single treatment with the experimental results of FIG. 5 in the same manner as compound 19 or A440_ cases were treated with retinol at the same time by treatment with retinol alone, it was confirmed that a much higher MMP-1 reduction ratio. 5 and 6, it was confirmed that the compound A440_19 not only inhibited the MMP-1 inhibitory activity even when treated with the retinol derivatives, but also had a superior anti-aging effect when compared with the retinol derivatives.

(5) compounds Of tetrapeptide (A440_19) UVA  Procollagen Synthesis Enhancement Effect Reduced by Irradiation

Human skin fibroblasts (Hs68) were dispensed in 6 well plates at a concentration of 1 * 10 ⁶ cells / well and incubated at 37 ° C. and 5% CO ₂ for 24 hours until they grew 70-80%. . 1 ml of PBS was added to each well plate and 30 J / cm ² by UVA irradiator (Sankyo, Japan). Ultraviolet rays were irradiated. Remove the phosphate buffer saline (PBS) and replace the serum-free medium with diluted sample and incubate for 48 hours. After removal of medium, cell lysis buffer (50 mM HEPES, pH 7.5, 150 mM NaCl, 10% glycerol, 5 mM EGTA, 1 mM sodium orthovanadate, 10 mM NaF, 12 mM bglycerophosphate, 1 mM DTT, 1 mM Cells were lysed with PMSF, 5 mg / ml aprotinin, and 1% NP40). The lysed cells were centrifuged at 26,000 g for 15 minutes, and then the cell extracts were separated. Bradford protein assay measures the amount of protein, adjusts the same amount, and heats at 95 ° C for 5 minutes to denature the protein. Walk Yeongdong. Electrophoresis gels were transferred to nitrocellulose membranes, and then incubated for 1 hour in 5% skim milk dissolved in TBST buffer, followed by procollagen 1 (Santa Cruze Biotechnology, CA). The antibodies are incubated with shaking for 3 hours in a solution diluted 1: 100 in TBST buffer. The antibody solution was removed, washed three times with Phosphate buffer saline added with 0.05% Tween20 for 5 minutes, and the HRP-conjugate secondary antibody was incubated in a solution diluted 1: 2000 in TBST buffer for 1 hour. After washing the membrane, spray the ECL solution with imaging densitometer [Labworks ver.4.6. (image acquisition and analysis software), UVP, CA].

An anti-tubulin antibody (Sigma) was used as a control.

FIG. 7 is a diagram illustrating reduced procollagen 1 biosynthesis enhancing effect of UVA irradiation of compound A440_19 by the above method as an embodiment of the present invention. In the negative control group, the group treated with Compound A440_19 , the test substance after UVA irradiation, increased the procollagen type 1 to 243% when the UVA irradiation group was based on 100% procollagen production. Therefore, it was confirmed that compound A440_19 promoted the amount of procollagen biosynthesis reduced by UVA irradiation.

(6) compounds Of tetrapeptide (A440_19)  Extracellular protein mRNA Manifestation of

Human skin fibroblasts (Hs68) were dispensed in 6 well plates at a concentration of 1 * 10 ⁶ cells / well and incubated at 37 ° C. and 5% CO ₂ for 24 hours until they grew 70-80%. . 1 ml of PBS was added to each well plate, and 80mJ / cm ² ultraviolet rays were irradiated with a UVB irradiation device (vilber loumet, France). Remove the phosphate buffer saline (PBS), replace the serum-free medium with diluted sample, and incubate for 24 hours. 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 mRNA expression levels of MMP-1, Procollagen 1 and Fibronectin. The primer sequences of GAPDH (Glyceraldehyde-3-phosphate dehydrogenase) and MMP-1 used are shown in Table 4 below. 1 μL (0.5 μg / μL) of each primer was added and the synthesized cDNA was added to the PCR-premix tube (Bionia, Korea) with DEPC-Water. Denaturation was performed at 95 ℃ for 30 seconds and annealing at 60 ℃. At 30 seconds, the extension proceeded to 30 cycles for 2 minutes at 72 ℃. The PCR product was confirmed to be non-specific PCR product or primer dimer in 1% agarose gel and the amount of product was confirmed by EC3 UV illuminator (UVP, USA).

PCR primer sequence of skin extracellular protein gene Gene name Forward primer (5 '→ 3') Reverse primer (5 '→ 3') MMP-1 ATT CTA CTG ATA TCG GGG CTT TGA ATG TCC TTG GGG TAT CCG TGT AG Procollagen Type 1 CTC GAG GTG GAC ACC ACC CT CAG CTG GAT GGC CAC ATC GG Fibronectin ACC ACG TAG GAG AAC AGT ACA GTA TTG CGG GCC AG GAPDH CAA AAG GGT CAT CAT CTC TG CCT GCT TCA CCA CCT TCT TG

Figure 8 is an embodiment of the present invention compared to the positive control group retinoic acid and TGF-β by the above method Compound A440_19 is an extracellular protein changed by UVA irradiation, MMP-1, Procollagen Type 1 (Procollagen Type I ), The effect of fibronectin mRNA expression was confirmed through PCR experiments. As a result of the experiment, when the concentration of Compound A440_19 was treated at 2, 5, 10, and 20 ppm, MMP-1 mRNA expression inhibitory effect was increased and concentration of procollagen type 1 and fibronectin mRNA increased by UVA was increased. It was confirmed. 9, 10 and 11 are data obtained by quantifying the bands shown in FIG. 8 using a densitometer. FIG. 9 shows MMP-1, FIG. 10 shows Procollagen type I, and FIG. 11 shows the expression level of Fibronectin.

Example 3 Skin Anti-inflammatory Effect

The anti-inflammatory effects of compound tetrapeptides were tested using human skin fibroblasts (Hs68) cell lines and epidermal keratinocytes (HaCaT) cells. The relationship between chronic inflammation of the skin and the production of several cancers is well known and has been shown in many tumor experiments. Keratinocytes induced to produce cytokines by various external stimuli lose cytokine control and eventually cause various skin diseases including cancer.

This experiment skin epidermal keratinocytes (human keratinocytes, HaCaT) in cell lines and using the UVB lamp Expression of Interleukin-1α (IL-1α), Interleukin-1β (IL-1β), Interleukin 6 (IL-6), and Interleukin 8 (IL-8) mRNA, which are representative cytokines expressed by UV irradiation, by establishing an in vitro assay system By evaluating the amount, the inhibitory effect of tetrapeptide was confirmed.

(1) Cell line and cell culture

The cell lines used in this study were human skin fibroblasts (Hs68) and HaCaT cells, a type of human Keratinocytes. Hs68 and HaCaT cells were DMEM (Dulbecco's modified Eagle) supplemented with 10% FBS (Gibco Co), 100 μg / ml streptomycin, and 100 U / ml penicillin at 37 ° C and 5% CO ₂ . medium) (Incubator; Thermo-scientific, USA). HaCaT cells are 100 cm ² After growing sufficiently in a flask (Corning, USA), the surface of the cultured cells was washed with PBS at intervals of 3 days, followed by 0.25% trypsin-EDTA solution, treated in the incubator for 3 minutes, and discarded the trypsin-EDTA solution at 5 ° C. Cells were detached by storage for a minute. The detached cells were subcultured at 37 ° C. and 5% CO ₂ at a split ratio of 1: 5 by transferring 10 ml of DMEM containing 10% FBS to a new culture vessel.

(2) UVB  Of genes related to skin inflammation caused by stimulation mRNA  Inhibitory effect

Human skin fibroblasts (Hs68) and Human Keratinocytes (HaCaT) cells were dispensed in 60 mm plates at 6 * 10 ⁵ cells / well concentrations, respectively, at 37 ° C and 5% CO ₂ until they grew 70-80%. After incubation for 24 hours, the medium of each well was removed and replaced with fresh serum-free DMEM. 3 ml of PBS was added to each well plate and 100mJ / cm ² by UVB irradiator (vilber loumet, France) UVB was irradiated. After removing the phosphate buffer saline (PBS), replace the serum-free medium diluted A440_19 with the culture medium and incubate for 24 hours. After removing the medium, RNA was isolated using an RNA extraction kit (Invitrogen, Korea), and cDNA was synthesized. RT-PCR was performed with cDNA synthesized to measure the expression levels of Interleukin-1α (IL-1α), Interleukin-1β (IL-1β), Interleukin 6 (IL-6), and Interleukin 8 (IL-8) mRNA. . The primer sequences of GAPDH (Glyceraldehyde-3-phosphate dehydrogenase, Interleukin-1α (IL-1α), Interleukin-1β (IL-1β), Interleukin 6 (IL-6), and Interleukin 8 (IL-8) were used. 1 μL (0.5 μg / μL) of each primer was added and the synthesized cDNA was added to the PCR-premix tube (Bionia, Korea) together with DEPC-Water, followed by denaturation at 95 ℃ for 30 seconds. 30 seconds at 56 ° C. and 2 cycles at 72 ° C. for 30 minutes at 50 ° C. PCR products were loaded onto a 1% agarose gel to confirm that there were no non-specific PCR products or primer dimers. UV illuminator (UVP, USA) was used to determine the amount of mRNA quantified by densitometer divided by GAPDH value.

PCR primer sequence of skin inflammation related gene Gene name Forward primer  (5 '→ 3') Reverse primer  (5 '→ 3') IL-1? CTC TCT GAA TCA GAA ATC CTT CTA TC CTA GTC AAA TTT CAC TGC TTC ATC C IL-1β TGG AGA ACA CCA CTT GTT GCT CCA AAA CAG ATG AAG TGC TCC TTC CAG G IL-6 GCC TTC GGT CCA GTT GCC TT GCA GAA TGA GAT GAG TTG TC IL-8 ATG ACT TCC AAG CTG GCC GTG GCT TCT CAG CCC TCT TCA AAA ACT TCT C GAPDH CAA AAG GGT CAT CAT CTC TG CCT GCT TCA CCA CCT TCT TG

The results of measuring the inhibitory effect of the genes mentioned in [Table 5] are shown in FIGS. 12 and 13. 12 is a diagram illustrating the mRNA expression inhibitory activity of cytokine (Cytokine) in human dermal cells (Hs68) and Figure 13 shows the mRNA expression inhibition of cytokine (Cytokine) by UV stimulation in keratinocytes (HaCat).

As a result, the compound A440_19 effectively inhibited the mRNAs of the cytokines IL-1α, IL-1β, IL-6 and IL-8 increased by UVB irradiation, which shows that the compound compound A440_19 shows an excellent anti-inflammatory effect. You can check it.

Example 5 Wrinkle Improvement Effect in Clinical

The lotion was prepared with the composition shown in Table 6 below, and the effect of improving wrinkles on the human body was evaluated through actual test.

Lotion Formulation for evaluation of wrinkle improvement effect Paid Awards ingredient Content (% by weight) ingredient Content (% by weight) Mineral Oil 6.0 Glycerin 4.0 Stearic acid 2.7 Methyl paraben 0.5 Cetyl alcohol 2.0 TEA 0.5 Propyl paraben 0.05 Distilled water 82.647 Tween 60 1.2 Aracel 83 0.4 Compound a440_19 0.003

(1) compound Of tetrapeptide (A440_19)  Wrinkle improvement effect evaluation

Twenty-three healthy Korean women aged 30-65 years were allowed to absorb a lotion of compound tetrapeptides every morning and evening twice a day for three months in a row.

The area to measure wrinkles was the area around the subject's eye area, and the left and right sides were measured as the measurement area. In order to measure the wrinkles, the subject washes the measuring area with water, and then stabilizes the skin surface temperature and humidity according to the environment of the measurement space by restoring for 30 minutes in an air-conditioned room with a room temperature of 20-25 ° C and a humidity of 40-60%. The water intake during the rest was limited. For the purpose of objective measurement, one researcher measured the same site for each measurement of visit 1 before visit, and visit 2, visit 3, and visit 4, 4, 8, and 12 weeks after product use.

Wrinkle improvement effect of each subject's wrinkle improvement before, during and after 4, 8 and 12 weeks using the Visiometer SV600 (Courage-Khazaka electronic GmbH, Germany) Transparancy profilometry analysis was performed to analyze the wrinkle parameters R1, R2, R3, R4, and R5 (R1: Skin roughness, R2: Maximum roughness, R3: Average roughness, R4: Smoothness depth, R5: Arithmetic average roughness) .

This test was conducted through the P & K Skin Clinical Research Center, a Korean clinical company, and the results are shown in FIG.

As shown in FIG. 14, it was confirmed that the skin wrinkle parameters R1, R2, R3, R4, and R5 were all significantly reduced in the example containing the compound A440_19, and the skin wrinkle improvement effect was very excellent.

Although the above-described anti-wrinkle effect and safety of the present invention have been described in detail with respect to the above-described embodiments and embodiments, it will be apparent to those skilled in the art that various modifications and modifications can be made within the technical spirit of the present invention. Naturally, the modifications belong to the appended claims.

Claims (10)

Tetrapeptide represented by the following formula (1) or (2).
[Chemical Formula 1]
RR ¹ -Gly-Leu-Phe
(2)
R-Gly-Leu-Phe-R ²
(In Formula 1 and Formula 2, R is tetradecanoyl, Leu may be D-Leu or L-Leu, Phe may be D-Phe or L-Phe. In addition, R ¹ in Formula 1 and Formula 2 is Selected from amino acids consisting of glycine, leucine, lysine and serine, and R ² is cysteine, glutamic acid, phenylalanine, glycine , Isoleucine (isoleucine), valine (valine), proline (proline), leucine (leucine), serine (serine), trionine (thrionine), tryptopane (tryptopane), tyrosine (tyrosine) The C-terminus may be in carboxylic acid or amide form.) delete delete delete The method of claim 1,
Wherein R ¹ is glycine and R ² is selected from the group consisting of phenylalanine, proline, proline, threonine, tryptopane, and tyrosine. . The method of claim 1,
The tetrapeptide of the above-described tetrapeptide amino acids Leu is L-Leu, Phe is L-Phe, and the C-terminus is a carboxylic acid form. An anti-aging, anti-wrinkle or anti-inflammatory cosmetic composition comprising the tetrapeptide according to any one of claims 1, 5 and 6 as an active ingredient. The method of claim 7, wherein
The composition is a cosmetic composition having a skin, lotion, cream, foundation, essence, gel, pack, for cleansing, body oil, lipstick, mascara, makeup base or soap form. delete The method of claim 7, wherein
Cosmetic composition comprising the tetrapeptide in an amount of 0.0001 to 10% by weight based on the total weight of the composition.

Images