RU2773534C1 - Peptide for the prevention of skin damage caused by atmospheric pollution and for anti-aging therapy, as well as its use - Google Patents

Abstract

FIELD: cosmetic industry.

SUBSTANCE: invention relates to a new peptide SYSGVLFFLK (SEQ ID NO: 1), characterized by the ability to prevent skin damage caused by pollutants, anti-aging activity for the skin and the ability to reduce the formation of wrinkles; as well as to the use of the peptide that is associated with the prevention or treatment of skin diseases caused by pollutants, or to improve skin anti-aging therapy. In particular, the new peptide of the present invention inhibits the entry of pollutants into skin cells, such as dioxin, suspended particles and cigarette smoke, and regulates the expression of skin aging-related genes, and thus can be effectively used as an active ingredient in a pharmaceutical composition or as an active ingredient in a cosmetic composition.

EFFECT: expansion of the range of skin anti-aging solutions.

12 cl, 8 dwg, 4 tbl, 8 ex

Description

The technical field to which the invention belongs

The present invention relates to a novel peptide capable of preventing skin damage caused by pollution, as well as having a rejuvenating effect on the skin, and to the use of this peptide.

Prior Art

In today's society, air pollution caused by the formation of large cities and industrialization is a serious social problem, the results of various studies have shown that air pollution can negatively affect the general health of a person, including the respiratory system. In particular, in Korea, the concentration of particulate matter in the air has increased significantly compared to previous years, the media constantly pays attention to it, and thus the public's interest and concern about the severity of the air pollution problem has increased.

Although among all the internal organs of the human body, the respiratory system is the most exposed to atmospheric pollution, but the skin, which has a large surface area in direct contact with pollution and is continuously exposed to air, is one of the main targets of pollutants. Usually the problem of deterioration of the health of the skin caused by the action of the external environment was associated with the action of ultraviolet radiation in the composition of sunlight, but the results of studies of increased environmental pollution and the increasing amount of pollution in the atmosphere can no longer be neglected. There is a need for research and development of materials or cosmetic products containing them that can protect the skin from atmospheric pollution and restore it.

The aromatic hydrocarbon receptor (AhR) is a key receptor that induces skin damage by transmitting damaging signals into the cell when air pollution comes into contact with the skin. When exposed to human skin, dioxins such as 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and polyaromatic hydrocarbons (PAH) bind to AhR and move to the cell nucleus, as in the mechanism of action of steroid hormones, which causes toxic effects. In the nucleus, the AhR dioxin complex binds to the AhR nuclear translocator protein (Arnt), and the AhR/Arnt complex binds to a nucleotide sequence called the drug/xenobiotic responsive element (DRE or XRE) in the transcriptional regulatory region of the target a gene that includes cytochromes P450s (P450 or cyps; CYP1A1, CYP1A2 and CYP1B1), which induces the transcription of several groups of genes and leads to toxicity.

On the other hand, skin cells may undergo aging due to various reasons, and due to the aging of skin cells, wrinkles may occur. Aging can be divided into age-related aging (endogenous aging), which is a natural aging that occurs over time due to biological processes, and photoaging (photogenic aging), when degenerative changes that occur in a place exposed to sunlight and age-related aging have a combined effect. . If the expression or activity of various factors associated with cell growth is inhibited, natural aging can occur, photoaging of skin cells can occur under the action of light with a wavelength of 280 to 400 nm in the composition of sunlight. In particular, damage to the skin or fibers can occur due to exposure to ultraviolet rays, such as UVB with a wavelength in the range of 280 to 320 nm, whereby the skin may become tanned and, when tanned, its color may become darker. UVB exposure facilitates the accumulation of reactive oxygen species (ROS) and free radicals in skin cells, and the radicals stimulate intracellular signaling systems that can cause oxidative stress on biomolecules such as DNA, proteins, and lipids, which can lead to damage to skin tissue.

Increased oxidative stress of skin cells can lead to stimulation of epidermal keratinocytes or dermal fibroblasts, can increase the expression of genes such as the matrix metalloproteinase (MMP) gene, which degrades collagen through a series of intracellular signaling processes, can cause a decrease in collagen, which makes up 90% of the dermis, and as the main component of the skin protecting it from external influences or forces, ensuring its durability and strength; thus, skin aging and wrinkling may result. Thus, by regulating the expression of genes involved in the synthesis or degradation of fibrillar proteins such as collagen, one can expect to prevent skin cell aging and reduce the number of wrinkles.

The effect of cell aging due to exposure to ultraviolet radiation in sunlight or atmospheric pollution as described above causes not only cosmetic problems due to skin damage, but also serious health problems such as cancer due to damage to the DNA of cells, the central and peripheral nervous system, destruction of the immune system, disorders of the reproductive organs, disorders in the development of infants and children of preschool age, and chloracne, and thus finding a solution to these problems is an urgent task.

To solve the problems described, various materials have been developed, and attempts have been made to overcome these problems using extracts obtained from various plants or microorganisms. Often, however, the composition of the extracts is not known, since it is not possible to specifically identify which substance in the extract of a natural product can have an effect on the protection of skin cells and their recovery after aging, and thus all unknown substances in the composition of the extract are used in their entirety. Using the characteristics of the material to be used in direct contact with the skin or applied to human skin, the side effects of the material should be minimized and the safety of the human body should be guaranteed. Thus, it is necessary to accurately identify individual substances capable of solving the above problem, and then apply them to human skin.

Description of embodiments

Technical problem

The present invention provides a peptide that can be used to protect skin cells by preventing skin damage caused by pollution sources by inhibiting the entry of contaminants such as dioxins, particulate matter and cigarette smoke into the cells.

What is provided is a peptide that can be used to reduce the phenomenon of skin cell aging occurring naturally or caused by ultraviolet radiation in the composition of sunlight and thereby reducing the number of wrinkles in the skin.

What is provided is a pharmaceutical composition for preventing or treating diseases caused by pollutants, or for improving skin rejuvenation therapy by including a peptide as an active ingredient.

A cosmetic composition is provided for preventing skin damage caused by pollutants or for improving skin rejuvenation therapy by including a peptide as an active ingredient.

Solving a technical problem

In order to achieve the above object, according to an aspect of the present invention, there is provided a peptide for preventing skin damage caused by pollutants, the peptide comprising the amino acid sequence of SEQ ID No: 1.

According to another aspect of the present invention, there is provided a peptide for skin rejuvenation therapy, the peptide comprising the amino acid sequence of SEQ ID No: 1.

According to another aspect of the present invention, there is provided a pharmaceutical composition for the prevention or treatment of diseases caused by pollutants, the pharmaceutical composition comprising the amino acid sequence of SEQ ID No: 1.

According to another aspect of the present invention, there is provided a pharmaceutical composition for improving skin rejuvenation therapy, the pharmaceutical composition comprising the amino acid sequence of SEQ ID No: 1.

According to another aspect of the present invention, there is provided a cosmetic composition for preventing skin damage caused by pollutants and for improving skin rejuvenation therapy, the cosmetic composition comprising a peptide comprising the amino acid sequence of SEQ ID No: 1 as an active ingredient.

Benefits of Disclosing an Invention

The peptide of the present invention is able to prevent airborne pollutants such as dioxins, particulate matter and cigarette smoke, and inhibit skin cell aging phenomena; thus it can be used as an active ingredient of a pharmaceutical composition for the prevention or treatment of cancer which may be caused by pollutants, skin diseases, lung diseases, or to reduce skin aging, and can be used as an active ingredient of a cosmetic composition for protecting skin from pollutants. substances and to reduce skin aging and wrinkle formation.

However, the effects of the present invention are not limited to those indicated, and other effects will be easily understood by those skilled in the art in light of the following description.

Brief description of the drawings

Fig. 1A-1C show Western blot results that confirm the number of AhR translocations to the nucleus by TCDD, PM and CSE, respectively, on HaCaT cells treated with a peptide comprising the amino acid sequence of SEQ ID NO: 1; Further "Contra." means control, and "Neg. Control." means negative control;

fig. 1D shows Western blot results that confirm the number of AhR translocations to the nucleus by CSE on A549 cells treated with a peptide comprising the amino acid sequence of SEQ ID NO: 1;

fig. 2A-2C show RT-PCR results that confirm expression levels of CYP1A1 and COX-2 genes by TCDD, PM, and CSE, respectively, on HaCaT cells treated with a peptide comprising the amino acid sequence of SEQ ID NO: 1;

fig. 3A and 3B show Western blot results that confirm the amount of MMP-1 and COX-2 protein by TCDD and PM, respectively, on HaCaT cells treated with a peptide comprising the amino acid sequence of SEQ ID NO: 1;

fig. 4A and 4B are images that show whether a peptide comprising the amino acid sequence of SEQ ID NO: 1 precipitates upon absorption of PM and CSE, respectively;

fig. 5A shows the result of a Western blot that confirms the amount of p-Akt protein and p-ERK of HaCaT cells treated with a peptide comprising the amino acid sequence of SEQ ID NO: 1;

fig. 5B shows the result of RT-PCR which confirms the expression levels of the SIRT1 gene of HaCaT cells treated with a peptide comprising the amino acid sequence of SEQ ID NO: 1;

fig. 6A shows the result of a Western blot that confirms the amount of p-Akt protein and p-ERK of NIH3T3 cells treated with a peptide comprising the amino acid sequence of SEQ ID NO: 1;

fig. 6B shows RT-PCR results that confirm gene expression levels for collagen (Col1a1), fibronectin, and elastin in NIH3T3 cells treated with a peptide comprising the amino acid sequence of SEQ ID NO: 1;

fig. 7 shows RT-PCR results that confirm the expression levels of the SIRT1 and AQP3 genes of HaCaT cells exposed to UVB and treated with a peptide comprising the amino acid sequence of SEQ ID NO: 1;

fig. 8A shows RT-PCR results that confirm gene expression levels for collagen (Col1a1), fibronectin, and elastin in NIH3T3 cells exposed to UVB and treated with a peptide comprising the amino acid sequence of SEQ ID NO: 1; and

fig. 8B shows Western blot results that confirm the amount of MMP-1 protein of NIH3T3 cells exposed to UVB and treated with a peptide comprising the amino acid sequence of SEQ ID NO: 1, and gelatin zymography results that confirm the amount of MMP-2 protein of NIH3T3 cells. , which were affected by UVB and treated with a peptide comprising the amino acid sequence of SEQ ID NO: 1.

Detailed description

The following is a detailed description of the present invention.

1. Peptide for the prevention of skin damage caused by pollutants or anti-aging therapy.

According to an aspect of the present invention, there is provided a novel peptide capable of preventing skin damage caused by pollutants.

In accordance with another aspect of the present invention, there is provided a novel peptide capable of inhibiting skin aging.

Peptide means a polymer consisting of two or more amino acids linked by a peptide bond. The size of the peptide is too large to effectively penetrate into target tissues or cells, or it has a short half-life and thus disappears in the body in a short time. The peptide of the present invention consists of 20 or less, for example, 15 or less or 10 or less amino acids, which can inhibit the entry of contaminants into the cell.

The peptide of the present invention may include the amino acid sequence of SEQ ID NO: 1 and may include amino acid variants or fragments with different sequences formed by deletion, insertion, substitution or combination of amino acid residues in a range that does not affect the ability to inhibit skin damage caused by pollutants, as well as the anti-aging properties of the peptide. Amino acid substitutions in a peptide are known in the art that do not fully modify the ability to inhibit skin damage caused by pollutants and the anti-aging activity of the peptide. Sometimes the peptide can be modified by phosphorylation, sulfation, acrylation, glycosylation, methylation and farnesylation. Accordingly, the present invention includes a peptide comprising an amino acid sequence that is substantially identical to the sequence of a peptide comprising the amino acid sequence of SEQ ID NO: 1, as well as a variant or active fragment thereof. Substantially identical protein means one in which the amino acid sequence has 75% or more, for example, 80% or more, 90% or more, 95% or more sequence homology with the amino acid sequence of SEQ ID NO: 1. In addition, the peptide may further include a target sequence, label, labeled residue, or amino acid sequence prepared for the specific purpose of increasing the stability or half-life of the peptide.

Additionally, the peptide of the present invention can be obtained by various methods well known in the prior art. For example, a peptide can be prepared by polynucleotide recombination and protein expression systems, in vitro synthesis by chemical synthesis such as peptide synthesis, and cell-free protein synthesis.

Additionally, to achieve better chemical stability, improved pharmacological properties (half-life, absorption capacity, potency, potency, etc.), specificity modifications (e.g., broad spectrum of biological activity), and reduced antigenicity with the N- or C-terminus of the peptide, one can link a protecting group. For example, the protecting group may be an acetyl group, a fluorenylmethoxycarbonyl group, a formyl group, a palmitoyl group, a myristyl group, a stearyl group, or a polyethylene glycone (PEG) group, but may, without limitation, include any element capable of enhancing peptide modification, such as peptide stability. The term "stability", as used in the present invention, means storage stability (eg, storage stability at room temperature), as well as in-vivo stability, meaning protection of the peptide of the present invention from attack by protein-cleaving enzymes in vivo.

The term "contaminants" as used in the present invention may mean dioxin (TCDD), particulate matter (PM), or cigarette smoke extract (CSE), but embodiments are not limited to these, and contaminants may include any xenobiotics that enter the cell from outside and have a toxic effect on growth and normal cell metabolism. Prevention of skin damage by pollutants can be achieved by preventing the penetration of pollutants into the cells, which makes it impossible for the toxic effect of the pollutants on the cells.

Additionally, the peptide of the present invention can inhibit the entry of contaminants into the cell by adsorption to them. When pollutants such as dioxin enter the cell, they can bind to AhR, inducing the expression of several groups of genes, and, accordingly, the expression of CYP1A1, CYP1A2, CYP1B1, COX-2, and MMP can increase. In this regard, the cytoprotective effect of the peptide of the present invention by inhibiting the entry of contaminants into the cell can be determined by checking the change in the level of expression of these genes.

The term "skin rejuvenation therapy" used in the present invention may mean the effect of suppressing the aging of skin cells. Aging may be the natural aging of cells over time, may be photoaging caused by exposure to sunlight, and in particular may be photoaging caused by exposure to ultraviolet radiation. Additionally, aging can be induced by intracellular oxidative stress, which can be caused by various causes, and lead to inhibition of cell growth and apoptosis, as well as inhibition of the synthesis of various fibrillar proteins that make up skin cells, and an increase in the expression of destructive enzymes. In particular, when irradiated with UV light, the expression of genes such as SIRT1, AQP3, Col1a1, fibronectin and elastin is suppressed in skin cells, and the expression of MMP-1 and MMP-2 genes is increased, which can cause photoaging of skin cells and the formation of wrinkles.

The peptides of the present invention can increase the expression of skin cell proliferation cascade genes, increase the expression of fibrillar protein genes, or induce suppression of gene expression of enzymes that cause fibrillar protein proteolysis. Thus, the peptide can reduce wrinkle formation and improve skin elasticity by enhancing the synthesis of fibrillar proteins, and exhibit skin rejuvenating properties by improving the skin barrier. The ability of the peptide of the present invention to exhibit an anti-aging effect and reduce the formation of wrinkles on the skin can be determined by measuring the expression levels of genes associated with proteins involved in cell proliferation, genes that mediate the anti-aging effect, as well as genes for fibrillar proteins and their degrading enzymes, where the expression levels in skin cells change with the progression of skin aging.

In order to determine the effect of preventing pollutant-induced skin damage of the peptide of the present invention, according to one specific embodiment of the present invention, as a result confirming the level of AhR translocation to the nucleus and the expression levels of CYP1A1, COX-2 and MMP-1 after treatment of keratinocytes and fibroblasts with peptides comprising the amino acid sequence of SEQ ID NO: 1 and pollutants such as dioxin, PM and CSE, it was shown that the entry of pollutants such as dioxin, PM and CSE was suppressed and that the level of AhR translocation into the nucleus and expression levels of CYP1A1, COX-2 and MMP-1 in response to the intake of pollutants decreased depending on the treatment using the peptide of the present invention (see Fig. 1A - 3B).

In addition, in order to determine the adsorption between a peptide and a contaminant, in another specific embodiment of the present invention, by observing with the naked eye as a result of mixing PM and CSE with a peptide comprising the amino acid sequence of SEQ ID NO: 1 of the present invention, it was shown that the peptide of the present of the invention precipitated upon adsorption with contaminants such as PM and CSE, and that adsorption was dependent on the amount of peptide (see FIGS. 4A and 4B).

In addition, in order to determine the inhibitory effect of the peptide of the present invention on the natural aging of skin cells, in another specific embodiment of the present invention, by detecting sufficient to confirm the amount of phosphorylated Akt and ERK and the expression levels of SIRT1, collagen, fibronectin and elastin, it was shown that after treatment of keratinocytes and fibroblasts with the peptide, including the amino acid sequence of SEQ ID NO: 1, the amount of phosphorylated Akt and ERK, the amount of SIRT1 protein, and the expression levels of fibrillar proteins such as collagen, fibronectin, and elastin were increased by treatment with the peptide of the present invention (see Fig. 5A - 6B).

In addition, in order to determine the repairing effect of the peptide of the present invention on skin cells after photoaging due to exposure to ultraviolet (UV) radiation, in another specific embodiment of the present invention, in an experiment to test whether the reduced expression level of SIRT1, AQP3, collagen, fibronectin and elastin increase again and whether the increased expression level of MMP1 and MMP2 will decrease again after treatment of keratinocytes and fibroblasts with a peptide comprising the amino acid sequence of SEQ ID NO: 1, it was shown that the expression level of SIRT1, AQP3, collagen, fibronectin and elastin increased after treatment peptide of the present invention, which confirmed the restoration of the expression levels of SIRT1, AQP3, collagen, fibronectin and elastin, which decreased after UV irradiation (see Fig. 7 and Fig. 8A), and that the expression levels of MMP-1 and MMP-2 decreased depending on the treatment with the peptide of the present invention, which was confirmed in restoring the expression levels of MMP-1 and MMP-2, which increased after UV irradiation (see. fig. 8B).

Thus, the peptide of the present invention prevents skin damage by inhibiting the entry of pollutants such as TCDD, PM and CSE into cells, and controls the expression of genes associated with natural aging of skin cells or UV-induced photoaging, and thereby it is clear that the peptide is able to reduce the number of wrinkles on the skin, showing anti-aging activity; thus, the peptide of the present invention can be effectively used as an active ingredient of a composition for preventing skin damage caused by pollutants, for anti-aging therapy, and for reducing skin wrinkles.

2. Pharmaceutical composition for preventing or treating diseases caused by pollutants and for reducing skin aging

According to another aspect of one or more embodiments of the present invention, there is provided a pharmaceutical composition for the prevention or treatment of diseases caused by pollutants, wherein the pharmaceutical composition comprises a peptide as an active ingredient comprising the amino acid sequence of SEQ ID NO: 1.

According to another aspect of one or more embodiments of the present invention, there is provided a pharmaceutical composition for reducing skin aging, wherein the pharmaceutical composition comprises, as an active ingredient, a peptide comprising the amino acid sequence of SEQ ID NO: 1.

The peptide comprising the amino acid sequence of SEQ ID NO: 1 is the same peptide as described in section “1. Peptide for prevention of skin damage caused by pollutants or anti-aging therapy", and thus a detailed description of the peptide comprising the amino acid sequence of SEQ ID NO: 1 can be made by reference to section "1. Peptide for the prevention of skin damage caused by pollutants, or for anti-aging therapy. Hereinafter, only the unique characteristics of a pharmaceutical composition for preventing or treating diseases caused by pollutants and for reducing skin aging will be described.

Since the peptide of the present invention can effectively inhibit the entry of pollutants into skin cells and suppress the aging of skin cells, the pharmaceutical composition comprising the peptide as an active ingredient can be used to prevent or treat diseases caused by pollutants, as well as to reduce wrinkles and improve skin elasticity by reduce the aging of skin cells.

Pollutant-induced diseases means diseases caused by atmospheric pollutants such as dioxin, particulate matter, and cigarette smoke and includes skin diseases, lung diseases, nervous system diseases, reproductive system diseases, and cardiovascular diseases, which may include cancer, atopic dermatitis, contact dermatitis, seborrheic dermatitis, acne, dry skin, psoriasis, death, immunotoxicity, peripheral nervous system damage, central nervous system damage, endocrine system disorders, developmental disorders in infants and preschool children, anemia, bronchitis, emphysema, decreased lung function, asthma, chronic bronchitis, arrhythmia, acute coronary syndrome, angina or myocardial infarction.

The pharmaceutical composition of the present invention can be used to prevent the occurrence of a disease caused by pollutants by inhibiting the entry of pollutants into cells, to reduce symptoms by suppressing additional entry of pollutants into the cells of patients with diseases, and to treat diseases by inhibiting deterioration.

Skin aging refers to a negative change in the condition of the skin, which can be caused by natural aging or photoaging of skin cells. Skin aging can be physiological and (or) photoaging, which includes wrinkles, mimic wrinkles, decrease in elasticity and (or) skin tension, wrinkling, thinning and reduction in luster and gloss of the skin, can be an internal degradation of the skin, in which the dermis after exposure to ultraviolet light becomes thin, or may represent an external degradation of the skin, in particular, caused by aging due to the destruction of collagen fibers.

The pharmaceutical composition of the present invention can be used to reduce skin aging when the elasticity of the skin can be restored to a medically acceptable skin condition, such as normal, compared to natural skin aging or light-induced aging, since the pharmaceutical composition of the present invention suppresses the phenomenon of aging, which can take place in skin cells.

A pharmaceutical composition containing a peptide of the present invention as an active ingredient may be mixed using a pharmaceutically acceptable carrier and/or additive well known in the art to form a unit dosage form or to be included in a multi-dose container. Here, the preparation may be a solution in an oily or aqueous medium, a suspension, an emulsifying solution, an extract, a powder, a granule, a tablet, a capsule, or a gel (eg a hydrogel), and may further include a dispersing or stabilizing agent.

In addition, the peptide of the pharmaceutical composition may be in pharmaceutically acceptable carriers such as colloidal suspensions, powders, saline, lipids, liposomes, microspheres or spherical nanoparticles. These peptides can form or be complexed with a carrier and can be carried in vivo using carrier systems known in the art such as lipids, liposomes, microparticles, gold, nanoparticles, polymers, condensing agents, polysaccharides, polyamino acids, dendrimers, saponin, adsorption enhancers or fatty acids.

Additionally, a pharmaceutically acceptable carrier may include lactose, dextrose, sucrose, sorbitol, mannitol, starch, gum arabic, calcium phosphate, alginate, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, methylcellulose, methylhydroxybenzoate, propylhydroxybenzoate, talc, stearate magnesium and mineral oil, which is usually used in dosage forms, but embodiments are not limited to these compounds. Additionally, the pharmaceutical composition may include a lubricant, a wetting agent, a sweetener, a flavor, an emulsifier, a suspending agent, and a preservative, in addition to the above ingredients.

The pharmaceutical composition of the present invention may be administered orally or parenterally during clinical use and may be incorporated into a standard pharmaceutical formulation. This means that in actual clinical administration, the pharmaceutical composition of the present invention can be administered in various oral and parenteral dosage forms, but when mixed, diluents or excipients such as fillers, diluents, binders, wetting agents, disintegrants, or surfactants can be used. substances that are commonly used in medicines. Solid dosage forms for oral administration may include tablets, pills, powders, granules and capsules, and these solid dosage forms are prepared by mixing a plant extract or plant fermented product with at least one excipient, for example, starch, calcium carbonate, sucrose, lactose or gelatin. In addition, lubricants such as magnesium stearate or talc can be used in addition to simple excipients. Liquid dosage forms for oral administration may include suspensions, solutions, emulsions and syrups, the aforementioned dosage forms may contain various adjuvants such as wetting agents, sweeteners, flavors and preservatives in addition to commonly used simple diluents such as water and liquid paraffin. Dosage forms for parenteral administration may include sterilized aqueous solutions, water-soluble excipients, suspensions, emulsions, lyophilized forms and suppositories. Water soluble excipients and suspensions may include propylene glycol, polyethylene glycol, vegetable oil such as olive oil, and an injectable ester such as ethylolate. Suppositories may include witepsol, macrogol, tween 61, cocoa butter, lauric oil, glycerin, and gelatin.

The pharmaceutical composition of the present invention is administered in a pharmaceutically effective dose. In the present invention, the term "pharmaceutically effective dose" means a sufficient amount to treat a disease with a reasonable balance of benefit and risk applicable to medical treatment, and the effective dose can be determined according to factors including the type and severity of the patient's disease, drug activity, drug sensitivity , time of administration, route of administration, rate of elimination, duration of treatment and concomitant drugs used, as well as other factors well known in medicine. The pharmaceutical composition of the present invention can be administered as a single therapeutic agent or in combination with other therapeutic agents for the treatment of diseases caused by pollutants or to reduce skin aging, and can be administered simultaneously, separately or sequentially with conventional therapeutic agents, and can also be administered once or multiple times. It is important to consider all factors and administer an amount sufficient to obtain the maximum effect with minimal or no side effects; such an amount can be easily determined by a person skilled in the art.

In particular, the effective dose of the pharmaceutical composition of the present invention may depend on the age, sex, condition and body weight of the patient, on the absorption of the active ingredient in vivo, on the rate of inactivation, the rate of excretion, the type of disease and concomitant drugs, and may increase or decrease according to with route of administration, degree of obesity, sex, body weight and age. For example, the peptide of the present invention may be administered in an amount of from about 0.0001 μg to 500 mg, eg, from about 0.01 μg to 100 mg per kg of the patient's body weight per day. Additionally, the pharmaceutical composition of the present invention can be administered several times a day, for example, 2-3 times a day at regular time intervals, in accordance with the decision of the doctor or pharmacist.

3. Cosmetic composition for preventing skin damage caused by pollutants or for reducing skin aging

According to another aspect of the present invention, there is provided a cosmetic composition for preventing skin damage caused by pollutants or for reducing skin aging, the pharmaceutical composition comprising a peptide comprising the amino acid sequence of SEQ ID NO: 1. A peptide comprising the amino acid sequence of SEQ ID NO: 1 , this is the same peptide as described in section “1. Peptide for prevention of skin damage caused by pollutants or anti-aging therapy", and thus a detailed description of the peptide comprising the amino acid sequence of SEQ ID NO: 1 can be made by reference to section "1. Peptide for the prevention of skin damage caused by pollutants, or for anti-aging therapy. In the following, only the unique characteristics of a cosmetic composition for treating skin damage caused by pollutants and for reducing skin aging will be described.

Since the peptide of the present invention can effectively inhibit the entry of pollutants into skin cells and suppress the aging of skin cells, the cosmetic composition comprising the peptide as an active ingredient can be used to prevent skin damage caused by pollutants, as well as to reduce wrinkles and improve skin elasticity by reducing aging skin cells.

The peptide may be included in its composition in an amount in the range from about 0.001 to 30 wt%, for example, from about 0.1 to 20 wt%, from about 0.1 to 10 wt%, from about 1 to 10 wt%. . or approximately 2 to 5 wt %, based on a total weight of 100 wt %. cosmetic composition, however, embodiments are not limited to these values.

The cosmetic composition of the present invention containing the peptide as an active ingredient may, for example, include additional ingredients with characteristics that enhance the activity of the peptide in the range in which it does not show activity in inhibiting the entry of contaminants into cells and in reducing the aging of skin cells. For example, the cosmetic composition may further include adjuvants such as tallow-containing substances, organic solvents, solubilizers, thickeners, gelling agents, emollients, antioxidants, suspending agents, stabilizers, foaming agents, perfume, surfactants, water, ionic emulsifiers, non-ionic emulsifiers, bulking agents, binding agents, chelators, preservatives, vitamins, blocking agents, humectants, fragrance oils, colorants, pigments, hydrophilic active agents, lipophilic active agents, lipid vesicles, or any ingredients commonly used in cosmetic or dermatological formulations, and these the components may be present in amounts normally used in dermatology.

The cosmetic composition of the present invention can be formulated into any formulation commonly used in the art, and examples of formulations may include cosmetic products such as solution, suspension, emulsion, gel, lotion, essence, cream, powder, soap, shampoo, conditioner, mask , surfactant-containing cleanser, cleansing water, oils, liquid foundation, cream base or spray.

If the form is a solution or emulsion, a solvent, solubilizer or emulsifier can be used as a carrier component, and for example, water, standard, isopropanol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butyl glycol oil can be used. , an aliphatic ether of glycerol, polyethylene glycol, or a fatty acid ester of sorbitan. If the form is a suspension, a liquid diluent such as water, ethanol or propylene glycol, ethoxylated isostearyl alcohol, a suspending agent such as polyoxyethylene sorbitol ether and polyoxyethylene sorbitan ether, aluminum metahydroxide, microcrystalline cellulose, bentonite, agar or tracanth can be used as a carrier. . If the form is a cream or gel, wax, paraffin, trakant, animal oil, starch, cellulose derivatives, silicone, bentonite, polyethylene glycol, silica, zinc oxide or talc can be used as a carrier. If the form is a powder or spray, a propellant such as silica, talc, aluminum hydroxides, lactose, calcium silicate, chlorofluorocarbon, propane/butane or dimethyl ether can be included as a carrier. If the form is a surfactant-containing cleanser, aliphatic alcohol sulphate, aliphatic alcohol ether sulphate, sulfosuccinic acid monoester, imidazole derivatives, isethionate, methyl taurate, sarcosinate, fatty acid amide ether sulphate, aliphatic alcohol, alkylamidobetaine, fatty acid glyceride, fatty acid diethanolamide, vegetable oil, lanolin derivative or ethoxylated glycerol fatty acid ester.

Hereinafter, the present invention will be described in more detail by examples.

However, these examples are intended to illustrate in detail what has been described, and the present invention is not limited to them.

[Preparation Example] Peptide preparation

The peptide with the amino acid sequence of SEQ ID NO: 1 shown in Table 1 below was synthesized using an automated peptide synthesizer (Milligen 9050, available from Millipore, USA), the synthesized peptide was isolated and purified by reverse phase high performance liquid chromatography (HPLC). ) with C18 (equipment available from Waters Associates, USA). For HPLC, an ACQUITY UPLC BEH300 C18 column (2.1 mm x 100 mm, 1.7 mm, available from Waters Co, USA) was used.

Table 1 SEQID NO: Peptide sequence one SYSGVLFFLK

[Experimental Example 1] Confirmation of the decrease in the number of AhR translocations to the nucleus after peptide treatment

In order to determine the effects of inhibiting the entry of contaminants into cells, the number of translocations of AhR to the nucleus after cell treatment with dioxin (hereinafter also referred to as "TCDD"), particulate matter (hereinafter also referred to as "PM"), and cigarette smoke extract (hereinafter referred to as also "CSE"). When xenobiotics enter the cell from outside, AhR can bind to them, act as a transcription factor, and migrate to the cell nucleus, and thus the degree of penetration of contaminants into the cell can be determined by the number of AhR translocations to the nucleus. The number of AhR translocations to the nucleus of human keratinocytes (HaCaT cells) was determined after treatment of cells with TCDD, PM and CSE, respectively, and the number of AhR translocations to the nucleus of human alveolar epithelial cells (A549 cells) was determined after treatment of cells with CSE. Each of TCDD, PM and CSE was used after mixing with the peptide of the present invention and pre-incubation of the mixture of cells in serum-free medium for approximately 1 hour.

In order to obtain results for cells treated with TCDD, first passage 74 HaCaT cells were plated on a 6-well plate at a cell density of 3×10 ⁵ /well and cultured for about 16 hours, then the culture solution was replaced with serum-free medium and preliminarily added to the mixture. the reacted mixture of TCDD and peptide for approximately 1 hour. The mixture included TCDD at approximately 0.1 μM and peptide at approximately 10 μM, 50 μM, and 100 μM, respectively. The negative control was not treated with the peptide, it only reacted with TCDD, and the positive control reacted with CH223191 at about 10 μM and with TCDD. After the cells interacted with the mixture for about 1 hour, only nucleoproteins were isolated using a kit capable of splitting cells and separating cytoplasm and nucleoproteins (Nuclear and Cytoplasmic Extraction Reagents Kit, available from Thermo scientific, USA), after which a Western blot was performed AhR using anti-AhR antibodies (available from Santacruz biotechnology, USA) and anti-HDAC antibodies (available from Santacruz biotechnology, USA). HDAC was determined to compare the total number of nucleoproteins used in the experiment.

Experiments for samples treated with PM and CSE were performed in the same manner as for samples treated with TCDD, with PM used at a concentration of about 50 μg/cm ² and CSE at a concentration of about 2%, respectively. CSE was prepared by filling a reduced pressure flask with 50 ml PBS and connecting it to a vacuum pump, followed by burning 40 cigarettes. The experimental procedure for the effect on A549 cells (human alveolar epithelial cells) of CSE was similar to those described above, 48-passage A549 cells were plated in a 6-well plate at a cell density of about 5×10 ⁵ /well and treated with CSE. After that, nucleoproteins were isolated from the resulting cells and then Western blot was performed.

As a result, as shown in FIG. 1A-1D, when cells were treated with the peptide, the amount of AhR in the nucleus decreased with increasing peptide concentration, compared to a negative control treated with TCDD, PM, or CSE alone and not treated with a peptide comprising the amino acid sequence of SEQ ID NO: 1, for which the amount AhR in cell nucleoproteins was large.

It can be concluded that the entry of contaminants such as TCDD, PM and CSE into HaCaT and A549 cells is inhibited by the peptide comprising the amino acid sequence of SEQ ID NO: 1, and thus the number of AhR translocations to the nucleus is also reduced.

[Experimental Example 2] Confirmation of a decrease in expression levels of CYP1A1 and COX-2 after treatment with a peptide

In order to determine the effect of inhibiting the entry of contaminants into cells in another way, the cells were treated with TCDD, PM and CSE and the expression levels of CYP1A1 and COX-2 were determined. If AhR activity is stimulated by pollutants such as TCDD, AhR acts as a transcription factor and increases CYP1A1 and COX-2 expression, and thus the degree of entry of pollutants into the cell and AhR activation can be determined by measuring the expression levels of CYP1A1 and COX genes. -2. Each of TCDD, PM and CSE was used after mixing with the peptide of the present invention and pre-incubation of the mixture of cells in serum-free medium for approximately 1 hour.

To obtain results for TCDD-treated cells, first passage 78 HaCaT cells were plated in a 6-well plate at a cell density of 3×10 ⁵ /well and cultured for about 16 hours, then the culture solution was replaced with serum-free medium and preliminarily added to the mixture. the reacted mixture of TCDD and peptide for approximately 2 hours. The mixture included TCDD at approximately 0.1 μM and peptide at approximately 10 μM, 50 μM, and 100 μM, respectively. The negative control was not treated with the peptide, it only reacted with TCDD, and the positive control reacted with CH223191 at about 10 μM and with TCDD. After the cells interacted with the mixture for about 2 hours, RNA was isolated. After removal of the culture solution, the plate on which the cells were cultured was treated with TRIzol (available from Thermo Fisher Scientific, USA) and the RNA containing sample was removed with a scraper. The sample was then treated with chloroform and centrifuged to obtain a supernatant solution. The supernatant solution was treated with isopropanol, centrifuged again, and the presence of RNA at the bottom of the sediment was verified. The precipitate was washed with 70% ethanol and then dried sufficiently to isolate the RNA. The isolated RNA was dissolved in RNase-free water (available from Corning, USA). The isolated RNA was reverse transcribed into cDNA and RT-PCR was performed (cDNA polymerization kit and PCR master mix available from Intron, Korea) using CYP1A1 and COX-2 primers (Table 2). Primer GAPDH, the sequence of which is shown in table 2, was used to compare the total amount of RNA in each of the control groups.

Experiments for samples treated with PM and CSE were performed in the same manner as for samples treated with TCDD, with PM used at a concentration of about 50 μg/cm ² and CSE at a concentration of about 2%, respectively. CSE was prepared by filling a reduced pressure flask with 50 ml PBS and connecting it to a vacuum pump, followed by burning 40 cigarettes.

table 2 SEQID NO: Sequence name Nucleotide sequence 2 CYP1A1 direct 5'-GGATCTTTCTCTGTACCCTGG-3' 3 CYP1A1 reverse 5'-AGCATGTCCTTCAGCCCAGA-3' four COX-2 straight 5'-ATCATTCACCAGGCAAATTG-3' 5 COX-2 reverse 5'-GGCTTCAGCATAAAGCGTTTG-3' 6 GAPDH straight 5'-GGTGTGAACGGATTTGGCCGTATTG-3' 7 GAPDH reverse 5'-CCGTTGAATTTGCCGTGAGTGGAGT-3'

As a result, as shown for RT-PCR in FIG. 2A-2C, when cells were treated with the peptide, CYP1A1 and COX-2 expression levels decreased with increasing peptide concentration, compared to a negative control treated with TCDD, PM, or CSE alone and not treated with a peptide comprising the amino acid sequence of SEQ ID NO: 1, for which expression levels of CYP1A1 and COX-2 genes were high.

It can be concluded that the entry of contaminants such as TCDD, PM and CSE into HaCaT cells is inhibited by the peptide comprising the amino acid sequence of SEQ ID NO: 1, and thus the expression levels of CYP1A1 and COX-2 are also reduced.

[Experimental Example 3] Confirmation of reduction in MMP-1 and COX-2 proteins after peptide treatment

HaCaT cells were treated with TCDD and PM, and the amount of MMP-1 and COX-2 proteins was measured to confirm the effect of inhibiting the entry of contaminants into the cells in a different way. MMP-1 is a collagenase. If AhR activity is stimulated by pollutants such as TCDD, AhR acts as a transcription factor and increases MMP-1 and COX-2 expression, and thus the degree of entry of pollutants into the cell and AhR activation can be determined by measuring MMP gene expression levels. -1 and COX-2. Each of TCDD and PM was used after mixing with the peptide of the present invention and pre-incubating the mixture of cells in serum-free medium for approximately 1 hour.

To obtain results for TCDD-treated cells, first passage 80 HaCaT cells were seeded in a 6-well plate at a cell density of 3×10 ⁵ /well and cultured for about 16 hours, then the culture solution was replaced with serum-free medium and preliminarily added to the mixture. the reacted mixture of TCDD and peptide for approximately 24 hours. The mixture included TCDD at approximately 0.1 μM and peptide at approximately 10 μM, 50 μM, and 100 μM, respectively. The negative control was not treated with the peptide, it only reacted with TCDD, and the positive control reacted with CH223191 at about 10 μM and with TCDD. After the cells were exposed to the mixture for about 24 hours, a cell lysate was prepared and Western blotting of MMP-1 and COX-2 was performed using antibodies to MMP-1 (available from Cell Signaling, USA) and to COX-2 (available at Santacruz biotechnology, USA).

β-actin (anti-β-actin antibody available from Santacruz biotechnology, USA) was used to compare the total amount of proteins in the study.

Experiments for samples treated with PM were performed in the same manner as for samples treated with TCDD, with PM used at a concentration of about 50 μg/cm ² .

As a result, as shown for the Western blot in FIG. 3A and 3B, when cells were treated with the peptide, the amount of MMP-1 and COX-2 decreased with increasing peptide concentration, compared to a negative control treated with TCDD or PM alone and not treated with a peptide comprising the amino acid sequence of SEQ ID NO: 1, for which the amount MMP-1 and COX-2 proteins were large.

It can be concluded that the entry of contaminants such as TCDD and PM into HaCaT cells is inhibited by the peptide comprising the amino acid sequence of SEQ ID NO: 1, and thus the amount of MMP-1 and COX-2 proteins is also reduced.

[Experimental Example 4] Confirmation of the Adsorption Effect of Peptide Contaminants

The deposition profile of the mixture of peptide and contaminants was observed with the naked eye to determine that the peptide of the present invention adsorbs contaminants.

First, to determine the degree of adsorption of suspended particles, 100 µg/ml of PM and 200 µM and 200 µM of the peptide were mixed together and studied the deposition profile. CSE prepared from 40 cigarette smoke and 200 μM peptide were also mixed and observed with the naked eye.

As a result, as shown in FIG. 4A and 4B, when cells were treated with the peptide, the degree of gradual precipitation increased with increasing peptide concentration, compared to cells treated with PM or CSE alone and not treated with a peptide comprising the amino acid sequence of SEQ ID NO: 1, in which case PM and CSE remained in mixtures.

It can be concluded that a peptide comprising the amino acid sequence of SEQ ID NO: 1 captures and adsorbs pollutant molecules such as PM or CSE, and thus, an effect of inhibiting the entry of pollutant molecules into the skin is observed.

[Experimental Example 5] Confirmation of stimulation of keratinocyte activity by natural aging-inhibiting peptide treatment

The amount of proteins and expression levels of genes involved in natural aging were measured to determine whether the peptide of the present invention can inhibit the naturally progressive aging of cells, as exemplified by keratinocytes.

<5-1> Confirmation of phosphorylation of signal proteins associated with proliferation

Since Akt and ERK are proteins involved in the signal transduction process associated with cell proliferation and exhibit activity in their phosphorylated forms (p-Akt and p-ERK, respectively), we determined whether an increase in Akt and ERK phosphorylation could suppress cellular aging when cells are treated with the peptide of the present invention.

First passage 81 HaCaT cells were seeded in a 6-well plate at a cell density of 3×10 ⁵ /well and cultured for about 24 hours, then the cells were treated with the peptide of the present invention at a concentration of approximately 10 μM, 50 μM and 100 μM, respectively, about 15 minutes. The positive control was treated with growth factors, 100 nM bFGF and 100 nM IGF, respectively, and not with the peptide. The reaction lasted about 15 minutes, then a cell lysate was prepared and Western blotting of p-Akt and p-ERK was performed using antibodies against p-Akt (available from Cell Signaling, USA) and against p-ERK (available from Cell Signaling, USA). USA).

β-Actin (anti-β-actin antibodies, available from Santacruz biotechnology, USA) was used to compare total protein.

As a result, as shown for the Western blot in FIG. 5A found that the degree of phosphorylation of Akt and ERK gradually increased with increasing concentration of the peptide treated with the cells.

It can be concluded that the degree of phosphorylation of Akt and ERK in HaCaT cells increases under the action of a peptide comprising the amino acid sequence of SEQ ID NO: 1, which characterizes the stimulation of cell growth, and thus it has been shown that the phenomenon of natural aging can be suppressed by treating cells with a peptide .

<5-2> Confirmation of increased expression of SIRT1 (anti-aging gene)

The ability to suppress cell aging by treatment with the peptides of the present invention was determined by checking whether the level of expression of the SIRT1 gene, which prevents cell aging, is increased.

Passage 78 HaCaT cells were plated in a 6-well plate at a cell density of 3×10 ⁵ /well and cultured for about 24 hours, then the cells were treated with the peptide of the present invention at a concentration of approximately 10 μM, 50 μM and 100 μM, respectively, for approximately 24 hours. The positive control was treated with growth factors, 100 nM bFGF and 100 nM IGF, respectively, and not with the peptide. After reacting for approximately 24 hours, RNA was isolated from the cells in the same manner as described in Experimental Example 2, and the isolated RNA was reverse transcribed into cDNA, followed by RT-PCR (cDNA polymerization kit and PCR master mix available from Intron). , Korea) using the SIRT1 primer (Table 3). Primer GAPDH, the sequence of which is shown in table 2, was used to compare the total amount of RNA in each of the control groups.

one.

Table 3 SEQID NO: Sequence name Nucleotide sequence eight SIRT1 straight 5'-TCAGTGGCTGGAACAGTGAG-3' 9 SIRT1 reverse 5'-TCTGGCATGTCCCACTATCA-3'

As a result, as shown for RT-PCR in FIG. 5B found that the expression level of the SIRT1 gene increased with increasing concentration of the peptide treated with the cells.

It can be concluded that the level of expression of the anti-aging SIRT1 gene in HaCaT cells increases under the action of a peptide comprising the amino acid sequence of SEQ ID NO: 1, and in this way, the natural aging of cells can be suppressed.

[Experimental Example 6] Confirmation of stimulation of fibroblast activity by natural aging-inhibiting peptide treatment

The amount of proteins and expression levels of genes involved in natural aging were measured to determine whether the peptide of the present invention can suppress the naturally progressive aging of cells, using fibroblasts as an example.

<6-1> Confirmation of phosphorylation of signal proteins associated with proliferation

Since Akt and ERK are proteins involved in the signal transduction process associated with cell proliferation and exhibit activity in their phosphorylated forms (p-Akt and p-ERK, respectively), we determined whether phosphorylated Akt and ERK can suppress cellular senescence. when the cells are treated with the peptide of the present invention.

First passage 38 NIH3T3 cells (mouse fibroblasts) were seeded in a 6-well plate at a cell density of 3×10 ⁵ /well and cultured for about 24 hours, then the cells were treated with the peptide of the present invention at a concentration of approximately 10 μM, 50 μM and 100 μM , respectively, about 15 minutes. The positive control was treated with growth factors, 50 nM bFGF and 25 nM IGF, respectively, rather than the peptide. The reaction lasted about 15 minutes, then a cell lysate was prepared and Western blotting of p-Akt and p-ERK was performed using antibodies against p-Akt (available from Cell Signaling, USA) and against p-ERK (available from Cell Signaling, USA). USA). β-Actin (anti-β-actin antibodies, available from Santacruz biotechnology, USA) was used to compare total protein.

As a result, as shown for the Western blot in FIG. 6A found that the degree of Akt and ERK phosphorylation gradually increased with increasing concentration of the peptide treated with the cells.

It can be concluded that the degree of Akt and ERK phosphorylation in NIH3T3 cells increases under the action of a peptide comprising the amino acid sequence of SEQ ID NO: 1, which characterizes the stimulation of cell growth, and thus it has been shown that the natural aging phenomenon can be suppressed by treating cells with the peptide .

<6-2> Confirmation of increased expression of skin components (collagen, fibronectin and elastin)

The ability to suppress the aging of cells by their treatment with the peptides of the present invention was determined by checking whether the level of expression of the collagen, fibronectin and elastin genes associated with the formation of fibers, where collagen, fibronectin and elastin are components that make up the dermis, is increased. The genome encoding collagen I is COL1A1.

Passage 26 NIH3T3 cells were plated in a 6-well plate at a cell density of 3×10 ⁵ /well and cultured for about 24 hours in serum-free medium, then the cells were treated with the peptide of the present invention at a concentration of approximately 10 μM, 50 μM and 100 μM, respectively , approximately 24 hours. The positive control was treated with growth factors, 100 nM bFGF and 5 nM IGF ^β1 , respectively, rather than the peptide. After reacting for approximately 24 hours, RNA was isolated from the cells in the same manner as described in Experimental Example 2, and the isolated RNA was reverse transcribed into cDNA, followed by RT-PCR (cDNA polymerization kit and PCR master mix available from Intron). , Korea) using primers Col1a1, fibronectin and elastin (Table 4). Primer GAPDH, the sequence of which is shown in table 2, was used to compare the total amount of RNA in each of the control groups.

Table 4 SEQID NO: Sequence name Nucleotide sequence 16 AQP3 straight 5'-CCTTCTTGGGTGCTGGAATA -3' 17 AQP3 reverse 5'-ACACGATAAGGGAGGCTCTG-3'

As a result, as shown for RT-PCR in FIG. 7 found that the level of expression of the UVB-inhibited SIRT1 and AQP3 genes gradually increased with increasing concentration of the peptide treated with the cells.

It can be concluded that the level of expression of the anti-aging gene SIRT1 and the skin barrier component gene AQP3, which decreased in HaCaT cells, can be increased and restored under the action of a peptide comprising the amino acid sequence of SEQ ID NO: 1, and in this way photoaging of cells can be suppressed.

[Experimental Example 8] Confirmation of Recovery of Fibroblast Activity Suppressed by Photoaging-Inducing UV Light

<8-1> Confirmation of restoration of expression of dermal components (collagen, fibronectin and elastin)

When irradiated with UV light, the expression of the genes of collagen, fibronectin and elastin, which are part of the dermis, is inhibited, and thus the ability to suppress photoaging of cells was tested by determining whether the expression levels of collagen, fibronectin and elastin are restored and increased after treatment of cells with the peptide of the present invention .

First, the 34th passage NIH3T3 cells were seeded in a 6-well plate at a cell density of 5×10 ⁵ /well and cultured for about 16 hours in serum-free medium, then the cells were treated with the peptide of the present invention at a concentration of approximately 10 μm, 50 μm and 100 μm, respectively, about 1 hour. The precipitate obtained was washed with PBS, filled with PBS and previously irradiated with UVA at an intensity of 6 J/cm ² for 1 hour. The cells were then treated with the peptide of the present invention at the indicated concentrations for about 6 hours. The positive control was treated with 2.5 mM NaC and 50 μM quercetin, respectively, rather than with the peptide. After the cells reacted with the peptide for approximately 6 hours, RNA was isolated from the cells in the same manner as described in Experimental Example 2, and the isolated RNA was reverse transcribed into cDNA, followed by RT-PCR (cDNA polymerization kit and PCR master mix are available). from Intron, Korea) using primers Col1a1, fibronectin, and elastin (Table 4). Primer GAPDH, the sequence of which is shown in table 2, was used to compare the total amount of RNA in each of the control groups.

As a result, as shown for RT-PCR in FIG. 8A found that the gene expression level of collagen (Col1a1), fibronectin, and elastin gradually increased with increasing concentration of the peptide treated with the cells.

It can be concluded that the gene expression levels of collagen (Col1a1), fibronectin and elastin, which are reduced after UVA irradiation, in NIH3T3 cells can be increased and restored by the action of a peptide comprising the amino acid sequence of SEQ ID NO: 1, and in this way, as a result of suppression photoaging of cells under the action of proteins expressed by the above genes, the effect of reducing the number of wrinkles on the skin can be observed.

<8-2> Confirmation of MMP-1 and MMP-2 expression inhibition

Since the expression of genes encoding MMP-1 and MMP-2 proteins is associated with degradation of collagen by UV irradiation, it was checked whether cell aging can be suppressed by suppressing the synthesis of MMP-1 and MMP-2 proteins by treating cells with the peptide of the present invention.

First, the 31st passage of NIH3T3 cells were seeded in a 6-well plate at a cell density of 5×10 ⁵ /well and cultured for about 16 hours in serum-free medium, then the cells were treated with the peptide of the present invention at a concentration of approximately 10 μm, 50 μm and 100 μm, respectively, about 1 hour. The precipitate obtained was washed with PBS, filled with PBS and previously irradiated with UVA at an intensity of 6 J/cm ² for 1 hour. The cells were then treated with the peptide of the present invention at the indicated concentrations for about 48 hours. The positive control was treated with 2.5 mM NaC and 50 μM quercetin, respectively, rather than with the peptide. After the cells reacted with the mixture for approximately 48 hours, a cell lysate was obtained and an MMP-1 Western blot was performed using anti-MMP-1 antibodies (available from Cell Signaling, USA). β-Actin (anti-β-actin antibodies, available from Santacruz biotechnology, USA) was used to compare total protein.

In addition, to analyze the amount of MMP-2, a lysate was prepared from the resulting cells and zymographed with gelatin. Using gelatin (2 mg/ml) as a substrate, protein electrophoresis (SDS-PAGE) was performed. Thereafter, the gel was immersed in 2.5% Triton X-100 for 30 minutes and then incubated in a buffer containing 50 mM Tris-HCl, 0.2 M NaCl and 5 mM CaCl ₂ and 1% Triton X-100 at 37°C for 24 hours. After incubation, the gel was stained with Coomassie Brilliant Blue R250 (available from Sigma), then treated with a decolorization buffer containing 5% ethanol, 7.5% acetic acid and distilled water. Thereafter, empty bands of the gel phase formed during gelatin hydrolysis were observed, confirming the activity of MMP-2.

As a result, as shown for Western blotting and zymography in FIG. 8B found that the amount of MMP-1 and MMP-2 protein gradually decreased with increasing concentration of the peptide treated with the cells.

It can be concluded that the amount of MMP-1 and MMP-2 protein, which increased under the action of UVA irradiation in NIH3T3 cells, can decrease and recover under the action of a peptide comprising the amino acid sequence of SEQ ID NO: 1, and the amount of collagen can increase again, which leads to to reduce the number of wrinkles on the skin as photoaging decreases.

As described above, although the present invention has been described in detail with reference to one or more embodiments described herein, it will be apparent to those skilled in the art that various changes and modifications are possible within the scope of the technical concept of the present invention, and it is also apparent that such changes and modifications are in the spirit and areas described in the following claims.

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<110> CAREGEN CO., LTD.

<120> Peptides for protecting skin damage by pollutants and

anti-aging and use thereof

<130> 2018DPA3173

<160> 17

<170> KoPatentIn 3.0

<210> 1

<211> 10

<212> PRT

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<220>

<223> Peptide 1

<400> 1

Ser Tyr Ser Gly Val Leu Phe Phe Leu Lys

1 5 10

<210> 2

<211> 21

<212> DNA

<213> Artificial Sequence

<220>

<223> CYP1A1 forward primer sequence

<400> 2

ggatctttct ctgtaccctg g 21

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<211> 20

<212> DNA

<213> Artificial Sequence

<220>

<223> CYP1A1 reverse primer sequence

<400> 3

agcatgtcct tcagcccaga 20

<210> 4

<211> 21

<212> DNA

<213> Artificial Sequence

<220>

<223> COX-2 forward primer sequence

<400> 4

atcattcacc aggcaaattg c 21

<210> 5

<211> 21

<212> DNA

<213> Artificial Sequence

<220>

<223> COX-2 reverse primer sequence

<400> 5

ggcttcagca taaagcgttt g 21

<210> 6

<211> 25

<212> DNA

<213> Artificial Sequence

<220>

<223> GAPDH forward primer sequence

<400> 6

ggtgtgaacg gatttggccg tattg 25

<210> 7

<211> 25

<212> DNA

<213> Artificial Sequence

<220>

<223> GAPDH reverse primer sequence

<400> 7

ccgttgaatt tgccgtgagt ggagt 25

<210> 8

<211> 20

<212> DNA

<213> Artificial Sequence

<220>

<223> SIRT1 forward primer sequence

<400> 8

tcagtggctg gaacagtgag 20

<210> 9

<211> 20

<212> DNA

<213> Artificial Sequence

<220>

<223> SIRT1 reverse primer sequence

<400> 9

tctggcatgt cccactatca 20

<210> 10

<211> 20

<212> DNA

<213> Artificial Sequence

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<223> Col1a1 forward primer sequence

<400> 10

caccctcaag agcctgagtc 20

<210> 11

<211> 20

<212> DNA

<213> Artificial Sequence

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<223> Col1a1 reverse primer sequence

<400> 11

agacggctga gtagggaaca 20

<210> 12

<211> 20

<212> DNA

<213> Artificial Sequence

<220>

<223> Fibronectin forward primer sequence

<400> 12

cggaaccg agtaccacat 20

<210> 13

<211> 20

<212> DNA

<213> Artificial Sequence

<220>

<223> Fibronectin reverse primer sequence

<400> 13

atacccaggt tgggtgatga 20

<210> 14

<211> 20

<212> DNA

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<223> Elastin forward primer sequence

<400> 14

ggacccctga ctcgcgacct 20

<210> 15

<211> 20

<212> DNA

<213> Artificial Sequence

<220>

<223> Elastin reverse primer sequence

<400> 15

ggggaggtgg gactgcccaa 20

<210> 16

<211> 20

<212> DNA

<213> Artificial Sequence

<220>

<223> AQP3 forward primer sequence

<400> 16

ccttcttggg tgctggaata 20

<210> 17

<211> 20

<212> DNA

<213> Artificial Sequence

<220>

<223> AQP3 reverse primer sequence

<400> 17

acacgataag ggaggctctg 20

<---

Claims (12)

1. A peptide for preventing skin damage caused by pollutants, wherein the peptide consists of the amino acid sequence of SEQ ID No: 1. 2. The peptide according to claim 1, wherein the peptide is characterized by the ability to inhibit the entry of contaminants into the skin. 3. The peptide according to claim 1, wherein the peptide adsorbs contaminants. 4. The peptide according to any one of paragraphs. 1-3, with contaminants selected from the group consisting of dioxin, particulate matter, and cigarette smoke extract. 5. A skin rejuvenation peptide, wherein the peptide consists of the amino acid sequence of SEQ ID No: 1. 6. The peptide according to claim 5, wherein the skin rejuvenation is a reduction in skin wrinkles or an improvement in skin elasticity. 7. A pharmaceutical composition for the prevention or treatment of diseases caused by pollutants, wherein the pharmaceutical composition contains a peptide according to claim 1 as an active ingredient. 8. The pharmaceutical composition according to claim 7, wherein the diseases caused by contaminants are at least those selected from the group consisting of cancer, atopic dermatitis, contact dermatitis, seborrheic dermatitis, acne, dry skin, psoriasis, death, immunotoxicity, damage to the peripheral nervous system, damage to the central nervous system, disorders of the endocrine system, disorders of the reproductive system, disorders in the development of infants and preschool children, anemia, bronchitis, emphysema, decreased lung function, asthma, chronic bronchitis, arrhythmias, acute coronary syndrome, angina or myocardial infarction. 9. A composition for improving skin rejuvenation, the composition comprising a peptide according to claim 5 as an active ingredient. 10. Composition according to claim 9, wherein the improvement in skin rejuvenation is the reduction of wrinkles. 11. Cosmetic composition for the prevention of skin damage caused by pollutants or to improve skin rejuvenation, and the cosmetic composition contains a peptide according to any one of paragraphs. 1 or 5 as active ingredient. 12. Cosmetic composition according to claim 11, wherein the cosmetic composition is represented by at least one composition selected from the group consisting of a solution, suspension, emulsion, gel, lotion, essence, cream, powder, soap, shampoo, conditioner, mask, containing surfactant of a cleanser, cleansing foam, cleansing water, oil, liquid foundation, cream base or spray.

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