Peptide having skin condition improving activity and use thereof

Abstract

The present application relates to a peptide having a skin condition improving activity and a use thereof, and provides a peptide comprising the amino acid sequence of SEQ ID NO: 1 and a cosmetic composition for improving a skin condition comprising the peptide comprising the amino acid sequence of SEQ ID NO: 1 as an active ingredient.

Description

Technical Field

[0001] The present application relates to a peptide having skin condition improving activity and its use.

Background Art

[0002] Human skin undergoes continuous changes, and the most representative ones are the reduction of skin functions caused by aging and the decrease in visual beauty. Aging causes skin wrinkles, and as representative factors for forming wrinkles, exposure to ultraviolet rays and the reduction of collagen biosynthesis can be cited. Skin aging is roughly classified into endogenous aging caused by genetic factors and exogenous aging caused by external environmental factors such as sunlight. Among them, for exogenous aging, it is known that aging can be prevented, treated or delayed by removing reactive oxygen species, proliferating fibroblasts, and promoting collagen biosynthesis.

[0003] Collagen, which is the main component of the extracellular matrix, is the main matrix protein produced in fibroblasts of the skin. Collagen forms most of the organic substances in the skin, tendon, bone and teeth, especially in high content in bone and skin (dermis). This collagen decreases due to age and photoaging caused by ultraviolet irradiation, which is known to be closely related to the formation of skin wrinkles. Also, collagen plays an important role in wound treatment, promoting collagen synthesis in damaged epithelium, so that the wound can be quickly and thoroughly restored. In addition, it has been reported that as the biosynthesis of collagen is promoted and the density of the basal layer and the like becomes higher, the concentration of melanin per unit skin density decreases, so a skin brightening effect can be expected.

[0004] Under this technical background, currently, various studies are being conducted to improve the skin condition through mechanisms such as promoting collagen biosynthesis, proliferating fibroblasts, etc. and enhancing activity (Korean Registered Patent No. 10-1813629), but it is still not perfect.

Summary of the Invention

[0005]

Technical Problem

[0006] On the one hand, a peptide consisting of the amino acid sequence of SEQ ID NO: 1 is provided.

[0007] On the other hand, a cosmetic composition for improving skin condition is provided, which comprises a peptide having the amino acid sequence of SEQ ID NO: 1 as an active ingredient.

[0008] Other objects and advantages of the present application will become more apparent in conjunction with the appended claims, the accompanying drawings, and the following detailed description. Regarding the content not described in this specification, as long as it can be fully recognized and analogized by those of ordinary skill in the art to which the present application belongs or those of ordinary skill in a similar technical field, the relevant description thereof is omitted.

[0009]

Technical Solution

[0010] The various descriptions and embodiments disclosed in the present application can also be applied to various other descriptions and embodiments. That is, all combinations of the various elements disclosed in the present application fall within the scope of the present application. Moreover, it cannot be determined that the scope of the present application is limited to the specific descriptions set forth below.

[0011] On the one hand, a peptide is provided, which consists of the amino acid sequence of SEQ ID NO: 1.

[0012] The term "peptide" used in this specification may refer to a linear molecule formed by multiple amino acid residues bound to each other through peptide bonds. The peptide can be prepared by chemical synthesis methods well known in the art, especially solid-phase synthesis technology or liquid-phase synthesis technology (US Registered Patent No. 5,516,891). As a result of efforts to develop peptides with biologically effective activities, the inventors have identified a peptide consisting of the amino acid sequence of SEQ ID NO: 1. Here, the biologically effective activity may refer to any one of the following characteristics: (a) promoting the proliferation of fibroblasts or keratinocytes; (b) enhancing the expression of type I collagen (Col1a1), fibronectin, or elastin, which are extracellular matrix constituent factors; (c) enhancing the expression of the skin barrier factors, namely sirtuin-1 (SIRT-1) or aquaporin-3 (AQP3); and (d) inhibiting the apoptosis of fibroblasts or keratinocytes and reducing the level of reactive oxygen species. Therefore, the peptide can be used for improving skin condition or antioxidation.

[0013] In the peptide, in order to obtain chemical stability, enhanced pharmacological properties (half-life, absorbability, potency, efficacy, etc.), altered specificity (e.g., a wide range of biological activities), and reduced antigenicity, a protecting group may be bound to the N- or C-terminus at the end of the peptide. In one embodiment, the N-terminus of the peptide may be bound to any protecting group selected from the group consisting of an acetyl group, a fluorenylmethoxycarbonyl group, a formyl group, a palmitoyl group, a myristyl group, a stearyl group, a butoxycarbonyl group, an allyloxycarbonyl group, and a polyethylene glycol (PEG); and / or the C-terminus of the peptide may be bound to any protecting group selected from the group consisting of an amino group (-NH2), a tertiary alkyl group, and a hydrazino group (-NHNH2). Further, the peptide may optionally further include a targeting sequence, a tag, a labeled residue, and an amino acid sequence prepared for a specific purpose of increasing the half-life or peptide stability.

[0014] The peptide is artificially synthesized or non-naturally occurring or engineered, and the "non-naturally occurring or engineered" refers to a state generated by artificial modification, rather than the state of the existence itself produced in the natural state. Herein, the artificial modification may include artificially synthesizing an amino acid sequence by mimicking the structures of multiple amino acids, or may include engineering as described above to obtain chemical stability, enhanced pharmacological properties, altered specificity, or reduced antigenicity.

[0015] The term "stability" used in this specification may refer not only to the in vivo stability that protects the peptide from the attack of in vivo proteases but also to storage stability (e.g., stability at room temperature).

[0016] On the other hand, a cosmetic composition for improving skin condition is provided, which includes a peptide having the amino acid sequence of SEQ ID NO: 1 as an active ingredient.

[0017] Among the terms or elements mentioned in the description of the peptide, the same content as that already mentioned is as described above.

[0018] The term "improvement" as used in this specification may refer to all actions that at least mitigate parameters related to the alleviation or treatment of a condition, such as the degree of symptoms.

[0019] The term "skin condition improvement" as used in this specification may broadly refer to the process of treating, alleviating, or mitigating skin damage caused by endogenous or exogenous factors in the skin, or its effects, etc. For example, it can be interpreted as wrinkle improvement, skin elasticity improvement, wound recovery, skin barrier strengthening, or skin aging inhibition, but is not limited thereto.

[0020] Here, "wrinkle improvement", "skin elasticity improvement", and "wound recovery" may refer to all actions that increase the total amount of extracellular matrix factors, including promoting collagen synthesis, etc. And, "skin barrier strengthening" may refer to strengthening the original function of the skin, that is, preventing the leakage of water and nutrients in the skin to the outside and preventing the invasion of harmful substances such as bacteria or viruses into the skin. And, "skin aging inhibition" may refer to inhibiting the reduction of skin functions such as wrinkles, skin sagging, and elasticity reduction. At this time, the skin aging may be photoaging, for example, skin aging caused by ultraviolet rays.

[0021] The disadvantage of existing functional peptides is that even if they have effective biological activities, due to the size of the peptide itself, they cannot be effectively introduced into target tissues or cells, or disappear from the body in a short time because of their short half-life. In contrast, according to one embodiment, a cosmetic composition includes a peptide composed of 10 or fewer amino acids as an active ingredient. Therefore, the skin permeability of the active ingredient is very excellent. For example, when locally applied to the skin, it can effectively improve the skin condition.

[0022] According to one embodiment, it can promote the proliferation of fibroblasts and keratinocytes and enhance the synthesis of extracellular matrix components and skin barrier factors. And, it has also been found that the peptide can restore the reduced activity of fibroblasts and keratinocytes and enhance their antioxidant efficacy. Therefore, the peptide can be used as an active ingredient of a cosmetic composition for skin condition improvement.

[0023] The cosmetic composition may include a cosmetically effective amount of the peptide; and / or a cosmetically acceptable carrier, but is not limited thereto.

[0024] The term "cosmetically effective amount" as used in this specification refers to an amount sufficient to achieve the skin condition improvement efficacy of the cosmetic composition.

[0025] The weight ratio between the peptide and the cosmetically acceptable carrier can be, for example, from 500:1 to 1:500. For example, the weight ratio can be from 450:1 to 1:450, from 400:1 to 1:400, from 350:1 to 1:350, from 300:1 to 1:300, from 250:1 to 1:250, from 200:1 to 1:200, from 150:1 to 1:150, from 100:1 to 1:100, from 80:1 to 1:80, from 60:1 to 1:60, from 40:1 to 1:40, from 20:1 to 1:20, from 10:1 to 1:10, from 8:1 to 1:8, from 6:1 to 1:6, from 4:1 to 1:4, or from 2:1 to 1:2, but is not limited thereto.

[0026] The cosmetic composition can be prepared in any dosage form commonly prepared in the art. For example, it can be prepared as a solution, suspension, emulsion, dough, gel, skin cream, skin lotion, powder, soap, surfactant-containing cleaner, oil, powder foundation, liquid foundation, wax foundation, and spray, etc., but is not limited thereto. For example, the cosmetic composition can be prepared as a soft lotion, nutritious lotion, nutritious cream, massage cream, essence, eye cream, cleansing cream, cleansing foam, cleansing water, facial mask, spray, or powder dosage form.

[0027] When the dosage form of the cosmetic composition is dough, skin cream or gel, animal oil, vegetable oil, wax, paraffin wax, starch, tragacanth, cellulose derivative, polyethylene glycol, silicon, bentonite, silica, talc or zinc oxide, etc. can be used as carrier components.

[0028] In the case where the dosage form of the cosmetic composition is foundation or spray, lactose, talc, silica, aluminum hydroxide, calcium silicate or polyamide powder can be used as carrier components. For example, if it is a spray, a promoter such as chlorofluorocarbon, propane / butane or dimethyl ether can be further included.

[0029] In the case where the dosage form of the cosmetic composition is solution or emulsion, a solvent, solubilizer or emulsifier can be used as carrier components. For example, it can include water, ethanol, isopropanol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol oil, glycerol fatty acid ester, polyethylene glycol or sorbitan fatty acid ester.

[0030] In the case where the dosage form of the cosmetic composition is suspension, a liquid phase diluent such as water, ethanol or propylene glycol, a suspending agent such as ethoxylated isostearyl alcohol, polyoxyethylene sorbitan ester and polyoxyethylene sorbitan ester, microcrystalline cellulose, aluminum hydroxide methyl, bentonite, agar or tragacanth, etc. can be used.

[0031] In the case where the dosage form of the cosmetic composition is a surfactant-containing cleanser, fatty alcohol sulfates, fatty alcohol ether sulfates, sulfosuccinic acid monoesters, hydroxyethyl sulfonates, imidazoline derivatives, methyl taurates, sarcosinates, fatty acid amide ether sulfates, alkylamide betaines, fatty alcohols, fatty acid glycerol esters, fatty acid diethanolamides, vegetable oils, lanolin derivatives, or ethoxylated glycerol fatty acid esters can be used as carrier components.

[0032] The peptide can be included in nanobodies or nanoparticles to further improve skin penetration problems or stability problems. For example, the nanobodies can be prepared using a microfluidizer with lecithin as a raw material and can be included in lecithin particles. As a method for preparing the nanobodies, any known method can be used. The size of the nanobody particles is preferably 30 to 200 nm. In the case where the size of the nanobody particles is less than 30 nm, skin penetration may be very fast, resulting in skin side effects. In the case where the size of the nanobody particles is greater than 200 nm, it may not be easy to penetrate the skin, making it difficult to obtain the usage effect of the nanobodies.

[0033] In addition to the peptide and the carrier component as the active ingredient, the components included in the cosmetic composition include conventional components for cosmetic compositions, such as conventional adjuvants like antioxidants, stabilizers, solubilizers, vitamins, pigments, and fragrances.

[0034] The content of the peptide as the active ingredient included in the cosmetic composition can be appropriately and non-restrictively selected according to the product form, desired use, etc. For example, it can be added in an amount of 0.01 to 15% by weight of the total weight of the cosmetic composition. And, for example, the cosmetic composition can include 1.0% to 3.0% by weight, preferably 2.0% to 3.0% by weight of the peptide based on the total weight.

[0035] On the other hand, a method for improving skin condition is provided, which includes the step of applying a cosmetic composition including a peptide consisting of the amino acid sequence of SEQ ID NO: 1 as an active ingredient to the skin of an individual; and the use of the peptide consisting of the amino acid sequence of SEQ ID NO: 1 in the preparation of a composition for improving skin condition.

[0036] Among the terms or elements mentioned in the description of the cosmetic composition, the same content as that already mentioned is as described above.

[0037] In the terms used in this specification, the "individual" refers to an object in need of improving skin condition. More specifically, it refers to mammals such as humans or non-human primates, mice, dogs, cats, horses, and cows.

[0038] As used in this specification, the terms "apply", "administer", and "coat" may be used interchangeably and may refer to at least partially localizing a composition according to an embodiment at a desired site, or disposing a composition according to an embodiment within an individual by an administration route.

[0039] On the other hand, a composition for antioxidation is provided, which comprises a peptide consisting of the amino acid sequence of SEQ ID NO: 1 as an active ingredient.

[0040] Among the terms or elements mentioned in the description of the peptide, the same content as that already mentioned is as described above.

[0041] The composition for antioxidation may be in the form of a pharmaceutical composition, a quasi-drug composition, or a cosmetic composition. As an example, the composition may be used as a cosmetic composition for improving skin conditions, or a pharmaceutical composition for improving or treating disease states related to skin damage.

[0042] According to an embodiment, it is shown that the peptide can restore the reduced activity of fibroblasts or keratinocytes and enhance their antioxidant efficacy. Therefore, the peptide can be used as an active ingredient of the composition for antioxidation.

[0043] As an example, the antioxidant composition may be provided in the form of a pharmaceutical composition. The pharmaceutical composition may include a pharmaceutically effective amount of the peptide; and / or a pharmaceutically acceptable carrier, but is not limited thereto.

[0044] The term "pharmaceutically effective amount" used in this specification may refer to the amount of the pharmaceutical composition sufficient to achieve a prophylactic or therapeutic effect on diseases related to skin damage.

[0045] The pharmaceutically acceptable carrier is commonly used in preparation and includes lactose, dextrose, sucrose, sorbitol, mannitol, starch, gum arabic, calcium phosphate, alginate, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, methylcellulose, methylparaben, propylparaben, talc, magnesium stearate, and mineral oil, etc., but is not limited thereto. Suitable pharmaceutically acceptable carriers and formulations are described in detail in Remington's Pharmaceutical Sciences (19th ed., 1995).

[0046] The weight ratio between the peptide and a pharmaceutically acceptable carrier can be, for example, from 500:1 to 1:500. As an example, the weight ratio can be from 450:1 to 1:450, from 400:1 to 1:400, from 350:1 to 1:350, from 300:1 to 1:300, from 250:1 to 1:250, from 200:1 to 1:200, from 150:1 to 1:150, from 100:1 to 1:100, from 80:1 to 1:80, from 60:1 to 1:60, from 40:1 to 1:40, from 20:1 to 1:20, from 10:1 to 1:10, from 8:1 to 1:8, from 6:1 to 1:6, from 4:1 to 1:4, or from 2:1 to 1:2, but is not limited thereto.

[0047] In addition to the above ingredients, the pharmaceutical composition may further include a lubricant, a wetting agent, a sweetening agent, a flavoring agent, an emulsifying agent, a suspending agent, a preservative, etc., but is not limited thereto.

[0048] The pharmaceutical composition can be administered enterally or parenterally, preferably parenterally. For parenteral administration, it can be administered by intramuscular injection, intravenous injection, subcutaneous injection, intraperitoneal injection, topical administration, transdermal administration, etc., but is not limited thereto.

[0049] The dosage of the pharmaceutical composition can be 0.0001 to 1000 μg (micrograms) per day, 0.001 to 1000 μg, 0.01 to 1000 μg, 0.1 to 1000 μg, or 1.0 to 1000 μg, but is not limited thereto, and can be differently prescribed according to factors such as the formulation method, the administration mode, the age, weight, gender, medical condition, diet, administration time, administration route, excretion rate, and reaction sensitivity of the patient.

[0050] The pharmaceutical composition can be formulated into a unit volume form by using a pharmaceutically acceptable carrier and / or excipient according to a method easily implemented by those of ordinary skill in the technical field to which the present invention pertains, or can be prepared by being filled into a multi-volume container.

[0051] The dosage form can be in the form of a solution, a suspension, or an emulsion in an oily or aqueous solvent, can also be in the form of an extractant, a powder, a granule, a lozenge, or a capsule, and can further include a powder and / or a stabilizer.

[0052] On the other hand, a food composition for improving skin condition is provided, which includes a peptide consisting of the amino acid sequence of SEQ ID NO: 1 as an active ingredient.

[0053] Among the terms or elements mentioned in the description of the peptide, the same content as that already mentioned is as described above.

[0054] The content of the peptide, which is an active ingredient included in the food composition, can be appropriately and non - restrictively selected according to the food form, desired use, etc. For example, it can be added at 0.01 to 15% by weight of the total weight of the food. For example, for a health drink composition, based on 100 ml, it can be added at a ratio of 0.02 to 10 g, preferably 0.3 to 1 g.

[0055]

Beneficial effects

[0056] Through the peptide according to one aspect, it is possible to promote the proliferation of fibroblasts and keratinocytes and enhance the synthesis of extracellular matrix - forming factors and skin barrier factors. Therefore, it can be applied to the improvement of skin conditions, including wrinkle improvement, skin elasticity improvement, wound recovery, skin barrier strengthening, or skin aging inhibition.

[0057] Through the peptide according to one aspect, it is possible to restore the reduced activity of fibroblasts and keratinocytes and enhance their antioxidant efficacy, thus contributing to the recovery of skin damage that may be caused by external environments such as ultraviolet rays.

[0058] Therefore, the peptide according to one aspect can be used as an active ingredient of a composition for improving skin conditions or an antioxidant composition.

Description of the drawings

[0059] Figure 1 Shows the results of confirming the cell proliferation level based on the change in cell viability after adding the peptide composed of the amino acid sequence of SEQ ID NO: 1 to NIH3T3 cells.

[0060] Figure 2 Shows the results of confirming the increased expression of extracellular matrix - forming factors, namely type I collagen (Col1a1), fibronectin, and elastin, after adding the peptide composed of the amino acid sequence of SEQ ID NO: 1 to NIH3T3 cells.

[0061] Figure 3 Shows the results of quantitatively evaluating the expression level of extracellular matrix - forming factors after adding the peptide composed of the amino acid sequence of SEQ ID NO: 1 to NIH3T3 cells, where Figure 3 A shows the results of confirming the expression level of Col1a1, Figure 3 B shows the results of confirming the expression level of fibronectin, Figure 3 C shows the results of confirming the expression level of elastin.

[0062] Figure 4 Shows the results of confirming the increased expression of skin barrier factors, namely silent information regulator 1 (SIRT - 1) and aquaporin 3 (AQP3), after adding the peptide composed of the amino acid sequence of SEQ ID NO: 1 to HaCaT cells.

[0063] Figure 5 It shows the results of quantitatively evaluating the expression levels of skin barrier factors after adding the peptide composed of the amino acid sequence of SEQ ID NO: 1 to HaCaT cells, where, Figure 5 Panel A shows the results of confirming the expression level of SIRT-1, Figure 5 Panel B shows the results of confirming the expression level of AQP3.

[0064] Figure 6 It shows the results of confirming the inhibitory effect on skin cell apoptosis induced by ultraviolet irradiation through viability assessment after adding the peptide composed of the amino acid sequence of SEQ ID NO: 1 to NIH3T3 cells.

[0065] Figure 7 It shows the results of confirming the inhibitory effect on skin cell apoptosis induced by ultraviolet irradiation through viability assessment after adding the peptide composed of the amino acid sequence of SEQ ID NO: 1 to HaCaT cells.

[0066] Figure 8 It shows the results of quantitatively confirming the changes in the level of reactive oxygen species increased by ultraviolet rays after adding the peptide composed of the amino acid sequence of SEQ ID NO: 1 to NIH3T3 cells.

[0067] Figure 9 It shows the results of quantitatively confirming the changes in the level of reactive oxygen species increased by ultraviolet rays after adding the peptide composed of the amino acid sequence of SEQ ID NO: 1 to HaCaT cells.

DETAILED DESCRIPTION

[0068] Hereinafter, the present invention will be described in more detail by way of examples. However, these examples are only used to exemplarily describe the present invention, and the scope of the present invention is not limited to these examples.

[0069]

Example 1: Synthesis of Peptide

[0070] A peptide having the amino acid sequence of SEQ ID NO: 1 described in Table 1 below was synthesized using an automatic peptide synthesizer (Milligen 9050, Millipore, USA), and the synthesized peptide was purified by C18 reversed-phase high performance liquid chromatography (HPLC) (Waters Associates, USA). ACQUITY UPLC BEH300 C18 (2.1 mm × 100 mm, 1.7 μm, Waters Co., USA) was used as the column.

[0071]

Table 1

[0072] SEQ ID NO: Sequence (N-terminal → C-terminal) 1 EVVDI

[0073]

Example 2: Confirmation of the effect of promoting the proliferation of skin cells

[0074] This example aims to confirm the effect of the peptide according to the example on the proliferation of skin cells by evaluating the change in the survival rate of mouse fibroblasts, i.e., NIH3T3 cells.

[0075] Specifically, NIH3T3 cells were seeded in a 96-well plate at a density of 1×10 4 cells / well and cultured for 24 hours. Then, the cells were washed once with serum-free DMEM media, and the peptide composed of the amino acid sequence of SEQ ID NO: 1 was aliquoted at 50 μM or 100 μM into 200 μL of the serum-free media, and then cultured in a CO2 incubator at 37 °C for 72 hours. Then, after washing the culture twice with PBS, MTT solution was aliquoted into each well at 0.5 mg / ml. Then, after shading, it was cultured in a CO2 incubator at 37 °C for 4 hours, and the absorbance at 540 nm was measured using a microplate reader (Molecular Devices, USA).

[0076] As a result, it was confirmed that, as Figure 1 shown, the proliferation of fibroblasts was promoted by the peptide composed of the amino acid sequence of SEQ ID NO: 1.

[0077]

Example 3: Confirmation of the effect of improving wrinkles and enhancing elasticity

[0078] This example aims to confirm the effect of the peptide according to an example on improving endogenous skin aging, including improving wrinkles and enhancing elasticity, by evaluating the expression levels of type I collagen (Col1a1), fibronectin, or elastin, which are known as dermal components.

[0079] Specifically, NIH3T3 cells were seeded at 3×10 5The cells were seeded at a density of cells / well in a 6-well plate and cultured for 24 hours. Then, after washing the cells once with serum-free DMEM media, the peptide consisting of the amino acid sequence of SEQ ID NO: 1 was dispensed at 10 μM or 50 μM into the 1 mL of the media, and it was cultured in a CO2 incubator at 37 °C for 24 hours. Then, after washing the culture twice with PBS, easy blue (iNtRON, Cat. No: 17061, Korea) was used to isolate ribonucleic acid (RNA) from the culture product. Then, the isolated RNA was reverse transcribed using an RT kit (Enzynomics, Cat. NO: RT200, Korea) to synthesize each complementary deoxyribonucleic acid (cDNA). Then, polymerase chain reaction (PCR) was performed using the synthesized 1 μg of cDNA and primers for Col1a1, fibronectin, or elastin and a PCR kit (enzynomics, Cat. No: P581T, Korea). In this example, the control group was the untreated group, and the group supplemented with transforming growth factor-β1 (TGF-β1) was used as the positive control group. The nucleotide sequences of the primers used in this example are shown in Table 2 below.

[0080]

Table 2

[0081]

[0082]

[0083] As a result, it was confirmed that, as shown in Figure 2 and 3 , due to the peptide consisting of the amino acid sequence of SEQ ID NO: 1, the expression of the extracellular matrix components, namely Col1a1, fibronectin, and elastin, increased.

[0084]

Example 4: Confirmation of Skin Barrier Strengthening Effect

[0085] This example aims to confirm the effect of the peptide according to an example on strengthening the skin barrier by evaluating the expression levels of sirtuin 1 (SIRT-1) or aquaporin 3 (AQP3).

[0086] Specifically, HaCaT cells were seeded at 3×10 5Cells were seeded at a density of [X] cells / well in a 6-well plate and cultured for 24 hours. Then, after washing the cells once with serum-free DMEM media, the peptide consisting of the amino acid sequence of SEQ ID NO: 1 was added to the 1 mL of media at 50 μM or 100 μM, and the cells were cultured in a CO2 incubator at 37°C for 24 hours. Then, after washing the culture twice with PBS, ribonucleic acid (RNA) was isolated from the culture product using easy blue (iNtRON, Cat. No: 17061, Korea). Then, reverse transcription was performed on the isolated RNA using an RT kit (Enzynomics, Cat. NO: RT200, Korea) to synthesize complementary deoxyribonucleic acid (cDNA). Then, polymerase chain reaction (PCR) was performed using 1 μg of the synthesized cDNA, primers for SIRT-1 or AQP3, and a PCR kit (enzynomics, Cat. No.: P581T, Korea). In this example, the control group was the untreated group, and the group supplemented with EGF was used as the positive control group. The nucleotide sequences of the primers used in this example are shown in Table 3 below.

[0087]

Table 3

[0088]

[0089] As a result, it was confirmed that, as shown in Figure 4 and Figure 5 , the expression of SIRT-1 and AQP3, which are skin barrier factors, increased due to the peptide consisting of the amino acid sequence of SEQ ID NO: 1. From the above results, it can be seen that the peptide according to an embodiment helps to strengthen the skin barrier and anti-aging of the skin by increasing skin barrier factors.

[0090]

Example 5: Confirmation of the inhibitory effect on ultraviolet-induced skin cell apoptosis

[0091] This example aims to confirm the inhibitory effect or cell recovery effect of the peptide according to an embodiment on ultraviolet-induced skin cell apoptosis by evaluating the change in the survival rate of skin cells induced to apoptosis by ultraviolet irradiation.

[0092] Specifically, NIH3T3 cells or HaCaT cells were seeded at a density of 1×10 4The cells were seeded at a density of cells / well in a 96-well plate and cultured for 24 hours. Then, the cells were washed once with serum-free DMEM media, and the peptide consisting of the amino acid sequence of SEQ ID NO: 1 was dispensed into the 200 μL of the media at 50 μM or 100 μM.

[0093] The media containing the dispensed peptide was cultured in a CO2 incubator at 37 °C for 1 hour, and then the media was transferred to an e-tube and mixed with 100 μl of PBS and dispensed into the well plate. Then, NIH3T3 cells were irradiated with ultraviolet light at 6 J / cm 2 using a UV irradiator (VILBER LOURMAT, France), and HaCaT cells were irradiated with ultraviolet light at 15 mJ / cm 2 . Then, the PBS in the well plate was removed, and an MTT (Sigma, Cat. No.: M2003, USA) solution was dispensed into each well at 0.5 mg / ml and cultured in a CO2 incubator at 37 °C for 72 hours. Then, the absorbance of the culture at 540 nm was measured using a microplatereader (Molecular Devices, USA). In this example, the control group was the untreated group, and the group supplemented with Trolox (Trolox) was used as the positive control group.

[0094] As a result, it was confirmed that Figure 6 and 7 as shown, the cell viability of fibroblasts and keratinocytes reduced due to ultraviolet irradiation was restored by the peptide consisting of the amino acid sequence of SEQ ID NO: 1. From the above results, it can be seen that the peptide according to one embodiment contributes to the inhibition of apoptosis of skin cells induced by ultraviolet light.

[0095]

Example 6: Confirmation of the effect of reducing reactive oxygen species increased by ultraviolet light

[0096] This example aims to confirm the antioxidant effect of the peptide according to one embodiment on damaged skin cells by evaluating the change in the level of reactive oxygen species in skin cells increased due to ultraviolet irradiation.

[0097] Specifically, NIH3T3 cells or HaCaT cells were seeded at 5×10 5The cells were seeded at a density of cells / well in a 6-well plate and then cultured for 24 hours. Then, after washing the cells once with serum-free DMEM media, the peptide consisting of the amino acid sequence of SEQ ID NO: 1 was dispensed into the 1 mL of the media at 50 μM or 100 μM, and it was cultured in a CO2 incubator at 37 °C for 1 hour. Then, the culture was transferred to an e-tube and mixed with 1 mL of PBS and dispensed into a well plate. Then, NIH3T3 cells were irradiated with ultraviolet light of 6 J / cm 2 and HaCaT cells were irradiated with ultraviolet light of 15 mJ / cm 2 . Then, the PBS in the well plate was removed, 900 μL of the media containing the dispensed peptide was added, and it was cultured in a CO2 incubator at 37 °C for 24 hours. Then, the culture product was treated with 10 μM of 2′,7′-dichlorofluorescin diacetate (DCFG-DA), the culture product was wrapped with foil, and the culture product was cultured in a CO2 incubator at 37 °C for 30 minutes. Then, the culture product was washed twice with PBS, treated with 500 μL of 1× trypsin / ethylenediaminetetraacetic acid (EDTA) to obtain cells, and then centrifuged. The centrifuged cells were washed with PBS, and the FL1 fluorescence value was measured using a flow cytometer (FACS, BD, USA). In this example, the control group was the untreated group, the comparison group was the group irradiated only with ultraviolet light, and the group supplemented with Trolox (Trolox) was used as the positive control group.

[0098] As a result, it was confirmed that, as shown in Figure 8 and 9 , the peptide consisting of the amino acid sequence of SEQ ID NO: 1 reduced the level of reactive oxygen species in fibroblasts and keratinocytes increased by ultraviolet irradiation. From the above results, it can be seen that the peptide according to one example contributes to the antioxidant effect of skin cells induced by ultraviolet light.

[0099]

Formulation Example 1: Preparation of Peptide Nanobody

[0100] 50 mg of the peptide of Example 1 was dissolved by sufficiently stirring with 500 ml of distilled water. After mixing the complex solution with 5 g of lecithin, 0.3 ml of sodium oleate, 50 ml of ethanol, and a small amount of oil phase, the volume was adjusted with distilled water until the total volume was 1 L, and then emulsified under high pressure using a high-pressure microfluidizer to prepare peptide nanobodies with a size of about 100 nm.

[0101]

Formulation Example 2: Soft Lotion

[0102] A soft lotion containing the peptide according to an embodiment and having the following composition was prepared by a method well known in the art.

[0103]

Table 4

[0104]

[0105]

[0106]

Formulation Example 3: Nutritional Cream

[0107] A nutritional cream containing the peptide according to an embodiment and having the following composition was prepared by a method well known in the art.

[0108]

Table 5

[0109] Ingredient Content (wt%) Peptide 2.5 Limnanthes alba seed oil 3 Cetearyl alcohol 1.5 Stearic acid 1.5 Glyceryl stearate 1.5 Liquid paraffin 10 Beeswax 2 Polysorbate 60 0.6 Sorbitan sesquioleate 2.5 Squalene 3 1,3-Butanediol 3 Glycerol 5 Triethanolamine 0.5 Tocopheryl acetate 0.5 Preservative, pigment Appropriate amount Fragrance Appropriate amount Purified water Balance Total 100

[0110]

Formulation Example 4: Nutritional Lotion

[0111] A nutritional lotion containing the peptide according to an embodiment and having the following composition was prepared by a method well known in the art.

[0112]

Table 6

[0113] Ingredient Content (wt%) Peptide 3.0 1,3-Butanediol 4 Glycerol 4 Cetearyl alcohol 0.8 Glyceryl stearate 1 Triethanolamine 0.13 Tocopheryl acetate 0.3 Liquid paraffin 5 Squalene 3 Oil 2 Polysorbate 60 1.5 Sorbitan sesquioleate 0.5 Carboxyvinyl polymer 1 Preservative, pigment Appropriate amount Fragrance Appropriate amount Purified water Balance Total 100

[0114]

Formulation Example 5: Serum

[0115] A serum containing the peptide according to an embodiment and having the following composition was prepared by a method well known in the art.

[0116]

Table 7

[0117]

[0118]

[0119] The description of the present invention is only for providing examples, and those of ordinary skill in the art should be able to understand that the present invention can be easily modified into other specific forms without changing the technical idea or essential features of the present invention. Therefore, the embodiments described above should be understood as exemplary in all aspects and not restrictive.

Claims

1. A peptide consisting of the amino acid sequence of SEQ ID NO:

1.

2. The peptide according to claim 1, wherein, The N-terminus of the peptide is bound to any one of protecting groups selected from the group consisting of acetyl, fluorenylmethyloxycarbonyl, formyl, palmitoyl, myristoyl, stearoyl, butoxycarbonyl, allyloxycarbonyl, and polyethylene glycol.

3. The peptide according to claim 1, wherein, The C-terminus of the peptide is bound to any one of protecting groups selected from the group consisting of amino, tertiary alkyl, and hydrazino.

4. The peptide according to claim 1, wherein The peptide exhibits at least any one of the following properties: (a) Promoting the proliferation of fibroblasts or keratinocytes; (b) Enhancing the expression of type I collagen, fibronectin, or elastin, which are extracellular matrix components; (c) Enhancing the expression of sirtuin-1 or aquaporin-3, which are skin barrier factors; and (d) Inhibiting the apoptosis of fibroblasts or keratinocytes and reducing the level of reactive oxygen species.

5. A cosmetic composition for improving skin condition, comprising the peptide according to any one of claims 1 to 4 as an active ingredient.

6. The cosmetic composition according to claim 5, wherein, The peptide is prepared in the form of a nanobody.

7. The cosmetic composition according to claim 5, wherein, The improvement of skin condition is improvement of wrinkles, improvement of skin elasticity, wound healing, strengthening of skin barrier, or inhibition of skin aging.

8. The cosmetic composition according to claim 7, wherein, The skin aging is skin aging caused by ultraviolet rays.

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