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] The 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 the fibroblasts of the skin. Collagen forms most of the organic substances in the skin, tendon, bone, and teeth, especially in high amounts 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] Pigments affecting skin color include melanin, pheomelanin, carotene, hemoglobin, and carotenoids, and the various colors of the skin, hair, and eyes depend on these pigments. Among them, the most important pigment determining skin color is melanin. Specifically, skin color depends on the amount and distribution of melanin. Melanin is produced by cells called melanocytes under the epidermis and moves to the keratin surface and exfoliates due to skin metabolism. Regardless of skin color, the number of melanocytes is almost the same, and only the production, type, and distribution of melanin result in different skin colors.

[0005] In this technical background, currently, various studies are being conducted to improve skin conditions through mechanisms such as promoting collagen biosynthesis, proliferating fibroblasts, etc. and enhancing activity (Korean Registered Patent No. 10-1813629), but they are still not perfect.

Summary of the Invention

[0006]

Technical Problem

[0007] On the one hand, a peptide is provided, which consists of the amino acid sequence of SEQ ID NO: 1.

[0008] 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.

[0009] On the other hand, a pharmaceutical composition for preventing or treating hyperpigmentation diseases is provided, which comprises a peptide consisting of the amino acid sequence of SEQ ID NO: 1 as an active ingredient.

[0010] Other objects and advantages of the present application will become more apparent in conjunction with the appended claims, 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 similar technical fields, the relevant description thereof is omitted.

[0011]

Technical Solution

[0012] 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. And the scope of the present application cannot be determined by the specific descriptions recorded hereinafter.

[0013] On the one hand, a peptide is provided, which consists of the amino acid sequence of SEQ ID NO: 1.

[0014] The term "peptide" used in this specification may refer to a linear molecule formed by multiple amino acid residues bonded 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 present inventors have identified a peptide consisting of the amino acid sequence of SEQ ID NO: 1. Among them, the biologically effective activity may refer to at least any one selected from the following characteristics: (a) promoting the expression of type I collagen (Col1a1), fibronectin, elastin, or hyaluronic acid, which are extracellular matrix components; (b) promoting the expression of silent information regulator-1 (SIRT-1) or aquaporin-3 (AQP3), which are skin barrier factors; (c) reducing the level of reactive oxygen species; (d) reducing the expression of matrix metalloproteinase-1 (MMP-1); and (e) inhibiting melanogenesis. Therefore, the peptide can be used for improving skin condition, skin whitening, or antioxidant.

[0015] 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 one of the protecting groups 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 one of the protecting groups 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.

[0016] The peptide is artificially synthesized, or non-naturally occurring or engineered. The "non-naturally occurring or engineered" refers to a state generated by artificial modification, rather than the state of the existence itself produced in a 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.

[0017] The term "stability" used in this specification may refer not only to the in vivo stability that protects the peptide from the attack of proteolytic enzymes in the body, but also to storage stability (e.g., stability at room temperature).

[0018] 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.

[0019] Among the terms or elements mentioned in the description of the peptide, the same content as that already mentioned is as described above.

[0020] The term "improvement" as used in this specification may refer to all actions that at least alleviate parameters related to the alleviation or treatment of a condition, such as the degree of symptoms.

[0021] The term "improvement of skin condition" as used in this specification may refer to the process of treating, alleviating, or moderating skin damage caused by endogenous or exogenous factors of the skin, or the effects thereof. For example, it may be interpreted as improvement of wrinkles, improvement of skin elasticity, wound recovery, strengthening of the skin barrier, or inhibition of skin aging, but is not limited thereto.

[0022] Herein, "improvement of wrinkles", "improvement of skin elasticity", and "wound recovery" may refer to all actions that increase the total amount of extracellular matrix factors, including promotion of collagen synthesis, etc. And, "strengthening of the skin barrier" may refer to strengthening the original function of the skin, that is, preventing the leakage of moisture and nutrients in the skin to the outside and preventing the invasion of harmful substances such as bacteria or viruses into the skin. And, "inhibition of skin aging" may refer to inhibition of the reduction of skin functions such as wrinkles, skin sagging, and loss of elasticity. At this time, the skin aging may be photoaging, for example, skin aging caused by ultraviolet rays.

[0023] Herein, "skin whitening" can be interpreted not only as brightening the skin tone by hindering melanin synthesis, but also as improving hyperpigmentation of the skin caused by ultraviolet rays, hormones, or genetics. Herein, the hyperpigmentation of the skin may include freckles, dark spots, senile lentigines, brown spots, or age spots, etc., but is not limited thereto.

[0024] 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 in the body in a short time because of their short half-life. In contrast, according to an 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 topically applied to the skin, it can effectively improve the skin condition.

[0025] According to an embodiment, it is possible to promote the synthesis of extracellular matrix components and skin barrier factors. And, the peptide can not only restore the blocked activities of fibroblasts and keratinocytes and enhance their antioxidant efficacy, but also significantly inhibit melanogenesis. Therefore, the peptide can be used as an active ingredient of a cosmetic composition for improving skin condition.

[0026] The cosmetic composition may include a cosmetically effective amount of the peptide; and / or a cosmetically acceptable carrier, but is not limited thereto.

[0027] As used herein, the term "cosmetically effective amount" means an amount sufficient to achieve the efficacy of improving the skin condition of the cosmetic composition.

[0028] The weight ratio between the peptide and the cosmetically acceptable carrier may be, for example, from 500:1 to 1:500. For example, the weight ratio may 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.

[0029] The cosmetic composition can be prepared into any dosage form commonly prepared in the art. For example, it can be prepared into 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 into a soft lotion, nourishing lotion, nourishing cream, massage cream, essence, eye cream, cleansing cream, cleansing foam, cleansing water, facial mask, spray or powder dosage form.

[0030] When the dosage form of the cosmetic composition is dough, skin cream or gel, animal oil, vegetable oil, wax, paraffin, starch, tragacanth, cellulose derivative, polyethylene glycol, silicon, bentonite, silica, talc or zinc oxide, etc. can be used as carrier components.

[0031] When 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.

[0032] When 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.

[0033] In the case where the dosage form of the cosmetic composition is a suspension, liquid phase diluents such as water, ethanol or propylene glycol, suspending agents such as ethoxylated isostearyl alcohol, polyoxyethylene sorbitan esters and polyoxyethylene sorbitan esters, microcrystalline cellulose, aluminum hydroxide methyl, bentonite, agar or tragacanth can be used.

[0034] 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 glycerides, fatty acid diethanolamides, vegetable oils, lanolin derivatives or ethoxylated glycerol fatty acid esters can be used as carrier components.

[0035] 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.

[0036] In addition to the peptide and the carrier component as the active ingredients, the components included in the cosmetic composition include conventional components for cosmetic compositions, for example, conventional adjuvants such as antioxidants, stabilizers, solubilizers, vitamins, pigments and fragrances can be included.

[0037] 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.

[0038] On the other hand, a method for improving the 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; the use of the peptide consisting of the amino acid peptide of SEQ ID NO: 1 in the preparation of a composition for improving the skin condition.

[0039] Among the terms or elements mentioned in the description of the cosmetic composition, the same content as that already mentioned is as described above.

[0040] As used in this specification, the term "individual" refers to an object in need of improving the skin condition, and more specifically, refers to mammals such as humans or non-human primates, mice, dogs, cats, horses, and cows.

[0041] As used in this specification, the terms "apply", "administer", and "coat" can be used interchangeably and can refer to localizing at least part of the composition according to an embodiment on a desired site, or disposing the composition according to an embodiment within an individual through an administration route.

[0042] On the other hand, there is provided a pharmaceutical composition for preventing or treating hyperpigmentation diseases, which comprises a peptide consisting of the amino acid sequence of SEQ ID NO: 1 as an active ingredient.

[0043] Among the terms or elements mentioned in the description of the peptide, the same content as that already mentioned is as described above.

[0044] In this specification, the term "prevention" refers to any act of inhibiting or delaying the occurrence of a disease by administering the composition.

[0045] In this specification, the term "treatment" refers to any form of treatment that provides an effect to an individual suffering from or at risk of suffering from a disease, and the effects include improving the condition of the individual (e.g., at least one symptom), delaying the progression of the disease, delaying the onset of symptoms, or slowing down the progression of symptoms, etc. Therefore, the "treatment" and "prevention" do not mean the cure or complete elimination of symptoms.

[0046] The "hyperpigmentation disease", that is, the disease to be prevented or treated with the pharmaceutical composition, can be a general term for diseases in which melanin is overproduced and deposited in the skin, such as melasma, freckles, senile lentigines, or solar lentigines, but is not limited thereto.

[0047] The pharmaceutical composition may include a pharmaceutically effective amount of the peptide; and / or a pharmaceutically acceptable carrier, but is not limited thereto.

[0048] As used in this specification, the term "pharmaceutically effective amount" may refer to an amount sufficient to achieve the preventive or therapeutic efficacy of the pharmaceutical composition against hyperpigmentation diseases.

[0049] The pharmaceutically acceptable carriers are those commonly used in preparation, including lactose, dextrose, sucrose, sorbitol, mannitol, starch, acacia, calcium phosphate, alginate, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, methylcellulose, methylparaben, propylparaben, talc, magnesium stearate, and mineral oil, etc., but not limited thereto. Suitable pharmaceutically acceptable carriers and formulations are described in detail in Remington's Pharmaceutical Sciences (19th ed., 1995).

[0050] The weight ratio between the peptide and the 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 not limited thereto.

[0051] In addition to the above components, the pharmaceutical composition may further include lubricants, wetting agents, sweeteners, flavoring agents, emulsifying agents, suspending agents, preservatives, etc., but not limited thereto.

[0052] 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 not limited thereto.

[0053] 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 not limited thereto, and can be formulated differently according to factors such as the formulation method, administration method, patient's age, weight, gender, disease condition, diet, administration time, administration route, excretion rate, and reaction sensitivity.

[0054] The pharmaceutical composition can be formulated into unit volume form by using pharmaceutically acceptable carriers and / or excipients according to methods easily implemented by those of ordinary skill in the technical field to which the present invention pertains, or can be prepared by injecting into multi-volume containers.

[0055] The dosage form may be in the form of a solution, suspension or emulsion in an oily or aqueous solvent, may also be in the form of an extractant, powder, granule, lozenge or capsule, and may further include a powder and / or a stabilizer.

[0056] On the other hand, a method for preventing and treating hyperpigmentation diseases is provided, which includes the step of administering a therapeutically effective amount of the pharmaceutical composition to an individual, wherein the pharmaceutical composition includes a peptide consisting of the amino acid sequence of SEQ ID NO: 1 as an active ingredient.

[0057] Among the terms or elements mentioned in the description of the pharmaceutical composition, the same content as that already mentioned is as described above.

[0058] On the other hand, an antioxidant composition is provided, which includes a peptide consisting of the amino acid sequence of SEQ ID NO: 1 as an active ingredient.

[0059] Among the terms or elements mentioned in the description of the peptide, the same content as that already mentioned is as described above.

[0060] The antioxidant composition may be in the form of a pharmaceutical composition, a pharmaceutical external product composition, or a cosmetic composition. As an example, the composition may be used as a cosmetic composition for improving skin condition, or a pharmaceutical composition for improving or treating skin damage-related disease states.

[0061] According to one 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 antioxidant composition.

[0062] 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. Among the terms or elements mentioned in the description of the pharmaceutical composition, the same content as that already mentioned is as described above.

[0063] 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.

[0064] Among the terms or elements mentioned in the description of the peptide, the same content as that already mentioned is as described above.

[0065] The content of the peptide, which is the 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.

[0066]

Beneficial effects

[0067] The peptide according to one aspect can be applied to improving skin conditions, including wrinkle improvement, skin elasticity improvement, wound recovery, skin barrier strengthening, skin aging inhibition, or skin whitening, etc., by promoting the synthesis of extracellular matrix - forming factors and skin barrier factors.

[0068] The peptide according to one aspect can help restore skin damage that may be caused by external environments such as ultraviolet rays by restoring the reduced activities of fibroblasts and keratinocytes and promoting the antioxidant effects of fibroblasts and keratinocytes.

[0069] Therefore, the peptide according to one aspect can be used as an active ingredient of a composition for improving skin conditions or a pharmaceutical composition for preventing or treating hyperpigmentation diseases.

Description of the drawings

[0070] Figure 1 Shows the results of confirming an increase in the expression of extracellular matrix - forming factors, namely type I collagen (Col1a1), fibronectin, and elastin, after adding the peptide consisting of the amino acid sequence of SEQ ID NO: 1 to NIH3T3 cells.

[0071] Figure 2 Shows the results of quantitatively evaluating the expression levels of extracellular matrix - forming factors after adding the peptide consisting of the amino acid sequence of SEQ ID NO: 1 to NIH3T3 cells, where Figure 2 A of shows the results of confirming the expression level of Col1a1, Figure 2 B of shows the results of confirming the expression level of fibronectin, Figure 2 C of shows the results of confirming the expression level of elastin.

[0072] Figure 3 Shows the results of quantitatively evaluating the expression level of procollagen type I alpha after adding the peptide consisting of the amino acid sequence of SEQ ID NO: 1 to NIH3T3 cells.

[0073] Figure 4 Shows the results of quantitatively evaluating the expression level of hyaluronic acid after adding the peptide consisting of the amino acid sequence of SEQ ID NO: 1 to HaCaT cells.

[0074] Figure 5 Shows the results of confirming the expression levels of extracellular matrix components, namely fibronectin and elastin, after adding the peptide composed of the amino acid sequence of SEQ ID NO: 1 and the control peptide to NIH3T3 cells.

[0075] Figure 6 Shows the quantitative evaluation of the expression levels of extracellular matrix components after adding the peptide composed of the amino acid sequence of SEQ ID NO: 1 and the control peptide to NIH3T3 cells. Figure 6 Panel A shows the results of confirming the expression level of fibronectin. Figure 6 Panel B shows the results of confirming the expression level of elastin.

[0076] Figure 7 Shows the results of confirming the increased expression of skin barrier factors, namely SIRT-1 and AQP3, after adding the peptide composed of the amino acid sequence of SEQ ID NO: 1 to HaCaT cells.

[0077] Figure 8 Shows the quantitative evaluation of 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 8 Panel A shows the results of confirming the expression level of SIRT-1. Figure 8 Panel B shows the results of confirming the expression level of AQP3.

[0078] Figure 9 Shows the results of quantitatively evaluating the changes in the level of reactive oxygen species increased by ultraviolet light after adding the peptide composed of the amino acid sequence of SEQ ID NO: 1 to NIH3T3 cells.

[0079] Figure 10 Shows the results of quantitatively evaluating the changes in the level of reactive oxygen species increased by ultraviolet light after adding the peptide composed of the amino acid sequence of SEQ ID NO: 1 to HaCaT cells.

[0080] Figure 11 Shows the results of quantitatively evaluating the changes in the level of reactive oxygen species increased by ultraviolet light after adding the peptide composed of the amino acid sequence of SEQ ID NO: 1 and the control peptide to HaCaT cells.

[0081] Figure 12 Shows the results of confirming the changes in the expression levels of SIRT-1 and AQP3 decreased by ultraviolet light after adding the peptide composed of the amino acid sequence of SEQ ID NO: 1 to HaCaT cells.

[0082] Figure 13It shows the quantitative confirmation of the changes in the expression levels of SIRT-1 and AQP3 reduced by ultraviolet rays after adding the peptide composed of the amino acid sequence of SEQ ID NO: 1 to HaCaT cells. Figure 13 A of Figure 13 shows the result of confirming the expression level of SIRT-1. Figure 13 B of Figure 13 shows the result of confirming the expression level of AQP3.

[0083] Figure 14 It shows the result of confirming the change in the expression level of MMP-1 increased by ultraviolet rays after adding the peptide composed of the amino acid sequence of SEQ ID NO: 1 to NIH3T3 cells.

[0084] Figure 15 It shows the result of confirming the change in the expression level of MMP-1 increased by ultraviolet rays after adding the peptide composed of the amino acid sequence of SEQ ID NO: 1 to HaCaT cells.

[0085] Figure 16 It shows the result of quantitatively confirming the change in the melanogenesis level after adding the peptide composed of the amino acid sequence of SEQ ID NO: 1 and the comparative group peptide to B16F10 cells.

Detailed Description of the Invention

[0086] 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.

[0087]

Example 1, Synthesis of Peptide

[0088] 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 (HighPerformanceLiquid 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.

[0089]

Table 1

[0090] SEQ ID NO. Sequence (N-terminal → C-terminal) 1 GPRGDP

[0091]

Example 2, Confirmation of Wrinkles Improvement and Elasticity Enhancement Effects

[0092] 【2-1, Evaluation of the Efficacy of the Peptide According to an Embodiment】

[0093] This example aims to confirm the effect of a peptide according to an example on improving endogenous skin aging, including wrinkle improvement and elasticity enhancement, through the expression levels of type I collagen (Col1a1), fibronectin, elastin, and hyaluronic acid, which are known as dermal components.

[0094] Specifically, NIH3T3 cells were seeded in a 6-well plate at a density of 3×10 5 cells / well and cultured for 24 hours. Then, after washing the cells once with serum-free DMEM media, the peptide composed of the amino acid sequence of SEQ ID NO: 1 was aliquoted into the 1 mL of the media at 10 μM, 50 μM, or 100 μM, and it was 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 using easy blue (iNtRON, Cat. No: 17061, Korea). Then, reverse transcription of the isolated RNA was performed 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.

[0095]

Table 2

[0096]

[0097] As a result, it was confirmed that, as shown in Figure 1 and Figure 2 , the expression of extracellular matrix components, namely Col1a1, fibronectin, and elastin, increased with the peptide composed of the amino acid sequence of SEQ ID NO: 1.

[0098] Furthermore, to evaluate the expression levels of procollagen type I alpha and hyaluronic acid at the protein level, NIH3T3 cells or HaCaT cells were seeded at 1×10 4After inoculating the cells at a density of cells / well into a 24-well plate, they were cultured for 24 hours. Then, the cells were washed once with serum-free DMEM media. The peptide consisting of the amino acid sequence of SEQ ID NO: 1 was dispensed at 10 μM, 50 μM, or 100 μM into the 2 ml of the media, and they were cultured in a CO2 incubator at 37 °C for 72 hours. Then, the culture was centrifuged to precipitate the suspension, and the supernatant was transferred to an e-tube. Then, Collagen ELISA (abcam, Cat No.: ab210579, UK) and HA ELISA (ECHELON bioscience, Cat No.: K-1200, USA) were performed on the centrifuged culture. In this example, the control group was the untreated group, and the groups supplemented with TGF-β1 or EGF were used as the positive control groups.

[0099] As a result, it was confirmed that, as Figure 3 and Figure 4 shown, the expression of procollagen α of type I of collagen precursors and hyaluronic acid related to maintaining skin elasticity and skin moisture was increased at the protein level 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 example contributes to improving endogenous skin aging including wrinkle improvement and elasticity enhancement by increasing extracellular matrix components.

[0100] 【2-2. Evaluation of the efficacy between the peptide according to one example and the comparison group】

[0101] This example aims to make a more specific comparison of the effect of the peptide according to one example on improving endogenous skin aging. For this purpose, peptides consisting of the amino acid sequences of SEQ ID NO: 2, 3, or 4, which have sequences similar to the peptide according to one example, were set as the comparison groups, and the expression levels of fibronectin and elastin were evaluated and compared in the same manner as in Example 2-1. In this example, the control group was the untreated group, the groups supplemented with the peptides consisting of the amino acid sequences of SEQ ID NO: 2, 3, or 4 (50 μM, 100 μM) were used as the comparison groups, and the groups supplemented with TGF-β1 or EGF were used as the positive control groups. The nucleotide sequences of the primers used in this example are shown in Table 2 as described above.

[0102] As a result, as Figure 5 and Figure 6 shown, the expression of extracellular matrix components, namely fibronectin and elastin, was increased by the peptide consisting of the amino acid sequence of SEQ ID NO: 1, and such an effect was superior to the expression levels of the comparison groups consisting of sequences similar to the peptide according to one example.

[0103]

Example 3. Confirmation of Skin Barrier Strengthening Effect

[0104] This example aims to confirm the effect of the peptide according to an embodiment on skin barrier strengthening by evaluating the expression levels of sirtuin 1 (SIRT-1) or aquaporin 3 (AQP3).

[0105] Specifically, HaCaT cells were seeded in a 6-well plate at a density of 3×10 5 cells / well 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 the medium at 10 μM, 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 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 each complementary deoxyribonucleic acid (cDNA). Then, polymerase chain reaction (PCR) was performed using 1 μg of the synthesized cDNA, primers for SIRT1-1 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.

[0106]

Table 3

[0107]

[0108]

[0109]

Example 4. Confirmation of the Effect of Reducing Reactive Oxygen Species Increased by Ultraviolet Rays

[0110] 【4-1. Evaluation of the Efficacy of the Peptide According to an Embodiment】

[0111] This example aims to confirm the effect of the peptide according to an embodiment on the antioxidant effect in damaged skin cells by evaluating the change in the level of reactive oxygen species in skin cells increased by ultraviolet irradiation.

[0112] Specifically, NIH3T3 cells or HaCaT cells were seeded at 5×105 The cells 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 the medium at 10 μM, 50 μM, or 100 μM, and the mixture was cultured in a CO2 incubator at 37°C for 1 hour. Then, the culture was transferred to an e-tube, mixed with 1 mL of PBS, and aliquoted into a well plate. Then, using a UV irradiator (VILBER LOURMAT, Cat NO.: 3102-BSU), NIH3T3 cells were irradiated with UV at 6 J / cm 2 and HaCaT cells were irradiated with UV at 15 mJ / cm 2 . Then, the PBS in the well plate was removed, 900 μL of the medium containing the aliquoted peptide was added, and the cells were cultured in a CO2 incubator at 37°C for 24 hours. Then, the culture was treated with DCFG-DA (2',7'-dichlorofluorescin diacetate), wrapped with foil, and cultured in a CO2 incubator at 37°C for 30 minutes. Then, the cultured product was washed twice with PBS, and 500 μL of 1× trypsin / EDTA was used to obtain the cells, which were then centrifuged. The centrifuged cells were washed with PBS, and the FL1 fluorescence value was measured using flow cytometry (FACS, BD, USA). In this example, the control group was the untreated group, the negative control group was the group irradiated only with UV, and the group treated with NaCl after UV irradiation was used as the positive control group.

[0113] As a result, it was confirmed that, as shown in Figure 9 and Figure 10 , the levels of reactive oxygen species in fibroblasts and keratinocytes increased by UV irradiation were reduced by the peptide consisting of the amino acid sequence of SEQ ID NO: 1. From these results, it can be seen that the peptide according to an embodiment contributes to the antioxidant effect of skin cells induced by UV.

[0114] 【4-2. Comparison of the efficacy between the peptide according to an embodiment and the control group】

[0115] Please note that the value in "[X]" in the translation of line ID=1 needs to be filled in according to the actual density value in the original text which is not provided completely in the given content. Also, the specific values in "6 J / cm" and "15 mJ / cm" should be adjusted according to the correct original values if there are any specific unit notations or missing parts in the original text.This example aims to make a more specific comparison of the antioxidant effect of a peptide on damaged skin cells according to an embodiment. To this end, peptides consisting of the amino acid sequences of SEQ ID NO: 2, 3, or 4, which have sequences similar to the peptide according to an embodiment, were set as the comparison group, and the intracellular reactive oxygen species level in skin cells increased by ultraviolet irradiation was evaluated and compared for HaCaT cells in the same manner as in Example 4-1. In this example, the control group was the untreated group, the negative control group was the group irradiated only with ultraviolet rays, the comparison group was the group to which the peptide consisting of the amino acid sequences of SEQ ID NO: 2, 3, or 4 (50 μM, 100 μM) was added after ultraviolet irradiation, and the positive control group was the group to which Trolox was added after ultraviolet irradiation.

[0116] As a result, as Figure 11 shown, the intracellular reactive oxygen species level in keratinocytes increased by ultraviolet irradiation decreased with the peptide consisting of the amino acid sequence of SEQ ID NO: 1, and such an effect was superior to the expression level of the comparison group consisting of a sequence similar to the peptide according to an embodiment.

[0117]

Example 5. Confirmation of the recovery effect of skin barrier factors reduced by ultraviolet rays

[0118] This example aims to confirm the effect of the peptide according to an embodiment on the recovery of skin barrier factors in damaged skin cells by evaluating the level of skin barrier factors in skin cells reduced by ultraviolet irradiation.

[0119] Specifically, HaCaT cells were seeded in a 6-well plate at a density of 5×10 5 cells / well 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 into the 1 mL of the medium at 10 μM, 50 μM, or 100 μM, and the cells were cultured in a CO2 incubator at 37 °C for 1 hour. Then, the culture was transferred to an e-tube, mixed with 1 mL of PBS, and dispensed into a well plate. Then, the HaCaT cells were irradiated with 15 mJ / cm 2Ultraviolet rays. Then, after removing the PBS in the orifice plate, 900 μL of the medium in which the peptide has been subcultured is added, and the cells are cultured in a CO2 incubator at 37 °C for 8 hours. Then, after washing the culture twice with PBS, easyblue (iNtRON, Cat. No: 17061, Korea) is used to isolate ribonucleic acid (RNA) from the culture. Then, reverse transcription is performed on the isolated RNA using an RT kit (Enzynomics, Cat. NO: RT200, Korea) to synthesize each complementary deoxyribonucleic acid (cDNA). Then, polymerase chain reaction (PCR) is performed using 1 μg of the synthesized cDNA, primers for SIRT1-1 or AQP3, and a PCR kit (enzynomics, Cat. No.: P581T, Korea). In this example, the control group is the untreated group, the comparison group is the group irradiated only with ultraviolet rays, and the group added with NaC is used as the positive control group. The nucleotide sequences of the primers used in this example are shown in Table 3 as described above.

[0120] As a result, it was confirmed that Figure 12 and Figure 13 as shown, the expression of SIRT-1 and AQP3 reduced by ultraviolet irradiation increased or recovered due to the peptide composed 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 contributes to skin barrier strengthening and skin anti-aging by increasing skin barrier factors in damaged skin cells.

[0121]

Example 6. Confirmation of the inhibitory effect on matrix metalloproteinases increased by ultraviolet rays

[0122] It has been reported that matrix metalloproteinase (MMP-1, matrix metalloproteinase-1) forms skin wrinkles and causes a decrease in elasticity by promoting collagen cleavage and elastin reduction in the dermis. Therefore, this example aims to confirm the effect of the peptide according to an embodiment on improving extrinsic skin aging, including wrinkle improvement and elasticity enhancement, by evaluating the change in the expression level of MMP-1 increased by ultraviolet irradiation.

[0123] Specifically, NIH3T3 cells or HaCaT cells are seeded at 5×10 5The cells were seeded at a density of [number of cells per 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 10 μM, 50 μM, or 100 μM, and the mixture was cultured in a CO2 incubator at 37°C for 1 hour. Then, the culture was transferred to an e-tube, mixed with 1 mL of PBS, and aliquoted into a well plate. Then, a UV irradiator (VILBER LOURMAT, Cat NO.: 3102-BSU, France) was used to irradiate the NIH3T3 cells with 6 J / cm 2 of ultraviolet light and the HaCaT cells with 20 mJ / cm 2 of ultraviolet light. Then, the PBS in the well plate was removed, 900 μL of the media containing the aliquoted peptide was added, and the mixture was cultured in a CO2 incubator at 37°C for 24 hours. Then, the culture was transferred to an e-tube, washed twice with PBS, and lysed by adding lysis buffer. Then, the lysed culture was treated with 5X sample buffer to prepare the sample, the proteins were separated by SDS-PAGE, and transferred to a PVDF membrane. Then, the PVDF membrane was blocked with 5% skim milk at room temperature for 1 hour, and the anti-MMP-1 antibody (Abcam, Cat.No.: ab137332, UK) was diluted 1:500 in 5% skim milk and reacted with the membrane for 2 hours. Then, after washing with 0.1% PBS-T (0.1% Tween-20 in PBS) for 10 minutes, 3 times, Goat anti-rabbit IgG (Jackson Immume Research, Cat.NO.: 111-035-033, USA) was diluted 1:3000 in 5% skim milk and reacted with the membrane for 2 hours. Then, 15.ECL solution (GE Healthcare, Cat.No.: RPN2232, USA) was used to detect MMP-1. In this example, the control group was the untreated group, the comparison group was the group irradiated only with ultraviolet light, and the group added with NaC was used as the positive control group.

[0124] As a result, it was confirmed as Figure 14 and Figure 15As shown, the expression of MMP-1 increased by ultraviolet irradiation was decreased or restored by the peptide consisting of the amino acid sequence of SEQ ID NO: 1. From the above results, it was found that the peptide according to an embodiment contributes to the improvement of aging such as wrinkle formation and elasticity reduction caused by external factors through the level of collagen-degrading enzyme, i.e., MMP-1.

[0125]

Example 7. Confirmation of Inhibitory Effect on Melanin Production

[0126] Mouse melanoma cells, i.e., B16F10 cells, were inoculated into a 6-well plate at a density of 5×10 4 cells / well and cultured for 16 hours. Then, after replacing the medium with a medium containing 2% serum, 200 ng / ml of α-melanocyte-stimulating hormone (α-MSH) was added thereto to promote melanin production. Meanwhile, each peptide consisting of the amino acid sequence of SEQ ID NO: 1 was added at 50 μM or 100 μM, and the cells were cultured for 72 hours. Then, the cultured cells were lysed with 1 N NaOH, and the absorbance at 450 nm was measured. The untreated group (Con) was used as a control group, the group with only α-MSH added was used as a negative control group, the groups with the peptide consisting of the amino acid sequence of SEQ ID NO: 2 or 3 and α-MSH added (50 μM, 100 μM) were used as comparison groups, and the groups with arbutin (200 μM, 500 μM), which is known as a skin whitening agent, and α-MSH added were used as positive control groups.

[0127] As a result, as Figure 16 shown, melanin production increased in the comparison groups consisting of sequences similar to the peptide according to an embodiment. On the contrary, melanin production decreased by adding the peptide consisting of the amino acid sequence of SEQ ID NO: 1. From the experimental results, it was found that the peptide consisting of the amino acid sequence of SEQ ID NO: 1 according to an embodiment not only has the above-described skin condition improving effect but also has a skin whitening effect.

[0128]

Formulation Example 1. Preparation of Peptide Nanobody

[0129] 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 to 1 L with distilled water, and then emulsification was performed at high pressure using a high-pressure microfluidic homogenizer to prepare peptide nanobodies with a size of about 100 nm.

[0130]

Formulation Example 2, Soft Lotion

[0131] A soft lotion comprising a peptide according to an embodiment and having the following composition was prepared by a method well known in the art.

[0132]

Table 4

[0133] Ingredient Content (wt%) Peptide 1.0 1,3-Butanediol 6 Glycerol 4 Polyethylene Glycol 1500 1 Sodium Hyaluronate 1 Polysorbate 20 0.5 Ethanol 8 Benzophenone-9 0.05 Preservative, pigment Appropriate amount Fragrance Appropriate amount Purified water Balance Total 100

[0134]

Formulation Example 3, Nutritional Cream

[0135] A nutritional cream comprising a peptide according to an embodiment and having the following composition was prepared by a method well known in the art.

[0136]

Table 5

[0137]

[0138]

[0139]

Formulation Example 4, Nutritional Lotion

[0140] A nutritional lotion comprising a peptide according to an embodiment and having the following composition was prepared by a method well known in the art.

[0141]

Table 6

[0142] 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

[0143]

Formulation Example 5, Serum

[0144] A serum comprising a peptide according to an embodiment and having the following composition was prepared by a method well known in the art.

[0145]

Table 7

[0146] Ingredient Content (wt%) Peptide 2.0 Glycerol 10 1,3-Butanediol 5 Polyethylene Glycol 1500 2 Allantoin 0.1 DL-Panthenol 0.3 EDTA-2Na 0.02 Hydroxyethyl Cellulose 0.1 Sodium Hyaluronate 8 Carboxyvinyl Polymer 0.2 Triethanolamine 0.18 Octyldodeceth-16 0.4 Ethanol 6 Preservative, pigment Appropriate amount Fragrance Appropriate amount Purified water Balance Total 100

[0147] The description of the present invention is provided only for 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, rather than restrictive.

Claims

1. A cosmetic composition for improving skin condition, which comprises a peptide consisting of the amino acid sequence of SEQ ID NO: 1 as an active ingredient.

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

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

4. The cosmetic composition according to claim 1, wherein, The peptide exhibits at least any one selected from the group consisting of the following characteristics: (a) Enhancing the expression of type I collagen, fibronectin, elastin, or hyaluronic acid, which are extracellular matrix components; (b) Enhancing the expression of sirtuin-1 or aquaporin-3, which are skin barrier factors; (c) Reducing the level of reactive oxygen species; (d) Reducing the expression of matrix metalloproteinase-1; and (e) Inhibiting melanogenesis.

5. The cosmetic composition according to claim 1, wherein, The peptide is prepared in the form of a nanobody.

6. The cosmetic composition according to claim 1, wherein The improvement of skin condition is improvement of wrinkles, improvement of skin elasticity, wound healing, strengthening of skin barrier, inhibition of skin aging, or skin whitening.

7. The cosmetic composition according to claim 6, wherein, The skin aging is skin aging caused by ultraviolet rays.

8. A pharmaceutical composition for preventing or treating hyperpigmentation diseases, which comprises a peptide consisting of the amino acid sequence of SEQ ID NO: 1 as an active ingredient.

9. The pharmaceutical composition according to claim 8, wherein, The hyperpigmentation diseases are melasma, freckles, senile lentigines, or solar lentigines.

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