Peptides having skin condition improving activity and uses thereof

Abstract

The present application relates to a peptide having skin condition-improving activity and its uses, and provides a peptide consisting of the amino acid sequence of SEQ ID NO: 1, and a cosmetic composition for improving skin condition that contains the peptide consisting of 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 uses thereof. [Background technology]

[0002] Human skin undergoes constant changes, the most common of which are a decline in skin function and a loss of visual beauty due to aging. Aging causes wrinkles on the skin, and typical factors that contribute to wrinkle formation include exposure to ultraviolet light and a decrease in collagen biosynthesis. Skin aging can be broadly divided into intrinsic aging, which is caused by genetic factors, and extrinsic aging, which is caused by external environmental factors such as sunlight. It is known that extrinsic aging can be prevented, treated, or delayed by removing reactive oxygen species, promoting fibroblast proliferation, and stimulating collagen biosynthesis.

[0003] Collagen, a major component of the extracellular matrix, is the primary matrix protein produced by skin fibroblasts. Collagen forms most of the organic material in skin, tendons, bones, and teeth, with particularly high concentrations in bone and skin (dermis). Collagen decreases with age and photoaging due to UV exposure, which is known to be closely related to the formation of skin wrinkles. Collagen also plays an important role in wound healing, promoting collagen synthesis in damaged epithelium and enabling wounds to heal quickly and without scars. Furthermore, it has been reported that promoting collagen biosynthesis increases the density of the basal layer and other layers, thereby reducing the melanin pigment concentration per unit skin density, resulting in a lighter skin tone.

[0004] Pigments that affect skin color include melanin, melanoids, carotene, hemoglobin, and carotenoids, and the various colors of skin, hair, and eyes are determined by these pigments. Among these, melanin is the most important pigment that determines skin color, and specifically, color is determined by the amount and distribution of melanin. Melanin is produced by cells called melanocytes beneath the epidermis of the skin and migrates to the stratum corneum surface and peels off through skin metabolism. Regardless of skin color, the number of melanocytes is roughly the same, but the amount, type, and distribution of melanin produced vary, resulting in differences in skin color.

[0005] Under such technical background, various researches are being conducted to improve skin condition through mechanisms such as promoting collagen biosynthesis and increasing the proliferation and activity of fibroblasts (Korean Patent Registration No. 10-1813629), but the reality is that there are still shortcomings. Summary of the Invention [Problem to be solved by the invention]

[0006] One embodiment is to provide a peptide consisting of the amino acid sequence of SEQ ID NO:1.

[0007] Another aspect of the present invention is to provide a composition for improving skin conditions, which comprises a peptide consisting of the amino acid sequence of SEQ ID NO: 1 as an active ingredient.

[0008] Another aspect of the present invention is to provide a pharmaceutical composition for preventing or treating melanin hyperpigmentation diseases, which comprises a peptide consisting of the amino acid sequence of SEQ ID NO: 1 as an active ingredient.

[0009] Other objects and advantages of the present application will become more apparent from the following detailed description taken in conjunction with the claims and drawings. The contents not described in this specification are fully understood and can be inferred by those skilled in the art of the present application or a similar art, and therefore, the description thereof will be omitted. [Means for solving the problem]

[0010] Each description and embodiment disclosed in this application may also be applied to each other description and embodiment. In other words, all combinations of various elements disclosed in this application belong to the scope of this application. In addition, the specific descriptions described below are not intended to limit the scope of this application.

[0011] One embodiment provides a peptide consisting of the amino acid sequence of SEQ ID NO:1.

[0012] The term "peptide" as used herein refers to a linear molecule formed by amino acid residues linked to each other by peptide bonds. The peptide can be prepared by chemical synthesis methods known to those skilled in the art, particularly solid-phase synthesis or liquid-phase synthesis (U.S. Patent No. 5,516,891). The present inventors have made extensive efforts to develop peptides with biologically effective activities, and have identified a peptide having the amino acid sequence of SEQ ID NO: 1. Here, the biologically effective activity may be any one or more of the following: (a) enhanced expression of extracellular matrix components such as collagen type 1 (Col1a1), fibronectin, elastin, or hyaluronic acid; (b) enhanced expression of skin barrier factors such as sirtuin-1 (SIRT-1) or aquaforin-3 (AQP3); (c) reduced levels of reactive oxygen species; (d) reduced expression of matrix metalloproteinase-1 (MMP-1); and (e) suppression of melanin production. Therefore, the peptide can be used for improving skin condition, whitening skin, or as an antioxidant.

[0013] The peptides may also have protecting groups attached to their N- or C-termini to achieve chemical stability, enhanced pharmacological properties (half-life, absorbency, potency, efficacy, etc.), altered specificity (e.g., a broader spectrum of biological activity), or reduced antigenicity. In one embodiment, the N-terminus of the peptide may be conjugated with any one 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 polyethylene glycol (PEG); and / or the C-terminus of the peptide may be conjugated with any one protecting group selected from the group consisting of an amino group (-NH), a tertiary alkyl group, and an azide (-NHNH). The peptide may also optionally further comprise a targeting sequence, a tag, a labeled residue, or an amino acid sequence specifically engineered to increase half-life or peptide stability.

[0014] The peptides are artificially synthesized or non-naturally occurring or engineered, where "non-naturally occurring or engineered" refers to a state in which the peptide is produced by artificial modification, rather than a state in which it exists in nature. Here, the artificial modification can include artificially synthesizing an amino acid sequence by mimicking multiple amino acid structures, or engineering to obtain chemical stability, enhanced pharmacological properties, altered specificity, or reduced antigenicity, as described above.

[0015] The term "stability" as used herein may refer not only to in vivo stability, which protects the peptide from attack by in vivo proteolytic enzymes, but also to storage stability (eg, storage stability at room temperature).

[0016] Another aspect provides a cosmetic composition for improving skin conditions, which comprises a peptide consisting of 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 as those already mentioned are as described above.

[0018] As used herein, the term "amelioration" can refer to the alleviation of a condition or any action that at least reduces a parameter of treatment, such as the severity of symptoms.

[0019] The term "skin condition improvement" as used herein comprehensively refers to the process or effect of treating, reducing, or alleviating skin damage induced by intrinsic or extrinsic factors, and may be interpreted as meaning, for example, wrinkle improvement, skin elasticity improvement, wound recovery, skin barrier strengthening, skin aging inhibition, or skin whitening, but is not limited thereto.

[0020] Here, "wrinkle reduction," "skin elasticity improvement," and "wound recovery" may refer to any action that increases the total amount of extracellular matrix factors, including promoting the synthesis of collagen or hyaluronic acid. Furthermore, "strengthening the skin barrier" may refer to strengthening the skin's natural functions of preventing the leakage of moisture and nutrients from within the skin and preventing the penetration of harmful substances such as bacteria and viruses into the skin. Furthermore, "inhibiting skin aging" may refer to inhibiting declines in skin function, such as wrinkles, skin sagging, and loss of elasticity. Here, skin aging also refers to photoaging, for example, skin aging caused by ultraviolet rays.

[0021] Here, "skin whitening" can be interpreted as not only lightening skin tone by inhibiting melanin synthesis, but also improving hyperpigmentation of the skin caused by ultraviolet rays, hormones, or genetics, which may include, but are not limited to, freckles, age spots, dark spots, brown spots, and age spots.

[0022] Although conventional functional peptides have effective biological activities, they have drawbacks such as ineffective delivery to target tissues or cells due to their size, or a short half-life that causes them to disappear in the body within a short period of time. In contrast, a cosmetic composition according to one embodiment contains a peptide consisting of 10 or fewer amino acids as an active ingredient, which provides excellent skin penetration of the active ingredient. For example, when applied topically to the skin, the cosmetic composition can effectively improve skin condition.

[0023] According to one embodiment, the peptide was able to enhance the synthesis of extracellular matrix components and skin barrier factors. Furthermore, the peptide not only restored the inhibited activity of fibroblasts and keratinocytes and improved their antioxidant activity, but also significantly inhibited melanin production. Therefore, the peptide can be used as an active ingredient in cosmetic compositions for improving skin conditions.

[0024] The cosmetic composition may contain, but is not limited to, a cosmetically effective amount of the peptide; and / or a cosmetically acceptable carrier.

[0025] As used herein, the term "cosmetically effective amount" means an amount sufficient to achieve the skin condition improving effect of the cosmetic composition.

[0026] The weight ratio between the peptide and the cosmetically acceptable carrier may be, for example, 500:1 to 1:500. By way of example, the weight ratio may be, but is not limited to, 450:1 to 1:450, 400:1 to 1:400, 350:1 to 1:350, 300:1 to 1:300, 250:1 to 1:250, 200:1 to 1:200, 150:1 to 1:150, 100:1 to 1:100, 80:1 to 1:80, 60:1 to 1:60, 40:1 to 1:40, 20:1 to 1:20, 10:1 to 1:10, 8:1 to 1:8, 6:1 to 1:6, 4:1 to 1:4, or 2:1 to 1:2.

[0027] The cosmetic composition may be prepared in any formulation commonly prepared in the art, such as, but not limited to, a solution, suspension, emulsion, paste, gel, cream, lotion, powder, soap, surfactant-containing cleanser, oil, powder foundation, emulsion foundation, wax foundation, spray, etc. For example, it may be prepared in the form of a softening lotion, nourishing lotion, nourishing cream, massage cream, essence, eye cream, cleansing cream, cleansing foam, cleansing water, pack, spray, or powder.

[0028] When the cosmetic composition is in the form of a paste, cream, or gel, the carrier component may be animal oil, vegetable oil, wax, paraffin, starch, tragacanth, cellulose derivatives, polyethylene glycol, silicone, bentonite, silica, talc, zinc oxide, or the like.

[0029] When the cosmetic composition is in the form of a powder or spray, lactose, talc, silica, aluminum hydroxide, calcium silicate, or polyamide powder is used as a carrier component, and in the case of a spray, for example, a propellant such as chlorofluorohydrocarbon, propane / butane, or dimethyl ether may be further included.

[0030] When the cosmetic composition is in the form of a solution or emulsion, a solvent, solubilizer, or emulsifier is used as a carrier component, and may include, for example, water, ethanol, isopropanol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butyl glycol oil, glycerol fatty acid esters, polyethylene glycol, or sorbitan fatty acid esters.

[0031] When the cosmetic composition is in the form of a suspension, a liquid diluent such as water, ethanol, or propylene glycol, a suspending agent such as ethoxylated isostearyl alcohol, polyoxyethylene sorbitol ester, or polyoxyethylene sorbitan ester, microcrystalline cellulose, aluminum metahydroxide, bentonite, agar, or tracant may be used as a carrier component.

[0032] When the cosmetic composition is in the form of a surfactant-containing cleanser, fatty alcohol sulfates, fatty alcohol ether sulfates, sulfosuccinic acid monoesters, isethionates, imidazolinium derivatives, methyl taurates, sarcosinates, fatty acid amide ether sulfates, alkylamidobetaines, fatty alcohols, fatty acid glycerides, fatty acid diethanolamides, vegetable oils, lanolin derivatives, or ethoxylated glycerol fatty acid esters may be used as carrier components.

[0033] To further improve skin penetration or stability, the peptide can be incorporated into nanosomes or nanoparticles. For example, the nanosomes can be prepared using lecithin as a raw material using a microfluidizer, and then incorporated into lecithin particles. Any known method can be used to prepare the nanosomes. The nanosome particle size is preferably 30 to 200 nm. Nanosome particle sizes less than 30 nm can penetrate the skin too quickly, resulting in side effects. Nanosome particle sizes greater than 200 nm can penetrate the skin too quickly, making it difficult to achieve the benefits of using the nanosome structure.

[0034] The components contained in the cosmetic composition include, in addition to the peptide as an active ingredient and a carrier component, components commonly used in cosmetic compositions, such as common adjuvants such as antioxidants, stabilizers, solubilizers, vitamins, pigments, and fragrances.

[0035] The content of the peptide as an active ingredient contained in the cosmetic composition is appropriately selected without limitation depending on the product form, desired use, etc., and may be added in an amount of, for example, 0.01 to 15 wt % of the total cosmetic composition weight. Also, for example, the cosmetic composition may contain the peptide in an amount of 1.0 to 3.0 wt %, preferably 2.0 to 3.0 wt %, based on the total weight.

[0036] Yet another aspect provides a method for improving skin condition, comprising applying to the skin of an individual a cosmetic composition containing as an active ingredient a peptide having the amino acid sequence of SEQ ID NO: 1; and a use of the peptide having the amino acid sequence of SEQ ID NO: 1 for the manufacture of a composition for improving skin condition.

[0037] Among the terms and elements mentioned in the description of the cosmetic composition, the same as those already mentioned are as described above.

[0038] As used herein, the term "individual" refers to a subject in need of skin condition improvement, and more specifically refers to mammals such as human or non-human primates, mice, dogs, cats, horses, and cows.

[0039] As used herein, the terms "apply," "administer," and "apply" are used interchangeably and may refer to causing at least partial localization of a composition according to an embodiment to a desired site, or to placing a composition according to an embodiment within an individual by a route of administration.

[0040] In yet another embodiment, there is provided a pharmaceutical composition for preventing or treating melanin hyperpigmentation diseases, which comprises a peptide consisting of the amino acid sequence of SEQ ID NO: 1 as an active ingredient.

[0041] Among the terms or elements mentioned in the description of the peptide, the same as those already mentioned are as described above.

[0042] As used herein, the term "prevention" refers to any action that inhibits or delays the onset of a disease by administering the composition.

[0043] As used herein, the term "treatment" refers to any form of care that provides a benefit to an individual suffering from or susceptible to a disease, including improvement of the individual's condition (e.g., one or more symptoms), delay in disease progression, delay in the onset of symptoms, or slowing of symptom progression, etc. Thus, the terms "treatment" and "prevention" are not intended to mean a cure or complete elimination of symptoms.

[0044] The "melanin hyperpigmentation disease," which is a target disease to be prevented or treated by the pharmaceutical composition, is a general term for diseases in which melanin pigment is produced excessively and deposited in the skin, such as, but not limited to, age spots, freckles, age spots, and solar lentigines.

[0045] The pharmaceutical composition may comprise, but is not limited to, a pharmaceutically effective amount of the peptide; and / or a pharmaceutically acceptable carrier.

[0046] The term "pharmaceutically effective amount" as used herein may refer to an amount sufficient to achieve the efficacy of the pharmaceutical composition in preventing or treating melanin hyperpigmentation diseases.

[0047] The pharmaceutically acceptable carriers are those commonly used in formulations, and include, but are not limited to, lactose, dextrose, saccharose, sorbitol, mannitol, starch, acacia gum, calcium phosphate, alginate, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, methylcellulose, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, mineral oil, etc. Suitable pharmaceutically acceptable carriers and formulations are described in detail in Remington's Pharmaceutical Sciences (19th ed., 1995).

[0048] The weight ratio between the peptide and the pharmaceutically acceptable carrier may be, for example, 500:1 to 1:500. By way of example, the weight ratio may be, but is not limited to, 450:1 to 1:450, 400:1 to 1:400, 350:1 to 1:350, 300:1 to 1:300, 250:1 to 1:250, 200:1 to 1:200, 150:1 to 1:150, 100:1 to 1:100, 80:1 to 1:80, 60:1 to 1:60, 40:1 to 1:40, 20:1 to 1:20, 10:1 to 1:10, 8:1 to 1:8, 6:1 to 1:6, 4:1 to 1:4, or 2:1 to 1:2.

[0049] The pharmaceutical composition may further contain, in addition to the above ingredients, lubricants, wetting agents, sweeteners, flavoring agents, emulsifiers, suspending agents, preservatives, etc., but is not limited to these.

[0050] The pharmaceutical composition may be administered orally or parenterally, preferably parenterally. In the case of parenteral administration, it may be administered by intramuscular injection, intravenous injection, subcutaneous injection, intraperitoneal injection, topical administration, transdermal administration, etc., but is not limited thereto.

[0051] The dosage of the pharmaceutical composition may be, but is not limited to, 0.0001 to 1000 μg (0.001 to 1000 μg, 0.01 to 1000 μg, 0.1 to 1000 μg, or 1.0 to 1000 μg) per day, and may be variously prescribed depending on factors such as formulation method, administration route, age, weight, sex, pathological condition, diet of the patient, administration time, administration route, excretion rate, and reaction sensitivity.

[0052] The pharmaceutical composition may be prepared in a unit dose form or in a multi-dose container by formulating it with pharmaceutically acceptable carriers and / or excipients in a manner that can be easily carried out by a person skilled in the art to which the invention pertains.

[0053] The dosage form may be in the form of a solution, suspension or emulsion in an oily or aqueous medium, or in the form of an extract, powder, granules, tablet or capsule, and may additionally contain a dispersing agent and / or a stabilizer.

[0054] Yet another embodiment provides a method for preventing or treating a melanin hyperpigmentation disease, comprising administering to an individual a pharmaceutical composition containing, as an active ingredient, a therapeutically effective amount of a peptide consisting of the amino acid sequence of SEQ ID NO: 1.

[0055] Among the terms or elements mentioned in the description of the pharmaceutical composition, the same as those already mentioned are as described above.

[0056] Yet another embodiment provides an antioxidant composition comprising 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 peptide, the same as those already mentioned are as described above.

[0058] The antioxidant composition may be in the form of a pharmaceutical composition, a quasi-drug composition, or a cosmetic composition. For example, the composition may be used as a cosmetic composition for improving skin conditions or a pharmaceutical composition for improving or treating the condition of a disease associated with skin damage.

[0059] According to one embodiment, the peptide was shown to be able to restore the inhibited activity of fibroblasts and keratinocytes and improve their antioxidant activity, and therefore, the peptide can be used as an active ingredient in antioxidant compositions.

[0060] The antioxidant composition may be provided in the form of a pharmaceutical composition, for example, which may contain a pharmaceutically effective amount of the peptide and / or a pharmaceutically acceptable carrier, and the terms and elements mentioned in the description of the pharmaceutical composition that are the same as those already mentioned are as described above.

[0061] Yet another aspect provides a food composition for improving skin conditions, which comprises a peptide consisting of the amino acid sequence of SEQ ID NO: 1 as an active ingredient.

[0062] Among the terms or elements mentioned in the description of the peptide, the same as those already mentioned are as described above.

[0063] The content of the peptide as an active ingredient contained in the food composition is appropriately selected without limitation depending on the food form, desired use, etc., and may be added at, for example, 0.01 to 15 wt% of the total food weight. For example, in the case of a health drink composition, the peptide may be added at a ratio of 0.02 to 10 g, preferably 0.3 to 1 g, based on 100 ml. [Effects of the Invention]

[0064] According to one embodiment, the peptide can improve the synthesis of extracellular matrix constituent factors and skin barrier factors, and can be applied to improving skin conditions, including wrinkle improvement, skin elasticity improvement, wound recovery, skin barrier strengthening, skin aging inhibition, or skin whitening.

[0065] According to one embodiment, the peptide can restore the inhibited activity of fibroblasts and keratinocytes and improve their antioxidant efficacy, thereby contributing to the recovery of skin damage that may be caused by external environmental factors such as ultraviolet rays.

[0066] Therefore, the peptide according to one embodiment can be used as an active ingredient in a composition for improving skin conditions or a pharmaceutical composition for preventing or treating melanin hyperpigmentation diseases. [Brief explanation of the drawings]

[0067] [Figure 1] This shows the results of confirming increased expression of extracellular matrix components Col1a1, fibronectin, and elastin after adding a peptide consisting of the amino acid sequence of SEQ ID NO: 1 to NIH3T3 cells. [Figure 2] A peptide consisting of the amino acid sequence of SEQ ID NO: 1 was added to NIH3T3 cells, and the expression levels of extracellular matrix components were quantitatively evaluated. Figure 2A shows the results of confirming the expression levels of Col1a1, Figure 2B shows the results of confirming the expression levels of fibronectin, and Figure 2C shows the results of confirming the expression levels of elastin. [Figure 3]1 shows the results of quantitatively evaluating the expression level of procollagen I alpha after adding a peptide consisting of the amino acid sequence of SEQ ID NO: 1 to NIH3T3 cells. [Figure 4] This shows the results of quantitatively evaluating the expression level of hyaluronic acid after adding a peptide consisting of the amino acid sequence of SEQ ID NO: 1 to HaCaT cells. [Figure 5] 1 shows the results of confirming the expression levels of fibronectin and elastin, which are components of the extracellular matrix, after adding a peptide consisting of the amino acid sequence of SEQ ID NO: 1 and a comparison peptide to NIH3T3 cells. [Figure 6] A peptide consisting of the amino acid sequence of SEQ ID NO: 1 and a comparison peptide were added to NIH3T3 cells, and the expression levels of extracellular matrix components were quantitatively evaluated. Figure 6A shows the results of confirming the expression level of fibronectin, and Figure 6B shows the results of confirming the expression level of elastin. [Figure 7] This shows the results of confirming increased expression of skin barrier factors SIRT-1 and AQP3 after adding a peptide consisting of the amino acid sequence of SEQ ID NO: 1 to HaCaT cells. [Figure 8] A peptide consisting of the amino acid sequence of SEQ ID NO: 1 was added to HaCaT cells, and the expression level of skin barrier factors was quantitatively evaluated. Figure 8A shows the results of confirming the expression level of SIRT-1, and Figure 8B shows the results of confirming the expression level of AQP3. [Figure 9] This shows the results of quantitatively confirming the change in the level of reactive oxygen species increased by ultraviolet light after adding a peptide consisting of the amino acid sequence of SEQ ID NO: 1 to NIH3T3 cells. [Figure 10] This is the result of quantitatively confirming the change in the level of reactive oxygen species increased by ultraviolet light after adding a peptide consisting of the amino acid sequence of SEQ ID NO: 1 to HaCaT cells. [Figure 11] The results show that the peptide consisting of the amino acid sequence of SEQ ID NO: 1 and a comparative peptide were added to HaCaT cells, and then the change in the level of reactive oxygen species increased by ultraviolet light was quantitatively confirmed. [Figure 12] The results show that after adding a peptide consisting of the amino acid sequence of SEQ ID NO: 1 to HaCaT cells, changes in the expression levels of SIRT-1 and AQP3, which were reduced by ultraviolet light, were confirmed. [Figure 13] After adding a peptide consisting of the amino acid sequence of SEQ ID NO: 1 to HaCaT cells, the changes in the expression levels of SIRT-1 and AQP3, which were reduced by ultraviolet light, were quantitatively confirmed. Figure 13A shows the results of confirming the expression level of SIRT-1, and Figure 13B shows the results of confirming the expression level of AQP3. [Figure 14] The results show that after adding a peptide consisting of the amino acid sequence of SEQ ID NO: 1 to NIH3T3 cells, changes in the expression level of MMP-1, which was increased by ultraviolet light, were observed. [Figure 15] This is the result of confirming the change in the expression level of MMP-1, which was increased by ultraviolet light, after adding a peptide consisting of the amino acid sequence of SEQ ID NO: 1 to HaCaT cells. [Figure 16] 1 shows the results of quantitatively confirming changes in melanin production levels after adding a peptide consisting of the amino acid sequence of SEQ ID NO: 1 and a comparison peptide to B16F10 cells. DETAILED DESCRIPTION OF THE INVENTION

[0068] The present invention will be described in more detail below with reference to examples. However, these examples are for illustrative purposes only and the scope of the present invention is not limited to these examples.

[0069] Example 1. Synthesis of peptides The peptide having the amino acid sequence of SEQ ID NO: 1 in Table 1 was synthesized using an automated peptide synthesizer (Milligen 9050, Millipore, USA), and the synthesized peptide was purified and separated using C18 reverse-phase high-performance liquid chromatography (HPLC) (Waters Associates, USA) on an ACQUITY UPLC BEH300C18 column (2.1 mm ∘ 100 mm, 1.7 μm, Waters Co., USA).

[0070] [Table 1]

[0071] Example 2. Confirmation of wrinkle improvement and elasticity improvement effects 2-1. Evaluation of efficacy of peptides according to one embodiment In this example, the expression levels of collagen type 1 (Col1a1), fibronectin, elastin, and hyaluronic acid, which are known to be components of the dermis, were evaluated to confirm the effect of the peptide according to one embodiment on improving intrinsic skin aging, including wrinkle improvement and elasticity improvement.

[0072] Specifically, NIH3T3 cells were cultured in a 6-well plate at 3 x 10 5After seeding at a density of 1000 cells / well, the cells were cultured for 24 hours. After washing the cells once with serum-free DMEM media, 1 mL of the medium was supplemented with 10 μM, 50 μM, or 100 μM of the peptide consisting of the amino acid sequence of SEQ ID NO: 1, and the culture was cultured for 24 hours in a CO2 incubator at 37°C. The culture was then washed twice with PBS, and RNA was isolated from the culture using Easy Blue (iNtRON, Cat. No.: 17061, Korea). The isolated RNA was then reverse transcribed using an RT kit (Enzynomics, Cat. No.: RT200, Korea) to synthesize the respective cDNAs. Then, polymerase chain reaction (PCR) was performed using 1 μg of the synthesized cDNA and primers for Col1a1, fibronectin, or elastin and a PCR kit (Enzynomics, Cat. No.: P581T, Korea). In this example, an untreated group was used as the control group, and a group treated with 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.

[0073] [Table 2]

[0074] As a result, as shown in Figures 1 and 2, it was confirmed that the peptide consisting of the amino acid sequence of SEQ ID NO: 1 increased the expression of extracellular matrix components Col1a1, fibronectin, and elastin.

[0075] To evaluate the expression levels of procollagen I alpha and hyaluronan at the protein level, NIH3T3 cells or HaCaT cells were cultured in 24-well plates at 1 x 10 4The cells were seeded at a density of 1000 cells / well and cultured for 24 hours. The cells were then washed once with serum-free DMEM media, and 2 ml of the medium was supplemented with 10 μM, 50 μM, or 100 μM of the peptide consisting of the amino acid sequence of SEQ ID NO: 1. The cells were then cultured in a CO2 incubator at 37°C for 72 hours. The culture was then centrifuged to precipitate the supernatant, and the supernatant was transferred to an e-tube. Collagen ELISA (Abcam, Cat No.: ab210579, UK) and HA ELISA (ECHELON biosciences, Cat No.: K-1200, USA) were then performed on the centrifuged culture. In this example, an untreated group served as the control, and TGF-β1 or EGF-treated groups served as positive controls.

[0076] As a result, as shown in Figures 3 and 4, it was confirmed that the peptide consisting of the amino acid sequence of SEQ ID NO: 1 increases the expression of procollagen I alpha, a collagen precursor, and hyaluronic acid, which is related to skin elasticity and skin moisture maintenance, at the protein level. From the above results, it was found that the peptide according to one embodiment contributes to improving intrinsic skin aging, including wrinkle improvement and elasticity improvement, by increasing extracellular matrix constituent factors.

[0077] 2-2. Comparison of efficacy between peptides and comparison groups according to an embodiment In this example, the effects of the peptide of an example on improving intrinsic skin aging were compared in more detail. To this end, peptides consisting of the amino acid sequences of SEQ ID NOs: 2, 3, or 4, which have sequences similar to those of the peptide of an example, were used as comparison groups, and the expression levels of fibronectin and elastin were evaluated and compared in the same manner as in Example 2-1. Meanwhile, in this example, the control group was an untreated group, the comparison group was a group to which the peptide consisting of the amino acid sequence of SEQ ID NOs: 2, 3, or 4 was added (50 μM, 100 μM), and the positive control group was a group to which TGF-β1 or EGF was added. The nucleotide sequences of the primers used in this example are as shown in Table 2.

[0078] As a result, as shown in Figures 5 and 6, the peptide consisting of the amino acid sequence of Sequence No. 1 increased the expression of fibronectin and elastin, which are components of the extracellular matrix, and such an effect was superior to the expression level of the comparison group consisting of a sequence similar to that of the peptide according to one embodiment.

[0079] Example 3. Confirmation of skin barrier strengthening effect In this example, the expression levels of sirtuin-1 (SIRT-1) or aquaforin-3 (AQP3) were evaluated to confirm the effect of a peptide according to one example on strengthening the skin barrier.

[0080] Specifically, 3 x 10 HaCaT cells were plated in a 6-well plate. 5 The cells were seeded at a density of 1000 cells / well and cultured for 24 hours. After washing the cells once with serum-free DMEM media, 1 mL of the medium was supplemented with 10 μM, 50 μM, or 100 μM of the peptide consisting of the amino acid sequence of SEQ ID NO: 1. The cells were then cultured in a CO2 incubator at 37°C for 24 hours. The culture was then washed twice with PBS, and RNA was isolated from the culture using Easy Blue (IntRON, Cat. No.: 17061, Korea). The isolated RNA was reverse transcribed using an RT kit (Enzynomics, Cat. No.: RT200, Korea) to synthesize the respective cDNAs. Polymerase chain reaction (PCR) was then performed using 1 μg of the synthesized cDNA, primers for SIRT-1 or AQP3, and a PCR kit (Enzynomics, Cat. No.: P581T, Korea). Meanwhile, in this example, an untreated group was used as the control group, and an EGF-added group was used as the positive control group. The nucleotide sequences of the primers used in this example are as shown in Table 3 below.

[0081] [Table 3]

[0082] Example 4: Confirmation of the effect of reducing reactive oxygen species increased by ultraviolet rays 4-1. Evaluation of efficacy of peptides according to one embodiment In this example, the effect of a peptide according to one embodiment on the antioxidant effect in damaged skin cells was confirmed by evaluating changes in the level of reactive oxygen species in skin cells that increased due to UV irradiation.

[0083] Specifically, 5 x 10 NIH3T3 or HaCaT cells were cultured in a 6-well plate. 5 After seeding at a density of 1000 cells / well, the cells were cultured for 24 hours. After that, the cells were washed once with serum-free DMEM media, and then 10 μM, 50 μM, or 100 μM of the peptide consisting of the amino acid sequence of SEQ ID NO: 1 was dispensed into 1 mL of the medium, and the resultant was cultured for 1 hour in a CO2 incubator at 37°C. After that, the culture was transferred to an e-tube, mixed with 1 mL of PBS, and dispensed into well plates. Then, a UV irradiator (VILBER LOURMAT, Cat No.: 3102-BSU, France) was used to irradiate NIH3T3 cells with 6 J / cm. 2 HaCaT cells were exposed to 15 mJ / cm of ultraviolet light. 2The wells were then irradiated with UV light for 10 min. Thereafter, the PBS in the well plate was removed, and 900 μL of culture medium containing the peptide was added. The cells were then cultured for 24 hours in a CO2 incubator at 37°C. The cultures were then treated with DCFG-DA (2',7'-dichlorofluorescin diacetate), wrapped in foil, and cultured for 30 minutes in a CO2 incubator at 37°C. The cells were then washed twice with PBS and treated with 500 μL of 1× trypsin / EDTA to obtain cells, which were then centrifuged. The centrifuged cells were washed with PBS and then measured for FL1 fluorescence intensity using flow cytometry (FACS, BD, USA). In this example, the control group was an untreated group, the negative control group was a group exposed to UV light only, and the positive control group was a group to which NaC was added after UV light exposure.

[0084] As a result, as shown in Figures 9 and 10, it was confirmed that the peptide consisting of the amino acid sequence of SEQ ID NO: 1 reduces the reactive oxygen levels in fibroblasts and keratinocytes that are increased by UV irradiation. From the above results, it was found that the peptide according to one embodiment contributes to the antioxidant effect on skin cells induced by UV rays.

[0085] 4-2. Comparison of efficacy between peptides and comparison groups according to an embodiment In this example, the effects of the peptide of an example on the antioxidant effect in damaged skin cells were compared in more detail. To this end, peptides consisting of the amino acid sequences of SEQ ID NOs: 2, 3, or 4, which have sequences similar to those of the peptide of an example, were used as comparison groups, and the increased intracellular ROS levels in HaCaT cells due to UV irradiation were evaluated and compared in the same manner as in Example 4-1. In this example, the control group was an untreated group, the negative control group was a group exposed to UV light alone, the comparison group was a group to which the peptide consisting of the amino acid sequence of SEQ ID NOs: 2, 3, or 4 was added (50 μM, 100 μM) after UV irradiation, and the positive control group was a group to which Trolox was added after UV irradiation.

[0086] As a result, as shown in Figure 11, the peptide consisting of the amino acid sequence of SEQ ID NO: 1 reduced the level of reactive oxygen species in keratinocytes that increased due to UV irradiation, and this effect was superior to the expression level of a comparison group consisting of a sequence similar to the peptide of one embodiment.

[0087] Example 5. Confirmation of the effect of restoring skin barrier factors reduced by ultraviolet rays In this example, the effect of a peptide according to one embodiment on the recovery of skin barrier factors in damaged skin cells was confirmed by evaluating changes in the levels of skin barrier factors in skin cells that were reduced by UV irradiation.

[0088] Specifically, 5 x 10 HaCaT cells were plated in a 6-well plate. 5 After seeding at a density of 1000 cells / well, the cells were cultured for 24 hours. After washing the cells once with serum-free DMEM media, 10 μM, 50 μM, or 100 μM of the peptide consisting of the amino acid sequence of SEQ ID NO: 1 was dispensed into 1 mL of the medium, and the resulting mixture was cultured for 1 hour in a CO2 incubator at 37°C. The culture was then transferred to an e-tube, mixed with 1 mL of PBS, and dispensed into well plates. The HaCaT cells were then exposed to 15 mJ / cm2 UV irradiation using a UV irradiator (VILBER LOURMAT, Cat No.: 3102-BSU, France). 2The wells were irradiated with UV light for 10 min. Then, the PBS in the well plate was removed, and 900 μL of the culture medium containing the peptide was added. The cells were then cultured in a CO2 incubator at 37°C for 8 hours. The cultures were then washed twice with PBS, and RNA was isolated from the cultures using Easy Blue (IntRON, Cat. No.: 17061, Korea). The isolated RNA was then reverse transcribed using an RT kit (Enzynomics, Cat. No.: RT200, Korea) to synthesize the respective cDNAs. 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). Meanwhile, in this example, the control group was an untreated group, the comparison group was a group exposed to UV light only, and the positive control group was a group to which NaC was added. The nucleotide sequences of the primers used in this example are as shown in Table 3 above.

[0089] As a result, as shown in Figures 12 and 13, it was confirmed that the expression of SIRT-1 and AQP3, which had been decreased by UV irradiation, was increased 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 one embodiment contributes to strengthening the skin barrier and skin anti-aging by increasing skin barrier factors in damaged skin cells.

[0090] Example 6. Confirmation of UV-induced matrix metalloproteinase inhibitory effect It has been reported that MMP-1 (matrix metalloproteinase-1) promotes collagen degradation and elastin reduction in the dermis, causing skin wrinkles and loss of elasticity. Therefore, in this example, the effect of a peptide according to one embodiment on improving extrinsic skin aging, including wrinkle reduction and elasticity improvement, was confirmed by evaluating changes in MMP-1 expression levels increased by UV irradiation.

[0091] Specifically, 5 x 10 NIH3T3 or HaCaT cells were cultured in a 6-well plate. 5 After seeding at a density of 1000 cells / well, the cells were cultured for 24 hours. After that, the cells were washed once with serum-free DMEM media, and then 10 μM, 50 μM, or 100 μM of the peptide consisting of the amino acid sequence of SEQ ID NO: 1 was dispensed into 1 mL of the medium, and the resultant was cultured for 1 hour in a CO2 incubator at 37°C. After that, the culture was transferred to an e-tube, mixed with 1 mL of PBS, and dispensed into well plates. Then, a UV irradiator (VILBER LOURMAT, Cat No.: 3102-BSU, France) was used to irradiate NIH3T3 cells with 6 J / cm. 2 HaCaT cells were exposed to ultraviolet light at 20 mJ / cm 2The wells were then irradiated with UV light for 10 min. After removing the PBS from the well plate, 900 μL of the culture medium containing the peptide was added and cultured for 24 hours in a CO2 incubator at 37°C. The culture was then transferred to an e-tube, washed twice with PBS, and lysed by adding lysis buffer. The lysed culture was then treated with 5X sample buffer to prepare a sample, followed by SDS-PAGE to separate proteins and transferring them to a PVDF membrane. The PVDF membrane was then blocked with 5% skim milk at room temperature for 1 hour, and an anti-MMP-1 antibody (Abcam, Cat. No.: ab137332, UK) diluted 1:500 in 5% skim milk was reacted with the membrane for 2 hours. After washing three times for 10 minutes with 0.1% PBS-T (0.1% Tween-20 in PBS), goat anti-rabbit IgG (Jackson Immune Research, Cat. No.: 111-035-033, USA) was diluted 1:3000 in 5% skim milk and incubated with the membrane for 2 hours. MMP-1 was then detected using 15.ECL solution (GE Healthcare, Cat. No.: RPN2232, USA). The control group was an untreated group, the comparison group was a group exposed to UV light alone, and the positive control group was a group containing NaC.

[0092] As a result, as shown in Figures 14 and 15, it was confirmed that the expression of MMP-1, which was increased by UV irradiation, was reduced 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 one embodiment contributes to improving aging symptoms such as wrinkle formation and loss of elasticity caused by external factors by regulating the level of collagen-degrading enzyme, i.e., MMP-1.

[0093] Example 7. Confirmation of melanin production inhibitory effect Mouse melanoma B16F10 cells were plated in a 6-well plate at 5 × 104 The cells were seeded at a density of 1000 cells / well and cultured for 16 hours. The culture medium was then replaced with medium containing 2% serum, and 200 ng / ml of α-melanocyte-stimulating hormones (α-MSH) was added to promote melanin production. The peptides consisting of the amino acid sequence of SEQ ID NO: 1 were also added at 50 μM or 100 μM, and the cells were cultured for 72 hours. The cultured cells were then lysed with 1N NaOH, and the absorbance at 450 nm was measured. The control group was an untreated group, the negative control group was a group containing α-MSH alone, the comparative group was a group containing the peptides consisting of the amino acid sequence of SEQ ID NO: 2 or 3 and α-MSH (50 μM, 100 μM), and the positive control group was a group containing arbutin (200 μM, 500 μM), a known skin whitening agent, and α-MSH.

[0094] As a result, as shown in Figure 16, it was confirmed that melanin production increased in the comparison group consisting of a sequence similar to that of the peptide according to one embodiment, while melanin production decreased with the addition of the peptide consisting of the amino acid sequence of SEQ ID NO: 1. From the above experimental results, it was found that the peptide consisting of the amino acid sequence of SEQ ID NO: 1 according to one embodiment not only has the above-mentioned skin condition improving effect, but also has skin whitening effect.

[0095] Dosage Form Example 1. Production of Peptide Nanosomes 50 mg of the peptide from Example 1 was dissolved in 500 ml of distilled water with thorough stirring. The resulting solution was mixed with 5 g of lecithin, 0.3 ml of sodium oleate, 50 ml of ethanol, and a small amount of oil, and then the mixture was adjusted to 1 L with distilled water. The mixture was then emulsified under high pressure in a microfluidizer to produce peptide nanosomes with a size of approximately 100 nm.

[0096] Dosage form example 2: Softening lotion A softening lotion containing a peptide according to one embodiment and having the following composition was prepared by a method known in the art.

[0097] [Table 4]

[0098] Dosage form example 3: Nutritious cream A nourishing cream containing a peptide according to one embodiment and having the following composition was prepared using a method known in the art.

[0099] [Table 5]

[0100] Dosage form example 4: Nutritious lotion A nutritious lotion containing a peptide according to one embodiment and having the following composition was prepared using a method known in the art.

[0101] [Table 6]

[0102] Dosage form example 5. Essence An essence containing a peptide according to one embodiment and having the following composition was prepared using a method known in the art.

[0103] [Table 7]

[0104] The above description of the present invention is for illustrative purposes only, and those skilled in the art will understand that the present invention can be easily modified into other specific forms without changing the technical spirit or essential features of the present invention. Therefore, it should be understood that the above-described embodiments are illustrative in all respects and are not limiting.

Claims

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

2. 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 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 polyethylene glycol (PEG).

3. The C-terminus of the peptide is an amino group (-NH 2 ), tertiary alkyl groups and azides (-NHNH 2 2. The cosmetic composition according to claim 1, wherein the protective group is any one selected from the group consisting of:

4. The cosmetic composition according to claim 1, wherein the peptide exhibits one or more properties selected from the group consisting of the following: (a) Enhanced expression of extracellular matrix components such as collagen type 1 (Col1a1), fibronectin, elastin, or hyaluronic acid; (b) enhanced expression of skin barrier factors sirtuin-1 (SIRT-1) or aquaforin-3 (AQP3); (c) reduction of reactive oxygen species levels; (d) a decrease in matrix metalloproteinase-1 (MMP-1) expression; and (e) Inhibition of melanin production.

5. The cosmetic composition according to claim 1 , wherein the peptide is formulated in the form of nanosomes.

6. The cosmetic composition according to claim 1, wherein the improvement of skin condition is wrinkle improvement, improvement of skin elasticity, wound recovery, 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 melanin hyperpigmentation diseases, comprising a peptide consisting of the amino acid sequence of SEQ ID NO: 1 as an active ingredient.

9. The melanin hyperpigmentation disease is age spots, freckles, age spots, or solar lentigines.

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