Peptides having skin condition-improving activity and uses thereof

Abstract

This application relates to a peptide with skin condition-improving activity and its use, providing a peptide consisting of the amino acid sequence of SEQ ID NO: 1 and a cosmetic composition for improving skin condition, said cosmetic composition comprising the peptide consisting of the amino acid sequence of SEQ ID NO: 1 as an active ingredient.

Description

Technical Field

[0001] This application relates to a peptide with skin condition-improving activity and its uses. Background Technology

[0002] Human skin undergoes constant changes, most notably the decline in skin function and visual appeal caused by aging. Aging leads to wrinkles, with factors such as ultraviolet radiation exposure and reduced collagen biosynthesis being representative examples. Skin aging can be broadly categorized into endogenous aging caused by genetic factors and extrinsic aging caused by external environmental factors such as sunlight. For extrinsic aging, it is known that methods can prevent, treat, or delay aging 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 in fibroblasts of the skin. It forms a large portion of the organic matter in the skin, tendons, bones, and teeth, with particularly high concentrations in bone and the dermis. This collagen decreases with age and due to photoaging caused by ultraviolet radiation, and its reduction is known to be closely related to the formation of wrinkles. Furthermore, collagen plays a crucial role in wound healing, promoting collagen synthesis in damaged epithelium, thus enabling rapid and thorough wound recovery. Additionally, it has been reported that as collagen biosynthesis is promoted, the density of the basal layer and other layers increases, leading to a decrease in melanin concentration per unit skin density, thus potentially resulting in skin rejuvenation.

[0004] Against this technological backdrop, extensive research has been conducted on mechanisms to improve skin condition by promoting collagen biosynthesis, fibroblast proliferation, and enhancing activity (Korean Patent Registration No. 10-1813629), but it is still not perfect. Summary of the Invention

[0005] Technical issues

[0006] One aspect provides a peptide consisting of the amino acid sequence of SEQ ID NO: 1.

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

[0008] Other objects and advantages of this application will become clearer in conjunction with the appended claims and drawings, as well as the detailed description below. Regarding matters not described in this specification, which can be fully understood and deduced by those skilled in the art or similar fields, their descriptions are omitted.

[0009] Technical solution

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

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

[0012] As used in this specification, the term "peptide" can refer to a linear molecule formed by multiple amino acid residues linked together by peptide bonds. The peptide can be prepared using chemical synthesis methods known in the art, particularly solid-phase or liquid-phase synthesis techniques (US Patent No. 5,516,891). As a result of efforts to develop peptides with biologically effective activity, the inventors have identified a peptide consisting of the amino acid sequence of SEQ ID NO: 1. Here, biologically effective activity can refer to any of the following properties: (a) promoting the proliferation of fibroblasts or keratinocytes; (b) enhancing the expression of type I collagen (Col1a1), fibronectin, or elastin, which are components of the extracellular matrix; (c) enhancing the expression of skin barrier factors, namely, silent regulatory protein 1 (SIRT-1) or aquaporin 3 (AQP3); and (d) inhibiting apoptosis of fibroblasts or keratinocytes and reducing reactive oxygen species levels. Therefore, the peptide can be used for skin condition improvement or anti-oxidation.

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

[0014] The term "stability" as used in this specification may refer not only to the in vivo stability of the peptide against attack by biological proteases, but also to storage stability (e.g., room temperature storage stability).

[0015] On the other hand, a cosmetic composition for improving skin condition is provided, comprising a peptide consisting of the amino acid sequence of SEQ ID NO: 1 as an active ingredient.

[0016] In the description of the peptide, the same terms or elements as those already mentioned are as described above.

[0017] The term “improvement” as used in this specification may refer to parameters related to the mitigation or treatment of a condition, such as all behaviors that at least reduce symptoms.

[0018] The term "skin condition improvement" as used in this specification may refer to the process or effect of treating, reducing, or alleviating skin damage caused by endogenous or exogenous factors of the skin. For example, it may be interpreted as wrinkle improvement, skin elasticity improvement, wound healing, skin barrier strengthening, or skin aging inhibition, but is not limited to these.

[0019] Here, "wrinkle improvement," "skin elasticity improvement," and "wound healing" can refer to all effects of increasing the total amount of extracellular matrix factors, including promoting collagen synthesis. Furthermore, "skin barrier strengthening" can refer to strengthening the skin's inherent functions, namely preventing the leakage of moisture and nutrients from the skin and preventing harmful substances such as bacteria or viruses from invading the skin. Additionally, "skin aging inhibition" can refer to inhibiting the decline in skin function, such as wrinkles, sagging skin, and reduced elasticity. In this case, skin aging can be photoaging, such as skin aging caused by ultraviolet radiation.

[0020] The drawback of existing functional peptides is that, even if they possess effective biological activity, they cannot be effectively delivered to target tissues or cells due to their size, or they are eliminated from the body in a short time due to their short half-life. In contrast, a cosmetic composition according to one embodiment includes a peptide composed of 10 or fewer amino acids as an active ingredient, thus exhibiting excellent skin penetration and other properties, effectively improving skin condition when partially applied to the skin.

[0021] According to one embodiment, the peptide can promote the proliferation of fibroblasts and keratinocytes and enhance the synthesis of extracellular matrix constituents and skin barrier factors. Furthermore, it has been found that the peptide can restore the reduced activity of fibroblasts and keratinocytes and enhance their antioxidant efficacy. Therefore, the peptide can be used as an active ingredient in cosmetic compositions for improving skin condition.

[0022] The cosmetic composition may include, but is not limited to, a cosmetically effective amount of the peptide and / or a cosmetically acceptable carrier.

[0023] The term "cosmetically effective amount" as used in this specification refers to an amount sufficient to achieve the skin condition-improving effect of the cosmetic composition.

[0024] The weight ratio between the peptide and the cosmetically acceptable carrier can be, for example, 500:1 to 1:500, such as 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, but is not limited thereto.

[0025] The cosmetic composition can be prepared in any manner commonly prepared in the art, such as solutions, suspensions, emulsions, doughs, gels, creams, lotions, powders, soaps, surfactant-containing cleansers, oils, foundation creams, liquid foundations, wax foundations, and sprays, but is not limited thereto. For example, the cosmetic composition can be prepared as softening toners, nourishing toners, nourishing creams, massage creams, serums, eye creams, cleansing creams, cleansing foams, cleansing waters, masks, sprays, or powder formulations.

[0026] When the dosage form of the cosmetic composition is dough, skin cream or gel, animal oil, vegetable oil, wax, paraffin, starch, tragacanth gum, cellulose derivatives, polyethylene glycol, silicon, bentonite, silica, talc or zinc oxide may be used as carrier components.

[0027] When the dosage form of the cosmetic composition is a foundation or a spray, lactose, talc, silica, aluminum hydroxide, calcium silicate, or polyamide powder may be used as a carrier component. For example, when the dosage form of the cosmetic composition is a spray, it may further include accelerators such as chlorofluorocarbons, propane / butane, or dimethyl ether.

[0028] When the dosage form of the cosmetic composition is a solution or emulsion, solvents, solubilizers or emulsifiers may be used, such as water, ethanol, isopropanol, ethyl carbonate, ethyl acetate, benzyl alcohol, methyl benzoate, propylene glycol, 1,3-butylethylene glycol oil, glycerol aliphatic esters, polyethylene glycol or sorbitan fatty acid esters.

[0029] When 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 ester and polyoxyethylene sorbitan ester, microcrystalline cellulose, methyl aluminum hydroxide, bentonite, agar or tragacanth gum, etc., can be used.

[0030] When the dosage form of the cosmetic composition is a surfactant-containing cleanser, fatty alcohol sulfates, fatty alcohol ether sulfates, sulfosuccinate monoesters, hydroxyethyl sulfonates, imidazoline derivatives, methyl taurate, sarcosinates, fatty acid amide ether sulfates, alkylamide betaine, fatty alcohols, fatty acid glycerides, fatty acid diethanolamides, vegetable oils, lanolin derivatives, or ethoxylated glycerol fatty acid esters may be used as carrier components.

[0031] The peptides may be incorporated into nanocapsules or nanoparticles to further improve skin penetration or stability. For example, the nanocapsules may be prepared using lecithin as a raw material via a microfluidic apparatus and may be incorporated into lecithin particles. Any known method may be used to prepare the nanocapsules. The size of the nanocapsule particles is preferably between 30 nm and 200 nm. If the size of the nanocapsule particles is less than 30 nm, skin penetration may be very rapid, leading to skin side effects; if the size of the nanocapsule particles is greater than 200 nm, it may not easily penetrate the skin, making it difficult to achieve the desired therapeutic effect.

[0032] In addition to peptides and carrier components that are active ingredients, the cosmetic composition includes conventional ingredients used in cosmetic compositions, such as antioxidants, stabilizers, solubilizers, vitamins, pigments, and fragrances.

[0033] The content of peptides as active ingredients included in the cosmetic composition can be appropriately and non-limitingly selected according to the product form, desired use, etc. For example, peptides can be added at 0.01 to 15% by weight of the total weight of the cosmetic composition. Furthermore, for example, the cosmetic composition may include 1.0% to 3.0% by weight, preferably 2.0% to 3.0% by weight, of peptides based on the total weight.

[0034] On the other hand, a method for improving skin condition is provided, comprising the step of applying a cosmetic composition comprising a peptide consisting of the amino acid sequence of SEQ ID NO: 1 as an active ingredient to an individual's skin; and the use of the peptide consisting of the amino acid peptide of SEQ ID NO: 1 in the preparation of a composition for improving skin condition.

[0035] In the description of the cosmetic composition, the terms or elements that are the same as those already mentioned are as described above.

[0036] As used in this specification, the term "individual" refers to an object whose skin condition needs improvement, and more specifically, to humans or non-human primates, mice, dogs, hairy animals, horses, and other mammals such as cattle.

[0037] The terms “application,” “apply,” and “coat” used in this specification are used interchangeably and can refer to causing the composition according to an embodiment to be at least partially positioned at a desired site, or to the arrangement of the composition according to an embodiment within an individual via an application route.

[0038] On the other hand, an antioxidant composition is provided, comprising a peptide consisting of the amino acid sequence of SEQ ID NO: 1 as an active ingredient.

[0039] In the description of the peptide, the same terms or elements as those already mentioned are as described above.

[0040] The antioxidant composition may be in the form of a pharmaceutical composition, a quasi-pharmaceutical composition, or a cosmetic composition. As an example, the composition may be used as a cosmetic composition for improving skin condition or as a pharmaceutical composition for improving or treating the condition of skin damage-related diseases.

[0041] According to one embodiment, it was found that the peptide can restore the reduced activity of fibroblasts and keratinocytes and enhance the antioxidant efficacy of fibroblasts and keratinocytes. Therefore, the peptide can be used as an active ingredient in antioxidant compositions.

[0042] As an example, the antioxidant composition may be provided in the form of a pharmaceutical composition. The pharmaceutical composition may include, but is not limited to, a pharmaceutically effective amount of the peptide and / or a pharmaceutically acceptable carrier.

[0043] The term "pharmaceuticalally effective amount" as used in this specification may refer to an amount of the pharmaceutical composition sufficient to achieve preventive or therapeutic effects against skin lesion-related diseases.

[0044] Pharmaceutically acceptable carriers are those commonly used in preparation, including, but not limited to, lactose, dextran, sucrose, sorbitol, mannitol, starch, gum arabic, calcium phosphate, alginate, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, methylcellulose, methylparaben, propylparaben, talc, magnesium stearate, and mineral oil. Suitable pharmaceutically acceptable carriers and formulations are detailed in Remington's Pharmaceutical Sciences (Page 19, 1995).

[0045] The weight ratio between the peptide and the pharmaceutically acceptable carrier can be, for example, 500:1 to 1:500, such as 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, but is not limited thereto.

[0046] In addition to the ingredients mentioned above, the pharmaceutical composition may further include, but is not limited to, lubricants, humectants, sweeteners, flavorings, emulsifiers, suspending agents, preservatives, etc.

[0047] The pharmaceutical composition can be administered via the gastrointestinal tract or parenterally, preferably via parenteral administration. For parenteral administration, it can be administered via intramuscular injection, intravenous injection, subcutaneous injection, intraperitoneal injection, local application, percutaneous application, etc., but is not limited thereto.

[0048] The dosage of the pharmaceutical composition may be 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, but is not limited thereto, and may be prescribed differently depending on factors such as formulation method, administration method, patient's age, weight, gender, symptoms, diet, administration time, route of administration, excretion rate and reaction sensitivity.

[0049] The pharmaceutical composition may be formulated and prepared in a unit volume form using pharmaceutically acceptable carriers and / or excipients in a manner readily practiced by those skilled in the art, or may be prepared by injection into multi-volume containers.

[0050] The dosage form may be a solution, suspension or emulsion in an oily or aqueous solvent, or it may be an extractant, powder, granule, tablet or capsule, and may further include powder and / or stabilizer.

[0051] On the other hand, a food composition for improving skin condition is provided, comprising a peptide consisting of the amino acid sequence of SEQ ID NO: 1 as an active ingredient.

[0052] In the description of the peptide, the same terms or elements as those already mentioned are as described above.

[0053] The content of peptides as active ingredients included in the food composition may be appropriately and non-limitingly selected according to the food form, intended use, etc., for example, they may be added at a rate of 0.01 to 15% by weight of the total food. For example, for a health beverage composition, they may be added at a rate of 0.02 to 10 g, preferably 0.3 to 1 g, based on 100 ml.

[0054] Beneficial effects

[0055] By using peptides of one type, it is possible to promote the proliferation of fibroblasts and keratinocytes and enhance the synthesis of extracellular matrix constituents and skin barrier factors, thus it can be applied to improve skin condition, including wrinkle improvement, skin elasticity improvement, wound healing, skin barrier strengthening, or skin aging inhibition.

[0056] By targeting a specific peptide, it is possible to restore the reduced activity of fibroblasts and keratinocytes and enhance their antioxidant effects, thereby helping to restore skin damage that may be caused by external environmental factors such as ultraviolet radiation.

[0057] Therefore, peptides can be used as active ingredients in compositions for improving skin condition or in antioxidant compositions. Attached Figure Description

[0058] Figure 1 The diagram illustrates the confirmation of cell proliferation levels after adding a peptide consisting of the amino acid sequence of SEQ ID NO: 1 to skin cells, wherein... Figure 1 A represents the result of changes in the survival rate of mouse embryonic fibroblasts (NIH3T3). Figure 1 B represents the result of changes in the survival rate of human skin keratinocytes (HaCaT).

[0059] Figure 2 The results show that adding the peptide consisting of the amino acid sequence of SEQ ID NO: 1 to NIH3T3 cells resulted in increased expression of extracellular matrix components, namely type I collagen (Col1a1), fibronectin, and elastin.

[0060] Figure 3 This demonstrates a quantitative assessment of the expression levels of extracellular matrix components after adding a peptide consisting of the amino acid sequence of SEQ ID NO: 1 to NIH3T3 cells. Figure 3 A shows the results confirming the expression level of Col1a1. Figure 3 B shows the results confirming fibronectin expression levels. Figure 3 C indicates the results confirming the level of elastin expression.

[0061] Figure 4 The results show that adding the peptide consisting of the amino acid sequence of SEQ ID NO: 1 to HaCaT cells confirmed an increase in the expression of skin barrier factors, namely silencing regulatory protein 1 (SIRT-1) and aquaporin 3 (AQP3).

[0062] Figure 5This demonstrates a quantitative assessment of the expression levels of skin barrier factors after adding a peptide consisting of the amino acid sequence of SEQ ID NO: 1 to HaCaT cells. Figure 5 A shows the results confirming SIRT-1 expression levels. Figure 5 B shows the results confirming the AQP3 expression level.

[0063] Figure 6 The study demonstrates the inhibitory effect on UV-induced apoptosis in skin cells confirmed by viability assessment after the peptide, consisting of the amino acid sequence of SEQ ID NO: 1, was added to skin cells. Figure 6 A shows the results of changes in NIH3T3 cell survival. Figure 6 B shows the results of changes in HaCaT cell survival.

[0064] Figure 7 The study demonstrates the antioxidant effect on skin cells induced by ultraviolet radiation, confirmed by assessing reactive oxygen species levels after the peptide consisting of the amino acid sequence of SEQ ID NO: 1 was added to skin cells. Figure 7 Figure A shows the results of changes in reactive oxygen species levels in NIH3T3 cells. Figure 7 B shows the results of changes in reactive oxygen species levels in HaCaT cells. Detailed Implementation

[0065] The present invention will now be described in more detail through embodiments. However, these embodiments are merely illustrative of the invention, and the scope of the invention is not limited to these embodiments.

[0066] Example 1: Peptide Synthesis

[0067] Peptides with the amino acid sequence SEQ ID NO: 1 described in Table 1 below were synthesized using an automated peptide synthesizer (Milligen 9050, Millipore, USA), and the synthesized peptides were purified using a C18 reversed-phase high-performance liquid chromatography (HPLC) system (Waters Associates, USA). An ACQUITY UPLC BEH300 C18 column (2.1 mm × 100 mm, 1.7 µm, Waters Co., USA) was used as the column.

[0068] Table 1

[0069]

[0070] Example 2: Confirmation of the effect on promoting skin cell proliferation

[0071] This embodiment aims to confirm the effect of a peptide according to one embodiment on skin cell proliferation by evaluating the survival rate of mouse fibroblasts (NIH3T3 cells) or human keratinocytes (HaCaT cells).

[0072] Specifically, NIH3T3 cells or HaCaT cells were administered at a rate of 5 × 10⁻⁶. 3 Cells were seeded at a density of 96-well plates and cultured for 24 hours. The cells were then washed once with serum-free DMEM media, which was then used to replace the medium. A peptide consisting of the amino acid sequence of SEQ ID NO: 1 was then aliquoted into the medium at 50 µM or 100 µM and cultured in a CO2 incubator at 37°C for 72 hours. The culture products were then washed twice with phosphate buffered saline (PBS) and fixed with 4% paraformaldehyde (PFA) solution for 15 minutes. The culture products were then washed twice with distilled water, stained with sulforhodamine B (SRB) for 1 hour, washed with 1% acetic acid solution, and dried. The dried culture products were then dissolved in 20 mM Tris buffer, and the absorbance at 560 nm was measured using a microplate reader (Molecular Devices, USA). In this embodiment, the control group was the untreated group, and the group supplemented with insulin-like growth factor (IGF) was used as the positive control group.

[0073] As a result, it was confirmed that... Figure 1 As shown, a peptide consisting of the amino acid sequence of SEQ ID NO: 1 promotes the proliferation of fibroblasts and keratinocytes.

[0074] Example 3: Confirmation of wrinkle improvement and elasticity enhancement effects

[0075] This embodiment aims to confirm the effect of a peptide according to one embodiment on improving endogenous skin aging, including wrinkle reduction and improved elasticity, by measuring the expression levels of type I collagen (Col1a1), fibronectin, or elastin, which are known dermal components.

[0076] Specifically, NIH3T3 cells were used at a rate of 3 × 10⁻⁶. 5Cells were seeded at a density of 6-well plates and cultured for 24 hours. The cells were then washed once with serum-free DMEM media, and the medium was replaced with the same serum-free medium. A peptide consisting of the amino acid sequence of SEQ ID NO: 1 was then aliquoted into the medium at 50 µM or 100 µM and cultured in a CO2 incubator at 37°C for 24 hours. The culture was then washed twice with PBS, and ribonucleic acid (RNA) was isolated from the culture product using easy blue (iNtRON, Korea). Complementary deoxyribonucleic acids (cDNAs) were synthesized from 1 µg of the isolated RNA using an RT kit (Enzynomics, Korea). Polymerase chain reaction (PCR) was then performed using the cDNA, Col1a1, fibronectin, and elastin primers. The control group was the untreated group, and the group with added 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.

[0077] Table 2

[0078]

[0079] As a result, it was confirmed that... Figure 2 and 3 As shown, the peptide, composed of the amino acid sequence of SEQ ID NO: 1, increased the expression of Col1a1, fibronectin, and elastin, which are extracellular matrix building blocks. These results indicate that the peptide according to one embodiment contributes to the improvement of endogenous skin aging, including wrinkle reduction and increased elasticity, by increasing extracellular matrix building blocks.

[0080] Example 4: Confirmation of the skin barrier strengthening effect

[0081] This embodiment aims to confirm the effect of a peptide according to one embodiment on skin barrier strengthening by evaluating the expression levels of silencing regulatory protein 1 (SIRT-1) or aquaporin 3 (AQP3).

[0082] Specifically, HaCaT cells were prepared at a rate of 3 × 10⁻⁶. 5Cells were seeded at a density of 6-well plates and cultured for 24 hours. The cells were then washed once with serum-free DMEM media, and the medium was replaced with the same serum-free medium. A peptide consisting of the amino acid sequence of SEQ ID NO: 1 was then aliquoted into the medium at 50 µM or 100 µM and cultured in a CO2 incubator at 37°C for 24 hours. The cultures were then washed twice with PBS, and RNA was isolated from the culture products using easy blue (iNtRON, Korea). 1 µg of the isolated RNA was reverse transcribed using an RT kit (Enzynomics, Korea) to synthesize cDNAs. Polymerase chain reaction (PCR) was then performed using the cDNAs, silencing regulatory protein-1, and aquaporin-3 primers. A control group served as the untreated group, and a group supplemented with epidermal growth factor (EGF) served as the positive control group. The nucleotide sequences of the primers used in this embodiment are shown in Table 3 below.

[0083] Table 3

[0084]

[0085] As a result, it was confirmed that... Figure 4 and Figure 5 As shown, the expression of SIRT-1 and AQP3, skin barrier factors, was increased by a peptide composed of the amino acid sequence of SEQ ID NO: 1. These results indicate that the peptide according to one embodiment contributes to skin barrier strengthening and anti-aging by increasing skin barrier factors.

[0086] Example 5: Confirmation of the inhibitory effect of ultraviolet-induced skin cell apoptosis

[0087] This embodiment aims to confirm the effect of a peptide according to one embodiment on the inhibition or cell recovery of skin cell apoptosis induced by ultraviolet radiation by evaluating changes in the survival rate of skin cells that have undergone apoptosis induced by ultraviolet radiation.

[0088] Specifically, NIH3T3 cells or HaCaT cells were administered at a rate of 1×10⁻⁶. 4Cells were seeded at a density of 96-well plates and cultured for 24 hours. The cells were then washed once with serum-free DMEM media, and the serum-free medium was used to replace the culture medium. A peptide consisting of the amino acid sequence of SEQ ID NO: 1 was aliquoted into the serum-free medium at 50 µM or 100 µM. The aliquots containing the peptide were cultured in a CO2 incubator at 37°C for 1 hour, and then transferred to an e-tube and mixed with 100 µl of PBS for aliquoting into the wells. The cells were then irradiated with 6 J / cm² of UV light using a NIH3T3 cell irradiator (VILBER LOURMAT, France). 2 The ultraviolet light was used to irradiate HaCaT cells at 15 mJ / cm². 2 The cells were then exposed to ultraviolet light. After removing the PBS from the wells and adding 900 µL of culture medium containing the peptide, the cells were incubated at 37°C in a CO2 incubator for 72 hours. The culture products were then washed twice with phosphate buffered saline (PBS) and fixed with 4% paraformaldehyde (PFA) solution for 15 minutes. The culture products were then washed twice with distilled water, stained with SRB solution for 1 hour, washed with 1% acetic acid solution, and dried. The dried culture products were then dissolved in 20 mM Tris buffer, and the absorbance at 560 nm was measured using a microplate reader (Molecular Devices, USA). In this example, the control group was the untreated group, and the group supplemented with Trolox (Trolox) served as the positive control group.

[0089] As a result, it was confirmed that... Figure 6 As shown, a peptide consisting of the amino acid sequence of SEQ ID NO: 1 restored the cell viability of fibroblasts and keratinocytes reduced by ultraviolet radiation. The results indicate that the peptide according to one embodiment helps to inhibit apoptosis of skin cells induced by ultraviolet radiation.

[0090] Example 6: Confirmation of the antioxidant effect of ultraviolet light induced in skin cells

[0091] This embodiment aims to confirm the effect of a peptide according to one embodiment on the antioxidant effect in skin cells induced by ultraviolet radiation by evaluating the increased level of reactive oxygen species in skin cells due to ultraviolet radiation.

[0092] Specifically, NIH3T3 cells or HaCaT cells were administered at a rate of 5 × 10⁻⁶. 5Cells were seeded at a density of 6-well plates and cultured for 24 hours. The cells were then washed once with serum-free DMEM media, and the medium was replaced with the same serum-free medium. Peptides consisting of the amino acid sequence of SEQ ID NO: 1 were aliquoted into the serum-free medium at 50 µM or 100 µM. The aliquoted peptides were cultured in a CO2 incubator at 37°C for 1 hour, and the medium was then transferred to an e-tube and mixed with 100 µl of PBS for aliquoting into the wells. The cells were then irradiated with 6 J / cm² of UV light using a NIH3T3 cell irradiator (VILBER LOURMAT, France). 2 The ultraviolet light was used to irradiate HaCaT cells at 15 mJ / cm². 2 The PBS was then removed from the wells, and 900 µL of culture medium containing the peptide was added. The culture was then incubated at 37°C in a CO2 incubator for 24 hours. The culture was then treated with 2',7'-dichlorofluorescin diacetate (DCFG-DA), the culture was wrapped in foil, and incubated at 37°C in a CO2 incubator for 30 minutes. The culture was then washed twice with PBS, and cells were obtained using 500 µL of 1×trypsin / EDTA and 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, and the group supplemented with Trolox (Trolox) served as the positive control group.

[0093] As a result, it was confirmed that... Figure 7 As shown, the peptide, composed of the amino acid sequence of SEQ ID NO: 1, reduces the levels of reactive oxygen species in fibroblasts and keratinocytes that are increased by ultraviolet radiation. The results indicate that the peptide according to one embodiment contributes to the antioxidant effect of ultraviolet-induced skin cells.

[0094] Dosage form example 1: softening lotion

[0095] A soft toner comprising a peptide according to one embodiment and having the following composition was prepared using methods known in the art.

[0096] Table 4

[0097]

[0098] Dosage Form Example 2: Nourishing Cream

[0099] A nourishing cream comprising a peptide according to one embodiment and having the following composition was prepared using methods known in the art.

[0100] Table 5

[0101]

[0102] Dosage form example 3: Nourishing toner

[0103] A nourishing toner comprising a peptide according to an embodiment and having the following composition was prepared using methods known in the art.

[0104] Table 6

[0105]

[0106] Dosage form example 4: serum

[0107] An essence comprising a peptide according to one embodiment and having the following composition was prepared using methods known in the art.

[0108] Table 7

[0109]

[0110] The description of this invention is for illustrative purposes only, and those skilled in the art should understand that the invention can be readily modified into other specific forms without changing the technical concept or essential features of the invention. Therefore, the embodiments described above should be understood as exemplary in all respects, and not as limiting. sequence list <110> CAREGEN CO., LTD. <120> Peptides with skin condition-improving activity and their uses <130> PX067297PCT <160> 13 <170> KoPatentIn 3.0 <210> 1 <211> 9 <212> PRT <213> Artificial sequence <220> <223> Peptide 1 <400> 1 Glu Ala Leu Lys Tyr Trp Tyr Glu Asn 1 5 <210> 2 <211> 20 <212> DNA <213> Artificial sequence <220> <223> Col1a1_F <400> 2 caccctcaag agcctgagtc 20 <210> 3 <211> 20 <212> DNA <213> Artificial sequence <220> <223> Col1a1_R <400> 3 agacggctga gtagggaaca 20 <210> 4 <211> 20 <212> DNA <213> Artificial sequence <220> <223> Fibronectin_F <400> 4 ccaggaaccg agtacaccat 20 <210> 5 <211> 20 <212> DNA <213> Artificial sequence <220> <223> Fibronectin_R <400> 5 atacccaggt tgggtgatga 20 <210> 6 <211> 20 <212> DNA <213> Artificial sequence <220> <223> Elastin_F <400> 6 gcaagacctg gctttggact 20 <210> 7 <211> twenty one <212> DNA <213> Artificial sequence <220> <223> Elastin_R <400> 7 gggagtttct ggttagggct g 21 <210> 8 <211> 20 <212> DNA <213> Artificial sequence <220> <223> GAPDH_F <400> 8 ggagccaaaa gggtcatcat 20 <210> 9 <211> 20 <212> DNA <213> Artificial sequence <220> <223> GAPDH_R <400> 9 gtgatggcat ggactgtggt 20 <210> 10 <211> 20 <212> DNA <213> Artificial sequence <220> <223> SIRT1_F <400> 10 tcagtggctg gaacagtgag 20 <210> 11 <211> 20 <212> DNA <213> Artificial sequence <220> <223> SIRT1_R <400> 11 tctggcatgt cccactatca 20 <210> 12 <211> 20 <212> DNA <213> Artificial sequence <220> <223> AQP3_F <400> 12 ccttcttggg tgctggaata 20 <210> 13 <211> 20 <212> DNA <213> Artificial sequence <220> <223> AQP3_R <400> 13 acacgataag ggaggctgtg 20

Claims

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

1.

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

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

4. A cosmetic composition for improving skin condition, comprising a peptide as an active ingredient according to any one of claims 1 to 3.

5. Use of the peptide consisting of the amino acid sequence of SEQ ID NO: 1 in the preparation of a composition for improving skin condition, said skin condition improvement being wound healing or inhibition of skin aging.

6. The use according to claim 5, wherein, The skin aging mentioned refers to skin aging caused by ultraviolet radiation.

7. Use of a peptide consisting of the amino acid sequence of SEQ ID NO: 1 in the preparation of a composition for wrinkle improvement.

8. Use of a peptide consisting of the amino acid sequence of SEQ ID NO: 1 in the preparation of a composition for improving skin elasticity.

9. Use of the peptide consisting of the amino acid sequence of SEQ ID NO: 1 in the preparation of compositions for strengthening the skin barrier.

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