Application of bolete peptide and salt thereof in cosmetics

Abstract

Disclosed is the use of a bolete peptide or a salt thereof in cosmetics, the bolete peptide comprising an amino acid selected from the group consisting of leucine (Leu), isoleucine (Ile), tyrosine (Tyr), alanine (Ala), glutamine (Gln), glycine (Gly), phenylalanine (Phe), serine (Ser), and lysine (Lys). In cosmetics, in order to achieve multiple effects, multiple functional peptides often need to be used at the same time, the formula is complicated, the cost is increased, the application range of the functional peptides such as blue copper peptide has certain limitation, and the bolete peptide or the salt thereof has the advantages of promoting generation of hyaluronic acid, promoting synthesis of AQP3 protein, improving the immunity and the like. Under the irradiation of UVA, the generation of MMP-1 is inhibited, the I-type collagen is protected, and the proliferation and migration of keratinocytes are promoted, so that certain moisturizing, anti-aging and repairing effects are simultaneously exerted on the skin.

Description

Technical Field

[0001] The invention relates to cosmetics, dermatological products and hygiene and personal care products, and in particular to use of boletin or a salt thereof in cosmetics. Background Art

[0002] As we age, our skin will show a series of signs of aging, including a decrease in the rate of epidermal renewal, weakened skin barrier function, reduced activity of keratinocytes, and reduced hydration capacity of the stratum corneum. These changes lead to flaking and dryness of the skin. At the same time, reduced sebum secretion and increased water loss make skin cells more susceptible to wrinkling and aging.

[0003] There are many kinds of anti-aging products on the market, and their functions include anti-oxidation, anti-glycation, collagen supplementation, etc. However, in order for all these products to fully exert their effects, the premise is that the skin must have sufficient moisture so that these products can penetrate deeply into the skin. Therefore, the reduction of skin water content will not only lead to dry skin, dull skin, sagging and wrinkles, but also affect the efficacy of other products. Therefore, if you want to improve the anti-aging state of the skin, replenishing moisture and keeping the skin moist is an essential step.

[0004] The stratum corneum is the outermost layer of the skin surface. Its main function is to prevent water evaporation and avoid the invasion of external harmful substances and microorganisms. The thickness and density of the stratum corneum of different individuals may vary due to various factors such as genetics, environment, and age. A weak or damaged stratum corneum will make the skin more sensitive and dry, and susceptible to damage and infection. In addition, the loss of the stratum corneum will also lead to the rapid evaporation of skin moisture, causing the skin to lose softness and elasticity, and accelerate the aging process of the skin.

[0005] In the field of cosmetics, one solution used for moisturizing is to combine a peptide that can increase the content of aquaporin 3 with a peptide that promotes the production of hyaluronic acid and / or collagen:

[0006] Aquaporin 3 (AQP3) can transport small molecules such as water and glycerol across the membrane to the epidermis and participate in the hydration of the stratum corneum. Hyaluronic acid (HA) can strengthen the retention of water in the dermis and form a water storage reservoir. Using ingredients that promote the production of aquaporin 3 and ingredients that promote the production of hyaluronic acid at the same time, multiple targets work synergistically, which can fully, instantly, efficiently and lastingly hydrate and moisturize the skin, and also play a positive role in anti-aging and maintaining skin health.

[0007] In addition to aging as we age, our skin also ages faster due to exposure to UV rays. When the skin is exposed to UV rays (especially UVA), it increases the production of MMP-1 in skin cells. Matrix metalloproteinase-1 (MMP-1) is an important enzyme in the skin, and its main function is to break down collagen in the dermis. Collagen itself is an important substance for maintaining skin elasticity and smoothness. Therefore, too much MMP-1 can cause skin sagging and the appearance of fine lines, wrinkles and other signs of aging.

[0008] In the cosmetics field, a peptide widely used to solve the problem of weak stratum corneum and resist the production of matrix metalloproteinase MMP-1 under ultraviolet radiation is copper peptide (CopperTripeptide). Copper peptide has a strong healing and regenerative ability, which can stimulate the skin to produce collagen and elastin, enhance the firmness and elasticity of the skin, help to increase the thickness of the stratum corneum, and prevent the loss of collagen caused by excessive production of MMP-1.

[0009] However, due to the structural characteristics of blue copper peptide itself, there is still a limitation of low skin permeability. Although the molecular weight of blue copper peptide is less than 500Da, its hydrophilicity and charged state lead to its relatively low permeability, which is not easy to pass through the skin. Blue copper peptide cannot be used at the same time as some other active ingredients (such as vitamin C) because they may offset each other's effectiveness. The use of blue copper peptide and some other ingredients (such as antioxidants) may cause instability and shorten the shelf life of the product. The formula composition is limited to a certain extent. Compared with other more common ingredients, the manufacturing cost of blue copper peptide is higher, so products containing blue copper peptide are usually more expensive.

[0010] The efficacy or effect of the amino acid sequence of Leu-Ile-Tyr-Ala-Gln-Gly-Phe-Ser-Lys as shown in SEQ ID NO: 1, or the salt of the amino acid sequence claimed by the present invention has not been reported or disclosed in the prior art. Summary of the invention

[0011] Based on this, the present invention provides a use of a bolete peptide or a salt thereof in cosmetics, wherein the bolete peptide comprises amino acids selected from the group consisting of leucine (Leu), isoleucine (Ile), tyrosine (Tyr), alanine (Ala), glutamine (Gln), glycine (Gly), phenylalanine (Phe), serine (Ser) and lysine (Lys).

[0012] According to another aspect of the present invention, there is provided a use of a boletin or a salt thereof as an active ingredient in a non-therapeutic cosmetic treatment of the skin and / or its appendages, wherein the boletin comprises amino acids selected from the group consisting of leucine (Leu), isoleucine (Ile), tyrosine (Tyr), alanine (Ala), glutamine (Gln), glycine (Gly), phenylalanine (Phe), serine (Ser) and lysine (Lys).

[0013] Furthermore, the boletin contains at least one, for example, 1 to 2, leucines (Leu).

[0014] Furthermore, the boletin contains at least one, for example, 1 to 2 isoleucines (Ile).

[0015] Furthermore, the boletin contains at least one, for example, 1 to 2, tyrosines (Tyr).

[0016] Furthermore, the boletin contains at least one, for example, 1 to 2, alanines (Ala).

[0017] Furthermore, the boletin contains at least one, for example, 1 to 2, glutamines (Gln).

[0018] Furthermore, the boletin contains at least one, for example, 1 to 2 glycine (Gly).

[0019] Furthermore, the boletin contains at least one, for example, 1 to 2, phenylalanine (Phe).

[0020] Furthermore, the boletin contains at least one, for example, 1 to 2, serines (Ser).

[0021] Furthermore, the boletin contains at least one, for example, 1 to 2 lysines (Lys).

[0022] Furthermore, the boletin contains at least one, for example, 1 to 2 Leu-Ile-Tyr sequences.

[0023] Furthermore, the boletin contains at least one, for example, 1 to 2, Ile-Tyr-Ala sequences.

[0024] Furthermore, the boletin contains at least one, for example, 1 to 2, Tyr-Ala-Gln sequences.

[0025] Furthermore, the boletin contains at least one, for example, 1 to 2, Ala-Gln-Gly sequences.

[0026] Furthermore, the boletin contains at least one, for example, 1 to 2, Gln-Gly-Phe sequences.

[0027] Furthermore, the boletin contains at least one, for example, 1 to 2, Gly-Phe-Ser sequences.

[0028] Furthermore, the boletin contains at least one, for example, 1 to 2, Phe-Ser-Lys sequences.

[0029] Furthermore, the boletin contains 8 to 10 amino acids, for example, 9 amino acids.

[0030] Further, the boletus peptide comprises the amino acid sequence of Leu-Ile-Tyr-Ala-Gln-Gly-Phe-Ser-Lys as shown in SEQ ID NO: 1, optionally the amino acid sequence of SEQ ID NO: 1 with one or more amino acid residues substituted, deleted and / or inserted, optionally the amino acid sequence having an identity of about 88.9% with the amino acid sequence of SEQ ID NO: 1.

[0031] The amino acid sequence of Leu-Ile-Tyr-Ala-Gln-Gly-Phe-Ser-Lys (LIYAQGFSK) of the present invention as shown in SEQ ID NO: 1 is commercially available or derived from peptide chain synthesis.

[0032] Furthermore, the amino acid sequence of Leu-Ile-Tyr-Ala-Gln-Gly-Phe-Ser-Lys shown in SEQ ID NO: 1 is obtained by artificially synthesizing a peptide chain.

[0033] Furthermore, the use includes the use of moisturizing the skin, the use of anti-skin aging and / or the use of repairing the skin.

[0034] Furthermore, the moisturizing includes promoting the production of hyaluronic acid (HA) and / or promoting the synthesis of aquaporin 3 (AQP3).

[0035] Furthermore, the anti-skin aging comprises inhibiting the generation of matrix metalloproteinase-1 (MMP-1) under ultraviolet irradiation and / or protecting type I collagen produced by skin fibroblasts under ultraviolet irradiation.

[0036] Further, the repair includes promoting keratinocyte proliferation and / or migration.

[0037] Further, the cosmetic treatment includes improving dryness of the skin, improving peeling of the skin, improving a weak stratum corneum of the skin, reducing fine lines on the skin, reducing wrinkles on the skin and / or preventing skin aging.

[0038] According to another aspect of the present invention, there is provided a use of a cosmetic composition comprising boletin or a salt thereof in non-therapeutic cosmetic treatment of the skin and / or its appendages, wherein the weight range of the boletin or a salt thereof is 0.000001% to 10%, preferably 0.000001% to 5%, for example 0.000001% to 1%, based on the total weight of the cosmetic composition, wherein the boletin comprises an amino acid selected from the group consisting of leucine (Leu), isoleucine (Ile), tyrosine (Tyr), alanine (Ala), glutamine (Gln), glycine (Gly), phenylalanine (Phe), serine (Ser) and lysine (Lys).

[0039] Furthermore, the cosmetic composition further comprises an active agent for enhancing the effect of the boletin or its salt.

[0040] Furthermore, the active agent is selected from one or more of the following: peptides, vitamin C and its derivatives, vitamins from group B, dehydroepiandrosterone (DHEA), phytosterols, salicylic acid and its derivatives, retinoids, flavonoids, amino sugars, azoles and metal salts.

[0041] Furthermore, the cosmetic further comprises cosmetic auxiliary materials.

[0042] Furthermore, the cosmetic auxiliary material is selected from one or more of the following: thickener, surfactant, co-surfactant, skin conditioner, preservative, flavor, lubricant, disintegrant, wetting agent, adhesive and filler.

[0043] According to another aspect of the present invention, there is provided a method for improving the aesthetic appearance of the skin, the method comprising topically applying an effective amount of a cosmetic composition comprising a boletin peptide or a salt thereof, the weight range of the boletin peptide or a salt thereof being 0.000001% to 10%, preferably 0.000001% to 5%, for example 0.000001% to 1%, based on the total weight of the cosmetic composition, wherein the boletin peptide comprises an amino acid selected from the group consisting of leucine (Leu), isoleucine (Ile), tyrosine (Tyr), alanine (Ala), glutamine (Gln), glycine (Gly), phenylalanine (Phe), serine (Ser) and lysine (Lys).

[0044] Beneficial effects of the present invention:

[0045] In cosmetics, in order to achieve multiple functions, it is often necessary to use multiple functional peptides at the same time, which not only complicates the formula but also increases the cost. In addition, the application scope of functional peptides such as blue copper peptides is limited. The amino acid sequence of Leu-Ile-Tyr-Ala-Gln-Gly-Phe-Ser-Lys or the salt of the amino acid sequence as shown in SEQ ID NO: 1 of the present invention has a multi-functional single peptide that replaces multiple peptides. It has the effects of promoting the production of hyaluronic acid (HA), promoting the synthesis of AQP3 protein, protecting type I collagen (UVA irradiation) and inhibiting the production of MMP-1 (UVA irradiation), promoting the proliferation of keratinocytes and the migration of keratinocytes, thereby exerting certain moisturizing, anti-aging and repairing effects on the skin at the same time. BRIEF DESCRIPTION OF THE DRAWINGS

[0046] In order to more clearly illustrate the technical solutions in the embodiments of the present invention, the drawings required for use in the description of the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without exceeding the scope of protection required by the present invention.

[0047] Figure 1 The figure is a schematic diagram of the results of the boletus peptide of the present invention promoting the synthesis of hyaluronic acid (HA). Among them, BC: blank control (HaCaT cells), PC: positive control (0.0001 mg / mL TGF-β1), compared with BC, * represents P < 0.05, ** Represents P < 0.01.

[0048] Figure 2 The figure is a schematic diagram of the results of the boletus peptide promoting the synthesis of aquaporin 3 (AQP3) of the present invention. Among them, BC: blank control (HaCaT cells), PC: positive control (0.375 mol / mL CaCl2), compared with BC, * represents P < 0.05, ** Represents P < 0.01.

[0049] Figure 3 The figure is a schematic diagram of the results of the boletus peptide protecting type I collagen (UVA) synthesis of the present invention. BC: blank control (HFF-1 cells), NC: negative control (9J / cm 2 UVA), PC: positive control (100μg / mL VC + 7μg / mL VE), compared with BC, # represents P < 0.05, * represents P < 0.05, ** Represents P < 0.01.

[0050] Figure 4 The figure is a schematic diagram of the results of the boletus peptide of the present invention inhibiting the synthesis of MMP-1 (UVA). BC: blank control (HFF-1 cells), NC: negative control (9J / cm 2 UVA), PC: positive control (100μg / mL VC+7μg / mL VE), compared with BC, # represents P < 0.05, * represents P < 0.05, ** represents P < 0.01, *** Represents P < 0.001.

[0051] Figure 5 The figure is a schematic diagram of the results of the bovine peptide promoting keratinocyte proliferation of the present invention. BC: blank control (HaCaT cells), PC: positive control (10% FBS), compared with BC, * represents P < 0.05, ** Represents P < 0.01.

[0052] Figure 6 The figure is a schematic diagram of the results of the bovine peptide promoting keratinocyte migration of the present invention. BC: blank control (HaCaT cells), PC: positive control (10% FBS), compared with BC, * represents P < 0.05, ** Represents P < 0.01. DETAILED DESCRIPTION

[0053] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative work are within the scope of protection of the present invention.

[0054] Unless otherwise indicated, all technical and scientific terms and abbreviations used herein have the meanings commonly understood by those of ordinary skill in the field of the invention or the field in which the terms are used. Although any methods, conditions, substances or materials similar to or equivalent to those disclosed herein may be used in the practice of the present invention, preferred methods, conditions, substances or materials are described herein.

[0055] The present invention comprises multiple aspects, features and embodiments, wherein such multiple aspects, features and embodiments can be combined and modified in any desired manner. These and other aspects, features and embodiments of the present invention will become apparent when referring to the rest of the present invention, including the following detailed description.

[0056] Unless the context clearly indicates otherwise, as used herein, the singular form of a word includes the plural form and vice versa. Thus, "a", "an" and "the" generally include the plural form of the corresponding term. For example, "a boletin or a salt thereof" includes the plural form of the "boletin or a salt thereof". Similarly, the term "for example", especially when preceding a series of terms, is merely exemplary and illustrative and should not be considered exclusive or comprehensive.

[0057] The present invention is intended to encompass all alternatives, variations and equivalents that may be included in the present invention as defined by the claims. Those skilled in the art will recognize many methods and materials similar or equivalent to those described herein that can be used in the practice of the present invention. The present invention is in no way limited to the methods and materials described.

[0058] It should be noted that any process not specifically described below can be implemented or understood by those skilled in the art with reference to the prior art. Reagents and instruments used without manufacturer indicated are considered to be conventional products that can be purchased commercially.

[0059] Unless otherwise specified, the practice of the present invention will adopt the conventional techniques of cell biology, cell culture, molecular biology, transgenic biology, microbiology, recombinant DNA and immunology. Unless otherwise defined, all technical and scientific terms used herein are identical with the meanings generally understood by the technicians belonging to the technical field of the present invention. The terms used in the specification of the present invention herein are only for the purpose of describing specific embodiments and are not intended to limit the present invention.

[0060] To the extent that the terms "comprises," "including," and "having," "has," and "with," or variations thereof, are used in the detailed description and / or claims, these terms are intended to be inclusive in a manner similar to the term "comprising."

[0061] As described in the background technology section, with the increase of age, the skin has technical problems such as weak stratum corneum, skin damage caused by ultraviolet rays, and skin dehydration, and the application scope of functional peptides such as blue copper peptides has certain limitations. In order to solve the above problems, the present invention provides a use of boletin or its salt in cosmetics, wherein the boletin contains amino acids selected from the group consisting of: leucine (Leu), isoleucine (Ile), tyrosine (Tyr), alanine (Ala), glutamine (Gln), glycine (Gly), phenylalanine (Phe), serine (Ser) and lysine (Lys).

[0062] According to another aspect of the present invention, there is provided a use of a boletin or a salt thereof as an active ingredient in a non-therapeutic cosmetic treatment of the skin and / or its appendages, wherein the boletin comprises amino acids selected from the group consisting of leucine (Leu), isoleucine (Ile), tyrosine (Tyr), alanine (Ala), glutamine (Gln), glycine (Gly), phenylalanine (Phe), serine (Ser) and lysine (Lys).

[0063] In a preferred embodiment, the boletin contains at least one, for example, 1 to 2, leucines (Leu).

[0064] In a preferred embodiment, the boletin contains at least one, for example, 1 to 2 isoleucines (Ile).

[0065] In a preferred embodiment, the boletin contains at least one, for example, 1 to 2, tyrosines (Tyr).

[0066] In a preferred embodiment, the boletin contains at least one, for example 1 to 2, alanines (Ala).

[0067] In a preferred embodiment, the boletin contains at least one, for example, 1 to 2, glutamines (Gln).

[0068] In a preferred embodiment, the boletin contains at least one, for example, 1 to 2 glycine (Gly).

[0069] In a preferred embodiment, the boletin contains at least one, for example, 1 to 2, phenylalanine (Phe).

[0070] In a preferred embodiment, the boletin contains at least one, for example, 1 to 2, serines (Ser).

[0071] In a preferred embodiment, the boletin contains at least one, for example, 1 to 2 lysines (Lys).

[0072] In a preferred embodiment, the boletin contains at least one, for example, 1 to 2 Leu-Ile-Tyr sequences.

[0073] In a preferred embodiment, the boletin contains at least one, for example, 1 to 2, Ile-Tyr-Ala sequences.

[0074] In a preferred embodiment, the boletin contains at least one, for example, 1 to 2, Tyr-Ala-Gln sequences.

[0075] In a preferred embodiment, the boletin contains at least one, for example, 1 to 2, Ala-Gln-Gly sequences.

[0076] In a preferred embodiment, the boletin contains at least one, for example, 1 to 2, Gln-Gly-Phe sequences.

[0077] In a preferred embodiment, the boletin contains at least one, for example, 1 to 2, Gly-Phe-Ser sequences.

[0078] In a preferred embodiment, the boletin contains at least one, for example, 1 to 2, Phe-Ser-Lys sequences.

[0079] In a preferred embodiment, the boletin contains 8 to 10 amino acids, such as 9 amino acids.

[0080] In a preferred embodiment, the boletin comprises the amino acid sequence of Leu-Ile-Tyr-Ala-Gln-Gly-Phe-Ser-Lys as shown in SEQ ID NO: 1, optionally the amino acid sequence of SEQ ID NO: 1 with one or more amino acid residues substituted, deleted and / or inserted, optionally the amino acid sequence having a degree of identity of about 88.9% with the amino acid sequence of SEQ ID NO: 1.

[0081] Compared with blue copper peptide, the boletus peptide or its salt of the present invention will not have lower permeability due to charge. There are many related studies on boletus in the food industry, and its safety has been fully proven. At present, the king boletus small molecule active peptide has not been applied to the cosmetics field in China.

[0082] The amino acid sequence of Leu-Ile-Tyr-Ala-Gln-Gly-Phe-Ser-Lys shown in SEQ ID NO: 1 of the present invention is commercially available or derived from artificial peptide chain synthesis.

[0083] Synthesis scheme: Place 2mmol CTC resin in a 100mL solid phase synthesis reactor, add 4mmol Fmoc-Lys(Boc)-OH, 20mL DCM, 2mL DIEA, react at 25°C for 3 hours, add 3mL methanol, react for 5 minutes. Filter, wash the resin with 20mL DCM, methanol, and DMF twice each. Add 20mL of 20% Pip / DMF solution, stir and react for 30min, filter to remove the deprotection solution, then wash 6 times with 20mL DMF solution, and drain for use.

[0084] Take 4mmol Fmoc-Ser(tBu)-OH, 4mmol HOBt in a 50mL beaker, cool to 2-8℃, add 2mL DMF, 4mmol DIC and let stand for 10-20 minutes, add the solution in the beaker to the solid phase synthesis reactor, stir and react for 1.5 hours, and the reaction is complete. Wash the resin with DMF solution three times, 20mL each time. After washing, add 20% Pip / DMF solution 20mL, stir and react for 30min, filter and remove the deprotection solution, then wash 6 times with 5mL DMF solution, and drain for standby use. According to the above operation, Fmoc-Phe-OH, Fmoc-Gly-OH, Fmoc-Gln(Trt)-OH, Fmoc-Ala-OH, Fmoc-Tyr(tBu)-OH, Fmoc-Ile-OH, Fmoc-Leu-OH are coupled sequentially.

[0085] Then, 5 mL of methanol was added to wash twice, DCM was added twice, and methanol was added twice, and vacuum dried to obtain peptide resin H-Leu-Ile-Tyr(tBu)-Ala-Gln(Trt)-Gly-Phe-Ser(tBu)-Lys(Boc)-CTC.

[0086] The obtained peptide resin was cut with 40 mL of cutting solution TFA / thioanisole / PhOH / H2O / EDT=87.5 / 5 / 2.5 / 2.5 / 2.5 at 30°C for 2.5 hours, and the cutting solution was added to 800 mL of ether (2-8°C) solution to precipitate a white solid. After centrifugation, a white solid crude peptide was obtained. After vacuum drying, a crude peptide powder H-Leu-Ile-Tyr-Ala-Gln-Gly-Phe-Ser-Lys-OH was obtained.

[0087] In a preferred embodiment, the use includes use for moisturizing the skin, use for resisting skin aging and / or use for repairing the skin.

[0088] In a preferred embodiment, the moisturizing includes promoting the production of hyaluronic acid (HA) and / or promoting the synthesis of aquaporin 3 (AQP3).

[0089] Among them, hyaluronic acid (HA), also known as hyaluronic acid or sodium hyaluronate, is present in many tissues and fluids of the human body, including the skin. Hyaluronic acid is well known for its strong water storage capacity. It can form hydrogen bonds with water to bind a large amount of water, forming a gel-like matrix that can absorb and lock up to 1,000 times its own water. It is an important water storage reservoir for the entire skin and is very effective in maintaining the moisture of the skin. In addition, hyaluronic acid also has the effect of improving fine lines and wrinkles on the skin. By replenishing the moisture of the skin, it can help the skin improve and maintain its structure, making the skin look fuller and reducing fine lines and wrinkles; aquaporin 3 (AQP3) plays a vital role in skin moisturizing. AQP3 is a special transmembrane protein, most of which is located in the basal layer and spinous layer of the epidermis. It is mainly responsible for regulating and controlling the transport of water molecules and glycerol between cells, and its function is to help maintain the moisture balance of the skin. It can transport water molecules from the body to the epidermis, promote the hydration of the stratum corneum, thereby helping to maintain the moisture of the skin and prevent dryness and peeling of the skin. In addition, AQP3 can also help maintain the skin's barrier function. The skin's barrier function is an important mechanism to prevent water loss and the invasion of harmful external substances. AQP3 can help maintain the integrity of the skin barrier by regulating the water balance inside and outside the skin cells, thereby protecting the skin from damage by the external environment.

[0090] In a preferred embodiment, the anti-skin aging comprises inhibiting the production of matrix metalloproteinase-1 (MMP-1) under ultraviolet irradiation and / or protecting type I collagen produced by skin fibroblasts under ultraviolet irradiation.

[0091] Among them, ultraviolet radiation can cause skin cells to produce too much matrix metalloproteinase MMP-1, which leads to the degradation of collagen, accelerating skin aging and the formation of wrinkles. The use of the amino acid sequence of Leu-Ile-Tyr-Ala-Gln-Gly-Phe-Ser-Lys or the salt of the amino acid sequence as shown in SEQ ID NO: 1 of the present invention can inhibit the generation of MMP-1 under ultraviolet radiation and prevent collagen from being enzymatically hydrolyzed; direct testing of type I collagen content also proves that the collagen of the skin can be protected, thereby preventing skin aging and the formation of wrinkles, and solving the technical problem of skin wrinkles caused by ultraviolet radiation.

[0092] In a preferred embodiment, the repair is to promote keratinocyte proliferation and / or migration.

[0093] Among them, the proliferation and migration of skin cells are the key processes of keratin formation. However, due to the influence of age, environmental factors and other factors, the skin often has the problem of a weak stratum corneum. The use of the amino acid sequence of Leu-Ile-Tyr-Ala-Gln-Gly-Phe-Ser-Lys or the salt of the amino acid sequence as shown in SEQ ID NO: 1 of the present invention can stimulate the proliferation and migration of skin cells, thereby promoting the formation and thickening of the stratum corneum, repairing the stratum corneum barrier, and solving the technical problem of a weak stratum corneum. The thickness and health of the stratum corneum will also affect the moisturizing ability of the skin. By promoting the proliferation and migration of keratinocytes, the polypeptide can indirectly help maintain the moisturizing ability of the skin and alleviate problems such as dry skin.

[0094] In a preferred embodiment, the cosmetic treatment includes inhibiting skin sagging, keeping skin firm, improving skin dryness, improving skin peeling, improving the weakness of the stratum corneum of the skin, reducing skin fine lines, reducing skin wrinkles and / or preventing skin aging.

[0095] According to another aspect of the present invention, there is provided a use of a cosmetic composition comprising boletin or a salt thereof in non-therapeutic cosmetic treatment of the skin and / or its appendages, wherein the weight range of the boletin or a salt thereof is 0.000001% to 10%, preferably 0.000001% to 5%, for example 0.000001% to 1%, based on the total weight of the cosmetic composition, wherein the boletin comprises an amino acid selected from the group consisting of leucine (Leu), isoleucine (Ile), tyrosine (Tyr), alanine (Ala), glutamine (Gln), glycine (Gly), phenylalanine (Phe), serine (Ser) and lysine (Lys).

[0096] In a preferred embodiment, the cosmetic composition further comprises an active agent for enhancing the effect of the boletin or its salt.

[0097] In a preferred embodiment, the active agent is selected from one or more of the following: peptides, vitamin C and its derivatives, vitamins from group B, dehydroepiandrosterone (DHEA), phytosterols, salicylic acid and its derivatives, retinoids, flavonoids, amino sugars, azoles and metal salts.

[0098] In a preferred embodiment, the cosmetic composition further comprises cosmetic excipients.

[0099] In a preferred embodiment, the cosmetic auxiliary material is selected from one or more of the following: thickener, surfactant, co-surfactant, skin conditioner, preservative, flavor, lubricant, disintegrant, wetting agent, binder and filler.

[0100] According to another aspect of the present invention, there is provided a method for improving the aesthetic appearance of the skin, the method comprising topically applying an effective amount of a cosmetic composition comprising a boletin peptide or a salt thereof, the weight range of the boletin peptide or a salt thereof being 0.000001% to 10%, preferably 0.000001% to 5%, for example 0.000001% to 1%, based on the total weight of the cosmetic composition, wherein the boletin peptide comprises an amino acid selected from the group consisting of leucine (Leu), isoleucine (Ile), tyrosine (Tyr), alanine (Ala), glutamine (Gln), glycine (Gly), phenylalanine (Phe), serine (Ser) and lysine (Lys).

[0101] An "effective" amount depends on a variety of factors, such as age, severity of the condition, and the manner of administration. An effective amount refers to a non-toxic amount sufficient to achieve the desired effect.

[0102] The composition according to the invention can be applied to all parts of the body, and more particularly to the face, body, decollete or scalp, as recommended by the indication, in any form or vehicle known to a person skilled in the art, in particular in the form of a solution, dispersion, emulsion, paste or powder, alone or in a premix or transported alone or premixed with a carrier (for example macrocapsules, microcapsules or nanocapsules, macrospheres, microspheres or nanospheres, liposomes, oil bodies or chylomicrons, macroparticles, microparticles or nanoparticles, macrosponges, microsponges or nanosponges, microemulsions or nanoemulsions, or adsorbed on powdered organic polymers, talc, bentonites, spores or exines and other mineral or organic carriers).

[0103] In cosmetics in particular, application may be mentioned in particular in the range of skin care for the face, body, hair and scalp as well as in the range of cosmetic care.

[0104] In general, the boletins according to the invention can be used in any form, in bound form, incorporated or adsorbed on macro-, micro- and nanoparticles or on macro-, micro- and nanocapsules, for the treatment of textiles, natural or synthetic fibers, wool and any material intended to come into contact with the skin and which can be used in clothing, underwear (day or night), handkerchiefs or tissues, in order to exert their cosmetic or therapeutic effect through this skin / textile contact and allow continuous topical delivery.

[0105] The present invention will be further described below in conjunction with specific examples. It should be understood that these examples are only used to illustrate the present invention and are not intended to limit the scope of the present invention. The experimental methods in the following examples without specifying specific conditions are usually carried out according to conventional conditions or conditions recommended by the manufacturer.

[0106] Unless otherwise defined, all professional and scientific terms used herein have the same meanings as those familiar to those skilled in the art. In addition, any methods and materials similar or equivalent to those described herein can be applied to the methods of the present invention. The preferred implementation methods and materials described herein are for demonstration purposes only.

[0107] The above features mentioned in the present invention or the features mentioned in the embodiments can be combined in any way. All the features disclosed in this patent specification can be used in combination with any combination form, and each feature disclosed in the specification can be replaced by any alternative feature that can provide the same, equal or similar purpose. Therefore, unless otherwise specified, the disclosed features are only general examples of equal or similar features.

[0108] Example

[0109] Efficacy Example

[0110] 1. Promote the synthesis of hyaluronic acid (HA) (moisturizing)---determination of hyaluronic acid content

[0111] 1.1 Experimental instruments and reagents

[0112] Instruments such as microplate reader, vortex mixer, cell counter, fluorescence inverted microscope, low-speed centrifuge, etc.

[0113] DMEM culture medium, trypsin, PBS, bovine serum, dimethyl sulfoxide, ELISA detection kit, TGF-β1, bFGF and other reagents.

[0114] 1.2 Experimental Methods

[0115] Sample preparation: cell digestion, inoculation, experimental setting of blank control group, positive control group and sample group. Prepare sample solutions of different concentrations. Among them, weigh 5 mg of boletin sample powder with a purity of >95%, dissolve it in 10 mL of deionized water to prepare a 0.5 mg / mL sample solution, and then dilute it with deionized water to 0.1 mg / mL, 0.05 mg / mL, and 0.001 mg / mL concentrations respectively.

[0116] Drug administration: Discard the culture medium in the 24-well plate and carry out drug administration. Add the culture medium containing the sample to the sample group, the culture medium containing the positive control to the positive control group, and the cell culture medium to the blank control group, 1 mL per well. After drug administration, place the 24-well plate in an incubator for culture. Collect the cell supernatant.

[0117] Standard preparation: Prepare HA standards according to the instructions of the kit. The standards will be used to establish a standard curve between HA content and absorbance.

[0118] Sample loading: Add the sample to be tested and the standard to the microplate wells pre-coated with HA antibody. Usually multiple experiments are performed with repeated controls.

[0119] Incubation: Incubate according to the instructions of the kit. During the process, the HA antigen binds to the antibody on the microplate to form an antigen-antibody complex.

[0120] Washing: Use washing solution to wash away unbound substances to reduce background interference. Add enzyme-labeled secondary antibody: Add enzyme-labeled secondary antibody to each well, which can bind to HA in the original sample.

[0121] Re-incubation and plate washing: After incubating the microplate at an appropriate temperature for a period of time, wash away the unbound enzyme-labeled secondary antibody with washing solution again.

[0122] Adding substrate: Adding substrate allows it to react with the enzyme label to form a visible chromatogram. The color change of the substrate is positively correlated with the antigen concentration.

[0123] Termination reaction: Add stop solution to stop the reaction and reduce errors.

[0124] ELISA reading: Use an ELISA reader to measure the light absorption value of each microwell. By comparing the absorbance value with the standard curve, the HA content in the sample can be calculated.

[0125] 1.3 Experimental Results

[0126] like Figure 1 As shown, compared with the BC group, the HA content secreted by HaCaT cells in the PC group was significantly increased, indicating that the experimental system is effective. Compared with the BC group, the amino acid sequence of Leu-Ile-Tyr-Ala-Gln-Gly-Phe-Ser-Lys of the present invention as shown in SEQ ID NO: 1 increased the HA content secreted by HaCaT cells at concentrations of 0.05 mg / mL and 0.1 mg / mL, indicating that the bolete peptide has the ability to promote hyaluronic acid synthesis, thereby proving that the bolete peptide has a moisturizing effect.

[0127] 2. Promote the synthesis of aquaporin 3 (AQP3) (moisturizing) --- determination of the content of aquaporin 3

[0128] 2.1 Experimental instruments and reagents

[0129] PCR experimental instruments, capillary electrophoresis instruments, protein immunoblotting equipment, fluorescence inverted microscope, low-speed centrifuge and other instruments.

[0130] DMEM culture medium, immunoblotting reagent, PBS, bovine serum, dimethyl sulfoxide, immunofluorescence staining reagent, CaCl2 and other reagents.

[0131] 2.2 Experimental Methods

[0132] Cell inoculation: Inoculate cells into 24-well plates and incubate overnight in an incubator (37°C, 5% CO2); Solution preparation: Prepare the working solution of the test substance; Add the test substance: After culturing in an incubator (37°C, 5% CO2) for 24 hours, add the test substance and continue culturing for 24 hours; Sample collection: Discard the supernatant and rinse the cells 3 times with PBS; Immunofluorescence staining: Add methanol to fix the cells, rinse 3 times with PBS, and then add 1mL BSA to each well for 1 hour; Discard the blocking solution, add the primary antibody to each well, and put it in a 4°C refrigerator overnight. Discard the primary antibody and rinse 3 times with PBS; Add the secondary antibody to each well for 2 hours, discard the secondary antibody, and rinse 3 times with PBS; Add DAPI to each well for nuclear staining, act for 10 minutes, discard DAPI, rinse 3 times with PBS, and then take pictures using a fluorescence microscope; Result analysis: The fluorescence intensity of AQP3 was quantitatively analyzed using Image Pro Plus software.

[0133] 2.3 Experimental Results

[0134] like Figure 2 As shown, compared with the BC group, the relative fluorescence intensity of AQP3 synthesized by HaCaT cells in the PC group was significantly increased after detection, indicating that the experimental system is effective. Compared with the BC group, the amino acid sequence of Leu-Ile-Tyr-Ala-Gln-Gly-Phe-Ser-Lys as shown in SEQ ID NO: 1 of the present invention has increased fluorescence intensity at concentrations of 0.05 mg / mL and 0.1 mg / mL, indicating that the bolete peptide has the ability to promote the synthesis of AQP3 protein, thereby proving that the bolete peptide has a moisturizing effect.

[0135] 3. Protecting type Ⅰ collagen (UVA) (anti-aging) --- Determination of type Ⅰ collagen content in UVA-stimulated fibroblasts

[0136] 3.1 Experimental instruments and reagents

[0137] Adjustable pipette, CO2 incubator, microplate reader, biological safety cabinet, micro-oscillator, analytical balance, constant temperature incubator, cell counter, ultra-low temperature refrigerator (-80℃), inverted microscope, low-speed centrifuge and other instruments.

[0138] DMEM culture medium, bovine serum, PBS, dimethyl sulfoxide, ELISA detection kit, type I collagen primary antibody, secondary antibody, TGF-β1 and other reagents.

[0139] 3.2 Experimental Methods

[0140] The determination of type I collagen content adopts enzyme-linked immunosorbent assay (ELISA) to carry out the detection of type I collagen content. The specific principle is: type I collagen specifically binds to the collagen antibody coated on the ELISA plate, and then binds to the antibody with substrate labeling. After the substrate is catalyzed by the enzyme, a colored product is generated. The type I collagen content is positively correlated with the color depth of the colored product. The absorbance (OD value) is measured at a wavelength of 450nm using an ELISA instrument to calculate the type I collagen content.

[0141] 3.3 Experimental Results

[0142] like Figure 3 As shown, compared with the BC group, the type I collagen content of the NC group was significantly reduced, and the type I collagen content of the PC group was significantly increased, indicating that the experimental system is effective. Compared with the NC group, the amino acid sequence of Leu-Ile-Tyr-Ala-Gln-Gly-Phe-Ser-Lys of the present invention as shown in SEQ ID NO: 1 increased the type I collagen content at concentrations of 0.05 mg / mL and 0.1 mg / mL, indicating that the bolete peptide has the ability to protect and promote the regeneration of cell type I collagen, thereby proving that the bolete peptide has anti-wrinkle effect.

[0143] 4. Inhibition of MMP-1 synthesis (UVA) (anti-aging) --- Determination of MMP-1 content in fibroblasts stimulated by UVA

[0144] 4.1 Experimental instruments and reagents

[0145] Adjustable pipette, CO2 incubator, microplate reader, biological safety cabinet, micro-oscillator, analytical balance, constant temperature incubator, cell counter, ultra-low temperature refrigerator (-80℃), inverted microscope, low-speed centrifuge and other instruments.

[0146] DMEM culture medium, bovine serum, PBS, dimethyl sulfoxide, ELISA detection kit, VC, VE and other reagents.

[0147] 4.2 Experimental Methods

[0148] Sample preparation: Take the sample to be tested (such as cell culture medium, serum, tissue solution, etc.) and dilute it appropriately. If the sample is prepared in the laboratory, it may be necessary to dilute the sample to an appropriate concentration range during the processing. Sample loading: Add samples and controls to the microplate pre-coated with MMP-1 antibodies one by one, and incubate at 37°C. During this process, the antibody will bind to the MMP-1 antigen in the sample. Washing: Use washing solution to wash away unbound substances and reduce background interference. Add enzyme-labeled secondary antibody: The enzyme-labeled secondary antibody commonly used in ELISA is an antibody with an enzyme label, which can bind to MMP-1 in the original sample. Incubate and wash the plate again: After incubating the microplate at 37°C for a period of time, wash the unbound enzyme-labeled secondary antibody again with washing solution. Add substrate: The enzyme-labeled substrate reacts with the enzyme to produce a visible chromatogram. Generally, the darker color of the substrate indicates a higher concentration of the antigen. Stop the reaction: Add the reaction stop solution to stop the reaction process. Enzyme label reading: Use an enzyme reader to read each microwell. The measured light absorbance value can represent the level of MMP-1 in each sample.

[0149] 4.3 Experimental Results

[0150] like Figure 4 As shown, compared with the BC group, the MMP-1 content of the NC group was significantly increased, and the MMP-1 content of the PC group was significantly reduced, indicating that the experimental system is effective. Compared with the NC group, the amino acid sequence of Leu-Ile-Tyr-Ala-Gln-Gly-Phe-Ser-Lys of the present invention as shown in SEQ ID NO: 1 reduced the MMP-1 content at a concentration of 0.05 mg / mL and significantly reduced the MMP-1 content at a concentration of 0.1 mg / mL, indicating that the bolete peptide has the ability to inhibit the generation of MMP-1, and also proves that the bolete peptide has a certain anti-wrinkle effect.

[0151] 5. Promote keratinocyte proliferation (repair) --- keratinocyte proliferation assay

[0152] 5.1 Experimental instruments and reagents

[0153] Incubator, clean bench, microscope, MTT detector, high-speed centrifuge and other instruments.

[0154] DMEM culture medium, MTT reagent, PBS, bovine serum, dimethyl sulfoxide, FBS and other reagents.

[0155] 5.2 Methods:

[0156] Cell culture: culturing keratinocytes in a culture medium suitable for their growth; adding samples: after the cells proliferate to an appropriate density, adding samples of different concentrations for treatment; determining cell proliferation: using CCK-8 or MTT methods to determine the proliferation of keratinocytes after sample treatment, usually adding CCK-8 or MTT reagents after a set time point (such as 24 hours, 48 ​​hours, etc.), after a period of culture, using a photometer to read the absorbance value, and calculating the cell proliferation according to the formula; data analysis: statistically analyzing all data to obtain the effect of the sample on the proliferation of keratinocytes.

[0157] 5.3 Results

[0158] like Figure 5 As shown, compared with the BC group, the cell viability of the PC group was significantly improved, indicating that the experimental system is effective. Compared with the BC group, the amino acid sequence of Leu-Ile-Tyr-Ala-Gln-Gly-Phe-Ser-Lys of the present invention as shown in SEQ ID NO: 1 increased cell viability at concentrations of 0.05 mg / mL and 0.1 mg / mL, indicating that the boletin has the ability to promote the proliferation of keratinocytes, thereby proving that the boletin has a certain repair effect.

[0159] 6. Promote keratinocyte migration (repair) --- keratinocyte migration assay

[0160] 6.1 Experimental instruments and reagents

[0161] Incubator, clean bench, microscope, MTT detector, high-speed centrifuge and other instruments.

[0162] DMEM culture medium, MTT reagent, PBS, bovine serum, dimethyl sulfoxide, FBS and other reagents.

[0163] 6.2 Experimental Methods

[0164] Scratch test: This test evaluates cell migration by creating a "scratch" or exposed area in the cell monolayer culture and then observing how quickly the cells migrate to that area. Metastatic ability test: This is performed using a Boyden Chamber device. The cells are placed in one chamber of the device, and the sample is placed in another chamber. There is a membrane with micropores between the two chambers of the device to separate them, and then observe whether the cells can migrate to the location of the sample. Data statistics: Using an image analysis system, the images of the treatment group and the control group are compared by digital technology to evaluate the cell migration.

[0165] 6.3 Experimental Results

[0166] like Figure 6As shown, compared with the BC group, the healing rate of the PC group was significantly improved, indicating that the experimental system is effective. Compared with the BC group, the amino acid sequence of Leu-Ile-Tyr-Ala-Gln-Gly-Phe-Ser-Lys shown in SEQ ID NO: 1 of the present invention has improved healing rates at concentrations of 0.001 mg / mL and 0.05 mg / mL, and significantly improved healing rates at a concentration of 0.1 mg / mL, indicating that the boletin has the ability to promote keratinocyte migration, thereby proving that the boletin has a certain repair effect.

[0167] The embodiments of the present invention are described in detail above. Specific examples are used herein to illustrate the principles and implementation methods of the present invention. The description of the above embodiments is only used to help understand the method of the present invention and its core idea. At the same time, changes or deformations made by those skilled in the art based on the ideas of the present invention, the specific implementation methods and application scope of the present invention, all belong to the scope of protection of the present invention. In summary, the content of this specification should not be understood as limiting the present invention.

Claims

1. A use of boletin or its salt in cosmetics, characterized in that: The boletin comprises amino acids selected from the group consisting of leucine (Leu), isoleucine (Ile), tyrosine (Tyr), alanine (Ala), glutamine (Gln), glycine (Gly), phenylalanine (Phe), serine (Ser) and lysine (Lys).

2. A use of boletin or its salt as an active ingredient in non-therapeutic cosmetic treatment of skin and / or its appendages, characterized in that: The boletin comprises amino acids selected from the group consisting of leucine (Leu), isoleucine (Ile), tyrosine (Tyr), alanine (Ala), glutamine (Gln), glycine (Gly), phenylalanine (Phe), serine (Ser) and lysine (Lys).

3. The use according to claim 1 or 2, characterized in that: The boletin contains at least 1, for example 1 to 2, leucines (Leu); Preferably, the boletin contains at least 1, for example 1 to 2 isoleucines (Ile); Preferably, the boletin contains at least 1, for example 1 to 2, tyrosines (Tyr); Preferably, the boletin contains at least 1, for example 1 to 2, alanines (Ala); Preferably, the boletin contains at least 1, for example 1 to 2, glutamine (Gln); Preferably, the boletin contains at least 1, for example 1 to 2, glycine (Gly); Preferably, the boletin contains at least 1, for example 1 to 2, phenylalanine (Phe); Preferably, the boletin contains at least 1, for example 1 to 2, serines (Ser); Preferably, the boletin contains at least 1, for example 1 to 2, lysines (Lys); More preferably, the boletin contains at least one, for example, 1 to 2 Leu-Ile-Tyr sequences; More preferably, the boletin contains at least one, for example, 1 to 2, Ile-Tyr-Ala sequences; More preferably, the boletin contains at least one, for example, 1 to 2, Tyr-Ala-Gln sequences; More preferably, the boletin contains at least one, for example, 1 to 2, sequences of Ala-Gln-Gly; More preferably, the boletin contains at least one, for example, 1 to 2, Gln-Gly-Phe sequences; More preferably, the boletin contains at least one, for example, 1 to 2, Gly-Phe-Ser sequences; More preferably, the boletin contains at least one, for example, 1 to 2, Phe-Ser-Lys sequence; Particularly preferably, the boletin contains 8 to 10 amino acids, such as 9 amino acids.

4. The use according to claim 1 or 2, characterized in that: The boletin comprises the amino acid sequence of Leu-Ile-Tyr-Ala-Gln-Gly-Phe-Ser-Lys as shown in SEQ ID NO: 1, optionally the amino acid sequence of SEQ ID NO: 1 with one or more amino acid residues substituted, deleted and / or inserted, optionally the amino acid sequence having an identity of about 88.9% with the amino acid sequence of SEQ ID NO:

1.

5. The use according to claim 1 or 2, characterized in that: The amino acid sequence of Leu-Ile-Tyr-Ala-Gln-Gly-Phe-Ser-Lys shown in SEQ ID NO: 1 is obtained by artificially synthesizing a peptide chain.

6. The use according to any one of claims 1 to 5, characterized in that The uses include moisturizing the skin, resisting skin aging and / or repairing the skin; Preferably, the moisturizing comprises promoting the production of hyaluronic acid (HA) and / or promoting the synthesis of aquaporin 3 (AQP3); More preferably, the anti-skin aging comprises inhibiting the production of matrix metalloproteinase-1 (MMP-1) under ultraviolet irradiation and / or protecting type I collagen produced by skin fibroblasts under ultraviolet irradiation; Also preferably, the repairing comprises promoting keratinocyte proliferation and / or migration; Particularly preferably, the cosmetic treatment includes improving dryness of the skin, improving peeling of the skin, improving a weak stratum corneum of the skin, alleviating fine lines of the skin, alleviating wrinkles of the skin and / or preventing skin aging.

7. Use of a cosmetic composition comprising boletin or a salt thereof for non-therapeutic cosmetic treatment of the skin and / or its appendages, characterized in that: Based on the total weight of the cosmetic composition, the weight range of the boletin or its salt is 0.000001% to 10%, preferably 0.000001% to 5%, for example 0.000001% to 1%, wherein the boletin comprises an amino acid selected from the group consisting of leucine (Leu), isoleucine (Ile), tyrosine (Tyr), alanine (Ala), glutamine (Gln), glycine (Gly), phenylalanine (Phe), serine (Ser) and lysine (Lys).

8. The cosmetic composition according to claim 7, characterized in that The cosmetic composition further comprises an active agent for enhancing the effect of the boletin or its salt; Preferably, the active agent is selected from one or more of the following: peptides, vitamin C and its derivatives, vitamins from group B, dehydroepiandrosterone (DHEA), phytosterols, salicylic acid and its derivatives, retinoids, flavonoids, amino sugars, azoles and metal salts.

9. The cosmetic composition according to claim 7 or 8, characterized in that The cosmetic further comprises cosmetic excipients; Preferably, the cosmetic auxiliary material is selected from one or more of the following: thickener, surfactant, co-surfactant, skin conditioner, preservative, flavor, lubricant, disintegrant, wetting agent, binder and filler.

10. A method for improving the aesthetic appearance of the skin, characterized in that The method comprises topically applying an effective amount of a cosmetic composition comprising a boletin or a salt thereof, wherein the weight of the boletin or a salt thereof is in the range of 0.000001% to 10%, preferably 0.000001% to 5%, for example 0.000001% to 1%, based on the total weight of the cosmetic composition, wherein the boletin comprises an amino acid selected from the group consisting of leucine (Leu), isoleucine (Ile), tyrosine (Tyr), alanine (Ala), glutamine (Gln), glycine (Gly), phenylalanine (Phe), serine (Ser) and lysine (Lys).

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