KR20240023412A - Fragmented peptide from Botulinum toxin, and cosmetic composition for ameliorating wrinkles comprising thereof - Google Patents

Abstract

Conventional cosmetic compositions for wrinkle improvement using botulinum toxin use botulinum toxin protein with a molecular weight of 150 kDa or more as is, so there is a problem in that storage, distribution, and management, including the pharmaceutical preparation process, are not easy as protein preparations. This is due to the instability of proteins, and the problem is serious in the case of protein preparations that are medicated at extremely low concentrations, such as botulinum toxin.
The present invention relates to a botulinum toxin-derived peptide fragment and a cosmetic composition for improving skin wrinkles containing the same. The peptide of the present invention has a remarkable wrinkle-improving effect, and cosmetic compositions containing it have high stability, reduced manufacturing costs, and are easy to distribute/storage, so it is expected to be widely used in the beauty field.

Description

Peptide fragment derived from botulinum toxin, and cosmetic composition for improving skin wrinkles comprising the same {Fragmented peptide from Botulinum toxin, and cosmetic composition for ameliorating wrinkles comprising the same}

The present invention relates to a botulinum toxin-derived peptide fragment and a cosmetic composition for improving skin wrinkles containing the same.

Skin care has become a daily lifestyle, coupled with modern people's desire to preserve beauty for a long time due to rising income levels, stress, worsening environmental pollution, and extension of lifespan. The global cosmetics market, which has grown to approximately $225.7 billion, proves this. As the cost spent on beauty is gradually increasing in Korea, the domestic cosmetics market size is USD 6.304 billion, ranking 11th in the world, and has been growing by more than 15% every year since 2011. Wrinkles, which are the most important area among the skin care items above, are caused by internal factors such as increasing age and stress, and/or external environmental factors such as air pollution and ultraviolet rays, and are the most prominent external skin aging feature. Wrinkles occur on various parts of the body, such as the forehead, around the eyes, between the eyebrows, and around the mouth, or on the neck, neck circumference, elbows, armpits, hands, and feet. They mostly start to become noticeable around the age of 30 and become noticeable with age. Their number, depth, and scope are increased. Wrinkles occur when reactive oxygen species generated during the skin aging process crosslink the collagen and elastic fibers that make up most of the dermis and change the production and decomposition of these fibers. Therefore, to suppress skin aging, antioxidants or radical scavengers such as vitamin E, beta-carotene, vitamin C, and glutathione, or substances that enhance dermal collagen synthesis such as vitamin A are used as cosmetic compositions for wrinkle improvement. Although they have been developed, most of these cosmetic active ingredients have the disadvantage of being strongly irritating to the skin or being unstable within the formulation, causing discoloration, off-odor, and precipitation, thereby reducing the inherent activity of the raw materials.

Meanwhile, a technology has recently been developed to improve skin wrinkles by using botulinum toxin as a kind of "competitive inhibitor" that interferes with nerve transmission by inhibiting the formation of the SNARE complex (Am Fam Physician. 2014 Aug 1;90(3):168 -75.). However, conventional cosmetic compositions for wrinkle improvement using botulinum toxin (Korean Patent Publication No. 10-2017-0089798 and Korean Patent Registration No. 10-0571444) use botulinum toxin protein with a molecular weight of 150 kDa or more as is, so they are medicated as protein preparations. There was a problem that storage, distribution, and management, including the process, were not easy. This is due to the instability of proteins, and the problem is serious in the case of protein preparations that are medicated at extremely low concentrations, such as botulinum toxin.

Accordingly, the present inventors completed the present invention to solve the conventional problems. The cosmetic composition of the present invention has excellent stability in the formulation compared to existing skin wrinkle improvement agents using botulinum toxin, reduces manufacturing cost, is easy to distribute/storage, and has a significant effect on wrinkle improvement, so it is used in the beauty field. It is expected that it will be greatly used.

The present invention was devised to solve the problems in the prior art as described above, and relates to a peptide fragment derived from botulinum toxin and a cosmetic composition containing the same for improving skin wrinkles.

However, the technical problem to be achieved by the present invention is not limited to the problems mentioned above, and other problems not mentioned will be clearly understood by those skilled in the art from the description below.

DETAILED DESCRIPTION OF THE INVENTION Various embodiments described herein are described below with reference to the drawings. In the following description, various specific details, such as specific forms, compositions, and processes, are set forth in order to provide a thorough understanding of the invention. However, certain embodiments may be practiced without one or more of these specific details or in conjunction with other known methods and forms. In other instances, well-known processes and manufacturing techniques are not described in specific detail so as not to unnecessarily obscure the invention. Reference throughout this specification to “one embodiment” or “an embodiment” means that a particular feature, form, composition or characteristic described in connection with the embodiment is included in one or more embodiments of the invention. Accordingly, the phrases “in one embodiment” or “an embodiment” expressed in various places throughout this specification do not necessarily refer to the same embodiment of the invention. Additionally, particular features, shapes, compositions, or properties may be combined in any suitable way in one or more embodiments.

Unless there is a special definition in the specification, all scientific and technical terms used in the specification have the same meaning as commonly understood by a person skilled in the art in the technical field to which the present invention pertains.

In one embodiment of the present invention, a peptide refers to a polymer of amino acids, where the linkage between amino acids consists of an amide bond or a peptide bond.

In the present invention, the peptide refers to a fragment peptide isolated from Botulinum Toxin, which can be artificially synthesized by biological/chemical methods known in the art. The peptide preferably consists of an amino acid sequence of less than 200 amino acids, more preferably of 3 to 180 amino acids, more preferably of 6 to 150 amino acids, and more preferably of 9 to 120 amino acids. More preferably, it consists of a 12 to 90 amino acid sequence, and even more preferably a 15 to 60 amino acid sequence, but is not limited thereto.

In another embodiment of the present invention, the peptide is preferably a peptide having a sequence of at least 70% homology to the sequence of SEQ ID NO: 1, 2, 3, 4, 5, or 6, and has at least 75% homology. It is more preferable that it is a peptide with a sequence having more than 80% homology, and even more preferably it is a peptide with a sequence having more than 85% homology, and it is more preferable that it is a peptide with a sequence having more than 90% homology. It is more preferable that it is a peptide of, and more preferably, it is a peptide of a sequence having at least 95% homology, and most preferably a peptide represented by SEQ ID NO: 1, 2, 3, 4, 5, or 6, but is limited thereto. It doesn't work.

The above-mentioned “Botulinum Toxin” is a neurotoxin protein produced by Clostridium botulinum bacteria. There are more than 127 species in the Clostridium genus, classified according to their form and function. The anaerobic, Gram-positive bacterium Clostridium botulinum produces botulinum toxin, a powerful polypeptide neurotoxin that causes the nerve paralysis disease botulism in humans and animals. Symptoms of botulinum toxin poisoning can progress to gait difficulties, swallowing and speech difficulties, respiratory muscle paralysis, and death.

The seven generally immunologically distinct botulinum neurotoxins are characterized by neurotoxin serotypes A, B, C1, D, E, F, and G, respectively, which are distinguished by neutralization with type-specific antibodies. The molecular weight of the botulinum toxin protein molecule is approximately 150 kDa for all seven known botulinum toxin serotypes. Botulinum toxin is released by clostridial bacteria as a complex containing a 150 kDa botulinum toxin protein molecule along with associated avirulent proteins. Therefore, botulinum toxin type A complex can be produced in 900kDa, 500kDa and 300kDa types by Clostridial bacteria. Botulinum toxin types B and C1 appear to be produced only as 700 kDa or 500 kDa complexes. Botulinum toxin type D is produced as 300 kDa and 500 kDa complexes. Finally, botulinum toxin types E and F are produced only as approximately 300 kDa complexes. These complexes (i.e., those with a molecular weight greater than about 150 kDa) are believed to contain a non-toxic hemagglutinin protein and a non-toxic and non-toxic non-hemagglutinin protein. These two non-toxin proteins (which together with the botulinum toxin molecule contain the associated neurotoxin complex) will act to provide stability against denaturation of the botulinum toxin molecule and protection against digestive acids when the toxin is ingested. Additionally, the larger the botulinum toxin complex (molecular weight greater than about 150 kDa), the more slowly the botulinum toxin complex will diffuse from the site where the botulinum toxin complex was intramuscularly injected. Therefore, in the present invention, botulinum toxin may include both a form that does not contain complexed proteins or a complex form that contains complexed proteins. The different serotypes of botulinum toxin differ depending on the animal species on which they act and the degree and duration of paralysis they cause. The molecular weight of the botulinum toxin protein from Clostridium botulinum types A, B, C, D, E, F or G, if it does not contain naturally occurring complexing proteins, is approximately 150 kDa.

In one embodiment of the present invention, nucleic acid is a polymeric organic substance in which nucleotides are polymerized into a long chain, and is genetic information encoding the peptide of the present invention.

In one embodiment of the present invention, “skin wrinkles” refer to fine lines caused by deterioration of the skin.

It is known that skin wrinkles are influenced by various factors such as skin moisture content, collagen content, and immunity to the external environment. Of these, the greatest influence on the formation of wrinkles is the amount of collagen produced and the matrix protein, a collagen decomposition enzyme. Expression and activity of decomposition enzymes.

More specifically, the main functions of skin fibroblasts include the regeneration of damaged skin tissue through extracellular matrix synthesis and proliferation, and cell aging due to extrinsic aging factors such as photoaging, accumulation of damage caused by free oxygen, and telomere disruption. The resulting endogenous aging factors reduce the physiological activity of skin fibroblasts within the dermal layer. Type I collagen, which accounts for more than 95% of the skin's extracellular matrix and plays a very important part in the physiological activity of fibroblasts through interaction and signal exchange with cell adhesion molecules and cytoskeleton When the synthesis of collagen decreases, the amount of collagen in the skin tissue decreases, which leads to a decrease in surface tension, which not only reduces skin elasticity and forms skin wrinkles, but also reduces physiologically active functions, including cell proliferation and regeneration functions. It becomes the cause of a vicious cycle. Therefore, collagen in skin fibroblasts can be considered to be directly related to skin regeneration, skin elasticity, skin wrinkle formation, and tissue repair or regeneration when skin is damaged.

In other words, when the synthesis of collagen or procollagen in skin fibroblasts is promoted or the synthesis of matrix metallo-proteinase (hereinafter referred to as 'MMP'), which decomposes collagen, is inhibited, skin elasticity is improved, skin regeneration, It can achieve effects such as improving skin wrinkles, healing wounds, repairing and regenerating damaged skin tissue, and preventing skin aging.

In one embodiment of the present invention, “prevention or improvement of skin wrinkles” means suppressing or inhibiting the formation of wrinkles on the skin or alleviating wrinkles that have already been formed.

The botulinum toxin-derived peptide fragment of the present invention and the cosmetic composition for improving skin wrinkles containing the same are characterized by increasing the expression of type I procollagen protein or suppressing the expression of MMP-1 protein.

Specifically, the botulinum toxin-derived peptide fragment of the present invention and the cosmetic composition for improving skin wrinkles containing the same increase the production of type I procollagen without showing cytotoxicity in normal human fibroblasts (HDFn) irradiated with ultraviolet rays. While suppressing the expression of matrix metallo-proteinase-1 (matrix metallo-proteinase), which promotes collagen decomposition, increases skin elasticity, skin regeneration, improvement of skin wrinkles, wound healing, repair and regeneration of damaged skin tissue, and skin It may have effects such as anti-aging. Specifically, it may have the effect of preventing or improving skin wrinkles.

The botulinum toxin-derived peptide fragment of the present invention and the cosmetic composition for improving skin wrinkles containing the same have the effect of removing skin wrinkles by themselves and are mixed with substances known to be effective in preventing and improving skin wrinkles. Even when used, the effect of preventing and improving skin wrinkles can be continuously maintained. For example, the cosmetic composition of the present invention further contains water-soluble vitamins, fat-soluble vitamins, high molecular weight peptides, high molecular weight polysaccharides, sphingo lipids, and seaweed extract. It can be included.

The water-soluble vitamins may be any that can be mixed into cosmetics, but are preferably vitamin B1, vitamin B2, vitamin B6, pyridoxine, pyridoxine hydrochloride, vitamin B12, pantothenic acid, nicotinic acid, nicotinic acid amide, folic acid, vitamin C, and vitamin H. and the like, and their salts (thiamine hydrochloride, sodium ascorbate, etc.) or derivatives (sodium ascorbic acid-2-phosphate, magnesium ascorbic acid-2-phosphate, etc.) can also be used in the present invention. Included in water-soluble vitamins. The water-soluble vitamins can be obtained by conventional methods such as microbial transformation, purification of microbial cultures, enzyme methods, or chemical synthesis.

The fat-soluble vitamins can be any one as long as they can be mixed in cosmetics, but preferably include vitamin A, carotene, vitamin D2, vitamin D3, vitamin E, etc., and their derivatives (ascorbin palmitate, ascorbic acid stearate) Bean, ascorbic acid dipalmitate, dl-alpha tocopherol acetate, dl-alpha tocopherol nicotinic acid (vitamin E, DL-pantothenyl alcohol, D-pantothenyl alcohol, pantothenyl ethyl ether, etc.) are also included in the fat-soluble vitamins used in the present invention. do.

The polymer peptide may be any one as long as it can be mixed into cosmetics, but preferably includes collagen, hydrolyzed collagen, gelatin, elastin, hydrolyzed elastin, and keratin. The above-mentioned high molecular peptides can be purified and obtained by conventional methods such as purification of microbial culture broth, enzyme methods, or chemical synthesis, or they can usually be purified and used from natural products such as dermis of pigs or cows or silk fibers of silkworms.

The above-mentioned high molecular weight polysaccharide can be anything as long as it can be mixed into cosmetics, but preferably includes proteoglycan, hyaluronic acid, hydroxyethylcellulose, xanthan gum, chondroitin sulfate or its salt (sodium salt, etc.). For example, chondroitin sulfate or its salt can usually be purified and used from mammals or fish.

The sphingolipid may be anything that can be mixed into cosmetics, but preferably includes ceramide, phytosphingosine, and sphingosaccharide lipid. The sphingolipids can usually be purified by conventional methods from mammals, fish, shellfish, yeast, or plants, or obtained by chemical synthesis.

The seaweed extract can be anything as long as it can be mixed in cosmetics, but preferably includes brown algae extract, red algae extract, green algae extract, etc., and also calrageenan, arginic acid, and arginic acid purified from these seaweed extracts. Sodium, potassium alginate, etc. are also included in the seaweed extract used in the present invention. Seaweed extract can be obtained by purifying seaweed by a conventional method.

In addition to the above-mentioned essential ingredients, the cosmetic of the present invention can be blended with other ingredients that are usually blended in cosmetics as needed.

Other ingredients that may be added include oils and fats, moisturizers, emollients, ultraviolet absorbers, pH adjusters, fragrances, blood circulation promoters, cooling agents, antiperspirants, and purified water.

The oil and fat components include ester-based fats and oils, hydrocarbon-based fats and oils, silicone-based fats and oils, fluorine-based fats and oils, animal fats and vegetable oils, and the like.

The ester-based oils include glyceryl tri2-ethylhexanoate, cetyl 2-ethylhexanoate, isopropyl myristate, butyl myristate, isopropyl palmitate, ethyl stearate, octyl palmitate, isocetyl isostearate, Butyl stearate, ethyl linoleate, isopropyl linoleate, ethyl oleate, isocetyl myristate, isostearyl myristate, isostearyl palmitate, octyldodecyl myristate, isocetyl isostearate, diethyl sebacate, Diisopropyl adipate, isoalkyl neopentanoate, tri(caprylic, capric acid)glyceryl, trimethylolpropane tri2-ethylhexanoate, trimethylolpropane triisostearate, pentaeli tetra2-ethylhexanoate Slitol, cetyl caprylate, decyl laurate, hexyl laurate, decyl myristate, myristyl myristate, cetyl myristate, stearyl stearate, decyl oleate, cetyl ricinooleate, lauric acid Sostearyl, isotridecyl myristate, isocetyl palmitate, octyl stearate, isocetyl stearate, isodecyl oleate, octyldodecyl oleate, octyldodecyl linoleate, isopropyl isostearate, 2-ethylhexane acid wash. Tostearyl, 2-ethylhexanoate stearyl, hexyl isostearate, ethylene glycol dioctanate, ethylene glycol dioleate, propylene glycol dicaprate, propylene glycol dicaprylate, neopentyl glycol dicaprate, neopentyl dicaprate. Glycol, glyceryl tricaprylate, glyceryl triundecylate, glyceryl triisopalmitate, glyceryl triisostearate, octyldodecyl neopentanoate, isostearyl octanoate, octyl isononanoate, hexyl neodecanoate. Decyl, octyldodecyl neodecanoate, isocetyl isostearate, isostearyl isostearate, octyldecyl isostearate, polyglycerol oleic acid ester, polyglycerol isostearic acid ester, triisocetyl citrate, triisoalkyl citrate, Triisooctyl citrate, lauryl lactate, myristyl lactate, cetyl lactate, octyldecyl lactate, triethyl citrate, acetyltriethyl citrate, acetyltributyl citrate, trioctyl citrate, diisostearyl malate, hydroxystearic acid 2- Ethylhexyl, di2-ethylhexyl succinate, diisobutyl adipate, diisopropyl sebacate, dioctyl sebacate, cholesteryl stearate, cholesteryl isostearate, hydroxycholesteryl stearate, cholesteryl oleate. Steryl, dihydrocholesteryl oleate, phytsteryl isostearate, phytsteryl oleate, isocetyl 12-stealoyl hydroxystearate, 12-stealoyl hydroxystearate, 12-stealo Ester systems, such as isostearyl hydroxystearate, etc. are mentioned.

Examples of the hydrocarbon oil include squalene, liquid paraffin, alpha-olefin oligomer, isoparaffin, ceresin, paraffin, liquid isoparaffin, polybudene, microcrystalline wax, and petroleum jelly.

The silicone oils include polymethyl silicone, methylphenyl silicone, methylcyclopolysiloxane, octamethylpolysiloxane, decamethylpolysiloxane, dodecamethylcyclosiloxane, dimethylsiloxane methylcetyloxysiloxane copolymer, dimethylsiloxane methylstealoxysiloxane copolymer, alkyl Modified silicone oil, amino-modified silicone oil, etc. can be mentioned.

Examples of the fluorine-based oil include perfluoropolyether.

The animal or plant oils include avocado oil, almond oil, olive oil, sesame oil, rice bran oil, soybean oil, corn oil, rapeseed oil, apricot oil, palm kernel oil, palm oil, castor oil, sunflower oil, grape seed oil, cottonseed oil, palm oil, Cuckoo nut oil, wheat germ oil, rice germ oil, shea butter, moonshine colostrum, marker damia nut oil, egg yolk oil, beef tallow, horse oil, mink oil, orange rape oil, jojoba oil, candelier wax, carnaba wax, liquid ranol. and animal or plant oils such as hydrogenated castor oil.

Examples of the moisturizing agent include water-soluble low-molecular-weight moisturizers, oil-soluble molecular moisturizers, water-soluble polymers, and oil-soluble polymers.

The water-soluble low-molecular-weight moisturizers include serine, glutamine, sorbitol, mannitol, sodium pyrrolidone-carboxylate, glycerin, propylene glycol, 1,3-butylene glycol, ethylene glycol, polyethylene glycol B (degree of polymerization n = 2 or more), Examples include polypropylene glycol (degree of polymerization n = 2 or more), polyglycerin B (degree of polymerization n = 2 or more), lactic acid, and lactate salts.

Examples of the fat-soluble low-molecular-weight moisturizer include cholesterol and cholesterol ester.

The water-soluble polymers include carboxyvinyl polymer, polyaspartate, tragacanth, xanthan gum, methylcellulose, hydroxymethylcellulose, hydroxyethylcellulose, hydroxypropylcellulose, carboxymethylcellulose, water-soluble chitin, chitosan, dextrin, etc. can be mentioned.

Examples of the oil-soluble polymer include polyvinylpyrrolidone eicosene copolymer, polyvinylpyrrolidone hexadecene copolymer, nitrocellulose, dextrin fatty acid ester, and polymer silicone.

Examples of the emollient agent include long-chain acyl glutamic acid cholesteryl ester, hydroxystearic acid cholesteryl, 12-hydroxystearic acid, stearic acid, rosin acid, and lanolin fatty acid cholesteryl ester.

The ultraviolet absorbers include para-aminobenzoic acid, ethyl para-aminobenzoate, amyl para-aminobenzoate, octyl para-aminobenzoate, ethylene glycol salicylate, phenyl salicylate, octyl salicylate, benzyl salicylate, butylphenyl salicylate, homomenthyl salicylate, Benzyl cinnamic acid, paramethoxycinnamic acid-2-ethoxyethyl, octyl paramethoxycinnamic acid, mono-2-ethylhexane glyceryl diparamethoxycinnamic acid, isopropyl paramethoxycinnamic acid, diisopropylㆍdiisopropylcinnamic acid ester mixture , Urocanic acid, ethyl urocanic acid, hydroxymethoxybenzophenone, hydroxymethoxybenzophenone sulfonic acid and its salts, dihydroxymethoxybenzophenone, dihydroxymethoxybenzophenone disulfonate sodium, dihydroxy Benzophenone, tetrahydroxybenzophenone, 4-tert-butyl-4'-methoxydibenzoylmethane, 2,4,6-trianilino-p-(carbo-2'-ethylhexyl-1'-oxy) -1,3,5-triazine, 2-(2-hydroxy-5-methylphenyl)benzotriazole, etc. are mentioned.

Examples of the pH adjusting agent include citric acid, sodium citrate, malic acid, sodium malate, fumal acid, sodium fumalate, succinic acid, sodium succinate, sodium hydroxide, sodium monohydrogen phosphate, and the like.

In addition, the mixing components that may be added are not limited to this, and any of the above components can be mixed within a range that does not impair the purpose and effect of the present invention, but is preferably 0.01 to 5 parts by weight, based on the total weight. Preferably, it can be mixed in 0.01 to 3 parts by weight.

Cosmetics manufactured containing the composition of the present invention may take the form of a solution, emulsion, viscous mixture, etc.

In addition, the ingredients included in the cosmetic composition of the present invention may include ingredients commonly used in cosmetic compositions in addition to the ingredients as active ingredients, for example, conventional auxiliaries such as stabilizers, pigments, and natural fragrances, and It may further contain a carrier.

The composition of the present invention can be used in a variety of ways, such as cosmetics or facial cleansers that have the effect of preventing and improving skin wrinkles.

Products to which the composition of the present invention can be added include, for example, skin lotion, skin softener, skin toner, astringent, lotion, milk lotion, moisture lotion, nutritional lotion, massage cream, nutritional cream, moisture cream, and hand cream. , cosmetics such as foundation, essence, nutritional essence, and packs, as well as soap, cleansing foam, cleansing lotion, cleansing cream, body lotion, and body cleanser.

When the formulation of the present invention is a paste, cream or gel, animal fiber, plant fiber, wax, paraffin, starch, tracant, cellulose derivative, polyethylene glycol, silicone, bentonite, silica, talc or zinc oxide may be used as the carrier ingredient. You can.

When the formulation of the present invention is a powder or spray, lactose, talc, silica, aluminum hydroxide, calcium silicate, or polyamide powder can be used as the carrier ingredient. In particular, when the formulation is a spray, chlorofluorohydrocarbon and propane may be used as carrier ingredients. , butane or dimethyl ether.

When the formulation of the present invention is a solution or emulsion, a solvent, solvating agent or emulsifying agent is used as a carrier component, such as water, ethanol, isopropanol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1 , 3-butyl glycol oil, glycerol aliphatic esters, polyethylene glycol or fatty acid esters of sorbitan.

When the formulation of the present invention is a suspension, the carrier ingredients include water, a liquid diluent such as ethanol or propylene glycol, a suspending agent such as ethoxylated isostearyl alcohol, polyoxyethylene sorbitol ester, and polyoxyethylene sorbitan ester, and microcrystalline Cellulose, aluminum metahydroxide, bentonite, agar, or tracant may be used.

According to another embodiment of the present invention, the present invention provides a pharmaceutical composition for preventing or treating skin wrinkles containing a botulinum toxin-derived peptide fragment as an active ingredient.

As an active ingredient of the pharmaceutical composition, the botulinum toxin-derived peptide fragment is characterized by increasing the expression of type I procollagen protein or inhibiting the expression of MMP-1 protein. The composition of the present invention is used in the production of a pharmaceutical composition. It may further include commonly used appropriate carriers, excipients, and diluents.

The pharmaceutical dosage form of the composition of the present invention can be used in the form of its pharmaceutically acceptable salt, and can also be used alone or in combination with other pharmaceutically active compounds.

Carriers, excipients and diluents that may be included in the composition of the present invention include lactose, dextrose, sucrose, oligosaccharides, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, gum acacia, alginate, gelatin, calcium phosphate, calcium. Silicates, cellulose, methyl cellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil.

When formulating the composition of the present invention, it can be formulated and used in the form of oral dosage forms such as powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols, external preparations, suppositories, or sterile injection solutions according to conventional methods. You can. In detail, when formulating, it can be prepared using diluents or excipients such as commonly used fillers, extenders, binders, wetting agents, disintegrants, and surfactants. Solid preparations for oral administration include tablets, pills, powders, granules, capsules, etc. These solid preparations contain polymer polysaccharides or taro extract and at least one excipient such as starch, calcium carbonate, water, etc. It can be prepared by mixing sucrose, lactose, gelatin, etc. Additionally, in addition to simple excipients, lubricants such as magnesium stearate and talc can also be used. Liquid preparations for oral use include suspensions, oral solutions, emulsions, and syrups. In addition to the commonly used simple diluents such as water and liquid paraffin, various excipients such as wetting agents, sweeteners, fragrances, and preservatives may be included. there is. Preparations for parenteral administration include sterile aqueous solutions, non-aqueous solutions, suspensions, emulsions, lyophilized preparations, and suppositories. Non-aqueous solvents and suspensions may include propylene glycol, polyethylene glycol, vegetable oil such as olive oil, and injectable ester such as ethyl oleate. As a base for suppositories, witepsol, macrogol, tween 61, cacao, laurel, glycerogelatin, etc. can be used.

The preferred dosage of the mixed composition of the present invention varies depending on the patient's condition and weight, degree of disease, drug form, administration route and period, but can be appropriately selected by a person skilled in the art, and is preferably 0.01㎍/㎖ to 0.01㎍/㎖. It can be administered at a dose of 25 μg/ml. External administration can be administered once a day or in several divided doses, and can be administered systemically or topically.

According to another embodiment of the present invention, the present invention provides a food composition for preventing or improving skin wrinkles containing a botulinum toxin-derived peptide fragment as an active ingredient.

As an active ingredient of the food composition, the botulinum toxin-derived peptide fragment is characterized by increasing the expression of type I procollagen protein or suppressing the expression of MMP-1 protein. The food composition of the present invention is a health functional food. can be used The above “health functional food” refers to food manufactured and processed using raw materials or ingredients with functionality useful to the human body in accordance with Act No. 6727 on Health Functional Food, and “functionality” refers to the structure of the human body. It means ingestion for the purpose of controlling nutrients for function or obtaining useful effects for health purposes such as physiological effects.

The food composition of the present invention may contain common food additives, and its suitability as a “food additive” is determined in accordance with the general provisions and general test methods of the food additive code approved by the Food and Drug Administration, unless otherwise specified. It is determined based on the specifications and standards for the relevant item.

Items listed in the "Food Additives Code" include, for example, chemical compounds such as ketones, glycine, potassium citrate, nicotinic acid, and cinnamic acid; natural additives such as subchromic pigment, licorice extract, crystalline cellulose, high-liquid pigment, and guar gum; Examples include mixed preparations such as sodium L-glutamate preparations, noodle additive alkaline preparations, preservative preparations, and tar coloring preparations.

Among capsule-type health functional foods, hard capsules can be manufactured by filling a regular hard capsule with a mixture of additives such as excipients or their granules or peeled granules, while soft capsules can be manufactured by filling a mixture with additives such as excipients. It can be manufactured by filling a capsule base such as gelatin. The soft capsule may contain plasticizers such as glycerin or sorbitol, colorants, preservatives, etc., if necessary.

Health functional foods in the form of pills can be prepared by forming a mixture of excipients, binders, disintegrants, etc. into herbal extracts in an appropriate manner. If necessary, they can be coated with sucrose or other suitable coating agents, or with starch, talc, or other suitable substances. You can also wear a fancy dress.

Health functional foods in the form of granules can be manufactured into granules by mixing the herbal extract with excipients, binders, disintegrants, etc., using an appropriate method, and may contain flavoring agents, flavoring agents, etc. as needed. Definitions of the excipients, binders, disintegrants, lubricants, corrigants, flavoring agents, etc. of the present invention are described in literature known in the art and include those with the same or similar functions (Korean Pharmacopoeia Commentary, Text) Seongsa, Korea Association of Colleges of Pharmacy, 5th revised edition, p33-48, 1989).

When the food composition of the present invention is provided in liquid form, the peptide of the present invention can be included in a dissolved or undissolved state, and for effective flavor masking, oligosaccharides, white sugar, fructose, syrup sugar, fruit extract, Flavoring agents, etc. may be included alone or in combination thereof, and are not limited to these, and ingredients suitable for food may be appropriately used.

In one embodiment of the present invention, a botulinum toxin-derived peptide is provided, wherein the botulinum toxin-derived peptide has at least 70% homology to the sequence represented by SEQ ID NO: 1, 2, 3, 4, 5, or 6. Provided is a botulinum toxin-derived peptide, wherein the botulinum toxin-derived peptide includes a sequence represented by SEQ ID NO: 1, 2, 3, 4, 5, or 6, but consists of an amino acid sequence of less than 200 amino acids. provides.

In another embodiment of the present invention, a nucleic acid encoding the botulinum toxin-derived peptide is provided.

In another embodiment of the present invention, a cosmetic composition for preventing or improving skin wrinkles is provided comprising the botulinum toxin-derived peptide, wherein the botulinum toxin-derived peptide increases the expression of type I procollagen protein. Provided is a cosmetic composition for preventing or improving skin wrinkles, wherein the botulinum toxin-derived peptide inhibits the expression of MMP-1 protein, and the cosmetic composition is used for skin lotion, skin softener, and skin wrinkles. Toner, astringent, lotion, milk lotion, moisture lotion, nutritional lotion, massage cream, nutritional cream, moisture cream, hand cream, foundation, essence, nutritional essence, pack, soap, cleansing foam, cleansing lotion, cleansing cream, body lotion, and Provided is a cosmetic composition for preventing or improving skin wrinkles, which is a formulation selected from the group consisting of body cleansers.

In another embodiment of the present invention, a pharmaceutical composition for preventing or treating skin wrinkles is provided comprising the botulinum toxin-derived peptide, wherein the botulinum toxin-derived peptide increases the expression of type I procollagen protein. Provided is a pharmaceutical composition for prevention or treatment, wherein the botulinum toxin-derived peptide inhibits the expression of MMP-1 protein, and the pharmaceutical composition is provided as a cream, gel, patch, or spray. It provides a pharmaceutical composition for preventing or treating skin wrinkles, which is any one formulation selected from the group consisting of ointments, warning agents, lotions, liniment agents, pasta agents, and cataplasma agents.

In another embodiment of the present invention, a food composition for preventing or improving skin wrinkles is provided, comprising the botulinum toxin-derived peptide of claim 1, wherein the botulinum toxin-derived peptide increases the expression of type I procollagen protein on the skin. A food composition for preventing or improving wrinkles is provided, wherein the botulinum toxin-derived peptide inhibits the expression of MMP-1 protein, and the food composition is provided in the form of capsules, pills, granules, It provides a food composition for preventing or improving skin wrinkles, which is any one formulation selected from the group consisting of a liquid agent.

Hereinafter, the present invention will be described in detail step by step.

The present invention relates to a botulinum toxin-derived peptide fragment and a cosmetic composition for improving skin wrinkles containing the same. The peptide of the present invention inhibits the expression of MMP-1 in human dermal fibroblasts and increases the production of type I procollagen, thereby preventing the occurrence of skin wrinkles and improving wrinkles in damaged skin. Overall, the composition of the present invention can prevent and significantly improve skin wrinkles.

Figure 1 shows the results confirming that the botulinum toxin-derived peptide fragment of the present invention does not exhibit toxicity in human fibroblasts (HDFn), according to an embodiment of the present invention.
Figure 2 shows the results confirming that the botulinum toxin-derived peptide fragment of the present invention has a collagen synthesis effect in human fibroblasts (HDFn), according to an embodiment of the present invention.
Figure 3 shows the results confirming that the botulinum toxin-derived peptide fragment of the present invention has an effect of suppressing MMP-1 expression in human fibroblasts (HDFn), according to an embodiment of the present invention.

Hereinafter, the present invention will be described in more detail through examples. These examples are only for illustrating the present invention in more detail, and it will be apparent to those skilled in the art that the scope of the present invention is not limited by these examples according to the gist of the present invention. .

Example 1: Synthesis of fragment peptide derived from botulinum toxin

The sequence of the region predicted to be the active site from botulinum toxin type A (BoNT/A) or expected to affect fibroblast growth factor receptor 3 is divided into 15 to 60 amino acid sequence units. By peptide truncation, various botulinum toxin-derived peptide fragments were prepared. The sequences of the peptide fragments are listed in Table 1 below.

Manufacturing example sequence number amino acid sequence Location in BoNT/A Peptide 1 One DPAVTLAHELIHAGHRL 216-232 peptide 2 2 LFEFYKLLCVRGIITSKTKSLDKGYNKALNDLCIKV 422-457 peptide 3 3 LRAQEFEHGKSRIALTNSVNEA 554-575 peptide 4 4 IPYIGPALNIGNMLYKDDFVGA 634-655 peptide 5 5 ATKAIINYQYNQYTEEEKNNINFNIDDLSSKL 742-773 peptide 6 6 GSVMTTNIYLNSSLYRGTKFIIKKYASGNKDNIVRNNDRV 1141-1180

Example 2: Confirmation of cytotoxicity of botulinum toxin-derived fragment peptide

Human fibroblasts (HDFn) were cultured and obtained with 1x trypsin/EDTA, the centrifuged cell pellet was resuspended with new medium, and then seeded at 2 × 10 ⁴ cells/well in a 96 well plate and incubated in a 37°C CO ₂ incubator. Cultured. Once the cells were stabilized, they were treated with each of the six peptides synthesized in Example 1 for 24 hours, and 20 μl of MTT reagent at a concentration of 5 mg/ml was dispensed per well and cultured for an additional 3 hours. Afterwards, the supernatant was removed and formazan was added. It was dissolved in DMSO and absorbance was measured at a wavelength of 540 nm. The results of the conversion of the measured results compared to the negative control group (no peptide treatment) are shown in Figure 1.

As a result of the experiment, it was confirmed that peptides 1 to 6 did not exhibit cytotoxicity up to the highest concentration tested. Considering the tested concentration range, peptides were found to be stable in cells in the following order: 5, 6 > 2, 4 > 1, 3.

Example 3: Confirmation of collagen synthesis effect of botulinum toxin-derived fragment peptide

Human fibroblasts (HDFn) were seeded at 2 × 10 ⁴ cells/well in a 24-well plate and stabilized for 24 hours, then replaced with FBS-free medium and treated with the six types of peptides synthesized in Example 1 for 24 hours each. Afterwards, the supernatant was separated and the amount of synthesized collagen was measured using the Procollagen Type I C-Peptide (PIP) EIA Kit. The collagen is a protein that maintains the mechanical properties of skin tissue to maintain elasticity and prevent the skin from sagging. For collagen measurement, the collagen synthesis rate (%) was obtained by comparing the average of three repetitions for each concentration with the negative control group (no peptide treatment), and the biological statistical standard (significance level (α): 5%) was determined using the two sample t-test method. ) was evaluated for significance. The results are shown in Figure 2.

As a result of the experiment, peptides 1 to 5 had little effect on collagen synthesis, but peptide number 6 showed a 24.22% increase in collagen synthesis at a concentration of 3 ug/ml.

Example 4: Confirmation of the inhibitory effect of botulinum toxin-derived fragment peptide on MMP-1 expression

Human fibroblasts (HDFn) were seeded at 2 × 10 ⁴ cells/well in a 24-well plate and stabilized for 24 hours, then washed twice with PBS and irradiated with UV at 6 J/cm ² . The medium was replaced with FBS free medium, and each of the six peptides synthesized in Example 1 was treated for 48 hours. Afterwards, the supernatant was separated and the expression of MMP-1 was measured using a human total MMP-1 ELISA Kit. The MMP-1 is an enzyme that decomposes extracellular matrix such as collagen and elastin, which are involved in skin elasticity. For MMP-1 measurement, the MMP-1 inhibition rate (%) was obtained by comparing the average of three repetitions for each concentration with the negative control group (no peptide treatment), and the biological statistical standard (significance level (α) was determined using the two sample t-test method. ): 5%) to evaluate significance. The results are shown in Figure 3.

As a result of the experiment, peptide 1 was 30.61% at a concentration of 300 ug/ml, peptide 3 was 22.72% at a concentration of 300 ug/ml, peptide 4 was 29.72% at a concentration of 100 ug/ml, and peptide 5 was 24.30% at a concentration of 3 ug/ml. It was found to have an inhibitory effect on MMP-1 expression. In the case of peptides 2 and 6, the inhibitory effect on MMP-1 expression was minimal. Therefore, it was found that the MMP-1 inhibitory effect was good in the order of peptide 1 > 4 > 5 > 3.

As the specific parts of the present invention have been described in detail above, it is clear to those skilled in the art that these specific techniques are merely preferred embodiments and do not limit the scope of the present invention. Accordingly, the actual scope of the present invention will be defined by the appended claims and their equivalents.

Claims (8)

A peptide derived from botulinum toxin consisting of the amino acid sequence of SEQ ID NO: 4.
A nucleic acid encoding the botulinum toxin-derived peptide of claim 1.
A cosmetic composition for preventing or improving skin wrinkles, comprising the botulinum toxin-derived peptide of claim 1.
According to clause 3,
A cosmetic composition wherein the botulinum toxin-derived peptide inhibits the expression of MMP-1 protein.
A pharmaceutical composition for preventing or treating skin wrinkles, comprising the botulinum toxin-derived peptide of claim 1.
According to clause 5,
A pharmaceutical composition wherein the botulinum toxin-derived peptide inhibits the expression of MMP-1 protein.
A food composition for preventing or improving skin wrinkles, comprising the botulinum toxin-derived peptide of claim 1.
According to clause 7,
A food composition wherein the botulinum toxin-derived peptide inhibits the expression of MMP-1 protein.