KR102449119B1 - Fragmented peptide from Botulinum toxin, and cosmetic composition for ameliorating wrinkles comprising thereof - Google Patents

Abstract

The conventional cosmetic composition for wrinkle improvement using botulinum toxin uses a botulinum toxin protein having a molecular weight of 150 kDa or more as it is, so there is a problem in that storage, distribution, and management, including the pharmaceutical process, are not easy as a protein preparation. This is due to the instability of the protein, and in the case of a protein preparation that is formulated at an extremely low concentration, such as botulinum toxin, the problem is serious.
The present invention relates to a botulinum toxin-derived peptide fragment and a cosmetic composition for improving skin wrinkles comprising the same. The peptide of the present invention has a remarkable wrinkle improvement effect, and the cosmetic composition comprising the same has high stability, reduced manufacturing cost, and easy distribution/storage, so it is expected to be widely used in the cosmetic field.

Description

Fragmented peptide from Botulinum toxin, and cosmetic composition for ameliorating wrinkles comprising thereof

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

In line with the desire of modern people to preserve beauty for a long time due to the increase in income level, stress, deepening environmental pollution, and life extension, skin care has become a daily life form. The global cosmetics market, which has grown to about $225.7 billion, proves this. As the cost of beauty is gradually increasing in Korea, the size of the domestic cosmetics market is $6.34 billion, the 11th largest in the world, and has been growing by more than 15% every year since 2011. Wrinkles, which are the most significant areas of the skin care items, are caused by intrinsic factors such as increase in age, stress, or/and external environmental factors such as air pollution and UV irradiation, and are the most prominent appearance of skin aging. Wrinkles occur on the face, neck, neck, elbows, armpits, hands and feet, such as the forehead, around the eyes, between the forehead and around the mouth. The number, depth, and range are increased. Wrinkles are caused by reactive oxygen species generated in the process of skin aging, crosslinking collagen fibers and elastic fibers that make up most of the dermis, and changing the generation and decomposition of these fibers. Therefore, in order to suppress skin aging, antioxidants or radical scavengers such as vitamin E, beta-carotene, vitamin C, and glutathione, or substances that promote dermal collagen synthesis such as vitamin A are used as cosmetic compositions for wrinkle improvement. Although developed, these cosmetic active ingredients have a disadvantage in that most of them have strong skin irritation or are themselves unstable in the formulation, resulting in discoloration, discoloration, and precipitation, thereby reducing the intrinsic activity of the raw material.

On the other hand, recently, a technique for improving skin wrinkles by using botulinum toxin as a kind of "competitive inhibitor" that interferes with neurotransmission by inhibiting the formation of the SNARE complex has been developed (Am Fam Physician. 2014 Aug 1;90(3):168 -75.). However, the conventional cosmetic composition for wrinkle improvement using botulinum toxin (Korean Patent Publication No. 10-2017-0089798, and Korean Patent No. 10-0571444) uses botulinum toxin protein with a molecular weight of 150 kDa or more as it is, so it is formulated as a protein preparation. There was a problem in that storage, distribution, and management including the process were not easy. This is due to the instability of the protein, and the problem is serious in the case of a protein preparation, such as botulinum toxin, which is formulated at an extremely low concentration.

Accordingly, the present inventors have completed the present invention to solve the problems of the prior art. The cosmetic composition of the present invention has excellent stability in the formulation, reduced manufacturing cost, easy distribution/storage, and a remarkable effect on wrinkle improvement, compared to existing skin wrinkle and wrinkle improving agents using botulinum toxin. It is expected to be of great use.

The present invention has been devised to solve the problems in the prior art, and relates to a botulinum toxin-derived peptide fragment, and a cosmetic composition for improving skin wrinkles comprising the same.

However, the technical task to be achieved by the present invention is not limited to the tasks mentioned above, and other tasks not mentioned will be clearly understood by those of ordinary skill in the art from the following description.

BRIEF DESCRIPTION OF THE DRAWINGS Various embodiments described herein are described below with reference to the drawings. In the following description, various specific details are set forth, such as specific forms, compositions and processes, and the like, for a thorough understanding of the present 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 have not been described in specific detail in order not to unnecessarily obscure the present 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. Thus, references to "in one embodiment" or "an embodiment" in various places throughout this specification do not necessarily refer to the same embodiment of the invention. Additionally, the particular features, forms, compositions, or characteristics may be combined in any suitable manner in one or more embodiments.

Unless otherwise defined in the specification, all scientific and technical terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

In one embodiment of the present invention, the peptide refers to a polymer of amino acids, wherein the linkage between amino acids is composed 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 200 or less, more preferably of 3 to 180 amino acid sequence, more preferably 6 to 150 amino acid sequence, and more preferably consisting of 9 to 120 amino acid sequence. More preferably, it is more preferably composed of a sequence of 12 to 90 amino acids, and more preferably composed of a sequence of 15 to 60 amino acids, but is not limited thereto.

Also in another embodiment of the present invention, the peptide is preferably a peptide of a sequence having at least 70% homology with the sequence of SEQ ID NO: 1, 2, 3, 4, 5, or 6, and has at least 75% homology More preferably, it is a peptide having a sequence having at least 80% homology, more preferably a peptide having a sequence having at least 80% homology, more preferably a peptide having a sequence having at least 85% homology, and a sequence having at least 90% homology It is more preferable that it is a peptide of it's not going to be

The above “botulinum toxin” is a neurotoxin protein produced by the Clostridium botulinum bacteria. There are more than 127 species of Clostridium, and they are classified according to their shape and function. The anaerobic, gram-positive bacterium Clostridium botulinum produces botulinum toxin, a potent polypeptide neurotoxin that causes a neuroparalytic disease called botulism in humans and animals. Symptoms of botulinum toxin poisoning may progress to gait disturbance, dysphagia and speech disturbance, paralysis of respiratory muscles, and death.

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 about 150 kDa for all seven known botulinum toxin serotypes. Botulinum toxin is released by Clostridial bacteria as a complex comprising a 150 kDa botulinum toxin protein molecule along with an associated non-toxic protein. Therefore, the botulinum toxin type A complex can be produced by Clostridial bacteria in 900 kDa, 500 kDa and 300 kDa types. 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 about 300 kDa complexes. These complexes (ie, those having 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 (comprising the associated neurotoxin complex with the botulinum toxin molecule) will act to provide stability against botulinum toxin molecular denaturation and protection against digestive acids when the toxin is ingested. Also, the larger the botulinum toxin complex (molecular weight is greater than about 150 kDa), the slower the botulinum toxin will diffuse from the site where the botulinum toxin complex was injected intramuscularly. Therefore, in the present invention, the botulinum toxin may include both a form not containing a complexed protein or a complex form containing a complexed protein. The different serotypes of botulinum toxin differ depending on the animal species they act on and the degree and duration of the paralysis they cause. The molecular weight of a botulinum toxin protein derived from Clostridium botulinum types A, B, C, D, E, F, or G is approximately 150 kDa unless it contains the naturally-forming complexing protein.

In one embodiment of the present invention, nucleic acid is a high molecular organic material in which nucleotides are polymerized in a long chain form, and is genetic information encoding the peptide of the present invention.

In one embodiment of the present invention, "skin wrinkles" refers to fine lines caused by deterioration of the skin.

It is known that skin wrinkles are affected by various factors such as skin moisture content, collagen content, and immunity to external environment. expression and activity of degrading enzymes.

More specifically, the main function of skin fibroblasts is regeneration of damaged skin tissue through extracellular matrix synthesis and proliferation. Accordingly, the endogenous aging factors reduce the physiologically active function of dermal fibroblasts in the dermal layer. Type I collagen, which accounts for more than 95% of the skin extracellular matrix, and plays a very important role in the physiological activity of fibroblasts through interaction and signal exchange with cell adhesion molecules and cytoskeleton, etc. When the synthesis of cause a vicious cycle. Therefore, it can be seen that collagen of dermal fibroblasts is directly related to skin regeneration, skin elasticity, skin wrinkle formation, and tissue repair or regeneration during skin damage.

That is, when the synthesis of collagen or procollagen in skin fibroblasts is promoted or the synthesis of matrix metallo-proteinase (hereinafter referred to as 'MMP') that decomposes collagen is inhibited, skin elasticity improvement, skin regeneration, It is possible to obtain effects such as skin wrinkle improvement, wound healing, repair and regeneration of damaged skin tissue, and prevention of skin aging.

In one embodiment of the present invention, "preventing or improving skin wrinkles" means inhibiting or inhibiting the generation of wrinkles on the skin, or alleviating the wrinkles already generated.

The botulinum toxin-derived peptide fragment of the present invention, and a cosmetic composition for improving skin wrinkles comprising the same, are characterized by increasing the expression of type I procollagen protein or inhibiting 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 comprising the same, increase the production of type I procollagen without showing cytotoxicity in normal human fibroblasts (HDFn) irradiated with UV light. while suppressing the expression of matrix metallo-proteinase, which promotes collagen degradation, increases skin elasticity, skin regeneration, skin wrinkle improvement, wound healing, repair and regeneration of damaged skin tissue, and skin It may have effects such as anti-aging. Specifically, it may have an effect of preventing or improving skin wrinkles.

The botulinum toxin-derived peptide fragment of the present invention and the cosmetic composition for skin wrinkle improvement comprising the same have an effect of removing skin wrinkles by themselves, and are mixed with substances known to be effective in preventing and improving skin wrinkles The effect of preventing and improving skin wrinkle can be continuously maintained even when used as a skin wrinkle. For example, the cosmetic composition of the present invention further contains water-soluble vitamins, fat-soluble vitamins, polymeric peptides, polymeric polysaccharides, sphingolipids, and seaweed extracts. may include

The water-soluble vitamin may be any type of vitamin that can be formulated in cosmetics. Preferably, vitamin B1, vitamin B2, vitamin B6, pyridoxine, pyridoxine hydrochloride, vitamin B12, pantothenic acid, nicotinic acid, nicotinic acid amide, folic acid, vitamin C, vitamin H and the like, and their salts (thiamine hydrochloride, sodium ascorbate salt, etc.) and derivatives (ascorbic acid-2-phosphate sodium salt, ascorbic acid-2-phosphate magnesium salt, etc.) can also be used in the present invention. Included in water-soluble vitamins. The water-soluble vitamin can be obtained by a conventional method such as a microbial transformation method, a microbial culture purification method, an enzymatic method or a chemical synthesis method.

The fat-soluble vitamin may be any type of vitamin that can be formulated in cosmetics, but preferably includes vitamin A, carotene, vitamin D2, vitamin D3, vitamin E, and the like, and their derivatives (ascorbine palmitate, ascor stearate). Bean, dipalmitate ascorbine, dl-alpha tocopherol acetate, dl-alpha tocopherol nicotinic acid, vitamin E, DL-pantothenyl alcohol, D-pantothenyl alcohol, pantothenyl ethyl ether, etc.) are included in the fat-soluble vitamins used in the present invention. do.

The polymer peptide may be any compound as long as it can be formulated in cosmetics, but preferably includes collagen, hydrolyzed collagen, gelatin, elastin, hydrolyzed elastin, and keratin. The polymer peptide can be purified and obtained by a conventional method such as a purification method of a microorganism culture solution, an enzyme method, or a chemical synthesis method, or can be purified from natural products such as the dermis of pigs or cattle and silk fibers of silkworms.

The polymer polysaccharide may be any compound as long as it can be formulated in cosmetics, but preferably proteoglycan, hyaluronic acid, hydroxyethyl cellulose, xanthan gum, chondroitin sulfate or a salt thereof (sodium salt, etc.). For example, chondroitin sulfate or a salt thereof can be used after purification from mammals or fish.

The sphingo lipid may be any as long as it can be formulated in cosmetics, but preferably includes ceramide, phytosphingosine, sphingosaccharide lipid, and the like. The sphingo lipid may be purified by a conventional method from mammals, fish, shellfish, yeast or plants, or may be obtained by chemical synthesis.

The seaweed extract may be any one as long as it can be formulated in cosmetics, but preferably brown algae extract, red algae extract, green algae extract, etc. Sodium, potassium alginate, etc. are also included in the seaweed extract used in the present invention. The seaweed extract can be obtained by purification from seaweed by a conventional method.

In the cosmetic of the present invention, in addition to the above essential ingredients, other ingredients commonly formulated in cosmetics may be blended as needed.

In addition, as a compounding component which may be added, an oil-fat component, a moisturizing agent, an emollient agent, a ultraviolet absorber, a pH adjuster, a fragrance|flavor, a blood circulation promoter, a cooling agent, a restrictor, purified water, etc. are mentioned.

Examples of the fats and oils include ester fats and oils, hydrocarbon oils, silicone oils, fluorine oils, animal fats, and vegetable oils.

The ester-based fats and oils include tri2-ethylhexanoate glyceryl, 2-ethylhexanoate cetyl, 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(capryl, capric acid) glyceryl, trimethylolpropane triethylhexanoate, trimethylolpropane triisostearate, pentaeli tetra2-ethylhexanoate Slitol, cetyl caprylate, decyl laurate, hexyl laurate, decyl myristate, myristyl myristate, cetyl myristate, stearyl stearate, decyl oleate, cetyl ricinoleate, laurate Sostearyl, isotridecyl myristate, isocetyl palmitate, octyl stearate, isocetyl stearate, isodecyl oleate, octyldodecyl oleate, octyldodecyl linoleate, isostearate isopropyl, 2-ethylhexanoic acid Tostearyl, 2-ethyl stearyl 2-ethylhexanoate, hexyl isostearate, ethylene glycol dioctanoate, ethylene glycol dioleate, propylene glycol dicaprylate, propylene glycol dicaprylate, neopentyl glycol dicaprate, neopentyl dioctanoate Glycol, glyceryl tricaprylate, glyceryl triundecyl acid, glyceryl triisopalmitate, glyceryl triisostearate, octyldodecyl neopentanoate, isostearyl octanoate, octyl isononanoate, hexyl neodecanoate Decyl, neodecanoate octyldodecyl, isocetyl isostearate, isostearyl isostearate, octyldecyl isostearate, polyglycerol oleate ester, polyglycerol isostearate ester, triisocetyl citrate, triisoalkyl citrate, Triisooctyl citrate, lauryl lactate, myristyl lactate, cetyl lactate, octyldecyl lactate, triethyl citrate, acetyl triethyl citrate, acetyl tributyl citrate, trioctyl citrate, diisostearyl malate, 2-hydroxystearic acid Ethylhexyl, di2-ethylhexyl succinate, diisobutyl adipate, diisopropyl sebacate, dioctyl sebacate, cholesteryl stearate, isostear Cholesteryl acid, cholesteryl hydroxystearate, cholesteryl oleate, dihydrocholesteryl oleate, phytsteryl isostearate, phytsteryl oleate, 12-stealoylhydroxystearate isocetyl, 12 -ester systems, such as stearyl stealoyl hydroxy stearate and 12-stealoyl hydroxy stearate isostearyl, etc. are mentioned.

Examples of the hydrocarbon-based fats and oils include hydrocarbon-based fats and oils such as squalene, liquid paraffin, alpha-olefin oligomer, isoparaffin, ceresin, paraffin, liquid isoparaffin, polybudene, microcrystalline wax, petrolatum.

The silicone-based fats and oils include polymethylsilicone, methylphenylsilicone, methylcyclopolysiloxane, octamethylpolysiloxane, decamethylpolysiloxane, dodecamethylcyclosiloxane, dimethylsiloxane methylcetyloxysiloxane copolymer, dimethylsiloxane methylstealloxysiloxane copolymer, alkyl Modified silicone oil, amino modified silicone oil, etc. are mentioned.

Examples of the fluorine-based oils and fats include perfluoropolyethers.

The animal or vegetable oils include avocado oil, almond oil, olive oil, sesame oil, rice bran oil, soybean oil, corn oil, rapeseed oil, sesame 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, moonflower colostrum, marker damia nut oil, egg yolk oil, beef tallow, horse oil, mink oil, orange rafi oil, jojoba oil, candelaer wax, carnauba wax, liquid ranol , and animal or plant oils and fats such as hydrogenated castor oil.

Examples of the moisturizing agent include a water-soluble low-molecular moisturizer, a fat-soluble molecular moisturizer, a water-soluble polymer, and a fat-soluble polymer.

The water-soluble low molecular weight moisturizer includes serine, glutamine, sorbitol, mannitol, pyrrolidone-sodium carboxylate, glycerin, propylene glycol, 1,3-butylene glycol, ethylene glycol, polyethylene glycol B (polymerization degree n = 2 or more), Polypropylene glycol (polymerization degree n = 2 or more), polyglycerol B (polymerization degree n = 2 or more), lactic acid, lactic acid salt, etc. are mentioned.

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

Examples of the water-soluble polymer include carboxyvinyl polymer, polyaspartate, tragacanth, xanthan gum, methyl cellulose, hydroxymethyl cellulose, hydroxyethyl cellulose, hydroxypropyl cellulose, carboxymethyl cellulose, water-soluble chitin, chitosan, dextrin, etc. can be heard

Examples of the fat-soluble polymer include polyvinylpyrrolidone eicocene copolymer, polyvinylpyrrolidone hexadecene copolymer, nitrocellulose, dextrin fatty acid ester, and high molecular weight silicone.

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

The UV 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 cinnamate, para-methoxycinnamic acid-2-ethoxyethyl, para-methoxycinnamic acid octyl, dipara-methoxycinnamic acid mono-2-ethylhexane glyceryl, para-methoxycinnamic acid isopropyl, diisopropyl diisopropyl cinnamic acid ester mixture , urocanic acid, ethyl urokanate, hydroxymethoxybenzophenone, hydroxymethoxybenzophenonesulfonic acid and its salts, dihydroxymethoxybenzophenone, dihydroxymethoxybenzophenonedisulfonate 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 adjuster include citric acid, sodium citrate, malic acid, sodium malate, fmalic acid, sodium fumarate, succinic acid, sodium succinate, sodium hydroxide, sodium monohydrogen phosphate, and the like.

In addition, blending components that may be added are not limited thereto, and any of the above components can be blended within a range that does not impair the object and effect of the present invention, but preferably 0.01 to 5 parts by weight based on the total weight, more Preferably, it may be blended in an amount of 0.01 to 3 parts by weight.

Cosmetics prepared by containing the composition of the present invention may take the form of solutions, emulsions, viscous mixtures, and the like.

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

The composition of the present invention can be used in various ways, such as cosmetics or face washes having 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, nourishing lotion, massage cream, nourishing cream, moisture cream, hand cream. , foundation, essence, nourishing essence, and pack, 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, vegetable fiber, wax, paraffin, starch, tracanth, cellulose derivative, polyethylene glycol, silicone, bentonite, silica, talc or zinc oxide may be used as a carrier component. can

When the formulation of the present invention is a powder or a spray, lactose, talc, silica, aluminum hydroxide, calcium silicate or polyamide powder may be used as a carrier component. In particular, in the case of a spray, additional chlorofluorohydrocarbon, propane , butane or propellants such as 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, for example, water, ethanol, isopropanol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1 ,3-butylglycol oil, glycerol fatty esters, fatty acid esters of polyethylene glycol or sorbitan.

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

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

The botulinum toxin-derived peptide fragment as an active ingredient of the pharmaceutical composition is characterized in that it increases the expression of type I procollagen protein or inhibits the expression of MMP-1 protein. It may further include suitable carriers, excipients and diluents commonly used.

The pharmaceutical dosage form of the composition of the present invention may be used in the form of a pharmaceutically acceptable salt thereof, or may 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, acacia gum, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil.

When formulating the composition of the present invention, according to a conventional method, powder, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols, etc., oral dosage forms, external preparations, suppositories, or sterile injection solutions may be formulated and used. can Specifically, in the case of formulation, it may be prepared using a diluent or excipient such as a filler, an extender, a binder, a wetting agent, a disintegrant, a surfactant, etc. normally used. Solid preparations for oral administration include tablets, pills, powders, granules, capsules, etc., and these solid preparations include at least one excipient in a high molecular weight polysaccharide or taro extract, for example, starch, calcium carbonate, water It can be prepared by mixing cross (sucrose), lactose (lactose), gelatin, and the like. In addition to simple excipients, lubricants such as magnesium stearate and talc may also be used. Liquid formulations for oral use include suspensions, solutions, emulsions, and syrups. In addition to commonly used simple diluents such as water and liquid paraffin, various excipients such as wetting agents, sweeteners, fragrances, and preservatives may be included. have. Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solutions, suspensions, emulsions, lyophilized formulations and suppositories. Non-aqueous solvents and suspending agents include propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate. As a base of the suppository, witepsol, macrogol, tween 61, cacao butter, laurin, glycerogelatin, and the like can be used.

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

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

The botulinum toxin-derived peptide fragment as an active ingredient of the food composition is characterized in that it increases the expression of type I procollagen protein or inhibits the expression of MMP-1 protein. The food composition of the present invention is a health functional food. can be used The term "health functional food" means a food manufactured and processed using raw materials or ingredients useful for the human body in accordance with Health Functional Food Act No. 6727, and "functionality" refers to the structure of the human body. And it means ingestion for the purpose of obtaining useful effects for health purposes such as regulating nutrients for function or physiological action.

The food composition of the present invention may contain conventional food additives, and the suitability as the "food additive" is determined according to the general rules and general test methods of the Food Additives Code approved by the Food and Drug Administration, unless otherwise specified. It is judged according to the standards and standards for the relevant item.

The items listed in the “Food Additives Code” include, for example, chemical compounds such as ketones, glycine, potassium citrate, nicotinic acid, and cinnamic acid; Mixed preparations such as sodium L-glutamate preparation, noodle-added alkali agent, preservative agent, and tar color agent can be mentioned.

Among health functional foods in the form of capsules, hard capsules can be prepared by filling conventional hard capsules with a mixture of additives such as excipients, or their granules or coated granules. It can be prepared by filling in a capsule base such as gelatin. The soft capsules may contain a plasticizer such as glycerin or sorbitol, a colorant, a preservative, and the like, if necessary.

A health functional food in the form of a pill can be prepared by molding a mixture of excipients, binders, and disintegrants into herbal extracts in an appropriate way. You can also dress up.

The health functional food in the form of granules may be prepared in a granular form by a mixture of excipients, binders, disintegrants, etc. in the herbal extract in an appropriate manner, and may contain flavoring agents, flavoring agents, etc. as necessary. The definitions of terms for the excipients, binders, disintegrants, lubricants, flavoring agents, and the like of the present invention are those described in documents known in the art and include those having the same or similar functions (Korean Pharmacopoeia commentary, text Seongsa, Korea Pharmaceutical University Association, 5th edition, p33-48, 1989).

When the food composition of the present invention is provided in a liquid form, the peptide of the present invention may be included in a dissolved or undissolved state, and for effective flavor masking, oligosaccharides, sucrose, fructose, syrup sugar, fruit extract, It may further include a flavoring agent or the like alone or a combination thereof, and without being limited thereto, ingredients suitable for food may be appropriately used.

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

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

In another embodiment of the present invention, there is provided a cosmetic composition for preventing or improving skin wrinkles comprising the botulinum toxin-derived peptide, wherein the botulinum toxin-derived peptide increases the expression of type I procollagen protein. It provides a cosmetic composition for prevention or improvement, wherein the botulinum toxin-derived peptide inhibits the expression of MMP-1 protein, and provides a cosmetic composition for preventing or improving skin wrinkles, the cosmetic composition comprising: a skin lotion, a skin softener, a skin Toner, Astringent, Lotion, Milk Lotion, Moisture Lotion, Nourishing Lotion, Massage Cream, Nourishing Cream, Moisture Cream, Hand Cream, Foundation, Essence, Nourishing Essence, Pack, Soap, Cleansing Foam, Cleansing Lotion, Cleansing Cream, Body Lotion and It provides a cosmetic composition for preventing or improving skin wrinkles that is in any one formulation selected from the group consisting of body cleanser.

In another embodiment of the present invention, there is provided a pharmaceutical composition for preventing or treating skin wrinkles comprising the botulinum toxin-derived peptide, wherein the botulinum toxin-derived peptide increases the expression of type I procollagen protein. It provides a pharmaceutical composition for prevention or treatment, wherein the botulinum toxin-derived peptide inhibits the expression of MMP-1 protein, and provides a pharmaceutical composition for preventing or treating skin wrinkles, wherein the pharmaceutical composition is a cream, gel, patch, 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, liniments, pastas, and cataplasmas.

In another embodiment of the present invention, there is provided a food composition for preventing or improving skin wrinkles comprising the botulinum toxin-derived peptide of claim 1, wherein the botulinum toxin-derived peptide increases the expression of type I procollagen protein. It provides a food composition for preventing or improving wrinkles, wherein the botulinum toxin-derived peptide inhibits the expression of MMP-1 protein. And it provides a food composition for preventing or improving skin wrinkles that is any one formulation selected from the group consisting of liquids.

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 comprising 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 the wrinkles of damaged skin. Collectively, the composition of the present invention can prevent and greatly improve skin wrinkles.

1 is a result 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.
2 is a result confirming that the botulinum toxin-derived peptide fragment of the present invention has an effect on collagen synthesis in human fibroblasts (HDFn) according to an embodiment of the present invention.
3 is a result confirming that the botulinum toxin-derived peptide fragment of the present invention has an effect of inhibiting 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 botulinum toxin-derived fragment peptide (peptide)

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

production example SEQ ID NO: amino acid sequence Location within 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 (peptide)

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

As a result of the experiment, it was confirmed that all of the peptides No. 1 to No. 6 did not exhibit cytotoxicity up to the highest concentration tested. Considering the tested concentration range, it was found that peptides 5, 6 > 2, 4 > 1, 3 were stable to cells in the order.

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

Human fibroblasts (HDFn) were seeded in a 24 well plate at 2 × 10 ⁴ cells/well and stabilized for 24 hours, then exchanged with FBS-free medium and treated with 6 types of peptides synthesized in Example 1 for 24 hours, respectively. Then, the amount of collagen synthesized by separating the supernatant was measured using a Procollagen Type I C-Peptide (PIP) EIA Kit. The collagen is a protein that maintains the mechanical properties of the 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 repeated three times for each concentration with the negative control group (no peptide treatment), and the biological statistical standard (significance level (α): 5%) was obtained by the two sample t-test method. ) to evaluate the significance. The results are shown in FIG. 2 .

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

Example 4: Confirmation of MMP-1 expression inhibition effect of botulinum toxin-derived fragment peptide (peptide)

Human fibroblasts (HDFn) were seeded in a 24-well plate at 2 × 10 ⁴ cells/well, stabilized for 24 hours, washed twice with PBS, and irradiated with UV at 6 J/cm ² . It was exchanged with FBS-free medium and each of the six peptides synthesized in Example 1 was treated for 48 hours. Then, 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 repeated three times for each concentration with the negative control group (no peptide treatment), and the biological statistical standard (significance level (α) ): 5%) to evaluate significance. The results are shown in FIG. 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. was shown 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 insignificant. Therefore, it was found that the MMP-1 inhibitory effect was good in the order of peptide 1 > 4 > 5 > 3.

As described above in detail a specific part of the present invention, for those of ordinary skill in the art, this specific description is only a preferred embodiment, and it is clear that the scope of the present invention is not limited thereto. Accordingly, the substantial scope of the present invention will be defined by the appended claims and their equivalents.

<110> HUGEL INC. <120> Fragmented peptide from Botulinum toxin, and cosmetic composition for ameliorating wrinkles comprising thereof <130> PDPB194306 <160> 6 <170> KoPatentIn 3.0 <210> 1 <211> 17 <212> PRT <213> Artificial Sequence <220> <223> Fragmented peptide from Botulinum toxin <400> 1 Asp Pro Ala Val Thr Leu Ala His Glu Leu Ile His Ala Gly His Arg 1 5 10 15 Leu <210> 2 <211> 36 <212> PRT <213> Artificial Sequence <220> <223> Fragmented peptide from Botulinum toxin <400> 2 Leu Phe Glu Phe Tyr Lys Leu Leu Cys Val Arg Gly Ile Ile Thr Ser 1 5 10 15 Lys Thr Lys Ser Leu Asp Lys Gly Tyr Asn Lys Ala Leu Asn Asp Leu 20 25 30 Cys Ile Lys Val 35 <210> 3 <211> 22 <212> PRT <213> Artificial Sequence <220> <223> Fragmented peptide from Botulinum toxin <400> 3 Leu Arg Ala Gln Glu Phe Glu His Gly Lys Ser Arg Ile Ala Leu Thr 1 5 10 15 Asn Ser Val Asn Glu Ala 20 <210> 4 <211> 22 <212> PRT <213> Artificial Sequence <220> <223> Fragmented peptide from Botulinum toxin <400> 4 Ile Pro Tyr Ile Gly Pro Ala Leu Asn Ile Gly Asn Met Leu Tyr Lys 1 5 10 15 Asp Asp Phe Val Gly Ala 20 <210> 5 <211> 32 <212> PRT <213> Artificial Sequence <220> <223> Fragmented peptide from Botulinum toxin <400> 5 Ala Thr Lys Ala Ile Ile Asn Tyr Gln Tyr Asn Gln Tyr Thr Glu Glu 1 5 10 15 Glu Lys Asn Asn Ile Asn Phe Asn Ile Asp Asp Leu Ser Ser Lys Leu 20 25 30 <210> 6 <211> 40 <212> PRT <213> Artificial Sequence <220> <223> Fragmented peptide from Botulinum toxin <400> 6 Gly Ser Val Met Thr Thr Asn Ile Tyr Leu Asn Ser Ser Leu Tyr Arg 1 5 10 15 Gly Thr Lys Phe Ile Ile Lys Lys Lys Tyr Ala Ser Gly Asn Lys Asp Asn 20 25 30 Ile Val Arg Asn Asn Asp Arg Val 35 40

Claims (16)

A botulinum toxin-derived peptide consisting of the amino acid sequence of SEQ ID NO: 6.
delete delete 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.
delete delete delete A pharmaceutical composition for preventing or treating skin wrinkles, comprising the botulinum toxin-derived peptide of claim 1.
10. The method of claim 9,
The botulinum toxin-derived peptide increases the expression of type I procollagen protein, a pharmaceutical composition.
delete delete A food composition for preventing or improving skin wrinkles, comprising the botulinum toxin-derived peptide of claim 1.
delete delete delete

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