KR102672416B1 - Novel peptide with antibacterial and anticancer efficacy against multidrug-resistant pathogens - Google Patents

Abstract

The inventors of the present invention directly isolated the actinomycete Micromonospora sp. The common presence of SapB peptide (lantipeptide) was confirmed in the genomes of 10 species, and a common gene was discovered in actinomycetes isolated from different regions and organisms, leading to research. Lantipeptide is widely known as an antibiotic peptide and is actually used commercially (e.g. nisin), but research on SapB peptide is insufficient. This is because SapB peptides tend to aggregate together and do not dissolve well in water, making it difficult to evaluate their antibiotic efficacy and conducting research. Therefore, in the present invention, we began research to determine whether the aggregation/non-hydrophilic nature of SapB peptide affected the antibiotic efficacy. As a result, we developed a new SapB peptide and confirmed its antibiotic and cancer cell growth inhibitory efficacy. The present invention has been completed. Based on the sequence of this research result, it will be possible to develop a more improved bioactive material.

Description

Novel peptide with antibacterial and anticancer efficacy against multidrug-resistant pathogens}

The present invention relates to a novel peptide with antibacterial and anticancer efficacy against multidrug-resistant pathogens.

The inventors of the present invention directly isolated the actinomycete Micromonospora sp. The common presence of SapB peptide (lantipeptide) was confirmed in the genomes of 10 species, and a common gene was discovered in actinomycetes isolated from different regions and organisms, leading to research. Lantipeptide is widely known as an antibiotic peptide and is actually used commercially (e.g. nisin), but research on SapB peptide is insufficient. This is because SapB peptides tend to aggregate together and do not dissolve well in water, making it difficult to evaluate their antibiotic efficacy and conducting research. Therefore, in the present invention, we began research to determine whether the aggregation/non-hydrophilic nature of SapB peptide affected the antibiotic efficacy. As a result, we developed a new SapB peptide and confirmed its antibiotic and cancer cell growth inhibitory efficacy. The present invention has been completed. Based on the sequence of this research result, it will be possible to develop a more improved bioactive material.

More specifically, in order to develop a new peptide of the present invention, the structural characteristics of SapB were maintained, but the amino acid sequence was changed and synthesized, and it was confirmed that it has antibacterial effect against multidrug-resistant bacteria. In particular, the antibacterial efficacy was evaluated against multidrug-resistant pathogens that are difficult to control (E. coli, Pseudomonas aeruginosa, Bacillus cereus, and Staphylococcus aureus), and it was confirmed that it exhibits a wide range of antibacterial efficacy against Gram-positive and Gram-negative bacteria. In addition, the new improved peptides showed growth inhibitory effects on cancer cells (liver cancer, colon cancer, lung cancer, breast cancer, and melanoma cancer). In other words, existing lantipeptides cannot be synthesized and are produced through biosynthesis, but the improved new peptide in this study is very easy to synthesize and has a short sequence, making production costs very economical.

In addition, lantipeptide is amphiphilic and can exhibit high hemolysis in red blood cells, but the improved new peptides of the present invention showed low hemolysis (less than 5% on average) even at high concentrations.

Therefore, the new peptides of the present invention exhibit antibacterial efficacy against multi-drug-resistant pathogens and growth inhibitory efficacy against cancer cells, are much simpler to synthesize, are easy to produce, and are shorter than existing sequences by more than 50% on average, so the synthesis cost is economical, so they can be used in industry. It is thought to be easier to do. This study is the first to confirm the antibiotic and cancer cell growth inhibitory effects of SapB peptide, and it will be possible to develop an improved bioactive material based on the sequence of this study result.

10-2158205

To solve the above problems, one object of the present invention is to provide a novel peptide with antibacterial or anticancer efficacy against multidrug-resistant pathogens.

One object of the present invention to solve the above problems is to provide an antibacterial or anticancer pharmaceutical composition containing the novel peptide as an active ingredient.

One object of the present invention to solve the above problems is to provide an antibacterial quasi-drug composition, cosmetic composition, agricultural and livestock feed composition, or health functional food composition containing the novel peptide as an active ingredient.

In order to achieve the above object, the present invention provides a novel peptide having antibacterial or anticancer activity consisting of an amino acid sequence represented by XCRAXLLC, wherein X in the amino acid sequence is R, K or H. to provide.

In one embodiment of the present invention, the “peptide” may be improved to improve the aggregation problem of the SapB peptide and enable synthesis, but is not limited thereto.

In one embodiment of the present invention, the “peptide” may be characterized as each peptide consisting of the amino acid sequence of SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3, but is not limited thereto.

In one embodiment of the present invention, the “peptide” may have antibacterial effect against multi-drug resistant pathogens.

In one embodiment of the present invention, the “multi-drug-resistant pathogen” may be Escherichia coli, Pseudomonas aeruginosa, Bacillus cereus, or Staphylococcus aureus.

In one embodiment of the present invention, the “peptide” may have anti-cancer efficacy.

In one embodiment of the present invention, the “anticancer” may have anticancer efficacy against liver cancer, colon cancer, lung cancer, breast cancer, or melanoma cancer.

In one embodiment of the present invention, the “peptide” is improved to improve the aggregation problem of the SapB peptide and enable synthesis, and may have a low hemolysis rate and thus be low toxic.

Additionally, in order to achieve the above object, the present invention provides an antibacterial or anticancer pharmaceutical composition containing the novel peptide as an active ingredient.

In addition, in order to achieve the above object, the present invention provides an antibacterial quasi-drug composition containing the novel peptide as an active ingredient.

Additionally, in order to achieve the above object, the present invention provides an antibacterial cosmetic composition containing the novel peptide as an active ingredient.

In addition, in order to achieve the above object, the present invention provides an antibacterial concentrated acid feed composition containing the novel peptide as an active ingredient.

Finally, in order to achieve the above object, the present invention provides an antibacterial health functional food composition containing the novel peptide as an active ingredient.

The inventors of the present invention directly isolated the actinomycete Micromonospora sp. The common presence of SapB peptide (lantipeptide) was confirmed in the genomes of 10 species, and a common gene was discovered in actinomycetes isolated from different regions and organisms, leading to research. Lantipeptide is widely known as an antibiotic peptide and is actually used commercially (e.g. nisin), but research on SapB peptide is insufficient. This is because SapB peptides tend to aggregate together and do not dissolve well in water, making it difficult to evaluate their antibiotic efficacy and conducting research. Therefore, in the present invention, we began research to determine whether the aggregation/non-hydrophilic nature of SapB peptide affected the antibiotic efficacy. As a result, we developed a new SapB peptide and confirmed its antibiotic and cancer cell growth inhibitory efficacy. The present invention has been completed. Based on the sequence of this research result, it will be possible to develop a more improved bioactive material.

Figure 1 is a diagram showing the original sequence of SapB peptide in one embodiment of the present invention.
Figure 2 is a diagram showing the chemical formula of the novel peptide SEQ ID NO: 1 of the present invention, in one embodiment of the present invention.
Figure 3 is a diagram showing the chemical formula of the novel peptide SEQ ID NO: 2 of the present invention in one embodiment of the present invention.
Figure 4 is a diagram showing the chemical formula of the novel peptide SEQ ID NO: 3 of the present invention in one embodiment of the present invention.
Figure 5 is a diagram showing the hemolysis rate of the novel peptide SEQ ID NOs: 1, 2, and 3 of the present invention in one embodiment of the present invention.

Hereinafter, the present invention will be described in detail through embodiments of the present invention with reference to the attached drawings. However, the following implementation examples are provided as examples of the present invention, and if it is judged that a detailed description of a technology or configuration well known to those skilled in the art may unnecessarily obscure the gist of the present invention, the detailed description may be omitted. , the present invention is not limited thereby. The present invention is capable of various modifications and applications within the description of the patent claims described below and the scope of equivalents interpreted therefrom.

In addition, terminology used in this specification is a term used to appropriately express preferred embodiments of the present invention, and may vary depending on the intention of the user or operator or the customs of the field to which the present invention belongs. Therefore, definitions of these terms should be made based on the content throughout this specification. Throughout the specification, when a part "includes" a certain component, this means that it may further include other components rather than excluding other components, unless specifically stated to the contrary.

Throughout this specification, '%' used to indicate the concentration of a specific substance is (w/w) % for solid/solid, (w/v) % for solid/liquid, and Liquid/Liquid is (v/v) %.

In one aspect, the present invention provides a new peptide that is an improved version of the SapB peptide.

The "novel peptide" of the present invention has excellent antibacterial efficacy against various multi-drug resistant pathogens (E. coli, Pseudomonas aeruginosa, Bacillus cereus and Staphylococcus aureus) that are difficult to control, and is therefore useful in cosmetic compositions, quasi-drug compositions, pharmaceutical compositions, agricultural and livestock feed compositions or health products. It can be used as a functional food composition.

The “novel peptide” of the present invention has excellent growth inhibitory effects on various cancer cells (liver cancer, colon cancer, lung cancer, breast cancer, and melanoma cancer), so it can be used as a pharmaceutical composition.

In one aspect, the present invention can be used as a cosmetic composition.

The novel peptide of the present invention can be used in a variety of products to exhibit antibacterial efficacy. Products to which this composition can be added include, for example, cosmetics such as various creams, lotions, skins, and essences, as well as shampoos, rinses, cleansers, face washes, soaps, treatments, packs, and beauty essences.

The cosmetic composition of the present invention may include a composition selected from the group consisting of water-soluble vitamins, oil-soluble vitamins, high molecular weight peptides, high molecular weight polysaccharides, sphingolipids, and seaweed extracts.

At this time, any water-soluble vitamin may be used as long as it can be mixed in cosmetics, but 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, etc. and their salts (thiamine hydrochloride, sodium ascorbate, etc.) or derivatives (sodium ascorbic acid-2-phosphate, magnesium ascorbic acid-2-phosphate, etc.) are water-soluble vitamins that can be used in the present invention. included in Water-soluble vitamins can be obtained by conventional methods such as microbial transformation, purification from microbial cultures, enzymatic methods, or chemical synthesis.

Oil-soluble vitamins may be any that can be mixed into cosmetics, but preferably include vitamin A, carotene, vitamin D2, vitamin D3, vitamin E (d1-alpha tocopherol, d-alpha tocopherol, d-alpha tocopherol), etc. , their derivatives (ascorbine palmitate, ascorbine stearate, ascorbine dipalmitate, dl-alpha tocopherol acetate, dl-alpha tocopherol nicotinate, vitamin E, DL-pantothenyl alcohol, D-pantothenyl alcohol, pantothenyl ethyl ether, etc.) are also included in the oil-soluble vitamins used in the present invention. Oil-soluble vitamins can be obtained by conventional methods such as microbial transformation, purification from microbial cultures, enzymatic or chemical synthesis.

The polymer peptide may be any peptide that can be blended into cosmetics, but preferably includes collagen, hydrolyzed collagen, gelatin, elastin, hydrolyzed elastin, and keratin. High molecular weight peptides can be purified and obtained by conventional methods such as purification from microbial culture media, enzymatic methods, or chemical synthesis, or they can usually be purified and used from natural products such as dermis of pigs or cows or silk fiber of silkworms.

The polymer polysaccharide may be any one that can be blended into cosmetics, but preferably includes hydroxyethyl cellulose, xanthan gum, sodium hyaluronate, chondroitin sulfate or its salts (sodium salt, etc.). For example, chondroitin sulfate or its salt can usually be purified and used from mammals or fish.

The sphingolipid may be any one that can be blended into cosmetics, but preferred examples include ceramide, phytosphingosine, and sphingolipid. 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 any one that can be mixed into cosmetics, but preferably includes brown algae extract, red algae extract, green algae extract, etc. Also, calrageenan, arginic acid, sodium alginate, etc. purified from these seaweed extracts. Potassium arginate and the like 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 composition of the present invention may contain other ingredients normally blended in cosmetics, if necessary. Other ingredients that may be added include oils and fats, moisturizers, emollients, surfactants, organic and inorganic pigments, organic powders, ultraviolet absorbers, preservatives, disinfectants, antioxidants, plant extracts, pH adjusters, alcohol, colorants, fragrances, Examples include blood circulation promoters, cooling agents, antiperspirants, and purified water. Examples of 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 oils, and vegetable fats and oils.

Ester-based oils include glyceryl tri2-ethylhexanoate, cetyl 2-ethylhexanoate, isopropyl myristate, butyl myristate, isopropyl palmitate, ethyl stearate, octyl palmitate, isocetyl isostearate, and stearic acid. Butyl, ethyl linoleate, isopropyl linoleate, ethyl oleate, isocetyl myristate, isostearyl myristate, isostearyl palmitate, octyldodecyl myristate, isocetyl isostearate, diethyl sebacate, adipine. Diisopropyl acid, isoalkyl neopentanoate, tri(caprylic, capric acid)glyceryl, trimethylolpropane tri2-ethylhexanoate, trimethylolpropane triisostearate, pentaelislitol tetra2-ethylhexanoate , cetyl caprylate, decyl laurate, hexyl laurate, decyl myristate, myristyl myristate, cetyl myristate, stearyl stearate, decyl oleate, cetyl ricinooleate, isostear laurate. Reyl, isotridecyl myristate, isocetyl palmitate, octyl stearate, isocetyl stearate, isodecyl oleate, octyldodecyl oleate, octyldodecyl linoleate, isopropyl isostearate, cetoste 2-ethylhexanoate. Aryl, 2-ethylhexanoate stearyl, hexyl isostearate, ethylene glycol dioctanate, ethylene glycol dioleate, propylene glycol dicapric acid, di(capryl, capric acid) propylene glycol, propylene glycol dicaprylate, dicapric acid Neopentyl glycol prinate, neopentyl glycol dioctanate, glyceryl tricaprylate, glyceryl triundecylate, glyceryl triisopalmitate, glyceryl triisostearate, octyldodecyl neopentanoate, isostear octanoate. Reyl, octyl isononanoate, hexyldecyl neodecanoate, 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, malic acid. Diisostearyl acid, 2-ethylhexyl hydroxystearate, di2-ethylhexyl succinate, diisobutyl adipate, diisopropyl sebacate, dioctyl sebacate, cholesteryl stearate, cholesteryl isostearate. , hydroxycholesteryl hydroxystearate, cholesteryl oleate, dihydrocholesteryl oleate, phytsteryl isostearate, phytsteryl oleate, 12-stealoyl hydroxystearate isocetyl, 12-steallo. Ester systems such as stearyl monohydroxystearate and isostearyl 12-stealoylhydroxystearate can be mentioned.

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

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

Examples of fluorine-based fats and oils include perfluoropolyether.

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

Moisturizers include water-soluble low-molecular-weight moisturizers, oil-soluble molecular moisturizers, water-soluble polymers, and fat-soluble polymers.

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), and polypropylene. Examples include glycol (degree of polymerization n = 2 or more), polyglycerin B (degree of polymerization n = 2 or more), lactic acid, lactate, etc.

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

Water-soluble polymers include carboxyvinyl polymer, polyaspartate, tragacanth, xanthan gum, methyl cellulose, hydroxymethyl cellulose, hydroxyethyl cellulose, hydroxypropyl cellulose, carboxymethyl cellulose, water-soluble chitin, chitosan, and dextrin. You can.

Examples of oil-soluble polymers include polyvinylpyrrolidone and eicosene copolymers, polyvinylpyrrolidone and hexadecene copolymers, nitrocellulose, dextrin fatty acid ester, and polymer silicone. Examples of emollients include long-chain acyl glutamic acid cholesteryl ester, hydroxystearic acid cholesteryl, 12-hydroxystearic acid, stearic acid, rosin acid, and lanolin fatty acid cholesteryl ester.

Examples of surfactants include nonionic surfactants, anionic surfactants, cationic surfactants, and amphoteric surfactants.

Nonionic surfactants include self-emulsifying glycerin monostearate, propylene glycol fatty acid ester, glycerin fatty acid ester, polyglycerin fatty acid ester, sorbitan fatty acid ester, POE (polyoxyethylene) sorbitan fatty acid ester, POE sorbitan fatty acid ester, POE. Glycerin fatty acid ester, POE alkyl ether, POE fatty acid ester, POE hydrogenated castor oil, POE castor oil, POE and POP (polyoxyethylene and polyoxypropylene) copolymer, POE and POP alkyl ether, polyether modified silicone, lauric acid. Examples include alkanolamide, alkylamine oxide, and hydrogenated soybean phospholipid.

Anionic surfactants include fatty acid soap, alpha-acyl sulfonate, alkyl sulfonate, alkyl allyl sulfonate, alkyl naphthalene sulfonate, alkyl sulfate, POE alkyl ether sulfate, alkyl amide sulfate, alkyl phosphate, POE alkyl phosphate industry, alkyl amide. Phosphate, alkyloyl alkyl taurine salt, N-acylamino acid salt, POE alkyl ether carboxylate, alkyl sulfosuccinate, sodium alkyl sulfoacetate, acylated hydrolyzed collagen peptide salt, perfluoroalkyl phosphate ester, etc. there is.

Cationic surfactants include alkyltrimethylammonium chloride, stearyltrimethylammonium chloride, stearyltrimethylammonium bromide, cetostearyltrimethylammonium chloride, distearyldimethylammonium chloride, stearyldimethylbenzylammonium chloride, behenyltrimethylammonium bromide, and chloride. Examples include benzalkonium, diethylaminoethylamide stearate, dimethylaminopropyl stearate, and quaternary ammonium salts of lanolin derivatives. Amphoteric surfactants include carboxy beta type, amide beta type, sulfo beta type, hydroxy sulfo beta type, amide sulfo betaine type, phosphobetain type, aminocarboxylate type, imidazoline derivative type, and amidamine type. Amphoteric surfactants, etc. can be mentioned.

Organic and inorganic pigments include silicic acid, anhydrous silicic acid, magnesium silicate, talc, sericite, mica, kaolin, bengala, clay, bentonite, titanium-coated mica, bismuth oxychloride, zirconium oxide, magnesium oxide, zinc oxide, titanium oxide, and aluminum oxide. Inorganic pigments such as calcium sulfate, barium sulfate, magnesium sulfate, calcium carbonate, magnesium carbonate, iron oxide, ultramarine blue, chromium oxide, chromium hydroxide, calamine and complexes thereof; Polyamide, polyester, polypropylene, polystyrene, polyurethane, vinyl resin, urea resin, phenol resin, fluorine resin, silicon resin, acrylic resin, melamine resin, epoxy resin, polycarbonate resin, divinylbenzene and styrene copolymer, Examples include organic pigments such as silk powder, cellulose, CI pigment yellow, and CI pigment orange, and complex pigments of these inorganic pigments and organic pigments.

Examples of organic powder include metal soap such as calcium stearate; Alkyl phosphate metal salts such as sodium zinc cetilate, zinc laurylate, and calcium laurylate; Acyl amino acid polyvalent metal salts such as N-lauroyl-beta-alanine calcium, N-lauroyl-beta-alanine zinc, and N-lauroyl glycine calcium; Amidesulfonic acid polyvalent metal salts such as N-lauroyl-taurine calcium and N-palmitoyl-taurine calcium; N such as N-epsilon-lauroyl-L-lysine, N-epsilon-palmitoylizine, N-alpha-paritoylolnithine, N-alpha-lauroylarginine, N-alpha-hydrogenated beef tallow fatty acid acylarginine, etc. -Acyl basic amino acid; N-acyl polypeptides such as N-lauroylglycylglycine; Alpha-amino fatty acids such as alpha-aminocaprylic acid and alpha-aminolauric acid; Examples include polyethylene, polypropylene, nylon, polymethyl methacrylate, polystyrene, divinylbenzene and styrene copolymers, and ethylene tetrafluoride.

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, and benzyl cinnamate. , paramethoxycinnamic acid-2-ethoxyethyl, octyl paramethoxycinnamic acid, mono-2-ethylhexane glyceryl diparamethoxycinnamic acid, isopropyl paramethoxycinnamic acid, diisopropyl and diisopropylcinnamic acid ester mixture, uro Kaninic acid, ethyl urocanate, hydroxymethoxybenzophenone, hydroxymethoxybenzophenone sulfonic acid and its salts, dihydroxymethoxybenzophenone, dihydroxymethoxybenzophenone disulfonate sodium, dihydroxybenzophenone , 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.

Disinfectants include hinokitiol, triclosan, trichlorohydroxydiphenyl ether, chlorhexidine gluconate, phenoxyethanol, resorcin, isopropylmethylphenol, azulene, salicylic acid, zincphyllithione, benzalkonium chloride, and photosensitive. Sub-No. 301, sodium mononitroguaiacol, undecirenic acid, etc. can be mentioned.

Antioxidants include butylhydroxyanisole, propyl gallate, and elisorbic acid.

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

Examples of alcohol include higher alcohols such as cetyl alcohol.

Additionally, the ingredients that may be added are not limited to these, and any of the ingredients can be blended within the range that does not impair the purpose and effect of the present invention.

The cosmetic composition of the present invention may take the form of a solution, emulsion, viscous mixture, etc.

The ingredients included in the cosmetic composition of the present invention may include ingredients commonly used in cosmetic compositions as active ingredients, for example, conventional auxiliaries and carriers such as stabilizers, solubilizers, vitamins, pigments, and fragrances. Includes.

The functional cosmetic composition of the present invention can be manufactured in any formulation commonly manufactured in the art, and examples include emulsions, creams, lotions, packs, foundations, lotions, beauty essences, hair cosmetics, etc.

Specifically, the cosmetic composition of the present invention includes skin lotion, skin softener, skin toner, milk lotion, astringent, lotion, moisture lotion, nutritional lotion, massage cream, nutritional cream, moisture cream, hand cream, foundation, essence, nutritional essence, It includes formulations of packs, soaps, cleansing foams, cleansing lotions, cleansing creams, hair lotions, hair tonics, hair essences, hair shampoos, hair rinses, hair treatments, body lotions, and body cleansers.

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. /May contain propellants such as 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.

When the formulation of the present invention is a surfactant-containing cleansing agent, the carrier ingredients include aliphatic alcohol sulfate, aliphatic alcohol ether sulfate, sulfosuccinic acid monoester, isethionate, imidazolinium derivative, methyl taurate, sarcosinate, and fatty acid amide. Ether sulfate, alkylamidobetaine, fatty alcohol, fatty acid glyceride, fatty acid diethanolamide, vegetable oil, linoline derivative, or ethoxylated glycerol fatty acid ester can be used.

In one aspect, the present invention can be used as a quasi-drug composition.

Ingredients included in the quasi-drug composition of the present invention may include ingredients commonly used in quasi-drug compositions in addition to the peptide as an active ingredient, such as abrasives, wetting agents, binders, foaming agents, sweeteners, preservatives, medicinal ingredients, flavoring agents, and colorants. , it may include a solvent, brightener, solubilizer, or pH adjuster.

In one aspect, the present invention can be used as a pharmaceutical composition.

The pharmaceutical composition of the present invention may further include adjuvants in addition to the peptide, which is an active ingredient. The auxiliary agent may be any one known in the art without limitation, but its functionality may be increased by further including, for example, Freund's complete auxiliary agent or incomplete auxiliary agent.

The pharmaceutical composition according to the present invention can be prepared by incorporating the active ingredient into a pharmaceutically acceptable carrier. Here, pharmaceutically acceptable carriers include carriers, excipients, and diluents commonly used in the pharmaceutical field. Pharmaceutically acceptable carriers that can be used in the pharmaceutical composition of the present invention include, but are not limited to, lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, gum acacia, alginate, gelatin, Examples include calcium phosphate, calcium silicate, cellulose, methyl cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil.

The pharmaceutical composition of the present invention 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. .

When formulated, 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., and such solid preparations contain the active ingredient plus at least one excipient, such as starch, calcium carbonate, sucrose, lactose, and gelatin. It can be prepared by mixing etc. Additionally, in addition to simple excipients, lubricants such as magnesium stearate and talc may also be used. Liquid preparations for oral administration include suspensions, oral solutions, emulsions, and syrups. In addition to the commonly used diluents such as water and liquid paraffin, they contain various excipients such as wetting agents, sweeteners, fragrances, and preservatives. You can. Preparations for parenteral administration include sterile aqueous solutions, non-aqueous solutions, suspensions, emulsions, lyophilized preparations, and suppositories. Non-aqueous solvents and suspensions include propylene glycol, polyethylene glycol, vegetable oil such as olive oil, and injectable ester such as ethyl oleate. As a base for suppositories, witepsol, tween 61, cacao, laurin, glycerogelatin, etc. can be used.

The pharmaceutical composition according to the present invention can be administered to an individual through various routes. All modes of administration are contemplated, for example, by oral, intravenous, intramuscular, subcutaneous, or intraperitoneal injection.

The dosage of the pharmaceutical composition according to the present invention is selected taking into account the age, weight, gender, physical condition, etc. of the individual. It is obvious that the concentration of the active ingredient included in the pharmaceutical composition can be selected in various ways depending on the target, and is preferably included in the pharmaceutical composition at a concentration of 0.01 to 1,000 μg/ml. If the concentration is less than 0.01 ㎍/ml, pharmaceutical activity may not appear, and if it exceeds 1,000 ㎍/ml, it may be toxic to the human body.

The pharmaceutical composition may be formulated into various oral or parenteral dosage forms.

Dosage forms for oral administration include, for example, tablets, pills, hard and soft capsules, solutions, suspensions, emulsifiers, syrups, granules, etc. These dosage forms contain diluents (e.g. lactose, dextrose, water) in addition to the active ingredient. crose, mannitol, sorbitol, cellulose and/or glycine), lubricants (e.g. silica, talc, stearic acid and magnesium or calcium salts thereof and/or polyethylene glycol). Additionally, the tablets may contain binders such as magnesium aluminum silicate, starch paste, gelatin, tragacanth, methylcellulose, sodium carboxymethylcellulose and/or polyvinylpyrrolidine, and in some cases starch, agar, alginic acid. or disintegrants such as sodium salts thereof or effervescent mixtures and/or absorbents, colorants, flavoring and sweetening agents. The formulation can be prepared by conventional mixing, granulating or coating methods.

In addition, representative formulations for parenteral administration are injectable formulations, and solvents for injectable formulations include water, Ringer's solution, isotonic saline solution, or suspension. The sterile fixed oil of the injectable preparation can be used as a solvent or suspending medium, and any non-irritating fixed oil, including mono- and di-glycerides, can be used for this purpose.

Additionally, the injectable preparation may use fatty acids such as oleic acid.

In one aspect, the present invention can be used as a food/health functional food composition.

In addition to containing peptide as an active ingredient, the food composition of the present invention may contain various flavoring agents or natural carbohydrates as additional ingredients like a typical food composition.

Examples of the above-mentioned natural carbohydrates include monosaccharides such as glucose, fructose, etc.; Disaccharides such as maltose, sucrose, etc.; and polysaccharides, such as common sugars such as dextrin and cyclodextrin, and sugar alcohols such as xylitol, sorbitol, and erythritol. The above-described flavoring agents include natural flavoring agents (thaumatin), stevia extracts (e.g., rebaudioside A, glycyrrhizin, etc.), and synthetic flavoring agents (saccharin, aspartame, etc.). The food composition of the present invention can be formulated in the same way as the pharmaceutical composition and used as a functional food or added to various foods. Foods to which the composition of the present invention can be added include, for example, beverages, meat, chocolate, foods, confectionery, pizza, ramen, other noodles, gum, candy, ice cream, alcoholic beverages, vitamin complexes, health supplements, etc. There is.

In addition, the food composition contains, in addition to the peptide as the active ingredient, various nutrients, vitamins, minerals (electrolytes), flavoring agents such as synthetic and natural flavors, colorants and fillers (cheese, chocolate, etc.), pectic acid and its salts, and alginic acid. and its salts, organic acids, protective colloidal thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohol, carbonating agents used in carbonated beverages, etc. In addition, the food composition of the present invention may contain pulp for the production of natural fruit juice, fruit juice beverages, and vegetable beverages.

The health functional food composition of the present invention can be manufactured and processed in the form of tablets, capsules, powders, granules, liquids, pills, etc. In the present invention, 'health functional food composition' refers to a 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 has It means taking it for the purpose of controlling nutrients or obtaining useful health effects such as physiological effects. The health functional food of the present invention may contain common food additives, and its suitability as a food additive is determined in accordance with the general provisions of the food additive code and general test methods approved by the Food and Drug Administration, unless otherwise specified. Judgment is made according to specifications and standards. Items listed in the 'Food Additive Code' include, for example, chemical compounds such as ketones, glycine, calcium citrate, nicotinic acid, and cinnamic acid; Natural additives such as dark pigment, licorice extract, crystalline cellulose, kaoliang pigment, and guar gum; Examples include mixed preparations such as sodium L-glutamate preparations, noodle additive alkaline preparations, preservative preparations, and tar coloring preparations. For example, the health functional food in the form of a tablet is made by granulating a mixture of the active ingredient of the present invention with excipients, binders, disintegrants and other additives in a conventional manner, and then adding a lubricant and compression molding, or The mixture can be directly compression molded. Additionally, the health functional food in the form of tablets may contain flavoring agents, etc., if necessary. Among capsule-type health functional foods, hard capsules can be manufactured by filling a regular hard capsule with a mixture of the active ingredient of the present invention mixed with additives such as excipients, and soft capsules can be prepared by mixing the active ingredient of the present invention with additives such as excipients. It can be manufactured by filling the mixture with a capsule base such as gelatin. The soft capsule may contain plasticizers such as glycerin or sorbitol, colorants, preservatives, etc., if necessary. The health functional food in the form of a pill can be prepared by molding a mixture of the active ingredient of the present invention and excipients, binders, disintegrants, etc., using a known method. If necessary, it can be coated with white sugar or other coating agent. Alternatively, the surface can be coated with substances such as starch or talc. Health functional food in the form of granules can be manufactured into granules by mixing a mixture of excipients, binders, disintegrants, etc. of the active ingredients of the present invention by a known method, and may contain flavoring agents, flavoring agents, etc., if necessary. You can.

Hereinafter, embodiments of the present invention will be described in more detail with reference to the attached drawings. However, the following examples are only intended to embody the content of the present invention, and the present invention is not limited thereto.

<Preparation example> Improvement and synthesis of peptide aggregation problem

In the present invention, when improving a new peptide, we designed a peptide that maintains the structural characteristics of SapB, can be synthesized by changing the amino acid sequence, and has improved aggregation problems. Afterwards, among the peptides designed according to the above method, only peptides that could be synthesized due to improved aggregation problems were selected.

The synthesis of the peptide was carried out by Peptron (Daejeon, Korea) using the solid phase method. Wang resin was used as the C-terminal, and coupling of Fmoc-L-amino acids was performed with O-Benzotriazole-N,N,N, HBTU (N'-tetramethyl-uronium-hexafluoro-phosphate). Amino acid side chains were protected with tert-butyl and tert-butyloxycarbonyl and protected with trifluoroacetic acid, water, thioanisole and 1,2- Deprotection and cleavage were performed using a mixture of 1,2-ethandithiol (87.5, 5.0, 5.0, 2.5, v/v). Cyclization was completed by disulfide bonding of cysteine residues in the sequence. Crude peptides were washed repeatedly with diethylether, dried under vacuum, and purified using RP-HPLC and Shimadzu 5-μm Shimpak ODS C18 column (20Х250 mm). The purified peptide was confirmed by analytical RP-HPLC and Shimpak ODS C18 column (4.6Х250 mm). The molecular masses of the synthesized peptide were confirmed using a matrix-assisted laser desorption ionization MALDI-mass spectrometer (Shimadzu Axima Assurance).

The improved peptide sequences and information on which the original sequence and aggregation problems were improved according to the above method are shown in Table 1 below (see Figures 1 to 4).

amino acid sequence Molecular weight (Da) Structure designation SEQ ID NO: 1 RCRARLLC 1100.61 Cyclic, C2-C9 ML-A1 SEQ ID NO: 2 KCRAKLLLC 1044.59 Cyclic, C2-C9 ML-A2 SEQ ID NO: 3 HCRAHLLLC 1062.52 Cyclic, C2-C9 ML-A3 Origin GCRA-Dha-LLLCGDCSL-Dha-ITTCD Not possible to synthesize Origin part 1 GCRA-Dha-LLLCG Not possible to synthesize Origin part 2 DCSL-Dha-ITTCD Not possible to synthesize note Due to the aggregation and non-hydrophilic nature of SapB of the original sequence, it was difficult to evaluate the antibacterial efficacy, making it difficult to present results (synthesis was attempted several times, but complete synthesis was impossible due to the aggregating nature of the original sequence).

<Experimental Example 1> Measurement of antibacterial activity of new peptide against multidrug-resistant bacteria

To measure the antibacterial activity of the new peptides shown in Table 1 against multidrug-resistant bacteria, an experiment was performed as follows.

Strains were purchased and used from the Culture Collection of Antimicrobial Resistant Microbes. The strains used in the experiment were Escherichia coli CCARM 1G440, Pseudomonas aeruginosa CCARM 0219), Bacillus cereus CCARM 0120, and Staphylococcus aureus CCARM 3090 (Table 2).

All strains were cultured in LB medium, then diluted to a cell concentration of 10 ⁶ cells/100 ㎕ and inoculated into 96-well plates. Then, the synthesized peptides were diluted in each 96-well and added to the plate at 50 ㎕ each, and the final concentration was adjusted to 0.1~1.8mM. After culturing the plate at 37°C for 16 hours, the MIC value for each strain was determined by measuring absorbance at 600 nm using a spectrophotometer (Tecan, Mannedorf, Switzerland). As a negative control, the same amount of PBS buffer as the peptide was used.

scientific name Antibiotic Resistance Information E. coli ( Escherichia coli CCARM 1G440) Amikacin, Ampicillin, Azetreonam, Ceftazidime, Ciprofloxacin, Cefepime, Cefotaxime, Cefoxitin, Gentamicin, Imipenem, Levofloxacin, Meropenem, Piperacillin, Tobramycin Pseudomonas aeruginosa ( Pseudomonas aeruginosa CCARM 0219) Ampicillin, Cephalothin, Gentamicin, Norfloxacin Bacillus cereus ( Bacillus cereus CCARM 0120) - Staphylococcus aureus ( Staphylococcus aureus CCARM 3090) Cephalothin, Erythromycin, Norfloxacin, Oxacillin, Vancomycin

The experimental results are shown in Table 3 below.

sequence number MIC(mM) E. coli Pseudomonas aeruginosa bacillus cereus Staphylococcus aureus One 0.45 1.8 0.9 0.45 2 0.9 >1.8 1.8 1.8 3 >1.8 >1.8 >1.8 1.8

It was confirmed that among the new peptides of the present invention, SEQ ID NOs: 1, 2, and 3, 1 exhibited the most excellent antibacterial activity. Contrary to reports that the original sequence SapB showed antibacterial activity only against Bacillus cereus , the modified peptides showed excellent antibacterial activity against both Gram-positive and Gram-negative bacteria. In particular, it was confirmed that it exhibits antibacterial activity against Staphylococcus aureus, which has resistance to vancomycin, a glycopeptide-based antibiotic.

<Experimental Example 2> Measurement of anticancer activity of new peptide

Next, an experiment was conducted as follows to measure the anticancer activity of the new peptides shown in Table 1 above.

The anticancer activity was measured using liver cancer cell Hep G2 (ATCC), colon cancer cell SW480 (ATCC), lung cancer cell A549 (ATCC), breast cancer cell MDA-MB-231 (ATCC), and melanoma cancer cell B16F10 (ATCC) cell lines. Cells were cultured under sterile conditions at 37°C in a humid environment containing CO ₂ (5%). Culture medium consisted of RPMI 1640 supplemented with FBS (10%), glutamine (4 mM), and penicillin/streptomycin.

For MTT assays, cell lines were distributed to each well in a 96-well plate at a concentration of 1 × 10 ⁵ cells (single-cell suspension), and each peptide was treated at different concentrations (0.1 ~ 2mM) for 24 hours. . CCK8 solution was added to each well, and the plate was incubated at 37°C for 1 hour to allow the reaction to occur before removing the culture medium. Cell viability was measured at absorbance at 450 nm using a spectrophotometer (Tecan, Mannedorf, Switzerland). Cell viability of each group was calculated as a ratio (%) to the control group. The results are shown in Table 4 below.

sequence number LD50(mM) liver cancer cells
(HepG2) colon cancer cells
(SW480) lung cancer cells
(A549) breast cancer cells
(MDA-MB-231) melanoma cancer cells
(B16F10) One 1.14 1.49 0.80 1.30 1.61 2 1.47 1.45 2.27 1.42 2.03 3 1.23 3.32 1.33 1.21 2.09

As shown in Table 4, the novel peptide of the present invention had cancer cell growth inhibition effect, and in particular, SEQ ID NO: 1 showed excellent growth inhibition in Hep G2 and A549 cell lines, SEQ ID NO: 2 showed excellent growth inhibition in SW480 cell line, and SEQ ID NO: 3 showed MDA-MB. It showed excellent growth inhibition in -231 cell line. This is because the characteristics of each cancer cell and the cancer cell membrane are different, so it is predicted that the peptide with the best growth inhibition effect will be different.

<Experimental Example 3> Measurement of red blood cell hemolysis rate of new peptide

Finally, the following experiment was conducted to measure the red blood cell hemolysis rates of the new peptides shown in Table 1 above.

To measure the hemolysis rate of the peptide, Sheep Red Blood Cells (10% Suspension, MP Biomedicals) were purchased and used. Red blood cells were washed several times with PBS buffer and dispensed into 20 ㎕ 96-well plates. After sufficiently dissolving the peptide in the PBS buffer, 20㎕ each was treated with red blood cells and the final concentrations were adjusted to 4, 2, 1, and 0.5mM. The supernatant was left in a 37°C incubator for 3 hours, centrifuged at 500 xg, and the absorbance of the recovered supernatant was measured at 405 nm to calculate the hemolysis rate. The hemolysis rate was calculated as a relative rate, taking the positive control treated sample as 100% (Equation 1 below). The positive control group used 0.1% Triton X-100, and the negative control group used PBS buffer in the same amount as the peptide.

[Equation 1]

The results are shown in Table 5 and Figure 5 below.

sequence number Hemolysis(%), when treated with 4mM One 8.6 2 5.6 3 7.0

The peptides of the present invention showed an overall low red blood cell hemolysis rate, and it was confirmed that they showed a low hemolysis rate even when treated at a high concentration of 4mM, which is higher than the concentration showing antibacterial or anticancer effects. As can be seen in Figure 5, there was no concentration-dependent hemolysis rate up to 0.5mM and 2mM.

As seen above, specific embodiments of the present invention have been described in detail, but those skilled in the art who understand the spirit of the present invention may add, change, delete, etc. other components within the scope of the same spirit, thereby creating other regressive inventions. However, other embodiments that are included within the scope of the present invention can be easily proposed. Therefore, the embodiments described above should be understood in all respects as illustrative and not restrictive. The scope of the present invention is indicated by the scope of the claims described below rather than the detailed description above, and all changes or modified forms derived from the meaning and scope of the claims and their equivalent concepts are included in the scope of the present invention. It should be interpreted as

Claims (8)

A novel peptide with antibacterial or anticancer activity consisting of the amino acid sequence represented by SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3.
According to paragraph 1,
The new peptide is characterized by improving the aggregation problem of the SapB peptide and improving it to enable synthesis.
delete An antibacterial or anticancer pharmaceutical composition comprising the novel peptide of claim 1 or 2 as an active ingredient.
An antibacterial quasi-drug composition comprising the novel peptide of any one of claims 1 or 2 as an active ingredient.
An antibacterial cosmetic composition comprising the novel peptide of claim 1 or 2 as an active ingredient.
An antibacterial agricultural and livestock feed composition comprising the novel peptide of any one of claims 1 or 2 as an active ingredient.
An antibacterial health functional food composition comprising the novel peptide of any one of claims 1 or 2 as an active ingredient.