WO2014030950A1 - Oyster hydrolysate-derived novel peptides having effects of inhibiting activity of collagenase, and use thereof - Google Patents

Oyster hydrolysate-derived novel peptides having effects of inhibiting activity of collagenase, and use thereof Download PDF

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WO2014030950A1
WO2014030950A1 PCT/KR2013/007557 KR2013007557W WO2014030950A1 WO 2014030950 A1 WO2014030950 A1 WO 2014030950A1 KR 2013007557 W KR2013007557 W KR 2013007557W WO 2014030950 A1 WO2014030950 A1 WO 2014030950A1
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collagenase
peptide
present
pgn
group
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PCT/KR2013/007557
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French (fr)
Korean (ko)
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최영준
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경상대학교 산학협력단
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Priority claimed from KR1020130098938A external-priority patent/KR101530125B1/en
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Publication of WO2014030950A1 publication Critical patent/WO2014030950A1/en

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K8/00—Cosmetics or similar toiletry preparations
    • A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
    • A61K8/30—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds
    • A61K8/64—Proteins; Peptides; Derivatives or degradation products thereof
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
    • A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
    • A23L33/17—Amino acids, peptides or proteins
    • A23L33/18—Peptides; Protein hydrolysates
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/56—Materials from animals other than mammals
    • A61K35/618—Molluscs, e.g. fresh-water molluscs, oysters, clams, squids, octopus, cuttlefish, snails or slugs
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/01—Hydrolysed proteins; Derivatives thereof
    • A61K38/012—Hydrolysed proteins; Derivatives thereof from animals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/04—Peptides having up to 20 amino acids in a fully defined sequence; Derivatives thereof
    • A61K38/06—Tripeptides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
    • A61Q19/00—Preparations for care of the skin
    • A61Q19/08—Anti-ageing preparations
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
    • C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
    • C07K5/06—Dipeptides
    • C07K5/06008—Dipeptides with the first amino acid being neutral
    • C07K5/06017—Dipeptides with the first amino acid being neutral and aliphatic
    • C07K5/06034—Dipeptides with the first amino acid being neutral and aliphatic the side chain containing 2 to 4 carbon atoms
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
    • C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
    • C07K5/06—Dipeptides
    • C07K5/06008—Dipeptides with the first amino acid being neutral
    • C07K5/06078—Dipeptides with the first amino acid being neutral and aromatic or cycloaliphatic
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
    • C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
    • C07K5/08—Tripeptides
    • C07K5/0802—Tripeptides with the first amino acid being neutral
    • C07K5/0804—Tripeptides with the first amino acid being neutral and aliphatic
    • C07K5/0806—Tripeptides with the first amino acid being neutral and aliphatic the side chain containing 0 or 1 carbon atoms, i.e. Gly, Ala
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
    • C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
    • C07K5/08—Tripeptides
    • C07K5/0802—Tripeptides with the first amino acid being neutral
    • C07K5/0804—Tripeptides with the first amino acid being neutral and aliphatic
    • C07K5/081—Tripeptides with the first amino acid being neutral and aliphatic the side chain containing O or S as heteroatoms, e.g. Cys, Ser
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
    • C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
    • C07K5/08—Tripeptides
    • C07K5/0802—Tripeptides with the first amino acid being neutral
    • C07K5/0812—Tripeptides with the first amino acid being neutral and aromatic or cycloaliphatic
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
    • C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
    • C07K5/08—Tripeptides
    • C07K5/0821—Tripeptides with the first amino acid being heterocyclic, e.g. His, Pro, Trp
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
    • A23V2002/00—Food compositions, function of food ingredients or processes for food or foodstuffs

Definitions

  • the present invention is a novel peptide derived from oyster hydrolyzate having the effect of inhibiting the activity of collagenase, collagenase mediated pharmaceutical composition for the prevention or treatment of collagenase mediated disease comprising the same, collagenase mediated comprising the same It relates to a functional food for preventing or improving disease and cosmetic composition for improving wrinkles comprising the same as an active ingredient.
  • Matrix metalloproteinase is a family of enzymes involved in extracellular matrix (ECM) and basal membrane degradation. Interstitial collagenase, stromelysin and gelatinase, depending on their structural and functional properties. (gelatinase), membrane-type MMP (membrane-type MMP, MT-MMP) is divided into four subfamily (subfamily). More than 20 MMPs are known to date in various species including mammals, birds, amphibians, plants and nematodes. MMPs also differ in substrate specificity but have structural and functional similarities. Thus, the names of all MMPs are generally numbered.
  • Collagenase-1, 2, 3 is MMP-1, 8, 13, gelatinase-A, B is MMP-2, 9, stromelysin-1, 2, 3 is MMP-3, 10, 11, metalloelastase is MMP-12, matlylysin is MMP-7, membrane-type MMPs (MT-MMPs) are MMP-14, 15, 16, 17 , 24, ennamelysin (enamelysin) is called MMP-20, MMP-19, MMP-23 and the like.
  • These enzymes are capable of degrading their respective substrates, such as collagen, fibronectin, laminin, proteoglycans, entaxin, and elastin.
  • MMP is a metalloproteinase having zinc in its active center and is secreted in the form of an inactive precursor (zymogen) in vivo.
  • enzyme enzyme
  • structural modifications occur and the amino terminus must be cleaved, and activated MMPs are controlled by inhibitors such as 2-macroglobulin or TIMPs (Tissue inhibitors of metalloproteinase).
  • Each MMP contains a specific amino acid sequence, exhibits specific cell and tissue distribution, and hydrolyzes a specific subset of target substrate proteins.
  • normal substrates of MMPs are extracellular or cell surface proteins.
  • cleavage by MMP inactivates or activates the substrate (if the substrate is an inert protein precursor). Since MMPs can activate and inactivate other proteins, MMPs often play an important role in the regulation of extracellular signaling, extracellular matrix remodeling and metabolism. Thus, proper regulation of the activity of MMPs is important for the normal development and maintenance of cells and tissues.
  • the pathological condition is generally for example tissue destruction, fibrotic disease, pathological matrix weakness, defective injury repair, cardiovascular disease, respiratory disease, kidney disease, liver Diseases, and central nervous system diseases.
  • Such conditions include, for example, rheumatoid arthritis, osteoarthritis, purulent arthritis, multiple sclerosis, pressure sores, corneal ulcers, epidermal ulceration, gastric ulceration, tumor metastasis, tumor invasion, tumors Angiogenesis, periodontal disease, cirrhosis of the liver, fibrotic lung disease, emphysema, otosclerosis, atherosclerosis, proteinuria, myocardial infarction, dilated cardiomyopathy, congestive heart failure, aortic aneurysm , Bullous epidermolysis bullosa, bone disease, Alzheimer's disease, defective wound repair (eg, weak repair, stenosis such as post-operative stenosis, and scarring), chronic obstructive pulmonary disease and perfusion Post myocardial infarction is included.
  • defective wound repair eg, weak repair, stenosis such as post-operative stenosis, and scarring
  • collagenase is a protease stored latent in neutrophil specific granules corresponding to a kind of matrix metalloproteinase (MMP) [Hasty, K, A., et al., J. Biol. Chem. 261: 5645-5650 (1986) and Hasty, K.A., et al., J. Biol Chem. 262: 10048-10052 (1987).
  • MMP matrix metalloproteinase
  • These diseases and conditions include, but are not limited to, bone resorption diseases such as osteoporosis and metastatic bone marrow cancer, corneal ulcers, periodontal disease, inflammatory joint disease, skin inflammatory diseases and wounds, and burns [Harris, ED, et al., NEJM 291: 605-609 (1074) and Harris, ED, et al., NEJM 291: 652-660 (1974).
  • Korean Patent Publication No. 1991-0016326 discloses the use of Tedidap and its pharmaceutically acceptable base salts to inhibit the activation of collagenase
  • Korean Patent Publication No. 2001-0031773 Discloses aromatic sulfone hydroxamic acid metallopronease inhibitors and methods for treating pathological MMP activity mediated diseases using the same
  • Korean Patent Laid-Open No. 1999-0040964 discloses MMP containing cetylpyridinium chloride.
  • compositions for inhibiting activity and wound, cancer metastasis, rheumatoid arthritis, inflammation, parathyroidism, diabetes, corneal ulcer, osteoporosis, gastric ulcer, trauma, wrinkle, acne, AIDS, burns, periodontal disease, arteriosclerosis, fracture Disclosed is a method of preventing and treating a disease.
  • Another object of the present invention to provide a pharmaceutical composition for the prevention or treatment of collagenase-mediated disease comprising the novel peptide as an active ingredient.
  • Still another object of the present invention is to provide a nutraceutical for preventing or improving collagenase mediated disease comprising the novel peptide as an active ingredient.
  • Another object of the present invention to provide a cosmetic composition for improving wrinkles comprising the novel peptide as an active ingredient.
  • the present invention provides a peptide for inhibiting collagenase activity having one amino acid sequence selected from the group consisting of GPN and PGN.
  • the present invention also provides a pharmaceutical composition for preventing or treating collagenase mediated disease comprising the peptide as an active ingredient.
  • the collagenase mediated disease may be one disease selected from the group consisting of osteoporosis, metastatic bone marrow cancer, corneal ulcer, periodontal disease, inflammatory joint disease, skin inflammatory disease, wound and burn .
  • the peptide may be included in a concentration of 0.1ug / ml ⁇ 5000mg / ml with respect to the composition.
  • the present invention also provides a dietary supplement for preventing or improving collagenase mediated diseases comprising the peptide as an active ingredient.
  • the food is selected from the group consisting of beverages, meat, chocolate, foods, confectionery, pizza, ramen, other noodles, gums, candy, ice cream, alcoholic beverages, vitamin complexes and health supplements Can be.
  • the present invention provides a cosmetic composition for improving wrinkles comprising the peptide as an active ingredient.
  • the cosmetic composition is a solution, suspension, emulsion, paste, gel, cream, lotion, powder, soap, surfactant-containing cleansing, oil, powder foundation, emulsion foundation, wax foundation and It can be formulated into any one selected from the group consisting of sprays.
  • the composition comprising the same as an active ingredient can be useful for preventing, ameliorating or treating collagenase mediated diseases as well as for improving wrinkles.
  • the novel peptide of the present invention can be widely used as a material of various fields such as pharmaceutical industry, food industry and cosmetics industry.
  • the novel peptide of the present invention is based on the amino acid sequence of the peptide derived from natural products, and has stability without cytotoxicity, the composition of the present invention comprising it as an active ingredient has a safe advantage even for long-term use.
  • Figure 2 is a graph measuring the degree of inhibition of collagenase activity of novel peptides (GPN, PGN) of the present invention.
  • Figure 3 is a graph measuring the residual activity of collagenase over time after treatment of the novel peptide PGN of the present invention.
  • Figure 4 is a graph measuring the thermal stability of the novel peptide PGN of the present invention.
  • Figure 5 is a graph measuring the concentration of novel peptide PGN required to inhibit 50% collagenase activity of the present invention.
  • FIGS. 6 and 7 are graphs of the degree of inhibition of collagenase activity according to the concentration of novel peptides (GPN, AFN, PGN) of the present invention.
  • FIG. 8 is a graph measuring the residual activity of collagenase over time after treatment of the novel peptide PGN of the present invention.
  • FIG. 9 is a graph measuring the inhibitory activity of collagenase over time after treatment of the novel peptide PGN of the present invention.
  • FIG. 10 is a graph measuring the inhibitory activity of collagenase according to the treatment concentration of the novel peptide PGN of the present invention.
  • the present invention relates to a peptide for inhibiting collagenase activity having one amino acid sequence selected from the group consisting of GPN and PGN.
  • Collagenase is a protease that is stored latent in neutrophil-specific granules, a type of matrix metalloproteinase (MMP).
  • MMP matrix metalloproteinase
  • the active form of collagenase is known to mediate various diseases and disorders in mammals. have.
  • These diseases and conditions include, but are not limited to, bone resorption diseases such as osteoporosis and metastatic bone marrow cancer, corneal ulcers, periodontal disease, inflammatory joint disease, skin inflammatory diseases and wounds, and burns.
  • oysters were hydrolyzed using proteolytic enzymes, and the hydrolysates obtained by the hydrolysis were purified through functional chromatography through several steps of chromatography. It was first identified that synthetic peptides (GPN and PGN) effectively inhibited collagenase activity (see Figure 6). In addition, as a result of cytotoxicity test of the synthetic peptide, it was confirmed that there is no intracellular toxicity, and thus it was confirmed that the composition can be stably used as a composition for preventing, improving or treating diseases (see FIG. 5 and Table 4).
  • novel peptides of the present invention can be prepared by direct isolation from oysters.
  • oysters are hydrolyzed using Protamex and Neutrase, and then functional peptides with ACE inhibitory activity in the hydrolyzate are purified by ultrafiltration, anion exchange resin chromatography, size exclusion chromatography, and reverse phase chromatography. Can be separated directly through (see Examples below for details).
  • novel peptides of the present invention can be synthesized chemically.
  • chemical synthesis it may be prepared by chemical synthesis well known in the art (Creighton, Proteins; Structures and Molecular Principles, W. H. Freeman and Co., NY, 1983).
  • Representative methods include, but are not limited to, liquid or solid phase synthesis, fragment condensation, F-MOC or T-BOC chemistry (Chemical Approaches to the Synthesis of Peptides and Proteins, Williams et al., Eds., CRC Press) , Boca Raton Florida, 1997; A Practical Approach, Athert on & Sheppard, Eds., IRL Press, Oxford, England, 1989).
  • novel peptides of the present invention can be prepared by genetic engineering methods.
  • a DNA sequence encoding the peptide is constructed according to a conventional method.
  • DNA sequences can be constructed by PCR amplification using appropriate primers.
  • DNA sequences may be synthesized by standard methods known in the art, such as using automated DNA synthesizers (eg, sold by Biosearch or Applied iosystems).
  • the constructed DNA sequence is inserted into a vector comprising one or more expression control sequences (eg, promoters, enhancers, etc.) operably linked to and regulate expression of the DNA sequence, and recombinant expression formed therefrom. Transform the host cell with the vector.
  • expression control sequences eg, promoters, enhancers, etc.
  • substantially pure peptide means that the peptide according to the invention is substantially free of any other protein derived from the host.
  • the present invention relates to a pharmaceutical composition for preventing or treating collagenase mediated disease comprising the novel peptide (peptide having one amino acid sequence selected from the group consisting of GPN and PGN) as an active ingredient.
  • novel peptide peptide having one amino acid sequence selected from the group consisting of GPN and PGN
  • the collagenase mediated disease prevented or treated by the composition of the present invention may be a disease such as osteoporosis, metastatic bone marrow cancer, corneal ulcer, periodontal disease, inflammatory joint disease, skin inflammatory disease, wound or burn.
  • the pharmaceutical composition of the present invention may be prepared using a pharmaceutically suitable and physiologically acceptable adjuvant in addition to the active ingredient, and the adjuvant may include excipients, disintegrants, sweeteners, binders, coating agents, swelling agents, lubricants, Lubricants, flavors and the like can be used.
  • the pharmaceutical composition may be preferably formulated into a pharmaceutical composition including one or more pharmaceutically acceptable carriers in addition to the above-described active ingredient for administration.
  • Formulation forms of the pharmaceutical composition may be granules, powders, tablets, coated tablets, capsules, suppositories, solutions, syrups, juices, suspensions, emulsions, drops or injectable solutions.
  • the active ingredient may be combined with an oral, nontoxic, pharmaceutically acceptable inert carrier such as ethanol, glycerol, water and the like.
  • suitable binders, lubricants, disintegrants and coloring agents may also be included in the mixture.
  • Suitable binders include but are not limited to natural and synthetic gums such as starch, gelatin, glucose or beta-lactose, corn sweeteners, acacia, trackercance or sodium oleate, sodium stearate, magnesium stearate, sodium Benzoate, sodium acetate, sodium chloride and the like.
  • Disintegrants include, but are not limited to, starch, methyl cellulose, agar, bentonite, xanthan gum, and the like.
  • Acceptable pharmaceutical carriers in compositions formulated in liquid solutions are sterile and physiologically compatible, including saline, sterile water, Ringer's solution, buffered saline, albumin injectable solutions, dextrose solution, maltodextrin solution, glycerol, ethanol and One or more of these components may be mixed and used, and other conventional additives such as antioxidants, buffers and bacteriostatic agents may be added as necessary. Diluents, dispersants, surfactants, binders and lubricants may also be added in addition to formulate into injectable formulations, pills, capsules, granules or tablets such as aqueous solutions, suspensions, emulsions and the like. Furthermore, the method disclosed in Remington's Pharmaceutical Science, Mack Publishing Company, Easton PA can be formulated according to each disease or component, as appropriate in the art.
  • the novel peptide of the present invention (peptide having one amino acid sequence selected from the group consisting of GPN, AFN and PGN) at a concentration of 0.1ug / ml ⁇ 50mg / ml with respect to the composition May be included.
  • the present invention may be a food composition for preventing or ameliorating collagenase mediated disease comprising a novel peptide (peptide having one amino acid sequence selected from the group consisting of GPN, AFN and PGN) as an active ingredient. .
  • such food compositions may further contain various flavors or natural carbohydrates and the like like conventional food compositions.
  • Examples of the above-mentioned natural carbohydrates include monosaccharides such as glucose, fructose and the like; Disaccharides such as maltose, sucrose and the like; And conventional sugars such as polysaccharides such as dextrin, cyclodextrin, and sugar alcohols such as xylitol, sorbitol, and erythritol.
  • the aforementioned flavoring agents can advantageously be used natural flavoring agents (tautin), stevia extracts (for example rebaudioside A, glycyrrhizin, etc.) and synthetic flavoring agents (saccharin, aspartame, etc.).
  • the food composition of the present invention may be formulated in the same manner as the pharmaceutical composition, used as a functional food, or added to various foods.
  • Foods to which the composition of the present invention may be added include, for example, beverages, meat, chocolate, foods, confectionery, pizza, ramen, other noodles, gums, candy, ice creams, alcoholic beverages, vitamin complexes, and health supplements. There is this.
  • the food composition is not only a novel peptide (peptide having one amino acid sequence selected from the group consisting of GPN and PGN) as an active ingredient, but also various flavors such as vitamins, minerals (electrolytes), synthetic flavors and natural flavors.
  • a novel peptide peptide having one amino acid sequence selected from the group consisting of GPN and PGN
  • various flavors such as vitamins, minerals (electrolytes), synthetic flavors and natural flavors.
  • the food composition of the present invention may contain a fruit flesh for producing natural fruit juice and fruit juice beverage and vegetable beverage.
  • novel peptide (peptide having one amino acid sequence selected from the group consisting of GPN and PGN), which is an active ingredient of the present invention, is based on the amino acid sequence of a peptide derived from natural products, and is stable without cytotoxicity. It can be used with confidence even for long-term use for the purpose of preventing or ameliorating a mediated disease.
  • the present invention provides a dietary supplement for preventing or ameliorating collagenase mediated diseases comprising a novel peptide (peptide having one amino acid sequence selected from the group consisting of GPN and PGN) as an active ingredient. .
  • the health functional food of the present invention can be prepared and processed in the form of tablets, capsules, powders, granules, liquids, pills and the like for the purpose of improving collagenase mediated diseases.
  • the health functional food refers to foods manufactured and processed using raw materials or ingredients having functional properties useful for the human body according to Act No. 6767 of the Health Functional Food Act, and control nutrients to the structure and function of the human body. Or ingestion for the purpose of obtaining useful effects in health applications such as physiological effects.
  • the health functional food of the present invention may include a conventional food additive, and the suitability as a food additive, unless otherwise specified, in accordance with the General Regulations of the Food Additives and General Test Methods approved by the Food and Drug Administration, etc. Judging by the standards and standards.
  • Examples of the items loaded on the food additive revolution include, for example, chemical compounds such as ketones, glycine, calcium citrate, nicotinic acid and cinnamon acid; Natural additives such as dark blue pigment, licorice extract, crystalline cellulose, high color pigment and guar gum; And mixed preparations such as sodium L-glutamate, algae additives, preservatives and tar dyes.
  • chemical compounds such as ketones, glycine, calcium citrate, nicotinic acid and cinnamon acid
  • Natural additives such as dark blue pigment, licorice extract, crystalline cellulose, high color pigment and guar gum
  • mixed preparations such as sodium L-glutamate, algae additives, preservatives and tar dyes.
  • a dietary supplement may contain a novel peptide (peptide having one amino acid sequence selected from the group consisting of GPN, AFN, and PGN) as an active ingredient of the present invention with excipients, binders, disintegrants and other additives.
  • the mixed mixture may be granulated by a conventional method, and then compression-molded with a lubricant or the like, or the mixture may be directly compression-molded.
  • the health functional food in the form of tablets may contain a mating agent or the like as necessary.
  • the hard capsule agent is a mixture of a novel peptide (peptide having one amino acid sequence selected from the group consisting of GPN and PGN), which is an active ingredient of the present invention, into an ordinary hard capsule and an additive such as an excipient. It can be prepared by the filling, soft capsules capsules such as gelatin gelatin mixture mixture of a novel peptide (peptide having one amino acid sequence selected from the group consisting of GPN, AFN and PGN) of the present invention with additives such as excipients It can be prepared by filling the base.
  • the soft capsule agent may contain a plasticizer such as glycerin or sorbitol, a colorant, a preservative, and the like, as necessary.
  • the cyclic health functional food includes a mixture of a novel peptide (peptide having one amino acid sequence selected from the group consisting of GPN, AFN, and PGN), an excipient, a binder, a disintegrant, and the like, which is an active ingredient of the present invention. It may be prepared by molding in a known manner, and may be coated with sucrose or another coating agent as needed, or the surface may be coated with a material such as starch or talc.
  • the health functional food in the form of granules is a mixture of a novel peptide (peptide having one amino acid sequence selected from the group consisting of GPN, AFN and PGN), an excipient, a binder, a disintegrant, etc. It can be manufactured granularly by a well-known method, and can contain a flavoring agent, a mating agent, etc. as needed.
  • Health functional foods comprising the novel peptide of the present invention as an active ingredient can significantly inhibit collagenase activity, as confirmed in the following examples, and thus are effective in improving collagenase mediated diseases.
  • the health functional food may be beverages, meat, chocolate, foods, confectionery, pizza, ramen, other noodles, gum, candy, ice cream, alcoholic beverages, vitamin complexes and health supplements.
  • the present invention comprises a novel peptide (peptide having one amino acid sequence selected from the group consisting of GPN and PGN) for the manufacture of a medicament or food for the prevention or treatment of collagenase mediated diseases as an active ingredient
  • a novel peptide peptide having one amino acid sequence selected from the group consisting of GPN and PGN
  • the composition of the present invention comprising the novel peptide as an active ingredient may be used for the manufacture of a medicament or food for the prevention or treatment of collagenase mediated diseases.
  • the present invention provides a method for preventing or treating collagenase mediated disease comprising administering to a mammal a novel peptide (peptide having one amino acid sequence selected from the group consisting of GPN and PGN).
  • mammal refers to a mammal that is the subject of treatment, observation or experimentation, preferably human.
  • the term “therapeutically effective amount” means an amount of an active ingredient or pharmaceutical composition that induces a biological or medical response in a tissue system, animal or human, as thought by a researcher, veterinarian, doctor or other clinician, which Amounts that induce alleviation of the symptoms of the disease or disorder being treated. It will be apparent to those skilled in the art that the therapeutically effective dosages and frequency of administrations for the active ingredients of the present invention will vary depending on the desired effect. Therefore, the optimal dosage to be administered can be readily determined by one skilled in the art and includes the type of disease, the severity of the disease, the amount of active ingredients and other ingredients contained in the composition, the type of formulation, and the age, weight, general health of the patient. It may be adjusted according to various factors including the condition, sex and diet, time of administration, route of administration and the rate of secretion of the composition, the duration of treatment, and the drugs used simultaneously.
  • the composition comprising the novel peptide of the present invention as an active ingredient is conventionally oral, rectal, intravenous, intraarterial, intraperitoneal, intramuscular, intrasternal, transdermal, topical, intraocular or intradermal. It may be administered in a phosphorous manner.
  • the present invention may be a cosmetic composition for improving wrinkles comprising a novel peptide (peptide having one amino acid sequence selected from the group consisting of GPN and PGN) as an active ingredient.
  • a novel peptide peptide having one amino acid sequence selected from the group consisting of GPN and PGN
  • composition of the present invention is made of a cosmetic composition
  • the composition of the present invention is generally used in cosmetic compositions as well as the novel peptides of the present invention (peptides having one amino acid sequence selected from the group consisting of GPN and PGN).
  • Ingredients used may include, for example, conventional auxiliaries such as antioxidants, stabilizers, solubilizers, vitamins, pigments and flavors, and carriers.
  • Examples of products to which the cosmetic composition of the present invention may be added include, for example, cosmetics such as astringent cosmetics, soft cosmetics, nourishing cosmetics, various creams, essences, packs, foundations, and the like, cleansing agents, face washes, soaps, treatments, and cosmetics Etc.
  • cosmetics such as astringent cosmetics, soft cosmetics, nourishing cosmetics, various creams, essences, packs, foundations, and the like
  • cleansing agents face washes, soaps, treatments, and cosmetics Etc.
  • compositions of the present invention include skin lotion, skin softener, skin toner, astringent, lotion, milk lotion, moisturizing lotion, nutrition lotion, massage cream, nutrition cream, moisture cream, hand cream, essence, nutrition essence, pack, Formulations such as soaps, shampoos, cleansing foams, cleansing lotions, cleansing creams, body lotions, body cleansers, emulsions, lipsticks, makeup bases, foundations, press powders, loose powders, eye shadows and the like.
  • the cosmetic composition according to the present invention may be formulated by containing a novel peptide (peptide having one amino acid sequence selected from the group consisting of GPN and PGN) inside the nanoliposomes.
  • a novel peptide peptide having one amino acid sequence selected from the group consisting of GPN and PGN
  • the components of the peptide are stabilized to solve problems such as precipitation formation and modification during formulation, and the solubility and transdermal absorption of the components can be increased to maximize the efficacy expected from the extract. Can be expressed as.
  • the frozen oysters are first steamed in boiling water for 3 minutes, and then ground using a meat grinder (meat grinder: M-12S, Hankook Fujee Inc, Korea), and the oyster weight 4 It was homogenized at 10,000 rpm (T-25 Basic, Ika Works, NC) by adding 1.5% (w / v) sodium chloride solution corresponding to the culture. The pH was adjusted to pH 6.5-6.7 using 1M NaOH. Thereafter, 0.1% (w / v) transglutaminase was added, followed by reaction at 30 ° C. for 1 hour, followed by 1% (w / v) of Protamex and 1% (w / v).
  • meat grinder M-12S, Hankook Fujee Inc, Korea
  • the pH was adjusted to pH 6.5-6.7 using 1M NaOH.
  • 0.1% (w / v) transglutaminase was added, followed by reaction at 30 ° C. for 1 hour, followed by 1% (w / v) of Protamex
  • hydrolysis was performed at 40 ° C. for 1 hour, and then heated at 100 ° C. for 1 hour to remove the activity of the hydrolase.
  • the hydrolyzate obtained through the above process was centrifuged at 8,000xg for 25 minutes (Supra 22K, Hanil Sci. Inc., Korea) to remove insoluble matters, and the supernatant was recovered.
  • the supernatant was desalted through electrodialysis, ultrafiltration with a membrane of 10 kDa, and lyophilized under reduced pressure to serve as a sample for functional peptide purification.
  • the functional peptide having collagenase inhibitory activity in the oyster hydrolyzate obtained through Example 1 was purified by anion exchange resin chromatography, size exclusion chromatography, and reverse phase chromatography.
  • the conditions of the column chromatography for peptide purification having collagenase inhibitory activity are shown in Table 2 below. Namely, 0.5 g of the sample prepared in Example 1 was dissolved in 12 mL of 20 mM Tris-Cl (pH 8.0) to purify the collagenase inhibitory peptide, and 5 parts were placed on a HiLoad 16/10 Q-Sepharose column (16x100 mm). mL was injected.
  • the unbound material was washed with one column volume of 20 mM Tris-Cl (pH 7.5) at a flow rate of 1 mL / min, then linearly sterilized with 20 mM Tris-Cl (pH 7.5) containing 100 mL of 0.75 M NaCl. The substance bound to the resin was eluted. Peptides were detected at 226 nm and fractionated 2 mL.
  • Example 2 Fractions obtained by reverse phase chromatography of Example 2 were dried completely in Micro-Centvac to determine the amino acid sequence of the functional peptide.
  • the dry sample was dissolved in distilled water containing 20 uL of 0.1% TFA and loaded into ZipTip C18 (Pierce # 87782) paralleled with distilled water containing 0.1% TFA, activated with 50% acetonitrile to remove salts in the sample. After washing 2-3 times with distilled water containing 0.1% TFA, peptide bound to ZipTip C18 was eluted with a 70% acetonitrile solution containing 0.1% TFA.
  • the eluted peptide solution was loaded with 10 uL of microb pretreated with biobrene (AB Systems Co., U.S.A.) and dried with argon gas.
  • the dried filter was mounted on the cartridge to determine the amino acid sequence by pulsed-liquid method of ABI492 automated protein sequencer (Applied Biosystem, Foster, CA, U.S.A.).
  • the molecular weight of the purified peptide was confirmed by detecting molecular weight ions and decomposed ions using a mass spectrometer using electrospray ionization (ESI).
  • ESI electrospray ionization
  • Mass spectra were obtained by Q-TOF2 (Micromass, U.K.) mass spectrometry using a data-dependent MS / MS method using the Nano-ESI interface.
  • Peptides were subjected to Fmoc solid phase peptide synthesis using ASP48S (Peptron Inc., Dajeon, Korea) and purified by reversed phase HPLC using Vydac Everest C18 column (22x250 mm, 10 um). That is, the first amino acid attached to the resin at the C-terminus of the peptide was attached to the Na-terminus of Fmoc-protected amino acids in order, and a protecting group was used to remove residues from the TFA.
  • HBTU / HOBt / NMM was used as the coupling reagent, and 20% piperidine / DMF was used to remove Fmoc.
  • Synthetic peptides were separated from the resin and TFA / EDT / thioanisole / TIS / H 2 O (90 / 2.5 / 2.5 / 2.5 / 2.5, v / v) was used to remove the protecting groups of the residues.
  • Peptides were eluted by linear sterilization with 40% acetonitrile solution containing 0.1% TFA. The molecular weight of the purified peptide was confirmed by LC / MS (Agilent HP1100 series).
  • the synthesized peptides showed a single peak in HPLC, and the synthesized peptide showed a molecular weight of a single peak in MALDI-TOF mass spectrometer.
  • two synthetic peptides having the functionalities as described above are shown in Table 3 below.
  • Chang cells normal hepatocytes. That is, Chang cells cultured in the cell culture flasks were cultured in MEM medium containing 10% FBS. The cells were dispensed into 96 well plates at a concentration of 1 ⁇ 10 4 cells / well and incubated in a CO 2 incubator adjusted to 35 ° C., 95% humidity, CO 2 , 5% for 24 hours. Samples were dissolved in fresh medium to a final concentration of 50, 100, 200 and 300 ug / mL, treated with cell lines and incubated for 24 hours.
  • Cell proliferation was measured at 490 nm using a MicroTiter 96 Aqueous One Solution Cell Proliferation Assay purchased from Promega using a microplate reader (Perkin Elmer 1420, VICTORTM X Multilabel Plate Readers, Waltham, MA, USA). Cell viability was expressed by the absorbance of the sample treated group compared to the control group not treated with the sample.
  • Collagenase activity was inhibited by the purchase of Invitrogen's EnzChekGelatinase / Collagenase Assay Kit-250-2000 Assays reagent.
  • 1.0 mL of DDW was added to 1 mg DG gelatin vial to make DG gelatin stock solution (1 mg / mL).
  • 18 mL of DDW was added to 2 mL of 10 x reaction buffer to dilute the reaction buffer.
  • collagenase enzyme reagent was prepared. The working solution was diluted with reaction buffer so that the final concentration was 0.2 unit / mL.
  • Samples to be used in the experiment was prepared so that the concentration is 0.01, 0.1 1, 10 mg / mL, and the samples were prepared in triple by 20 uL in a 96 well plate. 10 uL of DQ gellatin and 100 uL of reaction buffer in the sample blank and 100 uL of working solution in the sample were added to the sample, and left for 1 hour at room temperature in a light-blocked state. The fluorescence intensity excition wavelength 495 nm and emission wavelength 515 Fluorescence intensity was measured at nm with an ELAZA plate reader (SpectraMax M2 & M2e Multi-Mode Microplate Reader. Molecular Devices Corporation. USA).
  • Collagenase activity using bovine achilles tendon as a substrate was measured according to Worthington enzyme manual (1972). Accurately weigh the substrate collagen (Sigma C9879) into the capped test tube and add 50 mL TES buffer (pH 7.5) and collagenase (0.2 mg / mL, Sigma C0130) containing 2.0 mL of 0.36 mM CaCl 2 . The solution was hydrolyzed for 5 hours while shaking in a constant temperature bath at 37 ° C. The reaction was stopped by adding 0.2 mL of the hydrolyzate to a capped test tube containing 1 mL of 2% ninhydrin solution. The reaction was stopped for 20 minutes, and the absorbance was measured at 600 nm.
  • the amount of free amino acid was quantified according to the standard curve made with L-leucine and the collagenase activity was expressed as uM leucine / hr / g-collagen.
  • the inhibition of collagenase by synthetic peptide was measured according to the method of measuring collagenase activity after fully reacting at low temperature for 15 hours after mixing so that ratio of synthetic peptide and collagenase was 8.3 times and 12.5 times, respectively. Residual activity was expressed as% of residual activity, calculated as the ratio of activity with or without inhibitor addition.
  • GPN and PGN in the synthetic peptide showed a concentration-dependent inhibitory activity against collagenase.
  • GPN and PGN showed 58..1% and 100% of collagenase activity as shown in FIG. Inhibition effect was shown.
  • the functional peptide of the present invention In order to measure the collagenase activity inhibitory ability of PGN, the functional peptide of the present invention, after treating PNG with collagenase, the enzyme residual activity was measured over time.
  • the commercial collagenase and the synthetic peptide PNG of the present invention were mixed and reacted at 37 ° C. for 10 minutes in 10 minutes to inhibit collagenase activity. Then, the calcanase activity was measured by calf Achilles tendon as a substrate.
  • the functional peptide of the present invention In order to measure the collagenase activity inhibitory ability of PGN, the functional peptide of the present invention, the enzyme inhibitory activity over time was measured in the same manner as in ⁇ 3-1> after treating the collagenase with PNG.
  • the functional peptide of the present invention In order to measure the collagenase activity inhibitory ability of PGN, the functional peptide of the present invention, collagenase inhibitory activity according to concentration-dependent treatment of PNG to collagenase was measured.
  • CCD 986sk cells In order to examine the anti-wrinkle effect of the functional peptide of the present invention, CCD 986sk cells, the fibroblast line CCD 986sk cells, were cultured in a cell culture flask, and the cells were removed from the flask with a 10 ml pipette, and the cells contained 10% FBS. Incubated with IMDM medium. CCD 986sk cells were aliquoted into 96 well plates at a concentration of 2.5x10 4 cells / well and incubated in a CO 2 incubator controlled at 35 ° C., 95% humidity, 5% CO 2 for 24 hours. The sample was dissolved in fresh medium to a final concentration of 1, 10 ug / ml, treated with cells, cultured for 24 hours, and the supernatant was collected and used as a sample.
  • MMP-1 was measured by purchasing a SensoLyte 490 MMP-1 Assay Kit (AnaSpec, MI, USA). 50 ⁇ l of MMP-1 substrate was added to 50 ⁇ l of the culture supernatant of CCD 986sk, which was treated and cultured for 30 seconds, and then stirred for 10 seconds at a wavelength of 340 nm and emission wavelength of 490 nm using a microplate reader (Molecular Devices, VersaMax ELISA Microplate Reader, USA). The fluorescence value was measured for 60 minutes, and the inhibitory activity was measured by the following formula.
  • MMP-1 inhibitory activity [1- (S-B) / (C-B)] X 10
  • oyster-derived peptides PGN and GPN showed high inhibitory activity of MMP-1 at 53.9% and 41.4% as shown in Table 6 below at the concentration of 10ug / ml of the sample.
  • Procollagen production was measured to determine the skin regeneration effect of the functional peptides of the present invention GPN and PGN.
  • Procollagen was measured using a type 1 C-peptide (PIP) EIA kit.
  • PIP type 1 C-peptide
  • 40 ul of the sample diluent was added to 10 ul of the supernatant of the CCD 986sk and diluted 5 times to use the sample solution.
  • Add 100 ul of antibody-POD conjugate to the coated microplate add 20 ul of diluted sample solution, cover with a foil, and incubate at 37 ° C for 3 hours. After incubation, remove all the liquid, wash 4 times with 400 ul of PBS, remove the water completely, add 100 ul of TMBZ with substrate solution and leave at room temperature for 15 minutes.
  • CCD 986sk cells used for cell proliferation were available from American Type Culture Collection (ATCC, VA, USA). CCD 986sk cells cultured in cell culture flasks were removed from the flasks using a 10 mL pipette and passaged, and the cells were passaged with Iscove's Modified Dulbeccos Medium (IMDM, Lonza, Valais, Switzerland) containing 12% FBS (Lonza, Valais, Switzerland). Switzerland) was incubated in an incubator at 37 °C, 5% CO 2 maintained. At this time, antibiotics for culture medium (Penicillin streptomycin, Gibco, CA, USA) were used to inhibit the contamination or proliferation of microorganisms. Cells were washed with phosphated-buffered saline-EDTA (PBS-EDTA) and passaged with trypsin when the cells covered the dish about 80%, and the culture medium was exchanged every 48 hours.
  • IMDM Iscove's Modified Dulbeccos
  • CCD 986sk cells were injected at a concentration of 2.5x10 4 cells / well in IMDM medium containing 10% FBS in a 96 well plate and stabilized for 24 hours. Samples in fresh medium were dissolved in medium so that the final concentrations were 0.01, 0.1, 1 and 10 ug / ml, treated with cell lines, and then cultured for a period of time. Absorbance was measured at 450 nm with a microplate reader (Molecular Devices, VersaMax ELISA Microplate Reader, USA) using an MTS reagent to determine the viability of the cell line. Cell viability was expressed by the absorbance of the sample treated group compared to the control group not treated with the sample.
  • the cell regeneration ability of peptide YAK was determined using the CCD 986sk, which is a skin fibroblast. Nearly 70% of cell proliferation was observed at the concentration.

Abstract

The present invention relates to oyster hydrolysate-derived novel peptides having the effects of inhibiting the activity of collagenase, to a pharmaceutical composition comprising the novel peptides as active ingredients for preventing or treating collagenase-mediated diseases, to a functional health food comprising the novel peptides as active ingredients for preventing or improving collagenase-mediated diseases, and to an anti-wrinkle cosmetic composition comprising the novel peptides as active ingredients. The novel peptides according to the present invention may effectively inhibit the activity of collagenase, and therefore the composition comprising the novel peptides of the present invention as active ingredients can be effectively used not only in the prevention, improvement or treatment of collagenase-mediated diseases, but also in reducing wrinkles. Thus, the novel peptides of the present invention can be widely used as a material for various fields such as the drug industry, food industry and cosmetic industry. Particularly, the novel peptides of the present invention are based on the amino acid sequence of the peptides derived from a natural product, and may be safe without exhibiting cytotoxicity. Therefore, the composition of the present invention comprising the novel peptides as active ingredients may have the advantage of being safe over a long period of use.

Description

콜라게나제의 활성 저해 효과를 갖는 굴 가수분해물 유래 신규 펩타이드 및 그의 용도Novel peptide derived from oyster hydrolyzate and its use having inhibitory effect of collagenase
본 발명은 콜라게나제의 활성을 저해하는 효과를 가진 굴 가수분해물 유래 신규 펩타이드, 이를 유효성분으로 포함하는 콜라게나제 매개 질환 예방 또는 치료용 약제학적 조성물, 이를 유효성분으로 포함하는 콜라게나제 매개 질환 예방 또는 개선용 건강기능식품 및 이를 유효성분으로 포함하는 주름 개선용 화장료 조성물에 관한 것이다.The present invention is a novel peptide derived from oyster hydrolyzate having the effect of inhibiting the activity of collagenase, collagenase mediated pharmaceutical composition for the prevention or treatment of collagenase mediated disease comprising the same, collagenase mediated comprising the same It relates to a functional food for preventing or improving disease and cosmetic composition for improving wrinkles comprising the same as an active ingredient.
MMP(Matrix metalloproteinase)는 세포외 기질(Extracellular matrix, ECM)과 기저막 분해에 관여하는 효소군으로 구조와 기능적 특성에 따라 간질성 콜라게나제(interstitial collagenase), 스트로멜리신(stromelysin), 젤라티나제 (gelatinase), 막-타입 MMP(membrane-type MMP, MT-MMP) 등 네 개의 아과(subfamily)로 나누어진다. MMP들은 포유류, 조류, 양서류, 식물류 및 선충 등 다양한 종에서 현재까지 20종류 이상이 알려져 있다. 또한 MMP들은 기질 특이성에서는 서로 차이가 나지만 구조적, 기능적 유사성을 가지고 있다. 따라서 모든 MMP들의 명칭은 일반적으로 번호를 붙여서 부르고 있다. 즉, 콜라게나제-1, 2, 3은 MMP-1, 8, 13으로, 젤라티나제-A, B는 MMP-2, 9로, 스트로멜리신-1, 2, 3은 MMP-3, 10, 11로, 메탈로엘라스타제(metalloelastase)는 MMP-12로, 마트릴리신(matrilysin)은 MMP-7로, 막-타입 MMP(MT-MMPs)는 MMP-14, 15, 16, 17, 24로, 에나멜리신(enamelysin)은 MMP-20, MMP-19, MMP-23 등으로 부른다. 이 효소들은 각각 해당되는 기질, 즉 콜라겐, 피브로넥틴, 라미닌(laminin), 프로테오글리칸, 엔탁틴, 엘라스틴 등의 단백질들을 분해할 수 있다. MMP는 활성 중심부에 아연을 가지는 금속단백분해효소로 생체 내에서 비활성 전구체(zymogen) 형태로 분비된다. 효소활성을 가지기 위해서 구조적 변형이 일어나 아미노 말단부위가 절단되어야 하며, 활성화된 MMP는 2-마크로글로불린(2-macroglobulin)이나 TIMPs(Tissue inhibitors of metalloproteinase)와 같은 저해제에 의해 활성이 조절된다.Matrix metalloproteinase (MMP) is a family of enzymes involved in extracellular matrix (ECM) and basal membrane degradation. Interstitial collagenase, stromelysin and gelatinase, depending on their structural and functional properties. (gelatinase), membrane-type MMP (membrane-type MMP, MT-MMP) is divided into four subfamily (subfamily). More than 20 MMPs are known to date in various species including mammals, birds, amphibians, plants and nematodes. MMPs also differ in substrate specificity but have structural and functional similarities. Thus, the names of all MMPs are generally numbered. Collagenase-1, 2, 3 is MMP-1, 8, 13, gelatinase-A, B is MMP-2, 9, stromelysin-1, 2, 3 is MMP-3, 10, 11, metalloelastase is MMP-12, matlylysin is MMP-7, membrane-type MMPs (MT-MMPs) are MMP-14, 15, 16, 17 , 24, ennamelysin (enamelysin) is called MMP-20, MMP-19, MMP-23 and the like. These enzymes are capable of degrading their respective substrates, such as collagen, fibronectin, laminin, proteoglycans, entaxin, and elastin. MMP is a metalloproteinase having zinc in its active center and is secreted in the form of an inactive precursor (zymogen) in vivo. In order to have enzymatic activity, structural modifications occur and the amino terminus must be cleaved, and activated MMPs are controlled by inhibitors such as 2-macroglobulin or TIMPs (Tissue inhibitors of metalloproteinase).
각각의 MMP 는 특이적인 아미노산 서열을 포함하며, 특이적인 세포 및 조직 분포를 나타내며, 표적 기질 단백질의 특이적인 서브세트를 가수분해한다. 일반적으로, MMP 의 정상적인 기질은 세포외 또는 세포 표면 단백질이다. 기질에 따라서, MMP 에 의한 절단은 기질을 불활성화시키거나 또는 활성화시킨다(기질이 불활성 단백질 전구체인 경우). MMP가 기타 단백질을 활성화 및 불활성화할 수 있으므로, MMP 는 종종 세포외 신호전달, 세포외 매트릭스 리모델링 및 대사의 조절에 중요한 역할을 한다. 따라서 MMP 의 활성의 적절한 조절은 세포 및 조직의 정상적인 발생 및 유지에 중요한다.Each MMP contains a specific amino acid sequence, exhibits specific cell and tissue distribution, and hydrolyzes a specific subset of target substrate proteins. In general, normal substrates of MMPs are extracellular or cell surface proteins. Depending on the substrate, cleavage by MMP inactivates or activates the substrate (if the substrate is an inert protein precursor). Since MMPs can activate and inactivate other proteins, MMPs often play an important role in the regulation of extracellular signaling, extracellular matrix remodeling and metabolism. Thus, proper regulation of the activity of MMPs is important for the normal development and maintenance of cells and tissues.
MMP 활성이 잘못 조절되어 연결 조직의 과도한 분해되는 경우 다수의 병리학적 특징을 나타낼 수 있다. 상기 병리학적 상태는 일반적으로, 예를 들어 조직 붕괴 (tissue destruction), 섬유종 질환 (fibrotic disease), 병리학적 매트릭스약화, 결함 상처 수복 (defective injury repair), 심혈관계 질환, 호흡기 질환, 신장 질환, 간장 질환, 및 중추 신경계 질환이 포함된다. 상기 상태의 구체적인 예에는, 예를 들어, 류마티스 관절염, 골관절염, 화농성 관절염, 다발경화증, 욕창성 궤양, 각막 궤양, 표피 궤양 (epidermal ulceration), 소화관 궤양 (gastric ulceration), 종양 전이, 종양 침입, 종양 혈관형성, 치주 질환, 간 경화, 섬유성 폐질환 (fibrotic lung disease), 폐기종, 이 경화증 (otosclerosis), 죽상동맥경화, 단백뇨, 심근경색, 확장성 심근증 (dilated cardiomyopathy), 울혈성 심부전, 대동맥류, 수포성 표피박리증 (epidermolysisbullosa), 골 질환, 알츠하이머병, 결함 상처 수복 (예를 들어, 약한 수복 (weak repair), 협착증, 예컨대 수술후 협착증, 및 스케어링 (scarring)), 만성 폐쇄성 폐질환 및 관류후 심근 경색 (post myocardial infarction) 이 포함된다. Misregulation of MMP activity can lead to a number of pathological features when the tissue is overly degraded. The pathological condition is generally for example tissue destruction, fibrotic disease, pathological matrix weakness, defective injury repair, cardiovascular disease, respiratory disease, kidney disease, liver Diseases, and central nervous system diseases. Specific examples of such conditions include, for example, rheumatoid arthritis, osteoarthritis, purulent arthritis, multiple sclerosis, pressure sores, corneal ulcers, epidermal ulceration, gastric ulceration, tumor metastasis, tumor invasion, tumors Angiogenesis, periodontal disease, cirrhosis of the liver, fibrotic lung disease, emphysema, otosclerosis, atherosclerosis, proteinuria, myocardial infarction, dilated cardiomyopathy, congestive heart failure, aortic aneurysm , Bullous epidermolysis bullosa, bone disease, Alzheimer's disease, defective wound repair (eg, weak repair, stenosis such as post-operative stenosis, and scarring), chronic obstructive pulmonary disease and perfusion Post myocardial infarction is included.
한편, 콜라게나제는 이러한 MMP(Matrix metalloproteinase)의 일종에 해당되는 호중구 특이적 과립 내에 잠복형으로 저장되는 단백질 분해효소이다[Hasty, K, A., et al.,J. Biol. Chem.261:5645-5650(1986) and Hasty, K.A., et al., J. Biol Chem. 262:10048-10052(1987)]. 활성 형태의 콜라게나제는 포유동물에 있어 다양한 질병과 질환을 매개한다. 이들 질병 및 질환에는 골다공증 및 전이성 골수암과 같은 골 흡수병, 각막 궤양, 치주질환, 염증성 관절 질환, 피부 염증성 질환과 창상, 및 화상이 포함되나 이에 제한되지 않는다[Harris, E.D., et al., NEJM 291:605-609(1074) and Harris, E.D., et al., NEJM 291:652-660(1974)].On the other hand, collagenase is a protease stored latent in neutrophil specific granules corresponding to a kind of matrix metalloproteinase (MMP) [Hasty, K, A., et al., J. Biol. Chem. 261: 5645-5650 (1986) and Hasty, K.A., et al., J. Biol Chem. 262: 10048-10052 (1987). The active form of collagenase mediates various diseases and disorders in mammals. These diseases and conditions include, but are not limited to, bone resorption diseases such as osteoporosis and metastatic bone marrow cancer, corneal ulcers, periodontal disease, inflammatory joint disease, skin inflammatory diseases and wounds, and burns [Harris, ED, et al., NEJM 291: 605-609 (1074) and Harris, ED, et al., NEJM 291: 652-660 (1974).
따라서 상기와 같은 특징을 갖는 콜라게나제(MMP-1)의 과도한 활성을 억제하는 물질을 이용하여, 콜라게나제 매개 질병 또는 질환을 치료하려는 시도가 계속적으로 이루어지고 있다. 뿐만 아니라, 이러한 콜라게나제는 일회의 UV 조사시에도 피부 내의 활성이 증가하여 콜라겐을 현저하게 감소시킬 수 있으므로, 콜라게나제는 광노화에 주요 인자로 보고되어 있으며, 이에 주름을 개선하기 위한 화장품 산업에서는 이러한 콜라게나제의 활성 억제에 초점을 맞추어 연구가 많이 진행되고 있는 실정이다.Therefore, attempts have been made to treat collagenase mediated diseases or disorders using substances that inhibit excessive activity of collagenase (MMP-1) having the above characteristics. In addition, such collagenase may increase the activity in the skin significantly during a single UV irradiation, thereby significantly reducing collagen. Collagenase has been reported as a major factor in photoaging, thereby improving the cosmetic industry. In recent years, a lot of research is being conducted focusing on the inhibition of collagenase activity.
이와 관련하여, 한국공개특허 제1991-0016326호에서는 콜라게나제의 활성화를 저해하기 위한 테니댑(Tedidap) 및 그의 약학적으로 허용되는 염기 염의 용도를 개시하고 있으며, 한국공개특허 제2001-0031773호에서는 방향족 술폰 히드록삼산 메탈로프로네아제 억제제 및 이를 이용하여 병리학적인 MMP 활성 매개된 질병을 치료하는 방법을 개시하고 있고, 또한 한국공개특허 제1999-0040964호에서는 세틸피리디늄 클로라이드를 함유한 MMP 활성 억제용 약제 조성물 및 이를 이용하여 상처, 암전이, 류마토이드 관절염, 염증, 부갑상선항진증, 당뇨병, 각막궤양, 골다공증, 위궤양, 외상, 주름, 여드름, 에이즈, 화상, 치주질환, 동맥경화, 골절과 같은 질환을 예방하고 치료하는 방법에 대한 내용이 개시되어 있다.In this regard, Korean Patent Publication No. 1991-0016326 discloses the use of Tedidap and its pharmaceutically acceptable base salts to inhibit the activation of collagenase, and Korean Patent Publication No. 2001-0031773 Discloses aromatic sulfone hydroxamic acid metallopronease inhibitors and methods for treating pathological MMP activity mediated diseases using the same, and Korean Patent Laid-Open No. 1999-0040964 discloses MMP containing cetylpyridinium chloride. Pharmaceutical composition for inhibiting activity and wound, cancer metastasis, rheumatoid arthritis, inflammation, parathyroidism, diabetes, corneal ulcer, osteoporosis, gastric ulcer, trauma, wrinkle, acne, AIDS, burns, periodontal disease, arteriosclerosis, fracture Disclosed is a method of preventing and treating a disease.
그러나 이러한 종래기술들은 합성 화합물을 이용하여 콜라게나제 또는 MMP 활성을 억제하는 조성물에 대한 것으로, 합성 화합물의 장기적인 사용 시 전신성 결합 조직 독성과 같은 부작용이 유발되는 문제점을 가지고 있다.However, these conventional techniques are directed to compositions that inhibit collagenase or MMP activity using synthetic compounds, and have a problem of causing side effects such as systemic connective tissue toxicity in the long term use of synthetic compounds.
이러한 배경 하에, 본 발명자들은 부작용이 없으면서 콜라게나제의 활성을 억제할 수 있는 물질을 찾고자 예의 노력한 결과, 굴(oyster) 가수분해산물에서 펩타이드를 분리한 후, 분석된 아미노산 서열을 바탕으로 합성 펩타이드를 제조하였으며, 이렇게 제조된 합성 펩타이드가 세포독성 없이 콜라게나제의 활성을 효과적으로 억제시킬 수 있음을 확인함으로써 본 발명을 완성하게 되었다.Under these circumstances, the present inventors have diligently tried to find a substance capable of inhibiting the activity of collagenase without side effects. After separating the peptide from the oyster hydrolyzate, the inventors synthesized the peptide based on the analyzed amino acid sequence. The present invention was completed by confirming that the synthetic peptide thus prepared can effectively inhibit the activity of collagenase without cytotoxicity.
따라서 본 발명의 목적은 콜라게나제 활성을 효과적으로 억제할 수 있는 신규 펩타이드를 제공하는 것이다.It is therefore an object of the present invention to provide novel peptides that can effectively inhibit collagenase activity.
본 발명의 다른 목적은 상기 신규 펩타이드를 유효성분으로 포함하는 콜라게나제 매개 질환 예방 또는 치료용 약제학적 조성물을 제공하는 것이다.Another object of the present invention to provide a pharmaceutical composition for the prevention or treatment of collagenase-mediated disease comprising the novel peptide as an active ingredient.
본 발명의 또 다른 목적은 상기 신규 펩타이드를 유효성분으로 포함하는 콜라게나제 매개 질환 예방 또는 개선용 건강기능식품을 제공하는 것이다.Still another object of the present invention is to provide a nutraceutical for preventing or improving collagenase mediated disease comprising the novel peptide as an active ingredient.
본 발명의 또 다른 목적은 상기 신규 펩타이드를 유효성분으로 포함하는 주름 개선용 화장료 조성물을 제공하는 것이다.Another object of the present invention to provide a cosmetic composition for improving wrinkles comprising the novel peptide as an active ingredient.
상기와 같은 본 발명의 목적을 달성하기 위해서, 본 발명은 GPN 및 PGN로 이루어진 군으로부터 선택된 1종의 아미노산 서열을 갖는 콜라게나제 활성 억제용 펩타이드를 제공한다.In order to achieve the object of the present invention as described above, the present invention provides a peptide for inhibiting collagenase activity having one amino acid sequence selected from the group consisting of GPN and PGN.
또한, 본 발명은 상기 펩타이드를 유효성분으로 포함하는 콜라게나제 매개 질환 예방 또는 치료용 약제학적 조성물을 제공한다.The present invention also provides a pharmaceutical composition for preventing or treating collagenase mediated disease comprising the peptide as an active ingredient.
본 발명의 일실시예에 있어서, 상기 콜라게나제 매개 질환은 골다공증, 전이성 골수암, 각막 궤양, 치주질환, 염증성 관절 질환, 피부 염증성 질환, 창상 및 화상으로 이루어진 군으로부터 선택된 1종의 질환일 수 있다.In one embodiment of the present invention, the collagenase mediated disease may be one disease selected from the group consisting of osteoporosis, metastatic bone marrow cancer, corneal ulcer, periodontal disease, inflammatory joint disease, skin inflammatory disease, wound and burn .
본 발명의 일실시예에 있어서, 상기 펩타이드는 상기 조성물에 대해 0.1ug/ml~5000mg/ml의 농도로 포함될 수 있다.In one embodiment of the present invention, the peptide may be included in a concentration of 0.1ug / ml ~ 5000mg / ml with respect to the composition.
또한, 본 발명은 상기 펩타이드를 유효성분으로 포함하는 콜라게나제 매개 질환 예방 또는 개선용 건강기능식품을 제공한다.The present invention also provides a dietary supplement for preventing or improving collagenase mediated diseases comprising the peptide as an active ingredient.
본 발명의 일실시예에 있어서, 상기 식품은 음료류, 육류, 초코렛, 식품류, 과자류, 피자, 라면, 기타 면류, 껌류, 사탕류, 아이스크림류, 알코올 음료류, 비타민 복합제 및 건강보조식품류로 이루어진 군으로부터 선택될 수 있다.In one embodiment of the invention, the food is selected from the group consisting of beverages, meat, chocolate, foods, confectionery, pizza, ramen, other noodles, gums, candy, ice cream, alcoholic beverages, vitamin complexes and health supplements Can be.
또한, 본 발명은 상기 펩타이드를 유효성분으로 포함하는 주름 개선용 화장료 조성물을 제공한다.In addition, the present invention provides a cosmetic composition for improving wrinkles comprising the peptide as an active ingredient.
본 발명의 일실시예에 있어서, 상기 화장료 조성물을 용액, 현탁액, 유탁액, 페이스트, 겔, 크림, 로션, 파우더, 비누, 계면활성제-함유 클린싱, 오일, 분말 파운데이션, 유탁액 파운데이션, 왁스 파운데이션 및 스프레이로 이루어진 군으로부터 선택되는 어느 하나로 제형화될 수 있다.In one embodiment of the present invention, the cosmetic composition is a solution, suspension, emulsion, paste, gel, cream, lotion, powder, soap, surfactant-containing cleansing, oil, powder foundation, emulsion foundation, wax foundation and It can be formulated into any one selected from the group consisting of sprays.
본 발명에 따른 신규 펩타이드는 콜라게나제 활성을 효과적으로 억제할 수 있으므로, 이를 유효성분으로 포함하는 조성물은 콜라게나제 매개 질환의 예방, 개선 또는 치료에 유용하게 사용될 수 있을 뿐만 아니라, 주름 개선에 유용하게 사용할 수 있는바, 본 발명의 신규 펩타이드는 의약품 산업과 식품산업 및 화장품 산업과 같은 다양한 분야의 소재로서 폭넓게 사용될 수 있다. 특히 본 발명의 신규 펩타이드는 천연물로부터 유래된 펩타이드의 아미노산 서열에 바탕을 두고 있으며, 세포독성 없이 안정성을 가지므로, 이를 유효성분으로 포함하는 본 발명의 조성물은 장기적 사용에도 안전한 이점을 가진다.Since the novel peptides according to the present invention can effectively inhibit collagenase activity, the composition comprising the same as an active ingredient can be useful for preventing, ameliorating or treating collagenase mediated diseases as well as for improving wrinkles. As it can be used, the novel peptide of the present invention can be widely used as a material of various fields such as pharmaceutical industry, food industry and cosmetics industry. In particular, the novel peptide of the present invention is based on the amino acid sequence of the peptide derived from natural products, and has stability without cytotoxicity, the composition of the present invention comprising it as an active ingredient has a safe advantage even for long-term use.
도 1은 굴 가수분해물의 펩타이드 정제과정으로서 Q-Sepharose 크로마토그램, 크기배제 크로마토그램과 역상크로마토그램을 나타낸 것이다. 1 shows Q-Sepharose chromatogram, size exclusion chromatogram and reverse phase chromatogram as peptide purification process of oyster hydrolyzate.
도 2는 본 발명의 신규 펩타이드(GPN, PGN)의 콜라게나제 활성 억제정도를 측정한 그래프이다.Figure 2 is a graph measuring the degree of inhibition of collagenase activity of novel peptides (GPN, PGN) of the present invention.
도 3은 본 발명의 신규 펩타이드 PGN의 처리 후 시간 경과에 따른 콜라게나제의 잔여 활성을 측정한 그래프이다.Figure 3 is a graph measuring the residual activity of collagenase over time after treatment of the novel peptide PGN of the present invention.
도 4는 본 발명의 신규 펩타이드 PGN의 열안정성을 측정한 그래프이다.Figure 4 is a graph measuring the thermal stability of the novel peptide PGN of the present invention.
도 5는 본 발명의 콜라게나제 활성을 50% 억제하는 데 필요한 신규 펩타이드 PGN의 농도를 측정한 그래프이다.Figure 5 is a graph measuring the concentration of novel peptide PGN required to inhibit 50% collagenase activity of the present invention.
도 6 및 도 7은 본 발명의 신규 펩타이드(GPN, AFN, PGN)의 농도에 따른 콜라게나제 활성 억제정도를 측정한 그래프이다.6 and 7 are graphs of the degree of inhibition of collagenase activity according to the concentration of novel peptides (GPN, AFN, PGN) of the present invention.
도 8은 본 발명의 신규 펩타이드 PGN의 처리 후 시간 경과에 따른 콜라게나제의 잔여 활성을 측정한 그래프이다.8 is a graph measuring the residual activity of collagenase over time after treatment of the novel peptide PGN of the present invention.
도 9는 본 발명의 신규 펩타이드 PGN의 처리 후 시간 경과에 따른 콜라게나제의 억제 활성을 측정한 그래프이다.9 is a graph measuring the inhibitory activity of collagenase over time after treatment of the novel peptide PGN of the present invention.
도 10은 본 발명의 신규 펩타이드 PGN의 처리 농도에 따른 콜라게나제의 억제 활성을 측정한 그래프이다.10 is a graph measuring the inhibitory activity of collagenase according to the treatment concentration of the novel peptide PGN of the present invention.
하나의 양태로서, 본 발명은 GPN 및 PGN로 이루어진 군으로부터 선택된 1종의 아미노산 서열을 갖는 콜라게나제 활성 억제용 펩타이드에 관한 것이다.In one embodiment, the present invention relates to a peptide for inhibiting collagenase activity having one amino acid sequence selected from the group consisting of GPN and PGN.
"콜라게나제는 MMP(Matrix metalloproteinase)의 일종에 해당되는 호중구 특이적 과립 내에 잠복형으로 저장되는 단백질 분해효소로서, 활성 형태의 콜라게나제는 포유동물에 있어 다양한 질병과 질환을 매개하는 것으로 알려져 있다."Collagenase is a protease that is stored latent in neutrophil-specific granules, a type of matrix metalloproteinase (MMP). The active form of collagenase is known to mediate various diseases and disorders in mammals. have.
이들 질병 및 질환에는 골다공증 및 전이성 골수암과 같은 골 흡수병, 각막 궤양, 치주질환, 염증성 관절 질환, 피부 염증성 질환과 창상, 및 화상이 포함되나 이에 제한되지 않는다.These diseases and conditions include, but are not limited to, bone resorption diseases such as osteoporosis and metastatic bone marrow cancer, corneal ulcers, periodontal disease, inflammatory joint disease, skin inflammatory diseases and wounds, and burns.
본 발명에서는 굴(oyster)을 단백질 가수분해효소를 이용하여 가수분해하였으며, 이렇게 가수분해해서 얻은 가수분해산물을 여러 단계의 크로마토그래피를 통해 기능성 펩타이드를 정제하였고, 이들의 아미노산 서열을 바탕으로 제조한 합성 펩타이드(GPN 및 PGN)가 콜라게나제 활성을 효과적으로 억제하는 것을 최초로 규명하였다(도 6 참조). 또한, 상기 합성 펩타이드의 세포 독성 실험 결과 세포 내 독성이 없는 것을 확인하여 질환의 예방, 개선 또는 치료를 위한 조성물로도 안정성 있게 사용 가능함을 확인하였다(도 5 및 표 4참조).In the present invention, oysters were hydrolyzed using proteolytic enzymes, and the hydrolysates obtained by the hydrolysis were purified through functional chromatography through several steps of chromatography. It was first identified that synthetic peptides (GPN and PGN) effectively inhibited collagenase activity (see Figure 6). In addition, as a result of cytotoxicity test of the synthetic peptide, it was confirmed that there is no intracellular toxicity, and thus it was confirmed that the composition can be stably used as a composition for preventing, improving or treating diseases (see FIG. 5 and Table 4).
본 발명의 신규 펩타이드는 굴(oyster)로부터 직접 분리하여 제조할 수 있다. 간략하게는 굴(oyster)을 Protamex 및 Neutrase를 이용하여 가수분해한 후, 가수분해물 중 ACE 저해능을 갖는 기능성 펩티드를 한외여과, 음이온 교환수지 크로마토그래피, 크기 배제 크로마토그래피 및 역상 크로마토그래피로 정제하는 방법을 통해서 직접 분리할 수 있다(자세한 내용은 하기 실시예 참조).The novel peptides of the present invention can be prepared by direct isolation from oysters. In brief, oysters are hydrolyzed using Protamex and Neutrase, and then functional peptides with ACE inhibitory activity in the hydrolyzate are purified by ultrafiltration, anion exchange resin chromatography, size exclusion chromatography, and reverse phase chromatography. Can be separated directly through (see Examples below for details).
또한 본 발명의 신규 펩타이드는 화학적으로 합성할 수 있다. 화학적으로 합성하여 제조하는 경우, 당 분야에 널리 공지된 화학적 합성(Creighton, Proteins; Structures and Molecular Principles, W. H. Freeman and Co., NY, 1983)에 의해 제조될 수 있다. 대표적인 방법으로서 이들로 한정되는 것은 아니지만 액체 또는 고체상 합성, 단편 응축, F-MOC 또는 T-BOC 화학법이 포함된다 (Chemical Approaches to the Synthesis of Peptides and Proteins, Williams et al., Eds., CRC Press, Boca Raton Florida, 1997; A Practical Approach, Athert on & Sheppard, Eds., IRL Press, Oxford, England, 1989).In addition, the novel peptides of the present invention can be synthesized chemically. When prepared by chemical synthesis, it may be prepared by chemical synthesis well known in the art (Creighton, Proteins; Structures and Molecular Principles, W. H. Freeman and Co., NY, 1983). Representative methods include, but are not limited to, liquid or solid phase synthesis, fragment condensation, F-MOC or T-BOC chemistry (Chemical Approaches to the Synthesis of Peptides and Proteins, Williams et al., Eds., CRC Press) , Boca Raton Florida, 1997; A Practical Approach, Athert on & Sheppard, Eds., IRL Press, Oxford, England, 1989).
또한 본 발명의 신규 펩타이드는 유전공학적 방법에 의해 제조될 수 있다. 우선, 통상적인 방법에 따라 상기 펩타이드를 코딩하는 DNA 서열을 작제한다. DNA 서열은 적절한 프라이머를 사용하여 PCR 증폭함으로써 작제할 수 있다. 다른 방법으로 당업계에 공지된 표준 방법에 의해, 예컨대, 자동 DNA 합성기(예를 들면, Biosearch 또는 Applied iosystems사에서 판매하는 것)를 사용하여 DNA 서열을 합성할 수도 있다. 제작된 DNA 서열은 이 DNA 서열에 작동가능하게 연결되어 그 DNA 서열의 발현을 조절하는 하나 또는 그 이상의 발현 조절 서열 (예: 프로모터, 인핸서 등)을 포함하는 벡터에 삽입시키고, 이로부터 형성된 재조합 발현 벡터로 숙주세포를 형질전환시킨다. 생성된 형질전환체를 상기 DNA 서열이 발현되도록 하기에 적절한 배지 및 조건 하에서 배양하여, 배양물로부터 상기 DNA 서열에 의해 코딩된 실질적으로 순수한 펩타이드를 회수한다. 상기 회수는 당업계에 공지된 방법(예컨대, 크로마토그래피)을 이용하여 수행할 수 있다. 상기에서 '실질적으로 순수한 펩타이드'라 함은 본 발명에 따른 펩타이드가 숙주로부터 유래된 어떠한 다른 단백질도 실질적으로 포함하지 않는 것을 의미한다. 본 발명의 펩타이드 합성을 위한 유전공학적 방법은 다음의 문헌을 참고할 수 있다: Maniatis et al., MolecularCloning; A laboratory Manual, Cold Spring Harbor laboratory, 1982; Sambrook et al., Molecular Cloning: A Laboratory Manual, ColdSpring Harbor Press, N.Y., Second(1998) and Third(2000) Edition; Gene Expression Technology, Method in Enzymology, Genetics and Molecular Biology, Method in Enzymology, Guthrie & Fink (eds.), Academic Press, San Diego, Calif, 1991; 및 Hitzeman et al., J. Biol. Chem., 255:12073-12080, 1990.In addition, the novel peptides of the present invention can be prepared by genetic engineering methods. First, a DNA sequence encoding the peptide is constructed according to a conventional method. DNA sequences can be constructed by PCR amplification using appropriate primers. Alternatively, DNA sequences may be synthesized by standard methods known in the art, such as using automated DNA synthesizers (eg, sold by Biosearch or Applied iosystems). The constructed DNA sequence is inserted into a vector comprising one or more expression control sequences (eg, promoters, enhancers, etc.) operably linked to and regulate expression of the DNA sequence, and recombinant expression formed therefrom. Transform the host cell with the vector. The resulting transformants are cultured under media and conditions appropriate to allow the DNA sequence to be expressed to recover substantially pure peptides encoded by the DNA sequences from the culture. The recovery can be carried out using methods known in the art (eg chromatography). As used herein, "substantially pure peptide" means that the peptide according to the invention is substantially free of any other protein derived from the host. For genetic engineering methods for peptide synthesis of the present invention, reference may be made to Maniatis et al., Molecular Cloning; A laboratory Manual, Cold Spring Harbor laboratory, 1982; Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Press, N.Y., Second (1998) and Third (2000) Edition; Gene Expression Technology, Method in Enzymology, Genetics and Molecular Biology, Method in Enzymology, Guthrie & Fink (eds.), Academic Press, San Diego, Calif, 1991; And Hitzeman et al., J. Biol. Chem., 255: 12073-12080, 1990.
또 다른 양태에서, 본 발명은 상기 신규 펩타이드(GPN 및 PGN로 이루어진 군으로부터 선택된 1종의 아미노산 서열을 갖는 펩타이드)를 유효성분으로 포함하는 콜라게나제 매개 질환 예방 또는 치료용 약제학적 조성물에 관한 것이다.In another aspect, the present invention relates to a pharmaceutical composition for preventing or treating collagenase mediated disease comprising the novel peptide (peptide having one amino acid sequence selected from the group consisting of GPN and PGN) as an active ingredient. .
본 발명의 조성물에 의해 예방 또는 치료되는 상기 콜라게나제 매개 질환은 골다공증, 전이성 골수암, 각막 궤양, 치주질환, 염증성 관절 질환, 피부 염증성 질환, 창상 또는 화상 등과 같은 질환일 수 있다.The collagenase mediated disease prevented or treated by the composition of the present invention may be a disease such as osteoporosis, metastatic bone marrow cancer, corneal ulcer, periodontal disease, inflammatory joint disease, skin inflammatory disease, wound or burn.
본 발명의 약제학적 조성물은 상기 유효성분 이외에 약제학적으로 적합하고 생리학적으로 허용되는 보조제를 사용하여 제조될 수 있으며, 상기 보조제로는 부형제, 붕해제, 감미제, 결합제, 피복제, 팽창제, 윤활제, 활택제 또는 향미제 등을 사용할 수 있다.The pharmaceutical composition of the present invention may be prepared using a pharmaceutically suitable and physiologically acceptable adjuvant in addition to the active ingredient, and the adjuvant may include excipients, disintegrants, sweeteners, binders, coating agents, swelling agents, lubricants, Lubricants, flavors and the like can be used.
상기 약제학적 조성물은 투여를 위해서 상기 기재한 유효성분 이외에 추가로 약제학적으로 허용 가능한 담체를 1종 이상 포함하여 약제학적 조성물로 바람직하게 제제화할 수 있다.The pharmaceutical composition may be preferably formulated into a pharmaceutical composition including one or more pharmaceutically acceptable carriers in addition to the above-described active ingredient for administration.
상기 약제학적 조성물의 제제 형태는 과립제, 산제, 정제, 피복정, 캡슐제, 좌제, 액제, 시럽, 즙, 현탁제, 유제, 점적제 또는 주사 가능한 액제 등이 될 수 있다. 예를 들어, 정제 또는 캡슐제의 형태로의 제제화를 위해, 유효 성분은 에탄올, 글리세롤, 물 등과 같은 경구, 무독성의 약제학적으로 허용 가능한 불활성 담체와 결합될 수 있다. 또한, 원하거나 필요한 경우, 적합한 결합제, 윤활제, 붕해제 및 발색제 또한 혼합물로 포함될 수 있다. 적합한 결합제는 이에 제한되는 것은 아니나, 녹말, 젤라틴, 글루코스 또는 베타-락토오스와 같은 천연 당, 옥수수 감미제, 아카시아, 트래커캔스 또는 소듐올레이트와 같은 천연 및 합성 검, 소듐 스테아레이트, 마그네슘 스테아레이트, 소듐 벤조에이트, 소듐 아세테이트, 소듐 클로라이드 등을 포함한다. 붕해제는 이에 제한되는 것은 아니나, 녹말, 메틸 셀룰로스, 아가, 벤토니트, 잔탄 검 등을 포함한다. 액상 용액으로 제제화되는 조성물에 있어서 허용 가능한 약제학적 담체로는, 멸균 및 생체에 적합한 것으로서, 식염수, 멸균수, 링거액, 완충 식염수, 알부민 주사용액, 덱스트로즈 용액, 말토 덱스트린 용액, 글리세롤, 에탄올 및 이들 성분 중 1 성분 이상을 혼합하여 사용할 수 있으며, 필요에 따라 항산화제, 완충액, 정균제 등 다른 통상의 첨가제를 첨가할 수 있다. 또한 희석제, 분산제, 계면활성제, 결합제 및 윤활제를 부가적으로 첨가하여 수용액, 현탁액, 유탁액 등과 같은 주사용 제형, 환약, 캡슐, 과립 또는 정제로 제제화할 수 있다. 더 나아가 해당분야의 적절한 방법으로 Remington's Pharmaceutical Science, Mack Publishing Company, Easton PA에 개시되어 있는 방법을 이용하여 각 질환에 따라 또는 성분에 따라 바람직하게 제제화 할 수 있다.Formulation forms of the pharmaceutical composition may be granules, powders, tablets, coated tablets, capsules, suppositories, solutions, syrups, juices, suspensions, emulsions, drops or injectable solutions. For example, for formulation in the form of tablets or capsules, the active ingredient may be combined with an oral, nontoxic, pharmaceutically acceptable inert carrier such as ethanol, glycerol, water and the like. In addition, if desired or necessary, suitable binders, lubricants, disintegrants and coloring agents may also be included in the mixture. Suitable binders include but are not limited to natural and synthetic gums such as starch, gelatin, glucose or beta-lactose, corn sweeteners, acacia, trackercance or sodium oleate, sodium stearate, magnesium stearate, sodium Benzoate, sodium acetate, sodium chloride and the like. Disintegrants include, but are not limited to, starch, methyl cellulose, agar, bentonite, xanthan gum, and the like. Acceptable pharmaceutical carriers in compositions formulated in liquid solutions are sterile and physiologically compatible, including saline, sterile water, Ringer's solution, buffered saline, albumin injectable solutions, dextrose solution, maltodextrin solution, glycerol, ethanol and One or more of these components may be mixed and used, and other conventional additives such as antioxidants, buffers and bacteriostatic agents may be added as necessary. Diluents, dispersants, surfactants, binders and lubricants may also be added in addition to formulate into injectable formulations, pills, capsules, granules or tablets such as aqueous solutions, suspensions, emulsions and the like. Furthermore, the method disclosed in Remington's Pharmaceutical Science, Mack Publishing Company, Easton PA can be formulated according to each disease or component, as appropriate in the art.
본 발명의 일실시예에 있어서, 본 발명의 신규 펩타이드(GPN, AFN 및 PGN로 이루어진 군으로부터 선택된 1종의 아미노산 서열을 갖는 펩타이드)는 상기 조성물에 대해 0.1ug/ml~50mg/ml의 농도로 포함될 수 있다.In one embodiment of the present invention, the novel peptide of the present invention (peptide having one amino acid sequence selected from the group consisting of GPN, AFN and PGN) at a concentration of 0.1ug / ml ~ 50mg / ml with respect to the composition May be included.
또 다른 양태에서, 본 발명은 신규 펩타이드(GPN, AFN 및 PGN로 이루어진 군으로부터 선택된 1종의 아미노산 서열을 갖는 펩타이드)를 유효성분으로 포함하는 콜라게나제 매개 질환 예방 또는 개선용 식품 조성물일 수 있다.In another embodiment, the present invention may be a food composition for preventing or ameliorating collagenase mediated disease comprising a novel peptide (peptide having one amino acid sequence selected from the group consisting of GPN, AFN and PGN) as an active ingredient. .
이러한 식품 조성물은 본 발명의 신규 펩타이드를 함유하는 것 외에 통상의 식품 조성물과 같이 여러 가지 향미제 또는 천연 탄수화물 등을 추가로 함유할 수 있다.In addition to containing the novel peptides of the present invention, such food compositions may further contain various flavors or natural carbohydrates and the like like conventional food compositions.
상술한 천연 탄수화물의 예는 모노사카라이드, 예를 들어, 포도당, 과당 등; 디사카라이드, 예를 들어 말토스, 슈크로스 등; 및 폴리사카라이드, 예를 들어 덱스트린, 시클로덱스트린 등과 같은 통상적인 당, 및 자일리톨, 소르비톨, 에리트리톨 등의 당알콜이다. 상술한 향미제는 천연 향미제 (타우마틴), 스테비아 추출물(예를 들어 레바우디오시드 A, 글리시르히진 등) 및 합성 향미제 (사카린, 아스파르탐 등)를 유리하게 사용할 수 있다.Examples of the above-mentioned natural carbohydrates include monosaccharides such as glucose, fructose and the like; Disaccharides such as maltose, sucrose and the like; And conventional sugars such as polysaccharides such as dextrin, cyclodextrin, and sugar alcohols such as xylitol, sorbitol, and erythritol. The aforementioned flavoring agents can advantageously be used natural flavoring agents (tautin), stevia extracts (for example rebaudioside A, glycyrrhizin, etc.) and synthetic flavoring agents (saccharin, aspartame, etc.).
본 발명의 식품 조성물은 상기 약제학적 조성물과 동일한 방식으로 제제화되어 기능성 식품으로 이용하거나, 각종 식품에 첨가할 수 있다. 본 발명의 조성물을 첨가할 수 있는 식품으로는 예를 들어, 음료류, 육류, 초코렛, 식품류, 과자류, 피자, 라면, 기타 면류, 껌류, 사탕류, 아이스크림류, 알코올 음료류, 비타민 복합제 및 건강보조식품류 등이 있다.The food composition of the present invention may be formulated in the same manner as the pharmaceutical composition, used as a functional food, or added to various foods. Foods to which the composition of the present invention may be added include, for example, beverages, meat, chocolate, foods, confectionery, pizza, ramen, other noodles, gums, candy, ice creams, alcoholic beverages, vitamin complexes, and health supplements. There is this.
또한 상기 식품 조성물은 유효성분인 신규 펩타이드(GPN 및 PGN로 이루어진 군으로부터 선택된 1종의 아미노산 서열을 갖는 펩타이드) 외에 여러 가지 영양제, 비타민, 광물 (전해질), 합성 풍미제 및 천연 풍미제 등의 풍미제, 착색제 및 중진제 (치즈, 초콜릿 등), 펙트산 및 그의 염, 알긴산 및 그의 염, 유기산, 보호성 콜로이드 증점제, pH 조절제, 안정화제, 방부제, 글리세린, 알콜, 탄산음료에 사용되는 탄산화제 등을 함유할 수 있다. 그밖에 본 발명의 식품 조성물은 천연 과일 쥬스 및 과일 쥬스 음료 및 야채 음료의 제조를 위한 과육을 함유할 수 있다.In addition, the food composition is not only a novel peptide (peptide having one amino acid sequence selected from the group consisting of GPN and PGN) as an active ingredient, but also various flavors such as vitamins, minerals (electrolytes), synthetic flavors and natural flavors. Carbonate, used in colorants and neutralizers (cheese, chocolate, etc.), pectic acid and salts thereof, alginic acid and salts thereof, organic acids, protective colloidal thickeners, pH adjusting agents, stabilizers, preservatives, glycerin, alcohols, carbonated drinks And the like. In addition, the food composition of the present invention may contain a fruit flesh for producing natural fruit juice and fruit juice beverage and vegetable beverage.
본 발명의 유효성분인 신규 펩타이드(GPN 및 PGN로 이루어진 군으로부터 선택된 1종의 아미노산 서열을 갖는 펩타이드)는 천연물로부터 유래된 펩타이드의 아미노산 서열에 바탕을 두고 있으며, 세포독성 없이 안정성을 가지므로 콜라게나제 매개 질환의 예방 또는 개선을 목적으로 장기간 복용시에도 안심하고 사용할 수 있다.The novel peptide (peptide having one amino acid sequence selected from the group consisting of GPN and PGN), which is an active ingredient of the present invention, is based on the amino acid sequence of a peptide derived from natural products, and is stable without cytotoxicity. It can be used with confidence even for long-term use for the purpose of preventing or ameliorating a mediated disease.
또 다른 양태에서, 본 발명은 신규 펩타이드(GPN 및 PGN로 이루어진 군으로부터 선택된 1종의 아미노산 서열을 갖는 펩타이드)를 유효성분으로 포함하는 콜라게나제 매개 질환의 예방 또는 개선용 건강기능식품을 제공한다.In another aspect, the present invention provides a dietary supplement for preventing or ameliorating collagenase mediated diseases comprising a novel peptide (peptide having one amino acid sequence selected from the group consisting of GPN and PGN) as an active ingredient. .
본 발명의 건강기능식품은 콜라게나제 매개 질환 개선을 목적으로, 정제, 캅셀, 분말, 과립, 액상, 환 등의 형태로 제조 및 가공할 수 있다.The health functional food of the present invention can be prepared and processed in the form of tablets, capsules, powders, granules, liquids, pills and the like for the purpose of improving collagenase mediated diseases.
본 발명에서 건강기능식품이라 함은 건강기능식품에 관한 법률 제6727호에 따른 인체에 유용한 기능성을 가진 원료나 성분을 사용하여 제조 및 가공한 식품을 말하며, 인체의 구조 및 기능에 대하여 영양소를 조절하거나 생리학적 작용 등과 같은 보건 용도에 유용한 효과를 얻을 목적으로 섭취하는 것을 의미한다.In the present invention, the health functional food refers to foods manufactured and processed using raw materials or ingredients having functional properties useful for the human body according to Act No. 6767 of the Health Functional Food Act, and control nutrients to the structure and function of the human body. Or ingestion for the purpose of obtaining useful effects in health applications such as physiological effects.
본 발명의 건강기능식품은 통상의 식품 첨가물을 포함할 수 있으며, 식품 첨가물로서의 적합 여부는 다른 규정이 없는 한, 식품의약품안전청에 승인된 식품 첨가물 공전의 총칙 및 일반시험법 등에 따라 해당 품목에 관한 규격 및 기준에 의하여 판정한다.The health functional food of the present invention may include a conventional food additive, and the suitability as a food additive, unless otherwise specified, in accordance with the General Regulations of the Food Additives and General Test Methods approved by the Food and Drug Administration, etc. Judging by the standards and standards.
상기 식품 첨가물 공전에 수재된 품목으로는 예를 들어, 케톤류, 글리신, 구연산칼슘, 니코틴산, 계피산 등의 화학적 합성물; 감색소, 감초추출물, 결정셀룰로오스, 고량색소, 구아검 등의 천연첨가물; L-글루타민산나트륨제제, 면류첨가알칼리제, 보존료제제, 타르색소제제 등의 혼합제제류 등을 들 수 있다.Examples of the items loaded on the food additive revolution include, for example, chemical compounds such as ketones, glycine, calcium citrate, nicotinic acid and cinnamon acid; Natural additives such as dark blue pigment, licorice extract, crystalline cellulose, high color pigment and guar gum; And mixed preparations such as sodium L-glutamate, algae additives, preservatives and tar dyes.
예를 들어, 정제 형태의 건강기능식품은 본 발명의 유효성분인 신규 펩타이드(GPN, AFN 및 PGN로 이루어진 군으로부터 선택된 1종의 아미노산 서열을 갖는 펩타이드)를 부형제, 결합제, 붕해제 및 다른 첨가제와 혼합한 혼합물을 통상의 방법으로 과립화한 다음, 활택제 등을 넣어 압축성형하거나, 상기 혼합물을 직접 압축 성형할 수 있다. 또한 상기 정제 형태의 건강기능식품은 필요에 따라 교미제 등을 함유할 수도 있다.For example, a dietary supplement may contain a novel peptide (peptide having one amino acid sequence selected from the group consisting of GPN, AFN, and PGN) as an active ingredient of the present invention with excipients, binders, disintegrants and other additives. The mixed mixture may be granulated by a conventional method, and then compression-molded with a lubricant or the like, or the mixture may be directly compression-molded. In addition, the health functional food in the form of tablets may contain a mating agent or the like as necessary.
캅셀 형태의 건강기능식품 중 경질 캅셀제는 통상의 경질 캅셀에 본 발명의 유효성분인 신규 펩타이드(GPN 및 PGN로 이루어진 군으로부터 선택된 1종의 아미노산 서열을 갖는 펩타이드)를 부형제 등의 첨가제와 혼합한 혼합물을 충진하여 제조할 수 있으며, 연질 캅셀제는 본 발명의 신규 펩타이드(GPN, AFN 및 PGN로 이루어진 군으로부터 선택된 1종의 아미노산 서열을 갖는 펩타이드)를 부형제 등의 첨가제와 혼합한 혼합물을 젤라틴과 같은 캅셀기제에 충진하여 제조할 수 있다. 상기 연질 캅셀제는 필요에 따라 글리세린 또는 소르비톨 등의 가소제, 착색제, 보존제 등을 함유할 수 있다.In the capsule-type health functional food, the hard capsule agent is a mixture of a novel peptide (peptide having one amino acid sequence selected from the group consisting of GPN and PGN), which is an active ingredient of the present invention, into an ordinary hard capsule and an additive such as an excipient. It can be prepared by the filling, soft capsules capsules such as gelatin gelatin mixture mixture of a novel peptide (peptide having one amino acid sequence selected from the group consisting of GPN, AFN and PGN) of the present invention with additives such as excipients It can be prepared by filling the base. The soft capsule agent may contain a plasticizer such as glycerin or sorbitol, a colorant, a preservative, and the like, as necessary.
환 형태의 건강기능식품은 본 발명의 유효성분인 신규 펩타이드(GPN, AFN 및 PGN로 이루어진 군으로부터 선택된 1종의 아미노산 서열을 갖는 펩타이드)와 부형제, 결합제, 붕해제 등을 혼합한 혼합물을 기존에 공지된 방법으로 성형하여 조제할 수 있으며, 필요에 따라 백당이나 다른 제피제로 제피할 수 있으며, 또는 전분, 탈크와 같은 물질로 표면을 코팅할 수도 있다.The cyclic health functional food includes a mixture of a novel peptide (peptide having one amino acid sequence selected from the group consisting of GPN, AFN, and PGN), an excipient, a binder, a disintegrant, and the like, which is an active ingredient of the present invention. It may be prepared by molding in a known manner, and may be coated with sucrose or another coating agent as needed, or the surface may be coated with a material such as starch or talc.
과립 형태의 건강기능식품은 본 발명의 유효성분인 신규 펩타이드(GPN, AFN 및 PGN로 이루어진 군으로부터 선택된 1종의 아미노산 서열을 갖는 펩타이드)와 부형제, 결합제, 붕해제 등을 혼합한 혼합물을 기존에 공지된 방법으로 입상으로 제조할 수 있으며, 필요에 따라 착향제, 교미제 등을 함유할 수 있다.The health functional food in the form of granules is a mixture of a novel peptide (peptide having one amino acid sequence selected from the group consisting of GPN, AFN and PGN), an excipient, a binder, a disintegrant, etc. It can be manufactured granularly by a well-known method, and can contain a flavoring agent, a mating agent, etc. as needed.
본 발명의 신규 펩타이드를 유효성분으로 포함하는 건강기능식품은 하기 실시예에서도 확인한 바와 같이 콜라게나제 활성을 유의적으로 억제시킬 수 있기 때문에, 콜라게나제 매개 질환 개선에 효과적이다.Health functional foods comprising the novel peptide of the present invention as an active ingredient can significantly inhibit collagenase activity, as confirmed in the following examples, and thus are effective in improving collagenase mediated diseases.
상기 건강기능식품은 음료류, 육류, 초코렛, 식품류, 과자류, 피자, 라면, 기타 면류, 껌류, 사탕류, 아이스크림류, 알코올 음료류, 비타민 복합제 및 건강보조식품류 등일 수 있다.The health functional food may be beverages, meat, chocolate, foods, confectionery, pizza, ramen, other noodles, gum, candy, ice cream, alcoholic beverages, vitamin complexes and health supplements.
또 다른 양태에서, 본 발명은 콜라게나제 매개 질환 예방 또는 치료용 의약 또는 식품의 제조를 위한 신규 펩타이드(GPN 및 PGN로 이루어진 군으로부터 선택된 1종의 아미노산 서열을 갖는 펩타이드)를 유효성분으로 포함하는 조성물의 용도를 제공한다. 상기한 신규 펩타이드를 유효성분으로 포함하는 본 발명의 조성물은 콜라게나제 매개 질환 예방 또는 치료용 의약 또는 식품의 제조를 위한 용도로 이용될 수 있다.In another embodiment, the present invention comprises a novel peptide (peptide having one amino acid sequence selected from the group consisting of GPN and PGN) for the manufacture of a medicament or food for the prevention or treatment of collagenase mediated diseases as an active ingredient Provides use of the composition. The composition of the present invention comprising the novel peptide as an active ingredient may be used for the manufacture of a medicament or food for the prevention or treatment of collagenase mediated diseases.
또 다른 양태에서, 본 발명은 포유동물에게 신규 펩타이드(GPN 및 PGN로 이루어진 군으로부터 선택된 1종의 아미노산 서열을 갖는 펩타이드)를 투여하는 것을 포함하는 콜라게나제 매개 질환 예방 또는 치료방법을 제공한다.In another aspect, the present invention provides a method for preventing or treating collagenase mediated disease comprising administering to a mammal a novel peptide (peptide having one amino acid sequence selected from the group consisting of GPN and PGN).
여기에서 사용된 용어 "포유동물"은 치료, 관찰 또는 실험의 대상인 포유동물을 말하며, 바람직하게는 인간을 말한다.As used herein, the term "mammal" refers to a mammal that is the subject of treatment, observation or experimentation, preferably human.
여기에서 사용된 용어 "치료상 유효량"은 연구자, 수의사, 의사 또는 기타 임상에 의해 생각되는 조직계, 동물 또는 인간에서 생물학적 또는 의학적 반응을 유도하는 유효 성분 또는 약학적 조성물의 양을 의미하는 것으로, 이는 치료되는 질환 또는 장애의 증상의 완화를 유도하는 양을 포함한다. 본 발명의 유효 성분에 대한 치료상 유효 투여량 및 투여횟수는 원하는 효과에 따라 변화될 것임은 당업자에게 자명하다. 그러므로, 투여될 최적의 투여량은 당업자에 의해 쉽게 결정될 수 있으며, 질환의 종류, 질환의 중증도, 조성물에 함유된 유효성분 및 다른 성분의 함량, 제형의 종류, 및 환자의 연령, 체중, 일반 건강 상태, 성별 및 식이, 투여 시간, 투여 경로 및 조성물의 분비율, 치료기간, 동시 사용되는 약물을 비롯한 다양한 인자에 따라 조절될 수 있다.As used herein, the term “therapeutically effective amount” means an amount of an active ingredient or pharmaceutical composition that induces a biological or medical response in a tissue system, animal or human, as thought by a researcher, veterinarian, doctor or other clinician, which Amounts that induce alleviation of the symptoms of the disease or disorder being treated. It will be apparent to those skilled in the art that the therapeutically effective dosages and frequency of administrations for the active ingredients of the present invention will vary depending on the desired effect. Therefore, the optimal dosage to be administered can be readily determined by one skilled in the art and includes the type of disease, the severity of the disease, the amount of active ingredients and other ingredients contained in the composition, the type of formulation, and the age, weight, general health of the patient. It may be adjusted according to various factors including the condition, sex and diet, time of administration, route of administration and the rate of secretion of the composition, the duration of treatment, and the drugs used simultaneously.
본 발명의 치료방법에서 본 발명의 신규 펩타이드를 유효성분으로 포함하는 조성물은 경구, 직장, 정맥내, 동맥내, 복강내, 근육내, 흉골내, 경피, 국소, 안구내 또는 피내 경로를 통해 통상적인 방식으로 투여할 수 있다.In the method of treatment of the present invention, the composition comprising the novel peptide of the present invention as an active ingredient is conventionally oral, rectal, intravenous, intraarterial, intraperitoneal, intramuscular, intrasternal, transdermal, topical, intraocular or intradermal. It may be administered in a phosphorous manner.
또 다른 양태에서, 본 발명은 신규 펩타이드(GPN 및 PGN로 이루어진 군으로부터 선택된 1종의 아미노산 서열을 갖는 펩타이드)를 유효성분으로 포함하는 주름 개선용 화장료 조성물일 수 있다.In another embodiment, the present invention may be a cosmetic composition for improving wrinkles comprising a novel peptide (peptide having one amino acid sequence selected from the group consisting of GPN and PGN) as an active ingredient.
본 발명의 조성물이 화장료 조성물로 제조되는 경우, 본 발명의 조성물은 상술한 본 발명의 신규 펩타이드(GPN 및 PGN로 이루어진 군으로부터 선택된 1종의 아미노산 서열을 갖는 펩타이드)뿐만 아니라, 화장료 조성물에 통상적으로 이용되는 성분들을 포함할 수 있으며, 예컨대 항산화제, 안정화제, 용해화제, 비타민, 안료 및 향료와 같은 통상적인 보조제, 그리고 담체를 포함할 수 있다.When the composition of the present invention is made of a cosmetic composition, the composition of the present invention is generally used in cosmetic compositions as well as the novel peptides of the present invention (peptides having one amino acid sequence selected from the group consisting of GPN and PGN). Ingredients used may include, for example, conventional auxiliaries such as antioxidants, stabilizers, solubilizers, vitamins, pigments and flavors, and carriers.
본 발명의 화장료 조성물을 첨가할 수 있는 제품으로는, 예를 들어, 수렴화장수, 유연화장수, 영양화장수, 각종크림, 에센스, 팩, 파운데이션 등과 같은 화장품류와 클렌징, 세안제, 비누, 트리트먼트, 미용액 등이 있다.Examples of products to which the cosmetic composition of the present invention may be added include, for example, cosmetics such as astringent cosmetics, soft cosmetics, nourishing cosmetics, various creams, essences, packs, foundations, and the like, cleansing agents, face washes, soaps, treatments, and cosmetics Etc.
본 발명의 화장료 조성물의 구체적인 제형으로서는 스킨로션, 스킨 소프너, 스킨토너, 아스트린젠트, 로션, 밀크로션, 모이스처 로션, 영양로션, 맛사지크림, 영양크림, 모이스처 크림, 핸드크림, 에센스, 영양에센스, 팩, 비누, 샴푸, 클렌징폼, 클렌징로션, 클렌징크림, 바디로션, 바디클렌저, 유액, 립스틱, 메이컵 베이스, 파운데이션, 프레스파우더, 루스파우더, 아이섀도 등의 제형을 포함한다.Specific formulations of the cosmetic composition of the present invention include skin lotion, skin softener, skin toner, astringent, lotion, milk lotion, moisturizing lotion, nutrition lotion, massage cream, nutrition cream, moisture cream, hand cream, essence, nutrition essence, pack, Formulations such as soaps, shampoos, cleansing foams, cleansing lotions, cleansing creams, body lotions, body cleansers, emulsions, lipsticks, makeup bases, foundations, press powders, loose powders, eye shadows and the like.
한편, 본 발명에 따른 상기 화장료 조성물은 신규 펩타이드(GPN 및 PGN로 이루어진 군으로부터 선택된 1종의 아미노산 서열을 갖는 펩타이드)를 나노리포좀 내부에 함유시켜 안정화하여 제형화할 수도 있다. 상기 신규 펩타이드를 나노리포좀 내부에 함유시키면, 펩타이드의 성분이 안정화되어 제형화시 침전형성, 변형 등의 문제점을 해결할 수 있으며, 성분의 용해도 및 경피흡수율을 높일 수 있어 상기 추출물로부터 기대되는 효능을 최대로 발현시킬 수 있다.Meanwhile, the cosmetic composition according to the present invention may be formulated by containing a novel peptide (peptide having one amino acid sequence selected from the group consisting of GPN and PGN) inside the nanoliposomes. When the new peptide is contained inside the nanoliposomes, the components of the peptide are stabilized to solve problems such as precipitation formation and modification during formulation, and the solubility and transdermal absorption of the components can be increased to maximize the efficacy expected from the extract. Can be expressed as.
본 발명의 이점 및 특징, 그리고 그것들을 달성하는 방법은 상세하게 후술되어 있는 실시예들을 참조하면 명확해질 것이다. 이하, 본 발명을 실시예에 의해 상세히 설명하기로 한다. 그러나 이들 실시예들은 본 발명을 구체적으로 설명하기 위한 것으로서, 본 발명의 범위가 이들 실시예에 한정되는 것은 아니다.Advantages and features of the present invention and methods for achieving them will be apparent with reference to the embodiments described below in detail. Hereinafter, the present invention will be described in detail by way of examples. However, these examples are intended to illustrate the present invention in detail, and the scope of the present invention is not limited to these examples.
<실시예 1><Example 1>
굴(oyster)로부터 가수분해물 제조Hydrolyzate from Oyster
본 발명의 굴 유래 기능성 펩타이드를 동정하기 위하여, 먼저 냉동굴을 끓는 물에 3분간 찐 후, 미트그라인더(meat grinder: M-12S, Hankook Fujee Inc, Korea)를 이용하여 마쇄하고, 굴 무게의 4배 양에 해당하는 1.5%(w/v) 염화나트륨 용액을 첨가하여 10,000rpm(T-25 Basic, Ika Works, NC)에서 균질화하였다. pH는 1M NaOH를 이용하여 pH 6.5 내지 6.7로 조정하였다. 이 후 0.1%(w/v) 트랜스글루타미네이즈(transglutaminase)를 첨가한 다음 30℃에서 1시간 동안 반응시켰으며, 여기에 순차적으로 1%(w/v)의 Protamex 및 1%(w/v)의 Neutrase를 첨가한 후 40℃에서 1시간 동안 가수분해한 후, 가수분해 효소의 활성을 제거하기 위해 100℃에서 1시간 동안 가열하였다. 상기 과정을 통해 얻은 가수분해물은 8,000xg에서 25분간 원심분리(Supra 22K, Hanil Sci. Inc, Korea)하여 불용성 물질을 제거하고, 상등액은 회수하였다. 상등액은 전기투석을 통해 탈염하고, 10kDa의 멤브레인으로 한외 여과한 다음, 감압 하에 동결건조하여 기능성 펩티드 정제를 위한 시료로 사용하였다.In order to identify the oyster-derived functional peptide of the present invention, the frozen oysters are first steamed in boiling water for 3 minutes, and then ground using a meat grinder (meat grinder: M-12S, Hankook Fujee Inc, Korea), and the oyster weight 4 It was homogenized at 10,000 rpm (T-25 Basic, Ika Works, NC) by adding 1.5% (w / v) sodium chloride solution corresponding to the culture. The pH was adjusted to pH 6.5-6.7 using 1M NaOH. Thereafter, 0.1% (w / v) transglutaminase was added, followed by reaction at 30 ° C. for 1 hour, followed by 1% (w / v) of Protamex and 1% (w / v). After adding Neutrase), hydrolysis was performed at 40 ° C. for 1 hour, and then heated at 100 ° C. for 1 hour to remove the activity of the hydrolase. The hydrolyzate obtained through the above process was centrifuged at 8,000xg for 25 minutes (Supra 22K, Hanil Sci. Inc., Korea) to remove insoluble matters, and the supernatant was recovered. The supernatant was desalted through electrodialysis, ultrafiltration with a membrane of 10 kDa, and lyophilized under reduced pressure to serve as a sample for functional peptide purification.
<실시예 2><Example 2>
콜라게나제 저해 펩티드의 정제Purification of Collagenase Inhibiting Peptides
상기 실시예 1을 통해 수득한 굴 가수분해산물 중 콜라게나아제 저해능을 갖는 기능성 펩티드를 음이온 교환수지 크로마토그래피, 크기 배제 크로마토그래피 및 역상 크로마토그래피로 정제하였다.The functional peptide having collagenase inhibitory activity in the oyster hydrolyzate obtained through Example 1 was purified by anion exchange resin chromatography, size exclusion chromatography, and reverse phase chromatography.
<2-1> MMP 저해능의 측정<2-1> Measurement of MMP Inhibitory Activity
SensoLyte Generic MMP assay kit를 사용하여 측정하였다. 콜라게나아제 활성은 Worthingtom manual(Worthington Biochmical Co., 1988)에 따라 측정하였다. Assay kit에 의한 MMP 형태에 따른 저해활성은 표 1과 같다.It was measured using the SensoLyte Generic MMP assay kit. Collagenase activity was measured according to the Worthingtom manual (Worthington Biochmical Co., 1988). Inhibitory activity of MMP type by assay kit is shown in Table 1.
표 1 1 mg/mL 농도에서 MMP 형태에 따른 GPN과 PGN의 저해활성(%)
MMP type GPN PGN
mmp-1 30.69±7.26 10.87±2.28
mmp-2 79.57±3.04 59.03±3.81
mmp-9 34.35±2.45 non-detection
Table 1 Inhibitory Activity of GPN and PGN by MMP Type at 1 mg / mL (%)
MMP type GPN PGN
mmp-1 30.69 ± 7.26 10.87 ± 2.28
mmp-2 79.57 ± 3.04 59.03 ± 3.81
mmp-9 34.35 ± 2.45 non-detection
<2-2> 콜라게나아제 저해 펩타이드의 정제<2-2> Purification of Collagenase Inhibiting Peptides
콜라게나아제 저해능을 가진 펩타이드 정제를 위한 칼럼 크로마토그래피의 조건은 하기 표 2과 같다. 즉 콜라게나아제 저해 펩타이드를 정제하기 위해 실시예 1을 통해 제조한 시료 0.5 g을 12 mL의 20 mM Tris-Cl (pH 8.0)에 용해시키고 HiLoad 16/10 Q-Sepharose 칼럼(16x100 mm)에 5 mL를 주입하였다. 1 mL/min의 유속에서 1 칼럼 부피의 20 mM Tris-Cl(pH 7.5)로 비결합 물질을 세척한 후 100 mL의 0.75 M NaCl을 포함하는 20 mM Tris-Cl(pH 7.5)으로 선형 균배하여 수지에 결합한 물질을 용출하였다. 펩타이드는 226 nm에서 검출하였고, 2 mL를 분획하였다.The conditions of the column chromatography for peptide purification having collagenase inhibitory activity are shown in Table 2 below. Namely, 0.5 g of the sample prepared in Example 1 was dissolved in 12 mL of 20 mM Tris-Cl (pH 8.0) to purify the collagenase inhibitory peptide, and 5 parts were placed on a HiLoad 16/10 Q-Sepharose column (16x100 mm). mL was injected. The unbound material was washed with one column volume of 20 mM Tris-Cl (pH 7.5) at a flow rate of 1 mL / min, then linearly sterilized with 20 mM Tris-Cl (pH 7.5) containing 100 mL of 0.75 M NaCl. The substance bound to the resin was eluted. Peptides were detected at 226 nm and fractionated 2 mL.
그 결과 콜라게나제저해활성을 보이는 한 개의 분리 peak를 얻었다. 이를 Micro-Centrivac(NB-503CIR, N-Biotech Inc., Seoul, Korea)에서 농축한 후 Superdex peptide 칼럼(10x300 mm)에 200 uL를 injection하여 분자량 크기 배제 크로마토그래피를 수행하였다.As a result, one separation peak showing collagenase inhibitory activity was obtained. This was concentrated in Micro-Centrivac (NB-503CIR, N-Biotech Inc., Seoul, Korea) and 200 μL injection was performed on a Superdex peptide column (10x300 mm) to perform molecular weight size exclusion chromatography.
그 결과 첫 번째 피크에서 콜라게나제 저해활성을 보였으며, 이를 Source 5 RPC ST 칼럼(4.6x150 mm)에서 0.09% TFA를 포함한 60% 아세토니트릴 용매로 균배용출하여 최종적으로 3-1-1의 분획을 수득하였다. As a result, it showed collagenase inhibitory activity at the first peak, and it was eluted with 60% acetonitrile solvent containing 0.09% TFA in Source 5 RPC ST column (4.6x150 mm) and finally fraction of 3-1-1. Obtained.
표 2 바이오기능성 펩티드의 정제를 위한 컬럼 크로마토그래피의 조건
Chromatography Column Solvent Flow rate Detection
Anion-exchange HiLoad Q-Sepharose(16x600 mm) A: 20 mM Tris-Cl, pH 8.0B: 20% CH3CN in 0.1%TFA 1 mL/min 226 nm
Size exclusion Superdex peptide(10x300 mm) A: 20 mM Tris-Cl, pH 7.5 0.5 mL/min 216 nm, 254 nm
Reversed phase Source 5RPC ST(4.6x150 mm) A: 0.1% TFA/waterB: 0.1% TFA/60% ACN 1 mL/min 216 nm254 nm
TABLE 2 Conditions of Column Chromatography for Purification of Biofunctional Peptides
Chromatography Column Solvent Flow rate Detection
Anion-exchange HiLoad Q-Sepharose (16x600 mm) A: 20 mM Tris-Cl, pH 8.0B: 20% CH 3 CN in 0.1% TFA 1 mL / min 226 nm
Size exclusion Superdex peptide (10x300 mm) A: 20 mM Tris-Cl, pH 7.5 0.5 mL / min 216 nm, 254 nm
Reversed phase Source 5RPC ST (4.6x150 mm) A: 0.1% TFA / water B: 0.1% TFA / 60% ACN 1 mL / min 216 nm 254 nm
표 3 정제 단계별 수득한 분획들의 콜라게나제 억제능(%)
Purification step Pooled name Fraction number Collagenase inhibition, % Suggested peptide
Q-Sepharose ion exchange chromatopgraphy 3 30,31 6.7 -
Superdex peptide size exclusion chromatography 3-1 20 18.3
Source 5RPC ST reversed-phase chromatography 3-1-1 18,19 35.2 PGN, GPN
TABLE 3 Collagenase inhibitory activity (%) of the fractions obtained for each purification step
Purification step Pooled name Fraction number Collagenase inhibition,% Suggested peptide
Q-Sepharose ion exchange chromatopgraphy 3 30,31 6.7 -
Superdex peptide size exclusion chromatography 3-1 20 18.3
Source 5RPC ST reversed-phase chromatography 3-1-1 18,19 35.2 PGN, GPN
<실시예 3><Example 3>
정제 펩타이드의 분자량 분포와 아미노산 서열 분석Molecular Weight Distribution and Amino Acid Sequence Analysis of Purified Peptides
기능성 펩타이드의 아미산 서열을 결정하기 위해 상기 실시예 2의 역상 크로마토그래피로 얻은 분획물을 Micro-Centvac에서 완전히 건조시켰다. 건조한 시료를 20 uL의 0.1% TFA를 포함한 증류수에 용해시키고 시료 중의 염을 제거하기 위해 50% 아세토니트릴로 활성화시켜 0.1% TFA를 포함하는 증류수로 평행시킨 ZipTip C18(Pierce #87782)에 로딩하였다. 0.1% TFA를 포함하는 증류수로 2-3회 세척하고 ZipTip C18에 결합된 펩타이드를 0.1% TFA를 포함하는 70% 아세토니트릴 용액을 용출하였다. 용출한 펩타이드 용액을 biobrene(AB Systems Co., U.S.A.)이 전처리된 마이크로필터에 10 uL를 로딩하고 아르곤 가스로 건조시켰다. 건조가 끝난 필터를 카트리지에 장착하여 아미노산 자동 서열기(ABI492 automated protein sequencer, Applied Biosystem, Foster, CA, U.S.A.)의 pulsed-liquid 법을 통해 아미노산 서열을 결정하였다. 그리고 전기분무 이온화(ESI)법을 이용하여 질량분석기로 분자량 이온 및 분해된 이온을 검출하여 정제된 펩타이드 분자량을 확인하였다. 질량스펙트럼은 Q-TOF2(Micromass, U.K.) 질량분석기에 Nano-ESI interface를 이용하여 data dependant MS/MS 방법으로 얻었다.Fractions obtained by reverse phase chromatography of Example 2 were dried completely in Micro-Centvac to determine the amino acid sequence of the functional peptide. The dry sample was dissolved in distilled water containing 20 uL of 0.1% TFA and loaded into ZipTip C18 (Pierce # 87782) paralleled with distilled water containing 0.1% TFA, activated with 50% acetonitrile to remove salts in the sample. After washing 2-3 times with distilled water containing 0.1% TFA, peptide bound to ZipTip C18 was eluted with a 70% acetonitrile solution containing 0.1% TFA. The eluted peptide solution was loaded with 10 uL of microb pretreated with biobrene (AB Systems Co., U.S.A.) and dried with argon gas. The dried filter was mounted on the cartridge to determine the amino acid sequence by pulsed-liquid method of ABI492 automated protein sequencer (Applied Biosystem, Foster, CA, U.S.A.). The molecular weight of the purified peptide was confirmed by detecting molecular weight ions and decomposed ions using a mass spectrometer using electrospray ionization (ESI). Mass spectra were obtained by Q-TOF2 (Micromass, U.K.) mass spectrometry using a data-dependent MS / MS method using the Nano-ESI interface.
그 결과 ACE 저해 펩타이드의 정제물은 모두 500 Da 미만의 분자량에서 CHCA matrix와 차이를 보이는 주요 피크를 확인하였고, 2개의 펩타이드의 아미노산 결합서열을 확인하였다(하기 표 4 참조). 확인한 아미노산 서열 PGN 및 GPN이 가수분해물에서 새로이 확인하였고, GPN과 PGN의 콜라게나제 저해활성은 각각 0.43 mg/ml와 0.60 mg/ml의 농도에서 58.1%와 100%였다. As a result, all of the purified ACE inhibitory peptides had a major peak different from the CHCA matrix at a molecular weight of less than 500 Da, and the amino acid binding sequences of the two peptides were confirmed (see Table 4 below). The identified amino acid sequences PGN and GPN were newly identified in the hydrolyzate, and collagenase inhibitory activities of GPN and PGN were 58.1% and 100% at 0.43 mg / ml and 0.60 mg / ml, respectively.
<실시예 4><Example 4>
본 발명의 기능성 펩타이드 합성 및 합성된 펩타이드의 정제Functional Peptide Synthesis and Purification of Synthetic Peptides of the Invention
앞서 수행한 실험을 통해 콜라게나제 저해 활성을 갖는 펩타이드의 서열을 확인하고, 이러한 펩타이드의 또 다른 기능을 확인하기 위해 펩타이드를 합성하였다.Through the experiments performed above, the sequence of the peptide having collagenase inhibitory activity was identified, and the peptide was synthesized to confirm another function of the peptide.
펩타이드는 ASP48S(Peptron Inc., Dajeon, Korea)를 이용하여 Fmoc 고상 펩타이드 합성을 수행하고 Vydac Everest C18 칼럼(22x250 mm, 10 um)를 사용하여 역상 HPLC로 정제하였다. 즉 펩타이드의 C-말단에 첫 번째 아미노산이 수지에 부착된 것을 사용하여 Na-말단이 Fmoc로 보호된 아미노산을 순서에 따라 부착하였으며 잔기는 TFA에서 제거되는 보호기를 사용하였다. coupling 시약으로 HBTU/HOBt/NMM을 사용하였으며, Fmoc의 제거는 20% piperidine/DMF를 사용하였다. 합성 펩타이드를 수지에서 분리하고 잔기의 보호기 제거를 위해 TFA/EDT/thioanisole/TIS/H2O(90/2.5/2.5/2.5/2.5, v/v)를 사용하였다. 펩타이드는 0.1% TFA를 포함하는 40% 아세토니트릴 용액으로 선형 균배하여 용출하였다. 정제한 펩타이드의 분자량은 LC/MS (Agilent HP1100 시리즈)로 확정하였다.Peptides were subjected to Fmoc solid phase peptide synthesis using ASP48S (Peptron Inc., Dajeon, Korea) and purified by reversed phase HPLC using Vydac Everest C18 column (22x250 mm, 10 um). That is, the first amino acid attached to the resin at the C-terminus of the peptide was attached to the Na-terminus of Fmoc-protected amino acids in order, and a protecting group was used to remove residues from the TFA. HBTU / HOBt / NMM was used as the coupling reagent, and 20% piperidine / DMF was used to remove Fmoc. Synthetic peptides were separated from the resin and TFA / EDT / thioanisole / TIS / H 2 O (90 / 2.5 / 2.5 / 2.5 / 2.5, v / v) was used to remove the protecting groups of the residues. Peptides were eluted by linear sterilization with 40% acetonitrile solution containing 0.1% TFA. The molecular weight of the purified peptide was confirmed by LC / MS (Agilent HP1100 series).
그 결과 합성한 펩타이드들은 HPLC에서 단일의 peak를 나타내었으며, 합성 펩타이드는 MALDI-TOF 질량 분석기에서 단일 peak의 분자량을 나타내고 있었다. 본 발명에서는 상기와 같이 기능성을 갖는 2개의 합성 펩타이드를 하기 표 3에 나타내었다.As a result, the synthesized peptides showed a single peak in HPLC, and the synthesized peptide showed a molecular weight of a single peak in MALDI-TOF mass spectrometer. In the present invention, two synthetic peptides having the functionalities as described above are shown in Table 3 below.
표 4 본 발명의 기능성 펩타이드
순번 합성 펩타이드
1 GPN
2 PGN
Table 4 Functional Peptides of the Invention
turn Synthetic peptides
One GPN
2 PGN
<실험예 1>Experimental Example 1
본 발명의 기능성 펩타이드의 세포독성 측정Cytotoxicity Measurement of Functional Peptides of the Invention
본 실험에서는 상기 실시예 4를 통해 합성된 기능성 펩타이드의 세포독성을 평가하였다(표 5 참조).In this experiment, the cytotoxicity of the functional peptide synthesized in Example 4 was evaluated (see Table 5).
세포독성은 정상 간세포인 Chang cell로 확인하였다. 즉 세포 배양용 플라스크에 배양한 Chang cell을 10% FBS가 함유된 MEM 배지로 배양하였다. 1x104 cells/well의 농도로 96 웰 플레이트에 분주하고, 24시간 동안 35℃, 습도 95%, CO2, 5%로 조절된 CO2배양기에서 배양하였다. 새로운 배지에 시료를 최종 농도가 50, 100, 200 및 300 ug/mL이 되도록 녹여 세포주에 처리한 후 24시간 동안 배양하였다. 세포 증식률은 모두 Promega사에서 구입한 CellTiter 96 Aqueous One Solution Cell Proliferation Assay를 이용하여 microplate reader(Perkin Elmer 1420, VICTORTM X Multilabel Plate Readers, Waltham, MA, USA)로 490 nm에서 흡광도를 측정하였다. 세포의 생존율은 시료를 처리하지 않은 대조군에 대비한 시료 처리군의 흡광도로 표시하였다.Cytotoxicity was confirmed by Chang cells, normal hepatocytes. That is, Chang cells cultured in the cell culture flasks were cultured in MEM medium containing 10% FBS. The cells were dispensed into 96 well plates at a concentration of 1 × 10 4 cells / well and incubated in a CO 2 incubator adjusted to 35 ° C., 95% humidity, CO 2 , 5% for 24 hours. Samples were dissolved in fresh medium to a final concentration of 50, 100, 200 and 300 ug / mL, treated with cell lines and incubated for 24 hours. Cell proliferation was measured at 490 nm using a MicroTiter 96 Aqueous One Solution Cell Proliferation Assay purchased from Promega using a microplate reader (Perkin Elmer 1420, VICTORTM X Multilabel Plate Readers, Waltham, MA, USA). Cell viability was expressed by the absorbance of the sample treated group compared to the control group not treated with the sample.
표 5 본 발명의 기능성 합성 펩타이드의 세포 독성
합성 펩타이드 Cell viability, %
GPN 108.3±1.0
PGN 103.9±6.2
Table 5 Cytotoxicity of Functional Synthetic Peptides of the Invention
Synthetic peptides Cell viability,%
GPN 108.3 ± 1.0
PGN 103.9 ± 6.2
그 결과 상기 표 5에서 나타낸 바와 같이, 본 발명의 기능성 합성 펩타이드는 정상 간세포에 대하여 세포 독성을 보이지 않는 것을 확인할 수 있었다. 따라서 이를 통해 본 발명의 기능성 펩타이드는 세포에 유해작용 없이 안전하게 사용할 수 있음을 알 수 있었다.As a result, as shown in Table 5, it was confirmed that the functional synthetic peptide of the present invention does not show cytotoxicity to normal hepatocytes. Therefore, it was found that the functional peptide of the present invention can be safely used without harmful effects on the cells.
<실험예 2>Experimental Example 2
본 발명의 기능성 펩타이드의 콜라게나제 활성 저해능의 측정Determination of Collagenase Activity Inhibitory Activity of Functional Peptides of the Invention
본 실험에서는 상기 실시예 4를 통해 합성된 기능성 펩타이드의 콜라게나제 활성 저해능의 측정하였다.In this experiment, the collagenase activity inhibitory ability of the functional peptide synthesized in Example 4 was measured.
콜라게나제(collagenase) 활성 저해능은 Invitrogen사의 EnzChekGelatinase/Collagenase Assay Kit-250-2000 Assays 시약을 구입하여 사용하였다. 먼저 1 mg DG gelatin vial에 1.0 mL의 DDW를 가해 DG gelatin stock solution(1 mg/mL)을 만든 후, 10 x reaction buffer 2 mL에 DDW 18 mL를 가해 reaction buffer를 희석하였다. 다음으로 콜라게나제 효소 시약을 제조하였다. Working solution으로 최종 농도가 0.2 unit/mL이 되도록 reaction buffer로 희석하였다. 실험에 사용할 시료는 농도가 0.01, 0.1 1, 10 mg/mL가 되도록 제조하여, 시료를 96 웰 플레이트에 20 uL씩 triple로 준비하였다. DQ gellatin 10 uL와 sample blank에는 reaction buffer 100 uL, 샘플에는 working solution 100 uL를 시료에 가하고, 빛을 차단한 상태로 실온에서 1-2시간 동안 방치한 후 형광 강도 excition wavelength 495 nm 및 emission wavelength 515 nm에서 ELAZA plate reader(SpectraMax M2 & M2e Multi-Mode Microplate Reader. Molecular Devices Corporation. USA)로 형광강도를 측정하였다.Collagenase activity was inhibited by the purchase of Invitrogen's EnzChekGelatinase / Collagenase Assay Kit-250-2000 Assays reagent. First, 1.0 mL of DDW was added to 1 mg DG gelatin vial to make DG gelatin stock solution (1 mg / mL). Then, 18 mL of DDW was added to 2 mL of 10 x reaction buffer to dilute the reaction buffer. Next, collagenase enzyme reagent was prepared. The working solution was diluted with reaction buffer so that the final concentration was 0.2 unit / mL. Samples to be used in the experiment was prepared so that the concentration is 0.01, 0.1 1, 10 mg / mL, and the samples were prepared in triple by 20 uL in a 96 well plate. 10 uL of DQ gellatin and 100 uL of reaction buffer in the sample blank and 100 uL of working solution in the sample were added to the sample, and left for 1 hour at room temperature in a light-blocked state. The fluorescence intensity excition wavelength 495 nm and emission wavelength 515 Fluorescence intensity was measured at nm with an ELAZA plate reader (SpectraMax M2 & M2e Multi-Mode Microplate Reader. Molecular Devices Corporation. USA).
그리고 bovine achilles tendon을 기질로 사용한 콜라게나제 활성은 Worthington enzyme manual(1972)에 따라 측정하였다. 즉 capped test tube에 기질인 collagen(Sigma C9879)의 무게를 정확히 달고, 2.0 mL의 0.36 mM CaCl2를 포함하는 50 mM TES 완충액(pH 7.5)과 collagenase(0.2 mg/mL, Sigma C0130) 0.1 mL를 가하여 37℃의 항온조에서 흔들어 주면서 5시간 가수분해하였다. 1 mL의 2% ninhydrin 용액이 들어있는 capped test tube에 가수분해물 0.2 mL를 가하여 반응을 중지시키고, 100℃의 건조기에서 20분 동안 발색 시킨 후, 600 nm에서 흡광도를 측정하였다. 유리된 아미노산의 함량은 L-leucine으로 작성한 표준곡선에 따라 정량하였으며 콜라게나제 활성은 uM leucine/hr/g-collagen으로 표시하였다. 한편 합성 펩타이드에 의한 콜라게나제의 저해는 합성 펩타이드와 콜라게나제의 비가 각각 8.3배와 12.5배가 되도록 혼합한 후 저온고에서 15시간 충분히 반응 시킨 후, 콜라게나제 활성 측정방법에 따라 측정하였으며, 잔여활성은 저해제 첨가 유무에 따른 활성의 비로 계산하여 잔여 활성의 %로 표시하였다.Collagenase activity using bovine achilles tendon as a substrate was measured according to Worthington enzyme manual (1972). Accurately weigh the substrate collagen (Sigma C9879) into the capped test tube and add 50 mL TES buffer (pH 7.5) and collagenase (0.2 mg / mL, Sigma C0130) containing 2.0 mL of 0.36 mM CaCl 2 . The solution was hydrolyzed for 5 hours while shaking in a constant temperature bath at 37 ° C. The reaction was stopped by adding 0.2 mL of the hydrolyzate to a capped test tube containing 1 mL of 2% ninhydrin solution. The reaction was stopped for 20 minutes, and the absorbance was measured at 600 nm. The amount of free amino acid was quantified according to the standard curve made with L-leucine and the collagenase activity was expressed as uM leucine / hr / g-collagen. On the other hand, the inhibition of collagenase by synthetic peptide was measured according to the method of measuring collagenase activity after fully reacting at low temperature for 15 hours after mixing so that ratio of synthetic peptide and collagenase was 8.3 times and 12.5 times, respectively. Residual activity was expressed as% of residual activity, calculated as the ratio of activity with or without inhibitor addition.
그 결과 도 6에 나타낸 바와 같이, 합성 펩타이드 중 GPN 및 PGN은 콜라게나제에 대하여 농도 의존적인 저해활성을 보였다. 그리고 콜라게나제의 기질로 bovine achilles tendon을 사용하여 효소 대 합성 펩타이드의 저해활성을 측정한 결과, 도 7에서 나타낸 바와 같이, GPN과 PGN은 콜라게나제 활성의 58..1%와 100%를 억제하여 제해 효과를 보였다. As a result, as shown in Figure 6, GPN and PGN in the synthetic peptide showed a concentration-dependent inhibitory activity against collagenase. As a result of measuring the inhibitory activity of the enzyme versus the synthetic peptide using bovine achilles tendon as a substrate of collagenase, GPN and PGN showed 58..1% and 100% of collagenase activity as shown in FIG. Inhibition effect was shown.
<실험예 3>Experimental Example 3
본 발명의 기능성 펩타이드 PGN의 콜라게나제 활성 저해능Inhibition of Collagenase Activity of Functional Peptides PGN of the Present Invention
<3-1> 콜라게나제에 PNG 처리에 따른 시간경과별 효소 잔존 활성<3-1> Enzyme Remaining Activity over Time by PNG Treatment of Collagenase
본 발명의 기능성 펩타이드인 PGN의 콜라게나제 활성 저해능을 측정하기 위하여 콜라게나제에 PNG를 처리한 후 시간경과에 따른 효소 잔존 활성을 측정하였다.In order to measure the collagenase activity inhibitory ability of PGN, the functional peptide of the present invention, after treating PNG with collagenase, the enzyme residual activity was measured over time.
시판 콜라게나제와 본 발명의 합성 펩타이드 PNG를 혼합하여 37℃에서 10분 단위로 60분까지 반응시켜 콜라게나제의 활성을 저해시킨 후, 송아지 아킬레스 건을 기질로 콜라게나제 활성을 측정하였다.The commercial collagenase and the synthetic peptide PNG of the present invention were mixed and reacted at 37 ° C. for 10 minutes in 10 minutes to inhibit collagenase activity. Then, the calcanase activity was measured by calf Achilles tendon as a substrate.
그 결과 도 8에서 나타낸 바와 같이, 시판 콜라게나아제는 항온시간의 증가와 더불어 활성이 급속히 감소하였으며, 저해제로서 PNG를 첨가한 경우는 항온 시간에 상관없이 콜라게나아제 활성을 저해하여 잔여 활성은 거의 나타나지 않았다.As a result, as shown in FIG. 8, commercial collagenase rapidly decreased with increasing incubation time, and when PNG was added as an inhibitor, collagenase activity was inhibited regardless of the incubation time. Did not appear.
<3-2> 콜라게나제에 PNG 처리에 따른 시간경과별 효소 억제능 변화<3-2> Changes of Enzyme Inhibitory Capacity of Collagenase by PNG Treatment with Time
본 발명의 기능성 펩타이드인 PGN의 콜라게나제 활성 저해능을 측정하기 위하여 콜라게나제에 PNG를 처리한 후 시간 경과에 따른 효소 억제 활성을 상기 <3-1>과 동일한 방법으로 측정하였다.In order to measure the collagenase activity inhibitory ability of PGN, the functional peptide of the present invention, the enzyme inhibitory activity over time was measured in the same manner as in <3-1> after treating the collagenase with PNG.
그 결과 도 9에서 나타낸 바와 같이, 콜라게나제에 본 발명의 PNG 펩타이드를 처리한 후 37℃에서 전배양하는 동안 콜라게나제의 억제 활성이 85% 이상 유지되는 것을 확인할 수 있었으며, 특히 1시간이 경과한 시점에도 90% 이상인 것으로 나타났다.As a result, as shown in Figure 9, after treating the collagenase PNG peptide of the present invention, it was confirmed that the inhibitory activity of collagenase is maintained at 85% or more during pre-culture at 37 ℃, especially 1 hour Over 90% of the time passed.
<3-3> 본 발명의 PNG 펩타이드의 IC<3-3> IC of PNG peptide of the present invention 5050 값 value
본 발명의 기능성 펩타이드인 PGN의 콜라게나제 활성 저해능을 측정하기 위하여 콜라게나제에 PNG를 농도별 처리에 따른 콜라게나제 억제 활성을 측정하였다.In order to measure the collagenase activity inhibitory ability of PGN, the functional peptide of the present invention, collagenase inhibitory activity according to concentration-dependent treatment of PNG to collagenase was measured.
그 결과 도 10에서 나타낸 바와 같이, 콜라게나제에 본 발명의 PNG 펩타이드를 농도별로 처리한 경우 콜라게나제 억제 활성이 농도에 의존하여 증대되는 것을 확인할 수 있었으며, 특히 본 발명의 PNG 펩타이드의 IC50 값이 8.29ug으로 나타나 매우 높은 콜라게나제 억제 활성을 보여주었다.As a result, as shown in Figure 10, when collagenase was treated with PNG peptide of the present invention by concentration, the collagenase inhibitory activity was confirmed to increase depending on the concentration, in particular IC 50 of the PNG peptide of the present invention The value was 8.29 ug, indicating very high collagenase inhibitory activity.
<실험예 4>Experimental Example 4
본 발명의 기능성 펩타이드의 피부재생(주름개선) 효과Skin regeneration (wrinkle improvement) effect of the functional peptide of the present invention
<4-1> CCD 989sk cell 배양 및 시료 추출<4-1> CCD 989sk cell culture and sample extraction
본 발명의 기능성 펩타이드의 주름 개선 효과를 실험하기 위해 섬유아세포주인 CCD 986sk cell을 세포 배양용 플라스크에 배양한 CCD 986sk cell을 10ml 피펫으로 세포를 플라스크에서 떼어내어 계대하였고, 세포는 10% FBS가 함유된 IMDM 배지로 배양하였다. CCD 986sk cell를 2.5x104 cells/well의 농도로 96 well plate에 분주하고, 24시간 동안 35℃, 습도 95%, CO2 5%로 조절된 CO2 배양기에서 배양하였다. 새로운 배지에 상기 시료를 최종 농도가 1, 10ug/ml가 되도록 녹여 세포에 처리하여 24시간 배양 후, 상등액을 모아 시료로 사용하였다.In order to examine the anti-wrinkle effect of the functional peptide of the present invention, CCD 986sk cells, the fibroblast line CCD 986sk cells, were cultured in a cell culture flask, and the cells were removed from the flask with a 10 ml pipette, and the cells contained 10% FBS. Incubated with IMDM medium. CCD 986sk cells were aliquoted into 96 well plates at a concentration of 2.5x10 4 cells / well and incubated in a CO 2 incubator controlled at 35 ° C., 95% humidity, 5% CO 2 for 24 hours. The sample was dissolved in fresh medium to a final concentration of 1, 10 ug / ml, treated with cells, cultured for 24 hours, and the supernatant was collected and used as a sample.
<4-2> MMP-1 저해활성의 측정<4-2> Measurement of MMP-1 Inhibitory Activity
MMP-1은 SensoLyte 490 MMP-1 Assay Kit(AnaSpec, MI, USA)를 구입하여 측정하였다. 시료를 처리하여 배양한 CCD 986sk의 배양 상등액 50ul에 MMP-1기질을 50ul가하여 30초간 교반 후 microplate reader(Molecular Devices, VersaMax ELISA Microplate Reader, USA)로 흡수파장 340nm, 방출파장 490nm에서 10분 간격으로 60분 동안 형광값을 측정하여, 하기의 식으로 저해 활성을 측정하였다.MMP-1 was measured by purchasing a SensoLyte 490 MMP-1 Assay Kit (AnaSpec, MI, USA). 50 μl of MMP-1 substrate was added to 50 μl of the culture supernatant of CCD 986sk, which was treated and cultured for 30 seconds, and then stirred for 10 seconds at a wavelength of 340 nm and emission wavelength of 490 nm using a microplate reader (Molecular Devices, VersaMax ELISA Microplate Reader, USA). The fluorescence value was measured for 60 minutes, and the inhibitory activity was measured by the following formula.
MMP-1 저해활성 = [1-(S-B)/(C-B)] X 10MMP-1 inhibitory activity = [1- (S-B) / (C-B)] X 10
S : 시료의 흡광도, B : 공시험군의 흡관도, C : 대조군의 흡광도S: absorbance of the sample, B: absorbance of the blank test group, C: absorbance of the control group
MMP-1 활성 저해능 평가 결과 굴 유래 펩타이드인 PGN과 GPN은 시료 10ug/ml의 농도에서 MMP-1의 활성이 하기 표 6과 같이 53.9%와 41.4%로 높은 저해 활성을 보여주었다.As a result of evaluation of MMP-1 activity inhibitory activity, oyster-derived peptides PGN and GPN showed high inhibitory activity of MMP-1 at 53.9% and 41.4% as shown in Table 6 below at the concentration of 10ug / ml of the sample.
표 6 MMP-1 활성 측정 결과
Peptides(/) MMP-1 inhibition activity(%)
PGN 1 29.1±17.5*
10 53.9±7.6*
GPN 1 53.9±7.6*
10 41.4±7.8*
Table 6 MMP-1 activity measurement result
Peptides (/) MMP-1 inhibition activity (%)
PGN One 29.1 ± 17.5 *
10 53.9 ± 7.6 *
GPN One 53.9 ± 7.6 *
10 41.4 ± 7.8 *
*p<0.05: compared with control group* p <0.05: compared with control group
<4-3> Procollagen 생성량 측정<4-3> Procollagen Production
본 발명의 기능성 펩타이드인 GPN 및 PGN의 피부재생 효과를 측정하기 위하여 procollagen 생성량을 측정하였다. Procollagen은 type 1 C-peptide(PIP) EIA kit를 이용하여 측정하였다. CCD 986sk의 상등액 10 ul에 시료 희석제 40 ul를 넣어 5배 희석하여 시료액으로 사용하였다. 코팅된 microplate에 antibody-POD conjugate 100 ul를 넣고 희석된 시료액 20 ul를 가하여 뚜껑을 덮고 호일로 싼 후, 37℃에서 3시간 동안 배양한다. 배양한 후 모든 액을 제거하고 PBS를 400 ul씩 넣어 4번 세척후 물기를 완전히 제거하고 기질용액으로 TMBZ를 100 ul 넣고 실온에서 15분 동안 방치한다. 정지용액으로서 1 normal 황산 용액 100 ul를 넣고 반응을 중지시킨 다음 microplate reader(Molecular Devices, VersaMax ELISA Microplate Reader, USA)로 450 nm에서 흡광도를 측정하였다. 표준 용액의 농도는 0, 10, 20, 40, 80, 160, 320 및 640 ng/ml를 사용하였다. 피부재생효과는 표 7와 같다.Procollagen production was measured to determine the skin regeneration effect of the functional peptides of the present invention GPN and PGN. Procollagen was measured using a type 1 C-peptide (PIP) EIA kit. 40 ul of the sample diluent was added to 10 ul of the supernatant of the CCD 986sk and diluted 5 times to use the sample solution. Add 100 ul of antibody-POD conjugate to the coated microplate, add 20 ul of diluted sample solution, cover with a foil, and incubate at 37 ° C for 3 hours. After incubation, remove all the liquid, wash 4 times with 400 ul of PBS, remove the water completely, add 100 ul of TMBZ with substrate solution and leave at room temperature for 15 minutes. 100 ul of 1 normal sulfuric acid solution was added as a stop solution, the reaction was stopped, and the absorbance was measured at 450 nm with a microplate reader (Molecular Devices, VersaMax ELISA Microplate Reader, USA). Concentrations of standard solutions were used at 0, 10, 20, 40, 80, 160, 320 and 640 ng / ml. Skin regeneration effect is shown in Table 7.
표 7 CCD 986sk 피부 fibroblast 세포배양에 의한 본 발명의 기능성 합성 펩타이드의 피부 재생효과
펩타이드 농도 (mg/ml) 세포재생효과(%)
1일 3일
GPN 0.01 226.1±8.4 259.6±12.9
0.1 209.8±12.7 250.0±9.3
1 222.6±9.7 245.7±14.8
10 233.2±12.7 221.9±27.9
PGN 0.01 177.3±8.2 246.0±1.2
0.1 153.2±7.0 191.5±19.5
1 208.5±3.8 228.8±12.0
10 230.8±9.0 210.1±12.8
대조군 100.0±5.6 100.0±7.7
TABLE 7 Skin regeneration effect of functional synthetic peptides of the present invention by CCD 986sk skin fibroblast cell culture
Peptide Concentration (mg / ml) Cell renewal effect (%)
1 day 3 days
GPN 0.01 226.1 ± 8.4 259.6 ± 12.9
0.1 209.8 ± 12.7 250.0 ± 9.3
One 222.6 ± 9.7 245.7 ± 14.8
10 233.2 ± 12.7 221.9 ± 27.9
PGN 0.01 177.3 ± 8.2 246.0 ± 1.2
0.1 153.2 ± 7.0 191.5 ± 19.5
One 208.5 ± 3.8 228.8 ± 12.0
10 230.8 ± 9.0 210.1 ± 12.8
Control 100.0 ± 5.6 100.0 ± 7.7
<실험예 5> 피부섬유아세포(CCD 986sk)를 통한 피부세포 재생능 측정Experimental Example 5 Measurement of Skin Cell Regeneration Using Skin Fibroblasts (CCD 986sk)
<5-1> 세포의 배양<5-1> Cell Culture
세포 증식능에 사용한 CCD 986sk cell은 American Type Culture Collection (ATCC, VA, USA)에서 분양받아 사용하였다. 세포 배양용 플라스크에 배양한 CCD 986sk cell을 10 mL 피펫으로 세포를 플라스크에서 떼어내어 계대하였고, 세포는 12% FBS(Lonza, Valais, Switzerland)가 함유된 Iscove's Modified Dulbeccos Medium(IMDM, Lonza, Valais, Switzerland)배지를 사용하여 37℃, 5% CO2가 유지되는 배양기에서 배양하였다. 이때 미생물의 오염이나 증식을 억제하기 위해 배지용 항생제(Penicillin streptomycin, Gibco, CA, USA)를 사용하였다. 세포가 80% 정도 dish를 덮으면 phosphated-buffered saline-EDTA(PBS-EDTA)로 세척한 후 트립신 처리하여 계대 배양하였으며, 배지는 48시간마다 교환하여 세포를 배양하였다. CCD 986sk cells used for cell proliferation were available from American Type Culture Collection (ATCC, VA, USA). CCD 986sk cells cultured in cell culture flasks were removed from the flasks using a 10 mL pipette and passaged, and the cells were passaged with Iscove's Modified Dulbeccos Medium (IMDM, Lonza, Valais, Switzerland) containing 12% FBS (Lonza, Valais, Switzerland). Switzerland) was incubated in an incubator at 37 ℃, 5% CO 2 maintained. At this time, antibiotics for culture medium (Penicillin streptomycin, Gibco, CA, USA) were used to inhibit the contamination or proliferation of microorganisms. Cells were washed with phosphated-buffered saline-EDTA (PBS-EDTA) and passaged with trypsin when the cells covered the dish about 80%, and the culture medium was exchanged every 48 hours.
<5-2> 세포 증식능<5-2> cell proliferation ability
세포증식능을 확인하기 위하여 CCD 986sk cell을 96 well plate에 10% FBS가 함유된 IMDM배지에 2.5x104 cells/well의 농도로 100ul씩 분주하고 24시간 동안 안정화시켰다. 새로운 배지에 시료를 최종 농도가 0.01, 0.1, 1 및 10 ug/ml이 되도록 배지에 녹여 세포주에 처리한 후 일정기간 동안 배양하였다. 세포주의 생존률을 측정하기 위해 MTS 시약을 이용하여 microplate reader(Molecular Devices, VersaMax ELISA Microplate Reader, USA)로 450 nm에서 흡광도를 측정하였다. 세포의 생존율은 시료를 처리하지 않은 대조군에 대비한 시료 처리군의 흡광도로 표시하였다.To confirm cell proliferation, 100 μl of CCD 986sk cells were injected at a concentration of 2.5x10 4 cells / well in IMDM medium containing 10% FBS in a 96 well plate and stabilized for 24 hours. Samples in fresh medium were dissolved in medium so that the final concentrations were 0.01, 0.1, 1 and 10 ug / ml, treated with cell lines, and then cultured for a period of time. Absorbance was measured at 450 nm with a microplate reader (Molecular Devices, VersaMax ELISA Microplate Reader, USA) using an MTS reagent to determine the viability of the cell line. Cell viability was expressed by the absorbance of the sample treated group compared to the control group not treated with the sample.
그 결과 피부 섬유 아세포인 CCD 986sk를 사용하여 펩타이드 YAK의 세포 재생능을 알아본 결과 하기 표 8과 같이 1일 배양시 농도에 따라 세포의 증식이 증가함을 알 수 있었고, 3일 배양시에는 모든 농도에서 70%에 가까운 세포 증식능을 확인할 수 있었다.As a result, the cell regeneration ability of peptide YAK was determined using the CCD 986sk, which is a skin fibroblast. Nearly 70% of cell proliferation was observed at the concentration.
표 8 세포 재생능 측정 결과
Peptides (ug/ml) Cell proliferating activity (%)
1 day
control 100.0±5.6
PGN 0.01 135.2±4.9*
0.1 124.2±4.0*
1 149.4±2.4*
10 159.6±5.9*
GPN 0.01 150.1±0.5*
0.1 142.5±12.6*
1 155.8±6.2*
10 160.7±8.4*
Table 8 Cell regeneration capacity measurement result
Peptides (ug / ml) Cell proliferating activity (%)
1 day
control 100.0 ± 5.6
PGN 0.01 135.2 ± 4.9 *
0.1 124.2 ± 4.0 *
One 149.4 ± 2.4 *
10 159.6 ± 5.9 *
GPN 0.01 150.1 ± 0.5 *
0.1 142.5 ± 12.6 *
One 155.8 ± 6.2 *
10 160.7 ± 8.4 *
*p<0.05: compared with control group * p <0.05: compared with control group
이제까지 본 발명에 대하여 그 바람직한 실시예들을 중심으로 살펴보았다. 본 발명이 속하는 기술 분야에서 통상의 지식을 가진 자는 본 발명이 본 발명의 본질적인 특성에서 벗어나지 않는 범위에서 변형된 형태로 구현될 수 있음을 이해할 수 있을 것이다. 그러므로 개시된 실시예들은 한정적인 관점이 아니라 설명적인 관점에서 고려되어야 한다. 본 발명의 범위는 전술한 설명이 아니라 특히 청구범위에 나타나 있으며, 그와 동등한 범위내에 있는 모든 차이점은 본 발명에 포함된 것으로 해석되어야 할 것이다.So far I looked at the center of the preferred embodiment for the present invention. Those skilled in the art will appreciate that the present invention can be implemented in a modified form without departing from the essential features of the present invention. Therefore, the disclosed embodiments should be considered in descriptive sense only and not for purposes of limitation. The scope of the present invention is shown not in the above description but in particular in the claims, and all differences within the scope will be construed as being included in the present invention.

Claims (9)

  1. GPN 및 PGN로 이루어진 군으로부터 선택된 1종의 아미노산 서열을 갖는 콜라게나제 활성 억제용 펩타이드.Peptide for inhibiting collagenase activity having one amino acid sequence selected from the group consisting of GPN and PGN.
  2. GPN 및 PGN로 이루어진 군으로부터 선택된 1종의 아미노산 서열을 갖는 펩타이드를 유효성분으로 포함하는 콜라게나제 매개 질환 예방 또는 치료용 약제학적 조성물.Pharmaceutical composition for the prevention or treatment of collagenase mediated disease comprising a peptide having one amino acid sequence selected from the group consisting of GPN and PGN as an active ingredient.
  3. 제2항에 있어서,The method of claim 2,
    상기 콜라게나제 매개 질환은 골다공증, 전이성 골수암, 각막 궤양, 치주질환, 염증성 관절 질환, 피부 염증성 질환, 창상 및 화상으로 이루어진 군으로부터 선택된 1종의 질환인 것을 특징으로 하는 콜라게나제 매개 질환 예방 또는 치료용 약제학적 조성물.The collagenase mediated disease is one or more diseases selected from the group consisting of osteoporosis, metastatic bone marrow cancer, corneal ulcer, periodontal disease, inflammatory joint disease, skin inflammatory disease, wound and burn or Therapeutic pharmaceutical composition.
  4. 제2항에 있어서,The method of claim 2,
    상기 펩타이드는 상기 조성물에 대해 0.1ug/ml~5000mg/ml의 농도로 포함되는 것을 특징으로 하는 콜라게나제 매개 질환 예방 또는 치료용 약제학적 조성물.The peptide is a pharmaceutical composition for the prevention or treatment of collagenase mediated disease, characterized in that it comprises a concentration of 0.1ug / ml ~ 5000mg / ml with respect to the composition.
  5. GPN 및 PGN로 이루어진 군으로부터 선택된 1종의 아미노산 서열을 갖는 펩타이드를 유효성분으로 포함하는 콜라게나제 매개 질환 예방 또는 개선용 건강기능식품.Health functional food for preventing or improving collagenase mediated disease comprising a peptide having one amino acid sequence selected from the group consisting of GPN and PGN as an active ingredient.
  6. 제5항에 있어서,The method of claim 5,
    상기 식품은 음료류, 육류, 초코렛, 식품류, 과자류, 피자, 라면, 기타 면류, 껌류, 사탕류, 아이스크림류, 알코올 음료류, 비타민 복합제 및 건강보조식품류로 이루어진 군으로부터 선택되는 것을 특징으로 하는 건강기능식품.The food is a health functional food, characterized in that selected from the group consisting of beverages, meat, chocolate, food, confectionery, pizza, ramen, other noodles, gum, candy, ice cream, alcoholic beverages, vitamin complexes and health supplements.
  7. GPN 및 PGN로 이루어진 군으로부터 선택된 1종의 아미노산 서열을 갖는 펩타이드를 유효성분으로 포함하는 주름 개선용 화장료 조성물.Wrinkle improvement cosmetic composition comprising a peptide having one amino acid sequence selected from the group consisting of GPN and PGN as an active ingredient.
  8. 제7항에 있어서,The method of claim 7, wherein
    상기 화장료 조성물을 용액, 현탁액, 유탁액, 페이스트, 겔, 크림, 로션, 파우더, 비누, 계면활성제-함유 클린싱, 오일, 분말 파운데이션, 유탁액 파운데이션, 왁스 파운데이션 및 스프레이로 이루어진 군으로부터 선택되는 어느 하나로 제형화되는 것을 특징으로 하는 주름 개선용 화장료 조성물.The cosmetic composition is any one selected from the group consisting of solutions, suspensions, emulsions, pastes, gels, creams, lotions, powders, soaps, surfactant-containing cleansing, oils, powder foundations, emulsion foundations, wax foundations and sprays. Wrinkle improvement cosmetic composition characterized in that it is formulated.
  9. GPN 및 PGN로 이루어진 군으로부터 선택된 1종의 아미노산 서열을 갖는 펩타이드를 유효성분으로 포함하는 피부재생용 기능성 화장료 조성물. Functional cosmetic composition for skin regeneration comprising a peptide having one amino acid sequence selected from the group consisting of GPN and PGN as an active ingredient.
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