KR20130113996A - Novel peptide with collagenase inhibitory activity and use thereof - Google Patents

Abstract

PURPOSE: A novel peptide which is derived from natural products and suppresses collagenase activity and a composition containing the same are provided to show stability without cytotoxicity and to enable long-term use. CONSTITUTION: A peptide for suppressing collagenase activity has one kind of amino acid sequence selected among sequence numbers 1-5. A pharmaceutical composition for preventing or treating collagenase-mediated diseases contains 0.1 ug/ml to 50 mg/ml of the peptide as an active ingredient. The collagenase-mediated diseases are selected among osteoporosis, metastatic bone marrow cancer, corneal ulcer, periodontal diseases, inflammatory joint diseases, skin inflammatory diseases, wound, and burn.

Description

Novel Peptide with collagenase inhibitory activity and use thereof

The present invention is a novel peptide having the effect of inhibiting the activity of collagenase, a pharmaceutical composition for the prevention or treatment of collagenase mediated disease comprising the same as an active ingredient, preventing or improving collagenase mediated disease comprising the same as an active ingredient Health functional foods and relates to a cosmetic composition for improving wrinkles comprising the same as an active ingredient.

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).

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.

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 Post-perfusion myocardial infarction is included.

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).

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.

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.

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.

Against this background, the present inventors have diligently tried to find a substance capable of inhibiting the activity of collagenase without side effects. As a result, the peptide was isolated from the hydrolyzate of mechanical deboned chicken meat (MDCM) and analyzed. Synthetic peptide was prepared based on the amino acid sequence, and thus, the present invention was completed by confirming that the synthetic peptide thus prepared can effectively inhibit collagenase activity without cytotoxicity.

It is therefore an object of the present invention to provide novel peptides that can effectively inhibit collagenase activity.

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.

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 SEQ ID NO: 1 to SEQ ID NO: 5.

The present invention also provides a pharmaceutical composition for preventing or treating collagenase mediated disease comprising the peptide as an active ingredient.

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 .

In one embodiment of the present invention, the peptide may be included in a concentration of 0.1μg / ml ~ 50mg / 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.

In one embodiment of the present invention, the food is selected from the group consisting of beverage, meat, chocolate, foods, confectionery, pizza, ram noodles, gums, candy, ice cream, alcoholic beverages, .

In addition, the present invention provides a cosmetic composition for improving wrinkles comprising the peptide as an active ingredient.

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.

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.

Figure 1 shows the Q-Sepharose chromatogram of mechanically deboned chicken (MDCM) hydrolysate.
Figure 2 shows a size exclusion chromatogram of Q-Sepharose fraction 2.
Figure 3 shows a size exclusion chromatogram of Q-Sepharose fraction 8.
Figure 4 shows reverse phase HPLC chromatograms of 2-20 fractions (DPPH active fractions) obtained from size exclusion chromatograms.
FIG. 5 shows reverse phase chromatograms of 2-29 fractions showing ACE inhibitory activity obtained in size exclusion chromatograms.
FIG. 6 shows reverse phase chromatograms of 2-36 fractions showing ACE inhibitory activity obtained in size exclusion chromatograms.
Figure 7 shows the mass spectrum of peptides exhibiting DPPH radical scavenging ability determined from MDC hydrolyzate.
Figure 8 shows the mass spectrum of the peptide showing the ability to inhibit ACE determined from the mechanically deboned chicken (MDCM) hydrolysate.
Figure 9 is a graph measuring the cytotoxicity after treatment of hepatocytes by concentration of novel peptides (YAK, LTK, LY, TYP, FQNPD) of the present invention.
10 is a graph showing the degree of inhibition of collagenase activity according to the concentration of the novel peptide (YAK, LTK, LY, TYP, FQNPD) of the present invention.
Figure 11 is a graph measuring the residual activity of collagenase after the treatment of novel peptides (YAK, LTK, LY, TYP, FQNPD) of the present invention.

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 SEQ ID NO: 1 to SEQ ID NO: 5.

"Collagenase" is a protease stored latent in neutrophil-specific granules, a type of matrix metalloproteinase (MMP). The active form of collagenase mediates various diseases and disorders in mammals. Known.

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.

In the present invention, mechanical deboned chicken meat (MDCM: mechanically deboned chicken meat (also called lung meat or chicken meat recovery protein)), which recovers meat attached to bone after bone from chicken, is purified using proteolytic enzymes. The hydrolyzate obtained by the hydrolysis was purified, and the functional peptide was purified through several steps of chromatography, and the synthetic peptides (SEQ ID NO: 1 to SEQ ID NO: 5) prepared based on their amino acid sequences were subjected to collagenase activity. Was first identified to effectively inhibit (see FIG. 10). 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. 9).

The novel peptide of the present invention can be prepared by directly isolating from the milled bone meats (MDCM). Briefly, mechanically deboned chicken meat (MDCM) is hydrolyzed using Protamex and bromelain, followed by ultrafiltration, anion exchange resin chromatography, size exclusion chromatography, and reverse phase chromatography of ACE inhibitory functional peptides. It can be separated directly by the method of purification (see Examples below for details).

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).

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. Genetic engineering methods for peptide synthesis of the present invention can be found in the following references: 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.

In another embodiment, the present invention is a pharmaceutical for preventing or treating collagenase mediated disease comprising the novel peptide (peptide having one amino acid sequence selected from the group consisting of SEQ ID NO: 1 to SEQ ID NO: 5) as an active ingredient It relates to a composition.

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 can be prepared by using pharmaceutically acceptable and physiologically acceptable adjuvants in addition to the above-mentioned active ingredients. Examples of the adjuvants include excipients, disintegrants, sweeteners, binders, coating agents, swelling agents, lubricants, A lubricant or a flavoring agent can be used.

The pharmaceutical composition may be formulated into a pharmaceutical composition containing at least one pharmaceutically acceptable carrier in addition to the above-described active ingredients for administration.

The pharmaceutical composition may be in the form of granules, powders, tablets, coated tablets, capsules, suppositories, liquids, syrups, juices, suspensions, emulsions, drops or injectable solutions. For example, for formulation into tablets or capsules, the active ingredient may be combined with an oral, non-toxic pharmaceutically acceptable inert carrier such as ethanol, glycerol, water, and the like. Also, if desired or necessary, suitable binders, lubricants, disintegrants and coloring agents may also be included as a 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 for compositions that are formulated into a liquid solution include sterile water and sterile water suitable for the living body such as saline, sterile water, Ringer's solution, buffered saline, albumin injection solution, dextrose solution, maltodextrin solution, glycerol, One or more of these components may be mixed and used. If necessary, other conventional additives such as an antioxidant, a buffer, and a bacteriostatic agent may be added. In addition, diluents, dispersants, surfactants, binders, and lubricants may be additionally added to formulate into injectable solutions, pills, capsules, granules or tablets such as aqueous solutions, suspensions, emulsions and the like. Further, it can be suitably formulated according to each disease or ingredient, using the method disclosed in Remington's Pharmaceutical Science, Mack Publishing Company, Easton PA as an appropriate method in the field.

In one embodiment of the present invention, the novel peptide of the present invention may be included in a concentration of 0.1μg / ml ~ 50mg / ml with respect to the composition.

In another embodiment, the present invention is a food composition for preventing or ameliorating collagenase mediated disease comprising the novel peptide (peptide having one amino acid sequence selected from the group consisting of SEQ ID NO: 1 to SEQ ID NO: 5) as an active ingredient. Can be.

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.

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 above-described flavors can be advantageously used as natural flavorings (tau martin), stevia extracts (e.g., rebaudioside A, glycyrrhizin, etc.) and synthetic flavors (saccharin, aspartame, etc.).

The food composition of the present invention can be formulated in the same manner as the above pharmaceutical composition and used as a functional food or added to various foods. Foods to which the composition of the present invention can be added include, for example, beverages, meat, chocolates, foods, confectionery, pizza, ram noodles, other noodles, gums, candy, ice cream, alcoholic beverages, vitamin complexes, .

In addition, the food composition is a novel peptide (peptide having one amino acid sequence selected from the group consisting of SEQ ID NO: 1 to SEQ ID NO: 5) as an active ingredient, various nutrients, vitamins, minerals (electrolytes), synthetic flavors and natural flavors Flavoring agents, coloring and neutralizing agents (such as cheese, chocolate), pectic acid and salts thereof, alginic acid and salts thereof, organic acids, protective colloidal thickeners, pH adjusting agents, stabilizers, preservatives, glycerin, alcohols, carbonated drinks The carbonation agent used etc. can be contained. In addition, the food composition of the present invention may contain natural fruit juice and pulp for the production of fruit juice drinks and vegetable drinks.

The novel peptide (peptide having one amino acid sequence selected from the group consisting of SEQ ID NO: 1 to SEQ ID NO: 5), which is an active ingredient of the present invention, is based on the amino acid sequence of a peptide derived from natural products, and has stability without cytotoxicity. Because of this, it can be used safely even for long-term use for the purpose of preventing or ameliorating collagenase mediated diseases.

In another embodiment, the present invention provides a health function for the prevention or improvement of collagenase mediated disease comprising a novel peptide (peptide having one amino acid sequence selected from the group consisting of SEQ ID NO: 1 to SEQ ID NO: 5) as an active ingredient Provide food.

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.

In the present invention, "health functional food" refers to a food prepared and processed using raw materials or ingredients having functional properties useful for the human body according to the Health Functional Food Act No. 6767, and nutrients for the structure and function of the human body. It is meant to be consumed for the purpose of regulating or obtaining a useful effect for health use such as physiological action.

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 listed in the above-mentioned "food additives" include chemical compounds such as ketones, glycine, calcium citrate, nicotinic acid, and cinnamic acid; Natural additives such as persimmon extract, licorice extract, crystalline cellulose, high color pigment and guar gum; L-glutamic acid sodium preparations, noodle-added alkalis, preservative preparations, tar coloring preparations and the like.

For example, a dietary supplement may contain a novel peptide (peptide having one amino acid sequence selected from the group consisting of SEQ ID NO: 1 to SEQ ID NO: 5), which is an active ingredient of the present invention, as an excipient, a binder, a disintegrant, and other The mixture mixed with the additive 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 and the like as necessary.

Hard capsules among the health functional foods in the form of capsules are additives, such as excipients, with a novel peptide (peptide having one amino acid sequence selected from the group consisting of SEQ ID NO: 1 to SEQ ID NO: 5), which is an active ingredient of the present invention, in a conventional hard capsule. It can be prepared by filling a mixture mixed with, soft capsules can be prepared by filling the capsule base such as gelatin mixture mixture of the baekwool extract with additives such as excipients. The soft capsule may contain a plasticizer such as glycerin or sorbitol, a coloring agent, a preservative and the like, if necessary.

The health functional food in a cyclic form is a mixture of a novel peptide (peptide having one amino acid sequence selected from the group consisting of SEQ ID NO: 1 to SEQ ID NO: 5), an excipient, a binder, a disintegrant, etc., which is an active ingredient of the present invention. It may be molded and prepared by a conventionally known method, 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 SEQ ID NO: 1 to SEQ ID NO: 5), an excipient, a binder, a disintegrant, etc. It can be prepared in granules by a conventionally known method, and may contain a flavoring agent, a copper, and the like as necessary.

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 a beverage, a meat, a chocolate, a food, a confectionery, a pizza, a ramen, a noodle, a gum, a candy, an ice cream, an alcoholic beverage, a vitamin complex and a health supplement food.

In another aspect, the present invention provides a novel peptide (peptide having one amino acid sequence selected from the group consisting of SEQ ID NO: 1 to SEQ ID NO: 5) for the manufacture of a medicament or food for the prevention or treatment of collagenase mediated disease Provided is the use of a composition comprising as a component. 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.

In another embodiment, 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 SEQ ID NO: 1 to SEQ ID NO: 5). To provide.

The term "mammal " as used herein refers to a mammal that is the subject of treatment, observation or experimentation, preferably a human.

The term "therapeutically effective amount " as used herein refers to the amount of active ingredient or pharmaceutical composition that induces a biological or medical response in a tissue system, animal or human, as contemplated by a researcher, veterinarian, physician or other clinician, The amount that induces the relief of the symptoms of the disease or disorder being treated. It will be apparent to those skilled in the art that the therapeutically effective dose and the number of administrations of the active ingredient of the present invention will vary depending on the desired effect. Thus, the optimal dosage to be administered can be readily determined by those skilled in the art and will vary with the nature of the disease, the severity of the disease, the amount of active and other ingredients contained in the composition, the type of formulation, and the age, The age, body weight, sex, diet, time of administration, route of administration and fraction of the composition, duration of treatment, concurrent medication, and the like. In the treatment method of the present invention, in the case of an adult, when a new peptide (peptide having one amino acid sequence selected from the group consisting of SEQ ID NO: 1 to SEQ ID NO: 5) of the present invention is administered once to several times a day, 1 It is preferable to administer at a dose of mg / kg to 250 mg / kg.

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.

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 SEQ ID NO: 1 to SEQ ID NO: 5) as an active ingredient.

When the composition of the present invention is made of a cosmetic composition, the composition of the present invention is not only a novel peptide of the present invention (peptide having one amino acid sequence selected from the group consisting of SEQ ID NO: 1 to SEQ ID NO: 5), but also a cosmetic It may include components conventionally used in the composition, such as conventional adjuvants such as antioxidants, stabilizers, solubilizers, vitamins, pigments and flavors, and carriers.

Examples of products to which the cosmetic composition of the present invention can be added include cosmetics such as astringent lotion, softening longevity lotion, nutrition lotion, various creams, essences, packs, foundation and the like, cleansing, cleanser, soap, .

As a specific formulation of the cosmetic composition of the present invention, there may be mentioned a skin lotion, a skin softener, a skin toner, an astringent, a lotion, a milk lotion, a moisturizing lotion, a nutrition lotion, a massage cream, It includes formulations such as soap, shampoo, cleansing foam, cleansing lotion, cleansing cream, body lotion, body cleanser, latex, lipstick, makeup base, foundation, press powder, loose powder, eye shadow.

On the other hand, 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 SEQ ID NO: 1 to SEQ ID NO: 5) inside the nano-liposomes. 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.

Advantages and features of the present invention and methods of achieving them will become apparent with reference to the embodiments described in detail below. Hereinafter, the present invention will be described in detail with reference to examples. However, these examples are for illustrating the present invention specifically, and the scope of the present invention is not limited to these examples.

≪ Example 1 >

Preparation of Two-stage Hydrolysates from Mechanical Bony Chicken (MDCM)

Mechanical deboned chicken meat (MDCM), also called lung poultry or poultry recovery protein, was purchased as a packaging unit of 25 kg freezing block at the waste poultry processing plant in Jinju, Gyeongsangnam-do, and used as a raw material for the recovery of poultry protein. . Freeze MDCM is thawed while supplying tap water at room temperature. At the same time, it removes blood and some suspended substances, and removes tap water sufficiently from a tray coated with a single layer of gauze and freezes it at -20 ℃ for protein recovery. Used as a sample. Two-stage hydrolase, Protamex 1.5 MG and bromelain (1200 GDU), were purchased from Biosis (Pusan, Korea). For both acetonitrile and distilled water for separation purification, HPLCrmq (Fisher Scientific Co., U.S.A.) was used.

In order to prepare two-stage hydrolyzate from MDCM, 10 times of distilled water was added to thawed machine bone, and homogenized with custom-made industrial homogenase (Myung Sung Electric Co., Dajeon, Korea). While stored in a low temperature room at 4-7 ℃ to remove the fat floating in the upper layer with a double layer of gauze until the fat layer is not visible. Protamex 1.5 MG was added to the homogenate to a final concentration of 5%, hydrolyzed at 43 ° C. for 1 hour, and then adjusted to pH 7.0 with 6 N Hcl solution. Bromelain 1200GDU was added to the pH-controlled primary hydrolyzate to a final concentration of 1% and hydrolyzed at 50 ° C. for 1 hour and deactivated at 85 ° C. for 15 minutes. The supernatant was obtained by removing the floating upper fat while cooling in a low temperature room and centrifuging (Supra 22K plus, Hanil Scientific Co., Dajeon, Korea) for 20 minutes at 5,000xg. Ethanol was added to the secondary hydrolyzate recovered in the supernatant so as to have a ratio of 1: 1 to precipitate insoluble protein, filtered in a filter panel (Toyo No. 2), and evaporated ethanol with a rotary vacuum evaporator at 40 ° C. or lower, The concentrate was lyophilized and used as a sample for antioxidant and ACE inhibitory peptide purification.

< Example  2>

Purification of Functional Peptides

The functional peptide having antioxidant and ACE inhibitory activity in the hydrolyzate obtained through Example 1 was purified by ultrafiltration, anion exchange resin chromatography, size exclusion chromatography, and reverse phase chromatography.

<2-1> DPPH Radical Scatters  Measure

DPPH radical scavenging activity was measured according to the method of Park et al. (2005). That is, 500 μL of the sample solution was added to the test tube, 500 μL of ethanol and 250 μL of 0.5 mM 1,1-diphenyl-2-picrylhydrazyl (DPPH) / ethanol solution were added thereto, and left at room temperature of 25 ° C. for 30 minutes. Absorbance was measured at 517 nm. The control group used 500 μL of deionized water instead of the sample, and blank test used 750 μL of ethanol instead of DPPH and distilled water. Antiradical activity was calculated as% of DPPH bleaching according to the following equation.

<2-2> ACE Low performance  Measure

Angiotensin I-converting enzyme (ACE) inhibitory activity was measured by a slightly modified method of Wu et al. (2002). That is, 5 mM N-benzoyl-Gly-His-Leu (HHL, Sigma H1635) and 0.25 mUnit of ACE were prepared using 0.1 M borate buffer (pH 8.3) containing 0.3 M NaCl as a solvent. 40 uL of sample and 150 uL of ACE were added to the Eppendorf tube and reacted for 30 minutes in a 37 ° C. constant temperature water bath. After completion of the reaction, 150 uL of 1 M HCl was added to stop the enzymatic reaction, followed by centrifugation (5415C, Eppendorf, Hamburg, Germany) for 10 minutes at 10,000 rpm to obtain a supernatant. The content of free hippuric acid was measured by injecting 20 uL into HPLC (Hitachi, Tokyo, Japan) equipped with a reversed phase column (Watchers 120 ODS-AP, 4.6x250 mm, 5 um, Daiso, Japan). Distilled water containing 0.1% TFA was used as the solvent A for separation, and 100% acetonitrile containing 0.1% TFA was used as the solvent B. Concentration gradients were detected by absorbance at 228 nm with a linear gradient under the condition of 5-60% B solvent / 20 min. For the control solution, 20 uL of solvent A was used instead of the sample, and the flow rate was eluted under the condition of 1 mL / min. Enzyme inhibitory activity was calculated by the following equation, and the inhibitory activity of each concentration was measured by linear regression. And the concentration of the sample required to inhibit 50% of the ACE inhibitory activity was defined as IC50.

ACE inhibitory ability (%) = [(HA of HA-sample of control) / HA of control] * 100

<2-3> DPPH Radical Scatters  And ACE  Inhibition Of peptide  refine

The column chromatography conditions for purification of peptides having DPPH radical scavenging activity and ACE inhibitory activity are shown in Table 1 below. That is, 0.5 g of the lyophilized sample prepared in Example 1 to purify DPPH radical scavenging activity and ACE inhibitory peptide was dissolved in 12 mL of 20 mM Tris-Cl (pH 8.0), and a HiLoad 16/10 Q-Sepharose column (16x100). mm) was injected into 5 mL. 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 254 nm and fractionated 2 mL.

As a result, as shown in Figure 1, two peaks (Fr 1 and Fr2) in the non-adsorption region and six separation peaks were obtained in the eluent culture (FR 3-8), Fr 2 44.3% DPPH activity scavenging ability is In contrast, Fr 6, 7, 8 showed 0.4%, 6.1%, and 24.2% of ACE inhibitory activity, respectively. These results indicate that there is a difference in the electrical properties of peptides showing DPPH radical scavenging activity and ACE inhibitory activity.

In addition, fractions 2 and 8, which showed the greatest DPPH scavenging activity and ACE inhibitory activity, were collected in an anion exchange column and concentrated in a Micro-Centrivac (NB-503CIR, N-Biot Inc., Seoul, Korea), followed by a Superdex peptide column (10x300 mm). Molecular weight size exclusion chromatography was performed by injection into 200 uL.

As a result, Q-Sepharose fraction 2 showing DPPH radical scavenging activity was separated into a total of six peaks (2-20, 2-21, 2-23, 2-26, 2-29, 2-36) on size exclusion chromatography. (See FIG. 2), Q-Sepharose Fraction 8 showing ACE inhibition showed a total of six peaks on size exclusion chromatography (8-19, 8-22, 8-23, 8-26, 8-29, 36-6). (See FIG. 3). In size exclusion chromatography, DPPH radical scavenging activity showed 2.57% activity only at peaks of 2-20, whereas ACE inhibition showed 2-20, 2-29, 2-36, 8-19, 8-22, 8-23, The 8-24 peak showed 5.1%, 12.7%, 15.5%, 11.1%, 11.2%, 11.4%, and 13.0%, respectively.

In addition, seven peaks showing DPPH radical scavenging activity and ACE inhibition in size exclusion chromatography were eluted with 60% acetonitrile solvent containing 0.09% TFA in a Source 5 RPC ST column (4.6x150 mm), respectively, at 214 nm and 254 nm. Peptides isolated at were detected and the bioactivity of each peak was measured.

As a result, the reverse phase HPLC chromatogram phase of 2-20 obtained from the size exclusion chromatogram was as shown in FIG. 4, and the DPPH radical scavenging activity and the AC inhibitory activity of the main peak were 2-20-3 fractions and 2-20-21 fractions, respectively. DPPH radical scavenging ability of 4.85% and 3.95% was shown, but fraction of 2-20-3 was eluted in 2.95 mL after injection, which could be contaminated, resulting in amino acid sequence analysis of DP-20 radical scavenging ability. Twenty-one fractions were taken (see Table 2).

In contrast, reverse phase chromatography (29 and 6, respectively) of 2-29 and 2-36 fractions showing relatively high ACE inhibition of 12.7% and 15.5% in size exclusion chromatography confirmed the ACE inhibition of the main peak. Fractions 17 and 19 of 2-29 and 17 and 19 of 2-36 showed 2%, 4%, 1% and 0.7% of ACE inhibition, respectively. This very weak ACE inhibitory activity is presumed to be due to the relatively low concentration of peptide during the fractionation step due to the fact that the protein concentration is not detected by the Lowry method.

Conditions of Column Chromatography for Purification of Biofunctional Peptides Chromatography Column Solvent Flow rate Detection Anion-exchange HiLoad Q-Sepharose
(16x100 mm) A: 20 mM Tris-Cl, pH 7.5
B: 0.75 M NaCl-20 mM Tris-Cl, pH 7.5 0.5 mL / min 254 nm Size exclusion Superdex peptide
(10x300 mm) A: 20 mM Tris-Cl, pH 7.5 1 mL / min 216 nm, 254 nm Reversed phase Source 5RPC ST
(4.6 x 150 mm) A: 0.1% TFA / water
B: 0.09% TFA / 60% ACN 1 mL / min 216 nm
254 nm

DPPH radical scavenging activity and ACE inhibition (%) after reverse phase chromatography Fraction DPPH radical scavenging ability ACE inhibitory ability 2-20-3 4.85 - 2-20-21 ~ 22 3.95 - 2-1-55 - 1.7 2-1-67 - 5.2 2-1-68 - 2.5 2-1-70 - 2.6

< Example  3>

refine Of peptide  Molecular Weight Distribution and Amino Acid Sequence Analysis

Fractions obtained by reversed phase chromatography of Example 2 were completely dried in Micro-Centvac to determine DPPH radical scavenging activity and the amide acid sequence of the ACE inhibitory 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 a cartridge and the amino acid sequence was determined by the pulsed-liquid method of an amino acid automatic sequencer (ABI492 automated protein sequencer, Applied Biosystem, Foster, Calif., U.S.A.). The molecular weight and the decomposed ions were detected by mass spectrometry using electrospray ionization (ESI) to confirm the purified peptide molecular weight. Mass spectra were obtained by Q-TOF2 (Micromass, U.K.) mass spectrometry using a data-dependent MS / MS method using the Nano-ESI interface.

As a result, as shown in Figure 7, the peptide showing the purified DPPH radical scavenging ability was not able to observe the main peak distinguished from the CHCA matrix up to 500 Da on MALDI-TOF. These results suggest that the molecular weight of the peptide purified for amino acid sequence analysis is equivalent to 500 Da or more. However, the molecular weight of the purified peptide was estimated to be 700 Da or more based on the fact that a few minor peaks distinguished from the CHCA matrix were observed above 700 Da, and the amino acid sequences showing DPPH activity are shown in Table 3 below. However, it was impossible to estimate the exact amino acid sequence due to the large number of duplicated or unknown sequences during amino acid sequence analysis. This result suggests that several more purification steps are required to confirm the exact amino acid sequence of DPPH radical scavenging ability.

On the other hand, as shown in Figure 8, all of the purified ACE inhibitory peptides identified a major peak that differs from the CHCA matrix at a molecular weight of less than 500 Da, and confirmed the amino acid binding sequence of a total of five peptides (see Table 3 below) ). However, leucine in the amino acid sequence could not be confirmed by the mass spectrometer used in this study as an isomer having the same molecular weight as isoleucine. Thus, all synthetic peptides were synthesized with leucine residues. Among the identified amino acid sequences, LY was purified and purified from thermolysin hydrolysates of calf gelatin by Herregods et al. And 1.908 uM.

Amino Acid Sequences of Peptides Purified from MDCM Hydrolysates DPPH scavenging peptide ACE inhibition peptide Peptide EC50 Peptide IC50, uM K (G) L (G) G (S, L) XX - Yak 0.197 GGXXX - LTK 0.171 KG (L, Q) G (Q) G (E) X - LY 2.344 XG (L, P) XR (Q) Y - TYP 22.585 XQPQX - FQNPD 1.908 XQPQV -

< Example  4>

Functionality of the Invention Peptides  Synthesized and synthesized Of peptide  refine

Through the experiments performed above, peptide sequences having DPPH scavenging activity and ACE inhibitory activity were identified, and peptides were synthesized to confirm another function of these peptides.

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).

As a result, the synthesized ACE inhibitory peptide showed a single peak in HPLC, and retention times of YAK, LTK, LY, TYP and FQNPD were 2.300, 2.208, 9.875, 4.617 and 6.025 minutes, respectively. On the other hand, the synthetic peptide showed a molecular weight of a single peak on a MALDI-TOF mass spectrometer. In the present invention, five synthetic peptides having the functionalities as described above are represented by the following SEQ ID NOs: 1 to 5 (see Table 4 below).

Functional Peptides of the Invention Peptides SEQ ID NO: Yak One LTK 2 LY 3 TYP 4 FQNPD 5

< Experimental Example  1>

Functionality of the Invention Of peptide  Cytotoxicity measurement

In this experiment, the cytotoxicity of the functional peptide synthesized in Example 4 was evaluated (see Table 4).

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 μg / 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.

As a result, as shown in Figure 9, 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.

< Experimental Example  2>

Functionality of the Invention Of peptide Collagenase  activation Inferior  Measure

In this experiment, the collagenase activity inhibitory ability of the functional peptide synthesized in Example 4 was measured.

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.

As a result, as shown in FIG. 10, TYP and FQNPD of the synthetic peptides showed a concentration-dependent inhibitory activity against collagenase of 33.7% and 53.6% at a concentration of 1 mg / mL, respectively. In addition, as a substrate of collagenase using bovine achilles tendon to measure the inhibitory activity at the ratio of enzyme to synthetic peptide 8.3 and 12.5, as shown in Figure 11, LY and TYP at the enzyme to inhibitor ratio 8.3 is collagenase FQNPD showed no inhibitory effect, while 69.1% and 70.3% of the activity was inhibited. On the other hand, LY and TYP completely inhibited collagenase activity and FQNPD inhibited 60.9% at an enzyme to inhibitor ratio of 12.5.

< Experimental Example  3>

Peptides Yak Wrinkle improvement and skin regeneration effect

Experiments were carried out to determine the wrinkle improvement and skin regeneration efficacy of YAK among the synthesized peptides obtained in Example 4.

<3-1> wrinkle improvement effect

i) CCD  986 sk cell  Culture and Sampling

CCD 986sk cells, fibroblasts cultured in cell culture flasks, were detached from the flasks with a 10 mL pipette and passaged, and the cells were cultured in IMDM medium containing 10% FBS. CCD 986sk cells were dispensed into 96 well plates at a concentration of 2.5 × 10 ⁴ cells / well and incubated in a CO ₂ incubator controlled at 35 ° C., 95% humidity, 5% CO ₂ for 24 hours. In a fresh medium, the final concentration of the sample (YAK peptide) was dissolved at 1, 10 μg / mL, and the cells were treated. After 24 hours of incubation, the supernatant was collected and used as a sample.

ii ) MMP Measurement of -1 Inhibitory Activity

MMP-1 was measured by purchasing a SensoLyte 490 MMP-1 Assay Kit (AnaSpec, MI, USA). After adding 50 uL and stirring for 30 seconds, the fluorescence value was measured at 10 minutes intervals at 340 nm absorption wavelength and 490 nm emission wavelength for 60 minutes using a microplate reader (Molecular Devices, VersaMax ELISA Microplate Reader, USA). Measured.

S: absorbance of sample, B: absorbance of blank test group, C: absorbance of control group

iii ) Procollagen  Production measurement

Procollagen was measured using a Type 1 C-peptide (PIP) EIA Kit. A sample diluent 40 uL was added to 10 uL of the culture supernatant of the CCD 986sk, which was treated and cultured. Add 100 uL of Antibody-POD Conjugate to the coated microplate, add 20 uL of the diluted sample solution, cover with a foil, and incubate at 37 ° C for 3 hours. After incubation, remove all the liquid, wash 4 times by adding 400 uL PBS each time to completely remove the water, and put 100 uL Substrate solution (TMBZ) and leave at room temperature for 15 minutes. 100 uL of Stop solution (1 NH ₂ SO ₄ ) was added to stop the reaction, and the yield was calculated by measuring absorbance at 450 nm with a microplate reader (Molecular Devices, VersaMax ELISA Microplate Reader, USA).

<3-2> Skin fibroblasts (CCD 986sk)  through Skin cell Regenerative  Measure

i) culture of cells

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.

ii ) cell Proliferative ability  analysis

To confirm cell proliferation, CCD 986sk cells were dispensed in 100 μl at a concentration of 2.5 × 10 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.

Table 5 shows the results of measuring the activity inhibition of MMP-1 according to the concentration of the synthetic peptide YAK.

MMP-1 activity inhibition Peptides ( ug Of mL ) MMP -One inhibition activity (%) Yak One 39.4 ± 3.5 * 10 42.2 ± 1.7 *

* p <0.05: compared with control group

YAK, the synthetic peptide of the present invention, inhibited the activity of MMP-1 by 39.4% at a concentration of 1ug / ml, and showed an inhibitory effect of 42.2% at a concentration of 10ug / ml, thereby showing the possibility of using an antiwrinkle cosmetic raw material.

Procollagen Production Peptide
(ug / ml) Procollagen  formation ( ng Of ml )  (%) Control 12.67 ± 0.72 100.0 ± 5.7 YAK 1 13.20 ± 1.70 104.2 ± 12.9 YAK 10 13.95 ± 0.54 110.1 ± 3.8 *

^* p <0.05: compared with control group

In addition, Table 6 is a result of measuring the collagen precursor procollagen production capacity, even in the production capacity of collagen precursor procollagen YAK is 13.95 ± 0.54 ng / ml at a concentration of 10 ug / ml compared to the control group It showed an increase in production of about 10.1%, indicating a significant increase in procollagen production.

In addition, Table 7 shows the results of measuring the cellular regeneration of the peptide YAK using a CCD 986sk cell, a skin fibroblast.

Cell regeneration Peptide
(占 퐂 / ml) Cell regeneration activity (%) 1 day 3 day Control 100.0 ± 5.6 100.0 ± 7.7 YAK 0.01 99.6 ± 7.8  173.9 ± 6.6 * YAK 0.1 126.4 ± 10.7 *  168. ± 10.8 * YAK 1 133.4 ± 10.3 *  169.4 ± 11.3 * YAK 10 118.4 ± 16.2  181.6 ± 26.9 *

^* p <0.05: compared with control group

The cell renewal ability of peptide YAK was determined using CCD 986sk, a skin fibroblast. It was found that the proliferation of the cells increased with the concentration in the daily culture, and the cell proliferation ability was nearly 70% at all concentrations in the three-day culture.

Based on the results of Experiment 3, the YAK peptide of the present invention showed the ability to inhibit MMP-1 activity, procollagen production, and proliferation of dermal fibroblasts, thereby confirming the availability of cosmetic raw materials having ultimately wrinkle improvement and skin regeneration efficacy. .

For reference, matrix metalloproteinase (MMP) inhibitors can significantly reduce the growth rate of primary and secondary cancers and prevent metastasis in breast cancer model tests. Several synthetic MMP inhibitors are currently being developed to Clinical trials have been conducted in patients with preclinical and advanced malignancies (Denis and Verweij, 1997; Brown, 1998). Several hydroxamate derivatives have been demonstrated to be effective collagenase / gelatinase inhibitors depending on the type of ring in sulfonamide and are 100-500 times more active than the corresponding carboxylate (Scozzafava and Supuran, 2000; Scozzafava et al., 2000). Most of the synthetic MMP inhibitors are substituted peptide derivatives, in which thiol, sulphydryl, carboxyl or hydroxamate groups, which are metal bonds, bind to the zinc element at the active site of MMP (Denis and Verweij, 1997). In view of these reports, the collagenase inhibitory effect of the functional peptides LY, TYP, and FQNPD of the present invention is presumed to be due to the binding of collagenase to metal ions, and the activity of the hydroxamate derivatives of these synthetic peptides is further developed. It was supposed to be.

So far I looked at the center of the preferred embodiment for the present invention. It will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the spirit and scope of the invention as defined by the appended claims. Therefore, the disclosed embodiments should be considered in an illustrative rather than a restrictive sense. The scope of the present invention is defined by the appended claims rather than by the foregoing description, and all differences within the scope of equivalents thereof should be construed as being included in the present invention.

<110> Gyeongnam National University Of Science And Technology Industry-Academic Cooperation Foundation <120> Novel Peptide with collagenase inhibitory activity and use          the <130> PN1204-141 <160> 5 <170> Kopatentin 2.0 <210> 1 <211> 3 <212> PRT <213> Artificial Sequence <220> <223> Novel peptide 1 sequence inhibiting collagenase activity <400> 1 Tyr ala lys   One <210> 2 <211> 3 <212> PRT <213> Artificial Sequence <220> <223> Novel peptide 2 sequence inhibiting collagenase activity <400> 2 Leu Thr Lys   One <210> 3 <211> 2 <212> PRT <213> Artificial Sequence <220> <223> Novel peptide 3 sequence inhibiting collagenase activity <400> 3 Leu Tyr   One <210> 4 <211> 3 <212> PRT <213> Artificial Sequence <220> <223> Novel peptide 4 sequence inhibiting collagenase activity <400> 4 Thr Tyr Pro   One <210> 5 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> Novel Peptide 5 Sequence Inhibiting Collagenase Activity <400> 5 Phe Gln Asn Pro Asp   1 5

Claims (8)

Peptide for inhibiting collagenase activity having one amino acid sequence selected from the group consisting of SEQ ID NO: 1 to SEQ ID NO: 5. A pharmaceutical composition for preventing or treating collagenase mediated diseases comprising the peptide of claim 1 as an active ingredient. 3. The method of claim 2,
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. 3. The method of claim 2,
The peptide is a pharmaceutical composition for the prevention or treatment of collagenase mediated disease, characterized in that it comprises a concentration of 0.1μg / ml ~ 50mg / ml with respect to the composition. Health functional food for preventing or improving collagenase mediated disease comprising the peptide of claim 1 as an active ingredient. The method of claim 5,
Wherein said food is selected from the group consisting of beverage, meat, chocolates, foods, confectionery, pizza, ramen, other noodles, gums, candy, ice cream, alcoholic beverages, vitamin complexes and health supplement foods. Wrinkle improvement cosmetic composition comprising the peptide of claim 1 as an active ingredient. The method of claim 7, wherein
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.

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