WO2018147517A1 - Composition for preventing or treating bone diseases comprising cockle protein-derived peptide as active ingredient - Google Patents
Composition for preventing or treating bone diseases comprising cockle protein-derived peptide as active ingredient Download PDFInfo
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- WO2018147517A1 WO2018147517A1 PCT/KR2017/008288 KR2017008288W WO2018147517A1 WO 2018147517 A1 WO2018147517 A1 WO 2018147517A1 KR 2017008288 W KR2017008288 W KR 2017008288W WO 2018147517 A1 WO2018147517 A1 WO 2018147517A1
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- peptide
- bone
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Images
Classifications
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
- C07K7/04—Linear peptides containing only normal peptide links
- C07K7/06—Linear peptides containing only normal peptide links having 5 to 11 amino acids
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- 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
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- 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
- A61K38/07—Tetrapeptides
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- 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
- A61K38/08—Peptides having 5 to 11 amino acids
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P19/00—Drugs for skeletal disorders
- A61P19/02—Drugs for skeletal disorders for joint disorders, e.g. arthritis, arthrosis
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P19/00—Drugs for skeletal disorders
- A61P19/08—Drugs for skeletal disorders for bone diseases, e.g. rachitism, Paget's disease
-
- 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
- C07K5/10—Tetrapeptides
-
- 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
- C07K5/10—Tetrapeptides
- C07K5/1002—Tetrapeptides with the first amino acid being neutral
- C07K5/1016—Tetrapeptides with the first amino acid being neutral and aromatic or cycloaliphatic
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- 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
-
- 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
- A23V2200/00—Function of food ingredients
- A23V2200/30—Foods, ingredients or supplements having a functional effect on health
- A23V2200/306—Foods, ingredients or supplements having a functional effect on health having an effect on bone mass, e.g. osteoporosis prevention
-
- 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
- A23V2250/00—Food ingredients
- A23V2250/54—Proteins
- A23V2250/55—Peptide, protein hydrolysate
Definitions
- the present invention relates to a composition for the prophylaxis or treatment of bone diseases comprising the peptide protein-derived peptide as an active ingredient.
- Osteoporosis is postmenopausal osteoporosis, which is indicated by increased bone resorption due to the activation of osteoclasts due to rapid hormonal changes following menopause, and senile osteoporosis, in which osteoblasts decrease due to aging and osteoblasts decrease. Can be classified as Since osteoporosis fractures lead to severe activity limitations and are associated with a high mortality rate of about 15-35% in hip fractures, the diagnosis and treatment of osteoporosis is important before osteoporotic fractures occur (osteoporosis diagnosis). And Treatment Guidelines 2007, 2008).
- osteoporosis treatments include bisphosphonate-based drugs, which are deposited on the mineral component of the bone and form an ATP analog that does not hydrolyze when osteoclasts phage on the bone where the bisphosphonate is deposited. It is known to reduce the bone resorption and thereby increase the bone density by causing toxicity to cells or causing osteoclast activity and apoptosis in various ways in osteoclasts. These drugs are known to be relatively safe, but in recent years, their long-term use affects bone remodeling or bone regeneration after bone fracture due to normal bone resorption and bone formation. Concerns have been raised about the impact, and in fact, there are reports of fatigue fractures in many patients. Therefore, there is an urgent need for the discovery of new mechanisms of bone metabolism associated with the development of osteoporosis and the need for development of preventive or therapeutic agents for osteoporosis.
- An object of the present invention is to provide a peptide for promoting bone formation comprising SEQ ID NO: 1 (AWLNH) or SEQ ID NO: 2 (PHDL).
- Another object of the present invention to provide a pharmaceutical composition for the prevention or treatment of bone diseases comprising the peptide.
- Another object of the present invention to provide a composition for promoting osteoblast differentiation comprising the peptide.
- Another object of the present invention to provide a food composition for improving bone function comprising the peptide.
- Still another object of the present invention is to provide a method for preventing and treating osteoporosis, comprising administering to a subject a pharmaceutically effective amount of the peptide of claim 1.
- the present invention provides a peptide for promoting bone formation comprising SEQ ID NO: 1 (AWLNH) or SEQ ID NO: 2 (PHDL).
- the peptide may be derived from chock protein.
- the peptide may be to promote differentiation of osteoblasts.
- the present invention also provides a pharmaceutical composition for the prevention or treatment of bone diseases comprising the peptide.
- the bone disease may be any one selected from the group consisting of osteoporosis, osteoarthritis, rheumatoid arthritis, osteomalacia, rickets, fibrous osteoarthritis, aplastic bone disease and metabolic bone disease.
- composition for promoting osteoblast differentiation comprising the peptide.
- the present invention also provides a food composition for improving bone function comprising the peptide.
- the present invention also provides a method for preventing and treating osteoporosis, comprising administering a pharmaceutically effective amount of the peptide of claim 1 to a subject.
- Peptide according to the present invention is a peptide for promoting bone formation comprising SEQ ID NO: 1 (AWLNH) or SEQ ID NO: 2 (PHDL), the peptide has the effect of promoting the differentiation of osteoblasts, preventing and treating bone diseases, especially osteoporosis It can be usefully used.
- Figure 1 shows the results of measuring the ALP activity on these fractions after separation of the fractionated peptide protein-derived peptides by SP-Sephadex C-25 cation exchange chromatography ( af p ⁇ 0.05).
- Figure 2 shows the result of the separation of the fractions separated in cation exchange chromatography on HPLC.
- A shows results separated on HPLC equipped with Hypersil Gold C18 column (20 ⁇ 250 mm)
- B shows results separated on HPLC equipped with Hypersil Gold C18 column (4.6 ⁇ 250 mm).
- Figure 3 shows the results of confirming the peptide sequence by De-novo sequencing (Q-TOF LC-MS / MS).
- Figure 4 shows the results of measuring the ALP activity after treating the peptide of SEQ ID NO: 1 (AWLNH) or SEQ ID NO: 2 (PHDL) of the present invention.
- Figure 5 shows the results confirmed by Western blotting whether the protein expression involved in osteoblast differentiation after processing the peptide of SEQ ID NO: 1 (AWLNH) or SEQ ID NO: 2 (PHDL) of the present invention.
- Figure 6 shows the results confirmed by Western blotting whether the expression of MAPKs involved in osteoblast differentiation after processing the peptide of SEQ ID NO: 1 (AWLNH) or SEQ ID NO: 2 (PHDL) of the present invention.
- Figure 7 shows the result of confirming whether the mineral formation of osteoblasts after treatment of the peptide of SEQ ID NO: 1 (AWLNH) or SEQ ID NO: 2 (PHDL) of the present invention ( ac p ⁇ 0.05).
- Figure 8 shows the results of activation of the BMP signaling pathway according to the presence or absence of the BMP antagonist and the peptide of SEQ ID NO: 1 (AWLNH) or SEQ ID NO: 2 (PHDL) of the present invention.
- Figure 9 shows the results of activation of the MAPKs signaling pathway according to the presence or absence of the BMP antagonist and the peptide of SEQ ID NO: 1 (AWLNH) or SEQ ID NO: 2 (PHDL) of the present invention.
- Figure 10 shows the results of activation of the BMP signaling pathway after the treatment of the peptide of the present invention SEQ ID NO: 1 (AWLNH) or SEQ ID NO: 2 (PHDL) and MAPKs inhibitors.
- Figure 11 shows the results of ALP activity after the peptide and MAPKs inhibitor of the SEQ ID NO: 1 (AWLNH) or SEQ ID NO: 2 (PHDL) of the present invention.
- Figure 12 shows the overall bone density change by micro CT when administering a peptide or estrogen (17 ⁇ -estradiol, Est) of SEQ ID NO: 1 (AWLNH), SEQ ID NO: 2 (PHDL) of the present invention in the osteoporosis model, and micro-density BMD Measured by measurement and shown as a quantitative graph.
- the present invention provides a peptide for promoting bone formation comprising SEQ ID NO: 1 (AWLNH) or SEQ ID NO: 2 (PHDL).
- bone formation of the present invention refers to the formation of bone tissue by the deposition of lime salt on the bone tissue.
- the peptide is derived from the Scapharca subcrenata protein and promotes, but is not limited to, differentiation of osteoblasts.
- osteoblast is a cell having the ability to calcify bone tissue by synthesizing and secreting bone matrix and depositing inorganic salts such as calcium and magnesium ions on a substrate. It can be seen at the site of the neonatal.
- bone formation in the advanced state is a cell that is buried in the bone tissue formed by itself become bone cells.
- novel peptides of the invention may comprise their amino acid sequence variants.
- a variant herein refers to a protein (peptide) in which the natural amino acid sequence and one or more amino acid residues have different sequences by deletion, insertion, non-conservative or conservative substitution, or a combination thereof.
- Such variants include peptides with modifications that increase or decrease functional homologues or physicochemical properties with activities substantially equivalent to the wild type.
- the variant is a modified physicochemical property of the peptide.
- novel peptides of the present invention can be prepared by direct isolation from the proteins of the ileum, and their separation and purification can be carried out by many known methods.
- the peptide of the present invention can be synthesized chemically.
- chemical synthesis it can be obtained using a peptide synthesis method well known in the art.
- Peptides can be prepared using conventional stepwise liquid or solid phase synthesis, fragment condensation, F-MOC or T-BOC chemistry.
- the preferred method of preparing the peptide is to use solid phase syntheses.
- the novel peptides of the present invention can be synthesized in a conventional solid phase method by condensation reaction between protected amino acids, starting from the C-terminus and proceeding sequentially according to the sequence. After the condensation reaction, the carrier to which the protecting group and the C-terminal amino acid are linked can be removed by a known method such as acid decomposition or aminolysis.
- the peptide synthesis described above is described in detail in the related books.
- the present invention also provides a pharmaceutical composition for the prevention or treatment of bone diseases comprising the peptide.
- prevention means any action that inhibits or delays the development of a bone disease by the administration of the pharmaceutical composition of the present invention. Any action that improves or beneficially changes symptoms.
- the bone disease is any one selected from the group consisting of osteoporosis, osteoarthritis, rheumatoid arthritis, osteomalacia, rickets, fibrous osteoarthritis, aplastic bone disease and metabolic bone disease, preferably osteoporosis is not limited thereto.
- the pharmaceutical composition of the present invention may further include an adjuvant in addition to the peptide.
- the adjuvant may be used without limitation as long as it is known in the art, but may further include, for example, Freund's complete adjuvant or incomplete adjuvant to increase its immunity.
- compositions according to the present invention may be prepared in a form in which the active ingredient is incorporated into a pharmaceutically acceptable carrier.
- pharmaceutically acceptable carriers include carriers, excipients and diluents commonly used in the pharmaceutical art.
- Pharmaceutically acceptable carriers that can be used in the pharmaceutical compositions of the present invention include, but are not limited to, lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia rubber, alginate, gelatin, Calcium phosphate, calcium silicate, cellulose, methyl cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil.
- compositions of the present invention may be used in the form of powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols and the like, oral formulations, external preparations, suppositories, or sterile injectable solutions, respectively, according to conventional methods. .
- Solid preparations for oral administration include tablets, pills, powders, granules, capsules, and the like, and such solid preparations contain at least one excipient in the active ingredient, for example starch, calcium carbonate, sucrose, lactose, gelatin It can be prepared by mixing.
- lubricants such as magnesium stearate, talc can also be used.
- Liquid preparations for oral administration include suspensions, solvents, emulsions, and syrups.In addition to commonly used diluents such as water and liquid paraffin, various excipients such as wetting agents, sweeteners, fragrances, and preservatives may be included. Can be.
- Formulations for parenteral administration include sterile aqueous solutions, water-insoluble solvents, suspensions, emulsions, lyophilized formulations and suppositories.
- the non-aqueous solvent and suspending agent propylene glycol, polyethylene glycol, vegetable oil such as olive oil, injectable ester such as ethyl oleate and the like can be used.
- As the base of the suppository witepsol, tween 61, cacao butter, laurin butter, glycerogelatin and the like can be used.
- composition according to the present invention can be administered to a subject by various routes. All modes of administration can be expected, for example by oral, intravenous, intramuscular, subcutaneous, intraperitoneal injection.
- the dosage of the pharmaceutical composition according to the present invention is selected in consideration of the age, weight, sex, physical condition, etc. of the individual.
- the concentration of the peptide included in the pharmaceutical composition may be variously selected according to a subject, and preferably, the pharmaceutical composition is included in a concentration of 0.01 to 5,000 ⁇ g / ml. If the concentration is less than 0.01 ⁇ g / ml, the pharmaceutical activity may not appear, and when the concentration exceeds 5,000 ⁇ g / ml, the human body may be toxic.
- the present invention provides a composition for promoting osteoblast differentiation comprising the peptide.
- the present invention also provides a food composition for improving bone function comprising the peptide.
- Food composition of the present invention can be used in a variety of food and beverages for the improvement of bone function.
- Foods that may include the peptides of the present invention include various foods, such as beverages, gums, teas, vitamin complexes, dietary supplements, and the like, and are pills, powders, granules, acupuncture tablets, capsules or beverages. Available in form.
- the amount of the extract in the food or beverage in general, may be added to 0.01 to 15% by weight of the total food weight in the case of the health food composition of the present invention, 0.02 to 10g, based on 100ml for the health drink composition, preferably It may be added at a ratio of 0.3 to 1g.
- the health beverage composition of the present invention is not particularly limited in the liquid component except for containing the peptide as an essential ingredient in the indicated ratio, and may contain various flavors or natural carbohydrates, etc. as additional ingredients, as in general beverages.
- natural carbohydrates include monosaccharides such as glucose, fructose and the like; Disaccharides such as maltose, sucrose and the like; Polysaccharides such as dextrin, cyclodextrin; Conventional sugars such as and the like and sugar alcohols such as xylitol, sorbitol, and erythritol.
- natural flavoring agents such as, tauumatin, stevia extract (e.g., Rebaudioside A, glycyrrhizin, etc.) and synthetic flavoring agents (saccharin, aspartame, etc.) can be advantageously used.
- the proportion of natural carbohydrates is generally about 1-20 g, preferably about 5-12 g per 100 ml of the composition of the present invention.
- the composition of the present invention includes various nutrients, vitamins, minerals (electrolytes), flavors such as synthetic flavors and natural flavors, coloring and neutralizing agents (such as cheese and chocolate), pectic acid and salts thereof, alginic acid and salts thereof. , Organic acids, protective colloidal thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohols, carbonation agents used in carbonated beverages, and the like.
- the compositions of the present invention may also contain pulp for the production of natural fruit juices and fruit juice beverages and vegetable beverages. These components can be used independently or in combination. The proportion of such additives is not so critical but is generally selected from the range of 0 to about 20 parts by weight per 100 parts by weight of the composition of the present invention.
- food supplements which may further add food additives, include food additives conventional in the art, such as flavoring agents, flavoring agents, coloring agents, fillers, stabilizers, and the like.
- food additives conventional in the art such as flavoring agents, flavoring agents, coloring agents, fillers, stabilizers, and the like.
- 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.
- natural flavoring agents tauumatin, stevia extract (e.g., rebaudioside A, glycyrrhizin, etc.) and synthetic flavoring agents (saccharin, aspartame, etc.) can be advantageously used. .
- the functional food composition of the present invention includes various nutrients, vitamins, minerals (electrolytes), flavors such as synthetic and natural flavors, coloring and neutralizing agents (such as cheese and chocolate), pectic acid and salts thereof, alginic acid And salts thereof, organic acids, protective colloidal thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohols, carbonation agents used in carbonated beverages, and the like.
- Others may contain pulp for the production of natural fruit juices and fruit juice drinks and vegetable drinks. These components can be used independently or in combination.
- the present invention also provides a method for preventing and treating osteoporosis, comprising administering a pharmaceutically effective amount of the peptide of claim 1 to a subject.
- the pharmaceutical composition of the present invention is administered in a therapeutically effective amount or in a pharmaceutically effective amount.
- pharmaceutically effective amount means an amount sufficient to treat a disease at a reasonable benefit / risk ratio applicable to medical treatment, and an effective dose level is determined by the type and severity of the subject, age, sex, activity of the drug, drug Sensitivity, time of administration, route of administration and rate of release, duration of treatment, factors including concurrent use of drugs, and other factors well known in the medical arts.
- the ark shell ( Scapharca subcrenata ) used in the experiment was purchased from the traditional market in Suncheon.
- Other materials used for cell culture were purchased from Gibro-BRL (Gaithersburg, MD, USA).
- Antibodies for protein analysis p-Smad1 / 5, Smad1 / 5/8, Dlx5, Runx2, osterix, p-ERK, p-JNK, p-p38 and ⁇ -actin
- p-Smad1 / 5, Smad1 / 5/8, Dlx5, Runx2, osterix, p-ERK, p-JNK, p-p38 and ⁇ -actin are described in Santa Cruz Biotechnology (Santa Cruz, CA, USA).
- Other reagents used in this study were purchased from Sigma-Aldrich.
- the keratoplasm was lyophilized and then digested with pepsin.
- the enzyme and substrate ratio were 1: 500, and the reaction time was 2 hours.
- the degradation product was prepared by using a membrane separation method fractions of 1 kDa or less.
- the recovered peptides were lyophilized by removing NaCl through dialysis. Fractions separated by ion exchange chromatography were separated on a high performance liquid chromatography (HPLC) using a Hypersil Gold C18 column (20 ⁇ 250 mm, Thermo Scientific, PA, USA) and measured after osteoblast differentiation activity. Final separation into a C18 column (4.6 ⁇ 250 mm). A separate peptides was identified a sequence of Q-TOF LC-MS / MS mass peptide to de-novo sequencing method at spectrometer.
- the mouse-derived mesenchymal stem cells used in the experiment were purchased from ATCC (D1 cell, CRL-12424) and incubated in DMEM medium containing 10% FBS and 1% penicillin / streptomycin at 5% CO 2 and 37 ° C. .
- DMEM + 50 ⁇ g / mL ascorbic acid, 10 mM ⁇ -glycerolphosphate and 10 -7 M dexamethasone was exchanged every other day.
- BMP antagonist noggin, 100 ng / mL
- MAPKs inhibitors 10 ⁇ M SB203580, 20 ⁇ M PD98059 and 10 ⁇ M SP600125
- the cytotoxicity of the peptide was measured using MTT (3- (4,5-dimethythiazol-2-yl) -2,5diphenyltetrazolium bromide).
- MTT 3- (4,5-dimethythiazol-2-yl) -2,5diphenyltetrazolium bromide.
- ALP activity was calculated as follows.
- ALP activity (%) (A- A 0 ) / A 0 ⁇ 100
- A peptide treated group
- a 0 untreated group
- the peptide was treated for 7 days, and then the medium was removed and washed twice in PBS. After fixing the cells for 5 minutes by treatment with 10% formalin solution, washed again in PBS, BCIP / NBT substrate solution was added to the well plate and incubated for 15 minutes at 37 °C and observed under a microscope.
- the cells were cultured in 12-well plates and treated with peptides for 21 days. After the incubation, the cells were washed in PBS and fixed with 70% ethanol solution at 4 ° C. for 1 hour. 2% Alizarin red S (pH 4.2) solution was treated for 15 minutes at room temperature, dyed and washed 4 times with distilled water and dried completely. The mineral formation of the cells was observed under a microscope, and the absorbance was measured at 562 nm by decolorizing the Alizarin red S solution stained using 10% cetylpyridinium chloride solution for quantification. The degree of mineral formation was calculated using the following formula.
- A peptide treated group
- a 0 untreated group
- mice used in the experiment were 8-week-old C57BL / 6N female mice, purchased from Envigo (USA) and undergoing a 1 week accrual period.
- mice A total of 18 mice, 4-5 mice per group, were subjected to ovarian extraction surgery (OVX) to create an osteoporosis model, and one week later, SEQ ID No. 1 (AWLNH) and 2 (PHDL) peptides (5 ⁇ g / 25 g, respectively).
- PBS Probco
- the femur was separated from the mouse to remove muscle, washed with physiological saline, fixed in 4% formalin for 24 hours, and bone density was measured.
- Microscopic tomography of the femur was measured using a micro-CT scanner (Inveon preclinical CT, Siemens Healthcare, USA) with a sample length of 1.9 cm, width 2 cm, photon energy of 80 keV, and a current of 500 ⁇ A.
- a volume portion of 2 mm 3 of the same site per group was measured using Siemens Inveon Software.
- fractionated small protein peptides were isolated by ion exchange chromatography, and the fractions were examined for their effects on ALP activity in osteoblasts. As a result, it was confirmed that the fraction eluted at a concentration of 0.6-1.0M NaCl showed 210% higher ALP activity than the blank (untreated group) (Fig. 1).
- the flow rate was 1.0 mL / min, linear phase gradient (10-30% acetonitrile, 0-17 min) isolated in the same mobile phase, resulting in the best ALP activity in fraction 1 (FIG. 2B), again under the same conditions. Separated. As shown in FIG. 2C, the best fraction of ALP was observed in fraction 1, and the fraction was separated by a flow rate of 0.9 mL / min and a linear concentration gradient method (10-20%, 0-17min). It was.
- the two peptides were synthesized by Fmoc solid phase peptide synthesis (SPSS) method to confirm osteoblast differentiation promoting ability (Peptron Inc. Seoul). Two synthesized peptides were evaluated for their effect on ALP activity in osteoblast differentiation. As a result, it showed a very strong increase in ALP activity, in particular, the peptide of SEQ ID NO: 1 of the present invention (AWLNH) 210%, the peptide of SEQ ID NO: 2 of the present invention (PHDL) of 187% at 4 ⁇ M concentration was shown (FIG. 4).
- SPSS Fmoc solid phase peptide synthesis
- BMP-2 / 4 promotes the phosphorylation of Smad1 / 5 in downstream signaling systems to promote intracellular signaling, while phosphorylated Smad1 / 5 plays an important role in producing specific proteins involved in osteoblast differentiation and formation.
- the transcriptional regulators Dlx-5, Runx-2 and osterix were confirmed to be activated.
- the two peptides of the present invention increase the expression of BMP-2 / 4 so that the downstream signaling agents Smad1 / 5 and the transcriptional regulators Dlx-5, Runx-2 and Osterix are expressed or By increasing the activity, it was confirmed to promote the differentiation of osteoblasts (Fig. 5). It was also confirmed that the expression of type I collagen, an early biomarker for osteoblast differentiation, was strongly increased (FIG. 5).
- mitogen-activated protein kinases are known to play an important role in osteoblast differentiation, and the present inventors conducted an experiment to confirm whether the two types of peptides of the present invention have an effect of activating MARKs. As a result, it was confirmed that phosphorylation of MARKs p-p38, p-ERK and p-JNK was increased to promote osteoblast differentiation (FIG. 6).
- the two peptides of the present invention are involved in the signaling pathways that affect the phosphorylation of ERK and JNK, and can activate osteoblast differentiation by activating BMPs signaling pathways, thereby treating bone diseases, in particular osteoporosis. It was confirmed that the effect on.
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Abstract
The present invention relates to an osteogenic promotion peptide comprising sequence number 1 (AWLNH) or sequence number 2 (PHDL), and a composition, for preventing or treating bone diseases, comprising the peptide. The peptide promotes osteoblast differentiation and thus can be utilized for preventing and treating bone diseases, particularly osteoporosis.
Description
본 발명은 꼬막단백질 유래 펩타이드를 유효성분으로 포함하는 골질환의 예방 또는 치료용 조성물에 관한 것이다. The present invention relates to a composition for the prophylaxis or treatment of bone diseases comprising the peptide protein-derived peptide as an active ingredient.
골다공증은 폐경에 따른 급격한 호르몬의 변화에 의한 파골세포(osteoclast)의 활성화에 따른 골흡수 증가로 나타나는 폐경 후 골다공증과, 노화가 되면서 조골세포(osteoblast)의 기능이 감소하여 골형성이 감소하는 노인성 골다공증으로 분류할 수 있다. 이러한 골다공증으로 인한 골절은 심각한 활동 제한에 이르게 되고, 고관절 골절의 경우 약 15-35%의 높은 사망률과 관련되어 있기 때문에, 골다공증성 골절이 발생하기 이전에 골다공증의 진단과 치료가 중요하다(골다공증 진단 및 치료 지침 2007, 2008).Osteoporosis is postmenopausal osteoporosis, which is indicated by increased bone resorption due to the activation of osteoclasts due to rapid hormonal changes following menopause, and senile osteoporosis, in which osteoblasts decrease due to aging and osteoblasts decrease. Can be classified as Since osteoporosis fractures lead to severe activity limitations and are associated with a high mortality rate of about 15-35% in hip fractures, the diagnosis and treatment of osteoporosis is important before osteoporotic fractures occur (osteoporosis diagnosis). And Treatment Guidelines 2007, 2008).
기존에 알려진 골다공증 치료제로는 비스포스포네이트(bisphosphonate)계열의 약물이 있는데, 비스포스포네이트는 골의 무기질 성분에 침착되며, 비스포스포네이트가 침착된 골을 파골세포가 탐식할 경우 가수분해되지 않는 ATP 유사체(analogue)를 형성하여 세포에 독성을 나타내거나 파골세포 내에서 다양한 방식으로 파골세포의 활성 감소 및 세포사멸(apoptosis)을 초래해 골 흡수를 줄이고 이를 통해 골밀도를 증가시키는 것으로 알려져 있다. 이러한 약물들은 비교적 안전한 것으로 알려져 있으나, 최근 장기간 사용시 정상적인 골 흡수 및 골 형성에 의한 골의 재형성(remodeling)이나 골절 이후 골 재생(healing)과정에 영향을 주어 골의 탄성이 떨어져 오히려 골강도에 좋지 않은 영향을 줄 수 있다는 우려들이 제기되고 있으며, 실제로 이로 인해 많은 환자에서 피로 골절을 일으킨다는 보고가 있다. 따라서, 골다공증 발병과 관련된 새로운 골대사 기전의 발견 및 골다공증 예방 또는 치료제 개발의 필요성이 절실하게 요구되고 있는 실정이다.Known osteoporosis treatments include bisphosphonate-based drugs, which are deposited on the mineral component of the bone and form an ATP analog that does not hydrolyze when osteoclasts phage on the bone where the bisphosphonate is deposited. It is known to reduce the bone resorption and thereby increase the bone density by causing toxicity to cells or causing osteoclast activity and apoptosis in various ways in osteoclasts. These drugs are known to be relatively safe, but in recent years, their long-term use affects bone remodeling or bone regeneration after bone fracture due to normal bone resorption and bone formation. Concerns have been raised about the impact, and in fact, there are reports of fatigue fractures in many patients. Therefore, there is an urgent need for the discovery of new mechanisms of bone metabolism associated with the development of osteoporosis and the need for development of preventive or therapeutic agents for osteoporosis.
본 발명의 목적은 서열번호 1(AWLNH) 또는 서열번호 2(PHDL)를 포함하는 골 형성 촉진용 펩타이드를 제공하는 것이다. An object of the present invention is to provide a peptide for promoting bone formation comprising SEQ ID NO: 1 (AWLNH) or SEQ ID NO: 2 (PHDL).
본 발명의 또 다른 목적은 상기 펩타이드를 포함하는 골질환의 예방 또는 치료용 약학 조성물을 제공하는 것이다. Another object of the present invention to provide a pharmaceutical composition for the prevention or treatment of bone diseases comprising the peptide.
본 발명의 또 다른 목적은 상기 펩타이드를 포함하는 조골세포 분화 촉진용 조성물을 제공하는 것이다. Another object of the present invention to provide a composition for promoting osteoblast differentiation comprising the peptide.
본 발명의 또 다른 목적은 상기 펩타이드를 포함하는 골기능 개선용 식품 조성물을 제공하는 것이다.Another object of the present invention to provide a food composition for improving bone function comprising the peptide.
본 발명의 또 다른 목적은 약학적으로 유효한 양의 제1항의 펩타이드를 개체에 투여하는 단계를 포함하는 골다공증 예방 및 치료방법을 제공하는 것이다.Still another object of the present invention is to provide a method for preventing and treating osteoporosis, comprising administering to a subject a pharmaceutically effective amount of the peptide of claim 1.
상기 목적을 달성하기 위하여, 본 발명은 서열번호 1(AWLNH) 또는 서열번호 2(PHDL)를 포함하는 골 형성 촉진용 펩타이드를 제공한다. In order to achieve the above object, the present invention provides a peptide for promoting bone formation comprising SEQ ID NO: 1 (AWLNH) or SEQ ID NO: 2 (PHDL).
본 발명의 일실시예에 있어서, 상기 펩타이드는 꼬막단백질 유래인 것일 수 있다. In one embodiment of the present invention, the peptide may be derived from chock protein.
본 발명의 일실시예에 있어서, 상기 펩타이드는 조골세포의 분화를 촉진시키는 것일 수 있다. In one embodiment of the invention, the peptide may be to promote differentiation of osteoblasts.
또한, 본 발명은 상기 펩타이드를 포함하는 골질환의 예방 또는 치료용 약학 조성물을 제공한다. The present invention also provides a pharmaceutical composition for the prevention or treatment of bone diseases comprising the peptide.
본 발명의 일실시예에 있어서, 상기 골질환은 골다공증, 골관절염, 류미티스 관절염, 골연화증, 구루병, 섬유성 골염, 무형성 골질환 및 대사성 골질환으로 이루어진 그룹에서 선택되는 어느 하나인 것일 수 있다. In one embodiment of the present invention, the bone disease may be any one selected from the group consisting of osteoporosis, osteoarthritis, rheumatoid arthritis, osteomalacia, rickets, fibrous osteoarthritis, aplastic bone disease and metabolic bone disease.
또한, 상기 펩타이드를 포함하는 조골세포 분화 촉진용 조성물을 제공한다. In addition, it provides a composition for promoting osteoblast differentiation comprising the peptide.
또한, 본 발명은 상기 펩타이드를 포함하는 골기능 개선용 식품 조성물을 제공한다. The present invention also provides a food composition for improving bone function comprising the peptide.
또한, 본 발명은 약학적으로 유효한 양의 제1항의 펩타이드를 개체에 투여하는 단계를 포함하는 골다공증 예방 및 치료방법을 제공한다.The present invention also provides a method for preventing and treating osteoporosis, comprising administering a pharmaceutically effective amount of the peptide of claim 1 to a subject.
본 발명에 따른 펩타이드는 서열번호 1(AWLNH) 또는 서열번호 2(PHDL)를 포함하는 골 형성 촉진용 펩타이드로서, 상기 펩타이드는 조골세포의 분화를 촉진시키는 효과가 있어 골질환 특히 골다공증의 예방 및 치료에 유용하게 사용될 수 있다. Peptide according to the present invention is a peptide for promoting bone formation comprising SEQ ID NO: 1 (AWLNH) or SEQ ID NO: 2 (PHDL), the peptide has the effect of promoting the differentiation of osteoblasts, preventing and treating bone diseases, especially osteoporosis It can be usefully used.
도 1은 분획된 꼬막단백질 유래 펩타이드를 SP-Sephadex C-25 양이온 교환 크로마토그래피에 의하여 분리한 후, 이들 획분에 대한 ALP 활성을 측정한 결과를 나타낸 것이다(a-fp<0.05).Figure 1 shows the results of measuring the ALP activity on these fractions after separation of the fractionated peptide protein-derived peptides by SP-Sephadex C-25 cation exchange chromatography ( af p <0.05).
도 2는 양이온 교환 크로마토그래피에서 분리된 획분을 HPLC 상에서 분리한 결과를 나타낸 것이다. (A)는 Hypersil Gold C18 column(20×250mm)이 장착된 HPLC 상에서 분리된 결과이고, (B) 내지 (D)는 Hypersil Gold C18 column(4.6×250mm)이 장착된 HPLC 상에서 분리된 결과를 나타낸 것이다(a-fp<0.05).Figure 2 shows the result of the separation of the fractions separated in cation exchange chromatography on HPLC. (A) shows results separated on HPLC equipped with Hypersil Gold C18 column (20 × 250 mm), and (B) to (D) shows results separated on HPLC equipped with Hypersil Gold C18 column (4.6 × 250 mm). Af p <0.05).
도 3은 De-novo sequencing (Q-TOF LC-MS/MS)에 의한 펩타이드 서열을 확인한 결과를 나타낸 것이다. Figure 3 shows the results of confirming the peptide sequence by De-novo sequencing (Q-TOF LC-MS / MS).
도 4는 본 발명의 서열번호 1(AWLNH) 또는 서열번호 2(PHDL)의 펩타이드를 처리한 후, ALP 활성을 측정한 결과를 나타낸 것이다. Figure 4 shows the results of measuring the ALP activity after treating the peptide of SEQ ID NO: 1 (AWLNH) or SEQ ID NO: 2 (PHDL) of the present invention.
도 5는 본 발명의 서열번호 1(AWLNH) 또는 서열번호 2(PHDL)의 펩타이드를 처리한 후, 조골세포 분화에 관여하는 단백질 발현 여부를 웨스턴 블럿팅으로 확인한 결과를 나타낸 것이다. Figure 5 shows the results confirmed by Western blotting whether the protein expression involved in osteoblast differentiation after processing the peptide of SEQ ID NO: 1 (AWLNH) or SEQ ID NO: 2 (PHDL) of the present invention.
도 6은 본 발명의 서열번호 1(AWLNH) 또는 서열번호 2(PHDL)의 펩타이드를 처리한 후, 조골세포 분화에 관여하는 MAPKs 발현 여부를 웨스턴 블럿팅으로 확인한 결과를 나타낸 것이다. Figure 6 shows the results confirmed by Western blotting whether the expression of MAPKs involved in osteoblast differentiation after processing the peptide of SEQ ID NO: 1 (AWLNH) or SEQ ID NO: 2 (PHDL) of the present invention.
도 7은 본 발명의 서열번호 1(AWLNH) 또는 서열번호 2(PHDL)의 펩타이드를 처리한 후, 조골세포의 미네랄 형성 여부를 확인한 결과를 나타낸 것이다(a-cp<0.05). Figure 7 shows the result of confirming whether the mineral formation of osteoblasts after treatment of the peptide of SEQ ID NO: 1 (AWLNH) or SEQ ID NO: 2 (PHDL) of the present invention ( ac p <0.05).
도 8은 본 발명의 서열번호 1(AWLNH) 또는 서열번호 2(PHDL)의 펩타이드와 BMP 길항제의 처리 유무에 따른 BMP 신호전달경로의 활성화 결과를 나타낸 것이다. Figure 8 shows the results of activation of the BMP signaling pathway according to the presence or absence of the BMP antagonist and the peptide of SEQ ID NO: 1 (AWLNH) or SEQ ID NO: 2 (PHDL) of the present invention.
도 9는 본 발명의 서열번호 1(AWLNH) 또는 서열번호 2(PHDL)의 펩타이드와 BMP 길항제의 처리 유무에 따른 MAPKs 신호전달경로의 활성화 결과를 나타낸 것이다. Figure 9 shows the results of activation of the MAPKs signaling pathway according to the presence or absence of the BMP antagonist and the peptide of SEQ ID NO: 1 (AWLNH) or SEQ ID NO: 2 (PHDL) of the present invention.
도 10은 본 발명의 서열번호 1(AWLNH) 또는 서열번호 2(PHDL)의 펩타이드와 MAPKs 저해제를 처리한 후, BMP 신호전달경로의 활성화 결과를 나타낸 것이다. Figure 10 shows the results of activation of the BMP signaling pathway after the treatment of the peptide of the present invention SEQ ID NO: 1 (AWLNH) or SEQ ID NO: 2 (PHDL) and MAPKs inhibitors.
도 11은 본 발명의 서열번호 1(AWLNH) 또는 서열번호 2(PHDL)의 펩타이드와 MAPKs 저해제를 처리한 후, ALP 활성 결과를 나타낸 것이다. Figure 11 shows the results of ALP activity after the peptide and MAPKs inhibitor of the SEQ ID NO: 1 (AWLNH) or SEQ ID NO: 2 (PHDL) of the present invention.
도 12는 본 발명의 서열번호 1(AWLNH), 서열번호 2(PHDL)의 펩타이드 또는 에스트로겐(17β-estradiol, Est)을 골다공증 모델에 투여 시 전체적인 골밀도 변화를 micro CT로 촬영하고, 골밀도를 micro CT 측정로 측정하여 이를 그래프로 정량화하여 나타낸 것이다. Figure 12 shows the overall bone density change by micro CT when administering a peptide or estrogen (17β-estradiol, Est) of SEQ ID NO: 1 (AWLNH), SEQ ID NO: 2 (PHDL) of the present invention in the osteoporosis model, and micro-density BMD Measured by measurement and shown as a quantitative graph.
본 발명은 서열번호 1(AWLNH) 또는 서열번호 2(PHDL)를 포함하는 골 형성 촉진용 펩타이드를 제공한다. The present invention provides a peptide for promoting bone formation comprising SEQ ID NO: 1 (AWLNH) or SEQ ID NO: 2 (PHDL).
본 발명의 용어, "골 형성"은 골조직의 바탕에 석회염이 침착하여 골조직을 이루는 일을 말한다. The term "bone formation" of the present invention refers to the formation of bone tissue by the deposition of lime salt on the bone tissue.
상기 펩타이드는 꼬막(Scapharca subcrenata) 단백질 유래이고, 조골세포의 분화를 촉진시키나, 이에 제한되지 않는다. The peptide is derived from the Scapharca subcrenata protein and promotes, but is not limited to, differentiation of osteoblasts.
본 발명의 용어, "조골세포(osteoblast)"는 골기질을 합성, 분비하고, 기질에 칼슘, 마그네슘 이온 등의 무기염을 침착시킴으로서 골조직을 석회화시키는 능력을 갖고 있는 세포로서, 골 형성 등에 의해 뼈의 신생이 이루어지는 부위에서 볼 수 있다. 또한, 골 형성이 진행된 상태에서는 스스로 형성된 골조직 속에 묻혀 골세포가 되는 세포이다. The term "osteoblast" is a cell having the ability to calcify bone tissue by synthesizing and secreting bone matrix and depositing inorganic salts such as calcium and magnesium ions on a substrate. It can be seen at the site of the neonatal. In addition, bone formation in the advanced state is a cell that is buried in the bone tissue formed by itself become bone cells.
본 발명의 신규한 펩타이드는 이들의 아미노산 서열 변이체를 포함할 수 있다. 여기서 변이체란 천연 아미노산 서열과 하나 이상의 아미노산 잔기가 결실, 삽입, 비보전적 또는 보전적 치환 또는 이들의 조합에 의하여 상이한 서열을 가지는 단백질(펩타이드)을 의미한다. 이러한 변이체는 야생형과 실질적으로 동등한 활성을 갖는 기능적 상동체 또는 물리 화학적 성질을 증가 또는 감소시키는 변형을 가지는 펩타이드를 포함한다. 바람직하게는 펩타이드의 물리 화학적 성질이 변형된 변이체이다. 예를 들어, 온도, 수분, pH, 전해질, 환원당, 가압, 건조, 동결, 계면장력, 광선, 동결과 해동의 반복, 고농도 조건 등의 물리적 요인과 산, 알카리, 중성염, 유기용매, 금속이온, 산화환원제, 프로티아제 등의 화학적 요인의 외부 환경에 대한 구조적 안정성이 증대된 변이체이다. 또한 아미노산 서열상의 변이로 효소 활성이 증대된 변이체일 수 있다.The novel peptides of the invention may comprise their amino acid sequence variants. A variant herein refers to a protein (peptide) in which the natural amino acid sequence and one or more amino acid residues have different sequences by deletion, insertion, non-conservative or conservative substitution, or a combination thereof. Such variants include peptides with modifications that increase or decrease functional homologues or physicochemical properties with activities substantially equivalent to the wild type. Preferably the variant is a modified physicochemical property of the peptide. For example, physical factors such as temperature, moisture, pH, electrolytes, reducing sugars, pressurization, drying, freezing, interfacial tension, light rays, freezing and thawing, high concentration conditions, acids, alkalis, neutral salts, organic solvents, metal ions It is a variant with enhanced structural stability to the external environment of chemical factors such as redox, proteases and the like. In addition, it may be a variant in which the enzyme activity is enhanced by the mutation on the amino acid sequence.
본 발명의 신규한 펩타이드는 꼬막의 단백질로부터 직접 분리하여 제조할 수 있으며, 이의 분리 및 정제는 많은 공지 방법에 의해 실시할 수 있다.The novel peptides of the present invention can be prepared by direct isolation from the proteins of the ileum, and their separation and purification can be carried out by many known methods.
또한 본 발명의 펩타이드는 화학적으로 합성할 수 있다. 화학적으로 합성하여 제조하는 경우, 당 분야에 널리 공지된 펩타이드 합성법을 이용하여 얻을 수 있다. 펩타이드는 통상의 단계적인 액체 또는 고체상 합성, 단편 응축, F-MOC 또는 T-BOC 화학법을 이용하여 제조할 수 있다. 특히, 바람직한 펩타이드의 제조방법은 고체상 합성방법(solid phase syntheses)을 이용하는 것이다. 본 발명의 신규한 펩타이드는 보호된 아미노산간의 응축반응(condensation reaction)에 의한 통상의 고체상 방법으로, C-말단으로부터 시작하여 그 서열에 따라 순차적으로 진행하면서 합성할 수 있다. 응축 반응 후 보호기 및 C-말단 아미노산이 연결된 담체를 산분해(acid decomposition) 또는 아미놀리시스(aminolysis)와 같은 공지의 방법에 의해 제거할 수 있다. 상기 언급된 펩타이드 합성법은 관련 서적에 상세히 기술되어 있다.In addition, the peptide of the present invention can be synthesized chemically. When prepared by chemical synthesis, it can be obtained using a peptide synthesis method well known in the art. Peptides can be prepared using conventional stepwise liquid or solid phase synthesis, fragment condensation, F-MOC or T-BOC chemistry. In particular, the preferred method of preparing the peptide is to use solid phase syntheses. The novel peptides of the present invention can be synthesized in a conventional solid phase method by condensation reaction between protected amino acids, starting from the C-terminus and proceeding sequentially according to the sequence. After the condensation reaction, the carrier to which the protecting group and the C-terminal amino acid are linked can be removed by a known method such as acid decomposition or aminolysis. The peptide synthesis described above is described in detail in the related books.
또한, 본 발명은 상기 펩타이드를 포함하는 골질환의 예방 또는 치료용 약학 조성물을 제공한다.The present invention also provides a pharmaceutical composition for the prevention or treatment of bone diseases comprising the peptide.
본 발명의 용어, "예방"이란 본 발명의 약학 조성물의 투여에 의해 골 질환을 억제시키거나 발병을 지연시키는 모든 행위를 의미하고, "치료"란 본 발명의 약학 조성물의 투여에 의해 골 질환의 증상을 호전 또는 이롭게 변경하는 모든 행위를 의미한다. As used herein, the term "prevention" means any action that inhibits or delays the development of a bone disease by the administration of the pharmaceutical composition of the present invention. Any action that improves or beneficially changes symptoms.
상기 골질환은 골다공증, 골관절염, 류미티스 관절염, 골연화증, 구루병, 섬유성 골염, 무형성 골질환 및 대사성 골질환으로 이루어진 그룹에서 선택되는 어느 하나이고, 바람직하게는 골다공증이나 이에 제한되지 않는다.The bone disease is any one selected from the group consisting of osteoporosis, osteoarthritis, rheumatoid arthritis, osteomalacia, rickets, fibrous osteoarthritis, aplastic bone disease and metabolic bone disease, preferably osteoporosis is not limited thereto.
본 발명의 약학 조성물에는 상기 펩타이드 이외에 보조제(adjuvant)를 추가로 포함할 수 있다. 상기 보조제는 당해 기술분야에 알려진 것이라면 어느 것이나 제한 없이 사용할 수 있으나, 예를 들어 프로인트(Freund)의 완전 보조제 또는 불완전 보조제를 더 포함하여 그 면역성을 증가시킬 수 있다. The pharmaceutical composition of the present invention may further include an adjuvant in addition to the peptide. The adjuvant may be used without limitation as long as it is known in the art, but may further include, for example, Freund's complete adjuvant or incomplete adjuvant to increase its immunity.
본 발명에 따른 약학 조성물은 유효성분을 약학적으로 허용된 담체에 혼입시킨 형태로 제조될 수 있다. 여기서, 약학적으로 허용된 담체는 제약 분야에서 통상 사용되는 담체, 부형제 및 희석제를 포함한다. 본 발명의 약학 조성물에 이용할 수 있는 약학적으로 허용된 담체는 이들로 제한되는 것은 아니지만, 락토스, 덱스트로스, 수크로스, 솔비톨, 만니톨, 자일리톨, 에리스리톨, 말티톨, 전분, 아카시아 고무, 알지네이트, 젤라틴, 칼슘 포스페이트, 칼슘 실리케이트, 셀룰로스, 메틸 셀룰로스, 폴리비닐 피롤리돈, 물, 메틸히드록시벤조에이트, 프로필히드록시벤조에이트, 탈크, 마그네슘 스테아레이트 및 광물유를 들 수 있다.The pharmaceutical composition according to the present invention may be prepared in a form in which the active ingredient is incorporated into a pharmaceutically acceptable carrier. Here, pharmaceutically acceptable carriers include carriers, excipients and diluents commonly used in the pharmaceutical art. Pharmaceutically acceptable carriers that can be used in the pharmaceutical compositions of the present invention include, but are not limited to, lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia rubber, alginate, gelatin, Calcium phosphate, calcium silicate, cellulose, methyl cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil.
본 발명의 약학 조성물은 각각 통상의 방법에 따라 산제, 과립제, 정제, 캡슐제, 현탁액, 에멀전, 시럽, 에어로졸 등의 경구형 제형, 외용제, 좌제 또는 멸균 주사용액의 형태로 제형화하여 사용될 수 있다.The pharmaceutical compositions of the present invention may be used in the form of powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols and the like, oral formulations, external preparations, suppositories, or sterile injectable solutions, respectively, according to conventional methods. .
제제화할 경우에는 통상 사용하는 충진제, 증량제, 결합제, 습윤제, 붕해제, 계면활성제 등의 희석제 또는 부형제를 사용하여 조제될 수 있다. 경구투여를 위한 고형제제에는 정제, 환제, 산제, 과립제, 캡슐제 등이 포함되며, 그러한 고형 제제는 유효성분에 적어도 하나 이상의 부형제, 예를 들면 전분, 칼슘 카르보네이트, 수크로스, 락토오스, 젤라틴 등을 섞어 조제될 수 있다. 또한, 단순한 부형제 이외에 마그네슘 스테아레이트, 탈크 같은 윤활제들도 사용될 수 있다. 경구투여를 위한 액상 제제로는 현탁제, 내용액제, 유제, 시럽제 등이 해당되는데, 일반적으로 사용되는 희석제인 물, 리퀴드 파라핀 이외에 여러 가지 부형제, 예를 들면 습윤제, 감미제, 방향제, 보존제 등이 포함될 수 있다. 비경구 투여를 위한 제제에는 멸균된 수용액, 비수용성용제, 현탁제, 유제, 동결건조 제제 및 좌제가 포함된다. 비수용성용제, 현탁제로는 프로필렌 글리콜, 폴리에틸렌 글리콜, 올리브유와 같은 식물성 기름, 에틸올레이트와 같은 주사 가능한 에스테르 등이 사용될 수 있다. 좌제의 기제로는 위텝솔(witepsol), 트윈(tween) 61, 카카오지, 라우린지, 글리세로젤라틴 등이 사용될 수 있다.When formulated, it may be prepared using conventional diluents or excipients such as fillers, extenders, binders, wetting agents, disintegrating agents, surfactants, and the like. Solid preparations for oral administration include tablets, pills, powders, granules, capsules, and the like, and such solid preparations contain at least one excipient in the active ingredient, for example starch, calcium carbonate, sucrose, lactose, gelatin It can be prepared by mixing. In addition to simple excipients, lubricants such as magnesium stearate, talc can also be used. Liquid preparations for oral administration include suspensions, solvents, emulsions, and syrups.In addition to commonly used diluents such as water and liquid paraffin, various excipients such as wetting agents, sweeteners, fragrances, and preservatives may be included. Can be. Formulations for parenteral administration include sterile aqueous solutions, water-insoluble solvents, suspensions, emulsions, lyophilized formulations and suppositories. As the non-aqueous solvent and suspending agent, propylene glycol, polyethylene glycol, vegetable oil such as olive oil, injectable ester such as ethyl oleate and the like can be used. As the base of the suppository, witepsol, tween 61, cacao butter, laurin butter, glycerogelatin and the like can be used.
본 발명에 따른 약학 조성물은 개체에 다양한 경로로 투여될 수 있다. 투여의 모든 방식이 예상될 수 있는데, 예를 들면 경구, 정맥, 근육, 피하, 복강내 주사에 의해 투여될 수 있다.The pharmaceutical composition according to the present invention can be administered to a subject by various routes. All modes of administration can be expected, for example by oral, intravenous, intramuscular, subcutaneous, intraperitoneal injection.
본 발명에 따른 약학 조성물의 투여량은 개체의 연령, 체중, 성별, 신체 상태 등을 고려하여 선택된다. 상기 약학 조성물 중 포함되는 펩타이드의 농도는 대상에 따라 다양하게 선택할 수 있음은 자명하며, 바람직하게는 약학 조성물에 0.01 ~ 5,000 ㎍/ml의 농도로 포함되는 것이다. 그 농도가 0.01 ㎍/ml 미만일 경우에는 약학 활성이 나타나지 않을 수 있고, 5,000 ㎍/ml를 초과할 경우에는 인체에 독성을 나타낼 수 있다.The dosage of the pharmaceutical composition according to the present invention is selected in consideration of the age, weight, sex, physical condition, etc. of the individual. Obviously, the concentration of the peptide included in the pharmaceutical composition may be variously selected according to a subject, and preferably, the pharmaceutical composition is included in a concentration of 0.01 to 5,000 μg / ml. If the concentration is less than 0.01 μg / ml, the pharmaceutical activity may not appear, and when the concentration exceeds 5,000 μg / ml, the human body may be toxic.
또한, 본 발명은 상기 펩타이드를 포함하는 조골세포 분화 촉진용 조성물을 제공한다. In addition, the present invention provides a composition for promoting osteoblast differentiation comprising the peptide.
또한, 본 발명은 상기 펩타이드를 포함하는 골기능 개선용 식품 조성물을 제공한다. The present invention also provides a food composition for improving bone function comprising the peptide.
본 발명의 식품 조성물은 골기능의 개선을 위한 식품 및 음료 등에 다양하게 이용될 수 있다. 본 발명의 펩타이드를 포함할 수 있는 식품으로는, 각종 식품류, 예를 들어, 음료, 껌, 차, 비타민 복합제, 건강보조 식품류 등이 있으며, 환제, 분말, 과립, 침제, 정제, 캡슐 또는 음료인 형태로 사용할 수 있다.Food composition of the present invention can be used in a variety of food and beverages for the improvement of bone function. Foods that may include the peptides of the present invention include various foods, such as beverages, gums, teas, vitamin complexes, dietary supplements, and the like, and are pills, powders, granules, acupuncture tablets, capsules or beverages. Available in form.
이때, 식품 또는 음료 중의 상기 추출물의 양은, 일반적으로 본 발명의 건강식품 조성물의 경우 전체 식품 중량의 0.01 내지 15 중량%로 가할 수 있으며, 건강 음료 조성물의 경우 100㎖를 기준으로 0.02 내지 10g, 바람직하게는 0.3 내지 1g의 비율로 가할 수 있다.At this time, the amount of the extract in the food or beverage, in general, may be added to 0.01 to 15% by weight of the total food weight in the case of the health food composition of the present invention, 0.02 to 10g, based on 100ml for the health drink composition, preferably It may be added at a ratio of 0.3 to 1g.
본 발명의 건강 음료 조성물은 지시된 비율로 필수 성분으로서 상기 펩타이드를 함유하는 외에는 액체성분에는 특별한 제한점은 없으며 통상의 음료와 같이 여러 가지 향미제 또는 천연 탄수화물 등을 추가 성분으로서 함유할 수 있다. 상술한 천연 탄수화물의 예로는 모노사카라이드, 예를 들어, 포도당, 과당 등; 디사카라이드, 예를 들어 말토스, 슈크로스 등; 폴리사카라이드, 예를 들어 덱스트린, 시클로덱스트린; 등과 같은 통상적인 당 및 자일리톨, 소르비톨, 에리트리톨 등의 당알콜이다. 상술한 것 이외의 향미제로서 천연 향미제(타우마틴, 스테비아 추출물(예를 들어 레바우디오시드 A, 글리시르히진 등) 및 합성 향미제(사카린, 아스파르탐 등)를 유리하게 사용할 수 있다. 상기 천연 탄수화물의 비율은 본 발명의 조성물 100 ㎖당 일반적으로 약 1 내지 20g, 바람직하게는 약 5 내지 12g이다.The health beverage composition of the present invention is not particularly limited in the liquid component except for containing the peptide as an essential ingredient in the indicated ratio, and may contain various flavors or natural carbohydrates, etc. as additional ingredients, as in general beverages. Examples of the above-mentioned natural carbohydrates include monosaccharides such as glucose, fructose and the like; Disaccharides such as maltose, sucrose and the like; Polysaccharides such as dextrin, cyclodextrin; Conventional sugars such as and the like and sugar alcohols such as xylitol, sorbitol, and erythritol. As flavoring agents other than those described above, natural flavoring agents (tauumatin, stevia extract (e.g., Rebaudioside A, glycyrrhizin, etc.) and synthetic flavoring agents (saccharin, aspartame, etc.) can be advantageously used. The proportion of natural carbohydrates is generally about 1-20 g, preferably about 5-12 g per 100 ml of the composition of the present invention.
상기 외에 본 발명의 조성물은 여러가지 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, 탄산 음료에 사용되는 탄산화제 등을 함유할 수 있다. 그밖에 본 발명의 조성물들은 천연 과일 쥬스 및 과일 쥬스 음료 및 야채 음료의 제조를 위한 과육을 함유할 수 있다. 이러한 성분은 독립적으로 또는 조합하여 사용 할 수 있다. 이러한 첨가제의 비율은 그렇게 중요하진 않지만 본 발명의 조성물 100 중량부 당 0 내지 약 20 중량부의 범위에서 선택되는 것이 일반적이다. 본 발명의 기능성 식품 조성물에서 포함할 수 있는 필수 성분으로서 상기 펩타이드 또는 약학적으로 허용되는 이의 염을 포함하는 신경계 질환에 효과를 갖는 조성물 외에는 다른 성분에는 특별한 제한이 없으며 통상의 식품과 같이 여러 가지 생약 추출물, 식품 보조 첨가제 또는 천연 탄수화물 등을 추가 성분으로서 함유할 수 있다.In addition to the above, the composition of the present invention includes various nutrients, vitamins, minerals (electrolytes), flavors such as synthetic flavors and natural flavors, coloring and neutralizing agents (such as cheese and chocolate), pectic acid and salts thereof, alginic acid and salts thereof. , Organic acids, protective colloidal thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohols, carbonation agents used in carbonated beverages, and the like. The compositions of the present invention may also contain pulp for the production of natural fruit juices and fruit juice beverages and vegetable beverages. These components can be used independently or in combination. The proportion of such additives is not so critical but is generally selected from the range of 0 to about 20 parts by weight per 100 parts by weight of the composition of the present invention. There are no special limitations on the other ingredients other than the composition having an effect on the nervous system diseases including the peptide or a pharmaceutically acceptable salt thereof as essential ingredients that may be included in the functional food composition of the present invention, and various herbal medicines such as conventional foods. Extracts, food supplement additives or natural carbohydrates and the like may be included as additional ingredients.
또한, 상기 언급한 바와 같이 식품보조첨가제를 추가로 첨가할 수도 있는 바 식품보조첨가제는 당업계에 통상적인 식품보조첨가제, 예를 들어 향미제, 풍미제, 착색제, 충진제, 안정화제 등을 포함한다. 상기 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 것 이외의 향미제로서 천연 향미제(타우마틴, 스테비아 추출물(예를 들어 레바우디오시드 Ａ, 글리시르히진등) 및 합성 향미제(사카린, 아스파르탐 등)를 유리하게 사용할 수 있다.In addition, as mentioned above, food supplements, which may further add food additives, include food additives conventional in the art, such as flavoring agents, flavoring agents, coloring agents, fillers, stabilizers, and the like. . Examples of such 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. As flavoring agents other than those mentioned above, natural flavoring agents (tauumatin, stevia extract (e.g., rebaudioside A, glycyrrhizin, etc.) and synthetic flavoring agents (saccharin, aspartame, etc.) can be advantageously used. .
상기 외에 본 발명의 기능성 식품 조성물은 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, 알코올, 탄산 음료에 사용되는 탄산화제 등을 함유할 수 있다. 그밖에 천연 과일쥬스 및 과일 쥬스 음료 및 야채 음료의 제조를 위한 과육을 함유할 수 있다. 이러한 성분은 독립적으로 또는 조합하여 사용할 수 있다.In addition to the above, the functional food composition of the present invention includes various nutrients, vitamins, minerals (electrolytes), flavors such as synthetic and natural flavors, coloring and neutralizing agents (such as cheese and chocolate), pectic acid and salts thereof, alginic acid And salts thereof, organic acids, protective colloidal thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohols, carbonation agents used in carbonated beverages, and the like. Others may contain pulp for the production of natural fruit juices and fruit juice drinks and vegetable drinks. These components can be used independently or in combination.
또한, 본 발명은 약학적으로 유효한 양의 제1항의 펩타이드를 개체에 투여하는 단계를 포함하는 골다공증 예방 및 치료방법을 제공한다.The present invention also provides a method for preventing and treating osteoporosis, comprising administering a pharmaceutically effective amount of the peptide of claim 1 to a subject.
본 발명의 약학 조성물은 치료적 유효량 또는 약학으로 유효한 양으로 투여한다. 용어 "약학적으로 유효한 양"은 의학적 치료에 적용 가능한 합리적인 수혜/위험 비율로 질환을 치료하기에 충분한 양을 의미하며, 유효 용량 수준은 개체 종류 및 중증도, 연령, 성별, 약물의 활성, 약물에 대한 민감도, 투여 시간, 투여 경로 및 배출 비율, 치료기간, 동시 사용되는 약물을 포함한 요소 및 기타 의학 분야에 잘 알려진 요소에 따라 결정될 수 있다.The pharmaceutical composition of the present invention is administered in a therapeutically effective amount or in a pharmaceutically effective amount. The term “pharmaceutically effective amount” means an amount sufficient to treat a disease at a reasonable benefit / risk ratio applicable to medical treatment, and an effective dose level is determined by the type and severity of the subject, age, sex, activity of the drug, drug Sensitivity, time of administration, route of administration and rate of release, duration of treatment, factors including concurrent use of drugs, and other factors well known in the medical arts.
이하, 본 발명을 실시예를 통하여 더욱 상세히 설명하기로 한다. 이들 실시예는 본 발명을 보다 구체적으로 설명하기 위한 것으로서, 본 발명의 범위가 이들 실시예에 한정되는 것은 아니다.Hereinafter, the present invention will be described in more detail with reference to Examples. These examples are intended to illustrate the present invention more specifically, but the scope of the present invention is not limited to these examples.
실시예 1. 실험재료 및 방법Example 1 Experimental Materials and Methods
1.1. 재료1.1. material
실험에 사용된 꼬막(ark shell, Scapharca subcrenata)은 순천에 위치한 재래시장에서 구입하였다. 그 외 세포 배양에 사용된 재료는 Gibro-BRL(Gaithersburg, MD, USA)에서 구입하였다. 단백질 분석을 위한 항체(p-Smad1/5, Smad1/5/8, Dlx5, Runx2, osterix, p-ERK, p-JNK, p-p38 및 β-actin)는 Santa Cruz Biotechnology(Santa Cruz, CA, USA)에서 구입하였다. 본 연구에 사용된 그 외 시약은 Sigma-Aldrich 社에서 구입하였다.The ark shell ( Scapharca subcrenata ) used in the experiment was purchased from the traditional market in Suncheon. Other materials used for cell culture were purchased from Gibro-BRL (Gaithersburg, MD, USA). Antibodies for protein analysis (p-Smad1 / 5, Smad1 / 5/8, Dlx5, Runx2, osterix, p-ERK, p-JNK, p-p38 and β-actin) are described in Santa Cruz Biotechnology (Santa Cruz, CA, USA). Other reagents used in this study were purchased from Sigma-Aldrich.
1.2. 꼬막단백질 유래 저분자 가수분해물 제조1.2. Preparation of low molecular weight hydrolyzate derived from corticoprotein
조골세포 분화 촉진 펩타이드를 제조하기 위해서 꼬막을 동결건조한 후 펩신을 이용하여 분해하였다. 효소와 기질비는 1:500으로 하였고, 반응시간은 2시간으로 하였다. 분해물은 막분리 방법을 이용하여 분자량이 1 kDa 이하의 분획물을 제조하였다.To prepare osteoblast differentiation promoting peptides, the keratoplasm was lyophilized and then digested with pepsin. The enzyme and substrate ratio were 1: 500, and the reaction time was 2 hours. The degradation product was prepared by using a membrane separation method fractions of 1 kDa or less.
1.3. 조골세포 분화 펩타이드의 분리, 정제 및 구조 동정1.3. Isolation, Purification and Structure Identification of Osteoblast Differentiation Peptides
조골세포 분화 촉진 펩타이드를 분리하기 위해서 먼저 SP-Sephadex C-25 양이온 교환 크로마토그래피를 수행하였다. 1L 비이커에 미리 양이온 교환 수지를 50 mM sodium acetate buffer (pH 4.0)으로 평형화시킨 다음 꼬막단백질 유래 저분자 펩타이드를 첨가하여 양이온 교환 수지와 반응시켰다. 1시간 후에 비흡착된 펩타이드는 필터과정을 통해 모으고 나머지 흡착부분은 NaCl을 이용하여 순차적 방법으로 용출 시켰다. 즉, NaCl 농도를 0-0.2, 0.2-0.4, 0.4-0.6 및 0.6-1.0 M로 처리하여 흡착된 펩타이드를 얻었다. 회수한 펩타이드는 투석(dialysis)을 통하여 NaCl을 제거하고 동결건조하였다. 이온교환 크로마토그래피에서 분리한 분획물을 다시 고성능액체크로마토그래피(HPLC) 상에서 Hypersil Gold C18 column(20×250mm, Thermo Scientific, PA, USA)을 이용하여 분리하였고 조골세포 분화 촉진 활성을 측정한 후에 Hypersil Gold C18 column(4.6×250mm)으로 최종 분리하였다. 분리한 펩타이드는 Q-TOF LC-MS/MS mass spectrometer에서 de-novo sequencing방법으로 펩타이드의 서열을 동정하였다.In order to separate osteoblast differentiation promoting peptides, SP-Sephadex C-25 cation exchange chromatography was first performed. The cation exchange resin was previously equilibrated with 50 mM sodium acetate buffer (pH 4.0) in a 1 L beaker and then reacted with the cation exchange resin by addition of a small molecule peptide derived from the chocoprotein. After 1 hour, the non-adsorbed peptide was collected through a filter process, and the remaining adsorption portion was eluted with sequential method using NaCl. In other words, NaCl concentrations were treated with 0-0.2, 0.2-0.4, 0.4-0.6 and 0.6-1.0 M to obtain adsorbed peptides. The recovered peptides were lyophilized by removing NaCl through dialysis. Fractions separated by ion exchange chromatography were separated on a high performance liquid chromatography (HPLC) using a Hypersil Gold C18 column (20 × 250 mm, Thermo Scientific, PA, USA) and measured after osteoblast differentiation activity. Final separation into a C18 column (4.6 × 250 mm). A separate peptides was identified a sequence of Q-TOF LC-MS / MS mass peptide to de-novo sequencing method at spectrometer.
1.4. 세포배양 및 시약 처리1.4. Cell Culture and Reagent Processing
실험에 사용된 마우스 유래 중간엽 줄기세포는 ATCC (D1 cell, CRL-12424)에서 구입하였고, 10% FBS 및 1% penicillin/streptomycin이 포함된 DMEM 배지에서 5% CO2 및 37℃ 조건에서 배양하였다. 조골세포 분화를 위해서 분화용 배지(DMEM + 50 μg/mL ascorbic acid, 10 mM β-glycerolphosphate 및 10-7M dexamethasone)는 이틀에 한번 씩 교환하였다.The mouse-derived mesenchymal stem cells used in the experiment were purchased from ATCC (D1 cell, CRL-12424) and incubated in DMEM medium containing 10% FBS and 1% penicillin / streptomycin at 5% CO 2 and 37 ° C. . For osteoblast differentiation, differentiation medium (DMEM + 50 μg / mL ascorbic acid, 10 mM β-glycerolphosphate and 10 -7 M dexamethasone) was exchanged every other day.
BMP antagonist(noggin, 100 ng/mL) 또는 MAPKs 저해제(10 μM SB203580, 20 μM PD98059 및 10 μM SP600125)는 2시간 동안 세포에 처리한 후 펩타이드가 포함된 분화용 배지를 처리하여 지정된 시간동안 배양하였다.BMP antagonist (noggin, 100 ng / mL) or MAPKs inhibitors (10 μM SB203580, 20 μM PD98059 and 10 μM SP600125) were treated with cells for 2 hours and then incubated for a specified time period with the differentiation medium containing peptides. .
1.5. 세포독성 측정1.5. Cytotoxicity Measurement
펩타이드의 세포독성 측정은 MTT(3-(4,5-dimethythiazol-2-yl)-2,5diphenyltetrazolium bromide)를 이용하였다. 24-well plate에 세포가 70-80% confluent한 상태가 되었을 때 펩타이드를 농도별로 처리한 후 24 또는 48시간 후에 1 mg/mL MTT 용액을 첨가하였다. 4시간 동안 인큐베이터에서 배양한 후, 배지를 제거하여 생성된 formazan은 DMSO에 녹여 540nm에서 흡광도를 측정하였다.The cytotoxicity of the peptide was measured using MTT (3- (4,5-dimethythiazol-2-yl) -2,5diphenyltetrazolium bromide). When cells became 70-80% confluent in 24-well plates, 1 mg / mL MTT solution was added 24 or 48 hours after the peptide was treated by concentration. After incubation for 4 hours incubator, formazan produced by removing the medium was dissolved in DMSO and the absorbance was measured at 540nm.
1.6. ALP(alkaline phosphatase) 활성 및 염색1.6. ALP (alkaline phosphatase) activity and staining
ALP 활성 측정을 위해서 96-well plate에 세포를 배양 및 펩타이드를 7일 동안 처리한 후 배지를 제거하였다. 세포를 PBS에 세척한 다음 0.1% Triton X-100가 포함된 25 mM sodium carbonate buffer (pH 10)를 이용하여 세포를 분해시키고, 상층액 100 μL를 효소반응액(1.5 mM MgCl2, 3.8 mM p-nitrophenyl phosphate가 포함된 25 mM sodium carbonate buffer) 100 μL를 혼합하여 37℃에서 90분간 반응시킨 후 405nm에서 흡광도를 측정하였다. ALP 활성은 아래와 같이 계산하였다. In order to measure ALP activity, cells were cultured in 96-well plates and treated with peptide for 7 days, and then the medium was removed. The cells were washed in PBS and then digested with 25 mM sodium carbonate buffer (pH 10) containing 0.1% Triton X-100, and 100 μL of the supernatant was digested with enzyme reaction solution (1.5 mM MgCl 2 , 3.8 mM p). 100 μL of 25 mM sodium carbonate buffer) containing -nitrophenyl phosphate was mixed and reacted at 37 ° C. for 90 minutes, and the absorbance was measured at 405 nm. ALP activity was calculated as follows.
ALP activity (%) = (A- A0)/A0 × 100ALP activity (%) = (A- A 0 ) / A 0 × 100
A: 펩타이드 처리군, A0: 무처리군A: peptide treated group, A 0 : untreated group
ALP 염색을 위해서 12-well plate에 세포를 배양한 후 펩타이드를 7일 동안 처리한 후 배지를 제거하고 PBS에 2번 세척하였다. 10% formalin 용액을 처리하여 5분간 세포를 고정한 후 다시 PBS에 세척하고 BCIP/NBT 기질 용액을 well plate에 첨가한 후 37℃에서 15분간 배양하여 현미경으로 관찰하였다.After culturing the cells in a 12-well plate for ALP staining, the peptide was treated for 7 days, and then the medium was removed and washed twice in PBS. After fixing the cells for 5 minutes by treatment with 10% formalin solution, washed again in PBS, BCIP / NBT substrate solution was added to the well plate and incubated for 15 minutes at 37 ℃ and observed under a microscope.
1.7. Alizarin red S 염색(미네랄화 측정)1.7. Alizarin red S staining (mineralization measurement)
조골세포 분화의 마지막 단계인 석회화를 측정하기 위해서 12-well plate에 세포를 배양한 후 21일 동안 펩타이드를 처리하였다. 배양이 끝난 후 세포를 PBS에 세척하고 70% 에탄올 용액을 4℃에서 1시간 처리하여 고정시켰다. 2% Alizarin red S(pH 4.2) 용액을 상온에서 15분간 처리하여 염색 후 증류수로 4번 세척하여 완전히 건조시켰다. 현미경으로 세포의 미네랄 형성을 관찰하고, 정량을 위해서 10% cetylpyridinium chloride 용액을 사용하여 염색된 Alizarin red S 용액을 탈색 시켜 반응한 양을 562nm에서 흡광도를 측정하였다. 미네랄 형성 정도는 하기 수식을 이용하여 계산하였다. To measure calcification, the final stage of osteoblast differentiation, the cells were cultured in 12-well plates and treated with peptides for 21 days. After the incubation, the cells were washed in PBS and fixed with 70% ethanol solution at 4 ° C. for 1 hour. 2% Alizarin red S (pH 4.2) solution was treated for 15 minutes at room temperature, dyed and washed 4 times with distilled water and dried completely. The mineral formation of the cells was observed under a microscope, and the absorbance was measured at 562 nm by decolorizing the Alizarin red S solution stained using 10% cetylpyridinium chloride solution for quantification. The degree of mineral formation was calculated using the following formula.
Mineralization (%) = (A- A0)/A0 × 100Mineralization (%) = (A- A 0 ) / A 0 × 100
A: 펩타이드 처리군, A0: 무처리군A: peptide treated group, A 0 : untreated group
1.8. 웨스턴 블럿팅(Western blotting)1.8. Western blotting
펩타이드를 7일 동안 처리한 후 RIPA buffer(Sigma Chemical Co.)를 이용하여 세포를 파괴한 후 단백질을 추출하여 정량하였다(Pierce BCA assay kit, Thermo Fisher Scientific, MA, USA). 동량의 단백질을 10% SDS-PAGE를 이용하여 분리한 후 PVDF 멤브레인으로 옮겼다. 5% 스킴밀크로 블러킹한 후, 1차 항체를 처리하여 4℃에서 오버나잇하였고, 이후 2차 항체를 처리하여 상온에서 2-3시간 반응시킨 후 ECL(enhanced chemiluminescence) assay kit (Pierce Biotechnology, IL, USA)를 이용하여 PVDF 멤브레인 상의 단백질을 검출하였다. After peptide treatment for 7 days, the cells were disrupted using RIPA buffer (Sigma Chemical Co.) and protein was extracted and quantified (Pierce BCA assay kit, Thermo Fisher Scientific, MA, USA). Equal amounts of protein were separated using 10% SDS-PAGE and then transferred to the PVDF membrane. After blocking with 5% skim milk, the primary antibody was treated overnight at 4 ° C., and the secondary antibody was then reacted at room temperature for 2-3 hours, followed by an enhanced chemiluminescence assay kit (Pierce Biotechnology, IL). , USA) was used to detect proteins on PVDF membranes.
1.9. 마우스 준비 및 약물처리 프로토콜1.9. Mouse Preparation and Drug Handling Protocols
실험에 사용된 모든 마우스는 8주령의 C57BL/6N 암컷 마우스이며 Envigo社 (미국)로부터 구입하였고 1주일간 순화기간을 거쳤다.All mice used in the experiment were 8-week-old C57BL / 6N female mice, purchased from Envigo (USA) and undergoing a 1 week accrual period.
골다공증 모델을 만들기 위해 한 그룹 당 4-5마리 총 18마리의 마우스에게 난소적출 수술(OVX)을 실시하였고, 일주일 후, 서열번호 1(AWLNH) 및 2(PHDL) 펩타이드 (각각 5 μg/25 g 마우스), 양성대조군으로 17β-estradiol (Est, 0.5 μg/25 g 마우스) 및 PBS(Gibco) 100 μL를 하루에 한 번 2달간 복강 투여하였다. 대조군으로는 골다공증의 Sham 모델로 한 그룹당 4마리의 암컷 마우스를 피하절개만 실시하였고, 100 μL PBS를 복강 투여하였다.A total of 18 mice, 4-5 mice per group, were subjected to ovarian extraction surgery (OVX) to create an osteoporosis model, and one week later, SEQ ID No. 1 (AWLNH) and 2 (PHDL) peptides (5 μg / 25 g, respectively). Mice), 17β-estradiol (Est, 0.5 μg / 25 g mice) and 100 μL of PBS (Gibco) were intraperitoneally administered once a day for 2 months as a positive control. As a control group, four female mice per group were subjected to subcutaneous incision only in the Sham model of osteoporosis, and 100 μL PBS was intraperitoneally administered.
난소적출 수술 후 일주일 후에 골다공증의 효율적 유발을 위하여 칼슘결핍 식이(Envigo社, 미국)를 제공하였다. 정상 마우스는 수술 없이 정상 식이를 제공하였다.One week after ovarian extraction, a calcium deficiency diet (Envigo, USA) was provided for efficient induction of osteoporosis. Normal mice received a normal diet without surgery.
1.10. 마이크로 씨티(micro CT)1.10. Micro CT
마우스로부터 대퇴골을 분리하여 근육을 제거하고 생리식염수로 세척한 후 24시간 동안 4% 포르말린에 고정시키고 골밀도를 측정하였다. 대퇴골의 미세 단층 촬영은 micro-CT scanner (Inveon preclinical CT, Siemens Healthcare, 미국)를 사용하여 샘플 길이 1.9 cm, 폭 2 cm, 80 keV의 광자 에너지, 500 μA의 전류를 흘려 측정하였다. 골밀도 측정을 위해서 그룹당 동일한 부위의 2 mm3의 부피 부분을 Siemens Inveon Software를 이용하여 측정하였다.The femur was separated from the mouse to remove muscle, washed with physiological saline, fixed in 4% formalin for 24 hours, and bone density was measured. Microscopic tomography of the femur was measured using a micro-CT scanner (Inveon preclinical CT, Siemens Healthcare, USA) with a sample length of 1.9 cm, width 2 cm, photon energy of 80 keV, and a current of 500 μA. For bone density measurement, a volume portion of 2 mm 3 of the same site per group was measured using Siemens Inveon Software.
실시예 2. 꼬막단백질 유래 저분자 펩타이드의 ALP(alkaline phosphatase) 활성 결과Example 2 Results of Alkaline Phosphatase Activity of Small Molecule Peptides Derived from Corticoproteins
분획된 꼬막단백질 유래 저분자 펩타이드는 이온 교환 크로마토그래피에 의해서 분리되었고, 분리된 획분에 대하여 조골세포에서 ALP 활성에 미치는 영향은 조사하였다. 그 결과, 0.6-1.0M NaCl 농도에서 용출된 획분에서 blank(무처리군)보다 210% 높은 ALP 활성을 보임을 확인하였다(도 1).The fractionated small protein peptides were isolated by ion exchange chromatography, and the fractions were examined for their effects on ALP activity in osteoblasts. As a result, it was confirmed that the fraction eluted at a concentration of 0.6-1.0M NaCl showed 210% higher ALP activity than the blank (untreated group) (Fig. 1).
이후, 양이온 교환 크로마토그래피에서 분리된 획분을 Hypersil Gold C18 column(20×250mm)이 장착된 HPLC 상에서 분리하였다. 유속은 2.5 mL/min, 이동상은 0.1% TFA/H2O와 0.1% TFA/acetonitrile하여 선형상 농도구배법(0-60% acetonitrile, 2-25min)으로 용출시킨 다음, 각 분획물을 분리하였다. 분리한 결과 16-18min 사이에서 용출된 획분에서 가장 우수한 ALP 활성을 보였다(도 2A). 이 획분을 Hypersil Gold C18 column(4.6×250mm)이 장착된 HPLC 상에서 다시 분리하였다. 유속은 1.0 mL/min, 동일한 이동상에서 선형상 농도구배법(10-30% acetonitrile, 0-17min) 분리하였고, 그 결과 1번 획분에서 가장 우수한 ALP 활성을 나타내어(도 2B), 동일한 조건으로 다시 분리하였다. 도 2C에서 나타나듯이 1번 획분에서 가장 우수한 ALP 활성을 보여 이 획분을 유속 0.9 mL/min, 선형상 농도구배법(10-20%, 0-17min)으로 분리한 결과, 도 2D와 같이 최종 분리하였다.The fractions separated in cation exchange chromatography were then separated on HPLC equipped with Hypersil Gold C18 column (20 × 250 mm). The flow rate was 2.5 mL / min, the mobile phase was eluted with 0.1% TFA / H 2 O and 0.1% TFA / acetonitrile and eluted with a linear phase gradient method (0-60% acetonitrile, 2-25min), and each fraction was separated. The separation showed the best ALP activity in fractions eluted between 16-18 min (FIG. 2A). This fraction was separated again on HPLC equipped with Hypersil Gold C18 column (4.6 × 250 mm). The flow rate was 1.0 mL / min, linear phase gradient (10-30% acetonitrile, 0-17 min) isolated in the same mobile phase, resulting in the best ALP activity in fraction 1 (FIG. 2B), again under the same conditions. Separated. As shown in FIG. 2C, the best fraction of ALP was observed in fraction 1, and the fraction was separated by a flow rate of 0.9 mL / min and a linear concentration gradient method (10-20%, 0-17min). It was.
분리된 최종 획분을 Q-TOF LC-MS/MS를 이용하여 de-novo sequencing한 결과 두 개의 펩타이드를 확인할 수 있었고 이들의 펩타이드 서열은 Ala-Try-Leu-Asn-His(AWLNH, 640.3Da: 서열번호 1) 및 Pro-His-Asp-Leu(PHDL, 480.2Da: 서열번호 2)로 확인되었다(도 3). De-novo sequencing of the isolated final fractions using Q-TOF LC-MS / MS identified two peptides, and their peptide sequences were Ala-Try-Leu-Asn-His (AWLNH, 640.3Da: sequence). No. 1) and Pro-His-Asp-Leu (PHDL, 480.2 Da: SEQ ID NO: 2) (FIG. 3).
상기 두 개의 펩타이드는 조골세포 분화 촉진능을 확인하기 위해서 Fmoc SPSS(solid phase peptide synthesis) 방법으로 합성하였다(Peptron Inc. 서울). 합성된 두 개의 펩타이드는 조골세포 분화에서 ALP 활성에 미치는 영향을 측정하였다. 그 결과, 아주 강력한 ALP 활성 증가를 보였는데, 특히, 4 μM 농도에서 본 발명의 서열번호 1의 펩타이드(AWLNH)는 210%, 본 발명의 서열번호 2의 펩타이드(PHDL)은 187%의 ALP 활성을 보였다(도 4).The two peptides were synthesized by Fmoc solid phase peptide synthesis (SPSS) method to confirm osteoblast differentiation promoting ability (Peptron Inc. Seoul). Two synthesized peptides were evaluated for their effect on ALP activity in osteoblast differentiation. As a result, it showed a very strong increase in ALP activity, in particular, the peptide of SEQ ID NO: 1 of the present invention (AWLNH) 210%, the peptide of SEQ ID NO: 2 of the present invention (PHDL) of 187% at 4 μM concentration Was shown (FIG. 4).
실시예 3. 조골세포 분화에 관여하는 단백질 발현 결과Example 3 Results of Protein Expression Involved in Osteoblast Differentiation
ALP 활성 측정에서 본 발명의 두 종류의 펩타이드가 4 μM 농도에서 아주 우수하였기 때문에 4 μM 농도를 처리하여 다음 실험을 진행하였다. 세포주에 7일간 본 발명의 펩타이드를 처리한 후 단백질을 추출하여 웨스턴 블럿팅을 실시하였다. 그 결과, 두 종류의 펩타이드는 조골세포 분화에 관여하는 BMP-2/4(bone morphogenetic protein)의 발현을 증가시키는 것으로 확인되었다(도 5). Since two peptides of the present invention were very good at 4 μM concentration in the ALP activity measurement, the following experiment was conducted by treating the 4 μM concentration. After treatment with the peptide of the present invention for 7 days to the cell line, the protein was extracted and subjected to Western blotting. As a result, the two peptides were found to increase the expression of BMP-2 / 4 (bone morphogenetic protein) involved in osteoblast differentiation (Fig. 5).
또한, BMP-2/4는 다운스트림 신호체계에 있는 Smad1/5의 인산화를 촉진시켜 세포내 신호전달을 촉진시키고 인산화된 Smad1/5는 조골세포 분화 및 형성에 관여하는 특정 단백질을 생산하는데 중요한 역할을 하는 전사조절인자인 Dlx-5, Runx-2 및 osterix를 활성화 여부를 확인하였다. 그 결과, 본 발명의 두 종류의 펩타이드는 BMP-2/4의 발현을 증가시켜 다운스트림에 있는 신호전달물질들인 Smad1/5 및 전사조절인자인 Dlx-5, Runx-2 및 Osterix가 모두 발현 또는 활성을 증가시킴으로써, 조골세포의 분화를 촉진시키는 것을 확인하였다(도 5). 또한, 조골세포 분화의 초기 바이오마커인 type I collagen의 발현을 강하게 증가시키는 것도 확인하였다(도 5). In addition, BMP-2 / 4 promotes the phosphorylation of Smad1 / 5 in downstream signaling systems to promote intracellular signaling, while phosphorylated Smad1 / 5 plays an important role in producing specific proteins involved in osteoblast differentiation and formation. The transcriptional regulators Dlx-5, Runx-2 and osterix were confirmed to be activated. As a result, the two peptides of the present invention increase the expression of BMP-2 / 4 so that the downstream signaling agents Smad1 / 5 and the transcriptional regulators Dlx-5, Runx-2 and Osterix are expressed or By increasing the activity, it was confirmed to promote the differentiation of osteoblasts (Fig. 5). It was also confirmed that the expression of type I collagen, an early biomarker for osteoblast differentiation, was strongly increased (FIG. 5).
또한, MAPKs(mitogen-activated protein kinases)는 조골세포 분화에 있어서 중요한 역할을 하는 것으로 알려져 있어, 본 발명자들은 본 발명의 두 종류의 펩타이드가 MARKs에 활성시키는 효과가 있는지를 확인하는 실험을 진행하였다. 그 결과, MARKs인 p-p38, p-ERK 및 p-JNK의 인산화를 증가시켜 조골세포 분화를 촉진시키는 것을 확인하였다(도 6).In addition, mitogen-activated protein kinases (MAPKs) are known to play an important role in osteoblast differentiation, and the present inventors conducted an experiment to confirm whether the two types of peptides of the present invention have an effect of activating MARKs. As a result, it was confirmed that phosphorylation of MARKs p-p38, p-ERK and p-JNK was increased to promote osteoblast differentiation (FIG. 6).
실시예 4. 펩타이드에 의한 조골세포의 미네랄 형성 결과Example 4 Mineral Formation of Osteoblasts by Peptides
본 발명의 두 종류의 펩타이드(AWLNH 및 PHDL)를 21일 동안 처리한 후 미네랄 형성을 Alizarin red S 염색으로 관찰하였다. 그 결과, 본 발명의 두 종류의 펩타이드는 조골세포의 미네랄 형성을 강하게 촉진하는 것으로 확인되었다. 구체적으로 서열번호 1의 펩타이드(AWLNH)은 대조군에 비해 289%, 서열번호 2의 펩타이드(PHDL)는 259%의 미네랄 형성이 증가되었음을 확인하였다(도 7). After treatment with two kinds of peptides (AWLNH and PHDL) of the present invention for 21 days, mineral formation was observed by Alizarin red S staining. As a result, it was confirmed that two kinds of peptides of the present invention strongly promote mineral formation of osteoblasts. Specifically, the peptide of SEQ ID NO: 1 (AWLNH) was 289% compared to the control group, the peptide of SEQ ID NO: 2 (PHDL) was confirmed that the mineral formation of 259% increased (Fig. 7).
실시예 5.Example 5.
BMP 길항제(noggin) 및 MAPKs 저해제 처리에 따른 BMP 신호전달경로 및 MAPKs 인산화에 미치는 영향Effects of BMP Antagonists and MAPKs Inhibitors on BMP Signaling Pathway and MAPKs Phosphorylation
본 발명의 두 종류의 펩타이드가 BMP 신호전달경로 및 MAPKs 인산화 경로의 활성화에 따른 조골세포 분화 촉진을 규명하기 위해, 먼저 BMP의 길항제인 noggin을 처리한 후 관련 단백질 발현을 웨스턴 블럿팅으로 확인하였다. 그 결과, noggin과 펩타이드를 함께 처리한 군에서는 펩타이드만 처리한 군에 비해 BMP-2/4 및 다운스트림 신호전달물질의 발현이 현저히 감소함을 확인하였다(도 8).In order to investigate osteoblast differentiation according to activation of BMP signaling pathway and MAPKs phosphorylation pathway, two kinds of peptides of the present invention were first treated with noggin, an antagonist of BMP, and then related protein expression was confirmed by Western blotting. As a result, it was confirmed that in the group treated with noggin and peptide, the expression of BMP-2 / 4 and downstream signal transducers was significantly reduced compared to the group treated with peptide alone (FIG. 8).
일반적으로 BMPs의 활성화는 MAPKs의 인산화 증가와 관련이 있다고 알려져 있으나, 본 발명에서는 BMP 길항제를 처리한 경우 펩타이드에 의한 MAPKs(p-p38, p-ERK 및 p-JNK) 인산화에 거의 영향을 주지 않는 것을 확인하였다(도 9).In general, activation of BMPs is known to be associated with increased phosphorylation of MAPKs, but the present invention has little effect on the phosphorylation of MAPKs (p-p38, p-ERK and p-JNK) by peptide when treated with BMP antagonists. It was confirmed (Fig. 9).
반면, MAPKs 저해제인 SB203585(p38 저해제), PD98059(ERK 저해제) 및 SP600125(JNK 저해제)를 처리한 결과, 본 발명의 펩타이드만 처리한 군에서는 BMP-2/4, 인산화 Smad1/5 및 전자조절인자인 Runx-2의 발현이 증가하였으나, MAPKs 저해제를 함께 처리한 군에서는 BMP-2/4, 인산화 Smad1/5 및 전자조절인자인 Runx-2의 발현이 억제됨을 확인하였다(도 10). 특히, PD98059(ERK 저해제) 및 SP600125(JNK 저해제) 처리에 의해서 BMP 신호가 강하게 저해됨을 확인하였다.On the other hand, as a result of treatment of MAPKs inhibitors SB203585 (p38 inhibitor), PD98059 (ERK inhibitor) and SP600125 (JNK inhibitor), BMP-2 / 4, phosphorylated Smad1 / 5 and electron regulators in the peptide-treated group Phosphorus Runx-2 expression was increased, but in the group treated with the MAPKs inhibitor, it was confirmed that the expression of BMP-2 / 4, phosphorylated Smad1 / 5 and the electronic regulator, Runx-2 (Fig. 10). In particular, it was confirmed that the BMP signal was strongly inhibited by PD98059 (ERK inhibitor) and SP600125 (JNK inhibitor) treatment.
MAPKs 저해제가 조골세포 분화에 미치는 영향을 확인하기 위해서 조골세포 분화 초기 바이오마커인 ALP의 발현을 염색을 통하여 확인하였다. 그 결과, 본 발명의 두 종류의 펩타이드를 각각 처리한 세포에서는 ALP의 발현이 증가하여 아주 강하게 염색되는 것을 확인할 수 있었으나, MAPKs 저해제 중 PD98059(ERK 저해제) 및 SP600125(JNK 저해제)에 의해서는 ALP의 발현이 현저히 감소하는 것을 확인하였다(도 11). In order to confirm the effect of MAPKs inhibitors on osteoblast differentiation, the expression of ALP, an early biomarker for osteoblast differentiation, was confirmed by staining. As a result, it was confirmed that the cells treated with the two types of peptides of the present invention increased the expression of ALP and stained very strongly. However, PD98059 (ERK inhibitor) and SP600125 (JNK inhibitor) among MAPKs inhibitors showed that It was confirmed that the expression was significantly reduced (FIG. 11).
따라서, 본 발명의 두 종류의 펩타이드는 ERK 및 JNK의 인산화에 영향을 주는 신호전달경로에 관여하고, BMPs 신호전달경로의 활성화를 시킴으로써, 조골세포 분화를 촉진시킬 수 있어 골질환, 특히 골다공증의 치료에 효과가 있음을 확인하였다.Thus, the two peptides of the present invention are involved in the signaling pathways that affect the phosphorylation of ERK and JNK, and can activate osteoblast differentiation by activating BMPs signaling pathways, thereby treating bone diseases, in particular osteoporosis. It was confirmed that the effect on.
실시예 6.Example 6.
골다공증 모델에서 펩타이드가 골형성에 미치는 영향Effect of Peptides on Bone Formation in Osteoporosis Model
본 발명의 서열번호 1(AWLNH (P1)) 및 서열번호 2(PHDL (P2)) 펩타이드가 난소적출 및 칼슘결핍식이로 유도한 골다공증 모델에서 골형성에 미치는 영향을 측정하였다. Micro CT 촬영 사진과 골밀도 변화 결과에 대한 그래프는 도 12에 나타내었다. 골다공증을 유발한 모델에 서열번호 1(AWLNH (P1)) 및 서열번호 2(PHDL (P2))의 펩타이드를 각각 투여한 결과 PBS만 주입한 마우스보다 골밀도가 유의적으로 증가한 것으로 확인되었고, 양성대조군으로 사용된 17β-estradiol(Est, 에스트로겐)과 유사한 골형성 정도를 나타냄을 확인하였다.The effects of the SEQ ID NO: 1 (AWLNH (P1)) and SEQ ID NO: 2 (PHDL (P2)) peptides of the present invention on bone formation in the osteoporosis model induced by ovarian extraction and calcium deficiency diet were measured. A micro CT photograph and a graph of bone mineral density change results are shown in FIG. 12. When the peptides of SEQ ID NO: 1 (AWLNH (P1)) and SEQ ID NO: 2 (PHDL (P2)) were respectively administered to the model causing osteoporosis, it was confirmed that BMD was significantly increased compared to mice injected with PBS alone. It was confirmed that the degree of bone formation similar to 17β-estradiol (Est, estrogen) used as.
Claims (8)
- 서열번호 1(AWLNH) 또는 서열번호 2(PHDL)를 포함하는 골 형성 촉진용 펩타이드.Peptides for promoting bone formation comprising SEQ ID NO: 1 (AWLNH) or SEQ ID NO: 2 (PHDL).
- 제 1 항에 있어서, The method of claim 1,상기 펩타이드는 꼬막(Scapharca subcrenata) 단백질 유래인 것을 특징으로 하는 펩타이드.The peptide is a peptide, characterized in that derived from Scapharca subcrenata protein.
- 제 1 항에 있어서, The method of claim 1,상기 펩타이드는 조골세포의 분화를 촉진시키는 것을 특징으로 하는 펩타이드.The peptide is characterized in that to promote the differentiation of osteoblasts.
- 제 1 항 내지 제 3 항 중 어느 한 항의 펩타이드를 포함하는 골질환의 예방 또는 치료용 조성물.A composition for preventing or treating bone diseases comprising the peptide of any one of claims 1 to 3.
- 제 4 항에 있어서,The method of claim 4, wherein상기 골질환은 골다공증, 골관절염, 류미티스 관절염, 골연화증, 구루병, 섬유성 골염, 무형성 골질환 및 대사성 골질환으로 이루어진 그룹에서 선택되는 어느 하나인 것을 특징으로 하는 조성물.The bone disease is a composition characterized in that any one selected from the group consisting of osteoporosis, osteoarthritis, rheumatoid arthritis, osteomalacia, rickets, fibrous osteoarthritis, aplastic bone disease and metabolic bone disease.
- 제 1 항 내지 제 3 항 중 어느 한 항의 펩타이드를 포함하는 조골세포 분화 촉진용 조성물. Osteoblast differentiation promoting composition comprising the peptide of any one of claims 1 to 3.
- 제 1 항 내지 제 3 항 중 어느 한 항의 펩타이드를 포함하는 골기능 개선용 식품 조성물. Food composition for improving bone function comprising the peptide of any one of claims 1 to 3.
- 약학적으로 유효한 양의 제1항의 펩타이드를 개체에 투여하는 단계를 포함하는 골다공증 예방 및 치료방법.A method for preventing and treating osteoporosis, comprising administering to a subject a pharmaceutically effective amount of the peptide of claim 1.
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| HYUNG, JUN-HO ET AL.: "steoblastogenic Activity of Ark Shell Protein Hydrolysates with Low Molecular Weight in Mouse Mesenchymal Stem Cells", ROYAL SOCIETY OF CHEMISTRY ADVANCES, vol. 6, 2016, pages 29365 - 29370 * |
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