WO2018062895A1 - Composition comprising osmundacetone or pharmaceutically acceptable salt thereof for preventing or treating bone disease - Google Patents

Composition comprising osmundacetone or pharmaceutically acceptable salt thereof for preventing or treating bone disease Download PDF

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Publication number
WO2018062895A1
WO2018062895A1 PCT/KR2017/010822 KR2017010822W WO2018062895A1 WO 2018062895 A1 WO2018062895 A1 WO 2018062895A1 KR 2017010822 W KR2017010822 W KR 2017010822W WO 2018062895 A1 WO2018062895 A1 WO 2018062895A1
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WIPO (PCT)
Prior art keywords
bone
osmundacetone
disease
extract
osteoporosis
Prior art date
Application number
PCT/KR2017/010822
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French (fr)
Korean (ko)
Inventor
박길홍
손그림
홍성수
최춘환
최윤혁
김우중
김한경
강재우
이은정
박석인
이기호
최민지
Original Assignee
주식회사 코팜
고려대학교 산학협력단
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
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Publication date
Priority claimed from KR1020160142422A external-priority patent/KR101848489B1/en
Application filed by 주식회사 코팜, 고려대학교 산학협력단 filed Critical 주식회사 코팜
Priority to CN201780074399.XA priority Critical patent/CN110167539A/en
Priority to US16/338,354 priority patent/US20200129450A1/en
Publication of WO2018062895A1 publication Critical patent/WO2018062895A1/en

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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
    • 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
    • A23L33/105—Plant extracts, their artificial duplicates or their derivatives
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/12—Ketones
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
    • A61K36/11—Pteridophyta or Filicophyta (ferns)

Definitions

  • compositions for the prevention or treatment of bone diseases including osmundacetone or pharmaceutically acceptable salts thereof
  • the present invention relates to a composition for preventing or treating bone diseases, including pharmaceutically acceptable salts of osmundacetoiie or ° ⁇ , more specifically osmundacetone or a pharmaceutically acceptable salt or fern thereof (Osmunda japonica
  • the present invention relates to a pharmaceutical composition for the prevention or treatment of bone diseases, extracts, food compositions for improvement, uses for the preparation of a prophylactic or therapeutic agent, and a method of treatment.
  • osteoporosis is caused when the balance between absorption and production of the beet is broken by activation of osteoclasts, and the absorption amount is greater than the production amount. Osteoporosis increases the frequency of fractures by decreasing the density of bone parenchyma. It occurs most often in women with hormonal balance, including middle-aged and older women. It also occurs in patients who are unable to move due to fractures or severe disease. Recently, the incidence has increased in older men and older men.
  • osteoclast progenitor cells proliferate and survive through the ERK and PI3K / Akt pathways (Mancini et al., 1997).
  • RANK.L (0PGL, 0DF, TRANCE) and RAN also regulate osteoclast formation and function (Anderson DM et al., 1997; Dougal 1 WC et al., 1999; Kong YY et al., 1999) .
  • TNF receptor-associated f actors such as TRAFs 1, 2, 3, 5, and 6 bind RANK (Darnay BG et al., 1998; Walsh MC and Choi Y, 2003). TRAF6 is most important for osteoclast formation and function (Loniaga MA et al., 1999; Naito A et al., 1999).
  • TRAF6 modulates RANKL / RANK signals to NF ⁇ , c-Jun N-—terminal kinase (JNK), extracellular s igna ⁇ regulated kinase (ERK), p38, Akt, and Nuclear Factor Of Activated T-Cel Is l (NFATcl). And osteoclast proliferation, fusion, and differentiation (Kobayashi N et al., 2001; Loniaga MA et al., 1999; Naito A et al., 1999; Takayanagi H et al., 2002; Wong BR et al. , 1998; Wong BR et al., 1999)
  • the existing direction of developing a treatment for osteoporosis has been to find a substance that can prevent the loss of bone parenchyma by inhibiting the absorption of osteoclasts.
  • the representative drug is bisphosphonate family Fosainax.
  • Leukotriene—B4 is one of the metabolites of the 5-1 ipoxygenase pathway, a metabolic pathway of arachidonate (Ford-Hutchinson, AW et al., 1980).
  • C433 an interstitial cell obtained from giant cell tumors, has been reported to increase the number and activity of osteoblasts by increasing 5-1 ipoxygenase vs. aquatic products (Mundy, GR et al., 1993). Increased bone resorption has been observed when LTB4 is administered during bone tissue culture (Bonewald, LF et al., 1996). In w? And in In vivo studies have shown that LTB4 induces bone resorption by increasing the production of osteoclasts (Bonewald, LF et al., 1996). Accordingly, many LTB4 receptor antagoni sts have been developed for the treatment of osteoporosis, but they have not been successful in inhibiting the bone parenchymal uptake of osteoclasts.
  • Forsteo / Forteo is a drug for hypersensitivity reactions, pregnant women, breastfeeding, hypercalcemia, renal failure, hyperparathyroidism and Paget 'disease.
  • the patient population is not large because it cannot be used for unexplained elevations of alkaline phosphatase, radiation therapy patients, bone marrow cancer or bone metastases.
  • the present inventors studied natural ingredients to develop a bone-related disease treatment agent with low side effects, safety, and excellent effects, and thus, the extracts of ferns, which have been used as food for a long time, have bone loss inhibitory activity. Confirmed to complete the present invention.
  • a pharmaceutical for the prevention or treatment of any one or more bone diseases selected from the group consisting of osteoporosis, rheumatoid arthritis, joint pain, Paget's disease, bone metastases and fractures, including Osmundacetone or a pharmaceutically acceptable salt thereof Provide ever composition It is.
  • Another object of the present invention is to provide a third object of the present invention.
  • An effective amount of Osmundacetone or a pharmaceutically acceptable salt thereof is administered to a subject in need thereof.
  • a bone disease treatment method selected from the group consisting of osteoporosis, rheumatoid arthritis, arthralgia, Pajet disease, bone metastases and fractures.
  • Osteoporosis, rheumatoid arthritis, arthralgia, Paget's disease characterized by administering an effective amount of the fern (fls / w // M3 ⁇ 4 japonica) extract to a subject in need thereof. It is to provide a method for treating any one or more bone diseases selected from the group consisting of bone metastases and fractures.
  • the present invention provides
  • a pharmaceutical for the prevention or treatment of any one or more bone diseases selected from the group consisting of osteoporosis, rheumatoid arthritis, joint pain, Paget's disease, bone metastases and fractures, including Osmundacetone or a pharmaceutically acceptable salt thereof Provides a composition.
  • a pharmaceutical for the prevention or treatment of any one or more bone diseases selected from the group consisting of osteoporosis, rheumatoid arthritis, arthralgia, arthritis, bone metastases and fractures, wherein Osmundacetone or a pharmaceutically acceptable salt thereof is an active ingredient Provides a composition.
  • Osmundacetone or a pharmaceutically acceptable salt thereof for the prevention or treatment of any one or more bone diseases selected from the group consisting of osteoporosis, rheumatoid arthritis, joint pain, Paget's disease, bone metastases and fractures consisting essentially of an active ingredient Provide pharmaceutical compositions. '
  • ⁇ 53> Food for preventing or ameliorating any one or more bone diseases selected from the group consisting of osteoporosis, rheumatoid arthritis, joint pain, Paget's disease, bone metastases and fractures, including Osmundacetone or a pharmaceutically acceptable salt thereof Provide composition for do.
  • Osteoporosis, rheumatoid arthritis, arthralgia, Paget's disease, bone metastasis cancer and fractures which are selected from the group consisting of Osmunda j apon i ca extracts for the prevention or treatment of any one or more bone diseases It provides a pharmaceutical composition.
  • Osteoporosis, rheumatoid arthritis, arthralgia, Paget's disease, bone metastases and bone fractures which are composed essentially of the extract of Osmunda j aponi ca. It provides a pharmaceutical composition.
  • the present invention is
  • food compositions for preventing or improving one or more bone diseases are provided.
  • the present invention provides
  • Osteoporosis, rheumatoid arthritis, arthralgia, Paget's disease, bone metastases and fractures which are composed essentially of the extract of Osmunda j aponi ca.
  • a composition for food which are composed essentially of the extract of Osmunda j aponi ca.
  • the present invention provides
  • Osteoporosis rheumatoid arthritis, joint pain.
  • Osmundacetone or a pharmaceutically acceptable salt thereof for use in the manufacture of an agent for the prophylaxis or treatment of any one or more bone diseases selected from the group consisting of Paget's disease, bone metastases and fractures.
  • the present invention provides
  • Osteoporosis rheumatoid arthritis, characterized in that an effective amount of Osmundacetone or a pharmaceutically acceptable salt thereof is administered to a subject in need thereof.
  • Fertilizer ( ⁇ »3 ⁇ 4 / ⁇ 3 ⁇ 4 japonica for use in the manufacture of a preparation for the prevention or treatment of any one or more bone diseases selected from the group consisting of osteoporosis, rheumatoid arthritis, arthralgia, Paget's disease, bone metastases and fractures ) Provides the use of extracts.
  • the present invention is
  • ⁇ 89> At least one selected from the group consisting of osteoporosis, rheumatoid arthritis, arthralgia, Paget's disease, metastatic cancer and section 3 ⁇ 4, which is administered to an individual in need thereof an effective amount of the fern 7 ⁇ / ⁇ japonica) extract. Providing a way to treat bone disease
  • the present invention will be described in detail.
  • the invention relates to the prevention or prevention of any one or more bone diseases selected from the group consisting of osteoporosis, rheumatoid arthritis, arthralgia, Paget's disease, bone metastases and fractures, including osmundacetone or a pharmaceutically acceptable salt thereof.
  • bone diseases selected from the group consisting of osteoporosis, rheumatoid arthritis, arthralgia, Paget's disease, bone metastases and fractures, including osmundacetone or a pharmaceutically acceptable salt thereof.
  • the pharmaceutical composition according to the present invention may be a composition containing osmundacetone as an active ingredient, a composition consisting of osmundacetone as an active ingredient, or a composition consisting essentially of osmundacetone as an active ingredient. It may be.
  • the term 'comprising' is used in the same sense as 'including' or 'characterized by', and the composition or method according to the present invention. Does not exclude additional components or steps of the method not specifically mentioned.
  • the term 'consisting of' means to exclude additional elements, steps or components, etc., unless otherwise noted.
  • the term 'essentially consisting of' means that, within the scope of the composition or method, may include the materials or steps described, as well as materials or steps that do not substantially affect the basic properties thereof. .
  • Osmundacetone is represented by the molecular formula of C 10 H 10 3 ⁇ 4 (molecular weight 178.184Da).
  • Osmundacetone included in the composition of the present invention may be used on its own or in the form of a salt, preferably a pharmaceutically acceptable salt.
  • 'pharmaceutically acceptable 1' means a physiologically acceptable and, when administered to a human, usually does not cause an allergic reaction or a similar reaction.
  • an acid addition salt formed with a pharmaceutically acceptable free acid is preferable.
  • Organic acids and inorganic acids may be used as the free acid.
  • the organic acid is not limited thereto, citric acid, acetic acid, lactic acid, tartaric acid, maleic acid, fumaric acid, formic acid, propionic acid, oxalic acid, tripoloroacetic acid, benzoic acid, gluconic acid, metasulfonic acid, glycolic acid, succinic acid, 4-frame Luenesulfonic acid, glutamic acid and aspartic acid.
  • the inorganic acid includes, but is not limited to, hydrochloric acid, bromic acid, sulfuric acid and phosphoric acid.
  • the osmundacetone may be chemically synthesized or may be separated from natural products.
  • the osmundacetone may be separated from the Osmundaceae plant when using osmundacetone collected from natural products.
  • Most preferably in the fern It may be separated from belong to L ⁇ (OsmuncIa ' a / 3 ⁇ 4 / ca).
  • Osniundales is a group of old ferns that originated in the Mesozoic, Triassic, about 210 million years ago, and is classified as Leptosporangiate fern.
  • the fern belonging to the fern family ((3 ⁇ 4 (7 ⁇ japonica) scientific name is Osmunda japonica Thunb.
  • ⁇ - he is called 'The Os inda nipponica Makino, Latin people have a Osmundae Rhizoma. It is also called Japanese royal fern or Japanese flowering fern. It is native to East Asia and Russia in Japan, China, Korea and Taiwan. Fern young leaves have been used as food ingredients for a long time, as well as medicinal plants that have been used for various diseases in traditional medicine and folk medicine. Ferns are osmunda lactone, osmundacetone, osmunda 1 in, di hydro i soomunda 1 in. It contains parasorboside and morphogenetic hormones such as ponasterone A, ecdysone and ecdysterone.
  • root stems are called porcelain ( ⁇ or spectators), and they have some poisons, but they are gray, chonbaek, urine, etc., antiviral, antibacterial, etc. ( ⁇ ⁇ ⁇ ) ), Is used for the treatment of bloated hemorrhoids (0 ⁇ ⁇ ⁇ ⁇ ), wind-heat sensation, hyperthermia, bleeding, bleeding, and hemorrhoids caused by hemostasis, severity, and sputum (Chinese medicine dictionary).
  • the extract of ferns effectively inhibits the differentiation of osteoclasts, which serve to destroy and reabsorb bone tissue.
  • monocyte cells, stem cells progenitor cells of osteoclasts were isolated and stimulated with differentiation-promoting factors, RANKL and M—CSF, and the treatment of fern extracts confirmed the effects on osteoclast differentiation.
  • Fertilized hot water extract or ethyl acetate extract effectively inhibited the differentiation of myeloid cells into multinucleated osteoclasts.
  • Osmundacetone is a single compound that has been identified as isolated from fern hydrothermal extracts and ethyl acetate extracts / fractions. Osmundacetone has excellent osteoclast differentiation inhibitory activity and osteoblast activation activity. As well as having cytotoxicity, it was shown to be very low and safe. Therefore, the skilled person is using the above-described activity of fern extract and osmundacetone, which the inventors have identified, as the balance of bone absorption by osteoclasts, formation of new bone matrix by osteoblasts, and bone dashing of the subsequent mineralization process is broken.
  • the bone diseases in the present invention are osteoporosis, rheumatoid arthritis, vascular tube. It may be nodal pain, Paget's disease, bone metastasis cancer, or fracture, and the application of the present specification for the correlation between each disease and osteoclast is referred to. Means a clinical procedure to change the process, but can also be performed to prevent clinical pathology.
  • the desirable effects of treatment include: inhibiting the occurrence or recurrence of the disease, alleviating the symptoms, reducing any direct or indirect pathological consequences of the disease, decreasing the rate of disease progression, improving, improving, alleviating, or improving the prognosis of the disease.
  • prevention refers to any action that inhibits or delays the progression of a disease
  • the dosage of the pharmaceutical composition of the present invention is the route of administration, time of administration, frequency of treatment, treatment
  • a person skilled in the art can determine the appropriate effective amount for the specific use described above, taking into account various factors such as duration, age, weight, health condition, sex, severity of the disease, drug sensitivity, diet and excretion rate, etc.
  • the term “effective amount” refers to an amount sufficient to exert an effect of improving, treating, preventing, detecting, or diagnosing a bone disease when administered to an individual. It may preferably be a mammal, most preferably an animal, including a human, and may be cells, tissues, organs, etc. derived from the animal, etc.
  • the subject may be a patient with bone disease (pat i ent) in need of treatment.
  • the administration may be administered once or several times a day.
  • the pharmaceutical composition of the present invention may be administered alone or in combination with other therapeutic agents known to be effective in the prevention or treatment of bone diseases, and when administered in combination, may be administered sequentially or simultaneously with other therapeutic agents.
  • the foot when administered alone or in combination
  • the dosage of the pharmaceutical composition of the title is preferably administered in an amount that can achieve the maximum effect in a minimum amount without side effects, which can be easily determined by those skilled in the art.
  • the total effective amount of the composition of the present invention may be administered to a patient in a single dose, and may be administered by a fractionated treatment protocol that is administered at multiple doses for a long time. Can be.
  • the pharmaceutical composition of the present invention may vary the content of the active ingredient depending on the extent of the disease.
  • the total dose of the pharmaceutical composition of the present invention may be about 0.0 to 10,000 mg, most preferably 0.1 to 50 ng per kg of patient body weight per day.
  • the dosage of the pharmaceutical composition may be determined by taking into consideration the various factors such as the age, weight, health condition, sex, severity of the disease, diet and excretion rate, as well as the formulation method, route of administration and frequency of treatment. As will be determined, one of ordinary skill in the art will be able to determine the appropriate effective dosage of the compositions of the present invention.
  • the pharmaceutical composition according to the present invention is not particularly limited to the formulation, route of administration and method of administration as long as the effect of the present invention is shown.
  • the pharmaceutical composition of the present invention may be variously formulated according to the route of administration by a method known in the art together with a pharmaceutically acceptable carrier.
  • a pharmaceutically acceptable carrier e.g., a non-toxic composition that is physiologically acceptable and, when administered to humans, does not inhibit the action of the active ingredient and usually does not cause gastrointestinal disorders, allergic reactions such as dizziness or similar reactions.
  • Such carriers include all kinds of solvents j, dispersion media, oil-in-water or water-in-oil emulsions, aqueous compositions, liposomes, microbeads and microsomes.
  • the route of administration may be administered orally or parenterally.
  • Parenteral administration methods include, but are not limited to, intravenous and intramuscular. Intraarterial, intramedullary, intradural, intracardiac, transdermal, subcutaneous, intraperitoneal, intranasal, intestinal, topical, sublingual or rectal administration.
  • the pharmaceutical composition of the present invention may be prepared using powders, granules, tablets, according to methods known in the art, together with a suitable oral carrier. It can be formulated in the form of pills, dragees, capsules, liquids, gels, syrups, suspensions, wafers and the like.
  • suitable carriers include lactose, textrose, sucrose, sorbbi, mannitl.
  • Fillers such as celluloses, gelatin, polyvinylpyridone, and the like, including methyl salose, sodium carboxymethyl cellulose, hydroxypropyl methyl cellulose, and the like.
  • crosslinked polyvinylpyridone, agar, alginic acid or sodium alginate may be added as a disintegrating agent.
  • the pharmaceutical composition may further include an anticoagulant, a lubricant, a humectant, a perfume, an emulsifier, and a preservative.
  • the pharmaceutical composition of the present invention may be injected with a suitable parenteral carrier.
  • a suitable parenteral carrier may be formulated according to methods known in the art in the form of transdermal and nasal inhalants. Injectables must be sterile and protected from contamination of microorganisms such as bacteria and fungi.
  • suitable carriers include, but are not limited to, solvents including water, ethanol, poly (e.g. glycerin, propylene glycol and liquid polyethylene glycols), combinations thereof and / or vegetable oils, or It may be a dispersion medium.
  • suitable carriers include Hanks' solution, Ringer's solution, phospliate buf fered salin (PBS) containing triethanol amine or sterile water for injection, 10% ethane, 40% propylene glycol and 5% dextrose.
  • PBS phospliate buf fered salin
  • the same isotonic solution and the like can be used.
  • various antibacterial and antifungal agents such as parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like may be further included.
  • the injection may in most cases further comprise an isotonic agent such as sugar or sodium chloride.
  • transdermal administration ointments, creams, lotions, gels, external liquid crabs, pasta preparations, liniment preparations, and air preparations are included.
  • 'transdermal administration' means that the pharmaceutical composition is locally administered to the skin so that an effective amount of the active ingredient contained in the pharmaceutical composition is delivered into the skin.
  • the pharmaceutical composition of the present invention may be injectable.
  • the compounds used according to the present invention may be prepared by using a suitable propellant, for example, dichlorofuluromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas. Therefore, it can be conveniently delivered in the form of aerosol spray from pressurized pack or nebulizer.
  • a pressurized aerosol the dosage unit can be determined by providing a valve to deliver a metered amount.
  • gelatin capsules and cartridges for use in inhalers or blowers may be formulated to contain a compound and powdered mixture of suitable powder based such as lactose or starch.
  • compositions according to the invention may comprise one or more laxative agents (eg saline or PBS), carbohydrates (eg glucose, mannose, sucrose or texran), antioxidants Agents, bactericides, chelating agents (eg EDTA or glutathione), adjuvants (eg aluminum hydroxide), suspending agents, thickening agents and / or preservatives.
  • laxative agents eg saline or PBS
  • carbohydrates eg glucose, mannose, sucrose or texran
  • antioxidants Agents eg glucose, mannose, sucrose or texran
  • bactericides eg EDTA or glutathione
  • adjuvants eg aluminum hydroxide
  • suspending agents eg thickening agents and / or preservatives.
  • compositions of the present invention may be formulated using methods known in the art to provide rapid, sustained or delayed release of the active ingredient after administration to a mammal.
  • the present invention also provides osteoporosis comprising osmundacetone or a pharmaceutically acceptable salt thereof as an active ingredient.
  • composition for food is a functional food (functional food), nutritional supplements
  • the food composition itself of the present invention is used as a health food. It can be prepared in the form of juices and drinks for drinking, or ingested by granulation, encapsulation and powdering.
  • the food composition of the present invention may be prepared in the form of a composition by mixing with a known substance or active ingredient known to have the effect of preventing or improving bone diseases.
  • functional foods include beverages (including alcoholic beverages), fruits and processed foods (e.g. canned fruit, canned foods, jams, marmalade, etc.), fish, meat and processed food products (e.g. For example, ham, sausage cornebi, etc., breads and noodles (e.g. udon, soba noodles, ramen, spaghetti, macaroni, etc.), juices, various drinks, cookies, candy, dairy products (e.g. butter, cheese) It can be prepared by adding the food composition of the present invention to edible vegetable fats and oils, margarine, vegetable protein, retort food, frozen food, various seasonings (for example, miso, soy sauce, sauce, etc.).
  • the preferred content of the food composition according to the present invention is not limited thereto, but is preferably 0.01 to 50% by weight of the total weight of the finally prepared food.
  • it may be prepared and used in powder or concentrate form.
  • the present invention is at least one selected from the group consisting of osteoporosis, rheumatoid arthritis, arthralgia, Paget's disease, bone metastases and fractures, including fern (fls / wm / a japonica) extract as an active ingredient
  • a pharmaceutical composition for preventing or treating bone diseases is provided.
  • the effects of the prophylaxis or treatment against bone diseases expected from the fern extracts identified by the inventors are as described herein above.
  • the compound osmundacetone contained in the fern extract inhibits the proliferation and differentiation of osteoclasts responsible for absorption and osteoblasts. It has been shown to have activity to activate cell differentiation.
  • the Di Osi nda japonica extract can be prepared from fresh ferns. Fertilizers that have been processed for storage, such as freezing and drying, can also be used. There is no restriction on the form or property of the fern extract, and may be a solution, a concentrate, or may be a solid or powder from which the solvent used for preparing the extract is removed.
  • the ferns and extracts can be selected and used without limitation, as long as it is known as a natural product extraction method, in particular, it is most preferable to use an extraction method for producing an extract containing osmundacetone.
  • an extraction method for producing an extract containing osmundacetone For example, acid / base extraction, hot water extraction, room temperature stirring, cold immersion, reflux cooling extraction, supersonic extraction, pressurized heating extraction, low silver high pressure extraction, enzyme treatment extraction known in the art by prepacking a suitable extraction solvent. It can be prepared using an extraction method such as solvent extraction.
  • the extraction solvent may be water, ethanol, alcohol, methane, propanol, isopropanol, butanol, acetone, ether, chloroform, ethyl acetate, methylene chloride,
  • solvents selected from the group consisting of nucleic acids, cyclonucleic acids, petrol ether, diethyl ether, and benzene can be used.
  • the fern extract according to the present invention may be an extract first extracted by the solvent extraction method, or may be mixed with the primary extract by re-extracting the extraction residue after the first extraction in order to increase the efficiency of extraction.
  • various methods such as separation, fractionation, diatom filtration, and ultrafiltration (membrane separation, ul traf il terat i on) according to methods known in the art are additionally performed. It may be through one purification or filtration process.
  • the final extract can be concentrated using well-known concentration methods such as precipitation concentration, evaporation concentration, azeotropic concentration, reduced pressure concentration, distillation concentration, centrifugation, reverse osmosis method and concentration equipment, and freeze drying, spray drying, hot air drying, etc.
  • concentration methods such as precipitation concentration, evaporation concentration, azeotropic concentration, reduced pressure concentration, distillation concentration, centrifugation, reverse osmosis method and concentration equipment, and freeze drying, spray drying, hot air drying, etc.
  • concentration methods such as precipitation concentration, evaporation concentration, azeotropic concentration, reduced pressure concentration, distillation concentration, centrifugation, reverse osmosis method and concentration equipment, and freeze drying, spray drying, hot air drying, etc.
  • the solvent can be removed and solidified by the process to prepare in powder form.
  • Carriers that may be included in the pharmaceutical composition comprising the fern extract as an active ingredient, formulation of the pharmaceutical composition, the method of administration such as the route of administration and the dosage is as described above.
  • the present invention is at least one bone disease selected from the group consisting of osteoporosis, rheumatoid arthritis, arthralgia, Paget's disease, bone metastases and fractures, including fern (Osmunda j aponi ca) extract as an active ingredient
  • a composition for preventing or improving food is provided.
  • the fern extract for producing the food composition is as described above. Moreover, it is as having mentioned above about the illustration and content of the said food composition.
  • the present invention also relates to Osniundacetone or a pharmaceutical thereof, which is used for the preparation of an agent for the prevention or treatment of any one or more bone diseases selected from the group consisting of osteoporosis, rheumatoid arthritis, joint pain, Paget's disease, bone metastases and fractures. To provide for the use of acceptable salts.
  • 'pharmaceutically acceptable refers to a physiologically acceptable and generally does not cause an allergic reaction or the like when administered to a human, and the salt is pharmaceutically acceptable. Acid addition salts formed with free acid are preferred. Organic acids and inorganic acids may be used as the free acid. The organic acid is not limited thereto, but citric acid, acetic acid, lactic acid, tartaric acid, maleic acid, fumaric acid, formic acid, propionic acid, oxalic acid, trifluoroacetic acid, benzoic acid, gluconic acid, metasulfonic acid, glycolic acid, succinic acid, 4-frame Luenesulfonic acid, glutamate.acid and aspartic acid.
  • the inorganic acid includes, but is not limited to, hydrochloric acid, bromic acid, sulfuric acid and phosphoric acid.
  • Dosage of the therapeutic agent of the present invention is the route of administration, time of administration, frequency of treatment, duration of treatment, age, weight, health condition, sex, and severity of the subject in need of treatment.
  • Various factors, such as sensitivity to the drug, diet and excretion rate, etc. may be taken into consideration to those skilled in the art to determine an appropriate effective amount for the particular use described above.
  • the term "effective amount" refers to an amount sufficient to exhibit an effect of improving, treating, and preventing the above-described bone diseases when administered to an individual. do.
  • the term 'individual' may be an animal, preferably a mammal, most preferably an animal including a human, and may be a cell, tissue, organ or the like derived from the animal.
  • the subject may be a patient with bone disease (pat i ent) in need of treatment.
  • the administration may be administered once or several times a day.
  • the therapeutic preparation of the present invention may be administered alone or in combination with other therapeutic agents known to be effective in the prevention or treatment of bone diseases, and when administered in combination, they may be administered sequentially or simultaneously with other therapeutic agents.
  • the dosage of the therapeutic preparation of the present invention is preferably administered in an amount that can achieve the maximum effect in a minimum amount without side effects, which can be easily determined by those skilled in the art.
  • the osmudacetone is characterized in that the separation from plant OSMUNDACEAE (Osmundaceae), method for separation is as described above.
  • the present invention also comprises osteoporosis, rheumatoid arthritis, arthralgia, Paget's disease , bone metastases and fractures, comprising administering to a subject in need thereof an effective amount of Osmundacetone or a pharmaceutically acceptable salt thereof.
  • treatment 1 refers to a clinical procedure for altering the natural process of the individual or cell being treated, and may also be performed for the prevention of clinical pathology. Desirable effects of treatment include: inhibiting the occurrence or recurrence of the disease, alleviating symptoms, reducing any direct or indirect pathological consequences of the disease, decreasing the rate of disease progression, improving the disease state, improving, alleviating or improving the prognosis, etc. It includes. The term 'prevention' also means any action that inhibits or delays the development of a disease.
  • the present invention also provides an extract of the fern japonica extract for use in the manufacture of any one or more of the osteoporosis, rheumatoid arthritis, arthralgia, Paget's disease, bone metastases and fractures selected from the group consisting of one or more bone diseases Provides use.
  • the extract of the fern is known as a natural product extraction method can be selected and used without limitation, in particular to produce an extract containing osmundacetone
  • an extraction method is used.
  • acid / base extraction hot water extraction, phase silver stirring, cold immersion, reflux cooling extraction, ultrasonic extraction, pressurized heating extraction, low temperature, high pressure extraction, enzyme treatment extraction, solvent extraction It can manufacture using extraction methods, such as these.
  • the extraction solvent may be water, ethane, spirits, methane, propane, isopropanol, butanol, acetone, ether, chloroform, ethyl acetate, methylene chloride.
  • One or more solvents selected from the group consisting of nucleic acids, cyclonucleic acids, petrol ether ethers, diethyl ether and benzene can be used.
  • the fern extract according to the present invention may be an extract extracted primarily by an additive solvent ' extraction method, and may be mixed with the primary extract by re-extracting the extraction residue after the primary extraction in order to increase the extraction efficiency. It may be.
  • a variety of separation, fractionation, diatom filtration, ultrafiltration (membrane separation, u! Traf n terat ion), etc. according to methods known in the art are additionally performed. It may be through one purification or filtration process.
  • the final extract can be concentrated using well-known concentration methods such as precipitation concentration, evaporation concentration, azeotropic concentration, reduced pressure concentration, midstream concentration, centrifugation, reverse osmosis, and concentration equipment, and freeze drying, spray drying, hot air drying, etc.
  • concentration methods such as precipitation concentration, evaporation concentration, azeotropic concentration, reduced pressure concentration, midstream concentration, centrifugation, reverse osmosis, and concentration equipment, and freeze drying, spray drying, hot air drying, etc.
  • concentration methods such as precipitation concentration, evaporation concentration, azeotropic concentration, reduced pressure concentration, midstream concentration, centrifugation, reverse osmosis, and concentration equipment, and freeze drying, spray drying, hot air drying, etc.
  • the solvent can be removed and solidified by the process to prepare in powder form.
  • a method for treating bone diseases Any one selected from the group consisting of osteoporosis, rheumatoid arthritis, arthralgia, Paget's disease, metastatic cancer and fracture, characterized by administering an effective amount of high aV) s mda japonica) extract to a subject in need thereof.
  • the present invention is any one selected from the group consisting of osteoporosis, rheumatoid arthritis, arthralgia, Paget's disease, bone metastases and fractures, including os ⁇ ndacetone or its derivatives, or extracts of ferns containing the same as an active ingredient.
  • compositions for preserving, ameliorating or treating at least one bone disease Although the composition according to the present invention has very low cytotoxicity, it showed an effect of inhibiting strong proliferation and differentiation against osteoclasts causing bone loss, and at the same time, it showed the effect of activating osteoblast differentiation.
  • FIG. 1 shows the HPLC results of high-purity water (hot water) or ethyl acetate (EA) extract for separating and identifying the osteoclast differentiation inhibitory substances contained in the fern (detection wavelength 280 nm).
  • EA-2 is the second of EA extracts separated into seven fractions. Osniundacetone peaks are indicated by black arrows. Peaks marked with red ovals indicate the position of the same peaks observed during purification.
  • Figure 2 shows the results of TRAP assay using mouse bone marrow cells to confirm the proliferation and differentiation inhibitory activity of the fern extract and fractions, osmundacet one osteoclasts.
  • Figure 3 shows the osteoclasts of osmundacetone (Al fa Aesar, Thermo Fischer Sci entific) 1, 4, 7, ⁇ purchased commercially in order to obtain IC 50 , an osteoclast differentiation inhibitory ability of osmundacetone.
  • osmundacetone Al fa Aesar, Thermo Fischer Sci entific
  • Figure 4A is to compare the osteoclast differentiation inhibitory effect of osmundacetone and Fosaraax, a known drug, after inducing differentiation by treating various concentrations of osmundacetone and Fosamax in bone marrow cells, and confirmed the 1 (: 50)
  • Figure 4B shows the results confirming the ability to co-culture osteoclasts and osteoblastic progenitor cells and osmundacetone inhibits osteoclast differentiation and activate osteoblast differentiation.
  • Figure 5 shows the result of confirming the expression of 0CN by Western blot after treatment with osmundacetone in order to confirm the effect on the 0CN production of osteoblasts of osmundacet one.
  • Figure 6 shows the results of confirming the expression of RUNX2 by Western blot after treatment with osmundacetone in order to confirm the effect of osmundacetone on the expression of RUNX2.
  • the Gobi extract is washed with 200 ⁇ 250g of Gobi collected from Gangwon-do and steamed in a steaming vessel (OSK-2002, Red Ginseng, Well sosanaTM, Daewoong Pharmaceutical) and steamed for 24 hours by adding 1.5L of water. In addition, 3.5L of water was further added and aged for 72 hours, and the cold storage was used as a hot water extract. The same volume of ethyl acetate (EA) was added to the hot water extract and mixed well. The EA layer was dried with a rotary evaporator and used as an EA extract.
  • OSK-2002 Red Ginseng, Well sosanaTM, Daewoong Pharmaceutical
  • the EA extract was dissolved in a minimum amount of DMS0 and then diluted with water, where the dilution was diluted to 70% of the original volume of the hydrothermal extract in anticipation of a yield of approximately 70%.
  • the second fraction was dried with a rotary evaporator and used as an EA-2 extract.
  • the second fraction (EA-2) was observed to have osteoclast differentiation activity (see Figure 1, ⁇ Example 2>).
  • NIH3T3 mouse embryonic fibroblast #KCLB 21658
  • RAW264.7 murine macrophage preosteoclast cell line, #KCLB 40071
  • HCT116 human colon cancer #KCLB 10247
  • PC3 human prostate adenocarcinoma cell #state) KCLB 21435
  • IIT1080 human fibrosarcoma cells #KCLB 1121
  • ⁇ 16 ⁇ 0 mouse melanoma cells CLB 80008
  • AGS human gastric adenocarcinoma (#KCLB 21739), A549 human lung carcinoma cell (#KCLB 10185), Caki-1 human carcinoma cell (#KCLB 30046), T24 human bladder cancer Species (bladder carcinoma cell, #KCLB 30004), TC-1 P3 HPV-16E7-expressing mouse pulmonary epithelial cells, MHC class I (Professor Tae-Woo Kim, Korea University), RPMKRosewell Park Memorial Institute) 1640 (#RP TA, Capricorn Scientific , Ebsdor f er ground, Germany) medium at 37 ° C, 5% C0 2 conditions.
  • ADMSCs (CEF0 Bio, Seoul, Korea), which are human adipocyte-derived mesenchymal stem cells, were prepared at 37 ° C. using CB-ADMSC-GM (CEFO Bio, Seoul, Korea) medium. C, C0 2 was incubated in the conditions.
  • Bone marrow cells were collected from the femurs and shins of male C57BL / 6 mice 5-8 weeks old. Muscles were removed from bone and stored in cold phosphate saline (PBS; # CAP08-050, GenDEPOT, Katy, TX, USA).
  • PBS cold phosphate saline
  • Both ends of each bone were cut and flushed with bone marrow (serum-free ⁇ - ⁇ , 2 ⁇ ethylenediaminetetraacet ic acid) kept cold using a 25G needle. .
  • Bone marrow cells were collected by centrifugation for 3 minutes and then resuspended in washing medium. The collected 8 niL of bone marrow cells were then superimposed on 6 mL of lymphocyte separation medium (LSM; # 50494, MP Biomedicals, Santa Ana, CA, USA) and centrifuged at 1,600 rpm for 20 minutes to separate monocytes. It was.
  • LSM lymphocyte separation medium
  • ⁇ 7 23> purchased in order to promote differentiation of osteoclasts, a 48-well flat Tate 1 X 10 5 cells in 0.5 mL per well of times if in the osteoclast differentiation factor PeproTec (Seoul, Korea) -CSFC60 ng / mL) and RANKL (150 ng / mL).
  • PeproTec Seoul, Korea
  • RANKL 150 ng / mL
  • raehan macrophages / monocytes are after ', 6, as is well known has been completely differentiated into mature multinuclear osteoclasts (Gurt et al., 2015) .
  • ⁇ 240> 1 ⁇ 10 5 C57BL / 6 rat bone marrow mononuclear cells per 48 well plate wells were prepared in the same manner as above, and 1.5> ⁇ 1 MC3T3-E1 murine osteoblasts and 10 Using 500 yL Q-MEM medium with% FBS and 1% antibiotics (100 U / mL penicillin G and 100 mg / mL strattomycin) at 37 ° C, 5% C0 2 , every 3 days The cultures were cocultured with replacement.
  • Positive control cells were osteoclast differentiation factors M-CSF (60 ng / mL) and RANKL (150 ng / mL) and osteoblast differentiation factor ascorbic acid (ascorbic acid, 50 ⁇ g / niL). And incubated with lOmM ⁇ glycerophosphate.
  • Negative control cells were cultured with the addition of M ⁇ CSF as the differentiation factor.
  • osmundacetone was administered to positive control cells at a final concentration of 10 ⁇ M on day 1 after dispensing cells into plates.
  • the positive control group treated with Osmundacetone was cultured from 8 days after dispensing to 21 days with only osteoblast differentiation factor because osteoclasts completely disappeared after 6-7 days after dispensing.
  • This assay can directly measure matrix metal loproteinase release into osteoclasts of osteoclasts (Delaisse et al., 2003).
  • 2 ⁇ 10 4 mouse bone marrow cells prepared as above were dispensed into 96-well OsteoLyse TM cell culture plates coated with fluorescent-bound human bone collagen (europium-conjugated collagen). Incubated for 6 days at 37 ° C, 5% C3 ⁇ 4 in 0.1 niL of the complete ⁇ - ⁇ per well in the presence of M-CSFC60 tig / mL) and RANKL (150 ng / mL). The medium was then changed on the third day of dispensing.
  • Bone resorption rate (%) was obtained by calculating the ratio of bone resorption according to the presence of osinundacetone relative to the untreated control group and normalized by cellular DNA.
  • 3X10 5 MC3T3-E1 cells were prepared with complete ⁇ - ⁇ 10 containing ascorbic acid (50 yg / niL) and 10 mM-glycerophosphate per well. Dispense into 100 mm culture plates with mL. 50 ⁇ Osmundacetone was added or unadded, and cultured for 21 days in a 3G C, 5% C0 2 incubator, and medium was changed every 3-4 days.
  • Negative control cells were cultured without differentiation factors, and osteoblasts and their cultures were collected 7 days, 14 days or 21 days after dispensing.
  • Run-related transcription factor 2 (RUNX2) was analyzed.
  • culture medium was collected and normalized to cellular DNA to analyze 0CN secretion.
  • Proteins were obtained from 12.5% polyvinyl idene-Tr is gels.
  • PVDF polyvinylidene difluoride
  • the membrane was blocked using skim milk, anti-RUNX2 (DlH7) rabbit monoclonal antibody (# 8486, Cell Signaling Technology, M, USA) or anti-0CN (FL-95) antibody (#sc ⁇ ). 30045, Santa Cruz Biotechnology, TX, USA).
  • Anti-actin anti The sieve (# ⁇ 77-3, MEDICAL & BIOLOGICAL LABORATORIES CO., LTD., Nagoya, Aichi, Japan) was used as an internal standard.
  • Protein bands on the blot were visualized by an enhanced chemiluminescent detection kit (# EBP-1073, PicoEPD Western Reagent, ELPIS-BIOTECH, Dae j eon, Korea).
  • ALP activity is determined by Quant ichrom ALP Assay Kit (Bioassay Systems, Hayward, CA,
  • ascorbic acid 50 yg / mL
  • 3X10 MC3T3-E1 cells were added to each well.
  • the tibia and femur were aseptically excised from 6 to 8 week old male C57BL / 6 mice, and bone marrow cells were collected aseptically with a syringe (21G, Korea Green Cross). Bone marrow cells were suspended in 48well plates in 500 uL of ⁇ - ⁇ medium (Gibco BRL Co.) containing sodium bicarbonate (2.0 g / L), streptomycin (100 mg / L) and peniciinn (100,000 units / mL). , The assay was carried out by triplicate. Monocyte progenitor cells, osteoclasts, were differentiated into osteoclasts within 5 to 7 days with RANKL and M-CSF.
  • Osteoclasts were defined as TRAP-positive multinucleated cells stained with TRAP.
  • TRAP staining solution was dissolved 5 mg of naphthol AS-MS phosphate (Sigma N-4875) as a substrate and 25 mg of Fast Red Violet LB salt as a coloring reagent in about 0.5 mL of N, N-dimethylformamide, and 0.1 N containing 50 mM tartaric acid. NaIIC0 3 buffer solution (50 mL). The reaction reagents were stored in the refrigerator until use.
  • the bone marrow cells were cultured in a medium containing a differentiation-promoting factor for 7 days, and then the medium was removed, washed with PBS, and fixed in mulberry eyes for 2 to 5 minutes with PBS containing 10% formal in. Thereafter, the mixture was fixed for 1 minute with a 1: 1 mixed solution of ethanol and acetone, and dried. The fixed cells were treated with TRAP staining solution for 15 minutes, washed with PBS, and observed for the degree of staining of the cells under a microscope.
  • ⁇ 5 27> was determined for cells having two or more nuclei, TRAP- of positive cells in the microscope field of view by osteoclasts, and measuring the number of cells.
  • the osteoclast differentiation inhibitory effect of the fern extract was calculated as 1 (: 50 ).
  • the cells having two or more nuclei among the TRAP-positive cells were determined as osteoclasts and the number of cells was measured. Osmundacetone's osteoclast differentiation inhibitory effect was calculated as 1 (: 50 ).
  • the MTT assay method is as follows.
  • ⁇ 287> Cells were cultured at 5% C0 2 , 37 ° C in 96 well plates containing DMEM at a concentration of 1 X 10 3 eel ls / well and 10% FBSCfetal bovine serum), and then osiiiundacetone was cultured. And incubated for 3 ⁇ 4 hours. Thereafter, 100 ⁇ L of MTT (0.5 mg / ml PBS) was administered, followed by incubation for 2 hours. Then, the medium was removed from each well and 100MS DOO was added. After incubation for 10 minutes, the absorbance was measured at 570 nm using a micro crop late reader (SPCTRA MAX 340PC, Molecular Devices, USA). Absorbance is an indicator of the number of surviving cells, calculated by the equation below, and reproducible in three experiments.
  • MTT 0.5 mg / ml PBS
  • the extracts and fractions of the ferns prepared in ⁇ Example 1> are isolated from the osteoclast-specific staining method TRAP assay (tart rate-resist ant acid phosphatase) to inhibit the proliferation and differentiation of osteoclasts was confirmed.
  • TRAP assay mitogen-resist ant acid phosphatase
  • the bone marrow cells treated with DMS0 normally formed giant osteoclasts as in the positive control group (groups in which only the differentiation factor was added to the culture medium without the fern extract).
  • the control group was similar to the negative control group (both fern extract and differentiation promoter were not added to the culture medium).
  • the formation of giant osteoclasts which are multinucleated cells, was significantly inhibited, and in addition to the negative control group, the proliferation of osteoclast progenitor cells was also significantly suppressed, and the effect of inhibiting osteoclast differentiation and proliferation was also very large. .
  • Osmundacetone inhibited the bone resorption function of mature osteoclasts to 58.7 ⁇ 13% of untreated osteoclasts at the oocyte concentration based on the method of Example 1.
  • bone marrow mononuclear cells and osteoblast progenitors collected from C57B176 mice Co-cultured at 1 ⁇ 10 ° bone marrow cells and 3 ⁇ 10 3 MC3T3-E1 cell concentrations per 48 well pooled wells.
  • Mononuclear / macrophage lineage cells of bone marrow cells among co-cultured bone marrow cells and osteoblast progenitor cells were 6-7 days in the presence of M-CSF and RANKL . Within a few minutes, they differentiated into mature multinuclear osteoclasts.
  • Osmundacetone 10 ⁇ M completely inhibited the proliferation and differentiation of osteoclasts (Fig. 4B, 0s). Osteoblasts, on the other hand, continued to proliferate and differentiate. 3 ⁇ 4 activation of ALP activity on coculture 7, 14 and 21 days with Osmundacetone 10 ⁇ was 104%, 111% and 95%, respectively, compared to the non-administration of ALP activation of osteoblasts cultured alone after osmundacetone 10 ⁇ . There was no significant difference in comparison with.
  • Osmundaceton showed similar inhibitory activity on the differentiation of osteoclasts in the presence and absence of osteoblasts.
  • On day 6 after administration of 10 ⁇ M of Osmundacetone mature osteoclasts disappeared completely, while osteoblasts continued to proliferate. Therefore, osmundacetone did not inhibit the activity and proliferation of coexisting osteoblasts, as shown in the co-culture of preosteoclasts and preosteoblasts. Based on these results, osmundacetone was found to be capable of simultaneously inhibiting osteoclast differentiation and activating osteoblast differentiation.
  • osteoclasts and osteoblasts continued to proliferate and differentiate in the presence of osteoclasts and osteoblast differentiation factors after 7 days of co-culture of propagating osteoblasts and progenitor osteoblasts.
  • FIG. 4B positive control group
  • the size of differentiated osteoclasts was rather small compared to those grown in the absence of osteoblasts, which may be due to the increased cell density due to the coexistence of osteoclasts and osteoblasts.
  • osmundacetone is capable of independently inhibiting osteoclast differentiation and activating osteoblast differentiation at the same time.
  • ALP production in MC3T3-E1 cells was significantly weighted compared to ALP production in untreated control cells.
  • osteoclasts were found not to inhibit the differentiation of co-existing osteoclasts.
  • osteoblast differentiation has been shown to promote osteoblast differentiation and at the same time maintain its ability to inhibit osteoclast differentiation.
  • the major non-collagenous matrix protein, 0CN, has been reported to increase in expression only at or near mineralization, 21 days after induction of differentiation of MC3T3-E1 cells. (Young et al., 1992).
  • MC3T3-E1 cells were cultured in the presence of 50 ⁇ osmundacetone.
  • the negative control progenitor osteoblasts grown without osteoblast differentiation were found to have a significant effect of 0CN production up to the level of positive control cells with osteoclasts at days 14 and 21, compared to osteoblasts grown without osteoclasts. Showed an increase.
  • RUNX2 is known as a transcription factor that stimulates the transcription of ALP and 0CN in osteoblasts and increases bone formation (Phimphl ai et al., 2006).
  • Osmundacetone showed specific toxicity with high selectivity index values for cancer cells compared to non-cancer cells.
  • the cells used in this experiment are as follows.
  • HaCaT Human epidermal kerat inocytes
  • ADMSC Human Adipose-derived mesenchymal stem celKCEFO, Korea
  • RAW264.7 mouse macrophage cell line (preosteoclast); NIH3T3, Mouse embryo fibroblast; AGS, Human stomach adenocarcinoma; A549, Human lung carcinoma; HepG2, Human liver hepatoblastoma; HCT116, Human colon carcinoma; PC3, Human prostate adenocarcinoma; Caki-1, Human kidney carcinoma; T24, human bladder carcinoma; HT1080, human fibrosarcoma (Human fibrosarcoma); B16F10, mouse melanoma (Mouse melanoma); TOl P3, HPV-16E7-expressing mouse lung epithelial cells (-MHC class I)
  • osmundacetone was found to be 2, 760 ⁇ 220, 3510 ⁇ 110 and> 5,000 for ADMSC human adipocyte derived mesenchymal stem cells, HaCaT human epidermal keratinocytes and NIH3T3 mouse embryo fibroblasts, respectively. Insignificant cell virulence with an IC 50 value of ⁇ was shown.
  • osimmdacetone showed relatively significant cytotoxicity with an LD 50 of 507 cm 98 ⁇ against RAW264.7 mouse macrophage cell line.
  • osmundacetone was found to be 59.9 ⁇ 6.1, 65.5 5.9 8.7 and 8 for cancer cell lines including AGS human gastric cancer (stomach adenocarcinoma), PC3 human prostate adenocarcinoma and B16F10 mouse melanoma.
  • a low LD 50 of 75.8 ⁇ m 9.2 ⁇ was shown to indicate moderate cytotoxic activity.
  • the cancer selectivity index of osmundacetone in killing human cancer cell line (AGS ⁇ PC) compared to human normal cell line (HaCaT, ADMSC) is 45 to Very high at 60.
  • IC 5 ⁇ of osmundacetone for inhibition of osteoclast differentiation was 8 ⁇
  • the concentration of 280% activating osteoblasts was 50 ⁇
  • LD 50 for the normal cell line ranges from 2,500 to 5,000 ⁇ , and based on this, it was confirmed that the osmundacetone has a great safety when used as a therapeutic agent.
  • Bone is osteoclasts and osteoblasts that produce bone metabolically
  • Bone remodeling cycles are maintained throughout the balance. However, when osteoclasts and osteoblasts lose their balance and the osteoclasts become too active, the balance between bone absorption and production breaks down, leading to osteoporosis as the absorption is greater than the amount produced (Kim JH and Kim N, 2016; Shiozawa Y et. a /., 2011).
  • osmundacetone of the present invention exhibits an effect of inhibiting the proliferation and differentiation of osteoclasts and an effect of activating osteoblasts
  • the osmundacetone may have an effect of preventing or treating osteoporosis.
  • Rheumatoid arthritis is an autoimmune disease in which autoimmune antibodies promote osteoclast differentiation. The resulting excessive bone absorption exacerbates rheumatoid arthritis (Takayanagi H, 2007).
  • the mechanism is as follows. NFAT precursors (NFATc: i / c2 / c3 / c4), pivotal transcription factors related to osteoclast differentiation, are primarily activated by calcium / calmodulin signaling (Takayanagi H et a /., 2002).
  • tyrosine-based activation motifs such as the immunomodulatory proteins DNAX-activating protein 12 (DAP12) and the immune antibody Fc receptor common ⁇ chain (FcRx), bear calcium molecules in immune cells. Stimulates (Pitcher LA and van Oers NS, 2003). Osteoclasts also activate NFATcl through DAP12 and FcR ⁇ A calcium signaling. Thus, imniunoglobulin-like receptors associated with DAP12 and FcRy Plays an important role in osteoclast differentiation (Koga T et al., 2004; Mocsai A et al., 2004).
  • FcRy interacts with osteoclast-associated receptor (OSCAR) and paired immunoglobul in-1 ike receptor (PIR-A) in osteoclasts.
  • Phosphorylation of ITAM activates phospholipase C ⁇ ( ⁇ ), which releases intracellular calcium, which activates calcineurin, a calmodul in-dependent phosphatase.
  • Calcineurin directly dephosphorylates and activates NFATcl serine into the nucleus.
  • immune antibodies promote osteoclast differentiation, and excessive bone uptake by osteoclasts exacerbates rheumatoid arthritis.
  • inhibition of osteoclast differentiation in patients with rheumatoid arthritis may not correct the abnormalities of autoimmune mechanisms themselves.
  • skeletal symptoms such as arthritis and pain can be treated.
  • osmundacetone of the present invention exhibits an effect of inhibiting the proliferation and differentiation of osteoclasts, it can exhibit a prophylactic or therapeutic effect of rheumatoid arthritis.
  • Osteitis deformans also causes abnormal bone resorption of osteoclasts (Singer FR, 2016). Then, abnormal bone formation of osteoblasts progresses and this process is repeated, resulting in bone malformation, resulting in pain, headache, and hearing loss. It affects the arms, legs, pelvis, spine and skull. Newly formed bones are weak and have a high frequency of fractures. Hypercalcemia, heart attack, and incompetence can be caused (Ralstone SH, 2016). The cause is unknown, but genetic predisposition and childhood viral infections are suspected to be the cause. Medication can help to control the progression of the disease. Currently, the most used therapeutic agents are osteoclast differentiation inhibitor Fosamax and calcitonin to regulate bone metabolism.
  • Fosamax has limited side-effects in some patients as a side effect.
  • the pain is used to "severe surface Acetaminophen (Tylenol) or nonsteroidal ant i-inf lammatory drugs (NSAIDs).
  • osmundacetone of the present invention exhibits an effect of inhibiting the proliferation and differentiation of osteoclasts, it can have a prophylactic or therapeutic effect of Paget's disease.
  • Osteoclasts also promote bone metastasis in solid tumors. Bones of Cancer I This is the most common site. Cancer metastasis in the cortex causes severe pain and fractures of the bones, significantly reducing the likelihood of cure. OVeilbaecher N et al. , 2011). Systemic cancer cells are found at sites of blood stem cell proliferation in the bone marrow (Shiozawa Y et al., 2013). Cancer cells significantly promote the differentiation of osteoclasts from bone marrow cells to promote bone metastasis, cancer growth, and bone destruction. Therefore, osteoclasts play a key role in bone metastasis of cancer. Inhibiting osteoclast differentiation reduces bone metastasis.
  • Many solid cancer metastases are bone metastases, and blood stem cells are driven from the blood stem cell proliferation centers, and then fed to the blood and then metastasized.
  • the most common cancer of bone metastasis is prostate cancer, where bone metastasis worsens the cancer, making it harder to cure and is the leading cause of death.
  • Direct primary targets of human prostate cancer cells also serve as a base for metastatic cancer as a place of blood stem cell proliferation (Shiozawa Y et al., 2011).
  • Osteoclasts also promote cancer growth by promoting blood vessel formation within the prostate cancer tissue (Bruni-Carcloso A et al., 2010).
  • Breast cancer cells also promote osteoclast differentiation, and osteoclasts promote cancer recurrence through bone metastasis in breast cancer patients undergoing mastectomy (Danilin S et al., 2012; Lu X et al., 2011).
  • Bone-targeted therapeutics to prevent bone metastases are currently used in clinical trials, and osteoclasts are one of the key mechanisms for cancer metastasis. Accordingly, the only bisphosphonate-based drug currently approved by the US FDA for the purpose of inhibiting osteoclast differentiation is Zoledronic acid (E ⁇ Amm J et al., 2013). Zoledronic add preserves bones and increases survival rates. Zoledronic Acid significantly reduced bone metastasis in high risk nonmetastat i prostate cancer (Wirth M et al., 2014). The administration of Zoledronic acid with parathyroid hormone, which activates osteoblasts, further reduced bone metastasis (Schneider A et a., 2005).
  • Denosumab a monoclonal antibody to RANKL, a signaling agent for osteoclast differentiation, also inhibits bone metastasis in prostate cancer, again demonstrating that osteoclast inhibition is important for inhibiting bone metastasis in cancer (Smith MR et. aL, 2012).
  • Zoledronic acid inhibits hyper-osteoblast differentiation and significantly inhibits bone metastasis (Zhuang J et al., 2012). In other words, if a side effect low cost osteoclast inhibitor is developed, it can be administered long-term to suppress metastasis in cancer patients.
  • osmundacetone of the present invention exhibits an effect of inhibiting the proliferation and differentiation of osteoclasts and at the same time has an osteoblast activating effect, thus preventing or preventing bone metastasis.
  • Costimulatory signals mediated by the ITAM motif cooperate with RANKL for bone homeostasis. Nature 2004; 428: 758-63.
  • OPGL osteoclastogenesis f lymphocyte development and lymph-node organogenesis. Nature 1999: 397: 315 ° 23.
  • Lacey DL Timms E, Tan HL, Kel ley MJ, Duns tan CR, Burgess T, et al.
  • TNF ⁇ alpha tumor necrosis factor-alpha
  • converting enzyme- 1 ike protease in shedding of TRANCE a TNF family member involved in os t eoc 1 as t ogenes i s and dendritic eel 1 survival. J Biol Chem 1999; 274: 13613-8.
  • NFATcl transcription factor 2
  • TRANCE a TNF family member, activates Akt / PKB through a signaling complex involving TRAF6 and c—Src. Mol Cell 1999; 4: 1041-9.
  • composition according to the present invention exhibits potent proliferation and differentiation inhibitory activity against osteoclasts causing bone loss and simultaneously activates osteoblasts, it is useful for developing safe and effective osteoporosis therapeutics or foods for improvement.

Abstract

The present invention relates to a composition comprising osmundacetone or a pharmaceutically acceptable salt thereof for preventing or treating bone diseases. More specifically, the present invention relates to: a composition comprising osmundacetone, a pharmaceutically acceptable salt thereof, or an osmunda japonica extract as an effective ingredient, for preventing or treating osteoporosis, rheumatoid arthritis, arthralgia, Paget disease, bone metastatic cancer, or fracture; a food composition for improvement; a use of a salt; and a treatment method. The composition according to the present invention shows a strong inhibitory activity against proliferation and differentiation of osteoclast which causes bone loss, and activation of differentiation of osteoblast, and thus can be usefully utilized in developing medicines for safe and effective treatment of bone diseases or functional foods for improving symptoms of bone diseases.

Description

【명세서】  【Specification】
【발명의 명칭】  [Name of invention]
오스문드아세톤 또는 이의 약학적으로 허용가능한 염을 포함하는 골 질환 예 방또는 치료용조성물  Compositions for the prevention or treatment of bone diseases, including osmundacetone or pharmaceutically acceptable salts thereof
. ■'■ '【가술분야】 ' ' . ■ ' ■ ' 【Science Field】 ''
<ι> 본 출원은 2016년 9월 30일에 출원된 대한민국 특허출원 제 10— 2016-0126767 호 및 2016년 10월 28일에 출원된 대한민국 특허출원 제 10-2016— 0142422호를 우선 권으로 주장하고， 상기 명세서 전체는본출원의 참고문헌이다. <ι> This application claims the priority of Korean Patent Application No. 10—2016-0126767, filed September 30, 2016, and Korean Patent Application No. 10-2016— 0142422, filed October 28, 2016. The entire specification is a reference of the present application.
<2>  <2>
<3> 본 ^명은 osmundacetoiie 또는 °ί의 약학적으로 허용가능한 염을 포함하는 골 질환의 예방 또는 치료용 조성물에 대한 것으로, 보다 구체적으로는 osmundacetone 또는 이의 약학적으로 허용가능한 염 또는 고비 (Osmunda japonica) 추출물을 유효성분으로 포함하는 골 질환의 예방 또는 치료용 약학적 조성물, 개선 용 식품용조성물， 예방 또는 치료용 제제의 제조에 사용하는 용도, 치료방법에 대 한 것이다.  <3> The present invention relates to a composition for preventing or treating bone diseases, including pharmaceutically acceptable salts of osmundacetoiie or ° ί, more specifically osmundacetone or a pharmaceutically acceptable salt or fern thereof (Osmunda japonica The present invention relates to a pharmaceutical composition for the prevention or treatment of bone diseases, extracts, food compositions for improvement, uses for the preparation of a prophylactic or therapeutic agent, and a method of treatment.
【배경기술】 Background Art
<5> 뼈는 몸의 골격 구조를 형성하고 혈중 칼슘 (Ca?+) 수준을 유지하는 데 매우 중요한 역할을 한다. 뼈는 대사적으로 뼈를 흡수하는 파골세포 (osteoclast)와 생성 하는 조골세포 (osteobl ast) 간의 뼈 리모델 ¾ 순환 (bone remodel l ing cycle)의 균 형을 통해 유지된다. 뻐의 흡수와 생성 간의 균형이 파괴되어 흡수량이 생성량보다 많아지면 다양한 뼈 관련 질환이 발생하며, 과골세포의 분화 및 활성화와 관련된 대표적인 질환은 골다공증, 류마티스관절염, 관절통, 파제트 병， 골 전이암 및 골 절 등을 들 수 있다. (Kim JH and Kim N, 2016； Shiozawa Y et al. , 2011； Singer FR, 2016) . Bones play an important role in shaping the body's skeletal structure and maintaining blood calcium (Ca ? + ) Levels. Bone is maintained through the balance of the bone remodeling cycle between osteoclasts (osteoclasts) and osteoblasts that metabolize bone metabolically. When the balance between absorption and production of the Cuck is broken and the absorption is higher than the production amount, various bone-related diseases occur. Representative diseases related to the differentiation and activation of osteoclasts are osteoporosis, rheumatoid arthritis, joint pain, Paget's disease, bone metastases and Fractures, and the like. (Kim JH and Kim N, 2016; Shiozawa Y et al., 2011; Singer FR, 2016).
<6>  <6>
<7> 이 중에서, 골다공증은 파골세포의 활성화로 삐의 흡수의 · 생성 간의 균형이 파괴되어 흡수량이 생성량보다 많아지면 유발된다. 골다공중은 뼈 실질의 밀도를 감소시켜서 골절 빈도를 증가시킨다. 중년 및 노년 여성 등 호르몬 균형이 무너진 여성에서 가장 빈번하게 발생하며. 골절이나 중증 질환으로 거동을 못하는 환자에 서도 발병한다. 최근에는 중장년층 이상의 남성에서도 발생 빈도가 증가하고 았다.  Among these, osteoporosis is caused when the balance between absorption and production of the beet is broken by activation of osteoclasts, and the absorption amount is greater than the production amount. Osteoporosis increases the frequency of fractures by decreasing the density of bone parenchyma. It occurs most often in women with hormonal balance, including middle-aged and older women. It also occurs in patients who are unable to move due to fractures or severe disease. Recently, the incidence has increased in older men and older men.
대체용지 (규칙 제 26조) <8> ' Alternative Site (Article 26) <8>'
<9> 골수 대식세포 /단핵세포 세포주 (macrophage/monocytes lineage) 세포가 파골 세포로 분화하는 분자적 기전에는 먼저 아래 2개의 cytokine이 중요한 역할을 한다 (Teitelbaum SL and Ross FP, 2003) . ( i ) Macrophage colonyᅳ st imulat ing factor(M-CSF)가 그 수용체인 c— Fms와 결합하면 파골세포 전구세포들이 증식하고 생존한다. Receptor activator of nuclear factor- Β 1 igand(RAML)이 그 수용체 인 RANK에 결합하면 파골세포의 분화와 골 흡수 기능을 활성화하고 성숙한 파골세 포가 생존하게 한다 (Lacey DL et al. , 1998； Lum L et al. , 1999； Sherr CJ, 1990； Suda T et al. , 1999； Wong BR et al. , 1999). (ii) M-CSF가 cᅳ Fms의 활성화를 유 도하면 파골세포 전구세포가 ERK 및 PI3K/Akt 경로를 통하여 증식하고 생존한다 (Mancini et al. , 1997). (iii) RANK.L(0PGL, 0DF, TRANCE)과 RAN 역시 파골세포 형성과 기능을 조절한다 (Anderson DM et al. , 1997； Dougal 1 WC et al. , 1999； Kong YY et al. , 1999). RANKL이 RANK에 결합하면 TRAFs 1, 2, 3, 5, 6 등의 TNF receptor-associated f actor(TRAF)들이 RANK와 결합한다 (Darnay BG et al. , 1998； Walsh MC and Choi Y, 2003). 이중 TRAF6가 파골세포 형성과 기능에 가장 중요하다 (Loniaga MA et al . , 1999； Naito A et al. , 1999). TRAF6는 RANKL/RANK 신호를 NF一 κΒ, c-Jun Ν·— terminal kinase(JNK) , extracellular s igna卜 regulated kinase(ERK), p38, Akt , Nuclear Factor Of Activated T-Cel Is l(NFATcl)에 전달하 여 파골세포 증식 , 융합, 분화가 이루어진다 (Kobayashi N et al . , 2001； Loniaga MA et al. , 1999； Naito A et al. , 1999； Takayanagi H et al. , 2002； Wong BR et al. , 1998； Wong BR et al. , 1999)  In the molecular mechanism by which myeloid macrophage / monocytes lineage cells differentiate into osteoclasts, the following two cytokines play an important role (Teitelbaum SL and Ross FP, 2003). (i) When the macrophage colony st imulating factor (M-CSF) binds to its receptor c—Fms, osteoclast progenitor cells proliferate and survive. Binding of the receptor activator of nuclear factor-β 1 igand (RAML) to its receptor RANK activates osteoclast differentiation and bone resorption and allows mature osteoclasts to survive (Lacey DL et al., 1998; Lum L et. al., 1999; Sherr CJ, 1990; Suda T et al., 1999; Wong BR et al., 1999). (ii) When M-CSF induces activation of c ᅳ Fms, osteoclast progenitor cells proliferate and survive through the ERK and PI3K / Akt pathways (Mancini et al., 1997). (iii) RANK.L (0PGL, 0DF, TRANCE) and RAN also regulate osteoclast formation and function (Anderson DM et al., 1997; Dougal 1 WC et al., 1999; Kong YY et al., 1999) . When RANKL binds to RANK, TNF receptor-associated f actors (TRAFs) such as TRAFs 1, 2, 3, 5, and 6 bind RANK (Darnay BG et al., 1998; Walsh MC and Choi Y, 2003). TRAF6 is most important for osteoclast formation and function (Loniaga MA et al., 1999; Naito A et al., 1999). TRAF6 modulates RANKL / RANK signals to NF κΒ, c-Jun N-—terminal kinase (JNK), extracellular s igna 卜 regulated kinase (ERK), p38, Akt, and Nuclear Factor Of Activated T-Cel Is l (NFATcl). And osteoclast proliferation, fusion, and differentiation (Kobayashi N et al., 2001; Loniaga MA et al., 1999; Naito A et al., 1999; Takayanagi H et al., 2002; Wong BR et al. , 1998; Wong BR et al., 1999)
<\ϋ>  <\ ϋ>
<ll 기존의 골다공증의 치료제 개발 방향은 파골세포의 삐 흡수를 억제하여 뼈 실질의 손실을 예방할 수 있는 물질을 발굴하는 것이었다. 그 대표적인 약품이 bisphosphonate 계열의 Fosainax이다. 같은 맥락에서 arachidonate 대사산물이 삐 조직 대사에 미치는 영향에 대하여서도 많은 연구가 이루어졌다 (Lee Sung-eun , 1999). Leukotriene— B4(LTB4)는 arachidonate의 대사 경로인 5-1 ipoxygenase pathway의 대사산물 중 하나이다 (Ford-Hutchinson, A. W. et al. , 1980). Giant cell tumor에서 얻은 간질세포주 (interstitial cell)인 C433은 5-1 ipoxygenase 대 사산물을 증가시켜서 조골세포의 수와 활성을 증가시키는 것으로 보고되었다 (Mundy, G. R. et al. , 1993). 뼈 조직 배양 과정에서 LTB4를 투여하면, 뼈 흡수가 증가하는 것이 관찰되기도 하였다 (Bonewald, L. F. et al. , 1996) . In w ?와 in vivo 연구에서 LTB4는 파골세포의 생성을 증가시켜서 뼈 흡수를 유도한다는 연구 결과도 있다 (Bonewald, L. F. et al . , 1996) . 이에 따라, LTB4 수용체 억제제 (LTB4 receptor antagoni st)들이 골다공증 치료를 위하여 많이 개발되었지만， 파골세포의 뼈 실질 흡수를 층분히 억제하는 데는 성공하지 못했다. <ll The existing direction of developing a treatment for osteoporosis has been to find a substance that can prevent the loss of bone parenchyma by inhibiting the absorption of osteoclasts. The representative drug is bisphosphonate family Fosainax. In the same vein, a great deal of research has been conducted on the effects of arachidonate metabolites on skin tissue metabolism (Lee Sung-eun, 1999). Leukotriene—B4 (LTB4) is one of the metabolites of the 5-1 ipoxygenase pathway, a metabolic pathway of arachidonate (Ford-Hutchinson, AW et al., 1980). C433, an interstitial cell obtained from giant cell tumors, has been reported to increase the number and activity of osteoblasts by increasing 5-1 ipoxygenase vs. aquatic products (Mundy, GR et al., 1993). Increased bone resorption has been observed when LTB4 is administered during bone tissue culture (Bonewald, LF et al., 1996). In w? And in In vivo studies have shown that LTB4 induces bone resorption by increasing the production of osteoclasts (Bonewald, LF et al., 1996). Accordingly, many LTB4 receptor antagoni sts have been developed for the treatment of osteoporosis, but they have not been successful in inhibiting the bone parenchymal uptake of osteoclasts.
<12>  <12>
<13> 뿐만 아니라, 기존 골다공증 치료제의 부작용과 고가의 약값도 환자의 치료 에 충분한 용량으로 투여하는데 큰 장애가 되고 있다. Fosamax의 주요 부작용으로 는 중증 식도염, 신 손상， 간 손상, 저칼슘혈증. 근육 경련 등이 있고, 로슈 (Roche)의 Bonviva는 전신근육통, 몸살 등의 부작용이 있다. 노바티스 (Novart is)의 Aclasta(zoledronate)와 일라이 릴리 (Eli Lilly)의 anabolic 치료제인 parathyroid hormone인 Porsteo와 Forteo(ter iparat ide)는 효과는 좋으나 가격이 너무 비싸서 사용이 매우' 제한적이다. 특히 Forsteo/Forteo는 약물 과민반웅 환자， 임산부, 모 유수유， 고칼슘혈증 (hypercalcemia), 신부전, 부갑상선 기능항진증 (hyperparathyroidism)과 파제트 병 (Paget' disease) 등의 대사성 골 질환， 원인미 상의 알칼리포스파타제 활성 증가 (unexplained elevations of alkaline phosphatase), 방사선 치료 환자, 골수암 혹은 골 전이암 환자 등에게는 사용할 수 없기 때문에 적용 가능한 환자군이 크지 않다. In addition, the side effects and costly medications of existing osteoporosis treatments are becoming a major obstacle to the administration of sufficient doses for the treatment of patients. The main side effects of Fosamax include severe esophagitis, kidney damage, liver damage and hypocalcemia. Muscle cramps, and Roche Bonviva has side effects such as systemic muscle pain and body aches. Novartis (Novart is) Aclasta (zoledronate) and Eli Lilly (Eli Lilly) anabolic drug of the parathyroid hormone (ter iparat ide) Porsteo Forteo and the effect is good or a very 'limited use price too expensive. In particular, Forsteo / Forteo is a drug for hypersensitivity reactions, pregnant women, breastfeeding, hypercalcemia, renal failure, hyperparathyroidism and Paget 'disease. The patient population is not large because it cannot be used for unexplained elevations of alkaline phosphatase, radiation therapy patients, bone marrow cancer or bone metastases.
<14>  <14>
<15> 따라서 보다 효과가 강력하고 안전하며, 기존 치료제보다 저비용으로 생산가 능한 골 관련 질환 치료제 개발이 시급히 요구되고 있다.  Therefore, there is an urgent need to develop a therapeutic agent for bone-related diseases that is more effective, safer, and can be produced at lower cost than existing therapeutics.
<16>  <16>
【발명의 상세한 설명】  [Detailed Description of the Invention]
【기술적 과제】  [Technical problem]
<17> 이에 본 발명자들은 부작용이 적고 안전하며 효과가 뛰어난 골 관련 질환 치 료제를 개발하기 위하여 천연물 성분에 대하여 연구한 결과， 오랫동안 식품으로 이 용되어 온 고비의 추출물에 골 손실 억제 활성이 있는 것을 확인하여 본 발명을 완 성하였다..  Therefore, the present inventors studied natural ingredients to develop a bone-related disease treatment agent with low side effects, safety, and excellent effects, and thus, the extracts of ferns, which have been used as food for a long time, have bone loss inhibitory activity. Confirmed to complete the present invention.
<18>  <18>
<19> 따라서 본 발명의 목적은  Therefore, the object of the present invention
<20> Osmundacetone 또는 이의 약학적으로 허용가능한 염을 유효성분으로 포함하 는 골다공증, 류마티스 관절염, 관절통, 파제트 병, 골 전이암 및 골절로 이루어진 군에서 선택된 어느 하나 이상의 골 질환 예방 또는 치료용 약학적 조성물을 제공 하는 것이다. <20> A pharmaceutical for the prevention or treatment of any one or more bone diseases selected from the group consisting of osteoporosis, rheumatoid arthritis, joint pain, Paget's disease, bone metastases and fractures, including Osmundacetone or a pharmaceutically acceptable salt thereof Provide ever composition It is.
<2l>  <2l>
<22> 본 발명의 다른 목적은  <22> Another object of the present invention
<23> Osmundacetone 또는 이의 약학적으로 허용가능한 염을 유효성분으로 포함하 는 골다공증, 류마티스 관절염, 관절통, 파제트 병， 골 전이암 및 골절로 이루어진 군에서 선택된 어느 하나 이상의 골 질환 예방 또는 개선용 식품용 조성물을 제공 하는 것이다.  <23> Food for preventing or ameliorating any one or more bone diseases selected from the group consisting of osteoporosis, rheumatoid arthritis, joint pain, Paget's disease, bone metastases and fractures, including Osmundacetone or a pharmaceutically acceptable salt thereof It is to provide a composition for.
<24>  <24>
<25> 본 발명의 또 다른 목적은  <25> Another object of the present invention
<26> L ^ )S nda japonica) 추출물을 유효성분으로 포함하는 골다공증， 류마티 스 관절염， 관절통， 파제트 병, 골 전이암 및 골절로 이루어진 군에서 선택된 어느 하나 이상의 골 질환 예방 또는 치료용 약학적 조성물을 제공하는 것이다. <26> A pharmaceutical for the prevention or treatment of any one or more bone diseases selected from the group consisting of osteoporosis, rheumatoid arthritis, arthralgia, Paget's disease, bone metastases and fractures, including L ^) S nda japonica) extract as an active ingredient To provide a composition.
<27>  <27>
<28> 본 발명의 또 다른 목적은  <28> Another object of the present invention
<29> 고^ ( Os/m!nda japonica) 추출물을 유효성분으로 포함하는 골다공증， 류마티 스 관절염, 관절통， 파제트 병, 골 전이암 및 골절로 이루어진 군에서 선택된 어느 하나 이상의 골 질환 예방 또는 개선용 식품용 조성물을 제공하는 것이다.  <29> prevention of any one or more bone diseases selected from the group consisting of osteoporosis, rheumatoid arthritis, joint pain, Paget's disease, bone metastases and fractures containing the extract (Os / m! Nda japonica) as an active ingredient or It is to provide a composition for food for improvement.
<30>  <30>
<31> 본 발명의 또 다른 목적은 .  <31> Another object of the present invention.
<32> 골다공증, 류마티스 관절염， 관절통， 파제트 병, 골 전이암 및 골절로 이루 어진 군에서 선택된 어느 하나 이상의 골 질환 예방 또는 치료용 제제의 제조에 사 용되는 Osmundacetone 또는 이의 약학적으로 허용가능한 염의 용도를 제공하는 것 이다.  <32> Osmundacetone or a pharmaceutically acceptable salt thereof for use in the manufacture of an agent for the prevention or treatment of any one or more bone diseases selected from the group consisting of osteoporosis, rheumatoid arthritis, arthralgia, Paget's disease, bone metastases and fractures To provide a use.
<33>  <33>
<34> 본 발명의 또 다른 목적은  Another object of the present invention
<35> Osmundacetone 또는 이의 약학적으로 허용가능한 염의 유효량을 이를 필요로 하는 개체에 투여하는 것을 특징으로. 하는 골다공증， 류마티스 관절염, 관절통, 파 제트 병, 골 전이암 및 골절로 이루어진 군에서 선택된 어느 하나 이상의 골 질환 치료 방법을 제공하는 것이다.  An effective amount of Osmundacetone or a pharmaceutically acceptable salt thereof is administered to a subject in need thereof. To provide at least one bone disease treatment method selected from the group consisting of osteoporosis, rheumatoid arthritis, arthralgia, Pajet disease, bone metastases and fractures.
<36>  <36>
<37> 본 발명의 또 다른 목적은  <37> Another object of the present invention
<38> 골다공증， 류마티스 판절염， 관절통, 파제트 병, 골 전이암 및 골절로 이루 어진 군에서 선택된 어느 하나 이상의 골 질환 예방 또는 치료용 제제의 제조에 사 용하기 위한 고비 ( fls/ffl/ 70¾ japonica) 추출물의 용도를 제공하는 것이다. <38> Osteoporosis, rheumatoid arthritis, arthralgia, Paget's disease, bone metastases and fractures To provide a use of a high-fertilizer (fls / ffl / 70¾ japonica) extract for the manufacture of a formulation for the prevention or treatment of any one or more bone diseases selected from the group.
<39>  <39>
<40> 본 발명의 또 다른 목적은  <40> Another object of the present invention
<41> 고비 ( fls/w//M¾ japonica) 추출물의 유효량을 이를 필요로 하는 개체에 투여하 는 것을 특징으로 하는 골다공증, 류마티스 관절염, 관절통, 파제트 병 . 골 전이암 및 골절로 이루어진 군에서 선택된 어느 하나 이상의 골 질환 치료 방법을 제공하 는 것이다.  Osteoporosis, rheumatoid arthritis, arthralgia, Paget's disease, characterized by administering an effective amount of the fern (fls / w // M¾ japonica) extract to a subject in need thereof. It is to provide a method for treating any one or more bone diseases selected from the group consisting of bone metastases and fractures.
<42> <4 2 >
【기술적 해결방법]  Technical Solution
<43> 상기와 같은 목적을 달성하기 위하여 , 본 발명은  In order to achieve the above object, the present invention provides
<44> Osmundacetone 또는 이의 약학적으로 허용가능한 염을 유효성분으로 포함하 는 골다공증, 류마티스 관절염, 관절통, 파제트 병 , 골 전이암 및 골절로 이루어진 군에서 선택된 어느 하나 이상의 골 질환 예방 또는 치료용 약학적 조성물을 제공 한다.  <44> A pharmaceutical for the prevention or treatment of any one or more bone diseases selected from the group consisting of osteoporosis, rheumatoid arthritis, joint pain, Paget's disease, bone metastases and fractures, including Osmundacetone or a pharmaceutically acceptable salt thereof Provides a composition.
<45> - <46> 상기와 같은 목적을 달성하기 위하여， 본 발명은  <45>-In order to achieve the above object, the present invention
<47> Osmundacetone 또는 이의 약학적으로 허용가능한 염이 유효성분으로 구성되 는 골다공증， 류마티스 관절염, 관절통， 과제트 병, 골 전이암 및 골절로 이루어진 군에서 선택된 어느 하나 이상의 골 질환 예방 또는 치료용 약학적 조성물을 제공 한다.  <47> A pharmaceutical for the prevention or treatment of any one or more bone diseases selected from the group consisting of osteoporosis, rheumatoid arthritis, arthralgia, arthritis, bone metastases and fractures, wherein Osmundacetone or a pharmaceutically acceptable salt thereof is an active ingredient Provides a composition.
<48>  <48>
<49> 상기와 같은 목적을 달성하기 위하여 , 본 발명은  In order to achieve the above object, the present invention is
<so> Osmundacetone 또는 이의 약학적으로 허용가능한 염이 유효성분으로 필수적 으로 구성되는 골다공증, 류마티스 관절염, 관절통, 파제트 병, 골 전이암 및 골절 로 이루어진 군에서 선택된 어느 하나 이상의 골 질환 예방 또는 치료용 약학적 조 성물을 제공한다. ' <so> Osmundacetone or a pharmaceutically acceptable salt thereof for the prevention or treatment of any one or more bone diseases selected from the group consisting of osteoporosis, rheumatoid arthritis, joint pain, Paget's disease, bone metastases and fractures consisting essentially of an active ingredient Provide pharmaceutical compositions. '
<51>  <51>
<52> 본 발명의 다른 목적을 달성하기 위하예 본 발명은  <52> In order to achieve another object of the present invention
<53> Osmundacetone 또는 이의 약학적으로 허용가능한 염을 유효성분으로 포함하 는 골다공증, 류마티스 관절염， 관절통, 파제트 병， 골 전이암 및 골절로 이루어진 군에서 선택된 어느 하나 이상의 골 질환 예방 또는 개선용 식품용 조성물을 제공 한다. <53> Food for preventing or ameliorating any one or more bone diseases selected from the group consisting of osteoporosis, rheumatoid arthritis, joint pain, Paget's disease, bone metastases and fractures, including Osmundacetone or a pharmaceutically acceptable salt thereof Provide composition for do.
<54>  <54>
<55> 본 발명의 다른 목적을 달성하기 위하여 , 본 발명은  In order to achieve the other object of the present invention,
<56> Osmundacetone 또는 이의 약학적으로 허용가능한 염이 유효성분으로 구성되 는 골다공증， 류마티스 관절염, 관절통， 파제트 병， 골 전이암 및 골절로 이루어진 군에서 선택된 어느 하나 이상의 골 질환 예방 또는 개선용 식품용 조성물을 제공 한다.  <56> Food for preventing or ameliorating any one or more bone diseases selected from the group consisting of osteoporosis, rheumatoid arthritis, joint pain, Paget's disease, bone metastases and fractures, wherein Osmundacetone or a pharmaceutically acceptable salt thereof is an active ingredient It provides a composition for.
<57>  <57>
<58> 본 발명의 다른 목적을 달성하기 위하여 , 본 발명은  In order to achieve the other object of the present invention,
<59> ■ Osmundacetone 또는 이의 약학적으로 허용가능한 염이 유효성분으로 필수적 으로 구성되는 골다공증, 류마티스 관절염, 관절통, 파제트 병, 골 전이암 및 골절 로 이루어진 군에서 선택된 어느 하나 이상의 골 질환 예방 또는 개선용 식품용 조 성물을 제공한다. ■ Preventing or ameliorating any one or more bone diseases selected from the group consisting of osteoporosis, rheumatoid arthritis, joint pain, Paget's disease, bone metastases and fractures, in which Osmundacetone or a pharmaceutically acceptable salt thereof is essentially an active ingredient. Provide food compositions for food.
<60>  <60>
<61> 본 발명의 또 다른 목적을 달성하기 위하여， 본 발명은  In order to achieve another object of the present invention, the present invention
<62> 고비 (Osmunda j aponi ca) 추출물을 유효성분으로 포함하는 골다공증, 류마티 스 관절염， 관절통， 파제트 병， 골 전이암 및 골절로 이루어진 군에서 선택된 어느 하나 이상의 골 질환 예방 또는 치료용 약학적 조성물을 제공한다.  <62> A pharmaceutical for the prevention or treatment of any one or more bone diseases selected from the group consisting of osteoporosis, rheumatoid arthritis, arthralgia, Paget's disease, metastatic cancer and fractures, including Osmunda j aponi ca extract as an active ingredient To provide a composition.
<63>  <63>
<64> 본 발명의 또 다른 목적을 달성하기 위하여， 본 발명은  In order to achieve another object of the present invention,
<65> 고비 (Osmunda j apon i ca) 추출물이 유효성분으로 구성되는 골다공증, 류마티 스 관절염， 관절통， 파제트 병， 골 전이암 및 골절로 이루어진 군에서 선택된 어느 하나 이상의 골 질환 예방 또는 치료용 약학적 조성물을 제공한다.  Osteoporosis, rheumatoid arthritis, arthralgia, Paget's disease, bone metastasis cancer and fractures, which are selected from the group consisting of Osmunda j apon i ca extracts for the prevention or treatment of any one or more bone diseases It provides a pharmaceutical composition.
<66>  <66>
<67> 본 발명의 또 다른 목적을 달성하기 위하여 , 본 발명은  In order to achieve the another object of the present invention,
<68> 고비 (Osmunda j aponi ca) 추출물이 유효성분으로 필수적으로 구성되는 골다공 증, 류마티스 관절염， 관절통， 파제트 병, 골 전이암 및 골절로 이루어진 군에서 선택된 어느 하나 이상의 골 질환 예방 또는 치료용 약학적 조성물을 제공한다. Osteoporosis, rheumatoid arthritis, arthralgia, Paget's disease, bone metastases and bone fractures, which are composed essentially of the extract of Osmunda j aponi ca. It provides a pharmaceutical composition.
<69> <69>
<70> 본 발명의 또 다른 목적을 달성하기 위하여 , 본 발명은  In order to achieve another object of the present invention, the present invention is
<71> 고비 (Osmunda j apon i ca) 추출물을 유효성분으로 포함하는 골다공증, 류마티 스 관절염， 관절통， 파제트 병， 골 전이암 및 골절로 이루어진 군에서 선택된 어느 하나 이상의 골 질환 예방 또는 개선용 식품용 조성물을 제공한다. .<71> Any one selected from the group consisting of osteoporosis, rheumatoid arthritis, arthralgia, Paget's disease, bone metastases and fractures, which contains the extract of Osmunda j apon i ca as an active ingredient Provided are food compositions for preventing or improving one or more bone diseases. .
<72> <7 2 >
<73> 본 발명의 또 다른 목적을 달성하기 위하여， 본 발명은  In order to achieve another object of the present invention, the present invention
<74> . 고비 (Osmunda j aponi ca) 추출물이 유효성분으로 구성되는 골다공증, 류마티 스 관절염， 관절통, 파제트 병， 골 전이암 및 골절로 이루어진 군에서 선택된 어느 하나 이상의 골 질환 예방 또는 개선용 식품용 조성물을 제공한다.  <74>. Osteoporosis, rheumatoid arthritis, arthralgia, Paget's disease, bone metastases and fractures, wherein the extract of Osmunda j aponi ca is composed of an active ingredient to provide.
<7 >  <7>
<76> 본 발명의 또 다른 목적을 달성하기 위하여 , 본 발명은  In order to achieve another object of the present invention, the present invention provides
<77> 고비 (Osmunda j aponi ca) 추출물이 유효성분으로 필수적으로 구성되는 골다공 증， 류마티스 관절염， 관절통, 파제트 병, 골 전이암 및 골절로 이루어진 군에서 선택된 어느 하나 이상의 골 질환 예방 또는 개선용 식품용 조성물을 제공한다. Osteoporosis, rheumatoid arthritis, arthralgia, Paget's disease, bone metastases and fractures, which are composed essentially of the extract of Osmunda j aponi ca. Provided is a composition for food.
<78> <78>
<79> 본 발명의 또 다른 목적을 달성하기 위하여 , 본 발명은  In order to achieve another object of the present invention, the present invention provides
<80> 골다공증， 류마티스 관절염, 관절통. 파제트 병, 골 전이암 및 골절로 이루 어진 군에서 선택된 어느 하나 이상의 골 질환 예방 또는 치료용 제제의 제조에 사 용되는 Osmundacetone 또는 이의 약학적으로 허용가능한 염의 용도를 제공한다. Osteoporosis, rheumatoid arthritis, joint pain. Provided is the use of Osmundacetone or a pharmaceutically acceptable salt thereof for use in the manufacture of an agent for the prophylaxis or treatment of any one or more bone diseases selected from the group consisting of Paget's disease, bone metastases and fractures.
<81> <81>
<82> 본 발명의 또 다른 목적을 달성하기 위하여， 본 발명은  In order to achieve another object of the present invention, the present invention provides
<83> Osmundacetone 또는 이의 약학적으로 허용가능한 염의 유효량을 이를 필요로 하는 개체에 투여하는 것을 특징으로 하는 골다공증， 류마티스 관절염. 관절통, 파 제트 병, 골 전이암 및 골절로 이루어진 군에서 선택된 어느 하나 이상의 골 질환 치료 방법을 제공한다.  Osteoporosis, rheumatoid arthritis, characterized in that an effective amount of Osmundacetone or a pharmaceutically acceptable salt thereof is administered to a subject in need thereof. Provided are methods for treating any one or more bone diseases selected from the group consisting of arthralgia, fajet disease, bone metastases and fractures.
<84>  <84>
<85> 본 발명의 또 다른 목적을 달성하기 위하여， 본 발명은  In order to achieve another object of the present invention, the present invention
<86> 골다공증， 류마티스 관절염, 관절통, 파제트 병, 골 전이암 및 골절로 이루 어진 군에서 선택된 어느 하나 이상의 골 질환 예방 또는 치료용 제제의 제조에 사 용하기 위한 고비 ( β»¾/ ί¾ japonica) 추출물의 용도를 제공한다 .  <86> Fertilizer (β »¾ / ί¾ japonica for use in the manufacture of a preparation for the prevention or treatment of any one or more bone diseases selected from the group consisting of osteoporosis, rheumatoid arthritis, arthralgia, Paget's disease, bone metastases and fractures ) Provides the use of extracts.
<87>  <87>
<88> 본 발명의 또 다른 목적을 달성하기 위하여 , 본 발명은  In order to achieve another object of the present invention, the present invention is
<89> 고비 7Η/ ί japonica) 추출물의 유효량을 이를 필요로 하는 개체에 투여하 는 것을 특징으로 하는 골다공증， 류마티스 관절염， 관절통， 파제트 병, 골 전이암 및 ¾절로 이루어진 군에서 선택된 어느 하나 이상의 골 질환 치료 방법을 제공한 <91> 이하 본 발명을 상세히 설명한다 . <89> At least one selected from the group consisting of osteoporosis, rheumatoid arthritis, arthralgia, Paget's disease, metastatic cancer and section ¾, which is administered to an individual in need thereof an effective amount of the fern 7Η / ί japonica) extract. Providing a way to treat bone disease Hereinafter, the present invention will be described in detail.
<92> <92>
<93> 본발명은 osmundacetone 또는 이의 약학적으로 허용가능한 염을유효성분으 로 포함하는 골다공증， 류마티스 관절염， 관절통， 파제트 병， 골 전이암 및 골절로 이루어진 군에서 선택된 어느 하나 이상의 골 질환 예방 또는 치료용 약학적 조성 물을 제공한다. The invention relates to the prevention or prevention of any one or more bone diseases selected from the group consisting of osteoporosis, rheumatoid arthritis, arthralgia, Paget's disease, bone metastases and fractures, including osmundacetone or a pharmaceutically acceptable salt thereof. Provide a therapeutic pharmaceutical composition.
<94>  <94>
<9 > 본 발명에 따른 상기 약학적 조성물은 osmundacetone을 유효성분으로 포함하 는 조성물일 수도 있고, 유효성분으로서 osmundacetone으로 구성되는 조성물일 수 도 있으며, 또는 유효성분으로서 osmundacetone이 필수적으로 구성되는 조성물일 수도 있다. The pharmaceutical composition according to the present invention may be a composition containing osmundacetone as an active ingredient, a composition consisting of osmundacetone as an active ingredient, or a composition consisting essentially of osmundacetone as an active ingredient. It may be.
<96>  <96>
< 7> 본 명세서에서 용어 '〜을 포함하는 (comprising)' 이란 '함유하는 (including)' 또는 '특징으로 하는 (character ized by)' 과 동일한 의미로 사용되 며 , 본 발명에 따른 조성물 또는 방법에 있어서, 구체적으로 언급되지 않은 추가적 인 구성 성분 또는 방법의 단계 등을 배제하지 않는다. 또한 용어 '로 구성되는 (consisting of)' 이란 별도로 기재되지 않은 추가적인 요소, 단계 또는 성분 등을 제외하는 것을 의미한다. 용어 '필수적으로 구성되는 (essentially consisting of) ' 이란， 조성물 또는 방법의 범위에 있어서, 기재된 물질 또는 단계와 더불어 이의 기본적인 특성에 실질적으로 영향을 미치지 않는 물질 또는 단계 등을 포함할 수 있는 것을 의미한다. In the present specification, the term 'comprising' is used in the same sense as 'including' or 'characterized by', and the composition or method according to the present invention. Does not exclude additional components or steps of the method not specifically mentioned. In addition, the term 'consisting of' means to exclude additional elements, steps or components, etc., unless otherwise noted. The term 'essentially consisting of' means that, within the scope of the composition or method, may include the materials or steps described, as well as materials or steps that do not substantially affect the basic properties thereof. .
Figure imgf000010_0001
Figure imgf000010_0001
<99> <99>
<100> Osmundacetone은 C10H10¾의 분자식 (분자량 178.184Da)으로 표시되며， 상단의 화학식의 구조를 갖는 화합물로, 본 발명자들에 의하여 고비 추출물로부터 파골세 포의 중식 및 분화 억제 활성과 조골세포의 분화 활성화 능력을 갖는 것으로 분리 동정된 성분이다. Dihydroxybenzyl ideneacetone, ( 3E ) -4- ( 3 , -d i hydr oxypheny 1 ) -3- buten-2-one 또는 IUPAC 이름으로 5， 7-cH hydr oxy-2- ( 4-hydr oxypheny 1 )-8- [(2S, 3R, 4R, 5S, 6R)-3 ,4, 5-tr ihydroxy-6-(hydroxymethyl )oxan-2-yl ]-6-[ (2S , 3R, 4S, 5S)— 3,4,5-trihydroxyoxari-2-yl]-4H-chromen-4-one으로 .불리며， 상온에서 무색 또는 황 색에서 갈색의 유색 결정으로 존재한다. <100> Osmundacetone is represented by the molecular formula of C 10 H 10 ¾ (molecular weight 178.184Da). A compound having the structure of the formula, which has been identified and identified by the present inventors from the fern extract as having the ability to inhibit the differentiation and differentiation of osteoclasts and to activate the differentiation of osteoblasts. Dihydroxybenzyl ideneacetone, (3E) -4- (3, -di hydr oxypheny 1) -3-buten-2-one or 5, 7-cH hydr oxy-2- (4-hydr oxypheny 1) -8- under IUPAC name [(2S, 3R, 4R, 5S, 6R) -3,4, 5-tr ihydroxy-6- (hydroxymethyl) oxan-2-yl] -6- [(2S, 3R, 4S, 5S) — 3,4 , 5-trihydroxyoxari-2-yl] -4H-chromen-4-one . It is a colorless or yellow to brown colored crystal at room temperature.
<ioi> Osmundacetone은 다수의 미국 유해독성물질 규제기관에서 인체독성, 자극성, 발암성, 환경독성 등 모든 검사에서 무해하며 안전하다는 관정을 받았다 (참고: TSCA: Not Listed; CLEAN WATER ACT(CWA): Not Listed; SARA313: Not Listed; MARINE POLLUTANT: Not Listed; RIGHT TO KNOW LIST(NEW JERSEY): Not Listed; RIGHT TO KNOW L I ST ( MASSACHUSETTS ) : Not Listed; RIGHT TO KNOW LIST(PENNSYLVANIA)： Not Listed; ILLINOIS TOXIC AIR CONTAMINANTS: Not Listed; CLEAN AIR ACT(CAA)： Not Listed; DHS CHEMICALS OF INTEREST: Not Listed; CALIFORNIA PROP 65: Not Listed; OSHA: Not Listed; CALIFORNIA PROP 65 TOXICITY TYPECCANCER, DEVELOPMENTAL, FEMALE, MALE): None; OSHA HAZ CLASS (CARCINOGEN, CORROSIVE, FLAMABLE, REACTIVE, TOXIC)： None 둥). <ioi> Osmundacetone has been ruled by many US Hazardous Substances Regulators as harmless and safe for all tests, including human toxicity, irritation, carcinogenicity and environmental toxicity (see TSCA: Not Listed; CLEAN WATER ACT (CWA): Not Listed; SARA313: Not Listed; MARINE POLLUTANT: Not Listed; RIGHT TO KNOW LIST (NEW JERSEY): Not Listed; RIGHT TO KNOW LI ST (MASSACHUSETTS): Not Listed; RIGHT TO KNOW LIST (PENNSYLVANIA): Not Listed; ILLINOIS TOXIC AIR CONTAMINANTS: Not Listed; CLEAN AIR ACT (CAA): Not Listed; DHS CHEMICALS OF INTEREST: Not Listed; CALIFORNIA PROP 65: Not Listed; OSHA: Not Listed; CALIFORNIA PROP 65 TOXICITY TYPECCANCER, DEVELOPMENTAL, FEMALE, MALE): None; OSHA HAZ CLASS (CARCINOGEN, CORROSIVE, FLAMABLE, REACTIVE, TOXIC): None.
<102>  <102>
<i03> 본 발명의 조성물에 포함되는 osmundacetone은 그 자체 또는 염 , 바람직하게 는 약학적으로 허용가능한 염의 형태로 사용될 수 있다. 본 발명에서 '약학적으로 허용가능한1이란 생리학적으로 허용되고 인간에게 투여될 때, 통상적으로 알레르기 반웅 또는 이와 유사한 반웅을 일으키지 않는 것을 말하며 . 상기 염으로는 약학적 으로 허용가능한 유리산 (free acid)에 의하여 형성된 산 부가염이 바람직하다. 상 기 유리산으로는 유기산과 무기산을 사용할 수 있다. 상기 유기산은 이에 제한되는 것은 아니나, 구연산， 초산， 젖산, 주석산, 말레인산, 푸마르산, 포름산, 프로피온 산， 옥살산, 트리폴로오로아세트산, 벤조산， 글루콘산， 메타술폰산, 글리콜산, 숙 신산, 4-틀루엔술폰산, 글루탐산 및 아스파르트산을 포함한다. 또한 상기 무기산은 이에 제한되는 것은 아니나, 염산, 브름산, 황산 및 인산을 포함한다. <i03> Osmundacetone included in the composition of the present invention may be used on its own or in the form of a salt, preferably a pharmaceutically acceptable salt. In the present invention, 'pharmaceutically acceptable 1' means a physiologically acceptable and, when administered to a human, usually does not cause an allergic reaction or a similar reaction. As the salt, an acid addition salt formed with a pharmaceutically acceptable free acid is preferable. Organic acids and inorganic acids may be used as the free acid. The organic acid is not limited thereto, citric acid, acetic acid, lactic acid, tartaric acid, maleic acid, fumaric acid, formic acid, propionic acid, oxalic acid, tripoloroacetic acid, benzoic acid, gluconic acid, metasulfonic acid, glycolic acid, succinic acid, 4-frame Luenesulfonic acid, glutamic acid and aspartic acid. In addition, the inorganic acid includes, but is not limited to, hydrochloric acid, bromic acid, sulfuric acid and phosphoric acid.
<104>  <104>
<105> 상기 osmundacetone은 화학적으로 합성된 것일 수도 있고, 천연물에서 분리 된 것일 수도 있는데， 천연물에서 ^리된 osmundacetone을 사용하는 경우에는 특히 고비과 (Osmundaceae) 식물에서 분리된 것일 수 있다 . 가장 바람직하게는 고비과에 속하는 L^(OsmuncIa 'a/¾ /ca)로부터 분리된 것일 수 있다. The osmundacetone may be chemically synthesized or may be separated from natural products. In particular, the osmundacetone may be separated from the Osmundaceae plant when using osmundacetone collected from natural products. Most preferably in the fern It may be separated from belong to L ^ (OsmuncIa ' a / ¾ / ca).
<106>  <106>
<107> 고비과 (Osmundaceae)의 식물은 고비목 (Osniundales)에 속한 유일한 과목 식물 이다. 고비목 (Osniundales)은 약 2억 1000만 년 전 중생대, 트라이아스기에서 기원 한 오래된 양치식물군으로서, 박벽포자낭고사리류 (Leptosporangiate fern)로 분류 된다.  The plant of the Osmundaceae is the only subject plant belonging to Osniundales. Osniundales is a group of old ferns that originated in the Mesozoic, Triassic, about 210 million years ago, and is classified as Leptosporangiate fern.
<108>  <108>
<109> 고비과에 속하는 고비 ((¾歸 7<ώ japonica) 학명이 Osmunda japonica Thunb. <109> The fern belonging to the fern family ((¾ (7 <ώ japonica) scientific name is Osmunda japonica Thunb.
≡—는 ' Os inda nipponica Makino이며, 라틴명으로는 Osmundae Rhizoma라고 한다. 영 문으로 Japanese royal fern 또는 Japanese flowering fern으로도 불리는 식물로, 일본, 중국, 한국, 대만 등지의 동아시아와 러시아 등에 자생하는 식물이다. 고비 의 어린잎이 오랫동안 식재료로 이용되어 왔을 뿐만 아니라， 전통의학 및 민간요법 으로 다양한 질병에 사용되어 온 약용식물이다. 고비는 osmunda lactone, osmundacetone, osmunda 1 in, d i hydro i soomunda 1 i n . parasorboside 및 ponasterone A, ecdysone, ecdysterone 등 탈피호르몬 성분이 함유되어 있다. 전통의학에서는 뿌리줄기를 자기 (紫 또는 자기관중 (紫^貫衆)이라 하여 , 독이 조금 있으나 회 층， 촌백층， 요층 등 구층， 항바이러스， 항균 등 살층 (殺蟲), 청열해독 (熱解毒), 거어지혈 0ΐ去疲止血)， 풍열감모, 온열반진， 토혈， 비혈, 암치질로 인한 변혈, 적리, 대하증 등의 치료에 사용한다 (중약대사전). ≡- he is called 'The Os inda nipponica Makino, Latin people have a Osmundae Rhizoma. It is also called Japanese royal fern or Japanese flowering fern. It is native to East Asia and Russia in Japan, China, Korea and Taiwan. Fern young leaves have been used as food ingredients for a long time, as well as medicinal plants that have been used for various diseases in traditional medicine and folk medicine. Ferns are osmunda lactone, osmundacetone, osmunda 1 in, di hydro i soomunda 1 in. It contains parasorboside and morphogenetic hormones such as ponasterone A, ecdysone and ecdysterone. In traditional medicine, root stems are called porcelain (紫 or spectators), and they have some poisons, but they are gray, chonbaek, urine, etc., antiviral, antibacterial, etc. (청 解 청) ), Is used for the treatment of bloated hemorrhoids (0ΐ 去 疲 止血), wind-heat sensation, hyperthermia, bleeding, bleeding, and hemorrhoids caused by hemostasis, severity, and sputum (Chinese medicine dictionary).
<110>  <110>
<πι> 본 발명자들은 일실시예에서 고비의 추출물이 뼈 조직을 파괴하고 재흡수하 는 역할을 하는 과골세포 (osteoclast)의 분화를 효과적으로 억제하는 것을 확인하 였다. 마우스에서 분리한 골수세포에서 파골세포의 줄기세포 전구세포인 단핵구 세 포를 분리하여 분화촉진인자인 RANKL과 M— CSF로 자극하고, 고비 추출물을 처리하여 파골세포의 분화에 미치는 영향을 확인한 결과, 고비의 열수 추출물 또는 에틸 아 세테이트 추출물이 골수세포가 다핵 파골세포로 분화하는 것을 효과적으로 억제하 였다.  <πι> The inventors confirmed that in one embodiment, the extract of ferns effectively inhibits the differentiation of osteoclasts, which serve to destroy and reabsorb bone tissue. From the bone marrow cells isolated from the mouse, monocyte cells, stem cells progenitor cells of osteoclasts, were isolated and stimulated with differentiation-promoting factors, RANKL and M—CSF, and the treatment of fern extracts confirmed the effects on osteoclast differentiation. Fertilized hot water extract or ethyl acetate extract effectively inhibited the differentiation of myeloid cells into multinucleated osteoclasts.
<112>  <112>
<Π3> 본 발명자들은 HPLC와 NMR을 이용하여 고비 추출물에서 파골세포 분화 억제 및 조골세포 분화 활성화 효과가 있는 성분을 분리 동정하였다. 고비의 열수 추출 물과 에틸 아세트 추출물 /분획물로부터 분리 동정된 단일 화합물이 osmundacetone 이다. Osmundacetone은 뛰어난 파골세포 분화 억제 활성과 조골세포 활성화 활성을 가지고 있을 뿐만 아니라, 세포독성은 매우 낮아 안전한 것으로 나타났다. 따라서 통상의 기술자는 본 발명자들이 규명한 고비 추출물과 osmundacetone 의 상기 활성을 이용하여 파골세포에 의한 골 흡수와 조골세포에 의한 새로운 골 기질의 형성 및 이후의 무기질화 과정의 골 대시" 과정의 균형이 깨지면서 골밀도와 강도가 감소하여 발생하는 다양한 골 질환에 대한 효과적인 예방, 증상의 개선 또 는 치료의 효과를 기대할 수 있음을 이해할 수 있다. 바람직하게는， 본 발명에서 상기 골 질환은 골다공증, 류마티스 관절염， 관 절통， 파제트 병 , 골 전이암 또는 골절일 수 있으며， 상기 각각의 질환과 파골세포 와의 상관관계에 대해서는 본 명세서의 적용예를 참고한다. 본 명세서에서 '치료 '란 치료되는 개체 또는 세포의 자연적 과정을 변경시키 려는 임상적 시술을 의미하몌 임상적 병리의 예방을 위해서도 수행될 수 있다. 치 료의 바람직한 효과는 질병의 발생 또는 재발 억제, 증상의 완화, 질병의 임의의 직접 또는 간접적인 병리학적 결과의 감소 , 질병 진행 속도의 감소 , 질병 상태의 개선, 호전, 완화 또는 개선된 예후 둥을 포함한다. 또한 용어 '예방'은 질병의 발 병을 억제시키거나 진행을 지연시키는 모든 행위를 의미한다. 본 발명의 약학적 조성물의 투여량은 투여 경로. 투여 시간， 치료 횟수, 치 료 기간, 치료가 필요한 개체의 연령, 체중, 건강 상태, 성별, 질환의 중증도, 약 물에 대한 민감도, 식이 및 배설율, 등 다양한 요인을 고려하여 당업자가 상술한 특정 용도에 따른 적절한 유효량을 결정할 수 있다. 상기 "유효량" 이란 개체에게 투여하였을 때， 골 질환의 개선, 치료, 예방， 검출 또는 진단 효과를 나타내기에 층분한 양을 말한다. 상기 '개체' 란 동물, 바람직하게는 포유동물, 가장 바람직하 게는 인간을 포함하는 동물일 수 있으며, 동물에서 유래한 세포, 조직， 기관 등일 수도 있다. 상기 개체는 치료가 필요한 골 질환 환자 (pat i ent ) 일 수 있다. 상기 투여는 하루에 한번 또는 수회 나누어 투여할 수도 있다. 본 발명의 약 학 조성물은 단독으로 투여되거나 골 질환의 예방 또는 치료에 효과가 있는 것으로 알려진 다른 치료제와 병용하여 투여될 수 있고, 병용하여 투여할 경우 다른 치료 제와 순차적 또는 동시에 투여될 수 있다. 상기 단독 투여 또는 병용 투여시 본 발 명의 약학 조성물의 투여량은 부작용 없이 최소한의 양으로 최대 효과를 얻을 수 있는 양을 투여하는 것이 바람직하며， 이는 당업자에 의해 용이하게 결정될 수 있 다. The inventors separated and identified components having osteoclast differentiation inhibition and osteoblast differentiation activating effects in fern extracts using HPLC and NMR. Osmundacetone is a single compound that has been identified as isolated from fern hydrothermal extracts and ethyl acetate extracts / fractions. Osmundacetone has excellent osteoclast differentiation inhibitory activity and osteoblast activation activity. As well as having cytotoxicity, it was shown to be very low and safe. Therefore, the skilled person is using the above-described activity of fern extract and osmundacetone, which the inventors have identified, as the balance of bone absorption by osteoclasts, formation of new bone matrix by osteoblasts, and bone dashing of the subsequent mineralization process is broken. It can be understood that effective prevention, improvement of symptoms, or treatment of various bone diseases caused by decreased bone density and strength can be expected. Preferably, the bone diseases in the present invention are osteoporosis, rheumatoid arthritis, vascular tube. It may be nodal pain, Paget's disease, bone metastasis cancer, or fracture, and the application of the present specification for the correlation between each disease and osteoclast is referred to. Means a clinical procedure to change the process, but can also be performed to prevent clinical pathology. The desirable effects of treatment include: inhibiting the occurrence or recurrence of the disease, alleviating the symptoms, reducing any direct or indirect pathological consequences of the disease, decreasing the rate of disease progression, improving, improving, alleviating, or improving the prognosis of the disease. In addition, the term "prevention" refers to any action that inhibits or delays the progression of a disease The dosage of the pharmaceutical composition of the present invention is the route of administration, time of administration, frequency of treatment, treatment A person skilled in the art can determine the appropriate effective amount for the specific use described above, taking into account various factors such as duration, age, weight, health condition, sex, severity of the disease, drug sensitivity, diet and excretion rate, etc. The term “effective amount” refers to an amount sufficient to exert an effect of improving, treating, preventing, detecting, or diagnosing a bone disease when administered to an individual. It may preferably be a mammal, most preferably an animal, including a human, and may be cells, tissues, organs, etc. derived from the animal, etc. The subject may be a patient with bone disease (pat i ent) in need of treatment. The administration may be administered once or several times a day. The pharmaceutical composition of the present invention may be administered alone or in combination with other therapeutic agents known to be effective in the prevention or treatment of bone diseases, and when administered in combination, may be administered sequentially or simultaneously with other therapeutic agents. The foot when administered alone or in combination The dosage of the pharmaceutical composition of the title is preferably administered in an amount that can achieve the maximum effect in a minimum amount without side effects, which can be easily determined by those skilled in the art.
<124>  <124>
<125> 본 발명의 조성물의 총 유효량은 단일 투여량 (single dose)으로 환자에게 투여될 수 있으며， 다중 투여량 (multiple dose)으 장기간 투여되는 분할 치료 방 법 (fractionated treatment protocol)에 의해 투여될 수 있다. 본 발명의 약학적 조성물은 질환의 정도에 따라 유효성분의 함량을 달리할 수 있다.  The total effective amount of the composition of the present invention may be administered to a patient in a single dose, and may be administered by a fractionated treatment protocol that is administered at multiple doses for a long time. Can be. The pharmaceutical composition of the present invention may vary the content of the active ingredient depending on the extent of the disease.
<126> 바람직하게 본 발명의 약학적 조성물의 전체 용량은 1일당 환자 체중 1kg 당 약 0.0 내지 10,000mg, 가장 바람직하게는 0.1 내지 50 ng일 수 있다. 그러나 상기 약학적 조성물의 용량은 제제화 방법， 투여 경로 및 치료 횟수뿐만 아니라 환 자의 연령, 체중, 건강 상태, 성별， 질환의 중증도， 식이 및 배설율 등 다양한 요 인들을 고려하여 환자에 대한 유효 투여량이 결정되는 것이므로, 이러한 점을 고려 할 때 당 분야의 통상적인 지식을 가진 자라면 본 발명의 조성물의 적절한 유효 투 여량을 결정할 수 있을 것이다. 본 발명에 따른 약학적 조성물은 본 발명의 효과를 보이는 한 그 제형 , 투여 경로 및 투여 방법에 특별히 제한되지 아니한다.  Preferably the total dose of the pharmaceutical composition of the present invention may be about 0.0 to 10,000 mg, most preferably 0.1 to 50 ng per kg of patient body weight per day. However, the dosage of the pharmaceutical composition may be determined by taking into consideration the various factors such as the age, weight, health condition, sex, severity of the disease, diet and excretion rate, as well as the formulation method, route of administration and frequency of treatment. As will be determined, one of ordinary skill in the art will be able to determine the appropriate effective dosage of the compositions of the present invention. The pharmaceutical composition according to the present invention is not particularly limited to the formulation, route of administration and method of administration as long as the effect of the present invention is shown.
<127>  <127>
<128> 본 발명의 약학적 조성물은 약학적으로 허용되는 담체와 함께 당업계에 공지 된 방법으로 투여경로에 따라 다양하게 게형화될 수 있다 . '약학적으로 허용되는 ' 이란 생리학적으로 허용되고 인간에게 투여될 때, 활성성분의 작용을 저해하지 찮으며 통상적으로 위장 장애 , 현기증과 같은 알레르기 반응 또는 이와 유사한 반 응을 일으키지 않는 비독성의 조성물을 말한다. 상기 담체로는 모든 종류의 용미 j , 분산매질, 수중유 또는 유중수 에멀견， 수성 조성물, 리포좀， 마이크로비드 및 마 이크로좀이 포함된다.  The pharmaceutical composition of the present invention may be variously formulated according to the route of administration by a method known in the art together with a pharmaceutically acceptable carrier. 'Pharmaceutically acceptable' is a non-toxic composition that is physiologically acceptable and, when administered to humans, does not inhibit the action of the active ingredient and usually does not cause gastrointestinal disorders, allergic reactions such as dizziness or similar reactions. Say Such carriers include all kinds of solvents j, dispersion media, oil-in-water or water-in-oil emulsions, aqueous compositions, liposomes, microbeads and microsomes.
<12 >  <12>
<130> 투여 경로로는 경구적 또는 비경구적으로 투여될 수 있다 . 비경구적인 투여 방법으로는 이에 한정되지는 않으나 정맥내, 근육내. 동맥내, 골수내， 경막내, 심 장내, 경피， 피하， 복강내, 비강내, 장관, 국소, 설하 또는 직장내 투여일 수 있 다.  The route of administration may be administered orally or parenterally. Parenteral administration methods include, but are not limited to, intravenous and intramuscular. Intraarterial, intramedullary, intradural, intracardiac, transdermal, subcutaneous, intraperitoneal, intranasal, intestinal, topical, sublingual or rectal administration.
<131>  <131>
<132> 본 발명의 약학적 조성물을 경구투여하는 경우 본 발명의 약학적 조성물은 적합한 경구투여용 담체와 함깨 당업계에 공지된 방법에 따라 분말， 과립, 정제, 환제， 당의정제 , 캡슐제， 액게 , 겔제 , 시럽게 , 현탁액 , 웨이퍼 등의 형태로 제형화 될 수 있다. 적합한 담체의 예로는 락토즈, 텍스트로즈, 수크로즈, 솔비를, 만니 틀. 자일리톨, 에리스리틀 및 말티를 둥을 포함하는 당류와 옥수수 전분, .밀 전분, 쌀 전분 및 감자 전분 등을 포함하는 전분류， 셀를로즈. 메틸 샐롤로즈, 나트륨 카 르복시메틸셀롤로오즈 및 하이드록시프로필메틸셀를로즈 등을 포함하는 셀를로즈 류, 겔라틴, 폴리비닐피를리돈 등과 같은 충전제가 포함될 수 있다. 또한， 경우에 따라 가교결합 폴리비닐피를리돈， 한천， 알긴산 또는 나트륨 알기네이트 등을 붕해 제로 첨가할 수 있다. 나아가, 상기 약학적 조성물은 항응집제, 윤활제, 습윤제, 향료, 유화제 및 방부제 등을 추가로 포함할 수 있다. In the case of oral administration of the pharmaceutical composition of the present invention, the pharmaceutical composition of the present invention may be prepared using powders, granules, tablets, according to methods known in the art, together with a suitable oral carrier. It can be formulated in the form of pills, dragees, capsules, liquids, gels, syrups, suspensions, wafers and the like. Examples of suitable carriers include lactose, textrose, sucrose, sorbbi, mannitl. Starch, celrose, including sugars and corn starch, wheat starch, rice starch and potato starch, including xylitol, erythritol and malty. Fillers such as celluloses, gelatin, polyvinylpyridone, and the like, including methyl salose, sodium carboxymethyl cellulose, hydroxypropyl methyl cellulose, and the like. In some cases, crosslinked polyvinylpyridone, agar, alginic acid or sodium alginate may be added as a disintegrating agent. Furthermore, the pharmaceutical composition may further include an anticoagulant, a lubricant, a humectant, a perfume, an emulsifier, and a preservative.
<1 33>  <1 33>
<134> 또한, 비경구적으로 투여하는 경우 본 발명의 약학적 조성물은 적합한 비경 구용 담체와 함께 주사제. 경피투여제 및 비강 흡입제의 태로 당업계애 공지된 방법에 따라 제형화될 수 있다 . 상기 주사제의 경우에는 반드시 멸균되어야 히-며 박테리아 및 진균과 같은 미생물의 오염으로부터 보호되어야 한다. 주사제의 경우 적합한 담체의 예로는 이에 한정되지는 않으나, 물, 에탄올， 폴리을 (예를 들어, 글 리세를， 프로필렌 글리콜 및 액체 폴리에틸렌 글리콜 등)， 이들의 흔합물 및 /또는 식물유를 포함하는 용매 또는 분산매질일 수 있다 . 보다 바람직하게는, 적합한 담 체로는 행크스 용액, 링거 용액, 트리에탄올 아민이 함유된 PBS(phospliate buf fered sal ine) 또는 주사용 멸균수, 10% 에탄을， 40% 프로필렌 글리콜 및 5% 덱 스트로즈와 같은 등장 용액 등을 사용할 수 있다. 상기 주사제를 미생물 오염으로 부터 보호하기 위해서는 파라벤, 클로로부탄올， 페놀, 소르빈산, 티메로살 등과 같 은 다양한 항균제 및 항진균제를 추가로 포함할 수 있다. 또한, 상기 주사제는 대 부분의 경우 당 또는 나트륨 클로라이드와 같은 등장화제를 추가로 포함할 수 있 다.  In addition, when administered parenterally, the pharmaceutical composition of the present invention may be injected with a suitable parenteral carrier. It may be formulated according to methods known in the art in the form of transdermal and nasal inhalants. Injectables must be sterile and protected from contamination of microorganisms such as bacteria and fungi. In the case of injectables, examples of suitable carriers include, but are not limited to, solvents including water, ethanol, poly (e.g. glycerin, propylene glycol and liquid polyethylene glycols), combinations thereof and / or vegetable oils, or It may be a dispersion medium. More preferably, suitable carriers include Hanks' solution, Ringer's solution, phospliate buf fered salin (PBS) containing triethanol amine or sterile water for injection, 10% ethane, 40% propylene glycol and 5% dextrose. The same isotonic solution and the like can be used. In order to protect the injection from microbial contamination, various antibacterial and antifungal agents such as parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like may be further included. In addition, the injection may in most cases further comprise an isotonic agent such as sugar or sodium chloride.
<1.35>  <1.35>
< 136> 경피투여제의 경우 연고제, 크림제, 로션제, 겔제, 외용액게, 파스타제, 리 니맨트제 , 에어를제 등의 형태가 포함된다. 상기에서 ᅳ경피투여 ' 는 약학적 조성 물을 국소적으로 피부에 투여하여 약학적 조성물에 함유된 유효한 양의 활성성분이 피부 내로 전달되는 것을 의미한다. 예컨대, 본 발명의.약학적 조성물을 주사형 제 In the case of transdermal administration, ointments, creams, lotions, gels, external liquid crabs, pasta preparations, liniment preparations, and air preparations are included. In the above, 'transdermal administration' means that the pharmaceutical composition is locally administered to the skin so that an effective amount of the active ingredient contained in the pharmaceutical composition is delivered into the skin. For example, the pharmaceutical composition of the present invention may be injectable.
. 형으로 제조하여 이를 30게이지의 가는 주사 바늘로 피부를 가볍게 단자 (pr i ck)하 거나 피부에 직접적으로 도포하는 방법으로 투여될 수 있다. 이들 제형은 제약 화 학에 일반적으로 공지된 처방서인 문헌 (Remington ' s Pharmaceut i cal Sci ence , 15th Edition, 1975, Mack Publishing Com any , Easton, Pennsylvania)에 기술되어 있 다. . It can be administered in the form of a 30-gauge thin needle with light needles (pr i ck) or by direct application to the skin. These formulations are formulated in Remington's Pharmaceut i cal Science, 15th, which is a commonly known prescription in pharmaceutical chemistry. Edition, 1975, Mack Publishing Com any, Easton, Pennsylvania.
<138> 흡입투여제의 경우, 본 발명에 따라 사용되는 화합물은 적합한 추진제， 예를 들면, 디클로로풀루오로메탄, 트리클로로플루오로메탄, 디클로로테트라플루오로에 탄, 이산화탄소 또는 다른 적합한 기체를 사용하여， 가압 팩 또는 연무기로부터 에 어로졸 스프레이 형태로 편리하게 전달할 수 있디-. 가압 에어로졸의 경우， 투약 단 위는 계량된 양을 전달하는 밸브를 제공하여 결정할 수 있다. 예를 들면, 흡입기 또는 취입기에 사용되는 젤라틴 캡술 및 카트리지는 화합물 및 락토오즈 또는 전분 과 같은 적합한 분말 기제의 분말 흔합물을 함유하도록 제형화할 수 있다. In the case of inhalation agents, the compounds used according to the present invention may be prepared by using a suitable propellant, for example, dichlorofuluromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas. Therefore, it can be conveniently delivered in the form of aerosol spray from pressurized pack or nebulizer. In the case of a pressurized aerosol, the dosage unit can be determined by providing a valve to deliver a metered amount. For example, gelatin capsules and cartridges for use in inhalers or blowers may be formulated to contain a compound and powdered mixture of suitable powder based such as lactose or starch.
<139>  <139>
그 밖의 약학적으로 허용되는 담체로는 다음의 문헌에 기재되어 있는 것을 참고로 할 수 있다 (Remington' s Pharmaceutical Sciences, 19th ed. , Mack Publishing Company, Easton, PA, 1995) .  Other pharmaceutically acceptable carriers may be referred to those described in the following literature (Remington's Pharmaceutical Sciences, 19th ed., Mack Publishing Company, Easton, PA, 1995).
<142> 또한， 본 발명에 따른 약학적 조성물은 하나 이상의 완층제 (예를 들어， 식염 수 또는 PBS), 카보하이트레이트 (예를 들어, 글루코스, 만노즈， 슈크로즈 또는 텍 스트란), 항산화제， 정균제, 킬레이트화제 (예를 들어, EDTA 또는 글루타치온), 아 쥬반트 (예를 들어, 알루미늄 하이드록사이드)， 현탁제, 농후제 및 /또는 보존제를 추가로 포함할 수 있다. In addition, the pharmaceutical compositions according to the invention may comprise one or more laxative agents (eg saline or PBS), carbohydrates (eg glucose, mannose, sucrose or texran), antioxidants Agents, bactericides, chelating agents (eg EDTA or glutathione), adjuvants (eg aluminum hydroxide), suspending agents, thickening agents and / or preservatives.
<143>  <143>
또한， 본 발명의 약학적 조성물은 포유동물에 투여된 후 활성 성분의 신속, 지속 또는 지연된 방출을 제공할 수 있도록 당업계에 공지된 방법을 사용하여 제형 화될 수 있다.  In addition, the pharmaceutical compositions of the present invention may be formulated using methods known in the art to provide rapid, sustained or delayed release of the active ingredient after administration to a mammal.
<145> <14 5 >
<146> 또한 본 발명은 osmundacetone 또는 이의 약학적으로 허용가능한 염을 유효 성분으로 포함하는 골다공증. 류마티스 관절염, 관절통. 파제트 병. 골 전이암 및 골걸로 이루어진 군에서 선택된 어느 하나 이상의 골 질환 .예방 또는 개선용 식품 용 조성물을 제공한다.  The present invention also provides osteoporosis comprising osmundacetone or a pharmaceutically acceptable salt thereof as an active ingredient. Rheumatoid arthritis, arthralgia. Paget's bottle. Provided is a food composition for preventing or ameliorating any one or more bone diseases selected from the group consisting of bone metastasis cancer and bone bone.
<147>  <147>
<148> 본 발명자들이 규명한 osmundacetone의 상기 골 질환에 대한 예방 또는 개선 의 효과는 본 명세서에서 전술한 바와 같다. <149> The effects of the prevention or amelioration of osmundacetone on the bone disease, as identified by the present inventors, are as described above in the present specification. <149>
<i50> 상기 식품용 조성물은 기능성 식품 (functional food) , 영양보조제 <i50> The composition for food is a functional food (functional food), nutritional supplements
(nutritional sup lement), 건강식품 (health food) 및 식품첨기-제 (food additives) 등의 모든 형태를 포함한다. 상기 유형들은 당업계에 공지된 통상적인 방법에 따라 다양한 형태로 제조할 수 있다. It includes all forms such as nutritional supplement, health food and food additives. These types can be prepared in various forms according to conventional methods known in the art.
<151>  <151>
<152> 예를 들면, 건강식품으로는 본 발명의 식품용 조성물 자체를 차. 쥬스 및 드 링크의 형태로 제조하여 음용하도록 하거나, 과립화, 캡슐화 및 분말화하여 섭취할 수 있다. 또한 본 발명의 식품용 조성물은 골 질환 예방 또는 개선의 효과가 있다 고 알려진 공지의 물질 또는 활성 성분과 함께 흔합하여 조성물의 형태로 제조할 수 있다.  For example, as a health food, the food composition itself of the present invention is used. It can be prepared in the form of juices and drinks for drinking, or ingested by granulation, encapsulation and powdering. In addition, the food composition of the present invention may be prepared in the form of a composition by mixing with a known substance or active ingredient known to have the effect of preventing or improving bone diseases.
<153> <1 5 3>
<'54> 또한 기능성 식품으로는 음료 (알콜성 음료 포함), 과실 및 그의 가공식품 ( 예를 들어 과일 통조림 , 병조림 , 잼， 마아말레이드 등) , 어류 , 육류 및 그 가공식 품 (예를 들어 햄, 소시지콘비이프 등)， 빵류 및 면류 (예를 들어 우동, 메밀국수, 라면, 스파게티, 마카로니 등), 과즙， 각종 드링크， 쿠키， 엿, 유제품 (예를 들어 버터， 치이즈 등)， 식용식물유지， 마아가린, 식물성 단백질, 레토르트 식품, 냉동 식품, 각종 조미료 (예를 들어 된장， 간장, 소스 등) 등에 본 발명의 식품용 조성물 을 첨가하여 제조할 수 있다.  <'54> In addition, functional foods include beverages (including alcoholic beverages), fruits and processed foods (e.g. canned fruit, canned foods, jams, marmalade, etc.), fish, meat and processed food products (e.g. For example, ham, sausage cornebi, etc., breads and noodles (e.g. udon, soba noodles, ramen, spaghetti, macaroni, etc.), juices, various drinks, cookies, candy, dairy products (e.g. butter, cheese) It can be prepared by adding the food composition of the present invention to edible vegetable fats and oils, margarine, vegetable protein, retort food, frozen food, various seasonings (for example, miso, soy sauce, sauce, etc.).
<1S5>  <1S5>
<156> 본 발명에 따른 식품용 조성물의 바람직한 함유량으로는 이에 한정되지 않지 만 바람직하게는 최종적으로 제조된 식품 총 중량 중 0.01 내지 50증량%이다. 본 발명의 식품용 조성물을 식품첨가제의 형태로 사용하기 위해서는 분말 또는 농축액 형태로 제조하여 사용할 수 있다.  The preferred content of the food composition according to the present invention is not limited thereto, but is preferably 0.01 to 50% by weight of the total weight of the finally prepared food. In order to use the food composition of the present invention in the form of a food additive, it may be prepared and used in powder or concentrate form.
<157>  <157>
<158> 또한 본 발명은 고비 (fls/wm/a japonica) 추출물을 유효성분으로 포함하는 골 다공증， 류마티스 관절염， 관절통， 파제트 병， 골 전이암 및 골절로 이루어진 군에 서 선택된 어느 하나 이상의 골 질환 예방 또는 치료용 약학적 조성물을 제공한다. In addition, the present invention is at least one selected from the group consisting of osteoporosis, rheumatoid arthritis, arthralgia, Paget's disease, bone metastases and fractures, including fern (fls / wm / a japonica) extract as an active ingredient Provided is a pharmaceutical composition for preventing or treating bone diseases.
<159> <159>
<160> 본 발명자들이 규명한 고비 추출물에서 기대되는 골 질환에 대한 예방 또는 치료의 효과는 본 명세서에서 전술한 바와 같다. 특히 고비 추출물에 포함된 화합 물 osmundacetone이 뻐 흡수를 담당하는 파골세포의 증식 및 분화를 억제하고 조골 세포 분화를 활성화하는 활성을 갖는 것으로 규명한 바 있다 . The effects of the prophylaxis or treatment against bone diseases expected from the fern extracts identified by the inventors are as described herein above. In particular, the compound osmundacetone contained in the fern extract inhibits the proliferation and differentiation of osteoclasts responsible for absorption and osteoblasts. It has been shown to have activity to activate cell differentiation.
<161>  <161>
<162> 상기 Di Osi nda japonica) 추출물은 신선한 고비로부터 제조할 수 있으 며. 냉동, 건조 등 보관을 위한 가공 과정을 거친 고비를 사용할 수도 있다. 상기 고비 추출물의 형태나 성상에는 제한이 없으며, 용액, 농축물일 수도 있고， 추출물 제조에 사용된 용매를 제거한 고형분 또는 분말일 수도 있다.  The Di Osi nda japonica extract can be prepared from fresh ferns. Fertilizers that have been processed for storage, such as freezing and drying, can also be used. There is no restriction on the form or property of the fern extract, and may be a solution, a concentrate, or may be a solid or powder from which the solvent used for preparing the extract is removed.
<163>  <163>
<164> 상기 고비와추출물은 천연물 추출 방법으로 알려진 것이라면 제한없이 선택 하여 사용할 수 있으며, 특히 osmundacetone이 포함되는 추출물을 제조할 수 있는 추출 방법을 이용하는 것이 가장 바람직하다. 예를 들어， 적절한 추출 용매를 선팩 하여 당업계에 공지된 산 /염기추출, 열수추출, 상온교반추출， 냉침, 환류냉각추출, 초음과추출, 가압가열추출, 저은고압추출， 효소처리추출. 용매추출 등의 추출 방법 을 사용하여 제조할 수 있다.  The ferns and extracts can be selected and used without limitation, as long as it is known as a natural product extraction method, in particular, it is most preferable to use an extraction method for producing an extract containing osmundacetone. For example, acid / base extraction, hot water extraction, room temperature stirring, cold immersion, reflux cooling extraction, supersonic extraction, pressurized heating extraction, low silver high pressure extraction, enzyme treatment extraction known in the art by prepacking a suitable extraction solvent. It can be prepared using an extraction method such as solvent extraction.
<165>  <165>
<166> 상기 추출 용매로는 물， 에탄올， 주정 , 메탄을 , 프로판올 (propanol ) , 이소프 로판을 ( i sopropanol ) , 부탄올 (but anol ) , 아세톤, 에테르, 클로로포름, 에틸 아세테 이트, 메틸렌클로라이드, 핵산, 시클로핵산, 석유에테르 (pet ro l eum ether ) , 디에틸 에테르, 벤젠으로 이루어진 군에서 선택된 하나 이상의 용매를 사용할 수 있다. The extraction solvent may be water, ethanol, alcohol, methane, propanol, isopropanol, butanol, acetone, ether, chloroform, ethyl acetate, methylene chloride, One or more solvents selected from the group consisting of nucleic acids, cyclonucleic acids, petrol ether, diethyl ether, and benzene can be used.
<167> <167>
<168> 또한 본 발명에 따른 고비 추출물은 상기 용매 추출법에 의하여 1차로 추출 된 추출물일 수도 있고, 추출의 효율을 높이기 위하여 1차 추출 후 추출 잔류물을 재추출하여 1차 추출물과 흔합할 것일 수도 있다. 또한 불순물을 제거하고 활성 성 분의 농도를 높이기 위하여 당업계에 공지된 방법에 따라 추가적으로 크로마토그래 피에 의한 분리, 분획， 규조여과, 한외여과 (막분리, ul traf i l terat i on) 등의 다양 한 정제 또는 여과 과정을 거친 것일 수도 있다. 최종 추출물은 침전농축, 증발농 축 , 공비농축， 감압농축 , 증류농축， 원심분리 , 역삼투법 등 공지된 농축방법과 농 축장치를 이용하여 농축할 수 있으며， 동결건조, 분무건조, 열풍건조 등의 방법에 의해 용매를 제거하고 고형화하여 분말 형태로 제조할 수 있다.  In addition, the fern extract according to the present invention may be an extract first extracted by the solvent extraction method, or may be mixed with the primary extract by re-extracting the extraction residue after the first extraction in order to increase the efficiency of extraction. have. In addition, in order to remove impurities and increase the concentration of active ingredients, various methods such as separation, fractionation, diatom filtration, and ultrafiltration (membrane separation, ul traf il terat i on) according to methods known in the art are additionally performed. It may be through one purification or filtration process. The final extract can be concentrated using well-known concentration methods such as precipitation concentration, evaporation concentration, azeotropic concentration, reduced pressure concentration, distillation concentration, centrifugation, reverse osmosis method and concentration equipment, and freeze drying, spray drying, hot air drying, etc. The solvent can be removed and solidified by the process to prepare in powder form.
<169>  <169>
<170> 상기 고비 추출물에서 osnmndacetone의 함량을 높이기 위해서는 먼저 고비로 부터 열수 추출물을 제조하고， 열수 추출물을 다시 에틸 아세테이트 등 유기용매로 추출물 및 분획물을 제조하는 것이 바람직하다. <171> In order to increase the content of osnmndacetone in the fern extract, it is preferable to first prepare a hydrothermal extract from the fern, and then extract and fractionate the hydrothermal extract with an organic solvent such as ethyl acetate. <171>
<1 V2> 상기 고비 추출물을 유효성분으로 포함하는 약학적 조성물에 포함될 수 있는 담체 , 약학적 조성물의 제형화, 투여 경로와 투여량 등의 투여 방법 등에 대하여서 는 전술한 바와 같다.  <1 V2> Carriers that may be included in the pharmaceutical composition comprising the fern extract as an active ingredient, formulation of the pharmaceutical composition, the method of administration such as the route of administration and the dosage is as described above.
<173>  <173>
<17 > 또한 본 발명은 고비 (Osmunda j aponi ca) 추출물을 유효성분으로 포함하는 골 다공증, 류마티스 관절염, 관절통， 파제트 병, 골 전이암 및 골절로 이루어진 군에 서 선택된 어느 하나 이상의 골 질환 예방 또는 개선용 식품용 조성물을 제공한다. In addition, the present invention is at least one bone disease selected from the group consisting of osteoporosis, rheumatoid arthritis, arthralgia, Paget's disease, bone metastases and fractures, including fern (Osmunda j aponi ca) extract as an active ingredient Provided is a composition for preventing or improving food.
<175> <17 5 >
<I V6> 상기 식품용 조성물을 제조하기 위한 고비 추출물에 대하여서는ᅳ전술한 바와 같다. 또한 상기 식품용 조성물의 예시 및 함유량에 대하여서도 전술한 바와 같다. <I V6> The fern extract for producing the food composition is as described above. Moreover, it is as having mentioned above about the illustration and content of the said food composition.
<177> <177>
< 1 78> 또한 본 발명은 골다공증, 류마티스 관절염, 관절통, 파제트 병， 골 전이암 및 골절로 이루어진 군에서 선택된 어느 하나 이상의 골 질환 예방 또는 치료용 제 제의 제조에 사용되는 Osniundacetone 또는 이의 약학적으로 허용가능한 염의 용도 를 제공한다.  The present invention also relates to Osniundacetone or a pharmaceutical thereof, which is used for the preparation of an agent for the prevention or treatment of any one or more bone diseases selected from the group consisting of osteoporosis, rheumatoid arthritis, joint pain, Paget's disease, bone metastases and fractures. To provide for the use of acceptable salts.
<1 79>  <1 79>
<1 80> 본 발명에서 '약학적으로 허용가능한'이란 생리학적으로 허용되고 인간에게 투여될 때, 통상적으로 알레르기 반응 또는 이와 유사한 반응을 일으키지 않는 것 을 말하며, 상기 염으로는 약학적으로 허용가능한 유리산 ( f ree ac i d)에 의하여 형 성된 산 부가염이 바람직하다. 상기 유리산으로는 유기산과 무기산을 사용할 수 있 다. 상기 유기산은 이에 제한되는 것은 아니나, 구연산, 초산, 젖산， 주석산， 말레 인산, 푸마르산， 포름산， 프로피온산， 옥살산, 트리플로오로아세트산, 벤조산, 글 루콘산， 메타술폰산, 글리콜산, 숙신산, 4-틀루엔술폰산， 글루탐.산 및 아스파르트 산을 포함한다. 또한 상기 무기산은 이에 제한되는 것은 아니나， 염산， 브름산, 황 산 및 인산을 포함한다.  <1 80> In the present invention, 'pharmaceutically acceptable' refers to a physiologically acceptable and generally does not cause an allergic reaction or the like when administered to a human, and the salt is pharmaceutically acceptable. Acid addition salts formed with free acid are preferred. Organic acids and inorganic acids may be used as the free acid. The organic acid is not limited thereto, but citric acid, acetic acid, lactic acid, tartaric acid, maleic acid, fumaric acid, formic acid, propionic acid, oxalic acid, trifluoroacetic acid, benzoic acid, gluconic acid, metasulfonic acid, glycolic acid, succinic acid, 4-frame Luenesulfonic acid, glutamate.acid and aspartic acid. In addition, the inorganic acid includes, but is not limited to, hydrochloric acid, bromic acid, sulfuric acid and phosphoric acid.
<181 >  <181>
<182> 본 발명의 치료용 제제의 투여량은 투여 경로, 투여 시간, 치료 횟수， 치료 기간， 치료가 필요한 개체의 연령, 체중， 건강 상태， 성별, 질환의 중증도. 약물에 대한 민감도, 식이 및 배설율, 등 다양한 요인을 고려하여 당업자가 상술한 특정 용도에 따른 적절한 유효량을 결정할 수 있다. 상기 "유효량" 이란 개체에게 투여 하였을 때， 상기한 골 질환의 개선, 치료， 예방 효과를 나타내기에 충분한 양을 말 한다. 상기 '개체' 란 동물, 바람직하게는 포유동물, 가장 바람직하게는 인간을 포 함하는 동물일 수 있으며, 동물에서 유래한 세포, 조직， 기관 등일 수도 있다. 상 기 개체는 치료가 필요한 골 질환 환자 (pat i ent ) 일 수 있다. Dosage of the therapeutic agent of the present invention is the route of administration, time of administration, frequency of treatment, duration of treatment, age, weight, health condition, sex, and severity of the subject in need of treatment. Various factors, such as sensitivity to the drug, diet and excretion rate, etc. may be taken into consideration to those skilled in the art to determine an appropriate effective amount for the particular use described above. The term "effective amount" refers to an amount sufficient to exhibit an effect of improving, treating, and preventing the above-described bone diseases when administered to an individual. do. The term 'individual' may be an animal, preferably a mammal, most preferably an animal including a human, and may be a cell, tissue, organ or the like derived from the animal. The subject may be a patient with bone disease (pat i ent) in need of treatment.
<183>  <183>
<1 84> 상기 투여는 하루에 한번 또는 수회 나누어 투여할 수도 있다. 본 발명의 치 료용 제제는 단독으로 투여되거나 골 질환의 예방 또는 치료에 효과가 있는 것으로 알려진 다른 치료제와 병용하여 투여될 수 있고, 병용하여 투여할 경우 다른 치료 제와 순차적 또는 동시에 투여될 수 있다. 상기 단독 투여 또는 병용 투여시 본 발 명의 치료용 제제의 투여량은 부작용 없이 최소한의 양으로 최대 효과를 얻을 수 있는 양을 투여하는 것이 바람직하며， 이는 당업자에 의해 용이하게 결정될 수 있 다.  The administration may be administered once or several times a day. The therapeutic preparation of the present invention may be administered alone or in combination with other therapeutic agents known to be effective in the prevention or treatment of bone diseases, and when administered in combination, they may be administered sequentially or simultaneously with other therapeutic agents. When administered alone or in combination, the dosage of the therapeutic preparation of the present invention is preferably administered in an amount that can achieve the maximum effect in a minimum amount without side effects, which can be easily determined by those skilled in the art.
<185> 상기 osmudacetone은 고비과 (Osmundaceae) 식물에서 분리되는 것을 특징으로 하며， 분리하는 방법은 전술한 바와 같다. <5 18> the osmudacetone is characterized in that the separation from plant OSMUNDACEAE (Osmundaceae), method for separation is as described above.
<186>  <186>
<V87> 또한 본 발명은 Osmundacetone 또는 이의 약학적으로 허용가능한 염의 유효 량을 이를 필요로 하는 개체에 투여하는 것을 특징으로 하는 골다공증， 류마티스 관절염， 관절통， 파제트 병,, 골 전이암 및 골절로 이루어진 군에서 선택된 어느 하 나 이상의 골 질환 치료 방법을 제공한다. <V87> The present invention also comprises osteoporosis, rheumatoid arthritis, arthralgia, Paget's disease , bone metastases and fractures, comprising administering to a subject in need thereof an effective amount of Osmundacetone or a pharmaceutically acceptable salt thereof. Provided are one or more methods for treating bone disease selected from the group.
<188>  <188>
<189> 본 명세서에서 '치료 1란 치료되는 개체 또는 세포의 자연적 과정을 변경시키 려는 임상적 시술을 의미하며, 임상적 병리의 예방을 위해서도 수행될 수 있다. 치 료의 바람직한 효과는 질병의 발생 또는 재발 억제, 증상의 완화， 질병의 임의의 직접 또는 간접적인 병리학적 결과의 감소, 질병 진행 속도의 감소, 질병 상태의 개선， 호전， 완화 또는 개선된 예후 등을 포함한다. 또한 용어 '예방 '은 질병의 발 병을 억제시키거나 진행을 지연시키는 모든 행위를 의미한다. As used herein, "treatment 1" refers to a clinical procedure for altering the natural process of the individual or cell being treated, and may also be performed for the prevention of clinical pathology. Desirable effects of treatment include: inhibiting the occurrence or recurrence of the disease, alleviating symptoms, reducing any direct or indirect pathological consequences of the disease, decreasing the rate of disease progression, improving the disease state, improving, alleviating or improving the prognosis, etc. It includes. The term 'prevention' also means any action that inhibits or delays the development of a disease.
<190>  <190>
<191> 또한 본 발명은 골다공증, 류마티스 관절염, 관절통, 파제트 병, 골 전이암 및 골절로 이루어진 군에서 선택된 어느 하나 이상의 골 질환 예방 또는 치료용 제 제의 제조에 사용하기 위한 고비 japonica) 추출물의 용도를 제공한다. <191> The present invention also provides an extract of the fern japonica extract for use in the manufacture of any one or more of the osteoporosis, rheumatoid arthritis, arthralgia, Paget's disease, bone metastases and fractures selected from the group consisting of one or more bone diseases Provides use.
<192> <192>
<193> 상기 고비의 추출물은 천연물 추출 방법으로 알려진 것이라면 제한없이 선택 하여 사용할 수 있으며， 특히 osmundacetone이 포함되는 추출물을 제조할 수 있는 추출 방법을 이용하는 것이 가장 바람직하다. 예를 들어, 적절한 추출 용매를 선텍 하여 당업계에 공지된 산 /염기추출， 열수추출, 상은교반추출, 냉침, 환류냉각추출, 초음파추출, 가압가열추출, 저온고압추출， 효소처리추출， 용매추출 등의 추출 방법 을 사용하여 제조할 수 있다. If the extract of the fern is known as a natural product extraction method can be selected and used without limitation, in particular to produce an extract containing osmundacetone Most preferably, an extraction method is used. For example, acid / base extraction, hot water extraction, phase silver stirring, cold immersion, reflux cooling extraction, ultrasonic extraction, pressurized heating extraction, low temperature, high pressure extraction, enzyme treatment extraction, solvent extraction It can manufacture using extraction methods, such as these.
<194>  <194>
<195> 상기 추출 용매로는 물, 에탄을, 주정, 메탄을, 프로판을 (propanol ) , 이소프 로판올 ( i sopropanol ) , 부탄올 (butanol ) , 아세톤, 에테르, 클로로포름， 에틸 아세테 이트, 메틸렌클로라이드, 핵산， 시클로핵산, 석유에테르 (pet rol eum ether ) , 디에틸 에테르， 벤젠으로 이루어진 군에서 선택된 하나 이상의 용매를 사용할 수 있다. The extraction solvent may be water, ethane, spirits, methane, propane, isopropanol, butanol, acetone, ether, chloroform, ethyl acetate, methylene chloride. One or more solvents selected from the group consisting of nucleic acids, cyclonucleic acids, petrol ether ethers, diethyl ether and benzene can be used.
<196> . <196>.
<197> - 또한 본 발명에 따른 고비 추출물은 상가 용매'추출법에 의하여 1차로 추출 된 추출물일 수로 있고， 추출의 효율을 높이기 위하여 1차 추출 후 추출 잔류물을 재추출하여 1차 추출물과 흔합할 것일 수도 있다. 또한 불순물을 제거하고 활성 성 분의 농도를 높이기 위하여 당업계에 공지된 방법에 따라 추가적으로 크로마토그래 피에 의한 분리, 분획， 규조여과， 한외여과 (막분리. u! traf n terat ion) 등의 다양 한 정제 또는 여과 과정을 거친 것일 수도 있다. 최종 추출물은 침전농축, 증발농 축, 공비농축， 감압농축, 중류농축， 원심분리 , 역삼투법 등 공지된 농축방법과 농 축장치를 이용하여 농축할 수 있으며, 동결건조, 분무건조, 열풍건조 등의 방법에 의해 용매를 제거하고 고형화하여 분말 형태로 제조할 수 있다. In addition, the fern extract according to the present invention may be an extract extracted primarily by an additive solvent ' extraction method, and may be mixed with the primary extract by re-extracting the extraction residue after the primary extraction in order to increase the extraction efficiency. It may be. In addition, in order to remove impurities and increase the concentration of the active ingredient, a variety of separation, fractionation, diatom filtration, ultrafiltration (membrane separation, u! Traf n terat ion), etc. according to methods known in the art are additionally performed. It may be through one purification or filtration process. The final extract can be concentrated using well-known concentration methods such as precipitation concentration, evaporation concentration, azeotropic concentration, reduced pressure concentration, midstream concentration, centrifugation, reverse osmosis, and concentration equipment, and freeze drying, spray drying, hot air drying, etc. The solvent can be removed and solidified by the process to prepare in powder form.
<198> .  <198>.
<199> 상기 고비 추출물에서 osi mdacet one의 함량을 높이기 위해서는 먼저 고비로 부터 열수 추출물을 제조하고， 열수 추출물을 다시 에틸 아세테이트 등 유기용매로 추출물 및 분획물을 제조하는 것이 바람직하다.  In order to increase the content of osi mdacet one in the fern extract, it is preferable to first prepare a hydrothermal extract from fern, and then extract and fractionate the hydrothermal extract with an organic solvent such as ethyl acetate.
<200>  <200>
<2ϋΐ> 또한 고 aV )s mda japonica) 추출물의 유효량을 이를 필요로 하는 개체에 투여하는 것을 특징으로 하는 골다공증， 류마티스 관절염, 관절통， 파제트 병, 골 전이암 및 골절로 이루어진 군에서 선택된 어느 하나 이상의 골 질환 치료 방법을 제공한다.  <2ϋΐ> Any one selected from the group consisting of osteoporosis, rheumatoid arthritis, arthralgia, Paget's disease, metastatic cancer and fracture, characterized by administering an effective amount of high aV) s mda japonica) extract to a subject in need thereof Provided above is a method for treating bone diseases.
<202>  <202>
<203> 상기 고비 추출물의 유효량， 투여 경로와 투여량 등의 투여 방법 등에 대하 여서는 전술한 바와 같다. 【발명의 효과】 The effective amount of the fern extract, the route of administration and the method of administration such as dosage are as described above. 【Effects of the Invention】
<204> 따라서 , 본 발명은 os匪 ndacetone 또는 그 유도체， 또는 이를 포함하는 고비 의 추출물을 유효성분으로 포함하는 골다공증， 류마티스 관절염， 관절통, 파제트 병, 골 전이암 및 골절로 이루어진 군에서 선택된 어느 하나 이상의 골 질환 예빙- , 개선 또는 치료용 조성물을 제공한다. 본 발명에 따른 조성물은 세포독성이 매우 낮으면서도, 뼈 손실을 야기하는 파골세포에 대하여 강력한 증식 및 분화를 억제하 는 효과를 보였으며， 동시에 조골세포 분화를 활성화하는 호과를 보였다.  Therefore, the present invention is any one selected from the group consisting of osteoporosis, rheumatoid arthritis, arthralgia, Paget's disease, bone metastases and fractures, including os 匪 ndacetone or its derivatives, or extracts of ferns containing the same as an active ingredient. Provided are compositions for preserving, ameliorating or treating at least one bone disease. Although the composition according to the present invention has very low cytotoxicity, it showed an effect of inhibiting strong proliferation and differentiation against osteoclasts causing bone loss, and at the same time, it showed the effect of activating osteoblast differentiation.
<205> <20 5 >
【도면의 간단한 설명】  [Brief Description of Drawings]
<206> 도 1은 고비에 포함된 파골세포 분화억제 활성 물질을 분리 동정하기 위한 고비의 물 (열수) 또는 에틸 아세테이트 (EA) 추출물의 HPLC 결과를 나타낸다 (검출파 장 280nm) . EA-2는 7개 분획으로 분리된 EA 추출물 중 2번째 분획이다. Osniundacetone의 peak은 검정색 화살표로 표시하였다. 적색 타원형으로 표시한 peak들은 정제 과정에서 관찰되는 동일한 peak의 위치를 나타낸다.  FIG. 1 shows the HPLC results of high-purity water (hot water) or ethyl acetate (EA) extract for separating and identifying the osteoclast differentiation inhibitory substances contained in the fern (detection wavelength 280 nm). EA-2 is the second of EA extracts separated into seven fractions. Osniundacetone peaks are indicated by black arrows. Peaks marked with red ovals indicate the position of the same peaks observed during purification.
<207> 도 2는 고비 추출물과 분획물, osmundacet one의 파골세포의 증식 및 분화억 제 활성을 확인하기 위해 마우스 골수세포를 이용한 TRAP assay 결과를 나타낸다. Figure 2 shows the results of TRAP assay using mouse bone marrow cells to confirm the proliferation and differentiation inhibitory activity of the fern extract and fractions, osmundacet one osteoclasts.
<208> 도 3은 osmundacetone의 파골세포 분화 억제능력인 IC50을 구하기 위하여, 상업적으로 구입한 osmundacetone(Al f a Aesar , Thermo Fi sher Sci ent i f i c) 1， 4 , 7 , ΙΟ μ Μ의 파골세포의 증식 및 분화억제 활성을 확인하기 위한 마우스 골수세포를 이용한 TRAP assay 결과를 나타낸다. Figure 3 shows the osteoclasts of osmundacetone (Al fa Aesar, Thermo Fischer Sci entific) 1, 4, 7, ΙΟμΜ purchased commercially in order to obtain IC 50 , an osteoclast differentiation inhibitory ability of osmundacetone. The results of TRAP assay using mouse bone marrow cells to confirm proliferation and differentiation inhibitory activity.
<209> 도 4A는 osmundacetone과 공지된 약물인 Fosaraax의 파골세포 분화 억제효과 를 비교하기 위하여, 골수세포에 다양한 농도의 osmundacetone과 Fosamax를 처리하 여 분화를 유도한 후, 이의 1(:50을 확인한 결과를 나타낸다. 도 4B는 파골세포와 조 골세포 전구세포들을 공동배양하며 osmundacetone이 동시애 파골세포 분화는 억제 하고 조골세포 분화는 활성화하는 능력을 확인한 결과를 나타낸다. Figure 4A is to compare the osteoclast differentiation inhibitory effect of osmundacetone and Fosaraax, a known drug, after inducing differentiation by treating various concentrations of osmundacetone and Fosamax in bone marrow cells, and confirmed the 1 (: 50) Figure 4B shows the results confirming the ability to co-culture osteoclasts and osteoblastic progenitor cells and osmundacetone inhibits osteoclast differentiation and activate osteoblast differentiation.
<2io> 도 5는 osmundacet one의 조골세포의 0CN 생성에 미치는 영향을 확인하기 위 하여, osmundacetone을 처리한 후 웨스턴 블롯을 수행하여 0CN의 발현을 확인한 결 과를 나타낸다. <2io> Figure 5 shows the result of confirming the expression of 0CN by Western blot after treatment with osmundacetone in order to confirm the effect on the 0CN production of osteoblasts of osmundacet one.
<2U> 도 6은 osmundacetone이 RUNX2의 발현에 미치는 영향을 확인하기 위하여， osmundacetone을 처리한 후 웨스턴 블롯을 수행하여 RUNX2의 발현을 확인한 결과를 나타낸다. 【발명의 실시를 위한 형태】 <2U> Figure 6 shows the results of confirming the expression of RUNX2 by Western blot after treatment with osmundacetone in order to confirm the effect of osmundacetone on the expression of RUNX2. [Form for implementation of invention]
<213> 이하 본 발명을 상세히 설명한다 .  Hereinafter, the present invention will be described in detail.
<214> 단, 하기 실시예는 본 발명을 예시하는 것일 뿐， 본 발명의 내용이 하기 실 시예에 한정되는 것은 아니다.  However, the following examples are merely to illustrate the invention, but the content of the present invention is not limited to the following examples.
<21 >  <21>
<216> <실시예 1> 실험방법  Example 1 Experimental Method
<217> T. T7비 추출물과 분획물 제 ^ 및 학합물 분리 동정  <217> Isolation and Separation of T7 Ratio Extracts and Fractions
<218> 고비 (Osmundae Rhizoma 또는 Osmunda japonica)를 열수, 에틸 아세테이트 Hydrothermal (Esmundae Rhizoma or Osmunda japonica) hydrothermal, ethyl acetate
(ethyl acetate)의 순서로 추출한 후， 에틸 아세테이트 추출물 (EA 추출물)을 HPLC 로 7개의 분획으로 분리하였다. After extraction in the order of (ethyl acetate), ethyl acetate extract (EA extract) was separated into seven fractions by HPLC.
<219> 구체적으로, 고비 추출물은 강원도에서 채취한 고비 200~250g을 깨끗이 씻어 증숙용기 (OSK-2002, 홍삼박사, Well sosanaTM, 대웅제약)에 넣고 물 1.5L를 첨가하 여 24시간 증숙한 후, 물 3.5L를 추가로 첨가하여 72시간 동안 숙성시키고 냉장보 관한 것을 열수 추출물로 사용하였다. 열수 추출물에 동일한 부피의 에틸 아세테이 트 (ethyl acetate, EA)를 첨가하고 잘 혼합한 후 , EA 층을 rotary evaporator로 건 조하여 EA 추출물로 사용하였다. EA 추출물을 최소량의 DMS0에 녹인 후 물로 회석 하였는데, 이 때 회석 배수는 수율을 대략 70%로 예상하여 열수 추출물 원래 부피 의 70%까지 희석하였다. EA 추출물을 HPLC로 7개의' 분획으로 나눈 후， 두 번째 분 획을 rotary evaporator로 건조하여 EA-2 추출물로 사용하였다. 7개 분획물 중, 2 번째 분획물 (EA-2)이 파골세포 분화억제 활성을 갖는 것으로 관찰되었다 (도 1, <실 시예 2> 참고). Specifically, the Gobi extract is washed with 200 ~ 250g of Gobi collected from Gangwon-do and steamed in a steaming vessel (OSK-2002, Red Ginseng, Well sosanaTM, Daewoong Pharmaceutical) and steamed for 24 hours by adding 1.5L of water. In addition, 3.5L of water was further added and aged for 72 hours, and the cold storage was used as a hot water extract. The same volume of ethyl acetate (EA) was added to the hot water extract and mixed well. The EA layer was dried with a rotary evaporator and used as an EA extract. The EA extract was dissolved in a minimum amount of DMS0 and then diluted with water, where the dilution was diluted to 70% of the original volume of the hydrothermal extract in anticipation of a yield of approximately 70%. After dividing the EA extract into 7 ' fractions by HPLC, the second fraction was dried with a rotary evaporator and used as an EA-2 extract. Of the seven fractions, the second fraction (EA-2) was observed to have osteoclast differentiation activity (see Figure 1, <Example 2>).
<220>  <220>
<22i> 파골세포 분화억제 활성을 갖는 물질을 동정하기 위하여 EA-2 분획물에서 9 개의 단일 화합물을 분리， 정제하고， 핵자기공명 (匪 R)과 칠량분석 (MS)으로 각 화합 물의 종류와 화학구조를 규명하였다 . EA-2 분획물에서 단일 화합물을 분리하기 위 한 HPLC의 조건은 표 1에 나타낸 바와 같다. 분리된 화합물 중, 파골세포 분화억제 활성을 나타내는 물질은 osmundacetone인 것으로 확인되었다 (<실시예 2> 참고). 고 비 추출물에서 동정된 osmundacetone의 구체적인 NMR과 MS 분석 결과는 다음과 같 다:  In order to identify substances with osteoclast differentiation activity, nine single compounds were isolated and purified from the EA-2 fraction, and the type and chemistry of each compound were determined by nuclear magnetic resonance (匪 R) and seventh analysis (MS). The structure was identified. The conditions of HPLC for separating single compounds from EA-2 fractions are shown in Table 1. Among the compounds isolated, the substance showing the osteoclast differentiation activity was confirmed to be osmundacetone (see <Example 2>). Specific NMR and MS analyzes of osmundacetone identified from fern extracts are as follows:
<222> 匪 R(700 MHz, methanol-o^) δ 7.52(1Η, d, J=16.1, H— 7)， 7.08(1H, d, (R (700 MHz, methanol-o ^) δ 7.52 (1Η, d, J = 16.1, H-7), 7.08 (1H, d,
7=2.1 Hz, H-2), 6.99C1H, dd, J=7.7, 2.1 Hz, H-6) , 6.79(1H, d, J=7.7 Hz, H-5), 6.55 1H, d, J=16.1 Hz, H-8) , 2.34(3H, s, II- 10)； "C-NMR 175 MHz, methanol-^) δ 201.7(09)， 150.1(04), 147.1(03), 147.0(07), 127.9(01), 124.9(C-8), 123.7 C-6), 116.7(C-5), 115.4(C-2), 27.2(010); ESI -MS (negative mode) m/z 1777 = 2.1 Hz, H-2), 6.99C1H, dd, J = 7.7, 2.1 Hz, H-6), 6.79 (1H, d, J = 7.7 Hz, H-5), 6.55 1H, d, J = 16.1 Hz, H-8), 2.34 (3H, s, II-10); "C-NMR 175 MHz, methanol- ^) δ 201.7 (09), 150.1 (04), 147.1 (03), 147.0 (07), 127.9 (01), 124.9 (C-8), 123.7 C-6), 116.7 (C-5), 115.4 (C- 2), 27.2 (010); ESI -MS (negative mode) m / z 177
[MH]; ESIMS(positive mode) /z 179 [M+H]+, 201 [M+Na]+. [MH]; Positive mode (ESIMS) / z 179 [M + H] + , 201 [M + Na] + .
<223> 【표 1】  <223> [Table 1]
EA-2 fraction HPLC separation method  EA-2 fraction HPLC separation method
Figure imgf000024_0001
Figure imgf000024_0001
Figure imgf000024_0002
Figure imgf000024_0002
<224> <224>
<225> <225>
<226 2. 세포 tij^  <226 2. Cell tij ^
<227> MC3T3-E1 서브클론 4 뮤린 조골세포 (Subclone 4 murine osteoblastic cells, <227> MC3T3-E1 subclone 4 murine osteoblastic cells,
#CRL-2593TM, ATCC)는 37'C, 5% C¾ 조건으로 a -MEM(Mimmum Essential Medium# CRL-2593TM, ATCC) is a -MEM (Mimmum Essential Medium) at 37'C, 5% C¾
Eagle-Alpha Modification, #LM00853, WELGENE, Seoul, Korea) 배지를 이용하여 배 양하였다. Eagle-Alpha Modification, # LM00853, WELGENE, Seoul, Korea) culture medium was used.
<22s> HaCaT 인간 상피 각질형성세포 (human epidermal kerat inocyte, #ATCC PCS- <22s> HaCaT human epidermal kerat inocyte, #ATCC PCS-
200-011)는 DMEM(Dulbecco' s modified Eagle' s medium, #DME -HPA, Capricorn Scientific, Ebsdorfergrund, Germany) 배지를 이용하여 37'C, 5% C02 조건으로 배 양하였다. NIH3T3 마우스 배이ᅳ 섬유아세포 (embryo fibroblast, #KCLB 21658) , RAW264.7 murine macrophage(preosteoclast cell line, #KCLB 40071) , HCT116 인간 결장암 (#KCLB 10247), PC3 인간 전립선 샘암종 세포 (prostate adenocarcinoma cell, #KCLB 21435), IIT1080 인간 섬유육종세포 (fibrosarcoma cell, #KCLB 1121) 및 Β16Π0 마우스 악성 혹색종세포 (melanoma cell, CLB 80008)는 한국세포주은행 200-011) was incubated in DMEM (Dulbecco's modified Eagle's medium, # DME-HPA, Capricorn Scientific, Ebsdorfergrund, Germany) medium at 37'C, 5% C0 2 conditions. NIH3T3 mouse embryonic fibroblast (#KCLB 21658), RAW264.7 murine macrophage (preosteoclast cell line, #KCLB 40071), HCT116 human colon cancer (#KCLB 10247), PC3 human prostate adenocarcinoma cell (#state) KCLB 21435), IIT1080 human fibrosarcoma cells (#KCLB 1121) and Β16Π0 mouse melanoma cells (CLB 80008)
RECTIFIED SHEET (RULE 91) iSA/KR 에서 구매하였고, DMEM 배지를 이용하여 37°C, 5% C02 조건으로 배양하였다. RECTIFIED SHEET (RULE 91) iSA / KR It was purchased from and incubated at 37 ° C, 5% C0 2 conditions using DMEM medium.
<229> AGS 인간 위선암세포 (stomach adenocarcinoma, #KCLB 21739) , Α549 인간 폐 암종세포 (lung carcinoma cell, #KCLB 10185)， Caki-1 인간 신장암세포 (kidney carcinoma cell , #KCLB 30046), T24 인간 방광암종세포 (bladder carcinoma cell, #KCLB 30004) , TC-1 P3 HPV-16E7-발현 마우스 폐상피세포 , MHC class I(고려대학교 김태우 교수 제공)는 RPMKRosewell Park Memorial Institute) 1640(#RP T-A, Capricorn Scientific, Ebsdor f ergrund, Germany) 배지를 이용하여 37 °C , 5% C02 조 건에서 배양하였다. <229> AGS human gastric adenocarcinoma (#KCLB 21739), A549 human lung carcinoma cell (#KCLB 10185), Caki-1 human carcinoma cell (#KCLB 30046), T24 human bladder cancer Species (bladder carcinoma cell, #KCLB 30004), TC-1 P3 HPV-16E7-expressing mouse pulmonary epithelial cells, MHC class I (Professor Tae-Woo Kim, Korea University), RPMKRosewell Park Memorial Institute) 1640 (#RP TA, Capricorn Scientific , Ebsdor f ergrund, Germany) medium at 37 ° C, 5% C0 2 conditions.
<230> 인간 지방세포 -유래 중간엽줄기세포 (adipose-derived mesenchymal stem cells)인 ADMSCs(CEF0 Bio, Seoul , Korea)는 CB-ADMSC-GM(CEFO Bio, Seoul , Korea) 배지를 이용하여 37 °C, C02조건에서 배양하였다. <230> ADMSCs (CEF0 Bio, Seoul, Korea), which are human adipocyte-derived mesenchymal stem cells, were prepared at 37 ° C. using CB-ADMSC-GM (CEFO Bio, Seoul, Korea) medium. C, C0 2 was incubated in the conditions.
<231> < 2 31>
<232> 3.파골세포의 일차배양 (primary culture)  Primary culture of osteoclasts
<233> 골수세포를 5-8 주령의 수컷 C57BL/6 마우스의 대퇴골 및 정강이뻐로부터 수 집하였다. 뼈에서 근육을 제거한 후 차가운 인산완층식염수 (PBS; # CAP08-050, GenDEPOT, Katy, TX, USA)에 보관하였다.  Bone marrow cells were collected from the femurs and shins of male C57BL / 6 mice 5-8 weeks old. Muscles were removed from bone and stored in cold phosphate saline (PBS; # CAP08-050, GenDEPOT, Katy, TX, USA).
<234> 각 뼈의 양쪽 끝을 절단하고， 골수를 25G 바늘을 사용하여 차갑게 유지한 폴 러싱 배지 (serum-free α-ΜΕΜ, 2 πιΜ 에틸렌디아민테트라아세트산 (ethylenediaminetetraacet ic acid))로 플러시하였다 (flushed) .  Both ends of each bone were cut and flushed with bone marrow (serum-free α-ΜΕΜ, 2πιΜ ethylenediaminetetraacet ic acid) kept cold using a 25G needle. .
<235> Labogene 1248R (Labogene, Lynge, Denmark)를 이용하여 3,000 rpii의 속도로 235 using a Labogene 1248R (Labogene, Lynge, Denmark) at 3,000 rpii
3 분간 원심분리하여 골수세포를 수집한 다음 세척용 배지에 재현탁하였다. 그런 후 , 수집한 8 niL의 골수세포를 6 mL의 림프구 분리 배지 (LSM; # 50494, MP Biomedicals, Santa Ana, CA, USA) 위에 겹쳐 놓았고 1,600 rpm의 속도로 20 분간 원심분리하여 단핵세포를 분리하였다. Bone marrow cells were collected by centrifugation for 3 minutes and then resuspended in washing medium. The collected 8 niL of bone marrow cells were then superimposed on 6 mL of lymphocyte separation medium (LSM; # 50494, MP Biomedicals, Santa Ana, CA, USA) and centrifuged at 1,600 rpm for 20 minutes to separate monocytes. It was.
<236> 단핵세포의 밴드는 메디아 인터페이스 (media interface)에서 수집되었고， 완 전한 α— MEM 배지 (10% 소태아혈청 (FBS; Capricorn Scientific, Ebsdor f ergrund, Germany) 및 1%(ν/ν)의 항생제 (100 U/mL 페니실린 G(pemcillin G) 및 100 mg/niL 스트렙토마이신 (streptomycin) 포함)로 37°C , 5% C02의 조건에서 3일마다 배지를 갈 아주며 배양하였다. Bands of monocytes were collected at the media interface, complete α-MEM medium (10% fetal bovine serum (FBS; Capricorn Scientific, Ebsdor f ergrund, Germany) and 1% (ν / ν) The medium was incubated every 3 days at 37 ° C, 5% C0 2 with antibiotics (including 100 U / mL penicillin G and 100 mg / niL streptomycin).
<237> 파골세포의 분화를 촉진하기 위해， 48-웰 플테이트에서 well 당 0.5 mL의 배 지에서 1 X 105 세포를 파골세포 분화인자인 PeproTec(Seoul， Korea)에서 구매한 -CSFC60 ng/mL)와 RANKL(150 ng/mL)의 존재 하에 배양하였다. 하나의 골수에서 유 래한 대식세포 /단핵세포는 공지된 바와 같이', 6일 후에 성숙 다핵 파골세포로 완전 히 분화되었다 (Gurt et al. , 2015). <7 23> purchased in order to promote differentiation of osteoclasts, a 48-well flat Tate 1 X 10 5 cells in 0.5 mL per well of times if in the osteoclast differentiation factor PeproTec (Seoul, Korea) -CSFC60 ng / mL) and RANKL (150 ng / mL). In one marrow oil raehan macrophages / monocytes are after ', 6, as is well known has been completely differentiated into mature multinuclear osteoclasts (Gurt et al., 2015) .
<238>  <238>
<239> 4. 파골세포 (osteoclast) 및 ^골세 (osteoblast)와 공동배양  4. Coculture with osteoclasts and osteoblasts
<240> 48 웰 (well) 플레이트 웰 당 1X105의 C57BL/6 쥐 골수 단핵세포 (bone marrow mononuclear cell)를 상기와 같은 방법으로 준비하였고, 1.5><1으의 MC3T3- E1 뮤린 조골세포와 10% FBS 및 1% 항생제 (100 U/mL 페니실린 G 및 100 mg/mL 스트 랩토마이신)를 포함한 500 yL Q-MEM 배지를 이용하여 37°C , 5% C02의 조건으로， 3 일 간격으로 배지를 교체하며 공동배양하였다. <240> 1 × 10 5 C57BL / 6 rat bone marrow mononuclear cells per 48 well plate wells were prepared in the same manner as above, and 1.5><1 MC3T3-E1 murine osteoblasts and 10 Using 500 yL Q-MEM medium with% FBS and 1% antibiotics (100 U / mL penicillin G and 100 mg / mL strattomycin) at 37 ° C, 5% C0 2 , every 3 days The cultures were cocultured with replacement.
<241> 양성 대조군 세포는 파골세포 분화인자 (osteoclast differentiation factors)인 M-CSF(60ng/mL) 및 RANKL(150ng/mL)과 조골세포 분화인자인 아스코르브 산 (ascorbic acid, 50 μ g/niL) 및 lOmM β글리세로포스페이트 ( β glycerophosphate) 와 함께 배양하였다. Positive control cells were osteoclast differentiation factors M-CSF (60 ng / mL) and RANKL (150 ng / mL) and osteoblast differentiation factor ascorbic acid (ascorbic acid, 50 μg / niL). And incubated with lOmM βglycerophosphate.
<242> 음성 대조군 세포는 분화인자로 Mᅳ CSF만 첨가하여 배양하였다.  Negative control cells were cultured with the addition of M ᅳ CSF as the differentiation factor.
Osn ndacetone이 파골세포와 조골세포의 분화에 미치는 영향을 시험하기 위해, 세 포를 플레이트에 분주한 후 1일째 osmundacetone을 10μΜ의 최종 농도로 양성대조 군 세포에 투여하였다.  To test the effect of Osn ndacetone on the differentiation of osteoclasts and osteoblasts, osmundacetone was administered to positive control cells at a final concentration of 10 μM on day 1 after dispensing cells into plates.
<243> Osmundacetone을 처리한 양성대조군은 분주 후 6-7일이 지나면 파골세포가 완전히 사라졌기 때문에 분주 8일 후부터 조골세포 분화인자만 투여한 상태에서 21 일 까지 배양하였다.  The positive control group treated with Osmundacetone was cultured from 8 days after dispensing to 21 days with only osteoblast differentiation factor because osteoclasts completely disappeared after 6-7 days after dispensing.
<244>  <244>
<245> 5. ■§· 흡수 분석 <245> 5. ■ § · Absorption Analysis
<246> 인간 골 콜라겐의 in vitro 파골세포 매개 분해 (osteocl ast—medi atecl degradation)의 정량적 측정은 OsteoLyse TM Assay Kit((Lonza Walkersvi I le, Inc. Walkersville, MD, USA)를 사용하여 제조자의 지시에 따라 수행하였다  <246> Quantitative determination of in vitro osteoclast-mediated degradation of human bone collagen was determined by the manufacturer using the OsteoLyse ™ Assay Kit (Lonza Walkersvi Ile, Inc. Walkersville, MD, USA). Was performed according to
<247> 이 분석법은 파골세포의 흡수 열공 (lacuna) 내로의 금속단백분해효소 (matrix metal loproteinase) 방출을 직접적으로 측정할 수 있다 (Delaisse et al. , 2003). This assay can directly measure matrix metal loproteinase release into osteoclasts of osteoclasts (Delaisse et al., 2003).
<248> 간략하게, 상기와 같이 준비한 2 X 104 마우스 골수세포를 형광물질—결합 인간 골 콜라겐 (europium-conjugated collagen)이 코팅된 96-웰 OsteoLyse TM 세포 배양 플레이트에 분주하였다. <249> M-CSFC60 tig/ mL)와 RANKL (150 ng /mL)의 존재 하에 각 well 당 0.1 niL의 완전한 α-ΜΕΜ 안에서 37°C, 5% C¾의 조건으로 6일 동안 배양하였다. 그 후 분주 3 일째에 배지를 교환하였다. Briefly, 2 × 10 4 mouse bone marrow cells prepared as above were dispensed into 96-well OsteoLyse ™ cell culture plates coated with fluorescent-bound human bone collagen (europium-conjugated collagen). Incubated for 6 days at 37 ° C, 5% C¾ in 0.1 niL of the complete α-ΜΕΜ per well in the presence of M-CSFC60 tig / mL) and RANKL (150 ng / mL). The medium was then changed on the third day of dispensing.
<250> 성숙한 파골세포 기능에 대한 저해 능력을 측정하기 위해, 6일 째에 배지를 교체하며 osmundacetone을 TRAP분석으로부터 얻은 IC50농도로 첨가하였다. To determine the inhibitory capacity for mature osteoclast function, media was changed on day 6 and osmundacetone was added at IC 50 concentrations obtained from TRAP analysis.
<25i> 성숙한 파골세포에 osmundacetone을 3일간 처리한 후, 세포 배양액의 상등액 <25i> Supernatant of cell culture after treatment with mature osteoclasts osmundacetone for 3 days
K iL를 취하여 Fhiorophore— Releasing 시약이 담긴 두 번째 96-웰 분석 플레이트 에 넣고, 분해된 콜라겐은 시간 결정 형광물질 형광 측정기 (time-resolved fluorescence f luorimeter)인 Wal lac Victor (Perkin Elmer , Wal tham, MA, USA)를 이용하여 측정하였다. 이때, 400 μ초의 초기 지연 후에 400y초의 시간 간격 동안 340nm에서 여기 (excitat ion) 및 615ηιιι에서 방출 (emi ssion)을 이용하여 측정하였다.Take K iL and place it in a second 96-well assay plate containing Fhiorophore—Releasing reagent, and the degraded collagen is Wal lac Victor (Perkin Elmer, Wal tham, MA, time-resolved fluorescence fluorimeter). , USA). At this time, it was measured using excitation at 340 nm and emission at 615 ηιιι for a time interval of 400 y seconds after an initial delay of 400 μsec.
<252> 골 재흡수율 (%)은 처리하지 않은 대조군 대비 osinundacetone의 존재에 따른 골 재흡수 양의 비율을 계산함으로써 얻었고, 세포 DNA에 의해 정상화하였다.Bone resorption rate (%) was obtained by calculating the ratio of bone resorption according to the presence of osinundacetone relative to the untreated control group and normalized by cellular DNA.
<253> <253>
<254> 6. 웨 ^턴 붙랏  6. The wet turn
<25> 분화과정에서 조골세포 분화 마커의 발현을 분석하기 위해， 3X105 MC3T3-E1 세포를 각 well당 아스코르브산 (50 yg/niL)과 10 mM -글리세로포스페이트가 함유 된 완전한 α-ΜΕΜ 10 mL과 함께 100mm 배양 플레이트에 분주하였다 . 이에 50 μΜ의 Osmundacetone을 첨가 또는 미첨가하고 3그 C , 5% C02 배양기에서 21일 동안 배양하 며 매 3-4 일마다 배지를 교환하였다. In order to analyze the expression of osteoblast differentiation markers during the differentiation process, 3X10 5 MC3T3-E1 cells were prepared with complete α-ΜΕΜ 10 containing ascorbic acid (50 yg / niL) and 10 mM-glycerophosphate per well. Dispense into 100 mm culture plates with mL. 50 μΜ Osmundacetone was added or unadded, and cultured for 21 days in a 3G C, 5% C0 2 incubator, and medium was changed every 3-4 days.
<256> 음성 대조군 세포는 분화인자 없이 배양하였고, 조골세포와 이의 배양액은 분주한 후， 7일， 14일 또는 21일에 수집하였다.  Negative control cells were cultured without differentiation factors, and osteoblasts and their cultures were collected 7 days, 14 days or 21 days after dispensing.
<257> 상기 세포를 RIPA 버퍼를 이용하여 용해한 후, RUNX2(Runt— related transcription factor 2) 발현을 분석하였다. 더불어 0CN 분비를 분석하기 위해 배 양액을 채취하고 세포 DNA로 정상화시켰다 . After the cells were lysed using RIPA buffer, the expression of Run-related transcription factor 2 (RUNX2) was analyzed. In addition, culture medium was collected and normalized to cellular DNA to analyze 0CN secretion.
<258> 단백질은 12.5 % 풀리비닐리덴트리스 젤 (polyvinyl idene-Tr is gels)에서Proteins were obtained from 12.5% polyvinyl idene-Tr is gels.
SDS-PAGE로 분석하였고, PVDF(polyvinylidene difluoride) 막에 전기영동을 통해 이동시켰다. It was analyzed by SDS-PAGE and transferred by electrophoresis to polyvinylidene difluoride (PVDF) membrane.
<2?9> 상기 막은 탈지유를 이용하여 블록하였고, anti-RUNX2(DlH7) 토끼 단클론항 체 (#8486, Cell Signaling Technology, M, USA) 또는 anti-0CN(FL-95) 항체 (#sc一 30045, Santa Cruz Biotechnology, TX, USA)를 이용하여 조사하였다. Anti-액틴 항 체(#^77— 3， MEDICAL & BIOLOGICAL LABORATORIES CO. , LTD. , Nagoya, Aichi , Japan)는 내부 기준으로써 이용하였다. The membrane was blocked using skim milk, anti-RUNX2 (DlH7) rabbit monoclonal antibody (# 8486, Cell Signaling Technology, M, USA) or anti-0CN (FL-95) antibody (#sc 一). 30045, Santa Cruz Biotechnology, TX, USA). Anti-actin anti The sieve (# ^ 77-3, MEDICAL & BIOLOGICAL LABORATORIES CO., LTD., Nagoya, Aichi, Japan) was used as an internal standard.
<260> 블랏 상의 단백질 밴드는 강화된 화학발광 검출 키트 (enhanced chemi luminescent detection kit) (#EBP-1073, PicoEPD Western Reagent , ELPIS- BIOTECH, Dae j eon, Korea)를 이^하여 가시화하였다. Protein bands on the blot were visualized by an enhanced chemiluminescent detection kit (# EBP-1073, PicoEPD Western Reagent, ELPIS-BIOTECH, Dae j eon, Korea).
<261> < 2 61>
<262> 7. ALP 활성 측정  7. ALP Activity Measurement
<263> ALP 활성은 Quant ichrom ALP Assay Kit (Bioassay Systems, Hayward , CA, <263> ALP activity is determined by Quant ichrom ALP Assay Kit (Bioassay Systems, Hayward, CA,
USA)을 이용하여， 제조사의 지침에 따라 조사하였다. USA), according to the manufacturer's instructions.
<264> 간략하게， 3X10 MC3T3-E1 세포를 각 well당 아스코르브산 (50 yg/mL) 및 Briefly, ascorbic acid (50 yg / mL) and 3X10 MC3T3-E1 cells were added to each well.
10 ηιΜ β-글리세로포스페이트를 포함하는 100 의 완전한 α-ΜΕΜ과 함께 96웰 플 레이트에서 배양하였다. 이때, osmundacetone LO μΜ 및 50 μΜ)을 첨가 또는 미첨 가하였고, 배지는 3~4일 간격으로 교체하였다. Incubated in a 96-well plate with 100 complete α-ΜΕΜ containing 10 ηιΜ β-glycerophosphate. At this time, osmundacetone LO μM and 50 μM) were added or not added, and the medium was replaced every 3-4 days.
<265> 배양 14일째, 세포 분획에서 ALP 활성으로 인한 비색 변화를 분광 광도계 플 레이트 판독기 (Molecular Devices, Sunnyvale, CA, USA)를 이용하여 405ηιιι에서 측 정하였다 (Kim et al., 2016). ALP 활성의 % 활성화는 실험 화합물을 처리한 세포의 ALP 활성과 처리하지 않은 대조군 세포의 ALP 활성을 비교하여 나타내었다.  At 14 days of culture, colorimetric changes due to ALP activity in the cell fractions were measured at 405ηιιι using a spectrophotometer plate reader (Molecular Devices, Sunnyvale, Calif., USA) (Kim et al., 2016). The% activation of ALP activity was shown by comparing the ALP activity of the cells treated with the experimental compound with that of untreated control cells.
<266>  <266>
<267> 8. TRAP assay분석 (파골세포의 증식 및 분화억제 활싀 측정 J_  8. TRAP assay analysis (determination of the activity of inhibiting the proliferation and differentiation of osteoclasts J_
<268> 1) ¾수세포 배양  1) ¾ water cell culture
<269> 6~8주령 수컷 C57BL/6 마우스의 정강이뼈 (tibia)와 대퇴골 (femur)을 무균적 으로 절제하고 골수세포를 syringe(21G, Korea Green Cross)로 무균적으로 채취하 였다. 골수세포를 sodium bicarbonate(2.0g/L), streptomycin(100mg/L) , peniciinn(100,000unit/mL)을 포함하는 α-ΜΕΜ 배지 (Gibco BRL Co.) 500 uL에 부 유하여 48well plate에 분주하고， triplicate으로 assay를 진행하였다. 파골세포의 전구세포인 단핵구세포를 분화촉진인자인 RANKL과 M-CSF로 처리하면 5~7일 내에 파 골세포로 분화되었다.  The tibia and femur were aseptically excised from 6 to 8 week old male C57BL / 6 mice, and bone marrow cells were collected aseptically with a syringe (21G, Korea Green Cross). Bone marrow cells were suspended in 48well plates in 500 uL of α-ΜΕΜ medium (Gibco BRL Co.) containing sodium bicarbonate (2.0 g / L), streptomycin (100 mg / L) and peniciinn (100,000 units / mL). ， The assay was carried out by triplicate. Monocyte progenitor cells, osteoclasts, were differentiated into osteoclasts within 5 to 7 days with RANKL and M-CSF.
<270> ？) 파골세포 분학 억제 측정  <270>？) osteoclast differentiation inhibition measurement
<27i> 2-1) 시료 준비: 고비의 열수 추출물, EA 추출물 또는 분획물은 <실시예. 1> 의 1.과 동일한 방법으로 제조하였다. EA-2 추출물은 최소량의 DMS0에 녹인 후 EA- 2 추출 과정의 수율을 대략 70%로 예상하여 원래 열수 추출물 부피의 70%까지 물로 회석하였다. <272> 2-2) 시료 투여 : 시료는 골수세포 배양 첫날부터 l:20(v/v; 배지 500 μ L당 시료 25 yL)로 지속적으로 배지에 투여하고， 배지는 2~3일 간격으로 교체하였다.'<27i> 2-1) Sample preparation: Fertilized hydrothermal extract, EA extract or fraction is shown in <Example . 1> was prepared in the same manner as 1. The EA-2 extract was dissolved in a minimum amount of DMS0, and then distilled with water up to 70% of the original hydrothermal extract volume in anticipation of a yield of approximately 70% of the EA-2 extraction process. 2-2) Sample administration: Samples are continuously administered to the medium at l: 20 (v / v; 25 yL per 500 μL of medium) from the first day of bone marrow cell culture. Replaced. '
<273> 2-3) 파골세포 분화 측정: 파골세포는 TRAP으로 염색된 TRAP-양성 다핵세포 로 정의하였다. TRAP 염색 용액은 기질인 naphthol AS-MS phosphate (Sigma N-4875) 5mg과 발색시약인 Fast Red Violet LB salt 25mg을 약 0.5mL의 Ν,Ν- dimethylformamide에 녹인 후， 50mM tartaric acid를 포함하는 0.1N NaIIC03 buffer solution(50mL).과 흔합하였다. 반응시약은 사용 전까지 냉장고에 보관하였다. 2-3) Measurement of Osteoclast Differentiation: Osteoclasts were defined as TRAP-positive multinucleated cells stained with TRAP. TRAP staining solution was dissolved 5 mg of naphthol AS-MS phosphate (Sigma N-4875) as a substrate and 25 mg of Fast Red Violet LB salt as a coloring reagent in about 0.5 mL of N, N-dimethylformamide, and 0.1 N containing 50 mM tartaric acid. NaIIC0 3 buffer solution (50 mL). The reaction reagents were stored in the refrigerator until use.
<274> 골수세포는 분화촉진인자를 포함하는 배지에서 7일간 배양한 후, 배지를 제 거하고 PBS로 세척한 후 10% formal in을 포함한 PBS로 2~5분 뽕안 고정하였다. 이 후 ethanol과 acetone의 1:1 흔합용액으로 1분간 고정하고， 건조하였다. 고정된 세 포는 TRAP 염색용액으로 15 분간 처리하고 PBS로 세척한 후, 세포의 염색 정도를 현미경으로 관찰하였다. The bone marrow cells were cultured in a medium containing a differentiation-promoting factor for 7 days, and then the medium was removed, washed with PBS, and fixed in mulberry eyes for 2 to 5 minutes with PBS containing 10% formal in. Thereafter, the mixture was fixed for 1 minute with a 1: 1 mixed solution of ethanol and acetone, and dried. The fixed cells were treated with TRAP staining solution for 15 minutes, washed with PBS, and observed for the degree of staining of the cells under a microscope.
<275> 현미경 시야에서 TRAP-양성세포 중， 2개 이상의 핵을 갖는 세포를 파골세포 로 판정하고 세포의 수를 측정하였다. 고비 추출물의 파골세포 분화억제 효과는 대 조군 대비 50% 억제 농도를 1(：50로 계산하였다. <5 27> was determined for cells having two or more nuclei, TRAP- of positive cells in the microscope field of view by osteoclasts, and measuring the number of cells. The osteoclast differentiation inhibitory effect of the fern extract was calculated as 1 (： 50 ).
<276>  <276>
<277> 9. TRAP assay분석 (파골세포의 증식 및 분하 억제 활성 Κ:5ο~£·¾  9. TRAP assay analysis (proliferation and inhibition of osteoclasts Κ: 5ο ~ £ · ¾
<278> 1) 파골세포 분하 측정  1) Measurement of osteoclast division
<279> 1-1) 入 1료 준비: Osmundacetone을 Alfa Aesar , Thermo Fisher Scientific에 서 구입하여 최소량의 DMSO(dimethylsulfoxide)에 용해하였다. Fosamax는 CaymanCAnn Arbor, MI, USA)에서 구입하여 최소량의 무균 증류수에 용해하였다. 1-1) Preparation Preparation: Osmundacetone was purchased from Alfa Aesar, Thermo Fisher Scientific and dissolved in a minimum amount of dimethylsulfoxide (DMSO). Fosamax was purchased from Cayman CAnn Arbor, MI, USA) and dissolved in a minimal amount of sterile distilled water.
<280> 1-2) 시료 투여: Osmundacetone과 Fosanᅵ ax는 골수세포 배양 첫날부터 l:20(v/v; 배지 500nL당 시료 25μϋ으로 최종농도 각각 1, 4, 7, 10 μ Μ이 되도록 지속적으로 배지에 투여하고, 배지는 2~3일 간격으로 교체하였다. 1-2) Sample administration: Osmundacetone and Fosan Ax from the first day of bone marrow cell culture, l: 20 (v / v; 25μϋ of sample per 500nL of medium, sustained to 1, 4, 7, 10μM respectively. To the medium, and the medium was replaced every 2-3 days.
<281> 1-3) 파골세포 분화 측정: <실시예 ᅵ>의 8.의 방법으로 수행하였다.  1-3) Measurement of osteoclast differentiation: It was carried out by the method of <8> of the Example.
<282> 현미경 시야에서 TRAP-양성세포 중， 2개 이상의 핵을 갖는 세포를 파골세포 로 판정하고 세포의 수를 측정하였다. Osmundacetone의 파골세포 분화억제 효과는 대조군 대비 50% 억제 농도를 1(：50로 계산하였다. In the microscope field, the cells having two or more nuclei among the TRAP-positive cells were determined as osteoclasts and the number of cells was measured. Osmundacetone's osteoclast differentiation inhibitory effect was calculated as 1 (： 50 ).
<283>  <283>
<284> 10. 세포독성 확인  10. Confirmation of cytotoxicity
<285> <실시예 1>의 1.에서 제조한 고비에서 분리된 화합물은 MTT assay를 이용하 여 세포독성을 확인하였다. <285> Compounds isolated from the fertilizer prepared in Example 1 of <1> were subjected to MTT assay. Cytotoxicity was confirmed.
<286> MTT assay 방법은 다음과 같다.  The MTT assay method is as follows.
<287> 세포들을 1 X 103 eel ls/well의 농도로 10% FBSCfetal bovine serum)가 첨가 된 DMEM를 포함하는 96 well plate에서 5% C02 , 37°C에서 배양한 후， osiiiundacetone 을 세포배지에 첨가하고 ¾시간 배양하였다. 그 후 MTT(0.5mg/ml PBS) 100 μ L를 투 여한 후, 2시간 배양하였다. 그 후 각 well에서 배지를 제거하고 lOOyL의 DMS0를 가하였다. 10분간 배양 후 마이크로플레이트 리더 (mi crop late reader; SPCTRA MAX 340PC, Molecular Devices, USA)를 이용하여 570nm에서 흡광도를 측정하였다. 흡광 도는 생존한 세포의 수를 나타내는 지표로서 하단의 식으로 계산되며, 3회의 실험 으로 재현성을 확인하였다. <287> Cells were cultured at 5% C0 2 , 37 ° C in 96 well plates containing DMEM at a concentration of 1 X 10 3 eel ls / well and 10% FBSCfetal bovine serum), and then osiiiundacetone was cultured. And incubated for ¾ hours. Thereafter, 100 μL of MTT (0.5 mg / ml PBS) was administered, followed by incubation for 2 hours. Then, the medium was removed from each well and 100MS DOO was added. After incubation for 10 minutes, the absorbance was measured at 570 nm using a micro crop late reader (SPCTRA MAX 340PC, Molecular Devices, USA). Absorbance is an indicator of the number of surviving cells, calculated by the equation below, and reproducible in three experiments.
<288> 세포 증식율 (%)=0D550(sample)/0D550(control) <288> Cell proliferation rate (%) = 0D550 (sample) / 0D550 (control)
<289>  <289>
<290> <실시예 2>실험결과  <Example 2> Experimental Results
<291> 1. 파골세포의 증식 및 분화억제 활성 확인  1. Confirmation of osteoclast proliferation and differentiation inhibitory activity
<292> 상기 <실시예 1>에서 제조한 고비의 추출물, 분획물과 분리된 화합물은 파골 세포 특이적인 염색법인 TRAP assay( tart rate-resist ant acid phosphatase)를 이용 하여 파골세포에 대한 증식 및 분화억제의 활성을 확인하였다.  The extracts and fractions of the ferns prepared in <Example 1> are isolated from the osteoclast-specific staining method TRAP assay (tart rate-resist ant acid phosphatase) to inhibit the proliferation and differentiation of osteoclasts Was confirmed.
<293>  <293>
<294> 도 2에서 확인할 수 있듯이, DMS0를 처리한 골수세포는 양성대조군 (배양 배 지에 고비 추출물 없이 분화촉진인자만 첨가한 군)과 같이 정상적으로 거대한 파골 세포를 형성하였다. 이와 대조적으로, 같은 부피의 고비의 물 추출물， EA 추출물 EA-2 분획물 및 os醒 ndacetone(lOuM)으로 처리한 군에서는 음성대조군 (배양 배지 에 고비 추출물과 분화촉진인자 모두 첨가하지 않은 군)과 유사하게 다핵세포인 거 대한 파골세포 형성이 현저히 억제되었을 뿐만 아니라， 음성대조군의 결과에 부가 적으로， 파골세포 전구세포의 증식도 현저히 억제되어 파골세포의 분화뿐만 아니라 증식 억제효과도 매우 큰 것으로 관찰되었다. 특히 osmundacetone을 처리한 경우에 는 10 μΜ농도에서 골수의 단핵구 세포가 증식하고 융합하여 다핵 세포인 파골세포 로 분화하는 과정이 95% 이상 거의 완전히 억제된 것으로 나타났다. Osmundacetone ΙμΜ을 처리한 군에서는 거대한 파골세포가 형성되었으나 양성대조군에 비하여 그 수가 적었다. Fosamax ΙμΜ을 처리한 군 역시 거대한 파골세포가 형성되었으니- 양 성대조군에 비하여 수가 적었으며 osmundacetone ΙμΜ 처치군과 파골세포 밀도가 유사하였다. 더불어 파골세포의 증식 및 분화 억제 활성 IC 도출하였다. 그 결과 도As can be seen in FIG. 2, the bone marrow cells treated with DMS0 normally formed giant osteoclasts as in the positive control group (groups in which only the differentiation factor was added to the culture medium without the fern extract). In contrast, in the same volume of fern water extract, EA extract EA-2 fraction, and os 醒 ndacetone (lOuM), the control group was similar to the negative control group (both fern extract and differentiation promoter were not added to the culture medium). In addition, the formation of giant osteoclasts, which are multinucleated cells, was significantly inhibited, and in addition to the negative control group, the proliferation of osteoclast progenitor cells was also significantly suppressed, and the effect of inhibiting osteoclast differentiation and proliferation was also very large. . In the case of treatment with osmundacetone, more than 95% of the proliferation and fusion of bone marrow monocytes and differentiation into osteoclasts, which are multinuclear cells, were almost completely inhibited at 10 μΜ. In the group treated with Osmundacetone ΙμΜ, huge osteoclasts were formed, but the number was smaller than in the positive control group. Fosamax ΙμΜ treated groups also had large osteoclasts-fewer than the positive control group, and the osmundacetone ΙμΜ treated group had similar osteoclast density. In addition, IC was derived to inhibit the growth and differentiation of osteoclasts. As a result
3에서 확인할 있듯이, osmundacetone(Alfa Aesar, Thermo Fisher Scientific)을 ΙΟμΜ농도로 처리한 경우에는 골수의 단핵구 세포가 증식하고 융합하여 다핵 세포 인 파골세포로 분화하는 과정이 거의 완전히 억제되었으며 7μΜ농도로 처리한 경우 에는 약 3-40% 억제된 것으로 나타났다 (IC50=~8yM). 공지된 골다공증 치료제로서 positive control로 사용된 FosamaxC alendronate)^ IC 는 ~4μΜ이었다 (도 4Α) . As shown in Fig. 3, the treatment of osmundacetone (Alfa Aesar, Thermo Fisher Scientific) at ΙΟμΜ concentration almost completely inhibited the proliferation and fusion of bone marrow monocytes and differentiation into multinucleated osteoclasts. In this case, about 3-40% was inhibited (IC 50 = ~ 8yM). The FosamaxC alendronate) ^ IC used as a positive control as a known osteoporosis treatment was ˜4 μΜ (FIG. 4A).
Osmundacetone이 완전히 분화된 성숙한 파골세포의 골 흡수 기능을 억제하는 지 확인하기 위해, 상기 IC50 농도인 8μΜ의 osmundacetone 촌재 하에서 성숙한 파 골세포를 이용한 골 흡수 분석을 수행하였다. To confirm whether Osmundacetone inhibits bone resorption function of fully differentiated mature osteoclasts, bone uptake analysis using mature osteoclasts was performed under the IC 50 concentration of 8 μM osmundacetone.
Osmundacetone은 <실시예 1> 5. 방법에 근거하여 뗴농도에서 성숙한 파골 세포의 골 흡수 기능을 처리되지 않은 파골세포의 58.7±13 %까지 억제하였다.  Osmundacetone inhibited the bone resorption function of mature osteoclasts to 58.7 ± 13% of untreated osteoclasts at the oocyte concentration based on the method of Example 1.
2. Osmundacetone의 조골세포의 활성화 및 파골세포 분화 억제효과 확인2. Confirmation of Osmundacetone's Osteoblast Activation and Inhibition of Osteoclast Differentiation
Osmundacetone이 파골세포의 분화를 억제함과 동시에 조골세포 분화를 활성 화하는 능력을 가지고 있는지 조사하기 위해, C57B176 마우스로부터 수집한 단핵세 포 (bone marrow mononuclear cell)와 조골세포 전구세포인 MC3T3-E1을 48웰 (well) 풀레이트 웰 당 1X10°의 골수세포와 3X103 MC3T3-E1 세포 농도로 공동배양하였디-. 공동배양한 골수세포와 조골세포 전구세포 중 골수세포의 단핵세포 /대식세포 계통 세포는 M-CSF와 RANKL 존재 하에 6-7 일 .이내에 성숙한 다핵 파골세포로 분화되었 다. To investigate whether Osmundacetone has the ability to inhibit osteoclast differentiation and to activate osteoblast differentiation, bone marrow mononuclear cells and osteoblast progenitors, MC3T3-E1, collected from C57B176 mice Co-cultured at 1 × 10 ° bone marrow cells and 3 × 10 3 MC3T3-E1 cell concentrations per 48 well pooled wells. Mononuclear / macrophage lineage cells of bone marrow cells among co-cultured bone marrow cells and osteoblast progenitor cells were 6-7 days in the presence of M-CSF and RANKL . Within a few minutes, they differentiated into mature multinuclear osteoclasts.
Osmundacetone 10 μ M을 투여한 경우에는 파골세포의 증식 및 분화를 완전히 억제하였다 (도 4Β, 0s). 반면 조골세포는 증식과 분화를 지속하였다. Osmundacetone 10μΜ을 투여한 경우 공동배양 7, 14, 21일째 ALP 활성의 ¾활성화는 투여 안 한 경우와 비교하여 각각 104%, 111%, 95%로서 osmundacetone 10 μΜ 투여 후 단독배양한 조골세포의 ALP 활성화와 비교하여 큰 차이가 없었다.  Osmundacetone 10 μM completely inhibited the proliferation and differentiation of osteoclasts (Fig. 4B, 0s). Osteoblasts, on the other hand, continued to proliferate and differentiate. ¾ activation of ALP activity on coculture 7, 14 and 21 days with Osmundacetone 10 μΜ was 104%, 111% and 95%, respectively, compared to the non-administration of ALP activation of osteoblasts cultured alone after osmundacetone 10 μΜ. There was no significant difference in comparison with.
Osmundaceton은 조골세포의 존재 및 부재 하에 파골세포의 분화에 대해 유사 한 저해 활성을 나타내었다. Osmundacetone 10 μ M을 투여한 후 6일째에 성숙한 파 골세포는 완전히 사라진 반면, 조골세포는 지속적으로 증식하는 것으로 나타났다. 따라서 전과골세포 (preosteoclasts)와 전조골세포 (preosteoblast)의 공동배양에서 나타난 바와 같이, osmundacetone은 공존하는 조골세포의 활성 및 증식에는 억제활 성을 보이지 않았다. <303> 이러한 결과를 바탕으로 , osmundacetone은 동시에 파골세포 분화는 억제하고 조골세포 분화는 활성화하는 능력을 가진 것을 확인했다. Osmundaceton showed similar inhibitory activity on the differentiation of osteoclasts in the presence and absence of osteoblasts. On day 6 after administration of 10 μM of Osmundacetone, mature osteoclasts disappeared completely, while osteoblasts continued to proliferate. Therefore, osmundacetone did not inhibit the activity and proliferation of coexisting osteoblasts, as shown in the co-culture of preosteoclasts and preosteoblasts. Based on these results, osmundacetone was found to be capable of simultaneously inhibiting osteoclast differentiation and activating osteoblast differentiation.
<304> 대조적으로， Osmundacetone이 존재하지 않는 경우， 전파골세포 및 전조골세 포 공동배양 7 일 후， 파골세포 및 조골세포 분화인자의 존재 하에서 파골세포와 조골세포가 모두 증식 및 분화를 계속하였다. (도 4B , 양성대조군) .  In contrast, when Osmundacetone was not present, osteoclasts and osteoblasts continued to proliferate and differentiate in the presence of osteoclasts and osteoblast differentiation factors after 7 days of co-culture of propagating osteoblasts and progenitor osteoblasts. (FIG. 4B, positive control group).
<305> 그러나 분화된 파골세포의 크기는 조골세포 부재 하에 성장한 것들과 비교하 여 다소 작았는데, 이는 파골세포와 조골세포의 공존 때문에 중가된 세포 밀도에 기인한 것일 수 있다.  However, the size of differentiated osteoclasts was rather small compared to those grown in the absence of osteoblasts, which may be due to the increased cell density due to the coexistence of osteoclasts and osteoblasts.
<306> 음성 대조군 배양에서 증식한 세포는 M-CSF만을 분화인자로 이용하였기 때문 에 대식세포 /단핵세포 계통 세포로 구성될 가능성이 가장 높았다 (도 4B, 음성대조 군) .  Cells grown in negative control cultures were most likely composed of macrophages / monocyte lineage cells because only M-CSF was used as a differentiation factor (FIG. 4B, negative control group).
<307> 종합적으로， 이를 통해 osmundacetone은 독립적으로 동시에 파골세포 분화는 억제하고 조골세포 분화는 활성화하는 능력이 있음올 보여 주었다.  Overall, it has been shown that osmundacetone is capable of independently inhibiting osteoclast differentiation and activating osteoblast differentiation at the same time.
<308>  <308>
<309> 3. Osmundacetone의 조골세포에 의한 ALP와 0CN 생성 증가 효과 확인  3. Confirmation of Osmundacetone's Osteoblast-induced Increase in ALP and 0CN Production
<3 io> Osmundacetone의 골 생성 유도 능력을 평가하기 위해, 조골세포 분화의 조기 <3 io> Early assessment of osteoblast differentiation to assess Osmundacetone's ability to induce bone formation
/중간 단계 마커인 ALP(alkal ine phosphatase)의 활성을 증가시키는지 여부를 확인 했다. We confirmed whether or not to increase the activity of alkal ine phosphatase (ALP).
<311> 【표 2】  <311> [Table 2]
<3 i2> MC3T3ᅳ E1에서 os匪 ndacetone의 ALP 활성화의 활성 ¾  <3 i2> Activity of ALP activation of os \ ndacetone in MC3T3 \ E1
% Activation of  % Activation of
ALP activity  ALP activity
Compounds  Compounds
10 u 50 u M  10 u 50 u M
Osmundacetone 115 ± 9.4 279 ± 61* Osmundacetone 115 ± 9.4 279 ± 61 *
Parathyroid hormone-related pept i de 138 + 19 Parathyroid hormone-related pept i de 138 + 19
(1 u M)  (1 u M)
<313>  <313>
<314> 상기 값은 3번의 독립적인 실험의 mean土 s . d .이다. 상기 *는 PO .05를 나타 낸다. 부갑상선 호르몬 관련 펩타이드 (Parathyroid hormone-rel ated pept ide)를 양 성대조군으로 사용하였다. <315> The value is the mean 土 s of 3 independent experiments. d. * Indicates PO.05. Parathyroid hormone-related peptide (Parathyroid hormone-related peptide) was used as a positive control. <31 5 >
<3i6> 상기 표 1에 나타낸 바와 같이, osmundacetone을 처리한 조골세포 유사 As shown in Table 1 above, osmundacetone-treated osteoblast-like
MC3T3-E1 세포에서의 ALP 생산은 미처리된 대조군 세포의 ALP 생산에 비해 유의하 게 중가되었다. ALP production in MC3T3-E1 cells was significantly weighted compared to ALP production in untreated control cells.
<3i7> Osmundacetone은 기존 공지된 문헌 (Lyu et al. , 2008)의 결과에 기재된 바와 유사하게, 50 μΜ의 농도에서 ALP 생산을 279 土 61 %자극하였다 (Ρ <0.05). . Osmundacetone stimulated ALP production 279 土 61% at a concentration of 50 μΜ (Ρ <0.05), similar to that described in the results of the previously known literature (Lyu et al., 2008). .
<318> 더불어 osmundacetone은 공존하는 파골세포의 분화 억제에 관계없이 MC3T3-In addition, osmundacetone was found not to inhibit the differentiation of co-existing osteoclasts.
E1 세포에 의한 ALP 생성을 증가시키는 경향을 보였다. 조골세포와 파골세포 전구 체는 10 μΜ osmundacetone 및 파골세포 및 조골세포에 대한 분화인자와 공동 배양 하였을 때, 6일 째에서 성숙한 파골세포가 완전히 사라지는 결과를 보였다 (도 4B, 0s). It showed a tendency to increase ALP production by E1 cells. When osteoblasts and osteoclast precursors were co-cultured with 10 μΜ osmundacetone and differentiation factors for osteoclasts and osteoblasts, mature osteoclasts disappeared completely on day 6 (Fig. 4B, 0s).
<3i9> 그 후 osmundacetone으로 처리한 세포는 조골세포 분화인자의 존재 하에서 계속 성장하였다.  <3i9> The cells treated with osmundacetone then continued to grow in the presence of osteoblast differentiation factors.
<J20> 조골세포 및 파골세포 전구세포의 공동배양 후 7일, 14일 및 21일째에, osmundacetone이 처리된 세포의 ALP 활성의 ¾ 활성화는 처리되지 않은 대조군 세포 에 비해 각각 104 %, 111 % 및 95 %이었고, 이는 공존하는 파골세포가 없을 때 얻 은 % 활성화 값과 거의 유사하였다 (표 1).  <J20> At 7, 14 and 21 days after coculture of osteoblast and osteoclast progenitor cells, ¾ activation of ALP activity of osmundacetone treated cells was 104%, 111% and 95%, which is close to the% activation value obtained in the absence of coexisting osteoclasts (Table 1).
<32i> 따라서 osmundacetone은 조골세포 분화를 촉진하고, 이와 동시에 파골세포 분화를 억제하는 능력을 유지한다는 것을 보여 주었다.  Thus, osmundacetone has been shown to promote osteoblast differentiation and at the same time maintain its ability to inhibit osteoclast differentiation.
<322> 주요 비-콜라겐 매트릭스 단백질 (noncollagenous matrix protein)인 0CN은 광물화 (mineralization) 시점 또는 그에 근접한 시점에서만, 즉 MC3T3-E1 세포의 분화 유도 후 21일 경에 발현이 가장 증가하는 것으로 보고되었다 (Young et al., 1992).  The major non-collagenous matrix protein, 0CN, has been reported to increase in expression only at or near mineralization, 21 days after induction of differentiation of MC3T3-E1 cells. (Young et al., 1992).
<323> 조골세포에 의한 0CN 생산에 대한 osmundacetone의 효과를 조사하기 위해 To investigate the effect of osmundacetone on 0CN production by osteoblasts
MC3T3-E1 세포를 50 μΜ osmundacetone 존재 하에 배양하였다 . MC3T3-E1 cells were cultured in the presence of 50 μΜ osmundacetone.
<324> 웨스턴 분석에 따르면， osmundacetone 투여 후 14일과 21일에 조골세포에 의 한 0CN 생성이 처리되지 않은 양성 대조군 세포와 비교하여 각각 2.9와 1.2 배 증 가한 것으로 나타났다 (도 5A. 0s). According to Western analysis, 0CN production by osteoblasts increased 2.9 and 1.2 fold, respectively, at 14 and 21 days after osmundacetone administration (Fig. 5A.0s).
<325> 이를 통해 osmundacetone은 조골세포에 의한 0CN 생산의 현저한 중가를 조기 에 유도하는 것을 확인하였다. This confirmed that osmundacetone induces a significant increase in the 0CN production by osteoblasts early.
<326 전조골세포와 전파골세포의 공동배양에서도， osmundacetone은 0CN의 생산을 초기에 증가시키면서, 파골세포의 분화를 억제하는 능력은 유지했다 (도 5B, 0s). <327> Osmundacetone을 투여하지 않은 공동배양 환경에서. 조골세포와 파골세포 분 화인자의 존재 하에 성장한 양성 대조군 조골세포는， 7일부터 분주한 후 21일까지 osmundacetone이 처리된 공동배양 환경의 조골세포와 유사한 수준까지 0CN 생산을 초기에 유도하여 증가시키는 것을 보여주었다 (도 5B . P . O . 이는 파골세포와의 상 호작용에 의하여 0CN 생산의 증가가 초기에 유도된 것으로 사료된다. Even in coculture of <326 precursor and osteoblasts, osmundacetone initially increased the production of 0CN while maintaining the ability to inhibit osteoclast differentiation (FIGS. 5B, 0s). <327> In a co-culture without Osmundacetone. Positive control osteoblasts grown in the presence of osteoblast and osteoclast differentiation factors initially induced and increased 0CN production to levels similar to osteoblasts in osmundacetone-treated coculture environments from day 7 to day 21 after dispensing. (FIG. 5B. P. O. This suggests that an increase in 0CN production was initially induced by interaction with osteoclasts.
<328>  <328>
<329> 흥미롭게도， 조골세포 분화인자 없이 성장한 음성 대조군 전조골세포는 파골 세포 없이 성장한 조골세포의 경우와 비교하여, 14 일과 21일에 파골세포가 존재한 양성 대조군 세포의 수준까지 0CN 생산의 현저한 증가를 보였다.  Interestingly, the negative control progenitor osteoblasts grown without osteoblast differentiation were found to have a significant effect of 0CN production up to the level of positive control cells with osteoclasts at days 14 and 21, compared to osteoblasts grown without osteoclasts. Showed an increase.
<330> . 이러한 결과는, 조골세포 분화인자가 없는 경우， 골수 단핵세포 또는 성숙한 파골세포와의 상호작용이 조골세포에서의 0CN 생성의 증가에 기여할 가능성을 시사 하였다. <330>. These results suggested that in the absence of osteoblast differentiation factor, the interaction with bone marrow monocytes or mature osteoclasts may contribute to the increase of 0CN production in osteoblasts.
<3 > 따라서 osmundacetone은 파골세포 분화를 억제하면서 조골세포에서 ALP와 <3> Osmundacetone inhibits osteoclast differentiation, while ALP and ALP in osteoblasts
0CN의 발현을 증가시키는 능력을 유지하는 것을 확인하였다. It was confirmed to maintain the ability to increase expression of 0CN.
<332>  <332>
<333> 4. Osmundacetone의 RUNX?, 발현 증가 효과  4. Osmundacetone's RUNX ?, Increased expression effect
<334> 다음으로 osmundacetone이 조골세포의 조기 분화 마커인 RUNX2의 발현을 증 가시키는지 '확인하였다. 상기 RUNX2는 조골세포에서 ALP와 0CN의 전사를 자극하여 골 형성을 증가시키는 전사인자로 알려져 있다 (Phimphl a i et al. , 2006) . <334> Next, it was confirmed that osmundacetone early differentiation marker expression increase visibility of key RUNX2 of osteoblast cells. The RUNX2 is known as a transcription factor that stimulates the transcription of ALP and 0CN in osteoblasts and increases bone formation (Phimphl ai et al., 2006).
<335> Osmundacetone을 투여한 MC3T3-E1 세포의 RUNX2의 발현은， 처리되지 않은 양 성 대조군과 비교하였을 때， 투여 후 7일, 14일 및 21일째에 각각 1 . 1 , 1. 1 및 2. 1 배 증가하였다 (도 6) .  Expression of RUNX2 in MC3T3-E1 cells treated with Osmundacetone was 1, 7, 14 and 21 days after administration, respectively, compared to untreated positive controls. 1, 1.1 and 2. 1-fold increase (FIG. 6).
<336> Osmundacetone이 처리된 조골세포에서 RUNX2 발현은 분주 후 21 일까지 현저 하게 감소하지 않은 반면， 처리되지 않은 조골세포에서 RLINX2 발현은 21일째에 14 일 최대 발현량의 거의 절반으로 감소했다.  In osmundacetone-treated osteoblasts, RUNX2 expression did not decrease significantly until 21 days after dispensing, whereas RLINX2 expression in untreated osteoblasts decreased to almost half of the maximum expression on day 14.
<337> 이를 통해, osmundacetone은 조골세포에서 RUNX2 발현 기간을 연장시킬 수 있는 능력을 가지고 있음을 확인하였다.  This confirms that osmundacetone has the ability to prolong RUNX2 expression in osteoblasts.
<338>  <338>
<339> 5. Osmundacetone의 암세포 -특이적 세포독상  5. Cancer Cell-Specific Cytotoxicity of Osmundacetone
<340> Osmundacetone은 정상세포 (non-cancer ee l I s )에 비해 암세포에 대해 높은 선 택성 지수값을 갖는 특이적 독성을 보였다.  Osmundacetone showed specific toxicity with high selectivity index values for cancer cells compared to non-cancer cells.
<34i> IC50이 100 μ Μ 미만인 화합물은 세포 사멸 /항 증식 활성 측면에서 활성으로 간주 될 수 있다 (Boyd, 2003). <34i> Compounds with an IC 50 <100 μΜ are active in terms of cell death / antiproliferative activity. Can be considered (Boyd, 2003).
이에 따라, 하기 표 3을 참고하면, osmundacetone은 정상세포주에 대해 세포 독성을 나타내지 않았다. Accordingly, referring to Table 3 below, osmundacetone did not show cytotoxicity against normal cell lines.
Figure imgf000036_0001
Figure imgf000036_0001
【】5.3 값은 3 번의 독립적인 실험에 대한 평균 土 표준 편차 (average土 standard deviation)이다. Fosamax는 참고 (reference) 화합물로 사용하였다. 【】 5.3 The value is the average 土 standard deviation for 3 independent experiments. Fosamax was used as a reference compound.
<348> 본 실험에 사용한 세포는 하기와 같다. The cells used in this experiment are as follows.
<349> HaCaT, 인간 표피 각질형성세포 (Human epidermal kerat inocyte)； ADMSC, 인 간 지방세포 -유래 중간엽 줄기세포 (Human Adipose-derived mesenchymal stem celKCEFO, Korea)); RAW264.7, 마우스 대식세포 세포주 (Mouse macrophage cell line (preosteoclast)); NIH3T3, 마우스 배아 섬유아세포 (Mouse embryo fibroblast)； AGS, 인간 위 선암 (Human stomach adenocarcinoma); A549, 인간 폐암 (Human lung carcinoma)； HepG2 , 인간 간 간모세포종 (Human liver hepatoblastoma); HCT116, 인간 결장암 (Human colon carcinoma); PC3, 인간 전립선 샘암종 (Human prostate adenocarcinoma)； Caki-1, 인간 신장암종 (Human kidney carcinoma); T24, 인간 방광암 (Human bladder carcinoma); HT1080 , 인간 섬유육종 (Human fibrosarcoma); B16F10, 마우스 혹색종 (Mouse melanoma); TOl P3, HPV- 16E7-발현하는 마우스 폐 상피세포 (HPV-16 E7-expressing mouse lung epithelial cell(- MHC class I))  HaCaT, Human epidermal kerat inocytes; ADMSC, Human Adipose-derived mesenchymal stem celKCEFO, Korea); RAW264.7, mouse macrophage cell line (preosteoclast); NIH3T3, Mouse embryo fibroblast; AGS, Human stomach adenocarcinoma; A549, Human lung carcinoma; HepG2, Human liver hepatoblastoma; HCT116, Human colon carcinoma; PC3, Human prostate adenocarcinoma; Caki-1, Human kidney carcinoma; T24, human bladder carcinoma; HT1080, human fibrosarcoma (Human fibrosarcoma); B16F10, mouse melanoma (Mouse melanoma); TOl P3, HPV-16E7-expressing mouse lung epithelial cells (-MHC class I)
<350>  <350>
<35i> 특히， MTT 분석에 따르면, osmundacetone은 ADMSC 인간 지방세포 유래 중간 엽 줄기세포, HaCaT 인간 표피 각질세포 및 NIH3T3 마우스 배아 섬유아세포에 대해 각각 2， 760 ± 220, 3510 土 110 및 > 5,000 μΜ의 IC50 값을 갖는 미미한 세포 독 성을 나타냈다. <3 5 i> In particular, according to MTT analysis, osmundacetone was found to be 2, 760 ± 220, 3510 土 110 and> 5,000 for ADMSC human adipocyte derived mesenchymal stem cells, HaCaT human epidermal keratinocytes and NIH3T3 mouse embryo fibroblasts, respectively. Insignificant cell virulence with an IC 50 value of μΜ was shown.
<352> 한편, osimmdacetone은 RAW264.7 마우스 대식세포 세포주에 대해 507 士 98 μΜ의 LD50으로， 상대적으로 유의한 세포독성을 나타내었다. 따라서 osimmdacetone 이 고농도에서 일부 탐식세포의 생존력을 억제할 수 있는 가능성을 제시하였다. <353> 흥미롭게도 MTT 분석에 따르면 osmundacetone은 AGS 인간 위암 (stomach adenocarcinoma), PC3 사람 전립선 선암 (prostate adenocarcinoma) 및 B16F10 마우 스 흑색종 (melanoma)을 포함한 암 세포주에 대해 59.9 土 6.1, 65.5 士 8.7 및 75.8 士 9.2 μΜ의 낮은 LD50을 나타내 중등도의 세포독성 활성을 나타내는 것을 알 수 있었다. On the other hand, osimmdacetone showed relatively significant cytotoxicity with an LD 50 of 507 cm 98 μΜ against RAW264.7 mouse macrophage cell line. Thus, the possibility of osimmdacetone could inhibit the viability of some phagocytes at high concentrations. Interestingly, according to MTT analysis, osmundacetone was found to be 59.9 土 6.1, 65.5 5.9 8.7 and 8 for cancer cell lines including AGS human gastric cancer (stomach adenocarcinoma), PC3 human prostate adenocarcinoma and B16F10 mouse melanoma. A low LD 50 of 75.8 μm 9.2 μΜ was shown to indicate moderate cytotoxic activity.
<354>  <354>
<355> 특히， 인간 정상세포주 (HaCaT, ADMSC)와 비교하여 인간 암 세포주 (AGSᅳ PC) 를 사멸시키는 데 있어서 osmundacetone의 세포 독성의 암 선택도 지수가 45 내지 60으로 매우 높았다. In particular, the cancer selectivity index of osmundacetone in killing human cancer cell line (AGS ᅳ PC) compared to human normal cell line (HaCaT, ADMSC) is 45 to Very high at 60.
<356> < 35 6>
<357> 결론적으로 파골세포 분화 억제에 대한 osmundacetone의 IC5。는 8 μΜ, 조골 세포를 280% 활성화시키는 농도는 50 μΜ, 및 몇 암세포주에 대한 LD50값 50 내지In conclusion, IC 5。 of osmundacetone for inhibition of osteoclast differentiation was 8 μΜ, the concentration of 280% activating osteoblasts was 50 μΜ, and the LD 50 value 50 to several cancer cell lines.
70μΜ에 비하여, 정상세포주에 대한 LD50는 2，500 내지 5,000μΜ 범위를 나타내므 로, 이를 기반으로 osmundacetone을 치료제로 사용하는 경우 큰 안전성을 갖는 것 을 확인하였다. Compared to 70μΜ, LD 50 for the normal cell line ranges from 2,500 to 5,000μΜ, and based on this, it was confirmed that the osmundacetone has a great safety when used as a therapeutic agent.
< 58>  <58>
<359> <적용예 1> <3 5 9><Application example 1>
<360> ¾다공증  <360> ¾ porosity
<36i> 뼈는 대사적으로 뼈를 흡수하는 파골세포 (osteoclast)와 생성하는 조골세포 <36i> Bone is osteoclasts and osteoblasts that produce bone metabolically
(osteoblast) 간의 뼈 리모델링 순환 (bone remodelling cycle)의 균형을 통해 유지 된다. 그러나, 파골세포와 조골세포의 균형이 깨어지고 과골세포가 지나치게 활성 화되면 뼈의 흡수와 생성 간의 균형이 파괴되어 흡수량이 생성량보다 많아지면서 골다공증이 유발된다 (Kim JH and Kim N, 2016; Shiozawa Y et a/., 2011). Bone remodeling cycles (osteoblast) are maintained throughout the balance. However, when osteoclasts and osteoblasts lose their balance and the osteoclasts become too active, the balance between bone absorption and production breaks down, leading to osteoporosis as the absorption is greater than the amount produced (Kim JH and Kim N, 2016; Shiozawa Y et. a /., 2011).
<362> 따라서 , 본 발명의 osmundacetone은 파골세포의 증식 및 분화를 억제하는 효 과와 동시에 조골세포를 활성화하는 효과를 나타내기 때문에, 골다공증 예방 또는 치료효과를 나타낼 수 있다.  Therefore, since osmundacetone of the present invention exhibits an effect of inhibiting the proliferation and differentiation of osteoclasts and an effect of activating osteoblasts, the osmundacetone may have an effect of preventing or treating osteoporosis.
<363>  <363>
<364> <적용예 2>  <364> <Application Example 2>
<365> 류마티 관¾염  <365> Rheumatoid Tube Salt
<366> 류마티스관절염은 자가면역질환인데 자가면역항체가 파골세포 분화를 촉진한 다. 그로 인한 과도한 골 흡수는 류마티스관절염을 악화시킨다 (Takayanagi H, 2007) . 그 기전은 다음과 같다. 파골세포 분화 관련 중추적인 전사인자인 NFAT 전 사인자들은 (NFATc:i/c2/c3/c4) 기본적으로 calcium/calmodulin signaling에 의하여 활성화된다 (Takayanagi H et a/., 2002) . 완전한 활성화를 위해서는 면역조절 단 백질들인 DNAX-activating protein 12(DAP12) 및 면역항체 Fc receptor common γ chain(FcRx)과 같은 tyrosine一 based activation motif ( ITAM)— bear ing molecule들 이 면역세포에서 calcium signaling을 자극한다 (Pitcher LA and van Oers NS, 2003) . 파골세포에서 역시 DAP12와 FcR γ A calcium signaling을 통하여 NFATcl을 활성화시킨다. 따라서 DAP12 및 FcRy와 연계된 imniunoglobulin-like receptor가 파골세포 분화에서 중요한 역할을 한다 (Koga T et al. , 2004； Mocsai A et al . , 2004) . 즉, FcRy는 파골세포에서 osteoclast-associated receptor (OSCAR) 및 paired immunoglobul in-1 ike receptor (PIR— A)와 상호작용한다. ITAM이 인산화되면 phospho lipase C χ (ΡΙ χ )를 활성화하고 이는 세포 내 calcium을 유리하고 이는 calmodul in-dependent phosphatase인 calcineurin을 활성화한다 . Calcineurin은 직 접 NFATcl의 serine을 탈인산화하여 핵 내로 보내고 활성화시킨다. 결과적으로 면 역항체는 파골세포 분화를 촉진하게 되고, 파골세포에 의한 과도한 골 흡수는 류마 티스 관절염을 악화시키게 된다. 결국， 류마티스 관절염 환자에서 파골세포 분화 억제는 자가면역기전 자체의 이상을 교정하지는 못 해도 그 결과.유발된 관절염과 통증 등 골격계 증상은 치료할 수 있다. Rheumatoid arthritis is an autoimmune disease in which autoimmune antibodies promote osteoclast differentiation. The resulting excessive bone absorption exacerbates rheumatoid arthritis (Takayanagi H, 2007). The mechanism is as follows. NFAT precursors (NFATc: i / c2 / c3 / c4), pivotal transcription factors related to osteoclast differentiation, are primarily activated by calcium / calmodulin signaling (Takayanagi H et a /., 2002). For full activation, tyrosine-based activation motifs (ITAMs), such as the immunomodulatory proteins DNAX-activating protein 12 (DAP12) and the immune antibody Fc receptor common γ chain (FcRx), bear calcium molecules in immune cells. Stimulates (Pitcher LA and van Oers NS, 2003). Osteoclasts also activate NFATcl through DAP12 and FcR γ A calcium signaling. Thus, imniunoglobulin-like receptors associated with DAP12 and FcRy Plays an important role in osteoclast differentiation (Koga T et al., 2004; Mocsai A et al., 2004). In other words, FcRy interacts with osteoclast-associated receptor (OSCAR) and paired immunoglobul in-1 ike receptor (PIR-A) in osteoclasts. Phosphorylation of ITAM activates phospholipase C χ (ΡΙχ), which releases intracellular calcium, which activates calcineurin, a calmodul in-dependent phosphatase. Calcineurin directly dephosphorylates and activates NFATcl serine into the nucleus. As a result, immune antibodies promote osteoclast differentiation, and excessive bone uptake by osteoclasts exacerbates rheumatoid arthritis. As a result, inhibition of osteoclast differentiation in patients with rheumatoid arthritis may not correct the abnormalities of autoimmune mechanisms themselves. As a result, skeletal symptoms such as arthritis and pain can be treated.
<367> 따라서 , 본 발명의 osmundacetone은 파골세포의 증식 및 분화를 억제하는 효 과를 나타내기 때문에， 류마티스 관절염의 예방 또는 치료효과를 나타낼 수 있다.Therefore, since osmundacetone of the present invention exhibits an effect of inhibiting the proliferation and differentiation of osteoclasts, it can exhibit a prophylactic or therapeutic effect of rheumatoid arthritis.
<368> <368>
<369> <적용예 3>  <369> <Application Example 3>
<370> 파제트 병 (Osteitis deformans)  Page 370 Paget's disease (Osteitis deformans)
<37i> 파제트 병 (Osteitis deformans) 역시 파골세포의 비정상적 골 흡수가 유발한 다 (Singer FR, 2016). 그러면 조골세포의 비정상적 골 생성이 진행되고 이 과정이 반복되면서 골의 기형과 그로 인한 통증， 두통， 청력손실 등이 초래된다. 팔, 다 리, 골반， 척추, 두개골에 호발한다. 새로 생성된 골은 약하여 골절 빈도가 높다. 고칼슘혈증, 심장마비， 반신불수가 유발될 '수 있다 (Ralstone SH, 2016). 발병 원인 은 밝혀져 있지 않으나 유전적 소인과 어릴 적 바이러스 감염이 그 원인으로 의심 된다. 약물치료가 병의 진행을 억제하는데 도움이 된다. 현재 가장 많이 사용하는 치료제는 파골세포 분화 억제제인 Fosamax 및 골 대사를 조절하는 calcitonin이다. 하지만 Fosamax는 부작용으로 일부 환자에서 장기 복용이 제한적이다. 통증이' 심하 면 Acetaminophen(Tylenol)이나 nonsteroidal ant i-inf lammatory drugs(NSAIDs)를 사용한다. Osteitis deformans also causes abnormal bone resorption of osteoclasts (Singer FR, 2016). Then, abnormal bone formation of osteoblasts progresses and this process is repeated, resulting in bone malformation, resulting in pain, headache, and hearing loss. It affects the arms, legs, pelvis, spine and skull. Newly formed bones are weak and have a high frequency of fractures. Hypercalcemia, heart attack, and incompetence can be caused (Ralstone SH, 2016). The cause is unknown, but genetic predisposition and childhood viral infections are suspected to be the cause. Medication can help to control the progression of the disease. Currently, the most used therapeutic agents are osteoclast differentiation inhibitor Fosamax and calcitonin to regulate bone metabolism. However, Fosamax has limited side-effects in some patients as a side effect. The pain is used to "severe surface Acetaminophen (Tylenol) or nonsteroidal ant i-inf lammatory drugs ( NSAIDs).
<372> 따라서 , 본 발명의 osmundacetone은 파골세포의 증식 및 분화를 억제하는 효 과를 나타내기 때문에 , 파제트 병의 예방 또는 치료효과를 나타낼 수 있다.  Therefore, since osmundacetone of the present invention exhibits an effect of inhibiting the proliferation and differentiation of osteoclasts, it can have a prophylactic or therapeutic effect of Paget's disease.
<373>  <373>
<374> <적용예 4>  <374> <Application example 4>
<37 > 골 전이암  <37> bone metastases
<376> 파골세포는 또한 고형암 (solid tumor)의 골 전이를 촉진한다. 뼈는 암의 전 이가 가장 호발하는 부위이다. 뻐에 암이 전이되면 극심한 통증과 함께 뼈가 부서 져서 완치 가능성이 현저히 낮아진다 OVeilbaecher N et al. , 2011). 전신에 퍼진 암세포들은 골수 내의 혈액 줄기세포 증식 장소에서 발견된다 (Shiozawa Y et al., 2013). 암세포는 골수세포로부터 파골세포의 분화를 현저히 촉진하여 골 전이와 암 성장， 골 파괴를 촉진한다. 따라서 파골세포는 암의 골 전이에서 핵심적인 역할올 하아 파골세포 분화를 억제하면 골 전이가 감소한다. 많은 고형암의 전이가 골 전 이인데 혈액 줄기세포 증식 장소를 거점으로 혈액 즐기세포를 몰아내고 중식하다가 다시 혈액으로 나와 다른 곳으로 전이하기도 한다. 골 전이가 가장 흔한 암은 전립 선암으로서 골 전이가 암을 악화시켜서 완치를 어렵게 하고 사망의 주요 원인이다. 사람 전립선 암세포의 직접적인 주요 표적 역시 혈액 줄기세포 증식 장소로서 전이 암의 거점으로 사용한다 (Shiozawa Y et al. , 2011). 또한 파골세포는ᅳ전립선 암 조 직 내에 혈관 형성을 촉진하여 암 성장을 촉진한다 (Bruni-Carcloso A et al., 2010) . 유방암 세포도 파골세포 분화를 촉진하여 , 유방 절제술을 시행한 유방암 환 자에서 파골세포가 골 전이를 통한 암 재발을 촉진한다 (Danilin S et al. , 2012； Lu X et al . , 2011) . Osteoclasts also promote bone metastasis in solid tumors. Bones of Cancer I This is the most common site. Cancer metastasis in the cortex causes severe pain and fractures of the bones, significantly reducing the likelihood of cure. OVeilbaecher N et al. , 2011). Systemic cancer cells are found at sites of blood stem cell proliferation in the bone marrow (Shiozawa Y et al., 2013). Cancer cells significantly promote the differentiation of osteoclasts from bone marrow cells to promote bone metastasis, cancer growth, and bone destruction. Therefore, osteoclasts play a key role in bone metastasis of cancer. Inhibiting osteoclast differentiation reduces bone metastasis. Many solid cancer metastases are bone metastases, and blood stem cells are driven from the blood stem cell proliferation centers, and then fed to the blood and then metastasized. The most common cancer of bone metastasis is prostate cancer, where bone metastasis worsens the cancer, making it harder to cure and is the leading cause of death. Direct primary targets of human prostate cancer cells also serve as a base for metastatic cancer as a place of blood stem cell proliferation (Shiozawa Y et al., 2011). Osteoclasts also promote cancer growth by promoting blood vessel formation within the prostate cancer tissue (Bruni-Carcloso A et al., 2010). Breast cancer cells also promote osteoclast differentiation, and osteoclasts promote cancer recurrence through bone metastasis in breast cancer patients undergoing mastectomy (Danilin S et al., 2012; Lu X et al., 2011).
골 전이암을 예방하기 위한 골 표적 치료제가 현재 임상에서 사용되고 있으 며 파골세포가 암 골 전이의 핵심 기전 중 하나이므로 항암 신약개발의 주요 표적 이 되고 있다. 이에 따라 파골세포 분화를 억제하기 위한 목적으로 현재 미국 FDA 승인을 받은 유일한 bisphosphonate 계열 약제가 Zoledronic acid이다 (E卜 Amm J et al. , 2013). Zoledronic add는 뼈를 보존하고 생존률을 높인다. 고 위험 비전이 전립선암 (high risk nonmetastat i c prostate cancer)에서 Zoledronic Acid는 골 전 이를 크게 감소시켰다 (Wirth M et al. , 2014). Zoledronic acid를 조골세포를 활성 화하는 부갑상선호르몬 (parathyroid hormone)과 함께 투여하면 골 전이가 더욱 감 소하였다 (Schneider A et a/., 2005). 파골세포 분화의 신호전달물질인 RANKL에 대 한 단클론 항체 (monoclonal antibody)인 Denosumab 역시 전립선암의 골 전이를 억 제하여 파골세포 억제가 암의 골 전이 억제에 중요함이 다시 입증되었다 (Smith MR et aL , 2012). Multiple myeloma 환자에서도 Zoledronic acid를 투여하면 과골세 포 분화가 억제되어 골 전이가 현저히 억제된다 (Zhuang J et al. , 2012). 즉, 부작 용 적고 저렴한 파골세포 억제제가 개발되면 암 환자에서 전이를 억제하기 위하여 장기 투여할 수 있다.  Bone-targeted therapeutics to prevent bone metastases are currently used in clinical trials, and osteoclasts are one of the key mechanisms for cancer metastasis. Accordingly, the only bisphosphonate-based drug currently approved by the US FDA for the purpose of inhibiting osteoclast differentiation is Zoledronic acid (E 卜 Amm J et al., 2013). Zoledronic add preserves bones and increases survival rates. Zoledronic Acid significantly reduced bone metastasis in high risk nonmetastat i prostate cancer (Wirth M et al., 2014). The administration of Zoledronic acid with parathyroid hormone, which activates osteoblasts, further reduced bone metastasis (Schneider A et a., 2005). Denosumab, a monoclonal antibody to RANKL, a signaling agent for osteoclast differentiation, also inhibits bone metastasis in prostate cancer, again demonstrating that osteoclast inhibition is important for inhibiting bone metastasis in cancer (Smith MR et. aL, 2012). In patients with multiple myeloma, the administration of Zoledronic acid inhibits hyper-osteoblast differentiation and significantly inhibits bone metastasis (Zhuang J et al., 2012). In other words, if a side effect low cost osteoclast inhibitor is developed, it can be administered long-term to suppress metastasis in cancer patients.
따라서 , 본 발명의 osmundacetone은 파골세포의 증식 및 분화를 억제하는 효 과를 나타내며 동시에 조골세포 활성화 효과가 있기 때문에, 골 전이암의 예방 또
Figure imgf000041_0001
Therefore, osmundacetone of the present invention exhibits an effect of inhibiting the proliferation and differentiation of osteoclasts and at the same time has an osteoblast activating effect, thus preventing or preventing bone metastasis.
Figure imgf000041_0001
¾,:net o., a o w -nosw^d、?l linsf Brl.- ¾,: ne t o. , aow -nosw ^ d ,? l li ns f Br l .-
y,,g,g,yCgs BiseL Doua Wetoeasovs El Wll Tlk 1VE Adrso Makkllinnnr <34> y ,, g , g , yCgs B i seL Doua We t oeasovs E l W ll T l k 1V E Adrso Makk lli nnnr <34>
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<463>  <463>
【산업상 이용가능성】  Industrial Applicability
<464> 본 발명에 따른 조성물은 뼈 손실을 야기하는 파골세포에 대하여 강력한 증 식 및 분화 억제 활성을 나타내고 동시에 조골세포를 활성화하기 때문에， 안전하고 효과적인 골다공증 치료제 또는 개선용 식품을 개발하는데 유용하게 이용될 수 있 다，  Since the composition according to the present invention exhibits potent proliferation and differentiation inhibitory activity against osteoclasts causing bone loss and simultaneously activates osteoblasts, it is useful for developing safe and effective osteoporosis therapeutics or foods for improvement. Can be

Claims

【청구의 범위】 [Range of request]
【청구항 1】  [Claim 1]
<1> Osmundacetone 또는 이의 약학적으로 허용가능한 염을 유효성분으로 포함하는 골다공증， 류마티스 관절염， 관절통， 파제트 병， 골 전이암 및 골 절로 이루어진 군에서 선택된 어느 하나 이상의 골 질환 예방 또는 치료용 약학적 조성물.  <1> A pharmaceutical for the prevention or treatment of any one or more bone diseases selected from the group consisting of osteoporosis, rheumatoid arthritis, joint pain, Paget's disease, bone metastases and fractures, including Osmundacetone or a pharmaceutically acceptable salt thereof Composition.
<2>  <2>
【청구항 2】  [Claim 2]
<3> 계 1항에 있어서， 상기 osmudacetone은 고비과 (Osmundaceae) 식물에서 분리되는 것을 특징으로 하는 조성물.  <3> The composition according to claim 1, wherein the osmudacetone is separated from the fern family Osmundaceae.
<4>  <4>
【청구항 3】  [Claim 3]
<5> 제 2항에 있어서, 상기 고비과 식물은 고비 0 ζ/? japonica) QA 것을 특징으로 하는 조성물. <5> The method of claim 2, wherein the fern is a fern 0 ζ /? japonica) Q A composition, characterized in that.
<6>  <6>
【청구항 4】  [Claim 4]
<7> Osmundacetone 또는 이의 약학적으로 허용가능한 염을 유효성분으로 포함하는 골다공증， 류마티스 관절염， 관절통， 파제트 병， 골 전이암 및 골 절로 이루어진 군에서 선택된 어느 하나 이상의 골 질환 개선용 식품 조성  <7> Food composition for improving one or more bone diseases selected from the group consisting of osteoporosis, rheumatoid arthritis, arthralgia, Paget's disease, bone metastases and fractures, including Osmundacetone or a pharmaceutically acceptable salt thereof
<8> <8>
【청구항 5】  [Claim 5]
<9> LBK Osffiunda japonica) 추출물을 유효성분으로 포함하는 골다공증， 류마티스 관절염， 관절통， 파제트 병, 골 전이암 및 골절로 이루어진 군에 서 선택된 어느 하나 이상의 골 질환 예방 또는 치료용 약학적 조성물.  <9> A pharmaceutical composition for the prevention or treatment of any one or more bone diseases selected from the group consisting of osteoporosis, rheumatoid arthritis, arthralgia, Paget's disease, bone metastases and fractures comprising LBK Osffiunda japonica) extract as an active ingredient.
<10>  <10>
【청구항 6]  [Claim 6]
<11> 제 5항에 있어서, 상기 추출물은 물， 에탄올, 주정， 메탄을， 프로판을 (propanol ) , 이소프로판올 ( i sopropanol )， 부탄을 (butanol )， 아세톤， 에테르, 클로로포름， 에틸 아세테이트， 메틸렌클로라이드， 핵산， 시클로핵산， 석유 에테르 (petrol eum ether ) , 디에틸에테르, 벤젠으로 이루어진 군에서 선택된 하나 이상의 용매로 추출된 것을 특징으로 하는 조성물. 6. The extract of claim 5, wherein the extract is water, ethanol, alcohol, methane, propane, isopropanol, butanol, acetone, ether, chloroform, ethyl acetate, methylene chloride. ， Nucleic acid, cyclonucleic acid, petroleum ether (petrol eum ether), diethyl ether, benzene is a composition characterized in that extracted with one or more solvents selected from the group consisting of.
<12>  <12>
【청구항 7】  [Claim 7]
<13> 고비 ( <¾»ί /7ώ japonica) 추출물을 유효성분으로 포함하는 골다공증， 류마티스 관절염， 관절통， 파제트 병， 골 전이암 및 골절로 이루어진 군에 서 선택된 어느 하나 이상의 골 질환 개선용 식품 조성물. <14> <13> Food for improving one or more bone diseases selected from the group consisting of osteoporosis, rheumatoid arthritis, arthralgia, Paget's disease, bone metastases and fractures, which contains extracts of <fern »</» ί / 7ώ japonica Composition. <14>
【청구항 8]  [Claim 8]
<15> 골다공증， 류마티스 관절염， 관절통， 파제트 병， 골 전이암 및 골절 로 이루어진 군에서 선택된 어느 하나 이상의 골 질환 예방 또는 치료용 제 제의 제조에 사용되는 osmundacetone 또는 이의 약학적으로 허용가능한 염 의 용도.  <15> Osmundacetone or a pharmaceutically acceptable salt thereof for use in the manufacture of a preparation for the prevention or treatment of any one or more bone diseases selected from the group consisting of osteoporosis, rheumatoid arthritis, arthralgia, Paget's disease, bone metastases and fractures. Usage.
<16>  <16>
【청구항 9】  [Claim 9]
<17> 제 8항에 있어서， 상기 osmudacetone은 고비과 (Osmundaceae) 식물에서 분리되는 것을 특징으로 하는 염의 용도.  Use of a salt according to claim 8, wherein said osmudacetone is isolated from an Osmundaceae plant.
<18>  <18>
【청구항 10】  [Claim 10]
<19> Osmundacetone 또는 이의 약학적으로 허용가능한 염의 유효량을 이를 필요로 하는 개체에 투여하는 것을 특징으로 하는 골다공증， 류마티스 관절 염， 관절통, 파제트 병， 골 전이암 및 골절로 이루어진 군에서 선택된 어느 하나 이상의 골 질환 치료 방법 .  <19> Any one selected from the group consisting of osteoporosis, rheumatoid arthritis, arthralgia, Paget's disease, metastatic cancer and fractures, comprising administering to a subject in need thereof an effective amount of Osmundacetone or a pharmaceutically acceptable salt thereof Treatment of Abnormal Bone Disease.
<20>  <20>
【청구항 11】  [Claim 11]
<21> 골다공증， 류마티스 관절염， 관절통， 파제트 병， 골 전이암 및 골절로 이루어진 군에서 선택된 어느 하나 이상의 골 질환 예방 또는 치료용 제제의 제조에 사용하기 위한 고비 ( (¾ / /a japonica) 추출물의 용도.  Fertilizer (¾ / a japonica) extract for use in the manufacture of a preparation for the prevention or treatment of any one or more bone diseases selected from the group consisting of osteoporosis, rheumatoid arthritis, arthralgia, Paget's disease, bone metastases and fractures Use of
<22> ·  <22> ·
【청구항 12]  [Claim 12]
<23> 제 11항에 있어서， 상기 추출물은 물， 에탄을， 주정， 메탄올, 프로판올 (propanol ) , 이소프로판올 ( i sopropanol ) , 부탄을 (butanol )， 아세톤, 에테르， 클로로포름， 에틸 아세쩨이트， 메틸렌클로라이드， 핵산， 시클로핵산， 석유에테르 (petro l eum ether ) , 디에틸에테르， 벤젠으로 이루어진 군에서 선택된 하나 이상의 용매로 추출된 것을 특징으로 하는 용도.  12. The extract of claim 11, wherein the extract comprises water, ethane, alcohol, alcohol, methanol, propanol, isopropanol, butanol, acetone, ether, chloroform, ethyl acetate, methylene. Use of at least one solvent selected from the group consisting of chloride, nucleic acid, cyclonucleic acid, petroleum ether, diethyl ether, benzene.
<24>  <24>
【청구항 13]  [Claim 13]
<25> 고비 ( //2ώ japonica) 추출물꾀 유효량을 이를 필요로 하는 개체에 투여하는 것을 특징으로 하는 골다공증， 류마티스 관절염, 관절통， 파제트 병， 골 전이암 및 골절로 이루어진 군에서 선택된 어느 하나 이상의 골 질환 치료 방법.  At least one selected from the group consisting of osteoporosis, rheumatoid arthritis, arthralgia, Paget's disease, bone metastases and fractures, which comprises administering an effective amount of fern (// 2ώ japonica) extract to an individual in need thereof How to treat bone disease.
<26>  <26>
【청구항 14】 <27> 제 13항에 있어서， 상기 추출물은 물， 에탄올, 주정， 메탄올， 프로판올 (propanol), 이소프로판올 (isopropanol ) , 부탄을 (butanol )， 아세톤 에테르， 클로로포름， 에틸 아세테이트， 메틸렌클로라이드， 핵산, 시클로핵산, 석유에테르 (petroleum ether), 디에틸에테르， 벤젠으로 이루어진 군에서 선택된 하나 이상의 용매로 추출된 것을 특징으로 하는 치료 방법. [Claim 14] The extract of claim 13, wherein the extract is water, ethanol, alcohol, methanol, propanol, isopropanol, butanol, acetone ether, chloroform, ethyl acetate, methylene chloride, nucleic acid, cyclo A therapeutic method characterized in that extracted with at least one solvent selected from the group consisting of nucleic acid, petroleum ether, diethyl ether, benzene.
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