WO2021040416A1 - Pharmaceutical composition, for preventing or treating bone loss induced by metabolic bone diseases, comprising artemisia scoparia extract as active ingredient - Google Patents

Pharmaceutical composition, for preventing or treating bone loss induced by metabolic bone diseases, comprising artemisia scoparia extract as active ingredient Download PDF

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WO2021040416A1
WO2021040416A1 PCT/KR2020/011430 KR2020011430W WO2021040416A1 WO 2021040416 A1 WO2021040416 A1 WO 2021040416A1 KR 2020011430 W KR2020011430 W KR 2020011430W WO 2021040416 A1 WO2021040416 A1 WO 2021040416A1
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bone
extract
pharmaceutical composition
preventing
loss caused
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French (fr)
Korean (ko)
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박종환
조정용
최주희
송지은
김영민
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전남대학교산학협력단
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    • 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
    • A61K36/18—Magnoliophyta (angiosperms)
    • A61K36/185—Magnoliopsida (dicotyledons)
    • A61K36/28—Asteraceae or Compositae (Aster or Sunflower family), e.g. chamomile, feverfew, yarrow or echinacea
    • A61K36/282—Artemisia, e.g. wormwood or sagebrush
    • 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
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P19/00—Drugs for skeletal disorders
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
    • A23V2002/00—Food compositions, function of food ingredients or processes for food or foodstuffs
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
    • A23V2200/00—Function of food ingredients
    • A23V2200/30—Foods, ingredients or supplements having a functional effect on health
    • A23V2200/306—Foods, ingredients or supplements having a functional effect on health having an effect on bone mass, e.g. osteoporosis prevention

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  • the present invention relates to a pharmaceutical composition for the prevention or treatment of bone loss caused by bone metabolic disease, comprising the extract of Mugwort as an active ingredient.
  • Bone is bone cells such as osteocytes, osteoclasts, and osteoblasts, hydroxyapatite crystals, collagen fibers, and glycosaminoglycans.
  • the bone matrix and the bone marrow cavity, blood vessels (vascular canals), canaliculi, and bone marrow cavity (lacunae) are composed of the same space. Bone mechanically supports the body, protects vital organs, provides a microenvironment necessary for hematopoiesis, and stores calcium and various minerals.
  • Bone growth, development, and maintenance occur continuously throughout life. Aged bones are destroyed and new bones are regenerated in place. This turnover occurs mainly in BMU (Basic Multicellular Units) composed of osteoclasts and osteoblasts, and this process plays a role in restoring microscopic damage to bones caused by growth and stress and maintaining bone function. The destruction or absorption of aged bone is performed by osteoclasts. On the other hand, osteoblasts are responsible for the formation of new bones. The osteoclasts attach to the surface of the bones and secrete acid and degrading enzymes, thereby destroying the bone by removing the bone matrix such as apatite crystals and collagen, and the osteoblasts synthesize and secrete the bone matrix. It forms a skeleton by controlling the concentration of and phosphorus.
  • BMU Basic Multicellular Units
  • Bone metabolic disease occurs when the balance between osteoclasts and osteoblasts is broken in vivo.
  • a representative example of a bone metabolic disease is osteoporosis.
  • Osteoporosis is a disease in which the bone mass decreases due to various causes and the risk of fracture continues to increase due to the deterioration of the microstructure of the bone tissue.
  • osteoporosis is a state in which minerals (especially calcium) and substrates that make up bones are reduced, and the balance of bone remodeling is broken, resulting in a state in which the osteoclast action is increased than the osteoblast action.
  • Normal bones have a dense structure like a mesh, but in the case of osteoporosis, the gap between the structures becomes wider and the microstructures become thinner and weaken, so that the bones can easily be fractured even with a small impact.
  • Osteoporosis is largely classified into primary osteoporosis and secondary osteoporosis.
  • Primary osteoporosis includes postmenopausal osteoporosis, age-related osteoporosis, and idiopathic osteoporosis.
  • Secondary osteoporosis is caused by diseases or drugs regardless of age.
  • bone metastasis occurs in various cancer patients.
  • bone metastasis observed in breast cancer patients is osteolytic metastasis, which destroys most of the bones.
  • Osteolytic bone metastasis is known to occur by stimulating osteoclasts rather than by breast cancer cells directly affecting bones.
  • the present inventors have made intensive research efforts to develop natural materials that are effective for bone loss caused by bone metabolic diseases. As a result, by examining the effect of preventing or treating bone loss caused by bone metabolic disease by inhibiting the differentiation of osteoclasts and the activity of NFATc1, the present invention was completed.
  • a pharmaceutical composition for preventing or treating bone loss caused by bone metabolic disease comprising a wormwood extract as an active ingredient.
  • Another object of the present invention is to provide a food composition for preventing or improving bone loss caused by bone metabolic diseases, containing a wormwood extract as an active ingredient.
  • the present invention provides a pharmaceutical composition for preventing or treating bone loss caused by bone metabolic disease, comprising a wormwood extract as an active ingredient.
  • Artemisia scoparia is a perennial plant belonging to Artemisia of the family Compositae. It is a perennial plant that grows in sandy beaches and resembles wormwood, but has a different smell. Mugwort does not die even in winter because its stem is like a tree, but wormwood is characterized by the fact that the stem dries completely in winter. Mugwort is a rare herb that is rarely used in oriental medicine.
  • Osteoclasts are cells derived from mononuclear cells of hematopoietic stem cells, and RAW264.7 monocytes from mice are fused by RANKL (receptoractivator of nuclear factor ⁇ (RANK) ligand) to differentiate into multinucleated osteoclasts.
  • RANKL receptoractivator of nuclear factor ⁇ (RANK) ligand
  • RANKL outside the cell binds to RANK and promotes the activity of mitogen-activated protein kinase (MAPK), which is a transcription factor called NF- ⁇ that enters the nucleus, which is a TRAP related to osteoclast differentiation.
  • MAPK mitogen-activated protein kinase
  • RANKL binds to RANK, it promotes the activity of TRAF6 (tumor necrosis factor receptor associated factor 6), thereby promoting the activity of transcription factors such as MAPK, NF- ⁇ AP-1, and NFATc1.
  • TRAF6 tumor necrosis factor receptor associated factor 6
  • MAPK MAPK
  • NF- ⁇ AP-1 NF- ⁇ AP-1
  • NFATc1 transcription factors
  • AP-1 or NFAT specific for osteoclast differentiation can be regulated by ERK and NF- ⁇ , which have been identified as signaling molecules that play an important role in the survival and bone resorption of osteoclasts. Therefore, blocking of the signaling pathway activated by RANKL is recognized as one of the therapeutic approaches for the treatment of bone diseases including osteoporosis.
  • the bone metabolic disease is osteoporosis (osteoporosis), Paget disease (paget disease), periodontal disease (periodontal disease), bone metastatic cancer (metastatic cancer), rheumatoid arthritis (rheumatoid arthritis), bone Bone metastatic cancer, solid cancer bone metastasis, musculoskeletal complications due to solid cancer bone metastasis, hypercalcemia due to malignant tumor, multiple myelosis, primary bone tumor, degenerative arthritis, inflammatory alveolar bone resorption disease and inflammatory bone resorption It is selected from the group consisting of diseases.
  • the above effect is caused by having an effect of inhibiting differentiation of osteoclasts by the extract of Mugwort, and is not particularly limited to the above types of diseases.
  • treatment refers to all actions in which symptoms of bone metabolic diseases are improved or cured by administration of the composition according to the present invention.
  • prevention refers to any action of suppressing or delaying symptoms of bone metabolic disease by administration of the composition according to the present invention.
  • extract as used herein has a meaning commonly used as a crude extract in the art, but broadly includes a fraction obtained by further fractionating the extract. That is, the extract of Mugwort includes not only those obtained by using an extraction solvent, but also those obtained by additionally applying a purification process thereto. For example, fractions obtained by passing the extract through an ultrafiltration membrane having a constant molecular weight cut-off value, separation by various chromatography (sized for separation according to size, charge, hydrophobicity or affinity), etc. Fractions obtained through the purification method are also included in the extract of the present invention.
  • the term "comprising as an active ingredient” means including an amount sufficient to achieve the efficacy or activity of the wormwood extract.
  • the wormwood extract contained in the composition of the present invention is a natural plant material and does not have cytotoxicity.
  • the upper limit of the quantity of the wormwood extract contained in the composition of the present invention can be selected and carried out by a person skilled in the art within an appropriate range.
  • the pharmaceutical composition of the present invention may contain a pharmaceutically acceptable carrier.
  • the pharmaceutically acceptable carrier is commonly used in the formulation, lactose, dextrose, sucrose, sorbitol, mannitol, starch, gum acacia, calcium phosphate, alginate, gelatin, calcium silicate, microcrystalline cellulose, Polyvinylpyrrolidone, cellulose, water, syrup, methyl cellulose, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, mineral oil, and the like, but are not limited thereto.
  • the pharmaceutical composition of the present invention may further include a lubricant, a wetting agent, a sweetening agent, a flavoring agent, an emulsifying agent, a suspending agent, a preservative, and the like in addition to the above components.
  • a lubricant e.g., a talc, a kaolin, a kaolin, a kaolin, a kaolin, a kaolin, kaolin, kaolin, sorbitol, sorbitol, sorbitol, sorbitol, sorbitol, sorbitol, sorbitol, sorbitol, sorbitol, sorbitol, sorbitol, sorbitol, sorbitol, a talct, a talct, a talct, a stea, stevia, glycerin, glycerin, glycerin, g
  • the pharmaceutical composition of the present invention may be administered orally or parenterally, and in the case of parenteral administration, it may be administered by intravenous injection, subcutaneous injection, intramuscular injection, intraperitoneal injection, transdermal administration, or the like.
  • a suitable dosage of the pharmaceutical composition of the present invention varies depending on factors such as formulation method, mode of administration, age, weight, sex, pathological condition, food, administration time, route of administration, excretion rate and response sensitivity of the patient, Usually the skilled practitioner can readily determine and prescribe a dosage effective for the desired treatment or prophylaxis.
  • the daily dosage of the pharmaceutical composition of the present invention is 0.001-1000 mg/kg.
  • the pharmaceutical composition of the present invention is prepared in unit dosage form by formulating using a pharmaceutically acceptable carrier and/or excipient according to a method that can be easily carried out by a person skilled in the art. Or it can be prepared by incorporating it into a multi-dose container.
  • the formulation may be in the form of a solution, suspension or emulsion in an oil or aqueous medium, or may be in the form of an extract, powder, granule, tablet or capsule, and may additionally include a dispersant or a stabilizer.
  • administering means providing a given substance to a subject in any suitable way.
  • the administration route of the composition of the present invention may be administered orally or parenterally through any general route as long as it can reach the target tissue.
  • the composition of the present invention may be administered using any device capable of delivering the active ingredient to target cells, tissues or organs.
  • the term "subject” is not particularly limited, but, for example, human, monkey, cow, horse, sheep, pig, chicken, turkey, quail, cat, dog, mouse, mouse, rabbit, or It includes guinea pigs, and preferably means a mammal, more preferably a human.
  • a polar solvent or a non-polar solvent may be used.
  • Suitable as polar solvents are (i) water, (ii) alcohols (preferably methanol, ethanol, propanol, butanol, normal-propanol, iso-propanol, normal-butanol), (iii) acetic acid, (iv) DMFO. (dimethyl-formamide) and (v) dimethyl sulfoxide (DMSO).
  • Suitable non-polar solvents include acetone, acetonitrile, ethyl acetate, methyl acetate, fluoroalkane, pentane, hexane, 2,2,4-trimethylpentane, decane, cyclohexane, cyclopentane, diisobutylene, 1- Pentene, 1-chlorobutane, 1-chloropentane, o-xylene, diisopropyl ether, 2-chloropropane, toluene, 1-chloropropane, chlorobenzene, benzene, diethyl ether, diethyl sulfide, chloroform, dichloro Methane, 1,2-dichloroethane, aniline, diethylamine, ether, carbon tetrachloride and THF.
  • the Mugwort extract is a Mugwort extract extracted by treating Mugwort with any one extraction solvent selected from the group consisting of water, alcohol, or a mixed solvent thereof.
  • the alcohol is methanol, ethanol, propanol, butanol, normal-propanol, iso-propanol, and normal-butanol.
  • the wormwood extract is an ethanol solution extract.
  • the ethanol solution means an aqueous ethanol solution unless otherwise specified.
  • the concentration of the ethanol solution is 30 to 100% by volume.
  • the concentration of the ethanol solution is 40 to 100% by volume, 50 to 100% by volume, 60 to 100% by volume, 70 to 100% by volume, 80 to 100% by volume, 90 to 100% by volume, 30 to 95% by volume %, 40 to 95 vol%, 50 to 95 vol%, 60 to 95 vol%, 70 to 95 vol%, 80 to 95 vol%, 90 to 95 vol%, 30 to 90 vol%, 40 to 90 vol%, 50 to 90% by volume, 60 to 90% by volume, 70 to 90% by volume, 80 to 90% by volume, 30 to 80% by volume, 40 to 80% by volume, 50 to 80% by volume, 60 to 80% by volume, 70 to 80% by volume, 30 to 70% by volume, 40 to 70% by volume, 50 to 70% by volume, 60 to 70% by volume.
  • the concentration of the ethanol solution is 90 to 100% by volume.
  • the wormwood extract is a hot water extract extracted with hot water at 80 °C to 100 °C. More specifically, the wormwood extract is 70 to 100 °C, 60 to 100 °C, 50 to 100 °C, 80 to 110 °C, 70 to 110 °C, 60 to 110 °C, 50 to 110 °C, 80 to 120 °C, 70 to 120 It is a hot water extract extracted with hot water at °C, 60 to 120 °C, 50 to 120 °C, 80 to 130 °C, 70 to 130 °C, 60 to 130 °C, 50 to 130 °C.
  • the wormwood extract is a hot water extract extracted with hot water for 10 to 20 minutes at 80 °C to 100 °C. More specifically, the wormwood extract is 80 °C to 100 °C for 10 minutes to 25 minutes, 80 °C to 100 °C for 10 minutes to 30 minutes, 80 °C to 100 °C for 5 minutes to 20 minutes, 80 °C to 100 It is a hot water extract extracted with hot water for 5 to 25 minutes at °C and 5 to 30 minutes at 80 to 100 °C.
  • the wormwood extract inhibits the differentiation of osteoclasts.
  • the extract of Mugwort inhibits the activation of NFATc1.
  • activation means that the gene is expressed or expressed to produce a protein of the gene having functionality.
  • the meaning of “inhibiting activation” means that the gene is not expressed, or even if expressed, does not produce a protein of the gene having a function, or the expression level is significantly low, and the expression is not substantially expressed.
  • the wormwood extract inhibits the expression of a gene selected from the group consisting of TRAP, DC-STAMP, and Cathepsin K.
  • the present invention provides a food composition for preventing or improving bone loss caused by bone metabolic disease, containing a wormwood extract as an active ingredient.
  • the food composition of the present invention may be prepared in the form of powder, granules, tablets, capsules or beverages.
  • various foods such as candy, beverages, gum, tea, vitamin complexes, or health supplement foods.
  • the food composition of the present invention may include not only a wormwood extract as an active ingredient, but also ingredients commonly added during food production, and, for example, include proteins, carbohydrates, fats, nutrients, flavoring agents, and flavoring agents.
  • examples of the aforementioned carbohydrates include monosaccharides such as glucose, fructose, and the like; Disaccharides such as maltose, sucrose, oligosaccharides, and the like; And polysaccharides, for example, common sugars such as dextrin and cyclodextrin, and sugar alcohols such as xylitol, sorbitol, and erythritol.
  • natural flavoring agents [taumatin, stevia extract (eg, rebaudioside A, glycyrrhizin, etc.]) and synthetic flavoring agents (saccharin, aspartame, etc.) can be used.
  • synthetic flavoring agents sacharin, aspartame, etc.
  • the food composition of the present invention when it is prepared as a drink, it may further include citric acid, liquid fructose, sugar, glucose, acetic acid, malic acid, fruit juice, cephalic extract, jujube extract, licorice extract, etc. in addition to the wormwood extract of the present invention. have.
  • the wormwood extract contained in the composition of the present invention is a natural plant material and has been used for a long time as an edible and folk medicine, so it can be expected that the extract of the present invention extracted therefrom also has no problems such as toxicity and side effects. Therefore, the wormwood extract can be used not only as a pharmaceutical, food composition, but also can be developed as an animal medicine and functional feed.
  • the contents of the extract of the wormwood extract included in the food composition of the present invention are the same as the contents of the extract of the wormwood extract included in the pharmaceutical composition, the contents in common between the two are to avoid excessive complexity of the present specification. , The description is omitted.
  • Mugwort extract exhibits the efficacy of preventing or treating bone loss caused by bone metabolic diseases by inhibiting the differentiation of osteoclasts and the activity of NFATc1.
  • a pharmaceutical composition for preventing or treating bone loss caused by bone metabolic diseases that can be safely used in the human body without toxicity and side effects, or for preventing or improving bone loss caused by bone metabolic diseases
  • a food composition can be provided.
  • Figure 2 shows the results of observation under a microscope for the effect of inhibiting osteoclast differentiation of the ethanol extract of Mugwort in mouse macrophages.
  • Figure 3a is a graph showing the inhibitory effect on the expression of the osteoclast differentiation-related gene TRAP of the extract of hot water in mouse macrophages.
  • Figure 3b is a graph showing the inhibitory effect on the expression of the osteoclast differentiation-related gene Cathesin K of the extract of hot water in mouse macrophages.
  • Figure 3c is a graph showing the inhibitory effect on the expression of the osteoclast differentiation-related gene DC-STAMP of the extract of hot water in mouse macrophages.
  • Figure 3d is a graph showing the inhibitory effect on the expression of the osteoclast differentiation-related gene NFATc1 of the extract of hot water in mouse macrophages.
  • Figure 4a is a graph showing the inhibitory effect on the expression of the osteoclast differentiation-related gene TRAP of the ethanol extract of wormwood in mouse macrophages.
  • Figure 4b is a graph showing the inhibitory effect on the expression of the osteoclast differentiation-related gene Cathesin K of the ethanol extract of wormwood in mouse macrophages.
  • Figure 4c is a graph showing the inhibitory effect on the expression of the osteoclast differentiation-related gene DC-STAMP of the ethanol extract of wormwood in mouse macrophages.
  • Figure 4d is a graph showing the inhibitory effect on the expression of the osteoclast differentiation-related gene NFATc1 of the ethanol extract of Mugwort in mouse macrophages.
  • a pharmaceutical composition for the prevention or treatment of bone loss caused by bone metabolic diseases comprising a wormwood extract as an active ingredient.
  • % used to indicate the concentration of a specific substance is (weight/weight)% for solids/solids, (weight/volume)% for solids/liquids, and Liquid/liquid is (vol/vol) %.
  • Example 1 Method for preparing a wormwood extract
  • the wormwood above-ground part was collected and used in Jungdo-myeon, Sinan-gun, Jeollanam-do, and prepared wormwood extract through the manufacturing methods described in Examples 1-1 and 1-2.
  • Example 2 Experiment on the effect of preventing or treating bone disease using hot water and ethanol extract
  • Example 2-1 Isolation and culture of bone marrow-derived macrophages (BMDMs, bone marrow-derived macrophages)
  • M-CSF Macrophage-colony
  • FBS Fetal bovine serum
  • BMDMs Bone marrow-derived macrophages
  • the bone marrow-derived macrophages were cultured in a 12 well-plate at a density of 2 ⁇ 10 5 cells/well for 24 hours.
  • Mouse macrophages were incubated in a 12 well plate at a density of 2x10 5 cells/well in a medium containing 10% FBS, 1% PS, and M-CSF (25 ng/ml) for 24 hours, and then replaced with a medium of the same composition. After doing, hot water and ethanol extract were treated at concentrations of 6.125, 12.5, 25, and 50 ⁇ g/ml, respectively, for 2 hours. Then, RANKL was treated and maintained for 24 hours. Cells were differentiated for 4 days in the same way as above.
  • the nucleus was stained by adding a chromogenic substrate to the cytochemical labeling enzyme of osteoclast cells, TRAP (Tartrate resistance acid phosphatase), and cells with three or more nuclei multinucleated were observed and quantified.
  • TRAP Sterate resistance acid phosphatase
  • the macrophages When the macrophages are treated with RANKL, they bind to RANK and differentiate into TRAP-positive cells. When these TRAP-positive cells are stimulated with inflammatory factors such as RANKL and TNF- ⁇ , the cells are fused to each other and differentiate into multinuclear TRAP-positive cells.
  • Example 2-3 Evaluation of gene expression inhibition on osteoclast differentiation of Mugwort extract (Real-time PCR)
  • Mouse macrophages were cultured for 24 hours in a medium containing 10% FBS, 1% PS, and M-CSF (25 ng/ml) at a density of 2x10 5 cells/well in a 12 well plate, and then replaced with a medium of the same composition. After, 50 ⁇ g/ml of hot water extract and 20 ⁇ g/ml of ethanol extract were treated for 2 hours. Then, RANKL was treated and reacted for 24 hours.
  • RNA was isolated using Easy-blue solution, and cDNA was synthesized using RT premix based on the RNA quantification value.
  • the synthesized cDNA was amplified through real-time PCR using primers.
  • the primers used were mouse TRAP, Cathesin K, DC-STAMP, NFATc1 (Table 1), and the relative amounts were compared with the control gene GAPDH.
  • the macrophages When the macrophages are treated with RANKL, the macrophages bind to RANK and differentiate into TRAP-positive multinucleated osteoclasts. In addition, macrophages activated NFATc1, an important transcription factor for differentiation into osteoclasts, and increased expression of TRAP, Cathepsine K, and DC-STAMP, which are involved in osteoclastogenesis.
  • the above ingredients are mixed and filled in an airtight cloth to prepare a powder.
  • tablets are prepared by tableting according to a conventional tablet manufacturing method.
  • the above ingredients are mixed and filled into gelatin capsules to prepare a capsule.
  • Vitamin A acetate 70 ⁇ g
  • Vitamin B6 0.5 mg
  • Vitamin B12 0.2 ⁇ g
  • composition ratio of the vitamin and mineral mixture is relatively suitable for health food, but it may be arbitrarily modified, and the above ingredients are mixed according to a conventional health food manufacturing method.
  • Granules can be prepared, and used for preparing health food compositions according to conventional methods.
  • the resulting solution is filtered and obtained in a sterilized 2 L container, sealed and sterilized, and then stored in a refrigerator. It is used in the manufacture of the health beverage composition of the invention.
  • composition ratio is a mixture of ingredients suitable for a relatively preferred beverage in a preferred embodiment, but the mixing ratio may be arbitrarily modified according to regional and ethnic preferences such as the demand class, the country of demand, and the purpose of use.
  • the present invention relates to a pharmaceutical composition for the prevention or treatment of bone loss caused by bone metabolic disease, comprising the extract of Mugwort as an active ingredient.

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Abstract

The present invention relates to a pharmaceutical composition, for preventing or treating bone loss induced by metabolic bone diseases, comprising an Artemisia scoparia extract as an active ingredient. The Artemisia scoparia extract exhibits efficacy for preventing or treating bone loss induced by metabolic bone diseases by inhibiting osteoclast differentiation and NFATc1 activation. Due to the efficacy, provided is a pharmaceutical composition, for preventing or treating bone loss induced by metabolic bone diseases, or a food composition, for preventing or relieving bone loss induced by metabolic bone diseases, which can be safely used for the human body without toxicity and side effects when using the Artemisia scoparia extract of the present invention.

Description

비쑥 추출물을 유효성분으로 포함하는 골대사성 질환으로 유발된 골소실 예방 또는 치료용 약제학적 조성물Pharmaceutical composition for the prevention or treatment of bone loss caused by bone metabolic diseases comprising a wormwood extract as an active ingredient
본 발명은 비쑥 추출물을 유효성분으로 포함하는 골대사성 질환으로 유발된 골소실 예방 또는 치료용 약제학적 조성물에 관한 것이다.The present invention relates to a pharmaceutical composition for the prevention or treatment of bone loss caused by bone metabolic disease, comprising the extract of Mugwort as an active ingredient.
뼈는 골세포 (osteocyte), 파골세포 (osteoclast), 조골세포 (osteoblast)와 같은 뼈세포 (bone cell), 수산화인회석 (hydroxyapatite crystal), 교원질 섬유 (collagenous fibers), 글리코스아미노글리칸 (glycosaminoglycans)과 같은 뼈 기질 (bone matrix) 및 골수의 공동 (bone marrow cavity), 혈관 (vascular canals), 소관 (canaliculi), 골소강 (lacunae) 과 같은 공간으로 구성되어 있다. 뼈는 몸을 기계적으로 지지하고, 중요장기를 보호하며, 조혈작용 (hemopoeisis)에 필요한 미세 환경을 제공하고, 칼슘 및 여러 미네랄을 저장하는 역할을 한다.Bone is bone cells such as osteocytes, osteoclasts, and osteoblasts, hydroxyapatite crystals, collagen fibers, and glycosaminoglycans. The bone matrix and the bone marrow cavity, blood vessels (vascular canals), canaliculi, and bone marrow cavity (lacunae) are composed of the same space. Bone mechanically supports the body, protects vital organs, provides a microenvironment necessary for hematopoiesis, and stores calcium and various minerals.
뼈의 성장, 발달 및 유지는 일생을 거쳐 연속적으로 일어난다. 노화된 뼈는 파괴되고 이를 대신하여 새로운 뼈가 재형성(regeneration)된다. 이러한 교체(turnover)는 파골세포와 조골세포로 구성된 BMU(Basic Multicellular Units)에서 주로 일어나는데, 이 과정은 성장과 스트레스로 인한 뼈의 미세한 손상을 회복시키고 뼈의 기능을 유지하게 하는 역할을 한다. 노화된 뼈의 파괴 또는 흡수는 파골세포가 수행한다. 반면, 새로운 뼈의 형성은 조골세포가 담당한다. 파골세포는 뼈의 표면에 부착하여 산과 분해효소를 분비함으로서 뼈를 구성하는 인회석 결정 및 교원질과 같은 뼈 기질 (bone matrix)을 제거하여 뼈를 파괴하고, 조골세포는 뼈 기질을 합성하여 분비하고 칼슘과 인의 농도를 조절하여 골격을 형성한다.Bone growth, development, and maintenance occur continuously throughout life. Aged bones are destroyed and new bones are regenerated in place. This turnover occurs mainly in BMU (Basic Multicellular Units) composed of osteoclasts and osteoblasts, and this process plays a role in restoring microscopic damage to bones caused by growth and stress and maintaining bone function. The destruction or absorption of aged bone is performed by osteoclasts. On the other hand, osteoblasts are responsible for the formation of new bones. The osteoclasts attach to the surface of the bones and secrete acid and degrading enzymes, thereby destroying the bone by removing the bone matrix such as apatite crystals and collagen, and the osteoblasts synthesize and secrete the bone matrix. It forms a skeleton by controlling the concentration of and phosphorus.
골대사성 질환은 생체 내에서 파골세포와 조골세포의 평형이 깨짐으로써 발생한다. 골대사성 질환의 대표적인 예로는 골다공증을 들 수 있다.Bone metabolic disease occurs when the balance between osteoclasts and osteoblasts is broken in vivo. A representative example of a bone metabolic disease is osteoporosis.
골다공증(osteoporosis)은 여러 가지 원인에 의하여 뼈의 질량이 감소하고 뼈 조직의 미세구조의 퇴화로 골절 위험이 지속적으로 증가하는 질환이다. 특히， 골다공증은 뼈를 구성하는 미네랄(특히 칼슘)과 기질이 감소한 상태이며， 골재형성의 균형이 깨져서 파골작용이 조골작용보다 증가된 상태에서 발생한다.Osteoporosis (osteoporosis) is a disease in which the bone mass decreases due to various causes and the risk of fracture continues to increase due to the deterioration of the microstructure of the bone tissue. In particular, osteoporosis is a state in which minerals (especially calcium) and substrates that make up bones are reduced, and the balance of bone remodeling is broken, resulting in a state in which the osteoclast action is increased than the osteoblast action.
정상적인 뼈 내부는 그물망처럼 치밀한 구조를 이루고 있으나， 골다공증의 경우에는 구조 사이의 간격이 넓어지고 미세구조가 얇아져 약해짐으로써 조그만 충격에도 뼈가 쉽게 골절될 수 있는 상태로 진행된다.Normal bones have a dense structure like a mesh, but in the case of osteoporosis, the gap between the structures becomes wider and the microstructures become thinner and weaken, so that the bones can easily be fractured even with a small impact.
골다공증은 크게 일차성 골다공증(primary osteoporosis)과 이차성 골다공증 (secondary osteoporosis)으로 분류된다. 일차성 골다공증은 폐경 후 골다공증 (postmenopausal osteoporosis), 노인성 골다공증 (age-related osteoporosis), 및 특발성 골다공증 (idiopathic osteoporosis)을 포함한다. 이차성 골다공증(secondary osteoporosis)은 연령에 상관없이 질병이나 약물로 인해 발생한다.Osteoporosis is largely classified into primary osteoporosis and secondary osteoporosis. Primary osteoporosis includes postmenopausal osteoporosis, age-related osteoporosis, and idiopathic osteoporosis. Secondary osteoporosis is caused by diseases or drugs regardless of age.
갱년기의 여성에는 여러 폐경 증후들이 나타나는데， 특히 에스트로겐의 감소로 인한 뼈에서 칼슘이 빠져나가 뼈의 질량이 감소하고 골 손실의 증가 등으로 골다공증의 발병률이 높아지게 된다. 최근 보건복지부 국민 건강 통계에 따르면 30세 이상에서 골다공증 유병률이 남성은 1 %, 여성은 9 %로 나타나， 여성 유병률이 남성 대비 9 배나 높게 나타났다.In menopausal women, several menopause symptoms appear. In particular, the incidence of osteoporosis increases due to the loss of calcium from the bones due to a decrease in estrogen, resulting in a decrease in bone mass and an increase in bone loss. According to recent national health statistics from the Ministry of Health and Welfare, the prevalence of osteoporosis in men over 30 years old was 1% for men and 9% for women, and the prevalence of women was 9 times higher than that of men.
한편， 다양한 암 환자에게서 골전이가 일어난다. 그 중, 유방암 환자에서 관찰되는 골 전이는 대부분 뼈를 파괴하는 골 용해성 골 전이(osteolytic metastasis)이다. 골 용해성 골 전이는 유방암 세포가 뼈에 직접적인 영향을 미치는 것이 아니라 파골 세포를 자극함으로써 일어나는 것으로 알려져 있다.Meanwhile, bone metastasis occurs in various cancer patients. Among them, bone metastasis observed in breast cancer patients is osteolytic metastasis, which destroys most of the bones. Osteolytic bone metastasis is known to occur by stimulating osteoclasts rather than by breast cancer cells directly affecting bones.
위와 같은 골 질환을 치료하기 위해 천연물로부터 질병을 예방 또는 억제할 수 있는 생리활성 물질에 대한 연구가 진행되고 있다. 특히 생리활성물질의 보고로 알려진 해양 식물의 탐색에 관한 연구 및 해조류로부터 질병을 억제하거나 개선시킬 수 있는 소재를 찾으려는 연구가 활발히 진행되고 있다.In order to treat the above bone diseases, studies on physiologically active substances that can prevent or suppress diseases from natural products are being conducted. In particular, studies on the search for marine plants known as reports of physiologically active substances and studies to find materials that can suppress or improve diseases from seaweed are actively being conducted.
비쑥(Artemisia scoparia)은 바닷가 모래땅에서 자라는 다년초로서 사철쑥과 닮았으나 냄새가 다르다. 사철쑥은 줄기가 나무처럼 되어 있어 겨울철 에도 죽지 않지만 비쑥은 겨울철에 줄기가 완전히 말라 죽는 것이 특징이다. 비쑥은 한방에서는 거의 쓰지 않는 희귀한 풀이며， 인진쑥 및 큰비쑥 둥과 같은 종류의 쑥에 비해 연구가 거의 이루어지지 않았다.Artemisia scoparia is a perennial plant that grows in sandy beaches and resembles wormwood, but has a different smell. Mugwort does not die even in winter because its stem is like a tree, but wormwood is characterized by the fact that the stem dries completely in winter. Mugwort is a rare herb that is rarely used in oriental medicine, and little research has been conducted compared to the types of mugwort such as injinsuk and large Mugwort.
본 발명자들은 골대사성 질환으로 유발된 골소실에 유효한 천연물 소재를 개발하고자 예의 연구 노력하였다. 그 결과, 비쑥 추출물이 파골세포의 분화 및 NFATc1의 활성을 억제하여 골대사성 질환으로 유발된 골소실에 대한 예방 또는 치료 효과를 규명함으로써, 본 발명을 완성하게 되었다. The present inventors have made intensive research efforts to develop natural materials that are effective for bone loss caused by bone metabolic diseases. As a result, by examining the effect of preventing or treating bone loss caused by bone metabolic disease by inhibiting the differentiation of osteoclasts and the activity of NFATc1, the present invention was completed.
따라서, 본 발명의 목적은 비쑥 추출물을 유효성분으로 포함하는 골대사성 질환으로 유발된 골소실 예방 또는 치료용 약제학적 조성물을 제공하는 것이다. Accordingly, it is an object of the present invention to provide a pharmaceutical composition for preventing or treating bone loss caused by bone metabolic disease, comprising a wormwood extract as an active ingredient.
본 발명의 다른 목적은 비쑥 추출물을 유효성분으로 함유하는 골대사성 질환으로 유발된 골소실 예방 또는 개선용 식품 조성물을 제공하는 것이다.Another object of the present invention is to provide a food composition for preventing or improving bone loss caused by bone metabolic diseases, containing a wormwood extract as an active ingredient.
본 발명의 일 양태에 따르면, 본 발명은 비쑥 추출물을 유효성분으로 포함하는 골대사성 질환으로 유발된 골소실 예방 또는 치료용 약제학적 조성물을 제공한다.According to one aspect of the present invention, the present invention provides a pharmaceutical composition for preventing or treating bone loss caused by bone metabolic disease, comprising a wormwood extract as an active ingredient.
비쑥(Artemisia scoparia)은 국화과(Compositae)의 쑥속(Artemisia)에 속하는 다년초 식물로서, 바닷가 모래땅에서 자라는 다년초로서 사철쑥과 닮았으나 냄새가 다르다. 사철쑥은 줄기가 나무처럼 되어 있어 겨울철에도 죽지 않지만 비쑥은 겨울철에 줄기가 완전히 말라 죽는 것이 특징이다. 비쑥은 한방에서는 거의 쓰지 않는 희귀한 풀이다.Artemisia scoparia is a perennial plant belonging to Artemisia of the family Compositae. It is a perennial plant that grows in sandy beaches and resembles wormwood, but has a different smell. Mugwort does not die even in winter because its stem is like a tree, but wormwood is characterized by the fact that the stem dries completely in winter. Mugwort is a rare herb that is rarely used in oriental medicine.
파골세포는 조혈모세포의 단핵구로부터 유래되는 세포로서, 마우스의 RAW264.7 단핵구 세포는 RANKL(receptoractivator of nuclear factor κ(RANK) ligand)에 의해 융합하여 다핵 파골세포(multinucleated osteoclasts)로 분화된다. 이러한 분화 과정은 세포 외부의 RANKL이 RANK에 결합하여 미토겐 활성 단백질 키나아제(mitogen-activated protein kinase, MAPK)의 활성을 촉진하고, 이는 NF-κ라는 전사인자가 핵 내로 들어가서 파골세포 분화와 관련된 TRAP(tartrate-resistant acid phosphatase), MMP-9(matrix metalloproteinase-9), c-Src 티로신 키나아제(tyrosine kinase) 등의 발현을 증가시킴으로써 가능한데, 이러한 과정으로 형성된 다핵 파골세포는 무기질 골(mineralized bone)을 흡수할 수 있다. 또한, RANKL이 RANK에 결합하면 TRAF6(tumor necrosis factor receptorassociated factor 6)의 활성을 촉진시켜 MAPK, 또는 NF-κAP-1, NFATc1과 같은 전사인자들의 활성을 촉진시킨다. 특히, 파골세포의 생존 및 골 흡수 능력에서 중요한 역할을 하는 신호전달 분자로 밝혀진 ERK와 NF-κ에 의해 파골세포 분화에 특이적인 AP-1 또는 NFAT가 조절될 수 있다고 보고되어 있다. 따라서, RANKL에 의해 활성화되는 신호전달 경로의 차단은 골다공증을 비롯한 골 질환의 치료를 위한 치료적 접근 방법 중의 하나로 인지되고 있다.Osteoclasts are cells derived from mononuclear cells of hematopoietic stem cells, and RAW264.7 monocytes from mice are fused by RANKL (receptoractivator of nuclear factor κ (RANK) ligand) to differentiate into multinucleated osteoclasts. In this differentiation process, RANKL outside the cell binds to RANK and promotes the activity of mitogen-activated protein kinase (MAPK), which is a transcription factor called NF-κ that enters the nucleus, which is a TRAP related to osteoclast differentiation. (tartrate-resistant acid phosphatase), matrix metalloproteinase-9 (MMP-9), c-Src tyrosine kinase, etc. Can be absorbed. In addition, when RANKL binds to RANK, it promotes the activity of TRAF6 (tumor necrosis factor receptor associated factor 6), thereby promoting the activity of transcription factors such as MAPK, NF-κAP-1, and NFATc1. In particular, it has been reported that AP-1 or NFAT specific for osteoclast differentiation can be regulated by ERK and NF-κ, which have been identified as signaling molecules that play an important role in the survival and bone resorption of osteoclasts. Therefore, blocking of the signaling pathway activated by RANKL is recognized as one of the therapeutic approaches for the treatment of bone diseases including osteoporosis.
본 발명의 일 구현예에 있어서, 상기 골대사성 질환은 골다공증(osteoporosis), 파제트병(paget disease), 치주질환(periodontal disease), 골전이암(metastatic cancer), 류마티스 관절염(rheumatoid arthritis), 뼈전이암(bone metastatic cancer), 고형암 뼈전이, 고형암 뼈전이에 의한 근골격 합병증, 악성종양으로 인한 과칼슘혈증, 다발성 골수증, 원발성(primary) 뼈종양, 퇴행성 관절염, 염증성 치조골 흡수 질환 및 염증성 뼈흡수 질환으로 이루어진 군으로부터 선택된다.In one embodiment of the present invention, the bone metabolic disease is osteoporosis (osteoporosis), Paget disease (paget disease), periodontal disease (periodontal disease), bone metastatic cancer (metastatic cancer), rheumatoid arthritis (rheumatoid arthritis), bone Bone metastatic cancer, solid cancer bone metastasis, musculoskeletal complications due to solid cancer bone metastasis, hypercalcemia due to malignant tumor, multiple myelosis, primary bone tumor, degenerative arthritis, inflammatory alveolar bone resorption disease and inflammatory bone resorption It is selected from the group consisting of diseases.
상기 효과는 비쑥 추출물이 파골세포 분화 억제 효과를 가짐으로써 발생하는 것으로 특별히 상기 종류의 질환에만 한정되는 것은 아니다.The above effect is caused by having an effect of inhibiting differentiation of osteoclasts by the extract of Mugwort, and is not particularly limited to the above types of diseases.
본 명세서에서 용어 "치료"는 본 발명에 따른 조성물의 투여로 골대사성 질환의 증상이 호전되거나 완치되는 모든 행위를 의미한다. As used herein, the term "treatment" refers to all actions in which symptoms of bone metabolic diseases are improved or cured by administration of the composition according to the present invention.
또한, 본 명세서에서 용어 "예방"은 본 발명에 따른 조성물의 투여로 골대사성 질환의 증상을 억제 또는 지연시키는 모든 행위를 의미한다.In addition, the term "prevention" as used herein refers to any action of suppressing or delaying symptoms of bone metabolic disease by administration of the composition according to the present invention.
본 명세서에서 사용되는 용어 "추출물"은 당업계에서 조추출물(crude extract)로 통용되는 의미를 갖지만, 광의적으로는 추출물을 추가적으로 분획(fractionation)한 분획물도 포함한다. 즉, 비쑥 추출물은 추출용매를 이용하여 얻은 것뿐만 아니라, 여기에 정제과정을 추가적으로 적용하여 얻은 것도 포함한다. 예컨대, 상기 추출물을 일정한 분자량 컷-오프 값을 갖는 한외 여과막을 통과시켜 얻은 분획, 다양한 크로마토그래피 (크기, 전하, 소수성 또는 친화성에 따른 분리를 위해 제작된 것)에 의한 분리 등, 추가적으로 실시된 다양한 정제 방법을 통해 얻어진 분획도 본 발명의 비쑥 추출물에 포함되는 것이다.The term "extract" as used herein has a meaning commonly used as a crude extract in the art, but broadly includes a fraction obtained by further fractionating the extract. That is, the extract of Mugwort includes not only those obtained by using an extraction solvent, but also those obtained by additionally applying a purification process thereto. For example, fractions obtained by passing the extract through an ultrafiltration membrane having a constant molecular weight cut-off value, separation by various chromatography (sized for separation according to size, charge, hydrophobicity or affinity), etc. Fractions obtained through the purification method are also included in the extract of the present invention.
본 명세서에서 용어 "유효성분으로 포함하는"이란, 비쑥 추출물의 효능 또는 활성을 달성하는 데 충분한 양을 포함하는 것을 의미한다. As used herein, the term "comprising as an active ingredient" means including an amount sufficient to achieve the efficacy or activity of the wormwood extract.
본 발명의 조성물에 포함된 비쑥 추출물은 천연식물재료로서 세포 독성이 없다. 본 발명의 조성물에 포함된 비쑥 추출물의 양적 상한은 당업자가 적절한 범위 내에서 선택하여 실시할 수 있다.The wormwood extract contained in the composition of the present invention is a natural plant material and does not have cytotoxicity. The upper limit of the quantity of the wormwood extract contained in the composition of the present invention can be selected and carried out by a person skilled in the art within an appropriate range.
본 발명의 약제학적 조성물은 약제학적으로 허용되는 담체를 포함할 수 있다. 상기 약제학적으로 허용되는 담체는 제제시에 통상적으로 이용되는 것으로서, 락토스, 덱스트로스, 수크로스, 솔비톨, 만니톨, 전분, 아카시아 고무, 인산 칼슘, 알기네이트, 젤라틴, 규산 칼슘, 미세결정성 셀룰로스, 폴리비닐피롤리돈, 셀룰로스, 물, 시럽, 메틸 셀룰로스, 메틸히드록시벤조에이트, 프로필히드록시벤조에이트, 활석, 스테아르산 마그네슘 및 미네랄 오일 등을 포함하나, 이에 한정되는 것은 아니다. 본 발명의 약제학적 조성물은 상기 성분들 이외에 윤활제, 습윤제, 감미제, 향미제, 유화제, 현탁제, 보존제 등을 추가로 포함할 수 있다. 적합한 약제학적으로 허용되는 담체 및 제제는 Remington's Pharmaceutical Sciences (19th ed., 1995)에 상세히 기재되어 있다.The pharmaceutical composition of the present invention may contain a pharmaceutically acceptable carrier. The pharmaceutically acceptable carrier is commonly used in the formulation, lactose, dextrose, sucrose, sorbitol, mannitol, starch, gum acacia, calcium phosphate, alginate, gelatin, calcium silicate, microcrystalline cellulose, Polyvinylpyrrolidone, cellulose, water, syrup, methyl cellulose, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, mineral oil, and the like, but are not limited thereto. The pharmaceutical composition of the present invention may further include a lubricant, a wetting agent, a sweetening agent, a flavoring agent, an emulsifying agent, a suspending agent, a preservative, and the like in addition to the above components. Suitable pharmaceutically acceptable carriers and formulations are described in detail in Remington's Pharmaceutical Sciences (19th ed., 1995).
본 발명의 약제학적 조성물은 경구 또는 비경구로 투여할 수 있고, 비경구 투여인 경우에는 정맥내 주입, 피하 주입, 근육 주입, 복강 주입, 경피 투여 등으로 투여할 수 있다.The pharmaceutical composition of the present invention may be administered orally or parenterally, and in the case of parenteral administration, it may be administered by intravenous injection, subcutaneous injection, intramuscular injection, intraperitoneal injection, transdermal administration, or the like.
본 발명의 약제학적 조성물의 적합한 투여량은 제제화 방법, 투여방식, 환자의 연령, 체중, 성, 병적 상태, 음식, 투여 시간, 투여 경로, 배설 속도 및 반응 감응성과 같은 요인들에 의해 다양하며, 보통으로 숙련된 의사는 소망하는 치료 또는 예방에 효과적인 투여량을 용이하게 결정 및 처방할 수 있다. 본 발명의 바람직한 구현예에 따르면, 본 발명의 약제학적 조성물의 1일 투여량은 0.001-1000 ㎎/㎏이다.A suitable dosage of the pharmaceutical composition of the present invention varies depending on factors such as formulation method, mode of administration, age, weight, sex, pathological condition, food, administration time, route of administration, excretion rate and response sensitivity of the patient, Usually the skilled practitioner can readily determine and prescribe a dosage effective for the desired treatment or prophylaxis. According to a preferred embodiment of the present invention, the daily dosage of the pharmaceutical composition of the present invention is 0.001-1000 mg/kg.
본 발명의 약제학적 조성물은 당해 발명이 속하는 기술분야에서 통상의 지식을 가진 자가 용이하게 실시할 수 있는 방법에 따라, 약제학적으로 허용되는 담체 및/또는 부형제를 이용하여 제제화 함으로써 단위 용량 형태로 제조되거나 또는 다용량 용기 내에 내입시켜 제조될 수 있다. 이때 제형은 오일 또는 수성 매질중의 용액, 현탁액 또는 유화액 형태이거나 엑스제, 분말제, 과립제, 정제 또는 캅셀제 형태일 수도 있으며, 분산제 또는 안정화제를 추가적으로 포함할 수 있다.The pharmaceutical composition of the present invention is prepared in unit dosage form by formulating using a pharmaceutically acceptable carrier and/or excipient according to a method that can be easily carried out by a person skilled in the art. Or it can be prepared by incorporating it into a multi-dose container. In this case, the formulation may be in the form of a solution, suspension or emulsion in an oil or aqueous medium, or may be in the form of an extract, powder, granule, tablet or capsule, and may additionally include a dispersant or a stabilizer.
본 명세서에서 용어 "투여"는 임의의 적절한 방법으로 대상(subject)에게 소정의 물질을 제공하는 것을 의미한다. 본 발명의 조성물의 투여경로는 목적 조직에 도달할 수 있는 한 일반적인 모든 경로를 통하여 경구 또는 비 경구 투여될 수 있다. 또한, 본 발명의 조성물은 유효성분을 표적 세포, 조직 또는 기관으로 전달할 수 있는 임의의 장치를 이용해 투여될 수도 있다.As used herein, the term "administering" means providing a given substance to a subject in any suitable way. The administration route of the composition of the present invention may be administered orally or parenterally through any general route as long as it can reach the target tissue. In addition, the composition of the present invention may be administered using any device capable of delivering the active ingredient to target cells, tissues or organs.
본 명세서에서 용어 "대상(subject)"은, 특별히 한정되는 것은 아니지만, 예를 들어, 인간, 원숭이, 소, 말, 양, 돼지, 닭, 칠면조, 메추라기, 고양이, 개, 마우스, 쥐, 토끼 또는 기니아 피그를 포함하고, 바람직하게는 포유류, 보다 바람직하게는 인간을 의미한다.In the present specification, the term "subject" is not particularly limited, but, for example, human, monkey, cow, horse, sheep, pig, chicken, turkey, quail, cat, dog, mouse, mouse, rabbit, or It includes guinea pigs, and preferably means a mammal, more preferably a human.
본 발명의 조성물에서 이용되는 비쑥 추출물은 비쑥에 추출용매를 처리하여 수득하는 경우에는, 다양한 추출용매가 이용될 수 있다. 바람직하게는, 극성용매 또는 비극성 용매를 이용할 수 있다. 극성 용매로서 적합한 것은, (i) 물, (ii) 알코올(바람직하게는, 메탄올, 에탄올, 프로판올, 부탄올, 노말-프로판올, 이소-프로판올, 노말-부탄올), (iii) 아세트산, (iv) DMFO(dimethyl-formamide) 및 (v) DMSO(dimethyl sulfoxide)를 포함한다. When the wormwood extract used in the composition of the present invention is obtained by treating the wormwood extract with an extraction solvent, various extraction solvents may be used. Preferably, a polar solvent or a non-polar solvent may be used. Suitable as polar solvents are (i) water, (ii) alcohols (preferably methanol, ethanol, propanol, butanol, normal-propanol, iso-propanol, normal-butanol), (iii) acetic acid, (iv) DMFO. (dimethyl-formamide) and (v) dimethyl sulfoxide (DMSO).
비극성 용매로서 적합한 것은, 아세톤, 아세토나이트릴, 에틸 아세테이트, 메틸 아세테이트, 플루오로알칸, 펜탄, 헥산, 2,2,4-트리메틸펜탄, 데칸, 사이클로헥산, 사이클로펜탄, 디이소부틸렌, 1-펜텐, 1-클로로부탄, 1-클로로펜탄, o-자일렌, 디이소프로필 에테르, 2-클로로프로판, 톨루엔, 1-클로로프로판, 클로로벤젠, 벤젠, 디에틸 에테르, 디에틸 설파이드, 클로로포름, 디클로로메탄, 1,2-디클로로에탄, 어닐린, 디에틸아민, 에테르, 사염화탄소 및 THF를 포함한다.Suitable non-polar solvents include acetone, acetonitrile, ethyl acetate, methyl acetate, fluoroalkane, pentane, hexane, 2,2,4-trimethylpentane, decane, cyclohexane, cyclopentane, diisobutylene, 1- Pentene, 1-chlorobutane, 1-chloropentane, o-xylene, diisopropyl ether, 2-chloropropane, toluene, 1-chloropropane, chlorobenzene, benzene, diethyl ether, diethyl sulfide, chloroform, dichloro Methane, 1,2-dichloroethane, aniline, diethylamine, ether, carbon tetrachloride and THF.
본 발명에서 비쑥 추출시, 열수 추출법, 냉침 추출법, 환류추출법, 상온 추출법, 또는 초음파 추출법을 사용할 수 있다.In the present invention, when extracting wormwood, hot water extraction, cold needle extraction, reflux extraction, room temperature extraction, or ultrasonic extraction may be used.
본 발명의 일 구현예에 있어서, 상기 비쑥 추출물은 물, 알코올 또는 이들의 혼합용매로 구성된 군으로부터 선택되는 어느 하나의 추출 용매를 비쑥에 처리하여 추출한 비쑥 추출물이다.In one embodiment of the present invention, the Mugwort extract is a Mugwort extract extracted by treating Mugwort with any one extraction solvent selected from the group consisting of water, alcohol, or a mixed solvent thereof.
본 발명의 일 구체예에 있어서, 상기 알코올은 메탄올, 에탄올, 프로판올, 부탄올, 노말-프로판올, 이소-프로판올, 노말-부탄올이다. 본 발명의 일 구체예에 있어서, 상기 비쑥 추출물은 에탄올 용액 추출물이다. 상기 에탄올 용액은 특별한 언급이 없는 한 에탄올 수용액을 의미한다. In one embodiment of the present invention, the alcohol is methanol, ethanol, propanol, butanol, normal-propanol, iso-propanol, and normal-butanol. In one embodiment of the present invention, the wormwood extract is an ethanol solution extract. The ethanol solution means an aqueous ethanol solution unless otherwise specified.
본 발명의 일 구체예에 있어서, 상기 에탄올 용액의 농도는 30 내지 100 부피%이다.In one embodiment of the present invention, the concentration of the ethanol solution is 30 to 100% by volume.
보다 구체적으로는 상기 에탄올 용액의 농도는 40 내지 100 부피%, 50 내지 100 부피%, 60 내지 100 부피%, 70 내지 100 부피%, 80 내지 100 부피%, 90 내지 100 부피%, 30 내지 95 부피%, 40 내지 95 부피%, 50 내지 95 부피%, 60 내지 95 부피%, 70 내지 95 부피%, 80 내지 95 부피%, 90 내지 95 부피%, 30 내지 90 부피%, 40 내지 90 부피%, 50 내지 90 부피%, 60 내지 90 부피%, 70 내지 90 부피%, 80 내지 90 부피%, 30 내지 80 부피%, 40 내지 80 부피%, 50 내지 80 부피%, 60 내지 80 부피%, 70 내지 80 부피%, 30 내지 70 부피%, 40 내지 70 부피%, 50 내지 70 부피%, 60 내지 70 부피%이다. 바람직하게는 상기 에탄올 용액의 농도는 90 내지 100 부피%이다.More specifically, the concentration of the ethanol solution is 40 to 100% by volume, 50 to 100% by volume, 60 to 100% by volume, 70 to 100% by volume, 80 to 100% by volume, 90 to 100% by volume, 30 to 95% by volume %, 40 to 95 vol%, 50 to 95 vol%, 60 to 95 vol%, 70 to 95 vol%, 80 to 95 vol%, 90 to 95 vol%, 30 to 90 vol%, 40 to 90 vol%, 50 to 90% by volume, 60 to 90% by volume, 70 to 90% by volume, 80 to 90% by volume, 30 to 80% by volume, 40 to 80% by volume, 50 to 80% by volume, 60 to 80% by volume, 70 to 80% by volume, 30 to 70% by volume, 40 to 70% by volume, 50 to 70% by volume, 60 to 70% by volume. Preferably, the concentration of the ethanol solution is 90 to 100% by volume.
본 발명의 일 구현예에 있어서, 상기 비쑥 추출물은 80 ℃내지 100 ℃에서 열수 추출한 열수 추출물이다. 더욱 구체적으로 상기 비쑥 추출물은 70 내지 100 ℃, 60 내지 100 ℃, 50 내지 100 ℃, 80 내지 110 ℃, 70 내지 110 ℃, 60 내지 110 ℃, 50 내지 110 ℃, 80 내지 120 ℃, 70 내지 120 ℃, 60 내지 120 ℃, 50 내지 120 ℃, 80 내지 130 ℃, 70 내지 130 ℃, 60 내지 130 ℃, 50 내지 130 ℃에서 열수 추출한 열수 추출물이다. In one embodiment of the present invention, the wormwood extract is a hot water extract extracted with hot water at 80 ℃ to 100 ℃. More specifically, the wormwood extract is 70 to 100 ℃, 60 to 100 ℃, 50 to 100 ℃, 80 to 110 ℃, 70 to 110 ℃, 60 to 110 ℃, 50 to 110 ℃, 80 to 120 ℃, 70 to 120 It is a hot water extract extracted with hot water at ℃, 60 to 120 ℃, 50 to 120 ℃, 80 to 130 ℃, 70 to 130 ℃, 60 to 130 ℃, 50 to 130 ℃.
본 발명의 일 구체예에 있어서, 상기 비쑥 추출물은 80 ℃ 내지 100 ℃에서 10분에서 20분동안 열수 추출한 열수 추출물이다. 더욱 구체적으로 상기 비쑥 추출물은 80 ℃ 내지 100 ℃에서 10분에서 25분동안, 80 ℃ 내지 100 ℃에서 10분에서 30분동안, 80 ℃ 내지 100 ℃에서 5분에서 20분동안, 80 ℃ 내지 100 ℃에서 5분에서 25분동안, 80 ℃ 내지 100 ℃에서 5분에서 30분동안 열수 추출한 열수 추출물이다. In one embodiment of the present invention, the wormwood extract is a hot water extract extracted with hot water for 10 to 20 minutes at 80 ℃ to 100 ℃. More specifically, the wormwood extract is 80 ℃ to 100 ℃ for 10 minutes to 25 minutes, 80 ℃ to 100 ℃ for 10 minutes to 30 minutes, 80 ℃ to 100 ℃ for 5 minutes to 20 minutes, 80 ℃ to 100 It is a hot water extract extracted with hot water for 5 to 25 minutes at °C and 5 to 30 minutes at 80 to 100 °C.
본 발명의 일 구현예에 있어서, 상기 비쑥 추출물은 파골세포의 분화를 억제한다.In one embodiment of the present invention, the wormwood extract inhibits the differentiation of osteoclasts.
본 발명의 일 구현예에 있어서, 상기 비쑥 추출물은 NFATc1의 활성화를 억제한다.In one embodiment of the present invention, the extract of Mugwort inhibits the activation of NFATc1.
본 명세서에서 용어 “활성화”란 해당 유전자가 발현되거나, 발현되어 기능성이 있는 해당 유전자의 단백질을 생산하는 것을 의미한다. 또한 “활성화를 억제”한다는 것의 의미는 해당 유전자가 발현되지 않거나 발현되더라도 기능성이 있는 해당 유전자의 단백질을 생산하지 않는 것을 의미하거나, 그 발현수준이 현저하게 낮아 실질적으로 발현되지 않는 것도 포함한다. In the present specification, the term “activation” means that the gene is expressed or expressed to produce a protein of the gene having functionality. In addition, the meaning of “inhibiting activation” means that the gene is not expressed, or even if expressed, does not produce a protein of the gene having a function, or the expression level is significantly low, and the expression is not substantially expressed.
본 발명의 일 구현예에 있어서, 상기 비쑥 추출물은 TRAP，DC-STAMP 및 Cathepsin K로 이루어진 군에서 선택되는 유전자의 발현을 억제한다.In one embodiment of the present invention, the wormwood extract inhibits the expression of a gene selected from the group consisting of TRAP, DC-STAMP, and Cathepsin K.
본 발명의 다른 양태에 따르면, 본 발명은 비쑥 추출물을 유효성분으로 함유하는 골대사성 질환으로 유발된 골소실 예방 또는 개선용 식품 조성물을 제공한다.According to another aspect of the present invention, the present invention provides a food composition for preventing or improving bone loss caused by bone metabolic disease, containing a wormwood extract as an active ingredient.
본 발명의 식품 조성물은 분말, 과립, 정제, 캡슐 또는 음료 등의 형태로 제조될 수 있다. 예컨대 캔디류의 각종 식품류, 음료, 껌, 차, 비타민 복합제, 또는 건강보조 식품류 등이 있다.The food composition of the present invention may be prepared in the form of powder, granules, tablets, capsules or beverages. For example, there are various foods such as candy, beverages, gum, tea, vitamin complexes, or health supplement foods.
본 발명의 식품 조성물은 유효성분으로서 비쑥 추출물뿐 만 아니라, 식품 제조 시에 통상적으로 첨가되는 성분을 포함할 수 있으며, 예를 들어, 단백질, 탄수화물, 지방, 영양소, 조미제 및 향미제를 포함한다. 상술한 탄수화물의 예는 모노사카라이드, 예를 들어, 포도당, 과당 등; 디사카라이드, 예를 들어 말토스, 슈크로스, 올리고당 등; 및 폴리사카라이드, 예를 들어 덱스트린, 사이클로덱스트린 등과 같은 통상적인 당 및 자일리톨, 소르비톨, 에리트리톨 등의 당알콜이다. 향미제로서 천연 향미제 [타우마틴, 스테비아 추출물 (예를 들어 레바우디오시드 A, 글리시르히진 등]) 및 합성 향미제(사카린, 아스파르탐 등)를 사용할 수있다. 예컨대, 본 발명의 식품 조성물이 드링크제로 제조되는 경우에는 본 발명의 비쑥 추출물 이외에 구연산, 액상과당, 설탕, 포도당, 초산, 사과산, 과즙, 두충 추출액, 대추 추출액, 감초 추출액 등을 추가로 포함할 수 있다.The food composition of the present invention may include not only a wormwood extract as an active ingredient, but also ingredients commonly added during food production, and, for example, include proteins, carbohydrates, fats, nutrients, flavoring agents, and flavoring agents. . Examples of the aforementioned carbohydrates include monosaccharides such as glucose, fructose, and the like; Disaccharides such as maltose, sucrose, oligosaccharides, and the like; And polysaccharides, for example, common sugars such as dextrin and cyclodextrin, and sugar alcohols such as xylitol, sorbitol, and erythritol. As flavoring agents, natural flavoring agents [taumatin, stevia extract (eg, rebaudioside A, glycyrrhizin, etc.]) and synthetic flavoring agents (saccharin, aspartame, etc.) can be used. For example, when the food composition of the present invention is prepared as a drink, it may further include citric acid, liquid fructose, sugar, glucose, acetic acid, malic acid, fruit juice, cephalic extract, jujube extract, licorice extract, etc. in addition to the wormwood extract of the present invention. have.
발명의 조성물에 포함된 비쑥 추출물은 천연식물재료로서, 오랫동안 식용 및 민간약으로 사용되어 왔던 것이므로 이로부터 추출된 본 발명의 추출물 역시 독성 및 부작용 등의 문제가 없을 것으로 예상할 수 있다. 따라서, 비쑥 추출물은 의약품, 식품 조성물로 사용될 수 있을 뿐만 아니라, 동물의약품 및 기능성 사료로도 개발이 가능하다.The wormwood extract contained in the composition of the present invention is a natural plant material and has been used for a long time as an edible and folk medicine, so it can be expected that the extract of the present invention extracted therefrom also has no problems such as toxicity and side effects. Therefore, the wormwood extract can be used not only as a pharmaceutical, food composition, but also can be developed as an animal medicine and functional feed.
본 발명의 식품 조성물에 포함되는 비쑥 추출물의 추출에 관한 내용은 상기 약제학적 조성물에 포함되는 비쑥 추출물의 추출에 관한 내용과 동일하기 때문에, 이 둘 사이에 공통된 내용은 본 명세서의 과도한 복잡성을 피하기 위하여, 그 기재를 생략한다.Since the contents of the extract of the wormwood extract included in the food composition of the present invention are the same as the contents of the extract of the wormwood extract included in the pharmaceutical composition, the contents in common between the two are to avoid excessive complexity of the present specification. , The description is omitted.
본 발명의 특징 및 이점을 요약하면 다음과 같다:The features and advantages of the present invention are summarized as follows:
비쑥 추출물은 파골세포의 분화 및 NFATc1의 활성을 억제하여 골대사성 질환으로 유발된 골소실을 예방 또는 치료하는 효능을 나타낸다. Mugwort extract exhibits the efficacy of preventing or treating bone loss caused by bone metabolic diseases by inhibiting the differentiation of osteoclasts and the activity of NFATc1.
상기 효능으로 인해 본 발명의 비쑥 추출물을 이용하는 경우 독성 및 부작용 없이 인체에 안전하게 사용할 수 있는 골대사성 질환으로 유발된 골소실 예방 또는 치료용 약제학적 조성물 또는 골대사성 질환으로 유발된 골소실 예방 또는 개선용 식품 조성물을 제공할 수 있다.Due to the above efficacy, when the extract of the present invention is used, a pharmaceutical composition for preventing or treating bone loss caused by bone metabolic diseases that can be safely used in the human body without toxicity and side effects, or for preventing or improving bone loss caused by bone metabolic diseases A food composition can be provided.
도 1은 마우스 대식세포에서 비쑥 열수 추출물의 파골세포 분화 억제 효과에 대해 현미경으로 관찰한 결과를 나타낸다.1 shows the results of observation under a microscope for the inhibitory effect of hot water extract on osteoclast differentiation in mouse macrophages.
도 2는 마우스 대식세포에서 비쑥 에탄올 추출물의 파골세포 분화 억제 효과에 대해 현미경으로 관찰한 결과를 나타낸다.Figure 2 shows the results of observation under a microscope for the effect of inhibiting osteoclast differentiation of the ethanol extract of Mugwort in mouse macrophages.
도 3a는 마우스 대식세포에서 비쑥 열수 추출물의 파골세포 분화 관련 유전자 TRAP의 발현에 대한 억제 효과를 그래프로 나타낸 것이다.Figure 3a is a graph showing the inhibitory effect on the expression of the osteoclast differentiation-related gene TRAP of the extract of hot water in mouse macrophages.
도 3b는 마우스 대식세포에서 비쑥 열수 추출물의 파골세포 분화 관련 유전자 Cathesin K의 발현에 대한 억제 효과를 그래프로 나타낸 것이다.Figure 3b is a graph showing the inhibitory effect on the expression of the osteoclast differentiation-related gene Cathesin K of the extract of hot water in mouse macrophages.
도 3c는 마우스 대식세포에서 비쑥 열수 추출물의 파골세포 분화 관련 유전자 DC-STAMP의 발현에 대한 억제 효과를 그래프로 나타낸 것이다.Figure 3c is a graph showing the inhibitory effect on the expression of the osteoclast differentiation-related gene DC-STAMP of the extract of hot water in mouse macrophages.
도 3d는 마우스 대식세포에서 비쑥 열수 추출물의 파골세포 분화 관련 유전자 NFATc1 발현에 대한 억제 효과를 그래프로 나타낸 것이다.Figure 3d is a graph showing the inhibitory effect on the expression of the osteoclast differentiation-related gene NFATc1 of the extract of hot water in mouse macrophages.
도 4a는 마우스 대식세포에서 비쑥 에탄올 추출물의 파골세포 분화 관련 유전자 TRAP의 발현에 대한 억제 효과를 그래프로 나타낸 것이다.Figure 4a is a graph showing the inhibitory effect on the expression of the osteoclast differentiation-related gene TRAP of the ethanol extract of wormwood in mouse macrophages.
도 4b는 마우스 대식세포에서 비쑥 에탄올 추출물의 파골세포 분화 관련 유전자 Cathesin K의 발현에 대한 억제 효과를 그래프로 나타낸 것이다.Figure 4b is a graph showing the inhibitory effect on the expression of the osteoclast differentiation-related gene Cathesin K of the ethanol extract of wormwood in mouse macrophages.
도 4c는 마우스 대식세포에서 비쑥 에탄올 추출물의 파골세포 분화 관련 유전자 DC-STAMP의 발현에 대한 억제 효과를 그래프로 나타낸 것이다.Figure 4c is a graph showing the inhibitory effect on the expression of the osteoclast differentiation-related gene DC-STAMP of the ethanol extract of wormwood in mouse macrophages.
도 4d는 마우스 대식세포에서 비쑥 에탄올 추출물의 파골세포 분화 관련 유전자 NFATc1 발현에 대한 억제 효과를 그래프로 나타낸 것이다.Figure 4d is a graph showing the inhibitory effect on the expression of the osteoclast differentiation-related gene NFATc1 of the ethanol extract of Mugwort in mouse macrophages.
비쑥 추출물을 유효성분으로 포함하는 골대사성 질환으로 유발된 골소실 예방 또는 치료용 약제학적 조성물.A pharmaceutical composition for the prevention or treatment of bone loss caused by bone metabolic diseases, comprising a wormwood extract as an active ingredient.
이하, 실시예를 통하여 본 발명을 더욱 상세히 설명하고자 한다. 이들 실시예는 오로지 본 발명을 보다 구체적으로 설명하기 위한 것으로, 본 발명의 요지에 따라 본 발명의 범위가 이들 실시예에 의해 제한되지 않는다는 것은 당업계에서 통상의 지식을 가진 자에 있어서 자명할 것이다.Hereinafter, the present invention will be described in more detail through examples. These examples are only for describing the present invention in more detail, and it will be apparent to those of ordinary skill in the art that the scope of the present invention is not limited by these examples according to the gist of the present invention. .
본 명세서 전체에 걸쳐, 특정 물질의 농도를 나타내기 위하여 사용되는 “%”는 별도의 언급이 없는 경우, 고체/고체는 (중량/중량)%, 고체/액체는 (중량/부피) %, 그리고 액체/액체는 (부피/부피) %이다. Throughout this specification, “%” used to indicate the concentration of a specific substance is (weight/weight)% for solids/solids, (weight/volume)% for solids/liquids, and Liquid/liquid is (vol/vol) %.
실시예Example
실시예 1: 비쑥 추출물 제조 방법Example 1: Method for preparing a wormwood extract
비쑥 지상부는 2018년 9월에 전라남도 신안군 중도면에서 채취하여 사용하였고，실시예 1-1과 실시예 1-2에 기재된 제조방법을 통해 비쑥추출물을 제조하였다.The wormwood above-ground part was collected and used in Jungdo-myeon, Sinan-gun, Jeollanam-do, and prepared wormwood extract through the manufacturing methods described in Examples 1-1 and 1-2.
실시예 1-1. 비쑥 열수추출물의 제조Example 1-1. Preparation of hot water extract of Mugwort
신선 비쑥 지상부 50 g에 90 ℃의 열수 2.5 L를 가하여 호모게나이저로 분쇄한 다음 15분 동안 90 ℃를 유지하면서 열수 추출하였으며， 이후 부직포로 여과하여 열수추출여액을 얻었다. 또한 잔사에 90 ℃이상의 열수 1 L를 가하여 동일 방법으로 재차 추출 및 여과하였다. 2회에 걸쳐 얻어진 열수 추출 여액은 아스피레이터 (CCA-1110， Eyela, Tokyo, Japan)가 장착된 진공농축기 (A-3S ， Eyela, Tokyo, Japan)를 사용하여 45 ℃에서 감압 농축하여 비쑥 열수추출물을 제조하였다.2.5 L of hot water at 90° C. was added to 50 g of the above-ground part of fresh wormwood, pulverized with a homogenizer, and then extracted with hot water while maintaining at 90° C. for 15 minutes, and then filtered through a nonwoven fabric to obtain a hot water extract filtrate. In addition, 1 L of hot water of 90° C. or higher was added to the residue, followed by extraction and filtering again in the same manner. The hot water extraction filtrate obtained twice is concentrated under reduced pressure at 45 ℃ using a vacuum concentrator (A-3S, Eyela, Tokyo, Japan) equipped with an aspirator (CCA-1110, Eyela, Tokyo, Japan) The extract was prepared.
실시예 1-2. 비쑥 에탄올 추출물의 제조Example 1-2. Preparation of Mugwort Ethanol Extract
신선 비쑥 지상부 100 g를 95 % 에탄올 2.5 L를 가하여 호모게나이저로 분쇄한 다음 상온에서 24시간 동안 추출하였으며， 여과지 (No. 2, Whatman)로 여과를 행하여 에탄올추출여액을 얻었다. 또한 95 % 에탄올 1L를 가하여 동일 방법으로 재차 추출 및 여과하였다. 얻어진 에탄올 추출 여액은 아스피레이터 (CCA-1110, Eyela, Tokyo, Japan)가 장착된 진공농축기(A-3S， Eyela, Tokyo, Japan)를 사용하여 45 ℃에서 감압 농축하여 비쑥 에탄올 추출물을 제조하였다.100 g of the above-mentioned fresh wormwood was added to 2.5 L of 95% ethanol, pulverized with a homogenizer, and extracted for 24 hours at room temperature, followed by filtration with filter paper (No. 2, Whatman) to obtain an ethanol extract filtrate. In addition, 1 L of 95% ethanol was added, followed by extraction and filtration again in the same manner. The obtained ethanol extraction filtrate was concentrated under reduced pressure at 45° C. using a vacuum concentrator (A-3S, Eyela, Tokyo, Japan) equipped with an aspirator (CCA-1110, Eyela, Tokyo, Japan) to prepare an ethanol extract of Mugwort. .
실시예 2: 비쑥 열수 및 에탄올 추출물 이용한 골 질환 예방 또는 치료 효과에 대한 실험Example 2: Experiment on the effect of preventing or treating bone disease using hot water and ethanol extract
실시예 2-1. 골수 유래 대식세포(BMDMs， bone marrow-derived macrophages) 분리 및 배양Example 2-1. Isolation and culture of bone marrow-derived macrophages (BMDMs, bone marrow-derived macrophages)
8 내지 12 주령 사이의 C57BL/6 마우스의 대퇴골(femur)에서 골수를 분리하고, 10% FBS (Fetal bovine serum) 및 1% 페니실린과 대식세포(macrophage)로 분화시키기 위한 M-CSF (Macrophage-colony stimulating factor) 25 ng/ml가 첨가된 MEM alpha 배지에 도말하여 3 일 동안 37 ℃및 5% CO2 조건의 인큐베이터에서 배양하여 골수 유래 대식 세포 (BMDMs)를 얻었다.M-CSF (Macrophage-colony) for separating the bone marrow from the femur of C57BL/6 mice between 8 and 12 weeks of age, and for differentiation into 10% Fetal bovine serum (FBS) and 1% penicillin and macrophages. Bone marrow-derived macrophages (BMDMs) were obtained by spreading on MEM alpha medium to which 25 ng/ml of stimulating factor was added and culturing in an incubator at 37° C. and 5% CO 2 for 3 days.
상기 골수 유래 대식세포를 2x105 cells/well의 밀도로 12 well- plate에 24시간 동안 배양하였다.The bone marrow-derived macrophages were cultured in a 12 well-plate at a density of 2× 10 5 cells/well for 24 hours.
실시예2-2. 비쑥 추출물의 파골세포 분화에 대한 억제 평가 (TRAP staining)Example 2-2. Evaluation of inhibition of osteoclast differentiation of Mugwort extract (TRAP staining)
마우스 대식세포를 12 well plate에 2x105 cells/well의 밀도로 10% FBS, 1% PS 및 M-CSF (25 ng/ml)가 첨가된 배지에 24 시간 동안 배양 후， 같은 조성의 배지로 교체해준 후 비쑥 열수 및 에탄올 추출물을 각각 6.125, 12.5, 25, 50 μg/ml의 농도로 2 시간 동안 처리하였다. 이후 RANKL을 처리하여 24 시간 동안 유지하였다. 위와 같은 방법으로 세포를 4 일간 분화하였다. 이후 파골세포의 세포 화학적 표지 효소인 TRAP(Tartrate resistance acid phosphatase)에 발색성 기질을 첨가하여 핵을 염색하였고 핵이 3개 이상으로 다핵화 된 세포를 관찰하고 정량화 하였다.Mouse macrophages were incubated in a 12 well plate at a density of 2x10 5 cells/well in a medium containing 10% FBS, 1% PS, and M-CSF (25 ng/ml) for 24 hours, and then replaced with a medium of the same composition. After doing, hot water and ethanol extract were treated at concentrations of 6.125, 12.5, 25, and 50 μg/ml, respectively, for 2 hours. Then, RANKL was treated and maintained for 24 hours. Cells were differentiated for 4 days in the same way as above. Thereafter, the nucleus was stained by adding a chromogenic substrate to the cytochemical labeling enzyme of osteoclast cells, TRAP (Tartrate resistance acid phosphatase), and cells with three or more nuclei multinucleated were observed and quantified.
대식세포에 RANKL을 처리하면 RANK에 결합하며 TRAP 양성 세포로 분화하게 되는데，이 TRAP 양성 세포에 RANKL, TNF-α와 같은 염증인자로 자극하면 세포끼리 융합되어 다핵성 TRAP 양성세포로 분화한다.When the macrophages are treated with RANKL, they bind to RANK and differentiate into TRAP-positive cells. When these TRAP-positive cells are stimulated with inflammatory factors such as RANKL and TNF-α, the cells are fused to each other and differentiate into multinuclear TRAP-positive cells.
도 1 과 2에서 확인할 수 있듯이， 상기 대식세포에 RANKL을 처리했을 때 파골세포로의 분화가 증가하였고， 비쑥 열수 및 에탄올 추출물을 처리한 경우 농도 의존적으로 파골 세포의 수가 현저히 감소하였음을 확인하였다. 특히 열수 추출물 50 μg/ml와 에탄올 추출물 20 μg/ml농도에서 유의적으로 파골세포가 감소된 것을 확인할 수 있었다. 이로부터 비쑥 열수 및 에탄올 추출물은 파골세포로의 분화를 억제시킴을 확인할 수 있었다.As can be seen in Figures 1 and 2, when the macrophages were treated with RANKL, differentiation into osteoclasts increased, and when treated with hot water and ethanol extract, the number of osteoclasts was significantly reduced in a concentration-dependent manner. In particular, it was confirmed that osteoclasts were significantly reduced at concentrations of 50 μg/ml of hot water extract and 20 μg/ml of ethanol extract. From this, it was confirmed that hot water and ethanol extract inhibited the differentiation into osteoclasts.
실시예 2-3. 비쑥 추출물의 파골 세포 분화에 대한 유전자 발현 억제 평가 (Real-time PCR)Example 2-3. Evaluation of gene expression inhibition on osteoclast differentiation of Mugwort extract (Real-time PCR)
마우스 대식세포에 RANKL 및 비쑥 추출물을 처리했을 때 파골세포 분화에 관련하는 유전자인 TRAP, Cathepsin K, DC-STAMP, NFATc1에 대한 유전자 발현을 real-time PCR을 통해 확인하였다. 마우스 대식세포를 12 well plate에 2x105 cells/well의 밀도로 10% FBS, 1% PS 및 M- CSF (25 ng/ml)가 첨가된 배지에 24 시간 배양 후， 같은 조성의 배지로 교체해준 후 열수 추출물 50 μg/ml와 에탄올 추출물 20 μg/ml를 2 시간 동안 처리해 주었다. 이후 RANKL을 처리하여 24 시간 동안 반응시켰다. 위와 같은 방법으로 4 일간 분화시킨 후 Easy-blue 용액을 이용하여 세포내 RNA를 분리하고 RNA 정량값을 토대로 RT premix를 이용하여 cDNA를 합성하였다. 합성된 cDNA는 primer를 이용하여 real-time PCR을 통해 증폭시켰다. 사용된 primer는 mouse TRAP, Cathesin K, DC-STAMP, NFATc1 (표1)이고 control 유전자인 GAPDH와 비교하여 상대적 양을 비교하였다.Gene expression for TRAP, Cathepsin K, DC-STAMP, and NFATc1, genes related to osteoclast differentiation, when treated with RANKL and Mugwort extract on mouse macrophages was confirmed through real-time PCR. Mouse macrophages were cultured for 24 hours in a medium containing 10% FBS, 1% PS, and M-CSF (25 ng/ml) at a density of 2x10 5 cells/well in a 12 well plate, and then replaced with a medium of the same composition. After, 50 μg/ml of hot water extract and 20 μg/ml of ethanol extract were treated for 2 hours. Then, RANKL was treated and reacted for 24 hours. After differentiation for 4 days in the same manner as above, intracellular RNA was isolated using Easy-blue solution, and cDNA was synthesized using RT premix based on the RNA quantification value. The synthesized cDNA was amplified through real-time PCR using primers. The primers used were mouse TRAP, Cathesin K, DC-STAMP, NFATc1 (Table 1), and the relative amounts were compared with the control gene GAPDH.
서열번호Sequence number 명칭designation 서열 (5'-3')Sequence (5'-3')
1One TRAP 정방향 프라이머TRAP forward primer CTGGAGTGCACGATGCCAGCGACACTGGAGTGCACGATGCCAGCGACA
22 TRAP역방향 프라이머TRAP reverse primer TCCGTGCTCGGCGATGGACCAGATCCGTGCTCGGCGATGGACCAGA
33 DC-STAMP 정방향 프라이머DC-STAMP forward primer CCAAGGAGTCGTCCATGATTCCAAGGAGTCGTCCATGATT
44 DC-STAMP역방향 프라이머DC-STAMP reverse primer GGCTGCTTTGATCGTTTCTCGGCTGCTTTGATCGTTTCTC
55 Cathepsin K 정방향 프라이머Cathepsin K forward primer GGCCAACTCAAGAAGAAAACGGCCAACTCAAGAAGAAAAC
66 Cathepsin K 역방향 프라이머Cathepsin K reverse primer GTGCTTGCTTCCCTTCTGGGTGCTTGCTTCCCTTCTGG
77 NFATc1 정방향 프라이머NFATc1 forward primer CTCGAAAGACAGTGGAGCATCTCGAAAGACAGTGGAGCAT
88 NFATc1 역방향 프라이머NFATc1 reverse primer CGGCTGCCTTCCGTCTCATAGCGGCTGCCTTCCGTCTCATAG
대식세포에 RANKL을 처리하면 대식세포는 RANK에 결합하여 TRAP 양성인 다핵화된 파골세포로 분화한다. 또한 대식세포는 파골세포로 분화하는데 중요한 전사 인자인 NFATc1을 활성화시키고 파골세포분화기전 (osteoclastogenesis)에 관여하는 TRAP， Cathepsine K, DC-STAMP의 발현이 증가하였다.When the macrophages are treated with RANKL, the macrophages bind to RANK and differentiate into TRAP-positive multinucleated osteoclasts. In addition, macrophages activated NFATc1, an important transcription factor for differentiation into osteoclasts, and increased expression of TRAP, Cathepsine K, and DC-STAMP, which are involved in osteoclastogenesis.
도 3a 내지 4d와 표 2, 3에서 확인할 수 있듯이, RANKL처리에 의하여 증가된 TRAP， Cathepsin K, DC-STAMP， NFATc1의 발현은 비쑥 열수 및 에탄올 추출물의 처리에 의해서 감소하였다. 이로부터 상기 추출물이 전사인자인 NFATc1의 활성을 감소시키고， 파골세포분화기전 관련 유전자인 TRAP，DC-STAMP, Cathepsin K의 발현을 감소시킴으로써， 파골세포로의 분화를 억제시킴을 확인할 수 있었다.As can be seen in FIGS. 3A to 4D and Tables 2 and 3, the expression of TRAP, Cathepsin K, DC-STAMP, and NFATc1 increased by RANKL treatment was reduced by treatment with hot water and ethanol extract. From this, it was confirmed that the extract inhibited the differentiation into osteoclasts by reducing the activity of the transcription factor NFATc1 and reducing the expression of the genes related to the osteoclast differentiation mechanism, TRAP, DC-STAMP, and Cathepsin K.
RANKLRANKL -- -- ++ ++
비쑥 열수 추출물Mugwort Hot Water Extract -- ++ -- ++
유전자gene 유전자의 상대적인 발현량Relative expression level of gene
TRAPTRAP 1One 22 600600 113113
Cathepsin KCathepsin K 1One 22 58265826 771771
DC-STAMPDC-STAMP 1One 33 148148 3939
NFATc1NFATc1 1One 1One 44 33
RANKLRANKL -- -- ++ ++
비쑥 열수 추출물Mugwort Hot Water Extract -- ++ -- ++
유전자gene 유전자의 상대적인 발현량Relative expression level of gene
TRAPTRAP 1One 1One 465465 7070
Cathepsin KCathepsin K 1One 33 33613361 522522
DC-STAMPDC-STAMP 1One 1One 509509 117117
NFATc1NFATc1 1One 22 1414 44
하기에 본 발명의 추출물을 포함하는 조성물의 제제예를 설명하나, 본 발명은 이를 한정하고자 함이 아닌 단지 구체적으로 설명하고자 함이다.Hereinafter, an example of the formulation of the composition containing the extract of the present invention will be described, but the present invention is not intended to limit it, but is intended to be described in detail.
제제예Formulation example
제제예 1. 산제의 제조Formulation Example 1. Preparation of powder
실시예 1-1의 비쑥 열수 추출물 또는 실시예 1-2의 비쑥 에탄올 용액 추출물 500 mgMugwort hot water extract of Example 1-1 or 500 mg of Mugwort ethanol solution extract of Example 1-2
유당 100 mg100 mg lactose
탈크 10 mg10 mg of talc
상기의 성분들을 혼합하고 기밀포에 충진하여 산제를 제조한다.The above ingredients are mixed and filled in an airtight cloth to prepare a powder.
제제예 2. 정제의 제조Formulation Example 2. Preparation of tablets
실시예 1-1의 비쑥 열수 추출물 또는 실시예 1-2의 비쑥 에탄올 용액 추출물 300 mgMugwort hot water extract of Example 1-1 or 300 mg of Mugwort ethanol solution extract of Example 1-2
옥수수전분 100 mg100 mg corn starch
유당 100 mg100 mg lactose
스테아린산 마그네슘 2 mg2 mg of magnesium stearate
상기의 성분들을 혼합한 후 통상의 정제 제조방법에 따라서 타정하여 정제를 제조한다.After mixing the above ingredients, tablets are prepared by tableting according to a conventional tablet manufacturing method.
제제예 3. 캅셀제의 제조 Formulation Example 3. Preparation of Capsule
실시예 1-1의 비쑥 열수 추출물 또는 실시예 1-2의 비쑥 에탄올 용액 추출물 200 mg200 mg of wormwood hot water extract of Example 1-1 or 200 mg of wormwood ethanol solution extract of Example 1-2
결정성 셀룰로오스 3 mg3 mg of crystalline cellulose
락토오스 14.8 mg14.8 mg lactose
마그네슘 스테아레이트 0.2 mgMagnesium stearate 0.2 mg
통상의 캡슐제 제조방법에 따라 상기의 성분을 혼합하고 젤라틴 캡슐에 충전하여 캡슐제를 제조한다.According to a conventional capsule preparation method, the above ingredients are mixed and filled into gelatin capsules to prepare a capsule.
제제예 4. 액제의 제조Formulation Example 4. Preparation of liquid formulation
실시예 1-1의 비쑥 열수 추출물 또는 실시예 1-2의 비쑥 에탄올 용액 추출물 4 gMugwort hot water extract of Example 1-1 or 4 g of Mugwort ethanol solution extract of Example 1-2
이성화당 10 g10 g of isomerized sugar
만니톨 5 g5 g of mannitol
정제수 적량Purified water appropriate amount
통상의 액제의 제조방법에 따라 정제수에 각각의 성분을 가하여 용해시키고 레몬향을 적량 가한 다음 상기의 성분을 혼합한 다음 정제수를 가하여 전체를 정제수를 가하여 전체 100㎖로 조절한 후 갈색병에 충진하여 멸균시켜 액제를 제조한다.According to the usual preparation method of liquid preparation, add each ingredient to purified water to dissolve it, add lemon zest, mix the above ingredients, add purified water, add purified water, adjust the total to 100 ml, and fill it in a brown bottle. It is sterilized to prepare a solution
제제예 5. 건강 식품의 제조Formulation Example 5. Manufacture of health food
실시예 1-1의 비쑥 열수 추출물 또는 실시예 1-2의 비쑥 에탄올 용액 추출물 1,000 mgMugwort hot water extract of Example 1-1 or 1,000 mg of Mugwort ethanol solution extract of Example 1-2
비타민 혼합물 적량The right amount of vitamin mixture
비타민 A 아세테이트 70 ㎍Vitamin A acetate 70 ㎍
비타민 E 1.0 ㎎Vitamin E 1.0 mg
비타민 B1 0.13 ㎎Vitamin B1 0.13 mg
비타민 B2 0.15 ㎎Vitamin B2 0.15 mg
비타민 B6 0.5 ㎎Vitamin B6 0.5 mg
비타민 B12 0.2 ㎍Vitamin B12 0.2 ㎍
비타민 C 10 ㎎ Vitamin C 10 mg
비오틴 10 ㎍Biotin 10 ㎍
니코틴산아미드 1.7 ㎎Nicotinic acid amide 1.7 mg
엽산 50 ㎍Folic acid 50 ㎍
판토텐산 칼슘 0.5 ㎎0.5 mg of calcium pantothenate
무기질 혼합물 적량Suitable amount of inorganic mixture
황산제1철 1.75 ㎎Ferrous sulfate 1.75 mg
산화아연 0.82 ㎎Zinc oxide 0.82 mg
탄산마그네슘 25.3 ㎎Magnesium carbonate 25.3 mg
제1인산칼륨 15 ㎎Potassium monophosphate 15 mg
제2인산칼슘 55 ㎎Dicalcium phosphate 55 mg
구연산칼륨 90 ㎎ Potassium citrate 90 mg
탄산칼슘 100 ㎎100 mg of calcium carbonate
염화마그네슘 24.8 ㎎Magnesium chloride 24.8 mg
상기의 비타민 및 미네랄 혼합물의 조성비는 비교적 건강식품에 적합한 성분을 바람직한 실시예로 혼합 조성하였지만, 그 배합비를 임의로 변형 실시하여도 무방하며, 통상의 건강식품 제조방법에 따라 상기의 성분을 혼합한 다음, 과립을 제조하고, 통상의 방법에 따라 건강식품 조성물 제조에 사용할 수 있다.The composition ratio of the vitamin and mineral mixture is relatively suitable for health food, but it may be arbitrarily modified, and the above ingredients are mixed according to a conventional health food manufacturing method. , Granules can be prepared, and used for preparing health food compositions according to conventional methods.
제제예 6. 건강 음료의 제조Formulation Example 6. Preparation of healthy beverage
실시예 1-1의 비쑥 열수 추출물 또는 실시예 1-2의 비쑥 에탄올 용액 추출물 1,000 mgMugwort hot water extract of Example 1-1 or 1,000 mg of Mugwort ethanol solution extract of Example 1-2
구연산 1,000 ㎎Citric acid 1,000 mg
올리고당 100 g100 g oligosaccharides
매실농축액 2 g2 g of plum concentrate
타우린 1 g1 g taurine
정제수를 가하여 전체 900 ㎖Total 900 ml by adding purified water
통상의 건강음료 제조방법에 따라 상기의 성분을 혼합한 다음, 약 1시간동안 85℃에서 교반 가열한 후, 만들어진 용액을 여과하여 멸균된 2 L 용기에 취득하여 밀봉 멸균한 뒤 냉장 보관한 다음 본 발명의 건강음료 조성물 제조에 사용한다.After mixing the above ingredients according to the normal health drink manufacturing method, stirring and heating at 85°C for about 1 hour, the resulting solution is filtered and obtained in a sterilized 2 L container, sealed and sterilized, and then stored in a refrigerator. It is used in the manufacture of the health beverage composition of the invention.
상기 조성비는 비교적 기호음료에 적합한 성분을 바람직한 실시예로 혼합 조성하였지만, 수요계층, 수요국가, 사용용도 등 지역적, 민족적 기호도에 따라서 그 배합비를 임의로 변형 실시하여도 무방하다.The composition ratio is a mixture of ingredients suitable for a relatively preferred beverage in a preferred embodiment, but the mixing ratio may be arbitrarily modified according to regional and ethnic preferences such as the demand class, the country of demand, and the purpose of use.
본 발명은 비쑥 추출물을 유효성분으로 포함하는 골대사성 질환으로 유발된 골소실 예방 또는 치료용 약제학적 조성물에 관한 것이다.The present invention relates to a pharmaceutical composition for the prevention or treatment of bone loss caused by bone metabolic disease, comprising the extract of Mugwort as an active ingredient.

Claims (10)

  1. 비쑥 추출물을 유효성분으로 포함하는 골대사성 질환으로 유발된 골소실 예방 또는 치료용 약제학적 조성물.A pharmaceutical composition for the prevention or treatment of bone loss caused by bone metabolic diseases, comprising a wormwood extract as an active ingredient.
  2. 제 1 항에 있어서,The method of claim 1,
    상기 골대사성 질환은 골다공증(osteoporosis), 파제트병(paget disease), 치주질환(periodontal disease), 골전이암(metastatic cancer), 류마티스 관절염(rheumatoid arthritis), 뼈전이암(bone metastatic cancer), 고형암 뼈전이, 고형암 뼈전이에 의한 근골격 합병증, 악성종양으로 인한 과칼슘혈증, 다발성 골수증, 원발성(primary) 뼈종양, 퇴행성 관절염, 염증성 치조골 흡수 질환 및 염증성 뼈흡수 질환으로 이루어진 군으로부터 선택되는 것인, 골대사성 질환으로 유발된 골소실 예방 또는 치료용 약제학적 조성물.The bone metabolic diseases include osteoporosis, paget disease, periodontal disease, metastatic cancer, rheumatoid arthritis, bone metastatic cancer, and solid cancer. Bone metastasis, musculoskeletal complications due to solid cancer bone metastasis, hypercalcemia due to malignant tumors, multiple myelosis, primary bone tumors, degenerative arthritis, inflammatory alveolar bone resorption disease, and inflammatory bone resorption disease. , A pharmaceutical composition for preventing or treating bone loss caused by bone metabolic disease.
  3. 제 1 항에 있어서,The method of claim 1,
    상기 비쑥 추출물은 물, 알코올 및 이들의 혼합용매로 이루어진 군으로부터 선택되는 어느 하나의 추출 용매를 비쑥에 처리하여 추출한 비쑥 추출물인 것인, 골대사성 질환으로 유발된 골소실 예방 또는 치료용 약제학적 조성물.The wormwood extract is a wormwood extract extracted by treating a wormwood extract selected from the group consisting of water, alcohol, and a mixed solvent thereof, and a pharmaceutical composition for preventing or treating bone loss caused by bone metabolic diseases .
  4. 제 3 항에 있어서,The method of claim 3,
    상기 알코올은 메탄올, 에탄올, 프로판올, 부탄올, 노말-프로판올, 이소-프로판올, 노말-부탄올 및 이들의 혼합 용매로 이루어진 군으로부터 선택되는 것인, 골대사성 질환으로 유발된 골소실 예방 또는 치료용 약제학적 조성물.The alcohol is selected from the group consisting of methanol, ethanol, propanol, butanol, normal-propanol, iso-propanol, normal-butanol, and a mixed solvent thereof. Composition.
  5. 제 4 항에 있어서,The method of claim 4,
    상기 에탄올은 60 내지 100 부피%의 에탄올 용액인 것인, 골대사성 질환으로 유발된 골소실 예방 또는 치료용 약제학적 조성물.The ethanol is an ethanol solution of 60 to 100% by volume, a pharmaceutical composition for preventing or treating bone loss caused by bone metabolic disease.
  6. 제 1 항에 있어서,The method of claim 1,
    상기 비쑥 추출물은 80 내지 100 ℃에서 열수 추출한 열수 추출물인 것인, 골대사성 질환으로 유발된 골소실 예방 또는 치료용 약제학적 조성물.The wormwood extract is a hot water extract extracted with hot water at 80 to 100° C., a pharmaceutical composition for preventing or treating bone loss caused by bone metabolic disease.
  7. 제 1 항에 있어서,The method of claim 1,
    상기 비쑥 추출물은 파골세포의 분화를 억제하는 것인, 골대사성 질환으로 유발된 골소실 예방 또는 치료용 약제학적 조성물.The artemisiae extract is to inhibit the differentiation of osteoclasts, a pharmaceutical composition for preventing or treating bone loss caused by bone metabolic disease.
  8. 제 1 항에 있어서,The method of claim 1,
    상기 비쑥 추출물은 NFATc1의 활성화를 억제하는 것인, 골대사성 질환으로 유발된 골소실 예방 또는 치료용 약제학적 조성물.The artemisiae extract is to inhibit the activation of NFATc1, a pharmaceutical composition for the prevention or treatment of bone loss caused by bone metabolic disease.
  9. 제 1 항에 있어서,The method of claim 1,
    상기 비쑥 추출물은 TRAP，DC-STAMP 및 Cathepsin K로 이루어진 군에서 선택되는 유전자의 발현을 억제하는 것인, 골대사성 질환으로 유발된 골소실 예방 또는 치료용 약제학적 조성물.The wormwood extract is to inhibit the expression of a gene selected from the group consisting of TRAP, DC-STAMP and Cathepsin K, a pharmaceutical composition for preventing or treating bone loss caused by bone metabolic disease.
  10. 비쑥 추출물을 유효성분으로 함유하는 골대사성 질환으로 유발된 골소실 예방 또는 개선용 식품 조성물.A food composition for preventing or improving bone loss caused by bone metabolic diseases, containing a wormwood extract as an active ingredient.
PCT/KR2020/011430 2019-08-28 2020-08-26 Pharmaceutical composition, for preventing or treating bone loss induced by metabolic bone diseases, comprising artemisia scoparia extract as active ingredient WO2021040416A1 (en)

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