KR102430968B1 - Plant extract compositions for preventing or treating bone disease - Google Patents

Abstract

The present invention relates to a plant extract composition for the prevention or treatment of bone diseases, and more specifically, the composition according to the present invention is stable due to low cytotoxicity, and inhibits the formation of osteoclasts to suppress bone diseases caused by bone loss. Since it is effective in bone disease, it can be usefully used as a pharmaceutical composition for the prevention or treatment of bone disease and a food composition for preventing or improving bone disease.

Description

Plant extract compositions for preventing or treating bone disease

The present invention relates to a plant extract composition for the prevention or treatment of bone diseases, and more specifically, the composition according to the present invention is effective in suppressing bone diseases caused by bone loss by inhibiting the formation of osteoclasts, It can be usefully used as a pharmaceutical composition for the prevention or treatment of and as a food composition for the prevention or improvement of bone diseases.

Osteoclasts, which are multinucleated large cells, have the function of destruction and resorption of bone tissue, and are known to play a role in destroying bone matrix and decomposing bone minerals. Activated osteoclasts have three or more nuclei, and various hormones and factors are required to differentiate them from osteoclast precursor cells into mature multinucleated osteoclasts. Among them, the two most important factors are macrophage colony stimulating factor (M-CSF) and receptor activator of nuclear factor-kappa B ligand (RANKL) produced by osteoblasts (Mojtaba A., et al. , Cancer Biol Ther. , 7:1,3-9;1 (2008)]).

M-CSF is a cytokine expressed by osteoblasts and bone marrow stromal cells and plays an important role in the formation of osteoclasts, and mainly plays an important role in cell proliferation, survival, and cytoskeletal organization. known (Kim SY., et al ., J Korean Orthop Assoc ., 44:151-158 (2009)). Another important factor, RANKL, is expressed by osteoblasts and attaches to RANK receptors in osteoclast progenitors to induce osteoclast growth and differentiation (Mojtaba A., Cancer Biology & Therapy , 7:1,3). -9;1 (2008)]). In addition, RANKL promotes osteoclast differentiation by activating transcription factors such as c-fos, NFATc1 (nuclear Factor Of Activated T-Cells, cytoplasmic, calcineurin-dependent 1), and NF-kB (nuclear factor kappa B), It is known that PI-3K (phosphatidylinositol 3-kinase) and ERK (extracellular signal-regulated kinase), such as activating signaling systems, play a role in promoting the survival and function of osteoclasts (LEE ZH., et al . al ., Biochem Biophys Res Commun ., 305:211-213 (2003)]).

These osteoclasts cause the destruction and resorption of abnormal bone tissue due to imbalance with osteoblasts in the bone, and thereby, osteoporosis, in which bone mass and bone density decrease, and osteomalacia, in which lime is lost from the bone It is known to cause fibrous ostitis in which bone marrow is fibrous, periodontitis in which alveolar bone is lost, and rheumatoid arthritis that causes joint destruction and deformation.

Therefore, if the destruction and resorption of bone tissue by osteoclasts can be effectively inhibited, it is expected that various bone diseases can be treated. Accordingly, various drugs and treatments for osteoclasts are being actively studied. For example, in the treatment of bone damage caused by osteoclasts such as osteoporosis, bisphosphonates such as Fosamax (ingredient name: aledronate), Actonel (ingredient name: risedronate), Zometa (ingredient name: zoledronate), etc. Therapeutic agents are widely used. Most of these bisphosphonate agents act to delay or inhibit bone loss by weakening the function of osteoclasts that destroy bone and inducing the death of osteoclasts. However, the incidence of various side effects such as osteonecrosis, severe atrial fibrillation, bone or joint incapacity, and musculoskeletal pain in patients taking bisphosphonate drugs is increasing every year (Coleman RE., Br J Cancer , 98:1736-1740 (2008)]). In addition, the formation of osteoclasts is promoted by cancer cells that have metastasized to bone in breast cancer, prostate cancer, etc., and serious bone diseases occur, but drugs for treating them have not been developed.

As such, there is a need for development of drugs that can compensate for the disadvantages of the existing agents, have no toxicity, and effectively inhibit bone resorption by osteoclasts. Accordingly, the present inventors have studied natural products that can prevent and treat diseases induced or caused by bone loss by effectively inhibiting osteoclast formation to reduce bone loss, without side effects and toxicity, By suppressing the formation of osteoclasts, the extracts of ginseng ginseng, rhododendron, evening primrose, and lemon balm extracts have a preventive or therapeutic effect on bone diseases, thereby completing the present invention.

The present invention has been devised to solve the problems in the prior art as described above, and an object of the present invention is to provide various plant extract compositions for the prevention or treatment of bone diseases.

In addition, another object of the present invention is to provide a pharmaceutical composition for the prevention or treatment of bone diseases, each comprising a ginseng plant, rhododendron, evening primrose, ground algae, or lemon balm extract as an active ingredient.

In addition, another object of the present invention is to provide a food composition for the prevention or improvement of bone diseases, each comprising as an active ingredient an extract of ginseng ginseng, rhododendron, evening primrose, ground algae, or lemon balm.

Hereinafter, various embodiments described herein are described with reference to the drawings. In the following description, various specific details are set forth, such as specific forms, compositions, processes, and the like, for a thorough understanding of the present invention. However, certain embodiments may be practiced without one or more of these specific details, or in conjunction with other known methods and forms. In other instances, well-known processes and manufacturing techniques have not been described in specific detail in order not to unnecessarily obscure the present invention. Reference throughout this specification to “one embodiment” or “an embodiment” means that a particular feature, form, composition, or characteristic described in connection with the embodiment is included in one or more embodiments of the invention. Thus, references to "in one embodiment" or "an embodiment" in various places throughout this specification do not necessarily refer to the same embodiment of the invention. Additionally, the particular features, forms, compositions, or properties may be combined in any suitable way in one or more embodiments.

"Sedum middendorfianum Max." used in the present invention is a perennial herb in the family Sedumaceae and is mainly distributed in the alpine regions north of central Korea. The medicinal part mainly uses the above-ground part, and it is known to be used for smoothing blood circulation, relieving muscle tension, and reducing heat and removing poison.

"Rumex obtusifolius" used in the present invention is a perennial herb, and the medicinal site is mainly used for the root. It is known to be effective in insecticidal, diarrhea, antipyretic, eohyeol, dry stomach, beriberi, edema, jaundice, constipation, coma, postpartum pain, and skin diseases.

"Evening primrose (Oenothera odorata)" used in the present invention is a naturalized plant native to Chile, South America, and is a biennial herb belonging to the Achilles family. The roots are collected in spring or summer and dried in the sun, and the fully ripened seeds are collected in autumn and roasted for use as oil. Roots are used for high fever due to cold and sore throat, put in water and decocted, and seeds are used for hyperlipidemia. Unsaturated fatty acid called gamma-linolenic acid contained in evening primrose oil becomes the parent of physiologically active substances called prostaglandins, which are physiological actions of prostaglandins in the human body, such as constriction and expansion of blood vessels, contraction and relaxation of bronchial muscles, inhibition of gastric juice secretion, uterine muscle contraction, It is known to improve circulatory system disorders such as myocardial infarction, arteriosclerosis, hypertension, and angina pectoris or cardiovascular diseases through water excretion and induction or inhibition of platelet aggregation. It is also effective for rheumatoid arthritis and is also effective for skin care.

"Physalis angulata L." used in the present invention is a dicotyledonous annual plant of the Solanaceae family, native to tropical Africa. Anti-tumor activity, anti-inflammatory and pain suppression, anti-leukemia and anti-mutagenic activity have been reported.

"Lemon balm (Melissa officinalis)" used in the present invention is a perennial plant native to southern Europe and the Mediterranean coast. It is known for its soothing, digestive, sweating, and antipyretic effects, and the essential oil contained in the leaves is good for depression, neurological headache, memory loss, neuralgia, fever, etc. used as medicine Herbal tea made from the leaves boosts brain activity, improves memory, and even eliminates depression.

The term “above the ground” used in the present invention means a part exposed above the ground (soil) among parts constituting a plant. In general, elements belonging to the above-ground part include, but are not limited to, stems, leaves, flowers, bark, fruits, and the like.

As used in the present invention, the term “underground part” refers to a part below the ground (soil) among parts constituting a plant. In general, an element belonging to the underground part means a root, and the root includes stored roots such as radish, carrot, ginseng, arrowroot, and sweet potato. Even if it exists under the ground or stems such as bamboo shoots, taro, and lotus root, it may be included in the underground part.

As used herein, the term “extract” refers to an active ingredient separated from a natural product, for example, a preparation concentrated by evaporating the solvent after squeezing the natural product with an appropriate solvent and filtering it. In addition, it may be an extract obtained by extraction treatment, a diluted or concentrated solution of the extract, a dried product obtained by drying the extract, or a purified product thereof, but is not limited thereto. The extraction method is not limited thereto, but it can be obtained by an extraction method selected from the group consisting of a solvent extraction method, an ultrasonic extraction method, a supercritical extraction method, a fermentation method and a foaming method, and more specifically, it is preferable to use a solvent extraction method. In the solvent extraction method, extraction may be performed with a solvent selected from the group consisting of water, an organic solvent, and a mixed solvent thereof, and the organic solvent may preferably be ethanol.

As used herein, the term “bone disease” refers to a disease caused by an imbalance between osteoclasts and osteoblasts in the bone, and is a condition or disease caused by excessive generation and/or movement of osteoclasts. to be. The bone disease is preferably osteoporosis, osteomalacia, rickets, osteopenia, fibrous osteomyelitis, aplastic bone disease, osteogenesis imperfecta, bone atrophy, paget's disease, periodontitis, rheumatoid arthritis, metabolic There are bone diseases and bone damage caused by bone metastasis of cancer cells, but is not limited thereto as long as it is a disease caused by bone resorption by osteoclasts.

As used herein, the terms “prevention or treatment of bone disease” and “prevention or improvement of bone disease” are achieved by administering the composition according to the present invention to an individual. This includes the prevention, amelioration and treatment of bone disease, including reduction, amelioration, pain relief, reduction in incidence, and complete or partial treatment of bone disease symptoms.

As used herein, the term “individual” refers to all animals, including humans, that have or can develop bone disease. By administering the composition of the present invention to an individual, the bone disease can be effectively prevented or treated, and bone disease can be improved.

As used herein, the term “administration” means introducing a predetermined substance to an individual in an appropriate way, and the administration route of the composition of the present invention may be administered through any general route as long as it can reach the target tissue. can These include, but are not limited to, intraperitoneal administration, intravenous administration, intramuscular administration, subcutaneous administration, intradermal administration, oral administration, topical administration, intranasal administration, intrapulmonary administration, and rectal administration.

The term “pharmaceutical composition” used in the present invention refers to a composition administered for a specific purpose, and the pharmaceutical composition of the present invention contains the plant extract as an active ingredient and aims to prevent or treat bone diseases . The pharmaceutical composition may be in the form of capsules, tablets, granules, injections, ointments, powders or beverages, and oral dosage forms such as powders, granules, capsules, tablets, aqueous suspensions, etc., external preparations, suppositories and It may be formulated and used in the form of a sterile injectable solution, but is not limited thereto.

In addition, the pharmaceutical composition of the present invention may include a pharmaceutically acceptable carrier, and the pharmaceutically acceptable carrier is a binder, lubricating agent, disintegrant, excipient, solubilizer, dispersing agent, stabilizing agent, and suspending agent when administered orally. A topical agent, colorant, flavoring agent, etc. can be used, and in the case of injection, a buffer, preservative, analgesic agent, solubilizer, isotonic agent, stabilizer, etc. can be mixed and used. In the case of topical administration, a base, excipient, lubricant, Preservatives and the like may be used. The dosage form of the pharmaceutical composition of the present invention can be prepared in various ways by mixing with a pharmaceutically acceptable carrier as described above. For example, in the case of oral administration, it may be prepared in the form of tablets, troches, capsules, elixirs, suspensions, syrups, wafers, etc., and in the case of injections, it may be prepared in the form of unit dose ampoules or multiple doses. have. In addition, it may be formulated as a solution, suspension, tablet, capsule, sustained-release preparation, and the like. Further, examples of suitable carriers, excipients and diluents for formulation include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, malditol, starch, acacia gum, alginate, gelatin, calcium phosphate, calcium silicate , cellulose, methyl cellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate or mineral oil, etc. may be used, and fillers, anti-agglomeration agents, lubricants, Wetting agents, fragrances, emulsifiers, preservatives, and the like may be further included.

As used herein, the term “pharmaceutically effective amount” refers to an amount sufficient to treat a disease and not to cause side effects, and the effective dose level is determined by the sex, age, weight, health status, and disease of the individual. Depending on the type, severity, activity of the drug, sensitivity to the drug, administration method, administration time, administration route, and excretion rate, duration of treatment, factors including drugs used in combination or concurrently, and other factors well known in the medical field, the can be easily determined by In general, it may be administered at a dose of about 0.01 mg/kg/day to 1000 mg/kg/day. For oral administration, a range of 50 to 500 mg/kg may be suitable, and may be administered at least once a day, but is not limited thereto. In addition, the pharmaceutical composition according to the present invention may be formulated as pills, dragees, capsules, solutions, gels, syrups, slurries, suspensions, and the like.

The term “food composition” used in the present invention refers to a composition made by using the composition of the present invention as a raw material for food for the purpose of health supplementation, and is manufactured and processed by methods such as extraction, concentration, purification, and mixing to produce health food. can The health food manufactured in this way may perform functions related to disease prevention and improvement of diseases. There is no limitation on the type of health food, and the food composition of the present invention can be prepared by mixing appropriate other auxiliary ingredients and additives that may be contained in food according to those skilled in the art.

Examples of foods that can be added include meat, sausage, bread, chocolate, candy, snacks, confectionery, pizza, ramen, other noodles, gums, dairy products including ice cream, various soups, beverages, tea, drinks, alcoholic beverages and There are vitamin complexes and the like, and it can be prepared by adding the extract according to the present invention as a main component to juice, tea, jelly, juice, and the like. In addition, it includes all health foods in the ordinary sense. In addition, the food composition of the present invention may contain various flavoring agents or natural carbohydrates as additional ingredients, like conventional beverages. The natural carbohydrate may include monosaccharides such as glucose and fructose, disaccharides such as maltose and sucrose, and natural sweeteners such as dextrin and cyclodextrin, and synthetic sweeteners such as saccharin and aspartame. The ratio of the natural carbohydrate can be appropriately determined by the selection of those skilled in the art.

In addition, the food composition of the present invention includes various nutrients, vitamins, electrolytes, flavoring agents, coloring agents, pectic acid and its salts, alginic acid and its salts, organic acids, protective colloidal thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohol , a carbonation agent used in carbonated beverages, and the like. In addition, the composition of the present invention may contain pulp for the production of natural fruit juice, fruit juice beverage, and vegetable beverage. These components may be used independently or in combination, and the proportion of these additives may also be appropriately selected by those skilled in the art.

In one embodiment of the present invention, there is provided a pharmaceutical composition for the prevention or treatment of bone diseases, comprising an extract of a plant species such as ginseng ginseng, rhododendron, evening primrose, ground alfalfa, or lemon balm as an active ingredient, The plant provides a pharmaceutical composition of any one or more selected from the group consisting of Suyeong, Arabidopsis, rhododendron, sorijagi, mulberry, and dog rhubarb, and the extract is ginseng plant, rhododendron, evening primrose, ground algae. , or lemon balm root, rhizome, flower, leaf, stem, seed, fruit, fruit disease, pericarp, bark, or whole herb extract provides a pharmaceutical composition, wherein the bone disease is osteoporosis, osteomalacia, rickets, osteopenia, fibrous osteomyelitis , from the group consisting of bone damage caused by aplastic bone disease, osteoplasty, bone atrophy, Paget's disease, periodontitis, rheumatoid arthritis, metabolic bone disease, and bone metastasis of cancer cells It provides a pharmaceutical composition of any one or more selected, the pharmaceutical composition provides a pharmaceutical composition that inhibits the formation of osteoclasts.

In another embodiment of the present invention, it provides a food composition for the prevention or improvement of bone diseases, comprising an extract of a plant species such as ginseng herb, rhododendron, evening primrose, ground algae, or lemon balm as an active ingredient, Plants provide a food composition of any one or more selected from the group consisting of Suyeong, Arabidopsis, rhododendron, sorijae, mulberry, and dog rhubarb, and the extract is ginseng plant, rhododendron, evening primrose, ground algae. , or lemon balm root, rhizome, flower, leaf, stem, seed, fruit, fruit disease, pericarp, bark, or whole herb extract provides a food composition, wherein the bone disease is osteoporosis, osteomalacia, rickets, osteopenia, fibroostitis , from the group consisting of bone damage caused by aplastic bone disease, osteoplasty, bone atrophy, paget's disease, periodontitis, rheumatoid arthritis, metabolic bone disease, and bone metastasis of cancer cells It provides a food composition of any one or more selected, the pharmaceutical composition provides a food composition that inhibits the formation of osteoclasts.

In another embodiment of the present invention, (a) the step of powdering a plant material of ginseng giraffe, rhododendron, evening primrose, ground algae, or lemon balm; and (b) extracting the powder with an organic solvent in a solvent extraction device and then filtering it. It provides a manufacturing method that is °C, and the extracting step provides a manufacturing method that is extracted with ethanol or methanol, and the plant material is the root of a giraffe giraffe, rhododendron, evening primrose, groundnut, or lemon balm, It provides a method for producing a rhizome, flower, leaf, stem, seed, fruit, fruit disease, pericarp, bark, or outpost, wherein the bone disease is osteoporosis, osteomalacia, rickets, osteopenia, fibrous osteoarthritis, aplastic bone disease, and osteogenesis imperfecta. , bone atrophy, paget's disease, periodontitis, rheumatoid arthritis, metabolic bone disease, and bone damage caused by bone metastasis of cancer cells. to provide.

Hereinafter, the present invention will be described in detail step by step.

The composition comprising the plant extract according to the present invention as an active ingredient is stable with low cytotoxicity, and inhibits the differentiation of osteoclasts to prevent and treat bone diseases, pharmaceutical compositions for prevention and treatment, and food compositions for prevention and improvement of bone diseases, etc. It is expected that it will be usable.

1 is a view showing the measurement results of cytotoxicity according to the concentration of ginseng ginseng, rhododendron, evening primrose, ground algae, or lemon balm plant extract according to an embodiment of the present invention.
Figure 2 is a view showing the cytotoxicity measurement results for each concentration of the plant extract according to an embodiment of the present invention.
Figure 3 is a view showing the osteoclast count results according to the concentration of ginseng giraffe, rhododendron, evening primrose, ground algae, or lemon balm extract according to an embodiment of the present invention.
Figure 4 is a view showing the results of confirming the osteoclast differentiation inhibitory effect according to the concentration of the extract according to an embodiment of the present invention, ginseng ginseng, rhododendron, evening primrose, ground algae, or lemon balm extract.
5 is a view showing the osteoclast count results for each concentration of the plant extract according to an embodiment of the present invention.
FIG. 6 is a view showing the results of confirming the osteoclast differentiation inhibitory effect of an extract of rhubarb, which is a genus of rhododendrons, according to an embodiment of the present invention.
7 is a view showing the results of confirming the osteoclast differentiation inhibitory effect of the Arabidopsis Suyeong extract, which is a rhododendron plant according to an embodiment of the present invention.
FIG. 8 is a view showing the results of confirming the osteoclast differentiation inhibitory effect of the extract of Sorijai, which is an animal and plant belonging to the genus of the present invention, according to an embodiment of the present invention.
FIG. 9 is a view showing the results of confirming the osteoclast differentiation inhibitory effect of the Mukbatsorijae extract, which is an animal and plant belonging to the rhododendron in accordance with an embodiment of the present invention.
10 is a view showing the results of confirming the osteoclast differentiation inhibitory effect of the above-ground part and the underground part of the extract of the dolphinaceae according to an embodiment of the present invention.
11 is a view showing the results of confirming the osteoclast differentiation inhibitory effect of the Suyeong extract, which is a rhododendron plant, according to an embodiment of the present invention.

Hereinafter, the present invention will be described in more detail through examples. These examples are only for illustrating the present invention in more detail, and it will be apparent to those skilled in the art that the scope of the present invention is not limited by these examples according to the gist of the present invention. .

Preparation Example: Preparation of Plant Extract

Specimens of natural products selected as test subjects of the present invention, such as giraffe grass, rhododendron, evening primrose, ground algae, and lemon balm, were collected and dried. The plants were used to distinguish specific parts such as roots, rhizomes, flowers, seeds, or outposts. 2 kg of each dried sample powder was extracted and filtered with 80% ethanol in an accelerated solvent extractor (Accelerated Solution Extractor, Dinex, USA) at 50° C., and then the solvent was evaporated and concentrated to obtain each plant extract.

The flora and fauna of the Arabidopsis ginseng plant, rhododendron, evening primrose, ground algae, and lemon balm are shown in Table 1 below, and information on extracts of the flora and fauna used in the present invention is shown in Table 2. Plants listed in Table 2 all belong to the family Polygonaceae, and were extracted using 99.9% methanol (HPLC grade).

botanical name animal and plant name baby giraffe Sedum oryzifolium (Sedum oryzifolium) Giraffe grass (Sedum kamtschaticum) Sedum polystichoides Taebaek giraffe (Sedum latiovalifolium) Sedum sarmentosum (Sedum sarmentosum) Sedum viviparum (Sedum viviparum) Sedum takesimense Sedum spectabile (Sedum spectabile) Sedum rotundifolium Sedum bulbiferum (Sedum bulbiferum) boulder rocker Swimming (Rumex acetosa) Rumex conglomeratus (Rumex conglomeratus) Baby swimming (Rumex acetocella) Rumex crispus Rumex longifolious evening primrose Oenothera laciniate Evening primrose (Oenothera lamarckiana) big raven Solanum lyratum Solanum nigrum (Solanum nigrum) Eggplant (Solanum melongena)

country name scientific name part collection site collection date One muffler Rumex conglomeratus above ground Ulleungdo 2002.05.09. 2 muffler Rumex conglomeratus underground Ulleungdo 2002.05.09. 3 boulder rocker Rumex obtusifolius above ground Jeollanam-do 2003.04.15. 4 boulder rocker Rumex obtusifolius underground Jeollanam-do 2003.04.15. 5 screamer Rumex crispus above ground Jeollabuk do 2003.05.29. 6 screamer Rumex crispus underground Jeollabuk do 2003.05.29. 7 screamer Rumex crispus outpost Jeju Island 2003.05.22. 8 dog rhubarb Rumex longifolious above ground Gangwon-do 2006.05.17. 9 dog rhubarb Rumex longifolious underground Gangwon-do 2006.05.17. 10 baby swimming Rumex acetocella outpost Gangwon-do 2008.06.02. 11 swimming Rumex acetosa outpost Jeju Island 2007.12.14.

Example 1: Preparation of mouse bone marrow macrophages

For the preparation of mouse bone marrow macrophages, three-week-old male ICR mice (Nara Biotech, Korea) were cervical dislocated and then the skin of the hind legs was peeled off using a forceps, and the skin of the hind legs was cut with surgical scissors to remove the serum. It was soaked in unadded α-MEM (minimum essential medium alpha; Gibco, USA). Then, the bone in the muscle was separated using tweezers, transferred to a new α-MEM, and 600 μl of α-MEM was placed in a syringe, inserted into the central spinal cord of the separated leg bone, and injected 2 to 3 times to extract bone marrow cells. The extracted bone marrow cells were centrifuged to remove the supernatant, mixed with fresh α-MEM, and then bone marrow macrophages were isolated from the bone marrow cells using a separation medium histopaque (Sigma-Aldrich, USA). And in α-MEM 1% antibiotic-antimycotic (Gibco, USA), 10% fetal bovine serum (FBS); Gibco, USA), M-CSF (macrophage-colony stimulating factor) ; R&D system Inc., USA) After 30 ng/ml was added, the isolated mouse bone marrow macrophages were cultured.

Example 2: Cytotoxicity measurement experiment of plant extracts

In order to confirm the cytotoxicity of the plant extract described in Preparation Example, mouse bone marrow macrophages prepared by the method described in Example 1 were used. The plant extract was dissolved in DMSO (dimethyl sulfoxide; Sigma-Aldrich, USA) and diluted using α-MEM to which 1% antibiotic-antibacterial solution, 10% FBS, and 30 ng/ml M-CSF were added, It was diluted to a concentration of 5, 10, 20, 40, or 80 μg/ml, respectively. After that, 5x10 ⁴ mouse bone marrow macrophages were added to each well of the 96-well plate, and 200 μl of each of α-MEM to which the diluted extract was added was added and a cell incubator at 37° C., 5% CO ₂ condition. Mice bone marrow macrophages were cultured in Thereafter, the extract was replaced with a new α-MEM (1% antibiotic-antibacterial solution, 10% FBS, and 30 ng/ml of M-CSF added) every two days and cultured for 5 days. After 5 days, 20 μl of 5 mg/ml MTT (Thiazolyl Blue Tetrazolium Bromide; Sigma Aldrich, USA) solution was added to each well and put into a cell incubator at 37° C., 5% CO ₂ and reacted for 4 hours. After removing the medium, 100 μl of DMSO was added, dissolved in a shaker for 20 minutes, and absorbance was measured at 570 nm using an ELISA reader to confirm cell viability and toxicity of the sample. Cell viability was calculated as a percentage of the viability of the experimental group (each well treated with each extract) relative to the viability of the control group (well treated with medium only). The cell viability results of the Arabidopsis ginseng plant, rhododendron, evening primrose, ground algae, or lemon balm extract are shown in Table 3 and FIG. 1, and the cell viability results of the rhododendron plant extract are shown in Tables 4 to 9 and FIG. It was.

As a result of the experiment, the A. giraffe root extract, the rhododendron flower extract, and the evening primrose root extract showed higher cell viability than the control group at 40 μg/ml. Evening primrose seed extract showed more than 70% cell viability at concentrations of 20 μg/ml, above ground alfalfa extract, 40 μg/ml, and lemon balm root extract at 40 μg/ml, and it was confirmed that there was no significant cell viability inhibitory effect. Among the plant species of dolphinaceae, extracts of mukbatsorijae and sorijagi increased the cell viability. It was confirmed that these plant extracts do not exhibit toxicity to bone marrow macrophages. On the other hand, the above-ground parts of dolphins, rhubarb and Arabidopsis extract decreased the cell viability as the concentration increased. In subsequent experiments, the experiment was conducted at a concentration that did not show significant cytotoxicity.

botanical name part Concentration (μg/ml) absorbance value Cell viability (%) control - 1.206±0.101 100 baby giraffe Root 5 2.499±0.197 207 10 2.384±0.117 198 20 2.196±0.187 182 40 1.764±0.026 146 boulder rocker Flower 5 2.733±0.174 227 10 2.722±0.094 226 20 2.672±0.189 222 40 2.510±0.216 208 evening primrose Root 5 2.762±0.200 229 10 2.543±0.079 211 20 2.288±0.149 190 40 1.910±0.184 158 evening primrose strain 5 2.236±0.079 185 10 2.230±0.228 185 20 1.895±0.102 157 40 0.186±0.026 15 ground algae above ground 5 1.122±0.066 93 10 1.127±0.186 93 20 1.121±0.173 93 40 0.939±0.163 78 lemon balm Root 5 1.530±0.060 127 10 1.290±0.193 107 20 1.008±0.013 84 40 0.886±0.187 73

botanical name part Concentration (μg/ml) absorbance value Cell viability (%) control 0 1.433±0.085 100 muffler above ground 20 2.130±0.150 137 40 2.462±0.103 172 80 1.614±0.234 113 muffler underground 20 2.320±0.087 162 40 3.298±0.062 230 80 3.305±0.135 231

botanical name part Concentration (μg/ml) absorbance value Cell viability (%) control 0 1.282±0.073 100 boulder rocker
above ground 20 1.171±0.079 94 40 0.718±0.065 56 80 0.572±0.105 45 boulder rocker underground 20 1.465±0.122 114 40 1.402±0.070 109 80 1.705±0.092 133

botanical name part Concentration (μg/ml) absorbance value Cell viability (%) control 0 1.755±0.018 100 screamer above ground 20 3.138±0.206 166 40 2.881±0.310 164 80 2.041±0.148 116 screamer underground 20 2.107±0.014 120 40 2.735±0.057 156 80 3.265±0.077 186 screamer outpost 20 2.776±0.245 158 40 2.980±0.104 170 80 1.977±0.182 113

botanical name part Concentration (μg/ml) absorbance value Cell viability (%) control 0 1.820±0.149 100 dog rhubarb above ground 20 2.361±0.126 122 40 1.562±0.103 86 80 1.141±0.034 63 dog rhubarb underground 20 1.338±0.087 74 40 0.631±0.033 35 80 0.106±0.004 6

botanical name part Concentration (μg/ml) absorbance value Cell viability (%) control 0 1.749±0.034 100 baby swimming outpost above ground 20 2.411±0.172 131 40 1.686±0.195 96 80 1.018±0.054 58

botanical name part Concentration (μg/ml) absorbance value Cell viability (%) control 0 1.657±0.075 100 swimming outpost 20 1.954±0.062 114 40 1.702±0.136 103 80 2.075±0.100 125

Example 3: Confirmation of osteoclast formation inhibitory ability of plant extracts

In order to confirm whether the preparation according to the present invention inhibits osteoclast formation induced by RANK (receptor activator of nuclear factor kappa-B ligand), the literature [Park EK. et al ., Biochem Biophys Res Commun ., 325 (4): 1472-1480 (2004)], the osteoclast formation inhibitory ability was confirmed by the method described. Each plant extract was prepared by dilution to 10, or 20 μg/ml. After adding 5x10 ⁴ mouse bone marrow macrophages prepared by the method of Example 1 to each well of a 96-well plate, α-MEM (1% of antibiotic-antibacterial solution, 10% of each diluted extract) FBS, 30 ng/ml of M-CSF, and 50 ng/ml of RANKL) were added by 200 μl, respectively, and the mouse bone marrow macrophages were cultured in a cell incubator at 37° C. and 5% CO ₂ condition.

As a negative control group, cells were cultured using α-MEM supplemented with 1% antibiotic-antibacterial solution, 10% FBS, and 30 ng/ml M-CSF, and as a RANKL-treated group, 1% antibiotic-antibacterial solution , cells were cultured using α-MEM supplemented with 10% FBS, 30 ng/ml M-CSF and 50 ng/ml RANKL. And every two days, each extract was exchanged with a new α-MEM (1% antibiotic-antibacterial solution, 10% FBS, 30 ng/ml M-CSF, 50 ng/ml RANKL added) and cultured for 5 days. After 5 days, multinucleated osteoclasts were stained using a TRAP assay kit (tartrate resistant acid phosphatase assay kit; Sigma-Aldrich, USA), and the number of osteoclasts having three or more nuclei was counted using an optical microscope. The osteoclast counting results of the Arabidopsis ginseng plant, rhododendron, evening primrose, ground lichen, or lemon balm extract are shown in Table 10 and FIGS. 3 and 4, and the osteoclast counting results of the rhododendron plant extract are shown in Tables 11 to 16 , and are shown in FIGS. 5 to 11 .

As a result of the experiment, in the case of the group treated with 50ng/ml of RANKL, the number of osteoclasts was significantly increased, and when each extract was treated, it was confirmed that the formation of osteoclasts was reduced according to the concentration. Cylindrica root extract 98% at 10 μg/ml and 100% at 20 μg/ml, rhododendron flower extract at 86% at 10 μg/ml and 97% at 20 μg/ml, evening primrose root extract at 10 μg/ml 97% and 100% at 20 μg/ml, evening primrose seed extract 75% at 10 μg/ml and 100% at 20 μg/ml, ground alfalfa extract 100% at 10 and 20 μg/ml, lemon balm root extract 99% at 10 μg/ml and at 20 μg/ml, it was confirmed that 100% of osteoclast differentiation was inhibited.

Through the above results, it was confirmed that each extract has the ability to inhibit osteoclast formation, and through this, it was confirmed that each extract can be used for diseases caused by osteoclast formation.

botanical name part Concentration (μg/ml) Number of osteoclasts induced by RANKL Osteoclast formation inhibition rate (%) control - 0±0 - RANKL treatment group RANKL 229±2 0 baby giraffe Root RANKL+10 6±2 98 RANKL+20 0±0 100 boulder rocker Flower RANKL+10 32±8 86 RANKL+20 7±3 97 evening primrose Root RANKL+10 8±1 97 RANKL+20 0±0 100 evening primrose strain RANKL+10 57±16 75 RANKL+20 0±0 100 ground algae above ground RANKL+10 0±0 100 RANKL+20 0±0 100 lemon balm Root RANKL+10 3±1 99 RANKL+20 0±0 100

botanical name part Concentration (μg/ml) Number of osteoclasts induced by RANKL Osteoclast formation inhibition rate (%) control 0 304±25 0 muffler above ground 20 277±32 9 40 60±14 80 muffler underground 20 231±12 24 40 182±13 40

botanical name part Concentration (μg/ml) Number of osteoclasts induced by RANKL Osteoclast formation inhibition rate (%) control 0 267±14 0 boulder bum above ground 20 279±16 0 40 176±20 34 boulder rocker underground 20 199±35 25 40 10±1 96

botanical name part Concentration (μg/ml) Number of osteoclasts induced by RANKL Osteoclast formation inhibition rate (%) control 0 272±7 0 screamer above ground 20 259±8 5 40 5±3 98 screamer underground 20 272±14 0 40 328±36 0 screamer outpost 20 8±4 97 40 0±0 100

botanical name part Concentration (μg/ml) Number of osteoclasts induced by RANKL Osteoclast formation inhibition rate (%) control 0 300±14 0 dog rhubarb above ground 20 8±2 97 40 0±0 100 dog rhubarb underground 10 206±8 31 20 35±14 88

botanical name part Concentration (μg/ml) Number of osteoclasts induced by RANKL Osteoclast formation inhibition rate (%) control 0 306±16 0 baby swimming outpost above ground 20 80±12 74 40 1±1 100

botanical name part Concentration (μg/ml) Number of osteoclasts induced by RANKL Osteoclast formation inhibition rate (%) control 0 237±20 0 swimming outpost 20 195±6 18 40 69±1 71

As described above, as a result of confirming cytotoxicity and osteoclast formation inhibitory ability using the plant extract according to the present invention, it was confirmed that differentiation into osteoclasts was remarkably inhibited even under culture conditions inducing osteoclast differentiation, Since it does not show cytotoxicity, it was confirmed that it can be used as a pharmaceutical composition or a food composition for the prevention, improvement or treatment of bone diseases.

Claims (10)

As a pharmaceutical composition for the prevention or treatment of bone diseases caused by osteoclast hyperplasia, comprising the root extract of A. giraffe root as an active ingredient,
The bone disease is, osteoporosis, osteomalacia, rickets, osteopenia, fibrous osteomyelitis, osteogenesis imperfecta, bone atrophy, Paget's disease, periodontitis, rheumatoid arthritis, metabolic bone disease and bone metastasis of cancer cells Any one or more selected from the group consisting of bone damage caused thereby, a pharmaceutical composition.
delete delete A functional food composition for inhibiting osteoclast hyperplasia, comprising a ginseng root extract as an active ingredient.
5. The method of claim 4,
The composition is effective for osteoporosis, osteomalacia, rickets, osteopenia, fibrous osteomyelitis, osteogenesis imperfecta, bone atrophy, Paget's disease, periodontitis, rheumatoid arthritis, metabolic bone disease, and bone metastasis of cancer cells. A food composition for preventing or improving any one or more bone diseases selected from the group consisting of bone damage caused by
delete (A) step of pulverizing the root of the agarine giraffe; and
(b) as a method for producing a pharmaceutical composition for the prevention or treatment of bone diseases caused by osteoclast hyperplasia, comprising the step of extracting the powder with an organic solvent in a solvent extraction device and then filtering,
Bone diseases caused by osteoclast hyperplasia are osteoporosis, osteomalacia, rickets, osteopenia, fibrous osteomyelitis, osteogenesis imperfecta, bone atrophy, paget's disease, periodontitis, rheumatoid arthritis, metabolic bone Any one or more selected from the group consisting of disease and bone damage caused by bone metastasis of cancer cells, the manufacturing method.
8. The method of claim 7,
The temperature of the solvent extraction device is 30 ° C to 60 ° C, the manufacturing method.
8. The method of claim 7,
The extracting step is to extract with ethanol or methanol, the manufacturing method.
delete

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