KR101668845B1 - A pharmaceutical composition for preventing or treating bone diseases comprising Dendropanax morbifera extract - Google Patents

Abstract

The present invention relates to a pharmaceutical composition including a Dendropanax morbifera extract for preventing or treating a bone disease, and more particularly to use of a Dendropanax morbifera extract inhibiting the activity of receptor activator of nuclear factor kappa-B ligand (RANKL), which induces maturation of osteophages, a fraction of the extract, and compounds isolated from the fraction for treating a bone disease. Since the Dendropanax morbifera extract, the fraction thereof, the compounds isolated from the fraction or a pharmaceutically acceptable salt of the compound, according to the present invention, inhibits differentiation of osteophages, the pharmaceutical composition including the material as an active ingredient can prevent, alleviate, or treat various bone diseases.

Description

TECHNICAL FIELD The present invention relates to a pharmaceutical composition for preventing or treating bone diseases, which comprises extracts of Dendropanax morbifera extract,

The present invention relates to a pharmaceutical composition for preventing or treating osteoporosis, which comprises an extract of Ganoderma lucidum, and more particularly to a pharmaceutical composition for inhibiting the activity of RANKL (Receptor activator of nuclear factor kappa-B ligand) Extracts thereof, fractions thereof, and compounds isolated therefrom.

Reshaping of the bone occurs continuously in response to mechanical stress and hormonal control, while maintaining bone retention. To maintain normal bone mass, the bone is reabsorbed by osteoclasts through a tightly coupled process and new osteoblasts are accumulated by osteoblasts. As seen in many common bone diseases, imbalance in this process can lead to excessive bone resorption by osteoclasts and increased bone fragility.

It is known that osteoclasts are differentiated from hematopoietic precursors or myeloid precursors by the hormones secreted from T-cells, fibroblasts, osteoblasts, etc. existing around them. In osteoclast differentiation, signal transduction by RANKL plays an important role. RANKL is produced in osteoblasts, activated T cells and stromal cells, and activates osteoclast morphology, survival and bone resorption with M-CSF (macrophage colony-stimulating factor). When RANKL binds to the receptor RANK, TRAF6 (TNF receptor-associated factor 6) accumulates in the cytoplasmic domain of RANK, resulting in NF-κB and JNK (c-Jun N-terminal kinase), p38 (p38 MAP kinase) , And mitogen-activated protein kinase (MAPK) signaling pathways including ERK (extracellular signal-regulated kinase). Activated NF-κB induces the expression of downstream c-Fos and NFATc1, which are known to play a key role in osteoclast differentiation by RANKL. In addition, it is known that osteoclast differentiation is promoted by inflammatory cytokines such as IL-1, IL-3 and PGE.

Finally, the differentiated osteoclasts are in the form of multi-nulceated cells, which bind to the site of bone resorption and form a sealing zone using actin, vinculin, etc. And becomes airtight. After that, the pH is lowered by pumping the hydrogen ions to dissolve the hydroxyapatite component and secrete proteolytic enzymes such as MMP-9 or cathepsin K to decompose the collagen component, which accounts for about 10% of the bone. Since the final differentiated osteoclasts are known to specifically express phosphatase (tartrate-resistant acid phosphatase (TRAP), which is active in the presence of tartaric acid, osteoclast differentiation can be confirmed by measuring the activity of TRAP.

As described above, osteoclasts play an important role in bone metabolism in vivo. However, when the osteoclasts are over-differentiated due to various causes, diseases resulting in bone tissue disorders or bone tissue destruction may be induced. Osteoporosis, rheumatoid arthritis, periodontal disease, Paget disease, metastatic bone cancers, osteoporosis, osteoporosis, osteoporosis, osteoclastic bone resorption, And the like.

In order to treat the above-mentioned bone diseases, it is necessary to control the balance between osteoclasts and osteoblasts. Accordingly, there are a bone resorption inhibitor and a bone formation stimulator. In general, the therapeutic agent for bone diseases such as the bone resorption inhibitor or the bone formation stimulating agent should be administered to a patient for a long period of time, so that it should be low in toxicity. Oral administration is preferable to solve the complication of administration. It is true. On the other hand, studies on osteogenesis stimulants promoting osteogenesis by activating osteoblasts in the bone disease treatment agent have been actively carried out, but strengthening of bone mineral density due to osteogenesis stimulation does not necessarily mean reduction of fracture. On the other hand, Denosumab (Prolia ™, Amgen ^® ), which is currently being used to treat osteoclast activity, has been developed and various drugs have been developed. However, considering that the drug treatment should be continued for a long time, Side effects have not been sufficiently verified, and the exact mechanism of osteoclasts has not yet been elucidated.

Accordingly, the present inventors have found that Dendropanax morbifera extract, its fractions, and the compounds isolated therefrom inhibit the differentiation of osteoclasts while searching for natural products inhibiting osteoclast differentiation, thereby completing the present invention.

Accordingly, an object of the present invention is to provide a pharmaceutical composition for preventing or treating bone diseases, comprising Dendropanax morbifera extract, fractions thereof, a compound isolated therefrom, or a pharmaceutically acceptable salt of said compound as an active ingredient .

Another object of the present invention is to provide a food composition for preventing or ameliorating a bone disease comprising Dendropanax morbifera extract, a fraction thereof, a compound isolated therefrom, or a pharmaceutically acceptable salt of said compound as an active ingredient will be.

As one aspect for solving the above problems, the present invention provides a method for preventing bone diseases comprising Dendropanax morbifera extract, a fraction thereof, a compound isolated therefrom, or a pharmaceutically acceptable salt of the compound as an active ingredient Or a pharmaceutical composition for therapeutic use.

Dendropanax morbifera, an evergreen tree of the Araliaceae, is a species of dicotyledonous plant native to the southern coast of Korea and Jeju Island. It is a deciduous species in winter. The physiological activities such as anticancer activity and antioxidant activity of Hwangchulchu extract have been reported to be limited and they have been used in foods such as food additives and health functional supplements (Kim HR, et al .. Chemical characteristics of the leaves and the seeds (2000), Park SN, et al .. Cellular antioxidant activity and whitening effects of dendropanax morbifera leaf extracts, Korea ( Korean ) Dendropanax morphifera Lev., Korean Society for Applied Biological Chemistry Journal of Microbiology and Biotechnology, 41 (4): 407-415, 2013).

The extract of Huchilchia tree contained in the pharmaceutical composition of the present invention may be one obtained by extracting at least one selected from the group consisting of roots, stems, flowers, leaves and fruits of Huchilchia, or a stem, flower, Leaf, and fruit, and may be one obtained by extracting leaves of Hwangchujang.

In addition, the extract according to the present invention may be prepared by extracting Hwangchujang with water, C1-C4 lower alcohol or a mixed solvent thereof, preferably methanol extract.

The term "extract" of the present invention means a preparation in which a herbal medicine is extracted with an appropriate extraction solvent and the extraction solvent is evaporated to produce a concentrate. Although not limited thereto, the extract, the diluted solution or concentrate of the extract, Or a dried product obtained by drying the above, a controlled preparation thereof or a purified product thereof. The extract according to the present invention can be prepared by using common extraction, isolation and purification methods known in the art. The extraction method may be, but not limited to, hot water extraction, hot water extraction, cold extraction, reflux cooling extraction, or ultrasonic extraction.

According to a specific embodiment of the present invention, the extract of Tochigi extract of the present invention is extracted by adding 5 to 15 times of a solvent to a dried weedy tree of a certain weight. The solvent may be water, C1 to C4 lower alcohols or a mixture thereof, preferably methanol.

The pharmaceutical composition according to the present invention may contain the fractions of the Aspergillus oryzae extract of the present invention as an active ingredient.

The term "fraction " of the present invention means a product obtained by a fractionation method for separating a specific component or a specific group from a mixture containing various constituents. The separation of the specific component or the specific group can be achieved by a difference in solubility in these solvents using a concentration gradient of a mixed solvent having a specific property or a mixed solvent containing two or more kinds of solvents, But is not limited thereto. The fractions according to the present invention may be obtained by fractionating the extract of Hwigaechilkai using water, C1-C4 lower alcohol, chloroform, ethyl acetate, hexane, butanol or a mixed solvent thereof, preferably ethyl acetate, butanol or And may be fractionated using a mixed solvent of these.

In a specific example of the present invention, in order to obtain the fractions, the methanol extract of D. torus was sequentially extracted with ethyl acetate and n-butanol to obtain an ethyl acetate soluble fraction and an n-butanol soluble fraction, and a mixture of dichloromethane And methanol or a mixed solvent of n-hexane and ethyl acetate to obtain several fractions. At this time, the mixing ratio of the dichloromethane / methanol mixed solvent is sequentially increased from 100: 1 to 1: 1 or the mixed ratio of the n-hexane / ethyl acetate mixed solvent is gradually increased from 50: 1 to 1: 1 Concentration gradient elution system was applied.

The pharmaceutical composition according to the present invention may contain, as an active ingredient, a compound or a pharmaceutically acceptable salt thereof, which is isolated from the fragrant extract of Hwangcholguk tree or Hwangcholgak extract, and the compound is represented by the following general formulas And compounds represented by the following general formulas (1) and (2). The compounds represented by the general formulas (1) and (2) may be used alone or in combination of two or more.

[Chemical Formula 1]

(2)

(3)

(9Z, 16S) -16-acetyl-9,17-octadecadien-12,14-dinoic acid ((9Z, 16S) -16-acetyl-9,17-octadecadiene- (9Z, 16S) -16-hydroxy-9,17-octadecadiene-12,14-dinoic acid, and the compound of Formula 2 is (9Z, 16S) -16- 17-octadecadiene-12,14-diynoic acid), and the compound of Formula 3 is dendropanoxide.

In addition, the compound may be included in the pharmaceutical composition of the present invention in the form of a pharmaceutically acceptable salt. Salts are useful as acid addition salts formed by pharmaceutically acceptable free acids. The term "pharmaceutically acceptable salt" of the present invention means a concentration that has a relatively non-toxic, harmless and effective action on a patient, and that the side effects caused by the salt do not detract from the beneficial effects of the compounds isolated from the extract. Means any organic or inorganic addition salt of such a compound. In addition, the compounds of the present invention may be used alone or in combination with other pharmaceutically active compounds or in aggregates.

The pharmaceutically acceptable salts of the compounds of the present invention isolated from the extracts or fractions thereof refer to salts prepared according to a conventional method in the art, and such methods are well known to those skilled in the art. In particular, the pharmaceutically acceptable salts include, but are not limited to, salts derived from inorganic and organic acids and bases which are pharmacologically or physiologically acceptable.

The acid addition salt is prepared by a conventional method, for example, by dissolving the compound in an excess amount of an acid aqueous solution and precipitating the salt using a water-miscible organic solvent such as methanol, ethanol, acetone or acetonitrile. The same molar amount of the compound and the acid or alcohol (e.g., glycol monomethyl ether) in water may be heated and then the mixture may be evaporated to dryness, or the precipitated salt may be subjected to suction filtration. As the free acid, organic acids and inorganic acids can be used. As the inorganic acids, hydrochloric acid, phosphoric acid, sulfuric acid, nitric acid, tartaric acid and the like can be used. Examples of the organic acids include methanesulfonic acid, p- toluenesulfonic acid, acetic acid, trifluoroacetic acid, maleic acid, succinic acid, oxalic acid, benzoic acid, tartaric acid, fumaric acid, mandelic acid, propionic acid, citric acid, lactic acid, glycollic acid, gluconic acid, galacturonic acid, glutamic acid, glutaric acid, glucuronic acid, aspartic acid, ascorbic acid, carbonic acid, vanillic acid, hydroiodic acid and the like can be used , But are not limited to these.

In addition, bases can be used to make pharmaceutically acceptable metal salts. The alkali metal salt or alkaline earth metal salt is obtained, for example, by dissolving the compound in an excess amount of an alkali metal hydroxide or alkaline earth metal hydroxide solution, filtering the non-soluble salt salt, and evaporating and drying the filtrate. At this time, it is pharmaceutically acceptable to produce sodium, potassium, or calcium salt, but not limited thereto. The corresponding silver salt can also be obtained by reacting an alkali metal or alkaline earth metal salt with a suitable silver salt (e.g., silver nitrate).

The salt of the compound isolated from the extract of Tochigi leaf extract or its fractions of the present invention may be a pharmaceutically acceptable salt and any salt of the compound of the above-mentioned compound derivative exhibiting an osteoclast production or migration inhibitory effect equivalent to the compound derivative Available.

In addition, the pharmaceutical composition according to the present invention can prevent or treat bone diseases.

The bone diseases include, but are not limited to, osteoporosis, rheumatoid arthritis, periodontal disease, Paget disease, osteoporosis caused by growth retardation, fracture, excessive osteoclast bone resorption, And metastatic bone cancers. ≪ Desc / Clms Page number 2 >

As used herein, the term "bone disease" refers to a condition or disease caused by excessive production and / or migration of osteoclasts, including bone loss diseases. The term "lowering bone disease" refers to a condition or disease in which a decrease in bone mass accompanied by a symptom such as a decrease in bone density and a deterioration in bone tissue occurs, and bone loss caused by osteoclasts activated by cancer cells may be included in the scope of the present invention have.

Preferably, the composition of the present invention can be used for the prevention or treatment of osteoporosis or osteopenia. Specifically, the term "osteoporosis" used in the present invention means a condition in which bone mass is decreased and fracture is likely to occur due to a qualitative change, and "osteopenia" means an initial symptom of osteoporosis. In general, osteopenia is classified as -1.0 to -2.5 based on the bone mineral density (T value), and osteoporosis is classified as -2.5 or more.

As used herein, the term "prevention or treatment of bone disease" includes prevention and complete or partial treatment of the bone disease achieved by administering a composition according to the present invention to a subject. It also includes reduction or amelioration of symptoms of bone disease, relief of the symptoms of pain, reduction in the incidence of bone disease, or any other change in the patient that increases the outcome of the treatment.

As used herein, the term "individual" refers to all animals, including humans, who have developed or are capable of developing bone disease. The composition of the present invention can be administered to an individual to effectively prevent or treat the bone disease. The composition of the present invention may be administered in combination with a conventional bone disease therapeutic agent.

As used herein, the term "administering " means introducing a predetermined substance into a patient in an appropriate manner, and the administration route of the composition of the present invention is administered through any conventional route so long as it can reach the target tissue . But are not limited to, intraperitoneal, intravenous, intramuscular, subcutaneous, intradermal, oral, topical, intranasal, intrathecal, rectal.

The pharmaceutical composition of the present invention may be administered in a pharmaceutically effective amount. The term "pharmaceutically effective amount" means an amount sufficient to treat a disease at a reasonable benefit / risk ratio applicable to medical treatment and not causing side effects, and the effective dose level is determined by the patient's sex, Including, but not limited to, medicaments and other medical fields that are used in combination, or in combination with, or in combination with, a pharmaceutically acceptable carrier, excipient, Can be readily determined by those skilled in the art according to known factors. Generally, the active substance may be administered at a dose of from about 0.01 mg / kg / day to 1000 mg / kg / day. When administered orally, a dose of 50 to 500 mg / kg may be appropriate, and may be administered at least once a day.

Preferably, the composition of the present invention is characterized by inhibiting the production or migration of osteoclasts. As used herein, the term "osteoclast" is a large multinuclear cell that engages in bone resorption and is a cell that melts calcified cartilage and bone tissue to allow bone growth. It has 2 to 100 densely packed oval nuclei with a diameter of 20 to 100 μm, and typical osteoclasts are present in shallow, small grooves of the bone surface formed by oneself. The osteoclast located on the bone resorption surface has a bright band and a wave-like edge, and the absorption of the organic substrate by the hydrolytic enzyme secreted by the osteoclast, absorption of the inorganic matter by the acid, and absorption of the bone occur It is known. The activity of osteoblasts forming bones and osteoclasts reabsorbing bones is organically balanced to regulate the amount of bone tissue.

The osteoclasts migrate from the bone to the microfracture site by chemotaxis. It is formed by cell fusion of mononuclear-macrophage cell line and has the characteristic of expressing TRAP (Tartrate Resistant Acid Phosphatase) and cathepsin K at a high level. It is also characterized by cytoplasm with a homogeneous (foamy appearance) appearance due to high concentrations of vesicles and vacuoles. The fear is a lysosomes filled with acid phosphatase. The endoplasmic reticulum of osteoclasts is sparse, and the Golgi complex is extensive. The corrugated border that is useful for the removal of bone-like matrix is a morphological feature of osteoclasts that actively resorb bone. The mineral portion of the substrate, called hydroxyapatite, contains calcium and phosphate ions, which are absorbed into small vesicles by endocytosis and migrate across the cell, resulting in extracellular fluid extracellular fluid to increase the concentration of ions in the blood.

The osteoclast is regulated by parathyroid hormone (PTH) secreted from the parathyroid gland, calcitonin secreted from the thyroid gland, and growth factor interleukin 6 (IL-6). IL-6 is one of the factors of diseases such as osteoporosis due to an imbalance between aggregate absorption and osteogenesis. Also, the activity of osteoclasts is mediated by the interaction of two molecules, osteoprotegerin and RANK ligand, produced by osteoblasts, and osteoclast differentiation is regulated by these two molecules. The excessive production of osteoclasts causes various osteopathies. The osteoclast extract, its fractions, the compounds isolated therefrom, or the pharmacologically acceptable salts of the compounds or the compounds according to the present invention, Can be suppressed and treated.

Preferably, the pharmaceutical composition of the present invention contains 0.001 to 10% by weight, 0.01 to 10% by weight, 0.1 to 10% by weight of the extract, the fractions thereof, the compound isolated therefrom or the pharmaceutically acceptable salt of the compound or the compound. To 10% by weight, 0.001 to 1% by weight, 0.01 to 1% by weight or 0.1 to 1% by weight.

In addition, the pharmaceutical composition for preventing or treating bone diseases according to the present invention may further comprise a pharmaceutically acceptable carrier, excipient or diluent in addition to the above-mentioned effective ingredient. As used herein, the term "pharmaceutically acceptable" means that the composition is free of toxicity to an individual such as a cell or human being exposed to the composition. Such carriers may be used without limitation as long as they are known in the art such as buffers, preservatives, wetting agents, solubilizers, isotonic agents, stabilizers, bases, excipients and lubricants. Examples of carriers, excipients and diluents that can be included in the pharmaceutical composition of the present invention include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia rubber, alginate, gelatin, calcium phosphate, calcium Silicates, cellulose, methylcellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil.

When the composition of the present invention is formulated, it can be prepared using diluents or excipients such as fillers, extenders, binders, humectants, disintegrants, and surfactants which are usually used.

For oral administration, solid preparations such as tablets, pills, powders, granules, and capsules can be prepared. Such a solid preparation is prepared by mixing at least one excipient such as starch, calcium carbonate, sucrose, lactose, gelatin, etc. in addition to the above composition. In addition to simple excipients, lubricants such as magnesium stearate and talc are also used. Liquid preparations for oral administration include suspensions, solutions, emulsions, syrups, and the like. In addition to water and liquid paraffin which are commonly used simple diluents, various excipients such as wetting agent sweeteners, fragrances and preservatives are included . If the active ingredient is susceptible to degradation by an acid, the oral composition may be formulated to coat the active agent or protect it from degradation from above. Formulations for parenteral administration include sterilized aqueous solutions, non-aqueous solutions, suspensions, emulsions, freeze-dried preparations, and suppositories. Examples of the suspending agent include propylene glycol, polyethylene glycol, vegetable oil such as olive oil, injectable ester such as ethyl oleate, and the like. Examples of suppository bases include withexol, macrogol, tween 61, cacao butter, laurin, glycerogelatin and the like. Carbohydrates such as glucose, sucrose, and dextran, antioxidants such as ascorbic acid, glutathione, chelating agents, low molecular weight proteins, or other stabilizers may be used to increase stability or absorbency of the active ingredient.

The composition of the present invention may also be administered by any device capable of moving the active substance to the target cell. The preferred modes of administration and formulations are intravenous, subcutaneous, intradermal, intramuscular, and drip injections. The injectable solution may be a non-aqueous solvent such as an aqueous solvent such as a physiological saline solution or a ring gel solution, a vegetable oil, a higher fatty acid ester (e.g., oleic acid), an alcohol (e.g., ethanol, benzyl alcohol, propylene glycol, glycerin, etc.) (For example, ascorbic acid, sodium hydrogen sulfite, sodium pyrophosphate, BHA, tocopherol, EDTA and the like), an emulsifier, a buffer for pH control, a microbial growth inhibitor And a pharmaceutical carrier such as a preservative (e.g., mercury nitrate, thimerosal, benzalkonium chloride, phenol, cresol, benzyl alcohol, etc.).

In another aspect, the present invention provides a health functional food composition for preventing or ameliorating osteoporosis, comprising an extract of Huangchu tree, a fraction thereof, a compound isolated therefrom, or a pharmaceutically acceptable salt thereof, as an active ingredient .

The composition of the present invention can be used simultaneously or separately with a medicament for the treatment of a disease before or after the onset of osteopathy to prevent or ameliorate a bone disease.

As used herein, the term "improvement" means any action that at least reduces the degree of symptom associated with the condition being treated.

In addition, when the health functional food composition of the present invention is used as a food additive, the extract can be used as it is, or can be used in combination with other food or food ingredients, Lt; / RTI > The amount of the active ingredient to be mixed can be appropriately determined depending on the purpose of use such as prevention, health, or therapeutic treatment. In general, the composition of the present invention is added in an amount of not more than 15% by weight, preferably not more than 10% by weight based on the raw material, in the production of food or beverage. However, in the case of long-term ingestion intended for health and hygiene purposes or for the purpose of controlling health, it may be below the above range.

The term "health functional food" used in the present invention refers to a food prepared by processing a specific ingredient as a raw material for the purpose of health assisting or by extracting, concentrating, refining, or mixing a specific ingredient contained in the raw material Refers to foods that are designed and processed so that the body control function such as bio-defense, regulation of biorhythm, prevention and recovery of disease, and the like can be sufficiently exhibited to a living body by the above components. And the like.

There is no limitation on the kind of health food to which the composition of the present invention can be used. In addition, the composition comprising the compound of the present invention as an active ingredient can be prepared by mixing other suitable auxiliary ingredients and known additives, which may be contained in health functional foods, according to the selection of a person skilled in the art. Examples of foods that can be added include dairy products, such as meat, sausage, bread, chocolates, candies, snacks, confectionery, pizza, ramen, other noodles, gums, ice cream, various soups, drinks, tea, Vitamin complex, and the like, and can be prepared by adding to the juice, tea, jelly, and juice prepared from the extract of the present invention as a main component. In addition, it includes all the health foods in the ordinary sense.

The health beverage composition of the present invention may contain various flavors or natural carbohydrates as an additional ingredient such as ordinary beverages. Such natural carbohydrates include monosaccharides such as glucose and fructose, disaccharides such as maltose and sucrose, and natural sweeteners such as dextrin and cyclodextrin, synthetic sweeteners such as saccharine and aspartame, and the like . The ratio of the natural carbohydrate can be appropriately determined by a person skilled in the art.

In addition to the above, the composition of the present invention may further contain various nutrients, vitamins, electrolytes, flavors, colorants, pectic acids and salts thereof, alginic acid and its salts, organic acids, protective colloid thickeners, pH adjusting agents, stabilizers, preservatives, A carbonating agent used in a carbonated beverage, and the like. In addition, the composition of the present invention may contain flesh for the production of natural fruit juices, fruit juice drinks and vegetable drinks. These components may be used independently or in combination. The ratios of these additives can also be appropriately selected by those skilled in the art.

Since the extract of Huangchu tree, fractions thereof or the compounds separated therefrom of the present invention is a natural herbal medicine raw material, the use of the extract as a pharmaceutical composition or a food additive can be less adverse than general synthetic compounds. Therefore, It can be usefully included in foods.

The Dendropanax morbifera extract, its fractions, the compounds isolated therefrom, or the pharmaceutically acceptable salts of the compounds according to the present invention inhibit the differentiation of osteoclasts, and the inventive Can be used to prevent, ameliorate, or treat various bone diseases.

FIG. 1 (A) is an image showing the effect of inhibiting osteoclast differentiation according to the present invention by TRAP staining.
FIG. 1B is a graph showing the effect of inhibiting osteoclast differentiation according to the concentration of the extract according to the present invention.
FIG. 2 (A) shows an image of TRAP-stained osteoclast differentiation inhibitory effect of the fractions of the extract of Wiltrich tree extract according to the present invention.
FIG. 2B is a graph showing the effect of inhibiting the osteoclast differentiation according to the concentration of the fractions of the extract.
Fig. 3 shows the chemical formulas of the compounds 1 to 9 isolated from the woodchucking extract according to the present invention.
FIG. 4 shows images of TRAP-stained oocyte differentiation inhibitory effects of Compounds 1 to 9 isolated from the extracts of Euphorbiaceae according to the present invention.
FIG. 5 is a graph showing the effect of inhibiting osteoclast differentiation according to the concentration of the compounds 1 to 9 isolated from the extracts of H. grusia according to the present invention.
FIG. 6A shows images obtained by TRAP staining of the effect of inhibiting osteoclast differentiation according to the concentration of the compounds 1 and 2 isolated from the extract of D. torrillus according to the present invention.
FIG. 6B is a graph showing the effect of inhibiting osteoclast differentiation according to the concentration of the compounds 1 and 2 isolated from the extract of Wako et al.
FIG. 7 is a graph showing the cytotoxicity of Compounds 1 and 2 isolated from the extracts of Hexachia extract according to the present invention.

Hereinafter, the present invention will be described in more detail with reference to examples. However, these examples are for illustrative purposes only, and the scope of the present invention is not limited to these examples.

≪ Example > - Preparation of extract of Hwangchilchu and fractions thereof [

Example 1: Preparation of extract of Hwangchujang and fractions thereof

5 kg of dried Dendropanax morbifera was placed in an extraction container, and 50 L of 70% methanol was added thereto. The obtained extract was concentrated under reduced pressure using a vacuum rotary evaporator. The above extraction and concentration process was repeated twice to obtain 950 g of Hokutogi extract.

The resulting methanol extract of Huaculi wood was suspended in 5 L of distilled water and extracted with the same amount of ethyl acetate three times. The resulting extract was concentrated under reduced pressure to obtain 258 g of ethyl acetate fraction. The filtrate obtained from the above extraction procedure was repeatedly extracted three times with the same amount of n-butanol, and the extract was concentrated under reduced pressure to obtain 159 g of butanol fraction.

Example 2: Isolation and Identification of Active Ingredients from Hwangchilchu Extract

Nine kinds of compounds were isolated from the fragrant wood fractions prepared in Example 1, and the structural formula and the names of these compounds are shown in FIG.

Specifically, 220 g of the ethyl acetate fraction in the obtained fractions was taken, and the dichloromethane / methanol mixed solution was flowed into the mobile phase, and concentration gradient elution system (dichloromethane / methanol mixture ratio) was sequentially increased from 100: 1 to 1: To obtain a total of four fractions (first to fifth fractions).

The obtained second fraction (20 g) was again subjected to silica gel column chromatography (mobile phase: n-hexane / ethyl acetate (50: 1 to 1: 1)) to give 5 fractions (fractions 21 to 25) I divided it. Of these, the first 21 fractions recrystallization (solvent: n- hexane) to give the white powder The powder purified by silica gel column chromatography (mobile phase: dichloromethane / methanol (40: 1)) to an exemplary compound 3 (10.0 mg) and Compound 4 (14.5 mg) was isolated. The mother liquor after recrystallization was further purified by silica gel column chromatography (solvent: n-hexane / ethyl acetate (50: 1 to 1: 1) to obtain Compound 5 (120.0 mg) : 1)) was performed to divide into five small fractions (fractions 221 to 225). The 224 fraction was recrystallized (solvent: n-hexane) to obtain a white powder, which was confirmed to be a mixture (490.7 mg) of Compound 6 and Compound 7 in a ratio of 1: 3.5 through ¹ H NMR and ¹³ C NMR.

The obtained third fraction (34 g) was subjected to silica gel column chromatography (mobile phase: n-hexane / ethyl acetate (10: 1 to 1: 1)) to give five fractions (fractions 31 to 35) I divided it. The 32nd fraction was further purified twice by silica gel column chromatography (mobile phase: n-hexane / ethyl acetate (30: 1 to 5: 1)) to obtain 70.0 mg of white powder. ¹ H NMR and ¹³ C NMR showed that Compound 8 and Compound 9 were mixed in a ratio of 1: 1. 34 fraction was purified by silica gel column chromatography (mobile phase: n- hexane / ethyl acetate (20: 1 ~ 1: 1)) to separation / purification by reverse phase column chromatography after, 70% methanol (mobile phase), subjected to the compound 1 ( 201.0 mg) and Compound 2 (112.0 mg), respectively, and Compound 1 was confirmed to be a novel compound. The separated compounds were identified by ¹ H-NMR and ¹³ C-NMR.

<Experimental Example>

Experimental Example 1: Inhibitory effect on the osteoclast formation of the extract of Wuchulchia japonica and its fractions

Five-week-old male ICR mice were purchased and the femur and tibia were taken and washed with α-MEM containing antibiotics (100 units / ml penicillin and 100 μg / ml streptomycin) to form bone marrow cells ). The bone marrow cells were cultured in α-MEM containing 10% fetal bovine serum (FBS) and M-CSF (macrophage colony-stimulating factor; 10 ng / ml) for 1 day. Non-adherent bone marrow cells were seeded in Petri dishes and cultured for 3 days in the presence of M-CSF (30 ng / ml). Adherent cells were used as bone marrow-derived macrophages (BMM) after washing nonadherent cells. BMM cells were treated with DMSO or the extract of Wuchungchuck extract prepared in Example 1 or its fractions (1, 3 and 10 μg / ml) in the presence of RANKL (10 ng / ml) and M-CSF And cultured for 4 days. Negative controls used RANKL and cells not treated with Example 1. Multinucleated osteoclasts were fixed with 3.7% formalin for 10 minutes and treated with 0.1% Triton X-100 for 10 minutes to have permeability. Cells were stained with TRAP solution (Sigma-Aldrich). Multicucleated osteoclasts were visualized by TRAP (tartrate-resistant acid phosphatase) staining, which was shown in Figure 1 A (extract results) and Figure 2 A (fraction results) This was shown in Figure 1 B (extract results) and Figure 2 B (fraction results).

1 and 2 show that the extract of Hwangcholgwa extract and its fractions of Example 1 inhibit osteoclast formation in a concentration-dependent manner. The ethanol extracts of Hwangchul - myeon inhibited osteoclastogenesis significantly from the concentration of 3 ㎍ / ㎖, and the ethyl acetate fraction and n - butanol fraction of the methanol extract of Hwangchu - The inhibitory effect of the ethyl acetate fraction was noticeably better than that of the n-butanol fraction.

The significance test was performed by student t-test. The significance * *, p **, p *** and p *** 0.001 for the RANKL treatment group were p <0.05, ** and ***, respectively. ### represents the significance (p <0.001) as compared to the negative control group and the RANKL treatment group.

Experimental Example 2: Inhibitory effect on the osteoclast formation of the compound derived from the extract

<2-1> Inhibitory effect of compounds 1 to 9 on osteoclast formation

In order to examine the inhibitory effect of the compounds 1 to 9 isolated in Example 2 on osteoclast formation, the same method as in Experimental Example 1 was used, and the compounds 1 to 9 were treated at a concentration of 10 μM and 30 μM, respectively. The TRAP staining results are shown in Fig. 4, and the TRAP activity measurement results are shown in Fig. In FIG. 4, compounds 1, 2, 3, 4, 5, and 7 significantly inhibited osteoclastogenesis but did not significantly inhibit other compounds, . In addition, in FIG. 5, it was confirmed that compounds 1, 2 and 3 inhibited osteoclastogenesis significantly at both 10 μM and 30 μM concentrations. In particular, the inhibitory effect of compounds 1 and 2 on osteoclastogenesis was very significant I can confirm that it is excellent.

The significance test was carried out in the same manner as the test method of Experimental Example 1 above.

<2-2> Inhibitory effect of compounds 1 and 2 on osteoclast formation

To further examine the inhibitory effect of the compounds 1 and 2 isolated in Example 2 on osteoclastogenesis, the concentration was refined and the experiment was repeated. The same method as in Experimental Example 1 was used, and Compound 1 and Compound 2 were treated at concentrations of 0.1, 0.3, 1, 3, 10 and 30 μM, respectively. The result of TRAP staining is shown in Fig. 6A, and the result of TRAP activity measurement is shown in Fig. 6B.

Compound 1 (Formula 1) and 2 (Formula 2) inhibited osteoclast formation in a concentration-dependent manner as in the case of Experimental Example 2-1, and both of the compounds showed significant osteoclast formation from 1 μM concentration Respectively. In addition, the 50% inhibitory concentration (IC ₅₀ ) was calculated to be 2.41 μM, 3.11 μM for compounds 1 and 2, respectively.

Experimental Example 3: Cytotoxicity of Compounds 1 and 2

LDH cytotoxicity assay was performed to determine the cytotoxicity of compounds 1 and 2. The higher the degree of cell damage, the higher the level of LDH. BMM cells were divided into 96-well plates at a concentration of 1 × 10 ⁵ / ml using a medium containing 30 ng / ml of M-CSF and 10 ng / ml of RANKL, 0.3, 1, 3, 10, and 30 μM, and cultured for 4 days. Then, 50 μl of the supernatant and 50 μl of LDH substrate solution (CytoTox 96 Non-Radioactive Cytotoxicity Assay kit, Promega) were reacted at room temperature for 30 minutes. To stop the reaction, 50 μl of a stop solution (1M acetic acid) was added, and the absorbance at 490 nm was measured with a Microplate ELISA reader to confirm the cytotoxicity of the compound. The results are shown in FIG.

In FIG. 7, compounds 1 and 2 show LDH activity similar to the control group at all concentrations, indicating that they are not cytotoxic.

Claims (8)

A compound isolated from Dendropanax morbifera extract or a pharmaceutically acceptable salt of said compound as an active ingredient,
Wherein said compound is a compound represented by the following formula (1) or (2):
[Chemical Formula 1]

(2)

.
The method according to claim 1,
(9Z, 16S) -16-acetyl-9,17-octadecadien-12,14-dinoic acid ((9Z, 16S) -16-acetyl-9,17-octadecadiene- -dienoic acid). &lt; / RTI &gt;
The method according to claim 1,
(9Z, 16S) -16-hydroxy-9,17-octadecadiene-12, (9Z, 16S) -16-hydroxy-9,17-octadecadien- 14-diynoic acid). &Lt; / RTI &gt;
The method according to claim 1,
Wherein said compound inhibits osteoclast differentiation. &Lt; RTI ID = 0.0 &gt; 21. &lt; / RTI &gt;
5. The method of claim 4,
Wherein said compound inhibits the activity of RANKL (Receptor activator of nuclear factor kappa-B ligand) which induces osteoclast maturation.
delete The method according to claim 1,
The bone disease is selected from the group consisting of osteoporosis, rheumatoid arthritis, periodontal disease and Paget disease due to growth retardation, fracture, excessive bone resorption of osteoclast, osteoporosis, rheumatoid arthritis, RTI ID = 0.0 &gt; 1, &lt; / RTI &gt;
A compound isolated from Dendropanax morbifera extract or a pharmaceutically acceptable salt of said compound as an active ingredient,
Wherein the compound is a compound represented by the following formula (1) or (2):
[Chemical Formula 1]

(2)

.

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