WO2018038313A1 - Novel diynoic acid compound, and pharmaceutical composition for preventing or treating bone diseases including same - Google Patents

Abstract

The present invention relates to a 16-acetyl-9,17-octadecadiene-12,14-diynoic acid compound represented by chemical formula 1 or a pharmaceutically acceptable salt thereof; a method for preparing the compound; a pharmaceutical composition for preventing or treating bone diseases and including the compound or the pharmaceutically acceptable salt thereof; a quasi-drug composition for preventing or alleviating bone diseases; a health functional food composition; a cosmetic composition; and a method for treating bone diseases in non-human animals, wherein the method comprises a step for administrating the pharmaceutical composition to a subject suspected of a bone disease. The compound according to the present invention can be usefully used for preventing, treating, or alleviating bone diseases, and as a natural material-derived compound, can provide a safe therapeutic agent for which concerns about toxicity or other side effects are less than for synthetic drugs. In addition, a novel compound is provided, wherein the novel compound is effective for treating bone diseases, and can be used as a novel therapeutic agent for bone diseases.

Description

Novel dinoic acid compounds and pharmaceutical compositions for the prevention or treatment of bone diseases comprising the same

The present invention is 16-acetyl-9,17-octadecadiene-12,14-dinoic acid represented by Formula 1 (16-acetyl-9,17-octadecadiene-12,14-diynoic acid) compound or a pharmaceutically thereof Acceptable salts, methods for preparing the compounds, pharmaceutical compositions for the prevention or treatment of bone diseases, including the compounds or pharmaceutically acceptable salts thereof, quasi-drug compositions for the prevention or improvement of bone diseases, health functional food compositions, cosmetics The present invention relates to a method for treating bone diseases in animals other than humans, the method comprising administering the composition and the pharmaceutical composition to a suspected bone disease subject.

Bone remodeling occurs continually in life to maintain bone in response to mechanical stress and hormonal regulation. To maintain normal bone mass, bone is reabsorbed by osteoclasts and new bone matrix is accumulated by osteoblasts through a closely coupled process. As seen in many common bone diseases, imbalances in this process can lead to excessive bone resorption by osteoclasts and increased bone fragility.

Osteoclasts are known to differentiate from hematopoietic progenitors or myeloid progenitors by hormones secreted from T-cells, fibroblasts, osteoblasts, and the like. In the osteoclast differentiation process, 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 macrophage colony-stimulating factor (M-CSF). When RANKL binds to RANK, the receptor, TRAF6 (TNF receptor-associated factor 6) gathers in the cytoplasmic domain of RANK, thus NF-κB and JNK (c-Jun N-terminal kinase), p38 (p38 MAP kinase). And mitogen-activated protein kinase (MAPK) signaling pathways, including extracellular signal-regulated kinase (ERK), are activated. Activated NF-κB induces the expression of c-Fos and NFATc1 downstream, which are known to play a key role in osteoclast differentiation by RANKL. In addition, the osteoclast differentiation process is known to be promoted by inflammatory cytokines such as IL-1, Il-3, PGE.

Finally, differentiated osteoclasts have the form of multi-nulceated cells and bind to the site where bones are absorbed to form sealing zones using actin and vinculin. Sealed with the surroundings. Later, by pumping hydrogen ions, the pH is lowered to dissolve the hydroxyapatite component and secrete proteolytic enzymes such as MMP-9 or Kadipsin K to break down the collagen component, which accounts for about 10% of bone. As such, the differentiated osteoclasts are known to specifically express phosphatase (tartrate-resistant acid phosphatase: TRAP), which is active in the presence of tartaric acid. Thus, osteoclast differentiation can be confirmed by measuring the activity of TRAP.

As described above, osteoclasts play an important role in bone metabolism in vivo, but when excessively differentiated by various causes, it may cause disorders or destroy diseases of bone tissues. Growth stage development, fractures, osteoporosis due to bone resorption of excessive osteoclasts, rheumatoid arthritis, periodontal disease, Paget disease, metastatic bone cancers Etc. can be mentioned.

In order to treat such bone diseases, it is necessary to control the balance of osteoclasts and osteoblasts, and thus, the therapeutic agents for this include largely bone resorption inhibitors and stimulating bone formation. In general, a bone disease treatment agent such as a bone resorption inhibitor or a bone stimulating agent should be less toxic because it should be administered to patients for a long time, and it is preferable that oral administration is possible to solve the hassle of administration. It is true. On the other hand, research on bone stimulating agents that promote bone formation by activating osteoblasts among the bone disease treatments is being actively conducted, but bone mineralization by bone stimulating agents does not necessarily mean reduction of fractures. On the other hand, therapeutic agents for bone diseases that inhibit the activity of osteoclasts currently used include Denosumab (Denosumab; Prolia ™, Amgen ^® ), and various other drugs have been developed. However, the drug treatment should be continued for a long time. Adverse events have not been fully validated, and the exact mechanisms of osteoclasts are still unknown.

An object of the present invention is a 16-acetyl-9,17-octadecadiene-12,14-dinoic acid represented by the formula (1) or a pharmaceutical thereof To provide an acceptable salt.

Another object of the present invention (a) extracting the yellow lacquer with the extraction solvent, filtration and concentration to obtain a yellow lacquer extract; (b) fractionating the hwangchil extract of (a) with an organic solvent and performing column chromatography to obtain a fraction; And (c) separating the compound by performing high performance liquid chromatography from the fraction of (b).

Still another object of the present invention is to provide a pharmaceutical composition for preventing or treating bone diseases, including the compound or a pharmaceutically acceptable salt thereof.

Another object of the present invention to provide a quasi-drug composition for the prevention or improvement of bone diseases comprising the compound.

Still another object of the present invention is to provide a nutraceutical composition for preventing or improving bone diseases containing the compound.

Still another object of the present invention is to provide a cosmetic composition for preventing or improving bone diseases containing the compound.

Still another object of the present invention is to provide a method for treating bone diseases in animals other than humans, comprising administering the pharmaceutical composition to a suspected bone disease subject.

As one embodiment for solving the above problems, the present invention is 16-acetyl-9,17-octadecadiene-12,14-dinoic acid (16-acetyl-9,17-octadecadiene-12,14-diynoic acid) Compound or a pharmaceutically acceptable salt thereof. Specifically, (9Z, 16S) -16-acetyl-9,17-octadecadiene-12,14-dinoic acid ((9Z, 16S) -16-acetyl-9,17-octadecadiene represented by Formula 1 below: -12,14-diynoic acid) or a pharmaceutically acceptable salt thereof.

[Formula 1]

The compound represented by Chemical Formula 1 of the present invention is a novel dinoic acid compound, and is named a 16-acetyl-9,17-octadecadiene-12,14-dinoic acid compound. The compound of the present invention may be obtained by general chemical synthesis, but preferably, the compound may be separated from natural products such as Dendropanax morbifera , and more preferably, may be separated from a yellow lacquer extract or a fraction thereof. have. The hwangchil may be extracted from one or more selected from the group consisting of the roots, stems, flowers, leaves and berries of the hwangchil, or one selected from the group consisting of the stems, flowers, leaves and fruits of the hwangchil wood It may be extracted from the above, preferably may be extracted from the leaves of the hwangchil. In an embodiment of the present invention, a novel dinoic acid compound was isolated from the yellow lacquer, and the structural confirmation result by molecular weight measurement, molecular formula estimation and nuclear magnetic resonance (NMR) analysis of the compound, (9Z, 16S) It was confirmed that the compound was -16-acetyl-9,17-octadecadiene-12,14-dinoic acid.

As the pharmaceutically acceptable salt of the compound represented by Formula 1, an acid addition salt formed by a pharmaceutically acceptable free acid may be useful. Organic acids and inorganic acids may be used as the free acid, and hydrochloric acid, bromic acid, sulfuric acid, sulfurous acid, phosphoric acid, etc. may be used as the inorganic acid, and citric acid, acetic acid, maleic acid, fumaric acid, gluconic acid, metalsulfonic acid may be used as the organic acid. , Acetic acid, glycolic acid, succinic acid, tartaric acid, 4-toluenesulfonic acid, galacturonic acid, embonic acid, glutamic acid, citric acid, aspartic acid, and the like can be used.

The addition salt according to the present invention is conventionally used, that is, the compound of formula 1 is dissolved in a water-miscible organic solvent such as acetone, methanol, ethanol, or acetonitrile, and an equivalent or excess organic acid is added or an acid aqueous solution of an inorganic acid It can be prepared by addition, followed by precipitation or crystallization, or by evaporation of the solvent or excess acid followed by suction filtration of the dried or precipitated salt.

The present invention may include all of the compounds of Formula 1 and their pharmaceutically acceptable salts as well as the possible solvates, hydrates and stereoisomers that may be prepared therefrom within the scope of the invention.

In another aspect, the present invention provides a method for producing a yellow lacquer extract, comprising: (a) extracting a yellow lacquer with an extractant, filtration and concentrating to obtain a yellow lacquer extract; (b) fractionating the hwangchil extract of (a) with an organic solvent and performing column chromatography to obtain a fraction; And (c) separating the compound by performing high performance liquid chromatography from the fraction of (b).

In the present invention, the step (a) is a step of extracting the yellow lacquer with an extraction solvent, filtration and concentration to obtain a yellow lacquer extract.

In the present invention, the term "hwangchil" is a dicotyledonous plant native to the southern coast of Korea and Jeju Island, etc., an evergreen arborescent tree of the family Elmaceae (Dendropanax morbifera) is a species that does not fall even in winter when the bark is yellow The resin solution comes out is called Hwangchil. Physiological activities such as anticancer activity and antioxidant activity of Hwangchil-tree extract have been reported to some extent, and 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 ..... of Korean dendropanax (. dendropanax morvifera Lev) J. Korean Soc Agric Chem Biotechnol 43: 63-66 (2000), Park SN, et al .. Cellular antioxidant activity and whitening effects of dendropanax morbifera leaf extracts, South Korea Journal of Microbial Biotechnology, 41 (4): 407-415,2013). In the present invention, the hwangchil can be used without limitation, such as cultivated or commercially available, can be used both above ground, root, seed.

In the present invention, the extraction solvent may use an extraction solvent commonly used in the art, but is not limited thereto, and preferably water, alcohols having 1 to 6 carbon atoms, or a mixed solvent thereof may be used. The alcohol may be ethanol, methanol or butanol. The extraction solvent is not limited thereto, but may preferably be added 2 to 20 times the amount of water loss, more preferably 4 to 10 times, and even more preferably 5 times. The extraction method is not limited thereto, but preferably, hot water extraction, hot water extraction, cold needle extraction, reflux cooling extraction, or ultrasonic extraction may be used, and more preferably, hot water extraction or cold needle extraction may be used. . The temperature of the extraction solvent at the time of extraction is not limited thereto, but may be preferably 20 to 100 ° C, more preferably room temperature. Extraction time is not limited thereto, but may preferably be 12 hours to 10 days, more preferably 5 to 8 days, even more preferably 7 days. Extraction frequency is not limited thereto, but may be preferably 1 to 5 times, more preferably 3 times. Using the conditions of the extraction as described above can be extracted, filtered, and concentrated and lyophilized filtrate to obtain the yellow lacquer extract.

In the present invention, step (b) is a step of obtaining the fraction by fractionating the yellow lacquer extract of (a) with an organic solvent and performing column chromatography.

The organic solvent is a liquid organic compound that can dissolve solids, gases, and liquids, and organic solvents commonly used in the art may be used, and preferably methanol, chloroform, acetate, hexane, dichloromethane, acetone, acetonitrile , Benzene or a mixed solvent thereof can be used.

The column chromatography is a chromatographic method in which the stationary phase granules are filled into columnar columns in a chromatographic tube, and can be carried out several times by selecting an appropriate filler as necessary. Preferably, the filler is silica gel, Sephadex, RP. -18, polyamide, toyopearl or XAD resin can be used to isolate and purify.

In the present invention, step (c) is a step of separating the compound by performing high performance liquid chromatography from the fraction of (b).

The high performance liquid chromatography (HPLC) is a chromatographic method that enables high-speed and high-performance separation by improving and improving a column, a fixed bed filler filled in a column, and an apparatus. Finally, the compound represented by Chemical Formula 1 may be prepared. Can be.

As another aspect, the present invention provides a pharmaceutical composition for the prevention or treatment of bone diseases comprising the compound or a pharmaceutically acceptable salt thereof.

The pharmaceutical composition of the present invention is characterized by comprising the compound or a pharmaceutically acceptable salt thereof.

The compound or a pharmaceutically acceptable salt thereof is as described above. In one embodiment of the present invention, as a result of measuring the inhibitory effect of the osteoclast production of the compound, it was confirmed that the inhibitory effect of the osteoclast production of the compound according to the present invention is significantly superior to other compounds (Figs. 4 and 5). In particular, concentration-dependent osteoclast formation was inhibited (FIG. 6). In addition, the cytotoxicity was measured, it was confirmed that the compound according to the present invention is not cytotoxic at all concentrations (Fig. 7).

As used herein, the term "bone disease" refers to a condition or disease caused by excessive production and / or migration of osteoclasts, and includes bone loss diseases. The bone loss lowering disease refers to a condition or a disease in which a decrease in bone mass accompanied by symptoms such as a decrease in bone density and deterioration of bone tissues, and bone loss caused by osteoclasts activated by cancer cells may be included in the scope of the present invention. have. The bone disease is not limited to this, but the growth stage of development, fracture, osteoporosis (osteoprosis) due to excessive absorption of osteoclasts, rheumatoid arthritis, periodontal disease (periodontal disease), Paget disease (Paget disease) And it may be one or more diseases selected from the group consisting of metastatic bone cancers. 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 refers to a state in which bone fracture may occur due to a decrease in bone mass and qualitative changes, and "osteoporosis" refers to an early symptom of osteoporosis. Generally, it is classified as osteopenia in case of -1.0 to -2.5 and osteoporosis in case of -2.5 or more based on bone density (T).

As used herein, the term "prevention" refers to any action that inhibits or delays the development of bone disease by administration of the composition, and "treatment" refers to any action that improves or benefits the symptoms of bone disease by administration of the composition. it means.

The pharmaceutical composition of the present invention may comprise a pharmaceutically acceptable carrier. Compositions comprising a pharmaceutically acceptable carrier may be in various oral or parenteral formulations. When formulated, it may be prepared using diluents or excipients such as fillers, extenders, binders, wetting agents, disintegrating agents, surfactants, etc. which are commonly used. Solid preparations for oral administration may include tablet pills, powders, granules, capsules and the like, and such solid preparations may include at least one excipient such as starch, calcium carbonate, sucrose or lactose ( lactose), gelatin and the like can be mixed. In addition to the simple excipients, lubricants such as magnesium stearate, talc and the like can also be used. Liquid preparations for oral administration include suspensions, solution solutions, emulsions, and syrups, and various excipients, such as wetting agents, sweeteners, fragrances, and preservatives, in addition to commonly used simple diluents such as water and liquid paraffin, may be included. have. Formulations for parenteral administration may include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized preparations, suppositories. As the non-aqueous solvent and the suspension solvent, propylene glycol, polyethylene glycol, vegetable oil such as olive oil, injectable ester such as ethyl oleate, and the like can be used. As the base of the suppository, witepsol, macrogol, tween 61, cacao butter, laurin butter, glycerogelatin and the like can be used.

In addition, the pharmaceutical composition of the present invention is a group consisting of tablets, pills, powders, granules, capsules, suspensions, liquid solutions, emulsions, syrups, sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized preparations and suppositories It can have any one formulation selected from.

The pharmaceutical composition of the present invention is not particularly limited in its dosage, and may vary according to body absorption, weight, age, sex, health condition, diet, time of administration, administration method, excretion rate, and severity of disease. have. In general, the daily dose may preferably be from 0.1 to 100 mg / kg, more preferably from 30 to 80 mg / kg, even more preferably from 50 to 60 mg, based on the amount of the hwangchil extract / kg. The pharmaceutical compositions of the present invention may be prepared in consideration of the effective amount range, and the unit dosage form formulated in this way may be formulated using a specialized dosage method according to the judgment of a professional who monitors or observes the administration of the drug as required and the needs of the individual. It can be used or administered several times at regular time intervals.

As another aspect, the present invention provides a quasi-drug composition for the prevention or improvement of bone diseases comprising the compound. The compound is as described above, and may include a pharmaceutically acceptable salt thereof. More specifically, the composition of the present invention can be added to the quasi-drug composition for the purpose of preventing or improving bone diseases.

As used herein, the term "quasi drug" refers to a fiber, a rubber product or the like used for the purpose of treating, alleviating, treating or preventing a disease of a human or animal, has a weak action on the human body or does not directly act on the human body, Or non-machinery and the like, or any of the agents used for sterilization, insecticide and similar purposes for the purpose of preventing infection, for the purpose of diagnosing, treating, reducing, treating or preventing human or animal diseases. Means the goods used are not instruments, machines or devices, and the items used for the purpose of pharmacologically affecting the structure and function of humans or animals except those which are not devices, machines or devices.

When the composition of the present invention is used as an quasi-drug additive, the composition may be added as it is or used with other quasi-drugs or quasi-drug components, and may be appropriately used according to a conventional method. The mixing amount of the active ingredient may be appropriately determined depending on the intended use.

The quasi-drug composition of the present invention is not limited thereto, but may preferably be a disinfectant cleaner, a shower foam, a gagreen, a wet tissue, a detergent soap, a hand wash, a humidifier filler, a mask, an ointment, or a filter filler.

As another aspect, the present invention provides a nutraceutical composition for the prevention or improvement of bone diseases comprising the compound. The compound is as described above, and may include a pharmaceutically acceptable salt thereof. More specifically, the composition of the present invention can be added to the nutraceutical composition for the purpose of preventing or improving bone diseases.

When the composition of the present invention is used as a nutraceutical additive, the composition may be added as it is or used with other nutraceutical or nutraceutical ingredients, and may be appropriately used according to a conventional method. The mixing amount of the active ingredient may be appropriately determined depending on the intended use. In general, the composition of the present invention may be added in the amount of preferably 15 parts by weight or less, more preferably 10 parts by weight or less based on the raw material in the manufacture of food or beverage. However, in the case of long-term intake for the purpose of health control and hygiene, the amount may be below the above range, and since there is no problem in terms of stability, the active ingredient may be used in an amount above the above range.

There is no particular limitation on the type of dietary supplement of the present invention. Examples of health functional foods to which the composition can be added include meat, sausages, bread, chocolate, candy, snacks, confectionery, pizza, ramen, other noodles, gums, dairy products including ice cream, various soups, beverages, teas, Drinks, alcoholic beverages and vitamin complexes, and the like, may include all of the health functional foods in the conventional sense, may include foods used as feed for animals.

In addition, when the health functional food composition of the present invention is used in the form of a beverage, it may contain various sweetening agents, flavoring agents or natural carbohydrates, etc. as additional ingredients, as in the usual beverage. The natural carbohydrate can be glucose, monosaccharides such as fructose, maltose, disaccharides such as sucrose, polysaccharides such as dextrin, cyclodextrin, and sugar alcohols such as xylitol, sorbitol, erythritol. The ratio of the natural carbohydrate is not limited thereto, but may be preferably about 0.01 to 0.04 g, more preferably 0.02 to 0.03 g per 100 ml of the composition of the present invention. The sweetener may be a natural sweetener such as taumartin, stevia extract and a synthetic sweetener such as saccharin, aspartame.

In addition to the above, the nutraceutical composition of the present invention includes various nutrients, vitamins, electrolytes, flavors, coloring agents, pectic acid and salts thereof, alginic acid and salts thereof, organic acids, protective colloidal thickeners, pH regulators, stabilizers, preservatives, glycerin , Alcohols, carbonating agents used in carbonated drinks, and the like. Others may contain pulp for the production of natural fruit juices, fruit juice drinks and vegetable drinks.

As another aspect, the present invention provides a cosmetic composition for the prevention or improvement of bone diseases comprising the compound. The compound is as described above, and may include a pharmaceutically acceptable salt thereof. More specifically, the composition of the present invention can be added to the cosmetic composition for the purpose of preventing or ameliorating bone diseases.

The cosmetic composition of the present invention can be prepared in the form of general emulsion formulations and solubilized formulations. The emulsified formulations include nutrient cosmetics, creams, essences, etc., and the solubilized formulations include soft cosmetics. Suitable formulations include, but are not limited to, solutions, gels, solid or pasty anhydrous products, emulsions, suspensions, microemulsions, microcapsules, microgranules or ionics (liposomes) obtained by dispersing an oil phase in an aqueous phase, for example In the form of a vesicle dispersant, may be in the form of a cream, skin, lotion, powder, ointment, spray or cone stick. It may also be in the form of a foam or in the form of an aerosol composition further containing a compressed propellant.

The cosmetic composition additionally contains fatty substances, organic solvents, solubilizers, thickening and gelling agents, emollients, antioxidants, suspending agents, stabilizers, blowing agents, fragrances, surfactants, water, ionic or nonionic emulsifiers, fillers, metals Commonly used adjuvants such as ion blockers, chelating agents, preservatives, vitamins, blockers, wetting agents, essential oils, dyes, pigments, hydrophilic or lipophilic active agents, lipid vesicles or any other ingredients commonly used in cosmetic compositions It may contain.

As another aspect, the present invention provides a method for treating bone diseases in animals other than humans, comprising administering the pharmaceutical composition to a suspected bone disease subject.

In the present invention, the subject suspected of bone disease refers to any animal including a human having or may develop a bone disease, and a pharmaceutical composition comprising a compound of the present invention or a pharmaceutically acceptable salt thereof is a subject suspected of a bone disease. By administering to, the individual can be treated efficiently. The bone disease is as described above.

As used herein, the term "administration" refers to the introduction of the pharmaceutical composition of the present invention to a subject suspected of bone disease in any suitable manner, and the route of administration is through various routes, oral or parenteral, as long as the target tissue can be reached. May be administered.

The method of treatment of the present invention may comprise administering a pharmaceutical composition comprising said compound in a pharmaceutically effective amount. Suitable total daily doses may be determined by the treating physician within the scope of good medical judgment and may be administered once or in divided doses. However, for the purposes of the present invention, a specific therapeutically effective amount for a particular animal is determined by the specific composition, animal age, weight, general health, including the type and extent of the reaction to be achieved, whether or not other agents are used in some cases, It is desirable to apply differently depending on various factors and similar factors well known in the medical field, including sex and diet, time of administration, route of administration and rate of composition, duration of treatment, drugs used with or co-specific with the specific composition.

The compound according to the present invention can be usefully used for the prevention, treatment or amelioration of bone diseases, and can provide a safe therapeutic agent with no concern about toxicity or other side effects as a compound derived from natural products, compared to a synthetic medicine. Also provided are novel compounds, which are effective in treating bone diseases and can be used as therapeutic agents for new bone diseases.

Figure 1 A shows the image observed by TRAP staining the effect of inhibiting osteoclast differentiation by concentration of the Hwangchil wood extract according to the present invention.

1 B is a graph showing the effect of inhibiting osteoclast differentiation according to the concentration of Hwangchil-tree extract according to the present invention.

Figure 2 A shows the image observed by TRAP staining effect of the fraction of Hwangchil-tree extract according to the present invention to inhibit osteoclast differentiation by concentration.

2B is a graph showing the effect of inhibiting osteoclast differentiation by fractions of the extract of Hwangchil-tree according to the present invention.

Figure 3 shows the chemical formula of the compounds 1 to 9 isolated from the hwangchil wood extract according to the present invention.

Figure 4 shows the image observed by TRAP staining the effect of inhibiting osteoclast differentiation according to the concentration of compounds 1 to 9 are separated from the Hwangchil wood extract according to the present invention.

Figure 5 is a graph showing the effect of inhibiting osteoclast differentiation by the concentration of compounds 1 to 9 isolated from the extract of Hwangchil-tree according to the present invention.

Figure 6 A shows the image observed by TRAP staining the effect of inhibiting osteoclast differentiation according to the concentration of compounds 1 and 2 are separated from the Hwangchil wood extract according to the present invention.

6B is a graph showing the effect of inhibiting osteoclast differentiation according to the concentrations of Compounds 1 and 2 separated from the Hwangchil-tree extract according to the present invention.

7 is a graph showing the cytotoxicity of the compounds 1 and 2 isolated from the Hwangchil wood 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 Hwangchil Tree Extract and Fractions thereof>

Example 1 Preparation of Hwangchil Tree Extract and Fractions thereof

Dried Sacred Tree ( Dendropanax) morbifera ) 5 kg of the ground portion was placed in an extraction container, and 50% of 70% methanol was added thereto. The obtained extract was concentrated under reduced pressure using a vacuum rotary evaporator. The extraction and concentration process was repeated twice to obtain 950 g of Hilchi chile extract.

After the methanol extract of Hwangchil-tree was suspended in 5 L of distilled water, the extraction was repeated three times with the same amount of ethyl acetate. The extract was concentrated under reduced pressure to obtain 258 g of ethyl acetate. The filtrate obtained from the extraction process was extracted three times with the same amount of n-butanol and the extract was concentrated under reduced pressure to give 159 g of butanol fraction.

Example 2: Isolation and Identification of Active Ingredients from Hwangchil Tree Extracts

Nine compounds were isolated from the extract of Hwangchil tree prepared in Example 1, and the structural formulas and names of these compounds are shown in FIG. 3.

Specifically, 220 g of the ethyl acetate fraction of the obtained fractions, flowing a dichloromethane / methanol mixed solution to the mobile phase, but the concentration gradient elution method to sequentially increase the dichloromethane / methanol mixture ratio from 100: 1 to 1: 1 Silica gel column chromatography was performed to obtain a total of four fractions (first fraction to fifth fraction).

The obtained second fraction (20 g) was subjected to silica gel column chromatography again (mobile phase: n-hexane / ethyl acetate (50: 1 to 1: 1)) to 5 fractions (21 fractions to 25 fractions). Divided. Of this, the 21st fraction was recrystallized (solvent: n-hexane) to obtain a white powder, which was subjected to silica gel column chromatography (mobile phase: dichloromethane / methanol (40: 1)) to give compound 3 (10.0 mg). Compound 4 (14.5 mg) was isolated. In addition, the 22th fraction was recrystallized (solvent: n-hexane) to obtain Compound 5 (120.0 mg), and the mother liquor remaining after recrystallization was again subjected to silica gel column chromatography (solvent: n-hexane / ethyl acetate (50: 1 to 1). (1)) and divided into five small fractions (221 fractions-225 fractions). The 224th 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 by 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 5 fractions (31 fractions to 35 fractions). Divided. Of this, the 32nd fraction was purified two more times by silica gel column chromatography (mobile phase: n-hexane / ethyl acetate (30: 1 ~ 5: 1)) to obtain 70.0 mg of white powder, and ¹ H NMR and ¹³ C Through NMR, it was confirmed that the compound 8 and the compound 9 were mixed at a ratio of 1: 1. The 34th fraction was subjected to silica gel column chromatography (mobile phase: n-hexane / ethyl acetate (20: 1 to 1: 1)), and then separated / purified by reverse phase column chromatography with 70% methanol (mobile phase) to obtain Compound 1 ( 201.0 mg) and Compound 2 (112.0 mg) were obtained, and Compound 1 was identified as a novel compound. Each of the isolated compounds was identified by ¹ H-NMR and ¹³ C-NMR.

Experimental Example

Experimental Example 1 inhibitory effect of the production of osteoclasts of Hwangchil-tree extract and its fractions

Bone marrow cells were obtained by purchasing 5 week old male ICR mice, taking the femur and tibia and washing them with α-MEM containing antibiotics (100 units / ml penicillin and 100 μg / ml streptomycin). ) Was obtained. The bone marrow cells were cultured in α-MEM containing 10% fetal bovine serum (FBS) and macrophage colony-stimulating factor (M-CSF) for 10 days. Non-adherent myeloid cells were dispensed into Petri dishes and incubated for 3 days in the presence of M-CSF (30 ng / ml). After washing the non-adherent cells, adherent cells were used as bone marrow-derived macrophage (BMM). BMM cells were prepared in DMSO or in Example 1 from Hwangchil extract or fractions thereof in concentrations (1, 3 and 10 μg / ml) in the presence of RANKL (10 ng / ml) and M-CSF (30 ng / ml). Treated and incubated for 4 days. The negative control group used cells not treated with RANKL and 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 ensure permeability. Cells were stained with TRAP solution (Sigma-Aldrich). Multinucleated osteoclasts were visualized by TRAP (tartrate resistant acid phosphatase) staining and shown in FIG. 1A (extract result) and FIG. 2A (fraction result). This is shown in FIG. 1B (extract result) and FIG. 2B (fraction result).

Hwangchil wood extract of Example 1 and its fractions can be confirmed in Figure 1 and 2 to inhibit the production of osteoclasts in a concentration-dependent manner. The methanol extract of Hwangchil tree significantly inhibited the production of osteoclasts from the concentration of 3 μg / ml, and the ethyl acetate fraction and the n-butanol fraction of the Hwangchil methanol extract were significantly inhibited from the concentration of 10 μg / ml. The inhibitory effect of the ethyl acetate fraction was noticeably better than the n-butanol fraction.

The significance test was carried out through the student t-test, the significance * compared to the RANKL treatment group * p <0.05, ** p <0.01, *** *** p <0.001. ### represents the significance (p <0.001) when comparing the negative control group and RANKL treatment group.

Experimental Example 2 inhibitory effect of the production of osteoclasts of Hwangchil-tree extract-derived compound

<2-1> Inhibitory Effect of Compounds 1-9 on Osteoblast Production

In order to confirm the inhibitory effect of the osteoclast production of the compounds 1-9 isolated in Example 2, the same method as in Experimental Example 1 was used, and compounds 1-9 were treated at concentrations of 10 μM and 30 μM, respectively. TRAP staining results are shown in FIG. 4 and TRAP activity measurement results are shown in FIG. 5. In Figure 4, compounds 1, 2, 3, 4, 5, and 7 can be confirmed that significantly inhibits the production of osteoclasts, but other compounds do not significantly inhibit the osteoclast production is found to have no inhibitory effect Can be. In addition, compounds 1, 2 and 3 in Figure 5 it can be seen that significantly inhibiting the production of osteoclasts at both 10 μM, 30 μM concentration, in particular the effect of inhibiting the osteoclast production of compounds 1 and 2 significantly It can be confirmed that it is excellent.

Significance assay was performed in the same manner as the assay method of Experimental Example 1.

<2-2> Inhibitory Effects of Compounds 1 and 2 on Osteoclast Production

In order to confirm the inhibitory effect of the osteoclast production of the compounds 1 and 2 isolated in Example 2, the experiment was repeated by subdividing the concentration. The same method as Experimental Example 1 was used, and compounds 1 and 2 were treated at concentrations of 0.1, 0.3, 1, 3, 10, and 30 μM, respectively. TRAP staining results are shown in A of FIG. 6, and TRAP activity measurement results are shown in B of FIG. 6.

Compounds 1 (Formula 1) and 2 (Formula 2) inhibited osteoclast formation in a concentration-dependent manner in the same manner as the results of Experimental Example 2-1, both compounds significantly osteoclast formation from 1 μM concentration Suppressed. In addition, the 50% activity inhibitory concentration (IC ₅₀ ) was calculated to be 2.41 μM and 3.11 μM of compounds 1 and 2, respectively.

Experimental Example 3: Cytotoxicity of Compounds 1 and 2

In order to confirm the cytotoxicity of compounds 1 and 2, LDH cytotoxicity assay was performed. The higher the level of cellular damage, the higher the level of LDH. BMM cells were dispensed into 96-well plates at a concentration of 1 × 10 ⁵ / ml using a medium containing 30 ng / ml M-CSF and 10 ng / ml RANKL, and then Compounds 1 and 2 were respectively 0.1, The wells were treated at concentrations of 0.3, 1, 3, 10, 30 μM and incubated for 4 days. Thereafter, 50 µl of the culture medium supernatant and 50 µl of LDH substrate solution (CytoTox96 Non-Radioactive Cytotoxicity Assay kit, Promega) are reacted at room temperature for 30 minutes. To stop the reaction, 50 μl of stop solution (1M acetic acid) was added, and then the absorbance was measured at 490 nm using a Microplate ELISA reader to confirm the presence or absence of cytotoxicity of the compound. The results are shown in FIG. 7.

In FIG. 7, Compounds 1 and 2 show LDH activity similar to that of the control at all concentrations, indicating no cytotoxicity.

Claims (10)

16-acetyl-9,17-octadecadiene-12,14-dinoic acid (16-acetyl-9,17-octadecadiene-12,14-diynoic acid) compound or a pharmaceutically acceptable salt thereof.

(9Z, 16S) -16-acetyl-9,17-octadecadiene-12,14-dinoic acid ((9Z, 16S) -16-acetyl-9,17-octadecadiene-12,14 represented by Formula 1 below -diynoic acid) compound or a pharmaceutically acceptable salt thereof.

[Formula 1]

The method according to claim 1 or 2,

The compound is isolated from Dendropanax morbifera compound or a pharmaceutically acceptable salt thereof.

(a) extracting the yellow lacquer with an extractant, filtration and concentration to obtain a yellow lacquer extract;

(b) fractionating the hwangchil extract of (a) with an organic solvent and performing column chromatography to obtain a fraction; And

(c) separating the compound by performing high performance liquid chromatography from the fraction of (b).

A pharmaceutical composition for preventing or treating bone diseases, comprising the compound of claim 1 or 2 or a pharmaceutically acceptable salt thereof.

The method of claim 5,

The bone disease is a growth stage development, fracture, osteoporosis (osteoprosis) due to excessive absorption of osteoclasts, rheumatoid arthritis, periodontal disease (periodontal disease, Paget disease) and metastatic bone cancer (metastatic) one or more pharmaceutical compositions selected from the group consisting of bone cancers).

A quasi-drug composition for preventing or ameliorating bone diseases, comprising the compound of claim 1 or 2 or a pharmaceutically acceptable salt thereof.

A dietary supplement for the prevention or improvement of a bone disease, comprising the compound of claim 1 or 2 or a pharmaceutically acceptable salt thereof.

A cosmetic composition for preventing or improving bone diseases, comprising the compound of claim 1 or 2 or a pharmaceutically acceptable salt thereof.

A method of treating bone diseases in an animal other than a human, comprising administering the composition of claim 5 to a suspected bone disease subject.

Images