KR101854144B1 - Novel diynoic acid methyl ester compound and pharmaceutical composition for preventing or treating bone diseases comprising the same - Google Patents

Abstract

The present invention relates to novel dino acids acid methyl ester compounds, especially to 16-acetoxy-9,17-octadecadiene-12,14 -diynoic acid methyl ester compound is excellent in the osteoclast differentiation inhibitory effect even at a significantly smaller concentration than other extracts or compounds known to have an effect on bone diseases and thus is useful for prevention, A method of treating a bone disease of an animal other than a human, comprising the step of administering the pharmaceutical composition, the quasi-drug composition, the health functional food composition, the cosmetic composition, and the pharmaceutical composition to a suspected bone disease subject.

Description

TECHNICAL FIELD The present invention relates to a novel dienoic acid methyl ester compound and a pharmaceutical composition for preventing or treating bone diseases containing the same,

The present invention relates to a novel composition for the prevention or treatment of bone diseases, comprising a compound of the formula (I) or a pharmaceutically acceptable salt thereof, a process for producing the compound, a compound or a pharmaceutically acceptable salt thereof, To a quasi-drug composition for preventing or ameliorating a disease, a health functional food composition, a cosmetic composition, and a method for treating bone diseases in an animal other than a human, comprising the step of administering the pharmaceutical composition to a suspect bone disease subject.

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.

In addition, there is always a need for a compound having an effect superior to other extracts or compounds known to be effective for bone diseases.

It is an object of the present invention to provide a novel compound having superior osteoclast differentiation inhibitory effect even at a significantly lower concentration than other extracts or compounds known to be effective for bone diseases.

The present invention also relates to a pharmaceutical composition, a quasi-drug composition, a health functional food composition, a cosmetic composition and a pharmaceutical composition which can be usefully used for prevention, treatment or improvement of bone diseases through the novel compound, To provide a method for treating bone diseases in animals other than humans.

In order to solve the above-mentioned problems, the present invention provides a process for producing 16-acetoxy-9,17-octadecadiene-12,14-diynoic acid methyl ester (16-acetoxy-9,17-octadecadien- Or a pharmaceutically acceptable salt thereof.

Specifically, the compound is preferably represented by the following general formula (10).

[Chemical formula 10]

(9Z, 16S) -16-acetoxy-9,17-octadecadien-12,14-dinoic acid methyl ester ((9Z, octadecadiene-12,14-diynoic acid methyl ester).

The inventors of the present invention have confirmed that the compound of formula (10) has excellent osteoclast differentiation inhibitory effect even at a significantly lower concentration than other natural extracts known to be effective for bone diseases or other compounds separated therefrom, Respectively. That is, it was confirmed that the compound of formula (10) had about 10 times better effect than the other compounds isolated from Huanglong extract (see the formula (1) below, see FIG. 3).

The compound of formula (10) can be synthesized by reacting 16-acetoxy-9,17-octadecadiene-12,14-diynoic acid (16-acetoxy-9,17-octadecadiene- 1) can be obtained by methylation.

The above-mentioned 16-acetoxy-9,17-octadecadien-12,14-dinoic acid was isolated from the Huangchil extract of the present inventor and methylated to obtain the compound of formula (10) according to the present invention. That is, the compound of Formula 1 is contained in Huangchil or its extract, but the compound of Formula 10 is completely new, not included in Huangchil or its extract.

The present inventors have obtained the compound of formula (10) from the compound of formula (1) separated from the sulfuric acid, but the present invention is not limited thereto. The compound of formula (10) may be prepared by a chemical synthesis method or may be obtained from an artificially synthesized compound of formula (1).

Pharmaceutically acceptable salts of such compounds may be acid addition salts formed by pharmaceutically acceptable free acids. As the free acid, organic acid and inorganic acid can be used. As the inorganic acid, hydrochloric acid, bromic acid, sulfuric acid, sulfurous acid, phosphoric acid and the like can be used. As the organic acid, citric acid, acetic acid, maleic acid, fumaric acid, , Acetic acid, glycolic acid, succinic acid, tartaric acid, 4-toluenesulfonic acid, galacturonic acid, embonic acid, glutamic acid, citric acid and arpartic acid.

The addition salt according to the present invention can be obtained by a conventional method, that is, by dissolving the compound of formula (10) in a water-miscible organic solvent such as acetone, methanol, ethanol, acetonitrile or the like and adding an equivalent amount or excess amount of an organic acid, Followed by precipitation or crystallization, or by evaporating a solvent or excess acid, followed by drying or precipitation of the precipitated salt by suction filtration.

The present invention includes both the compounds of formula (10) and pharmaceutically acceptable salts thereof, as well as possible solvates, hydrates and stereoisomers thereof, which can be prepared therefrom, all within the scope of the invention.

In another aspect, the present invention relates to a method for producing a fermented soybean curd extract, comprising the steps of: (a) extracting Huangchil with an extraction solvent, filtering and concentrating the Huangchil extract; (b) fractioning the yellowish extract of (a) with an organic solvent and performing column chromatography to obtain a fraction; And (c) separating the compound from the fraction of (b) by performing high performance liquid chromatography; And (d) methylating the compound of (c).

In the present invention, the step (a) is a step of extracting Huangchil with an extraction solvent, filtering and concentrating to obtain Huangchil extract.

In the present invention, the term " Hwangchil " is a dicotyledonous plant native to the southern coast of Korea and Jeju Island, and Dendropanax morbifera, an evergreen tree of Araliaceae, Of the resin solution comes out. 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 ..... 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 Microbiology and Biotechnology, 41 (4): 407-415, 2013). In the present invention, the above-mentioned Huangchil can be cultivated or commercially available, and any of the above-ground parts, roots, and seeds can be used.

In the present invention, the extraction solvent may be an extraction solvent commonly used in the art, but not limited thereto, water, an alcohol 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 be added in an amount of 2 to 20 times, more preferably 4 to 10 times, and more preferably 5 times, in terms of the reduced amount. The method of extraction is not limited thereto, but it is preferable to use a method of hot water extraction, hot water extraction, cold extraction, reflux cooling extraction or ultrasonic extraction, more preferably hot water extraction or cold extraction . 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. The extraction time may be, but not limited to, preferably 12 hours to 10 days, more preferably 5 days to 8 days, and even more preferably 7 days. The number of times of extraction is not limited thereto, but may be preferably 1 to 5 times, more preferably 3 times. Using the above extraction conditions, the yellow crush is extracted and filtered, and the filtrate is concentrated and lyophilized to obtain a Huangchil extract.

In the present invention, the step (b) is a step of fractionating the Huangchil extract of (a) with an organic solvent and carrying out column chromatography to obtain fractions.

The organic solvent is a liquid organic compound capable of dissolving a solid, a gas and a liquid. An organic solvent commonly used in the art can be used, and preferably an organic solvent such as methanol, chloroform, acetate, hexane, dichloromethane, acetone, acetonitrile , Benzene, or a mixed solvent thereof.

The column chromatography is a chromatographic method in which a fixed-bed granule is filled in a column of a chromatographic column and proceeds. When necessary, an appropriate filler may be selected and performed several times. Preferably, the filler is silica gel, Sephadex, RP -18, polyamide, toyopearl or XAD resin.

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

The above-mentioned high performance liquid chromatography (HPLC) is a chromatography method in which high-speed and high-performance separation can be achieved by improvement and improvement of a column, a fixed-bed filler filled in a column and an apparatus, and finally, a compound represented by the above- .

In the present invention, step (d) is a step of methylating the above-mentioned (c). Methods and conditions for methylation are not particularly limited and may include all methods known in the art. Preferably, the compound of formula 1 is reacted with diazomethane and MeOH at 0 ° C to obtain 16-acetoxy-9,17-octadecadien-12,14-dinoic acid methyl ester (formula 10) .

The compound of formula (10) according to the present invention can be obtained through the above process, and the compound of formula (10) has a remarkably superior osteoclast differentiation inhibiting effect. This is a useful method.

In another aspect, the present invention provides a pharmaceutical composition for preventing or treating bone diseases, comprising the compound or a pharmaceutically acceptable salt thereof.

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

The compounds according to the present invention are excellent in the osteoclast differentiation inhibitory effect even at a significantly lower concentration than other extracts or compounds known to have an effect on bone diseases and thus can be usefully used for the prevention, It is effective.

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. 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 > 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.

In the present invention, the term " prevention " means all the actions of inhibiting or delaying the onset of bone disease by administration of the composition, and " treatment " it means.

The pharmaceutical composition of the present invention may comprise a pharmaceutically acceptable carrier. Compositions comprising a pharmaceutically acceptable carrier can be of various oral or parenteral formulations. In the case of formulation, it can be prepared using diluents or excipients such as fillers, extenders, binders, humectants, disintegrants, surfactants and the like which are usually used. Solid formulations for oral administration may include tablet pills, powders, granules, capsules and the like, which may contain one or more excipients such as starch, calcium carbonate, sucrose or lactose lactose, gelatin, and the like. In addition to simple excipients, lubricants such as magnesium stearate, talc, and the like may also be used. Liquid preparations for oral administration include suspensions, solutions, emulsions, syrups and the like. Various excipients such as wetting agents, sweeteners, fragrances, preservatives and the like may be included in addition to water and liquid paraffin, which are simple diluents commonly used. have. Formulations for parenteral administration may include sterile aqueous solutions, non-aqueous solutions, suspensions, emulsions, freeze-dried preparations, and suppositories. Examples of the non-aqueous solvent and the suspending agent include propylene glycol, polyethylene glycol, vegetable oil such as olive oil, and injectable ester such as ethyl oleate. Examples of the suppository base include witepsol, macrogol, tween 61, cacao paper, laurin, glycerogelatin and the like.

The pharmaceutical composition of the present invention may also be in the form of tablets, pills, powders, granules, capsules, suspensions, solutions, emulsions, syrups, sterile aqueous solutions, non-aqueous solutions, suspensions, emulsions, Or a pharmaceutically acceptable salt thereof.

The dosage form of the pharmaceutical composition of the present invention is not particularly limited and may be varied depending on the degree of absorption, body weight, age, sex, health condition, diet, administration time, administration method, excretion rate, have. In general, the daily dose may preferably be from 0.1 to 100 mg / kg, more preferably from 30 to 80 mg / kg, and even more preferably from 50 to 60 mg / kg, / kg. < / RTI > The pharmaceutical composition of the present invention is prepared in consideration of an effective dose range, and the unit dosage formulations thus formulated are classified according to the judgment of the expert who monitors or observes the administration of the drug, if necessary, Or may be administered several times at a predetermined time interval.

In another aspect, the present invention provides a quasi-drug composition for preventing or ameliorating a bone disease comprising the compound. Such compounds are as described above and may include pharmaceutically acceptable salts thereof. More specifically, the composition of the present invention can be added to a quasi-drug composition for the purpose of preventing or improving bone diseases.

The term " quasi-drug " in the present invention means a fiber, a rubber product or the like used for the purpose of treating, alleviating, treating or preventing a disease of a human or an animal, Or products similar to those that are not machinery, preparations used for sterilization, insecticides and similar uses for the prevention of infectious diseases, for the purpose of diagnosing, treating, alleviating, treating, or preventing diseases of human beings or animals Means goods, machinery, or apparatus other than the apparatus, machinery or equipment of the commodity being used and used for the purpose of giving pharmacological effects to the structure and function of humans or animals.

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

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

In another aspect, the present invention provides a health functional food composition for preventing or ameliorating a bone disease comprising the above compound. Such compounds are as described above and may include pharmaceutically acceptable salts thereof. More specifically, the composition of the present invention may be added to a health functional food composition for the purpose of preventing or improving bone diseases.

When the composition of the present invention is used as a health functional food additive, the composition may be added as it is or may be used together with other health functional foods or health functional food ingredients, and may be appropriately used according to a conventional method. The amount of the active ingredient to be mixed can be appropriately determined depending on the purpose of use. Generally, the composition of the present invention may be added in an amount of preferably not more than 15 parts by weight, more preferably not more than 10 parts by weight, based on the raw material, in the production of food or beverage. However, in the case of long-term intake intended for health control and hygiene, the amount may be less than the above range, and since there is no problem in terms of stability, the active ingredient may be used in an amount in the above range.

There is no particular limitation on the kind of the health functional food of the present invention. Examples of the health functional food to which the composition can be added include dairy products such as meat, sausage, bread, chocolate, candy, snack, confectionery, pizza, ramen and other noodles, gums, ice cream, Alcoholic beverages, and vitamin complexes, and may include all the health functional foods in the conventional sense, and foods used as feeds for animals.

In addition, when the health functional food composition of the present invention is used in the form of a drink, it may contain various sweetening agents, flavoring agents, or natural carbohydrates as additional components such as ordinary beverages. The natural carbohydrates may be polysaccharides such as disaccharides such as monosaccharides such as glucose and fructose, maltose, sucrose, dextrin, cyclodextrins, and sugar alcohols such as xylitol, sorbitol and erythritol. The ratio of the natural carbohydrate is not limited thereto, but may be 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 sweeteners may be natural sweeteners such as tau martin and stevia extract, and synthetic sweeteners such as saccharin and aspartame.

In addition to the above, the health functional food composition of the present invention may contain various nutrients, vitamins, electrolytes, flavors, colorants, pectic acid and salts thereof, alginic acid and salts thereof, organic acids, protective colloid thickening agents, pH adjusting agents, stabilizers, , Alcohols, carbonating agents used in carbonated drinks, and the like. It may also contain flesh for the production of natural fruit juices, fruit juice drinks and vegetable drinks.

In another aspect, the present invention provides a cosmetic composition for preventing or improving osteopathy comprising the above compound. Such compounds are as described above and may include pharmaceutically acceptable salts thereof. More specifically, the composition of the present invention can be added to a cosmetic composition for the purpose of preventing or improving bone diseases.

The cosmetic composition of the present invention can be produced in the form of a general emulsified formulation and a solubilized formulation. Examples of the emulsified formulations include nutritive lotions, creams, essences, and the like, and the solubilization formulations include softening longevity. Suitable formulations include, but are not limited to, solutions, gels, solid or paste anhydrous products, emulsions obtained by dispersing the oil phase in water, suspensions, microemulsions, microcapsules, microgranules or ionic (liposomes) A cream, a skin, a lotion, a powder, an ointment, a spray, or a conical 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 may further contain at least one selected from the group consisting of fatty substances, organic solvents, solubilizers, thickening and gelling agents, softening agents, antioxidants, suspending agents, stabilizers, foaming agents, perfumes, surfactants, water, ionic or nonionic emulsifiers, Auxiliaries such as ionic sequestering agents, chelating agents, preservatives, vitamins, blocking agents, moisturizers, essential oils, dyes, pigments, hydrophilic or lipophilic active agents, lipid vesicles or any other ingredients conventionally used in cosmetic compositions ≪ / RTI >

In yet another aspect, the present invention provides a method for treating bone diseases in an animal other than a human, comprising the step of administering the pharmaceutical composition to a subject suspected of having a bone disorder.

In the present invention, the suspected individual of bone disease refers to all animals, including humans, who have developed or may develop osteopathy. The pharmaceutical composition comprising the compound of the present invention or a pharmaceutically acceptable salt thereof may be administered to a subject suspected of having bone disease , The individual can be treated efficiently. The above-mentioned bone diseases are as described above.

The term " administering " in the present invention means introducing the pharmaceutical composition of the present invention to a subject suspected of having bone disease by any appropriate method, and the administration route may be selected from various routes of oral or parenteral administration ≪ / RTI >

The method of treatment of the present invention may comprise administering a pharmaceutical effective amount of a pharmaceutical composition comprising said compound. The appropriate total daily dose may be determined by the treatment within the scope of appropriate medical judgment, and may be administered once or several times. For purposes of the present invention, however, the specific therapeutically effective amount for a particular animal will depend upon the nature and extent of the reaction to be achieved, the specific composition, including whether or not other agents are used, the age, weight, Sex and diet of the patient, the time of administration, the route of administration and the rate of administration of the composition, the duration of the treatment, the drugs used or concurrently used with the specific composition, and similar factors well known in the medical arts.

The compounds according to the present invention are excellent in inhibiting osteoclast differentiation even at a significantly smaller concentration than other extracts or compounds known to be effective for bone diseases. That is, the compound according to the present invention has about 10 times better effect than the compound isolated from Huanglong extract. Therefore, the compound according to the present invention has an effect that can be effectively used for prevention, treatment, or improvement of bone diseases.

The present invention also relates to a pharmaceutical composition, a quasi-drug composition, a health functional food composition, a cosmetic composition and a pharmaceutical composition which can be usefully used for prevention, treatment or improvement of bone diseases through the novel compound, The present invention provides a method for treating bone diseases in animals other than humans.

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.
8 and 9 are graphs of NMR analysis of 16-acetoxy-9,17-octadecadien-12,14-dinoxymethylester obtained through methylation according to the present invention.
FIG. 10 is an image showing the effect of inhibiting the osteoclast differentiation of 16-acetoxy-9,17-octadecadien-12,14-dinoxymethyl ester 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 species of compounds were isolated from the extract of Wuchulia crassifolia prepared from 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.

Example  3: Dino acids methyl  Preparation of Ester (Formula 10) Compound

The compound 1 (16-acetoxy-9,17-octadecadiene-12,14-diynoic acid) obtained from the above Example 2 was dissolved at 0 ° C Lt; / RTI > with diazomethane and MeOH to give the new compound.

The results show that, as shown in the NMR analysis graphs of Figures 8 and 9, the new compound is 16-acetoxy-9,17-octadecadien-12,14-dinoic acid methyl ester -octadecadiene-12,14-diynoic methyl ester), which was found to have the structure of formula (10).

< Experimental Example  >

Experimental Example  1: Wortwood extract and Fraction  Inhibitory effect of osteoclast formation

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.

Experimental Example  4 : Dino acids methyl  Inhibitory effect of ester (Compound 10) on osteoclast differentiation

Raw 264.7 cells were plated in 96-well plates at a density of 1 × 10 ⁴ cells / well and 100 μl / well, and then cultured in a 5% CO 2 incubator at 37 ° C. After 2 hours, 100 ng / ml RANKL was added to the cell differentiation medium containing the compound 1 of Example 2 and the compound 10 of Example 3, followed by incubation for 5 days in a 5% CO 2 incubator at 37 ° C. Images were observed for each compound using a microscope.

As a result, as shown in FIG. 10, the experimental group treated with the compounds of formulas (1) and (10) showed similar cell shape to the normal control group (No RANKL), unlike the negative control group (100 ng / mL).

In addition, the compound of Formula 10 (lower picture of FIG. 10) showed similar cell morphology at a lower concentration of about 1/10 compared to the compound of Formula 1 (upper picture of FIG. 10). That is, the compound of formula (10) has an excellent effect of inhibiting osteoclast differentiation even at a significantly smaller concentration than the compound of formula (1), and has about 10 times better effect than the compound of formula (1).

Claims (10)

delete delete delete delete 16-acetoxy-9,17-octadecadiene-12,14-diynoic acid methyl ester compound represented by the following formula (10) or a 16-acetoxy-9,17- A pharmaceutical composition for the prevention or treatment of bone diseases comprising a pharmaceutically acceptable salt.
[Chemical formula 10]

6. The method of claim 5,
The above-mentioned bone diseases include osteoporosis, rheumatoid arthritis, periodontal disease, Paget disease and metastatic osteoclastic osteoclast due to growth retardation, fracture, excessive osteoclast bone resorption, osteoporosis, rheumatoid arthritis, periodontal disease, Paget disease, bone cancers). &lt; / RTI &gt;
16-acetoxy-9,17-octadecadiene-12,14-diynoic acid methyl ester compound represented by the following formula (10) or a 16-acetoxy-9,17- A quasi-drug composition comprising a pharmaceutically acceptable salt.
[Chemical formula 10]

16-acetoxy-9,17-octadecadiene-12,14-diynoic acid methyl ester compound represented by the following formula (10) or a 16-acetoxy-9,17- A health functional food composition for preventing or ameliorating a bone disease comprising a pharmaceutically acceptable salt.
[Chemical formula 10]

16-acetoxy-9,17-octadecadiene-12,14-diynoic acid methyl ester compound represented by the following formula (10) or a 16-acetoxy-9,17- A cosmetic composition comprising a pharmaceutically acceptable salt.
[Chemical formula 10]

A method for treating a bone disease in an animal other than a human, comprising the step of administering the composition of claim 5 to a suspect individual with a bone disorder.

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