JPWO2005085223A1 - Composition for prevention and / or treatment of bone disease, functional food or health food containing the composition, and pharmaceutical preparation comprising the composition as an active ingredient - Google Patents

Abstract

It aims at providing as a pharmaceutical, a functional food, or a health food which has the prevention and / or treatment action of the bone disease which uses the safe flavonoid compound derived from a natural product as an active ingredient. Pharmaceutical composition for prevention and / or treatment of bone disease comprising at least one compound represented by the following formula (I), or a physiologically acceptable salt, hydrate or glycoside thereof: Offer things. [Wherein R1 to R6 each represents a group selected from the group consisting of a hydrogen atom, a hydroxyl group and a methoxy group. Here, R1 or R3 may be linked to a sugar or a derivative thereof; When R1, R2, R3 and R5 are hydroxyl groups, sugars may be bonded to any of them to form a glycoside, and R1 to R6 simultaneously become hydrogen atoms. No.)

Description

  The present invention relates to the use of flavones represented by the following formula (I) among flavonoids derived from crude drug components. More specifically, a health food, a functional food, or a bone disease containing at least one flavone represented by the following formula (I) or a physiologically acceptable salt or hydrate thereof: The present invention relates to a pharmaceutical composition having a prophylactic and / or therapeutic effect, and a pharmaceutical preparation containing the pharmaceutical composition as an active ingredient.

(Wherein R ¹ , R ² , R ³ , R ⁵ and R ⁶ each represent a hydrogen atom or a hydroxyl group, and R ⁴ represents a functional group selected from the group consisting of a hydrogen atom, a hydroxyl group and a methoxy group. , R ¹ , R ² , R ³ and R ⁵ are hydroxyl groups, a sugar may be bonded to any of them to form a glycoside, R ^{1 to} R ⁶ Are not simultaneously hydrogen atoms.)

  Bone diseases include exogenous diseases such as traumatic fractures and fatigue fractures, osteoporosis, osteoporosis, hypercalcemia, hyperparathyroid hormone (hereinafter sometimes referred to as “PTH”), and bone paget. It is roughly divided into bone tissue weakness caused by diseases, arthritis, rheumatism, breast cancer bone metastasis, osteomalacia, malignant tumors and other diseases, and bone tissue weakness due to nutritional disorders.

  Human bones are constantly remodeling and resorbing with osteoblasts and osteoclasts. Therefore, when bone formation by osteoclasts exceeds bone resorption by osteoclasts, the bone tissue is weakened and endogenous bone diseases such as osteoporosis and osteoporosis as described above occur. It is said that.

  Among these endogenous bone diseases, osteoporosis and osteoporosis are increasing with the progress of an aging society. In addition, along with changes in dietary habits and a decrease in exercise, a decrease in calcium intake and a decrease in the rate of colonization of bone tissue occur, and the weakening of bone tissue is accelerated.

  A fracture refers to a state in which communication with a part of bone tissue is disconnected by an external force, and is accompanied by significant pain at the fracture site. In the case of a healthy person, for example, a fracture does not occur unless a considerable external force is applied due to a traffic accident, etc., but when the bone mass decreases and the bone tissue weakens, it falls while walking or running, etc. Even if an external force that is not so great is applied, a fracture occurs. In addition, when bone mass is reduced due to intrinsic diseases such as osteoporosis and osteoporosis as described above, fractures can be caused by slight external force applied when crawling or coughing on stairs. It comes to occur.

  When a fracture occurs, a treatment method is generally adopted in which the position of the displaced bone is corrected by traction, etc., and if it can be fixed with bolts or pins, it is fixed and then left to the patient's natural healing power. ing.

  On the other hand, in endogenous diseases such as osteoporosis and osteoporosis, both bone mineral and bone matrix decrease, so natural healing is delayed. There are many cases where people are bedridden.

Patent application 10-372842 Molteni, A., et al .: In vitro hormonal effects of soybean isoflavones, J. Nutr. 125, 3S, 751 (1995). Anthony, M.S., et al .: Soybean isoflavones improve cardiovascular risk factors without affecting the reproductive system of peripubertal rhesus monkeys, J. Nutr. 126, 43 (1996). Takuo Okuda: Flavonoids, Natural Product Chemistry Revision 2nd Edition (Hiroshi Mitsuhashi et al.), P.203, (Nanedo, 1986)

  Therefore, there is a strong social demand for shortening the time required for healing in the treatment of fractures. And in order to use it as a pharmaceutical, it must have a high therapeutic effect and a high safety such as few side effects.

Conventionally, active vitamin D ₃ , calcitonin and its derivatives, hormonal agents such as estradiol, various calcium preparations and the like have been clinically used.

  However, there are cases where such drugs cannot be administered orally due to absorption and metabolism, and there is a problem that the predictability of the effect is lacking due to large individual differences in the receptor level. For this reason, a new therapeutic agent to replace these has been demanded.

  On the other hand, the most effective means for reducing the number of patients with bone disease is prevention of bone disease. Generally, prevention of a disease involves administration of a prophylactic pharmaceutical preparation, intake of food such as a functional food, etc. depending on how close the disease is to the onset.

  In bone diseases as well, there is a social demand not only for therapeutic means for patients who have developed symptoms as described above, but also for effective preventive means for preventing unaffected or healthy people from being affected.

  The present invention has been completed under the above circumstances. That is, the inventors of the present invention have advanced screening of compounds having an inhibitory action on bone resorption, focusing on components contained in plants that have been used as herbal medicines for a long time, while paying attention to high safety. The present inventors have found that a known compound has an activity that has not been known so far, and thus completed the present invention.

  As a result of such screening, it was revealed that catechin and flavonol have an activity of inhibiting osteoclast differentiation. However, the above compounds have problems such as low inhibitory activity and damage to cellular DNA, and are not suitable for use as pharmaceuticals or food ingredients.

  On the other hand, soy-derived isoflavones such as daidzein, glycitin, and genistin have been found to have a female hormone-like action, and they have the effect of preventing myocardial infarction. It has been reported (Non-Patent Documents 1 and 2).

  As for flavones, baicalein, wogonin, and other flavonoids derived from the herbal medicine scutellariae radix have an inhibitory effect on the proliferation of vascular smooth muscle cells in cultured cells. (Patent Document 1).

  Furthermore, when flavones such as luteolin and apigenin are ingested as food ingredients, it is reported that metabolic diseases such as blood, circulatory organs, digestive organs, etc. are prevented (Non-patent Document 3). .

  However, there has been no report on flavones such as those described above that suppress the activity of osteoclasts or suppress bone resorption.

  Under the circumstances as described above, the inventors of the present invention proceeded with screening for a compound that suppresses bone resorption by osteoclasts, and found a naturally occurring compound having such an action and high safety. The present invention has been completed.

  That is, the present invention relates to prevention and prevention of bone diseases comprising at least one compound represented by the following formula (I), or a physiologically acceptable salt, hydrate or glycoside thereof. / Or a therapeutic pharmaceutical composition.

In the formula, R ^{1 to} R ⁶ each represent a group selected from the group consisting of a hydrogen atom, a hydroxyl group, and a methoxy group. Here, sugar or a derivative thereof may be bonded to R ¹ or R ^3, and when R ¹ , R ² , R ^3, and R ⁵ are hydroxyl groups, sugar is present in any of them. It may combine to form a glycoside. The sugar is a sugar selected from the group consisting of glucose, apiose-glucose, and glucopyranoside uronic acid. R ^{1 to} R ⁶ do not simultaneously become hydrogen atoms.

  Here, the compound represented by the above formula (I) is a compound represented by the following formula (II), or a physiologically acceptable salt, hydrate or glycoside thereof,

A compound represented by the following formula (III), or a physiologically acceptable salt, hydrate or glycoside thereof,

A compound represented by the following formula (IV), or a physiologically acceptable salt, hydrate or glycoside thereof,

And a compound represented by the following formula (V), or a physiologically acceptable salt, hydrate or glycoside thereof:

It is preferable to contain at least one compound selected from the group consisting of these, or a physiologically acceptable salt or hydrate thereof.

  Further, when two or more of the above-mentioned compounds, or physiologically acceptable salts or hydrates thereof are mixed to form a composition, the compounds of the above formulas (I) to (V), or these It preferably contains a physiologically acceptable salt or hydrate.

  Moreover, this invention is a pharmaceutical formulation for the prevention and / or treatment of the bone disease which uses the pharmaceutical composition mentioned above as an active ingredient.

  Here, the content of the active ingredient in the pharmaceutical preparation for the prevention and / or treatment of the bone disease is preferably 0.1 to 100 mg, more preferably 0.1 to 50 mg per dose of the preparation. Preferably, it is 0.3-10 mg.

  In addition, the pharmaceutical preparation for the prevention and / or treatment of bone disease is preferably an orally administrable dosage form, and is from the group consisting of tablets, powders, capsules, granules, pills, troches, and solutions. It is preferable to be selected.

  Here, the bone disease may be caused by an endogenous bone disease, an exogenous bone disease, or a nutritional disorder. In the case of the endogenous bone disease, osteoporosis, osteoporosis, high Due to weakening of bone tissue due to a disease selected from the group consisting of calciumemia, hyper-PTHemia, Paget's disease of bone, arthritis, rheumatism, bone metastasis of breast cancer, osteomalacia, malignant tumor, and nutritional disorder Therefore, the pharmaceutical preparation for prevention and / or treatment of bone disease of the present invention can be preferably used. Moreover, in the case of the said exogenous bone disease, it can use suitably in the case of a traumatic fracture, a fatigue fracture, etc.

  The present invention is also a functional food comprising a composition comprising the compound described above, a physiologically acceptable salt thereof, a glycoside thereof and a hydrate.

  Among the above-mentioned compounds, their physiologically acceptable salts, their hydrates and glycosides, those added to functional foods are those physiologically acceptable salts and hydrates. Are preferably selected from the group consisting of sodium salts, potassium salts, ammonium salts, magnesium salts and monohydrates or dihydrates thereof.

  The functional food is also preferably used for assisting in the prevention and / or treatment of bone diseases. Here, the content of the compound, a physiologically acceptable salt thereof, a glycoside and a hydrate is preferably 0.1 to 5 mg per 100 g, More preferably, it is ˜3 mg. When the content is 0.3 to 1 mg, the effect of assisting the prevention and / or treatment of bone disease is the highest.

  It is preferable that the health food is used for assisting the prevention and / or treatment of bone diseases. The content of the compound, those selected from the group consisting of physiologically acceptable salts, glycosides and hydrates thereof is preferably 0.1 to 5 mg per 100 g, preferably 0.1 to 3 mg. More preferably. When the content is 0.3 to 1 mg, the effect of assisting the prevention and / or treatment of bone disease is the highest.

  According to the present invention, functional food, health food, pharmaceutical composition for prevention and / or treatment of bone disease, health food, bone disease prevention, which have excellent osteoclast formation inhibitory activity and bone resorption inhibitory effect. And / or therapeutic agents are provided.

FIG. 1 is a graph showing the effect of luteolin concentration-inhibiting osteoclast differentiation on the MφRAW264 cell line stimulated with 50 ng / mL osteoclast differentiation inducing factor (ODF / RANKL). FIG. 2 is a graph showing the results of the concentration-dependent osteoclast differentiation inhibitory effect of luteolin on mouse bone marrow-derived M-CSF-dependent cells stimulated with 50 ng / mL of ODF / RANKL. FIG. 3A is a drawing-substituting photograph showing the results of confirmation and microscopic observation by a TRAP staining method after culturing the actin ring formed from the MφRAW264 cell line for 1 day in the absence of luteolin. FIG. 3B is a drawing-substituting photograph showing the results of confirmation by a TRAP staining method and microscopic observation after culturing the actin ring formed from the MφRAW264 cell line for 1 day in the presence of 3 μM luteolin. FIG. 3C is a drawing-substituting photograph showing the results of confirmation by a TRAP staining method and microscopic observation after culturing the actin ring formed from the MφRAW264 cell line for 1 day in the presence of 10 μM luteolin. FIG. 3D is a drawing-substituting photograph showing the results of confirmation by a TRAP staining method and microscopic observation after culturing the actin ring formed from the MφRAW264 cell line for 1 day in the presence of 30 μM luteolin. FIG. 4A is a drawing-substituting photograph showing the results of confirmation and microscopic observation by a TRAP staining method after culturing actin rings formed from mouse bone marrow-derived M-CSF-dependent cells for 1 day in the absence of luteolin. FIG. 4B is a photograph, which substitutes for a drawing, showing the results of confirmation by a TRAP staining method and microscopic observation after culturing an actin ring formed from mouse bone marrow-derived M-CSF-dependent cells in the presence of 3 μM luteolin for 1 day. FIG. 4C is a drawing-substituting photograph showing the results of confirmation and microscopic observation by a TRAP staining method after culturing an actin ring formed from mouse bone marrow-derived M-CSF-dependent cells in the presence of 10 μM luteolin for 1 day. FIG. 4D is a drawing-substituting photograph showing the results of confirmation and microscopic observation by a TRAP staining method after culturing an actin ring formed from mouse bone marrow-derived M-CSF-dependent cells in the presence of 30 μM luteolin for 1 day. FIG. 5A substitutes a drawing showing the formation of bone resorption pits after osteoclasts prepared from co-culture of mouse bone marrow cells and osteoblasts on ivory slices for 12 hours in the absence of luteolin. It is a photograph. FIG. 5B shows the formation of bone resorption pits after osteoclasts prepared from co-culture of mouse bone marrow cells and osteoblasts on ivory slices for 12 hours in the presence of 10 μM luteolin. It is a substitute photo. FIG. 6 is a diagram showing the results of the luteolin concentration-dependent pit formation inhibitory effect on osteoclasts prepared from co-culture of mouse bone marrow cells and osteoblasts.

  The present invention is described in further detail below. The compound contained in the pharmaceutical composition for prevention and / or treatment of bone disease of the present invention, or a physiologically acceptable salt, hydrate or glycoside thereof, belongs to a flavonoid, and is represented by the following formula (I): It is a compound called flavones represented.

In the formula, R ¹ , R ² , R ³ , R ⁵ and R ⁶ each represent a hydrogen atom or a hydroxyl group, and R ⁴ represents a functional group selected from the group consisting of a hydrogen atom, a hydroxyl group and a methoxy group. Here, when R ¹ , R ² , R ³ and R ⁵ are hydroxyl groups, sugars may be bonded to any of them to form a glycoside. R ^{1 to} R ⁶ do not simultaneously become hydrogen atoms.

Flavonoid is a general term for a group of compounds having a C ₆ -C ₃ -C ₆ carbon skeleton close to the phenylchroman ring widely distributed in the plant kingdom. As typical compound groups, flavones, isoflavones, flavonols, flavanones, flavans, flavan-3,4-diols, flavan-3-ols, anthocyanidins, neoflavanoids, aurones, chalcones, dihydrochalcones, etc. are known. .

  The flavones refer to a group of substances including a group of compounds in which a hydroxyl group and a methoxy group are bonded to the flavone skeleton and an alkoxy derivative. Most of the flavones are distributed as glycosides in almost all tissues of higher plants and lower plants. Unlike other flavonoids, flavones are known to have C-glycosides in addition to O-glycosides.

  Examples of the compound represented by the above formula (I) or a physiologically acceptable salt, hydrate or glycoside thereof include, for example, luteolin represented by the following formula (II),

Baicalein represented by the following formula (III)

Apigenin represented by the following formula (IV)

And ougonin represented by the following formula (V)

Or a physiologically acceptable salt, hydrate or glycoside thereof. In addition to these, the compounds shown in Table 1 below can be exemplified.

  Luteolin is found in the leaves and branches of Aspalathus linealis, a leguminous conifer, apigenin is found in the roots or shells of sorghum nervosum, and baicalein and ougonin are abundant in the roots of Labiatae Scutellaria baicalensis Georgi Flavones. Apiin is a flavone contained in parsley and celery leaves, and garteolin is contained in horsetail leaves.

  Examples of these physiologically acceptable salts include sodium salt, potassium salt, ammonium salt and the like. Examples of hydrates include monohydrate and dihydrate. Examples of sugars that are bonded to form glycosides include glucose, mannose, glucopyranose, and apiose-glucose (apiosylglucose). Examples of glycosides include luteolin 7 Examples include -glucoside and apigenin (7-D-glycosylapigenin), which is a glycoside of apigenin shown in Tables 1 and 2 above, apine (apigenin-7-apiosylglucoside), and the like.

  The various flavones described above can be used alone for the production of the pharmaceutical composition of the present invention, and two or more kinds may be used in appropriate combination as necessary. In addition, these compounds, or herbal medicines containing these physiologically acceptable salts, hydrates or glycosides, can be used alone or in appropriate combination of two or more.

  Examples of such herbal medicines include dragon (scutellariae radix) and jaundice.

  The above-mentioned flavones such as luteolin represented by formula (II), baicalein represented by formula (III), apigenin represented by formula (IV), and ougonin represented by formula (V) are known. It may be produced and obtained by a method or a method equivalent thereto, or a commercially available product may be purchased and used.

  Moreover, said flavones can also be obtained by extracting and isolating from plant material. For example, an example of a method for isolating luteolin from plant material is shown.

  Water-containing methanol or water-containing ethanol is added to the leaves of Aspalathus linealis (rooibos tea), which is a finely divided legume, to obtain an extract. After dissolving in the obtained extract water, it is degreased using a low polarity solvent such as petroleum ether or benzene. Next, by extracting with butanol, luteolin and its related compounds are extracted into the butanol phase. Here, the related compounds of luteolin include physiologically acceptable salts, hydrates or glycosides of luteolin.

  The luteolin and its related compounds extracted as described above can be used as a crude product, but can be further purified as necessary to obtain a purified sample. This purification can be performed by a conventional method. For example, luteolin and its related compounds are eluted and purified by step gradient column chromatography using silica gel as the stationary phase, chloroform / methanol as the mobile phase, and sequentially increasing the methanol content in the mobile phase. Alternatively, it can be obtained as yellow columnar crystals.

  The baicalein, apigenin, ougonin and other compounds represented by the above formula (I), or physiologically acceptable salts, hydrates or glycosides can be extracted and purified in the same manner.

  The compound represented by the above formula (I) obtained as described above, or a physiologically acceptable salt, hydrate or glycoside can be made into a pharmaceutical composition as follows. .

  When luteolin is used alone as a pharmaceutical composition, the crystals obtained as described above are treated according to a conventional method and mixed with excipients and the like described later to obtain a pharmaceutical composition.

  When luteolin and baicalein are used in combination, baicalein is appropriately mixed with luteolin 1 at a ratio of 0.1 to 10, and this mixture is made into a pharmaceutical composition in the same manner as luteolin alone. That's fine.

  Pharmaceutical preparations containing these pharmaceutical compositions as active ingredients include injections, suppositories, aerosols, transdermal absorption agents and other parenterals, tablets, powders, capsules, pills, troches, liquids and other Oral agents and the like can be mentioned. Here, the tablets include sugar-coated tablets, coated tablets, and buccal tablets, and the capsules include both hard capsules and soft capsules. Granules also include coated granules. The above liquid preparations include suspensions, emulsions, syrups, elixirs and the like, and syrups include dry syrups.

Note that each of the above-mentioned preparations includes not only those that have not been sustained-released but also those that have been sustained-released.

  Such preparations are produced according to known pharmacological production methods using carriers, excipients, disintegrants, lubricants, coloring agents, etc. described in the Japanese Pharmacopoeia that are pharmacologically acceptable in the production of the preparations. be able to.

  Examples of such carriers and excipients include lactose, glucose, sucrose, mannitol, potato starch, corn starch, calcium carbonate, calcium phosphate, calcium sulfate, crystalline cellulose, licorice powder, and gentian powder.

  Examples of the binder include starch, tragacanth gum, gelatin, syrup, polyvinyl alcohol, polyvinyl ether, polyvinyl pyrrolidone, hydroxypropyl cellulose, methyl cellulose, ethyl cellulose, carboxymethyl cellulose and the like.

  Examples of disintegrants include starch, agar, gelatin powder, sodium carboxymethylcellulose, calcium carboxymethylcellulose, crystalline cellulose, calcium carbonate, sodium bicarbonate, and sodium alginate. Examples of lubricants include magnesium stearate, talc, and hydrogenation. Vegetable oil, macrogol, etc. can be used.

  Any colorant can be used as long as it is allowed to be added to pharmaceuticals, and is not particularly limited. In addition to these, flavoring agents, flavoring agents, and the like can be appropriately used as necessary.

  In the case of tablets or granules, as required, sucrose, gelatin, hydroxypropylcellulose, purified shellac, gelatin, glycerin, sorbitol, ethylcellulose, hydroxypropylcellulose, hydroxypropylmethylcellulose, polyvinylpyrrolidone, cellulose phthalate acetate, It may be coated with hydroxypropylmethylcellulose phthalate, methyl methacrylate, methacrylic acid polymer, or the like, and may be coated with a plurality of layers.

  Furthermore, granules and powders can be packed into capsules such as ethyl cellulose and gelatin to form capsules.

  When preparing an injection using the above compounds, or physiologically acceptable salts, hydrates and glycosides thereof, a pH adjuster, a buffer, a stabilizer, Solubilizing agents can also be added.

  When a bone disease preventive and / or therapeutic agent as described above is administered to a patient, the dosage varies depending on various conditions such as the severity of the patient's symptoms, age, weight, and health status. In general, 1 mg / kg to 2,000 mg / kg, preferably 1 mg / kg to 1,000 mg / kg per day for an adult is administered orally or parenterally once or more times a day. Good. The frequency and amount of administration may be increased or decreased as appropriate according to the above conditions.

  When a pharmaceutical composition containing luteolin alone or luteolin and other flavones such as baicalein is contained in the preparation as an active ingredient, the content of luteolin in the composition is 0.1 to 100 mg It is preferably 0.1 to 50 mg, more preferably 0.3 to 10 mg.

  When the content of the active ingredient luteolin or other flavones is less than the lower limit value, the bone resorption suppressing action is not sufficiently exhibited. Conversely, even if the content exceeds the upper limit value, the effect corresponding to the added amount is not exhibited. Further, when the upper limit is exceeded, cytotoxicity may be exerted during administration, which may cause undesirable side effects on the living body.

  By appropriately adding the above-described composition of the present invention as necessary, a functional food or a health food having a bone disease prevention and / or treatment effect can be provided.

  In the present specification, the “functional food” refers to a food containing a component that can provide more benefits than can be provided to a person who has consumed the food by the nutrients originally contained in the food.

  “Health foods” refers to powders, granules, tablets, and capsules based on crude products or refined products produced by extracting ingredients that are useful for maintaining and improving health. It also includes supplements that assist in the intake of other nutritional components.

  The composition of the present invention includes, for example, bread, cookies and biscuits, cooked wheat and millet, udon, buckwheat, pasta and other noodles, cheese, yogurt and other dairy products, jam, mayonnaise, miso, soy sauce and other soy products. , Tea, coffee and cocoa, soft drinks, fruit drinks and other non-alcoholic drinks, medicinal liquors and other alcoholic drinks, candy, chocolate and other snacks, chewing gum, rice crackers, sheepskin and other sweets made from soybeans And it can be set as a functional food.

  In addition, when adding to the above-mentioned yogurt, soy sauce, beverages, etc., in order to prevent the composition of the present invention from crystallizing and precipitating among them, a dissolution aid or a stabilizer may be added as appropriate. it can.

  Moreover, it can be set as a health food by making the composition of this invention individually or in mixture of 2 or more types, and setting it as a powder agent, a granule, a tablet, and a capsule according to a conventional method.

  Here, in order to make the composition of the present invention a powder, the extract obtained in the production process is concentrated and dried using a method such as freeze drying, spray drying, vacuum drying, sample mill, blender, The dried solid may be pulverized with a mixer or the like. Moreover, you may add corn starch, potato starch, dextrin, cyclodextrin, oyster shell powder, etc. as needed.

  The powder obtained as described above may be appropriately added with the above-described binder and compressed into tablets. After making into tablets, the above-described coating agents such as sucrose or gelatin may be used to form sugar-coated tablets, or other coating agents may be used to make enteric solvents and the like.

  Furthermore, the powder obtained as described above can be made into granules according to a conventional method to produce granules. Moreover, it can also be set as a capsule by filling the above-mentioned capsule with the above-mentioned powder and granule in an appropriate amount.

  EXAMPLES Hereinafter, the present invention will be described in more detail with reference to examples, but the present invention is not limited to these examples.

Example 1
(1-1) Osteoclast Formation Test In the osteoclast formation test, there is a macrophage RAW264 cell line (RIKEN, cell number RCB0535, hereinafter referred to as “MφRAW264”) that differentiates into an osteoclast-like cell. )It was used. For the cultivation of MφRAW264, α-MEM (manufactured by INVITROGEN, catalog number 11900-024) containing 10% fetal bovine serum (Asahi Techno Glass Co., Ltd. (IWAKI), catalog number IWK-500) was used. As the osteoclast differentiation inducing factor (ODF / RANKL), catalog number 310-01 manufactured by PEPRO TECH INC was used.

  For luteolin, catalog number 129-04001 manufactured by Wako Pure Chemical Industries, Ltd. was used. Also, catalog numbers 161093 and 172958 manufactured by INVITROGEN were used for the 96-well microplate and the dish having a diameter of 100 mm, respectively.

  Ethanol, acetone, formaldehyde, and sodium chloride were purchased from Wako Pure Chemical Industries, Ltd. and used.

(1-2) Osteoclast formation test I
As an in vitro test method for evaluating the inhibition of bone resorption, an osteoclast formation test from the MφRAW264 cell line was performed. The osteoclast test was evaluated by measuring tartrate-resistant acid phosphatase (TRAP) activity, which is an early differentiation marker, and TRAP staining. By using these two indicators, the activity of flavones for differentiation of MφRAW264 cells into osteoclasts can be quantitatively evaluated.

The MφRAW264 cell line is suspended in α-MEM medium (hereinafter sometimes referred to as “10% FBS-MEM”) supplemented with 10% fetal bovine serum (FBS), and 1 × 10 ⁵ cells in a 100 mm diameter dish. / inoculated with 10 mL, 5% CO ₂ presence and cultured for 3 days at 37 ° C., the cells in the dish and the culture was terminated after confirming that become confluent (confluent, confluent). Then, the confluent cells were treated with Hanks buffer (0.05% Trypsin-0.53 mM EDTA / HBSS (INVITROGEN, catalog number 25300-054) containing 0.05% trypsin and 0.53 mM EDTA from the dish. Peeled off, suspended in 10% FBS-MEM, inoculated into each well of a 96-well microplate at 0.4 × 10 ⁴ cells / 0.1 mL, and cultured for 1 day under the same conditions as described above.

  Osteoclast differentiation inducing factor (ODF / RANKL) was dissolved in 10% FBS-MEM to prepare a 100 ng / mL solution. A solution obtained by dissolving luteolin in methanol at a concentration of 10 mM was prepared as a stock solution and stored at −20 ° C. When performing treatment with luteolin, dilute the stock solution and prepare solutions with concentrations 100 times the respective treatment concentrations (3 mM, 1 mM, 300 μM, 100 μM, and 3 μM) so that the final concentration of the solvent is less than 1%. (2 μL each) was added to each well. An equal amount of methanol was added to the control group. As the luteolin used in the following experiments, solutions having the respective concentrations described above were used.

  0.1 mL of ODF / RANKL was added to all 96 wells (final concentration of RANKL 50 ng / mL), and the luteolin solution was added to the wells (the final concentrations of luteolin were 0.3 μM, 1 μM, 3 μM, 10 μM, and 30 μM, respectively). ). An equal amount of methanol containing no luteolin was added to the control wells.

After culturing at 37 ° C. for 3 days in the presence of 5% CO ₂ , tartrate-resistant acid phosphatase (TRAP) activity was measured as follows.
Discard the culture solution in each well of the 96-well microplate, wash the plate with phosphate buffered saline solution (PBS), and then PBS containing 10% formaldehyde (hereinafter referred to as “10% HCHO-PBS”). Was filled into each well and the cells were fixed at room temperature for 15 minutes. Furthermore, it fixed for 1 minute at room temperature using the ethanol-acetone solution (1: 1). After fixing, the fixing solution was discarded and dried at room temperature. After drying, measurement of the tartrate-resistant acid phosphatase activity of the cells in each well (hereinafter sometimes referred to as “TRAP assay”) and staining were performed.

  The TRAP activity in the cultured cells was measured using a 50 mM citrate buffer (pH 4.6) containing 3.7 mM p-nitrophenol-disodium hydrogen phosphate and 10 mM tartaric acid. 0.1 mL of this buffer was added to each well and reacted at 37 ° C. for 30 minutes. After 30 minutes, the reaction was stopped by adding 0.1 mL of 0.1 M NaOH, and the released p-nitrophenol was measured at a measurement wavelength of 405 nm using a spectrophotometer (manufactured by Dainippon Pharmaceutical Co., Ltd.). .

  In addition, the result of the osteoclast formation test was expressed as a relative value (%) with respect to the control group when the TRAP activity of the control group (control) was 100.

  Furthermore, the state of intracellular tartrate-resistant acid phosphatase was confirmed by TRAP staining. The TRAP staining solution is obtained by dissolving 5 mg of Naphtol AS-MX phosphate (SIGMA, catalog number N-4875) as a substrate in 0.5 ml of N, N-dimethylformamide, and then using Fast red violet LB salt (SIGMA) as a dye. Manufactured and catalog number F-1625) dissolved in 50 ml of 50 mM sodium tartrate / 0.1 M sodium acetate buffer (pH 5.0) were used together. That is, as in the case of the TRAP assay, the medium was removed after culturing. After washing the cell layer with PBS, the cells were fixed with 10% HCHO-PBS for 15 minutes at room temperature. Subsequently, it was further fixed at room temperature for 1 minute using an ethanol-acetone solution (1: 1). After the fixation, the fixing solution was discarded and dried at room temperature. After drying, TRAP stain (added and stained for 20 to 30 minutes at room temperature. After staining, the stain was discarded, washed with running water, dried and observed with a microscope. The results are shown in FIG.

  In FIG. 1, the measurement results of intracellular TRAP activity obtained as a result of osteoclast formation test I are shown as black bar graphs. As apparent from FIG. 1, it was shown that the addition of luteolin suppresses TRAP activity in a concentration-dependent manner. This revealed that luteolin suppressed osteoclast formation from MφRAW264 cells.

(Example 2) Proliferation cell number measurement test (2-1) Materials, etc. Cell lines, medium, serum, 96-well microplate, dish with a diameter of 100 mm, luteolin, etc. are the same as those used in Example 1. used. A 10% FBS-MEM solution of osteoclast differentiation inducing factor (ODF / RANKL) and a methanol solution of luteolin were prepared in the same manner as in Example 1.

(2-2) Proliferating cell number measurement experiment Color reaction by XTT was used as a non-RI method for quantifying cell proliferation and cell viability. The MφRAW264 cell line was suspended in 10% FBS-MEM medium, inoculated into a 96-well microplate at 0.4 × 10 ⁴ cells / 0.1 mL, and cultured for 1 day. Thereafter, 0.1 mL of a 10% FBS-MEM solution (100 ng / mL) of osteoclast differentiation inducing factor (ODF / RANKL) was added to each well (final concentration of RANKL 50 ng / mL). At the same time, each concentration of luteolin was added to the wells. In the control group, only an equal amount of methanol containing no luteolin was added.

  Three days after the start of culture, cell proliferation was quantified using a commercially available Cell Proliferation Kit II (Roche Diagnostics, catalog number 1 465 015) to determine cell viability. The operation was performed according to the attached instruction manual, and the outline is as follows.

  The XTT labeling reagent and the electronic coupling reagent contained in the kit were mixed at a ratio of 50: 1, 50 μL was added per 0.1 mL of the medium added to each well, and the mixture was reacted at 37 ° C. for 4 hours. After completion of the reaction, absorbance at a wavelength of 492 nm was measured using an ELISA plate reader (Dainippon Pharmaceutical Co., Ltd.) with 690 nm as the control wavelength.

  The measurement results were expressed as a relative value (%) relative to the control group when the cell viability of the luteolin treatment group at each concentration was 100 as the cell viability of the control group. The results are shown in FIG.

In FIG. 1, the measurement results of the cell viability obtained as a result of the experiment for measuring the number of proliferating cells are shown as white bar graphs. As is apparent from FIG. 1, the cell viability decreased only to about 50% even when the concentration of luteolin was increased 100-fold. From this, it was clarified that luteolin does not show non-specific cytotoxicity to MφRAW264 cells and significantly suppresses differentiation of MφRAW264 cells into osteoclasts by RANKL depending on the concentration. (IC ₅₀ = 1 μM).

(Example 3) Osteoclast formation test II
(3-1) Materials In Example 3, instead of the MφRAW264 cells used in Examples 1 and 2, mouse bone marrow-derived M-CSF-dependent cells were used in order to evaluate inhibition of bone resorption in vitro. used. Mouse bone marrow-derived cells were collected from tibias and femurs excised from 6-9 week old ddY mice (male) (Chubu Scientific Materials Co., Ltd.).

  Streptomycin sulfate and penicin G sodium (INVITROGEN, catalog number 15140-122), tumor growth factor β (TGF-β, SIGMA, catalog number T-7039), macrophage colony stimulating factor (M-CSF, PEPRO TECH INC) Except for catalog number 300-25), the same one as in Example 1 was used. As in Example 1, the activity for differentiation into osteoclasts by luteolin was evaluated using the measurement of intracellular TRAP activity and TRAP staining as indicators.

(3-2) Osteoclast formation test II
The tibia and femur were removed from 6-9 week old ddY mice. Bone marrow cells were collected from these tibia and femur by extruding with a syringe equipped with 22G × 1 and 1/4 needle.

The obtained bone marrow cells were suspended in 10% FBS-MEM containing 100 U / mL penicillin G sodium salt, 100 μg / mL streptomycin sulfate, 50 ng / mL M-CSF, and 1 ng / mL TGF-β. A 96-well microplate was seeded at 1 × 10 ⁶ cells / 0.1 mL / well and cultured under conditions of 5% CO ₂ and 37 ° C.

Three days after the start of culture, the medium was replaced with a medium containing 50 ng / mL M-CSF and 50 ng / mL osteoclast differentiation inducing factor (ODF / RANKL), and at the same time, luteolin adjusted to the same concentration as in Example 1 was added. As a control group, the medium was replaced with the above medium containing M-CSF and ODF / RANKL but not containing luteolin. The cells were further cultured under the conditions of 5% CO ₂ and 37 ° C.

  1.5 days after the medium change, the tartrate-resistant acid phosphatase activity, which is an osteoclast early differentiation marker, was measured. The culture solution in each well was discarded, and the plate was washed with PBS. Then, cells in the well were fixed for 15 minutes at room temperature using 10% HCHO / PBS. Furthermore, it fixed for 1 minute at room temperature using the ethanol-acetone solution (1: 1). After fixing, the fixing solution was discarded and dried at room temperature. After drying, the measurement and staining of the tartrate-resistant acid phosphatase (TRAP) activity of the cells in each well were performed in the same manner as in Example 1. The results are shown in FIG.

  In FIG. 2, the measurement results of TRAP activity in bone marrow cells cultured in a medium supplemented with luteolin are shown as black bars.

  In addition, an experiment for measuring the number of proliferating cells using mouse bone marrow cells was performed in the same manner as in Example 2 to measure the cell viability. The results are shown in FIG. In FIG. 2, the cell viability was shown as a white bar graph. The results of the TRAP activity and proliferating cell count measurement experiments were all expressed as a relative value (%) relative to the control group when the control group was taken as 100.

As shown in FIG. 2, the addition of luteolin inhibited TRAP activity in a concentration-dependent manner. This indicates that osteoclast formation is inhibited in a concentration-dependent manner by luteolin. The results of XTT assay (expanded cell count measurement experiment) also revealed that luteolin did not show nonspecific cytotoxicity. From the above, it was shown that luteolin does not show non-specific cytotoxicity and significantly suppresses the differentiation of mouse bone marrow-derived M-CSF-dependent cells into osteoclasts by RANKL depending on the concentration ( IC ₅₀ = 3 μM).

(Example 4) Formation test I of an actin ring which is an osteoclast activation structure I
(4-1) Materials
MφRAW264 cells, fetal bovine serum, α-MEM, ODF / RANKL, 96-well microplate, and luteolin were prepared in the same manner as in Example 1 and used. PD98059 (CALBIOCHEM, catalog number 153000) was used.

(4-2) Actin ring formation test I
The MφRAW264 cell line was suspended in 10% FBS-MEM, inoculated into a 96-well microplate at 0.8 × 10 ⁵ cells / 0.1 mL / well, and cultured for 1 day under conditions of 5% CO ₂ and 37 ° C. Thereafter, 0.1 mL of the above medium containing 100 ng / mL ODF / RANKL and 20 μM PD98059 was added, and further cultured under the same conditions (final concentration of RANKL 50 ng / mL, final concentration of PD98059 10 μM).

  In order to promote differentiation to activated osteoclasts, the osteoclast activation structure is obtained by replacing with the above medium containing 50 ng / mL of ODF / RANKL 2 days after addition of ODF / RANKL and PD98059. The culture was continued under the same conditions until the formation of the actin ring was confirmed. When the actin ring was formed, a luteolin solution prepared in the same manner as in Example 1 was added, and further cultured for 1 day. In addition, only methanol containing no compound was added to the wells of the control group, and the culture was further continued for 1 day.

  After adding luteolin and culturing for 1 day, the cells in each well were stained with tartrate-resistant acid phosphatase (TRAP) in the same manner as in Example 1. The results are shown in FIGS.

  As shown in FIGS. 3A to 3D, actin ring formation was observed in the control group (FIG. 3A), but in the luteolin-treated group, the actin ring was destroyed in a concentration-dependent manner when treated at a concentration of 3 μM or more. (FIGS. 3B-D).

  Since the actin ring is an osteoclast activation structure formed by culturing the MφRAW264 cell line treated with ODF / RANKL and PD98059, luteolin is not only an osteoclast differentiation but also an activation structure It was thought to inhibit bone resorption by inhibiting maintenance.

(Example 5) Formation test II of an actin ring which is an osteoclast activation structure II
(5-1) Materials, etc. Except for streptomycin sulfate, penicillin G sodium, TGF-β, and M-CSF, the same ones as in Example 1 were used.

(5-2) Actin ring formation test II
In the same manner as in Example 3, bone marrow cells were collected from the tibia and femur of 6-9 week old ddY mice.

The obtained bone marrow cells were suspended in 10% FBS-MEM containing 100 U / mL penicillin G sodium salt, 100 μg / mL streptomycin sulfate, 50 ng / mL M-CSF, and 1 ng / mL TGF-β. A 96-well microplate was seeded at 1 × 10 ⁶ cells / 0.1 mL / well and cultured under conditions of 5% CO ₂ and 37 ° C.

  Three days after the start of the culture, the medium was replaced with a medium containing 50 ng / mL M-CSF and 50 ng / mL osteoclast differentiation inducing factor (ODF / RANKL), and the formation of an actin ring, an osteoclast activation structure, was observed. The culture was continued under the same conditions until confirmed. When an actin ring was formed, a luteolin solution prepared in the same manner as in Example 1 was added, and further cultured for 1 day. As a control group, only the solvent was added to the exchanged medium.

  After adding luteolin and culturing for 1 day, the cells in each well were stained with tartrate-resistant acid phosphatase (TRAP) in the same manner as in Example 1. The results are shown in FIGS.

  As shown in FIGS. 4A to D, actin ring formation was observed in the control group (FIG. 4A), but in the luteolin-treated group, the actin ring was destroyed in a concentration-dependent manner when treated at a concentration of 3 μM or more. (FIGS. 4B-D).

  Since the actin ring is an osteoclast activation structure formed by M-CSF and TGF-β or ODF / RANKL-treated mouse bone marrow-derived M-CSF-dependent cell culture, luteolin is an osteoclast. It was considered that bone resorption was suppressed by inhibiting not only cell differentiation but also maintenance of activated structures.

  Based on the above, luteolin does not exhibit non-specific cytotoxicity to these cells, either in the case of culturing cell lines derived from macrophages or in the case of culturing mouse bone marrow cells. It was proved that RANKL strongly inhibited osteoclast differentiation in a concentration-dependent manner. It was also shown that luteolin significantly inhibited bone resorption by activated osteoclasts because it inhibited actin ring maintenance in a concentration-dependent manner.

(Example 6) Osteoclast formation and bone resorption fossa formation method by co-cultivation Co-culture was performed by the method of O'Brien et al. (Charles A. O'Brien et al .; J. Biol. Chem. 274 (27), 19301- 19308 (1999)) and the bone marrow cells derived from mouse femur.

Active vitamin D ₃ of the mouse bone marrow cells prepared in Example 3 of the method (200 × 10 ⁴ cells / dish) and osteoblasts ^{UAMS-32 (100 × 10 4} cells / dish) ( Wako Pure Chemical Industries (Co. ), Catalog number 031-14281) 10 nM, prostaglandin E ₂ (manufactured by Wako Pure Chemical Industries, Ltd., catalog number 163-10814) suspended in α-MEM medium (20 ml) containing 1 mM. Subsequently, osteoclast differentiation was induced by culturing on a collagen gel coat culture dish having a diameter of 10 cm for 6 days under conditions of 5% CO ₂ and 37 ° C.

  After culturing for 6 days, osteoclasts were recovered by dissolving collagenase (manufactured by Wako Pure Chemical Industries, Ltd., catalog number 032-10534) to a final concentration of 0.1%.

  An ivory slice (thickness: about 0.2 m, diameter: about 4 mm) was placed in a 96-well plate, and α-MEM medium (100 μl) containing luteolin was added to a final concentration of 10 μM.

  Osteoclasts obtained in the above coculture system were appropriately diluted with α-MEM medium (about 10 ml / dish) and well suspended. This osteoclast suspension was carefully added to each well in an amount of 100 μl / well so that the cells adhered onto the ivory slices. After culturing for 12 hours, the bone resorption pits on the ivory sections were stained with toluidine blue solution (manufactured by Sigma, catalog number T3260-5G), and the resorption pits (pits) were counted.

  When osteoclasts prepared from co-culture of mouse bone marrow cells and osteoblasts were cultured on ivory slices, a number of bone resorption pits (pits) were formed as shown in FIG. 5A after 12 hours of culture. When cultured in the presence of 10 μM luteolin, the number of pits was significantly reduced as shown in FIG. 5B.

  A graph showing the relationship between the concentration of added luteolin and the number of pits is shown in FIG. In addition, the addition method and addition amount of luteolin in cultured cells were performed under the same conditions as in Example 1 above. As shown in FIG. 6, luteolin suppressed the formation of pits by osteoclasts in a concentration-dependent manner, and showed an inhibitory activity of 95% or more when added at 10 μM.

  From the above, it is clear that luteolin not only inhibits the initial differentiation of osteoclasts but also directly suppresses the bone resorption function of mature osteoclasts, and as a bone resorption inhibitor, it prevents and improves bone diseases such as osteoporosis The effect can be expected.

  Luteolin is a kind of flavonoid that has been used as a herbal medicine so far, indicating that flavones are useful as functional foods, pharmaceutical compositions, and medicines for the prevention and / or suppression of bone diseases. It was.

(Formulation example)
Next, although the formulation example containing the composition of this invention is shown, this invention is not limited to these.

(Formulation Example 1 Tablet)

  Each of the above components is weighed and mixed uniformly, and then compressed and compressed to produce a tablet having a weight of 300 mg.

(Formulation example 2 hard capsule)

  Each of the above components is weighed and uniformly mixed, and then hard capsules can be produced by filling hard capsules with 300 mg each. Here, the composition 1 is a mixture of luteolin and lactose at a ratio of 1: 1. In addition, the composition 1 used by the formulation examples 3-6 is the same as the above.

(Formulation example 3 soft capsule)

  Each of the above components is weighed and mixed uniformly, and then soft capsules can be produced by filling 100 mg each into soft capsules.

(Formulation Example 4 Granules)

  After each of the above components is weighed and mixed uniformly, a granule can be produced according to a conventional method.

(Formulation Example 5 Syrup)

  Each of the above components is weighed, sugar and saccharin are dissolved in 60 mL of distilled water for injection, and then a composition 2 and a seasoning solution dissolved in glycerin and ethanol are added. By adding purified water to this mixture to a final volume of 100 mL, a syrup for oral administration can be produced.

(Formulation example 6 granule)

  Each of the above components is weighed, and the composition 3 is adsorbed onto calcium silicate by a conventional method to form fine particles, whereby a powder can be produced.

It is useful in the fields of pharmaceutical compositions, pharmaceutical preparations, health foods, functional foods and the like containing the compound of formula (I) as an active ingredient.

Claims (7)

Pharmaceutical composition for prevention and / or treatment of bone disease comprising at least one compound represented by the following formula (I), or a physiologically acceptable salt, hydrate or glycoside thereof: object.

(In the formula, R ^{1 to} R ⁶ each represents a group selected from the group consisting of a hydrogen atom, a hydroxyl group and a methoxy group. Here, R ¹ or R ³ may be bound to a sugar or a derivative thereof; When R ¹ , R ² , R ³ and R ⁵ are hydroxyl groups, a sugar may be bonded to any of them to form a glycoside, which is glucose, apiose- (It is a sugar selected from the group consisting of glucose and glucopyranoside uronic acid, and R ^{1 to} R ⁶ do not simultaneously become hydrogen atoms.) A compound represented by the following formula (II), or a physiologically acceptable salt, hydrate or glycoside thereof,
A compound represented by the following formula (III), or a physiologically acceptable salt, hydrate or glycoside thereof,

A compound represented by the following formula (IV), or a physiologically acceptable salt, hydrate or glycoside thereof,

And a compound represented by the following formula (V), or a physiologically acceptable salt, hydrate or glycoside thereof:

The bone disease prevention and / or claim 1 comprising at least one compound selected from the group consisting of: or a physiologically acceptable salt, hydrate or glycoside thereof. A therapeutic pharmaceutical composition. A pharmaceutical preparation for preventing and / or treating a bone disease comprising the pharmaceutical composition according to claim 1 or 2 as an active ingredient. A functional food comprising a composition comprising the compound according to claim 1, a physiologically acceptable salt thereof, a glycoside thereof, and a hydrate. The functional food according to claim 4, wherein the functional food is used for assisting in the prevention and / or treatment of a bone disease. A health food comprising a composition comprising the compound according to claim 1, a physiologically acceptable salt thereof, a glycoside, and a hydrate. The health food according to claim 6, wherein the health food is used for assisting in the prevention and / or treatment of a bone disease.