JP5000214B2 - Novel compounds and osteoclast differentiation / proliferation inhibitors - Google Patents

Description

  The present invention relates to, for example, a novel compound that can be used as a raw material for foods and drinks, pharmaceuticals, and cosmetics, and an osteoclast differentiation / proliferation inhibitor.

  In the modern Japanese society where aging is progressing year by year, an increasing number of people suffer from various diseases associated with aging. For example, osteoporosis, which is one of the diseases associated with aging, refers to a condition in which the bone mass decreases and the bone becomes very brittle.

  In normal bone, bone resorption, in which osteoclasts break down old bone parts, and bone formation in which osteoblasts create new bone where bone resorption is performed, are balanced, resulting in a constant bone mass. It is kept. However, due to insufficient intake of calcium, a decrease in calcium absorption ability, disorder of hormone balance, etc., the balance of bone turnover is lost, and bone resorption exceeds bone formation, leading to osteoporosis. In particular, postmenopausal women are prone to osteoporosis due to hormonal balance disturbance.

  Therefore, for osteoporosis, it is considered necessary to suppress bone resorption or promote bone formation.

  Therefore, as active therapeutic agents for osteoporosis, active vitamin D3 preparation, vitamin K2 preparation, estrogen preparation, calcitocin preparation, calcium preparation, bisphosphonate preparation, etc. are used. There was a problem of side effects.

1) Active vitamin D3 preparation: Anorexia, malaise, renal failure, etc. due to excessive increase in blood calcium level.
2) Vitamin K2 preparation: Decreased effect of a drug called warfarin taken in the case of myocardial infarction.
3) Estrogen preparation: Increased risk of myocardial infarction and stroke, increased incidence of breast cancer and endometrial cancer.
4) Calcitocin preparation: nausea, hot flush, etc.
5) Calcium preparation: gastrointestinal disorders.
6) Bisphosphonate formulation: digestive symptoms, especially esophageal ulcers.
7) Synergistic effect of side effects by combined use of active vitamin D3 preparation and calcium preparation.

  Therefore, development of a safer osteoporosis treatment agent that has no side-effect problems even when ingested for a long period of time has been promoted. For example, Patent Document 1 listed below discloses a basic protein fraction derived from milk as an active ingredient. A bone strengthening agent is disclosed.

  Patent Document 2 listed below discloses an osteoclast differentiation inhibitor production promoter containing milk-derived basic protein fraction, particularly lactoferrin as an active ingredient.

  Patent Document 3 listed below discloses a bone strengthening agent containing casein phosphopeptide and genistein as active ingredients.

  Patent Document 4 listed below discloses an osteoporosis preventive / therapeutic agent containing collagen or an enzyme degradation product of collagen as an active ingredient.

Patent Document 5 below discloses a composition for promoting bone formation and preventing bone mineral content reduction, which contains isoflavone as a main active ingredient.
Japanese Patent No. 3112737 JP 2004-115509 A JP 2001-302539 A Japanese Patent Laid-Open No. 11-12192 Japanese Patent Laid-Open No. 10-114653

  Various osteoporosis therapeutic agents containing the ingredients derived from foods described in the above patent documents as active ingredients can be easily ingested as health foods, etc., and there are few side effects, but all can be expected to be sufficiently satisfactory. I could not say.

  Therefore, the present invention can be expected to have a sufficient effect of preventing or treating osteoporosis by inhibiting osteoclast differentiation / proliferation and suppressing bone resorption, and is used as a raw material for foods, beverages, pharmaceuticals, and cosmetics. It is an object of the present invention to provide a novel compound and an osteoclast differentiation / proliferation inhibitor that can be used.

  Makomotake is used as a raw material for traditional Chinese medicine, and various physiologically active effects such as suppression of dry mouth, fever reduction, bowel regulation, digestion and detoxification are known. The present inventors have investigated the further physiological activity of Momotake, which has been confirmed to be safe by abundant dietary experiences, and found the effect of preventing and treating osteoporosis as a new physiological activity of Momotake. As a result of further research on the physiologically active component, the present invention has been achieved.

  That is, the novel compound of the present invention is represented by the following chemical formula (1).

  It is preferable that the novel compound of this invention is used as a raw material of food-drinks, a pharmaceutical, or cosmetics.

  On the other hand, the osteoclast differentiation / proliferation inhibitor of the present invention contains a compound represented by the following chemical formula (1) and / or a compound represented by the following chemical formula (2) as an active ingredient.

  The osteoclast differentiation / proliferation inhibitor of the present invention preferably further contains a compound represented by the following chemical formula (3) as an active ingredient.

  In addition, the osteoclast differentiation / proliferation inhibitor of the present invention preferably contains a composition prepared from an organic solvent extract of Makomotake.

  Moreover, it is preferable that the osteoclast differentiation / proliferation inhibitor of the present invention contains a composition prepared from an organic solvent extract of the cervix of the fungus of Makomotake.

  The novel compound of the present invention can be used, for example, as a raw material for foods and drinks, pharmaceuticals, cosmetics and the like, and can be expected to have an effect of preventing and / or improving osteoporosis.

  Further, the osteoclast differentiation / proliferation inhibitor of the present invention contains a component derived from Makotake that has been confirmed to be safe by abundant dietary experience as an active ingredient, and is highly safe and further sufficient. Expected to be effective in preventing and treating osteoporosis.

  The novel compound of the present invention is represented by the following chemical formula (1).

  The compound represented by the above chemical formula (1) (hereinafter referred to as “compound (1)”) is a compound that is soluble in an organic solvent, and can be obtained, for example, by purifying a solvent extract of Makomotake.

  That is, first, the mushroom that is the raw material for extraction is dried or pulverized as necessary. Especially, since the fungal colon of Momotake has a high content of the compound (1), the recovery rate of the compound (1) can be improved by using the fungal colon of Momotake as an extraction raw material. Here, Makomotake is a cultivated shoot of Macomo, an aquatic plant belonging to the genus Macomo and Perennial, that grows sproutingly with Aspergillus oryzae, and is cultivated from East Asia to Southeast Asia, mainly in China and Taiwan. Vegetables. In recent years, cultivation has been carried out in Japan, and these commercial products can be easily obtained.

  Next, this extract raw material, Momotake, is immersed in an extraction solvent at room temperature to under heating to obtain a solvent extract. The extraction solvent is not particularly limited, and examples thereof include lower alcohols (methanol, ethanol, etc.), acetone, acetonitrile, chloroform, dichloromethane, ethyl acetate, and the like. These can be used alone or in combination of two or more. . Particularly preferred extraction solvents include methanol, ethanol, chloroform, dichloromethane, and mixed solvents thereof.

  Since this extraction solvent often contains a large amount of contaminants, for example, makomotake is soaked in a water-soluble organic solvent such as lower alcohols or acetone at room temperature to under heating to After obtaining the solvent-extracted fraction, it is preferable to collect the water-insoluble organic solvent-extracted fraction by separating it into a fat-soluble component and a water-soluble component using a water-insoluble organic solvent such as chloroform and dichloromethane. By carrying out like this, the solvent extract (non-water-soluble organic-solvent extraction fraction) of Makotake which has very few impurities can be obtained. Moreover, this solvent extract may be concentrated as needed, and may be further dried.

  Next, the solvent extract is purified by column separation purification, recrystallization, or the like, whereby the compound (1) can be obtained.

  For the purification of the solvent extract, first, a crude product obtained by primary purification using chromatography is subjected to at least one method selected from recrystallization, column chromatography, and thin layer column chromatography. Subsequent purification is preferred. By carrying out like this, the recovery rate of the said compound (1) can be improved.

  The chromatography used for the primary purification is not particularly limited, and conventional general chromatography such as normal phase column chromatography, reverse phase column chromatography, size exclusion column chromatography, and ion exchange column chromatography may be used. it can. The primary purification method is not particularly limited. For example, when chromatography using silica gel as a carrier, a water-insoluble organic solvent such as chloroform, dichloromethane, benzene, ethyl acetate, hexane, and a mixed solvent thereof, Furthermore, it is preferable to add an appropriate amount of a water-soluble organic solvent such as methanol, ethanol, acetone, etc., and change the ratio to elute while sequentially increasing the polarity, and a mixed solvent of hexane / ethyl acetate, chloroform / acetone, chloroform / methanol The method used is particularly preferred.

  Column chromatography used for the secondary purification includes normal phase column chromatography, diol column chromatography, reverse phase column chromatography, size exclusion column chromatography and the like, and purification conditions such as carrier and elution solvent are various chromatography. Can be selected as appropriate. These column chromatography can be employed singly or in combination, and can be appropriately applied to open columns, HPLC columns and the like.

  Examples of the thin layer chromatography used for the secondary purification include those using a reverse phase carrier or normal phase carrier or a chemically modified carrier modified with an amine or an optically active substance. It can be selected appropriately according to the chromatography.

  The novel compound of the present invention (compound (1)) is a component derived from Makotake that has been confirmed to be safe by abundant dietary experience, and its safety has already been proven, and is a raw material for various foods, pharmaceuticals, and cosmetics. Can be used as

  Examples of the food and drink include (a) soft drink, carbonated drink, fruit drink, vegetable juice, lactic acid bacteria drink, milk drink, soy milk, mineral water, tea-based drink, coffee drink, sports drink, alcoholic drink, jelly drink, and the like. (B) Processed vegetable products such as canned tomato puree, mushrooms, dried vegetables, pickles, (c) Processed fruit products such as dried fruit, jam, fruit puree, canned fruit, (d) curry powder, wasabi, Spices such as ginger, spice blend, seasoning powder, (e) noodles (including raw noodles, dried noodles) such as pasta, udon, soba, ramen, macaroni, (f) breads such as bread, confectionery bread, cooking bread, donut , (G) Alpha-ized rice, oatmeal, rice cake, batter flour, etc. (h) Baked confectionery, biscuits, rice confectionery, candy, chocolate, chewing gum , Snacks, frozen confectionery, candied confectionery, Japanese confectionery, Western confectionery, half confectionery, pudding, ice cream and other confectionery, (i) beans products such as red beans, tofu, natto, kinakome, yuba, boiled beans, peanuts, (j ) Honey, royal jelly processed foods, (k) meat products such as ham, sausage, bacon, (l) dairy products such as yogurt, pudding, condensed milk, cheese, fermented milk, butter, ice cream, (m) processed egg products, (N) Processed fish such as dried fish, sea bream, chikuwa, fish sausage, processed seaweed such as dried seaweed, kelp, and boiled fish, processed fish eggs such as octopus, scallop, salmon roe, and sea bream, (o) dashi stock, soy sauce, Condiments such as vinegar, mirin, consomme base, Chinese base, concentrated soup stock, dressing, mayonnaise, ketchup, miso, salad oil, sesame oil, linoleic oil, diacyl glycero , Edible fats and oils such as Benibana oil, (p) Cooking, semi-cooked foods such as soups (including powder and liquid), side dishes, retort foods, chilled foods, semi-cooked foods (eg cooked rice, crab Tamanomoto) and the like. And as addition amount to food / beverage products, if an effective amount can be ingested, it will not specifically limit, What is necessary is just to mix | blend suitably so that it may not impair to the taste and quality stability of food / beverage products, for example, 5-500 ppm mass, What is necessary is just to mix | blend so that it may become 10-100 ppm mass preferably.

  Examples of the cosmetics include lotions, emulsions, lotions, gels, foundations, creams, facial cleansers, body washing agents, and the like. And as addition amount to cosmetics, 5-500 ppm mass is preferable, and 10-100 ppm mass is more preferable. If the addition amount is less than the above range, sufficient physiological activity effect cannot be expected, and if it is more than the above range, there may be a problem in quality stability.

  Examples of the pharmaceuticals include osteoclast differentiation / proliferation inhibitors, immunostimulators, melanin production inhibitors, and the like. Examples of the form include dosage forms such as liquids, powders, tablets, pills, fine granules, granules, capsules, jelly, chewable and paste.

  Next, the osteoclast differentiation / proliferation inhibitor of the present invention will be described.

  The osteoclast differentiation / proliferation inhibitor of the present invention contains a compound represented by the following chemical formula (1) and / or a compound represented by the following chemical formula (2) as an active ingredient.

  Further, the osteoclast differentiation / proliferation inhibitor of the present invention preferably further contains a compound represented by the following chemical formula (3) (hereinafter referred to as “compound (3)”) as an active ingredient.

  The compound (3) has been reported to have an osteoporosis prevention / treatment effect, and by using in combination with the compounds (1) and (2), a more excellent osteoporosis prevention / treatment effect can be obtained.

  And, the osteoclast differentiation / proliferation inhibitor of the present invention preferably contains a composition prepared from an organic solvent extract of Makomotake, a composition prepared from an organic solvent extract of the fungus colony of Makomotake It is more preferable to contain.

  The above-mentioned compounds (1) to (3) are components contained in makomotake and are compounds that are soluble in organic solvents. For this reason, the organic solvent extract of Makomotake contains the above compounds (1) to (3), and a high osteoporosis prevention / treatment effect is obtained by the synergistic action of each compound. Especially, the organic solvent extract of the fungus colony of Momotake has a large amount of the above compounds (1) and (2). Therefore, the composition prepared from the organic solvent extract of the fungus mushroom of Momotake By using a thing, content of an active ingredient can be improved and the more excellent osteoporosis prevention and treatment effect is acquired.

  In addition, the osteoclast differentiation / proliferation inhibitor of the present invention preferably further contains one or more selected from isoflavones, collagen peptides, lactoferrin, casein phosphopeptides, fish bone calcium and shellfish calcium. These components are known to have osteoporosis prevention / treatment effects, and a synergistic effect can be expected. In addition, inorganic salts, organic acids, carbohydrates, proteins, peptides, amino acids, and lipids can be appropriately blended as necessary.

  Examples of the form of the osteoclast differentiation / proliferation inhibitor of the present invention include dosage forms such as solution, powder, tablet, pill, fine granule, granule, capsule, jelly, chewable and paste. And when each said compound (1) or (2) is ingested independently, the effective intake is 0.01-5 mg per kg body weight, respectively, and 0.05-0.5 mg is more preferable. In addition, when two or more of the above compounds (1) to (3) are used in combination, the effective intake is preferably 0.01 to 5 mg in total per kg of body weight, and 0.05 to 0.5 mg. Is more preferable.

  Hereinafter, the novel compound and osteoclast differentiation / proliferation inhibitor of the present invention will be described in detail.

In addition, the compound is identified by FAB-MS spectrum analysis, infrared absorption (IR) spectrum analysis, nuclear magnetic resonance (NMR) spectrum analysis, DEPT spectrum analysis, ¹ H- ¹ H COSY spectrum analysis, HMQC spectrum analysis, HMBC spectrum. The analysis was performed.

  FAB-MS spectral analysis was positive for compound (1), matrix was 3-nitrobenzyl alcohol, positive for compound (2), matrix was 3-nitrobenzyl alcohol or 2-nitrophenyl octyl ether, compound (3) Was positive and the matrix was 3-nitrobenzyl alcohol.

  Infrared absorption (IR) spectrum analysis was performed by the KBr method.

Nuclear magnetic resonance (NMR) spectrum analysis was performed under the conditions of 500 MHz for H ₁ -NMR, 125 MHz for C ₁₃ -NMR, and deuterated chloroform as a solvent.

(Production Example 1) <Production of novel compound>
Taiwan-grown mushrooms were cut into leaves and fungi. Bacteria cervix (16.7 kg) was sequentially extracted three times with 22 L of ethanol and once with 10 L of acetone. The obtained extract was concentrated under reduced pressure and subjected to solvent fractionation with dichloromethane and ethyl acetate to obtain a dichloromethane soluble part, an ethyl acetate soluble part, and a water soluble part.

  The obtained dichloromethane-soluble part was subjected to flash chromatography (filler; silica gel, 60N (295 g), 4 × 60 cm) and eluted with 1.2 liters of each of the following solvents (I) to (IV). The fraction was fractionated as 50 ml to obtain fractions a-1 to a-17.

(I) Normal hexane: ethyl acetate = 9: 1
(II) Methanol (III) Dichloromethane: acetone = 8: 2
(IV) Dichloromethane: methanol = 5: 5

  The (III) elution fraction a-11 (1.5502 g) was subjected to diol column chromatography (product name: Diol 40D, 50 × 300 mm), and 1.2 l of each of the following solvents (V) to (VIII) was added. And fractionation was carried out with one fraction being 50 ml to obtain fractions b-1 to b-18.

(V) Normal hexane: ethyl acetate = 9: 1
(VI) Normal hexane: ethyl acetate = 7: 3
(VII) normal hexane: ethyl acetate = 5: 5
(VIII) Methanol

  The (V) elution fraction b-5 was applied to an ODS column (product name: CAPCELLPAK C18 MG, 20 × 250 mm), eluted with methanol, and fractionated into 20 ml fractions c-1 to c-18 was obtained.

  The above fractions c-14 and c-16 were collected, applied to a silica gel column (product name; Senshu Pak. AQUASIL, 20 × 250 mm), and eluted with a mixed solvent of normal hexane: ethyl acetate = 9: 1. Fractions were fractionated as 20 ml to obtain fractions d-1 to d-7.

  Further, the fractions b-4, c-8 and c-13 were put together and subjected to a silica gel column (product name; Senshu Pak. AQUASIL, 20 × 250 mm), and a mixed solvent of normal hexane: ethyl acetate = 9: 1. Elution was carried out at a fraction of 20 ml to obtain fractions e-1 to e-5.

  Fractions d-2 and e-2 were combined, applied to an ODS column (product name; CAPCELLPAK C18 MG, 20 × 250 mm), eluted with methanol, fractionated as 20 ml, and fraction f− 1 to f-8 were obtained.

The above fraction f-7 was subjected to FAB-MS spectrum analysis, infrared absorption (IR) spectrum analysis, nuclear magnetic resonance (NMR) spectrum analysis, DEPT spectrum analysis, COSY spectrum analysis, and HMQC spectrum analysis. It was confirmed that the compound was the target novel compound represented by (1), and 1.1 mg of the novel compound (compound (1)) could be isolated. Fig. 1 shows the FAB-MS spectrum of this compound, Fig. 2 shows the IR spectrum, Fig. 3 shows the ¹ H-NMR spectrum, Fig. 4 shows the ¹³ C-NMR spectrum, Fig. 5 shows the DEPT spectrum (upper) and ¹³ C. FIG. 6 shows a ¹ H- ¹ H COSY spectrum diagram, FIG. 7 shows an HMQC spectrum diagram, and FIG. 8 shows an HMBC spectrum diagram.

-FAB-MS spectrum: A molecular ion peak of m / z 474 [M] ⁺ was observed.
¹ H-NMR spectrum: 3.10 δ ppm, 3.20 δ ppm with methoxy group-derived singlet, 0.79 δ ppm, 0.81 δ ppm, 0.90 δ ppm with doublet methyl group, 0.64 δ ppm, 0.72 δ ppm with singlet methyl group A triplet methyl group was observed at 0.83 δ ppm.
· ¹³ C-NMR spectrum: 31 signals were observed, a peak derived from the carbonyl was observed at 211.8Derutappm.

(Production Example 2) <Production of Compound (2)>
In Production Example 1, fractions d-5 and e-4 were combined, applied to an ODS column (product name; CAPCELLPAK C18 MG, 20 × 250 mm), eluted with methanol, and fractionated into 20 ml fractions. Fractions g-1 to g-4 were obtained.

  The above fraction g-3 was applied to a silica gel column (product name; Senshu Pak. AQUASIL, 20 × 250 mm), and eluted with a mixed solvent of normal hexane: ethyl acetate = 9: 1 to separate one fraction into 20 ml. The fractions h-1 to h-6 were obtained.

The above fraction h-4 was subjected to FAB-MS spectrum analysis, infrared absorption (IR) spectrum analysis, nuclear magnetic resonance (NMR) spectrum analysis, DEPT spectrum analysis, COSY spectrum analysis, and HMQC spectrum analysis. It was confirmed that the compound was represented by (2), and 2.7 mg of compound (2) was isolated. FAB-MS spectrum showing of this compound in 9 (upper part positive, 3-nitrobenzyl alcohol as a matrix, the lower the positive, 2-nitrophenyl octyl ether as a matrix), IR spectrum diagram in FIG. 10, FIG. 11 to ¹ H -NMR spectrum diagram, FIG. 12 shows ¹³ C-NMR spectrum diagram, FIG. 13 shows DEPT spectrum (top) and ¹³ C-NMR spectrum (bottom), FIG. 14 shows ¹ H- ¹ H COSY spectrum diagram, FIG. 15 shows an HMQC spectrum diagram, and FIG. 16 shows an HMBC spectrum diagram.

-FAB-MS spectrum: A molecular ion peak of m / z 429 [M + H] ⁺ was observed.
¹ H-NMR spectrum: doublet methyl group was observed at 0.79 δ ppm, 0.82 δ ppm and 0.91 δ ppm, singlet methyl group was observed at 0.67 δ ppm and 0.94 δ ppm, and triplet methyl group was observed at 0.83 δ ppm.
· ¹³ C-NMR spectrum: 29 signals were observed, a peak derived from carbonyl 209.1δppm and 211.3δppm were observed.

(Production Example 3) <Production of Compound (3)>
Taiwan-grown mushrooms were cut into leaves and fungi. The leaf part (7.9 kg) of the mushroom was sequentially extracted with 24 L of ethanol and 10 L of acetone. The obtained extract was concentrated under reduced pressure and subjected to solvent fractionation with trichloromethane and ethyl acetate to obtain a dichloromethane soluble part, an ethyl acetate soluble part, and a water soluble part.

  408.6 mg of the ethyl acetate soluble part was subjected to flash chromatography (filler; silica gel, 60 N (800 g), φ4 × 60 cm) and eluted with 1.2 L of each of the following solvents (IX) to (XII). And fractions were fractionated with 50 ml to obtain fractions i-1 to i-12.

(IX) Dichloromethane: methanol = 9: 1
(X) Dichloromethane: methanol = 7: 3
(XI) Methanol (XII) Methanol: Water = 9: 1

  Fraction (XI) elution fraction i-7 was subjected to ODS Sep-Pak and eluted with a mixed solvent of water: methanol = 6: 4, and one fraction was fractionated to 20 ml to obtain fraction j-1. ~ J-3 was obtained. The above fraction j-3 was applied to an ODS column (product name; CAPCELLPAK C18 MG, 20 × 250 mm), eluted with methanol, and eluted with a mixed solvent of water: methanol = 6: 4, and one fraction was obtained. Fractionation was carried out as 20 ml to obtain fractions k-1 to k-17.

The fraction k-12 was subjected to FAB-MS spectrum analysis, infrared absorption (IR) spectrum analysis, nuclear magnetic resonance (NMR) spectrum analysis, DEPT spectrum analysis, COSY spectrum analysis, and HMQC spectrum analysis. It was confirmed that the compound was represented by (3), and 1.9 mg of compound (3) was isolated. FIG. 17 shows an FAB-MS spectrum diagram of this compound, FIG. 18 shows an IR spectrum diagram, FIG. 19 shows a ¹ H-NMR spectrum diagram, FIG. 20 shows a ¹³ C-NMR spectrum diagram, FIG. 21 shows a DEPT spectrum (upper) and ¹³ C Corresponding diagram with -NMR spectrum (lower part), FIG. 22 shows ¹ H- ¹ H COSY spectrum, FIG. 23 shows HMQC spectrum, and FIG. 24 shows HMBC spectrum.

-FAB-MS spectrum: A molecular ion peak of m / z 543 [M + Na] ⁺ was observed.
· ¹ H-NMR spectrum: 3.85Derutappm, singlet from methoxy group, a signal derived from sugar regions and 4.87δppm of 3.39~3.50δppm was observed 3.86Ppm.
· ¹³ C-NMR spectrum: 26 signal was observed. In addition, 12 aromatic-derived signals were observed in the region of 111.0 to 151.0 δ ppm, and by DEPT spectrum analysis, 6 of these signals were quaternary carbon, and 6 signals were methine carbon. It was.

(Test Example 1)
[Evaluation of osteoclast differentiation / proliferation inhibitory activity]
Using the compounds (1) and (2) as test preparations, osteoclast differentiation / proliferation inhibitory activity was evaluated. The test preparations were dissolved in methanol so that each would be 100 mg / ml, and then diluted with a culture solution (α-MEM containing 10% FBS) to obtain 0.2 μg / ml, 0.4 μg / ml, 0 Those prepared to 0.8 μg / ml, 1.6 μg / ml, 3.2 μg / ml, 6.3 μg / ml, 12.5 μg / ml, 25 μg / ml, and 50 μg / ml were used.
Bone marrow cells 1.3 × 10 ⁸ cells collected from tibia and femurs of 5-7 week old female mice, and osteoblast-like stromal cells 1 × 10 ⁶ cells collected from the skull and cultured, (10% FBS-containing α-MEM) was suspended in 7.2 ml, and dispensed into a 48-well plate at 150 μl / well. Then, a sample solution 50 [mu] l of each concentration was added to _{1,25 (OH) 2 VD 3 (} 20ng / ml) culture medium with 50 [mu] l, the total volume to 250 [mu] l / well. Then, incubated with CO ₂ incubator (37 ° C., CO ₂ concentration of 5.0%) was. On the 3rd day from the start of culture, 100 μl of the supernatant of each well was removed, and 50 μl each of a test sample twice as much as the concentration added on the first day and 1,25 (OH) ₂ D ₃ (40 ng / ml) were added. Cultured for 1 week.
After co-culture, the culture solution was removed, washed with PBS (phosphate buffer solution), and 10% formalin-containing PBS solution was added at 0.5 ml / well and fixed for 10 minutes. Next, 0.5 ml / well of ethanol was added and refixed for 1 minute. Thereafter, it was dried, 0.3 ml / well of TRAP reaction solution was added, and staining was performed at room temperature for 30 minutes. After dyeing, it was washed with distilled water (0.5 ml / well) and dried. The TRAP reaction was positive, and cells having two or more nuclei were counted as TRAP-positive multinucleated cells, and the number per well was counted. The TRAP reaction solution was prepared by using 1.5 mg of Naphthol as-mx phosphate, 150 ml of N, N-dimethylformaldehyde, and 15 ml of buffer (0.1 M Na acetate buffer containing 50 mM Na tartrate, pH = 5. 0) and 9 mg of fast red violet lb salt.

  As shown in FIG. 25, the compound (1) was able to specifically inhibit the formation of osteoclasts in the range of 25 to 50 μg / ml.

  Further, as shown in FIG. 26, the compound (2) was able to specifically inhibit the formation of osteoclasts in the range of 12.5 to 50 μg / ml.

(Production Example 4)
A hard capsule was prepared by filling the composition prepared in the formulation shown in Table 1 into hard capsules at 230 mg / capsule according to a conventional method. The fish bone calcium used the brand name "baking Bonical" (made by Yaizu Suisan Chemical Co., Ltd.). By taking 10 capsules per day of this capsule, 10 mg of compound (1) can be taken.

(Production Example 5)
A jelly beverage was produced according to a conventional method with the formulation shown in Table 2. This jelly beverage had no particular problems in production, and was able to be eaten deliciously with no problems regarding quality and flavor. By taking one bottle of this jelly beverage per day, it is possible to take 5 mg each of compound (1) and compound (2).

(Production Example 6)
A milk beverage was produced according to a conventional method with the formulation shown in Table 3. This milk beverage had no particular problems in production, and was able to be eaten deliciously without any problems with respect to the quality and flavor of precipitation and the like. By taking one milk drink per day, it is possible to take 4 mg of compound (1), 3 mg of compound (2), and 3 mg of compound (3) each.

The novel compound of the present invention can be used as a raw material for foods and drinks, pharmaceuticals, cosmetics and the like.
In addition, the osteoclast differentiation / proliferation inhibitor of the present invention can be used for the prevention / treatment improvement of osteoporosis by ingesting it as it is or in combination with foods, beverages, pharmaceuticals, cosmetics and the like.

It is a FAB-MS spectrum figure of a compound (1). It is the same IR spectrum diagram. It is the same ¹ H-NMR spectrum figure. It is the ¹³ C-NMR spectrum figure. It is a corresponding figure of the DEPT spectrum (upper part) and a ¹³ C-NMR spectrum (lower part). It is the same ¹ H- ¹ H COSY spectrum figure. It is the same HMQC spectrum figure. It is the same HMBC spectrum figure. It is a FAB-MS spectrum figure of a compound (2). It is the same IR spectrum diagram. It is the same ¹ H-NMR spectrum figure. It is the ¹³ C-NMR spectrum figure. It is a corresponding figure of the DEPT spectrum (upper part) and a ¹³ C-NMR spectrum (lower part). It is the same ¹ H- ¹ H COSY spectrum figure. It is the same HMQC spectrum figure. It is the same HMBC spectrum figure. It is a FAB-MS spectrum figure of a compound (3). It is the same IR spectrum diagram. It is the same ¹ H-NMR spectrum figure. It is the ¹³ C-NMR spectrum figure. It is a corresponding figure of the DEPT spectrum (upper part) and a ¹³ C-NMR spectrum (lower part). It is the same ¹ H- ¹ H COSY spectrum figure. It is the same HMQC spectrum figure. It is the same HMBC spectrum figure. 4 is a chart showing osteoclast differentiation / proliferation inhibitory activity of compound (1). It is a graph which shows the osteoclast differentiation and proliferation inhibitory activity by a compound (2).

Claims (6)

A novel compound represented by the following chemical formula (1).
The novel compound according to claim 1, which is used as a raw material for foods, drinks, pharmaceuticals or cosmetics. An osteoclast differentiation / proliferation inhibitor comprising a compound represented by the following chemical formula (1) and / or a compound represented by the following chemical formula (2) as an active ingredient.

Furthermore, the osteoclast differentiation / proliferation inhibitor of Claim 3 which contains the compound shown by following Chemical formula (3) as an active ingredient.
5. The osteoclast differentiation / proliferation inhibitor according to claim 3 or 4, which comprises a composition prepared from an organic solvent extract of makomotake. 6. The osteoclast differentiation / proliferation inhibitor according to claim 5, comprising a composition prepared from an organic solvent extract of the fungus cervix of Macomotake.