JP2016069355A - Bone metabolism improver - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide agents, and foods and drinks having osteoclast differentiation inhibitory action and osteoblast differentiation promoting action, and useful for osteoporosis.SOLUTION: To provide an osteoclast differentiation inhibitor, a bone formation promoter, and an osteoporosis preventing or improving agent, comprising toddaculin as an active ingredient.SELECTED DRAWING: None

Description

  TECHNICAL FIELD The present invention relates to a health food and a pharmaceutical for preventing osteoporosis or improving bone metabolism, using todaculin extracted from salkakemikan, which is a plant belonging to the genus Sarcachemican, as an active ingredient.

  There are over 10 million osteoporosis patients in Japan, which is a big social problem. In addition, it is speculated that due to rapid aging, the population over 65 years old will increase, and osteoporosis patients will continue to increase. In the human body, bones are constantly resorbing and bone formation, and the cells involved in bone resorption are osteoclasts and the cells involved in bone formation are osteoblasts. Bone formation, maintenance, and repair depend on the balance between the formation and resorption of these cells. When this balance is lost, bone resorption exceeds bone formation, resulting in bone metabolic diseases such as osteoporosis. It has been known. Therefore, it is thought that it is important for the maintenance and enhancement of bone mass that bone formation does not fall below bone resorption.

  Since bone resorption is performed by osteoclasts, the greater the differentiation and activation of osteoclasts, the higher the bone resorption rate. On the other hand, osteogenesis is performed by osteoblasts, and the more osteoblasts are differentiated and activated, the higher the bone formation rate. Therefore, if a composition having a bone resorption inhibitory action for reducing the bone resorption rate and a bone formation promoting action for increasing the bone formation rate is obtained, it is effective for osteoporosis. As an example of reports of such effects, for example, a composition for improving bone metabolism comprising an extract of ginger containing 6-gingerol and 6-shogaol, which are pungent components of ginger (see Patent Document 1) Etc. are known.

JP 2014-043416

  Conventionally, drugs such as estrogen, bisphosphonate, vitamin D, and calcitonin have been clinically applied. However, these treatments are not sufficient, and new and better therapeutic agents are required.

  An object of the present invention is to provide an active ingredient that has been used for food and has an osteoclast differentiation inhibiting action and an osteogenesis promoting action, and is useful for the prevention and improvement of osteoporosis. .

  As a result of repeated search from various materials for active ingredients having an osteoclast differentiation inhibitory action and an osteogenesis promoting action, the present inventors have found that todaculin contained in Sarca kemican which is a plant belonging to the genus Sarcanaceae. The present inventors have found that the differentiation into osteoclasts is suppressed, and further, the bone formation of osteoblasts is promoted, and the present invention has been completed.

  The present invention is an osteoclast differentiation inhibitor, osteogenesis promoter, osteoporosis prevention and bone metabolism improving agent containing toddaculin.

  Moreover, this invention is the food-drinks containing a todaculin (toddaculin).

  The above-mentioned todacrine can be extracted and purified from the leaves, stems, berries, seeds, roots, or whole grasses of salka kekankan.

That is, the present invention is as follows.
[1] An osteoclast differentiation inhibitor containing toddaculin as an active ingredient.
[2] An osteogenesis promoter containing toddaculin as an active ingredient.
[3] A preventive or ameliorating agent for osteoporosis, containing toddaculin as an active ingredient.
[4] Contains toddaculin as an active ingredient, including extraction with sarcochemum as a raw material and extraction with hexane, ethyl acetate, methanol, ethanol, butanol, or diethyl ether, or any of these hydrates A method for producing the composition.
[5] A method for producing a composition containing toddaculin as an active ingredient, comprising purifying the composition obtained by the method of [4] to a single component by preparative HPLC. .

  The bone metabolism-improving agent of the present invention contains toddaculin, which is an extract component of salka kemikan, and suppresses bone resorption by inhibiting osteoclast differentiation, and further promotes osteoblast differentiation by promoting osteogenesis. Can prevent and improve osteoporosis.

  The food / beverage product of the present invention contains todaculin, which is an extract component of salka kemican, and can be taken orally to suppress differentiation into osteoclasts and promote bone formation, thereby preventing and improving osteoporosis.

It is a figure which shows the extraction protocol of a todaculin (toddaculin). It is a figure which shows the attribution data of the ¹ H- and ¹³ C-NMR spectrum of todaculin (toddaculin). It is a figure which shows the structural formula of todaculin (toddaculin), and the number in a figure corresponds to the position of FIG. It is a figure (photograph) which shows the differentiation inhibitory effect to the multinucleated cell using RAW264 cell of todaculin (toddaculin). It is a figure which shows the bone resorption suppression effect (TRAP activity suppression rate) using RAW264 cell of todaculin (toddaculin). It is a figure which shows the inhibition rate of NF- (kappa) B transcriptional activity using RAW264 cell of todaculin (toddaculin). It is a figure which shows the bone formation promotion effect (ALP activity promotion rate) using MC3T3-E1 cell of todaculin (toddaculin). It is a figure (photograph) which shows the bone formation promotion effect by the alizarin red dyeing | staining method using MC3T3-E1 cell of todaculin (toddaculin).

  The osteoclast differentiation inhibitor, osteogenesis promoter, and osteoporosis prevention and amelioration agent of the present invention contain todaculin as an active ingredient. Todacrine has the structural formula shown in the following formula (1), and the formal compound name is 5,7-dimethoxy-6- (3-methyl-2-butenyl) -2H-1-benzopyran-2-one ( 5,7-Dimethoxy-6- (3-methyl-2-butenyl) -2H-1-benzopyran-2-one).

  Toddaculin can be obtained by separating and purifying from an extract of salka kemikan. For example, general-purpose fractionation techniques such as liquid-liquid distribution and chromatography can be used. For example, todacrine is extracted from the plant body of salka kemikan using organic solvents such as acetone, hexane, ether, ethyl acetate, chloroform, butanol, propanol, methanol, ethanol, etc., and further todacrine is separated by chromatography using hydrophobic interaction, Can be purified. About 1 to 10 g of todacrine can be separated and purified from 1 kg of dried salkakemin plant. Examples of chromatography utilizing hydrophobic interaction include chromatography using Diaion (trademark) HP20 (Mitsubishi Chemical Corporation), which is a styrene-diphenylbenzene synthetic adsorbent.

Sarkakemikan is a plant (scientific name: Toddalia asiatica) belonging to the citrus family Sarkakemikan, and grows in the Okinawa region in Japan. Since it is a movement-restricted plant, it is difficult to obtain it as a live plant in Honshu, but processed products are sold and can be obtained as processed products. Since salka chewing mandarin is already used as a folk medicine and tea in Okinawa, etc., todaculin contained in salkake mikan is excellent in safety. Todaculin can be separated and purified from other species of plants containing todaculin. Examples of other species include Antitidesma venosum of the citrus family.
The chemical formula of todacrine is shown in the following formula (1).

  Todaculin may be synthetically produced. Those skilled in the art can easily synthesize based on the above chemical formula.

  The osteoclast differentiation inhibitor, osteoblast differentiation promoter, osteogenesis promoter, and osteoporosis prophylaxis and amelioration agent of the present invention contain todaculin, which is an extract of salka kemikan. As long as todaculin is contained, the extract of salkake mikan can be used as an osteoclast differentiation inhibitor, an osteoblast differentiation promoter, an osteogenesis promoter, and an osteoporosis prevention and improvement agent. The extract of salka kemikan can be obtained by uncooked or dried salka kemikan and extracting it with an extraction solvent. There are no particular limitations on the extraction solvent used for the extraction of salka kemikan as long as it can contain todaculin as an extraction component. For example, acetone, hexane, ether, ethyl acetate, chloroform, butanol, propanol, methanol, An organic solvent such as ethanol, a mixed solvent of an organic solvent, a mixed solvent of water and an organic solvent, or the like can be used. Preferably, hexane is used. For example, an extract extracted with hexane is called a hexane extract. Extraction can be performed by room temperature extraction or reflux extraction. As long as todaculin is included as an extraction component, any part of xylem, such as xylem, leaves, flowers, fruits, pericarps, stems, roots, root barks, can be used as the part of the salmon citrus used for extraction. In addition, other species of plants can be used as long as they contain toddaculin. Examples of other species include Antitidesma venosum of the citrus family.

  The extract of salka kemikan may be used as an extracted solution, but if necessary, a treatment such as filtration, a concentrated product, or a powdered product may be used.

  Toddaculin is useful for inhibiting bone resorption because it suppresses differentiation into osteoclasts. Todaculin is useful for increasing bone mass because it promotes osteoblast differentiation and promotes bone formation.

  It can be confirmed by the following method that todaculin has an osteoclast differentiation inhibitory action. Mammalian monocyte-derived cells such as RAW264 cells differentiate into osteoclasts while forming multinucleated cells by applying RANKL stimulation. Therefore, it is only necessary to confirm that monocyte-derived cells differentiate into osteoclasts by contacting and culturing monocyte-derived cells with differentiation promoting factors RANKL and toddaculin. For differentiation into osteoclasts, acid phosphatase (TRAP) activity, which is a differentiation marker for osteoclasts, may be confirmed. When having an osteoclast differentiation inhibitory effect, monocyte-derived cells do not differentiate into osteoclasts, and TRAP activity does not increase. Therefore, when TRAP activity does not increase, it can be evaluated that differentiation into osteoclasts is suppressed. In addition, it is known that the transcriptional activity of NF-κB increases when differentiation of mammalian monocyte-derived cells such as RAW264 cells into osteoclasts. Therefore, the action of promoting differentiation into osteoclasts may be evaluated by examining the NF-κB transcriptional activity of the cells.

  It can be confirmed by the following method that todaculin promotes osteoblast differentiation and has the activity of promoting bone formation. MC3T3-E1 cells are brought into contact with todacrine, and the degree of differentiation of MC3T3-E1 cells can be confirmed by the activity of alkaline phosphatase (ALP), which is a differentiation marker. When ALP activity increases, it can be evaluated that differentiation into osteoblasts is promoted. Further, differentiation into osteoblasts may be evaluated by, for example, an increase in calcium accumulated by the alizarin red staining method.

  In addition, the osteoclast differentiation inhibitory action may be confirmed by increasing expression of genes such as acid phosphatase (Acp5), cathepsin K (Ctsk), and Oscar, which are markers for osteoclast differentiation. On the other hand, the osteoblast differentiation promoting action may be confirmed by increasing the expression of genes such as alkaline phosphatase (Alpl) and osteocalcin (Bglap), which are markers for osteoblast differentiation.

  Todaculin can be used as an osteoclast differentiation inhibitor. The osteoclast differentiation inhibitor can also be used as a research reagent. Todaculin can be used as an osteogenesis promoter. The osteogenesis promoter can also be used as a research reagent.

  In addition, todaculin can be used as a medicament for preventing or ameliorating a disease caused by osteoporosis. The present invention prevents or ameliorates osteoporosis, rheumatoid arthritis, Paget's disease, bone metastasis of malignant tumors, retroactive thyroid function, and the like after an oophorectomy surgery, which contains an extract of toddaculin or salkake mikan A pharmaceutical composition for the treatment of

  The pharmaceutical composition may be administered orally or parenterally such as intravenous injection, intramuscular injection, subcutaneous administration, rectal administration, and transdermal administration. Preferably it is administered orally. The pharmaceutical composition may contain a pharmaceutically acceptable excipient, lubricant, binder, disintegrant, coating agent and the like. Moreover, a coloring agent, a fragrance | flavor, antiseptic | preservative, etc. may be included. Examples of excipients include lactose, glucose, corn starch, sorbit, crystalline cellulose, etc., examples of lubricants include talc, magnesium stearate, polyethylene glycol, hydrogenated vegetable oil, and examples of binders include dimethyl cellulose, polyvinyl alcohol, Polyvinyl ether, methyl cellulose, ethyl cellulose, gum arabic, gelatin, hydroxypropyl cellulose, polyvinyl pyrrolidone and the like, and examples of the disintegrant include starch, sodium alginate, gelatin powder, calcium carbonate, calcium citrate and dextrin. The form at the time of use is not specifically limited, It can be in the form of capsule, powder, granule, tablet, liquid and the like, and can also be used in the form of cream, paste, gel, etc. as an external preparation.

  The extract of toddaculin or salkake mikan of the present invention can be used as a supplement, or can be mixed with food or drink or used as a composition for eating or drinking.

  When used as a supplement, it can be used in the form of tablets or granules, and may be mixed with other useful ingredients.

  When used as a food or drink composition, it can be used as a health food or drink, a specific health food or drink, a nutritional function food or drink, a health supplement food or drink, and the like. These foods and drinks can be provided as functional foods that improve bone metabolism, and for specific health foods and drinks, other supplements, health foods and drinks, etc., osteoporosis, which suppresses bone resorption, promotes bone formation, It is possible to display functions such as prevention or improvement.

  Eating and drinking compositions are in various forms such as solids, liquids, powders, granules, pastes, etc., specifically, for example, soft drinks, lactic acid drinks, taste drinks, coffee, green tea, black tea, oolong tea, etc. Sweets such as candy, chocolate, biscuits, confectionery bread, cakes, rice cakes, rice confectionery, etc .; processed fruits and vegetables such as fruit drinks, vegetable drinks, jams, pastes; Japanese sake, shochu, wine Alcohol drinks such as Chinese liquor, whiskey, vodka, brandy, gin, rum, liquor, beer, soft alcoholic beverages, fruit liquor, liqueurs; dairy products such as yogurt, ice cream, butter, cheese, condensed milk, powdered milk ; Fats and fats processed products such as dressing, mayonnaise, tempura oil, salad oil; soy sauce, sauce, vinegar, mirin, dressing type seasoning, etc. Seasoning: Powdered juice, powdered soup, instant coffee, instant noodles, instant curry, instant miso soup, cooked food, cooked beverage, cooked soup and other dried foods, processed flour, processed starch, etc. Products. For example, it may be used as a solid material such as strawberries, cookies, chewing gum, biscuits, or a liquid such as soft drinks, milk, yogurt and syrup. When it is set as food and drink, additives usually used for food and drink such as citric acid, lactic acid, and casein can be blended.

  The content of todaculin in a pharmaceutical composition, supplement or food-and-drink composition containing toddaculin varies depending on the dosage form, but usually todaculin is present in all compositions. 0.01 to 50% by weight, preferably about 0.01 to 20% by weight. In the case of todaculin, the intake can be adjusted appropriately taking into account the age, gender, weight, type of symptoms, degree of symptoms, etc., but the daily intake is 0.01-300 mg / kg body weight It is preferable to set according to the form of food or drink. What is necessary is just to take by administration, feeding, etc. once a day or dividing into several times.

  The pharmaceutical composition containing the toddaculin of the present invention, the composition for food and drink such as supplement or food and drink is intended for mammals including humans. Examples of mammals other than humans include primates such as monkeys, rodents such as rats and mice, sheep, pigs, cows, cats and dogs.

  EXAMPLES The present invention will be described in detail below based on examples, but the present invention is not limited to these examples.

[Example 1] Extraction, separation, and purification of todaculin Todaculin was extracted from a stalk of dried salka kemikan. As shown in FIG. 1, a hexane extract (about 13 g) was prepared by pulverizing a dried product (dry weight about 500 g) and extracting with refluxing with hexane. The obtained extract was subjected to column chromatography using Diaion (trademark) HP-20 (Mitsubishi Chemical), and each fraction of Fr. 1-3 was separated with an acetonitrile gradient. Next, todacrine was isolated and purified by preparative HPLC. After concentration under reduced pressure, crystallization by freeze-drying isolated todaculin as a white plate crystal in a yield of about 2 g.

The assignment data of ¹ H- and ¹³ C-NMR spectra of todaculin are shown in FIG. 2 and FIG. The numbers in FIG. 3 correspond to the positions in FIG.

The compound is obtained as colorless prismatic crystals, and m / z 274 in the mass spectrum of GC-MS is considered to be a molecular ion, and m / z 275.12 is considered to be a proton adduct ion from the mass spectrum of FT-MS Thus, the mass number was determined to be 274.31, and the composition formula was estimated to be C ₁₆ H ₁₈ O ₄ . Using the two-dimensional correlation method, each signal observed in ¹ H and ¹³ C-NMR was assigned (FIG. 2). From the NMR results, it was inferred that the structure of this substance was as shown in FIG. When the structural formula was searched by Pub Chem, it was a known component called toddaculin. The ¹ H-NMR spectrum of the substance measured this time was highly consistent with the attribution data of toddaculin literature (Journal of Science, 111 (7), 365-375). Based on the above results, this substance was identified as todaculin.

[Example 2] Osteoclast differentiation inhibition test (TRAP activity inhibition)
Mouse macrophage-like RAW264 cells (referred to as RAW264 cells) (obtained from RIKEN) show a monocyte-like morphology under normal culture conditions. Since RAW264 cells are differentiated into osteoclasts efficiently while forming multinucleated cells by giving RANKL stimulation, the differentiation activity of osteoclasts by compounds is evaluated for TRAP activity, which is a differentiation marker. Thus, it is possible to evaluate the action of inhibiting differentiation induction into osteoclasts. Differentiation induction of RAW264 cells into osteoclasts was performed by the following method.

RAW264 cells were adjusted to 5.0 x 10 ⁴ cells / mL using medium (Eagle medium containing 10% FBS (fetal serum)) and dispensed at 0.5 mL / well into a 24-well multiwell plate. . The culture plate was cultured for 72 hours at 37 ° C. in the presence of 5% CO ₂ , and each well contained a differentiation-inducing agent consisting of 10 ng / mL RANKL and todaculin contained in salka kemican at a final concentration of 20 μM, 10%. It added so that it might become μM, 5 μM, and 2.5 μM. After 72 hours, the cells added with 20 μM todacrine were observed with a microscope. A result (microscope observation photograph) is shown in FIG. Fig. 4a shows the results without RANKL stimulation, Fig. 4b shows the results with RANKL stimulation, and Fig. 4c shows the results when todacrine was added in addition to the RANKL stimulation. As shown in FIG. 4b, multinucleated cells were formed by RANKL stimulation, but when todacrine was added, formation of multinucleated cells was not observed (FIG. 4c). This result indicates that todacrine suppresses differentiation into multinucleated cells. Next, the medium was extracted and washed twice with physiological saline, and then 1000 μL of cell lysate was added and sonicated to lyse the cells. After the cell lysate was centrifuged at 10,000 rpm for 3 minutes, the supernatant was collected, and TRAP activity was measured with a plate reader by adding an acid phosphatase substrate. Values are shown as mean ± standard error (n = 3). The significance test for each group was evaluated using Dunnett's test. The results are shown in FIG. In FIG. 5, BL indicates no RANKL stimulation, and CN indicates RANKL stimulation. As shown in FIG. 5, when todaculin was added, TRAP activity decreased in a concentration-dependent manner.

  From the results of FIG. 4 and FIG. 5, it was found that todaculin contained in salka kemican has an excellent osteoclast differentiation inhibitory action.

[Example 3] Osteoclast differentiation inhibition test (NF-κB transcriptional activity inhibition)
As a mechanism of osteoclast differentiation and activation, activation of Nuclear factor-κB (NF-κB) by RANKL stimulates the pathway of NF-κB, and differentiation and activation into osteoclasts It is known to be promoted. By evaluating the NFκB transcriptional activity inhibitory action by a reporter assay, the differentiation induction inhibitory action can be evaluated. Transformation of RAW264 cells with a reporter vector and measurement of luciferase activity were performed by the following methods.

  Mouse macrophage-like RAW264 cells were transformed with a reporter vector, and luciferase activity was measured using a specific substrate with a luminometer. At this time, a differentiation-inducing agent consisting of 10 ng / mL RANKL and todaculin contained in salka kemican were added to 20 μM, 10 μM, 5 μM or 2.5 μM. Transformation efficiency was corrected by measuring Renilla luciferase activity. Values are shown as mean ± standard error (n = 3). The significance test for each group was evaluated using Dunnett's test. The results are shown in FIG. In FIG. 6, BL indicates no RANKL stimulation, and CN indicates RANKL stimulation. As shown in FIG. 6, todacrine suppressed the transcriptional activity of NF-κB.

[Example 4] Osteoblast differentiation promotion test (ALP activity promotion)
Mouse skull-derived fibroblast MC3T3-E1 cells (referred to as MC3T3-E1 cells) (obtained from RIKEN) show a fibroblast-like morphology under normal culture conditions. MC3T3-E1 cells are induced to differentiate into osteoblasts upon stimulation with ascorbic acid. Therefore, by evaluating the differentiation activity to osteoblasts by the compound, ALP activity, which is a differentiation marker, It is possible to evaluate the effect of promoting differentiation induction into cells. Differentiation induction of MCT3-E1 cells into osteoblasts was performed by the following method.

Adjust MC3T3-E1 cells medium (Eagle's medium containing 10% FBS (fetal serum)) so as to be 1.0 x 10 ⁶ cells / mL with which minute at 1.0 mL / well in 24-well multiwell plates Noted. The culture plate is cultured for 72 hours at 37 ° C. in the presence of 5% CO ₂ , and each well contains a differentiation-inducing agent consisting of 50 μg / mL ascorbic acid and todaculin contained in salka kemican at a final concentration of 50 μM, It added so that it might become 20 micromol, 10 micromol, or 5 micromol. After 144 hours, the medium was removed and washed twice with physiological saline, and then 600 μL of a cell lysate was added and sonicated to lyse the cells. The cell lysate was centrifuged at 10,000 rpm for 3 minutes, and then the supernatant was collected and ALP activity was measured with a plate reader by adding an alkaline phosphatase substrate. Values are shown as mean ± standard error (n = 3). The significance test for each group was evaluated using Dunnett's test. The results are shown in FIG. In FIG. 7, BL indicates that there is no differentiation-inducing stimulus, and CN indicates that there is a differentiation-inducing stimulus.

  From the results shown in FIG. 7, the concentration-dependent increase in ALP activity increased in MC3T3-E1 cells due to todaculin contained in sarca chemist. In other words, it was shown that differentiation into osteoblasts was promoted.

[Example 5] Osteoblast differentiation promotion test (alizarin red staining)
It is possible to visually evaluate the calcification in MC3T3-E1 cells by the alizarin red staining method. Since alizarin red is adsorbed to calcium by a chelate reaction, the more the mineralization progresses, the deeper the cells are stained. Alizarin red staining of MCT3-E1 cells was performed by the following method.

Adjust MC3T3-E1 cells medium (Eagle's medium containing 10% FBS (fetal serum)) so as to be 1.0 x 10 ⁶ cells / mL with which minute at 1.0 mL / well in 24-well multiwell plates Noted. The culture plate was cultured for 72 hours at 37 ° C in the presence of 5% CO ₂ , and each well contained a differentiation inducer consisting of 50 μg / mL ascorbic acid and 10 mM β-groserophosphate and todacrine (toddaculin) ) Was added to a final concentration of 50 μM. Thereafter, the medium is changed every 2 days, and after 16 days, the medium is removed, the cells are washed with HBSS (-), fixed with ethanol, and lime with 20 mmol / L alizarin red staining (pH 4.8) solution. The matrix was stained and washed with HBSS (-). The alizarin red dye in the plate was dissolved with 10% cetylpyridinium chloride, and the absorbance at a wavelength of 562 nm was measured. The results are shown in FIG. 8a shows the result without differentiation-inducing stimulus, FIG. 8b shows the result with differentiation-inducing stimulus, and FIG. 8c shows the result of adding todaculin with differentiation-inducing stimulus.

  As shown in FIG. 8, the cells cultured with the addition of todaculin contained in salka kemican were more in comparison with the differentiation-inducing agent consisting of 50 μg / mL ascorbic acid and 10 mM β-gloelophosphate. You can see that the color is darker. That is, it was shown that the deposition (calcification) of calcium progresses by differentiation and activation into osteoblasts.

[Example 6] DNA microarray analysis Next, gene expression analysis of osteoclasts and osteoblasts was examined. To RAW264 cells, a differentiation inducer consisting of 10 ng / mL RANKL and todaculin contained in Sarkakekan were added to a final concentration of 20 μM, and RAW264 cells three days later were added to TRIzol (trademark) ) To obtain TotalRNA. Biotin-labeled aRNA was prepared from TotalRNA and hybridized to a focused array Genopal bone metabolism chip manufactured by Mitsubishi Rayon.

  Similarly, a differentiation-inducing agent consisting of 50 μg / mL ascorbic acid and todaculin contained in sarca chemist are added to MC3T3-E1 cells to a final concentration of 50 μM, and MC3T3-E1 cells 6 days later are added to TRIzol. Total RNA was obtained by dissolution with (trademark) (Life Technologies Japan). Biotin-labeled aRNA was prepared from TotalRNA and hybridized to a focused array Genopal bone metabolism chip manufactured by Mitsubishi Rayon.

  As a result of gene expression analysis, in osteoclasts, suppression of gene expression such as acid phosphatase (Acp5), cathepsin K (Ctsk), and Oscar, which are osteoclast differentiation markers, was remarkable in todaculin. In osteoblasts, the expression of genes such as alkaline phosphatase (Alpl) and osteocalcin (Bglap), which are differentiation markers of osteoblasts, was prominent in todaculin.

  From the results of DNA microarrays, it was found that todaculin contained in sarca chewing can inhibit osteoclast differentiation, promote osteoblast differentiation, and have an excellent bone metabolism improving effect even at the gene level. .

Claims (5)

  An osteoclast differentiation inhibitor containing toddaculin as an active ingredient.   An osteogenesis promoter containing toddaculin as an active ingredient.   An agent for preventing or improving osteoporosis, which contains toddaculin as an active ingredient.   A method for producing a composition containing todaculin, which comprises using salkake mikan as a raw material and performing extraction with hexane, ethyl acetate, methanol, ethanol, butanol, diethyl ether, or any one of these hydrates.   A method for producing a composition containing toddaculin, comprising purifying the composition obtained by the method according to claim 4 to a single component by preparative HPLC.

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