Composition for improving bone metabolism

Abstract

To provide compositions with an excellent effect of improving bone metabolism by utilizing Zingiberaceae plants.SOLUTION: Provided is a composition for improving bone metabolism, containing the fermented product of Zingiberaceae plants with Aspergillus oryzae.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to a composition for improving bone metabolism. [Background technology]

[0002] Ginger contains components such as gingerol and shogaol, which are known to have various beneficial effects such as antibacterial properties, antioxidant properties, and promoting blood circulation. Patent Document 1 discloses that an extract of ginger with normal hexane has the effect of improving bone metabolism. Patent Document 2 discloses a compound effective in preventing and treating bone diseases, and lists the roots of plants of the Zingiberaceae family as a source of this compound. However, with the recent progress of aging, there has been a demand for components with better bone metabolism improving properties.

[0003] Regarding Zingiberaceae plants, in addition to using extracts as in Patent Document 1, fermented products have also been studied. Patent Document 3 discloses that the flavor of a composition containing ginger is improved by fermenting Zingiberaceae plant materials with acetic acid bacteria or lactic acid bacteria. However, the bone metabolism improving effect of fermented Zingiberaceae plant materials has not been studied. [Prior art documents] [Patent documents]

[0004] [Patent Document 1] Japanese Patent Application Laid-Open No. 2014-043416 [Patent Document 2] International Publication WO2007 / 091707 [Patent Document 3] Japanese Patent Application Publication No. 2018-057377 Summary of the Invention [Problem to be solved by the invention]

[0005] An object of the present invention is to provide a composition that utilizes a plant of the Zingiberaceae family and has an excellent effect of improving bone metabolism. [Means for solving the problem]

[0006] The present inventors have conducted various studies on the conditions for treating plants of the Zingiberaceae family, and have found that products fermented with koji mold have an excellent effect of improving bone metabolism, leading to the completion of the present invention.

[0007] That is, the present invention relates to a composition for improving bone metabolism, which comprises a fermentation product of a plant material of the Zingiberaceae family with Aspergillus oryzae.

[0008] The composition for improving bone metabolism is preferably one for inhibiting differentiation of precursor cells into osteoclasts.

[0009] The koji mold is preferably a microorganism of the genus Aspergillus. [Effects of the Invention]

[0010] The bone metabolism improving composition of the present invention contains a product fermented by koji mold, and therefore has a superior bone metabolism improving effect compared to when an unfermented Zingiberaceae plant material is used. [Brief explanation of the drawings]

[0011] [Figure 1] Demonstrates the cytotoxicity of ginger koji. [Figure 2] Demonstrates ginger's cytotoxicity. [Figure 3] Demonstrates the cytotoxicity of rice koji. [Figure 4] Demonstrates rice cytotoxicity. [Figure 5] This shows the anti-inflammatory effects of ginger koji. [Figure 6] Ginger has anti-inflammatory properties. [Figure 7] Demonstrates the anti-inflammatory effects of rice koji. [Figure 8] Shows the anti-inflammatory properties of rice. [Figure 9] This shows the inhibitory effect of ginger koji on osteoclast differentiation (inhibition of NF-AT activation by RANKL stimulation). [Figure 10]The inhibitory effect of ginger koji on osteoclast differentiation (number of osteoclasts positive for TRAP staining) is shown. [Figure 11] The inhibitory effect of ginger koji on osteoclast differentiation (number of osteoclasts positive for TRAP staining) is shown. [Figure 12A] The inhibitory effect of ginger koji on osteoclast differentiation (TRAP expression level) is shown. [Figure 12B] The inhibitory effect of ginger koji on osteoclast differentiation (CTSK expression level) is shown. [Figure 12C] The figure shows the inhibitory effect of ginger koji on osteoclast differentiation (DC-STAMP expression level). [Figure 12D] The inhibitory effect of ginger koji on osteoclast differentiation (OC-STAMP expression level) is shown. [Figure 12E] The figure shows the inhibitory effect of ginger koji on osteoclast differentiation (RANK expression level). [Figure 12F] The inhibitory effect of ginger koji on osteoclast differentiation (c-Fos expression level) is shown. DETAILED DESCRIPTION OF THE INVENTION

[0012] The composition for improving bone metabolism of the present invention is characterized by containing a fermentation product of a plant material of the Zingiberaceae family with Aspergillus oryzae.

[0013] <Zingiberaceae plant ingredients> Examples of Zingiberaceae plants include golden ginger, Sanshu ginger, yellow ginger, Kintoki ginger, Yanaka ginger, etc. It is preferable to use the rhizomes of these.

[0014] The form of the Zingiberaceae plant raw material to be fermented is not particularly limited, and the rhizome can be used as is, or it can be used as a pulverized product, juice, extract, slices, etc. Examples of pulverized products include powder and granules, with powder being preferred, and dried powder being particularly preferred. The juice or extract may be in liquid form, but may also be in paste or powder form. The extract can be obtained using an appropriate solvent, such as water, ethanol, or aqueous ethanol.

[0015] <Koji mold> The koji mold used for fermentation is not particularly limited, and examples thereof include microorganisms of the genus Aspergillus. Examples of microorganisms of the genus Aspergillus include Aspergillus oryzae, Aspergillus sojae, Aspergillus luchuensis, Aspergillus awamori, Aspergillus niger, and Aspergillus glaucus. Among these, Aspergillus oryzae is preferred from the viewpoint of fermentation efficiency.

[0016] <Fermentation conditions> The fermentation method is not particularly limited, and either solid culture or liquid culture may be used. However, solid culture is preferred because it can reduce the overall volume of the fermented product and also allows the koji mold to express a variety of enzymes, resulting in efficient fermentation.

[0017] In solid culture, a solid Zingiberaceae plant material is inoculated with koji mold and fermented under temperature control. Before inoculation with koji mold, the Zingiberaceae plant material is preferably steamed at 100-120°C for 30-90 minutes. To ensure uniform fermentation, it is preferable to turn the fermented product over midway. After fermentation, a solid fermented product can be obtained by drying with hot air at 40-50°C.

[0018] In liquid culture, koji mold is inoculated into a medium containing a Zingiberaceae plant material, and fermentation is carried out while controlling the temperature. Examples of inoculation methods include adding koji mold or its culture solution to a medium containing a Zingiberaceae plant material. Specifically, examples include a method in which a Zingiberaceae plant material is dissolved or dispersed in a solvent to prepare a ginger solution or dispersion, a medium containing this solution or dispersion, and then adding koji mold or its culture solution to this medium. Another example includes a method in which a Zingiberaceae plant material is added to a Zingiberaceae plant material culture solution.

[0019] The medium used for liquid culture contains a plant material from the Zingiberaceae family, and may optionally contain carbon sources such as sugars, starch, and dextrin, nitrogen sources such as yeast extract and peptone, vitamins, minerals, etc. Examples of sugars include glucose, arabinose, sucrose, lactose, sorbitol, fructose, and trehalose. When sugars are used, the amount used in the medium is preferably 0.1 to 10% by weight, more preferably 0.2 to 5% by weight.

[0020] The means for liquid culture is not particularly limited, and examples include static culture, neutral culture at a constant pH, batch culture, and continuous culture.

[0021] After fermentation by liquid culture, the liquid component can be removed to obtain a solid fermented product by drying with hot air at 40 to 50° C. Alternatively, solid-liquid separation can be performed by filtration or centrifugation, and the resulting solid or liquid component can be dried to obtain a solid fermented product.

[0022] In either solid culture or liquid culture, the fermentation temperature is preferably 20 to 50° C., more preferably 30 to 40° C. The fermentation time is preferably 5 to 120 hours, more preferably 10 to 72 hours. The atmosphere during fermentation is not limited, but aerobic conditions are preferred.

[0023] <Fermented foods> The solid fermented product obtained by solid or liquid culture may be crushed or powdered as necessary. Active ingredients may also be extracted from the solid fermented product and used. When obtaining an extract, either a polar or nonpolar solvent can be used as the extraction solvent, and a mixture of these can also be used. Examples include water; alcohols such as methanol, ethanol, propanol, and butanol; polyhydric alcohols such as propylene glycol and butylene glycol; and ketones such as acetone and methyl ethyl ketone. Among these, alcohols such as methanol and ethanol are preferred. The extraction temperature is preferably room temperature. The extract may be used as is, or the extract obtained by filtering to remove solids may be dried and used.

[0024] The amount of the fermented product of Zingiberaceae plant material by Aspergillus oryzae in the composition for improving bone metabolism can be appropriately determined depending on the form of the composition and is not particularly limited, but for example, the amount of fermented product to be taken per dose is preferably 100 mg to 2 g, more preferably 200 mg to 800 mg. The number of doses can also be appropriately determined depending on the form of the composition for improving bone metabolism, but can be, for example, 1 to 3 times per day.

[0025] <Improvement of bone metabolism> Generally, bone density is maintained by the balance between bone formation by osteoblasts and bone resorption (calcium elution from bone) by osteoclasts. This balance is called bone metabolism. When bone metabolism is disrupted due to aging, menopause, calcium deficiency, vitamin D deficiency, etc., bone resorption by osteoclasts becomes dominant, resulting in a decrease in bone density and osteoporosis. Because bone resorption is promoted when osteoclasts differentiate and become activated, preventing or treating osteoporosis requires inhibiting osteoclast differentiation and activation. Furthermore, promoting bone formation requires promoting osteoblast differentiation and activation.

[0026] The composition of the present invention is useful for improving bone metabolism. Improving bone metabolism refers to suppressing the differentiation or activation of osteoclasts or promoting the differentiation or activation of osteoblasts.

[0027] The following pathway is known for the differentiation or activation of osteoclasts: inflammatory cytokines such as TNF-α, IL-1, IL-6, and IL-17 stimulate fibroblasts, T cells, osteoblasts, and the like. These stimulated cells produce RANKL (Receptor Activator of NF-κB Ligand), an osteoclast differentiation factor. When RANKL binds to RANK (Receptor Activator of NF-κB), a receptor present on macrophages, it activates the transcription factor NFAT (Nuclear Factor of Activated T cells) in the macrophages, promoting differentiation into osteoclasts and activation of differentiated osteoclasts. The mechanism of action of the composition of the present invention is not particularly limited, but examples include the suppression of inflammatory cytokine expression and the suppression of NFAT activation.

[0028] When measuring the ability to inhibit osteoclast differentiation or activation in vitro, test cells include mouse-derived macrophages such as RAW264.7. These test cells are stimulated with inflammatory cytokines, and the expression levels of marker genes for osteoclast differentiation or activation are measured in the presence of the composition of the present invention. Examples of marker genes include TRAP (tartrate-resistant acid phosphatase), cathepsin K, DC-STAMP (dendritic cell-specific transmembrane protein), OC-STAMP (osteoclast-stimulating transmembrane protein), and RANK. If the expression levels of these marker genes are reduced, it can be determined that the composition has the ability to inhibit osteoclast differentiation or activation.

[0029] When measuring the ability to promote osteoblast differentiation or activation, test cells include precursor osteoblast-like cells such as mouse-derived MC3T3-E1. These cells are cultured in the presence of the composition of the present invention, and the progress of mineralization is evaluated. Mineralization can be evaluated by alizarin red (AR) staining. If the progress of mineralization is promoted, it can be determined that the composition has the ability to promote osteoblast differentiation or activation.

[0030] The fermented composition of the present invention contains a ginger-derived component, and therefore has various effects such as a bactericidal effect, an antioxidant effect, a blood circulation promoting effect, an appetite stimulating effect, a body temperature increasing effect, an anti-inflammatory effect, an effect of activating energy metabolism, and an effect of preventing metabolic syndrome, in addition to an effect of improving bone metabolism. The fermented composition of the present invention may also have the flavor of ginger.

[0031] <Dosage form> The composition of the present invention can be used as a medicine, food, etc. for improving bone metabolism.

[0032] When used as a pharmaceutical, the dosage form may be oral or transdermal. Oral dosage forms include liquids; solids such as tablets, granules, fine granules, powders, and tablets; or various forms such as capsules, oral sprays, and troches containing the liquids or solids. Transdermal dosage forms include lotions and creams. When preparing compositions of such dosage forms, other pharmaceutically acceptable excipients, binders, bulking agents, disintegrants, surfactants, lubricants, dispersants, buffers, preservatives, flavoring agents, fragrances, coating agents, carriers, diluents, and the like may be used in appropriate combinations, as long as they do not interfere with the bone metabolism-improving effect.

[0033] When used as a pharmaceutical product, the content of the fermented product of a Zingiberaceae plant raw material by Aspergillus or an extract thereof in the composition is preferably 0.5% by mass to 50% by mass, more preferably 1% by mass to 20% by mass, of the total composition.

[0034] When the composition of the present invention is used as a food product, the form of the composition may be in the form of beverages such as fruit or vegetable juice drinks, carbonated drinks, tea drinks, dairy drinks, fermented milk, fermented fruit juice, fermented vegetable juice, alcoholic drinks, and soft drinks; various foods such as jelly foods, various snacks, baked goods, cakes, chocolate, jam, bread, gum, candy, soups, pickles, and tsukudani (foods boiled in soy sauce); and supplements such as tablets, capsules, and syrups. [Example]

[0035] (Production Example 1) Preparation of ginger koji Ginger chips dried with hot air were used as the raw material and koji was produced using the koji mold Aspergillus oryzae. Specifically, 30 kg of dried ginger chips were soaked in tap water for 1 hour and then steamed at 121°C for 60 minutes. After cooling to 30-35°C, 30 g of koji starter was added and seeded (product temperature 35-36°C). The maximum koji production temperature was set at 40±2°C. After 12 hours, the mixture was turned over once, and after 44 hours, the koji was released. This was then dried with hot air at 45°C for 24 hours, crushed, and powdered (passed through a 50 mesh). This was used as ginger koji.

[0036] (Production Example 2) Preparation of rice koji Koji was produced using Aspergillus oryzae in the same manner as in Production Example 1, and after the production, the koji was dried at 45°C, sterilized, and powdered for use.

[0037] (Production Example 3) Preparation of extract 10 g of each of the ginger koji and rice koji from Production Examples 1 and 2, as well as the raw ginger and rice, were weighed out, and 10 volumes of 1% TFA-containing methanol were added and extracted at room temperature for 1 hour. After extraction, the mixture was filtered, and the resulting extract was evaporated to dryness. DMSO was added to the dried extract to a concentration of 100 μg / ml, and the extract was dissolved. The dissolved extract was subjected to cell testing.

[0038] (Reference Example 1) Toxicity test [material] Cells: Mouse macrophages (RAW264.7) Medium: MEM Alpha (10% (v / v) FBS, 1% (v / v) Penicillin-Streptomycin), Gibco

[0039] [method] To evaluate the toxicity of each extract obtained in Production Example 3 to cells, the cell viability was confirmed in medium prepared to a predetermined concentration. The test solution was prepared by adding each extract to the medium at an adjusted concentration. RAW264.7 cells at an appropriate passage number were seeded and cultured in a 96-well plate. After confirming cell adhesion, the medium was removed and replaced with the test solution. To achieve the same conditions as those used in the anti-inflammatory effect test and osteoclast differentiation test, the cells were cultured for 24 hours or 4 days after replacement with the test solution. After culturing in the test solution, the cell viability in the test solution group was calculated using Cell Counting Kit-8 (Dojin Kagaku Kenkyusho) with the viability of cells cultured in extract-free medium set at 100%.

[0040] [result] When RAW264.7 cells were cultured in medium supplemented with ginger koji extract, a decrease in viability was first observed at a concentration of 200 μg / mL (Figure 1). Furthermore, a decrease in viability was observed at a concentration of 50 μg / mL for the raw material ginger extract. No toxicity was observed with rice or rice koji (Figures 2-4). Therefore, it was confirmed that ginger koji has lower cytotoxicity than the raw material ginger. The values in Figure 2 represent the mean ± standard error, with significant differences indicated by *: p<0.05 and **: p<0.01.

[0041] (Example 1) Anti-inflammatory test [material] The same cells and medium as in Reference Example 1 were used.

[0042] [method] In immune responses, NF-κB is known to be a transcriptional regulator whose level of nuclear translocation increases in response to inflammatory signals, regulating the expression of inflammatory cytokines. Using RAW264.7 (RAW / NFκB-luc) cells carrying a vector that inserted a luciferase protein gene downstream of the NF-κB-binding response element, the anti-inflammatory effect was confirmed using the luminescence intensity of the luciferase protein as an indicator.

[0043] The test solution was prepared by adding the extracts of each material obtained in Preparation Example 3 to the medium at adjusted concentrations. RAW / NFκB-luc cells at the desired passage number were seeded and cultured in a 96-well plate. After confirming cell adhesion, the medium was removed and replaced with the test solution. In addition to the test solution treatment group, other groups were treated with medium replacement without the test solution, and with medium containing the NF-κB inhibitor BAY11-7082 at a final concentration of 15 μM. After 24 hours, the cells were treated with the test solution containing the inflammatory stimulus lipopolysaccharide (LPS) at a final concentration of 100 ng / mL. The group that had only undergone medium replacement without treatment with the test solution the previous day was divided into two groups: one treated with LPS (LPS-treated group) and the other untreated. The BAY11-7082-treated group received the same concentration of BAY11-7082 as the previous day, followed by LPS. After LPS treatment, the cells were cultured for 3 hours. After 3 hours, luciferase activity was measured using a luciferase assay kit (Promega). Results were subjected to one-way analysis of variance followed by multiple comparisons using the Tukey-Kramer test. A p<0.01 value was considered significant compared to the LPS-treated group. Error bars represent the mean ± standard error.

[0044] [result] NF-κB activation induced by LPS stimulation was significantly suppressed by the addition of ginger koji extract in the range of 30-100 μg / mL, indicating that ginger koji has an inhibitory effect on NF-κB activation within this concentration range, suggesting that it has an anti-inflammatory effect that suppresses the inflammatory response caused by NF-κB activation (Figure 5).On the other hand, extracts of ginger, rice, and rice koji did not show any inhibitory effect, and no anti-inflammatory effect was observed (Figures 6-8).

[0045] (Example 2) Osteoclast differentiation inhibition test 1 [material] The same cells and medium as in Reference Example 1 were used.

[0046] During osteoclast differentiation, RANKL signaling increases the amount of NFAT translocated into the nucleus, and NFAT is one of the transcriptional regulators that activates transcription factors necessary for osteoclast differentiation. Using RAW264.7 (RAW / NFAT-luc) cells carrying a vector that inserted a luciferase protein gene downstream of the NFAT-binding response element, we confirmed the inhibitory effect of NFAT on osteoclast differentiation using the luminescence intensity of the luciferase protein as an indicator.

[0047] The test solution was prepared by adding ginger koji extract to the medium at various concentrations. RAW / NFAT-luc cells at an arbitrary passage number were seeded and cultured in a 96-well plate. After confirming cell adhesion, the medium was removed and the cells were treated with the test solution containing RANKL at a final concentration of 100 ng / mL. In addition to the group treated with the test solution, groups were also prepared in which the medium was replaced without the test solution, and groups treated with medium containing RANKL at a final concentration of 100 ng / mL.

[0048] After 24 hours of culture, luciferase activity was measured using a luciferase assay kit (Promega). Results were subjected to one-way analysis of variance followed by multiple comparisons using the Tukey-Kramer test. A p<0.01 value was considered significant compared to the RANKL-treated group. Error bars represent the mean ± standard error.

[0049] (result) RAW264.7 cells differentiate into osteoclasts upon stimulation with RANKL. During this process, the transcription factor NF-AT1c is activated, which in turn induces the expression of osteoclast differentiation-related genes. Therefore, just as NF-κB activation has been used as an indicator of inflammatory responses, NF-AT activation can serve as an indicator of osteoclast differentiation. Therefore, we investigated the effect of ginger koji on osteoclast differentiation using an NF-AT reporter assay system, which uses a luciferase gene designed to be induced by the NF-AT response element, similar to the NF-κB reporter assay. When RAW264.7 cells were cultured with RANKL for 6 days, NF-AT activity significantly increased. Adding ginger koji to RANKL at concentrations ranging from 30 to 100 μg / mL, which is the range where anti-inflammatory effects were observed, significantly suppressed RANKL-induced NF-AT activation (Figure 9). This indicates that ginger koji has the effect of inhibiting NF-AT activation induced by RANKL stimulation, and suggests that it suppresses osteoclast differentiation caused by NF-AT activation.

[0050] (Example 3) Osteoclast differentiation inhibition test 2 The promotion of osteoclast differentiation in RAW264.7 cells was confirmed using tartrate-resistant acid phosphatase (TRAP) activity, a differentiation marker for osteoclasts.

[0051] The test solution was prepared by adding ginger koji extract to the above medium at various concentrations. RAW264.7 cells of any passage number were cultured at 1 × 10 4 The cells were adjusted to 0.5 mL / well with medium and seeded onto a 24-well plate. The next day, the test solution was replaced with medium supplemented with NF-κB activator receptor activator ligand (RANKL), and the cells were cultured at 37°C and 5% CO for 4 days. The medium was not replaced during these 4 days. The same test was also performed using medium alone and medium supplemented with RANKL.

[0052] After 4 days of culture, the medium was removed and the cells were washed with 0.5 mL / well of PBS. This washing procedure was repeated twice. After washing the cells, 0.3 mL / well of Formalin in PBS (1:9) was added and the plate was left to stand for 10 minutes. After leaving the plate for 10 minutes, the solution in the wells was discarded, and 0.3 mL / well of EtOH Acetone Solution was added and the plate was left to stand for 1 minute. After leaving the plate for 1 minute, the solution in the wells was discarded, the plate lid was removed, and the cells were allowed to dry at room temperature.

[0053] The dried cells were added with 0.3 mL of TRAP staining solution per well, incubated at 37°C for 15 minutes, and then washed three times with sterile water. After washing, the cells were dried at room temperature and the number of osteoclasts was counted using an optical microscope. TRAP-positive cells with three or more nuclei were considered osteoclasts.

[0054] (result) When ginger koji was tested in the range of 30 to 100 μg / mL, which is the range where anti-inflammatory effects were observed, it strongly inhibited osteoclast differentiation at all concentrations, so a similar experiment was conducted at lower concentrations. As a result, ginger koji significantly reduced the number of TRAP-positive osteoclasts at concentrations of 3 to 30 μg / mL, which is lower than the range where anti-inflammatory effects were observed (Figures 10 and 11).

[0055] (Example 4) Osteoclast differentiation inhibition test 3 We analyzed genes that serve as indicators of osteoclast differentiation to confirm the details of the inhibitory effect on osteoclast differentiation.We analyzed the osteoclast differentiation marker genes TRAP, cathepsin K (CTSK), DC-STAMP, OC-STAMP, and RANK.

[0056] The test solution was prepared by adding ginger koji extract to the above medium at various concentrations. RAW264.7 cells at an arbitrary passage number were seeded onto a 6-well plate and cultured until they reached 90% confluence. After confirming that the cells were 90% confluent, the test solution was replaced with medium supplemented with RANKL and cultured at 37°C in 5% CO2 for 4 days. The medium was not replaced during these 4 days. Tests were also conducted using medium alone and medium supplemented with RANKL.

[0057] After 4 days of culture, the test solution and medium were removed, and the cells were washed with 1 mL / well of PBS. This washing procedure was repeated twice. After washing, 0.5 mL / well of Sepasol was added, and the cells were scraped off with a cell scraper and collected in a 1.5 mL tube.

[0058] 200 μL of chloroform was added to each collected cell solution, vortexed for 15 seconds, and then allowed to stand at room temperature for 3 minutes. After vortexing, the tubes were centrifuged at 12,000 rpm at 4°C for 15 minutes. The separated upper layers were transferred to new 1.5 mL tubes. 200 μL of chloroform was added to each tube, vortexed for 15 seconds, and centrifuged at 12,000 rpm at 4°C for 15 minutes. The separated upper layers were transferred to 1.5 mL tubes, and an equal volume of isopropanol was added. After mixing by inversion, the tubes were allowed to stand at room temperature for 5 minutes and then centrifuged at 12,000 rpm at 4°C for 10 minutes. The solution in the tubes was discarded, and 1 mL of 70% ethanol was added to each tube. After mixing by inversion, the tubes were centrifuged again at 12,000 rpm at 4°C for 10 minutes. The ethanol in the tubes was discarded, and the tubes were air-dried at room temperature to evaporate any remaining ethanol. Ultrapure water was added to the dried RNA, and the mixture was frozen at -80°C.

[0059] The frozen RNA solution was thawed on ice and the concentration was adjusted to 75 ng / μL. cDNA was synthesized by reverse transcription using the PrimeScript RT reagent Kit (TAKARA) in a thermal cycler. The reaction time and temperature were as follows: <Reverse transcription reaction> Repeats:1, 37℃ / 15min, 85℃ / 5sec, 4℃ / ∞ qPCR was performed using the cDNA as a template. THUNDERBIRD SYBR qPCR Mix (TOYOBO) and primers TRAP, CTSK, DC-STAMP, OC-STAMP, and RANK were used for the PCR reaction. The reaction time and temperature were as follows: Stage 1: Initial Denaturation Repeats: 1, 95°C / 30 sec <Stage 2: PCR Reaction> Repeats: 40, 95°C / 5 sec, 60°C / 1 min <Stage 3: Melt Curve> Repeats: 1, 95°C / 15 sec, 60°C / 1 min, 95°C / 15 sec

[0060] (result) To investigate the inhibitory effect of ginger koji on osteoclast differentiation in detail, we quantified the expression of osteoclast differentiation marker genes, which are indicators of osteoclast differentiation, using quantitative PCR. The results showed that the addition of ginger koji concentration-dependently suppressed the expression of TRAP, cathepsin K (CTSK), DC-STAMP, OC-STAMP, and RANK genes. However, no statistically significant suppression was observed for the osteoclast differentiation marker gene c-Fos (Figures 12A-F). 12 represent mean values ± standard error, and each letter indicates a significant difference at p<0.05. These results demonstrate that ginger koji has the effect of suppressing osteoclast differentiation.

Claims

1. A method for producing a composition for improving bone metabolism, comprising a step of fermenting ginger with koji mold.

2. A method for producing a composition for improving bone metabolism as described in claim 1, wherein the composition for improving bone metabolism is intended to inhibit the differentiation of precursor cells into osteoclasts.

3. 3. The method for producing a composition for improving bone metabolism according to claim 1, wherein the koji mold is a microorganism of the genus Aspergillus.

4. 4. The method for producing a composition for improving bone metabolism according to claim 3, wherein the koji mold is Aspergillus oryzae.

5. The fermentation is a solid culture fermentation. A method for producing the composition for improving bone metabolism according to claim 1 or 2.

6. The fermentation is performed by solid culture at 20 to 40 ° C. A method for producing the composition for improving bone metabolism according to claim 5.

Images