JP6031328B2 - Azuki-derived bone metabolism regulator - Google Patents

Description

The present invention relates to an azuki bean-derived bone metabolism regulator, and more particularly, to an azuki bean-derived extract that effectively acts in the prevention and treatment of osteoporosis, and relates to a prophylactic agent, a therapeutic agent , and a method for producing the same .

  The rapid progress of aging in Japan is a social problem of increasing medical costs, increasing diseases peculiar to the elderly as aging progresses. One such age-related disease is osteoporosis, the main cause of which is aging and post-menopausal hormone balance imbalance. Since osteoporosis frequently induces fractures, life behavior is restricted, and quality of life (QOL) is greatly reduced. Therefore, prevention and countermeasures are important so that symptoms are likely to get worse.

  Bone metabolism in the living body is not static but extremely dynamic, and is systematically regulated while maintaining homeostasis. Specifically, bone metabolism means that bone is formed by osteoblast differentiation and at the same time, bone lysis proceeds by osteoclasts. Metabolism by maintaining a suitable equilibrium between bone regeneration and destruction by both cells results in healthy bone remodeling. However, in addition to the decreased function of osteoblasts due to aging (low-rotation type), there is a function promotion (high-rotation type) in which osteoclast activity becomes superior to osteoblasts due to the decrease in postmenopausal estrogen content. It works in a complicated manner. For this reason, bone destruction progresses rather than bone formation, leading to a decrease in bone density.

Osteoporosis is a typical symptom caused by the above modulation of bone metabolism control. Currently, in the treatment of osteoporosis, calcium preparation, active vitamin D ₃ preparation, vitamin K ₂ preparation, parathyroid hormone (PTH), calcitonin preparation, ipriflavone, bisphosphonate preparation, estrogen preparation, selective estrogen receptor modulator (SERM), etc. Therapeutic drugs are used. However, hormonal agents found in estrogen preparations can induce side effects such as breast cancer, and their use requires caution. There are a number of side effects in the above-mentioned therapeutic agents. In addition, the progression of osteoporosis is greatly influenced by the lifestyle of the patient. For this reason, the component which can be used together with prevention of osteoporosis from the meaning which reduces the influence, or use of a therapeutic agent is calculated | required.

  Therefore, many attempts have been reported to use natural product extracts derived from animals and plants for osteoporosis countermeasures (Patent Documents 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, etc.). . According to these documents, it has been shown that the natural product extract exhibits a certain effect on osteoporosis countermeasures. However, they have only mentioned and clarified the limited effects of improving osteoblast function or suppressing osteoclast action.

  As described above, bone remodeling involves multiple cells and is complicatedly linked by various signal transductions. For example, the membrane-bound cytokine RANKL expressed on the surface of osteoblasts binds to the receptor on the surface of osteoclast precursor cells and promotes differentiation from osteoclast precursor cells to osteoclasts. ing. For this reason, when bone remodeling is comprehensively grasped, it can be considered that merely acting on either osteoblasts or osteoclasts is not sufficiently effective in improving osteoporosis.

  Apart from the above-mentioned circumstances, the inventors have been conducting various studies focusing on azuki. Azuki beans (Vigna angularis) are processed softly by heating mainly by cooking (cooking), and processed into cooking ingredients such as red rice or confectionery ingredients such as “Ogura An” with added sugar. . When processing azuki bean, a large amount of azuki soup is produced. It is known that azuki bean broth contains polyphenols, saponins, anthocyanins, and glycosides bound to sugar molecules. However, it is often discarded as it is without being used.

  Focusing on azuki bean broth, as an effective utilization of the bioactive ingredients contained therein, for example, the physiological action of alcohol extract of azuki bean broth is antiallergic activity, hypoglycemic activity, cell adhesion inhibition, high fat Effects such as prevention of septicemia have been reported (see Patent Document 11 etc., Non-Patent Documents 1, 2, 3, 4, 5 etc.).

  Based on the above-mentioned circumstances, the inventor conducted further earnest research on the physiological activity effect of components extracted from azuki bean, and as a result, obtained knowledge that acts on the differentiation of both osteoblasts and osteoclasts. That is, it suggests the possibility that azuki bean-derived components are effective in the prevention and treatment of osteoporosis.

JP 2007-137775 A JP 2008-214237 A JP 2008-303166 A JP 2009-107995 A JP 2009-256350 A JP 2010-30905 A JP 2010-215607 A JP 2010-235533 A JP 2010-235534 A Japanese Patent No. 3909391 JP 2011-178680 A

T. T. Itoh et al. , Biosci. Biotechnol. Biochem. 68 (12), 2421-2426 (2004). T. T. Itoh et al. , Biosci. Biotechnol. Biochem. 69 (3), 448-454 (2005) T. T. Itoh et al. , Nutrition, 25, 134-141 (2009) T. T. Itoh et al. , Nutrition, 25, 318-321 (2009) T. T. Itoh et al. Physother. Res. , 26 (7), 1003-1011 (2012)

  The present invention has been made in view of the above points, and focuses on an unused bioactive component of azuki bean, and a novel azuki bean-derived bone metabolism regulator (a bone metabolism-regulated azuki bean) that is effective in the prevention and treatment of osteoporosis and the like. And a prophylactic agent, a therapeutic agent, or a food containing the same.

That is, in the invention of claim 1, the supernatant is separated from the broth produced by ripening azuki bean in hot water, and the supernatant is adsorbed on the synthetic adsorbent, and then 10% on the synthetic adsorbent after the adsorption. An azuki bean-derived extract obtained by adding and elution with a 40% ethanol aqueous solution , wherein the azuki bean-derived extract promotes differentiation from osteoblasts to bone cells, and the azuki bean-derived extract is a precursor The present invention relates to an Azuki-derived bone metabolism regulator characterized by suppressing differentiation of osteoclasts into osteoclasts .

In the invention of claim 2, the supernatant is separated from the broth produced by boiling the azuki bean in hot water, and the supernatant is adsorbed on the synthetic adsorbent, and then 10% to 40% on the adsorbed synthetic adsorbent. Azuki bean-derived extract obtained by adding and elution with an aqueous ethanol solution, wherein the azuki bean-derived extract undergoes transcriptional expression in which BMP-2 binds to a BMP receptor on the surface of osteoblasts, and is expressed in the osteoblast. By increasing the transcription factors Runx2, OSX, and Dlx5, the azuki bean-derived extract promotes differentiation from osteoblasts to bone cells, and RANKL binds to RANK on the precursor osteoclast surface. In advancing transcriptional expression, the azuki bean-derived extract reduces the expression level of c-Fos in the progenitor osteoclast and inhibits signal transduction, whereby the azuki bean-derived extract is broken from the progenitor osteoclast. According to Azuki derived bone metabolism regulating agent characterized by inhibiting the differentiation into cells.

The invention of claim 3 relates to a preventive or therapeutic agent for osteoporosis comprising the azuki bean-derived bone metabolism regulator of claim 1 or 2 .

In the invention of claim 4, after separating the supernatant from the boiled broth of azuki bean cooked with hot water, adsorbing the supernatant to the synthetic adsorbent, the adsorbed synthetic adsorbent is 10% to 40%. A method for producing an azuki bean-derived bone metabolism regulator comprising, as an active ingredient, an azuki bean extract obtained by adding and elution with a% ethanol aqueous solution, wherein the azuki bean extract promotes differentiation from osteoblasts to bone cells And the said azuki bean-derived extract suppresses the differentiation from a precursor osteoclast to an osteoclast, It concerns on the manufacturing method of the azuki bean-derived bone metabolism regulator characterized by the above-mentioned .

In the invention of claim 5, after separating the supernatant from the broth produced by boiling the azuki bean in hot water and adsorbing the supernatant to the synthetic adsorbent, the adsorbed synthetic adsorbent is 10% to 40%. A method for producing an azuki bean-derived bone metabolism regulator comprising, as an active ingredient, an azuki bean extract obtained by adding and elution with a% ethanol aqueous solution, wherein the azuki bean extract extracts BMP-2 on a BMP receptor on the surface of osteoblasts. By increasing transcription factors Runx2, OSX, and Dlx5 in the osteoblasts in the transcriptional expression that proceeds by binding, the azuki bean extract promotes differentiation from the osteoblasts to bone cells, and In the transcriptional expression that proceeds when RANKL binds to RANK on the surface of progenitor osteoclasts, the extract derived from azuki bean reduces the expression level of c-Fos in the progenitor osteoclast and inhibits signal transduction. The azuki bean-derived extract according to the manufacturing method of the azuki bean-derived bone metabolism regulating agent characterized by inhibiting the differentiation of osteoclasts from the precursor osteoclasts.

According to the azuki bean-derived bone metabolism regulator according to the invention of claim 1, after separating the supernatant from the broth ripened with hot water and adsorbing the supernatant to the synthetic adsorbent, after the adsorption An azuki bean-derived extract obtained by adding 10% to 40% ethanol aqueous solution to the synthetic adsorbent and eluting , wherein the azuki bean-derived extract promotes differentiation from osteoblasts to bone cells, and Since the azuki bean-derived extract suppresses the differentiation from precursor osteoclasts to osteoclasts , focusing on the unused active ingredients of azuki bean that have been discarded so far, a novel and more active azuki bean-derived bone metabolism regulator An agent can be obtained, and it exhibits bone metabolic activity effective for prevention and treatment of osteoporosis and the like.

According to the azuki bean-derived bone metabolism regulator according to the invention of claim 2, after separating the supernatant from the broth produced by ripening the azuki bean in hot water, adsorbing the supernatant to the synthetic adsorbent, An extract derived from azuki bean obtained by adding 10% to 40% ethanol aqueous solution to the synthetic adsorbent and elution, wherein BMP-2 binds to the BMP receptor on the surface of osteoblasts. By increasing the transcription factors Runx2, OSX, and Dlx5 in the osteoblasts in advancing transcriptional expression, the Azuki bean extract promotes differentiation from the osteoblasts to bone cells, and progenitor osteoclasts In the transcriptional expression that proceeds with RANKL binding to the surface RANK, the azuki bean-derived extract reduces the expression level of c-Fos in the progenitor osteoclast and inhibits signal transduction. The new extract suppresses the differentiation from the precursor osteoclasts to osteoclasts, and therefore focuses on the unused active ingredients of azuki beans that have been discarded so far, and is a novel and more active azuki-derived bone metabolism regulator. And exhibits effective bone metabolic activity for the prevention and treatment of osteoporosis and the like.

According to the preventive or therapeutic agent for osteoporosis according to the invention of claim 3 , since it comprises the azuki bean-derived bone metabolism regulator of claim 1 or 2 , it has an effect in improving bone metabolism abnormalities such as osteoporosis. A drug can be obtained.

According to the method for producing azuki bean-derived bone metabolism regulator according to the invention of claim 4 , after separating the supernatant from the broth produced by boiling the azuki bean in hot water, and adsorbing the supernatant to the synthetic adsorbent, A method for producing an azuki bean-derived bone metabolism regulator comprising, as an active ingredient, an azuki bean-derived extract obtained by adding 10% to 40% ethanol aqueous solution to the synthetic adsorbent after the adsorption and eluting the adsorbate-derived extract. Promotes differentiation from osteoblasts to bone cells, and the azuki bean-derived extract suppresses the differentiation from the precursor osteoclasts to osteoclasts. Focusing on these active ingredients, a new and more active azuki bean-derived bone metabolism regulator can be obtained.

According to the method for producing an azuki bean-derived bone metabolism regulator according to the invention of claim 5 , after separating the supernatant from the broth produced by boiling the azuki bean in hot water, and adsorbing the supernatant to the synthetic adsorbent, A method for producing an azuki bean-derived bone metabolism regulator comprising, as an active ingredient, an azuki bean-derived extract obtained by adding 10% to 40% ethanol aqueous solution to the synthetic adsorbent after the adsorption and eluting the adsorbate-derived extract. Increases the transcription factors Runx2, OSX, and Dlx5 in the osteoblasts in the transcriptional expression that proceeds by binding BMP-2 to the BMP receptor on the surface of osteoblasts, whereby the azuki bean-derived extract becomes the osteoblast Expression of c-Fos in the precursor osteoclast in the transcriptional expression that promotes differentiation from bone to bone cells and proceeds with RANKL binding to RANK on the precursor osteoclast surface In order to suppress differentiation from the precursor osteoclasts to the osteoclasts by reducing the signal transduction by reducing the signal, attention is paid to the unused active ingredients of azuki beans that have been discarded so far Thus, a novel and more active azuki bean-derived bone metabolism regulator can be obtained.

It is a schematic process drawing which obtains the azuki bean-derived bone metabolism regulator of the present invention. It is a graph which shows alkaline phosphatase (ALP) activity. It is the photograph of the cultured cell which showed ALP activity. It is a schematic diagram of signal transduction and transcription in progenitor osteoblasts. It is a comparative photograph of a Western blot of transcription factor expression in osteoblasts. It is the graph which estimated the transcription | transfer factor amount in an osteoblast by quantitative real-time-PCR. It is a comparative photograph of the western blot of phosphorylated Smad. It is a comparative photograph of Western blotting such as P38 MAP kinase. It is a graph which shows a bone-type tartrate resistant acid phosphatase (TRACP) activity. It is a photograph of TRACP staining. It is a schematic diagram of signal transduction and transcription in precursor osteoclasts. It is a comparative photograph of Western blots of c-Fos and NFATc1.

  The process for obtaining the azuki bean-derived bone metabolism regulator of the present invention (the extract derived from a bone metabolism-regulated azuki bean) will be described with reference to the schematic process diagram of FIG. First, azuki bean (Adzuki beans: Vigna angularis) is appropriately washed with water and then simmered in hot water (hot water) at 90 ° C. to 95 ° C. for 20 to 60 minutes (boiled). Is also called boiling or cooking.) After the heating, azuki bean (boiled beans) and azuki bean broth (hot water extract) are separated. Usually, azuki bean broth is discarded as it is, and heated azuki bean is added with sugar or the like to be processed into an okura ann.

  As it is inferred from the seed coat color, azuki bean broth has a reddish brown color, so it is expected that some components are dissolved. Therefore, we decided to proceed with the elucidation in order to open the way for effective use of unused items.

  The broth produced when the azuki bean is ripened is cooled to room temperature, and the broth is divided into a supernatant and a precipitate. In addition to filter filtration, centrifugal separation under the conditions of Examples described later is also used for separating the broth.

  Various hot water soluble components are contained in the supernatant of the broth. Therefore, the supernatant is once adsorbed to the synthetic adsorbent for concentration and elution separation of the components in the supernatant. The synthetic adsorbent is an appropriate resin column, resin beads, or the like. In the examples described later, beads having a particle diameter of 0.25 mm or more provided with pores made of styrene-divinylbenzene resin were used.

  An aqueous solution of ethanol is added to the synthetic adsorbent adsorbing various components in the supernatant and sent. The adzuki bean extract adsorbed on the synthetic adsorbent is eluted in the aqueous ethanol solution. Thus, the component obtained by the process of azuki bean ripening, supernatant separation, adsorption, and elution is azuki bean-derived bone metabolism regulator defined in the present invention. The presence or absence of an elution component present in the aqueous ethanol solution can be easily grasped by confirming the absorbance at a predetermined wavelength (for example, 280 nm).

  The aqueous ethanol solution is prepared as a solution (v / v%) with a concentration gradient (gradient) in which the ethanol concentration is gradually increased gradually from the state of water, and is used for elution of the extract. In the examples, the concentration (v / v%) of the ethanol aqueous solution is from 0% (water only) to 10% to 40% concentration solution, 40% to 60% concentration solution, 60% to 80% concentration. There are four types of solutions. In terms of the accuracy of forming the concentration gradient, it is roughly divided into the concentration regions. Note that when the synthetic adsorbent is dried, the separation performance fluctuates, so that a necessary amount of water is also sent when the concentration is switched.

  By evaporating and drying moisture from the elution fraction (fraction) of each ethanol aqueous solution concentration, an extract sorted according to the concentration of the ethanol aqueous solution can be obtained. Among them, the expression of activity related to the regulation of bone metabolism of the extract of azuki bean in an aqueous ethanol solution in a concentration range of 10% to 40% is remarkable. Since the extract is a component derived from azuki bean broth, it is expected to contain polyphenol components such as glycosides, pigment components, and various catechins. Therefore, the Azuki bean-derived bone metabolism regulator of the present invention is considered to be a mixture containing the plurality of components.

  The reason why the elution fraction in the ethanol concentration range of 10% to 40% exhibits good activity is not necessarily clear. Since elution is performed using an ethanol aqueous solution having a concentration gradient, it is possible to separate a substance having a relatively good hydrophilicity and a part of hydrophobicity, not a completely hydrophilic or hydrophobic substance. In particular, when taking into consideration the good penetration into the cell after administration to the living body or after taking it, molecules with moderate amphipathy from the permeability through the cell membrane and diffusion within the cell are more physiological. The active effect is considered high.

  The azuki bean-derived bone metabolism regulator obtained by extraction from azuki bean is a component that acts on both progenitor osteoblasts and osteoclasts involved in bone remodeling (remodeling), as described in detail in Examples below. It is. The azuki bean-derived bone metabolism regulator (bone metabolism-regulated azuki bean-derived extract) enhances differentiation into osteoblasts and their calcification relative to progenitor osteoblasts. Moreover, the azuki bean-derived bone metabolism regulator suppresses differentiation into osteoclasts relative to precursor osteoclasts. In other words, bone destruction can be achieved by enabling both bone loss regulation of osteoblastic function (low rotation type) and function promotion (high rotation type) in which osteoclast activity is superior to osteoblasts. This suggests that bone formation is promoted while suppressing the above. For example, it is effective for the prevention and treatment of diseases such as osteoporosis and osteomalacia (rickets).

  Many of the natural product extracts that have been proposed for the prevention and treatment of osteoporosis have been aimed at acting on either the enhancement of bone formation or the suppression of bone destruction. In contrast, azuki bean-derived bone metabolism regulators act on both cells involved in bone formation and bone destruction in bone formation, while having the same components, so that bone mass and bone density are increased to improve symptoms. Effective in regulating bone metabolism. In addition, as a countermeasure against side effects caused by drugs used in the treatment of existing osteoporosis, etc., it is also used as an alternative drug for the prevention or treatment of osteoporosis, or as a preventive drug or treatment in terms of reducing the effect The advantage that can be used together with the use of drugs is great.

  When Azuki-derived bone metabolism-regulating agent (Bone metabolism-regulating Azuki-derived extract) is used as a prophylactic or therapeutic agent for improving bone metabolism abnormalities, the dosage forms for oral administration are tablets, capsules, powders, granules, liquids Etc. are appropriate. In addition, injections, suppositories, drops, sprays, nasal drops and the like can also be used. In the listed drugs, the azuki bean-derived bone metabolism regulator may be used alone or in combination with other drugs.

  The azuki bean-derived bone metabolism regulator of the present invention is a natural product and an extract derived from azuki bean that has been used for a long time. For this reason, the azuki bean-derived bone metabolism regulator is compatible with commonly used foods. Therefore, the azuki bean origin bone metabolism regulator containing food which added the azuki bean origin bone metabolism regulator newly can be made.

  Foods that can be supplemented with Azuki-derived bone metabolism regulator (Bone metabolism-regulated Azuki-derived extract) are specifically Mizuyokan, Yokan, Ogura An, Dorayaki, Azuki Mousse, Ice Cream, Candy, Caramel , Custard pudding, coffee jelly, gummy, bavaroa, marshmallow, castella, hot cake, cookies, fruit juice, carbonated drinks. Furthermore, the tablet used as a supplement is also mentioned.

  In producing the foods containing the Azuki-derived bone metabolism regulators listed, the content of the Azuki-derived bone metabolism regulator is the taste of the food itself and the change in taste after addition, other additive ingredients, the amount of food consumed per serving It is defined by comprehensively considering the eating frequency, seasonality, sales form, and the age group and consumer group taking into account gender. The amount of the Azuki-derived bone metabolism regulator added to the food is arbitrary, for example, 0.01 wt% to 100 wt% (that is, the food that is used as the total amount of Azuki-derived bone metabolism regulator), 1 wt% to 80 wt% Also good.

[Preparation of extract derived from azuki bean broth]
Azuki beans from Hokkaido were used as a raw material for adzuki bean (Vigna angularis). Azuki beans were put into an open-type steaming kettle, heated from water and boiled for 45 minutes. The boiled and softened azuki bean and its broth were separated. The broth was cooled to near room temperature (about 20 ° C.) and then centrifuged at 9000 rpm for 10 minutes. To 1 kg of the supernatant obtained by centrifugation, 300 g of an aromatic synthetic adsorbent (reverse phase adsorbent “DIAION HP-20” manufactured by Mitsubishi Chemical Corporation) was added and stirred at 4 ° C. for 12 hours. After dividing into a synthetic adsorbent and other liquids, the synthetic adsorbent was washed with distilled water and further eluted several times with 40% ethanol aqueous solution (v / v).

  The washed and eluted synthetic adsorbent was packed in a column (inner diameter 5 mm × total length 300 mm). A plurality of the synthetic adsorbent packed columns were prepared. First, only distilled water was sent to the column packed with the synthetic adsorbent, and a solution was prepared while gradually increasing the ethanol ratio. In the present embodiment, the elution of only distilled water from the beginning is carried out according to four types of concentrations: an aqueous ethanol solution of 10% to 40% concentration, an aqueous ethanol solution of 40% to 60% concentration, and an aqueous ethanol solution of 60% to 80% concentration. Elution was performed. Ethanol subjected to elution is accompanied by a concentration gradient (gradient). For this reason, when the density is switched, a range of the density inevitably occurs. Thus, elution conditions having a concentration range were used as described above.

  For each of elution from distilled water only, 10% to 40% ethanol aqueous solution, 40% to 60% ethanol aqueous solution, and 60% to 80% ethanol aqueous fraction (fraction). (Shibata Kagaku Co., Ltd.) was used to evaporate and dry the water in the fraction. Thus, four kinds of extracts derived from azuki bean broth with different elution conditions were obtained. In the following description and drawings, the extract obtained from the elution of distilled water alone is referred to as “WEx”, and the extract obtained from the elution of an aqueous ethanol solution having a concentration of 10% to 40% is referred to as “EtEx.40”, 40% to 60%. The extract obtained from the elution of the aqueous ethanol solution with a concentration of% is denoted as “EtEx.60”, and the extract obtained from the elution of an aqueous ethanol solution with a concentration of 60% to 80% is denoted as “EtEx.80”. The azuki bean extract was in the form of a dark purple powder.

[Verification of intracellular mechanism]
After preparing four kinds of extracts derived from azuki bean juice with different extraction concentrations, the mechanism of promoting differentiation into osteoblasts as a bone metabolism regulator and inhibiting the differentiation into osteoclasts was confirmed. The medicinal properties of the extract derived from the broth were confirmed. The verification experiment is described below.

[Effect of promoting differentiation into osteoblasts]
The mouse calvaria-derived MC3T3-E1 cells (osteoblast-like cells) used in the experiments are cells established by Sudo et al. (J. Cell Biol., Vol 96, 191-198 (1983)). MC3T3-E1 cells were selected because they showed relatively clear changes in alkaline phosphatase activity (hereinafter referred to as ALP activity) and calcification as described below as an osteoblast model.

MC3T3-E1 cells were suspended in α-MEM medium containing 10% fetal bovine serum (FBS) at a density of 1.0 × 10 ⁵ cells / mL and seeded in a 24-well multiwell plate. After culturing for 12 hours, osteoblast differentiation medium (ODM) containing ascorbic acid (manufactured by Wako Pure Chemical Industries, Ltd., final concentration 50 μg / mL), β-glycerophosphate (manufactured by Sigma Aldrich, final concentration 10 mM) ), Hepes (N-2-hydroxyethylperazine-N-ethane sulphonic acid) (Sigma Aldrich, final concentration 5 mM) was added, and the medium of MC3T3-E1 cells was changed to the prepared medium to induce differentiation. Hereinafter, the term “osteoblast differentiation medium” or “ODM” means a medium containing ascorbic acid.

  In the osteoblast differentiation medium, EtEx. 40 was added at different concentrations. 72 hours after the medium was changed, ALP activity was measured and Alizarin Red S staining was performed.

<Measurement of Alkaline Phosphatase (ALP) Activity>
When osteoblasts differentiate into bone cells, the expression of alkaline phosphatase (ALP; Alkaline Phosphatase) that degrades pyrophosphate that inhibits calcification increases in the cells. Therefore, the activity of alkaline phosphatase can be used as an index.

In measuring ALP activity, a Tris buffer (assay buffer) containing Tris-HCl (1.5 M), ZnCl ₂ (1 mM), and MgCl ₂ (1 mM) was prepared. At the same time, a surfactant-containing Tris buffer solution in which 1% of a surfactant (Triton X-100, manufactured by Wako Pure Chemical Industries, Ltd.) was dissolved in the same Tris buffer solution was also prepared.

  After culturing in the osteoblast differentiation medium (ODM), MC3T3-E1 cells were washed twice with ice-cooled PBS (−) (phosphate buffered saline) to remove the medium. Subsequently, a Tris buffer solution containing a surfactant was added, and the protein content in the cells was extracted by allowing to stand at room temperature for 30 minutes. Thereafter, the reaction mixture was diluted with an appropriate amount of Tris buffer, Tris buffer containing the same amount of pNPP (p-Nitrophenyl phosphate, Wako Pure Chemical Industries, Ltd., 10 mM) was added, and the mixture was incubated at 37 ° C. for 30 minutes. The amount of the reaction product (p-Nitrophenyl) produced at this time was measured by absorbance at a wavelength of 405 nm to evaluate ALP activity (n = 5 points). The unit of ALP activity was “units / mg protein amount”.

  The result is the graph of FIG. Cells cultured without using osteoblast differentiation medium (ODM) (ODM (−)) hardly increased ALP activity. In contrast, ALP activity increased in cells (Control) cultured using only osteoblast differentiation medium (ODM). Next, EtEx., Which is an adzuki-derived bone metabolism regulator of the present invention. In cells cultured in a medium supplemented with 40, ALP activity was significantly increased in a concentration-dependent manner at 5 μg / mL, 10 μg / mL, and 20 μg / mL, compared to culture using only osteoblast differentiation medium (Control). . From this, EtEx. 40 suggests an action of promoting differentiation from osteoblasts to bone cells depending on the concentration.

  EtEx. 40 other than WEx, EtEx. 60, and EtEx. 80 was cultured in the same manner. However, no significant difference could be found. For this reason, it is considered that the component that triggers the differentiation promoting action is lean, and EtEx. We decided to verify 40 below.

<Alizarin Red S staining>
The alizarin red S staining method was used for the evaluation of calcification in cultured cells. For MC3T3-E1 cells, osteoblast differentiation medium (ODM) is not used (ODM (−)), osteoblast differentiation medium (ODM) is used (Control, ODM (+)), cell differentiation medium (ODM) EtEx. 40 was added at 10 μg / mL (ODM (+) + EtEx.40, 10 μg / mL), and EtEx. 40 was cultured for 21 days under four types of medium conditions with addition of 20 μg / mL (ODM (+) + EtEx.40, 20 μg / mL). After culturing, the cells were washed twice with ice-cooled PBS (−) to remove the medium components, added with an appropriate amount of ethanol, and allowed to stand for 15 minutes to fix the cells. Thereafter, ethanol was removed, the cells were washed with distilled water, and alizarin red S aqueous solution was added for staining.

<Observation of cultured cells>
The upper part (ALP) of FIG. 3 is a photomicrograph (magnification 40 times) showing the medium components and the appearance of ALP activity. The lower part of FIG. 3 (Alizarin RedS) is a photomicrograph (magnification 40 times) showing the state of medium components and alizarin red S staining. In all the photographs, the ODM (-) in the leftmost column is a light color and has little expression and color development. From left to right, ODM (+), EtEx. The color became darker in the order of 40 density. That is, ALP activity and intracellular calcification are found in the Azuki bean extract EtEx. It was confirmed that it increased with the presence of 40, and further increased depending on its concentration.

[Expression of transcription factors in osteoblasts]
FIG. 4 is a schematic diagram schematically showing transcription factors, genes, protein expression, and the like related to differentiation into osteoblasts in osteoblasts. After the binding of BMP-2 (ligand) to the BMP receptor of osteoblasts, in the nucleus through signal transduction such as intracellular R-Smad, TAK1, etc., “Runx2” (Run-related transcription factor 2), “ The expression level of transcription factors represented by “OSX” (Osterix), “Dlx5” (distal-less homeobox 5), etc. is increased, translocates into the nucleus, adheres to the promoter region, and promotes transcription. Therefore, the expression of the transcription factor and the azuki bean-derived extract EtEx. 40 relationships were verified.

<Expression of osteoblast differentiation-related protein by Western blot>
First, azuki bean-derived extract EtEx. The presence or absence of 40, the proteins of Runx2, OSX, and Dlx5, which are transcription factors, and the expression of β-actin as an endogenous control were confirmed by Western blot. FIG. 5 shows a culture using osteoblast differentiation medium (ODM). 40 None (-) and EtEx. There were 40 (20 μg / mL) and (+) differences. As can be seen from the figure, the band was somewhat thicker and thicker, suggesting an increase in the amount of transcription factors such as Runx2. Runx2, OSX, and Dlx5 manufactured by Santacruz Biotechnology and β-actin manufactured by SIGMA were used as primary antibodies for protein labeling.

<Quantitative real-time-verification by PCR method>
In order to quantitatively grasp the expression of each transcription factor, a real-time PCR method (quantitative Real Time-PCR method) was used. The amount of the protein finally expressed can be estimated from the amount of mRNA generated upon translation into Runx2, OSX, and Dlx5 proteins.

MC3T3-E1 cells adjusted to 1 × 10 ⁵ cells / mL were seeded in a 6-well multiplate and pre-cultured for 12 hours. Thereafter, EtEx. 40 (20 μg / mL) was added, and after stimulation for 24 hours, the plate was washed 3 times with PBS (−). After washing, 1 mL / well of TRIzol reagent (manufactured by Life Technologies Japan Co., Ltd./Invitrogen) was added to lyse cells, and then transferred to a 1.5 mL tube with a lid (manufactured by Eppendorf Co., Ltd.).

  Next, 200 μL of chloroform was added, and after stirring for 30 seconds with a vortex mixer, the mixture was centrifuged at 11500 rpm for 15 minutes at 4 ° C., and the supernatant was transferred to a new 1.5 mL tube with a lid (made by the above company). 2-propanol was added and allowed to stand for 10 minutes. After standing for 10 minutes, the supernatant was carefully removed by centrifugation at 11500 rpm for 10 minutes at 4 ° C., and the residue was washed with 500 μL of 75% ethanol. The residue was collected by centrifugation at 9000 rpm for 5 minutes at 4 ° C., and the washing solution was carefully discarded and air-dried for 30 minutes. After air drying for 30 minutes, 180 μL of DEPC water (diethyl pyrocarbonate water) (manufactured by Invitrogen), 20 μL of × 10 Buffer (manufactured by Takara Bio Inc.), and 2 μL of DNase 1 were added and reacted at 37 ° C. for 1 hour. .

  Thereafter, 40 μL of sodium acetate (manufactured by Nippon Gene Co., Ltd., 3M, pH 5.2) and 150 μL of phenol chloroform solution (manufactured by Invitrogen) were added, and the mixture was vibrated and stirred with a vortex mixer. The tube containing phenol chloroform and the sample solution was centrifuged at 15000 rpm for 5 minutes, the supernatant was transferred to a new 1.5 mL capped tube (manufactured by the above company), and total RNA was extracted with 800 μL of ethanol. Thereafter, the mixture was centrifuged at 15000 rpm for 30 minutes at 4 ° C. to collect total RNA. Finally, the supernatant was discarded, air-dried, and dissolved in DNase-RNase-free water.

  After measuring the total RNA amount of the sample with a biophotometer, each RNA sample was adjusted to 100 ng / μL. The reverse transcription reaction was performed using “PrimeScript (registered trademark) RT reagent Kit” manufactured by Takara Bio Inc. The PCR conditions were “37 ° C .: 15 minutes, 85 ° C .: 2 minutes, 4 ° C .: continued”. Sample cDNA obtained by reverse transcription reaction and primers Runx2 (MA073758), OSX (MA117891), Dlx5 (MA090802) and GAPDH (MA095452) manufactured by Takara Bio Inc. were prepared, and “SYBR (registered trademark) manufactured by Takara Bio Inc. was prepared. According to “Premix Ex Taq II”, expression analysis was performed using real-time PCR (Applied Biosystems, Step One). The expression level was evaluated using GAPDH as an internal standard.

  FIG. 6 is a bar graph in which protein expression levels of transcription factors Runx2, OSX, and Dlx5 expressed in MC3T3-E1 cells are estimated from the copy number of mRNA based on the quantitative RT-PCR method. FIG. 6A shows Runx2, FIG. 6B shows OSX, and FIG. 6C shows Dlx5 mRNA copy number. It shows as a relative ratio with GAPDH (glyceraldehyde 3-phosphate dehydrogenase). The bar graph of each transcription factor shows whether or not osteoblast differentiation medium (ODM) is used (+/−), EtEx. The difference in the medium conditions with and without (+/−) of 40 (20 μg / mL) is shown. In both cases, p <0.05 and n = 5.

  As a result, the use of an osteoblast differentiation medium (ODM), and further the extract from the azuki bean EtEx. In the order of 40 administration, the increase in transcription factors Runx2, OSX, and Dlx5 involved in osteoblast differentiation was confirmed from the expression level of mRNA. In particular, azuki bean-derived extract EtEx. The expression level of cells cultured in the presence of 40 was significantly increased. As shown in FIG. 4 described above, OSX expression is regulated by Runx2 and Dlx5. From this, the extract from azuki bean EtEx. 40 promoted the expression of Runx2 and Dlx5, suggesting the possibility that the expression of OSX increased in synchronism with this. Therefore, the extract from azuki bean EtEx. 40 can be said to be a component having an action of regulating the expression of genes involved in osteoblast differentiation.

[Mechanism of action of azuki bean-derived extract on progenitor osteoblasts]
As shown in the schematic diagram of FIG. 4, the following two types are considered as pathways involved in OSX (Osterix) expression. After binding of BMP-2 to the BMP receptor, P38 MAP kinase is phosphorylated through a pathway in which Runx2 and the like are expressed through phosphorylation of R-Smad and TAK1 (TGF-β-activated kinase 1). It is a pathway expressed by OSX (Osterix).

  Therefore, activation (phosphorylation) of receptor type Smad1 / 5/8 after differentiation stimulation is caused by the extract of azuki bean derived from EtEx. It was verified whether it was influenced by the presence or absence of 40. MC3T3-E1 cells cultured in osteoblast culture medium were mixed with azuki bean-derived extract EtEx. 40 (20 μg / mL) was administered, and after 2 hours, azuki bean extract EtEx. Stimulated with osteoblast differentiation medium (ODM) containing 40. The Smad1 / 5/8 phosphorylation level immediately after ODM stimulation until 60 minutes was confirmed by Western blot. FIG. 7 is a blotting image at that time, and EtEx. 40 (−) is no administration, EtEx. 40 (+) is with administration. Phospho-Smad1 / 5/8 manufactured by Cell Signaling Technology was used as the primary antibody for labeling.

  As can be seen from the blotting image, azuki bean extract EtEx. Regardless of the presence or absence of 40, no difference was observed in the phosphorylation level of phosphorylated Smad1 / 5/8. In either case, the phosphorylation level increased with time from the induction of osteoblast differentiation medium stimulation. From this result, azuki bean-derived extract EtEx. It can be said that 40 does not affect the route of Smad.

  Next, the information transmission pathway of p38 MAP kinase (p38) via TAK1 is extracted from azuki bean extract EtEx. It was verified whether it was influenced by the presence or absence of 40. MC3T3-E1 cells cultured in osteoblast culture medium were mixed with azuki bean-derived extract EtEx. 40 (20 μg / mL) was administered, and after 2 hours, azuki bean extract EtEx. Stimulated with osteoblast differentiation medium (ODM) containing 40. The phosphorylation level of p38 MAP kinase from immediately after ODM stimulation to 60 minutes was confirmed by Western blot. P38 MAP kinase and Phospho-p38 MAP kinase manufactured by Cell Signaling Technology were used as primary antibodies for protein labeling.

  FIG. 8 is a blotting image of each protein by Western blotting. The phosphorylation level of p38 MAP kinase (p38) increased 15 minutes after ODM stimulation. Azuki bean extract EtEx. In the cells stimulated for differentiation in the ODM medium containing 40, the phosphorylation level was increased as compared with the cells stimulated for differentiation in the ODM medium not containing the azuki bean extract.

  That is, the azuki bean-derived extract EtEx. 40 can be said to promote phosphorylation of p38 MAP kinase via TAK1. Summarizing the findings obtained from the results of FIGS. 7 and 8, the extract of azuki bean derived EtEx. 40 (middle in the figure) acts on p38 MAP kinase (p38), and its phosphorous, Phospho-p38 MAP kinase (p-p38)) promotes the activation of transcription factors and newly attaches to the promoter region. Enhances transcriptional expression. Finally, OSX (Osterix) is expressed, and a mechanism that promotes differentiation from osteoblasts to bone cells by the transcription factor can be proposed (see FIG. 4).

[Inhibition of differentiation into osteoclasts]
Osteoclasts are multinucleated cells derived from hematopoietic stem cells and fused with multiple cells. Osteoclasts contain an acid hydrolase, bone-type tartrate-resistant acid phosphatase (TRACP: Tartrate-resistant Acid Phosphate (hereinafter referred to as TRACP for bone-type tartrate-resistant acid phosphatase)) in its cytoplasm. Since TRACP leaks from the cytoplasm when osteoclasts are absorbed into the bone, it is frequently used as a marker that is a direct indicator of the number of osteoclasts and their bone resorption activity. Therefore, TRACP activity serves as an index of the degree of differentiation into osteoclasts, and if TRACP activity is low, bone destruction is suppressed, resulting in suppression of osteoporosis.

  When progenitor osteoclasts differentiate into osteoclasts, RANKL (receptor activator of NFκB ligand) (sRANKL is also synonymous, s is an abbreviation for soluble) belongs to the TNF family and is secreted from osteoblasts. It binds to RANK and induces differentiation (see FIG. 11). Accordingly, RAW264.7 cells, which are progenitor osteoclast-like cells of a mouse-derived monocyte leukemia cell line, were used, and azuki bean-derived extract EtEx. In the presence of sRANKL stimulation. The effect of the presence or absence of 40 was examined. RAW264.7 cells can be differentiated without requiring macrophage colony-stimulating factor (hereinafter referred to as M-CSF), which is convenient as a control.

<Measurement of bone-type tartrate-resistant acid phosphatase (TRACP) activity>
RAW264.7 cells of mouse precursor osteoclast-like cells were suspended at a density of 5.0 × 10 ⁴ cells / mL in Dulbecco's Modified Eagle Medium medium (DMEM medium) containing 10% fetal bovine serum (FBS). And cultured for 24 hours. After this culturing, it is diluted stepwise with the same DMEM medium and seeded in a 24-well plate, and the azuki bean-derived extract EtEx. For 40, the dose was increased in the order of control, 5 μg / mL, 10 μg / mL, 20 μg / mL, and 50 μg / mL, and the cells were further cultured for 2 hours.

  In each well, sRANKL was administered at a concentration of 100 ng / mL, and M-CSF was administered at a concentration of 50 ng / mL to give differentiation stimulation. M-CSF (+) is administered and M-CSF (-) is not administered. After administration of each component, the cells were cultured for 5 days. After culturing, the supernatant was removed from the well plate and 10 μL / well of 10% formalin solution was added and fixed for 10 minutes. After fixation, the supernatant was removed, and 90% ethanol 5 μL / well was added and dried. TRACP activity measurement solution (p-Nitrophenylphosphate (5.5 mM), sodium tartrate (50 mM), citrate buffer (pH 4.5)) 0.4 mL was added to each well, and shaken at 37 ° C. for 1 to 2 hours. That ’s it. A 0.1N sodium hydroxide solution was dispensed in advance to another well for measurement in an amount of 20 μL per well, and 100 μL of the reaction solution in which the TRACP activity measurement solution was added and reacted was dispensed. About each well, the light absorbency of 405 nm was measured with the microplate reader.

  The result is a graph of TRACP activity in FIG. 9 (n = 5). Compared with control containing sRANKL and M-CSF, the extract from the azuki bean of the present invention EtEx. TRACP activity was significantly reduced under culture conditions containing 40. From this, the extract from azuki bean EtEx. 40 suggests the suppression of osteoclast differentiation even in the presence of M-CSF stimulation.

<TRACP staining>
For the purpose of TRACP staining, RAW264.7 cells contain sRANKL (100 ng / mL) and M-CSF (10 ng / mL), and azuki bean-derived extract EtEx. The culture was performed using DMEM medium according to the above-described culture conditions according to the presence or absence of 40 and the concentration of 10 μg / mL or 20 μg / mL. A staining kit (manufactured by Takara Bio Inc., TRACP & ALP double staining kit) was used for TRACP staining.

  The photograph of FIG. 10 is a cultured cell subjected to TRACP staining. The leftmost is a cultured cell containing no inducer or azuki bean-derived extract and is a control. In the presence of inducers of sRANKL and M-CSF, azuki bean-derived extract EtEx. If 40 is not present, reddish brown spots (black spots on the picture shown) appear frequently, indicating that TRACP activity has increased. That is, it is in a state where differentiation into osteoclasts is progressing. In contrast, azuki bean-derived extract EtEx. In the presence of 40, the number of spots decreased. In particular, it can be said to be concentration-dependent. This is consistent with the results of TRACP activity described above. That is, azuki bean-derived extract EtEx. 40 suggests the suppression of osteoclast differentiation even in an environment that promotes osteoclast differentiation.

[Mechanism of action of Azuki bean-derived extract on precursor osteoclasts]
As shown in the schematic diagram of FIG. 11, when RANKL (sRANKL) binds to RANK on the surface of osteoclasts, JNK, ERK, c-Fos, NF− start from TRAF6 (TNF receptor associated factor 6) in the same cell. AP-1 (activator protein 1), which is a transcription factor of heterodimeric protein, is formed from c-Jun and c-Fos via a signal transduction substance such as κB. NF-κB induces NFATc1 (cytoplasmic 1 protein nuclear factor of activated T-cell). The binding of AP-1 to NFATc1 further increases NFATc1. AP-1 and NFATc1 translocate into the nucleus with other nuclear translocation signals and attach to the promoter region to promote new transcriptional expression. Ultimately, it leads to the production of TRACP and cathepsin K, the expression of the receptor OSCAR, etc., and promotes differentiation into osteoclasts. As a result, bone metabolism (bone destruction) by osteoclasts is promoted.

  Therefore, if the pathway can be blocked, differentiation into osteoclasts can be suppressed, and bone destruction due to suppression of production of the causative substance of bone destruction that is the final target can be reduced.

  Based on the signal transduction in the precursor osteoclast of FIG. 11, the extract of the azuki bean of the present invention, EtEx. 40 verified which transmitter substance was acting on. RAW264.7 cells contained sRANKL (100 ng / mL) and M-CSF (10 ng / mL), and were cultured using the DMEM medium according to the culture conditions described above. Here, azuki bean-derived extract EtEx. 40 (20 μg / mL) was administered, and c-Fos and NFATc1 were confirmed by Western blot over time. FIG. 12 is a photograph of blotting at that time, and EtEx. 40 (−) is no administration, EtEx. 40 (+) is with administration. Β-actin was used as an index. An antibody manufactured by Cell Signaling Technology was used as the primary antibody for labeling c-Fos, and an antibody manufactured by abcam was used as the primary antibody for labeling NFATc1.

  As can be understood from the drawing, EtEx. Under the condition of 40 administrations, the expression level of c-Fos decreased. In synchronization with this, NFATc1 also decreased. Therefore, the formation of AP-1 can also be inhibited, which provides strong evidence that the amount of transcription can be reduced by suppressing the translocation of AP-1 and NFATc1 into the nucleus. From this knowledge, the extract of the azuki bean of the present invention, EtEx. 40 inhibits signal transduction in progenitor osteoclasts and acts transcriptionally. Therefore, it is suggested that it contributes to suppression of differentiation of precursor osteoclasts into osteoclasts.

[Summary of action mechanism of Azuki bean extract]
Considering the action of azuki bean-derived extract on osteoblasts and cells at the previous stage of osteoclasts, the action on both cells was clarified even though they were the same extract. For progenitor osteoblasts, differentiation into osteoblasts and calcification were clarified by promoting their differentiation. That is, it contributes to an increase in bone mass. At the same time, bone destruction is reduced by suppressing the differentiation of progenitor osteoclasts. Thus, the azuki bean-derived extract EtEx. No. 40 suitably has performance required for a bone metabolism regulator that promotes bone formation while suppressing bone destruction.

[Formulation example]
The extract derived from azuki bean broth of the present invention is considered to be effective as a bone metabolism regulator and effective in preventing and treating osteoporosis and the like. Therefore, the extract of azuki bean of the present invention, EtEx. A drug containing 40 was prototyped. The left column discloses the composition, and the right column discloses the blending ratio (in weight percent). Of course, the dosage form, blending amount, etc. are appropriate.

<Formulation Example 1: Tablet>
Cellulose 30.00
Calcium stearate 2.50
Silicon dioxide 1.50
Starch powder 49.40
Lactose 10.00
Azuki bean extract 6.60
(Total) 100.00

<Formulation Example 2: Capsule>
Lactose 50.00
Azuki bean extract 50.00
(Total) 100.00
The above ingredients are encapsulated in a known capsule.

[Application to food]
Azuki bean-derived extract EtEx. No. 40 is derived from azuki bean broth that has been eaten regularly by humans since ancient times. Therefore, it can be said that it is desirable to incorporate active ingredients from a meal by blending with food. Therefore, the azuki bean-derived extract (bone metabolism regulator) was blended into individual foods and actually cooked and manufactured. Below, the names of foods and beverages, their recipes (compositions): the left column, and the blending ratio (weight percent notation): the right column are disclosed. The foods and beverages listed are examples. Naturally, the addition object of the extract (bone metabolism regulator) derived from azuki bean broth of the present invention can be extended to other foods and beverages. Further, the blending ratio is not limited to the disclosed amount, and can be adjusted as necessary.

<1. Mizuyokan>
Agar 0.40
Water 45.35
Sugar 27.00
Raw Ann 27.00
Salt 0.10
Azuki bean extract 0.15
(Total) 100.00

<2. Yokan>
Agar 0.65
Water 24.00
Sugar 24.00
Raw Ann 48.00
Mizuame 3.20
Azuki bean extract 0.15
(Total) 100.00

<3. Anne Ogura>
Azuki 31.00
35.80 sugar
Salt 0.05
Water 33.00
Azuki bean extract 0.15
(Total) 100.00

<4. Dorayaki>
Soft flour 20.20
Super white sugar 17.00
Whole egg 17.50
Honey 2.50
Baking soda 0.20
Water 12.45
Azuki bean extract 0.15
Ogura Ann 30.00
(Total) 100.00

<5. Azuki Bean Mousse>
Raw bean paste 31.00
Powdered gelatin 1.40
Fresh cream 14.20
Egg white 10.50
Sugar 4.30
Water 38.45
Azuki bean extract 0.15
(Total) 100.00

<6. ice cream>
Yolk 11.00
Granulated sugar 17.80
Milk 53.20
Fresh cream 17.80
Vanilla flavor 0.05
Azuki bean extract 0.15
(Total) 100.00

<7. candy>
Mizuame 45.00
54.75 sugar
Azuki bean extract 0.15
(Total) 100.00

<8. caramel>
Mizuame 34.50
Sugar 19.70
Flour 4.85
Unsweetened condensed milk 34.60
Salt 0.20
Shortening 5.90
Azuki bean extract 0.25
(Total) 100.00

<9. Custard Pudding>
Milk 58.90
Egg 24.00
Sugar 17.00
Azuki bean extract 0.10
(Total) 100.00

<10. coffee jelly>
Coffee (liquid) 82.90
Sugar 8.30
Powdered gelatin 1.40
Coffee liqueur 0.30
Water 7.00
Azuki bean extract 0.10
(Total) 100.00

<11. Gummy>
Granulated sugar 39.00
Water 51.85
Gelatin 6.80
Citric acid 0.50
Fruit flavor 1.60
Azuki bean extract 0.25
(Total) 100.00

<12. Bavaroa>
Milk 47.40
Fresh cream 19.00
Egg yolk 12.60
Sugar 19.00
Gelatin 1.90
Azuki bean extract 0.10
(Total) 100.00

<13. marshmallow>
Mizuame 46.30
Sugar 50.90
Gelatin 2.70
Azuki bean extract 0.10
(Total) 100.00

<14. Castella>
Whole egg 34.30
Egg yolk 3.80
Sugar 34.30
Honey 4.80
Rice bran 1.90
Soft flour 18.00
Water 2.80
Azuki bean extract 0.10
(Total) 100.00

<15. Pancake>
Egg 19.70
Sugar 13.40
Milk 23.40
Baking powder 1.80
Soft flour 36.00
Butter 5.40
Salt 0.15
Vanilla flavor 0.05
Azuki bean extract 0.10
(Total) 100.00

<16. cookie>
Margarine 12.20
Shortening 12.20
Sugar 18.40
Whole egg 8.20
Weak flour 44.80
Powerful powder 4.10
Azuki bean extract 0.10
(Total) 100.00

<17. fruit juice>
Fruit juice 50.00
Fructose dextrose liquid sugar 10.00
Citric acid 0.30
Fruit flavor 0.20
Water 39.25
Azuki bean extract 0.25
(Total) 100.00

<18. soda drink>
Fructose glucose liquid sugar 11.00
Citric acid 0.20
Sodium citrate 0.05
Fruit flavor 0.20
Carbonated water 88.45
Azuki bean extract 0.10
(Total) 100.00

  Since the extract (bone metabolism regulator) derived from azuki bean broth of the present invention can be added to a wide variety of foods, it can be taken from a normal meal. Therefore, an effect for improving the quality of daily life (QOL) can be expected.

  Since the extract (bone metabolism regulator) derived from the azuki bean broth of the present invention can be added to a wide variety of food types, it can be easily taken from a normal meal. In addition, by increasing the active ingredient to obtain a drug, a drug that has an effect on improvement of abnormal bone metabolism such as osteoporosis and is effective for prevention and treatment can be obtained.

Claims (5)

The supernatant is separated from the broth produced by boiling the azuki bean in hot water, and the supernatant is adsorbed on a synthetic adsorbent, and then 10% to 40% ethanol aqueous solution is added to the adsorbed synthetic adsorbent. Azuki bean-derived extract obtained by elution ,
The azuki bean-derived extract promotes differentiation from osteoblasts to bone cells, and
The azuki bean-derived bone metabolism regulator is characterized in that the azuki bean-derived extract suppresses differentiation from precursor osteoclasts to osteoclasts . The supernatant is separated from the broth produced by boiling the azuki bean in hot water, and the supernatant is adsorbed on a synthetic adsorbent, and then 10% to 40% ethanol aqueous solution is added to the adsorbed synthetic adsorbent. Azuki bean-derived extract obtained by elution,
The azuki bean extract increases the transcription factors Runx2, OSX, and Dlx5 in the osteoblast in the transcriptional expression that proceeds when BMP-2 binds to the BMP receptor on the surface of the osteoblast. Promotes differentiation of the osteoblasts into bone cells, and
In transcriptional expression that proceeds by RANKL binding to RANK on the surface of progenitor osteoclasts, the Azuki bean-derived extract decreases the expression level of c-Fos in the progenitor osteoclasts, thereby inhibiting signal transduction, Azuki bean-derived extract suppresses the differentiation from precursor osteoclasts to osteoclasts
Azuki bean-derived bone metabolism regulator characterized by the above . A prophylactic or therapeutic agent for osteoporosis comprising the azuki bean bone metabolism regulator according to claim 1 or 2 . The supernatant is separated from the broth produced by boiling the azuki bean in hot water, and the supernatant is adsorbed on a synthetic adsorbent, and then 10% to 40% ethanol aqueous solution is added to the adsorbed synthetic adsorbent. A method for producing an azuki bean-derived bone metabolism regulator comprising, as an active ingredient, an azuki bean extract obtained by elution,
The azuki bean-derived extract promotes differentiation from osteoblasts to bone cells, and
The azuki bean-derived extract suppresses the differentiation from precursor osteoclasts to osteoclasts
A method for producing azuki bean bone metabolism regulator characterized by the above. The supernatant is separated from the broth produced by boiling the azuki bean in hot water, and the supernatant is adsorbed on a synthetic adsorbent, and then 10% to 40% ethanol aqueous solution is added to the adsorbed synthetic adsorbent. A method for producing an azuki bean-derived bone metabolism regulator comprising, as an active ingredient, an azuki bean extract obtained by elution,
The azuki bean extract increases the transcription factors Runx2, OSX, and Dlx5 in the osteoblast in the transcriptional expression that proceeds when BMP-2 binds to the BMP receptor on the surface of the osteoblast. Promotes differentiation of the osteoblasts into bone cells, and
In transcriptional expression that proceeds by RANKL binding to RANK on the surface of progenitor osteoclasts, the Azuki bean-derived extract decreases the expression level of c-Fos in the progenitor osteoclasts, thereby inhibiting signal transduction, Azuki bean-derived extract suppresses the differentiation from precursor osteoclasts to osteoclasts
A method for producing azuki bean bone metabolism regulator characterized by the above.

Images