KR20110122576A - A composition comprising diosgenin for differentiation osteoblast and use thereof - Google Patents

Abstract

PURPOSE: A composition containing diosgenin or pharmaceutically acceptable salt thereof is provided to promote proliferation, differentiation, and mineralization of osteoblast. CONSTITUTION: A composition for promoting osteoblast contains diosgenin or pharmaceutically acceptable salt thereof. Diosgenin is isolated from yam. The concentration of the diosgenin is 1-3 uM. A composition for preventing or treating bone metabolism contains diosgenin or pharmaceutically acceptable salt thereof. The bone metabolism is osteoporosis, bone metastatic lesion, rheumatic or degenerative arthritis, periodontal diseases, inflammatory bone absorption or Paget's disease. A food composition for improving bone function contains diogenin or pharmaceutically acceptable salt.

Description

A composition comprising diosgenin for differentiation osteoblast and use thereof

The present invention relates to a composition for promoting osteoblast differentiation comprising diosgenin or a pharmaceutically acceptable salt. The present invention also relates to a composition for preventing or treating bone metabolic disease and a composition for improving bone function, including diosgenin or a pharmaceutically acceptable salt.

Bone tissue is composed of cartilage and skeletal system, mechanical functions of supporting and muscle attachment, protecting the organs and bone marrow, and preserving them to maintain the homeostasis of calcium and phosphorus ions. These bone tissues are composed of cell substrates such as collagen and glycoproteins and various kinds of cells such as osteoblasts, osteoclasts and osteocytes. Osteoblasts derived from bone marrow stromal cells play a major role in bone formation. Osteoclasts derived from hematopoietic stem cells are responsible for the uptake of the broken aging bone, thereby maintaining the bone remodeling by balancing the osteoblasts and osteoclasts.

Bone metabolic disease occurs due to a breakdown of the balance between osteoclasts and osteoblasts in vivo. Osteoporosis is an example of a bone metabolic disease. Osteoporosis is a disease in which osteoclasts are more active than osteoblasts, resulting in a decrease in total bone mass and a breakage of bone even with a slight impact. In addition, metastatic cancer, rheumatoid arthritis, degenerative arthritis, and periodontal disease causing destruction of alveolar bone due to bacterial infection. Bone metabolic diseases, such as osteoclasts are excessively activated, resulting in the easy destruction of bone.

Osteoclast progenitors are hematopoietic cells of the monocyte / macrophage lineage originating in the bone marrow. Osteoclast precursors are differentiated into osteoclasts by growth factors and cytokines produced in the bone marrow (Roodman G. D., Endocr. Rev., 17, 308-332 (1996)). Osteoclasts act to destroy or absorb bone.

Osteoblasts originate from mesenchymal stem cells, and mineralization, including calcium, formed by the differentiation of osteoblasts not only maintains bone strength, but also plays an important role in the homeostasis of calcium and hormone metabolism throughout the body. Doing. Calcium formation by the differentiation of osteoblasts is regulated by vitamin D and parathyroid hormone, etc. In the cell, bone morphogenetic protein (BMP), Wnt, MAP kinase, calcineurin-calmodulin kinase Alkaline phosphatase (ALP), which is involved in the differentiation of osteoblasts, is synthesized at the early differentiation stage by cross-talk of various signaling systems such as calcinurin-calmodulin kinase, NF-κB and AP-1. After that, osteopontin, osteocalcin, type I collagen, and the like, which are involved in mineralization, are synthesized, and bone formation by osteoblast differentiation is known.

However, these human bones maintain homeostasis through the formation and remodeling process. Remodeling occurs in bone multicellular units (BMUs) consisting of osteoblasts and osteoclasts, and these cells are osteoblasts and bones, respectively. It is known to play an important role in the absorption process. Damage to the cooperative system between the two cells in this process leads to various bone metabolism diseases including osteoporosis. However, there is no effective treatment for cure to date and prevention is emphasized.

Recently, efforts to produce osteoblasts under in vitro culture conditions and to use them to strengthen bone tissue function or to treat injuries are increasing rapidly. Ascorbic acid, beta-glycerophosphate, and dexamethasone, which are currently used as osteogenic supplements, have been reported to be effective in inducing osteoblast differentiation (Karp JM et al., 2006 ; Cao T et al., 2005; Bielby RC et al., 2004; Sottile V et al., 2003), current activin, Bone Morphogenic Protein (BMP), inhibin, growth / differentiation factor Transforming Growth actor-beta (TGF-beta) subfamily, including / differentiation factor (GDF), is known to play an important role in the formation and maintenance of bone tissue, especially in culture of BMP2 or BMP4. Induction of differentiation into osteoblasts has been reported when added to the culture medium. However, the understanding of osteoblast differentiation mechanism and differentiation induction technology for securing cellular therapeutics that can perform clinically excellent function is still weak. To overcome this problem, it is necessary to understand factors and mechanisms involved in osteoblast differentiation. It is important. In addition, it can be said that it is very important to find a substance that modulates their activity and apply it to the differentiation induction technology.

Under this background, the present inventors have made diligent efforts to find a substance that not only proliferates osteoblasts but also induces differentiation. As a result, the present inventors confirmed that diosgenin extracted from yam is effective for differentiation and proliferation of osteoblasts. Was completed.

It is an object of the present invention to provide a composition for promoting osteoblast differentiation comprising diosgenin or a pharmaceutically acceptable salt.

Another object of the present invention is to provide a composition for preventing or treating bone metabolic diseases, including diosgenin or a pharmaceutically acceptable salt.

Still another object of the present invention is to provide a food composition for improving bone function, including diosgenin or a pharmaceutically acceptable salt.

As one aspect for achieving the above object, the present invention provides a composition for promoting osteoblast differentiation comprising a diosgenin or a pharmaceutically acceptable salt.

As used herein, the term "diosgenin" is a saponin-based compound having the following chemical structure, and is a substance known to be effective in regulating HER2 expression and cardiovascular diseases in anti-infection, anti-allergic, anti-flu, and breast cancer. Although it has been reported to have an effect of lowering the level of LDL cholesterol in serum, there is no known effect on osteoblast differentiation and proliferation. Diosgenin can be extracted from a variety of plants, preferably from yam (yam). Diosgenin may also be artificially synthesized using conventional techniques known in the art.

As used herein, the term "pharmaceutically acceptable salts" refers to salts prepared by conventional methods and can be used without limitation by methods known to those skilled in the art. "Pharmaceutically acceptable salts" include, but are not limited to, salts derived from the following pharmacologically or physiologically acceptable inorganic and organic acids and bases. Examples of suitable acids include hydrochloric acid, bromic acid, sulfuric acid, nitric acid, perchloric acid, fumaric acid, maleic acid, phosphoric acid, glycolic acid, lactic acid, salicylic acid, succinic acid, toluene-p-sulfonic acid, tartaric acid, acetic acid, citric acid, methanesulfonic acid, formic acid , Benzoic acid, malonic acid, naphthalene-2-sulfonic acid, benzenesulfonic acid, and the like. Salts derived from suitable bases may include alkali metals such as sodium, alkali cometals such as magnesium, ammonium and the like.

As used herein, the term "osteoblasts" refers to cells of a cuboidal cell forming bone, and cells that have bone formation and remodeling functions. Bone is composed of bone cells and bone matrix, bone cells are divided into osteoblasts and osteoclasts. Osteoclasts dissolve and break down existing bone constituents to absorb bone matrix for continued growth of bones. Osteoblasts form lattice-like collagen in the parts decomposed by osteoclasts, and calcium, magnesium and phosphorus Attach it to create a new bone matrix. The composition of the present invention can promote bone differentiation by promoting differentiation of osteoblasts.

As used herein, the term "yam" refers to a vine perennial herb of Liliaceae subfamily, about 1m high, with a long and thin stem. The leaves face or turn, which is a triangle with a heart shape under the leaves and a sharp tip. Petioles are long and have rounded shoots on the axils. Flower is white in June ~ July, and it is watery inflorescence. Fruits are capsules, with three wings and seeds with round wings. It is native to China and grows wild in the mountains. Other departments include D. japonica, D. tokoro, and D. quinqueloba.

Preferably, the concentration of diosgenin in the composition for promoting osteoblast differentiation comprising the diosgenin or the pharmaceutically acceptable salt of the present invention includes, without limitation, a concentration capable of differentiating osteoblasts, preferably 0.01 to 10 μM. , More preferably 1 to 3 μM, most preferably 1 μM.

According to a specific embodiment of the present invention, the present inventors treated the composition by culturing osteoblasts derived from mouse that the composition comprising diosgenin or a pharmaceutically acceptable salt has an effect of promoting the differentiation of osteoblasts. As a result of proliferation, collagen synthesis, and alkaline phosphatase activity related to osteoblastization, when the concentration of the composition containing diosgenin or a pharmaceutically acceptable salt was 10 μM or more, cytotoxicity was observed regardless of the culture period. It was confirmed that the concentration of the composition promotes the proliferation of osteoblasts without toxicity in the range of 1 to 3 μM, preferably 1 μM, and increases the amount of expression of intracellular proteins related to osteoblastization. .

In another aspect, the present invention relates to a composition for the prevention or treatment of bone metabolic diseases, including diosgenin or a pharmaceutically acceptable salt.

In the present invention, the term "diogeninin" is the same as described above.

In the present invention, the term "bone metabolic disease" includes a disease having a problem in bone formation due to the lack of osteoblasts, such as, without limitation, for example, bone destruction by interleukin-1 (IL-1) due to lack of estrogen and Inflammatory diseases, and the like, osteoporosis due to excessive osteoclasts (osteoprosis), bone metastatic lesions (primary), tumors formed in bone, rheumatoid or degenerative arthritis, periodontal disease Pathological bone diseases such as inflammatory alveolar bone resorption diseases, inflammatory bone resorption diseases and Paget's disease, and the like, including but not limited to the bone metabolic diseases.

As used herein, the term "prevention" refers to any action that inhibits or delays the onset of the disease by administration of a composition comprising a diosgenin or a pharmaceutically acceptable salt according to the present invention.

As used herein, the term "treatment" refers to any action that improves or beneficially alters the symptoms of the disease by administration of a composition comprising a diosgenin or a pharmaceutically acceptable salt according to the invention.

Preferably, the concentration of diosgenin in the composition for preventing or treating bone metabolic diseases including the diosgenin or a pharmaceutically acceptable salt of the present invention includes, without limitation, a concentration capable of differentiating osteoblasts, preferably 0.01 to 10 μM, more preferably 1 to 3 μM, most preferably 1 μM.

According to a specific embodiment of the present invention, the present inventors found that a composition containing diosgenin or a pharmaceutically acceptable salt is effective for the prevention and treatment of bone metabolic diseases, dios, which is a hemp extract in culture of mouse-derived MC3T3-E1 osteoblasts. As a result of confirming cell proliferation through MTT analysis after treating and culturing the composition comprising the gene composition, the composition containing diosgenin or a pharmaceutically acceptable salt is in the concentration range of 1 to 3 μM, preferably 1 When treated with a concentration of μM was found to promote osteoblast differentiation (Fig. 1), it was confirmed to promote collagen synthesis and secretion activity involved in the formation of osteogenic nodules (Fig. 2), intracellular osteoblastization It was confirmed that significantly increased the amount of protein expressed in relation to (Fig. 7). Therefore, it can be seen that diosgenin or a pharmaceutically acceptable salt thereof is useful for preventing and treating bone metabolic diseases.

Diosgenin or salts thereof of the present invention may further comprise a suitable carrier, excipient or diluent according to conventional methods.

As used herein, the term "carrier" refers to a carrier, excipient, or diluent that does not significantly irritate an organism and does not inhibit the biological activity and properties of the administered compound.

Examples of carriers, excipients and diluents that can be included in the composition of the present invention include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia rubber, alginate, gelatin, calcium phosphate, calcium silicate, Cellulose, methylcellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil.

Compositions containing the diosgenin or salts thereof according to the present invention may be prepared in the form of powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols, oral formulations, external preparations, suppositories, or sterile injectable solutions, respectively, according to conventional methods. It can be formulated and used in the form.

Specifically, when formulated, it may be prepared using diluents or excipients such as commonly used fillers, extenders, binders, wetting agents, disintegrating agents, surfactants, and the like. Solid preparations for oral administration include tablets, pills, powders, granules, capsules, and the like, and the solid preparations may include at least one excipient such as starch, calcium carbonate, sucrose in the extract. ), Lactose, gelatin and the like can be mixed. In addition to simple excipients, lubricants such as magnesium stearate, talc can also be used. Liquid preparations for oral use include suspensions, solvents, emulsions, and syrups, and include various excipients such as wetting agents, sweeteners, fragrances, and preservatives, in addition to commonly used simple diluents such as water and liquid paraffin. Can be. Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized preparations and suppositories. As the non-aqueous solvent and suspending agent, propylene glycol, polyethylene glycol, vegetable oil such as olive oil, injectable ester such as ethyl oleate, and the like can be used. As the base of the suppository, witepsol, macrogol, tween 61, cacao butter, laurin butter, glycerogelatin and the like can be used.

In another aspect, the present invention relates to a method for treating or preventing bone metabolism disease, comprising administering a composition comprising diosgenin or a pharmaceutically acceptable salt.

As used herein, the term "administration" refers to introducing a composition of the present invention to a patient in any suitable manner, wherein the route of administration of the composition of the present invention is via oral or parenteral various routes as long as the target tissue can be reached. It may be administered, and specifically, in a conventional manner via oral, rectal, topical, intravenous, intraperitoneal, intramuscular, intraarterial, transdermal, nasal, inhalation, intraocular or intradermal routes. The treatment method of the present invention includes administering a composition of the present invention in a pharmaceutically effective amount. It will be apparent to those skilled in the art that a suitable total daily dose may be determined by the practitioner within the correct medical judgment. The specific therapeutically effective amount for a particular patient may be based on the specific composition, including the type and severity of the reaction to be achieved, whether or not other agents are used in some cases, the age, weight, general health, sex and diet of the patient, time of administration, It is desirable to apply differently depending on the route of administration and the rate of release of the composition, the duration of treatment, and the various factors and similar factors well known in the medical arts, including drugs used with or concurrent with the specific composition. Therefore, the effective amount of the composition suitable for the purpose of the present invention is preferably determined in consideration of the above matters.

In addition, the method for preventing or treating bone metabolic disease of the present invention is applicable to any animal in which bone metabolic disease occurs, and animals include humans and primates, as well as domestic animals such as cattle, pigs, sheep, horses, dogs, and cats.

 As another aspect, the present invention provides a food composition for improving bone function comprising diosgenin or a pharmaceutically acceptable salt.

The composition containing the diosgenin or a pharmaceutically acceptable salt thereof of the present invention may be used in various ways, such as drugs, foods and beverages for the prevention of bone metabolism-related diseases. Foods to which the extract of the present invention may be added include various foods, for example, beverages, gums, teas, vitamin complexes, dietary supplements, and the like, which are pills, powders, granules, tablets, tablets, capsules or beverages. Available in form.

At this time, the amount of the extract in the food or beverage, in general, may be added to 0.01 to 15% by weight of the total food weight in the case of the health food composition of the present invention, 0.02 to 10g, based on 100ml for the health drink composition, preferably It may be added at a ratio of 0.3 to 1g.

The health beverage composition of the present invention has no particular limitation on the liquid component except for containing the extract as an essential ingredient in the indicated ratio, and may contain various flavors or natural carbohydrates as additional ingredients, such as ordinary drinks. Examples of the above-mentioned natural carbohydrates include monosaccharides such as glucose, fructose and the like; Disaccharides such as maltose, sucrose and the like; Polysaccharides such as dextrin, cyclodextrin; Conventional sugars such as and the like and sugar alcohols such as xylitol, sorbitol, and erythritol. As flavoring agents other than those described above, natural flavoring agents (tauumatin, stevia extract (e.g., Rebaudioside A, glycyrrhizin, etc.) and synthetic flavoring agents (saccharin, aspartame, etc.) can be advantageously used. The proportion of natural carbohydrates is generally about 1-20 g, preferably about 5-12 g per 100 ml of the composition of the present invention.

In addition to the above, the composition of the present invention includes various nutrients, vitamins, minerals (electrolytes), flavors such as synthetic flavors and natural flavors, coloring and neutralizing agents (such as cheese and chocolate), pectic acid and salts thereof, alginic acid and salts thereof. , Organic acids, protective colloidal thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohols, carbonation agents used in carbonated beverages, and the like. The compositions of the present invention may also contain pulp for the production of natural fruit juices and fruit juice beverages and vegetable beverages. These components can be used independently or in combination. The proportion of such additives is not so critical, but is generally selected in the range of 0 to about 20 parts by weight per 100 parts by weight of the composition of the present invention.

There is no particular limitation on the other ingredients other than the composition having an effect on the nervous system diseases including the diosgenin or a pharmaceutically acceptable salt thereof as essential ingredients that may be included in the functional food composition of the present invention, and various kinds of foods such as conventional foods. Herbal extracts, food supplements or natural carbohydrates and the like may be included as additional ingredients.

In addition, as mentioned above, food supplements, which may further add food additives, include food additives conventional in the art, such as flavoring agents, flavoring agents, coloring agents, fillers, stabilizers, and the like. .

Examples of such natural carbohydrates include monosaccharides such as glucose, fructose, and the like; Disaccharides such as maltose, sucrose and the like; And conventional sugars such as polysaccharides such as dextrin, cyclodextrin, and sugar alcohols such as xylitol, sorbitol, and erythritol. As flavoring agents other than those mentioned above, natural flavoring agents (tauumatin, stevia extract (e.g., rebaudioside A, glycyrrhizin, etc.) and synthetic flavoring agents (saccharin, aspartame, etc.) can be advantageously used. .

In addition to the above, the functional food composition of the present invention includes various nutrients, vitamins, minerals (electrolytes), flavors such as synthetic and natural flavors, coloring and neutralizing agents (such as cheese and chocolate), pectic acid and salts thereof, alginic acid And salts thereof, organic acids, protective colloidal thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohols, carbonation agents used in carbonated beverages, and the like. Others may contain pulp for the production of natural fruit juices and fruit juice drinks and vegetable drinks. These components can be used independently or in combination.

The present invention confirms the differentiation and calcification of osteoblasts involved in bone formation by promoting the proliferation, differentiation and mineralization of osteoblasts, and the composition comprising diosgenin isolated from hemp, which is useful for promoting osteoblast differentiation. Can be. In addition, the composition may be used as a composition for the prevention and treatment of bone metabolic diseases, it may be used as a food composition for improving bone function.

Figure 1 A shows the effect of diosgenin on osteoblast maturation, B shows the synthesis of proteins in MC3T3-E1 cells. Diosgenin was dissolved in DMSO (0.14 M) as a vehicle and treated in cells. (A) proliferation of cells AlamaBlue  Measurements were made using kits (Invitrogen) or MTS assay (Promega). There was no cytotoxicity in the vehicle compared to normal osteoblast differentiation normal medium (OSM). Diosgenin promoted the proliferation of osteoblasts up to 6 days in the 0.01-5 μM concentration range. Mean ± SEM (n = 6), abc: Analysis by single method ANOVA followed Tukey test method for mean comparison. Compared with the comparison criteria, ^* P <0.05, ^** P <0.01 and ^*** <0.001. (B) Cell protein activity was measured by BCA assay (Promega) and showed the same pattern as cell proliferation on days 5 and 10. Mean ± SEM (n = 4), abc: Tukey test, single method ANOVA, P <0.05.
Figure 2 is a diagram showing the quantification of collagen (B) of cells (A) and media by diosgenin treatment. Cells were treated with diosgenin at various concentrations, DMSO with vehicle, and treated for 1 day, 5 days and 15 days. Collagen expression of cells and media was measured by colorimetric method using Picro-Sirius Red. The collagen concentration of osteoblasts was expressed by unit standardizing the amount of protein in the cells. Mean values with different superscripts were due to ANOVA according to Tukey's post test (n = 8, mean ± 8), indicating different treatment concentrations of diosgenin at p <0.05 (OSM = osteoblast differentiation normal medium, vehicle = DMSO).
Figure 3 shows the cell substrate collagen staining by diosgenin treatment. The effect of diosgenin on the formation of collagen scaffolds for ossification was measured via staining and is represented by images (top) in the form of phase contrast microscopy and the cell layer (bottom) of each culture dish. Substrate collagen, which appears in red staining, is stimulated by dose and time dependent diosgenin treatment and is prominent on days 5 and 15. The image presented is representative of four wells per group.
4 is a diagram showing alkaline phosphatase activity in cells and media by diosgenin treatment. Cells were treated with various concentrations of diosgenin when it was time to sufficiently cover the cell culture plate with a monolayer, and cultured 1, 5 and 15 days after treatment with DMSO with vehicle. Alkaline phosphatase in cells and medium was measured by colorimetric method using PNPP as substrate. Alkaline phosphatase activity was normalized to cellular protein concentration. Mean values with different superscripts were by ANOVA according to Tukey's post test (n = 8, mean ± 8), indicating different treatment concentrations of diosgenin at p <0.05 (OSM = Coarse Differentiated Normal Medium, Comparative Criteria) = DMSO).
5 is a result of staining alkaline phosphatase activity of the cell substrate by diosgenin treatment. The effect of diosgenin on alkaline phosphatase activity in cell substrates is shown through staining of enzyme active products, shown in phase contrast microscopy images (top) and the cells placed in each culture dish (bottom). Cells were cultured in 6-well plates and staining was incubated in AS-MX phosphate solution with Fast Red salt substrate. Alkaline phosphatase activity of substrates stained in red is stimulated by diosgenin activity, depending on time and concentration, and is prominent on days 5 and 15.
Figure 6 is a diagram showing Alizarin and von Kossa staining Ca deposition (bone nodules) by diosgenin treatment in MC3T3-E1 cell line. Ca deposition of the extracellular matrix for ossification of the substrate was measured by Alizarin red S dye binding to Ca, and silver nitrate dye binding to P of the cell membrane substrate. Treatment of low concentrations (0.01-1 μM) of diosgenin resulted in increased ossification at 25 days of culture compared to the control group.
Figure 7 shows the effect of the treatment of bone matrix protein, bone specific transcription factor proteins Runx2 and osteopontin by the diosgenin treatment. (A) Expression of Bone Matrix Proteins and Early Osteoblast Marker Proteins, Type I Collagen and Alkaline Phosphatases were analyzed by Western blot analysis on days 3 and 10 after diosgenin treatment. (B) Runx2 and osteopontin were immunoprecipitated and expression of these proteins was analyzed by Western blot analysis 10 days after diosgenin treatment. The results of two repeated sets of immune blots and immune precipitation blots are shown.

Hereinafter, the present invention will be described in more detail with reference to the following examples. These examples are only for illustrating the present invention more specifically, but the scope of the present invention is not limited to these examples.

Example 1 Preparation of Materials

Antibodies against the desired protein (type I collagen, alkaline phosphatase, osteopontin and Runx2) were purchased from Santa Cruz, and antibodies for loading control (GAPDH) were purchased from Cell Signaling. All chemical and cell culture solutions were purchased from Sigma (St. Louis, MO) and Gibco, Invitrogen unless otherwise noted.

Example 2: Cell Culture

MC3T3-E1 cells, mouse osteoblasts, were inoculated at a concentration of 1 × 10 ⁴ cells / mL to give 10% fetal bovine serum (Gibco, USA), 1 mM sodium pyruvate (US, Sigma), 100 unit / L penicillin and 100 mg / L Incubation was carried out at 37 ° C., 5% CO ₂ conditions in a conventional growth culture medium containing α-minimum essential medium (Gibco, USA), added with streptomycin (Gibco). When the cells were sufficiently grown to 80%, the cells were cultured in differentiation medium to which 3 mM sodium phosphate (Sigma, USA) and 50 μg / mL ascorbic acid (Sigma, USA), which are osteoblast differentiation medium, were added. Diosgenin was dissolved in DMSO and ethanol and treated with cells at a concentration of 0-10 μM, and the medium was exchanged every two days. Cells cultured in the differentiation medium not treated with diosgenin were used as a control for osteoblastization (OSM).

Example 3: Cell Proliferation Assay

Cell proliferation was determined by the degree of reduction of synthetic metabolic substrate resazurin to resorufin (Invitrogen, Almer Blue kit) and MTT measurement (Promega) method according to the manufacturer's instructions. Specifically, cells (1 × 10 ⁴ cells / well) were incubated in growth medium for 24 hours at 37 ° C. supplied with 5% CO ₂ . When grown to about 90%, diosgenin at various concentrations (0, 0.01, 0.1, 1, 2, 3, 5 and 10 μΜ) was treated with cells and incubated for 1, 3 and 6 days. According to each treatment time, Rezazurin reagent or MTT was treated to culture cells and samples and incubated at 37 ° C. for 4 hours. Absorbance was measured using an optical 96 well plate reader (Tecan Austrica GmbH, Sunrise Absorbance Reader) at 570 nm using 600 nm as the reference wavelength.

Example 4: Collagen Measurement

4-1 Measurement of collagen concentration in cells and media

The amount of collagen in the cells and medium was measured using the Picro-Sirius red method. Cells were hydrolyzed at 4 ° C. after washing in PBS with 0.5 M acetic acid. 50 μl each of standard, cell fraction and media samples were added to 96 well plates, and then dried at 37 ° C. humidity for 24 hours. After drying, the cells were washed with 200 μl distilled water, and then 100 μl of 0.1% Picro-Sirius red dye dissolved in picric acid was added to each well. The samples and standards were then stained at 37 ° C. for 1 hour. After the reaction, samples and standards were washed three times with the addition of 200 μl of 10 mM hydrochloric acid to remove unbound dye. Finally, the bound collagen was dissolved by adding 200 μl of 0.1 M sodium hydroxide. Samples and standards were transferred to 96 well plates and absorbance was analyzed at 540 nm wavelength.

4-2 Cell Substrate Collagen Staining

Collagen synthesis was measured by Van Gieson staining. The cells were washed with distilled water and then completely dried. 2% formaldehyde was added and fixed at 4 ° C. for 15 minutes. The cells were then washed twice with distilled water and reacted with Van Gieson's reagent for 15 minutes. Excess dye was removed by washing with distilled water at least three times. Cell substrate collagen was stained red.

Example 5: ALP Activity Assay

ALP Activity of 5-1 Cells and Media

ALP activity of cells and medium was measured by culturing cells treated with 0, 1,3,5 and 10 μM diosgenin and control cells for 1, 5 and 15 days. After washing the cells with PBS, 25 mM Tris-Cl (pH 7.6), 150 mM NaCl, 1% Nonidet P-40 as proteolytic inhibitor. Cells were lysed using 0.1% SDS and 1 mM PMSF. Lysed cells were sonicated on ice for 30 seconds at 50% intensity. The ultrasonic digest was centrifuged at 12000 g for 15 minutes at 4 ° C. The supernatant was stored at 80 ° C. until analysis. Media was also recovered to measure ALP secretion. ALP activity of lysed cells and media was analyzed using p-nitrophenyl phosphate as substrate and absorbance was measured at a wavelength of 405 nm as described above. Protein concentration was measured by BCA analysis (Pierce). The ALP activity of the cells and medium is shown in nmol PNPP / mg protein / min and nmol PNPP / mL medium / min, respectively.

5-2 Cell Substrate ALP Staining

Cultured cells were washed with PBS and fixed in 2% formaldehyde. Naphthol As-Mx sodium diphosphate was used as a substrate, and N, N-dimethylformamide and fast red salt were used as dyes and stained at 37 ° C. for 30 minutes or until yellow color appeared. After washing with PBS, cells were taken. Alkaline phosphatase active products represent enzyme active products, which are dyed red.

Example 6: Extracellular Matrix Calcium Deposition Assay

6-1 Alizarin red S dyeing

To measure Ca deposition of extracellular matrix for bone nodule formation, the cell substrate was stained using an Alizarin red S stain that binds to Ca in the matrix. Cells were seeded (1 × 10 ⁴ cells / mL) in 48 well plates and incubated with treatment with 0, 0.01, 0.1, 1, 3 and 10 μM of diosgenin. At 15 and 25 days of differentiation, cells were washed twice with PBS and fixed with 2.0% formaldehyde. The cells were stained with 40 mM Alizarin red S solution for 45 minutes at room temperature and washed twice with distilled water.

6-2 von Kossa Dyeing

Due to the co-precipitation of phosphate ions and Ca in the substrate, ossification of the cultured cell nodules was measured using von Kossa staining. Cells were washed in PBS and then fixed in 2.0% formaldehyde for 15 minutes. After washing three times with distilled water, and treated with 3% silver nitrate at room temperature and reacted under UV light for 1 hour. After washing with distilled water, images of the stained cells were taken using a microscope.

Example 7: Western Blot Analysis

MC3T3 cells were incubated in a 10 cm dish and treated with 0, 0.01, 0.1, 1, 3 and 5 μM of diosgenin for 3 and 10 days. After cell recovery, use lysis buffer (150 mM NaCl with 1% proteolytic inhibitor, 1% Nonidet P-40, 1% sodium deoxycholate, 0.1% SDS, 25 mM Tris-Cl, pH 7.6) It was dissolved. Total cell lysates were separated by SDS-PAGE using a 12% gel and transferred to PVDF membranes (Millipore, Immobilon-P, USA). Fixed in 5% nonfat milk and reacted with primary antibodies against type I collagen and ALP (Santa Cruz Biotechnology). The secondary antibody (Santa Cruz Biotechnology) conjugated with horseradish peroxidase was then reacted. The membrane was developed using Super Signal West Pico Chemiluminescence detection reagent (Pierce Biotechnology, Rockford, IL).

Example 8 Immunoprecipitation of Runx2 and Osteopontin

Osteopontin and Runx2 were precipitated by reacting 1 μg of anti-Runx2 and anti-osteopontin antibodies with 100 μg of total protein at 4 ° C. for one day (Santa Cruze Biotechnology). Antigen-antibody complexes were added to Protein A / G resin and incubated for 2 hours at room temperature to facilitate the reaction. The bound protein was washed five times with IP buffer (25 mM Tris, 150 mM NaCl, pH 7.2). Finally, the binding protein was eluted using Ig elution buffer (0.2 M glycine-HCl, pH 2.8), separated by SDS-PAGE, and transferred to PVDF membrane. Membranes were immunoblotted using antibodies as indicated and proteins were visualized as described above.

Example 9: Statistical Analysis

The results were analyzed using software SPSS 17.0. Cell proliferation values, ALP activity and collagen synthesis values are expressed as mean ± SE values. Results analysis was performed using single path ANOVA and Tukey's HSD test was used as post hoc test when detecting significant among the treated groups at P <0.05 level.

Experimental Example 1: Analysis of Cell Proliferation

MC3T3-E1 treated with stepwise concentrations (0-10 μM) of diosgenin for 1, 3 and 6 days were analyzed for cellular stock using the Almer Blue assay. Preliminary analysis was performed to compare survival / proliferation rate using DMSO and ethanol as diosgenin solvents. These solvents did not show any toxicity in osteoblasts, but since diosgenin dissolved in DMSO showed higher cell proliferation activity than diosgenin dissolved in ethanol, DMSO was used as a solvent for future experiments (results not shown). There was no vehicle effect (DMSO) compared to the normal diosgenin control (OSM). Diosgenin has been shown to promote osteoblast proliferation in the concentration range of 0.01-5 μM.

In particular, it was confirmed that there is an excellent promoting effect in the concentration range of 1-2 μM (FIG. 1A) on days 1, 3 and 6 of the culture period. No toxic effects were observed at all concentrations when treated for 1 day, but at 10 μM when treated for 3 and 6 days. In addition, as a result of measuring the protein synthesis of osteoblasts during bone matrix maturation period (5-10 days), the promotion of protein synthesis by diosgenin increased the proliferation of cells after 5 and 10 days treatment at a diosgenin concentration range of 2-4 μM. It appeared in a pattern similar to (FIG. 1B). At day 5, during the initial substrate maturation, a decrease in protein synthesis was observed in the DMSO treated cell population. This temporary decrease may be due to cell regulation during this period, and the increase in protein synthesis by diosgenin recovered on day 10.

Experimental Example 2: Collagen Synthesis and Secretion

Type I collagen is a collagen protein that is deposited during the mineralization of the substrate into the inner portion of the extracellular matrix, which serves as a support that is a hydroxyapaptite crystal. Thus, the formation of collagen extracellular matrix is a prerequisite for bone formation.

As shown in FIGS. 2A and 2B, the cells (synthetic collagen) showed higher collagen synthesis than the control group (normal medium and DMSO medium) between diosgenin concentration 1-3 μM (FIG. 2A), with similar tendency in culture It was also shown in the secreted collagen concentration of (Fig. 2B). In addition, the collagen concentration of the cells and the culture medium showed a tendency to increase in proportion to time (Figs. A & B). Diosgenin was compared with normal OSM group when treated for 1, 5 and 15 days, significantly promoting collagen synthesis and secretion in concentration range of 1-3 μM in both cells and cultures. It was found that the dose-dependent decrease.

In addition, the effect of various concentrations of diosgenin on the extracellular matrix was confirmed by staining the cell substrate after 1, 5 and 15 days incubation. As a result, the cell substrate collagen level expressed by the intensity of the red dye in the cell layer was higher when treated with 1-3 μM diosgenin compared to the normal OSM group (FIG. 3). Collagen staining was also shown to accelerate time-dependently with respect to diosgenin treatment within 15 days of culture.

Experimental Example 3: ALP Activity of Cells and Medium

ALP is a representative osteoblast differentiation marker that is synthesized largely dependent on the synthesis and processing of type I collagen. In the present invention, ALP activity was measured in cells (FIG. 4A) and medium (FIG. 4B) for 1, 5 and 15 days, which are differentiation and proliferation periods of osteoblasts.

As a result, it was confirmed that the ALP activity of the cells (the degree of ALP synthesis by the cells) was significantly increased after 5-15 days in the 1-3 μM concentration range compared to the normal osteoblast differentiation medium (OSM) or the vehicle control. It was. There was no negative vehicle effect compared to normal osteoblast differentiation medium, and as a result, the ALP activity of the cells was much increased at 15 days (FIG. 4A).

ALP is mainly a cell membrane adhesion protein and thus secreted, ALP activity of the medium was also measured. As a result, extracellularly secreted media ALP activity was increased under conditions treated with low concentrations (1-3 μM) of diosgenin at days 1, 5 and 15 (FIG. 4B).

The effect of diosgenin treatment of extracellular stromal cell layer ALP activity was measured by ALP staining. As a result, ALP activity was shown to be promoted in a time-dependent manner with respect to the treatment of diosgenin on days 1, 5 and 15 of the culture period (FIG. 5). There was no significant difference in ALP activity by diosgenin after 1 day treatment, but weak staining was observed at 10 μM of diosgenin concentration. The proliferation result was not observed to be toxic to treatment for one day, but based on these results, it is clear that ALP activity is already compromised at an early stage. The concentration of diosgenin promoting ALP activity was found to be 1-3 μM concentration on days 5 and 15 compared to normal differentiation medium and vehicle control.

Experimental Example 4: Ossification Analysis

To determine the effect of diosgenin on ossification, it was analyzed whether diosgenin treatment could increase Ca deposition in culture.

As a result, Ca deposition of the extracellular matrix for osteogenic nodule formation was performed by Alizarin red S staining (FIG. 6A) binding to Ca ions and von Kossa staining using silver nitrate binding to normal proportions of phosphoric acid in Ca deposition (FIG. 6B). Confirmed. Calciumated nodules were stained bright red by Alizarin S staining and brown by von Kossa staining. Osteoblastization of MC3T3 cells was time-dependent within 25 days of culture, and dose-dependently promoted ossification of extracellular matrix at low concentrations (0.01-1 μM), and high concentrations (> 5 μM) No Ca deposition was observed in Alizarin Red S or von Kossa staining.

Experimental Example 5: Expression of Bone Related Protein

Similar to previous experiments, MC3T3 cells were incubated for 3 and 10 days after treatment with various concentrations of diosgenin. Cells were harvested, marked for treatment, and lysed, and homogenized whole cells were measured by Western blot analysis of bone matrix protein type I collagen and ALP. The expression patterns of ALP and type I collagen protein increased dose-dependently up to 1 μM, the low treatment concentration of diosgenin, but decreased at 3 and 10 days at high concentrations (3-5 μM). This pattern was confirmed in both collagen and ALP expression levels synthesized with cells.

Since osteoblast marker protein is regulated by Runx2, the major bone specific transcription factor is responsible for osteoblast marker gene transcription for osteoblast differentiation, while osteopontin is a Runx2-dependent osteoblast protein, so Runx2 and Osteo by treatment with diosgenin The expression of the opontin protein was measured primarily by Western blotting after immunoprecipitation of the cellular protein.

As a result, after 10 days of treatment, immunoprecipitated Runx2 protein expression was promoted at low treatment concentration of diosgenin (up to 0.1 μM) and decreased in the 1-3 μM range. The expression of osteopontin protein is regulated by Runx2, regulated by another osteogenic marker protein and observed in the same pattern as Runx2 expression.

These results indicate that diosgenin promotes osteoblast differentiation and induces the expression of bone-related proteins, which suggests that diosgenin may have therapeutic effects in diseases related to bone formation as well as osteoblast differentiation. The result suggests that.

Claims (10)

Diosgenin (diosgenin) or a composition for promoting osteoblast differentiation comprising a pharmaceutically acceptable salt.
The composition for promoting osteoblast differentiation according to claim 1, wherein the diosgenin is extracted from yam.
The composition for promoting osteoblast differentiation according to claim 1, wherein the concentration of diosgenin in the composition is 1 to 3 µM.
The composition for promoting osteoblast differentiation according to claim 1, wherein the concentration of diosgenin in the composition is 1 μM.
Diosgenin (diosgenin) or a composition for preventing or treating bone metabolic diseases comprising a pharmaceutically acceptable salt.
The composition for preventing or treating bone metabolic disease according to claim 5, wherein the diosgenin is extracted from yam.
The composition for preventing or treating bone metabolic disease according to claim 5, wherein the concentration of diosgenin in the composition is 1 to 3 µM.
The composition for preventing or treating bone metabolic disease according to claim 5, wherein the concentration of diosgenin in the composition is 1 μM.
6. The method of claim 5, wherein the bone metabolic disease is osteoporosis, bone metastatic lesions, primary tumors produced in the bone, rheumatoid or degenerative arthritis, periodontal disease, inflammatory alveolar bone resorption disease, inflammatory bone absorption Disease and Paguet disease (Paget's disease) composition for the prevention or treatment of bone metabolism disease selected from the group consisting of.
Diosgenin (diosgenin) or a food composition for improving bone function comprising a pharmaceutically acceptable salt.

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