KR102161111B1 - Pharmaceutical composition for preventing or treating osteoporosis containing linalool as an active ingredient - Google Patents

Abstract

The present invention relates to a pharmaceutical composition for the prevention or treatment of osteoporosis containing linarool as an active ingredient, and the pharmaceutical composition according to the present invention contains the expression levels of osteoblast differentiation marker genes Dlx5, Runx2, Id1, OC. By increasing it, it has the effect of promoting the differentiation of osteoblasts, so it can be widely used as a pharmaceutical or health food for preventing or treating osteoporosis.
In addition, it is possible to fundamentally treat osteoporosis by promoting bone formation by increasing the activity of osteoblasts rather than targeting osteoclasts, and has the advantage of showing a secondary fracture prevention effect.

Description

Pharmaceutical composition for preventing or treating osteoporosis containing linalool as an active ingredient {Pharmaceutical composition for preventing or treating osteoporosis containing linalool as an active ingredient}

The present invention relates to a pharmaceutical composition for preventing or treating osteoporosis, containing linarool as an active ingredient.

Recently, as various industrial accidents and traffic accidents increase and entering an aging society, diseases of the musculoskeletal system due to osteoporosis and resulting fractures are increasing. For the prevention and treatment of such bone diseases, various types of bone graft agents for inducing bone regeneration ability and new types of related disease treatments for improving the activity of osteoblasts and reducing the activity of osteoclasts are being developed. In addition, various studies have been attempted using stem cells and osteoblasts to actively induce new bones that can fill areas with severe bone tumors and bone defects.

Bone remodeling is a process in which old bones are periodically converted into new bones, and this process proceeds through steps such as proliferation, differentiation, and induction of calcification of the extracellular matrix. In general, the process of bone formation and resorption in adults is balanced by the interaction of osteoblasts derived from mesenchymal stem cells and osteoclasts derived from hematopoietic stem cells to maintain bone health, but bone formation decreases due to low osteoblast activity. If there is an active imbalance between osteoblasts and osteoclasts, such as increased bone resorption due to increased activity of osteoclasts or increased bone resorption, there is no significant change in the chemical composition in the tissue, but bone mass decreases, causing bone metabolic diseases such as osteoporosis. Can be.

Osteoporosis is a complex disease that is also affected by genetic factors, diet, and environmental factors such as lifestyle, and especially in women, bone loss rapidly progresses due to hormonal changes when menopause.

Currently marketed osteoporosis treatments include vitamin D, female hormones, bisphosphonates, selective estrogen receptor modulators, and calcitonin agents, and most of them are based on a method that prevents bone loss by reducing the activity of osteoclasts to regulate bone resorption. . However, because osteoporosis treatments through inhibition of osteoclast activity are not fundamentally curable treatments for osteoporosis, there is a limit to cure, and an increase in osteoblast activity that can substantially increase bone density and strengthen bone is required.

In addition, since osteoporosis cannot be cured by only short-term administration of drugs, and long-term administration of drugs is essential, development of new substances having no side effects even when long-term administration of drugs and having excellent efficacy is required.

Accordingly, in recent years, studies on alternative therapies using active ingredients derived from natural products such as herbal medicinal herbs and foods that can promote bone formation while minimizing side effects and bone loss of existing treatments have been actively conducted.

On the other hand, linarool is a naturally occurring terpene found in many flowers and spices, including lavender and coriander, and is one of the most widely used substances to reduce stress by emitting floral fragrance. It is naturally biosynthesized by plants such as herbs, spices, trees, and citrus fruits, and more than 95% of them are used in cosmetics, soaps and perfumes as fragrances and deodorants, and only 1% of them give flavor and taste to foods and beverages. It is being used to pay.

In addition, as a study related to the treatment of diseases of the linarool component, the angiogenesis inhibitory effect of linarool has been reported, and in Korean Patent No. 10-1189763, the effect of linarool in treating central nervous system disorders has been disclosed.

However, no research results have been reported on the effect of linarool on the proliferation and differentiation of osteoblasts.

Korean Patent Registration No. 10-1189763

The inventors of the present invention were trying to develop a new pharmaceutical composition that essentially increases the activity of osteoblasts to substantially increase bone density and strengthen bone to treat osteoporosis, and that has less side effects even when taken for a long time. The present invention was completed by confirming that it promotes cell proliferation and differentiation.

Accordingly, it is an object of the present invention to provide a pharmaceutical composition and a health functional food having excellent efficacy in preventing and treating osteoporosis and having fewer side effects even when taken for a long time.

In order to achieve the above object, the present invention provides a pharmaceutical composition for the prevention or treatment of osteoporosis containing a compound represented by the following formula (1) as an active ingredient.

The pharmaceutical composition may be characterized by promoting osteoblast differentiation.

The compound represented by Formula 1 is of any one or more of osteoblast differentiation marker genes of Runx2 (runt-related transcription factor 2), Id1 (inhibitor of DNA binding 1), Dlx5 (distal-less homeobox 5) and OC (osteocalcin). It may be characterized by increasing the expression level.

The concentration of the compound represented by Formula 1 may be 0.0001 to 5 μM.

In addition, the present invention provides a health functional food for preventing or improving osteoporosis containing a compound represented by the following formula (1) as an active ingredient.

[Formula 1]

The pharmaceutical composition according to the present invention has the effect of promoting the differentiation of osteoblasts by increasing the expression levels of the osteoblast differentiation marker genes Dlx5, Runx2, Id1, and OC, so it is widely used as a pharmaceutical or health food for the prevention or treatment of osteoporosis. Can be used.

In addition, it is possible to fundamentally treat osteoporosis by promoting bone formation by increasing the activity of osteoblasts rather than targeting osteoclasts, and has the advantage of showing a secondary fracture prevention effect.

1 is a result of an MTT experiment confirming the viability of cells according to linarool concentration.
Figure 2 is a result of confirming the osteoblast differentiation gene expression in MC3T3-E1 cells cultured for 0, 2, and 4 days after treatment with linarool at a concentration of 1 μM.
3 is a result of comparing osteoblast differentiation gene expression in MC3T3-E1 cells treated with linarool, ascorbic acid, and beta-glycerophosphate mixtures, and MC3T3-E1 cells treated by mixing all of them.
4 is a result of ALP staining in MC3T3-E1 cells treated with linarool, ascorbic acid, and beta-glycerophosphate mixture, and MC3T3-E1 cells treated by mixing all of them.
5 is a result of ARS staining in MC3T3-E1 cells treated with linarool, ascorbic acid, and beta-glycerophosphate mixture, and MC3T3-E1 cells treated by mixing all of them.

Hereinafter, the present invention will be described in detail. The terms or words used in the present specification and claims should not be construed as being limited to their usual or dictionary meanings, and the inventor may appropriately define the concept of terms in order to describe his own invention in the best way. It should be interpreted as a meaning and concept consistent with the technical idea of the present invention based on the principle that there is.

The present invention relates to a pharmaceutical composition and health functional food for the prevention or treatment of osteoporosis.

In the present specification, the term osteoporosis refers to a condition in which the amount of bone is decreased and the strength of the bone is weakened due to qualitative changes, and prevention, improvement and treatment of osteoporosis include all diseases caused by bone loss and bone loss. Is interpreted as doing.

The present invention provides a pharmaceutical composition for preventing or treating osteoporosis containing a compound represented by the following Formula 1 as an active ingredient.

[Formula 1]

The compound represented by Formula 1 may be named linalool, and C ₁₀ H ₁₈ O It has a molecular formula and a molecular weight of 154 g/mol.

The compound represented by Formula 1 may be used in the form of a pharmaceutically acceptable salt, and an acid addition salt formed by a pharmaceutically acceptable free acid is useful. Acid addition salts include inorganic acids such as hydrochloric acid, nitric acid, phosphoric acid, sulfuric acid, hydrobromic acid, hydroiodic acid, nitrous acid, phosphorous acid, etc., aliphatic mono and dicarboxylates, phenyl-substituted alkanoates, hydroxy alkanoates and alkanes. It is obtained from non-toxic organic acids such as dioate, aromatic acids, aliphatic and aromatic sulfonic acids, acetic acid, benzoic acid, citric acid, lactic acid, maleic acid, gluconic acid, methanesulfonic acid, 4-toluenesulfonic acid, tartaric acid, fumaric acid, and the like. Examples of such pharmaceutically non-toxic salts include sulfate, pyrosulfate, bisulfate, sulfite, bisulfite, nitrate, phosphate, monohydrogen phosphate, dihydrogen phosphate, metaphosphate, pyrophosphate chloride, bromide, i. Odide, fluoride, acetate, propionate, decanoate, caprylate, acrylate, formate, isobutyrate, caprate, heptanoate, propiolate, oxalate, malonate, succinate, sube Rate, sebacate, fumarate, maleate, butine-1,4-dioate, hexane-1,6-dioate, benzoate, chlorobenzoate, methylbenzoate, dinitro benzoate, hydroxybenzoate, Methoxybenzoate, phthalate, terephthalate, benzenesulfonate, toluenesulfonate, chlorobenzenesulfonate, xylenesulfonate, phenylacetate, phenylpropionate, phenylbutyrate, citrate, lactate, β-hydroxybutyrate, Glycolate, malate, tartrate, methanesulfonate, propanesulfonate, naphthalene-1-sulfonate, naphthalene-2-sulfonate, mandelate, and the like.

The acid addition salt according to the present invention can be prepared by a conventional method, for example, a precipitate produced by dissolving the derivative of Formula 1 in an organic solvent such as methanol, ethanol, acetone, dichloromethane, acetonitrile, etc. and adding an organic or inorganic acid May be prepared by filtration and drying, or may be prepared by distilling off a solvent and an excess of acid under reduced pressure and then drying to crystallize under an organic solvent.

In addition, a pharmaceutically acceptable metal salt can be made using a base. The alkali metal or alkaline earth metal salt is obtained, for example, by dissolving the compound in an excess alkali metal hydroxide or alkaline earth metal hydroxide solution, filtering the undissolved compound salt, and evaporating and drying the filtrate. At this time, it is pharmaceutically suitable to prepare sodium, potassium or calcium salt as the metal salt. In addition, the corresponding salt is obtained by reacting an alkali metal or alkaline earth metal salt with a suitable silver salt (eg, silver nitrate).

Linarool, a compound represented by Chemical Formula 1, may be extracted from natural products or synthesized by a known method, and its preparation method or origin is not limited.

Linarool can be prepared by saponifying essential oil containing linarool as a main component with alcoholic potassium, and distilling the oil washed to neutrality under reduced pressure.

In addition, if boric acid equivalent to the linarool contained in the refinery is added and heated slowly under reduced pressure to remove the water produced, linarool becomes a boric acid ester, so the unreacted material is distilled off under reduced pressure and then the boric acid ester is converted to steam or alkali. It can be prepared by a method of distilling after washing the decomposed and liberated oil.

The pharmaceutical composition may be characterized by promoting osteoblast differentiation.

Compounds that promote the differentiation of osteoblasts can be used not only to prevent or treat diseases and symptoms such as osteoporosis and fractures, but also to treat periodontal diseases for the purpose of strengthening bone (Zhang et al., ShanghaiKou Qiang Yi) Xue 7(2), pp99-103, 1998; Cahill et al, Biol Blood Marrow Transplant 10(10), pp709-717, 2004), also reported that it can be useful for treating bone growth disorders caused by burns. (Klein et al., Osteoporos Int. 16(6), pp631-635, 2005).

The pharmaceutical composition can be used without limitation for diseases having problems with bone formation due to lack of osteoblasts, etc., specifically, bone destruction and inflammatory diseases caused by interleukin-1 (IL-1) due to lack of estrogen. Osteoporosis due to excessive bone resorption of osteoclasts, bone metastatic lesions, primary tumors in bone, rheumatoid or degenerative arthritis, periodontal disease, inflammatory bone resorption disease, inflammatory bone resorption disease And diseases that promote bone destruction with pathological bone diseases such as Paget's disease.

In the compound represented by Formula 1, the compound represented by Formula 1 is any of Runx2 (runt-related transcription factor 2), Id1 (inhibitor of DNA binding 1), Dlx5 (distal-less homeobox 5), and OC (osteocalcin). It may be characterized by increasing the expression level of one or more osteoblast differentiation marker genes.

Osteoblast differentiation involves hormones, cytokines such as Bone Morphogenetic Proteins (BMPs), Runx2 (runt-related transcription factor 2), Id1 (inhibitor of DNA binding 1), Dlx5 (distal-less homeobox 5), OC It is regulated by various transcription factors such as (osteocalcin) and ALP (alkaline phosphatase). Among them, Id1, Dlx5, and Runx2 are essential genes that are expressed in the early stages of osteoblast differentiation. The expression of these genes means that osteoblast differentiation is accelerated.

Specifically, Runx2 (runt-related transcription factor 2) is widely known as an important regulator in osteoblast differentiation, and in multipotent cells or osteoblasts, ALP (alkaline phosphatase), OC (osteocalcin), and bone sialoprotein (bone sialoprotein), which promotes osteoblast differentiation by regulating genes.

Id1 (inhibitor of DNA binding 1) is a transcription factor that affects the balance between growth and differentiation of cells in osteoblasts, and is known to play an important role in the early stages in osteoblast-specific differentiation. In addition, when the myoblast and mesenchymal stem cells are converted to osteoblast differentiation, differentiation is promoted by regulating the expression of the Id1 (inhibitor of DNA binding 1) gene.

Dlx5 (distal-less homeobox 5) is induced by bone and directly binds to the promoter of Runx2 (runt-related transcription factor 2) to regulate transcription, and expression of the osteocalcin (OC) gene, a late marker gene for osteoblast differentiation. By regulating osteoblast differentiation.

The concentration of the compound represented by Formula 1 may be 0.0001 to 5 μM.

More specifically, the concentration of the compound represented by Formula 1 may be 0.0001 to 5 μM, preferably 0.0001 to 2 μM, and more preferably 0.0001 to 1 μM.

If the concentration of the compound represented by Formula 1 is less than the above-described range, the pharmaceutical effect may be insignificant, and if the concentration exceeds the above-described range, cytotoxicity may occur.

The pharmaceutical composition may be administered orally or parenterally during clinical administration, and may be used in the form of a general pharmaceutical formulation.

The pharmaceutical composition may be characterized in that it is formulated in the form of a powder, granule, tablet, hard capsule, soft capsule or injection.

More specifically, oral dosage forms such as powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols, etc., external preparations, suppositories, sterile injection solutions, pre-filled injection solutions, or freeze-dried It may be formulated in a form, but is not limited thereto.

In the case of formulation, it can be prepared using diluents or excipients such as commonly used fillers, extenders, binders, wetting agents, disintegrants, and surfactants.

Solid preparations for oral administration include tablets, pills, powders, granules, capsules, and the like, and such solid preparations include at least one excipient, such as starch, calcium carbonate, sucrose, lactose, gelatin, etc. in the active ingredient. It can be prepared by mixing. In addition, in addition to simple excipients, lubricants such as magnesium stearate and talc may be used.

Liquid formulations for oral administration include suspensions, liquid solutions, emulsions, syrups, etc., and various excipients, such as wetting agents, sweeteners, fragrances, preservatives, etc., may be included in addition to water and liquid paraffin, which are commonly used simple diluents. have. Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized formulations, suppositories, and the like.

A preferred dosage of the pharmaceutical composition of the present invention may be appropriately selected according to the condition and weight of the patient, the degree of symptoms, the form of the drug, the route and duration of administration.

In addition, the present invention provides a health functional food for preventing or improving osteoporosis containing a compound represented by the following formula (1) as an active ingredient.

[Formula 1]

The compound represented by Formula 1 may be named linalool, and C ₁₀ H ₁₈ O It has a molecular formula and a molecular weight of 154 g/mol.

The health functional food may be characterized by promoting osteoblast differentiation.

In the compound represented by Formula 1, the compound represented by Formula 1 is any of Runx2 (runt-related transcription factor 2), Id1 (inhibitor of DNA binding 1), Dlx5 (distal-less homeobox 5), and OC (osteocalcin). It may be characterized by increasing the expression level of one or more osteoblast differentiation marker genes.

The concentration of the compound represented by Formula 1 may be 0.0001 to 5 μM.

More specifically, the concentration of the compound represented by Formula 1 may be 0.0001 to 5 μM, preferably 0.0001 to 2 μM, and more preferably 0.0001 to 1 μM.

If the concentration of the compound represented by Formula 1 is less than the above-described range, the pharmaceutical effect may be insignificant, and if the concentration exceeds the above-described range, cytotoxicity may occur.

The health functional food may be prepared in the form of, but not limited to, various beverages, gum, tea, confectionery, vitamin complexes, health supplements, and the like.

In addition, the health functional food may be added to health food for the purpose of improving osteoporosis, and there is no particular limitation on the type of food. Examples of foods to which the above substances can be added include meat, sausage, bread, chocolate, candy, snacks, confectionery, pizza, ramen, other noodles, gums, dairy products including ice cream, various soups, beverages, tea, drinks, There are alcoholic beverages and vitamin complexes, and it includes all health foods in the usual sense.

The preferred intake amount of the health functional food depends on the condition and weight of the eater, the degree of symptoms, the food type, and the intake period, and may be appropriately selected.

The differentiation of osteoblasts in the process of bone formation is regulated by the expression of genotypes, and has unique characteristics depending on the culture method. Important signaling systems involved in bone cell differentiation and bone formation are typically transforming growth factor-β (TGF-β), bone morphogenetic protein (BMP), and wint/beta-catenin. (Wnt/β-catenin), fibroblast growth factor (FGF), hedgehog, and notch are known.

This signaling system induces the expression and activation of various transcription factors related to bone formation such as Runx2 (Runt-related transcription factor 2) and Dlx5 (Distalless-related homeobox 5) during the bone cell differentiation process.Alkaline phosphatase (ALP) , Id1 (inhibitor of DNA binding 1), OC (Osteocalcin) to express bone differentiation related genes. Ascorbic acid (AA), beta-glycerophosphate (β-GP), dexamethasone, and the like are known as substances that induce differentiation in osteoblasts.

In the present invention, an experiment was conducted to investigate the effect of linarool on the proliferation and differentiation of osteoblasts using MC3T3-E1 preosteoblasts similar to osteoblasts present in bone tissue. Was confirmed to have a positive effect on the proliferation and differentiation of osteoblasts.

Hereinafter, examples and experimental examples will be described in detail in order to describe the present invention in detail. However, the embodiments according to the present invention may be modified in various different forms, and the scope of the present invention should not be construed as being limited to the embodiments described below. Embodiments of the present invention are provided to more completely describe the present invention to those of ordinary skill in the art.

<Experimental Example 1> MTT cytotoxicity test

(1) Material preparation and cell culture

Linalool used in this experiment was purchased from Sigmal aldrich, and mouse-derived MC3T3-E1 cells were purchased from ATCC (American Type Culture Collection, Manassas, VA, USA) and used.

Alpha-minimum essential medium (α-MEM) used in MC3T3-E1 cell culture, fetal bovine serum (FBS), antibiotics, trypsin-EDTA, GibcoBRL (GibxoBRL, Grandisland, NY, USA) 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) reagent purchased from Sigma (chem, co, St.Louis) used for cytotoxicity test Mo. USA).

MC3T3-E1 cells were cultured using α-MEM, and MC3T3-E1 cells attached to the bottom of the dish were washed with 2 μL of FBS, and dropped with 500 μL of Trypsine.

Using new α-MEM, cells were placed in an e-tube and then centrifuged (Hano, science).After centrifugation was completed, the supernatant was removed and transferred to a 15 μL conical tube. .

The number of cells was calculated using a hemocytometer and a cover glass, and 200 μL of α-MEM was added to a 48-well tissue culture test plate.

The measured cells were placed in a 48-well and then stored in an incubator (Model3154, Forma Scientific, USA).

(2) MTT analysis

In order to evaluate the viability and toxicity of cells, MTT analysis using absorption was performed. MTT, a tetrazolium salt, is only degraded by living cells, and reduces water-soluble MTT to purple formazan, an insoluble precipitate, which requires succinate dehydrogenase, a mitochondrial enzyme. It can also be used to determine the sensitivity of a drug if a quantitative relationship is established between the number of cells and the formazan produced.

First, MC3T3-E1 cells were each dispensed into 48-wells and stored in an incubator for 24 hours.

Linarool and dimethyl sulfoxide (DMSO) were diluted, added to each well, and stored in an incubator for 1 hour. The purple dyed culture medium was removed and washed with FBS. 400 μL of DMSO was added to each well, and absorbance was measured at 570 nm with a UV/visible spectrophotometer (Uvikon 922, Kontron, Italy).

(3) Experiment result

The MTT analysis results are shown in FIG. 1. As shown, it was confirmed that the activity was rapidly lowered at a concentration of 5 μM of linarool.

<Experimental Example 2> Expression analysis of osteoblast differentiation markers according to linarool treatment

(1) Experiment method

1) RNA extraction and cDNA synthesis

Trizol solution (Bioscience technology) was used to extract RNA. The cells were collected with a scraper, chloroform was added to each of 60 μL, and then vortexing was performed for 15 seconds.

Subsequently, centrifugation (14500 rpm, 4° C., 15 min) was performed, the supernatant portion containing RNA was transferred, and 150 μL of isopropyl was treated, followed by reaction at room temperature for 30 minutes. After 30 minutes, centrifugation (14500 rpm, 4° C., 10 min) was performed, the supernatant was removed, and 200 μL of 70% ethanol was added and washed.

After washing, centrifugation (13000 rpm, 4°C, 10 min) was performed, and the supernatant was removed and dried. 20 μL of distilled water was added each, and reacted at 4° C. for 30 minutes, and RNA was quantified after 30 minutes. After that, absorbance was measured, RNA, tertiary distilled water, and emeraldAmp GT PCR Master Mix (Takara Bio Inc, Japan) were added to 25 μL, and absorbance was measured.

2) Real-time polymerase chain reaction (RT-PCR)

PCR buffer, primers (β-actin, Runx2, Id1, OC, Dlx5), and tertiary distilled water were prepared to prepare a mixture. A PCR tube was prepared, and 19 μL of the mixture and 1 μL of cDNA were added. The tube was vortexed, centrifuged, and then put into a PCR equipment (Mygenie96, Bioneer, USA) and the equipment was operated. Primer sequences and operating conditions are shown in Tables 1 and 2. After that, electrophoresis was performed using 1% agarose gel, and the result was measured using UV.

gene Primer sequence Forward Reverse β-actin 5'-TTC TTT GCT CCT TCG TTG CCG-3' 5'-TGG ATG GCT ACG　TAC ATG GGG-3' Runx2 5'-CCG CAC GAC AAC CGC ACC AT-3' 5'-CGC TCC GGC CCA CAA ATC TC-3' Id1 5'-ATG AAG GTC GCC AGT GGC AGT-3' 5'-ACT TTG CGG TTC TGG GGC AGG-3' OC 5'-GCA AAT AAG GTA AAC AGA CTC C-3' 5'-GTT TGT AGG CGG TCT TCA AGC-3' Dlx5 5'-CAG AAG AGT CCC AAG CAT CC-3' 5'-GAG CGC TTT GCC ATA AGA AG-3'

gene Reaction conditions Cycle Number Pre-
Denaturation Denaturation Annealing Extention Post-
Extension hold β-actine 25 cycle 95℃ for 5 minutes 95℃ for 30 seconds 66℃ for 30 seconds 72℃ for 30 seconds 72℃ for 5 minutes 4℃ Runx2 30 cycle 95℃ for 5 minutes 95℃ for 30 seconds 62℃ for 30 seconds 72℃ for 30 seconds 72℃ for 5 minutes 4℃ Id1 30 cycle 95℃ for 5 minutes 95℃ for 30 seconds 67℃ for 30 seconds 72℃ for 30 seconds 72℃ for 5 minutes 4℃ OC 30 cycle 95℃ for 5 minutes 95℃ for 30 seconds 60℃ for 30 seconds 72℃ for 30 seconds 72℃ for 5 minutes 4℃ Dlx5 30 cycle 95℃ for 5 minutes 95℃ for 30 seconds 63℃ for 30 seconds 72℃ for 30 seconds 72℃ for 5 minutes 4℃

(2) Experiment result

1) Expression of osteoblast differentiation gene according to linarool treatment time

After treatment with a concentration of 1 μM linarool, gene expression was confirmed through RT-PCR for each sample cultured for 0, 2, and 4 days, and the gene expression was highest on the 4th day. As a result, it was confirmed that the osteoblast differentiation gene expression appeared depending on the linarool treatment time (see FIG. 2).

2) Expression of osteoblast differentiation gene according to condition treatment

Ascorbic acid (AA) and beta-glycerophosphate (β-GP) are used as substances that induce osteoblast differentiation, and ascorbic acid, beta-glycerophosphate mixture, and linarool are used alone. The gene expression levels were compared between those treated with and those treated by mixing them all.

At this time, cells cultured for 4 days at a concentration of 1 μM linarool were used.

As a result of comparison, it was confirmed that Id1 and Runx2 were the most dark when only linarool was added, and Dlx5 was the most concentrated when ascorbic acid, beta-glycerophosphate, and linarool were all mixed and treated (see FIG. ).

<Experimental Example 3> ALP (Alkaline phosphatas) staining experiment

(1) Experiment method

Dispense MC3T3-E1 cells into 24 wells, control, ascorbic acid (AA, Sigma) and beta-glycerophosphate (β-GP, Sigma) mixture, linarool, ascorb A mixture of acid, beta-glycerophosphate and linarool was added to each well.

At this time, cells cultured for 4 days at a concentration of 1 μM linarool were used.

Thereafter, it was stored at room temperature, washed with distilled water, prepared BCIP/NBT (sigma aldrich) was added and left at room temperature for about 15 minutes, and then washed again with distilled water and the degree of dyeing was checked.

(2) Experiment result

The results of the experiment are shown in FIG. 4. As shown, the staining results in the linarool-treated wells were shown, indicating that linarool increased ALP expression, and was expected to be helpful in the early stages of forming multiple layers of bone in the cell proliferation stage.

In addition, when all of ascorbic acid, beta-glycerophosphate, and linarool were mixed and treated, it was confirmed that the darkest dyeing showed the highest activity.

<Experimental Example 4> Alizarin red s staining analysis

(1) Experiment method

As osteoblast differentiation progresses, calcium is deposited on the extracellular matrix and calcification proceeds.To check this, control, ascorbic acid and beta-glycerophosphate mixture, linarool, and ascorb in each well. ARS staining was performed by adding an acid, beta-glycerophosphate, and linarool mixture in order.

At this time, cells cultured for 21 days at a concentration of 1 μM linarool were used.

10% formaldehyde (Merck KGaA, Darmstadt, Germany) was added to the well and allowed to stand for 15 minutes to fix the cells. Afterwards, it was washed twice with 3rd distilled water. 200 μL of 2% ARS standard (alizarin red solution) was put into each well, wrapped in foil and shaken lightly. After 15 minutes, it was washed with third distilled water, and the result was confirmed.

(2) Experiment result

The ability to form calcified nodules can be confirmed by Alizarin Red S staining as an important biomarker for the differentiation of osteoblasts. Alizarin stains calcium in the mineralized cell matrix, so the degree of calcified nodule formation and the degree of staining are proportional.

As shown in Figure 5, it was confirmed that darker staining appeared when the ascorbic acid and beta-glycerophosphate and linarool mixture were treated than when the ascorbic acid and beta-glycerophosphate mixture was treated.

As a result, it can be seen that linarool can have a positive effect on osteoblast differentiation.

Meanwhile, the compound represented by Formula 1 according to the present invention can be formulated in various forms depending on the purpose. The following illustrates some formulation methods in which the compound represented by Formula 1 according to the present invention is contained as an active ingredient, and the present invention is not limited thereto.

<Preparation Example>

1-1. Preparation of powder

500 mg of the compound of formula 1

100 mg lactose

Talc 10 mg

The above ingredients are mixed and filled in an airtight cloth to prepare a powder.

1-2. Manufacture of tablets

500 mg of the compound of formula 1

Corn starch 100 mg

100 mg lactose

2 mg of magnesium stearate

After mixing the above ingredients, tablets are prepared by tableting according to a conventional tablet preparation method.

1-3. Preparation of capsules

500 mg of the compound of formula 1

Corn starch 100 mg

100 mg lactose

2 mg of magnesium stearate

According to a conventional capsule preparation method, the above ingredients are mixed and filled into gelatin capsules to prepare a capsule.

1-4. Preparation of injections

500 mg of the compound of formula 1

Suitable amount of sterile distilled water for injection

proper amount of pH adjuster

It is prepared with the above ingredients per ampoule (2 ml) according to a conventional injection preparation method.

1-5. Preparation of liquid

100 mg of the compound of formula 1

10 g of isomerized sugar

5 g of mannitol

Purified water appropriate amount

According to the usual preparation method of liquid preparation, add and dissolve each component in purified water, add lemon scent, mix the above ingredients, add purified water, add purified water, adjust the total to 100 ml, and fill in a brown bottle. The liquid is prepared by sterilization.

Claims (5)

In vitro (in vitro), osteoblast differentiation promoting method comprising the step of treating osteoblasts with a compound represented by the following formula (1).
[Formula 1]

delete The method of claim 1,
The compound represented by Formula 1 is of any one or more of osteoblast differentiation marker genes among Runx2 (runt-related transcription factor 2), Id1 (inhibitor of DNA binding 1), Dlx5 (distal-less homeobox 5), and OC (osteocalcin). Osteoblast differentiation promoting method, characterized in that to increase the expression level.
The method of claim 1,
The method for promoting osteoblast differentiation, characterized in that the concentration of the compound represented by Formula 1 is 0.0001 to 5 μM.
delete

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