KR20220027641A - A composition for prevention or treatment of bone disease comprising methyl-cinnamate - Google Patents

Abstract

The present invention relates to a composition for preventing, alleviating or treating bone diseases, including methyl-cinnamate, and a method for manufacturing the same. The composition comprising the methyl-cinnamate of the present invention has an effect of promoting apoptosis, regulating migration and differentiation of osteoblasts, thereby being able to be used for treatment or prevention of bone diseases.

Description

A composition for preventing or treating bone disease, including methyl-synamate {A COMPOSITION FOR PREVENTION OR TREATMENT OF BONE DISEASE COMPRISING METHYL-CINNAMATE}

The present invention relates to a composition for preventing, ameliorating or treating bone disease, including methyl-cinnamate, and a method for preparing the same.

Bone is an active tissue that constantly changes throughout life. Bone is visually divided into external cortical bone (compact bone) and internal trabecular bone (cancellous bone, spongy bone). It plays a role in keeping the change of

Even after bone growth has stopped, old bones are destroyed (bone resorption) and fixation (osteogenesis) to fill in the missing new bone is repeated throughout life. This phenomenon is called bone remodeling. When the interaction between osteoblasts and osteoclasts is balanced, and bone resorption and bone formation are balanced, bone homeostasis is maintained and the calcium concentration in the blood is kept constant. Bone calcium is released into the blood as absorption is increased, and if bone resorption continues, bones become weak and diseases such as osteoporosis occur.

In particular, osteoporosis is characterized by postmenopausal osteoporosis, which is caused by an increase in bone resorption due to the activation of osteoclasts due to rapid hormonal changes following menopause, and osteoporosis after menopause, in which the function of osteoblasts decreases with aging. It can be classified as senile osteoporosis. Fractures due to osteoporosis lead to severe activity limitation, and since hip fractures are associated with a high mortality rate of about 15-35%, diagnosis and treatment of osteoporosis before osteoporotic fractures occur is important.

In addition, Paget's disease is a disease in which bone resorption occurs due to abnormal bone metabolism and bone formation increases. Although the size of the bone increases, the formed bone is an irregular mosaic-shaped lamellar bone, and the bone tissue is structurally light, soft, and porous. Pathological fractures occur and various types of tumors and nocturnal lesions occur in the invaded bone as a complication. In particular, osteosarcoma associated with Paget's disease has a very poor prognosis, but effective drugs have not been developed to treat Paget's disease.

Therefore, for the fundamental treatment of bone diseases, there is an urgent need to develop small molecule bone control drugs that can not only restore lost bone mass but also control abnormalities in bone metabolism.

Korean Patent Registration No. 10-0895613

It is an object of the present invention to provide a pharmaceutical composition for preventing or treating bone disease, including methyl-cinnamate.

Another object of the present invention is to provide a method for preventing or treating bone disease using methyl-synamate.

Another object of the present invention is to provide a food composition for preventing or improving bone disease, including methyl-cinnamate.

Another object of the present invention is to provide a method for preparing a composition for regulating apoptosis and differentiation of osteoblasts, comprising the step of isolating methyl-synamate from the extract of A. katsumadai .

One aspect of the present invention relates to a pharmaceutical composition for preventing or treating bone disease, including methyl-cinnamate (methyl-cinnamate).

The methyl-cinnamate may be represented by Formula 1 below.

In the present invention, cinnamate is an organic compound having a chemical formula of C ₆ H ₅ CHCHCO ₂ H, and the methyl-cinnamate is methyl synthesized from cinnamic acid by cinnamic acid methyltransferase. It is a methyl ester compound. The synamic acid is an antioxidant phytochemical agent and is known as a safe flavoring agent used in the food industry.

Although the antibacterial and antifungal pharmacological properties of the methyl-synamate have been proven, the pharmacological effect on the biological action in osteoblasts has not been identified. Accordingly, the present inventors have identified that the methyl-synamate compound is involved in inhibiting bone resorption by regulating the function of osteoblasts. do.

Specifically, the bone disease is osteoporosis, periodontal disease, osteopetrosis, Paget's disease, osteomalacia, osteopenia, bone atrophy, fiber fibrous dysplasia, Paget's disease, hypercalcemia, neoplastic destruction of bone, osteolysis, osteoarthritis or rheumatoid arthritis .

The methyl-synamate may regulate apoptosis, migration and/or differentiation of osteoblasts.

In one embodiment of the present invention, methyl-synamate inhibits the expression of Bcl-2 family and survivin proteins, which are anti-apoptotic factors, while activating caspase-3 that induces PARP cleavage to induce apoptosis. It was confirmed that it is possible ( FIGS. 5 and 6 ).

In an embodiment of the present invention, it was confirmed that methyl-synamate effectively inhibited the migration of osteoblast progenitors (FIG. 8), and further reduced the activities of ERK1/2, JNK and p38 (FIG. 7). This shows that methyl-synamate can regulate cell migration and proliferation through ERK1/2, JNK and p38 signaling pathways.

In addition, in one embodiment of the present invention, it was confirmed that the expression level of ALP, an osteoblast differentiation-related marker, was reduced during methyl-synamate treatment, and it was confirmed that osteoblast differentiation could be inhibited through various pathways (FIG. 9). .

The above results suggest that methyl-synamate has excellent biological effects related to the regulation of cell death, migration and osteoblast differentiation, and suggests that it can be utilized for prevention, improvement and treatment of bone-related diseases.

For administration, the pharmaceutical composition of the present invention may include a pharmaceutically acceptable carrier, excipient or diluent in addition to the methyl-cinnamate of the present invention. The carrier, excipient and diluent include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, gum acacia, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil.

In addition, the pharmaceutical composition of the present invention can be applied in any dosage form, and oral dosage forms may be powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols, and the like. Formulations for parenteral use may be in the form of injections, coatings, sprays, such as aerosols.

The solid preparation for oral administration includes tablets, pills, powders, granules, capsules, etc., and these solid preparations include at least one excipient in the methyl-cinnamate, for example, starch, calcium, carbonate, sucrose, lactose, Alternatively, it can be prepared by mixing gelatin or the like. In addition to the above excipients, lubricants such as magnesium stearate and talc may be used.

As the liquid formulation for oral administration, suspensions, solutions, emulsions, syrups, etc. may be used, and various excipients such as wetting agents, sweeteners, fragrances, preservatives, etc. may be used in addition to simple diluents such as water, liquid, and paraffin. can

Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solutions, suspensions, emulsions, lyophilized formulations, and suppositories. Non-aqueous solvents and suspending agents include propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate.

Another aspect of the present invention relates to a method for treating a bone disease, comprising administering a pharmaceutical composition comprising methyl-cinnamate to a subject in need of treatment in a pharmaceutically effective amount. Specifically, bone diseases include osteoporosis, periodontal disease, osteopetrosis, Paget's disease, osteopenia, bone atrophy, and fibrous dysplasia. , hypercalcemia, neoplastic destruction of bone, osteolysis, osteoarthritis or rheumatoid arthritis.

In the present invention, "pharmaceutically effective amount" means an amount sufficient to treat a disease with a reasonable benefit/risk ratio applicable to medical treatment, and the effective dose level is the patient's sexually transmitted disease, age, type of disease, severity, drug activity, sensitivity to drugs, administration time, administration route and excretion rate, duration of treatment, factors including concurrent drugs, and other factors well known in the medical field.

The pharmaceutical composition of the present invention may be administered as an individual therapeutic agent or may be administered in combination with other therapeutic agents, and may be administered sequentially or simultaneously with a conventional therapeutic agent. In addition, the pharmaceutical composition of the present invention may be administered single or multiple. Taking all of the above factors into consideration, it is important to administer an amount that can obtain the maximum effect with a minimum amount without side effects, and can be easily determined by those skilled in the art.

As used herein, the term “subject” includes animals or humans with bone disease whose symptoms can be improved by administration of the pharmaceutical composition according to the present invention. By administering the composition for treatment according to the present invention to an individual, bone disease can be effectively prevented and treated.

The term "administration" of the present invention means introducing a predetermined substance into a human or animal by any suitable method, and the administration route of the therapeutic composition according to the present invention is through any general route as long as it can reach the target tissue. It may be administered orally or parenterally. In addition, the therapeutic composition according to the present invention may be administered by any device capable of moving the active ingredient to the target cell.

The preferred dosage of the pharmaceutical composition according to the present invention varies depending on the patient's condition and weight, the severity of the disease, the drug form, the route and duration of administration, but may be appropriately selected by those skilled in the art.

Another object of the present invention relates to a food composition for preventing or improving bone disease, including methyl-cinnamate.

The descriptions related to “methyl-synamate” and “bone disease” are the same as described above.

Foods to which the methyl-cinnamate of the present invention can be added include meat, bread, sausage, chocolate, snacks, candy, confectionery, ramen, pizza, other noodles, gum, dairy products including ice cream, various soups, beverages, There are teas, vitamin complexes, and health functional supplements.

The type of the food may specifically be a health functional food. The health functional food includes various nutrients, vitamins, minerals (electrolytes), synthetic flavoring agents and flavoring agents such as natural flavoring agents, coloring agents and enhancers (cheese, chocolate, etc.), pectic acid and salts thereof, organic acids, protective colloid increase Agents, pH adjusters, stabilizers, preservatives, glycerin, alcohol, carbonation agents used in carbonated beverages, and the like may be contained. These components may be used alone or in combination, and the proportion of these additives is generally selected in the range of 0.001 to 50 parts by weight based on the total weight of the composition.

The health functional food is a food that emphasizes the bioregulatory function of food, and is a food with added value to act and express for a specific purpose using a physical, biochemical, and bioengineering method. The ingredients of these health functional foods are designed and processed to sufficiently exert the body control functions related to the body defense, regulation of body rhythm, prevention and recovery of diseases, and are food supplementary additives, sweeteners or functionalities that are acceptable as food. It may contain raw materials.

The health functional food may be in any one formulation selected from the group consisting of tablets, granules, powders, capsules, liquid solutions and pills, but is not limited thereto. Specifically, the health functional food in the form of tablets is granulated with methyl-cinnamate, excipients, binders, disintegrants and other additive mixtures in a conventional manner, and then a lubricant is added and compression-molded, or the mixture is directly compression-molded. can be manufactured. In addition, the health functional food in the form of tablets may contain a bittering agent, etc., if necessary, and may be coated with a suitable skinning agent if necessary.

Among the health functional foods in the form of capsules, hard capsules can be prepared by filling conventional hard capsules with a mixture of methyl-cinnamate and additives such as excipients, or granules thereof or coated granules. Soft capsules can be prepared by filling a capsule base such as gelatin with a mixture of methyl-cinnamate and additives such as excipients. The soft capsules may contain a plasticizer such as glycerin or sorbitol, a colorant, a preservative, and the like, if necessary.

The health functional food in the ring form can be prepared by molding a mixture of methyl-cinnamate, excipients, binders, disintegrants, and the like in an appropriate way, and if necessary, the skin is coated with sucrose or other suitable skinning agent, or starch, talc, or You can also dress the gown with a suitable material.

The health functional food in the form of granules may be prepared in the form of a mixture of methyl-cinnamate, an excipient, a binder, a disintegrant, and the like, in a suitable manner, and may contain a flavoring agent, a bittering agent, etc. as necessary.

The definitions of the excipients, binders, disintegrants, lubricants, bittering agents, flavoring agents, etc. are known in the art and may include the same or similar ones for their functions.

In addition, the type of the food may be a food additive, and the food additive refers to a substance used by adding, mixing, infiltrating, or other methods to food for manufacturing, processing, or preservation of food. The food additives include natural products and synthetic products, and can be classified according to functions and uses. Currently, about 370 chemically synthesized products and 50 kinds of natural additives are approved as food additives in Korea. Preservatives, disinfectants, antioxidants, coloring agents, coloring agents, bleaching agents, seasonings, sweeteners, flavoring agents, expanding agents, strengthening agents, and improving agents are mainly used depending on the use. , emulsifier, thickener (fog) and stabilizer, film agent, gum base agent, defoaming agent, solvent, mold release agent, insect repellent, quality improver, and other additives for food manufacturing.

The form of the food additive may include powder, granule, tablet, capsule or liquid form, and specifically may be in the form of a capsule, but is not limited thereto.

When the methyl-cinnamate of the present invention is used as a food composition, the methyl-cinnamate may be added as it is or used with other foods or food ingredients, and may be appropriately used according to a conventional method. The mixing amount of the methyl-cinnamate may be appropriately determined depending on the purpose of its use (prevention, health or improvement, therapeutic treatment).

Another object of the present invention relates to a method for preparing a composition for regulating the differentiation of osteoblasts, comprising the step of isolating methyl-synamate from the extract of A. katsumadai .

In the present invention, "chodugu ( A. katsumadai )" is a drug used for the treatment of vomiting and stomach diseases. The seeds of the celery extract were used as antioxidants and placebos, and various biologically active compounds such as flavonoids, stilbenes, chalcones, monoterpenes and sesquiterpenoids holds the

The methyl-cinnamate may be isolated from the seeds of the extract of cinnamon. In addition, the methyl-cinnamate may be obtained by extraction with a single or a mixed solvent of the cinnamon extract, and any known solvent may be used.

Specifically, the extract is selected from the group consisting of water, C ₁ to C ₄ anhydrous or lower alcohol, a mixed solvent of water and lower alcohol, acetone, 1,3-butylene glycol, ethyl acetate, and chloroform It may be obtained by extraction with one or more single or mixed solvents, but is not limited thereto.

In addition, the 'extract' is not only the extract, but also a diluted or concentrated solution of the extract, a dried product obtained by drying the extract, a preparation or purified product of the extract, or a mixture thereof, etc. Using the extract itself and the extract Extracts of all formable formulations are included.

The extract may be extracted from the natural, hybrid, or mutated plants of the choucifera, and may also be extracted from the culture of plant tissues.

The method of extracting the extract of the present invention is not particularly limited, and may be extracted according to a method commonly used in the art. Non-limiting examples of the extraction method include hot water extraction, ultrasonic extraction, filtration, reflux extraction, and the like, and these may be performed alone or in combination of two or more methods.

In the present invention, the extract extracted with hot water or chilled is filtered to remove floating solid particles, for example, filtered using nylon, etc. or filtered using freeze filtration, etc. It can be used by drying it using spray drying or the like.

In an embodiment of the present invention, it was confirmed that methyl-synamate isolated from the extract of chodugoo was effective in promoting apoptosis of osteoblasts and regulating the migration and differentiation of osteoblasts. This suggests that the methyl-synamate isolated from the extract of chrysanthemum can be utilized for regulating the function of osteoblasts.

The composition comprising the methyl-synamate of the present invention has the effect of promoting apoptosis, regulating migration and differentiation of osteoblasts, and thus can be used for treatment or prevention of bone diseases. In addition, the methyl-cinnamate is a safe fragrance used in the food industry, and can improve the bone disease improvement effect while minimizing side effects.

The effect of the present invention is not limited to the above effect, but it should be understood to include all effects that can be inferred from the configuration of the invention described in the detailed description or claims of the present invention.

Figure 1 shows the structure and separation schematic diagram of methyl-cinnamate (methyl-cinnamate) (A: methyl-cinnamate structure, B: methyl-cinnamate separation schematic diagram).
2 shows the results of high performance liquid chromatography (HPLC) measurement for confirming the methyl-synamate structure.
3 shows the results of cytotoxicity analysis in osteoblast progenitor cells after treatment with methyl-synamate 1, 10, 30 and 100 μM (A: after treatment for 24 hours, B: after treatment for 48 hours).
4 shows the results of observing the morphological change of methyl-synamate with a phase-contrast microscope.
5 shows the results of methyl-synamate induction of apoptosis in osteoblastic progenitor cells (AC: expression levels of cleaved PARP and caspase-3, DF: expression levels of survivin and Bcl-2).
6 shows the results of TUNEL analysis of methyl-synamate.
7 shows the results of analyzing the effect of methyl-synamate on MAPKs activity in osteoblastic progenitor cells.
8 shows the results of analyzing whether or not methyl-synamate osteoblastic progenitor cells migrated.
9 shows the results of analyzing the differentiation effect of methyl-synamate on osteoblast progenitor cells (A: ALP staining, B: light microscopy detection, C: ALP activity measurement).

Hereinafter, embodiments of the present invention will be described in detail with reference to the accompanying drawings, but it is apparent that the present invention is not limited by the following examples.

Preparation Example 1. Separation of methyl-cinnamate (methyl-cinnamate)

10 kg of Cauliflower was extracted with 80% ethanol three times for 3 days at room temperature. 3 L of distilled water was added to the concentrated 80% ethanol extract and suspended. Solvent fractionation was performed using two separatory funnels using equal amounts of n -hexane, ethyl acetate (EtOAc) and n -butanol. Silica column chromatography was performed on 300 g of the ethyl acetate fraction. 300 g of the ethyl acetate fraction was adsorbed onto silica gel 70-230 mesh to solidify, and then loaded onto a column. As a developing solvent, normal hexane and ethyl acetate were used at a rate to increase the polarity, so that a total of 10 Fractions were obtained (hereinafter referred to as 'ALKAE-1 to ALKAE-10'). HPLC was performed on 670 mg of the obtained ALKAE-2, and the developing solvent was eluted with a gradient system of n-hexane and ethyl acetate to elute 10 small fractions (hereinafter 'ALKAE-2-1 ~ ALKAE-2-10'). named). Open column chromatography using Sephadex LH-20 filler was performed on 133 mg of ALKAE-2-3 in the small fraction, and a column having a diameter of 2.5 cm and a length of 40 cm was used. Only methylol was used as the developing solvent, and as a result, a total of six fractions were obtained (hereinafter referred to as 'ALKAE-2-3-1 ~ ALKAE-2-3-6'). At 40 mg of ALKAE-2-3-3, a pale off-white crystalline material was obtained, and as a result, the (E)-methyl-cinnamate ((E)-methyl-cinnamate) was obtained ( FIGS. 1 and 2 ). ).

colorless amorphous powder; electron ionization mass spectrometer (EI-MS) m/z 162 [M] ⁺ ; molecular formula C ₁₀ H ₁₀ O ₂ ; ¹ H-NMR (500 MHz, CD ₃ OD) δ 7.67 (1H, d, J = 16.1 Hz, H-7), 7.57 (2H, m, H-2, H-6), 7.38 (3H, m, H-3, H-4, H-5), 6.50 (1H, d, J = 16.0 Hz, H-8), 3.77 (3H, s, OCH ₃ ); ¹³ C-NMR (500 MHz, CD ₃ OD) δ 169.1 (C-9), 146.4 (C-7), 135.8 (C-1), 131.6 (C-4), 130.1 (C-2, C-6) ), 129.4 (C-3, C-5), 118.7 (C-8), 52.3 (OCH ₃ ).

Preparation Example 2. Osteoblastic progenitor cells MC3T3-E1 cell culture and osteoblast differentiation

Osteoblast progenitor MC3T3E-1 cells (#CRL-2593) purchased from the American Type Culture Collection (ATCC) (VA, Manassas, VA) were provided by the Bioevaluation Center (Korea Research Institute of Bioscience and Biotechnology).

The cells were cultured in α-minimum essential medium without L-ascorbic acid (WELGEME, Inc., Korea) supplemented with 10% fetal bovine serum (FBS), penicillin (100 units/mL) and streptomycin (10 g/mL) (WELGEME, Inc., Korea). α-minimum essential medium) in a humid atmosphere of 37 °C, 5% CO ₂ and 95% air.

Osteoblast differentiation was achieved by changing the osteogenic supplement medum (OS) containing 50 g/mL L-ascorbic acid and 10 mM β-glycerophosphate (Sigma-Aldrich, MO, St. Louis, MO). was induced by The medium was changed every 2 days during the incubation period.

Experimental Example 1. Confirmation of methyl-synamate cytotoxicity and morphological changes

Cell viability was determined using the MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) (Sigma-Aldrich, St.Louis, MO) assay for NADH-dependent dehydrogenase (NADH-dependent). dehydrogenase aictivity) activity was detected.

A solution of MTT in phosphate buffered saline (PBS) was added to the cells and then incubated for 2 hours to allow the MTT to be metabolized to formazan. After solubilization using 100% DMSO (Sigma-Aldrich, St. Louis, MO), absorbance was measured at a wavelength of 540 nm using a Thermo Scientific Multiskan GO microplate reader (Thermo Scientific, Waltham, MA, USA).

In addition, it was observed with a phase contrast microscope (phase contrast microscopy) after treatment with methyl-cinnamate for 48 hours.

As a result, methyl-synamate did not have a significant effect on the proliferation of the MC3T3E-1 cell line, and it was confirmed that methyl-synamate at a concentration of 10 to 30 μM was an appropriate concentration (FIG. 3).

Experimental Example 2. Confirmation of apoptosis-inducing effect in osteoblastic progenitor cells of methyl-synamate

In the progenitor cells of osteoblasts, an experiment was performed to confirm the apoptosis and anti-apoptotic protein expression regulating effect of methyl-synamate.

First, the cells were washed twice with ice-cold PBS, and a mixture of protease inhibitors (0.1 mM PMSF, 5 mg/mL aprotinin, 5 mg/mL pepstatin A, and 1 mg/ml kyestatin) was added. It was dissolved in 20 mM Tris-HCl buffer (pH 7.4) containing Protein concentration was determined using Bradford reagent (Bio-Rad, Hercules, CA). An equal amount of 20 μg of the lysate was resolved by sodium dodecyl-polyacrylamide gel electrophoresis (SDS-PAGE), and then transferred to a polyvinylidene fluoride (PVDF) membrane (Millipore, Bedford, MA). The membranes were then blocked with 1 × TBS containing 0.05% Tween 20 (TBST) and 5% skim milk for 1 h at room temperature. After blocking, the membranes were incubated overnight at 4° C. with each primary antibody, washed with 1 × TBST, and then diluted horseradish peroxidase (HRP)-conjugated secondary antibody (1 : 10,000, Jackson ImmunoResearch, West Grove, PA) was washed at room temperature for 2 h, and then bound antibody was detected using an enhanced chemiluminescence (ECL) kit (Millipore, Bedford, MA).

As a result, methyl-synamate activates caspase-3 in osteoblasts and induces cleavage of poly(ADP-ribose)polymer poly(PARP), while anti-apoptotic protein survivin and It was confirmed that the expression level of Bcl-2 was reduced (FIG. 5).

In addition, to detect DNA strand breakage and fragmentation, transferase-mediated FITC-Dudp nick-end labeling (TUNEL) analysis was performed. TUNEL analysis was performed using the in situ Cell Death Detection Kit (Roche Diagnostics GmbH, Mannheim, Germany) according to the manufacturer's instructions. For 4'6-diamidino-2-phenylindole (DAPI) (Sigma-Aldrich) staining, cells were incubated for 15 min in the dark at room temperature. TUNEL-positive and DAPI-stained cells were observed using a confocal microscope (K1-Fluo confocal laser scanning microscope, Korea).

As a result, TUNEL-positive and DAPI-stained cells, that is, apoptotic cells, were significantly increased during methyl-synamate treatment ( FIG. 6 ), which indicates that methyl-synamate is an apoptosis-related protein in osteoblastic progenitor cells. It suggests an important role in the regulation of programmed cell death through regulation.

Experimental Example 3. Effect of Methyl-Synamate on Reducing MAPKs Signaling and Cell Migration in Osteoblastic Progenitor Cells

Since mitogen-activated protein kinases (MAPKs) including ERK1/2, JNK and p38 cascades are involved in apoptosis, the effect of methyl-synamate was investigated using Western blot. The Western blot method is the same as the method of 2 in the above experiment.

In addition, it was evaluated using a wound-healing assay to determine whether apoptosis affects the migration of osteoblast progenitors. After the osteoblast progenitor cells were aliquoted in a 5-well culture plate, they were grown for 24 hours at 37° C. in a humidified atmosphere of 5% CO ₂ and 95% air. Cells were wound with a 200 μL pipette tip, washed with 1 X PBS to remove cell debris, and then incubated for 24 h in the absence and presence of methyl-synamate. Cell images were compared to quantify cell migration rates using light microscopy.

As a result, it was confirmed that phosphorylation of ERK1/2 was significantly inhibited, JNK and p38 were inactivated (FIG. 7), and migration of osteoblast progenitor cells was significantly inhibited (FIG. 8) during methyl-synamate treatment. . This shows that apoptosis of methyl-synamate can have a significant effect on cell migration inhibition.

Experimental Example 4. Confirmation of the inhibitory effect of methyl-synaptic on osteoblast differentiation (differentiation)

In order to confirm the effect of methyl-synamate on the differentiation of osteoblasts, osteoblast differentiation was induced as in Preparation Example 2.

First, the cells were washed with PBS and then fixed in 10% formalin for 15 minutes at room temperature. After washing with distilled water, the cells were incubated with a substrate solution for ALP reaction at 37° C. for 1 hour according to the manufacturer's protocol (Takara Bio Inc., Japan). ALP staining was measured with a digital camera and a colorimetric detector (ProteinSimple Inc., Santa Clara, CA).

Cell lysates were obtained using an alkaline phosphatase activity colorimetric assay kit (Biovision, Milpitas, CA). Absorbance was measured at 405 nm using a Multiskan GO microplate spectrophotometer (Thermo Fisher Scientific, Waltham, MA).

As a result, it was confirmed through an optical microscope that ALP-positive stained cells were reduced when methyl-synamate was treated (FIGS. 9A, B), and it was confirmed that ALP enzyme activity was significantly reduced under the same conditions (FIG. 9C).

Through the above results, it was confirmed that methyl-synamate can regulate apoptosis mechanism, increase apoptosis in osteoblast progenitors, and reduce cell migration and osteoblast differentiation by regulating MAPKs and activity. This proves that methyl-synamate plays a pharmacological and biological role in the survival, migration and osteoblast differentiation of osteoblastic progenitors, suggesting that methyl-synamate can be utilized in the treatment of bone diseases.

The description of the present invention described above is for illustration, and those of ordinary skill in the art to which the present invention pertains can understand that it can be easily modified into other specific forms without changing the technical spirit or essential features of the present invention. will be. Therefore, it should be understood that the embodiments described above are illustrative in all respects and not restrictive. For example, each component described as a single type may be implemented in a dispersed form, and likewise components described as distributed may also be implemented in a combined form.

The scope of the present invention is indicated by the following claims, and all changes or modifications derived from the meaning and scope of the claims and their equivalents should be construed as being included in the scope of the present invention.

Claims (7)

A pharmaceutical composition for preventing or treating bone disease, comprising methyl-cinnamate. The method of claim 1,
The methyl-synamate is a pharmaceutical composition for preventing or treating bone disease, which modulates osteoblast differentiation (differentiation). The method of claim 1,
The bone disease is osteoporosis, periodontal disease, osteopetrosis, Paget's disease, osteopenia, bone atrophy, fibrous dysplasia, Hypercalcemia (hypercalcemia), neoplastic destruction of bone (neoplastic destruction), osteolysis (osteolysis), osteoarthritis (osteoarthritis) or rheumatoid arthritis (rheumatoid arthritis) will, a pharmaceutical composition for the prevention or treatment of bone diseases. A food composition for preventing or improving bone disease, comprising methyl-synamate. 5. The method of claim 4,
The bone disease is osteoporosis, periodontal disease, osteopetrosis, Paget's disease, osteopenia, bone atrophy, fibrous dysplasia, Hypercalcemia (hypercalcemia), neoplastic destruction of bone (neoplastic destruction), osteolysis (osteolysis), osteoarthritis (osteoarthritis) or rheumatoid arthritis (rheumatoid arthritis) will, the prevention or improvement of bone disease food composition. Chodugu ( A. katsumadai ) A method for preparing a composition for regulating the differentiation of osteoblasts, comprising the step of isolating methyl-synamate from the extract. 7. The method of claim 6,
The extract is one or more solvents selected from the group consisting of water, C ₁ to C ₄ anhydrous or lower alcohol, a mixed solvent of water and lower alcohol, acetone, 1,3-butylene glycol, ethyl acetate, and chloroform. A method for preparing a composition for regulating the differentiation of osteoblasts, which is obtained by extraction.

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